Effect of cryopreservation on human cytotrophoblast cells in culture: hCG and PALP production.

Term villous cytotrophoblasts differentiate into syncytiotrophoblast during culture exhibiting characteristic changes in cellular morphology and protein expression profiles. Measurement of human chorionic gonadotropin (hCG) and placental alkaline phospatase (PALP) is often used to assess viability and syncytialisation of cultured cells. The objective of this study was to assess the effect of cryopreservation of isolated cytotrophoblasts on the expression hCG and PALP by cells during subsequent culture. Villous cytotrophoblasts isolated from term placentae from uncomplicated pregnancies were either cultured immediately after isolation or were cryopreserved (liquid nitrogen) prior to culture. Cells were cultured in identical conditions (5% CO(2) in air) for 96 h. Protein and DNA content of cells and HCG and PALP levels in culture medium were measured at 24 h intervals. Cryopreservation had no significant effect on the protein or DNA content of cultured cells but hCG levels in culture medium were significantly reduced after 72 h (P=0.025) compared to cultures of fresh cells. PALP levels were unchanged. Cryopreservation of cytotrophoblast cells prior to culture resulted in a decrease in basal secretion of hCG possibly caused by a failure or delay in the morphological and functional differentiation of cells.

Alphafetoprotein and alphafetoprotein receptor expression in the normal human placenta at term.

Alphafetoprotein (AFP) is detectable in maternal serum from around six weeks of gestation and is synthesised by the yolk sac and the fetal liver. The role of the placenta in the transport and possible synthesis of AFP is uncertain. The aim of this study was to investigate placental expression of AFP and the AFP receptor in uncomplicated pregnancies at term. Immunohistochemistry and Western blotting clearly demonstrated the presence of AFP in villous tissue at term. However, evidence of AFP mRNA expression or synthesis of AFP was not found following reverse transcription polymerase chain reaction of total RNA isolated from villous tissue and trophoblast cell cultures. The presence of a cell surface receptor for AFP in placental villous tissue, identified by immunohistochemistry and Western blotting, suggests a possible receptor-mediated mechanism for placental transport of AFP while the patterns of expression of AFP and its receptor may indicate a possible route by which AFP is transported across the placenta between the fetal and maternal circulations. These findings demonstrate that the placenta does not synthesise AFP at term and that the presence of AFP in the placenta is a reflection of transplacental transport of AFP possibly via a receptor-mediated mechanism.

Placental and maternal serum inhibin-A and activin-A levels in Down's syndrome pregnancies.

The objective of this study was to analyse the levels of inhibin-A and activin-A in maternal serum and placental tissue from Down's syndrome (DS) pregnancies. Inhibin-A and activin-A levels were determined by specific immunoassays and individual results were expressed as multiples of the control median (MoM) at the appropriate gestation. Immunohistochemistry was used to localize inhibin alpha and beta(A)-subunits in a selection of placental sections. In DS pregnancies, median inhibin-A levels were found to be significantly elevated to 1.46 MoM (P< 0.05) in placental extracts, and 2.06 MoM (P< 0.0001) in maternal serum, when compared with uncomplicated pregnancies. Median activin-A MoMs were also elevated in placental extracts and maternal serum to 1.62 MoM (P< 0.01), and 1.26 MoM (P< 0.05), respectively. Immunohistochemistry revealed that the alpha subunit of inhibin-A and the beta(A)subunit of inhibin-A and activin-A were mainly localized to the trophoblastic layer of placental villi. Semiquantitative studies of staining intensity revealed a trend towards stronger staining of placental trophoblasts and stroma of DS tissues, although this was statistically significant only for beta(A)subunit staining of trophoblasts (P< 0.05). These results support the hypothesis that maternal serum levels of inhibin-A and activin-A are elevated due to increased production in the placenta, and increased immunostaining of trophoblasts suggests that this may be due to increased production in the trophoblasts.

Postnatal development of the ob gene system: elevated leptin levels in suckling fa/fa rats.

The postnatal development of the ob gene system has been examined in Zucker fa/fa and +/fa plus +/+ (referred to as +/?) rats. White adipose tissue was taken from animals aged 1 to 28 days. Before weaning, white fat was predominantly subcutaneous, the amount increasing rapidly after birth. ob mRNA was detected by Northern blotting in samples of inguinal fat at 1 day of age and thereafter. Circulating leptin, measured by enzyme-linked immunosorbent assay, was also detectable from 1 day of age, the level rising to a peak by 10 days of age and then declining. The fa/fa genotype was determined from the size of the product after Msp I digestion of the Ob-receptor gene obtained by polymerase chain reaction amplification of genomic DNA. No statistically significant difference in ob mRNA level between fa/fa and +/? animals was obtained before 25 days of age. However, leptin levels were significantly higher in the fa/fa mutant by 10 days of age, despite the absence of any significant elevation in the weight of the major fat depots or in ob mRNA level. It is concluded that the ob gene is expressed and leptin produced early in postnatal life in rats; the elevation of circulating leptin in suckling fa/fa animals indicates that dysregulation of the leptin system occurs before the overt development of the obese phenotype.