Human amniotic epithelial cells improve fertility in an intrauterine adhesion mouse model
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Intrauterine adhesion (IUA) is an adhesion of the uterine cavity or cervical canal resulting from damage to the basal layer of the endometrium; this condition is usually accompanied by fibrosis of the endometrium. Previous studies have demonstrated that human amniotic epithelial cells (hAECs) have stem cell characteristics; however, it is unclear whether hAECs have the therapeutic potential to restore fertility after IUA.
A murine IUA model was established by mechanical injury to the uterus. Then, 106 hAECs were transplanted by intraperitoneal injection. The endometrium thickness, number of glands, and fibrosis area were measured by hematoxylin and eosin (H&E) staining and Masson staining. Molecules (including vWF, VEGF, PCNA, ER, PR, LC3, and p62) related to endometrial angiogenesis, cell proliferation, and autophagy were assayed by IHC staining. Pregnancy outcomes were also evaluated. Finally, hAECs were cocultured with human endometrial mesenchymal stem cells (hEnSCs) damaged by H2O2 to verify the paracrine effect on endometrial stromal cells in vitro.
The IUA uterine cavity presented with adhesion and even atresia, accompanied by a thinner endometrium, fewer glands, increased fibrosis area, and fewer microvessels. However, hAECs significantly improved the uterine structure after IUA. After hAEC treatment, the endometrium was thicker, the number of endometrial glands was increased, fibrosis was reduced, and more microvessels were generated. The expression levels of VEGF, PCNA, and ER were increased in the hAEC-treated endometrium, indicating improvements in angiogenesis and stromal cell proliferation. hAECs also increased pregnancy outcomes in IUA mice, and the pregnancy rate and fetus number increased. Furthermore, we observed altered autophagy in the IUA uterine model, and hAEC transplantation upregulated autophagy. An in vitro study showed that hAECs activated autophagy in (hEnSCs) treated with H2O2 in a paracrine manner.
Our results demonstrated that hAECs have the potential to repair the uterus after injury, providing a new strategy for the prevention and treatment of Asherman syndrome.
KeywordsHuman amniotic epithelial cells Intrauterine adhesion Asherman syndrome Autophagy Fibrosis
Endometrial stromal cells
Hematoxylin and eosin
Human amniotic epithelial cells
Human endometrial mesenchymal stem cells
Microtubule-associated protein light chain 3
Proliferating cell nuclear antigen
Vascular endothelial growth factor
von Willebrand factor
Intrauterine adhesion (IUA) is a consequence of endometrial trauma that leads to the complete or partial obstruction of the uterine cavity or cervical canal. IUA can also be defined as Asherman syndrome, which is characterized by symptoms such as hypomenorrhea, menopause, pelvic pain, recurrent abortion, or infertility . In general, a common cause of IUA is artificial trauma to the uterine cavity during operation procedures, such as curettage, cesarean section, and hysteromyomectomy, which may injure the endometrial basal layer. Tuberculosis and other infections may result in chronic inflammation of the endometrium, which is also a trigger for adhesion. Additionally, congenital Müllerian anomalies, such as uterus septus, increase the incidence of IUA. Trauma, infection, and genetic susceptibility result in the loss of spontaneous endometrium recovery and angiogenesis, initiating adhesion of the endometrium . The incidence of Asherman syndrome is approximately 1.5%. However, repeat curettage resulting from a miscarriage can increase the incidence of IUA to 39% . Currently, hysteroscopy adhesiolysis is the treatment of choice for Asherman syndrome . However, even after adhesiolysis, patients are still susceptible to pregnancy complications, such as preterm delivery and anomalous placenta development, because of impaired metabolism and angiogenesis in the endometrium . Because two thirds of women with Asherman syndrome have undergone post-abortion/miscarriage curettage, it is necessary to take actions to prevent adhesion after invasive operations in the uterine cavity . This can be accomplished via several methods, such as placing an intrauterine device (IUD)/Foley’s catheter balloon/hyaluronic acid in the uterine cavity or using conjugated estrogen treatment to facilitate endometrium recovery [7, 8, 9]. However, a foreign object may give rise to infection risks, and various therapies lack evidence from large-scale randomized clinical trials and lack further follow-up for pregnancy outcomes [10, 11]. Hence, to lower the incidence of Asherman syndrome, efficient efforts to prevent adhesion immediately after exposure to the risk factors would be of great benefit.
Stem cell therapy has been recognized as a potential treatment strategy for IUA. Kilic et al. established a rodent IUA model with trichloroacetic acid and then combined intraperitoneal injection of mesenchymal stem cells (MSCs) with oral estrogen for treatment. These authors showed that MSCs decreased the fibrotic area in the uterus and activated cell proliferation and angiogenesis . Zhang et al. found that in situ injections of induced pluripotent stem cells (iPSCs) could rescue IUA induced by either mechanical injury or lipopolysaccharide (LPS) in mice, thus reducing inflammation and improving fertility . Gan et al. reported that intramuscular injections of human amniotic mesenchymal stromal cells (hAMSCs) could induce endometrium regeneration in rodent IUA models . The efficacy of menstrual blood-derived stromal cells (menSCs) was initially demonstrated in clinical research. Seven severe Asherman syndrome patients were transplanted with their own menSCs. Five of these patients reached the endometrium thickness standard to receive frozen embryo transfer (FET) after transplantation. In the 3-year follow-up, three patients became pregnant spontaneously or through FET . Bone marrow-derived stem cells (BMDSCs) were also proven to be effective in both animal models and clinical trials [16, 17, 18].
In recent years, human amniotic epithelial cells (hAECs) derived from the placenta have been shown to have the multiplex differential potential of embryonic stem cells and the immune-regulating potential of adult stem cells. As perinatal stem cells, hAECs have attracted much attention in tissue regeneration because of their low mutation frequency, low immunogenicity, low tumorigenicity, and rich resources [19, 20]. However, whether hAECs could facilitate IUA recovery is unclear.
In this study, we aimed to demonstrate whether hAEC transplantation during endometrial repair in an IUA mouse model could improve reproductive performance.
Materials and methods
Culture and preparation of hAECs
hAECs were isolated as described previously . The protocol was approved by the Institutional Ethics Committee of the International Peace Maternity and Child Health Hospital. Human amniotic membranes were obtained from healthy women who provided informed consent. Briefly, the amnion was mechanically dissected into segments, digested with 0.25% trypsin/EDTA (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 25 min, filtered through a 40-μm filter, and then centrifuged for 5 min at 300g. Cells were seeded onto 100-mm plates containing Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Gibco) with 5% CO2 at 37 °C. For hAEC transplantation, the hAECs were digested with 0.25% trypsin-EDTA (Biological Industries, Kibbutz BeitHaemek, Israel) at 37 °C and then resuspended in phosphate-buffered solution (PBS) at 107/mL. hAECs used in the experiments had undergone fewer than two passages.
All animal procedures were approved by the Institutional Animal Care and Use Committee of Shanghai and were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals. Six-week-old Balb/c mice were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences and allowed to adapt to the new environment for at least a week. The mice were maintained in SPF conditions. Vaginal smears were obtained daily at 08:00 am to assess estrus cycles. Mice with consecutive 4-day estrus cycles were used to establish the animal model.
To establish an IUA model, mechanical injury of the uterus was performed at the diestrus stage [14, 17]. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium. The uterus was exposed by an excision in the low midline abdomen. A 7-gauge needle was inserted into the connection between the left and right uterus, and both areas were scratched back and forth carefully until the uterus became hyperemic to the naked eye. Subsequently, the abdominal cavity was closed. The surgical procedure was performed under sterile conditions. The control group did not undergo surgery. For the hAEC-treated group, 100 μL of a 107/mL hAEC (106 hAECs) suspension was injected intraperitoneally according to our previous research [21, 22]. The IUA group received 100 μL of PBS. The same amount of hAEC suspension was injected intraperitoneally on three subsequent days after the operation.
Uterus specimens were collected 8 days after the operation. Specimens were fixed in 4% paraformaldehyde, dehydrated, cleared in xylene, and finally embedded in paraffin. The embedded tissues were sliced into 5-μm-thick sections. Hematoxylin and eosin (H&E) staining was used to evaluate the morphological structure of the uterus. Masson staining was applied according to the manufacturer’s instructions (60532ES74, Yeasen, Shanghai, China). The fibrosis area, which was stained pale blue, was evaluated with ImageJ software (NIH, MD, USA).
For hAEC characterization, cells were harvested and incubated with labeled primary antibodies (SSEA4-FITC: 330410, isotype control 401317; CD324-APC: 324108, isotype control 400122; CD146-PE: 361006, isotype control 400114; HLADR-FITC: 11-9956-42, isotype control 11-4724-82; Biolegend, USA) at 4 °C for 30 min. Then, the cells were washed with PBS and analyzed with a BD Accuri C6 (BD Biosciences, NJ, USA).
For hAEC characterization, cells were plated on glass coverslips. After the cell density reached approximately 90%, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and washed with PBS. The cells were permeabilized with 0.5% Triton X-100 for 20 min, washed with PBS, and blocked with 3% goat serum for 30 min. Then, the cells were incubated with primary antibody (CK18: Boster Biological Technology, China; TRA-1-60: Cell Signaling Technology, USA) overnight at 4 °C. The cells were probed with Alexa 594, and the slides were mounted with medium with DAPI (H1200, Vector Laboratories).
For the cell tracking assay, hAECs were previously labeled with carboxyfluorescein succinimidyl ester (CFSE) according to the manufacturer’s instructions (CFDA SE Cell Proliferation and Cell Tracking Kit, Yeasen), and the IUA model was established in mice. For the CFSE group, the mouse uteruses were mechanically injured; then, 100 μL of 107 CFSE-labeled hAECs/mL was injected into the uterus cavity, and the same amount of hAECs in suspension was injected intraperitoneally for three following days. For the control group, murine uteruses were also injured, but the hAEC suspension was replaced with PBS. After 3 days, the uteruses were collected and fixed with optimal cutting temperature (OCT) compound (Sakura Finetek, Seattle, USA), and 10-μm fresh sections were generated. The slides were mounted with a medium with DAPI. Fluorescence images were obtained with a Leica DMI3000 microscope (Heidelberg, Germany).
Primary anti-VEGFA (ab52917, Abcam, USA), anti-vWF (PB0273, Boster, Wuhan, China), anti-PCNA (BM0104, Boster, Wuhan, China), anti-LC3 (WL01506, Wanlei, Shenyang, China), anti-p62 (WL02385, Wanlei, Shenyang, China), anti-ERα (ab32063, Abcam, USA), and anti-PR (sc-810, Santa Cruz, TX, USA) antibodies were diluted in 0.5% goat serum in PBS. The sections were heated in a microwave in sodium citrate solution for antigen recovery and pretreated with 0.3% H2O2 in methanol to quench endogenous peroxidase activity. Then, the samples were incubated with 3% goat serum to block nonspecific antibody binding sites. Subsequently, the samples were incubated with primary antibodies at 4 °C overnight. Immunoreactivity was visualized using a Mouse and Rabbit Specific HRP/DAB Detection IHC kit (ab64264, Abcam) according to the manufacturer’s instructions.
Nine days post-procedure, female mice were mated with male Balb/c mice whose fertility had been verified (8 weeks old). The morning of vaginal plug presence was considered to be gestational day 0.5. All female mice presented vaginal plugs in two estrus cycles. At gestation day 9, the female mice were sacrificed for uterus examination.
Human endometrial mesenchymal stem cells (hEnSCs) were isolated from the menstrual blood of a 40-year-old Chinese woman according to a protocol previously described . Briefly, menstrual blood was collected, and hEnSCs were acquired by density-gradient centrifugation. The procedure was approved by the Institutional Ethics Committee of the International Peace Maternity and Child Health Hospital (Shanghai, China). hEnSCs were cultured in Chang Medium (S-Evans Biosciences, Hangzhou, China) with 5% CO2 at 37 °C. For western blot assay and morphological assessment, 2 × 105 hEnSCs were seeded in the lower compartment of a 6-well plate. H2O2 was added to a terminal concentration of 1 μM to simulate IUA characteristics in vitro according to previous reports . After 2.5 h, the former medium was discarded and replaced with fresh medium. For the hAEC-treated group, 105 hAECs were seeded onto a Transwell insert with a 0.4-μm pore size membrane (Corning) and placed above the lower compartment for coculture. After 24 h, the hEnSCs were collected for protein extraction.
Cell viability assessment
For the cell viability assay, hEnSCs were seeded onto 24-well plates at 5 × 104 cells per well. Cells in the injury group were treated with 1 μM H2O2 for 2.5 h. Then, the former medium was discarded and replaced with fresh medium. After 24 h, cell viability was assessed using a Cell Counting Kit-8 (CCK8) (Yeasen) according to the manufacturer’s instructions.
Western blot analysis
Proteins were extracted from hEnSCs samples using RIPA buffer (Beyotime, Shanghai, China), and the protein concentrations were quantified using a Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific, USA). Proteins were separated in polyacrylamide gel and transferred to the PVDF membranes. Anti-p62 (88588, Cell Signaling Technology, USA), anti-LC3 (WL01506, Wanlei, Shenyang, China), and anti-β-tubulin (30303ES10, Yeasen, Shanghai, China) were used as primary antibodies. The blots were visualized using an Enhanced Chemiluminescence Kit (New Cell & Molecular Biotech Co., Suzhou, China), and the band intensity was quantified with ImageJ software.
The data are expressed as averages with standard deviations. Student’s t test or an ordinary one-way analysis of variance (ANOVA) was used to analyze the data. Significant differences were calculated using GraphPad Prism version 6 (GraphPad Software, La Jolla, USA). Differences were considered statistically significant when p < 0.05.
hAECs restore endometrial morphology in a mouse model of intrauterine adhesions
Fibrosis is a feature of endometrial adhesion. Masson staining was used to evaluate the extent of fibrosis. Compared with that in the control group (19.98 ± 2.92%), the fibrotic area in the endometrium of IUA model mice was significantly increased (43.18 ± 7.35%), but hAEC treatment led to a remarkable reduction in fibrosis (26.08 ± 7.73%) (Fig. 2D, E).
hAECs facilitated endometrial angiogenesis and stromal cell proliferation
Proliferating cell nuclear antigen (PCNA) is an objective index to evaluate the cell proliferation state. Quantitative analysis confirmed that PCNA expression decreased in the injured endometrium, while PCNA expression significantly increased in the hAEC-treated group (Fig. 4C, Fig. 5E).
Estrogen receptor (ER) and progesterone receptor (PR) are indicators of endometrial recovery in IUA . Our results showed that ER expression was significantly increased in the hAEC-treated group compared with that in the IUA group, while the expression of PR changed little among the three groups (Fig. 5A, B, F, G).
hAECs ameliorated pregnancy outcomes in the IUA mouse model
The IUA mice were mated with confirmed fertile male mice to assess the function of the injured uterus. The female mice were euthanized at 8.5 days after the presence of the vaginal plug, and the uterine horns were examined for the numbers and sizes of the fetuses.
hAECs rescue impaired autophagy in endometrial stromal cells in vivo and in vitro
Furthermore, we used an in vitro coculture system to confirm whether hAECs regulate autophagy in endometrial stromal cells in vitro. Human endometrial mesenchymal stem cells (hEnSCs) were isolated from menstrual blood and shown to have endometrial stromal cell characteristics .
Next, we investigated the effect of hAECs on the autophagy of hEnSCs by western blotting. Consistent with the IHC results, the protein level of p62 was significantly higher in H2O2-treated hEnSCs than in the control cells; however, the protein level of p62 in hEnSCs cocultured with hAECs was significantly decreased. In addition, H2O2 treatment significantly reduced the ratio of LC3-II/LC3-I in hEnSCs, whereas the ratio of LC3-II/LC3-I in hEnSCs cocultured with hAECs dramatically increased (Fig. 8D, E).
Several other proteins related to endometrium repair, including ER, PR, PCNA, and VEGF, were also evaluated by western blotting. The results showed that H2O2 treatment led to reduced levels of ER expression molecules and that hAECs could restore these expression levels in H2O2-treated hEnSCs. However, we did not observe similar changes in VEGF, PCNA, and PR expression in these three groups (Fig. 8D, E). These results indicate that hAEC transplantation could at least partly recover the damaged endometrium through autophagy induction.
The aim of this study was to determine whether hAEC transplantation could restore the fertility of an IUA mouse model. Herein, we successfully established an IUA model in female mice by mechanical injury. When IUA occurred, the murine uterus became adherent, the endometrium was thinner, the glands and microvessels decreased, and the fibrotic area increased, resulting in decreased fertility. By transplanting hAECs into IUA mice, the endometrial morphology improved, and the pregnancy rate and litter size increased. Furthermore, we observed autophagy suppression in endometrial stromal cells in IUA mice. Using a coculture system in vitro, we demonstrated that hAECs could activate autophagy in damaged endometrial stromal cells.
We conducted mechanical injury to the murine uterus to imitate uterine curettage. All intrauterine operations were performed by the same experimenter to minimize the bias resulting from the differences in personal techniques. We have previously shown that intraperitoneal hAEC injection improved ovarian function in a mouse model of primary ovarian insufficiency . Different methods, including tail vein injection, intraperitoneal injection, intramuscular injection, and intrauterine injection, have been used for stem cell transplantation to facilitate endometrium repair in animal models [12, 14, 16, 17]. As described in Kilic’s study, hAECs were transplanted into an IUA murine model by intraperitoneal injection immediately after the operation and for three subsequent days . Our cell tracking assay showed that hAECs migrated to the injured uterus, which demonstrated that hAECs homed to the damaged uterus (Fig. 3B). (Linefeed) Autophagy is a cellular degradation process in response to environmental stress that breaks down senescent organelles and provides energy when nutrition is lacking . Autophagy is closely relevant to physiological and pathological activities such as inflammation, apoptosis, cell proliferation, differentiation, and metabolism, and plays an important role in maintaining the endometrial function [28, 29]. Choi et al. collected endometrial samples from premenopausal/nonpregnant women and found that LC3-II gradually increased and reached its peak level at the late secretory phase within the menstrual cycle. These authors also found that autophagy worked in concert with apoptosis to rebuild the endometrium during the menstrual cycle . Tseng et al. analyzed an RNA array of endometrial samples from premenopausal women and found that two autophagy-related genes, γ-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1) and γ-aminobutyric acid receptor-associated protein-like 3 (GABARAPL3), showed the highest expression in the mid-secretory phase and the lowest expression in the late secretory phase . Xu et al. studied the repairing effect of temperature-sensitive heparin-modified poloxamer hydrogel with affinity to keratinocyte growth factor (KGF) in a rat IUA model. These authors demonstrated that LC3-II was decreased and that p62 was increased in the IUA model, but with hydrogel and KGF treatment; LC3-II relatively increased; and p62 decreased. This evidence suggested that endometrial autophagy was suppressed when IUA occurred, and the recovery process included autophagy rescue . Consistent with these findings, we showed that hAECs impaired autophagy in a murine IUA model and that hAECs activated autophagy in endometrial stromal cells in vitro.
Previously, we used a cytokine array assay and reported that concentrated hAEC medium contained abundant cytokines, including autophagy-related factors, such as IL-2, IL-6, and IL-1. IL-2 is an autophagy-activating cytokine . Recombinant IL-2 upregulated autophagy in liver injury in C57BL/6 mice . IL-2 activated autophagy in mouse embryonic fibroblasts and primary lung fibroblasts by regulating ATG5 and Beclin1 . Our Transwell coculture assay suggested that the autophagy-inducing effect of hAECs could occur through paracrine effects. However, whether hAECs induce autophagy by secreting cytokines, such as IL-2, should be further elucidated.
In the clinic, patients with Asherman syndrome are more likely to be infertile and suffer more pregnancy complications after surgical treatment. For example, the thin endometrium of IUA uteruses reduces the chance of pregnancy, and the low oxygen content impairs endometrial receptivity . Thus, adhesiolysis may not fully rescue the fertility of Asherman syndrome patients. For further fertility improvement, it is necessary to create a better endometrial environment in multiple aspects, including blood supply and oxidative stress. Therefore, we analyzed several molecules relevant to endometrial recovery in a murine model.
PCNA is responsible for accurate DNA duplication . Niklaus et al. compared endometrial samples from reproductive-age women and found that in both stroma and epithelial tissues, PCNA was most abundant at the proliferative phase but decreased at the secretory phase . In our study, hAECs significantly increased PCNA expression in the murine endometrium, indicating that hAECs might improve proliferation in the endometrium.
VEGF is expressed mostly during the menstrual period and proliferative phase and is relevant to the maintenance and formulation of microvessels and the reconstruction of endometrial tissue [37, 38]. Chen et al. found that hormone replacement therapy (HRT) combined with hysteroscopic adhesiolysis significantly increased endometrial VEGF expression and MVD in IUA patients. Additionally, those who had a better curative effect had higher VEGF expression and denser microvessels than those who responded poorly to the treatment. In our study, hAECs increased VEGF expression and MVD in the IUA model, indicating the angiogenesis effect of hAECs, which might facilitate endometrial injury recovery.
ER is a nuclear transcription factor that promotes metabolism and proliferation in endometrial cells in combination with estrogen . ER expression is significantly increased in the repaired endometrium of allogeneic UCMSC therapy-treated patients . Consistent with this finding, hAECs increased ER expression in damaged murine endometrium. Thus, the status of ER might regulate endometrial injury repair.
Recent studies have shown that stem cells could serve as a therapeutic agent for IUA caused by endometrial injury . In comparison with other stem cells, hAECs derived from human amnion, a type of medical waste, have a sufficient source and can be amplified easily in vitro. Some ethical problems related to embryonic stem cells could be avoided. Additionally, hAECs have low immunogenicity and lack tumorigenesis properties. Thus, hAECs have these advantages for broad application in regeneration medicine.
Here, we report for the first time that hAECs have the potential to restore fertility in an IUA mouse model and that hAECs promote endometrial injury repair by activating the autophagy pathway. Further studies should explore whether the autophagy pathway participates in angiogenesis and whether hAEC treatment combined with an autophagy inhibitor would improve endometrial regeneration.
The present study demonstrates that intraperitoneal transplantation of hAECs improved endometrium morphology in an IUA mouse model, contributing to a thicker endometrium, more glands, and less fibrotic area in an injured endometrium. hAECs also facilitated endometrial stromal cell proliferation and angiogenesis in an IUA model and promoted pregnancy in an IUA mouse model. Finally, hAECs promoted endometrial injury repair by activating the autophagy pathway.
LD and LB designed and conceived the study and drafted the manuscript. LB and SJ established the animal model. ZQ analyzed the cell characteristics. LB participated in the cell morphology assays, immunohistochemistry, and in vitro experiments. LB carried out the data analysis and manuscript revision. All authors read and approved the final manuscript.
This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800 and 2018YFC1004802), the Shanghai Municipal Council for Science and Technology (18410721200), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20152236), the National Natural Science Foundation of China (81741013), and the Opening Fund of the Key Laboratory for the diagnosis and treatment research of reproductive disorders of Zhejiang Province (2018001).
Ethics approval and consent to participate
This study was approved by the Institutional Ethics Committee of the International Peace Maternity and Child Health Hospital (Shanghai, China), and written informed consent was obtained from all participants.
Consent for publication
The authors declare that they have no competing interests.
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