CAKUTs are the most common cause of prenatal developmental malformations, and the leading cause of morbidity in pediatric and adolescent patient populations, accounting for approximately 50% of all cases of end-stage kidney disease in this demographic . Genetic counseling for prenatally diagnosed congenital urinary system abnormalities should be combined with ultrasound monitoring indicators and molecular genetic tests. Genetic analysis of fetuses with CAKUTs will help to uncover the fundamental pathways involved in urinary tract development.
4.1 Chromosomal karyotype analysis in fetuses with CAKUTs
The detection rate of chromosomal abnormalities was 9.52% (4/42) in our study. Furthermore, the incidence of chromosomal abnormalities was increased in fetuses with CAKUTs associated with extrarenal abnormalities; hence, it is necessary to conduct chromosome karyotype analysis to exclude related genetic diseases in such fetuses. In 2019, Li et al.  conducted a prospective study of 123 fetuses with CAKUTs, and found that 13 fetuses had chromosomal karyotype abnormalities, yielding a detection rate of 10.6%. Ryckewaert et al.  showed that the incidence of chromosomal abnormalities in CAKUT with extrarenal abnormalities was 18.6%. Currently, it is controversial whether karyotype analysis is necessary for fetuses with isolated CAKUTs.
4.2 CMA analysis in fetuses with CAKUTs
CMA has the advantage of detecting not only aneuploidies but also microdeletions and microduplications, which cannot be detected using tranditionanl chromosome karyotype analysis.
4.2.1 CMA detected aneuploidy in fetuses with CAKUTs
It has been reported that isolated mild hydronephrosis and ectopic kidney are not associated with an increased risk of chromosomal abnormalities [6, 7]. In this study, no aneuploidy was found in ectopic kidney cases. However, 2 cases of isolated hydronephrosis diagnosed at 24 weeks, was trisomy 21 syndrome. It has been reported that horseshoe kidney is present in 60% of Turner syndrome patients, 20% of trisomy 21 syndrome patients, and 20% of trisomy 18 syndrome patients . In this study, 5 cases of trisomy 18 (22.73%), 3 cases of trisomy 13 (13.64%), and 1 case of trisomy 21 (4.55%) were diagnosed in 22 fetuses with non-isolated horseshoe kidney. Thus, whether to do chromosomal karyotype analysis to detect aneuploidy for patients with prenatal solitary renal malformations depends on the specific type of urinary system abnormality.
4.2.2 Pathogenic CNVs associated with CAKUTs
The 2013 American College of Obstetricians and Gynecologists and Society for Maternal-Fetal Medicine guidelines recommend that CMA should be used to replace conventional karyotype analysis for the prenatal diagnosis of ultrasound-detected abnormalities in fetuses . In this study, CMA could increase the detection rate by 4%. Weber et al.  performed CMA on 30 fetus CAKUTs associated with extrarenal abnormalities, and obtained a diagnostic yield of 10%. A 2019 study performed CMA for 123 fetuses with normal karyotypes and CAKUTs, and reported a 10.6% detection rate of pathogenic CNVs . Among the 380 fetuses, the overall CMA detection rate was 10.52% (40/380) for pathogenic CNVs although isolated CAKUT significantly lower (3.47%, 11/317). Among fetuses with isolated MCDK, the positive detection rate was 13.33% (8/60), no significant difference between isolated unilateral MCDK and bilateral MCDK, which is consistent the results of Fu et al. .
We found 8 cases with 22q11.2 deletion syndrome, the most common pathogenic CNV in CAKUT fetuses, including 5 cases isolated renal abnormalities. This syndrome involves multi-system or multi-organ lesions, and mainly includes DiGeorge syndrome (OMIM: 188400), velocardiofacial syndrome (OMIM: 192430), and conotruncal anomaly face syndrome (OMIM: 217095), which are defined by different clinical subtypes. It has been reported that urinary system anomalies are present in up to 30–40% of patients with 22q11.2 deletions [12, 13]. This deletion has wide phenotypic variability. Some of the more well-characterized genes affected by this deletion are TBX1, DGCR6, CRKL, PRODH, and COMT. The deletion or mutation of the transcription factor TBX1 gene is the most important pathogenic gene of 22q11.2 deletion syndrome, and CRKL is a main genetic driver of kidney defects [14, 15]. In this study, the pathogenic genes involved in the deletion of this segment were mainly TBX1, COMT, and DGCR6 genes.
The 22q13.2q13.33 microdeletion mutation was detected in 2 cases. The 22q13.2q13.33 microdeletion was diagnosed as the Phelan-McDermid syndrome (OMIM: 606232), in which renal abnormalities are often present in this syndrome as high as 38%, including polycystic kidney, renal hypoplasia, hydronephrosis, vesicoureteral reflux, and horseshoe kidney. Chromosome 17q12 deletion syndrome was detected in one case of isolated bilateral hyperechoic renal parenchyma, and the other common renal presentation was hyperechogenic, multicystic, or enlarged kidneys. This syndrome usually comprises CAKUTs, maturity-onset diabetes of the young type 5, and neurodevelopmental or neuropsychiatric disorders. Congenital structural renal anomalies occur in 80–85% of the affected population. One study has indicated a strikingly high correlation between prenatally detected hyperechogenic kidneys and 17q12 deletion . A major pathogenic gene of 17q12 microdeletions is the hepatocyte nuclear factor 1-β (HNF1β) gene, also referred to as transcription factor 2 (TCF2; OMIM: 189907). This gene plays an important role in nephron development and regulates the development of the embryonic pancreas. Small-scale studies have suggested that 12–15% of individuals with 17q12 microdeletion syndrome may progress to end-stage renal disease [18, 19].
Among the pathogenic CNVs detected in our study, 5 variations were pathogenic fragments: 21q11.2q21.2 duplication, 2q21.2q32.1 duplication, 12p13.33p13.31 duplication with 14q32.33 deletion, 17q24.2q25.3 duplication, and 20p13p11.1 duplication with 20q13.31q13.33 duplication. The above pathogenic microdeletions/microduplications were first reported in CAKUTs through the online pathogenicity database, which enriches the genetic spectrum of CAKUTs. In addition, the chromosome 17q24.2q25.3 fragment involved many pathogenic genes such as SOX9 and PRKAR1A. The SOX9 gene is related to early embryonic development and plays an important role in chondrocyte and cartilage tissue differentiation. Studies have found that SOX9, as a testicular determinant gene, can induce the formation of supporting cells and the generation of anti-Mullerian hormone .
4.2.2 VOUSs in CAKUTs
In our study, the overall detection rate of VOUSs was 4.47% (17/380), while the detection rate of VOUSs in cases of isolated CAKUTs was 3.47% (11/317), which is consistent with the rate of 3.4% reported by the Euroscan Research Center , and higher than the 2.5% rate found among fetuses with isolated urinary system abnormalities by Cai et al.. The detection rates of pathogenic CNVs and VOUSs in different studies are different depending on the severity of the disease in the fetus and whether the parents agree to further testing. VOUSs pose a challenge to clinical genetic counseling. Therefore, antenatal technicians should fully communicate with clinicians when VOUSs are detected, and conduct sample testing of both parents for further expert lineage verification to clarify the source of the anomaly.
4.3 WES analysis in CAKUT fetuses
As prenatal WES is being applied more commonly in cases with fetal abnormalities and in nonviable pregnancies when the CMA results are normal, new genes have been identified to explain prenatal renal phenotypes [22, 23]. To date, approximately 40 single gene abnormalities have been identified to cause CAKUTs (25 dominant and 15 recessive), and approximately 18% of CAKUT cases can be explained by these identified single gene etiologies . PAX2 and HNF1B were the first two genes identified . In the 13 cases with positive mutations identified using WES in this study, the pathogenic genes were as follows: NOTCH2, PKHD1, BBS7, PIEZO2, TRRAP, ACE, TNXB, WASHC5, ANKH, LZTR1, PKD1, and HSPG2. In 3 cases with normal CMA results, WES detected abnormalities. In a 2021 study of 100 samples, the pathogenicity detection rate of WES in CAKUT was 22% . In our study, the pathogenicity detection rate of WES in CAKUT was 19.23%. WES has helped to identify candidate genes associated with fetal abnormalities, thereby further expanding our understanding of the clinical phenotypes of known genetic diseases. We identified 5 different monogenic genes (PKD1, PKHD1, TNXB, ANKH, and WASHC5) with mutations that can cause isolated or syndromic CAKUT phenotypes. WES has a high detection rate in renal cystic diseases. In this study, 4 cases involving the PKHD1 gene were detected. The PKHD1 gene is a pathogenic gene involved in autosomal dominant PKD (ADPKD). We detected the c.11117T > G, EX30_31Del, c.6840G > A, c.10058T > G, and c.8486T > C mutations, which have not been reported before. Pathogenic mutations in the PKD1 gene can lead to renal failure and end-stage renal disease in adulthood. The clinical manifestations of ADPKD are mainly caused by functional defects of the PKD1-encoded protein polycystin-1 or the PKD2-encoded protein polycystin-2. The loss of either protein leads to abnormalities in primary ciliary function, which in turn leads to abnormalities in cell signaling pathways that allow cell proliferation, resulting in cyst formation and progressive enlargement.
We detected the following syndromes associated with abnormal renal development: Hajdu-Cheney syndrome, BBS7, Marden-Walker syndrome, vesicoureteral reflux 8, and renal tubular dysgenesis. The nonsense variant c.7163C > A in exon 34 of NOTCH2 was detected in this study. NOTCH2 is associated with Hajdu-Cheney syndrome, which shows an autosomal dominant inheritance. NOTCH2 is required for glomerular renal development, and the downregulation of NOTCH2 in mice results in hypoplastic kidneys . Long bone deformities and polycystic kidneys are the characteristic manifestations of this disease. The variable expression of NOTCH2 demonstrates that the strong association between long bone deformities and renal cystic disease is just another manifestation of the disease, rather than multiple manifestations. Therefore, when CAKUT is detected prenatally along with bone dysplasia, WES is recommended for the detection of genetic abnormalities.
In our study, one fetus was diagnosed with BBS (OMIM: 209900), which is clinically characterized by polydactyly and kidney hypoplasia. When prenatal ultrasonography reveals fetal renal enlargement or renal cystic changes, it is necessary to carefully eliminate fetal fingers (or toes) abnormalities, and high vigilance for BBS is required when the two abnormalities coexist.
We detected a complex heterozygous variant of the ACE gene: C. 2593G > A and C. 2708C > T. The ACE gene is associated with renal tubular dysgenesis (RTD). RTD is a very rare but fatal disease caused by mutations in the renin-angiotensin-aldosterone system. The ACE gene mutation accounted for 64.6% of cases . Zhu et al.  reported and summarized 37 families in which the ACE gene caused RTD. The ACE complex heterozygous mutation site we found complements the new site, which is important for improving the detection of inherited RTD-related genes
WES often can provide finite reports that decrease the number of VOUSs reported to the clinician. The advantages of whole genome sequencing include not only the identification of non-coding pathogenic variations but also more complete exomic coverage than WES .
4.7 Prognostic analysis of fetuses with CAKUTs
Among those with negative genetic tests, 19.82% (66/333) of pregnant women chose to terminate the pregnancy; in 40.91% (27/66) of these cases had an extrarenal abnormality. The probability of poor prognosis in isolated CAKUT fetuses without genetic abnormalities was 2.56% (6/234). The operation rate among live-born infants with CAKUTs was 6.75% (51/755), of which 78.43% (40/51) underwent surgery for the correction of severe hydronephrosis, which was most commonly caused by ureteropelvic junction obstruction. Patients with removal of urinary tract obstruction had a better prognosis. The diseases detected among infants with CAKUTs who underwent postpartum urologic surgery were hydronephrosis (78.43%, 40/51), hypospadias (9.80%, 5/51), unilateral MCDK (5.88%, 3/51), kidney duplication (3.92%, 2/51), and renal dysplasia (1.96%, 1/51).
Hydronephrosis was the most common urinary system anomaly in our cohort. It has been reported that approximately 90% of fetal hydronephrosis cases undergo spontaneous regression with increase in gestational age or after birth . Our research is consistent with this report (214/251, 85.26%), generally, hydronephrosis was self-limiting or stabilized over time, with no pathological changes. Many studies have shown that APD in the third trimester is an important predictor of the need for surgery after delivery [33, 34]. We found that the optimal cutoff value of APD for predicting the need for postpartum surgery was 17.5 mm in the third trimester of pregnancy, with a sensitivity of 63.2% and a specificity of 94.7%. At present, the thresholds of APD in various studies are different, which may be related to the sample sizes or the lack of standardized ultrasonographic follow-up criteria for APD and surgical protocols. Therefore, a large-scale study is required to develop uniform ultrasound criteria and surgical indications and obtain a more accurate threshold. According to the British Association of Pediatric Urologists, surgical treatment for hydronephrosis should be performed if there are recurrent urinary tract infections, impaired renal function, and symptoms such as massive or progressive hydronephrosis .
Fetuses with the most kidney anomalies, such as isolated kidney duplication, horseshoe kidney, or single kidney, may have favorable outcomes. Poor outcomes were found in patients with renal parenchymal malformations and abnormalities of the urethra and bladder. Their prognosis often depends on the severity of complications such as cardiovascular, nervous, and other systemic abnormalities. Bilateral kidney agenesis and bilateral MCDK usually had unfavorable prognoses, while the prognosis of fetuses with unilateral kidney depends on the presence of the normal function of the contralateral urinary tract and the absence of other systemic anomalies. Posterior urethral valve was the primary cause of severe obstructive nephropathy in children, and was even associated with a poor prognosis. A fetal urine peptide signature predicted postnatal renal outcomes in fetuses with postnatally confirmed posterior urethral valve, with an accuracy of 90% . Abnormal amniotic volume in the presence of CAKUTs indicates that renal function may be affected and is associated with fetal prognosis.
Megabladder is the earliest and most common deformity, which is often accompanied by renal insufficiency, oligoamnios, and pulmonary dysplasia, and often has a poor prognosis. We found that 25% of cases with megabladder detected in the first trimester returned to normal with increasing gestational age. Therefore, it is necessary to perform dynamic observations. In addition, 30.56% (11/36) of the fetuses with megabladder were associated with multiple pregnancies, indicating early attention should be paid to ultrasonographic screening of megabladder in multiple pregnancies, and necessary early intervention should be undertaken to improve the pregnancy outcome. The incidence of chromosomal abnormalities in fetal megabladder is 15% , and the most common abnormalities are trisomy 13, trisomy 18, and trisomy 21 syndrome. One trisomy 21 syndrome (5.26%, 1/19) was found in fetus with megabladder and increased nuchal translucency (NT) A study has shown that chromosomal abnormalities in fetal megabladder are associated with increased NT . Therefore, chromosome karyotype analysis and genetic testing are recommended when fetal megacystis is found prenatally.
Bilateral hyperechoic kidneys are not necessarily a pathological change, and the renal echogenicity resolved in about one-third of the fetuses. Carr et al.  reported 8 cases of bilateral hyperechoic fetal kidneys, and during the postnatal follow-up, the renal echogenicity resolved in 4 cases, diminished in 1 case, and remained the same in 3 cases. Shuster et al.  performed CMA in 34 cases of isolated bilateral hyperechogenic kidneys, and found two inherited mutations: a 17q12 deletion inherited from an asymptomatic mother and a duplication at 3p26.1 inherited from a healthy father. CMA suggested 17q12 microdeletion syndrome in 1 fetus. It has been shown that bilateral marked hyperechoic kidneys are highly correlated with chromosome 17q12 deletion syndrome . CMA can help identify the genetic etiology in fetuses with bilateral hyperechoic kidneys.