Prenatal ultrasound findings associated with intrauterine CMV infection: a multicenter retrospective analysis of clinical and sonographic features

Abstract

Background

To assess the association between prenatal ultrasonography and intrauterine Cytomegalovirus (CMV) infection and to elucidate its clinical characteristics.

Methods

Cases were selected from three medical centers (2015–2024) in which amniotic fluid CMV-DNA testing was performed following prenatal ultrasound findings suggestive of CMV infection. CMV infection rates were analyzed in relation to distinct sonographic abnormalities. Associations between amniotic fluid viral load, imaging characteristics, and pregnancy outcomes were subsequently evaluated.

Results

Among 1,174 pregnant women, 45 (3.83%) tested positive for CMV‑DNA in amniotic fluid. Sonographic findings with CMV infection rates exceeding 50% included fetal intracranial hyperechogenicity, intraventricular adhesions, hepatosplenomegaly, and placental thickening. The infection rate was significantly higher in fetuses with multiple abnormalities (10.42%) than with isolated findings (2.14%) (P<0.001). Combined intracranial and extracranial abnormalities carried a higher infection rate (13.41%) than purely intracranial (3.28%) or purely extracranial (2.76%) abnormalities (P<0.001). In CMV‑positive cases, amniotic fluid VL ranged from 1.1 × 10² to 7.71 × 10⁷ copies/mL (median 3.86 × 10⁵ copies/mL). VL was significantly elevated in cases with intracranial, severe, or non‑isolated abnormalities (P<0.05). Of 14 cases tested for cord blood CMV‑IgM, 10 (71.43%) were positive; intracranial abnormalities were present in 8 of these, but in none of the IgM‑negative cases. Maternal CMV‑IgM was positive in only 2/19 (10.53%) infected cases. Pregnancy was terminated in 25 cases; among 13 live‑born infants with follow‑up, 9 had normal outcomes, 3 had hearing impairment, and 1 had severe neurological sequelae.

Conclusions

Specific prenatal ultrasound findings show a high rate of association with intrauterine CMV infection, especially when there are multiple abnormalities or when abnormalities involve both the intracranial and extracranial compartments. Amniotic fluid CMV-DNA load correlates positively with the presence of intracranial abnormalities and overall severity of ultrasound findings.

Background

Congenital Cytomegalovirus infection (cCMV) refers to an infection of the fetus with cytomegalovirus before birth. It is among the most common congenital infections, with a global incidence of approximately 0.67% [1]. In China, the prevalence of cCMV at birth is around 1.32% [2]. Infants with cCMV may range from being asymptomatic to exhibiting severe neurological abnormalities, such as mental retardation and motor nerve damage. Effective prenatal screening and diagnosis are crucial measures to reduce the incidence of severe cCMV.

Cytomegalovirus primarily infects fetuses through intrauterine vertical transmission, which can be either a primary or secondary infection in the mother. In China, the CMV-IgG positivity rate among women of childbearing age reaches as high as 95% [3], with most maternal infections during pregnancy being secondary. While serological antibody screening can help determine maternal infection status, the sensitivity and specificity of IgM antibodies are low [4]. The necessity of serological antibody screening in the general population remains a topic of debate [5]. Research indicates that fetal intracranial and extracranial abnormalities detected via prenatal ultrasound are associated with CMV infection [6]. Additionally, fetal cranial magnetic resonance imaging (MRI) can serve as a valuable supplement to ultrasound in diagnosing CMV infection [7]. However, the association of individual imaging features with infection requires further validation. Currently, China lacks substantial large-sample reports on this subject. This study retrospectively collected cases of prenatal diagnosis of CMV infection, indicated by ultrasound or MRI abnormalities, to explore the correlation between imaging features, intrauterine infection, and DNA load, and to analyze the clinical characteristics of intrauterine infection cases. This aims to provide references for prenatal diagnosis and consultation regarding CMV infection.

Methods

Subjects

This retrospective study involved cases with suspected CMV infection identified through prenatal ultrasound at the First Affiliated Hospital of Sun Yat-sen University, Guangzhou Women and Children Medical Center, and Peking University Shenzhen Hospital from 2015 to 2024. The inclusion criteria were: age between 18 and 45 years; presence of one or more manifestations of intrauterine CMV infection as specified in the 2020 ISUOG guidelines on prenatal ultrasound [6]; and performance of amniocentesis with CMV-DNA detection via PCR after 21 weeks of gestation. Exclusion criteria comprised fetal anomalies attributed to known etiologies, including chromosomal abnormalities, monogenic disorders, immune hemolytic anemia, and complications associated with monochorionic twin pregnancies. For cases with confirmed intrauterine CMV infection, participants were stratified based on the presence or absence of intracranial abnormalities, the severity of such abnormalities, and whether they occurred in isolation, to compare CMV-DNA levels across groups. This study received approval from the Research Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University.

Ultrasound examinations

Ultrasound examinations were performed in two phases: 21 to 24 weeks of gestation (the second trimester) and 28 to 32 weeks of gestation (the third trimester), as these are critical time points for screening for fetal structural abnormalities. All cases referred from other medical institutions underwent structural ultrasound re-evaluation at the three participating medical centers, and the results of these re-evaluations were taken as the definitive reference.

Fetal growth restriction (FGR) is defined as an estimated fetal weight or abdominal circumference below the 10th percentile for gestational age on ultrasound [8]. Microcephaly is diagnosed when the fetal head circumference measures more than 3 standard deviations below the mean for gestational age. Hydrops fetalis is characterized by the presence of two or more abnormal fetal fluid collections, such as ascites, pleural or pericardial effusion, or generalized skin edema (skin thickness > 5 mm) [9], which may be accompanied by placental thickening, polyhydramnios, or cardiomegaly. Fetal anemia is evaluated by Doppler measurement of the middle cerebral artery peak systolic velocity (MCA-PSV), with values ≥ 1.5 multiples of the median (MoM) indicating moderate to severe anemia [10]. Severity is further classified based on umbilical cord blood hemoglobin levels: 0.65–0.55 MoM corresponds to moderate anemia, and values < 0.55 MoM indicate severe anemia [11].

Given the heterogeneous sonographic manifestations of congenital CMV infection, we categorized pleural effusion, ascites, hepatosplenomegaly, placental thickening, cardiomegaly, pericardial effusion, and cutaneous edema as the “hydrops phenotype.” Echogenicity or calcification involving the bowel, heart, liver, kidneys, or placenta was classified as the “hyperechoic phenotype.” Severe fetal findings were defined as extreme FGR (estimated fetal weight <3rd percentile), severe fetal anemia, abnormal cerebral cortical development, severe ventriculomegaly, hydrops fetalis, microcephaly, or complex congenital heart defects.

Diagnosis of intrauterine CMV infection

Intrauterine CMV infection was confirmed through amniocentesis performed after 21 weeks of gestation, with detection of CMV DNA in amniotic fluid by PCR. The procedure was conducted within 7 days following the ultrasound examination. Amniocentesis for CMV-DNA testing was not performed routinely but was offered following prenatal counseling to pregnancies with ultrasound findings suggestive of possible fetal infection, as defined by the inclusion criteria.

Data collection

Maternal age, prenatal ultrasound findings, gestational age at amniocentesis, and amniotic CMV-DNA load were routinely recorded. Fetal MRI findings, CMV-IgM in cord blood, and maternal CMV-IgM and IgG levels were also collected when available. Fetal MRI was performed primarily when complex intracranial findings were detected on ultrasound or at the clinician’s discretion. For cases with confirmed intrauterine CMV infection, pregnancy outcomes, gestational age at delivery, and neonatal birth weight were documented. For live-born infants, this study acquired postnatal data from the established follow-up protocol, which included telephone interviews or outpatient visits at 1, 6, 12, 24, and 36 months to evaluate growth, hearing status (using Auditory Brainstem Response, ABR testing), and neurodevelopment (encompassing motor, sensory, and cognitive domains).

Statistical analysis

SPSS 24.0 statistical software (IBM, Armonk, NY, USA) was used for the analysis of the data. Categorical variables were expressed as n (%) and tested using Chi-square test between groups. The normally distributed variables were expressed as mean ± standard deviation and tested using student’s t-test, whereas non-normally distributed variables were expressed as median (interquartile range; IQR) and tested using the Mann–Whitney U test. P < 0.05 indicated a statistically significant difference.

Results

The result of baseline data

A total of 1,174 cases were included in this analysis (Fig. 1). The mean maternal age was 30.26 ± 2.1 years (range: 18–44 years). Multiple sonographic findings were observed in 240 cases, while isolated findings were present in 934 cases. Intracranial abnormalities alone were noted in 366 cases, exclusively extracranial findings in 726 cases, and combined intracranial and extracranial abnormalities in 82 cases. The mean gestational age at amniocentesis was 27.7 ± 3.7 weeks (range: 21–37 weeks). Among all cases, 45 tested positive for CMV DNA (Fig. 1), yielding an overall CMV infection rate of 3.83%.

Fig. 1.

Flow chart of this study

Of the total cohort, 605 cases were identified through second-trimester ultrasound, with 29 being CMV-positive (4.79%), and 569 cases were detected in the third trimester, among which 16 were CMV-positive (2.81%). Although the CMV positivity rate was numerically higher in cases detected during the second trimester compared with those detected in the third trimester, the difference did not reach statistical significance (χ² = 3.123, P = 0.077).

Infection rate varied between different types of abnormalities in imaging

The top five findings associated with CMV positivity were: intracranial hyperechogenicity (Fig. 2A and B), intraventricular adhesions (Fig. 2E and F), splenomegaly, placental thickening (Fig. 2K), and cortical dysplasia (Fig. 2A and H). Among intracranial findings, the five manifestations with the highest CMV detection rates were: intracranial hyperechogenicity, intraventricular adhesions, cortical dysplasia (Fig. 2A and H), arachnoid cyst, and intracranial hemorrhage. Regarding extracranial findings, the top five abnormalities by CMV detection rate included: hepatomegaly (Fig. 2J), splenomegaly, placental thickening (Fig. 2K), fetal anemia (Fig. 2L), and pleural effusion. Among commonly observed ultrasound indicators (prenatally diagnosed in ≥ 50 cases), the descending order of CMV infection rates was as follows: ventriculomegaly (Fig. 2G), fetal growth restriction, renal malformations, polyhydramnios, and cardiac malformations (Table 1).

Fig. 2.

Prenatal ultrasound findings in the cases with intrauterine CMV infection A intracranial hyperechoic focus (white arrow) and cortical dysplasia (red arrow); B intracranial hyperechoic focus (white arrow); C periventricular pseudocyst (white arrow); D Blake’s cyst (white arrow); E intraventricular adhesions (white arrow); F intraventricular adhesions (white arrow); G ventriculomegaly (white arrow); H cortical dysplasia (white arrow); I echogenic focus in bowel (white arrow); J ascites (white arrow) and hepatomegaly (red arrow); K placental thickening (white arrow); L fetal anemia [MCA-PSV 54.02 cm/s (1.46MOM)]

Table 1.

Rates of CMV infection according to different sonographic findings

US findings	N	No. of CMV-positive cases	Infection rate % (95% CI)
Intracranial findings
intracranial hyperechogenicity	7	5	71.43 (35.9–92.8)
intraventricular adhesion	3	2	66.67 (20.8–93.9)
cortical dysplasia	11	5	45.45 (21.3–71.9)
arachnoid cyst	5	1	20.00 (3.6–62.4)
intracranial hemorrhage	11	2	18.18 (5.1–47.7)
periventricular pseudocyst	23	3	13.04 (4.5–32.1)
agenesis of corpus callosum	20	2	10.00 (2.8–30.1)
microcephalus	36	3	8.33 (2.9–22.2)
Blake’s cyst	12	1	8.33 (1.5–35.4)
ventriculomegaly	293	21	7.17 (4.7–10.7)
cerebellar abnormalities	16	1	6.25 (1.1–28.3)
posterior fossa cyst	19	1	5.26 (0.9–24.8)
posterior fossa dilation	64	0	0.00 (0.0-5.7)
midline cyst	4	0	0.00 (0.0–49.0)
Extracranial manifestations
hepatomegaly	3	2	66.67 (20.8–93.9)
splenomegaly	3	2	66.67 (20.8–93.9)
placental thickening	4	2	50.00 (15.0–85.0)
elevated MCA-PSV (fetal anemia)	20	6	30.00 (14.5–51.9)
pleural effusion	17	4	23.53 (9.6–47.1)
cardiomegaly	17	4	23.53 (9.6–47.1)
echogenic focus in heart	5	1	20.00 (3.6–62.4)
ascites	33	6	18.18 (8.6–34.4)
echogenic focus in bowel	35	4	11.43 (4.5–26.0)
abdominal cyst	9	1	11.11 (2.0-43.5)
echogenic focus in liver	9	1	11.11 (2.0-43.5)
echogenic focus in kidney	11	1	9.09 (1.6–38.3)
hydrops	48	4	8.33 (3.3–19.4)
valve regurgitation	37	3	8.11 (2.8–21.4)
pericardial effusion	13	1	7.69 (1.4–33.3)
fetal arrhythmia	14	1	7.14 (1.3–31.5)
oligohydramnios	32	2	6.25 (1.7–20.7)
congenital cystic adenomatoid malformation	17	1	5.88 (1.0–27.0)
fetal growth restriction	352	16	4.55 (2.8–7.3)
renal abnormality	65	2	3.08 (0.8–10.6)
polyhydramnios	116	3	2.59 (0.9–7.4)
cardiac anomalies	110	1	0.91 (0.2-5.0)
meconium peritonitis	14	0	0.00 (0.0-22.8)

The CMV infection rate in cases with multiple sonographic findings (25/240, 10.42%) was significantly higher than that in cases with isolated findings (20/934, 2.14%) (χ² = 35.472, P < 0.001). In contrast, no statistically significant difference was observed between cases with purely intracranial findings (12/366, 3.28%) and those with purely extracranial findings (22/726, 3.03%) (P > 0.05).

The CMV infection rate in cases with combined extracranial and intracranial findings (13.41%) was significantly higher than in those with isolated intracranial (3.28%, P < 0.001) or isolated extracranial findings (2.76%, P < 0.001). Within both the purely intracranial and purely extracranial subgroups, cases presenting multiple findings demonstrated a significantly higher infection rate compared to those with a single finding (P < 0.05) (Table 2). Among cases exhibiting combined intracranial and extracranial abnormalities, the infection rate was markedly higher when multiple intracranial findings were present (58.33%) compared to cases with only a single intracranial finding (5.71%, P < 0.001). However, no statistically significant difference was observed between cases with multiple versus isolated extracranial findings (Table 2).

Table 2.

CMV infection rates in groups with intracranial versus extracranial findings

	without extracranial findings
		With extracranial findings (total)	With single extracranial finding	With Multiple extracranial findings
without intracranial findings	-	2.76% (20/724)c	1.96% (12/613)e	7.21% (8/111)e
With intracranial findings (total)	3.28% (12/366)a	13.41% (11/82)a, c	12.16% (9/74)	25.00% (2/8)
With single intracranial finding	1.88% (6/319)b	5.71% (4/70)d	4.62% (3/65)f	20.00% (1/5)
With multiple intracranial findings	12.77% (6/47)b	58.33% (7/12)d	66.67% (6/9)f	33.33% (1/3)

^a χ² = 12.127, P<0.001, ^b Fisher’s, P = 0.002, ^c χ² = 19.811, P<0.001, ^d χ² = 20.099, P<0.001, ^e χ² = 7.787, P = 0.005, ^f Fisher’s, P<0.001

The CMV infection rates associated with non-isolated presentations, including FGR, ventriculomegaly, hydrops phenotype, and hyperechoic phenotype, were all significantly higher than those of their isolated counterparts (P < 0.05) (Table 3). Specifically, the infection rate for isolated echogenic bowel was 4.55% (1/22), whereas it reached 21.43% (3/14) in cases where echogenic bowel occurred alongside other abnormalities.

Table 3.

Comparison of CMV infection rates for isolated versus non-isolated common ultrasound findings

	total	isolated findings	non-isolated findings	χ2	P
FGR	4.55% (16/352 )	2.68% (7/261)	9.89% (9/91)	6.504	0.011
ventriculomegaly	7.17% (21/293)	1.83% (4/218)	22.67% (17/75)	33.332	0.000
hydrops phenotype	7.27% (8/110)	1.59% (1/63)	14.89% (7/47)	-	0.020#
hyperechoic phenotypes	16.92% (11/65)	5.13% (2/39)	34.62% (9/26)	7.664	0.006

^#Fisher’s test is used

Results of intrauterine CMV infection

Forty-five cases with intrauterine infection are shown in (Table 4). The average age of these 45 pregnant women was 27.9 ± 4.07 years (18–38 years), and the average gestational age of the amniocentesis was 28.5 ± 4.5 weeks (21–35 weeks). The copy number of CMV-DNA ranged from 1.1 × 10² copies /mL to 7.71 × 10⁷ copies /mL. The median of virus load was 3.86 × 10⁵ copies /mL.

Table 4.

Clinical and diagnostic characteristics of forty-five intrauterine CMV infection cases in this study

case	age	gestational weeks	ultrasound findings	amniotic CMV-DNA (copies/mL)	CMV-IgM of cord blood (AU/mL)	maternal CMV-IgG (IU/mL)	maternal CMV-IgM (AU/mL)	pregnancy outcome
1	25	32	ventriculomegaly, intracranial hyperechoic focus, FGR	4.01 × 106	1.71	> 250.00	0.2	TOP
2	23	34+ 1	ventriculomegaly, periventricular pseudocyst	7.64 × 106	1.67	NA	NA	TOP
3	28	35	hydrops, enlarged spleen, ascites, pleural effusion, renal malformation, oligohydramnios	3.46 × 106	1.15	249.5	0.23	TOP
4	29	32+ 2	FGR	1.88 × 106	NA	196.70	0.88	TOP
5	34	21+ 4	congenital cystic adenomatoid malformation	1.49 × 104	NA	NA	NA	TOP
6	28	24+ 6	intracardial echogenic focus	1.45 × 105	NA	> 250.00	0.41	TOP
7	30	33+ 4	cardiovascular malformation, hydramnios	6.51 × 105	0.2	NA	NA	TOP
8	24	28+ 1	cortical dysplasia	3.50 × 105	1.3	NA	NA	TOP
9	26	27	ventriculomegaly, intracranial hyperechoic focus	8.65 × 106	1.29	> 250.00	0.46	TOP
10	29	26+ 1	ventriculomegaly, microcephaly, FGR	3.53 × 107	1.13	> 250.00	0.31	TOP
11	18	23+ 2	FGR, fetal anemia, echogenic bowel, ascites, pleural effusion, pericardial effusion	1.10 × 107	0.78	> 250.00	0.3	TOP
12	24	28+ 3	FGR, hydrops, fetal anemia, hepatomegaly, enlarged spleen, ascites, pleural effusion, cardiac enlargement, placental thickening	2.98 × 107	4.4	164.2	1.67	TOP
13	26	23+ 5	hydrops, ascites, pleural effusion	2.08 × 103	0.18	237.3	0.17	TOP
14	26	32	ventriculomegaly, intracranial hyperechoic focus, intraventricular adhesions, Blake’s cyst, FGR	1.66 × 106	2.35	> 250.00	0.33	TOP
15	32	30+ 6	FGR	5.69 × 103	NA	NA	NA	intrauterine death at 32 weeks
16	35	27+ 6	FGR, fetal anemia, oligohydramnios	1.63 × 103	NA	> 250.0	0.40	preterm delivery at 34 weeks, LOF
17	38	31+ 4	cardiac regurgitation	3.39 × 104	NA	156.5	0.19	preterm delivery at 36 weeks, normal outcome
18	34	31+ 4	fetal cardiac arrhythmias	1.25 × 103	NA	NA	NA	preterm delivery in 36 weeks, normal outcome
19	24	21	ventriculomegaly, FGR	3.37 × 102	NA	87.5	0.21	term delivery, LOF
20	30	26+ 4	renal malformation	1.10 × 102	NA	73.4	0.30	term delivery, normal outcome
21	22	37+ 3	periventricular pseudocyst	2.07 × 107	3.97	> 250.0	0.26	term delivery, normal outcome
22	29	33	FGR	1.01 × 103	0.05	NA	NA	term delivery, LOF
23	21	32+ 3	abdominal cyst	2.45 × 103	NA	NA	NA	term delivery, normal outcome
24	30	33+ 3	FGR	5.10 × 102	NA	NA	NA	term delivery, hearing impairment
25	26	33+ 5	ventriculomegaly, FGR	1.97 × 106	2.62	NA	NA	term delivery, normal outcome
26	26	20+ 1	fetal anemia, ascites, cardiac enlargement, cardiac regurgitation	3.86 × 105	NA	NA	NA	term delivery, hearing impairment
27	25	24+ 1	ventriculomegaly, intracranial hyperechoic focus, cortical dysplasia, agenesis of corpus callosum, FGR	9.97 × 106	NA	205.9	1.28	TOP
28	30	22+ 1	FGR	6.63 × 106	NA	positive	negative	term delivery, LOF
29	30	32+ 2	FGR	3.88 × 103	NA	positive	negative	term delivery, normal outcome
30	32	24+ 1	hydrops, echogenic focus in liver, ascites, pleural effusion	6.80 × 106	NA	NA	NA	TOP
31	24	25+ 4	ventriculomegaly, echogenic focus in kidney	2.96 × 104	NA	NA	NA	TOP
32	27	28+ 1	ventriculomegaly, cortical dysplasia, hydramnios	5.68 × 105	NA	109	negative	term delivery, hearing impairment
33	35	33+ 3	ventriculomegaly, FGR, fetal anemia, hepatomegaly, echogenic bowel, cardiac enlargement, oligohydramnios	2.17 × 105	NA	NA	NA	term delivery, LOF
34	32	27	ventriculomegaly, intracranial hemorrhage, fetal anemia, hydramnios	4.7 × 106	NA	NA	NA	TOP
35	31	32+ 3	echogenic bowel	2.25 × 103	NA	NA	NA	TOP
36	28	33+ 3	ventriculomegaly	7.71 × 107	NA	NA	NA	TOP
37	23	29+ 5	ventriculomegaly, periventricular pseudocysts*	6.70 × 105	NA	NA	NA	LOF
38	29	29+ 1	ventriculomegaly, microcephaly, intraventricular adhesions	3.75 × 105	NA	NA	NA	TOP
39	27	26+ 2	FGR	1.19 × 106	NA	NA	NA	TOP
40	31	29+ 5	ventriculomegaly	5.94 × 105	NA	NA	NA	term delivery, normal outcome
41	27	34+ 3	ventriculomegaly	1.93 × 104	NA	NA	NA	term delivery, normal outcome
42	25	32+ 2	ventriculomegaly, arachnoid cyst	1.94 × 105	NA	NA	NA	LOF
43	30	33+ 2	ventriculomegaly, intracranial hemorrhage, cortical dysplasia	1.35 × 105	NA	NA	NA	TOP
44	29	21+ 2	Ventriculomegaly, intraventricular adhesions	3.75 × 105	NA	NA	NA	TOP
45	24	25+ 4	ventriculomegaly, microcephaly, intracranial hyperechoic focus, cortical dysplasia*, cerebellar dysplasia*, agenesis of corpus callosum*, echogenic bowel	1.22 × 107	NA	NA	NA	term delivery, severe motor and language delay, and hearing impairment

CMV Cytomegalovirus, TOP Termination of pregnancy, NA No data available, LOF Loss of follow-up, FGR Fetal growth restriction

*Only found by MRI

Among the 45 infected fetuses, 23 presented with intracranial findings. Of these 23 cases, 11 were complicated by extracranial abnormalities, 10 exhibited severe abnormalities, and 17 showed non-isolated manifestations. Among the 22 cases without intracranial findings, 11 were associated with severe abnormalities, and 8 presented with non-isolated abnormalities.

Fetal MRI was performed prenatally in 16 infected cases. Findings were consistent with ultrasound in 11 cases (68.75%), which included six cases of ventriculomegaly (Fig. 3A) and posterior fossa dilation (Fig. 3B), one case of agenesis of the corpus callosum (Fig. 3C) with cortical dysplasia (agyria-pachygyria) (Fig. 3D). Intraventricular adhesions in cases 14 and 44 were visualized both on ultrasound (Fig. 2E and F) and MRI. In the remaining five cases (31.25%), MRI and ultrasound findings were discordant. Hyperechoic foci detected on ultrasound in cases 1 and 14 were not identified on MRI. Similarly, the intracranial hemorrhage observed on ultrasound in case 34 was not seen on MRI. In case 37, periventricular pseudocysts noted on MRI were not apparent on ultrasound. In case 45, MRI revealed cortical dysplasia, cerebellar dysplasia, and agenesis of the corpus callosum, none of which were detected by ultrasound.

Fig. 3.

Prenatal MRI findings in fetuses with intrauterine CMV infection A ventriculomegaly (white arrow) and cortical dysplasia (agyria-pachygyria) (black arrow) at 30 gestational weeks; B normal ventricle (white arrow) and normal cerebral cortex (black arrow) at 30 gestational weeks (as the control of A); C agenesis of corpus callosum (white arrow); D posterior fossa dilation (white arrow)

We next investigated the relationship between amniotic fluid viral load and the pattern of ultrasound findings. Amniotic CMV-DNA levels were significantly higher in fetuses with non-isolated findings, severe findings, or intracranial findings compared with those with isolated findings, without severe findings, or without intracranial findings, respectively (all P < 0.05) (Table 5).

Table 5.

Comparison of CMV-DNA levels across groups stratified by ultrasound findings

	CMV-DNA (copies/mL)	Z*	P
non-isolated findings (n = 25)	1.66 × 106(2.06 × 105 -8.15 × 106)	2.410	0.016
Isolated findings (n = 20)	2.66 × 104(2.30 × 103 -1.04 × 106)
with severe findings (n = 21)	3.46 × 106(2.96 × 105 -8.81 × 106)	2.764	0.006
without severe findings (n = 24)	8.95 × 104(2.30 × 103 -6.37 × 105)
with intracranial findings (n = 23)	6.70 × 105(2.17 × 105 -8.65 × 106)	2.384	0.017
without intracranial findings (n = 22)	2.44 × 104(1.74 × 103 -2.28 × 106)

^*Tested by Mann-Whitney U test

Of the 45 cases with confirmed intrauterine CMV infection, cord blood CMV-IgM was tested in 14 cases, with a positivity rate of 71.43% (10/14). The median viral load in IgM-positive cases was 5.83 × 10⁶ copies/mL, compared with 3.2 × 10⁵ copies/mL in the four IgM-negative cases. Intracranial abnormalities were present in 8 of the 10 IgM-positive cases (80.0%), whereas none were observed in the IgM-negative group. Maternal serum CMV-IgM and IgG were assessed in 19 cases. IgG was positive in all 19 cases, while IgM was detected in only two (10.53%).

The prognosis of CMV intrauterine infection and postpartum pathology examination

Among the 45 cases of intrauterine CMV infection, pregnancy was terminated in 25, while 18 resulted in live births. Follow-up on pregnancy outcome was unavailable for the remaining two cases (Fig. 1). Of the 18 live-born infants, 13 were successfully followed up for a mean duration of 20.7 months (range 1 to 36 months) for postnatal assessment, whereas 5 (27.8%) were lost to follow-up due to changes in contact information (Fig. 1). Normal growth, hearing, and neurodevelopment (motor, sensory, and cognitive functions) were observed in nine of the followed-up cases. The remaining four cases exhibited abnormalities: three (Cases 24, 26, and 32) presented with hearing impairment detected at 12, 6, and 36 months after birth, respectively; one (Case 45) showed severe motor and language delay along with hearing impairment identified at 12 months postpartum. The corresponding CMV-DNA levels in these four cases were 5.10 × 10², 3.86 × 10⁵, 5.68 × 10⁵, and 1.22 × 10⁷ copies/mL. The median CMV-DNA level was significantly higher in cases that ended in termination of pregnancy (1.66 × 10⁶ copies/mL) than in those with live births (2.66 × 10⁴ copies/mL) (Z = 2.462, P = 0.014). The termination rate was also significantly higher in cases with severe ultrasound findings (76.19%, 16/21) compared to those without severe findings (37.50%, 9/24) (χ² = 6.790, P = 0.009). Among the four live-born infants with severe prenatal findings, three exhibited normal postnatal development, while one (Case 45) presented with severe neurological abnormalities.

Placental pathological examination was performed in two cases (11 and 14), revealing enlarged eosinophilic/basophilic cells, viral inclusions, and positive CMV immunostaining in fetal and placental tissues (Fig. 4). Autopsy in two additional cases (12 and 14) demonstrated aortopulmonary septal defect, cardiomegaly, severe tricuspid regurgitation, ascites, and splenomegaly in one, and disseminated CMV infection in the lung, liver, kidneys, and pancreas in the other.

Fig. 4.

Histopathological features of CMV infection in fetal and placental tissue Immunohistochemistry showed CMV positive in fetal renal tissue (A, B) and placental tissue (C, D); cytomegalic inclusion bodies in alveolar epithelial cells of lung (E, F), renal tubular epithelial cells (G, H), hepatocyte (I, J) and epithelial cell of thyroid follicle (K, L)

Discussion

This retrospective analysis demonstrates that multiple ultrasound findings, particularly the combination of intracranial and extracranial abnormalities, are more strongly associated with intrauterine CMV infection. Uncommon prenatal sonographic markers, such as intracranial hyperechogenicity and intraventricular adhesions, showed a high rate of association with CMV infection in this small sample, warranting further investigation. In contrast, more common signs like FGR, ventriculomegaly, and echogenic bowel required co-occurring abnormalities to increase their association with CMV infection. Fetuses with intracranial, severe, or non-isolated features were correlated with elevated viral loads in amniotic fluid. Severe manifestations or high viral loads frequently resulted in pregnancy termination, consequently reducing the observed rate of abnormalities among live births. These findings provide valuable insights for the screening, diagnosis, and prognostic assessment of congenital CMV infection within the Chinese population.

The present findings suggest an intrauterine CMV infection rate of 3.83% among fetuses with abnormal ultrasound indicators. The association of different ultrasound indications with CMV intrauterine infection varies greatly. For the intracranial indicators, intracranial hyperechoic lesions, intraventricular adhesions, and cerebral cortex abnormalities showed a positive rate exceeding 45%, aligning with other studies [12, 13]. These rare intracranial findings can guide prenatal counseling and enhance prenatal diagnosis rates. Previous studies reported intracranial calcifications in 34%-70% of CMV-infected children [14] and lissencephaly-pachygyria malformations in 4/11 cases [13]. In our study, intraventricular adhesions showed a high rate of association (2/3). This was also observed in 4/8 and 3/8 cases reported by Dogan et al. [15] and Malinger et al. [12], respectively. Currently, the prenatal lateral ventricular adhesion band (intraventricular adhesion) is exclusively reported in cases with CMV infection, suggesting it may be a characteristic finding associated with CMV infection. In postnatal infants, the CMV positive rate for intracranial calcifications was 100% (3/3) [16]. Conversely, other common intracranial manifestations, such as ventriculomegaly, microcephaly, and posterior fossa cistern dilation, had CMV positive rates of less than 10%. Approximately 5% of ventriculomegaly is due to infections, predominantly caused by CMV, Toxoplasma gondii, and Zika virus [17]. In this study, the positive rate of isolated ventriculomegaly was only 1.83%, similar to another study in China (1.3%) [18], suggesting its limited association with CMV infection. When prenatal ultrasound detects ventriculomegaly, attention should be given to other intracranial or extracranial manifestations, particularly intracranial specific findings valuable in assessing CMV infection risk. The CMV infection rate among microcephaly fetuses in this study was 8.33%, higher than the 2.9% reported in 858 fetuses/infants from Brazil [19]. Congenital CMV infection significantly increased the incidence of microcephaly in live births compared to controls [20]. Another meta-analysis indicated that children with cCMV have a 2.283 times higher risk of microcephaly than controls [21], and 50% of children with symptomatic congenital infections exhibit microcephaly [22]. However, the proportions of microcephaly and periventricular cysts (6.7%) in this study were lower than those reported in SMFM guidelines, whereas the proportion of lateral ventricular dilatation (70%) was higher [23], possibly due to differences in the study population.

For extracranial manifestations, the CMV infection rate for hepatosplenomegaly, placenta thickening, fetal anemia, and pleural effusion was greater than 15%. Other sonographic findings, such as growth restriction, renal dysplasia, cardiac malformation, and polyhydramnios, showed a positivity rate of less than 5%. A large sample study reported similar results: CMV positivity rates were 1/4 in fetal pleural fluid, 8/12 in ascites, and 11/13 in hepatosplenomegaly, whereas in isolated FGR, the rate was only 2.6% (8/309), increasing to 5.7% when combined with other ultrasound abnormalities [16]. Similarly, another study found a CMV infection rate of 2.2% in FGR [24]. In this study, the CMV positivity rate in FGR was 2.68%, rising significantly to 9.89% with concurrent abnormalities. Isolated echogenic bowel had a low incidence of CMV infection (4.55%), although this was higher than the rates reported by D’Amico et al. [25] and Masini et al. [26], which were 1.4% and 1.3%, respectively. Nonetheless, its association with CMV infection was limited. Therefore, we combined various hyperechoic manifestations and classified them as hyperechoic phenotypes. When the hyperechoic phenotype was combined with other manifestations, the positivity rate significantly increased. Importantly, CMV infection rates were higher in patients with multiple intracranial manifestations or combined intracranial and extracranial manifestations. Enders et al. [27] also suggested that fetal multiple manifestations or intracranial ultrasound abnormalities indicate intrauterine infection. Simonazzi et al. [28] reported that among 9 cases of suspected maternal infection and fetal multiple ultrasound abnormalities, 4 fetuses were CMV positive. These results indicate that the presence of multiple ultrasound markers is more strongly associated with CMV infection. A comprehensive and systematic ultrasound or MRI examination can improve the identification of CMV infection.

In our study, ultrasound and MRI findings were consistent in 68.75% of cases, while complementary findings were noted in the remaining cases. However, due to the small and selected nature of the MRI subgroup (only 16 cases, with non-standardized timing and indications), these findings should be considered preliminary. The discordance for intracranial hemorrhage may be explained by the different sensitivities of the two modalities to blood products at various stages of evolution, with ultrasound being more sensitive to acute/subacute hemorrhages. MRI detected more intracranial cysts and parenchymal dysplasia, whereas ultrasound identified intracranial calcifications and hemorrhage not indicated by MRI, consistent with a large meta-analysis [29]. Buca et al. [30] proposed that around 6% of fetuses with negative ultrasound results could have additional findings on fetal MRI, but only in first-trimester infections. However, as these two examinations are typically not conducted simultaneously, direct comparison of their clinical utility for CMV infection is not feasible. The ultrasound positive rate also depends on whether neurosonography is performed, which correlates well with MRI [31].

Our data indicate that maternal IgM has limited clinical utility in assessing the severity of intrauterine infections. Kim et al. [32] discovered that among 8 cases of fetal infection, only one tested positive and one weakly positive. Nonetheless, the positive rate of maternal IgM positivity combined with prenatal ultrasound abnormalities for congenital infections was 61.1% [33]. A study reported that in 32 cases of maternal secondary infection with neonatal infections, maternal blood IgM was negative, and only 6% showed significantly elevated IgG levels in the first or the second trimester [34]. This indicates that elevated maternal IgM or IgG levels are not highly sensitive to secondary infections. Moreover, the clinical relevance of maternal serological antibody testing for isolated and non-specific ultrasound abnormalities is limited [35]. Based on the aforementioned research and the findings of this study, serological antibody screening is not recommended for low-risk pregnant women.

The present analysis suggests that cord blood CMV-IgM is negative in approximately 30% of confirmed intrauterine infection cases. Some studies suggest that the IgM positivity rate in umbilical cord blood is influenced by the timing of infection, with higher rates observed in the second trimester compared to the third trimester [36]. Among neonatal infections, the IgM positivity rate was only 40.7% [4]. This study found that IgM-positive infections had higher DNA levels than IgM-negative ones, possibly due to a correlation between higher viral loads and stronger immune responses [4]. Additionally, 80% of the IgM-positive cases exhibited intracranial manifestations, whereas none of the IgM-negative cases did. Although IgM positivity appears to be associated with intracranial lesions, the sample size is insufficient for statistically significant conclusions. For CMV-infected newborns, IgM positivity is associated with an increased risk of clinical symptoms and hearing impairment [37]. Enders et al. [27] reported that fetuses with ultrasound abnormalities had significantly higher IgM levels in umbilical cord blood compared to those without abnormalities. Bilavsky et al. [4] suggested that newborns with neurological abnormalities also had higher IgM levels. However, another study found no association between IgM positivity and fetal neurological abnormalities [36]. According to ISUOG guidelines, although fetal blood markers such as platelet count, beta-2 microglobulin, and cytomegalovirus IgM are linked to prognosis, the added value of fetal blood sampling for prognostic purposes remains uncertain [6]. Therefore, the relationship between IgM positivity and neurological manifestations remains unclear. The limited number of cases in this study underscores the need to increase the sample size to further investigate the clinical significance of fetal blood IgM in CMV.

Our findings indicate that severe and non-isolated developmental abnormalities are positively correlated with amniotic fluid viral DNA levels, suggesting that viral load may reflect the extent and severity of infection. Previous research has shown that viral load in amniotic fluid is higher in cases with significant fetal ultrasound abnormalities compared to those with normal ultrasound findings [38]. Among antenatally diagnosed infected fetuses, DNA levels were significantly higher in those with abnormal ultrasound findings than in those with normal ones [27]. However, another study found no association between viral load and sonographic signs [39]. These discrepancies could stem from differences in examination timing and methods, as amniotic fluid viral load fluctuates over time post-seroconversion [6]. The viral load in amniotic fluid reflects the combined effects of fetal virus excretion and amniotic fluid clearance [40]. Factors such as the gestational age at puncture and the interval between maternal infection and puncture also influence viral load [38]. Additionally, this study suggests that intracranial manifestations correlate with higher amniotic fluid DNA levels, likely due to their association with severe, non-isolated developmental abnormalities (17 of 23 intracranial cases in this study were non-isolated and 10 were severe). Viral load might reflect the severity of brain damage, although other research indicates it may not be linked with brain injury [41]. The relationship between viral load and nervous system damage warrants further investigation.

Among the successfully followed-up live birth cases in this study, 30.8% (4/13) had developmental abnormalities. A study in South Korea found that 43.5% (10/23) of infected infants were diagnosed with developmental delays, and 42.9% (9/21) also experienced hearing loss [32]. In comparison, the prognosis of live birth cases in this study was relatively better, likely because more severe cases opted for pregnancy termination, resulting in a lower level of amniotic fluid DNA. The most common issue observed in this study was hearing loss, which aligns with findings from other research [30]. A meta-analysis indicates that among isolated abnormalities, only microcephaly is associated with a risk of poor prognosis exceeding 95% [29].

Although DNA levels in cases with severe poor outcomes (1.22 × 10⁷/mL, case 45) were significantly higher than the mean DNA levels in cases with normal follow-up outcomes (3.86 × 10⁵/mL), DNA levels exceeded 1 × 10⁵/mL in two cases with normal outcomes (cases 25 and 40). This suggests that viral replication levels in amniotic fluid are not always correlated with prognosis, aligning with findings from previous studies [42]. Some fetuses with high amniotic viral loads were asymptomatic at birth, while others with low viral loads exhibited severe abnormalities [39]. The median viral load in amniotic fluid of symptomatic fetuses was higher than that of asymptomatic ones [6]. However, predicting the severity of neurological prognosis from viral load in amniotic fluid remains challenging [41]. Thus, the relationship between amniotic fluid viral load and postnatal symptoms and prognosis remains uncertain. Notably, DNA load in neonatal urine has been linked to sensorineural deafness (SNHL) and central nervous system damage [43]. Additionally, the rate of poor prognosis correlates with the length of follow-up. 10% of asymptomatic newborns show developmental impairments by age six [44]. The small number of follow-up cases and the short follow-up duration in this study limit the exploration of long-term prognosis.

This study has several important limitations that must be considered when interpreting the results. First, the most significant limitation is selection/referral bias. All three participating centers are tertiary referral centers. Our cohort exclusively includes cases where clinicians suspected CMV infection strongly enough to proceed with amniocentesis. Consequently, the reported CMV positivity rates (e.g., 3.83% overall, and 1.83% for isolated ventriculomegaly) likely overestimate the true prevalence of CMV infection among fetuses with similar ultrasound findings in the general population. Moreover, we do not know how many fetuses with mild or isolated findings such as ventriculomegaly, echogenic bowel, FGR, or polyhydramnios were never referred for CMV testing. Our results should be interpreted as associations within a high-risk, referred population, not as population-based estimates. Furthermore, referral criteria for amniocentesis were not standardized across centers and were subject to individual clinician judgment, introducing additional variability. Findings such as echogenic bowel, FGR, and polyhydramnios are particularly susceptible to such referral bias. Our study does not provide robust evidence to determine which findings should independently raise suspicion for CMV infection, when amniocentesis should be recommended, or how these findings should be incorporated into patient counseling. Such clinical decisions require prospective, population-based studies. Second, the precise timing of maternal infection was often unspecified, preventing a reliable analysis of its correlation with fetal infection rates or the timing of sonographic findings. Third, the number of cases with distinct imaging findings, such as intracranial hyperechogenicity and intraventricular adhesions, remains relatively small, leading to wide confidence intervals. Their association with CMV infection, while striking in this exploratory analysis, requires validation in larger, prospective cohorts. Fourth, the retrospective design limited data collection. All included cases had abnormal ultrasound findings, and complete maternal serological data (CMV-IgG/IgM) were unavailable for many, precluding a synchronous analysis of combined ultrasound and serological associations. Notably, CMV-IgG seroprevalence exceeds 90% among Chinese women of reproductive age, while CMV-IgM assays are prone to false positives with suboptimal sensitivity/specificity, diminishing their clinical utility–a key rationale for this study’s focus on specific ultrasound indicators. Fifth, the high proportion of terminated pregnancies (55.6%, 25/45) introduces significant survivorship bias. The live-born and followed-up infants (n = 13) likely represent a less severely affected subgroup, potentially underestimating the true prevalence of adverse neurodevelopmental and hearing outcomes associated with cCMV. The limited number of followed-up cases and the variable follow-up durations further restrict our ability to analyze long-term prognostic risk factors. Sixth, only a small subset (n = 16) of patients underwent fetal MRI, and the timing and indications were not standardized, introducing selection bias. Therefore, our MRI-related conclusions are preliminary and require validation in a prospective study with a standardized imaging protocol. Future prospective studies with larger, unselected cohorts and standardized imaging and follow-up protocols are warranted to address these limitations.

Conclusion

This study establishes a significant correlation between specific prenatal ultrasound features and intrauterine CMV infection. Distinctive sonographic markers, particularly when involving combined intracranial abnormalities, demonstrated a high rate of association. While isolated common findings showed a limited independent association with CMV infection, their co-occurrence with other anomalies substantially increased the likelihood of CMV infection. In confirmed cases, elevated CMV-DNA load in amniotic fluid was associated with the presence of intracranial, non-isolated, and severe fetal abnormalities. These findings underscore the clinical value of integrating detailed prenatal ultrasound and fetal MRI in the screening algorithm for congenital CMV infection. However, the relationship between specific imaging phenotypes, viral load, and long-term neurodevelopmental outcomes requires further validation in larger, prospective cohorts to refine risk stratification and counseling.

Abbreviations

CMV

Cytomegalovirus

VL

Viral load

FGR

Fetal growth restriction

TOP

Termination of pregnancy

LOF

Loss of follow-up

SNHL

Sensorineural deafness

MRI

Magnetic resonance imaging

MCA-PSV

The peak systolic velocity in the middle cerebral artery

MoM

Multiples of the median

Authors' contributions

XH designed and supervised the study. SLZ organized and analyzed the data and wrote the manuscript. HX, YF, RNZ, TGW, SLZ, and XMZ collected the clinical data. HNX and TL collected and analyzed the ultrasound examination results. HJW reviewed the features of the MRI images. HH and MZH collected the results of virological examinations. LC performed the histological examination. All authors reviewed the manuscript and approved the final manuscript.

Funding

No.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Requests to access these datasets should be directed to Xuan Huang, Email: huangx35@mail.sysu.edu.cn.

Declarations

Ethics approval and consent to participate

This study was in accordance with the Helsinki declaration. In addition, the study was approved by the Research Ethics Committee of The First Affiliated Hospital of Sun Yat-sen University. Data were obtained anonymously from medical records without the direct participation of subjects, hence written informed consent was waived, which was approved by the Research Ethics Committee of The First Affiliated Hospital of Sun Yat-sen University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.