5th International Andrology Symposium
5th International Andrology Symposium
Birgit Stallmeyer
Centre of Medical Genetics, Institute of Reproductive Genetics, University of Münster, Münster, Germany.
Correspondence: Dr. rer. nat. Birgit Stallmeyer, Centrum für Medizinische Genetik, 48149 Münster, DE
Defects in germline genome integrity and gene regulation frequently underpin male infertility. The PIWI–piRNA pathway safeguards the male germline against transposable elements (TEs) from fetal germ cells through the process of spermatogenesis. Over the past decade, particularly in recent years, exome and genome sequencing of large cohorts with clinically well-defined phenotypes has revealed that monogenic causes essentially contribute to the disease. Using data from the Münster-based MERGE cohort, we have shown that inherited defects in human piRNA biogenesis are also an important and actionable cause of spermatogenic failure. Accordingly, we have identified biallelic variants across core and accessory piRNA factors, established genotype–phenotype correlations, demonstrated loss of pachytene piRNAs in patient testis, and linked specific gene variants to transposon de-repression in human spermatogonia. Here, I summarise these recent findings and discuss the differences in the reproductive phenotypes between human variant carriers and knockout models of the mice orthologues.
Key words: Infertility, Transposon, piRNA
Les défauts d’intégrité génomique et de régulation génique de la lignée germinale constituent des causes majeures d’infertilité masculine. La voie PIWI–piRNA protège les cellules germinales masculines, depuis le stade fœtal jusqu’à la spermatogenèse, contre les éléments transposables (TEs). Au cours de la dernière décennie, et tout particulièrement ces dernières années, le séquençage d’exomes et de génomes dans de larges cohortes phénotypiquement bien caractérisées a mis en évidence une contribution substantielle de causes monogéniques.
À partir de la cohorte MERGE (Münster), nous avons montré que les anomalies héréditaires de la biogenèse des piARN humains constituent une cause importante et actionnable d’insuffisance spermatogénétique. Nous avons identifié des variants bialléliques affectant des facteurs centraux ou accessoires de la voie piRNA, établi des corrélations génotype–phénotype, démontré la perte des piARN pachytènes dans les testicules de patients et relié certains variants géniques à une dérégulation des transposons dans les spermatogonies humaines. Cette synthèse présente ces avancées récentes et discute les divergences phénotypiques entre porteurs humains et modèles murins invalidés pour les orthologues correspondants.
Semen analysis remains the starting point for male infertility diagnostics, and a substantial fraction of patients present with reduced sperm counts up to complete azoospermia, often reflecting intrinsic spermatogenic failure rather than obstructive causes. Historically, genetic testing focused on chromosomal aberrations (e.g., Klinefelter syndrome) and AZF microdeletions on the Y chromosome. Apart from CFTR for obstructive azoospermia, sequencing of single disease genes to identify monogenic causes remains underdetermined. However, more than three-quarters of human genes are expressed in testis, thousands are testis-enriched, and hundreds are testis-specific, implying that monogenic aetiologies could be expected. Indeed, mouse knockout studies have linked many genes to spermatogenic arrest in mice. However, the extent to which pathogenic genetic variants in human genes contribute to the disease remains unexplored. Recognising this, we established the MERGE cohort, comprising genome and exome sequencing data from approximately 2,800 infertile men, mainly affected by reduced sperm count to systematically uncover monogenic causes of male infertility and to translate valid disease genes into diagnostic practice [1,2]. By this approach, we increased the diagnostic yield in patients with azoospermia by 7%. Nevertheless, further research is required to expand the spectrum of valid disease genes.
To identify the key biological pathways that contribute to male infertility, we performed a Gene Ontology (GO) analysis on a list of genes in which homozygous loss-of-function variants were identified in the MERGE cohort. This revealed that piRNA processing is a key biological process contributing to the disease [3]. piRNAs represent the most abundant subgroup of regulatory small non-coding RNAs in the testis. They bind to PIWI proteins, a subclade of argonaute proteins, and guide them to silence transposable elements (TEs), mobile DNA sequences that are capable of copying and inserting themselves throughout the genome. Impaired piRNA activity can cause uncontrolled TE activity and threaten genome integrity. In mice, mutations in genes essential for piRNA biogenesis, such as Piwil1, Tdrd1, Mael, or Mili, lead to meiotic failure, and infertility [4]. In humans, two key genes of the piRNA pathway, PNLDC1 and FKBP6, had been linked to biallelic pathogenic variants in the piRNA pathway [5,6]. However, a comprehensive analysis of the contribution of a disturbed piRNA pathway to human male infertility has been lacking.
In 2024, our study on more than 2400 MERGE samples identified 31 infertile men with biallelic high-impact variants in 14 piRNA-pathway genes, including PIWIL1, GTSF1, TDRD1, TDRD12, GPAT2, MAEL, and TDRD9 [3]. Of these patients, 12 were carriers of biallelic loss-of-function variants, reflecting the phenotype of a human ‘knockout’. The variant carriers were affected by spermatogenic failure, with a significant proportion exhibiting azoospermia or cryptozoospermia. The testicular morphology of the variant carriers confirmed impaired spermatogenesis, displaying a broad spectrum of phenotypes ranging from hypospermatogenesis to a Sertoli cell-only type of histology. However, with respect to a single gene, the testicular phenotypes were mainly concordant. A gene-by-gene comparison of the testicular phenotypes in human variant carriers and knockout mice showed several gene-specific differences. For some genes, e.g. PIWIL2, the spermatogenic arrest occurred at an earlier stage in humans than in mice. For others, e.g. TDRD9, spermatogenesis progressed to later stages than those observed in the respective mouse knockout model. We demonstrated the absence of the affected protein in the testicular tissue of several homozygous stop-gain or frameshift variant carriers, proving the expected loss of protein function. Small-RNA sequencing of RNA samples derived from testicular biopsies of selected variant carriers resulted in reduced levels of pachytene piRNAs, thereby linking the genetic variations to disturbed piRNA biogenesis. Immunohistochemical staining for the LINE1 marker protein LINE1 ORF1p revealed upregulation of the protein in spermatogonia in several human variant carriers, including patients affected by biallelic variants in TDRD12, GPAT2 and FKBBP6. This LINE1 upregulation serves as a molecular signature of transposon de-silencing due to disrupted piRNA function. These findings redefine a subset of idiopathic male infertility as a distinct molecular entity: piRNA pathway deficiency–associated spermatogenic failure. Clinically, this work expands the catalogue of diagnostic targets for genetic testing panels.
This study provides definitive evidence that inherited defects in piRNA biogenesis genes are a direct cause of human male infertility. It establishes that the mechanisms underlying germline genome integrity, which were previously characterized mainly in model organisms, are conserved in humans. However, it also highlights that phenotypic findings from mice cannot be directly extrapolated to humans in the case of piRNAs. Further identification and functional characterisation of additional variants is required in order to systematically determine the reproductive phenotypes associated with impaired piRNA pathway function in humans.
a) Ethics Approval and Consent to Participate Investigations were carried out in accordance 326 with the Declaration of Helsinki of 1975, revised in 2008.
b) Consent for publication Not applicable
c) Availability of data and supporting material Not applicable
d) Competing interests Author/s declare that they have no competing interests
e) Funding: acknowledgments are as in the cited manuscripts.
I thank the patients participating to this study; the colleagues at the Institute of Reproductive Genetics (Münster) and collaborators contributing biopsies and small-RNA data.
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