Reduction of aquaporin-8 on fetal membranes under oligohydramnios in mice lacking prostaglandin F2 alpha receptor.

AIM: To investigate the association between aquaporin-8 (AQP-8: a water channel protein) expression in fetal membranes and oligohydramnios during near-term and postdate pregnancy, we set up an oligohydramnios model using prostaglandin F2 alpha receptor (FP)-deficient mice. METHODS: Pregnant FP-deficient mice from 14 to 21 gestational days (GD) were killed to measure the amniotic fluid volume (AFV), and fetal membranes were collected for the analysis of aquaporin-8 expression. RESULTS: The AFV was highest at 14 GD, and was significantly decreased to 28% and 0% at 20 GD and 21 GD, respectively, compared with the volume at 14 GD. Immunohistochemistry and immunoblot analysis showed that aquaporin-8 was expressed in the basal component of fetal membranes, and that the protein level was significantly decreased to 60% at 20 GD compared with that at 14 GD. CONCLUSIONS: We demonstrated that AQP-8 expression in the fetal membrane was decreased at post term in FP-deficient mice. Our findings suggest that aquaporin-8 in fetal membranes may be involved in the regulation of AFV, especially when oligohydramnios occurs.

Long-term neuroprotective effects of carbon dioxide on neonatal rat hypoxic-ischemic brain injury: an experimental study of skilled motor tasks.

OBJECTIVE: This study was undertaken to investigate the long-term effect of hypercapnia on neonatal hypoxic-ischemic brain injury, we tested its effect in a neonatal rat hypoxia-ischemia model. STUDY DESIGN: The rats were subjected to unilateral carotid artery ligation and exposure to 8% oxygen for 30 minutes. Six percent carbon dioxide was administered to the neonatal rats during unilateral hypoxia-ischemia, and the motor function and neurologic outcomes were determined 3 months later. RESULTS: Significant motor functional improvement was observed in the hypercapnic animals, as judged by the Montoya staircase test. The unilateral brain injury was significantly ameliorated in the hypercapnic animals, and this amelioration was well correlated with the motor functional performance. Cerebral blood flow during hypoxia-ischemia, monitored by laser Doppler flowmetry, was better preserved in the hypercapnic animals. CONCLUSION: Our results suggest that mild hypercapnia during hypoxia-ischemia may provide long-lasting motor functional as well as neurologic protection for immature brains, possibly by increasing cerebral blood flow during hypoxia.

Secretory leukocyte protease inhibitor levels in cervicovaginal secretion of elderly women.

OBJECTIVE: Secretory leukocyte protease inhibitor (SLPI) is a potent inhibitor of human leukocyte elastase. The aim of the present study was to examine whether there is an association between the SLPI concentration in the cervicovaginal secretion (CS) and vaginal complaints of post-menopausal women. METHODS: Uterine cervix tissues and CS of peri- or post-menopausal women were obtained. SLPI was assayed by ELISA. To determine the level of SLPI mRNA and the localization of SLPI protein in the uterine cervix, we performed RT-PCR and immunochemical staining, respectively. RESULTS: The levels of SLPI in the CS of post-menopausal women with vaginal complaints were significantly lower that those of post-menopausal women without vaginal complaints. The levels of SLPI in the CS of post-menopausal women were lower that those of peri-menopausal women and post-menopausal women treated with hormone replacement therapy. Positive staining was observed in epithelial cells of the cervix of elderly women, however, the intensity was weaker than that in peri-menopausal women. Positive staining was also observed in gland cells of the cervix of peri-menopausal women, but not in those of post-menopausal women. SLPI transcripts were detected in the cervix of post-menopausal women. The treatment of post-menopausal women with vaginal estrogen increased the concentrations of SLPI in CS of post-menopausal women. CONCLUSIONS: The present findings suggest that the decreased amount of SLPI in the CS of post-menopausal women might be one of the causes of the symptoms of post-menopausal women and contribute to the immunodefense mechanisms of the elderly women.

Doppler waveforms: the relation between ductus venosus and inferior vena cava.

This study was to assess whether or not there was correlation between the Doppler velocity waveform of the ductus venosus (DV) and inferior vena cava (IVC). A total of 142 healthy pregnant women were enrolled and divided into three groups according to the gestational weeks at the examination time. Group 1 was < or = 22 weeks; group 2 was between 22 and 28 weeks; and group 3 was > or = 28 weeks. Acuson 128xp was used to measure the Doppler velocity waveforms of DV and IVC by one experienced examiner. Doppler indices were used for analysis. Our results showed that, with the advance of pregnancy age, the resistance index of DV (DV-RI) and the preload index of IVC (IVC-PLI) were correlated with the gestational weeks, r = -0.247 and r = -0.540, respectively. There was a weak correlation between DV-RI and IVC-PLI, r = 0.202, p < 0.05; however, there was no significant correlation between DV-RI and IVC-PLI in group 1, group 2 or group 3. In conclusion, we report the first study on the correlation between DV-RI and IVC-PLI, which indicated the different roles of DV and IVC in fetal hemodynamics.

Contrast-enhanced power Doppler sonography of malignant ovarian tumors using harmonic flash-echo imaging: preliminary experience.

To investigate the potential usefulness of contrast-enhanced intermittent harmonic sonography in the diagnosis of ovarian cancer, we evaluated 4 patients with complex adnexal masses suspected of malignancy using intermittent harmonic sonography after injection of a contrast agent. Tumor and/or mural nodule tissue enhancement was detected in all cases of ovarian malignancy. Contrast-enhanced, intermittent harmonic sonography provides a satisfactory visualization of blood flow in the solid portion of the tumor tissue and may support a diagnosis of ovarian malignancy. Depiction of blood vessels using low MI techniques may be possible with other vascular ultrasonographic contrast agents.

Correlation of neuron-specific enolase and S100B with histological cerebral damage in fetal sheep after severe asphyxia.

Experimental brain damage was induced in 16 fetal sheep by umbilical cord occlusion, and the correlation of neuron-specific enolase (NSE) or S100B with the damage grade was investigated in seven fetuses. Significant correlations of damage degree with NSE (p = 0.016) and S100B (p = 0.018) in serum 2 h after insult were shown by Spearman's test. These findings suggest that they represent potentially useful markers for detecting brain damage at early stage after ischemic insult.

Effects of 4-hydroxy-2-nonenal, a marker of oxidative stress, on the cyclooxygenase-2 of human placenta in chorioamnionitis.

Chorioamnionitis (CAM) is one of the causes of preterm labour. A recent study has indicated that NADPH oxidase, a reactive oxygen species (ROS)-producing enzyme, is activated in CAM. CAM is thought to be closely associated with oxidative stress. We have hypothesized that oxidative stress in CAM may induce preterm labour. The purpose of this study is to examine the effect of 4-hydroxy-2-nonenal (HNE), which is a marker of oxidative stress, on human placenta during preterm labour. We initially examined the HNE-modified proteins in human placentas by immunoblotting and immunohistochemistry using anti-HNE antibody. To examine the effect of HNE on human placenta, we stimulated human placental tissue with HNE. The expressions of cyclooxygenase-2 (COX-2) mRNA and protein were observed by RT-PCR and western blot analysis respectively. Furthermore, we measured the peroxidase activity of COX-2 by COX activity assay kit. Prostaglandin E(2) (PGE(2)) in the supernatants of placental tissue was also determined by enzyme-linked immunosorbent assay. Immunoblotting and immunohistochemistry showed that the levels of HNE-modified proteins were increased in the placentas with CAM, compared to the normal placenta. HNE induced the expression of COX-2 mRNA, protein and activity in the placental tissue culture stimulated with HNE. In addition, PGE(2) was also released into the medium in a time-dependent fashion. These findings suggest that HNE-modified proteins, which were increased in the placenta with CAM, play an important role in preterm labour.

Expression of fractalkine in the Fallopian tube and of CX3CR1 in sperm.

BACKGROUND: Fractalkine is a CX(3)C chemokine that has chemoattractant activity for T cells, monocytes and natural killer (NK) cells. The objective of this study was 2-fold: to evaluate (i) the presence of fractalkine in the Fallopian tube and (ii) the existence of CX(3)CR1 (fractalkine receptor) in ejaculated sperm. METHODS AND RESULTS: Western blot analysis revealed that fractalkine protein was detected as a 95 kDa band in the isthmus, the ampulla and the infundibulum of the Fallopian tube. Immunohistochemistry revealed positive staining of epithelial cells in the Fallopian tube. RT-PCR demonstrated that fractalkine transcripts were expressed in all parts of the Fallopian tube. RT-PCR also revealed that CX(3)CR1-positive cells were present in the Fallopian tube. CX(3)CR1-positive cells were present in the stroma of the Fallopian tube. The villi of the ciliated cells were positively stained. To determine the function of fractalkine in the Fallopian tube, we examined whether CX(3)CR1 was present in ejaculated sperm. RT-PCR demonstrated that CX(3)CR1 transcripts were expressed in the ejaculated sperm. Immunohistochemistry demonstrated positive staining of the tail of the spermatozoa. CONCLUSIONS: The present findings suggest that fractalkine in the Fallopian tube contributes to the immunodefence mechanism during fertilization and to the sperm motion in the oviduct.

Postischemic hyperthermia induced caspase-3 activation in the newborn rat brain after hypoxia-ischemia and exacerbated the brain damage.

The effects of postischemic hyperthermia were investigated in the newborn rat brain after hypoxia-ischemia (HI). Seven-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 30 min, and divided into a hyperthermia group (rectal temperature at 39 degrees C for 6 h) and a normothermia group. Hyperthermia resulted in an approximately 5-fold increase in activated caspase-3 24 h after HI when compared with the normothermia group, and gross loss of brain tissue was observed only in the hyperthermia group at 7 and 30 days after HI. Our results show that postischemic hyperthermia exacerbates HI injury in immature brains, and that the mechanism is strongly associated with activation of an apoptotic pathway.

Serial transvaginal sonography of a case of complete hydatidiform mole.

Sonography has achieved a position of preeminence in the diagnostic evaluation of molar gestation. However, little information is available about the serial change of the transvaginal sonographic features of molar pregnancy. We describe a case of complete hydatidiform mole, and present serial transvaginal views taken during the early gestational period.

Neonatal Bartter syndrome with unilateral multicystic dysplastic kidney disease.

Neonatal Bartter syndrome is characterized by antenatal presentation with polyhydramnios. In this paper, we report a case of neonatal Bartter syndrome associated with unilateral multicystic dysplastic kidney disease. To our knowledge, this is the first case report of such an association.

Effects of hyperthermia on hypoxic-ischemic brain damage in the immature rat: its influence on caspase-3-like protease.

OBJECTIVE: Recent clinical studies suggested that intrapartum maternal fever is a strong independent risk factor for neonatal encephalopathy. With use of a well-studied rat model of neonatal hypoxic-ischemic encepalopathy, this study investigated the hypothesis that intraischemic hyperthermia accelerates and worsens brain injury in immature animals and examined whether apoptotic cell death machinery is involved in the underlying mechanisms. STUDY DESIGN: Seven-day-old rats underwent a combination of left common carotid artery ligation and exposure to 8% oxygen for 15 minutes (n = 32 rats). During the 15-minute hypoxic insult, body temperature was elevated to 40 degrees C in 16 animals (hyperthermic hypoxic insult group), and was maintained at 37 degrees C in 16 animals (normothermic hypoxic insult group). Then both groups were placed in the same chamber in a water bath at 37 degrees C for 24 hours and finally returned to the mothers. Caspase-3-like activity was assessed 36 hours after the hypoxic-ischemic insult. One week later, microtubule-associated protein-2 immunostaining was used to examine neuronal damage. RESULTS: Intraischemic hyperthermia was shown to activate the caspase-3 activity 36 hours after hypoxia-ischemia while caspase-3 was activated insignificantly in the normothermic hypoxic insult group at that time. The hyperthermic hypoxic insult group also showed a reduced microtubule-associated protein-2-positive area 7 days after hypoxia-ischemia compared with that in the normothermia group. CONCLUSION: Hyperthermia during hypoxia-ischemia makes the immature brain inordinately susceptible to hypoxic-ischemic insult and causes brain injury, even if hypoxic-ischemic insult is so mild that it causes no or little injury by itself. This effect may be mediated by the escalation of the apoptotic cell death pathway in the immature animal.

Long-term hospitalization during pregnancy is a risk factor for vitamin D deficiency in neonates.

In order to examine the effects of long-term hospitalization during pregnancy on vitamin D metabolism in pregnant women and neonates, we measured the serum 25-hydroxyvitamin D (25OHD) levels in pregnant women, as well as measuring 25OHD levels in cord blood and breast milk. In pregnant women hospitalized for longer than 1 month, the serum 25OHD levels were decreased at delivery compared with those in control subjects (10.9 +/- 2.6 ng/l vs 19.5 +/- 4.9 ng/l; P < 0.01). Although the levels of 25OHD in the cord blood were not significantly different between the long-term hospitalized and control pregnant women in this study (9.36 +/- 1.7 ng/l vs 11.1 +/- 3.0 ng/l), the 25OHD concentrations in the cord blood were significantly lower than the maternal levels in both groups; the ratios of the levels in cord blood to sera in the long-term hospitalized women and control subjects were 82.1% and 60.3%, respectively. Long maternal hospitalization does not always cause neonatal vitamin D deficiency, but could be one of its major risk factors. Therefore, sufficient sunlight exposure and intake of sufficient vitamin D are considered to be important to prevent vitamin D deficiency in long-term hospitalized pregnant women as well as their babies.

Apoptosis and related proteins in placenta of intrauterine fetal death in prostaglandin f receptor-deficient mice.

The present study investigated whether the increase of apoptosis in the placenta is associated with intrauterine fetal death in prostaglandin F receptor-deficient mice. Apoptosis was demonstrated within placental and decidual tissue by the TUNEL method. The majority of apoptosis was found in syncytiotrophoblast tissues. Enhanced TUNEL-positive staining in the syncytiotrophoblast layer was scattered in the placental tissues in clusters of apoptotic cells in the death group. Marked TUNEL-positive cells were identified in decidua of both groups. The rate of apoptosis in the placenta and decidua in the death group was higher than that in the survival group (P < 0.05). Immunohistochemical analysis showed that the level of active caspase-3 protein expression in the placenta in the death group was much higher than that in the survival group. The level of Bcl-2 protein expression in the placenta in the death group was much lower than that in the survival group. Western blot analysis demonstrated that increased expression of the active form of caspase-3 was detected in the placenta and decidua in the death group compared with that in the survival group. In contrast, a decrease in the expression of Bcl-2 was detected in the placenta and decidua in the death group compared with that in the survival group. Enhanced expression of Bax:Bcl-2 ratio was detected in placenta and decidua in the death group compared with that in the survival group. Thus, significantly increased apoptosis in the mouse placenta and decidua might be involved in the pathophysiologic mechanism of intrauterine fetal death.

Long-term neuroprotective effects of hypothermia on neonatal hypoxic-ischemic brain injury in rats, assessed by auditory brainstem response.

A method to assess long-term neurofunctional outcome of hypothermia on immature brains has not yet been clearly established. To investigate the effects of hypothermia on long-term neurofunctional outcome, we studied brainstem function using auditory brainstem response in adult rats after neonatal hypoxic-ischemic brain injury. Seven-day-old rats underwent a combination of left common carotid artery ligation and subsequent exposure to 8% O(2) for 1 h (n = 17). The rats were divided into three groups: hypothermia group (n = 6), normothermia group (n = 6), and sham control group (n = 5). During recovery from the hypoxic-ischemic insult, body temperature was reduced to 30 degrees C for 24 h in the hypothermia group, but was kept at 37 degrees C in the normothermia and sham control group. Three months later the rats were assessed by auditory brainstem response, then killed. The normothermia group showed increased III-V latencies and wave V abnormalities. Hypothermia significantly ameliorated wave V abnormalities. Injury to the ipsilateral inferior colliculus was also reduced in the hypothermia group compared with that in the normothermia group, and the degree of damage assessed histologically correlated well with auditory brainstem response findings. The current study demonstrates that postischemic hypothermia may provide effective and long-lasting neurofunctional as well as histopathologic protection to the immature brain.

Antenatal prediction of pulmonary hypoplasia by acceleration time/ejection time ratio of fetal pulmonary arteries by Doppler blood flow velocimetry.

OBJECTIVE: The purpose of this study was to develop a new method for the antenatal prediction of pulmonary hypoplasia by Doppler blood flow velocimetry. STUDY DESIGN: One hundred seventy-seven fetuses (160 normal fetuses and 17 fetuses with congenital anomalies that may affect fetal lung growth and/or development) were studied. Blood flow waveforms at the main branches of the pulmonary arteries were recorded by Doppler echocardiography from 20 to 39 weeks of gestation. The ratio of acceleration time to ejection time was calculated from the waveform as a parameter to predict pulmonary hypoplasia. RESULTS: Doppler waveform of normal fetal pulmonary artery showed a "spike-and-dome" pattern. The normal values of acceleration time/ejection time ratio from the right and left pulmonary arteries were 0.17 +/- 0.04 and 0.15 +/- 0.04, respectively. These values were not significantly altered through the gestational age observed in this study. The acceleration time/ejection time ratio of either right or left pulmonary artery was measured successfully in all cases of fetal congenital anomalies. In 8 of 17 fetuses, acceleration time/ejection time ratio was measured at both of the pulmonary arteries. Because of a congenital anomaly that affected the fetal lung or thorax asymmetrically (as in congenital diaphragmatic hernia or congenital cystic adenomatoid malformations of the lung), the acceleration time/ejection time ratio of both of the pulmonary arteries could be measured in only 5 of 13 fetuses. The technical difficulties for the measurement always existed in the affected side. Eleven of the 17 fetuses with congenital anomalies survived without signs of clinical pulmonary hypoplasia or persistent pulmonary hypertension of the newborn infant. The fetuses revealed normal acceleration time/ejection time ratio from at least one pulmonary artery. The remaining 6 fetuses died of pulmonary hypoplasia, and the diagnosis was confirmed by autopsy or clinical findings. Of those 6 fetuses, 5 fetuses demonstrated the acceleration time/ejection time ratio below normal in one side, and the ratio could not be obtained on the other side; 1 fetus showed the acceleration time/ejection time ratio below the normal range in both sides. CONCLUSION: The acceleration time/ejection time ratio by Doppler velocimetry that was obtained at the main branches of fetal pulmonary artery was consistent throughout gestational age from 20 to 39 weeks. This ratio appears to be an accurate parameter with which to predict the subsequent development of pulmonary hypoplasia and clinical outcomes of the newborn infants with high positive and negative predictive values (positive predictive value, 100%; negative predictive value, 100%).

Fetal gene transfer by intrauterine injection with microbubble-enhanced ultrasound.

Intrauterine injection of naked DNA expressing luciferase, green fluorescent protein (GFP), or beta-galactosidase (beta-gal) and fluorescein isothiocyanate-labeled oligodeoxynucleotide (FITC-ODN), in combination with microbubble-enhanced ultrasound (US), referred to as the "shotgun method" (SGM), produced high-level protein expression in fetal mice. With the SGM, luciferase expression increased approximately 10(3)-fold in comparison with expression after injection of naked DNA alone. Electron microscopic analysis demonstrated transient formation of pores on the skin surface after intraamniotic (i.a.) injection with the SGM. Widespread expression of GFP and beta-gal and delivery of FITC-ODN were observed in multiple fetal tissues adjacent to the injection points. PCR analysis indicated that germline transfection was only transient following intraperitoneal (i.p) injection, and there was no evidence of transfer of the reporter gene to the offspring. Thus, SGM might provide a useful means to clarify the molecular mechanisms of genetic diseases in utero, as well as a tool to develop gene therapies in utero.

Apoptosis and related proteins during parturition in prostaglandin F receptor-deficient mice.

This study investigated whether apoptosis and related proteins are involved in parturition by comparative observation of FP-deficient mice without labor and wild type mice with vaginal delivery. We examined the expression of apoptosis, Fas, FasL, active caspase-3 and bcl-2 proteins in the amnion, placenta and decidua. DNA laddering in the amnion, placenta and decidua tissue did not significantly differ between FP-deficient and wild type mice on day 18 of pregnancy. Similar TUNEL staining results were found in all tissues of FP-deficient mice compared with those of wild type mice. A higher intensity of apoptotic cells was found in the decidua basalis. The index of TUNEL-positive cells were not significantly different in the amnion, placenta and decidua of FP-deficient mice compared with that of wild type mice on day 18 of pregnancy. Specific bands for Fas were clearly observed in the amnion, placenta and decidua tissue. FasL specific bands were observed in the placenta and decidua, but a few in amnion tissue. A great number of active caspase-3 specific bands were detected in decidua, while a few such bands were detected in the placenta and few bands in the amniotic tissue. Bands for bcl-2 were detected in the amnion, placenta and decidua tissue. The weakest band was in decidual tissue. Fas, FasL, active caspase-3, and bcl-2 specific bands did not show any significant differences between the two groups. These findings demonstrate that apoptosis, Fas, FasL, caspase-3, and Bcl-2 occur in mouse term placenta that is not involved in parturition.

Investigation of intraplacental villous arteries by Doppler flow imaging in growth-restricted fetuses.

OBJECTIVE: The purposes of our study were to assess the ability of color and power Doppler sonography to depict the blood flow in the intraplacental villous arteries and to evaluate whether the blood flow of intraplacental villous arteries in a normal pregnancy is different from that in a pregnancy that is associated with intrauterine growth restriction. STUDY DESIGN: Eighty-five women with uncomplicated pregnancy and 16 women with intrauterine growth-restricted fetuses between 27 and 38 weeks of gestation were examined by color and power Doppler imaging. The blood flow of intraplacental villous arteries was analyzed comparatively. The pulsatility index and peak systolic velocity were measured. RESULTS: A unit of 1 intraplacental villous artery-1 and its branches were seen as 1 cotyledon by color and power Doppler imaging. The cotyledon was easily identified and counted. Each cotyledon contained only 1 intraplacental villous artery-1. This method can visualize the intraplacental villous artery-1 to intraplacental villous artery-4 in normal pregnancies. The terminal villous arteries beyond intraplacental villous artery-4 were not imaged. The number of detectable intraplacental villous artery-1 in 1 placenta in intrauterine growth restriction was significantly lower than that in normal pregnancy. The number of detectable branches in intrauterine growth restriction was also significantly lower than in normal pregnancy. No intraplacental villous artery-4 blood flow was found in women with intrauterine growth restriction. In examined arteries, pulsatility index decreased and peak systolic velocity increased significantly with advancing gestational age (P <.02). At any given gestational age, pulsatility index and peak systolic velocity in the peripheral arteries were significantly lower than those in the upstream arteries in normal pregnancy (P <.001). The pulsatility index value of each intraplacental villous artery was also lower than that of the umbilical artery in the women with intrauterine growth restriction (P <.05). There were no differences in pulsatility index in each artery between the groups, although there were a few high pulsatility index values in intrauterine growth restriction. CONCLUSION: Color Doppler and power flow sonography are valuable tools for the detection of the blood flow of intraplacental villous arteries. The decrease in the number of detectable intraplacental villous artery-1 and branches was associated with intrauterine growth retardation.

Effects of neonatal hypoxic-ischemic brain injury on skilled motor tasks and brainstem function in adult rats.

In an attempt to establish more sensitive long-term neurofunctional measurements for neonatal hypoxic-ischemic brain injury, we examined skilled motor task and brainstem functions in adult rats after neonatal cerebral hypoxia-ischemia (H-I), using a staircase test and auditory brainstem response (ABR), respectively. Seven-day-old rats underwent a combination of left common carotid artery ligation and exposure to 8% O(2) for 1 h (n=16). The control animals only received sham operation (n=16). At 3 months of age, the staircase test and ABR were performed. In the staircase test, H-I animals showed marked impairment of skilled forelimb use in the side contralateral to the occluded artery, and the degree of brain damage correlated significantly to skilled forelimb use. In the ABR, H-I animals showed brainstem dysfunction assessed by measuring interpeak latencies for waves III-V and I-V. We also examined the brainstem with antibodies specific for activated caspase-3, a protein involved in initiation of apoptosis, and observed that caspase-3 was activated in the ipsilateral inferior colliculus at 24 h after H-I. The present study shows that both the staircase test and ABR are sensitive and objective long-term neurofunctional measurements that can be used in future studies to assess therapeutic intervention in this neonatal cerebral H-I model.