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.

Thawing fertility: A View of Ovarian Tissue Cryopreservation Processes and Review of Ovarian Transplant Research.

Individuals with a disease or treatment that will increase their risk of premature gonadal insufficiency may opt to undergo fertility preservation. Those who are post-pubertal can often cryopreserve gametes, sperm or eggs, to expand their biological family using assisted reproductive technologies. Ovarian tissue cryopreservation (OTC) and testicular tissue cryopreservation may be an option for individuals who are unable to utilize 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 following OTC is through ovarian tissue transplantation (OTT). OTC and OTT have been successful for some patients. While OTC is no longer considered experimental by the American Society of 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 the 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 lab. The options and limitations within the research and analyses are briefly discussed. Literature focusing on techniques to improve OTT effectiveness and longevity was reviewed. OTT studies that performed xenograft experiments after pretreatment of the tissue graft by a ligand or drug, treatment of 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 for improving fertility preservation and restoration options for patients.

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 role of these proteins in folliculogenesis.

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.

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.

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.

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.

Utilizing 3D Printing Technology to Create Prosthetic Irises: Proof of Concept and Workflow.

PURPOSE: There are currently limited treatment options for aniridia. In this context, 3D printed iris implants may provide a cost-effective, cosmetically acceptable alternative for patients with aniridia. The purpose of this study was to develop a proof-of-concept workflow for manufacturing 3D printed iris implants using a silicone ink palette that aesthetically matches iris shades, identified in slit lamp images. METHODS: Slit lamp iris photos from 11 healthy volunteers (3 green; 4 blue; 4 brown) were processed using k-means binning analyses to identify two or three prominent colors each. Candidate silicone inks were created by precisely combining pigments. A crowdsourcing survey software was used to determine color matches between the silicone ink swatches and three prominent iris color swatches in 2 qualifying and 11 experimental workflows. RESULTS: In total, 54 candidate silicone inks (20 brown; 16 green; 18 blue) were developed and analyzed. Survey answers from 29 individuals that had passed the qualifying workflow were invited to identify "best matches" between the prominent iris colors and the silicone inks. From this color-match data, brown, blue, and green prototype artificial irises were printed with the silicone ink that aesthetically matched the three prominent colors. The iris was printed using a simplified three-layer five-branch starburst design at scale (12.8 mm base disc, with 3.5 mm pupil). CONCLUSIONS: This proof-of-concept workflow produced color-matched silicone prosthetic irises at scale from a panel of silicone inks using prominent iris colors extracted from slit lamp images. Future work will include printing a more intricate iris crypt design and testing for biocompatibility.

FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

STUDY QUESTION: Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. WHAT IS KNOWN ALREADY: During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. STUDY DESIGN, SIZE, DURATION: This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. WIDER IMPLICATIONS OF THE FINDINGS: FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Human ovarian gross morphology and subanatomy across puberty: insights from tissue donated during fertility preservation.

Objective: To study ovarian gross morphologic and subanatomic features across pubertal development. Design: Prospective cohort study. Setting: An academic medical center with specimens collected from 2018-2022. Patients: Tissue was obtained from prepubertal and postpubertal participants (0.19-22.96 years) undergoing ovarian tissue cryopreservation before treatment that put them at a significantly or high increased risk of developing premature ovarian insufficiency. Most participants (64%) had not received chemotherapy at tissue collection. Interventions: None. Main Outcome Measures: Ovaries procured for fertility preservation were weighed and measured. Ovarian tissue fragments released during processing, biopsies used for pathology, and hormone panels were analyzed for gross morphology, subanatomic features, and reproductive hormones. Graphical analysis of best-fit lines determined age at maximum growth velocity. Results: Prepubertal ovaries were significantly (1.4-fold and 2.4-fold) smaller than postpubertal ovaries by length and width and 5.7-fold lighter on average. Length, width, and weight grew in a sigmoidal pattern with age. Prepubertal ovaries were less likely to display a defined corticomedullary junction (53% vs. 77% in postpubertal specimens), less likely to have a tunica albuginea (22% vs. 93% in postpubertal specimens), contained significantly more (9.8-fold) primordial follicles, and contained primordial follicles at significantly deeper depths (2.9-fold) when compared with postpubertal ovaries. Conclusions: Ovarian tissue cryopreservation is a resource to study human ovarian biology and pubertal development. Maximum growth velocity occurs late within the pubertal transition (Tanner 3+) after changes in subanatomic features. This ovarian morphology model adds to foundational knowledge of human ovarian development and supports ongoing transcriptomics research.

Induced pluripotent stem cell line generated from a patient with differences in sex development (DSD) and multiple genetic variants including a large deletion in NR5A1.

The nuclear receptor subfamily 5, Group A, Member 1 (NR5A1) gene encodes steroidogenic factor 1 (SF1), which is necessary for development of steroid hormone-producing tissues including the gonad and adrenal gland. An induced pluripotent stem cell line (iPSC) LCHi002-B was generated from a participant with differences (disorders) of sex development (DSD) and multiple genetic variants including a large deletion in NR5A1, and three single nucleotide changes in DYNC2H1, PDE4D, and ZFPM2. The line presented typical morphology, expressed stem cell markers, differentiated into three germ layers, had normal karyotype, was mycoplasma-free, and carried mutations in NR5A1, DYNC2H1, PDE4D, and ZFPM2.

Workflow for Management of Gonadal Neoplasm in 2 Patients with Differences of Sex Development Enrolled in an Experimental Gonadal Tissue Cryopreservation Protocol.

OBJECTIVE: To outline our experimental gonadal tissue cryopreservation (GTC) protocol that does not disrupt the standard of care in medically-indicated gonadectomy for patients with differences of sex development, including highlighting the multidisciplinary collaborative protocol for when neoplasm is discovered in these cases. METHODS: Two patients with complete gonadal dysgenesis who were undergoing medically-indicated prophylactic bilateral gonadectomy elected to pursue GTC. Both were found to have germ cell neoplasia in situ on initial pathologic analysis, requiring recall of the gonadal tissue, which had been cryopreserved. RESULTS: Cryopreserved gonadal tissue was successfully thawed and transferred to pathology for complete analysis. No germ cells were identified in either patient nor were found to have malignancy, so further treatment beyond gonadectomy was not indicated. Pathologic information was communicated to each family, including that long-term GTC was no longer possible. CONCLUSION: Organizational planning and coordination between the clinical care teams, GTC laboratory, and pathology were key to handling these cases with neoplasia. Processes that anticipated the possibility of discovering neoplasia within tissue sent to pathology and the potential need to recall GTC tissue to complete staging included (1) documenting the orientation and anatomical position of tissue processed for GTC, (2) defining parameters in which tissue will be recalled, (3) efficiently thawing and transferring GTC tissue to pathology, and (4) coordinating release of pathology results with verbal communication from the clinician to provide context. GTC is desired by many families and at the time of gonadectomy and is (1) feasible for patients with DSD, and (2) did not inhibit patient care in 2 patients with GCNIS.

Factors within the Developing Embryo and Ovarian Microenvironment That Influence Primordial Germ Cell Fate.

BACKGROUND: Primordial germ cell (PGC) fate is dictated by the designation, taxis, and influence of the surrounding embryonic somatic cells. Whereas gonadal sex determination results from a balance of factors within the tissue microenvironment. SUMMARY: Our understanding of mammalian ovary development is formed in large part from developmental time courses established using murine models. Genomic tools where genes implicated in the PGC designation or gonadal sex determination have been modulated through complete or conditional knockouts in vivo, and studies in in situ models with inhibitors or cultures that alter the native gonadal environment have pieced together the interplay of pioneering transcription factors, co-regulators and chromosomes critical for the progression of PGCs to oocytes. Tools such as pluripotent stem cell derivation, genomic modifications, and aggregate differentiation cultures have yielded some insight into the human condition. Additional understanding of sex determination, both gonadal and anatomical, may be inferred from phenotypes that arise from de novo or inherited gene variants in humans who have differences in sex development. KEY MESSAGES: This review highlights major factors critical for PGC specification and migration, and in ovarian gonad specification by reviewing seminal murine models. These pathways are compared to what is known about the human condition from expression profiles of fetal gonadal tissue, use of human pluripotent stem cells, or disorders resulting from disease variants. Many of these pathways are challenging to decipher in human tissues. However, the impact of new single-cell technologies and whole-genome sequencing to reveal disease variants of idiopathic reproductive tract phenotypes will help elucidate the mechanisms involved in human ovary development.

Efficacy of Video-based Education in Improving Understanding of Pediatric Fertility Preservation.

Eighty percent of children diagnosed with cancer in childhood survive into adulthood. Fertility preservation (FP) is an important consideration, and procedures are available to reduce the risk of infertility following gonadotoxic therapies. Discussing FP options eases decision-making and minimizes regret; however, poor comprehension of these topics remains a challenge. This study evaluates if video-based educational tools increase understanding of FP options among pediatric patients and families. Videos were first tested among participants not at risk of infertility to ensure objective utility and optimize quality. In part 1, parents of pediatric surgical patients were randomized to view 2 publicly available educational videos on FP in differing orders. Each group completed pre-surveys and post-surveys assessing the comprehension and perception of video quality. In part 2, the parent and patient participants completed a comprehension assessment before and after viewing our institution-specific educational video, designed based on participant feedback from part 1. Part 1 results demonstrated a significant increase in participant knowledge and perceived understanding after viewing the videos ( P <0.001), regardless of order. In part 2, the post-test comprehension scores were significantly improved for all participants and all subgroups, P <0.01. Results suggest that video-based educational tools may help to reduce barriers to FP in pediatric oncology.

Anatomic nomenclature and 3-dimensional regional model of the human ovary: call for a new paradigm.

The ovaries are the female gonads that are crucial for reproduction, steroid production, and overall health. Historically, the ovary was broadly divided into regions defined as the cortex, medulla, and hilum. This current nomenclature lacks specificity and fails to consider the significant anatomic variations in the ovary. Recent technological advances in imaging modalities and high-resolution omic analyses have brought about the need for revision of the existing definitions, which will facilitate the integration of generated data and enable the characterization of organ subanatomy and function at the cellular level. The creation of these high-resolution multimodal maps of the ovary will enhance collaboration and communication among disciplines and between clinicians and researchers. Beginning in March 2021, the Pediatric and Adolescent Gynecology Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development invited subject-matter experts to participate in a series of workshops and meetings to standardize ovarian nomenclature and define the organ's features. The goal was to develop a spatially defined and semantically consistent terminology of the ovary to support collaborative, team science-based endeavors aimed at generating reference atlases of the human ovary. The group recommended a standardized, 3-dimensional description of the ovary and an ontological approach to the subanatomy of the ovary and definition of follicles. This new greater precision in nomenclature and mapping will better reflect the ovary's heterogeneous composition and function, support the standardization of tissue collection, facilitate functional analyses, and enable clinical and research collaborations. The conceptualization process and outcomes of the effort, which spanned the better part of 2021 and early 2022, are introduced in this article. The institute and the workshop participants encourage researchers and clinicians to adopt the new systems in their everyday work to advance the overarching goal of improving human reproductive health.

A Systematic Review of Ovarian Tissue Transplantation Outcomes by Ovarian Tissue Processing Size for Cryopreservation.

Ovarian tissue cryopreservation (OTC) is the only pre-treatment option currently available to preserve fertility for prepubescent girls and patients who cannot undergo ovarian stimulation. Currently, there is no standardized method of processing ovarian tissue for cryopreservation, despite evidence that fragmentation of ovaries may trigger primordial follicle activation. Because fragmentation may influence ovarian transplant function, the purpose of this systematic review was (1) to identify the processing sizes and dimensions of ovarian tissue within sites around the world, and (2) to examine the reported outcomes of ovarian tissue transplantation including, reported duration of hormone restoration, pregnancy, and live birth. A total of 2,252 abstracts were screened against the inclusion criteria. In this systematic review, 103 studies were included for analysis of tissue processing size and 21 studies were included for analysis of ovarian transplantation outcomes. Only studies where ovarian tissue was cryopreserved (via slow freezing or vitrification) and transplanted orthotopically were included in the review. The size of cryopreserved ovarian tissue was categorized based on dimensions into strips, squares, and fragments. Of the 103 studies, 58 fertility preservation sites were identified that processed ovarian tissue into strips (62%), squares (25.8%), or fragments (31%). Ovarian tissue transplantation was performed in 92 participants that had ovarian tissue cryopreserved into strips (n = 51), squares (n = 37), and fragments (n = 4). All participants had ovarian tissue cryopreserved by slow freezing. The pregnancy rate was 81.3%, 45.5%, 66.7% in the strips, squares, fragment groups, respectively. The live birth rate was 56.3%, 18.2%, 66.7% in the strips, squares, fragment groups, respectively. The mean time from ovarian tissue transplantation to ovarian hormone restoration was 3.88 months, 3.56 months, and 3 months in the strips, squares, and fragments groups, respectively. There was no significant difference between the time of ovarian function' restoration and the size of ovarian tissue. Transplantation of ovarian tissue, regardless of its processing dimensions, restores ovarian hormone activity in the participants that were reported in the literature. More detailed information about the tissue processing size and outcomes post-transplant are required to identify a preferred or more successful processing method. Systematic Review Registration: [https://www.crd.york.ac.uk], identifier [CRD42020189120].

Differences in gonadal tissue cryopreservation practices for differences of sex development across regions in the United States.

Objective: Some individuals with differences of sex development (DSD) conditions undergo medically indicated prophylactic gonadectomy. Gonads of individuals with DSD can contain germ cells and precursors and patients interested in future fertility preservation and hormonal restoration can participate in DSD-specific research protocols to cryopreserve this tissue. However, it is unclear how many providers or institutions offer gonadal tissue cryopreservation (GTC) and how widespread GTC for DSD is across the United States (US). The Pediatric Initiative Network (PIN) and Non-Oncologic Conditions committees of the Oncofertility Consortium sought to assess the current state of GTC for patients with DSD. Methods: An electronic survey was sent to providers caring for patients with DSD via special interest groups of professional societies and research networks. Results: The survey was administered between November 15, 2021 and March 14, 2022. A total of 155 providers responded to the survey, of which 132 respondents care for patients with DSD, and 78 work at facilities that offer medically indicated gonadectomy to patients with DSD diagnoses. They represented 55 US institutions including 47 pediatric hospitals, and 5 international sites (Canada, Denmark, Germany, Qatar). Of individual providers, 41% offer cryopreservation after prophylactic gonadectomy for patients with DSD (32/78). At an institutional level, GTC after medically indicated gonadectomy is available at 54.4% (24/46) of institutions. GTC is offered for a variety of DSD diagnoses, most commonly 45,X/46,XY DSD (i.e., Turner Syndrome with Y-chromosome material and mixed gonadal dysgenesis), ovotesticular DSD, complete androgen insensitivity syndrome (CAIS), and complete gonadal dysgenesis. Responses demonstrate regional trends in GTC practices with 83.3% of institutions in the Midwest, 66.7% in the Northeast, 54.6% in the West, and 35.3% in the South providing GTC. All represented institutions (100%) send gonadal tissue for pathological evaluation, and 22.7% preserve tissue for research purposes. Conclusions: GTC after gonadectomy is offered at half of the US institutions represented in our survey, though a minority are currently preserving tissue for research purposes. GTC is offered for several DSD conditions. Future research will focus on examining presence and quality of germ cells to support clinical decision making related to fertility preservation for patients with DSD.

Generation of two human induced pluripotent stem cell lines from a patient with complete androgen insensitivity syndrome with a hemizygous single nucleotide variant in the androgen receptor (AR) gene.

Complete Androgen Insensitivity Syndrome (CAIS) is a difference of sex development (DSD) caused by loss of function of the androgen receptor (AR) gene. Patients typically identify as female and have a 46,XY karyotype. Two induced pluripotent stem cell lines (iPSCs), LCHi001-A and LCHi001-B, were generated from a participant with CAIS with AR mutation: c.2698A>T (p.Ile900Phe). Both lines presented typical morphology, expressed stem cell markers, differentiated into three germ layers, had a normal 46,XY karyotype, were mycoplasma-free, and carried the expected mutation in AR. These iPSC lines are an important resource for studying CAIS pathogenesis and possible treatment options.