Human Reproduction Update, Vol.28, No.1, pp. 15–29, 2022 Advance Access Publication on September 8, 2021 doi:10.1093/humupd/dmab030 A systematic review of the validated monogenic causes of human male infertility: 2020 update and a discussion of emerging gene–disease relationships Brendan J. Houston 1,*, Antoni Riera-Escamilla2, Margot J. Wyrwoll3, Albert Salas-Huetos4,5, Miguel J. Xavier6, Liina Nagirnaja7,8, Corinna Friedrich3, Don F. Conrad7,8,9, Kenneth I. Aston4,8,9, Csilla Krausz8,9,10, Frank Tüttelmann 3,9, Moira K. O’Bryan1,8,9, Joris A. Veltman6,9, and Manon S. Oud 11,* 1 School of BioSciences and Bio21 Institute, The University of Melbourne, Parkville, VIC, Australia 2Andrology Department, Fundació Puigvert, Universitat Autònoma de Barcelona, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Barcelona, Catalonia, Spain 3 Institute of Reproductive Genetics, University of Münster, Münster, Germany 4Andrology and IVF Laboratory, Division of Urology, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, USA 5Department of Nutrition, Harvard T.H. Chan School of Public Health, Harvard University, Boston, MA, USA 6Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK 7Division of Genetics, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, USA 8Genetics of Male Infertility Initiative (GEMINI) 9International Male Infertility Genomics Consortium (IMIGC) 10 Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, Centre of Excellence DeNothe, University of Florence, Florence, Italy 11Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Nijmegen, the Netherlands *Correspondence address. School of BioSciences and Bio21 Institute, The University of Melbourne, 30 Flemington Rd, Parkville, VIC 3052, Australia. E-mail: brendan.houston@unimelb.edu.au (B.J.H.) https://orcid.org/0000-0002-1078-756X; Department of Human Genetics (route 848), Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Geert Grooteplein Zuid 10, 6525 GA Nijmegen, the Netherlands. E-mail: manon.oud@radboudumc.nl (M.S.O.) https://orcid.org/0000-0001-9513-3030 Submitted on June 21, 2021; resubmitted on August 05, 2021; editorial decision on August 12, 2021 TABLE OF CONTENTS ................................................................................................................................ • Introduction • Methods Search strategy and study selection Data extraction and assessment • Results Summary of included studies and design Systematic evaluation of evidence Insights into the genetic causes of male infertility • Discussion Recent developments in genetics of male infertility research and diagnostics Identifying the variant(s) causative for male infertility Dynamics of genotype-phenotype mapping Recommendations for genetic testing in male infertility Wider implications of male infertility genetics Strengths and limitations • Conclusion C The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. V This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Houston et al. BACKGROUND: Human male infertility has a notable genetic component, including well-established diagnoses such as Klinefelter syn- drome, Y-chromosome microdeletions and monogenic causes. Approximately 4% of all infertile men are now diagnosed with a genetic cause, but a majority (60–70%) remain without a clear diagnosis and are classified as unexplained. This is likely in large part due to a delay in the field adopting next-generation sequencing (NGS) technologies, and the absence of clear statements from field leaders as to what constitutes a validated cause of human male infertility (the current paper aims to address this). Fortunately, there has been a significant in- crease in the number of male infertility NGS studies. These have revealed a considerable number of novel gene–disease relationships (GDRs), which each require stringent assessment to validate the strength of genotype–phenotype associations. To definitively assess which of these GDRs are clinically relevant, the International Male Infertility Genomics Consortium (IMIGC) has identified the need for a system- atic review and a comprehensive overview of known male infertility genes and an assessment of the evidence for reported GDRs. OBJECTIVE AND RATIONALE: In 2019, the first standardised clinical validity assessment of monogenic causes of male infertility was published. Here, we provide a comprehensive update of the subsequent 1.5 years, employing the joint expertise of the IMIGC to systemati- cally evaluate all available evidence (as of 1 July 2020) for monogenic causes of isolated or syndromic male infertility, endocrine disorders or reproductive system abnormalities affecting the male sex organs. In addition, we systematically assessed the evidence for all previously reported possible monogenic causes of male infertility, using a framework designed for a more appropriate clinical interpretation of disease genes. SEARCH METHODS: We performed a literature search according to the PRISMA guidelines up until 1 July 2020 for publications in English, using search terms related to ‘male infertility’ in combination with the word ‘genetics’ in PubMed. Next, the quality and the extent of all evidence supporting selected genes were assessed using an established and standardised scoring method. We assessed the experi- mental quality, patient phenotype assessment and functional evidence based on gene expression, mutant in-vitro cell and in-vivo animal model phenotypes. A final score was used to determine the clinical validity of each GDR, across the following five categories: no evidence, limited, moderate, strong or definitive. Variants were also reclassified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines and were recorded in spreadsheets for each GDR, which are avail- able at imigc.org. OUTCOMES: The primary outcome of this review was an overview of all known GDRs for monogenic causes of human male infertility and their clinical validity. We identified a total of 120 genes that were moderately, strongly or definitively linked to 104 infertility phenotypes. WIDER IMPLICATIONS: Our systematic review curates all currently available evidence to reveal the strength of GDRs in male infertility. The existing guidelines for genetic testing in male infertility cases are based on studies published 25 years ago, and an update is far overdue. The identification of 104 high-probability ‘human male infertility genes’ is a 33% increase from the number identified in 2019. The insights generated in the current review will provide the impetus for an update of existing guidelines, will inform novel evidence-based genetic test- ing strategies used in clinics, and will identify gaps in our knowledge of male infertility genetics. We discuss the relevant international guide- lines regarding research related to gene discovery and provide specific recommendations to the field of male infertility. Based on our find- ings, the IMIGC consortium recommend several updates to the genetic testing standards currently employed in the field of human male infertility, most important being the adoption of exome sequencing, or at least sequencing of the genes validated in this study, and expand- ing the patient groups for which genetic testing is recommended. Key words: genetics / male infertility / clinical validity / gene panel / gene–disease relationship / next-generation sequencing / spermato- genic failure / multiple morphological abnormalities of the sperm flagella / systematic review .. Introduction .. variants and the assessment of definitive genotype–phenotype correla- .. tions, however, remains a challenge. Male infertility is a common condition, affecting at least 7% of men .. .. Advances in next-generation sequencing (NGS) have greatly facili- worldwide, and is often predicted to be largely genetic in origin .. tated the unbiased exome-wide (whole-exome sequencing (WES)) and (Krausz and Riera-Escamilla, 2018). A majority of all human (84%, .. .. genome-wide (whole-genome sequencing (WGS)) detection of any ge- 16 598; Uhlen et al., 2015) and mouse (90%, 18 037; Schultz et al., .. netic variants that may play a role in male infertility (e.g. Coutton et al., 2003) protein-coding genes are expressed in the testis, emphasising .. .. 2019; Alhathal et al., 2020; Chen et al., 2020; Liu et al., 2021). that sperm production is a complex process and involves many sepa- .. However, the population frequency of these variants is expected to be rate biological pathways. Furthermore, sperm must mature in the epi- .. .. very low given their negative effect on fertility. For this reason, the didymis and undergo a final step of activation in the female .. analysis of large patient and control cohorts is essential to identify re- reproductive tract, termed ‘capacitation’, before they are capable of .. .. currently mutated genes and detect statistical enrichments in patient fertilising an oocyte. By extension, there are many points during sperm .. cohorts. One major problem in the male infertility field is that genetic development and maturation that could be compromised by the effect .. .. testing strategies employed in andrology clinics are not standardised, of genetic variants. The identification of genes affected by these .. . Validated genetic causes of human male infertility 2020s 17 .. and in some countries/states even the most basic of tests (karyotype .. gene–disease evaluation and any conflicts of interest, reviewers and azoospermia factor deletion analysis) are still not routinely used. .. were not allowed to score any GDRs they had published. Scoring .. The male infertility field is currently catching up with other fields/dis- .. was separated into five categories: no evidence (<3 points), limited ease types with a strong genetic component, such as intellectual dis- .. (3–8 points), moderate (9–12 points), strong (13–15 points) or de- .. ability, neuromuscular disorders and hereditary hearing impairments .. finitive (>15 points), with a maximum of 17 points. After indepen- (Chiu et al., 2020; Markitantova and Simirskii, 2020; Whatley et al., .. dent scoring, the individual scores (for each GDR) for both .. 2020). The rapid uptake of NGS technologies in the male infertility re- .. reviewers were compared and any inconsistencies in scoring (>1 search field over the past 5–10 years, as well as the development of in- .. .. point difference or a difference in the final classification) were set- ternational consortia to collect and characterise clinical cohorts, is .. tled by the two assigned reviewers. Where this was not possible, aiding in the transition of findings into clinical practice. To assist in this .. .. the scoring was discussed with all non-conflicted reviewers. GDRs feedback of knowledge, clear direction is required for the validity of .. with a moderate or higher classification were deemed as confi- which individual genes to be screened and their relevance to certain .. .. dently linked to human male infertility and combined scoring sheets types of infertility. .. are available at http://www.imigc.org. All results in the study are .. In this article, we provide an updated clinical validity assessment of .. collated in Supplementary Table SII. the monogenic causes of male infertility (Oud et al., 2019), through .. .. the systematic analysis of newly published evidence from January 2019 .. to July 2020 for individual gene–disease relationships (GDRs). We .. .. Results have employed the joint expertise of the International Male Infertility .. Genomics Consortium (IMIGC) to systematically evaluate/re-evaluate .. .. Summary of included studies and design all available evidence for published monogenic causes of isolated or .. syndromic male infertility, endocrine disorders that impact male fertility .. We performed a literature search using terms related to ‘male infertil- .. and reproductive system abnormalities affecting the male sex organs. .. ity’ in combination with keywords related to the word ‘genetics’ in This analysis has resulted in the identification of 104 high-probability .. MEDLINE-PubMed and used the same inclusion and exclusion criteria .. ‘human male infertility genes’, a 33% increase from the number identi- .. as described previously (Oud et al., 2019). A total of 26 250 articles .. were identified, of which 2765 new articles were identified since the fied in 2019. .. .. previous search in 2018 and not covered by the previous review .. .. (Fig. 1). The total number of publications on genetic causes of male in- .. fertility has increased by 13% from 849 over the period 2010–2014 to Methods .. .. 963 articles in the period 2015–2019 (Fig. 2A). Furthermore, while .. the final search performed for this review was completed in mid-2020, Search strategy and study selection .. .. the total number of 2020 studies was estimated to overtake the 2019 A literature search was performed as described in Oud et al. (2019) .. to identify articles reporting on monogenic causes of male infertility or .. number by 20%. The absolute and relative contribution of publications .. on proposed monogenic causes of phenotypes related to male infertil- male reproductive system anomalies entered into MEDLINE-PubMed .. before 1 July 2020. Assessment of whether the articles met the inclu- .. ity is also growing (46% in 2017 versus 52% in 2020). The relative con- .. tribution of publications on association studies has declined by 42% sion or exclusion criteria (detailed in Supplementary Table SI) was per- .. formed by two independent reviewers (B.J.H. and M.S.O.). The .. (27% in 2017 versus 19% in 2020; Fig. 2B). The shift away from .. Sanger sequencing to NGS methods is continuing to take place, and present study and the corresponding search protocol were registered .. .. 71% of all sequencing studies used NGS in 2020 (Fig. 2C), which con- with the PROSPERO registry (http://www.crd.york.ac.uk/ .. PROSPERO) as PROSPERO 2021: CRD42021229164. .. stitutes an increase of 90% since 2017. While WGS has started to .. emerge in the field (recent examples: Bedoni et al., 2016; Wang et al., .. Data extraction and assessment .. 2017; Arafat et al., 2020; Yuan et al., 2020), WES is currently still the .. predominant method over panel sequencing and WGS (75% versus The clinical validity of each identified GDR was scored using a sys- .. .. 19% and 6%, respectively). Nevertheless, there are clinical presenta- tem published by Smith et al. (2017). Scoring was performed by .. two reviewers, randomly assigned from the pool of eight reviewing .. tions where Sanger/panel sequencing is still being utilised at a high fre- .. quency, e.g. 63% of studies on 46,XX and 46,XY disorders/ authors (B.J.H., A.R.-E., M.J.W., A.S.-H., M.J.X., L.N., C.F. and .. .. differences of sexual development (DSDs) in the period of 2019–2020. M.S.O.) using a standardised assessment template to extract gene .. names, inheritance patterns, patient phenotypes, method of discov- .. .. Systematic evaluation of evidence ery (sequencing method); annotate variants; and assess both func- .. tional evidence and clinical data, including the outcome of ART and .. A total of 1523 publications met all inclusion criteria and were used in .. recorded comorbidities. Expression of genes across human organs .. this systematic review. Of these, 186 publications were published in was assessed by consulting data available on the Human Protein .. the period from 2019 to mid-2020 and were thus not included in the .. Atlas website, NCBI’s RNA-seq dataset (Fagerberg et al., 2014), .. previous clinical validity assessment. Overall, 657 individual GDRs de- GTEx and an unpublished human testis single-cell RNA-seq library .. scribed in these 1523 publications were investigated, of which 136 .. tool (https://conradlab.shinyapps.io/HISTA). To avoid bias in . were novel and 521 were re-evaluated with our updated assessment 18 Houston et al. Identification of studies via databases and registers Identification of studies via other methods Identification Records identified from Pubmed Records removed before Records identified from: search on June 30th, 2020 screening: Reviews and reference lists n = 26,250 (+2,765)* None n = 43 Records excluded: Abstract and title screening n = 20,626 n = 26,250 (+2,765)* 1. Paper not in English: 2,654 2. Study not in humans: 5,060 3. Study topic irrelevant:12,912 Screening Records excluded: Reports assessed for eligibility: Reports excluded: Full text screening: n = 5,624 (+534)* n = 4,141 4. Association study: 745 n = 43 None 5. AZF deletion: 505 6. Multiple genes affected: 36 7. Karyotype anomaly: 1,209 8. Irrelevant: 981 9. Full text unavailable: 42 Included Papers included: Gene and disease names extracted from publications: n = 1,523 (+186) Number of genes = 596 (+109) Number of gene-disease relationships = 657 (+136) Definitive: Strong: Moderate: Limited: No evidence: Not evaluated: n=48 (+10) n=27 (+5) n=45 (+13) n=138 (+45) n=175 (+15) n=224 (+48) Evidence AR 28 (+5) 14 (+5) 26 (+10) 85 (+29) 114 (+14) AD 11 (+3) 9 (+2) 18 (+2) 39 (+10) 44 (-1) N/A XL 7 (+2) 4 (-2) 1 (+1) 13 (+6) 15 (+1) YL 2 (=) 0 (=) 0 (=) 1 (=) 2 (+1) Figure 1. PRISMA flowchart of search and assessment process. AZF, azoospermia factor; R, autosomal recessive; AD, autosomal domi- nant; XL, X-linked; YL, Y-linked. .. criteria in order to incorporate any additional supporting evidence .. score since 2019, 11 decreased in score and 9 changed from un- (Supplementary Table SII). .. able to be classified to no evidence (or vice versa). As detailed .. The quality and the extent of all evidence for the GDRs were .. above, GDRs were listed as ‘unable to classify’ when the predicted assessed by two independent reviewers, using a standardised scor- .. inheritance pattern or genotypes were not reported. In comparison .. ing method. This assessment was performed for GDRs that were .. to the previous assessment, 456 scores did not change because no new (n ¼ 136), existing with newly published evidence (n ¼ 145) or .. .. (or insufficient) new evidence was published during the search pe- previously confidently linked (moderate, strong or definitive) to .. riod (Supplementary Table SII). .. male infertility with no newly published evidence (n ¼ 19). This .. The novel and established GDRs combined resulted in a total of score was used to assign a clinical validity of each GDR as: no evi- .. 104 genes confidently linked to 120 human male infertility or abnormal .. dence, limited, moderate, strong or definitive. We assessed the ex- .. genitourinary development phenotypes with moderate (n ¼ 45), strong perimental quality, patient phenotype information, functional .. (n ¼ 27) or definitive (n ¼ 48) evidence (Tables I and II). An overview .. evidence based on gene expression and the study of in-vitro and in- .. of the organ or cell-level role(s) of these genes is detailed in Fig. 3. vivo loss-of-function animal/cell models. Variants were also reclassi- .. .. Another 138 GDRs were classified as ‘limited’ and are thus candidate fied according to the widely accepted the American College of .. genes whose dysfunction may result in a male infertility disorder Medical Genetics and Genomics-Association for Molecular .. .. (Supplementary Table SII). We propose that the latter (limited evi- Pathology (ACMG-AMP) standards and then recorded in spread- .. dence) group will be of particular interest for review in the next few .. sheets for each GDR. .. years. Of the newly identified GDRs, 17 were classified as having mod- .. .. erate evidence or higher (confidently associated with the pheno- .. Insights into the genetic causes of male type) and 43 were classified as having limited evidence. After .. .. infertility classification of the existing GDRs that had new evidence published .. and scoring of GDRs that were previously confidently linked to .. As outlined above, in recent years, the identification of genes confi- .. male infertility with our updated scoring criteria, 45 increased in . dently linked to a male infertility phenotype has risen by 150% from a Validated genetic causes of human male infertility 2020s 19 Figure 2. Breakdown of genetic testing approach and number of genes associated with male infertility phenotypes with limited or moderate and higher evidence classification, over time. (A) The fraction of studies assessed in this paper that used particular sequencing technologies to elucidate causes of male infertility from 1958 to 2020, including karyotyping, copy number variation assessment, azoospermia factor region deletion assessment, association studies and studies investigating monogenic causes. (B) The total number of studies assessed in this paper in- vestigating male infertility, from 1958 to 2020, based on sequencing approach. (C) The fraction of studies assessed in this paper using next-generation sequencing technology compared to Sanger sequencing, from 2010 to 2020. (D) The cumulative number of genes and their strength of evidence as linked to male infertility phenotypes based the scoring criteria used in this paper, from 1989 to 2020. CNV, copy number variant. 20 Houston et al. .. Table I Numbers of genes that are at least moderately .. (n ¼ 38), X-linked (n ¼ 12) and Y-linked (n ¼ 2) inheritance patterns .. were also reported. linked to male infertility or abnormal genitourinary devel- .. opment phenotypes. .. .. .. Description AR AD XL YL Total ...................................................................................................... .. Discussion .. Isolated infertility 27 5 4 0 36 .. .. The aim of this study was to provide an updated assessment of all ge- Acephalic sperm 3 0 0 0 3 .. netic variants reported as causative of human male infertility pheno- Globozoospermia 1 0 0 0 1 .. .. types. We also highlight male infertility phenotypes that have received Macrozoospermia 1 0 0 0 1 .. significant attention in the last few years and identify a group of promis- Multiple morphological 13 1 0 0 14 .. .. ing new candidate genes. These genes are currently classified as limited abnormalities of the .. evidence, requiring additional replication studies and/or functional evi- sperm flagella .. .. dence in order to be classified as strongly associated with male Non-obstructive azoo- 7 4 3 0 14 .. infertility. Based on our findings, the authors/the IMIGC consortium spermia or .. oligozoospermia .. recommend several updates to the genetic testing standards currently .. employed in the field of human male infertility, most important being Congenital bilateral ab- 1 0 1 0 2 .. sence of the vas .. the adoption of exome sequencing as the default sequencing approach. deferens .. .. Our clinical validity assessment revealed that as of 1 July 2020, a Fertilisation failure 1 0 0 0 1 .. total of 104 genes were linked to a total of 120 male infertility or .. Syndromic 13 3 1 0 17 .. abnormal genitourinary development phenotypes. This is a significant infertility .. increase from the 2019 report wherein 78 genes (33% increase to Primary ciliary dyskinesia 7 0 1 0 8 .. .. 104) were associated with 92 phenotypes (30% increase to 120) at a Other syndromes 6 3 0 0 9 .. moderate or higher level of supporting evidence. As this previous as- .. Endocrine disor- 28 30 7 2 67 .. sessment included all published reports from 1958 to 2018, this signifi- der/Reproductive .. cant increase in 2 years further emphasises the healthy uptake of system syndrome .. .. studies to elucidate genetic causes of male infertility, specifically those Disorders of sexual 14 13 4 2 33 .. development .. using NGS. .. Hypogonadotropic 14 17 3 0 34 .. hypogonadism* .. Recent developments in genetics of male .. AR, autosomal recessive; AD, autosomal dominant; XL, X-linked; YL, Y-linked. .. infertility research and diagnostics * .. We note that recent studies are identifying digenic/oligogenic causes for hypogona- .. The diagnostic rate of genetic tests for all types of isolated male infer- dotropic hypogonadism presentations. .. tility combined currently sits between 4% and 9.2% (Olesen et al., .. .. 2017; Punab et al., 2017; Tüttelmann et al., 2018). These rates are no- .. tably behind levels seen in other heterogeneous disorders with a large .. .. genetic contribution such as developmental delay (30%) and cardio- stable average of approximately four per year in the period of 2000– .. myopathies (30–40%; Rehm, 2017). The biggest difference between 2016 to 10 per year in the period of 2017–2019 (Fig. 2D). .. .. these diseases is the slow uptake of NGS approaches in the male infer- Additionally, the number of GDRs with limited (emerging) evidence is .. tility field in research, but even more so in diagnostics. WES and WGS growing quickly, at an average of 17 new genes per year in the period .. .. are now routinely being applied in the diagnostic follow-up of patients 2017–2019 (Fig. 2D). .. The majority of confident GDRs were endocrine disorders or repro- .. with other genetic disorders, resulting in the availability of very large .. cohorts for disease gene discovery (e.g. Bourinaris et al., 2020; ductive system syndromes (n ¼ 67) and isolated infertility phenotypes .. .. Cuvertino et al., 2020; Kaplanis et al., 2020). This, unfortunately, is still (n ¼ 36), while a minority were linked to syndromic infertility (n ¼ 17), .. not the case for severe forms of male infertility in most countries. including primary ciliary dyskinesia (PCD; Table I). The systematic .. .. However, as highlighted in Fig. 2C and D, the field of male infertility analysis of patients who present with multiple morphological abnormal- .. genetics is now expanding rapidly, largely due to the reduced costs ities of the sperm flagella (MMAF) phenotypes was also of particular .. .. and increasing accessibility of NGS, which allows for a more complete note. In 2018, there were five genes confidently linked to human .. and economic testing of patients. Consequently, the number of novel MMAF. This has now tripled to a total of 15 genes as of mid-2020. .. .. candidate genes and validated disease genes is rapidly growing, which Although representing only a small fraction of patients with isolated in- ... is essential for further diagnostic implementation of these approaches fertility, the use of NGS has the potential to diagnose up to 50% of .. .. in male infertility. MMAF patients (Toure et al., 2021). A further 15 genes were confi- .. dently linked to the most frequently presenting clinical presentations, .. Multiple morphological abnormalities of the sperm flagellum .. non-obstructive azoospermia (NOA) and oligozoospermia (Tables I .. The phenotype where most progress has been made in recent and II). The majority of all confident GDRs represented an autosomal .. years is MMAF. Of the 31 newly discovered genes confidently .. recessive inheritance pattern (n ¼ 68), while autosomal dominant . linked to male infertility, 9 were linked to MMAF (29%). When just Validated genetic causes of human male infertility 2020s 21 Table II List of genes linked to male infertility or abnormal genitourinary development phenotypes classified as moderate evi- dence or higher. Gene Location Disorder Inheritance Score Conclusion pattern ............................................................................................................................................................................................................................ Isolated infertility ADGRG2 Xp22.13 Congenital bilateral absence of the vas deferens; OMIM:300985 XL 16 Definitive AR Xq12 Non-obstructive azoospermia; OMIM:NA XL 17 Definitive ARMC2 6q21 Multiple morphological abnormalities of the sperm flagella; AR 11 Moderate OMIM:618433 AURKC 19q13.43 Macrozoospermia; OMIM:243060 AR 17 Definitive CFAP251 12q24.31 Multiple morphological abnormalities of the sperm flagella; AR 17 Definitive OMIM:NA (PS258150) CFAP43 10q25.1 Multiple morphological abnormalities of the sperm flagella; AR 17 Definitive OMIM:617592 CFAP44 3q13.2 Multiple morphological abnormalities of the sperm flagella; AR 17 Definitive OMIM:617593 CFAP65 2q35 Multiple morphological abnormalities of the sperm flagella; AR 15 Strong OMIM:618664 CFAP69 7q21.13 Multiple morphological abnormalities of the sperm flagella; AR 13 Strong OMIM:617959 CFAP91 3q13.33 Multiple morphological abnormalities of the sperm flagella; AR 9 Moderate OMIM:609910 CFTR 7q31.2 Congenital bilateral/unilateral absence of vas deferens; AR 17 Definitive OMIM:277180 DMRT1 9p24.3 Non-obstructive azoospermia; OMIM:NA (PS258150) AD 10 Moderate DNAH1 3p21.1 Multiple morphological abnormalities of the sperm flagella; AR 17 Definitive OMIM:617576 DNAH17 17q25.3 Multiple morphological abnormalities of the sperm flagella; AR 15 Strong OMIM:618643 DPY19L2 12q14.2 Globozoospermia; OMIM:613958 AR 16 Definitive FANCM 14q21.2 Oligozoospermia; OMIM:NA (PS258150) AR 13 Strong FSIP2 2q32.1 Multiple morphological abnormalities of the sperm flagella; AR 12 Moderate OMIM:618153 KLHL10 17q21.2 Oligozoospermia; OMIM:615081 AD 10.5 Moderate M1AP 2p13.1 Non-obstructive azoospermia; OMIM:619108 AR 12 Moderate MEI1 22q13.2 Non-obstructive azoospermia; OMIM:NA (PS258150) AR 13 Strong PLCZ1 12p12.3 Fertilization failure; OMIM:617214 AR 16 Definitive PMFBP1 16q22.2 Acephalic spermatozoa; OMIM:618112 AR 14 Strong QRICH2 17q25.1 Multiple morphological abnormalities of the sperm flagella; AR 12 Moderate OMIM:618341 SEPTIN12 16p13.3 Multiple morphological abnormalities of the sperm flagella; AD 11.5 Moderate OMIM:614822 SPEF2 5p13.2 Multiple morphological abnormalities of the sperm flagella; AR 14.75 Strong OMIM:618751 STAG3 7q22.1 Non-obstructive azoospermia; OMIM:NA (PS258150) AR 11.5 Moderate SUN5 20q11.21 Acephalic sperm; OMIM:617187 AR 16.75 Definitive SYCP2 20q13.33 Severe oligozoospermia; OMIM:258150 AD 10.75 Moderate SYCP3 12q23.2 Non-obstructive azoospermia; OMIM:270960 AD 14 Strong TEX11 Xp11 Non-obstructive azoospermia; OMIM:309120 XL 16 Definitive TEX14 17q22 Non-obstructive azoospermia; OMIM:617707 AR 10 Moderate TEX15 8p12 Non-obstructive azoospermia; OMIM:617960 AR 13.5 Strong TSGA10 2q11.2 Acephalic spermatozoa; OMIM:617961 AR 10.25 Moderate TTC29 4q31.22 Multiple morphological abnormalities of the sperm flagella; AR 14.5 Strong OMIM:618745 Continued 22 Houston et al. Table II Continued Gene Location Disorder Inheritance Score Conclusion pattern ............................................................................................................................................................................................................................ USP26 Xq26.2 Azoospermia or oligozoospermia; OMIM:NA (PS258150) XL 9.5 Moderate XRCC2 7q36.1 Non-obstructive azoospermia; OMIM: 617247 AR 10 Moderate Syndromic infertility APOA1 11q23.3 Testicular amyloidosis; OMIM:105200 AD 12 Moderate CATSPER2 15q15.3 Deafness infertility syndrome; OMIM: 611102 AR 11 Moderate CCDC39 3q26.33 Primary ciliary dyskinesia; OMIM:613807 AR 13 Strong CCDC40 17q25.3 Primary ciliary dyskinesia; OMIM:613808 AR 13.25 Strong CDC14A 1p21.2 Oligoasthenoteratozoospermia OMIM:608653 AR 9 Moderate CEP290 12q21.32 Leber congenital amaurosis; OMIM:611755 AR 9 Moderate DNAAF2 14q21.3 Primary ciliary dyskinesia; OMIM:612518 AR 12.25 Moderate DNAAF4 15q21.3 Primary ciliary dyskinesia; OMIM:615482 AR 13 Strong DNAAF6 Xq22.3 Primary ciliary dyskinesia: OMIM:300991 XL 15 Strong FANCA 16q24.3 Occult Fanconi anaemia; OMIM:NA (PS227650) AR 10 Moderate LRRC6 8q24.22 Primary ciliary dyskinesia; OMIM:614935 AR 13.5 Strong MNS1 15q21.3 Asthenoteratozoospermia; OMIM:NA (PS258150) AR 9.5 Moderate NLRP3 1q44 Muckle-Wells Syndrome; OMIM:191900 AD 9 Moderate PKD1 16p13.3 Polycystic kidney disease and asthenozoospermia; AD 11.25 Moderate OMIM:173900 RSPH3 6q25.3 Primary ciliary dyskinesia; OMIM:616481 AR 10.25 Moderate SPEF2 5p13.2 Primary ciliary dyskinesia with multiple morphological abnormali- AR 12 Moderate ties of the sperm flagellum; OMIM:618751 TRIM37 17q22 Mulibrey nanism; OMIM:253250 AR 10 Moderate Reproductive system syndrome/endocrine disorder AMH 19p13.3 Persistent Müllerian duct syndrome; OMIM:261550 AR 17 Definitive AMHR2 12q13.13 Persistent Müllerian duct syndrome; OMIM:261550 AR 17 Definitive ANOS1 Xp22.31 Kallmann syndrome; OMIM:308700 XL 16 Definitive ANOS1 Xp22.31 Isolated hypogonadotropic hypogonadism (normosmic); XL 13 Strong OMIM:308700 AR Xq12 Partial androgen insensitivity syndrome; OMIM:312300/300633 XL 17 Definitive BMP4 14q22.2 Hypospadias; OMIM:NA (PS300633). Micropenis; OMIM:NA AD 10.25 Moderate BMP7 20q13.31 Hypospadias; OMIM:NA (PS300633) AD 10.25 Moderate BNC2 9p22.3-p22.2 Hypospadias; OMIM:NA (PS300633) AD 10 Moderate CCDC141 2q31.2 Kallmann syndrome; OMIM:NA (PS147950) AR 12 Moderate CHD7 8q12.2 Kallmann syndrome without CHARGE phenotype; AD 16 Definitive OMIM:612370 CHD7 8q12.2 Isolated hypogonadotropic hypogonadism (normosmic) without AD 17 Definitive CHARGE phenotype; OMIM:612370 CYP11A1 15q24.1 Congenital adrenal insufficiency with partial 46,XY sex reversal AR 16 Definitive (Prader stage 4; 5 or 6); OMIM:613743 CYP11B1 8q24.3 46,XX Disorders of sexual development (Prader scale 4; 5 or 6) AR 17 Definitive due to congenital adrenal hyperplasia (11-beta-hydroxylase defi- ciency); OMIM: 202010 CYP17A1 10q24.32 46,XY Disorders of sexual development (Prader stage 4, 5 or 6) AR 16 Definitive due to 17-alpha-hydroxylase/17,20-lyase deficiency; OMIM:202110 CYP19A1 15q21.2 Aromatase excess syndrome with gynaecomastia; AD 17 Definitive OMIM:139300 CYP19A1 15q21.2 46,XX Disorders of sexual development (Prader scale 4; 5 or 6) AR 16 Definitive due to aromatase deficiency; OMIM:613546 CYP19A1 15q21.2 Male infertility in 46,XY men due to aromatase deficiency; AR 9.5 Moderate OMIM:613546 Continued Validated genetic causes of human male infertility 2020s 23 Table II Continued Gene Location Disorder Inheritance Score Conclusion pattern ............................................................................................................................................................................................................................ CYP21A2 6p21.33 Classic congenital adrenal hyperplasia; OMIM:201910 AR 17 Definitive CYP21A2 6p21.33 Non-classic adrenal hyperplasia (late onset or no CAH symp- AR 17 Definitive toms); OMIM: 201910 DHX37 12q24.31 46,XY Disorders of sexual development (Prader scale 4; 5 or 6); AD 11 Moderate OMIM:273250 FGF17 8p21.3 Kallmann syndrome; OMIM:615270 AD 9 Moderate FGF8 10q24.32 Kallmann syndrome; OMIM: 612702 AD 10 Moderate FGF8 10q24.32 Isolated hypogonadotropic hypogonadism (normosmic); AD 14 Strong OMIM:612702 FGFR1 8p11.23 Kallmann syndrome; OMIM:147950 AD 17 Definitive FGFR1 8p11.23 Isolated hypogonadotropic hypogonadism (normosmic); AD 17 Definitive OMIM:147950 FSHB 11p14.1 Isolated hypogonadotropic hypogonadism; OMIM:229070 AR 12.25 Moderate FSHR 2p16.3 Hypergonadotropic hypogonadism; OMIM:NA (PS147950) AR 11 Moderate GATA4 8p23.1 46,XY Disorders of sexual development (Prader scale 4; 5 or 6) AD 13 Strong resulting in anomalies of testicular development; OMIM:615542 GNRH1 8p21.2 Isolated hypogonadotropic hypogonadism; OMIM:614841 AR 13.5 Strong GNRHR 4q13.2 Isolated hypogonadotropic hypogonadism; OMIM:146110 AR 17 Definitive HS6ST1 2q14.3 Kallmann syndrome; OMIM:614880 AD 9.5 Moderate HSD17B3 9q22.32 46,XY Disorders of sexual development (Prader scale 4; 5 or 6) AR 16 Definitive resulting in anomalies of testicular development; OMIM:264300 HSD3B2 1p12 Adrenal hyperplasia due to 3b-hydroxysteroid dehydrogenase AR 16.5 Definitive deficiency; OMIM:201810 IGSF10 3q25.1 Delayed puberty; OMIM:NA (PS147950) AD 9.25 Moderate IL17RD 3p14.3 Kallmann syndrome with hearing loss; OMIM:615267 AD 14.5 Strong INSL3 19p13.11 Cryptorchidism; OMIM:219050 AD 12 Moderate KISS1R 19p13.3 Kallmann syndrome; OMIM:614837 AR 9 Moderate KISS1R 19p13.3 Isolated hypogonadotropic hypogonadism (normosmic); AR 17 Definitive OMIM:614837 LHB 19q13.33 Isolated Hypogonadotropic hypogonadism; OMIM:228300 AR 16.5 Definitive LHCGR 2p16.3 Leydig cell dysfunction with hypogonadism; OMIM:238320 AR 16.5 Definitive LHCGR 2p16.3 Male precocious puberty; OMIM:176410 AD 17 Definitive MAMLD1 Xq28 46,XY Disorders of Sex Development (Prader scale 4; 5 or 6); XL 15 Strong OMIM:300758 MYRF 11q12.2 46XY Disorders of Sex Development, OMIM gene 608329 AD 14.5 Strong NR0B1 Xp21.2 Congenital adrenal hypoplasia; OMIM:300200 XL 17 Definitive NR0B1 Xp21.2 Late-onset adrenal failure or isolated hypogonadotropic hypogo- XL 17 Definitive nadism; OMIM:NA (PS147950) NR5A1 9q33.3 46,XY Disorders of sexual development (Prader scale 4; 5 or 6); AD 17 Definitive OMIM:612965 NR5A1 9q33.3 46,XX Disorders of sexual development (Prader scale 4; 5 or AD 16 Definitive 6); OMIM:617480 NR5A1 9q33.3 Isolated spermatogenic failure; OMIM:184757 AD 14 Strong PLXNA1 3q21.3 Kallmann syndrome; OMIM:NA (PS147950) AD 13.5 Strong POU1F1 3p11.2 Combined pituitary hormone deficiency; OMIM:613038 AR 16 Definitive PROK2 3p13 Kallmann syndrome; OMIM:610628 AR 11.5 Moderate PROKR2 20p12.3 Kallmann syndrome; OMIM:244200 AR 17 Definitive PROP1 5q35.3 Pituitary hormone deficiency; OMIM:262600 AR 17 Definitive RSPO1 1p34.3 Palmoplantar hyperkeratosis with squamous cell carcinoma of AR 12 Moderate skin and sex reversal; OMIM:610644 SEMA3A 7q21.11 Kallmann syndrome; OMIM:614897 AD 16 Definitive Continued 24 Houston et al. Table II Continued Gene Location Disorder Inheritance Score Conclusion pattern ............................................................................................................................................................................................................................ SOX10 22q13.1 Kallmann syndrome; OMIM:NA (PS147950) AD 16 Definitive SOX2 3q26.33 Isolated hypogonadotropic hypogonadism (normosmic); AD 16 Definitive OMIM:NA (PS147950) SOX3 Xq27.1 46,XX Disorders of sexual development (Prader scale 4; 5 or XL 13 Strong 6); OMIM:NA SOX9 17q24.3 46,XY Disorders of sexual development (Prader scale 4; 5 or 6); AD 13.5 Strong OMIM:NA SRD5A2 2p23.1 46,XY Disorders of sexual development (Prader scale 4; 5 or 6); AR 17 Definitive OMIM:264600 SRY Yp11.2 46,XX Disorders of sexual development (Prader scale 4; 5 or YL 17 Definitive 6); OMIM:400045 SRY Yp11.2 46,XY Disorders of sexual development (Prader scale 4; 5 or 6); YL 17 Definitive OMIM:400044 STAR 8p11.23 Lipoid adrenal hyperplasia; OMIM:201710 AR 10 Moderate TACR3 4q24 Kallmann syndrome; OMIM:614840 AR 16.5 Definitive WDR11 10q26.12 Hypogonadotropic hypogonadism; OMIM:614858 AD 12 Moderate WDR11 10q26.12 Kallmann syndrome; OMIM:614858 AD 11 Moderate WT1 11p13 46,XY Disorders of sexual development (Prader scale 4; 5 or 6) AD 14.25 Strong without Wilm’s tumour; OMIM:NA (PS400044) We note that recent studies are identifying digenic/oligogenic causes for hypogonadotropic hypogonadism presentations including Kallman syndrome. considering genes likely associated with primary male infertility .. Azoospermia and oligozoospermia .. (i.e. spermatogenesis genes), this constitutes 47% of new, confi- .. NOA and severe oligozoospermia are expected to have extensive ge- dently classified genes (9 of 19). Currently, a total of 14 genes .. netic heterogeneity owing to the multiple phases of spermatogenesis .. (ARMC2, CFAP43, CFAP44, CFAP65, CFAP69, CFAP91, CFAP251, .. that can be affected to cause these presentations. Although a few .. DNAH1, DNAH17, FSIP2, QRICH2, SEPTIN12, SPEF2 and TTC29) are .. genes confidently linked to primary testicular failure are beginning to confidently linked to MMAF and another 9 (AK7, AKAP4, CEP135, .. emerge, including M1AP, STAG3, SYCP2 and TEX11, where data from .. CFAP70, DNAH2, DNAH6, DZIP1, TTC21A and WDR19) are listed .. animal models indicate each is essential for meiosis (Yang et al., 2006; as candidate genes, i.e. they have a limited classification. As a re- .. Arango et al., 2013; Winters et al., 2014; Yatsenko et al., 2015), large .. flection of collaborative research efforts, MMAF-associated genes .. cohort sizes are critical to reveal the full spectrum of disease genes. also comprise 42% of all confident genes that function during the .. The discovery and validation of three of these meiosis genes have .. spermiogenesis period. .. been possible through the collaborative efforts of the IMIGC/GEMINI Furthermore, and in line with the highly conserved core structure of .. consortia and the use of large infertile cohorts and replication studies. .. motile cilia and flagella across tissues, there is a clear phenotypic con- .. This has now led to the availability of WES data for >3000 men with tinuum of patients with phenotypes ranging from classical PCD, mani- .. NOA or severe oligozoospermia, which underscores the importance .. festing as complete sperm immotility but normal cytology, to severe .. of data sharing/collaboration for the continued identification of novel, forms of teratozoospermia. Sperm tail development is a remarkable .. rare genetic causes of male infertility. In addition, these large cohorts .. process that requires the expression of more than 1000 proteins .. help to validate and better estimate the role of previously implicated (Toure et al., 2021) and their co-ordinated transport into a distinct cili- .. .. variants/genes. For example, in 2017 variants in the D-box region of ary compartment originating from a modified centriole that docks to .. PIWIL1 were implicated as a recurrent cause of NOA (Gou et al., the sperm head (Pleuger et al., 2020). Thus, there are likely many addi- .. .. 2017). Using WES on 2740 men with NOA or severe oligozoosper- tional genes required for human/mammalian sperm tail development .. mia, the IMIGC/GEMINI consortia demonstrated that pathogenic var- to be discovered. Novel evidence has been identified from MMAF .. .. iants in the D-box region of PIWIL1 specifically, and variants elsewhere studies, where variants in SPEF2 cause PCD with MMAF (Tu et al., .. in the gene, are not a common cause of male infertility (Oud et al., 2020). While the origin of this commonality is largely unexplored, it .. .. 2021). may reflect shared protein transport pathways into the ciliary/sperm .. tail compartment (Pleuger et al., 2020). There are also genes that play .. .. Identifying the variant(s) causative for male important roles in axoneme function (e.g. DNAH17; Whitfield et al., .. 2019), whose loss of function results in an isolated infertility phenotype .. infertility .. where cilia are unaffected. .. Currently, 33% of all identified GDRs proposed in the literature fell .. into the ‘unable to classify’ category. For most, this classification is .. . given because the inheritance pattern is unclear. The list, however, Validated genetic causes of human male infertility 2020s 25 Figure 3. Overview of all genes associated with male infertility phenotypes at an organ/cell level. Definitive genes are labelled in red, strong in orange and moderate in yellow. Organs top to bottom: brain, adrenal gland (and kidney), testes and epididymides with vas deferens. Bottom left: sperm fertilising an oocyte surrounded by cumulus cells. Right: seminiferous tubule cross-section. Leydig cells (blue), Sertoli cells (purple), basement membrane (pink), spermatogonia (green), spermatocytes (brown) and spermatids (pink and orange). ** denotes germ cell arrest gene for FANCM and TEX14. Genes were classified as unclear when they were not clearly linked to a specific organ. .. likely contains multiple genes that play an important role in the aetiol- .. useful when it is the first time a damaging variant has been found in a ogy of monogenic male infertility. As with any disease, but perhaps ex- .. novel gene, where calculations are based on inheritance model (auto- .. acerbated by the wide range of individual biological processes required .. somal dominant, recessive, etc.) and allele frequency. As has been for male fertility, it can be difficult to determine the role of poorly .. highlighted by the GEMINI consortium, this tool is useful for discover- .. characterised genes, and whether loss of their function results in a dis- .. ing promising new candidate disease genes and individual pathogenic ease state. To assist in filtering for high confidence variants, members .. variants, but independent replication and functional validation is still .. of the GEMINI consortium have developed the ‘Population Sampling .. critically important before genes can be used in a diagnostic setting as Probability’ pipeline (PSAP; Wilfert et al., 2016). This is particularly .. a ‘validated disease gene’. . 26 Houston et al. Dynamics of genotype–phenotype mapping .. .. bilateral absence of the vas deferens (Krausz and Riera-Escamilla, Recent genetic male infertility studies suggest that a high percentage of .. 2018). These guidelines, or in some cases purely expert opinions, thus .. diagnoses can be revealed, especially in cohorts of men with matura- .. limit the genetic testing to the most common infertility presentations tion arrest. A clear determination of the point of arrest (i.e. at sperma- ... and exclude patients with other sperm phenotypes, such as teratozoo- .. spermia/asthenoteratozoospermia (e.g. globozoospermia and MMAF), togonia or spermatocytes) can assist in refining possibly relevant genes .. containing variants, based on any known biological function(s). .. and other sperm motility disorders (asthenozoospermia/OAT). .. According to the average clinical presentation of German men over However, very little so far has been done outside of meiotic and sper- .. matogonial arrest. Various genes have also been linked to multiple .. the last 30 years, this means that at least 40% of infertile men are not .. phenotypic outcomes; thus, the establishment of a clear genotype– .. being offered any form of genetic testing (Tüttelmann et al., 2018). phenotype correlation is not necessarily straightforward. One such ex- .. In line with the definitive evidence that pathogenic variants in at least .. ample is the TEX14 gene, where pathogenic variation can lead to a .. 36 genes may result in isolated infertility, we advocate for an update of Sertoli cell-only phenotype or spermatocyte arrest (Fakhro et al., .. genetic testing guidelines for male infertility. Ideally, the blood of infertile .. 2018). The progressive loss of germ cells may occur as a factor of age .. men should be used for WES to identify any protein-coding variants in a owing to a role in spermatogonial stem cell renewal, as suggested for .. non-biased manner. We appreciate there will often be an issue with a .. Nxf2 knockout mice (Pan et al., 2009). Alternatively, environmental .. lack of expertise in this technology, or access to sequencing platforms. exposures may interact with the genotype to lead to an exacerbation .. .. IMIGC members (imigc.org) are situated across the USA, Europe and of the phenotype. Variability in the genotype–phenotype relationship .. Australia, and are interested in forming additional collaborations for clini- has also been identified for single gene variants in reproductive system .. .. cal and research purposes. As a less impactful, backup approach, the ge- syndromes/endocrine disorders (Domenice et al., 2016; Goncalves .. netic targets of panels used in targeting sequencing should at least be et al., 2017) in 12 of the 13 (92%) confident genes—ANOS1, AR, .. .. updated to include the genes validated in this study (those moderately CHD7, CYP19A1, CYP21A2, FGF8, FGFR1, KISS1R, LHCGR, NR0B1, .. linked and stronger). Additionally, a wider subset of patients/pheno- NR5A1 and WDR11. These disorders are largely complex in that they .. .. types should be sequenced. Such approaches will help to achieve more effect multiple hormonal pathways and tissues beyond the testis. .. diagnoses, better-personalised treatment, improved risk assessment for .. Other presentations with variability in genotype and phenotype in- .. the transmission of infertility to offspring and better counselling for po- clude congenital hypogonadotropic hypogonadism (HH)/Kallmann syn- .. .. tential health risks in the infertile man. It will also help to improve drome. In addition to Mendelian causes, incomplete penetrance and .. counselling of azoospermic men prior to testicular biopsy, as finding a oligogenic forms are thought to be causative for these disorders .. .. genetic diagnosis may help in predicting the chances for successful testic- (Maione et al., 2018). One example is the PROK2 gene, for which au- .. ular sperm extraction, which is an incredibly invasive procedure. tosomal dominant, recessive and oligogenicity have been proposed to .. .. be causative (Leroy et al., 2008). For the DSD spectrum and HH, re- .. search groups are now actively exploring the role of oligogenic inheri- .. Wider implications of male infertility .. genetics tance in contributing to the disease (Cangiano et al., 2021). For DSDs, .. this genetic explanation has been proposed following the finding that .. Precise phenotyping of men with pathogenic variants will assist in eval- .. many patients present with at least one variant in two individual known .. uating potential future health risks. Mounting evidence suggests that or novel DSD genes, particularly when MAMLD1 is affected (Camats .. .. male infertility may pose as a predictive condition for life-threatening et al., 2020; Li et al., 2020). Similarly, variants in FGFR1 and other HH .. comorbidities in later life, such as various types of cancer and cardio- genes have been shown to cause Kallman syndrome in an oligogenic .. .. vascular disease (Jensen et al., 2009; Eisenberg et al., 2014; Ehrlich, manner (reviewed in Cangiano et al., 2021). .. 2015). While the precise mechanism(s) underpinning this observation .. .. are unknown, it is notable that many genes that are highly expressed Recommendations for genetic testing in .. in the testis and lead to male infertility when mutated are also .. male infertility .. expressed in other organs, but at considerably lower levels. These in- .. clude, for example, multiple DNA repair genes linked to infertility and Although NGS methods are frequently used in research laboratories .. .. various cancers (reviewed in Nagirnaja et al., 2018) and TEX11, which to study the genetics of male infertility, they have not yet been exten- .. sively employed in clinical genetic diagnostics for this condition. While .. is expressed in the testis and the pancreas according to RNA-seq data .. (GTEx Portal). As has been made clear above, this is the case for sev- there are no standardised international guidelines for clinical genetic .. testing in male infertility, a small number of guidelines have been pub- .. eral cilia genes, where men often present with chronic bronchitis and .. other lung conditions (reviewed in Leigh et al., 2009; Sironen et al., lished by professional andrology groups. Examples include guidelines .. for oligoasthenoteratozoospermia (OAT; Colpi et al., 2018) and .. 2020). Thus, additional studies where the health of infertile men is .. broadly investigated are urgently needed. Klinefelter syndrome (Zitzmann et al., 2021) by the European .. Academy of Andrology, and diagnostics/treatment prior to use of .. Although the topic could constitute an entire article itself, the rapidly .. ART (Toth et al., 2019) by the German Association of the Scientific .. increasing use of ART worldwide (de Mouzon et al., 2020) is resulting Medical Societies. However, sperm count cut-offs and genetic testing .. in the propagation of infertility-causing variants throughout the popula- .. approaches vary widely between countries. Patients with oligozoosper- .. tion. As we identify these variants in men who undergo ART, we will mia, <5 million sperm/ml (EU/USA), or azoospermia are generally of- .. also be faced with delivering this information back to the patients .. fered karyotyping and Y-chromosome microdeletion analysis, while .. through genetic counselling services. This is especially relevant for men CFTR testing is recommended for men with suspected congenital .. with Y-linked variants, where any sons will presumably be inflicted with Validated genetic causes of human male infertility 2020s 27 .. the same infertility phenotype as their affected fathers and thus also re- .. .. Funding quire ART if wanting their own biological child. .. National Health and Medical Research Council APP1120356 to .. Strengths and limitations ... M.K.O.B., J.A.V., D.F.C. and K.I.A. The Netherlands Organisation for .. Scientific Research (918-15-667) to J.A.V. as well as an Investigator We used the joint expertise of the IMIGC to systematically evaluate all .. Award in Science from the Wellcome Trust (209451) to J.A.V. .. available evidence (as of 1 July 2020) for monogenic causes of isolated .. German Research Foundation sponsored Clinical Research Unit ‘Male or syndromic male infertility, endocrine disorders or reproductive sys- .. .. Germ Cells: from Genes to Function’ (DFG, CRU326) to C.F. and F.T. tem abnormalities affecting the male sex organs. No other published .. National Institutes of Health: Genomics of Spermatogenic Impairment reviews in this field come close to including the 600 genes compre- .. .. (R01HD078641) to D.F.C. and K.I.A. Spanish Ministry of Health hensively evaluated here. We improved the quality of evidence scoring .. Instituto Carlos III-FIS-FEDER (Grant number: PI17/01822 PI20/ .. by including a larger number of reviewers from independent research .. 01562) to A.R.-E. and C.K. groups with broad expertise in male infertility. As all research groups .. .. have been involved in disease gene discovery of various genes de- .. .. scribed in this study, we prevented bias as much as possible by not .. Conflict of interest allowing scoring by reviewers who have worked on respective genes. .. .. The authors declare no conflicts of interest. .. .. .. Conclusion .. .. References Here, we describe an updated clinical validity assessment for a total of .. .. Alhathal N, Maddirevula S, Coskun S, Alali H, Assoum M, Morris T, 657 male infertility or abnormal genitourinary development GDRs, in- .. Deek HA, Hamed SA, Alsuhaibani S, Mirdawi A et al. A genomics volving 596 genes. We identified 104 genes linked to 120 phenotypes .. .. approach to male infertility. Genet Med 2020;22:1967–1975. with sufficient evidence for use in gene panels. These results may help .. Arafat M, Harlev A, Har-Vardi I, Levitas E, Priel T, Gershoni M, Searby .. to improve genetic testing in male infertility research and/or diagnostics. .. C, Sheffield VC, Lunenfeld E, Parvari R. Mutation in CATIP (C2orf62) .. causes oligoteratoasthenozoospermia by affecting actin dynamics. J .. .. Med Genet 2020. doi:10.1136/jmedgenet-2019-106825. .. Supplementary data .. Arango NA, Li L, Dabir D, Nicolau F, Pieretti-Vanmarcke R, Koehler .. C, McCarrey JR, Lu N, Donahoe PK. Meiosis I arrest abnormalities Supplementary data are available at Human Reproduction Update online. .. .. lead to severe oligozoospermia in meiosis 1 arresting protein .. (M1ap)-deficient mice. Biol Reprod 2013;88:76. .. .. Bedoni N, Haer-Wigman L, Vaclavik V, Tran VH, Farinelli P, Balzano .. Data availability .. S, Royer-Bertrand B, El-Asrag ME, Bonny O, Ikonomidis C et al. .. Mutations in the polyglutamylase gene TTLL5, expressed in photo- The present study and the corresponding search protocol were regis- .. tered with the PROSPERO registry (http://www.crd.york.ac.uk/ .. receptor cells and spermatozoa, are associated with cone-rod de- .. generation and reduced male fertility. Hum Mol Genet 2016;25: PROSPERO) as PROSPERO 2021: CRD42021229164. All scoring .. .. 4546–4555. spreadsheets are accessible via the IMIGC website (www.imigc.org/ .. Bourinaris T, Smedley D, Cipriani V, Sheikh I, Athanasiou-Fragkouli data-sharing). .. .. A, Chinnery P, Morris H, Real R, Harrison V, Reid E et al.; .. Genomics England Research Consortium. Identification .. Acknowledgements .. of UBAP1 mutations in juvenile hereditary spastic paraplegia in the .. .. 100,000 Genomes Project. Eur J Hum Genet 2020;28:1763–1768. We appreciate the feedback from Raul Pi~na-Aguilar for his feedback .. Camats N, Fluck CE, Audi L. Oligogenic origin of differences of sex during the initial discussion and planning of this study. .. .. development in humans. Int J Mol Sci 2020;21: 1809. .. Cangiano B, Swee DS, Quinton R, Bonomi M. Genetics of congenital .. .. hypogonadotropic hypogonadism: peculiarities and phenotype of .. an oligogenic disease. Hum Genet 2021;140:77–111. Authors’ roles .. .. Chen S, Wang G, Zheng X, Ge S, Dai Y, Ping P, Chen X, Liu G, B.J.H., A.R.-E., M.J.W., A.S.-H., M.J.X., L.N., C.F., M.S.O., M.K.O.B. .. Zhang J, Yang Y et al. Whole-exome sequencing of a large Chinese .. and J.A.V. designed and supervised the study; and D.F.C., K.I.A., C.K. .. azoospermia and severe oligospermia cohort identifies novel infer- and F.T. assisted in supervising this study. B.J.H. and M.S.O. selected .. .. tility causative variants and genes. Hum Mol Genet 2020;29: studies for the inclusion and evaluated their quality. The clinical validity .. 2451–2459. .. assessments were done by B.J.H., A.R.-E., M.J.W., A.S.-H., M.J.X., .. Chiu W, Hsun YH, Chang KJ, Yarmishyn AA, Hsiao YJ, Chien Y, L.N., C.F. and M.S.O., which were co-ordinated by B.J.H. and M.S.O. .. Chien CS, Ma C, Yang YP, Tsai PH et al. Current genetic survey .. B.J.H. and M.S.O. wrote the first draft of the manuscript. All authors .. and potential gene-targeting therapeutics for neuromuscular dis- contributed to the writing and revision process. .. eases. Int J Mol Sci 2020;21: 9589. 28 Houston et al. .. Colpi GM, Francavilla S, Haidl G, Link K, Behre HM, Goulis DG, .. Leroy C, Fouveaut C, Leclercq S, Jacquemont S, Boullay HD, Krausz C, Giwercman A. European Academy of Andrology guide- .. Lespinasse J, Delpech M, Dupont JM, Hardelin JP, Dode C. Biallelic .. line Management of oligo-astheno-teratozoospermia. Andrology .. mutations in the prokineticin-2 gene in two sporadic cases of 2018;6:513–524. .. Kallmann syndrome. Eur J Hum Genet 2008;16:865–868. .. Coutton C, Martinez G, Kherraf Z-E, Amiri-Yekta A, Boguenet M, .. Li L, Gao F, Fan L, Su C, Liang X, Gong C. Disorders of sex develop- Saut A, He X, Zhang F, Cristou-Kent M, Escoffier J et al. Bi-allelic .. ment in individuals harbouring MAMLD1 variants: WES and interac- .. Mutations in ARMC2 Lead to Severe Astheno-Teratozoospermia .. tome evidence of oligogenic inheritance. Front Endocrinol (Lausanne) Due to Sperm Flagellum Malformations in Humans and Mice. Am J .. 2020;11:582516. .. Hum Genet 2019;104:331–340. .. Liu C, Tu C, Wang L, Wu H, Houston BJ, Mastrorosa FK, Zhang W, .. Cuvertino S, Hartill V, Colyer A, Garner T, Nair N, Al-Gazali L, .. Shen Y, Wang J, Tian S et al. Deleterious variants in X- Canham N, Faundes V, Flinter F, Hertecant J et al.; Genomics .. linked CFAP47 induce asthenoteratozoospermia and primary male .. England Research Consortium. A restricted spectrum of .. infertility. Am J Hum Genet 2021;108:309–323. missense KMT2D variants cause a multiple malformations disorder .. Maione L, Dwyer AA, Francou B, Guiochon-Mantel A, Binart N, .. distinct from Kabuki syndrome. Genet Med 2020;22:867–877. .. Bouligand J, Young J. GENETICS IN ENDOCRINOLOGY: genetic de Mouzon J, Chambers GM, Zegers-Hochschild F, Mansour R, .. counseling for congenital hypogonadotropic hypogonadism and .. Ishihara O, Banker M, Dyer S, Kupka M, Adamson GD. .. Kallmann syndrome: new challenges in the era of oligogenism and International Committee for Monitoring Assisted Reproductive .. next-generation sequencing. Eur J Endocrinol 2018;178:R55–R80. .. Technologies world report: assisted reproductive technology 2012. .. Markitantova Y, Simirskii V. Inherited eye diseases with retinal mani- Hum Reprod 2020;35:1900–1913. .. festations through the eyes of homeobox genes. Int J Mol Sci 2020; .. Domenice S, Machado AZ, Ferreira FM, Ferraz-de-Souza B, Lerario .. 21: 1602. AM, Lin L, Nishi MY, Gomes NL, da Silva TE, Silva RB et al. Wide .. Nagirnaja L, Aston KI, Conrad DF. Genetic intersection of male infer- .. spectrum of NR5A1-related phenotypes in 46,XY and 46,XX indi- .. tility and cancer. Fertil Steril 2018;109:20–26. viduals. Birth Defects Res C Embryo Today 2016;108:309–320. .. Olesen IA, Andersson AM, Aksglaede L, Skakkebaek NE, Rajpert-de .. Ehrlich S. Effect of fertility and infertility on longevity. Fertil Steril 2015; .. Meyts E, Joergensen N, Juul A. Clinical, genetic, biochemical, and 103:1129–1135. .. testicular biopsy findings among 1,213 men evaluated for infertility. .. Eisenberg ML, Li S, Behr B, Cullen MR, Galusha D, Lamb DJ, .. Fertil Steril 2017;107:74–82.e7. Lipshultz LI. Semen quality, infertility and mortality in the USA. .. Oud MS, Volozonoka L, Friedrich C, Kliesch S, Nagirnaja L, Gilissen .. Hum Reprod 2014;29:1567–1574. .. C, O’Bryan MK, McLachlan RI, Aston KI, Tuttelmann F et al. Lack .. Fagerberg L, Hallstrom BM, Oksvold P, Kampf C, Djureinovic D, .. of evidence for a role of PIWIL1 variants in human male infertility. Odeberg J, Habuka M, Tahmasebpoor S, Danielsson A, Edlund K .. Cell 2021;184:1941–1942. .. et al. Analysis of the human tissue-specific expression by genome- .. Oud MS, Volozonoka L, Smits RM, Vissers L, Ramos L, Veltman JA. wide integration of transcriptomics and antibody-based proteo- .. A systematic review and standardized clinical validity assessment of .. mics. Mol Cell Proteomics 2014;13:397–406. .. male infertility genes. Hum Reprod 2019;34:932–941. Fakhro KA, Elbardisi H, Arafa M, Robay A, Rodriguez-Flores JL, Al- .. Pan J, Eckardt S, Leu NA, Buffone MG, Zhou J, Gerton GL, .. Shakaki A, Syed N, Mezey JG, Abi Khalil C, Malek JA et al. Point- .. McLaughlin KJ, Wang PJ. Inactivation of Nxf2 causes defects in of-care whole-exome sequencing of idiopathic male infertility. .. male meiosis and age-dependent depletion of spermatogonia. Dev .. Genet Med 2018;20:1365–1373. .. Biol 2009;330:167–174. Goncalves CI, Fonseca F, Borges T, Cunha F, Lemos MC. Expanding .. Pleuger C, Lehti MS, Dunleavy JE, Fietz D, O’Bryan MK. Haploid .. the genetic spectrum of ANOS1 mutations in patients with congenital .. male germ cells-the Grand Central Station of protein transport. hypogonadotropic hypogonadism. Hum Reprod 2017;32:704–711. .. Hum Reprod Update 2020;26:474–500. .. Gou LT, Kang JY, Dai P, Wang X, Li F, Zhao S, Zhang M, Hua MM, .. Punab M, Poolamets O, Paju P, Vihljajev V, Pomm K, Ladva R, Lu Y, Zhu Y et al. Ubiquitination-deficient mutations in .. Korrovits P, Laan M. Causes of male infertility: a 9-year prospective .. human Piwi cause male infertility by impairing histone-to-protamine .. monocentre study on 1737 patients with reduced total sperm .. exchange during spermiogenesis. Cell 2017;169:1090–1104.e13. .. counts. Hum Reprod 2017;32:18–31. Jensen TK, Jacobsen R, Christensen K, Nielsen NC, Bostofte E. .. Rehm HL. Evolving health care through personal genomics. Nat Rev .. Good semen quality and life expectancy: a cohort study of 43,277 .. Genet 2017;18:259–267. men. Am J Epidemiol 2009;170:559–565. .. Schultz N, Hamra FK, Garbers DL. A multitude of genes expressed solely .. Kaplanis J, Samocha KE, Wiel L, Zhang Z, Arvai KJ, Eberhardt RY, ... in meiotic or postmeiotic spermatogenic cells offers a myriad of contra- Gallone G, Lelieveld SH, Martin HC, McRae JF et al.; Deciphering .. ceptive targets. Proc Natl Acad Sci U S A 2003;100:12201–12206. Developmental Disorders Study. Evidence for 28 genetic disorders .. Sironen A, Shoemark A, Patel M, Loebinger MR, Mitchison HM. .. discovered by combining healthcare and research data. Nature .. Sperm defects in primary ciliary dyskinesia and related causes of 2020;586:757–762. .. male infertility. Cell Mol Life Sci 2020;77:2029–2048. .. Krausz C, Riera-Escamilla A. Genetics of male infertility. Nat Rev Urol .. Smith ED, Radtke K, Rossi M, Shinde DN, Darabi S, El-Khechen D, 2018;15:369–384. .. Powis Z, Helbig K, Waller K, Grange DK et al. Classification of .. Leigh MW, Pittman JE, Carson JL, Ferkol TW, Dell SD, Davis SD, .. genes: standardized clinical validity assessment of gene-disease Knowles MR, Zariwala MA. Clinical and genetic aspects of primary cil- .. associations aids diagnostic exome analysis and reclassifications. .. iary dyskinesia/Kartagener syndrome. Genet Med 2009;11:473–487. . Hum Mutat 2017;38:600–608. Validated genetic causes of human male infertility 2020s 29 Toth B, Baston-Bust DM, Behre HM, Bielfeld A, Bohlmann M, .. Whitfield M, Thomas L, Bequignon E, Schmitt A, Stouvenel L, .. Buhling K, Dittrich R, Goeckenjan M, Hancke K, Kliesch S et al. .. Montantin G, Tissier S, Duquesnoy P, Copin B, Chantot S et al. .. Diagnosis and treatment before assisted reproductive treatments. .. Mutations in DNAH17, encoding a sperm-specific axonemal outer guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF .. dynein arm heavy chain, cause isolated male infertility due to asthe- .. register number 015-085, February 2019)—part 2, hemostaseol- .. nozoospermia. Am J Hum Genet 2019;105:198–212. ogy, andrology, genetics and history of malignant disease. .. Wilfert AB, Chao KR, Kaushal M, Jain S, Zollner S, Adams DR, .. Geburtshilfe Frauenheilkd 2019;79:1293–1308. .. Conrad DF. Genome-wide significance testing of variation from sin- Toure A, Martinez G, Kherraf ZE, Cazin C, Beurois J, Arnoult C, Ray .. gle case exomes. Nat Genet 2016;48:1455–1461. .. PF, Coutton C. The genetic architecture of morphological abnor- .. Winters T, McNicoll F, Jessberger R. Meiotic cohesin STAG3 is re- malities of the sperm tail. Hum Genet 2021;140:21–42. .. quired for chromosome axis formation and sister chromatid cohe- .. Tu C, Nie H, Meng L, Wang W, Li H, Yuan S, Cheng D, He W, Liu .. sion. EMBO J 2014;33:1256–1270. G, Du J et al. Novel mutations in SPEF2 causing different defects .. Yang F, De La Fuente R, Leu NA, Baumann C, McLaughlin KJ, Wang .. between flagella and cilia bridge: the phenotypic link between .. PJ. Mouse SYCP2 is required for synaptonemal complex assembly MMAF and PCD. Hum Genet 2020;139:257–271. .. and chromosomal synapsis during male meiosis. J Cell Biol 2006; .. Tüttelmann F, Ruckert C, Ropke A. Disorders of spermatogenesis: .. 173:497–507. perspectives for novel genetic diagnostics after 20 years of .. Yatsenko AN, Georgiadis AP, Ropke A, Berman AJ, Jaffe T, .. unchanged routine. Med Genet 2018;30:12–20. .. Olszewska M, Westernstroer B, Sanfilippo J, Kurpisz M, Rajkovic A Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, .. et al. X-linked TEX11 mutations, meiotic arrest, and azoospermia .. Mardinoglu A, Sivertsson A, Kampf C, Sjostedt E, Asplund A et al. .. in infertile men. N Engl J Med 2015;372:2097–2107. Proteomics. Tissue-based map of the human proteome. Science 2015; .. Yuan P, Yang C, Ren Y, Yan J, Nie Y, Yan L, Qiao J. A novel homo- .. 347:1260419. .. zygous mutation of phospholipase C zeta leading to defective hu- Wang X, Jin H, Han F, Cui Y, Chen J, Yang C, Zhu P, Wang W, Jiao .. man oocyte activation and fertilization failure. Hum Reprod 2020; .. G, Wang W et al. Homozygous DNAH1 frameshift mutation causes .. 35:977–985. multiple morphological anomalies of the sperm flagella in Chinese. .. Zitzmann M, Aksglaede L, Corona G, Isidori AM, Juul A, T’Sjoen G, .. Clin Genet 2017;91:313–321. .. Kliesch S, D’Hauwers K, Toppari J, Słowikowska-Hilczer J et al. Whatley M, Francis A, Ng ZY, Khoh XE, Atlas MD, Dilley RJ, Wong .. European academy of andrology guidelines on Klinefelter .. EYM. Usher syndrome: genetics and molecular links of hearing loss .. Syndrome Endorsing Organization: European Society of and directions for therapy. Front Genet 2020;11:565216. .. Endocrinology. Andrology 2021;9:145–167. .