1

Title: The impact of examining the meiotic spindle by polarization microscopy on assisted reproduction

outcomes

Authors: Maria C. PICINATO1 B.Sc.; Wellington P. MARTINS1,2,3 Ph.D.; Roberta C. GIORGENON1 B.Sc.;

Camila K. B. SANTOS1 Ph.D.; Rui A. FERRIANI1,2 Ph.D.; Paula A. A. S. NAVARRO1,2 Ph.D., Ana C. J. S. ROSA E

SILVA1,2 Ph.D.

Affiliations: 1. Department of Gynecology and Obstetrics, Faculty of Medicine of Ribeirão Preto,

University of São Paulo (DGO-FMRP-USP), Ribeirão Preto – SP, Brazil; 2. National Institute of Science and

Technology – INCT/CNPq – Hormones and Women’s Health, Ribeirão Preto – SP, Brazil; 3. School of

Ultrasonography and Medical Recycling of Ribeirão Preto (EURP), Ribeirão Preto – SP, Brazil.

Correspondence: Ana Carolina Japur de Sá Rosa e Silva

Setor de Reprodução Humana, Departamento de Ginecologia e Obstetrícia da Faculdade de Medicina de

Ribeirão Preto - USP

Avenida Bandeirantes, 3900 - 8° andar- Monte Alegre, 14049-900

Ribeirão Preto SP, Brasil. e-mail: anasars@fmrp.usp.br

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Capsule

Use of polarization microscopy was associated with increased fertilization rate but reduced cleavage rate

and top quality embryos formation; no significant difference was observed for clinical pregnancy or live

birth.

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ABSTRACT

Objective: To examine the effect of submitting oocytes to polarization microscopy (PM) before

intracytoplasmic sperm injection (ICSI).

Design: Retrospective observational study.

Setting: University hospital in Brazil.

Patients: Couples undergoing ICSI.

Intervention: PM before ICSI (PM group) compared with no PM before ICSI (No-PM group)

Main outcomes measures: Fertilization and cleavage rates, formation of top quality embryos (TQE),

implantation, clinical pregnancy, miscarriage, and live birth rates.

Results The PM group consisted of 1,000 consecutive oocytes from 201 couples submitted to PM during

the year of 2008. The No-PM group consisted of 1,400 oocytes from 249 couples: 700 consecutive oocytes

retrieved before we started using PM and 700 consecutive oocytes retrieved after we stopped using PM. In

the PM Group, we observed an increased fertilization rate (79.7% vs. 72.5%, PM group vs. No-PM group

respectively), but reduced cleavage rate (86.2% vs. 92.5%) and TQE formation (33.1% vs. 49.9%).

Implantation (18.7% vs. 20.6%), clinical pregnancy (31.8% vs. 33.3%), miscarriage (21.9% vs. 15.7%), and live

birth (24.9% vs. 28.1%) rates were not significantly different between groups.

Conclusions: Use of polarization microscopy was associated with increased fertilization rate but reduced

cleavage rate and top quality embryos formation; no significant difference was observed for implantation,

clinical pregnancy, or live birth.

Keywords: Oocytes; Polarization microscopy; Assisted Reproductive Techniques; Comparative study.

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INTRODUCTION

The meiotic spindle (MS) of human oocytes in metaphase II (MII) stage is a temporary dynamic structure

consisting of microtubules which is associated with the oocyte cortex and its network of subcortical

microfilaments (1-3). The MS microtubules are linked to the kinetochores of the chromosomes (4) and

participate in segregation during meiosis. It is known that the oocyte meiotic spindle (MS) integrity is

important for chromosome segregation (5-7) and that it is extremely sensitive to various factors such as

aging, thermal changes, insufficient oxygen supply during culture and oocyte manipulation (8-10). Damage

to the MS may contribute to aneuploidy during the second meiotic division after fertilization and to the

development of embryos of poor quality, especially in women older than 40 years (7, 11).

Several studies have shown a correlation between the presence of MS identified by polarization

microscopy (PM) and an increased fertilization rate and embryo quality (12-18) in women undergoing

assisted reproductive techniques (ART). Additionally, the identification of the location of the MS in the oocyte

might prevent damage to this structure during ICSI (19, 20). However, some studies have questioned the

usefulness of MS evaluation by PM since it didn’t improve fertilization or early embryo quality (13, 21), and

there is a potential deleterious impact on embryo development since additional oocyte handling is

necessary.

The objective of the present study was to compare the reproductive outcomes of women undergoing ART

during a period when PM was routinely performed before ICSI to those observed when PM was not

employed.

METHODS

Study design and context

This was a retrospective observational study evaluating the data from women who were submitted to

ART in our assisted reproductive center. The study was approved by our local Ethics Committee. No

informed consent was asked for women, due to the nature of this study (retrospective analysis).

Participants

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In our assisted reproductive center, all oocytes with first PB extrusion determined by light microscopy

were routinely subjected to PM before ICSI during the year of 2008: we planned to compare the results

observed by 1,000 consecutive oocytes with first PB extrusion determined by light microscopy submitted to

PM during this year (PM group), with those observed in consecutive 1,400 oocytes with first PB extrusion

determined by light microscopy retrieved that were not submitted to PM: 700 consecutive oocytes retrieved

in the period before we started using PM and more 700 retrieved after we stopped using PM (No-PM group).

Variables

Fertilization rate: the number of oocytes that presented two distinct pronuclei and two polar bodies 18 -

20 hours after ICSI divided by the number of injected oocytes.

Cleavage rate: the number of cleaved embryos 40 - 44 hours after ICSI divided by the number of fertilized

oocytes.

Top quality embryo (TQE) formation: the number of embryos with 4-cell state, with symmetrical

blastomeres, and no fragmentation 40 - 44 hours after ICSI divided by the number of fertilized oocytes.

Implantation rate: the number of gestational sacs observed divided by the number of embryos

transferred.

Positive pregnancy test rate: the number of women who had a positive pregnancy test divided by the

number of women who had at least one MII oocyte.

Clinical pregnancy rate: the number of women who had at least one gestational sac observed by

ultrasonography divided by the number of women who had at least one MII oocyte.

Live birth rate: the number of women who delivered at least one living baby divided by the number of

women who had at least one MII oocyte.

Miscarriage rate: the number of women who had miscarriage (including ectopic pregnancy) divided by

the number of women who had a clinical pregnancy.

We also analyzed the causes of infertility as the proportion of included couples who had the following

diagnosis: endometriosis, anovulation, tubal, and male factor. These causes were not mutually exclusive; e.g.

one couple could have more than one cause.

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Controlled ovarian stimulation (COS), oocyte retrieval, and gamete preparation

All women were subjected to COS before oocyte retrieval according to standard long protocol, using

gonadotropins (150-300 IU/day) and GnRH analogues. Recombinant human chorionic gonadotropin (hCG)

was administered when two or more follicles reached a mean diameter ≥ 18 mm, and oocytes were

retrieved 34 to 36 hours after.

The retrieved oocytes were incubated in 5% CO2 at 37°C and 95% humidity for 2 hours, then denuded

and placed in 5 µl drops on the lower portion of a glass Petri dish (WillCo-dish; Willco Wells, Amsterdam, The

Netherlands). Approximately two hours after retrieval, the oocytes were denuded, placed individually in a 5

µl drop of HTF-Hepes with 10% synthetic serum substitute (SSS) and evaluated for the presence of the first

PB in order to classify their maturity.

Semen samples for the procedure were collected solely by masturbation. Fresh semen samples were

processed by the discontinuous gradient technique (90%-45%) and centrifuged at 1000 rpm for 30 minutes

(22).

Polarization microscopy (PM)

In the No-PM Group, ICSI was performed immediately after denudation.

In the PM group, the oocytes classified as mature were immediately analyzed by PM for the presence or

absence of the MS and its location in relation to the PB. The OCTAX ICSI GuardTM System, Medical

Technology Vertriebs-GmbH (Herborn, Germany) controlled by a computer using the OCTAX EyeWare™

software (Universal Imaging Corp., Boston, MA) was used to visualize the MS and to capture the image

immediately before ICSI. All readings were performed by the same observer (MCP), a biologist with 20 years

of experience in ART. This observer spent approximately 30-60 seconds to perform PM. ICSI was performed

just after PM.

The oocytes were classified by PM into 3 groups:

1. Unidentifiable MS.

2. Identifiable MS not completely located inside the oocyte cytoplasm: telophase I (TI).

3. Identifiable MS completely located inside the oocyte cytoplasm: metaphase II (MII).

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Additionally, the oocytes with identifiable MS in MII were subdivided into six groups according to the

angle detected between MS and PB: Position 1= 0-30°, Position 2=30-60°, Position 3=60-90°, Position 4=90-

120°, Position 5=120-150° and Position 6=150-180° in relation to the PB.

Evaluation of fertilization and cleavage

After the oocytes were subjected to ICSI, they were transferred to a 60mm plate with 25μl drops of

culture medium (HTF, LifeGLobal®, USA) covered with mineral oil (Irvine Scientific, Santa Ana, CA, USA) and

placed in an incubator (Forma™ Series II 3110 Water-Jacketed CO2 Incubators, Thermo Fisher Scientific Inc.,

Waltham, MA, USA). We removed the plates from the incubator 18-20 hours after ICS to examine

fertilization. We removed the plates from the incubator again 40-44 hours after ICSI to examine cleavage

and embryo morphology.

Embryo transfer and luteal support

One to three embryos were transferred 48–72 h after oocyte retrieval using transabdominal ultrasound

guidance and Sydney IVF catheters (Cook IVF, Eight Miles Plains, Queensland, Australia). The luteal phase

was supported by the administration of micronized progesterone (600 mg/day), which was stopped if the

pregnancy test (serum β-hCG test performed 14 days after embryo transfer) was negative or after week 12

of pregnancy.

Sample size

Considering that 60-70% of the oocytes with an identifiable MII are in position 1 and that 30% of these

oocytes will develop as TQE (23), 1000 oocytes subjected to PM (approximately 600-700 in MII at position 1)

and 1400 oocytes not subjected to PM would be necessary to demonstrate a relative difference of 20%

(from 30% to 36%) in the rate of TQE formation, with a power of 80%

Statistical analysis

Statistical analysis was performed using the GraphPad Prism software, version 5.0 for Windows

(GraphPad Software Inc., La Jolla, CA). We compared the groups using either the Fisher’s exact test or the χ²

test for the binary outcomes, and using unpaired t tests for the continuous outcomes. The significance level

was set as p < 0.05.

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RESULTS

Participants

We analyzed the results obtained after ICSI of 1000 consecutive oocytes submitted to PM (PM group)

obtained from 201 women, and of 1400 oocytes that did not undergo PM before ICSI retrieved from 249

women: 700 consecutive oocytes retrieved in the period before we started using PM and more 700 retrieved

after we stopped using PM (No-PM group).

Descriptive data

No significant difference was observed in age or subfertility cause between groups (Table 1).

Main results

Fertilization rates were significantly higher in the PM group; however, cleavage rate and TQE formation

were higher in the No-PM group (Table 2). Similar results were observed when comparing only the oocytes

with MS identified in MII in the PM Group with those in the No-PM group; only those MS in position 1 with

those in the No-PM Group (Supplemental Table 1).

No significant difference between groups was observed for the main reproductive outcomes

(implantation, positive pregnancy test, clinical pregnancy, miscarriage, and live birth, rates); however, all

results were slightly better in the “No-PM group” (Table 2).

Other analyses

When comparing the oocytes submitted to PM with different classifications (unidentified MS, MII, or TI),

only the fertilization rate was significantly different across groups; in the pairwise comparison, the only

significant difference observed was a higher fertilization rate in the oocytes with MS in MII compared with

those with MS in TI (Table 3). Additionally, no significant difference was observed in the oocytes submitted

to PM comparing to those with MS identified in MII but in different positions (Table 4).

DISCUSSION

Main results

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Use of PM was associated with increased fertilization rate but reduced cleavage rate and top quality

embryos formation. No significant difference was observed for implantation, clinical pregnancy or live birth,

but all the observed results for these outcomes were slightly better in the group not submitted to PM.

Limitations

The main limitation of this study is concerning its design: a retrospective observational study. The best

design to examine this question would be by performing a randomized controlled trial (RCT), which would be

at a lower risk of bias. However, while no proper RCT is available, reviews on this subject are being based on

the evidence from observational studies only (24).

Interpretation

To the best of our knowledge, all studies published so far have evaluated the clinical relevance of PM to

select the oocytes by comparing the results obtained by those oocytes with identifiable/normal MS, with

those with unidentifiable/abnormal MS (25).

Considering the evidence from these studies, in relation to the presence or absence of MS in oocytes

submitted to PM, differences between them were observed but with no clinical impact. We know that the

lack of MS visualization by PM does not necessarily indicate the absence of this structure. Several authors

have reported that the MS is a dynamic structure and that various environmental conditions can produce

temporary depolymerization without the occurrence of a real impairment of oocyte quality (8-10). This

depolymerization may be caused by changes in culture conditions, temperature and pH (26-30). In addition,

the lack of MS visualization does not prevent the occurrence of adequate fertilization and development (20).

Some authors have demonstrated that oocytes with MS might have higher fertilization rates (12, 14, 16, 17,

25, 31-34), higher proportion of blastocyst formation (12, 16) and better implantation and pregnancy rates

(17). On the other hand, some studies did not observe any significant difference in fertilization rates (18, 35)

or pregnancy/implantation (34).

Once identified de MS, its position can be also analyzed, the role of MS in relation to PB was thought to

be important because damaging the MS by transfixation during ICSI could be able to cause cell death (20).

Additionally, the misalignment between the MS and the first PB increases the risk of anomalous fertilization

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(13). Other studies have correlated the position of the MS close to the PB with better fertilization and

cleavage rates (35) and embryo morphology in the early stages of development (36). Despite these

previously published studies, we didn’t find significant differences in fertilization and cleavage rates or TQE

formation on D2 among oocytes with MS in different position. Therefore, in the present study, oocytes in

MII between positions 1 to 6 were analyzed jointly in a single group since the differences between them

were not statistically significant. However it is important to point out that had only one oocyte with MS in

the position 4, three in position 5 and none in position 6; thus, no meaningful conclusion can be drawn

regarding the reproductive potential of such oocytes.

There is a meta-analysis investigating the importance of identifying the MS by PM (37) that included

several of these previously published studies: the authors observed higher fertilization and cleavage rates,

and higher percentage of pro-nuclear-stage embryos with good morphology, day-3 top-quality embryos, and

blastocyst formation in oocytes submitted to PM with identifiable MS compared with those also submitted

to PM but with a non-identifiable MS. However no difference in clinical pregnancy was obtained with the use

of MP selection.

One of our main findings was a reduced TQE formation in the oocytes submitted to PM. We believe that

the additional oocyte handling necessary to perform PM is the main reason for such difference, since the

minimization of the manipulation time, and better control of temperature and pH of the oocytes is

associated with improved outcomes (38).

Indeed, the best evidence about the clinical relevance of this intervention (oocyte selection by PM) would

result from studies comparing performing PM for oocyte selection vs. not performing PM, preferentially by a

RCT. In the previously published studies, all oocytes were submitted to PM, and therefore the potential

deleterious effect of the additional oocyte handling was not examined. To the best of our knowledge, this is

the first study examining this aspect; however, the evidence of the present study is still limited because it is

based on a retrospective analysis rather than being based on a RCT.

Conclusions

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Although oocyte selection by PM can potentially increase the fertilization rate, this strategy reduces the

proportion of TQE and it is unlikely to improve the main reproductive outcomes, as live birth and clinical

pregnancy. Randomized controlled trials would provide better quality evidence about the effect of oocyte

selection by PM on the main ART outcomes.

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Acknowledgments

We wish to thank the employees of the Laboratory of Assisted Reproduction: Maria Aparecida Carneiro

Vasconcelos, Marilda Hatsumi Yamada Dantas, Sandra Aparecida Cavichiollo and Ricardo Perussi e Silva for

technical support.

Conflict of interest

We declare that none of the authors had a conflict of interest regarding the results of the present study.

Authors’ contributions

Maria C. PICINATO: Conception and design, data collection, interpretation of the results, writing, critical

revision, final approval of the article.

Wellington MARTINS: Conception and design, interpretation of the results, statistics, writing, critical

revision and final approval of the article.

Roberta C. GIORGENON: data collection and final approval of the article.

Camila K. B. SANTOS: data collection and final approval of the article.

Rui A. FERRIANI: critical revision and final approval of the article

Paula A. A. S. NAVARRO: critical revision and final approval of the article

Ana C. J. S. ROSA E SILVA: Conception and design, interpretation of the results, writing, critical revision

and final approval of the article.

Source of financial support

No institution provided financial support for this study.

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Table 1 Characteristics from the women included in this study.

PM group No-PM group

N 201 women 249 women

Age (years) 33.41±3.69 33.77±4.56

Transferred embryos 2.10±0.50 2.01±0.55

Number of oocytes retrieved 7.33±4.60 7.98±4.80

Number of oocytes MII 4.86±2.68 5.34±3.16

Subfertility cause

Endometriosis 65/201 (32.3%) 88/249 (35.3%)

Anovulation 30/201 (14.9%) 39/249 (15.6%)

Tubal Factor 49/201 (24.3%) 49/249 (19.7%)

Male factor 71/201 (35.3%) 79/270 (29.3%)

PM group = women whose oocytes were submitted to polarization microscopy before ICSI. No-PM group =

women whose oocytes were not submitted to polarization microscopy before ICSI. Data expressed as mean

± SD for age and number of transferred embryos, oocytes retrieved, and oocytes in metaphase II (MII). Data

expressed as numerator/denominator (percentage) for the subfertility cause. P-values were obtained by

either Fisher’s exact test or unpaired t test. The subfertility causes were not mutually exclusive; e.g. one

couple could have more than one cause.

Table 2 Reproductive outcomes observed in the group submitted to polarization microscopy before ICSI (PM

Group) and in the group not submitted to polarization microscopy before ICSI (No-PM group).

PM Group

1,000 oocytes

201 women

No-PM Group

1,400 oocytes

249 women

p

Fertilization rate 797/1,000 (79.7) 1,015/1,400 (72.5) <0.01

Cleavage rate 687/797 (86.2) 939/1,015 (92.5) <0.01

TQE formation 264/797 (33.1) 506/1,015 (49.9) <0.01

Implantation rate 79/423(18.7%) 103/501(20.6%) 0.51

Positive pregnancy test rate 74/201(36.8%) 101/249(40.6%) 0.44

Clinical pregnancy rate 64/201(31.8%) 83/249(33.3%) 0.76

Miscarriage rate 14/64(21.9%) 13/83(15.7%) 0.93

Live birth rate 50/201(24.9%) 70/249(28.1%) 0.46

Data presented as numerator/denominator (%). PM = polarization microscopy; MS = meiotic spindle; MII =

metaphase II; p-value obtained by Fisher’s exact test.

Table 3 Comparison of fertilization, cleavage, and top quality embryos (TQE) formation considering only the

oocytes submitted to polarization microscopy, comparing the oocytes with an unidentified meiotic spindle

(MS), those with a MS in metaphase II, and those with a MS in telophase I.

Unidentified MS Metaphase II Telophase I

Oocytes injected 134 827 39 p-value *

Fertilization rate 100/134 (74.6) 671/827 (84.14)a 26/39 (66.67)a 0.03

Cleavage rate 87/100 (87.0) 580/671 (86.4) 20/26 (76.9) 0.29

TQE 30/100 (30.0) 225/671 (33.5) 9/26 (34.6) 0.45

Data presented as numerator/denominator (%); * = p-value obtained by the χ² test; a = pairwise comparison,

p =0.04 by Fisher’s exact test.

Table 4 Fertilization, cleavage and top quality embryos (TQE) formation from oocytes submitted to

polarization microscopy (PM) comparing those with meiotic spindle (MS) identified in metaphase II (MII), but

in different positions regarding the polar body.

Meiotic spindle position on PM

1 2 3 4 5 6 p-value

N oocytes 668 126 29 1 3 0

Fertilized 542 (81.1) 103 (81.8) 24 (82.8) 0 (0.0) 2 (66.7) - 0.31

Cleaved 468 (86.3) 90 (87.4) 20 (83.3) - 2 (100.0) - 0.89

TQE 181 (33.4) 35 (34.0) 8 (33.3) - 1 (50.0) - 0.97

Oocytes in MII were divided into six groups according to the angle detected between MS and polar body:

Position 1= 0-30°, Position 2=30-60°, Position 3=60-90°, Position 4=90-120°, Position 5=120-150° and

Position 6=150-180° in relation to the PB. P-value obtained by the χ² test.

Supplemental Table 1 Fertilization, cleavage and top quality embryos (TQE) formation

comparing only the results obtained from oocytes submitted to polarization microscopy (PM

Group) with meiotic spindle identified in metaphase II, and only those with meiotic spindle

identified in position 1, with those in the group not submitted to polarization microscopy (No-

PM Group).

PM Group No-PM Group p

Oocytes classified as MII

827 oocytes

All oocytes

(N=1,400)

Fertilization rate 671/827 (81.1) 1,015/1,400 (72.5) <0.01

Cleavage rate 580/671 (86.4) 939/1,015 (92.5) <0.01

TQE 225/671 (33.5) 506/1,015 (49.9) <0.01

Oocytes with MS in position 1

668 oocytes

All oocytes

(N=1,400)

Fertilization rate 542/668 (81.1) 1,015/1,400 (72.5) <0.01

Cleavage rate 468/542 (86.3) 939/1,015 (92.5) <0.01

TQE 181/542 (33.4) 506/1,015 (49.9) <0.01

Data presented as Numerator/Denominator (%). PM = polarization microscopy; MS = meiotic

spindle; MII = metaphase II; p-value obtained by Fisher’s exact test.