Chronic hyperandrogenemia and western-style diet beginning at puberty reduces fertility and increases metabolic dysfunction during pregnancy in young adult, female macaques.

STUDY QUESTION: What are the impacts of elevated testosterone (T) and an obesogenic western-style diet (WSD), either independently or together, on fertility and metabolic adaptations of pregnancy in primates? SUMMARY ANSWER: Testosterone increases the time to achieve pregnancy, while a WSD reduces overall fertility, and the combination of testosterone and WSD additionally impairs glucose tolerance and causes pregnancy loss. WHAT IS KNOWN ALREADY: Both hyperandrogenemia and obesity are hallmarks of polycystic ovary syndrome, which is a leading cause of infertility among women worldwide. Female macaques receiving T and WSD beginning at puberty show increased metabolic, ovarian and uterine dysfunction in the non-pregnant state by 3 years of treatment. STUDY DESIGN, SIZE, DURATION: The same cohort of female rhesus macaques continued treatments from the time of puberty (2.5 years) to 4 years, including this fertility trial. There were four groups (n = 9-10/group): controls (C), T-treated (T; average total serum level 1.35 ng/ml), WSD-treated, and combined T and WSD-treated (T + WSD) females. PARTICIPANTS/MATERIALS, SETTING, METHODS: Females, which were typically having menstrual cycles, were paired for 4 days with a proven male breeder following the late follicular rise in circulating estradiol (≥100 pg/ml). The presence of sperm in the reproductive tract was used to confirm mating. Animals went through up to three successive rounds of mating until they became pregnant, as confirmed by a rise in circulating mCG during the late luteal phase and ultrasound evidence of a gestational sac at Day 30 post-mating (GD30). Placental vascular parameters were also measured at GD30. Metabolic measurements consisted of fasting levels of blood glucose and insulin at approximately GD30, 60, 90 and 115, as well as an intravenous (iv) glucose tolerance test (GTT) at GD115. MAIN RESULTS AND THE ROLE OF CHANCE: While all animals in the C and T groups eventually became pregnant, T-treated females on average had a greater interval to achieve pregnancy (P < 0.05). However, only ~70% of animals in the WSD and T + WSD groups became pregnant (P < 0.004). One pregnancy in T + WSD group resulted in an anembryonic pregnancy which miscarried around GD60, while another T + WSD female conceived with a rare identical twin pregnancy which required cessation due to impending fetal loss at GD106. Thus, the number of viable fetuses was less in the T + WSD group, compared to C, T or WSD. Placental blood volume at GD30 was reduced in all treatments compared to the C group (P < 0.05). Maternal P4 levels were elevated in the WSD (P < 0.03) group and E2 levels were elevated in T + WSD animals (P < 0.05). An increase in serum A4 levels throughout gestation was observed in all groups (P < 0.03) except WSD (P = 0.3). All groups displayed increased insulin resistance with pregnancy, as measured from the ivGTT during pregnancy. However, only the T + WSD group had a significant increase in fasting glucose levels and glucose clearance during the GTT indicating a worsened glucose tolerance. WSD treatment decreased female fetuses third trimester weights, but there was an interaction between WSD and T to increase female fetal weight when normalized to maternal weight. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The small number of pregnancies in the WSD and T + WSD groups hampers the ability to make definitive conclusions on effects during gestation. Also, the high fertility rate in the controls indicates the cohort was at their breeding prime age, which may impair the ability to observe subtle fertility defects. The low number of fetuses used for male and female analysis requires additional studies. WIDER IMPLICATIONS OF THE FINDINGS: The current findings strongly suggest that both hyperandrogenemia and obesity have detrimental effects on fertility and gestation in primates, which may be directly relevant to women with polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S): All ONPRC Cores and Units were supported by NIH Grant P51 OD011092 awarded to ONPRC. 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 R.L.S.). The authors have no competing conflict of interests to disclose.

Direct actions of androgen, estrogen and anti-Müllerian hormone on primate secondary follicle development in the absence of FSH in vitro.

STUDY QUESTION: What are effects of androgen, estrogen and anti-Müllerian hormone (AMH), independent of FSH action, on the development and function of primate follicles from the preantral to small antral stage in vitro? SUMMARY ANSWER: Androgen and estrogen, but not AMH, promote follicle survival and growth in vitro, in the absence of FSH. However, their growth-promoting effects are limited to the preantral to early antral stage. WHAT IS KNOWN ALREADY: FSH supports primate preantral follicle development in vitro. Androgen and estrogen augment follicle survival and growth in the presence of FSH during culture. STUDY DESIGN SIZE, DURATION: Nonhuman primate model; randomized, control versus treatment groups. Rhesus macaque (n = 6) secondary follicles (n = 24 per animal per treatment group) were cultured for 5 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicles were encapsulated in 0.25% (w/v) alginate and cultured individually in modified alpha minimum essential media with (i) FSH (1 ng/ml; control), (ii) no FSH, (iii) no FSH + estradiol (E2; 100 pg/ml)/dihydrotestosterone (DHT; 50 ng/ml) and (iv) no FSH + AMH (50 ng/ml). In a second experiment, follicles were cultured with (i) FSH (1 ng/ml), (ii) no FSH, (iii) no FSH + E2 (1 ng/ml), (iv) no FSH + DHT (50 ng/ml) and (v) no FSH + E2/DHT. Follicle survival, antrum formation and growth pattern were evaluated. Progesterone (P4), E2 and AMH concentrations in culture media were measured. MAIN RESULTS AND THE ROLE OF CHANCE: In the first experiment, FSH deprivation significantly decreased (P < 0.05) follicle survival rates in the no FSH group (16 ± 5%), compared to CTRL (66 ± 9%). E2/DHT (49 ± 5%), but not AMH (27 ± 8%), restored follicle survival rate to the CTRL level. Similarly, antrum formation rates were higher (P < 0.05) in CTRL (56 ± 6%) and E2/DHT groups (54 ± 14%), compared to no FSH (0 ± 0%) and AMH (11 ± 11%) groups. However, follicle growth rate after antrum formation and follicle diameter at week 5 was reduced (P < 0.05) in the E2/DHT group (405 ± 25 µm), compared to CTRL (522 ± 29 µm). Indeed, the proportion of fast-grow follicles at week 5 was higher in CTRL (29% ± 5), compared to E2/DHT group (10 ± 3%). No fast-grow follicles were observed in no FSH and AMH groups. AMH levels at week 3 remained similar in all groups. However, media concentrations of P4 and E2 at week 5 were lower (P < 0.05, undetectable) in no FSH, E2/DHT and AMH groups, compared to CTRL (P4 = 93 ± 10 ng/ml; E2 = 4 ± 1 ng/ml). In the second experiment, FSH depletion diminished follicle survival rate (66 ± 8% in control versus 45 ± 9% in no FSH, P = 0.034). E2 plus DHT (31.5 ± 11%) or DHT alone (69 ± 9%) restored follicle survival rate to the control (FSH) level as expected. Also, E2 plus DHT or DHT alone improved antrum formation rate. However, in the absence of FSH, E2 plus DHT or DHT alone did not support growth, in terms of follicle diameter, or steroid (P4 or E2) production after the antral stage. LIMITATIONS REASONS FOR CAUTION: This study is limited to in vitro effects of E2, DHT and AMH during the interval from the secondary to small antral stage of macaque follicular development. In addition, the primate follicle pool is heterogeneous and differs between animals; therefore, even though only secondary follicles were selected, follicle growth and developmental outcomes might differ from one animal to another. WIDER IMPLICATIONS OF THE FINDINGS: This study provides novel information on the possible actions of estrogen and androgen during early follicular development in primates. Our results suggest that sequential exposure of preantral follicles to local factors, e.g. E2 and DHT, followed by gonadotropin once the follicle reaches the antral stage, may better mimic primate folliculogenesis in vivo. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Center for Translational Research on Reproduction and Infertility 5P50HD071836, and the NIH Primate Centers Program 8P510D011092. There are no conflicts of interest.

Chronic combined hyperandrogenemia and western-style diet in young female rhesus macaques causes greater metabolic impairments compared to either treatment alone.

STUDY QUESTION: Does developmental exposure to the combination of hyperandrogenemia and western-style diet (WSD) worsen adult metabolic function compared to either treatment alone? SUMMARY ANSWER: Young female rhesus macaques treated for 3 years, beginning at menarche, with combined testosterone (T) and WSD have increased weight gain and insulin resistance compared to controls and animals treated with either T or WSD alone. WHAT IS KNOWN ALREADY: Hyperandrogenemia is a well-established component of polycystic ovary syndrome (PCOS) and can be observed in peripubertal girls, indicating a potential pubertal onset of the disease. Obesity is often associated with hyperandrogenemia in peripubertal girls, and overweight girls appear to be at higher risk for the development of PCOS later in life. STUDY DESIGN, SIZE, DURATION: Juvenile (2.5- year old) female rhesus macaques were divided into four groups (n = 10/group): control animals receiving cholesterol implants and a control diet with 15% of calories derived from fat (C), animals receiving T implants (mean serum levels: 1.35 ± 0.01 ng/ml) and a control diet (T), animals receiving a cholesterol implant and a WSD with 36% of calories derived from fat (WSD) and animals receiving a T implant and a WSD (T + WSD). Animals were maintained on the treatments for 36 months and were 5.5 years old at study completion. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metabolic testing consisted of body measurements including weight, dual-energy X-ray absorptiometry scans, activity monitoring, and glucose tolerance testing at zero months and at least once every 12 months for the remainder of the study. Indirect calorimetry and serum hormone assays were performed following 36 months of treatment. MAIN RESULTS AND THE ROLE OF CHANCE: Body weight and fat mass gain were significantly increased in T + WSD at 24 and 36 months of treatment compared to the other three groups. Log transformed fasting insulin and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) were significantly increased in T + WSD animals at 3 years of treatment compared to all other groups. T-treatment caused a greater rate of decline in activity after 18 months, while food intake and metabolic rate were largely unaffected by treatments. LIMITATIONS REASONS FOR CAUTION: Variability was present in the metabolic parameters measured; however, this is similar to the heterogeneity observed in human populations. WIDER IMPLICATIONS OF THE FINDINGS: Chronic hyperandrogenemia beginning at puberty may exacerbate metabolic dysfunction in women consuming a WSD and account for the increased rates of obesity and insulin resistance observed in PCOS patients. Counseling of female patient populations with elevated androgens about the potential benefit of consuming a lower fat diet could improve long-term metabolic health outcomes. STUDY FUNDING/COMPETING INTEREST(S): Eunice Kennedy Shriver National Institute of Child Health & Human Development P50HD071836 and Oregon National Primate Center Grant P51 OD011092. The authors have no competing conflict of interests to disclose.

Differential effects of estrogen and progesterone on development of primate secondary follicles in a steroid-depleted milieu in vitro.

STUDY QUESTION: What are the direct effects of progesterone (P4) and estradiol (E2) on the development and function of primate follicles in vitro from the pre-antral to early antral stage? SUMMARY ANSWER: In a steroid-depleted milieu, E2 improved follicle survival, growth, antrum formation and oocyte health, whereas P4 exerted minimal beneficial effects on follicle survival and reduced oocyte health. WHAT IS KNOWN ALREADY: Effects of P4 and E2 on follicle development have been studied primarily in large antral and pre-ovulatory follicles. Chronic P4 exposure suppresses antral follicle growth, but acute P4 exposure promotes oocyte maturation in pre-ovulatory follicles. Effects of E2 can be stimulatory or inhibitory depending upon species, dose and duration of exposure. STUDY DESIGN, SIZE, DURATION: Non-human primate model, randomized, control versus treatment. Macaque (n = 6) secondary follicles (n = 24 per animal per treatment group) were cultured for 5 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult rhesus macaque secondary follicles were encapsulated in 0.25% alginate and cultured individually in media containing follicle stimulating hormone plus (i) vehicle, (ii) a steroid-synthesis inhibitor, trilostane (TRL, 250 ng/ml), (iii) TRL + low E2 (100 pg/ml) or progestin (P, 10 ng/ml R5020) and (iv) TRL + high E2 (1 ng/ml E2) or P (100 ng/ml R5020). Follicles reaching the antral stage (≥750 µm) were treated with human chorionic gonadotrophin for 34 h. End-points included follicle survival, antrum formation, growth pattern, plus oocyte health and maturation status, as well as media concentrations of P4, E2 and anti-Müllerian hormone (AMH). MAIN RESULTS AND THE ROLE OF CHANCE: In a steroid-depleted milieu, low dose, but not high dose, P improved (P < 0.05) follicle survival, but had no effect (P > 0.05) on antrum formation and AMH production. Low-dose P increased (P < 0.05) P4 production in fast-grow follicles, and both doses of P elevated (P < 0.05) E2 production in slow-grow follicles. Additionally, low-dose P increased (P < 0.05) the percentage of no-grow follicles, and high-dose P promoted oocyte degeneration. In contrast, E2, in a steroid-depleted milieu, improved (P < 0.05) follicle survival, growth, antrum formation and oocyte health. E2 had no effect on P4 or E2 production. Follicles exposed to E2 yielded mature oocytes capable of fertilization and early cleavage, at a rate similar to untreated control follicles. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro effects of P and E2 during the interval from the secondary to small antral stage of macaque follicles. WIDER IMPLICATIONS OF THE FINDINGS: This study provides novel information on the direct actions of P4 and E2 on primate pre-antral follicle development. Combined with our previous report on the actions of androgens, our findings suggest that androgens appear to be a survival factor but hinder antral follicle differentiation, E2 appears to be a survival and growth factor at the pre-antral and early antral stage, whereas P4 may not be essential during early folliculogenesis in primates. STUDY FUNDING/COMPETING INTERESTS: NIH P50 HD071836 (NCTRI), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), ONPRC 8P51OD011092. There are no conflicts of interest.

Direct actions of androgens on the survival, growth and secretion of steroids and anti-Müllerian hormone by individual macaque follicles during three-dimensional culture.

STUDY QUESTION: What are the direct effects of androgens on primate follicular development and function at specific stages of folliculogenesis? SUMMARY ANSWER: Androgen addition altered primate follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, and oocyte quality in vitro, in a dose- and stage-dependent manner. WHAT IS KNOWN ALREADY: Androgens have local actions in the ovary, particularly in the developing follicles. It is hypothesized that androgen promotes early follicular growth, but becomes detrimental to the antral follicles in primates. STUDY DESIGN, SIZE, DURATION: In vitro follicle maturation was performed using rhesus macaques. Secondary (125-225 µm) follicles were mechanically isolated from 14 pairs of ovaries, encapsulated into alginate (0.25% w/v), and cultured for 40 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Individual follicles were cultured in a 5% O2 environment, in alpha minimum essential medium supplemented with recombinant human FSH. Follicles were randomly assigned to experiments of steroid ablation by trilostane (TRL), testosterone (T) replacement and dihydrotestosterone (DHT) replacement. Follicle survival and growth were assessed. Follicles with diameters ≥500 µm at Week 5 were categorized as fast-grow follicles. Pregnenolone (P5), progesterone (P4), estradiol (E2) and AMH concentrations in media were measured. Meiotic maturation and fertilization of oocytes from recombinant human chorionic gonadotrophin-treated follicles were assessed at the end of culture. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with controls, TRL exposure reduced (P < 0.05) follicle survival, antrum formation rate and follicle diameters at Week 5. While P5 concentrations increased (P < 0.05) following TRL treatment, P4 levels decreased (P < 0.05) in fast-grow follicles at Week 5. Few healthy oocytes were retrieved from antral follicles developed in the presence of TRL. T replacement with TRL increased (P < 0.05) follicle survival and antrum formation at Week 5, compared with TRL alone, to levels comparable to controls. However, high-dose T with TRL decreased (P < 0.05) diameters of fast-grow follicles. Although P4 concentrations produced by fast-grow follicles were not altered by T in the presence of TRL, there was a dose-dependent increase (P < 0.05) in E2 levels at Week 5. High-dose T with TRL decreased (P < 0.05) AMH production by fast-grow follicles at Week 3. More healthy oocytes were retrieved from antral follicles developed in TRL+T compared with TRL alone. DHT had the similar effects to those of high-dose T, except that DHT replacement decreased (P < 0.05) E2 concentrations produced by fast-grow follicles at Week 5 regardless of TRL treatment. LIMITATION, REASONS FOR CAUTION: This study reports T and DHT actions on in vitro-developed individual primate (macaque) follicles, which are limited to the interval from the secondary to small antral stage. WIDER IMPLICATION OF THE FINDINGS: The above findings provide novel information on the role(s) of androgens in primate follicular development and oocyte maturation. We hypothesize that androgens promote pre-antral follicle development, but inhibit antral follicle growth and function in primates. While androgens can act positively, excess levels of androgens may have negative impacts on primate folliculogenesis. STUDY FUNDING/COMPETING INTERESTS: NIH U54 RR024347/RL1HD058294/PL1EB008542 (Oncofertility Consortium), NIH U54 HD071836 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest.

Effects of hyperandrogenemia and increased adiposity on reproductive and metabolic parameters in young adult female monkeys.

Many patients with hyperandrogenemia are overweight or obese, which exacerbates morbidities associated with polycystic ovary syndrome (PCOS). To examine the ability of testosterone (T) to generate PCOS-like symptoms, monkeys received T or cholesterol (control) implants (n = 6/group) beginning prepubertally. As previously reported, T-treated animals had increased neuroendocrine drive to the reproductive axis [increased luteinizing hormone (LH) pulse frequency] at 5 yr, without remarkable changes in ovarian or metabolic features. To examine the combined effects of T and obesity, at 5.5 yr (human equivalent age: 17 yr), monkeys were placed on a high-calorie, high-fat diet typical of Western cultures [Western style diet (WSD)], which increased body fat from <2% (pre-WSD) to 15-19% (14 mo WSD). By 6 mo on WSD, LH pulse frequency in the controls increased to that of T-treated animals, whereas LH pulse amplitude decreased in both groups and remained low. The numbers of antral follicles present during the early follicular phase increased in both groups on the WSD, but maximal follicular size decreased by 50%. During the late follicular phase, T-treated females had greater numbers of small antral follicles than controls. T-treated monkeys also had lower progesterone during the luteal phase of the menstrual cycle. Although fasting insulin did not vary between groups, T-treated animals had decreased insulin sensitivity after 1 yr on WSD. Thus, while WSD consumption alone led to some features characteristic of PCOS, T + WSD caused a more severe phenotype with regard to insulin insensitivity, increased numbers of antral follicles at midcycle, and decreased circulating luteal phase progesterone levels.

Effects of steroid ablation and progestin replacement on the transcriptome of the primate corpus luteum during simulated early pregnancy.

Previous microarray analyses indicated that a portion of the transcriptome in the macaque corpus luteum (CL) of the menstrual cycle was regulated indirectly by luteinizing hormone via the local actions of steroid hormones, notably progesterone (P). The current study was designed to investigate this concept in the CL of early pregnancy by analyzing chorionic gonadotrophin (CG)-regulated genes that are dependent versus independent of local steroid action. Exogenous human chorionic gonadotropin treatment simulating early pregnancy (SEP) began on Day 9 of the luteal phase in female rhesus monkeys with and without concurrent administration of the 3-ß-hydroxysteroid dehydrogenase inhibitor trilostane (TRL) with or without the synthetic progestin R5020. Compared with SEP treatment alone, TRL altered 50 mRNA transcripts on Day 10, rising to 95 on Day 15 (P<0.05, ≥2-fold change in gene expression). Steroid-sensitive genes were validated; notably effects of steroid ablation and P replacement varied by day. Expression of some genes previously identified as P-regulated in the macaque CL during the menstrual cycle were not significantly altered by steroid ablation and P replacement during CG exposure in SEP. These data indicate that the majority of CG-regulated luteal transcripts are differentially expressed independently of local steroid actions. However, the steroid-regulated genes in the macaque CL may be essential during early pregnancy, based on previous reports that TRL treatment initiates premature structural regression of the CL during SEP. These data reinforce the concept that the structure, function and regulation of the rescued CL in early pregnancy differs from the CL of the menstrual cycle in primates.

A prostaglandin E2 receptor antagonist prevents pregnancies during a preclinical contraceptive trial with female macaques.

STUDY QUESTION: Can administration of a prostaglandin (PG) E2 receptor 2 (PTGER2) antagonist prevent pregnancy in adult female monkeys by blocking periovulatory events in the follicle without altering menstrual cyclicity or general health? SUMMARY ANSWER: This is the first study to demonstrate that a PTGER2 antagonist can serve as an effective non-hormonal contraceptive in primates. WHAT IS KNOWN ALREADY: The requirement for PGE2 in ovulation and the release of an oocyte surrounded by expanded cumulus cells (cumulus-oocyte expansion; C-OE) was established through the generation of PTGS2 and PTGER2 null-mutant mice. A critical role for PGE2 in primate ovulation is supported by evidence that intrafollicular injection of indomethacin in rhesus monkeys suppressed follicle rupture, whereas co-injection of PGE2 with indomethacin resulted in ovulation. STUDY DESIGN, SIZE, DURATION: First, controlled ovulation protocols were performed in adult, female rhesus monkeys to analyze the mRNA levels for genes encoding PGE2 synthesis and signaling components in the naturally selected pre-ovulatory follicle at different times after the ovulatory hCG stimulus (0, 12, 24, 36 h pre-ovulation; 36 h post-ovulation, n = 3-4/time point). Second, controlled ovarian stimulation cycles were utilized to obtain multiple cumulus-oocyte complexes (COCs) from rhesus monkeys to evaluate the role of PGE2 in C-OE in vitro (n = 3-4 animals/treatment; ≥3 COCs/animal/treatment). Third, adult cycling female cynomolgus macaques were randomly assigned (n = 10/group) to vehicle (control) or PTGER2 antagonist (BAY06) groups to perform a contraceptive trial. After the first treatment cycle, a male of proven fertility was introduced into each group and they remained housed together for the duration of the 5-month contraceptive trial that was followed by a post-treatment reversibility trial. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR, COC culture and expansion, immunofluorescence/confocal microscopy, enzyme immunoassay, contraceptive trial, ultrasonography, complete blood counts, serum biochemistry tests and blood lipid profiles. MAIN RESULTS AND THE ROLE OF CHANCE: Several mRNAs encoding proteins involved in PGE2 synthesis, metabolism and signaling increase (P < 0.05) in the periovulatory follicle after administration of an ovulatory hCG bolus. PGE2 signaling through PTGER2 induces cumulus cell expansion and production of hyaluronic acid, which are critical events for fertilization. Moreover, chronic administration of a selective PTGER2 antagonist resulted in a significant (P < 0.05 versus vehicle-treated controls) contraceptive effect without altering steroid hormone patterns or menstrual cyclicity during a 5-months contraceptive trial. Fertility recovered as early as 1 month after ending treatment. LIMITATIONS, REASONS FOR CAUTION: This is a proof-of-concept study in a non-human primate model. Further investigations are warranted to elucidate the mechanism(s) of PTGER2 antagonist action in the primate ovary. Although PTGER2 antagonist treatment did not produce any obvious undesirable effects, improvements in the mode of administration, as well as the efficacy of these compounds, are necessary to consider such a contraceptive for women. WIDER IMPLICATIONS OF THE FINDINGS: Monitoring as well as improving the efficacy and safety of female contraceptives is an important public health activity. Even though hormonal contraceptives are effective for women, concerns remain regarding their side-effects and long-term use because of the widespread actions of such steroidal products in many tissues. Moreover, some women cannot take hormones for medical reasons. Thus, development of non-hormonal contraceptives for women is warranted. STUDY FUNDING/COMPETING INTEREST(S): Supported by Bayer HealthCare Pharmaceuticals, The Eunice Kennedy Shriver NICHD Contraceptive Development and Research Center (U54 HD055744), NIH Office of the Director (Oregon National Primate Research Center P51 OD011092), and a Lalor Foundation Postdoctoral Basic Research Fellowship (MCP). The use of the Leica confocal was supported by grant number S10RR024585. Some of the authors (N.B., A.R., K.-H.F., U.F., B.B. and B.L.) are employees of Bayer Healthcare Pharma.

Vascular endothelial growth factor and angiopoietin production by primate follicles during culture is a function of growth rate, gonadotrophin exposure and oxygen milieu.

STUDY QUESTION: What is the time course of production of vascular endothelial growth factor-A (VEGF-A), angiopoietin (ANGPT)-1 and ANGPT-2 by primate follicles during encapsulated three-dimensional culture, and what conditions affect their production? SUMMARY ANSWER: Primate follicles produce VEGF-A and ANGPT-2 in vitro, particularly after developing to the antral stage, with VEGF production influenced by FSH concentration and O(2) tension. WHAT IS KNOWN ALREADY: Folliculogenesis, i.e. the development of primordial follicles into mature, antral follicles, requires the creation of a vascular network in the follicle wall via a process called angiogenesis. Angiogenic factors including VEGFs and ANGPTs have documented roles in angiogenesis. However, direct studies on the production and regulation of angiogenic factors by individual, growing follicles are limited. STUDY DESIGN, SIZE, DURATION: Ovaries (n = 9 pairs) were obtained from rhesus macaques during the early follicular phase of the menstrual cycle (cycle days 1-4). Secondary (125-225 µm) follicles were isolated mechanically, encapsulated into alginate (0.25% w/v) and cultured for 40 days. MATERIALS, SETTING, METHODS: Individual follicles were cultured in a 5 or 20% O(2) environment in alpha minimum essential medium supplemented with recombinant human (h) FSH. Half of the follicles had recombinant hLH added to the media from Days 30 to 40. Follicle diameters were measured weekly. Follicles were categorized at Week 5 as no-grow (NG; <250 µm in diameter), slow-grow (SG; 251-499 µm) and fast-grow (FG; >500 µm). VEGF-A, ANGPT-1 and -2 concentrations in media were measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: VEGF concentrations were low throughout the culture for NG follicles. SG and FG follicles had detectable VEGF concentrations at Week 2, which continued to rise throughout culture. VEGF concentrations were distinct (P < 0.05) among all three follicle categories during Weeks 4 and 5. VEGF concentrations were higher (P < 0.05) in SG follicles in the presence of high/mid-dose FSH at 5% O(2). In contrast, there were no dose-dependent differences in VEGF production for FG follicles based on FSH concentrations or O(2) tension. At Week 5, follicles that produced metaphase II oocytes, following exposure to an ovulatory hCG dose, secreted higher concentrations of VEGF than those containing germinal vesicle-intact oocytes. Media concentrations of ANGPT-1 were low throughout culture for all three follicle categories. ANGPT-2 concentrations were low throughout culture for NG follicles. In contrast, ANGPT-2 concentrations of SG and FG follicles continued to rise from Weeks 1 to 4. During Weeks 2-4, ANGPT-2 concentrations in FG follicles were significantly higher than those of SG and NG follicles (P < 0.05). LIMITATION, REASONS FOR CAUTION: This study reports VEGF-A, ANGPT-1 and -2 production by in vitro-developed individual primate (macaque) follicles, that is limited to the interval from the secondary to small antral stage. After VEGF and ANGPT-1 assays, the limited remaining samples did not allow assessment of the independent effects of gonadotrophin and O(2) on the ANGPT-2 production by cultured follicles. Findings await translation to human follicles. WIDER IMPLICATION OF THE FINDINGS: The above findings provide novel information on the process of primate follicle maturation. We hypothesize that a symbiotic relationship between elevated concentrations of ANGPT-2 and VEGF allows FG antral follicles to excel in follicle maturation, e.g. by promoting its vascularization. Elevated ANGPT-2 may also offer possible insight into future oocyte quality as early as Week 2, compared with Week 4 for VEGF and follicle size. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the following grants: NIH U54 RR024347/HD058294/PL1-EB008542 (Oncofertility Consortium), NIH U54-HD018185 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest to declare.

Fibrin promotes development and function of macaque primary follicles during encapsulated three-dimensional culture.

STUDY QUESTION: Does fibrin introduced into the extracellular matrix affect the growth and maturation of individual primate follicles during encapsulated three-dimensional (3D) culture? SUMMARY ANSWER: While not altering follicle survival, fibrin-alginate (FIBRIN) improves macaque primary, but not secondary, follicle development during encapsulated 3D culture in terms of growth, steroidogenesis, anti-Müllerian hormone (AMH)/vascular endothelial growth factor (VEGF) production and oocyte maturation. WHAT IS KNOWN ALREADY: Efforts to grow non-human primate ovarian follicles from the secondary to the antral stage during encapsulated 3D culture have been successful. However, the growth and maturation of primary follicles in vitro has not been reported in primates, especially in chemically defined conditions. STUDY DESIGN, SIZE, DURATION: In vitro follicle maturation was investigated using the rhesus macaque (Macaca mulatta). Ovaries (n = 7 pairs) were obtained during the early follicular phase of the menstrual cycle (cycle day 1-4). Primary (80-120 µm diameter) and secondary (125-225 µm diameter) follicles were isolated mechanically, randomly assigned to experimental groups, encapsulated into alginate (0.25% w/v) or FIBRIN (25 mg/ml fibrinogen-0.25% alginate) and cultured for 13 and 5 weeks, respectively. MATERIALS, SETTING, METHODS: Individual follicles were cultured in alpha minimum essential medium supplemented with FSH. Follicle survival and growth were assessed by microscopy. Follicles that reached the antral stage were treated with recombinant hCG. Metaphase II (MII) oocytes were inseminated via ICSI. Follicle morphology was evaluated by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed for cytochrome P450 family 17 subfamily A polypeptide 1 (CYP17A1) and 19 subfamily A polypeptide 1 (CYP19A1). Culture medium was analyzed for estradiol (E2) and progesterone by chemiluminescence, androstenedione (A4) by radioimmunoassay, as well as anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 105 primary and 133 secondary follicles were collected. The presence of fibrin in the alginate matrix had no effect on either primary or secondary follicle survival. Growing primary and secondary follicles formed an antrum at Weeks 9 and 3, respectively. The percentage of growing follicles was higher (P < 0.05) for primary follicles cultured in FIBRIN than alginate at Week 13. The diameters were larger for the growing secondary follicles cultured in alginate than FIBRIN at Week 5 (P < 0.05). H&E staining revealed the typical morphology for small antral follicles. CPY17A1 immunostaining was detected in theca cells, while CYP19A1 was observed in granulosa cells. E2 increased (P < 0.05) during antrum formation in growing follicles at Week 9 for primary and Week 3 for secondary follicles. AMH levels in medium from growing primary follicles increased (P < 0.05) after Week 4 with peak levels at Weeks 9-11. AMH increased (P < 0.05) in growing secondary follicles at Weeks 3-5. VEGF levels in medium were elevated (P < 0.05) in growing primary follicles at Week 9. VEGF increased (P < 0.05) in medium from growing secondary follicles at Weeks 3-5. E2, AMH and VEGF production was higher (P < 0.05) in primary follicle culture with FIBRIN than alginate alone. One primary follicle cultured in FIBRIN (1 of 5 follicles harvested) and a secondary follicle cultured in alginate alone (1 of 15 follicles harvested) yielded an MII oocyte. The fertilized oocyte from primary follicle culture arrested without cell division after fertilization, while the oocyte from secondary follicle culture cleaved and reached the morula stage. LIMITATIONS, REASONS FOR CAUTION: The study reports on in vitro development and function of individual macaque follicles, that is limited to the interval from the primary and secondary stage to the small antral stage. The findings await translation to human ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: The 3D model for primate follicle development offers a unique opportunity to investigate the growth and regulation of primate primary, as well as secondary follicles, and their enclosed oocytes, as they grow to the antral stage by monitoring and manipulating factors or signaling pathways in vitro. Since primate primary follicles, in addition to secondary follicles, can be cultured to the antral stage to provide mature oocytes, they represent an additional source of pre-antral follicles for in vitro follicle maturation with the potential to provide gametes for assisted reproductive technology as an option for fertility preservation in women, including patients with cancer. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by The Oncofertility Consortium (NIH U54 RR024347-HD058294, PL1-EB008542), NIH U54-HD18185 (Eunice Kennedy Shriver Specialized Cooperative Centers Program in Reproduction and Infertility Research), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), Oregon National Primate Research Center 8P51OD011092. There are no conflicts of interest.

Microarray analysis of the primate luteal transcriptome during chorionic gonadotrophin administration simulating early pregnancy.

To explore chorionic gonadotrophin (CG)-regulated gene expression in the primate corpus luteum (CL), adult female rhesus macaques were treated in a model of simulated early pregnancy (SEP). Total RNA was isolated from individual CL after specific intervals of exposure (1, 3, 6 and 9 days) to recombinant hCG in vivo and hybridized to Affymetrix™ GeneChip Rhesus Macaque Genome Arrays. The mRNA levels of 1192 transcripts changed ≥2-fold [one-way ANOVA, false discovery rate (FDR) correction; P< 0.05] during SEP when compared with Day 10 untreated controls. Real-time PCR validation indicated that 15 of 17 genes matched in expression pattern between PCR and microarray. Protein levels of three genes identified as CG-sensitive, CYP19A1 (aromatase), PGRMC1 (progestin-binding protein) and STAR (steroidogenic acute regulatory protein) were quantified by western blot analysis. To further analyze global changes in gene expression induced by CG exposure, luteal gene expression was compared between SEP (rescued) and regressing CL, utilizing previously banked GeneChip data from the luteal phase of the menstrual cycle. Expression patterns and mRNA levels were analyzed between time-matched intervals. Transcripts for 7677 mRNAs differed in expression patterns ≥2-fold (one-way ANOVA, FDR correction; P< 0.05) between the hCG-exposed (SEP) CL and regressing CL. Regressed CL (at menses) were most unlike all other CL. Pathway analysis of significantly affected transcripts was performed; the pathway most impacted by CG exposure was steroid biosynthesis. Further comparisons of the genome-wide changes in luteal gene expression during CG rescue and luteolysis in the natural menstrual cycle should identify additional key regulatory pathways promoting primate fertility.

Elevated androgens during puberty in female rhesus monkeys lead to increased neuronal drive to the reproductive axis: a possible component of polycystic ovary syndrome.

BACKGROUND: Hyperandrogenemia is associated with several clinical disorders in which both reproductive dysfunction and metabolic changes may coexist [i.e. polycystic ovary syndrome (PCOS), obesity and congenital adrenal hyperplasia]. Moreover, there is growing evidence that the elevated levels of circulating androgens in obese girls may lead to an increased neuroendocrine drive to the reproductive axis, similar to that associated with PCOS. METHODS: To test whether androgen exposure in the childhood and adolescent period could lead to pubertal alterations in LH secretory patterns, female rhesus monkeys received subcutaneous testosterone implants prepubertally beginning at 1 year of age, maintaining a 3.7-fold increase (P = 0.001) in circulating testosterone levels over cholesterol-implant controls (n = 6/group) into the post-pubertal period. In early adulthood, pulsatile secretion of LH was measured over 12 h during the early follicular phase of a menstrual cycle, and responsiveness of the pituitary to gonadotrophin-releasing hormone was determined. In addition, ultrasounds were performed to assess ovarian morphology and glucose tolerance testing was performed to assess insulin sensitivity. RESULTS: The timing of menarche was similar between groups. Testosterone-treated animals had a significantly greater LH pulse frequency during the early follicular phase compared with controls (P = 0.039) when measured at 5 years of age. There was a larger LH response to GnRH when testosterone-treated animals were 4 years of age (P = 0.042), but not when the animals were 5 years old (P = 0.57). No differences were seen in insulin sensitivity or ovarian morphology, and the groups showed similar rates of ovulation in early adulthood. CONCLUSIONS: Exposure to increased levels of androgens over the course of pubertal development appears to trigger physiological changes in the neural drive to the reproductive axis that resemble those of obese hyperandrogenemic girls in early adulthood and are characteristic of PCOS.

Decorin is a part of the ovarian extracellular matrix in primates and may act as a signaling molecule.

STUDY QUESTION: Is decorin (DCN), a putative modulator of growth factor (GF) signaling, expressed in the primate ovary and does it play a role in ovarian biology? SUMMARY ANSWER: DCN expression in the theca, the corpus luteum (CL), its presence in the follicular fluid (FF) and its actions revealed in human IVF-derived granulosa cells (GCs), suggest that it plays multiple roles in the ovary including folliculogenesis, ovulation and survival of the CL. WHAT IS KNOWN ALREADY: DCN is a secreted proteoglycan, which has a structural role in the extracellular matrix (ECM) and also interferes with the signaling of multiple GF/GF receptors (GFRs). However, DCN expression and action in the primate ovary has yet to be determined. STUDY DESIGN, SIZE, DURATION: Archival human and monkey ovarian samples were analyzed. Studies were conducted using FF and GC samples collected from IVF patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry, western blotting, RT-PCR, quantitative RT-PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) studies were complemented by cellular studies, including the measurements of intracellular Ca²âº, reactive oxygen species (ROS), epidermal GF receptor (EGFR) phosphorylation by DCN and caspase activity. MAIN RESULTS AND THE ROLE OF CHANCE: Immunohistochemistry revealed strong DCN staining in the connective tissue and follicular thecal compartments, but not in GCs of pre-antral and antral follicles. Pre-ovulatory follicles could not be studied, but DCN was associated with connective tissue of CL samples and the cytoplasm of luteal cells. DCN expression in monkey CL doubled (P < 0.05) towards the end of the luteal lifespan. DCN was found in human FF obtained from IVF patients (mean: 12.9 ng/ml; n = 20) as determined by ELISA. DCN mRNA and/or protein were detected in freshly isolated and cultured, luteinized human GCs. In the latter, exogenous human recombinant DCN increased intracellular Ca²âº levels and induced the production of ROS in a concentration-dependent manner. DCN, like epidermal GF, phosphorylated EGFR significantly (P < 0.05) and reduced the activity of caspase 3/7 in cultured GCs. The data indicate the expression of DCN in the theca of growing follicles, in FF of ovulatory follicles and in the CL. Therefore, DCN may exert paracrine actions via GF/GFR systems in multiple ovarian compartments. LIMITATIONS, REASONS FOR CAUTION: Functional studies were performed in cultures of human luteinized GCs, which are an apt model but may not fully mirror the pre-ovulatory GC compartment or the CL. Other human ovarian cells, including the thecal cells, were not available. WIDER IMPLICATIONS OF THE FINDINGS: In accordance with its evolving roles in other organs, ovarian DCN is an ECM-associated component, which acts as a multifunctional regulator of GF signaling in the primate ovary. DCN may thus be involved in folliculogenesis, ovulation and the regulation of the CL survival in primates. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Deutsche Forschungsgemeinschaft (DFG) MA1080/17-3 and in part DFG MA1080/21-1 (to AM), NIH grants HD24870 (S.R.O. and R.L.S.), the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement HD18185 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (S.R.O.) and 8P51OD011092-53 for the operation of the Oregon National Primate Research Center (G.A.D., J.D.H., S.R.O. and R.L.S).

Analysis of microarray data from the macaque corpus luteum; the search for common themes in primate luteal regression.

The factors and processes involved in regression of the primate corpus luteum (CL) are complex and not fully understood. Systemic identification of those genes that are differentially expressed utilizing macaque model systems of luteal regression could help clarify some of the important molecular events involved in loss of primate luteal structure and function during luteolysis. In addition, examining gene pathways involved in luteal regression may help elucidate novel approaches for overcoming infertility or designing ovary-based contraceptives. This review provides an overview of the current published microarray experiments evaluating the transcriptome of the macaque CL, and compares and contrasts the data from spontaneous, GnRH antagonist and prostaglandin F2α-induced luteal regression. In addition, further uses of these databases are discussed, as well as limitations of both array technology and the rhesus macaque genome array.

Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin.

BACKGROUND: An alginate-based matrix supports the three-dimensional (3D) architecture of non-human primate follicles and, in the presence of FSH, permits the in vitro development of pre-antral follicles to the small antral stage, including the production of ovarian steroids and paracrine factors. The current study investigated the ability of gonadotrophins, fetuin and oxygen (O2) to improve primate follicle growth and oocyte maturation in vitro. METHODS: Macaque secondary follicles were isolated from the early follicular phase ovaries, encapsulated in a sodium alginate matrix and cultured individually for 40 days in supplemented medium. The effects of recombinant human (rh) FSH (15, 3 and 0.3 ng/ml for high, medium and low FSH, respectively), bovine fetuin (1 or 0 mg/ml) and O2 (5 or 20% v/v) were examined. Half of the follicles in each culture condition received rhLH on Day 30-40. Follicles that reached antral stage were treated with rh chorionic gonadotrophin for 34 h to initiate oocyte meiotic maturation. Media were analyzed for ovarian steroids and anti-müllerian hormone (AMH). RESULTS: Improved culture conditions supported non-human primate, secondary follicle growth to the antral stage and, for the first time, promoted oocyte maturation to the MII stage. In the presence of fetuin at 5% O2, follicles had the highest survival rate if cultured with high or medium FSH, whereas follicles grew to larger diameters at Week 5 in low FSH. Oocyte health and maturation were promoted under 5% O2. High FSH stimulated steroid production by growing follicles, and steroidogenesis by follicles cultured with low FSH was promoted by LH. AMH biosynthesis was elevated with high compared with low FSH and for longer under 5% O2 than under 20% O2. CONCLUSIONS: This encapsulated 3D culture model permits further studies on the endocrine and local factors that influence primate follicle growth and oocyte maturation, with relevance to enhancing fertility preservation options in women.

Live birth after ovarian tissue transplant.

Radiation and high-dose chemotherapy may render women with cancer prematurely sterile, a side-effect that would be avoided if ovarian tissue that had been removed before treatment could be made to function afterwards. Live offspring have been produced from transplanted ovarian tissue in mice and sheep but not in monkeys or humans, although sex steroid hormones are still secreted. Here we describe the successful transplantation of fresh ovarian tissue to a different site in a monkey, which has led to the birth of a healthy female after oocyte production, fertilization and transfer to a surrogate mother. The ectopically grafted tissue functions without surgical connection to major blood vessels and sets the stage for the transplantation of cryopreserved ovarian tissue in humans.

Receptors for sex steroids in the primate corpus luteum New insight into gonadotropin and steroid action.

The evidence is compelling for progesterone receptor (PR) expression in the primate corpus luteum during the menstrual cycle, based on three experimental approaches: (a) immunocytochemistry, (b) radioligand binding to steroid-depleted tissue, and (c) reverse transcription-polymerase chain reaction or ribonuclease protection assay. This information is providing the impetus for studies on possible receptormediated roles for progesterone to control periovulatory events (including follicle rupture and luteal development) and the functional lifespan of the corpus luteum. Similar experiments suggest that estrogen receptors are nondetectable in the corpus luteum. Thus, "classic" receptor-mediated actions of estrogen, such as promoting PR expression, are not apparent; rather, the midcycle surge of LH assumes the role of stimulating PR expression in luteinizing granulosa cells. The recent discovery of androgen receptor expression in primate luteal tissue should lead to studies on the heretofore unsuspected actions of androgens in the corpus luteum.

Steroid receptors and action in the primate follicle.

Studies localizing steroid receptor (R) proteins and their mRNAs to the primate ovary are consistent with a local role for progesterone (P) and androgen (A) in modulating follicle growth and/or maturation via classic R-mediated pathways, but data in support of estrogen (E) action remain equivocal. Investigations of the hypothesis that steroids play a pivotal role in folliculogenesis or gametogenesis in macaques receiving inhibitors of steroid enzyme synthesis and in women with congenital steroid enzyme deficiencies reveal that (a) antral follicle growth, maturation, and luteinization, as well as oocyte meiosis, do not require high or increasing E levels; (b) elevated A to E ratios are detrimental to the gametogenic functions of the primate follicle, but do not alter growth and maturation of antral follicles; and (c) ovulation and luteinization of the mature follicle are dependent on local P actions. The genomic actions of steroids likely vary between compartments and with the developmental state of the follicle; however, potential nongenomic actions of steroids in follicles remain largely undefined. Future advances will rely on identification of discrete biochemical, morphological, and functional correlates of steroid hormone action in the somatic and gametogenic compartments of the primate follicle throughout its life cycle.

Identification of an ovarian voltage-activated Na+-channel type: hints to involvement in luteolysis.

An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24-48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24-48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results may be of clinical relevance.