Abstract
Introduction: Extracellular vesicles derived from the fetal central nervous system (FCNSEs) can be purified from maternal serum or plasma using the protein Contactin-2/TAG1that is expressed almost exclusively by developing neurons in the hippocampus, cerebral cortex and cerebellum. We hypothesized that fetal CNSEs could be used to non-invasively detect and quantify viral mediated in-utero brain injury in the first trimester.Materials and Methods: First trimester maternal samples were collected from a human clinical population infected with primary cytomegalovirus (CMV) and a non-human primate model of Zika (ZIKV) infection. In the CMV cohort, a nested case control study was performed comparing pregnancies with and without fetal infection. Cases of fetal infection were further subdivided into those with and without adverse neurologic outcome. ZIKV samples were collected serially following maternal inoculation or saline. All ZIKV cases had histopathologic findings on necropsy. Serum was precipitated with ExoQuick solution and FCEs were isolated with biotinylated anti-Contactin-2/TAG1 antibody-streptavidin matrix immunoabsorption. FCE Synaptopodin (SYNPO) and Neurogranin (NG) protein levels were measured using standard ELISA kits and normalized to the exosome marker CD81.Results: Fetal CNSE SYNPO and NG were significantly reduced in cases of first trimester fetal CMV infection compared to those with infection limited to the mother but could not discriminate between fetal infection with and without adverse neurologic outcome. Following ZIKV inoculation, fetal CNSE SYNPO was reduced by 48 h and significantly reduced by day 4.Discussion: These data are the first to suggest that first trimester non-invasive diagnosis of fetal viral infection is possible. Fetal CNSEs have the potential to augment clinical and pre-clinical studies of perinatal viral infection. Serial sampling may be needed to discriminate between fetuses that are responding to treatment and/or recovering due to innate defenses and those that have ongoing neuronal injury. If confirmed, this technology may advance the paradigm of first trimester prenatal diagnosis and change the calculus for the cost benefit of CMV surveillance programs in pregnancy.
Highlights
Extracellular vesicles derived from the fetal central nervous system (FCNSEs) can be purified from maternal serum or plasma using the protein Contactin2/TAG1that is expressed almost exclusively by developing neurons in the hippocampus, cerebral cortex and cerebellum
Based on comparisons of concentrations between pregnant and nonpregnant subjects, we have demonstrated that 97% of CTN2/TAG-1+ extracellular vesicles (ECVs) isolated from maternal serum are fetal in origin [16]; the 3% contamination by maternal neural ECVs reflects low levels of ongoing neural plasticity in adults
Our work demonstrates that CTN-2/TAG1 can be used to isolate fetal CNS derived ECV (CNSEs) from maternal blood to generate novel biomarkers
Summary
Extracellular vesicles derived from the fetal central nervous system (FCNSEs) can be purified from maternal serum or plasma using the protein Contactin2/TAG1that is expressed almost exclusively by developing neurons in the hippocampus, cerebral cortex and cerebellum. We hypothesized that fetal CNSEs could be used to non-invasively detect and quantify viral mediated in-utero brain injury in the first trimester. 10–15% of congenitally infected neonates will have symptoms; of those 6% will die and up to 90% will develop sequelae such as sensorineural hearing loss (SNHL), developmental delay, cognitive impairment, neuromuscular dysfunction (cerebral palsy), epilepsy, impaired vision function, and possibly autism spectrum disorder. Congenital CMV infection is responsible for more longterm sequelae than either Down syndrome or Fetal Alcohol Spectrum Disorder [2]. Zika virus (ZIKV) is a mosquito borne flavivirus that has been causally implicated in brain injury and microcephaly following in-utero infection [3]. The estimated risk of clinically detected central nervous system (CNS) injury following first trimester ZIKV infection ranges from 1 to 13% [4]. In an epidemiologic study based on Brazilian data, the peak risk of microcephaly appears to correlate with ZIKV infection between gestational week 14 and 17 [5], but findings of ventriculomegaly and cerebral calcifications have been seen with ZIKV infections as late as the 3rd trimester [6]
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