Milk sample collection, processing, and laboratory tests Colostrum samples were collected between 8 a.m. and 9 a.m. before infant feeding on the third day after delivery. Mature milk, including both foremilk and hindmilk, was delivered and collected from one breast before infant feeding using an electric pump (Medela, Baar, Switzerland) between 2 p.m. and 4 p.m. on days 42 and 90 in the clinics. The milk samples were frozen immediately in sterilized plastic tubes at -80°C. Before quantifying the hormones in BM, the samples were thawed at 4°C, sonicated, and centrifuged. The samples were sonicated using a sonicator (Braun-sonic sonicator, B. Braun, Melsungen, Germany) at 50 watts for 3 bursts with 10-second intervals, and centrifuged at 100,000g for 1 hour at 4°C. The supernatant fat was discarded and the skim milk was used for quantifying adiponectin, leptin, insulin, and ghrelin by ELISA at the Key Laboratory of Endocrinology in the Peking Union Medical College Hospital. The assay had an intra- and inter-assay CV of <5.4 and <8.5% for adiponectin, and <7.4% and <9.3% for leptin, respectively. The insulin assay had no cross-reactivity to proinsulin (<0.05%), and had sensitivity of 0.5 mU/L and an inter-assay CV of <9.0%. Total ghrelin was tested using the total human ghrelin ELISA kit (Millipore, USA). The intra- and inter-assay CVs for the ghrelin assay were <1.9% and <7.7%.