The search for biomarkers of human embryo developmental potential in IVF: a comprehensive proteomic approach

Abstract

The objective of these studies was to identify differentially expressed peptides/proteins in the culture media of embryos grown during in vitro fertilization (IVF) treatment to establish their value as biomarkers predictive of implantation potential and live birth. Micro-droplets of embryo culture media from IVF patients (conditioned) and control media maintained under identical culture conditions were collected and frozen at -80°C on Days 2-3 of in vitro development prior to analysis. The embryos were transferred on Day 3. The peptides were affinity purified based on their physico-chemical properties and profiled by mass spectrometry for differential expression. The identified proteins were further characterized by western blot and ELISA, and absolute quantification was achieved by multiple reaction monitoring (MRM). We identified up to 14 differentially regulated peptides after capture using paramagnetic beads with different affinities. These differentially expressed peptides were used to generate genetic algorithms (GAs) with a recognition capability of 71-84% for embryo transfer cycles resulting in pregnancy and 75-89% for those with failed implantation. Several peptides were further identified as fragments of Apolipoprotein A-1, which showed consistent and significantly reduced expression in the embryo media samples from embryo transfer cycles resulting in viable pregnancies. Western blot and ELISA, as well as quantitative MRM results, were confirmatory. These results demonstrated that peptide/protein profiles from the culture medium during early human in vitro development can discriminate embryos with highest and lowest implantation competence following uterine transfer. Further prospective studies are needed to establish validated thresholds for clinical application.

Non‐Invasive Assessment of the Embryo Viability via the Analysis of the Culture Media

“…With the emerging of sensitive and speciic new analytical techniques, it is possible to carry out a comprehensive analyses of the surrounding environment of pre-implantation embryos [21]. These molecular proiles are supposed to utilize with high accuracy the diferentiation of viable and non-viable embryos [22]. The identiication of new biomarkers of the embryonic secretome can result in signiicant improvements in the eiciency of IVF cycles, increasing pregnancy rate per transfer and decreasing the costs of the procedure.…”

“…It was found that the level of apolipoprotein A1 correlates with blastocyst grade, but it does not correlate with implantation and pregnancy rates. Contradictory to those indings, Nyalwidhe et al [22] used MS, Western-blot, and ELISA to identify 14 diferentially regulated peptides that were then used to generate genetic algorithms being able to identify embryo transfer cycles resulting in pregnancy and cycles with failed implantation. These genetic algorithms were able to recognize with 71-84% accuracy embryo transfer cycles, which resulted in pregnancy.…”

Predictive Roles of Proteomic Profiles in Assisted Reproduction-An Update

“…Improving assessment methods of embryonic viability will not only improve ART success rates, but will also lead to the transfer of single, rather than multiple embryos. Transferring multiple embryos in ART exposes patients to various maternal and fetal morbidities [16,48,52].…”

“…The ideal biomarker must be safe, easily, and accurately identified in a cost-effective and noninvasive or minimally-invasive manner [15]. In ART, biomarkers should have a significant potential of predicting fertilization, implantation and embryonic viability [16]. Proteomics is thus the key to improving ART success rates by identifying, quantifying, and characterizing protein profiles of the key players that determine ART success rates.…”

Raman spectrum: A potential biomarker for embryo assessment during in vitro fertilization

“…Accordingly, many different approaches have been suggested to reflect the embryo's function or physiological state. Several non-invasive methods assessing embryo viability and the potentiality of development have been established, such as proteomics (6), measurement of respiration rate (7), soluble human leukocyte antigen-G (8), pyruvate uptake (9) and glucose uptake (10). However, all of these methods have not been widely used in clinic because they were either expensive and required dedicated equipment, technical staff or do not provide results quickly enough to be used within the time frame of clinical IVF.…”