Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Describing patterns of familial cancer risk in subfertile men using population pedigree data.

STUDY QUESTION: Can we simultaneously assess risk for multiple cancers to identify familial multicancer patterns in families of azoospermic and severely oligozoospermic men? SUMMARY ANSWER: Distinct familial cancer patterns were observed in the azoospermia and severe oligozoospermia cohorts, suggesting heterogeneity in familial cancer risk by both type of subfertility and within subfertility type. WHAT IS KNOWN ALREADY: Subfertile men and their relatives show increased risk for certain cancers including testicular, thyroid, and pediatric. STUDY DESIGN, SIZE, DURATION: A retrospective cohort of subfertile men (N = 786) was identified and matched to fertile population controls (N = 5674). Family members out to third-degree relatives were identified for both subfertile men and fertile population controls (N = 337 754). The study period was 1966-2017. Individuals were censored at death or loss to follow-up, loss to follow-up occurred if they left Utah during the study period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Azoospermic (0 × 106/mL) and severely oligozoospermic (<1.5 × 106/mL) men were identified in the Subfertility Health and Assisted Reproduction and the Environment cohort (SHARE). Subfertile men were age- and sex-matched 5:1 to fertile population controls and family members out to third-degree relatives were identified using the Utah Population Database (UPDB). Cancer diagnoses were identified through the Utah Cancer Registry. Families containing ≥10 members with ≥1 year of follow-up 1966-2017 were included (azoospermic: N = 426 families, 21 361 individuals; oligozoospermic: N = 360 families, 18 818 individuals). Unsupervised clustering based on standardized incidence ratios for 34 cancer phenotypes in the families was used to identify familial multicancer patterns; azoospermia and severe oligospermia families were assessed separately. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to control families, significant increases in cancer risks were observed in the azoospermia cohort for five cancer types: bone and joint cancers hazard ratio (HR) = 2.56 (95% CI = 1.48-4.42), soft tissue cancers HR = 1.56 (95% CI = 1.01-2.39), uterine cancers HR = 1.27 (95% CI = 1.03-1.56), Hodgkin lymphomas HR = 1.60 (95% CI = 1.07-2.39), and thyroid cancer HR = 1.54 (95% CI = 1.21-1.97). Among severe oligozoospermia families, increased risk was seen for three cancer types: colon cancer HR = 1.16 (95% CI = 1.01-1.32), bone and joint cancers HR = 2.43 (95% CI = 1.30-4.54), and testis cancer HR = 2.34 (95% CI = 1.60-3.42) along with a significant decrease in esophageal cancer risk HR = 0.39 (95% CI = 0.16-0.97). Thirteen clusters of familial multicancer patterns were identified in families of azoospermic men, 66% of families in the azoospermia cohort showed population-level cancer risks, however, the remaining 12 clusters showed elevated risk for 2-7 cancer types. Several of the clusters with elevated cancer risks also showed increased odds of cancer diagnoses at young ages with six clusters showing increased odds of adolescent and young adult (AYA) diagnosis [odds ratio (OR) = 1.96-2.88] and two clusters showing increased odds of pediatric cancer diagnosis (OR = 3.64-12.63). Within the severe oligozoospermia cohort, 12 distinct familial multicancer clusters were identified. All 12 clusters showed elevated risk for 1-3 cancer types. An increase in odds of cancer diagnoses at young ages was also seen in five of the severe oligozoospermia familial multicancer clusters, three clusters showed increased odds of AYA diagnosis (OR = 2.19-2.78) with an additional two clusters showing increased odds of a pediatric diagnosis (OR = 3.84-9.32). LIMITATIONS, REASONS FOR CAUTION: Although this study has many strengths, including population data for family structure, cancer diagnoses and subfertility, there are limitations. First, semen measures are not available for the sample of fertile men. Second, there is no information on medical comorbidities or lifestyle risk factors such as smoking status, BMI, or environmental exposures. Third, all of the subfertile men included in this study were seen at a fertility clinic for evaluation. These men were therefore a subset of the overall population experiencing fertility problems and likely represent those with the socioeconomic means for evaluation by a physician. WIDER IMPLICATIONS OF THE FINDINGS: This analysis leveraged unique population-level data resources, SHARE and the UPDB, to describe novel multicancer clusters among the families of azoospermic and severely oligozoospermic men. Distinct overall multicancer risk and familial multicancer patterns were observed in the azoospermia and severe oligozoospermia cohorts, suggesting heterogeneity in cancer risk by type of subfertility and within subfertility type. Describing families with similar cancer risk patterns provides a new avenue to increase homogeneity for focused gene discovery and environmental risk factor studies. Such discoveries will lead to more accurate risk predictions and improved counseling for patients and their families. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by GEMS: Genomic approach to connecting Elevated germline Mutation rates with male infertility and Somatic health (Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): R01 HD106112). The authors have no conflicts of interest relevant to this work. TRIAL REGISTRATION NUMBER: N/A.

Changes in environmental exposures over decades may influence the genetic architecture of severe spermatogenic failure.

STUDY QUESTION: Do the genetic determinants of idiopathic severe spermatogenic failure (SPGF) differ between generations? SUMMARY ANSWER: Our data support that the genetic component of idiopathic SPGF is impacted by dynamic changes in environmental exposures over decades. WHAT IS KNOWN ALREADY: The idiopathic form of SPGF has a multifactorial etiology wherein an interaction between genetic, epigenetic, and environmental factors leads to the disease onset and progression. At the genetic level, genome-wide association studies (GWASs) allow the analysis of millions of genetic variants across the genome in a hypothesis-free manner, as a valuable tool for identifying susceptibility risk loci. However, little is known about the specific role of non-genetic factors and their influence on the genetic determinants in this type of conditions. STUDY DESIGN, SIZE, DURATION: Case-control genetic association analyses were performed including a total of 912 SPGF cases and 1360 unaffected controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants had European ancestry (Iberian and German). SPGF cases were diagnosed during the last decade either with idiopathic non-obstructive azoospermia (n = 547) or with idiopathic non-obstructive oligozoospermia (n = 365). Case-control genetic association analyses were performed by logistic regression models considering the generation as a covariate and by in silico functional characterization of the susceptibility genomic regions. MAIN RESULTS AND THE ROLE OF CHANCE: This analysis revealed 13 novel genetic association signals with SPGF, with eight of them being independent. The observed associations were mostly explained by the interaction between each lead variant and the age-group. Additionally, we established links between these loci and diverse non-genetic factors, such as toxic or dietary habits, respiratory disorders, and autoimmune diseases, which might potentially influence the genetic architecture of idiopathic SPGF. LARGE SCALE DATA: GWAS data are available from the authors upon reasonable request. LIMITATIONS, REASONS FOR CAUTION: Additional independent studies involving large cohorts in ethnically diverse populations are warranted to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Overall, this study proposes an innovative strategy to achieve a more precise understanding of conditions such as SPGF by considering the interactions between a variable exposome through different generations and genetic predisposition to complex diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the "Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)" (ref. PY20_00212, P20_00583), the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. PID2020-120157RB-I00 funded by MCIN/ AEI/10.13039/501100011033), and the 'Proyectos I+D+i del Programa Operativo FEDER 2020' (ref. B-CTS-584-UGR20). ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, is also partially supported by the Portuguese Foundation for Science and Technology (Projects: UIDB/00009/2020; UIDP/00009/2020). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Identification of two hidden clinical subgroups among men with idiopathic cryptozoospermia.

STUDY QUESTION: Are there subgroups among patients with cryptozoospermia pointing to distinct etiologies? SUMMARY ANSWER: We reveal two distinct subgroups of cryptozoospermic (Crypto) patients based on testicular tissue composition, testicular volume, and FSH levels. WHAT IS KNOWN ALREADY: Cryptozoospermic patients present with a sperm concentration below 0.1 million/ml. While the etiology of the severely impaired spermatogenesis remains largely unknown, alterations of the spermatogonial compartment have been reported including a reduction of the reserve stem cells in these patients. STUDY DESIGN, SIZE, DURATION: To assess whether there are distinct subgroups among cryptozoospermic patients, we applied the statistical method of cluster analysis. For this, we retrospectively selected 132 cryptozoospermic patients from a clinical database who underwent a testicular biopsy in the frame of fertility treatment at a university hospital. As controls (Control), we selected 160 patients with obstructive azoospermia and full spermatogenesis. All 292 patients underwent routine evaluation for endocrine, semen, and histological parameters (i.e. the percentage of tubules with elongated spermatids). Moreover, outcome of medically assisted reproduction (MAR) was assessed for cryptozoospermic (n = 73) and Control patients (n = 87), respectively. For in-depth immunohistochemical and histomorphometrical analyses, representative tissue samples from cryptozoospermic (n = 27) and Control patients (n = 12) were selected based on cluster analysis results and histological parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included two parts: firstly using clinical parameters of the entire cohort of 292 patients, we performed principal component analysis (PCA) followed by hierarchical clustering on principal components (i.e. considering hormonal values, ejaculate parameters, and histological information). Secondly, for histological analyses seminiferous tubules were categorized according to the most advanced germ cell type present in sections stained with Periodic acid Schif. On the selected cohort of 39 patients (12 Control, 27 cryptozoospermic), we performed immunohistochemistry for spermatogonial markers melanoma-associated antigen 4 (MAGEA4) and piwi like RNA-mediated gene silencing 4 (PIWIL4) followed by quantitative analyses. Moreover, the morphologically defined Adark spermatogonia, which are considered to be the reserve stem cells, were quantified. MAIN RESULTS AND THE ROLE OF CHANCE: The PCA and hierarchical clustering revealed three different clusters, one of them containing all Control samples. The main factors driving the sorting of patients to the clusters were the percentage of tubules with elongated spermatids (Cluster 1, all Control patients and two cryptozoospermic patients), the percentage of tubules with spermatocytes (Cluster 2, cryptozoospermic patients), and tubules showing a Sertoli cells only phenotype (Cluster 3, cryptozoospermic patients). Importantly, the percentage of tubules containing elongated spermatids was comparable between Clusters 2 and 3. Additional differences were higher FSH levels (P < 0.001) and lower testicular volumes (P < 0.001) in Cluster 3 compared to Cluster 2. In the spermatogonial compartment of both cryptozoospermic Clusters, we found lower numbers of MAGEA4+ and Adark spermatogonia but higher proportions of PIWIL4+ spermatogonia, which were significantly correlated with a lower percentage of tubules containing elongated spermatids. In line with this common alteration, the outcome of MAR was comparable between Controls as well as both cryptozoospermic Clusters. LIMITATIONS, REASONS FOR CAUTION: While we have uncovered the existence of subgroups within the cohort of cryptozoospermic patients, comprehensive genetic analyses remain to be performed to unravel potentially distinct etiologies. WIDER IMPLICATIONS OF THE FINDINGS: The novel insight that cryptozoospermic patients can be divided into two subgroups will facilitate the strategic search for underlying genetic etiologies. Moreover, the shared alterations of the spermatogonial stem cell compartment between the two cryptozoospermic subgroups could represent a general response mechanism to the reduced output of sperm, which may be associated with a progressive phenotype. This study therefore offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in cryptozoospermic patients. STUDY FUNDING/COMPETING INTEREST(S): German research foundation CRU 326 (grants to: SDP, NN). Moreover, we thank the Faculty of Medicine of the University of Münster for the financial support of Lena Charlotte Schülke through the MedK-program. We acknowledge support from the Open Access Publication Fund of the University of Münster. The authors have no potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

A loss-of-function variant in ZCWPW1 causes human male infertility with sperm head defect and high DNA fragmentation.

BACKGROUND: Male infertility is a global health issue. The more causative genes related to human male infertility should be further explored. The essential role of Zcwpw1 in male mouse fertility has been established and the role of ZCWPW1 in human reproduction needs further investigation to verify. METHODS: An infertile man with oligoasthenoteratozoospermia phenotype and his parents were recruited from West China Second University Hospital, Sichuan University. A total of 200 healthy Han Chinese volunteers without any evidence of infertility were recruited as normal controls, while an additional 150 infertile individuals were included to assess the prevalence of ZCWPW1 variants in a sporadic male sterile population. The causative gene variant was identified by Whole-exome sequencing and Sanger sequencing. The phenotype of the oligoasthenoteratozoospermia was determined by Papanicolaou staining, immunofluorescence staining and electron microscope. In-vitro experiments, western blot and in-silicon analysis were applied to assess the pathogenicity of the identified variant. Additionally, we examined the influence of the variant on the DNA fragmentation and DNA repair capability by Sperm Chromatin Dispersion and Neutral Comet Assay. RESULTS: The proband exhibits a phenotype of oligoasthenoteratozoospermia, his spermatozoa show head defects by semen examination, Papanicolaou staining and electron microscope assays. Whole-exome sequencing and Sanger sequencing found the proband carries a homozygous ZCWPW1 variant (c.1064C > T, p. P355L). Immunofluorescence analysis shows a significant decrease in ZCWPW1 expression in the proband's sperm. By exogenous expression with ZCWPW1 mutant plasmid in vitro, the obvious declined expression of ZCWPW1 with the mutation is validated in HEK293T. After being treated by hydroxyurea, MUT-ZCWPW1 transfected cells and empty vector transfected cells have a higher level of γ-H2AX, increased tail DNA and reduced H3K9ac level than WT-ZCWPW1 transfected cells. Furthermore, the Sperm Chromatin Dispersion assay revealed the proband's spermatozoa have high DNA fragmentation. CONCLUSIONS: It is the first report that a novel homozygous missense mutation in ZCWPW1 caused human male infertility with sperm head defects and high DNA fragmentation. This finding enriches the gene variant spectrum and etiology of oligoasthenoteratozoospermia.

A cryptozoospermic infertile male with Y chromosome AZFc microdeletion and low FSH levels due to a simultaneous polymorphism in the FSHB gene: a case report.

Genetic causes account for 10-15% of male factor infertility, making the genetic investigation an essential and useful tool, mainly in azoospermic and severely oligozoospermic men. In these patients, the most frequent findings are chromosomal abnormalities and Y chromosome long arm microdeletions, which cause a primary severe spermatogenic impairment with classically increased levels of FSH. On the other hand, polymorphisms in the FSH receptor (FSHR) and FSH beta chain (FSHB) genes have been associated with different FSH plasma levels, due to variations in the receptor sensitivity (FSHR) or in the production of FSH from the pituitary gland (FSHB). Here, we describe an unusual patient with a combined genetic alteration (classic AZFc deletion of the Y chromosome and TT homozygosity for the -211G>T polymorphism in the FSHB gene (rs10835638)), presenting with cryptozoospermia, severe hypospermatogenesis, and normal LH and testosterone plasma concentrations, but low FSH levels. The patient partially benefitted from treatment with FSH (150 IU three times/week for 6 months) which allowed him to cryopreserve enough motile spermatozoa to be used for intracytoplasmic sperm injection. According to our knowledge, this is the first report of an infertile man with AZFc microdeletion with low FSH plasma concentrations related to homozygosity for the -211G>T polymorphism in the FSHB gene.

Deleterious variants in X-linked RHOXF1 cause male infertility with oligo- and azoospermia.

Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.

Predictors of successful salvage microdissection testicular sperm extraction (mTESE) after failed initial TESE in patients with non-obstructive azoospermia: A systematic review and meta-analysis.

BACKGROUND: There has been no systematic review and meta-analysis to analyze and summarize the predictive factors of successful sperm extraction in salvage microdissection testicular sperm extraction. OBJECTIVES: We aimed to investigate the factors predicting the result of salvage microdissection testicular sperm extraction in patients with non-obstructive azoospermia who failed the initial microdissection testicular sperm extraction or conventional testicular sperm extraction. MATERIALS AND METHODS: We conducted a systematic literature search in PubMed, Web of Science, EMBASE, and the Cochrane Library for literature that described the characteristics of patients with non-obstructive azoospermia who underwent salvage microdissection testicular sperm extraction after failing the initial microdissection testicular sperm extraction or conventional testicular sperm extraction published prior to June 2022. RESULTS: This meta-analysis included four retrospective studies with 332 patients with non-obstructive azoospermia who underwent a failed initial microdissection testicular sperm extraction and three retrospective studies with 177 non-obstructive azoospermia patients who underwent a failed conventional testicular sperm extraction. The results were as follows: among non-obstructive azoospermia patients whose first surgery was microdissection testicular sperm extraction, younger patients (standard mean difference: -0.28, 95% confidence interval [CI]: -0.55 to -0.01) and those with smaller bilateral testicular volume (standard mean difference: -0.55, 95% CI: -0.95 to -0.15), lower levels of follicle-stimulating hormone (standard mean difference: -0.86, 95% CI: -1.18 to -0.54) and luteinizing hormone (standard mean difference: -0.68, 95% CI: -1.16 to -0.19), and whose testicular histological type was hypospermatogenesis (odds ratio: 3.52, 95% CI: 1.30-9.53) were more likely to retrieve spermatozoa successfully, while patients with Sertoli-cell-only syndrome (odds ratio: 0.41, 95% CI: 0.24-0.73) were more likely to fail again in salvage microdissection testicular sperm extraction. Additionally, in patients who underwent salvage microdissection testicular sperm extraction after a failed initial conventional testicular sperm extraction, those with testicular histological type of hypospermatogenesis (odds ratio: 30.35, 95% CI: 8.27-111.34) were more likely to be successful, while those with maturation arrest (odds ratio: 0.39, 95% CI: 0.18-0.83) rarely benefited. CONCLUSION: We found that age, testicular volume, follicle-stimulating hormone, luteinizing hormone, hypospermatogenesis, Sertoli-cell-only syndrome, and maturation arrest were valuable predictors of salvage microdissection testicular sperm extraction, which will assist andrologists in clinical decision-making and minimize unnecessary injury to patients.

Comparative studies on the chemical composition and pharmacological effects of vinegar-processed antler glue modified from Lei Gong Pao Zhi Lun and traditional water-processed antler glue.

ETHNOPHARMACOLOGICAL RELEVANCE: Antler glue is a classic medicinal to enhance sexual function in traditional Chinese medicine (TCM), which was first recorded in Shen Nong Ben Cao Jing (Shennong's Classic of the Materia Medica). Vinegar-processing is a classic method of processing traditional Chinese medicine. The method of preparing antler glue by boiling antlers in vinegar and then concentrating them is recorded in Lei Gong Pao Zhi Lun (Master Lei's Discourse on Medicinal Processing). In modern times, the typical processing method of antler glue is water extraction and concentration. However, it is not clear whether there is a difference in the effect of these two processing methods on the chemical composition and pharmacological activity of antler glue. AIM OF THE STUDY: The Chinese Pharmacopoeia (2020) records that the processing method of antler glue is water extraction and concentration. But Lei Gong Pao Zhi Lun differs in Chinese Pharmacopoeia (2020), which records the processing method of vinegar extraction and concentration. The effect of the two processing methods on antler glue's chemical composition and pharmacological activity is unknown. So this study aimed to elucidate the difference between different processing methods on the chemical composition and the treatment effect on oligoasthenospermia of antler glue. MATERIALS AND METHODS: So the automatic amino acid analyzer is used to determine the amino acid content of two different processing methods of antler glue. Proteomics was performed to detect the protein components of two different processing methods of antler glue and analyze them. Cyclophosphamide-induced mice models of oligoasthenospermia were used to study the different pharmacological effects of antler glue in two different processing methods. An automatic sperm analyzer observed the quantity and quality of sperm in mice epididymis. Serum sex hormone testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in mice were tested using the enzyme-linked immunosorbent assay (ELISA) kits. Hematoxylin-eosin (H&E) staining was used to analyze pathological alterations in mouse testicular tissue. The transcriptome has been used to reveal the potential mechanism of antler glue in treating oligoasthenospermia. Mitochondrial complex activity assay kits were used to assay the activity of mitochondrial respiratory chain complex I-V in mouse testicular tissue. Western blot was used to determine the expression of related proteins in mouse testicular tissue. RESULTS: Vinegar-processing can increase the alanine, proline, and glycine content in antler glue, reduce the length of protein peptides in antler glue, and produce a variety of unique proteins. Vinegar-processed antler glue (VAG) increased sperm density, sperm survival, sperm viability, and serum sex hormone levels in oligozoospermic mice. It reversed testicular damage caused by cyclophosphamide, and the effects were differently superior to those of water-processed antler glue (WAG). In addition, transcriptomics and related experiments have shown that VAG can increase the expression of Ndufa2, Uqcr11, Cox6b1, and Atp5i genes and proteins in mouse testis, thus promoting adenosine diphosphate (ATP) synthesis by increasing the activity of mitochondrial respiratory chain complexes I, III, IV and V. By promoting the oxidative phosphorylation process to produce more ATP, VAG can achieve the therapeutic effect of oligoasthenospermia. CONCLUSION: Vinegar-processing method can increase the content of active ingredients in antler glue. VAG increases ATP levels in the testes by promoting the process of oxidative phosphorylation to treat oligozoospermia.

EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: State of the art 2023.

Testing for AZoospermia Factor (AZF) deletions of the Y chromosome is a key component of the diagnostic workup of azoospermic and severely oligozoospermic men. This revision of the 2013 European Academy of Andrology (EAA) and EMQN CIC (previously known as the European Molecular Genetics Quality Network) laboratory guidelines summarizes recent clinically relevant advances and provides an update on the results of the external quality assessment program jointly offered by both organizations. A basic multiplex PCR reaction followed by a deletion extension analysis remains the gold-standard methodology to detect and correctly interpret AZF deletions. Recent data have led to an update of the sY84 reverse primer sequence, as well as to a refinement of what were previously considered as interchangeable border markers for AZFa and AZFb deletion breakpoints. More specifically, sY83 and sY143 are no longer recommended for the deletion extension analysis, leaving sY1064 and sY1192, respectively, as first-choice markers. Despite the transition, currently underway in several countries, toward a diagnosis based on certified kits, it should be noted that many of these commercial products are not recommended due to an unnecessarily high number of tested markers, and none of those currently available are, to the best of our knowledge, in accordance with the new first-choice markers for the deletion extension analysis. The gr/gr partial AZFc deletion remains a population-specific risk factor for impaired sperm production and a predisposing factor for testicular germ cell tumors. Testing for this deletion type is, as before, left at the discretion of the diagnostic labs and referring clinicians. Annual participation in an external quality control program is strongly encouraged, as the 22-year experience of the EMQN/EAA scheme clearly demonstrates a steep decline in diagnostic errors and an improvement in reporting practice.

Improvements in Sperm Motility Following Low- or High-Intensity Dietary Interventions in Men With Obesity.

INTRODUCTION: Obesity increases risks of male infertility, but bariatric surgery does not improve semen quality. Recent uncontrolled studies suggest that a low-energy diet (LED) improves semen quality. Further evaluation within a randomized, controlled setting is warranted. METHODS: Men with obesity (18-60 years) with normal sperm concentration (normal count) (n = 24) or oligozoospermia (n = 43) were randomized 1:1 to either 800 kcal/day LED for 16 weeks or control, brief dietary intervention (BDI) with 16 weeks' observation. Semen parameters were compared at baseline and 16 weeks. RESULTS: Mean age of men with normal count was 39.4 ± 6.4 in BDI and 40.2 ± 9.6 years in the LED group. Mean age of men with oligozoospermia was 39.5 ± 7.5 in BDI and 37.7 ± 6.6 years in the LED group. LED caused more weight loss than BDI in men with normal count (14.4 vs 6.3 kg; P < .001) and men with oligozoospermia (17.6 vs 1.8 kg; P < .001). Compared with baseline, in men with normal count total motility (TM) increased 48 ± 17% to 60 ± 10% (P < .05) after LED, and 52 ± 8% to 61 ± 6% (P < .0001) after BDI; progressive motility (PM) increased 41 ± 16% to 53 ± 10% (P < .05) after LED, and 45 ± 8% to 54 ± 65% (P < .001) after BDI. In men with oligozoospermia compared with baseline, TM increased 35% [26] to 52% [16] (P < .05) after LED, and 43% [28] to 50% [23] (P = .0587) after BDI; PM increased 29% [23] to 46% [18] (P < .05) after LED, and 33% [25] to 44% [25] (P < .05) after BDI. No differences in postintervention TM or PM were observed between LED and BDI groups in men with normal count or oligozoospermia. CONCLUSION: LED or BDI may be sufficient to improve sperm motility in men with obesity. The effects of paternal dietary intervention on fertility outcomes requires investigation.

Post-treatment sperm cryopreservation practices in childhood and young adult cancer survivors.

RESEARCH QUESTION: What are current practices of post-treatment fertility preservation in male childhood cancer survivors (CCS) who have not benefitted from pre-therapeutic fertility preservation in France and other European countries? DESIGN: A survey was conducted of all fertility preservation centres in France (n = 30) and European fertility specialists (n = 9) in five European countries. Eight clinical cases and 40 questions were included to assess the effect of age at diagnosis, type of treatment (alkylating-agents, orchidectomy, testicular radiotherapy) and sperm parameters on the probability of a post-treatment fertility preservation proposal. Demographic characteristics of the responding practitioner were also collected. RESULTS: Post-treatment sperm cryopreservation was proposed by 100% of fertility specialists in cases of severe oligoasthenoteratozoospermia, 77-88% in cases of moderate oligoasthenoteratozoospermia and in 65-77% in cases of sperm motility and vitality impairment. In cases of normal sperm parameters, 27-54% of fertility specialists would propose post-treatment sperm cryopreservation. These results did not differ significantly according to the type of treatment received or to responder-related factors. Practices of European specialists were also guided by sperm parameter results; 44-67% of specialists responding that they would propose sperm cryopreservation in cases of moderate to severe sperm parameter alterations. CONCLUSION: Post-treatment semen analysis could be widely proposed to CCS who have not benefitted from pre-therapeutic fertility preservation. Post-treatment fertility preservation could be proposed in cases of persistent moderate to severe sperm parameter alterations. Guidelines would be important to homogenize practices and to encourage oncologists to refer CCS for fertility assessments.

Interleukin 17 and Growth Differentiation Factor 9 Influence in a Sample of Iraqi Infertile Males.

Infertility is defined as the inability of couples to conceive after 1 year of regular unprotected intercourse, which affects 10-15% of couples. The present study aimed to investigate the influence of Interleukin-17 (IL-17) and growth differentiation factor 9 (GDF9) on three groups of infertile males, including control, azoospermia, and oligozoospermia. In total, this study was performed on 93 participants, consisting of 18, 65, and 10 subjects in the Azoospermia, oligozoospermia, and control groups, respectively. The mean plasma levels of IL-17 in the azoospermia and oligozoospermia groups were 21.317±3.605 and 15.101±2.416 ng/l, respectively, which were significantly higher than that in the control group (5.392±1.731 ng/l). Furthermore, the mean plasma levels of GDF9 in the azoospermia and oligozoospermia groups were 3.299±1.051 and 6.2603±2.621 ng/l, respectively, which was significantly higher than that in the control group (12.807±2.170 ng/l). One-way analysis of variance and least significant difference post-hoc test were performed to assess significant differences among means. R-squared measures how well the linear regression model fits the data. It can be interpreted as the proportion of variance of the outcome Y explained by the linear regression model. R-squared is a number between 0 and 1. In non-obstructive forms of severe oligozoospermia and azoospermia, like the case in the current study, intracytoplasmic sperm injection is suggested by using testicular biopsy for spermatozoa extraction, if viable spermatozoa are present.

[Epidemio-clinical and seminal profile of the man consulting for the desire to procreate: current situation in Lubumbashi, in the Democratic Republic of Congo]. / Profil épidémio-clinique et séminal de l'homme consultant pour désir de procréation: état des lieux à Lubumbashi, en République Démocratique du Congo.

Introduction: in Lubumbashi, as in upscale areas where explorations of fertility are very clever, the spermogram remains the essential analysis in the diagnosis of male infertility. This is the cause of 40% of couple infertility. The spermogram is the first step in identifying seminal abnormalities. The objective of this study was to determine the epidemiological-clinical and seminal profile of the man consulting for the desire to procreate in Lubumbashi. Methods: this was a cross-sectional study. We received 202 subjects in Lubumbashi, whose spermogram was performed from August 1st, 2020 to July 31st, 2021. The semen parameters were studied and interpreted according to WHO standards (2010) with studies of factors associated with their disturbance. Bivariate and multivariate analyzes had been carried out. The statistical significance threshold was set at p < 0.05. Results: the epidemiological-clinical profile of the respondents was as follows: the most represented age group was 30 to 39 years; infertility was primary in 80.69% of cases; the duration of the desire for paternity was 2 years at most in 44.55% of cases. The sperm abnormalities found were: oligozoospermia (40.09%), azoospermia (11.38%), asthenozoospermia (18.31%) and teratozoospermia (10.39%). Oligozoospermia was significantly associated with varicocele (ORa = 10.9 [3.0-39.5]; p < 0.0001), genital infection (ORa =2.7 [1.0-7, 2]; p = 0.041) and obesity (ORa = 2.6 [1.0-7.9]; p = 0.020) while azoospermia was the cure for inguinal hernia (ORa = 4.2 [1.0-17.2]; p = 0.049) and malnutrition (ORa =6.0 [1.2-29.7]; p = 0.027). Asthenozoospermia was significantly associated with the age group of 40 to 49 years (ORa = 6.6 [1.2-37.4]; p = 0.034), tobacco (ORa =7.5 [2.7 -21.0]; p = 0.000), undernutrition (ORa = 7.7 [1.0-61.9]; p = 0.045) and overweight (ORa =3.8 [1.3-11, 5]; p=0.019). Teratozoospermia was significantly associated with smoking (ORa = 5.6 [1.8-17.7]; p = 0.003) and overweight (ORa =5.3 [1.2-23.3]; p = 0.027). Conclusion: more than half of the respondents had, of the three main fertility parameters, at least one that was disturbed. Sperm count was the most affected parameter. Alcohol, tobacco, genital infection and malnutrition were the most common risk factors for the abnormalities observed.

Successful cryptozoospermia management with multiple semen specimen collection.

OBJECTIVE: To determine the prevalence of sperm suitable for intracytoplasmic sperm injection (ICSI) in fresh ejaculated semen samples provided by men scheduled for a microdissection testicular sperm extraction (mTESE) procedure. Secondary objectives included an evaluation of the effect of a short abstinence period on semen quality and ICSI outcomes for men with cryptozoospermia. DESIGN: Retrospective cohort study. SETTING: Academic medical center. PATIENTS: All men were scheduled to undergo a mTESE procedure by a single, high-volume surgeon at an academic center from September 1, 2015, to May 1, 2021. INTERVENTION: Presence of sperm suitable for ICSI in the ejaculate on the day of scheduled mTESE. MAIN OUTCOME MEASURES: Prevalence of sperm suitable for ICSI in the ejaculate among previously diagnosed men with azoospermia. Secondary outcomes included changes in semen parameters, clinical pregnancy rate, and live birth rate. RESULTS: Of 727 planned mTESE procedures, 69 (9.5%) were canceled because sperm suitable for ICSI were identified in a fresh ejaculated sample produced on the day of scheduled surgery (typically one day before oocyte retrieval). Overall, 50 men (50/727, 6.9%) used these rare, ejaculated sperm for ICSI. Semen samples obtained with <24 hours of abstinence were more likely to have better motility than the sample initially provided on the day of the planned mTESE. The live birth rate per ICSI attempt using these rare, ejaculated sperm was 36% (19/53). CONCLUSION: Providing a fresh ejaculated semen sample on the day of mTESE allows nearly 10% of men with azoospermia to avoid surgery with satisfactory ICSI outcomes. Providing multiple ejaculated samples over a short period of time does not adversely affect sperm concentration and may enhance sperm motility in men with cryptozoospermia.

HE4 overexpression in mice leads to leydig cell hyperplasia and spermatogensis impairment: Pathological implications for oligospermia.

Previous studies have shown that HE4 cancer biomarker promoted cancer cell proliferation and tumor growth in mouse xenograft models. Interestingly, HE4 levels are significantly increased in the seminal plasma of oligoasthenospermia patients, raising a question on HE4 role(s) in spermatogenesis. We constructed an HE4 overexpression mouse model (HE4-OE), and observed that HE4-OE male adult mice had small testes, low sperm counts, and elevated serum/testis testosterone levels. These mice exhibited disorganized seminiferous tubules and impaired spermatogenesis. HE4 overexpression concentrated in Leydig cells, and these cells had hyperplasia and increased testosterone biosynthesis. Mechanistic studies indicated that the impaired spermatogenesis was likely caused by a local and direct action of HE4 in the testis rather than by a hypothalamus/pituitary-initiated dysregulation. The new findings reveal a novel HE4 function in male reproductive system, and suggest the existence of a subtype of primary oligoasthenospermia characterized by HE4 overexpression, Leydig cell hyperplasia, and elevated testosterone levels.

Spontaneous alterations in semen parameters are associated with age, accessory gland function and the FSHB c.-211G>T variant.

BACKGROUND: There is a strong within-subject alteration of semen parameters in men with infertility. However, it remains unknown in which subgroup variations are likely to occur and which semen parameters are affected. OBJECTIVE: To evaluate parameters associated with spontaneous alterations in semen analysis. PATIENTS AND METHODS: We retrospectively selected 3456 men with infertility without known causes affecting spermatogenesis or sperm output for analysis of repeated ejaculate samples. Exclusion criteria comprised sperm concentration <1 million/mL, abnormal follicle-stimulating hormone or low testosterone, and low bitesticular volume (<10 mL). Grouped linear two-level nested mixed-effect models were applied. The analyzed parameters included abstinence time, bitesticular volume, age, accessory gland markers, follicle-stimulating hormone, and FSHB c.-211 variants. RESULTS: Groups include A (n = 397): ≥1.0 to <5.0 million/mL, B (n = 708): ≥5.0 to <15.0 million/mL, and C (n = 2351): ≥15.0 million/mL. Groups A, B, and C: changes in ejaculate volume were associated with alterations in total sperm count and motility (p < 0.003). Changes were, controlled for abstinence time (p < 0.001), related to α-glucosidase, fructose, or zinc (p = 0.005-0.02). Group A + B: fluctuations in follicle-stimulating hormone level influenced sperm concentration/count (p = 0.004-0.02), albeit only in men with FSHB c.-211 GG (p = 0.007-0.02). T-allele carriers did not show changes in follicle-stimulating hormone levels (p > 0.1). Group B: age <50 years (p = 0.007-0.01) and normal bitesticular volume (p = 0.008-0.02) were associated with spontaneous increases in sperm concentration, count, and motility. CONCLUSION: Semen parameters exhibit intra-individual alterations associated with organic, hormonal, and genetic variables. Changes are pronounced in younger men with normal bitesticular volume and oligozoospermia to almost normozoospermia. The effect is modulated by abstinence time, accessory gland function, and fluctuations in follicle-stimulating hormone level, which is bound to FSHB c.-211G>T variant. Judgment of semen analysis should be based on two semen samples, with abstinence times between 4 and 5 days. As a future perspective, it might be investigated whether younger men with normal bitesticular volume who are unable to elicit increases in serum follicle-stimulating hormone (FSHB c.-211 genotype of GT/TT) benefit from improving accessory gland function and increasing follicle-stimulating hormone.

The Loss-Function of KNL1 Causes Oligospermia and Asthenospermia in Mice by Affecting the Assembly and Separation of the Spindle through Flow Cytometry and Immunofluorescence.

KNL1 (kinetochore scaffold 1) has attracted much attention as one of the assembly elements of the outer kinetochore, and the functions of its different domains have been gradually revealed, most of which are associated with cancers, but few links have been made between KNL1 and male fertility. Here, we first linked KNL1 to male reproductive health and the loss-function of KNL1 resulted in oligospermia and asthenospermia in mice (an 86.5% decrease in total sperm number and an 82.4% increase in static sperm number, respectively) through CASA (computer-aided sperm analysis). Moreover, we introduced an ingenious method to pinpoint the abnormal stage in the spermatogenic cycle using flow cytometry combined with immunofluorescence. Results showed that 49.5% haploid sperm was reduced and 53.2% diploid sperm was increased after the function of KNL1 was lost. Spermatocytes arrest was identified at the meiotic prophase I of spermatogenesis, which was induced by the abnormal assembly and separation of the spindle. In conclusion, we established an association between KNL1 and male fertility, providing a guide for future genetic counseling regarding oligospermia and asthenospermia, and a powerful method for further exploring spermatogenic dysfunction by utilizing flow cytometry and immunofluorescence.

Touch print smear of testicular tissue with thionine stain for intraoperative diagnosis in azoospermic men.

PURPOSE: To compare the diagnostic value of testicular tissue touch print smear (TPS) conducted on azoospermic patients with results obtained from histopathology and in vitro fertility (IVF) lab findings. METHODS: Microdissection testicular sperm extraction was performed on a group of 148 azoospermic patients and testicular samples obtained intraoperatively. Using TPS, the samples were smeared onto a sterile slide, followed with staining using thionine. The testis tissue bulk samples were also transferred to the IVF lab, and determinations of sperm presence or absence obtained from IVF lab tests were compared with the TPS sample results. Needle testis biopsy was separately performed on a group of 360 azoospermic patients, and results of pathohistology review on the biopsies were further compared with determinations of spermatogenesis stage obtained from TPS for those patients. RESULTS: When compared with IVF lab results, TPS was found to have 100% (126/126) positive predictive value and 95.5% (25/26) negative predictive value for predicting sperm presence or absence, respectively. Furthermore, TPS was further found to have a 93.6% correlation (337 of 360 biopsies) with results of histological diagnoses performed by needle biopsy. Results from histology and TPS for the detection of sperm presence were concordant in 96.1% (346/360) of biopsies. Diagnosis of SCO by TPS shows the highest correlation with histopathology (98.6%), followed by complete spermatogenesis (97.5%), early maturation arrest (78.9%), and late maturation arrest (27.3%). CONCLUSIONS: The results support the continued use of TPS in testicular tissue analysis for more rapid assessment of spermatogenesis and for detection of spermatozoa in azoospermic subjects.

A Case of Oligoasthenoteratozoospermia Following Genital Tucking: Transgender Fertility Preservation.

Genital tucking is a behavioural practice facilitating gender expression by genitalia concealment. Genital tucking can be achieved by either pushing the testicles into the inguinal canal and/or securing the scrotum and penis between the legs towards the buttocks. Genital tucking has a negative impact on spermatogenesis, and therefore can result in a suboptimal sample and impaired sperm parameters. Fertility cryopreservation is recommended prior to hormone therapy or elective surgery, and therefore, the practice of genital tucking prior to fertility preservation could result in lesser quality and fewer samples being stored for future use. This case study presents a transwoman, aged 20-30, displaying severe oligoasthenoteratozoospermia derived from genital tucking. The case discusses how the cessation of genital tucking resulted in improved semen parameters, and therefore improved the quality and quantity of samples stored for future use. Fundamentally, the case highlights the necessity for health professionals to broaden their understanding and awareness of genital tucking, the impact it has on fertility, and how to better advise patients prior to fertility cryopreservation.