Paternally Expressed, Imprinted Insulin-Like Growth Factor-2 in Chorionic Villi Correlates Significantly with Birth Weight

<div><p>Context</p><p>Fetal growth involves highly complex molecular pathways. IGF2 is a key paternally expressed growth hormone that is critical for <i>in utero</i> growth in mice. Its role in human fetal growth has remained ambiguous, as it has only been studied in term tissues. Conversely the maternally expressed growth suppressor, <i>PHLDA</i>2, has a significant negative correlation between its term placental expression and birth weight.</p><p>Objective</p><p>The aim of this study is to address the role in early gestation of expression of <i>IGF1</i>, <i>IGF2</i>, their receptors <i>IGF1R</i> and <i>IGF2R</i>, and <i>PHLDA</i>2 on term birth weight.</p><p>Design</p><p>Real-time quantitative PCR was used to investigate mRNA expression of <i>IGF1</i>, <i>IGF2</i>, <i>IGF1R</i>, <i>IGF2R</i> and <i>PHLDA2</i> in chorionic villus samples (CVS) (n = 260) collected at 11–13 weeks' gestation. Expression was correlated with term birth weight using statistical package R including correction for several confounding factors.</p><p>Results</p><p>Transcript levels of <i>IGF2</i> and <i>IGF2R</i> revealed a significant positive correlation with birth weight (0.009 and 0.04, respectively). No effect was observed for <i>IGF1</i>, <i>IGF1R</i> or <i>PHLDA2</i> and birth weight. Critically, small for gestational age (SGA) neonates had significantly lower <i>IGF2</i> levels than appropriate for gestational age neonates (p = 3·6×10⁻⁷).</p><p>Interpretation</p><p>Our findings show that <i>IGF2</i> mRNA levels at 12 weeks gestation could provide a useful predictor of future fetal growth to term, potentially predicting SGA babies. SGA babies are known to be at a higher risk for type 2 diabetes. This research reveals an imprinted, parentally driven rheostat for <i>in utero</i> growth.</p></div

mRNA expression levels of <i>IGF2</i>, <i>IGF2R</i>, <i>PHLDA2</i>, <i>IGF1</i> and <i>IGF1R</i> in chorionic villi.

<p>The expression levels of <i>IGF2</i>, <i>IGF2R</i>, <i>PHLDA2</i>, <i>IGF1</i> and <i>IGF1R</i> after standardization to the endogenous control gene <i>L19</i>, in relation to birth weight corrected for parity, sex, GA at term, maternal BMI and smoking status. Significant associations were observed for CVS expression of A) <i>IGF2</i> (p = 0.009) and B) <i>IGF2R</i> (p = 0.04). No significant association was found for C) <i>PHLDA2</i> (p = 0.73), for D) <i>IGF1</i> (p = 0.48) or for E) <i>IGF1R</i> (p = 0.08).</p

mRNA expression levels of <i>IGF1/IGF1R</i>, <i>IGF2</i>/<i>IGF2R</i> and <i>IGF2/IGF1R</i> in chorionic villi.

<p>The expression levels of <i>IGF1/IGF1R</i>, <i>IGF2/IGF2R</i> and <i>IGF2/IGF1R</i> after standardization to the endogenous control gene <i>L19</i>, in relation to birth weight corrected for parity, sex, GA at term, maternal BMI and smoking status. No significant association was found for A) the ratio of <i>IGF1</i> to <i>IGF1R</i> (p = 0.76), or for B) the ratio of <i>IGF2</i> to <i>IGF2R</i> (p = 0.93). Significant association was observed for CVS expression of C) the ratio of <i>IGF2</i> to <i>IGF1R</i> (p = 0.005) and birth weight.</p

The human embryo does not elicit a secretory response in undifferentiated endometrium.

<p>Undifferentiated primary ESCs were co-cultured with embryos or not (control cultures, Con). Over the 72-hour co-culture period, 15 embryos arrested (Arr) whereas 6 continued to develop normally (Dev). Co-culture with either an arrested or developing embryo had no impact on the secreted levels of the indicated factors (<i>P</i>>0.05). The concentrations of IL-5, -12, -15, -17, -18, and eotaxin in culture supernatants of undifferentiated ESCs were below the level of detection.</p

Developmentally impaired human embryos inhibit the secretion of selective implantation modulators by decidualizing ESCs.

<p>Primary ESCs were first decidualized for 5 days and then co-cultured with human embryos or not (control cultures, Con). Over the 72-hour co-culture period, 30 embryos arrested (Arr) whereas 11 continued to develop normally (Dev). Analysis of the culture supernatants revealed that the presence of an arresting embryo inhibited the secretion of the indicated factors. The letters above the box plots indicate significant differences between groups. <i>P</i><0.01 for all comparisons except for IL-6 and IL-17 (<i>P</i><0.05).</p

Secreted decidual cytokines not regulated upon embryo co-culture.

<p>Primary ESCs were first decidualized for 5 days and then co-cultured with human embryos or not (control cultures, Con). Over the 72-hour co-culture period, 30 embryos arrested (Arr) whereas 11 continued to develop normally (Dev). Analysis of the culture supernatants revealed that the presence of an arresting or developing human embryo had no significant impact on the secretion of the indicated factors (<i>P</i>>0.05).</p

Human embryo development in co-culture.

<p>(<i>A</i>) Phase contrast images of decidualizing ESCs alone (control; left) or in the presence of an arrested (middle) or developing human embryo (right) (scale bar  = 100 µm). (<i>B</i>) Arrested embryos (Arr) express significantly less hCG than developing embryos (Dev). Secreted hCG levels were assayed after 72 hours of co-culturing human embryos and decidualizing ESCs (<i>P</i><0.01). (<i>C</i>) Formation of primitive endoderm in developing human embryos co-cultured with decidual ESCs. Optical cross-sections through a day 8 embryo, cultured first on a decidualizing ESC monolayer for 72 hours, demonstrates the presence of GATA-6 positive cells aligning predominantly to the blastocoelic surface of the inner cell mass, which corresponds to the location of the primitive endoderm (top panel). The lower panel represents z-projection of all the images in the stack from a top to bottom scan through the embryo (scale bar  = 50 µm).</p