Effect of metformin pretreatment on pregnancy outcome of in vitro matured oocytes retrieved from women with polycystic ovary syndrome Zhaolian Wei, M.D., a Yunxia Cao, M.D., Ph.D., a Lin Cong, M.D., a Ping Zhou, M.D., a Zhiguo Zhang, MSc., a and Jun Li, Ph.D. b a Reproductive Medicine Center, The First Affiliated Hospital, Anhui Medical University, Hefei, People s Republic of China; and b Pharmacological College, Anhui Medical University, Hefei, People s Republic of China Objective: To determine the effect of metformin pretreatment on in vitro maturation (IVM) in the patients with clomiphene citrate-resistant polycystic ovary syndrome. Design: Prospective randomized study. Setting: University affiliated hospital. Patient(s): Fifty-six women with clomiphene citrate-resistant polycystic ovary syndrome underwent 70 cycles of IVM treatment. Intervention(s): Metformin was administered at a dose of 500 mg twice per day for 12 weeks before IVM treatment. Main Outcome Measure(s): The number of immature oocytes, oocyte maturation, fertilization, cleavage, highquality embryo, and clinical pregnancy rates. Result(s): A significantly higher high-quality embryo rate (37.8%) was obtained in the metformin-treated group compared with the control group (24.3%). Accordingly, the clinical pregnancy and implantation rates were significantly higher in the metformin-treated group (38.2% and 15.3%) than the control group (16.7% and 6.2%). Conclusion(s): Pretreatment with metformin improves IVM outcome in terms of embryo quality and clinical pregnancy rate as well as implantation rate. (Fertil Steril Ò 2008;90:1149 54. Ó2008 by American Society for Reproductive Medicine.) Key Words: Metformin, polycystic ovary syndrome, in vitro maturation, pregnancy Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility, affecting 5% to 10% of women of reproductive age. The pathology character of PCOS is hyperandrogenism and chronic anovulation (1, 2). Traditionally clomiphene citrate (CC) has been used as the first-line therapy for ovulation induction in patients with PCOS. However, 20% to 30% of patients with PCOS are CC resistant in clinical practice (3). The role of insulin in the pathophysiology of PCOS has been established. Insulin resistance with compensatory hyperinsulinemia is common in women, especially in those with CC resistance. It has been believed that the high levels of insulin lead to an increase of ovarian androgen biosynthesis and also inhibit the liver s synthesis of sex hormone binding globulin and insulin-like growth factor binding protein-i, resulting in the increase of free testosterone in circulation blood, which aggravates the evolution the PCOS (4, 5). Received February 14, 2007; revised July 27, 2007. There was no conflict of interest with any product used in this study. Reprint requests to: Yunxia Cao, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, People s Republic of China (FAX: 86 511 2923 241; E-mail: caoyunxia6@126.com); or Jun Li, Pharmacological College, Anhui Medical University, Hefei, 230032, People s Republic of China. (FAX: 86 511 5161 115; E-mail: lijun@ahmu.edu.cn). Metformin, as a biguanide insulin-sensititiving agent, is capable of inhibiting insulin secretion, hyperinsulinism, and reducing the level of androgen secreted by the ovary and adrenal gland. Hence, metformin seems to be a perfect drug to treat patients with PCOS, including those with CC resistance. It was reported that metformin treatment for patients with PCOS improves a patient s menstrual cycle and increases the sensitivity for the ovulation induction drug reaction, especially in women with CC-resistant PCOS (6 8). Velazquez et al. (9) first reported on the use of metformin as a treatment drug for PCOS, and the result proved that metformin improves insulin sensitivity, lowered serum LH, total and free testosterone concentrations, and causes an elevation in serum FSH and sex hormone binding globulin levels in obese women with PCOS. In addition, Genazzani et al. (10) demonstrated that LH spontaneous episodic secretion was significantly modified and reproductive axis functioning was improved after 6 months metformin therapy in a group of nonobese PCOS patients. Furthermore, metformin administration has been shown to improve ovarian response to exogenous gonadotropin in women with CC-resistant PCOS (6, 8). It also has been reported that the dosage of gonadotropins administrated was reduced and a higher pregnancy outcome was achieved by the administration of metformin (11). 0015-0282/08/$34.00 Fertility and Sterility â Vol. 90, No. 4, October 2008 1149 doi:10.1016/j.fertnstert.2007.07.1385 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
Ovarian stimulation is usually associated with a potential risk of ovarian hyperstimulation syndrome (OHSS) in women with PCOS when they are undergoing IVF treatment. Immature oocyte retrieval followed by in vitro maturation (IVM) of those oocytes provides a new strategy to prevent OHSS. In fact, several groups worldwide have reported that IVM can be used successfully to treat infertile women with PCOS (12 14). Therefore, the advantages of IVM include avoidance of OHSS, simplification of treatment, and reduction in cost for the patients with CC-resistant PCOS. It has been reported that administration of metformin in patients with PCOS undergoing IVF cycle might improve the number of retrieved oocytes, the quality of embryos, and clinical pregnancy rate compared with that of the non-metformin-treated group (15, 16). However, it is unclear whether the clinical pregnancy rate can be improved by pretreatment with metformin in infertile women with CCresistant PCOS undergoing IVM treatment. We have designed a prospective and randomized study to determine whether pretreatment with metformin is able to improve the clinical outcome of IVM treatment for patients with CC-resistant PCOS to clarify the effect of pretreatment with metformin on IVM treatment cycles. MATERIALS AND METHODS Patients From October 2003 to March 2005, a total of 56 CC-resistant patients with PCOS were enrolled in this study. All patients had no any history of pregnancy. The mean age of patients was 28.7 2.7 years. The duration of infertility was 4.1 2.0 years. The patients were selected with the following criteria: [1] chronic anovulation or amenorrhea; [2] hyperandrogenism with clinical or biochemical evidence (testosterone R2.2 nmol/l); [3] R10 follicles (2 8 mm in diameter) in each ovary; [4] no other endocrine secretion diseases including thyroid disease, adrenal hyperplasia, and androgen-secreting tumor; [5] all the patients had taken CC at a dosage of 150 mg per day for 5 days and for at least three cycles, and had no mature follicle produced and ovulation. The study was approved by the institutional review board of the Affiliated Hospital of Anhui Medical University. All patients signed the informed consent forms before entering the trial. Pretreatment with metformin The patients were randomly divided into two groups: [1] metformin pretreatment group (M group): 28 patients were administrated metformin at a dose of 500 mg twice per day for 12 weeks before IVM treatment. Of those patients, two women became pregnant during the pretreatment with metformin and the remaining 26 patients underwent 34 cycles of IVM treatment following metformin pretreatment; [2] the control group (non-metformin group, NM group): 28 patients underwent 36 cycles of IVM treatment directly without metformin pretreatment. Hormonal Profiles The fasting plasma hormone levels in all the PCOS patients were measured on days 2 3 of the menstruation or amenorrhoea (follicular diameter <10 mm) between 8:00 A.M. and 10:00 a.m. The hormonal profiles (including LH, FSH, estradiol, testosterone, and prolactin) in serum under a fasting situation were assayed by an immune immunofluorometric method (ACS:180 SE, Bayer, Germany). The level of fasting plasma insulin was measured using an enzyme immunoassay system (IMMULITE and IMMULITE 1000 systems, DPC, BASF, Evans City, PA). Additionally, the concentration of fasting glucose was determined with the enzymatic colorimetric assay (Hitachi Modular Analytics, Roche, Switzerland). The intra- and interassay coefficients of variation of assay were expressed as 6.5% and 7.3%, respectively. hmg Priming and Immature Oocyte Retrieval From the commencement of day 3 to 5 of spontaneous menstrual cycle or the withdrawal-bleeding cycle induced by Medroxy-Progesterone, the patients were administrated hmg (Lizhu Ltd., China) at a dose of 75 150 IU per day for 5 days. Simultaneously, development status of the follicles and the thickness of endometrium were monitored by serial transvaginal ultrasonography between days 8 and 11 of the menstrual cycle. When the size of follicles in the ovary reached between 6 and 12 mm in diameter and the thickness of endometrium was R6 mm, 10,000 IU of hcg (Profasi, Serono, Switzerland) was injected to trigger oocyte maturation in vivo. Following 36 hours of hcg injection, the follicles were aspirated using a single lumen needle (17 G, Wallace, SIMS, UK) with a pressure of 100 mm Hg through the transvaginal ultrasound-guided route. The aspirates were collected into several 10-mL Falcon tubes prefilled with warmed DPBS supplemented with heparin (4 IU/mL). In Vitro Maturation of Immature Oocytes Cumulus oocyte complexes were identified under a stereomicroscope and then transferred to IVM medium for oocyte maturation in culture at 37 C in an atmosphere of 5% CO 2 in air for 24 to 48 hours. In vitro matruation medium, TCM199 medium (Sigma, St. Louis, MO, Lot: M4530) supplemented with 20% patient s serum (56 C 30-minute heat inactivated) and rec-fsh (0.075 IU/mL, Gonal-F, Serono, Geneva, Switzerland), estradiol (1.0 mg /ml, Sigma,), hcg (0.5 IU/mL, Profasi, Serono), and pyruvic acid (30 mol/l, Sigma). Before mature in culture, the maturity of oocytes were checked under an invert microscope (IX-71, Olympus, Japan). After 24 hours of culture, all oocytes were denuded from cumulus cells using hyaluronidase (80 IU/mL, Sigma) and mechanical pipetting with a fine glass pipette. Mature oocytes (metaphase-ii) were defined by the presence of the first polar body. The remaining immature oocytes were further cultured for 24 hours in IVM medium. 1150 Wei et al. Metformin for in vitro maturation Vol. 90, No. 4, October 2008
IVF and Embryo Transfer Mature oocytes were inseminated by intracytoplasmic sperm injection (ICSI), respectively, for 24 and 48 hours of the IVM oocytes. Following ICSI, the oocytes were transferred to fertilization medium (50 ml/droplet, G-FERT, Vitrolife, Kungsbacka, Sweden) equilibrated for 18 hours. Fertilization check was performed 17 to 19 hours after ICSI. The fertilized zygotes were further cultured in embryo developmental medium (G-1, Vitrolife). The embryos were cultured for 2 3 days after ICSI, and selected for embryo transfer. The embryo development and quality were evaluated on day 3 after ICSI based on the scoring criteria described by Tomas et al. (1998). Briefly, the embryos were scored as follows: Grade I, equal-sized blastomeres with no fragmentation; Grade II, equal-sized blastomeres with <20% fragmentation; Grade III, unequal-sized blastoberes with <20% fragmentation; Grade IV, equal and unequal blastomeres with 20% to 50% fragmentation; Grade V, equal and unequal blastomeres with >50% fragmentation. Goodquality embryos were comprised of Grade I and Grade II based on the embryonic morphology without or with less fragmentation. Preparation of Endometrium and Luteal Support Patients received 6 mg estradiol valerate per day for endometrial preparation, starting from the day of oocyte retrieval. Luteal support (60 mg progesterone) was given to patients (Xianju, China) by intramuscular injection daily, starting from the day of ICSI and continuing until 12 weeks of gestation. Urinary hcg test was performed on day 15 after embryo transfer to confirm pregnancy. Clinical pregnancy was determined when a gestation sac was seen by transvaginal ultrasound at 5 weeks after embryo transfer. Statistical Analysis A Mann Whitney U test and Student s t test were used to compare the data of the two groups. A P-value test criterion was defined as P¼.05, and P<.05 was considered as statistically significant. RESULTS A total of 54 patients underwent 70 IVM treatment cycles. Clinical information and hormonal profiles in two groups were similar (Table 1). The concentrations of fasting insulin (20.0 1.2 mmol/l vs. 20.21.3 mmol/l) and glucose (5.9 0.5 mmol/l vs. 5.8 0.4 mmol/l), FSH (5.3 1.4 IU/L vs. 5.6 1.7 IU/L), LH (10.8 4.5 IU/L vs. 11.2 5.1 IU/L), estradiol (181.7 72.2 nmol/l vs. 184.5 105.0 nmol/l), testosterone (2.5 0.3 nmol/l vs. 2.5 0.4 nmol/l), and prolactin (26.5 16.3 mg/l vs. 23.7 6.8 mg/l) in serum were not significantly different between groups. However, after 3 months of metformin treatment in the M group, serum basal fasting insulin (18.0 2.0 mmol/ L), testosterone (2.2 0.4 nmol/l), and body mass index (21.3 1.7 kg/m 2 ) were decreased significantly (P<.05) compared with before metformin treatment (fasting insulin 20.2 1.3 mmol/l, testosterone 2.5 0.4 nmol/l, and body mass index 23.3 1.6 kg/m 2, respectively). There were no significant changes in the levels of fasting glucose, FSH, LH, and Prolactin (Table 2). As shown in Table 3, there were no differences in the mean numbers of immature oocytes retrieved (9.4 6.2 vs. 12.4 6.3), oocyte maturation fertilization (70.4% vs. 76.7%), and cleavage rates (89.4% vs. 87.5%) between the M group and the NM group. However, the percentage of high-quality embryos in the M group (37.8%) was significantly higher (P<.05) than the NM group (24.3%). Furthermore, the TABLE 1 Clinical information and basal hormonal profiles in two groups of patients with PCOS. a NM group M group P No. of patients 28 26 NS Age (years) 28.6 3.3 28.6 2.5 NS Duration of infertility (years) 3.9 1.9 4.3 2.3 NS Body mass index (kg/m 2 ) 22.8 2.1 23.3 1.6 NS Fasting glucose (mmol/l) 5.9 0.5 5.8 0.4 NS Fasting insulin (mmol/l) 20.0 1.2 20.2 1.3 NS FSH (IU/L) 5.3 1.40 5.6 1.7 NS LH (IU/L) 10.8 4.5 11.2 5.1 NS Estradiol (nmol/l) 181.7 72.2 184.5 105.0 NS Testosterone (nmol/l) 2.5 0.3 2.5 0.4 NS Prolactin (mg/l) 26.5 16.3 23.7 6.8 NS Note: NS ¼ not significant between groups. a Values presented as mean SD. Wei. Metformin for in vitro maturation. Fertil Steril 2008. Fertility and Sterility â 1151
TABLE 2 Comparison of clinical and hormonal parameters in the patients with PCOS before and after Metformin treatment for 3 months. a Before treatment After treatment P No. of patients 26 26 NS Body mass index (kg/m 2 ) 23.3 1.6 21.3 1.7 <.05 Fasting glucose (mmol/l) 5.8 0.4 5.5 0.6 NS Fasting insulin (mmol/l) 20.2 1.3 18.0 2.0 <.05 FSH (IU/L) 5.6 1.7 5.3 1.6 NS LH (IU/L) 11.2 5.1 10.2 3.9 NS Estradiol (nmol/l) 184.5 105.0 180.4 83.7 NS Testosterone (nmol/l) 2.5 0.4 2.2 0.4 <.05 Prolactin (mg/l) 23.7 6.8 22.1 7.9 NS Note: NS ¼ not significant between groups; PCOS ¼ Polycystic ovary syndrome. a Values presented as mean SD. Wei. Metformin for in vitro maturation. Fertil Steril 2008. clinical pregnancy (16.7% vs. 38.2%) and implantation (6.2% vs. 15.3%) as well as live birth (16.7% vs. 23.1%) rates were also significantly different (P<.05) between these two groups. DISCUSSION The results of the present study demonstrate that pretreatment with metformin improves clinical pregnancy outcome in infertile women with CC-resistant PCOS, resulting from producing more good-quality embryos in the IVM treatment cycles. TABLE 3 Comparison of IVM outcome between M group and NM group. a Insulin resistance with compensatory hyperinsulinemia is common in women with CC-resistance PCOS. Metformin, as a treatment drug for PCOS, is capable of inhibiting insulin secretion, hyperinsulinism, and improving ovarian endocrinology. Genazzani et al. (10) reported that LH spontaneous episodic secretion was significantly modified and reproductive axis functioning was improved after 6 months metformin therapy in a group of nonobese PCOS patients. Recently, Genazzani et al. (17) reported that plasm LH, estradiol, and insulin were decreased significantly by metformin treatment in nonobese patients with PCOS. The results of the present study indicate that the basal fasting insulin and testosterone NM group M group P No. of patients (cycles) 28 (36) 26 (34) NS No. of immature oocytes (mean SD) 338 (9.4 6.2) 420 (12.4 6.3) NS No. of oocytes matured (%) 238 (70.4) 322 (76.7) NS No. of oocytes fertilized (%) 161 (67.7) 224 (70.0) NS No. of zygotes cleaved (%) 144 (89.4) 196 (87.5) NS No. of good-quality embryo (%) 35 (24.3) 74 (37.8) <.05 No. of embryos transferred (mean SD) 97 (2.7 1.4) 85 (2.6 1.7) NS No. of clinical pregnancy (%) 6 (16.7) 13 (38.2) <.05 No. of embryos implanted (%) 17(6.2) 40(15.3) <.05 No. of miscarriage (%) 1 (16.7) 3 (23.1) NS No. of live births (%) 5 (13.9) 10 (29.4) <.05 Singleton 5 9 Twin 0 1 Triplet 0 0 Note: IVM ¼ in vitro maturation. a Parentheses indicate the mean and percentage. Wei. Metformin for in vitro maturation. Fertil Steril 2008. 1152 Wei et al. Metformin for in vitro maturation Vol. 90, No. 4, October 2008
levels in serum as well body mass index were significantly reduced after 3 months of metformin pretreatment. This suggests that metformin pretreatment has improved the ovarian endocrinology and insulin sensitivity. Administration of metformin was shown to improve ovarian response to exogenous gonadotropin in women with CC-resistant PCOS (6, 8), the dosage of gonadotropins administrated was reduced, as well as a higher pregnancy outcome achieved, by the administration of metformin (11). Studies have elucidated the effect of metformin treatment before IVF in patients with PCOS, which show more mature oocytes collected and higher clinical pregnancy rates obtained than the control group (16, 18). It has also been indicated that metformin is associated with the changes in reduced FSH requirement to 10% in the IVF cycle (12) and helped in the prevention of OHSS (18, 19). However, Kjøtrød et al. (20) found that pretreatment with metformin before conventional IVF/ICSI in women with PCOS does not improve ovarian stimulation or clinical outcome among normal-weight PCOS women, but pretreatment with metformin tends to improve pregnancy rates. In 1994, Trounson et al. (21) reported the first pregnancy and delivery of a healthy baby after IVM of immature oocytes obtained from a patient with PCOS, and then IVM treatment became an option to treat patients with PCOS, because patients with PCOS have higher risk of OHSS (22). It has been reported that priming with recombinant FSH before harvesting of immature oocytes for patients with PCOS could improve the mature potential of oocytes and the subsequent clinical pregnancy rate in IVM treatment cycles (23). In the present study, we used a small dose of hmg to induce follicular development in both groups at the beginning of the menstrual cycle. Our experience indicated that the number of immature oocytes collected and the oocyte maturation rate are increased significantly following small dosages of hmg priming (authors unpublished data). Although it has been reported that 30% to 35% of the clinical pregnancy rates and 10% to 15% of the implantation rates are obtained from IVM treatment for infertile patients with PCOS (24), much effort is needed to improve the pregnancy outcome of IVM treatment. The results of the present study indicate that the percentage of high-quality embryos is significantly higher in the metformin-pretreated group than the control group, resulting in significantly higher clinical pregnancy and implantation rates. 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