aji13499-sup-0001-FigureS1.jpg79.7 KB FIGURE S1. Flow chart depicting clinical procedures and strategy to procure hE-SM samples for biomarker analysis from participating infertility (IVF) clinics. Shown were the (A) Clinical procedures performed at ART clinics. (B) Strategy for hE-SM samples, collected from (1) KMC, (2) MAARS and (3) AFC. Abbreviations: OPU , ovum pick-up; h, hour; ET = embryo transfer; sHLA-G , soluble human leukocyte antigen-G; KMC , Kasturba Medical College; MAARS , Manipal Ankur Andrology & Reproductive Services; AFC, Advanced Fertility Centre; hE-SM , human embryo spent medium. aji13499-sup-0002-FigureS2.jpg40.4 KB FIGURE S2. Shown are the representative photomicrographic images of in vitro cultured embryos captured at magnification 400X. Shown are embryos of transferrable quality or top quality embryos. (A) 8-cell embryo (72 h), (B) Morula (96 h) and (C) Blastocyst (120 h). Embryo scoring criteria followed in all the three participating clinics were as described elsewhere.8, 9, 11 Source: AFC (Dr. Varsha Samson Roy, Clinical Embryologist). aji13499-sup-0003-FigureS3.jpg20.1 KB FIGURE S3. sHLA-G levels in low and high secretors from three embryonic developmental stages. Histograms showing comparison of (Cumulative) sHLA-G levels in E-SMs between low secretors (n = 288) and high secretors (n = 157). Statistical analysis was performed using unpaired Student's t-test (two-tailed). *Indicates statistical significance at P < .05. p - values: ****P < .0001. Abbreviations: E-SMs , embryo spent medium samples; sHLA-G, soluble human leukocyte antigen-G. aji13499-sup-0004-FigureS4.jpg51.4 KB FIGURE S4. Correlation of sHLA-G levels in E-SMs with embryo quality scores from three developmental stages. Histograms showing developmental stage-wise, grade-wise comparison of levels of sHLA-G in E-SMs (Ai) cleavage-stage, (Aii) morulae and (Aiii) blastocysts. Statistical analysis was performed using one-way ANOVA, followed by Tukey's post-hoc test (Ai) unpaired Student's t-test (Aii & Aiii) and correlation study was performed with Pearson correlation. *indicates statistically significance at P < .05. ** P < .01. # indicates across comparisons statistically insignificant. Abbreviations: E-SMs, embryo-spent medium samples; sHLA-G, soluble human leukocyte antigen-G; G1-G4, Grades of cleavage-stage, morulae, and blastocysts; Blastocysts grades: G3 , expanded blastocysts; G4, fully expanded blastocysts; ns , not statistically significant. aji13499-sup-0005-FigureS5.jpg42.7 KB FIGURE S5. Comparison of sHLA-G levels in E-SMs obtained from fresh or frozen-ET (FET) categories. Histograms showing comparison of sHLA-G levels between cleavage vs blastocyst (Ai) fresh-ETs (Aii) Frozen-ETs, and among three developmental stages (Bi) fresh -ETs (Bii) Frozen-ETs category. Statistical analysis was performed using Student's t-test (Figures Ai & Aii) and one-way ANOVA followed by Tukey's post-hoc test (Figures Bi & Bii). *Indicates statistical significance at P < .05. p-values: ***P < .001, ****P < .0001. # indicates across comparisons statistically insignificant. Abbreviations: E-SMs, embryo spent medium samples; sHLA-G , soluble human leukocyte antigen-G; ET, embryo transfer; ns, not statistically significant. aji13499-sup-0006-FigureS6.jpg66.7 KB FIGURE S6. Comparison of sHLA-G levels in pregnancy outcomes obtained from transferred embryos from three developmental stages. Histograms showing (A) % of pregnancy outcomes, (B) sHLA-G levels in pregnancy outcomes i.e., live-births vs no-births obtained from three developmental stages, that is, (Bi) cleavage-stage ET, (Bii) morulae - ET and (Biii) blastocyst - ET categories. Values of the pregnancy outcomes (%) in terms of live birth, aborted and no birth were 24.60, 11.11 and 64.29, respectively for the cleavage-ET category. 43.4, 7.54 and 49.06 for morulae-ET category and 56.86, 5.88 and 37.25 for blastocyst-ET category. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test (Bi to Biii). *Indicates statistical significance at P < .05. p-values: *P < .05. Abbreviations: sHLA-G, soluble human leukocyte antigen-G; ET, embryo transfer; ns, not statistically significant. aji13499-sup-0007-TableS1.docx15.5 KB Table S1 aji13499-sup-0008-TableS2.docx16.7 KB Table S2 aji13499-sup-0009-TableS3.docx14.6 KB Table S3