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ObjectiveTo investigate whether serum 25-hydroxyvitamin D [25(OH)D] level is associated with ovarian reserve markers insecondary amenorrhea (SA) patients. MethodsSixty-three women diagnosed with SA were recruited during 12 months from the initiation of this prospectiveobservational study. Serum 25(OH)D levels, serum anti-Müllerian hormone (AMH) levels and antral follicle count (AFC)were estimated in study participants and ovarian reserve markers were compared between participants with vitaminD deficiency and those with normal vitamin D levels. ResultsOf the 63 participants, 27 (42.9%) were vitamin D deficient (<20 ng/mL) and 36 (57.1%) had normal vitamin D levels. The mean AMH levels and AFC were 10.86±8.94 µ/L and 15.23±7.65 in the vitamin D deficient group, and 7.24±5.62 µ/Land 12.30±6.95 in the normal vitamin D group. Univariate and multivariate linear regression analysis of log10transformed AMH and AFC with serum 25(OH)D adjusted for age and body mass index confirmed no associationbetween vitamin D levels and AMH levels or AFC. There was also no correlation between serum 25(OH)D and AMHlevels or AFC in all participants. However, participants with vitamin D deficiency had an increased chance of havingpolycystic ovarian syndrome (PCOS) as cause of SA than those with normal vitamin D levels (adjusted odds ratio, 7.559;95% confidence interval, 1.28–44.65; P=0.026) after adjustment for clinical factors by logistic regression model. ConclusionThere was no correlation between serum 25(OH)D levels and ovarian reserve markers in SA patients, but vitamin Ddeficiency may be linked to PCOS patients.