Study on the reference intervals of six androgen "backdoor pathway" steroids in healthy Chinese adults

Androgen backdoor pathway steroids are critical for identifying hyperandrogenism and differentiating polycystic ovary syndrome (PCOS) from non-classic congenital adrenal hyperplasia (CAH). However, reference intervals (RIs) for these steroids in healthy adults are lacking.

Between January and July 2025, 266 healthy adults (136 males and 130 females) aged 20-60 years were recruited from health check-up attendees at two hospitals in Beijing. Six backdoor pathway steroids, including 5α-dihydroprogesterone (5α-DHP), 17OH-dihydroprogesterone (17OH-DHP), 17OH-allopregnanolone (17OH-Allo), androsterone (AN), androstanediol (3α-diol), and 5α-dihydrotestosterone (DHT), were quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RIs were established according to sex and age.

Sex-stratified RIs (20-60 years) were established: in males, 5α-DHP <25.3-264 pmol/L, 17OH-DHP <24.1-50.9 pmol/L, 17OH-Allo <53.8-433 pmol/L, AN 293-1766 pmol/L, 3α-diol 184-1046 pmol/L, and DHT 350-1882 pmol/L; in females, 5α-DHP <25.3-725 pmol/L, 17OH-DHP <24.1-34.7 pmol/L, 17OH-Allo <53.8-258 pmol/L, AN 171-1210 pmol/L, 3α-diol 37.9-766 pmol/L, and DHT 31.3-437 pmol/L. Sex- and age-stratified RIs (20-40 / 41-60 years) were further established for selected steroids: in males, 5α-DHP <25.3-287 / <25.3-248 pmol/L, 17OH-Allo <53.8-514 / <53.8-415 pmol/L, and AN 250-2246 / 239-1263 pmol/L; in females, 5α-DHP <25.3-835 / <25.3-546 pmol/L, 17OH-Allo <53.8-281 / <53.8-273 pmol/L, AN 277-1357 / 79.3-1076 pmol/L, 3α-diol 60.6-838 / 37.4-733 pmol/L, and DHT 103-476 / 19.4-399 pmol/L.

This study established sex- and age-specific RIs for six androgen backdoor pathway steroids in healthy Chinese adults.