Characteristics of Included Studies
Our systematic review identified 4,106 records, from which 298 duplicates were removed and 3,808 titles and abstracts were screened. After this process, the reviewers identified 150 unique studies that seemed to be relevant to answer our question and those articles were assessed in full-text for eligibility. From those, 138 studies were excluded based on the exclusion criterias. After full-text review, a total of 12 observational studies were included in the synthesis of the litterature[29–40] (Figure 1). Of the 12 eligible studies, six authors provided the primary data [29,34,37–40], and thus were included in the meta-analysis. This process is summarized in Figure 1.
The twelve included studies were published from 2015 to 2018, ten of them had Research Ethics Board approval and 2 declared to be exempt [30,39]. Five were cohort studies[30,33–36], three were case-controls studies [31,32,38], three were case-series [29,37,39] and one was a cross-sectional study . The studies defined as case-series [29,37,39] were conducted based on surveillance data - local surveillance system and hospital based surveillance –, and reported only ZIKV infected cases with different outcomes. For that reason, since it was possible to compare the microcephaly group and the non-microcephaly group, for the meta-analysis we considered them as “prospective observational studies” in the same group as the cohort studies of PZIK.
All the 12 studies were conducted on the American Continent, three of them in the United States [30,36,38], one in the Caraibbean and eight in South America (Brazil, French Guiana and Colombia). The total population enrolled in the 12 included studies is 6,154 children/foetuses. From those, 1,120 (18.20%) had a diagnosis of ZIKV infection, of whom 509 (45.45%) were diagnosed with microcephaly. Most of the studies [30–34,36,38–40] addressed the link between PZIK and neurological findings or other outcomes in foetuses/children, however the laboratory confirmed ZIKV infection was not consistent in all the studies.
The assessment of PZIK varied among studies. From the 12 included studies included in this review, one  also included women that did not perform any laboratory test to determine ZIKV infection. In this study, the definition of ZIKV infection was due to epidemiological link and clinical characteristics, as accepted by the Ministry of Health. Regarding laboratory evidence, six tested the mothers/ pregnant women [33,35–39], and six tested both [29–32,34,40] (Table 1). RT-PCR was used in seven studies [29,30,33–37] in at least one phase of the diagnosis, but only two of them used this test as the confirmation tool for all the cases [29,33]. Plaque reduction neutralisation test (PRNT) was used in five studies [30–32,34,36] and 10 used serological test to detect either IgG or IgM antibodies [29–32,34–38,40].
The microcephaly definition varied across studies, along the time and between the studies. Regarding the moment of the detection, the microcephaly was diagnosed after delivery in all the 12 studies, but three of them also performed fetal ultrasounds [29,33,35] to detect microcephaly.
Based on the NOS, four studies [30,33,38,40] were deemed to be of good quality and seven [29,31,32,34–36,39] were of satisfactory quality. We summarized the characteristics of all the 12 selected studies in Table 1 and the characteristics of the population enrolled in Table 2.
Regarding the quantitative synthesis, six authors provided the primary data that was used in the meta-analysis. Three of them [34,37,39] were cohorts (total N = 1593), two [29,38] were case-controls (total N = 18) and one study with 32 cases was cross-sectional . The total number of participants that had microcephaly were 638 (40.05%) in the prospective studies and 12 (85.71%) in the retrospective studies. Ventura et al  also provided the primary data, but since it is the only cross-sectional study, we did not include their results in the meta-analysis. It was not possible to explore publication bias, since there was less than four studies included using the same methodology, most of them with small sample sizes.
The 12 selected studies reported a higher risk of microcephaly in the presence of ZIKV infection during gestation, with an Odds Ratio (OR) as high as 21.9 (95% Confidence Interval - CI of 7. 0, 109.3)  and a Relative Risk (RR) of 6.63 (95% CI, 0.78, 57.83)  when compared to no ZIKV infection during gestation. Whenan alysing only cases with ZIKV infection during gestation, microcephaly was prevalent in up to 54.82% of the children enrolled in one study . Considering the 705 foetuses/newborns diagnosed as ZIKV positive, whose mother’s had symptoms of ZIKV infection during pregnancy described in the published papers [24–32, 34, 35], we found a prevalence of microcephaly of 52.63% (CI95% = 48.3, 56.95) in the symptomatic group versus a prevalence of microcephaly of 45.64% (CI95% = 41.02, 50.26) in the asymptomatic group.
Schaub et al. , reported 14 cases of ZIKV infection during gestation. They found microcephaly in nine (64.28%) of them. But, only one of the pregnancies resulted in a livebirth (newborn at 40 weeks with microcephaly) and one case of intra-uterine death at 25 weeks. The 12 other participants had termination of pregnancy varying on 18 weeks and 3 days to 34 weeks of gestation. For that reason, the data on “gestational age at birth” of this study was not included in the analysis.
Assesed Risk Factors
The symptoms of ZIKV infection during pregnancy were assessed in all studies except in Kumar et al , which performed laboratory analyses of stored plasma samples from mothers who gave birth to babies with microcephaly and healthy babies collected prior to ZIKV became linked with microcephaly. Overall, symptoms of ZIKV infection during gestation were present in 705 of the 1116 pregnant women infected (63.17%). (n =.. From the studies with available informationta [31–35,37,39,40], 270 of the 513 women who reported symptoms during pregnancy (52.63%), delivered an infant with microcephaly.
The trimester of pregnancy when the infection occured was assessed in eight studies [30,31,33–35,37,39,40]. From the cases of ZIKV infection during the first trimester (N = 324), 42.59% exhibited microcephaly. Among those with ZIKV infection during other stages of pregnancy [second trimester (N = 332) and third trimester (N = 141)], 21.99% exhibited microcephaly.
Sanz Cortes et al.  and Schaub et al.  informed maternal nutritional status [mean maternal Body Mass Index (BMI) of 24.38 kg/m2 (SD 5.56) and 26.54 kg/m2 (SD 5.76), respectively]. The mean maternal BMI in the microcephaly group was 25.88 kg/m2 (SD 3.83) in the study of Sanz Cortes et al. , and 27.84 kg/m2 (SD 6.77) in the study of Schaub et al. ; while the mean maternal BMI for the non-microcephaly group was 19.89 kg/m2 (SD 8.43)  and 24.20 kg/m2 (SD 2.39) .
Although Vargas et al.  measured all the variables of interest, they mentioned that five (8.3% of the total population) cases were due to other congenital infection and did not explore the data separately. For that reason, it was not possible to use their data separately. For that reason, it was not possible to use their data for analysis of PZIK.
Concerning comorbidities, other infections were excluded in most of the studies (7/12). Infections known to have teratogenic effects such as syphilis, toxoplasmosis, rubella, cytomegalovirus and herpes simplex (STORCH) were excluded in six studies [29,33–35,37,40]; dengue virus infection in four [29,30,32,33]; HIV in four [29,33,35,40]; chikungunya in two [24, 28]; parvovirus in one ; and other sexually transmitted infections in one .
Three studies [33,35,40] provided information regarding the consumption of licit or illicit drugs during pregnancy. Sanz Cortes et al. used these exposures as exclusion criteria, Brasil et al.  informed that all included women reported no medication use and Ventura et al.  reported four cases (12.5%), all of them in the microcephaly group (13.79% of the microcephaly outcomes), but didn’t mention which particular drug was assessed.
Three studies reported the presence of singleton versus multiple gestation [30,33,34] and the delivery method among the PZIK cases was reported in two studies. Brasil et al. (2017), reported a C-section rate of 82.4% (N = 89/108), and Sanz Cortes et al. (2018), of 66.67% (N = 6/9).
Gestational age at birth in newborns with microcephaly due to PZIK was not provided in four studies [30,32,37,39]. One study  had only one newborn (7%), 40 weeks at birth, all other analysed cases (13 cases, 93%) had a termination of pregnancy at different times of the gestational outcome. Aragão et al. and Sanz Cortes et al. presented the mean and SD of all the population included, finding respectively 36.29 (SD 8.71) weeks of gestation and 37.8 (SD 1.15) weeks of gestation. The study of Brasil et al., provided the gestational age at birth of the 58 (43.3%) participants who had any abnormal finding at birth. From those, four (6.9%) were in the microcephaly group and two of them were born preterm. From the non-microcephaly group, 11.63% (n = 5) newborns were born preterm. Shiu et al. (2018), reported data of 86 women with laboratory evidence of PZIK. They did not provide the data regarding the presence or absence of microcephaly, but 34 (39.5%) of the participants were still pregnant by the time of the report, 8 (9.3%) had preterm delivery and 44 (51.1%) had term delivery. From the remaining population (N = 314) [34,38,40], the studies provided mean and SD. The weighted mean and SD from the microcephaly group (n = 60) was 37.91 (SD 2.72) gestational weeks at birth and from the infants in the non-microcephaly group was 38.06 (SD 2.42).
Ventura et al. provided data on 148 cases of PZIK, 140 (94.6%) of these infants with the microcephaly status (presence or absence) reported. From those, 124 (88.6%) infants presented microcephaly. In the microcephaly group, 56.45% (n = 70) were female, while in the non-microcephaly group, the majority were males (n = 9/16, 56,25%). The information on maternal symptomatology of PZIK was available for 132 infants (116 with microcephaly). From them, 108 women reported symptoms (such as rash, pruritus and conjunctivitis) being 97 (83.6%) of which belonged to the microcephaly group. The use of licit or ilicit drugs was available for 132 maternal (118 in the microcephaly group) and 13 mother’s reported the use of these substances. From the microcephaly group, 12 (10.2%) mothers reported this behavior and from the non-microcephaly group, one mother (7.1%) reported it. As for the gestational trimester of infection, most of the participants in the microcephaly group were infected in the first trimester (n = 48, from 100 with this information available) and the majority (n = 7, from 13 with this information available) of the non-microcephaly group had ZIKV infection in the second trimester. There was no sufficient data on maternal scholarity and previous vaccines (Yellow Fever vaccine or other vaccines) to conduct an analysis. Regarding the methodological quality, this study was assessed as good quality.
We thus, conducted meta-analysis to assess the rate of microcephaly detection according to seven identified characteristics: (i) sex (proportion of boys); (ii) maternal age; (iii) maternal ethnicity (proportion of non-white); (iv) gestational age at birth; (v) presence of symptoms during gestation; (vi) presence of comorbidities; (vii) gestational trimester of infection; and (viii) smoking habits and/or alcohol or other drugs consuption.
Regarding the meta-analysis of prospective studies, only three variables showed to be significant (presented in Figure 2). In relation to foetus/infant’s sex, females presented a lower risk of microcephaly compared to males (RR 0.79; 95% CI 0.70, 0.88; I2 = 0%) (Figure 2a). Infection in the first trimester of pregnancy (Figure 2b) was a risk factor (RR 1.42; 95% CI 1.09, 1.84, I2 = 0%) for microcephaly, compared to infection in the second and third trimesters of pregnancy. A decrease in the microcephaly detection rate risk was observed in women who did not presented symptoms of PZIK (RR 0.68; 95% CI 0.60, 0.77; I2 = 38%), such as conjunctivitis, prutitus and rash (Figure 2c).
Although not significant, the Maternal ethnicity—white (RR 0.91; 95% CI 0.77, 1.08; I2 = 0%) and no smoking habits and/nor alcohol nor other drugs consuption (RR 0.84, 95% CI 0.55, 1.29, I2 = 0%) had indicated, in a point estimate, to be protective. Maternal age and gestational age at birth—analysed using mean and SD, were similar between groups. The meta-analysis data of the factors that did not significantly increase the risk are illustrated in the Additional Figure 1.
As to methodological quality of the prospective studies included in the meta-analysis, França et al.  was the only included study assessed as low quality. Pomar et al. and Vargas et al.  were considered as satisfactory quality.
In relation to the retrospective studies[29,38], Kumar et al. tested archived blood samples collected at delivery at the Kapiolani Medical Center for Women and Children in Hawaii; and Schaub et al. investigated 12 cases diagnosed during pregnancy, but 11 cases resuted in pregnancy termination and only one live birth. Only infant’s sex could be tested as an exposure factor. There was a decrease of the Odds Ratio (OR) of microcephaly in females, even though it was not significant (OR 0.54; 95% CI 0.08, 3.66, I2 0%) (Additional Figure 2a). It was not possible to analyse the data on trimester of infection, presence of symptoms, smoking habits and vaccines exposure since only one study had this data vailable. Also, maternal age, maternal ethnicity and presence of comorbidities were not estimated, since the study of Kumar et al. had only one case without microcephaly and Schaub et al. included only non-white individuals without comorbidities (Additional Figure 2b). With reference to quality assessment, Kumar et al. was assessed as good methodological quality and Schaub et al. as satisfactory methodological quality.