Introduction: Leydig cells (LCs) are the principal source of testosterone in males, underpinning male reproductive health and androgendependent physiology. Precise isolation of viable human LCs is essential for mechanistic steroidogenesis research and cell-based therapeutic development. Existing protocols rely on density-gradient centrifugation, which is technically demanding and often compromises yield and viability. Here, we describe a simplified density-gradient-free approach using differential adhesion to enrich functional human LCs from testicular tissue. Methodology: Human testicular fragments (~5 mm³) were minced and enzymatically digested with collagenase IV (2 mg/mL) at 37°C for 20 min under gentle agitation (100 RPM). The suspension was filtered (45 µm) and plated onto poly-L-lysine-coated T25 flasks. After 24 hours, non-adherent cells were removed by PBS washing. Viability exceeded 90% by trypan blue exclusion. Cells were maintained in DMEM/F12 with 10% FBS, 1% Antibiotic-Antimycotic, and 10 ng/mL luteinizing hormone (LH) to preserve the mature LC phenotype. Characterization employed immunofluorescence and flow cytometry using antibodies against SF-1, StAR, LHCGR, PDGFRA, and TEM-1. Testosterone secretion was quantified by ELISA under basal conditions and LH-stimulated conditions (10 ng/mL). Results: The protocol yielded ~1.25 × 10⁶ LCs per gram of tissue with >80% purity. Adherent HLCs displayed dense cytoplasmic lipid granules and intercellular networks consistent with active steroidogenesis. Flow cytometry confirmed 84.7% StAR⁺ cells, indicative of a robust steroidogenic population. A distinct progenitor subpopulation (PDGFRα⁺/TEM-1⁺) comprising ~28% of primary cultures, suggests retention of regenerative capacity. Basal testosterone secretion averaged 8.88 ng/mL per 24 hours, confirming preserved functional activity post-isolation. Conclusion This differential-adhesion protocol efficiently isolates functional human LCs without density-gradient media. Multimodal validation integrating immunofluorescence, flow cytometry, and ELISA confirms both phenotypic identity and steroidogenic competence, providing a reproducible and accessible platform for LC research and translational applications in male hypogonadism and androgen replacement.
Leydig Cells

Testicular tumor is the most common solid malignancy in males under 40 years of age. This malignancy is known to have a negative impact on male fertility. Therefore, several techniques for sperm retrieval have been proposed, including microdissection testicular sperm extraction (mTESE). The objective of this study was to review the literature on the outcomes of oncological (Onco)-mTESE at the time of radical orchiectomy. We conducted a comprehensive literature search through PubMed, Scopus, and Cochrane Central Controlled Register of Trials. Only studies reporting ex vivo mTESE in patients with testicular tumor were considered. Twelve papers met the inclusion criteria and were included in this review. Tumor size was identified as the sole preoperative factor influencing spermatogenesis. The considered studies demonstrated a satisfactory success rate for Onco-mTESE, associated with a similarly valid percentage of live healthy births through assisted reproductive technology. Currently, no comparison has been made between Onco-mTESE and conventional Onco-TESE, hence further assessment is required. In cases where the tumor completely replaces the cancer-bearing testicle, a contralateral micro-TESE may be a viable alternative. However, the surgeon should evaluate associated risks and benefits preoperatively. In conclusion, Onco-mTESE at the time of radical orchiectomy appears to be a promising therapeutic option for young patients with testicular tumors. Nevertheless, additional studies are necessary to achieve a definitive conclusion.
Humans ; Male ; Azoospermia/etiology* ; Sperm Retrieval ; Orchiectomy/methods* ; Testicular Neoplasms/complications* ; Microdissection/methods* ; Testis/surgery* ; Adult

When microdissection testicular sperm extraction (micro-TESE) fails, a redo procedure may be the only option for patients who want a biological child. However, there are many gaps of knowledge surrounding the procedure, which need to be addressed to help clinicians and patients make informed decisions. This review explores redo micro-TESE in the context of nonobstructive azoospermia (NOA). Literature was searched using Google Scholar, Medline, and PubMed. Search terms were "NOA" AND "second microdissection testicular sperm extractions" AND "redo microdissection testicles sperm extraction" AND "repeat microdissection testicular sperm extractions" AND "failed microdissection testicular sperm extractions" AND "salvage microdissection testicular sperm extractions". Only original articles in English were included. A total of nine articles were included, consisting of four retrospective and five prospective studies. The time gap between the first and second micro-TESE varied from 6 months to 24 months. Most of the included studies reported successful surgical sperm retrieval (SSR) in the second micro-TESE in the range of 10%-21%, except in one study where it reached 42%. It has not been presented any definitive information about the use of hormonal treatment or the benefit of varicocelectomy prior to the second micro-TESE. Patients with hypospermatogenesis and Klinefelter syndrome (KS) had the highest chance of success in redo surgery. In conclusion, redo micro-TESE following a negative procedure can lead to sperm recovery in 10%-21%. Patients with hypospermatogenesis and KS have a higher chance of success. There is no enough evidence to conclude which is the best hormonal stimulation if any before a redo surgery.
Humans ; Male ; Azoospermia/surgery* ; Sperm Retrieval ; Microdissection ; Reoperation ; Treatment Failure ; Testis/surgery* ; Treatment Outcome

Nonobstructive azoospermia (NOA) is the most challenging and complex clinical scenario for infertile men. Besides circumstances such as hypogonadotropic hypogonadism, surgical sperm retrieval is typically necessary, and microdissection testicular sperm extraction (micro-TESE) is the procedure of choice for men with NOA desiring to father children with their own gametes. Micro-TESE results in the highest numbers of sperm cells retrieved for use with in vitro fertilization/intracytoplasmic sperm injection (ICSI) in comparison to all other techniques for surgical sperm retrieval in men with NOA. Several factors may affect sperm retrieval rate and ICSI outcomes, including the patient's age, testicular volume, histopathological and genetic profile, and serum hormone levels. This article aims to review the medical literature describing predictors of successful micro-TESE and the outcomes of ICSI in men with NOA.
Humans ; Azoospermia/surgery* ; Male ; Sperm Injections, Intracytoplasmic ; Sperm Retrieval ; Treatment Outcome ; Testis/pathology* ; Female ; Microdissection

One major challenge in male factor infertility is nonobstructive azoospermia (NOA), which is characterized by spermatozoa-deficient semen without physical duct blockage. This review offers a thorough overview of the histopathology of the testes in NOA cases, clarifying its complex etiology and emphasizing the possible value of histopathology inspection for both diagnosis and treatment. Variable histopathological findings have been linked to NOA, such as tubular hyalinization, Sertoli cell-only syndrome, hypospermatogenesis, and germ cell arrest. Understanding the pathophysiology and forecasting the effectiveness of treatment are further enhanced by both morphometric and ultrastructural analyses. The potential significance of testicular biopsy in forecasting reproductive outcomes is assessed, especially concerning assisted reproductive technologies like intracytoplasmic sperm injection (ICSI). Besides, testicular microlithiasis, serum hormone profiles, and testicular size are investigated concerning NOA histopathology. It is concluded that understanding the histopathological patterns in NOA is crucial for its accurate diagnosis and appropriate management. Further research is still warranted to improve understanding of the complex pathophysiology underlying NOA.
Humans ; Azoospermia/etiology* ; Male ; Testis/pathology* ; Sertoli Cell-Only Syndrome/pathology* ; Sperm Injections, Intracytoplasmic

Approximately 15% of men in the general population have varicoceles, and varicoceles are diagnosed in 40% of men presenting for fertility evaluations. One percent of men in the general population are azoospermic, and 15% of men presenting for fertility evaluations are diagnosed with azoospermia. This article aims to review the impact of varicoceles on testicular function in men with azoospermia, the impact of varicocele repair on the semen parameters of azoospermic men, and the impact of varicocele repair on sperm retrieval and pregnancy outcomes when the male partner remains azoospermic after varicocele repair.
Humans ; Varicocele/physiopathology* ; Azoospermia/physiopathology* ; Male ; Pregnancy ; Female ; Sperm Retrieval ; Semen Analysis ; Pregnancy Outcome ; Testis/physiopathology*

Testicular descent occurs in two consecutive stages: the transabdominal stage and the inguinoscrotal stage. Androgens play a crucial role in the second stage by influencing the development of the gubernaculum, a structure that pulls the testis into the scrotum. However, the mechanisms of androgen actions underlying many of the processes associated with gubernaculum development have not been fully elucidated. To identify the androgen-regulated genes, we conducted large-scale gene expression analyses on the gubernaculum harvested from luteinizing hormone/choriogonadotropin receptor knockout ( Lhcgr KO) mice, an animal model of inguinoscrotal testis maldescent resulting from androgen deficiency. We found that the expression of secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 ( Smoc1 ) was the most severely suppressed at both the transcript and protein levels, while its expression was the most dramatically induced by testosterone administration in the gubernacula of Lhcgr KO mice. The upregulation of Smoc1 expression by testosterone was curtailed by the addition of an androgen receptor antagonist, flutamide. In addition, in vitro studies demonstrated that SMOC1 modestly but significantly promoted the proliferation of gubernacular cells. In the cultures of myogenic differentiation medium, both testosterone and SMOC1 enhanced the expression of myogenic regulatory factors such as paired box 7 ( Pax7 ) and myogenic factor 5 ( Myf5 ). After short-interfering RNA-mediated knocking down of Smoc1 , the expression of Pax7 and Myf5 diminished, and testosterone alone did not recover, but additional SMOC1 did. These observations indicate that SMOC1 is pivotal in mediating androgen action to regulate gubernaculum development during inguinoscrotal testicular descent.
Animals ; Male ; Mice ; Testis/growth & development* ; Mice, Knockout ; Androgens/pharmacology* ; Testosterone/pharmacology* ; Receptors, LH/metabolism* ; Calcium-Binding Proteins/metabolism*

Nonobstructive azoospermia (NOA) is considered the most challenging clinical scenario for infertile men and current treatments leave many men unsuccessful at being able to achieve a pregnancy with their partner using their own sperm. Microdissection testicular sperm extraction (micro-TESE) is the choice for men with NOA desiring to father children with their own gametes. Micro-TESE results in the highest numbers of sperm cells retrieved for use with in vitro fertilization/intracytoplasmic sperm injection. With suboptimal micro-TESE success rates of sperm retrieval and then pregnancy and live birth using the retrieved sperm with in vitro fertilization/intracytoplasmic sperm injection, advances to improve outcomes are necessary. This article comprehensively reviews the technologies investigated to date to improve the outcomes for men undergoing micro-TESE.
Humans ; Male ; Sperm Retrieval ; Azoospermia/surgery* ; Pregnancy ; Female ; Sperm Injections, Intracytoplasmic ; Microdissection/methods* ; Fertilization in Vitro ; Testis/surgery*

Azoospermia is the complete absence of spermatozoa in the ejaculate in two or more semen analyses after centrifugation. Nonobstructive azoospermia (NOA) represents the most severe form of male factor infertility accounting for 10%-15% of cases and stems from an impairment to spermatogenesis. Understanding of the hypothalamic-pituitary-testicular axis has allowed NOA to be subcategorized by anatomic and/or pathophysiologic level. The etiologies of NOA, and therefore, the differential diagnoses when considering NOA as a cause of male factor infertility, can be subcategorized and condensed into several distinct classifications. Etiologies of NOA include primary hypogonadism, secondary hypogonadism, defects in androgen synthesis and/or response, defective spermatogenesis and sperm maturation, or a mixed picture thereof. This review includes up-to-date clinical, diagnostic, cellular, and histologic features pertaining to the multitude of NOA etiologies. This in turn will provide a framework by which physicians practicing infertility can augment their clinical decision-making, patient counseling, thereby improving upon the management of men with NOA.
Humans ; Azoospermia/diagnosis* ; Male ; Spermatogenesis/physiology* ; Hypogonadism/complications* ; Infertility, Male/etiology* ; Testis/pathology*

Spermatogenesis is a fundamental process that requires a tightly controlled epigenetic event in spermatogonial stem cells (SSCs). The mechanisms underlying the transition from SSCs to sperm are largely unknown. Most studies utilize gene knockout mice to explain the mechanisms. However, the production of genetically engineered mice is costly and time-consuming. In this study, we presented a convenient research strategy using an RNA interference (RNAi) and testicular transplantation approach. Histone H3 lysine 9 (H3K9) methylation was dynamically regulated during spermatogenesis. As Jumonji domain-containing protein 1A (JMJD1A) and Jumonji domain-containing protein 2C (JMJD2C) demethylases catalyze histone H3 lysine 9 dimethylation (H3K9me2), we firstly analyzed the expression profile of the two demethylases and then investigated their function. Using the convenient research strategy, we showed that normal spermatogenesis is disrupted due to the downregulated expression of both demethylases. These results suggest that this strategy might be a simple and alternative approach for analyzing spermatogenesis relative to the gene knockout mice strategy.
Spermatogenesis/physiology* ; Animals ; Male ; Mice ; Epigenesis, Genetic ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Histones/metabolism* ; RNA Interference ; Testis/metabolism* ; Methylation ; Mice, Knockout ; Histone Demethylases