Novel markers on microvascular flow imaging for identifying holoprosencephaly

Abstract

Objectives

To establish gestational age-specific reference ranges for two new indices, the forehead-anterior cerebral artery angle (FACAA) and forehead-anterior cerebral artery distance (FACAD), and to objectively evaluate their diagnostic effectiveness, measured using Doppler technology with microvascular flow imaging, for identifying holoprosencephaly, even the semilobar and lobar types, in early pregnancy.

Methods

We evaluated FACAA and FACAD in 462 normal fetuses between 12 and 34 gestational weeks (GW) to generate normative reference ranges. Additionally, we evaluated these two indices in 41 fetuses with holoprosencephaly and 34 fetuses with similar two-dimensional ultrasound features (the study group), including 20 fetuses with agenesis of the corpus callosum and 14 with isolated absent septum pellucidum/septo-optic dysplasia.

Results

In the normal group, FACAA was almost stable throughout pregnancy, whereas FACAD increased with GW. All fetuses with holoprosencephaly had both smaller FACAA (13.99–38.00°, ≤5th percentile) and shorter FACAD (1.6–10.5 mm, ≤5th percentile). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and negative likelihood ratio (LR–) of FACAA for the prediction of holoprosencephaly in fetuses with an intracranial malformation were 100% (95% confidence interval [CI]: 89.3–100.0%), 100% (95% CI: 87.4–100.0%), 100% (95% CI: 89.3–100%), 97.1% (95% CI: 82.9–99.8%), and 0, respectively. The sensitivity, specificity, PPV, NPV, and LR– of FACAD for the prediction of holoprosencephaly in fetuses with an intracranial malformation were 97.6% (95% CI: 85.9–99.9%), 100% (95% CI: 87.0–100.0%), 100% (95% CI: 89.3–100%), 97.1% (95% CI: 82.9–99.8%), and 0.02 (95% CI: 0.003–0.165), respectively.

Conclusion

FACAA and FACAD are sensitive, easy-to-measure indicators for the prenatal identification of holoprosencephaly, even in early pregnancy. These indices provide objective assessment tools for the diagnosis of fetuses with holoprosencephaly.

Trial registration

Not applicable.

Introduction

Holoprosencephaly is a severe cerebral developmental malformation characterized by incomplete separation of the cerebral hemispheres with a poor prognosis. It is classified into four subtypes: alobar, semilobar, lobar, and middle variant [1]. Among these, the alobar type is easily detected owing to the presence of one fused lateral ventricle and is typically diagnosed using basic ultrasound techniques. However, semilobar and lobar types often remain undetected until the midtrimester [2] because they present with subtle or absent abnormal features on gray-scale sonography. In addition, it is challenging to distinguish holoprosencephaly from abnormalities with similar appearances, such as isolated absent septum pellucidum (IASP), septo-optic dysplasia (SOD), or agenesis of the corpus callosum (ACC), as they all have abnormalities of the septum pellucidum [3].

Evaluation of vascular structures is crucial for diagnosing abnormal cerebral development and is greatly improved by microvascular flow imaging (MVFI), a doppler technology uses advanced filtering technology to remove low-frequency artifacts while retaining the low-speed blood signals in the micro vessels [4, –6]. Novel markers, such as the brain stem-tentorium (BST) angle [7] and the tela choroidea–anterior cerebral artery distance (TACAD) [8], have proven valuable for diagnosing open spina bifida (OSB) and ACC. Recently, we found that the angle between the vein of Galen and the straight sinus (AVGS) is a useful marker for the differential diagnosis of posterior fossa anomalies [9]. However, there is no quantitative marker for the differential diagnosis of holoprosencephaly.

In a clinical study, we found that in fetuses with holoprosencephaly, the anterior cerebral artery (ACA) appears to shift forward, positioning the ACA and pericallosal artery closer to the frontal skull. We hypothesized that quantifying this sign may help predict holoprosencephaly, especially for the semilobar and lobar types in early pregnancy, and aid in distinguishing it from intracranial abnormalities with similar sonographic appearances. In this study, two new markers, the forehead-anterior cerebral artery angle (FACAA) and forehead-anterior cerebral artery distance (FACAD), were measured using Doppler technology with microvascular flow imaging. This study aimed to establish reference ranges for FACAA and FACAD at different gestational ages and to objectively evaluate their effectiveness for the diagnosis of holoprosencephaly.

Methods

Participants

This was a retrospective analysis of data and scans from a single center performed between October 2019 and October 2024. We included 462 normal singleton pregnancies evaluated for a series of prenatal ultrasound scans (four scans) between 12 and 34 gestational weeks (GWs) as the control group. The study group comprised 20 fetuses with ACC, 14 fetuses with IASP/SOD, and 41 fetuses with holoprosencephaly. Gestation age was determined based on the last menstrual period and confirmed by ultrasound between 9 and 13 + 6 GWs, provided that the discrepancy was less than 7 days.

Inclusion criteria for the control group required: normal fetal growth, normal appearance on both gray-scale sonography and microvascular flow imaging, no fetal chromosomal abnormalities, normal amniotic fluid volume, no maternal complications, and normal pregnancy outcomes. Cases with fetal intracranial malformation < 18 GWs were confirmed with transabdominal or transvaginal ultrasound combined with genetic testing. According to fetal magnetic resonance imaging (MRI) guidelines, limited diagnostic information could be derived before 18 GWs [10]; therefore, cases with malformation identified at ≥ 18 GWs were further verified with MRI.

Holoprosencephaly was defined as the fusion of any part of the brain midline [1]. IASP/SOD was defined as the absence of a normal septum pellucidum in the coronal, sagittal, and transthalamus plane [11]. ACC was defined as the complete or partial absence of any of the four components of the corpus callosum (rostrum, genu, body, and splenium) [12]. Cases involving other severe intracranial malformations that can cause ACA displacement, such as hydrocephalus and large midline cysts, which can lead to significant variations in angle measurements, were excluded from this study. Fetuses with abnormal skull contours and shapes were excluded, as the FACAA is influenced by skull shape.

For both the control and study groups, the following data were collected: maternal age, obstetric history, gestation age at examination, associated intracranial or extracranial anomalies or syndromes, results of genetic testing (if available), and outcomes. All eligible patients were fully informed and signed an informed consent form for participation and use of the acquired data during the study period. The study was approved by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital (No.2022-KY-013-02).

Ultrasound image acquisition

Common systematic screening

High-resolution images of the fetal anatomy were acquired using the Samsung HERA10. For transabdominal ultrasound examinations, a probe with a frequency range of 1–8 MHz was used. The color Doppler flow gain was set between 50 and 65 dB, and the pulse repetition frequency (PRF) was configured at 0.13 kHz. The acoustic output power was set at 90%. The thermal index for bone (TIb), thermal index for soft tissue (TIs), and mechanical index (MI) were maintained at 0.4, 0.4, and 1.0, respectively. For transvaginal ultrasound examinations, a probe with a frequency range of 2–10 MHz was applied. The color Doppler flow gain ranged from 37 to 60 dB, with a PRF of 0.15 kHz. The acoustic output power was also set at 90%. Corresponding safety indices included a TIb of 0.3, a TIs of 0.3, and an MI of 0.98.

In all cases, a routine systematic screening according to the gestational age was performed following the guidelines of the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) [13, 14]. Standard biometric measurements, including biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL), were performed, followed by a more detailed structural assessment according to gestational age of the fetal central nervous system (CNS) as per ISUOG Practice Guidelines [15, 16]. For the first trimester scan, the detailed CNS scanning aimed to assess the contour and shape of the fetal skull, choroid plexus shape, lateral cerebral ventricles, third ventricle, aspect and position of the aqueduct of Sylvius (AS), brainstem, presence of the cisterna magna (CM), fourth ventricle, and choroid plexus of the fourth ventricle. For the second and third trimester scans, in addition to the above structures, the corpus callosum, sulci, parenchyma, and cerebellar vermis were evaluated.

Measurements

All measurements were performed in the midsagittal view, which was obtained through the anterior fontanelle, enabling optimal visualization of cerebral midline structures. This view included the root of the nose, forehead, corpus callosum (rostrum, genu, body, and splenium), cavum septi pellucidi, cavum vergae, third and fourth ventricles, cerebellar vermis, and brainstem. Microvascular flow imaging enabled clear visualization of the anterior edge and branches of the ACA and pericallosal arteries, and the images were saved for subsequent measurements.

Two parameters were evaluated in our study, which were measured by one of two examiners (Z.L. and L.S.), each with more than 5 years of experience in fetal neurosonography.

For FACAA, two tangent lines were made from the root of the nose to the forehead and the anterior borderline of the ACA and pericallosal artery. The acute angle between the two tangent lines was measured (Fig. 1).

Fig. 1.

Measurement of FACAA and FACAD in normal fetuses at 12 GWs. (A, D, G) Microvascular flow imaging; (B, C, E, F, H, I) corresponding images showing FACAA and FACAD measurements. FACAA (B) is defined as the acute angle between two tangent lines, one from the root of the nose (RoN) to the forehead and the other to the anterior border of the ACA. FACAD (C) is defined as the shortest distance between the two tangent lines and the parallel line (dotted line), which is marked with a double arrow line. Measurement of FACAA and FACAD for a normal fetus at 21 GWs (E, F). Measurement of FACAA and FACAD for a normal fetus at 29 GWs (H, I). ACA: anterior cerebral artery; PCA: pericallosal artery; RoN: root of the nose; FACAA: forehead-anterior cerebral artery angle; FACAD: forehead-anterior cerebral artery distance; GW: gestational weeks

For FACAD, a tangent line was made from the root of the nose to the forehead, then moving this line posteriorly in a parallel direction until it contacted the anterior border of the ACA/pericallosal artery. The shortest distance between the two parallel lines was measured (Fig. 1).

Statistical analysis

Data were analyzed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA), MedCalc Version 22.009 (MedCalc Software, Ostend, Belgium), and R project (R Core Team 2024, R Foundation for Statistical Computing, Vienna, Austria). A P-value < 0.05 was considered statistically significant.

Continuous data are expressed as mean ± standard deviation (SD). The association between FACAD and gestational age was analyzed by calculating the Spearman’s rank correlation coefficient. The curves of the relationship between FACAA and GWs and FACAD and GWs were fitted using the LMS method [17], and the reference values for the 5th, 50th, and 95th percentiles of FACAA and FACAD were calculated for each GW in the control group.

Intra- and inter-observer agreements were evaluated using 30 randomly chosen images. These images were measured by both observers (Z.L. and L.S.) to calculate inter-observer agreement and twice by one of the observers (Z.L.), with a 1-week interval between the measurements, to calculate intra-observer agreement. Intra- and inter-observer agreements were evaluated by calculating the mean difference between the two measurements using the Bland–Altman method [18] with 95% limits of agreement (LOA). The intraclass correlation coefficients (ICC) for intra- and inter-observer agreement were calculated, and an ICC > 0.8 was considered good.

Using MRI diagnosis as the gold standard, we evaluated the performance of the FACAA and FACAD in predicting holoprosencephaly in fetuses with intracranial malformations (fetuses with ACC and IASP/SOD). An abnormal value of FACAA and FACAD was defined as ≤5th percentile based on the normal reference range. Sensitivity, specificity, positive likelihood ratio (LR+), negative likelihood ratio (LR–), positive predictive value (PPV), and negative predictive value (NPV) were calculated. As FACAA is stable throughout pregnancy, receiver operating characteristic (ROC) curve analysis was used to determine the area under the curve (AUC) and optimal cut-off value of FACAA between the control and holoprosencephaly groups.

Results

Demographics

The demographic characteristics of the included fetuses are presented in Table 1.

Table 1.

Demographic characteristics

Characteristics/Group	Normal (n = 462)	Holoprosencephaly (n = 41)	IASP/SOD (n = 14)	ACC (n = 20)	P
Maternal age (years)	32.18 ± 3.53	32.65 ± 4.77	29.46 ± 4.48	31.71 ± 4.10	0.05
BMI (mean ± SD)	22.17 ± 2.99	23.54 ± 4.18	22.52 ± 1.68	23.97 ± 2.39	0.01
Gravidity	1 (0–6)	2 (1–5)	1 (1–3)	1 (1–4)	0.217
Parity	0 (0–2)	0 (0–2)	0 (0–1)	0 (0–1)	0.017
GW (weeks)	22.49 ± 6.91	14.08 ± 3.52	26.51 ± 4.17	24.45 ± 3.81	< 0.001
Termination of pregnancy (n)	0	41	10	19
Delivery (n)	462	0	4	1

Data are presented as mean±standard deviation (SD), median (range), or n

BMI, Body Mass Index; IASP, isolated absent septum pellucidum; SOD, sept-optic dysplasia; ACC, agenesis of the corpus callosum; GW, gestational week at ultrasonographic evaluation

Normal ranges of FACAA and FACAD at different gestational weeks

The reference ranges of FACAA and FACAD in normal fetuses at different GWs are presented in Table 2; Fig. 2, shown as the 5th, 50th, and 95th percentiles. FACAA values decreased with advancing gestational age (Spearman’s rho = − 0.377; P < 0.001), whereas FACAD values increased with advancing gestational age (Spearman’s rho = 0.959; P < 0.001). The intra-observer ICCs for FACAD and FACAA were 0.997 (95% confidence interval [CI]: 0.994–0.999) and 0.909 (95% CI: 0.817–0.955), respectively. The inter-observer ICCs for FACAD and FACAA were 0.950 (95% CI: 0.897–0.976) and 0.817 (95% CI: 0.651–0.909), respectively. All ICCs were > 0.8, indicating good intra- and inter-observer agreement for the FACAA and FACAD measurements (Fig. 3).

Table 2.

Normal ranges of FACAA and FACAD in normal fetuses

Gestational weeks	Number of fetuses	FACAA (degrees)			FACAD (mm)
		5th percentile	50th percentile	95th percentile	5th percentile	50th percentile	95th percentile
12	36	56.14	63.07	70.86	4.55	5.48	6.95
13	27	53.28	60.81	69.41	5.36	6.39	8.00
14	14	50.74	58.79	68.12	6.33	7.50	9.26
15	19	49.02	57.35	67.11	7.50	8.81	10.74
16	38	48.24	56.61	66.43	8.69	10.13	12.22
17	26	48.03	56.27	65.94	9.75	11.29	13.49
18	16	47.98	56.00	65.37	10.64	12.25	14.51
19	13	47.93	55.63	64.59	11.39	13.05	15.35
20	17	47.83	55.17	63.64	12.05	13.75	16.07
21	13	47.67	54.63	62.61	12.6	14.34	16.69
22	23	47.44	54.03	61.54	13.06	14.84	17.25
23	24	47.19	53.46	60.56	13.52	15.36	17.84
24	19	46.84	52.91	59.78	14.03	15.94	18.54
25	17	46.36	52.47	59.38	14.58	16.61	19.37
26	12	45.87	52.26	59.55	15.19	17.35	20.31
27	11	45.47	52.33	60.22	15.83	18.15	21.35
28	11	45.21	52.55	61.08	16.48	18.96	22.43
29	20	45.12	52.81	61.82	17.06	19.71	23.47
30	33	45.26	53.11	62.33	17.56	20.38	24.42
31	21	45.55	53.43	62.69	18.00	20.98	25.29
32	18	45.85	53.74	63.00	18.42	21.55	26.13
33	24	46.10	54.01	63.29	18.83	22.11	26.94
34	10	46.32	54.25	63.53	19.23	22.65	27.73

FACAA, Forehead-Anterior Cerebral Artery angle; FACAD, Forehead-Anterior Cerebral Artery distance

Fig. 2.

Measurements of FACAA and FACAD values. (A) Individual measurements of FACAA values in normal fetuses (dark circles), with reference ranges (5th, 50th, and 95th percentiles) plotted against each GW. In comparison, the values in 41 fetuses with holoprosencephaly were displayed (white triangle). (B) Individual measurements of FACAA values in normal fetuses (dark circles), with reference ranges (5th, 50th, and 95th percentiles) plotted against each GW. In comparison, the values in 14 fetuses with isolated absent septum pellucidum (IASP)/septo-optic dysplasia (SOD, white circle) and 20 fetuses with agenesis of the corpus callosum (ACC, white square) are displayed. (C) Individual measurements of FACAD values in normal fetuses (dark circles), with reference ranges (5th, 50th, and 95th percentiles) plotted against each GW. For comparison, the values for 41 fetuses with holoprosencephaly are shown (white triangles). (D) Individual measurements of FACAD values in normal fetuses (dark circles), with reference ranges (5th, 50th, and 95th percentiles) plotted against each GW. In comparison, the values in 14 fetuses with isolated absent septum pellucidum (IASP)/septo-optic dysplasia (SOD, white circle) and 20 fetuses with agenesis of the corpus callosum (ACC, white square) are displayed

Fig. 3.

Intraobserver and interobserver agreement in FACAA and FACAD. Bland–Altman plots showing intraobserver (A, B) and interobserver (C, D) agreement in the measurement of FACAA and FACAD. Thick lines (——) represent the mean difference and dotted lines (………….) represent 95% limits of agreement. Error bars indicate 95% confidence intervals (CIs)

FACAA remained relatively stable throughout pregnancy, whereas FACAD increased with gestational age. A quadratic equation provided a good fit for the relationship between FACAD and gestational age (R² = 0.959; P < 0.001). The regression equation was as follows:

FACAA and FACAD of fetuses with holoprosencephaly

In this study, we included 41 fetuses with holoprosencephaly, 20 with ACC, and 14 fetuses with IASP/SOD (Table 3). All fetuses with holoprosencephaly (41/41) had both reduced FACAA values (13.99–38.00°) and shortened FACAD values (1.6–10.5 mm), all below the 5th percentile of the normal reference range (Figs. 2 and 4). Compared to other groups, fetuses with holoprosencephaly had lower FACAA values (P < 0.001, Table 3; Fig. 5). No significant differences in FACAA and FACAD were observed between the holoprosencephaly subtypes (P > 0.05). Four fetuses with semilobar and lobar holoprosencephaly were diagnosed in the first trimester and early second trimester, including three cases with the semilobar type and one case with the lobar type, using FACAA and FACAD. The cases were suspected based on the measurements and confirmed in follow-up MRI examinations. ROC curve analysis showed the good diagnostic performance of FACAA, with an AUC of 1.00 for distinguishing the control group and the study group from the holoprosencephaly group (Figs. 6 and 7). The optimal cut-off values were 40.41° for the control vs. the holoprosencephaly comparison and 43.00° for the study group vs. holoprosencephaly comparison.

Table 3.

FACAA and FACAD of fetuses with intracranial anomaly included

Abnormalities	Number of fetuses	Subtypes (n)	FACAA (range, degrees)	FACAA (n, normal/decreased/increased)	FACAD (range, mm)	FACAD (n, normal/decreased/increased)
IASP /SOD	14	IASP/SOD (14)	48.00–64.67	13/0/1	11.90–22.60	13/0/1
ACC	20	partial(8)/complete (12)	48.00–70.00	15/0/5	14.10–28.90	10/1/9
Holoprosencephaly	41	alobar(33)/semilobar(4)/lobar (4)	13.99–38.00	0/41/0	1.60–10.50	0/41/0

IASP, isolated absent septum pellucidum; SOD, sept-optic dysplasia; ACC, agenesis of the corpus callosum

Decreased: below the 5th percentile of the normal reference; increased: above the 95th percentile of the normal reference; normal: between the 5th and 95th percentiles of the normal reference

Fig. 4.

Decreased FACAA and FACAD in the fetus with holoprosencephaly at 12 GWs. Brain imaging of a normal fetus at 12 GWs (A, B, C, G, H, I) and a fetus with holoprosencephaly at 12 GWs (D, E, F, J, K, L). (A, D) Microvascular flow imaging; (B, C, E, F) corresponding images with FACAA and FACAD measurements indicated. FACAA and FACAD were decreased in the fetus with holoprosencephaly (≤ 5th percentile). Figures G, H, and I are the midsagittal and transcerebral planes of the normal fetus. Figures J, K, and L are the midsagittal and transcerebral planes of the fetus with holoprosencephaly

Fig. 5.

Boxplot comparison of FACAA values across four groups. FACAA was relatively stable through pregnancy. Fetuses with holoprosencephaly exhibited significantly smaller FACAA values than normal fetuses and the study group (fetuses with IASP/SOD or ACC), P < 0.05

Fig. 6.

ROC curves of FACAA for predicting fetuses with holoprosencephaly. (A) ROC curves of FACAA for predicting the holoprosencephaly group from the control group (normal fetuses); (B)ROC curves of FACAA for predicting the holoprosencephaly group from the study group (fetuses with IASP/SOD or ACC)

Fig. 7.

ROC curves of FACAD for predicting fetuses with holoprosencephaly. (A) ROC curves of FACAD for predicting the holoprosencephaly group from the control group (normal fetuses); (B)ROC curves of FACAD for predicting the holoprosencephaly group from the study group (fetuses with IASP/SOD or ACC). Only decreased FACAD was listed as positive

Validation in 75 fetuses with intracranial anomaly

The sensitivity, specificity, PPV, NPV, and LR– of FACAA for the prediction of holoprosencephaly in fetuses with an intracranial malformation were 100% (95% CI, 89.3–100.0%), 100% (95% CI, 87.4–100.0%), 100% (95% CI, 89.3–100%), 97.1% (95% CI, 82.9–99.8%), and 0, respectively. The sensitivity, specificity, PPV, NPV, and LR– of FACAD for the prediction of holoprosencephaly in fetuses with an intracranial malformation were 97.6% (95% CI, 85.9–99.9%), 100% (95% CI, 87.0–100.0%), 100% (95% CI, 89.3–100%), 97.1% (95% CI, 82.9–99.8%), and 0.02 (95% CI, 0.003–0.165), respectively (Figs. 6 and 7).

Discussion

Principal findings

In this study, we measured two quantitative indices, the FACAA and FACAD, in 462 normal fetuses and established normal references at each GW. We then evaluated and found smaller FACAA and shorter FACAD (≤ 5th centile) in 41 fetuses with holoprosencephaly, which may be caused by the displacement of the ACA/pericallosal artery. Smaller FACAA and shorter FACAD could be sensitive indicators for the detection of holoprosencephaly, including semilobar and lobar types, particularly in early pregnancy. Moreover, they can aid in differentiating holoprosencephaly from similar malformations, such as ACC and IASP/SOD.

Our results indicated that all fetuses with holoprosencephaly exhibited smaller FACAA and shorter FACAD (Table 3; Figs. 2 and 8), suggesting forward displacement of the ACA/pericallosal arteries toward the skull. Previous case reports have noted this sign in the middle-variant and lobar subtypes [19–21]. We hypothesize that this anterior shift of the ACA may result from the obstacle constituted by midline interhemispheric parenchymatous fusion. Using FACAA and FACAD, we successfully diagnosed three cases of semilobar holoprosencephaly at 13–14 GWs and one case of lobar holoprosencephaly at 16 GWs. These diagnoses, typically challenging even in mid- or late pregnancy (when the fetal brain is more mature), were made during the first and early second trimesters with the aid of these indicators and were confirmed by follow-up MRI. Notably, the gray-scale images for the three semilobar holoprosencephaly cases diagnosed at 13–14 GWs showed no significant findings, whereas microvascular flow imaging showed anterior displacement of the ACA or pericallosal artery. In contrast, most fetuses with IASP/SOD or ACC had FACAA and FACAD values within or above the normal reference range. This highlights that decreased FACAA and FACAD caused by forward ACA displacement is a unique feature of holoprosencephaly and can serve as an important differentiator from other cerebral abnormalities. Thus, FACAA and FACAD enable the detection of fetuses with semilobar and lobar holoprosencephaly as early as the first trimester.

Fig. 8.

Decreased FACAA and FACAD in the fetus with holoprosencephaly at 23 GWs. Brain imaging for a normal fetus at 23 GWs (A, B, C), a fetus with lobar holoprosencephaly (D, E, F), a fetus with IASP/SOD (G, H, I), and a fetus with ACC (J, K, L). (A, D, G, J) Microvascular flow imaging; (B, C, E, F, H, I, K, L) corresponding images with FACAA and FACAD measurements indicated. FACAA and FACAD were decreased in the fetus with holoprosencephaly (≤ 5th percentile). FACAA and FACAD were within the normal range in the fetus with IASP/SOD, whereas FACAD was larger in the fetus with ACC

In this study, we found that the value of FACAA was relatively stable throughout pregnancy (Fig. 2), whereas FACAD increased with gestational age. This indicates that FACAD values vary across different GWs (Fig. 2), making FACAA a more stable, simple, and convenient indicator. Based on ROC analysis, holoprosencephaly should be suspected when the fetal FACAA is below 40.41°. When performing this measurement, it is important that the frontal cut line aligns with the bony edge of the forehead, but not the skin, which is especially important in fetuses with edema. Because of their different measurement methods, FACAA and FACAD are complementary. FACAA is easier to acquire and remains stable across gestation; however, in some cases, FACAD may be required for differential diagnosis.

Another notable finding was that some fetuses with ACC had larger FACAA or longer FACAD (Table 3; Fig. 2). We speculate that this may result from the absence of the corpus callosum, which results in severe retrogression of the ACA. Consequently, in these fetuses, the ACA may appear vertically oriented, totally different from the “ACA arch” seen in normal fetuses [8, 22]. Therefore, larger FACAA or longer FACAD may be good markers for the detection of ACC; however, owing to the limited sample size of this study, further validation with larger sample data is needed. Notably, one fetus with ACC who had a short FACAD also presented with complex cerebral abnormalities, including lateral ventricle dilatation and interhemispheric cysts, which can cause large variations in FACAA or FACAD.

Microvascular flow imaging (MVFI) utilizes the principle of time-space coherence, and advanced “algorithmic” filtering technology to extract low-velocity blood flow, which is more sensitive than power Doppler while ensuring high frame rates. It can precisely present slow micro-perfusion blood flow in peripheral structures. Up to now, MV has been applied to research on fetal cranial cerebrovascular development [23], prenatal renal diagnosis [24], diagnosis of fetal cranial brain malformations [9], placental development research [25], and so on.

Recently, novel markers such as the BST angle and the TACAD have shown promise in diagnosing intracranial abnormalities. These parameters evaluate maldeveloped cerebral vessels using microvascular flow imaging, which enables visualization of small and low-speed intracranial vessels. Calculation of the BST angle can help differentiate Dandy-Walker malformations (DWM) from OSB; fetuses at high risk for DWM exhibit a reduced BST angle (often < 20°), due to the elevated tentorium [23], while fetuses with OSB show a BST angle close to 90° due to the low position of the tentorium [7]. Karl et al. found that TACAD was significantly shorter in fetuses with partial ACC than in normal fetuses [8]. Vascular evaluation is crucial for the precise diagnosis of abnormal cerebral development, and microvascular flow imaging makes it easy to detect. Recently, we reported that the AVGS is a new and useful marker for prenatal evaluation of the fetal tentorium cerebelli (TC) position. An increased AVGS (≥ 95th percentile) suggests an abnormally elevated TC, whereas a decreased AVGS (≤ 5th percentile) suggests an abnormally low TC. AVGS may be particularly helpful in the differential diagnosis of posterior fossa anomalies [9].

Strengths and limitations

To the best of our knowledge, this is the first study to verify the anterior displacement of the ACA/pericallosal artery in fetuses with holoprosencephaly using quantifiable indices. In this study, we established gestational age-specific reference ranges for FACAA and FACAD in a large sample of the Chinese population, which significantly aids in the early diagnosis of holoprosencephaly, including the semilobar and lobar holoprosencephaly. Furthermore, the established indices simplify the diagnostic process and offer a timely prompt that a fetus is at a high risk of holoprosencephaly when the FACAA is < 40.41°. Given that the ACA and pericallosal artery were visualized in 98.8% of normal fetuses during the first trimester, we recommend that their evaluation be routinely conducted during early pregnancy to avoid missing fetuses with holoprosencephaly [26]. FACAA and FACAD demonstrated good discriminatory ability in differentiating holoprosencephaly from other abnormalities with similar appearances on gray-scale sonography. Additionally, in the future, these indices may be used to aid artificial intelligence (AI) in the assessment and diagnosis of fetal holoprosencephaly, as AI has recently been successfully applied to detect brain anomalies in the axial view [27, 28].

However, this study has several limitations. First, further confirmation of the findings is required through a prospective, multicenter study with a larger sample size, including a broader range of abnormal cases. Second, for fetuses with frontal bone defects or other severe malformations (e.g., hydrocephalus or large midline cysts) that can cause ACA displacement, accurate measurement of FACAA and FACAD may not be possible, limiting their applicability.

Conclusion

In this study, we measured two quantitative indices, FACAA and FACAD, to objectively assess the forward displacement of the ACA/pericallosal artery. Reference ranges were established, and both indices were found to be sensitive markers for predicting fetuses with holoprosencephaly, including the semilobar and lobar types, in early pregnancy. FACAA and FACAD can also aid in differentiating holoprosencephaly from malformations of similar appearance. We recommend routine assessment of the ACA and pericallosal artery during the first trimester to prevent missing fetuses with holoprosencephaly.

Abbreviations

ACA

Anterior cerebral artery

FACAA

Forehead-anterior cerebral artery angle

FACAD

Forehead-anterior cerebral artery distance

MVFI

Microvascular flow imaging

GW

Gestational weeks

IASP

Isolated absent septum pellucidum

SOD

Septo-optic dysplasia

ACC

Agenesis of corpus callosum

Author contributions

Zhen Li contributed to the conception and design of the work, the acquisition, analysis and interpretation of data; drafted the work or substantively revised it. Yuanjie Cui, Cuixia Guo, Yuting Wu and Juan Zhang contributed to acquisition, analysis and interpretation of data, revised the work; Lei Wang and Zhaoxia JIA contributed to analysis and interpretation of data; Qingqing Wu and Lijuan Sun contributed to the conception and design of the work, revised the work and approved the submitted version. All authors reviewed the manuscript.

Funding

We thank the families who participated in this study and allowed us to collect their clinical information. This study was funded by the Beijing Natural Science Foundation (No. 7222063), the National Natural Science Foundation of China (No. 81701704), the Beijing Municipal Science & Technology Commission (No. Z211100002921017), and a high-level public health technical talent construction project training plan (Subject Backstone-02-06).

Data availability

The patient data and ultrasound images supporting this study are not publicly available due to patient confidentiality restrictions under the ethical approval by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital. De-identified data may be made available from the corresponding author upon reasonable request, subject to institutional ethical approval and execution of a data access agreement.

Declarations

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital (No.2022-KY-013-02). All eligible patients were fully informed and signed an informed consent form for participation and use of the acquired data during the study period.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.