Monica M. Laronda

Purpose: To characterize structural access to pediatric ovarian and testicular tissue cryopreservation in the USA, including service availability, delivery models, and institutional research practices. Methods: We conducted a national cross-sectional survey of institutions identified from the FDA Human Cell and Tissue Establishment Registration database and the Oncofertility Consortium “Clinic Finder.” Institutions completed an online survey of multiple-choice and free-text items characterizing clinical delivery, case volume, and research practices. Descriptive analyses and geospatial mapping were performed. Results: Among 345 contact-eligible institutions, 36 responded (10.4%); this denominator was intentionally broad and substantially exceeded the number of institutions expected to provide these services. Twenty-three institutions reported offering ovarian tissue cryopreservation (OTC) and 23 reported offering testicular tissue cryopreservation (TTC); 21 provided pediatric OTC and 19 institutions provided pediatric TTC. Most functioned in a single role, commonly as surgical procurement sites rather than cryopreservation centers. Median annual case volume was 12.5 cases for OTC (interquartile range [IQR], 3.5–23.0) and 10.0 cases for TTC (IQR, 5.0–19.0). Pediatric patients accounted for a median of 100.0% of cases for both OTC and TTC, with prepubertal patients representing a substantial proportion, particularly for TTC. Research oversight and specimen utilization varied, with frequent external laboratory collaboration. Conclusion: Pediatric ovarian and testicular tissue cryopreservation in the USA functions through an informal, institutionally coordinated hub-and-spoke model of distributed surgical procurement and centralized cryopreservation. Variability in service roles, research oversight, and specimen utilization highlights opportunities to improve transparency, coordination, and access.

The female reproductive tract is essential for fertility, pregnancy, and overall health, yet many of its cellular components remain poorly defined. Recent advances in single-cell and spatial transcriptomics have begun to reveal this complexity, but inconsistent naming of cell types and states has limited the ability to compare findings across studies. To address this challenge, an international group of reproductive biologists and ontology experts collaborated to harmonize annotations within Cell Ontology, focusing on the ovary, fallopian tube, and uterus. Standardized terms are proposed for major epithelial, stromal, and germ cell populations, supported by marker gene sets and anatomical linkages. This framework provides a shared reference that can be used to harmonize existing datasets and guide annotation of future studies, enabling consistent classification of oocytes, granulosa, theca, luteal, epithelial, stromal, and immune cells across tissues. By establishing a unified taxonomy, this work lays the foundation for integrating datasets, supporting cross-tissue comparisons, and advancing the understanding of reproductive biology, health, and disease.

STUDY QUESTION: Are subtle changes in the paediatric adolescent/young adult ovarian follicles and microenvironment following low-dose acute alkylating chemotherapy detectable by granular assessment tools? SUMMARY ANSWER: Low-dose alkylating agents impact the ovaries of pre- and post-pubertal participants with significant alterations to the follicle and oocyte area, number of granulosa cells (GCs) per follicle, and number of immature vessels, when compared to ovaries from untreated participants. WHAT IS KNOWN ALREADY: The metric known as cyclophosphamide equivalent dose (CED) was developed to counsel patients on the risk of acute and long-term adverse outcomes following exposure to alkylating agents. Likewise, CED is used to evaluate the risk of developing premature ovarian insufficiency and can support decisions to undergo fertility preservation. It is well known that high levels of alkylating agents significantly impact the ovarian reserve and extrafollicular microenvironment. However, there is limited data on the impact of low levels of CED on the human ovary. STUDY DESIGN, SIZE, DURATION: This is a retrospective study of 75 participants aged from 0.53 to 22.82 years, all of whom had undergone ovarian tissue cryopreservation (OTC) at a children’s hospital. We assessed the short-term effects of low-dose alkylating agents (CED ≤8 g/m2) on the paediatric and adolescent ovary using immunohistochemical assessments and a comprehensive morphological assessment of ovarian follicles. The median time from first alkylating treatment to tissue collection was 1.35 months. The pre-pubertal cohort consisted of 50 participants; 4 had non-alkylating treatments (NA), 23 participants had alkylating treatments (A), and 23 were untreated (U). The post-pubertal cohort consisted of 25 participants: 5 with NA, 10 with A, and 10 who were U. PARTICIPANTS/MATERIALS, SETTING, METHODS: Biopsies for routine pathology were obtained during a unilateral oophorectomy performed for OTC. Formalin-fixed paraffin-embedded ovarian tissue was analyzed retrospectively. The extrafollicular environment was analyzed for collagen density, stromal fibre architecture, vascularity and vessel maturity, apoptosis, proliferation, and DNA damage. Digital scans of hematoxylin and eosin-stained pathology slides were used to classify and morphologically assess follicles in adjacent sections. MAIN RESULTS AND THE ROLE OF CHANCE: We first determined the effect of acute low-dose alkylating agents on the extrafollicular environment. Collagen was significantly more abundant in pre-pubertal ovarian tissue compared to post-pubertal (20.97 ± 3.30 vs. 7.11 ±1.82%, P ≤ 0.0008), but there was no significant difference in collagen abundance within either group based on exposure. Interestingly, collagen fibres in the cortex of post-pubertal ovaries exposed to non-alkylating agents were significantly narrower and less straight compared to untreated ovaries (7.85 ± 0.05 vs. 8.85 ± 0.13 µm, P = 0.01 and 92.24 ± 0.123 vs. 93.27 ± 0.252, P = 0.01). Consistent with previous work in reproductively aged individuals, there were significantly more immature vessels in pre-pubertal ovaries exposed to alkylating agents compared to untreated (3.29 ± 0.94 vs. 0.104 ± 0.07 vessel count/tissue area mm2, P = 0.037). Next, follicles within these cohorts were assessed using a granular and comprehensive morphological follicle analysis pipeline. Ovaries from pre-pubertal participants that were exposed to non-alkylating and alkylating therapy had significantly smaller primordial follicles (PMF) (1321 ± 14.52 (NA), 1379 ± 8.42 (A), 1533 ± 10.07 μm2 (U), P ≤ 0.0001) and transitional follicles (TF) (1356 ± 14.04 (NA), 1384 ± 10.05 (A), 1639 ± 14.04 μm2 (U), P ≤ 0.0001) with significantly fewer GCs per PMF (9.0 ± 0.15 (NA), 8.0 ± 0.08 (A), 10.0 ± 0.10 (U), P ≤ 0.0001) and TF compared to untreated ovaries (12.0 ± 0.16 (NA), 11.0 ± 0.09 (A), 13.0 ± 0.16 (U), P ≤ 0.0001, 0.0002). Ovaries from post-pubertal participants exposed to non-alkylating therapies had significantly smaller TFs (1437 ± 72.58 (NA), 1709 ± 131.8 (A), 2727 ± 308.9 μm2 (U), P = 0.021) and smaller primary follicles (1479 ± 71.26 (NA), 1709 ± 131.8 (A), 2981 ± 375.7 μm2 (U), P ≤ 0.0001). PMF (10.0 ± 0.26 (NA), 10.0 ± 0.28 (A), 8.0 ± 0.19 (U), P ≤ 0.0001, 0.0005) and TFs (12.0 ± 0.23 (NA), 12.0 ± 0.25 (A), 11.0 ± 0.20 (U), P ≤ 0.0001, 0.0013) had significantly more GCs per follicle, while primary follicles had significantly fewer GCs (16.0 ± 0.92 (NA), 17.0 ± 0.99 (A), 26.0 ± 2.85 (U), P ≤ 0.0001, 0.0003) compared to untreated ovaries. Then, we examined oocyte areas and found that oocytes from PMF (1032 ± 12.35 (NA), 1039 ± 9.29 (A), 1157 ± 10.81 μm2 (U), P ≤ 0.0001) and TFs (1030 ± 12.76 (NA), 1000 ± 12.53 (A), 1235 ± 18.53 μm2 (U), P ≤ 0.0001) of pre-pubertal ovaries exposed to non-alkylating and alkylating treatments were significantly smaller in area compared to untreated ovaries. In contrast, in post-pubertal ovaries, PMF (1051 ± 23.81 (NA), 1203 ± 18.74 (A), 980.0 ± 17.27 μm2 (U), P = 0.038, <0.0001) and TF (1257 ± 20.01 (NA), 1094 ± 19.43 (A), 963.2 ± 18.51 μm2 (U), P ≤ 0.0001, 0.0004) oocytes were significantly larger in exposed ovaries compared to untreated ovaries. In addition, there were significantly more anti-gamma H2A histone family member X (γH2AX)-expressing oocytes in ovaries from post-pubertal participants exposed to alkylating therapies compared to untreated participants (61.37 ± 9.13 (A) vs. 24.08 ± 8.37% (U), P = 0.0329). Finally, we found that PMFs containing γH2AX-expressing oocytes were significantly larger...

Study question: Can adeno-associated viruses (AAVs) efficiently and safely transduce human primary granulosa cells and human ovarian stromal fibroblasts to modulate the expression of genes involved in follicle recruitment and ovarian microenvironment regulation? Summary answer: AAVs can efficiently transduce human primary granulosa cells and human ovarian stromal cells with low toxicity, with the serotype DJ (AAV-DJ) demonstrating superior performance. AAVs enable targeted modulation of ovarian genes such as AMHR2 in human primary granulosa cells and TGFβ1 in human ovarian stromal cells, supporting the feasibility of gene-based approaches to improve ovarian function. What is known already: Infertility affects approximately 15% of couples, with diminished oocyte quality and quantity being major contributors, particularly with advanced reproductive age. Assisted reproductive technologies rely primarily on hormonal stimulation and do not directly target molecular pathways governing folliculogenesis or the ovarian microenvironment. Animal studies suggest that gene therapy using AAVs offers a promising strategy for tissue-specific and durable gene modulation, yet its application in human ovarian cells remains largely unexplored. Study design, size, duration: This was an experimental laboratory study using primary human granulosa cells from patients undergoing in vitro fertilization (IVF) and ovarian stromal cells obtained from post-pubertal patients who donated surplus ovarian tissue from ovarian tissue cryopreservation procedures for research. Sixteen AAV serotypes were evaluated for transduction efficiency, toxicity, and expression dynamics, followed by functional gene knockdown studies using AAV-DJ-based shRNA vectors. Participants/materials, setting, methods: Primary human granulosa cells and ovarian stromal cells were isolated from follicular aspirates and ovarian tissue, respectively. Cells were transduced with GFP-expressing AAV serotypes at varying multiplicities of infection (MOIs), incubation times, and culture days to assess transduction efficiency and cytotoxicity. Lead serotypes were further evaluated in dose-response studies. Functional gene modulation was assessed using AAV-DJ-mediated shRNA knockdown of AMHR2 in granulosa cells and TGFβ1 in stromal cells, quantified by RT-qPCR, immunofluorescence, and western blotting. Main results and the role of chance: Six of sixteen AAV serotypes (AAV2, AAV3, AAV6, AAV9, AAV-DJ, and AAV-X1.1) achieved greater than 10% transduction efficiency in human primary granulosa cells. Among these, AAV-DJ demonstrated the highest efficiency across all MOIs tested, reaching up to 48% transduction at 100,000 MOI, significantly outperforming AAV2 and AAV9. Toxicity increased with viral dose for all serotypes but remained comparable across vectors, with AAV-DJ exhibiting higher efficiency with comparable toxicity. The AAV-DJ-shRNA-AMHR2 construct produced a significant reduction in expression of AMHR2 mRNA by ~81% (p<0.05) in human primary granulosa cells as well as at the protein level in HEK293T-AMHR2-ZsGreen engineered cells (~85% knockdown, p<0.01). In human ovarian stromal cells, AAV-DJ preferentially transduced EMILIN1 and ACTA2-positive fibroblast and myofibroblast-like cells and enabled robust knockdown of TGFβ1 at both transcript (~89% knockdown, p<0.05) and protein levels (~67% knockdown, p<0.0001) using AAV-DJ-shRNA-TGFβ1 construct. Limitations, reasons for caution: These experiments were performed in vitro using primary human ovarian cells, which may not fully recapitulate the in vivo ovarian environment. Functional reproductive outcomes were not assessed, and future studies will be required to evaluate the effects of gene modulation on folliculogenesis and fertility. Wider implications of the findings: These findings establish AAV-DJ as a promising vector for targeted gene modulation in human ovarian cells. This approach introduces a novel framework for addressing infertility and ovarian function by directly targeting molecular pathways involved in follicle recruitment and ovarian stromal signaling, potentially enhancing current assisted reproductive technologies.

Testicular tissue cryopreservation (TTC) is currently the only option to preserve fertility in prepubertal individuals with testes. However, reimplantation of tissue collected during TTC has yet to result in a live human birth, despite recent progress in rhesus macaque models. Additionally, cell or tissue implantations are not options for individuals with metastatic disease, making it critical to develop alternative fertility restoration strategies using TTC specimens. Our goal was to generate primary cell-derived testicular assembloids with native-similar internal cell organization, enclosed seminiferous tubule-like structures (TLSs), and surviving germ cells. Organoids derived from primary testicular cells harvested from 5-day-old mice developed native-similar organization and enclosed TLSs when seeded at ~9300 cells/microwell. We assessed the fusion and internal organization of merged organoids (“assembloids”) in multiple open channel designs with differing physical constraints and access to media and oxygen. These conditions impacted assembloid ellipticity, cellular organization, TLS formation, TLS diameter, and germ cell survival. Open channels with 400- and 600-micron widths that were submerged within culture media most consistently produced assembloids with native-similar architecture. These data support a robust framework for the generation of murine testicular assembloids with germ cell-containing tubule-like structures and interstitial compartments. Optimization of this in vitro platform is an important step towards expanding fertility restoration options for prepubertal patients at increased risk for infertility.

Purpose: This protocol describes the procedure for cryopreservation of organ donor derived human ovarian tissue via slow freeze with a semi-automated controlled rate freezer in a research setting.

Paracrine signaling is critical for ovarian development and maintenance of non-growing, gonadotropin-independent follicles. During puberty the human ovary develops defined compartments as gonadotropin signaling triggers follicle activation and maturation. Adjacent follicles utilize paracrine signaling but also benefit from nearby interstitial cell populations. Here, we characterized human ovarian interstitial cells and their effects on follicle growth through a cross-species culture assay. Both prepubertal and postpubertal primary ovarian interstitial populations improved follicle growth, and postpubertal cells conferred a significant increase in follicle estradiol production but not maturation rates. In one cohort, interstitial cell-conditioned media was not sufficient for the growth advantage observed with co-culture. To investigate how follicle-interstitial cell crosstalk changes during puberty, single-cell RNA-sequencing was performed on 11 ovarian tissue cryopreservation samples (participants 0.34-22.8 years). Results confirmed the presence of heterogenous interstitial cell populations previously identified in the adult ovary, including abundant theca/stromal cells expressing genes enriched for ECM-related processes. Finally, over 100 proteins were identified in co-culture media using bottom-up proteomics representing the human ovarian interstitial secretome. This study uniquely characterized human ovarian interstitial cells across the pubertal transition and advances our understanding of the human pediatric and adolescent ovarian follicular microenvironment.