Unraveling the Intricacies of the Seminal Microbiome and Its Impact on Human Fertility.

Although the microbial communities from seminal fluid were an unexplored field some decades ago, their characteristics and potential roles are gradually coming to light. Therefore, a complex and specific microbiome population with commensal niches and fluctuating species has started to be revealed. In fact, certain clusters of bacteria have been associated with fertility and health, while the outgrowth of several species is potentially correlated with infertility indicators. This constitutes a compelling reason for outlining the external elements that may induce changes in the seminal microbiome composition, like lifestyle factors, gut microbiota, pathologies, prebiotics, and probiotics. In this review, we summarize the main findings about seminal microbiome, its origins and composition, its relationship with fertility, health, and influence factors, while reminding readers of the limitations and advantages introduced from technical variabilities during the experimental procedures.

The courtship choreography of homologous chromosomes: timing and mechanisms of DSB-independent pairing.

Meiosis involves deep changes in the spatial organisation and interactions of chromosomes enabling the two primary functions of this process: increasing genetic diversity and reducing ploidy level. These two functions are ensured by crucial events such as homologous chromosomal pairing, synapsis, recombination and segregation. In most sexually reproducing eukaryotes, homologous chromosome pairing depends on a set of mechanisms, some of them associated with the repair of DNA double-strand breaks (DSBs) induced at the onset of prophase I, and others that operate before DSBs formation. In this article, we will review various strategies utilised by model organisms for DSB-independent pairing. Specifically, we will focus on mechanisms such as chromosome clustering, nuclear and chromosome movements, as well as the involvement of specific proteins, non-coding RNA, and DNA sequences.

The impact of chromosomal fusions on 3D genome folding and recombination in the germ line.

The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination.

Sperm microRNA pairs: new perspectives in the search for male fertility biomarkers.

OBJECTIVE: To identify candidates of fertility biomarkers among pairs of human sperm microRNAs. DESIGN: Expression data of 736 sperm microRNAs from fertile and infertile individuals characterized in previous published studies by means of TaqMan quantitative polymerase chain reaction (PCR) were reexamined. A set of microRNA pairs with the best biomarker potential were selected and validated by means of quantitative real-time (qRT) PCR in an independent cohort. SETTING: University laboratory. PATIENT(S): Semen samples were obtained from fertile (n = 10) and infertile (asthenozoospermia, n = 10; teratozoospermia, n = 10; oligozoospermia, n = 10; unexplained male infertility [UMI], n = 8) individuals. The validation cohort included 9 fertile donors and 14 infertile patients with different seminal alterations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Spearman test was used to select microRNA pairs with a correlated expression in fertile individuals and a noncorrelated expression in each infertile group. The biomarker potential of these pairs was determined with the use of receiver operating characteristic curves. The differential relative expression of each pair in fertile and infertile populations was verified (Mann-Whitney test). Those pairs with best results were validated by qRT-PCR. RESULT(S): Forty-eight pairs showed significant correlations in the fertile group. The pairs that were uncorrelated in the infertile populations and displayed the best biomarker potential were hsa-miR-942-5p/hsa-miR-1208 (asthenozoospermia), hsa-miR-296-5p/hsa-miR-328-3p (teratozoospermia), hsa-miR-139-5p/hsa-miR-1260a (oligozoospermia), and hsa-miR-34b-3p/hsa-miR-93-3p (UMI). The hsa-miR-942-5p/hsa-miR-1208 pair showed the greatest potential for detecting seminal alterations in the validation cohort (85.71% true positives). CONCLUSION(S): The pairs hsa-miR-942-5p/hsa-miR-1208 and hsa-miR-34b-3p/hsa-miR-93-3p have the potential to become new molecular biomarkers that could help to diagnose male infertility, especially in cases of UMI or when seminal parameters are close to the threshold values.

The use of fluorescence in situ hybridization analysis on sperm: indications to perform and assisted reproduction technology outcomes.

PURPOSE: To determine the consequences of an altered sperm fluorescence in situ hybridization (FISH) result for ART outcomes and the indications for a sperm FISH analysis. METHODS: Data from 439 infertile men were collected. Bivariate analyses were performed to determine the association of men's age, seminal alterations, and sperm FISH indication, with the incidence of X, Y, 13, 18, and 21 sperm chromosomal abnormalities. A multivariate logistic regression analysis was performed to establish the most predictive variables for altered sperm FISH. Results from the IVF/ICSI cycles were collected for 248 out of 439 patients. Two distinct groups were established: 151 couples that used their own oocytes and 97 couples involved in egg donation programs. In both groups, ART outcomes were compared between normal and altered sperm FISH. RESULTS: Teratozoospermia and oligozoospermia were associated with sperm chromosome anomalies (p < 0.05). Indications for sperm FISH analysis with the highest predictability were teratozoospermia, male age, oligozoospermia, and implantation failure (AUC = 0.702). Embryo quality (p = 0.096), pregnancy rate (p = 0.054), and implantation rate (p = 0.089) were higher in own-oocytes couples with normal sperm FISH than in altered sperm FISH couples, although differences were not statistically significant. In donor-oocytes couples, in which high-quality embryos were transferred later than in own-oocytes couples (3.8 vs. 3.0 days), we did not identify differences in the ART outcome between normal and altered sperm FISH couples. In both groups, the possible interference of woman age was negligible. CONCLUSIONS: Sperm FISH is indicated in middle-aged oligoteratozoospermic patients with implantation failures in previous IVF/ICSI cycles. Sperm chromosome anomalies have a moderate detrimental impact on embryo quality, implantation, and pregnancy rates.

Chromosome positioning and male infertility: it comes with the territory.

The production of functional spermatozoa through spermatogenesis requires a spatially and temporally highly regulated gene expression pattern, which in case of alterations, leads to male infertility. Changes of gene expression by chromosome anomalies, gene variants, and epigenetic alterations have been described as the main genetic causes of male infertility. Recent molecular and cytogenetic approaches have revealed that higher order chromosome positioning is essential for basic genome functions, including gene expression. This review addresses this issue by exposing well-founded evidences which support that alterations on the chromosome topology in spermatogenetic cells leads to defective sperm function and could be considered as an additional genetic cause of male infertility.

Altered bivalent positioning in metaphase I human spermatocytes from Robertsonian translocation carriers.

PURPOSE: The study aims to determine whether there is an altered bivalent positioning in metaphase I human spermatocytes from Robertsonian translocation carriers. METHODS: Metaphase I human spermatocytes from three 45,XY,der(13;14)(q10;q10) individuals and a 45,XY,der(14;15)(q10;q10) individual were analyzed. Proximity relationships of bivalents were established by analyzing meiotic preparations combining Leishman staining and multiplex-FISH procedures. Poisson regression model was used to determine proximity frequencies between bivalents and to assess associations with chromosome size, gene density, acrocentric morphology, and chromosomes with heterochromatic blocks. The hierarchical cluster Ward method was used to characterize the groups of bivalents with preferred proximities in a cluster analysis. Bivalent groups obtained were individually compared with those obtained in normal karyotype individuals evaluated in a previous study. RESULTS: A total of 1288 bivalents were examined, giving a total of 2289 proximity data. Only four positive significant proximities were detected for each type of Robertsonian translocation. Significant bivalent associations were only observed by small-size chromosomes for MI,22,XY,III(13q14q). These results were clearly divergent from 46,XY individuals. Moreover, cluster analysis revealed that about 30 % of the bivalents showed changes in their proximity relationships in metaphase I. CONCLUSIONS: The territorial organization of bivalents in metaphase I human spermatocytes changes in the presence of a Robertsonian translocation.

Meiotic abnormalities in metaphase I human spermatocytes from infertile males: frequencies, chromosomes involved, and the relationships with polymorphic karyotype and seminal parameters.

The aim of this study was to look in depth at the relationship between meiotic anomalies and male infertility, such as the determination of the chromosomes involved or the correlation with patient features. For this purpose, a total of 31 testicular tissue samples from individuals consulting for fertility problems were analyzed. Metaphase I cells were evaluated using a sequential methodology combining Leishman stained procedures and multiplex fluorescence in situ hybridization protocols. The number of chromosomal units and chiasmata count per bivalent were established and a hierarchical cluster analysis of the individuals was performed. The relationship of the seminogram and the karyotype over recombination were evaluated using Poisson regression models. Results obtained in this study show a significant percentage of infertile individuals with altered meiotic behavior, mostly specified as a reduction in chiasmata count in medium and large chromosomes, the presence of univalents, and the observation of tetraploid metaphases. Moreover, the number and the type of anomalies were found to be different between cells of the same individual, suggesting the coexistence of cell lines with normal meiotic behavior and cell lines with abnormalities. In addition, chromosomal abnormalities in metaphase I are significantly associated with oligozoospermia and/or polymorphic karyotype variants.

Chromosome size, morphology, and gene density determine bivalent positioning in metaphase I human spermatocytes.

OBJECTIVE: To determine whether there is a preferential bivalent distribution pattern in metaphase I human spermatocytes and to analyze whether this positioning is influenced by chiasmata count, chromosome size, gene density, acrocentric morphology, and heterochromatic blocks. DESIGN: Proximity frequencies of bivalents were evaluated with the analysis of meiotic preparations combining sequentially standard techniques and multiplex fluorescence in situ hybridization. SETTING: University. PATIENT(S): Twenty-five men consulting for fertility problems. INTERVENTION(S): Unilateral testicular biopsies. MAIN OUTCOME MEASURE(S): Proximity analyses were performed for each bivalent considering as nearby bivalents those that were part of the first ring around the bivalent studied. Data were analyzed using Poisson regression models, multidimensional scaling, and cluster analysis. RESULT(S): Some bivalents have a preferential relative position. Significant associations among bivalents related to chromosome size, high gene density, and acrocentric morphology were observed. Chiasmata count and heterochromatic blocks were nonconditioning parameters of the bivalent organization. CONCLUSION(S): This study demonstrates that distribution in metaphase I is nonrandom and influenced by chromosome size, gene density, and acrocentric chromosome morphology. Results support that some features defining chromosome territories are maintained during meiosis.

A sequential methodology that allows apoptotic cell sorting and FISH analysis in human testicular cells.

The objective of this study was to develop a methodology that permits the detection and separation of apoptotic cells in human testicular tissue and their subsequent cytogenetic analysis by fluorescence in situ hybridization (FISH). The sequential methodology consisted of five steps: 1) enzymatic disaggregation of testicular tissue, 2) specific staining of apoptotic cells, 3) cell sorting by flow cytometry, 4) cell fixation, and 5) FISH. Enzymatic disaggregation yielded cell counts that ranged from 1.7x10(5) to 5x10(6) cells, and viability values greater than 72%. The apoptotic (mean ± SD: 22% ± 5.3%) and viable (45.5% ± 7.3%) populations were identified and selected by flow cytometry and demonstrated purity values ranging between 62% and 100%. The paraformaldehyde fixation of the selected fractions resulted in cell loss values of less than 10%. The application of three treatments before FISH (membrane permeabilization, elimination of cytoplasmic components, and re-fixation of the sample) resulted in hybridization frequencies of greater than 98%. In both selected fractions, cells of all spermatogenic stages and Sertoli cells were identified. The methodology developed has enabled the preparation of a cellular suspension with optimal viability and counting, the efficient selection of the apoptotic population, and its analysis by cytogenetic techniques. The application of this methodology in testicular cells should help establish whether there is a direct relationship between chromosome anomalies and apoptosis.

Acrocentric bivalents positioned preferentially nearby to the XY pair in metaphase I human spermatocytes.

OBJECTIVE: To analyze whether the preferential proximity between acrocentric bivalents and the XY pair described at pachytene was maintained in metaphase I human spermatocytes. DESIGN: Proximity frequencies of autosomic bivalents to the sex bivalent were evaluated with the analysis of meiotic preparations combining sequentially standard techniques and multiplex fluorescence in situ hybridization. SETTING: Assisted reproduction centers. PATIENT(S): Thirty-seven men consulting for fertility problems. INTERVENTION(S): Unilateral testicular biopsies. MAIN OUTCOME MEASURE(S): Proximity frequencies analysis to the XY pair, evaluated individually and grouping bivalents, was carried out using a logistical regression model with repeated measures. RESULT(S): Bivalents 22 and 15 were observed more frequently near to the sex bivalent than the others. Significant interindividual differences were not observed. CONCLUSION(S): Results suggest that bivalents distribution to the metaphase plate is nonrandom. The maintenance of the acrocentric chromosomes' proximity to the sex bivalent from pachytene to metaphase I would indicate that the relative bivalents position would be notably preserved. The observation of non-interindividual variability, despite different infertility etiology, suggests that the nuclear organization pattern remains largely unaffected even if spermatogenesis is compromised.

Role of sperm fluorescent in situ hybridization studies in infertile patients: indications, study approach, and clinical relevance.

OBJECTIVE: To determine the group of infertile patients that would benefit from sperm fluorescent in situ hybridization (FISH) analysis, the number of chromosomes to be analyzed, and the diagnostic interpretation of the results obtained. DESIGN: A retrospective study of sperm FISH analyses. SETTING: Universitat Autònoma de Barcelona. PATIENT(S): Three hundred nineteen infertile men. INTERVENTION(S): Semen samples were processed for FISH. MAIN OUTCOME MEASURE(S): The frequencies of chromosomal abnormalities for chromosomes 13, 18, 21, X, and Y were compared to the seminogram, the somatic and meiotic karyotype, and the age. RESULT(S): The highest percentages of patients with an increased rate of sperm chromosome abnormalities were found in the oligozoospermic (50%), oligoasthenozoospermic (33.3%), and oligoasthenoteratozoospermic (21%) individuals. Low sperm count was the only parameter correlated with the percentage of chromosome abnormalities. The 14% of the individuals with a normal somatic karyotype had an increased rate of sperm chromosome abnormalities. This percentage was higher in the group with an altered somatic karyotype (36%) and in patients with meiotic abnormalities (26%). CONCLUSION(S): Sperm FISH studies are indicated when the oligo condition is present and in individuals with an abnormal somatic or meiotic karyotype. The analysis of chromosomes 21, X, and Y is enough to identify at-risk individuals. Significant differences in the rates of chromosome abnormalities should be taken into consideration regardless of the numerical value.

Fluorescence in situ hybridization (FISH) protocol in human sperm.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring. For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice. To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21. This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

FISH on sperm: spot-counting to stop counting? Not yet.

OBJECTIVE: To evaluate the reliability and applicability of the spot-counting system (Cytovision Spot AX workstation) which offers an alternative to the tedious manual analysis of sperm fluorescence in situ hybridization (FISH). DESIGN: Manual and automatic analyses were performed and compared. SETTING: Universitat Autònoma de Barcelona. PATIENT(S): Twenty-four men who asked for information on infertility showing different seminal parameters. INTERVENTION(S): A semen sample for each patient was collected and prepared for FISH. MAIN OUTCOME MEASURE(S): A dual-color FISH using specific probes for chromosomes 13 and 21 and a triple-color FISH with centromeric probes for chromosomes 18, X, and Y were used (Vysis). Standard FISH analysis was carried out. Automatic analysis was subsequently performed using a Spot AX system. RESULT(S): Overall, we performed 120 comparisons. In 116 out of 120 (96.67%), the percentage of anomalies reported using manual counting fell within the incidence detected using the automatic system. In the remaining comparisons, statistical differences were detected (4 out of 120; 3.33%). Time consumed by the automatic analysis was always higher than the manual one, being influenced by the characteristics of the preparations. CONCLUSION(S): The spot-counting system has potential, but before the service is ready to be offered, we still need to overcome some limitations associated with it.

FISH studies of chromosome abnormalities in germ cells and its relevance in reproductive counseling.

Chromosome abnormalities are one of the major causes of human infertility. In infertile males, abnormal karyotypes are more frequent than in the general population. Furthermore, meiotic disorders affecting the germ cell-line have been observed in men with normal somatic karyotypes consulting for infertility. In both cases, the production of unbalanced spermatozoa has been demonstrated. Basically addressed to establish reproductive risks, fluorescence in situ hybridization (FISH) on decondensed sperm heads has become the most frequently used method to evaluate the chromosomal constitution of spermatozoa in carriers of numerical sex chromosome abnormalities, carriers of structural chromosome reorganizations and infertile males with normal karyotype. The aim of this review is to present updated figures of the information obtained through sperm FISH studies with an emphasis on its clinical significance. Furthermore, the incorporation of novel FISH-based techniques (Multiplex-FISH; Multi-FISH) in male infertility studies is also discussed.

Identification of meiotic anomalies with multiplex fluorescence in situ hybridization: Preliminary results.

OBJECTIVE: To characterize meiotic anomalies in infertile men by multiplex fluorescence in situ hybridization (M-FISH) and to determine whether synaptic problems affect specific bivalents or whether anomalies are random. DESIGN: Analysis of meiotic preparations with standard techniques and M-FISH. SETTING: Assisted reproduction centers and Universitat Autònoma de Barcelona. PATIENT(S): Three fertile men undergoing vasectomy, four sterile patients with oligoasthenoteratozoospermia, and one patient with a Robertsonian translocation t(13;14). INTERVENTION(S): Unilateral testicular biopsy in controls and patients with oligoasthenoteratozoospermia and collection of a semen sample from the translocation carrier. MAIN OUTCOME MEASURE(S): Identification of bivalents in metaphase I and chromosomes in metaphase II and characterization of chromosome abnormalities. RESULT(S): All bivalents in metaphase I and all chromosomes in metaphase II could be identified. In controls and in one patient with oligoasthenoteratozoospermia, meiosis was normal. Other patients with oligoasthenoteratozoospermia showed different types of anomaly: desynapsis, breaks, precocious XY separation, or cryptic reorganizations. The Robertsonian translocation t(13;14) was easily identified. CONCLUSION(S): Results confirm the high incidence of synaptic errors in oligoasthenoteratozoospermia patients. Bivalents in metaphase I and chromosomes in metaphase II were individually identifiable. Nondisjunctional errors or small reorganizations overlooked in classic meiotic preparations were identified. Synaptic anomalies seem to affect meiotic bivalents at random.