Heterogeneity of ovarian matrisome hydrogels elucidates factors that may influence follicle growth in vitro.

This work describes a valuable and reproducible method for generating optically clear bovine ovary-derived hydrogels that support in vitro murine follicle growth. These techniques are the foundation in which follicle growth dynamics and matrisome protein composition may be correlated to reveal the influence of matrisome proteins on folliculogenesis.

Comprehensive fertility preservation can be offered in a timely manner around gonadotoxic therapy in children and adolescents.

BACKGROUND: Treatment for certain childhood cancers and nonmalignant conditions can lead to future infertility and gonadal failure. The risk of treatment delay must be considered when offering fertility preservation (FP) options. We examined the timeline from FP referral to return to treatment (RTT) in pediatric patients who underwent FP due to iatrogenic risk for infertility. METHODS: A retrospective review was performed of patients with FP consultation due to an increased risk of iatrogenic infertility at Ann & Robert H. Lurie Children's Hospital of Chicago from 2018 to 2022. Data on diagnosis, age, treatment characteristics, and procedure were collected. RESULTS: A total of 337 patients (n = 149 with ovaries, n = 188 with testes) had an FP consultation. Of patients with ovaries, 106 (71.1%) underwent ovarian tissue cryopreservation (OTC), 10 (6.7%) completed ovarian stimulation/egg retrieval (OSER), and 33 (22.1%) declined FP. Of the patients with testes, 98 (52.1%) underwent testicular tissue cryopreservation (TTC), 48 (25.5%) completed sperm banking (SB), and 42 (22.3%) declined FP. Median time from referral to FP consultation was short (ovaries: 2 days, range: 0-6; testes: 1 day, range: 0-5). OSER had a significantly longer RTT versus OTC and no FP (52.5 vs.19.5 vs. 12 days, p = .01). SB had a significantly quicker RTT compared to TTC or no FP (9.0 vs. 21.0 vs. 13.5 days; p = .008). For patients who underwent OTC/TTC and those who declined FP, there was no significant difference in time from consultation to treatment. CONCLUSIONS: It is feasible to promptly offer and complete FP with minimal delay to disease-directed treatment.

Restoring Ovarian Fertility and Hormone Function: Recent Advancements, Ongoing Efforts and Future Applications.

The last 20 years have seen substantial improvements in fertility and hormone preservation and restoration technologies for a growing number of cancer survivors. However, further advancements are required to fill the gaps for those who cannot use current technologies or to improve the efficacy and longevity of current fertility and hormone restoration technologies. Ovarian tissue cryopreservation (OTC) followed by ovarian tissue transplantation (OTT) offers those unable to undergo ovarian stimulation for egg retrieval and cryopreservation an option that restores both fertility and hormone function. However, those with metastatic disease in their ovaries are unable to transplant this tissue. Therefore, new technologies to produce good-quality eggs and restore long-term cyclic ovarian function are being investigated and developed to expand options for a variety of patients. This mini-review describes current and near future technologies including in vitro maturation, in vitro follicle growth and maturation, bioprosthetic ovaries, and stem cell applications in fertility restoration research by their proximity to clinical application.

Thawing fertility: a view of ovarian tissue cryopreservation processes and review of ovarian transplant research.

Individuals with a disease or treatment that increases their risk of premature gonadal insufficiency may opt to undergo fertility preservation. Those who are postpubertal can often cryopreserve gametes, sperm, or eggs to expand their biologic family using assisted reproductive technologies. Ovarian tissue cryopreservation (OTC) and testicular tissue cryopreservation may be an option for individuals who are unable to use standard fertility preservation techniques. The development of OTC was critical for many patients, including prepubertal children with ovaries that do not yet produce eggs, adolescents who make few good-quality eggs, and adult women with ovaries who cannot undergo ovarian stimulation. The only option to restore fertility and hormone production after OTC is through ovarian tissue transplantation (OTT). Ovarian tissue cryopreservation and OTT have been successful for some patients. Although OTC is no longer considered experimental by the American Society for Reproductive Medicine, the process is far from standardized. Significant research needs to be done, especially at the point of OTT, to improve the success and longevity of ovarian tissue function. This article lists the main steps from surgical procurement of the ovarian tissue to transplantation and restoration of function. Our pediatric hospital program has had to decide which options in procurement, processing, cryopreservation, and warming will be used in our clinical laboratory. The options and limitations within the research and analyses are briefly discussed. Literature focusing on techniques to improve OTT effectiveness and longevity was reviewed. Ovarian tissue transplantation studies that performed xenograft experiments after pretreatment of the tissue graft by a ligand or drug, treatment of the host, or encapsulation of the ovarian tissue were identified. The intended effects of the treatments include increasing vascularization, reducing apoptosis, and directing activation or suppression of primordial follicles. Robust research in this area must continue with rigorous analyses to make strides in improving fertility preservation and restoration options for patients.

Encapsulation of Bovine Primordial Follicles in Rigid Alginate Does Not Affect Growth Dynamics.

The only fertility preservation and subsequent restoration option for many patients facing gonadotoxic treatments is ovarian tissue cryopreservation and transplantation. While this process is successful for some, there is significant room for improvement to extend the life of the transplant and to make it safe for patients that may have metastatic disease within their ovarian tissue. We need a deeper understanding of how the physical properties of the ovarian microenvironment may affect folliculogenesis to engineer an environment that supports isolated follicles and maintains primordial follicle quiescence. Bovine ovaries were used here as a monovulatory model of folliculogenesis to examine the effects of primordial follicle activation and growth under different physical conditions. We found that there were no differences in activation, growth or survival when primordial follicles were cultured in isolation or in situ (remaining in the tissue) under two significantly differently rigid alginate gels. To determine if the extra rigid environment did not affect activation in isolated follicles due to an immediate activation event, we used 5-ethynyl-2'-deoxyuridine (EdU) to track follicle activation during the isolation process. We identified EdU incorporation in granulosa cells after primordial follicles were isolated from the surrounding extracellular matrix (ECM). These findings support that isolation of primordial follicles from the ECM is an activating event and that the differentially rigid environments assessed here had no effect on follicle growth. Further work is needed to suppress activation in primordial follicles to maintain the ovarian reserve and extend the life of an ovarian tissue transplant.

A dozen years of ovarian tissue cryopreservation at a pediatric hospital: tracking program and patient metrics while adapting to increasing needs.

Objective: To review the program and patient metrics for ovarian tissue cryopreservation (OTC) within a comprehensive pediatric fertility preservation program in its first 12 years of development. Design: Retrospective review. Setting: A tertiary children's hospital in a large urban center between March 2011 and February 2023. Patients: Pediatric patients who underwent OTC. Interventions: Unilateral oophorectomy for OTC. Main Outcome Measures: Patient demographics and clinical course information were collected for analysis. Results: A total of 184 patients underwent OTC in the first 12 years. One hundred fifteen patients were prepubertal at the time of OTC, and 69 were postpubertal. In total, 128 patients (69.6%) received part of their planned therapy before OTC. Starting in 2018, 104 participants (92.0%) donated tissue to research, 99 participants (87.6%) donated blood, and 102 (90.2%) donated media to research. There was a decrease in the median age of patients who underwent OTC from 16.4-6.6 years and an overall increase in the proportion of patients per year that were prepubertal. Forty-eight (26.0%) patients who underwent OTC were outside referrals and traveled from as far as Seattle, Washington. Conclusion: During the first 12 years of this program, oncofertility research increased, annual tissue cryopreservation cases increased, and the median age of those who underwent OTC decreased. The program was adapted to build a stand-alone gonadal tissue processing suite and specialized in prepubertal ovarian tissue processing. The program will continue to adapt to patient needs in the upcoming decades because restoration technologies advance through research supported by this and collaborating programs.

Generation of Tailored Extracellular Matrix Hydrogels for the Study of In Vitro Folliculogenesis in Response to Matrisome-Dependent Biochemical Cues.

While ovarian tissue cryopreservation (OTC) is an important fertility preservation option, it has its limitations. Improving OTC and ovarian tissue transplantation (OTT) must include extending the function of reimplanted tissue by reducing the extensive activation of primordial follicles (PMFs) and eliminating the risk of reimplanting malignant cells. To develop a more effective OTT, we must understand the effects of the ovarian microenvironment on folliculogenesis. Here, we describe a method for producing decellularized extracellular matrix (dECM) hydrogels that reflect the protein composition of the ovary. These ovarian dECM hydrogels were engineered to assess the effects of ECM on in vitro follicle growth, and we developed a novel method for selectively removing proteins of interest from dECM hydrogels. Finally, we validated the depletion of these proteins and successfully cultured murine follicles encapsulated in the compartment-specific ovarian dECM hydrogels and these same hydrogels depleted of EMILIN1. These are the first, optically clear, tailored tissue-specific hydrogels that support follicle survival and growth comparable to the "gold standard" alginate hydrogels. Furthermore, depleted hydrogels can serve as a novel tool for many tissue types to evaluate the impact of specific ECM proteins on cellular and molecular behavior.

Is Routine Pathology Evaluation of Tissue Removed for Fertility Preservation Necessary?

BACKGROUND: For all fertility preservation (FP) cases at our institution, a biopsy is performed for routine pathology from all gonadal tissue removed. This is not standard at all centers. We reviewed our experience with biopsy for pathological evaluation of ovarian and testicular specimens in FP cases to determine clinical utility. METHODS: The medical records of individuals who underwent ovarian tissue cryopreservation (OTC) or testicular tissue cryopreservation (TTC) between 2011 and 2023 were retrospectively reviewed under an IRB-approved study at a free-standing tertiary care children's hospital. Patient demographics, diagnosis, operative characteristics, and pathology results were collected. RESULTS: One-hundred and eighty-three patients underwent OTC, and 134 patients underwent TTC. All patients had their gonadal tissue biopsied for routine pathology. Malignancy was identified in the biopsies of 4 OTC patients (2.2%) and 2 TTC patients (1.5%). Two OTC patients (1.1%) and 2 TTC patients (1.5%) did not have germ cells identified in their biopsy. All OTC and TTC patients and families elected to continue storing tissue for FP after discussion of pathology findings. CONCLUSIONS: Pathology results provide another data point to help inform patients and their families when making decisions on ovarian or testicular tissue storage and on how tissue may be utilized in the future to restore fertility and/or hormones. There is a low rate of identifying malignancy in gonadal tissue biopsies taken from FP specimens even in patients with known malignancy. However, when malignancy was identified, it could be unexpected and alter the diagnosis and treatment plan significantly for patients. LEVEL OF EVIDENCE: IV.

The oocyte microenvironment is altered in adolescents compared to oocyte donors.

Study question: Are the molecular signatures of cumulus cells (CCs) and follicular fluid (FF) of adolescents undergoing fertility preservation differ from that of reproductively adult oocyte donors? Summary answer: The microenvironment immediately surrounding the oocyte, including the CCs and FF, is altered in adolescents undergoing fertility preservation compared to oocyte donors. What is known already: Adolescents experience a period of subfecundity following menarche. Recent evidence suggests that this may be at least partially due to increased oocyte aneuploidy. Reproductive juvenescence in mammals is associated with suboptimal oocyte quality. Study design size duration: This was a prospective cohort study. Adolescents (10-19 years old, N=23) and oocyte donors (22-30 years old, N=31) undergoing ovarian stimulation and oocyte retrieval at the Northwestern Fertility and Reproductive Medicine Center between November 1, 2020 and May 1, 2023 were enrolled in this study. Participants/materials setting methods: Patient demographics, ovarian stimulation, and oocyte retrieval outcomes were collected for all participants. The transcriptome of CCs associated with mature oocytes was compared between adolescents (10-19 years old, n=19), and oocyte donors (22-30 years old, n=19) using bulk RNA-sequencing. FF cytokine profiles (10-19 years old, n=18 vs. 25-30 years old, n=16) were compared using cytokine arrays. Main results and the role of chance: RNA-seq analysis revealed 581 differentially expressed genes (DEGs) in cumulus cells of adolescents relative to oocyte donors, with 361 genes downregulated and 220 upregulated. Genes enriched in pathways involved in cell cycle and cell division (e.g., GO:1903047, p= 3.5 × 10-43; GO:0051983, p= 4.1 × 10-30; GO:0000281, p= 7.7 × 10-15; GO:0044839, p= 5.3 × 10-13) were significantly downregulated, while genes enriched in several pathways involved in cellular and vesicle organization (e.g., GO:0010256, p= 1.2 × 10-8; GO:0051129, p= 6.8 × 10-7; GO:0016050, p= 7.4 × 10-7; GO:0051640, p= 8.1 × 10-7) were upregulated in CCs of adolescents compared to oocyte donors. The levels of 9 cytokines were significantly increased in FF of adolescents compared to oocyte donors: IL-1 alpha (2-fold), IL-1 beta (1.7-fold), I-309 (2-fold), IL-15 (1.6-fold), TARC (1.9-fold), TPO (2.1-fold), IGFBP-4 (2-fold), IL-12-p40 (1.7-fold) and ENA-78 (1.4-fold). Interestingly, 7 of these cytokines have known pro-inflammatory roles. Importantly, neither the CC transcriptomes or FF cytokine profiles were different in adolescents with or without cancer. Large scale data: Original high-throughput sequencing data will be deposited in Gene Expression Omnibus (GEO) before publication, and the GEO accession number will be provided here. Limitations reasons for caution: This study aims to gain insights into the associated gamete quality by studying the immediate oocyte microenvironment. The direct study of oocytes is more challenging due to sample scarcity, as they are cryopreserved for future use, but will provide a more accurate assessment of oocyte reproductive potential. Wider implications of the findings: Understanding the underpinnings of altered immediate oocyte microenvironment of adolescent patients may provide insights into the reproductive potential of the associated gametes in the younger end of the age spectrum. This has implications for the fertility preservation cycles for very young patients. Study funding/competing interests: This project was supported by Friends of Prentice organization SP0061324 (M.M.L and E.B.), Gesualdo Family Foundation (Research Scholar: M.M.L.), and NIH/NICHD K12 HD050121 (E.B.). The authors have declared that no conflict of interest exists.

Turner Syndrome With Y Chromosome and Germ Cells: A Case Report Highlighting the Need to Prioritize Individualized Care.

Turner syndrome (TS) is a genetic condition in phenotypic females in which the individual has 1 intact X chromosome and the second sex chromosome is absent or structurally altered Components of Y chromosome (eg, 45,X/46,XY) have been found in 5%-15% of patients with TS; these patients are often referred to as having "Turner syndrome with Y" (TS+Y). The presence of Y chromosome material increases risk for development of gonadal tumors. Historically, prophylactic gonadectomy has been recommended in this population to prevent malignancy, and patients were presumed infertile due to the presence of streak gonads with no germ cells (GCs). More recently, studies have reported on spontaneous puberty and menarche in TS+Y patients suggesting the presence of viable GC and ovarian function. Our institution offers patients with TS+Y the option of experimental gonadal tissue cryopreservation (GTC) at the time of gonadectomy. We present a unique case of a young girl with TS+Y who had GCs present in her gonads and underwent experimental GTC at the time of gonadectomy.