The concentration of all plasma lipids and lipoprotein fractions increased over pregnancy, with the exception of NEFA. The changes in total cholesterol, total TG, VLDL and LDL represent the physiological hyperlipidaemic adaptation to pregnancy, where lipids are mobilized into the maternal circulation to supply fuel and essential nutrients for the fetus. Maternal obesity was associated with higher plasma levels of TG, VLDL-1 and VLDL-2 predominately at T1 and 17 weeks post delivery. By later gestations, plasma TG and TG-rich lipoproteins appeared to reach the same maximal level. This indicates that obese mothers are less metabolically flexible, in terms of the extent of the metabolic adaptation to pregnancy, than their normal weight counterparts. Estradiol incremental AUC was significantly associated with plasma TG, VLDL-cholesterol, VLDL-1 and VLDL-2 response to pregnancy with no change in VLDL-1 to VLDL-2 total mass ratio during pregnancy. These data indicate that estradiol up-regulates the secretion of both VLDL subfractions equally from the liver to promote the gestational increase in plasma TG. We are unable to assess the relative contributions to the gestational increase in VLDL-2 mass of direct synthesis of VLDL-2 by the liver and conversion of VLDL-1 to VLDL-2 by lipolysis. Kinetic studies in non-pregnant women show that production rates of both large and small VLDL fractions are increased by low dose oral contraceptives(26
). Our data are also consistent with the strong relationship observed between the gestational rise in estradiol and the increment in plasma TG in a small group of normal weight pregnant women(15
). We found an interaction between BMI and time for estradiol, with obese women having significantly lower estradiol levels in late pregnancy than normal weight women. This is likely due to the sequestration of estradiol in adipose tissue(27
). It is notable that measures of insulin resistance did not contribute to gestational change in VLDL concentration. This suggests that the effects of estradiol on VLDL production by the liver may override any insulin effects of VLDL production during pregnancy.
The VLDL compositional data suggest that as pregnancy progresses all TG-rich lipoprotein fractions carry more TG molecules per particle. Although plasma LPL mass was inversely correlated with TG at all time points, LPL incremental AUC (which is negative due to decreasing LPL levels during pregnancy) was not associated with plasma TG response to pregnancy. This finding was independent of the influence of the S447X LPL polymorphism which, for as yet unknown reasons, has a large influence on plasma LPL mass, post-heparin LPL activity and plasma TG levels(25
). LPL mass is a poor surrogate of total lipase activity, as it is mostly inactive(22
). It has been suggested in the non-pregnant, that low LPL mass is a marker of metabolic syndrome and that this reflects the low rate of LPL synthesis by adipocytes in the insulin resistant state(28
). However, we did not observe a BMI effect on LPL mass in pregnancy. We speculate that steady state plasma levels of TG are determined predominately by increased VLDL production in response to estradiol, rather than by LPL-mediated clearance.
During pregnancy, mass levels of LDL-I and LDL-II were unchanged but the mass of LDL-III increased, particularly in overweight and obese individuals. This was reflected in a significantly higher proportion of LDL-III in obese mothers in T3. Large LDL subfractions are remodelled into smaller, denser LDL-III as plasma TG rises above a threshold of 1.5mmol/L(12
), an effect that is facilitated by hepatic lipase and cholesteryl ester transfer protein(30
). Obese mothers have raised baseline TG and consequently reach the threshold concentration of TG earlier and more easily than normal weight women. A high proportion of the overweight and obese women had greater than 50% LDL-III in T3. This indicates a shift in their LDL subfraction profile from a healthy “Pattern A” phenotype to an atherogenic “Pattern B” phenotype(21
). Small dense LDL-III particles are susceptible to oxidation and oxidised LDL is inversely associated with vascular function in subjects exhibiting cardiovascular risk factors(31
). A predominance of LDL-III is a hallmark of conditions in which there is an accumulation of ectopic liver fat, such as non-alcoholic fatty liver disease(32
) and type 2 diabetes(14
). Thirty five percent of the obese pregnant women had an LDL “Pattern B” profile by T3. This suggests that a subgroup of obese women are less able to cope with the gestational increase in TG, are predisposed to store fat ectopically and are at risk of GDM and PE(9
). In theory, fatty liver has been linked to the formation of small, dense LDL through the production and release of TG-enriched VLDL(14
). However, there was no evidence in the present study to support a link between the increase in LDL-III and large TG-rich VLDL in obese women. Instead, the relative mass and proportion of LDL-III in T3 were associated with plasma adiponectin levels, and also maternal obesity, leptin and adiponectin, in respective multivariate models. The rise of leptin levels to a T2 peak and the fall of adiponectin to a T3 nadir in pregnancy have previously been reported(33
). While these changes are suggested to be due to gestational maternal fat accumulation, we found no evidence that maternal insulin resistance played a role, and others suggest that additional factors determine at least leptin levels in pregnancy(35
). Adiponectin levels are reduced in obesity and lower still in non-alcoholic fatty liver disease(36
). Adiponectin has been reported to reduce fatty acid synthesis via SREBP-1c, activate fatty acid oxidation via AMP kinase and to have anti-inflammatory and anti-oxidative effects at the liver(37
). The gestational levels of adiponectin reached by the obese pregnant women (mean 6.8, SD 2.3ug/mL) are in a similar range to obese individuals with nonalcoholic hepatic steatosis (5.6ug/mL), whereas normal weight pregnant women and obese individuals without non-alcoholic hepatic steatosis have similar levels of around 10-11ug/mL(36
Plasma NEFA levels were stable over pregnancy, as has been observed for non-pregnant obesity, insulin resistance and well-controlled type 2 diabetes(38
). Although NEFA concentration does not change this provides no information on NEFA flux; it merely suggests that rates of entry of NEFA into the blood via lipolysis and exit by uptake into tissues are equal. There were no associations between maternal obesity and cholesterol concentrations, levels and composition of cholesterol-rich lipoproteins or plasma NEFA. This observation is analogous to non-pregnant obese individuals, where the predominant phenotype is of metabolic syndrome characterised by raised TG, low HDL levels but often normal cholesterol levels.
The majority of lipid changes in response to pregnancy had resolved to baseline levels by three months after pregnancy. However, TG-enrichment of VLDL-1, and VLDL-2 remained high post-natally. Furthermore, LPL mass concentration was significantly higher in the post-natal period than at any time during pregnancy. This may represent an adaptation for breast-feeding, facilitating utilisation of TG by mammary tissue. Unfortunately, we do not have a record of breast-feeding activity in our women. We also showed that VLDL-1 levels remain significantly elevated in obese women post-pregnancy. This could be explained by the relative loss of estradiol and a re-emergence of the predominant effect of insulin resistance, with resulting failure to suppress VLDL-1 secretion from the liver(26
The strengths of this study are the longitudinal design, wide range of BMI, larger numbers than reported previously and concurrent measurements of lipids and hormones. There are a variety of methods for separating LDL subfractions ranging from the specialist analytical or density gradient ultracentrifugation and nuclear magnetic resonance, to more accessible gradient and tube gel electrophoresis. While a definitive classification of LDL subfractions has not been established, the method used here, density gradient electrophoresis, identifies the “classic” LDL-I, LDL-II and LDL-III subfractions that have an established link to metabolic disease risk(12
). A weakness is the lack of direct enzyme activity measurements. We were unable to measure activities of LPL (and hepatic lipase) in plasma as the intravenous injection of heparin to release the functional pool of lipases from the endothelium is ethically not acceptable in pregnant women. Furthermore, we lacked detailed dietary and breast-feeding data, and the absence of a kinetic analysis of lipoprotein metabolism left us unable to comment on the inter-conversion of VLDL-1 to VLDL-2.
Increased plasma levels of TG and cholesterol is an essential adaptation to pregnancy. Normal weight mothers enter pregnancy with a healthy lipid profile and increase levels of cholesterol and TG-rich lipoproteins to a maximum, which decline to pre-pregnancy levels post-natally. Obese mothers begin pregnancy with higher levels of TG-rich lipoproteins and these rise to the same level as normal weight women by late gestation. Although in quantitative terms, the plasma lipids of obese mothers are equivalent to that of their normal weight counterparts, the quality of their lipids may convey greater cardio-metabolic risk due to the formation of small, dense LDL. Not all obese women develop metabolic complications of pregnancy. We speculate that a subset of obese women are susceptible to the accumulation of ectopic fat in the liver, perhaps due to low adiponectin levels, and accompanying adverse changes in LDL composition. Small dense LDL has been observed in both PE and GDM(17
) and these conditions are susceptible to fatty liver in pregnancy(39
). Our data suggest that a subset of obese women, at high risk for PE and GDM, may be identified for targeted intervention. Such intervention may include specific dietary manipulations, such as long chain n-3 polyunsaturated fatty acid supplementation, which are known to exert a favourable impact on the pathways described herein. Response to pregnancy may also reveal at an early stage in a woman’s life her susceptibility to metabolic abnormalities and fatty liver later in life(40