Nuclear Magnetic Resonance Metabolomic Analysis of Spent Human Embryo Culture Media: Method Validation and Technical Considerations

Summary

Here, we present a protocol for collecting, preparing, and analyzing spent human embryo culture medium using nuclear magnetic resonance (NMR) spectroscopy, enabling reproducible detection of low-molecular-weight metabolites from microliter-scale samples under routine clinical culture conditions.

Abstract

Spent human embryo culture media contains low-molecular-weight metabolites that can provide a non-invasive readout of preimplantation embryo physiology. However, nuclear magnetic resonance (NMR)-based metabolomic analysis of these samples is technically challenging because routine embryo culture conditions involve microliter volumes, protein supplementation, and paraffin oil overlays that generate background signals and compromise spectral quality. Here, we present an optimized NMR metabolomics workflow for spent embryo culture media incorporating controlled droplet collection to minimize oil contamination, fluorinated ethylene propylene (FEP) tube liners to chemically isolate the sample from the external reference (TMSP) while accommodating microliter volumes, and acquisition using a Carr-Purcell-Meiboom-Gill pulse sequence to suppress macromolecular background signals. The protocol enables consistent detection and quantification of low-molecular-weight metabolites from individually cultured human embryos and supports reproducible analysis under routine laboratory conditions. Due to numerous technical challenges, the method must be properly validated before it can be used for research or clinical purposes.

Introduction

During preimplantation development in vitro., embryos continuously consume and release low-molecular-weight metabolites into the surrounding medium. The composition of spent embryo culture medium (SECM), therefore, reflects embryo metabolic activity and provides a non-invasive functional readout of embryo physiology and developmental competence^1,2. Analysis of this metabolic footprint may complement conventional assessment methods and improve embryo selection strategies in assisted reproductive technology^3,4. In particular, alterations in amin....

Protocol

This study was conducted between January and April 2023 at the Department of Reproductive Medicine and Gynaecological Endocrinology, University Medical Centre Maribor, Slovenia. NMR analysis was performed at the National Institute of Chemistry in Ljubljana, Slovenia. Written informed consent was obtained from all participants. It received approval from both the Institutional Ethics Committee (No: UKC-MB-KME-1/21) and the National Medical Ethics Committee (No: 0120-37/2021/15).

1. Study design

NOTE: The study analyzed human spent embryo culture media using NMR-based metabolomics.

1. Colle....

Results

The analytical workflow for untargeted NMR metabolomic profiling of spent embryo culture medium is summarized in Figure 1.

Removal of oil contamination during sampling
Figure 2 illustrates the sequential droplet transfer procedure used to reduce overlay oil carryover. Samples collected without careful pipetting exhibited broad hydrocarbon resonances spanning the aliphatic region of the spectrum (δ 0.5–2.3 ppm), resulting in substantial baselin.......

Discussion

In this study, we addressed key analytical challenges associated with identifying and quantifying low-molecular-weight metabolites in spent embryo culture media using untargeted NMR metabolomics. Our results show that the optimized workflow enables acquisition of high-quality, information-rich spectra suitable for reliable detection of low-molecular-weight metabolites.

Careful sequential droplet transfer proved essential for reducing oil contamination, which otherwise generated broad resonance.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5 mm FEP tube liner	SP Wilmad-LabGlass	4682701	Inner insert for coaxial NMR
5 mm NMR tube	SP Wilmad-LabGlass	535-PP-7-SB	Outer tube
6-well culture plate	Oosafe	OOPW-SW02	Embryo culture dish
Cryogenic liquid nitrogen	Messer	Contact manufacturer	Sample snap-freezing
Deuterium oxide (D2O)	Eurisotop	D214L	NMR solvent
Micropipette (10–100 µL)	Eppendorf	4924000053	Adjustable volume pipette
Microcentrifuge tubes, 1.5 mL, RNase-free	Eppendorf	N216966N	Sample collection and storage
Mineral oil	Vitrolife	VTL-10029	Culture droplet overlay
NMR spectrometer, 600 MHz	Bruker	https://www.bruker.com/en/products-and-solutions/mr/nmr/avance-nmr-spectrometer.html	Spectral acquisition
Pasteur pipette	Falcon	FAL-357575	Droplet transfer
Pipette, single-channel mechanical	Sartorius	728060	Sample handling
Pipette tips, sterile	Eppendorf	22491148	Compatible with micropipette
Pipette tips, sterile filter	Sartorius	790201F	Aerosol barrier
Sequential G-series culture media	Vitrolife	VTL-10143	Embryo culture medium
TMSP-d4 (sodium 3-trimethylsilylpropionate-d4)	Cambridge Isotope Laboratories	DLM-48-1	Chemical shift reference
Tubes, Safe-Lock	Eppendorf	0030121589	Storage tubes