{"paper_id":"1f4d806a-ed08-4526-8742-da442cec13ad","body_text":"Vol:.(1234567890)\nReproductive Sciences (2024) 31:1662–1673\nhttps://doi.org/10.1007/s43032-024-01458-2\nINFERTILITY: ORIGINAL ARTICLE\nA Novel Method to Repair Thin Endometrium and Restore Fertility \nBased on Menstruation‑Derived Stem Cell\nKai Chen1  · Huiru Wang1 · Xin Zhao1,2 · Jingxin Wang1,2 · Qi Jin1 · Xianhong Tong1 · Shengxia Zheng1 \nReceived: 5 September 2023 / Accepted: 8 January 2024 / Published online: 31 January 2024 \n© The Author(s) 2024\nAbstract\nThin endometrium (TE), which mainly occurs as a result of severe damage to the endometrial basalis, is one of the prominent \netiologies of menstrual abnormalities, infertility, and recurrent miscarriage in women. Previous studies have demonstrated \nthat mesenchymal stem cells (MSCs) are considered ideal cells with multipotency for regenerative medicine and exhibit \ntherapeutic effects on TE through their cellular secretions. However, there is limited research on strategies to enhance MSC \nsecretion to improve their therapeutic efficacy. Herein, we isolated menstrual blood–derived mesenchymal stem cells (Men-\nSCs) from menstruation and transformed them into decidualized stromal cells (DSCs), which are specialized cells with \nenhanced secretory functions. To assess the therapeutic potential of DSCs compared to MenSCs, we conducted a series of \nexperiments in cells and animals. The results demonstrated that DSCs exhibited changes in morphology compared to Men-\nSCs, with a decrease in cell proliferation but a significant improvement in secretion function. Furthermore, DSCs facilitated \nthe restoration of endometrial thickness and increased the number of glands and blood vessel formation. Most importantly, \nthe pregnancy rates in rats were effectively restored, bringing them closer to normal levels. These findings greatly contribute \nto our understanding of stem cell therapy for TE and strongly suggest that DSCs could hold significant promise as a potential \ntreatment option for TE.\nKeywords Thin endometrium · Menstrual blood stem cells (MenSCs) · Decidualization · Stem cell therapy · Animal model\nIntroduction\nHuman endometrium can be divided into approximately two-\nthirds of the functional layer located in the upper region and \none-third of the basal layer situated in the lower basal layer \n[1]. The periodic shedding of the functional layer, regulated \nby fluctuating levels of estrogen and progesterone, facilitates \nendometrial self-renewal [2]. Normal endometrium plays a \ncrucial role in the establishment and maintenance of preg -\nnancy [3]. However, invasive procedures performed within \nthe uterine cavity, such as curettage after abortion, can result \nin damage to the basal layer and subsequently lead to thin-\nning of the endometrium (TE) [4].\nTE can significantly impair endometrial receptivity, \nleading to embryo implantation failure or post-pregnancy \nmiscarriage [5]. Therefore, restoring TE to its normal thick-\nness is essential for the successful establishment and main-\ntenance of pregnancy [6, 7]. Currently, the primary clinical \napproach for treating TE involves stimulating endometrial \ngrowth through estrogen administration following adhesions \nunder hysteroscopy [8]. However, traditional hysteroscopic \ntreatment cannot fully achieve the desired effect and is often \naccompanied by a high recurrence rate. To prevent re-adhe-\nsion after hysteroscopy separation of adhesion, researchers \nhave explored the use of intrauterine devices (IUDs), bal-\nloons, and sodium hyaluronate to prevent adhesion after \nsurgery, but still could not fundamentally solve the problem \n[9, 10]. Thus, treatment of TE remains a thorny problem, and \nKai Chen and Huiru Wang contributed equally to this work and \nshould be considered co-first authors.\n * Xianhong Tong \n tong68xianhong@163.com\n * Shengxia Zheng \n zhengshengxia@ustc.edu.cn\n1 Reproductive Medicine Center & Department of Obstetrics \nand Gynecology, The First Affiliated Hospital of USTC, \nDivision of Life Sciences and Medicine, University \nof Science and Technology of China, Hefei 230001, Anhui, \nChina\n2 Wannan Medical College, Wuhu 241002, Anhui, China\n\n1663Reproductive Sciences (2024) 31:1662–1673 \nfuture investigations are imperative to enhance endometrium \nthickness and pregnancy outcomes in patients with TE.\nMesenchymal stem cell (MSC) has attracted more and \nmore attention in the treatment of TE, for MSC shows great \npotential in promoting cell proliferation, immune regulation, \nand angiogenesis in tissue repair [11]. It has been proved \nthat the main cause of TE is the reduction in the number of \nbasal layer stem cells of the endometrium [12]. Researchers \nextracted MSC from the endometrium of both TE patients \nand normal women, respectively, for comparative analysis. \nThe results showed that the MSC derived from TE endome-\ntrium had lower angiogenesis and cell proliferation capacity \n[13]. Consequently, restoring the population of basal stem \ncells plays an important role in improving endometrium \nthickness and enhancing pregnancy rates in TE patients.\nEndometrial decidualization refers to the transforma -\ntion of endometrial stromal cells into specialized secretory \ndecidualized cells with hormonal changes, which plays an \nindispensable role in successful embryo implantation and \nearly pregnancy maintenance [14]. The development of \nendometrial decidualization is the foundation of pregnancy \nas it promotes angiogenesis and immunomodulation [15]. It \nhas been shown in recent studies that abnormal endometrial \ndecidualization is strongly associated with early biochemical \npregnancy and abortion, and improvement of endometrial \ndecidualization will play a crucial role in embryo implanta-\ntion and successful pregnancy [16]. However, as decidu-\nalization occurs in the female endometrium, few research-\ners have recapitulated this process in vitro using menstrual \nblood-derived mesenchymal stem cells (MenSCs). It remains \nunclear whether decidualized stromal cells (DSCs) play a \ndefinite role in repairing TE.\nIn this study, we first induced MenSCs into DSCs in vitro \nand subsequently compared the morphological and func-\ntional characteristics between these two cell types. Secondly, \nthe two types of cells were compared in terms of their effects \non enhancing endometrial thickness, number of glands, and \nangiogenesis. Finally, the effects on fertility recovery of TE \nrats were observed by counting the number of embryos. \nAltogether, this study provides a new idea for the clinical \ntreatment of infertility patients caused by TE.\nMaterials and Methods\nAnimals\nEight- to 10-week-old Sprague-Dawley female rats weigh-\ning 160–200 g were used in all experiments, and all rats \nwere purchased from SLAC Laboratory Animals (Shanghai, \nChina). The animals had free access to water and food and \nwere maintained in a feeding room on a 12-h light and 12-h \ndark regimen with an average temperature of 22 °C and 70 to \n80% relative humidity. All the procedures were approved by \nthe Institutional Animal Care and Use Committee at the First \nAffiliated Hospital of USTC (code no. 2022-N(A)-119).\nIsolation of Menstrual Blood Stem Cells (MenSCs)\nMenstrual blood donors aged 25–40 years old, who were \ndiagnosed without any reproductive system-related dis-\neases (n =3). All donors gave consent, and all procedures \nwere approved by the Ethics Committee of First Affiliated \nHospital of USTC (IRB code NO. 2021KY015). MenSCs \nwere isolated using human lymphocyte isolation solution \n(abs930, Absin, China). The cells were routinely cultured \nin Dulbecco’s Modified Eagle’s Medium (DMEM,1-051-\n1ACS, BI, Israel) supplemented with 100 U/mL penicillin, \n100 μg/mL streptomycin (BL505A, Biosharp, China), and \n10% fetal bovine serum (FBS, F7524, Sigma, Germany) at \n37 °C under a humidified atmosphere containing 5% CO2. \nThe culture medium was changed every 3–4 days until \nadherent cells reached a confluence of approximately 90%; \nthen, the cells were passaged using 0.05% trypsin-EDTA \nsolution (25300-062, Gibco, USA).\nImmunofluorescence\nFor paraffin tissue sections, deparaffinization was performed \nusing a dewaxing solution (G1128, Servicebio, China) \nfollowed by rehydration in decreasing concentrations of \nethanol. For cells on culture dishes, the supernatant was \ndiscarded and the cells were fixed with paraformaldehyde \n(G1101, Servicebio, China) at room temperature for 20 min. \nAfter antigen retrieval and blocking of endogenous peroxi-\ndases, the samples were incubated in a wet box overnight at 4 \n°C with antibodies raised against Vimentin (VIM, SC-6260, \nSanta Cruz, USA, 1:200), cytokeratin7 (CK 7, 15539-1-AP, \nProteintech, China, 1:200), CD31 (28083-1-AP, Proteintech, \nChina, 1:200), and OCT-4 (ab19857, Abcam, USA). After \novernight incubation, the samples were washed with PBS \nand incubated with Alexa Fluor 488 or Alexa Fluor 594 sec-\nondary antibody (Jackson labs, USA, 1:200) for 3 h at room \ntemperature. This was followed by another wash in PBS \nand nuclear staining conducted with DAPI (1155MG010, \nBioFroxx, Germany, 1:1000). All procedures were in dark \nconditions. Images were collected by ECHO Revolve FL \n(ECHO, USA). All immunofluorescence tests were repeated \nat least three times.\nIdentification of MenSCs\nFlow cytometry was conducted using the Human MSC Anal-\nysis Kit (562245, BD Biosciences, USA). Briefly, MenSCs \nwere detached from the culture dish using Accutase (A6964, \nSIGMA, USA) at passage 3, and then resuspension in FACS \n\n1664 Reproductive Sciences (2024) 31:1662–1673\nbuffer at a concentration of 2 ×  104 cells/20 μL. Then, they \nwere incubated with the respective antibodies (1:200) in the \ndark at room temperature for 20 min: IgG1-PE, IgG1-FITC, \nIgG1-PC5.5, CD44-PE, CD73-APC, CD90-FITC, CD105-\nPC5.5, and CD11B/34/79a-PE. Then, the cells were washed \nwith FACS buffer and centrifuged at 600 rpm for 5 min. The \ncells were resuspended with 400 μL of FACS buffer. The \nantibody-labeled cells were analyzed with a BD FACSAriaII \ncell sorter (n=3).\nWe used a Human MSC Functional Identification kit \n(SC006, R&D systems, USA) to evaluate the ability of \nMenSCs to differentiate into multiple mesenchymal line-\nages (adipogenic, osteogenic, and chondrogenic). Briefly, \nthe MenSCs were cultured in a 24-well plate and incubated \nwith differentiation medium to induce the differentiation \nprocess. Following differentiation, the cells were fixed with \nparaformaldehyde. Ultimately, differentiation outcomes \n(n=3) were assessed through immunofluorescence staining \nof osteocalcin as an osteocyte marker, FABP-4 as an adipo-\ncyte marker, and aggrecan as a chondrocyte marker (undif-\nferentiated MenSCs as negative control).\nDecidualization of MenSCs\nUpon reaching 70% confluence in the petri dish, the Men-\nSCs were subjected to a medium change with differentia-\ntion media. Specifically, the differentiation medium was \nsupplemented with 10 nM/L 17-β-E2 (abs47006987, Absin, \nChina), 1 uM/L MPA (abs44122880, Absin, China), and \n0.2mM/L cAMP (D0627, Sigma, USA). After 14 days, the \ninduction of decidualization was brought to a halt (n =3).\nCell Proliferation Curve\nThe cells were seeded at a density of 100,000 cells per well \nin 6-well plates (n=3). Adherent cells were then digested and \ncounted every 2 days to generate the cell proliferation curves \nbased on the cell count.\nVEGF‑A Quantification\nMedia conditioned by MenSCs and DSCs were collected after \n24 hours (n=3). The concentration of VEGF-A (RK00023, \nABclonal, China) and PRL (KE00172, Proteintech, China) \nwas quantified using a commercially available enzyme-\nlinked immunosorbent assay kit. Firstly, samples were  \nadded and incubated at 37 °C for 2 h. Next, antibodies were \nadded and incubated at 37 °C for 1 h. Streptavidin-horserad-\nish peroxidase was then added for another incubation at 37 \n°C for 40 min. Finally, after adding the chromogenic solution \nand the terminating solution, the absorbance was measured, \nand a standard curve was constructed to determine the con-\ncentration of the sample under investigation.\nEndothelial Cell Scratch Healing Assay\nA total of 2 ×  105 human umbilical vein endothelial cells \n(HUVECs, Oricell) at passage 4 were seeded in a 6-well \nplate. Once the cells reached 100% confluence, scratches \nwere made by a 200-μL sterile pipette tip perpendicular to \nthe marking line. The culture medium was changed into con-\nditioned medium (MenSCs and DSCs for the experimental \ngroup and DMEM for the control group). All wells were \nimaged at 0 h and 24 h after creating a scratch. ImageJ soft-\nware was used to calculate the average area between cells \n(n = 3).\nDetermination of Estrus\nA sterile cotton swab was immersed in normal saline to col-\nlect the rat vaginal swab (n =9). The swab should be gen-\ntly rotated and rolled against the vaginal wall before being \nremoved. The collected cells are then delicately transferred \nTable 1  Primes used in this study\nPrime Forward prime Reverse prime\nHuman-derived primes\n  GAPDH ACA CCA TGG GGA AGG TGA AG GTG ACC AGG CGC CCA ATA \n  PRL CAA AGG ATC GCC ATG GAA CAC AGG AGC AGG TTT GAC AC\n  IGFBP-1 TTT TAC CTG CCA AAC TGC AACA CCC ATT CCA AGG GTA GAC GC\n  LIF CCA ACG TGA CGG ACT TCC C TAC ACG ACT ATG CGG TAC AGC \n  HOXA10 CTC GCC CAT AGA CCT GTG G GTT CTG CGC GAA AGA GCA C\n  OCT-4 GCT GGA GCA AAA CCC GGA GG TCG GCC TGT GTA TAT CCC AGG GTG \nRat-derived primes\n  GAPDH TTC CTA CCC CCA ATG TAT CCG CAT GAG GTC CAC CAC CCT GTT \n  VEGF ACA TCT TCA AGC CGT CCT GTG TGC AAA TGG CGA ATC CAG TCC CAC GAG \n\n1665Reproductive Sciences (2024) 31:1662–1673 \n\n\n1666 Reproductive Sciences (2024) 31:1662–1673\nonto a dry glass slide by gently sliding the swab across the \nsurface, followed by microscopic examination for identifica-\ntion of cell composition. When the vaginal swab contained \nmore anucleated keratinized epithelial cells and fewer neu-\ntrophils, it indicated that the rats’ uterus lining was thicker \nduring this period.\nEstablishment of the Rat Thin Endometrium (TE) \nModel\nRats in estrus were selected based on vaginal secretions \nand then anesthetized with Zoletil 50 (Virbac, France) via \nintramuscular injection at a concentration of 0.1 mL/100 g. \nFollowing shaving, the lower abdomen was disinfected with \niodophor. A midline incision was made across the skin and \nmuscles to expose the uterus. A precise longitudinal inci-\nsion measuring 5 mm in length was made near the uterine \nwall, close to the vaginal opening. Upon entering the uterine \ncavity, gentle rubbing was performed with the eye forceps \nfor a total of ten repetitions, resulting in a collective total of \n40 repetitions. Subsequently, the uterine cavity was washed \nwith 5 mL of normal saline thoroughly. Then, the uterus was \nsutured using 6-0 absorbable sutures, followed by suturing \nof the muscles using 5-0 absorbable sutures. Finally, the \nskin was sutured using non-absorbable sutures. All rats were \nrandomly assigned to the group.\nHistological Analysis\nHematoxylin and eosin (H&E) and Masson staining were \nemployed in the evaluation of rat endometrial tissue (n =6). \nParaformaldehyde was fixed at room temperature for at least \n24 h and then embedded in paraffin after removing the rat \nuterus. The sections were then cut into a thickness of 10 μm. \nThe endometrial thickness was measured from the luminal \nepithelium to the smooth muscle layer with imaging. The \ntwo perpendicular lines are averaged, and the average of the \nthree measured slices was taken.\nQuantitative Real‑Time Polymerase Chain Reaction\nTotal RNA was extracted from the adherent cells or excised \nuterine tissues by using the RNA-Quick purification kit \n(RN001, Esscience, China), and the reverse transcription \nwas employed using RT reagent kit (R-323-01, Vazyme, \nChina). Cham Q Universal SYBR qPCR Master Mix (Q511-\n02, Vazyme, China) was used for quantitative RT-PCR reac-\ntion. The primers used in this study are listed in Table  1. \nQuantitative RT-PCR was performed by LightCycler 96 \nInstrument (Roche, USA), with the following program set \nto 95 °C 30 s, 95 °C 10 s, 60 °C 30 s, 40 cycles, 95 °C 15 s, \n60 °C 60 s, 95 °C 15 s. GAPDH was used to normalize the \nrelative levels of the gene (n=3).\nFertility Test\nEight- to 10-week-old Sprague-Dawley female rats were \nused in the therapy experiment. Group 1 (6 VS 6) was the \nsham operation group on the left and the control (without \nany treatment) on the right. In group 2, the left uterus was \nthe sham-operated side, and the right side was the TE side \n(15 VS 15). In group 3, the left uterus was the sham-operated \nside, and the right side was the TE+MenSCs therapy side \n(15 VS 15). In group 4, the left uterus was the sham-operated \nside, and the right side was the TE+ DSCs therapy side (15 \nVS 15). In all cell-based therapy experiments, a total of 1 × \n 106 cells were suspended in 50 μL of DMEM. In the control \ngroup, 50 μL of DMEM was injected into the uterine cav -\nity. On the  14th day after treatment, the female rats were \nhoused together with healthy male rats at a ratio of 2:1. The \ndiscovery of the vaginal plug-in female rat is considered as \nday 0 of pregnancy [17]. The pregnant rats were sacrificed \non gestation days 14–18, and the number of embryos on both \nsides was counted.\nStatistics\nStatistical analysis was performed using GraphPad Prism \n7.0 (San Diego). The results are shown as mean ± SEM. In \nthis experiment, the one-way ANOVA test was used in the \nstatistical analysis of multiple comparisons between groups. \nThe two-tailed paired t-test was used in the comparative sta-\ntistical analysis of the two groups. *p  < 0.05, ** p < 0.01, \n***p < 0.001 are considered statistically significant.\nResults\nIsolation and Identification of MenSCs\nMenstrual blood–derived mesenchymal stem cells (MenSCs) \nwere isolated from female menstrual blood and cultured in \nFig. 1  Isolation and identification of MenSCs. A The morphology \nof MenSCs in the bright-field. Scale bar: 210 μm. B Cell composi-\ntion in primary cell (P0), passage 1 (P1), and passage 2 (P2). Green \nrepresents the stromal cell marker (VIM), red represents the epithelial \ncell marker (CK7), and blue represents the nuclear marker (DAPI). \nScale bar: 170 μm. C Isolated MenSCs at passage 4 were used for \nflow cytometry analyses, and the values represent the percentage \nof positive cells among all cells. CD44 (99.54%), CD73 (98.30%), \nCD90 (91.88%), CD105 (92.88%), CD11b/CD34/CD79a (0.04%). \n(D) Immunofluorescence staining verified the multipotent differentia-\ntion ability of the MenSCs, representing the ability of adipogenesis \n(FABP-4), osteogenesis (osteocalcin), and chondrogenic (aggrecan) \ndifferentiation, negative control was undifferentiated MenSCs. Nega-\ntive control scale bar: 50 μm. Multipotential differentiation scale bar: \n170 μm. E OCT-4 gene expression in the MenSCs, the control is \nfibroblast cells. F OCT-4 protein in the fibroblast (negative control) \nand MenSCs. Scale bar: 50 μm\n◂\n\n1667Reproductive Sciences (2024) 31:1662–1673 \n\n\n1668 Reproductive Sciences (2024) 31:1662–1673\nthe petri dish, exhibiting a fibroblast-like spindle morphol-\nogy (Fig.  1A). The endometrium typically consists of epi-\nthelial cells, stromal cells, vascular endothelial cells, and \nimmune cells [18]. Immunofluorescence was then performed \nto analyze the cell composition between different passages. \nThere were stromal and epithelial cells in P0 and P1, but \nepithelial cells disappeared at P2 with the application of \nDMEM (Fig.  1B). Cells after P2 were used for all the sub-\nsequent experiments to minimize any potential confounding \neffects from other cell types.\nTo evaluate the mesenchymal stem cell (MSC) properties \nof the MenSCs, we performed flow cytometry analysis to \nexamine the cell surface marker of MenSCs at P3 [19]. More \nthan 90% of cells were positive for CD44, CD73, CD90, and \nCD105, and less than 1% of MenSCs were positive for other \ncell surface markers, such as CD11b, CD34, and CD79a \n(Fig.  1C). In vitro differentiation experiments also proved \nthat the MenSCs can be induced into osteoblasts, adipocytes, \nand chondrocytes, respectively (Fig. 1D), demonstrating their \nmultipotent differentiation.\nPrevious studies have shown that endometrial stem cells \nhighly express the OCT-4 gene [20]. To investigate the expres-\nsion of the OCT-4 gene in MenSCs, we compared it with \nfibroblasts. According to the results, it is evident that Men-\nSCs express the OCT-4 gene at high levels and also exhibit \nsubstantial expression of the OCT-4 protein (Fig. 1E, F). These \nfindings provide evidence for the origin of MenSCs from the \nendometrium (compared with fibroblast). The experimental \nprocedure is summarized in Fig. 2A.\nDecidualization of MenSCs In Vitro\nThe P3 MenSCs were used to induce decidualization in vitro, \nresulting in a transformation of cell morphology from spin-\ndle-shaped to oblate (Fig. 2B). Apart from the morphological \nchanges, the proliferative ability of decidualized stromal cells \n(DSCs) was found to be weaker compared to that of MenSCs \n(Fig.  2C). The expression levels of decidual-related genes \n(PRL and IGFBP-1) significantly increased in DSCs, along \nwith an increase in endometrial receptivity-related genes (LIF \nand HOXA-10) (Fig.  2D). Furthermore, the ELISA experi-\nment revealed higher levels of PRL protein in the DSC cell \nsupernatant, indicating that DSC cells secrete a greater amount \nof PRL protein (Fig.  2E). These findings suggest significant \nfunctional differences between DSCs and MenSCs, as indi-\ncated by variations in cell morphology, proliferation capacity, \nand gene expression patterns.\nMenSCs and DSCs Secret More VEGF‑A In Vitro\nResearchers have proven that MSCs possess the ability to \ninduce angiogenesis, primarily through the secretion of VEGF-\nA [21]. Therefore, we verified the secretion of VEGF-A by \ndetecting the concentration of supernatant. Our results indicate \nthat MenSCs and DSCs secreted more VEGF-A compared to \nthe un-conditioned media. In addition, DSCs secrete twice as \nmuch VEGF as MenSCs (Fig.  3A). Subsequently, we found \nthat the supernatant from the MenSCs and DSCs significantly \npromoted scratch closure of endothelial cells compared to the \ncontrol (Fig. 3B, C). These results demonstrate that both DSCs \nand MenSC secrete VEGF and promote the proliferation and \nmigration of vascular endothelial cells.\nThe Therapeutic Effects of MenSCs and DSCs on TE \nRats\nBefore conducting the animal experiment, we determine \nwhether the rat is in estrus based on its vaginal secretion \n[22]. When the vaginal secretion contained more anucle-\nated keratinized epithelial cells, the uterus of the rats was \nthicker at this time, which was conducive to the establish-\nment of the TE model (Supplement Fig. 1A, B). Compared \nto normal endometrium, thin endometrium exhibits sig-\nnificantly reduced gland thickness and number, as well as \nan increased fibrosis area (Supplement Fig. 1C-E). Addi-\ntionally, the number of embryos successfully implanted is \nnotably reduced (Fig.  4G). Taking all of this into consid-\neration, we can conclude that our TE rat model has been \nsuccessfully established.\nAfterwards, we transplanted MenSCs and DSCs into the \nuterus of the rat for a duration of up to 14 days. Following \nthis, we extracted the gene from the rat endometrium and \ndiscovered that both groups showed a significant increase \nin the VEGF-A gene expression, with DSCs demonstrating \nsuperior efficacy compared to MenSCs (Fig. 4A). Addition-\nally, by IF staining, we observed a more pronounced angio-\ngenic response following transplantation of MenSCs and \nDSCs, and treatment with DSCs has more new blood ves-\nsels than treatment with MenSCs (Fig.  4B). To evaluate the \ntherapeutic efficacy of MenSCs and DSCs for TE, we exam-\nined the endometrial regeneration and fertility restoration \nafter transplantation of MenSCs and DSCs. It was observed \nthat the thickness of the endometrium and gland number \nrecovered to a level comparable to the control group after 14 \ndays of transplantation (Fig.  4E). The findings suggest that \nFig. 2  MenSCs induced into decidual stromal cells. A Schematic \noverview of MenSCs/DSCs treatment for thin endometrium. B The \nmorphology of MenSCs and decidual stromal cells (DSCs) in the \nbright-field: (i) Cell morphology on the 1st of the control group, \n(ii) cell morphology on the 14th of the control group, (iii) cell mor -\nphology on the 1st of the decidualization group, (iv) cell morphol-\nogy on the 14th of the decidualization group, Scale bar: 210 μm. C \nCell proliferation curve between MenSCs and DSCs. D Expression of \ndecidual-related genes (PRL and IGFBP-1) and endometrial receptiv -\nity-related genes (LIF and HOXA-10) after MenSCs-induced decidu-\nalization. E The PRL concentration in the supernatant was measured \nin different groups, with the control group being the DMEM medium\n◂\n\n1669Reproductive Sciences (2024) 31:1662–1673 \nFig. 3  MenSCs and DSCs secret more VEGF-A in vitro. A The con-\ncentration of soluble VEGF-A in control (un-conditioned media), \nMenSCs (MenSCs supernatant), and DSCs (DSCs supernatant). B \nThe impact of supernatants from different groups on the proliferation \nand migration of vascular endothelial cells was investigated. Scale \nbar: 530 μm. C Quantification of the wound healing assay results. \nValues are expressed as average ± SEM of three replicates\n\n1670 Reproductive Sciences (2024) 31:1662–1673\nboth MenSCs and DSCs exhibit beneficial effects on endo-\nmetrium regeneration in the TE model, with no significant \ndifference observed between the two groups.\nFurthermore, we performed a fertility test to evaluate the \ntherapeutic effects of MenSC and DSC transplantation on \nfertility restoration in the rat model of TE. Two weeks after \nthe fertility test, we examined well-developed embryos in \ndifferent treatment groups. There was no significant differ -\nence in the number of embryos between the sham and the \ncontrol groups, indicating that the sham operation did not \naffect the pregnancy of the rats (Fig.  4F). The TE groups \nexhibit less than 20% of implanted embryos (Fig.  4G). \nTransplantation of MenSCs has shown a notable therapeu-\ntic effect on TE and significantly improves the pregnancy \nrate in rats (Fig.  4H). Nevertheless, there is still a significant \nstatistical difference when compared to the sham-operated \ngroup on the opposite side. However, the transplantation \nof DSCs for treating TE proves to have a more pronounced \ntherapeutic effect, resulting in a greater similarity in the \nnumber of embryos when compared to the contralateral \nsham operation group (Fig.  4I). These results suggest that \nboth MenSC and DSC transplantation can promote endome-\ntrial regeneration and improve fertility. Most importantly, \ntransplantation of DSCs has a better effect on restoring rat \nfertility than MenSCs.\nTaken together, this study demonstrates that DSCs’ thera-\npeutic effects were superior to MenSCs, probably by pro-\nmoting angiogenesis, rather than due to their promotion of \ncell proliferation.\nDiscussion\nIn this study, we have demonstrated a novel approach to \nimprove the thickness and gland numbers of thin endome-\ntrium by inducing MenSC decidualization. Both MenSCs and \nDSCs were found to promote angiogenesis, but DSCs exhib-\nited superior efficacy in promoting angiogenesis compared \nwith MenSCs, thereby improving embryo implantation rates.\nInfertility is a reproductive health problem all over the \nworld, and the incidence of infertility patients has been \nincreasing in recent years [23]. The normal endometrium \nplays an irreplaceable role in female conception. However, \nTE can severely damage the endometrial morphology and \nfunction, resulting in implantation failure [24]. Cell trans-\nplantation therapy has considered the most convincing \ntreatment for moderate and severe TE [25]. Various tissue-\nderived stem cells have been used to treat TE, such as bone \nmarrow, adipose, and umbilical cord [26– 28]. However, \nthere are some risks and ethical constraints associated with \nthe acquisition of these MSCs (bone marrow and adipose). \nMenstruation-derived mesenchymal stem cells (MenSCs), \nwhich were first extracted from menstrual blood in 2007 \n[29], can be easily obtained through non-invasive surgery, \nthereby reducing many ethical controversies [ 30]. Most \nimportantly, MenSCs possess the properties of MSC and \nhave demonstrated powerful cell therapeutic capabilities in \nvarious diseases [24].\nBased on the previous protocol, we first isolated the Men-\nSCs from menstrual blood [12]. Before using flow cytom-\netry to identify the characteristics of MenSCs, we employed \nimmunofluorescence (IF) to determine the cell composition, \nfor endometrium not only contains stromal cells but also a \nlarge number of epithelial cells, vascular endothelial cells, \nand immune cells [18]. According to our results, a small \nnumber of epithelial cells were observed within the stromal \ncell population in the first two passages. However, the epi-\nthelial cells disappeared by passage 3, possibly due to the \napplication of a mesenchymal cell medium. Therefore, in \nthe follow-up experiments, we all used the cells after the \nthird generation to reduce the influence of other cells on the \nexperimental results. Subsequent experiments demonstrated \nthat these cells possess the characteristic traits of MSC and \noriginate from the endometrium.\nDecidualization refers to the morphological and func-\ntional changes of endometrial stromal cells in response to \nperiodic fluctuations in hormone levels [31]. At present, \nthere are primarily two methods for obtaining decidual \ncells: from decidual tissue derived from spontaneous abor -\ntion in vivo or by inducing endometrial-derived cell decidu-\nalization in vitro [32]. In this study, we induced MenSCs to \nundergo decidualized stromal cells (DSCs) in vitro, and the \nmorphology of the DSCs changed obviously after decidu-\nalization, but the proliferation ability of DSCs decreased. \nFurthermore, the expression levels of decidual-related genes \n(PRL and IGFBP-1) and endometrial receptivity-related \ngenes (LIF and HOXA10) all increased. We also detected a \nsignificant level of PRL protein secretion in the supernatant \nof decidualized cells through ELISA experiments. These \nfindings indicate that MenSCs can be effectively differenti-\nated into decidual cells in vitro, with distinct morphological \nand functional characteristics.\nEndometrial decidual cells can promote angiogenesis and \nimmunomodulatory effects, as well as positive regulatory \neffects on subsequent embryo implantation and placental \ndevelopment [14]. As we expected, the VEGF-A concentra-\ntion in the supernatant of different groups increased, with \nDSCs secreting more VEGF-A compared to control and \nMenSCs. Animal experiments have fully verified its func-\ntion in significantly promoting the formation of new blood \nvessels, as genes related to angiogenesis were found to be \nelevated 14 days after cell repair.\nTo directly observe the roles of MenSCs and DSCs in \nendometrial regeneration, the endometrial thickness and num-\nber of glands were measured after MenSC and DSC therapy. \nSimilarly, the results indicated that both MenSCs and DSCs \n\n1671Reproductive Sciences (2024) 31:1662–1673 \n\n\n1672 Reproductive Sciences (2024) 31:1662–1673\neffectively promoted the thickness of the endometrium and \nregeneration of glands, with no significant difference observed \nbetween the two cell types. The change in endometrial recep-\ntivity is mainly reflected by the pregnancy rate [33]. However, \nbased on the number of embryos, the therapeutic effect of \nDSCs was obviously better than that of MenSCs. Altogether, \nwe concluded that DSCs could improve endometrial receptiv-\nity primarily by promoting angiogenesis.\nHowever, there are some limitations to this study. Firstly, \nwe have only demonstrated that MenSCs can be induced to \ndecidualize in vitro, but it is not clear whether there were \nany differences between decidualization induced by Men-\nSCs in vitro and decidualization of endometrial stromal \ncells in vivo. Furthermore, how to control the efficiency of \nthe decidualization of MenSCs in vitro for clinical treat-\nment remains a challenge. In the future, further investigation \nshould focus on optimizing culture conditions and exploring \npotential molecular mechanisms to enhance the efficiency of \nMenSCs’ decidualization in vitro. This may provide a novel \ntherapeutic approach for the treatment of TE patients.\nConclusion\nIn conclusion, our study indicates that MenSCs can be induced \ninto decidual cells in vitro, similar to the process in the endo-\nmetrium. Both MenSCs and DSCs could increase endometrial \nthickness, number of glands, and fertility recovery in TE rats. \nThis effect was mainly achieved by promoting angiogenesis \nand cell proliferation. After MenSC-induced decidualization, \nthe proliferation ability of DSCs weakened, but their secre-\ntory ability was significantly enhanced. The fertility restoration \ncapability of DSCs in TE rats was better than that of MenSCs, \nit may be due to its stronger ability to promote angiogenesis \nrather than cell proliferation. Furthermore, our study provides \na new approach to restoring fertility with TE.\nSupplementary Information The online version contains supplemen-\ntary material available at https:// doi. org/ 10. 1007/ s43032- 024- 01458-2.\nThe datasets used and/or analyzed during the current study are avail‑\nable from the corresponding author upon reasonable request.\nAuthor Contributions Kai Chen: Methodology (lead); project adminis-\ntration (equal); resources (equal); writing—original draft (lead).\nHuiru Wang: Formal analysis (lead); validation (lead); resources \n(equal).\nXin Zhao: Investigation (equal); visualization (equal).\nJingxin Wang: Investigation (equal); visualization (equal).\nQi Jin: Software (equal); resources (equal).\nXianhong Tong: Supervision (lead); conceptualization (equal); \nsoftware (equal).\nShengxia Zheng: Conceptualization (equal); funding acquisition \n(lead); writing—review and editing (lead).\nFunding This study was funded by the National Natural Science Foun-\ndation of China (Grant No. 81971339)\nData Availability The datasets used and/or analyzed during the current \nstudy are available from the corresponding author upon reasonable \nrequest.\nDeclarations \nEthics Approval and Consent to Participate MenSCs were obtained \nfrom the First Affiliated Hospital of USTC, Division of Life Sciences \nand Medicine, University of Science and Technology of China, Hefei, \nAnhui, China. The collection of the MenSCs was approved under \nIRB code NO. 2021KY015, and all the patients signed informed con-\nsent before participating in the study. All procedures were conducted \naccording to the principles of the Helsinki Declaration.\nAll animal experiments were conducted by the Guide for the Care and \nUse of Animals for Research Purposes. All the procedures were ap-\nproved by the Institutional Animal Care and Use Committee at the First \nAffiliated Hospital of USTC (code no. 2022-N(A)-119, Title: Regula-\ntory network of Wnt/β-catenin signaling pathway in menstrual blood \nstem cells repairing thin endometrial fertility. Date of approval: 9 May \n2023), and followed the guidelines of the International Association for \nthe Study of Pain.\nCompeting Interests The authors declare no competing interests.\nConsent for Publication All authors gave consent for publication.\nOpen Access  This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. The images or other third party material in this article are \nincluded in the article's Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in \nthe article's Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. To view a \ncopy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.\nFig. 4  The therapeutic effects of MenSCs and DSCs on TE rats. A \nExpression of genes after treatment in different groups. VEGF-A: \nangiogenesis-related gene. B Expression of uterine vascular endothe-\nlial (CD31) in rats among different groups. Control, without any \ntreatment; TE, rat model of thin endometrium; TE+MenSCs, Men-\nSCs treat TE; TE+DSCs, DSCs treat TE. Scale bar: 170 μm. C H&E \nstaining of rat uterine after different treatments. Scale bar: 100 μm. D \nStatistical analysis of endometrial thickness after different treatments. \nValues are expressed as average ± SEM of three replicates. E Statisti-\ncal analysis of glands of the endometrium after different treatments. \nValues are expressed as average ± SEM of three replicates. F Image \nof embryo numbers on each side of the uterus after treatment (control \nvs sham), and statistical analysis of the embryo numbers. G Image \nof embryo numbers on each side of the uterus after treatment (sham \nvs TE), and statistical analysis of the embryo numbers. H Image of \nembryo numbers on each side of the uterus after treatment (sham \nvs TE+MenSCs), and statistical analysis of the embryo numbers. I \nImage of embryo numbers on each side of the uterus after treatment \n(sham vs TE+DSCs), and statistical analysis of the embryo numbers. \n*p < 0.05, **p < 0.01, ***p < 0.001\n◂\n\n1673Reproductive Sciences (2024) 31:1662–1673 \nReferences\n 1. Gargett CE. Uterine stem cells: what is the evidence? Hum Reprod \nUpdate. 2007;13(1):87–101. https:// doi. org/ 10. 1093/ humupd/  \ndml045.\n 2. Critchley HOD, Babayev E, Bulun SE, et al. 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