Analysis of 29 Targeted Genes for Non-obstructive Azoospermia: the Relationship Between Genetic Testing and Testicular Histology

BACKGROUND. Few studies have evaluated the relationship between testicular histology and pathogenic variations of genes regulating spermatogenesis.AIM. To analyze the presence of potentially pathogenic variants of 29 candidate genes known to cause spermatogenic failure (SPGF) in patients with non-obstructive azoospermia (NOA) who underwent testicular histology.PATIENTS AND METHODS. Sixty patients with NOA referred to the Department of Transfusion Medicine and Transplantation Biology, University Hospital Center Zagreb, Croatia, for testicular biopsy were consecutively assessed for eligibility. Twelve patients were excluded from the study because they had Klinefelter syndrome (n=1), Yq microdeletions (n=6), testicular trauma (n=2), or in-situ germ cell neoplasia (n=3). Therefore, 48 patients were considered eligible and included in this study. They were divided into three groups: those who had cryptorchidism (n=9), those with varicocele (n=14), and those with idiopathic NOA (n=25). All included patients underwent blood withdrawal for next-generation sequencing analysis and gene sequencing.RESULTS. We found a possible genetic cause in 4 patients with idiopathic NOA (16%) and in 2 with cryptorchidism (22%). No pathogenic or possibly pathogenic mutations were identified in patients with varicocele. Variants of undetermined significance (VUS) were found in 11 patients with idiopathic NOA (44%), 3 with cryptorchidism (33%), and 8 patients with varicocele (57%). VUSs of the USP9Y gene were the most frequently as they were found in 14 out of 48 patients (29%). In particular, the VUS USP9Y c.7434+14del was found in 11 patients. They showed varied histological pictures, including Sertoli cell-only syndrome, mixed atrophy, and hypospermatogenesis, regardless of cryptorchidism or varicocele. No direct correlation was found between the gene mutation/variant and the testicular histological picture. CONCLUSION. Different mutations of the same gene cause various testicular histological pictures. These results suggest that it is not the gene itself but the type of mutation/variation that determines the testicular histology picture. Based on the data presented above, it remains challenging to design a genetic panel with prognostic value for the outcome of testicular sperm extraction in patients with NOA.

Fertility of Cryptorchid Testis—An Unsolved Mistery

Cryptorchidism (undescended testis) is one of the most common diagnoses in the pediatric urologist office. Even in the modern era, there still are a lot of debates regarding the optimal time for surgery related to the expected results in relation with the testicular function, including fertility. The review below intends to clarify issues regarding the impact of cryptorchidism on testicular histology and function, semen analysis, the relation between hormonal and surgical treatment, future fertility, and paternity rate.

Oxidative Stress Disrupted Prepubertal Rat Testicular Development after Xenotransplantation

In the past two decades, testicular tissue grafting and xenografting have been well established, with the production of fertilization-competent sperm in some studies. However, few studies have been carried out to observe the development of grafted prepubertal testicular tissue of rats and compare the biological differences between in situ testis and grafted testis. In this study, we established the prepubertal testicular tissue xenografting model using a 22-day-old rat and evaluated certain parameters, including testicular histology, testosterone production, and ultrastructure of the grafted testes. We also assessed gene expression of cell proliferation markers, testicular cell markers, and antioxidative defense system. Our results showed that 47 days after transplantation, intratesticular testosterone concentration was not significantly altered; however, cell proliferation, spermatogenesis, and Sertoli cell markers in the transplanted testes were significantly disrupted compared with the control group, accompanied by aggravated apoptosis and oxidative damage. Moreover, the transplanted testes showed smaller tubular diameter and disrupted spermatogenic epithelium with apparent vacuoles, distorted and degenerated germ cells with obscure nuclear margin, and no spermatids in the center of the tubules. Although testis xenografting has been extensively tested and attained great achievement in other species, the prepubertal rat testicular tissue xenografting to immunodeficient mice exhibited obvious spermatogenesis arrest and oxidative damage. The protocol still needs further optimization, and there are still some unknown factors in prepubertal rat testes transplantation.

Testicular Histopathology and Spermatogenesis in Mice with Scrotal Heat Stress

Chronic heat stress-induced testicular damage and function therefore adversely affect their reproduction. Some research shows that heat stress has a negative effect on histopathological features of testicular tissue structure and spermatogenesis. An animal model was used to evaluate the effect of heat stress on testicular histology changes and spermatogenesis. The mouse model of heat stress was established by submerged in a pre-warmed incubator. The testes’ tissue was fixed and stained with hematoxylin–eosin (H&E) for quantitative analysis of histopathological alterations and spermatogenesis according to Johnson scoring system. Mice exposed to heat stress exhibited degenerated and disorganized features of spermatogenic epithelium and reduced spermatogenic cells. Heat stress exposure shows a significantly reduced Johnson score compared to the control condition. The percentage of high Johnsen score points was decreased in heat-stress exposure mice, while the ratio of low Johnsen score points was gradually increased. This chapter describes a mouse model for studying the male reproductive system and applies the Johnsen scores system to assess testicular histopathology in the seminiferous tubule cross-section. Collectively, this chapter indicated a negative impact of heat stress on mouse spermatogenesis as well as the human reproductive system.

The Abscopal Effects of Cranial Irradiation Induce Testicular Damage in Mice

To investigate whether the abscopal effects of cranial irradiation (C-irradiation) cause testicular damage in mice, male C57BL/6 mice (9weeks of age) were randomly divided into a sham irradiation group, a shielded group and a C-irradiation group and administered sham/shielded irradiation or C-irradiation at a dose rate of 2.33Gy/min (5Gy/d for 4 d consecutively). All mice were sacrificed at 4weeks after C-irradiation. We calculated the testis index, observed testicular histology by haematoxylin-eosin (HE) staining and observed testicular ultrastructure by transmission electron microscopy. Western blotting was used to determine the protein levels of Bax, Bcl-2, Cleaved caspase 3, glial cell line-derived neurotrophic factor (GDNF) and stem cell factor (SCF) in the testes of mice. Immunofluorescence staining was performed to detect the expression of Cleaved caspase 3 and 3β hydroxysteroid dehydrogenase (3βHSD), and a TUNEL assay was used to confirm the location of apoptotic cells. The levels of testosterone (T), GDNF and SCF were measured by ELISA. We also evaluated the sperm quality in the cauda epididymides by measuring the sperm count, abnormality, survival rate and apoptosis rate. The results showed that there was no significant difference in testicular histology, ultrastructure or sperm quality between the shielded group and sham group. Compared with the sham/shielded group, the C-irradiation group exhibited a lower testis index and severely damaged testicular histology and ultrastructure at 4weeks after C-irradiation. The levels of apoptosis in the testes increased markedly in the C-irradiation group, especially in spermatogonial stem cells. The levels of serum T and testicular 3βHSD did not obviously differ between the sham group and the C-irradiation group, but the levels of GDNF and SCF in the testes increased in the C-irradiation group, compared with the sham group. In addition, the sperm count and survival rate decreased in the C-irradiation group, while the abnormality and apoptosis rate increased. Under these experimental conditions, the abscopal effects of C-irradiation induced testicular damage with regard to both structure and function and ultimately decreased sperm quality in mice. These findings provide novel insights into prevention and treatment targets for male reproductive damage induced by C-irradiation.

How the Pregabalin (Lyrica) Administration as an Abuse Drug Can Affects the Reproductive Health of Male Wistar Rat

Drugs addiction considered a massive problem persist in diverse populations everywhere the world. Our work aimed to illustrate the correlation between drug addiction and reproductive function in an animal model, also the potential impact of pregabalin (Lyrica) intake on spermatozoa formation process and sexual hormone levels. In this study, we used 14 adult healthy rats that allocated into two groups (n = 7) as control and treated that orally administrated with pregabalin (23.7 mg/kg) for 30 subsequent days. Reproductive hormones, spermatozoa parameters (motility and morphology), lipid peroxidation (MDA), nitric oxide, total antioxidant activity, DNA damage and histopathological investigation were performed. The results revealed that pregabalin addiction had a harmful impact on the hypothalamus-pituitary-gonad axis of male rats through hindered hormones secretion, raised reactive oxygen species, affect antioxidant enzymes, triggered DNA damage and distorted testicular histology. Finally, we found that addiction of Lyrica caused adverse impact on the male reproductive health and subsequently affect fertility.

Phycobiliproteins Ameliorate Gonadal Toxicity in Male Mice Treated with Cyclophosphamide

Cyclophosphamide (CP)—which is used to treat autoimmune diseases and cancer—is related to gonadotoxicity attributed to oxidative stress. As phycobiliproteins (PBPs) are strong antioxidants that are unexplored as protective agents against male gonadotoxicity, our work aimed to investigate the effects of PBP crude extract on testicular damage and sperm parameter alterations caused by CP in mice. Three doses of PBP (50, 100, and 200 mg/kg) were tested in the experimental groups (n = 8 per group), administered concomitantly with 100 mg/kg CP. After 42 days receiving PBP daily and CP weekly, body and relative testicular weights, serum testosterone levels, testicular lipoperoxidation and antioxidant enzyme activity levels, and testicular histology and sperm parameter alterations were assessed. The results showed that PBP crude extract at 200 mg/kg prevented testosterone serum reduction, body weight loss, lipoperoxidation and enzyme activity increments, and sperm parameter alterations and partially ameliorated relative testicular weight reductions and histological damage in CP-treated mice. In conclusion, we showed that PBP crude extract (200 mg/kg) mitigated oxidative damage in the testes and ameliorated alterations in sperm parameters in mice treated with CP (100 mg/kg); therefore, PBP extract could be considered as a potential protective agent against CP toxicity.

Connexin43 in Germ Cells Seems to Be Dispensable for Murine Spermatogenesis

Testicular Connexin43 (Cx43) connects adjacent Sertoli cells (SC) and SC to germ cells (GC) in the seminiferous epithelium and plays a crucial role in spermatogenesis. However, the distinction whether this results from impaired inter-SC communication or between GC and SC is not possible, so far. Thus, the question arises, whether a GC-specific Cx43 KO has similar effects on spermatogenesis as it is general or SC-specific KO. Using the Cre/loxP recombinase system, two conditional KO mouse lines lacking Cx43 in premeiotic (pGCCx43KO) or meiotic GC (mGCCx43KO) were generated. It was demonstrated by qRT-PCR that Cx43 mRNA was significantly decreased in adult pGCCx43KO mice, while it was also reduced in mGCCx43KO mice, yet not statistically significant. Body and testis weights, testicular histology, tubular diameter, numbers of intratubular cells and Cx43 protein synthesis and localization did not show any significant differences in semi-quantitative Western blot analysis and immunohistochemistry comparing adult male KO and WT mice of both mouse lines. Male KO mice were fertile. These results indicate that Cx43 in spermatogonia/spermatids does not seem to be essential for successful termination of spermatogenesis and fertility as it is known for Cx43 in somatic SC, but SC-GC communication might rather occur via heterotypic GJ channels.

Changes in Testicular Biometry, Steroid Hormones and Receptor Expression in the Peripubertal Period of Indigenous Tenyi-vo Male Pigs of North-eastern Himalayan Region in India

Present study was conducted to characterize the testicular changes in the peripubertal period in Tenyi-vo, a miniature size pig of North-eastern Himalayan (NEH) region of India. A total of twenty-four male pigs were randomly selected and categorised for castration at different age groups, G1 (Days 30-45), G2 (Days 60-65), G3 (Days 80-100) and G4 (Days 150-160), n=6 each category. Paired testes and epididymis were used for the assessment of biometry, cauda epididymal spermiogram, testicular histology and relative expression of the androgen receptor (AR), estrogen receptors (ERα and ERβ), aromatase (CYP19A1), and insulin like growth factor-1β receptor (IGF-1R) in qPCR. Plasma testosterone (T), estradiol (E2), tri-iodothyronine (T3), thyroxine (T4), and cortisol concentrations were estimated on the day of castration in each group of male using commercial ELISA kits. In pigs of G2, a greater testicular weight, volume, epididymis weight was observed relative to G1. The presence of live spermatozoa at 1240.9±304.2×106/mLconcentration with 0.65% proximal droplets was recorded as early as day 60. The concentration of T increased steadily over the age of G1 to G4 and a significantly higher concentration was observed in G4 relative to the other categories. Among the transcripts analysed in the testis, the relative fold change of AR was 10.8 fold in G2, which was subsequently reduced in G3 and then down-regulated in G4. CYP19A1 was abundantly expressed in the testis and the fold change ranged from 41-54 fold, although it did not differ significantly from 60-150 days of age. Further, the presence of well-developed seminiferous tubules was evident in the Tenyi-vo male from day 60 onward with a body weight as low as 4.28 kg. The study concluded that the male of Tenyi-vo pig attained puberty at the earliest age of 60 days.