Amniocentesis (AC) is considered the best option for prenatal diagnosis (PD) of fetal cytomegalovirus (CMV) infection. To reduce the risk of false negative results, it is recommended to perform AC not before completed 21 weeks of gestation (WG) and >6 weeks from maternal infection. 1,2 In a recent publication, we described a single centre experience on invasive PD in a 5-year cohort of 115 women with CMV primary infection. 3 In this study, we did not observe a significant difference in sensitivity whether AC was performed ≥17 + 0 or ≥20 + 0 WG, as long as the interval between seroconversion and invasive PD was >8 weeks. However, the number of cases with intrauterine transmission and AC before 21 WG was limited.Therefore, we extended our investigation to all cases (n = 2706) with PD for CMV in amniotic fluid (AF) sampled between 17 + 0 and 22 + 6 WG during a 16-year period from 2001 to 2016. Women with multiple pregnancies were excluded from the study. Laboratory diagnosis of CMV primary infection was confirmed in 531 women either by CMV immunoglobulin G (IgG) seroconversion (n = 133) or a positive CMV immunoglobulin M (IgM) test in the presence of low CMV-IgG avidity (n = 398). PD was accomplished by rapid virus culture and/or by quantitative detection of CMV DNA in AF. In 338 cases, adequate specimen were available at termination of pregnancy or at birth for the diagnosis of congenital CMV infection (cCMV). Sixty-three of the 338 pregnancies were part of our previous study. 3 Time between seroconversion and invasive PD was estimated by patient's history and available laboratory results. In 184 cases, we received seropositive samples >8 weeks before AC and in 19 cases seronegative samples ≤8 weeks before AC. The other cases were categorized by combining onset of maternal symptoms, quantitative IgG and IgM results, IgG titre rises in follow-up sera and the results of VIDAS ® CMV IgG Avidity and recomBlot/recomLine CMV IgG [avidity] testing. 4,5 In total, an interval of ≤8 weeks and >8 weeks was warranted in 46 and 264 pregnancies, respectively. In 28 cases, the interval could not be determined.For the 338 cases, we observed an overall sensitivity of PD of 85.2% (95% CI: 77; 91). Sensitivity in the group with an interval of ≤8 weeks (n = 46) and >8 weeks (n = 264) was 58.3% (95% CI: 28; 85) and 89.4% (95% CI: 81; 95), respectively. This difference was statistically significant (p = 0.013; Fisher exact test). In cases with indeterminate interval (n = 28), the sensitivity was 75.0% (95% CI: 19; 99). Rapid virus culture and PCR were concomitantly performed in 320 of 338 AF samples. The agreement between the two assays was 99.7%. The discordant sample (obtained at 18 + 6 WG and ≤8 weeks after seroconversion) was culture negative and PCR positive (36.250 IU/mL) in an infant with confirmed cCMV at birth. Figure 1 shows the results of CMV-DNA detection in AF in pregnancies with an interval of >8 weeks between seroconversion and AC. Negative PD results in pregnancies affected by cCMV occurred at any gestational age. Sensi...