Length of Androgen Receptor–CAG Repeats in Fertile and Infertile Egyptian Men

Length of Androgen Receptor–CAG Repeats in Fertile andInfertile Egyptian Men

WAEL A. BADRAN,* IBRAHIM FAHMY,*{ WAEL M. ABDEL-MEGID,{ KAY ELDER,§

RAGAA MANSOUR,* AND MARIJO KENT-FIRST{

From *The Egyptian In Vitro Fertilization and Embryo Transfer Centre, Hadayk El-Maadi, Cairo, Egypt; �Andrology

Department, Faculty of Medicine, Cairo University, Cairo, Egypt; `Obstetrics & Gynecology Unit, University of

Wisconsin Medical School, Madison, Wisconsin; and §Bourn Hall Clinic, Cambridge, United Kingdom.

ABSTRACT: Androgens play key roles in spermatogenesis, and

they exert their effect via the androgen receptor (AR). The AR gene

has a repetitive DNA sequence in exon 1 that encodes a

polyglutamine tract. Instability in the glutamine (CAG) repeat unit

length is polymorphic across ethnic groups. Previous studies of the

relationship between the repeat unit length and male infertility have

been contradictory. To establish the range of wild-type alleles in

Egyptian men, we determined the range of repeat lengths in a

population of normally fertile, ethnically selected Egyptian men. We

also investigated the association between trinucleotide repeat length

within the AR gene and male factor infertility in a population of

ethnically selected Egyptian infertile men, who were compared with

fertile, ethnic group–matched and age-matched controls. The study

included 129 clinically selected infertile Egyptian men who were

scheduled for intracytoplasmic sperm injection and 52 ethnically

matched fertile controls. The experimental population was grouped

according to sperm counts ranging from nonobstructive azoosper-

mia to normozoospermia. The CAG repeat N-terminal domain

region of the AR gene was amplified in peripheral blood DNA, and

allele size was determined by fragment analysis. Allele size and

single-nucleotide polymorphism and mutation rates were deter-

mined by sequencing individual amplified alleles. The mean CAG

repeat length in the azoospermia group was 18.55 6 2.0; in the

severe oligozoospermia group it was 18.21 6 3.42; in the

oligozoospermia group it was 18.27 6 2.93; and in the infertile

with normal sperm count group it was 17.72 6 2.0. In the control

group, the mean CAG repeat length was 18.18 6 3.63. No

significant correlation was found between CAG repeat length and

the risk of male factor infertility in an ethnically defined population

of Egyptian men. However, a significant and positive correlation

between CAG repeat length and serum testosterone concentration

was demonstrated. This suggests the involvement of epigenetic

regulation linked to this region.

Key words: Male infertility, spermatogenesis, trinucleotide re-

peat.

J Androl 2009;30:416–425

Androgens control development of the male pheno-

type, including spermatogenesis (Yong et al, 2003).

The actions of androgens are mediated by the androgen

receptor (AR), a member of the steroid hormone

receptor subfamily of nuclear receptors (Molenda et

al, 2003; Verrijdt et al, 2006). The AR gene mutations

are linked to several pathologic conditions (Gottlieb,

2005; Gottlieb et al, 2005). Mutations that cause total

disruptions of AR function result in complete androgen

insensitivity syndrome (Gobinet et al, 2002). On the

other hand, mutations that do not result in complete

ablation of AR function can cause a wide spectrum of

milder phenotypes ranging from ambiguous genitalia in

the newborn to a normal male phenotype associated

with infertility (Hiort and Holterhus, 2003).

The AR gene is a single-copy gene located in the

pericentric region of the long arm of the X chromosome,

at locus Xq11-12 in the human (Brown et al, 1989). The

AR gene contains 8 exons. Toward the 59 end of exon 1

there is a polymorphic glutamine (CAG) repeat that

encodes a polyglutamine tract. This microsatellite locus

is the most variable region in the AR gene. In a healthy

individual, the number of CAG repeats ranges from 9 to

36, with an average of 21–22 repeats. The average

number of repeat lengths varies significantly between

different racial groups (Quigley et al, 1995). For

example, individuals of African descent have much

shorter average repeat lengths of CAG (18–20) com-

pared with Hispanics (CAG, 23). Studies of CAG repeat

lengths in whites and East Asians have reported the

repeat lengths to be 21–22 and 23, respectively.

Moreover, there are about 20 different allele lengths

reported across ethnic groups, and 90% of women are

heterozygous for the length of the repeat (Sartor et al,

1999; Bennett et al, 2002).

Spinal and bulbar muscular atrophy (SBMA; Online

Mendelian Inheritance in Man no. 313200), also known

Correspondence to: Dr Marijo Kent-First, Department of Biological

Sciences, Box GY, Mississippi State University, Mississippi State, MS

39672 (e-mail: [email protected]).

Received for publication May 24, 2008; accepted for publication

January 21, 2009.

DOI: 10.2164/jandrol.108.005843

Journal of Andrology, Vol. 30, No. 4, July/August 2009Copyright E American Society of Andrology

416

as Kennedy disease, is linked to an abnormal elongation

of more than 40 CAG repeats in exon 1 of the AR gene

(Quigley et al, 1995). SBMA is a neurodegenerativedisease characterized by progressive weakness and

atrophy of proximal muscles. Most affected men display

progressive androgen insensitivity in the form of

progressive testicular atrophy, gynecomastia, feminized

skin changes, and reduced fertility, which are either due

to azoospermia or severe oligozoospermia (Greenland

and Zajac, 2004).

Many studies have examined the possible correlationbetween the length of the polyglutamine repeat in the

AR gene and male factor infertility (Table 1). A number

of these reports suggested a link between male factor

infertility and expansion of the polymorphic trinucleo-

tide (CAG) repeat in the AR gene (Mifsud et al, 2001;

Patrizio et al, 2001; Madgar et al, 2002). Other reports

failed to demonstrate such an association (Giwercman et

al, 1998; Dadze et al, 2000; Tufan et al, 2005). The basisfor these investigations is the finding that the length of

the polyglutamine repeat is inversely proportional to the

degree of normal functionality of the AR (Kazemi-

Esfarjani et al, 1995). This observation led to the

hypothesis that significantly longer or significantly

contracted polyglutamine tracts should be considered

as a risk factor for male infertility. To test this

hypothesis in any population, it is critical that the rangeof alleles present in ethnically matched (relative to the

experimental population) fertile control men be deter-

mined (Zitzmann and Nieschlag, 2003). Unfortunately,

data interpretations in many studies are confusing

because of the general inherent instability of microsat-

ellite loci and the specific selective instability of loci

containing trinucleotide CAG repeats. Furthermore,

given fluid migration patterns in human populations,the wide variation in repeat lengths within an ethnically

mixed population(s) and the overlapping ranges between

defined ethnic groups make it difficult to assess data

interpretation and comparisons of data between ethnic

groups (Hiort and Holterhus, 2003).

The present study was designed to test the hypothesis

that significant deviation from the mean CAG repeat

length in the AR gene is correlated with male factor

infertility. To test this hypothesis, first the relativestability (including the range of allele sizes) of the AR-

CAG repeat length in an ethnically defined and

population of fertile Egyptian men with normal semen

parameters was determined. Second, the relationship of

the AR-CAG repeat length relative to male factor

infertility in an ethnically defined population of

Egyptian men who were intracytoplasmic sperm injec-

tion (ICSI) candidates was determined. An ethnicallyselected wild-type population of fertile Egyptian men

served as the control group in this study.

Materials and Methods

Subjects

A total of 129 infertile men presenting consecutively who were

scheduled for the ICSI program at the Egyptian In Vitro

Fertilization and Embryo Transfer Centre were prospectively

recruited for the study during the period from 2004 through

2005. Patients with obstructive azoospermia, hypogonadotro-

phic hypogonadism, genital tract infection, and genetic or

karyotype abnormalities were excluded from the study.

Infertile couples with definite female factor infertility were

also excluded. The control subjects were fertile volunteers and

patients attending urology clinics for complaints other than

infertility. Fifty-two men were recruited, all with proven

fertility, having fathered at least one child by natural

conception. These men had no previous history of infertility

or fertility treatment and had no identified genetic disorders.

All cases and controls were identified as Egyptians according

to self-categorization. The geographic regions of descent for all

subjects in the control and experimental populations were

derived from the Nile Delta and Upper Egypt, both along the

Nile River.

This study was conducted with institutional review board

approval in The Egyptian In Vitro Fertilization and Embryo

Transfer Centre and in the United States (Promega, Madison,

Wisconsin). Each participant (patients and controls) gave

written informed consent. The samples were made anonymous

throughout the study.

Clinical Evaluation

Each patient was subjected to history taking and clinical

examination. Determination of testicular volume was per-

formed by Orchidometer (Holtain Ltd, Crymych, United

Kingdom). Laboratory investigations included: semen analysis

according to World Health Organization (1999) guidelines,

measurement of serum levels of reproductive hormones

(follicle-stimulating hormone [FSH], luteinizing hormone

[LH], testosterone, and prolactin), and cytogenetic analysis.

The patient population was restricted according to the status

of the female partner’s age (younger than 40 years), normal

basal FSH level, no polycystic ovary syndrome, and normal

uterus with no hydrosalpinges detected by ultrasonography.

For each of the control subjects, at least one semen analysis

was performed, as well as clinical examination. For each

participant in the experimental and control groups, a

peripheral blood sample was collected for DNA analysis of

sequence of CAG repeat length within the AR gene. None of

the patients received infertility treatment during the 3 months

prior to these investigations

Molecular Analysis

Allele Sizing and Trinucleotide Repeat Allele Fragment

Analysis—DNA was purified from the peripheral blood of

patients and control subjects using a Wizard Genomic DNA

Purification System (Promega) and DNA IQ (Promega)

according to the manufacturer’s recommended protocol. The

CAG repeat within the N-terminal domain region was

Badran et al N AR-CAG in Fertile and Infertile Egyptians 417

Table

1.

Sum

mary

of

pre

vio

usly

publis

hed

stu

die

sin

vestigating

AR

-CA

Gre

peat

length

infe

rtile

and

infe

rtile

popula

tions

Sourc

e(L

ocatio

nof

Stu

dy)

Infe

rtile

Contr

ol

Sig

nific

ant

Diffe

rence

Betw

een

2G

roups

(P)

CA

GR

epeat

Cuto

ffP

oin

t

No.

of

Men,

n

Mean

6S

DR

ange

No.

of

Men,

nM

ean

6S

DR

ange

Azo

osperm

iaO

ligosperm

iaT

ota

l

Giw

erc

man,

1998

(Sw

eden)

617

33

21.9

16–27

294

23.2

8–30

No

n/a

Legiu

set

al,

1999

(Belg

ium

)22

201

223

21

(media

n)

15–30

181

21

(media

n)

14–29

Yes

$26

Dow

sin

get

al,

1999

(Austr

alia

)10

20

30

23.2

60.7

15–34

32

20.5

60.3

17–25

Yes

(.0001)

$26

Hio

rtet

al,

1999

(Germ

any)

13

167

180

23

13–30

53

24

17–39

No

n/a

Dadze

et

al,

2000

(Germ

any)

18

101

119

22

63.2

16–34

22

20.8

63.3

15–26

No

n/a

Patr

izio

et

al,

2001

(Unite

dS

tate

s)

16

53

69

23.5

63.4

18–39

45

22

62.8

12–30

Yes

(.03)

n/a

Walle

rand

et

al,

2001

(Fra

nce)

37

...

37

23.9

60.5

13–28

50

22.2

60.4

17–27

Yes

(.008)

n/a

Mifsud

et

al,

2001

(Unite

dS

tate

s)

23

72

95

21.9

56

0.3

114–31

55

20.7

26

0.5

28–27

Yes

(.034)

$26

Von

Eck

ard

ste

in,

2001

(Germ

any)

29

14

43

20.4

62.9

17–27

131

20

63.1

12–28

No

n/a

Rajp

ert

-De

Meyt

set

al,

2002

(Denm

ark

)30

89

119

21.5

62.9

15–29

110

21.8

63.4

14–33

No

n/a

Kukuvitis

et

al,

2002

(Gre

ece

)42

67

109

21

(media

n)

15–33

64

22

(media

n)

14–31

No

n/a

Van

Gold

eet

al,

2002

(The

Neth

erlands)

75

...

75

22.2

63.1

13–36

70

21.7

63.4

15–31

No

n/a

Era

sm

uson

et

al,

2003

(New

Zeala

nd)

...

105

105

21.4

66

0.3

12–30

93

20.9

96

0.2

89–26

No

n/a

Lund

et

al,

2003

(Fin

land)

...

192

192

22.1

66

2.8

16–30

149

22.4

62.8

15–29

No

n/a

Asa

tianiet

al,

2003

(Germ

any)

...

217

217

21.5

62.9

17–30

131

20

63.1

12–28

Yes

(.001)

n/a

Mengualet

al,

2003

(Spain

)102

...

102

23.2

56

2.7

18–32

96

22.4

622.8

15–34

Yes

(.033)

n/a

Ruhayelet

al,

2004

(Sw

eden)

36

49

85

...

14–29

223

...

12–30

No

n/a

Tufa

net

al,

2005

(Turk

ey)

25

22

47

22.2

86

0.3

718–29

32

22.4

16

0.5

416–29

No

n/a

Lavery

et

al,

2005

(Ire

land)

55

11

66

23.3

06

2.4

419–30

77

23.0

76

2.3

219–30

No

n/a

Kom

ori

et

al,

1999

(Japan)

...

59

59

21.2

64.2

14–32

36

21.4

63.5

16–21

Yes

(,.0

5)

,16

Yoshid

aet

al,

1999

(Japan)

41

...

41

26.5

63.5

20–34

48

23.9

62.9

17–30

Yes

(.0013)

31–40

Sasagaw

aet

al,

2001

(Japan)

30

...

30

23.4

62.9

19–30

51

23.7

63.2

17–28

No

n/a

Nakabaya

shiet

al,

2003

(Japan)

...

...

133

22.6

63

12–34

100

23.3

62.8

16–31

No

n/a

Tut

et

al,

1997

(Chin

a)

28

125

153

...

...

72

...

...

Yes

$27

Mifsud

et

al,

2001

(Chin

a)

33

87

120

23.8

26

0.4

14–33

87

22.3

86

0.3

211–29

Yes

(.043)

$26

Liet

al,

2005

(Chin

a)

31

52

...

22.1

6..

...

...

...

.Y

es

n/a

Thangara

jet

al,

2002

(India

)280

...

280

21.7

60.1

812–32

201

22.4

60.1

912–32

No

n/a

Dhill

on

et

al,

2003

(India

)103

80

183

22.2

61.5

13–33

59

21.4

61.4

12–33

No

n/a

Pan

et

al,

2002

(Taiw

an)

48

...

48

23

60.6

10–41

47

21

60.4

14–27

Yes

(.001)

.27

Tse

et

al,

2003

(Hong

Kong)

53

32

85

23

63.9

14–36

45

23

63.1

16–30

No

,16

to.

30

Kata

giriet

al,

2006

(United

Sta

tes)

757

64

22.2

63

17–32

13

19.3

65

10–24

Yes

(.02)

n/a

Madgar

et

al,

2002

(Isra

el)

46

15

61

18.6

63

13–24

50

16.6

62.6

11–22

Yes

(.003)

n/a

Hadjk

acem

et

al,

2004

(Tunis

ia)

27

38

129

20.8

60.2

712–28

98

21.1

36

0.3

114–29

No

n/a

West

erv

eld

et

al,

2008

(The

Neth

erlands)

...

...

700

21.5

63.1

...

...

...

...

No

n/a

Pre

sent

stu

dy

(Egypt)

44

74

118

+11

a18.2

96

36–26

52

18.1

86

3.6

38–26

No

n/a

Abbre

via

tions:

...,

an

absence

of

data

for

that

stu

dy;

n/a

,not

applic

able

.a

Ele

ven

infe

rtile

patients

with

norm

alsperm

count.

418 Journal of Andrology N July �August 2009

amplified using 2 flanking sets of primers: A(F) 59-TCC AGA

ATC TGT TCC AGA GCG TGC-39 and A1(R) 59-GCT GTG

AAG GTT GCT GTT CCT CAT-39. In set 1, the forward

primer, A(F), was fluorescently labeled and paired with the

unlabeled high-performance liquid chromatography (HPLC)–

purified reverse primer, A1(R). In set 2, the unlabeled HPLC–

purified forward primer was paired with a fluorescently labeled

reverse A1(R) primer. A total of 120 ng of DNA was amplified

in duplicate experiments designed to establish and to confirm

allele size. The PCR-amplified products were also sequenced as

needed to confirm CAG repeat length and polymorphism.

Conditions for large-pool PCR were as follows: primers were

diluted to 500 mM in a 40-mL total reaction volume consisting

of 4 mL of STAR buffer (Promega) and AmpliTaq Gold DNA

polymerase (0.5 mL/reaction; Applied Biosystems, Foster City,

California) PCR was performed using the ABI GeneAmp PCR

system 9600 (Perkin Elmer, Norwalk, Connecticut) according

to the Hot Start cycle protocol. The Hot Start cycle was 95uCfor 9 minutes, followed by 95u denaturation for 1 minute, 60uannealing for 1 minute, and 72u extension for 1 minute for 40

cycles, followed by a final extension at 72u for 5 minutes. The

products were separated by capillary electrophoresis on ABI

PRISM 3100 Genetic Analyzers with allele sizing using ILS-

600TM, 60–600 bp (Promega). PCR controls used in amplifica-

tion experiments included water, male DNA, and female DNA.

Fragment Size Confirmation and SNP Genotyping—PCR

products from a total of 75 individuals (40 samples from the

experimental group and 35 samples from the control group)

were gel purified according to standard methods. Both the

forward and reverse strands were sequenced to confirm the

length of the CAG repeat and to identify SNPs within the

region immediately flanking the CAG repeat.

Statistical Analysis

Statistical analysis was performed using the statistical package

SPSS for Windows (version 11.0; SPSS Inc, Chicago, Illinois).

The difference in the CAG repeat length between control,

combined infertile group, and various subgroups of infertile

men was evaluated by the Mann-Whitney U test. Spearman’s

correlation coefficient was used for bivariate regression

analysis to determine the correlation between CAG repeat

length, sperm count, bitesticular volume, and hormone data. A

probability of P , .05 was considered statistically significant.

Results

Clinical Findings

This study included 129 infertile, ethnically restricted

patients scheduled for an ICSI program. The group

included 44 patients with nonobstructive azoospermia

(absence of sperm in the ejaculate confirmed by

examination of sediment after centrifugation), 43

patients with severe oligozoospermia (sperm count

,5 million/mL), 31 patients with oligozoospermia

(sperm count range from 5 million/mL to less than

20 million/mL), and 11 patients with normal sperm

counts and varying degrees of asthenozoospermia/

teratozoospermia (sperm count $20 million/mL). A

summary of the clinical and molecular data is shown

in Table 2. The control group consisted of 52 fertile men

with fertility demonstrated by live births and sperm

counts ranging from 20 million/mL to 100 million/mL

(mean, 53.19 6 17.83 million/mL). The ethnically

selected control population was matched with the

experimental population according to both ethnicity

and age. In the combined infertile group, excluding

infertile patients with normal sperm counts, the sperm

concentrations ranged between 0 and 19 million/mL

(mean, 3.37 6 5.25 million/mL; Table 3). Bitesticular

volume in the infertile group ranged between 2 and

66 mL (mean, 28.97 6 14.3 mL), whereas in the control

group the range was between 34 and 70 mL (mean, 49 6

9.29 mL), which was significantly different (P , .05).

The sperm count was significantly correlated with

bitesticular volume (P 5 .01) and serum levels of FSH

(P 5 .001), LH (P 5 .001), and testosterone (P 5 .04).

High serum FSH was detected in 40 patients from the

experimental population, and an isolated high serum

FSH was detected in 18 infertile patients. High serum

LH was detected in 28 patients. Combined high serum

LH and serum testosterone were detected in only 3

patients. These 3 patients had sperm counts of 0

(azoospermia), 0.5 million/mL, and 6 million/mL, re-

spectively, and the lengths of their mean CAG repeats

were 19, 19, and 21, respectively. A total of 38 patients

(29.5%) had undergone bilateral or left varicocelectomy.

A total of 10 patients (7.8%) had an undescended testis.

One patient had a nonmalignant testicular tumor with

subsequent left orchidectomy. Parents’ consanguinity

was reported by 26 patients (20.2%), whereas another 9

patients (7%) had a history of infertility in first- or

second-degree relatives.

Molecular Findings: Length of AR-CAG Repeats

The means and ranges of CAG repeat lengths in control

and different infertile groups are summarized in Table 2.

The mean CAG repeat length did not differ statistically

between the combined infertile group (18.30 6 2.3) and

the control group (18.18 6 3.63). When infertile men

with normal sperm count were excluded, the mean CAG

repeat length of the remaining group (18.35 6 3.02) did

not differ significantly from that of the control group

(Table 3). The mean CAG repeat length did not differ

statistically between the control group and each of the

infertile subgroups in the experimental population.

A total of 40 patients with azoospermia were

subjected to testicular sperm extraction (TESE) as a

part of their ICSI schedule (another 4 patients were

scheduled for ICSI/TESE and dropped out before the


Table

2.

Sum

mary

of

clin

icaland

mole

cula

rdata

of

fert

ileand

infe

rtile

gro

ups

Range

(Mean

6S

D)

Cla

ssifi

cation

(n)

Age,

y

Sperm

Count,

Mill

ion

/mL

Bitestic

ula

r

Volu

me,

mL

Testo

ste

rone,

ng

/mL

aLH

,m

IU/m

La

FS

H,

mIU

/mL

aC

AG

Repeat

Length

Media

nof

CA

GLength

Contr

ol(5

2)

21–67

(38.7

96

10.2

4)

20–100

(53.1

96

17.8

3)

34–70

(49

69.2

9)

n/a

n/a

n/a

8–26

(18.1

86

3.6

3)

18

Tota

lin

fert

ile(1

29)

24–65

(38.6

26

7.5

1)

0–110

(7.6

66

16.5

1)

2–66

(28.9

76

14.3

0)

1.9

–9.5

(4.6

76

1.9

2)

1.3

–38.3

(8.2

46

5.9

1)

1.3

–73

(17.4

06

14.1

3)

6–26

(18.3

06

2.3

)

18

Azo

osperm

ia(4

4)

28–65

(40.2

56

8.0

5)

06–51

(24.1

36

12.7

3)

2.1

–9.5

(4.6

56

2.1

5)

1.3

–38.3

(10.3

66

8.1

4)

1.8

–73

(24.1

06

17.6

6)

15–26

(18.5

56

2.7

0)

18.5

Severe

olig

ozoosperm

ia

(43)

26–49

(36.9

86

5.2

1)

0.0

1–4

(1.0

96

1.1

7)

2–60

(25.6

76

13.6

1)

2.1

–8.4

(4.5

86

1.7

8)

1.6

–24.3

(7.6

46

4.0

0)

1.9

–43.2

5

(16.5

56

10.3

4)

6–26

(18.2

16

3.4

2)

18

Olig

ozoosp

erm

ia(3

1)

24–50

(37.7

86

7.6

9)

5–19

(11.3

26

4.0

1)

12–66

(35.7

76

12.9

3)

1.9

–8.3

(4.7

76

1.9

2)

2.1

–16.6

(6.6

56

3.7

1)

1.8

–36.6

(11.0

46

9.8

2)

10–22

(18.2

76

2.9

3)

19

Infe

rtile

with

norm

al

count

(11)

29–57

(40.9

16

8.5

7)

20–110

(53.6

46

25.0

1)

22.6

6

(42.0

612.6

2)

2.1

–6.9

(4.8

26

2.0

3)

2.7

–8.8

(5.4

26

2.4

3)

1.3

–13.2

(8.4

76

5.1

5)

11–21

(17.7

36

2.8

7)

18

Abbre

via

tions:

CA

G,

poly

morp

hic

glu

tam

ine;

FS

H,

folli

cle

-stim

ula

ting

horm

one;

LH

,lu

tein

zin

ghorm

one;

n/a

,not

applic

able

.a

Norm

alle

vels

are

2.4

1–8.2

7fo

rte

sto

ste

rone;

1.5

–9.3

for

LH

;and

1.4

–18.1

for

FS

H.

Table

3.

Sum

mary

of

clin

icaland

mole

cula

rdata

of

infe

rtile

subgro

ups

Range

(Mean

6S

D)

Cla

ssifi

cation

(n)

Age,

y

Sperm

Count,

Mill

ion

/mL

Bite

sticula

r

Volu

me,

mL

Testo

ste

rone,

ng

/mL

aLH

,m

IU/m

La

FS

H,

mIU

/mL

aC

AG

Repeat

Length

Media

nof

CA

GLength

Infe

rtile

without

patients

with

norm

alcount

(118)

24–65

(38.4

16

7.4

1)

0–19

(3.3

76

5.2

5)

2–66

(27.7

56

13.8

8)

1.9

–9.5

(4.6

66

1.9

4)

1.3

–38.3

(8.3

76

5.9

9)

1.8

–73

(17.8

16

14.2

9)

6–26

(18.3

56

3.0

2)

18.5

Infe

rtile

with

his

tory

of

undesce

nded

testis

(10)

28–49

(36.0

66.4

3)

0–6

(0.9

26

1.8

9)

11–22

(16.4

64.6

0)

2.6

–6.2

(4.0

36

0.9

5)

4.9

–13.7

(8.4

16

2.9

9)

8–37.2

(21.7

66

11.3

2)

15–26

(19.8

63.0

5)

19.5

Azoosperm

iaw

ith

positi

ve

sperm

retr

ieval

28–65

(40.4

76

8.5

)

06–51

(24.5

612.0

3)

2.2

–9.5

(4.8

62.2

3)

1.3

–38.3

(9.8

76

8.0

4)

2.3

–73

(22.2

56

16.6

9)

15–26

(18.4

46

2.3

9)

18.5

Azoosperm

iaw

ith

negative

sperm

retr

ieval

28–43

(36.6

76

5.0

5)

012–47

(20.8

36

14.2

5)

2.4

4–4.4

8

(3.5

86

0.6

9)

4.3

–20.9

(10.5

86

6.1

5)

8–54.2

(28.6

66

17.3

3)

17–26

(20.0

64.0

)

18.5

Abbre

via

tions:

CA

G,

poly

morp

hic

glu

tam

ine;

FS

H,

folli

cle

-stim

ula

ting

horm

one;

LH

,lu

tein

izin

ghorm

one.

aN

orm

alle

vels

are

2.4

1–8.2

7fo

rte

sto

ste

rone;

1.5

–9.3

for

LH

;and

1.4

–18.1

for

FS

H.


procedure). Sperm retrieval was successful in 34

patients. The mean length of CAG repeat did not reveal

a significant difference between azoospermic patients

with positive sperm retrieval and those with negative

sperm retrieval (Table 3).

There was a mild positive correlation between length

of CAG repeat and serum testosterone level. This

correlation was detected in the combined infertile group(P 5 .014, r 5 0.330) and the combined infertile group

after exclusion of patients with normal sperm counts (P

5 .027, r 5 0.312), and the correlation was strongly

positive in infertile men with normal sperm counts (P 5

.005, r 5 0.975), but the correlation was not detected in

the other infertile subgroups (azoospermia, severe

oligozoospermia, and oligzoospermia). However, the

strong positive correlation detected in infertile men withnormal sperm count may be affected by the small

number of this subgroup (11 patients).

None of the semen parameters or the other hormone

parameters was significantly correlated with length of

CAG repeat. On the other hand, there was no

correlation between length of CAG repeat and bitestic-

ular volume or sperm count in the fertile control group.

The distribution of CAG repeat lengths in the control

group and experimental infertile group is shown in theFigure. The CAG repeat lengths in the control, total

infertile group, and infertile subgroups overlap consid-

erably. The longest allele (26 repeats) was detected in 3

individuals: 1 person from the control group, 1 patient

with azoospermia, and 1 patient with severe oligozoo-spermia. The shortest allele (6 repeats) was detected in 1

patient with severe oligozoospermia.

In the combined infertile group, the most common

alleles were 17 and 19 repeats. Each of these respective

alleles was detected in 20 patients (15.5%). In the control

group, the most common allele (18 repeats) was detected

in 9 individuals (17.3%). No sequence variation or

polymorphism was observed in alleles isolated from 75individuals.

Sequence analysis of PCR products confirmed repeat

length in all of the sampled alleles.

Discussion

The present study examines the association between

expanded CAG repeat length within the AR gene and

defective spermatogenesis in 129 infertile and 52 control

men of matched Egyptian origin. The first experiment

established the common alleles present in an ethnically

restricted population of normally fertile men. This

population then served as the control for the clinicallyselected experimental population. There was no corre-

lation between CAG repeat length and defective

Figure. Distribution of CAG repeat length in normal fertile control and infertile Egyptian men. (A) Combined cases and control (number of men ingroup, 181; mean, 18.26 6 3.18; median, 18). (B) Control (number of men in group, 52; mean, 18.18 6 3.63; median, 18). (C) Combinedinfertile (number of men in group, 129; mean, 18.3 6 2.3). (D) Infertile after excluding patients with normal sperm count (number of men ingroup, 118; mean, 18.35 6 3.02; median, 18.5). (E) Azoospermia (number of men in group, 44; mean, 18.55 6 2.7; median, 18.5). (F)Oligozoospermia (number of men in group, 31; mean, 18.27 6 2.93; median, 19). (G) Severe oligozoospermia (number of men in group, 43;mean, 18.21 6 3.42; median, 18). (H) Infertile with normal sperm count (number of men in group, 11; mean, 17.73 6 2.87; median, 18).


spermatogenesis. The mean numbers of repeats in the

infertile population and fertile controls were similar

(18.30 6 2.30 and 18.18 6 3.63, respectively).The current study is the first to examine this

correlation in a defined Egyptian population, and this

adds to the data available for different ethnic back-

grounds. The mean CAG repeat length for the

combined control and infertile groups was 18.24 6

2.96, with a range of 6–26 repeats. Although several

studies have investigated the proposed association, these

reports have yielded conflicting results (Table 1). Theobserved variations in the results from previous studies

stem from several factors: 1) ethnically diverse popula-

tions; 2) the studied infertile men may represent a

heterogeneous group with respect to the causes of

infertility and may be under the effect of different

genetic mutations or even epigenetic phenomena; and 3)

different inclusion criteria in each study. The previously

studied infertile populations included various categoriesof infertility (eg, patients with varicocele, genital tract

obstruction, and/or genital tract infection) and semen

parameters (azoospermia, oligozoospermia, severe oli-

gozoospermia, or infertile men with normal sperm count

associated with varying degrees of asthenozoospermia/

teratozoospermia; Kukuvitis et al, 2002; Asatiani et al,

2003; Hadjkacem et al, 2004). Most importantly, the

control groups in many of these previous studies werenot well matched in terms of ethnicity and age. The

control groups in these studies often included not only

individuals with proven fertility, but also individuals

with normal sperm count but not proven fertility and/or

individuals from unselected populations (Legius et al,

1999; Erasmuson et al, 2003; Lund et al, 2003).

Unfortunately, these different reports render the

comparison between various studies inconclusive. Be-cause of racial variation and the impact of ethnicity, it is

possible that the results for AR-CAG repeat length in

selectively restricted racial/ethnic populations outweigh

those for a mixed population (Asatiani et al, 2003).

Some biomedical scientists assume that racial/ethnic

categorizations are inadequate descriptors of the distri-

bution of genetic variation in humans (Schwartz, 2001;

Hega and Venter, 2003). However, studies of different

populations have documented genetic, and therefore,biological differences among races (Keita et al, 2004).

Genetic variation tends to be geographically structured

in accordance with the classic common racial classifica-

tion, which is based on continental ancestry: African,

white, Asian, Pacific Islander, and Native American

(Jorde and Wooding, 2004; Tishkoff and Kidd, 2004). It

could be argued that population clusters identified by

genotype analysis seem to be more informative thanthose identified by self-declaration of race (Wilson et al,

2001). However, Risch et al (2002) demonstrated that

from both an objective and scientific (genetic and

epidemiologic) perspective, there is great validity in

racial/ethnic self-categorizations.The present work studied only an ethnically defined

population; all subjects were Egyptian by self-declara-

tion. Egypt’s geographic location has prompted its

population’s genetic diversity, but despite this, the

Egyptian population seems to be fairly homogenous.

This is supported by the findings of genetic studies that

characterize the Egyptian population. Studies of mito-

chondrial DNA variation, the Y-chromosome genepool, and different allelic variants in the modern

Egyptian reflect a mixture of European, Middle Eastern,

and African characteristics (Manni et al, 2002; Hamdy

et al, 2003). In addition, there is still a homogenous

genetic tie to ancient Egyptians (Kring et al, 1999). On

the other hand, these studies distinguished genetic

characteristics of the Egyptian from those of sub-

Saharan populations (Luis et al, 2004). This supportsthe concept that human populations are seldom

demarcated by precise genetic boundaries, and substan-

tial overlap can occur (Risch et al, 2002). The Egyptian

population, although fairly homogenous, is no excep-

tion in this regard.

A recent comprehensive meta-analysis included a

summary of 33 reports. This study investigated the

source of CAG variation between published results

(Davis-Dao et al, 2007). The report indicated that caseand control definitions likely influenced study results as

an important determinant of differences in repeat length

between cases and controls. In the present work, in

accordance with the recommendation of that meta-

analysis, we considered stringency of case and control

definitions. On the other hand, the analysis of the full set

of 33 reports revealed statistically significant longer

CAG repeat length among cases compared with controlpopulations. In an attempt to avoid methodologic

concerns, particularly small sample size and unmatched

case-control, Westerveld et al (2008) investigated AR-

CAG repeat in 700 men who presented for fertility

workup with varying degrees of semen quality. The

study concluded that there was no correlation between

CAG repeat length expansion in the AR gene and semen

quality. However, the study included an experimentalpopulation of men from mixed ethnic origins with no

matched control population. Exon 1 of the AR gene

contains another island of polymorphic microsatellite

GGC repeats. It was proposed that certain combina-

tions of CAG and GGC repeats may confer a risk of

infertility to the carriers. However, previous studies

showed that there was no association between AR

CAG/GGN microsatellites and impaired spermatogen-esis (Ruhayel et al, 2004; Rajender et al, 2006; Saare et

al, 2007).


In the present study, there was a mild positive

correlation between CAG repeat length and the level

of serum testosterone in 3 of the subgroups of theinfertile patients studied (combined infertile, infertile

excluding patients with normal sperm count, and

patients with normal sperm count; but not in azoosper-

mia, severe oligospermia, or oligspermia groups).

Although some studies found no significant association

between AR-CAG and hormonal levels, particularly

testosterone (Alevizaki et al, 2003; Harkonen et al,

2003), one study reported strikingly higher level of LHand androgen sensitivity index (ASI) in azoospermic

patients with short CAG repeats (Tse et al, 2003). Other

reports are in agreement with our findings and show a

positive correlation between AR-CAG repeats and

serum levels of LH, free testosterone, and ASI (Giwerc-

man et al, 2004). This hormone profile is often reported

in men with a range of clinical phenotypes associated

with aberrant secondary sex characteristics. Lim et al(2000) reported longer AR-CAG repeats to be associ-

ated with moderate to severe undermasculinized genita-

lia in XY men. In addition, Canale et al (2005)

investigated CAG repeat length in 3 groups: control,

infertile, and hypoandrogenized subjects. They conclud-

ed that hypoandrogenic traits, such as hypoplasia of the

prostate and seminal vesicles, and reduced beard and

body hair, are associated with longer CAG repeats.However, they found no difference in the mean of CAG

repeats in infertile populations compared with fertile

control populations.

In the present work, the secondary sexual characteristics

of infertile groups were comparable to those of controls.

Because there was no difference between the CAG repeat

length in the 2 different groups (infertile vs control), the

proposed effect of CAG repeat length on testosteronefunctionality may be limited to a subclinical level.

Thus, it might be assumed, at least in our studied

population, that the AR modulates its transactivation

potential via its N-terminal polyglutamine tract without

being associated with any pathologically apparent

disease (pathologic phenotype; Vogt, 1999).

In conclusion, our study established the range of

alleles in an ethnically and geographically restricted

population of Egyptian men with normal fertility. Therewas no significant correlation between CAG repeat

length and risk of male infertility in our ethnically

restricted experimental population compared with the

matched control population. Therefore, polymorphism

detected in the polyglutamine-rich region of the AR

gene is not a useful genetic indication of male factor

infertility. A significant positive correlation between

CAG repeat length and serum testosterone concentra-tion was detected, and this suggests that epigenetic

regulation linked to this region is involved.

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Length of Androgen Receptor–CAG Repeats in Fertile and Infertile Egyptian Men

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Transcript of Length of Androgen Receptor–CAG Repeats in Fertile and Infertile Egyptian Men