P-067 Characterization of seminal microbiota in patients with asthenozoospermic and normozoospermic using next-generation sequencing
Authors: Vajpeyee M , Tiwari S , Yadav L B , Chimote N 
Published: June 2023

Abstract

Study question
How are the semen microbiota compositions of patients with asthenozoospermic and normozoospermic different, According to data from 16S rRNA gene-specific Next-generation sequencing (NGS)?

Summary answer
Large numbers of microbes can be present in seminal fluid, and there are differences in the semen microbiota between asthenozoospermic and normozoospermic semen samples.

What is known already
Male factor is attributable in up to 50% of cases of infertility. Most studies on normal and abnormal sperm microbiota are based on 16S rRNA gene-specific next-generation sequencing (NGS). Microbiome analysis based on subunit 16S rRNA sequencing is a fast tool that can enable the identification of all the pathogenic microorganisms associated with semen in clinical pathology. Studies on the impact of semen micro-biomes in asthenozoospermic and normozoospermic samples could improve the results of assisted reproductive technologies. The major bacterial diversity in asthenozoospermic and normozoospermic samples belongs to the genera Lactobacillus, Prevotella, Staphylococcus, and Anaerococcus.

Study design, size, duration
Two eighty patients with their own semen samples were included in the study. The study population consists of patients attending to the “Pacific IVF Center” Pacific Medical University and Hospital (Udaipur, India) from November 2020 to October 2022. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 127) was asthenozoospermic, and Group 2 (n = 153) had normozoospermic semen samples, Patients were aged between (20-45 years). Ethical approval was obtained from the institute.

Participants/materials, setting, methods
Genomic DNA was extracted from samples using commercially available (Qiagen, DNeasy Power Soil kit). The amount of extracted DNA was measured using a Nanodrop spectrophotometer (Thermo Scientific). The microbiota of semen was analyzed using 16S ribosomal RNA (rRNA) gene amplification (MinION) Oxford Nanopore Ltd. Bioinformatics analysis was performed using QIIME2 and Microbiome Analyst packages. Alpha, beta diversity, and taxonomic characterization were compared for the seminal microbiome in asthenozoospermic and normozoospermic semen samples.

Main results and the role of chance
Different bacterial communities were detected when asthenozoospermic and normozoospermic semen samples were analyzed. In patients with asthenozoospermic parameters, a higher alpha diversity index tendency was found. In normozoospermic semen samples, a p-value of bacterial diversity was non-significant (p = 0.14 for the Shannon index), and significant differences in asthenozoospermic semen samples (p = 0.01 for the Shannon index). In the beta diversity analysis, no significant differences were observed between both groups
Relative abundance analysis identified the bacterial communities, Lactobacillus, Streptococcus, and Ureaplasma in normozoospermic semen samples, and Anaerococcus and Gardnerella in asthenozoospermic samples, Comparison of asthenozoospermic and normozoospermic samples with alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p &lt; 0.001) showed significant differences. Relative abundance in normozoospermic semen samples identified the bacterial communities, Lactobacillus (p = 3.70E-4), Prevotella (p = 8.11E-4), and in asthenozoospermic semen samples Anaerococcus (p = 0.004) and Gardnerella (p = 0.004).

Limitations, reasons for caution
The limited sample size is the primary constraint of this study. Larger studies with a larger sample size are needed to confirm the distinct microbiome patterns identified in asthenozoospermic and normozoospermic samples in connection to male infertility.

Wider implications of the findings
Further research could determine the detection rate of the described bacterial diversity in semen with other pathologies. Determining the relationship between sperm microbiota characteristics and infertility could enable the development of new algorithms for treating patients with reproductive disorders, An abnormal seminal microbiome appears to be strongly correlated with infertility.

Trial registration number
N/A