Introduction

Endometriosis is a complex gynecological disorder which occurs in women of reproductive age caused by a combination of several genetic and environmental factors characterized by a high migratory and invasive potential such as tumor metastasis [1, 2]. The prevalence of endometriosis is 10 percent of women of reproductive age regardless of ethnicity and social status [3]. Various data on the prevalence of vitamin D deficiency which occurs in women of reproductive age in European, American and Asian countries vary from 42%-90% vitamin D: An overview of vitamin D status and intake in Europe [4, 5], whereas the rate of vitamin D deficiency in Indonesia reaches 63% [6]. The features of women of reproductive age who have endometriosis are chronic type, thus affecting their quality of life with the main symptoms of pain and infertility [7], which interfere with psychological and social functions [8, 9]. Increased activity of cytokines and steroid hormones in endometrial tissue increases Matrix Metalloproteinase-9 (MMP-9) expression and plays an important role in invasion and metastasis [10]. Vascular Endothelial Growth Factor (VEGF) expression plays a role to form the vascular tissue cycle by stimulating the degradation of the extracellular matrix around endothelial cells [11]. MMP-9 is a group of proteolytic enzymes that degrade components of the extracellular matrix in their progression [12].  Vitamin D can suppress cyclooxygenase 2 (COX 2) expression, thereby reducing levels of IL-6, TNF, and PG [13, 14]. Vitamin D can reduce the inflammatory response induced by IL-1β- or TNF-α so that the expression of MMP-2 and MMP9 decreases through inhibition of nuclear factor-κB [15]. Vitamin D also reduces VEGF-A expression in the stroma of endometriosis lesions, and can reduce VEGF levels in diseases such as PCOS [16-18]. Research on nutrition and food groups that correlate with endometriosis is limited to determining potential of disease risk factors in the endometriosis pathogenesis to food groups and nutrition. Vitamins A, C, and E have been widely researched to be correlated with endometriosis. However, the results show that there is no statistically significant relationship, and only vitamin D shows significant results [13]. The researchers are interested in conducting research on endometriosis model mice that are administered vitamin D supplements at doses of 8, 16, and 24 iu a decrease in the expression of MMP-9 and VEGF and the relationship of the decreased expression with the administered graded doses.  

Materials and Methods

Experimental animals

The experimental animals were obtained from the Airlangga University Pusvetma. The research was carried out at the Laboratory of Veterinary Medicine Faculty, Airlangga University after obtaining a certificate of ethical due diligence no. 2.KE.144.12.2021.

Making endometriosis model mice

An endometriosis model mouse was mde by injecting cyclosporin A on the mice, and then intraperitoneal injection of 0.1 cc endometrial biopsy tissue was carried out on them and on the 5^th day, the mice were administered intramuscular injection of estrogen with a dose of 5.4 µg/mouse. After 14 days, endometriosis model mice were obtained.

Research design and stages

This type of research is true experimental using a completely randomized design posttest only control group design. Using the randomization technique, the samples were categorized into 4 groups, specifically the control group (C) which was not given vitamin D, the group treated with dose I (T1), dose II (T2), and dose III (T3). The need for vitamin D was calculated according to the Endocrine Society (21) and sampling was carried out so that the mice in group T1 were administered as much as 8 iu vitamin D that equals to 0.2 cc; those in group T2 were administered as much as16 iu Vitamin D that equals to 0.4 cc and those in group T3 received 24 iu vitamin D equals to 0.6 cc. Vitamin D was administered individually once daily. Treatment began on day 15 after the occurrence of endometriosis lesions by administering vitamin D according to the dose per group.

Examination on the expression of VEGF and MMP-9

Samples were taken from the peritoneal tissue of mice on day 37. The peritoneal tissue with endometriosis lesions was fixed in 10% formalin [19, 20]. VEGF expression was analyzed using immunohistochemical techniques. Staining was carried out using DAB Kit with Coomasie brilliant blu counterstaining. MMP-9 expression analysis was performed by immunohistochemical staining using MMP-9 polyclonal antibody. The sample was evaluated using the Remmele scale index (Immuno Reactive Score/IRS) in a semi-quantitative manner. The assessment involved multiplying the percentage score of immunoreactive cells by the color intensity score on these cells, as listed in Table 1. For each sample, the data represented the average IRS value observed across ten different field of views using the magnifications of 100x and 400x.

Results and Discussion

Effect of vitamin D on VEGF expression

Figure 1 displays the impact of administering vitamin D supplements to mice with an endometriosis model. This figure shows that there is VEGF expression in cell tissue of endometriosis lesions taken from the peritoneum of endometriosis model mice. Accordingly, group C has stronger expression and it is clustered, compared to group P1, P2, and P3 which have weaker expression and it is dispersed. The figure on VEGF expression is in accordance with the graph on average values which tends to decrease in Figure 2. VEGF expression in the control group had an average value of 5.6; the intervention group 1 with a dose of 8 iu had an average value of 5.5, intervention group 2 with a dose of 16 iu had an average value of 4.3, and the lowest was the intervention group 3 with a dose of 24 iu that had an average value of 3.8.

Table 1: Post Hoc Bonfferoni test of MMP-9 expression conducted on Endometriosis Model mice supplemented with vitamin D

C: A group that serves as a control; T1: A group that receives an intervention with 8 iu of vitamin D; T2: A group that receives an intervention with 16 iu of vitamin D; T3: A group that receives an intervention with 24 iu of vitamin D.

Figure 1: VEGF expression on endometriosis model mice tissue shown by red arrows

Figure 2: Distribution of VEGF expression average values ​​in endometriosis model mice          

Based on the graph, mice with an end