Fetus, Neonate and Infant

C H A P T E R

35 Fetus, Neonate and Infant Christopher S. Kovacs Memorial University of Newfoundland, Faculty of Medicine e Endocrinology, Health Sciences Centre, 300 Prince Philip Drive, St. John’s, Newfoundland A1B 3V6, Canada

OVERVIEW OF CALCIUM METABOLISM IN THE FETUS Adult calcium and bone homeostasis is largely regulated by the interactions of the classic calciotropic hormones: parathyroid hormone (PTH), 1,25dihydroxyvitamin D or calcitriol (1,25(OH)2D), calcitonin, FGF23, and the sex steroids. These hormones act to maintain a specific concentration of calcium (and other minerals) in the blood, a neutral calcium balance, and a fully mineralized skeleton. These hormones will also act to remove mineral from the skeleton when the demands for calcium outstrip the supply (lactation, renal insufficiency and other acidebase disturbances, hypercalciuria due to renal calcium leak, etc.). Deficiency of parathyroid hormone, vitamin D, or estradiol results in significant impairments in calcium and bone homeostasis. The blood calcium may be altered, skeletal mineralization becomes impaired, and a negative calcium balance results. Calcium and bone homeostasis is regulated differently during fetal development (reviewed in detail in [1,2]). The fetus has distinct goals from the adult, including that it must actively pump calcium from the maternal circulation against a concentration gradient, maintain a blood calcium higher than the maternal (adult) level, rapidly mineralize the skeleton during the final quarter of gestation, and achieve a positive calcium balance. Human babies accrete 80% of the required 30 g of calcium during the third trimester [3e5] while rats accrete 95% of the required 12.5 mg of calcium during the last 5 days of a 3-week gestation [6]. In achieving these goals fetuses do not appear to require vitamin D or the other calciotropic hormones listed above. There are limited data available from studies of human fetuses, and so human regulation of

Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10035-6

fetal mineral homeostasis must be largely inferred from studies in animals. A caveat to keep in mind is that some of what has been discovered in other mammals may not apply to humans. Within the developing human, the serum calcium, ionized calcium, magnesium, and phosphorus are raised above the maternal values [1,7]. Whether these elevated values have physiological importance is uncertain but studies in fetal mice indicate that an elevated serum calcium and magnesium are needed for the skeleton to accrete the normal amount of these minerals by term [8e10]. Studies in rodents have shown that if the fetal blood calcium is reduced to the maternal level or below it, fetal survival to term is unaffected [8,11,12]. The high fetal blood calcium may protect against severe hypocalcemia at birth, when the onset of breathing raises the pH and causes an obligatory fall in the blood calcium (see neonatal section, below). The elevated serum calcium in fetuses is robustly maintained despite significant hypocalcemia due to various causes in the mothers; this observation holds true for animals and apparently for humans as well [1,7,9]. Among the calciotropic hormones, parathyroid hormone and 1,25(OH)2D are low in human babies and other mammals while calcitonin is elevated [1]. Recent human data indicate that intact FGF23 levels are low in cord blood but reach adult values by 5 days after birth [13]. Despite its low levels, parathyroid hormone is physiologically important for fetal development because in its absence fetal mice have reduced blood calcium, undermineralized skeletons, and altered placental expression of genes involved in cation transport [8,9,12]. The high serum calcium and phosphorus, and low parathyroid hormone, likely all contribute to suppression of the renal 1a-hydroxylase (CYP27b1) and maintenance of low levels of 1,25(OH)2D. Animal

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studies have demonstrated that 25(OH)D readily crosses the placenta while 1,25(OH)2D does not [14,15]. Human data are consistent with these animal studies since cord blood 25(OH)D levels are within 75e100% of the maternal value whereas cord blood 1,25(OH)2D levels are well below maternal levels [16e20]. Studies of perfused human placentas also indicate that 25(OH)D readily crosses the placenta [21]. The elevated calcitonin derives from fetal sources but the physiological importance of calcitonin for calcium homeostasis is unknown. Fetal mice lacking the gene that encodes calcitonin had normal serum calcium, placental calcium transfer, and calcium content of the skeleton at term whereas the serum magnesium and skeletal magnesium content were modestly reduced [10]. The role of FGF23 in fetal mineral and bone homeostasis has not been studied, although it has been noted that Fgf23-null mice have normal length, weight, appearance, and serum calcium and phosphorus at birth [22]. Another hormone, parathyroid-hormone-related protein (PTHrP), circulates at high levels in fetal rodents, sheep, and humans [1,12,23e25]. When full-length parathyroid hormone and PTHrP were measured in cord blood, PTHrP levels were 2e4 pmol/l and up to 15fold higher than simultaneous values for PTH (0.2e0.5 pmol/L) [23e25]. PTHrP plays a significant role in regulating fetal mineral homeostasis and skeletal mineralization. Fetal mice lacking PTHrP are hypocalcemic; display abnormal endochondral bone development with short-limbed dwarfism, rounded skull, and shortened mandibles with protruding tongues; and have reduced placental calcium transfer [11,26]. In humans the equivalent to the PTHrP-null state has not been described but null mutations in the PTH receptor cause a similar and lethal condition called Blomstrand chondrodysplasia [27,28]. Calcium and other minerals enter the adult organism via intestinal absorption but this route is trivial in the fetus because only the amniotic fluid is available to be swallowed and absorbed. At this stage of development intestinal calcium absorption occurs by passive and nonsaturable mechanisms. Fetal urine is the major source of fluid and solute in the amniotic fluid. The main route of entry for minerals into the fetus is via the placenta (Fig. 35.1). Calcium, magnesium, and phosphorus are actively transported across the placenta, and this is required in order to fully mineralize the fetal skeleton by term. Studies in fetal sheep, rats, and mice have shown that PTHrP is a major regulator of calcium and possibly magnesium transfer while parathyroid hormone may also play a role [1,9,11,29e31]. Active transfer of calcium becomes evident in the last 7 days of gestation in the rat and mouse, coinciding with marked up-regulation of calcium-binding proteins, TRPV6 (transient receptor potential vanilloid 6), and

Ca2þ-ATPase within trophoblasts, and rapid accretion of mineral within the fetal skeleton [32e37]. A complete cartilaginous skeleton with digits and intact joints is present by the eighth week of gestation in humans. Primary ossification centers form in the vertebrae and long bones between the eighth to twelfth weeks, but it is not until the third trimester that the bulk of mineralization occurs. At the 34rd week of gestation, secondary ossification centers form in the femurs, but otherwise most epiphyses are cartilaginous at birth, with secondary ossification centers appearing in other bones in the neonate and child [38]. As in the adult, the fetal skeleton participates in the regulation of mineral homeostasis with calcium being resorbed to help maintain the concentration of calcium in the blood. If human mothers are severely hypocalcemic due to hypoparathyroidism, fetuses will develop secondary

FIGURE 35.1 Calcium sources in fetal life. The main flux of calcium is across the placenta and into fetal bone. Some calcium returns to the maternal circulation (backflux); calcium filtered by the kidneys is partly reabsorbed into the circulation; calcium excreted by the kidneys into the urine and amniotic fluid may be swallowed and absorbed by the intestine; calcium is also resorbed from the developing skeleton to maintain the circulating calcium concentration. Calcitriol and the VDR do not appear to be major regulators of placental calcium transfer. Reproduced from [7] Ó2003 by Academic Press, used with permission.

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hyperparathyroidism, skeletal demineralization, and fractures in utero [1,39].

ANIMAL DATA RELEVANT TO VITAMIN D AND THE FETUS The fetal demand for calcium is largely met by upregulated intestinal calcium absorption in the mother beginning early in pregnancy. Several studies indicate that this maternal adaptation does not require vitamin D. Pregnancy in vitamin-D-deficient rats and in mice lacking the vitamin D receptor (Vdr-null) results in a marked up-regulation of intestinal calcium absorption to the same high rate achieved during pregnancy by normal rats and mice [40e42]. This up-regulated calcium absorption is physiologically significant because both vitamin-D-deficient rats and Vdr-null mice experienced a significant increase in skeletal mineral content or BMD during pregnancy [42e44]. Additional animal studies suggest that the Vdr-null mouse compensates by up-regulating intestinal expression of the calcium channel TRPV6 [42], and that prolactin and placental lactogen may stimulate intestinal calcium absorption independently of 1,25(OH)2D, possibly by stimulating TRPV6 [45,46]. Although the animal data are compelling that 1,25(OH)2D or its receptor are not required to up-regulate intestinal calcium absorption during pregnancy, as yet no clinical study has examined intestinal calcium absorption in vitamin-D-deficient versus vitamin-D-replete pregnant women. The impact of altered vitamin D physiology on systemic calcium homeostasis during pregnancy has been examined in severely vitamin-D-deficient rats [43,47e49], pigs with a null mutation of the 1a-hydroxylase [50], and Vdr-null mice [37,42]. In each model the nonpregnant adult is unwell with hypocalcemia, hypophosphatemia, and rickets/osteomalacia. (1a-Hydroxylase-null mice also have hypocalcemia and hypophosphatemia but are infertile [51,52].) Females with vitamin D deficiency or with these mutations conceive less often than normal, maintain low blood calcium and phosphorus levels, and bear smaller litters. Sudden, sporadic (presumably hypocalcemia-induced) maternal deaths occur late in pregnancy during the interval of rapid calcium transfer to the fetus in vitamin-D-deficient rats and Vdr-null mice, possibly indicating that the mother has difficulty maintaining her own blood calcium in the face of rapid, active transfer of calcium across the placenta to multiple fetuses [37,42,48,53]. In contrast to their mothers who are so unwell as a consequence of disrupted vitamin D physiology, the fetuses and newborns show normal blood calcium,

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magnesium, phosphorus, parathyroid hormone, weight, skeletal mineral content, and skeletal calcium content. This includes pups born of severely vitamin-D-deficient rats [48,49,53,54], 1a-hydroxylase-null pigs [50] and both Vdr heterozygous and Vdr-null mice [37]. 1a-Hydroxylase-null mice are reportedly normal at birth but extensive studies of fetal chemistries and skeletal mineral content have not been published [51,52]. The normal, heterozygous, and null fetuses born of Vdr heterozygous mice were indistinguishable in all parameters of mineral homeostasis, weight, and skeletal size, histomorphometry, morphology, gene expression, and mineral content [37] (Fig. 35.2). Heterozygous and null fetuses born of Vdr-null mothers were smaller, weighed less, but maintained normal blood calcium and normal mineral content for their proportionately smaller skeletons [37]. This reduction in fetal size and weight in pups born of Vdr-null mice was not observed in models of vitamin D deficiency, and may indicate that absence of the vitamin D receptor has effects on fetal growth which vitamin D deficiency does not. Placental calcium transfer from mother to fetus was normal to increased, as assayed indirectly in pups born of vitamin-D-deficient rats [55] and directly in fetuses from Vdr heterozygous and Vdr-null mice [37]. The placenta provides calcium to the fetus without relying on vitamin D metabolites, and the “vitamin D-dependent” factors calbindinD-9K and Ca2þ-ATPase e which are important for intestinal calcium absorption in the adult e are expressed at normal levels in vitamin-D-deficient and Vdr-null placentas [37,55e57]. On the other hand, expression of PTHrP and CaT1 (TRPV6) were both up-regulated in placentas of Vdr-null mice versus wt, consistent with the observed increase in placental calcium transfer in Vdr-nulls [37]. In all of the aforementioned animal models the adults have florid rickets, hypocalcemia, hypophosphatemia, and marked secondary hyperparathyroidism. However, as noted below in the neonatal section, none of these abnormalities develop until after weaning in vitaminD-deficient rats [53,54], 1a-hydroxylase-null pigs [50], 1a-hydroxylase-null mice [51,52], and Vdr-null mice [58,59]. These observations underscore that 1,25(OH)2D is not needed to regulate placental calcium transfer or skeletal mineralization in the fetus. Although these studies suggested no role for 1,25 (OH)2D in fetal mineral homeostasis, several other reports have suggested otherwise. Prior nephrectomy in fetal lambs reduced placental calcium transfer as assessed by perfusing the placenta in situ after the fetus had been removed, and treatment with 1,25(OH)2D partly restored the rate of calcium transfer across the isolated placenta [60]. Further, pharmacological doses of 1,25(OH)2D or 1a-OHD increased calcium transfer across in situ perfused placentas of lambs and rats [61,62]. All of

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FIGURE 35.2 Resilience of fetal blood calcium regulation despite absence of VDR and marked maternal hypocalcemia. Ionized calcium was measured on whole blood in fetuses obtained from Vdrþ/e mothers (A) and Vdr-null mothers (B) on embryonic days 17.5 and 18.5 (1 or 2 days before expected birth). No difference in ionized calcium level was noted among the fetal genotypes, and the mother’s genotype or ionized calcium level did not affect the fetal ionized calcium level. The mean maternal ionized calcium is indicated by the horizontal line in each graph, with SE values displayed on the right. Double-headed arrows emphasize the maternalefetal calcium gradient which was strikingly increased in fetuses of Vdr-null mothers as compared to fetuses of Vdrþ/e mothers (P < 0.001). The number of observations is indicated in parentheses. Reproduced from [37] Ó2005 by the American Physiological Society, used with permission.

these reports appear to have demonstrated pharmacological rather than physiological actions of 1,25(OH)2D. The studies of Vdr-null mice described above indicated that removal of the effective actions of 1,25(OH)2D caused an increase in the rate of placental calcium transfer (and placental expression of PTHrP and TRPV6) in intact mice, not a decrease as predicted by these studies in perfused placentas from lambs and rats. As noted above in the overview, maternal hypocalcemia due to hypoparathyroidism causes fetal hypocalcemia, secondary hyperparathyroidism, skeletal demineralization, and fractures in utero [1,39,63]. This outcome has not been reported with any animal model of disrupted vitamin D physiology. The explanation may be that hypoparathyroidism usually causes more severe maternal hypocalcemia which is also aggravated by the accompanying hyperphosphatemia. When hypocalcemia occurs due to disrupted vitamin D physiology (vitamin D deficiency, absence of 1,25(OH)2D, or absence of VDR), compensatory secondary hyperparathyroidism and consequent hypophosphatemia both serve to lessen the magnitude of maternal hypocalcemia. Collectively these findings indicate that fetal calcium homeostasis and skeletal development/mineralization are independent of vitamin D, 1,25(OH)2D, and its receptor. The animal studies predict that human babies born of vitamin-D-replete and vitamin-D-deficient mothers should have similar blood calcium, phosphorus, and skeletal mineral content at birth. As noted in the next section, low maternal 25(OH)D levels during late pregnancy have been associated in humans with increased risk of a variety of maternal outcomes (preeclampsia, bacterial vaginosis, elective C-section) and fetal/neonatal outcomes (asthma, type 1 diabetes, etc.). Most of these associations have not been tested in animal models. Vdr-null mice are not

known to be predisposed to any adverse pregnancyrelated outcomes except hypocalcemia and sudden (presumed hypocalcemic) deaths near term [37,42,64]. In the nonobese diabetic (NOD) mouse, which is genetically predisposed to develop insulitis followed by type 1 diabetes, there is evidence that superimposed vitamin D deficiency causes the diabetes to develop sooner [65]. However, treatment of NOD mice with 1000 IU of supplemental vitamin D daily (a supraphysiological dose for the mouse) beginning in utero and continuing up to 10 weeks of age with follow-up to 32 weeks did not reduce the incidence of type 1 diabetes [66]. Treatment with pharmacological doses of 1,25(OH)2D or analogs of it have been shown to significantly reduce insulitis and the occurrence of diabetes, presumably through immune-mediated mechanisms [67,68]. A more recent study crossed the NOD mouse with Vdrnull mice but loss of the VDR did not alter the rate of development of insulitis and diabetes [69]. Overall these studies indicate that severe vitamin D deficiency increases the risk of type 1 diabetes in a genetically predisposed animal whereas loss of the VDR does not, and that pharmacological (hypercalcemic) doses of 1,25 (OH)2D are needed to prevent the diabetes. The studies do not address whether vitamin D deficiency or insufficiency significantly increase the risk of type 1 diabetes in an animal that is not genetically predisposed to develop the condition.

HUMAN DATA RELEVANT TO VITAMIN D AND THE FETUS Unlike the animal studies, there have been no systematic and comprehensive studies comparing serum calcium, calciotropic hormones, or skeletal mineral

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content among babies born of vitamin-D-replete, vitaminD-insufficient, and vitamin-D-deficient mothers. In the only attempt at a systematic examination of the fetal skeleton [70], investigators in China examined the ash weight and mineral content of 15 fetuses that had died due to obstetrical accidents: seven of these were born of mothers known to have osteomalacia and the other eight were born of otherwise healthy mothers. The first three fetuses analyzed appeared to have a lower ash weight versus controls but the latter four fetuses had an ash weight that equalled or exceeded the controls. Overall there were no significant differences in ash weight or calcium, phosphorus, and magnesium content of the ash between the two groups. The authors also described that centers of ossification were normal and that there were no radiographic signs of rickets, although they did note in two of the seven fetuses “an apparent cupping of the end of the [ulna].which at once suggested the possibility that this was rickety in nature” [70]. The authors concluded that “there is no evidence of pre-natal rickets” but “there are definite changes in the skeletal system of the foetus born from mothers who are the subject of osteomalacia” [70]. The sample size was quite low in this old report and the osteomalacia in the mothers was confounded by malnutrition. A few rare, isolated case reports have unequivocally indicated that craniotabes and other suggestive skeletal changes can be detected at birth [71e74], whereas in other reports that described craniotabes or rickets being present “at birth” the diagnosis was actually made within the first or second week [74e79]. In one such case convincing radiographic findings were not present at day 2 after birth but had developed by day 16 [79]. In many cases of congenital rickets the cause was not isolated vitamin D deficiency. Instead, the mothers had significant malnutrition, malabsorption (e.g., celiac disease, pancreatic insufficiency), or very low intakes of both calcium and vitamin D [72,74,78]. More recent clinical experience is that vitamin-D-deficient rickets usually does not develop (or become recognized) until weeks to months after birth with a peak incidence between 6e18 months, even in regions where severe vitamin D deficiency during pregnancy is endemic [80e83]. Consistent with this, in a clinical trial that resulted in 25(OH)D levels of 138 nmol/L in fetuses of vitamin-D-treated mothers versus 25(OH)D levels in the rachitic range of 10 nmol/L in fetuses of placebotreated mothers, there was no difference in cord blood calcium or phosphorus between groups and no radiologic evidence of rickets [84] The clinical courses of children born with genetic disorders of vitamin D physiology have also been reported in assorted cases, series and reviews. Babies with 1a-hydroxylase deficiency (vitamin-D-dependent rickets type I; VDDR-I) and those lacking the vitamin

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D receptor (vitamin-D-dependent rickets type II or hereditary vitamin-D-resistant rickets; VDDR-II) have been described as normal at birth [85e90]. In both conditions the child inevitably develops hypocalcemia, hypophosphatemia, and rickets. VDDR-I presents as early as the neonatal period to within the first year of life; VDDRII can also present in infancy but more often is diagnosed during the second year of life [85e90]. Thus the limited data from humans are in agreement with the animal data that hypocalcemia and rickets are not present at birth but develop postnatally. The clinical course of pregnancy in women with VDDR-I or VDDR-II is uneventful when normocalcemia is maintained in the mother [91,92]. Overall the clinical findings in term infants with vitamin D deficiency and in all infants with VDDR types I and II indicate that hypocalcemia and skeletal changes of rickets are not present at birth but develop days to months afterward. But a few isolated case reports (described above) of vitamin-D-deficient babies born to osteomalacic mothers have shown that craniotabes and other early rachitic changes may be present at birth; the etiology in these cases may be maternal malnutrition, malabsorption, hypophosphatemia, or inadequate intake of both calcium and vitamin D, and not isolated vitamin D deficiency. In animal studies intestinal calcium absorption in the fetus is trivial and occurs through passive, nonsaturable mechanisms. Preterm human infants, the developmental equivalent of fetuses, also demonstrate passive, nonvitamin-D-dependent absorption of calcium [93,94], while normal term and preterm infants show a postnatal increase in the efficiency of intestinal calcium absorption similar to the animal studies described above [93,95,96]. This maturation of intestinal calcium absorption to a vitamin-D-dependent process parallels and explains the aforementioned clinical observation that vitamin-D-deficient rickets does not develop (or become recognized) until several weeks to typically 6e18 months after birth, even in regions where severe vitamin D deficiency during pregnancy is endemic [80e82]. Observational and clinical studies of human pregnancy described in the next several paragraphs have shown no relation of cord blood 25(OH)D levels with cord blood calcium or parathyroid hormone. This is consistent with the animal studies mentioned earlier in which vitamin D deficiency and absence of the vitamin D receptor did not alter the fetal blood calcium. Large, double-blind, randomized, placebo-controlled trials provide the highest level of evidence, but no such studies of vitamin D have been done during human pregnancy. Instead, small randomized and often unblinded clinical trials have been reported. These have shown that increased maternal intake of vitamin D supplements results in higher maternal and fetal

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(cord blood) 25(OH)D levels, but no clear proof of any additional benefits.

Intervention Studies In one of the earliest such studies [84], Brooke conducted a double-blind randomized trial of vitamin D supplementation in an Asian population living in England. The study involved 59 women treated with vitamin D and 67 controls. This population was quite vitamin-D-deficient (mean 25(OH)D level 20 nmol/L at baseline) and therefore was an ideal group to test whether correction of vitamin D deficiency resulted in any maternal or fetal benefits. Although the treatment group nominally received 1000 IU of vitamin D per day, they achieved a substantial increase in maternal 25(OH) D levels to a mean level of 168 nmol/L, indicating that the dose must have been 10 000 IU per day or more, or that the assay yielded unrealistic results. The mean cord blood 25(OH)D value was 138 nmol/L in babies born of the treated mothers versus 10 nmol/L in control babies. Treatment with vitamin D caused a modest increase in maternal serum calcium but cord blood calcium remained not significantly different between the vitamin-D-supplemented and control infants. The treatment did not affect birth weight, crowneheel length, forearm length, triceps skinfold thickness, and head circumference, whereas the fontanelle area was reduced from 6.1  0.7 to 4.1  0.4 cm2 (p < 0.05). In another intervention trial [97], Mallet administered 1000 IU/day of vitamin D2 during the last trimester of pregnancy, or a single dose of 200 000 IU of vitamin D2 at month 7, or no supplementation, to three groups of women (N ¼ 21, 27, and 29 respectively) living in France. The treatments were equally effective in increasing maternal and cord blood 25(OH)D levels versus controls. Maternal, cord blood, and neonatal calcium levels did not differ among the three groups, nor was there any effect on skeletal or anthropometric parameters in the infants. Another clinical trial randomized 40 women to 1000 IU vitamin D3 supplementation daily versus placebo beginning at 6 months of pregnancy and found no effect on skeletal parameters or on the cord blood calcium [98]. In a study of vitamin-D-deficient Asian women which generated two separate reports (randomization methodology was unclear), 800 000 IU of vitamin D2 administered in the 7th and 8th months of pregnancy to 20 women resulted in a small increase in cord calcium but no other benefit compared to 75 women who received no supplement and 25 women who received 1200 IU of vitamin D2 per day [99,100]. Yu conducted a small randomized but unblinded intervention trial in London to determine the effects of daily vs. single-dose vitamin D supplementation on

pregnancy outcomes [101]. The doses of vitamin D given were 800 IU/d of vitamin D2 or a single dose of 200 000 IU of vitamin D2, each beginning at gestational week 27, as compared to no treatment. Maternal serum 25(OH)D levels at delivery were significantly greater in both treatment groups compared to the control group (mean 42, 34, and 27 nmol/L, respectively). Similarly, mean cord 25(OH)D levels were significantly greater in the treatment groups compared to the controls (26, 25, and 17 nmol/L, respectively). However there was no significant difference between treatment and control groups for gestational age at delivery, birth weight, or incidence of low birth weight. More recently Wagner and Hollis have completed an NIH-sponsored trial in which 494 women were randomized at 12e16 weeks of gestation to receive 400, 2000, or 4000 IU of vitamin D3 per day for the duration of pregnancy. Only 350 (70.9%) of women completed the study. The primary outcomes and intention-to-treat analysis of this potentially illuminating study remain unknown at the time of writing; the results have not been peer-reviewed or published, although preliminary abstracts were seen. Wagner and Hollis also completed a smaller study of 257 women who were enrolled at 12e16 weeks of gestation to receive 2000 or 4000 IU of vitamin D3 per day. Only 160 (62.3%) of women completed this study and the results of it also remain unknown. The large dropout rates and expected low numbers of events (e.g., prematurity, preeclampsia) make it unlikely that these two studies would provide conclusive data about benefits and risks of such levels of vitamin D supplementation during pregnancy. However, the results are expected to be more compelling than those of the various small trials completed to date.

Associational Studies Associational and epidemiological studies are hypothesis-generating but do not prove causality. Such studies in humans have led to numerous associations being made between serum 25(OH)D in the mother and various outcomes of pregnancy, but the evidence is inconsistent and confounded. Among maternal outcomes of pregnancy that can adversely affect the fetus, preeclampsia has been associated with low maternal 25(OH)D levels [102], while use of vitamin D supplements has been associated with a lower risk of preeclampsia [103,104]. Observational studies also suggest that low 25(OH)D levels increase the risk of gestational diabetes [105,106]. However, these observations are significantly confounded by such factors as increased maternal weight, lower socioeconomic status, and poorer nutrition, all of which are associated with lower 25(OH)D levels. Overweight and obesity directly

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predispose to gestational diabetes but also reduce 25 (OH)D levels due to sequestration in fat and possibly other mechanisms. In support of this notion, weight reduction studies show that serum 25(OH)D levels rise when obese individuals lose body fat but maintain the same vitamin D intake [107e110]. A similar association of low 25(OH)D levels during pregnancy with increased risk of elective or primary cesarian section may also be confounded by maternal overweight and obesity [111]. There are no randomized, controlled trials that have tested whether any of these associations are causative. There are numerous studies testing associations of maternal or cord blood 25(OH)D levels on fetal outcomes. In an observational study assessing the effects of maternal vitamin D status on fetal bone health, Mahon compared 25(OH)D levels at 36 weeks’ gestation with radiologic evidence for bone development in the fetus [112]. The analysis of 424 mothereinfant pairs found no association between maternal vitamin D status and femur length. However there was an association between maternal serum 25(OH)D levels below 50 nmol/L and greater distal metaphyseal crosssectional area (RR ¼ e0.10 (95% CI: e0.02 to 0.00)), and with a novel index derived by the authors called the “femoral splaying index” (the metaphyseal crosssectional area divided by the femur length) (RR ¼ e0.011 (95% CI: e0.21 to e0.01)). A longitudinal study by Javaid in England evaluated the effect of serum 25(OH)D levels in late pregnancy on pregnancy outcomes at birth, 9 months, and 9 years of age [113]. No associations were found between maternal serum 25(OH)D and birth weight, birth length, placental weight, abdominal circumference, or head circumference. There was also no association found between vitamin D status in late pregnancy and cord blood calcium level. At the 9-month follow-up there was still no association of the maternal 25(OH)D status during pregnancy with skeletal and anthropometric parameters in the children. However, at 9 years of age the investigators were able to show a significant correlation between skeletal parameters and the estimated maternal 25(OH) D value in late pregnancy. A maternal serum 25(OH)D level below 27.5 nmol/L was associated with a modest but statistically significant decrease in bone mineral content (BMC) in offspring at 9 years of age compared to offspring of mothers whose 25(OH)D levels were 50 nmol/L or higher (1.04  0.16 vs. 1.16  0.17 g) (P ¼ 0.04). These findings are compatible with the theory that vitamin D sufficiency during fetal development may program childhood or peak bone mass and mineralization [114,115]. However, there are important confounders. Low 25(OH)D status in any adult correlates with obesity, poorer nutrition, lower socioeconomic status, etc., and so the association may not be casual. Moreover much time elapsed between birth (when no

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effect was seen) and 9 years of age (when reduced BMC was observed); the lower maternal 25(OH)D status in late pregnancy may indicate that such factors as lower socioeconomic status and poorer nutrition in the mother were shared with the child. A similar study by Morley examined 374 mothereinfant pairs and found no association of maternal 25 (OH)D level at 28e32 weeks of gestation with any infant measurement (body weight, skeletal lengths, skinfold thickness) [116]. An additional analysis stratified the women into those with 25(OH)D levels below 28 nmol/L compared to those with levels greater than 28 nmol/L. The infants born to mothers with 25(OH)D levels below 28 nmol/L were found to have a slightly shorter kneeeheel length but the difference was not statistically significant. No association was seen with crownerump length, weight, or other parameters. A more recent but much smaller study in Finland planned to measure maternal and cord blood 25(OH)D and pQT of the left newborn tibia in 125 mothereinfant pairs [20]. Although 125 women enrolled in the first trimester, only 90 women remained to be stratified into two groups based on 25(OH)D levels below or above the median of 42.6 nmol/L while only 72 infants had cord blood measurements that could be stratified by their mother’s 25(OH)D levels. Left unreported was the number of infants that had pQCT measurements done during the first several days after birth. With these limitations in mind, the authors reported that babies born to mothers above the median 25(OH)D level had a slightly higher tibial cross-sectional area (11.5 mm2) and tibial BMC (0.04 g/cm) while there was no difference in birth weight, length, or BMD of the tibia [20]. Vitamin D supplementation has also been proposed to increase birth weight, but the evidence for this is largely negative. In the controlled trials described above, there was no effect of vitamin D supplementation on birth weight. However, in two follow-up analyses of the small clinical trial by Brooke [84], severely vitaminD-deficient women treated with vitamin D gained more weight during pregnancy than controls, and although birth weight did not differ, their babies gained more weight postnatally than control infants [117,118]. Several associational studies mentioned above showed no effect on birth weight [112,113,116]. One prospective study of 2251 low-income, minority gravidae found that maternal intake of less than 200 IU vitamin D per day was associated with significantly reduced birth weight, but the association may have been the result of globally reduced nutrition rather than a specific effect of low vitamin D intake [119]. In a cohort of 279 pregnant women stratified by milk intake (> or <250 ml per day) there was no significant effect on birth weight, length, or head circumference; but in a multivariate analysis milk intake and estimated vitamin D intake were significant

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predictors of birth weight [120]. Lastly, in a prospective study of 123 Gambian mothereinfant pairs there was no association between maternal 25(OH)D level and birth weight, length, head circumference, BMD, or BMC of the infants even though maternal 25(OH)D levels ranged from 51e189 nmol/L at 20 and 36 weeks of pregnancy [121]. Associational studies among older adults have suggested that vitamin D deficiency and insufficiency may increase the risk of chronic diseases such as type 1 diabetes, asthma, multiple sclerosis, and others. For most of these associations there are no specific data relating the disease outcome to fetal or maternal 25(OH)D levels during pregnancy. However, there is some evidence with respect to risk of type 1 diabetes. A higher dietary intake of vitamin D (but not the use of vitamin D supplements) during pregnancy is associated with decreased prevalence of islet cell antibodies and diabetes in the offspring [122], and a history of vitamin D supplementation of pregnant women [123,124] or the infant directly [125e127] is associated with a lower childhood incidence of type 1 diabetes. There are no clinical trials and so this evidence is not definitive. The association requires further investigation with randomized trials before vitamin D can be recommended as a treatment proven to reduce the incidence of type 1 diabetes. Similarly while some studies have associated higher maternal intake of vitamin D during pregnancy with lower risk of childhood asthma and allergy in the offspring [128,129], other studies have found supplemental vitamin D use during pregnancy to increase the risk of childhood asthma and atopy [130e132]. In a follow-up analysis of the Javaid study cited above, the investigators examined whether exposure to maternal serum 25(OH)D levels above 75 nmol/L during pregnancy had any other effects on the health of offspring at 9 years of age [133]. No correlation was found between very high maternal vitamin D status and offspring growth, cognitive function, or cardiovascular function. However, the babies of women whose serum 25(OH)D levels were greater than 75 nmol/L during pregnancy were five times more likely to have asthma at 9 years of age compared to offspring of mothers with lower 25(OH)D levels during pregnancy. The bottom line is that current evidence indicates the human fetus may suffer no skeletal problems as a consequence of vitamin D deficiency and insufficiency, but after birth hypocalcemia and progressive rickets will develop in those with severe vitamin D deficiency. It remains unclear whether 25(OH)D levels in the fetus or pregnant mother have a direct influence on childhood bone mass, or on nonskeletal conditions such as type 1 diabetes and asthma. Furthermore, some of the existing associative studies suggest harm to the offspring from higher 25(OH)D levels or maternal intake of vitamin D

during pregnancy. Large randomized controlled trials of vitamin D supplementation during pregnancy are sorely needed.

OVERVIEW OF CALCIUM METABOLISM IN THE NEONATE AND INFANT Upon cutting the umbilical cord the placental calcium infusion and placental sources of PTHrP are lost. A rapid adjustment in the regulation of mineral homeostasis is forced to begin and be completed over the coming hours to days. The neonate becomes dependent upon intestinal calcium intake, skeletal calcium stores, and renal calcium reabsorption, in order to maintain a normal blood calcium at a time of continued skeletal growth and mineral accretion. A positive mineral balance must be maintained until peak bone mass is achieved in the young adult. Parathyroid hormone, 1,25(OH)2D, and FGF23 become more important for neonatal calcium homeostasis while PTHrP becomes less involved. After birth the total and ionized calcium concentrations fall, provoked by loss of the placental calcium pump and placental-derived PTHrP, and by a rise in pH that the onset of breathing causes. Studies in rodents show a fall in total and ionized calcium levels to about 60% of the fetal value by 6e12 hours after birth, and a subsequent rise to the normal adult value over the succeeding week [1]. Although data are less complete for humans, the progression in ionized and total calcium values appears to be similar. The ionized calcium in normal neonates falls from the umbilical cord level of 1.45 mmol/L to a mean of 1.20 mmol/L by 24 hours after birth [134]. Babies delivered by elective C-section were found to have lower blood calcium and higher parathyroid hormone levels at birth compared to babies delivered by spontaneous vaginal delivery [135], indicating that the mode of delivery can affect early neonatal mineral homeostasis. Phosphorus initially rises over the first 24 hours of postnatal life in humans and then gradually declines. Parathyroid hormone rises from the lower fetal levels to within or near the normal adult range by 24e48 hours after birth [1]. The increase in parathyroid hormone follows the early postnatal drop in the serum ionized calcium, and precedes the subsequent fall in phosphorus and rise in ionized calcium and 1,25(OH)2D. 1,25(OH)2D rises to adult levels over the first 48 hours of postnatal life, likely in response to the increasing levels of PTH. Serum calcitonin rises during the same time interval and then declines to adult levels. Based on one human study, serum intact FGF23 levels rise over the first 5 days post delivery to equal to adult values [13]. FGF23 appears to play an important role

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in phosphorus regulation after weaning, when Fgf23null mice develop progressive hyperphosphatemia, modest hypercalcemia, elevated 1,25(OH)2D, low PTH, skeletal abnormalities, and soft tissue and vascular calcifications [22,136,137]. PTHrP secretion from placenta, amnion, and umbilical cord is lost at birth. Secretion from the parathyroid glands (if ever present) is also apparently lost sometime after birth, since PTHrP circulates at low to undetectable levels during normal adult life in humans and animals. Animal studies suggest that PTHrP may persist in the neonatal circulation for some time, whether secreted by the parathyroids or due to absorption of PTHrP from milk (milk contains PTHrP at concentrations 10 000-fold higher than the level in the fetal circulation). Whether PTHrP present in milk contributes to the regulation of neonatal mineral homeostasis is unknown. Intestinal calcium absorption in newborns is largely passive, nonsaturable, and not dependent on 1,25 (OH)2D. The high lactose content of milk increases the efficiency of intestinal calcium absorption and net bioavailability of dietary calcium, through effects on paracellular diffusion in the distal small bowel [138e140]. As postnatal age increases, enterocytes express higher levels of the vitamin D receptor and the calcium-binding protein calbindin9KD. At the same time, vitamin-D-dependent active transport of calcium increases and passive transfer of calcium through the paracellular route declines. In weaned rodents active transport is the major route by which calcium enters the intestinal mucosa. Data from newborn humans are less complete, but the onset of 1,25(OH)2D-dependent active transport of calcium follows a similar postnatal course. The programmed postnatal maturation of the neonatal intestine limits the ability of preterm humans to absorb sufficient calcium to regulate the blood calcium and facilitate skeletal mineral accretion. Although data are limited, urinary calcium excretion rises in humans over the first 2 weeks after birth, consistent with increased efficiency of intestinal calcium absorption. The human neonatal skeleton continues to accrete calcium at 120e150 mg per day, similar to late gestation. Premature infants are prone to develop metabolic bone disease of prematurity, a form of rickets precipitated by loss of the placental calcium infusion at a time when the skeleton needs to accrete calcium at a peak rate. This form of rickets is not due to vitamin D deficiency per se but is the consequence of inadequate calcium and phosphate intake to meet the demands of the mineralizing neonatal skeleton. The limiting factor is the immaturity of the intestine which is not ready to express the vitamin D receptor and respond to 1,25 (OH)2D. Special oral or parenteral formulas that are high in calcium and phosphorus content will increase

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passive absorption and enable normal skeletal accretion of these minerals. In the neonatal period the consequences of maternal hyperparathyroidism during pregnancy may become apparent with prolonged (and sometimes permanent) suppression of the neonatal parathyroid function, hypocalcemia, tetany, and even death. The mechanism for this is uncertain but maternal hypercalcemia may cause increased flux of calcium across the placenta, activation of the calcium-sensing receptor and suppression of the fetal parathyroid glands in utero. This postnatal suppression has also been observed in infants of women with hypercalcemia due to familial hypocalciuric hypercalcemia. Maternal hypoparathyroidism with hypocalcemia during pregnancy can result in fetal parathyroid gland hyperplasia but is usually not recognized until the neonatal period [1]. Sparse data are available but in affected neonates parathyroid hormone is increased while the serum calcium may be normal or elevated. The skeletal findings generally resolve over the first several months after birth, but acute interventions may be required to raise or lower the blood calcium in the neonate. In severe cases neonatal hyperparathyroidism has caused persistent hypercalcemia with subtotal parathyroidectomy required to correct it. Neonatal hypocalcemia typically presents as seizures that onset between 4 to 28 days of age. The preterm infant is particularly prone to hypocalcemia due to inefficient intestinal calcium absorption. Other causes of neonatal hypocalcemia include congenital hypoparathyroidism, magnesium deficiency, maternal diabetes, vitamin D deficiency or resistance, and hyperphosphatemia.

ANIMAL DATA RELEVANT TO VITAMIN D AND THE NEONATE AND INFANT Data described earlier indicate that skeletal development and blood calcium, magnesium, and phosphorus are normal in offspring of mothers with severely altered vitamin D physiology, including vitamin-D-deficient rats [48,53,54], 1a-hydroxylase-null pigs [50], Vdr heterozygous and Vdr-null mice [37], and probably 1a-hydroxylase-null mice as well [51,52]. However, maternal hypocalcemia due to disrupted vitamin D physiology is not without consequences for the neonate since sudden (presumably hypocalcemia-induced) maternal deaths have occurred in the puerperium and during lactation for severely vitamin-D-deficient rats [48,53] and Vdr-null mice [37,42]. As reviewed in greater detail elsewhere [1,2,141,142], resorption of the maternal skeleton appears to be the main source of calcium contained in milk and provided

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FIGURE 35.3 The breast regulates skeletal demineralization and milk calcium supply during lactation. Suckling induces release of prolactin. Suckling and prolactin both inhibit the hypothalamic GnRH pulse center, which in turn suppresses the gonadotropins (LH, FSH), leading to low levels of the ovarian sex steroids (estradiol and progesterone). PTHrP production and release from the breast is controlled by several factors, including suckling, prolactin, and the calcium receptor. PTHrP enters the maternal bloodstream and combines with systemically low estradiol levels to markedly up-regulate bone resorption. Increased bone resorption releases calcium and phosphate into the bloodstream, which then reaches the breast ducts and is actively pumped into the breast milk. PTHrP also passes into the milk at high concentrations, but whether PTHrP plays a role in regulating calcium physiology of the neonate is unknown. Reproduced from [141] Ó2005 with kind permission from Springer Science and Business Medi B.V.

to the suckling neonate (Fig. 35.3). PTHrP (secreted from mammary tissue) and low estradiol during lactation combine to stimulate bone resorption and osteocytic osteolysis. Lactating rodents also maintain an increased rate of intestinal calcium absorption, approximately double the nonpregnant value [40,143], and this may be necessary in order to meet the proportionately greater calcium demands of multiple suckling pups. Vitamin D contributes to the normal regulation of calcium homeostasis in lactating mothers. 1,25(OH)2D levels are elevated in lactating rodents and increase further in response to a low-calcium diet or larger litter sizes [144,145]. This may indicate a compensatory mechanism that increases intestinal calcium absorption even further when extra demands are placed on the mother. However, studies in vitamin-D-deficient rats and Vdrnull mice indicate that sufficiency of vitamin D or responsiveness to 1,25(OH)2D are not required for lactation. Vitamin-D-deficient rats and Vdr-null mice lactated normally and resorbed the normal amount of bone

[42,43,146], although one study in vitamin-D-deficient rats found that more skeletal mineral content was lost than normal [147]. Intestinal calcium absorption remained up-regulated to twice normal in lactating vitamin-D-deficient rats, confirming that vitamin D is not required for this adaptation to take place [40,143]. As mentioned earlier, prolactin may stimulate intestinal calcium absorption independently of 1,25(OH)2D, possibly by stimulating TRPV6 [45,46]. At the end of lactation a rapid phase of bone formation occurs during which the maternal skeleton regains its prepregnancy bone mineral content. Full post-weaning recovery occurred in Vdr-null mice while two studies of vitamin-D-deficient rats noted some recovery of mineral content after lactation, with the final value exceeding the prepregnancy value in one study [42,43,146]. These findings suggest that 1,25(OH)2D and its receptor are not needed for skeletal recovery post-weaning. The role of FGF23 in skeletal recovery has not been studied. The fetal section above described how all models of disrupted vitamin D physiology have shown no evidence of rickets or hypocalcemia in utero or at term. But the adults will develop florid rickets, hypocalcemia, hypophosphatemia, and marked secondary hyperparathyroidism. None of these abnormalities are present until after weaning. This is true for vitamin-Ddeficient rats [53,54], 1a-hydroxylase-null pigs [50], 1ahydroxylase-null mice [51,52], and Vdr-null mice [58,59]. The most careful studies have been done in vitamin-D-deficient rats and Vdr-null mice, in which serum calcium and skeletal mineral content remain normal for the first 2e3 weeks after birth. Shortly after weaning the animals develop progressive hypocalcemia, hypophosphatemia, and histomorphometric evidence of rickets. This parallels the maturation of calcium absorption in the intestine, which changes from a nonsaturable, passive process in the newborn to an active, saturable, 1,25(OH)2D-dependent process [148e150]. These observations illustrate the importance of 1,25(OH)2D to regulate intestinal calcium absorption and facilitate skeletal mineralization in the adolescent and adult, but not in the neonate. Additional studies in both Vdr-null and 1a-hydroxylase-null mice have shown that if a high-calcium, high-phosphorus, lactose-enriched diet is initiated prior to weaning, then rickets is completely prevented [58,151e155]. The total alopecia remains in Vdr-null mice. The prevention of rickets by increasing the calcium content of the diet suggests that the main role of 1,25(OH)2D in calcium homeostasis is to stimulate active intestinal calcium absorption and that this role can be completely bypassed by manipulating the content of the diet. Overall, the animal data indicate that calcium is supplied to milk through up-regulated resorption of the maternal skeleton. Neither resorption of the

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maternal skeleton nor intestinal absorption of calcium by the mother appears to require vitamin D or 1,25 (OH)2D. In animals with disrupted vitamin D physiology that leads to rickets and hypocalcemia as adults, the newborn pups maintain normal serum calcium and phosphorus, calciotropic hormones, and skeletal mineral content until near the time of weaning. At that stage intestinal calcium absorption has become dependent upon 1,25(OH)2D. After weaning rickets will develop unless the impaired intestinal calcium absorption is bypassed by substantially increasing the calcium content of the diet.

HUMAN DATA RELEVANT TO VITAMIN D AND THE NEONATE AND INFANT The clinical course of VDDR-I and II in women during lactation has not been reported in the literature. Consequently there are no human data on what effect lack of 1,25(OH)2D or the vitamin D receptor might have on breast milk calcium content or maternal calcium and bone homeostasis during lactation. No clinical study has compared vitamin-D-deficient to vitamin-Dsufficient women during lactation, but numerous clinical trials and observational studies summarized below have examined the effect of vitamin D supplementation in women across a wide range of baseline 25(OH)D levels. As will be shown, even high-dose vitamin D supplementation has no effect on the calcium or phosphorus content of milk. Available clinical data from pediatric and older-age VDDR type II patients is consistent with the animal data, indicating that the deficiency in intestinal calcium absorption can be bypassed. Repeated calcium infusions or high oral dose calcium (3.5e9 g/m2 body surface area) will correct the biochemical abnormalities and prevent or heal underlying rickets or osteomalacia in VDDR type II patients [88,156,157]. In one such case series growth velocity increased, bone pain reduced, and several pediatric patients were able to walk for the first time [157]. Serum calcium, parathyroid hormone, phosphorus, and alkaline phosphatase values returned to normal within a year. Radiologic signs of healing occurred more rapidly in younger patients and when intravenous calcium was administered. Similar to Vdrnull mice, the total alopecia in VDDR type II subjects is not corrected by normalizing mineral homeostasis, indicating that the roles of 1,25(OH)2D and the VDR in hair follicle growth and development are independent of systemic calcium homeostasis. For VDDR type I the high-calcium approach has not been used because physiological doses of 1,25(OH)2D (0.25e1.0 mg daily) or 1a-OHD (0.5e3.0 mg daily) will normalize intestinal calcium absorption [87].

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In one attempt at a systematic examination of bone mineral content and vitamin D status in newborns, Congdon studied infants born to 45 Asian women, 19 additional Asian women who had received 1000 IU of vitamin D during the last trimester, and 12 Caucasian women who did not take supplements. Mean cord blood concentrations of 25(OH)D were 5.9, 15.2, and 33.4 nmol/L, respectively; there were no significant differences in serum calcium, phosphorus, and alkaline phosphatase. Bone mineral content of the forearm was assessed by SPA within 5 days of birth and did not differ among groups. Craniotabes was present in three babies from the unsupplemented Asians, four babies from Asians who received vitamin D, and in none of the Caucasians. The authors concluded that vitamin D deficiency did not impair skeletal mineralization at this early postpartum time point and that craniotabes is a nonspecific finding that should not be used to indicate the presence of rickets [158]. In a more recent systematic study, Weiler obtained cord blood 25(OH)D levels and used DXA to measure bone mineral content of the lumbar spine, femur, and whole body within 15 days after birth in 50 healthy term infants [159]. DXA has sufficient accuracy and precision to reliably measure BMD in anesthetized mice but movement artifacts limit its usefulness in human babies; the authors did not indicate whether the babies were sedated or anesthetized for the procedure. Caucasian infants were separated into sufficient and insufficient groups based on 25(OH)D levels in order to compare to Asian and First Nations babies who had insufficient levels of 25(OH)D. Mean cord blood 25(OH)D was 73 and 36 nmol/L in the sufficient and insufficient Caucasians, as compared to 44 nmol/L in Asians and 27 nmol/L in First Nations. There was no difference in bone mineral content of vitamin-D-sufficient versus -insufficient Caucasians. Lumbar spine bone mineral content of Asians was lower than Caucasian while that of First Nations babies was intermediate between the two. Whole body and femur bone mineral content were no different among the groups. The authors concluded that ethnic differences explained the variation in spine bone mineral content and that vitamin D sufficiency or insufficiency did not affect mineralization of the newborn skeleton [159]. In the days after birth vitamin D deficiency increases the likelihood of neonatal hypocalcemia. This coincides with maturation of intestinal calcium absorption from a passive, nonsaturable process facilitated by lactose to one that is saturable and dependent upon 1,25(OH)2D. Preterm infants demonstrate passive, nonvitamin-Ddependent absorption of calcium [93,94]. The lactose content of breast milk has also been shown to increase the efficiency of passive intestinal calcium absorption in human infants [160,161], as it does in rodents [138e140]. As normal term and preterm infants mature,

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1,25(OH)2D-mediated intestinal calcium absorption increases while passive absorption declines, similar to the animal studies described above [93,95,96]. This maturation of intestinal calcium absorption to a 1,25 (OH)2D-dependent process parallels and explains the clinical observation that vitamin-D-deficient rickets does not usually develop (or become recognized) until several weeks to typically 6e18 months after birth, even in regions where severe vitamin D deficiency during pregnancy is endemic [80e82]. Time will pass before reduced intestinal calcium absorption leads to impaired skeletal mineralization. Two recent case series found no radiological evidence of rickets or skeletal demineralization (by DXA) among infants presenting with hypocalcemia and low 25(OH)D levels [162,163]. This suggests that, as in the animal studies, hypocalcemia will precede the development of rickets. Vitamin D and 25(OH)D have low penetrance into breast milk, together comprising 40e50 IU/L of antirachitic activity, of which most of this is 25(OH)D [164e167]. A recent USDA survey found the vitamin D content of human milk to be 4.3 IU per 100 kcal, as compared to the 40e100 IU per kcal mandated for infant formulas [168]. This means that in exclusively breastfed infants who do not receive supplemental vitamin D or sufficient sunlight exposure, the serum 25(OH)D level will drop over several months in accordance with its half-life. The lower the 25(OH)D value at birth, the sooner the infant’s 25(OH)D will reach levels that predispose to hypocalcemia and rickets. This is why breastfed infants born of women with low vitamin D stores are at the highest risk of developing rickets during the first 18 months of life [169]. The higher the 25(OH)D level at birth, the more likely the infant will have adequate vitamin D stores until the time that solid (vitamin-D-supplemented) foods are established around 6 months of age. In infants receiving vitamin-D-fortified formula the 25(OH)D levels should not decline. In sufficiently sun-exposed infants the 25(OH)D level should not decline but sunlight exposure is normally avoided over concerns about actinic damage and risks of skin cancer. Specker studied the effects of sunlight exposure in exclusively breastfed infants and determined that 30 minutes per week wearing only a diaper, or 2 hours per week fully clothed but without a hat, would provide adequate 25(OH)D levels [170]. There are no data that have established what the optimal 25(OH)D level should be in the neonate or infant, although a systematic review of the available literature noted that a 25(OH)D level above 30 nmol/L is generally protective against the development of rickets [171]. Rickets has also developed at higher levels of 25(OH)D but especially in infants with extremely low calcium intakes or consumption of diets that are high in calcium-binding phytate. In such infants the rickets is

essentially due to pure calcium deficiency: since rickets is ultimately a disorder of impaired mineralization of osteoid which can be corrected by calcium infusion or high calcium intakes, no amount of vitamin D will prevent or treat rickets if there is little or no calcium in the diet [172]. Several observational studies have found that in such children calcium is very effective in treating rickets while vitamin D is ineffective [173,174]. This is supported by a study of nutritional rickets due to very low calcium intakes in which vitamin D supplementation had no effect on the fractional intestinal calcium absorption, despite raising the mean 25(OH)D level from a baseline of 50 nmol/L to 75 nmol/L [175]. In the days to weeks after birth in vitamin-D-deficient neonates, the serum calcium and phosphorus can be expected to decline while the parathyroid hormone level should rise. A low serum calcium may be detectable within a few days of birth, as noted in the randomized trial by Brooke [84], and in an observational study by Zeghoud and colleagues [176]. Zeghoud took blood samples from 80 neonates at 3e6 days after delivery and stratified them by 25(OH)D levels. The serum calcium was higher in neonates with 25(OH)D levels greater than 30 nmol/L as opposed to 16e30 nmol/L or less than 16 nmol/L (2.51  0.11 vs. 2.44  0.22 and 2.42  0.21 mmol/L, respectively) [176]. In the subsequent weeks to months after birth, vitamin D deficiency may present in diverse ways, including hypocalcemic seizures, growth failure, lethargy, irritability, and a predisposition to respiratory infections during infancy [177]. This presentation may be confounded by globally inadequate nutrition in the mother and infant which contributed to the vitamin D deficiency. A UK retrospective chart review identified 65 cases of vitamin D deficiency in children ranging from less than 1 year to 13 years of age and discovered two patterns of presentation. The first was hypocalcemia during periods of rapid growth (less than 3 years and more than 10 years of age) in which 17/29 subjects had no radiological evidence of rickets. Hypocalcemia presented as seizures, neuromuscular irritability, apnea, or stridor. The second group had no hypocalcemia but all had chronic skeletal effects of rickets and demineralization, including bow legs, pain, swollen joints, and other bone abnormalities. In both presentations all children had 25(OH)D levels below 15 nmol/L [178]. The prevalence of vitamin D deficiency depends upon the definition that is used as well as latitude, fortification practices, skin pigmentation, diet, and other factors. In a recent study among largely African-American and Latino infants and toddlers in Boston (latitude 42 N), the authors used the 25(OH)D level to estimate prevalence of vitamin D deficiency; 1.9% of infants and toddlers had a 25(OH)D level <20 nmol/L, 12.1% had a level <50 nmol/L, and 40.0% had a level

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<75 nmol/L [179]. The prevalence was quite similar among a predominantly Caucasian population of infants and toddlers much further north in the province of Newfoundland and Labrador, Canada (latitude range 46 to 61 N) during 2008; 3.6% had a 25(OH)D level <25 nmol/L, 12.7% had <50 nmol/L, and 54.5% had <75 nmol/L (unpublished data). The Boston survey identified that the main risk factor among infants for low 25(OH)D levels was being breastfed with no vitamin D supplement administered, whereas among toddlers the high-risk factors were low milk consumption and a higher body mass index [179]. The incidence of rickets was 2.9 per 100 000 in a recent 2-year survey of Canadian pediatricians, and the mean age at diagnosis was 1.4 years (range 2 weeks to 6.3 years) [169]; 94% of children with rickets had been breastfed. Additional risk factors included dark skin, living in the far north, born of mother who took no vitamin supplements, limited sun exposure, emigrated from a region where vitamin D deficiency is endemic, and delayed initiation of solid foods [169].

Intervention Studies With respect to maternal outcomes during the neonatal period, a number of observational studies [180e185] and clinical trials [186e193] have examined the effects of vitamin D supplementation in lactating women. These studies have generally shown that providing vitamin D to lactating mothers increased their 25(OH)D levels but had no significant effect on any other maternal or neonatal outcome. However, many studies measured no outcome other than the achieved 25(OH)D level in mothers and neonates and were not powered to look at outcomes such as hypocalcemia or clinical rickets [186,187,190e192]. The effect of maternal dietary calcium intake on the skeletal resorption that occurs during lactation has been carefully examined through randomized trials and in observational studies. The consistent finding is that calcium intakes ranging from very low to supplemented well above normal had no effect on the degree of skeletal demineralization that occurred during lactation although it did increase urinary calcium excretion [194e200]. In a randomized clinical intervention that studied the effect of consuming dietary calcium in excess of the recommended daily allowance (2.4 g daily), lactating women still lost 6.3% of bone mineral density at the lumbar spine and up to 8% from the radius and ulna, as determined by DXA [194]. In another RCT that included 97 lactating women randomized to 1 g calcium supplement versus placebo, there was no effect of calcium on the magnitude of BMD decline during lactation [195]. The calcium content of mother’s milk is an important lactation outcome relevant to the neonate and infant. A

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survey of all available studies indicated that at 30 days’ postpartum, the average calcium content of milk is 259  59 mg/L; assuming 0.78 L ingested per day, the average intake per baby is 202  46 mg per day [201]. Since both the 1a-hydroxylase and the VDR are expressed by human mammary tissue [202,203], a role for calcitriol in regulating the calcium content of milk seems plausible. However, breast milk calcium content is unaffected by maternal vitamin D status or any amount of vitamin D supplementation [191,192,204]. This includes a study by Hollis in which lactating women received 6400 IU daily and had a mean 25(OH)D level of 168 nmol/L [192]. Similarly maternal calcium intake does not alter the breast milk calcium content as demonstrated in several clinical trials. In one study of 97 women randomized to take 1 g calcium supplement versus placebo, breast milk calcium was unaffected [195]. In a study of 125 Gambian women with habitually low calcium intakes treated with 1500 mg supplemental calcium versus placebo, there was no effect on breast milk calcium content and infant weight, growth, or BMD during the first year of life [205]. Another study confirmed that the breast milk output predicts the maternal BMD decline during lactation, whereas calcium intake, breast milk calcium concentration, and vitamin D receptor genotype have no effect [197]. While one might expect a priori that low maternal vitamin D and calcium intake would accentuate skeletal losses in order to maintain breast milk calcium content, the majority of studies cited above suggest that this is not the case. These results are consistent with the notion that the calcium content of the milk largely supplied by resorption of the maternal skeleton, while the final calcium concentration of secreted milk is determined by local regulation within mammary epithelial cells (a process that does not require calcitriol or its receptor). The skeletal resorption is programmed by the obligatory rise in PTHrP and fall in estradiol, and not influenced by vitamin D status. Increasing calcium and vitamin D intake during lactation may simply increase the urinary calcium excretion and, thereby, the risk of kidney stones. Lactation is followed by an interval of several months during which the skeletal mineral content returns to the prepregnancy value. No clinical trial has examined the effect of vitamin D deficiency or insufficiency on the ability of the skeleton to recover from lactational losses. Assuming that it is desirable for the infant to have a 25(OH)D level over 75 nmol/L, several clinical trials have demonstrated that 300 or 400 IU of vitamin D given directly to the baby is more than sufficient to achieve this level of 25(OH)D, and that typical maternal doses of vitamin D supplements (400 or 1000 IU per day) have little or no effect on infant 25(OH)D levels

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[186e188,190,206,207]. In several pilot studies Hollis and colleagues examined the possibility that mothers could be given enough vitamin D so that their babies would achieve 25(OH)D levels greater than 75 nmol/L from breast milk alone [190e192]. A maternal dose of 4000 IU per day brought the mean infant 25(OH)D level to 75 nmol/L, whereas a maternal dose of 6400 IU per day brought all infants to greater than 75 nmol/L. Infants treated with 300 IU directly also achieved the same 25(OH)D level of greater than 75 nmol/L [190e192]. No adverse effects of 6400 IU per day were observed in the mothers or infants during these trials but the numbers of subjects involved were small. Treating the infant directly with 300 or 400 IU per day remains the preferred method but a maternal dose of 6400 IU per day is an alternative if it is desired to have all infant nutrition come from breast milk. Apart from the studies demonstrating the amount of vitamin D intake required to achieve a certain infant 25 (OH)D level, no other functional outcomes of significance have been found when comparing breastfed to formula-fed babies. One study randomized 51 breastfed infants to receive 400 IU vitamin D or no supplement, and an additional 40 formula-fed infants were followed as controls. The 25(OH)D level was 50 nmol/L at 2 weeks of age in the breastfed babies who did not receive a supplement, and the value did not change significantly over the following 6 months (mean 42.5 nmol/L at 2 months to 47.5 nmol/L at 6 months). The 25(OH)D level was higher in the formula-fed infants (peak 67.5 nmol/L) and intermediate in the breastfed infants who received a supplement (peak 57.5 nmol/ L). The differences among the three groups were statistically significant solely at the 2-month time point [208]. There were also no differences among groups at any time point with respect to weight, length, and skeletal mineral content (measured by SPA of the radius). Although small, this study of 91 infants suggested that there was no benefit achieved by raising the infant 25 (OH)D level above 50 nmol/L. Beyond preventing rickets, only a transient benefit on bone mass has been seen through the use of vitamin D supplements in infancy. In a study of 18 healthy term infants randomized to receive 400 IU or placebo at 3 weeks of age, 25(OH)D levels increased to 95 nmol/L versus 50 nmol/L and the BMC of radius and ulna (measured by SPA) was 23% higher than in the placebo group by 12 weeks of age (79.3  3.0 vs. 64  3.0 mg/cm, P < 0.001) [206,209]. The treatment continued but the BMC was identical between the two groups at the 6- and 12-month time points [206]. In a separate study of 46 healthy breastfed infants randomized to receive 400 IU vitamin D or placebo, the mean 25(OH)D level increased to 92.4 versus 47 nmol/L in the placebo group, but there were no differences between groups in BMC

(by SPA of radius) at 3 or 6 months of age [207]. Overall these studies indicate no benefit of a 25(OH)D level over 50 nmol/L. In the Brooke study cited earlier, in which 59 women received probably 10 000 IU of vitamin D3 daily and were compared to 67 unsupplemented, vitamin-D-deficient controls, there was no difference in cord blood calcium. However, at 3 and 6 days postnatal the plasma calcium levels of infants from the treated mothers were modestly but statistically significantly higher than in infants from control mothers (2.30  0.04 compared to 2.18  0.04 mmol/L, P < 0.05) [84]. The control infants had a 25(OH)D level of only 10.2  2.0 nmol/L as compared to a level of 137.9  10.8 nmol/L in the infants born of vitamin-D-supplemented mothers. Five of 67 infants in the placebo group had symptomatic hypocalcemia (manifest by irritability and serum calcium <1.8 mmol/L) whereas none of the 59 infants in the treatment group had symptomatic hypocalcemia. No infants showed radiologic signs of rickets. At this low level of 25(OH)D in the control infants, 1,25(OH)2Ddependent intestinal calcium absorption was likely submaximal. Data from older children indicate that intestinal calcium absorption is maximal with a 25 (OH)D level in the range of 30e50 nmol/L [210,211].

Associational Studies There have been numerous epidemiological studies that examined the effect of a history of lactation or recalled months of lactation on the risk of developing low bone mass, fractures, or osteoporosis later in life (reviewed in [1,212]). The vast majority of these have shown no effect, or a protective effect, of lactation on subsequent risk of these adverse outcomes. None of these studies were sufficiently powered to test whether vitamin D sufficiency or deficiency impacts on the effect that a history of lactation has on BMD or fracture risk. Neonatal hypocalcemia has been associated with low 25(OH)D levels in the days to weeks after birth. In one recent case series all hypocalcemic infants had 25(OH) D levels below 25 nmol/L (range 10e25 nmol/L; mean 20.3  9.8 nmol/L) [163]. In another series, all cases of neonatal hypocalcemia attributed to low vitamin D status had 25(OH)D values below 30 nmol/L (mean 20.5  9.0); the remaining cases of hypocalcemia had 25(OH)D levels greater than 50 nmol/L and were attributed to other causes (e.g., hypomagnesemia) [162]. In both case series, none of the infants who had both hypocalcemia and low 25(OH)D levels showed radiological evidence of rickets. In a third series of 42 cases of vitamin D deficiency presenting in the first 3 months of life, most presented with hypocalcemic seizures and all had 25(OH)D levels less than 27 nmol/L. No clinical signs of rickets were detected but subtle changes

V. HUMAN PHYSIOLOGY

HUMAN DATA RELEVANT TO VITAMIN D AND THE NEONATE AND INFANT

consistent with early rickets were noted on radiographs [213]. These observational studies suggest that hypocalcemia will develop before there are any significant skeletal changes. The possible effect of lower 25(OH)D levels during fetal development on childhood bone mass has been examined in an associational study by Javaid cited earlier in the fetal section [113]. Although no significant associations were observed at birth or 9 months of age, at 9 years of age there was a significant reduction in BMC in the children born of mothers who had a 25(O) D level below 27.5 nmol/L during pregnancy as compared to children born of mothers who had a 25 (OH)D level of 50 nmol/L or higher during pregnancy. This possibly confounded association needs to be tested in a clinical trial. 25(OH)D may have been a marker for poor nutrition and socioeconomic status during pregnancy and childhood. One group of investigators has done several followup analyses of a cohort of 696 Tasmanian women (out of an eligible 735 consecutive admissions) whose newborn children were considered to be at high risk of sudden infant death syndrome in 1988. Within a few days of childbirth, and again at 1 and 3 months postpartum, the women completed questionnaires that addressed such factors as nutritional intake during pregnancy and breastfeeding intake or practice. In the first follow-up analysis, the investigators measured BMD by DXA in 47% of the children at eight years of age. Compared to bottle-fed babies, breastfed babies weighed less at birth and had a higher proportion of preterm births. When term babies only were analyzed, breastfeeding 3 months or longer was associated with a slightly higher BMD (0.2e0.29 SD) at all sites by eight years of age as compared to bottle-fed babies. Breastfeeding for less than 3 months did not show this association and preterm babies showed no difference in BMD by bottle or breastfed status [214]. In a second report correlations between maternal nutritional intake during the third trimester and BMD at eight years of age were made in 173 infants (24% of the original cohort). Several statistically significant associations were found between maternal intake of magnesium, phosphorus, and potassium and BMD of spine, hip, or total body. However, maternal calcium intake during pregnancy did not correlate with childhood BMD, and vitamin D intake or status was not assessed [215]. In the most recent report, 216 adolescents (31% of the original cohort) had BMD done at age 16, and maternal intake of milk, fat and magnesium (but not calcium) during the third trimester of pregnancy were predictive of BMD at age 16 [216]. Overall these studies provide some support to the notion that in utero nutrient exposure may program childhood BMD or peak bone mass. However, the low follow-up rate (70e76% lost to follow-up or inadequate

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data) and abnormal status of the children at birth (at high risk for sudden infant death syndrome) make this data questionable. In another prospective cohort study, 70 preterm infants were randomized into four groups for a three month study: to receive a vitamin D dose of 500 or 1000 IU/day, and either calcium- and phosphorussupplemented or unsupplemented breast milk. These doses of vitamin D resulted in 25(OH)D levels that ranged from 100e400 nmol/L regardless of the dose. At 3 months of age, infants with a calcium-and-phosphorus supplemented diet had a 36% higher BMD of the left forearm shaft as measured by SPA. At 9e11 years of age only 50% of the cohort could be assessed. There was no effect of the prior history of calcium-andphosphorus supplementation on BMC (measured by DXA) of the lumbar spine, left forearm, or distal radius. At neither 3 months nor 9e11 years of age did the history of vitamin D supplementation affect the BMC result [217]. The sample size per group was quite small, ranging from 12 to 23; at the later follow-up the sample size was 8e11 per group. The study suggests no effect of vitamin D supplementation on childhood BMD. As mentioned briefly in the fetal section, epidemiological studies suggest that vitamin D supplementation of the infant is associated with a lower childhood incidence of type 1 diabetes [125e127]. However, the evidence is not definitive. A study in Finland asked mothers to recall if they had given their infants a 1000 IU vitamin D supplement during the first year of life (a national campaign at the time promoted the use of a 1000 IU vitamin D supplement for all babies). Over 30 years later, the cohort was assessed again to determine the prevalence of type 1 diabetes. A statistically significant increase was found in the incidence of type 1 diabetes in offspring who had never received vitamin D supplementation, but this analysis relied on only two cases of type 1 diabetes among infants whose mothers recalled not administering the vitamin D supplement. In a subanalysis that examined children who had reportedly received vitamin D during their first year, there was a statistically significant reduction in diabetes among the cohort who received the recommended dose of vitamin D versus those who had received a lower dose, but this also relied on just two cases of diabetes in children who had reportedly received a lower dose of vitamin D. This study is subject to recall bias especially at the one year time point when compliance with the national policy was considered evidence of being “a good mother.” A chance result cannot be excluded since in both the main analysis and the subanalysis, one instead of two cases of type 1 diabetes among those who did not receive vitamin D, or among those who received less than 1000 IU of vitamin D, would have eliminated any statistical significance [125]. In Norway

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a caseecontrol study compared 545 cases of childhoodonset type 1 diabetes with 1668 control subjects. Use of cod liver oil in the first year of life was associated with a significantly lower risk of type 1 diabetes (adjusted odds ratio: 0.74; 95% CI: 0.56e0.99). However, use of other vitamin D supplements during the first year of life had no protective effect [126]. Oddly, maternal use of cod liver oil or other vitamin D supplements during pregnancy were not associated with type 1 diabetes, in contrast to an earlier study by the same authors which reported that cod liver oil use during pregnancy reduced the incidence of type 1 diabetes [123,126]. Finally, a metaanalysis of observational studies concluded that vitamin D supplementation given to the infant reduces the risk of type 1 diabetes by 29% [127]. There are no prospective, randomized, controlled trials of the effect of vitamin D supplementation on the incidence of type 1 diabetes. The existing evidence is suggestive but not definitive; these findings need to be confirmed by randomized, controlled trials to rule out substantial known confounding effects of socioeconomic status, lifestyle, nutrition, etc., in those children whose mothers chose to administer a vitamin D supplement versus those who did not.

CONCLUSIONS Animal data are quite convincing that vitamin D, 1,25 (OH)2D, and the VDR are not required during fetal development for normal regulation of calcium homeostasis, skeletal development, and skeletal mineral accretion. It is only during the post-natal period that disrupted vitamin D physiology will lead to impaired calcium and phosphorus absorption, hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and rickets or osteomalacia. These postnatal outcomes can be avoided by providing the animal with a high calcium diet, especially one supplemented with lactose to facilitate passive absorption of calcium. Human data are sparse but appear consistent with the animal data, specifically that babies with vitamin D deficiency or VDDR types I or II will be born with normal blood calcium and skeletal mineral content, and that vitamin D supplementation during pregnancy will not alter the cord blood calcium. It is in the weeks to months after birth that impaired intestinal absorption of calcium and phosphorus leads to hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and rickets or osteomalacia. The risk is substantially higher in babies born of vitamin D deficient and especially malnourished mothers, and highest in breastfed babies because the vitamin D and 25(OH)D content of breast milk is normally very low. Babies who receive vitamin D-fortified

formulas are at lower risk of developing vitamin D deficiency. Breastfed infants need to receive a vitamin D supplement directly; an alternative approach is to give the mother 6400 IU per day in order to ensure that the baby achieves a 25(OH)D of 75 nmol/L or greater. However, the long-term safety of this approach in women has not been examined nor has it been demonstrated that the infant or neonate requires a 25(OH)D level of 75 nmol/L or higher. The available data do not show any convincing additional benefits in neonates or infants when the 25(OH)D level is greater than 50 nmol/L. Children with VDDR type II have demonstrated that calcium infusions or a high oral intake of calcium can prevent and heal rickets or osteomalacia, bypassing the need for vitamin D. The available human data do not exclude the possibility that subtler skeletal effects of vitamin D deficiency (or even of VDDR type I and II) will be discovered by systematic comparison of affected to unaffected babies at birth, or that a sufficiently powered clinical trial might demonstrate increased bone mass or skeletal mineral content in the infant or child as a result of vitamin D supplementation during pregnancy, the neonatal period, or childhood. But at present these concepts remain unproven. Numerous nonskeletal benefits of vitamin D have been proposed and the associations may be strongest for a possible role during fetal development and early childhood in preventing type 1 diabetes. However, the evidence is conflicting and the associational studies are confounded. Proper randomized, controlled trials are needed to determine whether any of the postulated extra-skeletal benefits of vitamin D are real. The foregoing data were used to support the committee’s conclusion that maternal vitamin D requirements are not increased during pregnancy and lactation, Consequently, 600 IU of vitamin D should be consumed daily by non-pregnant, pregnant, and lactating adolescents and adults. Infants require 400 IU of vitamin D daily and special attention should be paid to breastfed infants who will normally receive little or no vitamin D through breast milk.

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