A Preclinical Evaluation towards the Clinical Application of Oxygen Consumption Measurement by CERMs by a Mouse Chimera Model

Abstract

We have developed an automated device for the measurement of oxygen consumption rate (OCR) called Chip-sensing Embryo Respiratory Measurement system (CERMs). To verify the safety and the significance of the OCR measurement by CERMs, we conducted comprehensive tests using a mouse model prior to clinical trials in a human in vitro fertilization (IVF) program. Embryo transfer revealed that the OCR measured by CERMs did not compromise the full-term development of mice or their future fertility, and was positively correlated with adenosine triphosphate (ATP) production and the mitochondrial membrane potential (ΔΨm), thereby indirectly reflecting mitochondrial oxidative phosphorylation (OXPHOS) activity. We demonstrated that the OCR is independent of embryo morphology (the size) and number of mitochondria (mitochondrial DNA copy number). The OCR correlated with the total cell numbers, whereas the inner cell mass (ICM) cell numbers and the fetal developmental rate were not. Thus, the OCR may serve as an indicator of the numbers of trophectoderm (TE) cells, rather than number or quality of ICM cells. However, implantation ability was neither correlated with the OCR, nor the embryo size in this model. This can probably be attributed to the limitation that chimeric embryos contain non-physiological high TE cells counts that are beneficial for implantation. CERMs can be safely employed in clinical IVF owing to it being a safe, highly effective, non-invasive, accurate, and quantitative tool for OCR measurement. Utilization of CERMs for clinical testing of human embryos would provide further insights into the nature of oxidative metabolism and embryonic viability.

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development

“…6 A,B). This agrees with a recent report from Kuno et al, in which chimeric mouse blastocyst OCR did not correlate with mtDNA copy number 23 . In this study, mtDNA copy number was not correlated to icO 2 in the samples measured.…”

“…However future use of an immunofluorescence approach using antibody markers of the ICM-specific Sox2 and TE-specific Cdx2 could improve specificity and reduce subjectivity 55 – 57 . Kuno et al recently reported a positive correlation between chimeric mouse blastocyst OCR measured using a chip-based electrochemical method and increasing total cell count measured by this immunocytochemical approach 23 .…”

“…Contemporary and emerging methods include the Unisense Nanorespirometer 15 , 18 , the self-referencing electrode 19 and bespoke electrochemical devices 20 , 21 . Recent developments include combining electrochemistry with microfluidic technologies 22 , 23 for embryo-compatible devices. Obeidat and colleagues 24 report detecting oxygen consumption alongside glucose consumption, lactate production and pH in individual equine blastocysts using multiple electrochemical sensors.…”

Exploration of the Cytoplasmic Function of Abnormally Fertilized Embryos via Novel Pronuclear-Stage Cytoplasmic Transfer

“…The measurement of OCR was performed according to previous studies using a Chip-sensing Embryo Respiration Monitoring system (CERMs) [21,22]. Briefly, preheated Cook ®® Sydney IVF Follicle Flushing Buffer (COOK Medical Australia Pty Ltd., Brisbane, Australia) supplemented with 10% Serum Substitute Supplement (SSS™; Irvine Scientific, Santa Ana, CA, USA) served as the measuring medium.…”

“…Immunocytochemical procedures were performed as previously described [22]. In brief, embryos were fixed in 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) and 0.1% Tween-20 (Sigma-Aldrich, Saint Louis, MO, USA).…”