Lactational performance in poorly nourished women

The problem of infant growth faltering in resource-poor regions of the world has long been recognised^{(Reference Shrimpton, Victora and de Onis1,Reference Victora, de Onis and Hallal2)} and remains an intense area of research and public health concern. In the past, because breast-feeding of infants was universally practised in these disadvantaged populations, it was assumed that growth faltering was a result of compromised pregnancy and lactation due to the mother's poor nutritional status. In the 1970s, Professor RG Whitehead, Director of the MRC Dunn Nutrition Unit in Cambridge, established an intensive series of programmes in Cambridge and The Gambia, West Africa, to provide the scientific evidence on which nutritional interventions to reduce infant growth faltering could be based. The Gambian research was based in the village of Keneba and its neighbouring villages in the province of West Kiang, a remote and resource-poor region of the country. This region had been the subject of longitudinal demographic and health surveys since 1950, primarily in connection with malaria research. In this rural Gambian society at that time, malnutrition and poor infant growth were already well documented, associated with marginal diets, seasonal food shortages and high infection rates^{(Reference Hennig, Unger and Dondeh3)}. Large family sizes were the norm and infants were customarily breast-fed for about 2 years with complementary foods introduced from about 4 months, making Keneba an ideal location for this research. In Cambridge, a much more affluent society, nutrition and health provision was generally good and breast-feeding, at least in the first year post-partum, was relatively common. Studies in Cambridge among pregnant and lactating women were conducted to provide comparative data for the Gambian studies.

The initial stages of the work involved establishing a small team of scientists, doctors and other staff in Keneba to create a rapport with the local community and develop culturally-sensitive research methods and protocols. This was especially important for the lactation studies in order to prevent any alteration in mother–infant behaviour that might interrupt breast-feeding, a problem that was suspected in many of the studies cited in the literature up to that time^{(Reference Prentice, Paul, Prentice, Hamosh and Goldman4)}. The ability to measure the breast-milk intake of babies in a non-intrusive manner was advanced by the development by the MRC Dunn Nutrition Unit scientists of innovative stable isotope methods using ²H oxide, and these were pioneered in the Gambian and Cambridge lactation studies^{(Reference Coward, Whitehead and Sawyer5,Reference Coward, Cole and Sawyer6)} . Unexpectedly, these studies demonstrated that Gambian infants had similar breast-milk intakes to those in Cambridge in the first months after birth when they were exclusively or predominantly breast-fed^{(Reference Prentice, Paul, Prentice, Hamosh and Goldman4,Reference da Costa, Haisma and Wells7)} .

The fact that the lactational performance of these Gambian women did not appear to be compromised by their poor diet was underscored by the results of maternal supplementation trials conducted during lactation. These trials used a specially prepared energy-protein-rich biscuit, made locally, and a vitamin-fortified tea drink. These trials demonstrated that improving the diet of the mother had no effect on the breast-milk intake or growth of the infant^{(Reference Prentice, Roberts and Prentice8)}. There were increases in the breast-milk content of certain vitamins present in the supplement but only marginal effects on the macronutrient content^{(Reference Prentice, Roberts and Prentice8)}. The most noteworthy effects were in mothers, who gained weight and reported fewer episodes of poor health. However, these women also had lower concentrations of several hormones associated with lactation, especially prolactin^{(Reference Lunn, Watkinson and Prentice9,Reference Prentice, Lunn and Watkinson10)} . This suggested that the supplement had produced a relaxation from a state of high metabolic efficiency and that much of the additional energy derived from the supplement was wasted in less efficient metabolic processes^{(Reference Prentice, Lunn and Watkinson10)}. Furthermore, the lower prolactin concentration of these mothers was associated with a shorter period of amenorrhoea^{(Reference Lunn, Watkinson and Prentice9)}. Even greater effects on lowering prolactin and shortening the period of amenorrhoea were seen when the Gambian mothers also received the dietary supplement during pregnancy^{(Reference Lunn, Austin and Prentice11)}.

As part of these early investigations, detailed studies of breast-milk composition revealed that the Gambian women had consistently lower breast-milk calcium concentrations than women in Cambridge, by about 25 %^{(Reference Laskey, Prentice and Shaw12,Reference Prentice, Laskey, Jarjou, Bonjour and Tsang13)} . Breast-milk calcium concentration was shown to decrease as lactation progressed, tracking within individuals, and was independent of the volume of breast-milk consumed by the infant^{(Reference Prentice, Laskey, Jarjou, Bonjour and Tsang13–Reference Prentice, Glorieux, Pettifor and Juppner15)}. These findings indicated, therefore, that, on average, the Gambian breast-fed infants consumed considerably less calcium than their Cambridge counterparts during exclusive breast-feeding and after the introduction of complementary foods^{(Reference Prentice, Paul, Atkinson, Hanson and Chandra16,Reference Jarjou, Goldberg and Coward17)} . With the exception of certain water-soluble vitamins whose concentration in breast-milk depends on maternal dietary intake, few components of breast-milk had been found to vary to this extent between populations^{(Reference Prentice, Jensen and Jenness18)}. A comparison of breast-milk calcium concentrations from different regions of the world showed that women in populations with a high customary calcium intake, such as in the UK and North America, tended to have higher breast-milk calcium than in African countries with a much lower dietary calcium intake^{(Reference Prentice, Laskey, Jarjou, Bonjour and Tsang13,Reference Prentice, Jensen and Jenness18)} . This led to the possibility that the calcium content of breast-milk might be dependent on the maternal intake.

Dietary calcium intake and breast-milk calcium

The unexpected finding of low breast-milk calcium concentrations among Gambian women led to a more thorough exploration of dietary calcium intake in this population. The Gambian diet at that time was based predominantly on millet, sorghum and rice as staples, with groundnut (peanut)-, leaf- and vegetable-based sauces. Fish were eaten occasionally, often as dried ingredients in sauces, but the diet rarely included meat or milk. Dietary assessments were conducted, using detailed weighed intakes conducted by trained research assistants, combined with laboratory analysis of local foods and condiments. Potential hidden sources of calcium, such as flavourings, bush foods and pica, were also analysed and included in the estimates.

These studies demonstrated that the customary diet in rural Gambia was, and still is, very low in calcium throughout life^{(Reference Prentice, Laskey and Shaw19,Reference Prentice, Burckhardt, Dawson-Hughes and Heaney20)} . This is largely ascribed to a lack of animal milk and milk products in the diet. Scrutiny of FAO balance sheets confirmed the much lower milk supply in The Gambia, and many other African countries, compared to the UK, Europe, Northern America and Australasia^{(Reference Prentice, Burckhardt, Dawson-Hughes and Heaney20)}. These studies provided the estimates of average calcium intake of 300–400 mg/d in women and children, and 200 mg/d in infants^{(Reference Jarjou, Goldberg and Coward17,Reference Prentice, Laskey and Shaw19)} . These intakes are considerably lower than international dietary reference values and recommendations^{(Reference Prentice21)}. Such low intakes were surprising given that they are very close to the daily biological requirements for fetal growth, breast-milk production and childhood mineral accretion, even before making allowance for the reduced amount of calcium that can be absorbed from the diet^{(Reference Prentice, Glorieux, Pettifor and Juppner15)}.

We conducted two randomised placebo-controlled supplementation trials to test whether raising the calcium intake of Gambian mothers would increase breast-milk calcium concentration and infant growth: the first during lactation, the second during pregnancy. The mothers were supplemented with 1000 mg Ca/d from 2 weeks post-partum for 12 months in the lactation trial^{(Reference Prentice, Jarjou and Cole22)} and with 1500 mg Ca/d from 20 weeks gestation to delivery in the pregnancy trial^{(Reference Jarjou, Prentice and Sawo23)}. In both trials, the supplement was orange-flavoured, chewable calcium carbonate. The supplement was well accepted by the mothers and compliance was high. Breast-milk samples were obtained serially at specific times post-partum using carefully standardised collection and assay protocols^{(Reference Laskey, Dibba and Prentice24)}. No significant differences were found between the calcium-supplemented and placebo groups in either trial (Fig. 1), thus demonstrating that breast-milk calcium concentration is not responsive to changes in maternal calcium intake. There was also no effect on the growth of the infants in terms of rates of increase in weight and length^{(Reference Prentice, Jarjou and Cole22,Reference Jarjou, Prentice and Sawo23,Reference Goldberg, Jarjou and Cole25)} . In addition, in the pregnancy trial, there were no differences in the mother's blood pressure between groups at any stage of pregnancy or lactation, nor was there an effect of the calcium supplement on infant birth size^{(Reference Goldberg, Jarjou and Cole25)}.

Fig. 1. Effects on breast-milk calcium concentration of maternal calcium supplementation during (a) lactation and (b) the preceding pregnancy. The data are expressed as mean (se) concentration (mg/l) in breast-milk collected serially from 60 and 125 rural Gambian mothers respectively during randomised placebo-controlled trials of calcium carbonate supplementation (lactation trial = 1000 mgCa/d for 12 months; pregnancy trial = 1500 mgCa/d from 20 weeks gestation to delivery)^{(Reference Prentice, Jarjou and Cole22,Reference Jarjou, Prentice and Sawo23)} . Dark bars, calcium-supplemented group; light bars, placebo group. The numbers of women in the calcium and placebo groups respectively who participated in the sub-study of breast-milk calcium were: lactation trial 30, 30; pregnancy trial 61, 64. There were no significant differences between the groups at any time.

Calcium requirements for maternal and infant bone health

During the 1980s, Dr Elsie Widdowson was awarded the prestigious Rank Prize in Nutrition, with which she endowed a fellowship at the MRC Dunn Nutrition Unit to work with me and the teams in Cambridge and Keneba to study the calcium requirements of mothers and children in Africa. This prompted the purchase of a single-photon absorptiometer, one of the first instruments designed to measure bone mineral content in vivo that could be used in healthy infants, children and adults. This was the start of the Nutrition and Bone Health Research Group based in the MRC Dunn Nutrition Unit and then in MRC Human Nutrition Research at the Elsie Widdowson Laboratory, and funds were obtained to set up parallel bone imaging facilities in Cambridge and The Gambia. The single-photon absorptiometer has been replaced over the years by new generations of bone scanning instruments as they became more sophisticated. Our more recent studies have predominantly used dual-energy X-ray absorptiometry and peripheral quantitative computed tomography.

To consider whether a low maternal calcium intake during lactation might necessitate mobilisation of skeletal calcium to support breast-milk production, we conducted a series of studies in Cambridge to investigate whether the bone mineral content of well-nourished breast-feeding mothers alters during and after lactation, and whether any changes are related to maternal calcium intake^{(Reference Laskey, Prentice and Shaw12–Reference Prentice14,Reference Laskey and Prentice26,Reference Laskey, Prentice and Hanratty27)} . These studies demonstrated that measurable decreases in size-adjusted bone mineral content (SA-BMC) occur in the first few months of lactation, predominantly at the lumbar spine and hip, and that these are reversed in later lactation or after breast-feeding stops^{(Reference Laskey and Prentice26)}. The magnitude of the decreases after 3 months of exclusive breast-feeding was shown to vary between individuals, depending on the volume of breast-milk produced and other factors^{(Reference Laskey, Prentice and Hanratty27)}. However, no correlations with maternal calcium intake or breast-milk calcium concentration were found, despite the wide range of calcium intakes between the study participants^{(Reference Laskey, Prentice and Hanratty27)}.

To investigate this further, bone scanning was conducted to chart the skeletal changes during lactation in both the Gambian lactation and pregnancy calcium supplementation trials described earlier. The expectations were that the skeletal response to lactation would be less among women in the calcium-supplemented group than in the placebo group because the requirement to mobilise bone calcium to support breast-milk production would be lower, given the greater amount of calcium available from the diet. The results showed that this was not the case. In the lactation trial, which was performed at a time when only forearm scanning by single-photon absorptiometer was available, the expected decrease and reversal in SA-BMC was observed, but there were no significant differences between the groups^{(Reference Prentice, Jarjou and Cole22)}. In the pregnancy trial, which was conducted several years after the lactation trial, whole-body and regional dual-energy X-ray absorptiometry scans were obtained to 12 months of lactation. The expected decrease in SA-BMC was observed in the first months of lactation but there was little sign of recovery of bone mineral by 12 months post-partum (Fig. 2)^{(Reference Jarjou, Laskey and Sawo28)}. The likely explanation for this was that, unlike most mothers in the Cambridge studies, all the mothers in the Gambian pregnancy trial were still breast-feeding on demand at 12 months and would not have begun to wean their infant from the breast. More surprisingly, and contrary to expectations, there were greater decreases in lumbar spine and whole-body SA-BMC in the mothers who had been in the calcium-supplemented group during pregnancy than the mothers who had been in the placebo group (Fig. 2), and they also had lower hip SA-BMC throughout the 12 months^{(Reference Jarjou, Laskey and Sawo28)}.

Fig. 2. Effects on size-adjusted bone mineral content (SA-BMC) of the lumbar spine (L1–4) of maternal calcium supplementation during pregnancy in Gambian mothers. The data are expressed as mean percentage difference (se) relative to the placebo group at 0⋅5 months post-partum. The scans were obtained using dual-energy X-ray absorptiometry (DXA) as part of a randomised placebo-controlled trial of 1500 mgCa/d from 20 weeks gestation to delivery with follow-up. Measurements in the DXA sub-study were made serially on each individual during the index lactation at 0⋅5, 3 and 12 months^{(Reference Jarjou, Laskey and Sawo28)} and approximately 5 years later at a time when the mother was neither pregnant nor lactating and at least 3 months since the end of a recent lactation period (NPNL)^{(Reference Jarjou, Sawo and Goldberg30)}. The numbers of measurements at each timepoint for the calcium and placebo groups respectively were 0⋅5 months = 23, 27; 3 months = 29, 29; 12 months = 40, 39; NPNL = 31, 28. Dark bars, calcium-supplemented group; light bars, placebo group. The changes over time were significantly different between the two groups: in the index lactation P for interaction = 0⋅05; in the follow-up study P for interaction = 0⋅002.

Follow-up studies of pregnancy calcium supplementation in The Gambia

Once the findings of the pregnancy calcium supplementation trial became apparent, those mothers who had been scanned during the trial were traced and invited to be scanned again. The time interval was approximately 5 years from their 12-month measurement; two-thirds of the women had had at least one more pregnancy-lactation cycle in the intervening years^{(Reference Jarjou, Sawo and Goldberg30)}. The scans were conducted either when the woman had been breast-feeding her latest child for 12 months or when she was neither pregnant nor lactating and at least 3 months after having stopped the most recent lactation period. The neither pregnant nor lactating women who had been in the placebo group during the trial were found to have had similar increases in SA-BMC post-lactation to those observed in Cambridge women, while no such skeletal recovery was seen in those who had received the pregnancy calcium supplement (Fig. 2)^{(Reference Jarjou, Sawo and Goldberg30)}. For those women in the follow-up study measured at 12 months lactation, the SA-BMC values in both groups were similar to their values at 12 months in the index lactation^{(Reference Jarjou, Sawo and Goldberg30)}.

Although the growth of the infants was not significantly affected by the maternal pregnancy supplement, there was an indication that the bone mineral accretion rate was lower in the offspring of mothers in the calcium-supplemented group^{(Reference Jarjou, Prentice and Sawo23)}. To investigate the possibility that the maternal supplement had influenced the skeletal growth of the offspring, regular follow-up measurements throughout childhood were introduced. dual-energy X-ray absorptiometry scans were possible once the children were 8–12 years old, an age which in rural Gambia represents late childhood but pre-puberty^{(Reference Prentice, Burckhardt, Dawson-Hughes and Heaney20)}. Unexpectedly, these studies demonstrated sex-specific effects of the pregnancy supplement, such that girls whose mothers had been in the calcium group were shorter, lighter and had smaller bones with less bone mineral than girls whose mothers had been in the placebo group^{(Reference Ward, Jarjou and Prentice31)}. The opposite effects were seen in the boys; those whose mothers had been in the calcium group tended to be larger with greater bone mineral than boys whose mothers had been in the placebo group^{(Reference Ward, Jarjou and Prentice31)}. A similar pattern was also seen in insulin-like growth factor 1 concentrations measured in plasma samples collected when the children were approximately 7 years old^{(Reference Prentice, Ward and Nigdikar32)}.

Calcium requirements in childhood and adolescence

One interpretation of the sex-specific effects noted in the offspring of mothers in the Gambian pregnancy trial is that the calcium supplement had increased the growth trajectory towards puberty faster in boys and more slowly in girls. This has resonances with the results of follow-up studies from our earlier calcium supplementation trial of prepubertal children in the same region of The Gambia. These children were 8–12 years of age and supplemented with 1000 mg Ca/d as calcium carbonate or placebo, 5 d weekly for 12 months. The calcium supplement increased forearm SA-BMC and decreased the bone turnover marker osteocalcin but with no increase in height or bone dimensions^{(Reference Dibba, Prentice and Ceesay34)}. Follow-up studies, however, demonstrated sex-specific effects on the passage through puberty for these children, such that the boys who had been in the calcium group entered their pubertal height spurt earlier than boys who had been in the placebo group and reached peak height velocity approximately 7–8 months earlier^{(Reference Prentice, Dibba and Sawo35)}. These boys were taller in mid-adolescence, but they stopped growing earlier and were shorter by an average of 3⋅5 cm in young adulthood (Fig. 3). Similar effects were seen in the boys on the timing of skeletal development and mineral accretion during adolescence measured by dual-energy X-ray absorptiometry^{(Reference Ward, Cole and Laskey36)}. There was no discernible effect on growth or skeletal development in girls (Fig. 3)^{(Reference Prentice, Dibba and Sawo35)}.

Fig. 3. Effects on height of calcium supplementation in pre-pubertal Gambian children. The data are expressed as the mean (se) difference in height between the calcium and placebo groups by year of the study obtained from regression models after adjustment for height and age at Y1. The measurements were made serially during adolescence in 160 children (80 boys, 80 girls) who had participated in a randomised placebo-controlled trial of 1000 mg Ca/d, 5 d weekly for 12 months at the age of 8–12 years^{(Reference Prentice, Dibba and Sawo35)}. The average age (years) of the boys at each measurement timepoint was approximately Y1 = 10⋅5; Y2 = 11⋅5; Y3 = 12⋅5; Y4 = 13⋅5; Y6 = 15⋅5; Y8 = 17⋅5; Y10 = 19⋅4; Y12 = 21⋅5; Y14 = 23⋅5. The average age of the girls was approximately 0⋅5 years younger at each timepoint. The supplementation was commenced after the Y1 measurement (baseline) and ceased after the Y2 measurement. The numbers of boys in the calcium and placebo groups respectively were: Y1–6 = 40, 40; Y8 = 39, 39; Y10 = 37, 39; Y12 = 34, 30; Y14 = 29, 25. The numbers for the girls were Y1–4 = 40, 40; Y6 = 38, 39; Y8 = 39, 40; Y10 = 29, 25; Y12 = 33, 30; Y14 = 25, 29. On the graph, XY = baseline for boys and girls respectively; significance of difference between the groups in boys *P = 0⋅04; **P = 0⋅01; ***P = 0⋅002. There were no significant differences between the groups in girls at any time.

These findings raise the question whether shortening the period of pre-pubertal growth by increasing the calcium intake of prepubertal Gambian boys to values closer to those recommended internationally confers a health or social benefit in the long-term, indeed the slightly shorter adult stature might be considered a disbenefit. However, the low calcium intake of young children in regions of Africa and Asia has been implicated in the aetiology of nutritional rickets that is not due to primary vitamin D deficiency^{(Reference Prentice, Ceesay and Nigdikar37–Reference Ahmed, Goldberg and Raqib39)} and, in rural Gambia, there is a higher prevalence of nutritional rickets among boys^{(Reference Jones, Jammeh and Owens40)}. This indicates that such low calcium intakes can be limiting for some children during periods of bone growth and development. However, other factors, such as iron deficiency and environmental contaminants, and secondary disturbances in the metabolism of phosphorus and vitamin D, have also been implicated in nutritional rickets, along with a very low calcium intake^{(Reference Prentice38)}. Furthermore, most studies have been conducted in children with active rickets or rickets-like bone deformities, and little is known about the predisposing factors that underlie the development of the condition. This includes whether or not the customary calcium intakes of affected children were lower before they developed rickets than those of their unaffected contemporaries^{(Reference Prentice38)}.

Calcium, vitamin D and osteoporosis risk

The complexity of issues surrounding the prevention and treatment of nutritional rickets is a reminder of the intimate inter-relationships between calcium and vitamin D metabolism and bone health^{(Reference Prentice, Goldberg and Schoenmakers41)}. The extent to which increasing dietary calcium intake and/or vitamin D supplementation in adults reduces the risk of osteoporosis and fracture risk in later life has been much debated^{(Reference Prentice, Goldberg and Schoenmakers41)}. It has long been recognised that the age-adjusted incidence of hip fracture in Africa is considerably lower than in Westernised countries, despite the lower calcium intakes throughout life^{(Reference Prentice42)}. Why this is the case is not known; detailed studies examining potential reasons, for example, higher physical activity, fewer environmental trip hazards and anatomical differences such as shorter hip axis length, have generally not provided clear answers, or have provided unexpected results^{(Reference Prentice42–Reference Dibba, Prentice and Laskey45)}. For example, our studies in The Gambia have shown that adult bone mineral density and bone mineral loss during menopause and ageing are very similar to Western countries^{(Reference Aspray, Prentice and Cole43,Reference Zengin, Fulford and Sawo44)} and that circulating concentrations of parathyroid hormone and bone turnover markers are greater^{(Reference Aspray, Yan and Prentice46)}, factors that are generally considered to be risk factors for osteoporosis and fracture.

It has been suggested from studies in Western countries that vitamin D deficiency resulting from increased metabolism might be responsible for the bone mineral loss seen in people living with HIV, especially in those receiving antiretroviral therapy (ART), and that supplementation might be beneficial^{(Reference Grant and Cotter47)}. The bone loss is particularly marked when the ART includes tenofovir disoproxil fumarate^{(Reference Grant and Cotter47)}. In Eastern and Southern Africa, where tenofovir disoproxil fumarate-based ART is commonly prescribed, women have the highest burden of HIV infection and, because of ART, women living with HIV are now likely to live into and beyond the menopause. Our recent study in Soweto, South Africa, among women with good vitamin D status, showed the expected bone mineral loss in those living with HIV after the initiation of ART^{(Reference Hamill, Pettifor and Ward48)}. However, we found no evidence of an effect on their vitamin D status. This suggests that vitamin D supplementation would be unlikely to provide any benefit for these women in terms of ameliorating the ART-related bone loss, although a randomised trial would be needed to confirm that finding.