IVF laboratory, Maria Infertility Hospital, Sinseol-dong, Dongdaemun-gu, Seoul 121742, Korea
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Abstract |
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Key words: blastocyst/hCG/immature oocytes/PCOS/vitrification
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Introduction |
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It has been reported that priming with hCG before immature oocyte retrieval in PCOS patients improves oocyte maturation, fertilization, embryo development and pregnancy rates (Chian et al., 1999, 2000
). In addition, it has also been reported that mature oocytes can be collected at the time of retrieval using only in-vivo hCG priming in women with PCOS, and clinical pregnancy can be established by transfer of blastocysts derived from the mature oocytes (Son et al., 2002
).
So far, only one case of the birth of an infant from cryopreserved embryos (zygotes) produced by IVM oocytes derived from an unstimulated patient with PCOS has been reported (Chian et al., 2001). However, it is still unclear whether the blastocysts produced by IVM oocytes can be frozen for further embryo transfer. In this case report, we present an ongoing pregnancy resulting from transfer of vitrified blastocysts produced from IVM oocytes. To the best of our knowledge, this is the first report of a pregnancy from cryopreserved blastocysts produced by IVM oocytes derived from the unstimulated ovary of a patient with PCOS.
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Case report |
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A 33 year old patient with PCOS presented with anovulation, polycystic ovaries visible on ultrasound, elevated serum testosterone concentrations (1.4 nmol/l on day 2) and12 IU/ml LH in serum on menstrual cycle day 2. The patient had failed to become pregnant after eight cycles of conventional IVF treatment and six cycles of cryopreservation over the past 5 years. The patient had experience of OHSS. In a previous COH cycle, 31 oocytes were retrieved from this patient. The patient had abdominal discomfort with ultrasound evidence of enlarged ovaries and ascites, corresponding to grade 3 moderate OHSS. The patient had given i.v. albumin to prevent severe OHSS. To initiate each treatment cycle, the patient received i.m. injections of progesterone (Progest; Samil Pharmacology, Seoul, Korea). Withdrawal bleeding occurred 3 days after the last dose. Ovarian follicle development was monitored by transvaginal ultrasonography (Aloka, Tokyo, Japan) beginning on cycle day 3. The patient was given 10 000 IU of hCG (IVF-C; LG chemical, Seoul, Korea) s.c. on cycle day 16 based on the cycle length and the endometrium thickness. After 36 h, oocytes were aspirated with a 19 gauge aspiration needle (Cook, Eight Mile Plains, Queensland, Australia) under the guidance of transvaginal ultrasound. A portable aspiration pump was used with a pressure of between 80 and 100 mmHg.
The aspirates were collected in 10 ml tubes containing prewarmed heparinized Hams F-10 medium. The follicular aspirates were filtered (70 µm mesh, Falcon 1060; Becton Dickinson, NJ, USA) and washed with Hams F-10 medium to remove erythrocytes and small cellular debris. The retained cells were then resuspended in the medium and the oocytes were isolated under a stereomicroscope. All oocyte handling procedures were conducted in a mini-chamber under 5% CO2 atmosphere at 37°C. Following oocyte collection, immature oocytes with a germinal vesicle (GV) were transferred to maturation medium for culture.
The IVM medium consisted of YS (Yoon Sanhyun) medium (Yoon et al., 2001) supplemented with 30% human follicular fluid (hFF), 1 IU/ml rFSH, 10 IU/ml hCG, and 10 ng/ml rhEGF (Son et al., 2002
). The hFF was prepared as previously described (Chi et al., 1998
). Immature oocytes were cultured in IVM medium at 37°C in an atmosphere of 5% CO2, 5% O2 and 90% N2. Twenty-four hours post-collection, the oocytes were denuded with hyaluronidase (Sigma Chemical Co., St Louis, MO, USA) and mechanical pipetting. Mature [metaphase II (MII)] oocytes were identified by the presence of the first polar body. Immature oocytes were further cultured in fresh IVM medium, and examined prior to ICSI and 48 and 72 h post-collection.
Sperm were prepared by 90% Percoll separation at 300x g for 20 min. After Percoll separation, the sperm pellet was washed twice (300 g) with 3 ml of Hams F10 medium containing 10% hFF and the motile sperm were collected by the swim-up method. Fertilization was assessed 1618 h after ICSI for the appearance of two distinct pronuclei and two polar bodies. Zygotes were co-cultured with cumulus cells in 10 µl YS medium supplemented with 10% hFF (Yoon et al., 2001). Embryos were transferred on day 4 after oocyte retrieval.
After embryo transfer, the surplus embryos were further co-cultured with cumulus cells in 10 µl YS medium supplemented with 10% hFF until day 7 and those embryos that developed to expanded blastocyst stage (with a diameter >160 µm) were cryopreserved using vitrification on an electron microscope (EM) grid. The freezing solution for vitrification, EFS40, was prepared according to a previously described method (Kasai et al., 1990), and consisted of 40% (v/v) ethylene glycol (EG; Sigma Chemical Co.), 18% (w/v) Ficoll (Ficoll 70, average MW: 70 000 kDa; Pharmacia Biotech, Uppsala, Sweden), 0.3 mol/l sucrose and 20% hFF in modified Dulbeccos phosphate buffered saline (m-DPBS). A pretreatment solution, m-DPBS (EG20) containing 20% ethylene glycol and 10% hFF, was prepared. The blastocysts were equilibrated in EG20 for 1.5 min before exposure to the vitrification solution at room temperature. The blastocysts were then incubated in EFS40 at room temperature, loaded onto the EM grid (IGC 400; Pelco International, CA, USA), excess cryoprotectant removed using sterilized filter paper, and were plunged directly in LN2 within 30 s. The EM grids containing the blastocysts were sealed in a cryovial that had previously been submerged under LN2. The cryovials were attached in canes and stored in LN2. Thawing was performed by first transferring the EM grids containing blastocysts to a 100 µl drop of 0.5 mol/l sucrose. After 3 min, the blastocysts were transferred sequentially to 100 µl drops containing 10% hFF in DPBS supplemented with 0.4, 0.3, 0.2, 0.1 and 0 mol/l of sucrose at intervals of 1.5 min at room temperature. The blastocysts were then washed three times in culture medium and co-cultured with cumulus cells in 10 µl YS medium containing 10% hFF. The post-thawing survival of blastocysts was observed about 1820 h after warming under a microscope, and blastocysts with a morphologically intact inner cell mass, trophectoderm and a re-expanding blastocoel were judged to have survived.
The endometrium was prepared for frozen embryo transfer to the anovulatory patient. Briefly, the patient received i.m. progesterone in a dose of 100 mg/night for 10 days to induce a withdrawal bleed. Menstrual bleeding commenced 3 days after stopping the progesterone. On day 5 of the cycle, the patient was given 100 mg clomiphene citrate (Clomiphene; Bando Pharmacology, Seoul, Korea) daily for 5 days, and monitored by ultrasound. Progesterone 100 mg was administrated daily from the day of embryo transfer. Progesterone was continued until a fetal heartbeat was positively identified.
For the IVM attempt, a total of 61 immature oocytes at GV stage was obtained, five, 27 and eight reached MII after 24, 48 and 72 h of culture respectively. Thirty-eight oocytes (five, 26 and seven in MII oocytes matured at 24, 48 and 72 h respectively) were fertilized after ICSI, and 31 cleaved. A total of four embryos (two eight-cell embryos of grade 1 and two six-cell embryos of grade 1) were transferred 4 days after oocyte collection. On the day of embryo transfer, the endometrial thickness was 10 mm at transvaginal ultrasonogram. No pregnancy occurred after embryo transfer. Out of remaining 27 embryos, eight were developed to expanded blastocysts and cryopreserved by vitrification on an EM grid. Two years after cryopreservation, four blastocysts were thawed for scheduled embryo transfer. Following thawing, three blastocysts re-expanded (75%) and were transferred (one grade 2, two grade 3). The endometrial thickness measured 10 mm on the day of embryo transfer. Nine days later, the serum ß-hCG concentration was 358.5 IU/l and 6 weeks after embryo transfer an ongoing intrauterine twin pregnancy with fetal heartbeat was confirmed by transvaginal ultrasonography. A subsequent scan at 30 weeks gestation confirmed viability of both fetuses.
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Discussion |
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Although Cha et al. (1991) were the first to describe a successful pregnancy by IVM, the immature oocytes used in that study were collected from ovarian biopsy specimens taken at the time of Caesarean section (Cha et al., 1991
). PCOS is a very common endocrine disorder. The first report of a successful of pregnancy following IVM of immature oocytes in a woman with anovulatory infertility was reported in 1994 (Trounson et al., (1994)
. Approximate clinical pregnancy rates of 2530% were recently reported from immature oocytes retrieved from patients with PCOS following IVM in several infertility centres (Cha et al., 2000
; Child et al., 2001
). Therefore, it seems possible that recovery of immature oocytes followed by IVM of these immature oocytes could be a promising treatment for women with PCOS (Chian et al., 2000
; Child et al., 2001
).
Recently, blastocyst transfer based on the improved culture system has been proven to be effective for increasing the pregnancy rate in IVF programmes (Gardner et al., 1998; Yoon et al., 2001
). However, so far, only one case of blastocyst embryo transfer and pregnancy derived from immature oocytes collected from unstimulated ovaries has been reported (Barnes et al., 1995
). This might be attributed to a poor quality oocyte caused by asynchrony in the cytoplasmic and nuclear maturation of the oocyte. Cytoplasmic maturity might not be complete despite a matured nucleus in IVM cycles. Aberrations in cytoplasmic maturation are more likely to be apparent as a failure in later stages of development (Moor et al., 1998
). Another possible explanation could be that good-quality embryos are obtained in relatively low numbers due to non-optimal culture condition in the IVM cycles, compared with conventional IVF cycles. However, Chian and his colleagues recently reported that rates of oocyte maturation and pregnancy could be improved by hCG priming 36 h before immature oocyte retrieval in women with PCOS (Chian et al., 2000
). We have also reported similar results for the effect of in-vivo hCG priming in patients undergoing IVM cycles (Son et al., 2001
). In addition, we improved the IVM/in-vitro culture (IVC) system, and obtained acceptable results (Son et al., 2002
). Our previous results also show that high clinical pregnancy rates (50.0%, 11/22) are achieved by the transfer of blastocysts produced from IVM oocytes after in-vivo hCG priming (Son et al., 2002
). In one patient in this study, 23.5% (8/34) 2PN embryos developed to the blastocyst stage, without counting the embryos transferred at cleavage stage. This indicates that hCG priming and an improved culture system can produce high quality human embryos from IVM cycles.
Cryopreservation of human embryos has become a routine procedure to increase cumulative pregnancy rates in IVF programmes. The success of human blastocyst vitrification has recently been improved by techniques using either EM grids (Choi et al., 2000) or cryo-loop (Mukaida et al., 2001
), that substantially increase the cooling rate. We also established a vitrification system on EM grids containing a 6-step cryoprotectant dilution method, and have reported the clinical usefulness of the system for the cryopreservation of human blastocysts (Cho et al., 2000
). In this study, we obtained a reasonable clinical pregnancy rate (34.1%) in the transfer cycle.
To date, only one case of pregnancy after transfer of a cryopreserved 2PN embryo derived from immature oocytes collected from unstimulated ovaries has been reported (Chian et al., 2001). However, there has been no report of clinical pregnancy of vitrified blastocysts derived from IVM-matured oocytes. Based on the experience of vitrification in blastocysts derived from our IVF programme, we vitrified the blastocysts derived from IVM-matured oocytes and report in this study an ongoing twin pregnancy currently at 35 weeks gestation with normal fetal development.
In conclusion, the blastocysts produced from IVM oocytes retrieved from unstimulated women with PCOS can be safely cryopreserved by vitrification on an EM grid and successful ongoing pregnancy can be achieved following embryo transfer. This report highlights the use of three advanced assisted reproduction techniques, namely IVM, blastocyst culture and vitrification, to overcome infertility problems.
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Note added in proof |
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Notes |
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References |
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Submitted on April 15, 2002; accepted on May 28, 2002.