| Objective:1.Establishment of reference intervals for six serum androgens in 20-40 year-old women in Zhejiang Province by liquid chromatography-tandem mass spectrometry(LC-MS/MS).2.To investigate the characteristics of each androgen index(testosterone,free androgen,free androgen index,androstenedione,dehydroepiandrosterone and dehydroepiandrosterone sulphate)in infertile women with different Kaohsiung-related phenotypes and to analyse the diagnostic sensitivity and accuracy of each index.Methods:1.Study 1: Healthy women of reproductive age from November 2021 to February2022 from 3 hospitals were recruited following inclusion and exclusion criteria.Their serum levels of total testosterone(T),free testosterone(FT),dihydrotestosterone(DHT),androstenedione(A4),dehydroepiandrosterone(DHEA)and dehydroepiandrosterone sulphate(DHEAS)were determined by LC-MS/MS.The percentile method(P97.5)was used to establish the reference intervals and the results were stratified by age and menstrual cycle.The correlation between androgen levels and metabolic indexes were also analyzed.2.Study 2: A total of 249 patients who visited the Zhejiang Provincial People’s Hospital from May 2022 to January 2023 were selected from the Department of Reproductive Endocrinology for ovarian polycystic changes combined with ovulatory disorder infertility,the Department of Endocrinology for obesity combined with insulin resistance and the Department of Dermatology for hirsutism or acne.There were 4 groups: obesity with ovarian polycystic pattern combined with ovulation disorder only,without obesity combined with insulin resistance and acne hirsutis(59 cases in group A);obesity combined with insulin resistance and ovarian polycystic pattern combined with ovulation disorder,but without hirsutism and acne(58 cases in group B);obesity combined with insulin resistance only,without ovarian polycystic pattern,ovulation disorder,hirsutism and acne(73 cases in group C);and hirsutism or acne,but without ovarian polycystic pattern,ovulation disorder,obesity and insulin resistance(59 cases in group D).The differences in each androgen index between the groups and normal healthy women of childbearing age in Study 1 were compared,and the diagnostic value of each androgen index was analysed by comparing the area under the curve with the ROC curve.3.Analysis methods: Data were analyzed using R software(version 4.1.2,Tusher et al.,2001)and SPSS(version 26,IBM Corporation,Armonk,NY,USA).the Shapiro-Wilk method was used to test the normality of the data distribution.Normally distributed data information is expressed as x ± s;non-normally distributed data are expressed as median and interquartile spacing,with the reference range expressed between the 2.5th and 97.5th percentiles.The Kruskal-Wallis method was used to analyze the correlation between androgens and age and menstrual cycle.spearman analysis was us ed to analyze the correlation between androgens and clinical serum metabolic parameters.p <0.05 is statistically significant difference.The area under the ROC curve was used to analyze the value of androgen indicators for the diagnosis of related diseases.Results:1.The reference intervals for T,FT,DHT,A4,DHEA and DHEAS for the total population(20-40 years)were 81.77-477.21 pg/ml,1.00-4.05 pg/ml,50.00-157.28 pg/ml,550.33-2172.06 pg/ml,1675.51-12054.90 pg/ml and0.92-4.05 ug/ml,respectively.T,FT and DHT reference intervals were significantly lower than those from the Mayo laboratory.When stratified by age,the differences in T,FT,FAI,DHT,A4 and DHEAS between the 20-30 and31-40 age groups were statistically significant,but the density curve overlaps highly.When stratified into the Early and middle follicles phase,late follicular phase and luteal phase,there were no significant differences in DHT,DHEA,or DHEAS(P > 0.05),while T,FT and A4 showed phase difference(P < 0.05),but the density curve overlaps highly.Various subtypes of androgens were correlated with BMI,HOMA-IR,HDL,LDL and TG to varying degrees in the total population(20-40 years).2.In group C patients: FT,FAI and A4 were higher than in the control group and SHBG was lower than in the control group(P<0.05).in group B patients: T,FT,FAI and A4 were higher than in the control group and SHBG was lower than in the control group(P<0.05).in group A patients: T,FT,FAI and A4 were higher than in the control group and SHBG was lower than in the control group;T and SHBG were higher than in group C patients(P<0.05).In group D: FT was higher than in the control group and lower than in group B(P<0.05).FAI was significantly lower than in the group of patients with combined metabolic disorders and SHBG was higher than in the group of patients with combined metabolic disorders(P<0.05),but not significantly different from normal controls.There was no significant change in DHEA and DHEAS,which are predominantly secreted from the adrenal glands.Notably,SHBG was significantly lower and FT was significantly higher in the group with metabolic disorders than in the control group(P<0.05).3.Using healthy subjects as a reference,the results of this study showed that the area under the curve(AUC)of the working characteristic curve for FT and FAI subjects was the largest among the four ABCD groups with 0.88 and 0.88,0.91 and 0.75,0.77 and 0.80,and 0.84 and 0.83,respectively,all with p-values < 0.05;cut-off values were 3.35 and 1023.04,3.15 and 886.98,2.85 and 750.75,3.25 and 829.05;sensitivities were 81% and 81%,84% and 58%,67% and 81%,80%and 88%,respectively;specificities were 93% and 94%,92% and 90%,85% and82%,93% and 87%,respectively.Except for significant high FT and FAI,serum T and A4 were diagnostic in both groups A and B with p-values <0.05.AUC was 0.79 and 0.69,cut-off values were 311.45 and 312.40,sensitivity was 74%and 58%,and specificity was 79% and 80%,respectively.Conclusion:The reference intervals for T,FT and DHT in women of reproductive age in Zhejiang are significantly lower than those in foreign populations.so local reference ranges are recommended for clinical application.Androgens in healthy women are stable throughout reproductive age and menstrual cycle,so a common reference range might be adopted.Some androgen subtypes levels are also strongly associated with body weight,insulin resistance index and lipid profiles in healthy women of reproductive age.In the diagnosis of hyperandrogenemia,we recommend the use of serum FT levels instead of T,which is routinely used in clinical practice,to reduce the rate of underdiagnosis,and FAI has a good reference value when direct FT testing is not available.The results of this study suggest that SHBG was significantly lower and T and FT were significantly higher in the combined metabolic disorder population,possibly due to an increase in androgens in the body as a result of the metabolic disorder inhibiting SHBG synthesis. |
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