Table 1. Table 1. Shepard’s Criteria for Proof of Teratogenicity in Humans as Applied to the Relationship between Zika Virus Infection and Microcephaly and Other Brain Anomalies.

In 1994, Thomas Shepard, a pioneer in the field of teratology, proposed a set of seven criteria for “proof” of human teratogenicity (Table 1) that incorporated both approaches.9 These criteria were an amalgamation of criteria developed by other teratologists and guided by methods that were used to identify previous teratogens. These criteria have been used to guide discussions about causation in teratology-related litigation30 and to assess other potential teratogens.10 We used Shepard’s criteria9 as a framework to evaluate whether the currently available evidence supports the hypothesis that prenatal Zika virus infection is a cause of microcephaly and other brain anomalies (Table 1).

According to these criteria, causality is established when either criteria 1, 3, and 4 (rare exposure–rare defect approach) or criteria 1, 2, and 3 (epidemiologic approach) are fulfilled. The first criterion states that a proven exposure to an agent must occur at a critical time during prenatal development. The severe microcephaly and other brain anomalies that have been observed in many infants are consistent with an infection occurring in the first or early second trimester of pregnancy. Several case reports and studies have shown that women who had fetuses or infants with congenital brain anomalies that were believed, on the basis of the mother’s symptoms or laboratory confirmation, to be due to Zika virus infection were infected in the first or early second trimester of pregnancy, as determined either according to the timing of the symptoms or according to the timing of travel to an area where Zika virus is endemic.14-20 An analysis of the timing of laboratory-confirmed Zika virus transmission in certain states in Brazil and of the increase in the cases of microcephaly identified the first trimester as the critical time period for infection.1 Zika virus infections that occur later in pregnancy have been associated with poor intrauterine growth, fetal death, or in some pregnancies, defects on prenatal imaging that have not yet been confirmed postnatally because the pregnancies are ongoing.14 We conclude that Shepard’s first criterion has been met.

Shepard’s second criterion requires that two epidemiologic studies of high quality support the association. Although ecologic data do not necessarily qualify as an epidemiologic study, data from Brazil regarding the temporal and geographic association between Zika virus infection and the later appearance of infants with congenital microcephaly are compelling.1,31,32 Two epidemiologic studies also provide support.2,14 In a study conducted during the outbreak in Brazil, 88 pregnant women who had had an onset of rash in the previous 5 days were tested for Zika virus RNA. Among the 72 women who had positive tests, 42 underwent prenatal ultrasonography, and fetal abnormalities were observed in 12 (29%); none of the 16 women with negative tests had fetal abnormalities. The abnormalities that were observed on ultrasonography varied widely, and some findings lacked postnatal confirmation because the pregnancies were ongoing.14

A retrospective analysis after the 2013–2014 outbreak of Zika virus disease in French Polynesia identified eight cases of microcephaly; the authors used serologic and statistical data and mathematical modeling to estimate that 1% of the fetuses and neonates who were born to mothers who had been infected with Zika virus in the first trimester had microcephaly2 — a prevalence that was approximately 50 times as high as the estimated baseline prevalence. However, this estimate was based on small numbers, confidence intervals were wide, and the risk of other adverse outcomes (e.g., other brain anomalies) was not assessed.2 Although these studies provide important evidence in support of a causal relationship between Zika virus and microcephaly and other brain anomalies, both have limitations as noted by their authors, such as a lack of control for confounding factors and relatively small numbers of cases, and therefore they do not meet the stringent criteria set by Shepard. Thus, we conclude that Shepard’s second criterion has not yet been satisfied.

The third criterion, careful delineation of clinical cases with the finding of a specific defect or syndrome, appears to be met. Previous teratogens have caused specific birth defects or syndromes rather than a broad range of birth defects.33 Many fetuses and infants with presumed congenital Zika virus infection have had a typical pattern, including severe microcephaly, intracranial calcifications, and other brain anomalies, sometimes accompanied by eye findings, redundant scalp skin, arthrogryposis, and clubfoot15,20-23; such findings have led authors to use the term “congenital Zika syndrome.”22,34,35 On the basis of clinical details from a limited number of cases, some infants with presumed congenital Zika virus infection have had features that were consistent with fetal brain disruption sequence,24 a phenotype involving the brain that is characterized by severe microcephaly, overlapping cranial sutures, prominent occipital bone, redundant scalp skin, and considerable neurologic impairment.20,22 For example, 11 of 35 infants (31%) with microcephaly whose cases were reported to a Brazil Ministry of Health registry had excessive and redundant scalp skin,20 a finding that is not typically seen in other forms of microcephaly.36 These findings suggest an interruption of cerebral growth, but not in that of the scalp skin, after an injury (e.g., viral infection, hyperthermia, or vascular disruption) that occurred after the initial formation of brain structures, followed by partial collapse of the skull. The fetal brain disruption sequence is rare; only 20 cases were identified in a literature review in 2001.24

Shepard’s fourth criterion refers to the association between a rare exposure and a rare defect; we conclude that this criterion also has been met. The concept behind this criterion is that a rare defect occurring after a rare exposure during pregnancy implies causation because of the unlikelihood of the two rare events occurring together.10 Microcephaly is a rare defect that is estimated to occur in 6 infants per 10,000 liveborn infants in the United States.25 Zika virus would not be a rare exposure among women living in Brazil during the Zika virus outbreak. However, reports of adverse birth outcomes among travelers who spent only a limited time period in an area where there is active Zika virus transmission are consistent with Zika virus being a rare exposure.16,18,19

A recent report is illustrative: a pregnant woman traveled for 7 days to Mexico, Guatemala, and Belize during her 11th week of gestation and had a positive test for Zika virus immunoglobulin M (IgM) antibodies 4 weeks later. On fetal ultrasonography and magnetic resonance imaging performed at 19 to 20 weeks of gestation, severe brain anomalies were diagnosed in the fetus, and the pregnancy was terminated at 21 weeks of gestation. Microcephaly was not present at the time of pregnancy termination, but the head circumference had decreased from the 47th percentile at 16 weeks of gestation to the 24th percentile at 20 weeks of gestation (a finding that is consistent with the timing of diminishing head sizes in previous cases),14 which suggests that microcephaly would have developed in the fetus had the pregnancy continued.16 In this woman, Zika virus would be considered a rare exposure, and her fetus had a rare outcome.

The last three criteria are helpful if they are present, but they are not considered to be essential. The fifth criterion, the need for an animal model that shows teratogenicity, has not been met. Although animal models have shown that Zika virus is neurotropic,27,28 no studies that tested for teratogenicity in an animal model have been published, although studies are under way. The sixth criterion, that the association should make biologic sense, is clearly met here. Other viral infections have had similar effects (microcephaly and eye problems).24,26 In addition, pathologic evidence supports this association: Zika virus RNA has been seen in damaged mononuclear cells (presumably glial cells and neurons) in the brains of newborns with microcephaly,17 and the virus appears to be neurotropic.17,19 Live Zika virus has been cultured from the brain of a fetus with severe brain anomalies after maternal infection at 11 weeks of gestation.16 Furthermore, Zika virus efficiently infects neural progenitor cells and produces cell death and abnormal growth, thus providing a possible mechanism for microcephaly.29 The seventh criterion, proof in an experimental system that the agent acts in an unaltered state, is aimed at medications or chemical exposures and does not apply to infectious agents. Thus, given Shepard’s criteria as a framework, criteria 1, 3, and 4 have been satisfied — evidence that is considered sufficient to identify an agent as a teratogen.