Vitamin D, Volume 1: Biochemistry, Physiology and Diagnostics, Fourth Editionhttp://dx.doi.org/10.1016/B978-0-12-809965-0.00042-2 © 2018 Elsevier Inc. All rights reserved.

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INTRODUCTION

Normal pregnancy and lactation demand that women adapt their physiology to provide sufficient calcium for fetal and neonatal skeletal development. Despite a similar magni-tude of demand for mineral within each of these periods, the adaptations differ significantly between pregnancy and lacta-tion (Fig. 42.1). Intestinal calcium absorption doubles during pregnancy, whereas skeletal resorption predominates during lactation. These adjustments enable the mother to meet her own needs as well as those of the fetus and infant without requiring increased intake of calcium, and without long-term adverse effects on the maternal skeleton.

In addition to human data from observational studies, clini-cal trials, and associational studies, this chapter also relies on data from animal models of disrupted vitamin D physiology.

The focus is on maternal physiology and outcomes; the related subjects of fetal and neonatal outcomes are addressed in Chapter 39. For a more detailed discussion and extensive cita-tion of primary literature, the reader is directed to two recent comprehensive reviews about pregnancy and lactation [1] and fetal and neonatal development [2].

OVERVIEW OF MINERAL PHYSIOLOGY DURING PREGNANCY

During pregnancy, maternal physiology adapts to supply mineral needed by the developing fetus. The magnitude of demand can be appreciated as the amount of mineral pres-ent in the average human skeleton at term, specifically 30 g of calcium, 20 g of phosphorus, and 0.80 g of magnesium [1].

O U T L I N E

Introduction 755

Overview of Mineral Physiology During Pregnancy 755Minerals and Calciotropic Hormones 756Intestinal Absorption of Calcium 758Renal Handling of Calcium 758Skeletal Metabolism 758

Animal Data Relevant to Vitamin D and Pregnancy 759

Human Data Relevant to Vitamin D and Pregnancy 761Randomized Interventions of Vitamin D Supplementation 761Genetic Disorders of Vitamin D Physiology 763Studies of Associations Between Obstetrical Outcomes

and Vitamin D Status 763

Overview of Mineral Physiology During Lactation and Postweaning Recovery 763

Minerals and Calciotropic Hormones 764Intestinal Absorption of Calcium 765

Renal Handling of Calcium 765Calcium Pumping in the Breast and Milk Formation 765Skeletal Metabolism During Lactation 767Skeletal Metabolism During Postweaning 768

Animal Data Relevant to Vitamin D, Lactation, and Postweaning Recovery 770

Human Data Relevant to Vitamin D, Lactation, and Postweaning Recovery 771

Conclusions 773

References 773

C H A P T E R

42Pregnancy, Lactation, and Postweaning Recovery

Christopher S. KovacsHealth Sciences Centre, St. John’s, NL, Canada

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This requirement for mineral is not met gradually during gestation. Instead, more than 80% of calcium, phosphorus, and magnesium is taken up by the fetal skeleton during the third trimester [1,2]. Calcium accretion increases from about 60 mg per day at week 24 to a peak rate of 300–350 mg per day between the 35th and 40th weeks [3]. Phosphorus accretion is about 60 mg per day at week 24 and 300–350 mg per day between the 35th and 40th weeks, whereas magnesium accre-tion is a modest 1.8 mg per day at week 24 and 5.0–7.5 mg per day during the last 5 weeks [3]. When considered as hourly rates of transfer from mother to fetus, the average fetus requires between 5% and 10% of the calcium and phosphorus present in the maternal circulation [4,5]. Therefore, the fetal demand for minerals has the potential to provoke maternal hypocalce-mia and hypophosphatemia, were it not for the physiological adaptations that are invoked to meet the combined needs of mother and fetus.

At first glance the ∼300 mg of calcium transferred daily across the placenta during the third trimester may seem easily obtainable from average dietary calcium intake and average efficiency of calcium absorption. However, normally about 25% of calcium is absorbed in healthy adults who consume adequate calcium [6]. With this rate of intestinal calcium absorption, pregnant women would have to consume an extra 1200 mg per day during the third trimester to meet the needs of their fetuses. Moreover, data from the US National Health and Nutrition Examination Survey (NHANES) and Statistics Canada show that between ages 18 and 50 years, the 50th percentile for calcium intake in North American women ranges from 800 to 1000 mg daily [7], for an expected fractional

absorption of 200–250 mg daily. The 25th percentile of intake is approximately 600–700 mg daily, for a fractional absorption of 140–175 mg daily [7]. Consequently, in the setting of a normal rate of intestinal calcium absorption, most women do not con-sume sufficient calcium to meet the combined needs of their babies and themselves.

However, increased intakes of calcium are not required in women who consume adequate calcium because the efficiency of intestinal calcium absorption doubles during pregnancy. This physiological adaptation normally meets the daily mineral requirements of the fetus and mother without adverse long-term adverse consequences for the maternal skeleton.

Minerals and Calciotropic HormonesNormal pregnancy is characterized by changes in serum

minerals and calciotropic hormone concentrations (Fig. 42.2). Total serum calcium progressively falls due to a dilutional decline in serum albumin, but the albumin-corrected calcium and ionized calcium remain normal. Serum magnesium and phosphorus are also unchanged. Intact parathyroid hormone (PTH) falls to the low-normal range or below during the first trimester in women from North America and Europe who consume adequate calcium [8–19]. PTH either remains low or slowly increases to the midnormal range by term [13,15–21]. In contrast, although some studies in women from Asia and Africa showed a similar fall in PTH during early pregnancy [18,19,22,23], others found that it does not decline and may even increase above normal [24–28]. In the studies where

FIGURE 42.1 Schematic illustration contrasting calcium homeostasis in human pregnancy and lactation, as compared to normal. The thickness of arrows indicates a relative increase or decrease with respect to the normal and nonpregnant state. Although not illustrated, the serum (total) calcium is decreased during pregnancy, whereas the ionized calcium remains normal during both pregnancy and lactation. Adapted from Kovacs CS, Kronenberg HM. Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocr Rev 1997;18:832–72, © 1997, The Endocrine Society.

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PTH increased, it may be a response to low calcium intake or high intake of phytate (which blocks calcium absorption).

Serum calcitriol has been shown in cross-sectional [9,13,20,29–36] and longitudinal [12,18,19,33,34,36,37] stud-ies to increase starting in the first trimester and reach double to triple nonpregnancy values by the third trimester. A modest (20%–40%) increase in vitamin D-binding protein, combined with the decline in serum albumin, indicates that free calcitriol is likely increased in all three trimesters [1,19,33,35,36,38,39]. It is unusual that calcitriol increases while PTH simultaneously declines to low values. Animal models indicate that calcitriol increases during pregnancy even in the absence of parathyroids [40] or the gene encoding PTH [41]. This is likely because the renal 1α-hydroxylase, cytochrome P450 family 27 subfamily B member 1 (Cyp27b1), is stimulated by nontraditional factors such as prolactin, placental lactogen, and high levels of estra-diol [1]. The placenta has often been assumed to be the source of increased calcitriol during pregnancy [42]; however, the mater-nal kidneys are the source, whereas the placental contributes lit-tle if any calcitriol to the maternal circulation. This is supported by a variety of studies [1], including that maternal kidneys in mice have 30-fold higher expression of Cyp27b1 versus the pla-centas [41]. Also, an anephric woman on hemodialysis had low calcitriol before and during a normal pregnancy, confirming that the placenta contributed little calcitriol to her circulation [43]. Furthermore, serum calcitriol increased equally in women with singleton and twin pregnancies, illustrating that two pla-centas do not result in higher calcitriol concentrations [44,45].

Serum 25-hydroxyvitamin D [25(OH)D] concentrations generally do not change during human pregnancy, as shown in numerous studies [12,25,36,39,44–50]. This includes the pla-cebo arm of a study in which women were severely vitamin D deficient at baseline (mean 25(OH)D level of 20 nmol/L or 8 ng/mL) [47]. Conversely, two small longitudinal studies found a modest decline in 25(OH)D during pregnancy [19,38]. These may have been influenced by seasonal and late-preg-nancy alterations in sunlight exposure and diet. A large cross-sectional study found that maternal 25(OH)D was consistently about 30% lower in women carrying twins as compared to women bearing singletons, whereas calcitriol was no differ-ent between groups [44]. Because the difference between mean 25(OH)D levels of women bearing singletons versus twins did not progress across the trimesters, it seems less likely that bearing two babies was the cause of lower maternal 25(OH)D and more likely that maternal differences between groups may have been relevant (e.g., vitamin D intake or sunlight expo-sure, maternal adiposity, ethnicity, etc.). Moreover, a longitu-dinal study found no difference in 25(OH)D levels between women bearing twins versus singletons at any time point [45]. Overall, the bulk of data indicate that maternal 25(OH)D does not usually change during pregnancy. Therefore, the higher concentrations of serum calcitriol do not use up its substrate 25(OH)D, nor does a fetus represent a substantial drain on maternal 25(OH)D stores.

The minimum level of 25(OH)D that indicates vitamin D sufficiency during pregnancy is arguable [1,7,51,52], but it should be evident that the normal suppression of PTH and

FIGURE 42.2 Schematic depiction of longitudinal changes in calcium, phosphorus, and calciotropic hormone levels during human pregnancy. Shaded regions depict the approximate normal ranges. PTH does not decline in women with low calcium or high phytate intakes and may even rise above normal. Calcidiol (25(OH)D) values are not depicted; most longitudinal studies indicate that the levels are unchanged by pregnancy but may vary due to seasonal variation in sunlight exposure and changes in vitamin D intake. FGF23 values cannot be plotted due to the lack of data. PTH, parathyroid hormone. Reproduced with permission from Kovacs CS. Maternal mineral and bone metabolism during pregnancy, lactation, and post-weaning recovery. Physiol Rev 2016;96:449–547.

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doubling or tripling of calcitriol during pregnancy are not the result of maternal vitamin D deficiency. Moreover, random-ized trials that supplemented women with 1000 international units(IU) to as much as 5000 IU of vitamin D daily during pregnancy did not alter the rise in calcitriol or the maternal serum calcium, albumin-corrected calcium, phosphorus, or PTH [25,39,48,53]. Instead, the increase in free and bound cal-citriol is a programmed response to pregnancy. In a random-ized trial in which 400, 2,000, and 4000 IU of supplemental vitamin D were administered, mean calcitriol levels reached a plateau at a 25(OH)D level of 100 nmol/L irrespective of vitamin D intake [39]. This plateau may reflect the maximal capacity of renal Cyp27b1 to convert 25(OH)D into calcitriol, balanced against expected increasing upregulation of Cyp24a1 to catabolize 25(OH)D and calcitriol. Moreover, such a plateau does not necessarily mean that 100 nmol/L is the optimal 25(OH)D level to be achieved in pregnancy, as the authors of that study asserted [39] because no benefit of this high level of 25(OH)D or calcitriol has been demonstrated.

Data from clinical use of PTH-related protein (PTHrP) assays are fraught with problems that have been detailed elsewhere, including which fragments of PTHrP are mea-sured and that PTHrP rapidly degrades in serum but more slowly in plasma that contains protease inhibitors [1]. Despite these problems, four longitudinal studies used the PTHrP1-86 assay and found a significant increase in PTHrP across the three trimesters, beginning as early as 3–13 weeks of gestation and reaching up to triple the first trimester value by term [16,19,54,55]. A cross-sectional study found that the mean PTHrP value at term was twice that of nonpregnant women [56]. Other studies have found elevated levels at term based on the expected values in nonpregnant adults [57,58]. In contrast, a few small cross-sectional studies found that PTHrP1−34 [59] or PTHrP1−86 [60,61] concentrations were no different than in nonpregnant controls or the expected normal range, whereas two longitudinal studies found no significant changes in PTHrP1-86 during pregnancy [21,62]. In some of these studies that showed no increase in PTHrP, the use of serum or inadequate collection and handling of plasma may have impacted the findings.

Calcitonin generally increases during pregnancy and may exceed the upper limit of normal [15,19,30,63–69]. The increase arises from the production of calcitonin by the breasts, pla-centa, and C-cells of the thyroid. A few studies have disputed this by finding no change in calcitonin [36] or even lower calci-tonin in all three trimesters [20].

Maternal fibroblast growth factor-23 (FGF23) levels have not been reported during human pregnancy but have been found to be normal at 24 hours after delivery [70].

Other hormones display altered basal concentrations dur-ing pregnancy and may influence maternal skeletal metabo-lism and mineral homeostasis. Estradiol increases up to 100-fold with smaller increases in estriol and estrone [20,71,72]. Progesterone increases up to 10-fold [71,72]. Prolactin increases at least 10-fold [13,19,20,72,73], whereas placental lactogen increases 10- to 100-fold [19,73,74]; both of these similar hor-mones activate prolactin receptors. Placental growth hormone

increases 25-fold, whereas pituitary growth hormone falls to low levels [75]. Insulin-like growth factor 1 (IGF-I) decreases to as low as 50% of nonpregnant values during the second tri-mester before increasing two- to three-fold above normal by term [20,21,74,75]. Oxytocin increases modestly during preg-nancy and reaches peak levels near term [76].

Intestinal Absorption of CalciumActive transport of calcium occurs in the duodenum and

proximal jejunum, whereas most calcium is absorbed through passive mechanisms in the distal jejunum and ileum [77,78]. Active transport is more rapid and becomes especially impor-tant when the dietary supply of calcium is low or the demand for calcium is high, such as during pregnancy. The use of sta-ble calcium isotopes (48Ca, 44Ca, and 42Ca) and mineral balance studies have consistently demonstrated that fractional absorp-tion of calcium doubles by as early as 12 weeks of pregnancy, and this increase is maintained to term [9,13,36,79–81]. Women are in a positive calcium balance by midpregnancy [79], which may prepare the mother to meet the peak fetal demands dur-ing the third trimester. Isotope studies have demonstrated a similar doubling of intestinal calcium absorption in pregnant rats and mice [1].

The doubling or tripling of serum calcitriol during preg-nancy likely contributes to the upregulation of intestinal cal-cium absorption. However, data from rodents indicate that calcitriol may not be required for this upregulation to take place; this is discussed in the section Animal Data Relevant to Vitamin D and Pregnancy.

Renal Handling of CalciumIn studies of women from North America who typically

consume a diet that is adequate in calcium, as early as the 12th week of gestation the 24-hour urine calcium excretion increases significantly and often reaches the hypercalciu-ric range [9,12,13,15,36,82,83]. The suppressed PTH con-centrations and high calcitonin levels of pregnancy may contribute to renal calcium excretion. This is absorptive hypercalciuria, which means that increased intestinal cal-cium absorption results in more calcium being absorbed than required, such that the excess is excreted by the kid-neys [1]. The fasting urine calcium remains normal or low in pregnant women [1], whereas a random urine sample will only detect an increased Ca/Cr ratio if it is collected postprandially.

Skeletal MetabolismThe doubling of intestinal calcium absorption during preg-

nancy appears to meet the fetal demand for calcium. However, the maternal skeleton may still be resorbed to some extent dur-ing pregnancy, thereby contributing mineral to the develop-ing fetal skeleton. There is a paucity of histomorphometric data from pregnant women. Bone biopsies obtained from 15 women at the time of elective first trimester abortions showed

increased bone resorption indices, as compared to biop-sies from nonpregnant women [84]; however, the study was confounded by poor age matching and small sample sizes. Multiple studies have demonstrated that bone resorption indi-ces (CTX, NTX, TRAP, and deoxypyridinoline) are normal or modestly reduced early in pregnancy but double by the third trimester [1]. Bone formation markers (P1NP, osteocalcin, and bone-specific alkaline phosphatase) are generally reduced in the first trimester before increasing to the midnormal range or above by term [1]. The placenta is responsible for a marked increase in total alkaline phosphatase, and so alkaline phos-phatase is not a reliable indicator of bone turnover during pregnancy.

If bone resorption increases significantly during pregnancy it should lead to bone loss, but the available data indicate no change to at most very modest losses. Bone density has been assessed using dual X-ray absorptiometry (DXA) with mea-surements obtained 1–18 months before planned pregnancy and 1–6 weeks postpartum, with readings during pregnancy avoided to prevent fetal radiation exposure [21,36,74,85–88]. These small studies found 0% to at most a 5% decrease in lum-bar spine bone density between the two measurements, with uncertainty as to when any observed decrease might have occurred [1]. The largest study involved 92 women who had DXA of hip, spine, and forearm done up to 8 months prior to planned pregnancy and within 15 days postpartum, whereas DXA of the forearm was also done in all three trimesters [89]. This is the only published study with serial DXA measure-ments during pregnancy, albeit only at a distal appendicular site. Seventy-three women completed the postpartum visit and were compared to 57 nonpregnant women who had DXA measurements done at similar intervals. During pregnancy, bone mineral density (BMD) decreased by 4% at the ultradis-tal radius but increased by 0.5% at the proximal 1/3 forearm [89]. A 1% lower BMD in the total forearm was discernible in pregnant women versus nonpregnant controls at the third trimester measurement. When prepregnancy and postpreg-nancy readings were compared, BMD was reduced by 1.8% at the lumbar spine, 3.2% at the total hip, and 2.4% at the whole body [89]. Overall, this study found small but statisti-cally significant declines in BMD at several skeletal sites. Such changes would not be distinguishable from error of measure-ment in an individual subject but were resolvable in this study due to the large cohort. Whether these small changes in BMD are completely attributable to pregnancy is uncertain because all women breastfed for up to 2 weeks before the postpartum measurement, and lactation induces rapid bone resorption (see section Overview of Mineral Physiology During Lactation and Postweaning Recovery).

In general the women in these studies consumed adequate calcium. But if dietary calcium is inadequate to meet the needs of the mother and fetus, notwithstanding the enhanced effi-ciency of intestinal calcium absorption, then skeletal resorp-tion must be induced and bone loss will be more substantial. Very low calcium intake explains some of the cases of osteo-porosis with fragility fractures that have presented at term in otherwise healthy women [90].

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increases 25-fold, whereas pituitary growth hormone falls to low levels [75]. Insulin-like growth factor 1 (IGF-I) decreases to as low as 50% of nonpregnant values during the second tri-mester before increasing two- to three-fold above normal by term [20,21,74,75]. Oxytocin increases modestly during preg-nancy and reaches peak levels near term [76].

Intestinal Absorption of CalciumActive transport of calcium occurs in the duodenum and

proximal jejunum, whereas most calcium is absorbed through passive mechanisms in the distal jejunum and ileum [77,78]. Active transport is more rapid and becomes especially impor-tant when the dietary supply of calcium is low or the demand for calcium is high, such as during pregnancy. The use of sta-ble calcium isotopes (48Ca, 44Ca, and 42Ca) and mineral balance studies have consistently demonstrated that fractional absorp-tion of calcium doubles by as early as 12 weeks of pregnancy, and this increase is maintained to term [9,13,36,79–81]. Women are in a positive calcium balance by midpregnancy [79], which may prepare the mother to meet the peak fetal demands dur-ing the third trimester. Isotope studies have demonstrated a similar doubling of intestinal calcium absorption in pregnant rats and mice [1].

The doubling or tripling of serum calcitriol during preg-nancy likely contributes to the upregulation of intestinal cal-cium absorption. However, data from rodents indicate that calcitriol may not be required for this upregulation to take place; this is discussed in the section Animal Data Relevant to Vitamin D and Pregnancy.

Renal Handling of CalciumIn studies of women from North America who typically

consume a diet that is adequate in calcium, as early as the 12th week of gestation the 24-hour urine calcium excretion increases significantly and often reaches the hypercalciu-ric range [9,12,13,15,36,82,83]. The suppressed PTH con-centrations and high calcitonin levels of pregnancy may contribute to renal calcium excretion. This is absorptive hypercalciuria, which means that increased intestinal cal-cium absorption results in more calcium being absorbed than required, such that the excess is excreted by the kid-neys [1]. The fasting urine calcium remains normal or low in pregnant women [1], whereas a random urine sample will only detect an increased Ca/Cr ratio if it is collected postprandially.

Skeletal MetabolismThe doubling of intestinal calcium absorption during preg-

nancy appears to meet the fetal demand for calcium. However, the maternal skeleton may still be resorbed to some extent dur-ing pregnancy, thereby contributing mineral to the develop-ing fetal skeleton. There is a paucity of histomorphometric data from pregnant women. Bone biopsies obtained from 15 women at the time of elective first trimester abortions showed

increased bone resorption indices, as compared to biop-sies from nonpregnant women [84]; however, the study was confounded by poor age matching and small sample sizes. Multiple studies have demonstrated that bone resorption indi-ces (CTX, NTX, TRAP, and deoxypyridinoline) are normal or modestly reduced early in pregnancy but double by the third trimester [1]. Bone formation markers (P1NP, osteocalcin, and bone-specific alkaline phosphatase) are generally reduced in the first trimester before increasing to the midnormal range or above by term [1]. The placenta is responsible for a marked increase in total alkaline phosphatase, and so alkaline phos-phatase is not a reliable indicator of bone turnover during pregnancy.

If bone resorption increases significantly during pregnancy it should lead to bone loss, but the available data indicate no change to at most very modest losses. Bone density has been assessed using dual X-ray absorptiometry (DXA) with mea-surements obtained 1–18 months before planned pregnancy and 1–6 weeks postpartum, with readings during pregnancy avoided to prevent fetal radiation exposure [21,36,74,85–88]. These small studies found 0% to at most a 5% decrease in lum-bar spine bone density between the two measurements, with uncertainty as to when any observed decrease might have occurred [1]. The largest study involved 92 women who had DXA of hip, spine, and forearm done up to 8 months prior to planned pregnancy and within 15 days postpartum, whereas DXA of the forearm was also done in all three trimesters [89]. This is the only published study with serial DXA measure-ments during pregnancy, albeit only at a distal appendicular site. Seventy-three women completed the postpartum visit and were compared to 57 nonpregnant women who had DXA measurements done at similar intervals. During pregnancy, bone mineral density (BMD) decreased by 4% at the ultradis-tal radius but increased by 0.5% at the proximal 1/3 forearm [89]. A 1% lower BMD in the total forearm was discernible in pregnant women versus nonpregnant controls at the third trimester measurement. When prepregnancy and postpreg-nancy readings were compared, BMD was reduced by 1.8% at the lumbar spine, 3.2% at the total hip, and 2.4% at the whole body [89]. Overall, this study found small but statisti-cally significant declines in BMD at several skeletal sites. Such changes would not be distinguishable from error of measure-ment in an individual subject but were resolvable in this study due to the large cohort. Whether these small changes in BMD are completely attributable to pregnancy is uncertain because all women breastfed for up to 2 weeks before the postpartum measurement, and lactation induces rapid bone resorption (see section Overview of Mineral Physiology During Lactation and Postweaning Recovery).

In general the women in these studies consumed adequate calcium. But if dietary calcium is inadequate to meet the needs of the mother and fetus, notwithstanding the enhanced effi-ciency of intestinal calcium absorption, then skeletal resorp-tion must be induced and bone loss will be more substantial. Very low calcium intake explains some of the cases of osteo-porosis with fragility fractures that have presented at term in otherwise healthy women [90].

Overall, the available human data indicate that bone turnover markers increase during pregnancy and that small losses of BMD may occur by the third trimester, which cor-responds to when the fetal demand for mineral is at its peak. Greater losses are more likely to occur in women with low calcium intake. Regardless of any short-term bone loss, over the long-term pregnancy does not usually impair skeletal strength or increase the risk of osteoporosis. Parity is not recognized as a significant risk factor in FRAX [91]. More than six dozen epidemiologic studies have found either no significant association, or a protective effect, of parity on BMD, fractures, or osteoporosis [1]. In contrast, fewer than one dozen studies found that parity increased the risk of low BMD or fractures [1]. One of the studies reporting a protec-tive effect of parity was especially strong because it involved 1852 twins and their female relatives and featured 83 identi-cal twins who were discordant for ever being pregnant [92]. An NHANES study of 819 women aged 20–25 years reported that BMD was no different among women who had expe-rienced an adolescent pregnancy, an adult pregnancy, or no prior pregnancies [93].

ANIMAL DATA RELEVANT TO VITAMIN D AND PREGNANCY

Calcitriol’s main role with respect to mineral and skeletal metabolism is to stimulate the active, saturable, transcellular mechanism of intestinal calcium delivery, as well as the pas-sive, paracellular mechanism of absorption [77]. As noted in Chapter 39, this role becomes evident at the time of wean-ing, when severe vitamin D deficiency and genetic absence of either calcitriol or the vitamin D receptor (VDR) lead to impaired intestinal calcium absorption, hypocalcemia, hypo-phosphatemia, and rickets. The fact that this is calcitriol’s main role has been confirmed by several lines of evidence. The abnormal mineral homeostasis and rachitic phenotype of Vdr null mice is rescued by expressing Vdr in intestinal cells [94,95]. Conversely, the Vdr null phenotype can be duplicated through selective ablation of Vdr solely from intestinal cells [95] or by severe dietary calcium restriction [95]. Moreover, ablating Vdr from chondrocytes, osteoblasts, or osteocytes, or Cyp27b1 from chondrocytes, do not cause rickets [95–98]. Furthermore, a diet high in calcium or parenteral calcium infusions bypass the need for calcitriol altogether and result in normalization of serum chemistries, skeletal morphology, and skeletal mineral content in severely vitamin D-deficient, Cyp27b1 null, and Vdr null models [99–111].

With this in mind, what impact do disorders of vita-min D physiology have on rodent pregnancy? A common finding is that, on a normal calcium diet, the mothers con-ceive less frequently and bear fewer pups in each litter. This includes severely vitamin D-deficient rats [112–115], Cyp27b1 null mice that cannot make calcitriol [116], and Vdr null mice that lack the receptor for calcitriol [117,118], but not Cyp27b1 null (Hannover) pigs which have only single-ton or twin pregnancies [119,120]. In fact, Vdr null mice and

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Cyp27b1 null mice were originally mistaken to be infertile. Pregnancies in Vdr null mice maintained on a normal cal-cium diet are otherwise normal [118], but the increased fre-quency of conception on a high calcium diet, and the larger litter sizes, facilitate studying the mice during reproductive cycles. Conversely, treatment of any of these rodent models with a “rescue diet” enriched in calcium, phosphate, and lactose corrects the low conception rate and the small litter sizes [108,111,116,118,121–123]. This confirms that it is some aspect of impaired calcium delivery, and not the actions of calcitriol, that leads to reduced fertility. It is unclear which of the stages of follicle development, ovulation, fertilization, implantation, and early embryonic delivery are affected, but the calcium concentration in extracellular fluids may be rel-evant at each of these steps.

In each of the models that have been studied (severely vitamin D-deficient rats [112,113,124,125], Cyp27b1 null pigs [119,120], Cyp27b1 null mice [116], and Vdr null mice [117,126,127]), occasional sudden deaths occur during late pregnancy. This likely indicates that rapid placental calcium transfer can overwhelm the mother’s ability to maintain her own ionized calcium level. The pregnancies are otherwise uneventful and result in fetuses that have normal ionized cal-cium, phosphorus, PTH, weight, skeletal development, and mineralization [2]. Offspring of Vdr null females were globally but proportionately smaller than offspring of their wild-type (WT) and Vdr+/− sisters, but this was not seen when compar-ing offspring of vitamin D-deficient and vitamin D-replete rats. This may indicate that maternal absence of VDR affects offspring growth independent of the fetal genotypes and that it is exerted by the receptor but not calcitriol. These issues are discussed in more detail in Chapter 39.

Increased fractional calcium absorption still occurs dur-ing pregnancy despite severe vitamin D deficiency in rats [128,129], absence of the VDR in mice [117], or maternal parathyroidectomy in rats [130]. In one study of severely vitamin D-deficient pregnant rats, an observed doubling to tripling of intestinal calcium, phosphorus, and magnesium absorption reached levels equivalent to that seen in normal pregnant rats [129], which indicates that full upregulation occurs during pregnancy despite the absence of calcitriol. In another study, intestinal calcium absorption doubled in severely vitamin D-deficient rats but peaked at 2/3 of the rate in pregnant vitamin D-replete rats [128]. And so, intesti-nal calcium absorption increases in the absence of calcitriol, but the peak rate may be blunted as compared to normal pregnancy.

The increased intestinal calcium and phosphorus absorp-tion observed in severely vitamin D-deficient rats con-tributed to significant increases in femoral ash weight and calcium content [112], and serum calcium and phosphorus [131], by the end of pregnancy. Similarly, pregnant Vdr null mice from the Boston strain (VdrBos) achieved a 55% increase in skeletal mineral content, reduction in osteoid by histo-morphometry, significant lessening in secondary hyperpara-thyroidism, normalization of serum calcium, and increased renal calcium excretion [117,126]. Pregnant Vdr null mice

in the Leuven strain (VdrLeu) also increased the serum cal-cium, trabecular BMD of the femur by quantitative computed tomography (qCT), and femoral ash weight, and showed his-tomorphometric evidence of reduced osteoid and osteoclast parameters [132]. A preliminary report in pregnant Cyp27b1 null mice has shown a 45% increase in bone mineral content by DXA, a marked reduction in secondary hyperparathyroid-ism, and normalization of serum calcium and phosphorus [116]. Intestinal calcium absorption has not yet been reported in pregnant Cyp27b1 null mice but is likely to be similar to normal pregnant mice, based on these observed improve-ments in bone mass and reduction in secondary hyperpara-thyroidism. In all of these studies the “rescue diet” enriched in calcium, phosphorus, and lactose was used, and this facili-tated calcium absorption despite the absence of calcitriol or the VDR. Nevertheless, it was not the initiation of the diet but the onset of pregnancy that resulted in the marked changes in skeletal metabolism and bone mass in these models.

It is uncertain what explains calcitriol-independent upregu-lation of intestinal calcium absorption during pregnancy. Vdr null mice display low intestinal expression of transient recep-tor potential cation channel subfamily V member 6 (TRPV6) when nonpregnant but a marked upregulation of TRPV6 dur-ing pregnancy on the rescue diet [117,133], while upregulation in calbindin-D9k has also been found in pregnant Vdr nulls on a normal calcium diet [132]. There is limited evidence that prolactin, placental lactogen, and growth hormone can stimu-late intestinal calcium absorption independently of calcitriol, possibly by increasing expression of TRPV6, transient receptor potential cation channel subfamily V member 5 (TRPV5), cal-bindin-D9k, and plasma membrane calcium ATPase isoform (PMCA1) [134–139]. Although growth hormone is normally suppressed during pregnancy, placental growth hormone is increased and conceivably may stimulate intestinal calcium absorption. Prolactin’s ability to stimulate intestinal calcium absorption was confirmed in pregnant, severely vitamin D-deficient rats [134]. Prolactin and placental lactogen both increased calcium absorption in everted gut sacs of nonpreg-nant, hypophysectomized rats [135,136]. Estradiol may stimu-late intestinal calcium absorption, and expression of TRPV6 and TRPV5, independent of calcitriol [133,140].

These data do not indicate that calcitriol plays no role in upregulating intestinal calcium absorption during pregnancy. Instead, the two- to five-fold increase in calcitriol quite likely contributes to the doubling in the efficiency of intestinal cal-cium absorption in vitamin D-replete animals. The additional factors (prolactin, placental lactogen, placental growth hor-mone, and possibly others) may provide additional stimula-tion of intestinal calcium absorption during normal pregnancy. The data from severe vitamin D-deficient rats, Vdr null mice, and Cyp27b1 null mice may indicate that these pregnancy-related factors can partly to fully compensate for the absence of calcitriol’s actions, such that intestinal calcium absorption reaches the normal peak levels of pregnancy in some rodents but falls below it in others.

Nonskeletal outcomes of pregnancy have not been exten-sively studied in these animal models. However, nonpregnant

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Cyp27b1 null mice were originally mistaken to be infertile. Pregnancies in Vdr null mice maintained on a normal cal-cium diet are otherwise normal [118], but the increased fre-quency of conception on a high calcium diet, and the larger litter sizes, facilitate studying the mice during reproductive cycles. Conversely, treatment of any of these rodent models with a “rescue diet” enriched in calcium, phosphate, and lactose corrects the low conception rate and the small litter sizes [108,111,116,118,121–123]. This confirms that it is some aspect of impaired calcium delivery, and not the actions of calcitriol, that leads to reduced fertility. It is unclear which of the stages of follicle development, ovulation, fertilization, implantation, and early embryonic delivery are affected, but the calcium concentration in extracellular fluids may be rel-evant at each of these steps.

In each of the models that have been studied (severely vitamin D-deficient rats [112,113,124,125], Cyp27b1 null pigs [119,120], Cyp27b1 null mice [116], and Vdr null mice [117,126,127]), occasional sudden deaths occur during late pregnancy. This likely indicates that rapid placental calcium transfer can overwhelm the mother’s ability to maintain her own ionized calcium level. The pregnancies are otherwise uneventful and result in fetuses that have normal ionized cal-cium, phosphorus, PTH, weight, skeletal development, and mineralization [2]. Offspring of Vdr null females were globally but proportionately smaller than offspring of their wild-type (WT) and Vdr+/− sisters, but this was not seen when compar-ing offspring of vitamin D-deficient and vitamin D-replete rats. This may indicate that maternal absence of VDR affects offspring growth independent of the fetal genotypes and that it is exerted by the receptor but not calcitriol. These issues are discussed in more detail in Chapter 39.

Increased fractional calcium absorption still occurs dur-ing pregnancy despite severe vitamin D deficiency in rats [128,129], absence of the VDR in mice [117], or maternal parathyroidectomy in rats [130]. In one study of severely vitamin D-deficient pregnant rats, an observed doubling to tripling of intestinal calcium, phosphorus, and magnesium absorption reached levels equivalent to that seen in normal pregnant rats [129], which indicates that full upregulation occurs during pregnancy despite the absence of calcitriol. In another study, intestinal calcium absorption doubled in severely vitamin D-deficient rats but peaked at 2/3 of the rate in pregnant vitamin D-replete rats [128]. And so, intesti-nal calcium absorption increases in the absence of calcitriol, but the peak rate may be blunted as compared to normal pregnancy.

The increased intestinal calcium and phosphorus absorp-tion observed in severely vitamin D-deficient rats con-tributed to significant increases in femoral ash weight and calcium content [112], and serum calcium and phosphorus [131], by the end of pregnancy. Similarly, pregnant Vdr null mice from the Boston strain (VdrBos) achieved a 55% increase in skeletal mineral content, reduction in osteoid by histo-morphometry, significant lessening in secondary hyperpara-thyroidism, normalization of serum calcium, and increased renal calcium excretion [117,126]. Pregnant Vdr null mice

in the Leuven strain (VdrLeu) also increased the serum cal-cium, trabecular BMD of the femur by quantitative computed tomography (qCT), and femoral ash weight, and showed his-tomorphometric evidence of reduced osteoid and osteoclast parameters [132]. A preliminary report in pregnant Cyp27b1 null mice has shown a 45% increase in bone mineral content by DXA, a marked reduction in secondary hyperparathyroid-ism, and normalization of serum calcium and phosphorus [116]. Intestinal calcium absorption has not yet been reported in pregnant Cyp27b1 null mice but is likely to be similar to normal pregnant mice, based on these observed improve-ments in bone mass and reduction in secondary hyperpara-thyroidism. In all of these studies the “rescue diet” enriched in calcium, phosphorus, and lactose was used, and this facili-tated calcium absorption despite the absence of calcitriol or the VDR. Nevertheless, it was not the initiation of the diet but the onset of pregnancy that resulted in the marked changes in skeletal metabolism and bone mass in these models.

It is uncertain what explains calcitriol-independent upregu-lation of intestinal calcium absorption during pregnancy. Vdr null mice display low intestinal expression of transient recep-tor potential cation channel subfamily V member 6 (TRPV6) when nonpregnant but a marked upregulation of TRPV6 dur-ing pregnancy on the rescue diet [117,133], while upregulation in calbindin-D9k has also been found in pregnant Vdr nulls on a normal calcium diet [132]. There is limited evidence that prolactin, placental lactogen, and growth hormone can stimu-late intestinal calcium absorption independently of calcitriol, possibly by increasing expression of TRPV6, transient receptor potential cation channel subfamily V member 5 (TRPV5), cal-bindin-D9k, and plasma membrane calcium ATPase isoform (PMCA1) [134–139]. Although growth hormone is normally suppressed during pregnancy, placental growth hormone is increased and conceivably may stimulate intestinal calcium absorption. Prolactin’s ability to stimulate intestinal calcium absorption was confirmed in pregnant, severely vitamin D-deficient rats [134]. Prolactin and placental lactogen both increased calcium absorption in everted gut sacs of nonpreg-nant, hypophysectomized rats [135,136]. Estradiol may stimu-late intestinal calcium absorption, and expression of TRPV6 and TRPV5, independent of calcitriol [133,140].

These data do not indicate that calcitriol plays no role in upregulating intestinal calcium absorption during pregnancy. Instead, the two- to five-fold increase in calcitriol quite likely contributes to the doubling in the efficiency of intestinal cal-cium absorption in vitamin D-replete animals. The additional factors (prolactin, placental lactogen, placental growth hor-mone, and possibly others) may provide additional stimula-tion of intestinal calcium absorption during normal pregnancy. The data from severe vitamin D-deficient rats, Vdr null mice, and Cyp27b1 null mice may indicate that these pregnancy-related factors can partly to fully compensate for the absence of calcitriol’s actions, such that intestinal calcium absorption reaches the normal peak levels of pregnancy in some rodents but falls below it in others.

Nonskeletal outcomes of pregnancy have not been exten-sively studied in these animal models. However, nonpregnant

and pregnant vitamin D-deficient rodents and Vdr null mice have been shown to be hypertensive [141–144], which is consistent with the possibility that vitamin D deficiency in humans may increase the risk of pregnancy-induced hypertension.

HUMAN DATA RELEVANT TO VITAMIN D AND PREGNANCY

Calcitriol-dependent active absorption of calcium repre-sents about 20% of net calcium absorption, with the rest occur-ring by passive, nonsaturable mechanisms. Recently, several dual- or single-isotope studies have been carried out in adults and adolescents to determine the level of 25(OH)D at which intestinal calcium absorption is maximized or reaches a pla-teau, and these have found that a plateau occurs by 20 nmol/L (8 ng/mL) of 25(OH)D, with little or no increase in intestinal calcium absorption above that value [7,145–149]. Secondary hyperparathyroidism begins to be induced as 25(OH)D declines from higher levels, and this stimulates formation of calcitriol, which may explain why vitamin D insufficiency does not reduce intestinal calcium absorption until the 25(OH)D level is around 20 nmol/L [148].

No study has measured intestinal calcium absorption during pregnancy in vitamin D-deficient women, and so it remains unknown whether intestinal calcium absorption doubles during pregnancy as it does in the animal models. However, since the increase in intestinal calcium absorption begins at 12 weeks, well before a more marked increase in cal-citriol and free calcitriol by the third trimester, this may indi-cate that the early increase in intestinal calcium absorption is driven by factors other than calcitriol. Moreover, as noted in Chapter 39, the extremes of severe vitamin D deficiency or genetic disorders of vitamin D physiology in the mother do not alter serum calcium, ionized calcium, phosphorus, PTH, or skeletal mineral content of the baby [2]. These normal fetal parameters imply that the mother is able to deliver adequate mineral during pregnancy, which should only happen if intes-tinal calcium absorption is upregulated.

The following subsections address whether maternal vita-min D deficiency or genetic disorders causing loss of calcitriol or VDR alter maternal mineral metabolism and obstetrical outcomes.

Randomized Interventions of Vitamin D Supplementation

Numerous randomized clinical trials, ranging from small to moderate sizes, have examined the effects of vitamin D supplementation during pregnancy. The fetal and neonatal outcomes of these studies have been described in another review [2] and in Chapter 39. In brief, no consistent change in cord blood calcium, phosphorus, PTH, birth weight, or anthropometric measurements was observed when babies of vitamin D-supplemented mothers were compared to babies of placebo-treated mothers. The incidence of neonatal

hypocalcemia after 48 hours was increased when babies of placebo-treated mothers had cord blood 25(OH)D lev-els below 20 nmol/L [2]. This is consistent with the afore-mentioned adult data that intestinal calcium absorption is impaired below that level, and the timing of when the intes-tines become an important source of mineral delivery to the postnatal human.

In the following paragraphs, the individual clinical trials will be described in sufficient detail to understand the doses of vitamin D tested, the sizes of the studies, the achieved incre-ment in 25(OH)D between groups, and any obstetrical (mater-nal) benefit observed.

Brooke studied 126 severely vitamin D-deficient women, beginning at 28–32 weeks of gestation [47]. At term the mean serum 25(OH)D was 16 nmol/L (6.4 ng/mL) in the placebo group and an exuberant 168 nmol/L (67 ng/mL) in women who nominally received 1000 IU of vitamin D daily but likely received a higher dose than that. A greater weight gain and a small increase in the unadjusted serum calcium were seen in the vitamin D-treated group, but no other obstetrical ben-efit was seen [47]. By contrasting severe vitamin D deficiency with an indisputably vitamin D-replete state, this study was well poised to demonstrate any maternal benefits of vitamin D repletion.

Cockburn randomized 164 women to 400 IU of vitamin D versus placebo beginning at the 12th week of pregnancy. Maternal 25(OH)D reached 42.8 versus 32.5 nmol/L (17 vs. 13 ng/mL) at term, but no obstetrical benefit was reported [150].

Mallet randomized 77 women to receive 1000 IU of vitamin D2 daily during the third trimester, or 200,000 IU of vitamin D2 at the start of the third trimester, or no supplementation [151]. The respective maternal 25(OH)D levels were 25.3, 26.0, and 9.4 nmol/L (10, 10.4, and 3.8 ng/mL) with no effect on mater-nal serum calcium or calcitriol, and no obstetrical benefits reported.

Delvin randomized 40 women in France to 1000 IU vitamin D3 daily versus placebo beginning at 6 months of pregnancy [53]. Maternal 25(OH)D increased to 56 nmol/L (22 ng/mL) versus 27.5 nmol/L (11 ng/mL) in the placebo group, but there was no difference in ionized calcium, serum calcium, calcitriol, or PTH, and no reported obstetrical benefit.

Marya performed two studies in presumed vitamin D-deficient Asian women in India, but no measurements of 25(OH)D were done [152,153]. The first study involved 20 women who received 800,000 IU of vitamin D2 in the seventh and eighth months of pregnancy, 25 women who received 1200 IU of vitamin D2 per day during the third trimester, and 75 women who received nothing [153]. No significant effect on serum calcium or phosphorus was noted. The second study gave 100 women 600,000 IU of vitamin D2 in the seventh and eighth months of pregnancy and compared the results to 100 women who received nothing [152]. The unadjusted serum calcium and phosphorus were slightly but statistically sig-nificantly higher in the women who received vitamin D. No obstetrical benefits were noted.

In the studies cited thus far, there are concerns about blind-ing and randomization methods, small sample sizes, and lack

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of detailed obstetrical data. The studies that follow were car-ried out within the past decade and reported more details about obstetrical outcomes.

Yu randomized 180 women in London to unblinded treat-ment starting at week 27 of pregnancy: 800 IU of vitamin D2 per day, a single dose of 200,000 IU of vitamin D2, or no treat-ment [154]. Mean 25(OH)D increased to 42 and 34 nmol/L (16.8 and 13.6 ng/mL) in the respective treatment groups at term versus 27 nmol/L (10.8 ng/mL) in untreated women. There was no effect on preterm delivery and no other obstetri-cal outcomes.

Roth randomized 160 women in Bangladesh to receive 35,000 IU of vitamin D3 per week or placebo beginning at 26–29 weeks [25]. Maternal 25(OH)D reached 134 nmol/L (53.6 ng/mL) in the supplemented women versus 38 nmol/L (15.2 ng/mL) in the placebo group. There was no effect on maternal serum calcium, but a borderline significant increase in albumin-adjusted calcium and decrease in PTH were found [155]. There was no effect on mode of delivery, C-section rates, adverse events, live versus stillbirths, or ges-tational age at delivery.

Hashemipour randomized 160 women in Iran to receive 50,000 IU vitamin D per week or 400 IU daily beginning at 26–28 weeks [156]. Maternal 25(OH)D reached 120 nmol/L (48 ng/mL) versus 40 nmol/L (16 ng/mL), and the unadjusted serum calcium was modestly higher in the vitamin-D treated women. There was no difference in gestational age of delivery; no other obstetrical outcomes were reported.

Grant randomized 260 women in New Zealand to placebo, 1000 IU vitamin D daily, or 2000 IU vitamin D daily [157]. Maternal 25(OH)D levels at term were 50, 98, and 103 nmol/L (20, 39, and 41 ng/mL), respectively. There was no difference in serum calcium or gestational age of delivery; no other obstetri-cal data were reported [157].

Kalra randomized 97 women in India to one dose of 60,000 IU vitamin D in the second trimester or 120,000 IU vitamin D in each of the second and third trimesters; 43 “usual care” women were the controls. Maternal 25(OH)D levels at term were 26.2, 58.7, and 39.2 nmol/L (10.5, 23.5, and 15.7 ng/mL), respectively [158]. There were no differences in obstetrical outcomes, including gestational age at delivery, intrauterine death, pregnancy-induced hypertension, cephalopelvic dis-proportion, nonprogression of labor, caesarean section, and placenta praevia [158].

Hollis and Wagner reported on 350 women randomized at 12–16 weeks of gestation to receive 400, 2,000, or 4000 IU of vitamin D3 per day [39]. Maternal 25(OH)D levels at term were 79, 98, and 111 nmol/L (31.6, 39.2, and 44.4 ng/mL), respec-tively. There was no significant difference in serum calcium across the treatment groups, but there was a borderline-signif-icant reduction in PTH when the high- and low-dose groups were compared. There were no differences in any obstetrical outcomes, including the mode of delivery, gestational age at delivery, preterm birth, preterm labor, preeclampsia, and infection [39,159].

A second study by Hollis and Wagner reported on 160 women randomized at 12–16 weeks to receive 2000 or 4000

IU of vitamin D3 daily [48]. Achieved maternal 25(OH)D was 90.5 (36.2 ng/mL) and 94.8 nmol/L (37.9 ng/mL), respectively. There was no effect on any obstetrical outcome, including the mode of delivery, preterm labor, preterm delivery, hyperten-sion, infection, gestational diabetes, or combinations of these outcomes [48].

Several post hoc analyses have been done by Hollis and Wagner on the data from these two separate trials, includ-ing analyses in which selective data from both studies were pooled, out of which some borderline significant results have been obtained [39,51,159–161]. These analyses suffer from the lack of adjustment for multiple comparisons, arbitrary group-ing of outcomes, and exclusion of certain ethnic groups from the analysis. For example, the combination of infection/pre-term labor/preterm birth/gestational diabetes/preeclamp-sia/hypertension/HELLP had a P value of .03 (not adjusted for multiple comparisons), whereas excluding preterm labor (which is linked to preterm birth and likely means women were counted twice) resulted in a P value of .06, including all ethnicities resulted in a P value of .17, while multiple other subgroupings and the individual outcomes were not statisti-cally significant [51,159].

Dawodu studied 162 Arab women who had mean 25(OH)D concentrations of 20.5 nmol/L (8.2 ng/mL) at baseline and were randomized to 400, 2,000, or 4000 IU of vitamin D per day [162]. Achieved 25(OH)D levels at delivery were 40 nmol/L (19.3 ng/mL), 65 nmol/L (25.9 ng/mL), and 90 nmol/L (35.9 ng/mL), respectively [162]. There were no differences in maternal serum calcium or urine calcium/creatinine between groups at any time point during pregnancy, PTH was reduced in the high-dose group but correlated poorly with 25(OH)D, no effect was seen on gestational age at delivery, and no obstet-rical outcomes were reported.

The most recent study was MAVIDOS, carried out by Cooper and Harvey in the United Kingdom [163]. The 900 women who completed the study randomly received either 1000 IU of vitamin D or placebo beginning at 14 weeks of pregnancy. Baseline 25(OH)D was 45 nmol/L (18 ng/mL), did not change in placebo-treated women, and rose to 68 nmol/L (27 ng/mL) near term in vitamin D-supplemented women. The primary and secondary out-comes were related to the neonates (see Chapter 39). There was no effect of supplementation on maternal hyperten-sion, preterm delivery, instrumental delivery, postpartum hemorrhage, intrauterine growth restriction, or intrauterine and neonatal death.

Overall, these clinical trials largely examined women who had 25(OH)D values well above the 20 nmol/L threshold that has been shown to result in normal intestinal calcium absorp-tion. Instead, various levels of vitamin D repleteness have been compared rather than vitamin D replete to deficient. Only Brooke’s study compared indisputable vitamin D deficiency throughout pregnancy to a vitamin D-replete state, with no obstetrical outcome differences reported. Dawodu’s study began with vitamin D-deficient women, but by delivery the mean 25(OH)D level was in the insufficient range at 40 nmol/L, and no obstetrical benefit was seen [162]. Therefore, the clinical

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IU of vitamin D3 daily [48]. Achieved maternal 25(OH)D was 90.5 (36.2 ng/mL) and 94.8 nmol/L (37.9 ng/mL), respectively. There was no effect on any obstetrical outcome, including the mode of delivery, preterm labor, preterm delivery, hyperten-sion, infection, gestational diabetes, or combinations of these outcomes [48].

Several post hoc analyses have been done by Hollis and Wagner on the data from these two separate trials, includ-ing analyses in which selective data from both studies were pooled, out of which some borderline significant results have been obtained [39,51,159–161]. These analyses suffer from the lack of adjustment for multiple comparisons, arbitrary group-ing of outcomes, and exclusion of certain ethnic groups from the analysis. For example, the combination of infection/pre-term labor/preterm birth/gestational diabetes/preeclamp-sia/hypertension/HELLP had a P value of .03 (not adjusted for multiple comparisons), whereas excluding preterm labor (which is linked to preterm birth and likely means women were counted twice) resulted in a P value of .06, including all ethnicities resulted in a P value of .17, while multiple other subgroupings and the individual outcomes were not statisti-cally significant [51,159].

Dawodu studied 162 Arab women who had mean 25(OH)D concentrations of 20.5 nmol/L (8.2 ng/mL) at baseline and were randomized to 400, 2,000, or 4000 IU of vitamin D per day [162]. Achieved 25(OH)D levels at delivery were 40 nmol/L (19.3 ng/mL), 65 nmol/L (25.9 ng/mL), and 90 nmol/L (35.9 ng/mL), respectively [162]. There were no differences in maternal serum calcium or urine calcium/creatinine between groups at any time point during pregnancy, PTH was reduced in the high-dose group but correlated poorly with 25(OH)D, no effect was seen on gestational age at delivery, and no obstet-rical outcomes were reported.

The most recent study was MAVIDOS, carried out by Cooper and Harvey in the United Kingdom [163]. The 900 women who completed the study randomly received either 1000 IU of vitamin D or placebo beginning at 14 weeks of pregnancy. Baseline 25(OH)D was 45 nmol/L (18 ng/mL), did not change in placebo-treated women, and rose to 68 nmol/L (27 ng/mL) near term in vitamin D-supplemented women. The primary and secondary out-comes were related to the neonates (see Chapter 39). There was no effect of supplementation on maternal hyperten-sion, preterm delivery, instrumental delivery, postpartum hemorrhage, intrauterine growth restriction, or intrauterine and neonatal death.

Overall, these clinical trials largely examined women who had 25(OH)D values well above the 20 nmol/L threshold that has been shown to result in normal intestinal calcium absorp-tion. Instead, various levels of vitamin D repleteness have been compared rather than vitamin D replete to deficient. Only Brooke’s study compared indisputable vitamin D deficiency throughout pregnancy to a vitamin D-replete state, with no obstetrical outcome differences reported. Dawodu’s study began with vitamin D-deficient women, but by delivery the mean 25(OH)D level was in the insufficient range at 40 nmol/L, and no obstetrical benefit was seen [162]. Therefore, the clinical

trial data do not support an obvious need for higher 25(OH)D levels during pregnancy with respect to maternal outcomes, but the lack of inclusion of vitamin D-deficient women is a significant limiting factor in what can be concluded from these results.

Genetic Disorders of Vitamin D PhysiologyWith respect to genetic disorders of vitamin D physiology,

pregnancies have been described as unremarkable in women with genetic deficiency of Cyp27b1 (vitamin D-dependent rickets type 1, VDDR1) or inactive VDRs (VDDR2) [164–166]. In one case of VDDR2, the pregnancy was unremarkable while the woman was maintained on her prepregnancy doses of calcium (800 mg) and high-dose calcitriol [165]. The clini-cians did increase the dose of calcitriol during that pregnancy “because of the knowledge that the circulating 1,25-(OH)2D concentration normally rises during pregnancy,” but appar-ently not because of any change in the albumin-adjusted serum calcium [165]. In multiple cases of VDDR1 the dose of calcitriol was unchanged in one-third of pregnancies and increased 1.5- to 2-fold in others, but the increases were not always objectively driven by a fall in the albumin-corrected serum calcium or hypocalcemic symptoms [1,164]. Instead, the physiological decline in serum calcium during pregnancy clearly led to some of the increases in supplemental calcium and/or calcitriol. In the normal management of both dis-orders during pregnancy, maternal hypocalcemia must be avoided due to the adverse effects it will have on the fetus [1]. This may require adjustments in calcium, calcitriol, or 1α-cholecalciferol to maintain a normal ionized or albumin-corrected serum calcium.

Genetic absence of Cyp24a1 leads to maternal hypercal-cemia during pregnancy [167,168]. Since calcitriol normally increases two- to three-fold during pregnancy, a reduced rate of catabolism enables relatively unopposed actions of the active hormone to stimulate intestinal calcium absorption and possibly osteoclast-mediated bone resorption.

Studies of Associations Between Obstetrical Outcomes and Vitamin D Status

Associational studies have suggested the possibility that vitamin D deficiency may have nonskeletal effects during pregnancy, such as increasing the risk of preterm delivery, C-sections, low birth weight, preeclampsia/preg-nancy-induced hypertension, and vaginal infections. One can find approximately as many studies suggesting these associa-tions [169–174] as there are studies indicating no association [11,175–180]. These studies generally have low power and are confounded by factors that may lead to lower 25(OH)D lev-els and to the outcome in question, including race/ethnicity, maternal overweight/obesity, lower socioeconomic status, poor nutrition, etc. For example, maternal overweight/obe-sity is a significant risk factor for preterm delivery, C-sections, low birth weight, preeclampsia/pregnancy-induced hyper-tension, vaginal infections, and other adverse obstetrical

outcomes [181]. But maternal overweight/obesity also cause low 25(OH)D by binding the fat-soluble vitamin D into the maternal fat stores and by additional associations with reduced vitamin D intake, less time spent outdoors, etc. Consequently, the studies reporting associations between obstetrical outcomes and vitamin D intake or maternal 25(OH)D levels may be demonstrating residual confounding from the links between obesity and these outcomes. To elimi-nate the confounding, large randomized trials that compare truly vitamin D-deficient to vitamin D-sufficient mothers are needed. However, such studies are unlikely to be carried out because of the general assumption that vitamin D defi-ciency may be harmful during pregnancy and that it would be unethical to leave women untreated.

OVERVIEW OF MINERAL PHYSIOLOGY DURING LACTATION AND POSTWEANING

RECOVERY

The neonatal requirement for mineral, as supplied through breast milk, has been determined from studies in which healthy neonates were weighed before and after each feed. These studies revealed an average daily intake of 780 mL of human milk [182–185]. The average calcium con-tent of milk is 260 mg/L during the first 6 months postpar-tum [186], which means that the average neonate consumes about 200 mg of calcium per day. Metabolic balance studies have determined that neonates absorb 60%–70% of calcium from human milk [187–190] and that it is facilitated by lac-tose and proportional to calcium intake [191,192]. This means that 120–140 mg of calcium is available for skeletal accretion each day in the average breast-fed baby. Fractional calcium absorption is significantly lower at 30%–40% of intake when formula (which has twice the calcium content of human milk) is consumed [193].

The Institute of Medicine used these calculations to determine that breast-feeding requires an average of 200 mg of calcium to be provided daily through milk to a singleton during the first 6 months, from which the neonatal skeleton will accrete about 100 mg of calcium daily [7]. On an indi-vidual basis the suckling demands of a neonate can mark-edly exceed these average values. Women who nurse twins and triplets have, respectively, more than double and triple the milk output and calcium losses of women who nurse singletons [194,195]. Moreover, individual women have been documented with daily milk outputs that average 2.4–3.1 L per day for more than 12 months of lactation [196,197]. The composition of milk is similar in women with average and high outputs [194,195], and so when greater volumes of milk are produced, there will be greater maternal losses of calcium.

Between 6 and 12 months of age, the infant normally consumes solid foods, and the proportion of nutrition coming from breast milk is usually less despite continued breast-feeding. The average calcium content of human milk during this interval is somewhat lower at 200 mg/L

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[186], and the output is reduced to an average of 600 mL/day [198]. This corresponds to the infant obtaining about 120 mg/day of calcium from human milk. An additional 140 mg/day of calcium comes from solid foods, for a total calcium intake of ∼260 mg/day [7]. The infant’s skeleton is expected to continue accreting about 100 mg per day dur-ing this interval [7].

As noted in the opening section about pregnancy, average daily intakes of calcium in reproductive age women would be insufficient to meet the combined needs of the mother and neonate in the setting of normal rates of intestinal cal-cium absorption. But in contrast to the doubling of intesti-nal calcium absorption that is invoked during pregnancy, a normal (nonpregnant) rate of absorption persists during lactation. Instead, the physiological adaptation to lactation is that the skeleton is resorbed by osteoclasts and probably osteocytes too, to provide the necessary calcium and other minerals. Greater milk output increases the magnitude of skeletal losses, which are also independent of maternal cal-cium intake. Although this physiological adaptation tempo-rarily reduces bone mineral density and skeletal strength, it does not appear to increase the long-term risk of osteoporosis and fractures.

Minerals and Calciotropic HormonesTypical mean changes that occur in serum minerals and

hormones during lactation are depicted in Fig. 42.3. The albumin-corrected serum calcium and ionized calcium remain normal, although longitudinal studies of exclu-sively lactating women have shown that these values do increase modestly while usually remaining within the nor-mal range [1]. Some otherwise normal women do become hypercalcemic while lactating [1]. Serum phosphorus increases and can exceed the normal range, likely because release of phosphorus from the skeleton overloads the abil-ity of the kidneys to excrete that filtered load.

Intact PTH is usually low or undetectable [11,13,199–209] during the first several months of exclusive lactation, that is, wherein all of the baby’s nutrition comes from breast milk. As the exclusivity and intensity of lactation lessens, PTH increases to normal [1]. In several studies, PTH increased above the nor-mal range after the baby was weaned [13,20,202,210]. These descriptions about changes in PTH during lactation apply to women from North America and Europe, whereas in some studies from women from Asia and Africa, PTH did not sup-press or even increased above normal [27,28,211–213]. As with what has been reported for some pregnant women from Asia and Africa (noted earlier), this may be due to a higher preva-lence of dietary calcium deficiency and higher dietary intake of phytate.

Free and total calcitriol concentrations are normal dur-ing lactation [13,20,28,34,36,201,202,207,208,210,214–216], in contrast to the two-fold to three-fold increased levels that are seen during pregnancy. Calcitriol levels were even higher in women nursing twins in one study, [217] whereas there was no difference between women with singletons or twins in another study [44]. A few small studies found that calcitriol

increased above normal during the postweaning interval [202,209].

Maternal 25(OH)D concentrations are not affected by up to 12 months of breast-feeding in most studies [19,28,36,202,204,207,208,210,216,218–222]. This is consistent with the fact that very little vitamin D and 25(OH)D normally escapes into milk.

PTHrP is present in breast milk at 1000 to 10,000 times the concentration found in patients with hypercalcemia of malig-nancy [59,223–227]. The inherent problems with measuring PTHrP in serum versus plasma, and limitations of the avail-able assays, apply to published studies of lactating women [1]. Despite these problems, most studies have found the circulating PTHrP1-86 or PTHrP1-34 level to be significantly increased in lactating women versus nonpregnant women or bottle-feeding controls [199,200,203,204,228]. A midregion sequence (PTHrP63-77) was also increased in lactating women [229]. PTHrP increases further with each suckling episode [203,204,230]. PTHrP concentrations correlate positively with ionized calcium and negatively with PTH and loss of bone density in lactating women [200,203,228]. Hypercalcemia dur-ing lactation has been shown to result from excess production of PTHrP by the breasts (a condition called pseudohyperpara-thyroidism) [1]. PTHrP declines during the postweaning inter-val [204] and becomes undetectable sometime in the weeks to months after weaning.

Calcitonin levels are increased above the normal range dur-ing the first 6 weeks of lactation, after which it may stay elevated [15,231] or return to normal [20,36,207,214,217]. Women nursing twins had higher serum calcitonin than those who nursed single-tons [217]. An increase in calcitonin is conceivably a physiologi-cally significant change that helps prevent excessive resorption of the maternal skeleton during lactation. Certainly, deletion of the gene encoding calcitonin caused mice to lose twice the bone mineral content during lactation as their WT sisters [232,233], but there are no human data. Women who lack calcitonin (the equivalent of a null mutation) may be predicted to lose more bone mass than normal during lactation and have increased risk of vertebral compression fractures. Thyroidectomized women are not calcitonin deficient due to substantial production of cal-citonin by lactating breast tissue [234–236].

No measurements of FGF23 have been reported in breast-feeding women.

Lactating women characteristically have increased prolactin with reduced estradiol and progesterone. Loss of the placenta causes a fall in progesterone and estradiol, which in turn trig-ger lactogenesis [237,238]. Failure to drain the breasts of milk (milk stasis) prompts apoptosis of mammary cells and lacta-tion ceases. Estradiol is reduced to menopausal levels during early lactation [36,73,207,208,239], especially when basal pro-lactin is at its highest [73,239]. Low estradiol may be present throughout lactation or return to normal values after several months, with the course being determined in large part by the intensity and exclusivity of lactation. Basal prolactin concen-trations gradually decline over succeeding weeks but continue to spike higher with each suckling episode [73,207,237,239]. Oxytocin similarly spikes in the maternal circulation when the baby suckles [237,240].

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Intestinal Absorption of CalciumIntestinal calcium absorption is normal in lactating

women [9,36,79–81,241–243], and this change appears to coincide with calcitriol falling to normal (nonpregnant) levels. Adding 1 g of calcium or placebo daily did not alter the fractional absorption of calcium in lactating women [242]. Intestinal calcium absorption is somewhat higher in women whose menses have resumed while they are still lactating [242].

Lactating women evidently do not need increased efficiency of intestinal calcium absorption to meet the calcium require-ments of milk production. This is supported by evidence from randomized trials and observational studies. Increased dietary intake of calcium does not reduce skeletal resorption during lactation, nor does it alter breast milk calcium content [201,244–248].

During the postweaning interval, intestinal calcium absorp-tion increased 20% in one study [242] but did not change sig-nificantly in another study [36].

Renal Handling of CalciumIn lactating women renal calcium excretion decreases such

that hypocalciuria has been documented in 24-hour urine col-lections [13,15,16,20,36,81,82,209,210,212,213,241,249]. PTH is often undetectable, and this would normally result in hyper-calciuria. It is likely the high concentrations of PTHrP that are responsible for increased renal tubular reabsorption of calcium, combined with the output of calcium into milk (the “open drain”) that in turn reduces the renal filtered load of calcium. Urine phosphorus excretion is increased [13,201,209,210,213,241,249], which may result from the phosphaturic actions of PTHrP and the increased filtered load of phosphorus.

The relative hypocalciuria and renal calcium conserva-tion persists during the postweaning interval in some studies [13,204,210], whereas the tubular maximum for phosphorus has been shown to decline to normal [13,201,204,210].

Calcium Pumping in the Breast and Milk Formation

Fig. 42.4 depicts the current understanding of how calcium enters mammary epithelial cells and is eventually secreted into milk [1]. It enters the basolateral membranes of mammary epithelial cells via stretch-activated and other calcium chan-nels. PMCA1 actively pumps calcium into the Golgi appa-ratus, wherein calcium becomes bound to proteins (casein and α-lactalbumin) or complexed to phosphate and citrate. Calcium is also carried by calbindin-D9k and other proteins within the cytoplasm [250] and transported directly to the api-cal membrane. The Golgi’s transepithelial secretion of calcium into milk [238,251] accounts for about 30% of milk calcium content. The other 70% of calcium in milk derives from plasma membrane calcium ATPase isoform 2 (PMCA2) pumping cal-cium across the apical membranes [252,253].

Animal studies have shown that the calcium and fluid con-tent of milk are tightly regulated by such factors as suckling, the calcium receptor, PTHrP, prolactin, calcitriol, and others

FIGURE 42.3 Schematic depiction of longitudinal changes in calcium, phosphorus, and calciotropic hormone levels during lactation and post-weaning skeletal recovery in women. Normal adult values are indicated by the shaded areas. PTH does not decline in women with low calcium or high phytate intakes and may even rise above normal. Calcidiol [25(OH)D] values are not depicted; most longitudinal studies indicate that the lev-els are unchanged by lactation but may vary due to seasonal variation in sunlight exposure and changes in vitamin D intake. PTHrP and prolactin surge with each suckling episode, and this is represented by upward spikes. FGF23 values cannot be plotted due to lack of data. Very limited data sug-gest that calcitriol and PTH may increase during postweaning, and the lines are dashed to reflect the uncertainty. FGF23, fibroblast growth factor-23; PTH, parathyroid hormone; PTHrP, PTH-related protein. Reproduced with permission from Kovacs CS. Maternal mineral and bone metabolism during preg-nancy, lactation, and post-weaning recovery. Physiol Rev 2016;96:449–547.

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[237,250,254,255] (Fig. 42.5). Lactating mammary epithelial cells also express the calcium receptor, PTHrP, calcitonin, the calcitonin receptor, Cyp27b1, VDR, and calbindin-D9k; how-ever, TRPV5 and TRPV6 are not expressed [126,255,256]. As discussed in more detail elsewhere [1], the calcium receptor

controls the calcium content of milk by inhibiting PTHrP and stimulating PMCA2. PMCA2 may also be locally regu-lated by PTHrP, serotonin, and calcitriol. PTHrP is intensely expressed by mammary epithelial cells, from where it reaches the maternal circulation to stimulate systemic bone

FIGURE 42.4 Calcium transport across mammary epithelial cells into milk. The basolateral (left) and apical (right) basement membranes of a mammary epi-thelial cell are depicted. Calcium enters the cell through channels that have not been defined; TRPV6 and TRPV5 are not expressed. About 20%–30% of calcium destined for secretion into milk is pumped into the Golgi apparatus via PMCA1, wherein it is packaged into secretory granules containing proteins (casein and α-lactalbumin) and complexes of calcium with phosphate and citrate. These granules are extruded from the Golgi apparatus into milk through transepithelial secretion at the apical membrane. About 70%–80% of calcium entering the cell becomes bound to carrier proteins such as calbindin-D9k and is shuttled to the apical membrane in the tran-scellular pathway, from where PMCA2 actively pumps the calcium into milk. Calcium ions binding to the calcium receptor on the basolateral membrane inhibit PTHrP and stimulate PMCA2 to pump calcium into milk. Calcitonin may also influence these processes, perhaps through regulation by the calcium receptor (which regulates calcitonin in C-cells of the thyroid), since the absence of calcitonin causes upregulation of PTHrP within mammary epithelial cells, and milk calcium concentration is also increased. PTHrP is released into milk at higher concentrations than it is released into the maternal circulation. PMCA1, plasma membrane calcium ATPase isoform 1; PMCA2, plasma membrane calcium ATPase isoform; PTHrP, PTH-related protein; TRPV5, transient receptor potential cation channel subfamily V member 5; TRPV6, transient receptor potential cation channel subfamily V member 6. Reprinted with permission from Kovacs CS. Control of mineral and skeletal homeostasis during pregnancy and lactation. In: Thakker RV, Whyte MP, Eisman JA, Igarashi T, editors. Genetics of bone biology and skeletal disease. 2nd ed. San Diego: Academic Press/Elsevier; 2017 [in press], © 2017 Elsevier.

FIGURE 42.5 The role of PTHrP and calcium receptor (CaSR) within the lactating breast. The calcium receptor (represented schematically) is expressed by lactating mammary epithelial cells. It monitors the systemic concentration of calcium to control PTHrP synthesis and, thereby, the supply of calcium to the breast. An increase in serum calcium or administration of a calcimimetic inhibits PTHrP expression (A), whereas a decrease in serum calcium or ablation of the calcium receptor from mammary epithelial cells stimulates PTHrP expression (B). The calcium receptor also directly regulates the calcium and fluid composition of milk independent of PTHrP. Administration of a calcimimetic stimulates calcium and water transport into the breast, whereas ablation of the calcium receptor results in low milk calcium despite increased PTHrP and systemic hypercalcemia. PTHrP produced by mammary epithelial cells enters the maternal circulation to stimulate maternal bone resorption and renal calcium conservation. It also enters milk at 1000 to 10,000-fold higher concentrations, from where it may influence neonatal accrual of calcium. PTHrP, PTH-related protein. Reprinted with permission from Kovacs CS. Control of mineral and skeletal homeostasis during pregnancy and lactation. In: Thakker RV, Whyte MP, Eisman JA, Igarashi T, editors. Genetics of bone biology and skeletal disease. 2nd ed. San Diego: Academic Press/Elsevier; 2017 [in press], © 2017 Elsevier.

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resorption and renal tubular conservation of calcium. In this way, PTHrP serves to supply the calcium needed to support milk production.

The regulation of milk calcium content has not been exten-sively studied in women. The PTHrP concentration in milk [225,257,258] and in the maternal circulation [257] correlates positively with total milk calcium content. A randomized trial found no effect of calcium supplementation on milk PTHrP content [227]. Diets that are high [201,244–246] or low [247,248,259,260] in calcium, or high [261] or low [260] in vita-min D, also do not affect milk calcium content.

Milk normally contains very little vitamin D or 25(OH)D, which explains why breast-feeding is a risk factor for vitamin D deficiency in exclusively breast-fed infants that receive no sun-light exposure [2]. This fact is often disbelieved because milk is generally considered a good source of vitamin D, but that is because commercial milk from cows and goats is fortified with vitamin D after pasteurization. Why does milk normally con-tain so little of vitamin D or its metabolites? Humans do not develop vitamin D toxicity from sunlight exposure because there is limited substrate produced in the skin each day, but it is possible to become vitamin D toxic from overingestion of vitamin D supplements, vitamin D-fortified foods, or vitamin D-rich fat/blubber. Therefore, a teleological argument is that the neonate preferentially obtains vitamin D from sunlight exposure, with the milk content of vitamin D kept low to pre-vent excess vitamin D intake by the baby.

Skeletal Metabolism During LactationMetabolic studies have revealed that women are in a

markedly negative calcium balance while breast-feeding, especially when milk output is greatest, and despite any

supplemental calcium intake [262,263]. Much of the calcium content of milk is resorbed from the maternal skeleton dur-ing lactation, with the excess phosphorus excreted in the urine.

This skeletal resorption has been well established in ani-mal models. During 3 weeks of lactation, bone resorption markers are markedly elevated. Rodents lose 20%–30% of ash weight, ash mineral content, and BMC or BMD as assessed by DXA [1]. Increased osteoclast number and sur-face, and osteocytic osteolysis, have been demonstrated by histomorphometry (Fig. 42.6), whereas microCT mea-surements have shown the resorption of trabecular bone and progressive thinning and porosity of cortical bone [1]. Skeletal strength (3-pt bend test of tibiae or femorae, and vertebral crush test) is reduced during lactation [1]. After weaning, these parameters (including skeletal strength) return to normal, although some microarchitectural changes persist in the tibiae or femora [1].

There are no comparable histomorphometric data from lac-tating women; instead, serial measurement of bone turnover markers, bone density, and bone structure by high-resolution peripheral QCT (HR-pQCT) has been carried out to confirm that skeletal resorption occurs.

Bone resorption markers (NTX, CTX, tartrate-resistant acid phosphatase, deoxypyridinoline/creatinine, and hydroxypro-line/creatinine) are elevated several fold during early lacta-tion [1]. Bone formation markers (P1NP, bone-specific alkaline phosphatase, and osteocalcin) have been normal in a few stud-ies but generally are also increased during lactation [1]. Total alkaline phosphatase falls at delivery due to loss of the placen-tal fraction but may remain elevated due to the bone-specific fraction [1]. These values indicate that bone turnover is signifi-cantly increased during lactation.

FIGURE 42.5 The role of PTHrP and calcium receptor (CaSR) within the lactating breast. The calcium receptor (represented schematically) is expressed by lactating mammary epithelial cells. It monitors the systemic concentration of calcium to control PTHrP synthesis and, thereby, the supply of calcium to the breast. An increase in serum calcium or administration of a calcimimetic inhibits PTHrP expression (A), whereas a decrease in serum calcium or ablation of the calcium receptor from mammary epithelial cells stimulates PTHrP expression (B). The calcium receptor also directly regulates the calcium and fluid composition of milk independent of PTHrP. Administration of a calcimimetic stimulates calcium and water transport into the breast, whereas ablation of the calcium receptor results in low milk calcium despite increased PTHrP and systemic hypercalcemia. PTHrP produced by mammary epithelial cells enters the maternal circulation to stimulate maternal bone resorption and renal calcium conservation. It also enters milk at 1000 to 10,000-fold higher concentrations, from where it may influence neonatal accrual of calcium. PTHrP, PTH-related protein. Reprinted with permission from Kovacs CS. Control of mineral and skeletal homeostasis during pregnancy and lactation. In: Thakker RV, Whyte MP, Eisman JA, Igarashi T, editors. Genetics of bone biology and skeletal disease. 2nd ed. San Diego: Academic Press/Elsevier; 2017 [in press], © 2017 Elsevier.

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Numerous longitudinal studies of breast-feeding women have confirmed a consistent decline in BMD or BMC occurs that is proportional to the intensity and duration of lactation. The greatest losses occur in the trabecular-rich spine and reach 5%–10% after 6 months, whereas more modest (0%–5%) losses occur at sites with more modest trabecular content (hip, femur, and distal radius), and the smallest (0%–2%) losses occur at purely cortical sites such as the whole body [1]. From all these studies reporting mean losses for the respective cohorts, it appears that the median loss from the lumbar spine is about 6%–8% of BMD, with half or less that amount lost from the appendicular skeleton [1]. Skeletal changes of individual women have ranged from a small gain to a 20% decline in BMD of the lumbar spine [88,264], and some women reach an osteoporotic level of BMD despite starting pregnancy with normal BMD [88].

HR-pQCT has been used in very few studies to date, and the technology cannot assess the spine, which is the site of the most intense resorption. The sites that can be assessed are appendicular ones, such as the radius and distal femur. These studies found 0%–2% reductions in trabecular thickness, and cortical thickness and volume, with the losses being even greater in those who lactate longer [265,266]. Trabecular num-ber and volumetric BMD appeared to increase, which may be an artifact from trabecularization of cortical bone [265,266].

Lactational bone loss is not reduced or prevented by increased intake of calcium [201,244–246], nor does low cal-cium intake accentuate bone loss or alter milk calcium content [247,248,259,260]. Women who breastfeed for a year or more may experience greater bone loss [267–269]. These observa-tions are consistent with most of the calcium content of milk coming from resorption of the mother’s skeleton and not from dietary calcium intake. This skeletal resorption is programmed by hormonal changes that occur during lactation, with greater loss of bone mass predicted by more intense lactation or greater breast milk output [270].

The skeletal resorption is understood to be driven, at least in part, by relative estradiol deficiency and the increased circulating concentrations of PTHrP (Fig. 42.7) [1]. Increased concentrations of prolactin, and suckling itself, act on the gonadotropin-releasing hormone (GnRH) pulse center in the hypothalamus to inhibit ovarian func-tion and ovulation, thereby leading to low sex steroid levels [1]. However, low estradiol is not the main cause of bone loss during lactation because it typically causes only 1%–2% annual losses of bone mass in recently menopausal women [271] or in young women who have been treated with GnRH analogs to induce marked estradiol deficiency (Fig. 42.8) [1]. This is contrasted with the 5%–10% reductions in bone mass that occur during 6 months of exclusive lactation. During lactation, PTHrP and low estradiol combine to stimulate osteoclast-mediated bone resorption and osteocytic oste-olysis (Fig. 42.6). Other hormones such as oxytocin, sero-tonin, and IGF-I may contribute to the regulation of skeletal metabolism during lactation, but there are no human data at present [1].

Skeletal Metabolism During PostweaningAfter weaning, bone resorption subsides and bone forma-

tion upregulates. Longitudinal DXA studies suggest that lacta-tional losses of BMD are restored by 12 months after weaning, whereas recovery is incomplete when assessed at 6 months or earlier after weaning [1]. HR-pQCT of the radius and ultradistal femur found recovery of trabecular microarchitecture and corti-cal parameters in women who lactate for shorter intervals but incomplete recovery in those who lactated for longer [265,266]. The follow-up may not have been long enough to determine if permanent loss of bone microarchitecture occurred. Some clini-cal studies found that the cross-sectional diameter of the femur was higher after lactation or postweaning recovery and that cor-tical bone area is normal or increased [272,273].

FIGURE 42.6 Osteocytic osteolysis and osteoclast-mediated bone resorption. (A) Quiescent bone with an osteocyte surrounded by its lacuna and canaliculi (gray halo and tentacles); (B) lactation with an osteoclast resorbing bone (resorption pit) while an osteocyte resorbs mineral from its lacuna and pericanalicular spaces (surrounding white regions); (C) postweaning phase, during which osteoblasts restore bone in areas previously resorbed by osteoclasts (hatched resorption pit) and osteocytes remineralize their lacuna and pericanalicular spaces (surrounding black regions). Evidence from osteocyte-specific ablation of the PTH/PTHrP receptor in mice suggests that bone resorption and osteocytic osteolysis may each account for about 50% of the mineral lost from the skeleton during lactation. PTH, parathyroid hormone; PTHrP, PTH-related protein. Reprinted with kind permission from Kovacs CS, Ralston SH. Presentation and management of osteoporosis presenting in association with pregnancy or lactation. Osteoporos Int 2015;26:2223–41, © 2015, Springer Science and Business Media B.V.

FIGURE 42.7 Breast-Brain-Bone circuit controls lactation. Suckling and prolactin (PRL) both inhibit the hypothalamic gonadotropin-releasing hor-mone (GnRH) pulse center, which in turn suppresses the gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]), leading to low levels of the ovarian sex steroids (estradiol [E2] and progesterone [PROG]). Prolactin may also have direct effects on its receptor in bone cells. PTHrP production and release from the breast are stimulated by suckling, prolactin, low estradiol, and the calcium receptor. PTHrP enters the bloodstream and combines with systemically low estradiol levels to markedly upregulate bone resorption and (at least in rodents) osteocytic osteolysis. Increased bone resorption releases calcium and phosphate into the blood stream, which then reaches the breast ducts and is actively pumped into the breast milk. PTHrP also passes into milk at high concentrations, but whether swallowed PTHrP plays a role in regulating calcium physiology of the neonate is uncertain. In addition to stimulating milk ejection, oxytocin (OT) may directly affect osteoblast and osteoclast function (dashed arrow). Calcitonin (CT) may inhibit skeletal responsiveness to PTHrP and low estradiol given that mice lacking calcitonin lose twice the amount of bone during lactation as normal mice. Not depicted is that calcitonin may also act on the pituitary to suppress prolactin release and within breast tissue to reduce PTHrP expression and lower the milk calcium content (see text). PTHrP, PTH-related protein. Adapted with kind permission from reference Kovacs CS. Calcium and bone metabolism during pregnancy and lactation. J Mammary Gland Biol Neoplasia 2005;10:105–118, © 2005, Springer Science and Business Media B.V.

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The skeletal resorption is understood to be driven, at least in part, by relative estradiol deficiency and the increased circulating concentrations of PTHrP (Fig. 42.7) [1]. Increased concentrations of prolactin, and suckling itself, act on the gonadotropin-releasing hormone (GnRH) pulse center in the hypothalamus to inhibit ovarian func-tion and ovulation, thereby leading to low sex steroid levels [1]. However, low estradiol is not the main cause of bone loss during lactation because it typically causes only 1%–2% annual losses of bone mass in recently menopausal women [271] or in young women who have been treated with GnRH analogs to induce marked estradiol deficiency (Fig. 42.8) [1]. This is contrasted with the 5%–10% reductions in bone mass that occur during 6 months of exclusive lactation. During lactation, PTHrP and low estradiol combine to stimulate osteoclast-mediated bone resorption and osteocytic oste-olysis (Fig. 42.6). Other hormones such as oxytocin, sero-tonin, and IGF-I may contribute to the regulation of skeletal metabolism during lactation, but there are no human data at present [1].

Skeletal Metabolism During PostweaningAfter weaning, bone resorption subsides and bone forma-

tion upregulates. Longitudinal DXA studies suggest that lacta-tional losses of BMD are restored by 12 months after weaning, whereas recovery is incomplete when assessed at 6 months or earlier after weaning [1]. HR-pQCT of the radius and ultradistal femur found recovery of trabecular microarchitecture and corti-cal parameters in women who lactate for shorter intervals but incomplete recovery in those who lactated for longer [265,266]. The follow-up may not have been long enough to determine if permanent loss of bone microarchitecture occurred. Some clini-cal studies found that the cross-sectional diameter of the femur was higher after lactation or postweaning recovery and that cor-tical bone area is normal or increased [272,273].

FIGURE 42.7 Breast-Brain-Bone circuit controls lactation. Suckling and prolactin (PRL) both inhibit the hypothalamic gonadotropin-releasing hor-mone (GnRH) pulse center, which in turn suppresses the gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]), leading to low levels of the ovarian sex steroids (estradiol [E2] and progesterone [PROG]). Prolactin may also have direct effects on its receptor in bone cells. PTHrP production and release from the breast are stimulated by suckling, prolactin, low estradiol, and the calcium receptor. PTHrP enters the bloodstream and combines with systemically low estradiol levels to markedly upregulate bone resorption and (at least in rodents) osteocytic osteolysis. Increased bone resorption releases calcium and phosphate into the blood stream, which then reaches the breast ducts and is actively pumped into the breast milk. PTHrP also passes into milk at high concentrations, but whether swallowed PTHrP plays a role in regulating calcium physiology of the neonate is uncertain. In addition to stimulating milk ejection, oxytocin (OT) may directly affect osteoblast and osteoclast function (dashed arrow). Calcitonin (CT) may inhibit skeletal responsiveness to PTHrP and low estradiol given that mice lacking calcitonin lose twice the amount of bone during lactation as normal mice. Not depicted is that calcitonin may also act on the pituitary to suppress prolactin release and within breast tissue to reduce PTHrP expression and lower the milk calcium content (see text). PTHrP, PTH-related protein. Adapted with kind permission from reference Kovacs CS. Calcium and bone metabolism during pregnancy and lactation. J Mammary Gland Biol Neoplasia 2005;10:105–118, © 2005, Springer Science and Business Media B.V.

FIGURE 42.8 Schematic depiction of the effects of acute, isolated estradiol deficiency from GnRH analog therapy versus lactation. Acute estrogen defi-ciency (e.g., GnRH analog therapy) increases skeletal resorption and raises the blood calcium; in turn, PTH is suppressed and renal calcium losses are increased. During lactation, the combined effects of PTHrP (secreted by the breast) and estrogen deficiency increase skeletal resorption, reduce renal calcium losses, and raise the blood calcium, but calcium is directed into breast milk. PTH, parathyroid hormone; PTHrP, PTH-related protein. Reprinted from Kovacs CS, Kronenberg HM. Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocr Rev 1997;18:832–72, © 1997, The Endocrine Society.

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Ovarian function and estradiol return to normal, which facilitates but does not fully explain postweaning recovery. Young women who lost BMD due to GnRH analog-induced estradiol deficiency did not recover the BMD after a year of normal menstrual function, whereas women do recover from the skeletal losses induced by lactation [1]. Furthermore, estra-diol suppresses bone formation and resorption when admin-istered as replacement therapy to estrogen-deficient women [274,275]. The factors that stimulate prompt skeletal recovery remain to be elucidated.

In rodents, forced weaning provokes widespread osteo-clast apoptosis within 24 hours [276,277]. This is followed by increased osteoblast activity and a surge in osteoblast num-bers [233,276], during which osteocytes now behave like osteo-blasts, laying down osteoid within their perilacunar matrices [278,279]. PTH, PTHrP, calcitriol, calcitonin, and estradiol are not required for skeletal recovery to be fully achieved in rodents [1,41,117,232,280]. The identity of the factors that stim-ulate this active phase of bone formation remains unknown but is the subject of current research [233].

As noted, although skeletal recovery in women appears to be complete by DXA of the spine, hip, and whole body, the skeletal microarchitecture may not be completely restored at all sites as determined by microCT of long bones [266,281]. In some studies of humans and animals the cross-sectional diameters of the long bones have increased by the end of lac-tation or postweaning recovery, whereas cortical bone area is restored after weaning to prepregnant values [273,282,283]. Such changes in bone diameters and cortical bone area may explain why DXA indicates full recovery despite μCT and his-tomorphometry showing that the trabecular microarchitecture does not recover fully. Increased bone diameters may compen-sate for the loss of trabecular microarchitecture and, thereby, maintain bone strength. A wider hollow cylinder is stronger than a narrower one [90].

Women who lactate for shorter durations recover bone mass sooner, likely because they lost less bone. Full recov-ery also occurs in women who lactate for much longer inter-vals or who become pregnant again without a postlactation recovery interval [267–269]. Vertebral compression frac-tures can occur rarely during lactation [90], but subsequent recovery of bone mass makes lactational bone loss clinically unimportant in the long term for most women. If BMD or skeletal strength was permanently reduced from lactation, then a history of breast-feeding should be a strong risk factor for low BMD, osteoporosis, or fragility fractures. However, more than five dozen epidemiologic studies of premeno-pausal, perimenopausal, and postmenopausal women have found a neutral effect, or a protective effect, of lactation on peak bone mass, BMD, and fracture risk [1]. In one study, an extended duration of breast-feeding per child conferred a progressively greater protection against hip fractures [284]. A study of 1852 twins and their female relatives, including 83 twins discordant for pregnancy and lactation, found no effect of breast-feeding history on BMD or BMC between twins [92]. However, parous women who had breast-fed had higher BMC and BMD than parous women who had never

breast-fed [92]. A study of 30 women who had had at least six pregnancies, and breast-fed each child for at least 6 months, found no effect of lactation on BMD [268]. The previously cited NHANES study of 819 women aged 20–25 years found that women who had breast-fed as adolescents had higher bone mass than those who had never breast-fed, indicat-ing a protective effect [93]. There are only a few studies that contest these findings by suggesting that lactation predicts lower BMD or higher fracture risk [1].

Overall, the bulk of the evidence suggests that the skeleton fully recovers BMD and strength after weaning, even if there are persistent changes in skeletal microarchitecture.

ANIMAL DATA RELEVANT TO VITAMIN D, LACTATION, AND POSTWEANING

RECOVERY

Lactation and postweaning recovery have been studied in Vdr null and Cyp27b1 null mice and vitamin D-deficient mice and rats. Parameters have included serum chemistries, intestinal calcium absorption, milk composition, and changes in BMC. A significant difference from human lactation is that intestinal calcium absorption normally doubles in lactating rodents, which require both intestinal delivery and skeletal resorption to supply the calcium needed for milk to sustain comparatively large litters [1].

VdrBos null mice that were maintained on a 2% calcium diet from the start of pregnancy lactated normally, had nor-mal serum calcium and phosphorus, and lost a similar amount of BMC as their WT sisters on the same diet [117,126]. Milk calcium content was no different between Vdr null and WT mice [126]. Within 14 days after weaning, the Vdr null and WT mice equally recovered whole body and lumbar spine BMC to achieve the end-of-pregnancy BMC value, which was 50% higher than prepregnancy value in Vdr nulls. This post-weaning increase in BMC implies that new bone formation occurred, but histomorphometric studies have not yet been carried out. After weaning, Vdr nulls had delayed involution and apoptosis of mammary epithelial cells [126], which indi-cate that calcitriol may be involved in regulating mammary gland involution after lactation.

VdrLeu null mice have also been studied on a 2% calcium diet during lactation [127]. WT and Vdr nulls resorbed a simi-lar amount of bone during lactation as confirmed by microCT and ash weight studies and lost a similar amount of breaking strength in the femora, with no significant differences between genotypes [127]. Postweaning recovery was not examined.

In a preliminary report, Cyp27b1 null and WT mice raised on a 2% calcium diet from the time of weaning resorbed a simi-lar amount of BMC during lactation and had a similar decline in tibial breaking strength, but Cyp27b1 null mice displayed a fall in serum calcium and phosphorus that was not seen in their WT sisters [116]. After weaning, Cyp27b1 null and WT mice regained the BMC and skeletal strength that had been lost during lactation, with the Cyp27b1 nulls achieving a BMC that was 40% higher than the prepregnancy value [116]. Serum

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calcium and phosphorus also normalized during postweaning in the Cyp27b1 nulls. It appears that Cyp27b1 nulls have dif-ficulty maintaining the serum calcium and phosphorus dur-ing lactation, unlike the Vdr nulls. But both achieved a marked postweaning increase in BMC that implies that substantial new bone formation occurred despite the absence of either cal-citriol or VDR.

Vitamin D-deficient mice were studied during lactation but without measurements of BMC or BMD being obtained. While maintained on a standard 1.2% calcium diet, lactating vitamin D-deficient CD1 mice displayed normal serum calcium and ionized calcium but lower serum phosphorus [285]. Milk from these mice showed normal nutritional and calcium content, but there was a 15% lower lactose content, and their pups grew at the same rate as those from vitamin D-sufficient mice [285]. Conversely, vitamin D-deficient mice in the Swiss background had 20% lower serum calcium and normal phosphorus and produced milk with lower protein content; the calcium content of milk was not determined [286].

Severely vitamin D-deficient rats have been studied dur-ing lactation and postweaning on diets ranging from 0.47% to 1.7% calcium. The lactating rats generally display hypo-calcemia (values 50% of normal and usually lower than in pregnancy) with serum phosphorus ranging from normal to modestly reduced [112,128,287–290], whereas during post-weaning recovery, serum calcium was closer to normal and phosphorus was consistently normal [112,128,287,288]. In several studies, lactating vitamin D-deficient and vitamin D-replete rats resorbed a similar amount of calcium from the femora [112,288,289], whereas one study found signifi-cantly widened osteoid seams and increased osteoblast sur-face, osteoclast number, and resorptive surface in vitamin D-deficient rats [290]. Postweaning skeletal recovery has been examined in two studies by the same investigators. Severely vitamin D-deficient Holtzman rats on a 0.47% calcium diet recovered lactational losses in mineralized bone volumes and cortical width, such that they equaled or exceeded the values that were present prior to pregnancy [288]. But in an earlier study, there was no recovery of ash weight or mineral content at 3 weeks [112]. The discrepancies between these two studies remain unexplained.

Intestinal absorption of calcium has also been examined in vitamin D-deficient rats. The rate doubled during lactation and declined to virgin values during postweaning recovery phase in rats consuming 0.47% to 1.2%–1.7% calcium diets [128,287]. Intestinal calcium absorption was also no different between vitamin D-deficient and vitamin D-replete rats dur-ing lactation or postweaning on the 1.2%–1.7% calcium diet [287]. Intestinal phosphorus absorption also increased two-fold on the 1.2%–1.7% calcium diet [287]. On a 0.47% calcium diet, vitamin D-deficient rats doubled the efficiency of intes-tinal calcium absorption during lactation; they were studied separately from vitamin D-replete rats and may have had a slightly lower peak efficiency of intestinal calcium absorption [128]. These studies in vitamin D-deficient rats indicate that calcitriol is not required for the efficiency of intestinal calcium and phosphorus absorption to double during lactation.

In contrast to prior findings in Vdr null or vitamin D-deficient mice, in one study, vitamin D-deficient rats pro-duced about 20% of the volume of milk as vitamin D-replete rats, as assessed after oxytocin stimulation and manual expression of milk. The nutritional content of the milk was enriched with more protein, calcium, and phosphorus, but somewhat less carbohydrate, than in milk from vitamin D-replete rats [291]. The pups of vitamin D-deficient rats had modestly reduced weight gain, whereas culling the lit-ters to a lower number enabled normal weight gain. This is consistent with the mother producing insufficient volumes of milk to meet the demands of larger litters [291]. However, a striking discrepancy is that the volume of expressed milk was markedly reduced, whereas pup weight gain was only modestly affected. The experimental method may have exag-gerated the true difference in milk volumes produced by vitamin D-deficient and vitamin D-replete rats. It is conceiv-able that, for example, there are differences between vitamin D-deficient rats and vitamin D-replete rats in the milk ejected in response to exogenous oxytocin but not in response to suckling.

As with all mammals, rat milk normally contains very low concentrations of vitamin D and 25(OH)D, which together total about 3–12 IU per liter [292].

Overall, the preceding rodent studies indicate that lacta-tion usually proceeds normally despite maternal loss of cal-citriol, VDR, or vitamin D, with the amount of bone resorbed being comparable to that of their respective WT or vitamin D-sufficient controls. Normal and vitamin D-deficient rats are more likely than mice to develop hypocalcemia while lactating, regardless of the calcium content of the diet [1]. After weaning, BMC increases in Vdr null and Cyp27b1 mice to a similar magnitude as in matched WT sisters, whereas two studies in vitamin D-deficient rats differ on the extent of skeletal recovery after lactation. Milk calcium content was normal to increased in vitamin D-deficient mice and rats, and in Vdr null mice, whereas one study of vitamin D-deficient rats found a significant reduction in the volume of milk pro-duced that was out of keeping with effects observed on pup growth.

HUMAN DATA RELEVANT TO VITAMIN D, LACTATION, AND POSTWEANING

RECOVERY

The effects of vitamin D deficiency versus sufficiency have not been as rigorously assessed in clinical studies as has been seen in the animal studies cited previously. There have been observational cohort studies [221,260,293,294] and randomized interventional trials [222,242,261,295–301], which did not find any effect of higher 25(OH)D concen-trations or vitamin D supplementation on parameters of mineral and skeletal homeostasis in otherwise healthy, lactating women. Vitamin D supplementation increases maternal 25(OH)D in lactating women with a similar dose response as observed in nonpregnant women. In many of

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the randomized trials the achieved 25(OH)D level was the outcome of interest, and few or no additional outcomes were assessed [222,261,295,297,298,301].

Consistent with all mammals, human milk normally contains little vitamin D or 25(OH)D, approximately 30–40 IU/L in total, with very low to undetectable concentrations of calcitriol [302–309]. Since milk contains a low content of vitamin D metabolites, lactation should not deplete mater-nal vitamin D stores, and numerous studies have confirmed that maternal 25(OH)D does not change during lactation [28,36,202,204,207,208,210,216,218–221]. The corollary of this is that breast milk is normally not a good source of vita-min D for the neonate or infant. Marketed milk and other dairy products in the United States and Canada contain about 10 times this concentration of vitamin D, that is, 400 IU/L or 100 IU per standard serving (250 mL or cup). But as noted earlier, that is a synthetic vitamin D supplement, which is added after pasteurization and not put there by the cow or goat.

Despite low content of vitamin D and 25(OH)D in human milk, there is a correlation between milk 25(OH)D content and neonatal serum 25(OH)D after the first month, indicat-ing that breast milk is an important determinant of neona-tal 25(OH)D [222,293,310]. Since the half-life of 25(OH)D in infants is 2–3 weeks [311], similar to adult values [312–316], the cord blood 25(OH)D value will only influence neona-tal levels over about the first 6 weeks. Randomized inter-vention studies found that typical doses of 400–1000 IU of vitamin D per day do not reliably increase breast milk con-tent of vitamin D or 25(OH)D, whereas maternal doses of 2000–4000 IU per day do measurably increase the milk con-tent of vitamin D and 25(OH)D and neonatal serum 25(OH)D [222,295–297]. Even higher intakes of vitamin D have been shown in randomized interventions to further increase the amount of vitamin D and 25(OH)D in milk [261,298,300,301]. With maternal intake of 6400 IU per day, maternal 25(OH)D increased to about 150 nmol/L (60 ng/mL), and the nursed babies achieved mean 25(OH)D values of 110–115 nmol/L (44–46 ng/mL), which is the same level achieved by babies who directly received 300 or 400 IU of vitamin D in the form of oral drops [298,317]. In another study, 5000 IU of vitamin D per day raised maternal 25(OH)D to 400 nmol/L (160 ng/mL) and neonatal 25(OH)D to 98 nmol/L (39 ng/mL), whereas a single maternal dose of 150,000 IU had a similar effect [300].

Are such high maternal or milk levels of 25(OH)D beneficial or necessary? These studies have used the achieved 25(OH)D level as the principal outcome and did not report any addi-tional skeletal or nonskeletal benefits. Recall that multiple stable isotope-based studies in children, adolescents, and adults have shown that intestinal calcium absorption appears to become maximal with a 25(OH)D level above 20 nmol/L [7,145–149]. In the breast-fed neonate and infant, intestinal cal-cium absorption is further boosted by the lactose content of milk [191,192]. Therefore, a 25(OH)D level of 100 nmol/L in a neonate or infant is far above the value needed for optimal intestinal calcium absorption.

Moreover, additional clinical data suggest that such levels of vitamin D intake are even unnecessary when the breast-fed neonate or infant has established vitamin D deficiency rickets that requires treatment. Such observational studies have been carried out in regions where severe vitamin D deficiency rick-ets is endemic. Exclusively breast-fed infants with rickets have been effectively treated by giving the mothers 150,000 IU of vitamin D every 3 months, which is equivalent to a daily dose of approximately 1800 IU [318,319]. In these studies the affected babies and their mothers were protected from sunlight expo-sure and had negligible vitamin D in the diet. Consequently, the parenterally administered vitamin D was the main source for the mother, whereas mother’s milk was the sole source for the infant. Baseline 25(OH)D was 6 nmol/L (2 ng/mL) or lower in mothers and babies, consistent with extreme vitamin D deficiency, whereas achieved 25(OH)D values were approxi-mately 50 nmol/L (20 ng/mL) in the mothers and 40 nmol/L (16 ng/mL) in their babies. The posttreatment 25(OH)D values were above the 20 nmol/L threshold that appears to maximize intestinal calcium absorption. Consequently, giving higher doses of 5000 or 6400 IU vitamin D each day to normal moth-ers seems unnecessary when far lower doses given to moth-ers have been shown to effectively treat established vitamin D deficiency rickets in the breast-fed infants. If it is desirable for all of infant nutrition to come from milk in breast-fed babies, such that vitamin D drops given directly to the baby are not necessary, then the equivalent maternal intake of 1800 IU of vitamin D per day may be all that is needed. An unanswered question is whether such high maternal vitamin D intakes or achieved 25(OH)D levels in the infants confer any nonskeletal benefits [52].

Although it remains arguable as to whether or not higher levels of vitamin D in milk are beneficial to the neonate, it is quite clear that higher maternal intakes of vitamin D do not alter the calcium content of milk. In fact breast milk calcium content is unaffected by maternal 25(OH)D levels ranging from 25 (10 ng/mL) to 160 nmol/L (64 ng/mL) [260,261]. These data come from cohort studies that examined low maternal vitamin D intakes and 25(OH)D concentrations [260] and randomized interventions which found that maternal intake as high as 2000–6400 IU of vitamin D per day failed to alter the calcium content of milk as compared to intakes of 400 IU per day [261,297,298]. On the other hand, a few individual cases from India found that milk calcium content was lower in mothers who had mean 25(OH)D values of 6 nmol/L (2.5 ng/mL) and clinically obvious vitamin D deficiency (hypocalce-mia, hypophosphatemia, and markedly elevated PTH) [319]. Taken together, the available data confirm that vitamin D and calcitriol do not play a direct role in causing calcium to enter milk, but the extreme situation of severe vitamin D deficiency (25(OH)D closer to 6 nmol/L) can impair milk production. Earlier, it was noted that high and low calcium intakes do not alter breast milk calcium content [201,244–246]. Instead, the cal-cium content of milk appears to come principally from resorp-tion of the maternal skeleton, with lesser contributions from maternal diet. In extreme vitamin D deficiency, skeletal resis-tance to the resorptive actions of PTH has been demonstrated

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[320–323]. Skeletal resistance to PTHrP should similarly occur during lactation in severely vitamin D-deficient women and contribute to maternal hypocalcemia and low breast milk cal-cium content.

The effect that vitamin D deficiency or insufficiency has on postweaning skeletal recovery has not been examined in women. This is the time frame when calcitriol may be espe-cially important to ensure that sufficient mineral is available to restore the skeleton. One study suggested that the FokI poly-morphism in Chinese women interacted with calcium intake to determine the magnitude of BMD increase 1 year after lacta-tion ended [324]. However, with just 40 subjects, the study was too small for generalizations or to be certain that the subgroup differences were not simply chance findings.

Earlier, it was mentioned that dozens of epidemiologic studies have found lactation to have a neutral or protective effect against long-term risk of osteoporosis or fragility frac-tures [1]. Vitamin D deficiency and insufficiency have not been explicitly examined in any of those studies. However, since most women in those studies are considered to be vitamin D insufficient or even deficient by some modern experts (where sufficiency may be considered to be 25(OH)D > 75 nmol/L (30 ng/mL), it seems probable that skeletal recovery after weaning is not impaired by vitamin D insuffi-ciency. Direct studies of the effect of vitamin D supplementa-tion during postweaning recovery are needed. Without such data, it remains prudent to recommend that lactating and postweaning women have the same intake of vitamin D as in nonpregnant women [7].

As noted previously, hereditary absence of Cyp24a1 reduces calcitriol catabolism and can lead to marked maternal hyper-calcemia during pregnancy, when calcitriol normally increases two-fold to three-fold. But during lactation, calcitriol levels are typically normal, and so, the effect of Cyp24a1 deficiency should be lessened during this time frame. Indeed, in one case of Cyp24a1 deficiency that was followed after pregnancy, the hypercalcemia was milder and serum calcitriol was normal, whereas the woman was breast-feeding [168].

CONCLUSIONS

During pregnancy, several animal models (severe vitamin D deficiency and absence of VDR or Cyp27b1) have consis-tently demonstrated that intestinal calcium absorption still doubles and mineral homeostasis improves significantly. This is consistent with calcitriol not being required for the adaptations that are normally invoked during pregnancy or that other physiological mechanisms compensate for its absence. Clinical data from pregnant women are not as extensive, but the results from randomized interventions do not show a clear benefit of high-dose vitamin D supplemen-tation on maternal mineral or skeletal homeostasis or obstet-rical and fetal outcomes. However, most of the clinical trials excluded women who were vitamin D deficient; and so, the ability to detect any benefit of vitamin D supplementation was likely lost.

During lactation, the same animal models have revealed that resorption of the maternal skeleton to support milk production is unaffected by the absence of VDR, calcitriol, and vitamin D. Postweaning recovery of the skeleton also occurs despite the absence of VDR, calcitriol, and vitamin D, although there are some inconsistent data from studies of vitamin D-deficient rats. Clinical data from lactating women are largely similar, with milk calcium content unaffected by extremes of severe vitamin D deficiency and excess, and lactational bone loss probably unaffected by vitamin D insufficiency and deficiency. Epidemiologic studies also support that postweaning recov-ery of maternal bone mass and strength likely occurs despite vitamin D insufficiency and deficiency. However, no studies have directly examined the effect of vitamin D sufficiency versus deficiency on skeletal losses during lactation and skel-etal recovery postweaning. Since milk normally contains little vitamin D or 25(OH)D, a theoretical benefit of high-dose vita-min D supplementation is that all of a baby’s nutrition could then come from breast milk, rather than requiring that breast-fed babies alone be stigmatized to directly receive vitamin D drops. However, since modest maternal doses of vitamin D have been sufficient to heal rickets in breast-fed infants, such high doses may not be necessary.

Overall, the available data suggest no compelling reason to recommend increased vitamin D intakes or 25(OH)D levels during pregnancy or breast-feeding. Instead, the most recent recommendations of the Institute of Medicine of the National Academies of Science USA remain reasonable, which are to maintain vitamin D intakes and target 25(OH)D levels at the same levels as in nonpregnant women from the same age group [7].

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