40 females adult NMRI mice (25-30 gr, 8-12 weeks) were purchased from the animal house of the university. To be acclimatized with the new living environment, the animals were kept in animal house for two weeks with no experimental treatment. Also, all standard living conditions (22 ± 2°C, 12-hour light/12-hour dark, and free access to water and food pletes) were provided.
Study animal groups
40 female mice were divided into 5 experimental groups (n=8 in each group); 1st sham (application of laparotomy followed by abdominal wall suture with no experimental treatments), 2nd mice-mice allograft uterus transplantation of mice to anterior abdominal wall of mice, 3rd mice-mice allograft uterus transplantation of mice to mesentery of mice, 4th rat-mice xenograft endometrial transplantation of rat to anterior abdominal wall of mice, 5th rat-mice xenograft endometrial transplantation of rat to mesentery of mice. In all treatments groups, laparotomy was applied in anterior midline region of animals. Due to the thin and inseparable layer of mice endometrium, uterus of mice with whole layers (endometrium, myometrium, and perimetrium) was dissected and transplanted in allografts. Also in rats, the endometrium was dissected from total uterus tissue and transplanted to recipient animals in xenograft procedure. Two transplantation locations were used; anterior abdominal wall and mesentery of small intestine.
Hormonal and estrous cycle synchronization in recipient animals
According to the Whitten effect and based on estrogen-depended nature of endometriosis in rodents, the female recipient animals were exposed to males (in nested cages) before and during of endometrial induction. Also, to estrus cycle synchronization, daily vaginal cytology was checked for a week before the surgery. Cotton swab impregnated with normal saline was used to collect vaginal smear from vaginal orifice. The samples were stained by Papa Nicola staining method, and endometriosis induction was applied in estrus stage for all animals. The mice were sexually receptive in estrus cycle characterized by estrogen secretion. In this study, the estrus cycle was detected by the presence of whole types of superficial cells through microscopic examination of vaginal cytology .
Preparation of donor animals
To increase the endometriosis thickness of rats and facilitated endometrium dissection, as the main modification in this method, three main conditions were provided for donor rodents; all animals were in adult age, they had a previous pregnancy experience, and they were exposed to male animals for two weeks. Exposure of female animals with males (divided by metal grid for mating prevention) was conducted with one male / two female ratio for sexual stimulation and endometrial growth.
Implantation of endometrium to abdominal wall and mesentery
Donor animals (mice and rats) were euthanized through cervical dislocation. Uterus (of mice) and endometrium (of rats) were dissected, and all surrounding attached connective tissues were removed under a loop microscope (1A). Dissected tissues were placed in DMEMF12 / FBS 5% cell culture solution to preserve cell viability (1B). Round grafts were prepared using a 3-mm diameter punch and were sutured (nylon, 5-0 USP, SUPA medical devices, Iran) to anterior abdominal wall or mesentery layer of small intestine in recipient animals (1C and 1D). Recipient mice were weighed and anaesthetized through intraperitoneal injection of ketamine/xylazine (20 IU per 25 gr animal of a mixture 10 IU ketamine/90 IU xylazine). Small intestine was explored, and the inner epithelial surface (containing endometrium) of grafts was sutured in direct contact with the peritoneal surface of mesentery or abdominal wall. Also, for preparation of blood supply, the grafts were transplanted exactly close to mesentery vessels. Peritoneal and muscular layers of abdominal wall were sutured using absorbable strings (chromic 5-0 USP, SUPA medical devices, Iran), and the skin was closed by nylon (Nylon, 5-0 USP, SUPA medical devices, Iran). A day after the recovery from surgery, the recipient animals were exposed to males (for 4 weeks) surrounded by metal cages (to prevent mating) for endometriosis induction (Fig. 1).
Animals weighting, dissection, and tissue sampling
4 weeks after surgery, the recipient mice were euthanized using cervical dislocation procedure. Immediately, 1 ml of distilled water was injected into the peritoneal cavity. 2 min later, the peritoneal fluid was aspirated. After laparotomy procedure, the endometrial lesions and uterus were dissected. Thoracotomy was also conducted, and the blood was aspirated from right ventricle and followed by centrifugation (3000 g, 15 min) to separated blood serum. All biological samples were frozen in nitrogen liquid for future biochemical and genetic analysis or were fixed in formaldehyde 10% for histopathological assessments. Total body weight was also recorded.
Morphometric assessments of endometrial lesions
During tissue sampling procedure, diameter of endometrial lesions was calibrated (Calliper, Sana`t Co, Iran), and the weight of the grafts was calculated (Laboratory scale, Model GR 202, Tajhizat Co, Iran) after complete excision of surrounding connective tissues. The presence or absence of pus-filled cysts was also detected. These factors were considered growth markers of endometrial lesions, and they were compared with primary size (3mm) and weight (0.01 gr) of endometrial lesions exactly prior to implantation.
Assessment of angiogenesis in endometrial lesions and peritoneal fluid
To evaluate angiogenesis rate in endometrial lesions, number of new generated vessels around endometrial lesions were counted using histopathological sections (Nicon biology microscope, E200). Also, peritoneal concentration of VEGF-A, as a peritoneal angiogenic biomarker, was assessed biochemically using ELISA kit (Abcam, ab100662, USA) according to the routine procedure and based on the manufacturer`s instruction .
Assessment of inflammation status in peritoneal fluid and blood serum
Concentration of TNF-α (Abcam, ab193687, USA) in peritoneal fluid was considered an inflammation statue induced by endometrial lesions and produced by peritoneal macrophages. Also, IL-37 (Abcam, ab213798, USA) was measured as a serum biomarker for endometriosis diagnosis. These measurements were done by ELISA kit according to the protocol .
Status of oxidative stress in peritoneal fluid following endometriosis induction
Generated oxidative stress following hyper-activation of macrophages and high rate proliferation of endometrial cells was measured in peritoneal fluid using ELISA kit. In this process, NO concentration (Abcam, ab272517, USA) was evaluated using Griess techniques, and MDA (Abcam, ab238537, USA) levels (representing lipid peroxidation status) were also measured .
Serum levels of CA-125
Cancer antigen 125 is a member of the mucin family glycoproteins. CA-125 is a biomarker which is elevated in the blood of some types of cancers and endometriosis. This factor was measured using ELISA kit (Abcam, ab108653, USA) .
Histopathological assessments using H&E and Perl`s staining
Right horn of uterus and half of endometriosis lesions were fixed in formaldehyde 10% for H&E and Perl staining. H&E staining was used for glandular and stromal assessments of endometriosis, and Perl's staining was also applied for hemosiderin deposition in macrophages as a crucial factor of endometriosis. Tissue processing was applied, and paraffin blocks were prepared. Thin sections (5 µm) were cut (Microtome, Leica RM 2125, Germany) and stained using H&E and Perl staining. Finally, the slides were assessed by a research microscope (Olympus, BX-51T-32E01) based on the histopathological variations, including; epithelium and stroma of endometrium, endometrial glands, blood vessels, and macrophages loaded with hemosiderin .
RNA extraction and Real-Time Quantitative PCR of HOX genes expression in uterus
Variation of HOX genes (HOXA 10 and 11) on endometriosis and as an indicator of embryo implantation during fertility was approved by literature. In order to assess the probable pathologic effects of endometriosis on uterus, the genes expression of HOXA10 (F: GCCCTTCCGAGAGCAGCAAAG, R: AGGTGGACGCTGCGGCTAATCTCTA) and HOXA11 (F: GATTTCTCCAGCCTCCCTTC, R: AGAAATTGGACGAGACTGCG) were assessed. Left horn of uterus was dissected, and total RNA was extracted (QIAGEN RNA purification kit), and a spectrophotometer checked the quality of the extracted RNA (UV1240, Shimadzu, Kyoto, Japan) in 260/280 nm wavelength absorbance ratio. DNA was synthesized using a commercial BioFact kit (BioFact RT Series, Korea). The genes expression was evaluated using High ROX BioFact™ 2X Real-Time PCR Smart mix SYBR Green PCR master mix. Also, β-actin (F: GGCACCACACCTTCTACAATG, R: GGGGTGTTGAAGGTCTCAAAC) was used as a housekeeping gene. Gene expression levels were measured by using Ct (2-ΔΔt) method (fold changes).
The present investigation was in compliance with ethical and human principles of research and approved by the Ethics Committee of the University of Medical Sciences.
After extracting the information, the Kolmogorov–Smirnov test was first conducted to confirm data compliance of the normal distribution. One-way analysis of variance (one-way ANOVA) was used for statistical analysis, and the Tukey post hoc test was used to determine the difference between the groups. Statistical Package for the Social Sciences 16 (SPSS Inc., Chicago, IL) was used for data analysis, and the results were expressed as mean ± standard error, and p < 0.05 was considered significant.