1 Service de Biologie et Génétique de la Reproduction and 2 Service de Gynécologie, Obstétrique et Médecine de la Reproduction, Hôpital Antoine Béclère, Clamart, France and 3 Department of Obstetrics and Gynecology, McGill University, Royal Victoria Hospital, Montréal, Québec, Canada 4 Present address: Department of Obstetrics and Gynecology, Saint-Luc Hospital (CHUM), Montréal, Québec, Canada
5 To whom correspondence should be addressed at: Service de Biologie et Génétique de la Reproduction, Hôpital Antoine Béclère, 157 rue de la porte de Trivaux, 92140 Clamart, France. Email: anne.ledu{at}abc.ap-hop-paris.fr
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Abstract |
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Key words: in vitro maturation/oocyte/ovarian hyperstimulation syndrome/polycystic ovary syndrome
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Introduction |
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Materials and methods |
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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 18). 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 cumulusoocyte complexes were collected in 15 ml NucleonTM (Nunc A/S, Denmark) tubes containing pre-warmed 3 ml sodium heparinate 2 IU/ml (SanofiSynthelabo, France). Follicular aspirates were not washed on a filter but tubes were spread onto sterile polystyrene culture dishes of 60 mm diameter NucleonTM (Nunc A/S). The cumulusoocyte 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 cumulusoocyte 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 cumulusoocyte 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 SpermTM (Nidacom, Sweden) preparation (90 and 45%) was performed and 90% Pure Sperm layer was washed in FerticultTM 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 (4144 h after insemination/injection) and on day 3 (6671 h post-insemination/injection): (i) fragmentation (A=no fragmentation; B=
20% by volume of anucleated fragments; C=2050% 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 610 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 -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).
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Results |
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Oocyte retrieval
Forty-five punctures were performed. The follicles obtained by puncture measured 39 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 cumulusoocyte complexes punctured, and the results of IVM and of IVF.
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Embryo freezing
Thirty supernumerary embryos from nine attempts were frozen. No pregnancy resulted in the four patients who received a total of 13 frozenthawed embryos from their previous cycle.
Pregnancy outcomes
Following embryo transfer, 11 women had a positive -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.
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Discussion |
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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 cumulusoocyte 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 cumulusoocyte 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
).
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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).
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barbieri R (2003) Metformin for the treatment of polycystic ovary syndrome. Obstet Gynecol 101, 785793.
Barnes F, Crombie A, Gardner D, Kausche A, Lacham-Kaplan O, Suikkari AM, Tiglias J, Wood C and Trounson AO (1995) Blastocyst development and birth after in vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum Reprod 10, 32433247.[Abstract]
Barnes FL, Kausche AB, Tiglias J, Wood C, Wilton L and Trounson A (1996) Production of embryos from in vitro matured primary human oocytes. Fertil Steril 65, 11511156.[ISI][Medline]
Bonduelle M, Ponjaert I, Steirteghem AV, Derde MP, Devroey P and Liebaers I (2003) Developmental outcome at 2 years of age for children born after ICSI compared with children born after IVF. Hum Reprod 18, 342350.
Campo S (1998) Ovulatory cycles, pregnancy outcome and complications after surgical treatment of polycystic ovary syndrome. Obstet Gynecol Surv 53, 297308.[CrossRef][Medline]
Cha K, Koo JJ, Choi DH, Han SY and Yoon TK (1991) Pregnancy after in vitro fertilization of human follicular oocytes collected from non stimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 55, 109113.[ISI][Medline]
Cha K and Chian RC (1998) Maturation in vitro of immature human oocytes for clinical use. Hum Reprod Update 4, 103120.
Cha K, Han S, Hyung M, Chung H, Choi D, Lim J, Lee W, Ko J and Yoon T (2000) Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome. Fertil Steril 73, 978983.[CrossRef][ISI][Medline]
Chian R, Güelkli B, Buckett W and Tan S (1999a) Priming with human chorionic gonadotropin before retrieval from unstimulated women with infertility due to the polycystic ovary syndrome. N Engl J Med 341, 16241626.
Chian R, Buckett W, Too L and Tan SL (1999b) Pregnancies resulting from in vitro matured oocytes retrieved from patients with polycystic ovary syndrome after priming with human chorionic gonadotropin. Fertil Steril 72, 639642.[CrossRef][ISI][Medline]
Chian R, Buckett W, Tulandi T and Tan SL (2000) Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 15, 165170.
Chian R, Gulekli B, Buckett W and Tan SL (2001) Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome. Hum Reprod 16, 17001702.
Chian RC and Tan SL (2002) Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles. Reprod BioMed Online 5, 125132.[Medline]
Chiesa-Montadou S, Rongieres C, Garbin O and Nisand I (2003) About two complications of ovarian drilling by fertiloscopy. Gynecol Obstet Fertil 31, 844846.[Medline]
Child T, Abdul-Jalil A, Gulekli B and Tan SL (2001) In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystics ovaries, and women with polycystic ovary syndrome. Fertil Steril 76, 936942.[CrossRef][ISI][Medline]
Child T, Phillips S, Abdul-Jalil A, Gulekli B and Tan S (2002) A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstet Gynecol 100, 665670.
Edwards R (1965) Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 20, 349351.
Edwards R, Bavister B and Steptoe P (1969) Early stages of fertilization in vitro of human oocytes matured in vitro. Nature 221, 632635.[ISI][Medline]
Fauser and Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group (2004) Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Hum Reprod 19, 4147.
Fernandez H, Alby J-D, Gervaise A, De Tayrac R and Frydman R (2001) Operative transvaginal hydrolaparoscopy for treatment of polycystic ovary syndrome: a new minimally invasive surgery. Fertil Steril 75, 607611.[CrossRef][ISI][Medline]
Gougeon A (1996) Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev 17, 121155.[ISI][Medline]
Kyono K, Fukunaga N, Haigo K, Chiba S, Ohira C, Nakajo Y and Araki Y (2002) Successful delivery following cryopreservation of zygotes produced by in vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome-like Disease: a case report. J Assist Reprod Genet 19, 390393.[CrossRef][ISI][Medline]
Lassalle B, Testart J and Renard JP (1985) Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil Steril 49, 283289.
Lin YH, Hwang JL, Huang LW, Mu SC, Seow KM, Chung J, Hsieh B, Huang SC, Chen CY and Chen PH (2003) Combination of FSH priming and hCG priming for in-vitro maturation of human oocytes. Hum Reprod 18, 16321636.
Liu J, Lu G, Qian Y, Mao Y and Ding W (2003) Pregnancies and births achieved from in vitro matured oocytes retrieved from poor responders undergoing stimulation in in vitro fertilization cycles. Fertil Steril 80, 447449.[CrossRef][ISI][Medline]
MacDougall MJ, Tan SL, Balen A and Jacobs HS (1993) A controlled study comparing patients with and without polycystic ovaries undergoing in vitro fertilization. Hum Reprod 8, 233237.[Abstract]
Mikkelsen A (2004) In vitro maturation of human ova. Int Congr Ser 1266, 160166.[CrossRef]
Mikkelsen A and Lindenberg S (2001) Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome: a randomized prospective study. Reproduction 122, 587592.
Nagele F, Sator M, Juza J and Huber J (2002) Successful pregnancy resulting from in-vitro matured oocytes retrieved at laparoscopic surgery in a patient with polycystic ovary syndrome: case report. Hum Reprod 17, 373374.
Son WY, Yoon SN, Park SJ, Yoon HJ, Lee WD and Lim JH (2002) ongoing twin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome. Hum Reprod 17, 29632966.
Trounson A, Wood C and Kausche A (1994) In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 62, 353362.[ISI][Medline]
Van Royen E, Mangelschots K, de Neubourg D, Valkenburg M, Van de Meerssche M, Ryckaert G, Eestermans W and Gerris J (1999) Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod 14, 23452349.
Veeck L, Worthman J, Witmyer J, Sandow B, Acosta A, Garcia J, Jones G and Jones H (1983) Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil Steril 39, 594602.[ISI][Medline]
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction. Cambridge University Press, Cambridge, UK.
Submitted on May 14, 2004; resubmitted on August 9, 2004; accepted on October 15, 2004.