Nuclear Magnetic Resonance (NMR) is a technique of tremendous structural power and innumerable advantages from an analytical point of view. Perhaps its greatest disadvantage is its low sensitivity when compared to techniques such as mass spectroscopy that are capable of detecting analytes in the part per billion and even part per trillion concentration range. To make up for this certain disadvantage, technology has brought to the market the so-called cryoprobes, which allow MRI to enter the clinical analytical world. In fact, more and more studies arise from the combination of both techniques that, when applied in a complementary manner, have led to the understanding of a large number of systems of biological, clinical or synthetic origin. The present study focuses on the application of this powerful technique to the study of D+2, D+3 and D+5 embryo culture media, with the aim of predicting embryo selection, at the time of transfer, which will directly lead to an improvement in the implantation rate, pregnancy and live newborn rate.
To build a new methodological and instrumental tool that combines NMR spectroscopy and multivariate analysis chemometric techniques for its application in predicting embryo implantation.
Materials and methods.
Methods: This is a multicenter, multidisciplinary study with a high analytical component, with a random selection and allocation of culture media. The contribution of the cases/sample is made by assisted reproduction clinics (private and public) from equidistant geographical points, providing a sample size of more than 500 cases/sample, as well as a wide range of ages and races, which will generate a high sample variability. The media used for embryo development have been the usual ones used in the clinical practice of any human assisted reproduction center, but both for culture or embryo development with sequential media and with single media (one-step). The quality of the pre-embryo is determined by visualization of its morphological characteristics, associating the highest implantation with the blastocyst stage. The culture medium, free of the pre-embryo, after incubation period (D+2: 43-45 hours post-insemination; D+3: 67-69 hours post-insemination; D+5: 114-118 hours post-insemination and/or D+6: 136-140 hours post-insemination), is thoroughly collected (20uL) and transferred to cryovials at -80 ºC. For NMR measurements, 400 uL of NaCl solution (0.09%) in D2O is added to each cryovial. Measurements are carried out on a Bruker Avance III 600 spectrometer equipped with a quadruple cryoprobe and using the 1H CPMG sequence. The characterization of the culture medium has been possible after the acquisition of one- and two-dimensional TOCSY, COSY, HMQC and HMBC experiments. The obtained spectra were processed through TOPSPIN 3.2 software (Bruker BioSpin GmbH) and then subjected to statistical analysis using SIMCA-P software (v. 14.0, Umetrics). Finally, several unsupervised and supervised multivariate data analysis techniques such as PCA, PLS, OPLS were applied on the NMR data for the identification of discriminant metabolites (biomarkers) of embryo development and embryo quality and pregnancy.
Results: After applying an OPLS-DA (Orthogonal Projections to Latent Structures Discriminant Analysis) model to the NMR data (Figure 1), a discrimination between embryo cultures that have generated positive pregnancy (blue) and negative pregnancy (green) has been observed. The loadings graph allows observing the spectral zones containing metabolites that exist in higher concentration in the samples of embryo cultures that have generated positive (blue) or negative (green) pregnancy. The green and blue dots in the loadings graph shown in Figure 1b show the spectral regions that cause differentiation and from whose structural identification the discriminating biomarkers or metabolites will be obtained.
The preliminary study yields a good classification of culture media of pre-embryos cultured both at D+2 and up to the blastocyst stage (D+5/D+6), which allows us to be optimistic about obtaining a solid pre-implantation prediction model with verified efficacy. In the future, we plan to add new cases to the study, which will allow us to obtain a more robust model and a more accurate classification of the metabolic biomarkers associated with a positive pregnancy that will improve the efficiency rate of IVF techniques and especially the number and quality of blastocysts obtained.
AUTHORS: A. C. Ralha de Abreu , C. González Navas, A. Gómez García, I. Fernández, M. A. Vilches Ferrón