The integrity of cfDNA in follicular fluid and spent medium from embryo culture is associated with embryo grade in patients undergoing in vitro fertilization.

PURPOSE: This study was conducted to verify if the cfDNA integrity (cfDI) in follicular fluid and subsequent spent embryo medium (SEM) could serve as potential non-invasive biomarker for high-grade embryo selection during IVF/ICSI. METHODS: Thirty-two follicular fluids, 32 subsequent corresponding cleavage embryo SEM, and 23 subsequent blastocyst SEM were collected from 11 patients undergoing IVF/ICSI. CfDI was measured by ALU gene amplicons with different sizes by qPCR, as the ratio of long to short fragments. RESULTS: CfDI in follicular fluid corresponding to subsequent high-grade cleavage embryos and blastocysts was significantly lower than that related to low-grade embryos (p = 0.018). Conversely, cfDI in SEM was significantly and positively correlated with high-grade embryos at both stages (p = 0.009). ROC curves of the analysis of cfDI in follicular fluid showed great potential in predicting subsequent embryogenesis and embryo grade (AUC > 0.927). Regardless of the cleavage embryo grade by morphology, cfDI in day 3 SEM could predict if the cleavage embryo could develop to a high-grade blastocyst (AUC = 0.820). A concordant shift pattern of cfDI from follicular fluid to subsequent day 3 SEM and day 5 SEM was found in 81.82% participants featured by various clinical characteristics. CONCLUSION: CfDI in follicular fluid and SEM was significantly correlated with embryogenesis and embryo grade and could serve as a potential non-invasive biomarker in high-grade embryo selection. Direct qPCR was proved as a labor-saving and sensitive method for the analysis of cfDI in low volume of SEM.

Agarose amplification based sequencing characterization cell-free RNA in preimplantation spent embryo medium.

BACKGROUND: The cell-free RNA (cf-RNA) of spent embryo medium (SEM) has aroused a concern of academic and clinical researchers for its potential use in non-invasive embryo screening. However, comprehensive characterization of cf-RNA from SEM still presents significant technical challenges, primarily due to the limited volume of SEM. Hence, there is urgently need to a small input liquid volume and ultralow amount of cf-RNA library preparation method to unbiased cf-RNA sequencing from SEM. (75) RESULT: Here, we report a high sensitivity agarose amplification-based cf-RNA sequencing method (SEM-Acf) for human preimplantation SEM cf-RNA analysis. It is a cf-RNA sequencing library preparation method by adding agarose amplification. The agarose amplification sensitivity (0.005 pg) and efficiency (105.35 %) were increased than that of without agarose addition (0.45 pg and 96.06 %) by âˆ¼ 90 fold and 9.29 %, respectively. Compared with SMART sequencing (SMART-seq), the correlation of gene expression was stronger in different SEM samples by using SEM-Acf. The cf-RNA number of detected and coverage uniformity of 3' end were significantly increased. The proportion of 5' end adenine, alternative splicing events and short fragments (<400 bp) were increased. It is also found that 4-mer end motifs of cf-RNA fragments was significantly differences between different embryonic stage by day3 spent cleavage medium and day5/6 spent blastocyst medium. (141) SIGNIFICANCE: This study established an efficient SEM amplification and library preparation method. Additionally, we successfully described the characterizations of SEM cf-RNA in preimplantation embryo using SEM-Acf, including expression features and fragment lengths. SEM-Acf facilitates the exploration of cf-RNA as a noninvasive embryo screening biomarker, and opens up potential clinical utilities of small input liquid volume and ultralow amount cf-RNA sequencing. (59).