Cells In Maternal Peripheral Blood Research Articles

16:00-16:10 Detection of microchimeric fetal cells in maternal peripheral blood during and after oocyte donation pregnancy

16:00-16:10 Detection of microchimeric fetal cells in maternal peripheral blood during and after oocyte donation pregnancy

Fetal nucleated red blood cells in maternal peripheral blood for non-invasive prenatal diagnosis

Fetal nucleated red blood cells in maternal peripheral blood for non-invasive prenatal diagnosis

Research Progress in Isolation and Enrichment of Fetal Cells from Maternal Blood

Prenatal diagnosis is an important means of early diagnosis of genetic diseases, which can effectively reduce the risk of birth defects. Free fetal cells, as a carrier of intact fetal genetic material, provide hope for the development of high-sensitivity and high-accuracy prenatal diagnosis technology. However, the number of fetal cells is small and it is difficult to apply clinically. In recent years, noninvasive prenatal diagnosis (NIPD) technology for fetal genetic material in maternal peripheral blood has developed rapidly, which makes it possible to diagnose genetic diseases by fetal cells in maternal peripheral blood. This article reviewed the current status of fetal cell separation and enrichment technology and its application in noninvasive prenatal diagnosis technology.

Abstract P3039: Altered Maternal and Fetal Vasopressin Receptors in Response to Hypertensive Stress in Pregnancy

Children born to women with hypertensive disorders in pregnancy have an elevated risk of developing hypertensive disorders later in life. The mechanisms for this fetal programming are unclear. Because vasopressin (AVP) is a hormone that functions to regulate blood pressure and volume homeostasis, we hypothesized that differences in expression of vasopressin receptors (AVPRs) and related genes are also altered in children born from preeclampsia-affected pregnancies. We measured the expression of receptors AVPR1a, AVPR1b, AVPR2, OXTR, and CUL5 as well as LNPEP, the major aminopeptidase that cleaves AVP. Expression in umbilical cord blood cells and maternal peripheral blood cells from women with 1) no hypertensive disorders in pregnancy (control subjects) (N=20) 2) pregnant women with chronic hypertension (N=20) and 3) women with preeclampsia (N=10) in the index pregnancy were compared. Coded cell samples and associated clinical data were obtained from the University of Iowa Maternal Fetal Tissue Bank (IRB# 200910784). RNA was purified from the cells and used for real-time quantitative PCR to assess variation in receptor expression in children born to women with hypertensive disorders of pregnancy relative to control subjects. In children born to women with chronic hypertension, AVPR2 (-8.2 Fold Change, P<0.001), LNPEP (-13 Fold Change, P<0.001), CUL5 (-14 Fold Change, P<0.001), AVPR1b (-3.1 Fold Change, P=0.001), and OXTR (-4.3 Fold Change) were all significantly under-expressed when compared to normotensive controls. Samples from children born to women with preeclampsia had significantly lower expression of AVPR1a (-4.7 Fold Change, P<0.001), AVPR1b (-2.5 Fold Change, P=0.045), AVPR2 (-2.5 Fold Change, P=0.001), and OXTR (-2.8 Fold Change, P<0.001) when compared to non-preeclamptic samples. Differences in expression were also mirrored early in pregnancy in maternal blood in women with chronic hypertension. For example, CUL5 and LNPEP were decreased in the first trimester (-3.01 Fold Change, P=0.004 and -3.64 Fold Change P=0.007, respectively). The differences in gene expression in cord blood may be related to the future risk of hypertension later in life for children born to women with hypertensive diseases in pregnancy.

Molecular genetic investigation of placental mesenchymal dysplasia

Molecular genetic investigation of placental mesenchymal dysplasia

Adhering maternal platelets can contribute to the cytokine and chemokine cocktail released by human first trimester villous placenta

Placental villous explant culture has been increasingly recognized as suitable model to study secretion of inflammatory and immune modulating factors by human placenta. Most of these factors likely derive from the syncytiotrophoblast, whereas extraplacental sources such as maternal peripheral blood cells are rarely considered. Due to their small size and absence of a nucleus, platelets adhering to perivillous fibrinoid of normal placenta are frequently ignored in routine immunohistochemistry. Here we demonstrate adhering maternal platelets on first trimester placental villi after explant culture and point out that platelet-derived factors must be considered when analyzing the inflammatory secretion profile of human placenta.

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Objectives Preeclampsia is a pregnancy-associated complex condition associated with inflammation, oxidative stress and angiogenic imbalance. Stress sensitive transcription factors nuclear factor-erythroid 2-like 1 (Nrf1) and nuclear factor-erythroid 2-like 2 (Nrf2) are involved in regulation of angiogenic, inflammatory and oxidative stress pathways. Recent evidence suggests a link between Nrf2 and angiogenic factor balance in preeclampsia though the degree to which maternal and placental DNA methylation contributes to disruption of Nrf pathways among women with preeclampsia is unknown. Our central hypothesis is that distinct patterns of DNA methylation in Nrf pathway and angiogenic genes contribute to altered angiogenic balance in preeclampsia. Methods Prospectively, we collected blood in each of three trimesters of pregnancy, maternal white blood cells in the first trimester of pregnancy and placental tissue at delivery from nulliparous women. We analyzed DNA methylation at individual CpG dinucleotides (Illumina Infinium) in Nrf and angiogenic (VEGFA, VEGFB, VEGC, FLT1, KDR, NRF1, PlGF) genes in maternal peripheral blood cells and placenta and circulating angiogenic factors (Millipore Bioplex) from women who developed preeclampsia and normotensive controls ( n = 6/group). Differences in average methylation beta scores (change in beta score >0.2 indicated significant gain of methylation; >−0.2 indicated significant loss of methylation) determined significant differences between groups. Results DNA methylation patterns in Nrf and angiogenic genes in maternal white blood cells and placenta were not significantly different in cases/controls. Among women with preeclampsia, PlGF was significantly lower across pregnancy and sFLT1 was significantly higher in the third trimester among women with preeclampsia. Conclusions DNA methylation in Nrf and angiogenic genes was not associated with altered angiogenic balance in women who developed late onset preeclampsia. Differences in DNA methylation may occur later in pregnancy when circulating factors differed between groups. Disclosures C.M. Anderson: None. J.L. Ralph: None. J.E. Ohm: None.

Prenatal detection of del(10)(q11.2) mosaicism in chorionic villus specimens likely caused by a common chromosomal fragile site FRA10G is associated with a normal phenotype

To summarize the pregnancy outcomes of cases with mosaicism for chromosome 10q11.2 deletion detected by chorionic villus sampling (CVS) and determine whether extensive cytogenetic work-up and follow-up amniocentesis are necessary in such cases. CVS was performed at 10-12 weeks of gestation. Chromosome analysis of chorionic villi was performed by standard G-banding techniques. Mosaicism of chromosome 10q11.2 deletion was observed in 24 out of 6063 CVS cases (0.39%). A common fragile site, FRA10G is located at the breakpoint region. The level of mosaicism ranged from 4% to 25%. No evidence of mosaic 10q11.2 deletion was found in follow-up amniocentesis, maternal peripheral blood cells, or from cytogenetic studies of other pregnancies from the same group of patients. All these cases resulted in the live birth of normal healthy infants. The presence of del(10)(q11.2) mosaicism in chorionic villus specimens most likely represents an in vitro culture artifact due to FRA10G fragile site in this region without any clinical consequences. If ultrasound results are normal, it is not necessary to perform follow-up amniocenteses and additional laboratory work-up for such cases.

Epigenomic markers for heritable risk of preeclampsia

Preeclampsia (PE) affects 8–10% of women in the US. Long‐term consequences include development of maternal hypertension and hypertension in offspring. The familial risk of PE is well established, though underlying mechanisms are unknown. In this study, our goal was to identify differentially methylated genes common to both maternal peripheral blood cells (MPBCs) and placental tissue of fetal origin in order to determine potential epigenomic markers of heritable PE risk. Methylation quantification of individual CpG dinucleotides in MPBCs and placental tissue from women with and without PE was determined using the Illumina Infinium methylation array (n=6/group). Gain of methylation was seen in 218 CpG dinucleotides from MPBCs, and ~34% of these changes were also present in placenta. Significant methylation loss was observed in 123 CpG dinucleotides, and ~28% of changes were also present in placenta. Validation of methylation was determined by bisulfite sequencing. Differentially methylated loci were primarily located in non‐CpG island regions of genes with known roles in angiogenesis, inflammation and metabolic regulation. This study provides novel evidence for the mechanisms underlying heritable risk for PE, implicating differential transgenerational DNA methylation in key genes associated with the etiology and consequences of PE and suggest that these may serve as novel biomarkers for PE screening.

Differences in lymphocyte subpopulation count and function in cord, maternal and adult blood.

Phenotypical characterization and functional activity of lymphocytes and natural killer (NK) cells in cord blood (CB) were investigated, and maternal peripheral blood (MPB) values were compared to those of adult peripheral blood (APB) (control). To determine cytotoxic activity target cells (K562) were labeled with carboxyfluorescein diacetate (CFDA) or fluorescein isothiocyanate (FITC), and propidium iodide (PI) was used to label dead cells. Cell surface expression in CB, APB, and MPB cells were analyzed using flow cytometry. CB and MPB mononuclear cells had similar CD45, CD34, CD4, and surface molecule for T helper cell expression, but had low-level expression of total T-lymphocyte surface molecules CD3 and CD8. CD19 and HLA-DR expression was higher in CB than in MPB. The same high-level of expression for CD19 and HLA-DR was observed in APB, as compared to MPB. All other cell surface expressions were similar in APB and MPB samples. NK (CD16+ and CD56+) cells in CB was similar to that in MPB and APB, and the level of inhibitory KIR receptors in NK cells was higher in venous CB than in MPB and APB. The only difference between MPB and APB was that the CD158a level was higher in MPB. No difference was observed in NK cells in CB and MPB, in terms of cytotoxicity. The present results show that there was numerical and proportional variability of lymphocytes and their subgroups in CB and APB, but no cytological difference.

Proportional changes of CD4+CD25+Foxp3+ Regulatory T cells in maternal peripheral blood during pregnancy and labor at term and preterm

To evaluate the proportional changes of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in maternal peripheral blood during pregnancy and labor at term and preterm. Peripheral blood was collected from 20 non-pregnant controls and 139 pregnant women (60 at different gestational ages, 48 at term with or without labor, and 31 in threatened or actual preterm labor). CD4+CD25+Foxp3+ Tregs in peripheral blood samples were analyzed in peripheral blood samples by flow cytometry. Placentas from preterm women were examined for the presence of histological chorioamnionitis (HC). The percentage of circulating CD4+CD25+Foxp3+ Tregs was significantly increased in women during the first trimester compared with non-pregnant controls (P < 0.0001), peaking during the second trimester and then declining slightly in the third trimester. There was a significantly lower level of CD4+CD25+Foxp3+ Tregs in women with term labor than in those at term without labor (P < 0.0001). Women admitted in preterm labor had a lower proportion of CD4+CD25+Foxp3+ cells than those admitted with threatening preterm labor (P < 0.0001). Preterm women with evidence of HC had decreased proportion of CD4+CD25+Foxp3+ cells than those without HC in preterm deliveries (P=0.008). Moreover, the percentages of CD4+CD25+Foxp3+ cells in preterm subjects, irrespective of the HC status, were significantly diminished compared with women with normal pregnancy at the same gestational age (P < 0.0001). Our data reveal a marked elevation of peripheral blood CD4+CD25+Foxp3+ Tregs during early pregnancy, but a dramatic decline during labor, either at term or preterm, suggesting their involvement in the maintenance of pregnancy and the initiation of labor.

Method for isolation of floating fetal RBCs from maternal circulation based on their inherent magnetic property

Isolation of floating fetal cells from maternal circulation has immense potential in diagnosing of various genetic alterations in the developing fetus. Currently, non-invasive fetal cell isolation methods include fluorescence/magnetic activated cell sorting that use antibodies specific to fetal cells. Apart from being complex and expensive the biggest challenge associated with these cell sorting methods is low concentration of fetal cells in maternal peripheral blood. In order to make the complete process much simpler and effective, we propose a novel method for isolation of floating fetal cells that uses a continuous flow of maternal blood for effectively harvesting a higher fetal cell volume compared to any other existing method. The isolation mechanism is based on the difference in the magnetic susceptibility of fetal hemoglobin (HbF- α 2 γ 2) and maternal hemoglobin (HbA- α 2 β 2). HbF has high oxygen saturation capacity (diamagnetic) compared to HbA (paramagnetic), and this difference in saturation is further enhanced by presence of 2,3-bisphosphoglycerate (2,3-BPG). When placed in magnetic field, these cells get separated based on the difference ( p ≤ 0.001) in their magnetophoretic mobility. This separation method may also be used for detection of fetomaternal hemorrhages and also treatment of Rh incompatibility.

Prenatal Diagnosis of Trisomy 21 with Fetal Cells in Maternal Blood Using Comparative Genomic Hybridization

Objective: This study was undertaken to determine the clinical use of comparative genomic hybridization (CGH) for detection of fetal trisomy 21 from fetal ceIls (nucleated red blood cells; nRBCs) isolated from maternal peripheral venous blood. Methods: Maternal peripheral venous blood samples were collected in sterile tubes containing heparin. After triple density gradient centrifugation, magnetic activated cell sorting using CD45 and CD71 was used to isolate the fetal nRBCs. Fetal nRBCs were successfully isolated from maternal peripheral blood in all cases. After laser-microdissecting fetal nRBCs, degenerate oligonucleotide-primed polymerase chain reaction, and nick translation, DNA size was suitable for hybridization. Results: By CGH analysis, we diagnosed one normal male, one normal female, and one trisomy 21 male fetus. These results were confirmed by amniocentesis. Conclusions: Prenatal diagnosis from fetal cells in maternal peripheral blood by CGH shows clinical promise as an alternative or as a supplement to fluorescence in situ hybridization with chromosome-specific probes but further studies are warranted.

Fetal nucleated red blood cells in maternal peripheral blood and gestational age

The theory that blood cells are transported between mother and fetus has been verified and inherited diseases can be diagnosed from fetal cells isolated from maternal peripheral blood. A total of 61 healthy pregnant women aged 21–35 years who were between the 11th and 34th weeks of pregnancy and who had not experienced any pregnancy complication such as serious anemia were enrolled in the study. Density gradient centrifugation was used to isolate the mononuclear cell layer from maternal blood and a fluorescence-activated cell sorter was used to isolate and enrich the nucleated red blood cell (NRBC) population. These procedures were performed to improve the sensibility and specificity of CD71+ cell detection and determine the value of these cells in prenatal diagnosis. An Olympus fluorescent microscope and an ordinary microscope were used for the observation. (excerpt)

Analysis of fetal DNA from maternal peripheral blood by lectin-polymerase chain reaction-single strand conformation polymorphism.

Fetal nucleated cells in maternal peripheral blood are a non-invasive source of fetal DNA for prenatal genetic diagnosis. However, the number of fetal cells present in maternal peripheral blood is very small. Therefore, fetal cell enrichment is generally considered necessary to allow detection and subsequent genetic analysis of the rare fetal cells. In the study presented here, we performed fetal cell separation from maternal blood using galactose-specific lectin to concentrate fetal nucleated red blood cells (FNRBCs), and attempted paternal diagnosis using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Fetal cell separation was performed using galactose-specific lectin on a PV-MeA coated slide. Twenty cells consisting of an NRBC and its surrounding 19 maternal cells were collected using laser microdissection for stable DNA amplification. DNA analysis was performed using three sequence tagged site markers (D13S270, D17S5, and D18S474) by PCR-SSCP. All seven cases were informative because they showed heterozygosity at least one locus in D13S270, D17S5, or D18S474, and paternal-specific bands were detected in all cases. These results suggest that our proposed lectin-laser-micromanipulation-PCR-SSCP method may contribute to the development of prenatal diagnosis.

Trophoblasts and soluble adhesion molecules in peripheral blood of women with pregnancy-induced hypertension.

The current hypothesis on the pathogenesis of pregnancy-induced hypertension (PIH) considers it as an endothelial disorder that is first local but with the potential of becoming general. The aim of the work was to investigate the relation of the number of trophoblast cells in maternal peripheral blood against the serum levels of soluble vascular and intercellular cell adhesion molecule (sVCAM-1 and sICAM-1) in PIH. Women with PIH were at 28th to 40th week of gestation. Control group were normotensive (NT) pregnant women at 28th to 41st week of gestation. Flow cytometry was used to assess the relative number of the trophoblasts in the peripheral blood. Trophoblasts were labeled with monoclonal anti-human trophoblast protein antibody MCA 277. The presence of sVCAM-1 and sICAM-1 was determined using the enzyme-linked immunosorbent assay method. Women with PIH had significantly higher trophoblasts number than NT women (median 19.0, range 5.0-57.0/400 microL versus median 7.0, range 0.0-18.0/400 microL; P = 0.000011) as well as plasma level of sVCAM-1 when compared with NT women (median 730.0, range 325.0-1525.0 ng/mL versus median 493, range 310-1075 ng/mL; P = 0.02). ICAM-1 level in the PIH group was slightly elevated (median 280.0, range 174.0-524.0 ng/mL) when compared with NT women (median 260.0, range 190.0-464.0 ng/mL, P = 0.322). Eight of 21 women with PIH had proteinuria but no correlation was found between this symptom and the laboratory findings. The increased number of trophoblast cells in maternal peripheral blood and higher levels of sVCAM-1 correlate with the presence of PIH. The differences of sVCAM levels were significantly higher than those observed for sICAM. The results indicate an association between circulating trophoblasts and vascular endothelium activation, during PIH.

Phenotype and function of lymphocytes from the neonatal umbilical cord compared to paired maternal peripheral blood cells isolated during delivery

In the present study, we analyzed the immunological characteristics of mononuclear cells (MNC) isolated from both neonatal umbilical cord blood (UCB) and maternal peripheral blood (MPB) during the delivery. The in vitro proliferative response of UCB T lymphocytes was significantly reduced compared to the maternal response to phytohemagglutinin A, pokeweed mitogen, and alloantigen stimulation, in correlation with the lower percentage of UCB than MPB lymphocytes, but not with that of B cells. The mean cytotoxic activity level of interleukin-2 (IL-2)-activated natural killer (NK) was higher in UCB than in MBP, whereas the percentage of CD56 + NK cell count was similar. Our results show differences in the immune reactivity of T and B lymphocytes from neonate and adult isolated under similar physiological conditions.

The application of magnetic cell sorter (MACS) to detect fetal cells in maternal peripheral blood.

The aim of the present study was to investigate the effectiveness of sorting fetal nucleated red blood cells (FNRBC) from maternal peripheral blood, particularly during early gestation periods, by a combination of specific gravity centrifugation and magnetic cell sorter (MACS). Without prior knowledge of the gender of the fetus, we determined gender by analyzing a Y-chromosome specific sequence by nested-PCR, using 10 ml of the peripheral blood of healthy primigravida women at different stages of gestation (first trimester: n = 17, second trimester: n = 13, and third trimester: n = 19). The results of this prenatal sex determination were compared to the sex of newborns. The specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of the present method during the first trimester were 100, 81.8, 100, and 75%, respectively; during the second trimester, 80, 50, 80, and 50%, respectively; and during the third trimester, 25, 63.6, 53.8, and 33.3%, respectively. The results show that this prenatal sex determination method has a highly accurate diagnostic rate during the first trimester, suggesting that it could be developed as a practical, non-invasive prenatal diagnostic technique for use during early gestation periods.

HLA-G expression in trophoblast cells circulating in maternal peripheral blood during early pregnancy

Objective: The aim of this study was to assess the use of circulating trophoblast cells in maternal peripheral blood for noninvasive prenatal diagnosis of numeric chromosomal aberrations. Study Design: A combined procedure for immunocytochemical identification and deoxyribonucleic acid fluorescence in situ hybridization was used after a single enrichment step consisting of density gradient centrifugation. A specific HLA-G monoclonal antibody was used in combination with X and Y chromosome specific probes in deoxyribonucleic acid fluorescence in situ hybridization to confirm fetal identity of cells bearing HLA-G in the case of a male fetus. Results: We detected fetal trophoblast cells expressing HLA-G in maternal blood starting at 9 weeks’ gestation. In addition to fetal sex prediction with X and Y chromosome–specific probes, fetal aneuploidy was confirmed in peripheral blood from a pregnancy complicated by trisomy 21. Conclusion: Although the numbers of fetal cells were extremely low, the proof of concept was demonstrated. Early noninvasive prenatal screening for numeric chromosomal abnormalities with fetal trophoblast cells is feasible. (Am J Obstet Gynecol 2001;184:991-7.)

The expression of IL-8 and IL-4 messenger RNAS in maternal peripheral blood cells during pregnancy

International Journal of Gynecology & ObstetricsVolume 70, Issue S2 p. B151-B151 Physiology of reproduction 2 The expression of IL-8 and IL-4 messenger RNAS in maternal peripheral blood cells during pregnancy C. Kato, C. KatoSearch for more papers by this authorT. Tamura, T. TamuraSearch for more papers by this authorT. Okuda, T. OkudaSearch for more papers by this authorH. Kojima, H. KojimaSearch for more papers by this authorM. Fukuoka, M. FukuokaSearch for more papers by this authorT. Yamamoto, T. YamamotoSearch for more papers by this authorH. Honjo, H. HonjoSearch for more papers by this author C. Kato, C. KatoSearch for more papers by this authorT. Tamura, T. TamuraSearch for more papers by this authorT. Okuda, T. OkudaSearch for more papers by this authorH. Kojima, H. KojimaSearch for more papers by this authorM. Fukuoka, M. FukuokaSearch for more papers by this authorT. Yamamoto, T. YamamotoSearch for more papers by this authorH. Honjo, H. HonjoSearch for more papers by this author First published: 10 December 2003 https://doi.org/10.1016/S0020-7292(00)83210-5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume70, IssueS22000Pages B151-B151 RelatedInformation