P-739 High-throughput Single-cell Survey Reveal a Pathological Niche in Developing Down Syndrome Testis
Authors: Chen Y , Yanyu X , Yanye Z , Dan Z 
Published: June 2023

Study question

What are the characteristics of developing testes in DS, and how do these alterations affect gamete development and androgen production?

Summary answer

Molecular alterations start accumulating in every cell-type of DS fetal testis. Different cell components, compromised functions, and deviated cellular communications make up a pathological niche.

What is known already

DS is the most common genetic chromosomal disorder. The flat or slightly decreased live birth and the significantly prolonged lifespan contribute to a growing DS population. Their needs for reproductive care come into view. Reproductive health is crucial for spermatogenesis, male characteristics, and even brain behaviors. DS males experience normal puberty, but their sex hormones and associated gonadotropins have altered since the infant, implying testis niche cell dysfunction. Male infertility may result from germ cell and/or niche cell malfunction. A single-cell resolution survey could help thoroughly uncover the heterogeneity of DS by cell type.

Study design, size, duration

We applied high-throughput single-cell RNA sequencing to capture the transcriptomes of DS testis (n = 1). In parallel, testis collected from the fetus reported no genetic disorders were set as control (n = 1). Molecular and histological verifications were conducted with independent samples (3 versus 3). Samples enrolled were around 22 weeks of gestation.

Participants/materials, setting, methods

With ethics approval and informed consent obtainment, DS and control samples were included. DS fetus was confirmed with amniocentesis, while no genetic abnormality was reported during the prenatal examination of the diploid fetus. Single-cell RNA sequencing was conducted with the Micro-well platform, pseudotime trajectory analysis, cellular interaction analysis, and weighted gene co-expression network analysis were performed. Transmission electron microscopy, immunohistochemistry, Westerns, and qPCR were used to verify the main differences.

Main results and the role of chance

A total of 15349 cell transcriptomes, DS and control pooled, were compared and clustered. Major cell types were unabridged in both karyotypes. DS testis contains more Sertoli cells with an atypical expression profile, and more immune cells. DS cells exhibit a much higher level of ribosomal protein (RP genes and a significantlylower level of marker genes for each cell type, such as INSL3 for DS Leydig cell and PECAM1 for DS endothelial cell, etc.

Corresponding to cell proportion difference, gene co-expression network and intercellular communication analysis also suggested an immune activation in DS.

Furthermore, pseudotime analysis suggested a relative retardation and malfunction of DS cells.

The main differences in gene expression were further verified. Cell proportion alteration was demonstrated with immunohistochemistry. Overwhelming RPs were shown by transmission electron microscopy. Western and qPCR were used to test the expression difference of RPs, cell-type specific functional genes, etc.

Limitations, reasons for caution

The sample size was limited, although verification experiments were applied with independent samples might increase the credibility. The sample size needs to be extended further. More precise molecular mechanisms of these alterations and clinical dysfunction remains to be elucidate.

Wider implications of the findings

This study described human fetal DS testis at single-cell level. It reveals a pathological niche of altered cell components, compromised cell function, and activated immune response. It provides further targets for complement and/or intervention to rescue the reproductive function and even improve brain behavior in DS patients, soon after birth.

Trial registration number

Not applicable