Association between vitamin D plasma concentrations and VDR gene variants and the risk of premature birth

A preterm birth is defined as a delivery that occurs before 37 weeks’ gestation, and can be classified as late preterm (36–34 weeks), very preterm (28–34 weeks), or extremely preterm (less than 28 weeks) [1, 2]. Prematurity is the main cause of death among children under 5 years of age [3], and preterm infants may present with early and late health complications involving the immune, respiratory, cardiovascular, gastrointestinal, and endocrine systems, as well as growth and developmental problems [3, 4]. The estimated overall global premature birth rate is 11.1% [5], and with an approximate rate of 11.8% [6], Brazil is in the top 10 countries with the highest rates [1, 2] .

Studies have associated prematurity with low serum levels of vitamin D during gestation, which is a period of risk for vitamin D deficiency and insufficiency. Vitamin D can be obtained in several ways: by endogenous synthesis in the skin driven by exposure to ultraviolet B rays in sunlight, during which 7-dehydrocholesterol is converted to cholecalciferol (vitamin D3) [7]; and from dietary sources such as fatty fish, which contain cholecalciferol [8], and plants and fungi containing ergocalciferol (vitamin D2) [9]. Both vitamin D2 and vitamin D3 undergo two hydroxylation reactions to become active: the first in the liver by 25-hydroxylase, which produces 25(OH) D (the circulating form); and the second in the kidneys by 1α-hydroxylase, which produces 1,25(OH)2D (the metabolically active form) [10].

An adequate level of vitamin D 25(OH) D is above 30 ng/mL (75 nmol/L); 21–29 ng/mL (51–74 nmol/L) is insufficient, and below 20 ng/mL (50 nmol/L) is low [11]. The global mean of vitamin D deficiency is 29.8% and the global mean of insufficiency during gestation is 87.0%. Vitamin D deficiency during pregnancy is associated with increased risk of gestational diabetes, preeclampsia, fetal growth restriction, bacterial vaginosis, caesarean section, and prematurity [12]. Vitamin D deficiency is also common among infants in several countries; prevalence ranges from 50 to 70% in the United States [13], and it is approximately 60.5% in Brazil [14].

Most of the biological activities attributable to vitamin D are mediated by its receptor (VDR), which is encoded by the VDR gene (vitamin D receptor; Gene ID 7421; MIM 601769) with a chromosomal locus of 12q13.11. It is a member of the steroid hormone receptor family that mediates the action of vitamin D by regulating the transcription of multiple genes [15]. Alterations in the VDR gene can lead to important defects in gene activation that can affect calcium metabolism [10], cell proliferation [8, 16], and immune function [8]. The most studied VDR gene single nucleotide variation (SNV) related to clinical outcomes are ApaI, BsmI, FokI, and TaqI [17‐22].

Studies have produced conflicting results regarding the association between VDR gene variants and prematurity [17‐22]. Different allelic frequencies among the populations arise from different genetic ancestries, so the racial origins of the studied population may be responsible for the divergent results. Hence, Brazilian population shows contributions from three main parental groups: Amerindian, European, and African [23, 24], this heterogeneity can produce different allele frequencies from those presented by non-mixed populations.

The data relating to the mothers and PTN and FTN newborns are shown in Table 1. The mean age of the mothers who experienced preterm births was 26.4 ± 7.1 years, compared to 25.8 ± 6.9 years for mothers who experienced full-term births. The mean pre-gestational body mass index for the mothers of PTNs was 27.2 ± 4.9 kg/m², compared to 26.2 ± 5.7 kg/m² for the mothers of FTNs. Two mothers (5.0%) in the case group and three (3.3%) mothers in the control group received 25(OH) D supplementation during gestation (P = 0.631). Considering pregnancy diseases, specific hypertension disease in pregnancy was found in 45.0% of mothers in the case group and 13.0% in the control group (P < 0.001); however, gestational diabetes mellitus (5.0% versus 2.2%) and urinary tract infection (22.5 and 33.7%) were not statistically different between groups. Only 10.0% of mothers in the case group reported regular solar exposure compared with 66.3% of mother of the control group (P < 0.001).

During gestation, the mother is the fetus’ only source of vitamin D, which is transmitted across the placenta [30]. The levels of vitamin D in the mother are directly related to those in the fetus, and vitamin D deficiency has been associated with an increased risk of certain childhood diseases, such as rickets, infectious diseases, noncommunicable chronic diseases, obesity, and asthma [13]. The reduction in the level of albumin and the increase in the hepatic production of vitamin D-binding protein (DBP) during pregnancy alter maternal vitamin D metabolism. This reduces the availability of free vitamin D and increases the conversion of 25(OH) D to 1,25(OH)2D3 through the placenta, which is the main extra-renal site of this conversion owing to the increased activity of 1α-hydroxylase [30]. Moreover, the late transfer of vitamin D to the fetus may be impaired in preterm birth, which has prompted various investigations of serum vitamin D levels in pregnant women [31, 32].

A meta-analysis including 10,098 patients from 10 studies found an increased risk of preterm birth for pregnant women with vitamin D deficiency (< 20 ng/mL; OR = 1.29; 95% IC = 1.16–1.45) [31]. A meta-analysis performed by Zhou et al. (2017) [33] including 6 randomized controlled trials and 18 observational studies, the increased risk of prematurity was associated to deficiency (OR = 1.25; 95% IC = 1.13–1.38) more than maternal vitamin D insufficiency (OR = 1.09; 95% IC = 0.89–1.35). Recently, the association between maternal vitamin D deficiency and preterm birth was evaluated in a study by Woo et al. [34] comprising studies from 2012 to 2018, being non conclusive despite analysis of research indicates that vitamin D deficiency is related to an increased risk for preterm birth [34].

The evidence suggests that preterm labor is a heterogeneous condition with many triggering and precipitating factors, especially infections and inflammatory causes [35]. 1,25(OH)2D promotes cytokine inhibition and the expression of potent antimicrobial peptides in various immune cells, such as macrophages and dendritic cells, and acts on placental tissue by modulating anti-inflammatory effects [31].

Preeclampsia is a risk factor for premature birth whose only treatment is birth [36, 37]. A large body of evidence indicates that increased inflammatory response is a feature of systemic vascular dysfunction in this disorder, although its cause is not completely known [38, 39]. Due to vitamin D anti-inflammatory effects, it may play a role in preeclampsia prevention [34, 40‐42]. A recent study [43] pointed that down-regulation of VDR expression and vitamin D deficiency may contribute to phenotypic changes of inflammatory patterns in maternal vasculature in preeclampsia. Otherwise, the VDR FokI variant was associated with decreased risk of preeclampsia in the dominant model in Iranian population [44].

Despite the evidence of an association between vitamin D deficiency and preterm birth and preeclampsia, data on the effect of vitamin D supplementation on preterm birth have not been consistent, probably due to the heterogeneity of studies regarding the differences in assay methodologies, definitions of vitamin D deficiency, timing of supplementation, and dose of supplementation [34]. A recent Cochrane review [39] showed evidence from 3725 pregnant women enrolled in 22 studies, suggesting that vitamin D supplementation alone during pregnancy potentially attenuates the risk of preeclampsia in comparison to placebo or no intervention. Also, it may have small or even no difference for the risk of premature birth. Other review raised evidence from nine studies involving 1916 pregnant women and suggested that the risk of preeclampsia is reduced when supplementation with vitamin D and calcium is performed, although may increases risk of preterm birth. Other than that, the benefits or harms of vitamin D supplementation alone or combined with calcium and other vitamins and minerals during pregnancy for mother and children remains unclear [39].

Currently, some countries and international scientific organizations recommend screening in pregnant women who are at risk for vitamin D deficiency and supplementation with doses ranging from 600 to 1000 IU/day [45‐47]. In Brazil there are no recommendation for vitamin D screening and supplementation during pregnancy. WHO [48] reported a need for more evidence for recommending vitamin D supplementation greater than 200 units per day.

It is important to note that there are several other pregnancy and neonatal complications associated with maternal vitamin D deficiency such as increased incidence of gestational diabetes mellitus, low birth weight, and possible epigenetic effects on offspring [34].

In the present study, specific hypertension disease in pregnancy (45.0% versus 13.0%, respectively) was significantly higher in PTN mothers in relation to the FTN group; whereas gestational diabetes mellitus (5.0% versus 2.2%, respectively) and urinary tract infection (22.5% versus 33.7%, respectively) were not significantly different between PTN and FTN mothers. In addition, no difference was found considering the supplementation of vitamin D, folic acid, and iron. Only 10% of PTN mothers regularly sunbathed in relation to 66.3% in FTN mothers; however the frequency of photo protection was not different between groups (20.0% in PTN mothers and in 15.2% of FTN mothers). Regarding the 25(OH) D levels, the present study revealed that the 25(OH) D levels in preterm mothers was significantly lower in relation to the full-term group, and there was vitamin deficiency in 47.5% of the PTN mothers. The 25(OH) D levels in preterm newborns was lower in relation to the full-term newborns, although this difference was not statistically significant. There was vitamin deficiency in 34.2% of the PTNs.

Considering the VDR variants, the findings of previous studies include strong indications of no association between VDR variants and the risk of prematurity (Table 6). Only six studies have associated VDR gene variants with the risk of preterm birth, and no study has associated these SNVs with serum vitamin D levels.

Manzon et al. (2014) [17] investigated 33 Caucasian Jewish Israeli mothers and their preterm newborns (24–35 weeks’ gestation), and 98 other mothers and their FTNs. They reported that the C allele of the Fokl SNV was more frequent, and the T allele of the TaqI SNV was less frequent, in preterm mothers. There was no association between the genotypes of the studied variants and preterm and full-term birth. Furthermore, Cai et al. (2016) [18] investigated 57 preterm and 84 full-term Chinese mothers, and reported that the FF genotype of the Fokl SNV was associated with an increased risk of preterm birth.

Baczyńska-Strzecha et al. (2016) [19] investigated 100 Polish preterm and 99 full-term mothers. They reported that the frequencies of the individual genotypes did not differ. However, the BsmI/bb-ApaI/AA-TaqI/TT and BsmI/BB-ApaI/aa-TaqI/tt genotype combinations were significantly more frequent in the preterm group, whereas the BsmI/Bb-ApaI/AA-TaqI/Tt and BsmI/BB-ApaI/Aa-TaqI/tt combinations reduced the risk of preterm birth. In contrast, in a study by Rosenfeld et al. (2017) [20] - which included 146 Israeli Jewish women and their preterm newborns, and 229 other women and their full-term newborns - the CC homozygous genotype of the ApaI SNV was associated with premature birth.

A Brazilian study by Javorski et al. (2018) [21] - which included 104 preterm and 85 full-term mothers—revealed that the T allele and TT wild genotype of the FokI SNV were more frequent in women who had preterm births. However, an Italian study by Barchitta et al. (2018) [22] - which comprised 17 pairs of mothers and their PTNs and 187 pairs of mothers and their FTNs - revealed that the Fokl variant genotype was associated with an increased risk for preterm birth.

In the newborns, the TT genotype of the BsmI, the AA genotype of the ApaI, the GG genotype of the Fokl SNVs, and the GAG (TaqI/G-ApaI/A-FokI/G) haplotype was significantly more frequent in the PTN group, whereas the frequencies of the CT genotype of the BsmI SNV and the GCA (TaqI/G-ApaI/C-FokI/A) haplotype were significantly higher in the FTN group. The PTN carriers of the GG genotype of the FokI SNV had significantly lower 25(OH) D levels.

The genotypes of the BsmI variant deviated from HWE in the PTNs mothers and their newborns. Hardy–Weinberg disequilibrium may indicate genotyping errors, population stratification, or selection bias [49]. However, the studied variants were identified by qPCR using TaqMan probes, which is a validated and robust methodology. This finding may indicate a possible association between the marker locus and the risk of prematurity.

Conflicting results can be attributed to important differences between studies. Ethnicity is an important factor in SNV prevalence among different geographic populations, and is especially important in admixed populations such as the Brazilian population. Brazil is a country of continental dimensions; it covers 8,516,000 km², and is inhabited by more than 200 million people. Brazilian population origins traces back to the former Amerindians and the main sources of immigration, specially western-european and African [50]. With regard to the five geographical regions of Brazil (North, Northeast, Central-West, Southeast, and South), ancestry from North Brazil is mostly amerindian, Northeast and Central-West mainly African, and Southern and Southeastern predominantly European. For decades, new immigrants and migrants from other parts of Brazil have flocked to Southeast Brazil, where intermarriage between individuals of different ancestries is very common [50]. This could explain the differences between the findings presented here - which are based on a population from the Southeast region of Brazil - and those of Javorski et al. (2018) [21] - which were also based on a Brazilian population, but from the Northeast region, although none of these studies evaluated the ancestral origin of the studied population.

Moreover, the variants studied in the different publications were not the same, and the genetic nomenclature used in some studies was diverse from described in the Human Genome Variation Society (HGVS) [29] - which hindered the interpretation of the results. Thus, the studied groups are heterogeneous, which may make their interpretation and comparison difficult.