New approaches in the analysis of spent embryo culture media in the IVF process

Abstract

Purpose: In vitro fertilization occurs in a controlled laboratory setting, where oocytes are fertilized by sperm, and the resulting embryos are cultured to the blastocyst stage before transfer to the uterus. The secreted/consumed substances by the embryo in the extracellular environment (secretome) contain a variety of molecules that may provide insights into embryo quality. This study presents new perspectives on the non-invasive and cost-effective assessment and evaluation of embryos during the IVF process, utilizing a spent embryo culture medium (SECM).

Methods: The SECM was used from blastocysts prepared for a single blastocyst transfer and was analyzed in two groups-the SECM with successful (F) (n = 30) and unsuccessful (N) (n = 36) embryo implantation in the woman's uterus. Building on our previous next-generation sequencing results, we decided to validate the expression levels of specific miRNAs, particularly hsa-miR-16-5p and hsa-miR-92a-3p, to assess their potential to predict embryo implantation success.

Results: Our results demonstrate different expression levels of miRNA molecules in the monitored groups, which could lead to their use in non-invasive analysis of the implantation potential of embryos in the IVF process. In this study, we employed a metabolomics approach using 3D fluorescence analysis of SECM to identify differences between the studied groups, F and N. Our preliminary results indicate a slightly increased metabolic activity in the group with unsuccessful embryo implantation group.

Conclusions: This is our pilot study where we demonstrated the use of two approaches in analyzing the SECM to predict the implantation potential of embryos in the IVF process which promises further development.

Keywords: Fluorescence; Implantation; Metabolomics; Secretome; Spent embryo culture media; miRNA.

Fig. 1

Relative expression of miR-16-5p (A) and 92a-3p (B) in SECM of 4D/5D embryos in the group with successful (F) and unsuccessful embryo implantation (N). Values are normalized to the free culture medium (M) (*p < 0.05). Error bars show SEM

Fig. 2

Fluorescence profiles A. Fluorescence profiles of average SECM spectra of samples in PBS. The fluorescence profile of a medium diluted with PBS (1: 2500) features two fluorescence emission maxima at 223 and 280 nm, which are attributable to tyrosine and tryptophan, respectively. B. Fluorescence profiles of average SECM spectra of samples in PBS:DMSO. C. The additive profile represents the average values of all samples in given groups with division into zones 1–5. The curves represent the maximum values in individual groups. F, SECM of successfully implanted blastocyst; N, SECM of unsuccessfully implanted blastocyst, M, free culture medium

Fig. 3

Display of individual zones for given groups (F/N). A. Fluorescence of zones after subtraction of free culture medium in PBS. B. Fluorescence of zones after subtraction of free culture medium (M) in PBS:DMSO. C. Sum of respective zones in PBS and PBS:DMSO. The values given are the average of all samples. F, SECM of successfully implanted blastocyst; N, SECM of unsuccessfully implanted blastocyst

Fig. 4

Statistical analysis of the individual zones normalized to free culture medium with p < 0.0001 (****). F, SECM of successfully implanted blastocyst; N, SECM of unsuccessfully implanted blastocyst

Fig. 5

Correlation graph of the dependence of the ratios of individual zones for the given groups. F, SECM of successfully implanted blastocyst; N, SECM of unsuccessfully implanted blastocyst; M, free culture medium