Keiko Koide

Attitudes toward non‐invasive prenatal diagnosis among pregnant women and health professionals in Japan

This study aims to assess the attitudes toward non‐invasive prenatal diagnosis (NIPD) and NIPD problems in clinical practice in Japan.

Recent advances in non-invasive prenatal DNA diagnosis through analysis of maternal blood.

Prenatal diagnosis of aneuploidy and single‐gene disorders is usually performed by collecting fetal samples through amniocentesis or chorionic villus sampling. However, these invasive procedures are associated with some degree of risk to the fetus and/or mother. Therefore, in recent years, considerable effort has been made to develop non‐invasive prenatal diagnostic procedures. One potential non‐invasive approach involves analysis of cell‐free fetal DNA in maternal plasma or serum. Another approach utilizes fetal cells within the maternal circulation as a source of fetal DNA. At the present time, fetal gender and fetal RhD blood type within RhD‐negative pregnant women can be reliably determined through analysis of maternal plasma. Furthermore, genetic alterations can be diagnosed in the maternal plasma when the mother does not have the alterations. However, the diagnosis of maternally inherited genetic disease and aneuploidy is limited using this approach. Non‐invasive prenatal diagnosis through examination of intact fetal cells circulating within maternal blood can be used to diagnose a full range of genetic disorders. Since only a limited number of fetal cells circulate within maternal blood, procedures to enrich the cells and enable single cell analysis with high sensitivity are required. Recently, separation methods, including a lectin‐based method and autoimage analyzing, have been developed, which have improved the sensitivity of genetic analysis. This progress has supported the possibility of non‐invasive prenatal diagnosis of genetic disorders. In the present article, we discuss recent advances in the field of non‐invasive prenatal diagnosis.

Comparison in gene expression of secretory human endometrium using laser microdissection.

BackgroundThe endometrium prepares for implantation under the control of steroid hormones. It has been suggested that there are complicated interactions between the epithelium and stroma in the endometrium during menstrual cycle. In this study, we demonstrate a difference in gene expression between the epithelial and stromal areas of the secretory human endometrium using microdissection and macroarray technique.MethodsThe epithelial and stromal areas were microdissected from the human endometrium during the secretory phase. RNA was extracted and amplified by PCR. Macroarray analysis of nearly 1000 human genes was carried out in this study. Some genes identified by macroarray analysis were verified using real-time PCR.ResultsIn this study, changes in expression <2.5-fold in three samples were excluded. A total of 28 genes displayed changes in expression from array data. Fifteen genes were strongly expressed in the epithelial areas, while 13 genes were strongly expressed in the stromal areas. The strongly expressed genes in the epithelial areas with a changes >5-fold were WAP four-disulfide core domain 2 (44.1 fold), matrix metalloproteinase 7 (40.1 fold), homeo box B5 (19.8 fold), msh homeo box homolog (18.8 fold), homeo box B7 (12.7 fold) and protein kinase C, theta (6.4 fold). On the other hand, decorin (55.6 fold), discoidin domain receptor member 2 (17.3 fold), tissue inhibitor of metalloproteinase 1 (9 fold), ribosomal protein S3A (6.3 fold), and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (5.2 fold) were strongly expressed in the stromal areas. WAP four-disulfide core domain 2 (19.4 fold), matrix metalloproteinase 7 (9.7-fold), decorin (16.3-fold) and tissue inhibitor of metalloproteinase 1 (7.2-fold) were verified by real-time PCR.ConclusionsSome of the genes we identified with differential expression are related to the immune system. These results are telling us the new information for understanding the secretory human endometrium.

Evaluation of physiological alterations of the placenta through analysis of cell-free messenger ribonucleic acid concentrations of angiogenic factors

OBJECTIVE Placental messenger ribonucleic acid (mRNA) has been shown to circulate in maternal plasma. We investigated concentrations of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1), and endoglin in subjects with preeclampsia, compared with normal pregnancies. STUDY DESIGN Peripheral blood samples were obtained from preeclampsia (n = 43) and control subjects (n = 41). Plasma ribonucleic acid was subjected to analysis by reverse transcription-polymerase chain reaction assay to examine the mRNA distribution among women with preeclampsia and control subjects during weeks 35-41 of gestation. RESULTS Concentrations of VEGF, VEGFR-1, and endoglin mRNA of women with preeclampsia were significantly increased. The mRNA values were observed to correlate directly with the severity of hypertension and proteinuria. VEGFR-1 mRNA was markedly elevated in women with preeclampsia and hemolysis, elevated liver enzymes, and low platelet syndrome. CONCLUSION The mRNA concentrations of VEGF, VEGFR-1, and endoglin were observed to correlate directly with the severity of preeclampsia.

Clinical Potential for Noninvasive Prenatal Diagnosis Through Detection of Fetal Cells in Maternal Blood

Fetal cells circulate in maternal blood and are considered a suitable means by which to detect fetal genetic and chromosomal abnormalities. This approach has the advantage of being noninvasive. Since the early 1990s, nucleated erythrocytes (NRBCs) have been considered good target cells for a number of techniques, including fluorescence-activated cell sorting and magnetic cell sorting, using antibodies such as anti-transferrin receptor and anti-gamma-hemoglobin antibodies, followed by analysis with fluorescence in situ hybridization or polymerase chain reaction. In the late 1990s, the National Institute of Child Health and Human Development Fetal Cell Isolation Study assessed the reliability of noninvasive prenatal diagnosis of fetal aneuploidy using NRBCs isolated from maternal circulation. This study revealed the limitations of NRBC separation using antibodies specific for NRBC antigens. A more recent study has demonstrated the efficiency and success of recovery of NRBCs using a galactose-specific lectin, based on the observation that erythroid precursor cells have a large quantity of galactose molecules on their cell surface. Thus, recent advances in this field enhance the feasibility of this diagnostic method. This review article focuses on various methods of detection of fetal cells within the maternal circulation, as well as the status of previous and current studies and the prospective view for noninvasive prenatal diagnosis using fetal cells from the maternal circulation.

Placental expression of microRNA-17 and -19b is down-regulated in early pregnancy loss.

OBJECTIVE First, to determine if microRNA-17 and -19b are expressed in villous samples at early stages of pregnancy. Second, to determine whether placental expressions of these microRNAs along with their main targets (PTEN, CREB-1, TGFβ-1 and TGFβ-RII) are altered in early pregnancy loss. STUDY DESIGN Expression levels of microRNAs and mRNA targets in villous samples from early pregnancy loss (n=11) and matched normal cases (n=20) by gestational age were determined by RT-PCR. RESULTS Both microRNA-17 and -19b were expressed in all cases of normal pregnancy. They were significantly down-regulated (relative ratios: 0.35 and 0.34 respectively) in early pregnancy loss. Their main target, PTEN mRNA, was significantly up-regulated in early pregnancy loss (relative ratio: 2.6, 95%CI: 0.2-29.8). TGF-β1, CREB-1 and TGFβ-RII were not significantly different between the two groups. CONCLUSION microRNA-17 and -19b are expressed in early stages of pregnancy. They are down-regulated in villous samples from early pregnancy loss. We suggest that these main members of the microRNA-17-92 cluster might be involved in placental invasion and its dysregulation might also be related to other conditions characterized by defective placentation.

Effects of Maternal Smoking on the Placental Expression of Genes Related to Angiogenesis and Apoptosis during the First Trimester

Objective Maternal cigarette smoking is reportedly associated with miscarriage, fetal growth restriction and placental abruption, and is paradoxically associated with a decreased risk of developing preeclampsia. In the present study, we investigated the gene expression levels of villous tissues in early gestation. We compared the expression levels of the genes related to angiogenesis and apoptosis in the villous tissues obtained from smoking and non-smoking pregnant women. Materials and Methods We collected villous tissue samples from 57 women requesting surgical termination due to non-medical reasons at 6–8 weeks of gestation. The maternal cigarette smoking status was evaluated by the level of serum cotinine and patients were divided into active smokers and non-smokers by the serum cotinine level. The placental levels of VEGFA, PGF, FLT1, HIF1A, TP53, BAX and BCL2 mRNA were quantified by real time PCR. Results The gene expression level of PGF and HIF1A in the active smoker group was significantly higher than that in the non-smoker group. We did not observe any significant differences in the VEGFA or FLT1 expression between the groups. In active smoker group, the gene expression levels of TP53 and BAX were significantly higher than those in the non-smoker group. The ratio of BAX/BCL2 mRNA in the active smoker group was significantly higher than that in the non-smoker group. Conclusions Our findings revealed that smoking might affect the placenta during early pregnancy. Maternal cigarette smoking in early pregnancy may be associated with villus hypoxia, which may influence angiogenesis and apoptosis.

Physiological changes in the pattern of placental gene expression early in the first trimester.

Objective: To assess the physiological changes in the placental expression pattern of a panel of genes related to angiogenesis and oxidative stress during the early part of the first trimester of pregnancy. Methods and Results: The expression of a selected panel of genes was quantified by reverse transcriptase–polymerase chain reaction in samples of villous trophoblasts obtained from women between 6 and 11 weeks of gestation undergoing elective artificial abortion. We found that the levels of messenger RNA (mRNA) expression of placental growth factor (PlGF), heme oxygenase 1(HO-1), and superoxide dismutase (SOD) increased significantly with gestational age (r = .37, P = .001; r = .24, P =.04; and r = .52, P < .001, respectively). Conversely, the mRNA expression level of fms-like tyrosine kinase 1 (FLT-1) decreased significantly (r = −.30, P = .009). Conclusion: During the early part of the first trimester of pregnancy, the placental gene expression levels of PlGF, HO-1, and SOD increase with gestational age, whereas the expression of FLT-1 decreases. The alteration in this pattern of gene expression in early pregnancy may therefore play an important role in placenta-related disorders such as preeclampsia.

Maternal smoking and placental expression of a panel of genes related to angiogenesis and oxidative stress in early pregnancy.

Objective: Maternal cigarette smoking is paradoxically associated with a decreased risk of developing preeclampsia. Since preeclampsia is thought to be associated with altered mechanisms of angiogenesis and oxidative stress, we aim to investigate the influence of maternal smoking on the early placental expression of a panel of genes related to angiogenesis and oxidative stress. Material and Methods: We collected villous tissue samples at 6-7 and 10-11 weeks of gestation from 31 women requesting surgical termination. Placental expression of the following genes were quantified by real-time PCR: vascular endothelial growth factor A (VEGFA), fms-like tyrosine kinase (Flt-1), soluble endoglin (sEng), placental growth factor (PlGF), heme oxygenase-1 (HMOX-1) and superoxide dismutase (SOD). Maternal smoking status was assessed by levels of serum cotinine. Results: Placental expression of VEGFA was significantly higher in smoking women at 10-11 weeks of gestation compared with nonsmoking women at the same gestational age. There was no significant difference at 6-7 weeks of gestation. There was no variation in the expression of the other genes explored related to smoking status. Conclusions: Here we report that VEGFA placental expression was higher in smoking women at 10-11 weeks of gestation. Increased VEGFA expression in the early stages of pregnancy in smoking women might contribute to the decreased risk of developing preeclampsia.

Increased Levels of Cell-Free Human Placental Lactogen mRNA at 28-32 Gestational Weeks in Plasma of Pregnant Women With Placenta Previa and Invasive Placenta:

We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P < .01 and P < .05, respectively. We found a sensitivity of 100% and a specificity of 61.5% for the prediction of invasive placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation.