MALDI Imaging Mass Spectrometry Reveals Lipid Alterations in Physiological and Sertoli Cell-Only Syndrome Human Testicular Tissue Sections.

Azoospermia, the absence of sperm cells in semen, affects around 15% of infertile males. Sertoli cell-only syndrome (SCOS) is the most common pathological lesion in the background of non-obstructive azoospermia and is characterised by the complete absence of germinal epithelium, with Sertoli cells exclusively present in the seminiferous tubules. Studies have shown a correlation between successful spermatogenesis and male fertility with lipid composition of spermatozoa, semen, seminal plasma or testis. The aim of this research was to discover the correlation between the Johnsen scoring system and phospholipid expressions in testicular cryosections of SCOS patients. MALDI imaging mass spectrometry is used to determine spatial distributions of molecular species, such as phospholipids. Phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and sphingomyelins (SMs) are the most abundant phospholipids in mammalian cells and testis. SMs, the structural components of plasma membranes, are crucial for spermatogenesis and sperm function. Plasmalogens, are unique PCs in testis with strong antioxidative properties. This study, using imaging mass spectrometry, demonstrates the local distribution of phospholipids, particularly SMs, PCs, plasmalogens and PEs in human testicular samples with SCOS for the first time. This study found a strong relationship between the Johnsen scoring system and phospholipid expression levels in human testicular tissues. Future findings could enable routine diagnostic techniques during microTESE procedures for successful sperm extraction.

Exosomes of mesenchymal stem cells and PRP restore spermatogenesis in the rat model of non-obstructive azoospermia.

In brief: Since available therapeutic approaches for chemotherapy-induced non-obstructive azoospermia (NOA) patients are not enough efficient, an urgent need for treatment alternatives is felt. This study shows that adipose tissue-derived mesenchymal stem cells-derived exosome (AD-Exo) treatment is more effective in ameliorating busulfan-induced NOA rat models compared to platelet-rich plasma (PRP). Abstract: Patients with non-obstructive azoospermia (NOA) are unable to have their children. Therefore, there is an urgent need for additional treatment alternatives for these patients. Recently, novel treatments based on the exosomes derived from mesenchymal stem cells (MSCs) as the agents responsible for exerting the paracrine effects and consequently biological functions of MSCs are proposed. Besides, platelet-rich plasma (PRP) as a significant blood byproduct has been therapeutically applied in several male infertility studies. In this study, we compared the effects of PRP and exosome treatment on spermatogenesis restoration in NOA rat models. Exosomes and PRP were isolated from the adipose tissue-derived MSCs (AD-MSCs) collected from conditioned medium and peripheral blood of human volunteers, respectively. Non-obstructive azoospermia (NOA) induction was done through two doses of busulfan at a 21-day interval. Thirty-five days after NOA induction, intratesticular injection of AD-MSCs-derived exosome (AD-Exo), PRP, and PBS was performed. The control group did not receive any treatment. Two months later, the rats were euthanized for further analysis. Our results revealed that both AD-Exo and PRP treatments improved the size and weight of testis, modulated the expression level of Dazl, Ddx4, Stra8, Pwil1, and Ccna1, and ameliorated the serum level of LDH, SOD, and GR enzymes in NOA rats. Moreover, the AD-Exo group showed improved testosterone, GPx, MAD, and CAT serum levels, sperm motility, and protein levels of DAZL and DDX4. This investigation verified the more efficient effects of AD-Exo treatment in comparison to PRP in ameliorating busulfan-induced NOA rat models.

Research progress on the fanconi anemia signaling pathway in non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is a disease characterized by spermatogenesis failure and comprises phenotypes such as hypospermatogenesis, mature arrest, and Sertoli cell-only syndrome. Studies have shown that FA cross-linked anemia (FA) pathway is closely related to the occurrence of NOA. There are FA gene mutations in male NOA patients, which cause significant damage to male germ cells. The FA pathway is activated in the presence of DNA interstrand cross-links; the key step in activating this pathway is the mono-ubiquitination of the FANCD2-FANCI complex, and the activation of the FA pathway can repair DNA damage such as DNA double-strand breaks. Therefore, we believe that the FA pathway affects germ cells during DNA damage repair, resulting in minimal or even disappearance of mature sperm in males. This review summarizes the regulatory mechanisms of FA-related genes in male azoospermia, with the aim of providing a theoretical reference for clinical research and exploration of related genes.

FANCM Gene Variants in a Male Diagnosed with Sertoli Cell-Only Syndrome and Diffuse Astrocytoma.

Azoospermia is a form of male infertility characterized by a complete lack of spermatozoa in the ejaculate. Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia, where no germ cells are found in the tubules. Recently, FANCM gene variants were reported as novel genetic causes of spermatogenic failure. At the same time, FANCM variants are known to be associated with cancer predisposition. We performed whole-exome sequencing on a male patient diagnosed with SCOS and a healthy father. Two compound heterozygous missense mutations in the FANCM gene were found in the patient, both being inherited from his parents. After the infertility assessment, the patient was diagnosed with diffuse astrocytoma. Immunohistochemical analyses in the testicular and tumor tissues of the patient and adequate controls showed, for the first time, not only the existence of a cytoplasmic and not nuclear pattern of FANCM in astrocytoma but also in non-mitotic neurons. In the testicular tissue of the SCOS patient, cytoplasmic anti-FANCM staining intensity appeared lower than in the control. Our case report raises a novel possibility that the infertile carriers of FANCM gene missense variants could also be prone to cancer development.

Cyto- and Histopographic Assessment of CPA3-Positive Testicular Mast Cells in Obstructive and Non-Obstructive Azoospermia.

Infertility is an important personal and society disease, of which the male factor represents half of all causes. One of the aspects less studied in male infertility is the immunological testicular microenvironment. Mast cells (MCs), having high potential for regulating spermatogenesis due to fine-tuning the state of the integrative buffer metabolic environment, are one of the most crucial cellular subpopulations of the testicular interstitium. One important component of the MC secretome is proteases that can act as proinflammatory agents and in extracellular matrix (ECM) remodeling. In the testis, MCs are an important cell component of the testicular interstitial tissue (TIT). However, there are still no studies addressing the analysis of a specific MC protease-carboxypeptidase A3 (CPA3)-in cases with altered spermatogenesis. The cytological and histotopographic features of testicular CPA3+ MCs were examined in a study involving 34 men with azoospermia. As revealed, in cases with non-obstructive azoospermia, a higher content of CPA3+ MCs in the TIT and migration to the microvasculature and peritubular tissue of seminiferous tubules were observed when compared with cases with obstructive azoospermia. Additionally, a high frequency of CPA3+ MCs colocalization with fibroblasts, Leydig cells, and elastic fibers was detected in cases with NOA. Thus, CPA3 seems to be of crucial pathogenetic significance in the formation of a profibrogenic background of the tissue microenvironment, which may have direct and indirect effects on spermatogenesis.

Interaction between Chromodomain Y-like Protein and Androgen Receptor Signaling in Sertoli Cells Accounts for Spermatogenesis.

Spermatogenesis is a highly regulated process dependent on androgen receptor (AR) signaling in Sertoli cells. However, the pathogenic mechanisms of spermatogenic failure, by which loss of AR impairs downstream target genes to affect Sertoli cell function, remain incompletely understood. By using microarray analysis, we identified several AR-regulated genes involved in the maturation of spermatogenesis, including chromodomain Y-like protein (CDYL) and transition proteins 1 (TNP-1), that were significantly decreased in ARKO mouse testes. AR and CDYL were found to co-localize and interact in Sertoli cells. The AR-CDYL complex bound to the promoter regions of TNP1 and modulated their transcriptional activity. CDYL acts as a co-regulator of AR transactivation, and its expression is decreased in the Sertoli cells of human testes from patients with azoospermia. The androgen receptor-chromodomain Y-like protein axis plays a crucial role in regulating a network of genes essential for spermatogenesis in Sertoli cells. Disruption of this AR-CDYL regulatory axis may contribute to spermatogenic failure. These findings provide insights into novel molecular mechanisms targeting the AR-CDYL signaling pathway, which may have implications for developing new therapeutic strategies for male infertility.

USP11 regulates proliferation and apoptosis of human spermatogonial stem cells via HOXC5-mediated canonical WNT/ß-catenin signaling pathway.

Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/ß-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/ß-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility.

Human benign Leydig cell tumor - Biochemical evaluation.

Recently we reported expressional alterations in 219 genes and their transcripts in Leydig cell tumors but nowadays there is still a lack of full basic biochemical characteristics of these tumors. The discovery of potential biochemical markers for tumor management from early detection, treatments, and control of therapy results may markedly supplement genetic data. Leydig cell micronodules were obtained from patients with azoospermia who were qualified for testicular biopsy. The biochemistry of Leydig cell tumors was analyzed using histological staining and spectrophotometric measurements of total proteins, carbohydrates, lipids, and nucleic acids. In addition, the levels of calcium (Ca2 +), copper (Cu2 +), zinc (Zn2 +), and selenium (Se2 +) ions were measured. When compared to healthy testis we revealed, for the first time, that in the interstitial tissue with Leydig cell tumors, great amounts of proteins, carbohydrates, lipids, and acids were dislocated from the seminiferous tubules. Measurements of organic compounds showed a decrease (P < 0.05) only in the Cu2 + content in Leydig cell tumors which may be related to their altered biochemical structure. This specific result may be promising for designing further approaches to manage this tumor based on combining morphological and molecular data.

Feasibility of high-frequency ultrasound for seminiferous tubule assessment and correlation of B-mode imaging with pathological findings in the testis in azoospermia.

PURPOSE: To determine the feasibility of high-frequency ultrasound (HFUS) for assessing seminiferous tubules and to understand high-resolution B-mode images of the testes in cases of azoospermia. METHODS: We verified how the histopathological images of testicular biopsy specimens can be observed using HFUS images and measurement analysis of seminiferous tubules was performed to 28 testes of 14 cases with azoospermia who underwent preoperative ultrasound and microdissection testicular sperm extraction (micro-TESE). The population consisted of obstructive azoospermia (OA) and non-obstructive azoospermia (NOA), including Sertoli cell-only syndrome (SCOS), and the other pathologies. Statistical verification of differences in seminiferous tubule diameters among preoperative ultrasound examination, ultrasound examination of pathological specimens, and histopathological specimens. We also examined the imagingpathology correlation via a case series presentation, aiming to identify imaging markers of testicular pathology and determine the possibility of predicting each condition. RESULTS: A comparison between HFUS images and histopathology from the same biopsy specimens suggested that ultrasonography could be seen as stereoscopic images due to its significantly greater slice thickness. The diameters of tubules were generally larger in pathological tissues as compared to ultrasonographic findings in OA and SCOS, but not in the other conditions. Comparisons provided insights into the predictability of SCOS and revealed imaging findings such as gaps between tubules and decreased diameter reflective of testicular damage. CONCLUSION: Seminiferous tubules can be observed however the diameter of seminiferous tubules varies in imaging and histopathology depending on the pathology. Imaging findings that reflect testicular damage and the predictability of SCOS were revealed in this study, but further verification is required.

Achieving an optimal pregnancy outcome through the combined utilization of micro-TESE and ICSI in cryptorchidism associated with a non-canonical splicing variant in RXFP2.

PURPOSE: To identify the genetic cause of a cryptorchidism patient carrying a non-canonical splicing variant highlighted by SPCards platform in RXFP2 and to provide a comprehensive overview of RXFP2 variants with cryptorchidism correlation. METHODS: We identified a homozygous non-canonical splicing variant by whole-exome sequencing and Sanger sequencing in a case with cryptorchidism and non-obstructive azoospermia (NOA). As the pathogenicity of this non-canonical splicing variant remained unclear, we initially utilized the SPCards platform to predict its pathogenicity. Subsequently, we employed a minigene splicing assay to further evaluate the influence of the identified splicing variant. Microdissection testicular sperm extraction (micro-TESE) combined with intracytoplasmic sperm injection (ICSI) was performed. PubMed and Human Genome Variant Database (HGMD) were queried to search for RXFP2 variants. RESULTS: We identified a homozygous non-canonical splicing variant (NM_130806: c.1376-12A > G) in RXFP2, and confirmed this variant caused aberrant splicing of exons 15 and 16 of the RXFP2 gene: 11 bases were added in front of exon 16, leading to an abnormal transcript initiation and a frameshift. Fortunately, the patient successfully obtained his biological offspring through micro-TESE combined with ICSI. Four cryptorchidism-associated variants in RXFP2 from 90 patients with cryptorchidism were identified through a literature search in PubMed and HGMD, with different inheritance patterns. CONCLUSION: This is the first cryptorchidism case carrying a novel causative non-canonical splicing RXFP2 variant. The combined approach of micro-TESE and ICSI contributed to an optimal pregnancy outcome. Our literature review demonstrated that RXFP2 variants caused cryptorchidism in a recessive inheritance pattern, rather than a dominant pattern.

A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies.

OBJECTIVE: To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS). DESIGN: Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA-sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes. SETTING: A laboratory study. PATIENTS: Three men with a diagnosis of obstructive azoospermia (age range, 30-40 years). INTERVENTION: None. MAIN OUTCOME MEASURES: Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue. RESULTS: Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%-8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1-/doublesex and Mab-3 related transcription factor 1-/STRA8+ spermatogonia as well as SYCP3+/protamine 2- spermatocytes. CONCLUSION: This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and in vitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

Genetic profiling of azoospermic men to identify the etiology and predict reproductive potential.

PURPOSE: To identify germline mutations related to azoospermia etiology and reproductive potential of surgically retrieved spermatozoa, and to investigate the feasibility of predicting seminiferous tubule function of nonobstructive azoospermic men by transcriptomic profiling of ejaculates. MATERIALS AND METHODS: Sperm specimens were obtained from 30 men (38.4 ± 6 years) undergoing epididymal sperm aspiration for obstructive azoospermia (OA, n = 19) acquired by vasectomy, or testicular biopsy for nonobstructive azoospermia (NOA, n = 11). To evaluate for a correlation with azoospermia etiology, DNAseq was performed on surgically retrieved spermatozoa, and cell-free RNAseq on seminal fluid (n = 23) was performed to predict spermatogenesis in the seminiferous tubule. RESULTS: Overall, surgically retrieved sperm aneuploidy rates were 1.7% and 1.8% among OA and NOA cohorts, respectively. OA men carried housekeeping-related gene mutations, while NOA men displayed mutations on genes involved in crucial spermiogenic functions (AP1S2, AP1G2, APOE). We categorized couples within each cohort according to ICSI clinical outcomes to investigate genetic causes that may affect reproductive potential. All OA-fertile men (n = 9) carried mutations in ZNF749 (sperm production), whereas OA-infertile men (n = 10) harbored mutations in PRB1, which is essential for DNA replication. NOA-fertile men (n = 8) carried mutations in MPIG6B (stem cell lineage differentiation), whereas NOA-infertile individuals (n = 3) harbored mutations in genes involved in spermato/spermio-genesis (ADAM29, SPATA31E1, MAK, POLG, IFT43, ATG9B) and early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Transcriptomic assessment of cell-free RNAs in seminal fluid from NOA men allowed the prediction of residual spermatogenic foci. CONCLUSIONS: Sperm genome profiling provides invaluable information on azoospermia etiology and identifies gene-related mechanistic links to reproductive performance. Moreover, RNAseq assessment of seminal fluid from NOA men can help predict sperm retrieval during testicular biopsies.

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 hemizygous loss-of-function variant in BCORL1 is associated with male infertility and oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a common type of male infertility; however, its genetic causes remain largely unknown. Some of the genetic determinants of OAT are gene defects affecting spermatogenesis. BCORL1 (BCL6 corepressor like 1) is a transcriptional corepressor that exhibits the OAT phenotype in a knockout mouse model. A hemizygous missense variant of BCORL1 (c.2615T > G:p.Val872Gly) was reported in an infertile male patient with non-obstructive azoospermia (NOA). Nevertheless, the correlation between BCORL1 variants and OAT in humans remains unknown. In this study, we used whole-exome sequencing to identify a novel hemizygous nonsense variant of BCORL1 (c.1564G > T:p.Glu522*) in a male patient with OAT from a Han Chinese family. Functional analysis showed that the variant produced a truncated protein with altered cellular localization and a dysfunctional interaction with SKP1 (S-phase kinase-associated protein 1). Further population screening identified four BCORL1 missense variants in subjects with both OAT (1 of 325, 0.31%) and NOA (4 of 355, 1.13%), but no pathogenic BCORL1 variants among 362 fertile subjects. In conclusion, our findings indicate that BCORL1 is a potential candidate gene in the pathogenesis of OAT and NOA, expanded its disease spectrum and suggested that BCORL1 may play a role in spermatogenesis by interacting with SKP1.

Timing of Testicular Biopsy in Relation to Oocyte Retrieval and the Outcomes of Intracytoplasmic Sperm Injection.

PURPOSE: We evaluate microscopic (micro) testicular sperm extraction (TESE) timing relative to oocyte retrieval on intracytoplasmic sperm injection outcome. MATERIALS AND METHODS: Couples with nonobstructive azoospermia who underwent intracytoplasmic sperm injection with freshly retrieved spermatozoa were analyzed based on whether micro-TESE was performed at least 1 day prior to oocyte retrieval (TESE-day-before group) or on the day of oocyte retrieval (TESE-day-of group). Embryology and clinical outcomes were compared. RESULTS: The percentage of patients who underwent a successful testicular sperm retrieval was significantly lower in the TESE-day-before cohort (62%) than in the TESE-day-of cohort (69%; odds ratio [OR] 1.4, 95% CI [1.1, 1.7], P < .001). The fertilization rate was also found to be significantly lower in the TESE-day-before group (45%) than in the TESE-day-of group (53%; OR 1.4, 95% CI [1.2, 1.7], P = .01). Although the association between the cleavage rate and TESE timing was not statistically significant, the implantation rate was found to be significantly higher in the day-before cohort (28%) than in the day-of cohort (22%; OR 0.7, 95% CI [0.6, 0.9], P = .01). Nevertheless, it was found that the clinical pregnancy and delivery rates were not statistically significantly associated with the TESE timing. CONCLUSIONS: Although sperm retrieval and fertilization rates were lower in the TESE-day-before cohort, the 2 cohorts showed comparable embryologic and clinical outcomes. Micro-TESE can be performed before oocyte harvesting to provide physicians ample time to decide between cancelling oocyte retrieval or retrieving oocytes for cryopreservation.

Testicular volume is a noninvasive predictor of sperm retrieval failure in idiopathic nonobstructive azoospermia.

We investigated the prognostic importance of noninvasive factors in predicting sperm retrieval failure in idiopathic nonobstructive azoospermia (iNOA). We studied 193 patients with nonobstructive azoospermia who underwent microsurgical testicular sperm extraction. The Chi-square test and Mann-Whitney U tests for clinical parameters and seminiferous tubule distribution were used for between-group comparisons. A logistic regression analysis was conducted to identify predictors of retrieval failure. Area under the receiver operating characteristic curve for each variable was evaluated, and the net clinical benefit was calculated using a clinical decision curve. Patients with iNOA had a lower sperm retrieval rate than those with known causes. Moreover, testicular volume was an independent factor affecting sperm extraction outcomes (odds ratio = 0.79, P < 0.05). The testicular volume cut-off value was 6.5 ml (area under the curve: 0.694). The patients with iNOA were categorized into two groups on the basis of the distribution of seminiferous tubules observed. The sperm retrieval rate and testicular volume were significantly different between the groups with a uniform or heterogeneous tubule distribution. There was also a significant association between a uniform tubule distribution and testicular volume. In conclusion, a testicular volume of more than 6.5 ml effectively predicts microsurgical testicular sperm extraction failure due to a uniform tubule distribution in patients with iNOA.

Single Cell Map of Human Azoospermia Testis Caused by Cyclophosphamide Chemotherapy.

Chemotherapeutic drugs will affect the process of spermatogenesis. However, most current studies on the effects of chemotherapeutic drugs on spermatogenesis are based on mouse models, with a shortage of human body evidence. In addition, the mechanism of chemotherapeutic drugs causing spermatogenesis disorder is not clear. Therefore, we have collected the testicular tissues of an inguinal-lipoma patient whose testes were resected after chemotherapy and a patient who had normal spermatogenesis disorder and underwent single-nucleus RNA sequencing (snRNA-Seq). After quality control, we obtained a total of 27,957 high-quality cells, including 18,612 normal cells and 9,345 drug-treated cells, which were all used in analyzing the mechanism of chemotherapeutic drugs causing spermatogenesis disorder. This study has provided data resources and references for exploring the mechanism of chemotherapeutic drugs causing spermatogenesis disorder with the insight of protecting the spermatogenic abilities of male tumor patients receiving chemotherapy.

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.