Ovulation: A consequence of acute inflammation cultivated by E2-induced reactive oxygen species and triggered by progesterone withdrawal

Following the LH surge, ROS production in granulosa cells intensifies and becomes essential for the structural and functional changes required for ovulation [ 7 21 ]. E2 rapidly increases intracellular ROS levels, predominantly H 2 O 2 , through integrin-dependent mitochondrial signaling. These ROS promote cumulus expansion and oocyte release. Experimental studies show that antioxidants suppress LH-induced cumulus mucification and progesterone production, indicating the necessity of ROS for follicular remodeling [ 7 ]. Moreover, H 2 O 2 can mimic LH activity by inducing cumulus-oocyte complex expansion and activating key ovulatory signaling pathways, including EGFR phosphorylation and the p42/44 MAPK cascade, which contributes to the proliferation, differentiation, and enzyme synthesis in the granulosa cells and is necessary for the resumption of meiosis in oocytes [ 7 22 23 ]. Notably, this ROS generation occurs independently of ER activation and is mediated through anchorage-dependent mitochondrial mechanisms [ 5 ].

Before the LH surge, estrogen (E2) primes the ovarian follicle for ovulation by establishing a delicate balance between oxidative signaling and antioxidant defense [ 5 6 ]. E2 promotes the generation of mitochondrial ROS), particularly hydrogen peroxide (H 2 O 2 ), which acts as a critical signaling molecule for ovulatory events such as cumulus expansion and steroidogenesis [ 7 ]. This ROS production is mediated by NADPH oxidase 4 (NOX4) and is triggered through anchorage- and integrin-dependent signaling, independent of classical estrogen receptor (ER) activation [ 5 8 ]. ROS accumulation is enhanced when antioxidant systems like catalase are inhibited [ 5 9 ], supporting its role as a regulated signaling mediator. However, excessive ROS can induce oxidative damage, necessitating tight control mechanisms. To counteract potential oxidative stress, E2 concurrently activates ER-dependent transcription of antioxidant enzymes, including catalase (CAT), extracellular superoxide dismutase, and manganese superoxide dismutase [ 10 11 12 13 14 15 ]. These enzymes function to neutralize superoxide and convert H 2 O 2 into water, thus preserving cellular integrity. In breast cancer cells, E2 was shown to increase superoxide dismutase (SOD) 1 expression, preventing ROS-induced protein damage [ 15 ]. Moreover, E2 directly protects mitochondria, which express ERs and respond to E2 by upregulating antioxidant pathways [ 12 16 17 ]. Proteomic studies of human follicular fluid confirm the enrichment of antioxidant enzymes such as CAT, SOD1, SOD3, glutathione transferase, and protein disulfide isomerase [ 18 19 20 ]. These systems work in concert to regulate ROS levels and protect granulosa cells during follicular maturation. Thus, E2 establishes a physiological redox balance that supports both signaling and cellular protection in preparation for ovulation [ Figure 1 ]. Before LH surge, Follicle-stimulating hormone (FSH) regulates E2 and antioxidants with a redox balance in granulosa cells. FSH binds to its receptor (FSHR) in granulosa cells, activating the protein kinase A signaling pathway, which subsequently regulates β-catenin (CTNNB1). CTNNB1 promotes the expression of aromatase, an enzyme responsible for converting androgens into E2. E2 directly promotes ROS production, leading to the activation of the NF-κB signaling pathway. Concurrently, by binding to ER, E2 enhances antioxidant capacity, thus maintaining a balance between ROS and antioxidants to ensure cellular stability and function

Advances in the molecular mechanism of ovulation shown above have led to multiple clinical implications, ranging from fertility, sterility, postoperative adhesions, and cancer formation. In addition, recognition of the inflammatory and oxidative nature of follicular rupture has unveiled new challenges in contraceptives, ovulation induction, and cancer prevention. The detailed understanding of ovulation mechanisms has led to the development of pharmacological agents that either induce or suppress ovulation, depending on clinical needs. These agents target key pathways involved in follicular development, luteinization, and ovulation. Pharmacologic agents designed to induce ovulation are critical tools in the management of anovulatory infertility. Clomiphene citrate, a selective estrogen receptor modulator (SERM), acts by competitively inhibiting estrogen feedback at the hypothalamus and pituitary, thereby enhancing the release of FSH and LH to stimulate follicular growth. Alternatively, letrozole, an aromatase inhibitor, suppresses estrogen biosynthesis, resulting in a similar upregulation of endogenous gonadotropins that promotes follicular recruitment and maturation [ 51 ]. In addition, exogenous gonadotropins – typically recombinant FSH and LH – are administered directly to stimulate multifollicular development and trigger ovulation, often as part of assisted reproductive technology (ART) protocols [ 52 ]. These pharmacological strategies target distinct checkpoints within the hypothalamic–pituitary–ovarian axis to optimize folliculogenesis and oocyte release in women with ovulatory dysfunction. (1) Combined oral contraceptives (COCs): COCs contain synthetic E2 and progestin. It is the progestin component in COCs that suppress ovulation by inhibiting the hypothalamic–pituitary–ovarian axis or P4 withdrawal. The former prevents follicular development and the LH surge, the latter prevents follicle rupture [ 53 ] (2) Morning-after pills: Progestins such as levonorgestrel and selective progesterone receptor modulators (SPRMs) such as ulipristal acetate are used to prevent pregnancy after unprotected intercourse. Progestins work primarily by avoiding progesterone withdrawal, thus delaying or inhibiting ovulation. Similarly, SPRMs block progesterone receptors, preventing progesterone effect after LH surge and follicular rupture [ 53 ]. These agents are most effective when taken within 72 h (levonorgestrel) or 120 h (ulipristal acetate) after intercourse. The progesterone receptor antagonist (mifepristone or RU486) is even more effective by competitively binding to the progesterone receptors. When administered prior to ovulation, it delays or blocks the LH surge, preventing the follicular rupture. When taken in the peri-ovulatory or post-ovulatory phase, it inhibits the decidualization of the endometrium, thus reducing the receptivity of the embryo. Compared to other emergency contraceptives, such as levonorgestrel, mifepristone demonstrates a broader window of efficacy and a more potent suppression of ovulatory and endometrial processes, making it a valuable tool in reproductive medicine for post-coital pregnancy prevention (3) COX-2 inhibitors as an alternative emergent contraceptive and their fertility risk. (1) Combined oral contraceptives (COCs): COCs contain synthetic E2 and progestin. It is the progestin component in COCs that suppress ovulation by inhibiting the hypothalamic–pituitary–ovarian axis or P4 withdrawal. The former prevents follicular development and the LH surge, the latter prevents follicle rupture [ 53 ] (2) Morning-after pills: Progestins such as levonorgestrel and selective progesterone receptor modulators (SPRMs) such as ulipristal acetate are used to prevent pregnancy after unprotected intercourse. Progestins work primarily by avoiding progesterone withdrawal, thus delaying or inhibiting ovulation. Similarly, SPRMs block progesterone receptors, preventing progesterone effect after LH surge and follicular rupture [ 53 ]. These agents are most effective when taken within 72 h (levonorgestrel) or 120 h (ulipristal acetate) after intercourse. The progesterone receptor antagonist (mifepristone or RU486) is even more effective by competitively binding to the progesterone receptors. When administered prior to ovulation, it delays or blocks the LH surge, preventing the follicular rupture. When taken in the peri-ovulatory or post-ovulatory phase, it inhibits the decidualization of the endometrium, thus reducing the receptivity of the embryo. Compared to other emergency contraceptives, such as levonorgestrel, mifepristone demonstrates a broader window of efficacy and a more potent suppression of ovulatory and endometrial processes, making it a valuable tool in reproductive medicine for post-coital pregnancy prevention (3) COX-2 inhibitors as an alternative emergent contraceptive and their fertility risk. COX-2 enzymes play a critical role in ovulation by mediating prostaglandin synthesis, which is essential for follicular rupture. Studies have demonstrated that COX-2 inhibitors can serve as safe, nonhormonal emergency contraceptives, effectively delaying ovulation by up to 5 days [ 54 55 56 ]. However, their efficacy as regular oral contraceptives appears suboptimal. A clinical trial evaluating meloxicam (even at high doses of 30 mg daily) found that ovulation still occurred in over 20% of cycles, limiting its reliability for routine contraception [ 57 ]. Conversely, COX-2 inhibitors pose a significant fertility risk for women attempting to conceive. By disrupting prostaglandin-dependent ovulation, these drugs can cause delayed or failed follicular rupture, leading to luteinized unruptured follicle (LUF) syndrome – a condition in which the follicle fails to release the oocyte despite CL formation [ 58 ]. Given these effects, women planning pregnancy should avoid COX-2 inhibitors to prevent unintended ovulation suppression and maximize their chances of conception [ 59 ]. Follicular fluid, the microenvironment surrounding the oocyte, contains a high level of ROS and a rich array of growth factors, cytokines, and coagulation proteins that play critical roles in ovulation and wound healing. Notably, repeated ovulatory cycles expose ovarian tissues to chronic oxidative stress, which has been implicated in DNA damage, genomic instability, and malignant transformation – key factors in the pathogenesis of ovarian cancer [ 60 61 ]. Epidemiological studies suggest that incessant ovulation may increase ovarian cancer risk [ 62 63 ], further supporting the hypothesis that ROS-mediated ovulatory mechanisms could contribute to tumor initiation. Recent studies have also revealed that follicular fluid can also contribute to postoperative intra-abdominal adhesions when it comes into contact with surgical wounds [ 47 ]. Follicular fluid, the microenvironment surrounding the oocyte, contains a high level of ROS and a rich array of growth factors, cytokines, and coagulation proteins that play critical roles in ovulation and wound healing. Notably, repeated ovulatory cycles expose the fimbrial epithelium of the fallopian tube to follicular fluid containing ROS, a known mutagen [ 64 65 ], and growth factor transforming agents such as IGF2, HGF, and EGF-like peptides [ 50 66 67 68 69 70 ]. The oncogenic potential of these ovulatory factors has been extensively demonstrated in vitro and in vivo in mouse and human systems [ 64 71 72 73 ]. Large-scale epidemiological studies have indicated that incessant ovulation increases ovarian cancer risk [ 62 ]. Consequently, ovulation suppression is considered one of the most effective strategies for ovarian cancer prevention [ 62 74 ]. FF is rich in coagulation cascade proteins (e.g., fibrinogen and thrombin) and growth factors (e.g., HGF [ 50 ], insulin-like growth factor 2 [IGF2] [ 66 ], and PDGF [ 75 ], which facilitate the four phases of wound healing: hemostasis, inflammation, proliferation, and remodeling [ 49 ]. For example, HGF promotes tissue repair and regeneration, whereas IGF2 supports stem cell proliferation and re-epithelialization. These factors ensure efficient healing of the ovulation wound, restoring ovarian surface integrity and preparing the tissue for subsequent cycles. While FF promotes the efficient healing of ovulation wounds, its spillage into the pelvic cavity during ovulation can have detrimental effects on surgical wounds. A 2024 study published in iScience [ 47 ] demonstrated that FF contains excessive coagulation and HGF signals, which, when exposed to surgical wounds, can lead to intra-abdominal adhesions. These adhesions are fibrous bands of scar tissue that form between abdominal tissues and organs, often causing chronic pain, bowel obstruction, and infertility. The study found that FF-induced activation of coagulation pathways and excessive HGF signaling promote fibroblast proliferation and ECM deposition, key processes in adhesion formation. This dual role of FF highlights its context-dependent effects: while beneficial for ovulation wound healing, it can be harmful when interacting with surgical wounds.

Studies in various animal models have demonstrated that progesterone levels exhibit a transient decline following the initial rise after the LH surge, reflecting the dynamic process of luteinization. In rabbit ovarian follicles, P4 levels rise within 1–3 h after human chorionic gonadotropin (hCG) administration, peaking around 3 h after mating and then decreasing [ 29 30 ]. In ruminants such as cattle and sheep, progesterone levels have been observed to fluctuate within 24–48 h postovulation. This transient decline is associated with the incomplete vascularization and steroidogenic enzyme activity during the early stages of luteal formation [ 31 32 ]. In rodent models, similar patterns have been reported, where progesterone secretion initially rises in response to the LH surge but temporarily decreases as luteal cells undergo functional maturation [ 3 ]. In vitro studies using granulosa cell cultures further support this phenomenon, showing that progesterone production may fluctuate due to the gradual upregulation of steroidogenic enzymes (e.g., 3β-HSD) and the dependence on sustained LH stimulation [ 33 ]. These findings highlight the transitional nature of luteinization, where the temporary decline in progesterone is a physiological response to the incomplete functional and structural maturation of the CL.

Ovulation concludes with follicular rupture, which is immediately followed by a tightly orchestrated process of tissue reorganization and wound healing. These events ensure not only the restoration of ovarian integrity but also the endocrine transition toward luteal function. Following follicle rupture, the remaining follicular cells undergo a highly regulated process of tissue reorganization, marked by the activation of promatrix factors such as pro-MMPs and the differentiation of granulosa and theca cells into luteal cells. This transformation leads to the formation of the CL, a transient endocrine structure essential for progesterone production. Progesterone is critical for maintaining endometrial receptivity and supporting early pregnancy. The CL also regulates pituitary gonadotropin secretion through negative feedback, ensuring hormonal balance during the luteal phase [ 3 43 ]. If implantation does not occur, the CL undergoes luteolysis, a process characterized by the infiltration of immune cells, increased oxidative stress, and the downregulation of steroidogenic enzymes. This regression allows the ovarian cycle to reset, enabling the development of a new cohort of follicles [ 3 43 ]. The healing of the ovulation wound resembles the process of ordinary wound healing, consisting of four overlapping phases: hemostasis, inflammation, proliferation, and remodeling [ 47 ]. The ovulatory follicular fluid (FF) is rich in coagulation cascade proteins and inflammatory cytokines which facilitate hemostasis and the inflammatory phase of wound healing [ 48 ]. In addition, FF contains abundant growth factors, such as hepatocyte growth factor (HGF), insulin-like growth factor 2 (IGF2), and platelet-derived growth factor (PDGF), which promote re-epithelialization during the proliferation phase [ 47 ]. Research has identified LGR5 + epithelial stem cells distributed throughout the ovarian surface epithelium (OSE), which likely respond to stemness and growth-promoting signals in FF to mediate postovulatory repair at the rupture site [ 49 ]. IGF2 has been demonstrated to promote stemness and clonal expansion (30852161), suggesting its importance in tissue regeneration. Following follicle rupture, thrombin activation converts the abundant HGF precursor into its active form [ 50 ], which may further stimulate re-epithelization at the wound site. Together, these stem cells and growth factors ensure efficient ovulation wound healing, maintaining ovarian integrity for subsequent reproductive cycles

The remodeling of the ovarian follicle ECM is a critical process for both ovulation and the subsequent vascularization of the CL. Under the influence of follicle-stimulating hormone (FSH), preovulatory follicles undergo rapid growth, forming a fluid-filled antral cavity. These follicles then become highly responsive to the mid-cycle LH surge, which coordinates oocyte maturation, cumulus expansion, follicular rupture, and luteinization [ 37 ]. At the LH surge peak, LH receptor activation on granulosa cells stimulates adenylyl cyclase, initiating the breakdown of the ECM across multiple follicular layers, including the theca interna, theca externa, and tunica albuginea. The degradation process is tightly regulated by MMPs and their inhibitors (TIMPs), produced by cumulus cells, oocytes, and surrounding stromal cells. These proteolytic activities are essential for weakening the follicular wall and enabling oocyte extrusion [ 38 39 40 41 ]. A key event in ovulation is the LH-induced mucification of the cumulus-oocyte complex (COC). In response to the LH surge, cumulus cells secrete large amounts of hyaluronic acid (HA), a glycosaminoglycan that forms a hydrated matrix around the oocyte. This process, known as cumulus expansion, is mediated by the upregulation of HA synthase 2 and the downregulation of HA-degrading enzymes. The accumulation of HA increases the hydrostatic pressure within the follicle, promoting rapid tissue remodeling and creating a pathway for the oocyte to exit the follicle. Cumulus expansion also ensures that the oocyte is surrounded by a protective matrix, which is crucial for its survival and fertilization [ 2 26 ]. Successful ovulation requires precise structural changes at the follicular apex, where a localized breach, known as the stigma, forms to release the oocyte. This process involves the breakdown of the ECM within the granulosa, theca, and ovarian surface epithelium (OSE) layers [ Figure 4 ]. The LH surge upregulates proteases such as MMPs and ADAMTS-1, which degrade collagen and other ECM components, weakening the follicular wall [ 40 41 ]. Concurrently, apoptosis of the OSE, mediated by pro-apoptotic factors like Fas ligand and caspase-3, creates a focal point for rupture [ 42 ]. Follicle apex decellularization and stigma formation before rupture (Modified from [ 44 ]). Representative electron micrographs showing the progressive changes in the follicle wall structure leading up to ovulation. (a) Before the LH surge: the follicle wall remains intact, with the ovarian surface epithelium (OSE), tunica albuginea (TA), theca externa (TE), theca interna (TI), basement lamina (BL), and granulosa cells (GC) visible. (b) 1–2 h before rupture: wall thinning and OSE disruption begin. (c) Just before rupture: ECM fragmentation facilitates oocyte release. Red arrows indicate rupture direction The stigma formation is further supported by localized inflammation and vascular changes. Leukocytes infiltrate the apical region, releasing additional proteases and ROS that enhance ECM degradation [ 43 ]. Increased hydrostatic pressure from peri-follicular vessel leakage, combined with enzymatic activity, culminates in follicular rupture [ 26 ]. These coordinated mechanisms ensure the precise and timely release of the oocyte during ovulation. Follicular rupture is initiated at the apex through coordinated ECM degradation and targeted cell death. The LH surge upregulates MMPs and ADAMTS-1, which disassemble collagen and proteoglycans at the granulosa, theca, and ovarian surface epithelium (OSE). Simultaneously, apoptosis of OSE cells – mediated by Fas ligand and caspase-3 – thins the apical region, forming a localized rupture site known as the stigma [ 40 41 42 ]. Leukocyte infiltration into this area amplifies inflammation by releasing cytokines, ROS, and proteolytic enzymes. This promotes further ECM breakdown and increases vascular permeability, which raises intrafollicular hydrostatic pressure. These combined processes culminate in the timely and site-specific rupture of the follicle, ensuring precise oocyte release [ 26 43 ]. While decellularization facilitates the formation of the apical rupture site, the remaining mural granulosa and theca cells undergo luteinization to form the CL. Granulosa cells cease proliferating, enlarge (hypertrophy), and accumulate lipid droplets that supply cholesterol for steroid hormone synthesis. The disruption of the granulosa cell basal lamina, coupled with the production of prostaglandins and vascular growth factors by granulosa and theca cells, attracts new capillary growth into the previously avascular granulosa cell layer. Stromal vessels invade the granulosa layer, forming an extensive capillary network that ensures the delivery of nutrients and hormones to the developing CL [ 2 3 ].

The LH surge plays a critical role in inducing luteinization and progesterone (P4) synthesis within the ovarian follicle. On binding to its G protein-coupled receptors on granulosa and theca cells, LH activates intracellular signaling pathways that elevate cyclic AMP (cAMP) levels. This, in turn, stimulates protein kinase A (PKA), leading to the upregulation of key steroidogenic enzymes, including cholesterol side-chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase (3β-HSD). These enzymatic changes drive the conversion of cholesterol into progesterone, marking the transition of theca and granulosa cells into luteal cells. The resulting increase in progesterone production is essential for preparing the endometrium for potential embryo implantation as well as the rupture of the follicle. P4 serves a vital anti-inflammatory role by mitigating the oxidative stress induced by E2-ROS accumulation within the follicle. By modulating redox-sensitive signaling cascades, P4 establishes a transiently controlled inflammatory milieu that supports ovulation without tissue damage. This dual role of LH-induced progesterone production – supporting both reproductive readiness and follicular homeostasis – highlights the hormone’s importance in ovarian physiology.

Ovulation is a highly coordinated biological process essential for female fertility, driven by the luteinizing hormone (LH) surge and a cascade of molecular events that lead to follicular rupture and oocyte release. This process shares key characteristics, including the generation of reactive oxygen species (ROS) by estrogen (E2), acute inflammation responses, immune cell recruitment, and tissue remodeling. Among them, E2 increases ROS generation in mitochondria. ROS plays a crucial role in regulating ovulatory signaling, such as steroidogenesis, cumulus expansion, and extracellular matrix (ECM) breakdown. Both ROS and antioxidants as well as E2 and progesterone (P4) work together to regulate ovulation. Imbalances between them can contribute to the development of female reproductive disorders and even cancers [ 1 ]. This review aims to explore two aspects of mechanisms involving ovulation, the mechanism of follicle maturation and rupture, and the mechanism of postovulatory wound repair, as well as the implications in the issues of fertility, sterility, and disease management. Understanding this dynamic molecular interplay before and after ovulation may offer new insights into fertility promotion, contraception, and the prevention and management of diseases and sequelae related to ovulation.

Ovulation is a hormonally regulated, inflammation-like process that represents a finely tuned interplay between estrogen-induced oxidative signaling and progesterone-mediated anti-inflammatory control. The LH surge initiates a cascade involving E2-driven ROS production, ECM remodeling, cumulus expansion, and transient P4 withdrawal, which collectively culminate in follicular rupture. Subsequent luteinization and ovulatory wound healing demonstrate the remarkable capacity of ovarian tissue for rapid regeneration, mediated by the coagulants, growth factors reside in the follicular fluid, and epithelial progenitor cells reside in the ovarian surface epithelium. Beyond its fundamental biological role, the ovulatory process carries significant clinical implications for reproductive medicine and women’s health. Our growing molecular understanding of ovulation has already yielded targeted treatments for both infertility and contraception. However, emerging research reveals potential risks associated with chronic ovulatory cycles – specifically, the cumulative effects of repeated inflammatory responses and tissue remodeling. These cyclical processes may contribute to several pathological conditions, including ovarian carcinogenesis, endometriosis development, and postoperative adhesion formation. The current study did not generate or analyze any data. There are no conflicts of interest.