Non-invasive molecular assessment of human embryo development and implantation potential

https://doi.org/10.1016/j.bios.2020.112144Get rights and content

Highlights

  • Impedimetric immunosensor for the quantitative quality assessment of single human embryos.

  • Quantification of chorionic gonadotropin, interleukin-8 and tumor necrosis factor alpha in human embryo conditioned media.

  • SPE EIS immunosensors achieve lower detection limits than commercial enzyme-linked immunosorbent assays.

  • Blastocysts with the same morphological grading have different molecular profiles.

Abstract

In vitro fertilization (IVF) is the most common assisted reproductive technology used to treat infertility. Embryo selection for transfer in IVF cycles relies on the morphological evaluation by embryologists, either by conventional microscopic assessment or more recently by time-lapse imaging systems. Despite the introduction of time-lapse imaging improvements in IVF success rates have failed to materialize, therefore alternative approaches are needed. Recent studies have shown that embryos resulting in successful pregnancy differ in their secretome and metabolism compared to embryos that fail to implant, suggesting that molecular analysis of embryo culture medium could assist in non-invasive single embryo selection. However, this approach has yet to be adopted clinically due to the lack of appropriate highly sensitive screening technologies needed to assess volume-limited samples. Here we report the detection of hCGβ, IL-8 and TNFα from conditioned culture media of single human embryos using electrochemical impedance spectroscopy. The impedimetric immunosensors revealed that morphologically non-viable embryos produce higher levels of IL-8 and TNFα, associated with abnormal cell division and cell death, respectively. More importantly, hCGβ detection was able to discriminate apparently morphologically identical viable embryos. This work brings an objective dimension to embryo selection, which could overcome the major limitations of morphology-based embryo selection for implantation. Future work should include the validation of these biomarkers in a large patient cohort.

Introduction

In vitro fertilization (IVF) is the most common form of assisted reproductive technology (ART) that has enabled effective treatment of millions of infertile couples worldwide. However, embryo implantation failure remains the rate-limiting step in IVF, as only ~35% of transferred embryos successfully implant (Armstrong et al., 2019). Embryo selection is determined using conventional or time-lapse imaging systems using standardized grading systems which offer no difference in IVF outcomes (Armstrong et al., 2019). As successful implantation requires the physical and biochemical interplay between the embryo and a receptive endometrium, the identification of biochemical signatures suggestive of invasion potential, as well as embryo viability at the blastocyst stage, could be used for the elective single embryo selection.

Molecular biomarker detection in embryo-conditioned media holds great promise for non-invasive analysis of embryo viability which might reflect physiological differences between embryos with the same morphological grading. Quantification of human chorionic gonadotropin (hCG) in embryo culture media has been suggested as a powerful indicator of trophectoderm differentiation and blastocyst viability as it acts as an important autocrine factor mediating and promoting trophoblast cell differentiation, invasion and placental growth (Butler et al., 2013). Conversely, human preimplantation embryos that secrete high levels of tumor necrosis factor alpha (TNFα) result in implantation failure (Zollner et al., 2012), possibly due to increased cell apoptosis and repressed inner cell mass development (Pampfer et al., 1994; Wuu et al., 1999). Interleukin-8 (IL-8) has been associated with the activation of cellular senescence mechanisms triggered by persistent DNA damage signaling in different model systems (Rodier et al., 2009), suggesting that it could serve as a potential predictor of abnormal cell cycle events.

To date, molecular profiling to identify biomarkers in embryo-conditioned media has been performed using techniques such as mass spectrometry (Cortezzi et al., 2013; Iles et al., 2019) and HPLC (Picton et al., 2010), however such technologies have not been used to quantify markers as part of a selection process. More effective, point-of-use and lower-cost technologies, with low sample volume requirements and low limits of detection, are required for the selection of embryos with the greatest developmental competence.

Here we show that gold electrochemical immunosensors can be used for the parallel quantification of hCGβ, IL-8 and TNFα in the conditioned media of day five human embryos of differing quality. In this proof-of-concept study, we show that an exacerbated inflammatory profile is exhibited in poor quality embryos, with elevated cellular degeneration and irregular cell division, and that total hCGβ levels were able to discriminate viable embryos with the same morphological grade. Future validation in a larger cohort is necessary to establish the optimal biomarker levels predictive of successful implantation.

Section snippets

Immunosensor functionalization

Biomarker quantification was performed using gold screen-printed electrodes (SPEs, DropSens, C223AT). The SPEs comprise one gold working electrode (WE) (1.6 mm diameter), a silver reference electrode (RE) and a gold counter electrode (CE). Electrodes were precleaned with isopropanol and deionized (DI) water, prior to functionalization. The WEs were further incubated with a 10 mg/mL solution of sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)propionamido)hexanoate) (ThermoFisher

EIS-based immunosensor for sensitive and selective biomarker detection

To evaluate secreted hCGβ, IL-8 and TNFα levels in culture media obtained from day five of embryo in vitro culture as part of an IVF procedure, we developed three independent SPEs with immuno-functionalized WEs to transduce the biomarker concentrations into electrochemical signals (Fig. 1). Biomarker detection was performed by electrochemical impedance spectroscopy (EIS) with impedance determined by applying a varying sinusoidal potential over a range of frequencies and measuring resultant

Conclusion

Here we have developed a simple biosensor screening methodology based on the quantification of three molecular markers (hCGβ, IL-8 and TNFα) associated with pregnancy outcomes to complement the currently performed embryo selection based on morphology. The targeted detection of hCG, IL-8 and TNFα is an area of much interest with EIS, differential pulse voltammetry (DPV) and field effect transistors (FET) immunosensors reported (Table S2). However, these systems have not been applied in the

CRediT authorship contribution statement

Catarina M. Abreu: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing - original draft, Writing - review & editing. Victoria Thomas: Resources, Validation, Writing - review & editing. Paul Knaggs: Resources, Validation, Writing - review & editing. Adnan Bunkheila: Resources, Writing - review & editing. Andrea Cruz: Investigation, Formal analysis. Sofia R. Teixeira: Formal analysis. Pedro Alpuim: Formal analysis, Writing - review & editing. Lewis

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors acknowledge Helena Sofia Domingues, Diogo Castro Fernandes and Jérôme Borme for critical reading of the manuscript, Katie Boothby and Rachel Walker for the preparation and shipment of the samples and the INL-Swansea University PhD program for mentoring to C.M.A. I.M.P. and R.S.C. acknowledge the financial support from the Marie Curie COFUND Programme “NanoTRAINforGrowth” from the European Union's Seventh Framework Programme for research, technological development and demonstration

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