High follicle-stimulating hormone increases aneuploidy in human oocytes matured in vitro

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High follicle-stimulating hormone increases aneuploidy in human oocytes matured in vitro

Yan-Wen Xu, M.D., Ph.D., Yue-Ting Peng, M.D., Bin Wang, M.D., Yan-Hong Zeng, B.Sc., Guang-Lun Zhuang, M.D., and Can-Quan Zhou, M.D.

Reproductive Medical Center, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China

Objective: To study the effect of FSH on the aneuploidy risk of human oocytes matured in vitro.

Design: Prospective study.

Setting: Hospital-based IVF center.

Patient(s): Patients with male factor infertility undergoing intracytoplasmic sperm injection (ICSI) cycles.

Intervention(s): Immature oocytes were put into five groups according to the FSH concentration (0, 5.5, 22, 100, and 2,000 ng/mL) in in vitro maturation (IVM) medium. Spindles were observed under a polarized microscope before polar body biopsy. Fixed polar bodies and corresponding oocytes were examined on chromosomes 13, 16, 18, 21, and 22 by fluorescence in situ hybridization. Oocytes matured in 5.5 and 2,000 ng/mL FSH were immunostained for tubulin and chromatin.

Main Outcome Measure(s): Aneuploidy rate, spindle visualization rate, and spindle morphology.

Result(s): The frequency rates of aneuploidy were 26.7%, 23.3%, 36.75%, 46.67%, and 63.3% in the five FSH groups, respectively. There was a significantly higher aneuploidy rate in oocytes matured in the 2,000 ng/mL FSH group. The spindle visualization rates assessed under PolScope were not significantly different between aneuploid and normal oocytes. There was no difference in spindle morphology between the 2,000 and 5.5 ng/mL FSH groups.

Conclusion(s): High-concentration FSH in IVM medium significantly increased the first meiotic division error, re- sulting in more aneuploid oocytes during IVM. (Fertil Steril^Ò2011;95:99–104. Ó2011 by American Society for Reproductive Medicine.)

Key Words: FSH, oocytes, in vitro maturation, aneuploidy

Aneuploidy is the most common chromosomal abnormality(1, 2).

The rate of occurrence is increased with maternal age, but the underlying causes of aneuploidy remain unclear. It has been hypothesized that an elevated gonadotropin level (either endogenous or exogenous) may be one underlying cause of aneuploidy(3). However, there is still debate about whether elevated FSH leads to an increase of aneuploidy in women with reduced ovarian reserve(4–6).

Exogenous FSH is often administered to induce multiple follicle growth in assisted reproductive technology (ART). For in vitro maturation (IVM), gonadotropins are routinely added to the culture medium. More and more evidence in animal studies demonstrates that FSH affects chromosomes and damages embryo quality(7–9).

The aneuploidy rate in human oocytes varies from 23% to 56%

(10–13). There have been no human studies showing a direct link between FSH and the genesis of aneuploidy.

Here we aimed to study whether FSH increases the aneuploidy rate in human IVM oocytes in a dose-dependent manner. We investigated the role of spindle visualization with a polarized microscope to predict aneuploidy. Then we evaluated the influence of high FSH on spindle morphology and chromosome configuration under a confocal microscope.

MATERIALS AND METHODS Patient Selection and Treatment

A total of 252 immature oocytes were collected from 86 intracytoplasmic sperm injection (ICSI) cycles from October 2008 to January 2010 in the Reproductive Medical Center of the First Affiliated Hospital, Sun Yat-sen University, China.

All patients received a long protocol for stimulation. Gonadotropin admin- istration with FSH (Gonal-F; Merck-Serono Company, Switzerland) 150–300 IU/day was initiated from days 3–5. Oocyte retrieval was performed 34–36 hours after 10,000 IU hCG was administered. Germinal vesicle (GV) or metaphase I (MI) stage oocytes were collected under patients’ written con- sent of donation for research. Our study excluded patients older than 40, patients with more than three treatment cycles, and patients with less than two available immature oocytes. This study was approved by Medical Ethical Committee of the First Affiliated Hospital, SUN Yat-sen University.

IVM

Cumulus-oocyte complexes (COCs) were observed under an inverted microscope (40) following 20 seconds of exposure in hyaluronidase. As shown in Figure 1, immature oocytes had tight and dense granulose cells surrounding Received September 24, 2009; revised April 15, 2010; accepted April 16,

2010; published online June 9, 2010.

Y.-W.X. has nothing to disclose. Y.-T.P. has nothing to disclose. B.W. has nothing to disclose. Y.-H.Z. has nothing to disclose. G.-L.Z. has nothing to disclose. C.-Q.Z. has nothing to disclose.

Yan-Wen Xu and Yue-Ting Peng contributed equally to this study.

This study was supported by the National Basic Research Program of China (grant no. 2007CB948101), the China Medical Board of New York Inc (grant no. 06840), and the National Natural Science Founda- tion of China (grant no. 30700910).

This study was presented at the Annual Meeting of the American Society for Reproductive Medicine, October 17–21, Atlanta, Georgia.

Reprint requests: Can-Quan Zhou, M.D., Reproductive Medical Center, the First Affiliated Hospital of Sun Yat-sen University, No. 58, Zhongshan Road 2, Guangzhou, People’s Republic of China (FAX:

0086-20-87755766-8365; E-mail:zhoucanquan@hotmail.com).

0015-0282/$36.00 Fertility and Sterility^âVol. 95, No. 1, January 2011

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the oocytes. In difficult cases, a holding pipette was used to rotate the COCs for the maturity assessment.

Tissue culture medium (TCM 199; Sigma, St. Louis, MO) was supple- mented with 10% serum protein substitute (SAGE In-Vitro Fertilization, Inc., Trumbull, CT), 50,000 IU/L penicillin (Sigma, St. Louis, MO), 50 mg/L streptomycin (Sigma, St. Louis, MO), and 25 mmol/L sodium pyruvate (Sigma, St. Louis, MO). Oocytes were allocated into five groups with 0, 5.5, 22, 100, and 2,000 ng/mL Gonal-F in IVM culture medium, respectively.

Spindle Visualization by Polarized Microscope

Oocytes with polar bodies (PBs) were biopsied 30–36 hours after IVM. Im- mediately before biopsy, a polarization-imaging software module (OCTAX ICSI Guard, MTG, Germany) was used to visualize the meiotic spindle (Fig. 2).

Fluorescence in Situ Hybridization (FISH)

The PB was biopsied and transferred onto a grease-free slide. After being completely dried, one drop of 3:1 mixture of methanol/acetic acid was added to fix PB chromosomes. The technique described by Dozortsev and Mcginnis (14)was used to fix the corresponding oocytes after PB biopsy (Fig. 1).

Five chromosomes were analyzed with commercial probes (MultiVy- sionTM PB; Abbott Laboratories, Chicago, IL). Denaturation and hybridization were performed according to the manufacturer’s recommendation. We used the scoring criteria described by Munne et al.(15).

Immunocytologic Staining

Oocytes matured in 5.5 and 2,000 ng/mL FSH were immunostained for tubulin and chromatin(9). Each group contained 20 oocytes. The localization of

FIGURE 1

IVM and PB biopsy. (A) GV oocyte; (B) MII oocyte; (C) zona opening; (D, E) PB aspiration; and (F, G) chromosomes spreading from PB and oocyte.

Xu. Influence of FSH on aneuploidy. Fertil Steril 2011.

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tubulin and chromatin was assessed with a Leica laser-scanning confocal microscope (Leica, TCS SPE, Germany).

Normal spindle configuration, where microtubules form two opposite poles, caused the chromosomes to be arranged on a compact plate at the equator. Slightly abnormal spindle configurations were identified when some microtubule structures displayed reduced dimensions of the spindle and lost normal poles, while abnormal spindles showed disorganized microtubule patterns with chromosome scattering (Fig. 3).

Statistical Analysis

Statistical comparisons between groups were performed with analysis of var- iance, c²test, Fisher’s exact test, and Student’s t-test in SPSS 10.0 (Chicago).

P<.05 was considered statistically significant.

RESULTS

A total of 171 oocytes were matured in vitro from 172 GV and 80 MI oocytes (Table 1). Together, 87.5% of the diagnosis based on the PBs was consistent with the results of the corresponding oocytes. Owing to PB fragmentation and chromosome condensa- tion, all the subsequent analyses were based on the results obtained from the oocytes.

There was no significant difference in aneuploidy rates between matured MI and GV oocytes (34.5% vs. 42.4%; P>.05).

However, the proportion of aneuploid oocytes increased dramati- cally with the increasing FSH concentration. The aneuploidy rate of the oocytes were 26.7%, 23.3%, 36.75%, 46.7%, and 63.3% for FSH levels of 0, 5.5, 22, 100, and 2,000 ng/mL, respectively..

Compared with the 0 and 5.5 ng/mL groups, there was a significantly higher aneuploidy rate in the 2,000 ng/mL FSH group (Table 2).

We observed unbalanced premature chromatid separation in 30 cases and chromosome nondisjunction in 12 cases. However, the difference was not statistically significant. The other 17 oocytes were of complex origin, with more than one chromosome error.

The overall spindle visualization rate was 52.0% (89/171), and 78 oocytes with visualized spindles had FISH results. The spindle visualization rate in aneuploid oocytes was 53.8% (29/59), similar to the rate of 49.2% (49/91) in normal oocytes. There was no difference in

abnormal gross spindle morphology and chromosomal configuration between the 2,000 and 5.5 ng/mL FSH groups (45.0% vs.

50.0%) after immunostaining.

DISCUSSION

There is growing evidence in animal studies to support the hypothesis that elevated FSH level is one underlying cause of aneuploidy(3). The FSH concentrations in different studies of FSH activity in IVM vary greatly, ranging from 0 to 2,000 ng/mL (9, 16, 17). In Roberts et al., high-level FSH exposure induces chromosomal abnormalities in a dose-dependent manner in a mouse IVM model(9).

However, a direct link between FSH and the genesis of aneuploidy in stimulation has not been established. In Xu et al.(18), there is no difference between in vivo and in vitro matured oocytes derived from both naturally cycling and stimulated mice. Only a single dose of 5.0 IU of recombinant FSH was injected for the purpose of stimulation or FSH priming before IVM. The aneuploidy rate is extremely low, ranging from 6.0% to 9.8%, which suggests that animal studies cannot be generalized to the human.

In our study, an FSH concentration of 5.5 ng/mL is equal to 0.075 IU/mL. The highest concentration of 2,000 ng/mL was chosen because it induced three-dimensional reconstructions of spindles in mouse oocytes(9). Clinically, the FSH level in follicular fluid in stimulated cycles has been reported to range from 5.54 to 7.80 IU/L(19), which is 1–2 IU/L lower than the serum FSH level. How- ever, it should be much higher on hCG day, since the serum FSH level drops 4–9 IU/L from hCG day to retrieval day in our prelimi- nary data.

Similar to the results in Roberts et al.(9), our results showed that aneuploidy rates increased with the FSH level. We observed a significant difference in the 2,000 ng/mL FSH group. However, our study is limited by the scarcity of human materials.

The exact mechanism by which FSH induces aneuploidy remains unclear, but there are several possible explanations. First, it may be due to the oocyte susceptibility to the environment during the first meiosis (20). According to a multicenter trial Spindle images visualized under OCTAX ICSI Guard. (A) MII oocyte with spindle image; and (B) MII oocyte without spindle image.

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that investigated the safety of follicular fluid meiosis-activating sterol for IVM of human oocytes, both prolonging the culture time from 30 to 36 hours and the 20-mM dose of follicular fluid meiosis-activating sterol increased the aneuploidy rate(21). Sec- ond, FSH may increase aneuploidy owing to its effects on microtubule (3, 22). Finally, FSH may rescue abnormal oocytes and facilitate the meiotic resumption of these oocytes that may otherwise be destined to atresia.

The development of PolScope has allowed a noninvasive way of assessing the spindle structure of oocytes(23). Our results demon- strated that spindle visualization under PolScope cannot predict the chromosomal status of in vitro matured human oocytes.

Immunocytologic staining for tubulin and chromatin is a classical method to evaluate spindle configuration in matured oocytes. In the study by Roberts et al.(9), FSH has no effect on gross spindle morphology; however, chromosomes are more scattered and farther

FIGURE 3

Spindle images visualized under a confocal microscope. (A–C) Normal spindle with microtubules (green) form barrel-shaped configuration, and chromosomes (blue) are arranged on a compact plate at the equator. (D–F) Slightly abnormal spindle configuration in which microtubules display reduction in the dimensions of the spindle. (G–I) Abnormal spindle with an aberrant, less condensed appearance of chromosomes and a disorganized microtubule pattern.

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apart in oocytes cultured in 2,000 ng/mL. We found no difference in chromosome configuration. The discrepancy may due to the limited sample size, and importantly, the software used to assess spindle three-dimensional morphology in Roberts et al. was not available in our study.

In summary, our results show that high FSH plays an important role in the aneuploidy formation of in vitro matured human oocytes.

Spindles birefringent cannot be used to predict the aneuploidy risk in human oocytes. More caution should be used when using a high dose of gonadotropins in human ART.

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Comparison of general characteristics among five groups.

Group 1 Group 2 Group 3 Group 4 Group 5

Mean age 34.04 4.90 31.57 4.40 31.39 4.58 32.54 4.32 31.39 4.22

Mean no. of ampoules 30.21 11.8 27.13 8.98 24.28 8.18 25.34 9.01 24.18 8.38

Mean basal FSH level (IU/L) 5.73 0.93 5.03 1.50 5.47 0.95 5.53 1.43 5.48 1.57

Mean FSH level on day of hCG (IU/L)

11.86 4.40 9.49 6.56 9.20 3.56 9.74 4.32 8.27 2.97

Mean no. of oocytes collected

18.58 11.12 18.15 9.12 17.71 7.02 18.29 10.31 16.19 10.10

No. of GV oocytes 39 30 32 32 39

No. of MI oocytes 16 17 18 16 13

GV maturation rate, % 56.4 66.7 62.5 65.6 56.4

MI maturation rate, % 75.0 88.2 83.3 87.5 76.9

No. of MII oocytes after IVM

34 35 35 35 32

No. of oocytes successfully biopsied

33 34 33 34 31

No. of fixed PBs 33 33 32 32 31

No. of fixed oocytes 32 33 31 32 30

No. of PBs with FISH results

27 31 31 29 31

No. of oocytes with FISH results

30 30 30 30 30

Note:Where appropriate, data are expressed as mean SD. Groups 1–5 had 0, 5.5, 22, 100, and 2,000ng/mL FSH supplementation, respectively. For the features surveyed, there were no significant differences among the five groups.

TABLE 2

Comparison of aneuploidy rates among groups.

Group 1 Group 2 Group 3 Group 4 Group 5

No. of oocytes with FISH results

30 30 30 30 30

No. of aneuploid oocytes

8 7 11 14 19

Pvalue NA NA >.05 >.05 .001

Note:Compared with group 1 and group 2, there was a significantly higher aneuploidy rate in group 5.

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