Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

Research question: Do patients presenting with flagella ultrastructural defects as assessed by electron microscopy, and defined within three phenotypes (dysplasia of the fibrous sheath [DFS], primary flagellar dyskinesia [PFD] and non-specific flagellar abnormalities [NSFA]), have decreased chances of success in intracytoplasmic sperm injection (ICSI) or adverse obstetric and neonatal outcomes?Design: Retrospective analysis of 189 ICSI cycles from 80 men with spermatozoa flagellum ultrastructural defects (DFS [n = 16]; PFD [n = 14]; NSFA [n = 50] compared with a control group (n = 97). Cycles were cumulatively analysed. All fresh and frozen embryo transfers resulting from each ICSI attempt were included. The effect of transmission electron microscopy (TEM) phenotype on the main ICSI outcomes was assessed by a multivariate logistic regression combined with a generalized linear mixed model to account for the non-independence of the observations.Results: No predictive value of TEM phenotype was found on the main outcomes of ICSI, namely fertilization rates, pregnancy and delivery rates, and cumulative pregnancy and delivery rates. Cumulative pregnancy rates ranged from 29.0-43.3% in the different TEM phenotype subgroups compared with 36.8% in the control group. Cumulative live birth rates ranged from 24.6-36.7% compared with 31.4% in the control group. No increase was found in miscarriages, preterm births, low birth weights or birth abnormalities.Conclusions: Data on the cumulative chances of success in ICSI of patients with ultrastructural flagellar defects, a rare cause of male infertility often associated with an underlying genetic cause, are reassuring, as are obstetrical and neonatal outcomes in this population.

AbstractBackgroundFew studies to date have attempted to measure serum anti-Müllerian hormone (AMH) levels in adult men, and solid references ranges have not yet been defined in a large cohort.ObjectiveIn this study, we aimed, first, to establish the reference ranges for serum AMH and AMH-to-total testosterone ratio (AMH/tT) in adult males. Second, we investigated the relationship between serum AMH and both reproductive hormones and semen parameters.MethodsThis single-center retrospective study included 578 normozoospermic adult men. Serum AMH concentrations were determined with an automated sandwich chemiluminescent immunoassay.ResultsThe median serum AMH was 43.5 pmol/L. The 2.5th and 97.5th percentile values for serum AMH and AMH/tT were 16.4 and 90.3 pmol/L and 0.45 and 3.43, respectively. AMH was positively correlated with inhibin B and sperm concentration and negatively correlated with age, follicle-stimulating hormone (FSH), and progressive sperm motility. Interestingly, using immunofluorescence, we documented for the first time that AMH type II receptor (AMH-R2) is expressed in ejaculated human spermatozoa and gonadotrophic cells in the postmortem pituitary gland.ConclusionsWe establish a new age-specific reference range for serum AMH and AMH/tT. Moreover, AMH-R2 expression in human spermatozoa and gonadotrophic cells, together with the relationship between serum AMH levels and sperm motility or mean FSH levels, highlight new potential functions of AMH in regulating sperm motility or FSH secretion in adult men.

Nous avons d’abord cherché à établir l’intervalle de référence pour les concentrations sériques d’AMH chez des hommes adultes. Deuxièmement, nous avons étudié la relation entre les concentrations sériques d’AMH et les gonadotrophines, la testostérone totale ainsi que les paramètres spermatiques. Au total, 578 hommes adultes normozoospermiques ont été inclus dans cette étude monocentrique. Pour les hommes âgés de 20 à 39 ans, les 2,5 e et 97,5 e percentiles des concentrations d’AMH étaient de 18,1–90,9 pmol/L, et pour ceux dont l’âge > 39 ans de 12,3–87,6 pmol/L. Les concentrations sériques d’AMH étaient positivement corrélées avec l’inhibine B et la concentration des spermatozoïdes, et négativement corrélées avec l’âge, la FSH et la mobilité progressive des spermatozoïdes. Ces corrélations persistent après ajustement sur l’âge, les concentrations d’inhibine B et/ou la FSH. En utilisant une approche par immunofluorescence, nous avons documenté pour la première fois que le récepteur d’AMH de type II (AMH-R2) est exprimé dans les spermatozoïdes humains et les cellules gonadotropes de l’hypophyse humaine. De nouveaux intervalles de référence des taux sériques d’AMH spécifiques à l’âge ont été établis. En outre, l’expression de l’AMH-R2 dans les spermatozoïdes et les cellules gonadotropes humaines, ainsi que les corrélations entre les taux sériques d’AMH et la mobilité des spermatozoïdes ou les taux de FSH, mettent la lumière sur de nouvelles potentielles fonctions de l’AMH dans la régulation de la motilité spermatique ainsi que la sécrétion de FSH chez les hommes adultes.

Testicular sperm extraction (TESE) is the method of choice for recovering spermatozoa in patients with azoospermia. However, the lack of reliable biomarkers makes it impossible to predict sperm retrieval outcomes at TESE. To date, little attention has been given to anti-Müllerian hormone (AMH) serum levels in adult men with altered spermatogenesis. In this study we aimed to investigate whether serum concentrations of AMH and the AMH to total testosterone ratio (AMH/T) might be predictive factors for sperm retrieval outcomes during TESE in a cohort of 155 adult Caucasian men with azoospermia. AMH serum levels were significantly lower in nonobstructive azoospermia (NOA) that was unexplained, cryptorchidism-related, cytotoxic and genetic (medians [pmol/l] = 30.1; 21.8; 26.7; 7.3; and p = 0.02; 0.001; 0.04; <0.0001, respectively]) compared with obstructive azoospermia (OA) (median = 44.8 pmol/l). Lowest values were observed in cases of genetic NOA (p < 0.0001, compared with unexplained NOA) and especially in individuals with non-mosaic Klinefelter syndrome (median = 2.3 pmol/l, p <0.0001). Medians of AMH/T values were significantly lower in genetic NOA compared to unexplained, cryptorchidism-related NOA as well as OA. Only serum concentrations of AMH differed significantly between positive and negative groups in men with non-mosaic Klinefelter syndrome. The optimal cut-off of serum AMH was set at 2.5 pmol/l. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of this cut-off to predict negative outcomes of SR were 100 %, 76.9 %, 66.6 %, 100 and 84.2 %, respectively. Serum AMH levels, but not AMH/T values, are a good marker for Sertoli and germ cell population dysfunction in adult Caucasian men with non-mosaic Klinefelter syndrome and could help us to predict negative outcomes of SR at TESE with 100 % sensitivity when serum levels of AMH are below 2.5 pmol/l.

Following publication of the original article [1], the authors reported that the author given names and family names had been transposed. The author names in this correction article are presented correctly.

Globozoospermia is a rare phenotype of primary male infertility inducing the production of round-headed spermatozoa without acrosome. Anomalies of DPY19L2 account for 50–70% of all cases and the entire deletion of the gene is by far the most frequent defect identified. Here, we present a large cohort of 69 patients with 20–100% of globozoospermia. Genetic analyses including multiplex ligation-dependent probe amplification, Sanger sequencing and whole-exome sequencing identified 25 subjects with a homozygous DPY19L2 deletion (36%) and 14 carrying other DPY19L2 defects (20%). Overall, 11 deleterious single-nucleotide variants were identified including eight novel and three already published mutations. Patients with a higher rate of round-headed spermatozoa were more often diagnosed and had a higher proportion of loss of function anomalies, highlighting a good genotype phenotype correlation. No gene defects were identified in patients carrying < 50% of globozoospermia while diagnosis efficiency rose to 77% for patients with > 50% of globozoospermia. In addition, results from whole-exome sequencing were scrutinized for 23 patients with a DPY19L2 negative diagnosis, searching for deleterious variants in the nine other genes described to be associated with globozoospermia in human ( C2CD6 , C7orf61 , CCDC62 , CCIN , DNAH17 , GGN , PICK1 , SPATA16 , and ZPBP1 ). Only one homozygous novel truncating variant was identified in the GGN gene in one patient, confirming the association of GGN with globozoospermia. In view of these results, we propose a novel diagnostic strategy focusing on patients with at least 50% of globozoospermia and based on a classical qualitative PCR to detect DPY19L2 homozygous deletions. In the absence of the latter, we recommend to perform whole-exome sequencing to search for defects in DPY19L2 as well as in the other previously described candidate genes.

L’hormone anti-Müllerienne (AMH) dont les taux sont plus élevés dans le syndrome des ovaires polykystiques est connue pour augmenter la sécrétion de LH/GnRH [1], [2]. Par ailleurs, les tanycytes, qui enchâssent les terminaisons des neurones à GnRH [3], [4], expriment le récepteur à l’AMH [2]. Ainsi, notre objectif est de tester les deux hypothèses suivantes dans un modèle animal et chez la femme. La physiopathologie du SOPK : – est-elle liée à une rétraction des tanycytes provoquée par l’AMH, entraînant l’augmentation de la sécrétion de LH/GnRH ? Et/ou – est-elle liée à une augmentation de l’activité des neurones à GnRH AMH-dépendante ? Nous avons étudié la modification de la plasticité neuro-structurale de l’hypothalamus en microscopie électronique chez des rats traités par de l’AMH et des contrôles. Puis, nous avons étudié de la même façon un modèle de souris SOPK en comparaison à des contrôles. Enfin, nous avons conduit une étude clinique par spectroscopie par résonance magnétique comparant les ratios de métabolites cérébraux entre des femmes SOPK et des témoins. Nous avons observé une augmentation significative du nombre de terminaisons de neurones à GnRH situées à proximité de l’espace péri-capillaire dans le groupe traité par l’AMH par rapport aux contrôles et nous avons fait les mêmes observations chez les souris SOPK comparées aux contrôles. Nos récents résultats chez les rongeurs SOPK, ont mis en évidence une augmentation des afférences excitatrices sur les neurones à GnRH [5]. Notre étude par IRM, chez les femmes SOPK et contrôles, vient conforter cette précédente étude, en montrant une augmentation de la viabilité/activité neuronale. Cette étude translationelle suggère que l’augmentation des sécrétions de LH/GnRH retrouvée dans le SOPK serait dépendante, d’une part, d’un rapprochement des terminaisons à GnRH vers l’espace péri-capillaire, et d’autre part, à une augmentation de l’activité neuronal hypothalamique.

Motile cilia and sperm flagella share an evolutionarily conserved axonemal structure. Their structural and/or functional defects are associated with primary ciliary dyskinesia (PCD), a genetic disease characterized by chronic respiratory-tract infections and in which most males are infertile due to asthenozoospermia. Among the well-characterized axonemal protein complexes, the outer dynein arms (ODAs), through ATPase activity of their heavy chains (HCs), play a major role for cilia and flagella beating. However, the contribution of the different HCs (γ-type: DNAH5 and DNAH8 and β-type: DNAH9, DNAH11, and DNAH17) in ODAs from both organelles is unknown. By analyzing five male individuals who consulted for isolated infertility and displayed a loss of ODAs in their sperm cells but not in their respiratory cells, we identified bi-allelic mutations in DNAH17. The isolated infertility phenotype prompted us to compare the protein composition of ODAs in the sperm and ciliary axonemes from control individuals. We show that DNAH17 and DNAH8, but not DNAH5, DNAH9, or DNAH11, colocalize with α-tubulin along the sperm axoneme, whereas the reverse picture is observed in respiratory cilia, thus explaining the phenotype restricted to sperm cells. We also demonstrate the loss of function associated with DNAH17 mutations in two unrelated individuals by performing immunoblot and immunofluorescence analyses on sperm cells; these analyses indicated the absence of DNAH17 and DNAH8, whereas DNAH2 and DNALI, two inner dynein arm components, were present. Overall, this study demonstrates that mutations in DNAH17 are responsible for isolated male infertility and provides information regarding ODA composition in human spermatozoa.

A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentration of proAKAP4 measured by ELISA in post-thawed semen. Frozen sperm from 13 different stallions were used. Semen samples (n = 17) were prepared using the INRA Freeze medium to reach a concentration of 150 million spermatozoa/mL, packaged and frozen in 0.5 mL straws. The precursor proAKAP4 and the mature protein AKAP4 both localize to the fibrous sheath of the principle piece of equine sperm flagellum. The concentrations of proAKAP4 were determined in the post-thawed semen using ELISA method (Horse 4MID® kits, 4BioDx, France). The mean concentration of proAKAP4 was then of 7.372 ± 0.79 ng/μL and was significantly correlated with the post-thawed total motility (Pearson coefficient r = 0.66, p = 0.002) and progressive motility (Pearson coefficient r = 0.76, p = 0.0002) and the amount of proAKAP4 represent the amount of spermatozoa that expressed proAKAP4. Taken together, these preliminary results confirm the interest to use proAKAP4 concentrations as a promising marker of stallion sperm quality as close correlation was observed between the proAKAP4 concentration and sperm motility parameters.

As with many other diseases, genetic testing in human azoospermia was initially restricted to karyotype analyses (leading to diagnostic chromosome rearrangement tests for Klinefelter and other syndromes). With the advent of molecular biology in the 1980s, genetic screening was broadened to analyses of Y chromosome microdeletions and the gene coding for the cystic fibrosis transmembrane conductance regulator ( CFTR ). Decades later, the emergence of whole-genome techniques has led to the identification of other genetic defects associated with human azoospermia. Although TEX11 and ADGRG2 defects are frequently described in men with azoospermia, most of the causal gene defects found to date are private (i.e. identified in a small number of consanguineous families). Here, we provide an up-to-date overview of all the types of genetic defects known to be linked to human azoospermia and try to give clinical practice guidelines according to azoospermia phenotype. Along with homozygous mutations, polymorphisms and epigenetic defects are also briefly discussed. However, as these variations predispose to azoospermia, a specific review will be needed to compile data on all the particular genetic variations reported in the literature.

Cryptorchidism is one of the most frequent causes of nonobstructive azoospermia (NOA) in adulthood. Although it is well known that spermatogenesis is more impaired in bilateral than in unilateral cryptorchidism, previous studies have only described small cohorts or inhomogeneous population. Consequently, we analyzed a cohort of 225 men with only a history of cryptorchidism as sole etiopathogenetic factor for NOA, and compared testicular sperm extraction (TESE) outcomes between men with bilateral versus unilateral cryptorchidism. Our results show no difference in follicle-stimulating hormone (FSH) levels and testicular volumes between men with a history of bilateral cryptorchidism compared to unilateral cryptorchidism (median: 21.3 IU l-1 vs 19.3 IU l-1, P = 0.306; and 7.2 ml vs 7.9 ml, P = 0.543, respectively). In addition, sperm retrieval rates were similar (66.2% vs 60.0%, P = 0.353). Using multivariate analysis, we have found that only a low inhibin B level (above the assay's detection limit) was positively associated with successful sperm retrieval (P < 0.05). Regarding intracytoplasmic sperm injection outcomes, we found that cumulative pregnancy rate and live birth rate per cycle were not statistically different between the two groups (17.4% vs 27.8%, P = 0.070; and 16.1% vs 26.4%, P = 0.067, respectively). Unexpectedly, there was no significant difference in hormonal profiles (FSH, luteinizing hormone [LH], testosterone, and inhibin B levels) and TESE outcomes between unilateral versus bilateral cryptorchidism. This suggests that a history of unilateral cryptorchidism could reflect a bilateral testicular impairment. Interestingly, inhibin B level might be a predictor of successful TESE.

Background The limitations of conventional sperm analyses have highlighted the need for additional means of evaluating sperm quality. Methods In a study of a cohort of 245 men with known conventional sperm parameters, one-dimensional PAGE was used to monitor protein content and quality in samples from individual ejaculates. Results The sperm protein content varied markedly from sample to another, especially in the high-molecular-weight range. The intensity of the 80–110 kDa bands was correlated with progressive motility ( r = 0.15, p = 0.015 ) and was significantly higher ( p = 0.0367) in the group of men with conventional parameters above the World Health Organization’s 2010 reference values than in the group with at least one subnormal parameter (i.e. semen volume, sperm concentration, sperm count per ejaculate, progressive motility, proportion of normal forms or multiple anomaly index below the lower reference value). Using mass spectrometry, the 80–110 kDa bands were found to correspond primarily to three proteins from the flagellum’s fibrous sheath: A-kinase anchor protein 4, A-kinase anchor protein 3, and spermatogenic cell-specific type 1 hexokinase. Conclusion One-dimensional PAGE constitutes a simple, rapid, reliable, inexpensive method for analyzing proteins associated with sperm motility in individual human ejaculates.

Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 ( n = 6), CFAP43 ( n = 10), and CFAP44 ( n = 6). CRISPR/Cas9 created homozygous CFAP43 / 44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human.

Dans le syndrome des ovaires polykystiques (SOPK), les taux d’hormone anti-Müllerienne (AMH) sont 2 à 3 fois plus élevés [1]. Or, l’AMH augmente la sécrétion de LH via une hyper-activation des neurones à GnRH [2]. Par ailleurs, les tanycytes, qui enchâssent les terminaisons des neurones à GnRH [3], [4], [5], expriment l’AMH et son récepteur [2]. Notre objectif est de déterminer si l’AMH pourrait conduire à la rétraction des tanycytes, entraînant l’augmentation de la sécrétion de GnRH/LH dans le SOPK. Nous avons réalisé des cultures cellulaires de tanycytes de rats en présence de doses croissantes d’AMH afin d’évaluer leur morphologie. Puis, nous avons étudié la modification de la plasticité neuro-structurale de l’hypothalamus en microscopie électronique. Nous avons comparé la distance entre les terminaisons des neurones à GnRH et l’espace péri-capillaire entre des éminences médianes issues de rats femelles en diestrus (au moment où ces terminaisons sont normalement enfouies par les tanycytes) traitées par de l’AMH et des contrôles. Nous avons confirmé que les tanycytes exprimaient le récepteur spécifique pour l’AMH (protéine et transcrit). En culture cellulaire, nous avons observé une rétraction significative des tanycytes en présence d’AMH à la concentration de 50 ng/mL par rapport aux contrôles (p < 0,05), avec une réduction de l’espace occupé par les tanycytes traités. En microscopie électronique, nos premiers résultats suggèrent une augmentation du nombre de terminaisons de neurones à GnRH situées à moins de 1 μm de l’espace péri-capillaire dans le groupe traité par l’AMH par rapport aux contrôles (en moyenne 11 % pour le groupe traité et 5 % pour les contrôles). Nos résultats suggèrent que l’AMH pourrait être responsable d’une rétraction des pieds tanycytaires au niveau de l’éminence médiane. Cette rétraction autoriserait la germination des terminaisons des neurones à GnRH vers l’espace péricapillaire et pourrait expliquer l’augmentation des sécrétions de GnRH/LH rencontrées dans le SOPK.

BACKGROUND:A-kinase anchor protein 4 (AKAP4) and its precursor pro-AKAP4 are two major proteins in spermatozoa of rodents and mammals. Although researchers have characterized the AKAP4 expression in various species, the protein's expression in humans has not been described in detail.OBJECTIVES:The objective of this study was to characterize human pro-AKAP4 more precisely (notably the definition of its localization and expression levels in human spermatozoa and testes).MATERIALS AND METHODS:pro-AKAP4 protein expression levels were assessed by Western blotting. The pro-AKAP4's localization in spermatozoa and testes was determined using immunofluorescence staining and immunogold electron microscopy. Furthermore, pro-AKAP4 protein expression levels were assessed in a series of 77 human semen samples, and associations with semen parameters were evaluated.RESULTS:Western blotting revealed a 100-kDa band in human sperm protein extracts. The pro-AKAP4 was immunolocalized in the fibrous sheath of the flagellum of ejaculated spermatozoa and in elongated spermatids in human testes. A Western blot analysis of 77 normozoospermic semen samples evidenced striking differences in pro-AKAP4 levels from one to another sample (median [interquartile range] integrated optical density = 305 [49-1038]). No correlations were found for pro-AKAP4 levels on one hand and semen volume, sperm concentration, sperm count, sperm motility, or sperm morphology on the other (p > 0.05 for all). However, pro-AKAP4 levels were positively correlated with motility after density gradient centrifugation of the semen (r = 0.224, p = 0.049).DISCUSSION:AKAP4 protein might be activated as an alternative pathway to rescue sperm motility. In human spermatozoa, pro-AKAP4 might therefore be a 'reservoir' of mature AKAP4.CONCLUSION:This study generated new knowledge about pro-AKAP4 in human semen, which may be of interest in the management of male infertility.