The rs2275913 polymorphism of the interleukin-17A gene is associated with the risk of ovarian endometriosis

Abstract The aim of this study was to explore the relationship between two genetic variants (rs2275913 and rs3748067) of interleukin-17A (IL-17A) and the risk of ovarian endometriosis in northern Chinese women. This study was performed in 316 patients with ovarian endometriosis and 328 female control subjects. The genotypes of the two polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The mRNA expression of IL-17A was detected by quantitative real-time PCR (qRT-PCR). There was a significant difference in the genotype distributions of the rs2275913 polymorphism between the patients and control subjects (p = .006). Compared with the GG genotype of rs2275913, the AA genotype was correlated with a higher susceptibility to the development of ovarian endometriosis (OR = 2.28, 95% confidence interval (CI) = 1.37–3.80). Furthermore, the levels of IL-17A mRNA were higher in the ectopic endometrium from ovarian endometriosis patients carrying the rs2275913 AA genotype than in those carrying the rs2275913 GG genotype (p = .007). This study suggested that the rs2275913 polymorphism at the promoter region of IL-17A may be a functional genetic variant, and the rs2275913 AA genotype is associated with a higher risk of developing ovarian endometriosis in northern Chinese women. IMPACT STATEMENT What is already known on this subject? Interleukin-17A (IL-17A) is a crucial proinflammatory and angiogenic cytokine that has been shown to participate in the pathogenesis of chronic inflammatory and autoimmune diseases. Genetic variants in IL-17A may alter the expression of the IL-17A, and are associated with the susceptibility to a wide range of human diseases. What do the results of this study add? This study suggested that the rs2275913 polymorphism at the promoter region of IL-17A may be a functional genetic variant that affects the expression of IL-17A mRNA in ectopic endometrial tissues. The AA genotype of rs2275913 was associated with a higher risk of developing ovarian endometriosis in northern Chinese women. What are the implications of these findings for clinical practice and/or further research? IL-17A may be a potential biomarker for early diagnosis and gene-targeted therapy of ovarian endometriosis.


Introduction
Endometriosis is a common chronic inflammatory disease affecting approximately 10% of women of reproductive age (Giudice 2010).The predominant symptoms of this disease include pelvic pain and infertility, which greatly affect the patients' health and quality of life.Sampson's retrograde menstruation is the most widely accepted theory to account for the pathogenesis of endometriosis.However, this hypothesis fails to explain why only 10% of females develop endometriosis when most women experience retrograde menstruation.Increasing evidence suggests that inflammation and immune responses also play an important role in the occurrence and development of endometriosis (Guo et al. 2016, Jiang et al. 2016).Interleukin-17A (IL-17A) is a crucial proinflammatory and angiogenic cytokine that has been shown to participate in the pathogenesis of chronic inflammatory and autoimmune diseases (Song and Qian 2013).Recent studies reported that the levels of IL-17A were significantly higher in the serum and peritoneal fluid of women with endometriosis than in the control subjects (Zhang et al. 2005, Sikora et al. 2018).IL-17A could promote the proliferation of endometriotic stromal cells by inducing the production of interleukin-8 and cyclooxygenase-2 (Hirata et al. 2008).In addition, IL-17A has been suggested to be critical in promoting angiogenesis and a proinflammatory environment in the peritoneal cavity for the establishment and maintenance of endometriosis lesions (Ahn et al. 2015).These reports indicated that IL-17A may be involved in the pathogenesis of endometriosis.
The human IL-17A gene is located on chromosome 6p12.2,and several single nucleotide polymorphisms (SNPs) have been identified in this gene.Among these SNPs, two widely studied polymorphisms (rs2275913 and rs3748067) have been found to be associated with susceptibility to a wide range of human diseases.For example, the A allele of the rs2275913 polymorphism may significantly increase the risk of colorectal cancer and asthma (Zhai et al. 2018, Zhang andWang 2020).The rs3748067 polymorphism was correlated with susceptibility to gastric cancer and cervical cancer (Qinghai et al. 2014, Niu et al. 2017).However, little is known regarding the contribution of IL-17A polymorphisms to the risk of endometriosis.
In this study, we explored the relationship between the two genetic variants of IL-17A and the risk of ovarian endometriosis in northern Chinese women.We also examined the mRNA levels of IL-17A in the ectopic endometrial tissues of ovarian endometriosis patients carrying different genotypes of the two polymorphisms.

Ethics statement
This case-control study was performed with the approval of the ethics committee of the People's Hospital of Xingtai.Written informed consent was acquired from all the participants according to the Helsinki declaration.The study was conducted in accordance with Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) criteria.

Study participants
A total of 316 patients with ovarian endometriosis and 328 control subjects were recruited for this study.All patients underwent surgical treatment of ovarian endometriosis at the People's Hospital of Xingtai from January 2016 to June 2020 and were confirmed by histological examination.Based on the revised American Fertility Society classification (1997), all patients were diagnosed with endometriosis stage III-IV.The control group included reproductive age females without endometriosis confirmed by either of the following methods: caesarean section (n ¼ 92), total laparoscopic hysterectomy for cervical intraepithelial neoplasia III (n ¼ 121), or laparoscopic surgery for hydrosalpinx (n ¼ 115).Women with a history of malignant disease or autoimmune disease were excluded from this study.All study participants were women of Han ethnicity in northern China, and detailed clinical information was extracted from their medical charts.
For gene expression analysis, 55 ectopic endometrial tissues and 26 eutopic endometrial tissues from above-mentioned ovarian endometriosis patients were obtained at the time of surgical treatment for ovarian endometriosis.In addition, 36 healthy endometrial tissues were collected from individuals without endometriosis.All samples were collected during the secretory phases of the menstrual cycle, which was estimated according to the date of the last menstrual phase or by histological evaluation (Noyes et al. 2019).None of them had received any hormonal treatment for a minimum of 6 months prior to surgery.All endometrial tissue samples were stored in RNAlater solution (Ambion, Carlsbad, CA, USA) immediately after surgical removal and then stored at -20 � C.

DNA extraction and genotyping
Five millilitres of venous blood was drawn from each participant into vacutainer tubes containing ethylenediaminetetraacetic acid and then stored at 4 � C. Genomic DNA was extracted from the collected blood samples using proteinase K digestion followed by a salting out procedure, as previously described by Miller et al. (1988).Genotypes of rs2275913 and rs3748067 were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.The primer sequences for PCR amplification of rs2275913 were (forward) 5 0 -GCA TAA CTC TTC TGG CAG CTG TA-3 0 and (reverse) 5 0 -TGC CCA CGG TCC AGA AAT AC-3 0 .The primer sequences for rs3748067 were (forward) 5 0 -TAT GGG GAA AAT GAA ACC-3 0 and (reverse) 5 0 -GTA GGG CAA GAC AGC ACA-3 0 .PCR was carried out in a 20 lL volume containing 50 ng of DNA, 2 lL of 10 X PCR buffer, 200 nM of each primer, 0.4 lL of 10 mM dNTPs, 1 U of Taq polymerase (Tiangen Biotech, Beijing, China), 2 lL of MgCl 2 (25 mM) and nuclease-free water.The cycling conditions for PCR were as follows: 94 � C for 5 min; followed by 35 cycles of 94 � C for 45 s, 57 � C for 45 s for rs2275913 and 60 � C for 45 s for rs3748067, 72 � C for 45 s; and a final elongation at 72 � C for 7 min.The PCR products were subjected to digestion for 16 h at 37 � C in a 10 lL reaction containing 10 U restriction enzyme XmnI (Thermo Scientific, Waltham, MA, USA) for the rs2275913 polymorphism and 10 U ApoI (Thermo Scientific, Waltham, MA, USA) for the rs3748067 polymorphism.Digested PCR products were observed on a 2% agarose gel and visualised under ultraviolet light.

Quantitative real-time reverse transcriptase-PCR
Total RNA was isolated from endometrial tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).Total cDNA was synthesised by the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions.Quantitative real-time PCR (qRT-PCR) was carried out on an ABI StepOnePlus Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using a QuantiNova TM SYBR V R Green PCR Kit (Qiagen, Hilden, Germany).The qPCR cycling conditions were 2 min at 95 � C followed by 40 cycles of 5 s at 95 � C, 10 s at 60 � C, and 25 s at 72 � C, with a final step at 95 � C for 15 s, 60 � C for 60 s, and 95 � C for 30 s. GAPDH was used as internal control.The PCR primers were as follows: (a) IL-17A: 5 0 -TCC CAC GAA ATC CAG GAT GC-3 0 (forward) and 5 0 -GGA TGT TCA GGT TGA CCA TCA C-3 0 (reverse); and (b) GAPDH: 5 0 -ACC ACA GTC CAT GCC ATC AC-3 0 (forward) and 5 0 -TCC ACC ACC CTG TTG CTG TA-3 0 (reverse).The relative amount of IL-17A mRNA was calculated with the 2 -DDCt method, and all experiments were repeated three times.

Statistical analysis
Statistical analysis was conducted using the SPSS software package (version 21.0; SPSS Inc., Chicago, IL, USA).Hardy-Weinberg equilibrium (HWE) was assessed to compare observed and expected genotype frequencies of the control subjects by chi-square test.Genotype frequencies in the case and control groups were analysed using the chi-square test with Bonferroni's correction.The odds ratio (OR) and 95% confidence interval (CI) were evaluated by unconditional logistic regression models.The comparisons of IL-17A mRNA levels in different groups and different genotypes were performed using the Mann-Whitney U-test with Bonferroni's correction.Statistical significance was set as a p value less than .05(two-sided).

General characteristics of study subjects
The clinical characteristics of study participants are shown in Table 1.There were no significant differences between patients and control subjects with regard to age, age at menarche, and smoking history (all p values >.05).However, the patients with ovarian endometriosis manifested a lower body mass index (p < .001),longer duration of menstruation period (p < .001),shorter length of menstrual cycle (p ¼ .011)and higher rate of infertility (p < .001).Genotype distributions of the two polymorphisms in the control subjects demonstrated no significant deviations from their expected for Hardy-Weinberg's equilibrium (p ¼ .988and .777,respectively).

Association of rs2275913 and rs3748067 with the risk of ovarian endometriosis
The genotype distribution frequencies of rs2275913 and rs3748067 in cases and controls are listed in Table 2.There was a significant difference in the genotype distributions of the rs2275913 polymorphism between the two groups (p ¼ .006).Compared with the GG genotype of rs2275913, the AA genotype was correlated with a higher susceptibility to the development of ovarian endometriosis (OR ¼ 2.28, 95% CI ¼ 1.37-3.80,p ¼ .001).After adjustment for BMI, the rs2275913 AA genotype was still associated with a higher risk of developing ovarian endometriosis than the rs2275913 GG genotype (OR ¼ 2.15, 95% CI ¼ 1.25-3.70,p ¼ .006).However, no significant difference was found in the frequency distributions of rs3748067 genotypes between the patients and control subjects (Table 2).

Expression of IL-17A in endometrial tissues from the cases and controls
To assess the IL-17A mRNA expression, we examined 55 ectopic endometrial tissues and 26 eutopic endometrial tissues from patients with ovarian endometriosis and 36 healthy endometrial tissue samples from control subjects using qRT-PCR.The mRNA levels of IL-17A were significantly higher in the ectopic endometrium than in the eutopic and control endometrium (Figure 1; p ¼ .010and .015,respectively), but were not significantly different between the eutopic endometrium and controls (Figure 1; p ¼ .875).

Effect of rs2275913 and rs3748067 on IL-17A expression
The qRT-PCR results showed that the expression of IL-17A mRNA was significantly different in the ectopic endometrium of ovarian endometriosis patients with different genotypes of rs2275913.The levels of IL-17A mRNA were higher in the ectopic endometrium of patients carrying the rs2275913 AA genotype than in those carrying the rs2275913 GG genotype (Figure 2(A); p ¼ .007).However, there were no significant differences in the levels of IL-17A mRNA between the different genotypes of rs3748067 (Figure 2(B)).

Discussion
Ovarian endometriosis is a common disorder observed in women of reproductive age.For presumed benign ovarian tumours, fertility sparing should be the primary concern, like in early stage endometrial cancer patients (Cavaliere et al. 2021, Gullo et al. 2021, Tanos et al. 2022).Although much work has been done in the field of endometriosis-related infertility, these approaches have associated failure rates, and do not completely remove the influence of this disease (Prapas et al. 2017, Gullo et al. 2020).Nowadays, research has focussed on identifying new molecular targets, which help to understand the systemic nature of endometriosis.In this case-control study, we examined the association between two genetic variants of the IL-17A gene and the risk of ovarian endometriosis.The results showed that the rs2275913 polymorphism was correlated with susceptibility to ovarian endometriosis.Compared to the GG genotype of rs2275913, the AA genotype was associated with a higher risk of developing ovarian endometriosis.Furthermore, the mRNA levels of IL-17A were significantly higher in the ectopic endometrium from ovarian endometriosis patients carrying the rs2275913 AA genotype than in those carrying the rs2275913 GG genotype.To the best of our knowledge, this is the first molecular epidemiological study to explore the relationship between genetic polymorphisms of IL-17A and ovarian endometriosis in northern Chinese women.The rs2275913 G/A polymorphism is located in the promoter region of the IL-17A gene.Considerable studies have suggested that this variant exerts an effect on the regulation of IL-17A production.Espinoza et al. (2011) revealed that the A allele of rs2275913 exhibited a higher affinity for the transcription factor NFAT and had a higher transcriptional activity of IL-17A than the rs2275913 G allele.Borilova Linhartova et al. (2016) demonstrated that rs2275913 AA þ AG genotype carriers had higher IL-17A levels in mononuclear cells than those with the rs2275913 GG genotype in chronic periodontitis patients.Similarly, the results of our study also showed that the levels of IL-17A mRNA were significantly increased in the ectopic endometrial tissues from ovarian endometriosis patients with the rs2275913 AA genotype compared with those with the rs2275913 GG genotype.These findings further indicated that the rs2275913 polymorphism may be a functional genetic variant.The relationship between rs2275913 and susceptibility to diseases has been investigated in numerous studies.However, the results were quite different.The A allele of rs2275913 was found to be correlated with a higher risk of preeclampsia and coronary artery disease (Ghaznavi andSoltanpour 2020, Lang et al. 2021).However, there was no significant association between this polymorphism and susceptibility to periodontitis and cervical cancer (Niu et al. 2017, Mazurek-Mochol et al. 2021).In the present study, we found that the frequency of the rs2275913 AA genotype was significantly higher in ovarian endometriosis patients than in control subjects.Moreover, the levels of IL-17A mRNA were significantly higher in the ectopic endometrium than in the eutopic and control endometrium.Our data suggest that the AA genotype of rs2275913 may act as a risk factor for the development of ovarian endometriosis by increasing the expression of IL-17A.The rs3748067 C/T polymorphism is located in the 3ometrium than in the eutopic and contIL-17A gene.Previous studies have demonstrated that the C allele of rs3748067 contributes to a higher risk of developing gastric cancer and coronary artery disease (Arisawa et al. 2012, Bao et al. 2016).However, no correlation was found between this genetic variant and the risk of breast cancer, laryngeal cancer and lung cancer (Wang et al. 2012, He et al. 2017, Si et al. 2017).Similarly, our study also showed that there were no significant differences in the genotype distributions of rs3748067 between ovarian endometriosis patients and control subjects.Furthermore, the mRNA levels of IL-17A were not significantly different in ectopic endometrium from ovarian endometriosis patients carrying the different genotypes of rs3748067.These data indicated that the rs3748067 polymorphism may have no effect on the risk of developing ovarian endometriosis in our study population.Of course, further studies are necessary to clarify the role of rs3748067 in the risk of diseases.
The main limitation of this study is the relatively small sample size, which involved only 316 patients with ovarian endometriosis and 328 control subjects.We could not exclude the possibility that the positive findings occurred by chance or selection bias.Therefore, larger multicentre and population-based studies are necessary to further verify our results.In addition, growing evidence suggests that endometriosis is a risk factor for developing ovarian cancer (Pearce et al. 2012, Lu et al. 2015).Several studies have shown a consistent epidemiological association between endometriosis and risk of ovarian cancer (Rueda-Mart� ınez et al. 2021).The genetic background of ovarian cancer has been extensively examined, and many validated genetic variants have been described (Pharoah et al. 2013, Earp et al. 2014).However, the effects of the two polymorphisms of IL-17A on the risk of ovarian cancer were not explored in this study.
In conclusion, our study suggested that the rs2275913 polymorphism at the promoter region of IL-17A may be a functional genetic variant that affects the expression of IL-17A mRNA in ectopic endometrial tissues.The AA genotype of rs2275913 was associated with a higher risk of developing ovarian endometriosis in northern Chinese women.Of course, well-designed and greater-scale studies are needed to strengthen these findings.

Figure 2 .
Figure 2. Effect of IL-17A polymorphisms on the expression of IL-17A in the ectopic endometrium of ovarian endometriosis patients.Data were expressed as median with interquartile range, and analysed by the Mann-Whitney U-test with Bonferroni's correction.A: rs2275913; B: rs3748067.

Figure 1 .
Figure 1.The mRNA expression of IL-17A in the 55 ectopic endometrial tissues and 26 eutopic endometrial tissues from patients with ovarian endometriosis and 36 healthy endometrial tissue samples from control subjects.Data were expressed as median with interquartile range, and analysed by Mann-Whitney's U-test with Bonferroni's correction.

Table 1 .
Clinical characteristics of women with ovarian endometriosis and controls.

Table 2 .
Association of rs2275913 and rs3748067 with the risk of ovarian endometriosis.
OR: odds ratio; CI: confidence interval.a Adjusted for body mass index.