Iranian Journal of

Iranian Journal of

Reproductive Medicine VOLUME 9 NUMBER 3 Summer 2011 ISSN: 1680-6433

Published by: Yazd Research & Clinical Center for Infertility

In collaboration with: Iranian Society for Reproductive Medicine

CHAIRMAN MANAGER Vahidi, Serajedin M.D. EDITOR-IN-CHIEF Aflatoonian, Abbas M.D. MANAGING EDITOR Anvari, Morteza Ph.D. EXCUTIVE BOARD Abdoli, Ali Mohammad M.D. Asadzadeh, Kobra B.S. Khani, Parisa M.D. Mortazavifar, Zahrasadat B.S. ENGLISH EDITOR Sheikhha, Mohammad Hasan M.D., Ph.D.

EDITORIAL BOARD

Ahmadi, Ali Ph.D. (USA)

Al-Hassani, Safa Ph.D. (GERMANY) Hosseini, Ahmad Ph.D. (IRAN) Hosseini, Seyed Jalil M.D. (IRAN) Kalantar, Seyed Mehdi Ph.D. (IRAN) Karimzadeh Meybodi, Mohammad Ali M.D. (IRAN) Kazemeyni, Seyed Mohammad M.D. (IRAN)

Khalili, Mohammad Ali Ph.D. (IRAN)

Lenton, Elizabeth Ann Ph.D. (UNITED KINGDOM)

Monsees, Thomas Ph.D. (GERMANY)

Moini, Ashraf M.D. (IRAN)

Nasr-Esfahani, Mohammad Hossein Ph.D. (IRAN)

Pour-Reza, Maryam M.D. (IRAN)

Pourmand, Gholamreza M.D. (IRAN)

Yasini, Seyed Mojtaba M.D. (IRAN)

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Iranian Journal of Reproductive Medicine

VIII

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IX

REVIEW ARTICLE

Hormonal treatment for endometriosis associated pelvic pain ................................................. 163

Wong WSF, Lim CED.

ORIGINAL ARTICLES

GnRH antagonist versus agonist in normoresponders undergoing ICSI: a randomized clinical

trial in Iran ..................................................................................................................................... 171

Tehraninejad E, Ghahghaei Nezamabadi A, Rashidi B, Sohrabi M, Bagheri M, Haghollahi F,

AzimiNekoo E, Jafarabadi M.

Effect of benzene extract of Ocimum sanctum leaves on cauda epididymal spermatozoa of

rats ................................................................................................................................................... 177

Ahmed M, Ahamed RN, Aladakatti RH, Ghodesawar MAG.

Icodextrin reduces adhesion formation following gynecological surgery in rabbits ............... 187

Khani B, Bahrami N, Mehrabian F, Naderi Naeni H.

Correlation between the level of cholesteryl ester transfer protein follicular fluid with

fertilization rates in IVF/ ICSI cycles ........................................................................................... 193

Mehdizadeh A, Rahimipour A, Farzadi L, Darabi M, Shahnazi V, Shaaker M, Vatankhah AM,

Golmohamadi Z, Nouri M.

Diagnostic value of saline contrast sonohysterography comparing with hysteroscopy for

detecting endometrial abnormalities in women with abnormal uterine bleeding ................... 199

Karimzadeh MA, Dehghani Firouzabadi R, Goharzad F.

Successful pregnancy following the transfer of vitrified blastocyst which developed from poor

quality embryos on day 3............................................................................................................... 203

Zhang X, Yang Y, Min L, Lv Q, Bai P, Li XJ, Liao M.

Maturation capacity, morphology and morphometric assessment of human immature oocytes

after vitrification and in-vitro maturation................................................................................... 209

Nazari S, Khalili MA, Esmaielzadeh F, Mohsenzadeh M.

Antifertility activity of aqueous ethanolic extract of Hymenocardia acida stem bark in female

rats ................................................................................................................................................... 217

Abu AH, Uchendu CN.

High plasma homocysteine and insulin resistance in patients with polycystic ovarian

syndrome ......................................................................................................................................... 223

Hemati T, Moghadami-Tabrizi N, Davari-Tanha F, Salmanian B, Javadian P.

http://www.ncbi.nlm.nih.gov/pubmed/18214508


X

Cotherapy of Tiron and selenium against vanadium induced toxic effects in lactating

rats ................................................................................................................................................... 229

Shrivastava S, Joshi D, Bhadauria M, Shukla S, Mathur R.

Evaluation of the effect of oral ritodrine on implantation rate in in-vitro fertilization-embryo

transfer cycles ................................................................................................................................. 239

Rabiee S, Farimani M, Ahmadi M.

CASE REPORTS

How should painful cystic degeneration of myomas be managed during pregnancy? a case

report and review of the literature ............................................................................................... 243

Kim TH, Lee HH.

Tubo-ovarian abscess in a virgin girl ........................................................................................... 247

Ashrafganjooei T, Harirchi I, Iravanlo G.

LETTER TO EDITOR

The association between iron status and some immunological factors in the pregnancy ....... 251

Sobhani SA, Etaati Z, Mirani S, Saberi P, Shiroodi M, Salmasian H, Naderi N.

PERSIAN ABSTRACTS ................................................................................................... 253

Iranian Journal of Reproductive Medicine Vol.9. No.3. pp:163-170, Summer 2011

Review article

Hormonal treatment for endometriosis associated

pelvic pain

Wu Shun Felix Wong1 M.D., Chi Eung Danforn Lim

2 M.B.B.S.

1 School of Women’s and Children’s Health, Faculty of Medicine, University of New South Wales,

Sydney, Australia.

2 South Western Sydney Clinical School, Faculty of Medicine, University of New South Wales, Sydney,

Australia.

Received: 28 August 2010; accepted: 10 March 2011

Abstract

Background: Endometriosis is a common gynecological problem associated with

chronic pelvic pain.

Objective: To evaluate the effectiveness of current hormonal treatments of

endometriosis associated pain.

Materials and Methods: Randomized Controlled studies identified from databases of

Medline and Cochrane Systemic Review groups were pooled. 7 RCTs were recruited

for evaluation in this review. Data from these studies were pooled and meta-analysis

was performed in three comparison groups: 1) Progestogen versus GnRHa; 2) Implanon

versus Progestogen (injection); 3) Combined oral contraceptive pills versus placebo and

progestogen. Response to treatment was measured as a reduction in pain score. Pain

improvement was defined as improvement ≥1 at the end of treatment.

Results: There was no significant difference between treatment groups of progestogen

and GnRHa (RR: 0.036; CI:-0.030-0.102) for relieving endometriosis associated pelvic

pain. Long acting progestogen (Implanon) and Mirena are not inferior to GnRHa and

depot medroxy progesterone acetate (DMPA) (RR: 0.006; CI:-0.142-0.162). Combined

oral contraceptive pills demonstrated effective treatment of relieving endometriosis

associated pelvic pain when compared with placebo groups (RR:0.321CI-0.066-0.707).

Progestogen was more effective than combined oral contraceptive pills in controlling

dysmenorrhea (RR:-0.160; CI:-0.386-0.066), however, progestogen is associated with

more side effects like spotting and bloating than the combined contraceptive pills.

Conclusion: Combined oral contraceptive pills (COCP), GnRHa and progestogens are

equally effective in relieving endometriosis associated pelvic pain. COCP and

progestogens are relatively cheap and more suitable for long-term use as compared to

GnRHa. Long-term RCT of medicated contraceptive devices like Mirena and Implanon

are required to evaluate their long-term effects on relieving the endometriosis associated

pelvic pain. Key words: Endometriosis associated pelvic pain, Medical treatments, Progestogen, Combined oral contraceptive pills, GnRH.

Introduction

Endometriosis is a common gynecological

Correspondent Author:

Chi Eung Danforn Lim, South Western Sydney Clinical

School, Faculty of Medicine, University of New South

Wales, Sydney, Australia. Email: [email protected]

disease, found in 70% of patient with chronic

pelvic pain (1). It is characterized by the presence

and growth of endometrial tissue outside the

uterine cavity (1). This condition is typically

associated with infertility; dyspareunia and

dysmenorrhea, with the latter being the most

frequent complaint by women with endometriosis

(2).

Wong et al

164 Iranian Journal of Reproductive Medicine Vol.9. No.3. pp:163-170, Summer 2011

The cyclic nature of pain associated with

endometriosis is probably attributed to the

response of endometrial tissue to cycling

reproductive hormones particularly estrogen (3).

Treatment of endometriosis associated pelvic

pain includes both medical and surgical options.

Current medical treatment options include

combined oral contraceptive pills, progestogens,

androgen hormone (e.g. Danazol) and

gonadotrophin-releasing hormone analogues

(GnRHa). Each treatment options have its own

systemic side effects, leading to no definite cure

for endometriosis. Associated pelvic pain

frequently recurs once medications are stopped due

to reactivation of ectopic endometrial implants.

Progestogen is most commonly used for

treatment of endometriosis (3). There aredifferent

types of progestogens including medroxy

progesterone acetate and 19-nortestosterone.

Their proposed mechanism of action is to stop

endometrial proliferation and to induce regressive

changes (3, 4). In a Cochrane review performed by

Prentice, Desary and Bland (2009) (5), they noted

that progestogen was an effective treatment for

endometriosis-associated pain.

However, the conclusion from their review was

based on limited data. Eight studies were recruited

and majority of the recruited studies had a relative

small sample size. The mean number of patients

recruited was 82.

Additionally, with the advanced therapeutic

development, apart from orally and

intramuscularly administered forms of delivering

progestogen, there are other forms such as

intrauterine device (Mirena) and subcutaneous

implant (Implanon).

They provide long- term release of

progestogens up to three to five years. Prentice,

Desary and Bland (2009) did not include these

long-term releases of progestogen in their review

(5).

This paper aims to compare and to determine

the effectiveness of current hormonal treatments of

endometriosis associated pelvic pain. Treatments

options included are progestogens, GnRHa and

combined contraceptive pills. They are compared

as following:

1. Progestogen versus GnRHa

2. Long acting progestogen versus GnRHa/

progestogen (injection)

3. Combined oral contraceptive pills versus

placebo and progestogen

Materials and methods

Inclusion criteria

Randomized controlled trials related to

endometriosis-associated pain and medical

treatments from the English literatures between

1995- 2009 were selected and pooled for analysis.

Exclusion criteria

Cohort studies, case control and case reports

were not considered. Studies which did not

measure pain improvement as a measure outcome

and did not match the above objectives were also

not recruited.

Search

Medline and Cochrane systemic review

databases search using keywords: endometriosis,

randomized controlled trial, pelvic pain,

dysmenorrhea, combined oral contraceptive pills,

progestagens and GnRHa were conducted.

Selection of studies

Only medical treatments aimed at symptomatic

improvement of pelvic pain were considered.

Treatments with any progestogens, combined oral

contraceptive pills, GnRHa and placebo were all

considered, irrespective of dosage, route of

administration or duration of treatment. Medical

treatments for painful symptoms after conservative

surgery were also considered in this review

because of the paucity of randomized controlled

studies.

This analysis considered women of

reproductive ages (18- 40 years) complaining of

pain symptoms related to endometriosis. The

endometriosis associated pain symptoms were:

Hormones in endometriosis pelvic pain

Iranian Journal of Reproductive Medicine Vol.9. No.3. pp: 163-170, Summer 2011 165

dysmenorrhoea, non-menstrual pelvic pain, chronic

pelvic pain and deep dyspareunia.

Studies where participants were asymptomatic

or presented with infertility alone were not

considered.

Data extraction process

This review included data from randomized

controlled studies comparing control, progestogens,

combined contraceptive pills and GnRHa in the

treatment of endometriosis- associated pain. Two

authors extract data independently concerning

details of study design, study population,

intervention and outcomes using a self-developed

data extraction form. Any differences in data

extraction were resolved by consensus, referring

back to the original article. Any disagreement of

data extraction was resolved by discussion with the

senior academic author.

Outcomes measures

Outcome measures were considered at the end

of treatment. The primary outcome measure

was pain improvements for each pain symptoms

where possible. Subjective pain relief measurement

was considered using both visual analogue scale

(VAS) and verbal rating scale (VRS). The

occurrence of side effects was also considered

as a secondary outcome measure.

Outcome definitions

It is defined that response to treatment was

considered as a reduction in pain scores. Pain

improvement was defined as improvement ≥1 at

the end of treatment.

Patient’s satisfaction with the treatment was

considered if they were very satisfied or satisfied.

Statistical analysis

Meta Analyst (6) Software was used in this

project. Statistical analyses were performed to use

the Relative Risk as the measure of effect for

dichotomous data.

There are many different existing methods to

assess pain, standardized the mean difference were

required.

Assessment of bias across studies

We assessed the methodological quality using

the standard as described by Kjaergard (2001)

generation of the allocation sequence, allocation

concealment, double blinding, and follow up (7).

Based on these criteria, the risk of bias with all

the features (random method, allocation

concealment, blinding and follow up) of the studies

was subdivided into the following three categories:

all quality criteria met leading to low risk of bias;

one or more of the quality criteria only partly met

leading to moderate risk of bias; and one or more

criteria not met leading to high risk of bias.

Jadad score was also used to assess the

methodology quality of the clinical trial articles. A

trial receives a score from zero to five. The

evidence may be biased by selection bias, poor

randomization and poor binding, which might

affect the results of a trial.

Funding support

There is no external funding support received

on this project.

Results

Study characteristics

Seven articles (3, 4, 8-12) were recruited for

further evaluation. Six (3, 4, 9- 12) out of seven

studies were identified comparing progestogen

versus other non-progestogen treatments.

One study (8) compared the effectiveness of

oral contraceptive pills versus control on relieving

endometriosis associated pelvic pain. Another

study (10) was evaluating the medical treatment in

controlling the endometriosis-associated painful

symptoms after conservative surgery. The main

characteristics of studies were summarized in table

I.

Sample size

A total of 1096 patients were recruited in seven

studies. The sample size varied between studies.

The mean numbers of patients included were 156

in the seven recruited randomized controlled trials.

Wong et al


Endometriosis was staged according to the

American Fertility Society classification in its

original or revised form in three studies, while the

remaining studies did not perform the staging of

endometriosis.

Measurement tools

Majority of the studies used objective scales,

such as verbal rating scale, a 10cm/100mm visual

analogue scale and five rating scale, to assess the

severity of pain. In two study (12, 13), patients

were asked to rate their satisfactory level to the

therapy; and treatment was considered beneficial if

patients rated themselves as very satisfied or

satisfied (12).

Treatment schedule

The mean duration of treatment was 7 months

(range 3 to 12 months).

Five recruited studies (3, 4, 9, 12, 13) used

progestogen (depot medroxy-progesterone acetate,

oral dinogest, implanon and levonorgestrel-

releasing intrauterine system), four studied (3, 4, 9,

10) used GnRHa (Buserelin acetate, tryporelin,

leuprerelin and lupron) and two studies (8, 10)

used combined oral contraceptive pills (ethinyl-

estradial 0.035mg+norethisterone 1mg; ethinyl

estradiol 0.02 g, desogestrel 0.15 mg) as treatment

intervention.

Risk of bias

Three studies (4, 8, 10) (scored five in Jadad

system which indicates having sufficient quality in

the methodological quality assessment.

Crosignani (2006) scored three in Jadad system

because the method of blinding was not clearly

stated (3). The evaluators were blinded but it was

not specified if the patients were known to the

medication that they were receiving.

Another two studies (12, 13) also scored three

and again there was no mention about the blinding.

Petta (2005) scored two because there was

unblended study with no clear description about

dropout rate (9).

Progestogen versus GnRHa

Progestogen (intrauterine device, DMPA and

oral contraceptive pills) was compared with

GnRHa in three of the seven randomized

controlled trials (3, 4, 9).

The three studies indicated prgestogens were as

effective as GnRHa. They did not show to have a

significant difference (Figure 2. RR: 0.036; CI -

0.03, 0.102).

In terms of side effects, GnRHa appeared to

cause more bone mineral density loss than

progestogens, therefore its use is usually limited to

a period of 6 months. Treatment with progestogens

was associated with a higher incidence of spotting.

Implanon versus Depot Medroxyprogesterone

Acetate (DMPA)

Implanon provides an alternative ways of

delivering progestogen. Comparing long acting

implanon and GnRHa/DMPA, there is no

significant difference in relieving endometriosis-

associated pain (Figure 3; RR: -0.006; CI: -0.142-

0.162).

Thus, the efficacy of implanon and Mirean is

similar to that of GnRHa and DMPA in

symptomatic endometriosis (13). Patients in both

treatment groups experienced similar side effects

such as weight gain, acne, loss of hair and breast

tenderness.

Combined oral contraceptive pills versus

control versus progestogen

Combined oral contraceptive pills was

compared with placebo in two randomized

controlled trials and compared with DMPA (150

mg) in a randomized controlled trial.

Combined oral contraceptive pills demonstrated

effective treatment of endometriosis- associated

pain when compared with placebo groups and

reduced the use of analgesia (RR: 0.562; CI: 0.396-

0.727).

It also showed that oral contraceptive pills as an

adjuvant therapy to surgery were more effective

than surgery plus placebo to provide pain relief in

patients with endometriosis stage 3- 4 (RR: 0.631;

CI: 0.390-0.664).

Vercellini (1996) study showed that long acting

progestogen is more effective than oral

contraceptive pills in controlling dysmenorrhea

despite all values in both groups were significantly

reduced from baseline (RR: -0.160; CI: -0.386-

0.066) (12).

Progestogen is however associated with more

spotting and bloating as side effects than the oral

contraceptive pills.

Overall, the meta- analysis of these studies

showed that oral contraceptive pills is more

effective to release pain than progestogen

treatment (RR: 0.321; CI: -0.066-0.707).



Ta

ble

I.

Mai

n c

har

acte

rist

ics

of

stu

die

s o

n t

he

use

of

med

ical

tre

atm

ent

of

end

om

etri

osi

s-as

soci

ated

pai

n.

Jad

ad

Score

5

5

3

2

5

3

3

Prim

ary e

nd

-poin

t of

measu

re &

foll

ow

-up

s

Res

ponse

to t

reat

men

t fo

r dysm

eno

rrhea

Ever

y 4

wee

ks

duri

ng t

reat

men

t

Chan

ge

in s

core

s of

subje

ctiv

e pai

n

sym

pto

ms

Ever

y 4

wee

ks

duri

ng t

reat

men

t

Chan

ge

in s

core

s of

subje

ctiv

e pai

n

sym

pto

ms

12 m

onth

s fo

llo

w-u

p p

ost

-tre

atm

ent

Impro

vem

ent

in e

ndo

met

riosi

s

asso

ciat

ed c

hro

nic

pel

vic

pai

n a

nd

qual

ity o

f li

fe

Foll

ow

up v

isit

s: e

ver

y 2

8+

/-3day

s fo

r at

leas

t si

x c

om

ple

ted v

isit

s

Impro

vem

ent

in p

elvic

pai

n a

nd h

ealt

h-

rela

ted q

ual

ity o

f li

fe

12 m

onth

s fo

llo

w u

p

Chan

ge

in p

ain s

core

aft

er 6

month

s

Foll

ow

up e

ver

y 3

mon

ths

for

1 y

ear

Impro

vem

ent

in p

ain s

ym

pto

ms

and

pat

ients

’ sa

tisf

acti

on a

t th

e en

d o

f

ther

apy

Ever

y 3

mon

ths

for

1 y

ear

Crit

eria

for p

ain

evalu

ati

on

Ver

bal

rat

ing s

cale

(VR

S)

and

vis

ual

anal

ogue

scal

e

(VA

S)

A f

ive-

level

rat

ing

scal

e, V

AS

Bib

eroglu

and

Beh

rman

modif

ied

VR

S s

cale

: 0 (

no

dis

com

fort

) to

3

(sev

ere

pai

n)

VA

S

VA

S

A 1

00m

m V

AS

A 1

0 c

m v

isual

anal

ogue

scal

e

En

do

metr

iosi

s st

age

No

t re

po

rted

No

t re

po

rted

No

t re

po

rted

All

sta

ges

Sta

ge

3 a

nd 4

end

om

etri

osi

s

(Rev

ised

AF

S)

All

sta

ges

(R

evis

ed

AF

S)

All

sta

ges

(R

evis

ed

AF

S)

No

. o

f w

om

en

wit

h

pa

in a

t en

try

10

0

20

5

30

0

82

22

2

41

80

Inclu

siv

e crit

eria

Mo

der

ate

to s

ever

e

dy

smen

orr

hea

No

n-m

enst

rual

Pel

vic

pai

n a

nd

ob

ject

ive

fin

din

gs

(in

du

rati

on

of

the

po

uch

of

Do

ug

las

& l

imit

ed u

teri

ne

mo

bil

ity

)

Pel

vic

pai

n,

Pre

men

op

ausa

l w

om

en

aged

18

-49

, w

ith

lap

aro

sco

pic

dia

gn

osi

s

18

-40

yea

rs o

ld

wo

men

,

dy

smen

orr

ho

ea,

chro

nic

pel

vic

pai

n

Dy

smen

orr

hea

an

d/o

r

no

n-m

enst

rual

pel

vic

pai

n a

nd

/or

dy

spar

eun

ia,

up

40

yea

rs a

t th

e ti

me

of

surg

ery

Dy

smen

orr

ho

ea,

no

nm

enst

rual

pel

vic

pai

n a

nd

dy

spar

eun

ia

18

-40

yea

rs o

ld

wo

men

, m

od

erat

e to

sev

ere

pel

vic

pai

n,

Pel

vic

pai

n >

6 m

on

ths

Sa

mp

le s

ize

10

0

27

1

30

0

82

22

2

41

80

Treatm

en

t sc

hed

ule

an

d

foll

ow

-up

Monophas

ic O

CP

(et

hin

yl-

estr

adia

l

0.0

35m

g +

nore

this

tero

ne

1m

g)

for

21 d

ays

plu

s 7 d

ays

of

pla

ceb

o, fo

r

4 c

ycl

es

Die

noges

t (2

mg/d

ay,

ora

lly

) o

r

Buse

reli

n a

ceta

te (

900µ

g/d

ay,

intr

anas

ally

) fo

r 24 w

eek

s

Med

roxypro

ges

tero

ne

acet

ate

(104m

g/0

.65m

l, s

ubcu

tan

eou

sly

,

ever

y 3

month

s) o

r le

up

roli

de

(3.7

5m

g m

on

thly

) fo

r 6 m

on

ths

Lev

ono

rges

trel

-rel

easi

ng

intr

aute

rine

syst

em (

Mir

ena,

19

-

nort

esto

ster

one

der

ivat

ive);

Gn

RH

-

anal

ogue

(Lu

pro

n d

epo

t 3.7

5m

g)

for

6 m

onth

s

1.

Pla

cebo o

r 2.

GnR

Ha

(try

po

reli

n

or

leupre

reli

n,

3.7

5m

g e

ver

y 2

9

day

s) o

r 3.

Conti

nu

ou

s

esto

pro

ges

tin

thynil

estr

adio

l

0.0

3m

g p

lus

ges

toden

0.7

5m

g)

or

4.

Die

tary

ther

apy (

vit

amin

s, m

iner

als

salt

s, l

acti

c fe

rmen

ts,

fish

oil

) fo

r 6

month

s Im

pla

no

n (

etonoges

trel

,

subder

mal

ly)

& d

epo

t

med

roxy

pro

ges

tero

ne

acet

ate

(150m

g,

intr

amusc

ula

rly,

ever

y 3

month

s) f

or

12 m

on

ths

DM

PA

(150m

g,

intr

amusc

ula

rly

) v

s

ora

l co

ntr

acep

tive

(eth

iny

l es

trad

iol

0.0

2g,

des

oges

trel

0.1

5m

g)

+

dan

azol

(50m

g a

day

for

21

day

s o

f

each

28 d

ay c

ycl

e) f

or

1 y

ear

Typ

e o

f st

ud

y

A p

lace

bo

-

contr

oll

ed

double

-bli

nd

random

ized

tria

l

A r

andom

ized

double

-bli

nd,

mult

icen

tre

contr

oll

ed t

rial

A m

ult

icen

tre,

eval

uat

or-

bli

nded

com

par

ato

r

contr

oll

ed

random

ized

study

Mult

icen

tre

random

ized

contr

oll

ed

clin

ical

tri

al

A r

andom

ized

com

par

ativ

e

tria

l

An o

pen

,

pro

spec

tive.

random

ized

,

contr

oll

ed

clin

ical

tri

al

A r

andom

ized

clin

ical

tri

al

Refe

ren

ce

Har

ada

et a

l

(20

08

)7

Har

ada

et a

l

(20

09

)4

Cro

sig

nan

i,

Lu

cian

o,

Ray

and

Ber

gq

vis

t

(20

06

)3

Pet

ta e

t a

l

(20

05

)8

Ses

ti e

t a

l

(20

07

)9

Wal

ch e

t a

l

(20

09

)12

Ver

cell

ini2

et a

l (1

99

6)1

1

Wong et al


Figure 1: A flow chart demonstrates the identification, recruitment and exclusion of studies.

Figure 2: Progestogen versus GnRHa.

Figure 3: LNG-IUS versus GnRHa and Implanon (etonogestrel)

versus DMPA.

Figure 4: Combined oral contraceptive pills versus placebo versus progestogens.

Discussion

There is a paucity of randomized controlled

trials in relating to endometriosis associated pain

symptoms. Disease staging was not always

uniformly employed in the studies, which limited

the evaluation of the severity of pain against the

effectiveness of medical treatment.

Furthermore, small sample sizes in some

studies (9, 12, 13) limited the ability to draw

definite conclusions. Overall, the results from our

analysis of pooled data from available randomized

controlled studies in the English literature suggest

that progestogens and long acting progestogen

might have slightly better result that GnRHa, but

oral contraceptive pills have a higher level of

efficacy than progestogen.

2 studies were review articles of medical

management of endometriosis

One study is a self-controlled trial

2 studies did not match our comparison objectives

GnRH versus control

GnRH versus Yiweining

550 studies were identified relating to management of pelvic pain in endometriosis

12 studies addressing the keywords search were identified

5 studies were excluded

7 RCTs were recruited for data analysis



The seven articles demonstrated a significant

reduction in pain scores after the commencement

of medical treatments. Five articles used Visual

analogue scale (VAS) to measure the severity of

pain pre- and post- treatment. VAS is a widely

used pain assessment tool, which provides a

continuous scale for subjective rating along the

line. The extremes carry a verbal description of

symptoms to be evaluated such as the most severe

pain and no pain. The advantages of using VAS are

time-saving and cross-culture. However, Langley

and Sheppeard (1985) question the validity of VAS

measurements due to its propensity to bias (14).

Firstly, the physical characteristics of the scale

might affect the accuracy of the scale. “No pain” is

influenced by subjective individual pain threshold

and some patients might have difficulties in

distinguishing discomfort and pain. Likewise, “the

most severe pain” is an infinite description. It can

be influenced by personal experience. Also,

patients’ behavior when completing the scale

might lead to bias. They tend to recall memory of

the previous pain scores. This might influence the

accuracy of the pain measurement (14).

One study included in this review used verbal

rating scale (VRS) which consists of a set of

descriptive words. A study comparing VRS and

VAS showed that the pain scores in the middle are

liner-related but not the upper and lower extremes

(15). Thus, there exists an inherent discrepancy in

pain measurements, due to the subjectivity of the

pain experience. Pain is influenced by multiple

factors such as personal belief, culture, past

experience and emotion, it is difficult to assess

pain as a whole. Nevertheless, VRS and VAS are

useful to measure the intensity of pain in short

term despite considerable uncertainty regarding

their long- term use as serial measurements.

Comparisons of progestogens and GnRHa have

been made. Two of the three studies have a

relatively large sample sizes, both proved that

progestoegn is as effective as GnRHa, although

neither treatment appears superior (3, 4). Apart

from looking at short-acting progestogen such as

oral and depot, we have included other long-acting

progestogen in treatment in endometriosis

associated pain. Mirena, the intrauterine device,

releases levonorgestrel directly into the uterine

cavity at a relative constant rate of 20 µg/ day for 5

years. Although its mechanism is still unclear, it

has been speculated that progestogen induced

endometrial atropy leading to amenorrhea (9).

Petta (2005) demonstrated the short term effect (6

months) of Mirena in controlling endometriosis-

associated pain (9). Since the release of

levonorgestrel may slowly reduce in the 5 years of

use, there is no evidence showing the efficacy of

Mirena in controlling endometriosis-associated

pain in long- term. Additionally, the longer the

effect of Mirena in pain control, the more cost-

effective it will be.

Implanon is a single rod etonogestrel-

containing contraceptive implant. It is inserted

subdermally and provides a slow release of

progestogen. It lasts for three years. Efficacy of

Implanon is not inferior to DMPA. A single

insertion of implanon is more convenient than an

injection every 3 months. Both Mirena and

Implanon are long- term treatment options in

women with symptomatic endometriosis who also

require contraception.

Complications and withdrawal of treatments are

other measures to look at the overall effectiveness

of the treatments. In some studies, side effects

were only considered if they were severe enough to

cause withdrawal of the patient. Many patients on

progestogen treatments experienced side effects

such as irregular bleeding, bloating and weight

gain.

Women on GnRHa complained of hot flushes

severe enough to stop treatment. Additionally,

GnRHa causes loss of bone mineral density which

limits its long-term use. Despite high reporting rate

of side effects, there was a relatively low dropout

rate in these studies. This could indicate that the

presence of the side effects could be well tolerated.

It is questionable whether the severity of side

effects significantly increased dropout rates,

impacting an overall effectiveness.

Compliance is always an issue in medical

management. Combined pills and oral

progestogens were taken everyday. The studies did

not measure the rate of compliance. Moreover, it

was often unclear if the recruited patients were

taking alternative medications, which may have a

significant confounding variable affecting

outcomes.

Follow- up is an important factor in monitoring

a chronic disease with high probability of

recurrence. Follow- up data in different studies

were referred to different lengths. Most studies

could not prove effectiveness in long- term

management of endometriosis-associated pain

since it is common to have symptoms recurrence

once the treatment stops. Similar with surgical

intervention, there is always a chance of relapse.

Wong et al


Conclusion

It appears that combined oral contraceptive pills,

GnRHa and progestogens are all effective and well

tolerated by patients in treating endometriosis

associated pain, though side effects have to be

considered. Combined oral contraceptive pills and

progestogens are relatively cheap and more

suitable for long-term use as compared to GnRHa.

Even though both Mirena and Implanon had a role

in controlling endometriosis pain, the conclusion

was drawn from a study with relatively small

sample sizes. Longer term follow up studies of

Mirena and Implanon are required to look at their

long-term effects on endometriosis associated pain.

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https://research.tufts-nemc.org/metaanalyst/index.html

https://research.tufts-nemc.org/metaanalyst/index.html

Iranian Journal of Reproductive Medicine Vol.9. No.3. pp: 171-176, Summer 2011

GnRH antagonist versus agonist in normoresponders

undergoing ICSI: a randomized clinical trial in Iran Ensieh Tehraninejad M.D., Akram Ghahghaei Nezamabadi M.D., Batool Rashidi M.D., Maryam

Sohrabi M.D., Maryam Bagheri M.Sc., Fedyeh Haghollahi M.Sc., Elham Azimi Nekoo M.D., Mina

Jafarabadi M.D.

Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran.

Received: 27 April 2010; accepted: 8 March 2011

Abstract

Background: General concern is that the pregnancy rate is higher with GnRH-agonist

as a protocol of pituitary suppression. GnRH-antagonist protocol provides a shorter

period of administration and an easy flexible protocol.

Objective: In this study, the outcomes of GnRH agonist and antagonist in ICSI cycles

are compared in normo responder patients.

Materials and Methods: In this randomized clinical trial, 300 normoresponders

undergoing ICSI were randomly divided to GnRh agonist (n=150) and GnRh antagonist

(n=150) groups. The main outcome measurements were chemical, clinical and ongoing

pregnancy rates (PR).

Results: The mean duration of stimulation were 9.6±1.6 and 8.2±1.6 days in agonist

and antagonist groups respectively (p=0.001). The mean number of MII oocyte

retrieved in agonist and antagonist groups were 7.7±4.0 and 6.9±4.3 respectively

(p=0.03). There was no significant difference between two groups regarding mean

number of gonadotrophin ampoules, follicles, occytes, total embryos and good quality

embryos, OHSS incidence, and abortion rate. Chemical pregnancy rate was 35.3% in

agonist and 39.3% in antagonist group. Clinical pregnancy rate was 35.3% in agonist

and 34% in antagonist group. Ongoing pregnancy rate was 45 (31.3%) in agonist and 44

(29.3%) in antagonist group. There was no significant difference between two groups in

pregnancy rates.

Conclusion: In this study antagonist protocol was shown to be an easy, safe and

friendly protocol in Iranian normoresponder patients, having similar outcomes with

standard agonist protocol but shorter period of stimulation.

Key words: IVF, GnRH agonist, GnRH antagonist, Normoresponder.

Registretion ID in IRCT: IRCT138902283950N1

Introduction

The first in vitro fertilization (IVF) therapy

was performed in a natural cycle. Gonadothropins

are given to induce multiple follicular

development and GnRH analogues are used for the

prevention of premature LH surges in IVF. LH

surges occur in about 20% of stimulated IVF

patients (1). Preventing LH surges using GnRH

analogues improves oocyte yielded with more


Mina Jafarabadi, Reproductive Health Research Center,

Imam Hospital Complex, Keshavarz Blvd., Tehran

14194, Iran.


embryos, allowing better selection and leading to

an increase in pregnancy rate.

GnRH agonist administration causes

gonadotrophin suppression via pituitary

desensitization, after an initial short period of

gonadotrophin hypersecretion. In contrast, GnRH

antagonist accuses immediate and rapid

gonadotrophin suppression by competitive

occupancy of GnRH receptor and therefore is a

choice to use in IVF for the prevention of

premature LH surge (2).

Several potential advantages of antagonists are

suggested over GnRH agonists. Among these

advantages are shorter duration of injectable drug

treatment, decreased gonadotropin requirement per

mailto:[email protected]

Tehraninejad et al


cycle, and lower overall treatment cost (3). Although agonist use is accompanied by a series of

disadvantages including hypoestrogenemia, cyst

formation, requirement for a prolonged period of

down-regulation and an increase in FSH and LH in

primary administration, agonist protocol was well

accepted in clinical practice, and general concern is

that the pregnancy rate was higher with agonist

protocol (4, 5).

The recent development of side- effect free

GnRH-antagonist protocol, with immediate

blockage of receptors and shorter period of

administration, provides physicians with an easy

flexible protocol and offers patients a side-effect

free, “friendly” protocol (4). Comparative studies

between GnRH analogues in IVF cycles have

suggested that the duration of stimulation in the

antagonist group was shorter with lower incidence

of OHSS, but in several outcomes the results of

studies remain controversial (6-11).

Several studies are done in different subgroups

of patients to recognize the best protocol of

pituitary suppression (11- 15). The aim of this

study was to compare outcomes of GnRH agonist

and antagonist stimulation protocols and to

evaluate the potential benefits of GnRH antagonist

utilization in ART cycles in normoresponder

Iranian patients. Normo-responder hints the group

of patients with neither decreased ovarian reserve

nor predisposition to hyperstimulation. The study

was approved by ethics committee of Tehran

University of Medical Sciences.


This randomized clinical trial was conducted at

Vali-e-Asr Reproductive Health Research Center

and Rooyan Institute, Tehran, Iran from January

2008 to January 2010. In total 300 patients

undergoing ICSI cycles with or without ICSI were

evaluated in this study. After obtaining informed

consent, patients were allocated to two groups

according to a sequence of computer generated

random numbers (0 or 1).

A total of 300 women were randomized, 150 in

each group. Inclusion criteria were: age<38 years,

normal basal serum FSH, 20≤ BMI< 30kg/ m2 and

regular menstrual cycle. Exclusion criteria were:

PCOS, severe endometriosis, history of poor

response in previous treatment cycles and history

of repeated IVF failure (more than 3 failed cycles).

The primary outcome measures were fertilization

and pregnancy rates. Additional outcomes of

interest were number of oocytes retrieved, number

of good quality embryos transferred, OHSS

incidence and patient's capacitance.

In the agonist group on cycle day 21,

Busereline acetate (Superfact, Aventis, Germany)

was started as 0.5 mg daily subcutaneous (S.C.)

injection until menstruation had begun and

adequate suppression was achieved (serum

estradiol level <50 pg/ml and no ovarian cystic

structures on ultrasound examination). At day 3 of

next menstrual cycle, the dose of Busereline was

diminished to 0.2 mg and rFSH (Gonal F, Merck

Serono, Switzerland) was started. The starting dose

for the first 5 days varied between 150-225 IU

daily by S.C. injection depending on the age (< or

>35 years) and history of patient. Thereafter,

transvaginal ultrasonography was done every other

day and the dose was adjusted on the basis of

follicle graph using Gonal-F and HMG

(Menoupour, Ferring, USA). Ovulation was

induced with 10000 IU, IM injection of HCG

(Profasi, Serono, Switzerland) when at least 2

follicles 18-20 mm were observed and serum

estradiol was between 1000 and 3000 pg/ml. In

the antagonist group, rFSH treatment was begun on

day 3 of menstrual cycle. The starting dose for the

first 5 days varied between 150-225 IU S.C.

depending on the patient's age and history.

Thereafter transvaginal ultrasonography was done

every other day and the dose was adjusted on the

basis of follicle graph using Gonal-F and HMG.

When there was one follicle 14mm in diameter,

antagonist (Cetrotide, Merck Serono, Germany),

0.25 mg S.C. daily dose was administrated until

the day of HCG administration. The time of

cetrotide injection was adjusted not to be more

than 30 hours apart from HCG administration.

When at least 2 follicles 18-20 mm in diameter

were seen, rFSH and HCG (10000 IU, IM) was

injected. Oocyte retrieval was performed 36h after

HCG administration, by transvaginal sonography

guided puncture of follicles. Two or three embryos

were transferred 72 hours after oocyte retrieval

using Cook catheter (Cook Medical Incorporated,

Bloomington, USA).

In both groups the luteal phase was supported

with vaginal suppository of cycleogest 400

mg/BD. Progesterone treatment was started on the

day of oocyte retrieval and continued until the day

of pregnancy test performed 14 days after the

embryo transfer. In the case of a positive test, this

regiment was continued during the first trimester

of pregnancy. Clinical pregnancy was defined as

the presence of a gestational sac with visible

heartbeat.

Embryos were scored based on the assessment

of the number and distribution of nucleoli

precursor bodies in the pronucleus to have good

and poor morphology (16). The OHSS

GnRH antagonist Vs Agonist

Iranian Journal of Reproductive Medicine Vol.9. No.3. pp:171-176, Summer 2011 173

classification utilized in this study was the one

proposed by Golan et al (17).

Statistical analysis:

All analyses were performed using SPSS

(version 16) with a two-sided 5% significance

level.

Results

In this study, 150 patients treated with agonist

protocol were compared with 150 patients treated

with the antagonist protocol. Two groups were

matched regarding age, BMI, duration of

infertility, cause of infertility, number of pervious

attempts and baseline FSH (Table I). Two groups

showed no significant difference regarding mean

number of gonadotropin ampoules used (p=0.63),

mean number of follicles ≥15mm on oocyte

retrieval day (p=0.12) and mean number of oocytes

retrieved (p=0.31) (Table II). Chemical, clinical

and ongoing pregnancy rates in two groups were

not significantly different (p=0.42, 0.83 and 0.71

respectively) (Table II).

There was no significant difference between

two groups regarding mean number of good

quality embryos (p=0.50), abortion rate (p=0.09)

and incidence of OHSS (p= 0.25) (Table II).

The duration of stimulation in agonist group

was significantly higher than antagonist group

(9.6±1.6 vs. 8.2±1.6 days, p=0.00).

This study showed significant difference

between two groups regarding endometrial

thickness on the day of HCC administration

(10.3mm in agonist vs. 9.3 mm in antagonist

group, p= 0.00). Mean number of M II oocytes

retrieved in agonist group was also significantly

higher than antagonist group (7.7±4 vs. 6.9±4.3,

p=0.03).

Table I. Demographic and clinical characteristics of patients in two groups.

Table II. Clinical and laboratory outcomes in two groups. GnRH agonist (n=150) GnRH antagonist (n=150) p-value

Duration of stimulation (Day, Mean± SD)

9.6±1.6 8.2±1.6 0.00

Number of gonadotropin ampoules (Mean±SD)

24.2±7.3 24.2±6.5 0.63

Number of mature follicle (Mean±SD)

11.3±6.3 10.7±6.6 0.12

Number of oocyte retrieved (Mean±SD)

9.2±4.2 8.6±4.3 0.31

Number of MII oocyte (Mean±SD)

7.7±4.0 6.9±4.3 0.03

Number of embryo (Mean±SD)

5.3±3.4 5.6±3.6 0.50

Number of good quality embryo (Mean±SD)

4±2.4 3.9±2.4 0.81

Chemical pregnancy rate (n %)

53 (35.3%) 59 (39.3%) 0.43

Clinical pregnancy rate (n %)

53 (35.3%) 51 (34%) 0.80

Ongoing pregnancy rate (n %)

47 (31.3%) 44 (29.3%) 0.72

Abortion (n %)

9 (17%) 18 (30%) 0.09

OHSS (n %)

26 (17.3%) 19 (12.7%) 0.25

Endometrial thickness (mm) (Mean±SD)

10.3±1.3 9.3±1.3 0.00

Mean cost of one cycle prior to oocyte retrieval

(visit+ sonography+ medication) (million Rials)

6.16 ± 0.23 5.90 ± 0.47 0.07

Characteristic GnRH agonist protocol (n=150) GnRH antagonist protocol (n=150) p-value

Age (yrs) 30.4 31.1 0.52

BMI (kg/m2) 26.7 24.7 0.41

Duration of infertility (yrs) 7.8 7.6 0.61

No. of previous attempts 0.95 0.85 0.09

Baseline FSH (mlU/mL) 6.4±1.0 6.5±1.2 0.06

Cause of infertility

Female factor [n (%)]

Male factor [n (%)]

Male and female [n (%)]

Unexplained [n (%)]

52 (34%)

70 (46%)

14 (9%)

14 (9%)

54 (36%)

68 (45%)

16 (10%)

12 (8%)

0.71

0.34

0.34

0.51

Tehraninejad et al


Discussion

In this study the results of GnRH antagonist

multiple doses protocol usage were compared

versus long protocol of GnRH agonist in ICSI

cycles in Iranian normoresponder patients.

Apart from significantly higher number of MII

oocyte in agonist group and shorter duration of

stimulation in antagonist group in our study there

was no difference in the number of follicles, total

retrieved oocytes, total embryos, good quality

embryos and mean cost of one cycle prior to

oocyte retrieval between two groups and as the

main outcome measurement the rates of chemical,

clinical and ongoing pregnancy were similar in two

groups.

Despite the result of some studies confirming

our results (9, 17-21), in meta-analysis of 5 RCTs

Aboulghar and Al-Inany reported that clinical

pregnancy rate was 5% lower in antagonist

protocol (5). In present study, the mean duration of

stimulation days was significantly longer in agonist

group. Many studies are in accordance with it

(Greco et al: 11.1±0.3 vs. 12.2±0.4, Kumbak et al:

11.7±1.2 vs. 12.9±1.6 and Xavier et al: 9.5±1.7 vs.

10.6±2.1) (17, 21, 22).

In present study, the mean dose of gonadotropin

used in two protocols had no significant difference

and this was similar to the result of the studies of

Berger (19), Xavier (18), Kumback (23) and

Kolibiakis meta-analysis (24). Although some

other researches reported the total dose of

gonadotrophin used in the agonist protocol was

significantly higher than antagonist protocol (10,

22). No sever OHSS occurred in either group

during our study and the incidence of mild OHSS

was higher in agonist protocol but this was not

statistically significant. We excluded PCOS

patients from the study and this can be interpreted

as the cause of absence of severs OHSS in our

study.

Using antagonist protocol for preventing OHSS

especially in PCOS patients is proved in several

studies (12-15). In present study the rate of

abortion was higher in antagonist group but this

was not statistically significant (p=0.09). Bahceci

et al, 2009, reported that the rate of early

pregnancy loss (EPL) was higher in antagonist

protocol (26).

The endometrial thickness in the antagonist

protocol was lower than agonist in the day of hCG

administration (10.3 mm vs. 9.3mm, p=0.00) in our

study. Xavier et al reported that there wasn’t any

significant difference between 2 protocols in this

variable (18) but Orveito et al reported that

endometrial receptivity and endometrial thickness

was higher in the agonist protocol (27).

GnRH antagonist molecules are potent

inhibitors of cell cycle, decreasing the synthesis of

locally produced growth factors. They can exhibit

this activity in all tissues presenting GnRH

receptors and consequently influence blastomere

formation, endometrium development and

fulliculogenesis and oocyte maturation (22). This

can explain the lower number of MII occyte and

lower endometrial thickness in antagonist protocol

and may reflect the cause of slight (but not

significant) increase in abortion rate in antagonist

group in present study.

The results of this study show that these two

protocols are very similar in outcomes in

normoresponder patients. Immediate mode of

action, flexibility of use, shorter duration of

administration, shorter duration of FSH

stimulation, and a lower incidence of hospital

admission due to sever OHSS make the antagonist

protocol an excellent approach for ovarian

stimulation in IVF. There was no significant

difference in the rate of live birth in GnRH

antagonist protocol comparing with agonist in the

study accomplished by Kolibinakis and Tarletzis in

2006 (24). Literature suggests that the side effect,

physiologic and psychological distress and

treatment burden is lower in antagonist protocol

(28), though these points were not concerned in

present study and is proposed to be evaluated in

further studies in Iranian patients.

On the basis of the results of this RCT on

Iranian normoresponder women, we offer using the

“GnRH Antagonist” as a patient friendly protocol

for the first choice in ART cycle with lower

incidence of side effects, similar pregnancy rate

and cost and time saving.

Acknowledgment

The authors wish to thank all academic

members and staff at the Vali-e-Asr Reproductive

Health Research Center for their sincere

cooperation.

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Effect of benzene extract of Ocimum sanctum leaves on

cauda epididymal spermatozoa of rats Mukhtar Ahmed

1,3 Ph.D., R. Nazeer Ahamed

1 Ph.D., Ravindranath H Aladakatti

1,2 Ph.D., Mukhtar

Ahmed G Ghodesawar1,4

Ph.D.

1 Department of Post-Graduate Studies and Research in Zoology, Karnatak University, Dharwad-580003,

India.

2 Central Animal Facility, Indian Institute of Science, Bangalore-560012, India.

3 Research Centre, Electron Microscope Unit, College of Science, King Saud University, Post Box 2455,

Riyadh 11451, Kingdom of Saudi Arabia.

4 Department of Zoology, Anjuman Arts, Sciences and Commerce College, Bijapur-586101, India.

Received: 9 December 2009; accepted: 23 May 2010 Abstract

Background: Recent studies have shown that benzene extract of Ocimum sanctum (O.

sanctum) leaves induces the ultrastructural changes in the epithelial cells of the cauda

epididymis, its subsequent recovery in the seminiferous epithelium and fertility of male

albino rats.

Objective: Our aim was to investigate the effect of benzene extract of O.sanctum leaves

on the cauda epididymal sperm parameters, morphology and their organelles at the

ultrastructural level in albino rats.

Materials and Methods: Wistar male rats (n=20) were allocated into two groups of

control (n=10) and test group (n=10). The test group received benzene extract of

O.sanctum leaves (250mg/kg/day) for 48 consequence days. Five animals from each

group were used for fertility test. Twenty-four hours after the last dose, the rest of the

control (n=5) and treated (n=5) animals were sacrificed by cervical dislocation and then

the cauda epididymal plasma was used for sperm analysis, scanning electron

microscopy (SEM) and transmission electron microscopic (TEM) studies.

Results: Sperm analysis of test group exhibited significant (p≤0.001) decrease in the

sperm count, motility, speed and increase in sperm anomalies when compare to control

group. SEM and TEM observation in treated animals indicated the morphological

changes in plasma membrane as well as in the acrosomal membrane of spermatozoa,

formation of a balloon-like cytoplasmic droplet in the mid-region of abnormal tail and

disorganization or degeneration of mitochondria of sperm mitochondrial sheaths.

Conclusion: The effects observed in this study may have resulted from a general

alteration in the cauda epididymal milieu, probably due to androgen deficiency

consequent to the anti-androgenic property of O.sanctum leaves.

Key words: Ocimum sanctum, Epididymis, Spermatozoa, Fertility, Electron microscope, Rats.

Introduction

Fertility control is an issue of global and

national public health concern. Through increased

public awareness, statements supporting research

Corresponding Author: Ravindranath H Aladakatti, Central Animal Facility,

Indian Institute of Sciences, Bangalore- 560012,

Karnataka, India.


on male methods and greater involvements of men

reproductive health have been forthcoming from

several quarters, including international women's

organizations. The clinical and scientific basis for

the research has been reviewed in recent years.

Apart from research for finding harmless chemical

drugs as effective oral contraceptives in the

western countries, the crude vegetal drugs used by

tribal people are being closely looked into for

their possible efficiency to find out safe and


Ahmed et al

178 Iranian Journal of Reproductive Medicine Vol.9. No.3. pp: 177-186, Summer 2011

effective oral drugs for controlling human

fertility. Many of the plants which are common in

India are reported to possess antifertility activity

as spermicidal, abortifacient or antiandrogenic in

nature (1-7). Therefore, it has become necessary

to use biologically active botanical substances or

fertility-regulating agents of plant origin which

are ecofriendly in approach and interfere with the

natural patterns of reproduction (8).

The plants of genus Ocimum belonging to

family Labiatae are very important for their

unique properties. Ocimum sanctum L. (Tulsi),

Ocimum gratissium (Ram Tulsi), Ocimum canum

(Dulal Tulsi), Ocimum basilicum (Ban Tulsi),

Ocimum kilimandscharicum, Ocimum

ammericanum, Ocimum camphora and Ocimum

micranthum are examples of known important

species of genus Ocimum which grow in different

parts of the world and are known to have

medicinal properties (9-11). Ocimum sanctum is, a

small herb seen throughout India, commonly

cultivated in gardens. In traditional systems of

medicine, different parts (leaves, stem, flower,

root, seeds and even whole plant) of Ocimum

sanctum, have been recommended for the

treatment of bronchitis, bronchial asthma, malaria,

diarrhea, dysentery, skin diseases, arthritis,

painful eye diseases, chronic fever, insect bite etc.

The Ocimum sanctum L. has also been suggested

to possess antifertility, anticancer, antidiabetic,

antifungal, antimicrobial, hepatoprotective,

cardioprotective, antiemetic, antispasmodic,

analgesic, adaptogenic and diaphoretic actions.

In addition, the leaves of O. sanctum

significantly altered the sperm count, motility,

velocity and fructose contained in the cauda

epididymis (12), reduce the mating behaviour of

both male and female albino rats (13-15). Recent

studies shown that benzene extract of Ocimum

sanctum leaves induces the ultrastructural changes

in the epithelial cells of the cauda epididymis, its

subsequent recovery, after withdrawal of

treatment, in the process of spermatogenesis and

fertility of male albino rats (16,17) and

morphological changes in the rat cauda

epididymal sperms upon graded dose treatment

(18).

As there is little information concerning the

influence of O. sanctum leaves on the cauda

epididymal sperm at the ultrastructural level, the

present investigation is designed to study whether

benzene extract of O.sanctum leaves could cause

some of the sperm parameters, morphological

alterations in cauda epididymal spermatozoa and

its organelles by electron microscopic studies and

fertility of male of albino rats as this medicinal

plant has anti-spermatogenic and anti-androgenic

like properties (12-15).


Preparation of test material

Fresh O.sanctum leaves were collected and

dried in shade. A voucher specimen

(Zoo/herb/File No.47-Acc.No.22) was deposited

at Zoology Department, Karnatak University,

Dharwad, India. The dried leaves were coarsely

powdered and subjected to soxheltation process to

get the benzene extract. Extract thus obtained was

allowed to dry and stored in a dessicator at 4ºC.

The benzene extract is then mixed with propylene

glycol as required and administered orally

(gavage) to the experimental animals (19).

Experimental Animals

Colony bred healthy adult male albino rats

(Wistar strain) weighing 190-200g were utilized

for experiments. All animals were proven fertility

and obtained from the rat colony maintained in the

department. They were housed at a temperature of

262oC and exposed to 13-14 h of daylight and

maintained on a standard rat pellet diet (Gold

Mohar, Hindustan Level Ltd., Hyderabad) and

water was given ad libitum. The animals were

acclimatized to the laboratory conditions before

conducting experiments and the care of the

laboratory animals was taken as per the

Committee for the Purpose of Control and

Supervision on Experiments on Animals

(CPCSEA) regulations.

Study protocol

The control group (n=10) were administered 1

ml propylene glycol/rat/day orally for 48 days and

test group (n=10), that received benzene extract of

Ocimum sanctum leaves (250mg/kg/day) orally

for 48 consequence days. The effective dose of

250mg/kg body weight has been arrived at after

preliminary studies on dose and duration of 48

days is concerned to the spermatogenic cycle of

rat in response studies in our laboratory and

reported (12).

Five animals from each group were used for

fertility test. Twenty-four hours after the last dose,

rest of the control (n=5) and treated (n=5) animals

were sacrificed by cervical dislocation and then

the cauda epididymal plasma was used for sperm

analysis, scanning and transmission electron

microscopic studies.

Effect of benzene extract of O. sanctum leaves on cauda epididymal spermatozoa


Sperm analysis

The cauda epididymis was chopped into

phosphate buffered glucose saline (PBGS)

[composition: NaCl 50 mM/l; Na2HPO4 200

mM/l; glucose 200 mM/l and KH2PO4 26 mM/l].

The debris was removed and a clear suspension,

the epididymal plasma was used for the analysis

of total sperm count, sperm motility, forward

velocity and relative percentage of abnormal

sperms in male albino rats. The total sperm count

and motility were calculated according to the

method of Besley et al (20) using Neubauer’s

haemocytometer. Briefly, to increase the accuracy

of sperm count, the epididymal plasma was

diluted with a spermicidal solution, prepared by

dissolving 5 g of sodium bicarbonate (NaHCO3)

and 1 ml of 40% formaldehyde in 100 ml of

normal saline.

A twenty times dilution was made using

W.B.C pipette, which was thoroughly mixed and

one drop was added to both sides of Neubauer

haemocytometer. The spermatozoa were allowed

to settle down in the haemocytometer by keeping

them in a humid chamber for one hour. The sperm

count was done in R.B.C counting 5 major

squares. The total number of sperms were counted

in all the major squares and calculated as follows.

Total number of sperms per square (x)

Total number of sperms = ______ __________ × dilution

/ml plasma Total volume per factor (20) square (10-4)

Similarly the total number of motile sperms

was calculated, using phosphate buffer saline

instead of spermicidal solution. The forward

velocity of the sperm was calculated according to

the method of Ratnasoorya (21). Briefly, the

epididymal plasma was suspended in phosphate

buffer saline, cleared the tissue debris and a clear

solution was used for the assessment of average

forward velocity of sperms. The assessment was

made under light microscope, fitted with a

movable mechanical stage and a calibrated ocular

micrometer, at 400 X magnification. A drop of

sperm suspension was transferred to a clean glass

slide and the initial place and time of each sperm

was recorded. The time taken for forward

movement of sperm from the initial place within

microscopic field was recorded using a stop

watch. The procedure was repeated for 10

spermatozoa in each sample and the average

forward velocity of sperm was calculated and

expressed as m/sec. The relative proportion of

abnormal sperms was analyzed according to the

method of Bauer et al (22). Briefly, equal volume

of cauda epididymal plasma and 5% NaHCO3

were taken in a centrifuge tube, mixed well and

centrifuged for 5 minutes at 4000g. The

supernatant was discarded and 5 ml of normal

saline was added to the precipitate, mixed well

and centrifuged again. The procedure was

repeated 2 to 3 times and a clear precipitate was

obtained. To the final precipitate few drops of

normal saline were added, mixed thoroughly and a

smear was prepared on a clean slide. The smear

was dried at room temperature, fixed by heating it

over the flame for two to three seconds. Then the

smear was flushed with 95% alcohol, drained and

dried. It was stained in Ziehl Neelson's Carbol

Fuchsin diluted with equal volume of 95% alcohol

for 3 minutes and counter stained with 1:3 (v/v)

aqueous solution of Loeffer's methylene blue for 2

minutes. After staining, the smear was rinsed in

water and dried in air. The abnormal sperms

included categories like double tailed, detached

head, detached tail, mid piece bending and

irregular head. The relative proportion of the

normal and abnormal sperms was from the smear

and expressed in terms of percentage.

Preparation of spermatozoa for SEM study

Preparation of rat spermatozoa for SEM

studies was performed as described elsewhere

(23). Briefly, a drop of cauda epididymal plasma

was fixed in 2% glutaraldehyde, centrifuged and

washed with 0.1 M Sodium cacodylate buffer

(pH=7.2), centrifuged again in distilled water till

the buffer solution was washed out and a thin film

was applied on a cover slip, dried, sputter coated

with gold and finally observed under scanning

electron microscope (Model. LEO 435 VP

Detector SL.1. LEO Electron Microscopy ltd

Cambridge, England).

Preparation of cauda epididymal spermatozoa

for TEM study

Preparation of rat spermatozoa for TEM

studies was performed as described elsewhere (3).

Briefly, the epididymis was removed, rapidly

fixed in 3% glutaraldehyde in phosphate buffer

(pH=7.4; 0.1 M) for 4 hr at 4°C, washed in

phosphate buffer and post-fixed in 1% osmium

tetraoxide in phosphate buffer (pH=7.4; 0.1M) for

6 hr. The fixed epididymis was washed several

times in distilled water, stained en bloc in 2%

Ahmed et al


aqueous uranyl acetate for 6 hr, dehydrated in

acetone series, infiltered in epon-araldite mixture

for 10 hr and embedded in the same media in a

beam capsule. The blocks were cut in LKB

Bromma ultramicrotome. Semithin sections of 1

µm thickness were stained with toludine blue for

identification of stages. Ultrathin sections were

cut at 100-300 A, mounted on copper grids and

stained with 1% aqueous uranyl acetate and lead

citrate (24). The stained sections were scanned in

Jeol-TEM 100 C X II electron microscope for

ultrastructural observations.

Fertility test

To assess the fertility rate with reference to the

number of implantations, the female rats of

proven fertility exhibiting regular estrous or early

proestrus stage were separately housed with the

control and treated males overnight. The

appearance of sperm in the vaginal smear next

morning confirmed the mating and is considered

as day1 of the pregnancy. After 8 days, the

females were laparotomized and the numbers of

implantations and pups were recorded.


The data were statistically analysed and

expressed as Mean± Standard error (25). The

comparison of data for statistically significant

differences was done using student's t-test and a

probability level of p≤0.001 was considered as

significant.

Results

Sperm analysis

Analysis of sperm parameters, such as total

sperm count, total number of motile sperm,

forward velocity of the sperm and percentage of

abnormal sperm of the cauda of epididymal

plasma were carried out in the control and all the

treated animals. The control rats showed

56.40×104

total numbers of sperm/ml epididymal

fluid, 52.40×104 numbers of motile sperm/ml

epididymal fluid with a speed of 127.63 m/sec

and 11.40% of abnormal sperm were recorded.

Whereas in the 250 mg/kg body weight of

benzene extract treated animals showed a highly

significant decrease (p≤0.001) in total sperm

count (56%), total number of motile sperm (45%),

forward velocity of sperm (49%) and a highly

significant increase (p≤0.001) in the percentage of

abnormal sperm (544%) when compared to

control animals (Table I).

Scanning electron microscopic (SEM)

observations of rat sperms from cauda

epididymal plasma

SEM observations of the cauda epididymal

sperms of control rats showed normal parts

(Figure 1A). Perforatorium and acrosome are

covered with the plasma membrane. A distinguish

acrosome is covered with acrosomal membrane.

The whole spermatozoon is intact with all the

membranes and organelles. However, in 250

mg/kg body weight of benzene extract, treated

animals showed disturbance in the plasma

membrane as well as in the acrosomal membrane

in the most of sperm heads. It is rather difficult to

differentiate the acrosomal membrane as well as

the plasma membrane. Serrations in the head

region of the spermatozoa are observed. The

shape and size of the sperm head has also changed

considerably.

There was acute dorsoventrally constriction in

the mid-head region of most sperms. The

perforatorium (Sub acrosomal material) is

bulged/swelled (Figure 1B). Most of the

spermatozoa showed a splitting of the tail and

distinct visibility of balloon-like cytoplasmic

droplets in the mid-region of the tail (Figure 1C).

Transmission electron microscopic (TEM)

observations of Cauda epididymal

spermatozoa

Observations with the TEM revealed that

different parts of the cauda epididymal sperm of

control rats exhibited normal features (Figure 2D-

G).Where as in the animals treated with 250mg/kg

body weight of benzene leaf extract, the sperm

heads exhibited disrupted plasma membrane,

acrosomal membrane and surface coating with

fuzzy material. The tip of the sperm head showed

disruption of the plasma membrane and acrosome.

The perforatorium was condensed and most of

its surface was covered by a thin portion of

acrosomal sac. The anterior and caudal portion of

the sperm heads revealed disruption or loss of

plasma membrane, acrosome, perforatorium and

small vesicles on the ventral surface of the

perforatorium, which is probably part of the

acrosomal system. Most of caudal portion of the

sperm head revealed the disturbance of lamellar

body and centrioles. The basal plate, posterior

ring and post-nuclear cap appeared normal (Figure

3H and I).

The middle part showed disruption and

degeneration of the mitochondrial sheath and a



loss of plasma membrane. The disorganization, or

commencement of degeneration of the

mitochondria, was observed in most of the

abnormal mitochondrial sheaths. Some showed a

displaced mitochondrial sheath on one or both

sides and there was abnormal pattern of outer

dense fibres. Different parts of the principle part

of the tail exhibited loss and discontinuation of

plasma membrane and fibrous sheath, respectively

(Figure 3J-L). Most of the tail sections showed

retention of cytoplasmic droplets around the

middle part on one side (Figure 3K).

Fertility test

Results of fertility performance test showed

that female rats mated with control male rats

illustrated that the numbers of implantations were

10.20±1.07 on day 8 of pregnancy and number of

pups obtained 9.60±1.08. However, no

implantations were observed in the female rats

mated with benzene extract O. sanctum leaves

treated male rats.

Table I. Effect of O.sanctum leaves (benzene extract) on various sperm parameters of cauda epididymal plasma in albino rats (values

are expressed as SEM of 5 animals).

Group Treatment Sperm count

(Total no. x 104/ml)

Motile sperm

(Total no. x 104/ml)

Forward velocity

(µm/sec)

Abnormal

sperms (%)

I 1 ml propylene glycol 56.40 ± 1.39

(100%)

52.40 ± 2.15

(100%)

127.63 ± 2.75

(100%)

11.40 ± 0.26

(100%)

II 250 mg/kg body weight of benzene extract O.sanctum

leaves

32.00 ±1.22***

(56%)

24.00 ±1.70***

(45%)

63.16 ±1.71***

(49%)

62.03 ± 1.95***

(544%)

*** p ≤ 0.001

Figure 1: Scanning electron micrographs (SEM) of cauda epididymal spermatozoa of control (Fig.A) and treated with 250mg/kg body weight of

benzene extract rat (Figs. B&C)

A. Spermatozoa of control rat exhibiting normal parts of acrosome (A); post nuclear cap (C); plasma membrane (M); nucleus (N) perforatium (P) and

tail region (T). 4.56kx. B. The perforatium (sub-acrosomal material), swells (PS) and the middle region of the sperm head is constricted dorsoventrally (arrows). There is

serration (S) at the connective piece of the spermatozoa. A5.56kx.

C. There is increase of swelling at tail region in the mid portion of the tail region rat spermatozoa (arrows, ST). 5.93 kx.

Ahmed et al


Figure 2. Transmission electron micrographs (TEM) of the control (D-G). D: A median section through the base of the sperm head illustrating nucleus (N) covered with perforatorium (P), basal plate (B), plasma membrane

(PM) and connecting piece of tail (CP). Caudal portion of the nucleus illustrating lamellar body (lb) and mitochondria (M) are seen, in oblique section,

in contact with connecting piece. Cross sections(CS) of mid-piece of spermatozoa flagellum with spatial arrangement of the 9 outer dense fibres to one another, to the centrally located axoneme composed of the 9 plus 2 arrangements of microtubules and to the mitochondrial sheath (MS) X 29,000.

E. C.S. of anterior portion of sperm head illustrate normal features of nucleus (N); perforatorium (P); acrosome (a); plasma membrane (PM) and

principal piece are with normal features of fibrous sheath (FS) and arrangement of outer fibres and axonemal component X 21,700. F-G: Longitudinal section (LS) of mid piece and principal piece illustrating well-preserved mitochondrial sheath (MS) and fibrous sheath (FS) and are

intact with plasma membrane. Mitochondria (M) appear normal. C.S. of principal piece is with normal features of longitudinal fibrous sheath (LFS) and

arrangement of outer fibres and axonemal component X 15,000.

Figure 3. Treated with 250mg/kg body weight of benzene extract rat (Figs. H-K).

H: C.S. of the different parts of sperm head (←) showing disruption in normal features plasma membrane, acrosome and perforatorium. Fuzzy material

is seen on their surface. X 15,000. I: L.S and C.S. of the mid piece shows the loss of plasma membrane. Most of mitochondria (M) are hypertrophied or have started degeneration. The

loss of plasma membrane is seen in the sperm head region (←) X 28,000.

J: C.S. of the mid piece showing abnormal pattern of mitochondrial sheath and loss of plasma membrane. Most of mid-piece show increased cytoplasmic droplets (CD) displaced on one side are clearly seen. Coating of fuzzy material is seen on the surface of head and tail sections X 10,300.

K: C.S of the mid piece shows abnormal pattern and degeneration of mitochondrial sheath (long arrows) along the length of the structure and displaced mitochondrial sheath on one side. The discontinuation of fibrous sheath is also noticed in principal piece X 17,500.



Discussion

In the present study of benzene extract of O.

sanctum leaves, treated animals exhibits significant

increase in sperm anomalies with reducing sperm

count, motility and sperm speed. The epididymis

plays an active role in sperm development, and

sperm maturation is dependent on the unique

luminal environment of the epididymis, including

specific proteins synthesized and secreted by the

epididymal epithelium (26). Although several

epididymis-specific secretory proteins have been

identified, little is known about the sperm

maturation events in the epididymis (27).

Various plants like methanol sub-fraction of the

seeds of Carica papaya (4); methanol extract of

Dendrophthoe falcate (5); ethanolic leaf extract of

Aegle marmelos (6); hydroalcoholic extract from

Lantana camara leaves (28); aqueous extracts of

Ruta graveolens L. solution (29); aqueous extract

of Peganum harmala (30); and marjoram volatile

oil and grape seed extract of Vitis vinifera (31)

have been reported to possess antifertility activity

by reducing above said sperm parameters in mice

and rats to support our results. It was suggested

that the extract causes androgen depletion at the

target level, particularly in the cauda epididymis

thereby affecting physiological maturation of the

sperm (32). Present observations of increased

abnormal sperms, reduced sperm count, motility

and sperm speed on treatment with benzene extract

of O. sanctum leaves, it can be suggested that

sperm anomalies in albino rats may have resulted

from the alteration in the epididymal milieu due to

androgen deficiency following antiandrogenic

property of the leaf extract.

Ultra study of cauda epididymal spermatozoa (SEM and TEM)

Androgens are essential for survival and

motility of spermatozoa in the rat epididymis.

Sperm motility is an important attribute of sperm

quality as there is a good correlation between

sperm motility on one hand and plasma membrane

integrity and conception rates on the other. There

is an evidence of acrosomal loss or damage as well

as other abnormalities observed in caudal region of

rat sperm due to this treatment suggesting that

these sperms were probably unable to fertilize the

ovum (33). From the literature of medicinal plants,

it has been shown that different parts of the plant

source affecting aspects of male reproduction,

brings about the effect through either of two

mechanisms namely, estrogenic or antiandrogenic

effect and extensively established that these plant

sources cause impairing sperm parameters results

exhibit androgen is essential for the maturation,

motility and survival of sperms in the epididymis

(1, 2, 32, 34).

The acrosome contains several enzymes which

are secreted by the Golgi apparatus and

endoplasmic reticulum. The production of

enzymes destined for the acrosome is regulated to

some degree by testosterone (35). From the

histochemical evidence, the presence of

carbohydrates or polysaccharides in the head of the

spermatozoa, which are associated with various

enzymatic activities, is indicated. Earlier studies

have been reported that morphological changes in

the head of spermatozoa in general and the

acrosome in particular may have resulted from an

alteration in the epididymal milieu of rats treated

with crude leaf extract of Azadirachta indica (23)

and alcohol seed extract of Momordica charantia

(2) suggested that these changes are due to a

general disturbance of carbohydrates or

polysaccharides present in the acrosome of the

sperm head (23). In the SEM, observation of

present study revealed that most of the sperms

shown deformity includes dorsoventrally

constricted with the bulged sub acrosomal material

in the mid region of sperm heads and disrupted

plasma membrane and acrosomal membrane

particularly at the anterior region on treatment with

benzene extract of O. sanctum leaves are probably

due to the general disturbance in the proteins.

Specialized structural features of the

spermatozoa are a reflection of its unique

functional activities. Various authors have

suggested that in spite of morphological variations,

the main structures present in the sperm head of

mammals are the nucleus, the acrosome or

acrosomal cap and the membranous envelops (36).

The acrosome is unique organelle (37) that is

required for fertilization in mammals. It has been

predicted that the post-nuclear cap plays a

protective role in maintaining the head shape and

this structure is very resistant to any extractive

agent. The mitochondrial sheath is believed to be

the source of energy for sperm motility and outer

dense fibres might be contractile because of their

close association with the axoneme and play an

active role in flagellar motion (37). These outer

dense fibres provide added strength to protect

sperm from damage by shear forces encountered

during epididymal transit or ejaculation (38).

Reports on different plants extract namely

gossypol, Solanum xanthocarpum, Carica papaya

cause the sperm abnormalities by exhibiting

acrosomal damage and mid-piece anomalies which

Ahmed et al


results in complete inhibition of fertility in rats and

mice (32, 39). Crude extract of Echeveria

gibbiflora on guinea pig sperm, results in the

formation of a huge bubble by distension of the

plasma membrane and dispersion of the acrosome

content with disappearance of the external

acrosomal membrane at the sperm head level (40).

Triptolide isolated from Tripterygium wilfordii

cause all cauda epididymal sperm to exhibit a

complete absence of the plasma membrane over

the entire middle and principal piece and

prematured decondensation of the nuclei in rats

(41). Similarly, crude extract of A.indica leaves

(3); aqueous decoction of Chenopodium album

seeds (7); chloroform extract of Carica papaya

seeds (42); and a hydroalcoholic extract of

Lantana camara leaves (28) were shown

morphological abnormalities in the head of

spermatozoa along with mid-piece anomalies in

rats and rabbits.

The spermatozoa of the cauda epididymis in the

present study of benzene extract of O. sanctum

leaves treated animals revealed several

abnormalities. Abnormal patterns of the outer

dense fibres and components of axoneme are

displaced on one side or both sides, complete

absence of plasma membrane of the entire middle

and principal pieces and disorganization of

mitochondrial sheath in several spermatozoa of

rats were observed. The missing segment of the

mitochondrial sheath probably represents a weak

point in the structural collar, which supports the

axial fibre-bundle during the contractional wave,

leading to splitting of the axial bundle and

subsequent dislocation of its fibres (37, 38). Recent

ultra study of aflatoxin B1, a food borne

mycotoxin, has shown that the sections of the testis

and cauda epididymidis revealed sperm flagella

missing one or more outer dense fibres and the

associated axonemal microtubule doublets. Severe

mitochondrial pathologies in spermatozoa and

elongated spermatids, suggesting a link between

AFB1-induced sperm mitochondrial pathology due

to possibility that AFB1 treatment would disrupt

the cytoskeletal proteins of the flagellum in male

albino rats (43). It has been suggested that the

extract might cause an androgen deprived effect to

target organs resulting in alterations in the internal

milieu, especially of cauda epididymis (32).

From both SEM and TEM observations of

treated animals showed that the tail portion has

balloon like cytoplasmic droplets. Ejaculates

containing a high proportion of spermatozoa with

attached cytoplasmic droplet can be correlated

with altered epididymal function and reduced

fertility (44, 45). The presence of high proportion

of spermatozoa with attached cytoplasmic droplets

in benzene extract of O. sanctum leaves treated

animals may be due to altered epididymal function.

Similar observations were made in studies of

combination of progestagen and androgen, Carica

papaya, vincristine and aflatoxin B1 treated

animals (1, 32, 33, 46). Agnes and Akbarsha (46)

showed in their experimental study a higher

percentage of cauda epididymal spermatozoa

retained the cytoplasmic droplets in albino mouse.

Cytoplasmic droplets contained electron-dense

spherical inclusions, which were hypothesized as

lipid inclusions produced from the lamellae

through the spherical vesicles of the cytoplasmic

droplets.

It is known that spermatozoa carrying

cytoplasmic droplets would be inhibited in motility

and may not fertilize the ova (47). Hence, in the

present study, in the light of the pathological

changes observed, it is suggested that benzene

extract of O.sanctum leaves damages the

spermatozoa in the epididymis, leading to reduced

fertilizing ability of the sperm. The fertility studies

reveal that the male rats treated with O.sanctum

leaves are unable to fertilize the female rats

probably because the male gametes are affected

thereby, establishing the antifertility property of

the plant studied.

Conclusion

It was demonstrated that the administration of

benzene extract of O. sanctum leaves can induce

the morphological changes in the head and tail

region of albino rat sperms.This benzene extract of

O. sanctum leaves was may be due to general

disturbance of proteins and alteration in the

epididymal milieu probably due to androgen

deficiency consequent upon the antiandrogenic

property of this plant. However, these conclusions

are based on the preliminary study, where the rats

are force fed with the benzene extract of O.

sanctum leaves. More refined and sophisticated

study is needed for identification of active

principles and their effects on androgen dependent

parts of the spermatozoa in albino rats which are

under progress.

Acknowledgement

Authors are thankful to Department of

Zoology, Karnatak University, India and Union

Grant Commission under SAP and COSIST

program, New Delhi, India for support to carry out



this research and also grateful to the whole TEM

unit, National Institute of Mental Health And

Neurosciences (NIMHANS) Bangalore and SEM

unit, Central Food Technological Research

Institute (CFTRI), Mysore, respectively, for

providing us the EM facility and their kind

encouragement.

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Icodextrin reduces adhesion formation following

gynecological surgery in rabbits Behnaz Khani

1 M.D., Nahid Bahrami

2 M.D., Ferdous Mehrabian

2 M.D., Hormoz Naderi Naeni

3 M.D.

1 Department of Obstetrics and Gynecology, Shahid Beheshti Hospital, Isfahan University of Medical

Sciences, Isfahan, Iran.

2 Department of Obstetrics and Gynecology, Alzahra Hospital, Isfahan University of Medical Sciences,

Isfahan, Iran.

3 Sepahan Hospital, Isfahan, Iran.

Received: 8 May 2010; accepted: 11 January 2011

Abstract

Background: Adhesion is a common complication of gynecology surgery so different

barrier agents and solutions have been used during these operations to separate and

protect tissues from adhesion after surgery. Adept is one of these solutions that have

been postulated to reduce the chance of adhesion following gynecolgy surgery.

Objective: To evaluate the effect of 4% icodextrin in reducing adhesion formation in

comparing with sterile water and human amniotic fluid in rabbits.

Materials and Methods: In this prospective experimental study 30 white Newzealand

female rabbits were selected and randomized in to three treatment groups. The rabbits

were anesthetized and an abdominal incison was made, uterine horns were abrated with

gauze until bleeding occurred. Before closing the abdomen, the traumatized area was

irrigated either by 30cc of sterile water, 30cc of 4% Adept or 30cc of human amniotic

fluid. The solutions were labeled only as solutions A (steriel water), B (icodextrin), or C

(human amniotic fluid). On the seventh day after surgery, second laparotomy was

performed to determine and compare adhesion formation in rabbits.

Results: There was significant difference between mean score of adhesions in 4%

icodextrin group (2.1±0.70) in comparison to sterile water group (10.4±0.60) and

amniotic fluid group (8.7±0.84). But the difference between mean score of adhesions in

amniotic fluid group in comparison to sterile water group was not significant (8.7±0.84)

versus (10.4±0.60).

Conclusion: The use of 4% icodextrin solution was more effective than human

amniotic fluid and sterile water in reducing adhesion formation in a gynecological

surgery model in rabbits Key words: Icodextrin solution, Human amniotic fluid, Adhesion formation, Rabbit.

Introduction

Adhesions are the most common cause of post

operative small bowel obstruction, infertility and

visceral pain (1). Pelvic surgery is associated with

high rates of pelvic adhesion formation. Careful

surgical techniques have been proved to reduce

adhesion formation. Application of fine, Non-

reactive suture materials and prevention of foreign-

body reaction, excision of necrotic tissue and


Behnaz Khani, Flezi Bridge, Shahid Beheshti Hospital,

Isfahan, Iran.


minimizing tissue and organ injury are effective in

reducing adhesion formation in a surgical

procedure (2).

Non surgical techniques such as application of

local and systemic anti-inflammatory agents and

peritoneal instillates have been used in this regard.

Anti adhesion barriers such as hyaluronic acid,

polyethylene glycol, fibrin glue, hyaluronic acid

film, and expanded polytetrafluoroethylene have

been shown to reduce the incidence and extent of

new and recurrent adhesions in different clinical

trials (3, 4).

The use of fluids in the peritoneal cavity to

separate surfaces and prevent adhesion formation

between organs is under investigation. One of

Khani et al


these fluids is human amniotic fluid which is a

hypotonic solution mainly contains albumin,

cholesterol and hyaluoronic acid, existance of

hyaluoronic acid in the peritoneal cavity shifts the

repair process into regeneration pathway and

decreases fibrosis and scar formation. Amniotic

fluid also contains some potent growth factors such

as insulin like growth factors that are involved in

repair process (5).

In our study, human amniotic fluid was selected

and compared with adept adhesion reduction

solution that is a pale yellow fluid (icodextrin w/v

4% solution) and is a non viscous, iso-osmotic,

clear solution, contains icodextrin, alpha-1, 4

linked glucose polymer with a molecular weight of

16,500 Daltons.

This product is not physiologically present in

the abdominal cavity; it remains in the peritoneal

cavity for 3- 5 days before absorption by the

lymphatic system and therefore resides longer

compared to other solutions such as saline and a

glucose-based peritoneal dialysis solution. The

existence of 4% icodextrin in the peritoneal cavity

during this critical period separates damaged

surfaces and minimizes adhesion formation

between organs. It gradually absorbs into the blood

stream and is broken down by amylase and

metabolized to glucose (6).

In a controlled pilot study, the safety and

efficacy of 4% icodextrin was evaluated after

laparoscopic gynecological surgery and the results

showed that it is effective in reducing adhesion

formation (7).

Also in another randomized blinded trial anti

adhesion efficacy of 4% icodextrin, ferric

hyaluronate gel and Ringer lactat were compared

in sever peritoneal damage caused by bipolar

coagulation in a laparoscopic rat model. Adhesins

were more filmier and easily separable in 4%

icodextrin group comparing with Ringer lactate

group (8).

In contrast to these research results some other

working groups found insufficient effects of 4%

icodextrin in animal models (9, 10). Two cases of

severe serosal fibrosis within a few days after usig

4% icodextrin for reducing adhesion in abdominal

surgery was reported (11). Numerous cases of

abdominal pain and sterile chemical peritonitis

have been contributed to 4% icodextrin (12).

Because the bio compatibility and efficacy of

4% icodextrin is the subject of controversial

discussion in the current literature, we planned our

study and used a rabbit model to evaluate the

effectiveness of 4% icodextrin in reducing

adhesion formation in comparing with sterile water

and human amniotic fluid.


This prospective experimental study was done

in Physiology Department of Isfahan University of

Medical Sciences, Isfahan, Iran and approved by

institutional review board and vice chancellery

research of this university by registry number of

386161.

30 white, Newzealand female rabbits, weighing

2000-2200g were randomly assigned to 3 groups.

Each group consisted ten non pregnant, 12 weeks

aged rabbits. They were fed with standard

laboratory rabbit food and water throughout the

study. Human amniotic fluid was taken in a sterile

condition during cesarean section of two pregnant

women who both were around 32 weeks pregnant

and had the same indication of cesarean section.

To remove red blood cells from the fluid it was

centrifuged for ten minutes (3000circules/min) and

kept in refrigerator for four hours before use. The

4% icodexterin solution was hydrochloride (Baxter

healthcare corporation, Deerfield IL, USA) and the

sterile water was from Daroupakhsh Company,

Tehran, Iran. The gauze was from Safa Company,

Isfahan, Iran.

The rabbits were anesthetized for surgery with

IM injection of 55 mg/kg of ketamine. The

abdominal ventral side was shaved and dis-infected

with povidone iodine. A vertical 5cm abdominal

incision was made. Uterine horns were exteriorized

and the serosal surfaces of the horns were abraded

with sterile gauze until bleeding occured. Up to

this point, all animals received the same procedure

but after that the injured area were irrigated with

different solutions. The solutions were labelled

only as A, B or C, so that the study personnel were

blinded to solution identity.

The first group acted as control group in which

30cc of A solution was poured over the

traumatized area. In the second group, the

damaged area was irrigated with 30cc of B solution

and the third group received 30cc of C solution

before closure of the abdomen.

Measurements

The second laparotomy was carried out in 30

rabbits after 7 days to assess adhesion formation.

The evaluations were blinded for three groups. The

formed adhesions were scored by qualitative and

quantitative parameters (Table I). Parameters

included extent, depth of adhesion, bursting

strength, and number of adhesion sites (13). The

score from four parameters were calculated and

added to define total adhesion score as the grade of

adhesion (Table II) (13).

Effect of 4% icodextrine on adhesion reduction



Adhesion scores were assessed by a blinded

surgeon and the mean scores of adhesion were

analyzed by SPSS software version 13 using

Mann-Whitney test p<0.05 was considered

statistically significant.

Results

In second laparotomy, 7 days later in the sterile

water group, the occurrence of severe adhesions

was evident. All rabbits in this group showed

adhesions, 7 cases (70%) had severe adhesion

(grade 3) and 3 cases (30%) had moderate

adhesion (grade 2). In 4% icodextrin group,

adhesions were found to develop only in 5 rabbits

(50%) and half of cases displayed no adhesions at

all. Adhesion in these 5 rabbits was merely low

grade (grade 1). Finally, in the human amniotic

fluid group all rabbits developed some extent of

adhesion, 4 rabbits (40%) displayed severe

adhesion, and another 4 cases (40%) showed

moderate adhesion and the rest, 2 cases (20%)

developed mild adhesion. The score of adhesion

was calculated for each rabbit as mentioned above.

Then the mean score for each group was measured

and compared as shown in table III. The mean

score of adhesion was (2.1±0.70) for 4% icodextrin

group while the mean score was (10.4±0.60) for

sterile water group, so the difference was

statistically significant (p=0.000) (Table III).

In human amniotic fluid group the mean score

was (8.7±0.84) and in comparison with sterile

water group (10.4±0.60), the difference was not

significant (p=0.10) (Table III).

Finally, there was a significant difference

between the mean score of adhesion in 4%

icodextrin group in comparison to amniotic fluid

group.

Table I. Qualitative and quantitative measurement. Score of adhesion

Adhesion type

Score

0

Score

1

Score

2

Score

3

Extent (mm)

0 <2 2-10 >10

Depth (mm)

0 <1 1-3 >3

Bursting strength

0 + ++ +++

Number of adhesion sites

0 1-2 3-4 >4

Table II. Total scoring of adhesion. Score

Adhesion

4-5

Mild (grade 1)

6-8

Moderate (grade 2)

9-12

Severe (grade 3)

Table III. Comparison of adhesion scores among groups. Groups No. of

rabbits

Mean ± SD p-value

vs. sterile water

Sterile water

10 10.4 ± 0.60 Adept

10 2.1 ± 0.70 p=0.000

Amnion fluid

10 8.7 ± 0.84 p=0.01

4% icodextrin Human amniotic fluid

Strile water

Figure 1. Gross view of adhesion with different treatments.

A B

C

Uterine horn

Uterus

Adhesion

Khani et al


Discussion

Because adhesion adveresely affects patient

morbidity and is a great burden to health system,

different techniques have been proposed and tested

to reduce adhesion formation (14). Fine surgical

techniques and use of laparoscopic surgery to

minimize tissue damage are to some extent

effective in this regard but Surgical and Clinical

Adhesions Research study data showed that this is

not sufficient to prevent adhesion formation (15).

Administration of specific fluids such as lactated

Ringer’s Solution (LRS), phosphate-buffered

saline (PBS) and normal saline in to the peritoneal

cavity during the surgery has been proposed to

reduce formation of adhesions. However these

solutions are absorbed in a short period of time and

therefore are not effective clinically in preventing

adhesion formation (16).

It was shown that administration of human

amniotic fluid in to the peritoneal cavity inhibits

production of expanded peritonitis. Human

amniotic fluid contains hyaluoronic acid that

promotes normal healing process (17) and also

contains hyaluoronic acid stimulating activator

(HASA) which stimulates scar cells to produce

hyaluoronic acid that inhibits migration of

lymphocytes and prevents chemotaxis and

phagocytosis of granulocytes and therefore inhibits

scar formation (18).

Four studies commented the prevalence of

adhesions at second look laparoscopy (19-22) and

showed evidence of decreased prevalence of

adhesions in patients who were treated with

hyaluronic acid compared with those given placebo

or no treatment.

Our results also indicate that human amniotic

fluid is more effective in reducing adhesion

formation in comparison with sterill water as

placebo. We used 4% icodextrin fluid because it

has a longer residual time in the abdominal cavity

in comparison to other solutions (23) and it was

compared with human amniotic fluid which

contains Hyaluronic acid that has been proposed to

reduce adhesion formation too. Rabbits were our

experimental model because the fluid dynamics of

icodextrin in this species is more closely similar to

human beings (24).

Early pre-clinical studies were performed (by

Verco et al 2000) to assess the efficacy of 4%

icodextrin in order to reduce adhesion in a rabbit

double uterine horn model (25). Their results

indicate that postoperative application of

icodextrin 4% causes a significant increase in

adhesion free sites (p=0.000).

In a randomized controlled study (by Dizerega

et al 2003) the safety and efficacy of 4% icodextrin

was evaluated. In order to compare icodextrin 4%

with Ringer’s lactated saline, 62 women who

required laparascopic adnexal surgery were

compared in two different treatment groups.

Results showed that lavage and instillation with

icodextrin 4% was effective in reducing adhesion

formation. The use of 4% icodextrin solution for

peri operative lavage and post operative instillation

in rabbit model of bowel anastomatic healing,

didn’t result in any difference from either LRS

treated or untreated surgical control (26).

In another study (by Muller et al 2005) the

effect of intraperitoneal anti adhesive fluids (4%

icodextrin, phospholipids, Ringer’s lactate) in a rat

peritonitis model was examined and the results of

4% icodextrin showed significantly enhancement

of adhesion and abscess formation, in comparison

with the other control groups. A case of

disseminated intra vascular coagulation after

laparoscopic multiple myomectomy with the use of

4% icodextrin solution was described by Santos et

al (2006). The possible cause might be

idiosyncratic immunologically mediated reaction

to icodextrin in the pelvic cavity, but no previous

case of DIC has been described in the published

litreture (27). Some cases of vulval edema, plural

effusion and even anaphylactoeid reaction related

to icodextrin 4% after laparoscopic and laparotomy

surgery have been reported (28).

But, ARILE (Adept registry for clinical

evaluation) was initiated in a number of centers in

the UK and then expanded to involve 253 centeres

(103 general-surgery and 150 gynecologyical-

surgery centers) in France, Germany, Italy, Spain,

Greece (gynecology only) and the UK. The

findings indicate that icodextrin 4% was well

tolerated by patients who underwent laparotomy or

laparoscopy (29).

In a recent study (by Colins et al 2007), 402

patients randomized intraoperatively to receive

either 4% icodextrin or LRS and then patients

returned for second laparoscopy within 4–8 weeks.

Incidence, severity, and extent of adhesions were

characterized for both groups and they

demonstrated that 4% icodextrin is a safe and

effective adhesion reduction agent in laparoscopy

(30).

Our study results support preclinical

observation (by Verco et al 2000) and recent study

(by Colin et al 2007), as mentioned before in adept

group, half of the cases were found with no

adhesion at all and the rest had mild adhesion.

Effect of 4% icodextrine on adhesion reduction


However in our study, no side effects, no

abscess formation and no other complication were

observed in contrast to Muller et al (2005). Our

data showed that lavage and instillation of 4%

icodextrin was not only safe but also effective in

reducing adhesion formation in rabbits, and with

regard to close relationship of fluid dynamics in

rabbits with human being this results suggest that

patients undergoing gynelocological surgery may

have a better prognosis for adhesion reduction after

using intra operative irrigation with 4% icodextrin.

Acknowledgment

This research was supported by Vice

Chancellery Research of Isfahan University of

Medical Sciences and there is no conflict of

interest in this article. The authors would like to

thank IUMS and Dr. Mehdi Nematbakhsh,

professor of Physiology Department of IUMS, and

Miss Mojdeh Ghasemi, midwife, and Miss Zahra

Samavatian for all their helps.

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Correlation between the level of cholesteryl ester

transfer protein in follicular fluid with fertilization

rates in IVF/ ICSI cycles Amir Mehdizadeh

1,2 M.Sc., Ali Rahimipour

3 Ph.D., Laya Farzadi

4 M.D., Masoud Darabi

2 Ph.D.,

Vahideh Shahnazi4 M.Sc., Maghsod Shaaker

2 B.Sc., Amir-Mansour Vatankhah

5 M.Sc., Zahra

Golmohamadi2 M.Sc., Mohammad Nouri

2 Ph.D.

1 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz,

Iran.

2 Laboratory of Chromatography, Department of Biochemistry and clinical Laboratories, Faculty of

Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.

3 Department of Laboratory Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

4 Women’s Reproductive Health Research Center, Alzahra Hospital, Tabriz University of Medical

Sciences, Tabriz, Iran.

5 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Received: 19 May 2010; accepted: 30 October 2010 Abstract

Background: Follicular fluid (FF) plays an important role in oocytes and embryo

development, which may contribute to IVF/ICSI success rate.

Objective: The aim of this study was to investigate the correlation between cholesteryl

ester transfer protein (CETP) level in FF and the success rate of IVF/ICSI.

Materials and Methods: In a cross-sectional study, FF samples, FF samples were

obtained from 100 patients referred to Tabriz Alzahra Hospital. Seventy-nine subjects

underwent IVF and the remaining 21 underwent ICSI. The levels of high-density

lipoprotein cholesterol (HDL-C), apolipoprotein A-I and CETP were measured using

enzymatic, turbidometric and ELISA methods respectively.

Results: Analysis of the subgroups with different levels of CETP showed a significant

lower level of CETP in the subgroup with the lowest number of mature oocytes

(p<0.05). The level of CETP was also considerably lower (18%, p=0.05) in subjects

with<50% oocytes fertilization ratio than subjects with >70% of this ratio.

Conclusion: While no association was found for pregnancy, the amount of CETP in FF

was associated positively to the maturity and the percentage of oocyte fertilization.

Key words: Infertility, IVF/ICSI, Apolipoprotein A-I, CETP.

Introduction

Infertility is a multifactorial disorder with

lifestyle and environmental traits playing important

role (1). Assisted conception techniques such as

IVF/ICSI are used to treat a range of infertilities.

However, these techniques are not always

successful and potential factors contributing to a

successful IVF/ICSI treatment are being


Mohammad Nouri, Department of Biochemistry,

Faculty of Medicine, Tabriz University of Medical

Sciences, Tabriz, Iran.


investigated (2).

Follicular fluid (FF) which is necessary for the

development of the female gamete, may also affect

the fertilization and embryonic development.

Therefore, FF is occasionally used as a medium

supplement in clinical IVF (3). High density

lipoprotein (HDL) particles of FF are the most

important agent for transferring cholesterol to

granulosa cells (4). So, HDL cholesterol (HDL-C)

is the most important resource of cholesterol for

progesterone synthesis after LH surge (5, 6).

Apolipoprotein A-I (apo A-I) is the major

apolipoprotein in FF, which is associated with

HDL (7). The cholesteryl ester transfer protein

mailto:%[email protected]

Mehdizadeh et al


(CETP), a hydrophobic glycoprotein transfers

cholesteryl esters from HDL to apolipoprotein B

(apoB)-containing lipoproteins (8). It seems that

the role and importance of CETP in blood is

different than other tissues. It has been

demonstrated that CETP is related to cell

membrane and affects on membrane physical and

chemical characteristics by facilitating selective

uptake of HDL-derived cholesteryl esters (9, 10).

Ranvik et al (11) reported that FF has lipid

transfer activity, and fractions of FF with this

activity stimulate human sperm capacitation and

acrosome reaction. It has been shown that this

process involves a sterol depletion of the

spermatozoa membrane (12).

The aim of the present study was to investigate

the correlation between CETP level in FF and the

success of IVF/ICSI techniques in infertile patients

undergoing these techniques.


A total of 100 patients referred to Tabriz

Alzahra Hospital in 2007-2009 were selected for

this cross-sectional study. All participants received

a screening history, physical examination,

measurements of serum hormone levels and blood

counts. The subjects' body mass index (BMI) was

calculated as measured weight divided by the

square of measured height (kg/ m2).

The mean age of subjects was 32±5.46 years

with no evidence of any disease. Healthy husband

with no smoking habit were defined as including

criteria. Uterus abnormalities, positive history of

endocrine disease and inflammatory disorders such

as thyroid and adrenal disorders, immune system

defect and sexual hormones disorders were

considered as exclusion criteria in this study.

Ovarian stimulation was achieved with a GnRH

agonist (Sereno, Switzerland) /FSH- long down

regulation protocol (13). Controlled ovarian

stimulation was started with recombinant human

follicle stimulating hormone (rFSH; Sereno,

Switzerland) at the third day of menstrual cycle.

The daily rFSH dose ranged between 150 and 300

IU, depending on body mass index, age of the

women, and the anticipated ovarian response. Dose

adjustment was done according to follicular

development and serum estradiol levels.

Intramuscular hCG (1000IU, Choriomon, Meizler,

Brazil) was administered when sonography

revealed the average diameter of 3 preovulatory

follicle had approached 18- 20mm.

Oocyte retrieval was done 36 h after hCG

administration by vaginal ultrasound-guided

puncture of the ovarian follicles. The collected

oocytes were incubated in 37ºc with 6% CO2 for

3-4 hours and then were used for IVF and ICSI. FF

was collected in a sterile tube, was centrifuged at

1500rpm for 5min and kept frozen at -70˚C until

analyses.

Oocytes with sporadic cumulus oophorus and

zona pellucida and also a clear ooplasm were

selected for insemination. The swim-up was used

to prepare sperm for IVF (14). Oocytes were

inseminated with 250000 sperm per oocytes in the

IVF technique (15). In the ICSI group, a single

motile sperm were injected into oocyes (16). After

24h, oocyets were separated from surrounding

granulosa cells. A maximum of three embryos

were transferred at 4-8 cells stages after 48h, under

ultrasound guidance. Clinical pregnancy assessed

by β-hCG test, 14 days after embryo transfer.

Proportion of the oocytes that had 2 pronuclei

was estimated as fertilization rate. This project was

approved by the ethics committee of Tabriz

University of Medical Sciences. Before the

sampling, we took a written consent from studied

subjects.

The levels of follicular HDL-C were measured

using enzymatic method by an Abbott Alycon 300

auto analyser. Apolipoprotein A-I concentration

determined by immune turbidometric method

(Diasys, Germany). The level of CETP was

measured by using an Alpco ELISA kit (catalogue

number 44-ADPRT-E01).


Values are presented as mean± S.D. The one

way ANOVA with Tukey post hoc pairwise

comparison and Multivariate analysis of variance

test were used for comparing means and ratios in

different groups. p-values of <0.05 were

considered statistically significant. Analysis was

carried out using SPSS 11.5 statistical software.

Results

General characteristics, husband spermogram

and FF parameters are shown in table I. The

average number of mature oocytes and fertilized

oocytes were 8.57±4.19 and 5.0±3.0, respectively.

The percentage of pregnancy in the studied

population was 24%.

As shown in table II the age of patients was

inversely associated with the number of mature

CETP in follicular fluid


oocytes (r=-0.21, p=0.04) but the level of CETP

was positively correlated with the number of

mature oocytes (r=0.24, p=0.02). We studied the

level of CETP and number of mature oocytes in 3

sub groups with various number of oocytes (group

1, <5; group 2, 6 to 9; and group 3, >10). The level

of CETP in group 1 was lower (25%, p=0.01) than

group 3 (Figure 1).

There was an inverse significant correlation

between BMI and the percentage of fertilized

oocytes (r=-0.27, p=0.02). We also found a

positive correlation between the percentage of

sperm motility and fertilized oocytes (r=0.27,

p=0.04). Correlation coefficients of fertilization

ratio with sperm count (r=0.23, p=0.05), HDL-C

(r=0.21, p=0.06) and CETP (r=0.21, p=0.08) were

positive and near to the significant levels (Table

III).

Our findings also showed that after matching

BMI, sperm count, sperm motility and HDL-C, the

level of CETP in patients with a percentage of

fertilized oocytes less than 50% is relatively lower

(18%, p=0.05) than in patients with a percentage of

fertilized oocytes> 70% (Figure 2). We found that

there were no significant difference between

patients with negative and positive pregnancy

(p=0.62) in the levels of follicular fluid CETP

concentration (Table IV).

Table I. Patients' clinical characteristics, biochemical profile of follicular fluid and semen parameters of husbands

Characteristics Mean ±SD (range)

General Age (years)

31.7±5.46 (21-43)

Body Mass Index (kg/m2)

25.5±3.13 (20-34)

IVF parameters

Mature oocytes

8.57±4.19 (1-19)

Fertilized oocytes

5.0±3.0 (0-13)

Biochemical Pregnancy 24%

Biochemical profile of follicular fluid

HDL-C

26±10 (9-24)

Apolipoprotein A-I (mg/dl)

105±19 (14-133)

CETP (µg/dl)

1.23±0.45 (0.1-2.6)

Semen characteristics

Sperm count (106/ml)

59.5±30.3 (28-120)

Sperm motility (%)

66.1±18.4 (10-90)

Values are expressed as mean± SD or percentage (ranges in parenthesis).

Table II. Association between parameters and number of mature oocytes.

p-value r

Age (years)

0.039 - 0.208

Body Mass Index (kg/m2)

0.74 0.034

Apolipoprotein A-I(mg/dl)

0.774 - 0.030

CETP(µg/dl)

0.019 0.240

r, Correlation coefficient

Mehdizadeh et al


Table III. Association between parameters and fertilization rate (FR) in patients undergoing IVF.

Parameter p-value r

Age (years)

0.436 -0.090

BMI(kg/m2)

0.016 - 0.273

Sperm count (106/ml)

0.051 0.235

Sperm motility (%)

0.049 0.266

HDL-C (mg/dl)

0.058 0.217

Apolipoprotein A-I(mg/dl)

0.329 0.113

CETP(µg/dl)

0.076 0.205

FR= (number of fertilized oocyte / number of mature oocytes) ×100 r, Correlation coefficient.

Table IV. Level of follicular fluid parameters in clinical positive and negative fertility.

Non-pregnant

(n=75)

Pregnant

(n=25) p-value

Age (years)

31.8±5.6 31.2±5.1 0.63

BMI (kg/m2)

25.5±3.2 25.7±3.1 0.74

HDL-C (mg/dl)

26.9±7.0 27.3±6.5 0.78

Apolipoprotein A-I (mg/dl)

103±20 106±15 0.66

CETP (µg/dl)

1.25±0.43 1.19±0.49 0.62

Values are expressed as mean± SD. Biochemical pregnancy was assessed by β-hCG test, 14 days after embryo transfer.

0

0.5

1

1.5

≤5 6-9 ≥10

CE

TP

, μ

g/d

l

Oocytes number

P=0.011

P=0.034

Figure 1. Levels of FF CETP in subgroups with various numbers of mature oocytes. Multivariate analysis of variance with post hoc pairwise

comparison. P-value adjusted for BMI and age.

CETP in follicular fluid


0

0.5

1

1.5

≤50 51-69 ≥70

CE

TP

, μ

g/d

l

Fertilization percentage

P=0.054

P=0.14

Figure 2. Level of follicular fluid CETP in subgroups with different rate of fertilization. Multivariate analysis of variance with post hoc pairwise

comparison. P-values adjusted for BMI, Sperm count, Sperm mobility.

Discussion

Our study showed that the proportion of the

number of fertilized oocytes to the number of

mature oocytes and the rate of positive pregnancy

were lower than previous studies on population of

other countries.

For example, Mutsubayashi et al study showed

that the proportion of the number of fertilized

oocyte to the number of mature oocytes was 70%

(17) and Von Wald et al (18) reported a 45% rate

of positive pregnancy. Since the success of

IVF/ICSI and the occurrence of pregnancy are

affected by different environmental factors, these

might be attributed to the presence of higher

burdens of environmental or genetic risk factors

among people of reproductive age in the local

population.

There were positive correlations between FF

CETP and the number of mature oocytes and

percentage of positive fertility. The present study

does not clarify the mechanism by which the CETP

affect infertility risk status. It can be explained by

the effect of CETP on the ability of oocytes to be

fertilized and develop into embryos. CETP is a

hydrophobic glycoprotein that plays a central role

in human HDL metabolism (19). CETP also play

an important role in the maintenance of vesicle

membrane integrity and/or in membrane fusion

events via the transportation of cholesterol from

the cytoplasm to the outside of the cell via the cell

membrane (20).

The oocyte membrane has been characterized as

important player in the control of sperm-oocyte

fusion on the basis of in vitro experiments (21).

One possible explanation for the relation of FF

CETP with fertilization rate is that CETP promote

membrane fusion during in vitro fertilization via

the modulating of oocyte membrane function. On

the other hand, Ishikawa et al found an interesting

relationship between cellular mRNA level of

CETP and maturation of B cell lymphocytes in

germinal centers (22).

These findings are consistent with the results of

the present study showing a positive relation

between number of mature oocytes and the level of

CETP in FF. It seems that CETP is also involved

in follicles and oocyte development via supplying

substrate for sexual hormones synthesis in

preovulatory ovarian follicles. Accordingly, the

importance of lipids metabolism in FF has been

identified in previous studies (23, 24).

No statistically significant correlation was seen

between clinical parameters and the rate of

pregnancy. Considering that the occurrence of

pregnancy is affected by factors beyond the

Mehdizadeh et al


characteristics of FF, the absence of a significant

relationship between the parameters and the

occurrence of pregnancy is probably because of the

indirect nature of the relationship and/or the low

number of subjects.

According to the results of this study, while

there was no association for pregnancy, the amount

of FF CETP was correlated positively with the

maturity and the percentage of oocyte fertilization.

These findings are consistent with the hypothesis

that CETP FF plays an important role in

cholesterol and lipid metabolism of follicles.

Acknowledgment

This work has been supported by Research

Center for Pharmaceutical Nanotechnology, Tabriz

University of Medical Sciences.

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Diagnostic value of saline contrast sonohysterography

comparing with hysteroscopy for detecting

endometrial abnormalities in women with abnormal

uterine bleeding Mohammad Ali Karimzadeh

1 M.D., Razieh Dehghani Firouzabadi

1 M.D., Farzaneh Goharzad

2 M.D.

1 Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, Shahid

Sadoughi University of Medical Sciences, Yazd, Iran.

2 Department of Obstetrics and Gynecology, Shahid Sadoughi Hospital, Shahid Sadoughi University of

Medical Sciences, Yazd, Iran.

Received: 14 June 2010; accepted: 3 October 2010

Abstract

Background: Abnormal uterine bleeding is a common presentation of uterine

abnormalities among premenopausal and postmenopausal women.

Objective: To evaluate and compare the diagnostic accuracy of saline contrast

sonohysterography and hysteroscopy for detecting the cause of abnormal uterine

bleeding.

Materials and Methods: A total of 65 women with abnormal uterine bleeding were

enrolled in this study. A prior saline contrast sonohysetrography followed by a

hysteroscopy was performed in all cases. Sensitivity, specificity, positive and negative

predictive value and test accuracy were calculated.

Results: As the most common abnormality, SCSH showed hyperplasia in 19 patients

while hysteroscopy diagnosed polyp in 15 cases. A sensitivity of 73.3%, 71.4% and

90.9% were reported for polyp, hyperplasia and submucous myoma respectively

whereas the specificity was calculated 96% for polyps, 82.3% for hyperplasia and

90.7% for submucous myoma. Conclusion: Comparing with hysteroscopy, sonohysterography showed a high

sensitivity and specificity for detecting submucous myoma but not for endometrial

polyp and endometrial hyperplasia.

Key words: Abnormal uterine bleeding, Uterine abnormalities, Sonohysterography, Hysteroscopy.

This article extracted from residential thesis

Introduction

Abnormal uterine bleeding (AUB) is a common

presenting symptom among women. AUB is

presented in 33% of women referred to

gynecologists and this pattern increases to 69% in

premenopausal and post menopausal women (1).

AUB can be caused by a variety of uterine

abnormalities such as polyp, submucous


Razieh Dehghani Firouzabadi, Department of

Obstetrics and Gynecology, Research and Clinical

Center for Infertility, Shahid Sadoughi University of

Medical Sciences, Yazd, Iran.


myoma, endometrial hyperplasia and endometrial

carcinoma. Cases of AUB require a good

diagnostic and therapeutic approach which can be

acquired by traditional dilatation and curettage or

recent and more effective diagnostic tools.

A variety of tools can be used for the diagnosis

of uterine abnormalities lead to AUB. Among

them, transvaginal sonography (TVS), saline

contrast sonohysterography (SCSH) and

hysteroscopy have been used commonly. TVS is

the first line investigation tool for diagnosis of

uterine abnormalities, whereas hysteroscopy has

become the gold standard for the evaluation of

patients with AUB. In postmenopausal women

TVS is an effective screening test for evaluation of





Karimzadeh et al


abnormal uterine bleeding caused by endometrial

atrophy (2). But in the figure of thickened and

inhomogeneous endometrium, TVS is presented as

a low specificity and limited diagnostic test which

can be replaced by SCSH (3, 4). SCSH can

distinguish between focal lesions such as polyps

and submucous myomas (5, 6) and diffuse lesions

like hyperplasia and cancer accurately (7, 8).

Furthermore, hysteroscopy is an effective but

expensive and invasive screening test for

evaluation of the uterine cavity in both pre and

postmenopausal women with AUB (9, 10).

Preoperative imaging of the uterine cavity is

very important and the results can be necessary for

the surgical management. A useful imaging

technique for accurate diagnosis should be highly

sensitive and specific, non invasive and cost-

effective. It seems that SCSH is a non invasive,

cheap and feasible technique with lower pain. In

order to compare SCSH and hysteroscopy, the

majority of women found that SCSH was not

painful, whereas only 25% said the same for

hysteroscopy (11). If it can be proven that the

sensitivity and specificity of SCSH and

hysteroscopy are the same, it can be recommended

as the first line detecting tool for uterine

abnormalities caused AUB.

The objective of this study was to evaluate the

sensitivity and specificity of SCSH compare with

hysteroscopy in the investigation of women of

reproductive age presenting with AUB.


Patients

65 consecutive women presenting AUB or

infertility were enrolled in this diagnostic study.

These patients were referred to both Yazd Shahid

Sadoughi and Yazd Madar Hospital from March

2006 to February 2007. The women who have any

evidence of systemic disease such as diabetes,

hypertension, PCO, thyroid disease, evidence of

pregnancy, evidence of pelvic inflammatory

disease and history of uterine surgeries were

excluded from the study. After obtaining informed

consent, saline contrast sonohysetrography

followed by a hysteroscopy was done in all cases.

The institutional Review Board at Yazd University

of Medical Sciences approved this prospective

study.

Imaging techniques Regarding SCSH, a sterile speculum was

passed, the cervix visualized and disinfected with Betadine solution. A flexible Foley catheter

number 8 with inflatable balloon (Supa, Tehran, Iran) was inserted through the cervical canal into the uterine cavity. After confirmation of the position of the catheter, 10ml of 0.9% sterile saline solution was injected into the uterine cavity slowly and continued to obtain optimal views of endometrial cavity. By using concomitant transvaginal sonography, the uterine cavity was evaluated for detecting any abnormality or pathological condition. This procedure was performed by a single investigator without the use of local anesthesia or prophylactic antibiotic therapy. All patients had diagnostic operative hysteroscopy under a general anesthesia. Hysteroscopy was performed using cervix dilatation, 2 Misoprostol tablet (6 hours before operation) and prophylactic antibiotic. The hysteroscopies were done by the expert operator who was blinded to the SCSH results. Endometrial biopsy was carried out directly after hysteroscopy. Statistical analysis

The Statistical Package for the Social Sciences 13.0 software was used to analyze data of all patients. Sensitivity, specificity, positive and negative predictive value and test accuracy were calculated for SCSH as compared with findings of hysteroscopy.

Results

Among 65 evaluated women, 78.5% presented AUB and 21.5% had infertility problem. The mean age of women presenting AUB was 37.02±7.85 years and infertile women had mean age of 25.50± 4.22 years. The most common abnormality in SCSH was hyperplasia (29.2%) while it was polyp (23.1%) in hysteroscopy. Hyperplasia was detected in 21.5% of cases by hysteroscopy and polyp was seen in 20% of patients using SCSH. As the second cause, SCSH suggested the presence of cancer in 23.1% of women whereas it was hyperplasia among 21.5% of cases in hysteroscopy group. The number and percentage of abnormalities detected in patients are listed in table I. According to hysteroscopy results the diagnosis of 36.9% of women remained unknown and it was 26.2% in SCSH. SCSH showed a sensitivity of 73.3%, 71.4% and 90.9% for polyp, hyperplasia and submucous myoma respectively whereas the specificity was reported 96% for polyps, 82.3% for hyperplasia and 90.7% for submucous myoma. Table II shows the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for SCSH compared with hysteroscopy as a gold standard.

Accuracy of sonohysterography among women with AUB


Table I. Number of uterine abnormalities diagnosed using SCSH and hysteroscopy.

SCSH no (%) Hysteroscopy no (%)

Polyps

12(20%) 15(23.1%)

Hyperplasia

19(29.2%) 14(21.5%)

Submucous myoma

1(1.5%) 1(1.5%)

Cancer

15(23.1%) 11(16.9%)

Unknown 17(26.2%) 24(36.9%)

Table II. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy for SCSH compared with

hysteroscopy.

Sensitivity Specificity Positive predictive value Negative predictive value Accuracy

Polyps

73.3% 96% 84.6% 92.3% 90.7%

Hyperplasia

71.4% 82.3% 52.6% 91.3% 90%

Submucous myoma 90.9% 90.7% 66.7% 98% 90.7%

Discussion

There are different methods for detecting causes of AUB as a common chief complain in premenopausal or post menopausal women. For many years, dilatation and curettage was performed as a first line approach, because the sonographical tools have limited accuracy specially unavailability of endometrial sampling (12, 13). Nowadays, this limitation has been overcome by TVS and SCSH followed by hysteroscopy and endometrial sampling. SCSH is a new evaluating method that makes distension in uterine cavity to visualize endometrial surface (14). In addition it has less pain in patients with minimal cost, and performs easier and faster with more safety comparing with hysteroscopy (15, 16). In this research we compared SCSH as an accurate method to distinguish local and diffuse lesions (5, 6) with hysteroscopy as a gold standard diagnostic method.According to our study, in detecting submucous myoma, SCSH has a good sensitivity of 90.9 and specificity of 90.7 compared with hysteroscopy. Regarding polyp and endometrial hyperplasia, hysteroscopy presented more sensitivity and specificity than SCSH. Some studies reported a similar diagnostic accuracy (17-20). One study found a sensitivity of 100% and specificity of 98.3% for myoma versus sensitivity of 87.5% and specificity of 95.9% for polyp; using sonohysterography compared with hysteroscopy as the reference test (17). In Kelekci et al study, for detecting endometrial polyp using saline infusion sonography; sensitivity, specificity, PPV and NPV were 70%, 100%, 100% and 90.9% retrospectively whereas all of these parameters were 100% in detecting submucous myoma (18). The current

study showed that sonohysterography is less sensitive and specific for detecting polyp and endometrial hyperplasia than hysteroscopy. In disagreement with our results, Soares et al indicated that sonohysterography had 100% sensitivity, 100% PPV and 100% diagnostic accuracy for endometrial polyps, fibroids and endometrial hyperplasia (19). In addition, Nanda et al reported that there is no missing in diagnosis of endometrial polyp using sonohysterography (21). In one study, 135 patients with AUB and subfertility were evaluated and the result showed that SCHS is a very accurate method for detecting focal endometrial pathology, compared to diagnostic hysteroscopy (22). Another study claimed that hysteroscopy can be replaced by Saline-infusion sonography in more than half of AUB cases (23) and also there is very good agreement between sonohysterography and hysteroscopy for diagnosis endometrial abnormalities in postmenopausal women (24). Mathew et al (25) concluded that saline infusion sonohysterography is a simple evaluating method with minimal invasiveness and cost which is more accurate than TVS and can be done as a screening tool before hysteroscopy. Saline infusion sono-hysterography also is a satisfactory method of identifying endometrial lesions which is less invasive alternative to hysteroscopy and result in less morbidity in the evaluation of AUB in women (26).

Conclusion

According to our result, comparing with

hysteroscopy; sonohysterography is sensitive and

specific for diagnosis of submucous myoma but

Karimzadeh et al


not for endometrial polyp and endometrial

hyperplasia. However hysteroscopy is a therapeutic

procedure and it is preferable for its therapeutic

role.

Acknowledgement

The authors thank the staff of operation room of

Madar Hospital and Shahid Sadoughi Hospital for

their cooperation.

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22Dijkhuizen%20FP%22%5BAuthor%5D

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22Mathew%20M%22%5BAuthor%5D

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22Yildizhan%20R%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ozkesici%20B%22%5BAuthor%5D

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Successful pregnancy following the transfer of vitrified

blastocyst which developed from poor quality embryos

on day 3 Xiao-jian Zhang M.Sc., Ye-zhou Yang Ph.D., Li-hua Min B.N., Qun Lv B.Sc. , Pin Bai B.N., Xiao-jie

Li B.S.M.T., Mei-xu Liao M.Sc.

Department of Assisted Reproductive Medical Center, Sichuan Academy of Medical Sciences and

Sichuan Provincial People’s Hospital, Sichuan, China.

Received: 5 July 2010; accepted: 11 January 2011

Abstract

Background: The selection of pre-embryos for transferred is based on morphological

appearance. But some poor quality cleaved embryos also can be cultured to the

blastocyst stage and implanted.

Objective: To assess the clinical pregnancy outcomes of blastocyst transfer which

developed from poor quality embryos.

Materials and Methods: A total of 109 cleaved embryos with poor quality were

cultured to day 5/day 6 and 27 (24.8%) blastocysts were collected from the 15

cycles/patients undergoing conventional IVF. All the blastocysts were cooling with fast-

freezing. Then the blastocysts were warmed for transfer.

Results: All of 25 vitrified blastocysts (92.6%) survived after warming and were

transferred to 15 patients. Five of the women became pregnant.

Conclusion: Our results suggest that vitrified human day 5/day 6 blastocyst transfer

which develop from poor quality embryo at day 3 can contribute to increasing

cumulative pregnancy rates in assisted reproduction.

Key words: Poor quality embryos, Vitified Blastocyst- transfer, Pregnancy.

Introduction

In IVF, the selection of pre-embryos for

transferred is based on morphological appearance

according to various scoring systems, including

blastomere uniformity, cell numbers, degree of

fragmentation, blastomere size and cytoplasmic

appearance. Only well developed, good quality

embryos will be transferred or cryopreserved. The

poor quality embryo is expected to have a

significantly lower chance of implanting and

usually will be discarded (1-3).

However, this scoring system has several

shortcomings. The viability and the implantation

potential of the poor quality embryo cannot be

determined accurately. Poor quality cleaved

embryos also can be cultured to the blastocyst

Corresponding Author: Xiao-jian Zhang, Department of Assisted Reproductive

Medical Center, Sichuan Academy of Medical Sciences

and Sichuan Provincial People’s Hospital, Sichuan,

China.


stage and implanted (4).

With the improvement of culture systems and

blastocyst cryopreservation techniques, some

pregnancies are achieved following the transfer of

cryopreserved day 5/day 6 blastocysts (5).

However, as far as is known, there were no reports

on pregnancies following the transfer of

cryopreserved day 5/day 6 blastocysts

development from poor quality cleaved embryos.

In this report, we present a successful

pregnancy after the transfer of a human vitrified

day 5/day 6 blastocyst developed from poor quality

cleaved embryos which inconcinnity transferred

and freezed on day 3.


The study retrospective comparative analysis

the pregnancy obtained after vitrified of human

blastocyst which developed from poor quality

embryos on day 3. It was carried out between

January 2009 and January 2010.

The treatment was conducted with patients

following informed consent and according to the


Xiao-jian Zhang et al


guidelines of the Ministry of Public Health of

China (MPH) and was approved by the

reproductive ethics committee at the Sichuan

Academy of Medical Sciences and Sichuan

Provincial People’s Hospital. Vitrified day 5/day 6

blastocysts were transferred to fifteen patients who

had previous failures of fresh and/or frozen day 3

embryo transfers before the transfer of day5 /day6

vitrified blastocysts. All the transferred embryos

were come from the same stimulated cycle on one

patient. The mean age of patients was 30.6 years

with a standard deviation of 3.1. Main causes of

infertility were female related in all of 15 cycles,

10 tubal, three tubo peritoneal, and two

endometriosis.

In seven cases clinical diagnosis was primary

infertility. Conventional IVF was performed in all

cycles. Over the study period, there were no

changes in the laboratory procedures and the

culture media utilized.

Women were treated with gonadotrophin-

releasing hormone agonist (triptorelin; diphereline,

France) using either the preceding mid-luteal phase

in a long treatment protocol or second day of the

cycle in a short treatment protocol. Ovarian

stimulation was carried out with human

menopausal gonadotrophin (Pergonal; Lizhu,

China) or recombinant follicle-stimulating

hormone (rFSH, GONAL-F, Serono, Germany).

Follicular development was monitored with serial

vaginal ultrasound examinations and serum E2

measurements. The human chorionic

gonadotrophin (hCG; Ovidrel, Serono, Germany)

were administered when at least two dominant

follicles reached a diameter of 18 mm. Oocytes

pick up (OPU) was performed 36–38h after hCG

administration.

Oocyte cumulus complexes were incubated in

human tubal fluid medium (HTF, Sage Biopharma,

USA) containing 5% (v/v) Human Serum Albumin

(Sage Biopharma, USA) at 37°C in an atmosphere

of 6% CO2. The day of oocyte retrieval was

considered as Day 0. Mature oocytes were

inseminated with sperm 4-6h after OPU at the

concentration of 50,000 to 100,000 motile sperm

per oocyte. Fertilization was confirmed at 16-18 h

after insemination by the presence of two

pronuclei. Fertilized oocytes transferred into

Cleavage Medium (Sage Biopharma, USA) until

Day 3.

The cleaved embryos were graded as described

by Balaban B (4), make adjustments as follow:

Class I： no more than 5% fragmentation with

equal sized homogenous blastomeres and the No.

of blastomeres≥ 6; Class II： no more than 20%

fragmentation with equal sized homogenous

blastomeres and the No. of blastomeres ≥6; Class

III： 20% to 50% fragmentation with unequal sized

blastomeres and the No. of blastomeres≥ 4; Class

IV： more than 50% fragmentation and /or very

unequal sized blastomeres or the number of

blastomeres<4.

In this study we aimed to determine the

feasibility and success of vitrified blastocyst-stage

transfer in patients yielding from poor (class III

and class IV) quality cleavage-stage embryos on

day 3. In all the patients, two or three excellent-

quality embryos were transferred on day 3 after

OPU. Supernumerary good quality embryos were

frozen as described previously (6). The others poor

(class III and class IV) quality embryo were

cultured to day 5 or day 6. For expanded

blastocysts, the development of the inner cell mass

(ICM) and trophectoderm (TE) was assessed. The

ICM grading was as follows: A: tightly packed,

many cells; B: loosely grouped, several cells and

C: very few cells. The TE grading was as follows:

A: many cells forming a tightly knit epithelium; B:

few cells and C: very few cells forming a loose

epithelium (7). Only expanded blastocysts scoring

B or higher for ICM and scoring C or higher for

TE grades were vitrified.

Fully expanded blastocysts were deflated by

gentle aspiration of the blastocoelic fluid using a

micromanipulator until the cavity collapses prior to

vitrification. The blastocysts were transferred to

the drop of MOPS buffered solution of modified

HTF which contained 7.5% (v/v) each of Dimethyl

Sulfoxide (DMSO) and ethylene glycol (EG) for 5

to 15 minutes. Then the blastocysts was washed

using 15% DMSO and 15% EG supplemented four

times, and transfer the blastocysts in <1 ul of the

solution from last drop to the tip of Cryoleaf

(CryoleafTM

Medicult, Denmark) which carefully

and immediately submerged into liquid nitrogen.

The warming procedure was done as follows.

The protective cover was removed in liquid

nitrogen and the end of the Cryoleaf was immersed

directly into 1 ml of 37°C 1.0 mol/l sucrose for 1

min. The blastocysts were then transferred into 1

ml of 0.5 mol/ l sucrose for 2 minutes and repeat

once again and washed thrice in the base medium

for 9 min. Finally, the blastocysts were transferred

to a dish of pre-equilibrated appropriate culture

medium (Sage Biopharma, USA) and incubate in a

6% CO2 incubator at 37°C for 3-4 hours to allow

for further recovery.

The laser hatching procedure was performed

using an inverted microscope (Nikon TE2000–U,

Japan) that was equipped with the Zona Infrared

Laser Optical System (Saturn Active Laser

System, Research Instrument, England). The

procedure was done as describe by Zhang et al (6).

Pregnancy obtained by vitrified blastocyst developed from poor quality embryos on day 3


All of the frozen day-3 embryos should be

warmed for transfer and no pregnancy occurred,

then the blastocyst were warmed. Warmed embryo

transfer was performed in hormonal replacement

treatment cycles. All women received oral

oestradiol (Progynova; Bayer, France) 2-4mg daily

for 2 days with gonadotrophin-releasing hormone

analogue begin at the tenth day of menstrual for

the preparation of the endometrium. The

administration of progesterone (Progesterone

Injection； Shanghai TongYong Pharmaceutical

Co., Ltd., Shanghai, China) was added transdermal

at a dose of 40mg/day when endometrial thickness

exceeded 8 mm. Embryo was warmed on the

morning of day 6 and transfer was scheduled after

the initiation of progesterone treatment irrespective

of whether they had been day 5 morula or day 6

blastocyst. A beta-hCG test was performed 2

weeks after ET. When two consecutive tests (at 2-

days intervals) showed elevated beta-hCG level,

pregnancy could be considered. Pregnancy was

confirmed when ultrasonograph revealed a

gestational sac or fetal heart beat 4 weeks after ET.

The implantation rates were calculated as the total

number of feta sacs expressed as a percentage of

the total number of transferred embryos.


The numerical variables of patients among

groups was compared by means of a two-tailed

unpaired Student’s t-test. Chi-squared test was

used to compare percentage of Cause of infertility,

Embryo cleaved, good/ poor quality embryos and

embryo implantation rates between transfers done

under the Pregnancy group and No pregnancy

group. A p-value of <0.05 was considered

statistically significant.

Results

Of 176 cleaving embryos from 15 cycles, 67

were judged to be of sufficient quality for use on

day 3 on the basis of traditional selection criteria.

Embryos-transfer were performed only on 14

patients. One patient was canceled for OHSS.

There was an average of 2.3 embryos per ET. The

remaining 35 good quality embryos would have

been cryopreserved. The other 109 poor quality

(class III and class IV) embryos undergoing

extended culture to day 5/day 6. 27 blastocysts

were obtained and vitrified either on day 5 or day

6.25 blastocysts were survived after thaw, and

transferred to 15 patients. With an average of 1.67

blastocysts per ET. Five of the women became

clinically pregnant, and two of them were twin

pregnancies. The data are summarized in table I.

The data for cyophoric patients are summarized

in table II. Eight blastocysts were vitrified on day 5

which were from, patient 3, patient 4 and patient 5.

Seven blastocysts were survived and transferred

after warming and were transferred into the

patient’s uterus. Five gestational sac with fetal

heartbeat was confirmed by ultrasound 30 days

after embryo replacement. Three blastocysts were

vitrified on day 6 which were from patient 2. Three

blastocysts were survived and transferred after

warming, only one gestational sac with fetal

heartbeat was confirmed. In patient 1, the two

blastocysts were vitrified both on day 5 and day 6.

One gestational sac with fetal heartbeat was

confirmed.

Table I. The outcome of demographic characteristics and IVF between No pregnancy group and pregnancy group.

Parameter

No pregnancy group Pregnancy group p-value

Cycles

10 5 -

No. of oocytes recovered (M±SD)

165 (16.5±4.89) 91 (18.2±5.81) 0.559

No. of oocytes fertilized (M±SD)

107 (10.70±3.56) 72 (14.40±5.73) 0.142

No. of embryos cleaved [n, (%)]

104 (97.20) 72 (100) 0.401

No. of class Ⅰ embryos

6 5 -

No. of class Ⅱ embryos

35 21 -

Percentage of good quality embryos [%, (n/n)]

39.42 (41/104） 36.11 (26/72) 0.752

No. of class Ⅲ embryos

30 22 -

No. of class Ⅳ embryos

33 24 -

Percentage of poor quality embryos [%, (n/n)]

60.58 (63/104) 63.89 (46/72) 0.752

No. of blastocysts obtained and vitrified

14 13 -

No. of blastocysts survived and transferred

13 12 -

Implantation rate per transferred cycle [%, (n/n)]

0 58.33 (7/12) -

No. of twin pregnancies (%)

0 2(40.0)* -

* All multiple pregnancies were dizygotic twins. NS= not significant



Table II. The study of five patients whom pregnancy following the transfer of vitrified human blastocysts developed from poor

quality embryos on day 3.

Parameter

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Total

Age

32 30 31 29 31 -

Cause of infertility

Tubal Tuboperitoneal Tubal Tubal Endometriosis -

Primary/secondary infertility

Primary Primary Secondary Primary Secondary -

No. of oocytes recovered

12 18 23 13 25 91

No. of oocytes fertilized (2PN)

7 14 21 11 19 72

No. of embryos cleaved to day 3

7 14 21 11 19 72

No. of embryos transferred on day 3

2 3 2 2 0** 7

No. of embryos freezing on day 3

2 2 5 1 9 19

No. of remaining embryos cultured on day 3

5 9 14 8 10 46

No. of blastocysts obtained and vitrified on day 5

1 0 4 2 2 9

No. of blastocysts obtained and vitrified on day 6

1 3 0 0 0 4

No. of blastocysts warmed

2 3 4 2 2 13

No. of blastocysts survived and transferred

2 3 3 2 2 12

No. of gestational sacs

1 1 1 2 2 7

No. of pregnancies ongoing or delivered

1 1 1 2 2 7

**ET were canceled for OHSS.

Discussion

We report five cases of successful pregnancies

following the transfer of vitrified human

blastocysts developed from poor quality cleaved

embryos from 15 patients who be treated with

hormone replacement. This study shows that

cleaved embryo morphology scores cannot predict

the development potential literally. The results of

our study have shown that extended culture of poor

quality embryos could result in blastocyst

development, therefore they should be considered

as viable and valuable embryos for vitrification

and transfer. Traditionally most of the IVF

laboratories transfer the embryos on day 3.

Unquestionably the morphology of embryos on

day 3 has some predictive value for implantation

potential. Until now the number of cells and grade

of fragmentation have been considered to be the

most important scoring factors, the uneven embryo

cleavage negatively affects both implantation and

pregnancy rate also (2); However, criteria for

embryo selection on day 3 seem to be inadequate,

this value may be limited by the fact that they are

still in part depending on the maternal genome.

The embryonic genome is fully activated after the

8-cell stage and the development potential will be

restored in the subsequent cultured process (8, 10).

The poor quality embryos are good sources for

deriving human embryonic stem cell lines (11, 12)

and also can be extended culture to blastocyst for

vitrification. There for, not all of the poor quality

embryos have development potential.

Research in the area of assisted reproduction

has resulted in significant improvements in

stimulation protocols and culture conditions

resulting in better quality and number of

blastocysts available for embryo transfer.

Blastocysts are generally considered to be

preimplantation embryos which have successfully

passed the genomic activation and have better

developmental potential thus allowing reducing the

number of embryos transferred (13).

With the introduction of sequential culture

system, blastocyst culture is being adopted by

many IVF clinics as a means to increase pregnancy

rates, while minimizing multiple gestations (14).

The vitrification technology has made rapid

progress in recent years (15, 16). Despite the

advances in human blastocyst vitrification, much

remains to be learned regarding the limits of

current extended human embryo culture techniques

and the clinical usefulness of later-stage

cryopreservation. Previous investigators have

found that pregnancies can be obtained after the

transfer of human day 7 re-vitrified blastocysts

Pregnancy obtained by vitrified blastocyst developed from poor quality embryos on day 3


developed from vitrified whole cleaved embryos

(7).

In our study, 27 (24.8%) blastocysts was

formed from 109 poor quality cleaved embryos

and 25 (92.59%) blastocysts was survived for

transfer. 5 (33.33%) cycles were obtained

pregnancy and the implanted rate was 28.00% , it

is lower than published article which from whole

embryo culture (17, 18).

We have thawed only 15 cycles of blastocysts

developed from poor quality embryos on day 3. It

will be difficult to increase the number of

treatment cycles in a short period. However, the

information of poor quality cleaved embryos

discard may have occurred in many IVF clinics.

That would have resulted in the loss of viable

supernumerary embryos.

Accordingly, although the number of treatment

cycles was small, the present report has profound

clinical value in knowing that blastocysts

developed from poor quality cleaved embryos can

be vitrified, successfully warmed, and result in

ongoing pregnancies.

This is the first report of successful pregnancies

after the transfer of vitrified human blastocysts

developed from supernumerary poor quality

cleaved embryos. Our results suggest that extended

culture poor quality cleaved embryos to day 5/day

6 and vitrified for transfer can contribute to

increasing cumulative pregnancy rates as a

supplementary method in assisted reproduction.

Acknowledgement

This study was supported by Scientific

Research found of Bureau of Health of Sichuan

Provincial (080342) and Advanced High-Tech of

Sichuan Academy of Medical Sciences and

Sichuan Provincial People’s Hospital (09-02).

References

1. Farhi J, Nahum H, Weissman A, Roybur M, Glezerman

M, Levran D. The value of culturing to the blastocyst

stage of supernumerary embryos not suitable for freezing

after embryo transfer at day 3. Fertil Steril 2001; 76

(Suppl.): 118.

2. Hardarson T, Hanson C, Sjögren A, Lundin K. Human

embryos with unevenly sized blastomeres have lower

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aneuploidy and multinucleation. Hum Reprod 2001; 16:

313-318.

3. Rijnders PM, Jansen CA. The predictive value of day 3

embryo morphology regarding blastocyst formation,

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following in-vitro fertilization or intracytoplasmic sperm

injection. Hum Reprod 1998; 13: 2869-2873.

4. Balaban B, Urman B, Alatas C, Mercan R, Aksoy S,

Isiklar A. Blastocyst-stage transfer of poor-quality

cleavage-stage embryos results in higher implantation

rates. Fertil Steril 2001; 75: 514-518.

5. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K.

Vitrification of human hatched blastocysts: a report of 4

cases. J Reprod Med 2007; 52: 413-415.

6. Zhang XJ, Yang YZ, Lv Q, Min LH, Li XJ, Bai P. Effect

of the size of zona pellucida thinning by laser assisted

hatching on clinical outcome of human frozen-thawed

embryo transfers. Cryo Letters 2009; 30: 455-461.

7. Hiraoka K, Fujimoto Y, Tateaki Y, Hiraoka K, Horiuchi

T, Okano S, et al. Case report: two successful pregnancies

following the transfer of re-vitrified human day 7

blastocysts developed from vitrified cleaved embryos. J

Assist Reprod Genet 2008; 25: 503-509.

8. Graham J, Han T, Porter R, Levy M, Stillman R, Tucker

MJ. Day 3 morphology is a poor predictor of blastocyst

quality in extended culture. Fertil Steril 2000; 74: 495-

497.

9. Braude P, Bolton V, Moore S. Human gene expression

first occurs between the 4-cell and 8-cell stages of

preimplantation development. Nature 1988; 332: 459-461.

10. Van Royen E, Mangelschots K, De Neubourg D,

Valkenburg M, Van de meerssche M, Ryckaert G, et al.

Characterization of a top quality embryo, a step towards

single-embryo transfer. Hum Reprod 1999; 14: 2345-2349.

11. Liu W, Yin Y, Long X, Luo Y, Jiang Y, Zhang W, et al.

Derivation and characterization of human embryonic stem

cell lines from poor quality embryos. J Genet Genomics

2009; 36: 229-239.

12. Raya A, Rodríguez-Pizà I, Arán B, Consiglio A, Barri PN,

Veiga A, et al. Generation of cardiomyocytes from new

human embryonic stem cell lines derived from poor-

quality blastocysts. Cold Spring Harb Symp Quant Biol

2008; 73: 127-135.

13. Ménézo YJ. Blastocyst freezing. Eur J Obstet Gynecol

Reprod Biol 2004; 115 (Suppl.): 12-15.

14. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T,

Stevens J, Hesla J. A prospective randomized trial of

blastocyst culture and transfer in in-vitro fertilization.

Hum Reprod 1998; 13: 3434-3440.

15. Yokota Y, Sato S, Yokota M, Ishikawa Y, Makita M,

Asada T, et al. Successful pregnancy following blastocyst

vitrification: case report. Hum Reprod 2000; 15: 1802-

1803.

16. Hiraoka K, Hiraoka K, Horiuchi T, Kusuda T, Okano S,

Kinutani M, et al. Case report: successful delivery

following the transfer of a human re-vitrified day-7

spontaneously hatched blastocyst developed from vitrified

cleaved embryos. J Assist Reprod Genet 2009; 26: 405-

409.

17. Zhang JQ, Li XL, Peng Y, Guo X, Heng BC, Tong GQ.

Reduction in exposure of human embryos outside the



incubator enhances embryo quality and blastulation rate.

Reprod Biomed Online 2010; 20: 510-515.

18. Van Landuyt L, De Vos A, Joris H, Verheyen G, Devroey

P, Van Steirteghem A. Blastocyst formation in in vitro

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cycles: influence of the fertilization procedure. Fertil

Steril 2005; 83: 1397-1403.


Maturation capacity, morphology and morphometric

assessment of human immature oocytes after

vitrification and in-vitro maturation Saeedeh Nazari

1 M.Sc., Mohammad Ali Khalili

1 Ph.D., Forouzan Esmaielzadeh

2 M.Sc., Mehdi

Mohsenzadeh3 M.Sc.

1 Researh and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd,

Iran.

2 Department of Animal Sciences, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran.

3 Shiraz University of Medical Sciences, Shiraz, Iran.

Received: 19 August 2010; accepted: 12 December 2010

Abstract

Background: In general, 15% of oocytes collected in ART cycles are immature. These

oocytes may be cryopreserved further for use in in-vitro maturation (IVM) program.

Objective: The aim of this study was to determine maturation capacity, morphometric

parameters and morphology of human immature oocytes in both fresh IVM (fIVM) and

vitrified-IVM (vIVM) oocytes.

Materials and Methods: 93 women who underwent controlled ovarian stimulation for

ART were included. The immature oocytes (n=203) were divided into two groups: the

first group (n=101) directly matured in vitro; and the second group (n=102) first

vitrified, then matured in vitro. All oocytes underwent IVM in Ham’s F10 supplemented

with LH+FSH and human follicular fluid. After 48h of incubation, the oocyte

maturation rates, as well as morphometric and morphologic characteristics were

assessed using cornus imaging and were compared.

Results: Oocyte maturation rates were reduced in vIVM, (40.4%), in comparison with

fIVM (59.4%, p<0.001). Following morphometric assessment, there was no difference

in the mean oocyte diameters (µm) between fIVM and vIVM, 156.3±6.8 and

154.07±9.9, respectively. Other parameters of perimeters, egg areas, as well as oocyte

and ooplasm volumes were similar in two groups. In addition, more morphologic

abnormalities, such as, vacuole, and dark oocyte were observed in vIVM oocytes.

Conclusion: fIVM was more successful than vIVM groups. No statistical differences

were noticed in morphometry assessment in two groups. This suggests that

morphometric parameters can not be applied as prognosis factor in oocyte maturation

outcome in IVM program. Key words: IVM, Oocyte, Vitrification, Maturation, Morphology, Morphometry.

Introduction

In-vitro maturation (IVM) and cryopreservation

of immature oocytes has been proposed as an

alternative for conventional IVF treatment. IVM

has several advantages of reducing the costs,


Mohammad Ali Khalili, Research and Clinical Center

for Infertility, Shahid Sadoughi University of Medical

Sciences, Yazd, Iran.


avoidance of the side effects of ovarian hyper

stimulation syndrome (OHSS) and simplified

treatment for certain infertile couples (1-3).

Currently, most IVM protocols have supplemented

with FSH and/or LH into the culture medium for

oocyte maturation (1). Cryopreservation could

benefit to patients who have specific situation such

as chemotherapy or radiation (4). Several factors

affect the survival and viability of human oocytes

and embryos such as exposure time of cells to

different cryoprotectant solutions, their different

concentrations and rate of ice crystal formation (3).

Nazari et al


One established method for the cryopreservation of

human oocyte and embryo is vitrification

technology (3, 5). During recent years, studies

have shown that vitrification offers new interesting

perspectives in the field of oocyte

cryopreservation, demonstrating less traumatic

than slow cooling (6, 7).

After ovarian stimulation, approximately 15%

of oocytes are immature at the GV and M1 stages.

In 1991, the first human birth resulting from IVM

was reported by Cha and associates (8). In 1994,

Trounson et al reported the first birth from

untreated polycystic ovarian (PCO) patient (9). In

many species, it has been demonstrated that a

suitable oocyte size is necessary for the

continuation of meiosis and maturation. Durinzi et

al (1995) demonstrated that the oocyte diameter at

the time of retrieval from un-stimulated cycles has

affected IVM in human oocytes to resume meiosis

and completion of their maturation (10).

In addition, Mikkelsen and Lindenbery (2001)

demonstrated that there was no difference in

morphology between the in-vitro matured oocytes

in normal ovaries and polycystic ovaries (11). In

1993, Albert et al reported that the diameter of a

normal human oocytes was 60-150µm (12).

Recently, Lasiene et al (2009) evaluated the size of

the zona pellucida (ZP) to vary from 10 to 31 µm,

which was not related to the cytoplasm diameter

(13). Bao et al (2000) showed that developmental

changes occurring during the final stages of oocyte

growth are critical for full developmental

competence (14).

Also, immature oocytes that underwent IVM

were smaller than in-vivo matured oocytes.

Therefore, the oocyte diameter grown in culture

are affected on maturation (15). Michelmman et al

(1995) examined micro morphometry and sperm

binding patterns between fertilized and unfertilized

human oocytes after in vitro fertilization. They

showed that there were no significant differences

in morphometry between fertilized and unfertilized

oocytes.

Though, differences demonstrated in the size of

perivetilline space and the ZP (16). Durizi et al

(1995) showed the size of human oocytes depend

with ability to resume meiosis and complete

maturation in IVM with un-stimulated cycles (10).

Although, there is some evidence available about

morphometry and morphology in human oocytes,

but there is no report on morphology and

morphometry of human immature oocytes

following fresh-IVM and vitrified-IVM in ART

cycles. Therefore, the main objective of this

prospective study was to investigate the success of

cryopreservation of immature human oocytes using

vitrification. This was followed by comparing

oocyte maturation rates, as well as morphology and

morphometry assessments.


Patients

A total of 93 infertile women were included in

this cross-sectional study. The investigation took

place over a period of 5 months in 2010 at

Research and Clinical Center for Infertility in

Yazd. This study was approved by ethics

committee of our institution. Oocytes were

allocated in two groups of fresh-IVM (fIVM;

n=101) and vitrified-IVM (vIVM; n=102).

Oocyte collection

The oocyte collection was performed 36h after

10,000 IU of hCG (IBSA Co, Switzerland)

injection. Transvaginal ultrasound was used for

oocytes collection with a single lumen aspiration

needle (Wallace, Smiths Medical International,

UK) with a reduced pressure of 150 mmhg. The

collected oocytes were assessed for nuclear

maturity under the stereo microscope (Olympus

Co, Japan). After denudation with 80 IU

hyaluronidase (Sigma Co, USA) and mechanical

pippetting, the oocytes were assessed for

maturity.The oocytes that extruded the first polar

body were considered mature (metaphase II) and

were inseminated using intracytoplasmic sperm

injection (ICSI) technique.

Follicular fluid preparation

Preparation of human follicular fluid (HFF) was

performed according to the method described

previously (17). HFF was obtained from women

who underwent follicular puncture. HFF was

centrifuged at 3500 RPM for 10 minutes. Blood

and granulosa cells were settled, and pure HFF was

transferred to new tube. The HFF was then

inactivated in water bath at 56°C for 30 min. At

last, HFF was filtered with 0.22 µm filters, then

aliquoted and stored at -20°C before use.

In vitro maturation

Immature oocytes were denuded of Cumulus-

oocyte complexes (COCs). The oocytes were then

washed in 3 drops of IVM medium and were

cultured in IVM medium containing Ham’s F10

(Biochrom Co, Germany) supplemented with 0.75

IU LH, 0.75 IU FSH (Ferring Co, Germany)with

40% HFF at 37°C in an incubator with 5% CO2

and 95% air with high humidity (98%). Oocytes

Vitrification of human immature oocytes and IVM


were observed under an inverted microscope

(Nikon Co, Japan) after 48h to determine maturity.

Vitrification of immature oocytes

Immature oocytes were frozen using a modified

vitrification method reported by Al-Hasani et al

(2007) (3). Initially, immature oocytes were placed

in an equilibration solution containing 7.5%

ethylene glycol (EG) (Merck Co, Germany), 7.5%

dimethyl sulphoxide (DMSO) (Merck Co,

Germany) in Ham’s F10 media supplemented with

20% human serum albumin (HSA) (Plasbumin Co,

USA) for 5-15 min at room temperature. Then,

oocytes were removed and placed into vitrification

solution containing 15% EG, 15% DMSO and

0.5M socruse (Sigma Co, USA) in Ham’s F10

medium supplemented with 20% HSA for 50-60

Sec at room temperature. After this stage, the

oocytes were loaded into cryotops that were

plunged into liquid nitrogen quickly. Next, the cap

was placed on the cryotop and put into the cane.

Finally, the samples were transferred to the liquid

nitrogen storage tank for 2 months.

Thawing of immature oocytes

Thawing of the oocytes was performed by

placing the cryotop in thawing solution in five

stages: thawing solution (Ham’s F10 supplemented

with 20% HSA and 1M sucrose) for 50-60 Sec,

dilution solution 1 (Ham’s F10 supplemented with

20% HSA and 0.5M sucrose) for 3 min, dilution

solution 2 (Ham’s F10 supplemented with 20%

HSA and 0.25 M sucrose) for 3 min, washing

solution 1 and 2 (Ham’s F10 supplemented with

20% HSA) each for 3-5 min. After this stage, the

oocytes were placed in IVM medium for 48h in

incubator.

Viability of vitrified oocytes was evaluated

microscopically 2-3 h after culture, based on the

morphology of cytoplasm (18). The dead oocytes

were shown with broken membrane and dark

ooplasm.

Morphometry and morphology evaluations

After a culture period of 48h, all oocytes were

assessed for maturity under an inverted

microscope; Matured oocytes were evaluated for

morphology and morphometry. The matured

oocytes were divided into two groups (Group

1=fIVM, Group 2=vIVM). Measurements

accomplished with inverted microscope equipped

with cornus imaging program (Research

instruments Ltd Co, UK). Measurements

comprised of the diameters of whole oocyte (µm),

ooplasm (µm), width of ZP (µm), perimeters and

areas of whole oocyte and ooplasm (µm2), and

volume of whole oocyte and ooplasm (µm3)

(Figure 2).

To calculate the average diameter of different

parts of each oocyte comprising of whole oocyte,

ooplasm and ZP were calculated in 4 different

parts. Also, the morphology of each mature oocyte

was determined by characteristics of refractile

body (RF), granularity, vacuole, smooth

endoplasmic reticulum (SER), bull’s eye, ZP and

perivite line space (PVS), coloration of ZP and

ooplasm (dark or normal), polar body shape

(normal or fragmented), oocyte shape (circular or

irregular) and PVS debris in both groups (Figure3).


Statistical analysis was carried out using t-test

for morphometrical data and χ2for morphological

data by SPSS (version 16). p<0.05 was considered

significant.

Results

As shown in table I, there were no significant

differences in characteristics of age, etiology of

infertility, total number of retrieved oocytes and

oocytes stage (GV, MI) between fIVM with vIVM

groups. The numbers of immature oocytes (Figure

1) were; GV=72, MI=29 in fIVM and GV=66,

MI=36 in vIVM group. There were no significant

differences in the numbers of GV and MI oocytes

in fIVM and vIVM.

The oocyte survival rate was 87.25% (89/102)

when the oocytes were vitrified. The oocyte

maturation rate was 40.4% (36/89) when the

oocytes were vitrified and then underwent IVM

after warming. This was significantly lower

(p<0.001) than the immature oocytes matured in

vitro without vitrification which was 59.4%

(61/101) (Table II). Also, a total of 16 immature

oocytes were collected from a patient with OHSS.

However, the rate of maturity after IVM protocol

was as low as 12.5%.

The data also showed that, morphometric and

morphologic analyses of matured oocytes were

possible in 96 oocytes (60 fIVM, 36 vIVM).

Although, there were no significant differences in

the diameters, areas, perimeters and volumes

between the oocytes vitrified at the immature stage

than those obtained using the oocytes without

vitrification for IVM (Table III). As shown in table

IV, there were no significant differences between

RF, fragmented polar body, regularity of shape,

granularity, PVS debris and width of PVS in fIVM

and vIVM groups. However, in vIVM oocytes, the

Nazari et al


rates of vacuoles and dark oocytes were

significantly higher than fIVM group. Both groups

didn’t show any SER and dark ZP in oocytes that

were matured in-vitro. In fIVM, the most common

morphological abnormality was presence of RF.

Table I. Characteristics of patients in fresh IVM and vitrified-IVM groups. Variables Fresh-IVM (n=101)

(GV=72,MI=29)

Vitrified-IVM (n=102)

(GV=66,MI=36) p-value

Age (years) (mean±SD)

29.3±5.9 32.2±5.5 NS

Female factor infertility

43(50.6) 42(49.4) 0.920

Male factor infertility

48(50.0) 48(50.0) 0.910

Both(male and female factors infertility)

10(45.5) 12(54.5) 0.724

NS= not significant

Values inside parentheses represent (%).

Table II. Comparison of maturation rates of human oocytes in two groups of IVM.

Values inside parentheses represents (%). * indicates significant difference between fresh IVM and vitrified-IVM

Table III. MII oocyte morphometric comparison between fresh IVM and vitrification groups in all in-vitro matured oocytes (n=96).

Variables

Fresh-IVM (n=60) Vitrified-IVM (n=36) Unit p value

Oocyte diameter

156.3 ± 6.8 154.07 ± 9.9 µm 0.183

Ooplasm diameter

115.3 ± 4.6 114.09 ± 7.5 µm 0.306

Zona pellucida width

17.8 ± 3.2 17.3 ± 2.6 µm 0.342

Oocyte area

188.8×102 ± 3.1×103 234.3×102 ± 2.9×104 µm2 0.353

Oocyte perimeter

4.9×102 ± 2.9×10 4.8×102 ± 3.1×10 µm2 0.343

Ooplasm area

1.07×104 ± 1.5×103 1.02×104 ± 1.6×103 µm2 0.196

Ooplasm perimeter

3×102 ± 2.3×10 3.6×102 ± 2.4×10 µm2 0.273

Oocyte volume

2×106 ± 2.7×105 1.9×106 ± 4.1×105 µm3 0.293

Ooplasm volume

1×106 ± 2.7×105 0.87×106 ± 0.58×106 µm3 0.373

Values are mean ± SD.

Table IV. MII oocyte morphologic comparisons between the two IVM groups.

Variables

Fresh IVM (n=60) Vitrified-IVM (n=36) p-value

Refractile body

37(60.7) 20(55.6) 0.673

Fragmented polar body

28(45.9) 19(52.8) 0.535

Regular shape

49(80.3) 30(83.3) 0.792

Ooplasm granularity

12(19.7) 11(30.6) 0.323

Vacuole

4(6.6) 9(25.0) <0.01*

Smooth endoplasmic reticulum

0(0) 0(0) -

PVS debris

7(11.5) 7(19.4) 0.371

Dark zona pellucida

0(0) 0(0) -

Dark oocyte

2(3.3) 11(30.6) <0.001*

Bull’s eye

5(8.2) 0(0) 0.154

Width zona pellucida (%)

3(4.9) 0(0) 0.293

Width PVS

9(14.8) 4(11.1) 0.762

NS= not significant.

Values inside parentheses represents (%). * indicates significant difference between fresh IVM and vitrified-IVM.

Variables

Fresh IVM (n=101) Vitrified-IVM (n=89) p-value

MII oocyte (matured)

60(59.4) 36(40.4) <0.001*

oocyte arrest

36(35.6) 14(15.7) -

Degeneration

3(3) 38(42.7) <0.001*

Parthenogenesis

2(2) 1(1.2) <0.001*



Figure 1. Morphological markers characterizing the meiotic status of human oocytes.

a) Immature germinal vesicle (GV) oocyte. b) Immature germinal vesicle breakdown (GVBD) oocyte (M1).

c) Mature oocyte (MII) .

Figure 2. Dimentions that were measured during mormorphometry evaluation of oocytes after IVM. a) Diameter of oocyte

b) Diameter of ooplasm

c) Diameter of ZP

Figure 3. Various morphological abnormalities exhibited by MII oocytes.

a) Fragmented polar body observed in vitrified-IVM group. b) Wide PVS without debris in one matured oocyte after fresh IVM.

c) A typical refractile body within ooplasm.

d) Central granularity in human oocytes after IVM.

e) Bull’s eye within ooplasm.

f) Several large vacuoles in oocyte. g) Degenerated oocyte after vitrification.

Nazari et al


Discussion

Microtubular spindle in MII oocyte is sensitive

to low temperature and cryopreservation.

However, GV oocytes with chromosomes within

the nuclear membrane are protected from these

disorders (19). We evaluated morphology and

morphometry of oocytes after maturation in fIVM

and vIVM in patients undergoing ART program.

The aim was to found out whether differences

existed in the morphology and morphometry of

human oocytes after IVM in two groups. In this

study, the maturity after IVM was demonstrated to

be higher in fIVM when compared with vIVM.

Also, the results showed that the rates of GV and

M1 arrest in fIVM were higher than vIVM.

However, the percentage of maturity (MII) was

higher in fIVM, when compared with vIVM. In

addition, rates of degeneration were significantly

increased in vIVM group. Therefore, immature

oocytes in ART cases should immediately undergo

the IVM technology, though they may be

cryopreserved successfully if it becomes necessary.

According to Kim et al (2000), almost 78.5% of

the immature oocytes retrieved in stimulated cycles

were matured, and 12.3% of oocytes remained at

the GV stage (17). Russel (1998) showed that

maturation rate was 63% in naked and compact

oocytes in unstimulated cycles which is similar to

our results (20). Later, Toth et al (1994)

demonstrated that 83.3% of the oocytes were

matured after thawing of cryopreserved oocytes

(21).

Recently, Cao et al (2009) reported the survival

rates of human immature oocytes in vitrified

oocytes as high as 85.4% after warming. This

survival rate was similar to our study. For this

reason, it seems that high survival rate can be

obtained when human oocytes are vitrified at the

immature stage. Also, Liu (2010) reported that, the

removal of granulosa cells would affect IVM of

immature oocytes (2).

In this study, the rates of maturation in vitrified

oocytes were somewhat similar to study done by

Cao et al (2009). But, rate of maturity following

IVM of immature oocytes without vitrification

were lower when compared to above (18). In

addition, morphometric comparison on fIVM and

vIVM showed only minor differences between two

groups. During growth phase, oocyte diameter

increases from 30 to 110 µm over a period of at

least 8 weeks in human (22). When oocytes are

matured in-vivo, the size of oocyte normality is

approximately 110-120 µm, excluding the ZP, also

whole size of oocyte was almost 150 µm (23).

We found that when immature oocytes were

vitrified and then matured using IVM technology,

no differences were noticed in parameters. This

evidence showed that vitrification seems to have

no effect on morphometric parameters when

compared with fresh IVM. Bertand et al (1995)

reported that human ZP varies from 10-30 µm with

a mean of 17.5 µm (24).

Cavilla (2008) found zona diameter from the

oocytes matured in vitro and then fertilized were

larger than diameters of the in-vivo matured

oocytes (12). In present study, all mature oocytes

had a ZP thickness of 17.8 and 17.3 µm in fIVM

and vIVM groups, respectively.

This was almost similar to the data reported by

Cavelia et al (15). Also, the mean oocyte diameter

in our study was statistically higher to the value

obtained by Salata and associates (23). This

difference could be credited to the large sample

size evaluated by Salata et al (23). In addition,

there were no significant differences in volume,

perimeter, area ooplasm and oocyte between fIVM

and vIVM. Michelman et al (1995) also found the

volume of human oocytes to be similar to our

results.

In addition, most of abnormalities were noticed

in the oocytes that underwent IVM technology.

This study demonstrated that rates of

vacuoalization and dark oocytes were significantly

increased in vIVM than fIVM. This may prove that

oocyte cryopreservation cause some irreversible

structural damage. Lasiene et al (2000) reported

the quality of oocytes deliberated by the structure

of COC.This procedure gives us some information

about the quality of oocytes(13).

Also, they showed that oocytes with less

cumulus compact, deficiency granularity and dark

ooplasms have higher developmental competence,

and the quality is increased when oocytes covered

by more layers of cells (25, 26). According to

defects in oocytes, they are categorized into single,

double, multiple and nodefects. About this

classification, no one reported any differences in

fIVM and vIVM groups. Balaban et al (2006)

classified oocytes abnormalities, that were similar

to our study (27). Xia (1997) showed that oocyte

with fragmented polar body have a lower

fertilization than normal polar body. Also, oocytes

with extensive and fragmented polar body have a

worse developmental after fertilization (28).

Ubalidi et al (2008) evaluated morphology of

oocytes and reported that such as fragmented polar

body and large PVS are rather higher than other

abnormalities (49% and 32%), respectively (29).

Our results showed that the rates of fragmented



polar body and SER are approximality similar to

theirs. Other defects, such as Bull’s eye, thick ZP,

SER and dark ZP were not observed in oocytes

under investigation.

Conclusion

The rates of human oocytes recovery following

vitrification concomitant with IVM program are

acceptable. However, morphometrical assessment

of oocyte does not seem to play a role in prognosis

of oocyte maturation outcome.

Acknowledgement

The authors would like to thank Mehrdad

Soleimani, Mohammad H. Razi, Leila

Motamedzadeh, Habibeh Gheisari, and Aazam

Agha Rahimi for their technical assistance during

the course of this study. The statistical assistance

of Farima Shams in also well appreciated. This

study was supported by a grant from research and

clinical center for infertility, Shahid Sadoughi

University of Medical Sciences, Yazd, Iran.

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Antifertility activity of aqueous ethanolic extract of

Hymenocardia acida stem bark in female rats Abu Adakole Hyacinth

1 Ph.D., Uchendu Chukwuka Nwocha

2 Ph.D.

1 Department of Veterinary Physiology, Pharmacology and Biochemistry, College of Veterinary

Medicine, University of Agriculture, P.M.B, 2373, Makurdi, Nigeria.

2 Department of Veterinary Physiology and Pharmacology, Faculty of Veterinary Medicine, University of

Nigeria, Nsukka, Nigeria.


Abstract

Background: Hymenocardia acida is traditionally used in African herbal medicine and

has numerous therapeutic benefits. But little is known about its potentially negative

effects on pregnant women.

Objective: The aim of the present study was to evaluate the antifertility effect of

aqueous ethanolic extract of Hymenocardia acida stem bark in female Wistar rats.

Materials and Methods: Four groups of rats were administered orally aqueous

ethanolic extract of Hymenocardia acida at doses of 100, 200 and 400 mg/kg body

weight daily for 19 days. The control group received distilled water. On day 20 of

gestation, each rat was laparatomised and number of corpora lutea of pregnancy,

number of live fetuses as well as the postcoitum fertility index, weights of the foetuses

and placentae were determined.

Results: Oral administration of the extract from days 1 to 19 of gestation showed

reduction (p<0.05) in the number of corpora lutea of pregnancy and number of live

fetuses. Weights of fetuses of extract treated female rats were also smaller (p<0.05)

compared with the control. Anti-implantation activity of the treatment groups were

41.4%, 48.3% and 51.7% for groups II to IV respectively, whereas antifertility activity

of the groups was found to be 40%, 60% and 60% in the same order.

Conclusion: The results suggest that aqueous ethanolic extract of Hymenocardia acida

stem bark could induce negative effects on reproductive functions in female albino rats.

Key words: Albino rats, Antifertility, Hymenocardia acida, Reproduction.

Introduction

There is an increasing trend in the use of

medicinal plants, botanicals or herbal preparations

particularly in developing countries where these

products are readily available. Several animal

studies have revealed anti zygotic, blastocytotoxic,

anti-implantation and abortifacient properties of

water and organic solvent extracts of many

commonly used medicinal plants, sometimes in

dose dependent manner. In an investigation of

antifertility property of a triterpenoid glycoside

Corresponding author: Abu Adakole Hyacinth, Department of Veterinary

Physiology, Pharmacology and Biochemistry, College

of Veterinary Medicine, University of Agriculture,

P.M.B, 2373, Makurdi, Nigeria.


isolated from Dalbergia saxatilis in female rats, a

decrease in maternal body weights and inhibition

of conception were observed (1). Similar

observations on antifertility, antiimplantation or

pregnancy interceptory properties suggestive of

anovulatory, antiprogesterogenic or estrogenic

effects have been made on extracts of Calotropis

gigantea (2) and Morinda citrifolia (3). However, other animal studies showed none

inhibitory effects of plant extracts on female

reproductive functions. For example, Carapa

guianensis seed oil administered orally during the

period of organogenesis failed to impair

implantation and induce the death of foetuses (4).

Sensitivity of experimental animals, dose of

extract used, period and route of administration as

well as physiological or pharmacological

mechanisms are some of the factors affecting the

implantation process.


Abu et al


Hymenocardia acida (Tul.) is a small browse tree or shrub with palatable foliage, widely distributed within the savanna region of Nigeria. It is called “Enache” by Idoma people of North Central Nigeria while the local or vernacular name among the Hausas in Nigeria is “Janyaro”. All parts of the plant are useful as remedies for many ailments. The powdered roots or stem bark decoction are used to treat fever, jaundice, muscular pains, diarrhoea, dysentery and sexual incapacity in males. It is commonly used as an agent for female genital hygiene. Experimental studies have also confirmed some of the claimed efficacy of this medicinal plant by the native healers. For example, crude extracts of the plant have been reported to possess anti-tumor, anti-HIV and anti-inflammatory (5), anti-sickling (6), anti-ulcer (7) and anti-diarrhoeal (8) activities. The leaf infusion is also used in the treatment of urinary tract infections (5) and as topical applications for skin diseases in Nigeria. Among the Idoma and Igede people of North Central Nigeria, the decoction of root and stem bark is used in the treatment of diabetes (9). Also there have been reports of in vitro antitrypanosomal efficacy of leaf (10) and root bark (11) as well as antiplasmodial (12) activities of Hymenocardia acida. Despite the popular use and numerous therapeutic benefits of Hymenocardia acida, little is known about the effect of crude extract of H. acida on reproduction. The aim of the present study was to evaluate the effect of aqueous ethanolic extract of H. acida stem bark on fertility of female Wistar rats.


Plant material

The stem bark of Hymenocardia acida was collected within the premises of University of Agriculture, Makurdi and authenticated by Mr. Patrick Ekwuno of College of Forestry, University of Agriculture, Makurdi, Nigeria Voucher specimen was deposited at the College herbarium. Preparation of crude extract

The stem bark was washed, air dried at room temperature for one week, pulverized and stored in air-tight container until required. As previously described (13), one hundred gram of powdered material was soaked in 500 ml of 70% ethanol and stirred intermittently for 48 hours at room temperature. The material was filtered using sterile cotton wool and Whatman (No. 1) filter paper; the residue was resuspended in the same amount of solvent and then filtered three more times. The pooled filtrates obtained were dried at room temperature under the electric fan. The extracts

were stored in air-tight containers at 4oC until

needed.

Animals Twenty white albino rats of weighing 150 g to

190 g were obtained from the College of Health Sciences, Benue State University, Makurdi, Nigeria. The animals were kept in polypropylene cages under room temperature, with 12-hour light and 12-hour dark cycle and were allowed to acclimatize for two weeks. The animals were provided commercial feed (Grand Cereals and Oil Mills Ltd, Bukuru, Jos, Nigeria) and clean water ad libitum. Protocols for this experiment was in accordance with the guidelines on the care and well being of research animals (14) and was approved by the Departmental Ethics Committee.

The rats were paired overnight with sexually active males in the ratio of 2:1. Successful mating was confirmed by the presence of vaginal plug and or sperm cells in the vaginal smear the following morning between 9.00 and 10.00 hours. The day sperm cells were found in the vaginal smear was considered as day 1 of pregnancy. Thereafter, the female rats were randomly divided into four groups of five rats each thus: Group 1 received by gavage distilled water (1ml/ 100g) daily for 19 days and served as control. Groups II–IV rats were given the aqueous ethanolic extract of Hymenocardia acida by gavage at doses of 100, 200 and 400 mg/kg body weight daily for 19 days respectively. The rats were weighed daily and observed for any untoward effects.

On day 20 of gestation, each rat was laparatomised under high ether anaesthesia. The uterine horns were exteriorized and incised at the greater curvature of the horns. The latter were examined for sites of implantation and resorption. Number of corpora lutea of pregnancy, number of live foetuses as well as weights of the foetuses and placentae were also determined. The postcoitum fertility index was evaluated using the following parameters according to the methods of Tafessel et al (16) and Uchendu et al (1). 1. Percentage of pregnant female animals in each

group (PPF) 2. Mean live foetal number per pregnant female

(LFN) 3. Mean day 20 foetal crown-rump length (FCRL) 4. Mean corpus luteum number per pregnant

female (CLN) The fertility index (FI) of each group was

calculated as

FI = LFN × FCRL × PPF

CLN

The anti-implantation and antifertility activities

of the extract were calculated as follows (16):

Antifertility of Hymenocardia acida stem bark in female rats


Anti-implantation activity= number of implants

in control minus number of implants in test group

divided by number of implants in control group

multiplied by 100. Antifertility activity= number of

rats showing no implantation divided by total

number of animals multiplied by 100.


Statistical evaluation of data was done using

one–way analysis of variance (ANOVA). Means

found to be significantly different at p<0.05 were

separated using Duncan multiple range test. The

results were expressed as mean± S.E.M. using

Graph Pad Prism Version 3.0 for Windows (Graph

Pad Software, San Diego, California).

Declaration

This is part of research work carried out by the

first author under the supervision of second author.

There is no conflict of interest. The work was

funded by the first author.

Results

The linear increases in maternal weights and

weights gained (Table I) were higher in the control

compared with the treatment groups. Oral

administration of the extract from days 1 to 19 of

gestation showed decrease (p< 0.05) in the number

of corpora lutea of pregnancy and number of live

foetuses (Table II).

Weights of foetuses of extract treated female

rats were also smaller (p<0.05) compared with the

control. However, no gross morphological

abnormalities were observed. All the females in the

control group became pregnant and on laparatomy,

were all found to have live foetuses. The extract

did not cause any abortion or vaginal bleeding. The

pregnant animals did not show signs of toxicity.

All rats survived till the termination day. There

were no foetal resorptions.

Significant differences (p<0.05) in the mean

foetal number per pregnant female (LFN), foetal

crown-rump length (FCRL) and mean corpus

luteum number per pregnant female (CLN) were

observed in all the treated groups relative to the

control (Table II). Antiimplantation activity of the

treatment groups were 41.4%, 48.3% and 51.7%

for groups II to IV respectively, whereas

antifertility activity of the groups was found to be

40%, 60% and 60% in the same order (Table III).

Table I. Effect of Hymenocardia acida stem bark extract on maternal and foetal weights. Values are expressed as means± S.E.M.;

N= 5.

Parameters

Control 100 mg/kg 200 mg/kg 400 mg/kg

Maternal weights (g)

Day 1

151. 40 ± 1. 40 152. 40 ± 1. 96 152. 40 ± 1. 40 152. 00 ± 1. 99

Day 7

162. 33 ± 2. 61a 159. 33 ± 3. 48b 152. 50 ± 3. 38b 152. 67 ± 3. 72b

Day 14

180. 50 ± 2. 59a 164. 00 ± 3. 56ab 156. 33 ± 2. 61b 157. 75 ± 2. 66b

Day 19

198. 75 ± 3. 68a 182. 00 ± 3. 00ab 166. 00 ± 2. 35b 165. 67 ± 2. 34b

Maternal weights (g)

Day 0 – 5

12. 60 ± 1. 80a 3. 00 ± 1.41b 3. 00 ± 1.18b 4. 33 ± 1.44ab

Day 6 – 14

10. 80 ± 3. 92a 8. 40 ± 3. 80ab 8. 25 ± 3. 30ab 7. 20 ± 2. 06b

Day 15 – 19

18. 80 ± 1. 71a 17. 67 ± 2. 29a 12. 20 ± 1. 12ab 7. 00 ± 1. 89b

Day 0 – 19

45. 00 ± 2. 86a 37. 33 ± 3. 41ab 22. 25 ± 3. 31b 17. 50 ± 2. 51b

Foetal weight (g)

2. 69 ± 0. 21a 1. 82 ± 0. 40ab 1. 56 ± 0. 20b 1. 65 ± 0. 40b

Placental weight (g)

0. 47 ± 0. 02a 0. 27 ± 0. 01ab 0. 14 ± 0. 02b 0. 17 ± 0. 01b

Means with different superscripts in a row are significantly different (p=0.05). “N” represents number of animals used in each group.

Table II. Effect of Hymenocardia acida stem bark extract on foetal score. Values are expressed as means ± S.E.M; “N” = 5.

Groups

No. of live foetuses(LFN) No. of dead

foetuses REN FCRL (cm) CLN

PPL

(%) F.I.

Control

7. 00 ± 0. 58a 0. 00 ± 0. 00 0.00 ± 0. 00 3. 56 ± 0. 21a 8. 40 ± 1. 03a 100a 990. 81

100 mg/kg

5. 67 ± 0.33b 0. 00 ± 0. 00 0.00 ± 0. 00 3. 50 ± 0. 31ab 5. 80 ± 1. 02ab 60b 486. 82

200 mg/kg

4. 50 ± 0. 50 b 0. 00 ± 0. 00 0. 00± 0. 00 2. 90 ± 0. 20b 5. 40 ±1. 34ab 40c 174. 00

400 mg/kg

3. 50 ± 0.50 b 0. 00 ± 0. 00 0. 00 ± 0. 00 2. 75 ± 0. 45b 4. 80 ± 1. 39b 40c 147. 64

Means with different superscripts in a row are significantly different (p= 0.05). “N” represents number of animals used in each group.

PPL- Percentage of Pregnant Females per Group; FCRl – Foetal Crown – Rump Length. F.I. – Fertility; CLN – Corpus Luteum Number. LFN- Live foetal Number; REN – Resorbed Embryo Number.

Abu et al


Table III. Antifertility and antiimplantation activities of Hymenocardia acida stem bark extract.

Groups No. pregnant/

No. tested

No. of dead

rats

No. of rats showing

implantations

Total No. of

implantations

Anti-implantation

activity (%)

Anti-fertility

activity (%)

Control

5/5 0 5 29 Nil Nil

100 mg/kg

5/3 0 3 17 41. 4 40. 0

200 mg/kg

5/2 0 2 9 48. 3 60. 0

400 mg/kg

5/2 0 2 7 51. 7 60. 0

Discussion

Administration of H. acida extract at the test

doses from days 1 to 19 of pregnancy resulted in

strong antiimplantation and antifertility activities

(Table III). At necropsy, no evidence of embryo

resorption was found in the non pregnant rats.

Several reports indicate antifertility activity of

crude extracts (17, 18) and active compounds in

animal models (19). Although there were neither

deaths nor clinically observable, treatment-related

inhibitory effects of H. acida on the pregnant rats,

changes in maternal body weights (Table I)

provide a good index of the integrity of maternal

homeostasis (20). In the present study, significant

decreases (p< 0.05) in maternal body weights were

observed when aqueous ethanolic extract of H.

acida stem bark was given to the rats.

The low body weight gain in pregnant rats

given the extract might suggest nil or few number

of implantations. It is well established that the

implantation index correlates with the number of

corpora lutea and indicates blastocyst implantation

in the endometrium (21) as well as normal

reproductive capacity (22). Hymenocardia acida

extract significantly reduced the number of

implantations at the test doses (Table III). The

mechanism of antiimplantation activity of

Monecha ciliatum has been explained by its strong

uterotonic property (23). Similarly, Musanga

cecropioides, a common Nigerian medicinal plant

used for its oxytocic effect has been reported to

increase uterine contraction in a dose - dependent

manner (24).

In a pilot study, the aqueous ethanolic extract of

Hymenocardia acida elicited contractile effect on

the isolated uterine muscle tissue (unpublished

observation). However, the mechanism of

antiimplantation activity of the extract requires

further investigation.

The rat endometrium is sensitive to blastocyst

signals in the morning of day 5 (25), but can

experience failure of blastocyst implantation due to

hostile and incompetent uterine environment (26),

antizygotic or blastocytotoxic property (27) and

expulsion of embryo (28). Previous reports on

antifertility effects of medicinal plants which

showed resorptions of embryos and abortions (29)

are not in agreement with the present study where

we observed neither resorption sites nor gross

malformations of the foetuses. Hymenocardia

acida perhaps did not act as an abortifacient since

there was no vaginal bleeding. However, there

were reductions in foetal and placental weights of

the treatment groups.

The endometrial environment might not be

conducive for implantation (30) as revealed by the

smaller litter size. In addition to the endometrial

microenvironment, hyper motility of the

myometrium can also prevent implantation,

especially in view of the effect of the extract on the

isolated tissue (data not shown).

The reduction in foetal crown- rump length

(FCRL) which is a parameter for foetal growth

agrees with previous findings of impaired placental

formation or placental insufficiency (31) and foetal

development (32). A reduction in weights of

foetuses of pregnant rats as observed in the present

study had also been reported when Acanthus

montanus leaves extract was administered to

pregnant rats during gestation (33). However, other

investigators showed that most pure compounds of

A. indica were of relatively low reproductive

toxicity compared with the crude seed oil (34).

Similarly, aqueous extract of Garcinia kola

administered to pregnant rats did not affect the

number and weights of foetuses as well as

implantation sites (35).

Previous reports indicate the presence of

flavonoids (36), alkaloids (37) and terpenoids (1)

in medicinal plants with contraceptive or

pregnancy interceptory effects. In the present

study, phytochemical screening of aqueous

ethanolic extract of H. acida stem bark revealed

the presence of alkaloids, tannins, flavonoids and

terpenoids.

Fertility of rats in groups treated with the

extract was significantly different (p<0.05) from

Antifertility of Hymenocardia acida stem bark in female rats


the control as reflected by the reduced pregnancy

and fertility index (Table II). A similar observation

on reduced fertility was made when pregnant rats

were treated with Dalbergia saxatilis (1).

The results suggest that aqueous ethanolic

extract of Hymenocardia acida stem bark could

induce inhibitory effects on reproductive functions

in female albino rats.

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High plasma homocysteine and insulin resistance in

patients with polycystic ovarian syndrome Tayebe Hemati

1 M.D., Nasrin Moghadami-Tabrizi

1 M.D., Fateme Davari-Tanha

1 M.D., Bahram

Salmanian2 M.D., Pouya Javadian

2 M.D.

1 Department of Obstetrics and Gynecology, Mirza Kouchak Khan Hospital, Tehran University of

Medical Sciences, Tehran, Iran.

2 School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Received: 6 June 2010; accepted: 11 January 2011

Abstract

Background: Polycystic ovarian syndrome (PCOS) is a common disease among

women in fertility ages and cause severe insulin resistance. Hyperhomocysteinaemia is

said to be among the features of PCOS that could influence its outcome.

Objective: This study aimed to investigate whether hyperhomocysteinaemia exists in

PCOS and if it is related to insulin resistance in the affected patients.

Materials and Methods: This prospective study was carried out in a university based

fertility clinic. Sixty four PCOS patients and 50 normo ovulatory controls were

reviewed for fasting glucose, insulin, homocysteine, luteinizing hormone (LH), and

follicle-stimulating hormone (FSH) plasma levels in the blood sample of the 3rd

day of

their menstrual cycle. Insulin resistance was determined with the fasting glucose

(mmol/L) to insulin (mIU/L) ratio and HOMA-IR (Homeostasis model assessment-

Insulin resistance). Independent-samples T-test and linear regression test were utilized

to analyze the obtained data.

Results: Homocysteine levels compared between PCOS patients and control group

showed a significant difference. PCOS group was divided into insulin resistant (IR)

(LogHOMA-IR≥0.57) and non insulin resistant (NIR) patients. The IR group had

significantly higher homocysteine (p-value=0.02), fasting insulin and glucose levels (p-

value<0.001) rather than NIR group.

Conclusion: PCOS patients have a leaning toward hyperhomocysteinaemia and insulin

resistance. Insulin resistant patients are found to have higher homocysteine level. Key words: PCOS, Homocysteine, Insulin resistance, Infertility.

Introduction

Polycystic ovarian syndrome (PCOS) is a

common disease among women in fertility age. It

causes ovulation disturbances, hyper androgenism,

infertility and increased abortion rate. Obesity,

hyper insulinemia, diabetes, hypertension,

dyslipidemia, atherosclerosis and vascular diseases

are other problems ascribed to PCOS (1-5). It has

been shown that PCOS can cause severe insulin


Fateme Davari-Tanha, Department of Obstetrics and

Gynecology, Mirza Kouchak Khan Hospital, Tehran

University of Medical Sciences, Tehran, Iran.


resistance and its secretion disturbances to some

extent.Homocysteine is an intermediate substance

in methionine metabolism. An increased

homocysteine plasma level classically happens due

to an enzymatic defect in the aforementioned

process.

The recent studies have shown there are so

many non-enzymatic factors affecting

homocysteine levels as well (6-8). According to

the insulin role in inhibition of hepatic cystathione

beta synthase, which is an enzyme involved in

methionine metabolism, insulin plasma level is

also introduced as a determining factor of

homocysteine levels (9, 10). Insulin resistance

seems to increase the homocysteine levels (6, 8,

11).

Hemati et al


There are several studies regarding the role of

homocysteine in pathogenesis of vascular diseases.

Homocysteine is found to be an effectual factor in

coronary artery diseases (CAD). Homocysteine

level higher than 11 µmol/L is detected in 30% of

CAD patients and increases the mortality rate by 3

times (12, 13). Herein, we aim to evaluate the

relation between homocysteine level and PCOS,

and its association with insulin resistance that

could affect the outcome of PCOS short and long

term management.


Sixty four patients referring to the infertility

unit at a university based fertility clinic, affiliated

to Tehran University of Medical Sciences, were

selected to be studied in a prospective study, in

2008-2009. The diagnosis of PCOS was made

according to the Rotterdam European Society of

Human Reproduction and embryology (ESHRE)/

American Society for Reproductive Medicine

(ASRM), sponsored PCOS consensus workshop

group guide lines (14). Those criteria are well

accepted for PCOS diagnosis (1). The study and its

methodology were approved by ethic committee of

Tehran University of Medical Sciences. All

possible risks were explained to patients and

informed consent was obtained from all

participants.

None of the patients were diagnosed with

diabetes or had any smoking habits. Those who

received metformin, folic acid or phenytoin were

also excluded.

Fifty other patients referring to the same unit

who had non ovarian problems were randomly

selected as the control group utilizing a pseudo-

random number generator. They had normal

ovarian and menstrual cycle; who did not present

with any of clinical and ultrasound signs and

symptoms of PCOS. They were matched with the

PCOS group regarding age and physical activity to

omit any known confounding factor.

Laboratory tests

All cases in both groups were weighed in two

different days and their height was measured. BMI

was calculated by dividing the weight (kg) by

squared height (m2). The basic plasma level of LH,

FSH and fasting glucose were measured in the 3rd

day of a spontaneous or progesterone induced

menstrual cycle before any treatments. Fasting

insulin and homocysteine total plasma levels were

also measured in the same day using Eliza enzyme

immunosorbent assay (Calbiotech Inc., CA).

Serum prolactin, thyroid hormone and 17-hydroxy

progesterone along with BUN and creatinine were

measured. Those were inspected not to exceed the

normal levels.

Insulin resistance is determined with several

indices, including: fasting insulin total plasma

level (mIU/L), the fasting glucose (mmol/L) to

insulin (mIU/L) ratio, and HOMA-IR

(Homeostasis model assessment-Insulin resistance)

calculated by Insulin (mIU/L) * glucose (mmol/L)/

22.5.

Fasting insulin total plasma level and fasting

glucose to insulin ratio have more restrictions

rather than HOMA-IR, are less sensitive

physiologically, and are not useful in abnormal

glucose levels. Therefore, we utilized the HOMA-

IR in the rest of this study. We converted HOMA-

IR into logarithmic scale, since it rarely has normal

distribution (15, 16). Insulin resistance was

inspected also by measuring the insulin levels 2

hours after glucose loading. 150 pmol/ L was

considered the cut-off.

To determine insulin resistance regarding each

index, the upper limit of the normal ranges was

considered mean±2SD of the control group.


We used SPSS software version 16 (SPSS Inc.

Chicago, IL, USA) for statistical analysis. p-value

of 0.05 and lower was considered significant.

Independent-samples T-test was utilized to

compare differences between parametric data

groups. In order to express the conditional

distribution of bivariate correlated items linear

regression test was done.

Results

Homocysteine levels compared between PCOS

patients and control group showed a significant

difference, 10.96±7.27 µmol/L in all PCOS vs.

6.8±1.95 µmol/L in controls (p-value <0.001). The

clinical and biochemical data of all patients

regarding their differences is shown in Table I.

Ninety five percent (Mean+2SD) of the control

group had homocysteine levels lower than

10.7µmol/L, while the normal range of

PCOS, insulin resistance (IR)


homocysteine is usually considered between 5 to

11 µmol/L (3). 35.94% of cases (23/64) in the

PCOS group had homocysteine levels higher than

10.7 µmol/L (p<0.001), who were regarded

hyperhomocysteinemic.

Insulin resistance determined with different

indices in our study. For each variable, the upper

limit was defined as mean±2SD of the normal

control group. Therefore 20 mIU/L, 0.3, and 0.57

were regarded as the cut-off levels for fasting

insulin level, glucose to insulin ratio, and Log

HOMA-IR respectively.

In the same order, 35.94%, 48.44% and 57.81%

of the patients had insulin resistance. Measuring

insulin level 2 hours after glucose loading, 45.31%

were resistant. All the aforementioned results were

found to be significantly different compared with

those of the control group (p<0.001, all groups).

Log HOMA-IR was utilized in the rest of the

study.

PCOS group was divided into insulin resistant

(IR) (Log HOMA-IR ≥ 0.57) and non insulin

resistant (NIR) (Log HOMA-IR < 0.57) patients.

The IR group had significantly higher

homocysteine (p-value=0.02), fasting insulin and

glucose levels (p-value<0.001) rather than NIR

group.

However, no association was found between

Log HOMA- IR and BMI (p-value=0.18) (Table

II). 18.52% (5/27) of NIR-PCOS patients had

increased levels of homocysteine (>10.7 µmol/L),

while 48.65% (18/37) of IR-PCOS were found

hyperhomocysteinemic (Homocysteine in IR-

PCOS 12.6±7.9 Vs. Homocysteine in NIR-PCOS

8.6±5.6, p-value= 0.02).

More number of PCOS group showed BMI≥ 23

in comparison with the control group (p< 0.06), but

the higher BMI did not have any significant

association neither with homocysteine nor with

insulin resistance in PCOS group, Homocysteine

and Log HOMA-IR 12.3±6.9 and 0.58±0.27 in

BMI ≥ 27 PCOS group respectively vs. 10.5±7.3

and 0.58±0.24 in BMI<27 PCOS group (p-value=

0.4 and 0.9). 12.88% of the PCOS group had BMI

≥ 27 Kg/m2.

Increased LH to FSH ratio was significantly

found in PCOS patients rather than the control

group (p<0.001), whether they were obese or

insulin resistant or none. Age was only related to

BMI (p-value= 0.001) but with no other variable.

All the insulin resistance indices were

correlated with homocysteine levels. (Fasting

insulin r= 0.513 p< 0.001, Glu/ Ins r=-0.3 p=

0.015, Log HOMA- IR r= 0.3 p= 0.015, Ins2hr r=

0.37 p= 0.002).

The correlation between homocysteine levels

and logarithmic transformation of HOMA-IR,

ANOVA linear regression test, is shown in figure

1.

Table I. Clinical and biochemical data of all patients.

Control (N=50) PCOS (N=64) p-value

Age (years)

29.1±3.2 31.09±8.9 0.08

BMI (kg/m2)

22.1±0.25 22.7±3.2 <0.06

Homocysteine (µmol/l)

6.8±1.95 10.9±7.2 <0.001

Insulin (mIU/l)

10.6±4.7 18.9±8.6 <0.001

LH/FSH

0.8±0.2 1.77±1 <0.001

Glucose/Insulin ratio

0.56±0.13 0.32±0.14 <0.001

Log HOMA-IR

0.23±0.17 0.58±0.24 <0.001

PCOS: polycystic ovarian syndrome, BMI: body mass index, Log HOMA-IR: logarithmic transformation of homeostasis index of insulin

resistance.

Table II. Variables and their differences in insulin resistant and non insulin resistant PCOS patients.

Number Homocysteine Fasting glucose Fasting insulin Glucose/Insulin BMI

PCOS-NIR

27 8.6±5.6 83.9±11 11.1±2.7 0.44±0.12 25.3±2.9

PCOS-IR

37 12.6±7.9 97.9±11.2 24.6±6.8 0.23±0.07 24.2±3.4

p value 0.02 <0.001 <0.001 <0.001 0.18

Values are mean±SD. PCOS: polycystic ovarian syndrome, NIR: non insulin resistant, IR: insulin resistant.

Hemati et al


Figure 1. Linear regression between homocysteine levels and logarithmic transformation of HOMA-IR

Discussion

There were significant differences found in

homocysteine levels and insulin resistance between

the PCOS patients and the control group. PCOS

group stratified into insulin resistant and non-

insulin resistant patients. Thereby, significant

difference was found between homocysteine levels

which propose significant correlation between

insulin resistance and homocysteine.

Recently, the local or systemic effects of insulin

resistance have been studied. Evidences provided

have shown that hyperinsulinemia and some

phenotypes of insulin resistance syndrome could

have several metabolic effects in general

population, including hyperhomocysteinemia (17).

Some previous studies had shown the relation

between increased homocysteine levels and insulin

resistance in particular groups of fertile women.

Laivuori et al investigated the association of

insulin resistance and hyperhomocysteinaemia in

preeclamptic pregnant women (18). They found

that such a relation exists in preeclampsia, but not

in the control group.

According to our findings insulin resistance is

higher in the PCOS patients. They have a leaning

toward hyper homocysteinaemia as well. However,

homocysteine levels were found to be significantly

higher in IR group than the NIR (p=0.026). That

could suggest the insulin resistance role in

hyperhomocysteinaemia, but not as the only factor.

Such increase could be due to relative

hyperandrogenism or steroidal sex hormones

influence in PCOS (19).

Bayraktar et al have shown a relation between

homocysteine plasma levels, insulin resistance and

androgen levels in PCOS. Such a relation is not

found in congenital adrenal hyperplasia patients

(20).

However, there was an investigation that could

not find any connection between polycystic ovaries

and insulin levels (21). The only criterion

considered by that study was ultrasound features.

None of the other PCOS criteria was taken into

consideration. That could give a reason to their

contradictory findings. Another study suggested

that hyperhomocysteinaemia in PCOS is

independent of insulin resistance and is due to

other factors. Their results may be affected by their

small sample size (n=29) (22).

Rosolová et al found an unexpected reverse

relation between insulin resistance and

homocysteine level in healthy people (23). To our

opinion, such controversial results are caused by

the difference in PCOS, insulin resistance and

hyperhomocysteinaemia definition among

populations studied.

As shown in several researches, homocysteine

is in a positive relation with the risk of

cardiovascular diseases (12, 13). Implantation

disturbances and early pregnancy losses are both

common in PCOS, even after the correction of

ovulation disturbances and increased LH levels or

hyperandrogenism. All those could be in

association with hyper homocysteinaemia due to

its effect on vascular structure.

Insulin resistance is independently a risk factor for

cardiovascular diseases, diabetes, nephropathy due

to hypertension, and dyslipidemia (24). All those

PCOS, insulin resistance (IR)


which are related to the metabolic syndrome are

intensified by hyperhomocysteinaemia. Then,

PCOS could be considered as a type of insulin

resistance syndrome or as an early sign of that

syndrome. However, it should be kept in mind that

the homocysteine plasma level is affected by

several factors.

If the PCOS is diagnosed, it’s better to have in

mind the other metabolic complications as well.

Gynecologists should consider both the short term

(infertility) and long term (cardiovascular and

metabolic diseases) outcomes in their management.

Further studies are needed to investigate the effect

of insulin sensitizers on homocysteine plasma

level.

Regarding the result of this study, approving

hyperhomocysteinaemia and its relation to insulin

resistance, and vascular abnormalities caused by

them shown in other studies, it seems that clinical

examination concerning those complications is a

matter of importance in PCOS management.

Active treatment of hyperhomocysteinaemia may

decrease PCOS morbidity. Prospective studies

could objectively show the promising role of such

treatments in PCOS.

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24. Goldstein BJ, Haffner SM, Hsueh WA. Insulin

resistance implications for metabolic and cardiovascular

diseases. CME Circle Diabetes and Endocrinology

continuing medical education online:

http://www.medscape.org/ viewarticle/412860. 2001.

http://www.medscape.org/%20viewarticle/412860


Cotherapy of Tiron and selenium against vanadium

induced toxic effects in lactating rats Sadhana Shrivastava Ph.D., Deepmala Joshi Ph.D., Monika Bhadauria Ph.D., Sangeeta Shukla D.Sc.,

Ramesh Mathur Ph.D.

UNESCO Satellite Center of Trace Element Research and Reproductive Biology and Toxicology

Laboratory, School of Studies in Zoology, Jiwaji University, Gwalior (MP), 474 011, India.


Abstract

Background: Vanadium is an important environmental and industrial pollutant. It has a

status of reproductive toxicant and is reported to cross placental barrier.

Objective: The current study was performed to assess the therapeutic efficacy of Tiron

and its combination with selenium against vanadium induced toxicity in lactating and

suckling rats.

Materials and Methods: Rats were exposed to vanadium at a dose of 7.5 mg/kg/day

(p.o.) for 20 days from 0 day of post partom (p.p.). Tiron (606 mg/kg/day, i.p.) and

selenium (0.5 mg/kg/day, p.o.) were administered for 5 days on 21-25 day PP.

Results: Vanadium exposure decreased blood sugar level while serum transaminases

and serum alkaline phosphatase showed increased values significantly (p<0.01).

Elevation in glycogen content of liver and kidney of suckling and kidney of lactating

rats was found after toxicant administration. Toxicant intoxication increased the

enzymatic activity of acid phosphatase in liver of suckling and lactating and kidney of

suckling rats. On the contrary alkaline phosphatase and adenosine triphosphatase

activities were inhibited significantly (p<0.01) in all the organs. Lipid peroxidation was

enhanced whereas glutathione was reduced significantly in liver of suckling and

lactating rats (p<0.01). Vanadium also caused histopathological lesions. Therapies of

Tiron per se and Tiron along with selenium maintained almost all blood and tissue

biochemical parameters towards normal. Tiron along with selenium reduced vanadium

induced lesions in lactating and sucklings rats.

Conclusion: Tiron along with selenium is more effective than Tiron alone against

vanadium induced toxic effect on lactating and suckling rats.

Key words: Vanadium, selenium, Suckling, lactating, Teratogenecity, Tiron.

Introduction

Industrial pollution is the biggest health hazard

in the 21st century. Vanadium is widely distributed

in the earth’s crust in a wide range of minerals and

in fossil fuels. The chemical gets into the air, water

and soil when fuel oil is burned, or when rocks and

soil containing vanadium are broken down (1-3).


Sadhana Shrivastava, UNESCO Satellite Center of

Trace Element Research and Reproductive Biology and

Toxicology Laboratory, School of Studies in Zoology,

Jiwaji University, Gwalior (MP), 474 011 India.


It has been recognized as essential nutrients in

higher animals (4).

It has a status of reproductive toxicant, as

microtubule damaging agent, reducing sperm

motility and damaging spermatozoa. Vanadium

decrease fertility and after expose to it, embryo

lethality, fetotoxicity and teratogenicity have been

reported to occur in rats, mice and hamsters (5-8).

It also increases malformations and abnormalities

in pups. It can pass the blood placenta barrier and

has been reported to be teratogenic in rodents and

affects sexual development in pre-pubertal

animals.

The development of the offspring was

significantly decreased from birth and during the

Shivastava et al


lactation period after vanadium intoxication (9,

10).

Tiron, 4, 5-dihydroxy-1, 3-benzene disulfonic

acid, has been known to be a widely used

antioxidant to rescue ROS-evoked cell death and a

non-toxic chelator to alleviate an acute metal

overload. It is a substrate in several enzyme

reactions and has small size which facilitates cell

entry and modulates intracellular electron transfer

reactions as an antioxidant by scavenging free

radicals (11, 12). It is also effective against various

metal intoxications of beryllium, aluminium, lead

and vanadium (13-16). Selenium (Se) is a trace

mineral and is needed in small amounts for good

health. It acts as a growth factor and is a powerful

antioxidant. It is a component of selenocysteine

and selenomethionine and cofactor for reduction

of antioxidant enzymes such as glutathione

peroxidases (17). Se protects toxic effects of

metals such as cadmium, and mercury, aluminium

(18-20). Se protects neuronal cells against

neurotoxic effects of vanadium (21). Thus the aim

of this study was to evaluate Tiron+Se as an

antidote against vanadium in sucklings and

lactating rats.


Detoxification of vanadium in rats. This study

comes under preclinical studies on laboratory

animals.

Chemicals

Vanadium sulphate and sodium selenite

procured from Glaxo Laboratory Ltd., Bombay,

India and sodium-4, 5-dihydroxybenzene-1, 3-

disulfonate (Tiron) procured from Sigma-Aldrich.

Other analytical grade laboratory reagents were

procured from Merck (Germany) and Glaxo

chemical (India).

Animals

Female albino rats of Sprague Dawley strain

(16010 g b.w.) from our departmental animal

facility were given a standard pellet diet (Pranav

Agro Industries, New Delhi, India having metal

contents in ppm dry weight Cu 10; Mn 33; Zn 45;

Co 5).

Drinking water was provided ad libitum.

Animals used in this study were treated and cared

for in accordance with the guidelines

recommended by the Committee for the Purpose of

Control and Supervision of Experiments on

Animals (CPCSEA), Government of India,

Ministry of Culture, Chennai. The dose of the

chelating agent was prepared daily and its pH was

adjusted to 6.4 with sodium bicarbonate before

administration.

Experimental design

Animals were selected on day 1 post partum

and were divided into four groups of six animals

each. Group 1 served as control. Groups 2, 3 and 4

were the experimental groups and received VOSO4

(7.5 mg/kg, p.o.) for 20 days. Group 2 served as

experimental control. Animals of group 3 were

taken individual treatment of Tiron (606 mg/kg,

i.p.) for 5 days on days 21-25 whereas group 4

were administered combination treatment of Tiron

(as in group 3)+ Se (0.5 mg/kg, p.o., through

catheter) for 5 days on days 21-25. Rats were

observed daily for mortality and morbidity through

out the study. 24 h after the final treatment animals

were sacrificed under light ether anesthesia. The

lactating and sucklings rats were examined for any

external and pathological lesions. The lactating’s

blood, liver, kidney, uterus and ovary were

investigated. Liver and kidney of suckling’s were

also processed for biochemical assays and

histopathological observation.

Biochemical assays

Blood was collected and serum was isolated for

various blood biochemical assays directly from the

heart by puncturing the retro-orbital venous sinus.

Blood sugar (22) aspartate aminotransferase

(AST), alanine aminotransferase (ALT) (23),

serum alkaline phosphatase (SALP) (24) and

serum protein (25) were studies. Standard

techniques were used to determine glycogen

contents in fresh tissue (26). A homogenate in

isotonic solution was processed for the

determination of protein (25) activities of acid and

alkaline phosphatases (ACP and ALP) (24) and

adenosine triphosphatase (ATP) (27). Lipid

peroxidation (LPO) (28) and reduced glutathione

(GSH) (29) were also estimated.

Histopathology

Liver, kidney, uterus, and ovary of lactating rats

and suckling’s liver and kidney were dissected out,

washed in saline and fixed in Bouin’s fluid,

embedded in paraffin, sectioned at 6μm and

stained with haemotoxylin and eosin for light

microscopical study.


Data were expressed as mean ± standard error

of mean (SEM). Statistical comparisons between

http://en.wikipedia.org/wiki/Selenocysteine

http://en.wikipedia.org/wiki/Selenomethionine

http://en.wikipedia.org/wiki/Cofactor_%28biochemistry%29

http://en.wikipedia.org/wiki/Redox

http://en.wikipedia.org/wiki/Antioxidant

http://en.wikipedia.org/wiki/Glutathione_peroxidase

http://en.wikipedia.org/wiki/Glutathione_peroxidase

Cotherapy of Tiron and Selenium against vanadium toxicity


all groups were performed by using one way

ANOVA followed by student’s t-test. P-value was

taken as significant at 0.01% level (30).

Results

Biochemical observation

Table I illustrates the protective effect of Tiron

and its combination with Se against vanadium

toxicity. Vandium induced significant elevation in

the activities of AST, ALT, SALP and serum

protein contents whereas the level of blood sugar

was decline significantly (p<0.01) in lactating rats.

Combination therapy (T+Se) effectively

recouped these values in all the parameters

compare with Tiron alone. After the injection of

vanadyl sulphate the wet weight of liver, kidney,

uterus, and ovary of lactatings and liver and kidney

of sucklings (Data not shown) showed no

significant variation. LPO showed enhance values

whereas glutathione showed inhibition in liver of

lactatings (p<0.01) and sucklings. Chelation

therapy showed improvement (Table IV).

Glycogen content of liver, uterus, ovary of

lactatings showed decreased values while kidney

of lactatings and liver and kidney of sucklings

showed elevated values after vanadium exposure.

With the combined treatment, values were restored

towards control (Table II). After administration of

vanadyl sulphate, protein content were decreased

in all the organs of lactatings and sucklings (Table

II). Activity of acid phosphatase showed increased

level in liver of lactatings, and liver and kidneyof

sucklings, while its activity decreased in uterus and

kidney of lactatings. With combined treatment,

values showed improvement. This improvement

was more effective with the Tiron+ Se (Table IV).

Activities of adenosine triphosphatase and alkaline

phosphatase showed inhibition after vanadium

exposure in liver, kidney, and uterus of lactatings

and liver and kidney of sucklings and values

restored with Tiron and Tiron+ Se. Tiron+ Se was

found effective (Table III and IV).

Histological observations

Vanadium induced proliferation in canaliculi,

hypertrophy and angular nuclei in hepatocytes and

sinusoidal spaces were filled with debris (Figure 1-

1).

Tiron protects the hepatocytes (Figure 1-2).

Tiron+ Se restore the structure of hepatocytes

showing normal sinusoidal spaces with mitotic

figures. Hepatocytes showed well-maintained

cytoplasm (Figure 1-3). Glomeruli showed

hypertrophy in the regions of cortex, disturbed

endothelial lining and degeneration in uriniferous

tubules (Figure 1-4).

Tiron showed better organization of kidney

(Figure 1-5). Treatment with Tiron+Se therapy

showed well form glomeruli and renal tubules

(Figure 1-6). Liver of sucklings, after injection of

vanadium in lactating rats, reduced sinusoidal

spaces and debris was observed in some sinuses

(Figure 2-7).

Treatment of Tiron showed normal portal triads

and well formed hepatocytes (Figure 2-8). Tiron+

Se therapy maintained hepatocytes and smooth

lining of sinus (Figure 2-9).

After administration of vanadium, glomeruli

occupied whole Bowman’s capsule and

degeneration in proximal and cortical tubules

(Figure 2-10). With the treatment of Tiron, kidney

showed rounded Glomeruli with maintained

tubules (Figure 2-11).

Tiron+ Se therapy improved proximal and

cortical tubules with Bowman’s capsule (Figure 2-

12). Toxicant exposed rats showed disorganized

uterine epithelium and uterine glands (Figure 3-

13). Tiron treatment showed recoupment (Figure

3-14).

The well formed endometrium, intact columnar

epithelium, well developed musculature prominent

vascularity and stroma was noted after treatment of

Tiron + Se (Figure 3-15). After vanadium

administration, decreased matured follicles,

disintegration in ovum, disorganized and

hypertrophied developing follicles were seen.

Stroma was loose and fibrotic (Figure 3-16).

Treatment of Tiron showed normal position of

follicles along with few and atretic follicles (Figure

3-17).

Combination therapy showed well organization

of developing follicles, freshly ovulated follicles

and maintain stroma (Figure 3-18).

Shivastava et al


Table I. Effect of Tiron (T) and selenium (Se) on liver function tests against vanadium (V) intoxication.

Treatments

Control V V+T V+T+Se ANOVA

Blood Sugar (mg/100 g)

101.6+5.49 69.1+3.52# 92.5+4.91 99.8+5.04* 11.6†

AST (IU/L)

64.6+4.28 124.8+6.55# 95.2+5.46* 84.2+5.24* 25.5†

ALT(IU/L)

41.6+2.15 83.6+4.40# 69.2+4.42 50.1+2.98* 32.6†

SALP(mg Pi/100 ml/h)

207.2+10.5 463.2+30.4# 393.4+20.1 311.8+16.2* 34.1†

Serum Protein (mg/100 ml)

36.4 +2.07 70.2+3.73# 69.8+3.65 66.4+3.74 27.8†

AST = aspartate aminotransferase, ALT = alanine aminotransferase, SALP = serum alkaline phosphatase. Values are mean+S.E., (n=6), ANOVA ‘F’

values, † = Significant at 1% level. # p< 0.01 compared to control group, * p < 0.01 compared to toxicant administered group.

Table II. Effect of Tiron (T) and selenium (Se) on protein (mg/100 mg) and glycogen (mg/100 g) contents against vanadium (V)

intoxication.

Treatments Control V V+T V+T+Se ANOVA

Sucklings

Protein (L) 10.76+0.66 9.86+0.50 10.60+0.53 10.70+0.79 0.52

Protein (K) 9.23+0.66 7.42+0.41 8.61+0.46 9.58+0.46* 4.12

Lactating

Protein (L) 16.78+0.94 12.24+0.62# 12.94+0.67 15.16+0.92 8.00†

Protein (K) 12.69+0.84 9.26+0.56# 9.65+0.52 9.86+0.56 7.23†

Protein (U) 10.28+0.60 7.98+0.42 8.15+0.43 9.58+0.64 5.23†

Sucklings

Glycogen (L) 2726.2+153 3648.4+206# 3066.8+161 2996.4+191 5.59†

Glycogen (K) 66.8+3.43 80.6+4.24 74.4+4.02 70.7+4.42 2.51

Lactating

Glycogen (L) 2892.3+151 1515.8+108# 2598.6+149* 2690.8+149* 23.0†

Glycogen (K) 62.20+3.45 87.8+5.60# 68.6+3.55 61.2+3.12* 11.0†

Glycogen (U) 123.4+8.28 73.5+4.12# 104.0+5.23* 118.4+8.04* 13.5†

L = Liver, K = Kidney, U = Uterus

Values are mean + S.E., (n = 6), ANOVA ‘F’ values, † = Significant at 1% level. # p< 0.01 compared to control group, * p< 0.01 compared to toxicant administered group.

Table III. Effect of Tiron (T) and selenium (Se) on acid (ACPase) and alkaline phosphatase (ALPase) (mg Pi/100 mg/h) against

vanadium (V) intoxication.


Suckling

ACPase (L)

237.2+14.9 265.4+13.7 238.7+13.5 237.2+14.8 1.12

ACPase (K)

207.7+12.5 271.0+18.7 232.2+14.8 226.4+12.0 3.87

Lactating

ACPase (L)

248.1+17.7 324.6+20.9 291.3+15.9 255.6+15.4 4.76

ACPase (K)

288.8+15.9 238.0+16.8 260.4+19.1 281.2+15.4 2.18

ACPase (U)

183.4+11.4 73.2+4.05# 158.2+8.82* 165.8+11.7* 31.7†

Sucklings ALPase (L)

80.6+4.29 57.4+3.30# 72.6+4.51 79.8+4.26* 8.19†

ALPase (K)

2306.8+118 1378.2+68.7# 1785.8+90.7* 2009.6+112* 18.5†

Lactating

ALPase (L)

79.7+4.77 66.1+3.58 69.4+4.60 74.1+4.30 2.23

ALPase (K)

2233.0+118 1046+60.4# 1636.2+109* 1892.1+98.7* 30.4†

ALPase (U)

472.9+26.0 233.6+15.0 301.6+22.0 324.2+17.2 28.8†





Table IV. Effect of Tiron (T) and selenium (Se) on adenosine triphosphatase (ATPase, mg Pi/100 mg/min), Lipid peroxidation

(LPO, n mole MDA/mg protein) and reduced glutathione (GSH, µ mole/g ) against vanadium (V) intoxication.


Suckling

ATPase (L)

844.4+43.1 625.8+44.8# 866.6+49.3* 880.4+52.7* 7.60†

ATPase (K) 2284.6+138 1325.2+87.9# 2142.6+124* 2153.4+122* 16.1†

Lactating

ATPase (L)

1804.0+102 1357.4+ 84.0# 1698.0+85.5 1896.6+95.5* 7.80†

ATPase (K)

2228.7+126 1570.8+85.5# 2132.4+115* 2102.0+125* 8.09†

ATPase (U) 774.2+51.4 535.4+27.8# 543.2+27.7 739.8+40.3* 13.1†

Sucklings

LPO (L)

0.40+0.02 0.49+0.02 0.43+0.02 0.41+0.02 2.79

GSH (L) 7.40+0.39 5.79+0.43 6.64+0.36 7.19+0.38 3.95

Lactating

LPO (L)

0.33+0.01 0.86+0.04# 0.46+0.02* 0.42+0.02* 67.50†

GSH (L) 8.36+0.43 4.81+ 0.25# 7.64+0.40* 7.90+0.42* 20.75†



Figure 1. 1. After V exposure, hepatocytes showed degeneration along with vacuolation and granulation (X 400). 2. Hexagonal hepatocytes were seen after conjoint treatment with Tiron (X 400).

3. After the treatment of T+ Se mitotic figures were seen in some hepatocytes (X 400). 4. After the administration of vanadyl sulphate kidney showed hypercellularity in glomeruli of Bowman’s capsule (X 400).

5. Tiron treatment after V exposure showed better organization of cortex region of kidney (X 400).

6. Conjoint treatment of T+Se showed well formed Bowman’s capsules with glomeruli (X 400).

Shivastava et al


Figure 2.

7. V caused hypertrophy in hepatocytes of liver of sucklings (X 400).

8. Hexagonal hepatocytes were seen after therapy with Tiron (X 400).

9. With the treatment of T + Se improvement was shown (X 400). 10. After administration of vanadyl sulphate kidney showed glomeruli occupied whole spaces of Bowman’s capsule (X 400).

11. Tiron treatment after vanadium exposure showed better organization of cortex region of kidney (X 400).

12. Conjoint treatment of T+ Se showed normal Bowman’s capsules with glomeruli in kidney of sucklings (X 400).

Figure 3.

13. Vanadium exposed rats showed degenerative changes in endometrium with degenerated uterine glands (X 120). 14. With the treatment of Tiron after V exposure endometrial cells were well formed with loose stroma (X 120).

15. Well formed uterine glands were noted with the co-therapy of T+ Se (X120).

16. V exposure caused degenerated primary, secondary follicles with loose stroma in ovary (X 120).

17. Tiron treatment after V exposure showed mostly normal secondary and tertiary follicles with normal lutin cells (X 120).

18. With the treatment of T+Se after vanadium exposure better organization of the follicles were seen (X 60).



Discussion

The efficacy of exogenous therapy of Tiron in

combination with Se in the acceleration of

vanadium elimination and in the reversal of

intoxication has been discussed. The aim of the

present study was to report the teratogenic effects

of vanadium and to develop an intervention

strategy of companion formula of chelators with

antioxidant effects against vanadium intoxication.

About 95% of the vanadium transported in the

blood is bound to transferrin as the vanadyl (IV), it

also complexs with lactoferrin to be transported to

sucklings during breast feeding. Vanadium is

present in placenta and competes with iron for both

gastrointestinal absorption and cellular receptor

sites (31-33).

Research indicates that ALT and AST can be

used as biomarkers of cellular damage in blood

plasma, protein degradation and liver damage with

loss of the functional integrity of cell membranes

(34), thus a direct correlation exists between rise in

the serum enzyme activity and severity of damage.

The ALT is localized mainly in the cytoplasm

whereas AST activity is fairly distributed between

cytoplasm and mitochondria. A significant increase

in AST, ALT and SALP activities was noted after

vanadium exposure in lactating rats. The elevated

activities are indicative of cellular leakage and loss

of the functional integrity of cell membranes, thus

necrosis or membrane damage which releases the

enzyme into circulation.

SALP mainly arises from the lining of the

canaliculi and also from the sinusoidal surface of

hepatocytes and is excreted via bile. The SALP is

closely connected with proximal tubules and

osteoblast and is involved in the active transport

across the capillary walls. The increased level of

SALP may be due to the de novo synthesis by the

liver cells.

Results of the present study clearly depicted

that vanadium administration enhanced

concentration of this soluble enzyme significantly

(34). An increase in the activities of these enzymes

is supported by various authors (34, 35).

The stabilization of AST, ALT and SALP

levels by therapy of chelating agent with and

without antioxidants is a clear indication of

improvement in the functional status of the liver

cells (13, 14).

Oral administration of Se attenuated

significantly the increased level of these enzymes

and caused a subsequent recovery towards

normalization, as seen from statistical analysis.

Combined therapy may combine with reactive

metabolites and lead to inactivate them, thus it may

prevent the acute organ dysfunction and cellular

injury thereby inhibiting the rapid leakage of these

enzymes.

A number of investigators have previously

demonstrated the antioxidative effect of

magnesium (36), zinc (37) and selenium (20) etc.

Activities of these enzymes were also recouped

significantly with the administration of Tiron

against other metals, such as, beryllium (13),

aluminum (14) and vanadium (16, 35). Lowering

of blood sugar level in the lactating rats may be

due to increase in the uptake of glucose in the

glycogen metabolism (34). In vanadate treated rats,

liver glycogen level elevated in sucklings whereas

decreased in lactating animals as seen in the

present study. This can be very well correlated

with the increased activity of glycogen synthetase

and decreased activity of glycogen phosphorylase

(38).

Improvement in blood sugar and serum protein

by Tiron treatment is also reported (13, 34).

Oxidative stress is a major pathway for vanadium

induced toxicity (39). The pro-oxidant effect of

vanadate was evident in our experiments by the

occurrence of LPO in liver (2, 40). A significant

increase in malaondialdehyde products was also

observed after exposure to metals such as mercury

(19), aluminium (14, 20) and beryllium (13). GSH

plays a major role in cellular defense and in the

maintenance of the thiol disulfide status. It showed

inhibition in liver of sucklings and lactating rats

because of bonding to sulfhydryl groups of

proteins.

The increased TBARS as seen in the present

study may be due to tissue injury and failure of

antioxidant defense mechanism. Therefor

increased accumulation of LPO products might

well be the consequences of a progressive

degradation of necrotic tissue. LPO mainly

damages Kupffer cells as also evident in

histological studies. The result suggests that

toxicant induced elevation in LPO might be

because of the lower level of GSH as observed in

this study. GSH depletion decreases the

Shivastava et al


GSH/GSSG ratio, which leads to the production of

free radicals (41).

Combined therapy clearly indicated the

supplementation of antioxidants increased

effectiveness of Tiron which help to regenerate and

maintain the normal functional status of the tissues.

Treatment with Se afforded better protection, this

may be due to the destruction of free radicals,

supplying a competitive substrate for unsaturated

lipids in the membrane and/or accelerating the

repair mechanism of damaged cell membrane.

Therefor combination therapy (Tiron+ Se)

effectively recouped these values than tiron per se.

These findings are substantiated by various studies

(13, 14, 42).

Significant decreased values were seen in

ALPase, ATPase and increased value noted in

ACPase due to the vanadyl ion formed may bind

with cell proteins. VO2+

resembles with size of

Mg2+

which is regulated for the activity of ALPase

(21, 33), thus was inhibited due to its structural

similarity with phosphate, vanadate (VO3) also

interferes with a large variety of phosphate

dependent enzymes (43). Vanadate is a potent

inhibitor of membrane bound ATPase (33). It is

also possible that accumulation of vanadium with

its concomitant reduction to vanadyl followed by

damage to biological membranes, lysosomal

enzymes releases and destruction of placental

tissue (34).

Fatty changes with partial cell necrosis in the

liver have been observed in rats as a result of

exposure to vanadium (34). In the present study

vanadium induced; proliferation in canaliculi,

hypertrophy and angular nuclei in hepatocytes,

hypertrophy in glomeruli and degenerated

uriniferous tubules, disorganized uterine

epithelium and disintegration in ovum. Vanadium

was accumulated within spheroidal electro dense

structures in the cytoplasm of these cells (44- 46).

Therapy with Tiron was found very effective and

this may be due to its diphenolic nature of Tiron

which forms water soluble complexes with a large

number of metals (13, 14). Thus, it is clearly

apparent that Tiron crosses the placental barrier as

reported for other metals as well (42). Se acts as

cofactor of many antioxidant enzymes, maintains

the availability of antioxidant nonprotein

sulfhydryl groups (47, 48) thus may induce binding

of the V-Se complexes to proteins. It is an

important nutrient and can pass through the milk,

thus ameliorating toxicity in lactating and

sucklings.

In conclusion, the present research has

identified the role of antioxidants, Se and Tiron in

mitigation of vanadium toxicity by acting

synergistically.

Acknowledgment

The author is thankful to University Grants

Commission, New Delhi for financial assistance.

(F.15-122/98 (PTRAW-S/SA-I).

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Evaluation of the effect of oral ritodrine on

implantation rate in in-vitro fertilization-embryo

transfer cycles

Soghra Rabiee M.D., Marziyyeh Farimani M.D., Maryam Ahmadi M.D.

Department of Obstetrics and Gynecology, School of Medicine, Hamadan University of Medical

Sciences, Hamadan, Iran.

Received: 29 November 2009; accepted: 6 May 2010

Abstract

Background: Pregnancy rate with IVF cycle is almost 22%. Many investigations

perform to increase this rate in IVF. Various factors affect the result of IVF cycles. One

of these factors could be uterine contractions that expel transferred embryo. Ritodrine is

a beta mimetic agent that can block and decrease uterine contractions.

Objective: The objective of this study was to determine ritodrine effectiveness for

increasing the implantation rate in IVF cycles, and its probable mechanisms in

decreasing uterine contractions as well.

Materials and Methods: A total of 100 patients of IVF-ET cycles were divided

randomly in two groups in a university hospital, Hamadan, Iran. The case group were

prescribed ritodrine 10 mg / bid orally after oocyte retrieval until 10 days. The control

group didn’t received ridotrine.

Results: In ritodrine group 14% of patients and in control group 16% had positive

β-hCG test (p-value>0.5).

Conclusion: Ritodrine did not improve the implantation rate in IVF-ET cycles.

Key words: Ritodrine, IVF-ET, Implantation rate.

Registretion ID in IRCT: IRCT201105316665N1

Introduction

Infertility is a common problem affecting up to

10% of married couples (1). A systematic

evaluation of aetiologic factors forms the basis for

choice of treatment and future fertility. On the

global perspective, assisted reproductive

technologies (ART) have become internationally

recognized treatment option for some infertile

couples. At present time, infertility treatments by

ART is progressing and improving considerably

(2). These treatments have improved the success

rate of IVF-ET up to 22% (2). However, infertile

couples spend much money and time especially in

ART cycles and they expect more success rate.

Presently , the maturation of follicles with


Soghra Rabiee, Department of Obstetrics and

Gynecology, School of Medicine, Hamadan University

of Medical Sciences, Hamadan, Iran.


gonadotropin, oocyte retrieval, in vitro fertilization

and embryo development are satisfactory, but the

main problem is why sometimes the embryo

doesn't implant in the uterus successfully (3)?

Different factors such as type of catheter for

embryo transfer, using tenaculum for grasping the

cervix, removing the obvious or excess cervical

mucus before transfer, volume of transfer media,

and embryo transfer under trans abdominal

ultrasound guidance avoiding the uterine

contraction at the time of embryo transfer, studied

previously (4, 5).

Ritodrine, as a beta- agonist, may improve the

implantation rate in IVF cycles with decreasing

uterine contraction and consequently decreasing

extraction of embryos which transferred to the

uterus (6).

However, the results of studies are controversial

in relation to controlling uterine contractions and

its effect on implantation rate (7, 8). Some other

chemicals such as hyoscine bromide, piroxicom

Rabiee et al


and oxytocin antagonists has investigated and

showed promising results (9-11).

The aim of this study was determination of

ritodrine effect on uterine contraction and

pregnancy rate in IVF-ET patient.


In a randomized controlled clinical trial, one

hundred women aged between 25 through 35 years

with a history of >5 year infertility enrolled to

study. Inclusion criteria were as follow: having a

morphologically normal uterus, infertility from

tubal, male, endometriosis or unexplained factor.

Excluding criteria were lack of any systemic

known diseases in both groups. These patients

were candidate for IVF-ET and were randomly

allocated into treatment (50 patients) and control

(50 patients) groups. Two groups had not

significantly differences in view of mean age,

causes of infertility and infertility duration. All the

patients were stimulated with a GnRH agonist and

exogenous gonadotropins with long protocol. On

the day after oocyte retrieval, all patients in the

case group received oral ritodrine 10 mg/ bid for

ten days. Other medications in both groups were

similar including ASA 80 mg/day, progesterone

100mg IM, erythromycin 400 mg/qid, and heparin

5000 u/bid. The luteal phase was supported by

progesterone injection. The successfulness

criterion was positive β-hCG after 2 weeks. At

least three good quality embryos transferred for

each patient and, the results of two groups (rate of

positive β-hCG) compared and were analyzed with

chi square test.

This study was done in a public center for

infertility treatment and was approved in the

Research Ethics Committee, Chancellor for

Research and Technology, Hamadan University of

Medical Sciences.


The pregnancy rate between two groups (rate of

positive β-hCG defined as pregnancy index),

compared and were analyzed with chi- square test.

The background variables between study group

and control group also were compared by t-student

test and chi-square test.

Results

The studied patients had no history of abortion

or pregnancy. Mean age were 29.5 and 30.8 years

in the study and control group respectively

(p>0.05) with the range of 20-40 years. The mean

duration of infertility in the study group were 8.3

and in the control group 7.5 years. The main

causes of infertility were as follow: tubal factors

31.5%, male factors 45%, ovulatory factors 18.5%

and unexplained factors 15%.

The minimum and maximum retrieved oocytes

from patients were 3 and 12. The numbers of

transferred embryo were between 2 and 4. This

study indicated embryo implantation followed by

positive β-HCG test in 7 (14%) patients in

ritodrine group and 8 (16%) patients in control

group (p>0.05). There was not a significant

difference in ritodrine group in comparison with

control group. One patient in ritodrine group had

minor side effects such as head ache and vertigo.

Discussion

As the results of this study indicate, ritodrine

has not significant effect on implantation rate in

IVF-ET cycles. Many chemicals were investigated

for increase implantation rate and clinical

pregnancy in IVF-ET (12, 13).

Gholami and colleagues (5) compared the

pregnancy outcome in patients undergoing IVF-ET

cycles, using human derived follicle-stimulating

hormone (FSH) or recombinant FSH for ovarian

stimulation protocols. They results did not

demonstrate a difference between the use of h-FSH

vs r-FSH for ovarian stimulation in terms of

pregnancy outcome, in good prognosis patients

undergoing their first IVF-ET procedure. Moon

and colleagues reported piroxicom increased the

implantation rate and clinical pregnancy in the

patients candidate for IVF-ET.

However, Dal Prato and Borini (10) indicated

that, administration of a single dose of piroxicam

before embryo transfer has no additional effect on

pregnancy outcome in patients receiving adequate

doses of progesterone for luteal phase

supplementation after IVF or ICSI.

On the other hand, in a limited trial, an oxitocine

antagonist (Atociban) was administered on 14th day

of endometrial synchronization for oocyste

donation. The treatment decreased the uterine

contractile activity and resulted in successful

embryo implantation and a normal twin diamniotic

pregnancy (7). It was concluded that Atosiban

may improve uterine receptivity during ET and

Oral ritodrine in IVF-ET


may increase success rates of advanced infertility

treatment procedures, but this report was only in

one case and could not be generalizes to all

patients. Gorkemli and colleagues in their study in

2004 concluded that adding estradiol to

progesterone in luteal phase may increase

implantation rate in IVF-ET cycles (12).

Amanda and Garas, in 2006 reported that

neither implantation rate nor pregnancy rate

increased with paracetamol and diclofenac (13). In

2003 Peinheiro and colleagues (14) found that

beta-2-adrenergic will not increase the

implantation rate in ICSI cycles. They applied both

ritodrine and terbutaline in divided groups. The

result of their study is in agreement with our study.

As we see different studies were done to identify

best medication for improving ART outcome, but a

definitive answer has not found yet.

NSAIDs such as paracetamol, indomethacin,

and diclofenacs, and beta agonists such as ritodrine

and terbutaline are the most examined drugs and

their positive effect is not proven definitely yet

(10, 13, 15) . Some reports indicated controversial

results on the effect of peritalsis of uterus and its

contractibility on the rate of implantation. Some

studies report anticholinergic agents significantly

suppress sporadic myometrial contractions and

uterine peristalsis (6) and even adequate uterine

contractility may provide for gamete/embryo

transportation through the utero-tubal cavities and

successful embryo implantation in spontaneous or

assisted reproduction. Inadequate uterine

contractility may lead to ectopic pregnancies,

miscarriages, retrograde bleeding with

dysmenorrhea and endometriosis (16). Therefore,

the questions about uterine contractions control

have some roles on the embryo implantation,

remained open for more studies.

Conclusion

This study indicated that, ritodrine has not

significant effect on the implantation rate in the

IVF-ET technique. We recommend extended

researches to find out a certain medication for

increasing ART outcomes.

Acknowledgement

Hereby, we thank all our colleagues in

Infertility Center of Fatemieh Hospital, Hamadan,

for kindly technical assistance in this study,

especially Mrs Ramezani for her honorable

assistance. This paper extracted from a thesis of

residency for Obstetrics and Gynecology specialty.

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Case report

How should painful cystic degeneration of myomas be

managed during pregnancy? a case report and review

of the literature

Tae-Hee Kim M.D., Hae-Hyeog Lee M.D., Ph.D.

Department of Obstetrics and Gynecology, College of Medicine, Soonchunhyang University, Bucheon,

420-767, Republic of Korea.


Abstract Background: Uterine myomas are common pelvic masses during pregnancy. The pain

and rapid growth of myomas are among the most common complications during

pregnancy. We evaluate management of painful cystic degeneration of myomas during

pregnancy.

Case: A 27-year-old primigravida had a pelvic mass. We have managed a case in which

the diagnosis of cystic degeneration of uterine myomas could not be easily

differentiated from an ovarian torsion or carcinoma. Differentiation between

degenerative pain of the myoma and an ovarian malignancy or torsion was necessary. A

complete aspiration of the cystic changes of the uterine myoma was performed without

performing a myomectomy.

Conclusion: We report a good result of aspiration of a cystic uterine myoma during

pregnancy with a review of the literature published for twenty years since 1 January

1988.

Key words: Leiomyomas, Uterine Fibroids, Pregnancy.

Introduction

Uterine leiomyomas are observed in pregnancy

more frequently now than in the past because many

women are delaying childbearing. Uterine myomas

during pregnancy increase the incidence of

spontaneous abortions, ectopic pregnancies,

preterm labor, premature rupture of the

membranes, placental abruptions, abnormal fetal

presentations, and red degeneration. Myomectomy

during pregnancy is rarely performed because of a

fear of pregnancy loss and bleeding (1). We report

a case of an aspiration of a painful uterine myoma

during pregnancy.


Hae-Hyeog Lee, Department of Obstetrics and

Gynecology, Soonchunhyang University Bucheon

Hospital. 1174 Jung-1-dong, Wonmi-gu, Bucheon-si,

Gyunggi-do, 420-767, Republic of Korea.

Email: [email protected]; [email protected]

Case Report

We performed a cystic myoma aspiration at 12

weeks gestation with preservation of the

pregnancy. A 27-year-old primigravida had an 8

cm pelvic mass on routine ultrasonography at 10

weeks gestation. She had a MRI at a local clinic.

The MRI demonstrated an 8 x 7 x 6 cm cystic and

solid mass with septa (Figure 1). The presumptive

diagnosis was a cystic borderline tumour or cyst

adenocarcinoma. She was admitted to the

emergency room for pain management.

Differentiation between degenerative pain of the

myoma and an ovarian malignancy or torsion was

necessary. We performed explolaparotomy. Intra-

operatively, the uterus was soft and the size was

appropriate for 12 weeks gestation. The

leiomyoma was situated in the broad ligament at

the left anterior aspect of the uterus. The left ovary

was located on the myoma of the broad ligament.

We performed a complete aspiration of the cystic



Tae-Hee Kim et al


changes of the uterine myoma, washing cytology,

and biopsy of the cyst wall of the uterus without

performing a myomectomy. The washing cytology

showed no malignant cells; the biopsy specimen

was a uterine leiomyoma. After cystic aspiration,

the pain was relieved. There were no other

complications during pregnancy. She admitted for

10 days for fetal monitoring and postoperative care

after explolaparotomy. She had no fever,

complication and preterm labor during pregnancy.

She had a myomectomy during caesarean section

and a healthy female weighing 2, 640 grams at 38

weeks gestation. Three years later, she had another

healthy baby and there were no other

complications.

Table I. Clinical characteristics of patients in articles including our case.

Age

(years) Parity

Gestational

age (week)

Diagnostic

method

Myoma

size (cm) Symptoms Delivery mode

Delivery

time

(weeks)

Newborn

weight

(g)

Michalas

1995 (2) 31 primi. 14 USG 23 cm respiratory pain C/S 39 2000 g

Mollica

1996 (3)

(18 cases)

33 (28-40)

primi.16 multi. 2

12 (10-19)

NM

5-10 cm;

9 cases 10 cm<

;9 cases

pain rapid growth

C/S 93.7%

N.M >2500 g; 17 cases <2500 g; 1 case

Majid 1997 (4)

35 primi. 17 USG 24 cm nausea

and vomiting C/S 17+5 NM

Ehligiegba

1999 (5) 28 NM 28 NM 6 cm umbilical hernia V/D 39 3400 g

Wittich 2000 (6)

31 primi. 11+4 MRI 2074 g pain C/S NM 3275 g

Danzer 2001 (7)

44 primi. 12 USG 10 cm pain, bleeding C/S 37 3235 g, 2810 g

Carolis

2001 (8)

(18 cases)

21-34 14-primi. 4-multi.

15.1±5.0 NM 14.3 ±7.9 cm pain-8 cases

pyrexia-4 cases C/S : 14 V/D : 2

37.6±4 3273.7 g ±399.3

Celik 2002 (9)

(5 cases)

31.4±3.5 primi.:2 multi.:3

17.8±3.4 NM 14 ±3.8 cm pain C/S:5 38.6±1.1 3220 g ±303.3

Lolis

2003 (10)

(13cases)

32.9±5.0 NM 16.2±1.3 NM 879.5 g±52.9

pain:10 cases

increase size:3

cases

C/S 37.1±2.7 3048.4 g ±528.2

Melgrati

2005 (11) 29 primi. 24 USG 7 cm pain, fever V/D 39 NM

Umesurike

2005 (12) 30 primi. 20

USG

Cyst

32 cm

7700 g pain V/D 38.9 3.560 g

Usifo

2007 (13) 31 primi. 13

USG

Cyst 2000 g

pain, nausea, vomiting,

diarrhea

C/S 38 3990 g

Bonito

2007 (14) (5cases)

34.4 11.8 NM 7 cm pain, rapid growing

C/S:2, V/D:3 38.2 3340 g

Okonkwo

2007 (15) 41 multi. 19 USG 1000 g

free fluid in

abdomen C/S 38 2600 g

Bhatla

2009 (16) 30 primi. 19+3 USG 3900 g pain V/D 38 2740 g

Our case

2009 27 primi. 12+2 MRI 8x7x6 cm pain C/S 38 2640 g

Primi= primi-parity, multi= multi-parity, NM= not mention, USG= ultrasonogram, MRI= magnetic resonance images, C/S=caesarean

section, V/D=vaginal delivery.

Cystic degeneration of uterine leiomyoma during pregnancy


Figure 1. T2-weighted image of MRI findings showed an 8×7×6 cm cystic and solid mass with septa. (A) Sagital view, (B) Coronal view.

Discussion

The PubMed search engine was used to identify

Korean and English language articles published

between 1 January 1988 and 1 January 2009 using

the search terms myomectomy, cyst aspiration, and

pregnancy. We found 18 articles, 2 of which could

not be accessed outside the web in Korea. In 71

cases, including the case herein, operative

procedures of myomas during pregnancy were

performed (2). The clinical characteristics of

published patients, including the patient presented

herein, are listed in table I (2). Our case had features

of an ovarian tumor or torsion similar to 5 other

cases.

Uterine leiomyomas occur in 1.6% ~ 2% of

pregnancies (16). Although leiomyomas during

pregnancy usually remain asymptomatic, they may

have complications. The most common complication

of uterine myomas during pregnancy is abdominal

pain, which is due to red or carneous

degeneration.The management of painful

leiomyomas during pregnancy is usually medical,

but a few myomectomies have been reported.

Myomectomies are generally avoided during

pregnancy because increased vascularity of the

uterus can lead to hemorrhagic complications.

Successful myomectomies during pregnancy have

been reported. The cases showed that the most

common indication for myomectomy during

pregnancy was severe abdominal pain to maintain

pregnancy without any procedure. In the article

review, there were gastrointestinal manifestations

such as nausea, vomiting and diarrhea due to

obstructive pressure of myomas on bowel. When we

have cases which require myomectomy during

pregnancy, addition of Doppler evaluation is

recommended. A sharp drop in residence index (RI)

in Doppler means an indication of some degree of

necrosis (17). The Doppler is a helpful modality to

decide doing myomectomy or not during pregnancy

(17). In 71 cases, only 2 pregnancies were

terminated after myomectomy (4, 8) and 2 cases had

preterm labor and preterm delivery respectively (3,

10). One case had intrauterine growth retardation

(4). There was one prospective large study. Among

15, 579 women registered at the prenatal clinic,

severe abdominal pain was seen in 16 patients, in 13

cases myomectomy was done. 12 cases has live

birth, 13 cases has no blood transfusion and other

complications (10).

There has been only one successful case of a

gasless laparoscopic myomectomy (11). In this case,

it could be difficult to differentiate a complex

ovarian mass from cystic degeneration of the

myoma. The prevalence of adnexal malignancy in

pregnancy is between 2% and 3% (18). The

likelihood of malignancy with a complex ovarian

mass is relatively low, but one must consider surgery

during pregnancy, and routine prenatal ultrasound

(US) to detect an ovarian mass. Magnetic resonance

imaging (MRI) can be safely used during pregnancy

to evaluate adnexal masses. But only one case was

evaluated by MRI. The severe abdominal pain

during pregnancy could be considered to perform

explolaparotomy or not. Degeneration of a myoma

may mimic an ovarian malignancy during pregnancy

and obstetricians have to be aware of the differential

diagnosis.

Our report provides suggestions for obstetricians

on the management of symptomatic uterine myomas

during pregnancy. A myomectomy during

pregnancy in carefully selected patients is a safe

procedure.

Tae-Hee Kim et al


From the literature review, there were rare cases

of cystic degeneration of uterine myomas during

pregnancy.

The severity of symptoms and suspicion of

malignant mass or torsion is the key in deciding

upon laparotomy. We recommend to perform cyst

aspiration rather than myomectomy in a myoma with

cyst degeneration and pain.

References 1. Parker WH. Uterine myomas: management. Fertil Steril

2007; 88: 255-271.

2. Michalas SP, Oreopoulou FV, Papageorgiou JS.

Myomectomy during pregnancy and caesarean section. Hum

Reprod 1995; 10: 1869-1870.

3. Mollica G, Pittini L, Minganti E, Perri G, Pansini F. Elective

uterine myomectomy in pregnant women. Clin Exp Obstet

Gynecol 1996; 23: 168-172.

4. Majid M, Khan GQ, Wei LM. Inevitable myomectomy in

pregnancy. J Obstet Gynaecol 1997; 17: 377-378.

5. Ehigiegba AE, Selo-Ojeme DO. Myomectomy in

pregnancy: incarcerated pedunculated fibroid in an umbilical

hernia sac. Int J Clin Pract 1999; 53: 80.

6. Wittich AC, Salminen ER, Yancey MK, Markenson GR.

Myomectomy during early pregnancy. Mil Med 2000; 165:

162-164.

7. Danzer E, Holzgreve W, Batukan C, Miny P, Tercanli S,

Hoesli I. Myomectomy during the first trimester associated

with fetal limb anomalies and hydrocephalus in a twin

pregnancy. Prenat Diagn 2001; 21: 848-851.

8. De Carolis S, Fatigante G, Ferrazzani S, Trivellini C, De

Santis L, Mancuso S, et al. Uterine myomectomy in

pregnant women. Fetal Diagn Ther 2001; 16: 116-119.

9. Celik C, Acar A, Cicek N, Gezginc K, Akyurek C. Can

myomectomy be performed during pregnancy? Gynecol

Obstet Invest 2002; 53: 79-83.

10. Lolis DE, Kalantaridou SN, Makrydimas G, Sotiriadis A,

Navrozoglou I, Zikopoulos K, Paraskevaidis EA. Successful

myomectomy during pregnancy. Hum Reprod 2003; 18:

1699-1702.

11. Melgrati L, Damiani A, Franzoni G, Marziali M, Sesti F.

Isobaric (gasless) laparoscopic myomectomy during

pregnancy. J Minim Invasive Gynecol 2005; 12: 379-381.

12. Umezurike C, Feyi-Waboso P. Successful myomectomy

during pregnancy: a case report. Reprod Health 2005; 2: 6.

13. Usifo F, Macrae R, Sharma R, Opemuyi IO, Onwuzurike B.

Successful myomectomy in early second trimester of

pregnancy. J Obstet Gynaecol 2007; 27: 196-197.

14. Bonito M, Gulemi L, Basili R, Roselli D. Myomectomy

during the first and second trimester of pregnancy. Clin Exp

Obstet Gynecol 2007; 34: 149-150.

15. Okonkwo JE, Udigwe GO. Myomectomy in pregnancy. J

Obstet Gynaecol 2007; 27: 628-630.

16. Bhatla N, Dash BB, Kriplani A, Agarwal N. Myomectomy

during pregnancy: a feasible option. J Obstet Gynaecol Res

2009; 35: 173-175.

17. Gojnic M, Pervulov M, Mostic T, Petkovic S. Doppler

ultrasound as an additional parameter for the evaluation of

myomas and the indication of myomectomy during

pregnancy. Fetal Diagn Ther 2004; 19: 462-464.

18. Leiserowitz GS, Xing G, Cress R, Brahmbhatt B, Dalrymple

JL, Smith LH. Adnexal masses in pregnancy: how often are

they malignant? Gynecol Oncol 2006; 101: 315-321.


Case report

Tubo-ovarian abscess in a virgin girl

Tahere Ashrafganjooei1 M.D., Iraj Harirchi

2 M.D., Giti Iravanlo

3 M.D.

1 Department of Obstetrics and Gynecology, Afzali-poor Hospital, Kerman University of Medical

Sciences, Kerman, Iran.

2 Department of Surgical Oncology, Tehran University of Medical Sciences, Tehran, Iran.

3 Department of Clinical Pathology, Tehran University of Medical Sciences, Tehran, Iran.

Received: 10 May 2010; accepted: 8 December 2010

Abstract

Background: Tubo-ovarian abscess as a serious complication of pelvic inflammatory

disease is very uncommon in sexually inactive girls.

Case: We report a case of tubo-ovarian abscess in a 24-year-old sexually inactive girl

with transverse vaginal septum who was presented with abdominal pain and a pelvic

mass and without prior surgical history and no evidences of appendicitis, inflammatory

bowel disease, or cancer. A huge unilateral tubo-ovarian abscess was recognized at

laparotomy. Unilateral salpingoophorectomy, hysterectomy and postoperative antibiotic

therapy cured the patient.

Conclusion: Early diagnosis and treatment are essential to prevent further sequel

including infertility, ectopic pregnancy, and chronic pelvic pain which cause morbidity.

Key words: Tubo-ovarian abscess, Virgin, Vaginal septum.

Introduction

Pelvic inflammatory disease (PID) is a complex

polymicrobial infection. It is thought to arise from

ascending of microorganisms from the lower

genital tract to the endometrium and fallopian

tubes, pelvic peritoneum and adjacent structures,

causing inflammation (1). It is generally a disease

of young, sexually active women of reproductive

age group (2). It has traditionally been thought that

sexual activity is a prerequisite for acquiring this

disease thus, PID is considered to be rare in

sexually inactive girls (3). Our review of the

literatures revealed 10 cases of tubo - ovarian


Tahere Ashrafganjooei, Department of Obstetrics and

Gynecology, Afzali-poor Hospital, Kerman University

of Medical Sciences, Kerman, Iran.


abscesses in virgins (3-8).

Tubo-ovarian abscess (TOA) is an end-stage

process and a serious manifestation of PID that

usually reflect an agglutination of pelvic organs

(tube, ovary, bowel) forming a palpable complex

(9). They may also occur after pelvic or abdominal

surgery by spreading of organisms from adjacent

gastrointestinal infections or occasionally form

super infection of a necrotic ovarian malignancy,

so; postmenopausal women presenting with TOAs,

should be thoroughly investigated to exclude a

concomitant pelvic malignancy and conservative

treatment of TOAs has no place during the

menopause (10). The correct diagnosis of TOA is

usually made during laparotomy and patient

history. Physical examination and diagnostic tests

are relatively non-specific (2).

Treatment of TOA involves the use of an

antibiotic regimen administered in a hospital.

Ashrafganjooei et al


About 75% of women with TOA respond to

antimicrobial therapy alone. Failure of medical

therapy suggests the need for drainage of the

abscess (1). Laparoscopic surgery which

minimizes postoperative complications can be the

first option in the treatment of TOA (5).

Percutaneous drainage guided by imaging studies

(ultrasound or computed tomography scan) has

also been used successfully (1).

Case report

A 24 year- old blind virgin, with history of

mental retardation, primary amenorrhea and

normal secondary sexual development, was

presented to infectious ward of Imam Khomaini

Hospital, Tehran, Iran; with severe right lower

quadrant pain, diarrhea and fever since two days

ago.

Her mother mentioned a history of lower

abdominal pain, low-grade fever, and medical

therapy in her since a few months ago. In physical

examination, external genitalia were grossly

normal and hymen was intact, but rectal

examination revealed blind vagina of about 1 cm

length and an ill-defined right pelvic mass.

Abdominal palpation revealed moderately

tenderness in lower abdomen particularly in right

lower quadrant.

Laboratory analysis showed a white blood cell

count of 188000/µl, hemoglobin level of 11.5g/dl,

ESR=121 and negative blood and stool culture.

The chest radiograph was negative. Preoperative

transabdominal sonogram and computed

tomographic scan of the abdomen and pelvis were

done.

The sonogram demonstrated a 7×7.5 cm

complex mass in right pelvis with irregular borders

and multiple septa. Dilation of the right urinary

collecting system due to extrinsic compression was

noted on sonograghy. Abdomino pelvic CT-Scan

confirmed ultra sonographic findings (Figure 1).

The patient was placed on intravenous

ceftriaxone, but subsequently developed spiking

fever after 5 days. On abdominal exam, she

developed severe tenderness and rebound. We

discussed with her family and emphasized the

clinical suspicion of pelvic tumor necrosis and

abscess formation or to lesser degree, apandicular

abscess and the need for surgical resection.

She underwent exploratory laparotomy. The

abdomen was free of frank pus and adhesions, but

there were severe adhesions in pelvis. After release

of adhesions, about 300 cc greenish yellow, pus

like foul smelling material was coming from right

adnexal swelling into the pelvic cavity. Pus was

drained and exploration revealed a10×12cm right-

sided pelvic mass with extensive adhesions to the

recto-sigmoid.

The mass was identified as a right tubo-ovarian

abscess, with no evidence of diverticular or

appendiceal diseases at surgical inspection. All

abdominal organs were found to be normal. Right

salpingo-oophorectomy, was performed. During

surgery pus was removed from TOA and

transported to the laboratory, cultured for both

anaerobic and aerobic organisms. Due to her

mental retardation, after consent of her mother, the

patient underwent total abdominal hysterectomy

and then peritoneal lavage was done. The thin

lower third vaginal septum also was resected. The

culture of pus was positive and mixed.

Postoperatively, the patient received ceftriaxone

1g i.v. every 8 hr plus clindamycin 900 mg i.v.

every 8 hr. She defervesced gradually and became

afebrile after three days. Parenteral antibiotics

were replaced with oral antibiotics 72 hours later

and recommended for 14 days. Histophatologic

analysis showed a right tubo-ovarian abscess with

severe inflammatory changes and necrosis with

marked neutrophil infiltration. Postoperative

recovery was uneventful and the patient was

discharged on the fifth postoperative day.

Tubo-ovarian abscess in a virgin


Figure 1. Preoperative CT-Scan demonstrating an ill-defined

pelvic mass.

Discussion

Tubo-ovarian abscess is an advanced form of

pelvic inflammatory disease, most often caused by

spread of bacteria from the lower genital tract (9).

TOA is a serious complication of pelvic

inflammatory disease rarely seen in sexually

inactive girls and it is generally the result of a

blood-borne or gastero-intestinal infection such as

appendicitis in these cases (5). It can also develop

as a complication of pelvic or abdominal surgery

and malignancy (8).

The infection in TOA is usually polymicrobial

with mixed aerobic-anaerobic organisms. The

clinical presentation of TOA tends to be non-

specific. Vaginal discharge is present in less than

5% of patients. The typical ultra sonographic

appearance of a tubo-ovarian abscess is a

multilocular, cystic and complex adnexal mass

often with debris and thick septations. Hartmann

reported two cases with purulent fluid collections

in the fallopian tubes that were not evident on

imaging, but the laparoscopic evaluation revealed

them (7).

Therefore he recommends the use of

laparoscopy for diagnosis in atypical cases.

Ruptured TOA is associated with a high risk of

development of septic shock, in case that urgent

surgical intervention is not undertaken (6). Broad

spectrum antibiotics should be given promptly to

cover polymicrobial mixed aerobic/anaerobic

infections. Prompt surgical treatment may be

associated with decreased morbidity and

postoperative hospitalization. Extensive

inflammation and edema makes the dissection of

pelvic abscess difficult.

Complications of tubo-ovarian abscesses

include tubal occlusion, increasing the risk of

infertility and ectopic pregnancy, on the other

hand, pelvic adhesions leading to chronic pelvic

pain. Early diagnosis and surgical treatment are

essential to prevent further sequela and improving

outcome.

We must consider TOA in the differential

diagnosis for abdominal pain and pelvic mass with

fever in adolescent females regardless of sexual

history.

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transmitted diseases. In: Berek JS: Novak's Gynecology,

14th Ed. Philadelphia; Lippincott Williams & Wilkins,

2007; 549-552.

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22Coskun%20M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hicsonmez%20A%22%5BAuthor%5D


http://www.ncbi.nlm.nih.gov/pubmed?term=%22Lerand%20SJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Jay%20MS%22%5BAuthor%5D

javascript:AL_get(this,%20'jour',%20'J%20Pediatr%20Adolesc%20Gynecol.');

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gensheimer%20WG%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Reddy%20SY%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Mulconry%20M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Greves%20C%22%5BAuthor%5D

javascript:AL_get(this,%20'jour',%20'J%20Pediatr%20Adolesc%20Gynecol.');

Ashrafganjooei et al


10. Khan NA, Maajeeni EH. Tubo-ovarian abscess in a

postmenopausal woman with underlying ovarian

carcinoma. Saudi Med J 2005; 26: 1010-1011.

Iranian Journal of Reproductive Medicine Vol. 9. No. 3. pp: 251-252, Summer 20111

Letter to Editor

The association between iron status and some

immunological factors in the pregnancy Seyed Alireza Sobhani

1 M.D., Zahra Etaati

2 M.D., Sepideh Mirani

3 M.D., Paknoosh Saberi

4 B.Sc.,

Mahnaz Shiroodi5 M.Sc., Hojjat Salmasian

6,7 M.D., Nadereh Naderi

5,8 Ph.D.

1 Department of Pathology, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.

2 Department of Obstetrics and Gynecology, Hormozgan University of Medical Sciences, Bandar Abbas,

Iran.

3 General physician, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.

4 Department of Biochemistry, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.

5 Center for research of Molecular Medicine, Hormozgan University of Medical Sciences, Bandar Abbas,

Iran.

6 Research Fellow, Tehran University of Medical Sciences, Tehran, Iran.

7 Research Fellow, Farzan Clinical Research Institute, Tehran, Iran.

8 Department of Immunology, Hormozgan University of Medical Science, Bandar Abbas, Iran.

Received: 7 August 2010; accepted: 18 May 2011

Iron deficiency anemia (IDA) is a common

problem in many developing countries. It is still

considered the most common nutrition deficiency

worldwide. Apart from its direct hematologic

importance, IDA affects cellular and humoral

immunity and predisposes the host to infections

(1).

Pregnant women are highly prone to IDA.

Controversial results are reported in studies

targeting this group of patients. Tang et al showed

a direct association between hemoglobin

concentration and the count of CD4+ T-cell

lymphocytes, serum levels of IL-2 and IgG, and an

inverse association with susceptibility to infection

(2). Ironically, Leush et al reported an increase in

IgM and IgG in the second and third trimesters of

pregnancy in women with IDA (3).

With regard to controversial results and the

scarcity of studies focusing on pregnant women,

we aimed to enlighten the relation between iron

status and some immunological factors include

some component of complement system, IgA,

IgM, IgG subclasses of immunoglobulins and pro-

inflammatory cytokines during the third trimester

of pregnancy.

In a descriptive-analytic study participants

were recruited using convenient sampling from

the women in the third trimester of pregnancy

Corresponding author:

Nadereh Naderi, Faculty of Medicine, First of Nabovat

minicity, opposite of Workers Welfare Community,

First of Imam Hossein Blv., Bandarabbas, Iran.


referred to the labor room of gynecology and

obstetrics ward of Dr. Shariati Hospital of Bandar

Abbas, Iran. Patients with signs and symptoms of

thalassemia, infectious diseases or autoimmune

diseases were excluded.

IDA were defined with two criteria,

hemoglobin concentration of less than 10 mg/dL

(its normal range during the third trimesters of

pregnancy is 11-14mg/dL) (4) and ferritin less than

40 ng/dL. Patients were categorized into two

groups: those with iron deficiency anemia (IDA)

and those without this condition (no IDA).

Red cell indices including hemoglobin (Hb)

levels, hematocrit (HCT), mean corpuscular

volume (MCV), red blood cell distribution width

(RDW), and mean corpuscular hemoglobin

(MCH), serum iron (SI) and total iron binding

capacity (TIBC), concentration of ferritin, C3 and

C4 complements and IgA, IgM and IgG subclasses

of immunoglobulins were determined. Data was

analyzed using SPSS version 11.5 using Student t-

test, Pearson’s correlation test and Kolmogorov-

Smirnov’s test of normal distribution.

Ninety-two patients were studied. They were

aged between 15 and 42 years (mean=25.69±6.2).

According to our definition of IDA in pregnancy,

21 patients (22.8%) had IDA.

Our analysis of differences between the two

groups in regard to immunologic markers showed

that C4 levels are lower in the IDA group

(p=0.009) and the levels of C3, IgM, IgG, IgA, IL-

1, IL-6 and TNF-α were not statistically different

in the two groups .


Sobhani et al

252 Iranian Journal of Reproductive Medicine Vol. 9. No. 3. pp: 251-252, Summer 20111

We noticed that higher levels of serum iron are

correlated with higher levels of C3, C4 and IgG1.

Due to important properties of IgG1 like

complement fixation and opsonic activity, this

subclass is dominant antibody to pneumococcal

capsular polysaccharides and its deficiency is

associated with current infections (5). Taken

together noticing key roles of C3, C4 in

complement-mediated bacteriolysis, opsonization,

facilitated ingestion immune adherence (6) and

association of C3, C4 with Iron serum levels found

in this study we suggest that decreased level of

Iron increases susceptibility of pregnant women to

infections like chronic bacterial respiratory

infections and recurrent genital herpes (5).

Analyzing immunologic parameters differences

between the two groups of IDA and no IDA we

found that C4 levels are lower in the IDA group

but not the levels of C3, IgM, IgG, IgA, IL-1, IL-6

and TNF-α .Our findings about IgM, IgG, IgA are

in contradiction to scanty studies in this field. Tang

et al (2) about significantly lower level of IgG,

CD3+ and CD4+ cells, the ratio of

CD4+/CD8+cells, serum IL-2 in second trimester

of IDA pregnant woman and Leush et al (3) study

showed increase of IgM and IgG in second and

third trimesters of IDA groups.

Our findings about non-significant difference in

C3 and significant difference in C4 levels in IDA

and no IDA groups is in agreement with Galan et

al report about significantly positive correlation of

C4 IgA, IgM and Serum ferritin (7). Despite of

mounting evidence that TNF, IL-1, and IL-6

cytokines affect hemopoiesis and iron metabolism

there was no significant association between IL-1,

IL-6 and TNF-α and serum Iron, ferritin, TIBC in

our study and inflammatory cytokines were

statistically indifferent in the two IDA and no IDA

groups. To our knowledge, there are no reports on

inflammatory cytokine levels and Iron parameters

in pregnant women, but a limited number of

reports exploring this field in children and adults.

Bergman et al which analyzed the in vitro

production of IL-1beta, IL-2, IL-6, IL-10, and

TNF-α by peripheral blood mononuclear cells from

20 patients with IDA report that the secretion of

the cytokines other than IL-2 did not differ from

that of controls (8). In another study there was no

difference in serum levels of IL-6 in iron

deficiency anemia before and after iron

supplementation in children with IDA but in the

iron-deficiency group the production of IL-2 was

found to be significantly lower than that in controls

and became normal after iron supplementation (9).

Safuanova et al work about adequate therapy by

iron-containing drugs in IDA patients resulted in

decreased concentrations of IL-1, IL-6, TNF-alpha

and INF-gamma and recovering the functional

status of the immune system (10). It is possible that

discrepancy seen between our results and

mentioned reports in non-pregnant patients is a

reflection of dramatic change of immune function

in pregnancy.

Analyze of the results of our study and similar

researches leads us to the conclusion that unlike

extensive immunological changes have been

observed in children, IDA has little effect on

humoral immunity system of pregnant women, but

decrease in serum iron could predispose them to

pyogenic infection and may predict increased

susceptibility IDA pregnant women to infections.

References

1. Ekiz C, Agaoglu L, Karakas Z, Gurel N, Yalcin I. The

effect of iron deficiency anemia on the function of the

immune system. Hematol J 2005; 5:579-583.

2. Tang YM, Chen XZ, Li GR, Zhou RH, Ning H, Yan H.

Effects of iron deficiency anemia on immunity and

infectious disease in pregnant women. Wei Sheng Yan Jiu

2006, 35: 79-81.

3. Leush SS, Futornyi SM. Humoral immunity in women

with a normally proceeding pregnancy and in pregnancy

complicated by iron-deficiency anemia. Lik Sprava 1997;

4: 107-110.

4. Andrews NC. Anemia of inflammation: the cytokine-

hepcidin link. J Clin Invest 2004; 113: 1251-1253.

5. Seppanen M, Meri S, Notkola IL, Seppala IJ, Hiltunen-

Back E, Sarvas H, et al. Subtly impaired humoral

immunity predisposes to frequently recurring genital

herpes simplex virus type 2 infection and herpetic

neuralgia. J Infect Dis 2006; 194: 571-578.

6. Molina H. Complement and immunity. Rheum Dis Clin

North Am 2004; 30: 1-18.

7. Galan P, Davila M, Mekki N, Hercberg S. Iron deficiency,

inflammatory processes and humoral immunity in

children. Int J Vitam Nutr Res 1988; 58: 225-230.

8. Bergman M, Bessler H, Salman H, Siomin D, Straussberg

R, Djaldetti M. In vitro cytokine production in patients

with iron deficiency anemia. Clin Immunol Dec 2004;

113: 340-344.

9. Sipahi T, Akar N, Egin Y, Cin S. Serum interleukin-2 and

interleukin-6 levels in iron deficiency anemia. Pediatr

Hematol Oncol 1998; 15: 69-73.

10. Safuanova GSH, Nikulicheva VI, Bakirov AB.

Comprehensive evaluation of the immune system and

various cytokines in patients with iron-deficient anemia.

Klin Lab Diagn 2004; 24: 33-35.


063 -079، صفحات: 09، تاتستاى 3فصلاه طة تليذ هثل ايراى، سال ن، شوار 253

مقاله مروری

درمان هورمونی درد لگنی در ارتباط با آندومتریوز

Wu Shun Felix Wong1 M.D., Chi Eung Danforn Lim

٭2 M.B.B.S.

1 School of Women’s and Children’s Health, Faculty of Medicine, University of New South Wales,

Sydney, Australia.

2 South Western Sydney Clinical School, Faculty of Medicine, University of New South Wales, Sydney,

Australia.

South Western Sydney Clinical School, Faculty of Medicine, University of Newآدرس نويسنده مسئول:٭

South Wales, Sydney Australia. [email protected]پست الكتزونيك:

99/91/89، پذيزش: 6/6/89دريبفت مقبله:

چكيده

هی تاشذ.در ارتثاط آذهتريز يک هشکل شايع زاى است ک تا دردای لگی : مقدمه

ز در ارتثاط تا درد هی تاشذ. ذهتريآدرهاى ای رهی تخشی اثرتررسی هيساى اثر ،هطالع ذف:هدف

Cochrane Systemic Review هذاليي هجد در اطالعات ،در ايي هطالع کترل شذ تصادفیمواد و روش هب:

groups گر اجام شذ. 3در آاآاليس در هرد اطالعات هتا. جت ايي تررسی فت هطالع اتخاب ذتررسی شذ

خراکی ضذ تارداری ای( قرص3در هقاتل تسريق پرشسترى؛ Implanon( 2؛ GnRHa( پرشسترى در هقاتل 0

هيساى تثد تيش از يک تعاى پالسث پرشسترى. پاسخ ت درهاى تسيل هقياس کاش درد ترکيثی در هقاتل

تثد درد در ظر گرفت شذ.

ذهتريز هشاذ گرديذ آاشی از در راتط تا کاش درد لگیGnRHaختالف هعی داری تيي پرشسترى انتبيج:

(RR: 0.036; CI:-0.030-0.102) .Implanon يا پرشسترى طيل االثرMirena اثر کوتری ازGnRHa DMPA شاى

ی در تردرهاى هثر در هقايس تا گر پالسث ضذ تارداری ترکيثی. قرصای (RR: 0.006; CI:-0.142-0.162)ذادذ

.وچيي پرشسترى سثت ت قرص ای (RR:0.321; CI:-0.066-0.707)ذهتريز شاى دادذآکاش درد اشی از

ی هاذ عارض جاثی تيشتر ،پرشسترى در حاليک. خراکی ضذ تارداری ترکيثی در کترل ديس هير هثرتر تد

ضذ تارداری ترکيثی داشت.قرص ای خراکی لخت سثت تلک تيی

در طر يکساى پرشسترى ت GnRHa، (COCP)خراکی ضذ تارداری ترکيثی ای قرصهصرف نتيجه گيزی:

ذهتريز هثر هی تاشذ. قرص ا پرشسترى سثتا ارزاى تد جت درهاى دراز هذت آکاش درد لگی اشی از

ياز Mirena Implanonهی تاشذ. هطالعات کترل شذ طالی هذت جت تررسی اثرات GnRHaهاسة تر از

اى دذ. ذهتريز شآاست تا اثرات هصرف دراز هذت آا را در کاش دردای لگی اشی از

ضذ تارداری ترکيثی.ذهتريز، درهاى طثی، پرشسترى، قرصای خراکی آ دردای لگی اشی ازکلمبت کليدی:

963-979 ، صفحبت:99 تببستبن،3فصلنبمه طب توليد مثل ايزان، سبل نهم، شمبره


171-176، صفحات: 90، تاتستاى 3فصلاه طة تليس هثل ايزاى، سال ن، شوار 254

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-برای خانمهای با پاسخ معمولی ICSIدرسیکلهای GnRHمقایسه آگونیست و آناگونیست

کارآزمایی بالینی در ایران

، فدیه حق اللهی.M.Sc، هزین ببقزی .M.D، هزین سهزابی .M.D، بتول رشیدی .M.D اکزم قنبعی نظبم آببدی ،.M.Dانسیه طبهزی نژاد

M.Sc. نکو الهبم عظیوی ،M.D.٭، هینب جعفزآببدی M.D..

هزکش تحقيقات تساشت تارری، زاشکس پششکی زاشگا تزاى، تزاى، ايزاى.

هزکش تحقيقات تساشت تارری، هجتوع تيوارستای اهام، تلار کشارس، تزاى، ايزاى.: آدرس نویسنده هسئول ٭

[email protected]پست الکتزونیك:

98/91/98پذیزش: ،7/5/98دریبفت هقبله:

چکیده

لف تيواراى تايج هتفاتی اس ذز شاى زاز است. تزر سيز گزای هر IVFزر GnRHاستفاز اس آالگای هقدهه:

تا آتاگيست آى زر سيکلای تزای تيواراى تا پاسد هعولی هقايسه GnRHزر ايي هطالع تاثيز استفاز اس آگيست هدف:

شس است.

اس تيي ذاوای تا پاسد هعولی اتراب شهس تطهر ICSI هرز کاسيس رز ت سيکل300زرايي کارآسهايی :هب واد و روشه

يی، کلييکهی اقزار گزفتس. زذای حاهلگی شيوي GnRHفز( آتاگيست 150) GnRHتصازفی زر ز گز آگيست

زرحال پيشزفت زر ز گز هقايس شس.

رس تهز 2/8±6/1 6/9±6/1هياگيي طل هست تحزيک تروک گذاری زر گهز آگيسهت آتاگيسهت ته تزتيهة :نتبیج

(001/0p-value= هياگيي تعساز تروک .)MII 7/7± 0/4تسست آههس زر ز گهز آگيسهت آتاگيسهت ته تزتيهة

(. زر هرز هياگيي تعساز آهپلای گازتزپيي استفاز شس، فليکلا، تروکا هجوع جيها =p 03/0تز ) 3/4±9/6

هيشاى سقط زر ز گز اذهتف هعهی زار هشهاس شهس. هيهشاى حهاهلگی OHSSجيای تا کيفيت هطلب يش شيع

زر 3/35%ز. هيشاى حاهلگی کلييکی زر گهز آگيسهت ت 4/39% زر گز آتاگيست 3/35% شيويايی زر گز آگيست

ههرز زر گهز آتاگيسهت زر (3/31)%45زر گهز آگيسهت زر زر حهال پيشهزفت تهز. حهاهلگی 34%گز آتاگيسهت

هرز اتفاق افتاز. زر يچکسام اس زخ ای حاهلگی اذتف هعی زار تيي ز گز جز ساشت. (%3/29)44

يش ت زر ايي هطالع شاى زاز شس ک پزتکل آتاگيست زر تيواراى ايزای رشی آساى ايوي است تيوارگیزی: نتیجه

آسای قازر ت اجام آى هيثاشس. تايج حاصل اس استفاز اس ايي پزتکل تا پزتکل استاسارز آگيست هشاتت زارز لی طل

يست کتاتز هيثاشس.هست تحزيک روک گذاری زر رش آتاگ

Normoresponderآتاگيست، GnRHآگيست، IVF، GnRH :کلوبت کلیدی

979-971 ، صفحبت:89 تببستبى ،3تولید هثل ایزاى، سبل نهن، شوبره فصلنبهه طب


677 -686، صفحات: 99،تابعتاى 3فصلاه طب تليذ هثل ايراى، ظال ن، شوار 255

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های موشدر دمی اپیدیدیمبر روی اسپرم های Ocimum sanctumعصاره بنسنی برگهای ثرا

بسرگ آزمایشگاهی

Mukhtar Ahmed1,3

Ph.D., R. Nazeer Ahamed1Ph.D., Ravindranath H. Aladakatti1,2٭

Ph.D., Mukhtar Ahmed

G. Ghodesawar1,4

Ph.D.

حش، داشگا کاراتاک، داراد، ذ. دپارتواى هطالعات حيات. 6

هرکس هطالعات حياات، اعتيت علم ذ، باگالر، ذ.. 2

ض، عربعتاى ظعدی.کالج علم، داشگا هلک ظعد، رياهرکس تحقيقاتی، احذ هيکرظکپ الکتری، . 3

دپارتواى حيات حش، اجوي ذ، کالج علم تجارت، بيجاپر، ذ. . 4

هرکس هطالعات حياات، اعتيت علم ذ، باگالر، ذ.5 آدرس نويسنده مسئول٭

[email protected] پست الكتزونيك5

7/8/34پذيزش5 ،72/2/33دريبفت مقبله5

چكيده

ييرات ظاختوای در ظلل باعث تغ Ocimum sanctum (O. sanctum)ک عصار بسی برگای اذ هطالعات اخير شاى داد مقدمه5

بارری هشای ر صاد آلبي هی شد. ظيوی فرضدر اپی تليم ، ببدی ثايcauda epididymisليايی در ای اپی ت

هرفلشی دهی اپيذيذين، یاپاراهترای اظپرم بر ری O. sanctumعصار بسی برگای در ايي بررظی، هطالع اثرذف ها هدف5

در رتای آلبي بد. آى ا ر ظاختوایل ای ظطحی تارگا

گر هرد عصار ارد هطالع گرديذذ.عذد( 69گر کترل هرد )ر کذام 2 ب پط از تقعين ر يعتار عذد رت 29 5هب مواد و روش

حياى از ر گر بعاى تعت بارری 5رز پشت ظر ن دريافت کردذ. 48( برای mg/kg/day259) O. sanctumبسی برگای

از شذذ کشت قطع خاع گردیعذد( بظيل 5عذد( هرد ) 5ظاعت بعذ از آخريي دز، بقي حياات کترل ) 24اظتفاد شذذ.

( SEM)اظکيگ هيکرظکپ الکتری اظتفاد شذ ظپط اظپرم ا تظط جت آاليس اظپرمآا احي دهی اپيذيذينپالظوای

هطالع شذذ. ( TEM)هيکرظکپ الکتری تراسيشي

ی آهاه افسايش دذ کاش هعی دار در تعذاد، حرکت ظرعت اظپرم ا آاليس اظپرم گر هرد در هقايع با گر شاذ شاى نتبيج5

کرزم آدذ تغييرات هرفلشيک در غشاء پالظوا غشاء در حياات گر هرد شاى SEM TEM. بد (p≤0.001)اظپرهی ای

ى در ظيتپالظن در احي هيای وچيي دم غيرطبيعی تحليل بن ريختگی هيتکذری الي اظپرم ا، تشکيل قطرات شبي بال

پرها بد. ای هيتکذريايی اظ

اثر عاقب درباشذ ک احتواال دم اپی ديذينتغييرات کلی در ای از اثرات هشاذ شذ در ايي هطالع هوکي اظت تيج نتيجه گيزی5

اظت. O. sanctumکاش آذشى اشی از اثرات ضذ آذرشی برگای

.لکتری، رتهيکرظکپ ا، اپيذيذين، اظپرم، بارری، O. sanctum 5کلمبت کليدی

622-5631 ، صفحبت49 ، تببستبن8ه فصلنبمه طب توليد مثل ايزان، سبل نهم، شمبر



121-132، صفحات4 30، تار 3ايزاى، سال ن، ضوار فصلاه طة تليذ هثل 256

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سبند گی متعاقب اعما ل جراحیچاز ایجاد ایکودکسترین

یري می کندگوش ها جلو گدرخر هاي رحمیلوله

2نبهید بهزامی ،.1M.D*بهنبس خبنی رببطیM.D. ، 2فزدوس محزابیبن M.D. هز مش نبدری ،:

M.D..

، ايزاى.صفاى، ادکتز تطتیتيوارستاى ضيذ داطگا علم پشضکی اصفاى، ،گز ساى هاهايی .1

اصفاى، ايزاى. پشضکی اصفاى، ، داطگا علمتيوارستاى الشزاء، گز ساى هاهايی .2

، ايزاى. اصفاى ،تيوارستاى سپااى .3

اى، ايزاى. ، اصفتيوارستاى ضيذ دکتز تطتی ،داطگا علم پشضکی اصفاى، پل فلشی ،گز ساى هاهايی آدرس نويسنده مسئول4*

[email protected]پست الكتزونیك4

71/77/23پذيزش4 ،82/8/23دريبفت مقبله4

چكیده

اس هاد هختلفی اعن اس خاهذ يا هايع تزای خذا کزدى سطح لذاچسثذ گی ا ضايع تزيي عارض اعوال خزاحی هی تاضذ مقدمه4

ول خزاحی استفاد ضذ است . يکی اس ايي هاد ک تصر ضذ است هی تاذ سج ت هظر خلگيزی اس ايداد چسثذگی در حيي ع

هی تاضذ. %4 ايکدکستزيي ،اس ايداد چسثذگی خلگيزی وايذ

در لل ای رحوی % تا آب هقطز هايع آهيتيک در کاص چسثذگی اضی اس اعوال خزاحی4هقايس اثز ايکدکستزيي 4 هدف

خزگش.

خزگش سفيذ اس ژاد يسلذ تطر راذم ت س گز د تايی تقسين ضذذ. لل ای 30در ايي هطالع کلييکی ب4مواد و روش ه

قثل اس سهاى خاری ضذى خى خزاضاذتزش تاس کزدى ضکن تسيل گاس استزيل تا ايداد رحوی خزگضا پس اس تيش ضذى،

هايع آهيتيک (B، ايکدکستزيي )هحلل ( Aهحللن هحل آسية ديذ تسيل يکی اسس هحلل آب هقطز )تست ضذى ضک

گز الپاراتهی هدذد ادام ضذ. ضذ، سپس فت رس تعذ ت هظر هقايس هيشاى چسثذگی تيي س ( ضستط دادC)هحلل

( هايع 4/10±00/0( در هقايس تا آب هقطز )1/2±10/0ي ايکدکستزيي ) اختالف قاتل تخی در هيشاى ايداد چسثذگی تي4 نتبيج4

در هاری هعی داری تيي هيشاى چسثذگی ت دثال استفاد اس هايع آهيتيک . اها اختالف آ( خد داضت1/2±24/0آهيتيک )

(.4/10±00/0در هقاتل 1/2±24/0)هقايس تا آب هقطزخد ذاضت

% در کاص چسثذگی اعوال خزاحی ساى در خزگش ا هثزتز اس 4ح طاى هی دذ استفاد اس ايکدکستزيي تاي نتیجه گیزی4

ايع آهيتيک آب هقطز هی تاضذ. ه

. تيک اسای، ايداد چسثذگی، خزگش، هايع آهيايکدکستزيي کلمبت کلیدی4 721-738 ، صفحبت394،بهبر 9تولید مثل ايزان، سبل نهم، شمبره فصلنبمه طب


257 103-108، صفحبت: 00، ببر 3فصلبه طب تليد هثل ايراى، سبل ن، ضوبر

مقبله اصیل

اووسیت ارتببط بین سطح کلسترول استر ترانسفر پروتئین مبیع فولیکولی و نسبت ببروری

IVF/ICSIدر سیکل هبی

، .M.Sc 4، يحیید هیىبسی .Ph.D 2، مسعد دارابیی .M.D 4، لعیب فزسدی.Ph.D 3، علی رحیمی پر.M.Sc 2ي1امیز مدی ساد

.Ph.D . 2٭، محمد وری.M.Sc 2، سزا گل محمدی.M.Sc 5خا، امیز مىصر يطه .B.Sc 2مقصد هبکز

ايراى. ،تبريس ، داطگب ػلم پسضکی تبريس،هرکس تحقيقبت بتکلشی داريی .1

ايراى. تبريس، سضکی، داطگب ػلم پسضکی تبريس، گر بيضيوی آزهبيطگب بی ببليی، آزهبيطگب کرهبتگرافی، داطکد پ .2

راى، ايراى.ت، داطگب ػلم پسضکی ضيد بطتی ،آزهبيطگبی گر ػلم .3

ايراى. تبريس، داطگب ػلم پسضکی تبريس، ،هرکس آهزضی درهبی السرا، هرکس تحقيقبت بداضت ببرری زبى .4

ايراى.تبريس، ،داطگب ػلم پسضکی تبريس ،هرکس تحقيقبت داريی .5

آزهبيطگب بی ببليی، آزهبيطگب کرهبتگرافی، داطکد پسضکی، داطگب ػلم پسضکی تبريس، گر بيضيویآدرس ويسىد مسئل: *

تبريس، ايراى.

[email protected]پست الكتزيویك:

9/9/92، پذيزش: 22/2/92دريبفت مقبل:

چكید

هوکي است در هيساى هفقيت در تکبهل سلل اسيت جيي ايفب هی کد ک : هبيغ فليکلی قص هویمقدم

IVF/ICSI .دخيل ببضد

ايي هطبلؼ بررسی ارتببط بيي هيساى کلسترل استر تراسفر پرتئيي هبيغ فليکلی هيساى هفقيت از دف دف:

IVF/ICSI .بد

90بيوبر هراجؼ کد ب بيوبرستبى السرای تبريزس بدسزت آهزد. 100از و بی هبيغ فليکلی ماد ي ريش ب:

، HDL-Cقزرار گرفتزد. هيزساى پبراهترزبی ICSIفر تحت درهبى بزب IVF 21فر از ايي بيوبراى تحت درهبى بب

Apo-AI CETP ضدد. هبيغ فليکلی ب ترتيب بب رش بی آسيوی ، تربيديوتری اليسا اداز گيری

در بيوبرای ک تؼداد اسيت بی CETP ، طبى داد ک هقبدير CETPآبليس زيرگر ب بب هقبدير هتفبت وتبيج:

(. وچيي در افراد بب سبت ببرری اسيت کوتر از >05/0pببلؾ کوتری دارد ب طر هؼی دار پبييي تر هی ببضد )

ب طر قببل هالحظ ای کوتر CETPهی ببضد، هقبدير ٪ 90ر آب بيص از در هقبيس بب افرادی ک ايي سبت د 50٪

%(.p ،18=05/0بد )

بر اسبس تبيج بدست آهد از ايي هطبلؼ، اگرچ ارتببطی بيي پبراهتربی هرد بررسی قع حبهلگی وتیج گیزی:

يت بی ببرر ضد ارتببط هثبت هؼی داری هبيغ فليکلی بب هيساى بلؽ درصد اس CETPيبفت طد، لی هقبدير

را طبى داد.

. A-I ،CETP، آپليپپرتئيي IVF/ICSIبببرری، کلمبت کلیدی: 123-12 9، صفحبت:29، تببستبن 3تلید مثل ايزان، سبل وم، همبر فصلىبم طب


199 -202، صفحات: 90، تابستاى 3فصلاه طب تليذ هثل ايراى، سال ن، ضوار 258

مقاله اصیل

مقایسه ارزش تشخیصی سالیه سو هیستريگرافی با هیستريسکپی در شىاسایی اختالالت آوذيمتر

در زوان با خوریسی های غیرطبیعی رحمی

1محمدعلی کزیم ساده

M.D.، 1٭راضیه دهقبنی فیزوسآببدیM.D.، 2فزسانه گوهزساد. M.D.

، يسد، ايراى.علم پسضکي ضيذ صذقيابارری، داطگا درهاي يهرکس تحقيقات .1

، يسد، ايراى.زايواى، بيوارستاى ضيذ صذقيبخص زاى .2

، يسد، ايراى.خياباى بعلي، صفايي، س تحقيقاتي درهاي ابارریهرکآدرس نویسنده مسئول:٭

[email protected]پست الكتزونیك:

11/7/98پذیزش: ،24/3/98دریبفت مقبله:

چكیده

خريسی غير طبيعي رحوي علت ضايع اختالالت رحوي در بيي زاى قبل بعذ از يائسگي هي باضذ.: مقدمه

ارزيابي هقايس ارزش تطخيصي ساليي سيسترگرافي يسترسکپي در ضاسايي خريسی ای غير طبيعي رحوي. :هدف

يسترگرافي پس ر و و ا ابتذا ساليي سرحوي ارد ايي هطالع ضذذ.دبيوار با خريسی غيرطبيعي 65 هب:مواد و روش

از آى يسترسکپي اجام ضذ. حساسيت، اختصاصي بدى، ارزش تطخيصي هثبت هفي صحت ر د رش هحاسب گرديذ.

15بيوار، در يسترسکپي آذهتر در 19در ب عاى ضايعتريي اختالل در ساليي س يسترگرافي يپرپالزی آذهتر نتبیج:

% برای پليپ يپرپالزی آذهتر هيم زير هخاطي ب ترتيب گسارش گرديذ. در 4/71% 3/73بيوار تطخيص داد ضذ. حساسيت

% گسارش گرديذ.7/90% هيم زير هخاطي 3/82%، يپرپالزی 96حاليک اختصاصي بدى برای پليپ

ر هقايس با يسترسکپي، ساليي س يسترگرافي حساسيت اختصاصي بدى بااليي را در ضاسايي هيم زير د نتیجه گیزی:

هخاطي سبت ب پليپ آذهتر يپرپالزی طاى داد.

کپي.سخريسی غير طبيعي رحوي، اختالالت رحوي، سيسترگرافي، يسترکلمبت کلیدی: 188-292 ، صفحبت:89 ، تببستبن3تولید مثل ایزان، سبل نهم، شمبرة فصلنبمه طب


259 203-208، صفحات: 90، تاتستاى 3فصلاه طة تليذ هثل ايراى، سال ن، شوار

مقاله اصیل

یت پايین با كیف بدست آمده از امبريو هاي حاملگي موفق به دنبال انتقال بالستوسیت هاي منجمد شده

در روز سوم

Xiao-jian Zhang M.Sc.*, Ye-zhou Yang Ph.D., Li-hua Min B.N.., QunLv B.Sc.Med., Pin Bai B.N., Xiao-jie Li

B.S.M.T., Mei-xu Liao M.Sc.

Department of Assisted Reproductive Medical Center, Sichuan Academy of Medical Sciences and Sichuan Provincial

People’s Hospital, Sichuan, China.

Department of Assisted Reproductive Medical Center, Sichuan Academy of Medical Sciences: آدرس نويسنده مسئول*

and Sichuan Provincial People’s Hospital, Sichuan, China.

[email protected]پست الكتزونيك:

23/41/98پذيزش: ،44/4/98دريبفت مقبله:

چكيده

ياي تا كيفيت پاييي هوكي تر اساس خصصيات هرفلشي اجام هي گيرد. اها تعضي از اهثر ،اتخاب اهثري جت اتقال: مقدمه

تا هرحل تالستسيت كشت داد شذ سپس جت ال گسيي اتخاب گردد. است

تذثال اتقال تالستسيتاي تذست آهذ از اهثرياي تا كيفيت پاييي. كلييكيتررسي تايج حاهلگي :هدف

تيوار تحت 15از ( %8/24)تالستسيت 27 تعذاد كشت داد 6 5ت پاييي تا رز اهثري تا كيفي 109تعذاد : مواد و روش هب

IVF سپس تالستسيت ا جت اتقال گرم شذذ. ام تالستسيت ا سريعا هجوذجوع آري شذ. تو

فر از تيواراى حاهل 5تيوار اتقال يافتذ. 15ت ك ذهاذزذ %( تعذ از گرم شذى 6/92تالستسيت هجوذ شذ ) 25تعذاد نتبيج:

شذذ.

يت پاييي در رز سم ت دست كيف هجوذ شذ ك از اهثرياي تا 6 5تايج ها شاى داد ك تالستسيت اي رز نتيجه گيزی:

تارري شذ. ش شاس حاهلگي در سيكل اي كوكهي تاذ تاعث افساي ،اذ آهذ

اهثري تا كيفيت پاييي، اجواد، اتقال تالستسيت، حاهلگي. کلمبت کليدی:

213-219: ، صفحبت81 ، تببستبن3ه توليد مثل ايزان، سبل نهم، شمبر فصلنبمه طب



209-216 ، صفحات:90، تابستاى 3فصلاه طب تليذ هثل ايزاى، سال ن، شوار 260

مقاله اصیل

بلوغ ورفولوشی و میسان بلوغ تخمک های نارس انسانی بعذ از انجام انجماد ارزیابی مرفومتری، م

آزمایشگاهی

1سعید وظزی

M.Sc.1*، محمدعلی خلیلیPh.D.2، فزيسان اسمبعیل سادM.Sc.3، مدی محسه ساد

M.Sc. .

صذقی، يشد، ايزاى. پششکی شيذعلم ، داشگا کش تحقيقاتی درهای ابارریهز1

داشگا آساد اسالهی، احذ هزدشت، گز علم داهی، هزدشت، ايزاى. 2

علم پششکی شيزاس، شيزاس، ايزاى.داشگا 3

، داشگا پششکی شيذ صذقی، يشد، ايزاى.ش تحقيقاتی درهای ابارریهزکآدرس ويسىد مسئل:٭

khalili [email protected]پست الكتزيویك:

21/8/28پذيزش: ،22/5/28دريبفت مقبل:

چكید

. (1% هتافاس4% صرهيال سيکل، 11) % تخوک ای جوع آری شذ ابالػ ستذ15در سيکل ای تکلصی کوک بارری : مقدم

جد دارد. IVM اهکاى اجواد ايي تخوک ا جت استفاد بيشتز در بزاه

fresh IVM هزفلصی تخوک ای ارس اسای در د گز هيشاى بلغ، پاراهتزای هزفهتزیذف اس ايي بزرسی تعييي :دف

(fIVM) vitrified-IVM (vIVM) .بد

بزای آا اجام شذ بد، شزکت کزدذ. اس ايي ARTتحزيک کتزل شذ تخوذای جت کسى 93در ايي بزرسی ب:ماد ي ريش

( ک ب طر هستقين در N;101( تخوک ای ابالػ )1ب د گز تقسين شذذ: ) تخوک ابالػ ب دست آهذ ک 203ساى تعذاد

، سپس در آسهايشگا بالػ شذذ. توام تخوک ا ( ک در ابتذا هجوذN;102( تخوک ای ابالػ )2شذذ ) IVMهحيط آسهايشگا

ساعت اس کشت، سبت بلغ 48 هايع فليکلی اسای شذذ. بعذ اس LH FSHهکول شذ با HAMʼs F10در IVMهتحول

اجام گزفت. Cornusتخوک ا بعال ارسيابی هزفهتزی هزفلصی سيتپالسوی با استفاد اس بزاه

(. در ارسيابی ای p<001/0، %4/59 در هقايس %4/40کاش يافت ) fIVMدر هقايس با vIVMدر سبت بلغ تخوک ا وتبيج:

±8/6 تزتيب گزديذ )ب هشاذ vIVM fIVM بيي (µm) هرفهتزيک بعذی تفات هعی داری در هياگيي قطز تخوک

(. ديگز پاراهتزا اس جول هحيط هساحت تخن بعال حجن تخوک اپالسن در د گز هشاب بدذ. 9/9±07/154 3/156

ديذ شذ. vIVMوچيي اجاری ای هزفلصی بيشتزی ظيز اکئل تخوک تيز در تخوک ای

قابل تجی در د گز هشاذ اختالف آهاری تز بدذ. يچ هفق vIVMاس ظز هيشاى بلغ سبت ب fIVMگز وتیج گیزی:

ب عاى يک فاکتر پيشگيی کذ در سزاجام بلغ تخوک در تاذ هی شد ک پاراهتزای هزفهتزی لذا پيش بيی ذ. گزدي

ذ.بکار بزد ش IVMبزاه

. زفهتزیبلغ آسهايشگای، تخوک، اجواد شيش ای، بلغ, هزفلصی، ه کلمبت کلیدی:

298-212 ، صفحبت:89 ، تببستبن3تلید مثل ايزان، سبل وم، شمبر فصلىبم طب


261 212-222 ، صفحات:09، تاتستاى 3فصلناهه طة توليد هثل ايراى، سال نهن، شواره

مقاله اصیل

Hymenocardia acida stem bark آبي الكلي عصاره ضدباروري اثرات

هاي مادهدر رت

Abu Adakole Hyacinth

1* Ph.D., UchenduChukwuka Nwocha

2 Ph.D.

1 Department of Veterinary Physiology, Pharmacology and Biochemistry, College of Veterinary Medicine, University

of Agriculture, P.M.B, 2373, Makurdi, Nigeria.

2 Department of Veterinary Physiology and Pharmacology, Faculty of Veterinary Medicine, University of Nigeria,

Nsukka, Nigeria.

Department of Veterinary Physiology, Pharmacology and Biochemistry, College of Veterinaryآدرس نويسنده مسئول:*

Medicine, University of Agriculture, P.M.B, 2373, Makurdi, Nigeria.



چكيده

يك گياه است كه ته طور سنتي در طة گياهي آفريقا استفاده هي شود و فوايد درهاني Hymenocardiaacida (HA): مقدمه

حاهله اطالعات زيادي در دسترس نوي تاشد. ره اثرات احتوالي آى تر روي زناىزيادي دارد. اها درتا

در رتهاي هاده نژاد ويستار هي تاشد. HAهدف هطالعه حاضر تررسي اثرات ضد تاروري عصاره الكلي آتي :هدف

هيلي گرم ته ازاي هر كيلوگرم 499و 299، 199تا دوزهاي HAسه گروه رت تحت رژين خوراكي عصاره الكلي آتي مواد و روش هب:

جسن تعدادو شده الپاراتوهيروز قرار گرفتند. گروه كنترل آب هقطر دريافت كردند. در روز تيستن حاهلگي، حيوانات 10وزى تراي

اي باريري بعد از وسديكي ب يزن جىيه ا ي جفت اوداز گيري شد. اي زود ي مچىيه اودكسجىيهحاملگي، هاي زرد

اي ي جىيه (>05/0pحاملگي ) جسن هاي زردنشاى دهنده كاهش تعداد حاهلگي 10تا اولاز روز HAعصاره تا درهاى خوراكي نتبيج:

2گسيىي در گري اي (. فعاليت ضد الو>05/0pكىترل كمتر بد )گري در مقايس با HA ا در حياوات تحت رشيم عصارزود بد. يزن جىيه % بد. 60% ي 60% ي 40% بد. در حاليك فعاليت ضد بارداري در ايه گري ا ويس ب ترتيب 7/51% ي 3/48 %،4/41 ، ب ترتيبتحت درمان 4تا

هاي هاده هي تواند تاعث اثرات هنفي تر روي عولكرد تاروري در رت HAنتايج هطالعه نشاى داد كه عصاره الكلي آتي نتيجه گيزی:

آلثينو گردد.

تليد مثل. ،Hymenocardia acidaرت آلثينو، ضد تاروري، کلمبت کليدی:

352-333 ، صفحبت:81 ، تببستبن2توليد مثل ايزان، سبل نهم، شمبره فصلنبمه طب


223-222 ، صفحبت:09، تببستبى 3فصلبه طب تليذ هثل ايراى، سبل ن، شوبر 262

مقبله اصیل

سطح ببالی همسیستئیه يمقبيمت به اوسلیه دربیمبران مبتال به سىذرم تخمذان پلی

کیستیک

. .M.D 2پیب جادیبى، .M.D 2بزام سلوبیبى ،.M.D 1٭فبطو داری تب ،.M.D 1تبزیشیهقدهی ، سزیي .M.D 1طیب وتی

.ایزاى، تزاى، شگب علم پششکی تزاىدا، بیوبرستبى هیزسا کچک خبى، سبى هبهبئی گز 1

پششکی، داشگب علم پششکی تزاى، تزاى، ایزاى. داشکد 2

.ایزاى، تزاى، داشگب علم پششکی تزاى، بیوبرستبى هیزسا کچک خبى، سبى هبهبئی گز آدرس یسد هسئل:٭

[email protected]پست الکتزیک:

21/10/98، پذیزش: 11/3/98دریبفت هقبل:

چکید

از بيوبری بی رايج دراى ببرری در هيبى زبى است هجب (PCOS)سيستيک بی پلی م تخوذاىسذر: هقده

م يپرهسيستئيوی هی ببشذ.د. از جول ديگر هشکالت ايي سذرهقبهت ب اسليي در هبتاليبى هی ش

ليي هی ارتببط آى بب هقبهت ب اس PCOSبيوبراى سی برز يپرهسيستئيوی درايي پژش ب برر دف:

پردازد.

بيوبر 64گر در يک کلييک داشگبی بببرری صرت گرفت. ی هقطعی آيذايي هطبلع هاد رش ب:

PCOS بی طبيعی قرار گرفتذ. سطح گلکز بشتب، اسليي فر افراد گر کترل بب تخوذاى 59در هقبيس بب

ی افراد هرد هطبلع ی خى رز سم سيکل قبعذگی و ر ود LH FSHبی بشتب، هسيستئيي، رهى

HOMA-IRبی سبت گلکز ب اسليي بشتب وچيي گيری شذ. هقبهت ب اسليي از طريق شبخصاذاز

بررسی شذ.

ب د PCOSشبى داد. گر هبتاليبى ب را ری اسطح پالسوبيی هسيستئيي در هيبى د گر تفبت هعبدتبیج:

گر هقبم غيرهقبم ب اسليي تقسين شذذ. گر هقبم ب اسليي ب صرت هعبداری سطح هسيستئيي،

(.p =0.02, <0.001, <0.001اسليي گلکز بشتبی ببالتری داشتذ )ب ترتيب

ذى تج ب هقبهت ب اسليي سطح پالسوبيی هسيستئيي ب PCOSبر ايي اسبس هبتاليبى ب تیج گیزی:

بی هقبهت، هقبهت ب اسليي يس در آب شبيع است. بر اسبس تبيج ايي ببالتری دارذ. وچيي بر اسبس شبخص

جد دارد. PCOSهعبداری هيبى يپرهسيستئيوی هقبهت ب اسليي در هبتاليبى ب هطبلع رابط

.، وسيستئيي، هقبهت ب اسليي، بببرری PCOS کلوبت کلیدی: 223-229 : ، صفحبت80، تببستبى 3تلید هثل ایزاى، سبل ن، شوبر فصلبه طب


263 220-232 ، صفحات:00، تابستاى 3فصلاه طب تليد هثل ايراى، سال ن، ضوار

مقاله اصیل

هاي شیردهدر رت واناديومبر علیه اثرات كمي ايجاد شده توسط سلنیومو تیروندرمان همزمان

SadhanaShrivastava* Ph.D., Deepmala Joshi Ph.D,. Monika Bhadauria Ph.D., SangeetaShuklaD.Sc., Ramesh

Mathur Ph.D.

UNESCO Satellite center, Trace Element Research and Reproductive Biology and Toxicology Laboratory, School of

Studies in Zoology, Jiwaji University, Gwalior (MP), 474 011, India.

UNESCO Satellite center of Trace Element Research and Reproductive Biology and Toxicologyآدرس نويسنده مسئول:*

Laboratory, School of Studies in Zoology, Jiwaji University, Gwalior (MP), 474 011, India.


59/51/98پذيزش: ،8/98/:6دريبفت مقبله:

چكيده

داضت گسارش ضد د كد هحيطي صعتي هن هي باضد. ايي هاد اثرات كوي بر ري تليد هثل ااديم يك هاد آل: مقدمه

رد هي ضد. سد جفتيك از

اي ضيرد ت تركيب آى با سليم بر علي اثرات سوي ااديم در ر تيرىهطالع حاضر جت بررسي اثرات درهاي :هدف

ر اجام ضد. اضيرخ

رز از رز صفر بعد از زايواى قرار گرفتد. از رز 20ااديم خراكي براي mg/kg/day 5/7ا تحت تأثير دز رت مواد و روش هب:

داد ضد. (.mg/kg/day, p.o 0.5)سليم ( .mg/kg/day, i.p 606)تيرى ا بعد از زايواى ب رت 25تا 21

. وچيي افسايص (p>10/1ضد. ) سرم فسفاتاز هيازا آلكالييتجيس ااديم باعث كاص سطح قد خى افسايص تراس آنتبيج:

اي اسيد فسفاتاز در كبد رت، افسايص فعاليت آسين اي ضيرد رت ير كليااي ضيرخرت يذخير گليكشى در كبد كلي

فسفاتاز ي تر ، فعاليت آلكاليي فسفاتاز آدزييضد. در حاليك برعكس ايي هرد ار ديداي ضيرخرت يير كلاضيرد ضيرخ

-ك گلتاسيى در كبد رتدر حالي ، تحريك گرديدپراكسيداسيى ليپيد .(>01/0pب طر اضحي در توام گر ا هار ضد بد )

ب Tironدرهاى با يستپاتلشيك ضد.(. ااديم وچيي باعث حساسيت >01/0pر كاص يافت بد )ااي ضيرد ضيرخ

با سليم باعث كاص ضايعات اضي از ااديم در رتاي ضيرخر ضيرد ضد. Tironتايي وچيي

اضي از ااديم در رتاي وي سب تايي بر علي اثرات Tironب ورا سليم داراي اثرات بيطتري از Tironنتيجه گيزی:

ضيرد ضيرخار است.

. تيرى، Teratogenecityااديم، سليم، ضيرخر، ضيرد، کلمبت کليدی:



264 230-242، صفحبت: 00 تببستبى،3 فصلبه طب تليذ هثل ايزاى، سبل ن، شوبر

مقاله اصیل

IVF-ETدر بیماران تحت درمان با میزان الوه گزیىیبر خوراكی بررسی اثر ریتودریه

..M.D احمدیدکتز مزیم ، .M.D مزضیه سنوئی فزیمبنیدکتز ، .M.D دکتز صغزا ربیعی

، ايزاى.، وذاىداشگب علم پششکی وذاى ،بيوبرستبى فبطوي ،هزکش تحقيقبتی بببرری

ى، ايزاى.، وذاداشگب علم پششکی وذاى ،بيوبرستبى فبطوي ،هزکش تحقيقبتی بببررینویسنده مسئول: آدرس

[email protected] پست الكتزونیك:


چكیده

ذببل دستکبری بی اجبم شذ بزای اتقبل يکی اس تئريبی هطزح بزای دفع جيي عذم ال گشيی، اقببضبت رحوی بمقدمه:

استفبد اس داربی هبر کذ اقببض رحوی، ب عاى عبهلی بزای افشايش احتوبل ال گشيی هطزح است.لذا .جيي هی ببشذ

در ايي تحقيق اثز ريتدريي ب عاى هبر کذ اقببضبت رحوی هرد بزرسی قزار گزفت. : هدف

شبذ(، پزتکل تحزيک 53هرد 53اجبم شذ ) IVF+ET بذيذک بيوبر 106ری کبرآسهبيی کتزل شذ ايي هب:مواد و روش

، پزصستزى، ASA، عال بز داربی هعول ) ETدر گز هرد بعذ اس ، بزای و يکسبى بد ETتخوک گذاری پبکچز

آسهبيش ETرس پس اس 12. تجيش شذبزای يک فت ريتدريي خراکی دببر در رس هيلی گزم 200فليک اسيذ، اريتزهبيسيي(

βHCG (هيشاى ال گشيیبزای بيوبراى اجبم هيشاى حبهلگی.) بزرسی شذ. در ز گز

تفبت اس ظز آهبری ايي % بد15 در گز شبذ %2/13 هيشاى ال گشيی هفقيت آهيش در گز دريبفت کذ ريتدريي نتبیج:

. (p>0.05) هعی دار بد

هطبلع حبضز شبى داد اقببضبت رحن وی تاذ در هوبعت اس ال گشيی تبثيزی داشت ببشذ. نتیجه گیزی:

. IVFريتدريي ، کبرآسهبيی ببليی، ال گشيی ، کلمبت کلیدی:

739-747، صفحبت: 90 تببستبن ،3تولید مثل ایزان، سبل نهم، شمبره فصلنبمه طب


423-426، صفحات: 09، تابستاى 3فصلاه طب تليذ هثل ايراى، سال ن، ضوار 265

گزارش مورد

چگونه بايد با دژنراسيون كيستيك دردناك ميوما در حين حاملگي برخورد كرد؟ گزارش مورد و

بررسي منابع

Tae-Hee Kim M.D., Hae-Hyeog Lee M.D., Ph.D*.

Department of Obstetrics and Gynecology, College of Medicine, Soonchunhyang University, Bucheon, 420-767,

Republic of Korea.

,Department of Obstetrics and Gynecology, College of Medicine, Soonchunhyang Universityآدرس نويسنده مسئول: *

Bucheon, 420-767, Republic of Korea.

[email protected] ; [email protected] پست الكتزونيك:


چكيده

هيهااي رحوي از تد اي ضايع لگي در حيي حاهلگي ستذ. درد رضذ سريع ايي هيهاا از ضايع تريي عارض :مقدمه

ستيل درداك هيها در حيي حاهلگي تضيح داد هي ضد. حاهلگي هي باضذ. در ايي گسارش برخرد با دشراسيى مي

ها ايي هرد را در حالي بررسي مردين م دشراسيى بد.يل تد لگي داراي سال با حاهلگي ضنن ال 42يل زى : مورد

راسيى درد اضي از دشخيص داد. بابرايي تطخيص بذخيوي تطميستيل هيهاي رحوي را وي تاى ب آساي از پيچص تخوذاى يا

ذ. هنتهي اجام ضسپراسيى ماهل از ميست هيهاي تخوذاي بذى هيص تخوذاى يا بذخيوي ضرري بد. آهيها از پيچ

سپراسيى ميست هيهاي رحوي را در حيي حاهلگي ب ورا بررسي هقاالت هتطر ضذ ها يل هرد هفقيت آهيس از آ نتيجه گيزی:

را گسارش مردين. 8011ل از ال شاي سا 49ب هذت

ليهيهاتز، فيبرئيذ اي رحوي، حاهلگي.: کليدی کلمبت





266 042 -059، صفحات: 09، تابستاى 3 طب تليذ هثل ايراى، سال ن، شوار فصلاه

گزارش مورد

آبسه لوله ای تخمذانی در یک خانم مجرد

. .M.D 3، دکتز گیتی ایزوانلو.M.D 2، دکتزایزج حزیزچی.M.D 1*گنجویی دکتز طبهزه اشزف

.بيوارستاى افضلی پر، داشگا علم پسشکی کرهاى، کرهاى ، ايراى ،بخش زاى هاهايی 1

.اى، تراى، ايراىبخش جراحی اکلشی ، داشگا علم پسشکی تر 2

.بخش کلييکال اکلشی، داشگا علم پسشکی تراى، تراى، ايراى 3

. بيوارستاى افضلی پر، داشگا علم پسشکی کرهاى، کرهاى ، ايراى ،بخش زاى هاهايی :آدرس نویسنده مسئول *

[email protected]پست الكتزونیك:


چكیده

از ظر جسی غير فعال ستذ دخترای ک التابی لگي در ای، ای تخوذای ب عاى يک عارض جذی بيواری آبس لل :مقدمه

ادر است. بسيار

ب هبتال از ظر جسی غير فعال. ايي دختر کينسال را هعرفی هی 04ای تخوذای در يک دختر : ها يک هرد آبس للمورد

شاذی از آپاذيسيت، بيواری التابی رد يا سرطاى قبلیبذى شرح حال ،با درد شکن تد لگی کسپتم عرضی اشى بد

سالپيگ با بيوار الپاراتهی تشخيص داد شذ. کطرف دريای تخوذای يک آبس بسرگ للاجام شذ ای در بررسی هراجع کرد.

ببد يافت. اى آتی بيتيکی بعذ از عوليسترکتهی دره ،يکطرف افرکتهی

درد هسهي لگي ک هجر ب اتای فرد حاهلگی خارج از رحن ب هظر پيشگيری از عارض بعذی از جول ازايی، ه گیزی:جنتی

ساهی است.لاايي بيواری تشخيص درهاى در هراحل الي ،هيشد

ای تخوذای، هجرد، سپتم اشى. : آبس للکلیدی کلمبت

241-250 ، صفحبت:80 ، تببستبن3تولید مثل ایزان، سبل نهم، شمبره فصلنبمه طب

Iranian Journal of Reproductive Medicine Vol.9. No.3. Summer 2011

Iranian Journal of Reproductive Medicine Vol.9. No.3. Summer 2011 267

BOARD OF CONSULTANTS

The Editor wishes to express his appreciation to the following scientists who

reviewed manuscripts submitted to the Iranian Journal of Reproductive Medicine

(Vol.9 No.3 Summer 2011) in a conscientious and objective manner.

Aflatoonian, Abbas M.D. (Iran)

Ahmadi, Ali Ph.D. (USA)

Ahmadi, Shahnaz M.D. (Iran)

Akhondi, Mohammad Mehdi Ph.D. (Iran)

Alborzi, Saeed M.D. (Iran)

Anvari, Morteza Ph.D. (Iran)

Asgharnia, Maryam M.D. (Iran)

Ayazi-Rozbahani, Maryam M.D. (Iran)

Babaie, Homayoun D.V.M., Ph.D. (Iran)

Dehghani Firoozabadi, Razieh M.D. (Iran)

Eftekhar, Maryam M.D. (Iran)

Ghaffari-Novin Maarefat Ph.D. (Iran)

Ghasemzadeh, Aalieh M.D. (Iran)

Ghazizadeh, Shirin M.D. (Iran)

Habibzadeh, Victoria M.D. (Iran)

Hojjat, Farzaneh M.D. (Iran)

Khaki, Amir Afshin Ph.D. (Germany)

Khalili, Mohammad Ali Ph.D. (Iran)

Madani, Tahere M.D. (Iran)

Mansuri Moghaddam, Fatemeh M.D. (Iran)

Mashayekhi, Mehri M.D. (Iran)

Mehrafza, Marzieh M.D. (Iran)

Modares- Mosaddegh, Monire Ph.D. (Iran)

Moein, Mohammad Reza M.D. (Iran)

Moeini, Ashraf M.D. (Iran)

Mohammadian Farnaz M.D. (Iran)

Mohiti, Mohammad Javad Ph.D. (Iran)

Mosaddegh, Mohammad Pharm M.D. (Iran)

Movaheddin, Mansoureh Ph.D. (Iran)

Nematollahi Mahani, Seyed Noureddin Ph.D. (Iran)

Oskoueian, Homa M.D. (Iran)

Rafaati, Ali Ph.D. (Iran)

Salehnia, Mojdeh Ph.D. (Iran)

Salehpour, Saghar M.D. (Iran)

Salami, Maryam M.D. (Iran)

Shahrokh Tehrani-Nejad, Ensieh M.D. (Iran)

Shamsa, Ali M.D. (Iran)

Tabibnejad, Nasim M.D. (Iran)

Taheripanah, Robabeh M.D. (Iran)

Talebi, Ali Reza Ph.D. (Iran)

Yousefnejad, Fariba M.D. (Iran)

Zolghadri, Jaleh M.D. (Iran)

Iranian Journal of Reproductive Medicine Vol.9. No.3. Summer 2011

Iranian Journal of Reproductive Medicine Vol.9. No.3. Summer 2011 268

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