Ovarian IL-1α and IL-1β levels are associated with primary ovarian insufficiency

Abstract

Primary ovarian insufficiency (POI) is characterized by premature ovarian failure and lack of menstrual periods in women under the age of 40. Ample evidence suggests that interleukin 1 (IL-1) family proteins are involved in the processes of ovarian follicular development and depletion. The association of these cytokines with POI disorder and the underlying molecular mechanism in regulation of the ovarian functions remain largely undetermined. In this study, follicular fluids and serum samples were harvested from POI patients and healthy women who underwent follicular aspiration during in vitro fertilization. Enzyme-linked immunosorbent assay (ELISA) and the quantitative real-time PCR (qPCR) were employed to assess the levels of protein secretion and mRNA expression of IL-1α and IL-1β, as well as the molecules that are critical for apoptotic pathways. Our results showed that IL-1α level in serum samples and follicular fluid of POI patients was significantly elevated in comparison to that in healthy women. Interestingly, the follicular levels of both IL-1α and IL-1β and TNF-α were significantly higher than their serum levels. Our qPCR analysis further revealed that there was a significant upregulation of apoptotic Bax mRNA expression, but expression of anti-apoptotic factor Bcl-2 mRNA expression was downregulated in POI patients. In conclusion, our studies revealed that the elevated level of IL-1 in follicle fluids of POI patients may be the major causal factor for follicular apoptosis that consequently impairs follicle reserve and ovarian functions by follicle depletion.

Introduction

Primary ovarian insufficiency (POI) is the clinical condition that is characterized by the absence of menstrual periods (amenorrhea) before the age of 40. It is primarily due to premature follicular cell depletion that consequently leads to a diminished ovarian reserve (DOR) and women’s infertility [1-3].

Early studies have shown that apoptosis plays essential roles in ovarian follicle reservation and atretic follicle formation. 99% of follicles appear to degenerate throughout a woman’s life. Apoptosis is a process of programmed cell death that is governed by pro- and anti-apoptotic regulators, such as BAX, BCL-2, TNF-α, and the Fas/Fas ligand (Fas/FasL) system. Early studies have shown that there are more primary follicles in adult ovaries lacking of Bax protein [4]. Bcl-2, on the other hand, reduces follicular atresia by inhibiting apoptosis of granulosa cells [5]. The Fas/FasL system has been shown to be one of the key mediators of apoptosis in atretic follicles. It was demonstrated that Fas/FasL is involved in p53-mediated apoptosis in granulosa cells during follicular development and atresia [6-8]. Interestingly, TNF-α level is negatively associated with the quality of oocytes [9]. Hence, the delicate balance between these critical molecules determines the fate of follicular cells to survival or death, and disruption of this balance will adversely influence the ovarian functions and ultimately affect women’s reproductive physiology [10].

Interleukin-1 (IL-1) family proteins are critical cytokines in regulation of inflammation, innate immunity, angiogenesis, and hematopoiesis [11]. IL-1α and IL-1β are the most studied members that regulate proliferation and apoptosis in a variety of cell types [11]. Early studies showed that IL-1 levels were closely associated with gonadotropin levels and pregnancy rates [12]. Patients with higher level of IL-1β exhibited a higher successful rate of pregnancy after embryo transplantation [12]. IL-1β has been shown to play an important role in follicle maturation and ovulation in mice [13] and rabbits [14]. By employing the genetic mutation mouse models, recent studies by Shiri Uri-Belapolsky et al [15] demonstrated that the capacity of ovarian reserve improved greatly in absence of IL-1α expression. These results indicate that IL-1 α plays a deteriorative role, but IL-1β plays a favorable role in follicle maturation and ovarian functions.

IL-1α and IL-1β are present in the serum and follicular fluid of patients who underwent IVF-ET. The clinical association between IL-1 production and POI condition remains unclear. The mechanism underlying the elevated level of IL-1 in regulation of human ovarian function requires further investigation.

Materials and methods

Recruitment criteria

The sample collection protocols were approved by the Human Reproduction Committee of the University, Changzhi Medical College. From May 2016 to September 2016, 42 patients who were undergoing IVF-ET at the Reproductive Medicine Center of Heping Hospital affiliated with Changzhi Medical College and Reproductive Medicine Centre of Maternal and Child Health Hospital of Shanxi province enrolled in this study. 22 patients who underwent IVF-ET due to male infertility were recruited as the control group. The POI group included 17 patients with DOR and 3 patients with POF. The DORs referred to hypomenorrhea, oligomenorrhea, amenorrhea, and infertility, and meet the following basal serum hormone criteria when diagnosed with transvaginal ultrasonography during the early follicular phase: follicle-stimulating hormone (FSH), 10~40 IU/L (2 intervals for more than one month); FSH/lutropin (LH), > 3.6 [16,17]; estradiol (E2), > 85 pg/ml [18], MOD (mean ovarian diameter), < 20 mm; and AFC (antral follicle count) ≤ 5 [19]. POFs with amenorrhea last for at least 4 months with perimenopausal symptoms and meet following basal serum hormone criteria: FSH: > 40 IU/L (2 intervals for more than one month) for 2 or more times; E2 < 20~30 pg/ml; and AMH (anti-Mullerian hormone): < 1.26 ng/ml [12]. All patients exhibited normal liver and kidney function without serious infection. All patients were informed of the study and provided consent.

Specimen collection

Fasting peripheral venous blood samples were obtained from the control group on days 2 to 4 of the menstrual period, and at any time from the POI patient group (no menstrual cycle). All the samples included one coagulant and one anticoagulant preparation. The coagulation sample was immediately centrifuged at 3000 rpm for 20 min and the supernatant was dispensed into 200 μl centrifuge tubes. The anticoagulation sample was immediately dispensed into 1 ml centrifuge tubes without RNase. Both samples were stored at -80°C.

After successful induction of ovulation, patients underwent follicular aspiration by transvaginal B-scan ultrasound-guided puncture of the posterior fornix to collect ootids on the day of ovulation. Follicular fluid was obtained from the follicular puncture (> 16 mm diameter), and the cumulus complex was identified and separated. The autologous follicular fluids (red blood cells < 10⁴/ml) were pooled from each patient, and the follicular fluid was centrifuged at 3000 rpm for 10 minutes. The supernatant was harvested, aliquoted, and stored at -80°C until the assays were performed.

Enzyme-linked immunosorbent assay

Follicular fluid and serum samples from patients were tested for IL-1α and IL-1β. The ELISA kits were used to quantitatively determine the levels of IL-1α (Abcam, Human Interleukin-1α ELISA kit, ab100560, British) and IL-1β (Abcam, Human Interleukin-1β ELISA kit, ab100562, British) according to the manufacturer’s specifications. Quantitation was performed with use of the standard dose-dependent curve, and the cytokine concentrations detected in the samples are given in pg/mL. The intra-assay and interassay coefficients of variation were < 10% and < 12% for the IL-1α and IL-1β assay, respectively. The detection limits were 0.5 pg/mL for IL-1α and 0.3 pg/mL for IL-1β.

RNA isolation and reverse transcription

Total RNA was isolated from peripheral venous blood samples which were stored at -80°C in 1 ml centrifuge tubes without RNase using Omega Blood RNA Kit (r6814-01) according to the manufacturer’s instructions and was quantified with the Microplate reader (TECAN Infinite M200 PRO NanoQuant). First-strand cDNA was synthesized by PCR Gene Amplification Instrument (Thermo Scientific) from a total of 4 μg of RNA using 1 μL of reverse transcriptase, incubating 15 minutes at 42°C and heating 5 seconds at 85°C.

Quantitative real-time PCR

cDNA was amplified (40 cycles) with 10 μM gene-specific primers (Supplementary Table 1) using Fluorescence Quantitative PCR (Applied Biosystems 7500 Real-Time PCR System). Changes in the level of mRNA expression were detected using gene-specific primers and fast SYBR green master mix reagent (TransGen Biotech Easy Taq DNA Polymerase kit, AP111-11 and Qiagen Quanti Fast SYBR Green PCR Kit). The thermal cycling conditions for the Fast SYBR Green reaction were 5 minutes at 95°C, followed by 40 cycles of 5 seconds at 95°C and 30 seconds at 60°C. PCR primers were designed by Primer 5.0 software and synthesized by Invitrogen Biotechnology Co., Ltd.

Statistical analysis

SPSS 17.0 software was used for statistical analysis. Data were shown as the mean ± standard deviation (x̅ ± SD). The Student t test was used as applicable. The Mann-Whitney test was used to assess differences for the means. The association among IL-1α, Bax, Bcl-2, Fas, FasL, and TNF-α was used as continuous variables on the Pearson correlation test. The criterion was set as α = 0.05. P < 0.05 was considered significant. All experiments were repeated at least three times and had similar results.

Results

We first compared the basic data of POI group with the control group. As shown in Table 1, no significant difference was observed in the ages and BMIs between these two groups (P > 0.05). Interestingly, there was a significant increase in the levels of FSH and FSH/LH in the POI group when compared to controls (P < 0.001).

Table 1.

Comparison of basic data between the POI group and the control group (x ± SD)

Group	Cases	Ages	BMI (kg/m2)	FSH (mIU/ml)	FSH/LH
POI group	20	32.49 ± 3.93	21.56 ± 3.97	22.06 ± 9.83	4.11 ± 1.51
Control group	22	30.91 ± 4.78	21.33 ± 4.05	6.99 ± 1.54	1.87 ± 0.71
P value	-	> 0.05	> 0.05	< 0.001*	< 0.001*

^*P < 0.05.

We next determined the levels of IL-1 in the serum and follicular fluid samples collected from these two groups. Our results showed that the IL-1α level in POI group was significantly higher compared to that in control group (P = 0.025, P < 0.001). There was no difference in the levels of IL-1β in these samples (P = 0.771, P = 0.054) (Table 2).

Table 2.

Comparison of IL-1α and IL-1β between the POI group and the control group (x̅ ± SD)

Item (pg/ml)	POI group (20)	Control group (22)	P value
Serum IL-1α	1.07 ± 1.61	0.48 ± 0.53	0.025*
Follicular fluid IL-1α	3.65 ± 4.16	1.67 ± 1.03	< 0.001*
Serum IL-1β	1.45 ± 1.84	1.17 ± 1.62	0.771
Follicular fluid IL-1β	3.11 ± 3.06	1.66 ± 1.12	0.054

^*P < 0.05.

We also compared IL-1 levels between serum and follicular fluid of POI patients and healthy women. Both IL-1α and IL-1β levels in follicular fluid were significantly higher than those observed in their serum levels (P < 0.001). The analyzed data for IL-1α and IL-1β levels in serum and follicular fluid samples harvested from the control and POI groups are summarized in Tables 3 and 4, respectively.

Table 3.

Comparison of IL-1α in serum and follicular fluid within each group

Item	Serum IL-1 (pg/ml)	Follicular fluid IL-1α (pg/ml)	P value
POI group	1.07 ± 1.61	3.65 ± 4.16	< 0.001*
Control group	0.48 ± 0.53	1.67 ± 1.03	< 0.001*

^*P < 0.05.

Table 4.

Comparison of IL-1β in serum and follicular fluid within each group

Item	Serum IL-1β (pg/ml)	Follicular fluid IL-1β (pg/ml)	P value
POI group	1.45 ± 1.84	3.11 ± 3.06	< 0.001*
Control group	1.17 ± 1.62	1.66 ± 1.12	0.009*

^*P < 0.05.

To further confirm the levels of IL-1α and IL-1β in POI patients, quantitative real-time PCR was employed to assess gene expression of these two cytokines. Our results revealed there was a significant increase in the levels of IL-1α mRNA expression in POI group in comparison to the control group (Figure 1A, P < 0.05), whereas IL-1β mRNA expression of remained unchanged (Figure 1B, P > 0.05).

Figure 1.

A. Comparison of mRNA expression levels of IL-1α between the two groups. B. Comparison of mRNA expression levels of IL-1β between the two groups. *, P < 0.05.

Interestingly, mRNA expression levels of Bax and TNF-α were significantly higher in the POI group (Figure 2A-C, P < 0.05), whereas the level of Bcl-2 mRNA expression was reduced significantly. No significant difference in the levels of Fas and FasL mRNA expression was observed (Figure 2D, 2E, P > 0.05).

Figure 2.

A. Comparison of mRNA expression levels of Bax between the control group and the POI group. B. Comparison of mRNA expression levels of Bcl-2 between the control group and the POI group. C. Comparison of mRNA expression levels of TNF-α between the control group and the POI group. D. Comparison of mRNA expression levels of Fas between the control group and the POI group. E. Comparison of mRNA expression levels of FasL between the control group and the POI group. *, P < 0.05.

Discussion

Early studies have shown that the proinflammatory cytokines IL-1α and IL-1β in follicle fluids are synthesized in ovarian follicle granulosa cells and oocytes. In this study, our results showed that these cytokines were elevated significantly in follicular fluids and serum samples of POI patients. Recent studies by Shiri Uri-Belapolsky showed that IL-1α-knockout female mice exhibited higher pregnancy rates and larger litter sizes in comparison to WT control females at advanced ages. In addition, the number of secondary and antral follicles and the level of serum anti-Müllerian hormone, a putative marker of ovarian reserve, was markedly higher in these mutants [15]. On the other hand, IL-1β-KO mice displayed a comparable but slightly longer ovarian lifespan [15]. It is plausible to envisage that the elevated level of IL-1 in follicle fluids of POI patients may elicit inflammation and impair ovarian functions by accelerating depletion of ovarian reserves.

Apoptosis is a process of programmed cell death that occurs in multicellular organisms, including the developing ovarian follicles. It is tightly controlled by apoptotic or antiapoptotic factors, such as BCL-2 family proteins. Bcl-2 is the founding member of the Bcl-2 family that regulate by either inducing (pro-apoptotic) or inhibiting (anti-apoptotic) apoptosis. Apoptosis regulator BAX (known as bcl-2-like protein 4) is also a member of the Bcl-2 gene family that forms a heterodimer with BCL2 and functions as an apoptotic activator [20]. Bax mediates the release of cytochrome C from mitochondria, which ultimately activates caspase and leads to cell death [10]. Ample evidence suggests that BCL-2 family proteins are critically involved in follicular maturation and atresia [4]. Loss of Bax expression led to a reduction in the average litter size and the number of oocytes ovulated in response to exogenous gonadotropin stimulation. These females also exhibited longer ovarian life in mice at old age [5,8,21,22]. Early studies have shown that the Bcl-2/Bax ratio controls ovarian cell fate determination of ovarian granulosa cells to apoptotic pathways [22]. In chemotherapy-induced POI mouse model, ovarian damage was associated with an increased level of Bax expression and decreased level of Bcl-2 expression [23,24]. Interestingly, our current study showed that mRNA expression levels of the proapoptotic factors Bax and TNF-α were significantly higher in POI patients, whereas the antiapoptotic factor Bcl-2 mRNA level was concomitantly reduced in these samples. Early studies indicate that IL-1 family proteins are involved in apoptotic pathways in many cell types by affecting BAX and Bcl-2 expression [25,26]. Hence, we postulate that IL-1-induced inflammation may promote ovarian follicle cell apoptosis by disturbing the BAX/BCL-2 balance. In support of this notion, we observed an increased level of TNF-α in POI patients. TNF-α is involved in apoptosis by means of the glutathione signaling pathway, ceramide signaling pathway, and calcium release in many cell types including ovarian follicular cells [27,28]. TNF-α regulates the process of follicular atresia by inducing the apoptosis of oocytes, stromal cells, and granulosa cells. In addition, TNF-α level in follicular fluid negatively correlated with embryo quality and pregnancy rate [29]. The number of apoptotic granulosa cells is closely associated with the serum levels of pro-inflammatory cytokines IL-6, IL-8, and TNF-α in chemotherapy-induced ovarian failure [30]. The apoptotic role of TNF-α in ovarian follicle cells of POI patients is under investigation in our laboratory.

The Fas/FasL system also plays an important role in regulation of granulosa cell apoptosis during the process of ovarian follicle atresia [6,31]. However, we did not observe any statistically significant changes in the Fas/FasL system between POI patients and healthy women. It remains elusive whether species heterogeneity contributes to this discrepancy.

Early studies showed that the IL-1β level in follicular fluid was positively correlated with the number of mature follicles and the number of eggs obtained [32]. IL-1β-KO female mice displayed a comparable but more subtle prolongation of ovarian lifespan [15]. In this study, the follicular IL-1β level in POI patients was higher than that in healthy women, but not statistically significant. The role of follicular IL-1 β on ovarian function in POI patient needs to be further investigated.

Our results also revealed that the levels of IL-1α and IL-1β in follicular fluids were significantly higher than those in serum. These results are consistent with the notion of “IL-1 ovarian origin” of these cytokines. Using western blot analysis and immunohistochemistry technology, Uri-Belapolsky [15] showed that IL-1α and IL-1β are detected in granulosa cells and oocyte cytoplasm throughout follicular development [33,34]. It remains intriguing to identify the causal factors that induce production IL-1α and IL-1β in ovarian follicles of POI patients [35].

In summary, our studies for the first time provided the clinical evidence that IL-1 levels in follicular fluids and serial samples of POI patients are significant higher than healthy women. We propose a mechanism by which IL-1 α-mediated inflammatory response promotes ovarian follicle apoptosis and adversely impact ovarian functions in POI patients. Our studies clearly indicate that IL-1α emerges as a new diagnostic and therapeutic target for POI and POI-associated disorders.

Disclosure of conflict of interest

None.