Endocrine disruptors (EDs) are implicated in adverse developmental and reproductive outcomes, yet their identification remains a major challenge in chemical safety assessment. Current testing strategies rely heavily on animal models, which are constrained by ethical concerns, interspecies differences, and limited mechanistic resolution but justified by the complexity of the endocrine system and its physiology. Capturing the complex biology of intact organisms and incorporating toxicokinetic properties in alternative test methods is challenging. To address this, the European Partnership for the Assessment of Risks from Chemicals (PARC) is advancing the development and regulatory integration of new approach methodologies (NAMs). This project specifically contributes by developing and validating human-relevant NAMs to identify key aspects of endocrine disruption relevant to developmental and reproductive toxicity (DART). Key innovative activities include predictive modeling, refinement of zebrafish and amphibian embryo assays, and establishment of advanced in vitro systems for assessing toxicity in the oocyte, testis, placenta, and brain. By combining mechanistic insights with multi-modality and high throughput testing strategies, this work aims to improve the predictive power and regulatory utility of NAMs for ED identification within the One Health paradigm.

Male reproductive health has declined over recent decades, characterized by rising rates of reproductive birth defects, reduced semen quality, and an increased risk of testicular cancer. This global trend has been linked to exposure to endocrine-disrupting chemicals (EDCs), which can interfere with the endocrine system including disrupting steroidogenesis, the process by which steroid hormones are synthesized. Steroid hormones, not least testosterone, are essential for male sex differentiation, masculinization, and spermatogenesis. During human fetal development, Leydig and Sertoli cells of the testis together produce androgens, whereas in adults, Leydig cells can synthesize testosterone alone. EDCs can disrupt this process through various mechanisms. Advances in in vitro models of the steroidogenic system, particularly three-dimensional (3D) cell cultures, have provided a more accurate representation of the in vivo environment, enabling more realistic responses to external stimuli. These models offer a powerful tool for studying the effects of EDCs on steroidogenesis and will deepen our understanding of the mechanisms underlying endocrine disruption, knowledge that is important in developing new strategies for chemical safety assessment. This review provides a comprehensive analysis of the steroidogenic system, with a focus on key enzymes involved. The current regulatory test strategies to reduce the impact of EDCs on male fertility is discussed. Furthermore, 3D testis models are compared, highlighting their strengths and limitations for testing effects on testis steroidogenesis. Emphasis is placed on the urgent need for innovative, practical, and cost-effective alternatives to traditional in vivo testing to improve chemical risk assessments.

STUDY QUESTION:Can cryopreserved primary testicular somatic cells from childhood cancer survivors with severely decreased fertility potential be reprogrammed into human-induced pluripotent stem cells (hiPSCs) competent for efficient specification into early human germ cells? SUMMARY ANSWER:Primary testicular somatic cells from cryopreserved testicular samples with severely compromised spermatogonial pools can be reprogrammed into hiPSCs using a non-genome-integrating, feeder-free approach and subsequently differentiated into human primordial germ cell-like cells (hPGCLCs) with high efficiency. WHAT IS KNOWN ALREADY:Infertility is one of the most concerning long-term side effects of cancer therapy in prepubertal boys, yet it remains largely unaddressed. Worldwide, biobanks storing cryopreserved immature testicular tissue are expanding to support the development of fertility preservation strategies, including future tissue and cell transplantation approaches. However, whether these samples can also serve as starting material to generate hiPSCs and subsequently differentiate into in vitro-derived germ cells remains largely unexplored. Currently, there are established protocols for non-genome-integrating reprogramming of somatic cells into hiPSCs and for hPGCLC specification that could be applied to cryopreserved testicular tissue, with special relevance for patient samples severely depleted of germ cells. STUDY DESIGN SIZE DURATION:Two biological replicates of cryopreserved prepubertal testicular tissue were used to obtain primary somatic cells, which were reprogrammed into hiPSCs and subsequently differentiated into hPGCLCs. This experimental pipeline had a duration of approximately four months. PARTICIPANTS/MATERIALS SETTING METHODS:Cryopreserved testicular tissue samples were obtained from two prepubertal cancer patients (6.2 and 6.3 years old) with depleted spermatogonial pools (spermatogonia count per round tubular cross-section of 0.04 and 0.02, and age-standardized Z-scores of -17.62 and -20.48). These samples were subsequently used to derive primary testicular somatic cell cultures, which were then reprogrammed into hiPSCs using a clinically compatible, non-genome-integrating mRNA-based method under feeder-free conditions. The resulting hiPSC lines were validated and subsequently differentiated into hPGCLCs using two different specification protocols. The transcriptomic profiles of the hiPSCs and their derived hPGCLCs were verified using bulk RNA sequencing. MAIN RESULTS AND THE ROLE OF CHANCE:Here, we present the first successful generation of hiPSCs from cryopreserved testicular somatic cells of childhood cancer patients with a severely depleted germ cell pool. We accomplished this using a non-genome-integrating mRNA-based reprogramming approach. We further demonstrated the specification of hPGCLCs from these patient-derived hiPSCs, effectively regenerating their germline. This provides proof-of-concept for a stem cell-based fertility regeneration strategy in childhood cancer survivors with non-functional or absent germ cells. LARGE SCALE DATA:N/A. LIMITATIONS REASONS FOR CAUTION:This proof-of-concept study was limited by a small sample size due to the restricted access to cryopreserved human prepubertal testicular tissues. Although multiple hiPSC clones were generated per individual, only one clone per patient was used for downstream analyses, precluding systematic assessment of intra-individual clonal variability, which remains an important aspect for future studies. While G-banding karyotyping was sufficient to validate genomic integrity in this proof‑of‑concept study, more comprehensive genetic and epigenetic analyses should be prioritized in future hiPSC and hPGCLC validation before any clinical application is considered. WIDER IMPLICATIONS OF THE FINDINGS:Childhood cancer patient-derived hiPSCs represent a powerful platform to investigate and address a broad range of long-term, cancer therapy-related complications. These hiPSCs and their derived germ cells may be used not only to develop in vitro gametogenesis protocols, but also to investigate mechanisms of cancer therapy toxicity and resistance among different patients, to identify early biomarkers of adverse outcomes, and to screen for germ cell protective agents. Importantly, cancer patient-specific hiPSCs have applications that extend well beyond germline, enabling regenerative strategies targeting other treatment-related sequelae through the generation of relevant somatic cell types. FUNDING:T.M. was supported by the Erasmus+ program, as part of the projects WORK4ALL 2023 and WORK4ALL 2024 (2023-1-PT01-KA131-HED-000121324 and 2024-1-PT01-KA131-HED-000214636). L.N.A. was supported by a Marie Skłodowska-Curie Actions Individual Fellowship (101278886: GERMFIT) from the European Commission. Y.B. was supported by the Scientific Fund Willy Gepts. K.J. was supported by the Foundation for Pediatric Research, the Finnish Cancer Society, the Swedish Childhood Cancer Foundation (KP2020-0012), and the Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00335, 2021-00079, and 2023-00380). J.-B.S. was supported by the Swedish Childhood Cancer Fund (PR2019-0123; PR2022-0115; TJ2020-0023) and the Swedish Research Council (2018-03094; 2021-02107). J.P.A.-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council, the Birgitta and Carl-Axel Rydbeck Research Grant for Paediatric Research 2024 (2024-00208), and the Scientific Fund Willy Gepts. DISCLOSURES:All authors declare no conflicts of interest.

STUDY QUESTION:Can testicular tissue from trans women (trans tissue) be used to create human testicular organoids? SUMMARY ANSWER:Testosterone-producing and cytotypic human testicular organoids with bicompartmental architecture can be successfully generated from trans tissue. WHAT IS KNOWN ALREADY:Testicular organoids are a promising tool for studying testicular function and the effects of toxicants. Immature testicular cells are currently the most efficient at forming organoids that closely recapitulate seminiferous tubule-like architecture and functions. However, the scarcity of immature human testicular tissue limits its use in high-throughput applications. Conversely, trans tissue is abundantly available and characterized by an immature phenotype. STUDY DESIGN SIZE DURATION:Trans tissue-derived organoids (trans organoids) were histologically and androgenically compared to reference organoids derived from immature (prepubertal and pubertal) and adult cisgender testicular tissues. Additionally, long-term testosterone production and gonadotrophic stimulation were assessed in trans organoids. To evaluate their cytotypic and transcriptomic resemblance to reference testicular tissue stages, trans organoids were compared at the gene expression level to prepubertal, pubertal, and adult cisgender tissues, along with their tissue of origin. PARTICIPANTS/MATERIALS SETTING METHODS:Testicular tissue samples from transgender women, as well as from prepubertal, pubertal, and adult cisgender donors, were used to generate testicular organoids and to compare organoid formation efficiency and testosterone production according to tissue origin. These samples also served as references for transcriptomic comparisons with organoids derived from transgender women's testicular tissue at Day 14 of culture. Testicular organoids were generated and cultured using 3D Petri Dish® platforms. Histochemistry and immunofluorescence staining were employed to characterize cellular composition and spatial organization. Testosterone production in culture media was assessed using electrochemiluminescence immunoassays. RNA was extracted and sequenced from organoids derived from transgender women, as well as from tissue samples of all donor groups. Deconvolution and differential gene expression analyses were performed to compare the organoids with testicular tissues across all groups. MAIN RESULTS AND THE ROLE OF CHANCE:Trans organoids form compartmentalized, cytotypic de novo tissues similar to those from pubertal testicular tissue. Additionally, trans organoids exhibit significant testosterone production, sustain this function over extended culture periods, and respond to gonadotrophic stimulation. Deconvolved bulk RNAseq data indicate that cell population proportions within these organoids are close to those in prepubertal and pubertal testicular tissues. Gene expression clusters trans organoids alongside prepubertal and trans tissues. Functional analysis reveals that trans organoids share with prepubertal, pubertal, and trans tissues varied cellular processes. Factors such as the duration of hormone therapy, the expression of anti-Müllerian hormone-an immaturity marker-within the tubules, and the proportion of peritubular myoid cells in the donor tissue were found to predict the success of trans organoid formation. LARGE SCALE DATA:The bulk RNA-seq raw and preprocessed data are stored under restricted access in the Vrije Universiteit Brussel (VUB) Institutional Data Repository (VUB/IVTD/1/000001) due to participant privacy concerns. Access to the data will be considered by contacting Prof. Yoni Baert (yoni.baert@vub.be). LIMITATIONS REASONS FOR CAUTION:Hormonal data from trans women donors were not acquired in a convenient manner for this study. Deconvolution data allow only cell proportions to be compared, not absolute numbers. WIDER IMPLICATIONS OF THE FINDINGS:This study highlights the potential of trans organoids as a novel and ethically sustainable human-based model for male reproductive health research, reproductive toxicology, and endocrine disruption studies. While trans tissue is a valuable replacement for immature tissue, further research should focus on optimizing organoid architecture, evaluating their utility in reprotoxicity testing, and promoting germ cell differentiation. STUDY FUNDING/COMPETING INTERESTS:This study was conducted with financial support from the VUB Research Council (OZR4004) to S.M.S., the Scientific Research Foundation-Flanders (G026223N) and the Scientific Fund Willy Gepts to Y.B., the Strategic Research Program 89 from the VUB to E.G., and the Mireille Aerens Chair to T.V. The authors declare no conflict of interest.

Although the impact of gender-affirming hormone therapy (GAHT) on spermatogenesis in trans women has already been studied, data on its precise effects on the testicular environment is poor. Therefore, this study aimed to characterize, through histological and transcriptomic analysis, the spermatogonial stem cell niche of 106 trans women who underwent standardized GAHT, comprising estrogens and cyproterone acetate. A partial dedifferentiation of Sertoli cells was observed, marked by the co-expression of androgen receptor and anti-Müllerian hormone which mirrors the situation in peripubertal boys. The Leydig cells also exhibited a distribution analogous to peripubertal tissue, accompanied by a reduced insulin-like factor 3 expression. Although most peritubular myoid cells expressed alpha-smooth muscle actin 2, the expression pattern was disturbed. Besides this, fibrosis was particularly evident in the tubular wall and the lumen was collapsing in most participants. A spermatogenic arrest was also observed in all participants. The transcriptomic profile of transgender tissue confirmed a loss of mature characteristics - a partial rejuvenation - of the spermatogonial stem cell niche and, in addition, detected inflammation processes occurring in the samples. The present study shows that GAHT changes the spermatogonial stem cell niche by partially rejuvenating the somatic cells and inducing fibrotic processes. These findings are important to further understand how estrogens and testosterone suppression affect the testis environment, and in the case of orchidectomized testes as medical waste material, their potential use in research.

In recent years, there has been a significant push towards the development of animal-free methods in toxicology. Despite this progress, the adoption of such methods in safety assessment practices remains limited, particularly in the context of gonadal and placental toxicity. This paper reviews current in vitro models relevant to gonadal (gametogenesis and steroidogenesis) and placental biology, and potentially applicable in developmental and reproductive toxicology (DART). Additionally, we present the results of a survey conducted among DART experts (n = 16), examining current practices and perceptions in industry regarding these in vitro methodologies. The findings indicate a predominant reliance on animal models, largely driven by regulatory requirements, despite concerns about their ability to reliably predict human outcomes. Respondents reported limited familiarity with and confidence in available in vitro models for gonadal and placental toxicity, yet expressed optimism about their future integration. These findings underscore the need for increased awareness of gonadal and placental in vitro models, particularly among DART risk assessors, to facilitate a shift toward more reliable and human relevant risk assessments.

To identify the sperm preparation procedure that selects the best sperm population for medically assisted reproduction. Prospective observational study comparing the effect of four different sperm selection procedures on various semen parameters. Unused raw semen after routine diagnostic analysis was split in four fractions and processed by four different methods: (1) density gradient centrifugation (DGC), (2) sperm wash (SW), (3) DGC followed by magnetic activated cell sorting (MACS), and (4) using a sperm separation device (SSD). Each fraction was analyzed for progressive motility, morphology, acrosome index (AI), and DNA fragmentation index (DFI). With DGC as standard of care in intraclass correlation coefficient analysis, only SSD was in strong disagreement regarding progressive motility and DFI [0.26, 95

This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.

Testes have a complex architecture that is compartmentalized into seminiferous tubules with a diameter of approximatively 200 μm in which the germ cells differentiate, surrounded by a basement membrane and interstitium. 3D bioprinting might be used to recreate the compartmentalized testicular architecture in vitro. Directed by a software program, pneumatic microextrusion printers can deposit 3D layers of hydrogel-encapsulated interstitial cells in a controlled manner by applying pressure. Once macroporous-shaped scaffolds resembling seminiferous tubules have been bioprinted with interstitial cells, the epithelial cell fraction can be seeded in the macropores to resemble the in vivo testicular architecture. Moreover, macropores can serve as a delimitation for all testicular cells to reorganize and improve the supply of nutrients to cells through the 3D constructs.

Gene editing in the murine germline is a valuable approach to investigate germ cell maturation and generate mouse models. Several studies demonstrated that CRISPR/Cas9 alters the genome of cultured male mouse germline stem cells delivered by electroporation of plasmids. Recently, we showed proof-of-principle that gene knockout can be effectively targeted in mouse germline stem cells by lipofecting Cas9:gRNA ribonucleoproteins. In this protocol, we describe a simple, fast, and cheap workflow for gene editing via the lipofection of non-integrative ribonucleoproteins in murine male germline stem cells.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available–and are currently in the process of regulatory validation–there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Gene editing in male germline stem (GS) cells is a potent tool to study spermatogenesis and to create transgenic mice. Various engineered nucleases already demonstrated the ability to modify the genome of GS cells. However, current systems are limited by technical complexity diminishing application options. To establish an easier method to mediate gene editing, we tested the lipofection of site-specific Cas9:gRNA ribonucleoprotein (RNP) complexes to knockout the enhanced green fluorescent protein (Egfp) in mouse EGFP-GS cells via non-homologous end joining. To monitor whether gene conversion through homology-directed repair events occurred, single-stranded oligodeoxynucleotides were co-lipofected to deliver a Bfp donor sequence. Results showed Egfp knockout in up to 22% of GS cells, which retained their undifferentiated status following transfection, while only less than 0.7% EGFP to BFP conversion was detected in gated GS cells. These data show that CRISPR/Cas9 RNP-based lipofection is a promising system to simply and effectively knock out genes in mouse GS cells. Understanding the genes involved in spermatogenesis could expand therapeutic opportunities for men suffering from infertility.

Abstract Study question Can our newly developed testicular organoid (TO) growth platform advance the robustness of murine TOs? Summary answer The platform resulted in more consistent TO histology. Moreover, improved germ cell survival was observed after a two-week culture with numbers comparable to fresh samples. What is known already Organ cultures have traditionally been used for in-vitro spermatogenesis (IVS) in rodents because they best preserve the testicular architecture which is pivotal in achieving IVS. However, organ cultures do not offer the ability to access and manipulate single cells, making it an inefficient model for mechanistic studies. Organoids made from testicular cell suspensions offer these features. Although TO cultures can result in organoids with compartmentalized testicular architecture, histological heterogeneity between individual TOs limits reproducibility of the results, offering unreliable readouts. Moreover, germ cell loss is characteristic during the reorganization phase. Study design, size, duration Here, we tested a new TO growth platform. Firstly, the focus was put on improving germ cell survival in TOs during tubulogenesis in the first two weeks of culture. For this, four different growth media (A-D), supplemented with other combinations or concentrations of growth factors, were compared. Next, five cell seeding densities (I-V) were tested for their ability to recreate the testicular architecture in TOs in the selected culture media. Participants/materials, setting, methods Testicular cells from 5 days old C57BL/6J mice were grown in our TO platform with alpha-MEM-based medium, previously found to support TO generation in mice (medium A). Three additional conditions were tested in their ability to improve germ cell survival during tubulogenesis (B-D). Finally, the ideal cell density (I-V) was determined based on histological resemblance to native tissue: one tubule-like structure and surrounding interstitium. Cellular reorganization and germ cell maintenance were characterized by (immuno)histochemistry. Main results and the role of chance During short-term cultures of 2 weeks, testicular cells self-assembled and compacted into organoids in our platform. Interestingly, media B and D resulted in the highest amount of germ cells (p < 0.05), comparable to the fresh control. Particularly TOs cultured in medium D also exhibited the largest surface area, indicative for better in-vitro growth. Finally, TOs that were cultured in condition D had the best histology when grown at cell density IV and V (p < 0.05). Limitations, reasons for caution Candidate factors have to be tested in their ability to elevate the meiotic blockage of germ cells typically observed in organ culture, but also in TOs. Finally, results obtained with rodents remain to be confirmed in further human studies. Wider implications of the findings The opportunities TOs offer to manipulate cells (genetic modification, inclusion and exclusion) are essential for the study of male infertility and the search for potential therapies. Moreover, they permit high-throughput screening of chemicals, thereby substantially reducing the number of animals for the high demanding reproductive toxicity and drug discovery studies. Trial registration number not applicable

Chronic exposure to arsenic may cause cancer. Many mechanisms have been suggested for arsenic carcinogenesis. Autophagy, an evolutionarily conserved cellular catabolic mechanism, has been implicated in cancer biology. Although being claimed as a type of cell death, autophagy may actually serve as a cell self-defense mechanism. In this review article, current understandings of the mechanisms of arsenic carcinogenesis, functions of autophagy and the role of autophagy in arsenic carcinogenesis are discussed.

Can improved culture conditions advance the functionality of murine testicular organoids (TOs)? Testicular cells formed spheroidal TOs resembling the functional unit of the testis and supporting meiotic entry of germ cells during long-term culture in printed macropores. Organ cultures at the air-medium interphase have traditionally been used for in-vitro spermatogenesis (IVS) in rodents because they best preserve the testicular architecture, which is pivotal in achieving IVS. However, organ cultures do not offer the ability to access and manipulate single cells, making it an inefficient model for mechanistic studies. Culturing testicular cell suspensions into organoids offer these features. Previously, testicular organoids in immersion culture resulted in testicular architecture, but only supported short-term survival of germ cells. Moreover, millimeter-sized organoids show signs of degeneration due to insufficient nutrient and oxygen supply. First, we focused on recreating the testicular architecture at air-medium interphase and determined whether higher cell densities could improve our previously developed 3D printed culture model during long-term culture using different mouse strains. Afterwards, the focus was put on improving TO morphology by adapting the scaffold design. Moreover, to expand the potential of TOs, the possibility to cultivate chimeric mixtures of testicular cells and germ line stem cells expressing a reporter transgene (EGFP) was assessed. Prepubertal testicular cells from C57BL/6J (n = 5) or CBAB6F1 (n = 3) mice were cultured in the macropores of 3D printed squared 1-layered scaffolds (1LSs) composed of Cellink-RGD (8x104 cells/mm²). Next, 1LS was modified with an additional layer of alginate (2LS) to culture a chimeric mixture of testicular cells of prepubertal C57BL/6J mice and EGFP-expressing germline stem cells (2:1). Cell reorganization and differentiation were characterized by immunohistochemistry and testosterone was quantified by electrochemiluminescence. During long-term cultures in 1LSs, testicular cells reorganized into organoids with restoration of testicular architecture and Leydig cell functionality supporting the differentiation of germ cells to the meiotic phase, regardless of the mouse strain. However, pore overgrowth and fusion of adjacent aggregates, resulted in irregularly shaped TOs. Based on these results, the design of 1LS was modified with an additional layer of alginate to entrap reorganizing cells (2LS). To non-invasively evaluate germ cell behavior, EGFP-expressing germline stem cells were mixed with testicular cells of prepubertal C57BL/6J mice in 2LS. This approach resulted in the formation of chimeric organoids with a more regular and spheroidal morphology. These improved TOs consisted typically of 1 tubule-like structure and surrounding interstitium, representing the functional unit of a testis. in contrast to primary germ cells, germline stem cells were not observed after the 3rd week of culture. Candidate factors have to be tested in their ability to elevate the meiotic blockage of germ cells in TOs. In addition, the culture medium needs further optimization to enhance maintenance of germline stem cells in chimeric models. Finally, results obtained with rodents remain to be confirmed in further human studies. Wider implications of the findings: The opportunities testicular organoids offer to manipulate cells through genetic modification, inclusion and exclusion, are essential for the study of male infertility and the search for potential therapies. Moreover, they permit high-throughput screening of chemicals, thereby substantially reducing the number of animals for the high demanding reproductive toxicity studies. Not applicable

Short-term germ cell survival and central tissue degeneration limit organoid cultures. Here, testicular organoids (TOs) were generated from two different mouse strains in 3D printed one-layer scaffolds (1LS) at the air-medium interface displaying tubule-like structures and Leydig cell functionality supporting long-term survival and differentiation of germ cells to the meiotic phase. Chimeric TOs, consisting of a mixture of primary testicular cells and EGFP+ germline stem (GS) cells, were cultured in two-layer scaffolds (2LSs) for better entrapment. They showed an improved spheroidal morphology consisting of one intact tubule-like structure and surrounding interstitium, representing the functional unit of a testis. However, GS cells did not survive long-term culture. Consequently, further optimization of the culture medium is required to enhance the maintenance and differentiation of germ cells. The opportunities TOs offer to manipulate somatic and germ cells are essential for the study of male infertility and the search for potential therapies.

Decellularized testicular matrix (DTM) enables researchers to focus on the specific composition of the testicular extracellular matrix (ECM) and elucidate its role in spermatogenesis. Furthermore, it provides the natural architectural arrangement that could guide the reorganization of dissociated testicular cells in vitro. This is a key consideration as the presence of an authentic nutritive and endocrine support has been proven to be essential for in vitro spermatogenesis, at least in the mouse (Oliver and Stukenborg in Andrology 8:825-834, 2020; Richer et al. in Andrology 12741, 2019). Hence, scaffolds of DTM could be harnessed for the development of a human in vitro spermatogenesis culture system, which is a missing link in male fertility preservation and could be a possible treatment for nonobstructive azoospermia (Gassei and Orwig in Steril 105:256-266, 2016).

STUDY QUESTION:Is it possible to co-culture and functionally link human liver and testis equivalents in the combined medium circuit of a multi-organ chip?SUMMARY ANSWER:Multi-organ-chip co-cultures of human liver and testis equivalents were maintained at a steady-state for at least 1 week and the co-cultures reproduced specific natural and drug-induced liver-testis systemic interactions.WHAT IS KNOWN ALREADY:Current benchtop reprotoxicity models typically do not include hepatic metabolism and interactions of the liver-testis axis. However, these are important to study the biotransformation of substances.STUDY DESIGN, SIZE, DURATION:Testicular organoids derived from primary adult testicular cells and liver spheroids consisting of cultured HepaRG cells and hepatic stellate cells were loaded into separate culture compartments of each multi-organ-chip circuit for co-culture in liver spheroid-specific medium, testicular organoid-specific medium or a combined medium over a week. Additional multi-organ-chips (single) and well plates (static) were loaded only with testicular organoids or liver spheroids for comparison. Subsequently, the selected type of medium was supplemented with cyclophosphamide, an alkylating anti-neoplastic prodrug that has demonstrated germ cell toxicity after its bioactivation in the liver, and added to chip-based co-cultures to replicate a human liver-testis systemic interaction in vitro. Single chip-based testicular organoids were used as a control. Experiments were performed with three biological replicates unless otherwise stated.PARTICIPANTS/MATERIALS, SETTING, METHODS:The metabolic activity was determined as glucose consumption and lactate production. The cell viability was measured as lactate dehydrogenase activity in the medium. Additionally, immunohistochemical and real-time quantitative PCR end-point analyses were performed for apoptosis, proliferation and cell-specific phenotypical and functional markers. The functionality of Sertoli and Leydig cells in testicular spheroids was specifically evaluated by measuring daily inhibin B and testosterone release, respectively.MAIN RESULTS AND THE ROLE OF CHANCE:Co-culture in multi-organ chips with liver spheroid-specific medium better supported the metabolic activity of the cultured tissues compared to other media tested. The liver spheroids did not show significantly different behaviour during co-culture compared to that in single culture on multi-organ-chips. The testicular organoids also developed accordingly and produced higher inhibin B but lower testosterone levels than the static culture in plates with testicular organoid-specific medium. By comparison, testosterone secretion by testicular organoids cultured individually on multi-organ-chips reached a similar level as the static culture at Day 7. This suggests that the liver spheroids have metabolised the steroids in the co-cultures, a naturally occurring phenomenon. The addition of cyclophosphamide led to upregulation of specific cytochromes in liver spheroids and loss of germ cells in testicular organoids in the multi-organ-chip co-cultures but not in single-testis culture.LARGE-SCALE DATA:N/A.LIMITATIONS, REASONS FOR CAUTION:The number of biological replicates included in this study was relatively small due to the limited availability of individual donor testes and the labour-intensive nature of multi-organ-chip co-cultures. Moreover, testicular organoids and liver spheroids are miniaturised organ equivalents that capture key features, but are still simplified versions of the native tissues. Also, it should be noted that only the prodrug cyclophosphamide was administered. The final concentration of the active metabolite was not measured.WIDER IMPLICATIONS OF THE FINDINGS:This co-culture model responds to the request of setting up a specific tool that enables the testing of candidate reprotoxic substances with the possibility of human biotransformation. It further allows the inclusion of other human tissue equivalents for chemical risk assessment on the systemic level.STUDY FUNDING/COMPETING INTEREST(S):This work was supported by research grants from the Scientific Research Foundation Flanders (FWO), Universitair Ziekenhuis Brussel (scientific fund Willy Gepts) and the Vrije Universiteit Brussel. Y.B. is a postdoctoral fellow of the FWO. U.M. is founder, shareholder and CEO of TissUse GmbH, Berlin, Germany, a company commercializing the Multi-Organ-Chip platform systems used in the study. The other authors have no conflict of interest to declare.

Research question: Which cryopreservation method better protects reproductive potential: the cryopreservation of a testicular cell suspension (TCS) or the cryopreservation of testicular tissue (TET)? Design: Two cryopreservation strategies for spermatogonial stem cells (SSCs) were compared in a mouse model: cryopreservation as TET or as TCS. Evaluated outcomes were number of viable cells after thawing, number and length of donor-derived colonies after spermatogonial stem cell transplantation (SSCT), number of litters, litter size and number of donor-derived pups after mating. Results: Compared with cryopreserving TCS, cryopreservation of TET resulted in significantly higher numbers of viable cells after thawing (TET: 13.4 x 10(4) +/- 72 x 10(4) versus TCS: 8.2 x 10(4) +/- 2.7 x 10(4); P = 0.0002), more (TET: 476 +/- 19.2 versus TCS: 18.5 +/- 13.0; P = 0.0039) and longer (TET: 5.2 +/- 1.0 mm versus TCS: 2.7 +/- 1.5 mm; P = 0.0016) donor-derived colonies, and more donor-derived pups per litter (TET: 2.2 +/- 0.2 versus TCS: 0.5 +/- 0.1; P = 0.0008). Conclusions: Cryopreservation of TET is the preferred method to cryopreserve SSCs prior to SSCT in a mouse model.

BACKGROUND:The testicular organoid concept has recently been introduced in tissue engineering to refer to testicular cell organizations modeling testicular architecture and function. The testicular organoid approach gives control over which and how cells reaggregate, which is not possible in organotypic cultures, thereby extending the applicability of in-vitro spermatogenesis (IVS) systems. However, it remains unclear which culture method and medium allow reassociation of testicular cells into a functional testicular surrogate in-vitro.OBJECTIVE:The aim of this paper is to review the different strategies that have been used in an attempt to create testicular organoids and generate spermatozoa. We want to provide an up-to-date list on culture methodologies and media compositions that have been used and determine their role in regulating tubulogenesis and differentiation of testicular cells.SEARCH METHOD:A literature search was conducted in PubMed, Web of Science, and Scopus to select studies reporting the reorganization of testicular cell suspensions in-vitro, using the keywords: three-dimensional culture, in-vitro spermatogenesis, testicular organoid, testicular scaffold, and tubulogenesis. Papers published before the August 1, 2019, were selected.OUTCOME:Only a limited number of studies have concentrated on recreating the testicular architecture in-vitro. While some advances have been made in the testicular organoid research in terms of cellular reorganization, none of the described culture systems is adequate for the reproduction of both the testicular architecture and IVS.CONCLUSION:Further improvements in culture methodology and medium composition have to be made before being able to provide both testicular tubulogenesis and spermatogenesis in-vitro.