Fetal aortic isthmus growth and morphology in late gestation.

OBJECTIVE: To establish normal values for fetal aortic isthmus diameter in late gestation and to identify any changes in aortic isthmus dimensions and morphology in pathological conditions. METHODS: In this prospective study, the fetal aortic isthmus was evaluated in 110 low-risk pregnant women at between 30 and 40 weeks of gestation and 42 pregnant women who were at high risk for congenital heart defects. From coronal echocardiographic images of the connection between the aorta and ductus arteriosus, the internal diameter of the aorta was measured at the middle of the isthmus, at the point of the isthmus just proximal to the entry of the ductus arteriosus and at the descending aorta below the entry of the ductus arteriosus. RESULTS: Correlation coefficients for the diameter of each aortic segment when related to gestational age varied from r = 0.60 to r = 0.80 (P < 0.001 for each), and growth curves were derived from the third and 97th percentiles about each linear regression analysis. The mean and the third percentile for the ratio of the isthmus just proximal to the entry of the ductus arteriosus to the middle of the isthmus were 1 and 0.81. In one fetus of the high-risk patients, a contraductal shelf and the accompanying area of tubular isthmic hypoplasia were suspected and a diagnosis of coarctation of the aorta was subsequently confirmed after birth. In two fetuses with growth restriction and one fetus with intestinal atresia, the isthmus diameters were below the third percentile but the ratios of the isthmus end to the middle of the aortic isthmus were all normal and no cardiac anomalies were detected after birth. CONCLUSIONS: We could establish normal values for aortic isthmus diameters in late gestation from a coronal view and identify even minimal changes in aortic isthmus dimensions and morphology in pathological conditions.

Transvaginal ultrasound-guided embryo transfer improves pregnancy and implantation rates after IVF.

BACKGROUND: Attempts are constantly being made to improve clinical pregnancy rates after IVF and embryo transfer. Since November 1998, we have gradually been adopting transvaginal ultrasound guidance during embryo transfer. We retrospectively examined the efficacy of this method on pregnancy and implantation rates. METHODS: The results of 846 cycles from our IVF-embryo transfer programme were analysed and comparisons were made between those carried out using ultrasound guidance and those by the clinical touch method. RESULTS: Higher pregnancy and implantation rates (28.9 and 15.2% respectively) were found in the group using the transvaginal ultrasound guidance during embryo transfer compared with those in the group using the clinical touch method (13.1 and 7.0% respectively). The differences were statistically significant (P < 0.01). There was no significant difference in ectopic pregnancy rates between the two groups. CONCLUSION: The use of transvaginal ultrasound-guided embryo transfer significantly improved both pregnancy and implantation rates. Although technically difficult, we suggest its use may maximize the chances of achieving a successful pregnancy outcome.

Antenatal diagnosis of velamentous umbilical cord insertion and vasa previa with color Doppler imaging.

OBJECTIVE: To determine whether cord insertion can be consistently visualized and whether velamentous cord insertion and vasa previa can be consistently identified with color Doppler imaging during routine sonography in the mid-trimester. DESIGN: A prospective study. SUBJECTS: A total of 587 fetuses at 18-20 weeks' gestation. METHODS: During routine ultrasound examinations, the sonographer was instructed to take additional time and to image the placental cord insertion with color Doppler imaging and classify this as normal, velamentous or 'not seen'. When the insertion was velamentous, the sonographer was instructed to indicate whether or not it was vasa previa. The sonogram obtained at 18-20 weeks' gestation was used for comparison with outcome data. RESULTS: Cord insertion was visualized by color Doppler imaging in 99.8% (586/587) of the fetuses in our study. The mean time required for examination was 20 s and, in 95% of the cases, cord insertion was visualized within 1 min. The sonographic identification of velamentous cord insertion had a sensitivity of 100% (5/5), a specificity of 99.8% (580/581), a positive predictive value of 83% (5/6) and a negative predictive value of 100% (580/580). In our study, vasa previa was diagnosed at 18 gestational weeks in two cases and, in one of the cases, vasa previa was confirmed at delivery. CONCLUSIONS: We could consistently identify cord insertion and velamentous cord insertion with color Doppler imaging during routine sonography in the mid-trimester. Transvaginal color Doppler imaging and serial scans were needed to identify vasa previa.

Local immune response in infertile patients with minimal endometriosis.

OBJECTIVE: To evaluate the role of local immunity in women with minimal endometriosis. METHODS: Uterine endometrium and endometrial implants were obtained simultaneously from 30 infertile women with minimal endometriosis and examined immunohistochemically using antibodies of T cell, B cell, macrophage, Langerhans cell, immunoglobulin (Ig)G, and complement (C) 3d. Serum IgG, IgA, IgM, C3, C4, antinuclear antibody and anti-DNA antibody were also examined in 24 of the women. Data from uterine endometrium and serum were compared with 10 fertile women without endometriosis as a control. RESULTS: Microscopic examination revealed that the endometrial implants were divided into two groups: group 1 (n = 13) showed lymphocytic infiltration in the endometrial implants and group 2 (n = 17) showed no or slight lymphocytic infiltration. The endometrial implants of group 1 showed significantly more dense T-cell infiltration than those of group 2. Other types of infiltrating cells and deposits of IgG and C3d revealed no significant differences between groups 1 and 2. The immunohistochemical examination of the uterine endometrium and the serum data revealed no significant differences among all three groups. Cumulative pregnancy rates showed no significant difference between groups 1 and 2. CONCLUSION: The difference of local immune response in endometrial implants did not affect systemic immunity.

Conservative treatment using a methotrexate-lipiodol emulsion containing non-ionic contrast medium for a cervical ectopic pregnancy.

We present a case in which a viable cervical pregnancy was successfully treated with preservation of the uterus. A 26 year old female presented with a 6 week cervical pregnancy with acute moderate bleeding. On the day of admission the patient underwent embolization of the descending uterine artery to decrease the bleeding. The following day, 4 mEq potassium chloride and methotrexate (MTX)(1 mg/kg) dissolved in saline was injected into the amniotic cavity using transvaginal sonographic guidance. The fetal heart beat ceased; however, the urinary beta-human chorionic gonadotrophin (HCG) concentration increased and the gestational sac size also increased. After a second injection of an emulsion of MTX (1 mg/kg) dissolved in a non-ionic contrast medium, iopamidol and lipiodol, the betaHCG concentration in the urine began to decrease. After a third injection with the same emulsion, the cervical mass became necrotic. The betaHCG concentrations in the urine and serum were undetectable and the external cervix returned to normal 44 days after admission. No vaginal bleeding or significant side effects of MTX were observed throughout the treatment. An in-vitro dissolution test revealed that the dissolution rate of MTX was slower from MTX-lipiodol-iopamidol with sonication than without sonication. The present study indicates that use of an MTX emulsion enables slow release of MTX and may be applicable for conservative treatment of ectopic pregnancies, including cervical pregnancy.

Transport mechanism of pirarubicin in human mononuclear cells.

We studied the transport mechanism of pirarubicin (THP) in mononuclear cells (MNCs) obtained from healthy human donors. The THP uptake was time-, temperature-, concentration- and energy (in part)-dependent. The uptake of daunorubicin (DNR) and doxorubicin (ADR) was also concentration-dependent, and the transport of ADR consisted of saturable and nonsaturable components. In cis-inhibition experiments, ADR inhibited both THP and DNR uptake noncompetitively, while DNR showed competitive inhibition of the uptake of THP. The THP uptake rate appeared to be increased by preloading DNR, indicating a trans-stimulatory effect, but not with ADR. These results suggest that THP and DNR were taken up into MNCs via a common carrier-mediated transport system, but that the carrier of ADR might differ from that of THP and DNR. Furthermore, apparent differences in the affinity for the carrier, transport efficacy and substrate specificity of the transporter between MNCs and the human leukemia cell lines (HL60 and K562) were indicated.

Transport mechanisms of anthracycline derivatives in human leukemia cell lines: uptake of pirarubicin, daunorubicin and doxorubicin by K562 and multidrug-resistant K562/ADM cells.

We studied the uptake mechanisms of anthracycline derivatives, pirarubicin (THP), daunorubicin (DNR) and doxorubicin (ADR), in K562 and multidrug-resistant K562/ADM cells, which overexpress a multidrug efflux pump P-glycoprotein (P-gp). The uptake of THP, DNR and ADR by K562 or K562/ADM cells was time-, temperature- and concentration-dependent. The THP and ADR uptake by the parental cells was not affected by treatment with 4 mM 2,4-dinitrophenol (DNP) alone or DNP plus a P-gp specific inhibitor, cyclosporin A (CyA, 10 microM), while the DNR uptake in the DNP treatment group was significantly greater than that in the control group. There was no difference in the uptake of THP between DNP-pretreated K562 cells and DNP plus CyA-pretreated K562/ADM cells. The uptake of DNR or ADR was almost equal in both types of cell treated with DNP alone. Every kinetic constant for THP, DNR and ADR uptake by the sensitive cells was approximately equal to that in the resistant cells, respectively, under the above conditions. THP uptake was noncompetitively inhibited and stimulated on simultaneous treatment and preloading, respectively, of DNR or ADR in each type of cell. ADR showed noncompetitive inhibition of DNR uptake by either type of cell. Therefore, it was suggested that a common carrier-mediated transport system was involved in the uptake of THP, DNR and ADR, and that their binding sites in the carrier might be different from one another in both K562 and K562/ADM cells.

Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells.

We studied the transport mechanism of pirarubicin (THP) in HL60 and its THP-resistant (HL60/THP) cells, which showed no expression of mdr1 mRNA on Northern blot analysis. Under physiological conditions, the uptake of THP by both types of cell was time- and temperature-dependent. The amount of drug transport in the resistant cells was significantly less than that in the parent cells within 3 min of incubation. THP uptake was significantly higher in the presence than in the absence of 4 mM 2,4-dinitrophenol (DNP) in glucose-free Hanks' balanced salt solution in both HL60 and HL60/THP cells and the increases were approximately equal. In the presence of DNP, the uptake of THP by both types of cell was concentration-dependent, and there were no significant differences in the apparent kinetic constants (Michaelis constant (Km), maximum velocity (Vmax) and Vmax/Km) for THP uptake between HL60 and HL60/THP cells. Additionally, THP transport was competitively inhibited by its analogue doxorubicin. The efflux of THP from HL60/THP cells was significantly greater than that from HL60 cells, and the release from both types of cell was completely inhibited by decreasing the incubation temperature to 0 degrees C and by treatment with DNP in glucose-free medium. In contrast, the P-glycoprotein inhibitors verapamil and cyclosporin A did not inhibit THP efflux. However, genistein, which is a specific inhibitor of multidrug resistance-associated protein (MRP), increased the THP remaining in the resistant cells, and the value was approximately equal to that of the control group in the sensitive cells. These results suggest that THP is taken up into HL60 and HL60/THP cells via a common carrier by facilitated diffusion, and then pumped out in an energy-dependent manner. Furthermore, the accelerated efflux of THP by a specific mechanism, probably involving MRP, other than the expression of P-glycoprotein, resulted in decreased drug accumulation in the resistant cells, and was responsible, at least in part, for the development of resistance in HL60/THP cells.

Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of daunorubicin and doxorubicin in HL60 and its resistant cells and comparison with those of pirarubicin.

We examined the transport mechanisms of daunorubicin (DNR) and doxorubicin (ADR) in HL60 and HL60/THP cells which were the non-P-glycoprotein-mediated resistant clone of the parent HL60 cells and showed a low degree of resistance, and compared them with those of pirarubicin (THP). In both lines, it appeared that the uptakes of DNR and ADR were time-, temperature- and concentration-dependent and energy independent, and the transport of DNR consisted of saturable and nonsaturable components. They were pumped out from the cells time-, temperature- and energy-dependently. There were no differences in the accumulation amount of either DNR or ADR between HL60 and HL60/THP cells. Comparing the transport of DNR or ADR with that of THP, the uptake amounts of DNR and THP were approximately equal, and were greater than that of ADR in both types of cell. In cis-inhibition experiments, DNR inhibited the THP uptake noncompetitively in the parent and resistant cells, in contradiction of the previously reported result in which ADR showed competitive inhibition (Nagasawa, K. et al., Cancer Chemother. Pharmacol., in press). The THP accumulation appeared to be increased by preload of DNR and ADR, indicating a counter transport. Thus, DNR and ADR as well as THP might be incorporated via a common carrier-mediated transport system, but DNR uptake in part appeared to follow a nonsaturable transport, and its binding site in the carrier might differ from that of THP and ADR in both HL60 and HL60/THP cells.

Transport mechanism of anthracycline derivatives in rat polymorphonuclear leukocytes: effect of sodium fluoride on pirarubicin uptake.

We previously revealed that pirarubicin (THP) was actively taken up by rat polymorphonuclear leukocytes via a carrier-mediated transport system. In the experiment on the effects of the metabolic inhibitors, rotenone, 2,4-dinitrophenol and sodium cyanide significantly decreased the THP transport. However, sodium fluoride (NaF) significantly increased the uptake, and this result is different from that in some reports. Therefore, we examined the action of NaF on THP uptake by the leukocytes to clarify the discrepancy in the effect of NaF on drug transport. The accelerating effect of 30 mM NaF on the THP uptake by the cells had an optimum period of action (15-20 min), and was concentration-dependent (5-30 mM). Thirty mM potassium fluoride, as well as NaF, increased the uptake amount. On the other hand, NaF (5-30 mM) dose-dependently decreased the ATP content in these cells. Additionally, the viable cells in the reaction suspension decreased by about 40% after incubation with 30 mM NaF for 15 min. Observing these leukocytes treated with NaF by optical microscopy, swelling of the cell and an alteration of the nuclei form occurred. On the basis of these results, we speculated that the increased THP transport in polymorphonuclear leukocytes by NaF, probably F-, might be due, at least in part, to an alteration of the morphological form.

Production and expression of granulocyte- and macrophage-colony-stimulating factors in newborns: their roles in leukocytosis at birth.

We analyzed the production and expression of three colony-stimulating factors (CSFs) in neonates to clarify the mechanism of leukocytosis at birth. Serial blood samples (n = 23) were collected from mothers, cord blood, and from newborn infants on days 1, 5, and 30 after birth. The serum levels of granulocyte-CSF (G-CSF), granulocyte/macrophage/CSF (GM-CSF) and macrophage-CSF (M-CSF) were measured by ELISA. The G-CSF levels on day 1 after birth were significantly higher than those thereafter, and they were also higher in the mothers than those on days 5 and 30 after birth. The GM-CSF levels did not change significantly during the neonatal period. The serum M-CSF levels were higher on postnatal day 1 than at other times, and gradually decreased thereafter. To confirm the production sites of G-CSF and M-CSF, the mRNA for these CSFs in peripheral mononuclear cells (MNCs) from healthy adults, mothers, and cord blood were analyzed by PCR. The expression of G-CSF and GM-CSF mRNA was undetectable in MNCs from adults, mothers, and cord blood, while these cells expressed low levels of M-CSF mRNA. After stimulation with lipopolysaccharide or phorbol myristate acetate, the MNCs expressed high levels of G-CSF and GM-CSF mRNA. The levels of G-CSF PCR products in cord MNCs were lower than those in adult and maternal MNCs. The expression of M-CSF mRNA was virtually unchanged by stimulation. To detect the localization of G-CSF and M-CSF in the placenta and umbilical cord, these tissues were immunocytochemically stained with anti-G-CSF and anti-M-CSF antibodies, G-CSF and M-CSF were expressed in trophoblasts and decidual stromal cells, whereas the umbilical cord did not express these CSFs. Moreover, large amounts of G-CSF and M-CSF were detected in the supernatant of cultured trophoblasts and decidual stromal cells. The expression of G-CSF and M-CSF in these cells was confirmed by PCR. These findings suggested that G-CSF and M-CSF produced in the placenta (trophoblasts and decidual stromal cells) are the major factors that induce leukocytosis in newborn infants at birth.

Transport mechanism of anthracycline derivatives in rat polymorphonuclear leukocytes: uptake of pirarubicin, daunorubicin and doxorubicin.

We performed experiments on the cis-inhibition and trans-stimulation effect on pirarubicin uptake in order to clarify the involvement of a carrier in the pirarubicin, daunorubicin and/or doxorubicin transport systems in rat polymorphonuclear leukocytes. The uptake of daunorubicin and doxorubicin was a saturable concentration-dependent process. Since the apparent kinetic constants, Michaelis constant (Km) and inhibition constant (Ki), were almost comparable, these drugs presented mutually competitive inhibition. Furthermore, the pirarubicin uptake by polymorphonuclear leukocytes was significantly elevated by increasing the preloaded amount of doxorubicin, indicating that there was a trans-stimulation effect on the pirarubicin transport in the leukocytes. These results suggest that carrier-mediated transport might be involved in the uptake of anthracycline derivatives, pirarubicin, daunorubicin and doxorubicin, by rat polymorphonuclear leukocytes.

Transport mechanism of anthracycline derivatives in rat polymorphonuclear leukocytes: uptake and efflux of pirarubicin.

The characteristics of pirarubicin transport have been investigated in polymorphonuclear leukocytes isolated from rats. The uptake of pirarubicin by leukocytes was time-, temperature- and concentration-dependent with the maximum velocity (Vmax) of 4.84 nmol/5 x 10(6) cells/min and the Michaelis constant (Km) of 13.4 microM. The uptake depended on the extracellular pH, indicating that the molecular form of pirarubicin is more permeable than its ionic form and/or that the transporter of pirarubicin has optimal pH range. When the intracellular space was changed by increasing the medium osmolarity with sucrose, the uptake was altered by osmolarity changes and it appeared that about 27% of the drug was bound to the membrane surface. The initial uptake was inhibited by the metabolic inhibitors rotenone, 2,4-dinitrophenol and sodium cyanide. Pirarubicin accumulation in the intracellular glucose-depleted cells diminished significantly compared with that in normal cells. The efflux of pirarubicin from leukocytes was also temperature-dependent and inhibited by a metabolic inhibitor. These results indicate that a specific mechanism is concerned in the transport of pirarubicin in polymorphonuclear leukocytes.