Akihide Ohkuchi

Human Villous Trophoblasts Express and Secrete Placenta-Specific MicroRNAs into Maternal Circulation via Exosomes

In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A. Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.

Guidelines for obstetrical practice in Japan: Japan Society of Obstetrics and Gynecology (JSOG) and Japan Association of Obstetricians and Gynecologists (JAOG) 2011 edition

Clinical guidelines for obstetrical practice were first published by the Japan Society of Obstetrics and Gynecology (JSOG) and the Japan Association of Obstetricians and Gynecologists (JAOG) in 2008, and a revised version was published in 2011. The aims of this publication include the determination of current standard care practices for pregnant women in Japan, the widespread use of standard care practices, the enhancement of safety in obstetrical practice, the reduction in burdens associated with medico‐legal and medico‐economical problems, and a better understanding between pregnant women and maternity‐service providers. These guidelines include a total of 87 Clinical Questions followed by several Answers (CQ&A), a Discussion, a List of References, and some Tables and Figures covering common problems and questions encountered in obstetrical practice. Each answer with a recommendation level of A, B or C has been prepared based principally on ‘evidence’ or a consensus among Japanese obstetricians in situations where ‘evidence’ is weak or lacking. Answers with a recommendation level of A or B represent current standard care practices in Japan. All 87 CQ&A are presented herein to promote a better understanding of the current standard care practices for pregnant women in Japan.

Hydroxysteroid (17-β) Dehydrogenase 1 Is Dysregulated by Mir-210 and Mir-518c That Are Aberrantly Expressed in Preeclamptic Placentas: A Novel Marker for Predicting Preeclampsia

In this study, to search for novel preeclampsia (PE) biomarkers, we focused on microRNA expression and function in the human placenta complicated with PE. By comprehensive analyses of microRNA expression, we identified 22 microRNAs significantly upregulated in preeclamptic placentas, 5 of which were predicted in silico to commonly target the mRNA encoding hydroxysteroid (17-&bgr;) dehydrogenase 1 (HSD17B1), a steroidogenetic enzyme expressed predominantly in the placenta. In vivo HSD17B1 expression, at both the mRNA and protein levels, was significantly decreased in preeclamptic placentas. Of these microRNAs, miR-210 and miR-518c were experimentally validated to target HSD17B1 by luciferase assay, real-time PCR, and ELISA. Furthermore, we found that plasma HSD17B1 protein levels in preeclamptic pregnant women reflected the decrease of its placental expression. Moreover, a prospective cohort study of plasma HSD17B1 revealed a significant reduction of plasma HSD17B1 levels in pregnant women at 20 to 23 and 27 to 30 weeks of gestation before PE onset compared with those with normal pregnancies. The sensitivities/specificities for predicting PE at 20 to 23 and 27 to 30 weeks of gestation were 0.75/0.67 (cutoff value=21.9 ng/mL) and 0.88/0.51 (cutoff value=30.5 ng/mL), and the odds ratios were 6.09 (95% CI: 2.35–15.77) and 7.83 (95% CI: 1.70–36.14), respectively. We conclude that HSD17B1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placenta and that reducing plasma level of HSD17B1 precedes the onset of PE and is a potential prognostic factor for PE.

Alterations in Placental Growth Factor Levels before and after the Onset of Preeclampsia Are More Pronounced in Women with Early Onset Severe Preeclampsia

It has been established that the serum placental growth factor (PlGF) decreases and the soluble fms-like tyrosine kinase-1 (sFlt-1) increases in women with preeclampsia. However, there have been no studies on the relation between preeclampsia onset time and the changes in PlGF and sFlt-1. Furthermore, the PlGF and sFlt-1 levels have not been evaluated using their reference values specific to each gestational age. In this study we reevaluated the serum PlGF and sFlt-1 levels before and after the clinical manifestation of early and late onset severe preeclampsia using the new reference values developed in our recent longitudinal study. Blood specimens were obtained immediately after the clinical manifestation of severe preeclampsia in 34 referred women, and both before and after the clinical manifestation in 8 women receiving a routine checkup at our institute. Both women with early and those with late preeclampsia showed decreased PlGF and increased sFlt-1 levels compared to normotensive controls at 28 and 37 weeks (n=68). However, those with early onset preeclampsia had a higher incidence of low PlGF (<5th percentile on the reference values) and high sFlt-1 (≥95th percentile) than those with late onset (low PlGF: 93% vs. 55%; high sFlt-1: 100% vs. 60%). log10PlGF (r=0.574, p<0.001) and log10(sFlt-1/PlGF) (r=−0.556, p<0.001) were correlated with the week of onset of preeclampsia. Before the onset of preeclampsia, the incidence rate of low PlGF in the women with early onset preeclampsia was 100% (5/5), whereas that in the women with late onset preeclampsia was 0% (0/2) (p=0.048). Therefore, alterations in the PlGF levels both before and after the onset of preeclampsia may be more pronounced in women with early onset than those with late onset severe preeclampsia.

Effect of maternal age on blood loss during parturition: a retrospective multivariate analysis of 10,053 cases.

Abstract Objective: An extensive study as to whether maternal age itself is a risk factor for blood loss during parturition. Method: A total of 10,053 consecutive women who delivered a singleton infant were studied.The excess blood loss was defined separately for women with vaginal and cesarean deliveries as ≥= 90th centile value for each delivery mode. The effects of 13 potential risk factors on blood loss were analyzed using multivariate analysis. Results: The 90th centile value of blood loss was 615 ml and 1,531 ml for women with vaginal and cesarean deliveries, respectively. A low lying placenta (odds ratio[OR] , 4.4), previous cesarean (3.1), operative delivery (2.6), leiomyoma (1.9), primiparity (1.6), and maternal age ≥= 35 years (1.5) were significant independent risk factors for excess blood loss in women with vaginal delivery. Placenta previa (6.3), leiomyoma (3.6), low lying placenta (3.3), and maternal age ≥= 35 years (1.8) were significant independent risk factors for excess blood loss in women with cesarean sections. Conclusion: A maternal age of ≥= 35 years was an independent risk factor for excess blood loss irrespective of the mode of delivery, even after adjusting for agerelated complications such as leiomyoma, placenta previa, and low lying placenta.

Evaluation of a new and automated electrochemiluminescence immunoassay for plasma sFlt-1 and PlGF levels in women with preeclampsia.

The first commercial automated immunoassays specific for soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) (Elecsys sFlt-1 and Elecsys PlGF, respectively) have recently been introduced. We constructed reference range values of plasma levels of sFlt-1 and PlGF, and the sFlt-1/PlGF ratio using Elecsys sFlt-1 and Elecsys PlGF during the second half of pregnancy and evaluated their sensitivity and specificity for the diagnosis of preeclampsia. Plasma samples were collected from 144 normal pregnant women at 19–25, 27–31 and 34–38 weeks of gestation and from 34 women with preeclampsia. The most appropriate reference range curves for plasma levels of sFlt-1 and PlGF, and the sFlt-1/PlGF ratio are presented as quadratic curves after logarithmic transformation. The sFlt-1/PlGF ratio showed the best diagnostic power for both early-onset and late-onset preeclampsia. In addition, a cutoff value of 45 for the sFlt-1/PlGF ratio resulted in the best sensitivity and specificity for the diagnosis of all preeclampsia (97 and 95%, respectively), and for the diagnosis of early-onset preeclampsia (100 and 95%, respectively). Using another 50 pairs of serum and plasma samples, including those from normal pregnant women and preeclamptic women, the plasma recovery rates of sFlt-1 and PlGF were 0.89 and 0.85, respectively; the correlation determinations between serum and plasma samples were 0.999 for sFlt-1, 0.990 for PlGF and 0.987 for sFlt-1/PlGF ratio. In conclusion, measurement of the plasma sFlt-1/PlGF ratio determined by Elecsys sFlt-1 and Elecsys PlGF and using a cutoff value of 45 might assist in the diagnosis of preeclampsia, especially for early-onset preeclampsia.

Effect of Recombinant Placental Growth Factor 2 on Hypertension Induced by Full-Length Mouse Soluble fms-Like Tyrosine Kinase 1 Adenoviral Vector in Pregnant Mice

The first aim of our study was to develop a pregnant mouse model for preeclampsia using adenoviral vector containing mouse full-length soluble fms-like tyrosine kinase 1 (sFlt-1) but not truncated sFlt-1. The second aim was to evaluate effects of recombinant mouse (rm) vascular endothelial growth factor (VEGF) and rm placental growth factor (PlGF) on a preeclampsia model induced by adenoviral vector containing mouse full-length sFlt-1. We injected adenoviral vector containing mouse full-length sFlt-1 on day 8.5 or 9.5 of gestation into pregnant Institute of Cancer Research mice, resulting in hypertension, proteinuria, and similar glomerular histological changes as those seen in human preeclamptic women with glomerular endotheliosis on day 16.5 or 17.5 of gestation. The preeclampsia models were treated with 100 &mgr;g/kg of rmVEGF164 (n=5), 100 &mgr;g/kg of rmPlGF-2 (n=5), or vehicle (n=7) twice a day for 2 days IP. The rmVEGF164 treatment significantly decreased the mean blood pressure on day 16.5 or 17.5 of gestation compared with the vehicle treatment (85±4 versus 97±2 mm Hg; P=0.018). The rmPlGF-2 treatment also significantly decreased the mean blood pressure on day 16.5 or 17.5 of gestation compared with the vehicle treatment (86±3 versus 97±2 mm Hg; P=0.018). However, proteinuria was not affected by either rmVEGF164 or rmPlGF-2. In conclusion, we, for the first time, created a mouse preeclampsia model using mouse full-length sFlt-1. VEGF and PlGF may be promising for ameliorating hypertension in women with preeclampsia. Additional study of PlGF as a potential drug for preeclampsia is warranted.

Uterine compression sutures for postpartum hemorrhage: an overview

In 1997, B‐Lynch pioneered the use of uterine compression sutures for postpartum hemorrhage. Since then, some researchers, including ourselves, have devised various uterine compression sutures. High‐level evidence has not been demonstrated as to whether compression sutures achieve better and safer hemostasis for postpartum hemorrhage than other methods, and, if they do, whether one suture is more efficient and safer than another. However, generally speaking, uterine compression sutures have achieved hemostasis while preserving fertility in many women and thus their efficacy and safety have been time‐tested. Each suture has both merits and drawbacks: obstetricians must be aware of the fundamental characteristics of various sutures. In this review, we summarize the technical procedures, efficacy, safety and complications of various uterine compression sutures. We add our own experiences and opinions where necessary.

Important surgical measures and techniques at cesarean hysterectomy for placenta previa accreta

For cesarean hysterectomy with placenta previa accreta, “universally achievable” measures are required. We propose eight measures: (i) placement of intra‐iliac arterial occlusion balloon catheters; (ii) placement of ureter stents; (iii) “holding the cervix” to identify the site to be transected; (iv) uterine fundal incision; (v) avoidance of uterotonics; (vi) “M cross double ligation” for ligating the ovarian ligament; (vii) “filling the bladder” to identify the bladder separation site and “opening the bladder” for placenta previa accreta with bladder invasion; and (viii) to continue to clamp the medial side of the parametrium or the cervix or employment of the “double edge pick‐up” to ligate it. These eight measures are simple, easy, effective, and thus “universally achievable”.

Normal and High-Normal Blood Pressures, but Not Body Mass Index, Are Risk Factors for the Subsequent Occurrence of Both Preeclampsia and Gestational Hypertension: A Retrospective Cohort Study

Blood pressure (BP) levels and body mass index (BMI) are known as risk factors for preeclampsia and gestational hypertension. However, there have been few investigations regarding the effects of BP and BMI levels on preeclampsia and gestational hypertension in the same cohort. In the present study, we conducted a retrospective cohort study using multiple logistic regression analysis. The cohort included 1,518 patients without nephritis. The unadjusted odds ratios (ORs) of preeclampsia and gestational hypertension were increased in pregnant women with normal BP (120–129 mmHg systolic or 80–84 mmHg diastolic), high-normal BP and hypertension in the second trimester compared to those with optimal BP. The unadjusted ORs of preeclampsia and gestational hypertension were also increased in obese women in the pre-pregnancy period compared to women with normal range BMI. When adjustment was made for both the BP levels and pre-pregnancy BMI levels, the ORs (95% confidence intervals) of normal BP, high-normal BP, hypertension and obesity for the subsequent occurrence of preeclampsia were 5.1 (2.2–12), 8.3 (3.1–22), 16 (5.0–50) and 2.0 (0.67–5.9), and those for the subsequent occurrence of gestational hypertension were 7.0 (2.6–19), 7.4 (2.1–25), 22 (6.1–83) and 1.3 (0.33–4.8), respectively. For the subsequent occurrence of preeclampsia or gestational hypertension, normal BP, high-normal BP and hypertension in the second trimester may be independent risk factors. Obesity in the pre-pregnancy period, however, may not be an independent risk factor.