Standardized First-Trimester Ultrasound Screening for Fetal Structural Abnormalities in A Non-Selective Population: A Single-Center Experience

Background : To explore the effectiveness of standardized first-trimester ultrasound screening (FTS) in detecting fetal structural abnormalities in a non-selective population. Methods : A retrospective study was performed on 7523 fetuses (6376 single and 569 twin pregnancies) who underwent FTS between 11 and 13 +6 weeks’ gestation. All fetuses received anatomy scans using a standardized protocol. Results : 147 fetuses (133 single and 7 twin pregnancies) were lost to follow up. Of the remaining 7376 fetuses, 119 (1.61%, 119/7376) developed structural malformations, with 64 cases (53.8%, 64/119) identified during the first trimester. The remaining cases were detected during the second trimester (24.4%, 29/119), the third trimester (1.68%, 2/119), and postnatally (20.2%, 24/119). There were 4 cases of suspected ventricular septal defect (VSD) by FTS, which were later confirmed to be normal. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for FTS were 54.2%, 99.9%, 94.1%, and 99.3%, respectively. Forty eight fetuses, accounting for 10.6% of the total (452), with thickened nuchal translucency (NT) (above the 95th percentile) showed structural malformations. This was significantly higher than the prevalence of structural abnormalities found in fetuses with normal NT (1.0%, 71/6924) ( p < 0.01). Conclusions : Standardized FTS is highly effective in detecting fetal structural malformations early, with impressive specificity, PPV, and NPV. Increased NT suggests detailed anatomy screening and helps guide treatment. However, while standardized FTS is an invaluable tool, it cannot fully replace the sensitivity of second-and third-trimester ultrasound screening. Clinical Trial Registration : The study was registered at https://www.chictr.org.cn (registration number ChiCTR-SOC-17010976).


Introduction
Since the incorporation of nuchal translucency (NT) in first-trimester ultrasound screening (FTS) for Down's syndrome [1], it has become an indispensable tool for screening for fetal aneuploidy in conjunction with maternal serology [2][3][4]. Although cell-free DNA testing has minimized the role of NT in first-trimester aneuploidy detection, the advancement of ultrasound technology and knowledge of fetal anatomy has enabled early pregnancy screening for fetal structural abnormalities. As more than 80% of fetal structural abnormalities manifest prior to the 12th week of gestation [5], careful examination of fetal structures during the first trimester could facilitate early detection of fetal structural anomalies. Despite the widespread use of FTS for detecting fetal structural abnormalities, there is no universally accepted standard for evaluating its performance. Differences in study populations, screening protocols, and criteria for inclusion of malformations have resulted in varying results being reported. To shed light on this issue, we conducted a study using a standardized scanning protocol in a non-selected Chinese population, with the goal of further exploring the role of FTS in detecting fetal structural abnormalities.

Study Population
This retrospective study analyzed data from women who underwent routine NT scan at the Affiliated Suzhou Hospital of Nanjing Medical University, China, between September 2017 and July 2021. The study was registered at https://www.chictr.org.cn (registration number ChiCTR-SOC-17010976). The study included a total of 7523 fetuses, comprising 6376 single pregnancies and 569 twin pregnancies. The study population had an average age of 29 years (interquartile range (IQR) [27][28][29][30][31] and the fetuses had a crown-rump length (CRL) of 45-84 mm, with a mean CRL of 66 mm (IQR 62-71). Prior to the examination, all study patients provided informed consent. In this study, a Philips Affiniti 70 color ultrasound machine was utilized, equipped with a convex probe of C9-2 (frequency 2-9 MHz). All examinations were conducted transabdominally by 5 ultrasound physicians who were accredited by the Fetal Medicine Foundation (FMF) for FTS.

Ultrasound Scan
All ultrasound scans were conducted in a transabdominal manner, adhering to the FMF standard for fetal CRL and NT measurements, as well as Doppler evaluation of the blood flow across the ductus venous and tricuspid valve [6]. Additionally, we followed a more detailed anatomy scanning protocol based on the guidelines of the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) [7]: (1) Transverse section of the fetal head to examine the skull, cerebral falx, lateral ventricle, and choroid plexus.
(3) Transverse section of the fetal eyes and retronasal triangle plane (RNT) to analyze the fetal eyes, palate, and upper lip.
(4) Two-dimensional and color-flow images of the 4-chamber cardiac view, the left ventricular outflow tract (LVOT) view, and the three-vessel-tracheal (3VT) view to examine the lungs, the fetal heart chambers, and great vessels.
(5) Parasagittal section of the upper chest to inspect the bilateral integrity of the diaphragm.
(6) Abdominal transverse section to assess fetal stomach and abdominal wall integrity and both cross and coronal sections of the lower abdomen to observe the fetal kidneys.
(7) Transverse section of the bladder to observe the fetal bladder and both umbilical arteries.
(8) Three-segment section of the upper and lower limbs to observe fetal limbs. (9) Sagittal and coronal views of the spine to observe the fetal spine.
(10) If the ultrasound image was not satisfactory, a repeat examination at intervals of 20-30 minutes was performed, and the total scanning time was limited to half an hour in normal fetuses and one hour in abnormal fetuses. If necessary, further genetic testing was suggested. All acquired ultrasound images were stored in an ultrasound database.

Follow-Up
Data on pregnancy outcomes in this study were obtained from the Jiangsu Maternal and Child Health Information System as well as by telephone surveys. Autopsies were performed on aborted fetuses. Severe structural malformations found during the first trimester, such as major cardiac malformations, were assessed by a specialized echocardiography physician, and then reviewed 2 weeks later to confirm the diagnosis. For ongoing pregnancies, results from later ultrasound examinations were documented, and the final pregnancy outcomes were monitored. Pediatricians conducted postnatal check-ups for all newborns, both immediately after birth and 42 days postdelivery.

Inclusion Criteria for Fetal Structural Abnormalities
Fetal findings considered to be structural abnormalities (regardless of chromosomal abnormalities): (1) Fatal malformations, i.e., anencephaly and body stalk anomalies.
(2) Malformations resulting in severe dysfunction or disability, i.e., complex heart malformations and abdominal wall defects.
(3) Malformations resulting in mild dysfunction, i.e., ventricular septal defect (VSD) and polydactyly. Only cases with complete follow-up results were enrolled.

Exclusion Criteria for Fetal Structural Abnormalities
Single umbilical artery, aberrant right subclavicular artery, and persistent left superior vena cava were considered anatomical variants; the absence of nasal bone, tricuspid regurgitation, and choroid cyst were considered ultrasound markers, none of which were included in the structural malformations of this study. Complications specific to twins, such as twin-to-twin transfusion syndrome (TTTS)   and twin reversed arterial perfusion syndrome (TRAPs), were not included in the category of structural malformations.

Statistical Analysis
The data was analyzed by SPSS 21.0 (IBM Corp., Chicago, IL, USA). The two-tailed Fisher's exact test was used to compare the prevalence of structural malformations between fetuses with increased NT (above the 95th percentile) and those with normal NT.

Overall Prevalence of Structural Malformations and Detection Rate by Prenatal Ultrasound
Among the 7523 fetuses screened for NT (6376 singletons and 569 twins), 147 were lost to follow-up (133 singletons and 7 twins), with the results of 7376 fetuses analyzed. One hundred nineteen cases had structural malformations, resulting in a prevalence of 1.61% (119/7376). The overall detection rate of prenatal ultrasound detecting fetal structural malformations was 79.8% (95/119). Notably, 24 fetuses with structural malformations were only identified through routine neonatal examination after being missed during prenatal ultrasound screening. Among the 119 cases, cardiac abnormalities were the most common (35/119, 29.4%), followed by genitourinary system abnormalities (14/119, 11.76%) and skeletal system abnormalities (13/119, 10.92%). Fig. 1 illustrates the timing of the abnormalities detected during screening.
All these structural malformations identified by FTS were displayed in Table 2.

The Performance of Standardized FTS in Our Study
In recent years, various studies have reported detection rates of fetal structural malformations in early pregnancy, with an average detection rate of approximately 50% [8,9]. The rates vary depending on the studied populations, the criteria for inclusion of malformations, and the existence of detailed standard protocols. Yimei Liao et al. [10]

Structural abnormalities (+) Structural abnormalities (-) Total
Increased NT (+)  48  404  452  Increased NT (-)  71  6853  6924  Total  119  7257  7376 NT, nuchal translucency. reported a more recent detection rate of 43.1% in the first trimester scan. Our study, which included a non-selective population and applied an established standard protocol, detected structural malformations in early pregnancy at a rate of 53.8% (64/119), regardless of chromosomal aberrations. The performance of FTS varied in different studies. In Fernando Felix Dulgheroff et al. [11] study, the sensitivity, specificity, PPV, and NPV of first-trimester ultrasound were reported as 14.06%, 98.65%, 39.13%, and 94.90%, respectively. The corresponding values in our study were as follows: 54.2%, 99.9%, 94.1%, and 99.3%, respectively. The FTS utilizing a standardized scanning protocol in our study had significantly higher sensitivity and PPV, suggesting a better performance in detecting malformations.

Early Structural Abnormalities Detected in Our Study
In the study by Syngelaki et al. [12], acrania, ectopia cordis, alobar holoprosencephaly, exomphalos, gastroschisis, tricuspid or pulmonary atresia, pentalogy of Cantrell, and body-stalk anomalies were all detected in the first trimester, indicating the high sensitivity of early screening. Ana Maria Vayna et al. [13] identified all cases of AVSD, right atrial isomerism, double-outlet right ventricle (DORV), megacystis, persistent cloaca, and apla-sia/hypoplasia of radius/ulna. Similarly, our study detected all alobar holoprosencephaly, encephalocele, exomphalos, hygroma, TOF, RAA, HLHS, AVSD, heterotaxy syndrome, megalocystis cases, limb reduction defects, severe short limb, and hemivertebrae cases. Due to a previous ultrasound examination prior to the first trimester NT screening in our center, no acrania cases were detected. The detection rate of the pentalogy of Cantrell and DORV was 50% (1/2) in our study, further highlighting the complexity of these conditions. One case of DORV was not proven until the mid-trimester due to poor image quality. Our study demonstrated a remarkable 100% detection rate for syndromes such as caudal regression syndrome and Larsen syndrome.
Furthermore, our investigation also revealed a high detection rate of ≥50% for CLP, complex heart abnormalities, CDH, club foot, and multiple system defects. We detected 71.4% (5/13) of CLP cases using the RNT plane, providing valuable information for early detection. De Robertis et al. [14] reported on the efficacy of the RNT plane indicating its superior sensitivity compared to the maxillary gap (MG) in detecting cleft palate. However, MG seemed to perform better in secondary cleft palate. Our study achieved a higher detection rate (71.4%) for CLP than the rates reported by Liao [10] and A. Syngelaki [12] (30.4%, 34.6%, respectively). This can be attributed to our acquisition of the RNT plane exclusively when the palate was perpendicular to the ultrasound beam, thereby avoiding the shadow of two frontal processes of the maxilla. Although soft tissue observation of the lip was challenging in early pregnancy, two cases of "cleft lip only" were eventually detected in the second trimester. A standardized protocol for fetal heart screening also proved to be a powerful tool in detecting congenital heart defects. Our study recorded a 57.1% (20/35) detection rate for such defects in the first trimester. The four-chamber view detected all the AVSD and HLHS cases, while the 3VT view and the LVOT view identified all the TOF and RAA cases. Interestingly, our findings suggest that the cardiac screening protocols utilized in the midtrimester, as summarized by Quaresima et al. [15], may also be suitable for the first trimester. Using a standardized screening strategy for early fetal heart assessment yielded remarkably high PPV and NPV (98.6% and 99.6%, respectively) in the Turan et al. [16] study. Karim et al. [17] advocated for the implementation of comprehensive structured anatomical assessment protocols to enhance the detection rate of cardiac defects. Studies have indicated that a  more detailed screening approach in early pregnancy is positively correlated with a higher detection rate of structural malformations [18]. Our study identified one of the two cases of CDH through the identification of anechoic stomach in the thorax, while the other case was missed in the first trimester, possibly due to the hernia's manifestation at a later stage. Lakshmy et al. [19] reported that the upturned course of the superior mesenteric artery (SMA) may be a diagnostic indicator for atypical CDH. While limb reduction defects were typically detected during early pregnancy, deformities of the hands and feet were not always immediately apparent. Vayna et al. [13] observed ectrodactyly in 75% (3/4) of cases, while Liao et al. [10] found no instances of syndactyly (0/11) and 25% (6/24) of cases involving poly-dactyly. In a previous study by Liao et al. [20] examining fetal limb abnormalities in the first trimester, they detected 1 of 2 cases of syndactyly, but no cases of polydactyly (0/4). Two cases of polydactyly and 1 case of syndactyly missed prenatal diagnosis in this study, underscoring the need for close assessment of the morphology of the hands and feet.
Our study found that certain abnormalities, including agenesis of the corpus callosum (ACC), arachnoid cyst, hydrocephalus, cardiac tumor, atrial septal defects (ASD), congenital chylothorax, congenital biliary atresia (CBA), as well as genitourinary system abnormalities such as multicystic dysplastic kidney (MCDK) and unilateral renal agenesis, were not detectable in the first trimester. This may be due to their late onset and subtle manifestation during this  period. However, our retrospective analysis revealed that 1 case of situs inversus, initially missed by FTS, could have been detected.
Although deformities relating to the corpus callosum and the posterior fossa were conventionally considered undetectable, several indirect signs have been reported to predict ACC [21][22][23][24] and posterior fossa malformations [22][23][24]. Also, for early detection of ventriculomegaly, various indicators have been extensively explored [25,26]. Through our study, we uncovered that the fetal kidneys are more discernible on the coronal plane than on the transverse plane, especially with the augmentation of ultrasound instrument gain. Nevertheless, the identification of MCDK in the first trimester remains unattainable, as corroborated by recent studies [10,12]. Assessing the urinary tract poses a challenge, as signs indicative of urinary tract deformities are not discernible in early pregnancy. As our study has shown, the presence of hydronephrosis and obstructive renal dysplasia suggests a gradual progression during fetal development.
Twenty-four cases with missed prenatal diagnosis exhibited deformities across various systems, including ear abnormalities, VSD and ASD, hypospadias and ambiguous genitalia, polydactyly, syndactyly, abnormalities of hand and foot joints, and hip dysplasia in the limbs and skeleton system. Additionally, 1 case exhibited multiple defects, namely coarctation of the aorta and pelvic kidney, while another case was diagnosed with Pierre Robin syndrome (PRS). Fortunately, none of these deformities proved fatal, and the majority can be surgically treated barring PRS. Our examination of the initial images in the PRS case revealed an overlooked sign, that being a small mandible.
Four cases of VSD diagnosed by FTS were later confirmed as being normal. Two explanations were proposed: either the VSD was present in the first trimester but healed spontaneously or the VSD was misdiagnosed altogether. Considering the small size of the heart and the limitations of two-dimensional and color imaging, diagnosis of VSD in early pregnancy requires careful consideration.
Several limitations of this study should be highlighted. Primarily, due to its retrospective nature and moderate sample size, the findings may not be fully representative. Additionally, the use of solely transabdominal ultrasound examination may have resulted in a lower detection rate compared to using a combination of transvaginal and transabdominal ultrasound. Moreover, a significant number of pregnant patients with fetal malformations underwent fetal genetic testing and exploration of the relationship between malformations and genetic results shall be pursued in subsequent studies.

Conclusions
Apart from the NT measurements, our study found that standardized FTS can identify over 50% of the structural anomalies present. Furthermore, the use of a standardized screening protocol enhances the detection of abnormalities that may not be readily discernable during the early stages of pregnancy, particularly in the identification of complicated cardiac malformations, CLP, and severe deformities. The elevated NT levels during the early stages of pregnancy serve as an indication for more detailed struc-tural screening. In addition, the use of standardized FTS provides earlier opportunities for genetic testing, specialist imaging, prognostic information, and management discussions. Nonetheless, screening during the second and third trimesters remains essential, as some malformations may have a delayed onset and certain organs develop later in pregnancy.

Availability of Data and Materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.