Presentation of a New Endometriosis Mice Model Through Rat to Mice Xenograft Transplantation of Endometrium with some Modifications; A Pilot Study


 Human endometriosis can lead to many complications, including permanent infertility. Rodent model of endometriosis is complicated due to the impractical isolation of myometrium. In this method, we defined a new model of endometriosis with some modifications. 40 female mice were grouped as; 1st sham, 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 recipient animals, estrous cycle was synchronized. Endometrium was dissected in rats and transplanted in abdominal wall or mesentery layer. Angiogenetic and inflammatory factors, serum levels of CA125, oxidative stress in peritoneal fluid, physical and histopathological features, and genes expression in uterus were assessed. Peritoneal concentrations of VEGF-A, TNF-α, NO, MDA, and serum levels of CA125, IL-37 were increased significantly (P < 0.05) in transplanted groups, especially in the 4th group than control. Total body weight of animals was decreased significantly (P < 0.05), while weight and size of endometrial lesions were increased significantly (P > 0.05). Genes expression of HOXA10 and HOXA11 were decreased in endometrium of uterus. Finally, histopathological features of endometrial lesions were confirmed by H&E and Perl staining. Xenograft transplantation of endometrium from rat to anterior abdominal wall of mice can potentially mimic the human endometriosis morphologically, histologically and genetically.


Introduction
The endometrium is the inner epithelial layer of the mammalian uterus divided into thin basal and thick functional layers. The presence of a functional layer strictly depends on the menstrual (in human) or estrous (in rodents) cycles.
Endometriosis is a pathologic condition in which the endometrial cells grow outside the uterus in abdominal or pelvic cavities. These cells reside on peritoneum layer located on organs (like ovaries, fallopian tubes, uterus, and intestine), attract adjacent vessels (angiogenesis), and grow by in ammation triggering. This pathologic condition leads to formation of new endometrial lesions. Thus, angiogenesis and in ammation are two crucial biological phenomena in endometriosis. Complications of this disease include presence of scar tissue, organs adhesions, pus-lled cysts, and adhesion-related obstruction (in lumen of the intestines or uterus). Also, following scar formation, anatomical disposition, and uterus obstruction, female infertility is probable, which is considered an end-stage outcome of endometriosis [1].
The most common theory of human endometriosis is a retrograde route of endometrial cells from intrauterine cavity to the peritoneal fossa. Although most studies in the eld of infertility and reproduction are performed in mice models than other animals, the presence of intrinsic endometriosis in mice is impossible because there are no menstrual cycles and endometrium abscission in rodents and these integrated cells are absorbed by adjacent tissues. Thus, endometriosis induction by a surgical procedure in mice seems necessary to assess human endometriosis aspects [2].
There are different animal models for endometriosis, especially in rodents. One of the most common types is transplantation of endometrial layer of uterus to various abdominal organs or abdominal wall in mice or rats. According to many studies, endometriosis induction is seen commonly in mice, during which the endometrium of uterus is dissected and transplanted to the abdominal wall or various organs. Endometrium in mice is a thin microscopic layer which its dissection is often impossible; besides the endometrium is a fragile tissue with no suturable property. However, endometrial layer in sexual stimulation is thick enough to be dissected and transplanted. Besides, transplantation to vital organs such as mesentery, as a routine protocol of endometriosis induction, can potentially damage vessels, leading to internal haemorrhage and animal death. These complications caused endometriosis induction a complicated process in mice.
According to the facts mentioned above, presenting a new animal endometriosis model and introducing a proper anatomical side of endometrial implantation with fewer complications that completely mimic human endometriosis conditions seems necessary. Thus, in the present study, we compared three models of animal endometriosis (rat-rat and mice-mice allografts, and rat-mice xenograft) transplanted to the anterior abdominal wall and mesentery layer. Finally, we introduced a new model of animal endometriosis through xenograft transplantation of endometrium of rat to anterior abdominal wall in mice. In this experimental animal study, we assessed the recipient animals biochemically, genetically, and histologically to ensure the accurate imitation of a new endometriosis model similar to human type.

Materials And Methods
Laboratory animals 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. 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 ori ce. 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 super cial cells through microscopic examination of vaginal cytology [3].

Preparation of donor animals
To increase the endometriosis thickness of rats and facilitated endometrium dissection, as the main modi cation 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 uid 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 xed 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-lled 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 uid
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 [4].

Assessment of in ammation status in peritoneal uid and blood serum
Concentration of TNF-α (Abcam, ab193687, USA) in peritoneal uid was considered an in ammation 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 [4]. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Status of oxidative stress in peritoneal uid following endometriosis induction Generated oxidative stress following hyper-activation of macrophages and high rate proliferation of endometrial cells was measured in peritoneal uid 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 [4].

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) [4].
Histopathological assessments using H&E and Perl`s staining Right horn of uterus and half of endometriosis lesions were xed 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 para n 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 [4].

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 puri cation 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).

Ethical Considerations
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.

Statistical analysis
After extracting the information, the Kolmogorov-Smirnov test was rst conducted to con rm 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 signi cant.

Angiogenesis rate
This value was assessed according to peritoneal concentration of VEGF-A and vessels count in microscopic sections. Peritoneal uid concentration of VEGF-A was increased signi cantly (p < 0.05) in mice-mice allograft and rat-mice xenograft transplantations in comparison with control group. Also, the levels of VEGF-A were accelerated signi cantly (p < 0.05) in rat-mice xenograft groups than mice-mice allograft animals representing high levels of VEGF-A in xenograft animals than allografts. Newly formed vessels also showed the incremental trend in whole groups of allograft and xenograft transplantations signi cantly (p < 0.05) compared to the control group. No signi cant (p > 0.05) differences were detected in vessels count in xenograft animals than allografts (Table 1). Serum concentration of CA-125 CA-125 factor, which represented the cancer antigen in blood serum, was elevated in allograft and xenograft transplantations in comparison with control animals signi cantly (p<0.05). Also, no signi cant (p>0.05) alteration was found among rat-mice xenograft transplantation than allograft group (Table 1).

In ammation status
In ammation was assessed using peritoneal concentration of TNF-α and serum levels of IL-37 as crucial in ammatory markers in endometriosis. In whole groups of treatments (allografts and xenografts), serum levels of TNF-α were increased signi cantly (p<0.05) in peritoneal uid than control group. Also, nonsigni cant (p>0.05) differences were detected in xenograft animals than allograft mice regarding TNF-α concentration. Serum levels of IL-37 were increased in all animals of allograft and xenograft transplantations signi cantly (p<0.05) in comparison with control group. This serum value showed signi cant (p<0.05) incremental changes in rat-mice xenograft group in comparison with mice-mice allograft animals than allograft cases (Table 1).

Oxidative stress status
Concentration of all oxidative stress markers (NO and MDA) was increased signi cantly (p<0.05) in whole transplanted animals (both treatment groups of allografts and xenograft) than control group. No signi cant (p>0.05) alteration was found among the animals in xenograft group than allografts (Table 1).

Physical features
Following endometriosis induction, total body weight of recipient animals was decreased signi cantly (p<0.05) in 2 nd (mice-mice allograft uterus transplantation to anterior abdominal wall) and 4 th (rat-mice xenograft endometrial transplantation of rat to anterior abdominal wall of mice) groups of treatment in comparison with control group. In other transplanted groups (transplanted to serous mesentery layer), non-signi cant (p>0.05) changes in the eld of animal`s weight were observed than control group. Also, the weight of endometrial lesions was increased signi cantly (p<0.05) in allograft and xenograft mice at the end of surgery than the primary weight (0.01 gr) at the start of induction. In this value, the animals in 4 th group of treatment (rat-mice xenograft endometrial transplantation of rat to anterior abdominal wall of mice) showed a signi cant (p<0.05) increase in weight of endometrial lesions than animals with allograft transplantation. Signi cant (p<0.05) increase in diameter of lesions was found in whole animals with allograft and xenograft transplanted tissues than the primary size of lesions prior to transplantation. More obviously, the size of the transplanted endometrial lesion was increased signi cantly (p<0.05) in rat-mice xenograft transplantation to anterior abdominal wall than allografts (Table 1).

Histopathological variations
As it was depicted in Fig. 2, the endometrial grafts of rat (with dissected layers of myometrium and perimetrium) were stained with H&E staining prior to implantation. Obvious epithelium (purple arrow) with compact stromal tissue (green rectangular) were seen located on lamina properia layer (2A). Also, stroma had coronal sections of endometrial glands (2A, black circle). After implantation of mice uterus to abdominal wall of recipient mice, pathologic features were found including; epithelial layer (2B, purple arrow) had no villi, stroma (2B, green rectangular) was detected thin layer with multiple cavities, and less number of coronal sections of blood vessels were found. Also, in Fig. 2B, the thick layer of myometrium and perimetrium were degenerated following uterus transplantation. These histopathological changes led to less secretion of pus in luminal space of endometrium, decreased size of grafts, and reduced weight of lesions after 4-week implantation (2B). Fig. 2C showed a complete section of rat-mice endometrial xenograft transplantation. Although the endometrial layer showed an irregular pattern than normal histology, thick and glandular epithelial formation caused the generation of a lumen full of secretion and haemorrhages (2C, yellow star). Outer part of this type of endometrial transplantation (xenograft rat-mice implantation) had a thick attachment layer as a common feature of endometriosis (2C) with many coronal sections of blood vessels (2C, red circle). There were many glandular sections (black circle) in thickness of stromal layer (2C, green rectangle), there were many glandular sections (black circle). Fig. 2D represented Perl`s staining in a part of connective tissue around endometrial lesions containing blood vessels. Black arrow (2D) showed macrophages loaded with hemosiderin following haemorrhages in endometrial lesions of xenograft transplantation to anterior abdominal wall.

HOX gene expression in uterus of recipient animals
Following induction of endometriosis in two recipient groups of 2 (mice-mice allograft uterus transplantation to anterior abdominal wall) and 4 (rat-mice xenograft endometrial transplantation of rat to anterior abdominal wall of mice), low decreased levels of HOXA10 and HOXA11 genes expression was found signi cantly (P<0.05). Also, in other treatment groups, no signi cant (P>0.05) alterations in genes expression of HOXA10 and HOXA11 were detected than βactin baseline (Fig. 3).

Discussion
As human endometriosis causes irreversible complications such as permanent female infertility, thus a comprehensive study in the eld of endometriosis and discovery of modern treatments seem necessary. For this purpose, application of animal endometriosis model for assessment of molecular pathways involved in this disease and innovation of modern treatments are crucial. The most common model of endometriosis is transplantation of endometrium to mesentery of recipient mice [5]. These mice to mice endometrial transplantations are practically di cult in two ways; it is impossible to separate the microscopic monolayer of endometrium, and thin mesentery layer as a background for endometrial implantation. Thus, in this pilot study, we particularly proposed a xenograft (rat to mice) model of endometriosis through implantation of endometrium to anterior abdominal wall. In this experiment, through exposure of adult female rats (with previous pregnancy experience) to males, estrogen production was stimulated, leading to increased thickness of endometrium. The recipient mice were also stimulated (using exposure to males) for estrogen production to achieve the transplantation of endometrial grafts.
Finally, we examined all histological, biochemical, and genetic factors to con rm the correct and proper induction of endometriosis. Finally, we concluded that due to the estrous cycle and induction of estrogen production in donor rat body, it is possible to increase the thickness of the rat endometrium, which can be dissected easily. Implantation of these dissected lesions to the anterior abdominal wall of a recipient mouse can successfully mimic histological and serological changes similar to human endometriosis.
Two main factors, as the basic features of endometriosis, are; angiogenesis and in ammation. Through the in ammation process, the successful implantation of endometrial lesions occurs, and the angiogenesis process approves the viability and the growth of endometrial lesions. A successful in ammatory process can potentially guarantee the precise implantation of endometriosis lesions. In ammation increases the capacity of lesion to attach to the peritoneal layer (in serous layer or abdominal wall) and increases the ability of endometrial cells to survive through induction of vessels formation. Macrophages located in the peritoneal layer are essential elements involved in onset of in ammation. With the permanent attachment of endometrium to peritoneal cells, macrophages begin to invade this area, leading to the release of several in ammatory factors. These in ammatory factors also enter into the peritoneal uid; thus in addition to triggering the in ammation in other parts of abdomen or pelvis, these substances can be measured within the peritoneal uid [6]. TNF-α is an important initiator of in ammation which is secreted by peritoneal macrophages into endometrial lesions and peritoneal uid. It has been shown that the higher level of TNF-α in peritoneal uid causes the severity of in ammation, which indicates a high level of endometriosis lesion attachment.
As it was seen in this study, the level of TNF-α in peritoneal uid was increased signi cantly in all transplanted groups. This incremental change indicated the successful existence of an in ammation model of endometriosis. Thus, allograft or xenograft endometrial transplantations can provide acceptable in ammation to initiate a successful endometriosis induction. In a meta-analysis study, Cao and coworkers investigated the association of TNF-α gene T-1031C polymorphism with endometriosis. As we found the high levels of TNF-α in this study, they also concluded that TNF-α is considered as an important factor in the advancement of endometriosis in females [7]. Although their study was conducted on human cases, the nal result was parallel to our ndings in which the peritoneal levels of TNF-α is increased in animals with endometriosis. TNF-α is an in ammatory cytokine secreted by peritoneal macrophages that induces proliferation, differentiation and migration of endometrial cells in endometrial pathophysiology. According to the in vitro studies, this cytokine stimulates the expression of matrix metalloproteinase. Functional studies in the eld of endometriosis modelling also suggest the vital role of TNF-α in endometriosis progression. Thus, it was proposed that the blockage of TNF-α activity can potentially reduce development of endometriosis [8]. Chronic in ammation following endometriosis induction, in addition to affecting the abdominal and pelvic organs, can also alter in ammatory factors in the blood serum. IL-37 is one of the important blood factors that indicate the presence of in ammation in a part of the body. Although this cytokine is considered as an anti-in ammatory agent, but various studies reported high serum levels of IL-37 in endometriosis conditions. Through examination of the blood serum of transplant recipients, the serum level of IL-37 showed a signi cant increase in allograft and xenograft animals. Also, the level of this factor (IL-37) in the blood of xenograft transplant recipients was considerably higher than the allograft group. This difference approved the higher in ammation rate in xenograft implantation than allografts implantation. The levels of IL-37 in blood serum correlates with endometriosis severity [9]. Thus, as we found in this experimental study, high levels of IL-37 are more prominent in xenograft transplanted animals than other allografts. Jiang and coworkers introduced IL-37 as a biomarker for endometriosis diagnosis because they found that this factor is increased in endometriosis cases [10]. The study of in ammatory cytokines following endometrial induction con rmed the important role of TNF-α and IL-37 in in ammatory phase of endometriosis. Totally it can be concluded that in ammation as a crucial factor can be induced through this xenograft transplantation.
Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Angiogenesis is another critical factor in successful induction of endometriosis. In this study, the rate of angiogenesis was determined by two factors; VEGF-A and the number of blood vessel sections per unit area. This glycosylated mitogen has proliferative effects on endothelial cells leading to angiogenesis. Thus, also the VEGF-A is essential during organ remodelling, but this type of growth factor is widely involved in pathologic condition with the high rate of blood vessels formation (such as endometriosis). The presence of macrophages at the site of vascular lesion transplantation has been suggested as a source of VEGF-A secretion. Thus, it seems that the higher presence of macrophages in the tissue can lead to the higher probability of growth factor secretion and angiogenesis rate. Thus, through initiation of in ammation following transplantation of endometrium, the angiogenesis subsequently occurs following VEGF-A secretion by macrophages. In all lesions induced by allograft and xenograft transplantation methods, the levels of VEGF-A were increased in peritoneal uid. This signalling factor was observed more prominent in both xenograft groups than allograft animals. In addition, the highest presence of VEGF-A in peritoneal uid belonging to the xenograft group was found in the anterior abdominal wall transplantation. With the presence of increased levels of VEGF-A in peritoneal uid, the rate of angiogenesis was also increased signi cantly around the lesions. This means that this growth factor by the effects on the vessels in the abdominal wall or mesenteric vessels induces their growth and accelerates the rate of angiogenesis. As it was obtained by microscopic examination, vessels sections in endometriosis lesions were signi cantly increased compared to the control group. Due to the tissue structure in the anterior wall of the abdomen, more blood ow and blood vessels were seen in this layer than the mesenteric vessels. This condition increased the rate of angiogenesis in endometrial lesions grafted to the anterior abdominal wall than implantation to mesentery. This study also observed that in the allograft and xenograft groups, the number of the blood vessel in tissue sections was much higher in grafts transplanted to the anterior abdominal wall than mesenteric sections. Based on the ndings of biochemical and microscopic studies, it can be mentioned that in xenograft transplant model, a high rate of angiogenesis and factors involved in this process were observed. As Takehara and coworkers found, high levels of VEGF-A were also detected in peritoneal uid. The results of Takehara`s study suggested that endometriosis may arise from eutopic endometrium with higher levels of angiogenic activity possibly induced by VEGF-A.
CA125 is one of the surface components of epithelia in female reproductive tract. This protein is highly glycosylated creating a hydrophilic barrier [11].
Cytoplasmic domain of CA125 interacts with cytoskeleton. In this case, it is affected from intracellular mechanisms and transmits extracellular signals to the intracellular matrix. CA125 has been shown to play a role in advanced tumorigenesis and tumor proliferation. This factor is considered a serum biomarker of endometriosis [12]. The expression of CA125 is altered in several types of cancers and endometriosis [13]. Since this protein is located on the cell surface of endometrial lesions, thus in high grades of endometriosis, serum levels of this protein had elevated concentration [14]. In the present study, signi cant accelerated serum levels of CA125 was also found in all cases of endometriosis (including allografts and xenografts). This nding showed that all implantations of endometrial lesions with a one-month duration of adaption could completely increase the serum levels of CA125 as a marker of endometriosis. CA125 also acts in cell-to-cell interaction for metastasis of cancerous tumors or proliferation of transplanted masses. This factor selectively binds to mesothelin (a glycoprotein expressed by mesothelial cells of peritoneum) to be implanted to peritoneal layer [15]. Rokhgireh et al. evaluated the probable role of CA125 as a non-invasive protocol for endometriosis diagnosis. They concluded that following endometriosis induction, high levels of CA125 in blood serum is also probable, which is parallel to our results [16].
Recent studies have focused on the role of oxidative stress on endometriosis pathophysiology. In oxidative stress condition, an imbalance among reactive oxygen species and antioxidants occurs causing a general in ammatory response in endometrial implanted region. Reactive oxygen species are intermediaries produced by normal oxygen metabolism and are physiological in ammatory mediators to modulate cell proliferation; besides, they have deleterious effects in uncontrolled activity or high concentration. The progression of endometriosis is clearly related to oxidative stress. The connection between endometriosis and ROS production is widely accepted and deeply studied [17]. In endometriotic cells, just similar to cancerous cells, increased ROS production is associated with an increase in cellular proliferation rate [18]. Peritoneal oxidative stress is a major constituent of endometriosis-associated in ammation regulating expression of numerous genes of immune-regulators, cytokines, and cell adhesion molecules which are necessary for ectopic endometrial lesions progression. Peritoneal aggregation of macrophages is at a higher rate in women with endometriosis. Macrophages release prostaglandins, cytokines, and growth factors. In the present study, we found high levels of NO in peritoneal uid. Osborn et al. assessed the expression of nitric oxide synthase by peritoneal macrophages in endometriosis-associated infertility. The higher levels of NOS2 and subsequently higher NOS enzyme activity in these cases. Osborn concluded that in addition to the effects of NO on reproduction, peritoneal NO levels could potentially serve in ammation in abdominal wall, the increase in size of endometrial lesions was at a maximal level compared to other treated groups. According to these ndings, it is concluded that by implantation of endometrial xenograft lesions to the anterior wall of the abdomen, the larger endometrial masses with more weight and pus secretions can be modelled, all of which physical features are crucial factors similar to endometriosis in humans.
A successful endometriosis transplantation should ultimately lead to the production of an endometriosis lesion with speci c histopathological features. Allimportant histopathological factors following endometriosis induction are related to the changes in epithelium, stroma, endometrial glands, surrounding connective tissues, blood vessels, serous or pus discharge into the lumen of endometriosis cavity, haemorrhage, and presence of hemosiderin disposition.
Epithelium in normal rat endometrial grafts prior to transplantation was regular with elongated columnar cells. Thick stroma also contained numerous coronal sections of endometrial glands (2A) which is responsible for secretion of pus or serous liquid by endometrium. After transplantation of this xenograft tissue to the anterior abdominal wall of recipient mice (2C), the normal and physiological arrangement of epithelium was disrupted, and the pathological form was obtained, including irregular epithelium formation and non-columnar epithelial layer. But in this way, high pus secretion still remained in xenograft transplantation to anterior abdominal wall animals. It showed that, although there were various pathological changes in endometrial glands in stroma of lesions, but the pus secretion continued. Besides, probably this deformed epithelium caused high levels of CA125, as a cell surface marker, in blood serum.
High proliferation rate in stromal cells also covered most of the thickness of grafts with different irregular sections of endometrial glands. In allograft endometriosis lesions (2B), a thin layer of primitive and immature epithelium and a thin and degenerated stroma were detected. As it was depicted in Fig. 2B, shrinkage stroma following uterine allograft transplantation contained numerous gaps instead of typical endometrial glands. This pathologic degenerated formation of allografts (2B) caused generation of a lumen of endometrial lesions with no pus or serous secretions in almost all cases. Growth of endometrial glands (2C) resulted in production of a large space containing multiple purulent secretions (2C, yellow star). While the analysis of epithelium and stroma in allograft lesions (2B) showed absence of purulent discharge into the cavity of endometrial lesions. In some cases, serous discharge was seen in a low inseparable amount. Examination of the outer layer of grafted specimens (2C) showed that transplantation of xenograft tissues to anterior wall of abdomen could induce proliferation of connective tissue in the form of scars or extensive dissectible adhesions. This connective tissue (2C) had many blood vessels supplying the nutrition and oxygen for lesion growth. While in the allograft groups, although the uterus was transplanted with all layers (endometrium, myometrium, and perimetrium), but the myometrium and perimetrium layers were degenerated. Besides, although myometrium and perimetrium layers were dissected in xenograft transplantation, but extensive outer connective tissue was generated. Presence of blood vessels in connective tissue (2C) led to vast haemorrhage in endometriosis lesions which were detected by Perl staining. As shown in Fig. 2D, following haemorrhage in endometriosis lesions, the macrophages were activated to regulate RBC phagocytosis. During this stage, hemosiderin granules were deposited in macrophages which were detected by pearl staining against Fe atoms. This feature, which is seen only in xenograft endometriosis lesions attached to the anterior abdominal wall, was a prominent feature of successful induced endometriosis lesions. Finally, it was concluded that based on the histopathological ndings, the xenograft endometrial lesions transplanted to anterior abdominal wall seems more resemble the morphology of endometriosis in human.
In high grades of endometriosis, the gene expression of HOX in uterus could be altered. HOXA10 and HOXA11 are homeobox genes that act as transcription factors essential to embryonic development. The role of each of these two genes in regulation of endometrial development was approved. Both HOXA10 and HOXA11 are essential for embryo implantation and appear to play a similar role in women [21]. Aberrant HOX gene expression following endometriosis contribute to the aetiology of female infertility in these patients. Thus it was approved that high grade and chronic presence of endometrial lesions can nally lead to changes in morphology and molecular characteristics of endometrium in uterus. In the present study, gene expression of HOXA10 and HOXA11 in uterus tissue were decreased in all allograft and xenograft transplanted to the anterior abdominal wall. This difference was more prominent in xenograft transplantation than allografts. Altered gene expression in uterus is considered as the main factor in the end stage of endometriosis pathology. As we found in the present study, genes expression of HOXA10 and 11 were altered, which represented successful implantation of endometrial lesions and endometriosis induction in xenograft transplantations.

Conclusions
This study aimed to determine the most similar model of animal to human endometriosis regarding molecular, histological, and genetic characteristics. Main modi cations to accelerate the success rate of xenigraft implantation were; previous pregnancy experience and exposure to male animals for two weeks. These important conditions led to increased levels of estrogen and high thickness of endometrium for proper dissection. In this study, blood and peritoneal factors affecting in ammation and angiogenesis, physical characteristics of lesions, oxidative stress levels, histopathological changes, and alteration in genes expression of uterus were analyzed and compared. Endometrial lesions isolated from stimulated uterus of a rat and implanted in the anterior abdominal wall of mice in a xenograft surgery could potentially be an appropriate option for endometriosis modelling in rodents. In this transplantation, the in ammatory process occurred, which led to a deep change in all in ammatory factors and oxidative stress in the peritoneal cavity. Subsequently, angiogenesis occurred with emergence of different layers of blood vessels to supply nutrition and oxygen to the implanted lesions. Thus, the lesions showed a higher growth rate. All these changes led to the histopathological alteration of lesions in which the initial and physiological form of lesions prior to implantation was shifted to the pathological form. At this stage, the lesion grew, scar tissue and adhesions appeared, and its lumen was lled with purulent or serous secretions. Eventually, this xenograft modelling also affected the uterine endometrium regarding the HOXA11 and HOXA12 genes expression. The whole above ndings con rmed that xenograft transplantation from rat endometrium to the anterior abdominal wall of mice can be considered a proper model for endometriosis in rodents. In this study, we evaluated the morphometrical, histological and genetic features of implanted endometrial lesions, followed by some laboratory examinations to con rm this type of modelling accuracy. Application of more laboratory protocols and comparison with actual human samples were important limitations we faced to. For future studies, conduction of high levels of laboratory assays like immunoassay and protein immunoblotting is strictly recommended. Also, the use of a human endometriosis sample to compare with rodent`s endometriosis lesions seems necessary to upgrade the level of study.
Declarations Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Rate of genes expression in uterus of graft-recipient animals. Number of exprimental groups; 1st sham, 2nd mice-mice allograft uterus transplantation to anterior abdominal wall, 3rd mice-mice allograft uterus transplantation to mesentery, 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. Data were presented as mean ± SD. * P < 0.05 compared to β-actine baseline