Changes in environmental exposures over decades may influence the genetic architecture of severe spermatogenic failure.

STUDY QUESTION: Do the genetic determinants of idiopathic severe spermatogenic failure (SPGF) differ between generations? SUMMARY ANSWER: Our data support that the genetic component of idiopathic SPGF is impacted by dynamic changes in environmental exposures over decades. WHAT IS KNOWN ALREADY: The idiopathic form of SPGF has a multifactorial etiology wherein an interaction between genetic, epigenetic, and environmental factors leads to the disease onset and progression. At the genetic level, genome-wide association studies (GWASs) allow the analysis of millions of genetic variants across the genome in a hypothesis-free manner, as a valuable tool for identifying susceptibility risk loci. However, little is known about the specific role of non-genetic factors and their influence on the genetic determinants in this type of conditions. STUDY DESIGN, SIZE, DURATION: Case-control genetic association analyses were performed including a total of 912 SPGF cases and 1360 unaffected controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants had European ancestry (Iberian and German). SPGF cases were diagnosed during the last decade either with idiopathic non-obstructive azoospermia (n = 547) or with idiopathic non-obstructive oligozoospermia (n = 365). Case-control genetic association analyses were performed by logistic regression models considering the generation as a covariate and by in silico functional characterization of the susceptibility genomic regions. MAIN RESULTS AND THE ROLE OF CHANCE: This analysis revealed 13 novel genetic association signals with SPGF, with eight of them being independent. The observed associations were mostly explained by the interaction between each lead variant and the age-group. Additionally, we established links between these loci and diverse non-genetic factors, such as toxic or dietary habits, respiratory disorders, and autoimmune diseases, which might potentially influence the genetic architecture of idiopathic SPGF. LARGE SCALE DATA: GWAS data are available from the authors upon reasonable request. LIMITATIONS, REASONS FOR CAUTION: Additional independent studies involving large cohorts in ethnically diverse populations are warranted to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Overall, this study proposes an innovative strategy to achieve a more precise understanding of conditions such as SPGF by considering the interactions between a variable exposome through different generations and genetic predisposition to complex diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the "Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)" (ref. PY20_00212, P20_00583), the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. PID2020-120157RB-I00 funded by MCIN/ AEI/10.13039/501100011033), and the 'Proyectos I+D+i del Programa Operativo FEDER 2020' (ref. B-CTS-584-UGR20). ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, is also partially supported by the Portuguese Foundation for Science and Technology (Projects: UIDB/00009/2020; UIDP/00009/2020). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Seminal extracellular vesicle sncRNA sequencing reveals altered miRNA/isomiR profiles as sperm retrieval biomarkers for azoospermia.

BACKGROUND: Non-invasive molecular biomarkers for classifying azoospermia by origin into either obstructive or non-obstructive/secretory azoospermia, as well as for inferring the spermatogenic reserve of the testis of non-obstructive/secretory azoospermia patients, are of great interest for testicular sperm retrieval outcome prediction for assisted reproduction. Prior analyses of semen small non-coding RNA expression in azoospermia have focused on microRNAs, but there has been a lack of attention on other regulatory small RNA species. In this regard, studying more in-depth expression changes of small non-coding RNA subtypes in small extracellular vesicles from semen of azoospermic individuals could be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes. MATERIAL AND METHODS: A high-throughput small RNA profiling analysis to determine the expression pattern of seminal small extracellular vesicle microRNAs (analyzed at the isomiR level), PIWI-interacting RNAs, and transfer RNA-derived small RNAs in normozoospermic (n = 4) and azoospermic (obstructive azoospermia because of pathological occurring obstruction in the genital tract, n = 4; secretory azoospermic individuals with positive testicular sperm extraction value, n = 5; secretory azoospermic individuals with negative testicular sperm extraction value, n = 4) individuals was carried out. Reverse transcriptase-quantitative real-time polymerase chain reaction validation analysis of selected microRNAs was additionally performed in a larger number of individuals. RESULTS AND DISCUSSION: Clinically relevant quantitative changes in the small non-coding RNA levels contained in semen small extracellular vesicles can be used as biomarkers for the origin of azoospermia and for predicting the presence of residual spermatogenesis. In this regard, canonical isoform microRNAs (n = 185) but also other isomiR variants (n = 238) stand out in terms of numbers and fold-change differences in expression, underlining the need to consider isomiRs when investigating microRNA-based regulation. Conversely, although transfer RNA-derived small RNAs are shown in our study to represent a high proportion of small non-coding RNA sequences in seminal small extracellular vesicle samples, they are not able to discriminate the origin of azoospermia. PIWI-interacting RNA cluster profiles and individual PIWI-interacting RNAs with significant differential expression were also not able to discriminate. Our study demonstrated that expression values of individual and/or combined canonical isoform microRNAs (miR-10a-5p, miR-146a-5p, miR-31-5p, miR-181b-5p; area under the receiver operating characteristic curve >0.8) in small extracellular vesicles provide considerable clinical value in identifying samples with a high likelihood of sperm retrieval while discriminating azoospermia by origin. Although no individual microRNA showed sufficient discriminating power on its own to identify severe spermatogenic disorders with focal spermatogenesis, multivariate microRNA models in semen small extracellular vesicles have the potential to identify those individuals with residual spermatogenesis. Availability and adoption of such non-invasive molecular biomarkers would represent a great improvement in reproductive treatment decision protocols for azoospermia in clinical practice.

3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation.

Small RNA-sequencing (small RNA-seq) has revealed the presence of small RNA-naturally occurring variants such as microRNA (miRNA) isoforms or isomiRs. Due to their small size and the sequence similarity among miRNA isoforms, their validation by RT-qPCR is challenging. We previously identified two miR-31-5p isomiRs-the canonical and a 3'isomiR variant (3' G addition)-which were differentially expressed between individuals with azoospermia of different origin. Here, we sought to determine the discriminatory capacity between these two closely-related miRNA isoforms of three alternative poly(A) based-RT-qPCR strategies in both synthetic and real biological context. We found that these poly(A) RT-qPCR strategies exhibit a significant cross-reactivity between these miR-31-5p isomiRs which differ by a single nucleotide, compromising the reliable quantification of individual miRNA isoforms. Fortunately, in the biological context, given that the two miRNA variants show changes in the same direction, RT-qPCR results were consistent with the findings of small RNA-seq study. We suggest that miRNA selection for RT-qPCR validation should be performed with care, prioritizing those canonical miRNAs that, in small RNA-seq, show parallel/homogeneous expression behavior with their most prevalent isomiRs, to avoid confounding RT-qPCR-based results. This is suggested as the current best strategy for robust biomarker selection to develop clinically useful tests.

Using Small Non-Coding RNAs in Extracellular Vesicles of Semen as Biomarkers of Male Reproductive System Health: Opportunities and Challenges.

Reproductive dysfunction and urogenital malignancies represent a serious health concern in men. This is in part as a result of the absence of reliable non-invasive tests of diagnosis/prognosis. Optimizing diagnosis and predicting the patient's prognosis will affect the choice of the most appropriate treatment and therefore increase the chances of success and the result of therapy, that is, it will lead to a more personalized treatment of the patient. This review aims firstly to critically summarize the current knowledge of the reproductive roles played by extracellular vesicle small RNA components, which are typically altered in diseases affecting the male reproductive tract. Secondly, it aims to describe the use of semen extracellular vesicles as a non-invasive source of sncRNA-based biomarkers for urogenital diseases.

Experimental validation of seminal miR-31-5p as biomarker for azoospermia and evaluation of the effect of preanalytical variables.

BACKGROUND AND OBJECTIVES: Predicting the origin of azoospermia with non-invasive biomarkers is clinically relevant for determining the chance of successful sperm retrieval from the testes before attempting assisted reproduction treatment. Here, the semen small extracellular vesicle microRNA miR-31-5p-based biomarker test to distinguish obstructive azoospermia from secretory azoospermia (previously described by our group) is validated for clinical use, and additionally, the sample source (seminal small extracellular vesicles vs. total seminal plasma) as a preanalytical variable is considered to optimize the procedure. RESULTS AND DISCUSSION: Our results provide evidence that altered miR-31-5p expression can be determined both from extracellular vesicles and from the whole seminal plasma to discriminate obstructive from secretory azoospermic samples. Not only have we validated this microRNA-based molecular model as a clinically useful test for predicting the origin of azoospermia in a sample from azoospermic individuals, but additionally, and more interestingly for the clinicians, we have evidenced its usefulness for predicting the presence of spermatogenic failure in azoospermic patients with follicle-stimulating hormone values <10 IU/L as a sensitive and specific biomarker (area under the receiver operating characteristic curve >0.88; p-value < 0.006). The use of total seminal plasma as an analytical sample would facilitate the use of a simplified technical procedure for miR-31-5p quantification and would represent a great improvement in reproductive treatment decision protocols for azoospermia in clinical practice.

Immune and spermatogenesis-related loci are involved in the development of extreme patterns of male infertility.

We conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DRß1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition.

Obesity Surgery Improves Hypogonadism and Sexual Function in Men without Effects in Sperm Quality.

(1) Background: Obesity is associated with hypogonadism, sexual dysfunction, and impaired fertility in men. However, its effects on semen parameters or sexual function remain debatable. (2) Methods: This paper involves a longitudinal study in men submitted for obesity surgery at a university tertiary hospital. Patients were studied at baseline and at 6, 12, and 18 months after obesity surgery. At each visit, anthropometry measures were collected and hormonal and semen parameters were studied. Sexual function was evaluated with the International Index of Erectile Function (IIEF). (3) Results: A total of 12 patients were included. The average body mass index of patients decreased from 42.37 ± 4.44 to 29.6 ± 3.77 kg/m2 at 18 months after surgery (p < 0.05). Hormonal parameters improved after obesity surgery. The proportion of sperm cells with normal morphology tended to decrease from baseline and became most significant at 18 months (5.83 ± 4.50 vs. 2.82 ± 2.08). No significant changes were found in the remaining semen parameters. Erectile function improved significantly at six months after surgery. (4) Conclusions: The authors believe that, in general, the effects of obesity surgery on fertility may be limited or even deleterious (at least in the short and midterm follow-up).

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Common Variation in the PIN1 Locus Increases the Genetic Risk to Suffer from Sertoli Cell-Only Syndrome.

We aimed to analyze the role of the common genetic variants located in the PIN1 locus, a relevant prolyl isomerase required to control the proliferation of spermatogonial stem cells and the integrity of the blood-testis barrier, in the genetic risk of developing male infertility due to a severe spermatogenic failure (SPGF). Genotyping was performed using TaqMan genotyping assays for three PIN1 taggers (rs2287839, rs2233678 and rs62105751). The study cohort included 715 males diagnosed with SPGF and classified as suffering from non-obstructive azoospermia (NOA, n = 505) or severe oligospermia (SO, n = 210), and 1058 controls from the Iberian Peninsula. The allelic frequency differences between cases and controls were analyzed by the means of logistic regression models. A subtype specific genetic association with the subset of NOA patients classified as suffering from the Sertoli cell-only (SCO) syndrome was observed with the minor alleles showing strong risk effects for this subset (ORaddrs2287839 = 1.85 (1.17-2.93), ORaddrs2233678 = 1.62 (1.11-2.36), ORaddrs62105751 = 1.43 (1.06-1.93)). The causal variants were predicted to affect the binding of key transcription factors and to produce an altered PIN1 gene expression and isoform balance. In conclusion, common non-coding single-nucleotide polymorphisms located in PIN1 increase the genetic risk to develop SCO.

Common genetic variation in KATNAL1 non-coding regions is involved in the susceptibility to severe phenotypes of male infertility.

BACKGROUND: Previous studies in animal models evidenced that genetic mutations of KATNAL1, resulting in dysfunction of its encoded protein, lead to male infertility through disruption of microtubule remodelling and premature germ cell exfoliation. Subsequent studies in humans also suggested a possible role of KATNAL1 single-nucleotide polymorphisms in the development of male infertility as a consequence of severe spermatogenic failure. OBJECTIVES: The main objective of the present study is to evaluate the effect of the common genetic variation of KATNAL1 in a large and phenotypically well-characterised cohort of infertile men because of severe spermatogenic failure. MATERIALS AND METHODS: A total of 715 infertile men because of severe spermatogenic failure, including 210 severe oligospermia and 505 non-obstructive azoospermia patients, as well as 1058 unaffected controls were genotyped for three KATNAL1 single-nucleotide polymorphism taggers (rs2077011, rs7338931 and rs2149971). Case-control association analyses by logistic regression assuming different models and in silico functional characterisation of risk variants were conducted. RESULTS: Genetic associations were observed between the three analysed taggers and different severe spermatogenic failure groups. However, in all cases, the haplotype model (rs2077011*C | rs7338931*T | rs2149971*A) better explained the observed associations than the three risk alleles independently. This haplotype was associated with non-obstructive azoospermia (adjusted p = 4.96E-02, odds ratio = 2.97), Sertoli-cell only syndrome (adjusted p = 2.83E-02, odds ratio = 5.16) and testicular sperm extraction unsuccessful outcomes (adjusted p = 8.99E-04, odds ratio = 6.13). The in silico analyses indicated that the effect on severe spermatogenic failure predisposition could be because of an alteration of the KATNAL1 splicing pattern. CONCLUSIONS: Specific allelic combinations of KATNAL1 genetic polymorphisms may confer a risk of developing severe male infertility phenotypes by favouring the overrepresentation of a short non-functional transcript isoform in the testis.

Genome-wide compound heterozygote analysis highlights DPY19L2 alleles in a non-consanguineous Spanish family with total globozoospermia.

RESEARCH QUESTION: Would the use of genome-wide genotyping be an advantageous strategy to identify the molecular aetiology of two brothers from a non-consanguineous family, clinically diagnosed with total globozoospermia? DESIGN: Two related Spanish globozoospermic patients were studied. Eight first- and second-degree family members were also included in the study. The clinical procedure included anamnesis, physical examination and semen analyses. Acrosome visualization was performed by fluorescein isothiocyanate-Pisum sativum agglutinin labelling and ultrastructural electron microscope sperm analysis. Sperm DNA fragmentation was determined by TUNEL and SCD. Molecular analysis included: the detection of deletion of the DPY19L2 gene by a BPa (break point "a") gap-polymerase chain reaction, and genotyping by using a high-throughput genome-wide genotyping platform and a genotype imputation strategy. RESULTS: The biological characteristics of the two globozoospermic siblings included round-headed spermatozoa without an acrosome; ultrastructural defects in spermatozoa; increased sperm fragmentation and aneuploidies, inability of spermatozoa to activate oocytes (correctable with artificial activation) and good developmental potential of embryos generated by IVF/intracytoplasmic sperm injection. This genetic study focused on a genome-wide compound heterozygote analysis that identified two deleterious rare coding variants in the DPY19L2 gene [rs771726551 (c.431T>A exon 3) and rs147579680 (c.869G>A exon 8)]. CONCLUSION: A genome-wide compound heterozygote analysis strategy should be considered for molecular screening in globozoospermia and other rare congenital diseases, particularly in cases from non-consanguineous families.

Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia.

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF. Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants. Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33-02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis. Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait.

Epidemiological, clinical and laboratory differences between male urethral infections due to Haemophilus spp. and those due to Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium and Ureaplasma urealyticum: A descriptive study.

OBJECTIVE: To describe the epidemiological, clinical and laboratory characteristics of male patients diagnosed with Haemophilus spp. urethral infection and to compare them with the characteristics of male patients diagnosed with N. gonorrhoeae, C. trachomatis, M. genitalium and U. urealyticum urethral infection. Over the past 2 years, an increase in urethral infections due to Haemophilus spp. was observed. MATERIALS AND METHODS: All male patients who attended our Department of Sexually Transmitted Infections between January 2018 and February 2019 were retrospectively studied; they underwent conventional bacteriological and multiplex PCR studies in the urethra at the same time. RESULTS: Of the 86 patients studied, a unique microorganism was detected in 76 cases, N. gonorrhoeae in 24, Haemophilus spp. in 21 (16 H. parainfluenzae and 5 H. influenzae), C. trachomatis in 19, M. genitalium in 8 and U. urealyticum in 4; 10 cases presented more than one microorganism. In case of multiple aetiological agents, sexual partnership was multiple. In the Haemophilus group, 81% reported only unprotected oral insertive sex; symptoms lasted for more than one week in 62% of the patients. CONCLUSIONS: Haemophilus is an aetiological agent of non-gonococcal urethritis whose incidence is clearly increasing; the main route of transmission is oral sex. The most common reason for consultation is dysuria and testicular pain, while urethral discharge was predominant for the other causes of urethral infection. Due to the high frequency of antibiotic resistance in the Haemophilus group, it is necessary to confirm eradication by performing a test of cure.

Effect and in silico characterization of genetic variants associated with severe spermatogenic disorders in a large Iberian cohort.

BACKGROUND: Severe spermatogenic failure (SpF) represents the most extreme manifestation of male infertility, as it decreases drastically the semen quality leading to either severe oligospermia (SO, <5 million spermatozoa/mL semen) or non-obstructive azoospermia (NOA, complete lack of spermatozoa in the ejaculate without obstructive causes). OBJECTIVES: The main objective of the present study is to analyze in the Iberian population the effect of 6 single-nucleotide polymorphisms (SNPs) previously associated with NOA in Han Chinese through genome-wide association studies (GWAS) and to establish their possible functional relevance in the development of specific SpF patterns. MATERIALS AND METHODS: We genotyped 674 Iberian infertile men (including 480 NOA and 194 SO patients) and 1058 matched unaffected controls for the GWAS-associated variants PRMT6-rs12097821, PEX10-rs2477686, CDC42BPA-rs3000811, IL17A-rs13206743, ABLIM1-rs7099208, and SOX5-rs10842262. Their association with SpF, SO, NOA, and different NOA phenotypes was evaluated by logistic regression models, and their functional relevance was defined by comprehensive interrogation of public resources. RESULTS: ABLIM1-rs7099208 was associated with SpF under both additive (OR = 0.86, p = 0.036) and dominant models (OR = 0.78, p = 0.026). The CDC42BPA-rs3000811 minor allele frequency was significantly increased in the subgroup of NOA patients showing maturation arrest (MA) of germ cells compared to the remaining NOA cases under the recessive model (OR = 4.45, p = 0.044). The PEX10-rs2477686 SNP was associated with a negative testicular sperm extraction (TESE) outcome under the additive model (OR = 1.32, p = 0.034). The analysis of functional annotations suggested that these variants affect the testis-specific expression of nearby genes and that lincRNA may play a role in SpF. CONCLUSIONS: Our data support the association of three previously reported NOA risk variants in Asians (ABLIM1-rs7099208, CDC42BPA-rs3000811, and PEX10-rs2477686) with different manifestations of SpF in Iberians of European descent, likely by influencing gene expression and lincRNA deregulation.

Impact of Extracellular Vesicle Isolation Methods on Downstream Mirna Analysis in Semen: A Comparative Study.

Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis.

Intronic variation of the SOHLH2 gene confers risk to male reproductive impairment.

OBJECTIVE: To evaluate whether SOHLH2 intronic variation contributes to the genetic predisposition to male infertility traits, including severe oligospermia (SO) and different nonobstructive azoospermia (NOA) clinical phenotypes. DESIGN: Genetic association study. SETTING: Not applicable. PATIENT(S): Five hundred five cases (455 infertile patients diagnosed with NOA and 50 with SO) and 1,050 healthy controls from Spain and Portugal. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genomic DNA extraction from peripheral blood mononuclear cells, genotyping of the SOHLH2 polymorphisms rs1328626 and rs6563386 using the TaqMan allelic discrimination technology, case-control association analyses using logistic regression models, and exploration of functional annotations in publicly available databases. RESULT(S): Evidence of association was observed for both rs6563386 with SO and rs1328626 with unsuccessful sperm retrieval after testicular sperm extraction (TESE-) in the context of NOA. A dominant effect of the minor alleles was suggested in both associations, either when the subset of patients with the manifestation were compared against the control group (rs6563386/SO: P=.021, odds ratio [OR] = 0.51; rs1328626/TESE-: P=.066, OR = 1.46) or against the group of patients without the manifestation (rs6563386/SO: P=.014, OR = 0.46; rs1328626/TESE-: P=.012, OR = 2.43). The haplotype tests suggested a combined effect of both polymorphisms. In silico analyses evidenced that this effect could be due to alteration of the isoform population. CONCLUSION(S): Our data suggest that intronic variation of SOHLH2 is associated with spermatogenic failure. The genetic effect is likely caused by different haplotypes of rs6563386 and rs1328626, which may predispose to SO or TESE- depending on the specific allelic combination.

Seminal plasma microRNAs improve diagnosis/prognosis of prostate cancer in men with moderately altered prostate-specific antigen.

There is an urgent need for accurate non-invasive biomarkers for prostate cancer (PCa) diagnosis and disease risk stratification. Previous data suggests that total seminal plasma (SP) represents a source of miRNAs for screening. We have evaluated a panel of eight PCa-associated miRNAs for their potential use as PCa biomarkers in SP by analyzing their levels using RT-qPCR. Multivariate logistic regression modelling and clinical risk assessment were performed for those SP miRNAs statistically altered between PCa and non-PCa (HCt and/or BPH) groups. Our results provide evidence that altered miRNA expression in PCa tissue can also be detected in total SP. We obtained a clinically useful SP miRNA-based combined model (PSA+miR-142-3p+miR-223-3p+miR-93-5p), which improves PCa specificity of the PSA test, for, firstly, predicting the presence of malignant tumors in a sample from the total population and secondly, and more interestingly for clinicians, for predicting PCa in samples from the positive PSA screening test (PSA>4 ng/ml). Additionally, [PSA+miR-30d-5p+miR-93-5p] and [PSA+miR-30d-5p] models have been shown to be useful for predicting the disease aggressiveness with diagnostic accuracy. In conclusion, our results provide evidence that miRNAs in total SP represent a useful target for evaluation for PCa, which technically simplifies the future use of semen miRNA-based models as non-invasive biomarkers to increase the efficiency of PCa diagnosis and prognosis.

Evaluation of Male Fertility-Associated Loci in a European Population of Patients with Severe Spermatogenic Impairment.

Infertility is a growing concern in developed societies. Two extreme phenotypes of male infertility are non-obstructive azoospermia (NOA) and severe oligospermia (SO), which are characterized by severe spermatogenic failure (SpF). We designed a genetic association study comprising 725 Iberian infertile men as a consequence of SpF and 1058 unaffected controls to evaluate whether five single-nucleotide polymorphisms (SNPs), previously associated with reduced fertility in Hutterites, are also involved in the genetic susceptibility to idiopathic SpF and specific clinical entities. A significant difference in the allele frequencies of USP8-rs7174015 was observed under the recessive model between the NOA group and both the control group (p = 0.0226, OR = 1.33) and the SO group (p = 0.0048, OR = 1.78). Other genetic associations for EPSTI1-rs12870438 and PSAT1-rs7867029 with SO and between TUSC1-rs10966811 and testicular sperm extraction (TESE) success in the context of NOA were observed. In silico analysis of functional annotations demonstrated cis-eQTL effects of such SNPs likely due to the modification of binding motif sites for relevant transcription factors of the spermatogenic process. The findings reported here shed light on the molecular mechanisms leading to severe phenotypes of idiopathic male infertility, and may help to better understand the contribution of the common genetic variation to the development of these conditions.

Clinical factors affecting semen improvement after microsurgical subinguinal varicocelectomy: which subfertile patients benefit from surgery?

BACKGROUND: The exact mechanism of varicocele-related infertility is still elusive, therefore, the current challenges for its management lie in determining which patients stand to benefit most from surgical correction. The authors aimed to assess the clinical factors affecting semen improvement after left microsurgical subinguinal varicocelectomy (MSV) in relation to patient age, ultrasound varicocele grading (USVG), and presence of a right subclinical varicocele (RSV). METHODS: From 2010 to 2017 a total of 228 infertile patients underwent left MSV for clinical varicocele. Descriptive statistics were used to describe the cohort and verify the surgical benefit in terms of semen improvement, in addition, subsets of patients were selected according to clinical covariates. Logistic regression modeling was applied to evaluate the presence of RSV, operative time, age, and USVG as explanatory variables. RESULTS: Sperm concentration (SC), progressive sperm motility (PSM), and normal sperm morphology (NSM) increased significantly after surgery (p = 0.002; p = 0.011; p = 0.024; respectively). Mean SC improved after MSV in ⩾35 year-old patients and the grade 3 USVG group (p = 0.01; p = 0.02; respectively). Logistic regression modeling showed a that the probability of SC improvement was 76% lower in subjects presenting RSV (p = 0.011). In addition, patients with a grade 3 USVG presented a three-times greater probability of SC improvement compared with patients with a lower USVG (p = 0.035). In addition, older patients showed a greater probability of SC improvement after MSV (p = 0.041). CONCLUSIONS: MSV is an effective varicocele-related infertility treatment that should also be offered to older patients. In addition, patients with a higher USVG benefit from surgery. In infertile men with an RSV in association with a left clinical disease, a bilateral varicocele repair should be considered.