Table 2.

CAUSAL INFERENCE APPROACH		APPROACH SUMMARY	BIASES ADDRESSED	STRENGTHS	POTENTIAL LIMITATIONS	EXAMPLE	SCHEMATIC
RANDOMIZED CONTROL TRIAL (RCT)		Subjects are randomly allocated to either exposure or control groups with assumption that there is no difference between the two groups except for the intervention they are receiving.	Confounding, reverse causality, selection bias, loss-to-follow up bias (using intention-to-treat analysis), measurement error	Gold standard for estimating causal effects. Any effect is very likely to be causal if study has large number and trial is reliably performed.	Generalizability may be questionable; impossible or unethical to randomize to certain exposures; can be expensive	Association between maternal breastfeeding behaviour and childhood outcomes 27–33	a
CROSS-COHORT COMPARISON		Associations are compared between two or more populations with markedly different confounding structures. If the observed association is causal, it should be present in both cohorts.	Confounding, Selection bias	Exploring residual confounding. Reliable findings if cohorts are markedly different and have large sample size.	Assumptions about different confounding structures may not be correct; need for variable harmonisation between cohorts	Association between breastfeeding and IQ, obesity and blood pressure in two cohorts 34	b
NEGATIVE CONTROL APPROACHES (approach used to rule out possible non-causal interpretation of results by performing study where hypothesised causal mechanism is removed; expected to produce null result)	CONTROL EXPOSURE	Effect of an exposure is compared with the effect of another exposure with similar confounding. Causal inference is strengthened if an association is seen with the exposure being tested and not with the similar exposure.	Confounding	Provides information on the specificity of the exposure being tested, and whether the observed association is simply due to confounding by other associated factors and study biases.	Assumption about the structure of confounding for negative control, some uncontrolled confounders	Association between maternal use of folic acid supplements (and comparison with fish oil) and risk of autism in children 37	c
	CONTROL OUTCOME	An outcome that shares similar confounding as the original outcome, but is unlikely to be influenced by the exposure of interest. Causal inference is strengthened if an association is seen with the outcome being tested and not with the similar outcome.	Confounding	Provides information on the specificity of the outcome being tested, and whether the observed association is simply due to confounding by other associated factors and study biases.	Assumption about the structure of confounding for negative control, some uncontrolled confounders	Association between use of folic acid supplements in pregnancy and severe language delay in children (and comparison with motor skills) 127
	NATURAL EXPERIMENT	Empirical study approach where a population is exposed to an external event or intervention at a specific time point. Associations are then compared with a similar cohort who was not exposed. The assumption is that exposure is caused by quasi-random assignment.	Confounding, reverse causality	Can study settings which would be impractical or unethical to produce by researchers	Selection bias as exposure cannot be manipulated by researcher, some unobserved confounding may remain	Association between imposed nutritional deprivation during early pregnancy on a number of health outcomes, compared with those who experienced famine at other stages in pregnancy 39
	PARENTAL COMPARISON	Maternal-child association is compared with paternal-child association for inferring causal effect of intrauterine exposure. If causal, maternal association is stronger than paternal association. Where associations are similar for both parents we assume that they are driven by genetic or postnatal environmental characteristics.	Confounding	Improves causal inference of intrauterine effect if exposures are measured in both parents at same time in pregnancy, and non-paternity is taken into account for phenotypic traits	Assumption that paternal exposures share same confounding structure as maternal exposure may not be correct; where parental associations are similar magnitude this may be due to offsetting paternal pathways rather than shared confounding	Association between parental smoking and birth weight 18
SIBLING COMPARISON		Compares outcomes when siblings are discordant for an exposure. If causal then there will evidence of a difference in outcome in relation to discordant exposure levels within sibships.	Confounding	Improves causal inference of intrauterine exposures. Controls for familial background and related confounding factors.	Assumes a stable family environment; confounding by factors not perfectly shared by siblings; potential for measurement error of exposure; limited power	Effect of maternal gestational diabetes on height, weight and BMI of offspring and their siblings 55	d
MENDELIAN RANDOMIZATION (MR)		MR is the use of a genetic variant robustly associated with an exposure/risk factor of interest as an instrumental variables to test and estimate the causal effect of that exposure/risk factor with a disease or health related outcome.	Confounding, reverse causality, selection bias, measurement errors, generalizability	Genetic instruments are not subject to confounding from environmental or lifestyle factor, are not influenced by the outcome, do not change over time and are measured with high accuracy	Low power, lack of instrumentation, pleiotropy and linkage disequlibrium, population stratification, non-linear associations, developmental canalisation	Association between maternal MTHFR variants and risk of neural tube defects (NTD) in offspring 74	a, e
NON-GENETIC INSTRUMENTAL VARIABLE (IV)		Similar to MR except that it uses a phenotype rather than a genetic variable as an IV for obtaining and estimating causal effects.	Sometimes confounding, reverse causality	IV is associated with the outcome only through association with exposure and will typically not be associated with confounding factors	True exogenous factors are generally rare or of small effect. When exposure in one family member is used as IV for exposure in another family member, residual confounding is likely.	Association between own BMI and mortality using son’s BMI as an IV 83	e