Insulin-like growth factor 2 is produced by antral follicles and promotes preantral follicle development in macaques†.

Insulin-like growth factors (IGFs) are known for their involvement in endocrine and paracrine regulation of ovarian function. Although IGF2 is the predominant circulating and intraovarian form of IGFs in primate species, the stage-specific follicular expression, action, and regulation of IGF2 are not well defined. Therefore, experiments were conducted to investigate the follicular IGF production in response to steroid hormone regulation and the direct IGF actions on follicular development and function in vitro. Preantral follicles were isolated from rhesus macaque ovaries and cultured to the antral stage in media supplemented with follicle-stimulating hormone and insulin. Follicles were randomly assigned to treatment groups: (a) control, (b) trilostane (a steroid synthesis inhibitor), (c) trilostane + estradiol, (d) trilostane + progesterone, and (e) trilostane + dihydrotestosterone. Media was analyzed for IGF concentrations, which were correlated to follicle growth. Follicles produced IGF2, but not IGF1, at the antral stage. Steroid depletion decreased, whereas steroid replacement increased, IGF2 production by antral follicles. Media IGF2 levels correlated positively with antral follicle diameters. Macaque preantral follicles and granulosa cells were subsequently cultured without (control) and with recombinant human IGF2 supplementation. Follicle survival, growth, and paracrine factor production, as well as granulosa cell proliferation and gonadotropin receptor gene expression, were assessed. IGF2 addition increased follicle survival rates, diameters and inhibin B production, as well as granulosa cell proliferation. These data demonstrate that IGF2 produced by antral follicles, in response to steroid hormone regulation, could act as a paracrine factor that positively impacts preantral follicle development and function in primates.

Maternal High-Fat Diet Consumption and Chronic Hyperandrogenemia Are Associated With Placental Dysfunction in Female Rhesus Macaques.

The risk of adverse perinatal outcomes with maternal polycystic ovary syndrome may differ among hyperandrogenic and nonhyperandrogenic phenotypes and is likely modulated by maternal obesity and diet. The relative contribution of maternal hyperandrogenism and nutritional status to placental dysfunction is unknown. Female rhesus macaques (N = 39) were assigned at puberty to one of four treatment groups: subcutaneous cholesterol implants and a standard chow diet (controls); testosterone (T) implants and a normal diet; cholesterol implants and a high-fat, Western-style diet (WSD); and testosterone implants in combination with a high-fat diet. After 3.5 years of treatment, contrast-enhanced and Doppler ultrasound analyses of placental blood flow were performed for a representative subset of animals from each treatment group during pregnancy, and placental architecture assessed with stereological analysis. Placental growth factors, cellular nutrient sensors, and angiogenic markers were measured with ELISA and Western blotting. WSD consumption was associated with a 30% increase in placental flux rate relative to that in animals receiving a normal diet. T and WSD treatments were each independently associated with increased villous volume, and T also was associated with an ∼ 40% decrease fetal capillary volume on stereological analysis. T treatment was associated with significantly increased mTOR and SOCS3 expression. WSD consumption was associated with decreased GLUT1 expression and microvillous membrane localization. Hyperandrogenemic and nonhyperandrogenemic phenotypes are associated with altered placental angiogenesis, nutrient sensing, and glucose transport. WSD and T appear to have distinct effects on vascular impedance and capillary angiogenesis.

Chronically elevated androgen and/or consumption of a Western-style diet impairs oocyte quality and granulosa cell function in the nonhuman primate periovulatory follicle.

PURPOSE: To investigate the impact of chronically elevated androgens in the presence and absence of an obesogenic diet on oocyte quality in the naturally selected primate periovulatory follicle. METHODS: Rhesus macaques were treated using a 2-by-2 factorial design (n = 10/treatment) near the onset of menarche with implants containing either cholesterol (C) or testosterone (T, 4-5-fold increase above C) and a standard or "Western-style" diet alone (WSD) or in combination (T+WSD). Following ~ 3.5 years of treatment, females underwent controlled ovulation (COv, n = 7-10/treatment) cycles, and contents of the naturally selected periovulatory follicle were aspirated. Follicular fluid (FF) was analyzed for cytokines, chemokines, growth factors, and steroids. RNA was extracted from luteinizing granulosa cells (LGCs) and assessed by RNA-seq. RESULTS: Only healthy, metaphase (M) I/II-stage oocytes (100%) were retrieved in the C group, whereas several degenerated oocytes were recovered in other groups (33-43% of T, WSD, and T+WSD samples). Levels of two chemokines and one growth factor were reduced (p < 0.04) in FF of follicles with a MI/MII oocyte in WSD+T (CCL11) or T and WSD+T groups (CCL2 and FGF2) compared to C and/or WSD. Intrafollicular cortisol was elevated in T compared to C follicles (p < 0.02). Changes in the expression pattern of 640+ gene products were detected in LGC samples from follicles with degenerated versus MI/MII-stage oocytes. Pathway analysis on RNAs altered by T and/or WSD found enrichment of genes mapping to steroidogenic and immune cell pathways. CONCLUSIONS: Female primates experiencing hyperandrogenemia and/or consuming a WSD exhibit an altered intrafollicular microenvironment and reduced oocyte quality/competency, despite displaying menstrual cyclicity.

Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys.

STUDY QUESTION: Does chronic hyperandrogenemia beginning at menarche, in the absence and presence of a western-style diet (WSD), alter ovarian and uterine structure-function in young adult rhesus monkeys? SUMMARY ANSWER: Phenotypic alterations in ovarian and uterine structure/function were induced by exogenous testosterone (T), and compounded in the presence of a WSD (T+WSD). WHAT IS KNOWN ALREADY: Hyperandrogenemia is a well-established component of PCOS and is observed in adolescent girls, indicating a potential pubertal onset of disease symptoms. Obesity is often associated with hyperandrogenemia and it is hypothesized that metabolic dysfunction exacerbates PCOS symptoms. STUDY DESIGN, SIZE, DURATION: Macaque females (n = 40) near the onset of menarche (~2.5 years of age) were assigned to a 2 by 2 factorial cohort design. Effects on reproductive characteristics were evaluated after 3 years of treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Rhesus macaques (Macaca mulatta) were fed either a normal balanced diet (n = 20) or a WSD (n = 20). Additionally, implants containing cholesterol (n = 20) or T (n = 20) were implanted subcutaneously to elevate serum T approximately 5-fold. This resulted in treatment groups of controls (C), T, WSD and T+WSD (n = 10/group). Vaginal swabbing was performed daily to detect menses. After 3 years of treatment, daily serum samples from one menstrual cycle were assayed for hormone levels. Ovarian structure was evaluated in the early follicular phase by 3D/4D ultrasound. Uterine endometrial size and ovarian/luteal vascular function was also evaluated in subgroups (n = 6/group) in the late follicular and mid-luteal phases by 3D/4D ultrasound and contrast-enhanced ultrasound, respectively. Expression of steroid hormone receptors and markers of decidualization and endometrial receptivity were assessed in endometrial biopsies at mid-luteal phase. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately 90% of menstrual cycles appeared ovulatory with no differences in frequency or duration between groups. Serum estradiol (E2) levels during the early follicular phase were greatest in the T alone group, but reduced in T+WSD (P < 0.02). Serum LH was elevated in the T group (P < 0.04); however, there were no differences among groups in FSH levels (P > 0.13). Ovarian size at menses tended to be greater in the WSD groups (P < 0.07) and antral follicles ≥1 mm were more numerous in the T+WSD group (P < 0.05). Also, females in T and T+WSD groups displayed polycystic ovarian morphology (PCOM) at greater frequency than C or WSD groups (P < 0.01). Progesterone (P4) levels during the luteal phase were reduced in the T+WSD group compared to C and T groups (P < 0.05). Blood volume (BV) and vascular flow (VF) within the corpus luteum was reduced in all treatment groups compared to C (P < 0.01, P = 0.03), with the WSD alone group displaying the slowest BV and VF (P < 0.05). C and WSD groups displayed endometrial glands at mid-luteal phase with low estrogen receptor 1 (ESR1) and progesterone receptor (PGR) mRNA and immunohistochemical staining in the functionalis zone, but appreciable PGR in the stroma. In contrast, T and T+WSD treatment resulted in glands with less secretory morphology, high ESR1 expression in the glandular epithelium and low PGR in the stroma. Endometrial levels of TIMP3 and MMP26 mRNA and immunostaining were also decreased in the T and T+WSD groups, whereas AR expression was unchanged. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Females are young adults, so effects could change as they reach prime reproductive age. The T level generated for hyperandrogenemia may be somewhat greater than the 3-4-fold increase observed in adolescent girls, but markedly less than those observed in male monkeys or adolescent boys. WIDER IMPLICATIONS OF THE FINDINGS: Alterations to ovarian and uterine structure-function observed in T and, in particular, T+WSD-treated female macaques are consistent with some of the features observed in women diagnosed with polycystic ovary syndrome (PCOS), and suggest impaired fertility. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under Award Number P50HD071836 (to RLS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Additional funding was provided by Office of the Director, NIH under Award Number P51OD011092 (Support for National Primate Research Center). Authors declare no competing interests.

Nonhuman Primates: A Vital Model for Basic and Applied Research on Female Reproduction, Prenatal Development, and Women's Health.

The comparative biology of reproduction and development in mammalian species is remarkable. Hence, because of similarities in environmental and neuroendocrine control of the reproductive axis, the cyclic function of the ovary and reproductive tract, establishment and control of the maternal-fetal-placental unit during pregnancy, and reproductive aging from puberty through menopause, nonhuman primates (NHPs) are valuable models for research related to women's reproductive health and its disorders. This chapter provides examples of research over the past 10+ years using Old World monkeys (notably macaque species), baboons, and to a lesser extent New World monkeys (especially marmosets) that contributed to our understanding of the etiology and therapies or prevention of: (1) ovarian disorders, e.g., polycystic ovary syndrome, mitochondrial DNA-based diseases from the oocyte; (2) uterine disorders, for example, endometriosis and uterine transplantation; and (3) pregnancy disorders, for example, preterm labor and delivery, environmental factors. Also, emerging opportunities such as viral (e.g., Zika) induced fetal defects and germline genomic editing to generate valuable primate models of human diseases (e.g., Huntington and muscular dystrophy) are addressed. Although the high costs, specialized resources, and ethical debate challenge the use of primates in biomedical research, their inclusion in fertility and infertility research is vital for continued improvements in women's reproductive health.

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.

Changes in immune cell distribution and their cytokine/chemokine production during regression of the rhesus macaque corpus luteum.

Our previous flow cytometry results demonstrated a significant increase in neutrophils, macrophages/monocytes, and natural killer (NK) cells in dispersed rhesus monkey corpora lutea (CL) after progesterone (P4) levels had fallen below 0.3 ng/ml for ≥3 days during the natural menstrual cycle. In this study, immunohistochemistry revealed the CD11b+ cells (neutrophils, macrophages/monocytes) present in the CL after luteal P4 synthesis ceased were distributed throughout the tissue. CD16+ cells (presumptive NK cells) were observed mainly near the vasculature in functional CL, until their numbers increased and they became widely distributed in regressing CL. To determine if the immune cells that enter luteal tissue during structural regression are functionally different from those that are present during peak function, CD11b+ or CD16+ populations were enriched from mid-late stage (functional) and regressing (days 1.8 ± 0.3 postmenses) CL using antibody-conjugated magnetic microbeads. Flow cytometry analyses revealed the majority of CD11b+ cells expressed CD14, a protein mainly produced by macrophages/monocytes. The antibody-enriched and depleted fractions were cultured for 24 h, and the media then analyzed for the production of 29 cytokines/chemokines. From the mid-late CL, the CD11b+-enriched fraction produced three cytokines/chemokines, whereas CD16+-enriched cells only produced the chemokine CCL2. However, CD11b +-enriched cells isolated from regressed CL produced eight cytokines/chemokines. The CD16+-enriched cells isolated from regressing CL produced significant levels of only three cytokines. Thus, the CD11b+ cells that appear in the rhesus macaque CL after functional regression produce several cytokines/chemokines that likely play a role in orchestrating structural regression.

Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production.

Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4-6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization.

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.

Dynamics of Immune Cell Types Within the Macaque Corpus Luteum During the Menstrual Cycle: Role of Progesterone.

The goal of the current study was to characterize the immune cell types within the primate corpus luteum (CL). Luteal tissue was collected from rhesus females at discrete intervals during the luteal phase of the natural menstrual cycle. Dispersed cells were incubated with fluorescently labeled antibodies specific for the immune cell surface proteins CD11b (neutrophils and monocytes/macrophages), CD14 (monocytes/macrophages), CD16 (natural killer [NK] cells), CD20 (B-lymphocytes), and CD3epsilon (T-lymphocytes) for analysis by flow cytometry. Numbers of CD11b-positive (CD11b(+)) and CD14(+) cells increased significantly 3 to 4 days after serum progesterone (P4) concentrations declined below 0.3 ng/ml. CD16(+) cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases and were 3-fold increased 3 to 4 days after serum P4 decreased to baseline levels. CD3epsilon(+) cells tended to increase 3 to 4 days after P4 decline. To determine whether immune cells were upregulated by the loss of luteotropic (LH) support or through loss of LH-dependent steroid milieu, monkeys were assigned to 4 groups: control (no treatment), the GnRH antagonist Antide, Antide plus synthetic progestin (R5020), or Antide plus the estrogen receptor agonists diarylpropionitrile (DPN)/propyl-pyrazole-triol (PPT) during the mid-late luteal phase. Antide treatment increased the numbers of CD11b(+) and CD14(+) cells, whereas progestin, but not estrogen, replacement suppressed the numbers of CD11b(+), CD14(+), and CD16(+) cells. Neither Antide nor steroid replacement altered numbers of CD3epsilon(+) cells. These data suggest that increased numbers of innate immune cells in primate CL after P4 synthesis declines play a role in onset of structural regression of primate CL.

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.

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.

Quantification of dynamic changes to blood volume and vascular flow in the primate corpus luteum during the menstrual cycle.

BACKGROUND: The objective of the current study was to determine changes to vascular parameters of nonhuman primate dominant ovarian structures by dynamic contrast-enhanced ultrasound (DCE-US). MATERIALS AND METHODS: Dynamic contrast-enhanced ultrasound with intravenous microbubble infusion was performed on the rhesus macaque ovary bearing the pre-ovulatory follicle and corpus luteum (CL) sequentially during the natural luteal phase (n = 8) and GnRH antagonist (antide)-induced luteal regression (n = 6). RESULTS: Changes in luteal blood volume (BV) and vascular flow (VF) were observed between stages of the luteal phase Luteal BV was highest in early stage CL, before decreasing 2.5-fold in late stage CL (P < 0.06); in contrast, luteal VF peaked at mid luteal stage (P < 0.01). Two females identified with luteal insufficiency trended toward lower peak BV, compared to typical CLs. Another female was identified with a luteal cyst on the contralateral ovary, and a CL that regressed before P levels declined. After 72 hours of antide exposure, BV was reduced 2.3-fold (P = 0.03). CONCLUSIONS: DCE-US provides a sensitive, non-invasive measurement of the dynamics of blood volume and flow in dominant ovarian structures.

Endocrine and local control of the primate corpus luteum.

The primate corpus luteum is a transient endocrine gland that differentiates from the ovulatory follicle midway through the ovarian (menstrual) cycle. Its formation and limited lifespan is critical for fertility, as luteal-derived progesterone is the essential steroid hormone required for embryo implantation and maintenance of intra-uterine pregnancy until the placenta develops. It is well-established that LH and the LH-like hormone, CG, are the vital luteotropic hormones during the menstrual cycle and early pregnancy, respectively. Recent advances, particularly through genome analyses and cellular studies, increased our understanding of various local factors and cellular processes associated with the development, maintenance and repression of the corpus luteum. These include paracrine or autocrine factors associated with angiogenesis (e.g., VEGF), and that mediate LH/CG actions (e.g., progesterone), or counteract luteotropic effects (i.e., local luteolysis; e.g., PGF2α). However, areas of mystery and controversy remain, particularly regarding the signals and events that initiate luteal regression in the non-fecund cycle. Novel approaches capable of gene "knockdown" or amplification", in vivo as well as in vitro, should identify novel or underappreciated gene products that are regulated by or modulate LH/CG actions to control the functional lifespan of the primate corpus luteum. Further advances in our understanding of luteal physiology will help to improve or control fertility for purposes ranging from preservation of endangered primate species to designing novel ovary-based contraceptives and treating ovarian disorders in women.

Primate follicular development and oocyte maturation in vitro.

The factors and processes involved in primate follicular development are complex and not fully understood. An encapsulated three-dimensional (3D) follicle culture system could be a valuable in vitro model to study the dynamics and regulation of folliculogenesis in intact individual follicles in primates. Besides the research relevance, in vitro follicle maturation (IFM) is emerging as a promising approach to offer options for fertility preservation in female patients with cancer. This review summarizes the current published data on in vitro follicular development from the preantral to small antral stage in nonhuman primates, including follicle survival and growth, endocrine (ovarian steroid hormone) and paracrine/autocrine (local factor) function, as well as oocyte maturation and fertilization. Future directions include major challenges and strategies to further improve follicular growth and differentiation with oocytes competent for in vitro fertilization and subsequent embryonic development, as well as opportunities to investigate primate folliculogenesis by utilizing this 3D culture system. The information may be valuable in identifying optimal conditions for human follicle culture, with the ultimate goal of translating the experimental results and products to patients, thereby facilitating diagnostic and therapeutic approaches for female fertility.

Ovarian cycle-specific regulation of adipose tissue lipid storage by testosterone in female nonhuman primates.

Previous studies in rodents and humans suggest that hyperandrogenemia causes white adipose tissue (WAT) dysfunction in females, although the underlying mechanisms are poorly understood. In light of the differences in the length of the ovarian cycle between humans and rodents, we used a nonhuman primate model to elucidate the effects of chronic hyperandrogenemia on WAT function in vivo. Female rhesus macaques implanted with testosterone capsules developed insulin resistance and altered leptin secretion on a high-fat, Western-style diet. In control visceral WAT, lipolysis and hormone-sensitive lipase expression were upregulated during the luteal phase compared with the early follicular (menses) phase of the ovarian cycle. Hyperandrogenemia attenuated elevated lipolysis and hormone-sensitive lipase activity in visceral WAT during the luteal phase but not during menses. Under control conditions, insulin-stimulated Akt and Erk activation and fatty acid uptake in WAT were not significantly affected by the ovarian cycle. In contrast, testosterone treatment preferentially increased fatty acid uptake and insulin signaling at menses. The fatty acid synthase and glucose transporter-4 genes were upregulated by testosterone during the luteal phase. In summary, this study reveals ovarian stage-specific fluctuations in adipocyte lipolysis and suggests that male sex hormones increase and female sex hormones decrease lipid storage in female WAT.

Human embryonic stem cells derived by somatic cell nuclear transfer.

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Future Directions in Oncofertility and Fertility Preservation: A Report from the 2011 Oncofertility Consortium Conference.

Fertility impairment and loss due to cancer or its treatment is a significant survivorship consideration for many pediatric, adolescent, and young adult cancer survivors. Chemotherapeutics, radiation, and surgery can impact the future fertility of men, women, and children with cancer. The field of oncofertility, founded to ensure the reproductive future of cancer survivors, gained momentum with 5 years of funding through a 2007 National Institutes of Health Roadmap Grant for Biomedical Research. This report from working group meetings at the fifth annual Oncofertility Consortium Conference speaks to the present state of oncofertility research and clinical care, existing gaps, and future directions for the field. This summary from conference participants and leaders in the field addresses the science, clinical specialties, and academic scholarship that can guide the field as the Roadmap Grant funding comes to a close.

Oocyte maturation and in vitro hormone production in small antral follicles (SAFs) isolated from rhesus monkeys.

PURPOSE: The small antral follicles (SAFs) from the ovarian medulla can be a potential source of oocytes for infertility patients, but little is known about their ability to yield mature oocytes. This study evaluated the response of these SAFs to a stimulatory bolus of human corionic gonadotropin (hCG) in vitro. METHODS: Oocyte nuclear maturation and hormone production (estradiol [E2], progesterone [P4]), antimullerian hormone [AMH]) by individual intact SAFs (n = 91; >0.5 mm; n = 5 monkeys) was evaluated after 34 h of culture in the absence (control) or presence of hCG. RESULTS: Of the total cohort (n = 91), 49 % of SAFs contained degenerating oocytes. The percentage of healthy oocytes able to reinitiate meiosis to the metaphase I (MI) and MII was greater (p < 0.05) after hCG compared to controls. E2, P4 and AMH levels were higher (p < 0.05) in SAF cultures containing germinal vesicle (GV) oocytes compared to those with MII oocytes regardless of hCG exposure. SAF with MI oocytes produced more E2, but less (p < 0.05) P4 and AMH compared to SAFs containing GV oocytes (p < 0.05). Follicles ≥1 mm produced more (p < 0.05) E2, whereas follicle diameter did not correlate with P4 or AMH levels. Only P4 increased (p < 0.05) in response to hCG, regardless of follicle size or oocyte maturity. SAFs containing degenerating oocytes produced similar levels of E2, P4 and AMH compared to SAFs containing healthy oocytes. CONCLUSIONS: These data indicate, for the first time, that oocytes within primate SAFs can reinitiate meiosis in vitro in the absence of hCG, but nuclear maturation is enhanced in SAFs cultured with hCG. Oocyte nuclear maturation within SAFs in is associated with decreased E2, P4 and AMH levels. Furthermore, hormone content within the culture media does not necessarily reflect oocyte quality.