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Non-invasive human preimplantation embryos sex determination using STR-based fluorescent multiplex PCR on days 3 and 5 post-fertilization

Published online by Cambridge University Press:  10 July 2025

Maryam Zare ,
Mehdi Totonchi ,
Hamid Gourabi ,
Mohammadreza Zamanian ,
Reza Mohammadi ,
Sirous Zeinali ,
Maryam Mohammadi  and
Poopak Eftekhari-Yazdi Open the ORCID record for Poopak Eftekhari-Yazdi [Opens in a new window]
Maryam Zare
Affiliation:
Department of Reproductive Biology, Faculty of Basic Sciences and Advanced Medical Technologies, Royan Institute, ACECR, Tehran, Iran Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Mehdi Totonchi
Affiliation:
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Hamid Gourabi
Affiliation:
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Mohammadreza Zamanian
Affiliation:
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Reza Mohammadi
Affiliation:
Genetic Laboratory of Shiraz Fertility Center, Shiraz, Iran
Sirous Zeinali
Affiliation:
Dr. Zeinali’s Medical Genetics Laboratory, Kawsar Human Genetics Research Center, Tehran, Iran
Maryam Mohammadi
Affiliation:
Department of Biostatistics, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Poopak Eftekhari-Yazdi*
Affiliation:
Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
*
Author for correspondence: Poopak Eftekhari-Yazdi. P.O. Box: 16635–148, Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. E-mail: eftekhari@royaninstitute.org
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Summary

This study aimed to investigate the efficacy of cell-free DNA (CF-DNA) in the spent cleavage and blastocyst medium versus blastomere biopsy for sex identification using short tandem repeat (STR) markers for the first time. In total, 39 samples of spent culture medium (SCM) from six couples were collected of which 28 samples were CF-DNA from blastocoel fluid + SCM (day 5) and 11 samples from SCM alone (day 3). The frequencies of allele dropout (ADO), fail rate and informativity markers were considered. The relationship between the morphology of embryos and ADO and the fail number of all markers was investigated. Sex identification rate between CF-DNA isolated from culture medium and fluorescence in situ hybridization (FISH) was then compared with measurement of Agreement Kappa (AK). The highest frequency of informative markers belonged to DXS6801 and HPRT. There was no relationship between the ADO number of all markers and embryo morphology. A significant difference was seen between embryo morphology and fail numbers. AK value between CF-DNA isolated from culture medium and FISH was 0.516, which is moderate. The ability of CF-DNA to detect the correct diagnosis of males and females showed that all values of specificity, sensitivity, positive predictive value, and negative predictive value were 100%. The presence of embryonic CF-DNA in the SCM on day 3 as well as blastocyst medium on day 5 using STR-based multiplex PCR is approximately consistent with FISH for sex identification. Advances in DNA extraction, amplification technique, and testing may allow for preimplantation genetic testing for aneuploidy (PGT-A) and monogenic/single-gene disorders (PGT-M) as a non-invasive approach without biopsy in the future either in sex determination or chromosomal abnormality.

Keywords

BlastocystCulture mediumMicrosatellite repeatsPreimplantation diagnosisSex determination analysis

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Type
Research Article
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Zygote , First View , pp. 1 - 11
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© The Author(s), 2025. Published by Cambridge University Press

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