FSHB Genotype Identified as a Relevant Diagnostic Parameter Revealed by Cluster Analysis of Men With Idiopathic Infertility.

Introduction and Objectives: About 30-75% of infertile men are diagnosed with idiopathic infertility, thereby lacking major causative factors to explain their impaired fertility status. In this study, we used a large cohort of idiopathic infertile men to determine whether subgroups could be identified by an unbiased clustering approach and whether underlying etiologic factors could be delineated. Patients and Methods: From our in-house database Androbase®, we retrospectively selected patients (from 2008 to 2018) with idiopathic male infertility (azoo- to normozoospermia) who fit the following selection criteria: FSH ≥ 1 IU/l, testosterone ≥ 8 nmol/l, ejaculate volume ≥ 1.5 ml. Patients with genetic abnormalities or partners with female factors were excluded.For the identified study population (n=2742), we used common andrologic features (somatic, semen and hormonal parameters, including the FSHB c.-211G>T (rs10835638) single nucleotide polymorphism) for subsequent analyses. Cluster analyses were performed for the entire study population and for two sub-cohorts, which were separated by total sperm count (TSC) thresholds: Cohort A (TSC ≥ 1 mill/ejac; n=2422) and Cohort B (TSC < 1 mill/ejac; n=320). For clustering, the partitioning around medoids method was employed, and the quality was evaluated by average silhouette width. Results: The applied cluster approach for the whole study population yielded two separate clusters, which showed significantly different distributions in bi-testicular volume, FSH and FSHB genotype. Cluster 1 contained all men homozygous for G (wildtype) in FSHB c.-211G>T (100%), while Cluster 2 contained most patients carrying a T allele (>96.6%). In the analyses of sub-cohorts A/B, two clusters each were formed too. Again, the strongest segregation markers between the respective clusters were bi-testicular volume, FSH and FSHB c.-211G>T. Conclusion: With this first unbiased approach for revealing putative subgroups within a heterogenous group of idiopathic infertile men, we did indeed identify distinct patient clusters. Surprisingly, across all diverse phenotypes of infertility, the strongest segregation markers were FSHB c.-211G>T, FSH, and bi-testicular volume. Further, Cohorts A and B were significantly separated by FSHB genotype (wildtype vs. T-allele carriers), which supports the notion of a contributing genetic factor. Consequently, FSHB genotyping should be implemented as diagnostic routine in patients with idiopathic infertility.

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA.

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

Does the FSHB c.-211G>T polymorphism impact Sertoli cell number and the spermatogenic potential in infertile patients?

BACKGROUND: A genetic variant within the FSHB gene can deviate FSH action on spermatogenesis. The c.-211G>T FSHB single nucleotide polymorphism impacts FSHB transcription and biosynthesis due to interference with the LHX3 transcription factor binding. This SNP was previously shown to be strongly associated with lowered testicular volume, reduced sperm counts, and decreased FSH levels in patients carrying one or two T-alleles. OBJECTIVE: To determine the impact of the SNP FSHB c.-211G>T on Sertoli cell (SC) number, Sertoli cell workload (SCWL) and thereby spermatogenic potential. MATERIAL AND METHODS: Testicular biopsies of 31 azoospermic, homozygous T patients (26 non-obstructive azoospermia (NOA), and five obstructive azoospermia (OA)) were matched to patients with GG genotype. Marker proteins for SC (SOX9), spermatogonia (MAGE A4), and round spermatids (CREM) were used for semi-automatical quantification by immunofluorescence. SCWL (number of germ cells served by one SC) was determined and an unbiased clustering on the patient groups performed. RESULTS: Quantification of SC number in NOA patients did not yield significant differences when stratified by FSHB genotype. SC numbers are also not significantly different between FSHB genotypes for the OA patient group and between NOA and OA groups. SCWL in the NOA patient cohort is significantly reduced when compared to the OA control patients; however, in neither group an effect of the genotype could be observed. The cluster analysis of the whole study cohort yielded two groups only, namely NOA and OA, and no clustering according to the FSHB genotype. DISCUSSION AND CONCLUSION: The FSHB c.-211G>T polymorphism does not affect SC numbers or SCWL, thereby in principle maintaining the spermatogenic potential. The previously observed clinical phenotype for the FSHB genotype might therefore be caused by a hypo-stimulated spermatogenesis and not due to a decreased SC number.

De Novo Mutations in FOXJ1 Result in a Motile Ciliopathy with Hydrocephalus and Randomization of Left/Right Body Asymmetry.

Hydrocephalus is one of the most prevalent form of developmental central nervous system (CNS) malformations. Cerebrospinal fluid (CSF) flow depends on both heartbeat and body movement. Furthermore, it has been shown that CSF flow within and across brain ventricles depends on cilia motility of the ependymal cells lining the brain ventricles, which play a crucial role to maintain patency of the narrow sites of CSF passage during brain formation in mice. Using whole-exome and whole-genome sequencing, we identified an autosomal-dominant cause of a distinct motile ciliopathy related to defective ciliogenesis of the ependymal cilia in six individuals. Heterozygous de novo mutations in FOXJ1, which encodes a well-known member of the forkhead transcription factors important for ciliogenesis of motile cilia, cause a motile ciliopathy that is characterized by hydrocephalus internus, chronic destructive airway disease, and randomization of left/right body asymmetry. Mutant respiratory epithelial cells are unable to generate a fluid flow and exhibit a reduced number of cilia per cell, as documented by high-speed video microscopy (HVMA), transmission electron microscopy (TEM), and immunofluorescence analysis (IF). TEM and IF demonstrate mislocalized basal bodies. In line with this finding, the focal adhesion protein PTK2 displays aberrant localization in the cytoplasm of the mutant respiratory epithelial cells.