Effects of Oral Contraceptives and Metformin on the Outcome of In Vitro Maturation in Infertile Women with Polycystic Ovary Syndrome

Abstract

Objective:

To evaluate the effects of oral contraceptives and metformin on the outcome of in vitro maturation (IVM) in infertile women with polycystic ovary syndrome (PCOS).

Methods:

This is a retrospective study of 108 women with PCOS, subject to 152 cycles of IVM treatment. The study was held at the Reproductive Medicine Center of the First Affiliated Hospital of Wenzhou Medical College, People's Republic of China. Before entering IVM treatment, 54 patients who received oral contraceptive pill (marvelon, 0.15 mg desogestrel, and 0.03 mg ethinylestradiol), one tablet every day, and metformin 500 mg twice or three times per day were defined as the pretreated group, and another 64 patients who were not administered any drugs as the control group. The main outcome measures were the rates of oocyte maturation, fertilization, cleavage, miscarriage, clinical pregnancy, and live birth.

Results:

There were no significant differences between the two groups in the rates of oocyte maturation, fertilization, cleavage, and clinical pregnancy (p > 0.05). A significantly lower miscarriage rate was obtained in the pretreated group than in the control group (16.13% vs 4.0%, p < 0.01). The live birth rate per embryo transfer seemed to be higher in the pretreated group than in the control group (37.70% vs 30.38%, p = 0.363), but was not statistically significant.

Conclusions:

Pretreatment with oral contraceptives and metformin improved the outcome of IVM related to the miscarriage rate and possibly also live birth rate.

Introduction

Polycystic ovary syndrome (PCOS) is the most common endocrine abnormality in reproductive-aged women with psychological, reproductive, and metabolic manifestations. In addition to healthy lifestyle changes, treatment options include cyclic progestin to induce withdrawal bleeds, oral contraceptives (OC), metformin, and targeted infertility therapies. The technology of in vitro maturation (IVM) has brought increased interest, because immature oocytes retrieved from unstimulated ovaries can avoid the side effects of medicine; it is also more economical and simple. It has been demonstrated that IVM is a successful treatment for women with infertility related to PCOS.^1–2 As we know, women with PCOS are considered to be at increased risk of miscarriage because of the abnormal endocrine environment. A prospective cohort study showed administration of metformin during pregnancy to women with PCOS was associated with a marked and significant reduction in the rate of early pregnancy loss.³ Another study reported that OC combined with metformin can rectify endocrine and metabolic disorders, which might lead to a better medical sensitivity to oocyte induction, higher ovulation rate, and higher pregnancy rate.⁴ Doldi et al.⁵ found that the use of metformin with a GnRH antagonist improved the outcome of ovarian stimulation in in vitro fertilization (IVF)-embryo transfer (ET) cycles, increased the number of mature oocytes, reduced gonadotropin dose needed, decreased serum estradiol level on the day of HCG, and lowered the incidence of ovarian hyperstimulation syndrome (OHSS) and the number of canceled cycles. Recently Wei et al.⁶ reported that pretreatment with metformin improved IVM outcome in terms of embryo quality and clinical pregnancy rate as well as implantation rate. However, no study has evaluated the effects of combination treatment with OC plus metformin on the outcome of IVM in PCOS patients.

The aim of the present study is to investigate the effects of OC plus metformin on the outcome of IVM and to provide a best treatment option for infertility in women with PCOS.

Materials and Methods

Patients and pretreatment

From March 2004 to October 2007, 118 women with PCOS underwent 152 IVM cycles. Before entering IVM treatment, according to the patients' inclination, 54 patients who received an oral contraceptive pill (marvelon, 0.15 mg desogestrel and 0.03 mg ethinylestradiol), one tablet every day, and antidiabetic biguanide metformin at a dose of 500 mg twice or three times per day for 3 months were defined as the pretreated group (Group A). The other 64 patients, who were eager to conceive, or who could not take the drugs for 3 months and were not administered any medicine, were defined as the control group (Group B). Some of the patients in Group B had previously taken OC or metformin or a combination, but they were not administered any drugs at least 2 months before they entered an IVM cycle. All patients were diagnosed with PCOS according to Rotterdam conference criteria.⁷ The menstrual patterns of the patients in the two groups included spontaneous menses (3.70% vs 4.69%), irregular menses (20.37% vs 21.88%), oligomenorrhea (66.67% vs 65.63%), and amenorrhea (9.26% vs 7.81 %). The patients in each group underwent comparable cycles, including one cycle in 42 cases (77.78%) in Group A and 44 cases (68.75%) in Group B, two cycles in 12 cases (22.22%) in Group A and 18 cases (28.13%) in Group B, and 2 cases (3.12%) in Group B underwent three cycles. The clinical characteristics of the two groups are shown in Table 1. Written, informed consent to participate was obtained from all of the patients before starting the treatment, and the research was approved by the Research Ethics Board of the Hospital.

Table 1.

Clinical Characteristics in Patients with Polycystic Ovary Syndrome in Group A and Group B

	Group
Parameter	A	B	p value
No. of start cycles	66	86
No. of transfer cycles	61	79
Ages (years)	30.27 ± 2.89	27.66 ± 3.11	<0.001
LH (IU/L)	10.53 ± 5.30	11.8 ± 6.24	0.2 ∼0.1
FSH (IU/L)	6.57 ± 1.66	6.71 ± 1.82	>0.05
T (nmol/L)	2.21 ± 0.86	2.57 ± 0.76	0.01 ∼0.005
BMI (kg/m²)	22.88 ± 3.75	21.30 ± 3.58	0.01 ∼0.005
Duration of infertility (years)	4.62 ± 2.53	5.22 ± 0.45	0.05 ∼0.02
Cycle day for oocyte retrieval (d)	12.01 ± 2.20	10.88 ± 2.57	0.01 ∼0.005
Endometrial thickness at transfer (mm)	8.74 ± 0.85	8.65 ± 0.79	>0.05

LH, luteinizing hormone; FSH, follicle-stimulating hormone; T, testosterone; BMI, body mass index.

Monitoring

All women underwent a baseline ultrasound scan (Aloka SSD-1700; Aloka, Tokyo, Japan) on days 3 to 5 of menstrual bleeding, which was spontaneous or induced. This was to ensure that no ovarian cysts were present and to measure the number of antral follicles and the endometrial thickness. At the same time, the baseline endocrine measurements were obtained from all the patients. No ovarian stimulation was carried out during the treatment cycle. The transvaginal ultrasound scan was repeated on days 9 to 14 of the cycle to ensure that all follicles were smaller than 10 mm in diameter prior to oocyte retrieval. If one or more follicles were larger than 10 mm in diameter, the IVM cycle would be canceled.

Endometrium preparation

The dose of estradiol valerate (EV, Progynova; Schering AG, Berlin, Germany) was adjusted according to the endometrial thickness. On days 3 to 5 of the cycle, 4 mg of EV was given daily if endometrial thickness was less than 5 mm. On the day of oocyte retrieval, 6 to 10 mg of EV was administrated daily if endometrial thickness was less than 7 mm; otherwise 4 mg of EV was given daily. On the day of embryo transfer, the dose of EV was reduced to 4 mg and was continued at 4 mg daily for 2 weeks.

Immature oocyte collection

In order to relieve the pain, 50 mg of pethidine hydrochloride was injected 30 minutes prior to the operation. Transvaginal ultrasound-guided oocyte collection was performed on days 9 to 14 of the cycle using a 17G single-lumen aspiration needle (Wallace, ONS 1733; Smiths Medical international, Hythe, Kent, U.K.), with a reduced aspiration pressure set between 75 and 80 mmHg. The visible follicles were punctured, and follicular flushing was not performed. Follicular aspirates containing cumulus-oocyte complexes (COCs) were collected into 10-mL Falcon tubes (BD Biosciences, San Jose, CA), which contained 2 mL of prewarmed Dulbecco's phosphate-buffered saline (DPBS; Sigma-Aldrich, St. Louis, MO) with 2 IU/mL of heparin. Follicle aspirates were filtered through a cell strainer with 70-um-sized mesh (S4145-5EA; Sigma-Aldrich). The collected cells were rinsed several times with prewarmed PBS plus 5% (v/v) heat inactivated (56°C, 30 minutes) fetal bovine serum (FBS; Hangzhou Sijiqing Bioengineering Material Co., Ltd., Hangzhou, Zhejiang, China). The retained cells were resuspended in the medium. The COCs were then isolated under a stereomicroscope (Stemi-2000, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) and washed twice with M-199 (Sigma-Aldrich) supplemented with 10% (v/v) FBS. After that, they were transferred to IVM medium for culture.

In vitro maturation, fertilization, embryo culture, and embryo transfer

The immature oocytes were incubated in the medium containing M-199 supplemented with 20% (v/v) FBS, 0.25 mM pyruvic acid (Sigma-Aldrich), 75 mIU/mL recombinant follicle-stimulating hormone (rFSH; Gonal-F; Serono, Switzerland), with or without 0.5 IU/mL hCG (Profasi, Serono, Switzerland). The COCs were cultured at 37°C in 6% CO₂ with high humidity. After 24 hours of culture, the maturity of the oocytes was determined under an inverted microscope every 6 to 8 hours for 48 hours. Only the oocytes with an extrusion of the first polar body were considered mature and were denuded of cumulus cells to prepare for intracytoplasmic sperm injection (ICSI). A single spermatozoon was injected into each metaphase II oocyte. After ICSI, each oocyte was transferred into a cell cleavage medium (Sage BioPharma, Bedminster, NJ). The fertilization was assessed 16 to 18 hours after ICSI by the appearance of two distinct pronuclei and two polar bodies. Embryos were selected and subject to laser-assisted hatching (LAH) according to the method provided by Mantoudi et al.⁸ These embryos were transferred on Day 2 or 3 following ICSI.

Luteal support and pregnancy confirmation

Luteal support was provided by daily intramuscular injection of 60 to 80 mg of progesterone starting on the day of ICSI. At the same time, 4 mg of EV was given daily for 2 weeks or until the tenth week of pregnancy if the hCG test was positive. If the vaginal bleeding occurred, hormonal treatment was continued until the bleeding ceased. A transvaginal ultrasound scan was performed to confirm the pregnancy and to determine the number of gestational sacs present. A chemical pregnancy is one that has miscarried before the gestational sacs become visible under an ultrasound scan. A clinical pregnancy is the presence of a fetal sac visible on ultrasound early in the pregnancy.

Statistical analysis

Statistical analysis was performed using the Fisher's exact test. SPSS version 11.0 was used for all statistical analysis. Values were considered significant with p < 0.05.

Results

In the present study, 118 women with PCOS underwent 152 cycles of IVM. No significant differences were found between the two groups in the basal level of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), duration of infertility, and the endometrial thickness on the day of embryo transfer. The patients in Group A were older than those in Group B (p < 0.001), and the body mass index (BMI) in Group A was higher than that in Group B (0.01 < p < 0.005).The serum level of T in Group A was much lower than that of Group B (0.01 < p < 0.005). The cycle day for oocyte retrieval in Group A was later than that in Group B.

Twelve cycles were canceled including 5 in Group A and 7 in Group B, and the rates of cycle cancellation in the two groups were not significantly different (7.6% vs 8.1%, p > 0.05). Among them, seven cycles produced less than 5 oocytes, and patients wanted to stop the therapy because they worried about no embryo to transfer; four cycles failed to produce mature oocytes after 48 h in culture, and one case failed by cleavage. The overall cancellation rate was 7.9% (12/152). A total of 140 cycles were transferred, including 61 in Group A and 79 in Group B. No significant differences were found in the number of retrieved oocytes, the rates of maturation, fertilization, cleavage, and the mean number of embryos between the two groups (p > 0.05). Five biochemical pregnancies and 56 clinical pregnancies were obtained. Both the pregnancy rates per transfer and the implantation rate were comparable between the two groups. Among the 56 clinical pregnancies, 2 ectopic pregnancies and 6 single pregnancies were spontaneously aborted during the first 8 to 22 weeks of gestation, two from Group A and 6 from Group B. The rate of miscarriage in Group A tended to be low, but there was no difference between the two groups (p = 0.145). One set of twins in Group B with premature rupture of membranes occurred in the 22nd week, and 3 subjects with triplets underwent selective fetal reduction successfully. So far, 38 boys and 27 girls were born, including 29 singleton infants and 18 sets of twins. The live birth rate per embryo transfer in Group A trended higher, but there was no statistically significant difference between them. The detailed results between the two groups are shown in Table 2.

Table 2.

Outcome of In Vitro Maturation with (Group A) or without (Group B) Oral Contraceptives and Metformin

Parameter	Group A	Group B	p value
No. of initiated cycles	66	86
No. of canceled cycles	5	7	0.898
No. of transferred cycles	61	79
Number of oocytes retrieved	18.52 ± 8.4	18.25 ± 8.23	>0.05
Maturation rate at 36 h (%)	61.07	59.56	0.471
Maturation rate at 48 h (%)	70.25	67.60	0.170
Fertilization rate (%)	70.40	70.10	0.895
Cleavage rate (%)	91.24	89.80	0.413
No. of transferred embryos	3.1 ± 0.71	3.2 ± 0.75	0.5 ∼0.2
Implantation rate (%)	15.86	15.12	0.605
No. of biochemical pregnancies (%)	1 (3.85)	4 (11.43)	0.286
No. of clinical pregnancies per transfer (%)	25 (40.98)	31 (39.24)	0.835
No. of early miscarriages (%)	1 (4.0)	5 (16.13)^*	0.145
No. of ectopic pregnancies (%)	1 (4.0)	1 (3.23)	0.685
No. of singleton pregnancies (%)	15 (60.0)	14 (45.16)	0.269
No. of twin pregnancies (%)	7 (28.0)	9 (29.03)	0.932
No. of triplet pregnancies (%)	1 (4.0)	2 (6.45)	0.685
Live birth rate (%)	92 (23/25)	77.42 (24/31)	0.14
No. of born babies	31	34
Live birth rate/embryo transfer (%)	37.70 (23/61)	30.38 (24/79)	0.363

p < 0.01, pretreatment vs. control groups.

Discussion

Polycystic ovary syndrome (PCOS) is a key cause of anovulatory infertility. It is characterized by hyperandrogenemia and disordered gonadotropin secretion and is often associated with insulin resistance. OC remains the mainstay of treatment for clinical hyperandrogenism, which suppresses the secretion of LH and leads to a decrease in ovarian androgen production. However, evidence has shown that OC may reduce insulin sensitivity and glucose tolerance in PCOS women.^9–10 Metformin is an insulin sensitizer widely used for the treatment of patients affected by type 2 diabetes mellitus. Because insulin resistance and secondary hyperinsulinemia affect approximately 65–70% of PCOS women,¹¹ metformin was introduced in clinical practice to treat these patients also. Velazquez et al.¹² first evaluated the effects of metformin administration in 26 obese PCOS patients at a dose of 1500 mg/d for 6 months and found a significant reduction in circulating androgen levels and body weight. Clinical evidence has shown that metformin is an effective first-line treatment for restoring ovulatory menstrual cycles, improving insulin sensitivity and, unlike OC, a lipoprotein pattern. On the contrary, OC might worsen insulin sensitivity.¹³ Currently, metformin is more widely used to treat PCOS patients, especially as a pretreatment with or without OC in ovulation induction and in IVF cycles. IVF is increasingly applied in PCOS patients. However, some studies have been noted to be associated with an increased likelihood of ovarian hyper-stimulation syndrome (OHSS), reduced fertilization rates, abnormal early embryo development, and high incidence of miscarriage because of specific endocrine abnormalities.^14
–16 The combination of estrogen-progestin contraceptives is known to be the predominant treatment for hirsutism and acne in PCOS, based on its ability to reduce testosterone in these patients.¹⁷ Androstenedione levels were suppressed more by the combination therapy of OC plus metformin than by OC alone.¹⁸ Treating obese patients with PCOS with metformin and clomiphene markedly increased the ovulatory response compared with clomiphene alone.¹⁹ Stadtmaur et al.²⁰ found that metformin had a beneficial effect on IVF with clomiphene-resistant PCOS patients, with respect to improving the rates of mature oocytes, fertilization, embryo cleavage, and pregnancy. It has also been demonstrated by Tang et al.²¹ that simultaneous treatment with metformin, which initiates down-regulation using a long protocol of gonadotropin-releasing hormone (GnRH) agonist, significantly improves pregnancy outcome and reduces the risk of OHSS in PCOS patients undergoing IVF treatment. Yet, another study reported pretreatment with metformin prior to conventional IVF/ICSI in women with PCOS did not improve stimulation or clinical outcome. However, among PCOS women with normal weight, pretreatment with metformin seemed to improve pregnancy rates.²² Recently, seventeen randomized controlled trials (RCTs) showed that metformin has no effect on the spontaneous abortion risk in PCOS patients when administered before pregnancy.²³

Our previous study demonstrated that the combined administration of OC with metformin for 3 months prior to ovulation induction not only improved imbalances in serum hormone levels and metabolic parameters, but also increased ovulation and decreased the risk of OHSS.⁴ We further extended our study by adding a combination of OC plus metformin to PCOS patients prior to IVM treatment. Our results showed that the mean numbers of oocytes, the rates of cancellation, oocyte maturation, fertilization, cleavage, implantation, biochemical pregnancy, and clinical pregnancy were not significantly different between the two groups (p > 0.05). However, it is worth mentioning that the live birth rate per embryo transfer tended to be higher in the pretreated group than in the control group, although the difference did not reach statistical significance. Moreover, the rate of miscarriage tended to be low in the pretreatment group, but there was no statistical difference between the two groups. It might be because the oocyte quality and the implantation environment were improved after the pretreatment. Buckett et al.²⁴ observed that there was a higher rate of clinical miscarriage after IVM when compared with IVF and ICSI, which appeared to be related to PCOS rather than to the IVM procedure. Further study is needed to evaluate the effect of this combination treatment on the pregnancy outcomes of PCOS patients treated by IVM.

Conclusions

In summary, our study demonstrated that IVM of unstimulated immature oocytes is a useful method of treatment for infertile Chinese women with PCOS. Pretreatment with oral contraceptives and metformin before IVM is promising, as suggested by a comparatively low miscarriage rate.

Footnotes

Acknowledgments

The authors wish to express their deep gratitude to Dr. Yuping Chen and Dr. Haiyun Xing for their review of the manuscript, and all the members of the IVM team for their hard work.

Disclosure Statement

The authors have no conflicts of interest to report.

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