Shuichiro Yoshimura

Detection of cell free placental DNA in maternal plasma: direct evidence from three cases of confined placental mosaicism

Fetal cells are consistently found in the maternal circulation, and polymerase chain reaction based studies have led to the identification of cell free fetal DNA (fetal DNA) in maternal blood. Approximately 1.2 nucleated fetal cells/ml of whole blood from women carrying a male fetus were detectable,1 and relative enrichment of fetal DNA was detected in the maternal plasma and serum.2 The amount of fetal DNA in the maternal blood increases with progression of pregnancy, and 3.4–6.2% of the total maternal plasma DNA during pregnancy was of fetal origin.3 Therefore, cell free fetal DNA in pregnant women’s plasma is useful for non-invasive prenatal diagnosis, especially for detection of fetal sex,3,4 RhD blood type,5–7 and gene mutations of paternal origin.8–10 Previous studies indicated that pregnant women with pre-eclampsia,11 placenta previa12 and fetal chromosome abnormalities13 tend to have elevated levels of fetal DNA in their plasma. Since functional or structural abnormalities of the placenta and destruction of the trophoblast may be associated with these diseases,14 it is suggested that cell free fetal DNA is of placental origin. This implies that quantitative analysis of fetal DNA may be valuable to screen for placental dysfunction. Ng et al15 recently reported that placental mRNA is present in the maternal circulation, and suggested that the same might occur for placental DNA,16 However, no direct evidence has been given for placenta derived cell free fetal DNA in the maternal blood, although its clinical use is growing.17 Confined placental mosaicism, which is defined by the presence of abnormal karyotypes only in the placenta while the fetus itself is usually diploid,18 may occur through a loss of the extra chromosome in a trisomic zygote during an early mitotic cell division in only the …

Ultrasonographic prediction of lethal pulmonary hypoplasia: Comparison of eight different ultrasonographic parameters☆☆☆★

OBJECTIVE The aim of this study was to determine the usefulness of eight different ultrasonographic fetal parameters for predicting fetal pulmonary hypoplasia. STUDY DESIGN Nomograms of eight different ultrasonographic fetal parameters were evaluated by studying uncomplicated single fetus pregnancies with well-established dates between 18 and 40 weeks of gestation. The eight parameters, which could reflect fetal lung mass, were as follows: thoracic circumference, thoracic area, thoracic area minus heart area, lung area, thoracic circumference/abdominal circumference ratio, thoracic area/heart area ratio, thoracic area minus heart area/thoracic area ratio and lung area/thoracic area ratio. The relative efficacy of the eight parameters was determined by studying 21 fetuses at high risk for development of lethal pulmonary hypoplasia and 30 fetuses with premature rupture of membranes within 1 week. RESULTS The lung area (gestational age-dependent parameter) and the thoracic circumference/abdominal circumference (gestational age-independent parameter) ratio had the best diagnostic accuracy (sensitivity 81.3% and 90.5%, specificity 100% and 90.0%, positive predictive value 100% and 86.4%, negative predictive value 90.9% and 93.1%, respectively). There were significant linear relationships between lung weight and lung area and between the lung weight/body weight ratio and the thoracic circumference/abdominal circumference ratio. CONCLUSION These data suggested that the application of lung area and the thoracic circumference/abdominal circumference ratio are clinically useful for the evaluation of fetal pulmonary hypoplasia.

Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid

AbstractCell-free fetal DNA (cffDNA) in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis. For rapid prenatal diagnosis of frequent chromosome abnormalities, for example trisomies 13, 18, and 21, and monosomy X, using cffDNA, we have developed a targeted microarray-based comparative genomic hybridization (CGH) panel on which BAC clones from chromosomes 13, 18, 21, X, and Y were spotted. Microarray-CGH analysis was performed for a total of 13 fetuses with congenital anomalies using cffDNA from their uncultured amniotic fluid. Microarray CGH with cffDNA led to successful molecular karyotyping for 12 of 13 fetuses within 5 days. Karyotypes of the 12 fetuses (one case of trisomy 13, two of trisomy 18, two of trisomy 21, one of monosomy X, and six of normal karyotype) were later confirmed by conventional chromosome analysis using cultured amniocytes. The one fetus whose molecular-karyotype was indicated as normal by microarray CGH actually had a balanced translocation, 45,XY,der(14;21)(q10;q10). The results indicated that microarray CGH with cffDNA is a useful rapid prenatal diagnostic method at late gestation for chromosome abnormalities with copy-number changes, especially when combined with conventional karyotyping of cultured amniocytes.

Diagnosis of fetal pulmonary hypoplasia by measurement of blood flow velocity waveforms of pulmonary arteries with Doppler ultrasonography.

OBJECTIVE The aim of the study was to determine the utility of ultrasonographically recorded blood flow waveforms of the pulmonary artery in the diagnosis of pulmonary hypoplasia. STUDY DESIGN The normal values of the pulsatility index and peak systolic flow of pulmonary arterial blood velocity waveforms were determined in 300 uncomplicated single fetus pregnancies with well-established gestational ages between 24 and 40 weeks. We also measured the same parameters in 5 fetuses at high risk for development of severe pulmonary hypoplasia. We also determined the radial alveolar count and microvessel density, representing the extent of angiogenesis, in tissue specimens collected at autopsy from normal and hypoplastic lungs and stained both with hematoxylin and eosin and immunohistochemically for factor VIII. RESULTS In healthy fetuses the pulsatility index values of both the right and left pulmonary arteries diminished significantly with advancing gestation, whereas the peak systolic velocity increased significantly with advancing gestation. In fetuses with pulmonary hypoplasia pulsatility index values were high and the peak systolic flow was significantly lower than in healthy fetuses. Histologic examination showed a lower radial alveolar count and poorer angiogenesis in fetuses with pulmonary hypoplasia than in healthy fetuses. CONCLUSION Doppler ultrasonographic determination of pulmonary artery blood velocity waveforms is a useful tool for the diagnosis of pulmonary hypoplasia.

Clinical application of fetal sex determination using cell-free fetal DNA in pregnant carriers of X-linked genetic disorders

As the first step in prenatal diagnosis of X-linked genetic disorders, chorionic villus sampling (CVS) for fetal sex determination is generally performed at 11–13 weeks of gestation. However, as the procedure-related miscarriage rate of CVS is 0.5–1.0%, non-invasive methods such as PCR of cell-free fetal DNA (cff-DNA) in maternal plasma are preferable. Here, we determined fetal sex at 9–12 weeks of gestation using PCR of cff-DNA in three pregnant carriers of Duchenne muscular dystrophy. The fetal sex was accurately determined in all three cases, as confirmed by ultrasound and amniocentesis at 16 weeks (for the two female fetuses) and CVS at 12 weeks (for the one male fetus). This procedure could avoid unnecessary CVS in female fetuses.

Methylation imprinting of H19 and SNRPN genes in human benign ovarian teratomas.

In humans, studies of female germ cells are very limited by ethics. The current study investigated the usefulness of benign ovarian teratomas as a substitute for ova in analyses of imprinted genes. Twenty-five human benign ovarian teratomas were typed with 45 microsatellite DNA markers and classified according to their genotypic features. Two oppositely imprinted genes, H19 and SNRPN, were then chosen for analysis of their methylation states in these tumors. These analyses revealed that benign ovarian teratomas consist of a mixture of genetically and epigenetically heterogeneous cell populations. In contrast to previous reports, we could document only one case rising from germ cells by meiosis-II nondisjunction. H19 and SNRPN were methylated in individual teratomas to various degrees, ranging from normal somatic cell to expected ovum levels. The allele with residual methylation of H19 was consistent with that methylated in the patients blood DNA, thus being of paternal origin. Degrees of H19 hypomethylation and SNRPN hypermethylation increased as the cellular origin of the tumors advanced in oogenesis and were closely correlated in individual teratomas. These results could be best explained by the assumption that the primary imprinting is a progressively organized process and suggest that the establishment of primary imprints on different genes might be mechanistically linked, even when those genes are oppositely imprinted.

Clinical outcome of infants with confined placental mosaicism and intrauterine growth restriction of unknown cause.

The purpose of this study was to know a role of confined placental mosaicism (CPM) in perinatal outcome and postnatal growth and development of infants with intrauterine growth restriction (IUGR). We selected 50 infants with IUGR (<−2.0 SD) from 3,257 deliveries in a regional medical center during the past 10‐year period, and carried out cytogenetic and molecular analyses in their placenta and cord blood. Of the 50 infants, 8 had CPM (CPM group) and were composed of five single (CPM2, 7, 13, 22, and 22), one double (CPM7/13), and one quadruple trisomy (CPM2/7/15/20), and one partial monosomy [del(2)(p16)]. The origin of an extra chromosome of trisomy was maternal in six cases of CPM, paternal in one, and undetermined in one. Uniparental disomy in disomic cell lines was ruled out in all these mosaics. We also compared clinical parameters for perinatal outcome between CPM group and infants without evidence of CPM (non‐CPM group), such as maternal and gestational age, birth weight, Apgar score, cord blood pH, gender, and uterine artery patterns by Doppler ultrasonography, as well as weight, height, and developmental quotient (DQ) by Denver Developmental Screening Test at age 12 months. Phenotypic abnormalities were noted in two infants with CPM and three infants of non‐CPM group: One with CPM22 had ASD and hypospadias, one with CPM7/13 had Russell–Silver syndrome (RSS), and one without CPM had polydactyly, and two without CPM had RSS. All but one infant with CPM are alive at age 12 months. Among the clinical parameters, the detection rate of a notch waveform pattern of the uterine artery was significantly higher in the CPM group (P < 0.05). However, no significant difference was noted in perinatal outcome of pregnancy and in DQ at age 12 months between the two groups. Interestingly, short stature (<−2 SD) at age 12 months was more frequently seen in CPM group (7/8 infants with CPM vs. 8/15 infants without CPM), although no statistically significant difference was obtained. The information obtained will be useful for perinatal care and genetic counseling for infants with IUGR and CPM.

The effects of oligohydramnios and cervical cord transection on lung growth in experimental pulmonary hypoplasia in rabbits

OBJECTIVE Our goal was to examine the effects of oligohydramnios and cervical cord transection on fetal lung development, pulmonary surfactant, and lung mechanics in rabbits. STUDY DESIGN The amniotic fluid was shunted into the maternal abdominal cavity in a group of 12 fetal rabbits. In another group (n = 12) high cervical cord transection was performed at day 24 of gestation. Another 12 littermates not operated on served as the control group. Fetuses were delivered on day 30 of gestation by cesarean section and immediately put to death. The body weight and wet lung and liver weights were measured. To determine the extent of fetal lung growth, we measured the size of lung acini, the number of terminal airspaces, and the diameter of alveoli. We also measured the dynamic compliance of the lung, the concentration of phosphatidylcholine, and the lecithin/sphingomyelin ratio in lung lavage fluid at birth. RESULTS Amniotic fluid shunting produced a significant reduction in amniotic fluid volume. Amniotic shunt and cervical cord transection significantly decreased wet lung weight and fetal lung/body weight ratio compared with the control. The concentration of phosphatidylcholine and the lecithin/sphingomyelin ratio in lung fluid lavage from fetuses with amniotic shunt were significantly higher than the values for control and cord transection fetuses. Histopathologic examination of the lungs showed significant reductions in the size of acini, the number of terminal airspaces, and the diameter of alveoli in shunted and cord transection groups compared with the control group. The dynamic compliance of transection fetuses was significantly reduced compared with control and shunted fetuses. CONCLUSION Our results indicate that oligohydramnios or cervical cord transections cause pulmonary hypoplasia. However, oligohydramnios-induced pulmonary hypoplasia is associated with increased pulmonary surfactant compared with control and cord transection fetuses.

Fetal redistribution of blood flow and amniotic fluid volume in growth-retarded fetuses.

Our purpose was to assess the relationship between the fetal redistribution of blood flow and the amount of amniotic fluid in appropriate-for gestational-age fetuses and growth-retarded fetuses. Blood flow velocity waveforms of the umbilical artery, descending aorta, middle cerebral artery, renal artery and uterine artery were recorded using pulsed Doppler ultrasonography in 100 appropriate-for gestational age fetuses and 39 growth-retarded fetuses. The pulsatility index (PI) values and the amount of amniotic fluid were compared between the two groups. The PI values of the umbilical artery and renal artery were significantly higher in appropriate for gestational-age-fetuses with oligohydraminos than in fetuses with an adequate amount of amniotic fluid. The PI values of the umbilical artery and renal artery were significantly higher and the PI of the middle cerebral artery was significantly lower in growth-retarded fetuses with oligohydramnios than in fetuses with an adequate amount of amniotic fluid. Furthermore, there was a significant negative correlation between the PI value of the renal artery and the vertical diameter of amniotic fluid, and between the PI value of the renal artery and the amniotic fluid index. The PI value of the renal artery was related to the amount of amniotic fluid in growth-retarded fetuses, and the same relationship was demonstrated in appropriate-for gestational age fetuses.

Effect of epidermal growth factor on lung growth in experimental fetal pulmonary hypoplasia.

The purpose of this study was (1) to compare the expression of epithelial growth factor receptor (EGFR) in the lung tissues of human fetuses with or without pulmonary hypoplasia, and (2) to investigate the effects of EGF on lung growth in experimental pulmonary hypoplasia in rabbits. Firstly, we investigated the expression of EGFR in lung tissues of human fetuses with or without pulmonary hypoplasia by immunohistochemistry. Secondly, the amniotic fluid was shunted into the maternal abdominal cavity in a group of 12 fetal rabbits, another group (n = 12) received EGF injection (5 microg, i.p.) at day 25 of gestation. The third group (n = 12) was only treated with EGF while littermates not operated on served as the control group (n = 12). On day 29 of gestation, fetuses were delivered by Cesarean section and the lungs removed. The body weight and wet lung and liver weights were measured. As a measure of fetal lung growth, we determined the size of lung acini, the number of terminal airspaces, and diameter of alveoli (n = 6, each groups). We also measured the concentration of phosphatidylcholine (PC) and the lecithin/sphingomyelin (L/S) ratio in lung lavage fluid at birth in some fetuses (n = 6, each groups). In human fetuses with pulmonary hypoplasia, there was a significant decrease in radial alveolar count and expression of EGFR compared with fetuses without pulmonary hypoplasia. Amniotic shunt significantly decreased fetal lung/body weight ratio compared with control. Injection of EGF in the shunted group significantly increased lung/body weight ratio to the control level. The concentration of PC and L/S ratio in lung fluid lavage from rabbit fetuses with hypoplastic lungs was significantly higher than the other three groups. Histopathological examination of fetuses with hypoplastic lungs treated with EGF showed no significant change in the size of acini, number of terminal airspaces or the diameter of alveoli compared with the control group. Our results suggested that EGF was associated with lung growth and maturation of human lung and that treatment of rabbit fetuses with hypoplastic lungs with EGF facilitated lung growth and development.