P-262 Magnetic Resonance spectroscopy of individual mammalian embryos and human microtissues
Authors: Sivelli G , Conley G.M , Herrera C , Marable K , Rodriguez K.J , Bollwein H , Sudano M.J , Grisi M 
Published: June 2022

Abstract

Study question
Can magnetic resonance unravel the metabolic fingerprint of samples at the embryo scale?

Summary answer
We successfully utilized Magnetic Resonance Spectroscopy (MRS) on single mammalian embryos and microtissues and observed a diverse range of lipids in less than 1 hour.

What is known already
Non-invasive selection of the best embryo to transfer is one of the most significant challenges for ART professionals. The chemical sensitivity, resolving power, and, more importantly, the non-invasive nature of MRS makes it an excellent candidate to investigate the building blocks of complex organisms. Although MRS is a well-established technique for the biochemical profiling of large organisms, handling small samples like embryos and 3D cell cultures and spheroids alongside sensitivity issues has prevented its adoption for clinical and research applications. Our group has overcome these limitations with microchip-based sensors to leverage non-invasive MRS technology down to the embryo scale.

Study design, size, duration
A NASH in vitro model of liver microtissues was used first to detect lipid-based metabolic biomarkers over multiple time points in complex 3D cell cultures such as microtissues. We could discriminate between healthy and diseased human micro-livers based on their lipid content. Once the MRS data acquisition and analysis were set up using stimulated or control samples, we moved to the single early or late arrested bovine embryo analysis. The whole experiment lasted one month.

Participants/materials, setting, methods
Early or late arrested bovine embryos were obtained through IVF, cryopreserved and measured shortly after thawing. The microtissues were provided by InSphero AG (Schlieren, CH) and individually measured fresh in a time frame of 14 days based on 117 measurements. The micro-MRS device was loaded with culture medium and a single sample, then loaded into the MRS magnet. Measurements were performed for a total time of 50 min per embryo or 15 min per microtissue.

Main results and the role of chance
Micro-MRS efficiently identified four different biomarkers that were significantly differently expressed (p &lt; 0.05) according to the biological state of microlivers simulating non-alcoholic fatty liver disease (NAFL). Three of these markers represent relative concentrations of lipid signatures from fully saturated, mono and poly-unsaturated fatty acids. In addition, we analyzed in vitro produced pre-implantation bovine embryos naturally arrested at different developmental stages. This choice arose from the necessity to analyze them at different and fixed developmental stages to investigate our technology discrimination potential. The spectra obtained from the embryos present up to 6 peak regions assignable to fatty acids as observed in microtissues. Furthermore, the amplitude of the peaks varies substantially within the same group, i.e., within the morulae and within the two 2-cell stage arrested embryos. Using both microtissues and mammalian embryos, we demonstrated that micro-MRS could be used to investigate variations in the lipid content of tiny samples non-invasively. Generally, the majority of the analytical assays used in lipid metabolism investigations rely on invasive/destructive methods such as histology and biopsies, which would hinder the continuation of embryonic development. A non-invasive in vivo assay like the one presented here would provide the means to reveal the role of embryonic lipids throughout development.

Limitations, reasons for caution
A vital step towards establishing MRS as a clinical and research tool is the reliable detection of a wide range of signals from cellular components. Our method is ready for R&D studies, while its clinical application requires further safety studies and protocol optimization.

Wider implications of the findings
A non-invasive quick assay would provide the means to reveal the role of embryonic lipids throughout development. Micro-MRS can further develop into a safe embryo assay for selection before embryo transfer. This would apply to both human and animal ART, whose success rate is relatively low.

Trial registration number
not applicable