Sodium thiosulfate does not protect ovarian reserve from cisplatin-induced gonadotoxicity†.

Cisplatin, a platinum-containing alkylating agent, is used in the treatment of various tumors owing to its potent antitumor activity. However, it causes permanent and adverse effects, particularly hearing loss and depletion of ovarian reserve. Until recently, there were no clinically available protective agents to mitigate the adverse side effects of cisplatin-induced cytotoxicity. In 2022, sodium thiosulfate (STS) was approved by the Food and Drug Administration for mitigating hearing loss in children and adolescents undergoing cisplatin treatment. Consequently, our investigation aimed to determine if STS could protect ovarian reserve against cisplatin-induced gonadotoxicity. In an ex vivo culture, the cisplatin-only group exhibited a loss of primordial follicles, while post-STS administration after cisplatin exposure effectively protected primordial follicles. However, when post-STS was administrated either 6 or 4 h after cisplatin exposure, it did not confer protection against cisplatin-induced gonadotoxicity in postnatal day 7 or adolescent mouse models. Immunofluorescence assays using γH2AX and cPARP revealed that oocytes within primordial follicles exhibited DNA damage after cisplatin exposure, irrespective of post-STS administration. This underscores the rapid and heightened sensitivity of oocytes to gonadotoxicity. In addition, oocytes demonstrated an increased expression of pCHK2 rather than pERK, suggesting that the pathway leading to oocyte death differs from the pathway observed in the inner ear cell death following cisplatin exposure. These results imply that while the administration of STS after cisplatin is highly beneficial in preventing hearing loss, it does not confer a protective effect on the ovaries in mouse models.

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.

Oocyte quality following in vitro follicle development†.

In vitro follicle development (IVFD) is an adequate model to obtain basic knowledge of folliculogenesis and provides a tool for ovarian toxicity screening. IVFD yielding competent oocytes may also offer an option for fertility and species preservation. To promote follicle growth and oocyte maturation in vitro, various culture systems are utilized for IVFD in rodents, domestic animals, wild animals, nonhuman primates, and humans. Follicle culture conditions have been improved by optimizing gonadotropin levels, regulatory factors, nutrient supplements, oxygen concentration, and culture matrices. This review summarizes quality assessment of oocytes generated from in vitro-developed antral follicles from the preantral stage, including oocyte epigenetic and genetic profile, cytoplasmic and nuclear maturation, preimplantation embryonic development following in vitro fertilization, as well as pregnancy and live offspring after embryo transfer. The limitations of oocyte quality evaluation following IVFD and the gaps in our knowledge of IVFD to support proper oocyte development are also discussed. The information may advance our understanding of the requirements for IVFD, with a goal of producing competent oocytes with genetic integrity to sustain embryonic development resulting in healthy offspring.

Sphingosine-1-phosphate and its mimetic FTY720 do not protect against radiation-induced ovarian fibrosis in the nonhuman primate†.

Oocytes are highly radiosensitive, so agents that prevent radiation-induced ovarian follicle destruction are important fertility preservation strategies. A previous study in rhesus macaques demonstrated that ovarian treatment with antiapoptotic agents, sphingosine-1-phosphate (S1P) and FTY720, its long-acting mimetic, preserved follicles following a single dose of 15 Gy X-ray radiation, and live offspring were obtained from FTY720-treated animals. However, it is unknown whether these antiapoptotic agents also protected the ovarian stroma from late effects of radiation, including vascular damage and fibrosis. Using ovarian histological sections from this study, we evaluated the vasculature and extracellular matrix in the following cohorts: vehicle + sham irradiation, vehicle + irradiation (OXI), S1P + irradiation (S1P), and FTY720 + irradiation (FTY720). One ovary from each animal was harvested prior to radiation whereas the contralateral ovary was harvested 10 months post-treatment. We assessed vasculature by immunohistochemistry with a PECAM1 antibody, hyaluronan by a hyaluronan binding protein assay, and collagen by picrosirius red and Masson's trichrome staining. Disorganized vessels were observed in the medulla in the OXI and S1P cohorts relative to the sham, but the vasculature in the FTY720 cohort appeared intact, which may partially explain fertoprotection. There were no differences in the hyaluronan matrix among the cohorts, but there was thickening of the tunica albuginea and fibrosis in the OXI cohort relative to the sham, which was not mitigated by either S1P or FTY720 treatment. Thus, the fertoprotective properties of S1P and FTY720 may be limited given their inability to protect the ovarian stroma against the late effects of radiation-induced fibrosis.

Impacts of different synthetic polymers on vitrification of ovarian tissue.

Type and concentration of cryoprotective agents (CPAs) are important factors which influence the likelihood of a successful ovarian tissue vitrification outcome. In an attempt to address this factor, the present study was conducted to evaluate the impacts of different synthetic polymers (Supercool X-1000, Supercool Z-1000 and PVP K-12) on vitrification of bovine ovarian tissue. From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification, either or not followed by in vitro culture for one or five days. Vitrification was performed using the ovarian tissue cryosystem (OTC) system. The ovarian tissues were intended for histological and viability analysis [Reactive oxygen species (ROS) production and degenerate cells assay (Ethidium homodimer-1)], as well as immunolocalization of AQP3 and AQP9 were measured. The results showed that during almost all the periods after warming, in treatment groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples. Furthermore, post-thawed X-1000 group revealed stronger labeling for AQP9 in primordial and transitional follicles, when compared with others. However, morphology after cryopreservation did not correlate with follicle viability and function where the levels of degeneration and tissue damage of PVP K-12 group were lower in comparison with X-1000 group and only in PVP K-12 group, ROS level was similar to that of the fresh control group. We believe that in addition to permeating CPAs, the addition of one (Supercool X-1000) or maybe a combination (Supercool X-1000 and PVP K-12) of non-permeating polymers could be useful to improve the outcome for vitrified bovine ovarian tissue.

A closed vitrification system enables a murine ovarian follicle bank for high-throughput ovotoxicity screening, which identifies endocrine disrupting activity of microcystins.

Increasing evidence reveals that a broad spectrum of environmental chemicals and pharmaceutical compounds cause female ovarian toxicity (ovotoxicity). The current gold standard of ovotoxicity testing largely relies on whole laboratory animals, but in vivo models are time consuming, costly, and present animal welfare concerns. We previously demonstrated that the 3D encapsulated in vitro follicle growth (eIVFG) is a robust in vitro model for ovotoxicity testing. However, the follicle preparation process is complex and highly dependent on technical skills. Here, we aimed to use vitrification method to cryopreserve murine immature follicles for a high-content eIVFG, chemical exposure, and ovotoxicity screening. Results indicated that a closed vitrification system combined with optimized vitrification protocols preserved mouse follicle viability and functionality and vitrified follicles exhibited comparable follicle and oocyte reproductive outcomes to freshly harvested follicles during eIVFG, including follicle survival and development, ovarian steroidogenesis, and oocyte maturation and ovulation. Moreover, vitrified follicles consistently responded to ovotoxic chemical, doxorubicin (DOX). We further used vitrified follicles to test the response of microcystins (MCs), an emerging category of environmental contaminants produced by cyanobacteria associated with harmful algal blooms (HABs), and found that different congeners of MCs exhibited differential ovotoxicities. In summary, our study demonstrates that vitrification enables a long-term-storage and ready-to-use ovarian follicle bank for high-throughput ovotoxicity screening, which identifies endocrine disrupting effects of MCs.

Use of synthetic polymers improves the quality of vitrified caprine preantral follicles in the ovarian tissue.

The aim of this study was to evaluate whether the addition of synthetic polymers to the vitrification solution affected follicular morphology and development and the expression of Ki-67, Aquaporin 3 (AQP3) and cleaved Caspase-3 proteins in ovarian tissue of the caprine species. Caprine ovaries were fragmented and two fragments were immediately fixed (Fresh Control) for morphological evaluation, while other two were in vitro cultured for 7 days (Cultured Control) and fixed as well. The remaining fragments were distributed in two different vitrification groups: Vitrified and Vitrified/Cultured. Each group was composed of 4 different treatments: 1) Sucrose (SUC); 2) SuperCool X-1000 0.2 % (X-1000); 3) SuperCool Z-1000 0.4 % (Z-1000) or 4) with polyvinylpyrrolidone K-12 0.2 % (PVP). Also, Fresh Control, Cultured Control, SUC and X-1000 were destined to immunohistochemical detection of Ki-67, AQP3 and cleaved Caspase-3 proteins. Morphologically, the treatment with X-1000 showed no significant difference with the Fresh Control group and was superior to the other treatments. After the cleaved caspase-3 analysis, X-1000 showed the lowest percentages of strong immunostaining while Cultured Control showed the highest. Also, a positive correlation was found between the percentages of degenerated follicles and the percentages of strong staining intensity follicles. Regarding the AQP3 analysis, the highest percentages of strong AQP3 staining intensity were found in X-1000. In conclusion, we have demonstrated that the addition of the synthetic polymer SuperCool X-1000 to the vitrification solution improved the current vitrification protocol of caprine ovarian tissue.

Can peri-ovulatory putrescine supplementation improve egg quality in older infertile women?

The aging-related decline in fertility is an increasingly pressing medical and economic issue in modern society where women are delaying family building. Increasingly sophisticated, costly, and often increasingly invasive, assisted reproductive clinical protocols and laboratory technologies (ART) have helped many older women achieve their reproductive goals. Current ART procedures have not been able to address the fundamental problem of oocyte aging, the increased rate of egg aneuploidy, and the decline of developmental potential of the eggs. Oocyte maturation, which is triggered by luteinizing hormone (LH) in vivo or by injection of human chorionic gonadotropin (hCG) in an in vitro fertilization (IVF) clinic, is the critical stage at which the majority of egg aneuploidies arise and when much of an egg's developmental potential is established. Our proposed strategy focuses on improving egg quality in older women by restoring a robust oocyte maturation process. We have identified putrescine deficiency as one of the causes of poor egg quality in an aged mouse model. Putrescine is a biogenic polyamine naturally produced in peri-ovulatory ovaries. Peri-ovulatory putrescine supplementation has reduced egg aneuploidy, improved embryo quality, and reduced miscarriage rates in aged mice. In this paper, we review the literature on putrescine, its occurrence and physiology in living organisms, and its unique role in oocyte maturation. Preliminary human data demonstrates that there is a maternal aging-related deficiency in ovarian ornithine decarboxylase (ODC), the enzyme responsible for putrescine production. We argue that peri-ovulatory putrescine supplementation holds great promise as a natural and effective therapy for infertility in women of advanced maternal age, applicable in natural conception and in combination with current ART therapies.

The National Physicians Cooperative: transforming fertility management in the cancer setting and beyond.

Once unimaginable, fertility management is now a nationally established part of cancer care in institutions, from academic centers to community hospitals to private practices. Over the last two decades, advances in medicine and reproductive science have made it possible for men, women and children to be connected with an oncofertility specialist or offered fertility preservation soon after a cancer diagnosis. The Oncofertility Consortium's National Physicians Cooperative is a large-scale effort to engage physicians across disciplines - oncology, urology, obstetrics and gynecology, reproductive endocrinology, and behavioral health - in clinical and research activities to enable significant progress in providing fertility preservation options to children and adults. Here, we review the structure and function of the National Physicians Cooperative and identify next steps.

Development of macaque secondary follicles exposed to neutral red prior to 3-dimensional culture.

PURPOSE: Neutral red (NR) may assist identification of preantral follicles in pieces of cortical tissue prior to cryopreservation in cancer patients requesting fertility preservation. This study is the first to analyze this effect by follicle growth rate after long-term culture in primates. METHODS: Ovarian cortex was obtained from adult rhesus macaques, was cut into fragments, and was incubated with NR. Secondary follicles were readily visualized following NR staining and then were encapsulated into alginate beads and cultured individually for 4 weeks in αMEM media supplemented with 10 ng/ml FSH at 5% O2. RESULTS: The survival rates of secondary follicles during culture were similar between those derived from control tissue (71 ± 13%) and those treated with NR (68 ± 9%). The proportion of surviving follicles that formed an antrum were also similar in both groups (70 ± 17% control; 48 ± 24% NR-treated). Follicle diameters were not different between control follicles (184 ± 5µm) and those stained with NR (181 ± 7 µm) on the day of isolation. The percentages of surviving follicles within three cohorts based on their diameters at week 4 of culture were similar between the control group and NR-stained tissue group, fast-grow follicles (24 ± 6% vs. 13 ± 10%), slow-grow follicles (66 ± 5% vs. 60 ± 9%), or no-grow (10 ± 9% vs. 27 ± 6%), respectively. There were no differences in follicle diameters between groups during the culture period. Pre-exposure of secondary follicles to NR diminished their capacity to produce both estradiol and androstenedione by week 4 of culture, when follicles are exhibiting an antrum. Inhibitory effects of NR on steroid production by slow-grow follicles was less pronounced. CONCLUSIONS: NR does not affect secondary follicle survival, growth, and antrum formation during long-term culture, but steroid hormone production by fast-grow follicles is compromised. NR can be used as a non-invasive tool for in situ identification of viable secondary follicles in ovarian cortex before tissue cryopreservation without affecting follicle survival and growth in vitro. Whether maturation or developmental competence of oocytes derived from antral follicles in 3D culture that were previously isolated from NR-stained tissue is normal or compromised remains to be determined. Likewise, the functional consequences of pre-exposure to NR prior to ovarian cortical tissue cryopreservation and transplantation are unknown.

Subcutaneous ovarian tissue transplantation in nonhuman primates: duration of endocrine function and normalcy of subsequent offspring as demonstrated by reproductive competence, oocyte production, and telomere length.

PURPOSE: The main purposes of the study were to investigate the endocrine function of ovarian tissue transplanted to heterotopic subcutaneous sites and the reproductive competence and telomere length of a nonhuman primate originating from transplanted tissue. METHODS: Ovarian cortex pieces were transplanted into the original rhesus macaques in the arm subcutaneously, in the abdomen next to muscles, or in the kidney. Serum estradiol (E2) and progesterone (P4) concentrations were measured weekly for up to 8 years following tissue transplantation. A monkey derived from an oocyte in transplanted ovarian tissue entered time-mated breeding and underwent controlled ovarian stimulation. Pregnancy and offspring were evaluated. Telomere lengths and oocytes obtained following controlled ovarian stimulation were assessed. RESULTS: Monkeys with transplants in the arm and abdomen had cyclic E2 of 100 pg/ml, while an animal with arm transplants had E2 of 50 pg/ml. One monkey with transplants in the abdomen and kidney had ovulatory cycles for 3 years. A monkey derived from an oocyte in transplanted tissue conceived and had a normal gestation until intrapartum fetal demise. She conceived again and delivered a healthy offspring at term. Controlled ovarian stimulations of this monkey yielded mature oocytes comparable to controls. Her telomere length was long relative to controls. CONCLUSIONS: Heterotopic ovarian tissue transplants yielded long-term endocrine function in macaques. A monkey derived from an oocyte in transplanted tissue was reproductively competent. Her telomere length did not show epigenetically induced premature cellular aging. Ovarian tissue transplantation to heterotopic sites for fertility preservation should move forward cautiously, yet optimistically.

Characterization of FOXO1, 3 and 4 transcription factors in ovaries of fetal, prepubertal and adult rhesus macaques.

The phosphoinositide 3-kinase/AKT (protein kinase B) signaling pathway negatively regulates follicle activation via the forkhead box O (FOXO) transcription factor in rodents. FOXO3 knockout mice exhibit global activation of primordial follicles leading to early depletion of ovarian follicles and subsequent infertility. Whether a similar mechanism for follicle activation exists in the primate ovary is unclear. In the current study, protein localization of FOXO1, 3, and 4 as well as their upstream regulator, AKT/p-AKT, was examined in rhesus macaque ovaries of three developmental stages: fetal, prepubertal, and adult. FOXO1 protein is expressed in granulosa cells of fetal, prepubertal, and adult ovaries. FOXO3 is distributed sparsely in the mitotically active germ cells, but its expression decreases following follicle formation in the macaque fetal ovary. In addition, FOXO3 is seldom with interanimal variation in the prepubertal ovary and is absent in the adult ovary. FOXO4 is nondetectable in fetal ovaries, although it is expressed in some theca cells of antral follicles and some stromal cells in prepubertal and adult ovaries. Our results suggest that the regulation and/or function of FOXO3 in the primate primordial follicle may differ than that of the rodent. Nevertheless, AKT/p-AKT is expressed in macaque primordial oocytes, suggesting that similar upstream events but different downstream effects may regulate primordial follicle activation in nonhuman primates compared to rodents. Elucidation of the mechanism responsible for follicle activation in primates will be crucial for understanding primary ovarian insufficiency, improving female fertility, and applying techniques for in vitro maturation of follicles for fertility preservation in cancer survivors.

Good manufacturing practice requirements for the production of tissue vitrification and warming and recovery kits for clinical research.

Products that are manufactured for use in a clinical trial, with the intent of gaining US Food and Drug Administration (FDA) approval for clinical use, must be produced under an FDA approved investigational new drug (IND) application. We describe work done toward generating reliable methodology and materials for preserving ovarian cortical tissue through a vitrification kit and reviving this tissue through a warming and recovery kit. We have described the critical steps, procedures, and environments for manufacturing products with the intent of submitting an IND. The main objective was to establish an easy-to-use kit that would ensure standardized procedures for quality tissue preservation and recovery across the 117 Oncofertility Consortium sites around the globe. These kits were developed by breaking down the components and steps of a research protocol and recombining them in a way that considers component stability and use in a clinical setting. The kits were manufactured utilizing current good manufacturing practice (cGMP) requirements and environment, along with current good laboratory practices (cGLP) techniques. Components of the kit were tested for sterility and endotoxicity, and morphological endpoint release criteria were established. We worked with the intended down-stream users of these kits for development of the kit instructions. Our intention is to test these initial kits, developed and manufactured here, for submission of an IND and to begin clinical testing for preserving the ovarian tissue that may be used for future restoration of fertility and/or hormone function in women who have gonadal dysgenesis from gonadotoxic treatment regimens or disease.

Western-style diet, with and without chronic androgen treatment, alters the number, structure, and function of small antral follicles in ovaries of young adult monkeys.

OBJECTIVE: To examine the small antral follicle (SAF) cohort in ovaries of adult rhesus monkeys after consumption of a Western-style diet (WSD), with or without chronically elevated androgen levels since before puberty. DESIGN: Cholesterol or T (n = 6 per group) implants were placed SC in female rhesus macaques beginning at 1 year of age (prepubertal), with addition of a WSD (high fat/fructose) at 5.5 years (menarche approximately 2.6 years). Ovaries were collected at 7 years of age. One ovary per female was embedded in paraffin for morphologic and immunohistochemical analyses. The SAFs (<2.5 mm) were dissected from the other ovary obtained at or near menses in a subgroup of females (n = 3 per group) and processed for microarray analyses of the SAF transcriptome. Ovaries of adult monkeys consuming a standard macaque diet (low in fats and sugars) were obtained at similar stages of the menstrual cycle and used as controls for all analyses. SETTING: Primate research center. ANIMAL(S): Adult, female rhesus monkeys (Macaca mulatta). INTERVENTION(S): None. MAIN OUTCOME MEASURES: Histologic analyses, SAF counts and morphology, protein localization and abundance in SAFs, transcriptome in SAFs (messenger RNAs [mRNAs]). RESULT(S): Compared with controls, consumption of a WSD, with and without T treatment, increased the numbers of SAFs per ovary, owing to the presence of more atretic follicles. Numbers of granulosa cells expressing cellular proliferation markers (pRb and pH3) was greater in healthy SAFs, whereas numbers of cells expressing the cell cycle inhibitor (p21) was higher in atretic SAFs. Intense CYP17A1 staining was observed in the theca cells of SAFs from WSD with or without T groups, compared with controls. Microarray analyses of the transcriptome in SAFs isolated from WSD and WSD plus T-treated females and controls consuming a standard diet identified 1,944 genes whose mRNA levels changed twofold or more among the three groups. Further analyses identified several gene pathways altered by WSD and/or WSD plus T associated with steroid, carbohydrate, and lipid metabolism, plus ovarian processes. Alterations in levels of several SAF mRNAs are similar to those observed in follicular cells from women with polycystic ovary syndrome. CONCLUSION(S): These data indicate that consumption of a WSD high in fats and sugars in the presence and absence of chronically elevated T alters the structure and function of SAFs within primate ovaries.

Expression of the beta-2 adrenergic receptor (ADRB-2) in human and monkey ovarian follicles: a marker of growing follicles?

BACKGROUND: ADRB-2 was implicated in rodent ovarian functions, including initial follicular growth. In contrast, ADRB-2 expression and function in nonhuman primate and human ovary were not fully known but innervation and significant levels of norepinephrine (NE), which is a ligand at the ADRB-2, were reported in the ovary. METHODS: We studied expression of ADRB-2 in human and rhesus monkey ovary (RT-PCR, immunohistochemistry; laser micro dissection) and measured levels of norepinephrine (NE; ELISA) in monkey follicular fluid (FF). 3D cultures of monkey follicles (4 animals) were exposed to NE or the ADRB-2 agonist isoproterenol (ISO), and follicular development (size) was monitored. Upon termination expression of ADRB-2, FSH receptor and aromatase genes were examined. RESULTS: Immunohistochemistry and RT-PCR of either human follicular granulosa cells (GCs) obtained by laser micro dissection or isolated monkey follicles revealed ADRB-2 in GCs of primordial, primary, secondary and tertiary follicles. Staining of GCs in primordial and primary follicles was intense. In large preantral and antral follicles the staining was heterogeneous, with positive and negative GCs present but GCs lining the antrum of large follicles were generally strongly immunopositive. Theca, interstitial, and ovarian surface epithelial cells were also positive. NE was detected in FF of preovulatory antral monkey follicles (0.37 + 0.05 ng/ml; n = 7; ELISA) but not in serum. We examined preantral follicles ranging from 152 to 366 µm in diameter in a 3D culture in media supplemented with follicle stimulating hormone (FSH). Under these conditions, neither NE, nor ISO, influenced growth rate in a period lasting up to one month. Upon termination of the cultures, all surviving follicles expressed aromatase and FSH receptors, but only about half of them also co-expressed ADRB-2. The ADRB-2 expression was not correlated with the treatment but was positively correlated with the follicular size at the beginning and at the end of the culture period. Hence, expression of ADRB-2 was found in the largest and fastest-in vitro growing follicles. CONCLUSIONS: The results imply ADRB-2-mediated actions in the development of primate follicles. Drugs interfering with ADRB-2 are used to treat medical conditions and may have unexplored effects in the human ovary.

Phosphodiesterase 3 (PDE3) inhibition with cilostazol does not block in vivo oocyte maturation in rhesus macaques (Macaca mulatta).

OBJECTIVE: Studies in mice suggest that cilostazol, an FDA-approved phosphodiesterase 3 (PDE3) inhibitor, might have a contraceptive effect within the approved dose range. We sought to evaluate the potential contraceptive effects of cilostazol in a nonhuman primate model. STUDY DESIGN: Adult female rhesus macaques were stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins, and oocytes were collected by follicle aspiration 36 h after an ovulatory stimulus (human chorionic gonadotropin). Monkeys received no further treatment (controls) or the PDE3 inhibitor cilostazol at the maximum approved human dose of 100mg twice daily starting 6 days prior to follicle aspiration. Recovered oocytes were scored for meiotic stage [germinal vesicle (GV) intact, GV breakdown], and metaphase II stage oocytes were fertilized in vitro and observed for normal embryo development. RESULTS: Similar proportions of GV stage oocytes were recovered from control (27%±4%) and cilostazol (27%±9%)-treated females, and the proportion of embryos that developed into blastocysts was also similar for both groups (7%±5% control vs. 15%±8% cilostazol). CONCLUSION: Oral dosing of cilostazol tablets during controlled ovarian stimulation protocols did not prevent oocyte maturation or embryo development in macaques. IMPLICATIONS: Since administration of the maximum approved human dose of cilostazol (an FDA-approved PDE3 inhibitor) to macaques did not prevent oocyte maturation or fertilization, it is not likely that this dose would be contraceptive in women.

Exposure of female macaques to Western-style diet with or without chronic T in vivo alters secondary follicle function during encapsulated 3-dimensional culture.

Increased adiposity and hyperandrogenemia alter reproductive parameters in both animal models and women, but their effects on preantral follicles in the ovary remain unknown. We recently reported that Western-style diet (WSD) consumption over 1 year, with or without chronic exposure to elevated circulating T, increased the body fat percentage, elicited insulin resistance, suppressed estradiol and progesterone production, as well as altered the numbers, size, and dynamics of antral follicles in the ovary during the menstrual cycle in female macaques. Therefore, experiments were designed to compare the WSD and WSD+T effects to age-matched controls on the survival, growth, and function of isolated secondary follicles during 5 weeks of encapsulated 3-dimensional culture. Follicle survival significantly declined in the WSD and WSD+T groups compared with the control (CTRL) group. Although media progesterone levels were comparable among groups, androstenedione and estradiol levels were markedly reduced in the WSD and WSD+T groups compared with the CTRL group at week 5. Anti-Müllerian hormone levels peaked at week 3 and were lower in the WSD+T group compared with the WSD or CTRL group. Vascular endothelial growth factor levels also decreased at week 5 in the WSD+T group compared with the WSD or CTRL group. After human chorionic gonadotropin exposure, only antral follicles developed from the CTRL group yielded metaphase II oocytes. Thus, WSD with or without T exposure affects the cohort of secondary follicles in vivo, suppressing their subsequent survival, production of steroid hormones and local factors, as well as oocyte maturation in vitro.