1 Department of Obstetrics and Gynecology, E-Da Hospital/I-Shou University, Kaohsiung County, 2 Institute of Clinical Medicine and 3 Institute of Physiology, National Yang-Ming University, Taipei, 4 Department of Medical Research & Education and 5 Department of Internal Medicine, Taipei Veterans General Hospital, Taipei, 6 Department of Obstetrics and Gynecology, Shin Kong Wu-Ho-Su Memorial Hospital, Taipei and 7 Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan
8 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Shin Kong Wu-Ho-Su Memorial Hospital, No. 95, Wen Chang Road, Shih Lin District, Taipei, Taiwan. Email: ed101174{at}edah.org.tw
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Abstract |
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Key words: follicular fluid/IVF/polycystic ovarian syndrome/resistin
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Introduction |
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In addition, women with PCOS have a higher rate of early pregnancy loss, poorer oocyte quality, and a lower fertilization and cleavage rate than normal women (Homburg et al., 1988; Smitz et al., 1992
; Balen et al., 1993
; Homburg et al., 1993
). Hyperinsulinaemia has been implicated as an independent risk factor for early pregnancy loss (Fedorcsak et al., 2000
). Pregnant PCOS women show a marked reduction in miscarriage rate if treated with metformin throughout pregnancy (Glueck et al., 2001
).
The hormone resistin, a 12.5 kDa cysteine-rich protein, is secreted by adipocytes (Steppan et al., 2001). Serum resistin levels are significantly increased in insulin-resistant mice and genetically or diet-induced obese mice (Steppan et al., 2001
). In addition, neutralization of endogenous resistin with antibodies significantly suppresses hyperglycaemia in diet-induced obese mice by increasing insulin sensitivity (Steppan et al., 2001
). Furthermore, resistin gene expression in obese mice is down-regulated by thiazolidinedione compounds, a new class of antidiabetic drugs with an insulin-sensitizing action. These results suggest that resistin may be an important link between increased fat mass and insulin resistance.
The association between PCOS and resistin has been discussed in several reports, which stated that serum resistin levels are not elevated in PCOS women with insulin resistance (Seow et al., 2004; Panidis et al., 2004
), and resistin was therefore not regarded as a major determining factor of PCOS-associated insulin resistance. However, resistin mRNA levels in adipocytes are increased 2-fold in PCOS patients (Seow et al., 2004
), suggesting that the resistin gene may be a local determining factor in the pathogenesis of PCOS. Follicular growth and maturation is a complex process regulated by autocrine and paracrine factors (Lédée-Bataille et al., 2001
) and provides the environment in which oocyte maturation occurs. Levels of cytokines such as leptin, tumour necrosis factor(TNF)-
and interleukin (IL)-6 in the follicular fluid affect the success of fertilization and early embryonic development (Brannström and Norman, 1993; Adashi, 1994
), including in women with PCOS (Mantzoros et al., 2000
; Lédée-Bataille et al., 2001
; Amato et al., 2003
).
The purpose of this study was to compare levels of serum and follicular fluid resistin in infertile women with PCOS undergoing IVF and in controls and to assess possible correlations between resistin levels and reproductive outcome.
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Materials and methods |
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Subjects
Twenty-one women who fulfilled the inclusion criteria for PCOS given below were enrolled. All were in good health and had not taken oral contraceptives within the last 3 months. Before entering the IVF programme, they had undergone more than six cycles of ovulation induction with increasing doses of clomiphene citrate or gonadotrophin, but had failed to conceive.
PCOS was defined by clinical, laboratory and ultrasound criteria according to the consensus criteria reported by the Rotterdam group (2004). The clinical criterion was oligomenorrhoea (menstrual interval >6 weeks) or amenorrhoea (no menstrual loss for
6 months) dating from menarche. None of the subjects had acanthosis nigricans. The biochemical criteria were increased LH levels (>6 mIU/ml, normal follicular range 16 mIU/ml), normal FSH levels, and increased total serum testosterone levels (>0.8 ng/ml, normal range 0.060.86 ng/ml). The ultrasound criteria were enlarged ovaries with an increased stroma and >10 subcapsular follicles of 38 mm diameter, arranged peripherally around a dense core of stroma, as described by Adams et al., (1986)
. Serum prolactin and thyroid hormone levels were checked in all patients and were within the normal limits. Cushing's syndrome and congenital adrenal hyperplasia were excluded by checking cortisol and serum 17-hydroxyprogesterone levels in the morning. The exclusion criteria also included patients aged >45 years or with serum FSH levels >12 mIU/ml.
Eighteen healthy, age- and body mass index (BMI)-matched, normally ovulating women diagnosed with male factor infertility served as controls. None was hirsute, and all had a normal regular cycling menstrual period. None was taking oral contraceptives. All had a normal appearance of the ovaries on ultrasound and normal LH and FSH levels, and none had elevated androgen levels.
Stimulation protocol and follicular fluid collection
Ovarian stimulation was induced with hMG (Pergonal; Serono, Italy) combined with FSH (Metrodin; Serono, Italy), or with GnRH analogues (Supremon; Hoechst, Germany). hCG (10 000 IU, Pregnyl; NY Organon, The Netherlands) was injected 36 h before oocyte retrieval when the leading follicle reached 18 mm. The aspirated oocytes were cultured and inseminated and the embryos transferred by transvaginal gamete transfer or zygote intra-Fallopian transfer. Follicular fluid was collected at the time of oocyte recovery. The fluid was aspirated from the follicles, avoiding contamination with the flushing medium. After oocyte removal, the follicular fluid from each follicle in the same cycle was pooled for each patient and centrifuged at 600 g for 5 min at 4°C and the supernatant stored at 80°C until assayed.
Oral glucose tolerance test and homeostasis model insulin resistance index
A 2 h oral glucose tolerance test (OGTT) with 75 g of glucose load was performed after overnight fast during the early follicular phase (days 37) on all women. Four blood samples were collected at 0, 30, 90 and 120 min and the serum stored at 20°C until assayed for glucose and insulin. The homeostasis model assessment of insulin resistance index (HOMAIR) was calculated as: fasting glucose (mmol/l) x fasting insulin (µIU/ml)/22.5 (Matthews et al., 1985). The HOMA applied to the OGTT (HOMAOGTT) was calculated as described by Matsuda and DeFronzo (1999)
. An insulin-resistant state was defined as a HOMAOGTT value <4.7.
Hormonal profile
The hormonal profile [FSH, LH, estradiol (E2), and testosterone] was measured in a blood sample drawn from the antecubital vein on the day of the OGTT. For patients with amenorrhoea, 75 mg of progesterone was given i.m. to induce withdrawal bleeding, then the blood sample was collected on cycle day 3 or 4. Serum levels of FSH, LH and testosterone were measured by immunoassay using an Immulite® kit (Diagnostic Products Corporation, USA). For FSH, the sensitivity was 0.1 mIU/ml and the intra- and inter-assay coefficients of variance (CV) 7.7 and 7.9% respectively. For LH, the sensitivity and intra- and inter-assay CV were 0.1 mIU/ml, 6.5 and 7.1% respectively. For testosterone, the sensitivity and intra- and inter-assay CV were 0.1 ng/ml (0.4 nmol/l), 4.0 and 5.6% respectively. E2 was measured by competitive immunoassay using an Immulite® kit, with sensitivity and intra- and inter-assay CV of 15 pg/ml (55 pmol/l), 6.3 and 6.4% respectively.
Blood collection for resistin assay
Blood samples (7 ml) were collected in Lavender Vacutainer tubes and transferred to a centrifuge tube containing aprotinin (0.6 TIU/ml of blood) and rocked several times to inhibit proteinease activity; the sample was then centrifuged at 1600 g for 15 min at 4°C and the serum stored at 70°C until assayed for resistin.
Resistin assay
Resistin was measured in fasting blood collected on the day of the OGTT and in follicular fluid samples collected on the day of transvaginal oocyte retrieval using a commercially available competitive enzyme-linked immunoassay (ELISA) kit (Phoenix Pharmaceuticals Inc., USA) with a low limit of sensitivity of 1.21 ng/ml (range: 0500 ng/ml) and intra- and inter-assay CV of <5 and <14% respectively. The plasma sample was diluted 2-fold with ELISA buffer prior to assay.
Statistics
The KolmogorovSmirnov test was applied to both groups to test the normal distribution for each variable. Since none of the variables showed a data normal distribution, all results are expressed as the median and range. Differences between the PCOS and control groups were compared using the non-parametric MannWhitney U-test. 2-Tests were used to compare the dichotomous data. Correlations between serum or follicular fluid resistin levels and each parameter were evaluated by bivariate correlation analysis (Spearman's rank test) using a two-tailed test. P<0.05 was considered significant.
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Results |
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Discussion |
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Women with PCOS are frequently insulin resistant, independent of obesity (Burghen et al., 1980). In the present study, although there was no difference in fasting glucose and fasting insulin levels between the PCOS women and the control group, the 2 h glucose and 2 h insulin levels were significantly higher in the PCOS group. Moreover, the women with PCOS had a significantly lower HOMAOGTT than the controls, showing that they were insulin-resistant (48% of the women with PCOS showed a HOMAOGTT <4.7). Several studies have reported that insulin resistance and compensatory hyperinsulinaemia in women with PCOS have direct effects on oocyte maturation and ovulation (Shepard et al., 1979
; Lobo et al., 1982
; Nestler et al., 1998
; Vandermolen et al., 2001
). However, in the present study, insulin resistance in PCOS showed no correlation with resistin levels in serum and follicular fluid during the IVF cycle, since these were no higher in the PCOS group as a whole or in the insulin-resistant subgroup than in the control group.
The role of cytokines in PCOS has been investigated in several studies. Panidis et al., (2004) reported that serum resistin levels were significantly higher in PCOS women with a BMI >25 kg/m2 than in lean PCOS women and controls. No difference was found in serum adiponectin levels between PCOS and control groups (Orio et al., 2004
). Compared to normal women, PCOS women on IVF have higher serum and follicular levels of TNF-
and IL-6, but lower follicular fluid E2 levels (Amato et al., 2003
). In addition, women with PCOS who become pregnant tend to have lower mean follicular fluid leptin levels than those who do not (Mantzoros et al., 2000
). Our present study did not find any difference in follicular fluid resistin levels between those women with PCOS who became pregnant and those who did not. Furthermore, there was no correlation between serum or follicular resistin levels and fertility rate, implantation rate, clinical pregnancy rate, or early miscarriage rate. These results suggest that resistin does not play a role in the maturation and development of oocytes in women with PCOS. Moreover, the fact that follicular fluid resistin levels were lower than serum levels may indicate that resistin does not have a paracrine effect in the process of oocyte development in women with PCOS and controls receiving IVF.
Several studies have reported that the raised LH levels in women with PCOS may reduce the fertilization rate in IVF cycles and contribute to the high early miscarriage rate in women with PCOS (Homburg et al., 1988; Smitz et al., 1992
; Balen et al., 1993
; Homburg et al., 1993
). We postulated that, in women with PCOS, resistin may have a detrimental effect on reproductive outcome similar to that of LH, but did not find any positive or negative effect of resistin on pregnancy outcome. We currently do not know whether the administration of GnRH analogue for ovulation induction reduces follicular resistin levels and whether it may be better to aspirate the follicular fluid before GnRH analogue administration.
The limitation of this study was that we pooled the follicular fluid from the different follicles in a single cycle for each individual patient and thus did not measure resistin levels in each individual follicle. However, this probably did not markedly affect the final results, since the overall follicular resistin levels were very low compared to the serum levels despite the samples being pooled.
In conclusion, the lack of a significant difference in serum resistin levels between PCOS and the control subjects and the fact that resistin levels in the follicular fluid compared to serum were lower in both groups of women during an IVF-stimulated cycle suggest that resistin is unlikely to be a major determining factor in the growth and maturation of oocytes during IVF-stimulated cycles in women with PCOS.
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Submitted on May 12, 2004; resubmitted on August 17, 2004; accepted on October 4, 2004.