The role of anti-Müllerian hormone as a therapeutic agent to preserve the ovarian follicle pool during chemotherapy

Purpose: Cancer treatments can deplete the ovarian follicle reserve, causing infertility and early menopause, with subsequent decline in cardiovascular, cognitive, and overall women’s health. Medical measures to prevent this chemotherapy-induced ovarian damage are currently not available. Anti-Müllerian hormone (AMH) is an inhibitory glycoprotein that plays a central role in regulating ovarian follicle development across the female lifespan, and in vitro, ex vivo, and gene therapy studies have demonstrated that AMH can protect the ovarian follicle pool during chemotherapy treatments.

Narrative review of available literature.

Experimental work has shown how AMH modulates folliculogenesis, notably through its signaling pathway that activates SMAD proteins, ultimately modulating the PI3K/AKT/FOXO3a pathway to help maintain primordial follicle dormancy and prevent premature depletion of the ovarian pool.

This review summarizes current understanding of AMH biosynthesis, AMH receptor 2 (AMHR2) signaling, and their genetic regulation, and examines emerging translational research on the use of recombinant AMH to protect the ovarian follicle reserve in models of accelerated ovarian damage, specifically chemotherapy-induced gonadotoxicity. Finally, this review highlights the potential of AMH-based therapies to preserve fertility and delay follicular depletion in conditions such as endometriosis, chronic inflammation, and natural aging. It distinguishes established findings from emerging hypotheses and outlines key challenges for translating these strategies into early-phase clinical trials. Similar content being viewed by others

Ovarian follicle development, or folliculogenesis, is a tightly regulated, multi-stage process that begins with activation of dormant primordial follicles and proceeds through clearly defined morphologic and molecular stages: primary, secondary (preantral), tertiary (antral), and preovulatory follicles [1]. Primordial follicles, each containing a primary oocyte arrested in prophase I of the meiotic division and surrounded by a single layer of flattened and metabolically quiescent granulosa cells, are maintained in dormancy by inhibitory factors such as forkhead box O3 subtype a (FOXO3a) and phosphatase and tensin homolog (PTEN) [2,3,4]. Follicle activation, termed “initial recruitment,” is the gatekeeper event controlling entry to folliculogenesis and is regulated by paracrine growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs) and by the intracellular pathways phosphoinositide 3-kinase-protein kinase B (PI3K/AKT) which leads to phosphorylation and nuclear export of FOXO3a (PI3K/AKT/FOXO3a), which relieves its inhibitory effect thus permitting follicle recruitment, and activation of mechanistic target of rapamycin (PI3K/AKT/mTOR), which enhances cellular metabolism and growth, further supporting follicle activation [5,6,7,8,9]. Once activated, follicles irreversibly enter the growth cycle: granulosa cells become cuboidal and proliferate, and the oocyte enlarges, marking the transition to the primary follicle stage. As granulosa-cell replication proceeds, secondary follicles acquire multiple granulosa layers, recruit surrounding theca cells, form a zona pellucida encircling the egg, and acquire gonadotropin responsiveness. Antral (tertiary) follicles form a fluid-filled cavity that increases in volume throughout the follicular phase in the selected dominant follicle [1, 10,11,12]. In humans, folliculogenesis is a continuous, unidirectional process that can span nearly a year from primordial activation to ovulation [1, 10]. Once a primordial follicle is activated, the transition from primary to secondary follicle represents the longest phase, requiring approximately 290 days (about 10 menstrual cycles), during which the process becomes progressively more dependent on gonadotropin support. In contrast, the interval from antrum formation to a mature preovulatory follicle is about 60 days (approximately two cycles) and becomes increasingly gonadotropin-dependent, with dominant follicle selection occurring roughly 20 days before ovulation, during the late luteal phase of the preceding menstrual cycle [1, 13,14,15]. The regulators that orchestrate each stage of folliculogenesis are not fully defined, but anti-Müllerian hormone (AMH), produced by granulosa cells of preantral and small antral follicles, has emerged as a key intra-ovarian regulator [16,17,18,19]. AMH is the main hormone responsible for inhibiting primordial follicle activation and early recruitment, thereby preserving the ovarian reserve and modulating the rate of follicle pool depletion. The other intra-ovarian mediators, GDF-9, BMP15, and signaling pathways such as PTEN and FOXO3a act as critical inhibitors that maintain dormancy [3, 12, 20]. The PI3K/AKT/mTOR pathway serves as the central signaling cascade mediating primordial follicle activation, fine-tune follicle growth, selection, and atresia across early and later stages [6, 21,22,23]. As follicles progress in development, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) become increasingly important: FSH promotes granulosa-cell proliferation and estradiol production, whereas LH stimulates theca-cell androgen synthesis [24, 25]. Upregulation of FSH and LH receptors at the antral stage permits gonadotropin-dependent growth and dominant follicle selection for ovulation [26,27,28]. Dysregulation of these pathways underlies several common clinical conditions. Low AMH levels, causing excessive activation of primordial follicles, can lead to depletion of the follicle pool with subsequent development of diminished ovarian reserve (DOR), characterized by subfertility or infertility, or premature ovarian insufficiency (POI), characterized by premature menopause [29,30,31,32]. Conversely, conditions characterized by elevated AMH levels, such as polycystic ovary syndrome (PCOS), are characterized by a slower depletion of the follicle reserve and a later onset of menopause, approximately 1.5–2 years later than women without the condition [33]. Thus, the balance between activation and inhibition of follicle recruitment and growth is essential for normal ovarian function and for maintaining a normal reproductive lifespan. This review summarizes the mechanisms of chemotherapy-induced primordial follicle depletion and the limitations of current fertility-preservation strategies. It then outlines the molecular biology, regulation, and physiological roles of AMH, with emphasis on its therapeutic role in protecting the ovarian follicle reserve in physiological and iatrogenic conditions. Finally, it discusses challenges in AMH production and delivery, and key considerations for translating AMH-based interventions into early-phase human clinical trials. Mechanisms and triggers of accelerated primordial follicle depletion Under normal physiological conditions, primordial follicle activation is tightly regulated to maintain an adequate ovarian follicle pool across a woman’s reproductive lifespan of approximately 40 years [34]. This balance can be disrupted by inflammation or gonadotoxic exposure, particularly chemotherapy and radiotherapy, which lead to a gradual or rapid loss of ovarian reserve [35,36,37]. Inflammation and oxidative stress, caused by pathological processes such as endometriosis, autoimmune and metabolic disorders, infections, environmental toxins, or allergens, have been linked to ovarian follicle loss and accelerated ovarian aging [35]. Importantly, oxidative stress is also a physiological consequence of normal aging. Single-cell transcriptomic profiling of primate ovaries identified inactivation of antioxidative pathways, accumulation of reactive oxygen species, and granulosa cell and oocyte apoptosis as core molecular features of age-related ovarian decline, independent of exogenous or pathological trigger [38]. Elevated levels of pro-inflammatory cytokines, including interleukin 1α (IL-1α), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6), promote oocyte damage and follicular atresia, contributing to progressive decline in ovarian function and fertility, and TNF-α in particular has been linked to oocyte death during the early stages of follicular development [35, 39]. Chemotherapy induces DNA double-strand breaks in oocytes, activating checkpoint kinases and apoptotic mediators, which lead to extensive apoptosis of oocytes and stromal injury, as demonstrated in murine models and human ovarian tissue [40,41,42,43]. Beyond direct cytotoxicity, chemotherapy also induces massive primordial follicle activation followed by rapid apoptosis and consequent follicle depletion. Granulosa cells of growing follicles are highly sensitive to alkylating agents, and their apoptosis leads to a rapid decline in circulating and intra-ovarian AMH, loss of inhibitory control over primordial follicle recruitment, and consequent massive activation of dormant follicles, a mechanism first proposed by Meirow and colleagues in cyclophosphamide-treated mice and conceptualized as “ovarian follicle burnout” [37, 44,45,46,47,48,49]. This acute over-recruitment of primordial follicles, followed by their subsequent atresia, results in accelerated depletion of the ovarian reserve and earlier onset of DOR and POI in animal models [50,51,52]. This model, supported by multiple preclinical studies, helps explain why the extent of follicle loss often exceeds what would be expected from apoptosis alone [53,54,55,56]. Recent reviews, however, emphasize that overactivation-mediated burnout, direct apoptosis of primordial and growing follicles, and stromal-microvascular damage likely coexist as parallel mechanisms of chemotherapy-induced ovarian failure, and they reach different conclusions about which pathway predominates in humans [37, 44,45,46,47, 49]. Figure 1 illustrates how chemotherapy disrupts orderly folliculogenesis through these concurrent processes, including apoptosis, dysregulated follicle activation, and microenvironmental injury, ultimately leading to depletion of the primordial follicle pool. AMH signaling acts restraining the intracellular pathways PI3K/AKT/FOXO3a and PI3K/AKT/mTOR, which promote the transition of primordial follicles from a dormant to a metabolically active and growing state, thus preventing primordial follicle activation and maintaining the follicular pool, as explained in Fig. 2 [49, 52]. Radiotherapy exacerbates follicle loss through both direct genotoxic injury and disruption of the ovarian microenvironment [57]. Dividing granulosa cells and oocytes are among the earliest targets, with prominent apoptosis occurring within hours of irradiation. It is estimated that a lethal dose 50% (LD50) of < 2 Gy and lower thresholds can cause permanent ovarian failure, especially in women of more advanced reproductive age [58]. Ionizing radiation also generates reactive oxygen species through water radiolysis, overwhelming antioxidant defenses and further inducing “indirect” oxidative DNA damage in oocytes and granulosa cells. Additionally, it activates tumor p53-mediated mitochondrial apoptosis cascades and MAPK signaling, thereby accelerating primordial follicle loss, stromal-vascular injury, and fibrotic atrophy [57, 58]. The accelerated ovarian follicle depletion caused by inflammation and cancer therapy can lead to DOR, POI, and early menopause, in a short period of time, resulting in long-term deleterious effects on women’s health. In fact, this results in long-term cardiovascular, cognitive, and overall health decline, with greater impact in younger women due to its abrupt onset, compounded by the adverse effects of cancer and its treatment [59]. These outcomes have prompted efforts to prevent ovarian damage and the associated health consequences [60,61,62]. Current fertility-preservation approaches and unmet needs in oncofertility Most established fertility-preservation strategies rely on surgical interventions such as oocyte or embryo cryopreservation, ovarian tissue cryopreservation and transplantation, and oophoropexy before pelvic irradiation [60, 63]. While effective for many patients, these approaches often require time-sensitive coordination and, in some cases, may delay initiation of cancer therapy; importantly, they preserve gametes or tissue before gonadotoxic exposure rather than preventing ovarian damage during cytotoxic treatment, and they do not guarantee recovery of fertility or endocrine function [60, 64, 65]. Access also remains uneven, with barriers including lack of insurance coverage, limited provider awareness, and geographic inaccessibility [66]. Oocyte cryopreservation, or egg freezing, enables women to safeguard reproductive potential before gonadotoxic therapy or for elective fertility preservation. It involves controlled ovarian stimulation, ultrasound-guided transvaginal oocyte retrieval, and vitrification, a rapid-freezing technique that has markedly improved outcomes compared with earlier slow-freeze methods [27]. Since its recognition as not experimental in 2012, it has become the preferred option for many postpubertal girls [67]. For patients who wish to preserve embryos rather than unfertilized oocytes, the retrieved oocytes can be fertilized with partner or donor sperm via conventional insemination or intracytoplasmic sperm injection (ICSI), followed by culture and vitrification of resulting embryos (embryo cryopreservation), providing an additional established pathway for fertility preservation [60, 68]. Ovarian tissue cryopreservation (OTC) is the only established fertility-preservation option for prepubertal girls and for some women in whom stimulation is not feasible, allowing later restoration of reproductive and endocrine function after autotransplantation. The procedure entails removal of one ovary and freezing of the ovarian cortex, which contains most primordial follicles, followed by cryopreservation by slow-freezing or vitrification until autotransplantation [69]. Many patients experience recovery of menses, normalization of gonadotropins, and pregnancies after transplantation. In a large multicenter European series of 285 women, resumption of endocrine function occurred in 88.7% of those with premature ovarian insufficiency, ranging from 77.6 to 97.2% across centers [70], although long-term graft survival depends on age and follicle density at the time of tissue collection [69, 71]. Key limitations of OTC include the risk of reintroducing malignant cells, particularly in cases of leukemia and hematologic malignancies [60, 72, 73], and rapid post-transplant primordial follicle loss, attributed to ischemia-related damage, delayed revascularization, and an initial absence of AMH leading to accelerated recruitment of dormant follicles [74,75,76]. In xenograft models of human ovarian cortex transplanted into ovariectomized nude mice, peri-transplant administration of recombinant AMH (rAMH) attenuated this accelerated recruitment, preserved primordial follicle numbers, and helped maintain ovarian reserve, highlighting a potential adjunctive role for rAMH in OTC protocols [74]. Gonadotropin-releasing hormone (GnRH) agonists are currently the only widely accepted medical strategy for fertility preservation during chemotherapy. By suppressing pituitary gonadotropins, they induce a reversible hypoestrogenic, prepubertal-like milieu that appears to render follicles less vulnerable to chemotherapy-induced damage [77,78,79]. Randomized trials and meta-analyses, including a Cochrane review by Chen et al. in 2019, suggest that GnRH agonists can decrease the risk of chemotherapy-induced POI and improve rates of post-treatment menstruation and pregnancy, particularly in women receiving cyclophosphamide-based regimens for breast cancer and autoimmune disease [60, 78, 80,81,82,83,84]. However, these agents induce profound, reversible hypoestrogenism with associated side effects, and animal data suggest they offer only partial and short-term protection, potentially mediated by supporting AMH production by secondary and early tertiary follicles rather than directly preventing primordial follicle activation [1, 85,86,87]. Therefore, GnRH agonists are best viewed as complementary measures rather than stand-alone strategies, emphasizing the need for ovary-focused, mechanism-based therapies, such as rAMH, that can restrain follicle recruitment during gonadotoxic exposure. Several other pharmacologic strategies have been evaluated to mitigate chemotherapy-induced ovarian damage. Non-hormonal approaches such as melatonin and metformin have demonstrated antioxidative and cytoprotective effects in animal studies [88,89,90,91]. Similarly, tamoxifen, antioxidant compounds (tocotrienol, quercetin, resveratrol), mTORC1/2 inhibitors (e.g., rapamycin), silibinin, chlorogenic acid, and sphingosine-1-phosphate have shown promise in preclinical models for preserving follicle viability [92,93,94,95,96,97,98,99,100]. Biological modulators such as exosomal miR-10a, ghrelin, and statins also influence follicular survival and cellular stress-response pathways [101, 102]. Luteinizing hormone (LH) enhances DNA repair responses and anti-apoptotic signaling [103,104,105,106]. Although these pharmacological therapies have shown at least partial efficacy in preclinical studies, they may raise concerns about interference with cancer treatment, are not universally accessible, and do not directly modulate the biological pathways driving accelerated primordial follicle depletion [91, 107]. By suppressing primordial follicle recruitment, AMH may represent an effective single-agent strategy to preserve ovarian follicle reserve during gonadotoxic therapy. To contextualize studies that have employed AMH as a therapeutic agent, it is essential to first understand its physiological role. AMH and regulation of ovarian reserve AMH, also known as Müllerian-inhibiting substance (MIS), is a glycoprotein member of the transforming growth factor-β (TGF-β) family that plays sex-specific roles in testicular and ovarian development [108,109,110]. AMH is encoded by the AMH gene located on chromosome 19p13.3, and its protein product signals via the AMH type II receptor (AMHR2), encoded by a gene on chromosome 12q13.13, together with a type I receptor—predominantly BMPR-IA/ALK3 in granulosa cells—which is essential for transmitting AMH-induced SMAD1/SMAD5 activation and downstream target-gene regulation (e.g., Id3) in a BMP-like signaling pathway distinct from other TGF-β family members [108, 109, 111, 112]. AMHR2 is a single-pass transmembrane serine/threonine kinase whose extracellular domain adopts a distinctive three-finger fold, conferring high ligand specificity within the AMH-AMHR2 axis (Fig. 3) [112,113,114]. Upon ligand binding, AMHR2 recruits a type I receptor to form a heteromeric signaling complex, leading to phosphorylation of SMAD1, SMAD5, and SMAD8 (the protein product of the SMAD9 gene), which associate with SMAD4 and translocate to the nucleus to regulate target-gene transcription [109, 115,116,117]; this canonical SMAD pathway is the principal signaling route in ovarian and other reproductive tissues, although AMH can also activate non-canonical cascades such as MAPK or PI3K/AKT in a context-dependent manner [118]. Unlike other TGF-β ligands, AMH does not require co-receptors [116], a property that emphasizes the therapeutic potential of selectively targeting this pathway [113, 114]. Upon binding AMHR2, AMH induces a conformational change in the receptor, activating AMHR1 (TGF-β receptors ALK2/ALK3/ALK6), eventually triggering the SMAD1/5/8 signaling pathways to regulate folliculogenesis in a stage-specific manner [119,120,121]. SMAD1/5/8 converge on the common mediator SMAD4 for nuclear translocation and target-gene regulation [119, 120]. In the ovary, oocyte-derived GDF-9 and BMP15, granulosa cell–derived activins, and theca- and stroma-derived BMPs collectively shape follicle growth, granulosa-cell differentiation, and the balance between quiescence and activation [119, 122, 123]. Notably, AMH opposes BMP-driven follicle activation at the primordial-to-primary transition despite utilizing the same SMAD1/5/8 pathway, highlighting ligand-specific effects within a shared signaling framework [119]. Collectively, these pathways position AMH within a coordinated TGF-β network governing follicle recruitment and ovarian reserve dynamics. AMHR2 is expressed in ovarian granulosa cells, as well as in reproductive tissues such as the mammary gland, endometrium and myometrium [124]. Beyond reproductive organs, AMHR2 expression has been identified in specific neuronal and glial populations within the central nervous system, notably in pituitary gonadotropes and hypothalamic GnRH neurons and related regions [125,126,127]. Specifically, AMHR2 is present in the hypothalamic olfactory region and median eminence, where AMH can modulate GnRH neuron migration, electrical activity, and hormone secretion, supporting broader regulatory roles across the hypothalamic-pituitary–gonadal axis [126,127,128,129]. Expression of AMH and AMHR2 is regulated by a complex interplay of transcriptional, epigenetic, post-transcriptional, and hormonal mechanisms. Key transcription factors include SRY-related HMG-box gene 9 (SOX9), steroidogenic factor-1 (SF1), Wilms tumor 1 (WT1), and GATA-binding protein 4 (GATA4), which are primarily involved in testicular AMH regulation, while forkhead box L2 (FOXL2) and steroidogenic factor-1 (SF1) are the key transcription factors in granulosa cells [130, 131]. Epigenetic modifications, such as histone changes and DNA methylation, further modulate promoter activity [132], while non-coding RNAs provide additional post-transcriptional regulation [133, 134]. Finally, pituitary hormones (FSH, LH) and oocyte-derived factors (GDF-9, BMP15), together with estradiol and activins, regulate AMH and AMHR2 expression through interconnected and partly opposing/counteracting mechanisms that link oocyte-granulosa communication to follicular maturation and control of the ovarian reserve. GDF-9 and BMP15 synergistically stimulate AMH expression, and BMP15 upregulates AMHR2, whereas FSH and activin enhance granulosa-cell differentiation and estradiol synthesis, and estradiol predominantly represses AMH transcription via ERβ in growing follicles, contributing to the decline in AMH expression as follicles approach selection [109, 132, 135]. Pathogenic variants in the AMH and AMHR2, as well as related signaling components such as BMP15, have been associated with inter-individual differences in ovarian reserve, susceptibility to ovarian dysfunction, including POI, and timing of natural menopause, supporting a genetic contribution to the regulation of follicle recruitment [136,137,138]. In females, AMH is secreted exclusively by granulosa cells of growing ovarian follicles [1, 109, 139,140,141]. AMH production begins in the second half of fetal life, with molecular and cellular studies suggesting onset as early as the 23rd week of gestation, though immunohistochemical detection in human ovarian tissue has been confirmed only from 36 weeks of gestation [140, 142, 143]. At birth, AMH shows higher cord-blood concentrations in female infants born to mothers with PCOS, denoting a unique production pattern in utero that will be perpetuated throughout the reproductive life [144]. After birth, AMH expression persists throughout the reproductive years, with the highest concentrations in preantral and small antral follicles; follicles measuring approximately 5–8 mm in diameter contribute the majority of circulating AMH, and both mural and cumulus granulosa cells participate in its synthesis [145,146,147]. AMH expression declines sharply in follicles larger than about 8 mm, coinciding with enhanced FSH-dependent growth and rising estradiol [145, 147]. Serum AMH levels show modest cyclical variation during the menstrual cycle, with slightly higher levels in the mid-follicular phase and a decrease around ovulation and in the luteal phase, but these fluctuations are not large enough to affect clinical interpretation [148, 149]. Serum AMH declines progressively with age, reflecting the gradual reduction in the follicle pool, and becomes undetectable after menopause [141, 150,151,152]. Rationale and preclinical evidence for AMH as a medical therapeutic agent The biological rationale for using AMH as a pharmacological agent to protect the ovarian follicle reserve was established by a series of foundational in vivo and in vitro studies. Durlinger et al. first demonstrated in AMH-null knockout mice that absence of AMH resulted in significantly more preantral and small antral follicles at 25days and 4 months of age, and almost complete depletion of follicles by 13 months, establishing that AMH prevents primordial follicle activation and regulates the rate of ovarian reserve exhaustion in vivo [153]. The intermediate phenotype observed in heterozygous AMH-null females further demonstrated a dose-dependent relationship between AMH signaling and follicle pool preservation. The same group showed that acute exogenous AMH administration to neonatal ovary reduced primordial follicle activation without altering AMHR2 expression of GDF-9 and zona pellucida glycoprotein 3, thus supporting a selective inhibitory effect at the earlier stages of folliculogenesis [154]. Preservation of GDF-9 expression indicates that AMH does not disrupt the oocyte-granulosa paracrine signaling required for subsequent follicular growth and maturation [155]. Building on these findings, Visser and Themmen proposed that AMH regulates folliculogenesis at two key developmental points by restraining the activation of primordial follicles and reducing granulosa cell sensitivity to FSH at later stages, thereby limiting follicle growth and dominant follicle selection [156]. Compelling evidence from preclinical studies has highlighted rAMH as a promising therapeutic agent for protecting ovarian follicles from chemotherapy-related damage [50, 53, 74, 86, 87, 157,158,159]. In human ovarian cortex xenografts and isolated human luteinized granulosa cells, rAMH reduced expression of endogenous AMH and inhibin B, as well as key granulosa cell receptors involved in follicular development, including AMHR2, FSH-R, LH-R, and IGF1-R1, consistent with a quiescence-promoting, anti-proliferative effect [74, 87]. In the human ovarian cortex, rAMH treatment suppressed steroid hormone production, reduced granulosa cell proliferation and apoptosis, and modulated stemness-related features [74, 160] while altering BMP15 and GDF-9, which regulate granulosa cell proliferation and differentiation and follicle growth [86, 87]. rAMH further influenced epigenetic regulation by modulating microRNAs (miRNAs), including upregulation of miR-181a1, miR-181a2, and miR-b181b2, and downregulation of miR-181b1, highlighting a multilayered control of granulosa cell function [86, 161]. Importantly, rAMH preserved the primordial follicle pool by preventing the transition from primordial to primary and secondary stages in both in vitro human and in vivo animal models [74, 157], even though the initial hypoxia-driven follicle loss after xenotransplantation was not prevented [157]. In the same ovarian cortex xenograft model, rAMH downregulated BRCA1 (but not BRCA2) expression, suggesting a role in double-strand DNA repair processes during graft revascularization and further supporting a contribution to maintenance of genomic integrity in transplanted tissue [162]. This data suggests AMH’s functional scope beyond follicular recruitment to DNA damage responses; however, it is possible that different rAMH exposure time and dosing could elicit different effects. Other murine models of chemotherapy exposure confirmed that rAMH potently suppresses primordial follicle activation and preserves the dormant follicle pool during gonadotoxic therapy [50, 53, 159, 163]. Kano et al. developed both adeno-associated virus (AAV9-MIS) gene therapy vector and recombinant human MIS protein (rhMIS) to achieve sustained supraphysiologic AMH levels in vivo. Elevated circulating AMH halted folliculogenesis by blocking primordial follicle recruitment, maintained the ovarian reserve during chemotherapy, and induced reversible ovarian quiescence with temporary infertility but no follicle depletion, with normal follicle development resuming after discontinuation of rhMIS treatment or ovarian cortex transplantation of AAV9-MIS mice into untreated hosts [159]. Even though endogenous production of AAV9-MIS cannot be stopped, these findings demonstrate AMH’s dual function as a reversible contraceptive and an ovarian-protective agent [159]. Roness et al. showed that preemptive rAMH administration prevented chemotherapy-induced primordial follicle “burn-out” and preserved long-term fertility after cyclophosphamide exposure, without compromising the anti-tumor efficacy of chemotherapy, supporting ovarian specificity and oncologic safety [53]. The authors highlighted that rAMH has a relatively short half-life and required high doses, which may pose a potential limitation for its future clinical application. Sonigo et al. provided additional mechanistic evidence that rAMH prevents cyclophosphamide-induced follicle loss by inhibiting PI3K/AKT/FOXO3 signaling: in pubertal mice, rAMH co-treatment preserved primordial follicles, normalized estrous cyclicity and ovulation rates, and maintained fertility during chemotherapy, with reduced phosphorylation of FOXO3, and a possible stimulation of autophagy for cell recycling and follicular protection, although the extent of this phenomenon and its direct activation were not conclusively demonstrated [50]. Recent work by Nguyen et al. has added new mechanistic insights into rAMH protective effects in a doxorubicin-treated mouse model. rAMH attenuated DNA damage and apoptotic pathways by reducing activation of tumor p53 target genes, downregulated Wingless-type MMTV integration site family 6 (Wnt6) signaling in granulosa cells to prevent activation, and increased Inhibitor of DNA binding/differentiation 3 (Id3) expression to support DNA repair and cell-cycle control in mouse prepubertal ovaries [163]. By stalling theca-cell differentiation and cell-cycle progression and promoting follicle survival, rAMH preserved the primordial follicle pool and sustained DNA integrity, with significantly improved fertility outcomes over six months [163]. Campbell et al. [164] found that in sheep, a monovulatory species like the human, AMH did not inhibit primordial follicle recruitment; however, AMH knockdown by active immunization significantly increased in both the number of gonadotropin-dependent antral follicles and ovulation rate, confirming that AMH exerts a net inhibitory role at the gonadotropin-responsive phase even in this species [164]. Other studies have reported mixed effects of AMH on follicle growth or recruitment. McGee et al. showed that MIS/AMH promoted growth of already-growing rat preantral follicles in an FSH-stimulated system, increasing follicle expansion without inducing cell differentiation or apoptosis [165]. Schmidt et al. [166] reported that high doses of rAMH enhanced the proportion of human primordial follicles advancing to primary and secondary stages in vitro; however, it also produced a pro-survival effect for the small growing follicles preventing them from undergoing atresia [166]. Furthermore, work in an AMH-overexpressing mouse model indicated that chronic moderately elevated AMH levels can impair preantral follicle survival [167]. These mixed stimulating/inhibitory effects likely reflect the inherent complexity of AMH signaling at doses and different follicle stages, animal species, and the specific hormonal environment, particularly FSH concentrations, all of which can modulate the direction and magnitude of AMH’s actions on granulosa cells. Overall, these preclinical studies show that elevated AMH levels, achieved through recombinant protein delivery or gene therapy, can reversibly suppress primordial follicle activation, preserve ovarian reserve, and maintain fertility potential during chemotherapy without compromising anti-cancer efficacy [50, 53, 159, 163]. rAMH’s protective effects are dose-dependent: low doses partially suppress activation, whereas robust preservation of the follicle pool requires sustained supraphysiological levels achieved through repeated dosing or infusion [50, 53, 86, 168], and very high doses may transiently suppress folliculogenesis, an effect that appears reversible after withdrawal [50, 159]. Limitations of the current evidence include reliance on animal and xenograft models, a relatively short in vivo half-life requiring high or repeated doses, incomplete characterization of potential off-target effects, particularly in AMHR2-expressing non-ovarian tissues, and uncertainty regarding optimal human dosing and timing relative to chemotherapy. Nonetheless, the breadth and consistency of these data support rAMH as a mechanism-driven, ovary-focused, candidate agent with potential to transform fertility preservation during gonadotoxic therapy [50, 53, 157, 159, 163]. Recombinant AMH production and delivery Recombinant AMH (rAMH) production was first established in the early 1990s, with mammalian expression systems favored because they provide appropriate protein folding and glycosylation required for full biological activity [169,170,171]. After translation, AMH is synthesized as a homodimeric glycoprotein precursor comprising an N-terminal prodomain, which mediates dimerization, proper folding, and secretion, and a C-terminal domain responsible for receptor binding and signaling [114, 172]. The prodomain remains associated with the mature dimer during secretion, stabilizing the circulating hormone until receptor engagement [112, 114]; these structural features explain why pro-mature isoforms produced in mammalian systems most closely recapitulate native AMH activity and inform current rAMH manufacturing strategies [170, 173]. However, large-scale manufacturing remains technically challenging due to the protein’s complex tertiary structure and relatively low expression yields; in contrast, bacterial and yeast systems tend to produce unstable or biologically inactive forms [169, 170]. These represent significant economic barriers to clinical translation. rAMH also presents pharmacokinetic and delivery challenges. In early studies, exogenous AMH displayed a short serum half-life of 27.6 ± 0.8 h, with complete elimination within approximately 1 week after injection, requiring frequent administration to maintain therapeutic levels [174]. Roness et al. further demonstrated that the in vivo half-life of exogenous rAMH at the ovarian tissue level is considerably shorter: biotin-labeled rAMH administered intraperitoneally was detectable in murine ovarian granulosa cells up to 7 h after injection but was no longer evident by 17 h, and the pharmacodynamic window of SMAD1/5/8 phosphorylation was observed between 3 and 6 h post-injection [53]. This short functional window necessitates frequent administration to maintain supraphysiologic, therapeutically active concentrations at the ovarian tissue target [174]. Dose-finding preclinical animal studies addressed this limitation through several strategies, including serial intraperitoneal injections every 6 h throughout the chemotherapy exposure window, subcutaneous twice-daily dosing at 1.5 mg/kg, intraperitoneally implanted osmotic pumps for continuous infusion, and adeno-associated virus serotype 9 (AAV9) gene therapy vectors delivering the optimized LR-MIS transgene to the liver, muscle, and pancreas for sustained systemic expression over many months [50, 53, 157, 159]. In mice, AAV9-MIS doses of ≥ 1 × 1011 particles per animal induced sustained AMH levels of approximately 0.2–2.7 µg/mL, levels an order of magnitude or more above those associated with physiological AMH activity and above the threshold associated with near-complete arrest of folliculogenesis [159]. Feline gene therapy studies extend these observations across species. Prepubertal female cats treated with AAV9-fcMISv2 exhibited sustained supraphysiologic AMH levels that peaked at 10–48 µg/mL and remained > 1 µg/mL for more than 3 years (> 200-fold above physiological adult concentrations), with resulting anovulation, reduced estrogen and progesterone, mild hypergonadotropic hypogonadism, and durable sterilization without overt systemic toxicity [175]. Conversely, adult cats receiving AAVrh91 vectors encoding more potent AMH analogs reached similarly high or higher circulating AMH (approximately 103-fold above baseline), developed progressive decreased antral follicle numbers, and mid-to-late-gestation pregnancy loss despite preserved ovulation of rare dominant follicles [176]. Together, these studies provide proof that the exposure levels required to suppress ovarian function remain far above physiological concentrations. While gene therapy delivery removes the need for repeated protein dosing and bypasses the pharmacokinetic limitations of the recombinant protein, it introduces its own regulatory, safety, and reversibility considerations that must be addressed before clinical use. To date, no human clinical trials have evaluated rAMH administration in a peri-chemotherapy setting, leaving key questions unanswered regarding optimal dosing, route, and schedule of delivery, reversibility of effects, long-term safety, and appropriate patient selection. Early-phase trials will need to systematically assess pharmacokinetics, pharmacodynamics, and safety, including potential off-target effects related to AMHR2 expression in non-ovarian tissues such as the endometrium, myometrium, pituitary, and hypothalamus. Careful dose escalation, integration with existing fertility-preservation strategies, and long-term follow-up for reproductive and oncologic outcomes will be essential steps in translating rAMH therapy from preclinical proof-of-concept to clinical practice [177]. In summary, the development of rAMH as a therapeutic agent faces several intertwined technical and economic barriers. The requirement for glycosylation-competent mammalian expression systems, low expression yields, and complex purification of a dimeric glycoprotein all contribute to high per-milligram production costs and limited availability of clinical-grade material. In parallel, the short in vivo half-life of current preparations, the need for intensive dosing or device-based delivery in preclinical models, and the absence of human pharmacokinetic, dosing, and safety data, particularly regarding AMHR2-expressing non-ovarian tissues, create substantial hurdles for translation into routine fertility-preservation practice. Careful dose escalation, integration with existing fertility-preservation strategies, and long-term follow-up for reproductive and oncologic outcomes will be essential steps in translating rAMH therapy from preclinical proof-of-concept to clinical practice [177]. Future directions and translational pathway At present, no clinical trials have evaluated peri-chemotherapy administration of rAMH in humans. To date, all studies investigating rAMH for fertility preservation during chemotherapy have been conducted in murine models using recombinant protein, osmotic pumps, or gene therapy vectors to maintain supraphysiologic AMH levels [50, 53, 157, 159, 163]. Off-target effects have not been reported in these models; however, the broad expression of AMHR2 in reproductive tissues and along the hypothalamic-pituitary axis necessitates careful evaluation of potential systemic effects and long-term safety in future human trials. Ex-vivo and xenograft studies using human ovarian tissue have focused primarily on feasibility, safety monitoring, and biomarker endpoints, including serum and intra-ovarian AMH, follicle counts, and granulosa-cell receptor expression [74, 157, 160, 178]. Nevertheless, the robust protection of the ovarian reserve observed in murine chemotherapy models positions rAMH as a potential breakthrough therapeutic strategy for fertility preservation in cancer patients and individuals at risk of accelerated ovarian reserve depletion [50, 86, 159, 163, 178, 179]. Beyond the chemotherapy setting, rAMH may hold therapeutic relevance in additional contexts, although the strength of supporting evidence varies considerably across these applications. AMH and AMHR2 are expressed in both normal endometrium and endometriotic lesions, and several preclinical studies suggest that exogenous AMH may help limit lesion growth by modulating cellular proliferation, inducing cell-cycle arrest, and enhancing apoptosis, in part through downregulation of the transcription factor E2F1 and aromatase activity [180]. Experimental models demonstrate reductions in endometriosis lesion size and cellularity with AMH treatment, and by transiently arresting folliculogenesis, rAMH may also reduce estrogen production while preserving the primordial follicle pool and fertility, although clinical confirmation is needed [180]. Similar principles may extend to women with chronic pelvic inflammatory conditions associated with autoimmune or metabolic disorders, infections, environmental toxins, or allergens, in whom rAMH could theoretically prevent or mitigate cytokine-driven follicular apoptosis, depletion, and premature primordial follicle activation [35, 38, 181]. This remains speculative, as no study has directly tested whether exogenous rAMH attenuates inflammation-associated follicle loss in these contexts. Emerging evidence also supports AMH-based approaches to extend reproductive lifespan, as prolonged AMH administration delays primordial follicle recruitment and can postpone menopause in animal models [152, 159, 179]. In fact, women with PCOS, who typically exhibit elevated AMH levels, further support this concept, as they tend to experience delayed menopause [33, 182]. Interestingly, AMH antagonists are being investigated as a potential therapeutic intervention to restore ovulation in women with PCOS [183,184,185], emphasizing the bidirectional therapeutic potential of modulating AMH/AMHR2 signaling across the reproductive lifespan. Despite this supporting biological plausibility of reproductive lifespan extension with rAMH, it does not constitute interventional evidence, as no preclinical or clinical study has demonstrated that pharmacological rAMH administration measurably postpones menopause in any species [186]. Translation of rAMH into clinical practice will require carefully designed phase I/II trials in well-defined high-risk populations, such as adolescents and young adults receiving highly gonadotoxic regimens after completion of standard fertility-preservation procedures, or patients undergoing ovarian tissue transplantation. Initial trials should prioritize safety, reversibility, and pharmacokinetics/pharmacodynamics, with primary endpoints including adverse events, serum AMH profiles, gonadotropin levels, antral follicle count, and menstrual cyclicity, and exploratory endpoints assessing ovarian volume, biomarkers of DNA damage and repair, and short-term fertility outcomes where feasible. Long-term follow-up will be essential to evaluate spontaneous conception, time to menopause, and other outcomes, and to define how rAMH should be combined with established fertility-preservation strategies such as GnRH agonists, oocyte and embryo cryopreservation, and ovarian tissue cryopreservation.

AMH exhibits robust preclinical efficacy in upholding follicle quiescence, preventing chemotherapy-induced primordial follicle activation, and preserving long-term fertility in animal and human xenograft models. As a mechanism-based, ovary-focused agent, AMH holds substantial promise for individuals at risk of ovarian reserve depletion due to iatrogenic causes such as chemotherapy. Beyond the chemotherapy setting, experimental data support its potential application in endometriosis, while its possible roles in cytokine-driven follicle loss and reproductive lifespan extension remain biologically plausible but speculative, warranting dedicated mechanistic and in vivo investigation. To translate these findings into clinical practice, future clinical trials must overcome challenges in large-scale production of bioactive pro-mature rAMH, optimize dosing and timing relative to gonadotoxic exposures, and develop practical delivery strategies that can sustain protective levels. Data availability No datasets were generated or analysed during the current study.

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The authors thank Bronwyn Sutherland, MLS, Research & Instruction Librarian at the Texas Medical Center Library, for her invaluable assistance with the literature search that supported this manuscript. Author information Authors and Affiliations Contributions Conceptualization: IPA, RZL, MS, LD Methodology: IPA, RZL, MS, LD Project administration: IPA, RZL, MS, LD Supervision: IPA, RZL, MS, LD, LMS, TLW, LJMK, JCH Writing- original draft: IPA, RZL, MS, LD Writing- review & editing: IPA, RZL, MS, LD, LMS, TLW, LJMK, JCH. Corresponding authors Ethics declarations Disclaimer Some figures have been made/modified using Biorender. AI and software Language editing for this manuscript was performed with the assistance of OpenAI’s ChatGPT, a language model designed for generating and refining text. All suggestions provided by the model have been proofread and verified by the authors. The use of this tool was for linguistic improvement only, and no generative content was created. No changes were made to the scientific content of the manuscript. Consent for publication Not applicable. Consent to participate Not applicable. Ethics approval Not applicable. Conflict of interest All authors declare that they have no competing interests related to this mini-review. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. About this article Cite this article Peregrin-Alvarez, I., Zemet, R., Woodard, T. et al. The role of anti-Müllerian hormone as a therapeutic agent to preserve the ovarian follicle pool during chemotherapy. J Assist Reprod Genet (2026). https://doi.org/10.1007/s10815-026-03941-y Received: Accepted: Published: Version of record: DOI: https://doi.org/10.1007/s10815-026-03941-y