Effect of estrogen receptor gene ESR1 polymorphism on development of premenstrual syndrome.

To identify risks of development of any disease is a priority of modern medicine. The aim of this study was to investigate the frequency of polymorphic variants of A-351G gene estrogen receptor ESR1 in patients with various forms of PMS. Molecular genetic analysis of ESR1 gene polymorphism in 50 women with PMS (25 women of them had edematous form of disease, 25 - neuropsychical, 25 - mild, 25 - severe form) was carried out. 25 women without diagnosis of PMS were examined as controls. The study A-351G polymorphism estrogen gene ESR1 showed no statistically significant differences in the frequency of distribution of genotypes and alleles between women with PMS and without this pathology. However, the frequency of GG genotype in women with severe PMS was significantly higher in 8.0 times compared with healthy women (χ2=4.87, p=0.03) and in women with edematous form of PMS - in 7.0 times (χ2=3.72, p=0.05). Thus, a polymorphic variant of A-351G gene ESR1 estrogen can be considered as a marker of PMS. Pathological variant GG genotype was significantly associated with the presence of edematous and severe forms of the disease.

Hormonal aspects of PMS are convincing. One of the first theories of development of this disease is the theory hyperestrogenemy. Estrogens exert their effects on cells by binding with α or β receptors. These receptors are encoded by genes ESR1 and ESR2. Expression of these genes is located not only female in genital tissues, but also in cells of the prostate, heart, lungs, kidneys, etc. [4]. There are only some reports about of ESR gene polymorphism in the development of gynecological pathology. This is possibly due to the relatively recent tendency of research and development of new technologies in medicine. There is a close relationship of A/G polymorphism of ESR1 with endometriosis and infertility on its background and ESR2 gene polymorphism is not associated with development of this disease [11]. In women with infertility and low response to ovarian stimulation genotype AG gene ESR1 was found most frequently [3]. ESR1 gene polymorphism may be associated with the development of dysmenorrhea [12], migraine [7,9], the regulation of behavior and mood of women [8,10].
The objective: investigate the frequency of polymorphic variants of A-351G gene estrogen receptor ESR1 in patients with various forms of PMS.

MATERIAL AND METHODS
The study included 200 women with premenstrual syndrome, which formed basic group. The control group consisted of 50 healthy women without diagnosis of PMS. Verification of diagnosis and severity of disease (mild and severe) was performed according to Order of Ministry of Health of Ukraine № 676 from 31.12.2004 [5]. Diagnosis of PMS was exhibited by presence of cyclical manifestations of the disease in luteal phase of menstrual cycle on the basis of history-taking and results of patient's self-observation diary for 2-3 menstrual cycles (R. Moos Menstrual Distress Questionnaire). Form of PMS (edematous, neuropsychical, cephalgic, crisis) was determined in accordance with the classification of V.P. Smetnik's [6]. Clinical examination was carried out on the basis of Ivano-Frankivsk clinical maternity hospital (Ivano-Frankivsk, Ukraine).
The criteria for inclusion of patients in research were: the reproductive age (18-44 years), regular menstrual cycles, presence of PMS, written consent of the patient. Exclusion criteria: women who had at the time of the study pregnancy or lactation, disorders of menstrual cycle, focal lesions of breast, dysfunctional uterine bleeding of unknown etiology, acute inflammation of pelvic organs, tumors of uterus and ovaries of unknown etiology, endometrial hyperplasia, genital endometriosis, severe somatic pathology in the history, organic pathology of the central nervous system, mental illness, hormonal tumors, diabetes, adrenal diseases, malignant tumors in the present or in anamnesis, premenstrual dysphoric disorder, women who took psychotropic medications or hormonal therapy within the last 3 months.
The average age of women in control and basic groups was not statistically different and was respectively 28.82±0.76 and 30.13±0.36 years (p=0.08). Age of menarche corresponded in two groups -12.94±0.13 and 12.86±0.06 years and had no differences depending on the form of PMS (p>0.05). We found The frequency of polymorphic variants of A-351G estrogen receptor gene ESR1 among examined women  Given that the most common forms of PMS are edematous and neuropsychical, we performed molecular genetic analysis of ESR1 gene polymorphism in 50 women of basic group, 25 of them had edematous form of disease, 25 -neuropsychical, 25 -mild, 25 -severe. Also 25 women of control group were examined.
A-351G polymorphism of the gene ESR1 estrogen was determined in the research laboratory, Department of Medical Genetics, Shupyk National Medical Academy of Postgraduate Education (Kyiv). Material for the study was peripheral blood, which was collected into tubes with EDTA in amount of 2.7 ml, then DNA was isolated using a commercial set «DNA-sorb-B» (Institute of Epidemiology of the Ministry of Health of Russian Federation). After the polymerase chain reaction with the reagents of the company Fermentas (Lithuania) in thermocycler «FlexCycler» (Analytik, Jena, Germany) restriction fragment length polymorphism was analyzed. The amplification products of A-351G gene ESR1 fragments were splitted using restriction endonuclease PvuII. Then, electrophoresis of resulting fragments in 2% agarose gel with the addition of ethidium bromide and visualization by computer system Vitran were performed.
For statistical analysis of the results we used the criterion χ 2 , odds ratio (OR), confidence interval (CI).

RESULTS AND THEIR DISCUSSION
We found no statistically significant differences in the distribution of allele and genotype frequencies of A-351G polymorphic estrogen receptor gene ESR1 between women of control and basic groups (see fig. 1). The frequency of heterozygous genotype AG was similar in both groups, but among healthy women met at 1.50 times more than in patients with PMS and reached 60.0% and 40.0%, respectively (χ 2 =1.93; p=0.16, OR=0.44, 95% CI 0.17-1.18; p=0.10).
In women with edematous, neuropsychical and mild forms of disease AG genotype was predominant (table 1).
In women of basic group GG genotype was determined at 5.50 times more than of control one (22.0% and 4.0%, respectively), but the differences did not reach statistical significance (χ 2 =2.79; p=0.09, OR=6.77, 95% CI 0.82-55.79, p=0.07). Mark OR of the distribution of GG genotype in women with PMS compared with healthy women greater than 1, namely 6.77, may indicate a possible association of AG polymorphism in the development of the disease (Table 3).
Women with edematous form of PMS had significantly higher in 7.0 times frequency of GG genotype (28.0%) than in control group (χ 2 =3.72; p=0.05). A frequency of GG genotype in women with severe PMS was significantly higher in the 8.0 times compared with healthy women, and accounted for 32.0% (χ 2 =4.87, p=0.03). The odds ratio of groups with edematous and severe forms of PMS compared with control group is high (OR=9.33-11.29, p=0.03-0.04), and points to the link of GG genotype with the occurrence of a certain form of the disease.
Thus, GG genotype may be regarded as a marker for increased risk of PMS, namely, its severe and edematous forms.
The frequency of the homozygous AA genotype was similar in the two groups of patients (38.0% of women with PMS and 36.0% of healthy women) and did not differ significantly depending on the clinical form and course of the disease.
G allele was determined in 31 (62.0%) women with PMS and 16 (64.0%) healthy women, A allele -in 49 (98.0%) and 24 (96.0%) persons, respectively. Distribution of G allele in women with various forms of PMS was approximately the same: in patients with edematous form it was determined in 16 (64.0%) women, neuropsychical -15 (60.0%), mild -17 (68.8%), severe -14 (56.0%). A allele was found in 18 (72.0%) women with edematous form of PMS, 21 (84.0%) -neuropsychical, 22 (88.0%) -mild, 17 (68.0%) -severe. Statistically significant differences between groups were not observed.  Table 3 Genotypes A-351G polymorphism of estrogen receptor gene ESR1 as risk markers of PMS Estrogen receptor gene ESR1 polymorphism as a factor of development of PMS is studied poorly. N.V. Aganezova indicates approximately the same distribution of genotypes A/G gene ESR between women with PMS and without this pathology, which corresponds to the results of our study. The author confirms that for women with PMS and genotype GG are characterized by mood swings, as well as such phenomena as affective lability, tendency to asthenic, hypochondriacal, anancastic features [1]. Therefore, this genotype she considers as «a marker which indirectly predisposes originality emotional and personal characteristics of women with PMS» [2].

CONCLUSION
A polymorphic variant of A-351G gene estrogen receptor ESR1 can be regarded as a marker for the development of PMS. Pathological variant GG genotype was significantly associated with the presence of edematous and severe forms of the disease.