Anti-Müllerian hormone promotes pre-antral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates.

What are the direct effects and physiological role of anti-Müllerian hormone (AMH) during primate follicular development and function at specific stages of folliculogenesis? AMH actions in the primate ovary may be stage-dependent, directly promoting pre-antral follicle growth while inhibiting antral follicle maturation and dominant follicle selection. AMH is expressed in the adult ovary, particularly in developing follicles. Studies in mice suggest that AMH suppresses pre-antral follicle growth in vitro, and inhibits primordial follicle recruitment and FSH-stimulated antral follicle steroidogenesis. For in vitro study, secondary follicles were isolated from ovaries of 12 rhesus macaques and cultured for 5 weeks. For in vivo study, intraovarian infusion was conducted on five monkeys for the entire follicular phase during two spontaneous menstrual cycles. For in vitro study, individual follicles were cultured in a 5% O2 environment, in alpha minimum essential medium supplemented with recombinant human FSH. Follicles were randomly assigned to treatments of recombinant human AMH protein or neutralizing anti-human AMH antibody (AMH-Ab). Follicle survival, growth, steroid production, steroidogenic enzyme expression, and oocyte maturation were assessed. For in vivo study, ovaries were infused with control vehicle or AMH-Ab during the follicular phase of the menstrual cycle. Cycle length, serum steroid levels, and antral follicle growth were evaluated. AMH exposure during culture weeks 0-3 (pre-antral stage) promoted, while AMH-Ab delayed, antrum formation of growing follicles compared with controls. AMH treatment during culture weeks 3-5 (antral stage) decreased (P < 0.05) estradiol (E2) production, as well as the mRNA expression of cytochrome P450 family 19 subfamily A polypeptide 1, by antral follicles relative to controls, whereas AMH-Ab increased (P < 0.05) follicular mRNA levels of the enzyme. Intraovarian infusion of AMH-Ab during the follicular phase of the menstrual cycle increased (P < 0.05) the average levels of serum E2 compared with those of the control cycles. Three of the five AMH-Ab-treated ovaries displayed multiple (n = 2-9) medium-to-large (2-8 mm) antral follicles at the mid-cycle E2 peak, whereas only one large (4-7 mm) antral follicle was observed in all monkeys during their control cycles. The average levels of serum progesterone were higher (P < 0.05) during the luteal phase of cycles following the AMH-Ab infusion relative to the vehicle infusion. The in vitro study of AMH actions on cultured individual macaque follicles was limited to the interval from the secondary to small antral stage. A sequential study design was used for in vivo experiments, which may limit the power of the study. The current study provides novel information on direct actions and role of AMH during primate follicular development, and selection of a dominant follicle by the late follicular phase of the menstrual cycle. We hypothesize that AMH acts positively on follicular growth during the pre-antral stage in primates, but negatively impacts antral follicle maturation, which is different from what is reported in the mouse model. NIH NICHD R01HD082208, NIH ORWH/NICHD K12HD043488 (BIRCWH), NIH OD P51OD011092 (ONPRC), Collins Medical Trust. There are no conflicts of interest. Not applicable.

Xu J ，Bishop CV ，Lawson MS ，Park BS ，Xu F ... -  《-》

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

What are the direct effects of androgens on primate follicular development and function at specific stages of folliculogenesis? 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. 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. 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. 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. 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. 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. 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. 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.

Rodrigues JK ，Navarro PA ，Zelinski MB ，Stouffer RL ，Xu J ... -  《-》

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

Xu J ，Lawson MS ，Yeoman RR ，Molskness TA ，Ting AY ，Stouffer RL ，Zelinski MB ... -  《-》

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

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? 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. FSH supports primate preantral follicle development in vitro. Androgen and estrogen augment follicle survival and growth in the presence of FSH during culture. 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. 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. 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. 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. 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. 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.

Baba T ，Ting AY ，Tkachenko O ，Xu J ，Stouffer RL ... -  《-》

Matrix-free 3D culture supports human follicular development from the unilaminar to the antral stage in vitro yielding morphologically normal metaphase II oocytes.

Can group culture with stage-specific anti-Müllerian hormone (AMH) modulation support human follicular development and oocyte maturation in vitro? In the presence of FSH, AMH supplementation at the secondary-to-early antral stage followed by AMH depletion promotes the coordinated growth and function of human follicles during group culture, thereby yielding mature oocytes. Stage-specific AMH modulation promotes in-vitro development of nonhuman primate follicles. The group culture method supports nonhuman primate follicle growth from the primary to antral stage, producing developmentally competent oocytes. Ovarian tissue samples were collected from 19 patients of reproductive age (22-47 years old having menstrual cycles) who underwent oophorectomy or hysterectomy for clinical purposes. Tissue pieces were cultured in a matrix-free system for 3 weeks followed by isolation of follicles for the subsequent 6-week individual or group culture. Pieces of ovarian cortical tissue were cultured to support primordial follicle activation and early-stage follicle growth. Secondary follicles isolated from cultured tissue were then randomly assigned to two groups for individual culture: control and AMH modulation, i.e., recombinant human AMH protein supplementation during the secondary-to-early antral stage followed by the addition of neutralizing anti-human AMH antibody. Secondary follicles were also cultured in groups with the same AMH modulation. Follicle survival, growth, steroid hormone and paracrine factor production, steroidogenic protein expression, as well as oocyte maturation and morphology were assessed. Follicles grew to the secondary stage during 3 weeks of ovarian tissue culture. In-vitro-developed follicles expressed AMH and levels of secreted AMH increased (P < 0.05) in the culture media over time. Secondary follicles isolated from cultured ovarian tissue survived and grew to the antral stage during 6 weeks of individual follicle culture. In-vitro-developed antral follicles produced granulosa and theca cell-derived steroid hormones and paracrine factors, which were detectable in the culture media. Germinal vesicle oocytes obtained from cultured follicles exhibited a perinucleolar chromatin rim configuration. AMH modulation did not alter follicle survival or oocyte maturation relative to those of the control follicles. However, follicle diameters, as well as steroid hormone and paracrine factor production, increased (P < 0.05) in the AMH-modulation group compared with the control group. Secondary follicles isolated from cultured ovarian tissue formed aggregates and grew to the antral stage during 6 weeks of group culture. In-vitro-developed antral follicles expressed steroidogenic enzymes and secreted steroid hormones were detectable in the culture media. Oocytes obtained from cultured follicle aggregates with AMH-modulation progressed to the metaphase II stage after IVM, containing a normal-sized first polar body and meiotic spindle. Oocytes exhibited a typical ultrastructure. Follicles were obtained from fresh ovarian tissue of adult patients. Oocyte maturation rates were relatively low and oocytes were assessed by morphological evaluation. Owing to the lack of a control group, the beneficial effects of AMH modulation remained undetermined for the group culture in this study. Stage-specific AMH modulation supports human follicular development in the matrix-free group culture, which is consistent with previously reported AMH actions on growing follicles in nonhuman primates. Oocytes generated by in-vitro-developed follicles achieve meiotic maturation with a typical morphology and ultrastructure, which supports in-vitro follicle maturation as a potential approach for fertility preservation in women. NICHD R01HD082208 and NIH Office of the Director P51OD011092. The authors have no competing interest to declare. N/A.

Xu F ，Lawson MS ，Bean Y ，Ting AY ，Pejovic T ，De Geest K ，Moffitt M ，Mitalipov SM ，Xu J ... -  《-》