In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience

Abstract

BACKGROUND: In vitro oocyte maturation (IVM) permits the use of immature oocytes in IVF. IVM does not require ovarian stimulation and so can be offered to patients at risk of ovarian hyperstimulation syndrome. METHODS: For this indication, we carried out 45 cycles of IVM in 33 women with polycystic ovarian syndrome (PCOS). RESULTS: A total of 509 cumulus–oocyte complexes was obtained; 276 (54.2%) oocytes matured in 24 h and 45 (8.8%) in 48 h. The normal fertilization (2PN) rate of oocytes matured in 24 and 48 h was 69.5 and 73.3% respectively. Among the 214 embryos obtained, 103 were transferred and 30 were frozen. Forty transfers were performed (2.5 embryos/transfer). Eleven women had a positive β-hCG test (26.2% of pregnancies/puncture, 27.5% of pregnancies/transfer) and nine women had a clinical pregnancy (20.0% of pregnancies/puncture, 22.5% of pregnancies/transfer). Five babies have been born and one pregnancy is ongoing. Results of the clinical examination carried out at birth were normal. CONCLUSIONS: Our results show that IVM may be offered as an alternative to conventional IVF and to ovarian drilling in women with PCOS. The role of IVM in the therapeutic armamentarium for this condition should be further clarified.

Introduction

In the follicular phase of the menstrual cycle, a single follicle grows up to the pre-ovulatory stage, then releases its oocyte for possible fertilization, while the other follicles undergo atresia. During gonadotrophin stimulation, the woman receives hormonal treatment intended to make several follicles grow so that several fertilizable oocytes are produced. These treatments, often preceded by down-regulation of the pituitary, are long and may cause side-effects that are more or less well tolerated. Hyperstimulation occurs in 2% of ovarian stimulations and may be serious, even life-threatening. The cost of these treatments is also very high, constituting a sizeable proportion of health care expenditure. This is why early puncture of immature follicles followed by in vitro maturation (IVM) of oocytes represents a feasible alternative for the management of infertile couples. The first IVM of human oocytes and the first fertilization of an in vitro-matured oocyte were reported by Edwards (1965) and Edwards et al. (1969) respectively. The first birth was achieved by Veeck et al. (1983), although in this case the immature oocytes were obtained during a cycle with ovarian stimulation. Since this time, other teams have reported cases of live births following IVM of oocytes obtained from non-stimulated ovaries (Cha et al., 1991; Trounson et al., 1994; Barnes et al., 1995). Cha et al. (1991) reported a triplet birth occurring after the donation of oocytes obtained by aspiration of follicles, themselves recovered by ovariectomy during a Caesarean section. Trounson et al. (1994) reported the first pregnancy achieved in patients with polycystic ovarian syndrome (PCOS) from immature oocytes recovered by puncture during non-stimulated cycles. Women with PCOS constitute the ideal population for this approach. These women are extremely sensitive to ovarian stimulation, which is required because of their ovulatory disorder, and frequently develop severe ovarian hyperstimulation syndrome (OHSS) (MacDougall et al., 1993). Follicular puncture prior to IVM is a new method for the management of this infertility (Child et al., 2001). We report the management of 33 couples during 45 cycles of IVM in women with PCOS.

Materials and methods

Patients

The report concerns 33 patients with PCOS managed at the Reproductive Medicine and Biology centre of Antoine Béclère Hospital between September 2002 and December 2003. All had signed a consent form after receiving information on the goals and modalities of the procedure. These patients consistently met both echographic criteria (≥24 follicles of 2–9 mm at the beginning of the follicular phase) and clinical criteria (oligomenorrhoea and/or hyperandrogenism) (Fauser et al., 2004) for the diagnosis of PCOS. The mean (±SD) age of the patients was 32.4 ± 3.4 years (range 22–39). The mean number of follicles of 2–9 mm at the beginning of the follicular phase was 40 (18–67). Seventeen women (51%) had at least one ovary of volume >10 ml. Twenty-four women (73%) had irregular cycles (35–90 days) and five (15%) had amenorrhoea. Thirteen women (39%) had clinical signs of hyperandrogenism and six (18%) had biological signs of hyperandrogenism.

Patients suffered from the following types of infertility: PCOS (n=9), PCOS/tubal infertility (n=5), PCOS/endometriosis (n=1), PCOS/diethylstilbestrol in utero exposure (n=1), PCOS/carrier of a Robertsonian translocation 45,X,der(13;14)(q10;10) (n=1), PCOS/oligoasthenoteratozoopermia (n=12), PCOS/azoospermia (n=2) and PCOS/antisperm antibodies (n=1), PCOS/tubal infertility oligoasthenoteratozoopermia (n=1).

All couples had previously received assisted reproductive therapy. Nine had only undergone intrauterine inseminations, without pregnancy. Twenty-four couples had attempted IVF at least once. The mean number of attempts was 2.9 ± 1.6 (range 1–8). OHSS had been experienced by 10 women, severe in six and moderate in four. Nine women had undergone ovarian drilling, without success.

Monitoring of IVM

The patients received a specialized consultation, as is offered to all patients with PCOS managed by our unit. A transvaginal ultrasound, hormonal measurements [FSH, LH, estradiol (E₂), prolactin, delta-4-androstenedione, 17-hydroxyprogesterone, sex hormone-binding globulin, insulinaemia, glycaemia, triglycerides and cholesterol] and karyotyping were performed on cycle day 3. If the cycle was highly irregular, dydrogesterone (Duphaston^®; Solvay Pharma, France) was also prescribed to correct it. The patient's consent was obtained during this consultation. The first ultrasound scan was scheduled for the third day of the cycle, as well as determination of E₂, LH and progesterone levels and measurement of endometrial thickness. These investigations were repeated around the sixth to eighth day of the cycle to exclude the development of a dominant follicle. The patient received 10 000 IU of hCG Chorionique Endo^® (Organon, France) s.c. when follicle size reached 7 mm, before selection of the largest follicle (Gougeon, 1996). The cycle is cancelled if the patient has a dominant follicle on the day of hCG administration. All patients received hCG according to Chian et al. (1999b, 2000) who have demonstrated in IVM cycles that hCG priming increases both the percentage and rate of immature oocyte maturation.

Oocyte retrieval

Oocyte recovery was performed 36 h after hCG injection. During the collection, patients received a mild i.v. sedation with propofol (Driprivan^®; AstraZeneca, France). Transvaginal ultrasonographically guided oocyte collection was done using a specially designed 19-Gauge single-lumen aspiration needle (K-OPS-7035-Wood; Cook, France). The aspiration pressure was set at 7.5 kPa. Follicular aspirates containing cumulus–oocyte complexes were collected in 15 ml Nucleon™ (Nunc A/S, Denmark) tubes containing pre-warmed 3 ml sodium heparinate 2 IU/ml (Sanofi–Synthelabo, France). Follicular aspirates were not washed on a filter but tubes were spread onto sterile polystyrene culture dishes of 60 mm diameter Nucleon™ (Nunc A/S). The cumulus–oocyte complexes were isolated under a stereomicroscope and then washed once in the culture medium, Universal IVF Medium^® (Medi Cult, Denmark), warmed to 37°C in a thermostatically controlled incubator under an atmosphere enriched to 5% CO₂.

IVM and fertilization

After two washes in the maturation culture medium (Chian and Tan, 2002), the cumulus–oocyte complexes were placed in the central wells of a Falcon 3037 culture dish (Becton Dickinson, USA) containing 1 ml maturation culture medium supplemented with 20% inactivated maternal serum at 56°C, 0.75 IU FSH and 0.75 IU LH Menopur^® (Ferring, Germany). The oocytes were incubated at 37°C under an atmosphere enriched to 5% CO₂. After 24 h of maturation, all the cumulus–oocyte complexes were decoronated with hyaluronidase solution (80 IU Syn Vitro Hyadase; Medi Cult). The mature oocytes were determined by the presence of a first polar body extrusion. Mature oocytes were fertilized by ICSI on the same day. Nude immature oocytes with a germinal vesicle or germinal vesicle breakdown were transferred for a further 24 h in the maturation culture medium at 37°C under an atmosphere enriched to 5% CO₂. Mature oocytes after 48 h of culture were fertilized by ICSI. Immature oocytes after 48 h of maturation were not fertilized. All oocyte handling procedures were conducted on warm stages and plates at 37°C.

Preparation of sperm

Semen samples were collected at the laboratory the day following follicular puncture. After liquefaction at 37°C, semen were analysed according to the World Health Organization (1999) guidelines. Discontinuous Pure Sperm™ (Nidacom, Sweden) preparation (90 and 45%) was performed and 90% Pure Sperm layer was washed in Ferticult™ medium (FertiPro NV, Belgium) by centrifugation at 600 g for 10 min. The pellet of motile sperm was used for the microinjection of oocytes matured for 24 h and preserved at room temperature for microinjection of oocytes matured for 48 h.

In one case, a testicular biopsy was carried out the day after follicular puncture. Sperm were collected from the supernatant after delaceration of the removed tissue. ICSI was carried out after 24 h of oocyte maturation.

Embryo culture

After ICSI, oocytes were individually cultured in microdrops of 35 μl of culture medium, ISM1 (Medi Cult) under paraffin mineral oil (Mineral Oil; Medi Cult) at 37°C under an atmosphere enriched to 5% CO₂. Fertilization was assessed 18 h after the injection for the appearance of two pronuclei and two polar bodies. The embryos were cultured for 2 or 3 days. The quality of the embryo was evaluated daily by taking into account development kinetics, the presence of anucleated exudates and the appearance of the blastomeres (Van Royen et al., 1999). All embryos were scored for three parameters on day 2 (41–44 h after insemination/injection) and on day 3 (66–71 h post-insemination/injection): (i) fragmentation (A=no fragmentation; B=≤20% by volume of anucleated fragments; C=20–50% by volume of anucleated fragments); (ii) number of blastomeres; (iii) number of multinucleated blastomeres.

Preparation of endometrium and embryo transfer

For endometrial preparation, E₂ Provames^® (Aventis, France) was administered daily at a dose of 6–10 mg depending on the endometrial thickness on the day of oocyte retrieval, starting on the day of oocyte retrieval. If the endometrial thickness on the day of oocyte retrieval was <5 mm, a 10 mg dose was administered; if it was >5 mm, a 6 mg dose was given. Progesterone Estima G^® (Effik, France), was prescribed on the day of the puncture. The estroprogesterone therapy was continued until the 12th week of pregnancy. At the same time, adjuvant treatments, such as folic acid, Aspegic^® (acetylsalicylic acid) and antibiotics were administered as in our IVF programme. The uterine transfer was carried out using a classic CCD catheter after a cervical wash.

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. Therefore, the developmental stage of each embryo transferred may have been different in individual women. The embryos were placed at 1 cm from the fundus. An endovaginal ultrasound scan was carried out on the day scheduled for the transfer to ensure that the endometrial thickness was >7 mm. If the endometrial thickness was <7 mm, the couples were offered embryo cryopreservation and transfer in a subsequent cycle. When the β-hCG tests were positive, E₂ and progesterone were prescribed until the 12th week of pregnancy. Clinical pregnancy was defined as an intrauterine gestation with a fetal heartbeat seen by transvaginal ultrasound scan. Concerning chromosome anomalies and malformations, non-invasive management of pregnancies using maternal serum markers and fetal ultrasound examinations were performed.

Freezing

Supernumerary embryos were frozen if their quality permitted this, using cryopreservation medium (Embryo freezing; Medi Cult, France). Embryo freezing was performed by the slow freezing protocol (Lassalle et al., 1985).

Results

Monitoring

The mean day of ovulation induction by hCG injection was day 11 ± 3 (range 8–18). The mean plasma concentrations of E₂, progesterone and LH on the day of induction were 54.1 ± 25.8 pg/ml, 0.22±0.2 ng/ml and 10.4±5.0 mIU/ml respectively. There were no cycles cancelled because of small follicle size or appearance of a dominant follicle. Fourteen women underwent two ultrasound examinations, 19 women underwent three and 12 women underwent four ultrasound examinations during the monitoring, before hCG administration.

Oocyte retrieval

Forty-five punctures were performed. The follicles obtained by puncture measured 3–9 mm. Three were blank. No severe adverse effects such as infection, bleeding or abdominal pain referred to as gynaecological were observed in this study.

IVM and embryo culture

Table I shows the number of cumulus–oocyte complexes punctured, and the results of IVM and of IVF.

Embryo transfer

Forty transfers were performed. In two cases, transfer did not take place because of failure of fertilization (n = 1) or poor embryo development (n = 1). There were no cycles cancelled because of thin endometrium, and all patients had an endometrial thickness of 8–13 mm. The transferred embryos were selected solely according to their quality. Five (4.9%) embryos were grade A, 44 (42.7%) were grade B, 52 (50.5%) were grade C and two (1.9%) were grade D. In 35 cases, the transferred embryos were from oocytes matured in 24 h. In one case, the transferred embryos were from oocytes matured in 48 h. In four cases, the transferred embryos were from oocytes matured in 24 and 48 h.

Embryo freezing

Thirty supernumerary embryos from nine attempts were frozen. No pregnancy resulted in the four patients who received a total of 13 frozen–thawed embryos from their previous cycle.

Pregnancy outcomes

Following embryo transfer, 11 women had a positive β-hCG test. Two pregnancies did not proceed beyond the sixth week of amenorrhoea, leaving nine clinical pregnancies. Three women had a miscarriage in the first trimester, and, in one of the three cases, cytogenetic analysis showed a normal female karyotype (46,XX). Six women had a normally developed pregnancy. Five children were delivered at full term, in good health. One pregnancy is ongoing. Among the six pregnant women, only one had a child previously by IVF. However, this number of patients was too small to detect a difference. Among the six pregnant women, five received embryos obtained from oocytes matured for 24 h and one woman received embryos obtained from oocytes matured for 24 and 48 h. The children are all followed up by examination when they are 1 and 2 years old. They are all healthy, the oldest child being 1 year old.

Discussion

This is the first report in France of births occurring after IVM of oocytes derived from non-stimulated cycles in women with PCOS.

Since the report by Trounson et al. (1994), there have been ∼100 reported births following IVM unstimulated cycles in women with PCOS (Barnes et al., 1995; Cha and Chian, 1998; Chian et al., 1999a, 2000, 2001; Cha et al., 2000; Abdul– ; Kyono et al., 2002; Nagele et al., 2002; Son et al., 2002; Lin et al., 2003). IVM is being developed as an assisted reproductive technique because of the many advantages it offers. Monitoring is simpler, both for patients and clinicians. It is shorter and gonadotrophins are not administered. Therefore patients do not experience side-effects such as hot flushes, local reactions at the injection site, mood disturbances, and, most importantly, there is no risk of ovarian stimulation. The cost is also reduced. Oocyte puncture during a non-stimulated cycle seems to be a worthwhile alternative to conventional IVF. However, it is technically much more difficult to recover immature oocytes obtained by follicular puncture that are <9 mm. The cumulus cells are not as expanded as they are during a stimulated cycle. The size of the cumulus–oocyte complex is similar to that of the oocyte (∼0.1 mm). Furthermore, the cumulus cells are not refringent, making it more difficult to identify the cumulus–oocyte complex. The puncture process also takes much longer because the follicles are not easily detected by ultrasound. Thus, in our study we used an i.v. sedation for the aspiration. No complications related to puncture have occurred.

In our study, the average number of immature oocytes recovered by puncture is comparable to that reported by other teams managing non-stimulated patients with PCOS (Trounson et al., 1994; Cha and Chian, 1998; Chian et al., 1999a, 2000; Cha et al., 2000; Child et al., 2001; Lin et al., 2003; Table II). The oocyte maturation percentage is comparable to that reported in the literature (Barnes et al., 1995; Cha et al., 2000; Chian et al., 2000), even though we use a different medium. We do not supplement the TCM-199 with hCG. The protein source is the patient's serum, not fetal calf serum. In series of >20 cycles of IVM, the maximum rate of pregnancy obtained was 27% following embryo co-culture with Vero cells (Cha et al., 2000).

PCOS involves a spectrum of aetiologies with various clinical manifestations. The first-line treatment is medical: management of excess body weight, dyslipidaemia and carbohydrate intolerance. Anovulation is usually treated with clomiphene citrate 100 mg/day in the follicular phase. Combination treatment with metformin has been suggested for its hypoinsulinaemic action (Barbieri, 2003). If clomiphene citrate alone or in combination with metformin leads to treatment failure, defined as absence of ovulation or pregnancy, several alternatives can be envisaged. One is stimulation by gonadotrophins, although the risk of hyperstimulation and/or multiple pregnancies remains, even when using the ‘step-up low-dose’ or ‘step-down’ protocols. Ovarian surgery may be offered: cuneiform resection by laparotomy, celioscopic electrocauterization, laser vaporization, and percelioscopic multiple biopsies (Campo, 1998). Recently, the value of fertiloscopy for ovarian drilling has been underlined (Fernandez et al., 2001). However, apart from the inherent risks of i.v. anaesthesia and of the surgical procedure itself (Chiesa-Montadou et al., 2003), the risk of treatment failure remains. Therefore, given the present situation, we believe that study of the feasibility of IVM is warranted. The role of IVM in the management of patients with PCOS should be re-examined in the context of a randomized controlled trial.

In previous human IVM investigations, the delivery of ∼300 babies has been reported after IVM followed by assisted reproduction treatments (Mikkelsen, 2004) and the birth of ∼100 healthy babies was reported after IVM in women with PCOS (Table II). In our series, no birth defects were noted. However, these babies must be followed up, as for babies born from ICSI-fertilized oocytes or from embryos biopsied for preimplantation genetic diagnosis (Bonduelle et al., 2003).

Our study has shown the effectiveness of IVM for the management of women with PCOS. Other patients, such as poor responders, could also benefit from IVM (Liu et al., 2003).

We are grateful to the technicians, the nurses, the secretary and Mrs Marie Dominique Prat for their assistance.

References

Author notes

1Service de Biologie et Génétique de la Reproduction and 2Service de Gynécologie, Obstétrique et Médecine de la Reproduction, Hôpital Antoine Béclère, Clamart, France and 3Department of Obstetrics and Gynecology, McGill University, Royal Victoria Hospital, Montréal, Québec, Canada 4Present address: Department of Obstetrics and Gynecology, Saint-Luc Hospital (CHUM), Montréal, Québec, Canada