Non-obstructive Azoospermia Patient Research Articles

Evaluation of an artificial intelligence-facilitated sperm detection tool in azoospermic samples for use in ICSI

Research questionCan artificial intelligence (AI) improve the efficiency and efficacy of sperm searches in azoospermic samples? DesignThis two-phase proof-of-concept study began with a training phase using eight azoospermic patients (>10,000 sperm images) to provide a variety of surgically collected samples for sperm morphology and debris variation to train a convolutional neural network to identify spermatozoa. Second, side-by-side testing was undertaken on two cohorts of non-obstructive azoospermia patient samples: an embryologist versus the AI identifying all the spermatozoa in the still images (cohort 1, n = 4), and a side-by-side test with a simulated clinical deployment of the AI model with an intracytoplasmic sperm injection microscope and the embryologist performing a search with and without the aid of the AI (cohort 2, n = 4). ResultsIn cohort 1, the AI model showed an improvement in the time taken to identify all the spermatozoa per field of view (0.02 ± 0.30 × 10–5s versus 36.10 ± 1.18s, P < 0.0001) and improved recall (91.95 ± 0.81% versus 86.52 ± 1.34%, P < 0.001) compared with an embryologist. From a total of 2660 spermatozoa to find in all the samples combined, 1937 were found by an embryologist and 1997 were found by the AI in less than 1000th of the time. In cohort 2, the AI-aided embryologist took significantly less time per droplet (98.90 ± 3.19 s versus 168.7 ± 7.84 s, P < 0.0001) and found 1396 spermatozoa, while 1274 were found without AI, although no significant difference was observed. ConclusionsAI-powered image analysis has the potential for seamless integration into laboratory workflows, to reduce the time to identify and isolate spermatozoa from surgical sperm samples from hours to minutes, thus increasing success rates from these treatments.

A bi-allelic REC114 loss-of-function variant causes meiotic arrest and nonobstructive azoospermia.

Nonobstructive azoospermia (NOA), the most severe manifestation of male infertility, lacks a comprehensive understanding of its genetic etiology. Here, a bi-allelic loss-of-function variant in REC114 (c.568C > T: p.Gln190*) were identified through whole exome sequencing (WES) in a Chinese NOA patient. Testicular histopathological analysis and meiotic chromosomal spread analysis were conducted to assess the stage of spermatogenesis arrested. Co-immunoprecipitation (Co-IP) and Western blot (WB) were used to investigate the influence of variant in vitro. In addition, our results revealed that the variant resulted in truncated REC114 protein and impaired interaction with MEI4, which was essential for meiotic DNA double-strand break (DSB) formation. As far as we know, this study presents the first report that identifies REC114 as the causative gene for male infertility. Furthermore, our study demonstrated indispensability of the REC114-MEI4 complex in maintaining DSB homoeostasis, and highlighted that the disruption of the complex due to the REC114 variant may underline the mechanism of NOA.

Can evaluation of seminal plasma and serum anti-Müllerian hormone be useful in the diagnosis of male infertility? (Literature review)

Background. Anti-Müllerian hormone (AMH) is secreted in men by Sertoli cells and plays an important role in the development of the male reproductive organs. High expression of AMH during embryonic development contributes to the regression of the Müllerian (paramesonephric) ducts; in the absence of AMH, the Müllerian ducts develop into the female internal genital organs. In adulthood, the clinical value of measuring AMH in serum and seminal plasma remains controversial. It is assumed that AMH, as a marker of the functional state of Sertoli cells, can be useful in the diagnosis of male infertility.Aim. To determine the clinical value of measuring serum and seminal plasma AMH levels in the diagnosis of male infertility by examining the current evidence base in the world literature.Materials and methods. A search was made for publications devoted to the study of the role of AMH in the diagnosis of male infertility in the international database PubMed, in the scientific electronic library eLIBRARY.RU and in the Google Scholar search engine. There were no restrictions regarding the status and period of publication.Results and conclusion. The results of the analysis indicate the absence of a significant relationship between the parameters of the ejaculate and the serum concentration of AMH, which does not allow using this indicator to assess male fertility. Serum AMH is of no value in the differential diagnosis of obstructive and non-obstructive azoospermia, the prediction of assisted reproductive technologies outcomes, and the prediction of testicular biopsy outcomes in the general non-obstructive azoospermia patient population. It is likely that serum AMH may be useful in predicting testicular biopsy results in patients with Klinefelter’s syndrome and idiopathic non-obstructive azoospermia.The clinical application of measuring the level of AMH in seminal plasma is limited by the high variability of this indicator, probably due to the dependence on the volume of the ejaculate and exposure to semen proteolytic enzymes. This indicator may be useful for the differential diagnosis of obstructive and non-obstructive azoospermia. In obstructive azoospermia, AMH is not detected in seminal plasma, but undetectable AMH concentrations can also be observed in patients with non-obstructive azoospermia, which reduces the differential value of this indicator. A promising direction is the use of AMH concentration in seminal plasma to predict the recovery of sperm motility after cryopreservation.

Factors associated with undergoing microdissection testicular sperm extraction among men with non-obstructive azoospermia following evaluation by a reproductive urologist.

Microdissection testicular sperm extraction (mTESE) is the gold standard treatment for men with non-obstructive azoospermia (NOA). However, many men do not elect to pursue this surgical intervention. We aimed to identify factors associated with NOA patients undergoing mTESE after initial evaluation by a reproductive urologist (RU) through a retrospective cohort study. We retrospectively reviewed NOA patient who underwent evaluation by a RU between 2002-2018. Demographic and clinical data were collected. Our primary outcome was electing to undergo mTESE. 44.4% (75/169) of NOA men underwent mTESE. These patients earned significantly higher median neighborhood income ($133,000 vs. $97,000, P<0.001), spent fewer years trying to conceive before seeking care {1.3 [interquartile range (IQR): 1-3] vs. 2.3 (IQR: 1-5), P=0.012}, and were more likely to be married (79.7% vs. 53.9%, P=0.001). On univariate analysis, married men [odds ratio (OR) 3.37, 95% confidence interval (CI): 1.67-6.79, P=0.001] and men with higher neighborhood income (OR 1.14, 95% CI: 1.06-1.21, P<0.001) were more likely to undergo mTESE, while couples attempting to conceive for a longer period of time prior to initial evaluation were less likely to undergo mTESE (OR 0.79, 95% CI: 0.68-0.92, P=0.003). On multivariable regression analysis, marital status and years attempting to conceive remained significantly associated with NOA patients undergoing mTESE (OR 4.61, 95% CI: 1.16-18.25, P=0.03; OR 0.67, 95% CI: 0.52-0.88, P=0.003, respectively). Higher neighborhood income and marital status were positively associated with patients undergoing mTESE, while couples who attempted to conceive for a longer period of time before seeking infertility care were less likely to undergo mTESE.

A 3-Gene Random Forest Model to Diagnose Non-obstructive Azoospermia Based on Transcription Factor-Related Henes.

Non-obstructive azoospermia (NOA) is one of the most severe forms of male infertility, but its diagnosis biomarkers with high sensitivity and specificity are largely unknown. Transcription factors (TFs) play essential roles in many pathological processes in different diseases. Herein, we aimed to identify the TFs showing high diagnosis ability for NOA through machine learning algorithms. The transcriptome data of the testicular tissue from 11 control and 47 NOA subjects were set as the training dataset; meanwhile, 1665 TFs were retrieved from the HumanTFDB. Through the feature extraction methods, including genomic difference analysis, Lasso, Boruta, SVM-RFE, and logistic regression, ETV2, TBX2, and ZNF689 were ultimately screened and then were included in the random forest (RF) diagnosis model. The RF model displayed high predictive power in the training (F-measure = 1) and two external validation (n = 31, F-measure = 0.902; n = 20, F-measure = 0.941) cohorts. The seminal plasma and testicular biopsy samples of 20 control and 20 NOA patients were collected from the local hospital, and the expression levels of ETV2, TBX2, and ZNF689 were measured via RT-qPCR and immunohistochemistry. The RF model could also distinguish the NOA samples in the local cohort (F-measure = 0.741). Single-cell RNA sequencing analysis, which was based on the 432 testicular cell samples from an NOA patient, showed that ETV2, TBX2, and ZNF689 were all significantly associated with spermatogenesis. In all, a 3-TF random forest diagnosis model was successfully established, providing novel insights into the latent mechanisms of NOA.

Novel exon mutation in SYCE1 gene is associated with non-obstructive azoospermia.

Non‐obstructive azoospermia (NOA) is a common cause of male infertility, and genetic problems, such as chromosomal abnormalities and gene mutations, are important causes of NOA. Our centre received a case of NOA, in which no mature sperm was found during microdissection testicular sperm extraction. A postoperative pathological examination revealed that testicular spermatogenesis was blocked. Target region capture combined with high‐throughput sequencing was used to screen for male infertility‐related gene mutations. Sanger sequencing further confirmed that the SYCE1 gene, a central component of the synaptonemal complex (SC) during meiosis, had a homozygous deletion mutation in the tenth exon (c.689_690del; p.F230fs). Through molecular biological studies, we discovered altered expression and nuclear localization of the endogenous mutant SYCE1. To verify the effects in vitro, wild‐ and mutated‐type SYCE1 vectors were constructed and transfected into a human cell line. The results showed that the expression and molecular weight were decreased for SYCE1 containing c.689_690del. In addition, mutated SYCE1 was abnormally located in the cytoplasm rather than in the nucleus. In summary, our research suggests that the novel homozygous mutation (c.689_690del; p.F230fs) altered the SYCE1 expression pattern and may have disturbed SC assembly, leading to male infertility and to a barrier to gamete formation. We reported for the first time that a frameshift mutation occurred in the exon region of SYCE1 in an NOA patient. This study is beneficial for accurate NOA diagnosis and the development of corresponding gene therapy strategies.

Deciphering the autophagy regulatory network via single-cell transcriptome analysis reveals a requirement for autophagy homeostasis in spermatogenesis

Background: Autophagy has been implicated as a crucial component in spermatogenesis, and autophagy dysfunction can lead to reproductive disorders in animal models, including yeast, C. elegans and mice. However, the sophisticated transcriptional networks of autophagic genes throughout human spermatogenesis and their biological significance remain largely uncharacterized.Methods: We profiled the transcriptional signatures of autophagy-related genes during human spermatogenesis by assessing specimens from nine fertile controls (including two normal persons and seven obstructive azoospermia (OA) patients) and one nonobstructive azoospermia (NOA) patient using single-cell RNA sequencing (scRNA-seq) analysis. Dysregulation of autophagy was confirmed in two additional NOA patients by immunofluorescence staining. Gene knockdown was used to identify the role of Cst3 in autophagy during spermatogenesis.Results: Our data uncovered a unique, global stage-specific enrichment of autophagy-related genes. Human-mouse comparison analysis revealed that the stage-specific expression pattern of autophagy-related genes was highly conserved in mammals. More importantly, dysregulation of some clusters of autophagy-related genes was observed in NOA patients, suggesting the association of autophagy with male infertility. Cst3, a human-mouse conserved and autophagy-related gene that is actively expressed in spermatogonia and early spermatocytes, was found to regulate spermatogonial stem cell (SSC) maintenance and subsequent male germ cell development. Knockdown of Cst3 increased autophagic activity in mouse SSCs and subsequently suppressed the transcription of SSC core factors such as Oct4, Id1, and Nanos3, which could be efficiently rescued by manipulating autophagic activity.Conclusions: Our study provides comprehensive insights into the global transcriptional signatures of autophagy-related genes and confirms the importance of autophagy homeostasis in SSC maintenance and normal spermatogenesis, opening new avenues for further dissecting the significance of the autophagy regulatory network in spermatogenesis as well as male infertility.

Disruption of human meiotic telomere complex genes TERB1, TERB2 and MAJIN in men with non-obstructive azoospermia.

Non-obstructive azoospermia (NOA), the lack of spermatozoa in semen due to impaired spermatogenesis affects nearly 1% of men. In about half of cases, an underlying cause for NOA cannot be identified. This study aimed to identify novel variants associated with idiopathic NOA. We identified a nonconsanguineous family in which multiple sons displayed the NOA phenotype. We performed whole-exome sequencing in three affected brothers with NOA, their two unaffected brothers and their father, and identified compound heterozygous frameshift variants (one novel and one extremely rare) in Telomere Repeat Binding Bouquet Formation Protein 2 (TERB2) that segregated perfectly with NOA. TERB2 interacts with TERB1 and Membrane Anchored Junction Protein (MAJIN) to form the tripartite meiotic telomere complex (MTC), which has been shown in mouse models to be necessary for the completion of meiosis and both male and female fertility. Given our novel findings of TERB2 variants in NOA men, along with the integral role of the three MTC proteins in spermatogenesis, we subsequently explored exome sequence data from 1495 NOA men to investigate the role of MTC gene variants in spermatogenic impairment. Remarkably, we identified two NOA patients with likely damaging rare homozygous stop and missense variants in TERB1 and one NOA patient with a rare homozygous missense variant in MAJIN. Available testis histology data from three of the NOA patients indicate germ cell maturation arrest, consistent with mouse phenotypes. These findings suggest that variants in MTC genes may be an important cause of NOA in both consanguineous and outbred populations.

Corrigendum. Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

Corrigendum. Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

Factors influencing sperm retrieval rate by Microdissection Testicular Sperm Extraction (mTESE) in non-obstructive azoospermia cases: a literature review

Background:Â Non-Obstructive Azoospermia (NOA) had dominating 60% of all azoospermia cases. Sperm Retrieval Techniques (SRT) continued with Intra-Cytoplasmic Sperm Injection (ICSI) are therapy modalities helping infertile azoospermia to be fertile. There are some options of SRT such as PESA, MESA, TESE, TESA and microdissection-TESE (mTESE) to collect sperm from azoospermia cases. This study aims to evaluate some factors influencing sperm retrieval rate by mTESE in non-obstructive azoospermia cases.Method:Â The data collection for eligible articles were conducted from 2011 to 2019. Different database and manual search methods were used to find the topic-related articles, particularly in some factors influencing the sperm retrieval rate of mTESE in NOA cases. A total of 21 relevant kinds of literature were studied regarding some factors that might be increasing or decreasing of sperm retrieval rate by mTESE procedure. However, four literature were elaborated further due to a similar approach to get the results.Results:Â A previous study from Saudi Arabia with 264 patients samples found significant different on testosterone level who had successful Sperm Retrieval Rate (SRR) 57.25%. SRR was related by several factors of testosterone, FSH, bilateral testis retrieval and seminiferous tubules diameter taken as the earliest study from India. Besides, taking bilateral testis sperm retrieval also had low SRR 14.4%, but serum FSH level was not influenced by increasing SRR. The previous study found a very weak correlation between FSH and SRR. A different study was conducted in Italy found that all NOA patient who undergone mTESE procedure found the Maturation Arrest (MA) has the significant higher success SRR until 67% after Sertoli Cell Only (SCO) syndrome and sclera-hyalinosis histology.Conclusion:Â SRR depends on some factors those influencing by hormonal baseline preoperative such as testosterone level and FSH serum level, technique and how mTESE done by the operator in mTESE procedure.

Does the FSHB c.‐211G&gt;T polymorphism impact Sertoli cell number and the spermatogenic potential in infertile patients?

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. To determine the impact of the SNP FSHB c.-211G>T on Sertoli cell (SC) number, Sertoli cell workload (SCWL) and thereby spermatogenic potential. 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. 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. 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.

FOXP3 pathogenic variants cause male infertility through affecting the proliferation and apoptosis of human spermatogonial stem cells.

Genetic causes of male infertility that is associated with aging are largely unknown. This study was designed to identify novel pathogenic variants of FOXP3 gene causing azoospermia. One homozygous (c.155 G > T) pathogenic variant of FOXP3 was identified in nine non-obstructive azoospermia patients, and one heterozygous (c.691 C > A) of FOXP3 was found in one non-obstructive azoospermia patient. Pedigrees studies indicated that the homozygous (c.155 G > T) FOXP3 pathogenic variant was inherited, while heterozygous (c.691 C > A) FOXP3 pathogenic variant was acquired. Human testis carrying pathogenic variant exhibited abnormal spermatogenesis. FOXP3 protein was expressed at a lower level or undetected in spermatocytes of mutant testis of non-obstructive azoospermia patients compared to obstructive azoospermia patients. FOXP3 stimulated the proliferation and inhibited the apoptosis of human spermatogonial stem cells, and we further analyzed the targets of FOXP3. We have identified two new pathogenic variants of FOXP3 in non-obstructive azoospermia patients with high incidence, and FOXP3 silencing inhibits the proliferation and enhances the apoptosis of human spermatogonial stem cells. This study provides new insights into the etiology of azoospermia and offers novel pathogenic variants for gene targeting of male infertility.

Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

What is the diagnostic potential of next generation sequencing (NGS) based on a 'mouse azoospermia' gene panel in human non-obstructive azoospermia (NOA)? The diagnostic performance of sequencing a gene panel based on genes associated with mouse azoospermia was relatively successful in idiopathic NOA patients and allowed the discovery of two novel genes involved in NOA due to meiotic arrest. NOA is a largely heterogeneous clinical entity, which includes different histological pictures. In a large proportion of NOA, the aetiology remains unknown (idiopathic NOA) and yet, unknown genetic factors are likely to play be involved. The mouse is the most broadly used mammalian model for studying human disease because of its usefulness for genetic manipulation and its genetic and physiological similarities to man. Mouse azoospermia models are available in the Mouse Genome Informatics database (MGI: http://www.informatics.jax.org/). The first step was to design of a 'mouse azoospermia' gene panel through the consultation of MGI. The second step was NGS analysis of 175 genes in a group of highly selected NOA patients (n = 33). The third step was characterization of the discovered gene defects in human testis tissue, through meiotic studies using surplus testicular biopsy material from the carriers of the RNF212 and STAG3 pathogenic variants. The final step was RNF212 and STAG3 expression analysis in a collection of testis biopsies. From a total of 1300 infertile patients, 33 idiopathic NOA patients were analysed in this study, including 31 unrelated men and 2 brothers from a consanguineous family. The testis histology of the 31 unrelated NOA patients was as follows: 20 Sertoli cell-only syndrome (SCOS), 11 spermatogenic arrest (6 spermatogonial arrest and 5 spermatocytic arrest). The two brothers were affected by spermatocytic arrest. DNA extracted from blood was used for NGS on Illumina NextSeq500 platform. Generated sequence data was filtered for rare and potentially pathogenic variants. Functional studies in surplus testicular tissue from the carriers included the investigation of meiotic entry, XY body formation and metaphases by performing fluorescent immunohistochemical staining and immunocytochemistry. mRNA expression analysis through RT-qPCR of RNF212 and STAG3 was carried out in a collection of testis biopsies with different histology. Our approach was relatively successful, leading to the genetic diagnosis of one sporadic NOA patient and two NOA brothers. This relatively high diagnostic performance is likely to be related to the stringent patient selection criteria i.e. all known causes of azoospermia were excluded and to the relatively high number of patients with rare testis histology (spermatocytic arrest). All three mutation carriers presented meiotic arrest, leading to the genetic diagnosis of three out of seven cases with this specific testicular phenotype. For the first time, we report biallelic variants in STAG3, in one sporadic patient, and a homozygous RNF212 variant, in the two brothers, as the genetic cause of NOA. Meiotic studies allowed the detection of the functional consequences of the mutations and provided information on the role of STAG3 and RNF212 in human male meiosis. All genes, with the exception of 5 out of 175, included in the panel cause azoospermia in mice only in the homozygous or hemizygous state. Consequently, apart from the five known dominant genes, heterozygous variants (except compound heterozygosity) in the remaining genes were not taken into consideration as causes of NOA. We identified the genetic cause in approximately half of the patients with spermatocytic arrest. The low number of analysed patients can be considered as a limitation, but it is a very rare testis phenotype. Due to the low frequency of this specific phenotype among infertile men, our finding may be considered of low clinical impact. However, at an individual level, it does have relevance for prognostic purposes prior testicular sperm extraction. Our study represents an additional step towards elucidating the genetic bases of early spermatogenic failure, since we discovered two new genes involved in human male meiotic arrest. We propose the inclusion of RNF212 and STAG3 in a future male infertility diagnostic gene panel. Based on the associated testis phenotype, the identification of pathogenic mutations in these genes also confers a negative predictive value for testicular sperm retrieval. Our meiotic studies provide novel insights into the role of these proteins in human male meiosis. Mutations in STAG3 were first described as a cause of female infertility and ovarian cancer, and Rnf212 knock out in mice leads to male and female infertility. Hence, our results stimulate further research on shared genetic factors causing infertility in both sexes and indicate that genetic counselling should involve not only male but also female relatives of NOA patients. This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS (grant number: FIS/FEDER-PI14/01250; PI17/01822) awarded to CK and AR-E, and by the European Commission, Reproductive Biology Early Research Training (REPROTRAIN, EU-FP7-PEOPLE-2011-ITN289880), awarded to CK, WB, and AE-M. The authors have no conflict of interest.

A new MEIOB mutation is a recurrent cause for azoospermia and testicular meiotic arrest.

Are there genetic variants that can be used for the clinical evaluation of azoospermic men? A novel homozygous frame-shift mutation in the MEIOB gene was identified in three azoospermic patients from two different families. Up to 1% of all men have complete absence of sperm in the semen, a condition known as azoospermia. There are very few tools for determining the etiology of azoospermia and the likelihood of sperm cells in the testis. The MEIOB gene codes for a single-strand DNA binding protein required for DNA double-strand breaks repair during meiosis. MEIOB appears to be exclusively expressed in human and mouse testis, and MeioB knockout mice are azoospermic due to meiotic arrest. Two brothers with non-obstructive azoospermia (NOA) underwent whole-exome sequencing followed by comprehensive bioinformatics analyses. Candidate variations were further screened in infertile and fertile men, as well as in public and local reference databases. This study included 159 infertile and 77 fertile men. The exomes of two Arab men were completely sequenced. In addition, 213 other men of the same Arab ethnicity (136 infertile and 77 fertile men) underwent restriction fragment length polymorphism (RFLP) screening, as did 21 NOA men, of other ethnicities, with testicular impairment of spermatocyte arrest. All of the infertile men underwent Y-chromosome microdeletion and CFTR gene mutation assessments. Comprehensive bioinformatics analyses were designed to uncover candidate mutations associated with azoospermia. A novel homozygous frame-shift mutation in the MEIOB gene was identified in two brothers of Arab ethnicity. This frame-shift is predicted to result in a truncated MEIOB protein, which lacks the conserved C-terminal DNA binding domain. RFLP screening of the mutation in 157 infertile men, including 112 NOA patients of Arab ethnicity, identified an additional unrelated NOA patient with the same homozygous mutation and a similar testicular impairment. This mutation was not found in available public databases (n > 160 000), nor in the 77 proven fertile men, nor in our database of local Israeli population variations derived from exome and genome sequencing data (n = 500). We have thus far screened for only two specific MEIOB probable pathogenic mutations in a relatively small local cohort. Therefore, the relative incidence of MEIOB mutations in azoospermia should be further assessed in larger and diverse cohorts in order to determine the efficiency of MEIOB sequence screening for clinical evaluations. The relatively high incidence of likely NOA-causing mutations in MEIOB that was found in our cohort supports the idea that a complete screening of this gene might be beneficial for clinical evaluation of NOA patients. This research was supported in part by a grant to EA from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC grant agreement (616088). There are no competing interests. N/A.

Testicular piRNA profile comparison between successful and unsuccessful micro-TESE retrieval in NOA patients.

PIWI-interacting RNA (piRNA) is a sub-group of small RNAs about 30 nucleotides length which specifically expressed in mammalian germ cells. Although piRNAs play pivotal roles in spermatogenesis regulation, little is known in the testicular tissues of infertile men. To explore whether piRNA profile could serve as a biomarker for male infertility diagnosis in a clinic, in this study, we systematically investigated the expression profile of piRNAs in testicular tissues from the patients with non-obstructive azoospermia (NOA) between successful and unsuccessful sperm retrieval before micro-dissection testicular sperm extraction (micro-TESE). The differential expression levels of piRNAs were evaluated using small RNA-Seq method. Ontologic analyses were performed to determine the presence of enriched biological processes. A total of 18,324 Homo sapiens piRNAs were identified by small RNA-Seq from NOA patient testicular tissues; among them, 959 piRNAs were significantly altered between successful and unsuccessful sperm retrieval groups, of which 951 testicular piRNAs were significantly downregulated and 8 piRNAs were upregulated in NOA patients with unsuccessful sperm retrieval (USR) groups compared to those with successful sperm retrieval (SSR) groups, respectively. Unexpectedly, 553 testicular piRNAs were found completely absent in USR but showing abundant in SSR, which suggests that those piRNAs might serve as a biomarker for micro-TESE application. A total of 20 significantly differential piRNAs (hsa-piR-20830, hsa-piR-4731, hsa-piR-6254, hsa-piR-419, hsa-piR-7152, hsa-piR-7548, hsa-piR-14195, hsa-piR-5026, hsa-piR-11482, hsa-piR-17765, hsa-piR-17102, hsa-piR-4484, hsa-piR-17260, hsa-piR-17098, hsa-piR-20511, hsa-piR-5802, hsa-piR-19121, hsa-piR-2510, hsa-piR-4745, hsa-piR-11873) were selected to further validate the RNA-Seq data by quantitative real-time polymerase chain reaction. In addition, bioinformatic analyses revealed that those altered piRNAs were involved in many important biological pathways, including apoptosis, cell proliferation, and differentiation. Our results demonstrate that testicular tissues from NOA patients with successful and unsuccessful spermatozoa retrieval exhibit differential piRNA profiles. This study provides a useful resource to further elucidate the regulatory role of piRNAs in spermatogenesis and provides a profound clue to identify useful biomarkers for predicting residual spermatogenic loci in NOA patients during assisted reproductive treatment.

Identification of Spermatogonial Stem Cell-Like Cell Differentiation In Vitro from Azoospermia Patient Using Modified Human Embryonic Stem Cell (HESC) Media

Background: Spermatogenesis is a process where proliferation and differentiation of spermatogonial stem cell (SSC) into haploid sperm cells in the testis. Azoospermia patients have impaired spermatogenesis where they have difficulty to get normal sperm. The purpose of this study is to proliferate and differentiate SSC-like cell in in vitro culture from azoospermia patient samples and to determine the pattern of gene expressions involved. Methods and Results: Testicular biopsy tissue obtained from obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) patients were dissociated and cultured in different human embryonic stem cell (HESC) media added with specific growth factors and reproductive hormones according to the day of culture using 24 well plate dishes. SSC-like cell colonies were formed after 5 weeks of culture. Gene expression analyses were done on day 49 and day 90 using quantitative reverse transcription polymerase chain reaction (q-RT PCR). OCT4 was detected in NOA patient and ITGB1 was identified in OA patient for 49 days of culture. Gene expression for all stages of spermatogenic cells in day 90 were mostly detected in OA patient; ITGB1, SCP3, H2B and TNP1. However, gene TNP1 was only expressed in NOA patient shown spermatid formation stage. Conclusions: Our result shown that there is a potential to develop sperm in vitro culture from testis biopsy of azoospermic patients for further clinical application.

Editorial commentary: How to predict fatherhood for men with non-obstructive azoospermia opting for TESE-ICSI?

The treatment of male factor infertility, due to non-obstructive azoospermia (NOA), by testicular sperm extraction (TESE)–ICSI is a major breakthrough in assisted reproduction. However, although the first patient series on this approach were published more than 15 years ago (Tournaye et al., 1995), to date, it still remains difficult to provide candidate patients with truthful data about their chances to eventually father their genetically own child. In this issue, Boitrelle et al. (2011) published a study trying to determine predictive parameters to improve patient counselling. Retrieval rates after testicular surgery reported in the literature differ considerably. While in unselected NOA patients, the surgical approach seems to have a limited effect as long as multiple biopsies are taken whenever needed, the extended processing of the wet preparations of the testicular tissue may have a greater impact on sperm recovery. Unfortunately, large well-designed controlled trials to support both assertions are lacking. But sperm retrieval rates are also subject to the (pre)selection of patients and can be biased either by re-allocating successful patients in case series, either by including patients showing almost normal spermatogenesis, e.g. mild-to-moderate hypospermatogenesis, or by inclusion of patients with no testicular histology available. As a result of the above, retrieval rates reported in the literature for NOA men may vary from about 30% to even more than 80%. Larger case studies in well-defined NOA populations report sperm recovery rates after a first TESE attempt around 50%. However, given the invasive character of TESE, NOA patients want to have a better prediction of their chances to retrieve testicular sperm than tossing a coin. Because testicular volume and serum FSH are routinely assessed in azoospermic men, these parameters are often used in studies on prediction either as a stand-alone parameter or in combination with other parameters. Unfortunately, their predictive power remains limited and is subject to the heterogeneity of the population of NOA patients studied. Idem dito for the predictive parameters published in the study by Boitrelle et al. (2011). The positive likelihood ratios for the stand-alone parameters are below 2 and hence not of a great diagnostic value in predicting testicular sperm recovery. With a positive likelihood ratio of 3, a predictive score combining testicular volume, FSH and inhibin-B looks more promising in their setting. But again, is this a robust predictive model applicable to every population of NOA men? All NOA men with all parameters included in the score available were selected in our Brussels database. Although we refrained from assessing inhibin-B routinely, after concluding that this parameter had a poor predictive value (Vernaeve et al., 2002), the score could be calculated in a population of 110 NOA men that had their first TESE attempt and showed neither normal spermatogenesis nor hypospermatogenesis on their testicular histologies. Compared with the study by Boitrelle et al. (2011), our sperm retrieval rates were, respectively, 50.0% compared with 77.4% in men with a score below 18.5 (n 1⁄4 38) and 57.1% compared with 39.7% in men with a score between 18.5 and 3700 (n 1⁄4 72). Our ‘test’ population did not include men with a score higher than 3700. This ‘test’ emphasizes that any blind application of a prediction model on a population of patients, other than the one it originated from, remains difficult due to patient heterogeneity. Whether out-of-routine assessmentsmay improve the prediction of a successful testicular recovery also remains to be proven. Doppler ultrasoundor even invasive assessments such as ‘testicularmapping’ by puncture provide low sensitivities. Since many NOA men do not accept donor insemination as an alternative for their fatherhood and because current predictive tests cannot exclude that any sperm will eventually be retrieved, to date, reliable counselling towards sperm retrieval remains problematic for all TESE candidates suffering from NOA. Counselling becomes even more difficult when an NOA patient would like to know what his chances are to have a child, which in the end is the true outcome of interest to candidate TESE–ICSI patients. At present, only fragmentary data exist either reporting on retrieval rates after TESE or reporting on the outcome of ICSI once testicular spermatozoa were obtained in different subsets of patients. Boitrelle et al. (2011) show that in their setting, the take-home baby rate in patients with a score over 3700 was low (7.7%) compared with lower-score patients (48.8%), a finding that still may suffer from bias because only a subset of patients was included in the subsequent ICSI analysis (89 out of 149 men in which testicular sperm were retrieved, i.e. 59%, eventually proceeded to ICSI). Again, the score can probably not be applied blindly in other TESE–ICSI programmes

Controversies in the management of nonobstructive azoospermia

The fertility potential of patients with nonobstructive azoospermia (NOA) depends on sperm extraction from the tissue sample and then in vitro fertilization with intracytoplasmic sperm injection (IVF/ICSI). Unfortunately, there is no consensus regarding predictors that can identify nonobstructive azoospermic men with a potentially high yield at the time of sperm extraction. This article analyzes two competing approaches to these patients: noninvasive and invasive. The noninvasive approach, based on clinical, laboratory, and ultrasonographic investigations, excludes from IVF/ICSI a significant number of patients owing to errors in predicting the presence of sufficient intratesticular spermatozoa. The invasive approach, with available percutaneous or surgical testicular biopsy techniques followed by morphologic examination and or sperm recovery, permits many patients with NOA to receive a favorable prognosis and therapeutic trial. However, the available testicular biopsy techniques are so variable that their performance parameters cannot be adequately compared. As a result, any progress in optimizing these techniques must involve delineation of specific selection criteria for each NOA patient.

Sperm retrieval and the predictive parameter of non-obstructive azoospermia: a meta-analysis of literatures 1990 to 2008

To investigate which sperm retrieval technique is suitable for the non-obstructive azoospermia (NOA) patient, and to identify the relevant predictive parameters. Literatures on NOA patients who had undergone sperm retrieval and pathological examination of testis were identified from Cochrane Library, CNKI and Medline (1990 to 2008) and analyzed. Twenty-five articles were enrolled. When testicular fine needle aspiration (TEFNA) was compared with testicular sperm extraction (TESE), the sperm retrieval rate of the former was 23.0%, significantly lower than that of the latter (52.2%, RR: 0.49, 95%CI: 0.41 - 0.60, P < 0.05); and when TESE was compared with micro-surgical testicular sperm extraction (mTESE), the sperm retrieval rate of the former was 35.7%, significantly lower than that of the latter (54.6%, RR: 0.70, 95%CI: 0.50 - 0.98, P < 0.05). Sperm retrieval rate was closely correlated with the testicular pathological category of the NOA patients. The sperm retrieval rates of the patients with hypospermatogenesis (HS), maturation arrest (MA), and Sertoli cell only syndrome (SCOS) were 76.7%, 46.2%, and 32.8% respectively (RR: 1.65, 2.40, 1.50; 95%CI: 1.21 - 2.91, 1.85 - 6.90, 1.02 - 2.26, P < 0.05). mTESE is the best sperm retrieval technique. A better to choice before deciding the treatment program of NOA patients is to identify the testicular pathological category in the NOA patients, and then to predict the outcome of TESE before assisted reproduction technology.

Testis Sperm Extraction (TESE) Is Effective in the Azoospermic Male with a History of Prior Chemotherapy

Objectives: Approximately 50% of the total group of men with non-obstructive azoospermia (NOA) will have sperm found within the testis parenchyma which can be used with ICSI to achieve biological paternity. Questions: what are the rates of sperm retrieval and pregnancy for subgroups with NOA, e.g., post-chemotherapy? The purpose of this study is to define the success of TESE and the pregnancy rates for only those men who are azoospermic due to chemotherapy so as to be able to counsel this type of NOA patient as best possible. Materials and Methods: 7 NOA men subsequent to chemotherapy and who underwent TESE comprise the study group. Harvested tissue was analyzed and cryopreserved if sperm were present and thawed homogenate used for subsequent cycles of ICSI. Age range: 28 to 43 years. legendAge at chemolegendC: cyclophosphamide; V: vincristine; D: doxorubicin; Chl: chlorambucil; Pro: procarbazine; P: prednisone; B: bleomycin; Cis: cisplatin.Reason for chemoChemo agents usedSperm present12Systemic LupusChlNo21Testis CancerCis, V, BNo4Nephrotic SyndromeC, P. ChlNo23Histiocytic lymphomaMechlorethamine, V, Pro, PYesPending24Hodgkin'sV, C, DYes7Twins lost at 25 weeks15Ewing's SarcomaV, D, Actinomycin DYes1Ongoing pregnancy15Testis CancerCis, Vp-16, BYes1Ongoing pregnancylegend C: cyclophosphamide; V: vincristine; D: doxorubicin; Chl: chlorambucil; Pro: procarbazine; P: prednisone; B: bleomycin; Cis: cisplatin. Open table in a new tab Results: Of the 7 men, 4 (57%) had sperm found during TESE. These 4 men were all pubertal or post-pubertal at the time of chemotherapy. The fertilization rate ranged from 58–80%. The twin aborteses were normal in appearance, premature rupture of membranes the cause. Conclusion: For the subgroup of men with NOA due to prior chemotherapy, TESE may be successful and pregnancy can be achieved. These are early and encouraging results but since there are so many types of chemotherapy agents used, large numbers of patients will be needed to give an individual patient an accurate assessment of his chances. In addition, the children will need to be followed carefully as there is no data as to their future health. Data that discusses naturally conceived offspring of post-chemotherapy men cannot be applied to this group as spermatogenesis in these men returned to good levels, unlike this study group who were more severely and permanently affected.