Expression Of Implantation-related Factors At The Maternal-fetal Interface During Tubal Pregnancy

The incidence of ectopic pregnancy has increased dramatically worldwide over the past few decades, currently accounting for 2% of all pregnancies. The most frequent sites (about 95%) of ectopic pregnancy are the fallopian tube (tubal pregnancy, TP) and the ampullary region within the tube. The increased incidence of ectopic pregnancy has occurred simultaneously with an increased incidence of pelvic inflammatory diseases, suggesting a causal relationship. The increased incidence of ectopic pregnancy is also a result of the progress in diagnostic modalities as highly sensitive human chorionic gonadotrophin radioimmunoassay and vaginal ultrasonography allowing early diagnosis of tubal pregnancies, some of which may have been unrecognized in the past. Pathologic damage of tube induced by salpingitis makes lumens not so easy and smooth, in the end lead to delay advance of blastocyst through uterinetuba. Certainly, the delayed advance of blastocyst makes TP possible. However the successful progression of TP relies on a pivotal event, i.e. implantation. Owing to limited by pellucid zone the diameter of blastocyst is simillar to single ovum, while the most straitness in isthmus fallopian tube is 2 millimeter. In the light of these, we presumed that change of local microenvironment in tuba caused by inflammation, such as related cytokine, adhesion molecule, makes the successful ectopic implantation.Implantation of the blastocyst into the lumenal epithelium of the endometrium is one of the most important steps in the establishment and maintenance of a viable pregnancy. Implantation can be divided into a series of steps known as apposition, adhesion, invasion and subsequently angiogenesis. The endometrium undergoescharacteristic morphological and biochemical changes under the influence of the ovarian steroid hormones in preparation for nidation. As a general regulator of implantation, the steroid hormones make effect through their receptors respectively; direct or indirect regulate the secretion of cytokines, growth factors and molecular adhesions, which may be necessary for successful implantation to occur. During the apposition and adhesion phase successful implantation is mediated by cytokines, growth factors and adhesion molecules such as leukemia inhibitory factor (LIF), heparin-binding epidermal growth factor (HB-EGF), interleukin-1 beta (IL-1β), colony-stimulating factor-1 (CSF-1), integrin, human oviduct specific glycoprotein (hOGP) and trophinin tastin bystin macula etc. Invasion then occurs allowing the trophoblast, through proteolytic processes, to penetrate into the maternal decidua and endometrial spiral arteries. In these process trophoblast cell plays the major role, while endometrium prevent them from overinvasion, and this is function of stroma. TIMP-3, produced by stroma cell periodically, is currently considered the most relevant factor for limiting trophoblast invasion. Furthermore, the higher expression of TIMP-3 was located at implantation site. Immediately after intimate contact to the endometrium, the embryo must induce angiogenesis to ensure its survival. VEGF is a potent angiogenetic growth factor, plays an important role during the implantation of intra uterine pregnancy (IUP).Tubal pregnancy is a type of abnormal gestation, in which an embryo implants in the Fallopian tube rather than the uterus. While chromosome analysis of embryo in TP shows similar to that of IUP. Most studies about TP have focused on cilinical therapy summary and related analysis of etiological factors, while few study on mechanism.We presumed that all implantation-related factors such as ER, PR, LIF, integrins, VEGF during IUP are involved in the TP implantation. In this study, we detected the above-mentioned factors in TP in order to identify its locations and whether involve in implantation, further whether change of these factors' expression under inflammation facilitates TP occurring by immunohistochemistry and Western blot analysis.Part one ER PR expression in chronic salpingitis, implantation site and non-implantation site during tubal pregnancyMethods: Thirty patients who were undergone salpingectomy for tubal pregnancy were enrolled; all the oviduct tissues were isolated from the implantation and nonimplantation sites. At the same time seventeen normal ampullary oviducts and twelve chronic inflammation ampullary oviducts during mid-secretory phase were included as control groups. The expression of ER and PR were measured by the routine two-step immunohistochemical technique.Results: 1 .Immunohistochemical study showed that ER and PR were mainly localized in karyon of epithelial and stroma cell, and also in smooth muscle cell in all specimen. Marked difference of intense staining was observed between implantation site and non-implantation site in the same patient, and the latter is stronger for both ER and PR . There are significant difference between implantation site and non-implantation site for ER expression or PR expression (t= - 3.895, p=0.001; t= - 3.574, p=0.001, respectively). 2. Semi-quantitative analysis also demonstrated that ER or PR expression level in implantation site was significantly lower than that of chronic inflammation group (both p<0.01) and normal group(p<0.01; p<0.05 respectively) but no significant difference between the non-implantation site and chronic inflammation group or between the non-implantation site and normal group respectively or between the chronic inflammation group and normal group (all p>0.05) .3. ER/PR values was calculated and performed by Mannu-White U test. The ER/PR values in inflammation group were significantly higher than that of implantation site (p<0.05), non-implantation site (p<0.01) and normal group (p<0.01). While there was no difference between implantation site group, non-implantation site and normal group each other (all p>0.05). 4. Tubal decidualizations in implantation site and non-implantation site were evaluated by pathologist. None had decidual reaction in non-implantation site group. While in implantation site group fifteen cases (50%) showed patchy decidual changes. No correlation was found between decidulization and hCG levels or PR expression except for positive association betweendecidulization and ER expression (r=0.398, P=0.029). 5. Pearson's analysis indicated ER and PR expressions have positive correlations in implantation site (r=0.629, P=0.000), nonimplantation site (r=0.402, P=0.028) and normal group (r=0.743, P=0.001), but no correlation in chronic inflammation group.Part two The role of LIF in tubal pregnancyMethods: The expression of LIF protein in implantation site and non-implantation site of tubal pregnancy, chronic inflammation ampullary oviducts and normal ampullary oviducts during mid-secretory phase were detected by immunohistochemical technique, Western blotting, respectively.Results: 1. Immunohistochemical study showed that LIF protein was mainly localized in epithelial cell of tuba, and faint in stromal cell. Staining in implantation was intense compared to corresponding non-implantation site. Immunostatining of tuba in chronic inflammation group was strong, in contrast, weak in normal tuba. Semi-quantitative analysis by H-score showed that statistical significant difference of LIF expression on tubal epithelium was found between implantation site and non-implantation site (t=3.062, P=0.005) or normal group (P=0.002). Yet there was no difference among inflammation group, normal group and non-implantation site each other. 2. Western blotting analysis showed a band at approximately 45KDa was detected in all samples. No bands were seen when primary antibody was replaced with normal goat serum or when the secondary antibody alone was used (data not shown). Semi-quantitative analysis demonstrated that LIF protein expression level in implantation site (median 2.38, range 0.31-6.71) was significantly higher than that of non-implantation site (median 0.87, range 0.32-4.6) ( t=2.871, P=0.009) and normal group (median 0.79, range 0.24-1.43) (P=0.003) but no significant difference between chronic inflammation group (median 1.39, range 0.55-3.99) and implantation site (P=0.381), the non-implantation site and chronic inflammation group (P=0.362), the non-implantation site and normal group (P = 0.098). Whereas there was statistic difference between chronic inflammation group and normal group (P=0.007). 3.When implantation site and non-implantation site combined together to make TP group, wefound that LIF expression in TP group was striking higher than normal group (P=0.008), while no significant difference between TP group and chronic inflammation group (P=0.985).Part three The expression of β₁, α₄, α_v in maternal-fetal interface during tubal pregnancyMethods: Thirty patients who were undergone salpingectomy for tubal pregnancy were enrolled; all the oviduct tissues were isolated from the implantation and nonimplantation sites. At the same time seventeen normal ampullary oviducts and twelve chronic inflammation ampullary oviducts during mid-secretory phase were included as control groups. The expression of integrin subunits β₁, α₄, α_v were measured by the routine two-step immunohistochemical technique.Results: 1.After staining with HE histological analysis of the implantation site showed that villus infiltrated the tube, next to them were presented normal tubal epithelium. While in the nonimplantation site there was only continuous normal epithelium, no any trophoblast cell. 2.Immunohistochemically, β₁ integrins were detected in the cytoplasm of epithelium and also weakly in stroma. Staining of β₁ was significantly more intense in inflammation group than in implantation site, nonimplantation site, normal group (H-score: 3.54 ± 0.51 versus 2.74±1.04, 2.56 ± 0.97, 2.67±1.12,P = 0.02,0.004,0.048 respectively, both p<0.05). While there were no significantly difference of β₁ expression in implantation site, nonimplantation site, normal group between each other (P>0.05). 3. Immunoreactivity of α₄ integrins located in epithelial cytoplasm, weak in stroma. And α_v integrins were detected in apical surface of luminal epithelium, none in stroma or muscle. There were no significant differences between each other (P>0.05).Part four The expression of VEGF in maternal-fetal interface during tubal pregnancyMethods: Thirty patients who were undergone salpingectomy for tubal pregnancy were enrolled; all the oviduct tissues were isolated from the implantation and non-implantation sites. At the same time seventeen normal ampullary oviducts during mid-secretory phase were included as control group. The routine two-step immunohistochemical technique was performed.Results: The expression of VEGF in implantation site of the human oviduct with ectopic gestation was significantly higher than those in the nonimplantation site(P<0.05) and normal ampullary oviducts during mid-secretory phase(P<0.05). There was no significant difference between the nonimplantation site and normal ampullary oviducts during mid-secretory phase (P>0.05).Conclusion:1. It is likely that down regulation of ER PR play a key role during implantation of tubal ectopic pregnancy2. Decidualization is not necessary for implantation during tubal pregnancy.3. The high values of ER/PR in fallopian tube maybe one of the aetiology of tubal pregnancy.4. Combining the known role of LIF in uterine implantation, we suggested high level of LIF may contribute to implantation of TP.5. Salpingitis leading to increased LIF expression supports a possible association between salpingitis and TP.6. There were no difference of β₁,α₄,α_v integrins expressions among implantation site, nonimplantation site, normal group. We cannot conclude those involved in TP implantation.7. The results suggest that VEGF play an pivotal role in the angiogenic factor responsible for the implantation and placentation of an ectopic pregnancy in the oviduct.8. The implantation site and non-implantation site marked by trophinin protein in this study, compared to traditional pathological implantation site, are easy to setup own control and raise reliability and science of study.9. This study is the first time to confirm that tubal ER/PR and LIF change may take an active part in TP occurring.10. Not only mechanical block induced by salpingitis but also local related-implantation factors may be one main cause of TP.