Despite considerable advances in the treatment of diabetes in the past decades, diabetic pregnancies can still be associated with fetal growth disturbances even when an optimized and intensive metabolic control has been achieved. Macrosomia of the fetus is a common and well-known consequence of maternal diabetes with an increased obstetric risk. The increase in fetal fat mass that accounts for the fetal overweight [1] is the result of fetal hyperinsulinism as a consequence of maternal and, therefore, fetal hyperglycaemia [2]. According to the dynamics of fetal growth, macrosomia is caused by metabolic alterations or hyperglycaemic insults predominantly in later stages of pregnancy [3,4]. In contrast diabetes-associated metabolic derangements in the first trimester of pregnancy can lead to malformations [5] and to delayed growth of the developing embryo [6,7]. Whereas the molecular mechanisms underlying embryo malformations have received enormous attention, no data are available to explain early fetal growth delay. Because of the close link between placental and fetal growth, early intrauterine growth delay could be caused by a reduced growth of the pla- Diabetologia (2001) Abstract Aims/hypothesis. Early intrauterine growth delay in diabetes could be caused by a reduced growth of the placenta. Our study investigates whether hyperglycaemia in vitro reduces trophoblast proliferation. Methods. First-trimester trophoblast cell models (BeWo, JAR and JEG-3 choriocarcinoma cells) were cultured for 24 and 48 h with 5.5 mmol/l d-glucose, 25 mmol/l d-glucose (hyperglycaemia) and with an osmotic control. Cell number, total protein and nucleic acid content and mitochondrial activity (tetrazolium salt assay) were measured, the cell cycle analysed (FACS, cyclin B1 levels) and apoptosis (Annexin-V) measured.Results. In BeWo cells hyperglycaemia reduced cell number, protein, nucleic acid and cyclin B1 levels. The reduced G 2 /M and increased G 0 /G 1 population after 24 h reflects growth arrest at G 0 /G 1 . In JAR cells after 24 h the population was arrested in G 0 /G 1 , whereas after 48 h the G 0 /G 1 block was abrogated and the cells were arrested at G 2 /M. The net effect was an unchanged cell number. In JEG-3 cells hyperglycaemia resulted in fewer cells after 24 h but not after 48 h indicating some adaptation. Mitochondrial activity was either stimulated (BeWo) or reduced (JAR, JEG-3) under hyperglycaemia. Some of these effects were also induced by hyperosmolarity alone. Conclusion/interpretation. Hyperglycaemia has the potential to inhibit the proliferation of first-trimester trophoblast cell models. The mechanisms leading to growth arrest and to changes in mitochondrial activity are complex and depend on differentiation. We hypothesise a hyperglycaemia-induced impairment of placental growth in the first trimester of a poorly controlled diabetic pregnancy. [Diabetologia (2001) 44: 209±219]

Several clinical situations require continuous glucocorticoid (GC) treatment during pregnancy. A well-known deleterious side effect of such treatment is the higher incidence of growth-restricted fetuses, for which a too shallow trophoblast invasion is presently hypothesised as the underlying cause. This study investigated whether the synthetic GC triamcinolone acetonide (TA) influences proliferation, invasion and endocrine activity of human trophoblast. BeWo and JEG-3 choriocarcinoma cell lines both express GC receptors (western blotting) and were used as models for human trophoblast. JAR devoid cells of GC receptor were used as negative control. The cells were cultured for 48 h without (control) or with 0.5, 5 and 50 mM TA. In the presence and absence of serum, proliferation was determined by cell counting and measuring the cell cycle regulating protein cyclin B1 (Western blotting); invasion was determined by a conventional Matrigel invasion assay and by measuring the secretion (ELISA) of matrix-metalloproteinases (MMP-2, MMP-9) into the culture medium; endocrine activity was assessed by measuring the levels of human chorionic gonadotropin (ELISA) into the culture medium. TA altered the number of viable and dead cells as well as cyclin B1 levels and, to a lesser extent, invasion of BeWo and JEG-3, with a strong influence of serum. BeWo and JEG-3 cells reacted differently and in most instances reverse. In the cell lines used as models of human trophoblast, TA alter some functions relevant to proliferation and invasion, and suggest that caution should be exercised when treating women with GCs during pregnancy.

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.

Objective: To investigate the mRNA expression of genes related to steroidogenesis and OHSS in granulosa cells (GCs) of patients triggered with GnRH agonist compared to patients triggered with hCG.Design: Mural GCs were obtained at the time of oocyte retrieval and gene expression was analyzed using quantitative real time RT-PCR.Settings: Single center, case control study. Patient(s):24 women who were treated with GnRH agonist or hCG for triggering of ovulation.Interventions: GC collection. Main Outcome Measure(s): The expression of genes related to steroidogenesis and OHSS in mural GCsResults: The fertilization rate was similar in the two groups. The mRNA expression of CYP19A1 (0.50 vs 1, arbitrary unit), CYP11A1 (0.6 vs. 1) and 3 beta hydroxysteroid-dehydrogenase (0.39 vs 1) was significantly lower in the GnRH group. The expression of VEGF (0.74 vs. 1) and inhibin b B (0.38 vs 1) was lower in the GnRH analog triggered group.Conclusion: Expression of genes related to steroidogenesis is lower at the time of oocyte retrieval in patients triggered with GnRH agonist. The decreased expression of VEGF and inhibin b B in the GnRH agonist group can explain the mechanism of early OHSS prevention.

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