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Research question The study aimed to determine the standard treatment dose of follitropin epsilon for ovarian stimulation in the context of IVF treatment. Design A total of 247 women aged 18-37 years were treated with either 52.5, 75, 112.5 or 150 IU follitropin epsilon daily, or 150 IU every other day, or 150 IU follitropin alfa daily in a long gonadotrophin-releasing hormone agonist protocol. The study was performed as a randomized, assessor-blinded, comparator-controlled, six-armed phase II trial in eight fertility clinics in two European countries. Results The primary results were as follow. First, none of the doses of follitropin epsilon showed superiority for the main outcome measure, i.e. number of follicles ≥12 mm in size. Follitropin epsilon 75 IU produced results most similar to those of follitropin alfa 150 IU. In terms of secondary results, stronger effects of follitropin epsilon 112.5 IU compared with follitropin alfa 150 IU were seen for secondary outcome measures such as hormone concentrations (oestradiol, inhibin B and progesterone) and oocyte number. Conclusions Follitropin epsilon 75 IU daily results in a similar ovarian response to a standard dose of 150 IU follitropin alfa. This dose could be tested in a phase III trial.

It is well accepted that pituitary follitropin is secreted into the circulation as a mixture of variants, which differ not in primary structure but rather at the level of glycosylation. These glycosidic forms vary in the number of glycosylation sites filled, complexity of glycosidic chains, and sialylation and sulfation. It is generally agreed that high sialylation, 2,3 sialic acid capping of terminal N-acetyl galactosamine or galactose leads to longer circulating half-life, by blocking binding of asialoglycoprotein receptor (ASGPR) in the liver. In contrast, 2,6 sialic acid found in humans does not prevent recognition of galactose and N-acetyl galactosamine by ASGPR. Few studies on clinical outcomes comparing differences in sialylation of follitropin found in commercially available preparations are available. Thus, there is a clear need for a consortium of open data to address this unmet need. Recently, FSH glycosylation, primarily on the β-subunit, which varies as women age, has emerged as a key modifier of follitropin action, with profound biological effects in vivo in animal models. To date, limited information of recombinant follitropin hormone preparations is available. Thus, most of the studies with FSH that is well characterized biochemically have been done in vitro, with engineered non gonadal host cells bearing recombinant receptors or in animal models. Since limited studies in human granulosa cells are available, a question is whether structural differences in glycosylation in commercially available follitropin affects biological function and clinical effect in humans. The presence of fucose, for example, has not been studied greatly even though, in the case of antibody therapy it has been shown to have a large effect on antibody targeting. This review on glycosidic variability of follitropin from the biochemical/structural point of view reflects on this question and presents an assessment in the context of available published data. If clinical differences are to be expected or not, the readers will have a better understanding of the evidence for and limitations of such expectations.

Introduction: Although active gonadotropin-secreting pituitary adenomas are considered very rare, the vast majority of pituitary tumours diagnosed as “non-functioning” express gonadotropins or their free β or α subunits. However, systemic investigations comparing the serum concentrations of follitropin (FSH), lutropin (LH), and α–subunit (αSU) before surgery with the immunoreactivity of the respective substances in the excised tumours are still lacking. Material and methods: Immunostaining of FSH, LH, and αSU was compared in 43 surgically removed gonadotropin — expressing pitu­itary adenomas with serum concentrations of the above-mentioned substances before surgery in the same patients. Results: The serum concentrations of FSH were elevated (> 11.6 mU/mL) in 8/12 (66.7%) cases of FSH-positive adenomas. By contrast, in FSH–negative tumours the elevation of FSH is absent. Moreover, only 1/25 (4%) patients with LH–positive adenoma had the elevated serum concentration of LH (51.5 mU/mL). The overproduction of LH was not observed in adenomas expressing free β LH or in LH-negative tumours. In patients with αSU-positive adenomas elevated serum levels of αSU were observed in 3/15 (20%) cases. No αSU elevations were observed in patients with αSU–negative adenomas. The mean serum FSH, LH, and αSU concentrations were higher in patients with FSH, LH, and/or αSU immunopositive tumours in comparison with immunonegative. However, the differences are not statistically significant. Conclusions: Although “silent” gonadotropinomas constitute a frequent subtype of pituitary adenomas, the “active” subtype (i.e. manifesting by gonadotropin excess) are rare (approx. 4% of all pituitary adenomas). Gonadotropinomas are difficult to diagnose before surgery. The measurement of gonadotropins including αSU is needed but often not sufficient for presurgical diagnosis.

Eighty-four subjects, premenopausal female patients (n=42, mean (SD) age: 26.4 (4.2) years) diagnosed with polycystic ovary syndrome (PCOS) and age-matched healthy volunteers (n=42, mean (SD) age: 27.6(3.4) years), were included in this study. Data on physical examination, anthropometric measurements and blood biochemistry analysis were recorded for each subject along with analysis for SOCS1-1478 CA/del polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The relation of SOCS1-1478 CA/del polymorphism to PCOS status and insulin resistance was analysed via logistic regression analysis. Mean (SD) levels for BMI (28.5(6.5) vs.22.5 (4.9) kg/m(2), p

The gonads (ovary, testis) produce gametes (oocytes and spermatozoa). The pituitary glycoprotein hormones luteinizing hormone (LH, lutropin) and follicle-stimulating hormone (FSH, follitropin) are gonadotropins because their targets are the gonads. Another hormone produced by the syncytiotrophoblast of the human placenta is human chorionic gonadotropin (hCG, choriogonadotropin), and its activity is like LH. This review describes the protein structure, gene structure, and regulation of biosynthesis of both the gonadotropins and their membrane bound receptors. The functions of both hormones and receptors are covered extensively, as well as naturally occurring mutations that have been identified in disease states and therapies derived from their study.

Follitropin (FSH) is a heterodimeric protein composed of an α subunit that is shared with the glycoprotein hormone family, including lutropin (LH), thyrotropin (TSH), human choriogonadotropin (hCG), and a unique β specific subunit. Both α and FSHβ subunits contain two sites of N-linked oligosaccharides, which are important for its function. FSH has a crucial function in the reproductive process in mammals. However, there are some clinical conditions, such as menopausal osteoporosis or adiposity, associated with increased FSH activity. Moreover, in some cases, carcinogenesis is evidently associated with activation of FSH receptor. Therefore, developing a follitropin antagonist might be beneficial in the treatment of these conditions. Here, we describe a novel, engineered, non-glycosylated single-chain FSH variant, prepared by site-directed mutagenesis and fusion of the coding genes of the α and β subunits. The designed variant was expressed in Chinese hamster ovary (CHO) cells and successfully secreted into the culture medium. We found that the non-glycosylated single-chain FSH analog binds with high affinity to FSH receptor and efficiently inhibits FSH activity in vitro. This variant acts at the receptor level and has the potential to serve as a follitropin antagonist for clinical applications in the future.

This study hypothesized that ovarian response for superovulation is better when using compounds containing luteinizing hormone (LH). To compare the ovarian responses and the ovarian blood flow using three gonadotropins, three equal groups of Holstein cows (N = 18) were treated with Pluset (P), Follitropin (F), and eCG (G). The ovarian response was evaluated from the day of starting treatments (Day -5) till Day 5 after ovulation using Doppler ultrasound. Results revealed that the ovarian arteries pulsatility index (PI), peak systolic velocity (PSV), and the blood flow volume (BFV) varied (P0.0001) between both ovarian arteries. Cows treated with P had low number of small and medium follicles and a high number of the large follicles on Day -1. Cows treated with F showed low (P = 0.0001) number and percentage of colored follicles and high % of ovulated follicles (P=0.0001) that obtained high colored areas (P = 0.043) and colored area % (P = 0.001). The G produced more (P = 0.001) colored follicles (Days -1, 0) of low area. Both P and G had the same higher (P = 0.0001) percentages of colored follicles (80 %) on Day-1 than F (60 %). F and P had higher ovulated colored follicles % than G. P obtained nearly the same ovulated follicles diameters and areas as F with higher antrum area (P = 0.029) but lower colored area (P = 0.043) and colored area % (P = 0.001). In conclusion, the number and the doses of gonadotropin injections and the side of the ovary influenced the ovarian response.

Objective: Deficiency of steroid 5-alpha reductase type 2 (5?RD2) is a rare autosomal recessive disorder caused by mutations in the SRD5A2 gene. A defect in the 5-alpha reductase enzyme, which ensures conversion of testosterone into dihydrotestosterone, leads to disorders of sex development. This study presents the clinical and genetic results of patients with 5?RD2 deficiency. Methods: 5?RD2 deficiency was detected in 6 different patients from 3 unrelated families. All patients were reared as girls. Two of the patients presented with primary amenorrhea, one with primary amenorrhea and rejection of female gender, and the others with masses in their inguinal canals. Chromosome and sex-determining region Y (SRY) gene analyses were performed in all patients. Additionally, five exons of the SRD5A2 gene were amplified with polymerase chain reaction in the obtained DNA samples and evaluated. Results: While 46,XY was identified in 5 patients, 47,XXY was detected in one patient. The SRY gene was positive in all patients. The p.Ala65Pro (c193G>C) mutation and V89L polymorphism were observed in exon 1 of the SRD5A2 gene in all patients. Conclusion: Identification of this mutation and polymorphism is a significant indicator of presence of 5?RD2 deficiency in Southeastern Turkey, a geographical region where consanguineous marriages are also highly common. © Journal of Clinical Research in Pediatric Endocrinology, Published by Galenos Publishing.

Background Individualization of the follicle-stimulating hormone (FSH) starting dose is considered standard clinical practice during controlled ovarian stimulation (COS) in patients undergoing assisted reproductive technology (ART) treatment. Furthermore, the gonadotropin dose is regularly adjusted during COS to avoid hyper- or hypo-ovarian response, but limited data are currently available to characterize such adjustments. This review describes the frequency and direction (increase/decrease) of recombinant-human FSH (r-hFSH) dose adjustment reported in clinical trials. Methods We evaluated the proportion of patients undergoing ART treatment who received ≥ 1 r-hFSH dose adjustments. The inclusion criteria included studies (published Sept 2007 to Sept 2017) in women receiving ART treatment that allowed dose adjustment within the study protocol and that reported ≥ 1 dose adjustments of r-hFSH; studies not allowing/reporting dose adjustment were excluded. Data on study design, dose adjustment and patient characteristics were extracted. Point-incidence estimates were calculated per study and overall based on pooled number of cycles with dose adjustment across studies. The Clopper–Pearson method was used to calculate 95% confidence intervals (CI) for incidence where adjustment occurred in Results Initially, 1409 publications were identified, of which 318 were excluded during initial screening and 1073 were excluded after full text review for not meeting the inclusion criteria. Eighteen studies (6630 cycles) reported dose adjustment: 5/18 studies (1359 cycles) reported data for an unspecified dose adjustment (direction not defined), in 10/18 studies (3952 cycles) dose increases were reported, and in 11/18 studies (5123 cycles) dose decreases were reported. The studies were performed in women with poor, normal and high response, with one study reporting in oocyte donors and one in obese women. The median day that dose adjustment was permitted was Day 6 after the start of treatment. The point estimates for incidence (95% CI) for unspecified dose adjustment, dose increases, and dose decreases were 45.3% (42.7, 48.0), 19.2% (18.0, 20.5), and 9.5% (8.7, 10.3), respectively. Conclusions This systematic review highlights that, in studies in which dose adjustment was allowed and reported, the estimated incidence of r-hFSH dose adjustments during ovarian stimulation was up to 45%.

International audience; FSH is synthesized in the pituitary by gonadotrope cells. By binding to and interacting with its cognate receptor [FSH receptor (FSHR)] in the gonads, this gonadotropin plays a key role in the control of gonadal function and reproduction. Upon activation, the FSHR undergoes conformational changes leading to transduction of intracellular signals, including dissociation of G protein complexes into components and activation of several associated interacting partners, which concertedly regulate downstream effectors. The canonical Gs/cAMP/protein kinase A pathway, considered for a long time as the sole effector of FSHR-mediated signaling, is now viewed as one of several mechanisms employed by this receptor to transduce intracellular signals in response to the FSH stimulus. This complex network of signaling pathways allows for a fine-tuning regulation of the gonadotropic stimulus, where activation/inhibition of its multiple components vary depending on the cell context, cell developmental stage, and concentration of associated receptors and corresponding ligands. Activation of these multiple signaling modules eventually converge to the hormone-integrated biological response, including survival, proliferation and differentiation of target cells, synthesis and secretion of paracrine/autocrine regulators, and, at the molecular level, functional selectivity and differential gene expression. In this mini-review, we discuss the complexity of FSHR-mediated intracellular signals activated in response to ligand stimulation. A better understanding of the signaling pathways involved in FSH action might potentially influence the development of new therapeutic strategies for reproductive disorders.

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r-/- mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreasedLHrelease in ovariectomized-sham replaced females, as documented forNK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons andGnRHneurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons. Copyright © 2015 by the Endocrine Society.

In this retrospective multicenter cohort study, women with congenital hypogonadotrophic hypogonadism (CHH) (n = 57) who underwent intra-cytoplasmic sperm injection in-between 2010-2014 were compared to age-matched controls with tubal factor infertility (n = 114) to assess ovarian stimulation cycle and pregnancy outcomes. Live birth rates (LBRs) per started cycle were 31.6 and 24.6% in CHH and controls groups, respectively (p = 0.36). Comparable success rates were also confirmed with the logistic regression analysis (OR: 1.44, 95% CI: 0.78-2.67, p = 0.24). Of the 57 women with CHH, 19 were stimulated with the gonadotropin-releasing hormone (GnRH) antagonist protocol, 13 with the long-GnRH-agonist protocol. Pituitary suppression (PS) was not employed in the remaining 25 cases. Compared to women with PS, women without PS had significantly higher embryo implantation rates (21.6 versus 52.6%, p = 0.03). Although there was a trend favoring no PS, LBRs (25.0 versus 40.0%, p = 0.26) per cycle were short of statistical significance. LBRs per cycle (57.1 versus 31.2%, p = 0.11) and miscarriage rates (11.1 versus 16.7%, p = 0.75) were similar between CHH women who were given estrogen + progesterone and progesterone alone to support the luteal phase. In conclusion, the optimal stimulation protocol appears to be exogenous gonadotropin stimulation alone, without PS, and progesterone-only luteal phase support in CHH patients.

Study question What are the differences and similarities of pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of the novel recombinant human FSH follitropin epsilon expressed in the human cell line GlycoExpress compared with a Chinese hamster ovary (CHO) derived compound and a urinary derived product? Summary answer Overall follitropin epsilon, with a fully human glycosylation, shows a comparable PK profile at single-dose as well as multiple-dose administration compared to recombinant CHO-derived FSH as well as urinary derived FSH, whereas the PD properties differ from product to product with follitropin epsilon being most active in PD parameters. What is known already Recombinant FSH produced in CHO and FSH obtained from the urine of postmenopausal women show comparable PK and PD properties. However, more recently a comparative study of a recombinant FSH produced in the human cell line PerC6 and a CHO-derived FSH preparation revealed differences in PK and PD properties of the molecule. Study design, size, duration Both studies were randomized, placebo- and comparator-controlled, single-blind phase I studies in healthy pituitary-suppressed female volunteers aged 18 and 40 years. The single-dose, dose escalation study included 19 women (April 2011 to September 2011) with three ascending dose levels per subject or placebo/comparators with a 14-day washout phase between dosings. The multiple-dose study included 57 women (October 2011 to April 2012) in five cohorts with three dose levels versus placebo and two comparators. Randomization to the respective treatment was performed after successful downregulation of the pituitary gland prior to Investigational Medicinal Product dosing. Participants/materials, setting, methods In the single-dose study, 12 subjects received follitropin epsilon (25, 75, 150 and 300 IU) in three of four possible ascending doses and seven subjects received one dose of two comparators (150 IU Bravelle and 150 IU Gonal-f) and placebo in random order in each treatment period. In the multiple-dose study, 30 subjects received follitropin epsilon (75 IU or 150 IU once daily [QD], or 150 IU every other day [QAD], 10 subjects each) and 27 subjects received 150 IU Gonal-f, 150 IU Bravelle, or placebo for 7 days (11/10/6 subjects). Blood samples for measuring PK as well as PD parameters were collected systematically before, during and after dosing. Adverse events (AEs) and other relevant safety parameters were recorded. Data were summarized using descriptive statistics. Main results and the role of chance The single- and multiple-dose PK parameters maximum concentration (Cmax) and area under the concentration-time curve (AUC0-last) increased in a linear fashion with increasing dose levels of follitropin epsilon. Follitropin epsilon showed PK characteristics comparable to the comparators indicating that well established treatment schemes could be applied. There was a dose-response effect of single and multiple doses of follitropin epsilon on follicular growth, which was shown for the biomarker inhibin B as well as for the mean number and size of follicles. Multiple doses of 75 IU follitropin epsilon given daily, as well as 150 IU follitropin epsilon every second day, showed a follicle growth comparable with 150 IU Gonal-f given daily, while in case of daily administration of 150 IU Bravelle only weak follicle stimulation was observed. Multiple doses of 150 IU follitropin epsilon induced a much higher follicle growth compared to the same dose of Gonal-f. All single and multiple follitropin epsilon doses tested were safe and well tolerated, and overall there were no relevant differences between follitropin epsilon and the comparators in terms of safety. The average number of AEs increased with increasing dose levels. No clinically relevant abnormalities were reported for any of the other safety parameters assessed. No follitropin epsilon anti-drug antibodies were observed. Limitations, reasons for caution The studies were conducted as a single-blind design. Hormone levels or other parameters assessed in serum are generally not considered as being subject to bias. Other assessments directly performed by the investigators, such as transvaginal ultrasound assessments, may have been subject to personal bias. No prospective calculations of statistical power had been made, as is common practice for first in human and early phase I studies in healthy volunteers. Wider implications of the findings These early development studies showed that follitropin epsilon exhibits comparable PK characteristics, as well as inducing stronger PD effects in terms of follicle growth and serum inhibin B, than the comparators. Follitropin epsilon induced a dose-dependent increase in follicular growth. The results warrant further studies with this new fully human recombinant FSH. Study funding/competing interest(s) The studies were sponsored by GLYCOTOPE GmbH, Berlin, Germany. K.A-E. is an employee of QPS-Netherlands, B.V., which received funding for the studies from Glycotope GmbH; I.D. and C.K. are employees of Dinox B.V., which received funding for the studies from Glycotope GmbH; L.S. and S.G. are employees and shareholders of Glycotope GmbH; B.D. and K.E. are employees of Glycotope GmbH. Trial registration number www.clinicaltrials.gov: NCT01354886 (single-dose); NCT01477073 (multiple-dose). Trial registration date The single-dose trial was registered on 11 May 2011 while the multiple-dose trial was registered on 09 November 2011. Date of first subject's enrolment First subject was enroled in the single-dose trial in 27 April 2011 and in the multiple-dose trial in 02 October 2011.

Congenital central hypothyroidism (CCH) is a very rare disease. Alterations in pituitary development genes as well as mutations of immunoglobulin superfamily member 1 and transducin β-like protein 1 can result in CCH and multiple pituitary hormone deficiencies. However, mutations of the thyrotropin-releasing hormone receptor or thyroid-stimulating hormone-beta (TSHB) gene are responsible for isolated CCH. In this paper, we present the cases of two siblings with a novel mutation of TSHB. Direct sequencing of the coding regions and exon/intron boundaries of the TSHB gene revealed two homozygous nucleotides changes. One of them was c.40A>G (rs10776792) which is a very common variation that is also seen in healthy individuals, the other was c.94G>A at codon 32 of exon 2 which resulted in a change from glutamic acid to lysine (p.E32K). Both patients were homozygous and the parents were heterozygous. © 2017 by Turkish Pediatric Endocrinology and Diabetes Society.

Pharmacokinetic and pharmacodynamic properties of a novel recombinant follicle-stimulating hormone (rFSH) preparation (FE 999049), expressed by a human cell line (PER.C6), was compared with an rFSH preparation (follitropin α) expressed by a Chinese hamster ovary (CHO) cell line in healthy pituitary-suppressed women. Following single intravenous administration of 225 IU (Steelman-Pohley assay), the clearance was lower, 0.31 versus 0.44 L/h, for FE 999049 than for follitropin α. Likewise, the apparent clearance after repeated daily subcutaneous administrations was lower, 0.58 versus 0.99 L/h, and AUC and C(max) higher, 1.7- and 1.6-fold. The absolute bioavailability after a single subcutaneous dose of 450 IU was similar for both preparations, 60-65%. After repeated subcutaneous administration the elimination half-life was approximately 30 and 24 hours for FE 999049 and follitropin α. The ovarian responses by number of follicles and serum concentrations of inhibin B and estradiol, were higher with FE 999049 than with follitropin α, AUC and C(max) for the two latter being >1.6-fold greater with FE 999049 than with follitropin α. These results indicate that administration of equal doses of FE 999049, expressed in a human cell line, and follitropin α, expressed in a CHO cell line, display different pharmacokinetic and pharmacodynamic properties in humans.

Background: Congenital leptin deficiency is a recessive genetic disorder associated with severe early-onset obesity. It is caused by mutations in the leptin (LEP) gene, which encodes the protein product leptin. These mutations may cause nonsense-mediated mRNA decay, defective secretion or the phenomenon of biologically inactive leptin, but typically lead to an absence of circulating leptin, resulting in a rare type of monogenic extreme obesity with intense hyperphagia, and serious metabolic abnormalities. Methods: We present two severely obese sisters from Colombia, members of the same lineal consanguinity. Their serum leptin was measured by MicroELISA. DNA sequencing was performed on MiSeq equipment (Illumina) of a next-generation sequencing (NGS) panel involving genes related to severe obesity, including LEP. Results: Direct sequencing of the coding region of LEP gene in the sisters revealed a novel homozygous missense mutation in exon 3 [NM_002303.3], C350G&gt, T [p.C117F]. Detailed information and clinical measurements of these sisters were also collected. Their serum leptin levels were undetectable despite their markedly elevated fat mass. Conclusions: The mutation of LEP, absence of detectable leptin, and the severe obesity found in these sisters provide the first evidence of monogenic leptin deficiency reported in the continents of North and South America.

Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus (Ctail) of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the abscence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and β-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these cysteine residues are S-palmitoylated, the data presented emphasize on this posttranslational modification as an important factor for both upward and downward trafficking of this receptor.

Objective: The aim of the presented study is to investigate the impact of progesterone change in the late follicular phase on the pregnancy rates of both agonist and antagonist protocols in normoresponders. Study design: A total of 201 normoresponder patients, who underwent embryo transfer were consecutively selected. 118 patients were stimulated using a long luteal GnRH agonist protocol and 83 using a flexible antagonist protocol. The level of change in late follicular phase progesterone was calculated according to the progesterone levels on the hCG day and pre-hCG day (1 or 2 days prior to hCG day) measurement. Results: Clinical pregnancy rates were comparable between long luteal and antagonist group (35.6 and 41%, respectively). The incidence of progesterone elevation on the hCG day was 11% in long luteal and 18% in antagonist group (p = 0.16). In pregnant cycles, p levels both on the hCG day and pre-hCG day measurement were significantly higher in antagonist than agonist cycles (p = 0.029, p = 0.038, respectively). The change of p level was statistically significant in non-pregnant cycles both for the agonist (-0.17 ± 0.07; 95% CI: −0.29 to −0.37) and antagonist groups (−0.18 ± 0.07; 95%CI: −0.31 to −0.04). Conclusions: Late follicular phase progesterone levels were stable during the cycles of pregnant patients irrespective of the protocols and were shown to be higher in pregnant patients in antagonist cycles when compared to agonist cycles.

Objective: Collagen type I is abundant in the outer layers of capsular stroma and theca exter-na in the ovary. C-terminal propeptide of Type I collagen (CICP) is the measurable form of type I procol-lagen in serum. Circulating CICP levels are indicative of collagen production. This study was designed to determine the serum levels of CICP and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in women with Polycystic Ovary Syndrome (PCOS). Material and Methods: This study included twenty-five women with PCOS in the study group and twenty healthy women in the control group. Serum lipid sub-fracti-ons, fasting glucose and insulin, hormone (gonadotropins, androgens), CICP and TIMP-1 levels were me-asured. Homeostasis model assessment (HOMA-IR) was used to estimate insulin resistance. Results: Serum luteinizing hormone (LH) and fasting insulin levels, LH/follicule stimulating hormone (FSH) ratio, free an-drogen index (FAI) and HOMA-IR values were higher in patients with PCOS compared with healthy wo-men. A significant increase in CICP level was observed in subjects with PCOS, and TIMP-1 level was found to be significantly decreased. HOMA-IR value was positively correlated with CICP level, but inver-sely with TIMP-1 level. The best cut-off values for CICP and TIMP-1 were >49.94 ng/mL (sensitivity 92.6% and specificity 65%) and

Background: Overweight or central obesity is generally associated with increases in fasting insulin levels, insulin resistance, and glucose intolerance and has been identified as a target for new therapeutic strategies, including early change in lifestyle. Early biochemical markers for identifying at-risk patients will be useful for prevention studies. The aim of this study is to investigate whether or not SHBG level is a useful index of hyperinsulinemia and/or insulin resistance in pre- and postmenopausal obese women. At the same time, the relationship between SHBG concentrations and features of the metabolic syndrome were evaluated. Methods: 229 women were eligible for this study. MetS was defined by using a modification of the ATP III guidelines. All patients were euthyroid, obese and overweight, 25 to 69 years of age. Subjects were divided into groups of premenopausal women (n = 125) and postmenopausal women (n = 104). Various fatness and fat distribution parameters, SHBG, sex hormones, FSH, LH, thyroid hormones, serum levels of fasting and postprandial glucose, lipid profile, uric acid and serum insulin, and blood pressure were measured. Results: No significant difference was found in mean SHBG levels between pre- and postmenopausal obese women in this study (p = 0.866). In premenopausal obese women, SHBG correlated negatively with BMI, waist circumference, fasting glucose, uric acid levels and FAI. In postmenopausal obese women, SHBG correlated negatively with fasting glucose, postprandial plasma glucose, fasting insulin, HOMA-IR and FAI and positively with HDL. SHBG had a significant inverse association with MetS parameters only in postmenopausal women, also after adjusting for BMI, age and estradiol. Conclusions: Obesity may influence the levels of endogenous sex steroid, especially after menopause. SHBG concentrations are correlated with features of the metabolic syndrome, particularly in postmenopausal obese women. These results suggest that SHBG may be an index of insulin resistance in postmenopausal obese women. © 2008 European Federation of Internal Medicine.

The objective of the present study was to characterize ovarian follicular dynamics and hormone concentrations during follicular deviation in the first wave after ovulation in Nelore (Bos indicus) heifers. Ultrasonographic exams were performed and blood samples were collected every 12h from the day of estrus until 120-144 h after ovulation in seven females. Deviation was defined as the point at which the growth rate of the dominant follicle became greater than the growth rate of the largest subordinate follicle. Deviation occurred approximately 65 h after ovulation. Growth rate of the dominant follicle increased (P0.05) after deviation, while growth rate of the subordinate follicle decreased (P0.05). Diameter of the dominant follicle did not differ from the subordinate follicle at deviation (approximately 5.4mm). The dominant follicle (7.6mm) was larger (P0.05) than the subordinate follicle (5.3mm) 96 h after ovulation or 24h after deviation. Plasma FSH concentrations did not change significantly during the post-ovulatory period. The first significant increase in mean plasma progesterone concentration occurred on the day of follicular deviation. In conclusion, the interval from ovulation to follicular deviation (2.7 days) was similar to that previously reported in B. taurus females, but follicles were smaller. Diameters of the dominant follicle and subordinate follicle did not differ before deviation and deviation was characterized by an increase in dominant follicle and decrease in subordinate follicle growth rate. Variations in FSH concentrations within 12-h intervals were not involved in follicular deviation in Nelore heifers.

Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with different regions of an FSHR/LHR chimera having only two unique LHR residues and that binds both hormones with high affinity. hCG and hFSH analogs dock with this receptor chimera in a manner similar to that in which they bind LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH.

We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

A chimeric recombinant human gonadotropin, termed C3, demonstrates both follitropic and lutropic bioactivities. The alpha-subunit construct for C3 is comprised of the recombinant wild-type human glycoprotein hormone alpha-subunit. The beta-subunit DNA construct for C3 encodes residues 1-145 from human chorionic gonadotropin (hCG)-beta with the exceptions that FSH beta amino acid 88 (D) is substituted for hCG beta amino acid 94 (R) and FSH beta amino acids 95-108 (TVRGLGPSYCSFGE) are substituted for hCG beta amino acids 101-114 (GGPKDHPLTCDDPR). C3 is a potent FSH and LH agonist able to bind and to signal through FSH and LH receptors in vitro. In in vivo bioassays optimized to quantify each type of activity, C3 was found to have lutropin and follitropin potencies at levels similar to those of recombinant human LH and recombinant human FSH, respectively. In immature rats, C3 was sufficient to support the maturation of normal ovarian follicles. Moreover, a significant portion of follicles matured by C3 ruptured in response to an ovulatory hCG stimulus and gave rise to morphologically normal oocytes. Furthermore, a low dose of C3 promoted weight gain in the rodent uterus, suggesting it also supported preparation for implantation without histological evidence of excessive luteinization of the ovary. In summary, the biological properties of C3 indicate that its chimeric nature has resulted in a fully functional, dual-acting human gonadotropin.

Background: Premature ovarian failure (POF) is characterized by hypergonadotropic amenorrhoea before the age of 40. Inhibin α-subunit (INHα) gene is proposed as a candidate gene due to its role in negative feedback control of FSH. Methods: Polymorphism -16 C>T of INHα gene was studied in 61 POF patients and 82 controls above 40 years old (C > 40). Substitution 769G>A was studied in 59 POF patients, 76 C > 40 and 73 controls below 40 years old (C < 40). Results: No significant difference in risk of POF development for -16T allele was found when comparing idiopathic POF (I-POF) with C > 40 (Odds ratio = 1.46; 95% confidence interval = 0.63-3.19). Implication of -16C>T polymorphism in serum inhibin levels was analysed in 46 controls, and no significant differences (P > 0.05) were found between CC and CT + TT genotype groups when comparing either mid-follicular phase Pro-αC and inhibin B values or mid-luteal phase Pro-αC and inhibin A values. Heterozygosity for substitution 769G>A was found in 1 of 59 POF woman, 2 of 76 C > 40 and 6 of 73 C < 40. Presence of this su bstitution in a relevant number of control subjects is herein described for the first time. Conclusion: Our results indicate that -16C>T and 769G>A variants in INHα gene may not be associated to POF disease. © 2006 Oxford University Press. Fil:Sundblad, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Charreau, E.H. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The revolutionary development of biotechnology-derived therapeutic proteins has provided the expected improvements in quality, purity and consistency, as demonstrated in recombinant human FSH (rhFSH). However, the development of urine-derived gonadotrophins has not always shown comparable improvements. More recently, highly purified urine-derived FSH (uFSH-HP) products have become widely available. The relative purity, level of urine-derived contaminants, and consistency of one such highly purified human uFSH (uhFSH) (urofollitropin) has been assessed and directly compared with rhFSH (follitropin alpha). It has been demonstrated that the highly purified urofollitropin contains variable levels of urine-derived contaminant proteins and demonstrates a variable level of FSH purity, FSH isoforms, and delivered dose. These variable factors may contribute to the control of ovarian stimulation. The relative purity, variable consistency and the presence of contaminants indicates that the urofollitropin is, at best, a partially purified uFSH that is not able to meet the quality attributes of follitropin alpha (rhFSH).

This study was designed to determine serum human leukocyte antigen-G (HLA-G) levels and establish whether serum HLA-G level is related with insulin resistance, oxidative stress, dyslipidemia and ovarian hyperandrogenism in women with polycystic ovary syndrome (PCOS). Twenty-five patients with PCOS and 23 healthy control women were evaluated in this study. Serum HLA-G, lipid fractions, glucose, insulin, malondialdehyde (MDA), glutathione (GSH), white blood cell (WBC), sex hormone-binding globulin (SHBG) and other hormone (gonadotropins and androgens) levels were measured. The estimate of insulin resistance was calculated by homeostasis model assessment (HOMA-IR). Serum luteinizing hormone (LH), total testosterone, fasting insulin, WBC levels and LH/follicle-stimulating hormone (FSH) ratio, free androgen index (FAI) and HOMA-IR values were significantly higher in patients with PCOS compared with healthy women. However, the women with PCOS had considerably lower serum FSH, SHBG, MDA, GSH and HLA-G levels than healthy subjects. HLA-G was inversely related with HOMA-IR, FAI, LH/FSH ratio and WBC, but positively with high-density lipoprotein cholesterol. Decreased serum HLA-G level may be related with insulin resistance, ovarian hyperandrogenism and oxidative stress in women with PCOS. Nevertheless, the exact role of HLA-G in the pathogenesis of the disease remains to be elucidated. © 2014 Informa UK Ltd.

The purpose of the present investigation was to study the extent to which season and the gonadotropin preparation interferes with the superovulatory response in the dromedary. Adult camels were treated for superovulation during the breeding (November to April) and non-breeding season (May to October). Animals were synchronized by daily i.m. injections of progesterone (125 mg/animal/day, Jurox, UK) for 10 to 14 days. Superovulation was induced by 400mg pFSH alone (Follitropin V, Vetrepharm, Canada) administered in eight descending doses at 12h intervals or a combination of PMSG (2000IU, Folligon, Intervet, The Netherlands), injected with last injection of progesterone and 400mg pFSH in eight descending doses. The follicular development was daily assessed by ultrasonography of the ovaries. The donors were classified as per their response to the superovulatory treatment into very good (10 follicles), good (5-10 follicle), poor (2-4 follicles) or no response (1 or no follicle) on each ovary. Ovulation was induced by injecting 3000 IU hCG (Chorulon, Intervet) at the time of first mating. The donors were mated twice at an interval of 12h when all or most of the follicles reached to a size of about 1.0-1.7 cm. Camels were flushed non-surgically on Day 6 or 7 after the ovulation. The proportion of camels showing very good response during the breeding as well as non-breeding season was higher (P0.05) when a combination of pFSH and eCG was used compared with pFSH only. There was no difference (P0.05) in the proportion of donors flushed successfully (embryos recovered) when treated either with a combination of pFSH and eCG or pFSH alone during the breeding and non-breeding season. The rate of recovery of ova/embryos and proportion of transferable embryos was higher (P0.05) when donors were treated with pFSH+eCG compared with pFSH only during the breeding as well as non-breeding season. The results may indicate that ova/embryo recovery rate of the dromedary is influenced by the gonadotropin preparation but is not appreciably affected by the season.

Recombinant follicle stimulating hormone (rFSH) has been in use in the management of infertility for the past decade. It is a virtually pure compound, with excellent batch-to-batch consistency and the ability to be produced in almost limitless quantities. A historical perspective leading to the development of recombinant products will be discussed, including a review of the two-cell, two-gonadotrophin theory of folliculogenesis. The two currently available recombinant follicle stimulating hormones (follitropin α and follitropin β) will be compared with each other and with urinary follicle stimulating hormone (uFSH) preparations. This comparison will highlight their safety, efficacy and side effect profiles. The use of recombinant follicle stimulating hormone in the special cases of polycystic ovarian syndrome and hypothalamic amenorrhea will be detailed. Finally, future directions in recombinant therapy will be considered, including the creation of long-acting preparations, the development of recombinant luteinizing hormone, and the potential future availability of more convenient administration systems.

Follitropin receptor (FSHR) in testicular Sertoli cells mediates signaling by pituitary follitropin (FSH) promoting intercellular communication with germ cells for normal spermatogenesis. Using receptor knockout mice we examined changes in sperm nucleoproteins and chromatin architecture. The expressions of transition proteins 1/2 (TP1/2) and protamine-2 (PRM-2) were greatly diminished at 21 days, but returned to normal at 35 days and 3 months after birth. However, protein components in chromatin were quite different. Western blots detected a reduction in PRM1/2 and prolonged retention of mono-ubiquitinated histone 2A (uH2A) in the epididymal sperm from adult mutants. Two forms of mono- and poly-uH2A were present in sonication-resistant testicular spermatids in normal mice, whereas only an elevated mono-uH2A was detectable in mutants. Decrease in PRM1/2 and retention of mono-uH2A was coincident with reduction in TP1/2 in premature spermatids. Thus lack of FSHR signaling impairs expression of TP1/2 and PRM-2 at an early stage of post-natal development causing delayed spermatogenesis. In the adult, absence of FSHR signaling prolongs retention of mono-uH2A, leading to impair transition of basic nucleoproteins and chromatin remodeling during mouse spermatogenesis.

Purpose: The aim of the current study was to evaluate potential relationships between serum adropin levels and metabolic parameters in polycystic ovary syndrome (PCOS). Materials and Methods: Twenty women with PCOS and 20 healthy, age and body mass index (BMI) matched controls were included in the study. All subjects underwent venous blood drawing on the early follicular phase after an overnight fasting. Serum adropin levels were measured with enzyme immunosorbent assay (EIA). The relationships between serum adropin levels and metabolic parameters were also assessed. Results: Serum adropin levels were found to be significantly lower in women with PCOS when compared to control group (p < 0.001). Serum adropin level was correlated negatively with fasting serum insulin levels, homeostasis model of assessment - insulin resistance (HOMA-IR) and serum lipid markers including cholesterol, very low-density lipoprotein, and triglycerides (TG) in PCOS patients (p < 0.05). Conclusion: The findings of current study suggest that women with PCOS have low serum adropin levels that may contribute to the underlying pathogenesis of metabolic disturbances in PCOS.

Purpose: To compare the efficiency and efficacy of two recombinant human FSH (r-FSH) and urinary (u-FSH) preparations in patients undergoing superovulation for IVF-ET using a short-term gonadotropin releasing hormone agonist (GnRH-a) (Triptorelin) protocol. Methods: A total of 88 women undergoing IVF-ET were included in this prospective study. They were randomized to receive u-FSH (150 IU/d), follitropin-α (100 IU/d), or follitropin-β (100 IU/d) for 2 days, and dosages were subsequently adjusted according to the ovarian response. Results: The FSH dose required for the overall stimulation was significantly lower in patients treated with r-FSH than in those treated with u-FSH while serum FSH values were higher in the latter group. There were no statistically significant differences in ovarian response and IVF outcome between r-FSH preparations. Conclusions: Recombinant FSH preparations have a higher efficiency than urinary ones in patients undergoing IVF-ET using a short-term GnRH-a protocol. In this situation, the two recombinant follitropins have comparable effectiveness.

Human reproduction requires specific interactions between follitropin (hFSH) and its receptor (FSHR) and between lutropin (hLH) or choriogonadotropin (hCG) and the lutropin receptor (LHR). Substitution of hFSH residues between hCG β-subunit cysteines 11–12 creates a bifunctional analog that binds both receptors. To understand the basis of this observation, we used antibody probes to compare the conformations of bifunctional analogs before and after they were complexed with each receptor. Introduction of hFSH residues between cysteines 11–12 changed a distant conformation-sensitive region created by the juxtaposition of the subunit aminotermini. This region, found not to contact either receptor, was altered further when bifunctional ligands bound FSHR. All other surfaces, detected in LHR complexes, were also recognized in FSHR complexes, an indication that bifunctional ligands bind both receptors in similar orientations. These observations suggest that unlike hCG or hFSH, bifunctional gonadotropins can acquire ‘lutropin’ and ‘follitropin’ conformations, a phenomenon accentuated by receptor contacts.

Bioassays constitute a unique approach to determine the functional aspects of gonadotropins. Indeed, these highly complex glycoprotein hormones, including pituitary lutropin (LH) and follitropin (FSH), are heterogeneous in terms of both peptidic and carbohydrate moieties, and, as a consequence, the bioactivity of the different molecular forms often does not match their immunoreactivity. In this article, we review the different types of LH and FSH bioassays. Conventional methods for measuring FSH bioactivity are first described and include the in vivo Steelman and Pohley bioassay, the radioligand receptor assays (RRAs), the in vitro Sertoli cell bioassay, the in vitro granulosa cell bioassay, and the inhibin immunoassay. Recent methods based on cell lines transfected with cloned receptors, particularly the human FSH receptor, are then described. Methods for developing these assays are presented, and the advantages and disadvantages of the different bioassays are discussed.

This study investigates whether chicken lutropin (LH) specifically binds to rat ovarian follitropin (FSH) receptor and exerts FSH-like bioactivity. Glycoprotein fraction, prepared from the chicken anterior pituitary gland, was fractionated using isoelectric focusing within a pH range of 3.5-11. Analysis of the focused fractions, by a radioreceptor assay (RRA) specific for FSH in rats using rat ovarian homogenate as receptor source, and 125I-labeled rat FSH as radioligand, detected a large component having an isoelectric point of 10.25. This focusing profile obtained by RRA was quite similar to that obtained by a specific radioimmunoassay (RIA) for chicken LH, but clearly different from that obtained by a specific RIA for chicken FSH, indicating this RRA specifically recognizes chicken LH. Chicken LH fraction prepared from the electrofocused material was used for further studies. The chicken LH preparation was three times more potent than rat FSH in the RRA in displacing the radioligand bound to rat ovarian receptor, while chicken LH facilitated an 8-fold less production of estradiol in dispersed rat granulosa cells than rat FSH. These results suggest that chicken LH acts like rat FSH in rat ovarian FSH receptor, but receptor-binding activity is much higher than biological activity.

Information on biological day-to-day variation is needed for detecting within-subject changes over time. In this study the daytime changes and the biological day-to-day variation of serum testosterone, follitropin, lutropin and oestradiol-17β concentrations were investigated in 31 healthy males. To analyse daytime changes, blood specimens were taken at 0800 h, 1200 h, 1600 h and 2000 h during one day (n=31) and two days (n=8). The day-to-day variation was analysed from blood specimens collected at 0800 h on days 1 and 2 (n=31) and additionally on days 3, 4, 6 and 9 (n=8). The evaluation of the day-to-day variation was based on calculations of the within-subject (CVA+I) and between-subject (CVG) coefficients of variation. When the within-subject day-to-day variances were not too heterogeneous, they were used for the calculation of 95 % reference change limits. Serum testosterone and oestradiol-17βconcentrations showed a significant daytime variation; testosterone had higher serum concentrations at 0800 and 1200 h. A peak in the serum concentration of oestradiol-17β occurred at 1200 h with a decrease towards the evening. There were no clear daytime changes in the serum concentrations of follitropin or lutropin. For different analytes the reference change limits were: serum testosterone ± 32.0 %, serum follitropin ± 24.1 % and serum oestradiol-17β ± 38.3 %. The reference change limit was not calculated for serum lutropin, as a high degree of heterogeneity and individuality was found. The interpretation of the results of hormone measurements requires recognition of the biological daytime and day-to-day changes of hormones. The reference change limits determine what changes are significant when monitoring the patient.

Recombinant gonadotrophins, in particular recombinant follitropin (follicle-stimulating hormone; FSH), are now available for ovarian hyperstimulation for assisted reproduction. In contrast with urinary FSH (urofollitropin), follitropin is available in virtually unlimited quantities. Follitropin is as potent as urofollitropin in all protocols of ovarian stimulation. Furthermore, it shows an improved purity without contamination by urinary proteins not related to FSH, and can be injected subcutaneously by the patients themselves. In patients with complete luteinising hormone (LH) deficiency, follitropin stimulates follicular development, although serum levels of estradiol remain low. For this group of patients the addition of LH is necessary. Ongoing phase III studies on the use of recombinant LH in this indication will provide an answer to how much LH is needed in order to guarantee sufficient follicular growth and hormonal response. Gonadorelin (gonadotrophin releasing hormone; GnRH) analogues are used to avoid the surge of endogenous LH in ovarian stimulation protocols. Gonadorelin antagonists are now in clinical testing for the same indication. Gonadorelin antagonists allow sufficient suppression of endogenous LH levels. In contrast with gonadorelin analogues, they avoid any flare-up effect, i.e. an initial release of gonadotrophins from pituitary reservoirs. Recombinant gonadotrophins and gonadorelin antagonists are new tools towards a more individual approach to ovarian stimulation.

9 pages, 7 figures The ability of TGF-α to regulate insulin-like growth factor binding protein-4 (IGFBP-4), was investigated. Primary cultures of rat granulosa cells (GC) were grown in serum-free medium with rat (r) TGF-α and/or rFSH, and secreted IGFBP-4 protein and its steady state mRNA levels were measured by Western immunoblotting and Northern blotting, respectively. Control (untreated) cells secreted IGFBP-4 spontaneously, and the levels were increased by rTGF-α in a dose- and time-dependent manner. rTGF-α abolished FSH-induced IGFBP-4 protease activity and suppressed FSH-dependent effects on IGFBP-4 production. IGFBP-4 mRNA levels were decreased and increased by FSH and TGF-α, respectively, and TGF-α blocked the FSH effects. These results demonstrate that TGF-α is a potent stimulator of IGFBP-4 expression in rat GC and can overcome the regulatory effects of FSH on IGFBP-4 production. The consequence of these TGF-α effects is a marked, sustained increase in the levels of IGFBP-4 in the microenvironment This work was supported by NICHD Grants HD-29008 and HD-30308. F.P. was supported by a NATO postdoctoral fellowship

This study was designed to determine serum Fetuin-A levels and establish whether serum Fetuin-A level is related with insulin resistance, oxidative stress, ovarian hyperandrogenism and dyslipidemia in women with polycystic ovary syndrome (PCOS). Twenty-two patients with PCOS and twenty-one healthy control women were evaluated in this controlled clinical study. Serum Fetuin-A, lipid fractions, glucose, insulin, malondialdehyde (MDA), myeloperoxidase (MPO), glutathione (GSH), superoxide dismutase (SOD) and other hormone (gonadotropins, androgens) levels were measured. The estimate of insulin resistance was calculated by homeostasis model assessment (HOMA-R). The women with PCOS had significantly higher serum fasting glucose, insulin, luteinizing hormone (LH), MDA, Fetuin-A levels, and LH/follicle-stimulating hormone (FSH) ratio, free androgen index (FAI), HOMA-IR than healthy women. However, sex hormone-binding globulin (SHBG) and GSH levels were significantly lower in patients with PCOS compared with controls. Fetuin-A was positively correlated with insulin, HOMA-IR and FAI. Multiple regression analysis revealed that FAI was strong predictor of serum Fetuin-A level. Serum Fetuin-A level was related with insulin resistance and ovarian hyperandrogenism in women with PCOS. These results suggest that Fetuin-A may have a role in triggering the processes leading to insulin resistance and androgen excess in PCOS. © 2013 Informa UK Ltd.

The ability of human chorionic gonadotropin (hCG) to distinguish lutropin (LHR) and follitropin (FSHR) receptors is controlled principally by β-subunit residues 94–117. To learn how residues 94–96 (Arg-Arg-Ser) influence LHR binding, we studied the effects of replacing them on the LH and FSH activities of a bifunctional hCG analog in which residues 101–109 were derived from FSH. Analogs containing 1–3 arginines and no aspartates at residues 94–96 bound LHR with 25–400% the potency of hCG. When residues 94–96 were neutral or contained 1–3 aspartates, LHR binding was reduced 6–100 fold but remained at least ten-fold greater than the negative control analog containing residues 94–117 derived from FSH. Residues 94–96 had little influence on FSHR binding. These observations support a model [Moyle et al. (1995) J. Biol. Chem. 270:20 020] in which residues 94–96 influence LHR binding specificity primarily through an effect on hormone conformation rather than by direct participation in essential high affinity receptor contacts.

The circadian rhythmic variations of the serum concentrations of follitropin, lutropin, sex-hormone-binding globulin and testosterone, the ratio between the serum concentrations of testosterone and sex-hormone-binding globulin, and the salivary concentration of testosterone were investigated in a group of 13 apparently healthy men. Venous blood and salivary specimens were collected at 4-h intervals over a 24-h period. The circadian rhythms were studied by using a periodic function resulting from the sum of two cosine functions with periods of 24 and 12 h. The serum concentrations of follitropin and lutropin showed no significant rhythmic variations. For the salivary concentration of testosterone and for the ratio between the serum concentrations of testosterone and sex-hormone-binding globulin, only the cosine function with a period of 24 h was significant. Serum concentrations of sex-hormone binding globulin and testosterone were significantly affected by 24- and 12-h rhythmic components. Of the quantities studied, the salivary concentration of testosterone showed the greatest daily rhythmic variation (28.8% of the mean estimated over rhythm).

Human chorionic gonadotropin (hCG) contains a beta-subunit N-terminal amino acid extension that contacts the alpha-subunit and is needed for efficient alpha and hCG beta-subunit combination. Here we report that an hCG beta-subunit analog, lacking residues 2-8, combined with the alpha-subunit more efficiently when positively charged residues between beta-subunit cysteines 10 and 11 were replaced with negatively charged residues found in the corresponding portion of follitropin. Residues 2-8 had no influence on binding of hCG to lutropin receptors. Positive charges between cysteines 10 and 11 are essential for high affinity binding of lutropins to their receptors. Therefore, the N-terminal extension found in all lutropin beta-subunits appears to have evolved to offset the inhibition of subunit combination by beta-subunit residues that are essential for lutropin activity. This beta-subunit extension is not found in follitropins or thyrotropins, hormones that have negatively charged residues between cysteines 10 and 11.

Theriogenology. 2012 Mar 15;77(5):967-78. Epub 2011 Dec 6. Effects of gonadotropin-releasing hormone administration on the pituitary-gonadal axis in male and female dogs before and after gonadectomy. de Gier J, Buijtels JJ, Albers-Wolthers CH, Oei CH, Kooistra HS, Okkens AC. Source Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. j.degier@uu.nl Abstract GnRH-stimulation tests were performed in 14 female and 14 male client-owned dogs of several breeds, before and 4 to 5 mo after gonadectomy. The aim of the study was to obtain more insight into the pituitary-gonadal axis in intact and neutered dogs and to establish reference values. Basal plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were increased significantly after gonadectomy in both bitches and male dogs. In both males and females ranges of the basal plasma FSH concentrations, before and after gonadectomy, did not overlap as opposed to the overlap in ranges of the basal plasma LH concentrations. Before gonadectomy basal plasma LH concentrations were lower and basal plasma FSH concentrations were higher in bitches than in male dogs. After gonadectomy these basal values did not differ significantly. GnRH administration before gonadectomy resulted in an increase in plasma LH and FSH concentrations in both genders. GnRH administration after gonadectomy produced an increase only in plasma LH concentrations in both genders, and a just significant increase in plasma FSH in castrated male dogs. GnRH administration before gonadectomy resulted in a significant increase in plasma testosterone concentration in both genders. In males ranges of basal and GnRH-stimulated plasma testosterone concentrations before and after gonadectomy did not overlap. Basal plasma estradiol concentrations were significantly higher in intact males than in castrated males and their ranges did not overlap. The basal estradiol concentrations in bitches before and after ovariectomy were not significantly different. At 120 min after GnRH administration, ranges of plasma estradiol concentration of intact and ovariectomized bitches no longer overlapped. In conclusion, basal plasma FSH concentration appears to be more reliable than basal plasma LH concentration for verification of neuter status in both male and female dogs. The basal plasma testosterone concentration appears to be reliable for verification of neuter status in male dogs. The plasma estradiol concentration at 120 min after GnRH administration can be used to discriminate between bitches with and without functional ovarian tissue. Copyright © 2012 Elsevier Inc. All rights reserved.

BACKGROUND Glucose-induced kinetics of bone marrow-derived stem cells in healthy females is presently unknown. The objectives of this study were to determine whether circulating levels of CD133(+), CD34(+) and CD133(+)CD34(+) cells increase in response to glucose load in healthy females and whether the kinetics is altered in amenorrhoeic women. The other objective of the work was to compare the endothelial differentiation potential of peripheral blood-derived endothelial progenitor cells (EPCs) from healthy versus amenorrhoeic women. METHODS In this case-control study, 44 amenorrhoeic subjects and 36 age-matched females with no menstrual disturbance were recruited at Apollo Hospitals, a Tertiary health care center in Chennai, India. Circulating bone marrow-derived stem cells were measured by two color direct flow cytometry. Cultured progenitor cells were characterized at Day 7 and 14 for expression of endothelial markers and production of nitric oxide (NO) via immunofluoroscence. RESULTS The amenorrhoeic subjects were insulin resistant with homeostatic model of assessment of insulin resistance values of 3.33 ± 0.3 versus 1.75 ± 0.148 observed for controls (P< 0.0001). Among the amenorrhoeic subjects, 38 subjects had polycystic ovaries with no signs of hyperandrogenism. Fasting levels of CD133(+), CD34(+) and CD133(+)CD34(+) cells were reduced in amenorrhoeic subjects (P< 0.001). There was a 1.5 to 2-fold increase in the circulating levels of these cells in response to 75 g oral glucose challenge at 1 and 2 h post-load conditions in controls, which was significantly blunted for CD133(+) (P< 0.001) and CD133(+)CD34(+) (P< 0.001) cells in amenorrhoeic subjects. A positive correlation was observed between estrogen and fasting CD133(+) (r= 0.205, P= 0.070), CD34(+) (r= 0.249, P= 0.027) and CD133(+)CD34(+) (r= 0.217, P= 0.055) cell counts. Additionally, fasting counts for CD34(+) and CD133(+)CD34(+) cells positively correlated with FSH and inversely correlated with LH and C-peptide in the polycystic group. Cultured cells from polycystic subjects exhibited reduced adherence to fibronectin and expressed lower levels of endothelial nitric-oxide synthase and NO. CONCLUSIONS Oral glucose-induced increase in circulating numbers of CD133(+) and CD133(+)CD34(+) cells and endothelial differentiation potential of peripheral blood-derived EPCs is attenuated in insulin resistant amenorrhoeic subjects.

The aim of this study was to investigate the relationship between insulin resistance and thyroid function in obese pre- and postmenopausal women with or without metabolic syndrome (MetS). 141 obese women were divided into two groups, HOMA-IR2.7, to evaluate relation with HOMA-IR and fatness, hormone and blood parameters. They were then divided into four groups as pre- and postmenopausal with or without MetS. Various fatness, hormone and blood parameters were examined. Statistically significant difference was found in weight, body mass index (BMI), waist circumference, fat%, fasting insulin, TSH, FT3, FT4, FSH, Anti-microsomal antibody (ANTIM) and triglycerides levels in HOMA-IR2.7 obese Turkish women. This study showed that age, weight, BMI, waist circumference, fat%, fasting insulin, FT3, ANTIM, FSH, LH, total cholesterol, triglycerides, HDL, HOMA-IR, systolic and diastolic blood pressure levels were related in preand post menopausal status in obese women with or without MetS. Obesity may influence the levels of thyroid hormones and increases the risk of MetS in women. Postmenopausal status with MetS is associated with an increased TSH, FT3 and FT4 levels and HOMA-IR in obese women. Strong relation was observed with MetS and TSH and FT3 levels. ©2012, Editrice Kurtis.

The family of pituitary/placental glycoprotein hormones includes follicle-stimulating hormone (follitropin, FSH), luteinizing hormone (lutropin, LH), chorionic gonadotropin (choriogonadotropin, CG), and thyroid-stimulating hormone (thyrotropin, TSH). These glycoproteins are heterodimeric, with an alpha-subunit of identical primary structure and a hormone-specific beta-subunit which is believed to confer receptor specificity. Previous studies demonstrated that substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta, residues 94-145, conferred to human (h) CG an FSH receptor binding specificity. To more finely map the LH/FSH receptor binding specificity determinant, hFSH beta residues 88DSDS91, within the small hydrophilic intercysteine loop, and residues 95TVRGLG100 COOH-terminal to the loop were switched to hLH beta residues 94RRST97 and residues 101GGPKDH106, respectively. Substitution of hLH beta residues 94RRST97 in place of hFSH beta residues 88DSDS91 did not affect FSH receptor binding or activation but, remarkably, conferred LH receptor binding activity to the chimera. This chimera retained the ability to stimulate progesterone production in a Yl cell line which expresses human FSH receptor. Replacing hFSH beta residues 95TVRGLG100 with hLH beta residues 101GGPKDH106 diminished FSH receptor binding activity but did not confer LH receptor binding to the chimera. This study suggests that amino acids within hFSH beta residues 95TVRGLG100 are important for FSH binding specificity and that hLH beta residues 94RRST97 are involved in LH receptor binding specificity. Thus, although the hFSH beta and hLH beta subunits may fold similarly, the loci of receptor binding specificity are not entirely homologous.

Myasthenia gravis (MG) is an autoimmune disorder characterized by autoantibodies against acetylcholine receptors. MG is generally an isolated disorder but may occur concomitantly with other autoimmune diseases. We describe an eighteen-year-old girl with MG who was admitted to our clinic with secondary amenorrhea and diagnosed as autoimmune oophoritis. Since her myasthenic symptoms did not resolve with anticholinesterase therapy, thymectomy was performed. After thymectomy, her menses have been regular without any hormonal replacement therapy. To our knowledge, this is the first report on a patient with autoimmune ovarian insufficiency and MG in whom premature ovarian insufficiency resolved after thymectomy, without hormonal therapy. Conflict of interest:None declared.

Congenital hypogonadotropic hypogonadism (CHH) causes pubertal failure and infertility in both women and men due to partial or total secretory failure of the two pituitary gonadotropins lutropin (LH) and follitropin (FSH) during periods of physiological activation of the gonadotropic axis. Men and women with CHH frequently seek treatment for infertility after hypogonadism therapy. Some etiologies, such as autosomal dominant or X-linked Kallmann syndrome, raise the question of hereditary transmission, leading to increasing demands for genetic counseling and monitoring of medically assisted pregnancies. Diagnosis and treatment of newborn boys is, therefore, becoming an increasingly important issue. In male individuals with complete forms of CHH, the antenatal and neonatal gonadotropin deficit leads to formation of a micropenis and cryptorchidism, which could undermine future sexual and reproductive functions. Standard treatments, usually started after the age of puberty, often only partially correct the genital abnormalities and spermatogenesis. The aim of this Review is to examine the possible additional benefits of neonatal gonadotropin therapy in male patients with CHH. Encouraging results of neonatal therapy, together with a few reports of prepubertal treatment, support the use of this novel therapeutic strategy aimed at improving sexual and reproductive functions in adulthood.