McGill Reproductive Center, Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada H3A 1A1
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Abstract |
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Key words: HCG/human/immature oocyte/in-vitro maturation/polycystic ovarian syndrome
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Introduction |
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Recovery of immature oocytes followed by in-vitro maturation (IVM) of these immature oocytes is a potentially useful treatment for women with PCOS-related infertility since the immature oocytes from these women retain their maturational and developmental competence (Trounson et al., 1994). In comparison with conventional IVF, the major benefits of IVM treatment include avoidance of the risk of OHSS, reduced cost, and less complicated treatment. However, the maturation rate of immature oocytes retrieved from women with PCOS is lower than that of those retrieved from women with normal menstrual cycles (Cha and Chian, 1998
). There is a paucity of information about the maturational capacity of immature oocytes derived from women with PCOS.
Several authors have reported pregnancies following the transfer of embryos produced from immature oocytes derived from stimulated ovaries (Veeck et al., 1983; Prins et al., 1987
; Nagy et al., 1996
; Edirisinghe et al., 1997
; Jaroudi et al., 1997
; Liu et al., 1997
; Tucker et al., 1998
). However, the pregnancy rate reported is correspondingly low. It has been demonstrated that morphological and molecular differences exist between immature oocytes retrieved from stimulated and unstimulated ovaries (Chian et al., 1997
). Although the time courses of germinal vesicle breakdown (GVBD) and oocyte maturation are different between immature oocytes retrieved from stimulated and unstimulated ovaries, the final rates of oocyte maturation are not different in these two groups (Cha and Chian, 1998
). It appears that the oocytes retrieved from small follicles in women undergoing ovarian stimulation respond to human chorionic gonadotrophin (HCG) which promotes the initiation of oocyte maturation. However, it remains unclear whether priming with HCG before immature oocyte retrieval in unstimulated ovaries could improve the rates and time course of oocyte maturation and fertilization, as well as the embryo quality.
The present study was designed to determine whether priming with HCG 36 h before immature oocyte retrieval alters the rates of oocyte maturation, fertilization and early embryo development in patients with PCOS.
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Materials and methods |
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The treatment cycle was initiated by the administration of intravaginal progesterone (Prometrium; Schering, Pointe-Claire, Quebec, Canada) in a dose of 300 mg daily for 10 days. The timing of the start of treatment was random, as all patients had irregular menstrual cycles. Withdrawal bleeding occurred within 3 days after the last dose. On day 2 or 3 following the onset of menstrual bleeding, the patients underwent a baseline ultrasound scan to ensure that no ovarian cysts were present. Transvaginal ultrasound scans were repeated on day 8 to exclude the development of a dominant follicle. The size of all follicles on ultrasound scan had to be <10 mm diameter on day 8 of the cycle.
Oocyte retrieval was performed on day 10 to 14 of the cycle. Using a computerized random table, the patients were assigned either to be primed with 10 000 IU HCG (Profasi; Serono, Oakville, Ontario, Canada) (group A), or not primed (group B). In group A the HCG was given s.c. to patients 36 h before oocyte retrieval. Transvaginal ultrasound-guided oocyte collection was performed using a specially designed 17G single-lumen aspiration needle (K-OPS-1235-Wood, Cook, Australia) with an aspiration pressure of 7.5 kPa. Aspiration of all small follicles was performed under spinal anaesthesia. Oocytes were collected in 10 ml culture tubes (Falcon, Franklin Lakes, NJ, USA) containing 2 ml warm 0.9% saline with 2 IU/ml heparin (Baxter, Toronto, Ontario, Canada).
Following oocyte collection, the oocytes were evaluated for the presence or absence of a germinal vesicle (GV) in the cytoplasm of the oocyte (Figure 1), and the immature oocytes were then transferred into maturation medium for culture. To identify whether GV was present in the oocyte cytoplasm, the special technique of observation called `sliding' was employed under the stereomicroscope. Briefly, the cumulusoocyte complex (COC) was allowed to slide slowly down from one side to the other on the bottom of the tissue culture dish (60x15 mm; Falcon), the COC being observed under the microscope. During COC sliding, it was possible to observe clearly whether or not the oocyte cytoplasm contained a GV. If no GV was seen in an immature oocyte, the oocyte was defined as germinal vesicle breakdown (GVBD). The mature oocytes were determined by the presence of a first polar body extrusion. All oocyte handling procedures were conducted on warm stages and plates at 37°C. COC were washed in TC-199 medium (Sigma Chemical Co., St Louis, MO, USA) with 10% inactivated (56°C, 30 min) fetal bovine serum (FBS; Sigma Chemical Co.). The immature oocytes were then incubated in an organ tissue culture dish (60x15 mm; Falcon) containing 1 ml of maturation medium, TC-199 medium supplemented with 20% FBS, 25 mol/l pyruvic acid (Sigma Chemical Co.), 75 mIU/ml follicle stimulating hormone (FSH) + LH (Humegon; Organon, Scarborough, Ontario, Canada) at 37°C in an atmosphere of 5% CO2 and 95% air with high humidity. Following culture, the maturity of the oocytes was determined under the microscope at 12 h intervals for up to 48 h.
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Following fertilization check, oocytes with two pronuclei (maximum five) were transferred into 1.0 ml of Medi-Cult IVF medium in the organ tissue culture dish (60x15 mm; Falcon) for further culture. Embryos were transferred on day 2 or 3 after ICSI. Since the oocytes were not matured and inseminated at the same time following maturation in culture, the developmental stages of embryos were variable both within and between patients. Before transfer, all embryos for each patient were pooled and selected for transfer. Therefore, the developmental stages of each embryo transferred may have been different in individual women (Figure 2). However, the range of variation for embryo growth in the two groups of patients should have been the same.
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The statistical significance of the differences between the means and percentages of the groups was determined and compared by StudentNewmanKeuls' test (Steel and Torrie, 1980). A P-value < 0.05 was considered statistically significant.
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Results |
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Discussion |
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The cyclic pattern of FSH and LH secretion in women with PCOS is typically absent, and there is often a disproportionately high secretion of LH with a relatively constant low rate of FSH secretion (Yen, 1980). The exact mechanism by which anovulation occurs in these women is unknown. There is a consensus that the common feature in women with PCOS is arrested follicular development at the stage when selection of the dominant follicle should normally occur (Erickson and Yen, 1984
). The initial steps of folliculogenesis, follicle recruitment, and follicle growth to the small antral stage, are functional in women with PCOS, but the terminal stepthe selection of a dominant follicle that is capable of ovulationdoes not occur regularly (Jakimiuk et al., 1997
). Normally, the mechanism of LH action on the ovary starts with the binding to specific LH receptors on the theca and granulosa cells, followed by a rapid stimulation of adenylate cyclase activity. Stimulation of adenylate cyclase results in elevated concentrations of cyclic adenosine monophosphate (cAMP) which then interacts with the regulatory subunits of protein kinase, resulting in production or activation of the appropriate enzymes (Marsh, 1976
). The action of oocyte maturation inhibitor (OMI) is itself inhibited by increased cAMP concentrations associated with the mid-cycle LH peak (Tsafriri and Pomerantz, 1986
). Excessive LH secretion is frequently encountered in women with PCOS (Waldstreicher et al., 1988
), and it has been shown that excessive LH secretion is associated with anovulation, infertility and miscarriage (Regan et al., 1990
; Balen et al., 1993
; Shoham et al., 1993
).
It is postulated that acquisition of optimal competence for maturation of immature oocytes in women with PCOS may require the presence of a mid-cycle LH surge for priming. It is known that GVBD is initiated by the pre-ovulatory surge of LH, suggesting that LH induces the loss of communication between the oocytecumulus cell complex, thus terminating the flow of OMI (Fagbohun and Downs, 1991). The results of this study show that there were 46.2 ± 5.2% of GVBD oocytes at the time of oocyte retrieval 36 h after HCG administration, which may suggest that the small follicles in polycystic ovaries possess some LH receptors, even though there is no direct evidence that LH receptors are present in the granulosa cells of these small follicles. This is an area that clearly requires further study.
In women with normal ovaries, the time courses of GVBD and oocyte maturation are different between immature oocytes retrieved from stimulated and unstimulated ovaries; however, the final rates of oocyte maturation are similar (Cha and Chian, 1998). The results of the present study indicate that the rate of oocyte maturation during the first 48 h of culture is increased in women with PCOS following HCG priming before immature oocyte retrieval. Three of the six women who achieved pregnancy and did not miscarry had failed to conceive following three cycles of conventional IVF treatment during the preceding 2 years. These results suggest that immature oocyte retrieval followed by IVM might be useful in 20% (Franks, 1989
) to 37% (Buckett et al., 1999
) of women undergoing IVF who have polycystic ovaries seen on ultrasound scan.
Extremely low fertilization rates are usually obtained after standard insemination of in-vitro-matured oocytes, suggesting that ICSI is the best option, even when the sperm parameters are not impaired (Nagy et al., 1996). Qualitative changes, including zona hardening, occur in the zona pellucida during oocyte maturation in vitro that may reduce the fertilization rates using conventional IVF (DeFelici and Siracusa, 1982
; Downs et al., 1986
; Choi et al., 1987
). There were no differences in the rates of fertilization and cleavage between the HCG-primed and non-HCG-primed groups. This suggests that although the time course and rate of oocyte maturation are hastened by HCG priming before immature oocyte retrieval, the rates of oocyte fertilization and development are not different in the two groups.
It has been suggested that the endometrial priming commencing at the mid-follicular phase may be optimal when embryos resulting from IVM are transferred in the same cycle in which the immature oocytes are retrieved (Russell et al., 1997). However, the results of this study indicate that endometrial priming can be started from the late follicular phase. In this regard, endometrial priming from the late follicular phase seems effective for embryo transfer in the same cycle of immature oocyte retrieval.
In conclusion, the time course of oocyte maturation in vitro, i.e. the maturation rate, has been shown to be hastened by priming with HCG before retrieval of immature oocytes. It is possible that pregnancy rate may potentially be improved by this means in women with PCOS. However, the numbers of patients in this study were too small to detect any such difference.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 24, 1999; accepted on October 8, 1999.