Single-cell RNA sequencing (scRNA-seq) was used to investigate the potential pathogenesis of cystic hygromas (CH) in fetuses with Turner's syndrome (TS) .

The posterior cervical tissues of the terminated pregnancy fetus, which ended in termination pregnancy at 11-13⁺⁶ weeks were collected due to CH at Guangzhou Women and Children Medical Care Center from January 2020 to December 2020. The posterior cervical tissues of normal fetuses who terminated pregnancy due to social factors were collected at the same gestational age. For those who did not receive prenatal genetic testing, placental tissue or skeletal muscle of the induced fetus should be taken for genetic etiology testing according to standard operating procedures. Genetic testing was used in all cases to determine the chromosomal abnormality type. According to the genetic etiology, we selected the TS fetuses as the experimental group, and fetuses with genetic results 46, XN were the control group in cases without CH. A total of six cases were collected. The experimental group and control group, respectively had 3 cases. In all enrolled patients, posterior cervical skin tissue was taken immediately after termination of pregnancy, HE staining to some samples for pathological analysis, and some samples were analyzed by scRNA-seq to analyze the cell types of cervical skin tissue and establish differential gene expression profiles. Furthermore, RT-qPCR was used to study the gene expression level in cells, and Western blot was used to detect the expression of related proteins to confirm the reliability of the single-cell sequencing results. An independent sample test was used to compare the differences between groups.

Pathological findings showed that pathological features of CH cases were significant lymphatic hyperplasia and dilation, increased lymph nodes and necrosis, decreased blood vessels, significantly increased and lengthened interstitial fibrofilaments, and a large number of cells infiltrated into the interstitial space. In the analysis of CH cases by scRNA-seq, a total of 54 488 cells were captured and 26 clusters were identified. Among them, fibroblasts accounted for 23.1%, and stromal cell protein CCN2 was found to have high expression. Further localization analysis showed that CCN2 was highly expressed in fibroblasts, endothelial cells, muscle cells, tissue stem cells, and mesenchymal stem cells in the experimental group. Furthermore, Western blot was to verify that CCN2 protein was significantly overexpressed in the experimental group (2.47 ± 0.49 vs 1.00 ± 0.08, P < 0.05) .

Pathological analysis confirmed that lymphatic abnormalities such as lymphatic hyperplasia and dilation, and increased lymph nodes and necrosis were the common pathophysiological basis of fetuses with TS. ScRNA-seq results suggested that abnormal overexpression of CCN2 may be related to CH in fetuses with TS.