Hypoxia and lactate production in trophoblast cells.

The etiology of preeclampsia is unknown but is thought to be related to hypoxia in the placenta. We previously reported that the enzyme lactate dehydrogenase (LDH) has increased activity and gene expression in placentas from preeclamptic pregnancies [Tsoi SCM, Zheng J, Xu F, Kay HH. Differential expression of lactate dehydrogenase isozymes (LDH) in human placenta with high expression of LDH-A(4) isozyme in the endothelial cells of pre-eclampsia villi. Placenta 2001;22:317-22]. LDH is responsible for pyruvate conversion to lactate through glycolysis. In this study, we further investigated the role of hypoxia in primary trophoblast cells and a cultured cell line, JEG3 cells, to obtain a better understanding of how it affects the activities of lactate dehydrogenase, lactate production and regulatory genes, as a possible model for preeclampsia. Primary trophoblast cells and JEG3 cells were cultured under 1% oxygen. At 6, 12 and 24h, cells were analyzed for LDHA and LDHB isozyme activities, mRNA and protein expression compared to standard culture conditions. Lactate was measured from cell medium. The hypoxia inducible transcription factor (HIF-1alpha) protein expression was confirmed by western blot. Two lactate transporters (MCT1 and MCT4) mRNA and protein expression were also studied under hypoxia. Finally, lactate was measured in plasma obtained from patients with severe preeclampsia. Under hypoxic conditions, LDHA mRNA is increased in primary trophoblast cells and JEG3 cells. The HIF-1alpha protein expression is higher in hypoxia-treated JEG3 cells than control. LDHA isozyme activity and its protein expression are increased most significantly at 24h of culture under hypoxia. However, LDHB protein is unchanged while its mRNA is decreased. Lactate secretion from JEG3 cells under hypoxia is increased, as is the lactate levels in the plasma from preeclampsia patients. Of the two lactate transporters studied, MCT4 mRNA and protein level are increased under hypoxia. Our findings support the role of hypoxia in inducing HIF-1alpha activity in trophoblasts and increasing LDH transcription as well as its activity. Higher levels of lactate are produced and secreted which may contribute to the higher lactate levels in plasma of preeclamptic patients. These mechanisms may be important in the pathophysiology of preeclampsia.

Influence of maternal diabetes on placental fibroblast growth factor-2 expression, proliferation, and apoptosis.

OBJECTIVE: Type I diabetes mellitus during pregnancy is associated with dysregulation of the oxygen and glucose metabolic pathways, both of which affect placental villous growth and function. Alteration of placental development in women with diabetes may contribute to the increased risk of preeclampsia, macrosomia, or fetal growth restriction. METHODS: To evaluate placental growth in the setting of maternal diabetes, immunohistochemical techniques were used to examine fibroblast growth factor-2 (FGF-2) expression, cell proliferation (Ki67), and apoptosis (Apo-Tag) in placentas from diabetic and nondiabetic patients. RESULTS: Immunostaining for FGF-2 in placentas from diabetic women demonstrated an increase in intensity within the villous stroma and syncytiotrophoblast (P<.05). Associated with these changes in FGF-2 expression, placentas from diabetic women showed no change in villous mitotic activity but did show decreased stromal compartment apoptosis. When expressed as a ratio of Ki67-positive:Apo-Tag-positive nuclei as an index of relative cell turnover, the stromal compartment showed a significant trend towards decreased nuclei turnover (P<.05), suggesting relative tissue growth in diabetic patients. CONCLUSION: Increased FGF-2 expression and decreased stromal cell compartment turnover in the diabetic placenta might be a compensatory mechanism in response to the altered physiologic milieu of maternal diabetes on placental function.

Color Doppler ultrasonography for distinguishing myomas from uterine contractions in pregnancy.

OBJECTIVE: To determine the applicability and reliability of color Doppler ultrasonography (US) for distinguishing a uterine myoma from a focal myometrial contraction. STUDY DESIGN: Images from 36 patients with uterine thickenings were classified as myomas when color Doppler US demonstrated no centralized flow with a circumscribed vessel pattern at the border. Thickenings were classified as focal myometrial contractions when there was demonstrable vascular flow throughout the thickening. RESULTS: Using these diagnostic criteria, images from 36 patients were reliably characterized as representing myomas or contractions. The diagnosis was made more reliable by using the lowest velocity settings and exclusion of power Doppler US in nonretroplacental lesions. CONCLUSION: Color Doppler US is a sensitive and reliable tool for distinguishing uterine myomas from focal myometrial contractions.

Differential expression of lactate dehydrogenase isozymes (LDH) in human placenta with high expression of LDH-A(4) isozyme in the endothelial cells of pre-eclampsia villi.

To evaluate the role of LDH isozymes in the human placenta during the third trimester, placentae were obtained from patients with normal pregnancy and pre-eclampsia. LDH-A(4)isozyme was immunolocalized primarily in the fetal endothelial cells while LDH-B(4)isozyme was predominantly present in syncytiotrophoblasts. This distinct cellular expression pattern of LDH isozymes was confirmed in HUVE and JEG cells. In addition to demonstrating the presence of five LDH isozymes in the placenta, zymograms showed that there was predominant activity of LDH-A(4)isozyme in HUVE cells and high activity of LDH-B(4)in JEG cells. Quantitative studies of LDH by agarose gel electrophoresis and Northern analysis in patients concluded that LDH-A(4)isozyme was increased in pre-eclampsia. The LDH-A(4)isozyme activity increased (P< 0.01) approx 1.6-fold in pre-eclampsia but there was no difference in the LDH-B(4)isozyme activity between placentae from normal compared to pre-eclampsia pregnancy. The level of LDH-A mRNA was increased (P< 0.05) approx twofold in pre-eclampsia. We conclude that the LDH-A gene in the endothelial cells of the placenta within the fetal microvasculature is increased in pre-eclampsia, probably as a result of hypoxia. LDH-A(4)isozyme activity and gene expression in placental endothelial cells, therefore, is a marker for the endothelial pathology seen in pre-eclampsia.

Antenatal steroid treatment and adverse fetal effects: what is the evidence?

This article reviews current animal and human data regarding possible adverse fetal effects from antenatal steroid treatment. Although it is now well accepted that such treatment is of benefit to fetal lung development, the potential for adverse fetal outcomes as a result of single or multiple glucocorticoid dosing has not been widely recognized. There are now growing concerns, based on animal and some human data, that repeated antenatal doses could lead to a decrease in birth weight, a decrease in fetal brain and other organ size, and abnormal neuronal development. Previous investigations have been hampered by nonstandardization in the type of glucocorticoid, route of delivery, timing of administration, and number of treatment courses. It is recommended that these concerns be addressed through large randomized, controlled clinical trials. In the meantime, it would be prudent to minimize antenatal steroid treatments to a single course with repeated dosing only if there is a persistent threat of preterm delivery. The practice of giving weekly injections of steroids starting at fetal viability and continuing into the third trimester is not supported.

Differential localization of placental extracellular superoxide dismutase as pregnancy progresses.

OBJECTIVE: The aim of this study was to determine placental localization and activity of extracellular superoxide dismutase, a nitric oxide modulator, during early gestation and to correlate these characteristics with fetal vascular development. STUDY DESIGN: First-trimester (n = 10) and second-trimester (n = 10) villi were obtained at elective pregnancy termination. Extracellular superoxide dismutase was localized by means of an immunoperoxidase method. Activity was measured by determining the inhibition of cytochrome c reduction at pH 10 and messenger ribonucleic acid expression by in situ hybridization. RESULTS: Extracellular superoxide dismutase was intracellular within villous trophoblasts until 17 weeks' gestation, when it relocated to the villous extracellular matrix. Activities were similar between first- and second-trimester villi. In situ hybridization confirmed extracellular superoxide dismutase messenger ribonucleic acid within trophoblasts throughout gestation. CONCLUSION: Extracellular superoxide dismutase is produced by trophoblasts early in pregnancy, but it remains intracellular until 17 weeks' gestation, which may be related to fetal vascular development.

Ethanol exposure induces oxidative stress and impairs nitric oxide availability in the human placental villi: a possible mechanism of toxicity.

OBJECTIVE: We undertook this investigation to explore the effects of ethanol exposure on nitric oxide synthase levels and nitric oxide release. Our hypothesis was that ethanol exposure modifies nitric oxide activity within the placenta as a result of oxidative stress. STUDY DESIGN: Four 10-g samples of term normal human placental villous tissue were perifused with nonrecirculating Dulbecco's modified Eagle's medium and 25-mmol/L N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] with 0-, 50-, 100-, or 200-mmol/L ethanol. After 2 hours of exposure, tissue was removed, fixed, and frozen for analysis. Immunohistochemical analysis was performed for subtype I or neuronal nitric oxide synthase (nNOS), subtype II or inducible nitric oxide synthase (iNOS), and subtype III or endothelial nitric oxide synthase (eNOS) localization. Western blot analysis was performed for eNOS quantitation. Cyclic guanosine monophosphate and copper-zinc superoxide dismutase levels were measured by electroimmunoassay and kinetic assay, respectively. Nitric oxide release was analyzed by a Sievers nitric oxide analyzer. RESULTS: Immunohistochemical examination confirmed that only eNOS was localized to the syncytiotrophoblasts. After ethanol exposure, eNOS protein expression increased 2.5- to 3.0-fold over that of the control. Tissue cyclic guanosine monophosphate content and nitric oxide release into the effluent were decreased, whereas superoxide dismutase levels were increased at higher ethanol levels (P <.05). CONCLUSION: Ethanol exposure appears to induce oxidative stress, which may account for the decreased nitric oxide release, because nitric oxide may be shunted toward scavenging free radicals. Increased eNOS protein expression may be a response to the increased demand for nitric oxide. Decreased nitric oxide availability could adversely affect placental blood flow regulation, which could, in turn, account for the growth restriction seen in ethanol-exposed fetuses.

A role for maternal serum screening in detecting chromosomal abnormalities in fetuses with isolated choroid plexus cysts: a prospective multicentre study.

A prospective multicentre study was performed to identify patients with fetal choroid plexus cysts and examine the association between choroid plexus cysts and chromosome abnormalities in the context of variables such as maternal age, serum triple-screen results, race, other prenatally-identified fetal anomalies and cyst characteristics. A total of 18 437 scans were performed in 5 centres and 257 fetuses were identified with choroid plexus cysts. Outcome was available on 250 patients, and of these, chromosomal abnormalities were detected in a total of 13 (5.2 per cent) fetuses. 26 patients in the group had additional ultrasound abnormalities, and 8 of these had fetal chromosome abnormalities. Among the 224 patients with isolated choroid plexus cysts, 5 (2.2 per cent) were found to have chromosomal abnormalities. All cases with identified chromosomal abnormalities were associated with an additional risk factor, such as other ultrasound findings, advanced maternal age or abnormal maternal serum triple-screen results.

Catheter-directed thrombolysis for thromboembolic disease during pregnancy: a viable option.

Anticoagulation with intravenous heparin has been the standard treatment for the management of gestational thromboembolic complications. Catheter-directed thrombolysis is an encouraging approach for the treatment of thromboembolic disease and has not been previously reported during pregnancy. One gravid woman with pulmonary embolism, critically ill, and hemodynamically compromised, and two gravid women with iliofemoral venous thrombosis, who failed to respond to standard treatment with intravenous heparin, were treated with catheter-directed urokinase. All three patients experienced rapid resolution of symptoms and successful pregnancy outcomes. In our three patients, catheter-directed thrombolysis for thromboembolic disease during pregnancy allowed rapid resolution of hemodynamic abnormalities and/or resolution of thrombus. Catheter-directed thrombolysis offered a reasonably safe alternative to prolonged medical management in these young, otherwise healthy, patients. Long-term, it may prevent the postphlebitic syndrome.

Prenatal diagnosis of thrombocytopenia absent radius (TAR) syndrome and vaginal delivery.

A fetus with bilateral radial aplasia was identified on routine ultrasound. The diagnosis of thrombocytopenia absent radius (TAR) syndrome was confirmed with cordocentesis. The differential diagnosis of radial aplasia and prenatal tests available to assist with management are discussed. Cordocentesis offered useful information in the management of this case for both diagnosis and in deciding the route of delivery. We believe our case represents the first prenatal diagnosis of TAR syndrome in which vaginal delivery of a liveborn infant was intentionally allowed. Caesarean delivery may not be necessary for all fetuses diagnosed with TAR syndrome.

Extracellular superoxide dismutase localization and activity within the human placenta.

Maintenance of low vascular tone within the placenta is mediated by nitric oxide (NO). The half-life of NO is very short, as superoxide anion (O2-) rapidly inactivates NO to form peroxynitrite. Superoxide dismutases compete with NO for O2-. By scavenging O2-, superoxide dismutase promotes the vasodilatory action of NO. Extracellular superoxide dismutase (EC-SOD) is present in high concentrations within the extracellular matrix of systemic arteries and has been proposed to mediate vascular smooth muscle tone by increasing NO bioavailability. The localization and activity of EC-SOD within the human placenta has not been determined. Placental EC-SOD may be involved in placental vascular tone, and abnormal activity may lead to pre-eclampsia secondary to increased O2--mediated inactivation of NO. To investigate this possibility, the activity and localization of human placental EC-SOD was determined in normal women, and then compared to pre-eclamptic women. Placental EC-SOD localized within the villous extracellular matrix around arterioles, and there were no differences in distribution between normal and pre-eclamptic women. There were no differences in placental EC-SOD activity between normal and pre-eclamptic subjects in either center (33.7+/-4.1 versus 33.1+/-2.5, P=0.6), or peripheral (34.3+/-5.6 versus 34.0+/-3.5, P=0.9) samples. EC-SOD localization around villous vessels suggests that EC-SOD serves potentially to protect the fetal vasculature from O2-, in both normal and pre-eclamptic pregnancies. Placental EC-SOD distribution and activity is not different between pre-eclamptic and normal women, suggesting that EC-SOD is not involved in the vascular changes seen in pre-eclampsia.

Is size discordancy an indication for delivery of preterm twins?

OBJECTIVE: Our goal was to determine the clinical significance of size discordancy in preterm twins. STUDY DESIGN: A retrospective study was performed to review outcomes of twins delivered between Jan. 1, 1988, and June 30, 1995. Maternal and neonatal records were assessed for demographic data, maternal medical history, and neonatal mortality and morbidity outcomes. Discordancy was defined as > or = 20% difference in birth weight. The chi 2 analysis was performed. RESULTS: There were 42 sets of discordant twins and 77 sets of concordant twins in the final analysis. The distribution of gestational ages in both groups was similar. We found no difference in maternal morbidity between the groups. Discordant sets had a significantly longer hospital stay (p = 0.003) and more cases of hyperbilirubinemia (p = 0.01), but there were no other differences in morbid outcomes. There were no differences in outcome variables between the two twins within discordant sets with respect to gender, size, birth order, growth restriction, or route of delivery. There were no stillbirths among any of the 238 infants. Of the 15 neonatal deaths, none occurred in infants delivered after 32 weeks' gestation or in infants weighing > 2000 gm at birth. Infants who were small for gestational age had a higher incidence of sepsis (p = 0.043) and longer hospital stays (p = 0.005) compared with infants who were appropriate for gestational age. CONCLUSIONS: Size discordancy alone does not appear to be an indication for preterm delivery of twins. When results of antenatal testing are normal and growth restriction is absent, attempts should be made to achieve a gestational age > 32 weeks and weight > 2000 gm before delivery is considered.

Placental villous glucose metabolism and hormone release respond to varying oxygen tensions.

OBJECTIVE: The effects of varying oxygen tensions on tissue metabolic behavior are not well understood, yet many intracellular pathways are influenced by them. In the placenta, optimal in vivo oxygen tension at the villous level is unknown. The purpose of this study was to determine effects of varying oxygen tensions on glucose metabolism and hormone release from perifused placental villous explants. METHODS: Placentas from term normal pregnancies (n = 8) were individually minced into villous fragments, placed into three parallel chambers for each placenta, and continuously perifused for 6 hours with nonrecirculating medium aerated with either 0%, 20%, or 95% oxygen yielding mean oxygen tensions of 76 mmHg, 167 mmHg, and 543 mmHg respectively. Outflow medium was removed at regular intervals and compared to the inflow medium to determine oxygen and glucose consumption as well as lactate, lactate dehydrogenase, hCG, estradiol, and progesterone release. RESULTS: Oxygen consumption was directly proportional to oxygen tension. Glucose consumption was lowest at low oxygen tension, while both lactate and LDH release were lowest at high oxygen tension. Both hCG and progesterone release rates were lowest at high oxygen tensions. Estradiol release demonstrated a trend similar to that of the other hormones although there was no statistically significant difference among the three different levels of oxygen tension. CONCLUSION: Varying oxygen tensions affect placental villous glucose metabolism and hormone release. Under lower oxygen tensions, glucose is metabolized through glycolysis rather than through oxidative phosphorylation and is associated with higher lactate release. Exposure to higher oxygen tensions results in reduced hCG and progesterone release. Higher oxygen tensions may be associated with tissue toxicity.

Simple ovarian cysts. Clinical features on a first-trimester ultrasound scan.

OBJECTIVE: To determine the clinical features of simple ovarian cysts, often seen on a first-trimester ultrasound scan. STUDY DESIGN: All initial examinations from patients scanned in 1993 (N = 1,001) were reviewed, and the clinical features of simple ovarian cysts were determined. Relationships were determined between the presence of a cyst and first-trimester pregnancy outcomes, including progression beyond the first trimester, blighted ovum, ectopic pregnancy and fetal demise. RESULTS: A simple ovarian cyst was observed in 29% of patients. Cysts were seen less often after 8 weeks' gestation, and there was an equal distribution between those on the right and those on the left side of the pelvis. Cyst diameters did not vary with gestational age, and most mean diameters were within a range of 1-3 cm. The absence of a cyst was more often associated with a blighted ovum on follow-up ultrasound scan (relative risk, 2.8), but its presence or absence did not correlate with failure to progress beyond the first trimester, ectopic pregnancy or intrauterine fetal demise. CONCLUSION: The presence of simple ovarian cysts in the first trimester, which may represent corpus luteal cysts, appears to support early pregnancy development due to its association with a lower incidence of blighted ova.

Third ventricle: size and appearance in normal fetuses through gestation.

PURPOSE: To define the size and appearance of the normal fetal third ventricle. MATERIALS AND METHODS: The third ventricle was prospectively assessed in 441 consecutive normal second- and third-trimester fetuses. The fetuses were divided into six gestational age ranges. Data regarding the size and configuration of the third ventricle were analyzed for each group. RESULTS: The third ventricle was seen in 435 of 440 (98.9%) fetuses. It appeared as a single echogenic line between the thalami in 171 (38.9%) fetuses, as parallel echogenic lines outlining a fluid-filled lumen in 243 (55.2%) fetuses, and as divergent lines delineating a V-shaped fluid-filled structure in 21 (4.8%) fetuses. The single-line configuration was most common early in the second trimester. Later in pregnancy, the ventricle walls could be discerned as separate parallel or divergent lines outlining a fluid-filled lumen. The average width of the ventricle was relatively constant at approximately 1 mm from 12 to 28 weeks. After this time, it enlarged, reaching a maximum 1.9 mm. CONCLUSION: The third ventricle can be imaged in most second- and third-trimester fetuses. Its size and configuration evolve through the second and third trimesters. This evolution must be considered in the evaluation of normality. At any gestational age, a third ventricle greater than 3.5 mm in width should be viewed with concern for abnormality.

Intravillous eicosanoid compartmentalization and regulation of placental blood flow.

OBJECTIVE: To determine the roles of the eicosanoids thromboxane and prostacyclin, and their compartmentalization, in the regulation of placental blood flow. METHODS: First, the sites of production of thromboxane and prostacyclin were determined within the placental villus using immunohistochemical staining for thromboxane and prostacyclin synthetase. Second, the production of both eicosanoids was studied in cultured trophoblasts and compared with that in the villous core by measuring the metabolites thromboxane B2 and 6-keto-prostaglandin F 1 alpha. Finally, eicosanoid production was assessed in intact villi after stimulation by an acute change in oxygen content, 5% to 95%. RESULTS: Immunohistochemical staining showed that thromboxane production was primarily within the trophoblasts, whereas prostacyclin production was localized to the endothelial cells within the villi. In culture, we found preferential production of prostacyclin by the villous core cells and increased production of thromboxane by trophoblasts. Perifusion of intact villi demonstrated increased production of thromboxane by trophoblasts in response to an increase in oxygen content. Prostacyclin levels were too low to be detected. CONCLUSIONS: Placental blood flow appears to be regulated by compartmentalized eicosanoids, with thromboxane affecting primarily the maternal side of the placental circulation and prostacyclin affecting primarily the fetal side.

Discrepancies in cytogenetic findings in chorionic villi.

We present a database and a review of published literature on discrepancies in chorionic villus (CV) diagnostic findings. The review includes 457 cases of discrepancies between CV findings (direct, culture, or both) and the fetus. One hundred and one cases reported normal by CV direct harvest included 30 with abnormal or mosaic abnormal fetal karyotype (30% false negatives). The corresponding number of false negatives for CV culture was only 4 cases out of 133 (3%). Assuming no bias in reporting cases based on site of discrepancy (assumption may not hold), these data imply that the probability of false-negative findings is 10-fold higher by CV direct method compared to CV culture method. We also reviewed recent studies reporting on large series of CV diagnosis. This review revealed that the reported overall frequencies of discrepancies as a percentage of abnormal and mosaic abnormal CV results ranged from 11 to 63% (a mean of 37%). These data, together with recent reports of survival of embryos with reported abnormal karyotypes because of confined placental mosaicism (CPM), raise several questions pertaining to the predictive value of CV sampling and the origin of the discrepancies in the fetal-placental unit. Caution is recommended in counseling patients undergoing CV sampling to provide appropriate follow-up studies in cases of possible discrepancies.

Plasma and erythrocyte profiles of nonesterified polyunsaturated fatty acids during normal pregnancy and labor.

This study was undertaken to determine the plasma and erythrocyte profiles of metabolically important nonesterified polyunsaturated fatty acids (PUFA), that is, free fatty acids, in each trimester of pregnancy and in labor. Blood was drawn from patients in the first, second, and third trimester and in labor. Nonesterified polyunsaturated fatty acids were extracted from erythrocytes with chloroform and methanol. The PUFAs from erythrocytes and plasma were then measured in their methylester form using high performance liquid chromatography (HPLC). Plasma levels of all PUFAs were similar in each trimester of pregnancy but levels of linoleic and linolenic acids were higher in laboring patients. Plasma levels of linoleic and arachidonic acid in the n-6 pathway (range 40 to 162 mg/L plasma) were higher than linolenic, eicosapentaenoic, and docosahexaenoic acids measured in the n-3 pathway (range 2.1 to 12.8 mg/L plasma). PUFA levels in erythrocytes were generally higher in the second trimester (range 2.6 to 79.7 mg/100-microL spun erythrocytes). In these erythrocytes, docosahexaenoic acid in the n-3 pathway and linoleic and arachidonic acids in the n-6 pathways were present in the highest amounts. Polyunsaturated fatty acids appear to be absorbed and mobilized in increasing amounts in plasma and erythrocytes with advancing gestational age and labor. This activity appears to be most pronounced in the second trimester. Further investigations into PUFA metabolism and the mechanisms which govern it could lead to a better understanding of the role of these important substances in normal and abnormal pregnancies as well as in the initiation of labor.

Dysmorphologic features of the fetal pelvis in Down syndrome: prenatal sonographic depiction and diagnostic implications of the iliac angle.

PURPOSE: To determine whether the axial pelvic profile is morphologically different in fetuses with Down syndrome from those with a normal karyotype. MATERIALS AND METHODS: Pelvic images were selected from ultrasound studies in 27 fetuses with trisomy 21 and in 135 fetuses with a normal karyotype. An observer blinded to study results measured the angle formed by the convergence of lines drawn tangent to the wing of the ilium. This angle was measured prospectively in 20 normal fetuses by four independent observers to estimate variability. RESULTS: The iliac bones could be assessed in 19 fetuses with trisomy 21 and in 87 fetuses with a normal karyotype. Between 15 and 20 weeks of gestation, the mean iliac angle was 60 degrees in normal fetuses and 75 degrees in fetuses with Down syndrome (P < .001). Intra- and interobserver correlation coefficients were .70 and .62, respectively. The greatest variability in results was among fetuses (estimated variance, 72.5); smaller variance was seen with repeat measurements in the same fetus (34.7) and with measurements by different observers (9.1). CONCLUSION: The mean iliac angle in fetuses with Down syndrome is larger than that in fetuses without Down syndrome and may aid in weighing the risks of trisomy 21 against the risks of performing amniocentesis.