Introduction

Endometriosis is defined as the presence of extrauterine endometrial tissue [1]. It affects approximately 10 % of reproductive-aged women and 20–50 % of infertile women [2]. Women with endometriosis present with characteristic signs and symptoms: dysmenorrhoea, dyspareunia, chronic pelvic pain or sub-fertility. Endometriosis is one of the most commonly encountered benign problems in gynecology [3].

Various theories have been proposed to explain how endometriosis develops. These include retrograde menstruation, coelomic metaplasia, lymphatic or hematogenous dissemination of endometrial cells, heredity and environmental toxins. Impaired immune function resulting in inadequate removal of refluxed menstrual debris has also been proposed as a possible causative factor in the development of endometriosis [4, 5]. Cytokine-mediated immune and inflammatory responses have been considered to play an important role in the pathogenesis of endometriosis [6–8] as well as oxidative stress [9], which occurs when the production of reactive oxygen species (ROS) overwhelms the body’s natural ability to neutralize them with the available antioxidant system [10]. Several studies indicate that antioxidant defenses may be altered in endometriosis and endometrial antioxidant enzymes are aberrantly expressed [11, 12].

Melatonin (N-acetyl-5-methoxy-tryptamine) is a potent antioxidant and free radical scavenger. It is the main pineal hormone synthesized from tryptophan, predominantly during the night [13]. It is critical for the regulation of circadian and seasonal changes in various aspects of physiology and neuroendocrine function [13–15]. It stimulates a number of antioxidative enzymes such as superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase and catalase. It also stabilizes microsomal membranes and helps them resist oxidative damage [16]. It acts as a direct scavenger of free radicals. It detoxifies both reactive oxygen and nitrogen species. It indirectly increases the activity of the antioxidative defense systems [17–19].

Melatonin also has anti-inflammatory effects. The immunologic activity of melatonin has been demonstrated in many experimental models [20–23]. The direct effect of melatonin on the human immune system is supported by the existence of specific melatonin binding sites on lymphocytes and monocytes [24–26]. Melatonin regulates lymphocyte and monocyte functions and Th1/Th2 cytokine balance through these receptors [27–29]. Melatonin reduces lymphocyte physiology in autoimmune-target organs and suppresses autoimmune diseases [30]. Recently, it has been reported that melatonin protects against immune ovarian failure induced by antiovarian antibodies in mice [31]. It has been shown that melatonin causes regression and atrophy of endometriotic lesions in rats [32]. In this study, we aimed to investigate the effects of different melatonin doses on endometriotic lesions in an oophorectomized rat model.

Material and method

Experimental animal model

This study was approved by the Experimental Animals Ethics Committee of Yeditepe University. All experiments were performed in compliance with international guidelines on the ethical use of animals.

Thirty-two reproductive-aged female non-pregnant, nulligravid Sprague–Dawley albino rats weighing 200–250 gr and bred at Yeditepe University Experimental Research Center (YUDETAM) were used in this study. The rats were caged individually in a controlled environment. The room temperature was 21 °C and the humidity was 60 %. Rats were fed ad libitum with 12-h light/dark cycles.

Oopherectomy was performed in the first operation on all rats to stabilize and standardize the estrogen levels in them throughout the study. The rats in all groups were administered high-dose subcutaneous estrogen (50 μg/kg, twice weekly) until the end of the study (6 weeks). Melatonin was given to both melatonin treatment groups for only 2 weeks to visualize melatonin-related endometriotic implant regression and recurrence after therapy cessation.

Surgical techniques

All the rats had four operations at 2 weeks intervals throughout the study. Rats were anesthetized in each operation with an intramuscular administration of 60-mg/kg ketamine hydrochloride (Ketalar; Eczacibasi Ilac Sanayi, Levent, Istanbul, Turkey) with 7-mg/kg xylazine hydrochloride (Rompun; Bayer Ilac Sanayi, Sisli, Istanbul, Turkey) as described previously [32, 33]. The abdominal cavity was entered by vertical abdominal incision. After the induction of endometriosis, rats were randomized into three groups using a randomization table (control, 10- and 20-mg/kg/day melatonin group). There were 12 rats in the control group, 10 rats in the 10-mg/kg/day melatonin group and 10 rats in the 20-mg/kg/day melatonin group.

One milliliter of peritoneal fluid was collected from all groups to assess biochemical parameters after each operation. The abdominal and skin incisions were closed in a continuous interlocking manner with 3–0 silk sutures after each operation.

First operation: endometriosis induction

In the first operation after oopherectomy, endometriosis was induced using homologous uterine horn auto-transplantation. According to the procedure after abdominal entry, bilateral ovaries and uterine horns were visualized and surgical specimens were excised in an en block manner by hemostatic clamps. 3/0 silk sutures were used to ligate ovarian and uterine branch vasculatures. Then uterine horns were placed in a phosphate buffer solution (PBS) at room temperature to separate from surrounding parametrial soft tissues. Two rectangular 6 × 3 × 1 mm uterine pieces with endometrium inside and myometrium outside were created from each uterine horns. A total of four pieces of uterine tissues—two on each side—were implanted with 6/0 non-absorbable polypropylene sutures on the highly vascular area at the right and left hypochondriac regions. After this operation, the rats were randomized into three groups: the control group, the 10- and the 20-mg/kg/day melatonin treatment groups. All the rats were given 50-mg/kg/day cefazolin sodium (IE Ulagay Ilac Sanayi, Istanbul, Turkey) intramuscularly for 3 days after the operation to prevent any intraperitoneal infection. Furthermore, high-dose subcutaneous estrogen (50 μg/kg; estradiol powder, ≥98 % Sigma-Aldrich®; twice weekly) therapy was started after the first operation and administered throughout the study.

Second operation: assessment of the endometriotic foci

Two weeks after the first operation (endometriosis induction), the second one was performed to assess the endometriotic lesions (Fig. 1). All the implants were measured by the same author (N.C) in three dimensions (length–width–height in millimeters) using a ruler, and 1 ml of peritoneal fluid was collected from each rat. After determining the presence of the four endometriotic lesions in the 2nd week with no significant difference in lesion volumes between the groups, one of them was removed in each group for histopathological analysis using a randomization table. Then, melatonin was administered daily using both intramuscular and intraperitoneal routes in treatment groups for 2 weeks (until the third operation).

Fig. 1
figure 1

White arrows indicate the endometriotic implants at the 2nd week

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Third operation: the effects of melatonin

The third operation was performed for the assessment of the effects of the melatonin treatment on the endometriotic foci. In the third operation, which was performed after melatonin treatment (at the end of 4th week), volumes of endometriotic foci were measured and peritoneal fluid was collected as previously done. One of the endometriotic lesions was randomly collected for histopathological examinations. Then, melatonin treatment was stopped while estrogen administration was continued.

Fourth operation–necropsy: evaluation of recurrence

Two weeks after the cessation of melatonin, all the rats were euthanized under general anesthesia. Endometriotic lesions were measured, fluids were collected and biopsies were performed to compare the recurrence of endometriosis in each group.

Volume analysis

The spherical volume of each sample of endometriotic tissue was calculated using the prolate ellipsoid formula: V (mm3) = 0.52 × length × width × height (all in millimeters) [2, 7].

Histopathologic analysis

Endometriotic biopsy samples were fixed in 10 % neutral buffered formaldehyde solution. After the dehydration procedure, all pieces were embedded in paraffin. Three-µm thick sections were made with microtome. The samples were stained with Hematoxylin and Eosin (HE). Slides were inspected under a light microscope. They were examined by a pathologist (FO) who was blinded to the treatment groups.

Under microscopic examination, the endometrial glands and stroma formation were inspected. Epithelial cells in the implants were evaluated as described by Keenan et al. [34]. Based on this scoring system, lesions were given points as follows: Point 3—if epithelial surface layers were well preserved, Point 2—if moderately leukocytic infiltration was seen, Point 1—if poor epithelial lining was detected and Point 0—if no epithelial lining was seen (Fig. 2).

Fig. 2
figure 2

A figure showing a histologic score 3 of an epithelial lining in an endometriotic implant (Hematoxylin and Eosin, ×40)

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Statistical analysis

Statistical analysis was performed using SPSS, version 11.5 (SPSS Inc, Chicago, IL, USA) for windows. Data were expressed as mean ± standard error of mean (SEM). When these parameters were compared between the groups, the Kruskal–Wallis test with Mann–Whitney U test as a post hoc test was performed. Friedman’s Test with Wilcoxon as a post hoc test was used for the evaluation of lesion volumes and histopathological scores throughout the study in each group. P < 0.05 was considered as statistically significant.

Results

Animals were controlled daily by a veterinary doctor and her assistant throughout the study period. Totally thirty-two rats were operated, and 128 uterine graft tissues were implanted. Endometriotic foci were seen in each of the implanted grafts at the second operation with no significant differences in lesion volumes between the groups, which means that standardized endometriotic lesions were created in all groups before the melatonin treatment. No graft failure was seen. Throughout the study, no complications related to operations or toxic effects of melatonin were observed. Some of the endometriotic foci were cystic in nature and the others were hyperpigmented thickened tissues. Study design gave us the opportunity to compare the rats in all the groups and also each group with itself as a separate entity.

Comparison between the groups

The mean volumes of endometriotic foci were 124.5 mm3 ± 14.8, 122.4 mm3 ± 23.1, 109.2 mm3 ± 12.8 and histopathological scores were 2.2 ± 0.2, 2.4 ± 0.3, 2.6 ± 0.2 in the control group at the end of the second, fourth and sixth weeks. The mean volumes of endometriotic foci were 98.8 mm3 ± 17.2, 54.1 mm3 ± 15.6, 42.8 mm3 ± 10.5 and histopathological scores were 2.2 ± 0.2, 2.6 ± 0.2, 2.6 ± 0.1 in the 10-mg/kg/day melatonin group at the end of the second, fourth and sixth weeks. The mean volumes of the endometriotic foci were 108.2 mm3 ± 17.5, 25.8 mm3 ± 3.6, 32.7 mm3 ± 6.0 and histopathological scores were 1.7 ± 0.1, 2.2 ± 0.2, 2.7 ± 0.2 in the 20-mg/kg/day melatonin group at the end of the second, fourth and sixth weeks.

The mean SOD levels were 0.07 U/ml ± 0.009, 0.08 U/ml ± 0.008, 0.05 U/ml ± 0.003 in the control group; 0.07 U/ml ± 0.005, 0.08 U/ml ± 0.007, 0.04 U/ml ± 0.003 in the 10-mg/kg/day melatonin group and 0.06 U/ml ± 0.007, 0.08 U/ml ± 0.011, 0.05 U/ml ± 0.005 in the 20-mg/kg/day melatonin group at the end of the second, fourth and sixth weeks.

The mean VEGF levels were 79.5 pg/ml ± 1.4, 83.5 pg/ml ± 1.2, 82.4 pg/ml ± 2.4 in the control group; 85.2 pg/ml ± 3.4, 86.4 pg/ml ± 3.3, 85.7 pg/ml ± 3 in the 10-mg/kg/day melatonin group and 85.7 pg/ml ± 2.2, 86 pg/ml ± 3.3, 84 pg/ml ± 2.4 in the 20-mg/kg/day melatonin group at the end of the second, fourth and sixth weeks.

The mean MDA levels were 453.8 pmol/ml ± 76.9, 381.3 pmol/ml ± 111.5, 573.5 pmol/ml ± 88.2 in the control group; 587.3 pmol/ml ± 101.5, 928.9 pmol/ml ± 98, 869.6 pmol/ml ± 79.1 in the 10-mg/kg/day melatonin group and 730.9 pmol/ml ± 105.6, 589.3 pmol/ml ± 103.7, 837.8 pmol/ml ± 82.2 in the 20-mg/kg/day melatonin group at the end of the second, fourth and sixth weeks.

At the second week investigations, there were no significant differences in the lesion volumes between the groups (P = 0.54), indicating that the groups were standardized. When the groups were compared, there were no significant differences in the histopathologic scores, SOD and VEGF levels between the groups at the end of the fourth and sixth weeks. However, endometriotic foci volumes were significantly decreased in both melatonin treatment groups with respect to the control group at the end of fourth and sixth weeks (Fig. 3). The mean MDA levels were significantly lower in the control group than in the 10-mg/kg/day melatonin group at the end of the fourth and sixth weeks (Table 1).

Fig. 3
figure 3

Black arrows indicate the endometriotic implants after the 20-mg/kg/day melatonin treatment

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Table 1 This represents the mean and standard error of mean (SEM) values of each parameter in the three different groups at the second, fourth and sixth weeks
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Comparison within each group

Control group

There were no statistically significant differences in lesion volumes (Volume A, B, C) and histopathology (Histology A, B, C) scores in the control group at the end of the second, fourth and sixth weeks. There were statistically significant differences in the SOD and MDA levels in the control group (P = 0.02). The mean SOD levels were significantly decreased (P = 0.004) at the end of sixth weeks compared to the fourth week, and the mean MDA levels were significantly increased (P = 0.004) at the end of sixth weeks according to fourth weeks. There were no significant differences in VEGF levels after the second, third and fourth operations (Table 2).

Table 2 This represents the mean and standard error of mean (SEM) values of the parameter in each group at the second, fourth and sixth weeks
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10-mg/kg/day melatonin group

Endometriotic volumes were significantly decreased (P = 0.001) at the end of the fourth and sixth weeks compared to the second week (P = 0.02 and P = 0.005), and there were no significant difference in the histopathological scores. The mean SOD levels were significantly decreased (P = 0.001) at the end of sixth weeks compared to the second and fourth weeks (P = 0.005 and P = 0.005 respectively). There were no significant differences in the levels of VEGF and MDA throughout the study in this group (Table 2).

20-mg/kg/day melatonin group

Endometriotic volumes were significantly decreased (P = 0.001) at the end of the fourth and sixth weeks compared to the second week (P = 0.003 and P = 0.003 respectively) and there was a significant increase (P = 0.009) in the histopathological scores at six weeks in comparison with the second week (P = 0.02), but not with the fourth week. There were no significant differences in the levels of SOD, VEGF and MDA throughout the study in this group (Table 2).

Discussion

Endometriosis is a benign disease which may have devastating effects such as severe peritoneal adhesions, pelvic distortions, secondary dysmenorrhea, dyspareunia and infertility [35]. Various therapies such as ovarian suppression therapy, surgical treatment, or a combination of these strategies have been used to treat endometriosis. Currently used medical treatments have adverse effects which limit their long-term use. Moreover, all of them are suppressive and not curative and the recurrence rates after the cessation of therapy are high [36, 37]. Therefore, researchers are now focusing on new treatment modalities.

Melatonin appears to be an effective agent for the treatment of endometriosis in experimental models [32, 33]. It is secreted from the pineal body in response to darkness. It is secreted from mononuclear cells and shows its effects by cell surface M1 and M2 receptors via inhibitory G protein complex. Its anti-inflammatory effects are through PGE2 inhibition due to COX-2 down-regulation. Experimental animal models show the anti-inflammatory, antioxidant, vasorelaxant effects of melatonin. It is also known that melatonin plays a role in oocyte maturation, ovulation, and corpus luteum function. Melatonin also has oncostatic, anti-proliferative and anti-estrogenic effects as it increases progesterone synthesis and secretion [38]. In a recent phase II placebo-controlled trial, melatonin treatment reduced daily pain scores and dysmenorrhea in endometriosis. It also improved sleep quality and reduced the risk of using an analgesic in the human setting [39].

We have previously shown that melatonin treatment resulted in regression of the endometriotic lesions [35]. In that study, the volumes of the endometriotic lesions were significantly decreased with the 20-mg/kg/day melatonin treatment. In another study, we compared the effects of letrozole (an aromatase inhibitor) and melatonin (an antioxidant) on surgically induced endometriosis in a rat model [33]. We showed that melatonin caused more pronounced regression of endometriotic foci when compared with letrozole in a rat model. After the cessation of melatonin treatment, the recurrence rate was lower than that observed after the cessation of letrozole treatment.

In the current study, we performed oophorectomy to inhibit fluctuation of endogen estrogen levels with respect to the physiology of rats. We compared the effects of the 10- and the 20-mg/kg/day melatonin treatment with respect to no therapy after the induction of standardized endometriotic lesions without significant differences in the lesion volumes at the second week. We measured the endometrial lesion volumes after therapy at fourth and sixth weeks, and we demonstrated that they were decreased in both groups. The regressions of the endometriotic implants were more pronounced in the 20-mg/kg/day melatonin group implying that melatonin was more effective on endometriotic lesions in higher doses. However, the difference was not statistically significant. We also analyzed the groups within themselves and demonstrated that lesion volumes were insignificantly changed at the sixth week compared to the fourth week, which meant lower recurrence rate after cessation of melatonin treatment. Our results were consistent with the previous studies [32, 33, 35, 40].

In our study, there were no statistically significant differences in the histopathological scores in the 10-mg/kg/day melatonin group. However, histopathological scores were significantly increased in the 20-mg/kg/day melatonin group at the end of the sixth week compared to the second week (P = 0.009), which might imply that higher levels of melatonin treatment can be more effective in improving histopathologic scores. We could not find any statistically significant differences in the SOD, VEGF and MDA levels between the groups.

In conclusion, we claim that melatonin might be a non-hormonal treatment alternative for endometriosis with obvious effects on minimizing endometriosis and with probable effects on reducing the recurrence rates or increasing the lesion differentiation after testing in a clinical setting. Higher levels of melatonin treatment tend to be more effective in the regression of endometriotic implants and the improvement of histopathologic scores in laboratory animals. Further studies are required using higher doses of melatonin (such as 50 and 100 mg/kg/day) and larger animal models to investigate the effects of melatonin on the distribution of SOD, MDA and VEGF levels and also its efficacy in the treatment of endometriosis in a clinical setting.