The development of new biomarkers for human male infertility is crucial to improve the diagnosis and the prognosis of this disease. Recently, seminal microbiota was shown to be related to sperm quality parameters, suggesting an effect in human fertility and postulating it as a biomarker candidate. However, its relationship to sperm DNA integrity has not been studied yet. The aim of the present study is to characterize the seminal microbiota of a western Mediterranean population and to evaluate its relationship to sperm chromatin integrity parameters, and oxidative stress. For that purpose, 14 samples from sperm donors and 42 samples from infertile idiopathic patients were obtained and were analyzed to assess the composition of the microbiota through full-length 16S rRNA gene sequencing (Illumina MiSeq platform). Microbial diversity and relative abundances were compared to classic sperm quality parameters (macroscopic semen parameters, motility, morphology and concentration), chromatin integrity (global DNA damage, double-stranded DNA breaks and DNA protamination status) and oxidative stress levels (oxidation-reduction potential). The seminal microbiota observed of these samples belonged to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The most abundant genera were Finegoldia, Peptoniphilus, Anaerococcus, Campylobacter, Streptococcus, Staphylococcus, Moraxella, Prevotella, Ezakiella, Corynebacterium and Lactobacillus. To our knowledge, this is the first detection of Ezakiella genus in seminal samples. Two clusters of microbial profiles were built based on a clustering analysis, and specific genera were found with different frequencies in relation to seminal quality defects. The abundances of several bacteria negatively correlate with the sperm global DNA fragmentation, most notably Moraxella, Brevundimonas and Flavobacterium. The latter two were also associated with higher sperm motility and Brevundimonas additionally with lower oxidative-reduction potential. Actinomycetaceae, Ralstonia and Paenibacillus correlated with reduced chromatin protamination status and increased double-stranded DNA fragmentation. These effects on DNA integrity coincide in many cases with the metabolism or enzymatic activities of these genera. Significant differences between fertile and infertile men were found in the relative presence of the Propionibacteriaceae family and the Cutibacterium, Rhodopseudomonas and Oligotropha genera, which supports its possible involvement in male fertility. Our findings sustain the hypothesis that the seminal microbiome has an effect on male fertility.

Killer cell immunoglobulin-like receptors (KIR), considered the most polymorphic natural killer (NK) cell regulators, bind HLA class-I molecules or still unknown ligands. Interest in KIR genotyping is increasing because of the importance of these receptors for identifying the best possible donor in hematopoietic stem cell transplantation to obtain a graft-versus-leukemia effect. Currently, routine protocols to determine the gene content of the KIR cluster are exclusively performed by PCR-SSO and PCR-SSP. To improve the study of these genes, we developed a multiplex, long-range PCR strategy suitable for simultaneous, high-resolution HLA class I and KIR genotyping by next generation sequencing (NGS). This protocol allows amplification of the 14 KIR genes, 2 KIR pseudogenes, and HLA class I genes, with subsequent sequencing on an Illumina platform. The bioinformatics analysis for KIR genotyping was performed by virtual hybridization of gene-specific probes, and HLA genotyping was done by GenDx NGSengine software. To validate the method reliability, 192 genomic DNA samples previously characterized by PCR-SSO were used. When a specific KIR gene was present, a large number of gene-specific virtual probes were detected, whereas when it was absent, very few or none were found, enabling cutoff establishment. Concordance for both the KIR and HLA assignments as compared with the previous characterization was 100%. In conclusion, the multiplex PCR NGS-based strategy presented could provide an efficient, less costly method for KIR-ligand genotyping by gene presence/absence. Furthermore, allele resolution will be possible when KIR-specific software becomes available.

Killer cell immunoglobulin-like receptors (KIR) belong to a diverse family of receptors present in NK cells and certain subsets of T cells. They bind to HLA class I molecules and, such as these, are highly polymorphic. This study is the first to determine KIR gene content diversity and perform high-resolution genotyping of corresponding HLA class I ligands in 445 unrelated blood donors resident in Catalonia (northeast Spanish region) and registered in the Blood and Tissue Bank of Barcelona between 2017 and 2018. The study was performed using a new next-generation sequencing method developed and validated by our group, with an important hands-on-time reduction and very cost-effectiveness. Thirty-eight KIR genotypes were observed. Haplotype combination frequencies were 24.49% for AA, 75.51% for BX. The most frequent genotype, found in 109 individuals (24.49%), was genotype ID 1. Seventeen genotypes occurred only once. Thirty individuals carried all KIR genes (genotype ID 6). We detected 46 HLA-A, 63 HLA-A-B, and 40 HLA-A-C alleles. A majority of individuals were heterozygous for C1/C2 (42.24%), 38.43% were homozygous for C1, and 19.33% were homozygous for C2. The most common KIR-HLA ligand pair was KIR3DL1-Bw4+, present in 75.73% of cases. No differences were found in KIR gene frequencies between the Catalan cohort and other Iberian Peninsula populations. Our findings will be useful for guiding further research evaluating the functional significance of KIR-ligand associations in specific diseases.