Article IVM the first choice for IVF in Italy

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1 RBMOnline - Vol 13 No Reproductive BioMedicine Online; on web 24 May 2006 Article IVM the first choice for IVF in Italy Maria Beatrice Dal Canto graduated in Biology cum laude in 1992 at the University of Pisa, Italy, and became a specialist in 1997 at the University of Milan. In 1994 she became involved in human IVF, achieving a Master s degree in Physiopathology of Assisted Reproduction. She was laboratory director of the IVF Unit at San Gerardo Hospital in Monza, Italy ( ) at the BIOGENESI Reproductive Medicine Centre in Monza since In September 2004 she also became Assistant Professor of Reproductive Biotechnologies at the University of Milan-Bicocca. Her current research interests include in-vitro maturation of human oocytes as well as oocyte cryopreservation. Dr Mariabeatrice Dal Canto MB Dal Canto 1,5 M Mignini Renzini 1, F Brambillasca 1, H Cepparo 1, R Comi 1, A Villa 1, G Rangoni 1, M Mastrolilli 1, M Crippa 1, E de Ponti 2, HI Nielsen 3,4, R Fadini 1 1 BIOGENESI Reproductive Medicine Centre, Istituti Clinici Zucchi, V. Zucchi, 24-Monza; 2 Department of Medical Physics, San Gerardo Hospital, Monza, Italy; 3 Essex Fertility Centre, Holly House Hospital, Essex, UK; 4 MediCult a/s, Denmark 5 Correspondence: Tel/Fax: ; biogenesi.zucchi@grupposandonato.it; mbdalcanto@hotmail. com Abstract In March 2004, a new law was introduced in Italy to regulate assisted reproduction; at present it is impossible to use more than a maximum of three oocytes per IVF cycle, nor can embryos or prezygotes (2PN cells) be selected or cryopreserved. The prohibitions introduced by the new law have, on the one hand, reduced the expectations of success of current techniques and, on the other hand, stimulated clinicians and embryologists to work on new therapeutic strategies so as to offer the highest chances of success with the lowest risks. In-vitro maturation (IVM) of oocytes fits very well with these new requirements: ovarian stimulation is avoided and the handling of spare oocytes is facilitated. The IVM protocol is an intriguing alternative to conventional IVF techniques, since it removes the side-effects of drug stimulation, especially ovarian hyperstimulation syndrome, and it also reduces the costs of the entire procedure, both in terms of time consumption and patient/society costs for drugs. In the authors IVF centre the IVM technique has been used for more than a year, with significant success in terms of maturation and fertilization rates, percentage of embryo transfers, number of pregnancies and, finally, healthy babies born. Keywords: ICSI, immature human oocytes, in-vitro maturation, polycystic ovarian syndrome Introduction The use of immature oocytes, cultured in vitro until metaphase II, for IVF is an important tool in assisted reproduction, which can avoid ovarian hyperstimulation and reduce risks and costs for patients (Edwards et al., 1996, 1997; Pellicer et al., 1996). In addition, cryopreservation of oocytes matured in vitro could be a realistic option to preserve female fertility before surgery/chemotherapy in cancer patients (Isachenko et al., 2004). To date, many teams around the world have reported cases of live births following in-vitro maturation (IVM) of oocytes (Mikkelsen et al., 1999; Chian et al., 2000, 2004; Lin et al., 2003), and recently Mikkelsen (2005) has published a study reporting the follow-up to 24 months of post-natal age of babies born from IVM. Patients with polycystic ovarian syndrome (PCOS) constitute the ideal population for this therapeutic strategy, since they are at high risk of developing ovarian hyperstimulation syndrome (OHSS) (MacDougall et al., 1993; Papanikolaou et al., 2005a,b). Several papers report excellent results with IVM in these patients (e.g. Le Du et al., 2005), whilst only a few studies have been carried out in women with regular menstrual cycles (Mikkelsen et al., 1999, 2000, Suikkari et al., 2000; Child et al., 2001; Son et al., 2002). In general, the pregnancy rate obtained with IVM is a little lower than that resulting from standard IVF because of the smaller number of oocytes and embryos obtained. This reduces the success rate when compared with standard IVF procedures, where selection and cryopreservation of embryos is possible. 159

2 160 In Italy, since March 2004, the law has forbidden the use of more than three oocytes per cycle and requires the transfer of all embryos obtained without any kind of selection. These restrictions have had a visible negative influence on the IVF success rates (Ragni et al., 2005; Rienzi et al., 2005; Ubaldi et al., 2005). Since, in such a context, women undergoing ovulatory induction obtain many more oocytes per cycle than they need, our center has decided to propose the IVM protocol as a first choice for all women with infertility problems, preferring not to use gonadotrophins. In 2004, IVM began to be used both in patients with regular cycles and in patients with polycystic ovaries. This report shows the results of 207 IVM cycles performed in the authors centre, in terms of pregnancy rate in relation to all the retrieved immature oocytes and to the number of oocytes that reached metaphase II stage. Materials and methods Patients and monitoring From July 2004 to September 2005, 152 patients underwent a total of 207 cycles of IVF by intracytoplasmic sperm injection (ICSI) after IVM of oocytes. The average age of patients was 32.2 years (range: 21 39). This study included all couples attending the centre with a maternal age below 40 years and in which the indication for IVF was infertility due to male factor (109 cycles/90 patients), PCOS (38 cycles/27 patients), or unexplained cause (60 cycles/35 patients); this also included patients who had already attempted conventional IVF/ICSI previously without obtaining a pregnancy. Written informed consent was obtained from all participating couples. All patients underwent transvaginal ultrasonography on day 3 of the menstrual cycle to investigate the absence of ovarian cysts and evaluate endometrial thickness. On the same day, a blood sample was taken to determine basal oestradiol serum concentrations. At this point, all patients with ovarian cysts >12 mm or endometrial thickness >5 mm or oestradiol serum concentration >55 pg/ml were excluded from the study. In women with regular menstrual cycles, no FSH stimulation was used prior to the retrieval of immature oocytes, whilst PCOS patients received priming with FSH (Organon, Rome, Italy) 150 IU/day for 3 days, starting on day 3 of the menstrual cycle. An ultrasound scan was repeated between days 6 and 8 of the cycle, so as to check follicular growth and exclude the development of a dominant follicle. Further transvaginal ultrasonographies were scheduled at 2-day to 3-day intervals from one another until a leading follicle of mm in diameter and an endometrium thickness of 5 mm could be seen, at which point oocyte retrieval was scheduled within 24 h. The cycle was cancelled if the patient showed a dominant follicle larger than 14 mm or endometrial thickness of less than 5 mm. No human chorionic gonadotrophin (HCG) priming was given prior to oocyte retrieval in accordance with protocols carried out by Mikkelsen et al. (1999, 2000, 2001). Oocyte retrieval The oocyte retrieval was performed by transvaginal ultrasoundguided follicle aspiration with a single lumen aspiration needle (no E2 17-gauge, 35 cm; Gynetics, Belgium) connected to a vacuum pump (Craft Pump-Rocket UK; pressure 80 mmhg). During the oocyte collection, patients received mild i.v. sedation with propofol (Astra-Zeneca, Milan, Italy). The follicular aspirate, containing oocytes, was collected in a single bottle (tissue culture flask 50 ml; Becton-Dickinson 3014, Mountain View, CA, USA) containing 15 ml of prewarmed flushing medium with heparin (Medi-Cult product no , Jillinge, Denmark). IVM and culture conditions The follicular aspirates were washed on a cell strainer with 70 µm of pore size (no ; Becton Dickinson Falcon, USA). The oocytes were isolated under a stereomicroscope and then washed once in Flushing Medium without heparin (Medi- Cult no ). The oocytes were examined, classified and then placed in a single-well Petri dish (Becton Dickinson, Falcon no. 3037) containing 0.5 ml of pre-equilibrated Lag Medium (vial 1 of IVM system medium; Medi-Cult no ) and incubated at 37 C and 5% CO 2 humidified atmosphere for 3 h. After 3 h, the oocytes were transferred into a 4-well culture dish with 0.5 ml of IVM Medium (vial 2 of IVM system medium; MediCult no ) supplemented with recombinant FSH IU/ml (Serono, Rome, Italy), HCG 0.1 IU/ml (Serono) and 10% maternal serum obtained on the day of aspiration and inactivated at 56 C. The oocytes were cultured in this medium for another 26 h. Following this period, the oocytes were treated with hyaluronidase solution 80 IU (hyaluronidase 80 IU/ml; Sage Media, USA), so as to remove the cumulus complex. The oocytes without cumulus were classified by determining the presence of the first polar body and their cytoplasmic characteristics. In accordance with the Italian law on Assisted Reproduction, ICSI was only performed on the three best mature oocytes. The semen samples were collected at 02:00 pm on the day after oocyte retrieval. After sample liquefaction at 37 C, discontinuous gradients (47.5 and 90%) of Sil-Select (FertiPro, Beernem, Belgium) were performed. The pellet containing motile spermatozoa was washed and resuspended in IVF Medium (MediCult no ) and stored in an incubator at 37 C and 5% CO 2 humidified atmosphere until use. Fertilization was assessed h after injection by the presence of two pronuclei. All the resulting prezygotes were individually cultured in micro-drops of 50 µl of IVF medium under mineral oil (Mineral Oil FertiPro). The embryos were cultured for 2 days. The embryo quality was evaluated daily by observing the percentage of fragmentation and the number of blastomeres.

3 All the resulting embryos were transferred without any selection on day 2 after ICSI, as determined by Italian law. Preparation of endometrium and embryo transfer For endometrial preparation, all patients received 6 mg/day of oestradiol (Novo-Nordisk, Denmark), starting on the day after oocyte retrieval. Luteal support was provided by intravaginal progesterone supplementation (Rottafarm, Italy) 600 mg/day, starting 2 days later. The embryo transfer was carried out using a Gynetics soft catheter (Semtrac set). All embryo transfers were performed 48 h after ICSI. On day 12 after transfer, an evaluation of serum β-hcg was performed. If the result was positive, oestradiol and progesterone were continued until week 12 of pregnancy. The presence of a clinical pregnancy was defined by the evidence of a fetal heartbeat by ultrasound. Statistical analysis Pregnancy rate versus biological characteristics of oocytes were analysed by chi-squared test. Stata software (Stata Corporation, 1999, College Station, TX, USA) was used for performing the statistical analysis. A level of P < 0.05 was adopted for significance. Results Oocyte retrieval, maturation and fertilization Table 1 shows details and biological data. A total of 897 immature oocytes were obtained: 744 (82.9%) with complete cumulus (defined as full cumulus), 98 (10.9%) with partial cumulus (sparse cumulus), 24 (2.7%) with no cumulus (nude) and 31 (3.5%) degenerate oocytes (atretic). The average number of oocytes retrieved per cycle was 4.7 ± 3.2 (range 1 16). Nude and atretic oocytes were discarded, whilst for all the others the maturation protocol was started. In 179 (94.2%) out of the 190 cycles mature oocytes were obtained. Of 842 retrieved oocytes, 475 reached metaphase II with a resulting maturation rate of 56.4%. In agreement with Italian law, only 383 metaphase II oocytes (not more than three per patient) were inseminated, resulting in 319 pre-zygotes with a fertilization rate of 83.3%. In 15 cycles (15/179, 8.4%), matured oocytes were obtained but none fertilized. Embryo transfers A total of 147 grade 1 embryos (completely lacking fragmentation) were obtained; there were 85 grade 2 embryos (with less than 10% of fragmentation); 38 grade 3 embryos (11 20% fragmentation); 29 grade 4 embryos (21 40% of fragmentation); and 20 grade 5 embryos (more than 40% of fragmentation) (Table 1). No embryo was selected and all the embryos were transferred. One hundred and sixty-four embryo transfers were therefore performed in 122 patients. Pregnancies A total of 29 β-hcg positive tests were achieved: 25 were clinical pregnancies, including 23 singletons, one twin and one triplet (Table 2). The pregnancy rates per embryo transfer (PRt) and per patient (PRp) were therefore 15.2 (25/164) and 20.5% (25/122) respectively. The recorded clinical miscarriage rate was 24.0% (6/25; five singletons and one triplet), whilst in 19 cases the pregnancies were ongoing. Up to now, seven healthy children have been born: six males and one female, all singletons. The mean birth weight of the infants was 3080 g ± 576 (range g). Two babies were born by elective Caesarean section at 38 and 39 weeks of gestation; two babies were born by Caesarean section at 35 and 36 weeks of gestational age, for dystocia and fetal distress respectively; three babies were born by vaginal delivery between 36 and 41 weeks of gestation. Only one complication was observed during oocyte retrieval, represented by peritoneal bleeding which necessitated a laparoscopy. No cases of infection or abdominal pain occurred. The observed pregnancy rate was related to the number of oocytes collected and matured (P = 0.03). When only one to three immature oocytes were recovered, the PRt was 6.6% (4/61); with four to six immature oocytes the PRt increased to 16.7% (10/60); with more than six immature oocytes the PRt was 25.6% (11/43) (Table 3). When only one metaphase II oocyte (MII) per cycle was obtained, the recorded PRt was 4.8% (2/42); with two metaphase II oocytes, the PRt was 11.1% (5/45); with three metaphase II oocytes it was 22.2% (8/36); and with more than three metaphase II oocytes the obtained PRt was 24.4% (10/41); the resulting P-value was 0.04 (Table 3). In 56 cycles, the transfer of a single embryo was performed, in 61 cases two embryos were transferred and only in 47 cycles were three embryos available for transfer. The average number of embryos per transfer was 2.1. When only one embryo was transferred, the obtained PRt was 8.9% (5/56) and the pregnancy rate/patient was 9.4% (5/53); with two embryos transferred the resulting PRt was 13.1% (8/61) and the PRp was 14.8 (8/54); with three embryos transferred the PRt was 25.5% (12/47) and the PRp was 30.0% (12/40). The resulting P- value was 0.03 (Table 4). Table 5 shows the results obtained with IVM, ICSI and IVF embryo transfer in terms of pregnancy rates during the study period. All cycles carried out in women >39 years of age and with endometriosis were excluded. 161

4 Table 1. Biological data and details of in-vitro oocyte maturation (IVM). No. % Cycles 207 Cycles with oocytes retrieved Oocytes retrieved a 897 Full cumulus Sparse cumulus Nude Atretic Cycles with MII oocytes MII oocytes/oocytes retrieved b 475/ Oocytes inseminated Oocytes fertilized (2PN) 319/383 Embryo transfers No. of transfers 164/ No. of patients 122 Embryos transferred 319 Embryo fragmentation (%) > Table 2. Clinical data for in-vitro oocyte maturation (IVM). No. % Age a (years) Cycles 207 Patients 152 No. of transfers b No. of patients 122 β-hcg positive test 29 Clinical pregnancies 25 Singleton Twins Triplets Pregnancy rate/transfer 25/ Pregnancy rate/patient 25/ Implantation rate 28/ Babies born Miscarriage HCG = human chorionic gonadotrophin. a Mean ± SD (range) = 32.2 ± 3 (21 39). b Calculated per cycle. MII = metaphase II; PN = pronuclear. a Mean ± SD (range) = 4.7 ± 3.2 (1 16). b Mean ± SD (range) = 2.7 ± 1.8 (1 8). Table 3. Association between number of immature and mature oocytes and pregnancy rate. No. of No. of No. of Pregnancy P-value cycles transfers pregnancies rate/transfer (%) (χ 2 test) Retrieved immature oocytes (n) > Metaphase II oocytes (n) >

5 Table 4. Association between number of embryos transferred and pregnancy rate. No. of No. of No. of PR per PR per P-value transfers patients pregnancies transfer (%) patient (%) (χ 2 test) No. of unselected embryos transferred a b PR = pregnancy rate. a Transfers. b Patients. Table 5. Pregnancy rate with standard IVM, ICSI and IVF embryo transfer using a maximum of three oocytes/cycle in women 39 years and without endometriosis (March 2004 to September 2005). No. of No. of No. of No. of PR/transfer PR/patient oocytes transfers patients clinical (%) (%) retrieved pregnancies IVM ICSI IVF ET ICSI = intracytoplasmic sperm injection; IVF ET = IVF and embryo transfer; IVM = in-vitro maturation; PR = pregnancy rate. There were no statistically significant differences between the groups. Discussion This is the first report in Italy of births occurring after IVF of oocytes matured in vitro. IVM can avoid the side effects of stimulation, in particular hyperstimulation syndrome in PCOS women (Papanikolaou et al., 2005a,b). IVM is also an interesting strategy in women with regular menstrual cycles, especially in the presence of severe legal restrictions, which can reduce the success rate of standard IVF protocols. The results demonstrate that with the IVM technique it is possible to obtain good success rates in terms of maturation and fertilization, a high percentage of embryo transfers, a good number of pregnancies and finally healthy babies born. The monitoring is simple, both for patients and clinicians; furthermore, gonadotrophins are not administered and so the costs are reduced. Oocyte retrieval is more complex than during collection from the stimulated ovary, because unstimulated ovaries have a higher degree of mobility, and when the follicular diameter is less than 10 mm, aspiration takes much longer. For this reason, it is preferable to use sedation during oocyte retrieval. In the authors experiences the aspiration of antral follicles allowed the retrieval of an average number of 4.3 oocytes per cycle, although in PCOS patients 6.9 ± 3.2 oocytes per cycle were recovered (260 immature oocytes in 38 cycles). This number is lower than that reported in the literature, but can be explained by the fact that most study groups have concentrated on PCOS patients and have been using a different protocol, with HCG priming (Chian et al., 2000, 2001, 2004; Chian, 2004; Le Du et al., 2005). Initial difficulties were experienced by the operators as they were learning, both during the immature oocyte retrieval procedure and during the identification of cumulus oocyte complexes under a microscope, although no major differences have been observed in the obtained results since starting with the IVM technique (Dal Canto et al., 2005). This aspect should be confirmed with further experience. The maturation rate was 56.4%, comparable with that reported in the literature (Mikkelsen et al., 1999, 2000, 2001; Chian et al., 2000, 2001, 2004; Suikkari et al., 2000; Child et al., 2001; Söderstrom-Anttila et al., 2005) and it ensures that the woman will obtain the right number of oocytes, according to Italian law. When only considering the oocytes retrieved from PCOS patients the obtained maturation rate was as low as 49.2% (120 MII/244 immature oocytes) and this was likely to be due to 163

6 164 the underlying ovarian pathology. If only oocytes retrieved from patients with male factor or unexplained infertility are considered, the achieved maturation rates were 61.8% (209 MII/338 immature oocytes) and 56.2% (146 MII/260 immature oocytes) respectively. Immature oocytes are maintained in culture for approximately h before the insemination procedure. The culture in specific media induces the resumption of meiosis until the stage of metaphase II and allows the onset of cytoplasmic competence. The high maturation rate confirms that the specific media are able to synchronize the process of cytoplasmic and nuclear maturation within 30 h of culture. It is preferable to use ICSI to fertilize oocytes because according to Italian law, no more than three oocytes per cycle can be used, so one should not jeopardize fertilization with standard IVF. The high percentage of good quality embryos confirms that in-vitro-matured oocytes have the same possibility as invivo-matured oocytes to progress in normal cleavage and development. Patients have a high chance of obtaining embryos for transfer and therefore a good pregnancy rate is achieved, comparable with the pregnancy rate obtained with standard IVF (IVF embryo transfer, ICSI), but with a substantial decrease in risks and costs (Table 5). The pregnancy rates reported in literature are higher than ours. A possible explanation of this incongruence is that we are obliged to use only three oocytes per cycle and can t perform morphological embryo selection. The pregnancy rate is significantly related to the number of oocytes collected at oocyte retrieval (P = 0.03), and with the number of oocytes that reach the stage of metaphase II after IVM (P = 0.04). The fact that only un-selected single embryo transfer could be performed in 34.1% of cases obviously influenced the general results in a negative way. When it was possible to choose the best metaphase II oocytes to be fertilized and three embryos could be transferred, the obtained PRt was 25.5% and the PRp was 30.0%. These results are in accordance with data published in the literature. The miscarriage rate (24.0%) could be explained by the absence of morphological embryo selection. In Italy, since March 2004, assisted reproduction law has limited the number of oocytes that can be inseminated per cycle, imposing that all embryos obtained are transferred with no kind of selection and forbidding embryo or prezygotes (2PN-cells) cryopreservation. These rules have had a visible influence on the success rates of IVF, and oocyte cryopreservation cannot compensate the patient who is obliged to repeat the treatment from the start in case of failure. This protocol is proposed as the safest alternative technique to conventional IVF for couples with infertility due to male factor and PCOS given the actual situation in Italy, where ICSI and IVM have the same success rates, as can be observed in Table 5. The pregnancy rates resulting from standard IVF embryo transfer are higher than those obtained with IVM and ICSI; this can be explained by the fact that conventional IVF protocols exclude male factor infertility, as this is only recommended for patients with ovulatory infertility that need stimulation, and couples with unexplained infertility in which previous IVM treatments failed. All over the world, a growing number of fertility centres are currently applying the IVM technique for assisted reproduction, and consider it effective and safe. IVM is particularly indicated in women at a high risk of developing an ovarian hyperstimulation syndrome, such as in patients with PCOS but it is also indicated in regularly cycling women. IVM is effective and also allows reduction of the costs in terms of time consumption and patient/society costs for drugs. IVM appears to be an effective and convenient option for infertile couples with male factor or unexplained infertility as well as for PCO patients and high responders. This technique can also become the first choice in those countries in which a restrictive legislation regarding IVF technologies reduces the success rates of standard IVF protocols. References Chian RC 2004 In-vitro maturation of immature oocytes for infertile women with PCOS. Reproductive BioMedicine Online 8, Chian RC, Buckett WM, Tan SL 2004 In-vitro maturation of human oocytes. Reproductive BioMedicine Online 8, Chian RC, Gulekli B, Buckett W, Tan SL 2001 Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from woman with polycystic ovarian syndrome. Human Reproduction 16, Chian RC, Buckett WM, Tulandi T, Tan SL 2000 Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Human Reproduction 15, Child TJ, Abdul-Jalil AK, Gulekli B, Tan SL 2001 In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertility and Sterility 76, Dal Canto MB, Fadini R, Mignini Renzini M et al The number of oocytes collected and matured affect IVM success. Abstracts of the 21st Annual Meeting of the ESHRE, Copenhagen, Denmark, June Human Reproduction 20, i119 [abstract P-326]. Edwards RG, Lobo R, Bouchard P 1997 Why delay the obvious need for milder forms of ovarian stimulation? Human Reproduction 12, Edwards RG, Lobo R, Bouchard P 1996 Time to revolutionize ovarian stimulation. 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7 comparing patients with and without polycystic ovaries undergoing in vitro fertilization. Human Reproduction 8, Mikkelsen AL 2005 Strategies in human in-vitro maturation and their clinical outcome. Reproductive BioMedicine Online 10, Mikkelsen AL, Andersson AM, Skakkebaek NE, Lindenberg S 2001 Basal concentrations of oestradiol may predict the outcome of in-vitro maturation in regularly menstruating women. Human Reproduction 16, Mikkelsen AL, Smith S, Lindenberg S 2000 Impact of oestradiol and inhibin A concentrations on pregnancy rate in in-vitro oocyte maturation. Human Reproduction 15, Mikkelsen AL, Smith SD, Lindenberg S 1999 In-vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulation hormone priming. Human Reproduction 14, Papanikolaou EG, Platteau P, Albano C et al. 2005a Immature oocytes in-vitro maturation: clinical aspects. Reproductive BioMedicine Online 10, Papanikolaou EG, Tournaye H, Verpoest W 2005b Early and late ovarian hyperstimulation syndrome: early pregnancy outcome and profile. Human Reproduction 20, Ragni G, Allegra A, Anserini P et al The 2004 Italian legislation regulating assisted reproduction technology: a multicentre survey on the results of IVF cycles. Human Reproduction 20, Rienzi L, Ubaldi FM, Ferrero S et al The Impact of new Italian law on ICSI results. Abstracts of the 21st Annual Meeting of the ESHRE, Copenhagen, Denmark, June Human Reproduction 20, i48 [abstract O-131]. Söderstrom-Anttila V, Makinen S, Tuuri T, Suikkari A-M 2005 Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients. Human Reproduction 20, Son WY, Park SJ, Hyun CS et al Successful birth after transfer of blastocysts derived from oocytes of unstimulated woman with regular menstrual cycle after IVM approach. Journal of Assisted Reproduction and Genetics 19, Suikkari A-M, Tulppala M, Tuuri T et al Luteal phase start low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes. Human Reproduction 15, Ubaldi F et al The effect of the Italian law on IVF: results from a multicentre study. Abstracts of the 21st Annual Meeting of the ESHRE, Copenhagen, Denmark, June Human Reproduction 20, i47 [abstract O-136]. Received 17 January 2006; refereed 23 February 2006; accepted 7 April