Androgen-responsive non-coding small RNAs extend the potential of HCG stimulation to act as a bioassay of androgen sufficiency

Abstract

Background: It is unclear whether a short-term change in circulating androgens is associated with changes in the transcriptome of the peripheral blood mononuclear cells (PBMC). Aims and methods:To explore the effect of hCG stimulation on the PBMC transcriptome, 12 boys with a median age (range) of 0.7 years (0.3, 11.2) who received intramuscular hCG 1500u on 3 consecutive days as part of their investigations underwent transcriptomic array analysis on RNA extracted from peripheral blood mononuclear cells before and after hCG stimulation. Results: Median pre-and post-hCG testosterone for the overall group was 0.7 nmol/L (<0.5, 6) and 7.9 nmol/L (<0.5, 31.5), respectively. Of the 12 boys, 3 (25%) did not respond to hCG stimulation with a pre and post median serum testosterone of <0.5 nmol/L and <0.5 nmol/L, respectively. When corrected for gene expression changes in the nonresponders to exclude hCG effects, all 9 of the hCG responders consistently demonstrated a 20% or greater increase in the expression of piR-37153 and piR-39248, non-coding PIWI-interacting RNAs (piRNAs). In addition, of the 9 responders, 8, 6 and 4 demonstrated a 30, 40 and 50% rise, respectively, in a total of 2 further piRNAs. In addition, 3 of the responders showed a 50% or greater rise in the expression of another small RNA, SNORD5. On comparing fold-change in serum testosterone with fold-change in the above transcripts, a positive correlation was detected for SNORD5 (P = 0.01). Conclusions: The identification of a dynamic and androgen-responsive PBMC transcriptome extends the potential value of the hCG test for the assessment of androgen sufficiency.

Genetic testing of XY newborns with a suspected disorder of sex development

“…However, there is a need to explore more effective methods of selecting cases that may display androgen insensitivity. Whilst in the past this has involved assessment of AR binding in genital skin fibroblasts [119,120] or measurement of circulating androgen responsive proteins in response to androgen stimulation [121,122], in the future it may be possible to use other methods such as measurement of apolipoprotein D in genital skin fibroblasts [117] or assessment of changes in an androgen responsive transcriptome within circulating polymorphonuclear blood cells [123]. Variants in several other genes, such as INSL3, AMH, AMHR2, MAMLD1, TAC3, WDR11, TACR3, HS6ST1, CHD7, may also contribute to DSD [124].…”

The Use of Genetics for Reaching a Diagnosis in XY DSD

“…An extended investigation where epigenetic regulation of AR was assessed at both transcription and translation levels resulted in molecular diagnosis of AIS type II due to the production of aberrant AR promoter transcripts [Hornig et al, 2018]. In another study, the existence of an androgen-responsive transcriptome was also hypothesised due to the identification of several non-coding RNAs that displayed different expression levels before and after hCG stimulation in boys with a DSD phenotype [Rodie et al, 2017]. Given the capability of WGS to iden-tify CNV, SNVs, and INDELs throughout the genome, it is possible to reach a better understanding of DSD mechanisms although the complexity of variants would render interpretation difficult [Bocher et al, 2020].…”

“…In the past, this has involved assessment of AR binding in genital skin fibroblasts [Evans et al, 1984], measurement of circulating androgen responsive proteins in response to androgen stimulation [Sinnecker et al, 1997;Bertelloni et al, 1997], or even clinical assessment of the genitalia in response to androgen stimulation [Stancampiano et al, 2022]. However, it may be possible to use other methods such as measurement of apolipoprotein D in genital skin fibroblasts [Hornig et al, 2016] or an androgen responsive transcriptome within circulating polymorphonuclear blood cells before and after androgen exposure [Rodie et al, 2017]. Although there are differences in the AR residual function among the AIS phenotypes, no difference has been observed in the hormonal levels across AIS phenotypes [Ahmed et al, 1999;Arnhol et al, 2011].…”

Society for Endocrinology UK Guidance on the initial evaluation of a suspected difference or disorder of sex development (Revised 2021)

“…Androgen sensitivity can be also assessed by measuring change in androgen responsive circulating proteins such as SHBG following androgen exposure but this is rarely performed in clinical practice as the response can be very variable. There may be other methods of assessing tissue responsiveness to androgens including the measurement of androgen responsive proteins in genital skin fibroblasts 100 or the assessment of the androgen responsive transcriptome 101 but their clinical utility requires further exploration. AR analysis may reveal a causative gene variant in over 90% of cases with a CAIS phenotype but given that only 20% of cases with a PAIS phenotype have a variant in the coding region of AR , 102 there is a need to improve the diagnosis of this condition especially as it has been reported that the gene variants in AR may exist beyond the coding sequence 100 .…”