Premature ovarian insufficiency (POI) is a common reproductive endocrine disorder affecting 1-5% of reproductive-aged women (1). POI is characterized by the early onset of ovarian dysfunction in women younger than the age of 40 years. It is often manifested by menstrual disorders like amenorrhea or oligomenorrhea, and hormone level disruption with increased follicle-stimulating hormone (FSH) whereas decreased estradiol (E₂) (1). Clinically, POI is regarded as a progressive state of ovarian insufficiency (2). It usually involves three consecutive stages from occult, and biochemical or precursor to overt ovarian insufficiency (2, 3). Ultimately, POI can lead to ovarian failure, female infertility, osteoporosis, cardiovascular disease and even neurodegenerative disorder (1). The known causes of POI involve a variety of aspects, such as genetic, metabolic, autoimmune, environmental pollution and iatrogenic factors. And yet a proportion of cases are idiopathic in origin (1). To date, there is, however, no therapy available to cure the POI patients (4). Moreover, the markers for diagnosis and prediction of POI, particularly for cases at early stage of POI, remain limited (3, 4). As such, the development of novel markers aimed at predicting and monitoring ovarian function in POI remains an alluring prospect.

While the pathogenesis of POI is not fully elucidated, it has been reported to be associated with a sterile chronic inflammation within the ovary (5–9). Along with POI arising from certain etiologies, like autoimmune, genetic, metabolic, and iatrogenic factors, both human and animal studies reveal that reactive oxygen species (ROS) accumulates, leading to excessive oxidative stress in ovaries (10–12). Subsequently, ROS triggers nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome activation, which boosts an excessive production of multiple pro-inflammatory cytokines, including IL-1β and IL-18 (13–15). Recent studies using mouse POI models indicate that over the course of POI, the levels of pro-inflammatory cytokines, involving IL-1α/β, IL-6 and TNF-α, were remarkably increased in both serum and ovary (16). In turn, these cytokines adversely affect follicle reserve and ovarian function (9, 16, 17). Consequently, the intensity and magnitude of ovarian inflammatory responses and their associations to the patient’s manifestation of POI seem to be relevant issues (2, 18, 19).

Interleukin 33 (IL-33) is a recently identified pro-inflammatory protein that belongs to the IL-1 family of cytokines. It has a molecular weight of 30 kDa (20). Its unique receptor is the suppressor of tumorgenicity 2 (ST2), also known as IL-33R and IL1RL1 (21). It exists in two forms: a transmembrane form known as ST2L, and a soluble form known as sST2. As a pleiotropic cytokine, IL-33 binds the ST2L that can subsequently induce either type 1 or type 2 helper T cell (Th) inflammation (21–24). It has been shown that both IL-33 and ST2 are constitutively expressed in both immune and non-immune cells within diverse tissues (22, 25). In response to tissue/cell damage, infection, necrosis, or oxidative stress, IL-33 is released by damaged or necrotic cells, which functions to regulate physiological and pathological processes related to chronic inflammation (20, 24, 26). Intriguingly, upon tissue-derived stimuli such as inflammatory factors, sST2 can be released from various cell types (25). It acts as a decoy receptor to inhibit the IL-33 signaling pathway (22, 25). Mounting evidence has revealed that IL-33 levels are elevated within the serum or tissue under inflammation-associated diseased conditions, such as heart failure, atherosclerosis, asthma, sepsis, trauma and pulmonary fibrosis (20, 25, 27). Similar changes are also observed in sST2 under these inflammatory conditions (25, 28). Thus, IL-33 seems to be an alarmin cytokine with essential roles in promoting chronic inflammation (22, 24, 26).

At the female reproductive system of mouse and human, IL-33 and ST2 are found to be expressed in uterus, endometrium as well as ovary (22, 29). So far, their roles in both reproductive physiologies and pathologies are not fully understood. Several studies have revealed that IL-33 and ST2 are associated with endometriosis, which is known as a chronic inflammatory gynecological disorder (30, 31). Their results showed that IL-33 levels were increased in both the plasma and peritoneal fluid of patients with severe endometriosis compared with healthy controls (30, 31). For sST2, an elevation was also found in peritoneal fluid of endometriosis patients in comparison to healthy controls (31). By contrast, although available data in the literature concerning the roles of IL-33/ST2 in POI is lacking, accumulated evidence has indicated vital roles of IL-33/ST2 in multiple aspects of ovarian physiology, including folliculogenesis, ovulation and follicular atresia (29, 32, 33). Given that IL-33 is closely associated with inflammation-associated diseases, its dynamic variation during the progression of ovarian insufficiency might mirror ovarian reserve and function for POI patients.

Therefore, in the current study, we aimed to investigate the levels of IL-33 and sST2 in both follicular fluid and paired serum during different stages of POI. Next, we explored whether their levels are correlated with POI progression. Finally, we attempted to determine whether IL-33 and sST2 levels are associated with embryo quality.

Due to the premature ovarian functional decay and unclear etiology, women with POI remain a challenging group of infertility patients (4). There are several therapies for POI in the clinical setting, including psychological and family genetic counseling, hormone-replacement therapy (HRT), oocyte donation and cryopreservation (1). However, these methods mainly focus on managing the symptoms of POI patients and have certain limitations. To date, there is no effective treatment for recovering ovarian function in women with POI (1, 4). These indications suggest that timely and accurate diagnosis of POI are crucial, especially for those patients within early stage of ovarian insufficiency. This can ensure adequate time for the clinician to make treatment options for POI patients. Currently, the primary biomarkers to predict and assess ovarian function of POI patients mainly originated from ovarian follicles and granulosa cells, like E₂, AMH and inhibin B (1, 3). Although chronic inflammatory milieu in ovaries have been reported in women with POI, the inflammatory markers identified for POI indications are still lacking (5, 7, 9, 36).

As a pro-inflammatory IL-1 family cytokine, IL-33 is initially identified as an alarmin or danger signal within a variety of tissues (22, 37). Normally, IL-33 is stored in the nucleus, but is frequently released from both the nucleus and cell in response to tissue damage, necrosis, oxidative stress, inflammation and infection (20, 22). Subsequently, it can stimulate a MyD88- and nuclear factor-kappa B (NF-κB)-dependent inflammatory reaction through its surface receptor called ST2 (20, 38). Aberrant expressions of IL-33 and/or ST2 has been well documented in plentiful inflammatory disorders, such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease (37). However, the roles of IL-33 and sST2 in patients with POI has not been elucidated (22). To our knowledge, the present study for the first time reveals that, IL-33 in follicular fluid, but not in serum, continuously increases, while sST2 has an opposing alteration along with the development of POI. This is further reinforced by their strong associations with other ovarian reserve markers, including FSH, AMH and AFC. In addition, the levels of IL-33 and sST2 in follicular fluid might serve as potential predictors for both bPOI and POI. Hence, our results indicate that follicular fluid IL-33 and sST2, as inflammation-associated factors, may be related with POI progression.

In females, IL-33 and ST2 are found to be present in the uterus, like cervical, endometrial, myometrial tissues of mice and humans (22, 32). Besides, they are constitutively expressed in ovarian tissues, including the ovarian follicles, granulosa cells of primordial, primary, secondary and antral follicles, ovarian stroma as well as corpus lutea (22, 32). Under the physiological condition, both IL-33 and ST2 expressions are shown to be modulated by fluctuations of ovarian hormones (e.g. estrogen and progesterone) along with menstrual cycles or estrous cycles (32). It has been revealed that within the ovary, the IL-33-ST2 signaling pathway plays an important role in folliculogenesis, ovulation and atretic follicle disposal (22, 29, 38). The physiological concentrations of IL-33 and ST2 appear to be required for these ovarian events (29, 32, 38). Notably, mice with IL-33 deficiency by blockage or knock-out of IL-33 exhibit reduced follicular pool, ovulation and phagocytotic activity, whereas accumulated catabolic waste in ovaries (29, 33, 38). Accordingly, the IL-33-ST2 signaling system might act to maintain the ovarian homeostasis and functions.

Given that POI is a continuum of compromised ovarian function, we wonder whether IL-33 is associated with the development of ovarian insufficiency. Thus, in our study, we assessed the levels of IL-33 in both follicular fluid and serum obtained from bPOI, POI patients and healthy controls. The data showed that comparing with controls, the concentration of follicular fluid IL-33 was greatly enriched with the progression of POI. By contrast, its concentration in serum was undetectable in most cases of bPOI, POI and control groups. So far, there is less data regarding IL-33 and POI in the literature, making it difficult to compare our results with others’. Nevertheless, IL-33 has been studied in other reproductive disorders including endometriosis and polycystic ovarian syndrome (PCOS) (30, 39, 40). Both of them are well understood to be characterized by chronic inflammation (40–42). An increased abundance of serum IL-33 was found in PCOS patients in comparison to healthy fertile controls (39). Similarly, several human studies have revealed that IL-33 levels were elevated in plasma, serum, peritoneal fluid and endometriotic tissues of women with endometriosis compared with healthy controls (30, 31, 40, 43). However, under the pathological condition, direct evidence in the literature to unravel the negative effects of IL-33 on ovarian function is currently lacking. Using human stromal cells derived from ovarian endometrioma (hOVEN-SCs), a recent work further demonstrated that IL-33 expression in these cells was upregulated by 17β-estradiol through estrogen receptor pathway. In turn, high levels of IL-33 enhanced invasion ability of hOVEN-SCs via ST2/mitogen-activated protein kinase (MAPK)/matrix metalloproteinase MMP-9 signaling pathway, ultimately contributing to ovarian dysfunctions and endometriosis progression (44). Remarkably, mouse model studies revealed that supplementation of recombinant IL-33 exacerbated ovarian dysfunctions and endometriosis via promoting inflammation and fibrosis within tissues (40, 43). Collectively, our results were consistent with these findings, indicating that IL-33 might drive inflammation in ovarian microenvironment, and its abundance may be in relation to disease activity during POI development. It was revealed that IL-33-ST2 axis can stimulate inflammation response by activating the MAPK and NF-κB pathway (24, 44, 45). Emerging evidence has suggested the roles of IL-33-ST2 axis in several inflammatory diseases like asthma, endometriosis, pulmonary and ovarian fibrosis (9, 44, 46, 47). As abundant IL-33 was present in POI ovaries, we speculate that IL-33 might drive the ovarian inflammation via MAPK signaling pathway during POI progression. This hypothesis is, however, not determined in our work, which represents a limitation of the present study. Therefore, further investigations to determine this issue as well as the source of IL-33 within the milieu of POI ovaries are required. Additionally, future studies will be necessary to delineate the functional role of IL-33 in the POI pathogenesis.

It is well known that sST2, a soluble form of ST, lacks the transmembrane and cytoplasmic regions (31, 48). It acts as a decoy receptor for inhibiting IL-33 activity by blocking IL-33 signaling through ST2L (20, 31). As such, sST2 often directs anti-inflammatory properties within diverse tissues (22, 31). In the present study, we found declined levels of follicular fluid sST2 in both bPOI and POI groups comparing with controls, albeit they remained abundant in all groups. But by comparison, serum sST2 levels were relatively lower, and no significant alteration was found among the three groups. Until now, the role of sST2 in the critical ovarian events like folliculogenesis remains poorly understood (22). A previous study analyzed sST2 expression in the follicular fluid obtained from infertility women who underwent in vitro fertilization or intra-cytoplasmic sperm injection (IVF/ICSI) due to female, male, mixed or unexplained factors (49). In agreement with our data, this study revealed that sST2 was more abundant in follicular fluid than those in plasma (approximately 7.9-fold) (49). Notably, higher levels of follicular fluid sST2 were found in larger follicles as well as in retrieval oocytes which developed to good-quality embryos than those which are in small size and graded average embryos, respectively (49). Furthermore, ex vivo experiments in this study showed that human granulosa cells secreted sST2. They might be a source of sST2 in ovaries during oocyte/embryo development (49). These findings suggest that physiological sST2 levels in the ovaries are associated with follicular development. Recent mice studies revealed that ovarian sST2 expressions were modulated by reproductive hormones, as reflected by their dynamic changes throughout the estrous cycle (32). Nevertheless, the molecular mechanisms underpinning sST2 modulation in ovarian physiological processes are still unknown. Future investigations are required to determine the precise roles of sST2 in ovaries.

Disturbed levels of sST2 have been reported in the context of reproductive pathologies, like endometriosis, pre-eclampsia (PE), recurrent spontaneous abortion (RSA), and preterm labor (PRL) (22, 50, 51). However, these studies have inconsistent results. Some studies showed an elevated serum level of sST2 in patients with RSA and PE (50, 52). Also, sST2 levels were increased in the peritoneal fluid of patients with endometriosis (31). Contrarily, another study found that women with PRL had diminished sST2 in the amniotic fluid (51). Current understanding of sST2 in reproductive pathologies is still limited, and as yet sST2 has not been investigated in POI (22). Our work is the first to examine the changes of follicular fluid sST2 concentration in POI patients. Unlike IL-33, a decreased level of sST2 in the follicular fluid was observed to be correlated with POI progression. This is in contrast to IL-33 which, as aforementioned, is elevated in the follicular fluid along with POI development. As a soluble receptor of IL-33, sST2 has anti-inflammatory properties through binding to IL-33, and limit the bioactivity of IL-33 (22, 53). Consequently, a possible explanation for our data is that sST2 deficiency in local ovarian environment fails to dampen the pro-inflammatory effects driven by IL-33, ultimately leading to POI occurrence. In line with our findings, a previous study has showed that preterm labor (PRL) patients caused by intra-amniotic infection or inflammation had diminished concentrations of sST2 in amniotic fluid comparing with those who delivered at term (51). This implicates that a decline in sST2 likely augments the inflammatory reaction. Hence, these findings further support our hypothesis that sST2 may function as a counter-regulatory mechanism for balancing IL-33 abundance involved in the POI pathogenesis. The deficiency of sST2 could induce exacerbated the inflammatory response within ovaries and aggravated POI progression. However, available data concerning potential adverse effects of sST2 overexpression on ovarian functions is still lacking. Accordingly, further studies are required to clarify this issue and the regulatory mechanisms responsible for sST2 secretion during POI.

Owing to the irreversible decline of ovarian function in women with POI and no curable approaches for these patients, timely diagnosis and even prediction of POI are of significance, which require multiple reliable markers throughout the spectrum of POI (1, 3, 4). In the clinical practice, the current markers applied for POI patients are mainly focused on ovarian reserve, involving AFC, FSH, E2, AMH and inhibin B (1, 4). Given that IL-33-sST2 system might reflect inflammatory status and involve in essential ovarian events within ovaries with POI development, it is of particular interest to determine whether follicular fluid IL-33 or sST2 could serve as a predictor for POI. Our data showed that comparing with sST2, follicular fluid IL-33 had a better predictive accuracy for bPOI and POI, but inferior to AMH. Furthermore, our results indicated that there were strong associations of follicular fluid IL-33 and sST2 with FSH, E₂, AMH and AFC. It’s well established that the follicular microenvironment is essential for folliculogenesis and subsequent oocyte development (9, 19). Previous studies have showed that high levels of pro-inflammatory cytokines in follicular fluid, like IL-1, IL-6 and IL-18, could compromise oocyte/embryo development (16, 17, 54). In agreement with these findings, the present study showed that follicular fluid IL-33 levels were negatively associated with the rates of Day-3 good-quality embryos, whereas sST2 did not. This indicates that elevated IL-33 derived local follicular inflammation was relevant with declined quality of oocytes and embryos. Additionally, it’s noteworthy that IL-33 or sST2, as a follicular fluid predictor, differs from the commonly used indicators for ovarian reserve. This is likely because they may specifically mirror the ovarian inflammatory milieu, which affects ovarian function and follicle developmental competence. Although our data are preliminary, they are intriguing, and suggest that IL-33 and sST2 in follicular fluid might become supplementary indicators for an overall evaluation of POI, as well as for a comprehensive assessment of the effectiveness of these patients’ treatment. Nevertheless, our study was limited by the small sample size, future work is still required to examine the potentials of IL-33 or sST2 becoming a biomarker for POI by using randomized clinical trial with a large sample size and suitable statistical models.

In summary, the present study first revealed an increased abundance of follicular fluid IL-33, whereas an sST2 deficiency in women with POI. This implied that IL-33 and sST2 might be associated with POI development. However, our conclusion was limited due to a small number of human samples analyzed. Future researches on the roles of IL-33-sST2 signaling system in regulating follicular development, steroidogenesis, and POI pathogenesis is warranted.