Variant PNLDC1, Defective piRNA Processing, and Azoospermia.

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).

The influence of body composition on the response to dynamic stimulation of the endocrine pituitary-testis axis.

BACKGROUND: Testosterone treatment is generally not recommended in men with obesity induced low serum testosterone. However, distinguishing this condition from overt testosterone deficiency in men with obesity where treatment should be initiated is a diagnostic challenge and tools to differentiate these conditions are scarce but could be of important clinical relevance. OBJECTIVES: To investigate the association between body composition and dynamic responses of the pituitary-testis axis in men. METHODS: Single-center cross-sectional study including 112 healthy men. Participants went through a full biochemical assessment of the pituitary-testis axis, and dynamic stimulatory tests of luteinizing hormone (LH) secretion (gonadotropin-releasing hormone (GnRH)-test) and testosterone secretion (choriogonadotropin (hCG)-test). A subset (N = 78) further had a DXA-scan performed. RESULTS: A higher body mass index (BMI) was associated with lower basal serum LH (BU = -0.44, 95% CI: -0.88--0.01, p = 0.04). The GnRH-stimulated LH increase was not significantly associated with BMI (BU = -0.10, 95% CI: -0.72-0.51, p = 0.74). Furthermore, a high BMI was associated with low basal testosterone (BU -0.02, 95% CI: -0.03--0.02, p < 0.001), and free testosterone (BU -15.0, 95% CI: -19.9--10.0, p < 0.001) and men with overweight and obesity had significantly lower testosterone (9%, p = 0.003 and 24%, p < 0.001) and free testosterone (25%, p = 0.006 and 50%, p < 0.001) concentrations compared to men with normal weight. The HCG-stimulated testosterone increase was significantly less dependent on BMI compared to the influence of BMI on basal testosterone concentrations (p = 0.04 for the interaction). CONCLUSIONS: Dynamic sex hormone responses following pituitary-testis axis stimulation were less dependent on BMI, compared to the influence of BMI on basal hormone concentrations and could potentially assist clinical decision making in patients with obesity suspected of testosterone deficiency.

Results from the first autologous grafting of adult human testis tissue: a case report.

Fertility restoration using autologous testicular tissue transplantation is relevant for infertile men surviving from childhood cancer and, possibly, in men with absent or incomplete spermatogenesis resulting in the lack of spermatozoa in the ejaculate (non-obstructive azoospermia, NOA). Currently, testicular tissue from pre-pubertal boys extracted before treatment with gonadotoxic cancer therapy can be cryopreserved with good survival of spermatogonial stem cells. However, strategies for fertility restoration, after successful cancer treatment, are still experimental and no clinical methods have yet been developed. Similarly, no clinically available treatments can help men with NOA to become biological fathers after failed attempts of testicular surgical sperm retrieval. We present a case of a 31-year-old man with NOA who had three pieces of testis tissue (each ∼2 × 4 × 2 mm3) extracted and cryopreserved in relation to performing microdissection testicular sperm extraction (mTESE). Approximately 2 years after mTESE, the thawed tissue pieces were engrafted in surgically created pockets bilaterally under the scrotal skin. Follow-up was performed after 2, 4, and 6 months with assessment of reproductive hormones and ultrasound of the scrotum. After 6 months, all engrafted tissue was extracted and microscopically analyzed for the presence of spermatozoa. Furthermore, parts of the extracted tissue were analyzed histologically and by immunohistochemical analysis. Active blood flow in the engrafted tissue was demonstrated by doppler ultrasound after 6 months. No spermatozoa were found in the extracted tissue. Histological and immunohistochemical analysis demonstrated graft survival with intact clear tubules and normal cell organization. Sertoli cells and spermatocytes with normal morphology were located near the basement membrane. MAGE-A and VASA positive spermatogonia/spermatocytes were detected together with SOX9 positive Sertoli cells. Spermatocytes and/or Sertoli cells positive for γH2AX was also detected. In summary, following autologous grafting of frozen-thawed testis tissue under the scrotal skin in a man with NOA, we demonstrated graft survival after 6 months. No mature spermatozoa were detected; however, this is likely due to the pre-existing spermatogenic failure.

High-dose vitamin D3 supplementation shows no beneficial effects on white blood cell counts, acute phase reactants, or frequency of respiratory infections.

BACKGROUND: Vitamin D has been suggested to influence the immune system, and vitamin D metabolites and the vitamin D receptor (VDR) are generated and expressed in white blood cells (WBC). Moreover, vitamin D status has been associated with incidence and prognosis of some respiratory tract infections (RTI). Therefore, we investigated the effect of vitamin D3 supplementation on WBC, acute phase reactants (APR), and the risk of developing RTIs. METHODS: A double-blinded, randomized, placebo-controlled clinical trial of 307 infertile men with multiple secondary immunological endpoints. The vitamin D3 group (n = 151) initially received 300,000 IU (7,500 µg) cholecalciferol once - followed by 1,400 IU (35 µg) daily for 150 days. The placebo group (n = 156) did not receive active ingredients. RESULTS: At baseline, stratification into clinically relevant groups of vitamin D status (< 25; 25-50; 50-75; >75 nmol/L), showed an inverse association with total leucocyte concentrations (7.0 vs. 6.0 vs. 6.0 vs. 5.5 (109/L); p = 0.007), lymphocytes (2.4 vs. 2.1 vs. 2.0 vs. 2.0 (109/L); p = 0.048), CRP (2.0 vs. 1.7 vs. 1.2 vs. 1.2 (mg/L); p = 0.037), and orosomucoid (0.82 vs. 0.77 vs. 0.76 vs. 0.70 (g/L); p = 0.015). After 150 days, no differences were detected in WBC counts or APRs between the vitamin D3 and the placebo group. However, vitamin D3 treated men had a higher prevalence of self-reported RTIs compared with the placebo group (55% vs. 39%; p = 0.005). CONCLUSIONS: High-dose vitamin D3 supplementation did not alter WBCs or APRs, but a higher prevalence of respiratory infections was observed in the vitamin D3 group. Serum 25(OH)D3 was negatively correlated with most WBCs, indicating that vitamin D status may be linked with inflammation and WBC turnover, but not an important determinant of developing RTIs. TRIAL REGISTRATION: NCT01304927 (ClinicalTrials.gov). Registered February 20, 2011.

Men treated with BEACOPP for Hodgkin lymphoma may be at increased risk of testosterone deficiency.

In the current study, we report the prevalence of male testosterone deficiency in a cohort of 60 male long-term survivors of malignant lymphoma with normal total testosterone but in the lower part of the reference level. Testosterone deficiency was defined as subnormal concentrations of total testosterone or subnormal concentrations of calculated free testosterone. The aim was to clarify whether total testosterone was sufficient for identification of testosterone deficiency in male survivors of malignant lymphoma. Hormonal analyses taken at follow-up were compared with samples taken at diagnosis for a subgroup of 20 survivors, for evaluation of changes in hormones over time. Another group of 83 similar survivors of malignant lymphoma with testosterone in the high end of reference levels were also used for comparison, to identify groups of increased risk of testosterone deficiency. A total group of 143 survivors were therefore included in the study. Our findings indicate that for screening purposes an initial total testosterone is sufficient in some survivors because sexual hormone binding globulin concentration was found stable over time. However, 15% were found with subnormal calculated free testosterone. Survivors intensely treated for Hodgkin lymphoma and older survivors were identified as high-risk groups for testosterone deficiency necessitating endocrinological attention during follow-up. Some evidence of pituitary downregulation was also found, because of uncompensated decreases in testosterone concentration over time. In conclusion, longitudinal measurements of total testosterone alone do not seem adequate for the screening of testosterone deficiency for all long-term lymphoma survivors.

APHRODITE criteria: addressing male patients with hypogonadism and/or infertility owing to altered idiopathic testicular function.

RESEARCH QUESTION: Can a novel classification system of the infertile male - 'APHRODITE' (Addressing male Patients with Hypogonadism and/or infeRtility Owing to altereD, Idiopathic TEsticular function) - stratify different subgroups of male infertility to help scientists to design clinical trials on the hormonal treatment of male infertility, and clinicians to counsel and treat the endocrinological imbalances in men and, ultimately, increase the chances of natural and assisted conception? DESIGN: A collaboration between andrologists, reproductive urologists and gynaecologists, with specialization in reproductive medicine and expertise in male infertility, led to the development of the APHRODITE criteria through an iterative consensus process based on clinical patient descriptions and the results of routine laboratory tests, including semen analysis and hormonal testing. RESULTS: Five patient groups were delineated according to the APHRODITE criteria; (1) Hypogonadotrophic hypogonadism (acquired and congenital); (2) Idiopathic male infertility with lowered semen analysis parameters, normal serum FSH and normal serum total testosterone concentrations; (3) A hypogonadal state with lowered semen analysis parameters, normal FSH and reduced total testosterone concentrations; (4) Lowered semen analysis parameters, elevated FSH concentrations and reduced or normal total testosterone concentrations; and (5) Unexplained male infertility in the context of unexplained couple infertility. CONCLUSION: The APHRODITE criteria offer a novel and standardized patient stratification system for male infertility independent of aetiology and/or altered spermatogenesis, facilitating communication among clinicians, researchers and patients to improve reproductive outcomes following hormonal therapy. APHRODITE is proposed as a basis for future trials of the hormonal treatment of male infertility.

High-dose cholecalciferol supplementation to obese infertile men is sufficient to reach adequate vitamin D status.

Obesity is associated with low vitamin D status, and the optimal supplement and dosage of cholecalciferol (vitamin D3) or calcidiol (25OHD) for individuals with obesity have been debated. We aimed to determine the effect of high-dose vitamin D3 supplementation on achieving adequate vitamin D levels among infertile men with normal weight v. obesity. Here, we present secondary end points from a single-centre, double-blinded, randomised clinical trial, comprising 307 infertile men randomised to active or placebo treatment for 150 days. Men in the active group initially received an oral bolus of 300 000 mg of vitamin D3, followed by daily supplementation with 1400 mg of vitamin D3 and 500 mg of calcium. Baseline BMI was listed as a predefined subgroup. At baseline, serum 25OHD was significantly higher in men with normal weight (BMI < 25 kg/m2) compared with men with overweight (BMI 25-30 kg/m2) and obesity (BMI > 30 kg/m2) (48 nmol/l v. 45 nmol/l and 39 nmol/l, respectively; P = 0·024). After the intervention, men with normal weight, overweight and obesity treated with vitamin D3 had a significantly higher serum 25OHD compared with corresponding placebo-treated men (BMI < 25 kg/m2: 92 nmol/l v. 53 nmol/l, BMI = 25-30 kg/m2: 87 nmol/l v. 49 nmol/l and BMI > 30 kg/m2: 85 nmol/l v. 48 nmol/l; P < 0·001 for all, respectively). In conclusion, we show that high-dose vitamin D3 supplementation to infertile men with obesity and low vitamin D status is sufficient to achieve adequate serum 25OHD levels.

Male Reproductive Disorders and Fertility Trends: Influences of Environment and Genetic Susceptibility.

It is predicted that Japan and European Union will soon experience appreciable decreases in their populations due to persistently low total fertility rates (TFR) below replacement level (2.1 child per woman). In the United States, where TFR has also declined, there are ethnic differences. Caucasians have rates below replacement, while TFRs among African-Americans and Hispanics are higher. We review possible links between TFR and trends in a range of male reproductive problems, including testicular cancer, disorders of sex development, cryptorchidism, hypospadias, low testosterone levels, poor semen quality, childlessness, changed sex ratio, and increasing demand for assisted reproductive techniques. We present evidence that several adult male reproductive problems arise in utero and are signs of testicular dysgenesis syndrome (TDS). Although TDS might result from genetic mutations, recent evidence suggests that it most often is related to environmental exposures of the fetal testis. However, environmental factors can also affect the adult endocrine system. Based on our review of genetic and environmental factors, we conclude that environmental exposures arising from modern lifestyle, rather than genetics, are the most important factors in the observed trends. These environmental factors might act either directly or via epigenetic mechanisms. In the latter case, the effects of exposures might have an impact for several generations post-exposure. In conclusion, there is an urgent need to prioritize research in reproductive physiology and pathophysiology, particularly in highly industrialized countries facing decreasing populations. We highlight a number of topics that need attention by researchers in human physiology, pathophysiology, environmental health sciences, and demography.

Low serum anti-Müllerian hormone is associated with semen quality in infertile men and not influenced by vitamin D supplementation.

BACKGROUND: Anti-Müllerian hormone (AMH) is released by testicular Sertoli cells and of great importance during fetal male sexual development, but less is known about the role of circulating AMH during adulthood. In vitro studies have shown that vitamin D may induce AMH transcription, but a controlled trial investigating the possible effect of vitamin D on serum AMH has not been conducted in men. METHODS: A single-center, double-blinded, randomized placebo-controlled clinical trial (NCT01304927) conducted in Copenhagen, Denmark. A total of 307 infertile men were included and randomly assigned (1:1) to a single dose of 300,000 IU cholecalciferol followed by 1400 IU cholecalciferol + 500 mg of calcium daily (n = 151) or placebo (n = 156) for 150 days. Difference in serum AMH was a predefined secondary endpoint. Explorative outcomes were associations between serum AMH and gonadal function in infertile men. The primary endpoint was difference in semen quality and has previously been published. RESULTS: Infertile men in the lowest AMH tertile had significantly lower sperm concentration (∆T3-1 16 mill/mL (228%); P < 0.001), sperm count (∆T3-1 55 million (262%); P < 0.001), motile sperm count (∆T3-1 28 million (255%); P < 0.001), progressive motile sperm count (∆T3-1 18 million (300%); P < 0.001), testis size (∆T3-1 2.7 mL (16%); P < 0.001), serum inhibin B (∆T3-1 72 pg/mL (59%); P < 0.001), inhibin B/FSH ratio (∆T3-1 48 (145%); P < 0.001), and higher FSH (∆T3-1 2.6 (38%); P < 0.001) than the tertile of infertile men with highest serum AMH. Vitamin D supplementation had no effect on serum AMH compared with placebo treatment. CONCLUSIONS: In infertile men, low serum AMH is associated with severely impaired gonadal function illustrated by poor semen quality and lower testosterone/LH ratio. Serum AMH in infertile men was not influenced by vitamin D supplementation.

Functional and cognitive rehabilitation interventions during intensive care admission: A protocol for a systematic integrative review.

BACKGROUND: Long-term cognitive impairment occurs in up to 60% of intensive care unit (ICU) survivors. Early use of functional and cognitive rehabilitation interventions, while patients are still in ICU, may reduce cognitive decline. We aim to describe the functional and cognitive interventions used during the ICU stay, the healthcare professionals providing interventions, and the potential impact on functional and cognitive rehabilitation. METHOD: In this integrative systematic review, we will include empirical qualitative, quantitative, mixed- and multiple-methods studies assessing the use of functional and cognitive rehabilitation provided in ICU. We will identify studies in relevant electronic databases from 2012 to 2022, which will be screened for eligibility by at least two reviewers. Literature reported as narrative reviews and editorials will be excluded. We will assess the impact of interventions evaluating a cognitive and functional function, quality of life, and all-cause mortality at 6-12 months after ICU discharge. The Revised Cochrane risk-of-bias Tool will be used for assessing risk of bias in clinical trials. For observational studies, we will use the National Institutes of Health Quality Assessment tool for Observational Cohort and Cross-Sectional Studies. Furthermore, we will use the critical appraisal skills programme for qualitative studies and the mixed methods appraisal tool for mixed methods studies. We will construct four matrices, including results describing which ICU patients and healthcare professionals were engaged in rehabilitation, which interventions were included in early rehabilitation in ICU, the potential impact on patient outcomes of rehabilitation interventions provided in ICU and a narrative synthesis of themes. A summary of the main results will be reported using modified GRADE methodology. IMPACT: This integrative review will inform the feasibility randomised clinical trial testing the development of a complex intervention targeting functional and cognitive rehabilitation for patients in ICU.

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.

Optimized detection of germ cell neoplasia in situ in contralateral biopsy reduces the risk of second testis cancer.

OBJECTIVE: To evaluate whether optimized and standardized diagnostic procedures would improve detection of germ cell neoplasia in situ (GCNIS) in the contralateral testis of patients with testicular germ cell tumour (TGCT) and decrease the rate of metachronous tumours, which in a nationwide Danish study was estimated to be 1.9%. PATIENTS AND METHODS: This was a retrospective analysis of outcomes in 655 patients with TGCT who underwent contralateral biopsies (1996-2007) compared with those in 459 non-biopsied TGCT controls (1984-1988). The biopsies were performed using a standardized procedure with immunohistochemical GCNIS markers and assessed by experienced evaluators. Initial histopathology reports were reviewed, and pathology and survival data were retrieved from national Danish registers. In 604/608 patients diagnosed as GCNIS-negative (four were lost to follow-up), the cumulative incidence of metachronous TGCT was estimated in a competing risk setting using the Grey method. All cases of metachronous TGCT were re-examined using immunohistochemistry. RESULTS: Germ cell neoplasia in situ was found in 47/655 biopsied patients (7.2%, 95% confidence interval [CI] 5.4-9.5%). During the follow-up period (median 17.3 years) five of the 604 GCNIS-negative patients developed a TGCT. In 1/5 false-negative biopsies, GCNIS was found on histological revision using immunohistochemistry and 2/5 biopsies were inadequate because of too small size. The estimated cumulative incidence rate of second tumour after 20 years of follow-up was 0.95% (95% CI 0.10%-1.8%) compared with 2.9% (95% CI 1.3%-4.4%) among the non-biopsied TGCT patients (P = 0.012). The estimates should be viewed with caution due to the small number of patients with metachronous TGCT. CONCLUSIONS: Optimized diagnostic procedures improved the detection rate of GCNIS in patients with TGCT and minimized their risk of developing metachronous bilateral cancer. Urologists should be aware of the importance of careful tissue excision (to avoid mechanical compression) and the need of adequate biopsy size. Performing contralateral biopsies is beneficial for patients' care and should be offered as a part of their management.

Familial resemblance in markers of testicular function in fathers and their young sons: a cross-sectional study.

STUDY QUESTION: Is testicular function associated within father-son pairs? SUMMARY ANSWER: Familial resemblance in testis volume and serum markers of spermatogenesis was observed in father-son pairs. WHAT IS KNOWN ALREADY: Studies suggest familial clustering of male subfertility and impaired spermatogenesis, but in men from the general population little is known about concordance in testicular function between fathers and sons. STUDY DESIGN, SIZE, DURATION: This cross-sectional study with simultaneous collection of data in fathers and sons included 72 pairs (144 fathers and sons), unselected regarding testicular function were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: A subgroup of men from the background population and participating in a study on testicular function were asked permission to invite their fathers to participate in a similar setup. Fathers (median age of 53 years) and sons (median age of 19 years) participated in the same study setup including assessment of testis size, having a blood sample taken and analysed for serum levels of reproductive hormones (FSH, inhibin B, LH, testosterone, oestradiol, sex hormone-binding globulin (SHBG) and calculated free testosterone) and delivering a semen sample for assessment of traditional semen parameters. Mixed-effects models were fitted to estimate the familial resemblance as the proportion of variance in markers of testicular function due to shared factors for fathers and sons accounted for using random-effects. Variance components were calculated from both unadjusted and adjusted models. MAIN RESULTS AND THE ROLE OF CHANCE: After adjustments, variance component analyses showed that familial resemblance between fathers and sons accounted for 48% (P < 0.001) of the variation in testicular volume, 32% (P = 0.009) of the variation in FSH, 31% (P = 0.009) of the variation in the inhibin B/FSH ratio, 33% (P = 0.007) and 45% (P < 0.001) of the variation in testosterone and free testosterone, respectively, and 31% (P = 0.009) of the variation in SHBG. None of the semen parameters were associated within father-son pairs. LIMITATIONS, REASONS FOR CAUTION: The present study may have lacked power to detect associations for semen quality, as large intra- and inter-individual variation occur in semen parameters. WIDER IMPLICATIONS OF THE FINDINGS: In this study, testis volume, serum testosterone and serum markers of spermatogenesis including FSH were associated in fathers and sons, suggesting an impact of paternal genetics for testicular function in the son. However, the estimated familial resemblance for spermatogenesis markers highlights that other factors, such as maternal genetics and prenatal as well as adult exposures, are also of major importance for testicular function. STUDY FUNDING/COMPETING INTEREST(S): The study has received funding from Danish Health Authority, Research Fund of the Capital Region of Denmark and Independent Research Fund Denmark (8020-00218B). None of the funders had any role in the study design, collection, analysis or interpretation of data, writing of the paper of publication decisions. The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.

Use of e-cigarettes associated with lower sperm counts in a cross-sectional study of young men from the general population.

STUDY QUESTION: Are use of e-cigarettes and snuff associated with testicular function as previously shown for conventional cigarettes and marijuana? SUMMARY ANSWER: Use of e-cigarettes is associated with reduced semen quality but not with higher serum testosterone level as observed for conventional cigarette use. Snuff use was not associated with markers of testicular function. WHAT IS KNOWN ALREADY: Cigarette smoking has previously been associated with higher testosterone levels and impaired semen quality, whereas it is unresolved whether use of e-cigarettes or snuff influence the testicular function. STUDY DESIGN, SIZE, DURATION: This cross-sectional population-based study included 2008 men with information on cigarette and marijuana use (enrolled between 2012 and 2018), among whom 1221 men also had information on e-cigarette and snuff use (enrolled between 2015 and 2018). PARTICIPANTS/MATERIALS, SETTING, METHODS: Men (median age 19.0 years) from the general population provided a semen and blood sample and filled out a questionnaire on lifestyle including information on smoking behaviour. Associations between different types of smoking (e-cigarettes, snuff, marijuana and cigarettes) and reproductive hormones (total and free testosterone, sex hormone-binding globulin, LH, oestradiol and ratios of inhibin B/FSH, testosterone/LH and free testosterone/LH) and semen parameters (total sperm count and sperm concentration) were examined using multiple linear regression analyses adjusted for relevant confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately half of the men (52%) were cigarette smokers, 13% used e-cigarettes, 25% used snuff and 33% used marijuana. Users of e-cigarettes and marijuana were often also cigarette smokers. Compared to non-users, daily e-cigarette users had significantly lower total sperm count (147 million vs 91 million) as did daily cigarette smokers (139 million vs 103 million), in adjusted analyses. Furthermore, significantly higher total and free testosterone levels were seen in cigarette smoking men (6.2% and 4.1% higher total testosterone and 6.2% and 6.2% higher free testosterone in daily smokers and occasional smokers, respectively, compared to non-smoking men), but not among e-cigarette users. Daily users of marijuana had 8.3% higher total testosterone levels compared to non-users. No associations were observed for snuff in relation to markers of testicular function. LIMITATIONS, REASONS FOR CAUTION: We cannot exclude that our results can be influenced by residual confounding by behavioural factors not adjusted for. The number of daily e-cigarette users was limited and findings should be replicated in other studies. WIDER IMPLICATIONS OF THE FINDINGS: This is the first human study to indicate that not only cigarette smoking but also use of e-cigarettes is associated with lower sperm counts. This could be important knowledge for men trying to achieve a pregnancy, as e-cigarettes are often considered to be less harmful than conventional cigarette smoking. STUDY FUNDING/COMPETING INTEREST(S): Funding was received from the Danish Ministry of Health (1-1010-308/59), the Independent Research Fund Denmark (8020-00218B), ReproUnion (20200407) and the Research Fund of the Capital Region of Denmark (A6176). The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: NA.

A history of cryptorchidism is associated with impaired testicular function in early adulthood: a cross-sectional study of 6376 men from the general population.

STUDY QUESTION: Is there a difference in testicular function in early adulthood between men born with cryptorchidism and men born with normally descended testes? SUMMARY ANSWER: In men from the general population, a history of cryptorchidism was associated with lower total testis volume and impaired semen quality as well as altered serum levels of reproductive hormones. WHAT IS KNOWN ALREADY: The association between cryptorchidism and testicular function is well documented in studies based on sub-fertile or infertile men recruited from a clinical setting. However, the association has not previously been investigated in men from the general population, who were unselected regarding fertility status. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional population-based study of 6376 young Danish men examined from 1996 to 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study is based on young men from the greater Copenhagen area, Denmark (median age of 19 years) who were unselected regarding fertility status and semen quality. The young men delivered a semen sample, had a blood sample drawn and underwent a physical examination including assessment of testis volume. Participants completed a questionnaire regarding cryptorchidism at birth, current lifestyle and their mother's pregnancy, after consulting their mother. The differences in markers of testicular function, including testis volume, semen parameters and reproductive hormones between men with and without a history of cryptorchidism were investigated with multiple linear regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The participation rate was 24% for the entire study period. Overall, a history of cryptorchidism was associated with reduced testicular function. In the adjusted models, a history of cryptorchidism was associated with a 3.5 ml lower total testis volume, determined by orchidometer (P < 0.001), 28% lower sperm concentration (95% CI: -37 to -20) and 26% lower inhibin B/FSH ratio (95% CI: -50 to -22) compared to men without a history of cryptorchidism, suggesting a reduced spermatogenetic capacity. Men with a history of cryptorchidism also had a slightly reduced Leydig cell function expressed as a 6% lower testosterone/LH ratio (95% CI: -12 to -0.7). The significant effect sizes and different markers of testicular function pointing in the same direction across the different models based on a large sample size support that the results are not chance findings. LIMITATIONS, REASONS FOR CAUTION: Information on cryptorchidism at birth and treatment modus was obtained by retrospective self-report, and each participant only delivered one semen sample. WIDER IMPLICATIONS OF THE FINDINGS: The results suggest that men with a history of cryptorchidism could be at increased risk of experiencing fertility problems. However, among these men there is a wide variation in semen quality and further research is needed in order to identify the subgroup of boys born with cryptorchidism who are at the greatest risk of impaired semen quality when reaching adulthood. STUDY FUNDING/COMPETING INTEREST(S): The study received financial support from the Research fund of Rigshospitalet, Copenhagen University Hospital; the European Union (Contract numbers BMH4-CT96-0314, QLK4-CT-1999-01422, QLK4-CT-2002-00603. FP7/2007-2013, DEER Grant agreement no. 212844); the Danish Ministry of Health; the Danish Environmental Protection Agency; A.P. Møller and wife Chastine McKinney Møllers Foundation; and Svend Andersens Foundation. None of the founders had any role in the study design, collection, analysis or interpretation of data, writing of the paper or publication decisions. The authors have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.

Age-related changes in human Leydig cell status.

STUDY QUESTION: What are the consequences of ageing on human Leydig cell number and hormonal function? SUMMARY ANSWER: Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. WHAT IS KNOWN ALREADY: There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. STUDY DESIGN, SIZE, DURATION: This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsies were obtained from 15 heart beating organ donors (age range: 19-85 years) and 24 patients (age range: 19-45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. MAIN RESULTS AND THE ROLE OF CHANCE: Increasing age was negatively associated with Leydig cell number (R = -0.49; P < 0.01) and concomitantly with the Sertoli cell population size (R= -0.55; P < 0.001). A positive correlation (R = 0.57; P < 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= -0.52; P < 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men. LIMITATIONS, REASONS FOR CAUTION: In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Semen quality in hypogonadal acromegalic patients.

OBJECTIVE: Growth hormone (GH) activity might be implicated in male reproductive function. One previous study has suggested significantly reduced semen quality in untreated acromegalic patients due to both reduced sperm counts and sperm motility. DESIGN AND METHODS: A retrospective study comprising ten uncontrolled hypogonadal acromegalic patients (median age 29 years) who delivered semen for cryopreservation before initiation of testosterone therapy. Semen variables and hormone concentrations were compared to those of ten non-acromegalic hypogonadal men with pituitary disease (age 31 years) and those of young healthy men. RESULTS: Acromegalic patients vs. non-acromegalic patients had a higher percentage of progressive motile spermatozoa (62 vs. 47%, p = 0.04). Eight of ten acromegalic patients and 82% of controls had total sperm counts above 39 million and progressive motile spermatozoa above 32% (p = 0.55), corresponding to the WHO 2010 reference levels for expected normal fertility for these variables. Non-acromegalic patients vs. healthy controls had reduced percentage of progressive motile spermatozoa (47 vs. 57%, p = 0.02) and only five of ten patients had semen quality above the WHO reference level, which was significantly lower than observed in healthy controls (p = 0.022). Total sperm counts were similar between both patient groups and controls. There were no differences in reproductive hormone levels between acromegalic patients vs. non-acromegalic patients (p-values between 0.10 and 0.61). Compared to healthy controls both patient groups had severely reduced serum testosterone, calculated free testosterone. CONCLUSIONS: Despite severe hypoandrogenism acromegalic patients had semen quality similar to healthy controls based on determination of the number of progressively motile spermatozoa. By contrast non-acromegalic pituitary patients had reduced sperm motility. Our data do not support reduced semen quality in acromegaly.

Occurrence of Cyanobacteria and microcystins in hydroelectric reservoirs used for fish farming.

Fish farming can have a negative impact on water quality and aquatic organisms due to emerging blooms of Cyanobacteria and the production of cyanotoxins. In this study, the effect of aquaculture in hydroelectric reservoirs in Brazil was evaluated in six fish farms and in upstream and downstream water through analysis of the microbiome, Cyanobacteria and microcystin concentrations. Synechococcus and Microcystis were observed at all six locations, while Limnothrix was also observed abundantly at two locations. An increase in the relative abundance of Cyanobacteria inside the fish farms was observed at two locations, while an increase of Cyanobacteria was observed in downstream at five of the six locations. Microcystins were detected in significant and high values in all locations, with concentrations up to 1.59 µg/L. The trend in microcystin concentrations was mirrored in copy numbers of the mcyE gene (encodes microcystin synthetase) and presence of Microcystis, but not in any of the other observed cyanobacterial groups. In summary, the study shows that aquaculture production influenced the water microbiome inside and downstream the fish farms, and a direct correlation was found between mcyE gene copies, microcystin production and abundance of Microcystis, but not for the total abundance of Cyanobacteria.

Adherence to diet quality indices in relation to semen quality and reproductive hormones in young men.

STUDY QUESTION: Is adherence to an a priori defined diet quality indices [Alternate Healthy Index 2010 (AHEI-2010), relative Mediterranean diet score (rMED) or dietary approaches to stop hypertension (DASH)] associated with semen quality and reproductive hormone levels in young men? SUMMARY ANSWER: Greater adherence to the DASH diet is related to higher sperm counts. WHAT IS KNOWN ALREADY: Studies assessing the relationship between dietary intake and male reproductive function have mainly been focused on specific nutrients, food groups or data-driven dietary patterns, but the evidence on a priori defined dietary indices is still scarce. STUDY DESIGN, SIZE, DURATION: Cross-sectional study of 209 male university students recruited from October 2010 to November 2011 in Murcia Region (Southern Spain). PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthy young men aged 18-23 years were included in this study. Diet was assessed using a validated food frequency questionnaire and three a priori-defined dietary indices (AHEI-2010, rMED and DASH) were calculated. Linear regression was used to analyze the relation between the three dietary indices and semen quality parameters and reproductive hormone levels accounting for potential confounders and covariates. MAIN RESULTS AND THE ROLE OF CHANCE: We found statistically significant positive associations between the DASH index and sperm concentration (P, trend = 0.04), total sperm count (P, trend = 0.04) and total motile sperm count (P, trend = 0.02). No associations were observed for other semen parameters or male reproductive hormones. LIMITATIONS, REASONS FOR CAUTION: Even though we adjusted for several known and suspected confounders we cannot exclude the possibility of residual or unmeasured confounding or chance findings. Subjects were blinded to the study outcomes thus reducing the potential influence on their report of diet. Our sample size may be too small to rule out associations with other semen parameters or reproductive hormones. Causal inference is limited, as usual with all observational studies. WIDER IMPLICATIONS OF THE FINDINGS: The results suggest that greater adherence to the DASH may help improve sperm counts. This study was carried out on young men from the general population. However, results may differ among other populations (e.g. infertile men). Therefore, further research is needed to confirm these findings and extend these results to other populations. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Fundación Séneca, grants No 08808/PI/08 and No 19443/PI/14; Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (AES), grants No PI10/00985 and No PI13/01237; and grant P30DK046200 from the National Institutes of Health. Authors have no competing interests to declare.