Testosterone

Circulating leptin, insulin, interleukin (IL) 1β, IL6, and tumor necrosis factor α (TNFα) were determined by multiplexed immunoassay according to the manufacturer’s instructions (Millipore, Billerica, MA) in a Bio-Plex suspension array system 200 (Bio-Rad Laboratories, Hercules, CA, USA). Mean fluorescence intensity was analyzed by using Bio-Plex Manager Software 4.1.
Serum adiponectin (Millipore, Billerica, MA, USA), testosterone (Cusabio, Wuhan, PR China), and 17β-estradiol (Cusabio) concentrations were determined by ELISA as indicated by the manufacturer.
All samples were run in duplicate and within the same assay for all analyses. The limit of detection and the intra- and inter-coefficients of variation for all analyses are shown in Table 1.Table 1

The limit of detection and intra- and inter-assay coefficients (CV) of variation for the hormone and cytokine assays

	Detection limit (pg/ml)	CV intra-assay (%)	CV inter-assay (%)
Leptin	21.5	8.2	8.5
Insulin	51	7.3	9.8
Adiponectin	400	1.8	7.3
testosterone	60	7.7	8.0
Estradiol	<25	11.4	13.2
IL 6	9.8	10.4	14.2
IL1β	2.4	7.6	12.4
TNFα	4.5	9.2	11.1

Serum levels of these hormones in dam were determined at PND 7. Blood was collected via decapitation and serum was separated. The levels of testosterone (CUSABIO, Wuhan, China), estradiol (CUSABIO, Wuhan, China), progesterone (CUSABIO, Wuhan, China), prolactin (CUSABIO, Wuhan, China), corticosterone (CUSABIO, Wuhan, China), oxytocin (Enzo life sciences, PA, USA) and AVP (Enzo life sciences, PA, USA) were assessed according to the kit instruction. All the 8 control dams in part II were included in this section. But among the 9 MIA dams, we failed to get blood in 1 MIA dam, and no enough serum was separated from another MIA dam. Finally, the serum hormone analytes had n = 8 per group except testosterone and corticosterone had n = 7 for the MIA group.