Akihiko Sekizawa

Prenatal DNA diagnosis of a single-gene disorder from maternal plasma.

Achondroplasia is a short-limb disorder caused by a point mutation in a single gene. To diagnose such a disorder prenatally requires the use of invasive procedures such as amniocentesis. However, using PCR and restriction fragment length polymorphism analysis, we were able to detect the mutation in the plasma of a woman carrying a fetus suspected of having achondroplasia. The detection of a fetus-derived mutant gene from maternal plasma may therefore permit non-invasive prenatal diagnosis of single-gene disorders.

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.

Lower Rate of Preeclampsia After Antioxidant Supplementation in Pregnant Women with Low Antioxidant Status

Objective: To investigate maternal and neonatal outcomes after antioxidant supplementation relatively early in pregnancy (8 to 12 weeks) for pregnant women with low antioxidant status. Methods: A randomized, double-blind, placebo-controlled trial of daily antioxidant supplementation was performed on pregnant women screening positive for low antioxidant status at 8 to 12 weeks of gestation. Low antioxidant status was defined as a superoxidedismutase (SOD) level below 1102 U/g Hb or 164 U/mL. The supplementation group received the following antioxidants daily: vitamins A (1000 IU), B6 (2.2 mg), B12 (2.2 μg), C (200 mg), and E (400 IU), folic acid (400 μg), N-acetylcysteine (200 mg), Cu (2 mg), Zn (15 mg), Mn (0.5 mg), Fe (30 mg), calcium (800 mg), and selenium (100 μg). The control group received Fe (30 mg) and folic acid (400 μg). Maternal (preeclampsia, abortion, and hypertension) and perinatal outcomes were assessed. Results: In the supplementation group (29 subjects), we observed 2 cases of preeclampsia (6.8%, 1 mild and 1 severe), 1 of IUGR (birth weight 2300 g at 38 weeks), and 1 preterm delivery. In the control group (31 subjects), there were 8 abortions, 9 cases of preeclampsia (29%, 6 mild and 3 severe) with perinatal outcome: 3 preterm delivery cases and 1 IUGR (birth weight 2030 g at 39 weeks). Preeclampsia was significantly less frequent in the supplementation group when compared to the control group (2 vs. 9 cases, p = 0.043, OR = 0.18 [95% CI: 0.03, 0.92]). Finally we focused on the prediction of preeclampsia at 8 to 12 weeks. Combined sensitivity of markers of antioxidant status (SOD slutathione peroxidase, [GPx], and total anti-oxidant status [TAS]) was 33% (false-positive rate of 4.5%). Conclusion: Antioxidant supplementation was associated with better maternal and perinatal outcome in pregnant women with low antioxidant status than control supplementation with iron and folate alone. In a selected population already screened positive for low SOD, preeclampsia can be detected in 33% of asymptomatic cases in the first trimester using SOD, GPx, and TAS. It seems feasible that panels of both biochemical and molecular markers may be clinically useful in the prediction of this disease.

p53 mutations and overexpression affect prognosis of ovarian endometrioid cancer but not clear cell cancer

OBJECTIVE Although ovarian clear cell adenocarcinoma (OCCA) and ovarian endometrioid adenocarcinoma (EC) are considered to be closely related to endometriosis, the mechanisms of carcinogenesis of these two malignancies and malignant transformation of endometriosis are unclear. In this study, we examined the biology of OCCA and EC by performing large-scale analysis of K-ras activation and p53 mutation and overexpression in these malignancies. The results were subsequently analyzed for correlation with the clinicopathologic data. METHODS In the present study of OCCA and EC, we obtained clinicopathological data and analyzed frequency of mutations and overexpression of K-ras and p53. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and target sequences were amplified in vitro by polymerase chain reaction. The DNA was analyzed for K-ras and p53 mutations by testing for single-strand conformation polymorphisms and by direct sequencing. Immunohistochemical staining was performed using p53 monoclonal antibody. Univariate analysis was performed using the Kaplan-Meier algorithm, and differences in survival were analyzed using the log rank test. The prognostic significance of the studied variables for survival was assessed using multivariate analysis with Cox regression analysis. RESULTS K-ras mutation was detected in 16.2% (6/37) of OCCA patients and 3.7% (1/27) of EC patients. No evidence of p53 mutation was detected in OCCA patients, but p53 mutation was detected in 63.0% of EC patients; these findings are consistent with the results of p53 immunohistochemistry. No statistical significance was observed for K-ras mutation in OCCA or EC. In EC patients, the absence of endometriosis and p53 overexpression was associated with a poorer survival. In OCCA patients tubulocystic and papillary histotype as well as stage II correlated with a worse survival. CONCLUSIONS p53 mutation, which was found in 63% of EC tumors, is an independent prognostic factor for EC patients. However, no p53 mutation was found in OCCA tumors. K-ras mutations did not affect survival of OCCA or EC patients.

Genetics of endometrial cancers.

Endometrial cancers exhibit a different mechanism of tumorigenesis and progression depending on histopathological and clinical types. The most frequently altered gene in estrogen-dependent endometrioid endometrial carcinoma tumors is PTEN. Microsatellite instability is another important genetic event in this type of tumor. In contrast, p53 mutations or Her2/neu overexpression are more frequent in non-endometrioid tumors. On the other hand, it is possible that the clear cell type may arise from a unique pathway which appears similar to the ovarian clear cell carcinoma. K-ras mutations are detected in approximately 15%–30% of endometrioid carcinomas, are unrelated to the existence of endometrial hyperplasia. A β-catenin mutation was detected in about 20% of endometrioid carcinomas, but is rare in serous carcinoma. Telomere shortening is another important type of genomic instability observed in endometrial cancer. Only non-endometrioid endometrial carcinoma tumors were significantly associated with critical telomere shortening in the adjacent morphologically normal epithelium. Lynch syndrome, which is an autosomal dominantly inherited disorder of cancer susceptibility and is characterized by a MSH2/MSH6 protein complex deficiency, is associated with the development of non-endometrioid carcinomas.

PP13 mRNA Expression in Trophoblasts From Preeclamptic Placentas

To assess the role of placental protein 13 (PP13) in the pathogenesis of preeclampsia, the messenger RNA expression of PP13 was investigated in the trophoblasts from first and third trimester placenta. First, syncytiotrophoblasts and extravillous trophoblasts were separated from frozen section of third trimester placenta obtained from pregnant women with and without preeclampsia by laser microdissection. The PP13 levels of syncytiotrophoblasts were significantly higher than those of extravillous trophoblasts. The expression levels in syncytiotrophoblasts from the preeclampsia were significantly lower than those from normal. Next, the PP13 expression was assessed in trophoblasts from residual samples of chorionic villus sampling at 11 weeks of gestation, who subsequently developed preeclampsia. The levels in the preeclampsia group were significantly lower than those of normal cases. These findings indicate that an alteration in the PP13-messenger RNA expression in the trophoblasts may be associated with the pathogenesis of preeclampsia.

Limitations of conservative treatment for repeat Cesarean scar pregnancy

delineate the level of fusion, connecting vessels and fetal contour (Figure 3). One week later, repeat scans by 3D transvaginal ultrasonography confirmed the diagnosis of conjoined twins. After counseling, the patient opted to terminate the pregnancy. After evacuation, two separate bodies and two separate upper and lower extremities were noted. The location of the conjoined site could not be identified on gross inspection of the abortus because the embryos had been destroyed during the evacuation procedure. Early diagnosis of conjoined twins is crucial for determining subsequent management and possibly decreasing maternal morbidity (evacuation vs. hysterotomy). Specific sonographic findings of conjoined twins examined during the first trimester include inseparable fetal bodies despite manipulation of the uterus with a transvaginal probe or prolonged continuous scanning, bifid appearance of the embryo, single yolk sac3, and a single umbilical cord with more than three vessels. Recently, some reports4–8 have described early diagnosis of conjoined twins by 3D ultrasound imaging combined with power Doppler, computed tomography and magnetic resonance imaging. In this case, prenatal diagnosis of conjoined twins was established by transvaginal 2D ultrasonography and power Doppler. However, 3D imaging with surfacerendering provided clearer images of the characteristic features of ischiopagus twins and helped the parents to understand the complex anomalies present in their fetuses. Furthermore, it improved our diagnostic confidence to provide adequate early intervention.

K-ras mutation may promote carcinogenesis of endometriosis leading to ovarian clear cell carcinoma

Endometriosis shares some features characteristic of malignancy; however, it remains unclear whether endometriosis is a precursor to malignant disease. The objective is to determine the genetic relationship between endometriosis and ovarian clear cell carcinoma (OCCA). Among 37 Japanese patients with OCCA who underwent primary surgery at Showa University Hospital between 1987 and 1999, K-ras mutations were detected in 6. Three of these patients had ectopic endometrial tissue adjacent to the site of carcinoma. These cases demonstrated areas of endometriosis and areas of OCCA bordered by atypical endometriosis. We retrieved cells from regions of endometriosis and atypical endometriosis, as well as OCCA cells, by laser microdissection in each case. K-ras mutations were analyzed in each specimen dissected. DNA analysis of each region revealed that K-ras mutations were detectable in OCCA but not in endometriosis or atypical endometriosis. It is thought that a number of genetic alterations are involved in malignant transformation. It is possible that K-ras mutations are associated with malignant transformation of atypical endometriosis into OCCA, although further research is needed to define this mechanism.

Prediction of preeclampsia by analysis of cell-free messenger RNA in maternal plasma

OBJECTIVE The purpose of this study was to predict the occurrence of preeclampsia in a series of patients at gestational week 15-20 weeks, with the use of a panel of messenger RNA markers. STUDY DESIGN Data from 62 patients with preeclampsia who were asymptomatic at the time of blood testing and 310 control subjects were analyzed. Multivariable analysis was performed with discriminant analysis. RESULTS Univariable analysis identified vascular endothelial growth factor receptor 1 as the marker with the highest detection rate; placenta-specific 1 with the lowest. Mean estimated score for preeclampsia was 9.4 for control subjects and 72.5 for subjects who experienced preeclampsia. A receiver operating characteristic curve that was obtained with the estimated score for preeclampsia as a test variable yielded a detection rate of 84% (95% CI, 71.8-91.5) at a 5% false-positive rate with an area under the curve of 0.927 (P < .001). Again, detection rate and score for each patient for classification as preeclamptic correlated with severity. CONCLUSION A panel of messenger RNA is able to detect subjects who will experience preeclampsia.

Prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from maternal blood

Abstract We have developed a method that allows the prenatal DNA diagnosis of ornithine transcarbamylase (OTC) deficiency by using a single fetal nucleated erythrocyte (NRBC) isolated from maternal blood. OTC gene analysis of a male patient (TF) with early onset OTC deficiency was performed by single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. To investigate the possible prenatal diagnosis of OTC deficiency, maternal blood was obtained at 13 weeks of gestation of a subsequent pregnancy, from the mother of patient TF. NRBCs in the maternal blood were separated by using the density gradient method and then collected with a micromanipulator. The entire genome of a single NRBC was amplified by primer extension preamplification (PEP). The human leukocyte antigen (HLA)-DQ alpha genotype and sex were determined from small aliquots of the PEP product. The HLA-DQ alpha genotype of each of the parents of the male patient was also determined. Once a single NRBC had been identified as being of fetal origin, the OTC gene was analyzed by using the restriction fragment length polymorphism (RFLP) method. DNA analysis revealed a point mutation in exon 9 of the OTC gene in the OTC-deficient patient (TF). All NRBCs retrieved from maternal blood were successfully identified as being of fetal origin by HLA-DQ alpha genotyping and sex determination. RFLP analysis demonstrated that the fetal OTC gene was normal. This is the first study to successfully diagnose OTC deficiency prenatally, by using a single fetal NRBC from the maternal circulation. Such prenatal DNA diagnosis is non-invasive and can be applied to other genetic diseases, including autosomal and X-linked diseases.