Cytogenomic investigation in 151 Brazilian infertile male patients: Experience of 14 years in a public genetic service

Cytogenomic investigation in 151 Brazilian infertile male patients: Experience of 14 years in a public genetic service | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Cytogenomic investigation in 151 Brazilian infertile male patients: Experience of 14 years in a public genetic service Márcia Regina Gimenes Adriano, Adriana Bortolai, Fabricia Andreia Rosa Madia, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3317746/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2024 Read the published version in BMC Research Notes → Version 1 posted 4 You are reading this latest preprint version Abstract Objectives Male infertility accounts for approximately 30% of cases of reproductive failure. The characterization of genetic variants using cytogenomic techniques is essential for the adequate clinical management of these patients. We aimed to conduct a cytogenetic investigation of numerical and structural rearrangements and a genomic study of Y chromosome microdeletions/microduplications in infertile men derived from a single centre with over 14 years of experience. Results We evaluated 151 infertile men in a transversal study using peripheral blood karyotypes and 15 patients with normal karyotypes through genomic investigation by multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) techniques. Out of the 151 patients evaluated by karyotype, 13 presented chromosomal abnormalities: 2 had numerical alterations, and 11 had structural chromosomal rearrangements. PCR-STS detected a BPY2 gene region and RBMY2DP pseudogene region microdeletion in one patient. MLPA analysis allowed the identification of one patient with CDY2B_1 and CDY2B_2 probe duplications ( CDY2B gene) and one patient with BPY2_1, BPY2_2, and BPY2_4 probe duplications ( BPY2 gene). male infertility Y chromosome cytogenetics karyotype multiplex polymerase chain reaction duplicate genes recurrent abortion Figures Figure 1 Figure 2 Introduction Infertility is the inability of a sexually active couple to generate and maintain a pregnancy that results in a live foetus after trying for one year [ 1 ]. Male infertility (MI) accounts for approximately 30% of cases of marital reproductive failure [ 2 ], thus justifying its investigation. The diagnosis of MI involves a series of laboratory and imaging tests. Among the genetic tests available, the G-banding karyotype is considered a valid technique for identifying numerical and structural alterations greater than 5 Mb [ 3 ]. However, as a diagnostic limitation, the G-banded karyotype does not allow the identification of minor DNA alterations. On the long arm of the Y chromosome, for instance, the azoospermia factor (AZF) regions have multiple genes associated with fertility [ 4 ] that are susceptible to microdeletion and/or microduplication due to their ampliconic sequences organized as palindromes prone to nonallelic homologous recombination [ 5 ]. In addition to pregestational infertility, which is related to failure to conceive, including azoospermic and oligozoospermic men, there is gestational infertility when the couple is able to conceive but the embryo/foetus is lost [ 6 ]. Thus, in addition to studying the genotype-phenotype relationship of patients with spermatic failure, it is also important to evaluate cases of repeated abortion presenting normal spermograms. There is strong evidence that normozoospermia and fertility are not synonymous [ 7 ]. In the present study, we analysed data from infertile patients who attended our medical genetics unit for 14 years (2006–2019). Initial screening was performed using classical cytogenetics, and in selected cases, multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) was performed to investigate single gene microduplications/microdeletions on the Y chromosome to assist in the genetic and infertility counselling of patients who seek, in a public service, to undergo assisted reproduction procedures. Subjects and Methods Genetic Service The medical genetics unit of the Hospital do Servidor Público do Estado de São Paulo in Instituto de Assistência Médica do Servidor Público do Estado (HSPE-IAMSPE) comprises a general genetic ambulatory and a cytogenetic laboratory. Due to this general service, suspected cases of genetic problems are referred from different units of the hospital, such as paediatrics, oncology, gynaecology, urology, and assisted reproduction. In 2006, the centre had three medical geneticists who provided genetic diagnosis and counselling and requested exams to be performed in our cytogenetics laboratory or in affiliated units. Over time, the outpatient clinic was reduced due to limited resources, and since 2014, only one medical geneticist provided care up to 2019. When that person retired, and the vacancy was not restored. Patients We selected 151 patients with MI who consecutively attended the Medical Genetics Service of HSPE - IAMSPE from January 1, 2006, to November 30, 2019. These patients were aged between 27 and 49 years. These patients/couples were referred by the gynaecology, urology, and assisted reproduction units of the hospital with the complaint of marital infertility of unknown aetiology after performing clinical, imaging, and laboratory tests. The female factor for infertility was excluded. All couples underwent karyotypic analysis. This study retrospectively analysed the medical records of infertile patients between 2006 and 2017. Additional data were obtained from exams performed between 2018 and 2019. In total, 15 patients were selected for Y chromosome genomic investigation by MLPA and/or PCR-STS methods. Among these patients, we included pregestational infertile men (with azoospermia or oligozoospermia) and gestational infertile men (with a history of recurrent miscarriages). Eligibility criteria for the genomic investigation encompassed couples that did not have a successful full-term pregnancy by natural methods for a minimum of one year. The men had a normal karyotype, sex hormone patterns within the reference values, and at least one spermogram. Their wives/partners had exams showing normal features of the reproductive system, a normal karyotype, and adequate hormonal patterns without any apparent or diagnosed cause that would justify marital infertility. Some of these women reported the birth of children with a previous partner. Exclusion criteria for genomic analysis consisted of patients presenting 1) chromosomal alterations identified by karyotype examination, 2) obstruction of the urogenital pathways, 3) previous disease that could justify infertility, such as varicocele, mumps, or any other condition indicated by the urologist, 4) no sperm test, and 5) normozoospermia, without the occurrence of recurrent spontaneous abortions. In addition, we excluded patients selected for cytogenetic screening but could not be contacted for genomic analysis (in the retrospective selection of patients: between 2006 and 2017). The research technical flowchart is presented in Fig. 1 . We carefully investigated patients before cytogenomic evaluation on their clinical condition, age, reproductive methods, success or failure, and spontaneous and recurrent abortions. All procedures were carried out in accordance with the Declaration of Helsinki. Methods We performed cytogenetic analysis using the Wright G-banding technique from peripheral blood lymphocyte culture. At least 20 metaphases were analysed for each patient using light microscopy. We performed genomic analysis by the MLPA technique using the SALSA MLPA probe-mix P360 version B1 and PCR-STS in an affiliated laboratory to evaluate, AZFa, AZFb, and AZFc regions (sY84, sY86, sY127, sY134, sY254, and sY255). Results and Discussion Karyotype Analysis Out of the 151 patients, 13 male patients presented with chromosomal abnormalities. Of these abnormalities, two were numerical alterations and 11 were structural anomalies or polymorphisms. Klinefelter syndrome (KS) is the most frequently identified genetic cause of MI [ 8 ]. We detected this syndrome in two patients with numerical chromosomal alterations: one was pure Klinefelter with a 47,XXY karyotype, and the other was a mosaic 47,XXY[46]/46,XY[04] karyotype. Amongst the structural anomalies observed, the chromosome 9 inversion was found in seven patients with karyotype 46,XY,inv(9)(p12;q13)[ 20 ]. The other structural alterations were 46,XX[100], 46,XY,t(3;4)(q13;q34)[ 20 ], 46,XY,t(X;3)(p22;p11)[ 20 ], and 46,XY,inv(Y)[ 20 ], each of which was identified in one different patient. Structural chromosomal alterations, such as reciprocal translocations, Robertsonian translocations, and chromosome 9 inversions, play a significant role in MI, similar to polymorphic structural alterations that affect fertility [ 9 ]. Genomic Analysis Regarding the genomic analysis, of the 11 cases with sperm failure (pregestational infertility), six patients had azoospermia, four had oligozoospermia, and one had oligoasthenozoospermia. Among these 11 cases, one azoospermic patient presented a duplication of the CDY2B gene probes (CDY2B_1 and CDY2B _2) analysed by the MLPA technique (Fig. 2 A). Another azoospermic patient presented a partial microdeletion of the AZFc region detected by PCR-STS at loci sY254 and sY255 (in the BPY2 gene and RBMY2DP pseudogene region). Of the four cases with normozoospermia and miscarriages (gestational infertility), one patient had Y chromosome duplications in BPY2 gene probes. This patient presented two spermograms with normal concentrations (58.0 and 61.2 million/mL), motility (66 and 38%), and viability (70 and 79%). He had a history of approximately six years of reproductive failure and four spontaneous abortions, all of which occurred in the gestational period of 4 to 8 weeks. The patient presented duplications in three of the five probes of the BPY2 gene (Fig. 2 B). The clinical characteristics and genomic analysis of patients with alterations on the Y chromosome are shown in Table 1. Table 1 - Clinical characteristics and the altered genomic results of selected infertile patients. It is interesting to point out that the Y chromosome microduplications observed would not be detected using the gold standard method for genomic analysis, which is the PCR-STS technique [ 10 – 12 ]. Although Y chromosome gene microdeletions are well established as one of the most common causes of male infertility [ 13 ], the consequences of its microduplications have not yet been fully established [ 14 ]. Noordam et al. (2011) found decreased sperm count and motility in men with primary AZFc microduplications (no microdeletions) [ 11 ]. Johansson et al. (2015) suggested that microduplications of regions on the Y chromosome could interfere with fertility by altering gene dosage [ 15 ]. Singh et al. (2019) pointed out that an overdose of genes on the Y chromosome and on specific autosome regions may impair spermatogenesis [ 16 ]. The CDY2B gene variation is classified as a variant of uncertain significance (VUS) for spermatogenesis (score 0) by the Franklin tool database ( http://franklin.genoox.com ) due to a lack of information associated with the region of the variant using ACMG 2020 criteria [ 17 ]. Ghorbel et al. (2014) found a significant difference in spermatogenesis between infertile men with CDY1B gene deletions and fertile control individuals [ 18 ]. Machev et al. (2004) suggested a strong association between CDY1 gene deletions and infertility [ 19 ]. Thus, alterations in the CDY gene family may affect fertility. Although we cannot claim that the duplication of the CDY2B gene detected in the present study is related to azoospermia, we believe that this alteration may have an effect on spermatogenesis. An increase in histone hyperacetylation in sperm DNA, a known function of the CDY2B gene [ 20 ], may promote increased chromatin decondensation, disrupting correct DNA condensation during meiosis. Previous studies have found significant differences between infertile and fertile patients, indicating that Y chromosome microduplications may interfere with normal spermatogenesis [ 14 , 21 – 23 ]. However, those reports on azoospermic and oligozoospermic men described partial AZFc microduplications of regions including several genes in clusters. Moreover, normozoospermic infertile men, such as those with a history of repeated spontaneous abortions, are not frequently reported in the literature. It is now known that male infertility is related to several factors, in addition to abnormal seminal parameters, since normozoospermic men may still be infertile [ 9 ]. The BPY2 gene variation is regarded as a VUS for infertility by the Franklin tool due to a lack of information associated with the region of the variant using ACMG criteria [ 17 ]. One of our patients presenting partial duplication of the BPY2 gene had a history of miscarriage. Although we cannot say that duplications of the BPY2 gene directly affect male fertility, we cannot exclude the possibility that this situation occurs because this gene encodes a protein that interacts with ubiquitin ligase E3A (UBE3A) in the testes [ 24 ]. UBE3A promotes ubiquitination, a normal process that catalyses molecules that are no longer needed and plays a crucial role in metabolizing substituted histones in late spermatids. In addition, microparticles formed during embryogenesis need to be degraded by ubiquitination for proper embryonic development [ 25 ]. Thus, if the catalysis process occurs in excess, it may affect the course of gestation [ 25 ] and result in embryo defects and/or spontaneous abortion. The patient with this alteration showed duplication of three out of the five BPY2 gene probes used in the MLPA kit, revealing partial duplication of this gene. Microduplications in regions of the BPY2 gene identified in the present study may be related to embryo development blockage. Another patient presented a microdeletion in the BPY2 gene region, which may be associated with his azoospermia. This is because the absence of the BPY2 gene may affect male fertility by decreasing catalysis processes during spermatogenesis. Additionally, the lack of degradation of histones released in the process results in the absence of adequate sperm formation or maturation, as observed in a testicular biopsy of this patient (data not shown). In addition to the Y chromosome, several autosomal genes are crucial for human spermatogenesis [ 26 ]. Even so, autosomal genes essential for male fertility may be modulated or regulated by genes on the Y chromosome, as their functions are not yet fully understood. In this way, the microduplications on the Y chromosome described here may have a regulatory role in other genes, causing spermatogenesis failure and repeated abortions. More studies on gene expression and sperm DNA fragmentation are necessary to understand the individual role of Y chromosome genes in male fertility. Epigenetic alterations and noncoding RNAs may also interfere with male fertility [ 27 ]. DNA methylation profile and noncoding RNA expression studies would help to clarify specific male infertility conditions. Moreover, the recent report of the entire sequence of the Y chromosome highlights the relevance of more studies of genomic variants related to male infertility, using the new reference data set T2T-CHM13 + Y [ 28 ]. In conclusion, the present work reinforces the importance of performing a cytogenomic investigation in a governmental institution as a routine practice. It represents an important diagnostic tool to aid in the genetic and infertility counselling of patients undergoing assisted reproduction procedures. Limitations The limitations of this study are the small number of samples for genomic investigation and the limited number of techniques used for genomic analysis. Abbreviations AZF azoospermia factor HSPE - IAMSPE Hospital do Servidor Público do Estado de São Paulo in Instituto de Assistência Médica do Servidor Público do Estado KS Klinefelter syndrome MI male infertility MLPA multiplex ligation-dependent probe amplification PCR-STS polymerase chain reaction of sequence-tagged sites VUS variant of uncertain significance Declarations Ethics approval and consent to participate The protocol for the research for genomic analysis project was approved by the Instituto de Assistência Médica ao Servidor Público Estadual (IAMSPE) Ethics Committee, São Paulo, Brasil under the number CAAE 79306117.7.0000.5463, and written informed consent was obtained from all participants. The retrospective study of medical records was approved under the number CAAE 36467620.1.0000.5463 by the IAMSPE Ethics Committee. All the procedures were followed in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1964 and its later amendments. Consent for publication Not applicate Availability of data and materials Data can be reached by contacting the corresponding author. Competing interests The authors declare they have no competing interests. Funding No external funding was available for the study. Author’s contributions ATD, LDK and JW conceived and designed the study. MRGA recruited the subjects for MLPA tests and with AB gathered patient data and undertook karyotypes. AB analysed and interpreted the karyotypes. FARM, GFSC, AMN and MRGA performed MLPA experiments and helped to analyse and interpret MLPA results. ATD with EAZ and BW analysed and interpreted MLPA results. MRGA and ABM performed critical discussion and text draft and ATD, ABM and LDK contributed to drafting the final version of the manuscript. All authors contributed to this research, revising it critically, approved the final manuscript to the submission and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Acknowledgements We are grateful to all patients and volunteers for their help and participation in the study. We thank the Hospital do Servidor Público do Estado de São Paulo and Faculdade de Medicina da Universidade de São Paulo. References Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al. International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009. Fertil Steril. 2009;92(5):1520-4. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37. 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Partial AZFc duplications not deletions are associated with male infertility in the Yi population of Yunnan Province, China. J Zhejiang Univ Sci B. 2013;14(9):807-15. Yang B, Ma YY, Liu YQ, Li L, Yang D, Tu WL, et al. Common AZFc structure may possess the optimal spermatogenesis efficiency relative to the rearranged structures mediated by non-allele homologous recombination. Sci Rep. 2015;5:10551. Wong EY, Tse JY, Yao KM, Tam PC, Yeung WS. VCY2 protein interacts with the HECT domain of ubiquitin-protein ligase E3A. Biochem Biophys Res Commun. 2002;296(5):1104-11. Alijotas-Reig J, Garrido-Gimenez C. Current concepts and new trends in the diagnosis and management of recurrent miscarriage. Obstet Gynecol Surv. 2013;68(6):445-66. Okutman O, Rhouma MB, Benkhalifa M, Muller J, Viville S. Genetic evaluation of patients with non-syndromic male infertility. J Assist Reprod Genet. 2018;35(11):1939-51. Gunes S, Arslan MA, Hekim GNT, Asci R. The role of epigenetics in idiopathic male infertility. J Assist Reprod Genet. 2016;33(5):553-69. Rhie A, Nurk S, Cechova M, Hoyt SJ, Taylor DJ, Altemose N, et al. The complete sequence of a human Y chromosome. Nature. 2023. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 05 Mar, 2024 Read the published version in BMC Research Notes → Version 1 posted Editorial decision: Major revision 08 Sep, 2023 Submission checks completed at journal 05 Sep, 2023 Editor assigned by journal 05 Sep, 2023 First submitted to journal 01 Sep, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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15:44:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3317746/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3317746/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-024-06710-1","type":"published","date":"2024-03-05T15:01:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":42841801,"identity":"425984e3-f89b-477a-aa55-25545baed7ca","added_by":"auto","created_at":"2023-09-08 16:59:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowchart containing the complete casuistry of all patients evaluated in the research\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3317746/v1/bd5cd7f5904fee0cd52489cf.png"},{"id":42841805,"identity":"00c8c62f-cf99-4846-bb5f-acd9fdbdbbd5","added_by":"auto","created_at":"2023-09-08 16:59:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":909959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLPA altered results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eGenomic analysis of the pregestational infertile patient with \u003cem\u003eCDY2B\u003c/em\u003e gene duplication. Histogram of the results of each probe containing the CDY2B_1 and CDY2B_2 probes above the reference values. MLPA is a comparative test, and the analysis was normalized to the number of suitable copies (x2) considering that all probes presented as duplicates have more than two copies of the gene. \u003cstrong\u003eB) \u003c/strong\u003eGenomic analysis of the gestational infertile patient with \u003cem\u003eBPY2\u003c/em\u003e gene duplication. Histogram of the results of each probe containing probes BPY2_1, BPY2_2 and BPY_4 above the reference values. MLPA analysis was normalized to the number of suitable copies (x3) considering that all probes presented as duplicates have more than three gene copies – Coffalyser software.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3317746/v1/e00dc98be00a678254df47fb.png"},{"id":52431871,"identity":"571bd765-05df-41cc-aadc-d317777b5781","added_by":"auto","created_at":"2024-03-11 15:08:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1427907,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3317746/v1/c00335f3-ebae-4ede-af46-f795b5861973.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytogenomic investigation in 151 Brazilian infertile male patients: Experience of 14 years in a public genetic service","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfertility is the inability of a sexually active couple to generate and maintain a pregnancy that results in a live foetus after trying for one year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Male infertility (MI) accounts for approximately 30% of cases of marital reproductive failure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], thus justifying its investigation.\u003c/p\u003e \u003cp\u003eThe diagnosis of MI involves a series of laboratory and imaging tests. Among the genetic tests available, the G-banding karyotype is considered a valid technique for identifying numerical and structural alterations greater than 5 Mb [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, as a diagnostic limitation, the G-banded karyotype does not allow the identification of minor DNA alterations. On the long arm of the Y chromosome, for instance, the azoospermia factor (AZF) regions have multiple genes associated with fertility [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] that are susceptible to microdeletion and/or microduplication due to their ampliconic sequences organized as palindromes prone to nonallelic homologous recombination [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to pregestational infertility, which is related to failure to conceive, including azoospermic and oligozoospermic men, there is gestational infertility when the couple is able to conceive but the embryo/foetus is lost [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, in addition to studying the genotype-phenotype relationship of patients with spermatic failure, it is also important to evaluate cases of repeated abortion presenting normal spermograms. There is strong evidence that normozoospermia and fertility are not synonymous [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, we analysed data from infertile patients who attended our medical genetics unit for 14 years (2006\u0026ndash;2019). Initial screening was performed using classical cytogenetics, and in selected cases, multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) was performed to investigate single gene microduplications/microdeletions on the Y chromosome to assist in the genetic and infertility counselling of patients who seek, in a public service, to undergo assisted reproduction procedures.\u003c/p\u003e"},{"header":"Subjects and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eGenetic Service\u003c/h2\u003e \u003cp\u003eThe medical genetics unit of the Hospital do Servidor P\u0026uacute;blico do Estado de S\u0026atilde;o Paulo in Instituto de Assist\u0026ecirc;ncia M\u0026eacute;dica do Servidor P\u0026uacute;blico do Estado (HSPE-IAMSPE) comprises a general genetic ambulatory and a cytogenetic laboratory. Due to this general service, suspected cases of genetic problems are referred from different units of the hospital, such as paediatrics, oncology, gynaecology, urology, and assisted reproduction.\u003c/p\u003e \u003cp\u003e In 2006, the centre had three medical geneticists who provided genetic diagnosis and counselling and requested exams to be performed in our cytogenetics laboratory or in affiliated units. Over time, the outpatient clinic was reduced due to limited resources, and since 2014, only one medical geneticist provided care up to 2019. When that person retired, and the vacancy was not restored.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eWe selected 151 patients with MI who consecutively attended the Medical Genetics Service of HSPE - IAMSPE from January 1, 2006, to November 30, 2019. These patients were aged between 27 and 49 years. These patients/couples were referred by the gynaecology, urology, and assisted reproduction units of the hospital with the complaint of marital infertility of unknown aetiology after performing clinical, imaging, and laboratory tests. The female factor for infertility was excluded. All couples underwent karyotypic analysis.\u003c/p\u003e \u003cp\u003eThis study retrospectively analysed the medical records of infertile patients between 2006 and 2017. Additional data were obtained from exams performed between 2018 and 2019.\u003c/p\u003e \u003cp\u003eIn total, 15 patients were selected for Y chromosome genomic investigation by MLPA and/or PCR-STS methods. Among these patients, we included pregestational infertile men (with azoospermia or oligozoospermia) and gestational infertile men (with a history of recurrent miscarriages).\u003c/p\u003e \u003cp\u003eEligibility criteria for the genomic investigation encompassed couples that did not have a successful full-term pregnancy by natural methods for a minimum of one year. The men had a normal karyotype, sex hormone patterns within the reference values, and at least one spermogram. Their wives/partners had exams showing normal features of the reproductive system, a normal karyotype, and adequate hormonal patterns without any apparent or diagnosed cause that would justify marital infertility. Some of these women reported the birth of children with a previous partner.\u003c/p\u003e \u003cp\u003eExclusion criteria for genomic analysis consisted of patients presenting 1) chromosomal alterations identified by karyotype examination, 2) obstruction of the urogenital pathways, 3) previous disease that could justify infertility, such as varicocele, mumps, or any other condition indicated by the urologist, 4) no sperm test, and 5) normozoospermia, without the occurrence of recurrent spontaneous abortions. In addition, we excluded patients selected for cytogenetic screening but could not be contacted for genomic analysis (in the retrospective selection of patients: between 2006 and 2017).\u003c/p\u003e \u003cp\u003eThe research technical flowchart is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe carefully investigated patients before cytogenomic evaluation on their clinical condition, age, reproductive methods, success or failure, and spontaneous and recurrent abortions. All procedures were carried out in accordance with the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cp\u003eWe performed cytogenetic analysis using the Wright G-banding technique from peripheral blood lymphocyte culture. At least 20 metaphases were analysed for each patient using light microscopy.\u003c/p\u003e \u003cp\u003eWe performed genomic analysis by the MLPA technique using the SALSA MLPA probe-mix P360 version B1 and PCR-STS in an affiliated laboratory to evaluate, AZFa, AZFb, and AZFc regions (sY84, sY86, sY127, sY134, sY254, and sY255).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKaryotype Analysis\u003c/h2\u003e \u003cp\u003eOut of the 151 patients, 13 male patients presented with chromosomal abnormalities. Of these abnormalities, two were numerical alterations and 11 were structural anomalies or polymorphisms.\u003c/p\u003e \u003cp\u003eKlinefelter syndrome (KS) is the most frequently identified genetic cause of MI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. We detected this syndrome in two patients with numerical chromosomal alterations: one was pure Klinefelter with a 47,XXY karyotype, and the other was a mosaic 47,XXY[46]/46,XY[04] karyotype.\u003c/p\u003e \u003cp\u003eAmongst the structural anomalies observed, the chromosome 9 inversion was found in seven patients with karyotype 46,XY,inv(9)(p12;q13)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The other structural alterations were 46,XX[100], 46,XY,t(3;4)(q13;q34)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], 46,XY,t(X;3)(p22;p11)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and 46,XY,inv(Y)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], each of which was identified in one different patient.\u003c/p\u003e \u003cp\u003eStructural chromosomal alterations, such as reciprocal translocations, Robertsonian translocations, and chromosome 9 inversions, play a significant role in MI, similar to polymorphic structural alterations that affect fertility [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGenomic Analysis\u003c/h2\u003e \u003cp\u003eRegarding the genomic analysis, of the 11 cases with sperm failure (pregestational infertility), six patients had azoospermia, four had oligozoospermia, and one had oligoasthenozoospermia. Among these 11 cases, one azoospermic patient presented a duplication of the \u003cem\u003eCDY2B\u003c/em\u003e gene probes (CDY2B_1 and CDY2B _2) analysed by the MLPA technique (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Another azoospermic patient presented a partial microdeletion of the AZFc region detected by PCR-STS at loci \u003cem\u003esY254\u003c/em\u003e and \u003cem\u003esY255\u003c/em\u003e (in the \u003cem\u003eBPY2\u003c/em\u003e gene and \u003cem\u003eRBMY2DP\u003c/em\u003e pseudogene region). Of the four cases with normozoospermia and miscarriages (gestational infertility), one patient had Y chromosome duplications in \u003cem\u003eBPY2\u003c/em\u003e gene probes. This patient presented two spermograms with normal concentrations (58.0 and 61.2\u0026nbsp;million/mL), motility (66 and 38%), and viability (70 and 79%). He had a history of approximately six years of reproductive failure and four spontaneous abortions, all of which occurred in the gestational period of 4 to 8 weeks. The patient presented duplications in three of the five probes of the \u003cem\u003eBPY2\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThe clinical characteristics and genomic analysis of patients with alterations on the Y chromosome are shown in Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1 - Clinical characteristics and the altered genomic results of selected infertile patients.\u003c/b\u003e \u003c/p\u003e\u003cp\u003e\u003cimg 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\" height=\"215\" width=\"638\"\u003e\u003c/p\u003e\u003cp\u003eIt is interesting to point out that the Y chromosome microduplications observed would not be detected using the gold standard method for genomic analysis, which is the PCR-STS technique [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough Y chromosome gene microdeletions are well established as one of the most common causes of male infertility [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], the consequences of its microduplications have not yet been fully established [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNoordam et al. (2011) found decreased sperm count and motility in men with primary AZFc microduplications (no microdeletions) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Johansson et al. (2015) suggested that microduplications of regions on the Y chromosome could interfere with fertility by altering gene dosage [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Singh et al. (2019) pointed out that an overdose of genes on the Y chromosome and on specific autosome regions may impair spermatogenesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eCDY2B\u003c/em\u003e gene variation is classified as a variant of uncertain significance (VUS) for spermatogenesis (score 0) by the Franklin tool database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://franklin.genoox.com\u003c/span\u003e\u003cspan address=\"http://franklin.genoox.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) due to a lack of information associated with the region of the variant using ACMG 2020 criteria [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Ghorbel et al. (2014) found a significant difference in spermatogenesis between infertile men with \u003cem\u003eCDY1B\u003c/em\u003e gene deletions and fertile control individuals [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Machev et al. (2004) suggested a strong association between \u003cem\u003eCDY1\u003c/em\u003e gene deletions and infertility [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, alterations in the \u003cem\u003eCDY\u003c/em\u003e gene family may affect fertility. Although we cannot claim that the duplication of the \u003cem\u003eCDY2B\u003c/em\u003e gene detected in the present study is related to azoospermia, we believe that this alteration may have an effect on spermatogenesis. An increase in histone hyperacetylation in sperm DNA, a known function of the \u003cem\u003eCDY2B\u003c/em\u003e gene [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], may promote increased chromatin decondensation, disrupting correct DNA condensation during meiosis.\u003c/p\u003e \u003cp\u003ePrevious studies have found significant differences between infertile and fertile patients, indicating that Y chromosome microduplications may interfere with normal spermatogenesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, those reports on azoospermic and oligozoospermic men described partial AZFc microduplications of regions including several genes in clusters. Moreover, normozoospermic infertile men, such as those with a history of repeated spontaneous abortions, are not frequently reported in the literature. It is now known that male infertility is related to several factors, in addition to abnormal seminal parameters, since normozoospermic men may still be infertile [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBPY2\u003c/em\u003e gene variation is regarded as a VUS for infertility by the Franklin tool due to a lack of information associated with the region of the variant using ACMG criteria [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. One of our patients presenting partial duplication of the \u003cem\u003eBPY2\u003c/em\u003e gene had a history of miscarriage. Although we cannot say that duplications of the \u003cem\u003eBPY2\u003c/em\u003e gene directly affect male fertility, we cannot exclude the possibility that this situation occurs because this gene encodes a protein that interacts with ubiquitin ligase E3A (UBE3A) in the testes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. UBE3A promotes ubiquitination, a normal process that catalyses molecules that are no longer needed and plays a crucial role in metabolizing substituted histones in late spermatids. In addition, microparticles formed during embryogenesis need to be degraded by ubiquitination for proper embryonic development [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Thus, if the catalysis process occurs in excess, it may affect the course of gestation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and result in embryo defects and/or spontaneous abortion. The patient with this alteration showed duplication of three out of the five \u003cem\u003eBPY2\u003c/em\u003e gene probes used in the MLPA kit, revealing partial duplication of this gene. Microduplications in regions of the \u003cem\u003eBPY2\u003c/em\u003e gene identified in the present study may be related to embryo development blockage.\u003c/p\u003e \u003cp\u003eAnother patient presented a microdeletion in the \u003cem\u003eBPY2\u003c/em\u003e gene region, which may be associated with his azoospermia. This is because the absence of the \u003cem\u003eBPY2\u003c/em\u003e gene may affect male fertility by decreasing catalysis processes during spermatogenesis. Additionally, the lack of degradation of histones released in the process results in the absence of adequate sperm formation or maturation, as observed in a testicular biopsy of this patient (data not shown).\u003c/p\u003e \u003cp\u003eIn addition to the Y chromosome, several autosomal genes are crucial for human spermatogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Even so, autosomal genes essential for male fertility may be modulated or regulated by genes on the Y chromosome, as their functions are not yet fully understood. In this way, the microduplications on the Y chromosome described here may have a regulatory role in other genes, causing spermatogenesis failure and repeated abortions. More studies on gene expression and sperm DNA fragmentation are necessary to understand the individual role of Y chromosome genes in male fertility. Epigenetic alterations and noncoding RNAs may also interfere with male fertility [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. DNA methylation profile and noncoding RNA expression studies would help to clarify specific male infertility conditions. Moreover, the recent report of the entire sequence of the Y chromosome highlights the relevance of more studies of genomic variants related to male infertility, using the new reference data set T2T-CHM13\u0026thinsp;+\u0026thinsp;Y [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, the present work reinforces the importance of performing a cytogenomic investigation in a governmental institution as a routine practice. It represents an important diagnostic tool to aid in the genetic and infertility counselling of patients undergoing assisted reproduction procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThe limitations of this study are the small number of samples for genomic investigation and the limited number of techniques used for genomic analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAZF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eazoospermia factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSPE - IAMSPE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHospital do Servidor P\u0026uacute;blico do Estado de S\u0026atilde;o Paulo in Instituto de Assist\u0026ecirc;ncia M\u0026eacute;dica do Servidor P\u0026uacute;blico do Estado\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKlinefelter syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emale infertility\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emultiplex ligation-dependent probe amplification\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR-STS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction of sequence-tagged sites\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVUS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evariant of uncertain significance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol for the research for genomic analysis project was approved by the Instituto de Assistência Médica ao Servidor Público Estadual (IAMSPE) Ethics Committee, São Paulo, Brasil under the number CAAE 79306117.7.0000.5463, and written informed consent was obtained from all participants. The retrospective study of\u0026nbsp;medical records\u0026nbsp;was approved under the number CAAE 36467620.1.0000.5463 by the IAMSPE Ethics Committee. All the procedures were followed in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1964 and its later amendments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData can be reached by contacting the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo external funding was available for the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eATD, LDK and JW conceived and designed the study. MRGA recruited the subjects for MLPA tests and with AB gathered patient data and undertook karyotypes. AB analysed and interpreted the karyotypes. FARM, GFSC, AMN and MRGA performed MLPA experiments and helped to analyse and interpret MLPA results. ATD with EAZ and BW analysed and interpreted MLPA results. MRGA and ABM performed critical discussion and text draft and ATD, ABM and LDK contributed to drafting the final version of the manuscript.\u0026nbsp;All authors contributed to this research,\u0026nbsp;revising it critically,\u0026nbsp;approved the final manuscript to the\u0026nbsp;submission and\u0026nbsp;agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to all patients and volunteers for their help and participation in the study. We thank the Hospital do Servidor Público do Estado de São Paulo and Faculdade de Medicina da Universidade de São Paulo.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al. International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009. Fertil Steril. 2009;92(5):1520-4.\u003c/li\u003e\n\u003cli\u003eAgarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37.\u003c/li\u003e\n\u003cli\u003eGouas L, Goumy C, Veronese L, Tchirkov A, Vago P. Gene dosage methods as diagnostic tools for the identification of chromosome abnormalities. Pathol Biol (Paris). 2008;56(6):345-53.\u003c/li\u003e\n\u003cli\u003eLin YM, Lin YH, Teng YN, Hsu CC, Shinn-Nan Lin J, Kuo PL. Gene-based screening for Y chromosome deletions in Taiwanese men presenting with spermatogenic failure. Fertil Steril. 2002;77(5):897-903.\u003c/li\u003e\n\u003cli\u003eKrausz C, Chianese C, Giachini C, Guarducci E, Laface I, Forti G. The Y chromosome-linked copy number variations and male fertility. J Endocrinol Invest. 2011;34(5):376-82.\u003c/li\u003e\n\u003cli\u003eLi D, Zhang H, Wang R, Zhu H, Li L, Liu R. Chromosomal abnormalities in men with pregestational and gestational infertility in northeast China. J Assist Reprod Genet. 2012;29(8):829-36.\u003c/li\u003e\n\u003cli\u003eKrausz C. Male infertility: pathogenesis and clinical diagnosis. Best Pract Res Clin Endocrinol Metab. 2011;25(2):271-85.\u003c/li\u003e\n\u003cli\u003eRutherford TR, P.; Matson, P. Klinefelter syndrome: phenotype, testicular function and infertility treatment. J Reprod Biotechnol Fertil. 2019;8:66-79.\u003c/li\u003e\n\u003cli\u003ePractice Committee of the American Society for Reproductive M. Diagnostic evaluation of the infertile male: a committee opinion. Fertil Steril. 2015;103(3):e18-25.\u003c/li\u003e\n\u003cli\u003eKrausz C, Hoefsloot L, Simoni M, Tuttelmann F, European Academy of A, European Molecular Genetics Quality N. EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 2013. Andrology. 2014;2(1):5-19.\u003c/li\u003e\n\u003cli\u003eNoordam MJ, Westerveld GH, Hovingh SE, van Daalen SK, Korver CM, van der Veen F, et al. Gene copy number reduction in the azoospermia factor c (AZFc) region and its effect on total motile sperm count. Hum Mol Genet. 2011;20(12):2457-63.\u003c/li\u003e\n\u003cli\u003eForesta C, Moro E, Ferlin A. Y chromosome microdeletions and alterations of spermatogenesis. Endocr Rev. 2001;22(2):226-39.\u003c/li\u003e\n\u003cli\u003eZhang YS, Dai RL, Wang RX, Zhang HG, Chen S, Liu RZ. Analysis of Y chromosome microdeletion in 1738 infertile men from northeastern China. Urology. 2013;82(3):584-8.\u003c/li\u003e\n\u003cli\u003eLu C, Jiang J, Zhang R, Wang Y, Xu M, Qin Y, et al. Gene copy number alterations in the azoospermia-associated AZFc region and their effect on spermatogenic impairment. Mol Hum Reprod. 2014;20(9):836-43.\u003c/li\u003e\n\u003cli\u003eJohansson MM, Van Geystelen A, Larmuseau MH, Djurovic S, Andreassen OA, Agartz I, et al. Microarray Analysis of Copy Number Variants on the Human Y Chromosome Reveals Novel and Frequent Duplications Overrepresented in Specific Haplogroups. PLoS One. 2015;10(8):e0137223.\u003c/li\u003e\n\u003cli\u003eSingh V, Bala R, Chakraborty A, Rajender S, Trivedi S, Singh K. Duplications in 19p13.3 are associated with male infertility. J Assist Reprod Genet. 2019;36(10):2171-9.\u003c/li\u003e\n\u003cli\u003eRiggs ER, Andersen EF, Cherry AM, Kantarci S, Kearney H, Patel A, et al. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet Med. 2020;22(2):245-57.\u003c/li\u003e\n\u003cli\u003eGhorbel M, Baklouti-Gargouri S, Keskes R, Chakroun N, Sellami A, Fakhfakh F, et al. Deletion of CDY1b copy of Y chromosome CDY1 gene is a risk factor of male infertility in Tunisian men. Gene. 2014;548(2):251-5.\u003c/li\u003e\n\u003cli\u003eMachev N, Saut N, Longepied G, Terriou P, Navarro A, Levy N, et al. Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility. J Med Genet. 2004;41(11):814-25.\u003c/li\u003e\n\u003cli\u003eLahn BT, Tang ZL, Zhou J, Barndt RJ, Parvinen M, Allis CD, et al. Previously uncharacterized histone acetyltransferases implicated in mammalian spermatogenesis. Proc Natl Acad Sci U S A. 2002;99(13):8707-12.\u003c/li\u003e\n\u003cli\u003eLin YW, Hsu LC, Kuo PL, Huang WJ, Chiang HS, Yeh SD, et al. Partial duplication at AZFc on the Y chromosome is a risk factor for impaired spermatogenesis in Han Chinese in Taiwan. Hum Mutat. 2007;28(5):486-94.\u003c/li\u003e\n\u003cli\u003eYe JJ, Ma L, Yang LJ, Wang JH, Wang YL, Guo H, et al. Partial AZFc duplications not deletions are associated with male infertility in the Yi population of Yunnan Province, China. J Zhejiang Univ Sci B. 2013;14(9):807-15.\u003c/li\u003e\n\u003cli\u003eYang B, Ma YY, Liu YQ, Li L, Yang D, Tu WL, et al. Common AZFc structure may possess the optimal spermatogenesis efficiency relative to the rearranged structures mediated by non-allele homologous recombination. Sci Rep. 2015;5:10551.\u003c/li\u003e\n\u003cli\u003eWong EY, Tse JY, Yao KM, Tam PC, Yeung WS. VCY2 protein interacts with the HECT domain of ubiquitin-protein ligase E3A. Biochem Biophys Res Commun. 2002;296(5):1104-11.\u003c/li\u003e\n\u003cli\u003eAlijotas-Reig J, Garrido-Gimenez C. Current concepts and new trends in the diagnosis and management of recurrent miscarriage. Obstet Gynecol Surv. 2013;68(6):445-66.\u003c/li\u003e\n\u003cli\u003eOkutman O, Rhouma MB, Benkhalifa M, Muller J, Viville S. Genetic evaluation of patients with non-syndromic male infertility. J Assist Reprod Genet. 2018;35(11):1939-51.\u003c/li\u003e\n\u003cli\u003eGunes S, Arslan MA, Hekim GNT, Asci R. The role of epigenetics in idiopathic male infertility. J Assist Reprod Genet. 2016;33(5):553-69.\u003c/li\u003e\n\u003cli\u003eRhie A, Nurk S, Cechova M, Hoyt SJ, Taylor DJ, Altemose N, et al. The complete sequence of a human Y chromosome. Nature. 2023.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"male infertility, Y chromosome, cytogenetics, karyotype, multiplex polymerase chain reaction, duplicate genes, recurrent abortion","lastPublishedDoi":"10.21203/rs.3.rs-3317746/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3317746/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eMale infertility accounts for approximately 30% of cases of reproductive failure. The characterization of genetic variants using cytogenomic techniques is essential for the adequate clinical management of these patients. We aimed to conduct a cytogenetic investigation of numerical and structural rearrangements and a genomic study of Y chromosome microdeletions/microduplications in infertile men derived from a single centre with over 14 years of experience.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe evaluated 151 infertile men in a transversal study using peripheral blood karyotypes and 15 patients with normal karyotypes through genomic investigation by multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) techniques. Out of the 151 patients evaluated by karyotype, 13 presented chromosomal abnormalities: 2 had numerical alterations, and 11 had structural chromosomal rearrangements. PCR-STS detected a \u003cem\u003eBPY2\u003c/em\u003e gene region and \u003cem\u003eRBMY2DP\u003c/em\u003e pseudogene region microdeletion in one patient. MLPA analysis allowed the identification of one patient with CDY2B_1 and CDY2B_2 probe duplications (\u003cem\u003eCDY2B\u003c/em\u003e gene) and one patient with BPY2_1, BPY2_2, and BPY2_4 probe duplications (\u003cem\u003eBPY2\u003c/em\u003e gene).\u003c/p\u003e","manuscriptTitle":"Cytogenomic investigation in 151 Brazilian infertile male patients: Experience of 14 years in a public genetic service","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-08 16:59:06","doi":"10.21203/rs.3.rs-3317746/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-09-08T14:45:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-09-05T14:13:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-05T14:13:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2023-09-01T15:36:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c59f5c1f-f1be-4172-9674-21e9539c7b7e","owner":[],"postedDate":"September 8th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-11T15:04:02+00:00","versionOfRecord":{"articleIdentity":"rs-3317746","link":"https://doi.org/10.1186/s13104-024-06710-1","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2024-03-05 15:01:09","publishedOnDateReadable":"March 5th, 2024"},"versionCreatedAt":"2023-09-08 16:59:06","video":"","vorDoi":"10.1186/s13104-024-06710-1","vorDoiUrl":"https://doi.org/10.1186/s13104-024-06710-1","workflowStages":[]},"version":"v1","identity":"rs-3317746","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3317746","identity":"rs-3317746","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}