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folate carrier (RFC1); MTR, methionine synthase; CUBN, cubilin. Numbers in bold show statistically significant differences.

### **4. Discussion**

The present study was carried out on pregnan<sup>t</sup> women from northwestern Spain, which showed diversity in the prevalence of one-carbon metabolism risk polymorphisms. Importantly, among the evaluated SNPs, those related to *MTHFR* were associated with a lower frequency of spontaneous gestation and higher frenquecy ofpreeclampsia, and PTB, while the *CUBN* polymorphism was associated with a lower frequency of complications during delivery. Detection of risk alleles in women may lead to personalised medicine with targeted treatment based on increased vitamin intake that can improve success in conception and maternal and foetal outcomes.

Different genetic variants have been related to vitamin deficiency. The population prevalence of most of the polymorphisms associated with altered vitamin levels is unknown. The most widely studied polymorphism of one-carbon metabolism is the *MTHFR* genotype 677 C > T (rs1801133), which is responsible for the synthesis of the *MTHFR* enzyme and whose activity is decreased in TT homozygosis (by 60%) and in CT heterozygosis (by 30%) with respect to the CC genotype [24]. The prevalence of the TT SNP, the highest risk genotype, represented between 14.2% and 19.9% [25,26] of the cases described in studies carried out in Europe. More recently, Aguilar-Lacasaña et al. (2021) showed a higher prevalence (38%) in pregnan<sup>t</sup> women in southern Spain [27]. In the current study, the prevalence in pregnan<sup>t</sup> women carriers of TT SNP was 57%. This alteration has grea<sup>t</sup> relevance because individuals with TT have lower levels of folic acid than those with CC and CT [28–30].

Several studies have shown that the homozygous TT genotype has a lower response to folic acid treatment than those with the homozygous CC genotype, suggesting that the TT genotype requires higher folic acid intake [31]. The impact of these genetic alterations on folic acid levels and their effect on different diseases [31,32] such as breast, lung, or colorectal cancer [33] is widely known. Specifically, in pregnan<sup>t</sup> women, the homozygous TT genotype has been associated with an increased risk of neural tube defects [34–37], reinforcing the idea of the well-known link between folic acid deficiency during pregnancy and the risk of neural tube defects in the newborn.

In our study, all women received supplements with folic acid or folic acid and B12. In this regard, Colson et al. (2017) [38] showed that a dose of 400 mcg daily of folic acid would be sufficient to overcome the deficits resulting from these polymorphisms. However, a recent study by Aguilar-Lacasaña et al. (2021) [27] showed that, although their population had received a correct folic acid intake during gestation, the prevalence of SGA and LGA was higher in pregnan<sup>t</sup> women with T or TT in relation to the hetero or homo polymorphism CC, suggesting that there must be other factors that influence these results.

In addition, different studies have shown that women with the *MTHFR* 677 TT genotype are predisposed to elevated homocysteine levels when folic acid intake is inadequate [39], endothelial damage, arterial constriction, and thrombosis [40,41], all of which can lead to placental hypoperfusion resulting in worse neonatal outcomes with PTB and LBW [42]. In our study, women with the *MTHFR* risk alleles had a higher frequency of PTB, even though they all received adequate folic acid, but no higher frequency of LBW was observed. In addition, this higher frequency of premature newborns is concomitant with a higher frequency of LBW, which is a frequent neonatal complication [43,44] that has a direct implication in adolescence and adulthood, with a higher prevalence of chronic diseases such as obesity, diabetes, metabolic syndrome, or cardiovascular pathology [45].

Elevated homocysteine levels, derived from the incorrect functioning of the *MTHFR* enzyme, have also been related to an increased risk of spontaneous abortions [46]. In our series, women with the *MTHFR* risk allele had a lower frequency of spontaneous pregnancy, i.e., a greater need for assisted fertilization, which may reflect a difficulty in gestation derived from the incorrect functioning of the one-carbon metabolism pathway. The *MTHFR* risk allele in our population was associated with a lower frequency of spontaneous pregnancy, which is known to have a negative impact on health care costs and maternal mental health.

In our series, women with risk alleles of *MTHFR* had a higher frequency of preeclampsia than those without. Preeclampsia is characterised by hypertension accompanied by proteinuria in pregnan<sup>t</sup> women over 20 weeks of gestation. This disease can affect both the foetus and the mother and, in extreme situations, can compromise the life of both [47–49]. A meta-analysis report by Wang et al. (2013) [50] showed a significant association between the *MTHFR* T allele and pre-eclampsia among Caucasians and people of Asian descent but not among people of African descent. However, a recent study carried out in Lagos, southwestern Nigeria, has shown an occurrence of preeclampsia was significantly associated with the presence of the T allele of *MTHFR* (OR = 1.855; *p* < 0.05) [51].

As for the cubilin gene (*CUBN*), the intrinsic factor-vitamin B12 receptor, we found that the allelic distribution of SNP rs1801222 was significantly different depending on complications during delivery—namely, a higher frequency of delivery complications was found among carriers of the wild-type allele. This suggests that carrying this genetic variant could reduce the risk of delivery complications. In this regard, previous studies have observed a significant increase in the risk of congenital heart disease for carriers of the wild-type allele of the *CUBN* SNP rs11254363 [52]. Cubilin favours the absorption of intrinsic factor-vitamin B12 complex in the intestinal mucosa. Polymorphisms in the cubilin gene were associated with variability in the binding and transport of vitamin B12. In this regard, it was demonstrated in a meta-analysis that participants homozygous for the rs1801222 G allele had higher plasmatic B12 levels [53]. Adequate maternal vitamin B12 status is associated with advantageous maternal and child health outcomes [54]. Therefore, the low frequency of delivery complications observed in G carriers in the current study could be related to higher plasmatic B12 levels in these participants. As far as we know, this is the first study that found a different distribution of *CUBN* SNPs in women with delivery complications, such as caesarean section and induced or instrumental delivery.

Other relevant polymorphisms involved in the one-carbon metabolism are *MTR* and *SLC19A1*. *MTR* encoded an enzyme involved in the synthesis of methionine through homocysteine methylation with the presence of vitamin B12 (Vit. B12) as a co-factor, and SNPs in this gene were associated with risk during pregnancy [55]. SLC19A1 gene encodes a typical transporter with 12 transmembrane domains involved in the active transport of 5-methyltetrahydrofolate from plasma to the cytosol and regulation of intracellular folate concentration. It may limit the absorption of folic acid by the developing foetus, thus affecting the growth of the foetus [56]. In the current study, we did not observe an association between the studies SPNs of these genes and pregnancy or newborn complications.

The present study has several limitations such as a small sample size and short followup time. Moreover, all women included in this study were prescribed folic acid and vitamin B12 supplementation during pregnancy which possibly mask the association between one-carbon polymorphisms and pregnancy or newborn complications. Among the mother complications, a higher prevalence of GDM than the general population was found in this cohort. This is because pregnan<sup>t</sup> women between 24 and 28 weeks of gestation who attended both the endocrinology and obstetrics departments were invited to participate in the study, and, in particular, the pregnan<sup>t</sup> women who attended the endocrinology department mainly visited for metabolic problems, mostly GDM. Another limitation of this study could be that there are no metabolic data to assess the consequences of polymorphisms on the metabolism of single carbons, which limits the analysis of the significance of the associations. However, this may be the beginning of future studies with larger sample sizes and longer follow-up times that include both pregnan<sup>t</sup> women as well as women with gestational desire to know the implication of these polymorphisms and their possible approach in the preconception stage.

### **5. Conclusions**

In conclusion, our data show a high prevalence of genetic variants related to folic acid and vitamin B12 metabolic genes in pregnan<sup>t</sup> women that may justify the difference in maternal and neonatal outcomes. This study warrants the need to perform further studies

to elucidate whether knowing the prevalence of these polymorphisms on an individual basis and its association with the mother and newborn health may lead to personalised medicine with a nutritional assessment of vitamin intake.

**Author Contributions:** Conceptualisation, A.B.C.; data curation, G.R.-C., P.M.L. and A.C.-B.; formal analysis, G.R.-C., P.M.L., A.B.C., M.A., M.Z. and L.M.; investigation, F.F.C. and A.B.C.; methodology, A.B.C., L.M., L.S., M.M.-C. and F.F.C.; supervision, A.B.C.; writing—original draft preparation, G.R.- C., P.M.L. and A.B.C.; writing—review and editing, G.R.-C., P.M.L. and A.B.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research work of the authors is supported by Fundación Paideia Galiza and Fundación de la Sociedad Gallega de Endocrinología, Nutrición y Metabolismo (SGENM), as well as Xunta de Galicia-Gain (IN607B2020/09) and Centro de Investigación Biomedica en Red fisiopatología de la obesidad y nutricion (CIBERobn), the Miguel Servet Project (CP17/0008) and research projects (PI20/00650; PI20/00628), under the initiative of Instituto de Salud Carlos III (ISCIII) and co-financed by the European Regional Development Fund (FEDER). PML is funded by a predoctoral gran<sup>t</sup> from Xunta de Galicia (IN606-2020/013). ABC is a Miguel Servet researcher (ISCIII; CP17/0008).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Autonomic Committee of Clinical Research Ethics of Galicia, Spain (protocol code 2017/622). The participants did not receive economic profit.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data are available upon reasonable request from the corresponding author.

**Acknowledgments:** The authors thank all participants in this study and the research group involved in the project, as well as the individuals who performed the fieldwork, such as the Laboratory Technician Jesus Iglesias and Data Manager Maribel Rendo.

**Conflicts of Interest:** The authors declare no conflict of interest.
