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Michal Zygarowicz,1,* Joanna Kacperczyk-Bartnik,2,3,* Janusz Sierdzinski,4 Cezary Wojtyla,5,6 Piotr Pierzynski,5,6 Grzegorz Manka,7 Mariusz Kiecka,7 Robert Z Spaczynski,8 Piotr Piekarski,9 Beata Banaszewska,10 Artur Jakimiuk,11,12 Tadeusz Issat,13 Wojciech Rokita14,15 ,† Jakub Mlodawski,14,15 Maria Szubert,3,16,17 Piotr Sieroszewski,16,18 Grzegorz Raba,19,20 Kamil Szczupak,19,20 Tomasz Kluz,21 Marek Kluza,21 Michal Lipa,3,22 Damian Warzecha,5,22 Miroslaw Wielgos,23,24 Ewa Koc-Zorawska,25,26 Marcin Zorawski,26,27 Piotr Laudanski5,6,28 1Students’ Scientific Group Affiliated to the Department of Obstetrics, Gynecology and Gynecological Oncology, Medical University of Warsaw, Warsaw, Poland; 2II Department of Obstetrics and Gynaecology, Medical University of Warsaw, Warsaw, Poland; 3Club 35. Polish Society of Gynecologists and Obstetricians, Wrocław, Poland; 4Department of Medical Informatics and Telemedicine, Medical University of Warsaw, Warsaw, Poland; 5OVIklinika Infertility Center, Warsaw, Poland; 6Women’s Health Research Institute, Calisia University, Kalisz, Poland; 7Angelius Provita Hospital, Katowice, Poland; 8Center for Gynecology, Obstetrics and Infertility Treatment Pastelova, Poznan, Poland; 9Division of Infertility and Reproductive Endocrinology, Department of Gynecology, Obstetrics and Gynecological Oncology, Poznan University of Medical Sciences, Poznan, Poland; 10Chair and Department of Laboratory Diagnostics, Poznan University of Medical Sciences, Poznan, Poland; 11Department of Obstetrics and Gynecology, Central Clinical Hospital of the Ministry of Interior, Warsaw, Poland; 12Center of Reproductive Health, Institute of Mother and Child in Warsaw, Warsaw, Poland; 13Department of Obstetrics and Gynecology, Institute of Mother and Child in Warsaw, Warsaw, Poland; 14Collegium Medicum Jan Kochanowski University in Kielce, Kielce, Poland; 15Clinic of Obstetrics and Gynecology, Provincial Combined Hospital in Kielce, Kielce, Poland; 16Department of Gynecology and Obstetrics Medical University of Lodz, Lodz, Poland; 17Department of Surgical Gynecology and Oncology, Medical University of Lodz, Lodz, Poland; 18Department of Fetal Medicine and Gynecology, Medical University of Lodz, Lodz, Poland; 19Clinic of Obstetrics and Gynecology, Provincial Hospital, Przemysl, Poland; 20Department of Obstetrics and Gynecology, University of Rzeszow, Rzeszow, Poland; 21Department of Gynecology, Gynecology Oncology and Obstetrics, Institute of Medical Sciences, Medical College of Rzeszow University, Rzeszow, Poland; 22Department of Obstetrics and Gynaecology, Warsaw Southern Hospital, Warsaw, Poland; 23Premium Medical, Warsaw, Poland; 24Faculty of Medicine, Lazarski University, Warsaw, Poland; 25II Department of Nephrology, Hypertension and Internal Medicine with Dialysis Unit, Medical University of Bialystok, Bialystok, Poland; 26The Academy of Applied Medical and Social Sciences, Elbląg, Poland; 27Department of Cardiology, Lipidology and Internal Medicine with Cardiac Intensive Care Unit, Medical University of Bialystok, Bialystok, Poland; 28Department of Obstetrics, Gynecology and Gynecological Oncology, Medical University of Warsaw, Warsaw, Poland*These authors contributed equally to this work†Professor Wojciech Rokita passed away on March 18, 2020Correspondence: Piotr Laudanski, Department of Obstetrics, Gynecology and Gynecological Oncology, Medical University of Warsaw, Kondratowicza Street 8, Warsaw, 03-242, Poland, Tel +48 22 326 53 80, Email piotr.laudanski@wum.edu.plIntroduction: Endometriosis is an inflammatory-related reproductive age disease characterized by the presence of endometrial cells outside the uterine cavity. Current laboratory practice does not provide specific markers for detecting and assessing the advancement of endometriosis in either plasma or peritoneal fluid. The severity of disease is assessed in stages from I to IV based on the results of laparoscopic inspection. The protein annexin A2 (ANXA2) has been reported to be associated with inflammatory processes.Aim of the Study: The study aimed to investigate and compare ANXA2 protein concentration using the ELISA method in plasma and peritoneal fluid in a group of women with endometriosis compared to controls.Materials and Methods: Biological material was collected during a multicenter, cross-sectional study, which was conducted at eight departments during elective laparoscopy from 53 women with and 40 women without endometriosis. Patients were divided by endometriosis stage and infertility status, and then compared with subgroups. Analysis included the Chi-square test for categorical variables, Mann–Whitney U-test and two-sided Wilcoxon rank-sum test for continuous variables.Results: Women with endometriosis had significantly elevated plasma ANXA2 levels compared to women without endometriosis (mean concentrations 28.69 vs 19.61 ng/L, p=0.01). Differences in peritoneal fluid ANXA2 levels were statistically insignificant (mean concentrations of 23.7 vs 22.97 ng/L, p=0.06). Plasma concentrations in patients with stage III and IV endometriosis were significantly higher compared to controls (mean concentrations of 24.19 vs 19.71 ng/L, p=0.03). No such differences were observed in plasma when comparing stages I–II vs III–IV, and stages I–II vs controls (mean concentrations of 33.82 vs 24.19 ng/L, p=0.72 and 33.82 vs 19.71 ng/L, p=0.12, respectively). Comparison of samples from patients with or without infertility, primary or secondary infertility, endometriosis with or without infertility, and non-endometriosis with or without infertility showed no significant differences in the plasma nor in the peritoneal fluid concentrations.Conclusion: ANXA2 is possibly involved in the pathogenesis of endometriosis, especially in advanced stages. Due to the limited group of tested samples, further studies are needed to confirm its role.Keywords: annexin A2, endometriosis, infertility

Polycystic ovary syndrome (PCOS) is the most common heterogeneous endocrine disorder among women of reproductive age. The pathogenesis of PCOS remains elusive; however, there is evidence suggesting the potential contribution of genetic interactions or predispositions combined with environmental factors. Among these, endocrine disrupting chemicals (EDCs) have been proposed to potentially contribute to the etiology of PCOS. Granulosa and theca cells are known to cooperate to maintain ovarian function, and any disturbance can lead to endocrine disorders, such as PCOS. This article provides a review of the recent knowledge on PCOS pathophysiology, the role of granulosa and theca cells in PCOS pathogenesis, and the evidence linking exposure to EDCs with reproductive disorders such as PCOS.

Background: Sex hormones influence the cardiovascular (CV) function in women. However, it is uncertain whether their physiological variation related to the regular menstrual cycle affects the CV system. We studied changes in the hemodynamic profile and body’s water content and their relation to sex hormone concentration in healthy women during the menstrual cycle. Material and methods: Forty-five adult women were examined during the early follicular, late follicular, and mid-luteal phases of the same menstrual cycle. The hemodynamic profile was estimated non-invasively by cardiac impedance while water content was estimated by total body impedance. Results were compared with repeated measures ANOVA with post-test, if applicable. Results: There were no significant changes in most hemodynamic and water content parameters between the menstrual cycle phases in healthy women. Left ventricular ejection time differed significantly among phases of the menstrual cycle, with shorter values in the mid-luteal phase (308.4 vs. 313.52 ms, p < 0.05) compared to the late follicular phase. However, the clinical relevance of such small differences is negligible. Conclusions: Changes in sex hormones during the physiological menstrual cycle appear to have no considerable effect on healthy women’s hemodynamic function and water accumulation.

Objectives: The purpose of this publication is to present data on the results and complications associated with infertility treatment using assisted reproductive technology (ART) and intrauterine insemination (IUI) in Poland between 2013 and 2016., Material and Methods: The report was prepared by the Polish Society of Reproductive Medicine and Embryology (PTMRiE) and the Fertility and Sterility Special Interest Group of the Polish Society of Gynaecologists and Obstetrics (SPiN PTGiP) as a part of the European IVF Monitoring program (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). Reporting was voluntary and the data was not subject to external control. The report presents the availability and structure of infertility treatment services, the number of procedures, their effectiveness and complications., Results: Between 2013 and 2016, a total of 106,718 treatment cycles using ART [64,413 classical in vitro fertilization and in vitro fertilization with intracytoplasmic sperm injection (IVF + ICSI), 36,041 frozen embryo replacements (FER)] and 51,405 IUI were recorded. The clinical pregnancy rates per embryo transfer in IVF, ICSI and FER were 38.3%, 38.1% and 32.4%, respectively. The effectiveness of IUI with husband/partner's semen (IUI-H) was 11.1% and with donor semen (IUI-D) 16.7%. Multiple delivery rates were 11.3% and 6.2% in IVF + ICSI and IUI, respectively. The most common complication was the ovarian hyperstimulation syndrome (OHSS) (0.34%)., Conclusions: PTMRiE and SPiN PTGiP report is the only national study documenting Polish reproductive medicine. The results of infertility treatment effectiveness in Poland are comparable with the European data, complications are less frequent than in other countries. The low percentage of multiple pregnancies, and so perinatal complications, is especially valuable. However, due to the lack of a central database and register, the possibility of external control and monitoring of pregnancies and births is limited. Thus, a fully reliable assessment of the treatment quality in our country is not possible.

Ovarian Granulosa Cells (GCs) are known to proliferate in the developing follicle and undergo several biochemical processes during folliculogenesis. They represent a multipotent cell population that has been differentiated to neuronal cells, chondrocytes, and osteoblasts in vitro. However, progression and maturation of GCs are accompanied by a reduction in their stemness. In the developing follicle, GCs communicate with the oocyte bidirectionally via gap junctions. Together with neighboring theca cells, they play a crucial role in steroidogenesis, particularly the production of estradiol, as well as progesterone following luteinization. Many signaling pathways are known to be important throughout the follicle development, leading either towards luteinization and release of the oocyte, or follicular atresia and apoptosis. These signaling pathways include cAMP, PI3K, SMAD, Hedgehog (HH), Hippo and Notch, which act together in a complex manner to control the maturation of GCs through regulation of key genes, from the primordial follicle to the luteal phase. Small molecules such as resveratrol, a phytoalexin found in grapes, peanuts and other dietary constituents, may be able to activate/inhibit these signaling pathways and thereby control physiological properties of GCs. This article reviews the current knowledge about granulosa stem cells, the signaling pathways driving their development and maturation, as well as biological activities of resveratrol and its properties as a pro-differentiation agent.

Context: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy affecting women of reproductive age. Hyperandrogenism is the central feature of PCOS. Studies on isolated ovarian theca-interstitial cells suggest that resveratrol, a natural polyphenol, reduces androgen production., Objective: This study was designed to evaluate endocrine and metabolic effects of resveratrol on PCOS., Design and Setting: This was a randomized (1:1) double-blind, placebo-controlled trial that evaluated the effects of resveratrol over a period of 3 months in an academic hospital., Patients and Other Participants: Subjects with PCOS were identified according to the Rotterdam criteria. Thirty-four subjects were enrolled and 30 subjects completed the trial. Evaluations were performed at baseline and repeated after 3 months of treatment., Intervention: Resveratrol (1,500 mg p.o.) or placebo were administered daily., Main Outcome Measure: Primary outcome was the change in the serum total T., Results: Resveratrol treatment led to a significant decrease of total T by 23.1% (P = .01). In parallel, resveratrol induced a 22.2% decrease of dehydroepiandrosterone sulfate (P = .01), a decrease of fasting insulin level by 31.8% (P = .007) and an increase of the Insulin Sensitivity Index (Matsuda and DeFronzo) by 66.3% (P = .04). Levels of gonadotropins, the lipid profile as well as markers of inflammation and endothelial function were not significantly altered., Conclusions: Resveratrol significantly reduced ovarian and adrenal androgens. This effect may be, at least in part, related to an improvement of insulin sensitivity and a decline of insulin level.

Free fatty acid binding protein-4 (FABP4) and retinol binding protein-4 (RBP4) contribute to metabolic syndrome. We investigated serum FABP4 and RBP4 responses to insulin sensitizing and lipid lowering therapies in polycystic ovary syndrome (PCOS). Sixty-two healthy, untreated women with PCOS (age 25.1 ± 3.6 years, BMI: 24.0 ± 4.7 kg/m(2)) were randomized to metformin (n = 24), simvastatin (n = 20) or metformin plus simvastatin (n = 18) for 3 months. Anthropometric measures, fasting blood tests and oral glucose tolerance tests (OGTT) were obtained at the baseline and the end. At the baseline serum FABP4 correlated with obesity (BMI: r = 0.63, p < 0.001), insulin resistance (fasting insulin: r = 0.44, p = 0.0002; QUICKI: r = -0.30, p = 0.02; OGTT-insulin sensitivity index: r = -0.27, p = 0.04), dyslipidemia (HDL: r = -0.26, p = 0.03) and hyperandrogenemia (free-testosterone: r = 0.23, p = 0.03; SHBG: r = -0.28, p = 0.03); while RBP4 correlated with total-cholesterol (r = 0.33, p = 0.009). Multiple regression analysis indicated that t best predictors of serum FABP4 and RBP4 were BMI (β = 1.02, p = 0.0003) and total cholesterol (β = 2326, p = 0.01), respectively. Simvastatin, alone or with metformin did not affect serum FABP4 or RBP4. Serum FABP4 related to the obesity, insulin resistance and inflammation while RBP4 related to lipids. Insulin sensitizing and lipid lowering therapies did not affect FABP4 or RBP4 levels in PCOS.

Context: A randomized trial on women with polycystic ovary syndrome (PCOS) compared simvastatin, metformin, and a combination of these drugs., Objective: The aim of the study was to evaluate long-term effects of simvastatin and metformin on PCOS., Design: Women with PCOS (n = 139) were randomized to simvastatin (S), metformin (M), or simvastatin plus metformin (SM) groups. Evaluations were performed at baseline and at 3 and 6 months., Setting: The study was conducted at a university medical center., Primary Outcome: We measured the change of serum total testosterone., Results: Ninety-seven subjects completed the study. Total testosterone decreased significantly and comparably in all groups: by 25.6, 25.6, and 20.1% in the S, M, and SM groups, respectively. Both simvastatin and metformin improved menstrual cyclicity and decreased hirsutism, acne, ovarian volume, body mass index, C-reactive protein, and soluble vascular cell adhesion molecule-1. Dehydroepiandrosterone sulfate declined significantly only in the S group. Total cholesterol and low-density lipoprotein cholesterol significantly declined only in the S and SM groups. Ongoing reduction of ovarian volume, decreased hirsutism, acne and testosterone were observed between 0 and 3 months as well as between 3 and 6 months. Improvement of lipid profile, C-reactive protein, and soluble vascular cell adhesion molecule-1 occurred only during the first 3 months of treatment, with little change thereafter. Treatments were well tolerated, and no significant adverse effects were encountered., Conclusions: Long-term treatment with simvastatin was superior to metformin. Improvement of ovarian hyperandrogenism continued throughout the duration of the study.

Context: Polycystic ovary syndrome (PCOS) is characterized by ovarian dysfunction and hyperandrogenism; it is also associated with increased cardiovascular risks such as adverse lipid profile and endothelial dysfunction. Metformin and, more recently, statins have been shown to improve endocrine and metabolic aspects of PCOS., Objective: The aim of the study was to compare effects of simvastatin and metformin on PCOS., Design: In a prospective trial, women with PCOS (n = 136) were randomized to simvastatin (S), metformin (M), or simvastatin plus metformin (SM) groups. Evaluations were performed at baseline and after 3 months., Setting: The study was conducted at an academic medical center., Primary Outcome: The change of serum total testosterone was measured., Results: The study was completed by 113 subjects. Total testosterone decreased significantly and comparably in all groups: by 17.1, 13.6, and 15.1%, respectively, in the S, M, and SM groups. Significant decreases were also observed in all groups with respect to body mass index, C-reactive protein, and soluble vascular cell adhesion molecule-1. DHEAS declined significantly only in the S group. None of the treatments were associated with significant changes in LH or FSH. Total cholesterol and low-density lipoprotein cholesterol significantly declined only in S and SM groups., Conclusions: Simvastatin treatment was superior to metformin alone, whereas a combination of simvastatin and metformin was not significantly superior to simvastatin alone.

Context: Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism and cardiovascular risks including dyslipidemia and systemic inflammation. In vitro, statins decrease proliferation and steroidogenesis of ovarian theca-interstitial cells., Objective: The study objective was to compare effects of two treatments of PCOS: simvastatin plus oral contraceptive pill (OCP) vs. OCP alone., Design: In a prospective, crossover trial, 48 women with PCOS were randomized to either simvastatin plus OCP for 12 wk followed by OCP alone for an additional 12 wk, or to OCP alone for 12 wk and, subsequently, simvastatin plus OCP for an additional 12 wk. Evaluations were performed at baseline, after 12 wk (crossover), and after 24 wk. Data were analyzed using a random effects model., Setting: The study was conducted in an academic medical center., Primary Outcome: Serum total testosterone was the primary outcome measure., Results: Total testosterone decreased by 38% after Statin + OCP, whereas OCP alone led to a 26% decrease; the statin-attributable effect was significant (P < 0.004). Free testosterone declined by 58% after Statin + OCP, significantly more than the 35% decline after OCP alone (P = 0.006). Hirsutism decreased by 8.1% after Statin + OCP, a greater effect than the 4.7% decrease after OCP alone (P = 0.02). Statin decreased LH, but not FSH or prolactin. Statin + OCP decreased total and low-density lipoprotein cholesterol by 7.5% and 20%, respectively. OCP alone led to a 5% increase of total cholesterol without effect on low-density lipoprotein cholesterol. Statin prevented OCP induced increase of triglycerides. C-reactive protein decreased by 45% after Statin + OCP, a significantly different effect (P = 0.006) than a 6% increase after OCP alone. Soluble vascular cell adhesion molecule 1 decreased by 18% after Statin + OCP, a greater decline than the 10% decrease after OCP alone (P = 0.01)., Conclusions: Simvastatin improved endocrine/clinical aspects of PCOS and had beneficial effects on lipid profile and markers of systemic inflammation.

Insulin and moderate oxidative stress stimulate proliferation of ovarian theca-interstitial cells. The effects of these agents on selected signal transduction pathways were examined. PD98059 (inhibitor of MAP2K1, also known as MEK-1, upstream of extracellular signal-regulated protein kinases MAPK3/1, also known as ERK1/2), wortmannin (inhibitor of PIK3C2A, also known as PI3K), and rapamycin (inhibitor of FRAP1, also known as mTOR, upstream of RPS6KB1) each significantly decreased insulin and oxidative stress-induced proliferation of theca-interstitial cells. The greatest inhibition was observed in the presence of rapamycin; this effect occurred without a significant change in cell viability. Phosphorylation of AKT was stimulated by insulin only, while phosphorylation of MAPK3/1 and RPS6KB1 was increased by insulin and oxidative stress. Insulin-induced and oxidative stress-induced phosphorylation of RPS6KB1 was partly inhibited by wortmannin and partly by PD98059; the greatest inhibition was observed in the presence of a combination of wortmannin plus PD98059. Effects of insulin and oxidative stress on phosphorylation of RPS6KB1 were confirmed by kinase activity assays. These findings indicate that actions of insulin and oxidative stress converge on MAPK3/1 and RPS6KB1. Furthermore, we speculate that activation of RPS6KB1 may be in part induced via the MAPK3/1 pathway.

Objective: Patients with polycystic ovary syndrome frequently suffer from hyperinsulinemia and adverse lipid profile. This study evaluated effects of metformin on lipid profile., Study Design: Lipid profiles in hyperinsulinemic (n = 43) and normoinsulinemic (n = 45) subjects were compared. Hyperinsulinemic subjects were treated with metformin (500 mg po tid) and lipid profiles were reevaluated at 3 and 6 months of treatment., Results: The hyperinsulinemic group had significantly lower high-density lipoprotein (HDL) (by 11%), higher total cholesterol to HDL ratio (by 23%), and greater triglycerides (by 57%) than the normoinsulinemic group. Six-month metformin therapy in hyperinsulinemic women was associated with a significant decrease of insulin level (by 35%), total cholesterol (by 11%), low-density lipoprotein (LDL) (by 12%), and triglycerides (by 33%). Levels of lipids in hyperinsulinemic women after metformin therapy were comparable to levels observed in normoinsulinemic subjects., Conclusion: The present study supports the notion that metformin may be considered as a prophylactic therapy lowering cardiovascular risk factors in hyperinsulinemic women with polycystic ovary syndrome.

Objective: To test the hypothesis that statins improve hyperandrogenemia in women with polycystic ovary syndrome (PCOS)., Design: Prospective, randomized trial., Setting: Academic medical center., Patient(s): Forty-eight women with PCOS., Intervention(s): Subjects were randomized to a statin group (simvastatin, 20 mg daily plus oral contraceptive pill [OCP]; n = 24) or an OCP group (OCP alone; n = 24)., Main Outcome Measure(s): Serum T., Result(s): Baseline parameters of both groups were comparable. After 12 weeks of treatment, serum T levels declined by 41% in the statin group and by 14% in the OCP group. In the statin group, there was a greater decrease of LH (43% decrease vs. 9% in the OCP group) and a greater decline of LH/FSH ratio (44% vs. 12%). In the statin group, total cholesterol declined by 10% and low-density lipoprotein (LDL) by 24%. In the OCP group, total cholesterol increased by 8%, and LDL was unchanged., Conclusion(s): This is the first study demonstrating that statin decreases T levels and normalizes gonadotropin levels in women with PCOS. Statin therapy might offer a novel approach, providing endocrine and cardiovascular benefits.

Theca-interstitial (T-I) cells play a fundamental role in the control of ovarian function. Steroidogenic activity and growth of the T-I cells are regulated by many paracrine and endocrine factors. However, little is known about the mechanisms controlling T-I death. In an in vitro model of apoptosis, purified rat T-I cells were cultured for 24 h with serum and subsequently for up to an additional 24 h with serum or in serum-free medium with or without insulin, insulin-like growth factors (IGF-I and IGF-II) and LH or 8-bromo-cyclic AMP (8Br-cAMP). Apoptosis was identified by histological assessment of nuclear morphology and by detection of internucleosomal cleavage and quantified by determination of [alpha32P]-dideoxy-ATP 3'-end labeling of low molecular weight DNA. Serum withdrawal resulted in nuclear condensation and fragmentation into apoptotic bodies of T-I cells and led to pronounced DNA cleavage. Insulin (10 nM) protected T-I cells from apoptosis, reducing DNA fragmentation by 39 +/- 8% compared to serum-free controls. IGF-I (10 nM) and IGF-II (10 nM) had comparable antiapoptotic effects, decreasing DNA fragmentation by 55 +/- 9% and 37 +/- 14%, respectively. In contrast, LH (100 ng/ml) and 8Br-cAMP (1 mM) augmented the pro-apoptotic effect of serum withdrawal, increasing DNA fragmentation by 85 +/- 55% and 72 +/- 42%, respectively. The antiapoptotic effects of insulin and IGFs and the pro-apoptotic effect of LH, acting via the cAMP system, may be important in the maintenance of T-I homeostasis. Moreover, excessive levels of insulin and free IGFs may lead to T-I cell hyperplasia characteristic of conditions such as polycystic ovary syndrome.

Objectives: Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism, insulin resistance, compensatory hyperinsulinemia, and increased levels of free insulin-like growth factor-I (IGF-I), presumably due to a decline in IGF binding protein 1 (IGFBP-1). This study was designed to evaluate effects of metformin therapy on serum levels of IGFBP-1 and IGF-I., Study Design: Twenty-seven obese, hyperandrogenic PCOS women with elevated fasting insulin were treated for 12 weeks with metformin (500 mg p.o., t.i.d.). Serum levels of insulin, testosterone, sex hormone binding globulin (SHBG), IGF-I, and IGFBP-1 were measured before and after treatment. Body mass index (BMI) and waist-to-hip ratio (WHR) were assessed at baseline and at the end of therapy., Results: Metformin therapy significantly increased IGFBP-1 concentration by 38% (P = 0.05) but had no demonstrable effect on the total IGF-I levels. Fasting insulin levels declined by 38% (P = 0.0001) while the glucose/insulin ratio increased by 72% (P = 0.0001) and quantitative insulin sensitivity check index (QUICKI) increased by 8% (P = 0.0001). Metformin treatment also significantly decreased testosterone (by 37%, P = 0.0001) and increased SHBG concentration (by 16%, P = 0.04). Multiple linear regression analysis revealed that baseline IGFBP-1 levels correlated inversely and independently with two baseline parameters: WHR (P = 0.003) and free testosterone index (P = 0.04)., Conclusions: The present study shows that metformin therapy not only restores normal levels of insulin and testosterone, but also decreases the pool of free-bioactive IGF-I by increasing the levels of circulating IGFBP-1. We provide further arguments in favor of metformin therapy in hyperinsulinemic women with PCOS.

Endometriosis is a common disorder of women of reproductive age, yet diagnosis of this condition is often problematic. The most frequent clinical presentations of endometriosis include dysmenorrhea, pelvic pain, dyspareunia, infertility, and pelvic mass. However, the correlation between these symptoms and the stage of endometriosis is poor. Currently available laboratory markers are of limited value. At present, the best marker, serum CA-125, is usually elevated only in advanced stages and therefore not suitable for routine screening. Transvaginal ultrasound and magnetic resonance imaging are often helpful, particularly in detection of endometriotic cysts. Recently, transrectal ultrasound and magnetic resonance imaging were shown to be valuable in detection of deep infiltrating lesions, especially in the rectovaginal septum. Although direct assessment of endometriotic foci at laparoscopy may be viewed as a "gold standard" for identifying endometriosis, the correlation of laparoscopic observations with histological findings is often low. Ultimately, diagnosis of endometriosis requires a careful clinical evaluation in combination with judicious use and critical interpretation of laboratory tests, imaging techniques, and, in most instances, surgical staging combined with histological examination of excised lesions.

Objective: To evaluate the effects of laparoscopic ovarian wedge resection on hormonal and metabolic parameters of polycystic ovary syndrome (PCOS) and to compare profiles of women who achieved pregnancy with those who did not., Design: Prospective study., Setting: University hospital., Patient(s): Thirty-three women with PCOS., Intervention(s): Laparoscopic ovarian wedge resection using harmonic scalpel., Main Outcome Measure(s): Pregnancy; levels of testosterone, DHEAS, gonadotropins, sex hormone-binding globulin (SHBG), and glucose and insulin during 2-hour glucose tolerance test; lipid profile; body mass index; and waist-to-hip ratio., Result(s): Twenty-two women (67%) achieved clinical pregnancy within the mean of 4.9 months after surgery. Baseline parameters of women who became pregnant differed from those who did not: those who became pregnant were less obese, had lower levels of total cholesterol, low-density lipoprotein, and triglycerides; higher levels of SHBG; lower levels of fasting insulin; lower insulin area under the curve; and higher insulin sensitivity index. Subjects not pregnant by 12 weeks after surgery underwent repeat endocrine and metabolic evaluations. In these women, wedge resection was followed by declines in testosterone, LH, and insulin sensitivity index. Wedge resection had no significant effect on SHBG, DHEAS, or lipid profile., Conclusion(s): Laparoscopic wedge resections are associated with the highest pregnancy rates among less obese subjects with favorable lipid profiles and lower insulin. In this study, the postoperative decline of serum testosterone and LH is not attributable to improvement of insulin sensitivity.

Objective: To evaluate the association between insulin/insulin-like growth factor I (IGF-I) systems and androgen levels in pregnancy., Design: Prospective cohort study., Setting: Yale University School of Medicine., Patient(s): Pregnant women undergoing a 100-gram 3-hour glucose tolerance test (GTT)., Intervention(s): Serum samples collected during GTT were analyzed for insulin, androgens, free IGF-I, insulin-like growth factor-binding protein (IGFBP) 1, and estriol., Main Outcome Measure(s): Observing the relationship between insulin/IGFs and androgen levels., Result(s): The insulin area under the curve (I(AUC)) during GTT correlated positively with total T and free T, but not with dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), or estriol. The peak insulin values (I(max)) during GTT also correlated positively with total T and free T, but not with DHEA, DHEAS, or estriol. There was no statistically significant correlation of T levels with free IGF-I, IGFBP-1, glucose, DHEAS, or estriol. Multiple linear regression analysis modeling showed that I(AUC) and I(max) did have a statistically significant correlation with free T levels., Conclusion(s): This study demonstrates for the first time that I(AUC) and I(max) measured in hyperinsulinemic states such as pregnancy correlate with T levels. In view of the lack of correlation between insulin and DHEAS or estriol, insulin-related T production during pregnancy is likely of ovarian origin.

There is growing evidence that the function of ovarian theca-interstitial (T-I) cells may be modulated by paracrine actions of activin, inhibin, and follistatin. Furthermore, either dysregulation, dysfunction, or both, of these peptides may play a role in conditions associated with T-I hyperplasia, such as polycystic ovary syndrome (PCOS) and hyperthecosis. This study was designed to evaluate the role of activin, inhibin, and follistatin in the modulation of T-I cell proliferation. Interaction of these peptides with insulin-like growth factor-I (IGF-I), a known stimulator of T-I cell proliferation, was also assessed. Purified rat T-I cells were cultured for 48 h in chemically defined media and with or without activin (3-30 ng/ml), inhibin (3-30 ng/ml), follistatin (100 ng/ml), and/or IGF-I (10 nM). T-I cell proliferation was assessed using radiolabeled thymidine incorporation assay. Activin alone stimulated proliferation of T-I cells in a dose-dependent fashion (by up to 320% above control; P < 0.001), whereas inhibin alone or follistatin alone had no significant effect. Inhibin had also no effect on activin-induced proliferation. Follistatin significantly reduced the stimulatory effects of activin and decreased proliferation by up to 46% (P < 0.01) below the level attained in the presence of activin alone. IGF-I (10 nM), at a dose producing a near-maximal effect, increased proliferation by 175% above control (P < 0.001); insulin (10 nM) increased proliferation by 52% above control (P < 0.03). A combination of IGF-I (10 nM) and activin (30 ng/ml) resulted in a 1090% increase of proliferation above control (P < 0.001); this stimulatory effect was significantly greater than that achieved in the presence of either activin alone or IGF-I alone (P < 0.001). Similarly, a combination of insulin (10 nM) and activin (30 ng/ml) increased proliferation by 506% above control levels. Flow cytometry evaluation revealed that activin increased the proportion of actively dividing cells (in S or G2/M phase of the cell cycle) by 42% (P < 0.02), whereas IGF-I had no effect on the proportion of actively dividing cells. The present findings indicate that an activin-follistatin system may be involved in the regulation of the size of ovarian thecal-stromal compartment. In view of the synergy between activin and IGF-I, and the difference in the effects on the cell cycle distribution, stimulation of T-I proliferation by these agents is likely to be mediated via separate transduction pathways. Excess activin or insufficient follistatin may contribute to T-I hyperplasia.

Objective: To evaluate the effects of 12 weeks of metformin therapy on hormonal and clinical indices in polycystic ovary syndrome (PCOS)., Design: Prospective study., Setting: University hospital., Patient(s): Thirty-nine women with PCOS and fasting hyperinsulinemia., Intervention(s): Twelve weeks of therapy with oral metformin (500 mg three times per day)., Main Outcome Measure(s): Levels of insulin, T, DHEAS, insulin-like growth factor-I (IGF-I), gonadotropins, and sex hormone-binding globulin (SHBG); and clinical symptoms including acne, hirsutism, and length of the menstrual cycle were assessed before and after treatment with metformin., Result(s): Metformin therapy resulted in a significant decrease in fasting insulin and total T and an increase in SHBG, leading to a decrease in the free T index. In addition, there was a significant decline in mean body mass index, waist-hip ratio, hirsutism, and acne, as well as an improvement in the menstrual cycle. No changes in LH and LH-FSH ratio were observed. Multiple regression analysis demonstrated that the greatest decline of T and free T index in response to metformin was observed among patients with the most pronounced hyperandrogenemia. Subjects with elevated DHEAS differed from those with normal DHEAS in their responses to metformin treatment. Women with high DHEAS exhibited less improvement of menstrual cycle regularity, no change in hirsutism, and an increase in levels of IGF-I after treatment., Conclusion(s): Metformin treatment of women with PCOS results in a decline of insulin as well as total and bioavailable T, leading to significant improvement of clinical manifestations of hyperandrogenism. Responses to metformin are related to the severity of hyperandrogenemia and to adrenal function.

Tumor necrosis factor-alpha (TNF-alpha) is a potent modulator of ovarian function, affecting steroidogenesis of both granulosa and theca-interstitial (T-I) cells. Women with polycystic ovary syndrome (PCOS) have increased levels of serum TNF-alpha. The present study evaluated the effects of TNF-alpha on T-I cell proliferation. Purified rat T-I cells were cultured for 48 h with or without TNF-alpha (0.001-1 nM), insulin-like growth factor I (IGF-I; 10 nM), and/or insulin (10 nM). Proliferation was measured by [(3)H]thymidine incorporation assay and by counting the steroidogenically active (stained positive for 3beta-hydroxysteroid dehydrogenase; 3beta-HSD) and inactive (3beta-HSD negative) cells. TNF-alpha stimulated thymidine incorporation in a dose-dependent fashion (up to 3.2-fold; P < 0.01). Insulin and IGF-I stimulated T-I proliferation (respectively, by up to 2.4- and 3.1-fold; P < 0.001). TNF-alpha potentiated effects of insulin and IGF-I in a dose-dependent and additive fashion (up to 6.7-fold; P < 0.001). TNF-alpha (1 nM) increased total cell count (by 80%, P < 0.05) and the proportion of 3beta-HSD-positive cells (by 19%, P < 0.05). Flow cytometry DNA analysis revealed that TNF-alpha (1 nM) increased the proliferative index by up to 16% (P = 0.05). The present findings demonstrate that TNF-alpha stimulates mitotic activity of T-I cells by increasing the proportion of actively dividing cells and preferentially increasing the number of steroidogenically active cells. The effects of TNF-alpha appear to be independent of those induced by insulin and IGF-I. We postulate that TNF-alpha may play a pathophysiologic role in disorders of the T-I compartment, such as PCOS.

Luteinizing hormone (LH) and insulin-like growth factor I (IGF-I) are recognized as major regulators of ovarian theca-interstitial (T-I) function. This study was designed to compare the effects of LH and IGF-I on T-I proliferation and steroidogenesis. Purified rat T-I cells were cultured in chemically-defined media. DNA synthesis was evaluated by a radiolabelled thymidine incorporation assay. The cells were also directly counted. Progesterone production was assessed using a specific radioimmunoassay. DNA synthesis of T-I cells was stimulated by IGF-I (10 nM) but modestly inhibited by LH (100 ng/ml). The inhibitory effect of LH was mimicked by 8Br-cAMP (10(-4) to 10(-3) M); forskolin (10(-5) M), cholera toxin (10 ng/ml) and 3-isobutyl-methylxanthine (10(-5) M). Stimulation of protein kinase C with phorbol 12-myristate 13-acetate (10(-7) M) had no significant effect on DNA synthesis. Furthermore, DNA synthesis was not affected by testosterone (10(-10) to 10(-9) M) or progesterone (10(-9) to 10(-8) M). Accumulation of progesterone was co-operatively stimulated by LH and IGF-I. These results suggest that LH-induced inhibition of T-I proliferation and/or survival is mediated via the cAMP system. IGF-I may be viewed as a co-gonadotrophin with respect to steroidogenesis but not with respect to proliferation/survival. The divergence of the effects on proliferation/survival versus steroidogenesis underscores the complexity of the interactions between LH and IGF-I signalling pathways.

This study was designed to evaluate mechanisms regulating proliferation of steroidogenically active and steroidogenically inactive theca-interstitial (T-I) cells, and, specifically, to evaluate the effects of platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I). T-I cells obtained from immature Sprague-Dawley rats were cultured in chemically defined media. Proliferation was assayed by thymidine incorporation and cell counting. Steroidogenically active cells were identified by the presence of 3beta-hydroxysteroid dehydrogenase activity. Flow cytometry facilitated separation of dividing cells (in S and G2/M phases of the cell cycle) from nondividing cells (in G0 and G1 phases of the cell cycle). PDGF alone (0.1-1 nM) produced a dose-dependent increase in DNA synthesis by up to 136%. IGF-I alone (10 nM) increased DNA synthesis by 56%. In the presence of both IGF-I (10 nM) and PDGF (0.1-1 nM), DNA synthesis increased by 108-214%. PDGF (1 nM) increased the total number of T-I cells by 43%; this effect was due to an increase in the number of steroidogenically inactive cells (47%). In contrast, the stimulatory effect of IGF-I (10 nM) was predominantly due to an increase in the number of steroidogenically active cells (163%). Separation of dividing cells from nondividing cells was accomplished with the aid of flow cytometry. In the absence of growth factors, the proportion of steroidogenically active cells was 35% lower among proliferating than resting cells. PDGF (1 nM) decreased the proportion of steroidogenically active cells among both proliferating and resting cells (by 43% and 16%, respectively). In contrast, IGF-I (10 nM) increased the proportion of steroidogenically active cells among proliferating cells by 56%. These findings indicate that differentiated/steroidogenically active cells divide; furthermore, PDGF and IGF-I may selectively stimulate proliferation of individual subpopulations of T-I cells, thereby providing a mechanism for development of structural and steroidogenically active components of the T-I compartment.

Objective: To determine whether insulin and insulin-like growth factor I (IGF-I) affect the proliferation of human theca-interstitial cells., Design: In vitro assays., Setting: University laboratory., Patient(s): Premenopausal women undergoing oophorectomy for benign conditions., Intervention(s): Purified theca-interstitial cells were cultured in chemically defined media with or without insulin and IGF-I., Main Outcome Measure(s): The proliferation of cells was evaluated by determination of [3H] thymidine incorporation and cell counting., Result(s): Insulin and IGF-I stimulated DNA synthesis by theca-interstitial cells in a dose-dependent fashion. Insulin-like growth factor I had a greater potency than did insulin. The effects of both approached, but did not reach, the level of DNA synthesis observed in cultures exposed to 10% fetal bovine serum. Direct counting of theca-interstitial cells revealed that IGF-I significantly increased the total number of cells (36% above control), whereas insulin induced a modest and statistically nonsignificant increase in the cell number (14% above control)., Conclusion(s): The present results support the hypothesis that insulin and IGF-I promote the mitotic activity of theca-interstitial cells. These effects may represent mechanisms that lead to hyperplasia of the thecal/stromal compartment in polycystic ovary syndrome.

Hyperplasia of the theca-interstitial (T-I) compartment, such as observed in polycystic ovary syndrome, is associated with ovarian dysfunction. Yet the mechanisms regulating proliferation of T-I cells are virtually unknown. This study was an investigation of the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on proliferation of rat T-I cells. Purified T-I cells were incubated in chemically defined media. Insulin (1-100 nM) and both IGFs (0.3-30 nM) dose-dependently stimulated DNA synthesis as determined by radiolabeled thymidine incorporation assay. IGF-I was most potent with EC50 = 1.4 +/- 0.4 nM, while IGF-II had EC50 = 4.3 +/- 0.18 nM and insulin had EC50 = 8.4 +/- 3.9 nM. The maximal effects of all three treatments were comparable. A combination of IGF-I at 10 nM (a concentration producing a near-maximal effect) with insulin or IGF-II resulted in DNA synthesis comparable to that achieved by IGF-I alone. IGF-I mutants with decreased affinity to IGF-binding proteins (IGFBPs)-long R3-IGF-I and des(1-3)IGF-I-produced greater effects on DNA synthesis than did IGF-I. The effects of insulin and IGFs on cell proliferation were confirmed by counting the steroidogenically active cells (stained positive for 3 beta-hydroxysteroid dehydrogenase [3 beta-HSD]) and steroidogenically inactive cells (3 beta-HSD negative). The number of steroidogenically active T-I cells was increased by insulin (by 3.7-fold, p < 0.001), IGF-I (by 3.2-fold, p < 0.001), and IGF-II (by 2.1-fold, p < 0.001). The number of steroidogenically inactive cells was not significantly altered. These findings indicate that 1) insulin- and IGF-dependent synthesis of DNA by T-I cells is stimulated via a common pathway, probably via type I IGF receptors; 2) endogenous IGFBPs may modify the effects of IGF-I; and 3) the increased DNA synthesis is reflected by an increase in the number of steroidogenically active cells. Insulin and the IGFs may play a role in the regulation of proliferation and differentiation of T-I cells under physiological and pathological conditions. In particular, the present observations may explain thecal and stromal hyperplasia accompanying hyperinsulinemic conditions such as polycystic ovary syndrome or hyperthecosis.