Construction And Verification Of Endometrial Organoids Derived From Endometrial Tissue And Uterine Fluid

Background:The endometrium,as the communication interface between mother and fetus,is one of the important factors for successful embryo implantation.Appropriate research models can help to explore the mechanism of human endometrium in the process of embryo implantation.Endometrial cell lines and animal models have limitations in reflecting the physiological status of human endometrium.In recent years,the emerging endometrial organoid(EO)model can imitate some physiological and pathological processes in vivo to a certain extent.However,at present,the clinical samples of endometrial organoids are mainly obtained through invasive operations.Therefore,we urgently need to establish a non-invasive and repeatable construction method of endometrial organoids.Objectives:1.To optimize the tissue-derived endometrial organoid(T-EO)culture system,construct T-EO and verify its epithelial characteristics.2.To explore methods for non-invasive sampling and culture of human endometrial organoids derived from uterine lavage fluid(UF-EO),and to verify their epithelial characteristics and reliability.3.To explore the reactivity of T-EO and UF-EO to E2,MPA and c AMP.Methods:1.Endometrial biopsy tissue was digested by collagenase IV to obtain cell suspension.To construct T-EO,endometrial gland cells were isolated,extracted and then embedded in Matrigel for 3D culture.We used Tryp LE digestive solution combined with 26 G syringe to blow and dissociate organoids to simplify the methods of organoid passage and cryopreservation.HE and IF staining were used to detect the morphological characteristics and markers of T-EO.2.Collect normal saline uterine lavage fluid,and then separate and enrich the exfoliated cells of it by low-temperature centrifugation.To construct the UF-EO,the exfoliated cells of the uterine lavage fluid were embedded in Matrigel for 3D culture.Matched EOs were constructed with endometrial tissue and uterine lavage fluid from the same patient.The morphological characteristics and markers of T-EO and UF-EO from the same patient were compared and detected by HE and IF staining,so as to verify the reliability of the construction method of UF-EO.Endometrial tissue and intrauterine lavage fluid were collected from patients with intrauterine adhesion(IUA)or repeated implant failure(RIF)to construct T-EO and UF-EO,so as to prove that the culture method of UF-EO is also applicable in these special populations.3.T-EO and UF-EO were divided into 4 groups and treated with E2,MPA and c AMP separately or sequentially :Group NC(hormone free treatment group),Group E(E2 treatment group),Group EP(E2 and MPA treatment group)and Group EPC(E2,MPA and c AMP treatment group).The morphological changes were observed by PAS staining and light microscope.The expression of endometrial receptivity related genes such as PAEP and SPP1 were detected by IHC and q RT-PCR.Results:1.Endometrial gland cells could self-organize into spherical structures after Matrigel embedding and 3D culture.The organoids were identified as three-dimensional structures by continuous paraffin section and HE staining.IF results showed that T-EO expressed epithelial markers such as CK7,Ep CAM and E-cadherin.The use of 26 G syringe made it easier for organoids to separate into single cells,simplifying the process of passage and cryopreservation.T-EO could be passaged to the8 th generation at least and expanded after cryoresuscitation.2.Endometrial cells enriched from uterine lavage fluid could self-organize into spherical structures after Matrigel embedding and 3D culture.UF-EO were identified as three-dimensional structures by continuous paraffin section and HE staining.The cells extracted from endometrial tissue and uterine lavage fluid from the same patient could form spherical structures after 3D culture.Under the microscope,UF-EO and T-EO had similar morphological structures.IF results showed that UF-EO also expressed endometrial epithelial markers CK7,Ep CAM and E-cadherin.These two culture methods were also suitable for patients with IUA or RIF.UF-EO could be passaged to the sixth generation at least and be expanded after cryoresuscitation.3.Compared with Group NC,Group EPC of T-EO and UF-EO had morphological changes such as gland cavity folding and bending under the microscope;PAS staining showed that compared with Group E,the glycogen secretion in the lumen of T-EO and UF-EO in EPC groups increased;IHC results showed that compared with Group E,the expression of PAEP in T-EO and UF-EO in Group EPC was enhanced.q RT-PCR results showed that compared with Group NC,the m RNA expression level of PAEP,SPP1 and 17HSDβ2 was up-regulated significantly in Group EPC and Ki67 gene expression was down regulated significantly in Group EP and EPC;Compared with Group NC,PGR gene expression of Group E,Group EP and EPC was up-regulated,but there was no significant difference between Group E and EP;Compared with Group NC,the expression of IHH in Group E and EP was up-regulated only in UF-EO of Donor 1;Compared with Group NC,the expression of OLFM4 in Group E was up-regulated.Conclusion:Endometrial organoid models derived from endometrial tissue and uterine lavage fluid had good response to sex hormone,and could reproduce some characteristics of endometrial proliferation and secretory phase,which were ideal models for studying pregnancy and gynecological diseases.This study contains 21 figures,17 tables and 66 references.