Chronic superphysiologic AMH promotes premature luteinization of antral follicles in human ovarian xenografts

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Chronic superphysiologic AMH promotes premature luteinization of antral follicles in human ovarian xenografts

https://www.science.org/doi/10.1126/sciadv.abi7315

Abstract

Anti-Müllerian hormone (AMH) is produced by growing ovarian follicles and provides a diagnostic measure of reproductive reserve in women; however, the impact of AMH on folliculogenesis is poorly understood. We cotransplanted human ovarian cortex with control or AMH-expressing endothelial cells in immunocompromised mice and recovered antral follicles for purification and downstream single-cell RNA sequencing of granulosa and theca/stroma cell fractions. A total of 38 antral follicles were observed (19 control and 19 AMH) at long-term intervals (>10 weeks). In the context of exogenous AMH, follicles exhibited a decreased ratio of primordial to growing follicles and antral follicles of increased diameter. Transcriptomic analysis and immunolabeling revealed a marked increase in factors typically noted at more advanced stages of follicle maturation, with granulosa and theca/stroma cells also displaying molecular hallmarks of luteinization. These results suggest that superphysiologic AMH alone may contribute to ovulatory dysfunction by accelerating maturation and/or luteinization of antral-stage follicles.

INTRODUCTION

Anti-Müllerian hormone (AMH), found in 1947 by Alfred Jost, was initially noted for its role in promoting male sexual development (1). AMH secreted by Sertoli cells in the fetal testes drives regression of Müllerian ducts, thereby enabling establishment of the Wolffian ducts and their derivatives in response to testicular androgens (2). Yet, AMH is also expressed by the correlate of Sertoli cells in the ovary, granulosa cells (GCs) that surround and foster the development of oocytes within ovarian follicles (3, 4). The exclusive production of AMH by GCs in growing follicles has made serum measurement of this protein a reliable diagnostic indicator of ovarian follicular reserve and a surrogate for antral follicle count (5, 6). While serum AMH measurement is routinely applied in reproductive medicine and assisted reproductive technologies, its physiological, and potentially therapeutic, function in the context of reproductive biology and fertility is not well understood.

AMH performs critical yet unclear functions during folliculogenesis. It is a glycoprotein belonging to the transforming growth factor–β (TGFβ) superfamily secreted by GCs of growing follicles beginning at primary stages (7). AMH is believed to exert its activity in a paracrine fashion, with production by growing follicles providing negative feedback on activation to preserve the primordial follicle (PrF) pool for an extended reproductive life span (8). AMH knockout mice exhibit precocious activation and growth of follicles and premature depletion of PrFs (4), which can be partially reversed in vitro with supplementation of neonatal mouse ovary culture with AMH alone (9). Previous studies have recapitulated this growth-suppressive effect in vitro in explanted ovaries of mouse (3), bovine (10), and human (11), and continuous application of recombinant or adenovirus-encoded AMH has been shown to confer both contraceptive and fertoprotective effects in mice (12, 13). Work from our group (14, 15) has extended this growth-suppressive effect to human xenografts, demonstrating the capacity for exogenous paracrine AMH to improve the retention of PrFs. However, following extended treatment with recombinant AMH, mouse ovaries display compensatory rebound folliculogenesis, with a threefold increase in oocyte production (16). Multiple studies have demonstrated a positive effect of AMH on follicle growth in rats, primates, and humans (17–20). For these reasons, the precise role and mechanistic function of AMH within the ovary remain controversial.

AMH activity has also been shown to intersect with other circulating factors that modulate the reproductive axis in women. Aromatase [cytochrome P450 family 19 subfamily A member 1 (CYP19A1)], the GC-specific enzyme that converts androgens to estrogens, is stimulated by follicle-stimulating hormone (FSH) derived from the pituitary. In cultured GCs, AMH has been shown to inhibit the expression of FSH receptor (FSHR) (21) and the catalytic activity of aromatase (22). Conversely, FSH induces, and estradiol represses, the expression of AMH (23). These relationships describe a feedback loop whereby stimulatory input from circulating FSH promotes increased estradiol production by GCs in growing follicles. At the same time, FSH also drives increased expression of AMH that tempers growth and aromatization until accelerated production of estradiol at later antral stages ultimately reaches a threshold that silences AMH expression. In parallel, androgens drive increased expression of FSHRs in the GCs of preantral follicles (23), augmenting their growth response and potentially increasing their expression of AMH. While the intersection of these signaling factors is fundamental to the homeostasis of the reproductive axis, their imbalance can result in endocrine dysfunction and infertility.

The most common cause of anovulatory infertility is polycystic ovary syndrome (PCOS), affecting greater than 5% of reproductive-aged women (24). The Rotterdam criteria for diagnosis of PCOS require two of the following three criteria: oligo-anovulation, clinical and/or biochemical signs of hyperandrogenism, and polycystic morphology of ovaries on ultrasound (25). While the etiology remains poorly understood, increasing evidence points to developmental (26) and epigenetic (27) influences that take root in the ovary. An early treatment of PCOS known as ovarian wedge resection effectively addressed the condition (28). Also, as patients approach menopause, they resume regular menstrual cycles with the waning of their ovarian reserve (29). Increased serum AMH in women with PCOS is believed to be a by-product of the increased volume of growing follicles that occurs either in parallel to or downstream of a hyperandrogenic milieu. However, the specific contribution of elevated AMH to the molecular pathology of PCOS and its defining clinical features is unclear, as no study, to date, has examined the effect of chronically elevated AMH in an experimentally controlled in vivo model.

Here, we use ovarian cortical xenografts with cotransplantation of engineered endothelial cells (ECs) to test the effect of chronic paracrine AMH stimulus on human folliculogenesis. We show that long-term xenografts exhibit an accelerated growth rate in the context of chronically elevated AMH and exhibit a molecular signature indicative of more mature stages, including that of luteinization. GCs and theca/stroma from follicles grown in the context of chronic AMH exhibited increased expression of a broad spectrum of factors related to cholesterol biosynthesis and metabolism, as well as factors that have been linked to PCOS. These data decouple elevated AMH from the metabolic and hyperandrogenic conditions that define PCOS and suggest that chronically elevated AMH induces a molecular cascade that contributes, at least in part, to the anovulatory phenotype in these patients.

RESULTSContinuous paracrine stimulus of ovarian xenografts increases follicular activation and growth

We have previously demonstrated a benefit conferred by cotransplantation of ECs with thawed ovarian cortex and used these ECs as a vector to convey a continuous paracrine source of superphysiological AMH that aids in the retention of PrFs in short-term xenografts (14, 15). Here, we applied the same approach (Fig. 1A) in long-term (8 and 14 weeks) xenografts; exogenous ECs transduced with lentivirus encoding AMH complementary DNA (cDNA) secreted high levels of AMH in vitro (Fig. 1B) and were observed in a broad distribution within the fibrin matrix surrounding the graft (Fig. 1C) while also exhibiting continued expression of AMH from ECs at late stages (Fig. 1D). As observed in short-term grafts previously, the ratio of primordial to growing follicles was increased in the context of AMH ECs at the short-term interval (2 weeks; Fig. 1E and table S1). However, long-term xenografts at 8 and 14 weeks showed the reverse trend (Fig. 1E and table S1), with the diameter of antral follicles observed in the AMH condition threefold greater than controls (1.5 mm versus 0.5 mm, P = 0.01; Fig. 1F)

Single-cell RNA sequencing of AMH-EC–conditioned antral follicles

Accelerated antral follicle growth in long-term xenografts with AMH (Fig. 1) suggested a role for this factor in regulating follicular homeostasis. To elaborate on the influence of chronic AMH on folliculogenesis, we performed single-cell RNA sequencing (scRNA-seq) on granulosa and stroma cells isolated from antral-stage follicles (Fig. 2A). Ovarian cortical fragments were transplanted under three conditions: (i) with control ECs (CTL ECs; n = 7 follicles), (ii) with a >95% purity population of ECs constitutively expressing AMH (high-AMH ECs, n = 7 follicles; Fig. 1B), or with a 1:9 ratio of high-AMH ECs to CTL ECs (low-AMH ECs, n = 3 follicles). We monitored xenograft-bearing mice via weekly magnetic resonance imaging (MRI; Fig. 2B), targeting follicles between 1 and 4 mm for recovery (Fig. 2C). Seven CTL EC follicles (1.93 ± 1.24 mm) were isolated from four mice at 136 ± 30 days after transplant, and 10 AMH EC (including 7 AMH-high and 3 AMH-low) follicles (2.28 ± 0.73 mm) were isolated from seven mice after 115 ± 18 days (P = 0.07; Fig. 2D).