The Action of Reproductive Fluids and Contained Steroids, Prostaglandins, and Zn2+ on CatSper Ca2+ Channels in Human Sperm.

The sperm-specific Ca2+ channel CatSper registers chemical cues that assist human sperm to fertilize the egg. Prime examples are progesterone and prostaglandin E1 that activate CatSper without involving classical nuclear and G protein-coupled receptors, respectively. Here, we study the action of seminal and follicular fluid as well of the contained individual prostaglandins and steroids on the intracellular Ca2+ concentration of sperm from donors and CATSPER2-deficient patients that lack functional CatSper channels. We show that any of the reproductive steroids and prostaglandins evokes a rapid Ca2+ increase that invariably rests on Ca2+ influx via CatSper. The hormones compete for the same steroid- and prostaglandin-binding site to activate the channel, respectively. Analysis of the hormones' structure-activity relationship highlights their unique pharmacology in sperm and the chemical features determining their effective properties. Finally, we show that Zn2+ suppresses the action of steroids and prostaglandins on CatSper, which might prevent premature prostaglandin activation of CatSper in the ejaculate, aiding sperm to escape from the ejaculate into the female genital tract. Altogether, our findings reinforce that human CatSper serves as a promiscuous chemosensor that enables sperm to probe the varying hormonal microenvironment prevailing at different stages during their journey across the female genital tract.

Diagnosis and Treatment of Endometriosis. Guideline of the DGGG, SGGG and OEGGG (S2k Level, AWMF Registry Number 015/045, August 2020).

Aims The aim of this official guideline published and coordinated by the German Society of Gynaecology and Obstetrics (DGGG) in cooperation with the Austrian Society for Gynaecology and Obstetrics (OEGGG) and the Swiss Society for Gynaecology and Obstetrics (SGGG) was to provide consensus-based recommendations for the diagnosis and treatment of endometriosis based on an evaluation of the relevant literature. Methods This S2k guideline represents the structured consensus of a representative panel of experts with different professional backgrounds commissioned by the Guideline Committee of the DGGG, OEGGG and SGGG. Recommendations Recommendations on the epidemiology, aetiology, classification, symptomatology, diagnosis and treatment of endometriosis are given and special situations are discussed.

The indication for fertility preservation in women with Turner syndrome should not only be based on the ovarian reserve but also on the genotype and expected future health status.

Fertility preservation in women with Turner syndrome is highly controversial. Some strongly recommend freezing of ovarian tissue at a young age, others do not. The controversy is partly due to different perspectives and professions. Biologists prefer to freeze young ovaries with high follicle density, reproductive physicians want to avoid risky operations and iatrogenic infertility by removing one ovary, and cardiologists and obstetricians warn against the risks of later pregnancies. Accordingly, fertility preservation in young women with Turner syndrome is more than just the freezing of ovarian tissue or oocytes. Fertility preservation requires a balanced decision considering the conservation of fertility, the protection of reproductive health, and future health consequences. Therefore, fertility preservation strategies should be based not only on the individual ovarian reserve but also on the genotype and the expected cardiac health status to decide what is the best option: to freeze tissue or alternatively to wait and see.

Diagnosis and Therapy Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 1, Basic Assessment of the Woman.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German/Austrian/Swiss interdisciplinary S2k guideline on "Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)" was published in February 2019. This guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aims One third of the causes of involuntary childlessness are still unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. At present, there is no standard treatment concept, as currently no standard multidisciplinary procedures exist for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnostic workup and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations The first part of this guideline focuses on the basic assessment of affected women, including standard anatomical and endocrinological diagnostic procedures and examinations into any potential infections. Other areas addressed in this guideline are the immunological workup with an evaluation of the patient's vaccination status, an evaluation of psychological factors, and the collection of data relating to other relevant factors affecting infertility. The second part will focus on explanations of diagnostic procedures compiled in collaboration with specialists from other medical specialties such as andrologists, human geneticists and oncologists.

Diagnosis and Treatment Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 2, Hemostaseology, Andrology, Genetics and History of Malignant Disease.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German-language interdisciplinary S2k guideline on "Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)" was published in February 2019. The guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aim In one third of cases, the cause of involuntary childlessness remains unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. There is no standard treatment concept for these patients at present, as there are currently no standard multidisciplinary procedures for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnosis and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations This second part of the guideline describes the hematological workup for women as well as additional diagnostic procedures which can be used to investigate couples and which are carried out in cooperation with physicians working in other medical fields such as andrologists, geneticists and oncologists.

FSHB -211 G>T Polymorphism as Predictor for TESE Success in Patients With Unexplained Azoospermia.

CONTEXT: Testicular sperm extraction (TESE) followed by assisted reproductive techniques often remains the only therapeutic option for men with azoospermia due to spermatogenic failure. Reproductive parameters, such as gonadotropin levels and testicular volume or histopathology, contribute to the prediction of sperm retrieval rate (SRR) in TESE. However, there is an eminent lack of noninvasive predictive factors for TESE outcome. OBJECTIVE: To clarify the impact of three common genetic variants affecting FSH and its cognate receptor on testicular histopathology patterns and SRR in TESE. DESIGN: We evaluated the association of the single-nucleotide polymorphisms (SNP) FSHB -211G>T (rs10835638), FSHR -29G>A (rs1394205), and FSHR c.2039A>G (rs6166) with testicular histopathology and SRR in patients with azoospermia. SETTING: Tertiary referral center for andrology. PATIENTS OR OTHER PARTICIPANTS: Men (n = 1075) with azoospermia who underwent TESE (grouped by clinical pathologies). INTERVENTION(S): All participants underwent TESE. MAIN OUTCOME MEASURE(S): Testicular histopathology, SRR, and reproductive hormone levels. RESULTS: FSHB -211G>T was significantly associated with reduced chances of sperm retrieval in patients with unexplained azoospermia. Indicating an additional mechanism, the association of the SNP with SSR could not be solely attributed to decreased FSH levels. CONCLUSION: A common genetic factor was significantly associated with SRR in TESE. In perspective, a calculator or score including the noninvasive parameters FSH level, testicular volume, and FSHB haplotype should be considered to estimate the chances for sperm retrieval in men with azoospermia.

Arrangement of myofibroblastic and smooth muscle-like cells in superficial peritoneal endometriosis and a possible role of transforming growth factor beta 1 (TGFß1) in myofibroblastic metaplasia.

PURPOSE: Superficial peritoneal endometriotic (pEM) lesions are composed of endometrial glands and stroma, in addition to a third component-myofibroblasts and smooth muscles (SM)-like cells. The latter develops secondary to a metaplasia. In this study, we characterised the third component cells in pEM according to differentiation markers in different micro-compartments. Furthermore, a possible effect of TGFß1 on myofibroblastic metaplasia in endometriotic epithelial cells was studied. METHODS: Seventy-six premenopausal patients were included. Peritoneal biopsies were excised from EM patients (n = 23), unaffected peritoneum (peritoneum from EM patients but without EM components, n = 5/23) and non-EM patients (n = 10). All peritoneal biopsies were immunolabeled for ASMA, calponin, collagen I, desmin, TGFß receptor 1 (R1), R2 and R3 in addition to ultrastructure examination by transmission electron microscopy (TEM) (n = 1). TGFß1 level was measured in peritoneal fluid (PF) (EM, n = 19 and non-EM, n = 13) collected during laparoscopy. Furthermore, TGFß1 effect on myofibroblastic metaplasia was studied in vitro. RESULTS: At the centre of pEM lesions, calponin immunolabeling outweighs the collagen I while in the periphery the reverse occurs. SM-like cells expressing desmin predominate at the periphery, while ASMA immunolabeling was detectable in all micro-compartments. Both indicate an abundance of myofibroblasts at the centre of pEM lesions and SM-like cells in the periphery. Although activated TGFß1 in PF did not differ between EM and non-EM, it inhibited the cell proliferation of the endometriotic epithelial cells and induced an upregulation in ASMA and collagen IA2 expression as well. CONCLUSION: The abundance of the myofibroblasts and SM-like cells points to a myofibroblastic metaplasia in pEM. Both cells are differentially arranged in the different micro-compartments of pEM lesions, with increasing cell maturity towards the periphery of the lesion. Furthermore, TGFß1 may play a role in the myofibroblastic metaplasia of the endometriotic epithelial cells. These findings provide a better insight in the micro-milieu in EM lesions, where most of the disease dynamics occur.

Fertility Preservation for Patients with Malignant Disease. Guideline of the DGGG, DGU and DGRM (S2k-Level, AWMF Registry No. 015/082, November 2017) - Recommendations and Statements for Girls and Women.

AIM: The aim of this official guideline published by the German Society of Gynecology and Obstetrics (DGGG) and coordinated with the German Society of Urology (DGU) and the German Society of Reproductive Medicine (DGRM) is to provide consensus-based recommendations, obtained by evaluating the relevant literature, on counseling and fertility preservation for prepubertal girls and boys as well as patients of reproductive age. Statements and recommendations for girls and women are presented below. Statements or recommendations for boys and men are not the focus of this guideline. METHODS: This S2k guideline was developed at the suggestion of the guideline commission of the DGGG, DGU and DGRM and represents the structured consensus of representative members from various professional associations (n = 40). RECOMMENDATIONS: The guideline provides recommendations on counseling and fertility preservation for women and girls which take account of the patient's personal circumstances, the planned oncologic therapy and the individual risk profile as well as the preferred approach for selected tumor entities.

The endometrial stem cell markers notch-1 and numb are associated with endometriosis.

Previous studies reported increased expression of the notch pathway-associated protein Musashi-1 in endometriosis. This case-control study investigates an association of the endometrial stem cell markers notch-1 and numb with endometriosis. Fifty-one endometriosis patients and 76 controls were recruited in the IVF unit and tertiary endometriosis referral centre of a university hospital. All subjects underwent transcervical endometrial biopsy and diagnostic laparoscopy. Expression of endometrial notch-1 and numb was assessed by immunostaining and correlated with clinical data. Association of stem-cell-marker expression with the presence of endometriosis was evaluated. Numb expression in the luminal epithelium was significantly higher in eutopic endometrium of endometriosis patients compared with controls (20.5% versus 16.5%, P = 0.033). Numb-positive single stromal cells were less frequent in endometrioma patients compared with other forms of endometriosis (0.3 versus 0.5 cells/visual field; P = 0.028). Notch-1 expression in endometrial glands was significantly higher in patients with deep infiltrating endometriosis compared with controls (39.1% versus 21.8%; P = 0.045). We conclude that stem cell markers notch-1 and numb of eutopic endometrium are associated with endometriosis and its clinical presentations, supporting the stem cell hypothesis of endometriosis. These findings could help develop promising research strategies applying endometrial stem cells as novel tools.

The Simultaneous measurement of serum testosterone and 5α-dihydrotestosterone by gas chromatography-mass spectrometry (GC-MS).

BACKGROUND: Simultaneous measurement of testosterone (T) and 5α-dihydrotestosterone (DHT) is important for diagnosing androgen deficiency states and hyperandrogenism in males and females, respectively. However, immunoassays used for T and DHT determination suffer from inadequate specificity and sensitivity, while tandem mass spectrometry is expensive and demanding in use. METHODS AND RESULTS: We developed a selective gas chromatography-mass spectrometry (GC-MS) method for parallel T and DHT measurement. The assay showed a linear response up to 46.5nmol/L, intra- and interassay imprecision and inaccuracy <15% and recoveries in spiked samples >90% for both analytes. The limit of quantitation was 0.117nmol/L for T and 0.168nmol/L for DHT. Comparison with immunoassays revealed good agreement for T in males, but a bias in favour of immunoassays at low concentrations for T in females and DHT in both sexes. We established reference ranges for T and DHT and suggest interval partitioning for T according to age in men and menstrual cycle in women. Assay validation in a clinical setting suggests that measuring DHT or T/DHT ratio may help identify patients with polycystic ovary syndrome. CONCLUSION: We developed a selective, simple and inexpensive GC-MS method for parallel measurement of T and DHT with potential use in the clinical laboratory.

Syndecan-4 expression is upregulated in endometriosis and contributes to an invasive phenotype.

OBJECTIVE: To study the expression and function of syndecan-4 in endometriosis. DESIGN: Histopathological investigation of eutopic endometrium and experimental laboratory study on a cell line derived from epithelial endometriotic cells (12Z). SETTING: University hospital laboratory. PATIENT(S): One hundred six women (62 controls/44 endometriosis) from the IVF center of Münster University Hospital aged 23-44 undergoing Pipelle biopsy and diagnostic exploratory laparoscopy. INTERVENTION(S): Eutopic endometrial tissue was investigated by immunohistochemistry for the expression of syndecan-4. The human endometriotic cell line 12Z was transiently transfected with syndecan-4 small interfering RNA and investigated for changes in cell behavior. MAIN OUTCOME MEASURE(S): Syndecan-4 expression in eutopic endometrium was evaluated immunohistochemically in endometrial glands and stroma. Scoring results were correlated with the stages of the menstrual cycle and presence or absence of endometriosis. Quantitative polymerase chain reaction was used to measure syndecan-4-dependent expression changes of MMP2, MMP3, MMP9, Rac1, and ATF2. Altered cell behavior was monitored by matrigel invasion assays and cell viability assays. RESULT(S): Syndecan-4 expression was significantly higher in the glands and stroma of patients with endometriosis compared with controls, whereas no menstrual cycle-dependent expression was observed. In 12Z cells, syndecan-4 depletion did not affect cell viability but resulted in a significantly reduced matrigel invasiveness and reduced expression of the small GTPase Rac1, the transcription factor ATF-2, and MMP3. CONCLUSION(S): The upregulation of syndecan-4 in the eutopic endometrium of endometriosis patients may facilitate the pathogenetic process by promoting invasive cell growth via Rac1, MMP3, and ATF-2.

microRNA miR-200b affects proliferation, invasiveness and stemness of endometriotic cells by targeting ZEB1, ZEB2 and KLF4.

Endometriosis is characterized by growth of endometrial tissue at ectopic locations. Down-regulation of microRNA miR-200b is observed in endometriosis and malignant disease, driving tumour cells towards an invasive state by enhancing epithelial-to-mesenchymal transition (EMT). miR-200b up-regulation may inhibit EMT and invasive growth in endometriosis. To study its functional impact on the immortalized endometriotic cell line 12Z, the stromal cell line ST-T1b, and primary endometriotic stroma cells, a transient transfection approach with microRNA precursors was employed. Expression of bioinformatically predicted targets of miR-200b was analysed by qPCR. The cellular phenotype was monitored by Matrigel invasion assays, digital-holographic video microscopy and flow cytometry. qPCR revealed significant down-regulation of ZEB1 (P < 0.05) and ZEB2 (P < 0.01) and an increase in E-cadherin (P < 0.01). miR-200b overexpression decreased invasiveness (P < 0.0001) and cell motility (P < 0.05). In contrast, cell proliferation (P < 0.0001) and the stemness-associated side population phenotype (P < 0.01) were enhanced following miR-200b transfection. These properties were possibly due to up-regulation of the pluripotency-associated transcription factor KLF4 (P < 0.05) and require attention when considering therapeutic strategies. In conclusion, up-regulation of miR-200b reverts EMT, emerging as a potential therapeutic approach to inhibit endometriotic cell motility and invasiveness.

Establishing reference intervals for sex hormones on the analytical platforms Advia Centaur and Immulite 2000XP.

Reliable reference intervals for sex hormones are indispensable in evaluations of the hypothalamo-pituitary-gonadal axis. This study established reference intervals for estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, and prolactin with the immunoassay platforms Advia Centaur and Immulite 2000XP (Siemens Healthcare, Germany). We recruited healthy men (n=220), women in the follicular (n=139) or luteal (n=87) phases of the menstrual cycle, and postmenopausal women (n=103). Data was analyzed according to CLSI EP28-A3c guidelines. Although reference intervals established with both platforms showed good agreement with ranges quoted by the assay manufacturer, two discrepancies were noted. First, intervals for prolactin in women were influenced by hormonal status, and the partition analysis supported their separation into subgroups based on menstrual cycle. Second, the upper limit for estradiol in the follicular phase was nearly a half of that provided by the manufacturer. This discrepancy was attributed to the stringent definition of the follicular phase (consistently set at days 3-5 after menstruation onset). Our findings suggest that reference values for prolactin should both be gender specific and account for menstrual cycle phase. The results also emphasize that clear-cut selection criteria are required when assembling populations for establishing endocrine reference intervals.

Head-To-Head Assessment of Diagnostic Performance of Testosterone Immunoassays in Patients With Polycystic Ovary Syndrome.

BACKGROUND: Determination of plasma testosterone is critical for the proper diagnosis of polycystic ovary syndrome (PCOS), but the interpretation of biochemical tests is hampered by inadequate specificity and precision of available immunoassays. We here compared the diagnostic performance of three testosterone immunoassays (Advia Centaur, Immulite 2000 XPi, Cobas e411) in PCOS patients using receiver operator characteristics curve analysis. METHODS AND RESULTS: Plasma levels of testosterone, androstendione, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, estradiol, progesterone, steroid hormone binding globulin, luteinizing hormone, and follicular stimulating hormone were determined in 188 patients with PCOS and 202 controls. Free testosterone (fT) levels and free androgen index (FAI) were calculated. Testosterone levels measured on Advia Centaur, Immulite 2000 XPi, and Cobas e411 showed clear linear relationship to each other. Testosterone measured with Advia Centaur showed discriminatory performance superior to Immulite 2000 XPi and Cobas e411. Calculation of fT or FAI improved the performance of Advia Centaur and Immulite 2000 XPi, which nevertheless performed better than Cobas e411. The performance of other parameters was inferior to that of testosterone, fT, and FAI. CONCLUSION: Present study documents striking differences between testosterone immunoassays with respect to their capacity to identify PCOS patients and favors the use of calculated parameters reflecting active testosterone in plasma.

Fertility-preservation counselling and treatment for medical reasons: data from a multinational network of over 5000 women.

Fertility-preservation techniques for medical reasons are increasingly offered in national networks. Knowledge of the characteristics of counselled patients and techniques used are essential. The FertiPROTEKT network registry was analysed between 2007 and 2013, and included up to 85 university and non-university centres in Germany, Austria and Switzerland; 5159 women were counselled and 4060 women underwent fertility preservation. In 2013, fertility-preservation counselling for medical reasons increased significantly among nullipara and women aged between 21 and 35 years (n = 1043; P < 0.001). Frequency of GnRH applications slowly decreased, whereas tissue, oocytes and zygote cryopreservation increased. In 2013, women with breast cancer mainly opted for tissue freezing, whereas women with lymphoma opted for GnRH agonist. Women younger than 20 years predominantly opted for GnRH agonists and ovarian tissue cryopreservation; women aged between 20 and 40 years underwent a variety of techniques; and women over 40 years opted for GnRH agonists. The average number of aspirated oocytes per stimulation cycle decreased as age increased (< 30 years: 12.9; 31-35 years: 12.3; 36-46: 9.0; > 41 years: 5.7). For ovarian tissue cryopreservation, removal and cryopreservation of fewer than one ovary was preferred and carried out in 97% of cases in 2013.

Xenotransplantation of cryopreserved human ovarian tissue--a systematic review of MII oocyte maturation and discussion of it as a realistic option for restoring fertility after cancer treatment.

OBJECTIVE: To systematically review the reporting of MII (MII) oocyte development after xenotransplantation of human ovarian tissue. DESIGN: Systematic review in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). SETTING: Not applicable. PATIENT(S): Not applicable. INTERVENTION(S): Formation of MII oocytes after xenotransplantation of human ovarian tissue. MAIN OUTCOME MEASURE(S): Any outcome reported in Pubmed. RESULT(S): Six publications were identified that report on formation of MII oocytes after xenotransplantation of human ovarian tissue. CONCLUSION(S): Xenografting of human ovarian tissue has proved to be a useful model for examining ovarian function and follicle development in vivo. With human follicles that have matured through xenografting, the possibility of cancer transmission and relapse can also be eliminated, because cancer cells are not able to penetrate the zona pellucida. The reported studies have demonstrated that xenografted ovarian tissue from a range of species, including humans, can produce antral follicles that contain mature (MII) oocytes, and it has been shown that mice oocytes have the potential to give rise to live young. Although some ethical questions remain unresolved, xenotransplantation may be a promising method for restoring fertility. This review furthermore describes the value of xenotransplantation as a tool in reproductive biology and discusses the ethical and potential safety issues regarding ovarian tissue xenotransplantation as a means of recovering fertility.

MicroRNA miR-145 inhibits proliferation, invasiveness, and stem cell phenotype of an in vitro endometriosis model by targeting multiple cytoskeletal elements and pluripotency factors.

OBJECTIVE: To study the function of miR-145, known to be dysregulated in endometriosis, and to identify its target genes in an in vitro endometriosis model. DESIGN: Experimental laboratory study. SETTING: University medical centers. PATIENT(S): Primary endometrial stroma cells were derived from eutopic endometrium of three American Society for Reproductive Medicine stage III endometriosis patients and from ectopic lesions of four patients with deep infiltrating endometriosis. INTERVENTION(S): The human endometriotic cell line 12Z and primary eutopic and ectopic endometrial stroma cells were transiently transfected with miR-145 precursors or anti-miR-145 inhibitors and investigated for posttranscriptional regulation of predicted target genes and changes in cell behavior. MAIN OUTCOME MEASURE(S): Predicted target expression was measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, and altered cell behavior was monitored by cell proliferation assays. The 12Z cells were additionally investigated by Matrigel invasion assays, cell cycle analysis, side population analysis, and aldehyde dehydrogenase activity assays. RESULT(S): In all cells investigated, miR-145 overexpression inhibited cell proliferation and induced down-regulation of FASCIN-1, SOX2, and MSI2. In 12Z cells miR-145 upregulation increased Matrigel invasiveness and reduced side population and aldehyde dehydrogenase-1 activity. Additional down-regulated targets in 12Z cells included OCT4, KLF4, PODXL, JAM-A, and SERPINE1/PAI-1. ACTG2 and TAGLN were up-regulated upon pre-miR-145 transfection. JAM-A, FASCIN-1, and PAI-I down-regulation in 12Z cells were confirmed by Western blotting. CONCLUSION(S): miR-145 inhibits endometriotic cell proliferation, invasiveness, and stemness by targeting multiple pluripotency factors, cytoskeletal elements, and protease inhibitors.

Effects of the FSH-ß-subunit promoter polymorphism -211G->T on the hypothalamic-pituitary-ovarian axis in normally cycling women indicate a gender-specific regulation of gonadotropin secretion.

CONTEXT: A polymorphism in the FSHB promoter (-211G→T, rs10835638) was found to be associated with decreased FSH, elevated LH, reduced testosterone, and oligozoospermia in males. Although FSH is pivotal for ovarian function, no data on consequences of FSHB -211G→T are available in females. OBJECTIVE: We studied the effects of FSHB -211G→T on the hypothalamic-pituitary-ovarian axis in women. DESIGN AND SETTING: In a university-based in vitro fertilization unit, women undergoing standardized diagnostics were genotyped and compared with a fertile control group. PATIENTS: The study group consisted of 365 thoroughly characterized women with normal menstrual cycle intervals and proven ovulation, with predominantly male-factor infertility. The independently recruited control group included 438 women with proven fertility and no history of abortions. MAIN OUTCOME MEASURES: Distribution of alleles and genotypes were compared between the study group and controls. In the study group, associations of endocrine parameters with FSHB -211G→T were assessed. RESULTS: Allele and genotype frequencies were not significantly different between the study population and controls (T-allele: 14.4 vs. 16.6%; TT-homozygotes: 2.5 vs. 3.2%). The FSHB -211G→T TT-genotype was strongly associated with elevated FSH (TT-homozygosity effect 2.05 U/liter, P = 0.003). LH increased with the number of T-alleles (1.30 U/liter per T-allele, P < 0.001). Additionally, FSHB -211G→T was associated with reduced progesterone (-1.96 ng/ml per T-allele, P = 0.047). CONCLUSIONS: This is a report on phenotypic consequences of FSHB -211G→T on the hypothalamic-pituitary-ovarian axis in women. The findings, partially contradictory to those in men, point to a gender-specific compensatory mechanism of gonadotropin secretion, probably involving progesterone.

Targeting of syndecan-1 by micro-ribonucleic acid miR-10b modulates invasiveness of endometriotic cells via dysregulation of the proteolytic milieu and interleukin-6 secretion.

OBJECTIVE: To study the function of syndecan-1 (SDC1) and its potential regulator miR-10b in endometriosis. DESIGN: Experimental laboratory study. SETTING: University medical center. PATIENT(S): Not applicable. INTERVENTION(S): The human endometriotic cell line 12Z was transiently transfected with SDC1 small interfering RNA or miR-10b precursors and investigated for changes in cell behavior and gene expression. 12Z and primary eutopic endometrial stroma cells of two American Society for Reproductive Medicine class III endometriosis patients were transfected with miR-10b precursors to investigate posttranscriptional regulation of SDC1. MAIN OUTCOME MEASURE(S): Quantitative polymerase chain reaction, Western blotting, flow cytometry, 3' untranslated region luciferase assays, and zymography were used to measure miR-10b-dependent targeting of SDC1 and SDC1-dependent expression changes of proteases and interleukin-6. Altered cell behavior was monitored by Matrigel invasion assays, cell viability assays, and mitogen-activated protein kinase activation blots. RESULT(S): Knockdown of SDC1 inhibited Matrigel invasiveness by >60% but did not affect cell viability. Interleukin-6 secretion, matrix metalloproteinase-9 expression, and matrix metalloproteinase-2 activity were reduced, whereas plasminogen activator inhibitor-1 protein expression was up-regulated. miR-10b overexpression significantly down-regulated SDC1, reduced Matrigel invasiveness by 20% and cell viability by 14%, and decreased mitogen-activated protein kinase activation in response to hepatocyte growth factor. CONCLUSION(S): Syndecan-1, a target of miR-10b, inhibits epithelial endometriotic cell invasiveness through down-regulation of metalloproteinase activity and interleukin-6.

Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells.

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1­7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6­7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6­7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH.