Increased expression of NLRP3 inflammasome components in granulosa cells and follicular fluid interleukin(IL)-1beta and IL-18 levels in fresh IVF/ICSI cycles in women with endometriosis

Abstract

The inflammasomes are a family of recently described multi-protein cytoplasmic sensors that orchestrate the inflammatory response and participate in a variety of inflammatory conditions. We hypothesized that the activation of pyrin domain‑containing protein 3 (NLRP3) inflammasome by granulosa cells (hGCs) may be activated in women with endometriosis and influence oocyte maturation and IVF outcomes. We performed a cross-sectional study to investigate the NLRP3 inflammasome status in follicular fluid (FF) and in hGCs from 44 women undergoing controlled ovarian stimulation for IVF/ICSI. Study subjects were divided into two groups according to the infertility etiology: group with tubal or male factor (control, n = 22) vs. group with endometriosis (n = 22). The FF IL-1beta and IL-18 levels in the endometriosis group were significantly higher than those in the non-endometriosis group, i.e., 5010 pg/mL and 2738 pg/mL, respectively (p < 0.05). No correlation was found between clinical pregnancy and live birth rate and analyzed inflammasome component levels (p > 0.05). In addition, the hGCs from endometriosis women demonstrated high expression of NLRP3 inflammasome at both protein and mRNA levels. Higher expression of inflammasome components within the ovary compartment may result from the exaggerated inflammatory state associated with endometriosis and thus impact the fertility of these women.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10815-022-02662-2.

Introduction

The follicular fluid (FF) is the natural environment in which an oocyte grows and differentiates. Most components of FF are contributed by oocytes and granulosa cells (hGCs) and change with the oocyte growth and maturation. As a result, the metabolite, ion, and enzymatic characteristics of FF and follicle or oocyte are highly correlated. Thus, variations in FF composition influence the fertility and up-growth potency of oocytes and thus indirectly affect women’s reproductive function.

Endometriosis is a chronic estrogen-dependent inflammatory disease and a cure does not exist. The exact prevalence rates of endometriosis in the general population are not known, but it is estimated that it affects about 10% of premenopausal women worldwide [15]. Moreover, women aged 40–44 years had the highest prevalence rate and infertility was documented in 37% of patients [7]. Endometriosis is associated with a variety of clinical presentations, ranging from asymptomatic women to patients suffering from dysmenorrhea, chronic pelvic pain, dyspareunia, and dyschezia. Several factors have been found to be associated with the presence of endometriosis such as body mass index, gravidity, elective abortions, and ectopic pregnancies than women seeking care for infertility who did not have endometriosis [3, 12]. Although the cause of endometriosis is unknown, endometriosis patients display an aberrant number of immune cells and higher amounts of cytokines and chemokines in plasma compared to healthy women [2]. Excessive local and systemic inflammation does not allow the removal of the debris but rather facilitates endometriotic lesion formation and menstrual tissue adhesion to peritoneal structures. Macrophage recruitment has been considered to play a pivotal role in the pathogenesis of endometriosis. But while M2 macrophages have been suggested to promote the development of endometriosis, M1 macrophages inhibit endometriosis. As follows, it was demonstrated that M1 polarised macrophages increased the production of anti-inflammatory molecules by hGCs [9]. Therefore, disruption of well-controlled inflammatory mechanisms may be responsible for infertility, spontaneous abortion, and recurrent pregnancy loss associated with endometriosis patients [14].

Few reports addressed the inflammatory mechanisms involved in pregnancy pathologies. The inflammasome is a large multiprotein complex found in the cytosol that mediates innate immune response via caspase-1 activation [31]. It plays key roles in the production of pivotal inflammatory cytokines, interleukin (IL)-1β and IL-18, and pyroptosis (proinflammatory cell death). It is considered a key regulator of the innate and adaptive host response that surveys the cytosol and other compartments of the cell. In particular, the nucleotide-binding domain and leucine-rich repeat pyrin domain containing protein-3 (NLRP3) inflammasomes bind to procaspase-1, which becomes active and cleaves pro–IL-1beta and pro–IL-18 to IL-1beta and IL-18, respectively [27]. When released to the extracellular medium, these proinflammatory mediators contribute to the development of inflammatory responses and immune defenses. The NLRP3 inflammasome is activated by a range of danger and stress signals and excessive activation contributes to the pathogenesis of a wide variety of diseases, such as atherosclerosis, type 2 diabetes, obesity, and inflammatory bowel disease [10]. Some studies suggest that abnormal activation of the NLRP3 inflammasome is also linked to the occurrence of endometriosis (Bullon et al., 2017), polycystic ovary syndrome [24], recurrent spontaneous abortion (M. D. [18], and pre-eclampsia [20].

Large amounts of inflammatory cell infiltration and inflammatory cytokines (IL-1beta, IL-6, and tumor necrosis factor-alpha) occurred in an animal model of endometriosis compared with the control (J. [29]. In humans, IL-1beta in the extrauterine tissues and peritoneal fluid from patients with endometriosis were significantly higher than those from healthy women. In addition, genes encoding caspase-1, IL-18, and NLRP3 are upregulated in ectopic endometrial tissues [1] as well as the inhibition of NLRP3 significantly restrained lesion progression and fibrogenesis in endometrial stromal cells and cyst-derived stromal cells with endometriosis [21]. The ability of IL-1beta to upregulate cyclooxygenase-2 expression and increase vascular endothelial growth factor (VEGF) and IL-6 release by endometrial stromal cells are probable explanations [11]. Collectively, these findings suggest that inflammasomes contribute to the development of endometriosis through NLRP3 inflammasome activation mediated by nuclear-initiated estrogen signalling pathway. The endometriosis lesions cause local inflammation, and the release of inflammatory factors through the circulation into the ovary may cause alterations in FF content. Despite such evidences, scant information is available about the activation of inflammasome within human ovary and their potential involvement in endometriosis-associated infertility. These new clues regarding the pathogenic mechanisms involving the inflammasome may be crucial in the future development for endometriosis-associated infertility therapy.

The aim of this study was, therefore, to better understand inflammasome status in women undergoing controlled ovarian stimulation for IVF/ICSI. For this purpose, we investigated the levels of IL-1beta and IL-18 levels in the follicular fluid (FF) from women with endometriosis and compare them to controls. Moreover, we determined NLRP3 inflammasome expression in human granulosa cells (hGCs) from these patients.

Materials and methods

Study design and eligibility criteria

The present cross-sectional study was conducted on 44 women undergoing IVF and embryo transfer procedures at the Fertility Unit of Centro Hospitalar Vila Nova de Gaia/Espinho E.P.E. (CHVNG/E), Portugal. Eligibility criteria included premenopausal women aged 18–39 years and body mass index (BMI) from 18 to 40 kg/m² that have been unable to conceive naturally for at least 1 year before entering the study. The study included 22 patients previously diagnosed with minimal or mild endometriosis endometriosis (stage I or II) by transvaginal ultrasound and/or laparoscopy. The control group included 22 women that undergone IVF/ICSI cycles due to male or tubal factors during the same time period and no history or evidence of pelvic inflammatory disease or malignancies. Patients with and without endometriosis were included with a 1: 1 ratio. They were consecutively invited by convenience before the procedure was performed by a research assistant. Informed consent was taken from all women who agreed to participate in our study. Consent forms and protocols were approved by the ethics committee of the CHVNG/E and by the National Data Protection Commission (authorization number 526/2017) and fulfilled the ethical considerations in accordance with the declaration of Helsinki for medical research involving the human subject. Details of the participants are shown in Table 1.

Table 1.

Demographics, clinical characteristics, and outcomes of the study population

Variable	Control (n = 22)	Endometriosis (n = 22)	p
Maternal age, years	35.3 (3.8)	33.4 (3.5)	0.0908
BMI, kg/m2	23.6 (3.4)	23.8 (3.5)	0.8890
Duration of infertility, years	3.5 (2.0–4.0)	3.0 (2.0–5.0)	0.8129
Follicle count (> 16 mm)	6.0 (4.5–7.0)	6.0 (2.0–11.3)	0.3985
Number of retrieved oocytes	6.0 (4.0–14.0)	8.5 (3.0–17.8)	0.7080
Fertilization rate, %	0.60 (0.50–0.83)	0.67 (0.50–0.96)	0.8213
Number of embryos transferred
0	1	2
1	11	7
≥ 2	9	10
Number of embryos implanted
0	13	10
1	10	5
≥ 2	0	4
Cycles protocol (39/44)
Short agonist protocol	18	7
Long agonist protocol	0	14
Antagonist protocol (29/44)	17	9
Serum basal levels
FSH, ng/ml	6.7 (5.7–7.3)	7.5 (6.1–9.5)	0.4014
LH, ng/ml	5.5 (3.9–7.2)	6.1 (5.2–6.8)	0.3179
TSH, ng/ml	1.4 (0.9–2.1)	1.1 (0.7–1.6)	0.2163
AMH, ng/ml	18.1 (11.3–26.6)	14.0 (5.3–26.3)	0.9814
E2, pg/ml	37.2 (26.5–57.0)	40.0 (29.2–81.2)	0.9280
Follicular fluid
IL1beta, pg/ml	29.8 (21.9–34.4)	33.1 (24.3–51.4)	0.0147 *
IL18, pg/ml	75.6 (65.8–85.7)	99.6 (69.7–112.2)	0.0341 *
Caspase 1 (RLU)	2037 (1153–2716)	2209 (1561–3227)	0.1192

Values are expressed as the mean (SD) or median (IQR). BMI body mass index

Study procedures

Depending on the women’s age, the antral follicle count and the basal serum follicle-stimulating hormone (FSH) concentration, pituitary downregulation was achieved with the long GnRH agonist [triptorelin (Decapeptyl) 0.1 mg; Ipsen Pharma Biotech], the short GnRH agonist, or GnRH antagonist protocol [Ganirrelix (Orgalutran), Schering-Plough]. Ovulation stimulations were conducted with subcutaneous injections of individual starting doses of recombinant FSH (Gonal-f®, Serono Pharma; Bemfola®, Gedeon Richter; Ovaleap®, Teva; Puregon®, Organon; Luveris®, Merck Europe; or Elonva®, Schering-Plough) or human menopausal gonadotropin (Menopur®, Ferring Pharmaceutical Co.) at appropriate doses (50–450 IU). When the leading follicle reached 16 mm in mean diameter, ovulation was triggered with 250 μg recombinant hCG (Ovitrelle; Merck Serono). Biochemical pregnancy was defined as the detection of β-HCG levels in serum but no signs of pregnancy by ultrasound, whereas clinical pregnancy was defined as the visualization of a gestational sac at ultrasound examination.

Human follicular fluid and granulosa cells collection

Follicles with colorless and homogenous textures were selected for follicular fluid (FF) aspiration. FF samples were pools of several follicles of individual ovaries and from individual patients. The FF was collected at the time of oocyte retrieval from women undergoing ovarian stimulation for classic IVF, intracytoplasmic sperm injection (ICSI), or both. FF without obvious blood contamination was centrifuged at 300 g for 10 min at 4ºC and supernatants were divided into aliquots and immediately stored at − 80 °C until the ELISAs were performed. The volume and the appearance of each FF sample were recorded. For primary human granulosa cells (hGC) isolation, after centrifugation, the cell pellet was then collected and washed with phosphate buffer saline (PBS). Following this step, cells were isolated by density gradient centrifugation with Percoll for 20 min at 900 g and 4ºC. The hGC in the interphase layer was collected and washed with PBS. Unless otherwise stated, all reagents were purchased from Invitrogen.

Enzyme-linked immunosorbent assay

The concentrations of cytokines IL-1beta and IL-18 in FF were determined using enzyme-linked immunosorbent assay (ELISA) kits. Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA) were employed for human IL-1beta (#DLB50) and IL-18 (#DL180) determination following the manufacturer’s instructions. Assay sensitivity limits were 1 pg/ml for IL-1beta and 5.15 pg/ml for IL-18. Results were expressed as pg/ml of follicular fluid.

Evaluation of the expression of transcripts related to inflammation

Total RNA was extracted from the hGCs using the TripleXtractor reagent (grisp, Research Solutions, Porto, Portugal) according to the manufacturer’s protocol. The quality and purity of all samples of the total RNA obtained were assessed by Experion (Bio-Rad Laboratories, USA) and the quantity by using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). Subsequently, the synthesis of complementary DNA (cDNA) for performing the polymerase chain reaction coupled reverse transcription was conducted with one microgram of the total RNA using the Xpert cDNA synthesis kit (grisp, Research solutions, Portugal; #GK34.0100), according to the manufacturer’s protocol. The quantification of gene expression of IL-1beta, IL-18, NLRP3, and caspase-1 was made using Xpert Fast SYBR Mastermix Kit (grisp, Research solutions, Portugal). The device used was StepOnePlus PCR Systems (Applied Biosystems, USA). Each reaction was set in duplicate in a total of 20 μL each, which contains 1 μL of each primer (0,3 μM; forward and reverse), 10 μL of master mix, 7 μL of nuclease-free water, and 1 μL of cDNA. Additionally, a control was inserted, also in duplicate, which was included in each reaction in order to prove that there is no contamination. The conditions for the RT-qPCR reactions and primer sequences used in this study are in Supplementary Table 1. The amplification of each particular transcript was confirmed by the melting curve-generated profile at the end of each reaction. Relative gene expression was normalized to the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), and mRNA abundance was quantified using the 2^−ΔΔCt method. To analyze the relative expression, after the analysis of gene expression, we chose an RNA sample obtained from the control group, which received a relative value of 100. All other samples received values for that sample.

Protein extraction and Western blot analysis

Following RNA extraction, after the addition of chloroform and phase separation with the removal of the aqueous phase containing the RNA, the interphase and the organic phase of the TripleXtractor reagent (grisp, Research Solutions, Porto, Portugal) was processed for protein extraction, according to the manufacturer’s protocol. After quantification by Bradford assay, 25 µg of protein was denatured and separated by SDS-PAGE on 10% gels. Then separated proteins were transferred to nitrocellulose membranes as previously described [8]. Non-specific binding sites were blocked by incubating the membrane with 5% (w/v) nonfat milk in Tris-buffered saline (TBS-T, 10 mM Tris, 0.15 M NaCl, 0.1% Tween-20, pH 7.4) to block non-specific binding sites, for 1 h at room temperature. For the detection of NLRP3, the membranes were incubated overnight at 4 °C with a rabbit anti-human NLRP3 antibody diluted 1:1000 (Cell Signaling Technology, Boston, USA,#15,101) in a blocking solution and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-rabbit IgG antibody diluted 1:500. The membranes were then washed again and immunoreactive proteins were visualized by enhanced chemiluminescence (Advansta, CA, USA) on an imaging system (ChemiDocTM Touch Imaging System; BioRad, Laboratories Melville, NY, USA) after incubation with enhanced chemifluorescence (Advansta, CA, USA). After detection, the membranes were stripped and reprobed for β-actin using anti-β-actin antibody at a dilution of 1:500 (#sc-47778) to control loading variation. Results were quantified by using the Image Lab v5.2.1 software (BioRad, Laboratories Melville, NY, USA).

Statistical analysis

The sample size was estimated based on the means and standard deviations of the pilot study using G*Power version 3.0.10 (Universitat Kiel, Kiel, Germany) software, considering the usual values of statistical power (80%) and significant level (5%) to achieve a small/medium effect size. A sample size of 22 patients per group was determined.

Patient and cycle characteristics data are expressed as mean ± SD (range) or median with interquartile ranges where appropriate, while categorical variables are shown as n (%). The compliance of the data to a normal distribution was evaluated with the Shapiro–Wilk test. Student’s t test was used to compare the means between the two groups. Correlations between variables of interest were performed using Spearman’s rank order tests. The variables were examined at a 95% confidence level, and a p value of less than 0.05 was considered statistically significant. Statistical analyses were performed using the GraphPad Prism Statistical software version 9.1.1 (GraphPad San Diego, Calif., USA).

Results

Expression of NLRP3 protein and associated genes in hGCs

A total of 44 patients participated in the current study with a mean age and BMI of 34.2 ± 3.8 years and 24.3 ± 3.8 kg/m2, respectively. There were no significant differences in terms of age, BMI, or baseline characteristics (E2, LH, AMH, TSH, FSH), between the endometriosis (n = 22) and non-endometriosis (n = 22) groups (p > 0.05 for all). In the endometriosis group, the peak E2 level was significantly higher than it was in the other group (p < 0.05). Other parameters including the duration of infertility, follicle number, number of oocytes retrieved, and number of embryos transferred were similar (p > 0.05 for all). The patient data assisted the reproductive technologies (ART) characteristics, implantation, and clinical pregnancy rates as well as the concentration of the inflammasome markers FF from women undergoing IVF/ICSI are summarized in Table 1.

We analyzed the mRNA expression of NLRP3, caspase-1, IL-1beta, and IL-18 in hGCs by using qRT-PCR. As shown in Fig. 2, the expression of NLRP3, caspase-1, IL-1beta, and IL-18 mRNA was detected in hGCs from both endometriosis and control group patients. The level of NLRP3 and caspase-1 mRNA was higher in hGCs from women with endometriosis than in control samples (Fig. 1). The expression of NLRP3 was further analyzed by the detection of the relevant protein in hGCS from endometriosis and control patients by western blot and the levels of NLRP3 protein were lower in normal patients than in the endometriosis group (Fig. 2).

Fig. 2.

Western blot and respective densitometry of NLRP3 protein expression in granulosa cells. A Representative western blot reflecting NLRP3 protein levels in homogenates of hGCs obtained from fluid from women undergoing IVF/ICSI with endometriosis and control patients. B The level of NLRP3 protein in hGCs from endometriosis patients was higher than that in the control group. Blots were stripped and re-probed for β-actin as a loading control. Bars represent the mean ± SEM. The independent samples t test was used to examine statistical differences between the means of the two groups. All samples were run in duplicate. *p < 0.05 vs control group

Fig. 1.

Gene expression of inflammasome markers in granulosa cells from women undergoing IVF/ICSI. Quantitative PCR analysis of IL-1beta, IL-18, NLRP3, and caspase-1 relative to GAPDH housekeeping gene expression from 22 women undergoing IVF/ICSI techniques with male factor (C) and from 22 women with endometriosis (E). Significant elevation of NLRP3 (p < 0.001) and caspase-1 (p = 0.025) expression was observed in FF from endometriosis patients but not for IL-1beta (p = 0.956) or IL18 (p = 0.323). Bars represent the mean ± SEM. The independent samples t test was used to examine statistical differences between the means of the two groups. All samples were run in duplicate. *p < 0.05 and ***p <0.001 vs control group

IL-1beta and IL-18 are detected in follicular fluid

We next examined differences in follicular concentrations of IL-1beta and IL-18 i. IL1-beta and IL18 were significantly higher in the pool from endometriosis patients (28.0 ± 10.2 and 76.5 ± 22.5 nmol/L) compared with control factors (38.0 ± 16.9 and 93.1 ± 28.2 nmol/L), respectively (Fig. 3A). We further measured caspase-1 activity directly in FF, a direct readout for inflammasome activation, though no differences were observed between endometriosis and control group of patients (Fig. 3B).

Fig. 3.

Concentration of inflammasome markers in follicular fluid from women undergoing IVF/ICSI. Follicular fluid IL-1beta and IL-18 concentrations in control (C) and endometriosis (E) women. B Relative light units (RLU) of caspase-1 activity does not show any differences between the control (C) and endometriosis (E) group. Horizontal bars indicate medians, and corresponding error bars indicate IQR. The independent samples t test was used to examine statistical differences between the means of the two groups. All samples were run in duplicate. *p < 0.05 vs control group

Follicular inflammasome-related markers and clinical outcomes in IVF/ICSI treatment

We also determined the correlation coefficients for follicular concentrations of inflammasome-related markers according to clinical parameters (n = 44). After Spearman’s correlation analysis, no significant correlation was observed between follicular inflammasome-related marker concentrations and patient age or BMI (Fig. 4). Similarly, there was an association in inflammasome component concentrations with either fertility treatment outcomes and ART characteristics. However, concerning hormonal levels, a negative correlation was found between AMH levels and caspase-1 (r =  − 0.33), whereas FSH levels correlated positively with IL1beta (r = 0.47). A statistically significant but weakly negative correlation was found for AMH with either E2 (r =  − 0.42) or FSH levels (r =  − 0.32). In addition, a statistically significant positive correlation was found for follicle count with the number of retrieved oocytes (r = 0.70) as well as between these parameters with age (r = 0.-35; r =  − 0.35), AMH (r = 0.49; r = 0.63) and FSH (r =  − 0.36; r =  − 0.53) (Fig. 4).

Fig. 4.

Correlation matrix for clinical, ART characteristics, and follicular inflammatory markers in women undergoing IVF/ICSI. The correlation matrix was made with Spearman ranking, using pair-wise–complete observations for all studied women (n = 44). The significant (p < 0.05) Spearman rho (r) values are provided inside the boxes. Blue color denotes genes showing positive correlations and red denotes genes showing negative correlations. Abbreviation: BMI, body mass index; FC, follicle count (> 16 mm); NRO, number of retrieved oocytes; FR, fertilization rate

Discussion

There are a myriad of factors that determine the success of ART. To date, research into the physiological mechanisms underlying infertility has been fragmented, but exacerbated and prolonged inflammation within reproductive tissues might be at least partly involved. The inflammatory response is highly complex and NLRP3 inflammasome has been implicated in different models of inflammation (M. [18, 26]. However, NLRP3 activation in hGCs is not clear and inflammasome studies in the ovarian tissue are scarce. In the present study, we investigated the NLRP3 inflammasome activation in hGCs from women undergoing IVF/ICSI treatments.

We found NLRP3 expression at both mRNA and protein in hGCs from either endometriosis or control patients, demonstrating that NLRP3 is continuously activated in clinical conditions and suggesting that NLRP3 inflammasomes activation may play an important role in the regulation of women’s ovulation. As inflammasome facilitates the cleavage and activation of caspase-1 in response to damage signals, one possible explanation questions the continued exposure of hGCs to the contents of inflammatory niche in the peritoneal cavity, ovaries, and uterus of endometriosis patients. In fact, we report that within hGCs, NLRP3 expression levels were higher in hGCs from endometriosis patients, as compared to control patients suggesting that unregulated inflammasome activation may be involved in the pathogenesis of infertility. In this regard, NLRP3 may be more activated in the stressful environment of exposure to the contents of endometriosis tissue than in normal endometrium. NLRP3 inflammasome activation requires priming and protein complex assembly. Although NLRP3 inflammasome priming is triggered by various pathogen-derived ligands and physiological aberrations, numerous molecular or cellular events, including mitochondrial dysfunction and reactive oxygen species generation, may be involved in the activation of NLRP3 inflammasome assembly [19],S. [30]. In addition, excessive or altered regulation of NLRP3 inflammasome activity is related to the pathogenesis of a wide variety of inflammatory, autoimmune, and degenerative diseases [25, 28]. In ovaries, the activation of the NLRP3 inflammasome has been implicated in age-associated inflammation and diminished ovarian reserve raising the possibility that ovarian aging could be delayed, and the fertile window prolonged, by suppressing inflammatory processes in the ovary [17]. Nevertheless, the mechanisms of noncanonical NLRP3 inflammasome activation are beyond the scope of this manuscript.

As per inflammasome-mediated activation of caspase-1 and its target substrates are physiologically important to regulate immune cell response and metabolic activities, we then quantified inflammasome markers in hGCs and FF. We also observed an increase in caspase-1 transcripts by hGCs from endometriosis patients. Once activated, caspase-1 leads to the maturation of IL-1beta. Notably, we detected increased IL-1beta and IL-18 levels in the FF from endometriosis patients, as compared to control samples, which is consistent with an inflammasome activation within the human ovary, particularly in hGCs, which mostly contributes to FF composition. The expression and activation of NLRP3 inflammasome in hGCs were simultaneous to those in the FF. Thus, as we did not observe an increase in IL-1beta and IL-18 transcript levels by respective hGCs, we can imply that other ovarian cells are contributing to IL-1beta and IL-18 follicular levels. In addition, we cannot exclude that FF milieu resultant from hGC activation by inflammatory cytokines released by the endometriotic tissues can activate the inflammasome. On the other hand and considering the heterogeneity in clinical conditions, it is possible that other inflammasome markers are also elevated in FF samples in response to endometriosis stimuli.

Recently, it was demonstrated that IL-1beta and IL-18 levels are higher in the FF of polycystic ovary syndrome (PCOS) patients than in controls [16]. The authors also found that NLRP3 inflammasome was formed in ovarian hGCs of PCOS patients involving the activation of the NF-κB pathway [16]. This evidence reinforces our hypothesis that inflammatory microenvironment alterations in the FF of patients undergoing IVF/ICSI lead to an activated inflammatory pathway in hGCs, providing a novel mechanism in the inflammatory process of endometriosis patients. In addition, the higher expression of NLRP3 in hGCs and increased levels of proinflammatory cytokines in FF from endometriosis women observed in the present study are in accordance with previous studies demonstrating that primary ovarian insufficiency leads to overexpression of NLRP3 and increased production of caspase-1 and IL-1beta proinflammatory cytokines in hGCs [22]. Notably, we observed a negative correlation for AMH levels with caspase-1. In rodents, a previous study observed that ovarian NLRP3 expression was inversely correlated with serum AMH levels [22]. As AMH is considered as the more reliable of the ovarian reserve assessment tests, it was suggested that NLRP3 expression in these tissues may be associated with ovarian aging. However, in our study, none of the inflammasome markers evaluated correlated with patients’ age. In addition, we observed that FSH positively correlated with IL1beta. Previously, it was documented that IL1beta and FSH increase lactate production in rat Sertoli cells (male counterpart of hGCs) by increasing glucose uptake [23]. Similarly, within ovaries, both FSH and IL1beta may regulate glucose transporter activation or translocation in hGCs. In fact, it was already demonstrated in hGCs that the transcript for glucose transporter 1 increases in response to IL1 stimulation [13]. Finally, the observation that AMH negatively correlated with FSH has been previously documented as well as the observed correlations between age, hormonal serum levels, and ART characteristics [4], which reinforces our results.

The study has a few limitations that should be considered. First, the limited sample size may impact the results and undermine statistical power to detect modest differences in associations between FF cytokine levels and IVF/ICSI outcomes. Additionally, women were assigned to different stimulation protocols for controlled ovarian stimulation; this is, however, according to previous studies since the stimulation protocol and medication dose are based on ovarian response prediction. In addition, FF samples were pooled for each patient, and thus, we are not able to correlate each follicle with oocyte and embryo development. Nevertheless, the analysis of the overall FF is a reliable and comprehensive method for detecting the differences in FF IL-1beta and IL-18 levels profile between the control and endometriosis groups. Another limitation is that women undergoing IVF/ICSI in a public center are selected with respect to age, and so, the generalizability of the results may be limited. This creates the possibility for selection bias, over- or under-estimating effects. However, we have no reason to believe that participation in the study would be different.

To our knowledge, this is the first report on inflammasome hyperactivity in hGCs from women with endometriosis. Collectively, our findings suggest that NLRP3 inflammasome activation by hGCs is normally activated but the endometriosis environment influences follicular inflammasome markers. Although we were not able to demonstrate a significant effect of inflammasome markers per se on pregnancy rates, this study found a significant association between high inflammasome activation in hGCs and endometriosis patients. Therefore, although up to 30 to 50% of women with endometriosis may experience infertility [5] and present higher expression of inflammasome components [6], it may result from the exacerbated inflammatory condition associated with endometriosis that is not directly associated with the fertility of these women. Nevertheless, these results should be interpreted with caution, because the number of participants was relatively small and the data regarding women’s stimulation protocols were heterogeneous. Thus, additional work is needed to understand the molecular mechanism that controls the assembly and activation of the NLRP3 inflammasome and the impact of their activation on pregnancy outcomes. s

Supplementary Information

Below is the link to the electronic supplementary material.

Authors’ contributions

All authors contributed substantially to this work. The authors collectively developed the original concept of this study. BMF wrote the manuscript. IR revised it critically. Data collection and analysis were performed by BMF; BP, EF, and LC helped with the collection of follicular fluid and contributed to the data analysis and study preparation and statistical analysis by BMF. The authors contributed to critical discussion and reviewed and approved the final version of the manuscript for submission.

Funding

This work was financially supported by FCT—Fundação para a Ciência e a Tecnologia, I.P., in the framework of the project PTDC/MEC-OUT/28931/2017.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials. The data that support the findings of this study are available from the corresponding authors, Bruno Fonseca or Irene Rebelo, upon reasonable request.

Declarations

Ethics approval

This study was approved by the Ethics Committee of Centro Hospitalar de Vila Nova de Gaia/Espinho) and by the National Data Protection Commission (authorization number 526/2017). Informed consent was signed by the patients.

Conflicts of interest

The authors declare no competing interests.