We conducted this analysis using maternal plasma samples collected from a prior investigation, “The Mothers, Omega-3, and Mental Health Study,” a 3-armed prospective, double-blinded, randomized controlled trial designed to test whether EPA- or DHA-fish oil supplementation would prevent perinatal depressive symptoms among women at risk. The full details of the trial have been previously described.12,13 In brief, patients with singleton pregnancies between 12- and 20-weeks gestation were recruited. Subjects were selected based on an elevated risk for depression, defined as a past history of major depressive disorder, a history of postpartum depression, or an Edinburgh Postnatal Depression Scale (EPDS) score between 9 and 19. Exclusion criteria included age <18, current major depressive disorder diagnosis, bipolar disorder or schizophrenia, substance abuse disorder, history of bleeding disorder, or clotting disorder requiring anticoagulation.13,14
Women who met eligibility criteria and who consented to participate were randomized to one of three arms: 1) an EPA-rich fish oil supplement (1060 mg EPA plus 274 mg DHA), 2) a DHA-rich fish oil supplement (900 mg DHA plus 180 mg EPA), or 3) a soy oil placebo. Because the DHA- and EPA-rich fish oil capsules were not identical in appearance, all participants took some placebo capsules (double-dummy design). Full details of the randomization procedure and the supplements are described elsewhere. 13,14
Maternal venous blood samples were collected after a three hour fast; baseline samples were collected at the time of study enrollment at 12-20 weeks gestation (visit 1), and again between 34- and 36-weeks’ gestation (visit 3). After delivery, a sample of fetal blood was collected from the umbilical cord (visit 4). Samples were centrifuged within 12 hours of collection and plasma aliquots were stored at -70 degrees Celsius.
Stored maternal and umbilical cord plasma samples from participants of the study were analyzed using commercially-available sandwich enzyme-linked immunosorbent assay kits (EMD Millipore, St. Charles, MO) to quantify leptin and adiponectin levels according to the manufacturer’s protocols.15,16 The only variation from the protocol was a dilution of fetal adiponectin samples to 1:1000 as opposed to 1:500 for maternal samples, based on higher fetal levels of adiponectin. Absorbance of both adiponectin and leptin were measured at 450 nm and 690 nm and the results compared. 25-hydroxyvitamin D levels, as well as DHA and EPA fractions in maternal and cord blood serum had been previously assayed.14,17
ETHICS:
The procedures followed were approved and conducted in accordance with the institutional review boards of the University of Michigan Medical Center, Ann Arbor, MI, St. Joseph Mercy Hospital in Ypsilanti, MI, and The University of New Mexico Health Sciences Center Human Research Protections Office. All subjects gave written informed consent to participate in the study. The trial was registered on 7/7/2008 at clinicaltrials.gov: NCT00711971 under the title: “Does Fish Oil Prevent Depression in Pregnancy and Postpartum”. The secondary blood sample analyses described in this manuscript were judged exempt by the University of New Mexico Health Sciences Center Human Research Protections Office.
Statistics: Demographic variables were compared among three randomized groups (EPA-rich fish oil, DHA-rich fish oil, and placebo) as means and standard deviations if continuous or ordinal scale using 1-way ANOVA and as frequencies using Fisher’s exact test. We computed medians, and interquartile ranges for each outcome parameter, adiponectin, leptin and the adiponectin:leptin ratio (ALR). Using raw (unadjusted) data, in order to test whether these parameters changed over time or were different between groups we performed repeated measures ANOVA with group as grouping factor and visit as repeated factor. The analysis of the ALR was also performed using repeated measures ANOVA with time (enrollment before supplementation, and after supplementation) as a repeated factor and 3 groups as a grouping factor. This was an intent-to-treat analysis in that all available data was used. Adiponectin and leptin, constituents of ALR, were analyzed similarly. Because box plot analysis of the data revealed non-normality, we also analyzed these data after square root transformation.
Covariate adjustments were done for BMI, maternal weight gain, age, and ethnicity. Post hoc analyses of trend in our measures of insulin sensitivity in each group were done by regression. In addition, multivariable regression analyses exploring the relationships between measured serum DHA and EPA fractions and 25-OH vitamin D concentrations with measures of insulin sensitivity were done in the three groups and two maternal time points pooled unless otherwise specified; other predictors included BMI and age at enrollment. We calculated the effect of a one unit increase in each of the predictive variables on the ALR using the two-tailed inverse student's T distribution.
We calculated the medians and interquartile ranges for adiponectin, leptin and the ALR in umbilical cord blood. We used raw unadjusted data, as well as square root transformation to evaluate difference according to group assignment using ANOVA. Covariate adjustments were done for birth weight and length of gestation. The association of DHA and EPA fractions, as well as 25-OH vitamin D concentrations in cord blood with measures of insulin sensitivity were analyzed by multivariable linear regression. P-values ≤ 0.05 were considered statistically significant.