5th International Andrology Symposium
5th International Andrology Symposium
Jörgen Thorup
Univ. Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendorine Brain, UMR_S1172, Lille, France
Correspondence: Dr. Vincent Prévot, Ph.D. UMR_S1172, Lille, France
Cryptorchidism affects ~2–5% of full-term male infants and remains a leading cause of impaired spermatogenesis in adulthood despite contemporary surgical timing. Quantitative histology shows that a subset of cryptorchid testes have markedly reduced germ cells per tubular cross-section (G/T) in early childhood and, in the most severe cases, Sertoli-cell-only (SCO) histology—changes that predict poor fertility regardless of orchiopexy or adjunctive hormonal therapy. Preservation of future reproductive potential for prepubertal boys who cannot produce sperm therefore demands strategies that bank the spermatogonial stem cell (SSC) compartment. Since 2002, hospital programs—first pioneered in Brussels—have offered testicular tissue cryopreservation (TTC) on an experimental basis to prepubertal boys at high risk of infertility, initially those facing gonadotoxic therapies and, progressively, selected boys with cryptorchidism. Parallel laboratory advances have established: (i) long-term survival of human SSCs after xenotransplantation to murine hosts; (ii) in vitro propagation of human spermatogonia; (iii) viability and endocrine functionality of cryopreserved prepubertal human testicular tissue; and (iv) full translational feasibility in a non-human primate model culminating in a live-born rhesus macaque from sperm derived after autologous grafting of cryopreserved prepubertal testis tissue.
Over 25 years, the Copenhagen program and collaborators have contributed key clinical and laboratory milestones: defining age-sensitive germ cell depletion, demonstrating FSH-responsive ex vivo cultures, developing human TTC protocols for boys with cryptorchidism, and characterizing biomarker correlations (notably inhibin B) with G/T to guide selection.
Recent prospective series from Copenhagen (2014–2022) show TTC can be integrated around orchiopexy with high parental acceptance and robust tissue quality, although clinical fertility restoration in humans remains unproven and ethically complex.
In this paper the author argues that TTC may reasonably be considered for a narrowly defined subset of boys with cryptorchidism—specifically those with bilateral disease and severely reduced G/T (≈≤0.2–0.3) or concordant biomarker profiles (very low inhibin B), after thorough counseling that emphasizes experimental status, uncertain timelines, and alternatives. The
author synthesizes historical context, mechanistic rationale, translational evidence, clinical selection frameworks, ethics, and a pragmatic pathway to implementation within pediatric andrology services. The conclusion balances prudence with progress: offer TTC selectively today while powering registries and translational pipelines that make tomorrow’s restoration options safe and real.
Key words Cryptorchidism, testis, cryopreservation
La cryptorchidie touche ~2–5 % des garçons nés à terme et demeure une cause majeure d’altération de la spermatogenèse à l’âge adulte malgré l’optimisation du calendrier opératoire. L’histologie quantitative montre qu’un sous-groupe de testicules cryptorchides présente, dès la petite enfance, une réduction marquée du nombre de cellules germinales par coupe tubulaire (G/T) et, dans les cas les plus sévères, un aspect Sertoli-cell-only (SCO), prédictifs d’une fertilité médiocre indépendamment de l’orchidopexie ou de toute hormonothérapie adjuvante. La préservation du potentiel reproducteur chez les garçons prépubères, incapables de produire du sperme, requiert donc des stratégies visant à conserver le compartiment des cellules souches spermatogoniales (SSCs).
Depuis 2002, des programmes hospitaliers—initiés à Bruxelles—proposent la cryoconservation de tissu testiculaire (TTC) à des garçons prépubères à haut risque d’infertilité, initialement avant traitements gonadotoxiques puis, progressivement, chez certains garçons cryptorchides. Parallèlement, des avancées précliniques ont démontré : (i) la survie à long terme de SSCs humaines après xénogreffe murine ; (ii) la propagation in vitro de spermatogonies humaines ; (iii) la viabilité et la fonctionnalité endocrine du tissu testiculaire prépubère cryoconservé ; et (iv) la faisabilité translationnelle complète dans un modèle de primate non humain, avec naissance d’un macaque à partir de sperme dérivé d’un greffon autologue.
Depuis 25 ans, le programme de Copenhague et ses partenaires ont apporté des contributions essentielles : définition de l’appauvrissement germinal selon l’âge, démonstration de cultures ex vivo FSH-sensibles, développement de protocoles TTC pour la cryptorchidie, et identification d’associations biomarqueurs–histologie (notamment inhibine B ↔ G/T) pour guider la sélection. Les séries prospectives récentes (2014–2022) montrent que le TTC peut être intégré autour de l’orchidopexie, avec une forte acceptabilité parentale et une qualité tissulaire satisfaisante, bien qu’aucune restauration de fertilité humaine ne soit encore démontrée.
L’auteur soutient que le TTC peut être envisagé chez un sous-ensemble restreint de garçons cryptorchides—ceux présentant une atteinte bilatérale et un G/T sévèrement réduit (≈≤0,2–0,3) ou des biomarqueurs concordants (inhibine B très basse)—après un conseil éclairé insistant sur le caractère expérimental, les incertitudes temporelles et les alternatives. Cette contribution intègre le contexte historique, la justification mécanistique, les données translationnelles, les critères de sélection, les considérations éthiques et un cadre pragmatique d’implantation en andrologie pédiatrique. La conclusion conjugue prudence et progrès : offrir un TTC sélectif aujourd’hui tout en alimentant les registres et pipelines translationnels nécessaires pour rendre les options futures sûres et effectives.
Mots-clés: Cryptorchidie, testicule, cryoconservation
Cryptorchidism—testicular maldescent—disrupts the temperature-sensitive milieu needed for neonatal gonocyte transformation and SSC establishment. Quantitative histology from classic and contemporary cohorts demonstrates that undescended testes often have reduced germ cells per tubular cross-section, with some progressing to SCO tubules if correction is delayed beyond infancy. These lesions are strongly associated with subfertility in adulthood. Early work from Copenhagen (and collaborators) quantified these deficits across thousands of boys and highlighted a steep drop in G/T after the first year of life, strengthening international guidance for orchiopexy within 6–12 months. [1].
Despite earlier surgery, 20–25% of boys with nonsyndromic cryptorchidism are still at risk of compromised fertility potential on histological and hormonal grounds. Bilateral disease and profoundly low G/T carry the greatest risk. These observations compel us to consider fertility preservation strategies before irreversible depletion of the SSC niche. [2,3].
Prepubertal boys cannot bank semen. For adolescents who can, semen cryopreservation remains first-line. But for those before spermarche, the only way to safeguard genetic fatherhood is to bank tissue that contains SSCs—either as intact tissue fragments or isolated cell suspensions—until technologies mature to reinstate spermatogenesis. This logic mirrors the path taken by ovarian tissue cryopreservation in girls, which evolved from experimental to accepted care in many jurisdictions. The male analogue is TTC, still experimental but rapidly systematized across leading centers since 2002. [4,5].
The Universitair Ziekenhuis (UZ) Brussel launched the world’s first clinical program to bank testicular tissue for prepubertal boys at risk of infertility in 2002, initially targeting pediatric oncology and bone marrow transplantation populations. Over two decades the Brussels group standardized slow-freezing protocols, built governance and ethics frameworks, and tracked long-term safety of biopsy and pubertal development. The program has now enabled the first approved re-implantations in adult survivors, reflecting a maturation of the translational pipeline. [6,7].
Laboratory tracks progressed in parallel across species: murine organotypic cultures established cryo-methods; xenografting of human testis tissue/cells into immunodeficient mice demonstrated long-term survival and proliferation of human spermatogonia (though not full meiosis); and adult human testis studies clarified propagation conditions for spermatogonia. These steps forged feasibility, safety, and readouts needed for clinical protocols. [8,9].
Well before the widespread adoption of TTC, the Copenhagen team-built ex vivo culture systems for cryptorchid testis biopsies, showing that FSH maintained tubule structure and transiently increased germ cell numbers, while LH did not confer the same benefit—suggesting a Sertoli-centric nursing effect pertinent to early human SSC biology. Subsequent work demonstrated that intact testicular tissue from young boys with cryptorchidism tolerates cryopreservation with surviving spermatogonia and sustained testis-specific hormone production in vitro-evidence that underpins the practical decision to bank tissue at the time of orchiopexy. [10,11].
The perinatal testis undergoes gonocyte-to-spermatogonia transformation, colonizing the basement membrane as AD (type A dark) spermatogonia, a proposed SSC surrogate in early childhood. In cryptorchidism, heat stress, endocrine milieu, and local paracrine disruption impede this transformation, reducing the SSC pool and the G/T metric. Multiple studies, including Thorup and colleagues, associate low counts of PLAP-positive gonocytes and AD spermatogonia with later infertility risk. If the SSC pool is small by 6–12 months, even perfect surgical repositioning cannot fully restore it. TTC aims to bank the residual SSCs before attrition completes.[12].
A critical milestone came in 2002, when adult human spermatogonial stem cells transplanted into nude mouse testes survived at least 6 months and proliferated in the first month.
Colonization rates were high (>70% of recipient testes), though meiosis did not occur in the murine environment. This confirmed that human SSCs can survive cryoprocessing and transplantation and offered an assay for potency. The Copenhagen group later confirmed the technique using cryopreserved testicular tissue from prepubertal boys. [8,13].
Techniques to isolate and expand human spermatogonia have advanced steadily—from early clusters characterized by germ-cell markers to xeno-free systems and bioengineered scaffolds. Collectively, they suggest that banked human SSCs can be amplified to clinically meaningful numbers, although genomic integrity, epigenetics, and differentiation competence remain under scrutiny. [14-16].
In 2019, a Science paper reported full translational proof in rhesus macaques: prepubertal testis tissue was cryopreserved, later autografted under scrotal/back skin at puberty, matured for ~1 year, and then used for TESE-ICSI to achieve a live birth—the female infant Grady. This closes the loop from cryostorage to offspring in a primate whose testicular physiology approximates humans, transforming TTC from concept to demonstrated reproductive potential (albeit not yet in humans).[17].
Slow-freezing protocols have been optimized for clinical-grade (GMP-adjacent) conditions suitable for pediatric programs. Controlled slow-freezing with DMSO-based cryoprotectants maintains morphology, SSC marker expression, and post-thaw viability. Recent reports emphasize xeno-free media for downstream clinical translation.[18].
From 2014 to 2022, Rigshospitalet (Copenhagen) implemented TTC around orchiopexy for 56 boys with cryptorchidism—predominantly bilateral cases—explicitly to preserve reproductive potential in those deemed at highest risk by histology and hormonal profile. Median age at orchiopexy was ~1.3 years. Germ cells were detected in ~98–100% of biopsies, with a median G/T ≈0.39 (range 0–2.88), indicating that even high-risk cryptorchid testes typically retain some SSCs to bank. [19].
Parental acceptance rates for TTC in Danish cohorts have been very high, reflecting the perceived value of “banking a chance” despite the technique’s experimental status. In a 2020 study focused on cryptorchid boys, over 90% of families offered TTC consented. This aligns with similar acceptance levels in broader pediatric cohorts internationally. The counseling model is therefore crucial: obtain informed consent that covers experimental status, storage logistics, costs, and re-implantation uncertainties. [20,21].
The Copenhagen program has long integrated histology (G/T, AD spermatogonia, SCO) and hormones to stratify risk. Inhibin B correlates positively with G/T and Sertoli-cell number; in bilateral cryptorchidism, very low inhibin B levels have a high predictive value for globally impaired G/T. These data support a biomarker-assisted selection model when biopsy is not favored or must be minimized. [22].
The Copenhagen work with long-term organ culture of cryptorchid biopsies demonstrated that FSH (but not necessarily LH) can rescue or maintain germ cell numbers over 1–3 weeks, preserving tubule structure. This is consistent with the concept that Sertoli cells orchestrate the microenvironment for SSC survival. Although ex vivo rescue is not a clinical therapy today, such data de-risk culture steps before cryopreservation and in future ex vivo amplification workflows. [10].
We subsequently showed that cryopreserved prepubertal testis tissue from boys with cryptorchidism preserves SSC viability and testis-specific steroidogenic function after thaw, again validating the biological plausibility of TTC in this population. [11].
Drawing on the translational and clinical evidence above, TTC should be considered in the following scenarios:
Combine TTC with orchiopexy under the same anesthetic to minimize burden: take a small wedge biopsy (typically a few 1–3 mm fragments) from the most affected testis (or from each in bilateral cases, per protocol) when a diagnostic biopsy is clinically indicated or when histology/biomarker risk is high. This mirrors established oncology TTC workflows and leverages operating room sterility and logistics. [26].
Most pediatric centers bank intact tissue fragments using controlled slow-freezing. Some also process cell suspensions enriched for spermatogonia. Both approaches have preclinical justification; intact tissue keeps niche architecture for future autografting, while suspensions may support intratubular transplantation or in vitro spermatogenesis once clinically validated. Programs should specify standard of procedures and chain-of-custody. [27].
Clinical protocols typically employ DMSO-based slow-freezing with stepwise cooling, validated by post-thaw viability, morphology, and marker assays. Emerging xeno-free media address regulatory concerns. Documentation must include fragment counts, dimensions, cryoprotectant exposure, and storage location, enabling future traceability for re-implantation or lab expansion. [18].
TTC for cryptorchidism is experimental. Families must receive transparent counseling: there is no human live birth from prepubertal human tissue to date, although the primate success suggests feasibility. Consent should cover unknowns, alternative strategies (e.g., donor sperm, adoption), biopsy risks (low but non-zero), storage obligations, and future decision points. High parental acceptance rates underscore demand but must not substitute for balanced information. [6].
Programs must guard against inequity. TTC should be embedded in publicly accountable pathways with clear selection criteria to avoid offering an experimental procedure only to the well-resourced. International collaborations (e.g., registries, shared standard of procedures) can harmonize standards and minimize therapeutic misconception. [26].
TTC creates decades-long custodianship of gametogenic tissue. Governance should codify ownership, consent at majority, disposition options, cross-border storage, and privacy. These are not abstract: Brussels’ 2002 cohort is now reaching reproductive age, and initial reimplantations have received ethical approval—a harbinger of what cryptorchid cohorts may face in 10–20 years. [31].
Pre-operative risk stratification: Bilateral cryptorchidism, delayed surgery (>1 year), abnormal testicular volume, very low inhibin B. Offer shared decision-making about TTC if risk is high. [22].
Intra-operative tissue acquisition: During orchiopexy, harvest 1–3 small fragments (~1–3 mm) with atraumatic technique from the most at-risk testis. Send a diagnostic piece for histology; allocate remaining to cryo. [26].
Cryopreservation: Controlled slow-freezing with validated standard of procedures; store in liquid nitrogen; log metadata for future traceability. Consider xeno-free adaptations [18].
Documentation and follow-up: Provide families with a TTC passport (what was stored, where, for how long), and enroll in longitudinal registries.
Adolescent/adult transition: Reassess fertility; discuss restoration options (to be determined by future clinical trials).
“No human live births yet—why offer TTC?”
True—but prepubertal ovarian tissue was once in the same position and is now standard in many settings. The rhesus primate proof-of-principle shows that tissue banking can lead to live offspring in a closely related species. In cryptorchidism, tissue is benign, simplifying later fertility restoration techniques compared to oncology. Offering TTC selectively preserves options for a group otherwise facing irreversible infertility. [17,23].
“Biopsy harms the small testis.”
Complication rates of small wedge biopsies are low in experienced hands, particularly when combined with planned surgery. Longitudinal studies (e.g., Brussels) track pubertal development without overt harm, though vigilance is warranted. [32].
“Markers are imperfect.”
Yes; G/T and inhibin B are not infallible. But convergent histology + hormonal profiles highlight those at highest risk—precisely where the risk-benefit calculus favors offering TTC with robust consent. [3].
“Costs and storage commitments are significant.”
Correct. Programs should ensure transparent funding, equitable access, and contingency plans for storage. Research funding should shoulder much of the burden while TTC remains experimental. [4].
The narrative from Vassalli Hall captures the arc of this field:
2002 Brussels program inaugurates TTC for prepubertal boys facing gonadotoxic therapies, inspired by ovarian tissue success. [6].
Early Copenhagen culture experiments show FSH supports germ cells in cryptorchid biopsies; subsequent cryopreservation studies demonstrate post-thaw viability and hormone production in vitro. [10,11].
Adult human work in Philadelphia demonstrates long-term survival of human SSCs in nude mouse testes, establishing the key potency readout. [8].
Propagation of human spermatogonia in vitro evolves to cluster-based systems and, later, xeno-free conditions in Copenhagen’s post-doctoral projects. [14,15].
Winston-Salem and other centers implement clinical TTC around 2014–2016, reporting high parental acceptance. Copenhagen’s cryptorchid cohort mirrors this pattern. [20,21].
The 2019 rhesus macaque breakthrough—Grady—establishes end-to-end feasibility from cryopreserved prepubertal tissue to offspring. [17].
Biomarker work consolidates inhibin B as a practical correlate of G/T, offering a less invasive path to selection. [22].
These points reinforce the thesis: selective TTC is a defensible, ethically sound offer to certain cryptorchid boys today, with a credible translational horizon.
Cryptorchidism continues to confer substantial infertility risk for a meaningful minority of affected boys, predominantly those with bilateral disease and severe early germ cell depletion. While orchiopexy by 6–12 months mitigates risk, it cannot restore an SSC reservoir that was never properly established. Over the last 25 years, a translational pipeline has cohered: human SSC survival in xenografts, in vitro propagation, validated cryopreservation, and a non-human primate live birth from cryopreserved prepubertal testis tissue. Clinical programs—first in Brussels and subsequently worldwide—have embedded TTC into pediatric care for high-risk groups, with Copenhagen extending this rationale to selected cryptorchid boys based on histology and biomarkers.
Given this evidence, it is reasonable today to offer TTC to a narrowly defined subgroup of boys with cryptorchidism—bilateral, severely low G/T (≈≤0.2–0.3) or very low inhibin B—provided families consent with full understanding that (i) TTC is experimental, (ii) no human births from such tissue have yet been reported, and (iii) restoration is most plausibly via autografting or SSC transplantation as clinical trials emerge. This approach preserves future options without overpromising, advances equitable access via structured protocols, and aligns with a 20-year horizon in which at least one restoration pathway is likely to become clinically usable. In other words, banking potential now is a rational step toward realizing fertility later for the most vulnerable cryptorchid boys.
I thank colleagues in pediatric urology, reproductive biology, and andrology in Copenhagen, Brussels, Philadelphia, Pittsburgh, Amsterdam, and Winston-Salem whose work has propelled this field; and the families who entrust us with safeguarding their sons’ future fertility.
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