The histopathological features of the surgical endometriosis model using systemic autoimmune disease-prone mice

As mentioned in Materials & Methods, the endometriosis lesions developed in two regions: the proximal lesions and the distal lesions ( Fig. 2A Fig. 2. Endometriosis lesions in MRL/MpJ (MRL/+) and MRL/MpJ- Fas lpr/lpr (MRL/lpr) mice. ( A ) The gross feature of the endometriosis lesions (white arrowheads). ( B ) The appearance rate of endometriosis lesions. ( C ) The appearance rate of the proximal lesions. ( D ) The appearance of the distal lesions. ND: not detected. n=5 for each group. ). Proximal lesions were observed in all mice, whereas distal lesions were observed only in MRL/lpr mice at 3 and 6 months of age. The appearance rates of endometriosis lesions were 80% (one of the five recipient mice did not develop any lesions) and 100% (all examined recipient mice developed distal and/or proximal lesions) in MRL/+ and MRL/lpr mice, respectively ( Fig. 2B ). Recipient MRL/+ and MRL/lpr mice with endometriosis lesions in more than two regions were also observed. Moreover, the appearance rates of proximal lesions were 80% in both mice at 3 months of age, 100% in MRL/+ at 6 months of age, and 40% in MRL/lpr at 6 months of age ( Fig. 2C ). Finally, the appearance rate of the distal lesions in MRL/lpr mice was 40% at 3 months of age and 100% at 6 months of age ( Fig. 2D ). Endometriosis lesions in MRL/MpJ (MRL/+) and MRL/MpJ- Fas lpr/lpr (MRL/lpr) mice. ( A ) The gross feature of the endometriosis lesions (white arrowheads). ( B ) The appearance rate of endometriosis lesions. ( C ) The appearance rate of the proximal lesions. ( D ) The appearance of the distal lesions. ND: not detected. n=5 for each group. Donor and recipient MRL/lpr mice had a higher S/B than MRL/+ mice at 3 and 6 months of age ( Fig. 3A and 3B Fig. 3. The ratio of spleen-to-body weight ratio (S/B) in the donor ( A ) and the recipient ( B ). * P <0.05, ** P <0.01 (Tukey’s test, n=5 for each group). MRL/+: MRL/MpJ, MRL/lpr: MRL/MpJ- Fas lpr/lpr . ). At 3 months of age, the recipient MRL/lpr mice had more severe splenomegaly than the donor MRL/lpr mice at the same age ( Fig. 3A and 3B ). The ratio of spleen-to-body weight ratio (S/B) in the donor ( A ) and the recipient ( B ). * P <0.05, ** P <0.01 (Tukey’s test, n=5 for each group). MRL/+: MRL/MpJ, MRL/lpr: MRL/MpJ- Fas lpr/lpr . At 3 and 6 months of age, the distribution of uterine glands in the lamina propria of the endometrium was lower in MRL/lpr mice than in MRL/+ mice. Estrogen and progesterone receptors were localized to the epithelial cells of the uterine glands and interstitial cells of the lamina propria in all mice. The expression and distribution of sex hormone receptors in the uterine horn did not differ across age and strain groups. CD3-positive T cells were scattered in the lamina propria of the uterine horn of MRL/+ mice at both 3 and 6 months of age and MRL/lpr mice at 3 months of age, whereas they were infiltrated in MRL/lpr mice at 6 months of age ( Fig. 4 Fig. 4. Histology of the uterine horn in donor. Asterisks: uterine lumen, black arrowheads: uterine glands, white arrowheads: immunoreactive positive cells, arrows: CD3-positive T cells. The dashed line shows the infiltration of CD3-positive T cells. HE: hematoxylin-eosin staining, Est R: immunohistochemistry of estrogen receptor, Prg R: immunohistochemistry of progesterone receptor, CD3: immunohistochemistry of CD3-positive T cells. MRL/+: MRL/MpJ, MRL/lpr: MRL/MpJ- Fas lpr/lpr . ). Histology of the uterine horn in donor. Asterisks: uterine lumen, black arrowheads: uterine glands, white arrowheads: immunoreactive positive cells, arrows: CD3-positive T cells. The dashed line shows the infiltration of CD3-positive T cells. HE: hematoxylin-eosin staining, Est R: immunohistochemistry of estrogen receptor, Prg R: immunohistochemistry of progesterone receptor, CD3: immunohistochemistry of CD3-positive T cells. MRL/+: MRL/MpJ, MRL/lpr: MRL/MpJ- Fas lpr/lpr . All mice had uterine-gland-like structures, blood vessels, and cysts in both proximal and distal endometriosis lesions. Estrogen and progesterone receptors are localized in the epithelium of the uterine-gland-like structures and interstitial cells. In all endometriosis lesions, CD3-positive T cells were scattered in the interstitium, with no differences in the expression and distribution among genotype or age groups ( Fig. 5 Fig. 5. Histology of the endometriosis lesions developed in recipient. Asterisks: cysts, black arrowheads: uterine glands-like structures, white arrowheads: immunoreactive positive cells, arrows: CD3-positive T cells. HE: hematoxylin-eosin staining, Est R: immunohistochemistry of estrogen receptor, Prg R: immunohistochemistry of progesterone receptor, CD3: immunohistochemistry of CD3-positive T cells. MRL/+: MRL/MpJ, MRL/lpr: MRL/MpJ- Fas lpr/lpr . ). Histology of the endometriosis lesions developed in recipient. Asterisks: cysts, black arrowheads: uterine glands-like structures, white arrowheads: immunoreactive positive cells, arrows: CD3-positive T cells. HE: hematoxylin-eosin staining, Est R: immunohistochemistry of estrogen receptor, Prg R: immunohistochemistry of progesterone receptor, CD3: immunohistochemistry of CD3-positive T cells. MRL/+: MRL/MpJ, MRL/lpr: MRL/MpJ- Fas lpr/lpr .

In this study, we established a surgical endometriosis model in autoimmune disease-prone mice. The endometriosis lesions developed in MRL-strain mice consist of uterine gland-like structures and interstitial cells expressing sex hormone receptors, indicating that these lesions originated from the uterine fragments transplanted from the donor. The gross and histological features of the endometriosis lesions observed in MRL-strain mice mirror those in previously established murine models of endometriosis [ 6 , 19 , 20 ]. Furthermore, uterine gland development, angiogenesis, and cystic lumen in MRL-strain mice also mirror the ones in humans [ 12 ]. In addition to previously established rodent models of endometriosis [ 5 , 18 , 19 ], the model established in this study presents a valid research approach to understand the relationship between autoimmune disease and endometriosis. The previously established surgical endometriosis models [ 21 , 31 ], in which the endometrial fragments were sutured on the peritoneum or intestinal mesentery, could not determine the transfer properties of the endometrium from the transplantation point to the distal regions of the peritoneal cavity. However, in the present and previously established models [ 5 , 18 , 19 ], mice were injected with fragmented uterine tissues, which was useful for examining the ectopic engraftment and proliferation properties of the endometrium. However, there have been no detailed reports on these properties in the latter models. In this study, we observed the differences in the regions of lesion development among mice genotypes. As reported in previous studies [ 16 , 17 ], MRL/lpr mice showed significant splenomegaly at both 3 and 6 months of age and exacerbated severe systemic autoimmune disease at 6 months of age when compared to MRL/+ mice. Although MRL/lpr mice exhibited systemic autoimmune abnormalities, the appearance rate of endometriosis in MRL/lpr mice at 6 months of age showed no differences between those in MRL/+ mice and MRL/lpr mice at 3 months of age. In contrast, the endometriosis lesions in MRL/lpr mice tend to develop distally from the transplantation site. These results suggest that systemic autoimmune abnormalities in mice do not affect the incidence of endometriosis but do affect the growth of ectopic endometrium. In humans, the common regions of endometriosis development are the ovaries, uterine serosa, and recto-uterine pouch. The clinical symptoms and fertile condition in patients vary depending on where the endometriosis lesions develop [ 33 ]. Systemic autoimmune abnormalities, such as SLE and rheumatoid arthritis, are associated with more severe endometriosis in female patients [ 13 ]. In humans, systemic autoimmune diseases might alter the clinical symptoms of endometriosis, owing to their effect on the region of retrograde endometrium development. MRL/lpr mice with surgically induced endometriosis potentially contribute to the elucidation of the pathogenesis of endometriosis lesions that develop far from the peritoneal opening of the fallopian tube (i.e., recto-uterine pouch and peritoneum) in humans. The histology and distribution of sex hormone receptors and T cells in endometriosis lesions did not differ across mice of different ages and genotypes. In contrast, uterine tissue collected and transplanted from the donor showed significant infiltration of T cells and lower distribution of uterine glands in MRL/lpr mice compared to MRL/+ mice. The uterine glands express a range of molecules that have been thought to be involved in endometriosis development, such as interleukin-11/its receptors [ 28 ] and distal-less [ 2 ]. These molecules are downregulated in endometriosis patients compared to healthy women and are involved in steroid production and endometriosis development [ 2 , 7 ]. Therefore, the lower distribution of the uterine glands in the uterine lamina propria of MRL/lpr mice is estimated to affect the expression levels of molecules regulating endometrial survival and proliferation. Furthermore, regulatory T cells in MRL/lpr mice have been reported to have a reduced capacity to suppress the secretion of inflammatory cytokines from effector T cells [ 24 ]. The decrease in activated regulatory T cell counts results in activation of effector T cells in endometriosis lesions in both humans and mice, indicating that dysregulation of regulatory T cells plays a role in the onset of endometriosis [ 29 ]. Therefore, in MRL/lpr mice, the activation of effector T cells originating from transplanted uterine tissues and the inactivation of regulatory T cells in the recipient peritoneal cavity exacerbated the inflammatory cytokine secretion, progressed the ectopic graft, and proliferated the endometrium, all of which contributed to the endometrium survival at the distal site from the transplantation point. In MRL/lpr mice, the lpr mutation in the Fas gene impairs the Fas-mediated apoptosis of immune cells [ 35 ]. In women with endometriosis, the ectopic endometrium evades scavenging by effector T cells, natural killer cells, and macrophages owing to the inhibition of Fas-mediated apoptosis, which is thought to be involved in the development of endometriosis [ 11 , 30 ]. Although the autoreactive immune cells, including effector T cells, natural killer cells, and macrophages lose Fas activity in MRL/lpr mice and are regarded to have a high ability to eliminate the ectopic endometrium, the distal endometriosis lesions observed in MRL/lpr mice might be caused by prolonged survival of ectopic endometrial cells. The aforementioned mechanisms, including the molecular alterations in uterine glands and activation of effector T cells in the transplanted uterine tissues under the dysregulated regulatory T cells in the recipient mice, seem to be more essential for the proliferation of ectopic endometrium in MRL/lpr mice than the scavenging ability of autoreactive immune cells. In MRL/lpr mice at 3 months of age, the recipients showed a higher spleen-to-body weight ratio than the donors, indicating that endometriosis exacerbates systemic autoimmune disease in mice. When combined with the acute inflammation of ectopic endometrial cells in the peritoneal cavity that transitions to the chronic phase, where autoreactive antibodies are highly produced in women with endometriosis [ 8 ], these results provide basic evidence that endometriosis triggers the progression of systemic autoimmune diseases. In conclusion, the established surgical endometriosis model using MRL/lpr mice revealed an interactive relationship between endometriosis and systemic autoimmune diseases. These findings expand the basic research and understanding of endometriosis pathogenesis.

The authors declare no conflicts of interest.

Animal experiments were approved by the Institutional Animal Care and Use Committee of Rakuno Gakuen University (No. VH21A8). Animals were managed in accordance with the Guide for the Care and Use of Laboratory Animals, Rakuno Gakuen University, Japan. Female MRL/lpr mice and MRL/MpJ (MRL/+) mice, as the wild type of MRL/lpr mice, were obtained at 3 and 6 months of age from Japan SLC, Inc. (Hamamatsu, Japan). The mice were housed in groups within plastic cages at 18–26°C in a 12 hr light/dark cycle with free access to a commercial diet and water. All mice were euthanized by cervical dislocation under anesthesia using a combination of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg). Endometriosis was surgically induced in mice as previously described, with minor modifications [ 5 , 18 , 21 ]. A schematic of the experimental design is shown in Fig. 1A Fig. 1. Scheme showing timeline of the surgical endometriosis model experiment. ( A ) All mice are ovariectomized (ovx) at day 0 and injected estradiol subcutaneously (s.c.) at day 7 and 14. At day 14, the donor mice are euthanized, and their uterine tissues are collected and fragmented. The fragmented uterine tissues are transplanted into the recipient mice. The recipient mice are injected estradiol s.c. on days 17, 20, 23, and 26, and euthanized at day 28. ( B ) The incision and transplantation point on the right dorsal abdominal wall are denoted by the cross mark. ( C ) The regions where the proximal lesions develop around the transplantation point are circled in white line (the peritoneum) and white dashed line (the adipose tissue). The region where the distal lesions develop is circled in black line. The cross mark denotes the sutured incision on the abdominal wall for uterine tissue transplantation. . All mice were ovariectomized under anesthesia by peritoneal injection of a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) and subcutaneous injection of buprenorphine (0.1 mg/kg) was administered for pain-control. Through a 1 cm incision on the right dorsal abdominal wall the ovaries, oviducts, and cranial section of the uterine horn were extracted. The oviducts were ligated and both ovaries were removed. The ligated female reproductive tract was reinserted into the abdominal cavity, and the abdominal wall and skin incisions were closed. All mice recovered after receiving a intraperitoneal injection of atipamezole (0.3 mg/kg) (day 0). On days 0 and 7, all mice were subcutaneously injected with β-estradiol (0.5 µg/head, FUJIFILM Wako Pure Chemical Co., Osaka, Japan) to facilitate the entopic growth of the endometrium as described in previous studies [ 18 ]. Ovariectomized mice were randomly divided into two groups: donor and recipient. Four combinations of donors and recipients were used in this study: (donor/recipient)=(MRL/+ at 3 months of age/MRL/+ at 3 months of age), (MRL/+ at 6 months of age/MRL/+ at 6 months of age), (MRL/lpr at 3 months of age/MRL/lpr at 3 months of age), and (MRL/lpr at 6 months of age/MRL/lpr at 6 months of age) (n=5 per group). Donor mice were euthanized on day 14, and the right uterine horn tissues were collected and fragmented in 400 µL of ampicillin/D-phosphate-buffered saline (PBS) (FUJIFILM Wako Pure Chemical Co.). The left uterine horn was collected, fixed with 4% paraformaldehyde (PFA) overnight at 4°C, and embedded in paraffin. The spleens of donor mice were collected, and the ratio of spleen weight to body weight (S/B) was measured as a marker of systemic autoimmune disease. On day 14, recipient mice were implanted with fragmented uterine tissues, under anesthesia by the peritoneal injection of a combination of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) and a subcutaneous injection of buprenorphine (0.1 mg/kg) was administered for pain-control. Through a 1 cm incision on the right dorsal abdominal wall, fragmented uterine samples collected from donor mice (400 µL/head) were transplanted into the peritoneal cavity of recipient mice ( Fig. 1B and 1C ). In this study, we did not suture the implanted uterine tissues with the abdominal wall. The wounds were closed, and the recipient mice were administered an intraperitoneal injection of atipamezole (0.3 mg/kg) for recovery. The recipient mice were subcutaneously injected with β-estradiol (0.5 µg/head) on days 17, 20, 23, and 26 and euthanized on day 28 to enhance ectopic growth of the endometrium and induce endometriosis [ 18 ]. The S/B ratio was measured, and the endometriosis lesions were collected, fixed with 4% PFA overnight at 4°C, and embedded in paraffin. The endometriosis lesions developed in two regions: the proximal lesions, which developed on the peritoneum or adipose tissue around the transplantation site (i.e., the incision point on the right dorsal abdominal wall) and the distal lesions, which developed on the gastrosplenic ligament or intestinal mesentery far from the transplantation site ( Fig. 1C ). Scheme showing timeline of the surgical endometriosis model experiment. ( A ) All mice are ovariectomized (ovx) at day 0 and injected estradiol subcutaneously (s.c.) at day 7 and 14. At day 14, the donor mice are euthanized, and their uterine tissues are collected and fragmented. The fragmented uterine tissues are transplanted into the recipient mice. The recipient mice are injected estradiol s.c. on days 17, 20, 23, and 26, and euthanized at day 28. ( B ) The incision and transplantation point on the right dorsal abdominal wall are denoted by the cross mark. ( C ) The regions where the proximal lesions develop around the transplantation point are circled in white line (the peritoneum) and white dashed line (the adipose tissue). The region where the distal lesions develop is circled in black line. The cross mark denotes the sutured incision on the abdominal wall for uterine tissue transplantation. The embedded right uterine horn collected from the donor and endometriosis lesions collected from the recipient were sliced into 3.5 µm-thick histological sections. Immunohistochemistry (IHC) and hematoxylin and eosin (HE) staining were performed on deparaffinized sections. Detailed information on the antibodies and serum blocking agents used for IHC is presented in Table 1 Table 1. Primary antibody information in immunohistochemistry Antigen Cat. No Source Host Dilution Blocking serum Biotinylated secondary antibody for immunohistochemistry Estrogen receptor Ab32603 Abcam Inc., Cambridge, UK Rabbit 1:400 10% goat normal serum Goat anti-rabbit IgG antibody, 426012, undiluted (Nichirei) Progesterone receptor MA1-410 Invitrogen, Carlsbad, CA, USA Mouse 1:1,000 10% rabbit normal serum Rabbit anti-mouse IgG+IgA+IgM antibody, 426031, undiluted (Nichirei) CD3 413591 Nichirei, Tokyo, Japan Rabbit No need 10% goat normal serum Goat anti-rabbit IgG antibody, 426012, undiluted (Nichirei) . The sections were then incubated for 15 min at 110°C in 20 mM Tris-HCl (pH 9.0). The sections were soaked in methanol containing 0.3% hydrogen peroxide, incubated with blocking serum for 60 min at room temperature, and then incubated overnight at 4°C with primary antibodies. After washing thrice in 0.01 M PBS, the sections were incubated for 30 min with secondary antibodies before being washed. The sections were incubated for 30 min at room temperature using a streptavidin-biotin complex (SABPRO Kit, Nichirei, Tokyo, Japan), incubated with a 3,3′-diaminobenzidine tetrahydrochloride-hydrogen peroxide solution, and lightly stained with hematoxylin. A Primostar 3 microscope (ZEISS Inc., Oberkochen, Germany) was used to examine the stained sections. The results are expressed as mean ± standard error (s.e.). Data from three or more groups were compared using Tukey’s test ( P <0.05).