Effect of Metformin Treatment on some Blood Biomarkers in Women with Endometriosis

Endometriosis is a common women health disorder that occurs when Endometrial-like tissue grows outside the uterus. This may lead to irregular bleeding , pelvic pain, infertility and other complications. Metformin, because of its activity to improve insulin sensitivity, it is used for the treatment of diabetes; it also has a modulatory effect on ovarian steroid production and has antiinflammatory properties, all may suggest its possible effect in treatment of endometriosis. This study was planned to determine the effect of metformin on serum levels of interleukineight(IL8), Tumor necrosis factor-alpha (TNF –α) and estradiol (E2) production , and related symptomatic changes that accompany with endometriosis (pelvic pain, dysmenorrhea and menorrheaga) after three months of study. Blood samples were obtained from those taking metformin and measure the serum levels of (IL-8) , TNF –α and (E2) were measured before and after three months of taking a metformin .Metformin therapy resulted in a significant reduction in the clinical symptoms of endometriosis (pelvic pain and dysmenorrhea) and insignificant changes in menorrhagia. Metformin therapy resulted in a significant reduction in the serum levels of IL-8, TNFα while insignificant reduction in estradiol E 2 in the study group after 3 months of treatment .In conclusion the results of this study, demonstrated that metformin may be a well-tolerated treatment for endometriosis that relieved pain and reduces menstrual disorders and serum levels of the inflammatory markers (IL8 and TNF-α) are decreased in study group treated with metformin after 3 months due to its antiinflammatory effects.


Introduction
Endometriosis is a common health problem in women .It got its name from the word endometrium, which is the tissue lining the uterus (1) .During a woman's menstrual period, this tissue thickens in preparation for a fertilized egg (pregnancy).If there is no fertilization, the tissue breaks down and bleeds with each menstrual period, allowing it to exit the body.
In endometriosis, the tissue that normally lines the uterus grows outside the uterus.Most commonly, it is found on the ovaries, fallopian tubes, tissue that holds the uterus in place, outer surface of the uterus, or the lining of the pelvic cavity.Other sites for growths can include the bladder, bowel, cervix, rectum and vagina, or vulva (2) .
The different theories involved in the pathogenesis of endometriosis indicate that the etiology of endometriosis is complex and multifactorial, involving hormonal,genetic immune and environmental components (3)   Although the etiology of disease is undetermined, four main hypotheses have been circulated as understandable causes (4) : A.
Sampson's theory of retrograde menstruation.B. Ceolomic metaplasia and induction theories (an extension of the ceolomic metaplasia theory).C. The embryonic rest theory.D.
Lymphatic and vascular metastasis theories.Several theories considering the pathogenesis of endometriosis are shown in Table -1 , retrograde menstruation may be one of the initiating steps in the pathogenesis of superficial endometriosis, genetic and micro environmental factors that prevent clearance of ectopic lesions and allow remodeling of peritoneum are essential for the propagation of endometriotic lesions (3,5) . (6)

Metaplasia
Estrogen-driven proliferation of endometrial lesions.Resistance to progesterone-mediated control of endometrial proliferation

Stem cells
Tumor necrosis factor-alpha TNF-α is the main pro-inflammatory cytokine known to impair glutathione (GSH) production by several ways, making an environment conducive to the development of oxidative stress(OS).This pathogenic cycle of GSH disturbances and enhanced TNF-α production may be active in the female reproductive tract in endometriosis.An in vitro study investigating endometriosisassociated infertility showed that the quality of spermatozoa may be decreased following incubation with TNF-α in a doseand timedependent manner (7).
TNF-α secretion is stimulated by IL-1 and bacterial endotoxin.When mediated by IL-8, TNF-α has been known to promote the growth of endometriotic cells (8) .The mechanisms connecting endometriosis and infertility involved:  Distorted pelvic anatomy, including adhesions resulting from endometriosis, which can impair oocyte release or prevent ovum pickup and transport, (9) as well as damaged or plugged fallopian tubes or acquired or congenital uterine defects. (10). Altered peritoneal function, including increases in fluid volume; concentration of activated microphages; prostaglandins; IL-1, IL-6, TNF-alpha, IgG, and IgA antibodies; lymphocytes; an ovum capture inhibitor preventing cumulus -fimbria interaction; (11). Endocrine and an ovulatory disorders, including luteinized unruptured follicle syndrome (LUF), luteal phase defect, abnormal follicular growth, and premature as well as multiple luteinized hormone surges.It has been hypothesized that LUF may not be a consequence of endometriosis, but, in fact, may be a cause or cofactor in the development of the disease (9) . Impaired implantation, with evidence suggesting that endometriosis may be responsible for reduced expression of the (alpha(v)beta(3) integrin)αvβ3 cell adhesion molecule during the time of implantation (11) .The diagnosis of endometriosis can be substantiated only by direct visualization during laparoscopy or laparotomy confirmed by tissue biopsy (12) Larger lesions may be seen within the ovaries as ovarian endometriomas or "chocolate cysts" as they contain a thick brownish fluid, mostly old blood.However, smaller endometriosis implants cannot be visualized with ultrasound technique (12) .Surgically, endometriosis can be staged I-IV according to the (Revised Classification of the American Society of Reproductive Medicine) (13) .In principle the various stages show these findings: Stage I (Minimal): Endometriosis restricted to only superficial lesions and possibly a few filmy adhesions, there are isolated incidents of endometrial tissue growth outside the uterus (14) .
Stage II (Mild): This diagnosis occurs when there are several small implants and a few small areas of scar tissue or adhesions and some deep lesions are present in the cul-desac. (15).

Stage III (Moderate):
As in stage II, plus presence of endometriomas on the ovary and more adhesions.The implants in stage three must be superficial and deep.
Stage IV (Severe): This is the most severe stage of endometriosis.Patients with stage IV endometriosis will have many superficial and deep implants as well as large adhesions found.
The purpose of medical management is to minimize proliferation/reduce pain, inhibit inflammation, minimize menstrual volume, frequency and oppose E2 action (16) .Medical treatment included :Initial therapy should include a non-steroidal antiinflammatory drugs (NSAIDs) such as : a-Naproxen 500 mg at first then followed by 250 mg orally three times daily, or b-Ibuprofen 800 mg as single dose, then 400 mg orally every 6 to 8 hours or c-Mefenamic acid 500 mg orally then followed by 250 mg every 6 hours. (17).Surgical treatment is highly effective for the alleviation of symptom, pain reduction and can increase fertility in sub-fertile women (18)   .Medical management is based on hormonal suppression of endometriotic lesions and is particularly effective when amenorrhea occurs via down -regulation of the hypothalamic -pituitary-ovarian axis (19) .As shown in table-2. (20).

Patients and methods
Thirty women were included in this study from AL-Elwiyah Maternity Teaching Hospital and Al-Yarmok Teaching Hospital for the period from January 2014 to June-2014.Verbal consent was obtained from all women prior to enrollment in the study.Inclusion criteria 1-Women undergo diagnostic laparoscopy for pelvic pain or treated for some causes of infertility.
2-Women undergo diagnostic laprotomy for ovarian cyst or acute abdominal pain who were found to have endometriosis.

Grouping of patients A-Control group
Fifteen patients diagnosed by laparoscopy to have different stages of endometriosis.These patients were complaining of one or more symptoms such as dysmenorrhea, pelvic pain or menorrhagia as seen in table -3.
They received classical drugs such as danazol capsules (isoxazolic derivative of a synthetic steroids,17 αethinyl testosterone) , some women treated with zoladex (goserelin acetate implant) ,and some women treated with oral contraceptive pills as seen in table -4.

Sampling
Blood samples were obtained for the estimation of the cytokines as (IL-8 (21) and TNF-α (22) )in addition to estradiol E2 (23 levels at the start of the study from the fasting patients in morning and also at the follow up visits after three months.Estimation of the cytokines IL-8, TNF-α level was performed using a commercially available enzyme -linked immunosorbent assay (ELISA) kits.

Estimation of the estradiol E2 was performed using Radioimmunoassay.
Five ml of venous blood was withdrawn aseptically into dry plastic tubes.Then the collected blood samples were centrifuged at 3000g for 15 min and the separated serum were stored at −20 °C until time of assay.

Statistical analysis
The collected data were tabulated, compared and proper statistical analyses were performed.A t-test is any statistical hypothesis test in which the test statistic follows a Student's t distribution if the null hypothesis is supported.It can be used to determine if two sets of data are significantly different from each other..When the scaling term is unknown and is replaced by an estimate based on the data, the test statistic (under certain conditions) follows a Student's t distribution (24) .Frequency, mean and standard deviation (SD) were used to describe data .P value was considered positive and significant if less than 0.05.

Results and Discussion
The study sample included (30)  Endometriosis classified into stages according to the revised criteria of the American Society of Reproductive Medicine (ASRM) (13)  Endometriosis is estimated to occur in 5/15(33.33%) of women presenting with pelvic pain for control groups , 9/15(60%) in study groups also showed that 5/15(33.33%) of women presenting menorrhagia in control groups and 2/15(13.33%) in study groups as shown table-3.
The serum levels of TNF-α , IL-8 and E2 in the control and study groups levels in the endometriosis before treatment and after 3 months of treatment.As shown in Table -8 in the present study there was a significant reduction in the number of cases with dysmenorrhea and pelvic pain (P< 0.05) after three months of metformin therapy.as seen in table -9.
Metformin significantly decreased pain and the level of C-reactive protein after 6 months of treatment in patient with endometriosis associated chronic pelvic pain (25) Rannou F et al 2007 , study demonstrated that IL-6, may be considered as a major regulator of C-reactive protein gene (25) tumor necrosis factor alpha (26) , nuclear factor ĸɃ (27) and transforming growth factor beta-one (TGFβ-1) (28) .The inflammation associated with endometriosis, through increased levels of peritoneal fluid Vascular Endometriosis growth factor (VEGF), may promote angiogenesis for progressive growth of endometriosis (29) .Thus , endometriosis had a direct effect on adhesion formation (30) .Moravek et al (2009)  ,provided a preliminary data about the effectiveness of rosiglitazone, an insulin sensitizer, in treating endometriosis -related pain in six patients and concluded that it was effective and promising for usage in endometriosis patients desiring the chance to conceive (31) .The result of this study showed that the level of IL-8 was significantly decreased after 3 months of metformin therapy (P< 0.05).IL-8 level was significantly higher in the endometriosis groups than in the control groups (p=0.01).Table 8.Arici et al 1998.reported" that IL-8 is produced in the human endometrium in vivo, especially in glandular cells" (32) .IL-8 raise the proliferation of endometrial stromal cells as a potential autocrine growth factor (36) .A previous study Y. Takemura et al (33) has shown that metformin in non decidualized ovarian endometriotic stromal cells could reduce IL-1b-induced IL-8 production, aromatase activation and proliferation.So far only a reduction of aromatase activation and a reduction of proliferation has been demonstrated in vitro in ovarian endometriotic stromal cells after treatment with metformin (Y.Takemura et al 2007) (33) .The results of Iwabe et al (8) clearly demonstrated that "IL-8 is a growthpromoting factor in normal endometrium as well as in endometriotic cells".The study of Baracz et al 2012. (34)excluded the primary role of IL-8 in the formation of endometriosis -associated peritoneal adhesions and confirmed the role of cytokines in the pathogenesis and progression of endometriosis.The result of the present study showed that there was significant reduction in TNF-α levels in women with endometriosis after 3 months of metformin treatment.The result of Iwabe et al 2001 (8) ,proved that it through induction of IL-8 gene and protein expression in endometriotic stromal cells ,TNF-α stimulated proliferation of endometriotic stromal cells in a dosedependent fashion.The addition of anti-TNF-α antibody or anti-IL-8 antibody, lead to neutralized the proliferated effect of the endometriotic stromal cells, and the stimulatory effects of TNF-α (34) Bedaiwy et al (2002) (35) demonstrated that serum TNF-α had a high degree of sensitivity and specificity so it could be surprisingly used to differentiate between patients with and without endometriosis.The result of the current study showed that there was no significant reduction in level of estradiol E2 in women with endomterosis treated with metformin for periods of 3 months and this disagree with the study of Mansfield et al 2003 (36) .Also table 8 showed that the significant reduction in estradiol E2 ,FSH ,insulin -stimulated progesterone production in granulosa cells Thus, metformin may be effective in treatment endometriosis through suppression both ovarian and local production of estrogen (36) .So the result from this study about serum estradiol E2 may be attributed to the small sample size and short time of study or the dose of metformin was not enough to lower of estradiol E2 level.

Conclusion
From the results of this study, it was found that metformin may be well tolerated treatment for endometriosis that relieved pain and reduced menstrual disorders.Serum levels of the inflammatory markers (IL-8 and TNF-α) are decreased in study groups with metformin after 3 months indicating that metformin may be have anti-inflammatory effect.