Background: Information about the use of flow cytometry in the diagnosis of male urethritis is scarce. The current study aims to evaluate the performance of flow cytometry on first-voided urine in males with infectious urethritis (Chlamydia trachomatis,Neisseria gonorrhoeae,Mycoplasma genitaliumandTrichomonas vaginalis).Methods: Male patients of the Andrology Centre (Tartu University Hospital, Estonia) were recruited during the period March 2015 –January 2018. Cases included 306 patients with infectious urethritis caused byChlamydia trachomatis,Neisseria gonorrhoeae,Mycoplasma genitaliumand/orTrichomonas vaginalis. The control group consisted of 192 patients without uro-genital complaints, negative tests forC.trachomatis,N.gonorrhoeae,M.genitaliumandT.vaginalisfrom first-voided urine and no inflammation in first-voided urine, mid-stream urine and urine after prostate massage.C.trachomatis,N.gonorrhoeae,M.genitaliumandT.vaginaliswere detected from first-voided urine using polymerase chain reaction (PCR) method. First-voided urine was analysed using urine particle analyzer Sysmex UF-500i.Results: The most prevalent infection was chlamydia (64.1%), followed byMycoplasma genitalium(20.9%), gonorrhoea (7.8%) and trichomoniasis (1.6%). Gonorrhoea caused the highest flow-cytometric leucocyte/bacteria count, followed by chlamydia andMycoplasma genitalium.Trichomonas vaginalisshowed nearly absent inflammation in first-voided urine. Using an empiric flow-cytometry diagnostic threshold for urethritis in first-voided urine (leucocytes ≥ 15/μl and bacteria ≥ 20/μl) the total calculated sensitivity was over 90%. However, when applying such criteria for deciding whether to perform first-voided urine PCR forC.trachomatis,N.gonorrhoeae,M.genitaliumandT.vaginalisor not, we could miss 23 cases with infectious urethritis that makes up 7,5% of all proven cases.Conclusions: Flow cytometry of first-voided urine can be considered as a rapid and objective screening method in case of suspected male infectious urethritis.

STUDY QUESTION: What is the load and profile of hereditary cancer-linked germline variants in infertile compared to fertile men? SUMMARY ANSWER: This study showed almost 5-fold enrichment of disease-causing findings in hereditary cancer genes in infertile compared to fertile men (6.9% vs 1.5%, P = 2.3 × 10−4). WHAT IS KNOWN ALREADY: Epidemiological studies have revealed that men with low sperm count have a 2-fold higher risk of developing cancer during their lifetime. Our recent study observed a 4-fold increased prevalence of cancer in men with monogenic infertility compared to the general male population (8% vs 2%). Shared molecular etiologies of male infertility and cancer have been proposed. STUDY DESIGN, SIZE, DURATION: This retrospective study analyzed germline likely pathogenic and pathogenic (LP/P) variants in 157 hereditary cancer genes in 522 infertile and 323 fertile men recruited to the ESTonian ANDrology (ESTAND) cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: All study participants (n = 845) had been recruited and phenotyped at an Andrology Clinic. Identification of LP/P variants in the cancer gene panel was performed from an exome sequencing dataset generated for the study cohort. All variants passed an automated filtering process, final manual assessment of pathogenicity, and experimental confirmation using Sanger sequencing. Retrospective general health records were available for 36 out of 41 (88%) men with LP/P findings. MAIN RESULTS AND THE ROLE OF CHANCE: Infertile men presented a nearly 5-fold higher load of LP/P findings (36 of 522 cases, 6.9%) compared to fertile subjects (5 of 323, 1.5%; odds ratio (OR) = 4.7, 95% CI 1.81–15.5; P = 2.3 × 10−4) spanning over 24 hereditary cancer genes. The prevalence of findings was not significantly different between azoospermic and oligozoospermic cases. There was also no enrichment of findings in men with a history of cryptorchidism. By the time of the study, six men carrying hereditary cancer variants had been diagnosed with a tumor. Family members affected with cancer had been documented for 10 of 14 cases with available pedigree health data. Nearly half of the infertile men with LP/P findings (17 out of 36) carried variants in genes belonging to the Fanconi anemia (FA) pathway involved in the maintenance of genomic integrity in mitosis and meiosis, repair of DNA double-stranded breaks, and interstrand crosslinks. Overall, FA-pathway genes BRCA2 (monoallelic) and FANCM (biallelic) were the most frequently affected loci (five subjects per gene). LP/P findings in pleiotropic genes linked to human development and hereditary cancer (TSC1, PHOX2B, WT1, SPRED1, NF1, LZTR1, HOXB13) were identified in several patients with syndromic phenotypes. Four cryptorchid infertile men were carriers of MLH1, MSH2, and MSH6 variants implicated in Lynch syndrome. Future studies will reveal whether this observation is a by chance or replicable finding. Most hereditary cancer genes with LP/P variants show high expression in one or more testicular cell types, and mouse models for 15 of 24 affected genes have been reported to exhibit male sub- or infertility. These data support shared genetic etiology of impaired spermatogenesis and cancer. A significantly increased fraction of cancer-linked variants in infertile compared to fertile men could also explain the reported high prevalence of cancer as a comorbidity in male infertility. LARGE SCALE DATA: All hereditary cancer-linked variants identified in this study have been submitted to the National Center for Biotechnology Information (NCBI) ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/). LIMITATIONS, REASONS FOR CAUTION: All recruited participants were of white European ancestry and living in Estonia. Thus, the results might not apply to other ethnic groups. Due to the young age of study participants (median age 34.4 years), the true incidence of cancer during lifetime could not be assessed. As retrospective clinical data were not available for all men, it was not possible to evaluate all possible genotype–phenotype links. The absence of genetic data from family members precluded the assessment of the hereditary nature of the variants or their potential de novo occurrence. WIDER IMPLICATIONS OF THE FINDINGS: Infertility affects about 7–10% of men worldwide. In this study, one in 15 men with spermatogenic failure carried germline LP/P variants in hereditary cancer genes. As exome sequencing is gradually entering the molecular diagnostics setup in andrology, analyzing hereditary cancer-linked variants in the workup of infertile men will offer additional clinical benefits. Male factor infertility is typically diagnosed in men in their 30s, often before the onset of cancer or its symptoms. Early knowledge of germline predisposition to cancer enables timely screening and multidisciplinary management options, potentially improving the prognosis. The study data provide support for the shared monogenic etiologies of hereditary cancer and spermatogenic failure. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Estonian Research Council grant PRG1021 (M.L. and M.P.). The authors declare no conflicts of interest.

The impact of sexually transmitted infections (STI) on male fertility is controversial. Aims: To investigate the prevalence of urethritis-associated STIs (chlamydia, gonorrhoeae, Mycoplasma genitalium, trichomoniasis) among infertile males; to analyze the effect of STIs on semen parameters and blood PSA. Case-control study. Study group (n = 2000): males with fertility problems or desire for fertility check. Control group (n = 248): male partners of pregnant women. Analyses: polymerase chain reaction for STI, seminal interleukin 6 (IL-6), semen and fractionated urine, blood analyses (PSA, reproductive hormones). The prevalence of M. genitalium and chlamydia in the study group was 1.1% and 1.2%, respectively. The prevalence of chlamydia in the control group was 1.6%, while there were no M. genitalium cases. No cases with gonorrhoeae or trichomoniasis or combined infections were observed in neither group. There was a higher seminal concentration of neutrophils and IL-6 among M. genitalium positives compared with STI negatives. There was a trend toward a lower total count of spermatozoa and progressive motility among STI positives. No impact of STIs on PSA was found. The prevalence of STIs among infertile males is low. M. genitalium is associated with seminal inflammation. The impact of STIs on semen parameters deserves further investigations.

To conduct longitudinal precision clinical phenotyping and management planning of 45,X/46,XY mosaicism case, including in-depth documentation of notable deviations at the male accessory glands level, monitoring and timely handling of potential comorbidities. Case report. A 29-year-old phenotypically male patient of European origin presenting for andrological evaluation due to sudden enlargement of the right hemiscrotum. We report a 29-year-old phenotypically male patient with 45,X/46,XY mosaicism and syndromic features, short stature (152 cm), inguinal hernia, azoospermia, moderate testicular hypotrophy (10 mL and 8 mL by orchidometer), distal hypospadias, and autoimmune thyroiditis. The patient presented normal intelligence quotient and cognition. As a novel clinical finding, notable deviations at the male accessory glands’ level were documented in follow-up investigations, reduced prostate volume, unilateral hypoplasia of the seminal vesicle, absence of a distal deferential duct and a dilatation of the contralateral counterpart, dilatation of the ejaculatory duct and an extensive prostatic microcalcification. Intriguingly, an initial cytogenetic analysis from the peripheral blood leucocytes showed a normal male karyotype. Incidentally, 45,X/46,XY mosaicism was uncovered using whole exome sequencing (WES) of peripheral blood deoxyribonucleic acid for molecular diagnostic purposes. This genetic diagnosis was confirmed subsequently by interphase fluorescence in situ hybridization (buccal smear), chromosomal microarray analysis, and repeated cytogenetic testing of peripheral blood cells, emphasizing the sensitivity and precision of WES in detecting chromosomal mosaicism. The estimated proportion of 45,X cells ranged from 55% in buccal smear to 60%–67% in peripheral blood. Wolffian anomalies may be present in 45,X/46,XY phenotypic male mosaics and warrant systematic evaluation, particularly in the context of infertility management. This case also demonstrates that using WES instead of conventional karyotyping may enhance the speed in reaching 45,X/46,XY diagnosis, and consequently, longitudinal management of the broad spectrum of comorbidities in this condition.

STUDY QUESTION: What is the impact of undiagnosed microdeletion and microduplication syndromes (MMS) for men with idiopathic low sperm count? SUMMARY ANSWER: Among idiopathic male infertility, ∼2% of cases harbour known disease-causing microdeletions and duplications linked to clinically well-established syndromes, representing ∼2.5-fold higher prevalence than in the general population. WHAT IS KNOWN ALREADY: While infertility affects up to 10% of men, a substantial proportion of cases remain with no identifiable underlying cause. Recurrent submicroscopic losses or gains cause MMS, some of which also impact reproductive phenotypes, including cryptorchidism and reduced fertility. STUDY DESIGN, SIZE, DURATION: This retrospective study investigated the proportion of undiagnosed MMS among idiopathic male infertility cases. Patients with unexplained low total sperm counts (TSC; defined as ≤39 million sperm per ejaculate) were recruited to the ESTonian ANDrology (ESTAND) cohort at the Andrology Clinic of Tartu University Hospital (AC-TUH) in Estonia. A total of 504 men were included in the analysis, and the study capitalized on available whole-exome sequencing (WES) data to explore large (>500 kb) chromosomal deletions and duplications. PARTICIPANTS/MATERIALS, SETTING, METHODS: Copy number variant (CNV) calling was executed on the WES dataset, followed by a stringent, custom-developed filtering pipeline that retained only high-confidence CNVs larger than 500 kb. Candidate CNVs were validated by chromosomal microarray analysis (CMA) or whole-genome sequencing (WGS). Prevalence of identified MMS-linked deletions and duplications in the ESTAND cohort was compared to general population literature data. MAIN RESULTS AND THE ROLE OF CHANCE: A total of nine patients (1.8%) carried losses and gains linked to clinically well-characterized MMS—recurrent microdeletions at 16p11.2 (two cases), 2q13-14.1, and 15q13.2-13.3, and microduplications at 22q11.21 (three cases), 16p11.2, and 8p23.1. The total burden of MMS among infertile men was ∼2.5-fold higher compared to the general population (P = 0.01, χ2 test). Cryptorchidism was a novel shared feature among all individuals with 16p11.2 rearrangements, suggesting a potential role in disrupting testicular development. Three subjects with MMS-linked microduplications, but none with a microdeletion, had achieved biological fatherhood. An oligozoospermia case (TSC 1.92 × 106/ej.) with 16p11.2 duplication had a naturally conceived child in youthhood. For two men carrying 22q11.21 duplication (TSC 0 and 4.2 × 106/ej., respectively), implementation of ARTs–ICSI with or without preceding testicular sperm aspiration—resulted in successful conception and childbirth. Evidence for a plausible link to male gonadal development and function has been reported for MAZ and KCTD13 at 16p11.2, and LZTR1 at 22q11.21. As an additional finding, a novel ∼3.8 Mb microduplication at 3p25.1 was identified in an oligozoospermia patient and his azoospermic son, conceived naturally at the age of 28 years. This region encompasses a triplosensitive gene, NR2C2, linked to affected meiosis and oligozoospermia in mouse models. LIMITATIONS, REASONS FOR CAUTION: WES may not detect all structural variations, such as inversions or balanced translocations. As some MMS CNVs were only identified in singleton cases (8p23.1 duplication, 2q13-14.1 and 15q13.2-13.3 deletions), the link to male infertility could not be clarified. Further data are needed about the novel large 3p25.1 microduplication to understand its effect on spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS: The yield of identified MMS in idiopathic cases with low sperm counts (∼2%) was close to the carriership of Y-chromosome microdeletions, tested in routine infertility workup. Early identification of MMS can inform genetic counselling regarding congenital health risks to the patient and future offspring and options for decision-making in ART. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Estonian Research Council (Grant number PRG1021 to M.L.). The authors declare that they have no conflict of interest in relation to the data in this paper. TRIAL REGISTRATION NUMBER: N/A