Keiichi Matsubara

Circulating Endothelial Progenitor Cells During Normal Pregnancy and Pre-Eclampsia

Endothelial progenitor cell (EPC), which mediates neovascularization of uterine endometrium may be involved in the neovascularization in the utero‐placental circulation. We evaluated whether EPC proliferation in pre‐eclampsia (PE) differed from that in normal pregnancy.

Nitric Oxide and Reactive Oxygen Species in the Pathogenesis of Preeclampsia

Preeclampsia (PE) is characterized by disturbed extravillous trophoblast migration toward uterine spiral arteries leading to increased uteroplacental vascular resistance and by vascular dysfunction resulting in reduced systemic vasodilatory properties. Its pathogenesis is mediated by an altered bioavailability of nitric oxide (NO) and tissue damage caused by increased levels of reactive oxygen species (ROS). Furthermore, superoxide (O2−) rapidly inactivates NO and forms peroxynitrite (ONOO−). It is known that ONOO− accumulates in the placental tissues and injures the placental function in PE. In addition, ROS could stimulate platelet adhesion and aggregation leading to intravascular coagulopathy. ROS-induced coagulopathy causes placental infarction and impairs the uteroplacental blood flow in PE. The disorders could lead to the reduction of oxygen and nutrients required for normal fetal development resulting in fetal growth restriction. On the other hand, several antioxidants scavenge ROS and protect tissues against oxidative damage. Placental antioxidants including catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) protect the vasculature from ROS and maintain the vascular function. However, placental ischemia in PE decreases the antioxidant activity resulting in further elevated oxidative stress, which leads to the appearance of the pathological conditions of PE including hypertension and proteinuria. Oxidative stress is defined as an imbalance between ROS and antioxidant activity. This review provides new insights about roles of oxidative stress in the pathophysiology of PE.

Significance of a diastolic notch in the uterine artery flow velocity waveform induced by uterine embolisation in the pregnant ewe

Objective To investigate the relation between placental embolisation and the diastolic notch in the uterine artery flow velocity waveform of pregnant ewes under general anaesthesia.

Estrogen and progesterone play pivotal roles in endothelial progenitor cell proliferation

BackgroundIt has been previously suggested that angiogenesis occurs during the menstrual cycle. Moreover, a rise in uterine blood flow is largely maintained by vasodilatation and substantial increases in angiogenesis. It is known that estradiol (E2) and progesterone (P4) are involved in angiogenesis. Recently, endothelial progenitor cells (EPCs) were found to be involved in neovascularization; however, their roles in uterine neovascularization have not been well characterized. We hypothesized that E2- or P4-mediated EPC proliferation plays important roles in uterine neovascularization during the menstrual cycle.MethodsThe number of EPCs in peripheral blood from subjects in the menstrual phase (n = 12), follicular phase (n = 8), and luteal phase (n = 16), was measured using flow cytometry. Peripheral blood mononuclear cells (PBMCs) were cultured for seven days with or without 17beta-estradiol (E2beta) or P4, followed by assessment of EPC proliferation based upon the uptake of acetylated low density lipoprotein (LDL) and lectin. The expression of estrogen receptor (ER) or progesterone receptor (PR) in EPCs was also evaluated using real-time PCR.ResultsE2beta and P4 significantly increased the proliferation of EPCs derived from the peripheral blood of subjects in menstrual phase, but not subjects in the luteal phase. In addition, the expression level of ERalpha was markedly higher than ERbeta in EPCs derived from women in menstrual phase.ConclusionsEPC proliferation is induced during the menstrual phase and proliferation can be affected by estrogen through ERalpha activation.

CONCENTRATIONS OF SERUM GRANULOCYTE- COLONY-STIMULATING FACTOR IN NORMAL PREGNANCY AND PREECLAMPSIA

OBJECTIVE In order to elucidate the potential role of granulocyte-colony-stimulating factor (G-CSF) during the course of normal pregnancy and preeclampsia, we measured the serum concentrations of G-CSF in both normal pregnant women and preeclamptic patients. METHODS Sera of 10 nonpregnant women, 34 normal pregnant women (n = 10, first trimester; n = 10, second trimester; n = 14, third trimester), 10 postpartum women, 10 mild preeclamptic patients, and 10 severe preeclamptic patients were collected. The serum concentrations of G-CSF were measured by enzyme immunoassay. RESULTS The serum level of G-CSF in normal pregnant women (third trimester: 38.3 +/- 15.3 pg/mL; mean +/- SD) was significantly increased when compared with the levels observed in nonpregnant women (20.3 +/- 10.1 pg/mL, p < 0.05), which was similar to the G-CSF concentrations in postpartum subjects (20.7 +/- 9.1 pg/mL). The mild and severe preeclamptic patients showed significantly higher levels of G-CSF (56.9 +/- 18.8 pg/mL, p < 0.05; 73.2 +/- 30.4 pg/mL, p < 0.0001, respectively) than those noted in the third trimester women. The preeclamptic patients who presented with edema had greater (p < 0.01) serum levels of G-CSF (75.5 +/- 25.0 pg/mL) compared with nonedematous patients (44.7 +/- 14.9 pg/mL). The serum levels of G-CSF significantly correlated with both weight gain (p < 0.05), diastolic blood pressure (p < 0.01), and systolic blood pressure (p < 0.01), but not with white blood cell counts. CONCLUSION Serum concentrations of G-CSF are increased in normal and, even more so, in preeclamptic pregnancies. Because there was no relationship between serum G-CSF concentration and the number of leukocytes, G-CSF might act not to promote the physiological leukocytosis of pregnancy, but to stimulate the function of leukocytes such as phagocytosis. Moreover, it might be that G-CSF plays important roles in the activation of granulocytes or vascular endothelial injury, which are considered to be important pathological conditions in the development of preeclampsia.

A clomiphene citrate and tamoxifen citrate combination therapy: a novel therapy for ovulation induction

OBJECTIVE To assess the efficacy of a clomiphene citrate (CC) and tamoxifen citrate (CC/tamoxifen) combination therapy in ovulation induction by comparing with a CC alone therapy. DESIGN A randomized cross-over study with CC alone and CC/tamoxifen combination therapies for 20 normoprolactinemic anovulatory women. Randomly selected 10 of the 20 women (group A) underwent a CC alone therapy (100 mg CC during cycle days 5 to 9) for the initial three consecutive treatment cycles and a CC/tamoxifen combination therapy (50 mg CC and 20 mg tamoxifen during cycle days 5 to 9) for the subsequent three consecutive treatment cycles. The remaining 10 (group B) were treated similarly but with the inverse sequence. Ovulation was documented when a high BBT phase persisted for 11 days or more, when midluteal serum P levels exceeded 7 ng/mL (22.3 nmol/L), or when pregnancy ensued. RESULTS The rates of ovulatory/treated, pregnant/treated, and pregnant/ovulatory cycles were 42 of 56 (75.0%), 3 of 47 (6.4%), and 3 of 35 (8.6%) for CC/tamoxifen, respectively. Those were 25 of 57 (43.9%), 1 of 48 (2.1%), and 1 of 21 (4.8%) for CC, respectively. Clomiphene citrate/tamoxifen was more effective in ovulation induction than CC. All the pregnancies were normal and single. None of the treatments was accompanied by any remarkable side effects. CONCLUSION Clomiphene citrate/tamoxifen is a novel treatment modality of ovulation induction with high efficacy.

Changes in serum concentrations of tumor necrosis factor α and adhesion molecules in normal pregnant women and those with pregnancy-induced hypertension

Aim:  To study whether serum tumor necrosis factor α gene (TNFα) and adhesion molecule levels are indicators of the onset of pregnancy‐induced hypertension (PIH), we compared levels of these molecules between normal pregnant women and PIH patients from the first to the third trimester.

Elevated Levels of Adhesion Molecules Derived from Leukocytes and Endothelial Cells in Patients with Pregnancy‐Induced Hypertension

Objective. Our purpose was to elucidate the role of adhesion molecules in the pathogenesis of pregnancy‐induced hypertension (PIH). Methods. Sera, peripheral lymphocytes, and polymorphonuclear cells (PMN) from PIH patients, normal pregnant women, and nonpregnant women were collected. Soluble intercellular adhesion molecule‐1 (sICAM‐1) in sera was measured by ELISA. ICAM‐1 expression on endothelial cells (EC) incubated with sera was analyzed by flow cytometry and RT‐PCR. CD11a, CD11b, and CD18 expression on lymphocytes and PMN were also measured by flow cytometory. Results. CD11a and CD18 expression levels on PMN and lymphocytes of PIH patients were significantly higher than those of normal pregnant women (p<0.05). The expression of CD11b was significantly increased in normal pregnancy compared with that in nonpregnant women (p<0.05). Serum sICAM‐1 in PIH patients was higher than that in normal pregnant women (p<0.05). ICAM‐1 expression level on EC incubated with PIH serum for 24 hr was significantly higher than that with normal pregnant serum (p<0.0005). ICAM‐1 mRNA expression after 12‐hr incubation with PIH serum was also significantly increased compared with serum from normal pregnant women (p<0.05). Conclusion. Adhesion molecules may play an important role in the pathogenesis of PIH.

Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy.

The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging.

Cytokine Regulation of Intercellular Adhesion Molecule-1 Expression on Trophoblasts in Preeclampsia

In normal pregnancy, trophoblast (TR) invasion plays a crucial role in remodeling the spiral arteries to develop uteroplacental circulation. Disruption of this invasion is associated with deficient uteroplacental circulation, which can lead to the development of preeclampsia (PE) through abnormal expression of adhesion molecules in the placenta and high serum causative factors such as cytokines. We aimed to evaluate whether serum factors in PE influence intercellular adhesion molecule-1 (ICAM-1) expression of TRs. ICAM-1 expression of TRs was measured using flow cytometry and immunohistochemistry was performed to examine the localization of tumor necrosis factor-α (TNFα) and ICAM-1 in placentas derived from women with normal pregnancies and women with PE. Sera from PE patients significantly increased ICAM-1 expression on TRs compared to sera from normal pregnant women; this increase was blocked with an antibody to TNFα. TNFα also enhanced ICAM-1 expression on TRs through nuclear factor-ĸB activation. We conclude that ICAM-1 expressed on TRs is involved in PE pathogenesis and is regulated by cytokines.