A retrospective analysis was conducted on 94 cases with fetal hyperechoic kidneys identified at Fujian Maternal and Child Health Hospital from November 2017 to January 2024. Cases further treated at a prenatal diagnosis center that provided informed consent for interventional prenatal diagnosis were included. The mean age of the pregnant women was 29.6 years (range, 23–41 years); the mean gestational age was 25.83 weeks (range, 15–34 + 4 weeks). According to whether other ultrasound abnormalities were combined, cases were categorized into isolated (16 cases) and non-isolated (78 cases) hyperechoic kidney groups. Postpartum follow-up included collection of pregnancy information, results of postpartum ultrasound examinations, and determination of the need for surgical treatment. This is a secondary analysis of data (Ethics Approval No.: 2014042), where all participants provided written informed consent explicitly permitting future research use of their de-identified data. The current study was separately approved by the Ethics Committee of Fujian Maternal and Child Health Hospital.

The Voluson E8 ultrasound diagnostic instrument from GE in the United States, with an abdominal probe frequency of 2.0–5.0 MHz. was employed. A systematic examination of the entire body and accessory structures of the fetus was performed along with routine biological measurements. Examination of fetal kidneys included assessments of kidney size and shape, echo, collecting system, a clear boundary between the cortex and medulla, and the presence of cysts. The criterion for diagnosing fetal hyperechoic kidney was renal echo during mid-to-late pregnancy brighter than the liver (Figure 1). The deepest vertical pocket and amniotic fluid index were used to evaluate amniotic fluid volume during mid and late pregnancy, respectively. Cases were categorized as isolated or non-isolated hyperechoic kidney based on the presence of additional abnormalities.

Under the guidance of B-ultrasound, we performed amniocentesis to extract amniotic fluid from pregnant women. Amniotic fluid specimens were placed in two sterile centrifuge tubes and centrifuged at 2,000 rpm for 10 min. The supernatant was discarded, and the precipitate was inoculated into two bottles of 4 ml amniotic fluid cell culture medium. The culture was then incubated at 37°C in a 5% CO₂ incubator for 8 days. After the cells grew well, the medium was changed, and the culture was continued for an additional day. Upon observing multiple cell clones under an inverted microscope and refractive amniotic fluid cells constituting ≥90% of the cloned cells, colchicine was added to arrest the culture. The adherent cells were digested with trypsin, and hypotonic, fixed, droplet, band, and Giemsa staining were performed. We counted ≥20 split cells cultured in double lines per sample, analyzed ≥5 split cells, and increased the count when chimerism was observed. The karyotype results are described according to the requirements detailed in the reference (10).

Genomic DNA extraction was performed on fetal samples using the column method with the Qiamp DNA Blood MiniKit (Qiagen, Germany). After extraction, the purity and concentration of the DNA were measured using a NanoDrop 2000 ultra microspectrophotometer. Employing the Affymetrix CYTOSSCAN 750K single nucleotide polymorphism microarray detection platform (Affymetrix), 300 ng of genomic DNA was subjected to enzymatic digestion, ligation, PCR, purification, fragmentation, labeling, hybridization, washing, and scanning according to the standard experimental procedure. Analysis was performed using Chromosome Analysis Suite software version 4.2. Interpretation involved a comprehensive analysis of the Online Mendelian Inheritance in Man (OMIM) database, Clinical Genomic Variation Database (ClinVar), DECIPHER database, Database of Genomic Variants, and Clinical Genomic Resources (ClinGen). Following guidelines from the American Society for Medical Genetics and Genomics (11, 12), CNVs were classified into five levels: benign CNV, pathogenic CNV, likely pathogenic CNV, likely benign CNV, and variant of uncertain significance (VUS).

WES was performed by Beijing BioChain Medical Laboratory. Fetal DNA was cut into millions of small DNA fragments to construct a genomic library, obtain exon sequences using targeted hybridization probes, and sequence the DNA. Following sequencing, the raw data were aligned using BWA software. Mutations, including single nucleotide polymorphisms (SNPs), insertions, and deletions, were identified and analyzed using GATK and VarScan software for detection and annotation. Annovar software was used to annotate variant sites from external databases and evaluate the impact of target sequence mutations. Mutations detected using whole-exome technology were classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) into pathogenic mutations, suspected pathogenic mutations, mutations of unknown significance, suspected benign mutations, and benign mutations (13). The DNA sequence obtained through the WES of the family was compared with that of the reference human genome hg19, and the coverage and sequencing quality of the target area were evaluated. Bioinformatics analysis was performed on the variations, and potential pathogenic homozygous and compound heterozygous single nucleotide variations and small variations were screened under quality control standards of target area coverage >99% and average depth >120×. Based on the site alignment of family sequencing data, the genetic patterns of sample variations were analyzed. The median turnaround time for WES results was 25 days (range: 20–28), with results typically available at a median gestational age of 28 weeks (range: 24–34).

The internal clinical information registration system of the hospital and telephone follow-ups were used to track fetal pregnancy outcomes and the postnatal growth and neurobehavioral development of infants. Outcomes included live births, fetal deaths in utero, pregnancy terminations, spontaneous abortions, and infant deaths. Follow-ups, including evaluations of postpartum imaging, surgical interventions, effectiveness of the surgery, and growth and intellectual development of the neonates, were conducted on all cases after birth.

The obtained data were analyzed using SPSS 25.0 statistical software, and Fisher's exact probability method was used for rate comparisons. All statistical tests were conducted using a two-sided design, with a significance level of α = 0.05. A P < 0.05 is considered statistically significant.

Among the 94 fetuses with hyperechoic kidneys, five were excluded from karyotype analysis owing to advanced gestational age, and one sample failed to culture. We analyzed karyotypes in 88 cases and detected six abnormal karyotypes (6.82%, 6/88), including four cases with abnormal chromosome numbers (three cases of trisomy 21 and one case of XXY) and two cases with abnormal chromosome structures. Chromosomal structural abnormalities comprised one case of a large segment deletion [46, X, del (X) (q28)] and one case of an unbalanced chromosomal translocation [46, X, add (13) (p11)]. In follow-up, except for cases carrying 46, X, del (X) (q28) who refused follow-up, all pregnancies involving the five fetuses with identified karyotype abnormalities were terminated.

Among the 94 cases, four were not assessed, and the results from 90 fetuses revealed 17 with abnormal CNVs (18.89%, 17/90), including 12 pathogenic CNVs (13.33%, 12/90), two likely pathogenic CNVs (2.22%, 2/90), and three VUS (3.33%, 3/90). Identified abnormalities included three cases of aneuploidy (two trisomy 21, one 47, XXY), six cases of 17q12 microdeletion, two cases of 1p36.33p36.32 microdeletion, and one case each of 16p11.2 microdeletion, Xq28 deletion, 13q31.1q34 duplication, 16q22.2q23.2 microdeletion, 8q11.23q24.3 uniparental disomy, and 2p25.3p11.2 uniparental disomy (Tables 1, 2). In the follow-up of pregnancy outcomes of 17 fetuses with hyperechoic kidneys carrying abnormal CNVs, follow-up was refused for four cases, six pregnancies were terminated, and seven fetuses with normal clinical phenotypes were successfully followed up after birth.

Conventional karyotype analysis and CMA both helped effectively detect chromosomal numerical abnormalities and large fragment deletions or duplications. We employed these methods to identify two simultaneous cases of trisomy 21: one involved an abnormal sex chromosome number, another a chromosome duplication, and a third involved a chromosome deletion. Routine karyotype analysis revealed trisomy 21 in a patient who did not undergo CMA testing. CMA helped detect an additional 10 abnormal CNVs in 82 fetuses with normal chromosome karyotypes, including seven pathogenic CNVs, one likely pathogenic CNV, and two VUS. This increased the detection rate of chromosomal abnormalities by 12.20% (10/82). Additionally, CMA helped detect two additional abnormal CNVs in four fetuses without chromosomal karyotyping, including one harboring pathogenic CNV and one with a likely pathogenic CNV.

Among the 94 fetuses with hyperechoic kidneys, 13 cases with normal conventional karyotype and CMA results were further subjected to WES; two cases of pathogenic mutations and one likely pathogenic gene mutation involving genes, HNF1B, NPHP3, and KMT2D, were identified (Tables 3, 4). The key clinical distinction between fetal hyperechoic kidneys with normal vs. abnormal whole-exome sequencing (WES) results primarily manifests in the presence of concurrent extrarenal anomalies (Table 5).

We categorized 94 cases into isolated and non-isolated fetal hyperechoic kidneys groups based on the presence of other ultrasound abnormalities. In the isolated fetal hyperechoic kidneys group (including 16 cases), we detected two cases of pathogenic CNV, one VUS, and one potential pathogenic gene mutation, resulting in a genetic abnormality detection rate of 25.00% (4/16). In the non-isolated fetal hyperechoic kidneys group (including 78 cases), we found 10 cases of pathogenic CNVs, two cases of potentially pathogenic CNVs, two cases of VUS, and two cases of pathogenic gene mutations. The detection rate of genetic abnormalities was 20.51% (16/78), with no statistically significant difference in the detection rate of genetic abnormalities between the two groups (P > 0.05).

Out of 94 cases of fetuses with hyperechoic kidneys, 78 completed pregnancy outcome follow-up; 16 were lost to follow-up. Of the followed-up cases, pregnancies were terminated in 25 cases, one fetal death occurred in utero, and 52 live births were recorded.

After follow-up observation of 52 live births, 2 cases experienced adverse outcomes post-birth, with an incidence rate of 3.85% (2/52). One fetus died within 24 h of birth despite normal genomic testing and an intrauterine ultrasound phenotype of hyperechoic kidney and oligohydramnios. Another exhibited fetal intellectual developmental delay post-birth with CMA, revealing a 17q12 microdeletion and intrauterine ultrasound phenotype of hyperechoic kidney and strong intestinal echo. Among the remaining 50 live births, 21 refused post-birth ultrasound follow-up, 24 had normal renal ultrasound results, and six maintained the same ultrasound conditions as before (these six cases within the non-isolated hyperechoic kidney).