E el 0033c

Blood samples were collected both at RW and after SD, which were performed after the HAMD assessment and MRI scan. Because the procedure of blood sample collection was invasive, only 17 paired samples were finally obtained. Blood was collected into ethylenediaminetetraacetic acid tubes (5 ml, for measurement of 5-HT and NE). Samples were left standing at room temperature for at least 30 min. The supernatant serum was removed after centrifuging at 3,000 × g for 10 min in a 4°C centrifuge. Then, a 600-μl liquid component was immediately transferred in Eppendorf tubes, which were stored at a −80°C refrigerator until further use. Enzyme-linked immunosorbent assay was performed to measure the concentrations of 5-HT (Elabscience, E-EL-0033c, China) and NE (Elabscience, E-EL-0047c, China) following the manufacturer's instruction (27 (link)).

After removing myometrium, the endometrium at implantation sites on day 5 of pregnancy was homogenized for serotonin measurement according to the manufacturer’s instructions (Elabscience, E-EL-0033c, Wuhan, China). This kit’s sensitivity is greater than 9.38 ng/ml. The standard working solution or samples of 50 μL were mixed with 50 μL biotinylated antibody in working solution and incubated at 37° C for 45 min. Following washing 3 times, 100 μL HRP enzyme conjugate working solution was added to each well and incubated at 37° C for 30 min. After washing 5 times, 90 μL of substrate solution was added to each well and incubated at 37° C for 15 min. Absorbance at 450 nm wavelength was measured following adding 50 μL of termination solution to each well. Each experiment was repeated at least three times.