Non-invasive molecular assessment of human embryo development and implantation potential
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
References (20)
- et al.
Anti-EpCAM modified LC-SPDP monolayer on gold microelectrode based electrochemical biosensor for MCF-7 cells detection
Biosens. Bioelectron.
(2013) - et al.
The biological function of hyperglycosylated hCG
Asian Pac. J. Reprod.
(2012) - et al.
Hyperglycosylated hCG (Invasive Trophoblast Antigen, ITA) a key antigen for early pregnancy detection
Clin. Biochem.
(2003) - et al.
Evaluation of in vitro fertilization outcomes using interleukin-8 in culture medium of human preimplantation embryos
Fertil. Steril.
(2017) - et al.
LIF and TNF alpha concentrations in embryo culture media are predictive for embryo implantation in IVF
Asian Pac. J. Reprod.
(2012) - et al.
Time-lapse systems for embryo incubation and assessment in assisted reproduction
Cochrane Database Syst. Rev.
(2019) - et al.
Wearable sweat sensors
Nat. Electron.
(2018) - et al.
Human chorionic gonadotropin (hCG) in the secretome of cultured embryos: hyperglycosylated hCG and hCG-free beta subunit are potential markers for infertility management and treatment
Reprod. Sci.
(2013) - et al.
Prediction of embryo implantation potential by mass spectrometry fingerprinting of the culture medium
Reproduction
(2013) - et al.
Electrochemical immunosensor for TNFα-mediated inflammatory disease screening
ACS Chem. Neurosci.
(2019)
Cited by (10)
A novel label-free electrochemical immunosensor for detection of surfactant protein B in amniotic fluid
2023, TalantaCitation Excerpt :After modifying the surface of the electrode with sulfo-LC-SPDP, a decrease of the oxidation peak current was observed, suggesting the binding of the cross-linker to the surface. This phenomenon is also obtained in the subsequent functionalization steps (anti-SPB and BSA immobilization), since it act as a diffusional barrier to the electron transfer between the redox probe and the electrode surface [31], indicating successful immobilization of the biomolecules. The EIS method was used to investigate the electron transfer on the interface of the gold electrode along the different modifications of its surface.
Microphysiological stem cell models of the human heart
2022, Materials Today BioCitation Excerpt :Other approaches include collecting tissue-conditioned media for biomarker detection or recent impedimetric immunosensors with antibody coated electrodes which transmit electrical signals when biomarkers or pathogens bind to them [112]. These methods are currently used for quality control of embryos derived from in vitro fertilization where invasive cell sampling carries high risk [113–116]. Clinical cardiac biomarkers [117,118] can also be used on conditioned media and all of these non-invasive methods allow long-term monitoring of live tissues.
Are we approaching automated assisted reproductive technology? Embryo culture, metabolomics, and cryopreservation
2021, F and S ReviewsCitation Excerpt :A number of target biomarkers have been investigated and related to embryo development, such as human chorionic gonadotropin (hCG) (79), Apolipoprotein A1 (80), human leukocyte antigen-G (81, 82), granulocyte-macrophage colony-stimulating factor (83), pregnancy-specific beta-1 glycoprotein (84), alpha interferone 2 (85), and platelet activating factor (86). In a recent work, using electrochemical impedance spectroscopy, hCGβ, IL-8, and tumor necrosis factor α (TNFα) were detected from culture media of single human embryos: nonviable embryos produced higher levels of IL-8 and TNFα; interestingly, hCGβ levels were able to discriminate between morphologically identical viable embryos (68). Another recent study used a bead-based digital microfluidic system to simultaneously measure IL-1β and TNFα in a single 520-nL droplet of culture medium in ≤40 minutes (87).
Nanotechnologies in Obstetrics and Cancer during Pregnancy: A Narrative Review
2022, Journal of Personalized MedicineImpedimetric Immunosensing for Neuroinflammatory Biomarker Profiling
2022, Neuromethods