A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Disease gene discovery in male infertility: past, present and future.

Identifying the genes causing male infertility is important to increase our biological understanding as well as the diagnostic yield and clinical relevance of genetic testing in this disorder. While significant progress has been made in some areas, mainly in our knowledge of the genes underlying rare qualitative sperm defects, the same cannot be said for the genetics of quantitative sperm defects. Technological advances and approaches in genomics are critical for the process of disease gene identification. In this review we highlight the impact of various technological developments on male infertility gene discovery as well as functional validation, going from the past to the present and the future. In particular, we draw attention to the use of unbiased genomics approaches, the development of increasingly relevant functional assays and the importance of large-scale international collaboration to advance disease gene identification in male infertility.

Toward Harmonized Treadmill-Based Validation of Step-Counting Wearable Technologies: A Scoping Review.

BACKGROUND: The authors conducted a scoping review as a first step toward establishing harmonized (ie, consistent and compatible), empirically based best practices for validating step-counting wearable technologies. PURPOSE: To catalog studies validating step-counting wearable technologies during treadmill ambulation. METHODS: The authors searched PubMed and SPORTDiscus in August 2019 to identify treadmill-based validation studies that employed the criterion of directly observed (including video recorded) steps and cataloged study sample characteristics, protocol details, and analytical procedures. Where reported, speed- and wear location-specific mean absolute percentage error (MAPE) values were tabulated. Weighted median MAPE values were calculated by wear location and a 0.2-m/s speed increment. RESULTS: Seventy-seven eligible studies were identified: most had samples averaging 54% (SD = 5%) female and 27 (5) years of age, treadmill protocols consisting of 3 to 5 bouts at speeds of 0.8 (0.1) to 1.6 (0.2) m/s, and reported measures of bias. Eleven studies provided MAPE values at treadmill speeds of 1.1 to 1.8 m/s; their weighted median MAPE values were 7% to 11% for wrist-worn, 1% to 4% for waist-worn, and ≤1% for thigh-worn devices. CONCLUSIONS: Despite divergent study methodologies, the authors identified common practices and summarized MAPE values representing device step-count accuracy during treadmill walking. These initial empirical findings should be further refined to ultimately establish harmonized best practices for validating wearable technologies.

Quantifying Physical Activity in Young Children Using a Three-Dimensional Camera.

The purpose of this study was to determine the feasibility and validity of using three-dimensional (3D) video data and computer vision to estimate physical activity intensities in young children. Families with children (2-5-years-old) were invited to participate in semi-structured 20-minute play sessions that included a range of indoor play activities. During the play session, children's physical activity (PA) was recorded using a 3D camera. PA video data were analyzed via direct observation, and 3D PA video data were processed and converted into triaxial PA accelerations using computer vision. PA video data from children (n = 10) were analyzed using direct observation as the ground truth, and the Receiver Operating Characteristic Area Under the Curve (AUC) was calculated in order to determine the classification accuracy of a Classification and Regression Tree (CART) algorithm for estimating PA intensity from video data. A CART algorithm accurately estimated the proportion of time that children spent sedentary (AUC = 0.89) in light PA (AUC = 0.87) and moderate-vigorous PA (AUC = 0.92) during the play session, and there were no significant differences (p > 0.05) between the directly observed and CART-determined proportions of time spent in each activity intensity. A computer vision algorithm and 3D camera can be used to estimate the proportion of time that children spend in all activity intensities indoors.

The role of miRNAs in male human reproduction: a systematic review.

BACKGROUND: The presence of miRNAs in human reproductive tissue is intriguing and suggests the possibility that these important regulatory molecules play a role in reproductive function. However, the regulatory role of miRNAs in reproductive tissue remains poorly understood with a significant amount of controversial and contradicting data. OBJECTIVES: To systematically review the high-quality studies published to date investigating miRNAs associated with male human reproduction in order to describe their roles and relations with infertility and update the knowledge in the field. MATERIALS AND METHODS: A comprehensive systematic review of the published literature in MEDLINE-PubMed and EMBASE databases from the earliest available online indexing year until June 2018 (complimentary search until July 2019) was performed, in accordance with the PRISMA guidelines. We have included descriptive, case-control, cross-sectional, and observational prospective and retrospective studies in which fertile/infertile men were well-defined. The primary outcome was the miRNA expression in testis, epididymis, sperm cells, seminal plasma, and extracellular vesicles (i.e., exosomes and microvesicles). RESULTS: We identified 25,204 articles, of which 42 were selected for qualitative analysis. Of the 42 articles included, 15 evaluated the miRNAs in testis, five in epididymis, 13 in spermatozoa, and 11 in seminal plasma and/or extracellular vesicles. Two studies tackled more than one sub-group. As far as miRNA presence and content, the results of this systematic review indicated that every tissue/cell contains a well-defined and stable population of miRNAs that could be potentially related to spermatogenesis and embryogenesis. DISCUSSION AND CONCLUSION: Our systematic review of descriptive and observational studies shows a consistent relationship between aberrant miRNA expression and infertility. Therefore, it seems reasonable that measuring the expression of particular miRNAs might be useful not only as infertility biomarkers, but also for developing therapeutic strategies.

Cadence-based Classification of Minimally Moderate Intensity During Overground Walking in 21- to 40-Year-Old Adults.

BACKGROUND: A walking cadence of ≥100 steps/min corresponds to minimally moderate intensity, absolutely defined as ≥3 metabolic equivalents (METs). This threshold has primarily been calibrated during treadmill walking. There is a need to determine the classification accuracy of this cadence threshold to predict intensity during overground walking. METHODS: In this laboratory-based cross-sectional investigation, participants (N = 75, 49.3% women, age 21-40 y) performed a single 5-minute overground (hallway) walking trial at a self-selected preferred pace. Steps accumulated during each trial were hand tallied and converted to cadence (steps/min). Oxygen uptake was measured using indirect calorimetry and converted to METs. The classification accuracy (sensitivity, specificity, overall accuracy, and positive predictive value) of ≥100 steps/min to predict ≥3 METs was calculated. RESULTS: A cadence threshold of ≥100 steps/min yielded an overall accuracy (combined sensitivity and specificity) of 73.3% for predicting minimally moderate intensity. Moreover, for individuals walking at a cadence ≥100 steps/min, the probability (positive predictive value) of achieving minimally moderate intensity was 80.3%. CONCLUSIONS: Although primarily developed using treadmill-based protocols, a cadence threshold of ≥100 steps/min for young adults appears to be a valid heuristic value (evidence-based, rounded, practical) associated with minimally moderate intensity during overground walking performed at a self-selected preferred pace.

Quantitative gait assessment in children with 16p11.2 syndrome.

BACKGROUND: Neurodevelopmental disorders such as 16p11.2 syndrome are frequently associated with motor impairments including locomotion. The lack of precise measures of gait, combined with the challenges inherent in studying children with neurodevelopmental disorders, hinders quantitative motor assessments. Gait and balance are quantifiable measures that may help to refine the motor phenotype in 16p11.2. The characterization of motor profile is useful to study the trajectories of locomotion performance of children with genetic variants and may provide insights into neural pathway dysfunction based on genotype/phenotype model. METHODS: Thirty-six children (21 probands with 16p11.2 deletion and duplication mutation and 15 unaffected siblings), with a mean age of 8.5 years (range 3.2-15.4) and 55% male, were enrolled. Of the probands, 23% (n = 6) had a confirmed diagnosis of autism spectrum disorder (ASD) and were all male. Gait assessments included 6-min walk test (6MWT), 10-m walk/run test (10MWR), timed-up-and-go test (TUG), and spatio-temporal measurements of preferred- and fast-paced walking. The Pediatric Evaluation of Disability Inventory-Computer Adaptive Tests (PEDI-CAT), a caregiver-reported functional assessment, was administered. Measures of balance were calculated using percent time in double support and base of support. Analyses of the six children with ASD were described separately. RESULTS: Thirty-six participants completed the protocol. Compared with sibling controls, probands had significantly lower scores on the 6MWT (p = 0.04), 10MWR (p = 0.01), and TUG (p = 0.005). Group differences were also identified in base of support (p = 0.003). Probands had significantly lower PEDI-CAT scores in all domains including the mobility scale (p < 0.001). Using age-matched subsamples, the ASD and non-ASD genetic variant groups had larger base of support compared to the controls. In the fast-paced condition, all participants increased their velocity, and there was a corresponding decrease in percent time in double support compared to the preferred-pace condition in all participants. Only the ASD group presented with upper limb arm/hand stereotypies. CONCLUSIONS: Children with 16p11.2, with and without ASD, present with balance impairment during locomotion activities. Probands performed worse on functional assessments, and quantitative measures revealed differences in base of support. These results highlight the importance of using precise measures to differentiate motor dysfunction in children with neurodevelopmental disorders.

Cardiometabolic thresholds for peak 30-min cadence and steps/day.

PURPOSE: To provide empirically-supported thresholds for step-based intensity (i.e., peak 30-min cadence; average of the top 30 steps/min in a day) and steps/day in relation to cardiometabolic health outcomes. METHODS: Receiver operating characteristic curve analysis was applied to the National Health and Nutrition Examination Survey (NHANES) 2005-2006 accelerometer-derived step data to determine steps/day and peak 30-min cadence as risk screening values (i.e., thresholds) for fasting glucose, body mass index, waist circumference, high blood pressure, triglycerides, and HDL cholesterol. Thresholds for peak 30-min cadence and steps/day were derived that, when exceeded, classify the absence of each cardiometabolic risk factor. Additionally, logistic regression models that included the influence of age and smoking were developed using the sample weights, primary sampling units (PSUs), and stratification variables provided by the NHANES survey. Finally, a decision tree analysis was performed to delineate criteria for at-risk versus healthy populations using cadence bands. RESULTS: Peak 30-min cadence thresholds across cardiometabolic outcomes ranged from 66-72 steps/min. Steps/day thresholds ranged from 4325-6192 steps/day. Higher thresholds were observed in men compared to women. In men, higher steps/day thresholds were observed in age ranges of 30-39, while in women, higher thresholds were observed in the age-range 50-59 years. Decision trees for classifying being at low risk for metabolic syndrome contained one risk-free leaf at higher cadence bands, specifically for any time accumulated at ≥120 steps/min. CONCLUSIONS: Minimum thresholds representing absence of cardiometabolic risk range from 4325-6192 steps/day and 66-72 steps/min for peak 30-min cadence. Any time accumulated at ≥120 steps/min was associated with an absence of cardiometabolic risk. Although based on cross-sectional data, these thresholds represent potentially important and clinically interpretable daily physical activity goals.

Validity and Reliability of the Exercise Vital Sign Questionnaire in an Ethnically Diverse Group: A Pilot Study.

The purpose of this study was to determine the validity and reliability of the Exercise Vital Sign (EVS) questionnaire in an ethnically diverse sample. Participants (N = 39) were asked to wear an accelerometer at the hip for at least 7 days and to complete the EVS at the beginning (T1) and end (T2) of the wear period. The EVS questionnaire validity was determined against accelerometry, and bias was calculated as the mean difference between measures. The sensitivity and specificity of the EVS questionnaire were also evaluated. The reliability of the questionnaire was calculated using intraclass correlation coefficient (ICC) between EVS responses at T1 and T2. The mean difference in EVS- and accelerometer-determined time in MVPA was 24 min/wk. The reliability for the questionnaire was excellent (ICC = 0.98). The EVS specificity and sensitivity at T2 were 56% and 78%, respectively. The EVS questionnaire may be an acceptable measure of weekly MVPA time compared to accelerometry in an ethnically diverse sample; however, further research is needed to confirm these findings.

SmartTots Outcomes Workshop 2017: Notes From a Round Table Discussion About Outcome Measures.

An important element of designing research studies is the selection of appropriate outcome measures to ensure that the question posed is properly answered given the evidence. The selection of outcome measures is especially important when tackling complex, interdisciplinary problems, where appropriate outcome measures may not be as simple as a blood test or a laboratory value. One such area of study is the research into neurodevelopmental outcomes after early exposure to anesthetic agents. Concern has arisen recently that certain anesthetic agents may be toxic to the developing brain; a public-private partnership, SmartTots, was formed in conjunction with the Food and Drug Administration and various stakeholders to develop safe anesthetic regimens for neonates and infants who require surgery. However, as research has progressed, questions have arisen regarding the best outcome measures to use in order to detect a true effect, as well as the optimal window in which to measure. These issues were discussed in a round table meeting during the SmartTots meeting in September 2017, and a summary of the discussion is presented here.

Interactive Dyadic Physical Activity and Spatial Proximity Patterns in 2-Year-Olds and Their Parents.

This study aimed to characterize daily physical activity (PA) behaviors in 2-year-old girls and boys and their parents, with and without an objective measure of dyadic spatial proximity. Urban-dwelling parent⁻toddler dyads (N = 110) wore accelerometers for 7 days, and parents completed a sociodemographic questionnaire. Accelerometers were initialized to collect PA and Bluetooth-based proximity data. After applying wear-time algorithms, n = 65 dyads were further analyzed using a dyadic analysis statistical methodology. Toddler⁻parent sedentary and light PA time were respectively interdependent, conditional on child sex and child-parent proximity, but moderate⁻vigorous physical activity (MVPA) time was not. Toddlers were significantly more active on weekdays and weekends than their parents, and no differences were found in daily PA volumes between girls and boys. In dyads with proximity data (n = 34), analyses of joint (i.e., proximal and mutual) PA time showed that girls participated in significantly more joint PA with their mothers than boys. Children who engaged in ≥60 min of MVPA/day participated in ~2 h of joint PA/day, on average, while children with <60 min of MVPA/day engaged in ~30 min less joint-PA time with their mothers. Boys and girls who participated in higher daily MVPA volumes engaged in joint PA with their mothers across greater relative distances, as compared to less active boys who engaged in joint PA at closer relative distances to their mothers. Toddlers who engaged in ≥60 min of daily MVPA participated in joint PA with their mothers at greater relative distances and for longer durations than less active children. Further research on the dyadic activity⁻proximity relationship is needed across early childhood development.

Semen parameters are unaffected by statin use in men evaluated for infertility.

The effects of statin use on conventional semen parameters in humans are largely unknown and have not been previously studied in subfertile men. We retrospectively reviewed data from 10,140 patients seen at our fertility clinic between 2002 and 2013 to assess the effects of statin use on semen parameters. Men who used any statins for >3 months before semen sample collection were included as cases. Data were gathered on patient age, medication use and conventional semen parameters. A total of 118 patients (126 samples) used statins for at least 3 months before semen sample collection. Data from 7698 patients (8,760 samples), who were not using any medications, were used as controls. In age-adjusted regression models, statin use was not associated with statistically significant changes in semen parameters. When used in combination with other nonspermatotoxic medications, it was associated with 0.3 ml decrease in semen volume (95% confidence interval: 0.02 to 0.58 ml, p-value = .04). In conclusion, statin use was not adversely associated with semen parameters other than semen volume in subfertile patients. These findings from our large-scale retrospective study suggest that there are no clinically relevant deleterious effects from statin use on conventional semen parameters.

Cigarette smoking significantly alters sperm DNA methylation patterns.

Numerous health consequences of tobacco smoke exposure have been characterized, and the effects of smoking on traditional measures of male fertility are well described. However, a growing body of data indicates that pre-conception paternal smoking also confers increased risk for a number of morbidities on offspring. The mechanism for this increased risk has not been elucidated, but it is likely mediated, at least in part, through epigenetic modifications transmitted through spermatozoa. In this study, we investigated the impact of cigarette smoke exposure on sperm DNA methylation patterns in 78 men who smoke and 78 never-smokers using the Infinium Human Methylation 450 beadchip. We investigated two models of DNA methylation alterations: (i) consistently altered methylation at specific CpGs or within specific genomic regions and (ii) stochastic DNA methylation alterations manifest as increased variability in genome-wide methylation patterns in men who smoke. We identified 141 significantly differentially methylated CpGs associated with smoking. In addition, we identified a trend toward increased variance in methylation patterns genome-wide in sperm DNA from men who smoke compared with never-smokers. These findings of widespread DNA methylation alterations are consistent with the broad range of offspring heath disparities associated with pre-conception paternal smoke exposure and warrant further investigation to identify the specific mechanism by which sperm DNA methylation perturbation confers risk to offspring health and whether these changes can be transmitted to offspring and transgenerationally.

Sperm DNA damage output parameters measured by the alkaline Comet assay and their importance.

The alkaline Comet assay has shown high diagnostic value to determine male reproductive health and prognostic ability to predict ART success. Here, spermatozoon was analysed in 47 fertile donors and 238 patients, including 132 couples undergoing ART [semen was collected: Group I - within 3 months of their treatment (n = 79); and Group II - 3 months prior to their treatment (n = 53)]. We introduce four Comet distribution plots (A, B1, B2 and C) by plotting the level of DNA damage (x-axis) and percentage of comets (y-axis). Fertile donors had low mean DNA damage, olive tail moment and per cent of spermatozoa with damage and increased type A plots. Comet parameters were associated with clinical pregnancies in Group I. About 66% of couples with type A distribution plot were successful after ART, whereas couples with type B1, B2 and C distribution plots achieved 56%, 44% and 33% pregnancies respectively. The efficiency of the Comet assay was due to complete decondensation process, where the compact sperm nuclear DNA (28.2 ± 0.2 µm3 ) is decondensed to ~63 µm3 (before lysis) and ~1018 µm3 (after lysis). A combinational analysis of all the Comet output parameters may provide a comprehensive evaluation of patient's reproductive health as these parameters measure different aspects of DNA damage within the spermatozoa.

Teratozoospermia and asthenozoospermia are associated with specific epigenetic signatures.

Semen analysis is commonly used as a tool to assess the fertility potential of a male, despite its relatively low predictive power. In this study, we have assessed associations between semen analysis findings (low count, low motility, low viability, poor sperm penetration assay results, poor morphology, and increased DNA damage) and DNA methylation patterns in mature spermatozoa. DNA methylation patterns in the mature spermatozoa are thought to be indicative of patterns in the adult germline stem cells and may offer insight into potential perturbations to cellular pathways involved in spermatogenesis. In this study, sperm DNA methylation at >480,000 CpGs was assessed in 94 men using the Illumina 450k HumanMethylation Array and compared to standard measures of sperm quality. We did not identify any global changes to methylation profiles that were associated with reduced semen parameters. Similarly, we found no significant difference in methylation variability that was associated with any abnormal semen analysis parameter, although sperm displaying abnormal parameters tended to have an increased coefficient of variability, suggesting that, in some samples, this may be a contributing factor. Analysis of methylation at single CpGs and genomic regions did identify associations for low viability and low motility, and to a smaller extent, low count. Interestingly, based on GO Term analysis, differentially methylated regions associated with low viability were over-represented in regions important in meiosis, spermatogenesis, and genomic imprinting. These results suggest that while there are not global alterations to the sperm methylome associated with semen abnormalites, some viability associated regional alterations do exist that may be indicative of perturbed cellular pathways during spermatogenesis.

The "omics" of human male infertility: integrating big data in a systems biology approach.

Spermatogenesis is a complex process in which >2300 genes are temporally and spatially regulated to form a terminally differentiated sperm cell that must maintain the ability to contribute to a totipotent embryo which can successfully differentiate into a healthy individual. This process is dependent on fidelity of the genome, epigenome, transcriptome, and proteome of the spermatogonia, supporting cells, and the resulting sperm cell. Infertility and/or disease risk may increase in the offspring if abnormalities are present. This review highlights the recent advances in our understanding of these processes in light of the "omics revolution". We briefly review each of these areas, as well as highlight areas of future study and needs to advance further.

Associations of single nucleotide polymorphisms in the Pygo2 coding sequence with idiopathic oligospermia and azoospermia.

Male infertility is often associated with a decreased sperm count. The Pygo2 gene is expressed in the elongating spermatid during chromatin remodeling; thus impairment in PYGO2 function might lead to spermatogenic arrest, sperm count reduction, and subsequent infertility. The aim of this study was to identify mutations in Pygo2 that might lead to idiopathic oligospermia and azoospermia. DNA was isolated from venous blood from 77 men with normal fertility and 195 men with idiopathic oligospermia or azoospermia. Polymerase chain reaction-sequencing analysis was performed for the three Pygo2 coding regions. Non-synonymous single nucleotide polymorphisms (SNPs) were detected and analyzed using SIFT, Polyphen-2, and Mutation Taster softwares to identify possible changes in protein structure that could affect phenotype. Pygo2 sequencing was successful for 178 patients (30 with mild or moderate oligospermia, 57 with severe oligospermia, and 91 with azoospermia). Three previously reported non-synonymous SNPs were identified in patients with azoospermia or severe oligospermic but not in those with mild or moderate oligozoopermia or normozoospermia. SNPs rs61758740 (M141I) and rs141722381 (N240I) cause the replacement of one hydrophobic or hydrophilic amino acid, respectively, with another, and SNP rs61758741 (K261E) causes the replacement of a basic amino acid with an acidic one. The software predictions demonstrated that SNP rsl41722381 would likely result in disrupted tertiary protein structure and thus could be involved in disease pathogenesis. Overall, this study demonstrated that SNPs in the coding region of Pygo2 might be one of the causative factors in idiopathic oligospermia and azoospermia, resulting in male infertility.

The safety and efficacy of clomiphene citrate in hypoandrogenic and subfertile men.

Our objective was to evaluate the safety and efficacy of clomiphene citrate (CC) in infertile and hypoandrogenic men through a retrospective study between September 2013 and May 2014. We identified 47 men between 18 and 55 years placed on 50 mg CC every other day. We evaluated the effect of CC on testosterone after 2 weeks, rates of adverse effects and predictors of CC response. Mean baseline testosterone, bioavailable testosterone and estradiol were 246.8 ng dl(-1), 125.5 ng dl(-1) and 20.8 pg dl(-1), respectively. At 2 weeks, mean testosterone, bioavailable testosterone and estradiol increased to 527.6 ng dl(-1), 281.8 ng dl(-1) and 32.0 pg dl(-1) (all P<0.001). Two patients at 2 weeks and one patient at 3 months had a paradoxical decrease in testosterone. Mean total motile count (TMC) and concentration increased from 59.7 million (s.e.m.: 16.5) and 50.7 millions ml(-1) (s.e.m.: 11.1) at baseline to 90.9 million (s.e.m.: 25.9) and 72.5 millions ml(-1) (s.e.m.: 17.5), respectively, at 3 months, although this was nonsignificant (P=0.09, 0.09). No patient on CC experienced a paradoxical decrease in TMC or sperm concentration. On age-adjusted regression analysis, age, BMI, longitudinal testis axis, baseline follicle-stimulating hormone, LH and estradiol did not correlate with improvement in bioavailable testosterone at 2 weeks. CC improves testosterone and may improve semen parameters, although a small percentage of men may not demonstrate improvement in testosterone.