Early- Life Effects of Vitamin D: A Focus on Pregnancy and Lactation
The initiation of human life at the moment of conception involves a myriad of ancient signaling hormones, which include vitamin D
Early- Life Effects of Vitamin D: A Focus on Pregnancy and Lactation
Long known for its role as a preprohormone in calcium and bone homeostasis, our understanding of vitamin D now extends to its functions in regulating innate and adaptive
immunity. From early in pregnancy, there is a rise in circulating levels of 1,25-dihydroxyvitamin D, but drop to prepregnancy levels after birth. A growing body of evidence indicates that vitamin D can affect gene expression, including genes associated with immune defense pathways. In turn, vitamin D metabolism during pregnancy is modulated by the individual’s genetic background. In the future, this knowledge may enable us to fine-tune the dosing of vitamin D supplements during pregnancy, as well as identify subgroups of women who may be at greater risk of vitamin D deficiency.
There are 2 forms of vitamin D: ergocalciferol (or vitamin D2, synthesized by plants and fungi) and cholecalciferol (or vitamin D3, synthesized in human skin and by animals). Humans are able to metabolize both forms of vitamin D. The initial step in metabolic activation of vitamin D is an enzyme-catalyzed insertion of an OH group at carbon 25, resulting in 25(OH)D, the most abundant form of vitamin D in the circulation. Parathyroid
hormone (PTH) is an important mediator of vitamin D status. When vitamin D levels decrease, PTH increases, affecting intestinal absorption of vitamin D and skin conversion from its precursor. Thus, measurement of intact PTH levels also has been used as an indicator of vitamin D deficiency.
When mother is vitamin D insufficient or deficient, breast milk has a relatively low vitamin D content. Consequently, all breastfed babies should receive a vitamin D supplement of
400 IU/day. Most infants in technologically dependent societies are not exposed to direct sunlight until after 6 months of age; therefore, endogenous synthesis is not a reliable source of vitamin D. Currently, a major challenge is lack of compliance among parents in giving vitamin D supplements to their breastfed infants. Where maternal compliance with taking a vitamin D supplement is greater than that of parental adherence to infant supplementation, maternal vitamin D supplementation remains a viable alternative that safely and effectively treats both the mother and her breastfeeding infant.
Wagner CL, Eidelman AI. The impact of vitamin D on the maternal and infant epigenome: the role of pregnancy and breastfeeding. Breastfeed Med. 2018 Jun;13(5):305–6.
• The active form of vitamin D – 1,25-dihydroxyvitamin D (1,25[OH]2 D) – increases during pregnancy, remains elevated throughout, and, unlike at other times during the lifecycle, is
directly affected by circulating 25-hydroxyvitamin D (25[OH] D) concentration with the optimal point of conversion of 25(OH)D to 1,25(OH)2 D at 100 nmol/L (40 ng/mL).
• Lactation has increased demands on the mother regarding nutrient intake delivered through her breast milk to her recipient infant: when a mother is vitamin D deficient, her milk is deficient, which can be remedied by direct infant supplementation; however, this treats only the infant.
• A safe alternative during lactation to infant supplementation is direct maternal vitamin D supplementation at higher doses than usual (6,400 IU/day), improving the vitamin D status of the mother, the content of the milk, and, consequently, the vitamin D status of the infant, effectively treating both the mother and the infant.
1,25-dihydroxyvitamin D · 25-hydroxyvitamin D · Cholecalciferol · Calcidiol · Clinical nutrition · Human nutrition · Infancy and childhood · Lactation · Pregnancy
Vitamin D is an endocrine regulator of calcium and bone metabolism. Yet, its effects include other systems, such as innate and adaptive immunity. Unique to pregnancy, circulating 1,25-dihydroxyvitamin D (1,25[OH]2 D) increases early on to concentrations that are 2–3 times prepregnant values. At no other time during the lifecycle is the conversion of 25-hydroxyvitamin D (25[OH]D) to 1,25(OH)2 D directly related and optimized at ≥ 100 nmol/L. Vitamin D deficiency appears to affect pregnancy outcomes, yet randomized controlled trials of vitamin D supplementation achieve mixed results depending on when supplementation is initiated during pregnancy, the dose and dosing interval, and the degree of deficiency at the onset of pregnancy. Analysis of trials on an intention-to treat basis as opposed to the use of 25(OH)D as the intermediary biomarker of vitamin D metabolism yields differing results, with treatment effects often noted only in the most deficient women. Immediately after delivery, maternal circulating 1,25(OH)2 D concentrations return to pre-pregnancy baseline, at a time when a breastfeeding woman has increased demands of calcium, beyond what was needed during the last trimester of pregnancy, making one question why 1,25(OH)2 D increases so significantly during pregnancy. Is it to serve as an immune modulator? The vitamin D content of mother’s milk is directly related to maternal vitamin D status, and if a woman was deficient during pregnancy, her milk will be deficient unless she is taking higher doses of vitamin D.
Because of this relative “deficiency,” there is a recommendation that all breastfed infants receive 400 IU vitamin D3 /day starting a few days after birth. The alternative – maternal supplementation with 6,400 IU vitamin D3 /day, effective in safely raising maternal circulating vitamin D, that of her breast milk, and effective in achieving sufficiency in her recipient breastfeeding infant – remains a viable option. Additional research is needed to understand vitamin D’s influence on pregnancy health and the effect of maternal supplementation on breast milk’s immune signaling.
From the moment of conception, there are tremendous changes that must occur for growth and shaping of a singlecell organism to billions of cells as the construct of diverse
systems, which function in concert to form a living human being. It is in the context of this timing, this concert of matter and energy transfer across cells, that we can appreciate what is happening surrounding conception. Conception does not occur in a hostile or nonnurturing environment, yet the very invasion of extravillous cytotrophoblasts into the uterine wall is an invasive and inflammatory process [1–3]. Pregnancy is a state of change and flux that must balance between negentropy—
organization of tissue and cellular death and apoptosis necessary for refinement of tissue and organ structure. The very event of conception is dependent upon a functional
neuroendocrine system in both the mother and father, a functioning uterus with a rich lining to allow for invasion of the extravillous cytotrophoblasts into the uterine wall, and a
dynamic synchrony of cell division and cell death. The initiation of human life at the moment of conception involves a myriad of ancient signalling hormones, which include vitamin D [4, 5].
Vitamin D as Preprohormone
Long known as an endocrine facilitator in its role as a preprohormone affecting calcium and bone metabolism and homeostasis, vitamin D is something more as well. Our understanding of vitamin D has expanded in the decades since its discovery in the early 20th century. There are provocative experimental models in animals that extend to observational and some clinical trials in humans, which suggest that vitamin D plays a role in both innate and adaptive immunity, affecting our ability to survive infectious insults as well as long-latency diseases, such as autoimmune diseases and cancers, all of
which depend on a balanced and functional immune system . There are 2 forms of vitamin D: ergocalciferol (or vitamin D2, which is synthesized by plants and fungi) and cholecalciferol (or vitamin D3, which is synthesized in the skin of humans
and animals). Humans can metabolize both forms of vitamin D. Pre-vitamin D3 is synthesized in the epidermal layer of the skin by keratinocytes mainly in the stratum basale and stratum spinosum when 7-dehyrocholesterol is exposed to ultraviolet
B light in the wavelength of 290–320 nm . Through this photolytic energy transfer, pre-vitamin D is formed, and with further thermally induced isomerization in the skin, the parent compound vitamin D3 is produced. Vitamin D3 is carried into the bloodstream bound to vitamin D-binding protein (VDBP) or, less frequently, to albumin. Once vitamin D (either form D2 or D3) enters the circulation, either through epidermal transfer or intestinal absorption, it associates with VDBP, a 58-kD globular protein that binds vitamin D and its metabolites with various affinities based on the number and position of polar functional groups and/or methyl groups . The initial step in the metabolic activation of vitamin D is the enzyme-catalyzed insertion of an OH group at carbon 25 this oxidation process is primarily a hepatic microsomal function mostly by CYP2R1, a 25-hydroxylase , producing 25-hydroxyvitamin D (25[OH]D), the most abundant circulating
form of vitamin D .
As shown in Figure 1 (from Hollis and Wagner  , with permission), following formation in the liver, 25(OH)D appears in the circulation – bound primarily to VDBP. The half-life of
the parent compound is 12–24 h, while that of its first metabolite 25(OH)D is 2–3 weeks. The conversion of 25(OH)D to the active hormone 1,25-dihydroxyvitamin D (1,25[OH]2 D) through the CYP27B1 enzyme mainly occurs in the proximal tubules of the kidney, and then it is carried throughout the body also bound to VDBP.
Unlike 25(OH)D, 1,25(OH)2 D has a much shorter half-life of 4–8 h. VDBP preferentially binds 25(OH)D with higher affinity than 1,25(OH)2 D or the parent compound . The high affinity of VDBP for the vitamin D and its metabolites, coupled with the excessive binding capacity, keeps “free” or unbound concentrations of vitamin D and its metabolites at quite low relative concentrations [13,14]. This is important because only the “free” concentrations of the vitamin and its metabolites have transmembrane diffusion capabilities, thus exerting their
biologic function. What influences vitamin D status throughout the lifecycle is parathyroid hormone (PTH). When circulating 1,25(OH)2 D concentrations decrease, PTH increases, affecting intestinal absorption of vitamin D and conversion of vitamin D from its precursor in the skin. The measurement of intact PTH (iPTH) has long been considered an indicator of vitamin D deficiency and is used as a marker .
All vitamin D moieties are capable of binding to the vitamin D receptor (VDR). As shown in Figure 1 , the conversion of vitamin D to 25(OH)D and of 25(OH)D to 1,25(OH)2 D in the nuclear membrane of the cell is not limited to the liver and kidneys, respectively keratinocytes and many cells throughout the body, including monocytes, macrophages, and prostate and breast cells, can convert vitamin D3 to 25(OH)D and then to the active form 1,25(OH)2 D [16, 17]. 1,25(OH)2 D’s endocrine effects include the following classic triad of action: (1) increase in intestinal calcium (as Ca2 + ions) absorption
through the actions of calbindin; (2) increase in urinary calcium reabsorption; and (3) regulation of PTH in a negative feedback loop that allows calcium to be absorbed from the gastrointestinal tract, reabsorbed from urine, and metabolized from bone in order to maintain calcium homeostasis within the body. Because calcium is essential to all tissues and organs, particularly the heart, skeletal muscle, and brain, the body will claim calcium, if necessary, from the skeleton. In individuals with vitamin D deficiency, only trace amounts of vitamin D will be found in the body because whatever comes into the circulation is quickly converted to 25(OH)D and then 1,25(OH)2 D to maintain calcium homeostasis . For this reason, 1,25(OH) 2 D is not the indicator of vitamin D status and why 25(OH)D with its longer half-life should be used.
Another important factor influencing the conversion rate of 25(OH)D to 1,25(OH)2 D is the tissue transport mechanism for these secosteroids referred to as the megalin-cubilin system. The megalin-cubilin endocytic system  serves as an essential delivery system of 25(OH)D to the 25(OH)D-1-α-hydroxylase in the kidney, necessary in the conversion of 25(OH)D to 1,25(OH)2 D  . This system also exists in the parathyroid glands and, therefore, plays an important role in the endocrine function of vitamin D to maintain calcium homeostasis. Interestingly, the megalin-cubilin system also functions in the placenta and likely orchestrates maternalfetal calcium homeostasis . For those tissues that lack this endocytic transport system, free circulating concentrations of vitamin D moieties reach target cells through passive diffusion. For additional information, there are excellent reviews available that detail vitamin D metabolism in the nonpregnant individual [17, 21–23].
Also of importance is that 1,25(OH) 2 D itself is responsible for reducing 1,25(OH) 2 D concentrations in cells primarily by stimulating its catabolism through the induction of 24-hydroxylase, 24CYP24A1. This enzyme hydroxylates both 25(OH) D and 1,25(OH)2 D in the 24 position to form 24,25(OH)2 D and 1,24,25(OH)3 D  . As is discussed next, during pregnancy, there is increased 1,25(OH)2 D concentration presumed to be
due to decreased catabolism.
Differences in Vitamin D Metabolism during Pregnancy
From early on in pregnancy, circulating 1,25(OH)2 D concentrations increase without the predicted surge in PTH that causes a rise in calcitriol in nonpregnant individuals. While calcitriol is synthesized by the placenta, during pregnancy it is mainly synthesized by the kidneys . There appears to be a slower rate of catabolism of 1,25(OH)2 D to 24,25(OH)2 D . What purpose does this early and sustained rise in 1,25(OH)2 D serve? There has been much speculation about this. It has been theorized for decades that this increase during pregnancy was due to increased fetal calcium requirements,
most notable during the last trimester . Elevated circulating 1,25(OH)2 D was also thought to continue during lactation , but later, with more sensitive assay methodology surrounding the measurement of 1,25(OH)2D, this was shown not to be the case [29, 30] . The return to pre-pregnancy circulating concentrations of 1,25(OH)2 D during lactation is poorly understood and suggests that the role of 1,25(OH)2 D during pregnancy may be for reasons that extend beyond calcium metabolism and which surround vitamin D’s role in immune function . The above occurs in the presence of a continued high calcium requirement of the breastfeeding infant of at least 200–350 mg/day for growth that is comparable to fetal requirements during the last trimester of pregnancy.
Specific to pregnancy, there are changes in states of inflammation: early in pregnancy, there is inflammation – to allow the conceptus to invade the uterine milieu and for a network of channels between maternal and embryo to develop that give rise to the placenta, following a time of relative quiescence of those inflammatory processes that facilitate fetal growth beginning in the middle of the first trimester toward the end of pregnancy, with a return to a relatively inflammatory state with the onset of labor and the expulsion of the placenta . Pregnancy represents tremendous change in numerous systems with most notable increases in estrogen, progesterone, human placental growth factor, the interleukins, as well as 1,25(OH)2 D. Each has its purpose, but with any system, various growth factors and cytokines do not operate in isolation, but there is much interaction.
There is evidence that maternal vitamin D deficiency – however this is defined – affects maternal and fetal outcomes. Although scientific inquiry on the topic with published
observational and clinical vitamin D supplementation trials did not consistently appear in the literature until the late 1970s/early 1980s , there is historical information as early
as the 1940s with halibut liver oil – rich in both vitamins A and D and other vitamins – given as a supplement to pregnant women that showed benefit . Specifically, a study conducted by the People’s League of Health in 1938–1939 involving over 5,000 pregnant women who were randomized to receive a cocktail of vitamins and halibut liver oil (a source of both vitamins A and D) or placebo was rediscovered by Olsen and Secher  and the results published in 1990. This nutritional supplement was superior compared to control in achieving reductions in preterm birth and preeclampsia. Since that time, studies that have focused on one nutrient instead of a combined nutritional supplement, with the exceptionof higher-dose vitamin D studies in the most deficient
women, and more recently in systematic reviews and meta-analyses, have failed to demonstrate this effect. Much research has occurred with far more studies published each year on the topic. With those trials, there have been mixed results, with some studies showing a positive effect and others showing a minimal or no effect. There are, however, indisputable findings surrounding gene expression on the basis of maternal vitamin D status.
Focusing on vitamin D, the metabolism of this important preprohormone during pregnancy is vastly different when compared to the nonpregnant state. As noted earlier,
1,25(OH)2 D increases 2- to 3-fold within days of conception, while 25(OH)D remains relatively stable within a certain range [33–35]. It is 25(OH)D which crosses the placenta to the fetus and, thus, is the main pool of vitamin D in the fetus, not 1,25(OH)2 D; the fetus must synthesize 1,25(OH)2 D from that pool. While the main source of the increased 1,25(OH)2 D during pregnancy comes from the kidney, its other source is the
placenta, with VDR and regulatory metabolic enzymes synthesized in the placenta and decidua. This is considered a potential critical point in the immunomodulation at the maternal-fetal interface and raises the question if maternal hypovitaminosis D during pregnancy leads to pregnancy-related disorders [36, 37].
Genetic Studies and Vitamin D Status
There is an increasing number of genetic studies to evaluate vitamin D’s effect on gene expression. One of the first was a study by Al-Garawi et al.  who, in their post hoc analysis of a randomized clinical trial of maternal vitamin D supplementation in women who themselves or of whom a firstdegree relative had allergy or asthma, sought to explore vitamin D’s effect on genomic changes during pregnancy, which is one of the first reports of its kind. Women were randomized at 10–18 weeks of gestation to 400 and 4,400 IU vitamin D 3 / day  with the primary outcome wheezing in the offspring at 3 and 6 years. An analysis of a subset of blood samples for RNA gene expression changes between the first and third trimesters was conducted. Using significance of analysis of microarrays (SAM) and clustered weighted gene co-expression network analysis (WGCNA) to identify major biological transcriptional profiles between those time points, 5,839 significantly differentially expressed genes were studied. Transcripts from these genes clustered into 14 co-expression modules, of which 2 (associated with immune defense pathways, extracellular matrix reorganization, and Notch signaling
and transcription factor networks) showed significant correlation with maternal 25(OH)D concentrations. The findings show that maternal gene expression changes during
pregnancy are affected by maternal vitamin D status, which, in turn, is a direct reflection of maternal vitamin D supplementation.
Additional evidence of vitamin D’s effect on gene expression comes from Baca et al.  and another from Barchitta et al.  in their focus on vitamin D-related genes. Baca et al.  conducted a meta-analysis of 2 large cohorts – the Epidemiology of Vitamin D Study (EVITA) and the Collaborative Perinatal Project (CPP) – where the combined analysis of more than 4,000 randomly selected samples showed that the maternal genotypes of 7 SNPs in VDR, 3 SNPs in GC (VDBP), and 1 SNP in the flanking regions of Cyp27B1 were associated with maternal vitamin D status as expressed as the log25(OH) D concentration. Adjusting for multiple comparisons, 1 SNP in VDR and 2 SNPs in GC remained significant. The investigators theorized that SNPs in VDR may influence circulating 25(OH) D by changing the rate at which 25(OH)D is hydroxylated either directly or indirectly through a negative feedback loop. The 2 SNPs in GC are likely related to the response of an individual to vitamin D supplementation, with certain GC polymorphisms associated with an attenuated or refractory response to supplementation compared to other genotypes, such as 1S or 2 .
Barchitta et al.  conducted a study to examine the association of VDR polymorphisms and preterm birth and neonatal anthropometric measures. Utilizing the Italian “Mamma and Bambino” cohort (n = 187), they studied the most common polymorphisms – BsmI, ApaI, FokI and TaqI. The investigators found that for the FokI polymorphism, gestational duration (age) and birth weight (that are clearly linked) were statistically significantly lower with increasing number of the A allele. In addition, when compared to mothers with the GG or GA genotype, those mothers who carried the AA genotype had a higher risk of preterm birth (OR 12.049, 95% CI 2.606–55.709, p = 0.001). Further, the BsmI polymorphism appeared to be protective against preterm birth, both allelic (A vs. G: OR 0.74, 95% CI 0.59–0.93) and recessive (AA vs. GG + AG: OR
0.62, 95% CI 0.43–0.89, p = 0.0001). Mothers with the AA genotype exhibited a 12-fold increased risk of preterm birth that was independent of sociodemographic characteristics, lifestyle, vitamin D intake/use of supplements, type of delivery, and parity. The results of this study were combined with earlier reported studies, which strengthened the robustness of these findings.
These genetic studies collectively suggest that genotyping of common allelic variants and polymorphisms may play an important role in vitamin D metabolism during pregnancy. The findings further suggest that certain functional genetic variants may contribute to vulnerability or risk of vitamin D deficiency. The findings suggest that there may be subgroups of women based on their genotype profile for relevant vitamin D-related genes who would benefit from certain dosing regimens while others would not. The changes in gene expression from the first trimester compared to the third may also suggest that the prescription of one vitamin D supplement dose throughout pregnancy does not meet the physiological needs of the pregnant woman and might be based more on convenience than what is needed for optimal vitamin D status.