Maternal diet influences foetus growth and is pivotal for the delivery of healthy, full-term newborns [1
]. Maternal metabolism and body composition change through the pregnancy to assure adequate supply of nutrients to the developing foetus. The first trimester is considered an anabolic period that serves to promote maternal fat storage, while the third trimester is deemed to be a catabolic status that favours nutrient supply to the foetus [1
During pregnancy, fatty acids (FAs) are required (i) as an energy source; (ii) to carry out structural functions (conformation, fluidity and permeability of cellular membranes); and (iii) to act as cellular signalling molecules or precursors of bioactive compounds (prostaglandins, leukotrienes, thromboxanes). These last two functions are provided in particular by essential fatty acids (EFAs), linoleic acid (18:2 n
-6, LA) and α-linoleic acid (18:3 n
-3, ALA), and their long-chain metabolites (long-chain polyunsaturated fatty acids, LC-PUFAs): dihomogamma linoleic acid (18:3 n
-6, DGLA), arachidonic acid (20:4 n
-6, AA), eicosapentaenoic acid (22:5 n
-3, EPA) and docosaexahenoic acid (22:6 n
-3, DHA) [2
AA, DHA, and EPA are the main components of neuronal membrane phospholipids. They are essential for visual and cognitive development [3
All the FAs are transported across the placenta from the maternal side to the foetus by specific fatty acid binding/transport proteins [4
]. This mechanism is specially effective for all the LC-PUFAs [5
], particularly for DHA. In physiological conditions, the phenomenon of “bioattenuation” protects the foetus from the excessive passage of maternal DHA through the placenta. Conversely, the “biomagnification” occurs when the DHA percentage in foetal erythrocytes is higher than in mothers [7
] and might actually reflect the maternal reduced DHA status [8
]. The efficiency of the LC-PUFA transport system has been found to be impaired in maternal dysmetabolic conditions, such as obesity, potentially leading to an altered foetal lipid profile [9
]. The Generation R study found obesity and excessive gestational weight gain (GWG) to affect maternal FA plasma profile but it did not investigate the effect on the foetal lipid profile [10
The main aim of this population study was to evaluate the nutritional FA status, measured in erythrocyte membrane phospholipids, in a large cohort of Italian pregnant women and their offspring. In particular, we aimed (i) to compare maternal and foetal fatty acids profile at birth; and (ii) to explore the effect of pre-pregnancy body mass index (BMI) and GWG on both maternal and foetal lipid profile.
2. Materials and Methods
2.1. Subjects and Study Design
The “Feeding Low-Grade Inflammation and Insulin Resistance of the Foetus” study is a population study of 1000 mother-infant pairs with the primary aim of evaluating the association at birth between maternal erythrocyte concentrations of FAs and the child’s insulin resistance and low-grade inflammation. Healthy pregnant women (aged 18–45 years) were enrolled, at the San Camillo Forlanini Hospital (SCH) in Rome, from February 2013 until June 2015 and followed up from the 1st trimester of pregnancy to childbirth with monitoring of lifestyle, blood testing, and ultrasonography, according to the guidelines of the Italian Society for Gynaecology and Obstetrics [11
Inclusion criteria were being at week 7–10 of gestation, folic acid supplementation from week 7, singleton pregnancy, no alcohol or medications, no systemic, chronic, or autoimmune disease, no previous diagnosis of Gestational Diabetes Mellitus (GDM) or miscarriage, no conception through ovulation induction or in vitro fertilization, planned delivery at SCH-Unit, maternal and foetal fatty acids erythrocyte profiles.
The “Feeding” study was approved by the Ethical Committees of the “Ospedale Pediatrico Bambino Gesù” (OPBG) and the SCH, in full agreement with the national and international regulations and the Declaration of Helsinki (2000). All the participants signed an informed consent.
2.2. Anthropometrics and Clinical Evaluation
Mothers’ body weight and height were measured at the enrolment following international guidelines [12
]. Repeated measures of body weight were performed during pregnancy until childbirth. Pre-pregnancy body weight was used as reference and, in case of incongruities, the patient’s general practitioner was consulted. Pre-pregnancy BMI was calculated as kg/m2
and classified according to the World Health Organization (WHO) [13
]. GDM was diagnosed according to the American Diabetes Association’s (ADA’s) Standards of Care [14
GWG was calculated by subtracting the pre-pregnancy weight to the weight reached at time of delivery. According to the Institute of Medicine (IOM) guidelines, we defined adequate gestational weight gain in relation to pre-pregnancy BMI (12.5–18 kg in underweight; 11.5–16.0 kg in normal weight; 7.0–11.5 kg in overweight and 5.0–9.0 kg in obesity). Otherwise, it was defined as inadequate or excessive if weight gain was below or exceeded values recommended for pre-pregnancy BMI classes, respectively.
At each trimester, women underwent a 40-minute interview with a nutritionist (GC) to estimate food consumption frequencies and received recommendations for healthy eating habits.
The following sociodemographic and anthropometric data for both the parents were collected to estimate socioeconomic status (SES); race; level of education; profession; smoking and parity.
Newborns’ anthropometrics (body weight, BW; body length, BL and head circumference, HC) were evaluated at birth according to standardized procedures [15
]. Standard deviation scores (SDS) for infant weight and height was calculated following the Italian INeS (Italian Neonatal Study) Chart [15
2.3. Samples Collection
Maternal blood samples were withdrawn at fasting, 12–24 h before giving birth, during the pre-partum foetal monitoring. Cord-blood samples (2.5 mL) were collected at birth by venipuncture from the placental portion of the umbilical cord immediately after clamping.
Blood was placed in ethylenediaminetetraacetic acid (EDTA) tubes. Erythrocyte membranes were isolated within 2 h after collection: plasma was separated by centrifugation (980 rpm, 18 min); whereas erythrocytes were added with acid citrate dextrose, washed with distilled water (10:1) and centrifuged (4000 rpm, 5 min) four times. Erythrocytes were frozen immediately at −80 °C and stored until lipid extraction.
2.4. Fatty Acid Analysis
2.4.1. Lipid Extraction from Erythrocyte Membranes
Erythrocytes were resuspended into hexane/methanol (1:3) solution and homogenized by vortexing for 1 min. Tubes were stored at 4 °C for 5 min and then 400 µL of acetyl chloride were added, by a careful handling, and incubated at 100 °C for 1 h. After incubation, the tubes were cooled down to 4 °C for 30 min, then 3 mL of K2CO3 (12%) were added.
At the end of CO2 production, the tubes were homogenized by manual inversion for 1 min. The mixture was then centrifuged at 3500 rpm for 5 min. The supernatant was transferred in gas-chromatography vials and the solvent was removed by using the GeneVac EZ-2 Plus evaporator (GeneVac, New York, NY, USA). The methyl-C11 (2 mg/mL) was added as internal standard.
2.4.2. Gas Chromatography Analysis of Fatty Acid Methyl Esters
Analyses of fatty acid methyl esters (FAME) were performed by a fast gas-chromatography/flame-ionization detector 2010 Plus with an autosampler AOC-20i (Shimadzu, Kyoto, Japan), equipped with a fused silica BPX70 capillary column (10 m × 0.1 mm I.D, 0.2 µm film thickness; SGE, Melbourne, Australia). Split injector (100:1) and flame-ionization detector system were operating at 250 °C.
The oven temperature programming at injection was 50 °C isothermal for 0.2 min, increased to 175 °C at 120 °C/min, then increased to 220 °C at 20 °C/min and finally to 250 °C at 50 °C/min.
The carrier gas (H2) flow was maintained at 0.8 mL/min and the volume injected was 0.30 µL. The method was optimized in house according to Destillas et al.
The chromatograms were integrated and identified by comparing the retention times and the peak area with those of a commercial lipid standard of 52 fatty acids (GLC 463 Nuchek; Elysian, MN, USA) and a conjugated linoleic acids mixture (UC-59M Nuchek; Elysian, MN, USA). Quantitative data were obtained by interpolation of the relative areas vs. internal standard (Methyl-C11) area. Data are shown as FAME concentration (ng/mL) or percentage (% of total FAME).
2.5. Statistical Analysis
Data are represented as number and percentage in parentheses (%) for categorical variables, or median and interquartile range (IRQ) for continuous variables. To evaluate differences and correlations between maternal and foetal fatty acid compositions, Wilkoxon Signed-Rank test was performed and Spearman correlation coefficient was calculated on concentration (ng/mL) and percentage of total FAs.
Quantile regression analyses were conducted on median percentage of total FAs to investigate the association between pre-pregnancy BMI and classes of GWG and maternal/foetal erythrocyte lipid profile. Multivariate quantile regression analyses, adjusted for maternal characteristics (GWG, pre-pregnancy BMI, smoking, age, educational level, offspring sex, gestational age, parity) were conducted on median maternal lipid profile. Multivariate quantile regressions, adjusted also for maternal lipid profile, were run on foetal lipid profile, in order to estimate the effect of these conditions on foetal erythrocyte fatty acid composition. All covariates were included in the multivariate models and the final one was determined through a backward approach.
Statistical analysis was performed through Stata 13.1 software (StataCorp, 4905 Lakeway Drive, College Station, TX, USA).
Findings of the present study demonstrated first that pre-pregnancy BMI affects maternal and foetal erythrocyte lipid profile, while GWG seems to influence only maternal profile. Pre-pregnancy BMI was negatively associated with maternal LA and MUFAs and positively with DHA. In foetal erythrocytes, pre-pregnancy BMI was inversely associated with PUFAs, DHA and n-6 FAs. Inadequate GWG was, conversely, correlated to an increased maternal DPA while the excessive GWG WAS associated with a decreased maternal DHA, albeit with no effect on foetus lipid profile in both cases.
Maternal lipid profile likely changes during the pregnancy to adapt to the developing foetus demand [1
]. By the end of full-term pregnancies (37–41 weeks), we found that the percentage of PUFAs, particularly n
-3 FAs and DHA, significantly increased with the gestational age (Table 2
). The Generation R study is the largest cohort study that investigated the influence of pre-pregnancy BMI and GWG in 5636 women on maternal lipid profile, but at mid-pregnancy and on plasmatic concentration of fatty acids. Hence, our findings are only partly comparable to those of the Generation R study, which found a higher pre-pregnancy BMI associated with total SFAs and n-6 PUFAs concentrations in women’s plasma and a decrease of LA [10
]. In keeping with the Generation R findings [10
], we observed a lower percentage of LA at the end of pregnancy in mothers whose pre-pregnancy BMI was higher. In addition, we found that pre-pregnancy BMI was positively associated with DHA percentage in mothers and negatively in foetuses. It is unlikely that the former association reflects a higher fish intake in obese mothers with respect to normal weight since most of them consumed less than three servings per week. On the contrary, it might suggest an impaired transfer of DHA from mother to foetus, which has been previously hypothesised in obese mothers [17
]. Obesity, indeed, has been associated with an impaired expression of FAs carriers on the placenta membranes [18
]. The clinical impact of the latter association is minimal since it means 0.03% difference in foetuses’ DHA percentage per maternal pre-pregnancy BMI point. As to excessive GWG, the Generation R study found an association with plasmatic concentrations of SFAs, MUFAs and n
-6 PUFAs in mothers [10
Very few and only small-sample studies compared maternal and foetal lipid profile on erythrocyte membranes [19
]. By comparing pairs’ profiles, it seems that AA and DGLA are preferentially transferred across the placenta with respect to LA, ALA n
-3 EPA and DPA [19
]. In our cohort, the distribution of LA and ALA in maternal and cord-blood erythrocytes, compared to AA, confirms the preferential LC-PUFAs transfer to the foetus [23
]. In a large proportion of mothers (55%), we observed a very low percentage of ALA but higher DHA content, supporting the notion of favoured ALA conversion to DHA at the end of pregnancy to supply the foetus’s demand. DHA is essential for the foetal neurodevelopment in late pregnancy [3
]. Even if foetal tissues are able to convert the precursor ALA into DHA, foetal ability is relatively low [24
], hence placental transfer from the mother is the major source of DHA [25
]. It has been supposed that in the case of excessive supply of DHA from the mother to the foetus, the passage of DHA through the placenta is inhibited in a process known as “bioattenuation”, possibly to prevent DHA competition with AA in infant organs [8
]. Conversely, whenever the maternal percentage of DHA is reduced (in our population, below 3.4% of the whole FAs content), the placental transport is favoured, resulting in higher foetal DHA (i.e.
, “biomagnification”) [2
]. Biomagnification might be confined to populations with low maternal DHA status [8
]. The percentages of DHA in our sample were comparable, and even lower than the ones detected in populations with low fish consumption [20
]. Indeed, only ~6% of the women included in our sample reported eating three or more portions of fish per week, whereas 6% stated not eating fish at all. Isolating the 27 women with the highest intake of fish, the percentage of DHA rose up to a mean value of ~5%. In keeping with this hypothesis by further dividing into two subsamples (Figure S1
), we found different trends in maternal and foetal percentage of DHA.
Our study, including 435 mother-infant pairs, was the largest one in the literature investigating the erythrocyte fatty acids profile of mothers and their offspring at the end of the pregnancy. Erythrocyte FAs reflect dietary intake in the last 40 days of pregnancy and provide a more accurate estimate of fat consumption than any dietary recall [27
]. To our knowledge, it was also the first study exploring the association between pre-pregnancy BMI and GWG and maternal and foetal lipid profile. As a major limitation, however, we recognize the lack of information on the eventual DHA supplementation during the pregnancy.
Furthermore, due to a delay in funding, it was not possible to complete the lipid profile of all the stored samples. A complete dataset of fatty acids in both mother and newborn was available in 460 pairs. There was no difference in anthropometrics, clinical and SES characteristics of pairs whose lipid profile was available with respect to mother-newborn pairs whose profile was not analysed.
In conclusion, our results suggest the obesity status more than excessive weight gain can favour an adverse foetal lipid profile at the end of pregnancy. In the present cohort, the pre-pregnancy BMI affected the foetal lipid profile, being associated with decreased PUFAs, both n-6 and DHA, and likely owing to an impaired transfer across the placenta. Even though no association was found between maternal weight gain and foetal lipid profile, caution must be paid to maternal DHA levels and future research is needed in this regard. Further studies are also required to investigate the underlying mechanisms that regulate nutrient sensing across the placenta.