Supplementary Table 1Top 50 most changed metabolites in T2 vs T1 ranked by VIP values (corrected with creatinine)

Primary ID / VIP / Match Accuracy (ppm) / HMDB / Theretical identity
4.83_995.3539 / 2.87 / 5.95 / HMDB01521 / Tetradecanoyl-CoA
8.45_405.2606 / 2.67 / 8.66 / HMDB00447 / 7a,12a-Dihydroxy-3-oxo-4-cholenoic acid
8.41_625.3921 / 2.60 / 3.61 / HMDB12937 / Dynorphin B (6-9)
5.36_557.2703 / 2.58 / 4.38 / HMDB03426 / Pantetheine
0.57_111.0567 / 2.56 / 7.76 / HMDB03905 / Imidazole-4-acetaldehyde
1.39_400.9923 / 2.50 / 7.46 / HMDB00843 / N-Acetylgalactosamine 4,6-disulfate
5.35_535.2879 / 2.48 / 1.82 / HMDB10389 / LysoPC(18:4(6Z,9Z,12Z,15Z))
0.50_501.0365 / 2.47 / 1.52 / HMDB01474 / 3,4-Dihydroxyphenylglycol O-sulfate
0.95_100.0763 / 2.42 / 0.61 / HMDB11749 / 2-Piperidinone
6.02_299.1155 / 2.41 / 8.16 / HMDB10350 / 2-Phenylethanol glucuronide
4.83_819.1932 / 2.40 / 8.27 / HMDB11612 / 6-Hydroxy flavin adenine dinucleotide
2.31_547.2473 / 2.34 / 5.46 / HMDB13129 / Glutaconylcarnitine
5.20_389.0654 / 2.31 / 9.65 / HMDB05008 / Lansoprazole
5.02_287.1263 / 2.30 / 7.01 / HMDB00894 / Vinylacetylglycine
7.93_277.1430 / 2.23 / 3.52 / HMDB04284 / Tyrosol
6.06_461.3238 / 2.19 / 1.06 / HMDB02126 / 27-Nor-5b-cholestane-3a,7a,12a,24,25-pentol
1.30_211.1198 / 2.19 / 6.29 / HMDB05973 / Dimethyltryptamine
0.61_243.0988 / 2.17 / 2.89 / HMDB00273 / Thymidine
2.49_302.2094 / 2.16 / 8.58 / HMDB03422 / Boldione
3.49_244.1538 / 2.14 / 4.44 / HMDB02366 / Tiglylcarnitine
8.80_692.4574 / 2.14 / 2.03 / HMDB07860 / PA(16:0/18:2(9Z,12Z))
2.46_420.0556 / 2.12 / 4.07 / HMDB01274 / dTDP
8.91_485.2165 / 2.11 / 2.77 / HMDB10337 / 6-Dehydrotestosterone glucuronide
3.48_190.0515 / 2.10 / 5.70 / HMDB00715 / Kynurenic acid
0.95_207.1121 / 2.10 / 6.04 / HMDB02302 / 3-Indolepropionic acid
2.26_276.1457 / 2.09 / 3.54 / HMDB13130 / Glutarylcarnitine
5.64_299.2375 / 2.08 / 8.06 / HMDB05830 / 5a-Androstan-3b-ol
5.32_555.2534 / 2.08 / 2.09 / HMDB03828 / D-Pantethine
8.93_571.4183 / 2.08 / 9.72 / HMDB12148 / 2-Hexaprenyl-6-methoxy-1,4-benzoquinol
6.54_256.2643 / 2.07 / 1.07 / HMDB12273 / Palmitic amide
8.82_653.3873 / 2.06 / 4.76 / HMDB03039 / Isoacitretin
4.93_607.2693 / 2.03 / 1.01 / HMDB01097 / Protoporphyrinogen IX
8.95_527.3909 / 2.03 / 7.87 / HMDB06455 / Arachidonyl carnitine
2.37_257.1149 / 2.02 / 4.51 / HMDB11648 / 1-(beta-D-Ribofuranosyl)-1,4-dihydronicotinamide
3.24_143.0726 / 2.02 / 9.49 / HMDB00097 / Choline
6.17_359.2557 / 2.01 / 1.44 / HMDB06070 / Pregnanetriol
4.67_327.1007 / 2.01 / 0.37 / HMDB05004 / Sodium lauryl sulfate
1.35_262.1300 / 2.00 / 3.61 / HMDB13133 / Methylglutarylcarnitine
3.25_188.0716 / 1.99 / 2.35 / HMDB00734 / Indoleacrylic acid
5.29_241.1455 / 1.99 / 4.49 / HMDB13224 / Caproylcholine
0.88_179.0483 / 1.99 / 4.18 / HMDB00078 / Cysteinylglycine
2.79_295.0860 / 1.98 / 5.48 / HMDB01786 / Ethenodeoxyadenosine
5.01_969.2751 / 1.97 / 7.24 / HMDB03571 / Lauroyl-CoA
8.45_395.2415 / 1.96 / 5.56 / HMDB12868 / 9'-Carboxy-gama-chromanol
3.99_258.1715 / 1.96 / 3.78 / HMDB13161 / 2-Hexenoylcarnitine
3.47_172.0417 / 1.96 / 5.14 / HMDB00696 / L-Methionine
1.05_551.0733 / 1.96 / 9.75 / HMDB12305 / UDP-L-rhamnose
3.59_250.0127 / 1.95 / 3.66 / HMDB11639 / Topaquinone
1.93_186.1017 / 1.94 / 7.63 / HMDB12897 / Beta-Carboline
2.36_233.0909 / 1.94 / 2.81 / HMDB00114 / Glycerylphosphorylethanolamine

ppm, parts per million.

Supplementary Table 2Top 50 most changed metabolites in T3 vs T2 ranked by VIP values (corrected with creatinine)

Primary ID / VIP / Match Accuracy (ppm) / HMDB / Theretical identity
2.61_210.0527 / 2.90 / 1.89 / HMDB00734 / Indoleacrylic acid
7.14_249.1494 / 2.89 / 1.35 / HMDB01931 / Gamma-CEHC
2.60_411.1148 / 2.87 / 0.82 / HMDB02873 / Deacetyldiltiazem
6.97_293.1744 / 2.75 / 7.60 / HMDB03701 / Dimethylbenzimidazole
1.54_226.0476 / 2.72 / 2.50 / HMDB12150 / 2-Keto-6-acetamidocaproate
5.62_246.1105 / 2.63 / 6.14 / HMDB00012 / Deoxyuridine
8.45_405.2603 / 2.57 / 9.34 / HMDB00447 / 7a,12a-Dihydroxy-3-oxo-4-cholenoic acid
2.93_356.0976 / 2.53 / 1.29 / HMDB01517 / AICAR
2.80_295.0447 / 2.51 / 3.72 / HMDB06809 / Nicotinate D-ribonucleoside
2.33_374.0257 / 2.46 / 2.94 / HMDB06824 / Dihydroneopterin phosphate
0.54_536.1232 / 2.46 / 2.28 / HMDB00632 / Dermatan
3.25_249.0636 / 2.45 / 1.72 / HMDB01555 / Pyridoxamine 5'-phosphate
3.12_354.0587 / 2.45 / 2.07 / HMDB00905 / Deoxyadenosine monophosphate
4.93_315.1303 / 2.44 / 5.62 / HMDB11687 / Phenylbutyrylglutamine
3.89_796.2115 / 2.44 / 9.74 / HMDB10366 / Hyaluronan
8.41_625.3920 / 2.41 / 3.51 / HMDB12937 / Dynorphin B (6-9)
6.00_377.1552 / 2.40 / 2.10 / HMDB10336 / Epinephrine glucuronide
0.88_239.1288 / 2.36 / 0.19 / HMDB13162 / 2-Hexenoylcholine
3.40_387.0653 / 2.34 / 7.49 / HMDB12467 / (-)-epicatechin sulfate
2.91_325.0792 / 2.33 / 3.85 / HMDB12134 / 1,2-Dihydroxy-3-keto-5-methylthiopentene
7.31_414.2159 / 2.33 / 3.24 / HMDB12868 / 9'-Carboxy-gama-chromanol
8.82_653.3873 / 2.32 / 4.76 / HMDB03039 / Isoacitretin
4.93_607.2694 / 2.31 / 1.21 / HMDB01097 / Protoporphyrinogen IX
3.12_239.0466 / 2.31 / 0.13 / HMDB04821 / Bisnorbiotin
6.60_135.1181 / 2.30 / 5.42 / HMDB05805 / p-Cymene
4.98_308.1850 / 2.27 / 3.96 / HMDB13324 / 2-Octenoylcarnitine
6.60_141.0200 / 2.25 / 8.66 / HMDB06804 / Propynoic acid
2.43_262.0701 / 2.25 / 3.49 / HMDB01265 / Fucose 1-phosphate
5.63_555.2562 / 2.25 / 7.15 / HMDB03828 / D-Pantethine
4.27_567.1511 / 2.24 / 0.22 / HMDB10356 / Estriol 3-sulfate 16-glucuronide
1.19_206.0424 / 2.22 / 2.52 / HMDB00017 / 4-Pyridoxic acid
5.70_299.2377 / 2.22 / 8.77 / HMDB05830 / 5a-Androstan-3b-ol
2.87_332.0716 / 2.20 / 9.10 / HMDB10319 / Inodxyl glucuronide
2.38_276.0955 / 2.20 / 6.74 / HMDB01940 / Triamterene
5.90_281.2274 / 2.16 / 9.66 / HMDB03424 / 1-Hexadecanol
7.32_421.3175 / 2.16 / 4.06 / HMDB12517 / 11'-Carboxy-gama-chromanol
7.31_773.4957 / 2.15 / 4.58 / HMDB00385 / 12a-Hydroxy-3-oxocholadienic acid
2.54_253.0628 / 2.13 / 1.09 / HMDB12111 / (+)-(1R,2R)-1,2-Diphenylethane-1,2-diol
4.84_447.2013 / 2.13 / 1.36 / HMDB04483 / Estrone glucuronide
4.84_253.1557 / 2.12 / 5.06 / HMDB00910 / Tridecanoic acid
0.95_221.0578 / 2.09 / 8.15 / HMDB02015 / Cystathionine ketimine
5.23_297.2219 / 2.09 / 8.32 / HMDB05935 / Androstenol
7.06_269.1154 / 2.07 / 0.45 / HMDB00623 / Dodecanedioic acid
3.28_345.1057 / 2.07 / 3.10 / HMDB01212 / Hydantoin-5-propionic acid
4.47_327.1005 / 2.06 / 1.03 / HMDB05004 / Sodium lauryl sulfate
4.93_899.4084 / 2.04 / 4.75 / HMDB13019 / Neuromedin C 1-8
8.80_692.4573 / 2.04 / 2.20 / HMDB07860 / PA(16:0/18:2(9Z,12Z))
6.79_495.2933 / 2.02 / 3.08 / HMDB13127 / Hydroxybutyrylcarnitine
5.02_947.2916 / 2.02 / 5.48 / HMDB06692 / Disialyllactose
0.51_235.1171 / 2.02 / 6.88 / HMDB00128 / Guanidoacetic acid

ppm, parts per million.

Supplementary Table3 Identification of markers involved in amino acid metabolism in the urine of healthy pregnant women

Marker / Ionization mode / tR
(min) / m/z / Chemical identity / Comparison / Mass error (ppm) / Related features / Major MS/MS fragment ions(m/z)
1 / ES+ / 3.48 / 190.0515 / [M+H]+ of
Kynurenic acid (HMDB00715) / T2 vs T1 / 5.70 / m/z 188.03
([M-H]-) / 144,116
2 / ES+ / 2.10 / 157.0778 / [M+H]+ of 3-Indoleacetonitrile (HMDB06524) / T2 vs T1, T3 vs T2 / 7.87 / m/z 201.07 ([M+HCOO]-) / 140, 130
3 / ES+ / 7.93 / 277.1430 / [2M+H]+ of
Tyrosol
(HMDB04284) / T2 vs T1, / 3.52 / m/z 137.06
([M-H]-), m/z 275.13
([2M-H]-) / 121
4 / ES+ / 3.28 / 345.1053 / [2M+H]+ of Hydantoin-5-propionic acid
(HMDB01212) / T2 vs T1 / 1.86 / m/z 195.04 ([M+Na]+), m/z 171.04 ([M-H]-), m/z 343.09 ([2M-H]-), m/z 217.05 ([M+HCOO]-) / 154, 127, 99
5 / ES+ / 0.57 / 111.0567 / [M+H]+ of Imidazole-4-acetaldehyde (HMDB03905) / T2 vs T1, / 7.76 / m/z 109.04 ([M-H]-) / 81, 67
6 / ES+ / 2.92 / 356.0961 / [M+NH4]+ of
AICAR
(HMDB01517) / T2 vs T1 / 2.91 / m/z 339.07 ([M+H]+), m/z 361.05 ([M+Na]+), m/z 337.05 ([M-H]-) / 127, 110
7 / ES+ / 0.88 / 179.0473 / [M+H]+ of Cysteinylglycine
(HMDB00078) / T3 vs T2 / 9.72 / m/z 198.03 ([(2M+H+K)/2]+), m/z 355.07 ([2M-H]-) / 104,76
8 / ES+ / 4.21 / 118.0666 / [M+H]+ of
Indole
(HMDB00738) / T3 vs T2 / 7.82 / m/z 140.05 ([M+Na]+), m/z 156.02 ([M+K]+), m/z 116.05 ([M-H]-), m/z 162.05 ([M+HCOO]-) / 118, 91
9 / ES+ / 0.89 / 148.0406 / [M+H]+ of Indole-5,6-quinone
(HMDB06779) / T3 vs T2 / 5.05 / m/z 165.07 ([M+NH4]+), m/z 146.02 ([M-H]-) / 119
10 / ES+ / 4.31 / 232.9873 / [(2M+H+K)/2]+ of L-Aspartyl-4-phosphate
(HMDB12250) / T3 vs T2 / 9.89 / m/z 214.01 ([M+H]+),m/z 251.97 ([M+K]+) / 196, 177, 168, 116, 99

tR, retention time; ppm, parts per million; M, intact molecule (e.g., [M+H]+ stands for the positive ion formed by the intact molecule adding a proton); “Related features” refers to the features generated from identical molecules including features of quasi or adduct molecular ions, diametric ions and so on which possess the similar retention time and chromatographic shape;T1, T2 and T3 denote the first trimester, the second trimester and the third trimester, respectively.

Supplementary figure 1. PLS-DA analysis between the second and first trimesters. a: PLS-DA scores for the second trimester vs the first trimester. T1 and T2 denote the first trimester and the second trimester, respectively; t[1], t[2] and t[3] denote the first principal component, the second principal component and the third principal component, respectively. b: Fit goodness of PLS-DA model. The R2Ycum value was 0.98, while Q2cum was 0.94, indicating that valid PLS-DA models were established.

Supplementary figure 2. PLS-DA analysis between the third and second trimesters. a: PLS-DA scores for the third trimester vs the second trimester. T2 and T3 denote the second trimester and the third trimester, respectively; t[1], t[2] and t[3] denote the first principal component, the second principal component and the third principal component, respectively. b: Fit goodness of PLS-DA model. The R2Ycum value was 0.98, while Q2cum was 0.95, indicating that valid PLS-DA models were established.