The elusive male microbiome: revealing the link between the genital microbiota and fertility. Critical review and future perspectives.

There is a growing focus on understanding the role of the male microbiome in fertility issues. Although research on the bacterial communities within the male reproductive system is in its initial phases, recent discoveries highlight notable variations in the microbiome's composition and abundance across distinct anatomical regions like the skin, foreskin, urethra, and coronary sulcus. To assess the relationship between male genitourinary microbiome and reproduction, we queried various databases, including MEDLINE (available via PubMed), SCOPUS, and Web of Science to obtain evidence-based data. The literature search was conducted using the following terms "gut/intestines microbiome," "genitourinary system microbiome," "microbiome and female/male infertility," "external genital tract microbiome," "internal genital tract microbiome," and "semen microbiome." Fifty-one relevant papers were analyzed, and eleven were strictly semen quality or male fertility related. The male microbiome, especially in the accessory glands like the prostate, seminal vesicles, and bulbourethral glands, has garnered significant interest because of its potential link to male fertility and reproduction. Studies have also found differences in bacterial diversity present in the testicular tissue of normozoospermic men compared to azoospermic suggesting a possible role of bacterial dysbiosis and reproduction. Correlation between the bacterial taxa in the genital microbiota of sexual partners has also been found, and sexual activity can influence the composition of the urogenital microbiota. Exploring the microbial world within the male reproductive system and its influence on fertility opens doors to developing ways to prevent, diagnose, and treat infertility. The present work emphasizes the importance of using consistent methods, conducting long-term studies, and deepening our understanding of how the reproductive tract microbiome works. This helps make research comparable, pinpoint potential interventions, and smoothly apply microbiome insights to real-world clinical practices.

PELP1 and SRC kinase as important molecules in the estrogen-mediated pathway in human testis and epididymis.

BACKGROUND: Estrogens have pleiotropic mechanisms of action, and their cellular transduction pathways can modulate various proteins with differential tissue expression. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is one such protein whose role seems important, although little is known about this protein. However, very little is known about the expression of modulators involved in the estrogen-mediated pathways in the tissues of the male reproductive tract. METHODS: In this study, we obtained autopsy specimens of testis and epididymis from 13 men of Caucasian descent. Expression levels were analyzed for both estrogen receptors (ESR1 and ESR2) and their co-regulators, including PELP1 and kinase c-Src (SRC). RESULTS: Protein expression was confirmed with western blot and immunocytochemistry techniques. The expression of both SRC and PELP1 was significantly higher in the testis compared to the epididymis (p=0.040 and p=0.002, respectively). Furthermore, a significant, positive correlation was observed between SRC and PELP1, regardless of tissue type p<0.0001, R=0.78). In the testis, PELP1 expression positively correlated with ESR1 expression (p=0.367, R=0.6). CONCLUSIONS: Our study suggests a possible relationship between PELP1, SRC, and ESR1 in the human testis and epididymis. This study makes a valuable contribution to the field of estrogen-mediated pathways in the male reproductive tract and describes trends of analyzed genes' expression and presence. We think our results may open some new research directions of the estrogen signaling in the male reproductive system.

Expression of estrogen receptors, PELP1, and SRC in human spermatozoa and their associations with semen quality.

Sperm cells are target cells for both estrogens and xenoestrogens. Due to the specific structure of spermatozoa, these hormonal compounds may act on sperm in a non-genomic mechanism only. However, the ESR-mediated signaling pathways are still poorly understood. In this study, we obtained 119 samples from male participants of Caucasian descent who donated semen for standard analysis. We analyzed gene expression of estrogen receptors (ESR1 and ESR2) and their coregulators-proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and cellular kinase c-Src (SRC). RNA level was established using reverse-transcribed RNA as a template, followed by a polymerase chain reaction. Proteins' presence was confirmed by western blot and immunocytochemistry techniques. "Normal" values of semen parameters were defined as follows: > 32% sperm with progressive motility, > 4% sperm cells with normal morphology, > 15 × 106 sperm per mL, > 58% live spermatozoa and leukocyte amount < 106 cells per mL, according to WHO 2010 reference. Semen parameters that deviated from these "normal" values were labeled as "abnormal". Gene expression ratios revealed significant, moderate, and negative correlations for ESR1/ESR2 and weak, negative ESR2/PELP1 correlations in the subgroup of patients with abnormal values of semen parameters. In addition, SRC/PELP1 was moderately and positively correlated in the subgroup with parameters within the reference values established by WHO 2010. Our study showed that both PELP1 scaffolding protein and SRC kinase might influence semen quality via ESRs. It seems that not the expression of a single gene may affect the sperm quality, but more gene-to-gene mutual ratio. Characterization of estrogen-signaling pathway-related genes' modulated expression in sperm cells could aid in better understanding sperm biology and quality.

Impact of Biometric Patient Data, Probiotic Supplementation, and Selected Gut Microorganisms on Calprotectin, Zonulin, and sIgA Concentrations in the Stool of Adults Aged 18-74 Years.

Alterations to the intestinal barrier may be involved in the pathogenesis of various chronic diseases. The diagnosis of mucosal barrier disruption has become a new therapeutic target for disease prevention. The aim of this study was to determine whether various patient demographic and biometric data, often not included in diagnostic analyses, may affect calprotectin, zonulin, and sIgA biomarker values. Stool markers' levels in 160 samples were measured colorimetrically. The analysis of twenty key bacteria (15 genera and 5 species) was carried out on the basis of diagnostic tests, including cultures and molecular tests. The concentrations of selected markers were within reference ranges for most patients. The sIgA level was significantly lower in participants declaring probiotics supplementation (p = 0.0464). We did not observe differences in gastrointestinal discomfort in participants. We found significant differences in the sIgA level between the 29-55 years and >55 years age-related intervals groups (p = 0.0191), together with a significant decreasing trend (p = 0.0337) in age-dependent sIgA concentration. We observed complex interdependencies and relationships between their microbiota and the analyzed biomarkers. For correct clinical application, standardized values of calprotectin and sIgA should be determined, especially in elderly patients. We observed a correlation between the composition of the gut community and biomarker levels, although it requires further in-depth analysis.

Commercial microbiota test revealed differences in the composition of intestinal microorganisms between children with autism spectrum disorders and neurotypical peers.

The early-life modifications of intestinal microbiota may impact children's subsequent emotional and cognitive development. Studies show that some bacteria species in gut microbiota, and the lack of others, may play a key role in autism spectrum disorders (ASD) development. Fecal samples were obtained from three groups of children: 16 healthy, 24 with allergies (ALG), and 33 with ASD (probiotics and non-probiotics users). The analysis was carried out according to the KyberKompakt Pro protocol. We observed a significantly higher level of Klebsiella spp. in the healthy children from the non-probiotics group, considering three groups. In the same group, Bifidobacterium spp. the level was lower in ASD compared to neurotypical individuals. In healthy children who did not use probiotics, strong positive correlations were observed in E. coli and Enterococcus spp. and Bacteroides and Klebsiella spp., and a negative correlation for Akkermansia muciniphila with both Klebsiella spp. and Bacteroides spp. In the ASD group who take probiotics, a strongly negative correlation was observed in Lactobacillus spp., and both Faecalibacterium prausnitzii and Akkermansia muciniphila levels. In the ALG group, the strongest, negative correlation was found between Enterococcus spp. and Lactobacillus spp. as in Akkermansia muciniphila and Bifidobacterium spp. The simple commercial test revealed minor differences in the composition of intestinal microorganisms between children with autism spectrum disorders and neurotypical peers.

Effect of low-frequency electric field screening on motility of human sperm.

INTRODUCTION: The human body is constantly exposed to an extremely low electromagnetic field (ELF-EMF), in particular at 50 Hz, emitted by power lines, domestic distribution lines, electrical appliances, etc. It is assumed that the increase in electromagnetic exposure may cause adverse effects upon human health, as well as raising concerns regarding the impact on human fertility. OBJECTIVE: The aim of this in vitro study was to investigate the influence of ELF-EMF with a frequency of 50 Hz on the motility of human sperm. At the same time, the effectiveness of the dielectric screen constructed by ADR Technology ® in absorbing the emitted radiation was examined. MATERIAL AND METHODS: Semen samples of 20 patients were exposed to the influence of an extremely low electromagnetic field. After 5, 15 and 30 min., spermatozoa motility was analysed using a computer-assisted spermatozoa motility analysis system. The following sperm motility parameters were examined: 1) velocity straight linear motility; 2) cross-beat frequency; 3) lateral head displacement; 4) homogeneity of progressive motility velocity. RESULTS: It was found that the ELF-EMF presented a negative effect on the motility of human spermatozoa. A significant decrease in spermatozoa motility speed and a significant increase in lateral head deviation values were observed under the influence of the electromagnetic field. ELF-EMF did not show an effect on either lateral head displacement or homogeneity of progressive motility velocity. CONCLUSIONS: A positive effect of the dielectric screen ADR Technology® was found. This effect compensated spermatozoa motility changes induced with ELF-EMF.

Increased expression of PELP1 in human sperm is correlated with decreased semen quality.

Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein involved in both genomic and nongenomic estrogen signal transduction pathways. To date, the role of PELP1 protein has yet to be characterized in human sperm and has not been associated with sperm parameters. To confirm the presence of PELP1 in human sperm, fresh semen samples were obtained from 178 donors. The study was designed to establish both mRNA and protein presence, and protein cellular localization. Additionally, the number of PELP1-positive spermatozoa was analyzed in men with normal and abnormal semen parameters. Sperm parameters were assessed according to the World Health Organization (WHO) 2010 standards. The presence of PELP1 in spermatozoa was investigated using four precise, independent techniques. The qualitative presence of transcripts and protein was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and western blot protocols, respectively. The cellular localization of PELP1 was investigated by immunocytochemistry. Quantitative analysis of PELP1-positive cells was done by flow cytometry. PELP1 mRNA and protein was confirmed in spermatozoa. Immunocytochemical analysis identified the presence of PELP1 in the midpieces of human sperm irrespective of sperm parameters. Becton Dickinson fluorescence-activated cell sorting (FACSCalibur™) analysis revealed a significantly lower number of PELP1-positive cells in males with normal semen parameters versus abnormal samples (42.78% ± 11.77% vs 61.05% ± 21.70%, respectively; P = 0.014). The assessment of PELP1 may be a time-saving method used to obtain information about sperm quality. The results of our study suggest that PEPL1 may be utilized as an indicator of sperm quality; thereby, PELP1 may be an additional biomarker useful in the evaluation of male infertility.

17ß-estradiol modifies human spermatozoa mitochondrial function in vitro.

BACKGROUND: It is assumed that spermatozoa are target cells for estrogens however, the mechanism of their action is not fully understood. The aim of this study was to investigate the influence of 17ß-estradiol (E2) on the human spermatozoa mitochondrial function. METHODS: The effects on spermatozoa of E2 at final concentrations of 10(-10), 10(-8) and 10(-6) M were studied regarding the following phenomena: (1) kinetics of intracellular free calcium ions changes (using Fluo-3), (2) mitochondrial membrane potential ΔΨm (using JC-1 fluorochrome), (3) production of superoxide anion in mitochondria (using MitoSOX RED dye), (4) spermatozoa vitality (propidium iodide staining) and (5) phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein). RESULTS: E2 initiated rapid (within a few seconds) dose dependent increase of intracellular free calcium ions concentration. E2 was changing the mitochondrial membrane potential: 10(-8) M initiated significant increase of percentage of high ΔΨm spermatozoa while the 10(-6) M induced significant decrease of high ΔΨm cells. In spermatozoa stimulated with E2 10(-6) M a significant increase of mitochondrial superoxide anion level was observed. 2 h incubation of spermatozoa with E2 did not alter cells vitality nor stimulated phosphatidylserine membrane translocation, for all three doses. CONCLUSIONS: 17ß-estradiol affected the human spermatozoa mitochondrial function. E2 in low concentration improved while in high concentration might deteriorate mitochondrial function.

17ß-estradiol and xenoestrogens reveal synergistic effect on mitochondria of human sperm.

OBJECTIVES: The aim of the study was to investigate the influence of 17ß-estradiol (main endogenous estrogen) and selected xenoestrogens (genistein, bisphenol-A), individually and in combination, on the mitochondrial function of human sper-matozoa. In natural environment, human beings are exposed to multiple xenoestrogens, so their impact is combined with endogenous steroids. MATERIAL AND METHODS: The effects of ligands on human spermatozoa were assessed regarding the following phenomena: spermatozoa vitality (propidium iodide staining), phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein), mitochondrial membrane potential (using JC-1 fluorochrome), and production of superoxide anion in mitochondria (using MitoSOX RED dye). RESULTS: Two-hour incubation of spermatozoa with 17ß-estradiol, genistein, and bisphenol-A neither altered cell vitality nor stimulated phosphatidylserine membrane translocation. Incubation of spermatozoa with 17ß-estradiol or bisphenol-A sepa-rately, as well as incubation with the three ligands simultaneously, resulted in altered mitochondrial membrane potential. Spermatozoa incubation with the three ligands significantly increased the mitochondrial superoxide anion level. CONCLUSIONS: It seems safe to conclude that human spermatozoa mitochondria are target cell structures for both, 17ß-estradiol and xenoestrogens. The reaction to the 17ß-estradiol and xenoestrogens mixture suggests a synergistic mechanism of action. Xenoestrogens may increase the sensitivity of spermatozoa to 17ß-estradiol.

Sperm midpiece apoptotic markers: impact on fertilizing potential in in vitro fertilization and intracytoplasmic sperm injection.

The aim of this study was to investigate the relationship between apoptotic markers present in human spermatozoa, namely phosphatidylserine translocation (PST) from the inner to the outer layer of the cytomembrane and the active form of caspase-3 (c3) versus the fertilizing potential of male gametes in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) models. A total of 116 male patients treated with their partners for infertility underwent basic semen analysis and an assessment of the presence of PST and the active c3 in sperm using flow cytometry. Forty patients underwent IVF, group A, while 76 patients underwent ICSI, group B. The fertilizing potential of the gametes was measured as the percentage of oocytes with pronuclei present after either procedure. PST and active c3 were identified in vital gametes, mainly in the midpiece area. Concentration, motility, morphology, and viability of spermatozoa strongly negatively correlated with both markers. In group A, a negative correlation between both markers and the success rate of conventional IVF was observed (r = -0.4, p = 0.04 for PST; r = -0.4, p = 0.02 for active c3, respectively). In group B, the success rate of ICSI did not correlate with either marker (r = -0.2, p = 0.85 for PST and r = 0.1, p = 0.51 for active c3). The two apoptotic markers localized in the sperm midpiece area may affect their function not only by decreasing basic andrologic parameters but also by reducing the probability of conception. Therefore, analysis of PST and active c3 in the sperm of patients undergoing infertility treatment should be recommended.

Hydroxyflutamide alters the characteristics of live boar spermatozoa.

Our previous study revealed that in vitro incubation of boar ejaculates with hydroxyflutamide (OH-Flu) causes changes in sperm plasma membrane integrity and its stability and sperm mitochondrial oxidative capability. To broaden the knowledge of cellular physiology of spermatozoa, we investigated direct effects of OH-Flu administered for 2 and 24 hours at concentrations of 5, 50, and 100 µg/mL, on sperm mitochondrial membrane potential and mitochondrial superoxide anion production using JC-1 dye and MitoSOX Red fluorescent probe, respectively. We further measured phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the sperm plasma membrane using an annexin-V binding assay. To provide new information of direct effects of OH-Flu on cell signaling pathway, we measured sperm intracellular calcium ion dynamics using Fluo-3. Finally, we assessed sperm motility using a computer-assisted spermatozoa analysis system. Motile sperm were highlighted using the "C-Ruch" computer program for detailed analysis of the straight line velocity distribution. For each functional test, boar spermatozoa were examined and analyzed by flow cytometry and/or confocal microscopy. The results revealed a significant decrease (P<0.05) in sperm mitochondrial membrane potential and a concomitant increase (P<0.05) in mitochondrial superoxide anion production after a 2-hour incubation with 50 µg OH-Flu compared with the respective controls and other doses used (P<0.05). The adverse effects of OH-Flu become strengthened over time (P<0.05). Notably, 50 and 100 µg OH-Flu appeared to be effective in decreasing sperm motility. Hydroxyflutamide significantly decreased (P<0.05) the fast sperm subpopulation percentage after 15 minutes and reduced the straight line velocity distribution (P<0.05). An assessment of PST revealed an increase in the percentage of PST-positive spermatozoa (P<0.05) only after exposure to OH-Flu for 24 hours. Moreover, OH-Flu at all concentrations induced a rapid increase in sperm intracellular calcium ion concentration. Altogether, the altered in vitro characteristics of live boar spermatozoa provide new insight into direct effects of OH-Flu on sperm mitochondrial membrane potential, superoxide anion production, translocation of membrane phosphatidylserine, free calcium ion dynamics, and sperm motility.

Decreased motility of human spermatozoa presenting phosphatidylserine membrane translocation-cells selection with the swim-up technique.

Phosphatidylserine membrane translocation (PST) is considered to be a marker of apoptosis; however, numerous studies have reported on its role in processes not related to cell death. The purpose of the study was to investigate: (1) what is the impact of PST on the motility of spermatozoa, and (2) does the swim-up isolation involve the percentage of cells presenting PST? Semen of 28 normozoospermic men (WHO criteria) was analyzed. High motility spermatozoa were isolated by the swim-up technique. The percentage of spermatozoa with PST in neat semen and after swim-up isolation was assessed with Annexin-V labeled with fluorescein, using flow cytometry technique. The spermatozoas' motility was measured with a computer-assisted analysis system. The kinetic subpopulations of spermatozoa were identified with dedicated software and analyzed regarding PST. Vital spermatozoa with PST demonstrated progressive movement. The motion analysis system revealed a very strong positive correlation between the percentage of vital spermatozoa with PST and the percentage of spermatozoa belonging to the slow subpopulation (r = 0.83; p < 0.05), as well as a very strong negative correlation between the percentage of vital spermatozoa with PST and the percentage of spermatozoa belonging to the rapid subpopulation (r = -0.86; p < 0.05). After the swim-up isolation, the percentage of vital spermatozoa presenting PST significantly decreased (2.4 ± 2.1% vs. 5.2 ± 2.4%; p < 0.05). Spermatozoa with PST present progressive movement; however, their motility is decreased. Isolation of spermatozoa with the swim-up technique eliminates the cells with PST.

Phosphatidylserine membrane translocation in human spermatozoa: topography in membrane domains and relation to cell vitality.

The complex structure of the human spermatozoa membrane comprises five topographic domains. Transmembrane asymmetry of the distribution of phospholipids including phosphatidylserine (PS) is considered a marker of cell activity. The objective of the study was to determine which cytomembrane domains of human spermatozoa are involved in PS membrane translocation and to identify the possible relationship of PS translocation with spermatozoa morphology and vitality. In normozoospermic semen of 35 donors, annexin-V labeling with fluorescein determined PS translocation. Propidium iodide staining distinguished between vital and dead spermatozoa. Three types of PS membrane translocation have been distinguished: (1) in the midpiece, (2) in the acrosomal part and (3) simultaneously in the midpiece and acrosomal part. In morphologically normal vital spermatozoa, PS translocation occurred in the midpiece but never in the equatorial region. In dead spermatozoa, simultaneous PS translocation in the midpiece and acrosomal part was most often observed. The difference between proportions of, respectively, vital and dead spermatozoa presenting PS translocation located in different domains was significant (P < 0.0001). In vital cells, there was no difference in PS translocation prevalence between morphologically normal and abnormal spermatozoa (P > 0.05). The strict relation of PS translocation to specific membrane domains indicates functional specificity. It seems doubtful to include this phenomenon in physiological mechanisms of elimination of abnormal spermatozoa.