Richard K. Miller

Second-trimester maternal serum placental growth factor and vascular endothelial growth factor for predicting severe, early-onset preeclampsia.

OBJECTIVE To determine whether alterations in second-trimester maternal serum cytokine concentrations can identify women at risk for developing severe, early-onset preeclampsia. METHODS Patients with severe preeclampsia requiring delivery prior to 34 weeks (n = 20) were each matched by gestational age, gravidity, parity, and sample freezing time with three healthy controls who delivered at term (n = 60). By using second-trimester maternal sera originally collected for fetal aneuploidy screening, the concentrations of placental growth factor, vascular endothelial growth factor, granulocyte colony-stimulating factor, endothelin-1, and human chorionic gonadotropin were compared between patients and controls. Logistic regression analysis was used to estimate odds ratios for high versus low (median split) cytokine concentrations with respect to the development of severe, early-onset preeclampsia. Receiver operating characteristic (ROC) curves based on a second logistic regression, using actual cytokine values, were plotted to illustrate reciprocal impact on sensitivity and specificity. RESULTS Placental growth factor and vascular endothelial growth factor levels were significantly lower in patients than in controls. No significant differences were observed for the other cytokines. The odds ratios (with 95% confidence intervals) were 15.54 (3.29, 73.40) for vascular endothelial growth factor and 4.20 (1.35, 13.06) for placental growth factor. Receiver operating characteristic analysis of placental growth factor and vascular endothelial growth factor confirmed that both were useful in discriminating between patients and controls. Models combining both vascular endothelial growth factor and placental growth factor provided the best performance for identifying patients at risk for developing severe, early-onset preeclampsia, according to both odds ratios and ROC analyses. CONCLUSION Combined analysis of placental growth factor and vascular endothelial growth factor is potentially useful as a tool for early identification of patients at risk for developing severe, early-onset preeclampsia.

Villous culture of first trimester human placenta-model to study extravillous trophoblast (EVT) differentiation

During implantation and subsequent placentation the human extravillous trophoblast (EVT) cells invade the endometrium and maternal vasculature within the uterus. The origin of the EVT and signals triggering its differentiation, migration and invasion are poorly understood. First and second trimester human chorionic villi explants were used as a source of EVT and a variety of substrates which resemble extracellular matrix (ECM) in vivo have been tested to induce EVT differentiation and migration. The obtained results demonstrate that villous explants from both 5-7 and 8-10 weeks of gestation give rise to EVT cells in vitro if maintained on the surface of Matrigel or decidual extract supplemented collagen gel. Fetal calf serum (FCS) supplemented media was essential for EVT differentiation and villous trophoblast viability. Immunostaining of both EVT cells and cells from the cytotrophoblastic column with monoclonal antibody Ki67 (cell proliferation marker) indicate that EVT cells differentiate in vitro by proliferation from the tip of anchoring villi. These mononucleated, round-shaped, migrating cells are HLA-A,B,C class I antigen (W6/32) antibody and low molecular weight cytokeratin positive, and do not immunostain with PAI-1 (plasminogen activator inhibitor) and HPL antibodies. Differentiation of EVT was restricted to first trimester villous tissue; explants from second trimester placentae did not give rise to EVT. Tissue viability as monitored by glucose utilization, lactate, progesterone and hCG production rates correlated with EVT differentiation. The production rates for hCG demonstrated significant variation among individual placentae and was maintained constant for 10 days consistently only in explants cultured on decidual extract supplemented collagen matrix. The described villous tissue culture system may be, therefore, a unique in vitro model to study proliferation and differentiation of EVT from cytotrophoblastic columns, the regulation of EVT proliferation and differentiation, the role of ECM in the induction of the migration and the interaction of extravillous and villous trophoblast at the level of the cytotrophoblastic column.

Teratogenic mechanisms of medical drugs

BACKGROUND Although prescription drug use is common during pregnancy, the human teratogenic risks are undetermined for more than 90% of drug treatments approved in the USA during the past decades. A particular birth defect may have its origins through multiple mechanisms and possible exposures, including medications. A specific pathogenic process may result in different outcomes depending upon factors such as embryonic age at which a drug is administered, duration and dose of exposure and genetic susceptibility. This review focuses on the teratogenic mechanisms associated with a number of medications. METHODS We used three methods to identify the teratogenic mechanisms of medications: the MEDLINE and EMBASE databases, two recent books on teratogenic agents and a list of drugs classified as U.S. Food and Drug Administration class D or X. Mechanisms were included only if they are associated with major structural birth defects and medications that are used relatively frequently by women of reproductive age. RESULTS We identified six teratogenic mechanisms associated with medication use: folate antagonism, neural crest cell disruption, endocrine disruption, oxidative stress, vascular disruption and specific receptor- or enzyme-mediated teratogenesis. Many medications classified as class X are associated with at least one of these mechanisms. CONCLUSIONS Identifying teratogenic mechanisms may not only be relevant for etiologic and post-marketing research, but may also have implications for drug development and prescribing behavior for women of reproductive age, especially since combinations of seemingly unrelated prescription and over the counter medications may utilize similar teratogenic mechanisms with a resultant increased risk of birth defects.

Toxicity of cadmium in the perfused human placenta

Cadmium, a placental toxicant in rodents, was studied in the in vitro isolated dually perfused human placental lobule for periods of up to 12 hr to determine if cadmium can also be toxic in the human placenta. Placental lobules were perfused with a modified M199 medium containing 0, 10, 20, or 100 nmol of cadmium chloride/ml added only initially to the maternal perfusate. Every 4 hr, the perfusates in both the maternal and the fetal circuits were replaced with fresh perfusate containing no Cd. Measurements during perfusion were oxygen consumption, net fetal oxygen transfer, fetal pressure, fetal volume loss, glucose utilization, lactate production, human chorionic gonadotropin (hCG), and zinc transfer. Postperfusion, morphology, and tissue slice studies were performed to evaluate cellular metabolic function and uptake of an amino acid (alpha-[14C]-aminoisobutyric acid). In all cadmium experiments, there were no significant alterations in oxygen consumption, lactate production, glucose utilization, or amino acid uptake compared with controls; however, there were dose-related changes in the synthesis and release of the protein hormone, hCG, beginning within 4 hr of initial exposure to Cd. There were also dose-related volume loss from the fetal vasculature (greater than 6 ml/hr) and ultrastructural changes (subsyncytiotrophoblastic vesiculations, stromal edema, vacuoles in Hofbauer cells), with necrosis at 100 nmol Cd/ml occurring between 5 and 8 hr. Cadmium (10 nmol/ml) reduced the placental transfer of zinc into the fetal circuit. Thus, the human placenta is a site for toxic action of cadmium and is at least as sensitive as the rodent placenta to the actions of cadmium. In addition, these human studies demonstrated a selectivity in the toxic effects with a maintenance of carbohydrate metabolism and amino acid uptake even after 12 hr of exposure with placental Cd burdens of 151 +/- 37 nmol/g, but with the earliest (within 4 hr) dose-related functional alterations occurring in protein hormone production and zinc transfer followed by later changes in morphology with a tissue Cd burden of 46.5 +/- 4.0 nmol/g.

Periconceptional vitamin a use: How much is teratogenic?

The objective of the review is to determine whether preformed vitamin A (retinol and retinyl esters) is teratogenic at dosages commonly used by women living in industrialized countries. Published human and animal data and research developed by the authors are reviewed. It is well known that vitamin A is essential for normal reproduction and development. Although doses of 10,000 IU/d or less of preformed vitamin A (retinyl esters and retinol) are considered safe, doses > 10,000 IU/d as supplements have been reported to cause malformations in a single epidemiologic study. Nonhuman primate data show no teratogenicity at doses of 30,000 IU/d. Daily periconceptional exposures greater than 25,000 IU/d of preformed vitamin A have not been sufficiently studied to establish specific risk. Because no study reports adverse effects of 10,000 IU/d preformed vitamin A supplements and this dose is more than the Recommended Dietary Allowance for pregnant women (2670 IU or 800 RE/d), we recommend that women living in industrialized countries or who otherwise have nutritionally adequate diets may not need to ingest more than the Recommended Dietary Allowance of preformed vitamin A as supplements. If periconceptional vitamin A exposures to levels up to 30,000 IU/d (9,000 micrograms RE/d) do occur unintentionally, multiple animal studies do support only very low risk. Human epidemiologic studies do not establish at what level vitamin A becomes teratogenic; however, pharmacokinetic data presented in this paper indicate that blood levels of retinoids from women taking 30,000 IU/d of preformed vitamin A are not greater than retinoid blood levels in pregnant women during the first trimester who delivered healthy babies. Interestingly, neither teratogenicity nor vitamin A toxicity has been observed in multiple species exposed to high doses of beta-carotene.

Fetal toxicity of cadmium in the rat: Decreased utero-placental blood flow

Abstract Previous studies have shown that fetuses can tolerate direct injections of CdCl 2 in amounts greater than those which cross the placenta following maternal injections. Thus, fetal death is not the result of direct effects of cadmium on the fetus. Alterations in utero-placental blood flow were investigated as a possible mechanism of fetal toxicity. Organ blood flow was measured using the microsphere technique. Utero-placental blood flow was unchanged from control values 8–10 hr following 40 μmol/kg CdCl 2 on Day 18 but was reduced 40 and 73% from control values at 12–16 and 18–24 hr, respectively. Fetal death increased from 5 to 20 to 60% during the same time periods. Blood flow to maternal organs was unchanged from control values with the exception of the adrenal after 12–16 hr and myometrium at 18–24 hr. Histologic examination of placentae indicated that the placental necrosis, fetal death, and utero-placental blood flow alterations occurred over a similar time period. Thus, a decrease in placental blood flow was associated with placental necrosis and fetal death. However, it is not apparent whether placental necrosis was a cause or an effect of the decrease in utero-placental blood flow.

An immunodeficiency characterized by defective signal transduction in T lymphocytes.

We studied a nine-year-old boy with severe, recurrent infections. The patient was exposed in utero to azathioprine and prednisone. He had autoimmune hemolytic anemia, bronchiectasis, and Hodgkins disease. The patients circulating lymphocytes were normal in number and phenotype, but stimulation of the T-cell receptor by antigens, mitogens, and monoclonal antibodies failed to induce interleukin-2-receptor expression, interleukin-2 synthesis, or lymphocyte proliferation. The early biochemical events necessary to initiate lymphocyte activation--accumulation of the second messenger diacylglycerol, activation of the enzyme protein kinase C, and elevation of the free intracellular calcium concentration--failed to occur in this patients lymphocytes. The defect in the lymphocyte could be corrected in vitro by two agents that bypass the receptor-mediated signal mechanism (the diacylglycerol analogue phorbol and the calcium ionophore ionomycin). Further studies localized the defect in signal transduction to the interaction between cell-surface receptors and the guanine nucleotide-binding protein. We conclude that this patients immunodeficiency was caused by a defective coupling of surface receptors to signal-transducing proteins in his T lymphocytes, resulting in failure of lymphocyte activation.

Transfer of PAMAM dendrimers across human placenta: prospects of its use as drug carrier during pregnancy.

Dendrimers offer significant potential as nanocarriers for targeted delivery of drugs and imaging agents. The objectives of this study were to evaluate the transplacental transport, kinetics and biodistribution of PAMAM dendrimers ex-vivo across the human placenta in comparison with antipyrine, a freely diffusible molecule, using dually perfused re-circulating term human placental lobules. The purpose of this study is to determine if dendrimers as drug carriers can be used to design drug delivery systems directed at selectively treating either the mother or the fetus. The transplacental transfers of fluorescently (Alexa 488) tagged PAMAM dendrimer (16 kDa) and antipyrine (188 Da) from maternal to fetal circulation were measured using HPLC/dual UV and fluorescent detector (sensitivity of 10 ng/mL for dendrimer and 100 ng/mL for antipyrine respectively). C(max) for the dendrimer-Alexa (DA) in maternal perfusate (T(max)=15 min) was 18 times higher than in the fetal perfusate and never equilibrated with the maternal perfusate during 5.5 h of perfusion (n=4). DA exhibited a measurable but low transplacental transport of 2.26±0.12 μg/mL during 5.5h, where the mean transplacental transfer was 0.84±0.11% of the total maternal concentration and the feto-maternal ratio as percent was 0.073%±0.02. The biochemical and physiological analysis of the placentae perfused with DA demonstrated normal function throughout the perfusion. The immunofluorescence histochemistry confirmed that the biodistribution of DA in perfused placenta was sparsely dispersed, and when noted was principally seen in the inter-villous spaces and outer rim of the villous branches. In a few cases, DA was found internalized and localized in nuclei and cytoplasm of syncytiotrophoblast and inside the villous core; however, DA was mostly absent from the villous capillaries. In conclusion, the PAMAM dendrimers exhibited a low rate of transfer from maternal to the fetal side across the perfused human placenta, which is similar to other investigations of large macromolecules, e.g., IgG. These overall findings suggest that entry of drugs conjugated to polymers, i.e., dendrimers, would be limited in their transfer across the human placenta when compared to smaller drug molecules alone, suggesting novel methods for selectively delivering therapeutics to the pregnant woman without significant transfer to the fetus, especially since the half life of the dendrimer in blood is relatively short.

Prenatal Cocaine Exposure to the Fetus: A Sheep Model for Cardiovascular Evaluation

Transplacental passage of cocaine in response to maternal administration of intravenous (IV) cocaine in doses of 1.0 and 2.0 mg/kg was studied in 6 pregnant ewes and fetuses and correlated with maximum changes in maternal and fetal blood pressures (BP), heart rates (HR) and fetal arterial blood gas values. Certain animals were given larger doses (3.0 and 5.0 mg/kg) of cocaine to examine cocaine-related cardiopulmonary and neurologic sequelae. Cocaine was extracted on C-18 sorbent columns and analyzed by gas chromatography. At 1.0 and 2.0 mg/kg, cocaine produced dose-dependent increases in maternal HR and BP which were maximum by 1 minute. The fetal response was characterized by maximum increases in BP and decreases in PO2 by 3 minutes and increases in HR by 15 minutes. Cocaine rapidly appeared in the fetal circulation, was approximately 15% of maternal concentrations by 5 minutes, and was undetectable in both circulations by 60 minutes. At cocaine doses of 3.0 and 5.0 mg/kg significant maternal cardiopulmonary and neurologic complications were encountered including bradyarrhythmias, respiratory distress, seizure and death. These data indicate that cocaine exerts direct drug actions upon maternal cardiovascular and neurologic function. In addition, cocaine affects fetal cardiovascular function directly via transplacental passage and indirectly by fetal hypoxemia from cocaine-induced uterine artery vasoconstriction. (NIDA 04415)

Effect of maternal diabetes on the embryo, fetus, and children: Congenital anomalies, genetic and epigenetic changes and developmental outcomes

INTRODUCTION Pregestational and gestational diabetes mellitus (PGDM; GDM) are significant health concerns because they are associated with an increased rate of malformations and maternal health complications. METHODS We reviewed the data that help us to understand the effects of diabetes in pregnancy. RESULTS Diabetic embryopathy can affect any developing organ system, but cardiovascular and neural tube defects are among the most frequent anomalies. Other complications include preeclampsia, preterm delivery, fetal growth abnormalities, and perinatal mortality. Neurodevelopmental studies on offspring of mothers with diabetes demonstrated increased rate of Gross and Fine motor abnormalities, of Attention Deficit Hyperactivity Disorder, learning difficulties, and possibly also Autism Spectrum Disorder. The mechanisms underlying the effects of maternal hyperglycemia on the developing fetus may involve increased oxidative stress, hypoxia, apoptosis, and epigenetic changes. Evidence for epigenetic changes are the following: not all progeny are affected and not to the same extent; maternal diet may influence pregnancy outcomes; and maternal diabetes alters embryonic transcriptional profiles and increases the variation between transcriptomic profiles as a result of altered gene regulation. Research in animal models has revealed that maternal hyperglycemia is a teratogen, and has helped uncover potential therapeutic targets which, when blocked, can mitigate or ameliorate the negative effects of diabetes on the developing fetus. CONCLUSIONS Tight metabolic control, surveillance, and labor management remain the cornerstone of care for pregnant women with diabetes, but advances in the field indicate that new treatments to protect the mother and baby are not far from becoming clinical realities.