Low intakes of dietary fiber and magnesium are associated with insulin resistance and hyperandrogenism in polycystic ovary syndrome: A cohort study.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) often have insulin resistance (IR) which may be worsened by obesity. The roles of dietary intake and activity are unclear. Our objectives were to determine whether (a) high caloric intake or inactivity explains obesity in PCOS, and (b) dietary composition is associated with PCOS phenotypes. METHODS: Eighty-seven women with PCOS and 50 women without PCOS participated in this cohort study at a reproductive medicine center. Data collected included 3-day food and physical activity records, anthropometrics, and metabolic and hormonal assays. RESULTS: Women with PCOS had increased body mass index (BMI) but similar caloric intake and activity to women without PCOS. There were no differences in protein, carbohydrates, fat, or glycemic load consumption, but women with PCOS consumed less fiber (medians: 19.6 vs. 24.7 g) and less magnesium (medians: 238.9 vs. 273.9 mg). In women with PCOS, those with IR consumed less fiber, less magnesium, and greater glycemic load than those without IR (medians: 18.2 vs. 22.1 g, 208.4 vs. 264.5 mg, 89.6 vs. 83.5). Fiber intake of women with PCOS was negatively correlated with IR, fasting insulin, glucose tolerance, testosterone, and dehydroepiandrosterone sulfate. Magnesium intake was negatively correlated with IR, C-reactive protein, and testosterone, but positively correlated with HDL cholesterol. Fiber intake and BMI accounted for 54.0% of the variance observed in IR. CONCLUSIONS: Obesity in women with PCOS could not be explained by overeating or inactivity. Increasing dietary fiber and magnesium intakes may assist in reducing IR and hyperandrogenemia in women with PCOS.

A randomized controlled trial comparing lifestyle intervention to letrozole for ovulation in women with polycystic ovary syndrome: a study protocol.

BACKGROUND: Polycystic ovary syndrome (PCOS) affects between 8 and 18% of women and is the leading cause of female anovulatory infertility. Unfortunately, common treatments for women trying to conceive can be ineffective as well as disruptive or harmful to patients' quality of life. Despite evidence that women with PCOS have expressed the need for alternative fertility treatments, lifestyle interventions incorporating a nutritional plan with supplementation, increased physical activity, and techniques for stress management have not been combined as a program and studied in this population. Literature suggests that each of these individual components can positively influence reproductive hormones and metabolic health. METHODS/DESIGN: This is a randomized controlled trial which will include 240 women diagnosed with PCOS, according to the Rotterdam criteria, who are trying to conceive. Participants will be randomized to either a comprehensive lifestyle intervention program or prescribed an oral fertility medication, letrozole. These two groups will be further randomized to consume either myo-inositol or a placebo. Participants will be between the ages of 18 and 37 years. Exclusion criteria include women who have already begun fertility treatment, who are currently using myo-inositol or have taken it within the past 3 months, or who are being treated for, or have a history of, an eating disorder. The primary outcome will be the ovulation rate, the secondary outcome will be conception. Other outcomes include miscarriage rates, validated rating measures of overall quality of life (including social, relational, mind/body and emotional sub-categories) and mental health scores (depression, anxiety, and stress). DISCUSSION: This trial will determine the effectiveness of a structured lifestyle-based comprehensive intervention program for women with PCOS experiencing infertility. In addition, it will determine whether supplementing with myo-inositol provides any further benefit. The objective of this study is to assess a possible non-pharmacological solution to ovulatory dysfunction in these patients and perhaps improve other associated features of PCOS. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT02630485 . Registered on 15 December 2015.

No. 362-Ovulation Induction in Polycystic Ovary Syndrome.

OBJECTIVE: To review current non-pharmacologic and pharmacologic options for ovulation induction in women with polycystic ovary syndrome (PCOS). OPTIONS: This guideline reviews the evidence for the various options for ovulation induction in PCOS. OUTCOMES: Ovulation, pregnancy and live birth rates, risks, and side effects are the outcomes of interest. EVIDENCE: Published literature was retrieved through searches of Medline using appropriate controlled vocabulary and key words spanning from 2000 to 2016. Results were restricted to systematic reviews, randomized control trials/controlled clinical trials, and observational studies. Grey (unpublished) literature was identified through searching the websites of health technology assessment and of health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. VALUES: The evidence gathered was reviewed and evaluated by the Reproductive Endocrinology and Infertility Committee of the Society of Obstetricians and Gynaecologists of Canada. The quality of evidence was quantified using the Canadian Task Force on Preventive Health Care. BENEFITS, HARMS, AND COSTS: Benefits include weight reduction and improvements in ovulation, pregnancy, and live birth rates. Potential harms include medication side effects and multiple pregnancies. VALIDATION: These guidelines have been reviewed and approved by the Reproductive Endocrinology and Infertility Committee of the SOGC. CONCLUSION: First line management of infertility once a diagnosis of PCOS is made should include weight loss and exercise with goals to below class 2 obesity (BMI <35 kg/m2) as applicable. Subsequently, first line medical therapy for ovulation induction should include aromatase inhibitors (now considered both safe and effective) and selective estrogen receptor modulators as available. Insulin sensitizers should not be used as first line therapy but as adjuncts as appropriate. Referral to a reproductive endocrinologist should be considered if there is failure or resistance to these approaches to consider ovulation induction with gonadotropins or IVF as appropriate. SPONSOR: The Society of Obstetricians and Gynaecologists of Canada.

Does Molimina Indicate Ovulation? Prospective Data in a Hormonally Documented Single-Cycle in Spontaneously Menstruating Women.

Approximately 33% of normal-length (21⁻35 days) cycles have subclinical ovulatory disturbances and lack sufficient progesterone, although their normal length ensures enough estrogen. Subclinical ovulatory disturbances are related to significant premenopausal spine bone loss (-0.86%/year). Molimina, non-distressing premenstrual experiences, may detect ovulation within normal-length cycles. This prospective study assessed the relationship between molimina and ovulation. After 1-cycle of daily diary and first morning urine collections, women answered the Molimina Question (MQ): "Can you tell by the way you feel that your period is coming?" and were invited to share (a) predictive premenstrual experience(s). A 3-fold increase in follicular-luteal pregnanediol levels confirmed ovulation. In 610 spontaneously menstruating women (not on hormonal contraception, mean age 31.5 ± 5.3, menarche age 12.7 ± 1.5, cycle length [CL] 29 days, MQ positive in 89%), reported premenstrual experiences which included negative moods (62%), cramps (48%), bloating (39%), and front (26%) or axillary (25%) breast tenderness. Of 432 women with pregnanediol-documented cycles, 398 (92%) were ovulatory (CL: 29 ± 5) and 34 (8%) had ovulatory disturbances (CL: 32 ± 14). Women with/without ovulatory cycles were similar in parity, body mass index, smoking, dietary restraint and the MQ; ovulatory-disturbed cycles were longer. Molimina did not confirm ovulation. A non-invasive, inexpensive ovulation indicator is needed to prevent osteoporosis.

Circulating metals and persistent organic pollutant concentrations in Canadian and non-Canadian born primiparous women from five Canadian centres: results of a pilot biomonitoring study.

The developing foetus is thought to be at increased risk from exposure to environmental contaminants; however, developmental exposure data is notably lacking for many contaminants. Moreover, potential regional differences or effect of place of birth on residue levels measured in pregnant women is also unknown. Therefore, as part of a multinational biomonitoring study, 125 primiparous pregnant Canadian women were recruited from five Canadian centres (Vancouver, Calgary, Hamilton, Ottawa, and Halifax). Metals in whole blood and persistent organic pollutants (POPs) in plasma were measured by inductively coupled plasma mass spectrometry (ICPMS) and gas chromatography-mass spectrometry (GCMS), respectively. Of the 125 women recruited to this study, complete data sets were available for 123 of which 103 were Canadian born. Data were analysed by analysis of covariance and linear mixed models using age and body mass index as covariates. The metals cadmium (Cd), cobalt (Co), lead (Pb), nickel (Ni), selenium (Se), and total mercury (Hg) were detected in more than 93% of the samples tested. ß-Hexachlorohexane (ß-HCH), oxychlordane, trans-nonachlor, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), polybrominated diphenyl ether (PBDE) congeners (PBDE-153, PBDE-47), polychlorinated biphenyl (PCB) congeners (PCB-138, -153, and -180), and the dioxin-like PCB congener PCB-118 were quantified in greater than 70% of the samples tested. Significant differences in the concentrations of Cd, Ni, PCB-153, and p,p'-DDE were found between the centres studied. Furthermore, foreign-born pregnant women had significantly higher concentrations of Cd, ß-HCH, PBDE-47, PCB-138, -153, -180, and p,p'-DDE compared to Canadian born pregnant women. Taken together, the data suggest that there are potential regional differences in contaminant body burden and place of birth may also contribute to differences in maternal residue concentrations.

Advanced reproductive age and fertility: no. 269, November 2011.

OBJECTIVE: To improve awareness of the natural age-related decline in female and male fertility with respect to natural fertility and assisted reproductive technologies (ART) and provide recommendations for their management,and to review investigations in the assessment of ovarian aging. OPTIONS: This guideline reviews options for the assessment of ovarian reserve and fertility treatments using ART with women of advanced reproductive age presenting with infertility. OUTCOMES: The outcomes measured are the predictive value of ovarian reserve testing and pregnancy rates with natural and assisted fertility. EVIDENCE: Published literature was retrieved through searches of PubMed or Medline, CINAHL, and The Cochrane Library in June 2010, using appropriate key words (ovarian aging, ovarian reserve, advanced maternal age, advanced paternal age, ART). Results were restricted to systematic reviews, randomized controlled trials/controlled clinical trials, and observational studies. There were no date or language restrictions. Searches were updated on a regular basis and incorporated into the guideline to December 2010. VALUES: The quality of evidence was rated using the criteria described in the Report of the Canadian Task Force on Preventive Health Care. Recommendations for practice were ranked according to the method described in that report (Table). BENEFITS, HARMS, AND COSTS: Primary and specialist health care providers and women will be better informed about ovarian aging and the age-related decline in natural fertility and about options for assisted reproductive technology.

The diagnosis and management of ovarian hyperstimulation syndrome: No. 268, November 2011.

OBJECTIVE: To review the clinical aspects of ovarian hyperstimulation syndrome and provide recommendations on its diagnosis and clinical management. OUTCOMES: These guidelines will assist in the early recognition and management of ovarian hyperstimulation. Early recognition and prompt systematic supportive care will help avert poor outcomes. EVIDENCE: Medline, Embase, and the Cochrane database were searched for relevant articles, using the key words "ovarian hyperstimulation syndrome" and "gonadotropins," and guidelines created by other professional societies were reviewed. VALUES: The quality of evidence was rated using the criteria described in the Report of the Canadian Task Force on Preventive Health Care. Recommendations for practice were ranked according to the method described in that report (Table 1).

Molecular and immuno-characteristics of immunoglobulin-like glycoproteins in cancer cell-expressed biomarker, CA215.

RP215 monoclonal antibody (Mab) was shown to recognize a specific carbohydrate-associated epitope found in cancer cell-expressed glycoproteins, known as CA215. The membrane-bound and soluble forms of CA215 were detected in almost all of the cancer cells in humans, but rarely found in normal tissues. Through MALDI-TOF MS analysis, it has been reported previously that as much as 40% of the detected tryptic peptides of CA215 showed high degrees of sequence homology to those found in immunoglobulin heavy chains. The cancer cell-derived immunoglobulins were further purified from CA215 by affinity column-linked with goat anti-human IgG for molecular characterizations. Semi-quantitative RT-PCR was used to determine the mRNA levels of various immunoglobulin genes expressed by cancer cells of single or multi-cell origins and compared with those found in normal human serum. The stability of CA215 was investigated under different experimental conditions. It was observed that the RP215-specific epitope in CA215 is stable at neutral pH, in human serum or in mice (half life of 5-18 days), but unstable at extreme pH's (pH ≤ 2.0; pH ≥ 12.0) or high temperatures. Enzyme immunoassays were performed with several secondary antibody probes related to human IgG. It was demonstrated that cancer cell-expressed immunoglobulins with RP215-specific epitope have much lower immunoactivity than that of normal human IgG (≤ 5%), despite the fact that both showed almost identical amino acid sequence in the respective Fc region reported previously. This could be the result of aberrant glycosylation of CA215 in cancer cells. Aberrant glycosylation of glycoproteins may have important biological implications on the proliferation of cancer cells in vitro or in vivo.

CA215 and GnRH receptor as targets for cancer therapy.

Two monoclonal antibodies (Mabs), RP215 and GHR106, were selected for the preclinical evaluations of anti-cancer drugs targeting various human cancers including those of the ovary, cervix, lung, and liver. Both Mabs were shown to react with pan cancer markers, which are over-expressed on the surface of almost all human cancers. RP215 Mab was shown to react with the carbohydrate-associated epitope(s) of cancer cell-expressed glycoproteins, mainly consisting of immunoglobulin superfamily (IgSF) proteins and mucins, generally known as CA215. GHR106 Mab was generated against the extracellular domain of human GnRH receptor, which is also highly expressed on the cancer cell surface. Preclinical studies were performed to evaluate the efficacy of these two Mabs as anti-cancer drugs for treating human cancers. High tumor specificity of RP215 Mab was demonstrated with immunohistochemical staining studies of various cancer cell lines, as well as normal and cancerous tissue sections. These two Mabs were shown to induce apoptosis as well as complement-dependent cytotoxicity upon treatment to many cultured cancer cells. Significant dose-dependent growth inhibition of tumor cells from several different tissue origins were demonstrated by nude mouse experiments. It was further demonstrated that GHR106 Mab can function as long-acting GnRH analogs in its biological actions. Efforts were made to generate human/mouse chimeric forms of the GHR106 Mab. Based on the results of these preclinical studies, we believe that these two Mabs, in chimeric or humanized forms, can be developed into suitable therapeutic agents for treatment of human cancers as anti-cancer drugs.

Gonadotropin-releasing hormone-I or -II interacts with IGF-I/Akt but not connexin 43 in human granulosa cell apoptosis.

BACKGROUND: We have recently demonstrated that GnRH-I or -II can induce apoptosis in immortalized human granulosa cells by activating the caspase signaling cascade. Whether GnRH-I or -II can affect other regulators such as Bcl-2 family members, IGF-I, or gap junctions and the mechanisms involved are unknown. METHODS: Immortalized human granulosa cells were treated with GnRH-I, GnRH-II, IGF-I, or antide (a GnRH-I receptor antagonist), in various combinations. Cell proliferation and apoptotic changes were evaluated by cell counting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunoblotting. Activated or total protein expression of IGF-I receptor, Akt, connexin 43 (Cx43), or caspase-3 with and without dominant-negative Akt (an Akt-suppressing vector), wortmannin (a phosphatidylinositol-3-kinase inhibitor), or Cx43 small interfering RNA transfection were assessed by immunoblotting. Gap junctional communication was determined by dye transfer assay. RESULTS: GnRH-I or -II inhibited cell proliferation, induced TUNEL-positive cells, and increased caspase-3 activities but had no effects on Bcl-2 family members. IGF-I increased cell proliferation, decreased TUNEL-positive cells and caspase-3 activities, and increased Akt activities, and these effects were attenuated by GnRH-I or -II. Effects of IGF-I on caspase-3 activities were attenuated by dominant-negative Akt or wortmannin. GnRH-I or -II decreased dye transfer, increased Cx43 phosphorylation, and increased caspases-3 activities even after Cx43 knockdown. CONCLUSION: GnRH-I or -II induces apoptosis in human granulosa cells through a caspase-3-dependent extrinsic pathway rather than a Bcl-2 family-dependent intrinsic pathway and attenuates the antiapoptotic action of IGF-I through Akt. Cx43-induced gap junctional changes do not initiate granulosa cell apoptosis but likely result from apoptosis induced by GnRH-I or -II.

Carbohydrate-associated immunodominant epitope(s) of CA215.

RP215 monoclonal antibody (Mab) was initially generated against OC-3-VGH ovarian cancer cells and was shown to react with a cancer-associated carbohydrate epitope in glycoproteins designated as CA215. Additional five high affinity Mabs, designated as RCA-10, -100, -104, -110 and -111, respectively, were generated by using affinity-purified CA215 as the immunogen in this study. All RCA Mabs were found to recognize periodate-sensitive carbohydrate-associated epitope(s) and to pair with RP215 in typical sandwich enzyme immunoassays for the quantification of CA215. When compared with those of RP215, the amino acid sequence homology of the Fab regions ranged from 100% for RCA-100 to 65% for RCA-110, based on which 3 distinct Mab groups were categorized. In vitro TUNEL apoptosis and complement-dependent cytotoxicity assays were performed with these Mabs and found to have comparable inhibitory efficacy to cancer cells. Results of biochemical and immunological assays revealed that RP215, RCA-100 and RCA-10 react with the linear carbohydrate-associated epitope, whereas the others recognize the conformational form of the epitope in CA215. This study has suggested that the unique carbohydrate-associated epitope(s) is immunodominant in mice when immunized with CA215. It remains to be demonstrated if the differential anti-cancer efficacy exists among the distinct groups of these anti-CA215 Mabs.

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.

BACKGROUND: We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin ß(B)-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A. METHODOLOGY: hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin ß(B)-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test. PRINCIPAL FINDINGS: GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin ß(B)-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin ß(B)-subunit mRNA and inhibin B production were attenuated by adding FST. CONCLUSION: GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin ß(B)-subunit mRNA expression and inhibin B production in hGL cells.

Ovulation induction in polycystic ovary syndrome: No. 242, May 2010.

OBJECTIVE: To review current non-pharmacologic and pharmacologic options for ovulation induction in women with polycystic ovary syndrome (PCOS). OPTIONS: This guideline reviews the evidence for the various options for ovulation induction in PCOS. OUTCOMES: Ovulation, pregnancy and live birth rates, risks, and side effects are the outcomes of interest. EVIDENCE: Published literature was retrieved through searches of Medline using appropriate controlled vocabulary and key words. Results were restricted to systematic reviews, randomized control trials/controlled clinical trials, and observational studies. Grey (unpublished) literature was identified through searching the websites of health technology assessment and of health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. VALUES: The evidence gathered was reviewed and evaluated by the Reproductive Endocrinology and Infertility Committee of the Society of Obstetricians and Gynaecologists of Canada. The quality of evidence was quantified using the Canadian Task Force on Preventive Health Care. BENEFITS, HARMS, AND COSTS: Benefits include weight reduction and improvements in ovulation, pregnancy, and live birth rates. Potential harms include medication side effects and multiple pregnancies. VALIDATION: These guidelines have been reviewed and approved by the Reproductive Endocrinology and Infertility Committee of the SOGC.

Growth differentiation factor 9 reverses activin A suppression of steroidogenic acute regulatory protein expression and progesterone production in human granulosa-lutein cells.

BACKGROUND: We have reported that growth differentiation factor 9 (GDF9) can enhance activin A (ß(A)ß(A))-induced inhibin B (αß(B)) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. METHODS: hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey's test. RESULTS: Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3ß-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of α-subunit siRNA, activin A level increased (P < 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P < 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone. CONCLUSION: GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

Ovulation induction in polycystic ovary syndrome.

OBJECTIVE: To review current non-pharmacologic and pharmacologic options for ovulation induction in women with polycystic ovary syndrome (PCOS). OPTIONS: This guideline reviews the evidence for the various options for ovulation induction in PCOS. OUTCOMES: Ovulation, pregnancy and live birth rates, risks, and side effects are the outcomes of interest. EVIDENCE: Published literature was retrieved through searches of Medline using appropriate controlled vocabulary and key words. Results were restricted to systematic reviews, randomized control trials/controlled clinical trials, and observational studies. Grey (unpublished) literature was identified through searching the websites of health technology assessment and of health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. VALUES: The evidence gathered was reviewed and evaluated by the Reproductive Endocrinology and Infertility Committee of the Society of Obstetricians and Gynaecologists of Canada. The quality of evidence was quantified using the Canadian Task Force on Preventive Health Care. BENEFITS, HARMS, AND COSTS: Benefits include weight reduction and improvements in ovulation, pregnancy, and live birth rates. Potential harms include medication side effects and multiple pregnancies. VALIDATION: These guidelines have been reviewed and approved by the Reproductive Endocrinology and Infertility Committee of the SOGC. SPONSOR: The Society of Obstetricians and Gynaecologists of Canada. RECOMMENDATIONS 1. Weight loss, exercise, and lifestyle modifications have been proven effective in restoring ovulatory cycles and achieving pregnancy in overweight women with PCOS and should be the first-line option for these women. (II-3A) Morbidly obese women should seek expert advice about pregnancy risk. (III-A) 2. Clomiphene citrate has been proven effective in ovulation induction for women with PCOS and should be considered the first-line therapy. Patients should be informed that there is an increased risk of multiple pregnancy with ovulation induction using clomiphene citrate. (I-A) 3. Metformin combined with clomiphene citrate may increase ovulation rates and pregnancy rates but does not significantly improve the live birth rate over that of clomiphene citrate alone.(I-A) Metformin may be added to clomiphene citrate in women with clomiphene resistance who are older and who have visceral obesity. (I-A) 4. Gonadotropin should be considered second-line therapy for fertility in anovulatory women with PCOS. The treatment requires ultrasound and laboratory monitoring. High costs and the risk of multiple pregnancy and ovarian hyperstimulation syndrome are drawbacks of the treatment. (II-2A) 5. Laparoscopic ovarian drilling may be considered in women with clomiphene-resistant PCOS, particularly when there are other indications for laparoscopy. (I-A) Surgical risks need to be considered in these patients. (III-A) 6. In vitro fertilization should be reserved for women with PCOS who fail gonadotropin therapy or who have other indications for IVF treatment. (II-2A).

Effects of endogenous growth differentiation factor 9 on activin A-induced inhibin B production in human granulosa-lutein cells.

BACKGROUND: We recently reported on the effects of exogenous growth differentiation factor 9 (GDF9) in enhancing activin A-induced inhibin beta(B)-subunit mRNA and inhibin B levels in human granulosa-lutein (hGL) cells by modulating key components of the activin signaling pathway. We undertook the following study to characterize the role of endogenous GDF9 in this regard. METHODS: We compared inhibin subunit (alpha, beta(A), and beta(B)) mRNA and inhibin B levels and activation of activin receptors (ACVRs) and Smad signaling pathway in hGL cells obtained from women undergoing in vitro fertilization and cultured with and without activin A treatment after GDF9-targeting small interfering RNA transfection. GDF9, inhibin subunits, ACVR2B/1B and Smad2/3/4/7 mRNA and/or protein levels, Smad phosphorylation, and inhibin B were assessed with RT-PCR, immunoblotting, and ELISA. Data were analyzed by ANOVA followed by Tukey's test. RESULTS: GDF9 was detected as mRNA and protein in hGL cells and protein in follicular fluid from all 11 patients tested. Reduced endogenous GDF9 expression after targeting small interfering RNA transfection was associated with decreased ACVR2B/1B and Smad2/3/4 but increased inhibitory Smad7 mRNA and protein levels and, consequently, reduced activin A-induced beta(B)-subunit mRNA and inhibin B levels. CONCLUSIONS: We report here for the first time autocrine roles for endogenous GDF9 in hGL cells in enhancing activin A-induced beta(B)-subunit mRNA and inhibin B levels via key components of the activin signaling pathway. However, the relative contributions of GDF9 in granulosa cells vs. oocyte as autocrine/paracrine regulators of beta(B)-subunit and inhibin B production in normal and abnormal human ovarian functions remain to be determined.

Growth differentiation factor 9 enhances activin a-induced inhibin B production in human granulosa cells.

Activin A or growth differentiation factor 9 (GDF9) alone can increase beta(B)-mRNA level in human granulosa-lutein cells from women undergoing in vitro fertilization, but their potential interactions and related cell signaling pathways involved are unknown. We therefore compared inhibin subunit and inhibin levels and activation of activin receptors (ACVRs) and Smad signaling pathway in these human granulosa-lutein cells with and without GDF9 and/or activin A treatment. Inhibin subunit (alpha, beta(A), beta(B)), ACVR, and Smad2/3/4/7 mRNA levels, inhibin A and B production, and Smad phosphorylation were assessed by real-time RT-PCR, ELISA, and immunoblotting, respectively. Data were analyzed by ANOVA followed by Tukey's test. Activin A (1-50 ng/ml) or GDF9 (1-200 ng/ml) alone had only little stimulatory effects on alpha- and beta(A)-mRNA levels. In contrast, GDF9 could stimulate beta(B)-subunit levels but to a lesser degree than the dose- and time-dependent effects of activin A. Compared with untreated cells, GDF9 pretreatment for 24 h significantly enhanced activin A-induced beta(B)-mRNA levels, inhibin B secretion, and Smad2/3 phosphorylation (effects attenuated by bone morphogenetic protein receptor 2 extracellular domain, a GDF9 antagonist); and induced ACVR2B/1B and Smad2/3 but reduced Smad7 (an inhibitory Smad) mRNA levels. We report here for the first time that GDF9 enhances cell response to activin A by modulating key components of the activin signaling pathway in regulating inhibin subunits and hence inhibin B production in human granulosa-lutein cells.