André Masse

ERK1/2 and p38 regulate trophoblasts differentiation in human term placenta

Mitogen‐activated protein kinases (MAPKs) control many cellular events from complex programmes, such as embryogenesis, cell differentiation and proliferation, and cell death, to short‐term changes required for homeostasis and acute hormonal responses. However, little is known about expression and activation of classical MAPKs, extracellular signal‐regulated kinase1/2 (ERK1/2) and p38 in human placenta. Therefore, we examined the expression of ERK1/2 and p38 in trophoblasts from human term placenta, and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultivated in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). This study shows that the level of ERK1/2 and p38 decreases with increasing days of culture, to reach an undetectable level after 5 days of culture. Moreover, pretreatment of cells with an ERK1/2‐specific inhibitor (PD98059) and/or a p38‐specific inhibitor (SB203580) suppressed trophoblast differentiation. Our results also demonstrate that the p38 pathway is highly solicited as compared to the ERK1/2 pathway in the differentiation process. Furthermore, ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. In summary, this study showed that ERK1/2 and p38 pathways are essential to mediate initiation of trophoblast differentiation.

Substance Use in Pregnancy

OBJECTIVE To improve awareness and knowledge of problematic substance use in pregnancy and to provide evidence-based recommendations for the management of this challenging clinical issue for all health care providers. OPTIONS This guideline reviews the use of screening tools, general approach to care, and recommendations for clinical management of problematic substance use in pregnancy. OUTCOMES Evidence-based recommendations for screening and management of problematic substance use during pregnancy and lactation. EVIDENCE Medline, PubMed, CINAHL, and The Cochrane Library were searched for articles published from 1950 using the following key words: substance-related disorders, mass screening, pregnancy complications, pregnancy, prenatal care, cocaine, cannabis, methadone, opioid, tobacco, nicotine, solvents, hallucinogens, and amphetamines. Results were initially restricted to systematic reviews and randomized control trials/controlled clinical trials. A subsequent search for observational studies was also conducted because there are few RCTs in this field of study. Articles were restricted to human studies published in English. Additional articles were located by hand searching through article reference lists. Searches were updated on a regular basis and incorporated in the guideline up to December 2009. Grey (unpublished) literature was also identified through searching the websites of health technology assessment and health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. VALUES The quality of evidence was rated using the criteria described in the Report of the Canadian Task Force on the Preventive Health Care. Recommendations for practice were ranked according to the method described in that report (Table 1). BENEFITS, HARMS, AND COSTS This guideline is intended to increase the knowledge and comfort level of health care providers caring for pregnant women who have substance use disorders. Improved access to health care and assistance with appropriate addiction care leads to reduced health care costs and decreased maternal and neonatal morbidity and mortality. RECOMMENDATIONS 1. All pregnant women and women of childbearing age should be screened periodically for alcohol, tobacco, and prescription and illicit drug use. (III-A) 2. When testing for substance use is clinically indicated, urine drug screening is the preferred method. (II-2A) Informed consent should be obtained from the woman before maternal drug toxicology testing is ordered. (III-B) 3. Policies and legal requirements with respect to drug testing of newborns may vary by jurisdiction, and caregivers should be familiar with the regulations in their region. (III-A) 4. Health care providers should employ a flexible approach to the care of women who have substance use problems, and they should encourage the use of all available community resources. (II-2B) 5. Women should be counselled about the risks of periconception, antepartum, and postpartum drug use. (III-B) 6. Smoking cessation counselling should be considered as a first-line intervention for pregnant smokers. (I-A) Nicotine replacement therapy and/or pharmacotherapy can be considered if counselling is not successful. (I-A) 7. Methadone maintenance treatment should be standard of care for opioid-dependent women during pregnancy. (II-IA) Other slow-release opioid preparations may be considered if methadone is not available. (II-2B) 8. Opioid detoxification should be reserved for selected women because of the high risk of relapse to opioids. (II-2B) 9. Opiate-dependent women should be informed that neonates exposed to heroin, prescription opioids, methadone, or buprenorphine during pregnancy are monitored closely for symptoms and signs of neonatal withdrawal (neonatal abstinence syndrome). (II-2B) Hospitals providing obstetric care should develop a protocol for assessment and management of neonates exposed to opiates during pregnancy. (III-B) 10. Antenatal planning for intrapartum and postpartum analgesia may be offered for all women in consultation with appropriate health care providers. (III-B) 11. The risks and benefits of breastfeeding should be weighed on an individual basis because methadone maintenance therapy is not a contraindication to breastfeeding. (II-3B).

Modulation of Fatty Acid Transport and Metabolism by Maternal Obesity in the Human Full-Term Placenta

ABSTRACT Knowledge of the consequences of maternal obesity in human placental fatty acids (FA) transport and metabolism is limited. Animal studies suggest that placental uptake of maternal FA is altered by maternal overnutrition. We hypothesized that high maternal body mass index (BMI) affects human placental FA transport by modifying expression of key transporters. Full-term placentas were obtained by vaginal delivery from normal weight (BMI, 18.5–24.9 kg/m2) and obese (BMI > 30 kg/m2) women. Blood samples were collected from the mother at each trimester and from cord blood at delivery. mRNA and protein expression levels were evaluated with real-time RT-PCR and Western blotting. Lipoprotein lipase (LPL) activity was evaluated using enzyme fluorescence. In vitro linoleic acid transport was studied with isolated trophoblasts. Our results demonstrated that maternal obesity is associated with increased placental weight, decreased gestational age, decreased maternal high-density lipoprotein (HDL) levels during the first and third trimesters, increased maternal triglyceride levels during the second and third trimesters, and increased maternal T3 levels during all trimesters, and decreased maternal cholesterol (CHOL) and low-density lipoprotein (LDL) levels during the third trimester; and increased newborn CHOL, LDL, apolipoprotein B100, and T3 levels. Increases in placental CD36 mRNA and protein expression levels, decreased SLC27A4 and FABP1 mRNA and protein and FABP3 protein expression, and increased LPL activity and decreased villus cytotrophoblast linoleic acid transport were also observed. No changes were seen in expression of PPARA, PPARD, or PPARG mRNA and protein. Overall this study demonstrated that maternal obesity impacts placental FA uptake without affecting fetal growth. These changes, however, could modify the fetus metabolism and its predisposition to develop diseases later in life.

Calcium uptake and calcium transporter expression by trophoblast cells from human term placenta

Placental transfer of maternal calcium (Ca(2+)) is a crucial step for fetal development although the biochemical mechanisms responsible for this process are largely unknown. This process is carried out in vivo by the placental syncytiotrophoblast layer. The aim of this study was to define the membrane gates responsible for the syncytiotrophoblast Ca(2+) entry, the first step in transplacental transfer. We have investigated the basal Ca(2+) uptake by primary culture of human term placenta syncytiotrophoblast. Kinetic studies revealed an active extracellular Ca(2+) uptake by cultured human syncytiotrophoblast. We demonstrated by Northern blot the presence of transcript for calcium transporter type 1 (CaT1) in cultured human syncytiotrophoblast and CaT1 expression was further confirmed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the expression of calcium transporter type 2 (CaT2) was revealed by RT-PCR in cultured human syncytiotrophoblast. It has been reported that the activity of this family of Ca(2+) channels is voltage-independent, and is not sensitive to L-type Ca(2+) channels agonist and antagonist. Interestingly, modulation of membrane potential by extracellular high potassium concentration and valinomycin had no effect on the basal Ca(2+) uptake of human syncytiotrophoblast. Moreover, the addition of L-type Ca(2+) channel modulators (Bay K 8644 and nitrendipine) to the incubation medium had also no effect on the basal Ca(2+) uptake, suggesting that the process is mainly voltage-independent and does not involved L-type Ca(2+) channels. On the other hand, we observed that two known blockers of CaT-mediated Ca(2+) transport, namely extracellular magnesium (Mg(2+)) and ruthenium red, dose-dependently inhibited Ca(2+) uptake by cultured human syncytiotrophoblast. Therefore, our results suggest that basal Ca(2+) uptake of human syncytiotrophoblast may be assured by CaT1 and CaT2.

Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study.

OBJECTIVE We sought to evaluate the association between inherited thrombophilia and preeclampsia. STUDY DESIGN From a multicenter cohort of 5337 pregnant women, we prospectively identified 113 women who developed preeclampsia and selected 443 control subjects who did not have preeclampsia or nonproteinuric gestational hypertension. Blood samples were tested for DNA polymorphisms affecting thrombophilia (factor V Leiden mutation, prothrombin G20210A mutation, methylenetetrahydrofolate reductase C677T polymorphism), homocysteine, and folate levels, and placentae underwent pathological evaluation. RESULTS Thrombophilia was present in 14% of patients and 21% of control subjects (adjusted logistic regression odds ratio, 0.6; 95% confidence interval, 0.3-1.3). Placental underperfusion was present in 63% of patients vs 46% of control subjects (P < .001) and was more frequent in women with folate levels in the lowest quartile (P = .04), but was not associated with thrombophilia. CONCLUSION We did not find evidence to support an association between inherited thrombophilia and increased risk of preeclampsia. Placental underperfusion is associated with preeclampsia, but this does not appear to be consequent to thrombophilia.

Src family kinases play multiple roles in differentiation of trophoblasts from human term placenta

Tyrosine phosphorylation plays a major role in controlling many biological processes in different cell types. Src family kinases (SFKs) are one of the most studied groups of tyrosine kinases and can mediate a variety of signalling pathways. However, little is known about the expression of SFKs in human term placenta and their implication in trophoblast differentiation. Therefore, we examined the expression profile of SFK members over time in culture and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultured in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that resemble phenotypically mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). In this study, we showed that trophoblasts expressed all SFK members and some of them are expressed as different splice variants. Moreover, using real‐time PCR, this study showed two different expression profiles of SFKs in human trophoblasts during culture. In addition, the protein level and phosphorylation status of Src were evaluated using specific antibodies. Src was rapidly phosphorylated at Tyr‐416 and dephosphorylated at Tyr‐527 after FBS addition. Surprisingly, inhibition of SFKs by 4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl) pyrazolo[3,4‐d] pyrimidine (PP2) or herbimycin A had different effects on trophoblast differentiation. While herbimycin A inhibited morphological and hormonal differentiation, PP2 stimulated hormonal differentiation and inhibited cell adhesion and spreading with no effect on cell fusion. In summary, this study showed that SFKs play different roles in trophoblast differentiation, probably depending on SFK members activated. Thus, this study increases our knowledge and understanding of pathology related to impaired trophoblast differentiation such as pre‐eclampsia and trophoblast neoplasm.

Fetal and perinatal autopsy in prenatally diagnosed fetal abnormalities with normal karyotype.

OBJECTIVE To review the information on fetal and perinatal autopsies, the process of obtaining consent, and the alternative information-gathering options following a prenatal diagnosis of non-chromosomal malformations, and to assist clinicians in providing postnatal counselling regarding fetal diagnosis and recurrence risks. OUTCOMES To provide better counselling about fetal and perinatal autopsies for women and families who are dealing with a prenatally diagnosed non-chromosomal fetal anomaly. EVIDENCE Published literature was retrieved through searches of PubMed or Medline, CINAHL, and The Cochrane Library in 2009 and 2010, using appropriate key words (fetal autopsy, postmortem, autopsy, perinatal postmortem examination, autopsy protocol, postmortem magnetic resonance imaging, autopsy consent, tissue retention, autopsy evaluation). Results were restricted to systematic reviews, randomized controlled trials/controlled clinical trials, and observational studies. Additional publications were identified from the bibliographies of these articles. There were no date or language restrictions. Grey (unpublished) literature was identified through searching the websites of health technology assessment and health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. BENEFITS, HARMS, AND COSTS This update educates readers about (1) the benefits of a fetal perinatal autopsy, (2) the consent process, and (3) the alternatives when the family declines autopsy. It also provides a standardized approach to fetal and perinatal autopsies, emphasizing pertinent additional sampling when indicated. VALUES The quality of evidence was rated using the criteria described in the Report of the Canadian Task Force on Preventive Health Care (Table 1). Recommendations 1. Standard autopsy should ideally be an essential part of fully investigating fetal loss, stillbirths, and neonatal deaths associated with non-chromosomal fetal malformations. (II-3A) 2. Clinicians and health care providers approaching parents for autopsy consent should discuss the options for a full, limited, or step-wise postmortem examination; the issue of retained fetal tissues; and the value of autopsy and the possibility that the information gained may not benefit them but may be of benefit to others. This information should be provided while respecting the personal and cultural values of the families. (III-A) 3. If parents are unwilling to give consent for a full autopsy, alternatives to full autopsy that provide additional clinical information must be presented in a manner that includes disclosure of limitations. (III-A) 4. External physical examination, medical photographs, and standard radiographic or computed tomography should be offered in all cases of fetal anomaly(ies) of non-chromosomal etiology. (II-2A) 5. Well-designed, large prospective studies are needed to evaluate the accuracy of postmortem magnetic resonance imaging. It cannot function as a substitute for standard full autopsy. (III-A) 6. The fetal and perinatal autopsies should be performed by trained perinatal or pediatric pathologists. (II-2A) 7. The need for additional sampling is guided by the results of previous prenatal and/or genetic investigations, as well as the type of anomalies identified in the fetus. Fibroblast cultures may allow future laboratory studies, particularly in the absence of previous karyotyping or if a biochemical disorder is suspected, and DNA analysis. (II-3A) 8. In cases requiring special evaluation, the most responsible health care provider should have direct communication with the fetopathologist to ensure that all necessary sampling is performed in a timely manner. (II-3A) 9. The most responsible health care providers must see the families in follow-up to share autopsy findings, plan for the management of future pregnancies, obtain consent for additional testing, and offer genetic counselling to other family members when appropriate. (III-A).

Modulation of placental protein expression of OLR1 : implication in pregnancy-related disorders or pathologies.

The lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR1) is a newly described receptor for oxidatively modified LDL. The human pregnancy is associated with hyperlipidemia and oxidative stress. It has been reported that modification in maternal lipid profile can induce disturbance during pregnancy. In this study, we have evaluated the expression protein level of OLR1 in human term placenta of women having plasma cholesterol level lower to 7 mM or higher to 8 mM and women of gestational diabetes mellitus (GDM) by western blot analysis. The present study demonstrates that the maternal lipid profile is associated with placental protein expression of OLR1. A significant increase in the protein expression of OLR1 was observed in placenta of women with elevated plasmatic total cholesterol level (>8 mM). In addition, the placental protein expression of OLR1 is increased in mothers having the highest pre-pregnancy body mass index (BMI) and low (<7 mM) plasmatic total cholesterol level at term. Interestingly, the placental protein expression of OLR1 is increased in the presence of GDM pregnancies compared with normal lipids level pregnancies, without the modification of mRNA expression. In conclusion, placental OLR1 protein expression is associated with maternal lipid profile, pre-pregnancy BMI, and pathology of GDM.

Modulation of Apolipoprotein D levels in human pregnancy and association with gestational weight gain

BackgroundApolipoprotein D (ApoD) is a lipocalin involved in several processes including lipid transport, but its modulation during human pregnancy was never examined.MethodsWe investigated the changes in the levels of ApoD in the plasma of pregnant women at the two first trimesters of gestation and at delivery as well as in the placenta and in venous cord blood. These changes were studied in 151 women classified into 9 groups in relation to their prepregnancy body mass index (BMI) and gestational weight gain (GWG).ResultsPlasma ApoD levels decrease significantly during normal uncomplicated pregnancy. ApoD is further decreased in women with excessive GWG and their newborns. In these women, the ApoD concentration was tightly associated with the lipid parameters. However, the similar ApoD levels in low cholesterol (LC) and high cholesterol (HC) women suggest that the plasma ApoD variation is not cholesterol dependant. A tight regulation of both placental ApoD transcription and protein content is most probably at the basis of the low circulating ApoD concentrations in women with excessive GWG. After delivery, the plasma ApoD concentrations depended on whether the mother was breast-feeding or not, lactation favoring a faster return to baseline values.ConclusionIt is speculated that the decrease in plasma ApoD concentration during pregnancy is an adaptive response aimed at maintaining fetal lipid homeostasis. The exact mechanism of this adaptation is not known.

Calcitonin gene-related peptide receptor in human placental syncytiotrophoblast brush-border and basal plasma membranes

Minerals, such as calcium and potassium, are essential for fetal development, but their transplacental transport, and in particular, the effect of hormones on this process has not been extensively studied. Human alpha-calcitonin gene-related peptide (h alpha CGRP), a hormone constituted of 37 amino acids, is obtained by the alternative splicing of the mRNA from the calcitonin gene, and could be implicated in placental ion transport. In order to study the presence of this receptor, brush-border and basal plasma membranes were purified, and membrane binding studies were conducted using [125I]h alpha CGRP. The initiation of binding of [125I]h alpha CGRP to both membranes was rapid and reached maximal value after 10 min of incubation at 37 degrees C. Scratchard analysis revealed single-affinity binding sites for h alpha CGRP with Kd equal to 4412.45 +/- 604.81 pM and 2673.24 +/- 552.51 pM for brush-border and basal plasma membranes, respectively, which were significantly different. Moreover, the maximal number of receptors was significantly different (P < 0.001) in both membranes, with Bmax of 627.94 +/- 31.40 fmol/mg protein for brush-border membranes and 343.70 +/- 43.52 fmol/mg protein in basal-plasma membranes. Competitive displacement of [125I]h alpha CGRP with other ligands showed the following potencies; h alpha CGRP approximately h beta CGRP approximately Cys (acm)2,7 CGRP > CGRP (8-37), but no competition was observed with human and salmon calcitonin. Half-maximal displacement for human alpha- and beta CGRP was reached at approximately 10(-10)M for brush-border and basal-plasma membranes. alpha- and beta CGRP, and their fragment and analogue, stimulated cyclic AMP production in placental homogenate ranging from 143-163 per cent. Thus, our results show the presence of CGRP-specific receptors in both the syncytiotrophoblast membranes of human placenta. The role(s) of this related peptide in placenta remains to be investigated.