The Expression Of TM4SF9at Human Maternal-fetal Interface And Its Role In Blastocyst Implantation

ObjectiveTo investigate the expression of Transmembrane4superfamily member9(TM4SF9) protein at human maternal-fetal interface and to explore its role inblastocyst implantation, in order to find a new way of the major problems inreproductive medicine, such as recurrent implantation failure (RIF), early pregnancylosses and ectopic pregnancy.Grouping and Methods1. Objects①Group of Spontaneous Abortion：52spontaneous abortion(SA) patients(gestational age,6-12weeks) were included in this prospective study design.Cytogenetic analysis of the products of conception (POC) was performed for all theSA patients. According to the test results,37SA patients with normal chromosome inthe POC were constituted the SA-1group, and15chromosomal abnormalities in thePOC were divided into SA-2group.②Group of ectopic pregnancy: Total of40ectopic pregnancy, who underwentsalpingectomy of the trouble side, and a diagnostic uterine curettage was performedbefore salpingectomy. All the cases were selected from archival material of theDepartment of Pathology of the Guangdong No.2People’s Hospital from2012to2014.2. Methods1) Immunohistochemistry was used to detect the expression of TM4SF9in villi,decidual or endometrial tissues and mucosa of the oviduct.2) Chromosome karyotype of products of conception by cytogenetic analysis. ResultsPart I The Expression Of TM4SF9at Human Maternal-Fetal Interface ofspontaneous abortion1. In human villi：1) Location:In human placental villi of normal or abnormal pregnancy, TM4SF9was detectedin the cytoplasm and cell membrane of cytotroblasts and Extravillous Cytotrophoblas(EVCT), which invading the endometrium during implantation and placentation. Butnot in the syncytiotrophoblasts.2) The expression levels:①The expression levels of TM4SF9in villi group of SA-1:The expression level decreased in the group of SA-1, compared with normalpregnancy group, showing significant differences between the samples(Mean ofIOD：53.22±35.47Vs89.04±36.44；t=4.39，P=0.000).②The expression levels of TM4SF9in villi group of SA-2:And the expression level of TM4SF9in the group of SA-2was significantly higherthan the group of SA-1(Mean of IOD：140.43±61.76Vs53.22±35.47，P=0.000).2. In human deciduas or endometrium:1) Location:In human deciduas of pregnancy or endometrium of non-pregnancy, TM4SF9wasexpressed in the cytoplasm and cell membrane of glandular epithelial cells andDecidual/Endometrial Stromal Cells (DSC/ESC), which showed the biologicalproperty of motility. But not in granulocytes.2) The expression levels:①The expression levels of TM4SF9in deciduas from group of SA-1:The intensity of TM4SF9was strongest in deciduas from group of SA-1(Mean ofIOD：184.49±119.69); the level in normal pregnancy group comes the second (Meanof IOD：93.43±67.10); And the intensity of TM4SF9was weakest in the proliferativeendometrium of non-pregnancy. Comparison of the3groups, there were significantdifference. ②The expression levels of TM4SF9in deciduas from group of SA-2:The expression level of TM4SF9in deciduas from group of SA-2is slightly lowerthan group of SA-1. But there was no significant difference (Mean of IOD:147.22±84.78Vs184.49±119.69；t=1.10，P=0.278).③The expression levels of TM4SF9in deciduas from normal pregnancy andendometrium of non-pregnancy:The expression levels of TM4SF9in deciduas from normal pregnancy was slightlyhigher, compared with endometrium in proliferative or secretory endometrium ofnon-pregnancy. But there was no significant difference (Mean of IOD:93.43±67.10Vs75.71±29.87、88.56±33.24; F=1.26，P=0.296).Part II The Expression Of TM4SF9at Human Maternal-Fetal Interface ofectopic pregnancy1. In human villi：1) Location:In human placental villi of ectopic pregnancy, TM4SF9was detected in thecytoplasm and cell membrane of cytotroblasts and Extravillous Cytotrophoblas(EVCT), but not in the syncytiotrophoblasts.2)The expression levels:The intensity of TM4SF9in villi of ectopic pregnancy was significantly higher thannormal pregnancy, showing significant differences (Mean of IOD：109.39±61.84Vs89.04±36.44；t=2.33，P=0.023).2. In human deciduas or endometrium:1) Location:In human deciduas of ectopic pregnancy or endometrium of non-pregnancy,TM4SF9was expressed in the cell cytoplasm and membrane of glandular epithelialcells and Decidual/Endometrial Stromal Cells (DSC/ESC), but not in granulocytes.2) The expression levels:①The expression levels of TM4SF9in endometrium from ectopic pregnancy:The intensity of TM4SF9was strongest in the group of ectopic pregnancy (Mean of IOD:144.18±106.22); group of normal pregnancy comes to the second (Mean ofIOD:93.43±67.10); the intensity of TM4SF9was weakest in the endometrium ofproliferative (Mean of IOD:88.56±33.24). Comparison of the3groups, there weresignificant difference (F=4.29，P=0.019).②The comparison of the TM4SF9levels between the two groups ofendometrium in ectopic pregnancy:In the40cases of endometrium from diagnostic curettage of the ectopicpregnancy patients, we found that21cases undergone decidualization and19casesshowed non-decidualization. Comparing the expression levels between the two groups,we discovered that the intensity of TM4SF9was stronger in the group of decidualendometrium, showed no statistically significant difference(Mean of IOD：166.73±106.11Vs119.25±100.31，t=1.47，P=0.151).3. In the mucosa of oviduct1) Location:TM4SF9was also detested in the fallopian tube mucosa from the patients ofectopic pregnancy and women who required tubal sterilization surgery. It localised tothe cytoplasm and cell membrane of ciliated cells, especially in the cilium. But not inthe secretory cells.2) The expression levels:The expression level of TM4SF9was highest in the fallopian tube mucosa fromthe patients of ectopic pregnancy (Mean of IOD：228.92±133.67. And the level inendometrium from diagnostic curettage of the ectopic pregnancy patients comes thesecond (Mean of IOD：144.18±106.22). In addition, the intensity of TM4SF9wasweakest in the fallopian tube mucosa from non-pregnancy (Mean of IOD：63.57±26.74). All showed significant differences (F=57.89，P=0.000).Conclusion1) This study is the first to investigate the role of TM4SF9in blastocyst implantation.Our result indicated that TM4SF9reflects the proliferation and invasiveness oftrophoblast, is expected to become a marker protein.2) We speculate that some factors in the environment of endometrium, including TM4SF and mechanism of immune tolerance, may inhibit the invasion behavior oftrophoblast. And due to escape from the inhibitory of decidua microenvironment, thetrophoblasts in ectopic pregnancy showed more proliferation and invasiveness.3) Presence of TM4SF9does not depend on the existence of the embryo. And webelieved that the level of TM4SF9in endometrial stromal cells may reflect the degreeof decidualization. If this protein abnormally high expression in the endometrium maygo against implantation of embryo.4) Taken together, this study found that TM4SF9was widely expressed at humanmaternal-fetal interface, and its abnormal expression level may relate to abnormalpregnancy or pregnancy loss.5)In addition, the expression of TM4SF9in ciliated cells of tubal mucosa indicatesthat the protein may participate in the regulation of tubal peristalsis and cilium swings.We speculated that TM4SF9may regulate these biological events. However, thishypothesis needs to be verified and explored in-depth study.