Current Fertility Preservation Steps in Young Women Suffering from Cancer and Future Perspectives.

Childhood cancer incidence, especially in high-income countries, has led to a focus on preserving fertility in this vulnerable population. The common treatments, such as radiation and certain chemotherapeutic agents, though effective, pose a risk to fertility. For adult women, established techniques like embryo and egg freezing are standard, requiring ovarian stimulation. However, for prepubescent girls, ovarian tissue freezing has become the primary option, eliminating the need for hormonal preparation. This review describes the beginning, evolution, and current situation of the fertility preservation options for this young population. A total of 75 studies were included, covering the steps in the current fertility preservation protocols: (i) ovarian tissue extraction, (ii) the freezing method, and (iii) thawing and transplantation. Cryopreservation and the subsequent transplantation of ovarian tissue have resulted in successful fertility restoration, with over 200 recorded live births, including cases involving ovarian tissue cryopreserved from prepubescent girls. Despite promising results, challenges persist, such as follicular loss during transplantation, which is attributed to ischemic and oxidative damage. Optimizing ovarian tissue-freezing processes and exploring alternatives to transplantation, like in vitro systems for follicles to establish maturation, are essential to mitigating associated risks. Further research is required in fertility preservation techniques to enhance clinical outcomes in the future. Ovarian tissue cryopreservation appears to be a method with specific benefits, indications, and risks, which can be an important tool in terms of preserving fertility in younger women.

Quantification of follicles in human ovarian tissue using image processing software and trained artificial intelligence.

Cancer survival rates in prepubertal girls and young women have risen in recent decades due to increasingly efficient treatments. However, many such treatments are gonadotoxic, causing premature ovarian insufficiency (POI), loss of fertility and ovarian endocrine function. Implantation of donor ovarian tissue encapsulated in immune-isolating capsules is a promising method to restore physiological endocrine function without immunosuppression or risk of reintroducing cancer cells harbored by the tissue. The success of this approach is largely determined by follicle density (FD) in the implanted ovarian tissue, which is analyzed manually from histologic sections and necessitates specialized, time-consuming labor. To address this limitation, we developed a fully automated method to quantify FD that does not require additional coding. We first analyzed ovarian tissue from 12 human donors between 16 to 37 years old using semi-automated image processing with manual follicle annotation and then trained artificial intelligence program based on follicle identification and object classification. One operator manually analyzed 102 whole slide images (WSIs) from serial histologic sections. Of those, 77 images were assessed by a second manual operator, followed with an automated method utilizing artificial intelligence (AI). Of the 1181 follicles the control operator counted, the comparison operator counted 1178, and the AI counted 927 follicles with 80% of those being correctly identified as follicles. The three-stage AI pipeline finished 33% faster than manual annotation. Collectively, this report supports the use of artificial intelligence and automation to select tissue donors and grafts with the greatest FD to ensure graft longevity for POI treatment.

In Vitro Growth of Human Follicles: Current and Future Perspectives.

Ovarian tissue cryopreservation is gaining importance as a successful method to restore fertility to girls and young women at high risk of sterility. However, there are concerns regarding the safety of transplantation after ovarian tissue cryopreservation due to the high risk of reintroducing cancer cells and causing disease recurrence. In these cases, the development of culture systems that support oocyte development from the primordial follicle stage is required. Notable achievements have been reached in human follicle in vitro growth in the past decade. Currently, systems for the in vitro culture of ovarian tissue are based on two-dimensional substrates that do not support the survival of follicles or recapitulate the mechanical heterogenicity in the mammalian ovary. Recognition of the importance of special arrangements between cells has spurred research in three-dimensional culture systems, and the provision of a precise culture system that maximizes the diffusion of nutrients and gases through the follicles has raised interest in advanced biomimetic models. The current review critically examines various culture systems employed for the in vitro development of follicles, with a particular focus on solutions utilizing Organ-on-a-Chip (OOC) technology. The emphasis on OOC technology underscores its role as a promising avenue in ensuring the successful cultivation and maintenance of follicular structures during the culture period.

In vitro fertilization outcomes in a mouse model of gender-affirming hormone therapy in transmasculine youth.

OBJECTIVE: To investigate in vitro fertilization (IVF) outcomes in an adolescent transmasculine mouse model mimicking gender-affirming hormone therapy in prepubertal youth, both on testosterone (T) and after T washout. DESIGN: Experimental laboratory study using a validated mouse model. SETTING: University-based basic science research laboratory. ANIMAL(S): A total of 80 prepubertal 26-day-old C57BL/6N female mice were used in this study. INTERVENTION(S): Animals (n = 10/group) were implanted subcutaneously with gonadotropin-releasing hormone agonist at 3.6 mg or received sham surgery. After 21 days, they were implanted with silastic tubing containing either T 10 mg or placebo for 6 weeks. After 6 weeks, a group of animals were superovulated for immediate IVF, and another group had the implant removed and went through superovulation for IVF after 2 weeks (washout IVF). The total number of oocytes yielded, oocyte maturity rate, fertilization rate, and numbers of 2-cell embryos, 4-8-cell embryos, morula, blastocysts, and hatching blastocysts were recorded. RESULT(S): Testosterone treatment negatively impacted IVF outcomes in animals stimulated when receiving T, but not after T washout. Pretreatment with gonadotropin-releasing hormone agonist did not affect IVF outcomes. CONCLUSION(S): Although current T had a negative impact on IVF outcomes compared with controls, animals were still able to produce viable oocytes for fertilization and develop into blastocysts. Future efforts to study the impact of long-term T exposure on oocyte quality, especially aneuploidy rates, pregnancy outcomes, and live birth rates, are necessary.

Encapsulated Allografts Preclude Host Sensitization and Promote Ovarian Endocrine Function in Ovariectomized Young Rhesus Monkeys and Sensitized Mice.

Transplantation of allogeneic donor ovarian tissue holds great potential for female cancer survivors who often experience premature ovarian insufficiency. To avoid complications associated with immune suppression and to protect transplanted ovarian allografts from immune-mediated injury, we have developed an immunoisolating hydrogel-based capsule that supports the function of ovarian allografts without triggering an immune response. Encapsulated ovarian allografts implanted in naïve ovariectomized BALB/c mice responded to the circulating gonadotropins and maintained function for 4 months, as evident by regular estrous cycles and the presence of antral follicles in the retrieved grafts. In contrast to non-encapsulated controls, repeated implantations of encapsulated mouse ovarian allografts did not sensitize naïve BALB/c mice, which was confirmed with undetectable levels of alloantibodies. Further, encapsulated allografts implanted in hosts previously sensitized by the implantation of non-encapsulated allografts restored estrous cycles similarly to our results in naïve recipients. Next, we tested the translational potential and efficiency of the immune-isolating capsule in a rhesus monkey model by implanting encapsulated ovarian auto- and allografts in young ovariectomized animals. The encapsulated ovarian grafts survived and restored basal levels of urinary estrone conjugate and pregnanediol 3-glucuronide during the 4- and 5-month observation periods. We demonstrate, for the first time, that encapsulated ovarian allografts functioned for months in young rhesus monkeys and sensitized mice, while the immunoisolating capsule prevented sensitization and protected the allograft from rejection.

A mouse model mimicking gender-affirming treatment with pubertal suppression followed by testosterone in transmasculine youth.

STUDY QUESTION: Can mice serve as a translational model to examine the reproductive consequences of pubertal suppression with GnRH agonist (GnRHa) followed by testosterone (T) administration, a typical therapy in peripubertal transmasculine youth? SUMMARY ANSWER: An implanted depot with 3.6 mg of GnRHa followed by T enanthate at 0.45 mg weekly can be used in peripubertal female mice for investigating the impact of gender-affirming hormone therapy in transmasculine youth. WHAT IS KNOWN ALREADY: There is limited knowledge available in transgender medicine to provide evidence-based fertility care, with the current guidelines being based on the assumption of fertility loss. We recently successfully developed a mouse model to investigate the reproductive consequences of T therapy given to transgender men. On the other hand, to our knowledge, there is no mouse model to assess the reproductive outcomes in peripubertal transmasculine youth. STUDY DESIGN, SIZE, DURATION: A total of 80 C57BL/6N female mice were used in this study, with n = 7 mice in each experimental group. PARTICIPANTS/MATERIALS, SETTING, METHODS: We first assessed the effectiveness of GnRHa in arresting pubertal development in the female mice. In this experiment, 26-day-old female mice were subcutaneously implanted with a GnRHa (3.6 mg) depot. Controls underwent a sham surgery. Animals were euthanized at 3, 9, 21 and 28 days after the day of surgery. In the second experiment, we induced a transmasculine youth mouse model. C57BL/6N female mice were subcutaneously implanted with a 3.6 mg GnRHa depot on postnatal day 26 for 21 days and this was followed by weekly injections of 0.45 mg T enanthate for 6 weeks. The control for the GnRH treatment was sham surgery and the control for T treatment was sesame oil vehicle injections. Animals were sacrificed 0.5 weeks after the last injection. The data collected included the day of the vaginal opening and first estrus, daily vaginal cytology, weekly and terminal reproductive hormones levels, body/organ weights, ovarian follicular distribution and corpora lutea (CL) counts. MAIN RESULTS AND THE ROLE OF CHANCE: GnRHa implanted animals remained in persistent diestrus and had reduced levels of FSH (P = 0.0013), LH (P = 0.0082) and estradiol (P = 0.0155), decreased uterine (P < 0.0001) and ovarian weights (P = 0.0002), and a lack of CL at 21 days after GnRHa implantation. T-only and GnRHa+T-treated animals were acyclic throughout the treatment period, had sustained elevated levels of T, suppressed LH levels (P < 0.0001), and an absence of CL compared to controls (P < 0.0001). Paired ovarian weights were reduced in the T-only and GnRHa+T groups compared with the control and GnRHa-only groups. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although it is an appropriate tool to provide relevant findings, precaution is needed to extrapolate mouse model results to mirror human reproductive physiology. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this study describes the first mouse model mimicking gender-affirming hormone therapy in peripubertal transmasculine youth. This model provides a tool for researchers studying the effects of GnRHa-T therapy on other aspects of reproduction, other organ systems and transgenerational effects. The model is supported by GnRHa suppressing puberty and maintaining acyclicity during T treatment, lower LH levels and absence of CL. The results also suggest GnRHa+T therapy in peripubertal female mice does not affect ovarian reserve, since the number of primordial follicles was not affected by treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Michigan Institute for Clinical and Health Research grants KL2 TR 002241 and UL1 TR 002240 (C.D.C.); National Institutes of Health grants F30-HD100163 and T32-HD079342 (H.M.K.); University of Michigan Office of Research funding U058227 (A.S.); American Society for Reproductive Medicine/Society for Reproductive Endocrinology and Infertility grant (M.B.M.); and National Institutes of Health R01-HD098233 (M.B.M.). The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core Facility was supported by the Eunice Kennedy Shriver NICHD/NIH grants P50-HD028934 and R24-HD102061. The authors declare that they have no competing interests.

Human Ovarian Follicles Xenografted in Immunoisolating Capsules Survive Long Term Implantation in Mice.

Female pediatric cancer survivors often develop Premature Ovarian Insufficiency (POI) owing to gonadotoxic effects of anticancer treatments. Here we investigate the use of a cell-based therapy consisting of human ovarian cortex encapsulated in a poly-ethylene glycol (PEG)-based hydrogel that replicates the physiological cyclic and pulsatile hormonal patterns of healthy reproductive-aged women. Human ovarian tissue from four donors was analyzed for follicle density, with averages ranging between 360 and 4414 follicles/mm3. Follicles in the encapsulated and implanted cryopreserved human ovarian tissues survived up to three months, with average follicle densities ranging between 2 and 89 follicles/mm3 at retrieval. We conclude that encapsulation of human ovarian cortex in PEG-based hydrogels did not decrease follicle survival after implantation in mice and was similar to non-encapsulated grafts. Furthermore, this approach offers the means to replace the endocrine function of the ovary tissue in patients with POI.

Cellular heterogeneity of human fallopian tubes in normal and hydrosalpinx disease states identified using scRNA-seq.

Fallopian tube (FT) homeostasis requires dynamic regulation of heterogeneous cell populations and is disrupted in infertility and ovarian cancer. Here, we applied single-cell RNA-seq to profile 59,738 FT cells from four healthy, pre-menopausal subjects. The resulting cell atlas contains 12 major cell types representing epithelial, stromal, and immune compartments. Re-clustering of epithelial cells identified four ciliated and six non-ciliated secretory epithelial subtypes, two of which represent potential progenitor pools: one leading to mature secretory cells and the other contributing to either ciliated cells or one of the stromal cell types. To understand how FT cell numbers and states change in a disease state, we analyzed 17,798 cells from two hydrosalpinx samples and observed shifts in epithelial and stromal populations and cell-type-specific changes in extracellular matrix and TGF-ß signaling; this underscores fibrosis pathophysiology. This resource is expected to facilitate future studies aimed at expanding understanding of fallopian tube homeostasis in normal development and disease.

TNFRSF13B genotypes control immune-mediated pathology by regulating the functions of innate B cells.

Host genes define the severity of inflammation and immunity but specific loci doing so are unknown. Here we show that TNF receptor superfamily member 13B (TNFRSF13B) variants, which enhance defense against certain pathogens, also control immune-mediated injury of transplants, by regulating innate B cells' functions. Analysis of TNFRSF13B in human kidney transplant recipients revealed that 33% of those with antibody-mediated rejection (AMR) but fewer than 6% of those with stable graft function had TNFRSF13B missense mutations. To explore mechanisms underlying aggressive immune responses, we investigated alloimmunity and rejection in mice. Cardiac allografts in Tnfrsf13b-mutant mice underwent early and severe AMR. The dominance and precocity of AMR in Tnfrsf13b-deficient mice were not caused by increased alloantibodies. Rather, Tnfrsf13b mutations decreased "natural" IgM and compromised complement regulation, leading to complement deposition in allografted hearts and autogenous kidneys. Thus, WT TNFRSF13B and Tnfrsf13b support innate B cell functions that limit complement-associated inflammation; in contrast, common variants of these genes intensify inflammatory responses that help clear microbial infections but allow inadvertent tissue injury to ensue. The wide variation in inflammatory reactions associated with TNFRSF13B diversity suggests polymorphisms could underlie variation in host defense and explosive inflammatory responses that sometimes enhance morbidity associated with immune responses.

Capitalizing on transcriptome profiling to optimize and identify targets for promoting early murine folliculogenesis in vitro.

In vitro ovarian follicle culture is an active area of research towards providing fertility options for survivors of childhood cancer. Late-stage murine follicles (multilayer secondary and onwards) can be cultured successfully to maturity to obtain a meiotically competent oocyte for fertilization, but primordial and primary follicles usually die in culture because many key components of early follicle development are still unknown and difficult to mimic in vitro. To engineer a biomimetic three-dimensional culture system with high efficacy and reproducibility for the clinic, detailed mechanisms of early folliculogenesis must be uncovered. Previous studies have shown that primary murine follicles co-cultured in groups, in contrast to single follicles cultured in isolation, can reach preovulatory size and produce competent oocytes, but the factors accounting for the synergy of follicle co-culture are still unknown. To probe the underlying mechanisms of successful follicle co-culture, we conducted a time-course experiment for murine follicles encapsulated in 0.3% alginate hydrogels and compared between two conditions: groups of 5 (5X) versus groups of 10 (10X). For every 2 days during the course of 12 days, follicles were dissociated and somatic cells were isolated for microarray-based gene expression analysis (n = 380 follicles for 5X and n = 430 follicles for 10X). Gene activities in follicles co-cultured in larger groups (10X) had a distinct transcriptomic profile of key genes and pathways such as prolactin signaling and angiogenesis-related genes when compared to cells from follicles co-cultured in the smaller cohort (5X). To benchmark the results for follicles grown in culture, we compared our microarray data to data from murine follicles freshly isolated from the ovary at comparable stages of development previously published by Bernabé et al. Comparison of these datasets identified similarities and differences between folliculogenesis in the native microenvironment and the engineered in vitro system. A more detailed understanding of follicle growth in vitro will not only allow for better culture methods but also advance the field towards providing improved fertility options for survivors of childhood cancer.

Sequestered cell-secreted extracellular matrix proteins improve murine folliculogenesis and oocyte maturation for fertility preservation.

Synthetic matrices offer a high degree of control and tunability for mimicking extracellular matrix functions of native tissue, allowing the study of disease and development in vitro. In this study, we functionalized degradable poly(ethylene glycol) hydrogels with extracellular matrix (ECM)-sequestering peptides aiming to recapitulate the native ECM composition for culture and maturation of ovarian follicular organoids. We hypothesized that ECM-sequestering peptides would facilitate deposition and retention of cell-secreted ECM molecules, thereby recreating cell-matrix interactions in otherwise bioinert PEG hydrogels. Specifically, heparin-binding peptide from antithrombin III (HBP), heparan sulfate binding peptide derived from laminin (AG73), basement membrane binder peptide (BMB), and heparan sulfate binding region of placental growth factor 2 (RRR) tethered to a PEG hydrogel significantly improved follicle survival, growth and maturation compared to PEG-Cys, a mechanically similar but biologically inert control. Immunohistochemical analysis of the hydrogel surrounding cultured follicles confirmed sequestration and retention of laminin, collagen I, perlecan, and fibronectin in ECM-sequestering hydrogels but not in bioinert PEG-Cys hydrogels. The media from follicles cultured in PEG-AG73, PEG-BMB, and PEG-RRR also had significantly higher concentrations of factors known to regulate follicle development compared to PEG-Cys. PEG-AG73 and PEG-BMB were the most beneficial for promoting follicle maturation, likely because AG73 and BMB mimic basement membrane interactions which are crucial for follicle development. Here we have shown that functionalizing PEG with ECM-sequestering peptides allows cell-secreted ECM to be retained within the hydrogels, restoring critical cell-matrix interactions and promoting healthy organoid development in a fully synthetic culture system. STATEMENT OF SIGNIFICANCE: Here we present a novel approach for sequestering and retaining cell-secreted extracellular matrix in a fully synthetic material for organoid culture. We have engineered a biomimetic poly(ethylene glycol) hydrogel functionalized with extracellular matrix-binding peptides to recapitulate the ovarian microenvironment. Incorporation of these peptides allows ovarian follicles to recreate their native matrix with the sequestered ECM that subsequently binds growth factors, facilitating follicle maturation. The novel design resulted in improved outcomes of folliculogenesis, potentially developing a fertility preservation option for young women undergoing sterilizing treatments for cancer. The fully synthetic and modular nature of this biomimetic material holds promise for other tissue engineering applications as it allows encapsulated cells to rebuild their native microenvironments in vitro.

Hormonal Stimulation of Human Ovarian Xenografts in Mice: Studying Folliculogenesis, Activation, and Oocyte Maturation.

Ovarian tissue cryopreservation and banking provides a fertility preservation option for patients who cannot undergo oocyte retrieval; it is quickly becoming a critical component of assisted reproductive technology programs across the world. While the transplantation of cryopreserved ovarian tissue has resulted in over 130 live births, the field has ample room for technological improvements. Specifically, the functional timeline of grafted tissue and each patient's probability of achieving pregnancy is largely unpredictable due to patient-to-patient variability in ovarian reserve, lack of a reliable method for quantifying follicle numbers within tissue fragments, potential risk of reintroduction of cancer cells harbored in ovarian tissues, and an inability to control follicle activation rates. This review focuses on one of the most common physiological techniques used to study human ovarian tissue transplantation, xenotransplantation of human ovarian tissue to mice and endeavors to inform future studies by discussing the elements of the xenotransplantation model, challenges unique to the use of human ovarian tissue, and novel tissue engineering techniques currently under investigation.

The ovarian stroma as a new frontier.

Historically, research in ovarian biology has focused on folliculogenesis, but recently the ovarian stroma has become an exciting new frontier for research, holding critical keys to understanding complex ovarian dynamics. Ovarian follicles, which are the functional units of the ovary, comprise the ovarian parenchyma, while the ovarian stroma thus refers to the inverse or the components of the ovary that are not ovarian follicles. The ovarian stroma includes more general components such as immune cells, blood vessels, nerves, and lymphatic vessels, as well as ovary-specific components including ovarian surface epithelium, tunica albuginea, intraovarian rete ovarii, hilar cells, stem cells, and a majority of incompletely characterized stromal cells including the fibroblast-like, spindle-shaped, and interstitial cells. The stroma also includes ovarian extracellular matrix components. This review combines foundational and emerging scholarship regarding the structures and roles of the different components of the ovarian stroma in normal physiology. This is followed by a discussion of key areas for further research regarding the ovarian stroma, including elucidating theca cell origins, understanding stromal cell hormone production and responsiveness, investigating pathological conditions such as polycystic ovary syndrome (PCOS), developing artificial ovary technology, and using technological advances to further delineate the multiple stromal cell types.

Ovarian tissue cryopreservation and novel bioengineering approaches for fertility preservation.

PURPOSE OF REVIEW: Breast cancer patients who cannot delay treatment or for whom hormone stimulation and egg retrieval are contraindicated require alternative methods of fertility preservation prior to gonadotoxic treatment. Ovarian tissue cryopreservation is an alternative approach that may offer patients the opportunity to preserve fertility and carry biologically-related children later in life. Various experimental approaches are being explored to obtain mature gametes from cryopreserved and thawed ovarian tissue for fertilization and implantation using biomimetic tissue culture in vitro. Here we review the most recent developments in ovarian tissue cryopreservation and exciting advances in bioengineering approaches to in vitro tissue and ovarian follicle culture. RECENT FINDINGS: Slow freezing is the most widely accepted method for ovarian tissue cryopreservation, but efforts have been made to modify vitrification for this application as well. Numerous approaches to in vitro tissue and follicle culture are in development, most prominently two-step culture systems for ovarian cortical tissue and encapsulation of ovarian follicles in biomimetic matrices for in vitro culture. SUMMARY: Refinements to slow freeze and vitrification protocols continue to address challenges associated with cryopreservation, such as ice crystal formation and damage to the stroma. Similarly, improvements to in vitro tissue and follicle culture show promise for utilizing patients' cryopreserved tissues to obtain mature gametes after disease treatment and remission. Development of an effective and reproducible culture system for human ovarian follicles will serve as a broad assisted reproductive technology for cancer survivors who cryopreserved tissue prior to treatment.

Encapsulation of ovarian allograft precludes immune rejection and promotes restoration of endocrine function in immune-competent ovariectomized mice.

Premature ovarian insufficiency (POI) is a significant complication of cytotoxic treatments due to extreme ovarian sensitivity to chemotherapy and radiation. POI is particularly devastating for young girls reaching puberty, because it irreversibly affects their physical and cognitive development. Changes occurring during puberty determine their height, bone health, insulin responsiveness, lipid metabolism, cardiovascular health and cognition. The only available treatment for POI during puberty is hormone replacement therapy (HRT), which delivers non-physiological levels of estrogen, lacks other ovarian hormones and pulsatility, and is not responsive to feedback regulation. Here we report that ovarian allografts encapsulated in a hydrogel-based capsule and implanted in ovariectomized mice restore ovarian endocrine function in immune competent mice. Ovarian tissue from BALB/c mice was encapsulated in poly(ethylene-glycol) (PEG) hydrogels, with a proteolytically degradable core and a non-degradable shell. The dual capsules were implanted subcutaneously in immune competent ovariectomized C57BL/6 mice for a period of 60 days. As expected, non-encapsulated ovarian allografts implanted in a control group sensitized the recipients as confirmed with donor-specific IgG in the serum, which increased 26-fold in the 3 weeks following transplantation (p = 0.02) and infiltration of the graft with CD8 T cells consistent with allo-immunity. In contrast, encapsulation in the Dual PEG capsules prevented sensitization to the allograft in all the recipients with no evidence of lymphocytic infiltration. In summary, the approach of hydrogel-based immunoisolation presents a minimally invasive and robust cell-therapy to restore hormonal balance in ovarian insufficiency. This report is the first to demonstrate the application of a tunable PEG-based hydrogel as an immunoisolator of allogeneic ovarian tissue to restore endocrine function in ovariectomized mice and prevent cell-mediated immune rejection in immune competent mice.

Adipose-derived stem cells promote survival, growth, and maturation of early-stage murine follicles.

BACKGROUND: Premature ovarian insufficiency is a common complication of anticancer treatments in young women and girls. The ovary is a complex, highly regulated reproductive organ, whose proper function is contingent upon the bidirectional endocrine, paracrine, and autocrine signaling. These factors facilitate the development of the follicles, the functional units of the ovary, to progress from the gonadotropin-independent, paracrine-controlled early stage to the gonadotropin-dependent, endocrine-controlled later stage. We hypothesized that the low survival rate of individually cultured early-stage follicles could be improved with co-culture of adipose-derived stem cells (ADSCs) that secrete survival- and growth-promoting factors. MATERIALS AND METHODS: Ovarian follicles ranging from 85 to 115 µm in diameter, from 10- to 12-day-old B6CBAF1 mice were mechanically isolated and co-encapsulated with ADSCs within alginate-based 3D culture system. The follicles were cultured for 14 days, imaged using light microscopy every 2 days, and matured at the end. Follicle media were changed every 2 days and collected for hormone measurements. Follicle diameter, morphology, number of transzonal projections, and survival and maturation rates were recorded. Statistical analyses using one- and two-way ANOVA were performed to compare hormone levels, survival of the follicles and ADSCs, oocyte maturation rates, and follicle growth. RESULTS: The co-encapsulation of the follicles with ADSCs increased follicle survival, ranging from 42.4% for the 86-95 µm to 86.2% for the 106-115-µm follicle size group. Co-culture also improved the follicle growth, the rate of antrum formation and oocyte maturation compared to the follicles cultured alone. The levels of androstenedione, estradiol, and progesterone of co-encapsulated follicles increased progressively with time in culture. CONCLUSIONS: To our knowledge, this is the first report of an in vitro system utilizing mouse adipose-derived stem cells to support the development of the mouse follicles. Our findings suggest that co-encapsulation of ADSCs with early-stage follicles supports follicular development, through secretion of cytokines that promote follicular survival, antrum formation, and meiotic competence. The unique 3D culture system that supports the survival of both cell types has translational implications, as ADSCs could be used as an autologous source for in vitro maturation of early-stage human follicles.

Immuno-Isolating Dual Poly(ethylene glycol) Capsule Prevents Cancer Cells from Spreading Following Mouse Ovarian Tissue Auto-Transplantation.

For female cancer survivors, premature ovarian insufficiency (POI) is a common complication of anticancer treatments. Ovarian tissue cryopreservation before treatment, followed by auto-transplantation after remission is a promising option to restore fertility and ovarian endocrine function. However, auto-transplantation is associated with the risk of re-introducing malignant cells harbored in the stroma of the ovarian autograft. To mitigate this risk, we investigated in this pilot study whether an immuno-isolating dual-layered poly(ethylene glycol)(PEG) capsule can retain cancer cells, while supporting folliculogenesis. The dual PEG capsule loaded with 1000 4T1 cancer cells retained 100% of the encapsulated cells in vitro for 21 days of culture. However, a greater cell load of 10,000 cells/capsule led to capsule failure and cells' release. To assess the ability of the capsule to retain cancer cells, prevent metastasis, and support folliculogenesis in vivo we co-encapsulated cancer cells with ovarian tissue in the dual PEG capsule and implanted subcutaneously in mice. Control mice implanted with 2000 non-encapsulated cancer cells had tumors formed within 14 days and metastasis to the lungs. In contrast, no tumor mass formation or metastasis to the lungs was observed in mice with the same number of cancer cells encapsulated in the capsule. Our findings suggest that the immuno-isolating capsule may prevent the escape of the malignant cells potentially harbored in ovarian allografts and, in the future, improve the safety of ovarian tissue auto-transplantation in female cancer survivors.

Retrievable hydrogels for ovarian follicle transplantation and oocyte collection.

Cancer survivorship rates have drastically increased due to improved efficacy of oncologic treatments. Consequently, clinical concerns have shifted from solely focusing on survival to quality of life, with fertility preservation as an important consideration. Among fertility preservation strategies for female patients, ovarian tissue cryopreservation and subsequent reimplantation has been the only clinical option available to cancer survivors with cryopreserved tissue. However, follicle atresia after transplantation and risk of reintroducing malignant cells have prevented this procedure from becoming widely adopted in clinics. Herein, we investigated the encapsulation of ovarian follicles in alginate hydrogels that isolate the graft from the host, yet allows for maturation after transplantation at a heterotopic (i.e., subcutaneous) site, a process we termed in vivo follicle maturation. Survival of multiple follicle populations was confirmed via histology, with the notable development of the antral follicles. Collected oocytes (63%) exhibited polar body extrusion and were fertilized by intracytoplasmic sperm injection and standard in vitro fertilization procedures. Successfully fertilized oocytes developed to the pronucleus (14%), two-cell (36%), and four-cell (7%) stages. Furthermore, ovarian follicles cotransplanted with metastatic breast cancer cells within the hydrogels allowed for retrieval of the follicles, and no mice developed tumors after removal of the implant, confirming that the hydrogel prevented seeding of disease within the host. Collectively, these findings demonstrate a viable option for safe use of potentially cancer-laden ovarian donor tissue for in vivo follicle maturation within a retrievable hydrogel and subsequent oocyte collection. Ultimately, this technology may provide novel options to preserve fertility for young female patients with cancer.

Detection of lindane and 7,12-dimethylbenz[a]anthracene toxicity at low concentrations in a three-dimensional ovarian follicle culture system.

Exposure to environmental toxicants that target ovarian follicles can have long-lasting effects on women's reproductive health and health of the offspring. Experiments in rodents have contributed knowledge about the effects of individual toxicants on ovarian follicles. However, little is known about the effects of mixtures of toxicants on ovarian follicular health. We studied the combined effects of low, physiologically- and environmentally-relevant concentrations of toxicants on murine secondary ovarian follicles cultured in an encapsulated three-dimensional (3D) system. Exposure to lindane and 7,12-dimenthylbenz(a)anthracene (DMBA) led to decreased follicle survival, impaired development and compromised maturation in a concentration-dependent manner. DMBA showed a greater toxicity to cultured follicles compared to lindane. The mixtures of lindane and DMBA did not produce a synergistic toxic effect on follicles. Rather, ovarian follicles exposed to the mixtures showed survival and growth patterns similar to the follicles exposed to the same concentrations of individual toxicants. Our findings regarding follicle toxicity at such low concentrations help informing what might be overlooked when regulating environmental toxicants. The proposed 3D culture system allowed studying the effects of mixtures of environmental toxicants in a physiological setting, providing much needed information on how simultaneous exposure to multiple toxicants affects complex and sensitive biological structures, such as ovarian follicles.

Immunoisolating poly(ethylene glycol) based capsules support ovarian tissue survival to restore endocrine function.

A common irreversible adverse effect of life-saving anticancer treatments is loss of gonadal endocrine function and fertility, calling for a need to focus on post-treatment quality of life. Here, we investigated the use of poly(ethylene glycol)-vinyl sulfone (PEG-VS) based capsules to support syngeneic donor ovarian tissue for restoration of endocrine function in mice. We designed a dual immunoisolating capsule (PEG-Dual) by tuning the physical properties of the PEG hydrogels and combining proteolytically degradable and nondegradable layers to meet the numerous requirements for encapsulation and immunoisolation of ovarian tissue, such as nutrient diffusion and tissue expansion. Tuning the components of the PEG-Dual capsule to have similar physical properties allowed for concentric encapsulation. Upon implantation, the PEG-based capsules supported ovarian tissue survival and led to a significant decrease in follicle stimulating hormone levels 60 days postimplantation. Mice that received the implants resumed regular estrous cycle activity and follicle development in the implanted grafts. The PEG-Dual capsule provided an environment conducive for tissue survival, while providing a barrier to the host environment. This study demonstrated for the first time that immunoisolating PEG-VS capsules can support ovarian follicular development resulting in the restoration of ovarian endocrine function and can be applied to future allogeneic studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1381-1389, 2018.