Polycystic ovary syndrome (PCOS) is a common endocrine
disorder in women of reproductive age, which is
mostly associated with hyperinsulinemia, hyperandrogenism
and anovulatory infertility (
Follicular arrest (FA) and dysregulation of paracrine activity
in follicles are noticeable ovarian signs in PCOS
Recently, it has been observed that insulin can modulate steroidogenesis through its own receptor. Moreover, in case of insulin resistance, the steroidogenesis appears to be preserved likely by various mechanisms of regulation of receptors receptivity in different tissue (
Excessive ovarian androgen production has also been implicated in the pathogenesis of PCOS. It has been postulated that hyperinsulinemia in case of insulin resistance, is associated with capacity of ovarian androgen production (
Folliculogenesis and oocyte maturation are complex processes that require the action of both LH and FSH (
In this study, we prospectively evaluated the association between different factors [age, body mass index (BMI), number of previous ART cycle, and LH/FSH ratio] and oocyte maturity in insulin-resistant and insulin-sensitive women with PCOS in comparison with control group.
In this case-control study, each of 40 patient’s case seeking assisted reproduction at Royan Institute from April 2014 to January 2015 was analyzed. Written informed consent was obtained from all the participants. Ethics approval was obtained from the local Ethics committee of Royan Institute (no.EC/93/1138). PCOS patients were allocated to one of the two groups formed based on the level of fasting insulin (FI): insulin resistant (PCOS-IR; FI≥12 mg/dl) and insulin sensitive (PCOS-IS; FI<12 mg/dl). In control group, 20 women with regular menstrual cycle without known diseases (i.e. fertile women with male infertility history) were included. Exclusion criteria were impaired thyroid, renal or hepatic function, congenital adrenal hyperplasia (CAH), endometriosis, premature ovarian insufficiency (POI), functional hypothalamic amenorrhea (FHA), unexplained infertility (UI) and age>36 years.
In order to controlled induce ovarian stimulation (COS), daily subcutaneous injection of recombinant human FSH (rFSH, Gonal F®; Serono Pharma, Switzerland) was started from the second day of the cycle. Starting dose of rFSH was adjusted individually depending on patients response measured by transvaginal ultrasonography, antral follicle count (AFC), levels of serum estradiol (E2) and AMH. A GnRH antagonist-cetrorelix (Cetrotide®, Merck Serono, Germany) was administered subcutaneously when at least two ovarian follicles reached 14 mm in diameter. The protocol consisted of daily subcutaneous injections of Cetrotide 0.25 mg, until the criteria for human chorionic gonadotropin (hCG) administration were met. For final oocyte maturation, when the dominant follicle reached ≥18 mm in diameter with the following two follicles ≥16 mm and E2 levels between 1000-4000 pg/mL, an intramuscular injection of 10.000 IU hCG (Pregnyl®, Organon, Holland) or subcutaneous injection of 250 μg hCG (Ovitrelle®, Merck Serono, France) was given.
Oocyte Pick-up (OPU) was done using transvaginal ultrasound-guided follicle aspiration, 36 hours after hCG administration to collection tubes. Following OPU, cumulus-oocyte complexes were washed several times in fertilization medium (G-IVF®, Vitrolife, Sweden) to remove blood and cell debris, and incubated for two hours in fertilization medium (G-IVF®, Vitrolife, Sweden). Retrieved oocytes were classified into metaphase II (MII) stage as mature and metaphase I (MI) or germinal vesicle (GV) stage as immature. Oocyte denudation was performed using 80 IU of hyaluronidase (Sigma, USA) (
Based on our laboratory standards, embryos were graded at the pronuclear and cleavage stages. The quality of the embryos at cleavage stage were classified according to the following criteria: [excellent quality (≥4 cells or ≥8 cells and <10 % fragmentation), good quality (≥ 4 cells or ≥8 cells and 10-20% fragmentation) and poor quality (<4 cells or <8 cells and >20 % fragmentation)] (
On the day of oocyte retrieval, luteal phase support included Cyclogest® 200 mg (Actavis, UK) vaginal suppositories, twice daily (bid) for 14 days. Endometrial thickness was between 8 to 11 mm and showed a triple-line pattern as examined by vaginal ultrasonography on the day of hCG injection. Gestation was confirmed by pregnancy test 14 days after ET. Clinical pregnancy confirmed when a gestational sac with fetal cardiac activity was detectable after 7 weeks of gestation. Biochemical gestation was not taken into consideration at any stage of the study.
In this study, categorical variables are presented as number (%) and continuous variables as mean ± SD or median (minimum-maximum; inter-quartile range) where appropriate. Statistical comparisons of means of the three study groups were performed using ANOVA or its nonparametric equivalent, Kruskal-Wallis test. Independent t-test was used to assess mean differences in FI between IR and IS- PCOS groups. Chi-square analysis was used for qualitative data. Univariate and backward multiple linear regression, including all variables, were used to evaluate the association between MII oocyte number and some demographic and clinical variables. Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 22.0 (IBM Crop., Armonk, NY, USA). All statistical tests were 2-tailed and a P<0.05 was considered statistically significant.
Clinical characteristics of participants are shown in Table 1. BMI was significantly higher in PCOS-IR group (29.97 ± 4.39) compared to control group (25.12 ± 4.21 P=0.023), this difference was not significant between PCOS-IS (26.31 ± 8.03) and either of PCOS-IR and control groups. PCOS-IR women had significantly fewer number of MII oocytes (8.10 ± 3.61) compared to controls (11.57 ± 5.11, P=0.028); but, the number of MII oocytes was not significantly different between PCOS-IS women (9.05 ± 3.37) and control subjects. There were no significant differences in MI, GV and dead oocytes between PCOS groups (IR and IS) and control group. Fasting insulin was significantly higher in PCOS-IR (19.30 ± 9.60 mg/dl) compared to PCOS-IS group (6.67 ± 2.89 mg/dl, P=0.006). Other criteria including age, previous ART history, number of retrieved oocytes, number of 2PN embryos, total number of embryo and success rate did not differ significantly among the three groups.
LH/FSH ratio was significantly higher in PCOS-IR women (1.67 ± 1.75) compared to controls (0.94 ± 0.68, P=0.047) but not significantly different from that of PCOS-IS group (1.45 ± 0.94) (
Boxplots of LH/FSH in PCOS-IR, PCOS-IS and control group. LH; Luteinizing hormone, FSH; Follicle-stimulating hormone, PCOS; Polycystic ovary syndrome, IR; Insulin resistant, and IS; Insulin sensitive. Extreme values are indicated using asterisks and outliers are indicated using circles.
Demographic and clinical characteristics of recruited patients
|PCOS-IR n=20||PCOS-IS n=20||Control n=20||P value|
|Age (Y)||30.04 ± 4.45||28.00 ± 4.00||29.05 ± 5.18||0.381|
|Yes||7 (25.9)||7 (25.9)||13 (48.2)||0.108|
|No||15 (42.9)||12 (34.3)||8 (22.8)|
|Fasting insulin (mg/dl)||19.30 ± 9.60||6.67 ± 2.89||-||0.006|
|BMI||29.97 ± 4.39a||26.31 ± 8.03ab||25.12 ± 4.21b||0.023|
|Oocyte retrievedMIIMIGVDegenerated/dead||11.90 ± 7.098.01 ± 3.61a0 (0-3; 1)0 (0-2; 0)0 (0-1; 0)||13.58 ± 11.269.05 ± 3.37ab0 (0-2; 0)0 (0-4; 1)0 (0-4; 1)||12.90 ± 5.59111.57 ± 5.11b0 (0-1; 1)0 (0-3; 1)0 (0-2; 0.5)||0.8140.0280.8060.4680.125|
|Total embryo||7.15 ± 4.29||9.44 ± 7.34||7.95 ± 4.07||0.415|
|2PN embryos||6 (0-14; 4.75)||7 (1-13; 3.25)||9 (2-18; 4.5)||0.723|
|Fertilization (%)||73.28 ± 25.43||75.90 ± 25.89||76.01 ± 17.24||0.914|
PCOS; Polycystic ovary syndrome, IR; Insulin resistant, IS; Insulin sensitive, ART; Assisted reproductive technology, BMI; Body mass index, MI; Metaphase I oocytes, MII; Metaphase II oocytes, GV; Germinal vesicle stage, 2PN; Two pronuclei. Continuous variables are presented as mean ± SD or median (minimum-maximum; inter-quartile range) when appropriate. Categorical variables are presented as number (%). Different letters indicate significantly different means (as evaluated by ANOVA and Tukey post-hoc test). A P<0.05 was considered significant.
Regression analysis for factors associated with MII oocytes number
|Univariate analysis||Multiple analysis|
|B||SE||P value||B||SE||P value|
|No. of previous ART cycle||-0.71||0.61||0.251||-1.23||0.59||0.043|
|PCOS-IR vs. control||-3.47||1.30||0.010||-4.09||1.29||0.003|
|PCOS-IS vs. control||-2.52||1.33||0.064||-3.21||1.34||0.020|
B; Unstandardized coefficient, SE; Standard error, BMI; Body mass index, LH; Luteinizing hormone, FSH; Follicle-stimulating hormone, ART; Assisted reproductive technology, PCOS; Polycystic ovary syndrome, IS; Insulin sensitive, and IR; Insulin resistant.
PCOS is a heterogeneous endocrinopathy; insulin resistance and elevated LH/FSH ratio play a potential role in the pathogenesis of the disorder (
According to recent studies, insulin signals relay through multiple pathways, many of them are active in ovarian follicular cells especially oocytes. These pathways are able to interact with each other and also with gonadotropins. Thus, insulin has a direct regulatory effect on ovarian physiology (
The strengths of this study were evaluation of the effects of increased LH/FSH level and insulin resistance on the oocyte maturation. This study has a prospective scheme and all patients underwent ICSI and received GnRH antagonist. Besides, the statistical calculations were accomplished by multivariable analysis. Finally, we observed lower number of MII oocytes in IR patients. The mechanisms underlying these results are unclear; although previous studies showed that ovarian folliculogenesis may disrupted by high LH levels (
According to some studies, insulin resistance is not a disease but it can develop numerous metabolic alterations (
According to previous studies, the success probability is essentially constant across the first three IVF attempts (
Based on the results of this study, for each unit increase in previous IVF/ICSI failure, the expected number of MII oocytes decreases by 1.23 in PCOS women. Moreover, to the best of our knowledge, the present study is the first report showing that the history of IVF/ICSI failure is associated with reduction of MII oocytes in PCOS-IR patients. Also, our results showed that retrieval of lower numbers of mature oocytes from PCOS women might be in part related to insulin resistance. Our study suggested that treatment of insulin resistance should be considered in PCOS-IR patients who have a history of canceled IVF cycles. This can help to achieve greater numbers of mature oocytes.
Insulin resistance is a common metabolic abnormality in PCOS patients and PCOS-IR women had lower MII oocytes than control group. Collectively, histories of ART failure and insulin resistance are two important factors in predicting the number of mature oocytes in PCOS-IR patients.