1. Introduction
Nutrient deficiencies may lead to undesirable health outcomes. Pregnant women are considered vulnerable, as the mother is the sole provider of nutrients for the fetus [
1,
2,
3]. During pregnancy and lactation, the maternal fatty acid status declines [
4,
5], which may lead to a suboptimal supply for the fetus, principally in cases where the dietary intake of these fatty acids is low or absent. In addition, fatty acids are released from maternal adipose tissue stores to the fetus, especially docosahexaenoic acid (DHA, 22:6ω-3), and marginally change blood levels [
3,
6]. The rapid growth of the fetal brain during pregnancy and the first two years of childhood demand adequate levels of nutrients, such as the omega-3 long-chain polyunsaturated fatty acids (ω-3 LCPUFA), eicosapentaenoic acid (EPA, 20:5ω-3), and DHA. Experimental evidence suggests that DHA is the major structural and functional fatty acid in the central nervous system [
5,
7]. Consequently, the maintenance of maternal fatty acid supply is crucial.
Norway recommends a daily intake of 200 mg DHA for pregnant women [
8]. Aquatic foods and ω-3 supplements are the main dietary sources of EPA and DHA [
9]. Pregnant women are advised to follow the general dietary recommendations, which is to consume 300–450 g of fish per week, corresponding to fish or fish products for dinner 2–3 times per week, of which a minimum of 200 g should be fatty fish. There is inconsistency regarding the effects of DHA supplementation during pregnancy and in the early phase of infant cognitive development. Some research suggests a beneficial effect of DHA supplementation during pregnancy and/or lactation on mental development and on long-term cognition [
10]. However, the evidence on cognitive development is inconclusive [
11,
12,
13,
14,
15]. Recent studies have also concluded that low levels of ω-3 LCPUFA in the blood are a risk factor for early preterm birth and that an increased intake of ω-3 LCPUFA (via fish or supplements) is advisable [
6,
16]. Some studies suggested that pregnant and lactating women should consume 225–350 g (8–12 oz.) per week (~250–375 mg/day of EPA and DHA) of a variety of seafood [
17]. However, a study on DHA and the increased risk for early preterm birth recommends a range of 600–800 mg/day of DHA for women with levels of DHA in red blood cells (RBC) lower than 5% [
6]. Some authors who support the supplementation of ω-3 LCPUFA as an effective strategy for reducing preterm birth advise that a follow-up of completed trials is needed to assess long-term outcomes [
18]. Lands and collaborators emphasize that careful handling of data on fatty acid composition is needed when interpreting evidence of dietary fatty acids on health outcomes [
19].
Determination of fatty acid levels in RBC is a well-known approach for assessing fatty acid status as it reflects the last 30–60 days of intake [
20]. EPA and DHA, accompanied by some other fatty acids, for example, short-chain fatty acids present in milk, are indirect biomarkers of specific foods as these foods are the primary dietary source of the respective fatty acids [
21].
Reference intervals provide information on specific biomarkers in population-based cohort studies and offer a clear understanding of the initial status, as well as provide the basis for comparison over time. Most laboratories and scientific reference tables offer information derived from healthy nonpregnant women, but lack reference intervals for pregnant women. During pregnancy, there are changes in many biological markers, and therefore, reliable reference values derived from a healthy pregnant population are of importance for correct clinical decisions. Without adequate reference intervals, there is an increased risk of missing important changes, due to pathological conditions and to erroneously interpretation of normal changes as pathological events [
22]. Hence, reference intervals are the most widely used tool for medical decision-making, therapeutic management decisions, and other physiological assessments [
23,
24]. The present study aims at suggesting reference intervals and cut-offs for fatty acids in maternal RBC on a sufficiently large healthy population that can be used in future studies to identify women who are at risk of adverse health outcomes as a result of under or overexposure to fatty acids. In addition, the relationship between the intake of seafood and ω-3 LCPUFA, generally characterized as poor in many pregnancy cohort studies [
25,
26], is thoroughly investigated using a principal component analysis.
4. Discussion
The applied e-FFQ was not focused on ω-3 fatty acids originating from plants, but from the habitual intake of seafood (fish and shellfish) and the use of dietary supplements, because the endogenous metabolization of ALA (18:3 n-3) from plants to ω-3 PUFA (e.g., EPA, DPA, and DHA) is minimal. Furthermore, the e-FFQ considered different forms of seafood individually. For example, the indexes for dinners were grouped into five categories comprising dinner items of oily fish, lean fish, shellfish, processed fish, and freshwater fish. Additionally, freshwater fish consumption was divided into two separate questions, frequency of perch/pike (lean fish) and frequency of char/whitefish (oily fish) [
28].
The e-FFQ indicated that 29.1% of the participants reported an intake of fish for dinner that was in accordance with dietary guidelines from the Norwegian Directorate of Health (
Table 2). However, a high percentage of participants from all the assessed groups (under and over seafood as dinner 2–3 times/week) reported the intake of ω-3 supplements. In addition, it was remarkable that the intake of ω-3 supplements was almost identical (around 77%) for all the observed groups, 1–3 times per month (27/35 × 100 = 77.14%), one time per week (74/96 × 100 = 77.08%) and 2–3 times per week (46/59 × 100 = 77.97%), as shown in
Table 2. The high intake of omega-3 supplements in this particular cohort of Norway is in accordance with global awareness towards the beneficial effects of these dietary products as they improve the levels of omega-3 PUFA by covering dietary seafood shortfalls, particularly for those who dislike the taste or smell of fish.
The observed frequencies for gestational weeks 16 and 32 of 68.97, 29.06 and 76.85% for the categories seafood intake under dietary guidelines (
n = 140), 2–3 times/week (
n = 59) and intake of ω-3 supplements (
n = 156), respectively (
Table 2) are consistent with those reported for gestational week 22 and 32 by The Norwegian Mother and Child Cohort Study (
n = 67007) of 60.06, 23.47 and 63.95 for the categories seafood intake under 2–3 servings/week (
n = 40244), seafood intake of 2–3 servings/week (
n = 15724) and intake of ω-3 supplements (
n = 428852), respectively [
34]. In addition, the observed 29.06% frequency (for those Norwegian pregnant women (30.1 ± 4.6 years) in accord with the national dietary guidelines), is in close agreement with the latest national dietary survey conducted among adults in Norway (2010–2011) where women in the age group 30–39 reported a frequency of 21% for the intake of seafood for dinner three times per week or more [
35]. The agreement with previous studies confirms the robustness of the semi-quantitative e-FFQ to assess the dietary intake of seafood and ω-3 supplements.
The PCA plot (
Figure 4) detected a correlation between the ω-3 PUFA and the e-FFQ variables, and it discriminated the ω-6 and ω-3 PUFA into three clusters that can be intuitively explained, as follow: The concentration levels of 20:3ω-6, 20:4ω-6, and 22:4ω-6 (inside the green frame in
Figure 4) and 20:5ω-3, 22:5ω-3 and 22:6ω-3 (inside the black frame in
Figure 4) reflect both endogenous (de novo lipogenesis) and exogenous (dietary intake) sources; whereas, the concentration levels of essential fatty acids, such as 18:2ω-6 and 18:3ω-3 (inside the blue frame in
Figure 4), exclusively reflect the dietary intake of the participants. In addition,
Figure 4 reveals that neither 18:2ω-6 nor 18:3ω-3 are correlated with any of the e-FFQ variables.
The associations between qualitative variables (e.g., frequency of consumption of fish, BMI, ethnicity, etc.) and fatty acids in plasma from pregnant adolescents (14–18 years old) by using PCA has been published elsewhere [
36]. Although this particular study did not discuss in detail the PCA results, an analysis of its reported PC1 and PC2 loadings revealed that the association 20:4ω-6/fish was stronger than the association ω-3 PUFA/fish (e.g., 18:3ω-3, EPA, DPA); and also the lack of correlation between essential fatty acids (e.g., 18:3ω-3, 18:2ω-6) which should exclusively reflect the dietary intake. In general, studies on the association between food intake variables from FFQ and fatty acids from pregnant women, by using techniques different to PCA, have consistently demonstrated poor correlations between dietary fatty acid intake and blood levels [
25,
26]. The present pregnant cohort study is the first to report a clear association between e-FFQ variables and fatty acids in RBC from the pregnant cohort by using PCA.
Except for 18:2ω-6, the sequence of most concentrated fatty acids reported in the present study (16:0, 18:1, 18:2ω-6, 22:6ω-3) has been also observed in studies with pregnant women from Belgium [
37], Netherlands [
38], Germany [
39], and Japan [
40,
41]. In these countries, the major ω-6 PUFA was 20:4ω-6, and its level was consistently higher than 18:2ω-6 by 69.6, 2.8, 1.0, and 28.2% (average from References [
40,
41]), respectively; whereas, in the present study 20:4ω-6 was lower than 18:2ω-6 by 9.5%. Possible explanations behind the observed reduction in the present study might be the high intake of ω-3 supplements (76.9%) compared to the studies from Belgium (24.6%), Netherlands (14.3%), Germany (20%), and Japan (2.2% in Reference [
41]). In addition, an analysis of the estimated global seafood consumption per country [
42] by the time these specific studies were performed indicated that Norway had the highest seafood consumption per capita (52.9 Kg in 2012) compared to Belgium (23.8 Kg in 2016), Netherlands (22.11 Kg in 2000), Germany (14.3 Kg in 2011) and Japan (48.6 Kg in 2013).
In the present study, the ω-3 PUFA sequence ranked from lowest to highest concentration was 18:3ω-3, 20:5ω-3, 22:5ω-3, and 22:6ω-3. This specific sequence is in agreement with similar studies from the Netherlands [
38], Germany [
39], and Japan [
41]. Other studies from Japan [
40], Belgium [
37], and Iceland [
43] have not reported the concentration levels of 18:3ω-3 or 22:5ω-3. However, in these studies, the declared ω-3 PUFA followed the aforementioned order.
In general, the range of concentrations for selected fatty acids in RBC from pregnant women in
Table 3 is in agreement with reported median or average values in similar studies from different countries, as indicated in
Table 4 in green color. However, in some countries, the levels of particular fatty acids were distinct from the 2.5 or 97.5 percentiles of the present study, as indicated in
Table 4 in yellow and red colors, respectively. The reasons behind the observed discrepancies are beyond the scope of the present article.
Some studies have indicated that values ≥8% or <5% are associated with the lowest risk for cardiovascular events [
44] or the highest risk of depressive episodes [
45], respectively. Despite these observations, an optimal range of omega-3 index for pregnant women has not been defined yet. A recent study has indicated that no human being has an omega-3 index <2% [
44]. Contrary to this observation, in the present study that involved only healthy pregnant women, a participant (hereinafter referred to as p#159) with an omega-3 index of 1.93% was recorded. The relative concentrations of EPA (0.43%) and DHA (1.50%) for p#159 were allocated inside the range and under the lowest percentiles for these fatty acids (
Table 3). In addition, p#159 exhibited the largest DPA concentration level (3.59%). A close inspection of the same fatty acids in µg/g units for p#159 revealed that EPA, DHA, and DPA were allocated in the 55, 35, and 55 percentiles, respectively, and consequently, the values in µg/g units are inside the range of the studied population. It is equally important to mention that the ω-6/ω-3 index is another key player in epidemiological studies that are generally associated with depression [
45] and cardiovascular events [
46]. Some studies have indicated that ω-6/ω-3 >9 is associated with postpartum depression [
47]; whereas, an ω-6/ω-3 around 4 exerts cardioprotective effects [
46]. Experimental evidence suggests that the optimum ω-6/ω-3 ratio must be kept around 4 and 5 and should not exceed 10 [
48]. The computed ω-6/ω-3 ratio for p#159 was 3.84 (95 percentile in
Table 3), and it can be regarded as optimum. The previous observations about the different indexes and measurement units, do not try to draw general conclusions based on the results of just one participant, but to highlight the importance of a comprehensive evaluation of the implications in human health of the different indexes and their corresponding threshold not only from the perspective of relative units (%), but also absolute units (mg/g). In addition, it is important to highlight that published randomized trials have not provided conclusive evidence yet about the effect of ω-3/ω-6 PUFA on postpartum depression.
In the present research, 42% of the pregnant women had an omega-3 index above 8%. It was mentioned that this index plays a pathophysiologic role in depressive symptoms [
45,
49,
50]. The International Society for Nutritional Psychiatry Research Practice Guidelines for ω-3 fatty acids has recently recommended therapeutic dosages of pure EPA or a combination of EPA and DHA (with net EPA starting from at least 1 up to 2 g/day) for at least eight weeks as a potential treatment for major depressive disorders [
51]. We have previously shown that low omega-3 index in pregnancy is a possible risk factor for postpartum depression [
52], with a cut-off at 4%. This cut-off is similar to the 2.5 percentile in
Table 3 and in accordance with the cut-off for those at high risk of developing coronary heart disease [
53]. Thus, the suggested reference values and omega-3 index cut-off could help to identify women who might benefit from increasing the dietary intake of EPA and DHA, like seafood and supplements that are important dietary sources of these long-chain PUFA, and hence, will influence their nutritional status. It must be mentioned that there are no specific recommendations on the intake of EPA or DHA for the general population, including prenatal women, in Norway [
54].
Cohort studies for establishing national reference intervals for fatty acids in RBC of pregnant women are largely dependent, among other things, on the number of participants, the number of health stations, the geographical distribution of the health stations along with their inherent infrastructure for collecting and preserving samples long-term at appropriate temperatures. For instance, fatty acids in RBC are susceptible to degradation and remain stable for 42 or 91 days at 1 °C or −20°C, respectively [
30,
31]. Failure to comply with these requirements might be regarded as a drawback. Some of the apparent limitations of the present study are the lack of blood collection/preservation facilities (namely seven well-equipped facilities). However, most of the studies in
Table 4 were performed in one specific geographical region by using just one blood collection facility. In some cases, the selected geographical regions represented a very low percentage of the total female population of the country in question. For example, the studies from Belgium [
37], the Netherlands [
38], and Germany [
39] represented ~1.71, ~0.71%, and ~0.13% of the total female population, respectively. Moreover, the studies from Iceland [
43] and Japan [
40,
41] constituted approximately 33.73 and 18.23% of the total female population, respectively, they were carried out in specific regions (Reykjavik and the Miyagi Prefecture), and they do not contain all the important characteristics of the country population from which they were drawn. The present study collected samples from the main geographical regions of Norway, which account for a ~91.5% of the targeted population. In addition, the present study with seven collection facilities has a higher level of enrolment per thousand pregnant women than Japan with 15 collection facilities, namely, 4.16‰ and 1.63‰ by the time these specific studies were performed, respectively. The expression
n = N/[1 + N(e/100)
2] (aka Slovin formula) [
55], that is generally considered to estimate the sample size (n) given the population size (N) and a percentage of margin error (e) was used to judge whether
n = 247 was an appropriate sample size. By the time the samples were collected (2011–2012), the parameter N was estimated using the Statistics Bureau of Norway’s records of the average number of births (59410 ± 10) between 2011–2012 [
56], while the parameter e was set at 7.5% (half the maximum margin of error of 15% proposed by IUPAC for monitoring fatty acid concentrations by gas chromatography [
57]). A minimum value of
n = 177 was calculated by introducing the aforementioned parameters in the Slovin expression, which in turn concluded that the sample size of the present research (
n = 247) was sufficient to determine reliable reference intervals for fatty acids in maternal RBC. An important feature of a selected sample size should be its ability to make projections or generalizations regarding an entire population. The information in
Table 1 and
Figure 2 indicates that pregnant women were recruited from all over the Norwegian territory, which emphasizes the strength and representativeness of the sample size, and consequently, the validity of the proposed reference values in the present study. The previous observations indicate that there is not any suspicion of misrepresentation of the population of interest in the present study.