A simple and rapid flow cytometry-based assay to identify a competent embryo prior to embryo transfer

Abstract

Multiple pregnancy is a risk for prematurity and preterm birth. The goal of assisted reproduction is to achieve a single pregnancy, by transferring a single embryo. This requires improved methods to identify the competent embryo. Here, we describe such a test, based on flow cytometric determination of the nucleic acid (PI+) containing extracellular vesicle (EV) count in day 5 embryo culture media. 88 women undergoing IVF were included in the study. More than 1 embryos were transferred to most patients. In 58 women, the transfer resulted in clinical pregnancy, whereas in 30 women in implantation failure. In 112 culture media of embryos from the "clinical pregnancy" group, the number of PI+ EVs was significantly lower than in those of 49 embryos, from the "implantation failure" group. In 14 women, transfer of a single embryo resulted in a singleton pregnancy, or, transfer of two embryos in twin pregnancy. The culture media of 19 out of the 20 "confirmed competent" embryos contained a lower level of PI+ EVs than the cut off level, suggesting that the competent embryo can indeed be identified by low PI+ EV counts. We developed a noninvasive, simple, inexpensive, quick test, which identifies the embryos that are most likely to implant.

Figure 1. Annexin V staining of embryo-derived EVs in embryo culture medium.

(a) Representative FSC-SSC dot plot shows the size distribution of EVs in embryo culture medium. (b) Representative dot plot shows the phycoerythrin (PE) fluorescence of EVs after AnnexinV-PE labelling. (c) Representative dot plot shows the PE fluoresce after Triton-X 100 differential detergent lysis.

Figure 2. Propidium iodide (PI) staining of embryo-derived EVs in embryo culture medium.

(a) Representative FSC-SSC dot plot shows the size distribution of EVs in embryo culture medium. (b) Representative dot plot shows the autofluorescence of embryo culture medium in FL2 channel (embryo culture medium + 4% formaldehyde solution +PI, without EVs. (c) Representative dot plot shows the PI fluorescence of 4% formaldehyde fixed embryo-derived EVs in embryo culture medium.

Figure 3. Propidium iodide (PI)+ EVs in culture medium of embryos resulting in clinical pregnancy or implantation failure.

***P < 0.001.

Figure 4. Propidium iodide (PI) staining of embryo-derived EVs in embryo culture medium.

Two transferred embryos resulting in singleton pregnancy. (a) Representative FSC-SSC dot plot shows the size distribution of EVs in embryo culture medium. (b,c) dot plots show the PI fluorescence of EVs transferred to the same mother.

Figure 5. Propidium iodide (PI) staining of embryo-derived EVs in embryo culture medium.

Two transferred embryos resulting in pregnancy failure. (a) Representative FSC-SSC dot plot shows the size distribution of EVs in embryo culture medium. (a,c) dot plots show the PI fluorescence of EVs transferred to the same mother.

Figure 6. Propidium iodide (PI) staining of embryo-derived EVs in embryo culture medium.

Two transferred embryos, resulting in twin pregnancy. (a) Representative FSC-SSC dot plot shows the size distribution of EVs in embryo culture medium. (b,c) dot plots show the PI fluorescence of EVs transferred to the same mother.

Figure 7. Culture media of embryos from clinical pregnancies with higher (N = 48) or lower (N = 64) number of PI+ EVs.

***p < 0.001 ****p < 0.0001.

Figure 8. Evaluation of optimal cut-off score and the diagnostic ability of test by ROC analysis.

(a) Data of confirmed competent embryos are plotted in function of data from implantation failure. (b) Data from presumed competent embryos (giving the lowest PI+ EV values among embryos from the same mother) are plotted against data from implantation failure. (c) Data from presumed competent embryos are plotted against data from presumed incompetent embryos in the clinical pregnancy group, plus data from the implantation failure group.