Whole-exome sequencing in patients with maturation arrest: a potential additional diagnostic tool for prevention of recurrent negative testicular sperm extraction outcomes.

STUDY QUESTION: Could whole-exome sequencing (WES) be useful in clinical practice for men with maturation arrest (MA) after a first testicular sperm extraction (TESE)? SUMMARY ANSWER: WES in combination with TESE yields substantial additional information and may potentially be added as a test to predict a negative outcome of a recurrent TESE in patients with MA. WHAT IS KNOWN ALREADY: At present, the only definitive contraindications for TESE in men with non-obstructive azoospermia (NOA) are a 46,XX karyotype and microdeletions in the azoospermia factor a (AZFa) and/or AZFb regions. After a first negative TESE with MA, no test currently exists to predict a negative outcome of a recurrent TESE. STUDY DESIGN, SIZE, DURATION: In a cohort study, we retrospectively included 26 patients with idiopathic NOA caused by complete MA diagnosed after a first TESE. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty-six men with MA at the spermatocyte stage in all seminiferous tubules, according to a histopathological analysis performed independently by two expert histologists, and a normal karyotype (i.e. no AZF gene microdeletions on the Y chromosome) were included. Single-nucleotide polymorphism comparative genomic hybridization array and WES were carried out. The results were validated with Sanger sequencing. For all the variants thought to influence spermatogenesis, we used immunohistochemical techniques to analyse the level of the altered protein. MAIN RESULTS AND THE ROLE OF CHANCE: Deleterious homozygous variants were identified in all seven consanguineous patients and in three of the 19 non-consanguineous patients. Compound heterozygous variants were identified in another 5 of the 19 non-consanguineous patients. No recurrent variants were identified. We found new variants in genes known to be involved in azoospermia or MA [including testis expressed 11 (TEX11), meiotic double-stranded break formation protein 1 (MEI1), proteasome 26s subunit, ATPase 3 interacting protein (PSMC3IP), synaptonemal complex central element protein 1 (SYCE1) and Fanconi anaemia complementation group M (FANCM) and variants in genes not previously linked to human MA (including CCCTC-binding factor like (CTCFL), Mov10 like RISC complex RNA helicase 1 (MOV10L1), chromosome 11 open reading frame 80 (C11ORF80) and exonuclease 1 (EXO1)]. LARGE SCALE DATA: Data available on request. LIMITATIONS, REASONS FOR CAUTION: More data are required before WES screening can be used to avoid recurrent TESE, although screening should be recommended for men with a consanguineous family background. WES is still a complex technology and can generate incidental findings. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirmed the genetic aetiology of MA in most patients: the proportion of individuals with at least one pathologic variant was 50% in the overall study population and 100% in the consanguineous patients. With the exception of MEI1 (compound heterozygous variants of which were identified in two cases), each variant corresponded to a specific gene-confirming the high degree of genetic heterogeneity in men with MA. Our results suggest that WES screening could help to avoid recurrent, futile TESE in men with MA in general and in consanguineous individuals in particular, but these results need to be confirmed in future studies before clinical implementation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Fondation Maladies Rares (Paris, France), Merck (Kenilworth, NJ, USA), IRSF (Montigny le Bretonneux, France) and Agence de la Biomédecine (Saint Denis, France). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

A-kinase anchor protein 4 precursor (pro-AKAP4) in human spermatozoa.

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.

[Necrozoospermia: From etiologic diagnosis to therapeutic management]. / La necrozoospermie : du diagnostic étiologique à la prise en charge thérapeutique.

This review describes necrospermia, its diagnosis, causes and management. Sperm vitality is commonly assessed in the laboratory of reproductive biology, with the eosin test or with the hypo-osmotic swelling test. Necrospermia is defined by a percentage of living spermatozoa inferior to 58%, and can be related to male infertility. Several pathological mechanisms may be involved and can be classified either in testicular causes (hyperthyroidism, local hyperthermia, varicocele), or post-testicular causes (epididymal necrospermia, dysregulation of seminal plasma, adult polycystic kidney disease, vasectomy reversal, anti-sperm antibodies) or both (infection, toxic, age, spinal cord injury). The first treatment is to correct the underlying cause, if possible. Repetitive ejaculation has demonstrated to be effective as well. Many drugs would also improve the sperm vitality (antioxidants, non-and-steroidal anti-inflammatory drugs) but there is currently no guideline to recommend their use. With necrospermia, fertilization rates are lower but in vitro fertilization (IVF) with Intracytoplasmic sperm injection (ICSI) improves the chances of conception.

Extreme spermatogenesis failure: andrological phenotype and intracytoplasmic sperm injection outcomes.

Patients with very low sperm count through direct sperm examination can exhibit extreme oligozoospermia or cryptozoospermia (after centrifugation). The management of these patients is a real challenge for both clinicians and biologists. In this retrospective and comparative cohort study, we compared the andrological phenotype of patients with extreme alterations of spermatogenesis and assessed whether the origin of spermatozoa (testicular or ejaculate) had any influence on intracytoplasmic sperm injection (ICSI) outcomes. A total of 161 ICSI cycles were performed using ejaculated spermatozoa from 75 patients with extreme oligozoospermia (EOS) or cryptozoospermia (CS) and 150 ICSI cycles using extracted testicular spermatozoa from 74 patients with non-obstructive azoospermia (NOA). Physical, hormonal, ultrasound assessments, and ICSI outcomes were performed in each group. Cryptorchidism was significantly more frequent in the NOA group (60.8% vs. 22.6%, p = 0.001). FSH levels were significantly higher [18.9 IU/L (5.9-27.0) vs. 15.3 IU/L (9.0-46.5), p = 0.001] and the majority of inhibin B levels measured were found mostly undetectable in the NOA group as compared to EOS/CS group (31.1% vs. 10.7%, p = 0.0004). Moreover, we found no significant differences in the respect to the fertilization rates (48.9% and 43.3%, p = 0.43), implantation rates (17.4% and 15.9%, p = 0.77), and percentage of top quality embryo (22.4% and 20.4%, p = 0.73) between the two groups. The clinical pregnancy rates per embryo transferred were comparable in both groups (28.3% and 27.4%, p = 0.89). In this study, we showed for the first time a different andrological phenotype between EOS/CS and NOA groups. Indeed, cryptorchidism was significantly more frequent with more severe endocrine parameters found in the NOA group. These results reflect a more profound alteration in spermatogenesis in NOA patients. However, there was no difference in ICSI outcomes between NOA and EOS/CS groups.

[Effects of father's age on fertility, results of ART and health of children]. / Conséquences de l'âge du père sur la fertilité, les résultats de l'AMP et la santé des enfants.

Many studies exist on the impact of female age on fertility, success of assisted reproductive technologies and on obstetric, fetal and neonatal adverse outcomes. Late paternity seems commonplace especially in the media… But there are reliable scientific data which confirm decline of fertility related to male age but also an increased risk of genetic diseases for the offspring. The objective of this article is to make a synthesis of the literature on this subject.

Pregnancy after intracytoplasmic sperm injection following extended sperm preparation and hormone therapy in an azoospermic man with maturation arrest and microlithiasis: a case report and literature review.

In the management of azoospermia, a combination of testicular sperm extraction and intracytoplasmic sperm injection (ICSI) is usually the most successful option for fatherhood. However, an outstanding question remains: How can at least a few spermatozoa be obtained from the ejaculate, thus avoiding the need for a surgical procedure? A 36-year-old man presented to Assisted Reproduction Unit with his 26-year-old wife. The ultrasound assessment revealed bilateral microlithiasis. Two spermograms revealed absolute azoospermia. Levels of follicle-stimulating hormone (FSH) and luteinising hormone were normal-low. The patient underwent 10 months of treatment with clomiphene citrate. A bilateral testicular sperm extraction failed to retrieve spermatozoa and revealed a maturation arrest at spermatocyte/spermatid stages depending on the tubules. Clomiphene citrate was replaced with recombinant FSH (rFSH). After 9-month treatment with rFSH, motile spermatozoa from droplets of ejaculate pellet were cryopreserved as a single straw. Ovarian stimulation was provided using classic antagonist protocol, and five mature oocytes were collected. Two consecutive fresh semen samples on the day of ICSI yielded seven motile spermatozoa, and fertilisation was achieved in all five oocytes. On day 3, two embryos were transferred, yielding positive beta-human chorionic gonadotropin and a healthy delivery of a boy and a girl.

Leydig cell tumor of the testis with azoospermia and elevated delta4 androstenedione: case report.

BACKGROUND: Secreting interstitial cell (Leydig cell) tumors are rare. In adults, the clinical picture and steroid levels are variable. CASE PRESENTATION: This paper presents a case of left testicular tumor, showing azoospermia with normal serum level of total testosterone, collapsed FSH and LH, and high delta4 androstenedione. Histopathological investigation revealed a Leydig cell tumor. TESE allowed spermatozoa extraction and freezing. Testicular histology found hypospermatogenesis and germ-cell aplasia with interstitial fibrosis. Surgical resection of the tumor resulted in normalization of gonadotropins and fall in serum delta4 androstenedione to subnormal levels in the postoperative period confirming that the tumor was secreting delta4 androstenedione. It was hypothesized that high delta4 androstenedione resulted in intra tumoral 17 ß-HSD overtaken by delta4 androstenedione or that 17 ß-HSD activity in the tumor was different from that of normal Leydig cells. Three months after surgery sperm analysis found a complete recovery of spermatogenesis. A spontaneous pregnancy occurred 3 months after surgery and a girl was born. CONCLUSIONS: In this case, the diagnosis of testicular Leydig cell tumor secreting delta4 androstenedione was made in a context of azoospermia.

INTRODUCTION: Les tumeurs testiculaires interstitielles (ou tumeurs testiculaires à cellules de Leydig) à expression endocrine sont rares. Chez l'adulte le tableau clinique et le bilan hormonal sont variables. PRÉSENTATION DU CAS: Cet article présente le cas d'une tumeur testiculaire gauche dans un contexte d'azoospermie. Le bilan hormonal montre des gonadotrophines effondrées, une testostéronémie normale et une delta4 androstenedione augmentée. L'examen anatomopathologique a mis en évidence une tumeur à cellule de Leydig. La TESE a permis l'extraction et la congélation de spermatozoïdes. L'histologie a retrouvé un aspect mixte d'hypospermatogenèse diminuée incomplète et d'aplasie. Dans les suites de l'orchidectomie partielle gauche les taux de gonadotrophines se sont normalisés ainsi que le taux de delta4 androstenedione. L'hypothèse physiopathologique est que l'augmentation de la delta4 androstenedione résulte de la sursaturation de la 17 ß-HSD intra-tumoral ou que l'activité de la 17 ß-HSD intra-tumoral est différente de celle dans les cellules de Leydig normales. Trois mois après la chirurgie, le spermogramme a montré une normalisation des paramètres spermatiques et une grossesse spontanée est survenue permettant la naissance d'une petite fille. CONCLUSION: Dans ce cas clinique, le diagnostic de tumeur testiculaire à cellule de Leydig sécrétant de la delta4 androstenedione a été fait dans un contexte d'azoospermie.

Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk.