Unexplained Male Infertility Research Articles

P-085 Transcriptomic and proteomic characterization of unexplained male infertility: alterations in energy metabolism

Abstract Study question Which are the transcriptomic and proteomic profiles of spermatozoa belonging to patients with Unexplained Male Infertility (UMI)? Summary answer Transcriptomic and proteomic profiles of UMI patients differ from the fertile men, having the energy metabolism altered. What is known already About 30% of infertile couples suffer from “Unexplained Infertility” (UI). Considering that the male factor contributes to 50% of infertility cases, it is estimated that half of all couple UI cases are due to unexplained male infertility (UMI). Men are diagnosed with UMI when despite having “normal” seminal parameters they are unable to conceive. Due to the lack of UMI classification criteria and the seminogram-only-based infertility diagnosis, little is known about the mechanisms underlying UMI. Study design, size, duration 80 normozoospermic human semen samples with proven fertility and 80 samples from patients with UMI were used for this study. Ejaculates were obtained from Clínica Quirón Bilbao between 2012 and 2018, and after capacitation they were pooled to perform protein and RNA extractions. Participants/materials, setting, methods For transcriptomic analysis, libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v3. Sequencing was performed on NextSeq 2000 system (1x 50 bp, 50 cycles, P3 kit) at Centre de Regulació Genómica (CRG). For the proteomic analysis, Tandem MassTag (TMT) 6-plex isotopic labeling strategy was adopted, and generated peptides were run in a Q Exactive mass spectrometer. To find differentially expressed genes and proteins between both groups EdgeR and Perseus softwares were employed. Main results and the role of chance In the present study, a total of 156.124 transcripts and 1722 proteins were identified in human sperm. Principal Component Analysis (PCA) confirmed the existence of distinguishable differences in the transcriptomic and proteomic profile of control and UMI samples. Specifically, 3112 differentially expressed transcripts and 385 differentially expressed proteins were identified. Among the differentially expressed proteins, 49 were correlated with RNA expression. Coherent RNA-protein signatures were mainly related to axonemal dynein complex assembly and flagellated sperm motility. Proteins involved in energy metabolism were enriched in UMI protein signatures but were not well correlated with the RNA. Furthermore, genes involved in protein transport and chromosome condensation were highly enriched in UMI group but were not detected by proteomics profiling. According to our results, there is a lack of RNA-protein correlation at human sperm. However, differential transcriptomic and proteomic profiles were found when comparing fertile normozoospermic samples to UMI samples. Concretely, our results suggest that altered energy metabolism may be one of the key factors in patients leading with UMI. Thus, the study of human sperm metabolism may be essential to find new targets of male infertility. Limitations, reasons for caution The lack of a consensus on the classification criteria of UMI makes this group of patients very heterogeneous. Further studies are needed to identify and validate specific transcripts and/or proteins that may alter specific metabolic processes and cause infertility. Wider implications of the findings The study of human sperm metabolism may be essential to understand the causes of many UMI cases. In this context, the identification of key protein or transcripts could be very helpful to predict the success of the assisted reproductive treatments. Trial registration number Not applicable

O-120 The role of CatSper in male infertility - from bench to bedsite and back

Abstract Infertility is on the rise, but the underlying causes still remain mostly unknown. In fact, for about one third of infertile men, semen parameters are within reference limits. This so called ‘unexplained male infertility’ seems to rest on elusive functional defects of the sperm undetectable by semen analysis, precluding early diagnosis of the infertility and evidence-based treatment by medically assisted reproduction (MAR). As a result, affected couples often experience failed MAR attempts, subjecting the ‘healthy’ female to recurring medical risks. We set out to gain insight into the pathomechanisms underlying unexplained male infertility. To this end, we systematically assessed the function of the sperm-specific multisubunit CatSper-channel complex in sperm of 2,300 men undergoing a fertility workup at our center, using a simple motility-based ‘CatSper-Test’ combined with [Ca2+]i fluorimetric and electrophysiological recordings. Thereby, we identified a group of men with normal semen parameters, but defective CatSper function. These men or couples failed to conceive naturally and upon MAR via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, we provide the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

P-047 Sperm moving pattern analysis with artificial intelligence solution predicts aneuploidy ratios of zygotes

Abstract Study question Can an analysis of sperm movements with artificial intelligence predict aneuploidy ratios of zygotes? Summary answer And sperm movement patterns analyzed by AI diagnosis predicted the aneuploidy ratios of zygotes. What is known already Sperm motilities present a specific pattern like strength, strength with curving, curving only and none progressive et al. Sperm movement patterns are determined by the sperm neck protein which is the centromere protein. Also, this centromere protein of the sperms was involved in the chromosome segregation in the zygote. Study design, size, duration A total of 8,000 clinical participants were recruited by CHA Infertility Center Seoul Station and 5 videos were taken from the semen sample of each participant. Machine learning with sperm moving clip for the diagnosis algorithm predicted aneuploidy ratios of fertilized embryos. Participants/materials, setting, methods We used YOLO_v8 and EfficientNet algorithm on the extracted sperm movement trajectory image to classify sperm into classes according to their motion pattern. This information was combined with the age of the patient to predict the possibility of infertility by analyzing the association with aneuploidy ratios of zygotes using deep learning model. Main results and the role of chance Deep learning AI algorithm utilized diagnosis for unexplained male infertility. No strength moving sperm pattern has shown association with male origin aneuploidy after fertilization. Especially high ratios of cycle moving sperm significantly increased the aneuploidy ratios in the zygote. But paternal aging has no correlation with aneuploidy ratios in the embryo. AI diagnosis have a 0.9 accuracy score. It is expected that the AI solution generated through this system can help overcome the problem of finding the cause of male infertility by predicting aneuploidy ratios of zygotes. Limitations, reasons for caution This study is designed for and performed with a minimum number of sperm move data. For clinical application, further mass data was set-up to increase accuracy of diagnosis. Wider implications of the findings Our AI solution was constructed to predict the possibility of aneuploidy ratios in the zygote by analyzing and classifying the movement patterns of sperm. Therefore, sperm movement-based AI diagnosis solution can be applied to define unexplained male infertility. Trial registration number non-clinical trials

Human fertilization in vivo and in vitro requires the CatSper channel to initiate sperm hyperactivation.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

Delayed impacts of COVID-19 infection on unexplained male infertility: 2-year follow-up of normal sperm parameters in unexplained male infertility in KSA.

The current study aimed to assess the long-term effect of COVID-19 infection on unexplained male infertility. A retrospective comparative study of 134 men attending the infertility outpatient clinic of our institution before exposing to COVID-19 infection in KSA from January 2019 to July 2022. Medical recorded data of these patients who were investigated before COVID-19 infection were retrospectively collected using the hospital's electronic database, including semen analysis, sex hormonal, and ultrasound testicular size, and their data were compared prospectively to collected data after 2-year follow-up. One hundred and thirty-four infertile males who got COVID-19 infection in KSA (median age, 33 years) were assisted retrospectively preinfection and delayed 2 years postinfection (median of 23 months). Of the 134 men, 44 (32.83%) were asymptomatic positive COVID-19 (Group A), 68 (50.74%) had mild-to-moderate symptomatic positive COVID-19 (Group B), and 22 (16.41%) had severe symptomatic positive COVID-19 (Group C). There was no significant change between pre- and postinfections in sperm parameters, sex hormonal level, and testicular size. Subgroup analyses were performed for patients regarding the severity of infections. None of the evaluated parameters differed significantly after infections up to 2 years. Results of this study demonstrate that COVID-19 infection does not have significant changes in sperm parameters, sex hormonal level, and testicular size. The long-term impact of COVID-19 infections has no significant effect on normal sperm parameters, sex hormones, and testicular size in male infertility in KSA.

Could the sperm epigenome become a diagnostic tool for evaluation of the infertile man?

Although male infertility is currently diagnosed when abnormal sperm parameters are found, the poor predictive ability of sperm parameters on natural fecundity and medically assisted reproduction outcome poses the need for improved diagnostic techniques for male infertility. The accumulating evidence about the role played by the sperm epigenome in modulation of the early phases of embryonic development has led researchers to focus on the epigenetic mechanisms within the sperm epigenome to find new molecular markers of male infertility. Indeed, sperm epigenome abnormalities could explain some cases of unexplained male infertility in men showing normal sperm parameters and were found to be associated with poor embryo development in IVF cycles. The present mini-review summarizes the current knowledge about this interesting topic, starting from a description of the epigenetic mechanisms of gene expression regulation (i.e. DNA methylation, histone modifications, and non-coding RNAs' activity). We also discuss possible mechanisms by which environmental factors might cause epigenetic changes in the human germline and affect embryonic development, as well as subsequent generations' phenotypes. Studies demonstrating sperm epigenome abnormalities in men with male infertility are reviewed, with particular emphasis on those with the more severe form of spermatogenic dysfunction. Observations demonstrate that the diagnostic and prognostic efficacy of sperm epigenome evaluation will help facilitate the management of men with male factor infertility.

New Insights on Sperm Function in Male Infertility of Unknown Origin: A Multimodal Approach.

The global trend of rising (male) infertility is concerning, and the unidentifiable causes in half of the cases, the so-called unknown origin male infertility (UOMI), demands a better understanding and assessment of both external/internal factors and mechanisms potentially involved. In this work, it was our aim to obtain new insight on UOMI, specifically on idiopathic (ID) and Unexplained male infertility (UMI), relying on a detailed evaluation of the male gamete, including functional, metabolic and proteomic aspects. For this purpose, 1114 semen samples, from males in couples seeking infertility treatment, were collected at the Reproductive Medicine Unit from the Centro Hospitalar e Universitário de Coimbra (CHUC), from July 2018-July 2022. Based on the couples' clinical data, seminal/hormonal analysis, and strict eligibility criteria, samples were categorized in 3 groups, control (CTRL), ID and UMI. Lifestyle factors and anxiety/depression symptoms were assessed via survey. Sperm samples were evaluated functionally, mitochondrially and using proteomics. The results of Assisted Reproduction Techniques were assessed whenever available. According to our results, ID patients presented the worst sperm functional profile, while UMI patients were similar to controls. The proteomic analysis revealed 145 differentially expressed proteins, 8 of which were specifically altered in ID and UMI samples. Acrosin (ACRO) and sperm acrosome membrane-associated protein 4 (SACA4) were downregulated in ID patients while laminin subunit beta-2 (LAMB2), mannose 6-phosphate isomerase (MPI), ATP-dependent 6-phosphofructokinase liver type (PFKAL), STAR domain-containing protein 10 (STA10), serotransferrin (TRFE) and exportin-2 (XPO2) were downregulated in UMI patients. Using random forest analysis, SACA4 and LAMB2 were identified as the sperm proteins with a higher chance of distinguishing ID and UMI patients, and their function and expression variation were in accordance with the functional results. No alterations were observed in terms of lifestyle and psychological factors among the 3 groups. These findings obtained in an experimental setting based on 3 well-defined groups of subjects, might help to validate new biomarkers for unknown origin male infertility (ID and UMI) that, in the future, can be used to improve diagnostics and treatments.

Measuring free radicals with relaxometry: Pioneering steps for measurements in human semen

A possible biological mechanism for unexplained male infertility is due to the effect of oxidative stress (OS), defined by the imbalance of reactive oxygen species (ROS) production, and the capacity of the antioxidant defence system to counteract it. In physiological concentrations, ROS and especially free radicals play an essential role in sperm maturation and fertilization, while an overabundance could lead to OS-induced damage to spermatozoa. To date, there are no direct detection techniques available that can measure the total amount of free radicals real time and identify where and when free radicals are generated. This study applies a quantum sensing technique using fluorescent nanodiamonds (FNDs), called T1 relaxometry, which is uniquely sensitive and specific for free radicals allowing measurements of the current radical load for nanoscale detection in living cells and body fluids. This proof-of-principle study investigates if we can use this technique to detect the free radical generation in human whole and separated, using density gradient centrifugation, semen. This method could be potentially used as new diagnostic measure for unexplained infertility or to track the effect of therapeutic interventions such as lifestyle changes. We adapted the existing relaxometry technique to measure free radicals in semen. The measured relaxation time (T1 time) was correlated to sperm concentration and progressive motility. Additionally, we explored the influence of the oxidative trigger hydrogen peroxide and the antioxidant glutathione on the free radical concentration measured. No significant correlations were found, which indicates that measurements in more proximity of the sperm cell are required to use relaxometry as a potential diagnostic tool for unexplained male infertility.

O-017 A novel diagnostic test identifies patients suffering from loss of CatSper function

Abstract Study question Is a loss of CatSper function in sperm a common cause of unexplained male infertility? Summary answer Loss of CatSper function leads to similar infertility phenotypes in mice and men and represents one of the most common causes of unexplained male infertility. What is known already Male infertility is often due to low sperm counts, impaired motility, and/or abnormal morphology. However, for a large fraction of infertile men, semen parameters are normal, suggesting that the infertility is rather due to an unexplained dysfunction of the sperm. There has been a growing body of evidence that human sperm dysfunction might involve the sperm-specific Ca2+ channel CatSper (cation channel of sperm) that translates changes in the chemical microenvironment of the female reproductive tract into changes in swimming behavior. Study design, size, duration In an observational study over the course of three years, a prototype of the “CatSper-Activity-Test” (CAT) was used to assess the function of CatSper in sperm from the left-over semen samples of 2286 unselected patients undergoing a semen analysis. Participants/materials, setting, methods The CatSper-Activity-Test (CAT) was developed at the Centre of Reproductive Medicine and Andrology, University Hospital Münster and subsequently performed by medical technical assistants in the diagnostic facility of this institution. Requiring only 40 µl of ejaculate, a standard light microscope, and a hands-on-time of a few minutes, the CAT enables a read-out of the activity of CatSper through a motility response: in the CAT buffer, CatSper-intact, but not CatSper-deficient sperm become immotile. Main results and the role of chance Using this CatSper-Activity-Test, we identified seven patients suffering from a loss of CatSper function in a cohort of men seeking medical advice for suspected male infertility. Standard semen analysis revealed that the CatSper-deficient patients are (with one exception) normozoospermic and were diagnosed with unexplained infertility. Notably, their sperm not only failed to fertilize the egg naturally but also upon intrauterine insemination (IUI) and in vitro fertilization (IVF), whereas intra-cytoplasmic sperm injection (ICSI) was successful. Two additional CatSper-deficient patients were identified among patients visiting our clinics for other reasons. We show that the loss of CatSper function is predominantly caused by a homozygous deletion of the CATSPER2 gene; one patient featured compound heterozygous variants of the CATSPERE gene. According to the study results, CatSper dysfunction underlies 1.7% of cases of unexplained male infertility, for which a female factor can be excluded. Limitations, reasons for caution Results from this observational study were obtained from a single institute. A multi-center study involving various countries would enable a more precise determination of the prevalence of CatSper-related male infertility. Wider implications of the findings The CatSper-Activity-Test reliably identifies CatSper-deficient patients with a hands-on-time of a few minutes and requires neither special equipment nor specific training. We envisage the CatSper-Activity-Test as a novel tool for the early diagnosis of male infertility, thus, contributing to evidence-based treatment decisions and sparing patients unnecessary medical and financial risks. Trial registration number not applicable

A Determination of p97/VCP (Valosin Containing Protein) and SVIP (Small VCP Interacting Protein) Expression Patterns in Human Testis.

Background and Objectives: The ubiquitin proteosome system (UPS) is a non-lysosomal pathway that functions in all eukaryotes. The transport of polyubiquitinated proteins to proteosomes takes place via the p97/Valosin-containing protein (VCP) chaperone protein. The p97/VCP binds to polyubiquitinated proteins, allowing these proteins to reach the proteasome and, thus, their destruction. In the case of p97/VCP deficiency, ubiquitinated proteins accumulate in the cell cytoplasm, and their subsequent failure to break down produces various pathological conditions. Small VCP interacting protein (SVIP) and p97/VCP proteins have not been studied in human testicular tissues from different postnatal periods. Therefore, in our study, we aimed to examine the expression of SVIP and p97/VCP in postnatal human testicular tissues. Our study aimed to contribute to further studies on the use of these proteins as testicular cell biomarkers in cases of unexplained male infertility. Materials and Methods: Immunohistochemical studies with the aim of determining the expression of p97/VCP and SVIP proteins in neonatal, prepubertal, pubertal, adult, and geriatric human testis tissues were performed. Results: In testicular sections obtained from a neonatal group, p97/VCP and SVIP were localized in different testicular and interstitial cells, and the lowest expression was observed in this group. While the expressions of these proteins were low in the neonatal period, they increased gradually in the prepubertal, pubertal and adult periods. The expression of p97/VCP and SVIP, which peaked in adulthood, showed a significant decrease in the geriatric period. Conclusions: As a result, the expression of p97/VCP and SVIP correlated with the increase in age, but it decreased significantly in older groups.

Loss-of-function mutations in IQCN cause male infertility in humans and mice owing to total fertilization failure.

Fertilization failure is a significant manifestation of unexplained male infertility. Previous work has suggested a genetic origin. In this study, we report on a man with unexplained infertility from a large consanguineous marriage family. Whole-exome sequencing and Sanger sequencing identified a homozygous frameshift variation of the IQ motif containing N (IQCN; GenBank: NM_001145304.1; c.1061_1062delAT; p.Y354Sfs*13) in the proband and one of his two brothers, who also remained infertile. Analyses of spermatozoa by quantitative RT-PCR indicated that the level of IQCN mRNA was significantly reduced compared to fertile men and the protein could not be detected by western blotting and immunofluorescent staining in the proband. Immunofluorescent staining of spermatozoa from fertile men showed that IQCN was located in the acrosomal region and translocated to the equatorial segment after the acrosome reaction. The proband spermatozoa had abnormal morphology and function. Finally, the proband couple underwent IVF with donor sperm and a healthy baby was born. Furthermore, we developed an Iqcn-KO mouse model using the CRISPR/Cas9 technique. Sperm quality, except for sperm motility, and the fertility of male Iqcn-/- mice were consistent with those of the proband. In conclusion, the findings in humans and mice demonstrate that the homozygous frameshift variant of IQCN causes male infertility owing to autosomal-recessive fertilization failure.

MP43-13 ENCLOMIPHENE IS ASSOCIATED WITH HIGHER PREGNANCY RATES WHEN COMPARED TO CLOMIPHENE IN INFERTILE MEN WITH LOW TESTOSTERONE

You have accessJournal of UrologyCME1 Apr 2023MP43-13 ENCLOMIPHENE IS ASSOCIATED WITH HIGHER PREGNANCY RATES WHEN COMPARED TO CLOMIPHENE IN INFERTILE MEN WITH LOW TESTOSTERONE Jordan Kassab, Juan Torres-Anguiano, John Lindsey, Jabez Gondokusumo, Sabine Itani, Mohamad Jabin, and Larry Lipshultz Jordan KassabJordan Kassab More articles by this author , Juan Torres-AnguianoJuan Torres-Anguiano More articles by this author , John LindseyJohn Lindsey More articles by this author , Jabez GondokusumoJabez Gondokusumo More articles by this author , Sabine ItaniSabine Itani More articles by this author , Mohamad JabinMohamad Jabin More articles by this author , and Larry LipshultzLarry Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003289.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Clomiphene citrate has been used for decades for female infertility; however, it has also been used off-label for many years to treat infertile males with low testosterone. Enclomiphene citrate is the trans isomer of clomiphene citrate and is another off-label medication for the same at-risk population but with no estrogenic agonism. The purpose of this study is to evaluate and compare the pregnancy outcomes of couples seeking fertility who have been treated with either clomiphene or enclomiphene as well as quantitating effects on serum hormones. METHODS: We studied patients with low testosterone and infertility who had been prescribed either clomiphene or enclomiphene at a university hospital within the observation range of 01/01/2021 - 08/01/2022. Dosage information as well as baseline serum hormone levels (LH, FSH, testosterone, estradiol) were captured into a patient database. T-tests were calculated in SPSS to compare means across the clomiphene and enclomiphene patient populations; a two-sample Z-test was used to compare pregnancy outcomes. RESULTS: 110 patients taking clomiphene were compared to 114 patients taking enclomiphene, with the former demonstrating higher testosterone levels (886 ng/dL vs. 547 ng/dL), [t(222)=4.25, p=.000031]. Those taking enclomiphene had significantly higher levels of both LH (6.08 IU/L vs. 4.26 IU/L) and FSH (6.46 IU/L vs. 4.17 IU/L), [t(195)=2.36, p=.019 and t(195)=2.29, p=.023, respectively]. Estradiol also demonstrated a difference between the groups: the mean for clomiphene patients was 35.34 pg/mL, whereas the mean for enclomiphene patients was 29.85 pg/mL [(213)=2.00, p=.047]. Most importantly, enclomiphene patients demonstrated significantly higher rates of pregnancy compared to the clomiphene patients (52% vs. 34%, p=.046). CONCLUSIONS: The empirical treatment of unexplained male infertility has for many years utilized clomiphene citrate in patients with concurrent low testosterone. This study evaluates the use of the single trans-isomer enclomiphene and demonstrates its association with better pregnancy outcomes when compared to clomiphene. Future studies should focus in the use of enclomiphene in a blinded prospective manner to better define its efficacy. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e606 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jordan Kassab More articles by this author Juan Torres-Anguiano More articles by this author John Lindsey More articles by this author Jabez Gondokusumo More articles by this author Sabine Itani More articles by this author Mohamad Jabin More articles by this author Larry Lipshultz More articles by this author Expand All Advertisement PDF downloadLoading ...

Lifestyle and environmental risk factors for unexplained male infertility: study protocol for Australian Male Infertility Exposure (AMIE), a case–control study

BackgroundApproximately 1 in 20 men are sub-fertile or infertile yet the aetiologies of male infertility remain largely unexplained. It is suggested that lifestyle choices and environmental factors contribute but research is limited. In particular, no study has evaluated early life exposures and subsequent male infertility. To address this knowledge gap, this study aims to characterise a cohort of men with idiopathic infertility and compare their general health, lifestyle choices and environmental exposures from teenage years onwards to men without reproductive abnormalities.MethodsTwo groups of men (N = 500 cases; N = 500 controls), matched for age and socio-economic status, will be recruited from fertility clinics around Australia between June 2021 and June 2024. Men will be eligible if they are between 18 and 50 years, with a female partner less than 42 years, and have identified idiopathic male infertility (case) or are part of a couple with diagnosed female factor infertility but with no indication of compromised male fertility (control). Participants will complete an in-depth survey on general health, lifestyle and environmental exposures, reporting from teenage years onwards. An online medical data capture form will be used to gather fertility assessment information from participant medical records. Biological specimens of saliva (all study participants), blood and urine (optional) will be collected and stored for future genetic and epigenetic analysis. Differences in outcome measures between cases and controls will be determined using appropriate between groups comparisons. The relationship between explanatory variables and infertility will be analysed using multilevel modelling to account for clustering within fertility clinics.DiscussionThis study addresses an important gap in research on the aetiology of male infertility and will provide a comprehensive profile of the lifestyle and environmental risk factors for male infertility, leading to provision of up-to-date health advice for male teenagers and adults about optimising their fertility.

Erkek İnfertilitesinin Cerrahi Dışı Tedavisi: Spesifik Olmayan Tedavi

In male infertility, all possible known causes should be carefully evaluated and if detected, targeted treatment options for the cause should be implemented. The known causes of male infertility such as hypogonadotropic hypogonadism, hyperprolactinemia, genital tract infections, disorders of ejaculation, thyroid hormone disorders and varicocele can be treated efficiently by targeted therapies or surgical corrections. Unfortunately, these known causes cover about 20% of male infertility and the rest remains idiopathic. On the other hand, management of idiopathic, unexplained male infertility, in which no etiological factors can be found, is a challenge for both the clinician and couples seeking solutions. In the era of assisted reproductive technology, few medical options in this regard are still available with limited benefits and low scientific foundation based on theoretical concepts but empirical medical therapy continues as a mostly off-label option for obtaining a natural pregnancy. Comprehending the hypothalamic-pituitary-gonadal axis and the regulation of hormones is crucial in this regard. Empirical therapies have the potential to overcome overtreatment with assisted reproductive technology yet clinicians and couples must be aware of the limitations of empirical therapies and should be counseled in this direction. In this review, non-specific medical treatment options for idiopathic male infertility were covered.

Male infertility: what on earth is going on? Pilot international questionnaire study regarding clinical evaluation and fertility treatment for men.

Infertility is a time-consuming and exhaustive process, which disproportionally affects women. Although concerns have been raised about deficiencies in the clinical evaluation of infertile men, there are currently little published data documenting this. A SurveyMonkey questionnaire was therefore created to capture the current clinical practice of fertility specialists working in in vitro fertilisation clinics. Responses were collected from May to July 2021. A total of 112 clinicians completed the pilot survey with respondents from Europe (n = 49; 43.8%), Africa (n = 39, 34.8%), North America (n = 6; 5.4%), Asia (n = 16; 14.3%), South America (n = 1; 0.9%) and Australasia (n = 1; 0.9%). Forty-one percent of fertility specialists (45/110) reported taking only a brief medical history and 24% reported that they never routinely examined infertile male patients. Fifty-four percent of fertility specialists also reported issues getting men to undertake diagnostic semen analysis. Treatment for male infertility spanned assisted reproductive technology (ART), with themes of individualised medicine influencing treatment recommendations. Of the clinicians, 48.2% clinicians reported using empirical medical therapy for unexplained male infertility. Notably, 3.6% respondents recommended testosterone treatment, despite the likely negative impact on spermatogenesis. However, high levels of opportunistic general health advice were reported, including discussion of life exposures thought to be important for male reproductive health. This study adds novel evidence and highlights current deficiencies in clinical practice relating to male infertility. Evaluation of the infertile male using simple medical tools (detailed history taking and clinical examination) has the potential to identify treatable or reversible conditions and should be an immediate focus for education and improvement in reproductive medicine. Investment in research and development is much needed in the field of andrology to develop effective non-ART treatment options for male infertility.Lay summaryPoor sperm quality (male infertility) significantly reduces the chance of natural conception and accounts for half of all cases of infertility, yet affected men are frequently overlooked when couples seek fertility investigations and treatment. Despite a growing awareness of men’s issues and a need to improve patient experience, there is very little documented about how fertility specialists (clinicians) routinely assess and treat male infertility. This study used a SurveyMonkey® questionnaire to capture current clinical practice, with 112 respondents from around the world. Forty-one percent of clinicians did not routinely consider male medical history in detail and 24% never routinely examined infertile men. This should be a focus for improvement in clinical care. As expected, fertility treatment recommended for male infertility was mostly in vitro fertilisation and intracytoplasmic sperm injection, where a single sperm is injected into each mature egg. However, 48.2% of clinicians also reported prescribing unproven medical therapy for unexplained male infertility. Of concern, a few clinicians routinely recommended testosterone treatment, which is likely to harm sperm production. However, advice regarding general health was universally delivered.

O-248 Cluster Analysis of men with idiopathic and unexplained male infertility identifies FSHB Genotype as relevant diagnostic parameter

Abstract Study question In a cohort of idiopathic and unexplained infertile men we aimed to identify subgroups with similar characteristics, and therewith underlying etiologic factors, by clustering approach. Summary answer We identified two distinct patient clusters. Across all diverse phenotypes of infertility, the strongest segregation markers were FSHB c.-211G>T, FSH, and bi-testicular volume. What is known already In about 30-75% of infertile men no major causative factors can be identified; leading to the diagnose of unexplained (normozoospermia) or idiopathic (abnormal semen parameters) male infertility. This cohort of men remains very heterogenous, albeit the detailed andrological characterization that is currently applied in infertility workup. New analysis tools such as machine learning and cluster analysis can provide a more in-depth approach. Such explorative analyses have the potential to uncover hitherto hidden patterns in data that might be difficult to spot for andrologists but become visible by these tools. Study design, size, duration A Cluster analysis was retrospectively performed in a clinically well characterized cohort of 2742 men with unexplained or idiopathic male infertility. These men had visited our Centre within a 10-year period (2008-2018) for infertility workup. Due to the well curated database (Androbase®) we were able to include up to 37 andrologic parameters in the unbiased cluster analysis. Participants/materials, setting, methods After applying strict selection criteria 2742, of initially 7627, infertile men remained for cluster analysis (exclusion: obstructive -, genetic -, other causative factors, female factor; inclusion: azoo- to normozoospermia, FSH ≥ 1IU/l, Testosterone ≥ 8nmol/l). For subsequent analyses the following parameters were included: somatic/semen/hormone parameters, testicular sonography and testis volume, genotyping of the FSHB c.-211G>T (rs10835638) single nucleotide polymorphism. For cluster analysis, partitioning around medoids method was employed based on Gower distance between patients. Main results and the role of chance The applied cluster approach for the study population yielded two separate clusters (average silhouette width ∼0.12). These clusters showed significantly different distributions in bi-testicular volume, FSH and FSHB genotype. Cluster 1 contained all men homozygous for G (wildtype) in FSHB c.-211G>T (100%), while Cluster 2 contained most patients carrying a T allele (>96.6%). Even in subgroup analysis (Total sperm count (TSC) <1Mill and TSC 1³Mill) two clusters each were formed too. Again, the strongest segregation markers between the respective clusters were FSHB c.-211G>T, bitesticular volume, and FSH, supporting the notion of a contributing genetic factor. Surprisingly, sperm parameters like TSC, motility and morphology played a minor role in cluster formation; as well as testicular maldescent, varicocele, smoking, and microlithiasis testes. The genetic parameter of FSHB c.-211G>T in combination with the established parameters FSH and testicular volume should attract more attention in future clinical workups of infertile men with unknown etiologic factors. Limitations, reasons for caution Categorical and numeric features contribute diversely to the calculation of patient dissimilarity. Potentially, categorical features can have a higher impact because patients are rated as completely different if they fall in different categories; for numeric features, the dissimilarity depends on the range of values. Wider implications of the findings The FSHB SNP was identified as an informative segregation marker; we therefore suggest introducing diagnostic genotyping into clinical routine in men with so far idiopathic or unexplained male infertility. This may reduce the high number of infertile men with so far unknown origin by nearly one-third. Trial registration number DFG Clinical Research Unit 326 Male Germ Cells

O-246 A drop in serum progesterone(P4) levels between 5th and 7th days of triggering ovulation is associated with lower ongoing pregnancy rate(OPR) in intrauterine insemination cycles

Abstract Study question Is there any association between early- and mid-luteal phase serum P4 levels and OPR in patients undergoing IUI due to unexplained or mild male-factor infertility? Summary answer A drop in serum progesterone (P4) levels between 5th and 7th day of triggering ovulation is independently associated with lower OPR in IUI cycles. What is known already The definition of luteal phase insufficiency in natural cycles is controversial and might be encountered ∼10% of infertile couples. Serum P4 level measurement is a still the most feasible method for luteal phase monitoring. Recently, some research groups have been focusing on monitoring luteal phase in in-vitro fertilization cycles, however, there is scarce of evidence on luteal phase characteristics and reproductive outcomes in IUI cycles. Study design, size, duration A prospective cohort study. A total of 179 consecutive patients (230 cycles) with unexplained and mild male factor infertility undergoing IUI treatment at Hacettepe University in between June 2020 and May 2021 were included. For ovarian stimulation, of the included 230 cycles, clomiphene citrate and gonadotropin were used consecutively in 161 cycles and only gonadotropin in the remaining 69 cycles. The primary outcome measure was the OPR. No progesterone was administered for luteal phase support. Participants/materials, setting, methods Serum P4 levels were measured on the 5th and 7th day of hCG triggering.P4 level were available in 183 cycles for hCG+5 day, in 161 cycles for hCG+7 day. In order to investigate the effect of P4 levels on OPR, patients were divided into two groups as ≤ 10th percentile and >10th percentile according to serum P4 levels measured on hCG+5 and hCG+7 days, with the cut off values ≤5.6 ng/ml and ≤8.46 ng/ml, respectively. Main results and the role of chance Of the 230 cycles 17 (7.4%) were resulted with ongoing pregnancy. In the univariate analysis, the OPRs of the hCG+5 and hCG+7 serum P4 ≤10% and >10% groups were 10.5% (2/19) versus 5.5% (9/164) (p = 0.80) and 0% (0/16) versus 7.5% (n = 11/145) (p = 0.24), respectively. There was no ongoing pregnancy in patient with ≤8.46 ng/ml serum P4 level on hCG+7 day. The ΔP4 value was calculated by subtracting the serum P4 level measured on hCG+5 from the serum P4 level measured on day hCG+7 (ΔP4= hCG+7 – hCG+5). While none of the 26 patients with negative ΔP4 had ongoing pregnancy, 8 (6.2%) of 130 patients with positive ΔP4 had ongoing pregnancy (p = 0.194). In the multivariate-GEE (generalized estimating equation) analysis, ΔP4 was found to be an independent predictor of ongoing pregnancy alone (ß:0.137, 95% CI = 0.020-0.254, p = 0.02). Limitations, reasons for caution The physiological circadian pulsatile secretion of P4 during the mid-luteal phase and limited sample size are limitations, however, blood sampling was standardized to reduce the impact of timing. Wider implications of the findings Low progesterone level with the threshold ≤8.46 ng/ml on the hCG+7 day is associated lower OPR. Two measurements (hCG+5 day, hCG+7 day) of P4may delineate those patients with a drop in P4, associated lower OPRs. Rescuing these IUI cycles with additional P4supplementation should be tested in future randomized controlled trials. Trial registration number NCT04707430

P-113 Association of Novel Mutations of TSPY1 Gene With Spermatogenic Failure Among Men

Abstract Study question Are the novel mutations of TSPY1 gene associated with idiopathic male infertility in men? Summary answer Sporadic TSPY1 gene mutation is one of the major causes of idiopathic male infertility among men. What is known already As per the published research, there are several studies including AZF micro-deletion, sex chromosome copy number variation but surprisingly studies on Y chromosome genes are not that much available. Due to the genetic cross-talks of different regulatory mechanisms involved in the spermatogenesis process, male infertility still remains idiopathic. Contradictory outcome of the analyses intrigues the association of the variations of TSPY1 gene with male infertility. Previous analyses did not reveal implication of TSPY1 gene as etiologic factor of male infertility. Study design, size, duration This was a population-based case-control study that includes 184 cases of spermatogenic failure and 197 age-matched fertile men as controls and the study was conducted between March, 2018 and February, 2021 to infer whether the genetic mutations are significantly associated with spermatogenic failure. Participants/materials, setting, methods The samples were collected from the Institute of Reproductive Medicine (IRM) and Genome-the fertility centre. All the selected individuals agreed to participate in the study and provided blood samples. We isolated DNA from the blood samples, designed specific primers and went for PCR. We performed Sanger’s dideoxy sequencing of TSPY1 gene reading frame followed by in silico analyses of detected variations to interpret their intuitive effects on transcript and protein level of TSPY1 gene. Main results and the role of chance We found one novel deletion namely MN734578 (NC_000024.10:g.9468830del) and one novel insertion MN719944 (NC_000024.10:g.9468815_9468816insA). Both these mutations exhibited strong association with male infertility. MN734578 showed compelling confederacy with male infertility increasing the risk factor against the odd 19.023 (P value 0.0027). Here the mutation was found in 8 out of 184 case individuals. MN719944 was found to be extremely correlated with male infertility increasing the risk factor by 14.547 folds (P value 0.0117). This mutation is found in 6 out of 184 case individuals. Both the mutations were not not found in the control individuals. In silico analyses using different software SIFT, PROVEAN, REGULATIONSPOTTER, HSF and SpliceAid suggest prospective disruption in splice sites and alteration in different isoforms of TSPY1 transcripts and amino acid sequence in TSPY1 protein. We calculated Odds ratios with respective 95% confidence interval (CI) by performing Fisher’s exact test. After Bonferroni's correction, a two-tailed P-value of less than 0.02 was considered statistically significant. Limitations, reasons for caution The origin of the novel mutations is unknown and the outcome does not provide mechanistic details how do de novo mutations imperil the process of spermatogenesis. Wider implications of the findings The study provides evidence in favour of implication of TSPY1 gene in male fertility. The outcome sheds light to get insight into the issue of unexplained male infertility in Bengali will help to manage fertility problem in men. Trial registration number Not applicable

O-016 Variability of sperm DNA fragmentation in a longitudinal trial of intrauterine inseminations

Abstract Study question Does variability in sperm DNA fragmentation (SDF) follow that observed in standard semen parameters and is this linked to outcome results? Summary answer Variability in SDF and in semen parameters is comparable but live birth rate tends to be lower in men with repetitive high SDF. What is known already Environmental, technical and biological factors are implicated in the intra-individual variability observed in routine semen parameters. This variation is not observed in SDF testing using the sperm chromatin structure assay. Most laboratories undertake SDF testing on one semen sample, assuming that one single measurement represents the patient’s condition and is both stable and associated with a good diagnostic value. Most variability observed in SDF tests relies essentially on paired observations with a short or long time interval between test and retest. Study design, size, duration A monocentric, prospective cohort study was conducted October 2017 - October 2020. Couples with a mild or unexplained male infertility initiated a natural cycle IUI protocol until pregnancy was achieved for a maximum of four cycles. 313 semen samples from 112 men were analyzed for SDF before and after semen preparation. This work was supported by the Fonds Wetenschappelijk Onderzoek (FWO), Research Foundation Flanders (grant number T007016N). Participants/materials, setting, methods Semen samples were analyzed and prepared by density gradient centrifugation. SDF was assessed via TUNEL assay before and after preparation. Clinical pregnancies and live births were registered. Intraclass Correlation Coefficients (ICC) were used to estimate the intra- and inter-variability. Main results and the role of chance The ICC for SDF (0.33 in the ejaculate, 0.54 after density gradient) and semen variables (0.42-0.62) were comparable. These values mean considerable intra-individual variation, both for semen variables and for SDF variables. Using fertile threshold SDF values (13% before and 8% after density gradient), 3 patient groups were distinguished: 13.4% of men showed high SDF in all ejaculates, 58.0% always showed low SDF and 28.6% fluctuated between the two during the trial. After density gradient more patients were found in the High group (42.3%) and less patients in the Low group (26.1%), while the proportion of Fluctuaters remained constant (31.5%). Most (66.7%) men with high SDF retain their high SDF after gradient, and also 56.3% of the Fluctuaters react with a high SDF after gradient. The Low SDF category, on the contrary, distributes itself evenly between the three categories after gradient. In the 112 couples, 26 (23.2%) clinical pregnancies were obtained with 21 (18.8%) live births. The live birth rate for the High, Fluctuaters and Low groups was 6.7%, 18.8% and 20.0% for ejaculate SDF and 12.8%, 20.0% and 27.6% for SDF after density gradient. These differences however did not reach significance (chi-square testing, p > 0.05). Limitations, reasons for caution Our results need to be substantiated and opportunities created to explore populations with an extreme male factor and the clinical implications in different assisted reproductive techniques. Wider implications of the findings SDF testing provides additional information and helps identify causes otherwise missed with traditional semen analysis alone. One out of 3 patients fluctuate in their SDF values. A second SDF testing is advocated to isolate these fluctuaters. The detection of DNA damage after semen preparation is indispensable for therapeutic purposes. Trial registration number NCT03319654

Predictive value of seminal oxidation-reduction potential analysis for reproductive outcomes of ICSI

Research questionIs seminal oxidation-reduction potential (ORP) clinically relevant to reproductive outcome? DesignProspective observational study including a total of 144 couples who had an intracytoplasmic sperm injection (ICSI) cycle between June 2018 and December 2020. The study included patients undergoing fresh ICSI cycles with autologous gametes. Cycles that had day 3 embryo transfers and cryopreservation cycles were excluded. There was no restriction on patients with severe male infertility; couples with unexplained infertility and unexplained male infertility were included, those with azoospermia were excluded. Semen analysis, seminal ORP as determined by means of the MiOXSYS system, sperm DNA fragmentation (SDF) and reproductive outcomes (fertilization, blastocyst development, clinical pregnancy and live birth) were determined. ResultsSeminal ORP was significantly negatively correlated with fertilization rate (r = –0.267; P = 0.0012), blastocyst development rate (r = –0.432; P < 0.0001), implantation/clinical pregnancy (r = –0.305; P = 0.0003) and live birth (r = –0.366; P < 0.0001). Receiver operating characteristic curve analysis showed significant predictive power for ORP for fertilization (≥80%; area under the curve [AUC] 0.652; P = 0.0012), blastocyst development rate (≥60%; AUC 0.794; P < 0.0001), implantation/clinical pregnancy (AUC 0.680; P = 0.0002) and live birth (AUC 0.728; P < 0.0001). Comparable results were obtained for SDF (fertilization: AUC 0.678; blastocyst development: AUC 0.777; implantation/clinical pregnancy: AUC 0.665; live birth: AUC 0.723). Normal sperm morphology showed the lowest predictive power for all reproductive outcome parameters. With male age as confounding factor, ORP (cut-off value of 0.51 mV/106 sperm/ml) has significant (P < 0.04667) effects on odds ratios for all reproductive outcome parameters. Multivariate logistic regression to investigate potential seminal and female confounding factors revealed that seminal ORP significantly (P < 0.0039; P < 0.0130) affects reproductive outcome. ConclusionSeminal ORP is relevant for good fertilization, blastocyst development, implantation, clinical pregnancy and live birth.