1 Laboratoire de Biologie de la Reproduction, Université Paris V Hôpital Cochin,123 Bd du Port Royal, 75014 Paris and 2 Service de Gynecologie Obstetrique, Hôpital Saint Vincent de Paul, 82 Ave, Denfert Rochereau, 75014 Paris, France
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Abstract |
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Key words: diethylstilboestrol/embryo/infertility/in-vitro fertilization/oocytes
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Introduction |
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The aim of this study was to investigate oocyte quality, fertilization and embryo quality in women exposed to DES by comparing such women participating in an in-vitro fertilization (IVF) programme to a control group.
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Materials and methods |
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Ovarian stimulation and oocyte recovery
The protocols for ovarian stimulation involved associating long or short gonadotrophin-releasing hormone agonist (GnRHa) treatment and administration of gonadotrophins. For the long protocol, the women were down-regulated with GnRHa (triptoreline or leuproreline) and thereafter stimulated with daily injections of human menopausal gonadotrophin (HMG) or purified follicle stimulating hormone (FSH). For the short protocol, GnRHa was started on day 1 of menstruation and HMG was added on day 3 (Frydman et al., 1988). The short protocol was used for 5% (n = 6) of the DES-exposed group and 14% (n = 12) of the control group. Ovulation was induced with 5000 IU of human chorionic gonadotrophin (HCG). Oocytes were harvested 3537 h after HCG injection using a transvaginal ultrasound procedure (Frydman et al., 1988
).
All the oocytecumulus complexes except those with `fractured zona pellucida' and degenerating oocytes were washed and incubated in B2 medium (CCD, Paris, France) before insemination.
Sperm preparation
Patients' semen was collected by masturbation on the day of IVF and allowed to liquefy for 30 min at 37°C. The sperm samples were prepared by a conventional swim-up procedure in 31% of cases and by centrifugation through a mini-Percoll gradient in 69% of cases (Ng et al., 1992).
IVF procedure
From 1989 to 1994, oocytes were classified as mature if they were fully enclosed by expanded radiant corona and cumulus cells (type I), or as immature if the cumulus was partly expanded (type II) or non-expanded with a compact layer of corona cells (type III). Four oocytes per well were inseminated with 30 00060 000 spermatozoa in 1 ml of B2 medium using a multidish four-well system. After 1994, the cumuli were placed into 30 µl drops of B2 medium under equilibrated mineral oil (Merck, Nogent sur Marne, France) and inseminated 3 h after the sperm preparation with 5000 motile spermatozoa. All oocytes were incubated and inseminated in separate microdrops to allow for individual examination and follow-up. Gametes were cultured in a humidified gas incubator at 37°C (5% CO2, 95% air). At ~1720 h post-insemination, the corona cells were removed mechanically by repeated gentle aspiration and expulsion of the oocyte through a Pasteur pipette. The zygotes were transferred into sperm-free B2 medium and observed to determine the pronuclear status of the zygote and the nuclear status of non-fertilized oocytes using a Nikon Diaphot inverted microscope with phase contrast optics, at x400 magnification. Abnormal oocytes with a fractured zona pellucida, atretic oocytes and immature oocytes at the germinal vesicle stage or at metaphase I were recorded. Unfertilized mature oocytes at metaphase II were recognized by the first polar body and the absence of pronuclei.
Forty-eight hours after insemination, cleaved embryos were graded according to their morphological appearance using the following scale: type A (no extracellular cytoplasmic fragments), type B (embryo fragmentation of 120%), type C (embryo fragmentation rate 2150%), and type D (embryo fragmentation rate >50%).
Statistical analysis
Student's t-test was used for statistical evaluation. Statistical significance was defined as a P value < 0.05.
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Results |
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Discussion |
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In mice, it has been demonstrated (MacLachlan et al., 1980) that an abnormally low number of oocytes are recovered and a large number of degenerating oocytes found after ovarian stimulation in females previously exposed to DES in utero. It has also been found (Iguchi et al., 1991
) that mouse oocyte maturation is affected by perinatally administered DES. Neonatal exposure leads to larger gap junctions in the granulosa cells of mature follicles and a stronger attachment among granulosa cells, which prevents disaggregation of the cumulusoophorus complex, even after ovulatory stimuli. DES-exposed mice also discharged a similar number of ova to control mice following stimulation by gonadotrophins, and oocytes from polyovular follicles in DES-exposed mice had a significantly decreased fertilization capacity in vitro.
Data concerning the effects of DES on women's fertility are conflicting. According to previous studies (Senekjian et al., 1988), primary infertility was significantly more frequent among women who had been exposed to DES than among unexposed women. Other studies (Barnes et al., 1980
; Cousins et al., 1980
; Stillman, 1982
) did not find differences in fertility rates between DES-exposed women and unexposed subjects, as estimated by the number of pregnancies per woman. It has been suggested (Bibbo et al., 1977
) that DES exposure may be associated with menstrual irregularities, but other studies (Barnes, 1979
) revealed no significant irregularities at either initial or follow-up examination. During IVF procedures some studies (Muasher et al., 1984
; Karande et al., 1990
) did not find significant differences in preovulatory, immature and degenerated oocytes between patients with tubal disease and DES-exposed women. However, other studies (Sangvai et al., 1996
) found a similar incidence of diminished ovarian reserve and similar follicular recruitment with gonadotrophins in DES-exposed and other women. In the group of in-utero DES-exposed women analysed here, the prevalence of ovulatory dysfunction and endometriosis was very high, but the number and quality of oocytes retrieved after stimulation were similar to those from women not exposed to DES. This suggests that the exposure to DES in utero did not alter the development of the oocytes in the ovary and that those oocytes are not refractory to the maturation stimulus and/or to the removal of inhibitory substances that maintain the oocyte in the germinal vesicle stage. The fertilization ability and the embryo formation also indicate a good qualitative maturation of the oocytes retrieved, but factors such as aneuploidy, embryonic genome expression and ultrastructure have not been assessed in this study.
The clinical pregnancy rate (number of cycles with fetal sacs on ultrasound) per embryo transfer was not significantly different between the two groups although lower in the IVF cycles performed in DES-exposed patients. Thus, lower delivery rate could be related to other important variables such as uterine defects and endometrial morphology and thickness as previously reported (Kaufman et al., 1977; Siegler et al., 1979
; Cabau, 1984
; Epelboin and Bulwa, 1993
; Noyes et al., 1996
; Salle et al., 1996
).
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Acknowledgments |
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Notes |
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References |
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Submitted on August 11, 1998; accepted on February 15, 1999.