Serum hyperglycosylated human chorionic gonadotropin to predict the gestational outcome in in vitro fertilization/intracytoplasmic sperm injection pregnancies

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted by the placenta in early pregnancy. Decreased H-hCG levels have been associated with abortion in spontaneous pregnancy. We retrospectively measured H-hCG and dimeric hCG in the sera of 87 in vitro fertilization patients obtained in the 3 weeks following embryo transfer and set the results in relation to pregnancy outcome. H-hCG and dimeric hCG were correlated (r(2) = 0.89), and were significantly decreased in biochemical pregnancy (2 microg/l and 18 IU/l, respectively) compared to early pregnancy loss (22 microg/l and 331 IU/l) and ongoing pregnancy (32 microg/l and 353 IU/l). Only H-hCG tended to discriminate between these last two groups.

Tolerance of human placental tissue to severe hypoxia and its relevance for dual ex vivo perfusion

In the dual ex vivo perfusion of an isolated human placental cotyledon it takes on average 20-30 min to set up stable perfusion circuits for the maternal and fetal vascular compartments. In vivo placental tissue of all species maintains a highly active metabolism and it continues to puzzle investigators how this tissue can survive 30 min of ischemia with more or less complete anoxia following expulsion of the organ from the uterus and do so without severe damage. There seem to be parallels between "depressed metabolism" seen in the fetus and the immature neonate in the peripartum period and survival strategies described in mammals with increased tolerance of severe hypoxia like hibernators in the state of torpor or deep sea diving turtles. Increased tolerance of hypoxia in both is explained by "partial metabolic arrest" in the sense of a temporary suspension of Kleiber's rule. Furthermore the fetus can react to major changes in surrounding oxygen tension by decreasing or increasing the rate of specific basal metabolism, providing protection against severe hypoxia as well as oxidative stress. There is some evidence that adaptive mechanisms allowing increased tolerance of severe hypoxia in the fetus or immature neonate can also be found in placental tissue, of which at least the villous portion is of fetal origin. A better understanding of the molecular details of reprogramming of fetal and placental tissues in late pregnancy may be of clinical relevance for an improved risk assessment of the individual fetus during the critical transition from intrauterine life to the outside and for the development of potential prophylactic measures against severe ante- or intrapartum hypoxia. Responses of the tissue to reperfusion deserve intensive study, since they may provide a rational basis for preventive measures against reperfusion injury and related oxidative stress. Modification of the handling of placental tissue during postpartum ischemia, and adaptation of the artificial reperfusion, may lead to an improvement of the ex vivo perfusion technique.

Cloning and characterization of a novel zinc finger protein (rZFP96) in the rat corpus luteum

The corpus luteum (CL) is a temporary organ involved in the maintenance of pregnancy. In the course of its life-cycle, the CL undergoes two distinct and consecutive processes for its inevitable removal through apoptosis: functional and structural luteolysis. We isolated a gene encoding for a novel rat zinc finger protein (ZFP), named rat ZFP96 (rZFP96) from an ovarian lambda cDNA library. Sequence analysis revealed close sequence and structural similarity to mouse ZFP96 and human zinc finger protein 305 (ZNF305). Quantitative reverse transcription-polymerase chain reaction analysis revealed a positive correlation with the end of pregnancy, that is, the onset of structural luteolysis of the CL. Messenger RNA levels increased 3-fold (P < 0.01) between days 13 and 22 of pregnancy and 8-fold (P < 0.01) between day 13 of pregnancy and day 1 post-partum. In addition, we detected rZFP96 expression in mammary, placenta, heart, kidney and skeletal muscle. Sequence analysis predicted that rZFP96 has a high probability of localizing to the nuclear compartment. The presence of both a perfect consensus TGEKP linker sequence between zinc fingers 2 and 3 as well as several similar sequences between the other zinc fingers suggests physical interaction with DNA. Speculatively, rZFP96 may therefore function as a transcription factor, switching-off pro-survival genes and/or upregulating pro-apoptotic genes and thereby contributing to the demise of the CL.

Fetal programming of pulmonary vascular dysfunction in mice: role of epigenetic mechanisms.

Insults during the fetal period predispose the offspring to systemic cardiovascular disease, but little is known about the pulmonary circulation and the underlying mechanisms. Maternal undernutrition during pregnancy may represent a model to investigate underlying mechanisms, because it is associated with systemic vascular dysfunction in the offspring in animals and humans. In rats, restrictive diet during pregnancy (RDP) increases oxidative stress in the placenta. Oxygen species are known to induce epigenetic alterations and may cross the placental barrier. We hypothesized that RDP in mice induces pulmonary vascular dysfunction in the offspring that is related to an epigenetic mechanism. To test this hypothesis, we assessed pulmonary vascular function and lung DNA methylation in offspring of RDP and in control mice at the end of a 2-wk exposure to hypoxia. We found that endothelium-dependent pulmonary artery vasodilation in vitro was impaired and hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in vivo were exaggerated in offspring of RDP. This pulmonary vascular dysfunction was associated with altered lung DNA methylation. Administration of the histone deacetylase inhibitors butyrate and trichostatin A to offspring of RDP normalized pulmonary DNA methylation and vascular function. Finally, administration of the nitroxide Tempol to the mother during RDP prevented vascular dysfunction and dysmethylation in the offspring. These findings demonstrate that in mice undernutrition during gestation induces pulmonary vascular dysfunction in the offspring by an epigenetic mechanism. A similar mechanism may be involved in the fetal programming of vascular dysfunction in humans.

Placental ABCA1 and ABCG1 expression in gestational disease: pre-eclampsia affects ABCA1 levels in syncytiotrophoblasts

INTRODUCTION Transplacental feto-maternal lipid exchange through the ATP-binding cassette transporters ABCA1 and ABCG1 is important for normal fetal development. However, only scarce and conflicting data exist on the involvement of these transporters in gestational disease. METHODS Placenta samples (n = 72) derived from common gestational diseases, including pre-eclampsia (PE), HELLP, intrauterine growth restriction (IUGR), intrahepatic cholestasis of pregnancy and gestational diabetes, were assessed for their ABCA1 and ABCG1 expression levels and compared to age-matched control placentas with qRT-PCR and immunohistochemistry. ABCA1 expression was additionally investigated with immunoblot in placental membrane vesicles. Furthermore, placental cholesterol and phospholipid contents were assessed. RESULTS ABCA1 mRNA levels differed significantly between preterm and term control placentas (p = 0.0013). They were down-regulated in isolated PE and PE with IUGR (p = 0.0006 and p = 0.0012, respectively), but unchanged in isolated IUGR, isolated HELLP and other gestational diseases compared to gestational age-matched controls. Correspondingly, in PE, ABCA1 protein expression was significantly reduced in the apical membrane of the villous syncytiotrophoblast (p = 0.011) and in villous fetal endothelial cells (p = 0.036). Furthermore, in PE there was a significant increase in the placental content of total and individual classes of phospholipids which were partially correlated with diminished ABCA1 expression. Conversely, ABCG1 mRNA and protein levels were stable in the investigated conditions. CONCLUSIONS In gestational disease, there is a specific down-regulation of placental ABCA1 expression at sites of feto-maternal lipid exchange in PE. At a functional level, the increase in placental lipid concentrations provides indirect evidence of an impaired transport capacity of ABCA1 in this disease.

Human TRPV5 and TRPV6: key players in cadmium and zinc toxicity

TRPV5 and TRPV6 are two major calcium transport pathways in the human body maintaining calcium homeostasis. TRPV5 is mainly expressed in the distal convoluted and connecting tubule where it is the major, regulated pathway for calcium reabsorption. TRPV6 serves as an important calcium entry pathway in the duodenum and the placenta. Previously, we showed that human TRPV6 (hTRPV6) transports several heavy metals. In this study we tested whether human TRPV5 (hTRPV5) also transports cadmium and zinc, and whether hTRPV5 together with hTRPV6 are involved in cadmium and zinc toxicity. The hTRPV5 mRNA and protein were expressed in HEK293 cells transiently transfected with pTagRFP-C1-hTRPV5. The overexpression of the hTRPV5 protein at the plasma membrane was revealed by cell surface biotinylation and immunofluorescence techniques. We observed that both cadmium and zinc permeate hTRPV5 in ion imaging experiments using Fura-2 or Newport Green DCF. Our results were further confirmed using whole-cell patch clamp technique. Transient overexpression of hTRPV5 or hTRPV6 sensitized cells to cadmium and zinc. Toxicity curves of cadmium and zinc were also shifted in hTRPV6 expressing HEK293 cells clones. Our results suggest that TRPV5 and TRPV6 are crucial gates controlling cadmium and zinc levels in the human body especially under low calcium dietary conditions, when these channels are maximally upregulated.

SLC13 family of Na⁺-coupled di- and tri-carboxylate/sulfate transporters

The SLC13 family comprises five genes (SLC13A1, SLC13A2, SLC13A3, SLC13A4, and SLC13A5) encoding structurally related multi-spanning transporters (8-13 transmembrane domains) with orthologues found in prokaryotes and eukaryotes. Mammalian SLC13 members mediate the electrogenic Na(+)-coupled anion cotransport at the plasma membrane of epithelial cells (mainly kidney, small intestine, placenta and liver) or cells of the central nervous system. While the two SLC13 cotransporters NaS1 (SLC13A1) and NaS2 (SLC13A4) transport anions such sulfate, selenate and thiosulfate, the three other SLC13 members, NaDC1 (SLC13A2), NaCT (SLC13A5) and NaDC3 (SLC13A3), transport di- and tri-carboxylate Krebs cycle intermediates such as succinate, citrate and α-ketoglutarate. All these transporters play a variety of physiological and pathophysiological roles in the different organs. Thus, the purpose of this review is to summarize the roles of SLC13 members in human physiology and pathophysiology and what the therapeutic perspectives are. We have also described the most recent advances on the structure, expression, function and regulation of SLC13 transporters.

Mammalian iron transporters: Families SLC11 and SLC40

This review is focused on the mammalian SLC11 and SLC40 families and their roles in iron homeostasis. The SLC11 family is composed of two members, SLC11A1 and SLC11A2. SLC11A1 is expressed in the lysosomal compartment of macrophages and in the tertiary granules of neutrophils, playing a key role in innate resistance against infection by intracellular microbes. SLC11A2 is a key player in iron metabolism and is ubiquitously expressed, most notably in the proximal duodenum, immature erythroid cells, brain, placenta and kidney. Intestinal iron absorption is mediated by SLC11A2 at the apical membrane of enterocytes, followed by basolateral exit via SLC40A1. To meet the daily requirement for iron, approximately 80% of the iron comes from the breakdown of hemoglobin following macrophage phagocytosis of senescent erythrocytes (iron recycling). Both SLC11A1 and SLC11A2 play an important role in macrophage iron recycling. SLC11A2 also transports iron into the cytosol across the membrane of endocytotic vesicles of the transferrin receptor-cycle. SLC40A1 is the sole member of the SLC40 family and is involved in the only cellular iron efflux mechanism described. SLC40A1 is highly expressed in several tissues and cells that play a critical role in body iron homeostasis. The signaling pathways that regulate SLC11A2 and SLC40A1 expression at transcriptional, post-transcriptional and post-translational levels are discussed. The roles of SLC11A2 and/or SLC40A1 in iron-associated disorders such as hemochromatosis, neurodegenerative diseases, and breast cancer are also summarized.

The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia.

Growth-restricted fetuses are at risk for a variety of lifelong medical conditions. Preeclampsia, a life-threatening hypertensive disorder of pregnancy, is associated with fetuses who suffer from intrauterine growth restriction (IUGR). Recently, emerging evidence indicates that preeclamptic women harbor AT(1) receptor agonistic autoantibodies (AT(1)-AAs) that contribute to the disease features. However, the exact role of AT(1)-AAs in IUGR and the underlying mechanisms have not been identified. We report that these autoantibodies are present in the cord blood of women with preeclampsia and retain the ability to activate AT(1) receptors. Using an autoantibody-induced animal model of preeclampsia, we show that AT(1)-AAs cross the mouse placenta, enter fetal circulation, and lead to small fetuses with organ growth retardation. AT(1)-AAs also induce apoptosis in the placentas of pregnant mice, human villous explants, and human trophoblast cells. Finally, autoantibody-induced IUGR and placental apoptosis are diminished by either losartan or an autoantibody-neutralizing peptide. Thus, these studies identify AT(1)-AA as a novel causative factor of preeclampsia-associated IUGR and offer two possible underlying mechanisms: a direct detrimental effect on fetal development by crossing the placenta and entering fetal circulation, and indirectly through AT(1)-AA-induced placental damage. Our findings highlight AT(1)-AAs as important therapeutic targets.

Analysis of a human placental lactogen gene promoter

Human placental lactogen (hPL) and human growth hormone (hGH) comprise a multigene family that share $>$90% nucleic acid sequence homology including 500 bp of 5$\sp\prime$ flanking sequence. Despite these similarities, hGH is produced in the anterior pituitary while hPL is expressed in the placenta. For most genes studied to date, regulation of expression occurs by alterations at the level of transcriptional initiation. Nuclear proteins bind specific DNA sequences in the promoter to regulate gene expression. In this study, the hPL$\sb3$ promoter was analyzed for DNA sequences that contribute to its expression. The interaction between the hPL$\sb3$ promoter and nuclear proteins was examined using nuclear extracts from placental and non-placental cells.^ To identify regulatory elements in the promoter of the hPL$\sb3$ gene, 5$\sp\prime$ deletion mutants were constructed by cleaving 1200 bp of upstream sequence with various restriction enzymes. These DNA fragments were ligated 5$\sp\prime$ to a promoterless bacterial gene chloramphenicol acetyltransferase (CAT) and transfected into JEG-3 cells, a human placental choriocarcinoma cell line. The level of CAT activity reflects the ability of the promoter mutants to activate transcription. Deletion of the sequence between $-$142 bp and $-$129 bp, relative to the start of transcription, resulted in an 8-fold decrease in CAT activity. Nuclear proteins from JEG-3, HeLa, and HepG2 (human liver cells), formed specific binding complexes with this region of the hPL$\sb3$ promoter, as shown by gel mobility shift assay. The $-$142 bp to $-$129 bp region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. Sp1-like proteins were identified by DNA binding assay, in the nuclear extracts of the three cell lines. A series of G nucleotides in the hPL$\sb3$ promoter regulatory region were identified by methylation interference assay to interact with the DNA-binding proteins and the pattern obtained is similar to that for other Sp1 binding sites that have been studied. This suggests that hPL$\sb3$ may be transcriptionally regulated by Sp1 or a Sp1-like transacting factor. ^