This systematic review suggests that the evidence for an association between water fluoride level and the incidence of Down's syndrome is weak, and that all the identified studies were of poor quality. All results, positive and non-positive, should therefore be considered together with the methodological weaknesses of the studies which could have lead to spurious results. In particular, the results of the two studies [16
] which showed a significant positive association with water fluoride level should be interpreted with extreme caution due to the methodological limitations of these studies discussed below.
The major weakness of these studies was the failure to control sufficiently for confounding factors. All six studies used study designs that measured population rather than individual exposure to fluoridated water and because of this are particularly susceptible to confounding. If the populations being studied differed in respect to other factors that are associated with the outcome under investigation then the outcome may differ between these populations leading to an apparent association with water fluoride level. [19
] The incidence of Down's syndrome is known to be strongly associated with maternal age. [20
] If the average maternal age of the high fluoride population is higher than that of the low fluoride population an association with water fluoride level would most likely be found, even if such an association does not in fact exist. Maternal age was considered by all but one of the included studies, however only two of the six studies appropriately controlled for the effects of maternal age. The two studies [16
] which found a positive association between Down's syndrome and water fluoride level were two of the studies which did not control appropriately for the possible confounding effects of maternal age and so the results of these studies should be interpreted with some degree of caution. Another factor which may affect the association of Down's syndrome with water fluoride level is maternal exposure to other sources of fluoride, such as fluoridated toothpaste, mouthwashes and fluoride tablets. None of the studies controlled for or measured any of these factors.
Other factors which could have led to misleading study results include selection of study areas, ascertainment of cases, population selected for the denominator, migration, classification of exposure and blinding of investigators to the fluoridation status of cases. If study areas are not selected at random there is a possibility that selection may be biased, for example, a fluoridated area with a relatively high incidence of Down's syndrome (possibly for reasons other than fluoride concentration of the water) and a non-fluoridated area with a relative low incidence may be selected which would result in biased results.
Case ascertainment must be as complete as possible and must be uniform across study areas otherwise cases in one area may be more likely to be identified than those in another area and possibly result in a misleading finding. All but one [16
] of the studies attempted to locate all cases born in the study areas during the study period by searching a variety of sources, these studies all state that they believe that they located the majority of cases. The other study [16
] limited case ascertainment to live cases living in institutions and hospitals. Limiting the cases in this way may result in a large proportion of cases (more than half) being missed. [14
] This would be a particular problem if the proportion of cases identified differed between the different areas, for example if a higher proportion of cases lived in institution in the fluoridated area compared to the control area this would result in a misleading association. Also, if there more deaths among people with Down's syndrome in one area than another this could result in fewer living people with Down's syndrome in one of the study areas, leading to a possibly biased association.
The population selected for the denominator may also affect the associations found. One of the two studies [16
] which found a positive association used the total population of the study areas as the denominator while all the other studies [13
] used the number of live births as the denominator. For studies of birth defects it is more usual to use the total number of births as the denominator. If the population structure of two areas differ, with one area having a higher proportion of women of childbearing age, then the birth rate in this area will also be higher and thus the incidence of birth defects, such as Down's syndrome, is likely to be higher. Using this figure as the denominator can thus lead to false conclusions.
Classification of exposure is another area where bias can be introduced. Down's syndrome is a genetic defect that occurs at around the time of conception [2
] and so water fluoride exposure should be classified according to the area in which the mother was resident at the time of conception. Only one study classified exposure at the time of conception, [15
] the others classified exposure at the time of birth, this may lead to the misclassification of births to mothers who moved during their pregnancy. The length of exposure to fluoride necessary to have an effect could be several years in which case the exposure should be classified as women exposed or not exposed to water fluoride for a certain number of years prior to conception. Exposure was not classified in this way in any of the included studies.
The effects of migration were not discussed in any of the studies. Whether migration could bias study results depends on when the water fluoride level is thought to have an effect on the woman: whether it is a long term build up or a short term effect around the time of conception. If it is the latter then as long as exposure status was identified as exposure at time of conception not birth this should not a problem. However, if there is a fluoride effect with a long induction period, any study of this effect would have to take account of migration.
Investigators should be blinded to the fluoridation status of the cases that they are identifying otherwise their views on fluoridation may affect the thoroughness of their search for cases. For example, if an investigator believes that there is an association between water fluoride level and down's syndrome, and knows that the sources they are searching to identify cases relate to cases whose mothers have been exposed to high levels of water fluoride, they may be more thorough in their search for cases. None of the studies mentioned blinding of investigators.
The studies included in the review were all conducted at least 20 years ago. This may be a problem in generalising results to the present time if factors that would affect the incidence of Down's syndrome, and especially its association with water fluoride levels, have changed in that time. It may be that if fluoride has an effect on the incidence of Down's syndrome the mother has to be exposed to fluoride over a long period of time. Fluoridation was first initiated in the 1940s [21
] thus many of the women included in these studies may only have been exposed to water fluoride for a short period of time. Another factor which has changed since most of these studies were conducted is the total fluoride exposure of the mothers, fluoride is now available from other sources, to which women would not have been exposed in the earlier studies. Other factors which may affect the incidence of Down's syndrome is the changing demographics of maternal age at birth, with women in the developed world now giving birth at older ages than they did 20 years ago. [22
] Abortion is now more acceptable [23
] and screening for Down's syndrome is routine, especially in older women, [24
] and so the option to terminate a birth if the child is diagnosed with Down's syndrome is now a possibility.