Association of maternal circulating 25(OH)D and calcium with birth weight: A mendelian randomisation analysis

BackgroundSystematic reviews of randomised controlled trials (RCTs) have suggested that maternal vitamin D (25[OH]D) and calcium supplementation increase birth weight. However, limitations of many trials were highlighted in the reviews. Our aim was to combine genetic and RCT data to estimate causal effects of these two maternal traits on offspring birth weight.Methods and findingsWe performed two-sample mendelian randomisation (MR) using genetic instrumental variables associated with 25(OH)D and calcium that had been identified in genome-wide association studies (GWAS; sample 1; N = 122,123 for 25[OH]D and N = 61,275 for calcium). Associations between these maternal genetic variants and offspring birth weight were calculated in the UK Biobank (UKB) (sample 2; N = 190,406). We used data on mother–child pairs from two United Kingdom birth cohorts (combined N = 5,223) in sensitivity analyses to check whether results were influenced by fetal genotype, which is correlated with the maternal genotype (r ≈ 0.5). Further sensitivity analyses to test the reliability of the results included MR-Egger, weighted-median estimator, ‘leave-one-out’, and multivariable MR analyses. We triangulated MR results with those from RCTs, in which we used randomisation to supplementation with vitamin D (24 RCTs, combined N = 5,276) and calcium (6 RCTs, combined N = 543) as an instrumental variable to determine the effects of 25(OH)D and calcium on birth weight. In the main MR analysis, there was no strong evidence of an effect of maternal 25(OH)D on birth weight (difference in mean birth weight −0.03 g [95% CI −2.48 to 2.42 g, p = 0.981] per 10% higher maternal 25[OH]D). The effect estimate was consistent across our MR sensitivity analyses. Instrumental variable analyses applied to RCTs suggested a weak positive causal effect (5.94 g [95% CI 2.15–9.73, p = 0.002] per 10% higher maternal 25[OH]D), but this result may be exaggerated because of risk of bias in the included RCTs. The main MR analysis for maternal calcium also suggested no strong evidence of an effect on birth weight (−20 g [95% CI −44 to 5 g, p = 0.116] per 1 SD higher maternal calcium level). Some sensitivity analyses suggested that the genetic instrument for calcium was associated with birth weight via exposures that are independent of calcium levels (horizontal pleiotropy). Application of instrumental variable analyses to RCTs suggested that calcium has a substantial effect on birth weight (178 g [95% CI 121–236 g, p = 1.43 × 10−9] per 1 SD higher maternal calcium level) that was not consistent with any of the MR results. However, the RCT instrumental variable estimate may have been exaggerated because of risk of bias in the included RCTs. Other study limitations include the low response rate of UK Biobank, which may bias MR estimates, and the lack of suitable data to test whether the effects of genetic instruments on maternal calcium levels during pregnancy were the same as those outside of pregnancy.ConclusionsOur results suggest that maternal circulating 25(OH)D does not influence birth weight in otherwise healthy newborns. However, the effect of maternal circulating calcium on birth weight is unclear and requires further exploration with more research including RCT and/or MR analyses with more valid instruments.

研究背景
针对随机对照试验（randomised controlled trials, RCT）的系统综述显示，母体补充维生素D（25[OH]D）与钙剂可提升新生儿出生体重。但上述综述指出了多项纳入试验的局限性。本研究旨在整合遗传数据与RCT数据，评估这两种母体特征对子代出生体重的因果效应。

研究方法与结果
我们依托全基因组关联研究（genome-wide association studies, GWAS）中已鉴定的、与25羟维生素D及钙剂相关的遗传工具变量，开展两样本孟德尔随机化（two-sample Mendelian randomisation, MR）分析（样本1：25羟维生素D对应样本量N=122123，钙剂对应样本量N=61275）。随后，我们在英国生物库（UK Biobank, UKB，样本2：N=190406）中计算了这些母体遗传变异与子代出生体重的关联。我们使用来自两个英国出生队列的母婴配对数据（合并样本量N=5223）开展敏感性分析，以检验结果是否受与母体基因型存在约0.5相关性的胎儿基因型的影响。为进一步验证结果的可靠性，我们还开展了MR-Egger分析、加权中位数估计量分析、留一法分析以及多变量MR分析。我们将MR分析结果与RCT研究结果进行三角验证：以维生素D补充（24项RCT，合并样本量N=5276）与钙剂补充（6项RCT，合并样本量N=543）的随机分配作为工具变量，以此推断25羟维生素D与钙剂对出生体重的效应。
在主MR分析中，未发现母体25羟维生素D对出生体重存在显著效应的强证据：母体25羟维生素D每升高10%，平均出生体重差值为-0.03g（95%置信区间：-2.48~2.42g，p=0.981）。该效应估计值在各项MR敏感性分析中保持一致。针对RCT的工具变量分析提示存在微弱的正向因果效应（母体25羟维生素D每升高10%，效应值为5.94g，95%置信区间：2.15~9.73g，p=0.002），但由于纳入的RCT存在偏倚风险，该结果可能被夸大。
针对母体钙剂的主MR分析同样未发现其对出生体重存在显著效应的强证据：母体钙剂水平每升高1个标准差，平均出生体重差值为-20g（95%置信区间：-44~5g，p=0.116）。部分敏感性分析提示，钙剂的遗传工具变量可能通过与钙剂水平无关的暴露因素影响出生体重，即水平多效性。针对RCT的工具变量分析提示，钙剂对出生体重存在显著效应（母体钙剂水平每升高1个标准差，效应值为178g，95%置信区间：121~236g，p=1.43×10^-9），该结果与所有MR分析结果均不一致。然而，由于纳入的RCT存在偏倚风险，该RCT工具变量估计值可能被夸大。
本研究的其他局限性包括：英国生物库的应答率较低，可能对MR估计结果造成偏倚；以及缺乏合适的数据以检验遗传工具变量对妊娠期间母体钙剂水平的效应，是否与非妊娠期间一致。

研究结论
本研究结果提示，母体循环中的25羟维生素D并不会对健康新生儿的出生体重产生影响。然而，母体循环钙剂对出生体重的效应尚不明确，需开展更多研究（包括RCT及/或使用更有效工具变量的MR分析）以进一步探索。