Takumi Yanaihara

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.

Plasma levels of atrial natriuretic peptide during normal pregnancy and in pregnancy complicated by hypertension

To clarify a possible role for atrial natriuretic peptide in the pathophysiology of pregnancy complicated by hypertension, we studied plasma levels of atrial natriuretic peptide in 176 pregnant women with or without hypertension. Plasma atrial natriuretic peptide levels in normal pregnant women showed a gradual increase as pregnancy advanced, but the mean (+/- SD) concentrations in women in each trimester (34.8 +/- 14.7 pg/ml in the first trimester, n = 35; 38.7 +/- 12.2 pg/ml in the second trimester, n = 34; and 43.1 +/- 20.0 pg/ml in the third trimester, n = 71) did not differ statistically from the mean plasma atrial natriuretic peptide level in nonpregnant women (38.2 +/- 13.6 pg/ml, n = 44). In contrast, plasma atrial natriuretic peptide levels were elevated in 9 of the 12 women who had hypertension. The mean plasma atrial natriuretic peptide concentration in these patients (162 +/- 95.2 pg/ml) was significantly (p less than 0.01) higher than in normal pregnant women and in nonpregnant controls. On the other hand, 11 pregnant women with proteinuria or edema but without hypertension had normal plasma atrial natriuretic peptide levels. These results suggest that plasma atrial natriuretic peptide levels are normal in women during uncomplicated pregnancy, while the levels are elevated in pregnancy complicated by hypertension. Increased atrial natriuretic peptide secretion in the latter condition may reflect a mechanism of compensation that operates in response to water and sodium retention.

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.

Osteoblast cells (MG‐63 and HOS) have aromatase and 5α‐reductase activities

Both aromatase and 5α‐reductase activities were found by whole‐cell assay in osteoblast‐like cells, MG‐63 and HOS. Aromatase activity was measured by the [3H] water release method, and the formation of 5α‐androstanedione from androstenedione was expressed as 5α‐reductase activity. When CGS16949A, an inhibitor of aromatase, was added to the incubation medium at a concentration of 2 × 10‐9 M, sufficient to completely inhibit placental aromatase activity, only 63% to 68% inhibitions were observed. When progesterone, a competitive inhibitor of 5α‐reductase, was added at a concentration of 10‐5 M, 28% to 40% inhibitions were recorded. Because the release of [3H] from [1β‐3H] androstenedione into water by 5α‐reductase is reported, results from the present study suggest that the measurement of aromatase activity in osteoblasts by the [3H] water release method may overestimate aromatase activity owing to the inclusion of 5α‐reductase activity. The results also suggest that osteoblast cells may play an important role in bone metabolism by transforming androgens into estrogens and more biologically active androgen derivatives.

Behavioral and adrenocortical responses to stress in neonates and the stabilizing effects of maternal heartbeat on them

Adrenocortical response to stress in neonates was assessed by estimating salivary concentrations of cortisol, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEA-S). Behavioral responses to stress were also scored clinically, according to the method of Lewis and Thomas [15]. The modulation of these measures by hearing maternal heartbeat was also evaluated. Levels of cortisol and DHEA in saliva in neonates correlated well with those of serum showing significant variation in painful stress. Hearing the maternal heartbeat significantly reduced the grade of variation, but the recorded sound of the Japanese drum with the same rhythm showed no significant effect. Scores of the behavioral response to stress also decreased significantly in neonates who were presented with the maternal heartbeat in comparison with those hearing no sound or the beat of a Japanese drum.

Serum steroid levels in children at birth and in early neonatal period

During the neonatal period, adrenal steroidogenic function was assessed by determining the peripheral levels of eight steroids by radioimmunoassay with the use of specific antisera. Serum concentrations of cortisol and cortisone in the umbilical artery were 92.2 +/- 13.3 ng/ml (m +/- SE) and 78.0 +/- 11.1 ng/ml, respectively, and these levels decreased to 37.4 +/- 8.2 and 47.9 +/- 14.7 ng/ml on the first day after birth. Serum concentrations of progesterone, 17 alpha-hydroxyprogesterone, total pregnenolone, and total dehydroepiandrosterone in the umbilical artery were 294 +/- 41, 8.7 +/- 1.0, 611 +/- 86, and 867 +/- 179 ng/ml (mean +/- SE), respectively, and decreased immediately after birth. In contrast, serum levels of total 16 alpha-hydroxydehydroepiandrosterone in the umbilical artery were 3,415 +/- 416 ng/ml, and those levels were maintained on the first day of life, whereas serum levels of 16 alpha-hydroxypregnenolone in the umbilical artery were 667 +/- 173 ng/ml and increased to 5,180 +/- 728 ng/ml. The significance of these changes in adrenal steroids during the neonatal period is discussed in the text.

The effect of vaginal delivery on the pelvic floor

This study was undertaken to determine the effects of vaginal deliveries on anorectal function, and to analyze the possible clinical, physiological, and radiological risk factors predisposing to damage of the pelvic floor musculature. We studied 25 consecutive women with a mean age of 32 years old, 3 months after vaginal delivery, 17 of whom were primiparae and 8, multiparae. The symptoms of anal incontinence were assessed, and anorectal manometry, rectal sensation, and radiological measurements of the anorectal angle and pelvic floor position at rest, on squeezing, and on straining were performed. As a control, six nulliparous women underwent the same examinations. Pelvic floor descent in both the primiparae and multiparae was significantly greater at rest and on squeezing than that in the nulliparous women. Furthermore, pelvic floor descent on straining was greater in the multiparae than in the nulliparous women (P=0.028). An analysis of the 17 primiparae showed that prolonged duration of the second stage of labor and third-degree perineal tears were important factors predisposing to pelvic floor descent. In fact, 3 of the 17 primiparae (17%) had anal incontinence. These findings indicate that vaginal delivery may cause pelvic floor descent, an obtuse anorectal angle, and bowel symptoms.

Regulation of estrogen activity in human endometrium: Effect of IL-1β on steroid sulfatase activity in human endometrial stromal cells

We investigated the effect of interleukin 1beta (IL-1beta) on steroid sulfatase (STS) activity and the expression of STS mRNA in human endometrial stromal cells. Endometrial tissue samples were obtained from patients undergoing hysterectomy to remove uterine fibroids. Stromal cells were isolated from the tissue preparation and cultured. IL-lbeta (1 approximately 100 ng/ml) was added into the culture medium and incubated for 24 h. The expression of STS mRNA was measured by competitive RT-PCR. The addition of IL-lbeta at 10 and 100 ng/ml suppressed STS mRNA expression to 55.2 +/- 12.8% and 25.1 +/- 10.9%, respectively, of the control sample to which no IL-lbeta had been added. STS activity was measured by radiolabelled steroid metabolite using thin layer chromatography, and this activity was also significantly suppressed in response to the administration of IL-lbeta in a dose-dependent manner. When IL-1 receptor antagonist (IL-1ra) was added together with IL-1beta to the culture medium, mRNA expression and STS activity were recovered. The present study is the first to demonstrate IL-1beta regulation of STS activity locally in human endometrium. IL-1beta suppressed mRNA and activity of STS in stromal cell culture. This initial demonstration of IL-1beta regulation of STS implies that IL-1beta may control the steroid microenvironment in human uterine endometrium by reducing biologic action of estrogen.

Effect of lactoferrin on lipopolysaccharide (LPS) induced preterm delivery in mice

Background. The present study attempted to confirm the preventive effect of lactoferrin on lipopolysaccharide (LPS) induced preterm delivery in mice.

Localization and expression of steroid sulfatase in human fallopian tubes

Localization of steroid sulfatase, a membrane-bound microsomal enzyme, in human fallopian tubes was immunohistochemically investigated, and expression of RNA was confirmed by competitive RT-PCR. Human fallopian tubes were obtained from 10 patients in follicular and early luteal phases during gynecological laparotomy. An anti-human rabbit polyclonal antibody was prepared against sulfatase protein purified from human placenta. Total RNA was isolated from epithelium of fallopian tubes. A heterologous RNA competitor was designed, and competitive RT-PCR was carried out. Steroid sulfatase was localized to the cytoplasm of epithelial cells. With respect to the positive staining of cells, the number of positive secretory cells was higher than that of ciliated cells. A significantly higher number of positive cells was found in tissue obtained from the early luteal phase than that found in tissue from the follicular phase. An abundant expression of sulfatase mRNA in early luteal phase was also observed. This study demonstrates, for the first time, that steroid sulfatase is localized to human epithelial cells and that steroid sulfatase staining and mRNA expression changes with the menstrual cycle. These results suggest that sulfatase in the fallopian tube may be involved in controlling the local steroid environment, which appears to regulate aspects of the physiological reproductive function of the fallopian tube.