Human Reproduction. 2016 July;31(7):1522-30.
Anti-Müllerian hormone promotes pre-antral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates.
Xu J1, Bishop CV1, Lawson MS1, Park BS2, Xu F1.
1Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Beaverton, Oregon 97006, USA; 2OHSU-PSU School of Public Health, Oregon Health & Science University, Portland, Oregon 97239, USA.
STUDY QUESTION: What are the direct effects and physiological role of anti-Müllerian hormone (AMH) during primate follicular development and function at specific stages of folliculogenesis?
SUMMARY ANSWER: 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.
WHAT IS KNOWN ALREADY: 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.
STUDY DESIGN, SIZE, DURATION: 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.
PARTICIPANTS/MATERIALS, SETTING, METHODS: 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.
MAIN RESULTS AND THE ROLE OF CHANCE: 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.
LIMITATIONS, REASONS FOR CAUTION: 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.
WIDER IMPLICATIONS OF THE FINDINGS: 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.
STUDY FUNDING/COMPETING INTERESTS: NIH NICHD R01HD082208, NIH ORWH/NICHD K12HD043488 (BIRCWH), NIH OD P51OD011092 (ONPRC), Collins Medical Trust. There are no conflicts of interest.
TRIAL REGISTRATION NUMBER: Not applicable.
Anti-Müllerian hormone (AMH) is originally discovered as a fetal hormone that regulates sexual differentiation. AMH is expressed in testes of male fetuses and causes the regression of Müllerian ducts, which are paired ducts of the embryo that develops into the female reproductive tract. Later, AMH is also identified in postnatal ovaries and is suggested to have a role in regulating follicular development in women.
In the ovary, once primordial follicles are activated, they grow to become preantral follicles. Upon the formation of an antral cavity, the follicle enters the antral stage. A model emerged from studies in mice suggests that AMH is inhibitory throughout the follicular development. AMH inhibits primordial follicle activation, preantral growth and antral follicle maturation. However, AMH expression in the developing follicles begins at the preantral stage, increases during preantral follicle growth, and peaks in small antral follicles. Therefore, we question why preantral follicles could keep growing while their AMH expression increases, if AMH is inhibitory? And we designed experiments to investigate the role of AMH in the ovary using adequate in vitro and in vivo models involving nonhuman primates to test the hypothesis that AMH actions on follicular development is stage-dependent by promoting preantral follicle growth and inhibiting antral follicle maturation.
Figure 1. Three-dimensional follicle culture. A rhesus macaque preantral follicle (diameter = 150 µm; A) develops to the small antral stage (diameter = 1,500 µm; B) during 5 weeks of culture.
We developed three-dimensional culture technique to grow individual primate follicles in vitro (1). Figure 1 shows a rhesus macaque preantral follicle (diameter = 150 µm) develops to the small antral stage (diameter = 1,500 µm) during 5 weeks of culture. Cultured follicles produce steroid hormones and peptide factors as those developed in vivo. Using this technique, we are able to study the direct actions of AMH from the preantral to the small antral stage of follicular development. Our current research indicated that AMH exposure during the preantral stage promoted follicle growth to the antral stage. In contrast, AMH treatment during the antral stage inhibited the steroidogenic function (estradiol production) of in vitro-developed follicles.
In women, only one of the antral follicles is selected as a dominant follicle for continued growth and maturation to become a preovulatory follicle, which produces mature oocyte for fertilization. Thus, we performed additional studies to investigate the role of AMH from the small antral to the preovulatory stage in vivo using an intraovarian infusion technique (2). As shown in Figure 2, an osmotic pump filled with treatment agent is placed subcutaneously in the abdomen of a rhesus macaque, which was connected to a catheter placed in the center of the ovary for constant local treatment. Doppler 3D ultrasonography was used to monitor antral follicle development during the menstrual cycle. Our data suggested that blocking endogenous AMH actions by a neutralizing antibody during the follicular phase of the menstrual cycle led to an increase in serum estradiol levels, formation of multiple medium-to-large antral follicles (diameter = 2-8 mm) at the mid-cycle estradiol peak, and an increase of serum progesterone levels during the luteal phase .
In summary, our studies suggest that the local actions of AMH on follicular development are stage-dependent, particularly in the switch from the preantral to the antral stage, which is different from findings in mice where AMH appears inhibitory to both the preantral follicle growth and the antral follicle maturation. AMH promotes preantral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates. Thus, AMH actions on folliculogenesis may be species-specific.
AMH actions could be related to the dysregulation of folliculogenesis, e.g., the pathology of Polycystic Ovarian Syndrome (PCOS). PCOS is a common endocrine system disorder and a major cause of infertility in women. PCOS patients have elevated circulating levels of AMH. Based on our findings, AMH promotes preantral follicle growth to the small antral stage and, at the same time, inhibits further antral follicle maturation to become a dominant follicle. Therefore, multiple follicles develop and arrest at the small-to-medium antral stage as observed in PCOS patients (Figure 3).
Figure 2. Intraovarian infusion. A catheter is placed in the center of a rhesus macaque ovary (A), which is connected to an osmotic pump filled with treatment agent for constant local treatment. Antral follicle development is monitored by Doppler 3D ultrasonography (B) during the menstrual cycle.
Figure 3. Hypothesized anti-Müllerian hormone (AMH) actions in the ovary of Polycystic Ovarian Syndrome (PCOS). Elevated AMH production promotes preantral follicle growth to the small antral stage and, at the same time, inhibits further antral follicle maturation to become a dominant follicle. Therefore, multiple follicles arrest at the small-to-medium antral stage as observed in PCOS patients.
- Xu J, Xu M, Bernuci MP, Fisher TE, Shea LD, Woodruff TK, Zelinski MB, Stouffer RL 2013 Primate follicular development and oocyte maturation in vitro. Adv Exp Med Biol 761:43-67.
- Xu J, Bishop CV, Lawson MS, Park BS, Xu F 2016 Anti-Müllerian hormone promotes pre-antral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates. Hum Reprod 31:1522-1530.
Acknowledgements: This study was supported by NIH NICHD R01HD082208 awarded to Jing Xu; NIH ORWH/NICHD K12HD043488 (BIRCWH) awarded to Dan Dorsa and Jeanne-Marie Guise , Program director; NIH OD P51OD011092 (core); Collins Medical Trust awarded to Jing Xu.
Jing Xu, Ph.D.
Division of Reproductive & Developmental Sciences
505 NW. 185th Ave.
Oregon National Primate Research Center
Oregon Health & Sciences University
Portland, OR 97006