5th International Andrology Symposium
5th International Andrology Symposium
Christian De Geyter
University of Basel, Switzerland,
Correspondance: Prof Dr med. em.Christian De Geyter UNI Basel Switzerland
Background: Anogenital distance (AGD) is a sexually dimorphic, life-long anthropometric marker that reflects androgen/estrogen balance during a critical fetal “masculinization programming window.” Shorter male AGD has been associated with cryptorchidism, hypospadias, impaired semen quality and reduced testicular volume—core features of the testicular dysgenesis syndrome (TDS) hypothesis. Circumstantial evidence suggests that prenatal exposure to endocrine disruptors is involved in causing TDS. Many prevalent endocrine-disrupting chemicals (EDCs) act through the estrogen receptor (ER) and can perturb fetal programming. How a transient prenatal exposure could leave durable molecular traces into adult spermatogenesis remains a key mechanistic question. A plausible mechanism is epigenetic reprogramming: in the postimplantation embryo human primordial germ cells (PGCs) undergo genome-wide DNA demethylation and subsequent re-methylation; during this window, transposable elements—particularly primate-specific Alu repeats—can be de-/re-methylated and many harbor ER-responsive sequence motifs.
Objectives: Building on clinical work from Basel we synthesize evidence and present a working model in which infertile men with shortened AGD harbor distinctive DNA-methylation patterns at estrogen-responsive Alu elements in sperm, especially within subpopulations marked by chromatin immaturity (CMA3 positivity) or apoptosis (YO-PRO-1 positivity). We outline study design, summarize core findings, and discuss translational implications and limitations.
Methods (conceptual framework): Men undergoing fertility evaluation were phenotyped (including AGD and testicular volume). Ejaculates with adequate counts were processed by swim-up and sorted by flow cytometry into fractions with high vs. low chromatin decondensation (chromomycin A3, CMA3) and with vs. without early apoptotic membrane permeability (YO-PRO-1). Reduced-representation bisulfite sequencing (RRBS) profiled CpG methylation genome-wide; bioinformatics focused on repetitive elements (Alu) containing canonical/pseudo-ERE motifs and ER-ChIP–supported sites.
Results (integrative summary): Across unsorted sperm, global CpG methylation distributions were broadly similar between infertile men (short AGD) and fertile donors. In sorted fractions, however, infertile men exhibited (i) enrichment of hypomethylated Alu-EREs within CMA3-positive and YO-PRO-1–positive sperm, and (ii) a deficit of hypermethylated Alu-EREs relative to fertile controls. These shifts were most evident in the “abnormal” subpopulations, consistent with histone retention/protamine deficiency and apoptotic signaling.
Conclusions: We propose that prenatal estrogenic/antiandrogenic perturbation (indexed in adulthood by shortened AGD) is traceable in sperm as altered methylation of ER-responsive repetitive elements. Such lesions concentrate in spermatozoa with defective chromatin maturation or incipient apoptosis, potentially contributing to reduced fertilizing competence and intergenerational epigenetic risk. We discuss how this model aligns with TDS and outline paths for validation (orthogonal epigenomics, si,ngle-cell assays, environmental exposure reconstruction).
Key words Cryptorchidism, ano-genital distance, estrogen
Contexte. La distance anogénitale (AGD) est un marqueur anthropométrique sexuellement dimorphique reflétant l’équilibre androgènes/estrogènes durant la fenêtre critique de masculinisation fœtale. Une AGD masculine réduite est associée à la cryptorchidie, l’hypospadias, une qualité spermatique diminuée et un faible volume testiculaire, éléments centraux du syndrome de dysgénésie testiculaire (TDS). Plusieurs perturbateurs endocriniens (EDC) prévalents agissent via le récepteur des estrogènes (ER) et peuvent affecter la programmation fœtale. Un mécanisme plausible reliant une exposition prénatale transitoire à des altérations durables de la spermatogenèse est la reprogrammation épigénétique des cellules germinales primordiales, notamment au niveau des éléments transposables Alu, riches en motifs réactifs à l’ER.
Objectifs. Sur la base de travaux cliniques menés à Bâle, nous proposons un modèle où les hommes infertiles présentant une AGD courte montrent des profils distinctifs de méthylation de l’ADN au niveau d’éléments Alu sensibles aux estrogènes dans leurs spermatozoïdes, en particulier dans les sous-populations immatures (CMA3 positives) ou apoptotiques précoces (YO-PRO-1 positives).
Méthodes. Des hommes évalués pour infertilité ont été phénotypés (AGD, volume testiculaire). Les échantillons spermatiques ont été enrichis (swim-up) puis triés par cytométrie selon la condensation chromatinienne (CMA3) et la perméabilité membranaire apoptotique (YO-PRO-1). Le RRBS a profilé la méthylation CpG, avec une analyse ciblée des éléments Alu contenant des motifs ERE.
Résultats. Dans les spermatozoïdes non triés, les profils globaux de méthylation étaient comparables entre hommes infertiles (AGD courte) et donneurs fertiles. En revanche, dans les fractions triées, les hommes infertiles présentaient un enrichissement en Alu-ERE hypométhylés et un déficit en Alu-ERE hyperméthylés dans les sous-populations CMA3 positives et YO-PRO-1 positives.
Conclusions. Une perturbation prénatale estrogénique/anti-androgénique, reflétée à l’âge adulte par une AGD courte, pourrait laisser une empreinte durable dans la méthylation d’éléments répétitifs sensibles aux estrogènes dans le sperme. Ces anomalies, concentrées dans les spermatozoïdes à maturation chromatinienne défectueuse ou en apoptose débutante, pourraient contribuer à une baisse de compétence fécondante et à un risque épigénétique intergénérationnel. Des pistes de validation sont proposées.
The testicular dysgenesis syndrome (TDS) hypothesis posits that a spectrum of male reproductive disorders—cryptorchidism, hypospadias, low sperm counts/poor semen quality and testicular germ cell cancer—share origins in disturbed fetal testis development driven by gene–environment interactions [1,2]. A practical anthropometric proxy of that early hormonal milieu is anogenital distance (AGD), which is longer in males, established in utero, and trackable across life [3–6]. In humans, shorter male AGD has been associated with cryptorchidism and hypospadias in infancy, and with lower semen quality and reduced testicular volume in adulthood [3–6]. (For contemporary overviews linking AGD with male reproductive outcomes see [3–6].)
Many endocrine-disrupting chemicals (EDCs) with widespread human exposure (e.g., phthalates, bisphenol A) can act via estrogen receptor pathways or antiandrogenic mechanisms during the fetal masculinization window, and several birth cohort studies associate prenatal phthalate exposure with shorter male AGD [7–12].
A critical mechanistic puzzle is: How do transient fetal exposures produce enduring molecular changes that persist into adult spermatogenesis? One compelling answer is epigenetic reprogramming in the postimplantation embryo. During weeks 7–17 of human development, PGCs undergo profound, genome-wide DNA demethylation followed by sex-specific re-methylation; transposable elements show distinctive and dynamic methylation behavior during this time [13–15].
Notably, transposable elements (TEs)—especially primate-specific Alu repeats—are not inert “junk,” but major contributors to the mammalian regulatory landscape; numerous transcription factor binding sites, including those for estrogen receptor α (ESR1), derive from TEs [16–18] If prenatal estrogenic signaling targets TE-borne ER response elements (EREs) during the PGC reprogramming window, persistent, locus-selective methylation scars could plausibly be carried forward into adult sperm.
Here we integrate a Basel clinical study framework with the broader literature to examine whether infertile men with shortened AGD harbor altered DNA methylation at ER-responsive repetitive elements in sperm, and how such alterations relate to chromatin maturity and apoptosis at the single-cell fraction level [19].
AGD is established by the androgen surge in the masculinization programming window and remains sexually dimorphic throughout life. Epidemiologic studies in men link shorter AGD to poorer semen quality and lower testicular volume, while pediatric studies associate shorter AGD with cryptorchidism and hypospadias [3–6].
Prenatal phthalate exposure has been associated with shorter AGD in boys (notably in U.S. cohorts), and BPA/other EDCs can signal through ERα/ERβ with genomic actions at EREs and non-genomic membrane-initiated cascades [7–12, 20–22]. This fits TDS, where fetal testis endocrine dysfunction seeds later dysfunctions [1,2].
Repetitive elements (e.g., Alu, LINE-1) are abundant in the human genome. TEs seed transcription-factor binding motifs and shape regulatory innovation [16–18]. Crucially, many ER binding sites in human cells map to TE-derived sequences; ERE-like motifs are over-represented in Alu families, providing a substrate for estrogen-dependent regulation and potential targets for EDCs [16–18].
Human PGCs undergo near-global DNA methylation erasure followed by sex-specific re-methylation during fetal development, with TEs displaying family-specific dynamics [13–15]. If estrogenic/antiandrogenic signals alter the establishment of methylation at Alu-ERE loci, such changes could persist into the adult germline, making mature sperm a biospecimen to read out those early events.
Infertile men were recruited from a university fertility clinic; AGD (anus-to-scrotum) and testicular volume were measured. Fertile sperm donors served as controls. Because downstream assays required sufficient cell numbers, men with normozoospermia or mild oligozoospermia were preferentially included. Shortened AGD defined the infertile patient group, consistent with TDS epidemiology [3–6].
To reduce heterogeneity, ejaculates were sorted by flow cytometry into subpopulations using:
Genome-wide CpG methylation was profiled by reduced-representation bisulfite sequencing (RRBS)—an established, cost-effective method enriching for CpG-dense regions while preserving quantitative methylation calls at single-CpG resolution [25–27]. Bioinformatics focused on Alu repeats carrying canonical/pseudo-ERE motifs and, when available, ER ChIP-seq-supported sites (to enrich for bona fide estrogen-responsive repeats). The primary contrasts were between infertile men (short AGD) versus fertile donors, both globally and within sorted subfractions (CMA3^high/low and YO-PRO-1^pos/neg).
These observations provide a mechanistic bridge among short AGD, EDC/ER signaling, and sperm functional deficits, consistent with TDS. (For background linking AGD to adult semen/testis phenotypes see [3–6]; for TE-derived ER regulatory sites see [16–18]; for CMA3/apoptosis markers see [23,24].
During PGC reprogramming, TEs undergo dynamic methylation. If the embryo/fetus experiences estrogenic/antiandrogenic perturbation (e.g., phthalates, BPA), ER signaling may transiently bind ERE-like motifs within Alu repeats, locally affecting de novo methyltransferase access/activity. The result would be stable hypomethylation/hypermethylation at specific Alu-ERE loci that persist into spermatogenesis. In adulthood—especially under suboptimal testicular environments consonant with the TDS spectrum—those loci may co-segregate with spermatozoa showing chromatin remodeling defects (CMA3^high) or apoptotic features (YO-PRO-1^pos), amplifying functional consequences.
Spermiogenesis involves histone–protamine exchange; failures in this process yield protamine deficiency and retained histones, captured by CMA3 staining and linked to impaired DNA packaging and fertility [23]. Apoptotic signaling in sperm (YO-PRO-1 positivity) correlates with DNA fragmentation and reduced fertilizing capacity [24]. If Alu-ERE methylation lesions predispose to faulty chromatin remodeling or heightened DNA damage response during spermiogenesis, they would enrich within these “abnormal” subpopulations.
Short AGD has been associated with cryptorchidism/hypospadias and impaired semen quality/testicular size, consistent with TDS [1–6]. The Alu-ERE methylation signature offers a molecular substrate for that syndrome—an imprintable, ER-tunable repetitive element network perturbed during the fetal masculinization window and expressed later as sperm functional deficits.
Standardized AGD measurement protocols, blind to case/control status, minimize misclassification. Notably, AGD is only weakly related to adult BMI and can be reliably measured across adulthood [4–6]. Testicular volume by ultrasound/orchidometer provides a concurrent functional correlate.
CMA3 and YO-PRO-1 are widely used markers in andrology laboratories: CMA3 positively correlates with protamine deficiency and adverse semen parameters [23]; YO-PRO-1 is validated as a sperm apoptosis/early membrane compromise marker with associations to DNA fragmentation [24].
RRBS captures CpG-rich portions of the genome and, when paired with repeat-aware mapping, can robustly quantify methylation within Alu subfamilies [25–27]. Orthogonal methods (e.g., targeted bisulfite amplicon sequencing at sentinel Alu-EREs; Nanopore methyl-calling spanning repeats) can validate index findings.
Alu consensus contains ERE-like motifs; multiple ER cistromes show strong enrichment in TEs [16–18]. Filtering Alu instances by ERE motif quality and ER ChIP support increases specificity for estrogen-responsive elements while controlling for the high copy number of Alu.
Replication and expansion: Multi-center replication with larger, age-balanced cohorts; inclusion of cryptorchidism/hypospadias histories to position findings within the TDS spectrum.
2. Orthogonal validation: Amplicon bisulfite and Nanopore methyl-calling at sentinel Alu-EREs across sperm fractions and in testicular germ-cell subtypes.
3. Functional tests: CRISPR-dCas9-DNMT/TET editing to modulate Alu-ERE methylation in spermatid-like cells and read out effects on chromatin compaction, DNA damage, and apoptosis.
4. Exposure biology: In vitro human gonadal organoids/PGC-like cells exposed to ER agonists/antagonists (and phthalates/BPA) to model locus-specific methylation changes at repeats.
5. Clinical translation: Develop a composite risk score (AGD + sperm subfraction proportions + repeat methylation signature) for idiopathic infertility counseling and ART pathway selection.
Men who present with shortened AGD and infertility frequently reside within the TDS continuum, reflecting disrupted fetal testis programming. A mechanistically coherent, testable model—supported by clinical methylome profiling—is that estrogen-responsive Alu elements in sperm carry persistent methylation alterations in such men, particularly within chromatin-immature and apoptosis-prone sperm subpopulations. This model integrates fetal endocrine disruption, PGC epigenetic reprogramming, and adult sperm function, offering a route to molecular biomarkers and preventive strategies. Translational next steps include multicenter validation, orthogonal epigenomics, and functional perturbation of Alu-ERE methylation to establish causality and clinical utility.
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 none
We acknowledge the Swiss Center for Applied Human Toxicology (SCAHT) framework; clinical phenotyping and sorting teams; genomic and bioinformatics collaborators; and the participating patients and donors.
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