e8dfe2cc34ea1f9077d563e5aa59ae26 hoaf049.pdf a79c145d90139e461c86b034f313e1b3d706cd9e hoaf049.pdf 408a941bfc7c8dd3e67ef7e3114fb0b3eebec68ed05b8f50cfa4c6100190db37 hoaf049.pdf Title: Genetic determinants of testicular sperm extraction outcomes: insights from a large multicentre study of men with non-obstructive azoospermia Subject: Doi: 10.1093/hropen/hoaf049 Human Reproduction Open, 2025, 3, 2025 Publication Date: 19/08/2025 Abstract STUDY QUESTION What is the diagnostic yield and the pre-testicular sperm extraction (TESE) prognostic value of a non-obstructive azoospermia (NOA)-specific virtual gene panel?SUMMARY ANSWER The diagnostic yield in our cohort was 6.1%, and by combining our data with published literature, we identified 11 genes compatible with testicular sperm production and 19 genes associated with no sperm retrieval in carriers of pathogenic (P) or likely pathogenic (LP) mutations.WHAT IS KNOWN ALREADY Azoospermia, the most severe form of male infertility, affects ∼1% of the male population, with TESE being the primary treatment option. However, in NOA, TESE fails in nearly 50% of cases and existing clinical parameters are unable to predict TESE failure. Over the past decade, next-generation sequencing (NGS) has identified several candidate NOA genes, but their diagnostic utility and impact on TESE outcomes have not been fully explored.STUDY DESIGN, SIZE AND DURATION A literature search was addressed to identify well-established NOA genes for designing a specific virtual gene panel for NOA. Our retrospective study analysed the diagnostic yield of the NGS-based virtual gene panel, comprising 145 genes, in 571 men affected by idiopathic NOA with known TESE outcomes. Subsequently, a second literature search was performed to identify carriers of LP/P variants in the genes where we identified mutations, focusing on individuals with known TESE outcomes. This approach allowed us to integrate the published data with our findings and predict a genotype–phenotype correlation between the affected genes and TESE success.PARTICIPANTS/MATERIALS, SETTINGS, METHODS 571 NOA patients with known TESE outcomes were recruited in two European and one Middle East centres. Variants were obtained from a whole-exome sequencing dataset and crossed with the 145 genes of the virtual gene panel. After a filtering process, variants were manually assessed and classified according to ACMG guidelines by using two methods: (i) In order to compare our data with previously published studies, we applied ACMG-AMP guidelines along with ClinGen recommendations used by other similar studies. (ii) A new approach was used to optimize ACMG-AMP guidelines with all ClinGen recommendations and incorporated NOA-specific rules addressing phenotypic, locus, and allelic heterogeneity. LP and P variants were confirmed by Sanger sequencing.MAIN RESULTS AND THE ROLE OF CHANCE By using the new variant classification approach adapted for NOA, we identified LP/P variants in 6.1% of patients, with a higher yield (9.4%) in cases with negative TESE outcomes and maturation arrest (11.7%). By integrating our findings with the literature, we highlight 19 genes recurrently associated with negative TESE outcomes and 11 genes associated with positive sperm retrieval either in the testis or in semen. TESE is recommended for patients with LP or P variants in the 11 specific genes. Notably, six of these genes are located on the X chromosome, therefore, these variants will be obligatorily transmitted to daughters, and potentially increase the risk of NOA-related infertility in male offspring. We observed that nine genes, in which we identified LP/P variants, have been previously described in individuals with premature ovarian insufficiency (POI). Of these, eight were associated with negative TESE outcomes in men. Furthermore, we propose seven additional genes mutated in our cohort of NOA patients as novel POI candidates. These genes have not yet been considered as POI candidates, but they result in female infertility when knocked out in mouse models.LARGE SCALE DATA LP/P variants have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/).LIMITATIONS, REASONS FOR CAUTION NOA is genetically heterogeneous, and our panel excludes those genes which were reported only in a single subject or single family. Although this can limit the diagnostic yield in our study, it ensures that only genes with clear relationship with NOA have been analysed. While in our cohort TESE outcomes are known for all patients, this information is often not available for mutation carriers in the published studies. Consequently, the total number of patients with P variants in the same gene remains relatively low, limiting our final conclusions. However, even if the number of carriers of genes associated with positive sperm retrieval is relatively low, it does not constrain our conclusions regarding TESE prediction. On the other hand, caution is warranted for genes linked to negative TESE outcomes, except for TEX11, SYCE1, and MSH4, each of which have 10 or more reported TESE-negative cases.WIDER IMPLICATIONS OF THE FINDINGS Our study was performed on the largest available NOA cohort with known TESE outcomes. It not only provides an estimate on the diagnostic potential of a NOA-specific virtual gene panel, but it also advances the understanding of genetic factors influencing TESE outcomes. Half of the genes mutated in our study and presenting TESE-positive outcomes are already informative for clinical decision-making. The observed genotype–phenotype correlations may help in personalized decision-making prior to TESE, in order to undergo the procedure or to avoid unnecessary invasive treatment. It provides valuable insights that can inform clinical management strategies and potentially offer personalized treatments based on genetic profiles. The use of two different variant classification methods highlights that previous studies may have over-estimated the diagnostic yield, underscoring the need for a standardized variant classification approach addressed specifically to male infertility. Our study also emphasizes the overlap between NOA- and POI-associated genes, which has important clinical implications for genetic counselling of female siblings of affected individuals.STUDY FUNDING/COMPETING INTEREST(S) This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS FONDOS FEDER (grant numbers PI20/01562 and PI23/00425) and the Fanconi Research Fund awarded to C.K. and A.R.-E. This article is based upon work from COST Action CA20119 (ANDRONET), supported by COST (European Cooperation in Science and Technology) (www.cost.eu). C.K., A.R.-E., G.F., M.J.X., M.S.O., C.A., M.S., and E.R.-C. are members of the Action. This research was also supported by the Qatar National Research Fund (QNRF) under grant NPRP12S-0318-190394, and by an Investigator Award in Science from the Wellcome Trust (209451 to J.A.V.). The authors declare no competing interests. Keywords: azoospermia; TESE; genetics; diagnostic; male infertility; spermatogenesis; exome; gene panel; POI Creator: Servigistics Arbortext Advanced Print Publisher 11.1.4667/W Producer: PDFlib+PDI 9.0.7p3 (C++/Win32) CreationDate: Tue Aug 26 11:36:18 2025 CEST ModDate: Tue Aug 26 11:37:02 2025 CEST Custom Metadata: yes Metadata Stream: yes Tagged: yes UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 612 x 792 pts (letter) Page rot: 0 File size: 2103909 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- EHJOFD+CaeciliaLTStd-Bold Type 1C Custom yes yes yes 1159 0 ZUWJWR+CaeciliaLTStd-Roman Type 1C Custom yes yes yes 1164 0 TeX_CM_Maths_Symbols Type 1 Builtin yes no yes 1169 0 TeX_CM_Roman Type 1 Builtin yes no yes 1173 0 DVREKZ+CaeciliaLTStd-Light Type 1C Custom yes yes yes 1177 0 WMAERC+CaeciliaLTStd-Heavy Type 1C Custom yes yes yes 1182 0 QWMDKA+STIX-Regular Type 1C Custom yes yes yes 1187 0 UROOFE+CaeciliaLTStd-Italic Type 1C Custom yes yes yes 1192 0 WSKGRJ+CaeciliaLTStd-LightItalic Type 1C Custom yes yes yes 66 0 MOJEFT+STIXTwoMath-Regular CID TrueType Identity-H yes yes yes 118 0 TeX_CM_Maths_Italic Type 1 Builtin yes no yes 123 0 FRBUGF+CaeciliaLTStd-BoldItalic Type 1C Custom yes yes yes 170 0 IYUISS+STIX-Bold Type 1C Custom yes yes yes 25 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-10-04 04:06:40 CEST RepresentationInformation: hoaf049.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-10-03 18:51:33 CEST Size: 2103909 Format: PDF Version: 1.5 Status: Well-Formed, but not valid SignatureMatches: PDF-hul ErrorMessage: Invalid indirect destination - referenced object 'lhoaf049-FM1' cannot be found ID: PDF-HUL-149 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'lhoaf049-FM1' cannot be found ID: PDF-HUL-122 Offset: 1982875 MIMEtype: application/pdf Profile: Tagged PDF PDFMetadata: Objects: 1270 FreeObjects: 1 IncrementalUpdates: 2 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: true NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseOutlines Language: en Outlines: Item: Title: Active Content List Destination: mkchap_artid Children: Item: Title: Introduction Destination: mkchap11__sec Item: Title: Materials and methods Destination: mkchap12__sec Item: Title: Results Destination: mkchap24__sec Item: Title: Discussion Destination: mkchap35__sec Item: Title: Conclusion Destination: mkchap36__sec Item: Title: Supplementary data Destination: mkchap37__sec Item: Title: Data availability Destination: mkchap38__sec Item: Title: Acknowledgements Destination: mkchap39_ack1_ack Item: Title: Authors' roles Destination: mkchap39__sec Item: Title: Funding Destination: mkchap40__sec Item: Title: Conflict of interest Destination: mkchap41__sec Item: Title: References Destination: mkchap42_ref1_ref-list Info: Title: Genetic determinants of testicular sperm extraction outcomes: insights from a large multicentre study of men with non-obstructive azoospermia Subject: Doi: 10.1093/hropen/hoaf049 Human Reproduction Open, 2025, 3, 2025 Publication Date: 19/08/2025 Abstract STUDY QUESTION What is the diagnostic yield and the pre-testicular sperm extraction (TESE) prognostic value of a non-obstructive azoospermia (NOA)-specific virtual gene panel?SUMMARY ANSWER The diagnostic yield in our cohort was 6.1%, and by combining our data with published literature, we identified 11 genes compatible with testicular sperm production and 19 genes associated with no sperm retrieval in carriers of pathogenic (P) or likely pathogenic (LP) mutations.WHAT IS KNOWN ALREADY Azoospermia, the most severe form of male infertility, affects ∼1% of the male population, with TESE being the primary treatment option. However, in NOA, TESE fails in nearly 50% of cases and existing clinical parameters are unable to predict TESE failure. Over the past decade, next-generation sequencing (NGS) has identified several candidate NOA genes, but their diagnostic utility and impact on TESE outcomes have not been fully explored.STUDY DESIGN, SIZE AND DURATION A literature search was addressed to identify well-established NOA genes for designing a specific virtual gene panel for NOA. Our retrospective study analysed the diagnostic yield of the NGS-based virtual gene panel, comprising 145 genes, in 571 men affected by idiopathic NOA with known TESE outcomes. Subsequently, a second literature search was performed to identify carriers of LP/P variants in the genes where we identified mutations, focusing on individuals with known TESE outcomes. This approach allowed us to integrate the published data with our findings and predict a genotype–phenotype correlation between the affected genes and TESE success.PARTICIPANTS/MATERIALS, SETTINGS, METHODS 571 NOA patients with known TESE outcomes were recruited in two European and one Middle East centres. Variants were obtained from a whole-exome sequencing dataset and crossed with the 145 genes of the virtual gene panel. After a filtering process, variants were manually assessed and classified according to ACMG guidelines by using two methods: (i) In order to compare our data with previously published studies, we applied ACMG-AMP guidelines along with ClinGen recommendations used by other similar studies. (ii) A new approach was used to optimize ACMG-AMP guidelines with all ClinGen recommendations and incorporated NOA-specific rules addressing phenotypic, locus, and allelic heterogeneity. LP and P variants were confirmed by Sanger sequencing.MAIN RESULTS AND THE ROLE OF CHANCE By using the new variant classification approach adapted for NOA, we identified LP/P variants in 6.1% of patients, with a higher yield (9.4%) in cases with negative TESE outcomes and maturation arrest (11.7%). By integrating our findings with the literature, we highlight 19 genes recurrently associated with negative TESE outcomes and 11 genes associated with positive sperm retrieval either in the testis or in semen. TESE is recommended for patients with LP or P variants in the 11 specific genes. Notably, six of these genes are located on the X chromosome, therefore, these variants will be obligatorily transmitted to daughters, and potentially increase the risk of NOA-related infertility in male offspring. We observed that nine genes, in which we identified LP/P variants, have been previously described in individuals with premature ovarian insufficiency (POI). Of these, eight were associated with negative TESE outcomes in men. Furthermore, we propose seven additional genes mutated in our cohort of NOA patients as novel POI candidates. These genes have not yet been considered as POI candidates, but they result in female infertility when knocked out in mouse models.LARGE SCALE DATA LP/P variants have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/).LIMITATIONS, REASONS FOR CAUTION NOA is genetically heterogeneous, and our panel excludes those genes which were reported only in a single subject or single family. Although this can limit the diagnostic yield in our study, it ensures that only genes with clear relationship with NOA have been analysed. While in our cohort TESE outcomes are known for all patients, this information is often not available for mutation carriers in the published studies. Consequently, the total number of patients with P variants in the same gene remains relatively low, limiting our final conclusions. However, even if the number of carriers of genes associated with positive sperm retrieval is relatively low, it does not constrain our conclusions regarding TESE prediction. On the other hand, caution is warranted for genes linked to negative TESE outcomes, except for TEX11, SYCE1, and MSH4, each of which have 10 or more reported TESE-negative cases.WIDER IMPLICATIONS OF THE FINDINGS Our study was performed on the largest available NOA cohort with known TESE outcomes. It not only provides an estimate on the diagnostic potential of a NOA-specific virtual gene panel, but it also advances the understanding of genetic factors influencing TESE outcomes. Half of the genes mutated in our study and presenting TESE-positive outcomes are already informative for clinical decision-making. The observed genotype–phenotype correlations may help in personalized decision-making prior to TESE, in order to undergo the procedure or to avoid unnecessary invasive treatment. It provides valuable insights that can inform clinical management strategies and potentially offer personalized treatments based on genetic profiles. The use of two different variant classification methods highlights that previous studies may have over-estimated the diagnostic yield, underscoring the need for a standardized variant classification approach addressed specifically to male infertility. Our study also emphasizes the overlap between NOA- and POI-associated genes, which has important clinical implications for genetic counselling of female siblings of affected individuals.STUDY FUNDING/COMPETING INTEREST(S) This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS FONDOS FEDER (grant numbers PI20/01562 and PI23/00425) and the Fanconi Research Fund awarded to C.K. and A.R.-E. This article is based upon work from COST Action CA20119 (ANDRONET), supported by COST (European Cooperation in Science and Technology) (www.cost.eu). C.K., A.R.-E., G.F., M.J.X., M.S.O., C.A., M.S., and E.R.-C. are members of the Action. This research was also supported by the Qatar National Research Fund (QNRF) under grant NPRP12S-0318-190394, and by an Investigator Award in Science from the Wellcome Trust (209451 to J.A.V.). The authors declare no competing interests. Keywords: azoospermia; TESE; genetics; diagnostic; male infertility; spermatogenesis; exome; gene panel; POI Creator: Servigistics Arbortext Advanced Print Publisher 11.1.4667/W Producer: PDFlib+PDI 9.0.7p3 (C++/Win32) CreationDate: Tue Aug 26 12:06:18 CEST 2025 ModDate: Tue Aug 26 12:07:02 CEST 2025 ID: 0xd1f8d97ef4a790e74928e9b265a74e9c, 0x7bbcd9518133a9e4312c7acc4d79215a Filters: FilterPipeline: DCTDecode Images: Image: NisoImageMetadata: FormatName: image/jpg CompressionScheme: JPEG ImageWidth: 2000 ImageHeight: 1144 ColorSpace: RGB BitsPerSample: 8 BitsPerSampleUnit: integer Image: NisoImageMetadata: FormatName: image/jpg CompressionScheme: JPEG ImageWidth: 2000 ImageHeight: 1208 ColorSpace: RGB BitsPerSample: 8 BitsPerSampleUnit: integer Image: NisoImageMetadata: FormatName: image/jpg CompressionScheme: JPEG ImageWidth: 1510 ImageHeight: 1934 BitsPerSample: 8 BitsPerSampleUnit: integer Image: NisoImageMetadata: FormatName: image/jpg CompressionScheme: JPEG ImageWidth: 1510 ImageHeight: 1990 BitsPerSample: 8 BitsPerSampleUnit: integer Fonts: Type0: Font: BaseFont: MOJEFT+STIXTwoMath-Regular Encoding: Identity-H ToUnicode: true Type1: Font: BaseFont: TeX_CM_Maths_Symbols FirstChar: 1 LastChar: 138 FontDescriptor: FontName: TeX_CM_Maths_Symbols Flags: Symbolic FontBBox: -50, -180, 1000, 720 FontFile: true ToUnicode: true Font: BaseFont: WSKGRJ+CaeciliaLTStd-LightItalic FontSubset: true FirstChar: 1 LastChar: 59 FontDescriptor: FontName: WSKGRJ+CaeciliaLTStd-LightItalic Flags: Nonsymbolic, Italic FontBBox: -166, -267, 1033, 909 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: QWMDKA+STIX-Regular FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: QWMDKA+STIX-Regular Flags: Nonsymbolic FontBBox: -970, -486, 2208, 1023 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: TeX_CM_Roman FirstChar: 1 LastChar: 127 FontDescriptor: FontName: TeX_CM_Roman Flags: Symbolic FontBBox: -50, -180, 1000, 720 FontFile: true ToUnicode: true Font: BaseFont: EHJOFD+CaeciliaLTStd-Bold FontSubset: true FirstChar: 1 LastChar: 64 FontDescriptor: FontName: EHJOFD+CaeciliaLTStd-Bold Flags: Nonsymbolic, ForceBold FontBBox: -166, -273, 1141, 938 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: UROOFE+CaeciliaLTStd-Italic FontSubset: true FirstChar: 1 LastChar: 46 FontDescriptor: FontName: UROOFE+CaeciliaLTStd-Italic Flags: Nonsymbolic, Italic FontBBox: -166, -270, 1053, 915 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: DVREKZ+CaeciliaLTStd-Light FontSubset: true FirstChar: 1 LastChar: 88 FontDescriptor: FontName: DVREKZ+CaeciliaLTStd-Light Flags: Nonsymbolic FontBBox: -164, -266, 1130, 908 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: IYUISS+STIX-Bold FontSubset: true FirstChar: 1 LastChar: 2 FontDescriptor: FontName: IYUISS+STIX-Bold Flags: Nonsymbolic, ForceBold FontBBox: -998, -552, 2000, 1055 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: FRBUGF+CaeciliaLTStd-BoldItalic FontSubset: true FirstChar: 1 LastChar: 6 FontDescriptor: FontName: FRBUGF+CaeciliaLTStd-BoldItalic Flags: Nonsymbolic, Italic, ForceBold FontBBox: -169, -273, 1082, 932 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: TeX_CM_Maths_Italic FirstChar: 1 LastChar: 127 FontDescriptor: FontName: TeX_CM_Maths_Italic Flags: Symbolic FontBBox: -50, -180, 1000, 720 FontFile: true ToUnicode: true Font: BaseFont: ZUWJWR+CaeciliaLTStd-Roman FontSubset: true FirstChar: 1 LastChar: 74 FontDescriptor: FontName: ZUWJWR+CaeciliaLTStd-Roman Flags: Nonsymbolic FontBBox: -167, -270, 1136, 926 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: WMAERC+CaeciliaLTStd-Heavy FontSubset: true FirstChar: 1 LastChar: 65 FontDescriptor: FontName: WMAERC+CaeciliaLTStd-Heavy Flags: Nonsymbolic, ForceBold FontBBox: -170, -277, 1146, 970 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true CIDFontType2: Font: BaseFont: MOJEFT+STIXTwoMath-Regular CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: MOJEFT+STIXTwoMath-Regular Flags: Nonsymbolic FontBBox: -978, -1641, 3072, 2627 FontFile2: true XMP: uuid:c248697d-5b8b-286d-5557-794cb61d98f8 uuid:21B09EAB-D33A-E5E6-35FA-CB4CC018A07C 2025-08-26T15:06:18+05:30 2025-08-26T15:07:02+05:30 Servigistics Arbortext Advanced Print Publisher 11.1.4667/W 2025-08-26T15:07:02+05:30 PDFlib+PDI 9.0.7p3 (C++/Win32) azoospermia; TESE; genetics; diagnostic; male infertility; spermatogenesis; exome; gene panel; POI Genetic determinants of testicular sperm extraction outcomes: insights from a large multicentre study of men with non-obstructive azoospermia Antoni Riera-Escamilla Mohamed M Arafa Ginevra Farnetani Miguel J Xavier Manon S Oud Ahmad A Majzoub Liliana Ramos Chiara Abrardo Matilde Spinelli Daniel Moreno-Mendoza Giuseppe Defazio Elisabet Ars Marc Pybus Josvany R Sánchez Curbelo Haitham T Elbardisi Shoaib Nawaz Najeeb Syed Eduard Ruiz-Castané Godfried W van der Heijden Khalid A Fakhro Joris A Veltman Csilla Krausz Doi: 10.1093/hropen/hoaf049 Human Reproduction Open, 2025, 3, 2025 Publication Date: 19/08/2025 Abstract STUDY QUESTION What is the diagnostic yield and the pre-testicular sperm extraction (TESE) prognostic value of a non-obstructive azoospermia (NOA)-specific virtual gene panel?SUMMARY ANSWER The diagnostic yield in our cohort was 6.1%, and by combining our data with published literature, we identified 11 genes compatible with testicular sperm production and 19 genes associated with no sperm retrieval in carriers of pathogenic (P) or likely pathogenic (LP) mutations.WHAT IS KNOWN ALREADY Azoospermia, the most severe form of male infertility, affects ∼1% of the male population, with TESE being the primary treatment option. However, in NOA, TESE fails in nearly 50% of cases and existing clinical parameters are unable to predict TESE failure. Over the past decade, next-generation sequencing (NGS) has identified several candidate NOA genes, but their diagnostic utility and impact on TESE outcomes have not been fully explored.STUDY DESIGN, SIZE AND DURATION A literature search was addressed to identify well-established NOA genes for designing a specific virtual gene panel for NOA. Our retrospective study analysed the diagnostic yield of the NGS-based virtual gene panel, comprising 145 genes, in 571 men affected by idiopathic NOA with known TESE outcomes. Subsequently, a second literature search was performed to identify carriers of LP/P variants in the genes where we identified mutations, focusing on individuals with known TESE outcomes. This approach allowed us to integrate the published data with our findings and predict a genotype–phenotype correlation between the affected genes and TESE success.PARTICIPANTS/MATERIALS, SETTINGS, METHODS 571 NOA patients with known TESE outcomes were recruited in two European and one Middle East centres. Variants were obtained from a whole-exome sequencing dataset and crossed with the 145 genes of the virtual gene panel. After a filtering process, variants were manually assessed and classified according to ACMG guidelines by using two methods: (i) In order to compare our data with previously published studies, we applied ACMG-AMP guidelines along with ClinGen recommendations used by other similar studies. (ii) A new approach was used to optimize ACMG-AMP guidelines with all ClinGen recommendations and incorporated NOA-specific rules addressing phenotypic, locus, and allelic heterogeneity. LP and P variants were confirmed by Sanger sequencing.MAIN RESULTS AND THE ROLE OF CHANCE By using the new variant classification approach adapted for NOA, we identified LP/P variants in 6.1% of patients, with a higher yield (9.4%) in cases with negative TESE outcomes and maturation arrest (11.7%). By integrating our findings with the literature, we highlight 19 genes recurrently associated with negative TESE outcomes and 11 genes associated with positive sperm retrieval either in the testis or in semen. TESE is recommended for patients with LP or P variants in the 11 specific genes. Notably, six of these genes are located on the X chromosome, therefore, these variants will be obligatorily transmitted to daughters, and potentially increase the risk of NOA-related infertility in male offspring. We observed that nine genes, in which we identified LP/P variants, have been previously described in individuals with premature ovarian insufficiency (POI). Of these, eight were associated with negative TESE outcomes in men. Furthermore, we propose seven additional genes mutated in our cohort of NOA patients as novel POI candidates. These genes have not yet been considered as POI candidates, but they result in female infertility when knocked out in mouse models.LARGE SCALE DATA LP/P variants have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/).LIMITATIONS, REASONS FOR CAUTION NOA is genetically heterogeneous, and our panel excludes those genes which were reported only in a single subject or single family. Although this can limit the diagnostic yield in our study, it ensures that only genes with clear relationship with NOA have been analysed. While in our cohort TESE outcomes are known for all patients, this information is often not available for mutation carriers in the published studies. Consequently, the total number of patients with P variants in the same gene remains relatively low, limiting our final conclusions. However, even if the number of carriers of genes associated with positive sperm retrieval is relatively low, it does not constrain our conclusions regarding TESE prediction. On the other hand, caution is warranted for genes linked to negative TESE outcomes, except for TEX11, SYCE1, and MSH4, each of which have 10 or more reported TESE-negative cases.WIDER IMPLICATIONS OF THE FINDINGS Our study was performed on the largest available NOA cohort with known TESE outcomes. It not only provides an estimate on the diagnostic potential of a NOA-specific virtual gene panel, but it also advances the understanding of genetic factors influencing TESE outcomes. Half of the genes mutated in our study and presenting TESE-positive outcomes are already informative for clinical decision-making. The observed genotype–phenotype correlations may help in personalized decision-making prior to TESE, in order to undergo the procedure or to avoid unnecessary invasive treatment. It provides valuable insights that can inform clinical management strategies and potentially offer personalized treatments based on genetic profiles. The use of two different variant classification methods highlights that previous studies may have over-estimated the diagnostic yield, underscoring the need for a standardized variant classification approach addressed specifically to male infertility. Our study also emphasizes the overlap between NOA- and POI-associated genes, which has important clinical implications for genetic counselling of female siblings of affected individuals.STUDY FUNDING/COMPETING INTEREST(S) This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS FONDOS FEDER (grant numbers PI20/01562 and PI23/00425) and the Fanconi Research Fund awarded to C.K. and A.R.-E. This article is based upon work from COST Action CA20119 (ANDRONET), supported by COST (European Cooperation in Science and Technology) (www.cost.eu). C.K., A.R.-E., G.F., M.J.X., M.S.O., C.A., M.S., and E.R.-C. are members of the Action. This research was also supported by the Qatar National Research Fund (QNRF) under grant NPRP12S-0318-190394, and by an Investigator Award in Science from the Wellcome Trust (209451 to J.A.V.). The authors declare no competing interests. © The Author(s) 2025. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. azoospermia TESE genetics diagnostic male infertility spermatogenesis exome gene panel POI application/pdf PStill version 1.84.42 True 3 A http://ns.adobe.com/pdf/1.3/ pdf Adobe PDF Schema internal A name object indicating whether the document has been modified to include trapping information Trapped Text http://ns.adobe.com/xap/1.0/mm/ xmpMM XMP Media Management Schema internal UUID based identifier for specific incarnation of a document InstanceID URI internal The common identifier for all versions and renditions of a document. 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