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Manuela Simoni and Livio Casarini

Objective

To assess the pharmacogenetic potential of FSH for infertility treatment.

Design

Review of the literature and genomic databases.

Methods

Single-nucleotide polymorphism (SNP) assessed: rs6166 (c.2039A>G, p.N680S), rs6165 (c.919A>G, p.T307A), rs1394205 (c.−29G>A) in FSHR, and rs10835638 (c.−211G>T) in FSHB. Literature search via PubMed. Blast analysis of genomic information available in the NCBI nucleotide database. Comparison of allele frequency and haplotype distribution using the http://spsmart.cesga.estool.

Results

All these SNPs appear first in Homo, result in reduced FSH action, and are present with variable frequencies and combinations worldwide. Stringent clinical studies demonstrate that the FSHR genotype influences serum FSH levels and gonadal response in both sexes. Serum FSH levels depend on the −211G>T SNP, influencing transcriptional activity of the FSHB promoter. Genotypes reducing FSH action are overrepresented in infertile subjects.

Conclusions

Although the clinical relevance of the FSHR polymorphisms alone is limited, the combination of FSHR and FSHB genotypes has a much stronger impact than either one alone in both sexes. About 20% of people are carriers of the alleles associated with lower serum FSH levels/reduced FSHR expression or activity, possibly less favorable for reproduction. Prospective studies need to investigate whether stratification of infertile patients according to their FSHR–FSHB genotypes improves clinical efficacy of FSH treatment compared with the current, naïve approach. A relative enrichment of less favorable FSHR FSHB genotypes may be related to changes in human reproductive strategies and be a marker of some health-related advantage at the cost of reduced fertility.

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Manuela Simoni, Friedrich Jockenhövel and Eberhard Nieschlag

The new international standard for FSH, IS 83/575, has been analyzed, after isoelectric focusing separation, by Sertoli cell in vitro bioassay, radioligand receptor assay and two highly specific immunometric assays. Its molecular composition was then compared with the isoelectric focusing profiles obtained from the fractionation of the reference preparation 2nd IRP 78/549 and from pools of human male and female pituitary extracts and male and female sera. The results showed that >80% of immunoreactive and bioactive FSH in the IS 83/575 has a pI value <4, while such very acidic material was represented much less in the other FSH preparations tested. All the immunoreactive material contained in the IS 83/575 was shown to be capable of receptor binding and bioactivity in vitro. A generally good correspondence between IEF profiles obtained by bioassay and by immunofluorimetric assay was evident in the case of IS 83/575, 2nd IRP 78/549 and pituitary extracts, although the profiles recorded by immunofluorimetric assay were rather smooth and more isoforms were detected by bioassay. A striking discrepancy between immunoreactive FSH and bioactive FSH was observed after isoelectric focusing fractionation of the serum pools, in which some bioactive material was not detected by immunofluorimetric assay and some of the immunoreactive FSH peaks were devoid of bioactivity, indicating that serum contains inhibitors of FSH action and that immunometric assays based on monoclonal antibodies may miss some bioactive FSH isoforms. Taken together, these results suggest that the IS 83/575 is not fully representative of pituitary and serum FSH, and its use for calibration of modern immunometric methods based on monoclonal antibodies is unlikely to resolve current problems of inaccuracy in measurements of serum FSH.

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Aris Junaidi, C Marc Luetjens, Joachim Wistuba, Axel Kamischke, Ching-Hei Yeung, Manuela Simoni and Eberhard Nieschlag

Objective: Norethisterone enanthate (NETE) is evaluated in trials of hormonal male contraception. It has been speculated that progestins may exert their contraceptive effects not only by suppressing gonadotropins but also by direct effects on male organs. NETE was given to monkeys in which endogenous gonadotropin secretion was suppressed by a gonadotropin releasing hormone (GnRH) antagonist, and replaced by human follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). If NETE has a direct effect on spermatogenesis and/or epididymal function, some changes in testicular histology, sperm motility and/or morphology should occur soon after exposure to NETE.

Methods: Fifteen adult intact male monkeys were grouped and treated for a 38-day period. Group I received GnRH antagonist, FSH, hCG and NETE while group II received a regime identical to group I without NETE and group III received only NETE and vehicle. Ejaculates, body weight, testicular biopsies and volume, and hormones were evaluated.

Results: There was a similar pattern of serum FSH and testosterone in groups I and II. Testicular volume and the proportion of tubuli exhibiting spermatids was significantly decreased in group III. There were no significant differences between group I and group II in any parameters measured. The forward progression of sperm was not affected by NETE treatment. The consistently low percentages of grade c sperm indicated no sign of hyperactivation. No changes in the gross morphology of the acrosome were detected.

Conclusions: Short-term NETE treatment has neither a direct effect on the testis nor on the epididymis in this nonhuman primate model and its contraceptive effects appear to be exerted exclusively through gonadotropin suppression.

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Manuela Simoni, Jörg Peters, Hermann M Behre, Sabine Kliesch, Eckhard Leifke and Eberhard Nieschlag

Simoni M, Peters J, Behre HM, Kliesch S, Leifke E, Nieschlag E. Effects of gonadotropin-releasing hormone on bioactivity of follicle-stimulating hormone (FSH) and microstructure of FSH. luteinizing hormone and sex hormone-binding globulin in a testosterone-based contraceptive trial: evaluation of responders and non-responders. Eur J Endocrinol 1996;135:433–9. ISSN 0804–4643

Only a proportion of normal men participating in testosterone-based contraceptive trials develop azoospermia (responders). This study analyzed whether serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and sex hormone-binding globulin (SHBG) are qualitatively different between responders and non-responders. Determination of in vitro bioactive FSH after stimulation with gonadotropin-releasing hormone (GnRH) and analysis of molecular heterogeneity of serum FSH. LH and SHBG was carried out by chromatofocusing and concanavalin-A affinity chromatography in eight men who had participated in a previous contraceptive study with testosterone buciclate. Blood was withdrawn at 15-min intervals on two basal occasions and 30, 45 and 60 min after iv administration of GnRH (100 μg). Pools of sera were separated by chromatofocusing in the pH range 3–6 and by lectin chromatography on concanavalin A. Immunoreactive FSH, LH and SHBG were assayed in the eluates. Bioactive FSH was analyzed by the rat Sertoli cell bioassay. Serum bioactive FSH increased after GnRH stimulation, without significant differences between responders and non-responders. The chromatofocusing profiles of serum FSH showed a significant shift towards the less acidic region after GnRH. The isoform distribution was similar in responders and non-responders. No significant differences were found in the relative proportion of FSH, LH and SHBG retained by concanavalin A. It is concluded that the extent of suppression of sperm production by androgen administration cannot be foreseen either on the basis of the response of bioactive FSH to GnRH administration or from the glycosylation pattern of serum FSH, LH and SHBG.

E Nieschlag, Institute of Reproductive Medicine of the University, Domagkstr. 11, D-48129 Münster, Germany

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Fabio Lanfranco, Jörg Gromoll, Sigrid von Eckardstein, Eva M Herding, Eberhard Nieschlag and Manuela Simoni

Objective: To determine the frequency of mutations of the gonadotropin-releasing hormone receptor (GnRHR) and of the G protein-coupled receptor 54 (GPR54) genes in normosmic idiopathic hypogonadotropic hypogonadism (IHH).

Methods: In a retrospective study we analyzed the GnRHR and the GPR54 genes of 45 IHH patients and 50 controls. Genomic DNA was amplified by PCR to obtain partially overlapping amplicons encompassing the exon–intron boundaries of the GnRHR and GPR54 genes and analyzed by single-stranded conformation polymorphism gel electrophoresis and/or DNA sequencing.

Results: One heterozygous R262Q mutation of the GnRHR gene was identified in one patient with familial IHH. The silent single-nucleotide polymorphism (SNP) 453C > T occurred at the same frequency in patients and controls. One patient with sporadic IHH and consanguineous parents showed a novel homozygous sequence variation of the GPR54 gene (1001_1002insC) resulting in an open reading frame shift and elongation of 43 amino acids with an increased number of proline residues in the intracellular receptor domain. This patient had delayed puberty, low testosterone (3.4 nmol/l), and low-normal LH and FSH levels responsive to GnRH. Pulsatile GnRH administration normalized testosterone levels and induced spermatogenesis sufficiently to induce a pregnancy with assisted reproduction. Two common SNPs in exon 1 and exon 5 of the GPR54 gene showed similar frequency distribution and hormonal profiles in IHH and controls.

Conclusions: Mutations of the GnRHR and of the GPR54 gene are rare in IHH and should be investigated especially in cases with autosomal recessive transmission. Common SNPs of the GnRHR and GPR54 genes do not play any role in IHH.

Free access

Daniele Santi, Elisa Giannetta, Andrea M Isidori, Cristiana Vitale, Antonio Aversa and Manuela Simoni

Objective

Diabetes mellitus (DM) is associated with endothelial dysfunction, reducing nitric oxide-dependent vasodilation, and increasing production of pro-inflammatory factors, leading to an increased risk of long-term cardiovascular disease. As the effects of phosphodiesterase 5 inhibitors (PDE5i) on endothelial function have not been systematically investigated, we conducted a meta-analysis of available randomized clinical trials (RCTs).

Design

A thorough search of the literature was carried out. Relevant studies were considered according to RCT study design, enrollment of men with type 2 DM, chronic administration of PDE5i, and evaluation of endothelial function through both hemodynamic and endothelial inflammation-related parameters.

Results

Fifteen studies fulfilled the eligibility criteria but only six RCTs met the inclusion criteria and were analyzed for 476 diabetic men, 239 randomized to Sildenafil, and 237 to placebo respectively. Four RCTs evaluated flow-mediated dilation (FMD), demonstrating a weighted mean increase of 2.19% (95% CI 0.48 to 3.90). This result showed a high heterogeneity (I 2: 98%). Thus, a further sub-group meta-analysis was performed and this analysis confirmed a significant, Sildenafil-related FMD improvement. Sildenafil improved endothelin 1 and high sensitivity C-reactive protein by ∼−0.94 pg/ml and −0.36 mg/l, respectively, not reaching statistical significance (P=0.69 and P=0.22 respectively). Finally, Sildenafil administration significantly reduced serum levels of interleukin 6 (IL6, −0.82 pg/ml; 95% CI −1.58 to −0.07).

Conclusion

This meta-analysis suggests a beneficial effect of chronic PDE5i administration on endothelial function. Chronic Sildenafil administration seems to improve hemodynamic (FMD) and serum pro-inflammatory makers (IL6) in diabetic men. Larger studies are needed to confirm the effects of chronic PDE5i on endothelial function.

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Ramesh K. Chandolia, Gerhard F. Weinbauer, Manuela Simoni, Hermann M. Behre and Eberhard Nieschlag

Abstract.

The effects of chronic blockade of androgen action by the antiandrogens flutamide and Casodex on serum and pituitary concentrations of LH and FSH, serum and testicular androgen levels, reproductive organ weights, and on spermatogenesis were compared in the adult rat. Animals were treated for 3 and 8 weeks with vehicle, Casodex (20 mg · kg−1 · (day)−1, flutamide (20 mg · kg−1 · (day)−1) and GnRH antagonist (150 μg/day, Detirelix). Treatment with GnRH antagonist suppressed gonadotropin and testosterone production, reduced the weights of testes, epididymides and seminal vesicles, and inhibited germ cell development. Flutamide administration markedly elevated serum and pituitary levels of gonadotropins as well as serum and testicular androgen concentrations. Casodex-induced elevation of gonadotropin concentrations was less pronounced and serum and testicular levels of androgens did not change significantly. The reduction of seminal vesicle weights was similar after Casodex and GnRH antagonist treatment, whereas flutamide was less effective. Testicular weight and spermatogenesis (assessed by light microscopical and flow-cytometric analysis) remained unaffected by Casodex and flutamide. It is concluded, that 1. Casodex, in contrast to flutamide, is a peripherally selective antiandrogen, and 2. Casodex influences release of gonadotropins into circulation less than flutamide. Therefore this antiandrogen might be useful clinically for selectively blocking androgen actions in the accessory sex glands.

Free access

Daniele Santi, Antonio R M Granata, Alessandro Guidi, Elisa Pignatti, Tommaso Trenti, Laura Roli, Roberto Bozic, Stefano Zaza, Chiara Pacchioni, Stefania Romano, Jerzy Roch Nofer, Vincenzo Rochira, Cesare Carani and Manuela Simoni

Objective

Type 2 diabetes mellitus (T2DM) is associated with endothelial dysfunction, characterized by a reduction of nitric oxide (NO)-mediated relaxation. Phosphodiesterase type 5 inhibitors (PDE5i) improve NO levels. The aim of the study was to investigate whether long-term, chronic treatment with the PDE5i vardenafil improves systemic endothelial function in diabetic men.

Design

A prospective, investigator-initiated, randomized, placebo-controlled, double-blind, clinical trial was conducted.

Methods

In total, 54 male patients affected by T2DM, diagnosed within the last 5 years, and erectile dysfunction were enrolled, regardless of testosterone levels. In all, 26 and 28 patients were assigned to verum and placebo groups respectively. The study consisted of an enrollment phase, a treatment phase (24 weeks) (vardenafil/placebo 10 mg twice in a day) and a follow-up phase (24 weeks). Parameters evaluated were as follows: International Index of Erectile Function 15 (IIEF-15), flow-mediated dilation (FMD), serum interleukin 6 (IL6), endothelin 1 (ET-1), gonadotropins and testosterone (measured by liquid chromatography/tandem mass spectrometry).

Results

IIEF-15 erectile function improved during the treatment (P<0.001). At the end of the treatment both FMD (P=0.040) and IL6 (P=0.019) significantly improved. FMD correlated with serum testosterone levels (R 2=0.299; P<0.001). Testosterone increased significantly under vardenafil treatment and returned in the eugonadal range only in hypogonadal men (n=13), without changes in gonadotropins. Chronic vardenafil treatment did not result in relevant side effects.

Conclusion

This is the first double-blind, placebo-controlled clinical trial designed to evaluate the effects of chronic treatment of vardenafil on endothelial health-related parameters and sexual hormones in patients affected by a chronic disease. Chronically administered vardenafil is effective and improves endothelial parameters in T2DM patient. Moreover, chronic vardenafil therapy improves hypogonadism in diabetic, hypogonadal men.

Free access

Marco Bonomi, Valeria Vezzoli, Csilla Krausz, Fabiana Guizzardi, Silvia Vezzani, Manuela Simoni, Ivan Bassi, Paolo Duminuco, Natascia Di Iorgi, Claudia Giavoli, Alessandro Pizzocaro, Gianni Russo, Mirella Moro, Letizia Fatti, Alberto Ferlin, Laura Mazzanti, Maria Chiara Zatelli, Salvo Cannavò, Andrea M Isidori, Angela Ida Pincelli, Flavia Prodam, Antonio Mancini, Paolo Limone, Maria Laura Tanda, Rossella Gaudino, Mariacarolina Salerno, Pregnolato Francesca, Mohamad Maghnie, Mario Maggi, Luca Persani and Italian Network on Central Hypogonadism

Objective

Isolated hypogonadotropic hypogonadism (IHH) is a rare disorder with pubertal delay, normal (normoosmic-IHH, nIHH) or defective sense of smell (Kallmann syndrome, KS). Other reproductive and non-reproductive anomalies might be present although information on their frequency are scanty, particularly according to the age of presentation.

Design

Observational cohort study carried out between January 2008 and June 2016 within a national network of academic or general hospitals.

Methods

We performed a detailed phenotyping of 503 IHH patients with: (1) manifestations of hypogonadism with low sex steroid hormone and low/normal gonadotropins; (2) absence of expansive hypothalamic/pituitary lesions or multiple pituitary hormone defects. Cohort was divided on IHH onset (PPO, pre-pubertal onset or AO, adult onset) and olfactory function: PPO-nIHH (n = 275), KS (n = 184), AO-nIHH (n = 36) and AO-doIHH (AO-IHH with defective olfaction, n = 8).

Results

90% of patients were classified as PPO and 10% as AO. Typical midline and olfactory defects, bimanual synkinesis and familiarity for pubertal delay were also found among the AO-IHH. Mean age at diagnosis was significantly earlier and more frequently associated with congenital hypogonadism stigmata in patients with Kallmann’s syndrome (KS). Synkinesis, renal and male genital tract anomalies were enriched in KS. Overweight/obesity are significantly associated with AO-IHH rather than PPO-IHH.

Conclusions

Patients with KS are more prone to develop a severe and complex phenotype than nIHH. The presence of typical extra-gonadal defects and familiarity for PPO-IHH among the AO-IHH patients indicates a common predisposition with variable clinical expression. Overall, these findings improve the understanding of IHH and may have a positive impact on the management of patients and their families.