Bacteriospermia in men among infertile couples in Nepalese community and its relation to sperm DNA integrity

Abstract

Abstract: Infertility has been an increasing problem in married couples worldwide. Both partners may contribute equally to the infertility. Male infertility can be caused by anatomical defects, hormonal imbalance, exposure to various other factors affecting spermatogenesis, and infection of the urogenital tract or accessory reproductive glands. Genitourinary or accessory gland infection results in the presence of bacteria in semen that may affect the semen quality. The fertility potential of semen is investigated by analysis of semen for sperm concentration, motility, morphology, and vitality. Sperm DNA fragmentation has now been known as a significant defect affecting the diagnostic sperm characteristics and function. Sperm DNA integrity plays a crucial role in normal fertilization, healthy embryo development and successful implantation, pregnancy, as well as in fetal development. The fragmentation of sperm DNA might be caused due to several factors. Bacteriospermia is considered one of the factors affecting sperm DNA integrity and other sperm characteristics. However, there is limited data available on bacteria causing sperm defects in Nepalese men. Therefore, this study aimed to assess the seminal bacteria and to determine their relation with sperm DNA integrity in men among infertile Nepalese couples. A cross-sectional study was carried out from June 2021 to August 2024, in which 217 semen samples were collected from male partners of couples for fertility consultation at Creator’s IVF Nepal, Lalitpur. The laboratory analysis of samples was done in the andrology laboratory of Creator’s IVF Nepal and the laboratory of the Central Department of Microbiology, Tribhuvan University. Semen analysis for ejaculate volume, sperm concentration, motility, morphology, vitality, leucocyte count, and sperm DNA fragmentation (SDF) was done following WHO guidelines for human semen analysis, 2021. Bacteria in semen were studied by culture-based and 16S rRNA gene-based approaches. Bacteriospermia was detected in 55 (25.35%) samples by semen culture and 148 (70.81%) samples by 16S rRNA gene amplification. Staphylococcus aureus was the most frequently isolated bacterium. Virulence genes for clumping factors (clfA, clfB) and the methicillin resistance gene (mecA) were detected in a significant proportion (66.67%) of S. aureus isolates. The sequencing of the 16S rRNA gene amplicon revealed diverse bacterial genera including Prevotella (14.2% relative abundance), Fenollaria (12.2%), Lactobacillus (9.6%), Veillonella (8.4%), Corynebacterium (7.8%), Acinetobacter (6.7%), Sneathia (5.7%), Gardnerella (5.1%), Porphyromonas (4.2%), Ezakiella (3.0%), Dialister (2.8%), Streptococcus (2.7%), Peptoniphilus (2.4%), Negativicoccus (1.7%), Finegoldia (1.6%), Anaerococcus (1.4%), Staphylococcus (1.0%), Megasphaera (1.0%), Moryella (0.9%), and Campylobacter (0.6%). Most of the bacterial genera were anaerobes. Semen analysis showed that 188 out of 217 had normal sperm concentration, among which 20.21% had high DNA fragmentation index (DFI ≥ 30%). The association of bacteriospermia with semen characteristics, including sperm DNA fragmentation, was studied. Culture-based bacteriospermia was significantly associated with decreased ejaculate volume (p = 0.001), whereas 16S rRNA-based bacteriospermia was significantly associated with lower sperm concentration (p = 0.02). However, no significant associations were found between bacteriospermia and sperm DNA fragmentation. Differential abundance analysis of 16S rRNA sequencing results depicted that Acinetobacter was negatively associated with multiple semen parameters, while Lactobacillus was consistently enriched in semen with normal parameters and lower sperm DNA integrity, indicating a potentially protective role. The bacterial community composition of semen obtained in this study concludes that a rich seminal microbiota may influence the characteristics of semen. This study provides baseline data for future in-depth studies on bacteria and their clinical implications in the seminal function that may help in improving the reproductive health of Nepalese men.