Ayşe Altun, Ateş Kadıoğlu, Murat Dursun, İlkay Özdemir, Sibel Bulgurcuoglu Kuran, İlknur Keskin
Objectives
In our study, it is aimed to show the current microbial diversity of testicular tissue taken by microscopic-testicular sperm extraction(m-TESE) from infertile patients with azoospermic and sperm production and its relationship with spermatogenesis by next generation sequencing(NGS) and histological process.
Materials and Methods
Testicular tissues of infertile male patients with known presence of sperm in the ejaculate were used as the control group (n=30), while testicular tissues obtained from patients with known azoospermia,TESE(+); containing sperm (n=30) and TESE(-);without sperm (n=30) were used as the study group.16Sr RNA sequencing was performed on testis tissues with ıllumina technology.In addition, tissues were stained with Hematoxylin-Eosin(HE) for histological examination and Johnsen scoring was performed.For Blood Testicular Barrier(BTB) analysis, ZO-1 protein staining was performed immunohistochemically and transmission electron microscopy analysis was performed.Immunfluorescent staining was performed for the presence of Neisseria gonorrhoeae bacteria and gram staining was performed for the presence of gram +/- bacteria.
Results
In our study result, no microbiota was observed in the testicular tissues of the control group. In the testicular tissues of the TESE(+) with sperm(n=15) and TESE(-) without sperm(n=15) study groups, microbiota were found in samples with impaired BTB.In alpha diversity analysis, according to Shannon test(p=0.51), Chao test(p=0,82) Simpson’s test(p=0.27), the TESE(+) group with TESE(-) group, but no significant difference was found.In beta diversity analysis, PCoA analysis compared to unifract test, TESE(-) and TESE(+) groups were shown to be phylogenetically similar in terms of density and diversity (p=0.43).It was found to be similar to the taxabare plot in terms of phylum and genus(Figure 1).No bacterial presence was found in the tissue extracts of the control group in gram staining.In the TESE(+) and TESE(-) groups, the presence of gram +/- bacteria was observed with similar density.According to our findings of HE staining while the seminiferous tubule structure preserved its integrity in the control group, the seminiferous tubule structure was disrupted in the TESE(+) and TESE(-).Johnsen skoru (p<0.05), ZO-1 reaction, which showed more expression in the control group, was observed significantly higher than the TESE(+) group and TESE(-) groups (p<0.05).ZO-1 semi-quantitative result;Control Group: 65.4% ± 2.2%, TESE(+) Group: 44.5% ± 2.02, TESE(-) Group: 23.4% ± 1.8.The electron microscopic findings on the intercellular tight junction structure supported our light microscopic findings.Neisseria gonorrhoeae bacteria, no staining was found in the testicular tissues of the control group.TESE(+) and TESE(-) groups, Neisseria gonorrhoeae bacterium immunofluorescently showed green light with FITC, thus its presence was demonstrated (Figure 2).
Conclusions
With the microbial identification of the testicular tissue, it will be proven that the testis is not sterile and it has brought innovation to the literature with high number patient studies by NGS method. Showing immunofluorescence in Neisseria gonorrhoea in the presence of testicular microbiota, which we think causes deterioration in the BTB, is the first researched subject in this direction.Depending on the presence of microbiota, the testicular structure and spermatogenesis may be affected to what extent, and spermatogenesis pauses due to expression of ZO-1, a tight junction protein involved in the BTB, have been clarified.