Predictive potential of combined secretomics and image-based morphometry as a non-invasive method for selecting implanting embryos

Background
Non-invasive selection of human embryos for in vitro fertilization purposes is still a major challenge to pursue. Therefore, this study aims to identify non-invasive morphometric and secretomic parameters that reliably select the embryos with the highest likelihood of implantation prior to embryo transfer (ET).

Methods
Prospective single-centre cohort study. Thirty-two day 5 blastocysts derived from 28 couples undergoing intracytoplasmic sperm injection (ICSI) and ET between January 2023 and April 2023. Patients were split according to their implantation outcome, confirmed with serum beta-human chorionic gonadotropin (b-hCG) levels > 5 mIU/mL nine days post-SET. Ninety-two proteins involved in embryonic developmental programming were measured in spent blastocyst media (SBM) using a protein extension assay. Sparse PLS-DA (sPLS-DA) was used for principal component analysis. Forty-seven morphometric parameters related to the trophoblast, inner cell mass and blastocele dimension were evaluated in microphotographs of day 5 embryos with ImageJ software.

T-test and Mann–Whitney tests were respectively used to compare morphometric measurements and normalized expression of secreted protein (NPx) levels between embryos that implanted or not. Predictive value of models of implantation based on embryo morphometric parameters and secreted proteins.

Results
Chi-squared tests showed no significant differences in transferred blastocyst stage, quality, and state between subgroups. Implanting blastocysts (n = 14) presented significantly different morphometric shape descriptors (i.e., internal circularity, internal roundness, internal axis ratio, internal angle and trophoblast mean width) than non-implanting blastocysts (n = 13). Among the quantifiable proteins (86/92) in SBM from eleven implanting and nine non-implanting blastocysts, NPx and sPLS-DA analysis revealed three differentially expressed proteins. Matrilin-2 (MATN2) and legumain (LGMN) were significantly elevated (p < 0.01 in both cases) while thymosin beta-10 (TMSB10) was significantly decreased (p < 0.05) in implanting embryos. Predictive models based exclusively on morphometric or secreted protein profiles accurately discriminated implantation outcomes (AUC > 0.71). The model integrating the blastocysts’ internal circularity, internal roundness, internal axis ratio and the NPx of MATN2 and TMSB10 in SBM had exceptional negative and positive predictive power for implantation outcomes (100% and 90.91%, respectively; AUC = 0.93).

Conclusions
Morphometric shape descriptors and NPx levels of MATN2 and TMSB10 in SBM emerge as promising candidate markers for non-invasive embryo selection.