Placental protein 13 and decidual zones of necrosis: an immunologic diversion that may be linked to preeclampsia.

We evaluated the role of placental protein 13 (PP13; galectin 13) in the process of trophoblast invasion and decidual necrosis. Immunohistochemical analysis for PP13, immune cells, human placental lactogen, cytokeratin, and apoptosis markers was performed on 20 elective pregnancy termination specimens between 6 and 15 weeks of gestation. Placental protein 13 was localized to syncytiotrophoblasts in the chorionic villi and to occasional multinucleated luminal trophoblasts within converted decidual spiral arterioles. Cytotrophoblasts, anchoring trophoblasts, and invasive trophoblasts did not stain for PP13. Extracellular PP13 aggregates were found around decidual veins associated with T-cell-, neutrophil- and macrophage-containing decidual zones of necrosis (ZONEs). We hypothesize that PP13 is secreted into the intervillus space, drains through the decidua basalis veins, and forms perivenous PP13 aggregates which attract and activate maternal immune cells. Thus, syncytiotrophoblast-derived PP13 may create a ZONE that facilitates trophoblast invasion and conversion of the maternal spiral arterioles.

Placental and trophoblastic in vitro models to study preventive and therapeutic agents for preeclampsia.

In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the syndrome already in the first trimester of pregnancy. At the same time, there is the need for in vitro models to test such biomarkers prior to their use in clinical trials. In addition, in vitro models may accelerate the development and evaluation of the benefit of any putative therapeutics. Therefore, in vitro systems have been established to evaluate the release of biomarkers and measure the effect of putative therapeutics using placental villous explants as well as the choriocarcinoma cell line BeWo. For explants, a cryogenic method to freeze, transport and thaw villous explants was developed to use such tissues for a multi-site tissue culture evaluation. Here we focus on three out of many in vitro models that have been established for human placental trophoblast. (1) Choriocarcinoma cell lines such as BeWo, Jeg-3 and Jar cells (2) isolated primary trophoblast cells, and (2) villous explants from normal placentas delivered at term. Cell lines were used to assess the effect of differentiation and fusion on the expression and release of a preeclampsia marker (placental protein 13; PP13) and beta-hCG. Moreover, cell lines were used to study the effect of putative preeclampsia therapeutics such as vitamins C and E, heparin and aspirin on marker release and viability. Cryopreservation of villous explants enabled shipment to a remote laboratory and testing of parameters in different countries using explants from one and the same placenta. Recently published data make it tempting to speculate that the choriocarcinoma cell line BeWo as well as fresh and cryogenically stored placental villous explants may well serve as in vitro models to study preventive and therapeutic agents in the field of preeclampsia.

Cryogenic and low temperature preservation of human placental villous explants - a new way to explore drugs in pregnancy disorders.

BACKGROUND: A major handicap in cell culture studies using human tissues is the insufficient availability of fresh material on site. A method was developed for cryogenic storage and low temperature preservation of human placental villous explants, facilitating multi-site distribution for functional studies. METHODS: Explants from term placentas were incubated with cryoprotectant agents (dimethyl-sulfoxide (DMSO), ethylene glycol, propanediol or Aedesta), frozen in liquid nitrogen, thawed and then cultured in-vitro. Viability was assessed by comparing frozen and thawed explants with non-frozen controls for morphological changes, lactate dehydrogenase (LDH) release, placenta protein 13 (PP13) secretion, and PCNA Western blotting. Functional studies determined the effect of oxygen and magnesium on explant viability. RESULTS: Cryoprotection by 3 M DMSO best maintained explants' viability, morphological integrity and PP13 release after freezing and thawing from liquid nitrogen. The effect of oxygen and magnesium was used to test the functional viability of cultured explants, after freezing in liquid nitrogen and transfer to dry ice for 1-5 days on site or for shipment to a remote lab. The tested parameters were similar between controls and cryogenically treated explants in the remote lab and the lab of origin, demonstrating the possibility of cryostoring explants for functional studies. CONCLUSION: Cryogenically stored placental villous explants shipped frozen can serve as a useful tool for comparative functional studies of placental villous tissues. The results of this pilot study also open the way for multi-site studies associated with drug tailoring for pregnancy disorders.

Effects of calcium, magnesium, low-dose aspirin and low-molecular-weight heparin on the release of PP13 from placental explants.

BACKGROUND: Preeclampsia is one of the leading causes for maternal and fetal morbidity. Attempts to prevent preeclampsia have already been made using low-dose aspirin, low-molecular-weight heparin (LMWH), and calcium supplementation. Magnesium sulphate is used at the time of disease to prevent eclampsia. Here we investigated the effect of these agents on PP13 release from placental explants. METHODS: Placentas harvested after C-section of term or preterm control and preeclampsia cases or first trimester terminations were used to obtain explants. Explants were incubated for 24h with/without respective agents, harvested, weighed and subjected to PP13 determination in the culture medium and the explant. LDH was used to determine viability. Dose response curves were obtained for each drug. P < 0.05 was considered significant. RESULTS: Exposure to magnesium (0.7-7g/day) slightly decreased PP13 release from controls, and slightly increased it in preeclampsia and first trimester termination. Calcium (0. 3-6g/day) showed a tendency to decrease the release in control and preeclampsia, whereas in first trimester release was increased in a bell-shaped manner. Aspirin (0-250 mg/day) tended to decrease the release in controls but increased it in a bell-shaped manner in first trimester and preeclampsia. LMWH showed no effect from 0 to 80 mg/day in controls but tended to decrease PP13 release in preeclampsia and first trimester. CONCLUSION: This data might point to a beneficial effect of aspirin and calcium supplementation in the first trimester of pregnancy and aspirin at the time of disease, although the interaction with the maternal system still needs to be elucidated.

Placental protein 13 as an early marker for pre-eclampsia: a prospective longitudinal study.

OBJECTIVE: To assess the value of placental protein 13 (PP13) as an early marker of pre-eclampsia. DESIGN: Sequential blood samples were obtained from women with singleton viable pregnancies at 6-10, 16-20 and 24-28 weeks of gestation. Samples were tested for PP13 using a solid-phase sandwich enzyme-linked immunosorbent assay. Levels were expressed as multiples of the medians (MoM) of the unaffected population. The slope or rate of change in PP13 concentration per week of gestation was also calculated. SETTING: Thirty-five prenatal care community clinics. SAMPLE: In total, 1,366 women were recruited, and subsequently, 20 were diagnosed with pre-eclampsia, 41 with gestational hypertension and 1,178 were unaffected. MAIN OUTCOME MEASURES: Sensitivity and specificity of screening with PP13 at each gestational period and of PP13 level combined with the slope of PP13 between two testing periods. RESULTS: At 6-10 gestational weeks, PP13 levels were significantly lower among the pre-eclampsia group with a median 0.28 MoM (95% CI 0.15-0.39, P < 0.004). Using a cutoff of 0.40 MoM, the sensitivity was 80%, false-positive rate (FPR) was 20% and odds ratio was 16.0 (95% CI 5.3-48.4). Combining MoM of 6-10 weeks and slope between 6-10 and 16-20 weeks, the sensitivity was 78%, the FPR was 6% and odds ratio was 55.5 (95% CI 18.2-169.2). The gestational hypertension group was not different from the normal group. CONCLUSIONS: PP13 in the first trimester alone or in combination with the slope between the first and the second trimesters may be a promising marker for assessing the risk of pre-eclampsia.