Immediate versus delayed single blastocyst transfer following the first stimulated IVF cycle in the freeze-all strategy: a study protocol for a randomised controlled trial.

INTRODUCTION: In recent years, the use of frozen embryo transfers (FET) has rapidly increased following the freeze-all strategy due to the advantages of increased maternal safety, improved pregnancy rates, lower ectopic pregnancy rates and better obstetric and neonatal outcomes. Currently, there is still no good scientific evidence to support when to perform FET following a stimulated in vitro fertilisation (IVF) cycle in the freeze-all strategy. METHODS/ANALYSIS: This will be a randomised controlled trial. A total of 828 women undergoing their first FET following their first stimulated IVF cycle in the freeze-all strategy will be enrolled and randomised into one of the following groups according to a computer-generated randomisation list: (1) the immediate group, in which FET will be performed in the first menstrual cycle following the stimulated IVF cycle; or (2) the delayed group, in which FET will be performed at least in the second menstrual cycle following the stimulated IVF cycle. The primary outcome will be live birth, which is defined as the delivery of any infants at ≥22 gestational weeks with heartbeat and breath. ETHICS/DISSEMINATION: Ethical approval was granted by the Ethics Committee of Assisted Reproductive Medicine at the Shanghai JiAi Genetics & IVF Institute (JIAI E2019-15). Written informed consent will be obtained from each woman before any study procedure is performed, according to good clinical practice. The results of this trial will be disseminated in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04371783.

Comparing blastocyst euploid rates between the progestin-primed and gonadotrophin-releasing hormone antagonist protocols in aneuploidy genetic testing: a randomised trial protocol.

INTRODUCTION: Progestin can inhibit the pituitary luteinising hormone (LH) surge during ovarian stimulation for in vitro fertilisation (IVF) and studies show progestin-primed ovarian stimulation (PPOS) is effective in blocking the LH surge in IVF. More and more centres are using PPOS because this regimen appears simpler and cheaper. This study aims to compare the euploidy rate of blastocysts following the PPOS protocol and the gonadotropin-releasing hormone antagonist protocol in women undergoing preimplantation genetic testing for aneuploidy (PGT-A). METHODS/ANALYSIS: This is a randomised trial. A total of 400 women undergoing PGT-A will be enrolled and randomised according to a computer-generated randomisation list to either (1) the antagonist group: an antagonist given once daily from day 6 of ovarian stimulation till the day of the ovulation trigger; or (2) the PPOS group: dydrogesterone from the first day of ovarian stimulation till the day of ovulation trigger. The primary outcome is the euploidy rate of blastocysts. ETHICS/DISSEMINATION: An ethical approval was granted from the ethics committee of assisted reproductive medicine in Shanghai JiAi Genetics and IVF institute (JIAIE2020-03). A written informed consent will be obtained from each woman before any study procedure is performed, according to good clinical practice. The results of this randomised trial will be disseminated in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04414748.

A prospective study to evaluate whether serum kisspeptin is a marker predictive of the first-trimester miscarriage of women who conceive in IVF.

PURPOSE: Kisspeptin is an emerging biomarker for the discrimination of viable pregnancy. The aim of the study is to determine whether serum kisspeptin can predict the first-trimester miscarriage and compare it with serum HCG in the prediction of the first-trimester miscarriage. METHODS: This study is a prospective case-control design including 178 women who had experienced early miscarriage (n = 21) and viable single pregnancy (n = 157), following frozen-thawed embryo transfer (FET) from May to December 2019. Serum samples on 14 days, 21 days, and 28 days after FET were collected for kisspeptin and HCG detection. TRIAL REGISTRATION NUMBER: NCT03940495. RESULTS: On day 21 after FET, serum kisspeptin levels were significantly lower in the early miscarriage group [0.260 (0.185-0.375)] vs in the viable single-pregnancy group [0.370 (0.280-0.495)] (p = 0.005). Similar results were shown on day 28 after FET, the serum kisspeptin levels were significantly lower in the early miscarriage group [0.270 (0.200-0.330)] vs in the viable single pregnancy group [0.670 (0.455-1.235)] (p < 0.001). But on day 14 after FET, serum kisspeptin levels were comparable in the early miscarriage group [0.260 (0.210-0.325)] and in the viable single-pregnancy group [0.280 (0.215-0.340)] (p = 0.551). Serum kisspeptin levels on days 21 and 28 have a poor predictive value of miscarriage compared with serum HCG levels. [Day 21: AUC = 0.687 (kisspeptin) and 0.816 (HCG); Day 28: AUC = 0.896 (kisspeptin) and 0.909 (HCG)]. CONCLUSIONS: Serum kisspeptin on day 14 failed to discriminate between miscarriage and ongoing pregnancies, and days 21 and 28 had poor predictive values of miscarriage.

A novel frameshift variant of LMX1A that leads to autosomal dominant non-syndromic sensorineural hearing loss: functional characterization of the C-terminal domain in LMX1A.

Non-syndromic sensorineural hearing loss (NSHL) is a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. There is, at present, no curative treatment for genetic hearing loss (HL). Early molecular diagnosis of progressive disorders and elucidation of the causes and pathomechanisms are essential for developing therapeutic strategies. Here, we identified a novel rare frameshift variant of LMX1A (c.915dup), which resulted in the C-terminal-altered and -truncated LMX1A (p.Val306Cysfs*32). This C-terminal frameshift mutation co-segregated with autosomal dominant (AD) NSHL in a four-generation Chinese family, suggesting that the LMX1A non-missense mutation is also contributed to ADNSHL. In this family, the affected individuals exhibited the variable auditory phenotypes ranging from profound congenital deafness at birth or to mild/moderate HL in adulthood. We also found that the embryonic cells carrying with the heterozygous variant significantly expressed several upregulated HL-associated genes at transcriptional level. In vitro splicing assay suggested that the LMX1A mRNA with c.915dup did not cause nonsense-mediated decay and was translated into a truncated LMX1A. In addition, electrophoresis mobility shift assay and luciferase assays have shown that the highly conserved C-terminal domain (amino acid 306-382) of the LMX1A was required for regulating the protein-DNA interaction and transactivation in vitro. Furthermore, apoptosis assays suggested that the C-terminal domain of the LMX1A was important for mediating apoptosis in the cochlear hair cells. Our work provided the multiline of the evidence to support that non-missense mutation of LMX1A leads to ADNSHL and the C-terminal domain of LMX1A is important for mediating transcriptional activity and associated with promoting apoptosis in the cells.

Endometrial transcriptome profiling of patients with recurrent implantation failure during hormone replacement therapy cycles.

Background: The molecular mechanisms underlying window of implantation (WOI) displacement in patients with recurrent implantation failure (RIF) remain unclear. This study aims to explore the transcriptomic signatures of endometrium with normal and displaced WOIs and to identify the causes of endometrial receptivity (ER) abnormalities and WOI displacement in RIF patients. Methods: In this study, 40 RIF patients were recruited and underwent personalized embryo transfer (pET) guided by the predicted results of endometrial receptivity diagnosis (ERD) model. Transcriptome analysis of endometrium from patients with clinical pregnancies after pET was performed to identify differentially expressed genes (DEGs) associated with WOI displacement. Gene expression data from HRT and natural cycle endometrium were compared to identify specific gene expression patterns of ER-related genes during WOI. Results: The ERD results indicated that 67.5% of RIF patients (27/40) were non-receptive in the conventional WOI (P+5) of the HRT cycle. The clinical pregnancy rate in RIF patients improved to 65% (26/40) after ERD-guided pET, indicating the effectiveness of transcriptome-based WOI prediction. Among the 26 patients with clinical pregnancy, the gene expression profiles of P+5 endometrium from advanced (n=6), normal (n=10) and delayed (n=10) WOI groups were significantly different from each other. Furthermore, 10 DEGs identified among P+5 endometrium of 3 groups were involved in immunomodulation, transmembrane transport and tissue regeneration, which could accurately classify the endometrium with different WOIs. Additionally, a large number of ER-related genes showed significant correlation and similar gene expression patterns in P+3, P+5, and P+7 endometrium from HRT cycles and LH+5, LH+7, and LH+9 endometrium from natural cycles. Conclusion: Our study shows that ER-related genes share similar gene expression patterns during WOI in both natural and HRT cycles, and their aberrant expression is associated with WOI displacements. The improvement of pregnancy outcomes in RIF patients by adjusting ET timing according to ERD results demonstrates the importance of transcriptome-based endometrial receptivity assessment and the clinical efficiency of ERD model.

The clinical efficiency of transcriptome-based endometrial receptivity assessment (Tb-ERA) in Chinese patients with recurrent implantation failure (RIF): A study protocol for a prospective randomized controlled trial.

Background: Today, approximately 10% of participants in assisted reproductive technology (ART) are defined as having recurrent implantation failure (RIF). Recent studies show that endometrial receptivity array can improve pregnancy and implantation rates by nearly 20% in women with RIF. However, these studies are limited, with little published data in the Chinese population. Recently, we have established a transcriptome-based endometrial receptivity assessment (Tb-ERA) method of predicting the endometrial window of implantation (WOI) using transcriptome-profiling data of different phases of the menstrual cycle from healthy fertile Chinese women by RNA-Seq. It is meaningful to conduct a randomized controlled trial (RCT) to assess the clinical efficiency of Tb-ERA in Chinese patients with RIF. Methods: In this RCT, a total of 200 RIF patients will be recruited and randomized into 2 groups. Patients in the Tb-ERA group will undergo a Tb-ERA test, after which embryo transfer time will be adjusted according to Tb-ERA results and embryo transfer will be performed again in the next cycle. Patients in the control group will not receive any interventions until the next transfer cycle. We will perform statistical analysis on both groups at the primary endpoint (clinical-pregnancy rate) and at secondary endpoints (rate of WOI displacement, embryo implantation, biochemical pregnancy, early abortion, and ectopic pregnancy). Implications: This study aims to evaluate the effectiveness of our Tb-ERA test in Chinese RIF patients and to determine that whether Tb-ERA could improve the clinical-pregnancy rate in these RIF patients. Trial registration: NCT04497558, registered August 4, 2020.

Immediate versus delayed frozen embryo transfer in patients following a stimulated IVF cycle: a randomised controlled trial.

STUDY QUESTION: Is there any difference in the ongoing pregnancy rate after immediate versus delayed frozen embryo transfer (FET) following a stimulated IVF cycle? SUMMARY ANSWER: Immediate FET following a stimulated IVF cycle produced significantly higher ongoing pregnancy and live birth rate than did delayed FET. WHAT IS KNOWN ALREADY: Embryo cryopreservation is an increasingly important part of IVF, but there is still no good evidence to advise when to perform FET following a stimulated IVF cycle. All published studies are retrospective, and the findings are contradictory. STUDY DESIGN, SIZE, DURATION: This was a randomised controlled non-inferiority trial of 724 infertile women carried out in two fertility centres in China between 9 August 2017 and 5 December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile women having their first FET cycle after a stimulated IVF cycle were randomly assigned to either (1) the immediate group in which FET was performed in the first menstrual cycle following the stimulated IVF cycle (n = 362) or (2) the delayed group in which FET was performed in the second or later menstrual cycle following the stimulated IVF cycle (n = 362). All FET cycles were performed in hormone replacement cycles. The randomisation sequence was generated using an online randomisation program with block sizes of four. The primary outcome was the ongoing pregnancy rate, defined as a viable pregnancy beyond 12 weeks of gestation. The non-inferiority margin was -10%. Analysis was performed by both per-protocol and intention-to-treat approaches. MAIN RESULTS AND THE ROLE OF CHANCE: Women in the immediate group were slightly younger than those in the delayed group (30.0 (27.7-33.5) versus 31.0 (28.5-34.2), respectively, P = 0.006), but the proportion of women ≤35 years was comparable between the two groups (308/362, 85.1% in the immediate group versus 303/362, 83.7% in the delayed group). The ongoing pregnancy rate was 49.6% (171/345) in the immediate group and 41.5% (142/342) in the delayed group (odds ratios 0.72, 95% CI 0.53-0.98, P = 0.034). The live birth rate was 47.2% (163/345) in the immediate group and 37.7% (129/342) in the delayed group (odds ratios 0.68, 95% CI 0.50-0.92, P = 0.012). The miscarriage rate was 13.2% (26 of 197 women) in the immediate group and 24.2% (43 of 178 women) in the delayed group (odds ratios 2.10; 95% CI 1.23-3.58, P = 0.006). The multivariable logistic regression, which adjusted for potential confounding factors including maternal age, number of oocytes retrieved, embryo stage at transfer, number of transferred embryos/blastocysts, reasons for FET, ovarian stimulation protocol and trigger type, demonstrated that the ongoing pregnancy rate was still higher in the immediate group. LIMITATIONS, REASON FOR CAUTION: Despite randomisation, the two groups still differed slightly in the age of the women at IVF. The study was powered to consider the ongoing pregnancy rate, but the live birth rate may be of greater clinical interest. Conclusions relating to the observed differences between the treatment groups in terms of live birth rate should, therefore, be made with caution. WIDER IMPLICATIONS OF THE FINDINGS: Immediate FET following a stimulated IVF cycle had a significantly higher ongoing pregnancy and live birth rate than delayed FET. The findings of this study support immediate FET after a stimulated IVF cycle. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used and no competing interests were declared. TRIAL REGISTRATION NUMBER: ClinicalTials.gov identifier: NCT03201783. TRIAL REGISTRATION DATE: 28 June 2017. DATE OF FIRST PATIENT'S ENROLMENT: 9 August 2017.

Transcriptomic analysis of endometrial receptivity for a genomic diagnostics model of Chinese women.

OBJECTIVE: To define the transcriptomic signature with respect to human endometrial receptivity in Chinese women by next-generation sequencing and to develop a more refined and customized bioinformatic predictive method for endometrial dating in Chinese women. DESIGN: Randomized. SETTING: A tertiary hospital-based reproductive medicine center. PATIENT(S): Ninety healthy, fertile Chinese women. INTERVENTION(S): Human endometrial biopsies. MAIN OUTCOME MEASURE(S): Gene expression of endometrial biopsies. RESULT(S): Ninety endometrial samples from healthy Chinese women during their menstrual cycles-including prereceptive (luteinizing hormone [LH] + 3 days/LH + 5 days), receptive (LH + 7 days), and post-receptive (LH + 9 days) phases-were subjected to transcriptomic analysis using messenger RNA (mRNA)-enriched RNA-Seq. Feature genes were obtained and used to train the predictor for endometrial dating, with 63 samples for the training set and 27 samples for the validation set. Differentially expressed genes (DEGs) were identified by comparing samples from different phases of the menstrual cycle. Based on the transcriptomic feature genes, we constructed a bioinformatic predictor for endometrial dating. The accuracy on assessment of the endometrium on days LH + 3, LH + 5, LH + 7, and LH + 9 was 100% in the training set and 85.19% in the validation set. CONCLUSION(S): Our transcriptomic profiling method can be used to monitor the window of implantation with regard to the endometrium in the Chinese population. This method potentially provides an evaluation of endometrial status, and can be used to predict a personal window of implantation by reproductive medicine clinicians.

17ß-estradiol promotes bone marrow mesenchymal stem cell migration mediated by chemokine upregulation.

Bone marrow-derived cells engraft to the uterine endometrium and contribute to endometriosis. This study sought to further confirm that estrogen can promote the migration of bone marrow mesenchymal stem cells (BMSCs) and to investigate the function of estrogen on the secretion of chemokines during BMSC migration. BMSCs were treated with or without 17ß-estradiol, cultured with or without endometrial stromal cells (ESCs), or pretreated with or without AMD 3100 (an antagonist of the SDF-1α receptor) before co-culture. A migration assay was used to investigate the changes in the migration of BMSCs. The secretion of chemokines in the co-culture medium was detected by chemokine analysis, and the mRNA expression of SDF-1α in cells was tested using quantitative real-time PCR. The results revealed that the migration of BMSCs was promoted by ESC, and the migration ability of BMSCs was enhanced after treatment with 17ß-estradiol (p < 0.05). Through chemokine analysis, we further showed that 17ß-estradiol promoted the secretion of chemokines especially for SDF-1α (p < 0.05). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these chemokines were mainly linked to the cytokine signaling pathway and interaction with cytokines receptors. Furthermore, the expression of SDF-1α mRNA was significantly increased in the 17ß-estradiol treatment group (p < 0.001), and the migration of BMSCs was blocked by the use of our SDF-1α antagonist (p < 0.01). Our results indicate that 17ß-estradiol could promote the chemotaxis and migration of BMSCs by up-regulating the secretion of chemokines, especially SDF-1α. Our study provides additional evidence to support and supplement the stem cell theory of endometriosis.

Expanded carrier screening in Chinese patients seeking the help of assisted reproductive technology.

BACKGROUND: Expanded carrier screening (ECS) has emerged as an effective approach to identify at-risk couples (ARCs)-before they initiate attempts at reproduction-who possess a high probability of having a child affected by severe recessive diseases. The objective of this study was to evaluate the clinical utility of ECS in Chinese patients seeking the help of assisted reproductive technology (ART). METHODS: An ECS test, which covers 201 genes implicated in 135 recessive (autosomal or X-linked) diseases, was routinely offered to all ART patients in a single genetics and in vitro fertilization clinic. Additional options for preimplantation or prenatal genetic diagnosis were discussed and offered to all ARCs. All ECS results were aggregated and the clinical decisions of the ARCs were surveyed. RESULTS: A total of 2,923 ART patients, representing 1,462 couples, were screened. Overall, 46.73% of the individuals were found to be the carriers for at least 1 of the 135 diseases. Of the tested couples, 2.26% (n = 33) were identified as ARCs. As of the completion of this study, 21 (63.6%) ARCs have decided to avert an affected pregnancy with the help of preimplantation genetic testing for monogenetic conditions. The cumulative carrier rate of the 187 autosomal recessive genes in the ECS panel for the 2,836 Han Chinese individuals without a family history was estimated to be 45.91%. The estimated at-risk couple rate indicates that the screening for only the top 31 genes with gene carrier rates >0.5% would identify more than 94% of the ARCs identified by screening all 187 genes. CONCLUSION: Our study demonstrates that ESC yields a significant clinical value for ART patients in China. In addition, by estimating the yields of the ECS panel, we identify genes that are appropriate for screening the Han population.

MicroRNA-451 is downregulated in the follicular fluid of women with endometriosis and influences mouse and human embryonic potential.

BACKGROUND: Previous work demonstrated that there are numerous miRNAs in human follicular fluids, some of which are associated with reproductive diseases. In the current study, we sought to determine whether microRNAs (miRNAs) in the follicular fluid (FF) are differentially expressed between women with and without endometriosis and to uncover the association of miRNAs with the oocyte and embryonic development potential. METHODS: FF was harvested from 30 women with endometriosis and 30 women without who underwent in vitro fertilization treatment at the University Hospital between February and December 2016. The FF samples were subjected to miRNA profiling and validation via quantitative reverse transcription polymerase chain reaction analysis. Mouse/human metaphase-I (MI) oocytes were harvested and micro-injected with an miR-451 inhibitor, and the effects of miR-451 knockdown on Wnt/WNT signalling genes were investigated. RESULTS: Oocyte number, fertilization rate, and number of available embryos were decreased significantly in women with endometriosis relative to those without endometriosis. Hsa-miR-451 in FF was downregulated in endometriosis patients relative to control subjects (P < 0.01). Moreover, the proportions of mouse/human MI oocytes that developed into 2-pronuclei (2PN), 2-cell, 8-10-cell and blastocyst-stage embryos were affected by miR-451 knockdown in mouse/human oocytes. Components of the Wnt signalling pathway were aberrantly expressed in the mouse/human oocytes and embryos in the miR-451 inhibitor-injected group. CONCLUSIONS: miR-451 was downregulated in FF samples from endometriosis patients and was modestly effective in distinguishing endometriosis patients from non-endometriosis patients. miR-451 downregulation in mouse and human oocytes affected pre-implantation embryogenesis by suppressing the Wnt signalling pathway. This miRNA might serve as a novel biomarker of oocyte and embryo quality in assisted reproductive treatment.

Expression of ENPP3 in human cyclic endometrium: a novel molecule involved in embryo implantation.

Ectonucleotide pyrophosphatase-phosphodiesterase 3 (ENPP3), a protein detected in the human uterus, has been found to play an important role in the development and invasion of tumours. It was recently discovered that ENPP3 was upregulated during the window of implantation in the human endometrium but its functional relevance remains elusive. The objective was to determine ENPP3 expression in human endometrium and its roles in endometrial receptivity and embryo implantation. ENPP3 expression was analysed using immunohistochemistry and western blot assay. The effects of ENPP3 on embryo implantation were evaluated using a BeWo cell (a human choriocarcinoma cell line) spheroid attachment assay and BeWo cells were dual cultured with Ishikawa cells transfected with lentiviral vectors (LV5-NC or LV5-ENPP3) to mimic embryo implantation in a Transwell model. The effects of endometrial ENPP3 on factors related to endometrial receptivity were also determined. The results showed that ENPP3 was expressed in human endometrial epithelial cells and its expression levels changed during the menstrual cycle, peaking in the mid-secretory phase, corresponding to the time of embryo implantation. The overexpression of endometrial ENPP3 not only increased the embryo implantation rate but also had positive effects on the expression of factors related to endometrial receptivity in human endometrial cells. The results indicate that ENPP3 levels undergo cyclic changes in the endometrium and affect embryo adhesion and invasion via altering the expression of implantation factors in the human endometrium. Therefore, ENPP3 may play an important role in embryo implantation and may be a unique biomarker of endometrial receptivity.

Non-invasive metabolomic profiling of embryo culture media and morphology grading to predict implantation outcome in frozen-thawed embryo transfer cycles.

PURPOSE: Assessment of embryo viability is a crucial component of in vitro fertilization and currently relies largely on embryo morphology and cleavage rate. Because morphological assessment remains highly subjective, it can be unreliable in predicting embryo viability. This study investigated the metabolomic profiling of embryo culture media using near-infrared (NIR) spectroscopy for predicting the implantation potential of human embryos in frozen-thawed embryo transfer (FET) cycles. METHODS: Spent embryo culture media was collected on day 4 after thawed embryo transfer (n = 621) and analysed using NIR spectroscopy. Viability scores were calculated using a predictive multivariate algorithm of fresh embryos with known pregnancy outcomes. RESULTS: The mean viability indices of embryos resulting in clinical pregnancy following FET were significantly higher than those of non-implanted embryos and differed between the 0, 50, and 100 % implantation groups. Notably, the 0 % group index was significantly lower than the 100 % implantation group index (-0.787 ± 0.382 vs. 1.064 ± 0.331, P < 0.01). To predict implantation outcomes, we examined the area under the ROC curve (AUCROC), which was significantly higher for the viability than for the morphology score (0.94 vs. 0.55; P < 0.01); however, the AUCROCs for the composite and viability scores did not differ significantly (0.92 vs. 0.94; P > 0.05). CONCLUSIONS: NIR metabolomic profiling of thawed embryo culture media is independent of morphology and correlates with embryo implantation potential in FET cycles. The viability score alone or in conjunction with morphologic grading is a more objective marker for implantation outcome in FET cycles than morphology alone.

In vitro differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in mouse: a proteomic analysis.

OBJECTIVE: Mouse bone marrow mesenchymal stem cells (BMSCs) have been demonstrated to differentiate into female endometrial epithelial cells (EECs) in vivo. Our previous studies demonstrated that BMSCs can differentiate in the direction of EECs when co-cultured with endometrial stromal cells in vitro. Here, we obtain and analyse differential proteins and their relevant pathways in the process of BMSCs differentiating into EECs by isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis. METHODS: A 0.4-µm pore size indirect co-culture system was established with female mice endometrial stromal cells (EStCs) restricted in the upper Transwell chamber and BMSCs in the lower well plate. After indirect co-culture for several days, the BMSCs were revealed to progressively differentiate towards EECs in vitro. Then, four groups were divided according to different co-culture days with single culture groups of BMSCs as controls. Proteins were detected using iTRAQ based on 2DLC-ESI-MS/MS and data were analysed by bioinformatics. RESULTS: A total number of 311 proteins were detected, of which 210 proteins were identified with relative quantitation. Among them, 107 proteins were differentially expressed with a 1.2-fold change as the benchmark, with 61 up-regulated and 46 down-regulated proteins. Differential proteins CK19 and CK8 were epithelial markers and upregulated. Stromal marker vimentin were downregulated. Top canonical pathways was "remodeling of epithelial adhesions junctions" and "actin cytoskeleton signaling". Top networks was "cell-to-cell signaling and interaction, tissue development and cellular movement" regulated by ERK/MAPK and α-catenin. CONCLUSION: To the best of our knowledge, this is the first preliminary study of differential protein expression in the differentiation process of BMSCs into EECs in vitro. We further elucidated BMSCs differentiated in the direction of EECs. In addition, ERK/MAPK and α-catenin played important roles by regulating core differential proteins in the "cell-to-cell signaling and interaction, tissue development and cellular movement" network.

A study in vitro on differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in mice.

OBJECTIVE: To investigate the differentiation conditions of bone marrow mesenchymal stem cells (BMSCs) into endometrial epithelial cells and to confirm the effect of 17ß-estradiol in this process. STUDY DESIGN: BMSCs were cultured alone or co-cultured with endometrial stromal cells (EStCs) in control/differentiation medium (17ß-estradiol, growth factors) and were co-cultured with EStCs in different concentrations of 17ß-estradiol. Flow cytometry and immunocytochemistry were used to identify the isolated cells. Real-time RT-PCR and immunofluorescence were used to test the expression of epithelial cell markers. RESULTS: The epithelial markers cytokeratin-7, cytokeratin-18, cytokeratin-19, and epithelial membrane antigen were elevated in real-time RT-PCR (P<0.05), and cytokeratin was strongly positive in immunofluorescence analysis in the differentiated BMSCs. Cytokeratin-7 and cytokeratin-19 expression levels were highest in the 1 × 10⁻8 mol/L 17ß-estradiol group, as shown in real-time RT-PCR (P<0.05). CONCLUSION: BMSCs could be differentiated in the direction of endometrial epithelial cells in appropriate conditions in vitro: 17ß-estradiol may play a key role in stimulating BMSCs' epithelial differentiation in the process of endometriosis. CONDENSATION: Bone marrow mesenchymal stem cells can differentiate in the direction of endometrial epithelial cells in a certain microenvironment and appropriate concentration of 17ß-E2 can facilitate this differentiation.