Introduction
Dysmenorrhea, painful cramps of uterine origin during menstrual periods (1), is the major cause of emergency department visits in reproductive-age women regardless of age, race, nationality, and socioeconomic status. It is classified as primary dysmenorrhea (PD) (menstrual pain with no organic cause) or secondary dysmenorrhea (menstrual pain due to pelvic disease) (2). The cause of PD is still not clear. Hyperproduction of uterine prostaglandin (PG), especially of PGF2a and PGF2 is held responsible for the pathophysiology. High levels of these PGs lead to increased uterine tone and high-amplitude myometrial contractions (3). PG synthesis is regulated by progesterone; when progesterone is low, as in right before the menstruation, PG levels rise (3). It has been demonstrated that PGs are involved in variety of biological actions both physiological and pathological such as pain, inflammation, body temperature, and sleep regulation (3). Strömberg et al. (4) reported, markedly higher plasma vasopressin and PGF2α metabolite levels on the first day of menstruation in women with severe PD. In their study including 43 women with dysmenorrhea and 51 controls, Yeh et al. (5) found that plasma oxytocin and interleukin-6 (IL-6) levels were significantly higher in patients with dysmenorrhea than in controls. Uterine contractility might be increased by these mediators playing important roles in PD pathophysiology (6).
Interleukin-4 (IL-4), a cytokine with several biological functions, is secreted chiefly by activated T cells and monocytes, basophilic granulocyte, and mast cells (7). It is the first discovered B-cell pleiotropic cytokine induces proliferation of T cells and antibody synthesis in B cells and has a significant role in immune system (8). The third intron of the IL-4 gene has variable number of tandem repeats (VNTR) polymorphism with 70 bp occurring as 2 to 4 repeats. The most frequent IL-4 VNTR allelic form has three repeats (allele 1), whilst there is a less common allele that has two repeats (allele 2), and a rarer allele with four repeats (allele 3) (9). Several studies have reported a link between VNTR P1 allele and inflammatory diseases (10).
Given possible defect in IL-4 function in PD, it was decided to examine the IL-4 VNTR polymorphism in patients with PD.
Methods
Study Population
Protocols of the study were in line with the principles of the Declaration of Helsinki. The present study was approved by Gaziosmanpaşa University, Medical Faculty (approval no: 18-KAEK-260). The study sample included unrelated female patients with the clinical diagnosis of PD admitted to the Emergency Medicine Department of Gaziosmanpaşa University Research Hospital. Patients with menstrual cycles lasting 21-35 days and menstrual period lasting 3-7-day, who experienced painful menstruations in the past five years with pain starting 1 day prior to or on the menstruation day, were enrolled in the study. Gynecological exam revealed no pathology, no gastrointestinal, gynecologic, or autoimmune diseases, and previous pelvic operation. Unrelated healthy women with no gynecological pathologies found in annual gynecologic examination were included in the study. They had no regularly used drugs (i.e. nonsteroidal anti-inflammatory drugs/oral contraceptives). The patients and healthy controls were Turkish, living in the Black Sea region of Turkey. All participants gave informed written consent before participating in the study.
Confounders
A clinical data collection form was created, and age, age at menarche, marital status, family history of dysmenorrhea, children, smoking status, alcohol consumption, history of rheumatoid arthritis (RA), familial Mediterranean fever, arthralgia and lumbar pain, effect of dysmenorrhea on school performance, oral drug use, intramuscular medication use and Life Satisfaction Scale score were recorded. Life satisfaction refers to as a person’s subjective, global evaluation of the positivity of his/her life as a whole or with specific domains of life (e.g. family life, school experiences). The Life Satisfaction Scale is used extensively as a measure of life satisfaction component of subjective well-being with a minimum score of 3 and maximum 8; the lower score indicates lower life satisfaction.
Genotyping
Genomic DNAs from whole blood of patients and the control group taken with standard procedures (Sigma-Aldrich, St. Louis, MI, USA) were kept at -20 °C. Analyses on VNTR polymorphism of IL-4 were made in line with previously described protocols (9). A 25 µL reaction mixture with 50 ng DNA, 0.8 µM of primers, 200 µM of each dNTP, 2.5 mMMgCl, 1.5 units of Taq polymerase, and 2.5 µL10× KCl buffer were used for polymerase chain reaction (PCR). Forward 5¢ AGG CTG AAA GGG GGA AAG C3¢ and reverse 5¢-CTG TTC ACC TCA ACT GCT CC-3¢ primers were used for amplification in the first denaturation at 95 °C for 5 minutes, 30 denaturation cycles at 94 °C for 30 s, annealing at 58 °C for 45 s, extension at 72 °C for 1 minute, and the last extension at 72 °C for 10 minutes. The separation of PCR products was carried out with 3% agarose gel, and visualization was carried out with ethidium bromide staining. PCR products were 183 bp for P1 allele, and 253 bp for P2 allele.
Statistical Analysis
The Statistical Package for the Social Sciences (IBM SPSS, version 20), and OpenEpi Info package v 3.01 (www.openepi.com) were used for statistical analyses. All data were expressed as mean ± standard deviation. The relationships between IL-4 VNTR polymorphism and clinical and demographical characteristics of patients were analyzed with c2 test, Fisher’s exact test, or ANOVA test. To evaluate risk factors, Odds ratios (ORs) and 95% confidence intervals (CIs) were employed. All p values were binary. A p value of less than 0.05 was considered statistically significant. Additionally, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis was added (Figure 1).
Results
A total of 236 subjects [120 PD females (mean age: 25.64±4.24 years) and 116 healthy females (mean age: 25.76±4.23 years)] were genotyped for IL-4 VNTR polymrphism. Baseline clinical and demographic features of the patient and control groups are given in Table 1.
The IL-4 VNTR polymorphism genotype and allele frequency distribution in study and control groups is given in Table 2. The 3 IL-4 genotypes were classified as: P1/P1 (183 bp), P2/P2 (253 bp) and P1/P2 (183 and 253 bp fragments). The frequencies of P1/P1, P1/P2, and P2/P2 genotypes of VNTR variant in patients were 1.72%, 34.4%, and 63.7% and in controls were 0.8%, 26.6%, and 72.5%, respectively. The distribution of genotype of IL-4 VNTR polymorphism was not statistically different between PD patient and control groups (p>0.05). P1 and P2 allele frequencies were; 18.9% and 81.0% in patient group, respectively; and 14.1% and 85.8% in the control group, respectively. There was no significant difference in the allele frequencies of IL-4 VNTR between the study and control groups (p>0.05, OR: 0.705, CI 95%: 0.43-1.15).
The STRING database collects scores and integrates all publicly available sources of protein-protein interaction information. Analyzing the IL-4 protein with STRING database, predicted the functional partners of the protein with high confidence (score: 0.7) as: IL-6, TNF, IL1-B, CXCL8, IL-4R, CCL2, IL-18, IL-13RA1, STAT6, IL-2RG.
Figure 1 shows the interaction network of these proteins.
Discussion
Menstruation is a physiological event that occurs in cycles. Various problems might occur (irregular menstruation, excessive hemorrhage, and dysmenorrhea). Dysmenorrhea is generally defined as severe, painful, cramping sensation in the lower abdomen frequently associated with perspiration, headache, nausea, vomiting, and diarrhea (11). Its worldwide prevalence varies between 28% and 71.7% (12,13). In a study conducted with university students in Turkey, the prevalence was found to be 87.7% (14). There is a scarce number of studies about genetic susceptibility to dysmenorrhea. A study involving monozygotic/dizygotic Australian twins by Treloar et al. (15) showed that genetic factors were responsible for 39% of longitudinally stable variation in menstrual flow, 55% for pain, and 77% for perceived limitations.
Pre-clinical studies assume that PG-dependent events induce dysmenorrhea in most women. The onset of menstruation is associated with simultaneous reduction in circulating progesterone and estradiol levels, promoting increased transcription of endometrial collagenase, matrix metalloproteinase, and inflammatory cytokines (16). Peripheral blood analysis in dysmenorrheal women showed extravagant production and levels of oxytocin, PGF2α, vasopressin and IL-6 (4,5,17,18). PGs are important inflammation mediators. Along with its role as a vasodilator, it has been reported that PGE2 synergizes with IL-8 (19), and that progesterone withdrawal, hypoxia, and PGE2 modulate endometrial IL-8 by acting through HIF-1 and NF-κB (20). Menstruation can be considered an inflammatory process manifested by tissue demolition and involvement of inflammatory cells. What is more, there is a significant increase in leukocytes, especially in neutrophils, uNK cells and macrophages in the endometrium where they constitute maximum 40% of stromal cells just before menstruation (21).
IL-4, a pleotropic cytokine encoded by a gene found on the long arm of chromosome 5, is believed to be important for Th2 responses because it controls the differentiation of precursor T helper cells into Th2 subset mediating humoral immunity and modulate antibody generation. Besides, IL-4 hinders the synthesis of the proinflammatory cytokines including TNF-a, IL-6 and IL-1a and destructive enzymes by monocytes and this emphasizes its strong anti-inflammatory effect (22). There are VNTRs in cytokine genes and various single nucleotide polymorphisms that could have impact on genetic predisposition to some disorders and carcinomas. The 70-base-pair (bp) VNTR variant in the third intron of the IL-4 gene may modify the expression level of this gene, with P1 allele increasing IL-4 expression compared to P2 allele (9). Studies on the relationship between VNTR variant of IL-4 gene have been conducted in different populations having several different morbidities with contradictory outcomes. Numerous research have reported the relationship IL-4 VNTR polymorphism with immunologic diseases such as RA (23), systemic lupus erythematosus (24), vitiligo (25), multiple sclerosis (26), alopecia areata (27), and recurrent aphthous stomatitis (28). There are some studies suggesting a relationship between dysmenorrhea and inflammation. In their study, Ma et al. (6) suggested that underlying inflammatory responses vary in patients with dysmenorrhea even in the lack of pain, which may be an etiological factor in dysmenorrhea. Dogru et al. (29) assessed the relationship of MIF-173 and TNF-308 genetic variants with clinical characteristics of PD. They found a statistically significant relationship between gene MIF-173G/C polymorphism and age at menarche and history of back pain in patients with dysmenorrheal (p=0.003 and p=0.042, respectively). Furthermore, they reported that genotype and allele frequencies of TNF-308G/A polymorphism were significantly different between dysmenorrhea patients and controls (p=0.023 and p=0.009, respectively). Erten et al. (30) reported that there was a significant increase in MEFV gene mutation frequency in dysmenorrhea patients compared with controls. MEFV gene encodes a protein, pyrin, involving in inflammatory response (31). However, Ozsoy et al. (32) suggested that there was no significant association between IL-6 gene promoter -572G/C and -597G/A polymorphisms and PD.
The study aimed to determine the relationship between 70-bp VNTR variant of intron-3 of IL-4 gene and PD development. It was found that all subjects included had two alleles (P1 and P2). No significant differences were detected in terms of genotype and allele frequencies in IL-4 VNTR polymorphism between the groups.
Study Limitations
This study has several limitations. One of the limitations of our study is the relatively small sample size. The population studied consisted of only Turkish subjects. Due to interethnic variability, further studies on other ethnic populations are needed for validation of our findings.
Conclusion
Dysmenorrhea is a common problem among young women. Our results suggest that IL-4 VNTR polymorphism does not contribute to the PD etiopathogenesis. Unexplained mechanisms and multiple factors involved in the pathogenesis of PD necessitate further studies on this issue.
Authorship Contributions
Concept: A.F.N., S.Y. Design: S.Y., M.E. Data Collection or Processing: M.E. Analysis or Interpretation: S.Y., E.D. Literature Search: A.F.N., S.Y. Writing: A.F.N.
Conflict of Interest: The authors declared no conflicts of interest concerning the authorship and publication of this article.
Financial Disclosure: The authors declared that this study received no financial support.