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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 INTEREST(S) 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. [ABSTRACT FROM PUBLISHER]

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 O2 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% O2 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% O2. In contrast, there were no dose-dependent differences in VEGF production for FG follicles based on FSH concentrations or O2 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 O2 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 ... [ABSTRACT FROM PUBLISHER]

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 ... [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]