Anna Kazienko

Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim‐up‐separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC‐1 test as well as the NADH‐dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.

Association study of folate-related enzymes (MTHFR, MTR, MTRR) genetic variants with non-obstructive male infertility in a Polish population

Spermatogenesis is a process where an important contribution of genes involved in folate-mediated one-carbon metabolism is observed. The aim of the present study was to investigate the association between male infertility and the MTHFR (677C > T; 1298A > C), MTR (2756A > G) and MTRR (66A > G) polymorphisms in a Polish population. No significant differences in genotype or allele frequencies were detected between the groups of 284 infertile men and of 352 fertile controls. These results demonstrate that common polymorphisms in folate pathway genes are not major risk factors for non-obstructive male infertility in the Polish population.

Novel Morphological Findings of Human Sperm Removal by Leukocytes in In Vivo and In Vitro Conditions: Preliminary Study

Biological significance of increased number of leukocytes in ejaculate remains a subject of controversy. The aim of this research was to describe the morphological characteristics of the sperm elimination by leukocytes in in vivo and in vitro conditions using natural stimulator of the immune system—uropathogenic Escherichia coli, O75:HNT, isolated from semen.

TGFβ3 (TGFB3) polymorphism is associated with male infertility

Factors affecting the blood-testis barrier function may be involved in testicular damage and male infertility. Two cytokines play an important role in the barrier regulation, namely transforming growth factor beta 3 (TGF-β3) and tumor necrosis factor (TNF-α). The aim of this study was to investigate the potential association between TGF-β3 (TGFB3) and TNF-α (TNF) gene polymorphisms and male infertility. A total of 846 subjects, 423 diagnosed with male infertility and 423 fertile men were enrolled. TGFB3 (rs2268626:T > C, rs3917158:C > T, rs2284792:A > G, rs2268625:T > C, rs3917187:C > T) and TNF (rs1800629:-308G > A) gene polymorphisms were genotyped. No association between TNF genotype and infertility was observed. As for TGFB3, the genotypes distribution was similar in infertile and fertile men. However, rs2284792 minor allele frequency was significantly higher among infertile subjects. Heterozygous rs2284792 AG genotype was associated with increased odds for infertility [OR = 1.40 (95% CI 1.05–1.86), p = 0.021] and similar results were observed for G allele carrier status [OR = 1.40 (95% CI 1.06–1.84), p = 0.017]. Heterozygosity in TGFB3 rs3917158 was also associated with the infertility [OR = 1.37 (95% CI 1.01–1.87), p = 0.041]. The TGFB3 variant genotypes were associated with lower spermatozoa motility parameters in fertile men. The results indicate that variants in TGFB3 gene may be associated with male infertility. However, the findings require further replication and validation.

MMP2, MMP9 and TIMP2 polymorphisms affect sperm parameters but not fertility in Polish males.

Proper function of the blood–testis barrier is pivotal to spermatogenesis. Synchronised action of matrix metalloproteinases (MMP) and their inhibitors (TIMP) is mandatory to maintain dynamic balance of the barrier. Therefore, the association of functional genetic variants of MMP‐2, MMP‐9 and TIMP‐2 and male infertility was studied. A total of 416 infertile males and 421 healthy subjects were genotyped for 7 SNPs within MMP2, MMP9 and TIMP2 genes, along with the assessment of semen parameters (concentration, motility and morphology of spermatozoa). No association was observed between the studied genotypes and male infertility. However, higher sperm concentration was associated with TIMP2 rs8080623 C and rs2277698 T variants among infertile men, and with MMP9 rs17576 A minor allele in controls (p < .05). TIMP2 rs9900972 T and rs2277698 T allele were associated with higher percentage of morphologically normal spermatozoa among controls. MMP2 rs2285053 TT homozygous infertile patients presented higher percentage of spermatozoa displaying nonprogressive motility. Haplotype analysis revealed strong linkage disequilibrium between the studied loci (5 of 8 possible TIMP2 haplotypes, and 3 of 4 possible MMP2 and MMP9 were found). None of the haplotypes showed association with infertility. This study results suggest an association between MMP9 and TIMP2 SNPs with sperm parameters, but not infertility.

Organic cation/carnitine transporter OCTN2 (SLC22A5) -207C>G (rs2631367) polymorphism is not associated with male infertility.

Carnitine is an important factor in the initiation of progressive sperm motility and end stage of sperm maturation. The compound is transported by an organic cation/carnitine transporter (OCTN2), which is expressed in the male reproductive system. OCTN2 is encoded by SLC22A5 gene with proven -207C>G functional polymorphism. The aim of the case-control study was to investigate a potential association between the -207C>G SLC22A5 polymorphism and male infertility. The -207C>G SLC22A5 polymorphism was determined by means of TaqMan assay in 206 infertile Caucasian males and 256 ethnically matched controls. Besides genotyping study, sperm mitochondrial function was assessed using NADH-dependent NBT assay. The distribution of SLC22A5 genotypes in infertile men was as follows: CC - 29.6%, CG - 53.9%, GG - 16.5% and in fertile men: CC - 32.0%, CG - 50.0%, GG - 18.0%, and was comparable in both evaluated groups. Likewise, the studied polymorphism did not affect sperm mitochondrial function. The results of the current study demonstrated that -207C>G polymorphism of the SLC22A5 gene is not associated with male infertility.