Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.

Autor:	Hammond ER; Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., McGillivray BC; Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., Wicker SM; Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., Peek JC; Fertility Associates, Auckland, New Zealand., Shelling AN; Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., Stone P; Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., Chamley LW; Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., Cree LM; Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand; Fertility Associates, Auckland, New Zealand. Electronic address: l.cree@auckland.ac.nz.
Jazyk:	angličtina
Zdroj:	Fertility and sterility [Fertil Steril] 2017 Jan; Vol. 107 (1), pp. 220-228.e5. Date of Electronic Publication: 2016 Nov 16.
DOI:	10.1016/j.fertnstert.2016.10.015
Abstrakt:	Objective: To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. Design: Prospective embryo cohort study. Setting: Academic center and private in vitro fertilization (IVF) clinic. Patient(s): Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. Intervention(s): Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. Main Outcome Measure(s): Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. Result(s): Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. Conclusion(s): Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin. (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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