Suarez, P. | bioRxiv Science

bioRxiv2026

“Mapping of Gonadal Development in Cryptorchidism: UTF-1 and Germ Cell Dysgenesis”

Study questionHow does cryptorchidism affect germ cell development and UTF-1-mediated pluripotency potential at the time of orchiopexy? Summary answerCryptorchidism was associated with the following germ cell abnormalities: germ cell clustering with many cords/tubules lacking germ cells and reduced UTF-1 expression, suggesting limited germ cell differentiation into spermatogonia What is known alreadyCryptorchidism, affecting 1.6-9% of male newborns, is known to increase the risk of infertility and testicular cancer due to abnormal germ cell development. Germ cells and pluripotent stem cell gene, UTF-1, play critical roles in spermatogonia differentiation, self-renewal, and spermatogenesis. No prior study has evaluated the testicular development by immunohistochemically mapping of these cell populations, Study design, size, durationA cross-sectional study of 31 postnatal cryptorchid testis and 5 age-matched scrotal testicular biopsies obtained from UCSFs pathology department performed between 1993-2023. Participants/materials, setting, methodsSpecimens were grouped by age at surgery (6-18 months, 19 months-7 years, 8-12 years, and [≥]13 years) and testis location (palpable vs. non-palpable). Scrotal prepubertal testis biopsies were sourced through the Pedi-LIFE program, a fertility preservation research biobank, with at least one control per age group. Immunohistochemistry was performed to stain specimens for germ cell (DDX4, OCT4, TSPY), pluripotent cell marker (UTF-1), as well as other key testis cell markers (A-actin, AR, P450, Sox-9), with staining graded based on control expression levels. The number of germ cells per seminiferous tubule was quantified and compared across anatomical locations using appropriate statistical analyses. Main results and the role of chanceThis study included 36 specimens, comprising 31 cryptorchid testes (86%) and 5 scrotal control testes (16%). The cryptorchid group exhibited testicular dysgenesis and reduced germ cell expression, correlated with increased age and testis location. Qualitative assessment revealed reduced germ cell expression across all ages in cryptorchid testes. The number of germ cells per tubule was markedly reduced in cryptorchid compared with scrotal testes after 19 months of age for DDX4, TSPY, and UTF-1. Germ cell clusters were identified in 15 out of 31 cryptorchid specimens (48%) stained for DDX4 and TSPY. UTF-1 expression was lower in cryptorchid testes across all age groups. No significant differences were noted in other testicular cell markers. Large scale dataNA Limitations, reasons for cautionFirst, the power and generalizability of the study are limited by the availability of specimens within each age group, particularly for scrotal testes, as biopsies of these tissues are not routinely performed. Second, a cross-sectional study design limits a longitudinal comparison to evaluate changes in marker expression, delayed maturation, or irreversible germ cell loss. Third, immunohistochemistry data is semi-quantitative, and protein detection is affected by antibody sensitivity and tissue preservation and influenced by antibody sensitivity. Lastly, scrotal testis used as controls were obtained from cryopreserved tissue from patients with other unrelated pathology, which may influence histological profiles. Wider implications of the findingsCollectively, our findings support a model in which cryptorchidism involves both germ cell depletion and disrupted SSC lineage formation, with UTF-1 downregulation and germ cell clustering as early signatures of testicular dysgenesis. These features may help identify high-risk patients for worsening gonadal dysgenesis and infertility and can provide a rationale for earlier orchiopexy or SSC-preserving strategies. Study funding/competing interest(s)The authors declare no conflicts of interest and received no funding for this study. Data Availability StatementThe data underlying this article cannot be shared publicly due to ethical and legal restrictions related to the use of human tissue specimens, which may compromise donor privacy and confidentiality. Data are available from the corresponding author upon reasonable request and subject to institutional and ethical approvals.

Developmental Biology↗

bioRxiv2025

Development and validation of an ultra-low-cost, open source normothermic ex vivo organ perfusion platform

BackgroundNormothermic ex vivo organ perfusion (NEVOP) promises to catalyze organ preservation, therapeutic discovery, and organ-specific disease modeling. Existing technology platforms remain inaccessible for research due to restricted access to commercial organ perfusion devices, high costs of both devices and proprietary consumables, and steep technical learning curves. Additionally, the available technology is not optimized to perfuse smaller organs such as the kidney. MethodsTo overcome these barriers, a custom NEVOP circuit was developed using recycled, repurposed, and low-cost components. Porcine kidneys and autologous blood were used to iteratively optimize circuit design. A porcine kidney autotransplantation protocol was adapted to evaluate in vivo kidney function after ex vivo perfusion. To pilot the flexibility of this system as a multi-organ platform for ex vivo human biology, non-transplantable human donor kidney, spleen, and pancreas specimens were stably perfused using human blood products and analyzed. ResultsAn ultra low-cost NEVOP system engineered to perfuse porcine kidneys and diverse human organs (kidney, pancreas, and spleen) sustained viable organs for up to 24 hours with evidence of both function and viability. Key innovations included a parallel flow resistor to facilitate low-flow perfusion in non-heparinized organs and a containment bag with adjustable magnets to provide vascular stability and recycling of venous overflow. The circuit costs less than 1,500USD to construct, and porcine kidneys perfused for 24 hours on this platform demonstrated healthy in vivo function upon autotransplantation. ConclusionsCustom NEVOP platforms constitute novel and potentially transformative research platforms which use low-cost and readily available materials. Paired with access to non-transplantable research organs from altruistic donors, this model provides a road map for investigators to advance biomedical discovery and human ex vivo biology.

bioengineering↗

bioRxiv2020

Self-antigen driven affinity maturation is required for pathogenic monovalent IgG4 autoantibody development

Pathogenic IgG4 autoantibodies in autoimmune myasthenia gravis (MG) are functionally monovalent as a result of Fab-arm exchange. The origin and development of these unique autoantibodies are not well understood. We examined MG patient-derived monoclonal autoantibodies (mAbs), their corresponding germline-encoded unmutated common ancestors (UCA) and monovalent antigen-binding fragments (Fabs) to investigate how antigen-driven affinity maturation contributes to both binding and immunopathology. Mature mAbs, their UCA counterparts and mature monovalent Fabs bound to the autoantigen and retained their pathogenic capacity. However, monovalent UCA Fabs still bound the autoantigen but lost their pathogenic capacity. The mature Fabs were characterized by very high affinity (sub-nanomolar) driven by a rapid on-rate and slow off-rate. However, the UCA affinity was approximately 100-fold less than that of the mature Fabs, which was driven by a rapid off-rate. Crystal structures of two Fabs shed light on how mutations acquired during affinity maturation may contribute to increased MuSK binding affinity. These collective findings indicate that the autoantigen initiates the autoimmune response in MuSK MG and drives autoimmunity through the accumulation of somatic hypermutation such that monovalent IgG4 Fab-arm exchanged MG autoantibodies reach a high affinity threshold required for pathogenic capacity. SummaryIgG4 autoantibodies in autoimmune myasthenia gravis are functionally monovalent, requiring a high affinity threshold featuring fast on/slow off rates, to reach pathogenic capacity. This capacity is dependent on self-antigen initiated and driven maturation, which includes the accumulation of indispensable somatic hypermutations that may alter electrostatic interactions with the antigen.

immunology↗