In vitro maturation (IVM) of immature

Transcription

1 Improved implantation and ongoing pregnancy rates after single-embryo transfer with an optimized protocol for in vitro oocyte maturation in women with polycystic ovaries and polycystic ovary syndrome Stephen M. Junk, Ph.D., and Doreen Yeap, M.B.B.S., F.R.A.N.Z.C.O.G. Fertility Specialists of Western Australia, Bethesda Hospital, Claremont, Western Australia, Australia Objective: To describe an optimized protocol for oocyte in vitro maturation (IVM) that achieves improved implantation and ongoing pregnancy rates in women with polycystic ovaries (PCO) and polycystic ovary syndrome (PCOS). Design: Prospective cohort study. Setting: Hospital fertility unit. Patient(s): Women with PCO and PCOS undergoing treatment for infertility. Intervention(s): Follicle-stimulating hormone (FSH) priming, IVM, blastocyst culture, hormone replacement therapy. Main Outcome Measure(s): Clinical pregnancy rates. Result(s): Our optimized IVM protocol achieves implantation and ongoing pregnancy rates comparable to in vitro fertilization. From 66 oocyte collections, 844 oocytes were collected (12.8 oocytes/cycle), 588 oocytes matured after IVM (69.7% maturation rate), 420 oocytes fertilized after ICSI (71.4% fertilization rate), and 175 blastocyst-stage embryos resulted (41.7% blastocyst-development rate). Of these, 62 blastocyst-stage embryos were transferred as single embryos, resulting in 29 clinical pregnancies (43.9%/oocyte collection, 46.7%/embryo transfer) and 28 live births (42.4%/oocyte collection, 45.2%/embryo transfer). Conclusion(s): In women with PCO or PCOS, improved implantation, clinical pregnancy, and live-birth rates can be achieved after single-embryo transfer by the use of an optimized IVM protocol. (Fertil Steril Ò Medicine.) Key Words: Blastocyst, hormone therapy, in vitro, oocyte, IVM, maturation 2012;98: Ó2012 by American Society for Reproductive Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/junksm-implantation-pregnancy-rates-embryo-transfer-pcos/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. In vitro maturation (IVM) of immature oocytes offers a number of benefits to patients, in particular avoiding the adverse effects associated with ovarian stimulation (1). Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic, potentially lifethreatening condition that results from excessive ovarian follicular responses to gonadotropin therapy during the Received December 4, 2011; revised June 17, 2012; accepted June 28, 2012; published online July 24, S.M.J. is a shareholder, director, and employee of Fertility Specialists of Western Australia, Bethesda Hospital, Claremont, Australia. D.Y. is a shareholder, director, and employee of Fertility Specialists of Western Australia, Bethesda Hospital, Claremont, Western Australia, Australia. Reprint requests: Doreen Yeap, M.B.B.S., F.R.A.N.Z.C.O.G., Fertility Specialists of Western Australia, Bethesda Hospital, 25 Queenslea Drive, Claremont, Western Australia, 6010 Australia ( doreen@fertilitywa.com.au). Fertility and Sterility Vol. 98, No. 4, October /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. course of an in vitro fertilization (IVF) cycle, with reported incidence ranging from 0.5% to 14% of IVF cycles (2, 3). Women with polycystic ovaries (PCO) or polycystic ovary syndrome (PCOS) are at an even higher risk for OHSS; a diagnosis of PCOS characterizes 37% to 63% of all women who undergo severe OHSS (4, 5). Numerous and varied IVF protocols have been used to reduce this risk, which nevertheless remains substantial (2, 3). The use of a gonadotropin-releasing hormone (GnRH) agonist for triggering 888 VOL. 98 NO. 4 / OCTOBER 2012

2 Fertility and Sterility oocyte maturation in an antagonist cycle is one option, and it has been used together with luteal support for the virtual elimination of OHSS (6, 7). The use of IVM is another alternative (8). In women with PCO or PCOS, IVM effectively eliminates the risk of OHSS (9 11), which makes it the treatment of choice for this group. Some studies have raised concerns about congenital abnormalities (12), but others have reported normal neonatal outcomes (13, 14), with rates of chromosomal abnormalities and other outcomes that are similar to the conventional IVF population (15, 16). The major reason why IVM has not been used more widely in women with PCO or PCOS is the reduced likelihood of success, particularly for clinical pregnancy rates, after singleembryo rather than multiple-embryo transfer (9, 10, 17 20). There are other indications for IVM, including oocyte donation (17, 21), oocyte preservation for women before cancer treatment (22), and oocyte development in women with reduced ovarian response (23). A number of clinical protocols have been employed for IVM with varying degrees of success. The use of low-dose follicle-stimulating hormone (FSH) in the early follicular phase, or FSH priming, has been used in a number of settings (18, 19). After FSH priming, Mikkelsen and Lindenberg (18) found improved rates of oocyte maturation, although Lin et al. (19) reported no significant benefit. Human chorionic gonadotropin (hcg) priming also has been used in a number of settings, with equally inconsistent results. Son et al. (24) found that it improved IVM, but Junk et al. (25) observed no difference in their animal model. Additionally, the size of the follicles when immature oocytes have been aspirated may influence IVM results and subsequent embryonic development (26 28). Most centers typically will schedule the IVM oocyte collection when the lead follicle is %8 mm (1, 9, 10, 18, 29) before the recruitment of the dominant follicle. Embryo development to the blastocyst stage has been shown to be reduced when using oocytes from IVM (30, 31). For this reason, the majority of centers performing IVM will transfer embryos at the cleavage stage, with few transferring blastocyst-stage embryos (1, 9, 10, 18). Despite the use of various clinical protocols to improve the rates of oocyte maturation and subsequent embryo development, the ideal conditions have yet to be elucidated. We report the use of IVM in combination with FSH priming, collection of oocytes when the leading follicle(s) is mm, transfer of blastocyst-stage embryos, and a modified hormone therapy (HT) regime to assist endometrial development, leading to improved implantation and clinical pregnancy outcomes. MATERIALS AND METHODS Study Participants and Procedures All women included in this study had PCO confirmed by ultrasound assessment or were diagnosed with PCOS using the Rotterdam ESHRE/ASRM classification (32, 33). Women were excluded if they were R42 years of age or had a body mass index of R35. Women were placed on an oral contraceptive pill (OCP) before treatment (Microgynon 30; Bayer Australia Ltd.). On cessation of the OCP, the women attended the clinic on day 2 of their menses. Transvaginal ultrasound was performed to ensure no cyst development, and estrogen and progesterone levels were tested to ensure that they were at baseline. If there was no cyst development and the estrogen and progesterone levels were at baseline, the women began FSH priming (100 IU or 150 IU, Gonal F; Merck-Serono Pty Ltd.) on the following day (day 3) of their cycle for 3 days. On day 6 of their menstrual cycle, the women received a transvaginal ultrasound scan of the ovaries. If the leading follicle(s) measured 10 to 12 mm, an oocyte collection was planned for day 7 or 8 of the cycle (i.e., 1 to 2 days later). If the follicles had not reached the appropriate size, the patient returned to the clinic for a transvaginal ultrasound examination every 2 days until the appropriate follicular size was reached, and oocyte collection was planned accordingly. The IVM protocol was approved by the Curtin University of Technology Human Research Ethics Committee, and all participants provided written informed consent. In Vitro Maturation The oocyte collection was done using a flushing technique with a standard 16-gauge aspiration needle (Cook Australia). Immature oocytes were identified visually under a dissecting microscope (Olympus SZX12, Olympus Australia Pty Ltd.) and were placed into single 20 ml droplets of G2 Plus medium (Vitrolife Sweden AB) supplemented with 10% of the patient's heat-inactivated serum, 0.1 IU/mL of FSH (Puregon; MSD), and 0.5 IU/mL of hcg (Pregnyl; MSD) under paraffin oil (Ovoil; Vitrolife Sweden AB). Culture was performed in a MINC incubator (Cook Australia) with 6% CO 2,5%O 2, and balance N 2 at 37 C. After 24 to 26 hours of culture, the oocytes were placed into 80 IU of hyaluronidase (Hyalase; Sanofi-Aventis Australia) for denudation, and their maturity status was recorded. Intracytoplasmic Sperm Injection Semen was prepared from the partner when it was established that there were metaphase 2 (MII) oocytes. Samples were produced by masturbation and were prepared using a gradient (Puresperm 100; Nidacon International AB). We performed ICSI on the MII oocytes 3 to 4 hours after their maturation status was established. The ICSI procedure was performed on an Olympus IX70 inverted microscope (Olympus Australia) by use of Eppendorff TransferMan NK micromanipulators. The injected oocytes were placed into G1 Plus medium (Vitrolife Sweden AB). Sixteen to 18 hours later, the injected oocytes were observed for signs of fertilization. The pronucleate oocytes were cultured for an additional 24 hours in G1 Plus medium. They were then transferred to G2 Plus medium (Vitrolife Sweden AB) for an additional 48 hours. After this period, the embryos were observed for evidence of blastocyst development. When one was available, a single blastocyst was transferred to each patient. Supernumerary blastocysts were cryopreserved by vitrification (K-SIBV-5000; Cook Australia) on CVM Fibreplugs with sleeve (Cryologic). VOL. 98 NO. 4 / OCTOBER

3 ORIGINAL ARTICLE: ASSISTED REPRODUCTION Hormone Therapy The estrogen therapy administered depended on endometrial thickness. If the endometrial thickness was R6 mm when the leading follicle was 10 to 12 mm (2 days before the oocyte collection), a woman was given 2 mg of estradiol valerate three times a day (Progynova; Schering). If the endometrial thickness was <6 mm at this time point, the estrogen dose was 3 mg three times a day, with a proportion of women also receiving estrogen patches at a dose of 50 mg per day (Estradot; Novartis Pharmaceuticals). On the day after oocyte collection, women commenced progesterone treatment at 90 mg twice per day (Crinone 8%; Merck-Serono). The Progynova tablets estrogen patches and progesterone treatment were continued until day 30 of the menstrual cycle; if the woman became pregnant, the regimen was continued until 12 weeks' gestation; if the woman did not become pregnant, the regimen was ceased immediately. Identification of Pregnancy Pregnancies were diagnosed by b-hcg level on day 30 of the menstrual cycle, and a live intrauterine pregnancy was confirmed by a heartbeat detectable at pelvic ultrasound at 7 weeks from the last menstrual period. The heartbeat on ultrasound determined a clinical pregnancy. Statistics The statistical program SPSS (SPSS, Inc.) was used for the analyses. P<.05 was considered statistically significant. RESULTS Patient Characteristics The study comprised 66 IVM cycles. No women had been previously treated by IVM. There were 19 women with PCO and 47 with PCOS (Table 1). The mean age of the women in this study was 31.6 years (standard deviation [SD] 4.1; range: 23 to 40 years). The mean body mass index of the women was 24.4 (SD 3.9; range: 18.1 to 29.2). The time trying to conceive before entering the study averaged 2.5 years (SD 2.3; range: 1 to 10 years), and the mean number of traditional IVF cycles that women had received before entering the study was 0.9 (SD 1.1; range: 0 to 4). TABLE 1 Characteristics of patients undergoing in vitro maturation stratified according to whether polycystic ovaries (PCO) or polycystic ovary syndrome (PCOS) was present. Characteristic PCO PCOS P value N NS Age (y) NS BMI (kg/m 2 ) NS Time trying to conceive (y) NS Prior IVF cycles (n) NS Oocytes/cycle (n) <.001 Note: Data are mean standard deviation. BMI ¼ body mass index; IVF ¼ in vitro fertilization; NS ¼ not statistically significant. Junk. Optimized IVM protocol for PCO/PCOS. Fertil Steril Response to IVM All women were observed to have an endometrial thickness of R8 mm on the day of oocyte collection. By use of the previously described protocols, we initiated 66 cycles, and all led to oocyte collection for a total of 844 oocytes collected, of which 588 matured to MII over 24 to 26 hours; 420 were fertilized using ICSI, and 175 blastocysts were generated (Table 2). This resulted in 62 cycles having a single blastocyst-stage embryo transferred, and 29 ongoing pregnancies that resulted in 28 live births (one pregnancy was ectopic). All live births occurred after 36 weeks' gestation, and no congenital abnormalities were noted. In four cycles, no blastocyst-stage embryos were available for transfer, so they were abandoned at that stage with no embryo transfer performed. There were no multiple pregnancies, and none of the women developed OHSS. As only a single embryo was transferred, the implantation rates were identical to the pregnancy rates: 43.9% per egg collection and 46.7% per embryo transfer. Results were comparable for the women with PCO when compared with the women with PCOS, with implantation rates of 44.4% versus 47.7% per embryo transfer, respectively. Women with PCOS had statistically significantly more oocytes collected (P<.001) than the women with PCO, but all other variables were similar. DISCUSSION The mean number of oocytes collected and the oocyte maturation and fertilization rates reported in this study are similar to the rates reported in the literature. However, blastocyst development, implantation, and clinical pregnancy rates were improved compared with the rates that have been previously reported for IVM (9, 10, 17 20). The implantation rate of 43.9% per egg collection and 46.7% per single-embryo transfer, which we achieved using this protocol, compares favorably with the results of standard IVF regimens for women of comparable ages with or without PCO and/or PCOS (34). The 29 clinical pregnancies resulted in 28 live births, with the single loss being an ectopic pregnancy. It was reassuring that no congenital birth defects were noted. Until now, pregnancy rates after IVM have continued to lag behind the rates reported with traditional IVF. We did not conduct a randomized clinical trial, so we are comparing results from our cohort study of 66 cycles with previously published studies where the implantation rates tended to be considerably lower (9, 10, 17 20). For example, Cha et al. (9) performed 85 cycles with a transfer of multiple embryos resulting in 23 pregnancies (27.1% pregnancy rate). Child et al. (10) reported implantation, pregnancy, and live-birth rates per transfer for women with PCO of 8.9%, 23.1%, 17.3%, respectively; for women with PCOS, the rates were 9.6%, 29.9%, and 14.9%, respectively. Mikkelsen and Lindenberg (18) reported an implantation rate of 21.6% and a pregnancy rate of 29% in FSH-primed women. Lin et al. (19) reported on 68 cycles with an average of 3.8 embryos transferred, achieving implantation rates of 9.7% and 11.3%, and pregnancy rates of 31.4% and 36.4% with and without FSH priming, respectively. Son et al. (20) reported on 106 cycles in which immature oocytes were recovered after hcg 890 VOL. 98 NO. 4 / OCTOBER 2012

4 Fertility and Sterility TABLE 2 Results of the in vitro maturation protocol in women with polycystic ovaries (PCO) or polycystic ovary syndrome (PCOS). Parameter PCO PCOS Total Cycles Oocytes collected MII oocytes after IVM 115 (68.9%) 473 (69.9%) 588 (69.7%) Normally fertilized 85 (73.9%) 335 (70.8%) 420 (71.4%) Blastocysts (day 5/6) 35 (41.1%) 140 (41.8%) 175 (41.7%) Embryo transfers Clinical pregnancies 8 (42.1%/EC; 44.4%/ET) 21 (44.7%/EC; 47.7%/ET) 29 (43.9%/EC; 46.7%/ET) Live births 8 (42.1%/EC; 44.4%/ET) 20 a (42.6%/EC; 45.5%/ET) 28 a (42.4%/EC; 45.2%/ET) Note: EC ¼ egg collection; ET ¼ embryo transfer; IVM ¼ in vitro maturation; MII ¼ metaphase 2. a One pregnancy was ectopic. Junk. Optimized IVM protocol for PCO/PCOS. Fertil Steril priming and were transferred at blastocyst stage; after transfer, the implantation rate was 26.8%, and the clinical pregnancy rate was 51.9%. Our results indicate that IVM with single-embryo transfer can yield results comparable to those of IVF, while avoiding the risk of complications such as OHSS and multiple pregnancy. The improvement in outcomes seen with our protocol may be due to several factors. First, we used FSH priming in all IVM cycles, which has been demonstrated in a number of centers to improve maturation rates (35) and implantation rates (18). Priming with FSH generates greater follicle recruitment, which results in a cohort of follicles growing in size at similar rates. Therefore, a number of follicles achieve a dominant status, rather than the single one observed in a natural cycle. It is possible that the increased rate of follicles achieving dominance results in the oocytes having improved viability, which may explain the improved blastocyst development rates and implantation rates observed. Second, the size of the follicles aspirated in this study was larger than has generally been reported. This may mean that the oocytes collected have attained a higher level of competence with improved embryonic potential. A number of investigators (36 38) have shown that the size of the follicle is important in the competence of the oocytes. Others (18, 27, 29) believe that it is important to aspirate follicles at a smaller size, particularly smaller than 10 mm, before the dominant follicle has been recruited. As we have used FSH priming, a cohort of several follicles develops at similar sizes, overcoming the potential drawback of a single dominant follicle developing. Third, all women had embryo transfer with a single blastocyst-stage embryo. Previous studies (39) have demonstrated that blastocyst transfer may improve implantation rates, as it provides a better tool for embryo selection for transfer. Finally, we used an HT regime that may have had a beneficial effect on the endometrial environment for the developing embryo. We commenced estrogen therapy 2 days earlier and at a higher dosage than cited in most of the literature. Typically, other centers performing IVM will commence estrogen therapy on the day of IVM oocyte collection and at a dose of 6 to 10 mg per day (24, 29, 40). The patients in our study commenced estrogen therapy 2 days before the IVM oocyte collection and dosages ranging from 6.05 mg to 9.15 mg daily, depending on the thickness of their endometrium. We have individualized estrogen therapy in our patients such that all have an endometrial thickness of at least 8 mm on the day of oocyte collection. Other studies not performing IVM have shown that increased endometrial thickness is associated with higher pregnancy rates (41). Our novel approach to IVM FSH priming, aspiration of follicles at the size of 10 to 12 mm, blastocyst-stage transfer, and improved HT has shown that improved rates of blastocyst development, implantation, clinical pregnancy, and live birth can be achieved. 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