Characteristics of Included Studies
Our systematic review identified 4106 records, from which 298 duplicates were removed and 3808 titles and abstracts were screened. After this process, considering the eligibility criteria, the reviewers identified 150 unique studies that seemed to be relevant to our question, and those articles were assessed in full-text for eligibility. From those, 138 studies were ruled out based on the exclusion criteria. After the full-text review, a total of 12 observational studies were included in the synthesis of the literature [29-40] (Figure 1). Of the 12 eligible studies, six authors provided the primary data [29, 34, 37-40], and therefore were included in the meta-analysis. This process is summarized in Figure 1.
The twelve included studies were published between 2015 and 2018; ten of them had Research Ethics Board approval and two declared themselves exempt from this approval [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 varied outcomes. For that reason, as 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 12 studies were conducted in the Americas: three of them in the United States [30, 36, 38], one in the Caribbean ,, and eight in South America, i.e., Brazil [31-33, 37, 39, 40], French Guiana , and Colombia .. The total population enrolled in the included studies is 6154 newborns/fetuses. From those, 1120 (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 newborns/fetuses; however laboratory-confirmed ZIKV infection was not consistent across all the studies.
The assessment of PZIK varied among studies. One of the included studies  also included women who did not undergo a laboratory test to determine ZIKV infection. In this study, ZIKV infection was defined based on 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 studies tested both mothers/pregnant women and newborns/fetuses [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]. The plaque reduction neutralization test (PRNT) was used in five studies [30-32, 34, 36] and 10 used serological testing to detect either IgG or IgM antibodies [29-32, 34-38, 40].
Microcephaly definitions varied across studies, changing over time. Regarding the moment of detection, microcephaly was diagnosed after delivery in all the 12 studies, but three of them [29, 33, 35] also performed fetal ultrasounds 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 summarize 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. Of these, three [34, 37, 39] were cohort studies (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 newborns/fetuses 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 these results in the meta-analysis. It was not possible to explore publication bias, as less than four included studies used the same methodology, most of them with small sample sizes.
The 12 selected studies reported a higher risk of microcephaly with the presence of ZIKV infection during gestation, with an Odds Ratio as high as 21.9 (95% Confidence Interval – CI of 7. 0, 109.3)  and a Relative Risk of 6.63 (95% CI, 0.78, 57.83)  when compared to no ZIKV infection during gestation. When analysing only cases with ZIKV infection during gestation, microcephaly was prevalent in up to 54.82% of the infants enrolled in one study . Considering the 705 newborns/fetuses diagnosed as ZIKV positive and whose mothers 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 live birth (born at 40 weeks with microcephaly), with one case of intra-uterine death at 25 weeks. The 12 other cases had a termination of pregnancy varying from 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.
Assessed Prognostic 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 microcephalic and healthy babies, collected before ZIKV was linked with microcephaly. Overall, symptoms of ZIKV infection during gestation were present in 705 of the 1116 pregnant women infected (63.17%). From the studies with this available information [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 occurred 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.  reported 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]. Sanz Cortes et al. reported the mean maternal BMI in the microcephaly group and non-microcephaly group as 25.88 kg/m2 (SD 3.83) and 19.89 kg/m2 (SD 8.43), respectively . Schaub et al. reported mean maternal BMI as 27.84 kg/m2 (SD 6.77) in the microcephaly group and 24.20 kg/m2 (SD 2.39) in the non-microcephaly group .
Although Vargas et al.  measured all the variables of interest, they mentioned that five (8.3% of the total population) cases of microcephaly were due to other congenital infections and did not explore the 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 substances 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 did not mention which particular substance 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, while all the 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 enrolled, finding 36.29 (SD 8.71) weeks of gestation and 37.8 (SD 1.15) weeks of gestation, respectively. Brasil et al.  provided the gestational age at birth of the 58 (43.3%) participants who had any abnormal finding at birth. Of these, four (6.9%) were in the microcephaly group, two of which were born preterm. From the non-microcephaly group, five (11.63%) newborns were born preterm. Shiu et al.  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 mothers were still pregnant by the time of the report, eight (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 in the microcephaly group (n=60) was 37.91 (SD 2.72) gestational weeks at birth and that of the infants in the non-microcephaly group was 38.06 (SD 2.42).
Studies that Provided Data to Conduct the Meta-Analysis
Six studies provided primary data to perform the meta-analysis [29, 34, 37, 38, 39, 40]. Three of them [34, 37, 39] had prospective designs, two [29, 38] had retrospective designs, and one  was a cross-sectional study. The study by Ventura et al.  was the only cross-sectional one, and therefore, we were not able to incorporate the data into the meta-analysis. For this reason, we describe it briefly, below.
Ventura et al.  provided data on 148 cases of PZIK. Microcephaly status (presence or absence) was reported for 140 (94.6%) of these infants. Of these, 124 (88.6%) presented microcephaly. In the microcephaly group, 56.45% (n=70) were female, while in the non-microcephaly group, the majority were male (n=9/16, 56,25%). The information on maternal symptomatology of PZIK was available for 132 infants (116 with microcephaly). The mothers of 108 of the infants reported symptoms (such as rash, pruritus, and conjunctivitis), 97 (83.6%) of whom belonged to the microcephaly group. Data on the use of licit or illicit substances was available for 132 mothers (118 in the microcephaly group) and 13 of them reported the use of these substances. In the microcephaly group, 12 (10.2%) mothers reported this behaviour, and in the non-microcephaly group, one mother (7.1%) reported it. As for the gestational trimester of infection, most of the mothers in the microcephaly group were infected in the first trimester (n=48, out of 100 with this information available), and the majority (n=7, out of 13 with this information available) in the non-microcephaly group had ZIKV infection in the second trimester. There was not enough data on maternal schooling and previous vaccines (yellow fever or other) to conduct an analysis. Regarding the methodological quality, this study was assessed as good quality.
We conducted meta-analysis to assess the rate of microcephaly detection according to seven identified characteristics: (i) sex (proportion of males); (ii) maternal age; (iii) maternal ethnicity (proportion of non-white); (iv) gestational age at the 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 drug consumption.
Regarding the meta-analysis of prospective studies, only three variables showed to be significant (presented in Figure 2). In relation to the sex of newborns/fetuses, 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 risk rate 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, pruritus, and rash (Figure 2c).
There was no statistically significant difference between groups regarding maternal ethnicity – white (RR 0.91; 95% CI 0.77, 1.08; I2=0%) – or the absence of tobacco, alcohol, and/or other substance consumption (RR 0.84, 95% CI 0.55, 1.29, I2=0%), although the point estimates indicated these characteristics as probable protective factors. Maternal age and gestational age at birth – analysed using mean and SD – were also 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 the 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, with only one live birth and 11 cases terminated in pregnancy. Only the infants’ sex could be tested as an exposure factor in the retrospective study design. There was a decrease of the Odds Ratio (OR) of microcephaly in females, although 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, substance consumption, and vaccine exposure, as only one study had this data available. Maternal age, maternal ethnicity, and presence of comorbidities were not estimated, as the study by Kumar et al.  had only one case without microcephaly and Schaub et al.  included only non-white individuals without comorbidities (Additional Figure 2b). Regarding quality assessment, Kumar et al.  was assessed as good methodological quality and Schaub et al.  as satisfactory methodological quality.