Discussion

To the best of our knowledge, this study is the first demonstrating an association of maternal FA levels with the occurrence of NTDs in fetuses in Tunisia.

It is well-documented that an optimal FA profile during pregnancy, particularly concerning DHA and EPA, is crucial for maternal and fetal health. ^{Reference Aparicio, Martín-Grau, Bedmar, Serrat Orus, Basora and Arija14} Despite the abundance of literature on fatty acid levels and congenital birth defects, including neurological abnormalities, this study uniquely dissects individual fatty acids, revealing significant associations not previously identified.

This study showed that cases had higher but not significant levels of total SFA. The only significant difference among studied SFA was found for heptadecanoic acid.

Currently, it is recognized that consuming large amounts of SFA alters metabolism in the central nervous system as well as in the peripheral organs. Even while the exact mechanisms and signaling pathways in the brain that are impacted by exposure to various SFA concentrations are unknown, there is evidence that involves altered energy metabolism, decreased neuronal viability, and neuroinflammation. As a result, high SFA intake is thought to pose a serious risk for Alzheimer’s disease, pathological brain aging, and cognitive decline. ^{Reference Sánchez-Alegría and Arias15}

In contrast, the amount of nervonic acid, ARA, adrenic acid, eicosapentaenoic acid, docosahexaenoic acid, and n-3 PUFA were significantly lower in cases (p < 0.05). Over the last decade, studies reported that ARA and DHA predominate in the composition of the brain. Both ARA and DHA are preferentially transported across the placenta during pregnancy and are stored in the developing brain from the early stages of its development. ^{Reference Crawford, Golfetto and Ghebremeskel16}

ARA accumulates rapidly in the brain during development, which occurs from the beginning of the third trimester of pregnancy until about 2 years of age. ^{Reference Martinez17}

Because NTDs are common major congenital anomalies that result from very early disruption in the development of the brain and spinal cord, ^{Reference Nasri, Midani and Kallel18} this may explain the reduced levels of ARA, DHA, and EPA in cases.

Various functions have been shown to be related to ARA. It is required for brain development, where it is involved in cell division and communication. It regulates neuronal firing, signaling, and long-term potentiation. ^{Reference Sanchez-Mejia, Newman and Toh19} It also contributes to the maintenance of membrane organization and hippocampal plasticity, protects the brain from oxidative stress in the hippocampus by activating the peroxisome proliferator-activated receptor gamma, and aids in the production of new protein in the tissue. ^{Reference Fukaya, Gondaira and Kashiyae20}

ARA is also involved in the hormonal regulation of appropriate bone formation and whole-body mineral metabolism during baby and childhood development. Eicosanoids convey cellular, organ, and systemic signals during skeletal development to balance calcium and phosphate requirements for bone formation and other metabolic activities. When bone tissue is formed during long bone growth, ARA mediates vitamin D3-regulated chondrocyte maturation and proliferation for the mineralization of skeletal growth plates. ^{Reference Brochhausen, Neuland, Kirkpatrick, Nusing and Klaus5}

On the other hand, EPA and DHA can function as competing inhibitors of ARA release from cell membranes and binding to the active site of COX-2. EPA also works as an alternate substrate for this enzyme, resulting in less production of pro-inflammatory and pro-carcinogenic ARA derivatives. ^{Reference DiNicolantonio, McCarty, Chatterjee, Lavie and O’Keefe21} This could explain the deduced suggestion of higher NTD risk with an increased ARA:(EPA + DHA) ratio.

In animal models, roughly half of the quantities of ARA and DHA accumulate in the rat brain before myelination and at 15 d after birth, when myelination has just begun. Myelination is principally responsible for the highest rate of brain growth, and the synthesis of myelin lipids, which are required early in brain development, has been demonstrated affected by low-PUFA diets. ^{Reference Crawford and Sinclair22}

Cases of this study had significantly lower levels of DHA than controls.

DHA has been shown to have a neuroprotective role during neonatal asphyxia in rats and in prematurely born pigs. In rats, a maternal diet enriched in DHA during pregnancy prevents neonatal brain injury. ^{Reference Herrera and Ortega-Senovilla4}

In other studies, lower levels of maternal DHA status during pregnancy were suggested to be involved in compromised maternal selective attention, which is a key component of cognition. ^{Reference De Groot, Adam and Hornstra23} A declining maternal DHA status has also been associated with a higher incidence of postpartum depression. ^{Reference Otto, de Groot and Hornstra24}

In our study, linolelaidic acid (a cis-trans isomer of linoleic acid (LA)) was significantly higher in cases than in controls.

According to brain kinetics in fetal baboons, in addition to maternal-produced ARA, LA may be transported across the blood-brain barrier despite its very low level in brain lipids. The brain contains an active desaturation/elongation machinery that converts LA to ARA. ^{Reference Su, Corso, Nathanielsz and Brenna25}

This may explain high linolelaidic acid levels in cases which may be due to its inability to be converted to ARA or to decreased levels of ARA found in our cases.

In the present study, n-3 PUFA levels were significantly lower in cases than in controls.

In the study of Koletzko et al. (2008), a low n-3 PUFA intake during pregnancy resulted in a little shorter gestation, a slightly lower birth weight, and an increased chance of premature delivery. ^{Reference Nandi, Wadhwani and Joshi26}

Contrariwise, the ingestion of n-3 PUFA has been shown to have a wide range of health benefits. The brain effect of n-3 PUFA is permanent, as neuropsychiatric illnesses such as Parkinson’s disease ^{Reference Farquharson, Jamieson, Abbasi, Patrick, Logan and Cockburn27} and mild cognitive impairment ^{Reference Bo, Zhang and Wang28} have been reversibly related to n-3 PUFA deficit.

In our study, nervonic acid, a MUFA, was found with significantly lower levels in cases.

It is commonly proved that MUFA have anti-inflammatory benefits and may improve endothelial function. ^{Reference Summerhill, Karagodin, Grechko, Myasoedova and Orekhov29}

Neuroinflammation, a complex process involving the activation of various immune cells within the central nervous system, was reported as an important pathological feature of NTDs. ^{Reference Wang, Ji and Dong30}

Adrenic acid levels were significantly lower in cases than in controls in our study group.

Adrenic acid is the third most abundant PUFA in the brain, and it is rich in myelin lipids, especially phosphatidylethanolamine. Like ARA, it accumulates rapidly in babies during the early postnatal period of the brain growth spurt. As an early precursor to adrenic acid, ARA plays a potentially important role in its metabolism in vivo. Adrenic acid conversion may be a key mechanism for ARA use in babies in order to fulfill the rapid increase in adrenic acid required for neural tissue development. ^{Reference Martinez31}

This could explain decreased adrenic acid levels in cases of our study, which may be due to the decreased levels of its precursor, ARA, found in our case group or a possible non-conversion of ARA to adrenic acid.

In this current research, we also studied the correlation between FA and vitamin D levels in cases. To our knowledge, this study for the first time reports the association of 25(OH)D levels with fatty acid levels in pregnancies affected by NTDs.

Maternal 25(OH)D levels were positively correlated with maternal PUFA and n-6 PUFA.

The same finding was observed in other studies, which demonstrated a positive association of vitamin D with total PUFA and n-6 PUFA levels in women with preeclampsia. However, in contrast with our results, they also showed that vitamin D was negatively associated with SFA and MUFA levels. ^{Reference Nandi, Wadhwani, Randhir, Wagh and Joshi32}

The anti-inflammatory and immune-regulating effects of both vitamin D and PUFA are well established. ^{Reference Chirumbolo, Bjørklund, Sboarina and Vella33,Reference Ghosh, Gao, Thakur, Siu and Lai34} Neuroinflammation was identified as the significant pathological feature in both the central nervous system and amniotic fluid from fetuses with NTDs. ^{Reference Wang, Ji and Dong30}

In the study of Nandy et al. (2019), lower levels of ALA and EPA were observed in vitamin D deficiency as compared to controls. However, ARA and n-3 PUFA levels were similar in both groups. ^{Reference Nandi, Wadhwani and Joshi26}

PUFA, such as ARA, have been demonstrated to be significant in the homeostatic regulation of vitamin D and parathyroid hormone during bone formation. ^{Reference Hadley, Ryan, Forsyth, Gautier and Salem35}

This could explain why bones are affected in some NTD subtypes in mothers with decreased levels of ARA.

Furthermore, an animal study demonstrates that maternal deficiency of vitamin D influences FA metabolism and alters FA levels. Nandi et al. (2019) observed that maternal vitamin D deficiency increases the ratio of vasoconstrictor and vasodilator lipid metabolites of PUFA by increasing Hcy and oxidative stress levels. ^{Reference Nandi, Wadhwani and Joshi36}

It is important to remind that significant low levels of vitamin D and increased Hcy levels were associated with the occurrence of NTDs in in this study group in previous studies. ^{Reference Nasri, Ben Fradj and Feki12,Reference Nasri, Ben Fradj and Touati13}

Our results have shown that maternal RBC folate levels were negatively correlated with maternal n-3 PUFA.

According to Kulkarni A et al. (2011), there was no correlation between folic acid or plasma vitamin B₁₂ and erythrocyte n-3 PUFA in either the normotensive or pre-eclamptic groups. ^{Reference Kulkarni, Mehendale and Pisal37}

These findings suggest an intrinsic link between biochemical parameters and FA profiles in the occurrence of NTDs.

We have also studied the distribution of FA levels by maternal/fetal characteristics in cases. Results have shown decreased SFA levels and increased UFA levels in cases aged less than 30 years.

The study of Carver JD et al. (2001) indicated that the relative levels of PUFA decreased with age with the exception of DHA. ^{Reference Carver, Benford, Han and Cantor38} In our previous study, DHA level was positively correlated with maternal age. ^{Reference Nasri, Ben Jamaa, Siala Gaigi, Feki and Marrakchi10}

In our study group, maternal n-3 PUFA levels were significantly higher in the group of multiparous cases and in the group of multigravida cases. This finding is in contrast with the study of Bokor et al. (2007), who reported a maternal PUFA depletion with increasing parity. ^{Reference Bokor, Koletzko and Decsi39}

This result may be due to that multiparous women are more likely exposed to health promotion messages regarding folate during previous pregnancies and thus have a greater awareness of the need for periconceptional folic acid. This suggestion is confirmed by our finding where cases supplemented with folic acid had higher levels of n-3 PUFA than cases not supplemented with folate (Table 5).

Further studies are needed to establish the association between FA levels and folic acid supplementation.

In addition, we have found that cases with previous history of NTDs had higher levels of SFA but lower levels of MUFA and UFA (Table 5).

This finding confirms our hypothesis of an association between increased SFA levels (heptadecanoic acid), related to altering metabolism in the central nervous system, decreasing neuronal viability, and inducing neuroinflammation, as well as decreased MUFA levels (nevronic acid), characterized by its anti-inflammatory benefits, as shown previously, in the occurrence of NTDs. Cases with fetal term ≤ 20 weeks of gestation had higher levels of n-6 PUFA. However, the absolute amount of n-6 PUFA increases in all organs with increasing gestational age, especially the absolute amount of ARA, which increases because of the brain growth. ^{Reference Kuipers, Luxwolda, Offringa, Boersma, Dijck-Brouwer and Muskiet40}

However, due to the absence of a major part of the brain, skull, and scalp (anencephaly) ^{Reference Nasri, Ben Fradj and Hamdi41} and to the very early disruption in the development of the brain and spinal cord in NTDs subtypes, we have observed decreased n-6 PUFA with increasing fetal term.

Finally, our study is limited by the lack of some mothers’ information resulting in varied sample numbers depending on the parameter and by the small sample size of case and control groups, which may be due to the fact that Tunisia is a small country. More research with a larger sample is required to support our findings.