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Objective Thyroid peroxidase antibodies ( TPOAb) and thyroglobulin antibodies ( TGAb) are frequently measured to investigate thyroid dysfunction in pregnancy. Despite the recognized fall of these autoantibodies in pregnancy, there is limited guidance on the timing of such testing. We assessed optimal test timing of TPOAb/ TGAb for the detection of Hashimoto's thyroiditis and post-partum thyroid dysfunction ( PPTD). Design Prospective longitudinal study with recruitment in Trimester 1. Patients Healthy women ≤13 weeks' gestation from Mercy Hospital for Women, a tertiary obstetric hospital in Melbourne. Measurements Serum TPOAb, TGAb, TSH and fT4 were measured at Trimester 1 (T1), Trimester 2(T2), Trimester 3(T3) and postpartum (PP) in each participant. Post-partum thyroid dysfunction (PPTD) was defined if TSH deviated from the assay's nonpregnant reference interval. Longitudinal random-effect logistic regression was used to investigate the association between time and positive/negative thyroid autoantibody status. Results Samples from 140 women at T1 (12·0: 10·3-13·0) (median: IQR weeks' gestation); 95 at T2 (24·3: 23·0-25·9), 79 at T3 (35·9: 34·8-36·7) and 83 at PP (12·4: 10·8-14·6 weeks post-partum) were attained. At T1, 13 (9%) and 15 (11%) women had positive TPOAb and TGAb, respectively. The odds of having a positive TPOAb were 96% lower at T2 [ OR = 0·04 (95% CI: 0·02-0·8; P = 0·03)] and 97% lower at T3 [ OR = 0·03 (95% CI: 0·001-0·6; P = 0·02)] than at T1. Similarly, the odds of having a positive TGAb were 99·4% lower [ OR = 0·006 (95% CI: 0-0·3; P = 0·01)] at T2, and 99·5% lower [ OR = 0·005 (95% CI: 0-0·4; P = 0·02)] at T3 than at T1. The ROC analysis diagnostic ORs for a positive TPOAb and/or TGAb to predict PPTD were 7·8 (95% CI: 2·2-27·6), 1·2 (95% CI: 0-8·9), 2·0 (95% CI: 0-16·8), and 12·2 (95% CI: 3·3-44·9) at T1, T2, T3 and post-partum, respectively. Conclusions A significant proportion of pregnant women lose their thyroid autoantibody positivity after T1. The gestation-dependent loss of TPOAb/ TGAb positivity and reduction in diagnostic accuracy for predicting PPTD limits the value of testing at T2 and T3. [ABSTRACT FROM AUTHOR]