Microcephaly is an abnormality that can be congenital or postnatal, primary or secondary, isolated or multiple. It is therefore a challenge to evaluate its etiology. 1 Both genetic and environmental factors, such as radiation, intrauterine infections, alcohol consumption, drug ingestion, and trauma have been linked to microcephaly, but for isolated microcephaly major etiologic factors remain unknown. 2 We used population-based data from the Hungarian Case-Control Surveillance of Congenital Abnormalities 3,4 to examine the etiologic factors, particularly environmental agents, related to isolated microcephaly.
Subjects and Methods
We identified cases from reports to the national dataset of the Hungarian Congenital Abnormality Registry between 1980 and 1996. 5 Notification by physicians of cases with congenital abnormalities is mandatory. Most reports are from obstetricians, given that in Hungary practically all deliveries occur in inpatient obstetric clinics, or from pediatricians who are working in the neonatal units of inpatient obstetric clinics and various inpatient and outpatient pediatric clinics. During the study period, autopsy was required for all infant deaths and was usual for stillborn fetuses. Pathologists sent a copy of each autopsy report to the Registry if congenital abnormalities were identified. The recorded total prevalence of cases with congenital abnormalities diagnosed from the second trimester of pregnancy through the age of 1 year was 35 per 1,000 informative offspring (liveborn infants and stillborn and malformed fetuses from electively terminated pregnancies). About 90% of major congenital abnormalities were reported to the Registry during 17 years of the study period. 5
The dataset of the Hungarian Case-Control Surveillance of Congenital Abnormalities includes cases with isolated and multiple congenital abnormalities reported during the first 3 months after birth or after termination of pregnancy. This shorter time between the birth or termination and data collection increases the accuracy of the information about pregnancy history without undue loss of precision, given that 77% of cases were reported during this time window to the Registry. We also excluded three mild congenital anomalies (congenital dislocation of hip based on Ortolani click, congenital inguinal hernia, and hemangiomas), minor variants (for example, umbilical hernia), and syndromes of known origin (except Down). Hungarian physicians usually diagnose microcephaly if the head circumference is below the tenth percentile for the given sex and age, using the Hungarian population standards. We selected cases with isolated microcephaly. In the majority of these cases, the diagnosis of primary microcephaly was made at birth. 6
We defined population controls as infants matched to each case according to sex, week of birth in the year when cases were born, and district of parental residence from the birth registry of the Central Statistical Office. From the years 1980 through 1985, we selected two controls for each case and, after 1985, three controls for each case. About 3% of matched controls had some congenital abnormalities, and we excluded these infants from the study. We also identified patient controls, cases with Down syndrome, from the Registry.
We collected data on etiologic risk factors from three sources. First, we sent a post-paid questionnaire to the parents of cases, population controls, and patient controls. The questionnaire requested information on pregnancy complications, family history, maternal diseases, and drugs taken during pregnancy, according to gestational months. We obtained no information regarding smoking and alcohol use because of the expected low quality of these data. We included a list of drugs and diseases as a memory aid and asked mothers to review these lists before they replied. Completed questionnaires were returned on average at 1.6 and 1.8 postnatal months for the cases and patient controls, respectively, and at 3.5 postnatal months for the population controls. Second, mothers were requested to send us the prenatal care logbook and all other medical documents concerning their child’s defect, as well as their diseases and drug intakes specified during the study pregnancy. In Hungary, prenatal care obstetricians are required to record all prescribed drugs, pregnancy complications, and diseases in the logbook, which is similar to the prenatal record sheet in the United States. Third, regional district nurses visited and interviewed the nonresponding families of cases and patient controls. In total, information was available for 82% of cases (74% from mailed responses and 8% from visits) and 81% of patient controls (72% from mailed responses and 9% from visits). The response rate was 65% from population controls; however, district nurses did not visit nonrespondent families of population controls, because the ethical committee considered this follow-up to be disturbing to the parents of healthy infants. Of 109 cases in the study, 69 had three matched population controls. In the analysis, we randomly selected two controls for each case. Thirty index cases had two matched population controls each. Because ten cases had only one matched population control each, we selected a second matched control from the rest of the 38,151 population controls.
We used Stata statistical software for data analysis. 7 For the continuous data, we calculated mean values. For categorical data, we estimated prevalence odds ratios (PORs) and their 95% confidence intervals. We used conditional logistic regression to adjust for potential confounders.
There were 109 infants with isolated microcephaly diagnosed at birth. Each case had two matched population controls (N = 218). There were 812 patient controls with Down syndrome.
Table 1 shows the demographic characteristics of newborn infants and their parents. Cases were more likely to be female, part of a twin birth, and of low birth weight.
Cases, population, and patient control groups had similar rates of pregnancy complications, such as excessive nausea/vomiting (10.1%, 8.3%, and 7.0%, respectively), threatened abortion (12.8%, 19.7%, and 14.4%), and preterm delivery (7.3%, 6.4%, and 7.8%). These groups were also similar with regard to the occurrence of common colds (13.8%, 17.4%, and 14.2%), influenza (9.2%, 5.5%, and 7.0%), pharyngitis/tonsillitis (5.5%, 8.3%, and 6.4%)/ and cystitis/pyelonephritis (8.3%, 8.7%, and 7.1%). We examined the effects of drugs that were used by at least three case mothers and adjusted these effects for maternal age, birth order, and maternal diseases (Table 2). Whereas we found a 70% decreased odds of microcephaly among children whose mothers used clotrimazole, this effect was not seen in the analysis using patient controls. We did see an approximate tripling of the odds of microcephaly among cases whose mothers used either nitrofurantoin or phenoxymethylpenicillin. We also saw a modest decrease (30–40%) in the odds of microcephaly associated with maternal use of penamecillin. For drugs related to pregnancy complications, none of the elevations of odds that we observed were found consistently in the analyses with both population and patient controls. We did see a modest decrease (30–40%) in the odds of microcephaly associated with maternal use of drotaverine.
The estimated effects of the fourth group of drugs included, the so-called nutritional supplements during pregnancy , were quite consistent across the analysis with the two control groups (Table 3). Maternal use of iron and folic acid decreased the odds of microcephaly. In Hungary, the available iron supplements included 50, 70, and 100 mg of ferrous sulfate in one tablet; however, 2 tablets of 50 mg (including 10 mg elementary Fe 2) per day were generally suggested. Only one kind of folic acid tablet (3 mg) was available during the study period. The general recommendation was 2 tablets per day after the first prenatal visit (8th through 12th week of gestation). Maternal use of vitamin B1 was associated with higher odds of microcephaly, but this effect was not estimated with precision. When evaluating only medically documented drug treatments, we found the adjusted POR of microcephaly to be lower with maternal use of folic acid (with both control comparisons), iron, and clotrimazole when comparing cases and population controls.
In general, folic acid and iron are used together during pregnancy; therefore, the supplementation of iron and particularly folic acid alone is rare. Thus, it is difficult to evaluate these supplement subgroups separately (Table 4). In comparing cases with matched population controls and patient controls, whose mothers took iron but did not take folic acid, the adjusted PORs were 0.5 and 0.7. On the other hand, the adjusted POR was 0.9 when mothers took only folic acid, but the numbers were too small. Iron and folic acid, however, were rarely used together by the mothers of cases compared with mothers of both matched population and patient controls.
When we evaluated family history of cases, we considered only microcephaly and first-degree relatives. Of 109 index cases, 7 (6.4%) had 9 affected first-degree relatives (2 index cases had affected mother/brother and brother/sister pairs). Of 109 mothers and fathers, 2 and 0 had microcephaly, respectively. Forty-four index cases had no siblings. The rest of the cases had 136 siblings, and 7 (5.1%) were affected with microcephaly. Of the 7 (2 males and 5 females) familial microcephalic cases, 2 of the mothers had taken both folic acid and iron, 1 only folic acid, and 1 only iron, and none had taken clotrimazole. The previously presented results were not changed after the exclusion of cases with positive family history, however.
This article provides estimates of the effects of a wide range of prescription drugs and nutritional supplements taken during pregnancy on the odds of isolated microcephaly. Data are from the population-based Hungarian Case-Control Surveillance of Congenital Abnormalities dataset. Our previous studies have indicated a misdiagnosis rate of 5.2% among recorded microcephalic cases of the Registry. 6
The female excess in cases with isolated microcephaly needs further study to determine whether it is a general characteristic or a chance effect. The higher rate of low-birth weight newborns and lower mean birth weight indicate intrauterine growth retardation, given that preterm birth and gestational age were not risk factors for microcephaly. The question is whether retardation of brain development is part of intrauterine growth retardation or is independent.
The evaluation of acute infectious maternal diseases did not indicate a detectable risk for isolated microcephaly; however, use of the antimycotic clotrimazole demonstrated some protective effect. The treatment of vulvovaginal candidosis and complementary infections during pregnancy can prevent preterm delivery 8 and related fetal complications, such as undescended testis. 9 Nevertheless, an association between intrauterine growth retardation and isolated microcephaly was found in this and other studies. 1 The possible effect of drugs such as nitrofurantoin, penicillins, and others on incidence of microcephaly is unclear, however.
Maternal use of folic acid and iron appears to have a preventive role in the development of isolated microcephaly. Periconceptional multivitamin (including physiological dose of folic acid, that is, less than 1 mg) supplementation can reduce both the first occurrence 10,11 and recurrence of neural-tube defects, 11,12 as well as reduce the occurrence of some other major congenital abnormalities, particularly malformations of the urinary tract, 13–15 cardiovascular system, 13,14,16,17 and limb deficiencies. 13,16,18,19 Although multivitamins, including 0.8 mg of folic acid, were not effective for the prevention of orofacial defects, 10,14 the high dose of folic acid alone was able to reduce the first occurrence 20,21 and recurrence 22 of cleft lip with or without cleft palate and cleft palate alone. Our study suggests that the pharmacological dose of folic acid (in general 6 mg per day) may be protective against microcephaly. A high rate (about 35%) of microcephaly has been found among aboriginal Australian children who had a 40% prevalence of iron deficiency anemia. Supervised iron and other nutritional supplementation may lead to catch-up growth in head circumference. 23
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