DEFB119 stratifies dysbiosis with distorted networks in the seminal microbiome associated with male infertility

doi:10.21203/rs.3.rs-2716767/v1

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Research Article

DEFB119 stratifies dysbiosis with distorted networks in the seminal microbiome associated with male infertility

https://doi.org/10.21203/rs.3.rs-2716767/v1

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Background

Infertility is associated with the alteration of the seminal microbiome. However, how the onset of dysbiosis remains controversial and the involvement of host factors remains elusive. This study investigates the alterations of the seminal microbiome in male infertility and examines the association and function of DEFB119, a reproductive-tract-specific host antimicrobial peptide, on the seminal microbiome and male fertility.

Results

We analyzed the seminal microbiome by 16S rRNA sequencing in 30 individuals with normal sperm parameters and 58 male-factor infertile patients. While we observed comparable genera, diversity and evenness of bacterial communities, a marked decrease in the modularity of the metacommunities was observed in patients with abnormal spermiogram. Analysis of the protein level of DEFB119 by ELISA revealed a marked elevation of DEFB119 in a subpopulation of male-infertile patients. Elevated seminal DEFB119 was associated with a decrease in the observed genera, diversity and evenness of bacterial communities and further distortion of the metacommunities. Mediation analysis suggests the involvement of elevated DEFB119 and dysbiosis of the seminal microbiome in mediating the abnormalities in the spermiogram. Functional experiments showed that recombinant DEFB119 significantly decrease the progressive motility of sperm in patients with abnormal spermiogram. Moreover, DEFB119 demonstrated species-specific antimicrobial activity against common seminal and non-seminal species.

Conclusions

Male infertility is associated with distorted bacterial networks. Elevation of the DEFB119 level in seminal plasma stratifies male infertile patients with dysbiosis of the seminal microbiome. Both elevated DEFB119 and dysbiosis contribute to the abnormal spermiogram. Our work identifies an important host factor that mediates the host-microbiome interaction and stratifies the seminal microbiome associated with male infertility. These results may lead to a new diagnostic method for male infertility and regimens for formulating the microbiome in the reproductive tract and other organ systems.

β-defensin

bacterial networks

host-microbe interaction

microbiota

The microbiomes of the reproductive tracts play pivotal roles in fecundity [1–5]. Defects of which represent an important cause of infertility that affects 15% of couples worldwide [6, 7]. The microbiome of the semen herein referred to as the seminal microbiome, has been shown to be modulated by both physiological, environmental and genetic factors [2]. Previous studies have shown that the major bacterial phyla in the seminal microbiome are Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes [2]. This balanced composition of the seminal microbiome is regulated by both the innate immune response and secretions by various glands in the male reproductive tract (MRT). Alteration in the healthy composition of the seminal microbiome is associated with sperm abnormalities such as motility and DNA damage [8, 9].

Apart from the involvement in regulating sperm functions and qualities, the seminal microbiome also alters the microenvironment of the female reproductive tract (FRT) after deposition. The seminal microbiome can be shared between heterosexual couples, giving complementary semino-vaginal microbiomes that affect the sperm migration along the FRT, the conception and embryo development in the oviduct, and the receptivity of the uterus for implantation [10, 11]. While several phyla of the seminal microbiome have been shown to be associated with sperm abnormalities, the onset of dysbiosis in infertile cases remains controversial. Moreover, the question as to whether there is a host factor involved in the host-microbes interactions remains unknown.

The β-defensin family is a group of small antimicrobial peptides mainly expressed by the epithelial cells [12]. Ubiquitously expressed β-defensins such as HBD1-4 play major roles in host defence and innate immune responses [13]. Intriguingly, a number of β-defensin family members are specifically expressed in the MRT. These reproductive-tract enriched/specific β-defensins play important functions in sperm functions and infertility [14, 15]. Surprisingly, although β-defensins are present in semen and possess antimicrobial activity, their physiological functions and involvement in modulating the seminal microbiome have not been explored.

DEFB119 is a reproductive tract-specific β-defensin highly expressed in the testis and the epididymis [16]. Single-cell sequencing analysis of testicular cells isolated from non-obstructive azoospermia men demonstrates an elevated expression of DEFB119 in the Sertoli cells [17]. Male mice lacking the mouse ortholog DEFB19 demonstrated subfertility in both sexes [18, 19]. DEFB119 is also expressed in sperm and is involved in sperm chemotaxis [19]. However, the antimicrobial activity of DEFB119 and its potential involvement in regulating the reproductive tract microbiome has not been reported.

In view of the antimicrobial activity conserved among members of the β-defensin family, we hypothesize that reproductive-tract β-defensin serves as a host factor in maintaining the seminal microbiome. Defects of which may lead to abnormal spermiogram and contribute to male infertility. In this study, we investigate the seminal microbiome of infertile couples with normal or abnormal spermiogram parameters. We also investigate the involvement and function of host factor DEFB119 on the dysbiosis of the seminal microbiome.

Study design and participants

Infertile men aged 25 - 44 years who attended the assisted reproduction clinic at the Prince of Wales Hospital of The Chinese University of Hong Kong were recruited. All participants provided written informed consent and local ethics approvals were obtained for this study (approval number: CREC2016.499). All participants were in good health and none of them was under antibiotic therapy during sampling. Semen samples were collected according to a standardized protocol after 2-7 days of abstinence. Specifically, men were instructed to void, wash their hands with soap and water, and cleanse the glans penis with water before collecting the sample. Samples were collected via masturbation into a sterile container, without the use of saliva or lubrication, and immediately provided to clinical staff for processing and sample storage.

For diagnosis of male-factor infertility, semen analysis was performed according to the World Health Organization (WHO) 2010 guidelines [20]. Briefly, semen samples were allowed to liquefy for up to 30 minutes at 37°C. The samples were manually evaluated for volume and then assessed using optical microscopy for concentration, percentage of total motility, percentage of progressive motility, total motile sperm count, and percentage of normal morphology. Infertile couples without a known cause of infertility were included as idiopathic infertility.

To examine the association between DEFB119 levels and male infertility, the levels of DEFB119 in seminal plasma were determined by ELISA. Infertile men were categorized according to the level of DEFB119. The seminal microbiome in the categorized patients was determined by 16S rRNA gene sequencing. The relationship between the level of DEFB119 and the phylotypes in the seminal microbiome of participants was analyzed.

Immunostaining analysis

Sperm smears were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton-100, then sperm were blocked with 1% BSA, 22.52 mg/ml glycine in PBST. These sperm were probed with Anti-DEFB119 antibody (ab157790) at 1:100 in PBST with 1% BSA and incubated overnight at 4°C. After washing in PBS, the sperm were incubated with Alexa-fluor 488 conjugated anti-rabbit IgG (ab21206) at 1:500 in PBST at RT in the dark. Slides were mounted with Vecta Mount Mounting Medium (H5000) after washing in PBS. Images were captured with a fluorescent microscope.

DEFB119 ELISA analysis

Seminal fluid was collected from the supernatant of semen after centrifugation at 2,000g for 15 min. and the assay was conducted according to the manufacturer’s instruction (Human DEFB119 ELISA Kit - LS-F13148). Briefly, The seminal fluid was diluted at 1:100 with sample Diluent, then the mixture was added to a coated Strip Plate and incubated for 2 h at 37°C. After washing, Detection reagent A was added and incubated for 1 hour at 37°C. After washing, Detection reagent B was added and incubated for 1 hr at 37°C. Finally, the TMB substrate was added and incubated for 20 minutes at 37°C in the dark at 37°C. The reaction was then quenched with the Stop solution and the absorbance was measured at 450 nm. The raw data was calculated by the online tool https://elisaanalysis.com.

DNA extraction and library preparation

Seminal plasma DNA was extracted from the seminal fluid using the QIAamp DNA mini kit (Qiagen#51306). Briefly, A total of 300ul semen was centrifuged at 167,000 g for 15 minutes, 180 ul Buffer ATL and 40 ul proteinase K were added into the pellet and incubated at 56°C overnight with agitation at 700 rpm. Then the Buffer AL was added to the mixture and incubated at 70°C for 30 minutes. The DNA was precipitated with 100% ethanol and loaded into QIAamp Spin Column. After washing with Buffer AW1 and AW2, the DNA was eluted with a total of 40 ul of prewarmed water. DNA concentration and quality were measured by Nanodrop spectrophotometer.

We adopted a nested PCR protocol to generate amplicon products (appendix for primer design). First, the full length of the 16S rRNA gene was amplified in primary PCR (20 cycles) using KAPA HiFi HotStart Readymix (Roche). The V3 and V4 hypervariable regions of the 16S rRNA were amplified by internal primers in secondary PCR (25 cycles) using the same reagents. The amplicons were purified by AMPure XP beads to remove unbound primers and dimers according to the manufacturer’s manual. The purified amplicons were attached to dual indices and Illumina sequencing adapters using the Nextera XT Index Kit (FC-131-2001). The indexed library was validated by Qubit™ dsDNA HS Assay Kit (Q32851) and Bioanalyzer Agilent DNA 1000 Kit (5067-1505).

Next-generation sequencing

The library was sequenced by the Illumina HiSeq system with ~31.93 M paired-end reads. The raw data was processed using a Singularity pipeline developed by the Microbiome Research Centre of The University of New South Wales) that was wrapped with Quantitative Insights into Microbial Ecology (QIIME2, v2020.8) software performed on a high-performance cluster [21]. Overall sequence quality was assessed with Fastp [22], and background noise and chimeric reads were removed with DADA2 [23]. Host decontamination was conducted by mapping the human genome. The amplicon sequence variants (ASVs), which allow precise identification of microbes [24], were aligned to reference sequences at a cutoff of ≥ 99% in Greengenes (v13.5) [25], and normalized using a single rarefaction at 2900 features. Environmental contaminants were identified by the negative controls and removed from subsequent analysis using the Decontam R package. Downstream analysis was completed by the R package microeco (v0.3.2) [26].

Recombinant DEFB119 treatment

Recombinant DEFB119 was commercially available (Cloud-Clone Corp, RP1653Hu01). The semen samples were liquefied at room temperature for 30 minutes, and semen analysis was performed as described above. Samples with ≥ 40% total sperm motility (40% were used to examine the effect of rDEFB119 on sperm motility. From each sample, two aliquots of 300 μl raw semen were incubated with vehicle control or 2 μg rDEFB119 at 37°C for 1 hour. Sperm total and progressive motility were manually evaluated after treatment.

Bactericidal assay

Streptococcus intermedius (JTH08 strain; isolate ID: CC00038), Streptococcus anginosis (isolate ID: CC00108), Streptococcus mitis (ATCC49456 strain; isolate ID: CC00095), Pseudomonas aeruginosa (SNP0614 strain; isolate ID: CC00104), Prevotella stercorea (strain CB35, isolate ID: CC00801), Prevotella copri (strain JCM 13464; isolate ID: CC00987) were kindly provided by Dr. Samuel Forster’s group at Hudson Institute of Medical Research [27]. Streptococcus and Pseudomonas were cultured on blood agar plates and colonies were sub-cultured in BHI broth 24 hours prior to the experiment. Prevotella was cultured on pre-reduced YCFA plates and subcultured in YCFA broth 48 hours prior to the experiment. On the day of the experiment, bacterial culture containing Pseudomonas aeruginosa (1.2-1.4x10⁷ CFU), Streptococcus mitis (2-2.2x10⁴ CFU), Streptococcus anginosus (0.5-1.4x10⁷ CFU), Streptococcus intermedius (2.4-2.8x10⁷ CFU), Prevotella stercorea (1.2x10⁵ CFU) and Prevotella copri (80 CFU) were mixed with 3-12 ug/ml rDEFB119 or vehicle control in a 96-well plate with a total volume of 200 ul and were incubated under aerobic condition for 24 hours for Pseudomonas or anaerobic conditions for 24 hours for Streptococcus and for 48 hours for Prevotella. Optical density at 620 nm was measured after the incubation.

Statistical analysis

Statistical analysis of the seminal microbiome was conducted in R 4.0.4. A series of α-diversity analyses were calculated by the Kruskal-Wallis rank-sum test among groups. β-diversity was calculated based on unweighted and weighted UniFrac distance metrics. The PERMANOVA test on β-diversity with 999 permutations was analyzed to compare the significant community dissimilarity among groups. The ANCOM analysis, another test for microbial composition, was performed to test differential bacteria among groups. The differential biomarkers among groups were picked by both the Random forest method and Linear discriminant analysis effect size (LEfSe) method with α equal to 0.05 and an LDA score threshold of 3.0. Demographic characteristics across groups were compared using Mann-Whitney tests (two groups) or Kruskal-Wallis test (three groups) with Dunn’s multiple comparison test for continuous variables and the Chi-square test for categorical variables. Mediation analyses were performed following the published protocol with minor modifications [28]. Briefly, significant mediation effects were assessed between sequential pairs of factors along the microbiome (M)–DEFB119 (D)–spermiogram(S) axis in the normal and infertile groups respectively using R mediate package, with subject age (A) being covariates. Six models of mediation were tested. For example, mediation analysis for Model 1: DEFB119(D)→ microbiome(M, ie. each ASVs)→spermiogram(S, each spermiogram) was performed by fitting into 2 linear models, M =α1+β1D+δ1A+εi1 and S =α2+β2D+γ1M+δ2A+εi2 PThe adjusted P values of the mediation models from Mediate R package were obtained for each model. P-value < 0.05 represents statistical significance.

Data availability

The 16s rRNA gene sequencing data are available in the Sequence Read Archive database with BioProject ID: PRJNA747100. Pre-publication access for reviewer is available at: https://dataview.ncbi.nlm.nih.gov/object/PRJNA747100?reviewer=vvq8rtf3tn7bisaafjtugpapse

Distorted bacterial network in male factor infertility

To compare the seminal microbiome in men with normal or abnormal spermiogram, we recruited 88 patients seeking assisted reproduction with either abnormal (male-factor infertility, n=58) or normal spermiogram (partners of female-factor infertility or idiopathic infertility, n=30) (Supplemental Table S1). Patients with abnormal spermiogram include oligozoospermia, asthenozoospermia, teratozoospermia or combined cases. The age of the patient, the age of the partner, the treatment outcome and semen volume were comparable. However, the sperm concentration, total motility, progressive motility and morphology were significantly lowered in the male-factor infertile group.

We then performed 16S rRNA gene sequencing on seminal plasma samples from this cohort of patients. After sequencing, a sequence curation pipeline optimized for analyses of amplicon libraries was performed for quality control with a low sequencing error rate [29]. In total, 2591 amplicon sequence variants (ASVs) were identified across all the seminal plasma samples. After removing all 178 contaminants in the negative controls, 2413 ASVs were maintained. Consistent with previous reports [2], Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes were the major phyla in the seminal microbiome that constituted at least 80% of the phyla identified (Fig 1A). We then compared the richness and evenness of the bacterial community using multiple indices. In our cohort, we observed a significant difference in Weighted Unifrac distance between the normozoospermia and male-factor infertile group but no significant difference in other α- and β- diversities between the normozoospermia and male-factor infertile group were observed (Fig 1B-D, Supplemental Fig S1-2). At the genus level, we observed an enrichment of Veillonella, Gardnerella and Lactobacillus in normozoospermia patients. However, the differential abundance of these genera was not statistically significant.

To further investigate the structure and interaction of seminal microbial communities, we performed community network analysis using the Weighted Correlation Network Analysis (WGCNA) algorithm [30], which has not been applied in the seminal microbiome. In this analysis, the nodes represent ASVs and the edges that connect these nodes represent correlations between ASVs. Notably, while the number of nodes was comparable, we observed a marked decrease in the number of edges in male-factor infertile patients (Fig 1E). In microbial community networks, the ASVs clustered into independent modules, a property known as modularity, with a small group of ASVs serving as module connectors. We observed the absence of a module hub and the disappearance of peripheral nodes in three phyla, Fusobacteria, TM7, and Spirochaetes in male-factor infertile patients which lead to the shrinkage of network diameter and heterogeneity (Fig 1E-F). These data suggest that the bacterial network is distorted in patients with abnormal spermiogram despite the comparable richness and evenness of the metacommunities.

Elevation of DEFB119 stratifies the dysbiosis in male-factor infertility

Previous studies have utilized sperm parameters as stratification factors in the analysis of seminal microbiomes. We speculated that a host factor potentially involved in the host-microbe interactions would provide a better stratification of the seminal microbiome and a higher resolution of the dysbiosis associated with male infertility. Therefore, we explored the involvement of β-defensins in regulating the seminal microbiome, we focused on DEFB119, a β-defensin that plays pivotal roles in sperm production and functions [18, 19]. We determined the protein level of DEFB119 in seminal plasma by enzyme-linked immunoassay with an antibody against the C-terminus of the protein. We observed a range of DEFB119 levels in subjects with normal spermiogram (mean 291.30, CI 210.50 – 372.00 ng/ml). Intriguingly, while the patients with abnormal spermiogram expressed a subtle increased level of DEFB119 in the seminal plasma (mean 393.60, CI 245·70 - 541·50 ng/ml), a subgroup of patients demonstrated a marked elevation of DEFB119 in seminal plasma above the 100th centile (>900 ng/ml) of subjects with normal spermiogram (Fig 2A & Table 1).

To examine the metacommunity structure associated with elevated DEFB119, we categorized the patients according to the following grouping: G1 - Normal spermiogram and low DEFB119 level (n=30); G2 - Abnormal spermiogram and low DEFB119 (n=52); and G3 - Abnormal spermiogram and elevated level of DEFB119 (n=5). We observed a lowered sperm concentration, motility and morphology in G2 and G3 as compared to G1. However, the spermiogram parameters were comparable in male-factor infertile patients with normal (G2) or elevated levels of DEFB119 (G3)(Table 1). The phylotypes in G3 were dominated by Firmicutes and Proteobacteria while the abundance of Actinobacteria and Bacteroidetes was diminished (Fig 2B). Comparing the top forty most abundant genera, the abundance of Bactobacillus, Gardnerella, and Prevotella decreased (Fig 2C). We have also compared the richness and evenness of the bacterial community among the three groups using multiple indices as in our previous analysis. The α-diversity was significantly reduced in patients with elevated levels of DEFB119 as compared to G1 and G2 groups (Shannon index p < 0.05, Fig 2D and Supplemental Fig S3). Similarly, the β-diversity as measured by Jaccard distance, Bray Curtis Distance and Weighted Unifrac distance showed that the bacterial community in patients with high levels of DEFB119 (G3) was significantly different from those in G1 and G2 groups (p < 0.05, Fig 2E-F, Supplemental Fig S4). No significant difference in α- and β- diversities except Weighted Unifrac distance were observed between G1 and G2 groups.

Differential abundance analysis at the genus level revealed a decrease in Clostridium and increases in eight genera in G3 (p < 0.05), including Sporosarcina, AF12, Helicobacter, Desulfovibrio, Phyllobacterium, Enterobacter, Anaerobacillus and Carnobacterium, despite their rare occurrence and low relative abundance (Supplemental Fig S5). WGCNA community network analysis showed that patients with elevated levels of DEFB119 (G3) showed a marked decrease in the numbers of both nodes and edges (Fig 2G). The network diameter and heterogeneity were further diminished in G3 as compared to G2. These data suggest that the elevated level of DEFB119 is associated with dysbiosis of the seminal microbiome and severe distortion of bacterial networks in male-factor infertile patients.

Mediators of abnormal sperm parameters in male infertility

Members of the β-defensin family are known to regulate sperm functions required for the migration in the female reproductive tract and successful fertilization [14, 15]. While our data showed that the elevated level of DEFB119 was associated with the dysbiosis of the seminal microbiome in male infertility, the infertile outcome could be attributed to the effect of DEFB119 on sperm functions per se, the indirect effect from the dysbiosis of the seminal microbiome or both. To study this, we examined the correlation of DEFB119 level with various sperm parameters in normozoospermia and male-factor infertile patients. Among the sperm parameters, a significant negative correlation was observed in progressive motility (Supplemental Fig S6, p < 0.05). In line with this, redundancy analysis (RDA) also revealed a negative correlation of DEFB119 with sperm motility and progressive motility (Fig 3A). Although genera such as Gardnerella and Campylobacter were positively associated with sperm concentration, Veillonella was positively associated with sperm motility and Prevotella was positively associated with sperm morphology and concentration, these associations were not significant (Fig 3A). Next, we set out to test if the abnormal sperm parameter was mediated by the elevation of DEFB119 and the dysbiosis of the seminal microbiome. Mediation analysis revealed a notable increase in the number of significant mediations in the male-factor infertile patients but not the normozoospermia control cohort (Fig 3B). Unexpectedly, significant mediations were observed in 4 out of 6 tested models, regardless of the initiators and mediators. These results suggest that the elevation of DEFB119 and the dysbiosis of the seminal microbiome could be the cause or the effect and vice versa. Nonetheless, the RDA and mediation analysis suggests the possible involvement of elevated DEFB119 and dysbiotic seminal microbiome in mediating the abnormal sperm parameters.

Elevated DEFB119 decreases sperm motility in male infertility

To validate the mediation analysis, we set out to examine the effect of elevated DEFB119 on sperm parameters. We mimicked the elevated level of DEFB119, as observed in G3, by recombinant DEFB119 (rDEFB119) treatment in a separate cohort of patients with normal or abnormal spermiogram profiles and normal level of DEFB119 in the seminal plasma i.e. being classified as G1 or G2. Since the decrease in sperm count and morphology are spermatogenic factors that would not be altered in a short incubation period of rDEFB119 treatment theoretically and both progressive motility and total motility demonstrated a negative correlation with DEFB119 in RDA (Fig 3A), we examined if the elevated DEFB119 affects the motility of sperm. Our results showed that rDEFB119 significantly decreased both total and progressive motility when compared with the vehicle control (Fig 4A-B, p<0·001). Interestingly, when the sample cohort was further categorized into normozoospermic and male-factor infertile groups based on the spermiogram profile, the decrease in progressive motility was only observed only in male-factor infertile patients (Fig 4C-D). These results, in corroboration with the mediation analysis, suggest that elevated DEFB119 level has detrimental effects on sperm motility, and may contribute to the infertile outcome of the male-factor infertile patients with disturbed seminal microbial networks.

Species-specific antimicrobial activity of DEFB119 shapes the seminal microbiome

It is well established that the β-defensins possess antimicrobial activity that contributes to the host defence against pathogens [31–35]. Of note, reproductive-tract-specific β-defensins play dual roles in host defence and sperm functions in the reproductive tract, their expressions are known to be induced by both physiological and pathological stimuli including hormone and bacterial toxin lipopolysaccharides [36, 37]. Therefore, the elevated DEFB119 in seminal plasma could be a cause or an effect of the dysbiosis of the seminal microbiome. To investigate if DEFB119 plays an active role in shaping the seminal microbiome and provoking dysbiosis, we performed bactericidal assay against two dominant genera observed in G1 and G2, Prevotella and Streptococcus, and the genus found in G3, Pseudomonas (Fig 5A-C). We include species that can be propagated in vitro and have either been reported in the semen, including Prevotella copri, Streptococcus mitis, Streptococcus anginosus and Pseudomonas aeruginosa [38–41], or found in other organ systems such as Prevotella stercorea in the gut microbiome and Streptococcus intermedius in the central nervous system [42, 43].

We treated the bacterial culture with various doses of rDEFB119 and observed the bacterial growth after culture. We observed a dose-dependent decrease in the amount of Prevotella stercorea, Streptococcus mitis, Streptococcus anginosus and Streptococcus intermedius after rDEFB119 treatments as compared to the vehicle controls, suggesting the bactericidal effect of DEFB119 on these species (Fig 5D-I). Intriguingly, rDEFB119 significantly promoted the growth of Streptococcus mitis at 3 ug/ml while higher doses demonstrate significant inhibitory effects. This result suggests that reproductive tract β-defensins can shape the seminal microbiome by exerting both promoting and inhibitory effects on specific species in a dose-dependent manner. Recombinant DEFB119 exerted a negligible effect on the growth of Pseudomonas aeruginosa and Prevotella copri regardless of the dosage used (Fig 5D-I), suggesting that these bacteria were resistant to DEFB119. Taken together, our results suggest that the elevation of DEFB119 in semen plays ab active role in provoking the dysbiosis of the seminal microbiome.

The present study is the first to characterize host-microbiome interaction in the seminal plasma of infertile patients via a reproductive tract-specific β-defensin. Members of the β-defensin family are involved in host defence and sperm functions, e.g. sperm motility and sperm-egg interaction, in the male and female reproductive tracts. Disorder in these β-defensins expressions, caused either by mutations or decreased expression, is associated with male infertility. Notably, our results showed that an elevated level of DEFB119 was significantly associated with male infertility. More importantly, we revealed a significant correlation between the elevated level of DEFB119 and the decrease in the abundance of genera, diversities and community networks of the seminal microbiome in male-factor infertile patients. In contrast, the elevated level of DEFB119 was not associated with the spermiogram parameters tested. Further functional analysis revealed that elevated DEFB119 decreases the progressive motility of sperm in male-infertile patients but not normozoospermic individuals. These results suggest that both the elevated level of DEFB119 and the dysbiosis of the seminal microbiome contribute to the infertile outcome. It should be noted that the spermiogram analysis does not include a complete profile of sperm functions, particularly those to be triggered in the female reproductive tract. Furthermore, the seminal microbiome can alter the microbiome in the female tract which affects fertilization and embryo development. Therefore, it is possible that the altered seminal microbiome stemming from elevated levels of DEFB119 may lead to deregulation in the event that occurs in the female reproductive tract that contributes to the infertile outcome. Nonetheless, the present study opens a new area of research on the etiology of male infertility attributed to the interplay between β-defensin and the seminal microbiome.

Previous studies by several groups have shown that specific phylotypes of the seminal microbiome are positively or negatively associated with abnormal spermiogram i.e. male infertility. For example, the Lactobacillus-predominant phylotype is observed in the normozoospermic patient, Pseudomonas-predominant and Prevotella-predominant phylotype is associated with abnormal spermiogram [8, 41]. In line with these findings, in the subgroup of patients with elevated levels of DEFB119, we also observed a decrease in Lactobacillus and an increase in Pseudomonas. At the species level, we observed both promoting or inhibiting roles of DEFB119 on Streptococcus and Prevotella but not Pseudomonas in a dose-dependent manner, suggesting that DEFB119 provokes the dysbiosis of the seminal microbiome. To this end, it is noteworthy that although we did not identify a phylotype associated with abnormal spermiogram in our cohort of male-factor infertility patients with normal levels of DEFB119, a decrease in the diameter of the microbial community networks and heterogeneity was observed. These findings suggest that a distorted communities network could be an event independent of the elevation of DEFB119. More importantly, dysbiosis was developed in patients with elevated DEFB119 (G3) but not those with normal levels of DEFB119 (G1 and 2). Therefore, our results have identified a previously unknown host factor that stratifies the dysbiosis from distorted microbial networks in male infertility. Further investigation of the stratification by a combination of host factors and sperm parameters will provide a better resolution on the phylotype that leads to the infertile outcome.

The human microbiota, particularly those in the gut, oral cavity and skin are known to play important roles in health and disease. The endogenously secreted anti-microbial peptides, including several members of the β-defensin family as well as other defensins, are known to be correlated with unique phylotypes in various organ systems. Our results showed that β-defensin DEFB119 specifically expressed in the reproductive tract caused a significant decrease in Actinobacteria, Bacteroidetes, Tenericutes and Fusobacteria phyla but an increase in Proteobacteria and Firmicutes. The increased phyla demonstrate resistance to the antimicrobial activity of DEFB119. Similar resistance is also observed at the species level against Pseudomonas aeruginosa. Moreover, at the species level, we observed the antimicrobial activity of DEFB119 against Prevotella stercorea and Streptococcus intermedius, both of which have not been found in the semen. These results suggest species-specific antimicrobial activity against bacteria commonly found in the semen, as well as those in other organ systems. In view of the strong expression of a plethora of β-defensin family members in the reproductive tract and the specificity of the antimicrobial activity of individual β-defensin, we postulate that the reproductive tract-specific β-defensins represent a valuable immunocompatible way to formulate desirable microbiome phylotypes in other organ systems so as to restore normal tissue homeostasis.

Taken together, our data indicate that Male infertility is associated with distorted bacterial networks. The elevation of DEFB119 in seminal plasma lower sperm motility in male infertile patients and provoke the dysbiosis of the seminal microbiome with a decrease in the number of observed genera, diversity, evenness and community networks. Our work has provided novel insight into the host-microbiome interaction via reproductive-tract-specific antimicrobial peptides, which shed light on the etiology of male infertility and may provide a valuable tool for formulating microbiomes in other organ systems.

MRT – male reproductive tract

DNA – deoxyribonucleic acid

FRT – female reproductive tract

rRNA – ribosomal ribonucleic acid

ELISA – enzyme-linked immunoassay

CI – confidence interval

ASV – amplicon sequence variant

RDA – redundancy analysis

WGCNA – Weighted Correlation Network Analysis

PCR – polymerase chain reaction

Ethics approval and consent to participate

All participants provided written informed consent and local ethics approvals were obtained for this study.

Consent for publication

Not applicable.

Availability of data and material

All sequencing data are available at Sequence Read Archive database.

Competing interests

The authors declare no conflict of interest.

Funding

This work was partially funded by Health and Medical Research Fund, Department of Health, Hong Kong SAR government (06170476 to EKLF, 17180701 to MBWC, DYLC, HCHY, EKLF and 06170246 to DYLC) and Lo Kwee Seong Start-up Fund to EKLF.

Authors’ contributions

HCHY, and EKLF conceived and design the study. JJ, HMEC, CSYC, OAA, and DYLC participated in sample collection the acquisition of data. JJ, HCHY, HMEC, YW, JL, XJ, EKLF analyzed and interpreted the data. JJ, HCHY, and EKLF drafted and revised the manuscript.

Acknowledgements

We would like to thank the Dr Samuel Forster (Hudson Institute of Medical Research, Australia) and Prof Georgina Hold (University of New South Wales, Australia) for providing the bacterial species. We would also like to thank the core facilities of the School of Biomedical Sciences for providing technical support.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

Table1.xlsx
Table 1: Clinical characteristics of patients with normozoospermia (G1) and male-factor infertility with (G3) or without (G2) elevation of DEFB119 levels. Data represent mean ± 95% confidence interval. N.A.: Not available
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DEFB119 stratifies dysbiosis with distorted networks in the seminal microbiome associated with male infertility

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