Prenatal predictors of adverse perinatal outcome in congenital cytomegalovirus infection: a retrospective multicenter study

Study design and participants

This was a multicenter, retrospective, cohort study involving 11 referral centers in Italy from 2012 to 2021 (Brescia, Chieti, Foggia, Modena, Naples, Padova, Rome–Catholic University of Sacred Heart, Rome; Sapienza University, Rome; University Tor Vergata, Treviso and Trieste). The study included pregnant women with primary or non-primary CMV infection confirmed by PCR analysis of amniotic fluid. Approval of the local Ethical Committees of the participating centers was obtained.

Inclusion criteria were fetuses with congenital CMV infection diagnosed by PCR analysis of amniotic fluid obtained by amniocentesis performed after 20 weeks of gestation or after 6–8 weeks after maternal seroconversion as recommended by national and international guidelines [10]. And normal karyotype, Exclusion criteria were the presence of CNS and extra-CNS anomalies detected at the time of the diagnosis.

The outcomes observed were:

1. Additional CNS or extra-CNS anomalies detected either at ultrasound or fetal MRI.

2. Additional anomalies detected exclusively post-natally.

3. Post-natal clinical symptoms related to congenital CMV infection, including microcephaly, neurological symptoms, hearing and ocular anomalies.

Longitudinal, detailed ultrasound follow-up after diagnosis of fetal infection was performed every 2–4 weeks, according to each local protocol, including a comprehensive evaluation of fetal growth, placenta, amniotic fluid volume, fetal Doppler and the assessment of potential ultrasound markers of fetal disease. Fetal MRI was performed according to the standardized planes for fetal brain examination according to ISUOG guidelines for fetal MRI [18].

Cases with ultrasound anomalies at diagnosis and chromosomal anomalies or genetic syndromes detected either before or after birth were excluded. The clinical records were examined, and data collected in a dedicated merged database. STROBE guidelines were followed [19].

Data analysis

The potential association between the recorded demographic and clinical characteristics and each of the three outcomes were initially evaluated using chi-squared test for categorical variables, t-test and Kruskal-Wallis test for normally distributed and non-normally distributed continuous variables (Shapiro-Wilk test), respectively. The potential independent predictors of each outcome were then evaluated using stepwise forward logistic regression. All covariates were tested for inclusion in the final model, in which only those significant at either univariate or adjusted analysis were retained. To reduce potential overfitting, the overall number of covariates was limited to 1/10 of the anomalies in all phases of model fitting. The goodness-of-fit was checked using Hosmer-Lemeshow test, and the predictive power assessed through C-statistics (area under the receiving operator curve). Given the low number of women with additional anomalies detected only on postnatal imaging (n=10) and with postnatal clinical symptoms (n=13), no multivariate analysis could be performed for these two outcomes, and raw ORs and 95% CIs were reported. Missing values were less than 1% for all variables, thus no missing imputation technique was adopted.

Statistical significance was defined as a two-sided p-value<0.05, and all analyses were carried out using Stata, version 13.1 (Stata Corp., College Station, Texas, USA, 2013).

Maternal characteristics

One hundred and four fetuses with congenital CMV were included in the study. General characteristics of the study population are reported in Table 1. Mean maternal age was 31.3 ± 5.8 years while mean BMI was 25.9 ± 3.9 years. 85.6% of women acquired infection in the first while 14.4% in the second trimester. 96.2% of congenital CMV infections were primary and only 3.8% secondary.

Additional anomalies were detected at follow-up ultrasound or prenatal imaging in 18.3% (19/104) cases. Table 2 reported the results of the univariate analysis comparing fetuses with and those without additional anomalies detected at prenatal imaging (either MRI or ultrasound). There was no difference in either maternal age (p=0.3), type (p=0.12) and trimester (p=0.4) of infection and prenatal therapy with either Immunoglobulin or Valacyclovir (p=0.4) between fetuses who compared to those who did not show additional anomalies at follow-up ultrasound or MRI. Conversely, median viral load in the amniotic fluid was higher in fetuses with compared to those without additional anomalies at follow-up (p=0.02). The rate of termination of pregnancy was significantly higher in fetuses with anomalies (0.012). Additional anomalies were found after birth in 11.9% (10/84) of cases. There was no significant difference in any of the main prenatal characteristics explored between fetuses with compared to those without additional anomalies detected exclusively after birth (Table 3).

After birth 15.5% (13/85) of newborns showed clinical symptoms related to CMV infection. At univariate analysis (Table 4), the presence of associated anomalies detected prenatally (p=<0.001) and postnatally, were the only predictors of symptomatic infection, while viral load in the amniotic fluid (p=0.05), type and timing at infection were not.

At multivariate logistic regression analysis, high viral load in the amniotic fluid, defined as a 100,000 copies/mL was the only independent predictor for the occurrence of anomalies detected exclusively at follow-up ultrasound assessment or MRI, with an OR of 3.12 (95% CI 1.0–9.4), while it was not possible to carry multivariate analysis for the other explored outcomes in view of the small number of events detected.

Summary of the main findings

The findings from this study show that, in fetuses with congenital CMV infection and normal ultrasound assessment at the time of diagnosis, additional anomalies at follow-up or after birth are detected in 18% and 11.9% of cases respectively, while 16% of newborns showed clinical symptoms related to the infection. Viral load in the amniotic fluid was independently associated with adverse fetal and post-natal outcome, irrespective of the presence of normal prenatal imaging, thus confirming the prognostic role of PCR in predicting the prognosis of fetuses with congenital CMV infection.

Strengths and limitations

The relatively large sample size, longitudinal assessment of fetal anatomy through ultrasound and MRI in each of the included cases and the multitude of pre and post-natal outcomes explored represents the main strengths of the present study.

The retrospective non-randomized design represents the main weakness of the present study. Furthermore, we could not elucidate the potential role of prenatal medical therapy because not all the included cases received valacyclovir at the time of maternal seroconversion. Finally, we could not assess the individual components of post-natal clinical symptoms in view of the small number of events reported for this outcome.

Implications for clinical practice and research

Prenatal prediction of adverse outcome in fetuses with congenital CMV infection is challenging [14]. CMV is associated with a multitude of adverse neurological and neurosensorial outcome, including epilepsy, mental retardation, hearing, and visual problems [20]. The presence of associated anomalies, mainly those involving the CNS, is among the main determinants of post-natal outcomes in fetuses with CMV infection. However, prenatal counselling in case no associated anomaly is detected prenatally is difficult [10].

CMV has a peculiar tropism for the neurons in the periventricular zone, thus potentially affecting neuronal migration and proliferation [21]. However, anomalies involving the cortical surface of the brain, including lissencephaly and microcephaly can be detected only later in gestation [22], [23], [24], [25], [26]. In the present study, additional anomalies detected only at follow-up imaging, either ultrasound or MRI, 18% and 11.9% of the cases respectively. These findings highlight the need for a thorough longitudinal imaging follow-up in fetuses with congenital CMV infection. Likewise, parents should be counselled on the high risk of detecting associated anomalies only later in pregnancy or after birth. High viral laod in the amniotic fluid has been anecdotally reported to predict the outcome of fetuses with CMV infection. However, previous studies did not differentiate between cases presenting compared to those not presenting with additional structural ultrasound [12]. This is crucial as the presence of a CNS anomaly is likely to be associated with a high risk of adverse neurological outcome. In the present study, mean viral load in the amniotic fluid was higher in fetuses with additional anomalies detected exclusively at follow-up or at birth, confirming the predictive role of viral load in identifying fetuses at risk of adverse outcome. Importantly, at multivariate logistic regression analysis, viral load was independently associated with the risk of associated anomalies, irrespective of type and gestational age at infection. In this scenario, parental counselling of pregnant women with congenital CMV infection should mention that the risk of anomalies detected only in the third trimester or after birth and potentially not identified with either ultrasound or MRI.

Despite its prevalence and morbidity among the neonatal population, there is not yet a standardized diagnostic test and therapeutic approach for congenital CMV infection. Literature analysis shows that preventive interventions other than behavioral measures during pregnancy are still lacking, although many clinical trials are currently ongoing to formulate a vaccination for women before pregnancy [27, 28]. Some evidence in literature describes attempts to use CMV hyperimmune globulin in order to decrease the incidence of congenital CMV infection or fetal or neonatal death among the offspring of women with primary CMV infection in pregnancy, without significant success [28, 29]. Secondary prevention of congenital cytomegalovirus infection with valacyclovir following maternal primary infection in early pregnancy seems to show some benefit in reducing vertical transmission [29, 30].

The burden of congenital CMV infection is an unmet public issue [30], for this reason the attention towards this infection must be destined to grow. The lack of evidence in the literature on any prenatal predictors of adverse outcomes in congenital CMV infections should be a pretext for new future studies focused on this aspect, in parallel with the attention for new therapeutic strategies in the case of confirmed infection. This study identifies the high viral load in the amniotic fluid as the only predictor of abnormalities at follow-up ultrasound assessment or MRI that were the only predictors of symptomatic infection (p=<0.001). This finding indicates that therapeutic strategies should focus on viral load reduction and that in the future viral load can be a useful tool for risk prediction and prenatal follow up of these fetuses.