1. Response to Reviewer #3 (Public Review):

    [...] The authors imply that the combination of the inversion+deletion risk allele favours a meiotic failure disease aetiology as opposed to a gene dosage aetiology. This is a potentially disruptive finding.

    Response: We thank the reviewer for these supportive remarks on our research and manuscript.

    Weaknesses:

    1. The authors do not replicate their association, raising the possibility of a false positive finding.

    Response: We acknowledge this weakness and have modified the Discussion (page 15, lines 447-459) to emphasize the importance of replication studies in other European populations with high R1a1-M458 frequency.

    1. The study is underpowered to reliably detect variants of small effect, and underpowered in general. This is a common challenge in reproductive genetics.

    Response: The current study is the largest and most detailed to date in terms of both the number of included patients with spermatogenic failure and reference samples with available andrological data from a single population, and in the detailed characterization of the genetic diversity of the AZFc region and phylogenetic background of the Y chromosomes.

    Our pre-study knowledge about genetic variability within the AZFc region and possible range of its genetic effects was largely limited to the risk estimates of gr/gr or b2/b3 deletion carriership in different populations. Before this study, there was inadequate information to design an approach that would have explicit high statistical power to also detect variants of small effect. The questions at the ‘start-line’ were: what type of genetic variation can be identified in AZFc region among Y chromosomes of a large study group of infertile and fertile men? Are these variations correlated with spermatogenic failure? Our study was implemented using in parallel several technologies to detect alternative genetic variation types – structural variants and Y-chromosomal haplogroups, intergenic SNV variability and copy number variation of duplicate genes in the region etc. Based on our seminal investigation, further studies can be focused on specific variants or variant types to test in larger sample sets their possible smaller effects on andrological parameters, an outcome which we see as a strength of our work.

    It has to be highlighted that an additional challenge in the reproductive genetics of Y-chromosomal variants is the ‘selfish’ nature of the Y-chromosome, supported by the findings of this study. Except for the pseudo-autosomal regions, it does not recombine with other chromosomes and there is an extremely high intra-chromosomal recombination rate and gene conversion between duplicated regions (Trombetta et al. 2017, doi: 10.1007/s00439-017-1777-8). Thus, every Y-chromosomal lineage may have its own genetic risk variants of both effect types, large or small. This needs to be assessed in a series of future and preferably long-range sequencing based studies focusing on infertile men representing specific Y-chromosomal haplogroups. The current accumulated knowledge suggests that choosing for the Y-chromosomal risk variant analysis study groups based on only clinical data, the association testing with haplogroup-specific variants with moderate effect is underpowered.

    1. The logical inferences (as opposed to direct measurement) made by the authors are elegant but add substantial uncertainty to the findings. Most notably, cytogenetic or long-read sequencing based validation of the inversion genotype would strengthen confidence in the study considerably.

    Response: We agree with the reviewer that validation and fine-scale mapping of inversion genotype with an independent method will be an important part of follow-up studies. Our Discussion section (page 12, lines 356-359) has been improved to point this out.

    If the genetic association is robust and the allele frequency estimates are well calibrated, the implications of this work are considerable. The locus could become a genetic biomarker for infertility. The locus could potentially account for a huge amount of variance in polygenic risk associated with infertility. The findings also raise a fascinating evolutionary conundrum as to how an allele associated with such an evolutionarily destructive phenotype could occur at such high frequencies. The authors briefly raise the possibility of age-dependent effects, but with extremely sparse data.

    Response: We thank the reviewer for this positive, supporting and stimulating concluding summary.

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  2. Response to Reviewer #2 (Public Review):

    [...] Some major strengths of this paper are the size of the groups recruited (1,190 patients and 1,134 reference individuals) from a single national population, the extensive accompanying andrological data, and the genomic characterisation of many individuals to elucidate the relationship between specific structural variants and effects on fertility.

    The discovery of the fixed inversion infertility risk factor on a specific Y haplogroup is a useful contribution that could aid genetic counselling efforts through carrier identification and risk mitigation.

    Response: We thank the reviewer for these supportive comments on our research and manuscript.

    However, I am seeking clarity on multiple testing correction for microdeletion association with specific andrological parameters. Besides this, the main conclusions of this paper are supported by other data presented.

    Response: The issue has been addressed and the relevant improvements to the manuscript were introduced.

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  3. Response to Reviewer #1 (Public Review):

    [...] The authors mentioned that the variant largely 'destroys' two palindromes, P1 and P2. One would like to see more discussion what are the structural and functional consequences - e.g. are any loci for e.g. non-coding RNA affected by a deletion in men with this inversion in comparison to those without?

    Response: We thank the reviewer for the comment and have extended the Discussion (page 12, lines 362-370) to address this.

    The authors also speculated in the discussion that deletion on this background might lead to progressive worsening of the reproductive phenotype. This is based on just one control individual, a young man with borderline reproductive parameters, and corroborating this hypothesis would require further studies, including repeated evaluation of the same individuals over a long period of time.

    Response: We fully agree with the reviewer that the data to support this hypothesis is currently thin and further longitudinal studies on several patients are needed. We have modified the Discussion section (page 13, lines 391-393) accordingly.

    This is a high quality study, performed by collaborators from the UK and Estonia, with an excellent track record in the analysis of the Y-chromosome structure and evolution, and in reproductive genetics and clinical andrology, respectively. The data presentation and figures are very informative and convincing. Among the strengths of the study, I have to emphasise a detailed phenotypic evaluation of the study subjects, including several parameters of testis function, semen analysis, and reproductive hormone profiles. Hence, the results and conclusions are valuable and add to the understanding of the consequences of the partial AZFc deletions. The authors also provided useful guidelines how to identify men with this variant in labs performing genetic analysis of infertile couples.

    Response: We thank the reviewer for their supportive opinion on our research and manuscript.

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  4. Reviewer #3 (Public Review):

    Hallast and coworkers identify a potentially novel complex Y chromosome structural rearrangement that is associated with male infertility in a carefully phenotyped European cohort. The authors interrogate the Y chromosome AFZc region in 1190 Estonian idiopathic male infertility cases of varying severity and 1134 controls (healthy young men or proven fathers). They replicate partial AFZc deletions and replicate a known gr/gr deletion association with comparable effect sizes. After conditioning on gr/gr deletion status, they identify an association with secondary b2/b4 duplications on case status, but with no accompanying observed effect on andrological sub-phenotypes.

    The authors identify multiple non-syntenic DAZ/CDY1 deletion patterns that are consistent with a large inversion followed by deletion. The authors further infer that this putative inversion is fixed in a Y chromosome sub-lineage. Based on population haplotype frequency estimates they infer that a surprisingly large number of individuals harbouring the r2/r3 inversion have a subsequent deletion. They show through detailed phenotyping shows that r2/r3 inversion+deletion cases in their cohort have more severe disease.

    Strengths:

    1. Despite being a very common disease, idiopathic infertility is severely understudied, due in large part to difficulties in sample acquisition. More generally, sex chromosome genetic associations for common disease as a whole are understudied owing to their structural complexity and other technical issues. The authors should be applauded for attempting to overcome these challenges.

    2. The putative finding of a large-effect common variant conferring risk to a common genetic disease is of great interest. The authors leverage the advantages of a logistically coherent health care system. The level of phenotypic detail of andrological parameters in both cases and controls is impressive and aid in biological interpretation of the genetic findings. For example, the distinction between azoo- versus oligozoo-spermia shed light on a potential meiotic disease aetiology. The endocrine values add important context.

    3. The authors imply that the combination of the inversion+deletion risk allele favours a meiotic failure disease aetiology as opposed to a gene dosage aetiology. This is a potentially disruptive finding.

    Weaknesses:

    1. The authors do not replicate their association, raising the possibility of a false positive finding.

    2. The study is underpowered to reliably detect variants of small effect, and underpowered in general. This is a common challenge in reproductive genetics.

    3. The logical inferences (as opposed to direct measurement) made by the authors are elegant but add substantial uncertainty to the findings. Most notably, cytogenetic or long-read sequencing based validation of the inversion genotype would strengthen confidence in the study considerably.

    If the genetic association is robust and the allele frequency estimates are well calibrated, the implications of this work are considerable. The locus could become a genetic biomarker for infertility. The locus could potentially account for a huge amount of variance in polygenic risk associated with infertility. The findings also raise a fascinating evolutionary conundrum as to how an allele associated with such an evolutionarily destructive phenotype could occur at such high frequencies. The authors briefly raise the possibility of age-dependent effects, but with extremely sparse data.

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  5. Reviewer #2 (Public Review):

    Hallast et al. have performed an extensive genetic analysis of a cohort of men with idiopathic infertility, for whom many have accompanying phenotypic data in the form of andrological parameters. The complex genetic architecture of repeating sequences on the Y chromosome gives rise to recurrent AZFc deletions that affect male infertility. However, while partial deletions of AZFc are reasonably frequent, they have less clear phenotypic effects. gr/gr and b2/b3 deletions seem to be a risk factor for spermatogenic impairment in some populations but are fixed in others. Hallast et al. focus on these partial AZFc deletions in a reference cohort and a cohort with idiopathic infertility from the same geographic population, characterising further structural and sequence variation, Y-chromosomal haplogroups, and gene dosage.

    While the gr/gr deletion is present in the reference group of individuals with normal andrological parameters, Hallast et al. show that this deletion is enriched among patients, with 2.2-fold increased susceptibility to infertility. As observed in other European populations, the prevalence of b2/b3 deletion was similar in the reference group and the patient group, suggesting that it is not a spermatogenic impairment risk factor for this Estonian population either.

    A quarter of Estonian gr/gr deletion carriers belonged to the Y chromosomal haplogroup R1a1-M458. Within this Y haplogroup, an inversion has occurred that promotes subsequent deletion, likely causing severe spermatogenic failure in the majority of carriers as this complex rearrangement is enriched 8.6-fold in individuals with severe spermatogenic impairment.

    Some major strengths of this paper are the size of the groups recruited (1,190 patients and 1,134 reference individuals) from a single national population, the extensive accompanying andrological data, and the genomic characterisation of many individuals to elucidate the relationship between specific structural variants and effects on fertility.

    The discovery of the fixed inversion infertility risk factor on a specific Y haplogroup is a useful contribution that could aid genetic counselling efforts through carrier identification and risk mitigation.

    However, I am seeking clarity on multiple testing correction for microdeletion association with specific andrological parameters. Besides this, the main conclusions of this paper are supported by other data presented.

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  6. Reviewer #1 (Public Review):

    In this study, Hallast and colleagues performed a detailed genetic analysis of the AZFc region of the Y-chromosome in a large cohort of 1190 Estonian men with idiopathic infertility and >1100 controls from the same population. They focused on partial deletions of the AZFc regions, because their clinical significance remains controversial and published reports are often contradictory. The authors performed a comprehensive genetic analysis, which in addition to a standard AZFc deletion protocol with gene dosage of the key AZFc genes, included also Y-haplogroup determination and re-sequencing of the retained DAZ, BPY2 and CDY genes. The authors showed that gr/gr deletions were enriched in infertile men, thus confirming that this deletion is a risk factor for impaired spermatogenesis. An important novel finding is identification of a previously unknown structural variant: a long r2/r3 inversion, which likely destabilizes two palindromes and leads to deletions. This variant is fixed in the Y lineage R1a1-M458, which is common in some Central European populations. In the Estonian study group, nearly all patients with this variant and a gr/gr deletion, had a severe impairment of spermatogenesis. The authors mentioned that the variant largely 'destroys' two palindromes, P1 and P2. One would like to see more discussion what are the structural and functional consequences - e.g. are any loci for e.g. non-coding RNA affected by a deletion in men with this inversion in comparison to those without?

    The authors also speculated in the discussion that deletion on this background might lead to progressive worsening of the reproductive phenotype. This is based on just one control individual, a young man with borderline reproductive parameters, and corroborating this hypothesis would require further studies, including repeated evaluation of the same individuals over a long period of time.

    This is a high quality study, performed by collaborators from the UK and Estonia, with an excellent track record in the analysis of the Y-chromosome structure and evolution, and in reproductive genetics and clinical andrology, respectively. The data presentation and figures are very informative and convincing. Among the strengths of the study, I have to emphasise a detailed phenotypic evaluation of the study subjects, including several parameters of testis function, semen analysis, and reproductive hormone profiles. Hence, the results and conclusions are valuable and add to the understanding of the consequences of the partial AZFc deletions. The authors also provided useful guidelines how to identify men with this variant in labs performing genetic analysis of infertile couples.

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  7. Evaluation Summary:

    This study presents extensive genetic analysis of a relatively large cohort of men with idiopathic infertility, with considerable accompanying andrological phenotypic data. Through careful step-by-step investigations, an inversion variant is identified as a risk factor for subsequent deletion variants that can lead to substantially increased risk of impaired spermatogenesis, on an age-structured basis, relative to non-carriers. This work will be of particular interest to the reproductive genetics field, but also has wide ranging implications for colleagues interested in common disease genetics, meiosis, structural variation, dosage sensitivity, and sex chromosome evolution. As part of the most comprehensive investigation of AZFc micro-deletions and structural variation to date, the authors have identified a novel structural variant of the Y-chromosome that predisposes to spermatogenic failure and provided clear guidelines for genetic counseling.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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