| Assisted reproductive technology (ART) is defined as a series of treatments usedto achieve pregnancy, in which both oocytes and sperm are handled in vitro, such asconventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).Early data suggested that infants conceived with ART would develop normally.However in the past decade, increasing amount of well-designed studies have beenshowing that ART pregnancies could produce detrimental perinatal outcomes,compared to those of non-assisted pregnancies including: preterm birth; congenitalabnormality; low birth weight; perinatal morbidity; mortality; negativeneurodevelopmental outcome; or even cancer. However, the mechanism of theincreasing maternal and offspring health risks of ART remains unclear. Researchersare having debate on whether the increased risk of ART is due to infertilitybackground of these patients or caused by assisted reproductive technology. Resultsfrom a population-based cohort study indicated that adverse outcomes associated withassisted fertilization could be attributed not to the reproductive technology itself, butrather to certain other factors that lead either to infertility or subfertility. Recently,more molecular biology studies have proven that ARTs are the main causes byshowing ART were associated with anomalies in sperm chromatin packaging andabnormal placentation. Thus, this issue becomes even more controversial and itbecomes more important to conduct in depth investigations.The placenta is a temporary organ with many physiological functions, which isclosely related to pregnancy outcomes. It maintains fetal homoeostasis by providingan immune interface between the maternal and fetal allograft, transports nutrients andwaste products between mother and fetus, and acts as source of many peptide andsteroid hormones that influence fetal, placental, and maternal metabolism anddevelopment. Disturbances of placental development, including the reducedutero-placental blood flow, decreased oxygen supply in the uterus or abnormal maternal nutrient status can alter the biological characteristics of trophoblast cells andbring about changes in normal placental function. Under such circumstances, theplacenta may adapt by altering its functional protein expression or changingepigenetic modification of placental gene expression in order to meet the requirementof fetal development. Once the placental structure and function is abnormal, it willnot only lead to a variety of pregnancy complications, such as Preeclampsia (PE),Gestational diabetes mellitus (GDM) and Fetal growth restriction (FGR) and so on,but also ’programmed’ the developing fetus for increased risk of developing variousendocrine diseases in later adult life.Variations in ART procedures such as: gonadotropins for superovulation;medication taken for pregnancy sustenance; intracytoplasmic sperm injection;blastocyst culture; assisted hatching; or preimplantation genetic diagnosis couldpossibly disturb placental development which is assumed might alter placentation andpossibly cause the harmful outcome in offspring derived from ART treatment.Therefore, it is essential to study ART derived placentae to ensure the maternal-fetalsafety. Animal studies have demonstrated that abnormal placentation includingincreasing inflammations, oxidative stress and apoptosis were investigated afterembryo in vitro culture and transfer. Researches of human placentae have also provedthe incidence of villous edema and microcalcifications were increased in ART derivedplacentae; proteomics study have revealed that abnormal protein profiles related toanomalous placental functions are involved in ART placentae. Although the abovestudies have been done, systematic studies on ART derived placentae are still required.Researches of placental morphology, gene expression profiling and the underlyingmolecular mechanisms will help further evaluation the effects of the ARTmanipulation on placental development and fetal development, and to explore thepotential causal relationship to pregnancy outcomes.Placental barrier transport is a vital function to maintain the maternal-fetalhomeostasis by transporting water, nutrients and metabolites. The tightness and theselectivity of paracellular transport is one of the important factors for the transportfunction of the placental barrier system, which is mainly dependent on the tightjunction structure between epithelial cells. Dysfunction of placental tight junctions would accompany by the alteration of transepithelial resistance (TER) andparacellular permeability (CPP), leading to dysfunction of transplacental transportand other functions, which involved in the occurrence and development of a varietyof placenta-related pregnancy complications (such as PE, hydatidiform moles, etc).However, mechanisms of tight junction in the placentae haven’t been deeplyinvestigated. Researchers conducted in blood-brain barrier, air blood barrier systemsuggest that tight junction were disturbed in oxygen depletion, estrogen and insulinstimulation and inflammatory environment, accompanied by the alteration ofexpression and location of tight junction proteins, leading to epithelial barrierdysfunction and changed paracellular transport capacity. Oxygen is the basiccondition for cell growth and differentiation. During early pregnancy thedevelopment of both placenta and fetus are established in a relatively hypoxicenvironment. Concentration of oxygen in various stages of pregnancy will affectplacental functions, and is believed as the key to a successful pregnancy. Placentalischemia and hypoxia have been proven to be the causal factors of a variety ofpregnancy complications. Therefore, hypoxia has been considered to be the mostcritical factors to affect placental morphology and function during the late pregnancy.The effects of hypoxia on tight junctions and tansplacental transport capacities havenot been carried out in human placentae and trophoblasts. The in-depth study of theplacental functions in tight junction will be of great significance for us to clarify theoccurrence of pregnancy related complications.Herein, by using transmission electron microscopy (TEM) and microarrayanalysis, we compared morphological appearance and global gene expression patternsof placentae from mothers in ART group with those from control group. The objectiveof this study is to discover the potential effects of ART treatment on themorphological appearance and gene expression in placentae and to possible causalrelationship between ART procedures and offspring health. Further, we chosetransplacental barrier transport as our main target based on the basis of morphologicaland microarray experiments, especially focused on placental tight junctions, toinvestigate whether there are differentiated expressed factors in the ART group.Moreover, we studied the effects and mechanisms of hypoxia on tight junction functions in human trophoblast cells, providing new clues to clarify the pathogenesisof some pregnancy related complications.Materials and Methods(1) Five hundreds and forty-three placental samples were collected from thewomen who had undergone ART treatment in our study to establish sample bank ofplacenta. Pregnancy outcomes have been compared between383ART singletonpregnancies and control group consisted of2981natural conceived pregnant womenin Jiangsu Province People Hospital in the same period.(2) In order to minimize the ambiguity of our results, placental samplessubjected to IVF-ET due to tubal factor or mild male factors were selected for furtherstudy. Morphological appearance has been compared between ART group and controlgroups using light microscope and TEM focused on the placental blood barrier,villous stroma, and the cytotrophoblasts (CT) and syncytiotrophoblasts (ST) withtheir substructures in terminal villi.(3) A GeneChip Affymetrix HG U133Plus2.0Array was utilized to analyze theglobal gene expression patterns between goups, which were further certified usingReal time PCR. Immunohistochemistry (IHC) was conducted to investigate thelocation of dysregulated genes in the placentae. The differentially expressed geneswere classified according to their biological processes.(4) Transplacental barrier transport was chosen as our main target based on themorphological and microarray results, especially focused on placental tight junctions.Real time PCR and Western Blot were utilized to analyze the expression of tightjunctions factors CLDN4, CLDN8and OCLN between ART and control groups bothat mRNA and protein level, respectively.(5) Millicell chambers were utilized for human trophoblast cell line BeWo cell invitro culture and250uM cobalt chloride was used to establish the chemical anoxiamodel of hypoxia. TER and CPP were detected at different time points (6h,12h,24h)after exposed to hypoxia, thus evaluating the alteration of tight junctions oftrophoblast cells under hypoxic conditions. Real time PCR and Western Blot wereemployed for the detection of both mRNA and protein expression of tight junctionrelated factors CLDN4, CLDN8, OCLN and ZO1of trophoblast cells at different time points (6h,12h,24h) after exposed to hypoxia, trying to elucidate the mechanism ofdisturbance of tight junction in hypoxia.Results(1) Our results showed ART singleton pregnancy was associated with higherincidence of preterm labor, PE, GDM, placenta pervia, pretern premature rupture ofmemberane and abnormal amniotic fluid volume than control group.(2) Light microscopy results showed that both ART derived and controlplacentae had normal microscopic histological features, indicating that the ARTprocedure did not affect the gross structure of the placentae, and also demonstratedthat the samples were of sufficient quality to be analyzed using TEM. Moreover,twenty-three placental samples including8in ART group and15in control groupwere underwent TEM examination to compare their ultrastructure. The present studyrevealed a mild alteration of the placental barrier in ART derived placentae, includinga thickening of the placental barrier, decreased density of syncytiotrophoblastic apicalmicrovilli, and increased vacuoles observed in ST, all of which are potentiallyinvolved in the downregulation of transplacental transports and exchanges.(3) After the microarray analysis, twenty-six differentially expressed genes wereidentified in the ART treated placentae:17up-regulated;9down-regulated. Eighteenof these were classified into six groups according to critical placental function:immune response; transmembrane transport; metabolism; oxidative stress; celldifferentiation; and other functions. Ten differentially expressed genes were furthercertified by Real time PCR and IHC showed some of these gene products wereexpressed in the placental villus tissues, either in the cytoplasm or in the membrane ofST.(4) Tight junctions in human placentae were detailed investigated in our furtherstudy and the structure of tight junction were conformed to be localized in the apicalpart of the syncytium and also between the cell-cell contacts of fetal blood vesselendothelial after TEM examination. After Real time PCR and Western Blot analysis,the mRNA level of CLDN4and CLDN8were significantly differentially expressed inART derived placentae when compared with control groups. However, the proteinlevels of these tight junctions did not differ between groups. (5) The in vitro monolayer culture model of trophoblast cells has been successfulestablished using Millicell chamber. Tight junction dysfunction of trophoblast cellswas found at different time points after exposed to250uM cobalt chloride whichmimic chemical hypoxia, with the observation of decreased TER and increased CPP.The results of Real time PCR and Western Blot showed both mRNA and proteinexpressions of ZO1, CLDN4of trophoblast cells were decreased, and CLDN8oftrophoblast cells were increased.Conclusions(1) The collection of large amount of placental samples both from ART andnatural conceived pregnancy in our study has provided a sample basis for furtherresearches on placental morphology and functions. Ultrastructural examinationsrevealed a mild alteration of the placental barrier in ART derived placentae, includinga thickening of the placental barrier, decreased density of syncytiotrophoblastic apicalmicrovilli, and increased vacuoles observed in ST. However major morphologicalstructure has not significantly changed, reflecting that the intrauterine environment ofART pregnancy is roughly the same as naturally conceived pregnancy. Therefore,ART technology is relatively safe based on the structure studies while theultrastructural alteration in ART placentae should also be continually investigated.(3) Microarray analysis have investigated26differentially expressed genes wereidentified in the ART treated placentae related to different placental functions. Thechanged gene expression files have been speculated to be associated with thealteration of some placental functions attributed to ART manipulations and thepossibly changed pregnancy outcomes.(4) The changed mRNA level of CLDN4and CLDN8have been demonstrated inART derived placentae, however their protein level has no alteration between groups,indicating the regulation of placental gene expression and its ability of compensation.Tight junction factors might have their possible roles in the regulation oftransplacental barrier transport after ART manipulation, which have provided the newaspect for the ART security studies. |
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