1. Objectives Methods Results Conclusions References Conclusions
Sexing Of Human Preimplantation Embryos Without Embryos Biopsy Through RT-PCR On Spent Culture Media
Mahnaz Esmaeili1, 2*, Masood Bazrgar2, Hamid Gourabi2, Bita Ebrahimi3, Parnaz Borjian-Borujeni2
1.  Department of Genetics, University of Science and Culture, ACECR, Tehran, Iran.
2.  Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
3. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Objectives
Methods
Results
In this double-blind study, two groups were evaluated, the first group had history of sexing by Fluorescent in Situ Hybridization (FISH) method through blastomere biopsy on the third day. These human embryos were cultured individually after biopsy. We received the spent media of the embryos on the fifth day. In the second group, embryos of ART candidates were individually cultivated on the third day until development to blastocyst stage; this group of embryos was not biopsied but the blastocysts were fixed for sexing by FISH. Each group was divided to 2 subgroups; in the first subgroup we extracted the RNA from the spent culture medium of each embryo. Following RNA extraction, the total RNA was immediately reverse transcribed to cDNA. In the second subgroup, PCR was performed directly on the culture medium. PCR was performed for SRY and GAPDH genes. The SRY positive embryos were considered as male embryos and those GAPDH positive and SRY negative were considered as females. Finally, the results of sexing based on culture media was compared with the results of sexing based on FISH.
Preimplantation genetic diagnosis/screening (PGD/PGS) is a routine clinical procedure in many IVF clinics in the world that requires biopsy of embryonic cells or oocyte polar bodies. This is an invasive method to prevent the birth of children suffering from x-linked genetic disorders(1). Sex selection also is used for non-medical reasons like family balancing(2). The aim of this study was noninvasive sexing of preimplantation embryos using reverse transcriptase-polymerase chain reaction (RT-PCR) based on the presence of SRY RNA in the spent culture medium as a biomarker in sexing of human preimplantation embryos.
For the first group, 12 samples were evaluated. After comparing the results, we correctly diagnosed all 12 samples. For this group, the ability for correctly diagnosis of male samples was 100% (95% confidence interval (CI): %) and the ability for correctly diagnosis of female samples was 100% (95% CI: %) and the test accuracy was 100% (95% CI: %). In the second group, we were able to correctly diagnose 12 of the 14 samples; both embryos with a false diagnosis were of direct PCR. For the second group, the ability of correctly diagnosis of male samples was 100% (95% CI: %) and the ability to correctly diagnosis of female sample was 80% (95% CI: 45-98%) and the test accuracy was 86% (95% CI: 58-99%).
Conclusions
By means of recombinant lentiviral vectors it is possible to integrate the gene of interest into the cell genome. This can lead to the final production of transgenic animals.
positive control(GAPDH)
Positive control(SRY)
Conclusions
GAPDH
Ladder(50 bp)
GAPDH
Blank
GAPDH
Male
Male
Female
Sexing of preimplantation embryos using RT-PCR technique on the spent culture medium without embryo biopsy seems to be a reliable tool for non-invasive preimplantation sexing. However, direct PCR-based diagnosis might be lead to false results; this is probably due to DNA contamination.
References
123 (bp)
97)bp)
References
1) Savulescu, J. and E. Dahl, Sex selection and preimplantation diagnosis: a response to the Ethics Committee of the American Society of Reproductive Medicine. Human Reproduction, (9): p
2) Malpani, A., A. Malpani, and D. Modi, Preimplantation sex selection for family balancing in India. Human reproduction, (1): p
RT-PCR for 3 samples