Placental Trophoblast Inclusions in Autism Spectrum Disorder George M. Anderson, Andrea Jacobs-Stannard, Katarzyna Chawarska, Fred R. Volkmar, and Harvey J. Kliman Background: Microscopic examination of placental tissue may provide a route to assessing risk and understanding underlying biology of autism. Methods: Occurrence of a distinctive microscopic placental morphological abnormality, the trophoblast inclusion, was assessed using archived placental tissue. The rate of occurrence of trophoblast inclusion-positive slides observed for 13 individuals with autism spectrum disorder (ASD) was compared to the rate in an anonymous consecutive birth cohort. Results: The occurrence of inclusion positive slides was significantly greater in the ASD group compared to the control group (6/27 slides, 22.2% vs. 12/154, 7.8%; Fisher Exact Test, two-tailed p = .033; relative risk 2.85). The proportion of positive cases was also greater in the ASD group (5/13 cases, 38.5% vs. 8/61, 13.1%; Fisher Exact, two-tailed p = .044; relative risk 2.93). Behavioral severity scores did not differ across groups of inclusion positive (N = 4) and negative (N = 8) ASD individuals. Conclusions: Although probably not functionally detrimental or causative, the greater occurrence of placental trophoblast inclusions observed in ASD individuals may reflect altered early developmental processes. Further research is required to replicate the basic finding, to understand the basis for the trophoblastic abnormality, and to determine the utility of the measure in early detection of ASD. Key Words: Autism, early screening, marker, placenta, trophoblast, trophoblast inclusion T win and family studies have provided convincing evi- dence that autism is largely genetically determined (Bailey et al 1995; Bolton et al 1994; Folstein and Rutter 1977). The clinical genetic data and recent genome-wide screening studies indicate that, in most cases, multiple genetic factors are involved, with each likely contributing only a small amount of risk. It also appears that these risk factors can vary substantially across individuals classified as having an autism spectrum disorder (ASD) (Bailey et al 1995; Cook 2001; Folstein and Rosen-Sheidley 2001; Jones and Szatmari 2002; Lauritsen and Ewald 2001; Maestrini et al 2000; Risch et al 1999; Szatmari 1999; Veenstra- Vanderweele et al 2003). This apparent polygenetic and hetero- genetic nature of autism, as well as the unclear role of environ- mental influences on expression, makes an elucidation of the molecular and neurobiological basis of autism extremely diffi- cult. Basic questions about the relative utility of considering autistic behavior from a categorical or dimensional perspective further complicate matters (Adrien et al 2001; Bolte and Poustka 2001; Szatmari et al 2002). Despite the inherent difficulties in understanding etiological aspects of autism, the identification of early autism-related markers, traits or endophenotypes offers a promising route of investigation (Gottesman and Gould 2003; Gottesman and Han- son 2005; Leboyer et al 1998; McBride et al 1996; Skuse 2001). Early disorder-associated phenotypes could be of great use in focusing neurobiological research, in suggesting candidate genes, and in early screening or risk assessment. Although most measurable biological phenotypes are expected to be multi- determined, they may actually offer advantages in reflecting convergent processes related to neurobiological abnormality. Even if sets of risk alleles can be identified, at best they can be expected to contribute in a probabilistic fashion to autism-related behavior. Thus, a relevant biomarker or endophenotype may be of particular value in light of the apparent genetic complexities. The placenta may offer a readily available tissue for the detection of developmental abnormality. The placental tropho- blasts are among the first fetal cells formed and they are essential to the implantation of the conceptus and the development of the placenta. The cytotrophoblast and the syncytiotrophoblast form a bilayer that surrounds the inner cell mass and serves to separate the fetal and maternal circulations (Boyd and Hamilton 1970; Kliman 1999). Cytotrophoblasts are known to be the proliferative stem cells of the placenta, while the overlying syncytiotropho- blast layer is formed by fusing cytotrophoblasts (Kliman et al 1986). The relative rates of cytotrophoblast proliferation and incorporation into the outer syncytiotrophoblast layer appear to determine the morphology of the finger-like chorionic villi (Huppertz et al 2001; Kliman and Segel 2003; Rejniak et al 2004). When these critical processes are altered, the bilayer can inap- propriately bulge inward into the villi, creating invaginations and trophoblast inclusions that can be readily detected upon histo- logical examination of sectioned placental tissue (Figure 1). Trophoblast inclusions have been associated with a number of frank genetic abnormalities including triploidy, trisomy and Turner’s syndrome (Honore et al 1976; Novak et al 1988; Silvestre et al 1996; Szulman et al 1981). The balance between cytotro- phoblast proliferation and fusion into the syncytiotrophoblast layer may offer a sensitive early (sentinel) marker for genetic vulnerability to disruptions in the regulation of basic proliferative and cell specification processes (Kliman and Segel 2003). In other words, although trophoblast inclusions probably have no effect on overall placental function, they may be a reflection of genetic diatheses that could have subtle, yet profound, effects in the developing embryo and the forming nervous system. The possibility that just such diatheses or vulnerabilities may influ- ence brain development in children with ASD prompted us to consider whether trophoblast inclusions might not occur at a The Yale Child Study Center (GMA, KC, FRV), Department of Laboratory Medicine (GMA), and Department of Obstetrics, Gynecology and Repro- ductive Sciences (AJ-S, HJK), Yale University School of Medicine, New Haven, Connecticut. Address reprint requests to Harvey J. Kliman, M.D., Ph.D., Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, 300 George Street, Suite 8100, New Haven, CT 06511; E-mail: harvey.kliman@yale.edu. Received December 6, 2005; revised March 21, 2006; accepted March 21, 2006. BIOL PSYCHIATRY 2007;61:487– 491 0006-3223/07/$32.00 doi:10.1016/j.biopsych.2006.03.068 © 2007 Society of Biological Psychiatry