Advanced maternal age is a frequent indication for performing chorionic villus sampling (CVS) and it might be a confounder of the association between transverse limb deficiencies (TLD) and early CVS. We have first analyzed the maternal age-specific rates of TLD in the population monitored by the Italian Multicentric Birth Registry; then we updated a case control study controlling for maternal age. The rate of all limb deficiencies (LD) was 5.9 per 10,000 births. No trend for an excess risk for TLD or other LD with advancing maternal age was found. The relative risk for women 35 years of age and older vs. those under 35 was 0.92 (95% CI, 0.72-1.19) for any LD and 0.99 (95% CI, 0.71-1.39) for TLD. In the case control study, 11 mothers of case patients with a TLD had been exposed to CVS out of a total of 206 (5.3%), compared to 54 mothers of control patients with defects other than TLD out of a total of 12,140 (0.4%). The risk estimate for TLD associated with CVS was high in the overall analysis (OR, 12.63) and did not decrease after stratification, both in the overall sample (Mantel-Haenszel OR, 14.01) and in each gestational age stratum. Thus, advanced maternal age does not explain the association between CVS and TLD found in this study and it is unlikely to explain that observed in the several other positive studies. We recommend that any study addressing the relationship between CVS and LD should include a careful evaluation of the type of LD and the timing of CVS, and present the results for specific gestational age periods.