Steroid Metabolome Profiling Of Women With Polycystic Ovary Syndrome And Association With IVF/ICSI Outcomes

Background:Polycystic ovary syndrome(PCOS)is the most common ovulation disorder in women of reproductive age.It is characterized by clinical or biochemical androgen excess,menstrual cycle irregularities(amenorrhea or oligomenorrhea),and polycystic ovarian morphology.Hyperandrogenism caused by dysregulation of steroidogenesis plays a central role in the pathogenesis of PCOS.The liquid chromatography-tandem mass spectrometry(LC-MS/MS)with high sensitivity and specificity has improved the performance of steroids measurement in human serum.However,it has not been widely used in clinical practice.To date,few studies have described the steroid metabolism profiling of PCOS patients and its correlation with assisted reproductive outcomes.Objective:To detect 21 serum steroid levels of PCOS patients and control group on oocyte retrieval day and explore differences in the steroidogenic pathway between two groups.To explore the correlation between serum steroid levels and IVF/ICSI outcomes and provide evidence for clinical therapeutics of PCOS patients.Materials and methods:From September 2018 to December 2019,100 patients with PCOS and 101 patients with tubal infertility or male factor infertility(control group)were enrolled.The serum of PCOS patients and control group on oocyte retrieval day were collected.Clinical information were collected,including age,BMI,indexes of endocrine and metabolic and IVF outcomes.LC-MS/MS was used to detect the serum steroid levels,including pregnenolones(P5 and 17OHP5),progestins(P and 17OHP),androgens(DHEA,DHEAS,AD,T,ADT and DHT),estrogens(E1,E2,and E3),glucocorticoids(S,21-DOF,F,and C),and mineralocorticoids(18-OHB,DOC,B,and Aldo).The differences of steroid hormones between the two groups were compared.Principal component analysis(PCA),partial least square discriminant analysis(PLS-DA)and orthogonal partial least squared discriminant analysis(OPLS-DA)were used to determine steroid changes in two groups.Steroid enzyme activities in the steroidogenic pathway were assessed by product-to-precursor concentration ratios.The corrleation between steroid hormone levels and the number of retrieved oocytes,the number of2 PN,the number of good quality embryos and endometrial thickness were analyzed.Predictive ability of steroid hormone levels on pregnancy outcomes and correlation with singleton birth weight in fresh cycles were analyzed.Continuous variables were compared by Mann Whitney U test.Spearman’s rank correlation coefficient was used for bivariate correlation analysis.Area under curve(AUC)of receiver operating characteristic curve(ROC)was used to compare predictive ability.Results:1.Compared with the control group,the patients in PCOS group had higher BMI,higher levels of basal LH,basal T and AMH,lower levels of basal FSH and lower dosage of Gn.PCOS patients had higher E2 on h CG day,more retrieved oocytes,2PN and good quality embryos.There was no difference in the cause of infertility,ART type and ovarian stimulation protocols.2.Compared with the control group,the levels of progesterone,progesterone,17-hydroxyprogesterone,dehydroepiandrosterone,androstenedione,testosterone,dihydrotestosterone,androsterone,estrone,estradiol,estriol,21-deoxycortisol and18-hydroxycortisol in PCOS group were significantly higher.3.PCA,PLS-DA and OPLS-DA analysis showed significant separation of steroid metabolome profiling of PCOS and control group.According to VIP scores of OPLS-DA>1 and P<0.05,androstenedione,17-hydroxyprogesterone and progesterone were was considered important in the discrimination.4.The activities of P450c17(17-hydroxyprogesterone/pregnenolone,androstenedione/17-hydroxyprogesterone)in PCOS group was lower than control group(P=0.004,P < 0.001),while the activities of P450c17(17-hydroxyprogesterone/progesterone,dehydroepiandrosterone/17-hydroxyprogesterone)in PCOS group was higher than control group(P=0.012,P=0.034).The activity of P450c21(11-deoxycorticosterone/progesterone)was significantly lower in PCOS group(P<0.001),but there was no significant difference in P450c21(21-deoxycorticosterone/17-hydroxyprogesterone).PCOS patients had decreased activity of P450c11β1(cortisol/11-deoxycortisol)compared with control women(P=0.032),but there was no difference in the activity of P450c11β2(corticosterone/11-deoxycortisol).PCOS group had lower activity of P450aro(estradiol/testosterone)than control group(P<0.001),but there was no significant difference in the activity of P450aro(estrone/ androstenedione).The activity of 3βHSD2(progesterone/pregnenolone)was significantly higher in PCOS patients,but no difference was observed in the activity of 3βHSD2(17-hydroxyprogesterone/17-hydroxypregnenolone).The activities of 11βHSD2(cortisol/cortisol)and 17βHSD3(testosterone/androstenedione)were significantly higher in PCOS patients(P<0.001).There was no significant difference in the activity of 5α-reductase(dihydrotestosterone/testosterone).5.In the control group,the number of oocytes retrieved were positively correlated with serum estradiol(r=0.456,P<0.001),estrone(r=0.384,P<0.001),testosterone(r=0.347,P<0.001),androstenedione(r=0.332,P<0.001),11-deoxycorticosterone(r=0.283,P=0.004),progesterone(r=0.262,P=0.008),and were negatively correlated with serum dehydroepiandrosterone sulfate(r=-0.214,P=0.031).The number of 2PN were positively correlated with serum androstenedione(r=0.270,P=0.006),estradiol(r=0.268,P=0.007)and estrone(r=0.211,P=0.034).The number of good quality embryos were positively correlated with levels of androstenedione(r=0.361,P<0.001),estradiol(r=0.334,P<0.001),testosterone(r=0.293,P=0.003),11-deoxycorticosterone(r=0.212,P=0.034)and testosterone(r=0.202,P=0.043).Only dehydroepiandrosterone sulfate was positively correlated with endometrial thickness on the day of oocyte retrieval(r=0.235,P=0.022).In PCOS group,the number of oocytes retrieved were positively correlated with serum estradiol(r=0.511,P<0.001),11-deoxycorticosterone(r=0.507,P<0.001),17-hydroxyprogesterone(r=0.506,P<0.001),estrone(r=0.492,P<0.001),testosterone(r=0.426,P<0.001),pregnenolone(r=0.399,P<0.001),androstenedione(r=0.346,P<0.001)and progesterone(r=0.332,P<0.001)and dihydrotestosterone X(r=0.295,P=0.003).The number of 2PN were positively correlated with the levels of serum 11-deoxycorticosterone(r=0.250,P=0.012),progesterone(r=0.211,P=0.035),estradiol(r=0.206,P=0.039),estrone(r=0.202,P=0.044)and 17-hydroxyprogesterone(r=0.199,P=0.047).The number of good quality embryos were positively correlated with serum testosterone(r=0.255,P=0.011),17-hydroxyprogesterone(r=0.220,P=0.029)and testosterone(r=0.208,P=0.038).There were no significant correlation between steroid levels and endometrial thickness on the day of oocyte retrieval.6.The levels of 18-hydroxycorticosterone and cortisol can predict pregnancy outcome in PCOS patients.The AUC of ROC was 0.778(95%CI: 0.618-0.938,P=0.005)and 0.742(95%CI: 0.585-0.898,P=0.016).The levels of estrone,estradiol and testosterone were the proteintial predictors of pregnancy outcome in control women.The AUC of ROC was 0.659(95%CI: 0.519-0.798,P=0.030),0.650(95%CI:0.499-0.800,P=0.040)and 0.644(95%CI: 0.496-0.792,P=0.048).7.In PCOS group,the levels of cortisol and 21-deoxycortisol showed negative correlation with the birth weight of singletons in fresh cycles(r=-0.560,P=0.020;r=-0.532,P=0.028).In the control group,estradiol level was negatively correlated with birth weight(r=-0.446,P=0.017).Conclusion:PCOS patients and control group had different steroid metabolome map in serum.Androstenedione,17-hydroxyprogesterone and progesterone were the top steroid hormones that resulted in the group separation.The changes in serum steroid hormone levels were related to the IVF/ICSI outcomes.The levels of 18-hydroxycorticosterone,cortisol,estrone,estradiol and testosterone could predictpregnancy outcome before transplantation.The levels of cortisol,21-deoxycortisol and estradiol were negatively correlated with the birth weight of singletons in fresh cycles.