Relationship Between Quantification Of Circulating Fetal Cells Marked By HLA-G Antibody And Hypertensive Disorder Complicating Pregnancy

Hypertensive disorder complicating pregnancy is one of the most common and dangerous pregnancy related complications. Although it occurrs in the third trimester, the onset factors exist in early pregnancy. Currently, there is no available method to predict the occurrence of the disease in the first trimester.The concept of noninvasive prenatal diagnosis based on isolation of rare fetal cells from maternal blood has been the source of anticipation and enthusiasm over the past few decades for researchers dedicated to this field. Cells of embryonic (nucleated red blood cells and lymphocytes) and extraembryonic origin (trophoblasts) circulate in the maternal peripheral blood during early pregnancy. After enrichment, these cells can be used for noninvasive prenatal diagnosis. Trophoblast cells are formed very early in gestation and circulate in the maternal blood as a consequence of their invasive and proliferative nature. In addition, because trophoblast cells are unlikely to persist after delivery, including subsequent pregnancies, these cells carry intrinsic advantages for prenatal diagnostic use. However, reports on the successful enrichment of circulating trophoblast cells are scarce and the results are not always reliable because of the lack of trophoblast-specific antibodies. In extravillous trophoblast cells, the classical major histocompatibility complex (MHC) class I and MHC class II molecules are completely absent. However, HLA-G, a nonclassical MHC class I antigen, is specifically expressed in extravillous trophoblasts. Because of its highly exclusive expression, HLA-G has been suggested to play a pivotal role in maternal-fetal interface. In this study, the extravillous cytotrophoblast cells marked by HLA-G-specific monoclonal antibody were identified by an immunocytochemical assay. Moreover, the numbers of circulating fetal trophoblast cells were compared between normal pregnant and hypertensive disorder complicating pregnant women, exploring the role of the quantitative abnormality in this disease. It will provide the basis for noninvasive prenatal diagnosis on the early stage of this disease.PartⅠInvestigation of Identifying Circulating Fetal Trophoblast Cells Marked by HLA-G AntibodyObjective To set up a method to identify extravillous cytotrophoblast cells with HLA-G-specific monoclonal antibody using an immunocytochemical assay.Methods The trophoblast cells were isolated by density gradient centrifugation from maternal blood samples of normal labor and hypertensive disorder complicating pregnancy women. After preliminary enrichment, the circulating fetal trophoblast cells were identified by immunocytochemical stain with MEM-G/9 (monoclonal antibody to HLA-G). The blood sample from a non-pregnant woman and a male fetus's cord blood served as negative and positive controls, respectively.Results In neonatal cord blood, maternal blood of hypertensive disorder complicating pregnancy women and maternal blood of control pregnant women, the HLA-G positive fetal cells were found. The morphological features of the positive cells were observed, which were characterized by mononucleated, cytotrophoblast-like cells with diameter larger than the negative cells (peripheral blood mononuclear cells), large nucleus with rather condensed chromatin and small cytoplasm. Conclusion The sorting method with HLA-G monoclonal antibody MEM-G/9 can effectively recognize the circulating fetal trophoblast cells, which serves as basis for the subsequent assessment of fetal cell origin.PartⅡVerification of the Fetal Origin of the Circulating HLA-G Positive Cells by Micromanipulation Sorting and Detecting the Sex-determining Region of Y-chromosome (SRY) GeneObjective To evaluate the applicability of sex-determining region of Y-chromosome (SRY) gene to identify of fetal cells and detect of fetal sex in noninvasive prenatal diagnosis, and to prove fetal origin of the HLA-G positive cells.Methods Peripheral blood samples were obtained from 8 male and 8 non-pregnant female subjects. Primer extension preamplification (PEP) and polymerase chain reaction (PCR) based on single cell were adopted to detect sex-determining region of Y-chromosome (SRY) gene. Afterwards, circulating fetal trophoblast cells from patients with hypertensive disorder complicating pregnancy were micromanipulated separately. PEP and PCR were adopted to prove the fetal origin of the positive cells.Results The specificity of the SRY primers were assessed by amplifying the DNA obtained from 8 men and 8 women. All of the male-derived samples, used as positive control, scored positive. Meanwhile, all of the female-derived samples scored negative. Applying PEP-PCR method to detect SRY gene from the HLA-G positive cells from the patients with hypertensive disorder complicating pregnancy, it was detected in all women with male fetus and not detected in the other women with female fetus. The sensitivity and specificity of this method are 100%, in concordance with the actual sex of the labored fetuses.Conclusion PEP and PCR method could effectively and exactly identify the origin of the single cell. HLA-G positive fetal cells from maternal peripheral blood were the circulating fetal trophoblast cells. High purity of fetal trophoblast cells can be obtained by using HLA-G as a sorting marker, combined with micromanipulation.PartⅢRelationship Between Quantification of Circulating Fetal Cells Marked by HLA-G Antibody and Hypertensive Disorder Complicating PregnancyObjective To determine if quantitative abnormalities of circulating fetal trophoblast cells are associated with hypertensive disorder complicating pregnancy.Methods The trophoblast cells were isolated by density gradient centrifugation from maternal blood samples of normal labor (n=16) and hypertensive disorder complicating pregnancy women (n=18). After preliminary enrichment, the circulating fetal trophoblast cells were identified by immunocytochemical stain with MEM-G/9. Levels of circulating fetal trophoblast cells numbers were calculated and compared using non-parametric methods with Mann–Whitney test.Results There were 6.88±1.54 and 30.56±5.16 HLA-G positive cells in 6 mL maternal blood from normal labor and hypertensive disorder complicating pregnancy women, respectively. The difference was statistically significant (P < 0.0001). Conclusion The increased number of trophoblast cells in maternal peripheral blood correlate with the presence of hypertensive disorder complicating pregnancy, which might be useful for noninvasive prenatal diagnosis of hypertensive disorder complicating pregnancy.