IN VITRO MATURATION OF HUMAN OOCYTES

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1 REVIEW ARTICLE IN VITRO MATURATION OF HUMAN OOCYTES Yu-Hung Lin 1,2, Jiann-Loung Hwang 1,3 * 1 Department of Obstetrics and Gynecology, Shin Kong Wu Ho-Su Memorial Hospital, 2 School of Medicine, Fu Jen Catholic University, and 3 Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan. SUMMARY Immature oocyte retrieval followed by in vitro maturation (IVM) opens a new horizon for modern assisted reproductive technologies (ART). Recent studies in IVM make it a feasible alternative to in vitro fertilization. Antral follicle count is correlated with the pregnancy rate, so women with polycystic ovarian syndrome or polycystic ovaries are the best candidates for IVM. IVM can also be offered to women at risk of ovarian hyperstimulation syndrome or poor responders. From the available information, IVM is a safe procedure and does not increase congenital anomalies or pregnancy complications. Further research is necessary in order to apply this technique to other patients. [Taiwanese J Obstet Gynecol 2006;45(2):95 99] Key Words: germinal vesicle, immature oocyte, in vitro maturation, polycystic ovarian syndrome Introduction Modern assisted reproductive technologies (ART) utilize ovarian stimulation to increase the number of available oocytes and the outcome. However, the use of ovarian stimulation increases the patients cost and suffering, and is associated with side effects including ovarian hyperstimulation syndrome (OHSS) and potential cancer risk. The recovery of immature oocytes followed by in vitro maturation (IVM) and fertilization is an attractive alternative to in vitro fertilization (IVF). Because few or no injections of gonadotropins are needed, IVM reduces the patients cost and suffering and avoids the side effects associated with ovarian stimulation. Another application of IVM is preservation of women s fertility; for example, cancer patients who are going to undergo cancer treatment [1]. Cryopreservation of immature oocytes or ovarian tissue, coupled with IVM, is a potential way to preserve fertility [2]. Besides, immature oocytes can be a new source of oocyte donation. Pregnancies resulting from immature *Correspondence to: Dr Jiann-Loung Hwang, Department of Obstetrics and Gynecology, Shin-Kong Wu Ho-Su Memorial Hospital, 95, Wen Chang Road, Shih Lin District, Taipei, Taiwan. m002179@ms.skh.org.tw Accepted: November 9, 2005 oocyte donation have been reported from oophorectomy specimen [3], during cesarean section [4], and from a woman with polycystic ovaries [5]. Oocyte Maturation Oocyte maturation is a complex process that comprises nuclear maturation and cytoplasmic maturation. After birth, human oocytes remain at prophase I of meiosis until they are stimulated by gonadotropin to resume meiosis before ovulation. Nuclear maturation refers to the resumption of meiosis and progression from germinal vesicle breakdown (GVBD) to metaphase II (MII). Cytoplasmic maturation refers to the preparation of oocyte cytoplasm for fertilization and embryonic development [6]. GVBD is initiated by the preovulatory surge of luteinizing hormone (LH). LH probably induces GVBD by an indirect action mediated by cumulus cells. The oocyte and the cumulus cells are coupled by gap junctions. Inhibitory factors are transported from the cumulus cells to the oocytes to maintain meiotic arrest of the oocytes. Some evidence suggests cyclic adenosine monophosphate as a potential inhibitor of meiotic resumption [7,8]. It was speculated that LH causes dissociation of the cumulus cells and the oocyte, thus terminating the flow of the meiosis-inhibiting substances into the oocyte [6]. It is also possible that LH induces Taiwanese J Obstet Gynecol June 2006 Vol 45 No 2 95

2 Y.H. Lin, J.L. Hwang the production of a GVBD-inducing signal in the cumulus cells that is subsequently transferred to the oocyte through the gap junctions [9]. Cytoplasmic maturation involves complicated processes that prepare the oocyte for activation, fertilization, and development. During this process, RNA molecules, proteins, and imprinted genes are accumulated in the cytoplasm to regulate oocyte meiosis and development [10]. Insufficient cytoplasmic maturation will fail to promote male pronucleus formation and increase chromosomal abnormalities after fertilization [11]. Human IVM In 1935, Pincus and Enzmann found that the rabbit oocytes, when liberated from Graffian follicles, would undergo spontaneous meiotic maturation in vitro [12]. Edwards demonstrated in 1965 that human oocytes removed from follicles could mature in medium supplemented with serum [13]. The first human birth resulting from IVM was reported by Cha et al in 1991 [3]. They obtained immature oocytes from oophorectomy specimen. After maturing in vitro, the oocytes were donated to a woman of premature menopause, and a set of triplets was born. In 1994, Trounson et al reported the first birth from IVM in women with polycystic ovarian syndrome (PCOS), where Trounson developed a special aspiration needle for immature oocytes [14]. However, the maturation rate of human IVM was about 30 50% [3,13,15], which was much lower than those of other species, and the pregnancies resulting from IVM were limited. Besides, Trounson et al have shown that the in vitro matured human oocytes had reduced developmental competence [16]. The poor outcome of human IVM was thought to be at least partly due to abnormalities of cytoplasmic maturation in in vitro-matured oocytes. Several treatment modalities have been proposed to improve the outcome of human IVM. FSH Priming The early studies on human IVM did not use gonadotropin before oocyte retrieval. Since follicle-stimulating hormone (FSH) acts on cumulus cells and promotes steroid production, oocyte RNA and protein synthesis [17], it has been postulated that pretreatment with FSH might increase either the number of immature oocytes recovered or the maturation potential and developmental competence of the oocytes. It has been shown in rhesus monkeys that after FSH priming for 6 7 days, greater percentages of oocytes completed meiotic maturation (74% vs. 41%), fertilized (85% vs. 61%), and cleaved from the 2 4 cell (79% vs. 38%) [18]. The effects of FSH priming on human IVM were contradictory. Wynn et al gave a truncated course of 600 IU FSH to normal women; after which, a significantly greater percentage of oocytes completed meiotic maturation in vitro (71.1% vs. 43.5%) and higher serum E 2 concentrations on the day of oocyte retrieval were found after FSH treatment (1,049 ± 241 pg/ml vs. 154 ± 17 pg/ml) [19]. Mikkelsen and Lindenberg pretreated PCOS women with 150 IU FSH per day for 3 days and found that the maturation rate was significantly higher in the FSH-primed group (59% vs. 44%), although the fertilization rates and embryo cleavage were not different between the two groups [20]. However, in their other similar study on normal cyclic women, FSH priming did not make any difference in the number of oocytes obtained, maturational potential and embryo development [21]. Similarly, Trounson et al also found no significant differences in the number of oocytes recovered, maturation rate, fertilization rate, and embryo development in patients pretreated with recombinant FSH compared to patients without treatment [16]. hcg Priming Since LH surge or human chorionic gonadotropin (hcg) injection induces final oocyte maturation, it is tempting to give hcg before oocyte retrieval in IVM cycles. In 1999, Chian et al reported a series of 25 IVM cycles in PCOS women [22]. By giving 10,000 IU hcg 36 hours before oocyte retrieval, they obtained an impressive pregnancy rate of 40%. In their other randomized study comparing the outcome of IVM with and without hcg priming, the maturation process was faster in the hcgprimed group [23]. The percentage of MII oocytes after 48 hours of culture was higher in the hcg-primed group (84.3%) than in the non-hcg-primed group (69.1%), although the rates of fertilization and cleavage and embryo quality were similar between the two groups. We have performed a randomized study on PCOS women to see if the combination of FSH priming and hcg priming would improve the outcome of IVM. Seventy-five IU rfsh was administered for 6 days in 35 cycles while 33 cycles did not have rfsh administered; 10,000 IU hcg were given 36 hours before oocyte retrieval. Serum estradiol level on the day of hcg injection was significantly higher in the FSH-primed group than in the non-fsh primed group ( vs pg/ml), but the number of oocytes, endometrial 96 Taiwanese J Obstet Gynecol June 2006 Vol 45 No 2

3 In Vitro Maturation of Human Oocytes thickness, and maturation, fertilization, and pregnancy rates were similar between the two groups [24]. From the above studies, we conclude that hcg priming plays a more important role in IVM, and FSH priming has no additional beneficial effect. Application of IVM PCOS or PCO The best candidates for IVM are women with PCOS or polycystic appearing ovaries (PCO) because these women have many antral follicles. Tan et al demonstrated that the pregnancy rates after IVM correlated with the number of immature oocytes retrieved, with the highest 26.8% in those with >10 immature oocytes [25]. Child et al demonstrated that the number of immature oocytes and pregnancy rates were higher in women with PCOS or PCO than in women with normal ovaries [26], and in PCOS women, IVM was comparable to IVF [27]. High responders Patients at risk of OHSS may also benefit from IVM. Lim et al reported IVM in a series of patients undergoing IVF who were at the risk of OHSS. hcg was administered when the follicles reached mm, and a pregnancy rate of 47.1% was obtained [28]. Poor responders Other potential candidates of IVM are poor responders. Although several stimulation protocols and strategies have been proposed for poor responders, some women may still respond poorly to ovarian stimulation. IVM may be an option for these women. Although the outcome is still dismal for these women because only a few immature oocytes can be obtained (usually <5, unpublished data and Chian RC, personal communication), at least they do not have to face the risk of cycle cancellation after vigorous ovarian stimulation. Liu et al reported three pregnancies out of eight patients (37.5%), suggesting that IVM may be an alternative for poor responders [29]. IVM in stimulated cycles After ovarian stimulation with gonadotropin and hcg, about 15% of oocytes are found in germinal vesicle (GV) or metaphase I (MI) at the time of oocyte retrieval. Attempts have been made to mature these oocytes in vitro but the results were very poor [30 33]. Recently Chian et al designed a new IVM medium, which has been shown to improve the maturation rate and blastocyst development of GV oocytes from stimulated cycles [34]. However, the GV oocytes obtained from stimulated ovaries have a lower fertilization rate even with intracytoplasmic sperm injection (ICSI) [35], and the resulting embryos have high incidence of noncleavage (21%) and chromosomal anomalies (78.5%) [36]. We speculate that the oocytes that remain at the GV stage in spite of ovarian stimulation are of inferior quality or have intrinsic defects in the oocytes or follicles. Further study is necessary to document the safety of IVM with GV oocytes from stimulated cycles. Combination of natural-cycle IVF and IVM Recently, Chian et al proposed a combination of natural-cycle IVF and IVM as a new approach in infertility treatment [37]. Ten thousand IU hcg were administered when the largest follicle was mm in natural cycle. After oocyte retrieval 36 hours later, MII oocytes were injected and GV oocytes were cultured in vitro, which then underwent ICSI when they were MII. Similar to natural-cycle IVF, this strategy is patient-friendly, but may increase the number of embryos available. Outcome of IVM The results of IVM from several centers are listed in the Table. The implantation rates of IVM are lower than IVF, Table. Outcome of IVM from the literature Authors No. of cycles Maturation Fertilization Implantation Pregnancy rate (%) rate (%) rate (%) rate (%) Chian et al (2004) [37] 254 NA NA Lim et al (2002) [28] 419 (day 3 transfer) NA NA (day 5 transfer) Lin et al (2003) [24] Le Du et al (2005) [40] Cha et al (2005) [38] 203 NA NA NA not available. Taiwanese J Obstet Gynecol June 2006 Vol 45 No 2 97

4 Y.H. Lin, J.L. Hwang but Lim et al reported a high implantation rate following blastocyst transfer [28]. Among our 23 pregnancies, there were two miscarriages (8.7%), one stillbirth at 17 weeks, 10 preterm deliveries (43.5%), and 10 term deliveries (43.5%). However, in the report of Cha et al, although there were 36.8% miscarriage and 7.3% preterm labor, the obstetric outcome of PCOS women treated by IVM was comparable with those treated by IVF [38]. Mikkelsen reported 46 pregnancies with only two preterm deliveries and one stillbirth [39]. We performed karyotyping in every case from amniocentesis or cord blood at delivery and all gave normal results. There was no congenital anomaly and their growth and development were all normal until 2 years of follow-up. In the report of Cha et al, there were three congenital anomalies out of 38 pregnancies (7.9%), including hydrops fetalis, omphalocele, and cleft palate [38]. Mikkelsen reported 47 children born after IVM in whom chromosomal analysis was performed in 34 cases. There was only one abnormal karyotype (46,XX, var(20) cenh ) but without clinical abnormalities. The only baby with malformation is a girl with cleft palate [39]. From the available data of about 300 babies delivered worldwide, IVM does not seem to increase the risk of congenital anomalies [39,40]. Conclusion Immature oocyte retrieval followed by IVM and fertilization is now a feasible approach in ART. With or without limited use of gonadotropin, IVM avoids the side effects of ovarian stimulation and reduces the patients expenses. This technique is better offered to those women with many antral follicles, such as women with PCOS or PCO. hcg priming initiates oocyte maturation in vivo and produces favorable outcome in IVM. The role of FSH priming is controversial. References 1. Durga Rao G, Chian RC, Son WS, Gilbert L, Tan SL. Fertility preservation in women undergoing cancer treatment. Lancet 2004;363: Tucker MJ, Wright G, Morton PC, et al. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil Steril 1998;70: Cha KY, Koo JJ, Ko JJ, et al. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991;55: Hwang JL, Lin YH, Tsai YL. Pregnancy after immature oocyte donation and intracytoplasmic sperm injection. Fertil Steril 1997;68: Hwang JL, Lin YH, Tsai YL, et al. Oocyte donation using immature oocytes from a normal ovulatory woman. Acta Obstet Gynecol Scand 2002;81: Cha KY, Chian RC. Maturation in vitro of immature human oocytes for clinical use. Hum Reprod Update 1998;4: Downs SM. Factors affecting the resumption of meiotic maturation in mammalian oocytes. Theriogenology 1993;39: Albertini DF, Carabatsos MJ. Comparative aspects of meiotic cell cycle control in mammals. J Mol Med 1998; 76: Downs SM, Daniel SA, Eppig JJ. Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin. J Exp Zool 1988;245: De Sousa PA, Caveney A, Westhusin ME, et al. Theriogenology 1998;49: Thibault C, Gerard M, Menezo Y. Preovulatory and ovulatory mechanisms in oocyte maturation. J Reprod Fertil 1975; 45: Pincus G, Enzmann EV. The comparative behavior of mammalian eggs in vivo and in vitro. J Exp Med 1935;62: Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 1965;208: Trounson A, Wood C, Kausche A. In vitro maturation and fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 1994;72: Tsuji K, Sowa M, Nakano R. Relationship between human oocyte maturation and different follicular sizes. Biol Reprod 1985;32: Trounson A, Anderiesz C, Jones GM, et al. Oocyte maturation. Hum Reprod 1998;13(Suppl 3): McGee E, Spears N, Minammi S, et al. Preantral follicles in serum-free culture: suppression of apoptosis after activation of the cyclic guanosine 3,5 -monophosphate pathway and stimulation of growth and differentiation by folliclestimulating hormone. Endocrinology 1997;138: Schramm RD, Bavister BD. Follicle-stimulating hormone primig of rhesus monkeys enhances meiotic and developmental competence of oocyte matured in vitro. Biol Reprod 1994;51: Wynn P, Picton HM, Krapez JA, et al. Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in vitro maturation. Hum Reprod 1998;13: Mikkelsen AL, Lindenberg S. Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome: a randomized prospective study. 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5 In Vitro Maturation of Human Oocytes 22. Chian RC, Gulekli B, Buckett WM, et al. Priming with human chorionic gonadotropin before retrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome. N Engl J Med 1999;341: Chian RC, Buckett WM, Tulandi T, et al. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000;15: Lin YH, Hwang JL, Huang LW, et al. Combination of FSH priming and hcg priming for in vitro maturation of human oocytes. Hum Reprod 2003;18: Tan SL, Child TJ, Gulekli B. In vitro maturation and fertilization of oocytes from unstimulated ovaries: predicting the number of immature oocytes retrieved by early follicular phase ultrasonography. Am J Obstet Gynecol 2002;186: Child TJ, Abdul-Jalil AK, Gulekli B, Tan SL. In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertil Steril 2001;76: Child TJ, Phillips SJ, Abdul-Jalil AK, Bulekli B, Tan SL. A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstet Gynecol 2002; 100: Lim KS, Son WY, Yoon SH, Lim JH. IVM/F-ET in stimulated cycles for the prevention of OHSS. Fertil Steril 2002; 78(Suppl 3S):S Liu J, Lu G, Qian Y, Mao Y, Ding W. Pregnancies and births achieved from in vitro matured oocytes retrieved from poor responders undergoing stimulation in in vitro fertilization cycles. Fertil Steril 2003;80: Jaroudi KA, Hollanders JMG, Elnour AM, et al. Embryo development and pregnancies from in vitro matured and fertilized oocytes. Hum Reprod 1999;14: Veeck L, Edwards J, Witmyer J, et al. Maturation and fertilization of morphologically immature oocytes in a program of in vitro fertilization. Fertil Steril 1983;39: Nagy Z, Cecile J, Liu J, et al. Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal vesicle stage oocytes: case report. Fertil Steril 1996;65: Jaroudi KA, Hollanders JMG, Sieck U, et al. Pregnancy after transfer of embryos which were generated from in vitro matured oocytes. Hum Reprod 1997;12: Chian RC, Tan SL. Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles. Reprod BioMed Online 2002;5: Kim BK, Lee SC, Kim KJ, et al. In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles. Fertil Steril 2000;74: Nogueira D, Staessen C, van de Velde H, et al. Nuclear status and cytogenetics of embryos derived from in vitro matured oocytes. Fertil Steril 2000;74: Chian RC, Buckett WM, Abdul Jalil AK, et al. Natural-cycle in vitro fertilization combined with in vitro maturation of immature oocytes is a potential approach in infertility treatment. Fertil Steril 2004;82: Cha KY, Chung HM, Lee DR, et al. Obstetric outcome of patients with polycystic ovary syndrome treated by in vitro maturation and in vitro fertilization-embryo transfer. Fertil Steril 2005;83: Mikkelsen AL. Strategies in human in vitro maturation and their clinical outcome. Reprod BioMed Online 2005;20: Le Du A, Kadoch IJ, Bourcigaux N, et al. In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience. Hum Reprod 2005;20: Taiwanese J Obstet Gynecol June 2006 Vol 45 No 2 99

IVM in PCOS patients. Introduction (1) Introduction (2) Michael Grynberg René Frydman

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