1 Department of Obstetrics and Gynecology, MBC #52 and 2 Department of Pathology and Laboratory Medicine MBC #10, King Faisal Specialist Hospital and Research Center, PO Box 3354, Riyadh, 11211, Saudi Arabia
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Abstract |
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Key words: embryo/human/ICSI/in-vitro maturation
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Introduction |
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Gonadotrophin stimulation is used to achieve multifollicular recruitment, enabling an increased number of embryos to be transferred. However, there are disadvantages associated with gonadotrophin stimulation. Some are economical and related to the cost of drugs, ultrasound, etc. The estimated cost of an in-vitro fertilization (IVF) cycle being in the region of $10 000. Other major side effects of superovulation are ovarian hyperstimulation and deep vein thrombosis (Roest et al., 1996; Steward et al., 1997
) and other possible long-term side effects of fertility drugs include ovarian cancer (Whittemore et al., 1992
). Hence, there is an increasing interest in retrieving oocytes without gonadotropins or with limited gonadotrophin exposure and then maturing them in vitro for embryo transfer purposes. The cost of an in-vitro (IVM) cycle may be ~$5000. A large pool of preantral and antral oocytes is theoretically available, both from `poor' responders and other types of infertility. Maturation and fertilization of human oocytes has been successfully performed in vitro (Edwards, 1965
; Edwards et al., 1969
; Shea et al., 1975
). However, pregnancies have not been established from embryos generated from such oocytes until more recently (Cha et al., 1991
; Trounson et al., 1994
; Barnes et al., 1995
; Jaroudi et al., 1997
; Russell et al., 1997
). Previously, we have reported that immature oocytes can be retrieved and matured in vitro from patients at risk of hyperstimulation after gonadotrophin injections (Coskun et al., 1998
). The aim of this report is to present our experience of fertilization, embryonic development and pregnancies in IVM cycles over the last 2 years from a similar patient population.
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Materials and methods |
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Oocyte retrieval and IVM
Immature oocyte recovery and maturation from a similar patient population were described previously (Coskun et al., 1998). Briefly, the aspirates obtained from all of the visible antral follicles were poured into 60 mm dishes as a thin layer. Immature oocytes were visualized under a stereo-dissecting microscope. A second search was also performed after sedimentation of red blood cells in order to obtain better visibility of the immature oocytes. The dishes were examined by at least two biologists. An average of 8.1 oocytes (171 immature oocytes from 21 cycles) was obtained, while no oocytes were recovered from a single patient. All of the oocytes together with some granulosa cells were transferred into HEPES-buffered media with 10% synthetic serum substitute (SSS, Irvine Scientific, Santa Ana, CA, USA) and washed twice. They were then cocultured in 50 µl of human tubal fluid (HTF, Irvine Scientific) supplemented with 10% SSS, 75 mIU/ml HMG and 500 mIU/ml HCG under pre-equilibrated mineral oil (R.E. Squibb & Sons Inc., Princeton, NJ, USA). Cumulusoocyte complexes were decoronated after 44 h in culture with a 160 µm capillary pipette following exposure to 80 U/ml of hyaluronidase (Sigma, St Louis, MO, USA) for 30 s. The stage of nuclear maturation of the oocytes was checked under an inverted microscope and classified as germinal vesicle, metaphase I, II or degenerated. Cumulus cells from decoronation and granulosa cells from maturation medium were collected, washed with fresh medium and resuspended in 200 µl HTF supplemented with 10% SSS. This suspension was used as 20 µl drops for the culture of individual injected oocytes.
Sperm preparation and ICSI
Semen was diluted with 10 ml HTF containing 10% SSS and centrifuged at 1800 g for 5 min. Discontinuous Percoll separation (95 and 47.5%) was performed and the 95% Percoll layer was washed in HTF containing 10% SSS by centrifugation at 1800 g for 5 min. ICSI was performed as previously described (Palermo et al., 1992). Injected oocytes were cultured as described above. Fertilization was checked 18 h later by the presence of two pronuclei and two polar bodies. Embryos were cultured for a further 2 days and development was monitored daily. Embryos were graded as good, fair and poor. Good embryos were defined as those with even-sized blastomeres and no obvious fragmentation, with even-sized blastomeres and <10% fragmentation or uneven-sized blastomeres with no obvious or <10% fragmentation. Fair embryos had 1030% fragmentation and poor embryos were heavily fragmented (>30%). Embryo transfer was performed 5 days after immature oocyte retrieval under abdominal ultrasound guidance using a Wallace® catheter (Sims Portex Ltd, Kent, UK).
Endometrial priming
From the day of immature oocyte retrieval, 8 mg of oestradiol valerate was administered. Progesterone 50 mg i.m. was administered daily starting 2 days after oocyte retrieval. Both medications were continued until either a negative pregnancy urine test or a positive fetal heartbeat was seen on ultrasound at 5 weeks of gestation.
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Results |
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Discussion |
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Cleavage, implantation and pregnancy rates were lower than those obtained with conventional IVF/ICSI in our clinic. Other studies have indicated low cleavage rates after fertilization of in-vitro matured oocytes. Rates of 59.2 and 65.2% from either PCO or regular cycling patients respectively were found (Barnes et al., 1996). Similarly, 56% cleavage rates in anovulatory PCO patients were obtained (Trounson et al., 1994
). The hormonal status of the patient during follicular growth may also affect the cleavage rate. A higher cleavage rate was shown in patients who received endometrial priming around the midfollicular phase of the menstrual cycle compared to those who received early follicular phase priming (Russell et al., 1997
).
Although total oocyte wastage was high in this study (118/171, 69%), the number of embryos transferred (mean 2.4) is comparable to regular IVF/ICSI cycles. However, this number did not translate into comparable pregnancy or implantation rates. This may be related to the low efficiency of the IVM system, since only 53 embryos were available for transfer out of 171 oocytes retrieved. There was a very limited selection of good quality embryos, since 83% of the available embryos had to be transferred. In regular IVF/ICSI cycles ~50% of the generated embryos are available for embryo transfer, enabling morphologically better embryos to be transferred. Only 10% pregnancy and 4.5% implantation rates were achieved in this study. These lower pregnancy and implantation rates are the major obstacles preventing IVM from entering the regular clinical practice. Other investigators have also reported lower pregnancy and implantation rates (Trounson et al., 1994; Barnes et al., 1997
; Russell et al., 1997
). This low success rate might be attributed to asynchrony in the cytoplasmic and nuclear maturation of the oocyte. Cytoplasmic maturity may not be complete despite the fact that nuclear maturation may be satisfactory. Another reason for the low success rate could be related to the patient population in this study. All the patients were at risk of hyperstimulation, with high concentrations of oestradiol and the majority had PCO or anovulation. It has been reported (Aboulghar et al., 1997
) that oocytes in hyperstimulated patients are of inferior quality and hence have lower fertilization rates, although the number of oocytes may be higher than in controls. When hyperstimulated and control groups were categorized in terms of prevalence of PCO, it was concluded that PCO rather than high response may affect oocyte quality. It has been also reported that immature oocytes recovered from PCO patients exhibit compromised developmental potential when compared to regular cycling patients (Barnes et al., 1996
).
During normal development of an antral follicle, the follicle and cumulus oocyte complex undergo a highly co-ordinated maturation process (Eppig, 1997). Assisted reproductive technology programs employ gonadotrophin stimulation to achieve multifollicular growth in order to increase the number of embryos for transfer (Edwards and Brody, 1995
). However, the low percentage of zygotes reaching blastocyst stage, as well as low implantation rates in stimulated cycles (Edwards and Brody, 1995
), suggest that the natural growth process is negatively affected. This adverse effect may be even more prominent in IVM generated oocytes.
In conclusion, IVM oocytes can undergo fertilization and the resulting embryos can establish pregnancies. However, the success rate is low. There must be a major improvement of IVM success rates in order to achieve results at least equivalent to those of current stimulation protocols prior to its routine use in daily clinical practice.
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Notes |
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References |
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Submitted on June 26, 1998; accepted on March 15, 1999.