Quantitative analysis of piwiRNAs in the culture medium of euploid and aneuploid blastocysts as an additional method of selecting a high-quality embryo for transfer to the uterine cavity in assisted reproductive technology programs

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Abstract

Objective: To identify piwiRNAs in the culture medium of a blastocyst associated with cell ploidy and the implanting ability of an embryo after transfer into the uterine cavity.

Materials and methods: The study included 73 couples whose culture medium of 93 embryos was analyzed; among them there were 53 euploid blastocysts (according to the results of PGT-A) with implantation (27 embryos) and without implantation (26 embryos) after transfer of cryopreserved embryos into the uterine cavity in ART programs using cyclic hormone therapy; 40 blastocysts were aneuploid. piwiRNAs were isolated from 20 µl of the culture medium with the miRNeasy Serum/Plasma Kit (Qiagen) and analyzed using deep sequencing on the NextSeq 500/550 platform (Illumina, USA). The obtained data were subsequently validated by quantitative polymerase chain reaction in real time with the miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany).

Results: Two logistic regression models were built after quantification of hsa_piR_020497 and hsa_piR_020829, or hsa_piR_016677 and hsa_piR_020829, which have 93% and 100% specificity, respectively, in identifying euploid blastocysts with high implantation potential. The miRanda algorithm was used to analyze potential target genes of piwiRNAs associated with aneuploidy, which are involved in the formation of the spindle apparatus, the development and functioning of kinetochore, and cytokinesis.

Conclusion: A noninvasive method of selecting euploid embryo with a high implantation potential for transfer into the uterine cavity in ART programs was developed. At this stage of the study, this method is additional but not alternative to the PGT-A method.

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About the authors

Angelika V. Timofeeva

Academician V.I. Kulakov National Medical Research Centre for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia

Author for correspondence.
Email: v_timofeeva@oparina4.ru

Ph.D. (Bio), Head of the Laboratory of Applied Transcriptomics

Russian Federation, 117997, Moscow, Academician Oparin str., 4

Ivan S. Fedorov

Academician V.I. Kulakov National Medical Research Centre for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia

Email: is_fedorov@oparina4.ru

Researcher at the Laboratory of Applied Transcriptomics

Russian Federation, 117997, Moscow, Academician Oparin str., 4

Guzel V. Savostina

Academician V.I. Kulakov National Medical Research Centre for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia

Email: savostina2324@gmail.com

postgraduate student

Russian Federation, 117997, Moscow, Academician Oparin str., 4

Alexey N. Ekimov

Academician V.I. Kulakov National Medical Research Centre for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia

Email: a_ekimov@oparina4.ru

Ph.D., Head of the Laboratory of Preimplantation Genetic Screening

Russian Federation, 117997, Moscow, Academician Oparin str., 4

Svetlana G. Perminova

Academician V.I. Kulakov National Medical Research Centre for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia

Email: s_perminova@oparina4.ru

Dr. Med. Sci., Leading Researcher at the Scientific and Clinical Department of ART named after F. Paulsen

Russian Federation, 117997, Moscow, Academician Oparin str., 4

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