Spermatocyte Maturation Arrest Research Articles

(183) The Predictive Value of AMH, FSH, and Inhibin B for Sperm Retrieval in Azoospermic Men

Abstract Introduction Several studies assessed the sensitivity and specificity of plasma concentrations of hormones such as FSH and AMH as biomarkers that can predict the presence of sperm cells, prior to testicular sperm extraction (TESE) in azoospermic men. However, the literature contains conflicting reports in this context. Objective To evaluate the potential of hormonal profile as a non-invasive predictor of mature sperm cells' presence in the testis of azoospermic men, and to assess if the source of these hormones- testicular or peripheral blood makes any difference. Methods Clinical data, hormonal profile, and histological classification of 57 azoospermic men were analyzed. They were categorized into 4 following groups by their testicular pathologies: obstructive azoospermia, hypospermatogenesis, spermatocyte maturation arrest, and Sertoli cells only. The level of hormones was measured by the enzyme-linked immunosorbent assay. Statistical analyses tested the predictability of hormone values for sperm retrieval. Results Overall, 28 patients (49%) had spermatozoa detected. FSH and Inhibin B measurements were statistically significantly different between the 4 groups with different testicular pathologies (p<0.001). AMH, FSH and Inhibin B were found to be independent predictor for positive sperm retrieval (p<0.05). However, a decision tree with random forest classifying the predictive ability of the variables to indicate the presence of sperm produced low accuracy. There was no significant difference between the hormone levels of peripheral blood source and testicular blood source hormone levels. Conclusions AMH, FSH, and Inhibin B are optional markers for positive sperm retrieval in TESE. Increasing the research group may contribute to their ability to predict the presence of mature sperm cells in the testis of azoospermic men. Disclosure No.

MicroRNAs expression in semen and testis of azoospermic men.

MicroRNAs are involved in the regulation of spermatogenesis, are detected in semen and may be useful as molecular markers for predicting residual complete spermatogenesis in azoospermic men. To study the biomarker potential of microRNAs that are detected in semen and testicular tissue. MicroRNA profiles were analyzed in semen fractions of normozoospermic (n=3) and azoospermic (n=6) men by small RNA deep sequencing. Specific microRNAs were further analyzed by reverse transcription and quantitative polymerase chain reaction in eight testicular samples and 46 semen supernatants. The semen supernatant samples included 18 normozoospermic and 28 azoospermic men with various pathologies. The sequenced microRNA profiles of semen supernatant fraction samples were distinct from the other fractions. Significant expression differences were observed between the semen supernatant of normozoospermic and azoospermic men. Further analysis by reverse transcription and quantitative polymerase chain reaction revealed that expression of miR-202-3p was considerably reduced (undetectable in most samples) in the azoospermic semen supernatants. The expression of miR-202-3p was significantly lower in the azoospermic specimens than in the normozoospermic specimens and a trend was observed for miR-629-5p (p=0.03 and 0.06, respectively). Differences in expression levels in the semen supernatant were observed among the various pathologies but not to a level of significance, possibly because of the small subgroups. miRNA-370-3p was significantly higher in semen supernatant samples from azoospermic men without sperm cells in testis (p=0.05). In testes, the three microRNAs were expressed at higher levels in the obstructive and spermatocyte maturation arrest pathologies than in mixed atrophy and Sertoli cell only. miR-202-3p was detected in all testicular samples. MicroRNA expression profiles in semen were distinguishable between azoospermic and normozoospermic men. The microRNA profile also diverged among azoospermic men subdivided according to their testicular pathologies. The levels of specific microRNAs in testis and in the semen supernatant were not directly correlated.

Questioning the utility of round spermatid injections in men with non-obstructive azoospermia.

Data on who among the infertile male population may benefit from round spermatid injections (ROSI) are lacking. To determine the probability of finding round spermatids suitable for ROSI in men with non-obstructive azoospermia (NOA) in whom no spermatozoa were retrieved at testicular sperm extraction. Four-hundred fifty-seven consecutive men with azoospermia underwent testicular sperm extraction. Clinical examination included age, secondary sexual characteristics, testicular size, reproductive hormone estimation, karyotyping, and Y chromosome microdeletion analyses. Histologic examination was performed, and histologic classification was determined by the most advanced spermatogenetic cell identified in the combined histologic and cytologic examination. Of the 457 azoospermic men, 342 were diagnosed with NOA, and 148 (148/342, 43%) had mixed atrophy on histopathology and retrievable spermatozoa. No spermatozoa were found in 194/342 men with NOA (57%). Histopathology diagnosed 145/194 (75%) of them with Sertoli cell only, 45/194 (23%) with spermatocyte maturation arrest, and 4/194 (2%) with spermatid maturation arrest. Histopathologically identified round spermatids without spermatozoa were rare in men with NOA. Only very few of them are likely to reap the benefits of ROSI, thus presenting the need to reconsider its actual clinical value.

A recurrent ZSWIM7 mutation causes male infertility resulting from decreased meiotic recombination.

Are mutations in the zinc finger SWIM domain-containing protein 7 gene (ZSWIM7) associated with human male infertility? The homozygous frameshift mutation (c.231_232del) in ZSWIM7 causes decreased meiotic recombination, spermatogenesis arrest, and infertility in men. ZSWIM7 is a SWIM domain-containing Shu2/SWS1 protein family member and a subunit of the Shu complex. Zswim7 knockout mice were infertile due to impaired meiotic recombination. However, so far there is no direct evidence that mutations of ZSWIM7 cause human infertility. Screening for mutations of ZSWIM7 was performed using in-house whole-exome sequencing data from 60 men with non-obstructive azoospermia (NOA). Mice with a corresponding Zswim7 mutation were generated for functional verification. Sixty Chinese patients, who were from different regions of China, were enrolled. All the patients were diagnosed with NOA owing to spermatocyte maturation arrest based on histopathological analyses and/or immunostaining of spermatocyte chromosome spreads. ZSWIM7 mutations were screened from the whole-exome sequencing data of these patients, followed by functional verification in mice. A homozygous frameshift mutation (c.231_232del) in ZSWIM7 was found in two out of the 60 unrelated NOA patients. Both patients displayed small testicular size and spermatocyte maturation arrest in testis histology. Spermatocyte chromosome spreads of one patient revealed meiotic maturation arrest in a pachytene-like stage, with incomplete synapsis and decreased meiotic recombination. Male mice carrying a homozygous mutation similar to that of our patients were generated and also displayed reduced recombination, meiotic arrest and azoospermia, paralleling the spermatogenesis defects in our patients. As Zswim7 is also essential for meiosis in female mice, future studies should evaluate the ZSWIM7 mutations more in depth and in larger cohorts of infertile patients, including males and females, to validate the findings. These findings provide direct clinical and functional evidence that the recurrent ZSWIM7 mutation (c.231_232del) causes decreased meiotic recombination and leads to male infertility, illustrating the genotype-phenotype correlations of meiotic recombination defects in humans. This work was supported by the National Natural Science Foundation of China (31890780, 31630050, 32061143006, 82071709, and 31871514), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000), and the National Key Research and Developmental Program of China (2018YFC1003900 and 2019YFA0802600). Not applicable.

Histopathological Evaluation of Non-Obstructive Azoospermic Males Using Testicular aspirate (TESA) biopsy

Background: Male infertility considers a real diagnostic challenge because of several factors have beeninvolved in its pathogenesis. Histopathology of the testicular biopsy consider the corner stone in thediagnosis. The aim of this study is to report the utility of using of Testicular aspirate biopsy (TESA) for theevaluation of the male infertility & compare the outcomes with different parameters.Materials & methods: we design prospective study for 205 infertile males with Non -Obstructive azoospermiawho referred to the Teeba IVF & genetic center for the period from December 2016 to December 2018, theyunderwent bilateral TESA for histopathological evaluation & 45 patients out of 205 male had previous opentesticular biopsy. The hormonal profile for all patient. were done. Results: among 205 patients underwentTESA procedure the commonest histopathological pattern was spermatocyte maturation arrest, present in113 patients (55.1%) followed by round maturation arrest in 41 (20%). There was a good degree of agreementbetween the spermatogenesis scoring according to TESA and open surgical biopsy (P=< 0.001). The plasmalevels for both LH & FSH were statistically showing significant differences in their levels according to theTESA spermatogenesis scoring. (P<0,001).Conclusion: the utility of TESA biopsy in the evaluation oftesticular biopsy from Azoospermic males is significantly associated with the histopathological pattern ofspermatogenesis & hormonal profile.

Olfactory Receptors in Semen and in the Male Tract: From Proteome to Proteins.

The estimated number of testicular olfactory receptors (ORs) in mammals range between 20 and 66. Previous data reported the role of hOR17-4 and mOR23 in sperm–oocyte chemiotaxis. Proteomic analysis was performed to understand which are the ORs expressed in seminal plasma. Seminal samples by four fertile men were analyzed by an Ultimate 3000Nano/Micro-HPLC apparatus coupled with an LTQ-Orbitrap XL hybrid mass spectrometer. Western blot analysis confirmed the expression of three identified ORs. The expression of ORs in sperm cells, testis, and epididymis was evaluated by confocal microscopy analysis. In seminal plasma eight different ORs were identified by proteomics and three ORs have been confirmed by western blot. Confocal microscopy analysis revealed that OR4S1, OR4C13, and OR1I1 are expressed on the surface of sperm cells. In testicular tissue, OR4S1 and OR1I1 are expressed in spermatocytes and spermatids and OR4C13 is expressed throughout all the tubules. In patients with spermatocyte maturation arrest OR4S1 and OR1I1 expression was reduced and a weak positivity for OR4C13 was detected in the spermatogonia. OR4S1, OR4C13, and OR1I1 had intense and diffuse staining in the epididymis. This study initiated a new methodology for screening OR repertoire in sperms, testis and epididymis. Our results open new insights into OR involvement in sperm maturation and migration.

New insights into the role of the Brdt protein in the regulation of development and spermatogenesis in the mouse

The bromodomain testis-specific (BRDT) protein belongs to the bromodomain extra-terminal (BET) family of proteins. It serves as a transcriptional regulator of gene expression during spermatogenesis, and is an essential factor for the normal spermatogenesis process. In this study, we characterized mice of several age groups who lacked the Brdt gene. The testes of Brdt mutant mice aged 8 weeks exhibited complete spermatocyte maturation arrest with a significantly increased number of apoptotic cells. The weights of the testes and accessory glands as well as the testosterone levels of the mutant mice were significantly lower compared to the normal mice. The mutant mice had delayed puberty, with normal levels of testosterone and accessory gland weights at the age of 14 and 28 weeks. The testes of the mutant mice at older ages also exhibited round spermatids. The presence of the BRDT protein was identified in the mice pituitary gland. Microarray analysis of mice pituitaries showed that 28 genes were down-regulated while 26 genes were up-regulated in the absence of the Brdt gene. Our results suggest that in addition to its critical role in the spermatogenesis process, the BRDT protein is also responsible for scheduling male puberty by regulation of the pituitary-gonad axis.

Screening of genomic regions affecting lawc/Trf2 gene expression during Drosophila melanogaster development

leg-arista-wing complex (lawc) mutations affect the expression of Drosophila proteins homologous to a human basic transcription factor, TBP (TATA-box binding protein) Related Factor 2 (TRF2), specifically involved in development.The paper for the first time reports the application of genetic screens for various genomic regions to identify genetic interactions between the lawc/Trf2 gene and other genes and genetic loci by using Deficiency Kit lines with small deletions in total providing maximal coverage of the genome. The deletion mapping allowed us to recover 26 genomic regions that, when deleted, are lethal or modify the mutant phenotype due to a decreased TRF2 expression level. These deletions could be useful in identifying both novel TRF2 targets and its positive and negative regulators.There is evidence that TRF2 can be a component of high molecular DNA Replication-related Element Factor (DREF) and Nucleosome Remodeling Factor (NURF)-containing complexes. The present study for the first time reports new genetic interactions of lawc/Trf2 with genes that encode basic and specific transcription factors. In most cases, if mutated, those genes caused developmental defects or death of progeny. However, in the case of the e(y)1 gene, coding for the Taf9 transcription factor, only the male reproductive system is impaired when the lawc/Trf2 phenotype is associated with a e(y)1 gene mutation. Mutant lawcp1e(y)1u1 males become infertile due to primary spermatocyte maturation arrest and impaired premeiotic chromosome condensation in germ cells.

Expression of BET genes in testis of men with different spermatogenic impairments

To characterize the BET gene expression in human testis with spermatogenetic impairments; to examine BRDT protein expression in testis and semen. Prospective study. Fertility clinic. Azoospermic men (n = 120) who underwent testicular sperm extraction and who were classified as either normal spermatogenesis, mixed atrophy, spermatocyte maturation arrest, or Sertoli cells only according to their combined histologic and cytologic testicular findings and three normozoospermic men who donated sperm. Evaluation of testicular biopsies by qualitative and quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical staining, and analysis of spermatozoa by immunofluorescence. Expression of the four BET genes in testis and localization of BRDT protein in testicular tissue and ejaculated spermatozoa. The BRDT gene was not expressed in testicular tissue from patients with Sertoli cells only, whereas the other three genes of the BET family retained expression in all the pathologies. The BRDT protein was localized in the nuclei of spermatocytes, spermatids, and ejaculated spermatozoa. Expression of BRDT protein was almost nil in testicular tissue specimens with spermatocyte maturation arrest despite normal transcript levels. Human BRDT expression pattern differs from mouse BRDT expression. In human, BRDT is the only BET gene expressed exclusively in testicular germ cells. Its expression in elongated spermatids and ejaculated spermatozoa raises the possibility that it is involved in unidentified additional functions.

CDY1 and BOULE transcripts assessed in the same biopsy as predictive markers for successful testicular sperm retrieval

To evaluate the molecular markers CDY1 and BOULE in the same testicular biopsy for predicting success of sperm retrieval in azoospermic men. Prospective study. University-affiliated medical center. Azoospermic men (n = 92) who underwent testicular sperm extraction (TESE) and who were classified as normal spermatogenesis, mixed atrophy, spermatocyte maturation arrest, or Sertoli cell only according to their combined histological and cytological testicular findings. Quantitative and qualitative evaluation of testicular biopsies by histological and qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) expression methodologies. CDY1 and BOULE expression and the presence of sperm cells in testicular tissue. Both transcripts significantly predicted the presence of sperm cells by qualitative and quantitative methodologies. Although CDY1 had the best sensitivity by qualitative RT-PCR (98.3%), assessing both transcripts simultaneously had an additive efficacy compared with assessing CDY1 alone, improving the specificity from 84.4% to 96.3%. Assessing the expression of both CDY1 and BOULE by qualitative RT-PCR is a sensitive and feasible test for predicting the presence of sperm cells in testicular tissue and may serve as a predictive tool if repeated TESE is required.

Role of poly(ADP-ribosyl)ation during human spermatogenesis

Genomic stability of cells is known to be linked to their poly(ADP-ribosyl)ation capacity. We aimed to demonstrate, for the first time, the patterns of poly(ADP-ribosyl)ation during human spermatogenesis. Retrospective case-control study. Teaching hospital. Azoospermic men who underwent testicular biopsy for sperm recovery. Testicular biopsy evaluation by immunohistochemistry for the expression of poly(ADP-ribose) polymerase-1 (PARP-1) enzyme and of poly(ADP-ribose) (PAR) (an indicator for PARP activity.) The subcellular localization of both markers in testes with full spermatogenesis (obstructive azoospermia), spermatocyte maturation arrest, or Sertoli cell-only syndrome. Expression of both markers was localized in germ cell nuclei in full spermatogenesis: PAR expression, indicating PARP activity, was exhibited in round and elongating spermatids and in a subpopulation of primary spermatocytes. Strong immunoreactivity for PAR was identified in all of the spermatocytes in maturation arrest at the spermatocyte level. Sertoli cells lacked immunoreactivity for both markers, whereas other somatic testicular cells were rarely immunostained. The detection of PAR expression in germ-line cells and its subcellular localization in meiotic and postmeiotic prophases demonstrates chromatin modifications occurring during spermatogenesis and establishes a key role for poly(ADP-ribosyl)ation in germ cell differentiation, presumably to safeguard DNA integrity.

Spermatogonial proliferation patterns in men with azoospermia of different etiologies

Objective To demonstrate the pattern(s) of spermatogonial proliferation in different spermatogenic disorders. Design Retrospective case-control study. Setting Teaching hospital. Patient(s) Azoospermic men who underwent testicular biopsy for sperm recovery and preparation for intracytoplasmic sperm injection. Intervention(s) Testicular biopsy evaluation by quantitative immunohistochemistry for proliferating cell nuclear antigen (PCNA). Main outcome measure(s) The expression of PCNA in spermatogonia as an index of proliferating activity in testes with focal spermatogenesis, spermatocyte maturation arrest, or normal spermatogenesis. Result(s) In biopsies with focal spermatogenesis (11 men), there was a statistically significant reduction of PCNA-labeled spermatogonia in seminiferous tubules showing spermatocyte arrest compared with the expression in adjacent tubules with advanced spermatogenic stage or in normal spermatogenesis (obstructive azoospermia, six men). However, PCNA expression in tubules of the group with complete maturation arrest (six men) was significantly higher compared with the same spermatogenic defect—spermatocyte arrest—within focal spermatogenesis biopsies. Conclusion(s) Different causes underlie the spermatogenic disorders reported in this study. In focal spermatogenesis, the disorder is associated with the presence of mitotic inactive spermatogonia. The detection of normal active spermatogonia in the spermatocyte arrest group indicates that the spermatogenic defect, which is accompanied by meiosis impairment, is not related to a malfunction of spermatogonial proliferation.

Sertoli cell maturation in men with azoospermia of different etiologies

Objective: To evaluate the involvement of Sertoli cell in different spermatogenic disorders. Design: Retrospective case-control study. Setting: Teaching hospital. Patient(s): Azoospermic men who underwent testicular biopsy for sperm recovery in preparation for intracytoplasmic sperm injection. Intervention(s): Testicular biopsy evaluation by quantitative immunohistochemistry for the immature Sertoli cell markers anti-Müllerian hormone and cytokeratin 18 (CK-18). Main Outcome Measure(s): Relative area of immature Sertoli cells in testes with focal spermatogenesis, spermatocyte maturation arrest, or normal spermatogenesis. Result(s): The relative area occupied by immature Sertoli cells, as revealed by anti-Müllerian hormone and CK-18 expression, was highest in the 11 men with focal spermatogenesis. In the group representing normal spermatogenesis (obstructive azoospermia, 6 men) and in the group characterized by spermatocyte maturation arrest (6 men), the areas occupied by anti-Müllerian hormone- and CK-18-positive cells were minimal. Conclusion(s): Different etiologies underlie the spermatogenic disorders reported in this study. In focal spermatogenesis with high anti-Müllerian hormone and CK-18 expression, the spermatogenic impairment is associated with the presence of immature Sertoli cells. The detection of normal mature Sertoli cells in the spermatocyte maturation arrest group indicates that the spermatogenic defect that is accompanied by an impairment of meiosis is intrinsic to the germ line without affecting Sertoli cell differentiation.

Evaluation of meiotic impairment of azoospermic men by fluorescence in situ hybridization

Objective: To identify predictive criteria for the existence of spermatogenesis in nonobstructive azoospermic men. Design: Prospective study. Setting: Andrology laboratory at a teaching hospital. Patient(s): Twenty-two azoospermic men were divided into three groups by qualitative testicular histopathology and the presence of spermatozoa in minced biopsies. Intervention(s): Testicular biopsies evaluation. Main Outcome Measure(s): The presence of spermatozoa and/or mature spermatids, the percentage of sex vesicle formation (X and Y chromosomes in proximity), and the pairing of the two 18 homologous chromosomes. Result(s): Spermatozoa and mature spermatids were found in 17 study patients. Whenever few mature spermatids and/or spermatozoa were found, the rates of X-Y and 18 bivalents were significantly higher (mean ± SD, 73% ± 13.3% and 91% ± 7.1%) than those in cases of spermatocyte maturation arrest (23% ± 8.0% and 60% ± 11.8%, respectively). Conclusion(s): Pairing of chromosomes during meiosis is apparently related to the progression of spermatogenesis. Consequently, high rates of bivalent formation increase the prospect of focal spermatogenesis in the testis, despite the failure to identify mature spermatids in the specific testicular biopsy under examination.

The role of autosomal cell apoptosis regulator genes in human spermatogenesis.

Immunohistochemical analysis of the expression of the autosomal apoptosis regulator genes, BAX, bcl-2, p53, and cyclic-AMP responsive element modulator (CREM) in testis biopsies from infertile men demonstrated that BAX, bcl-2, and p53 immunoreactivity was absent irrespective of seminiferous tubule histology. In contrast, cell-specific CREM immunoreactivity in round spermatids, with complete absence of CREM in patient biopsies showing spermatocyte maturation arrest and Sertoli cell only, was evident, suggesting a possible role of autosomal genes in the regulation of apoptosis in human spermatogenesis regulation.

Absence of Cyclic Adenosine 3′:5′ Monophosphate Responsive Element Modulator Expression at the Spermatocyte Arrest Stage

Objective: To test the hypotheses that variations in the expression of adenosine 3′:5′ monophosphate (cAMP) responsive element modulator are found in human seminiferous epithelium in men with impaired testicular function and subsequent infertility and that variations in apoptosis frequency are associated with differential cAMP responsive element modulator expression in male infertility states. Design: Standard immunohistochemical staining using a rabbit polyclonal antibody against the τ isoform of the cAMP responsive element modulator protein was performed on 5-μM sections of Bouin’s fixed, paraffin-embedded testicular tissue obtained from azoospermic or severely oligozoospermic men for routine clinical purposes. Histologic diagnosis was confirmed with computerized image analysis of Feulgen-stained sections. Setting: Tertiary male infertility referral center at a medical school. Patient(s): Forty-eight testis biopsies were performed in 38 azoospermic or severely oligozoospermic males. Intervention(s): Rabbit polyclonal cAMP responsive element modulator τ antibody was applied to the paraffin-embedded testis sections. Main Outcome Measure(s): Testis immunoreactivity to polyclonal cAMP responsive element modulator τ antibody and apoptotic indices. Result(s): Although cAMP responsive element modulator immunoreactivity was present in the round spermatid stage of meiosis in testis biopsy specimens showing normal spermatogenesis, spermatid maturation arrest, and hypospermatogenesis, there was complete absence of expression in biopsy specimens from patients with Sertoli cell only and spermatocyte maturation arrest states. In addition, significantly increased apoptotic indices were observed in the spermatocyte maturation arrest state in comparison with normal spermatogenesis and Sertoli cell only pattern. Conclusion(s): These data suggest that cAMP responsive element modulator may be important for spermatid development and a stage-specific regulator of human spermatogenesis. Absence of cAMP responsive element modulator may be a cause of testicular failure in various types of male infertility.

Immunohistochemical localization of calmodulin in the testes of patients with idiopathic male infertility.

The localization of calmodulin in testes of patients with idiopathic male infertility was studied using the indirect immunoperoxidase method. Specimens were obtained by testicular biopsy from 55 patients. They were divided into 26 cases of hypospermatogenesis, 11 cases of maturation arrest (8 of primary spermatocyte arrest and 3 of spermatid arrest) and 18 cases of Sertoli cell-only syndrome. Regardless of the type of testicular pathology, the types of immunoreactive cell and the intensities of staining were the same as those in the normal testis. That is, staining for calmodulin was first found to be positive in early pachytene primary spermatocytes. It became intense in late pachytene primary spermatocytes and round spermatids. By contrast, elongated spermatids and spermatozoa were not stained. Sertoli cells were stained slightly or not at all. A calmodulin-staining index (CaM-S index) was defined as the proportion of primary spermatocytes that were stained intensely for calmodulin relative to the total number of primary spermatocytes. The indices for the testes of men with complete spermatocyte maturation arrest were significantly lower than those for the testes of normal controls and of men with hypospermatogenesis. Degenerating late pachytene spermatocytes observed in the testes of men with spermatocyte arrest showed low calmodulin-specific immunoreactivity. Such a decrease in numbers of normal late pachytene spermatocytes might be responsible for the low CaM-S index in cases of complete spermatocyte arrest.