A prospective study to evaluate the gender prediction of blastocysts by using cell-free DNA within a culture medium


Background: Preimplantation genetic diagnosis (PGD) has been used as an option for couples with the possibility of having a baby with a genetic disorder. The common method for performing this test involves isolating 1 cell from day 3 or a few cells from day 5 embryos and performing genetic studies on the cell-extracted DNA. This method is invasive and can cause abortion after implantation in the uterus. Because of this, 2 noninvasive methods for performing a PGD have been studied: PGD using blastocyst fluid and PGD using embryo culture medium.

Objective: The aim of this study is to determine the sensitivity of the polymerase chain reaction (PCR) technique to detect the Y chromosome using cell-free DNA within a culture medium for gender prediction of blastocysts.

Materials and Methods: In this study, the gender of 30 embryos on day 5 was determined using embryonic DNA extraction from the culture medium and the PCR technique to evaluate the sex-determining region Y and fragile X mental retardation genes. Then, the accuracy was assessed using ultrasound.

Results: The results of the PCR technique showed that 7 embryos were male, but an ultrasound revealed that 13 were male.

Conclusion: The given results indicated that, because of the low amount of DNA extracted from the culture medium, the diagnosis of the existence of the Y chromosome by this method is still not accurate enough for detecting the gender of the embryo.

Key words: Preimplantation diagnosis, Embryo implantation, Culture media, Blastocyst, Polymerase chain reaction.

[1] Basille C, Frydman R, El Aly A, Hesters L, Fanchin R, Tachdjian G, et al. Preimplantation genetic diagnosis: State of the art. Eur J Obstet Gynecol Reprod Biol 2009; 145: 9-13.

[2] Tasca RJ, McClure ME. The emerging technology and application of preimplantation genetic diagnosis. J Law Med Ethics 1998; 26: 7-16.

[3] Baruch S, Javitt G, Scott J, Hudson K. Preimplantation genetic diagnosis: A discussion of challenges, concerns, and preliminary policy options related to the genetic testing of human embryos. USA: Genetics and Public Policy Center; 2004.

[4] Harper JC, SenGupta SB. Preimplantation genetic diagnosis: State of the art. Hum Genet 2012; 131: 175- 186.

[5] Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990; 344: 768-770.

[6] Handyside AH, Penketh R, Winston R, Pattinson J, Delhanty J, Tuddenham E. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989; 333: 347-349.

[7] Li P, Song Z, Yao Y, Huang T, Mao R, Huang J, et al. Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization. Sci Rep 2018; 8: 1-10.

[8] Yang L, Lv Q, Chen W, Sun J, Wu Y, Wang Y, et al. Presence of embryonic DNA in culture medium. Oncotarget 2017; 8: 67805-67809.

[9] Kuznyetsov V, Madjunkova S, Antes R, Abramov R, Motamedi G, Ibarrientos Z, et al. Evaluation of a novel non-invasive preimplantation genetic screening approach. PLoS One 2018; 13: e0197262.

[10] Petersen CG, Mauri AL, Vagnini LD, Renzi A, Petersen B, Matilla MC, et al. Randomized comparison of two commercial culture media (Cook and Vitrolife) for embryo culture after IMSI. JBRA Assist Reprod 2019; 23: 33-36.

[11] Vera-Rodriguez M, Diez-Juan A, Jimenez-Almazan J, Martinez S, Navarro R, Peinado V, et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Hum Reprod 2018; 33: 745-756.

[12] Galluzzi L, Palini S, De Stefani S, Andreoni F, Primiterra M, Diotallevi A, et al. Extracellular embryo genomic DNA and its potential for genotyping applications. Future Sci OA 2015; 1: FSO62.

[13] Ethics Committee of the American Society of Reproductive Medicine. Sex selection and preimplantation genetic diagnosis. Fertil Steril 2004; 82 (Suppl.): S245-S248.

[14] Rajan-Babu I-Sh, Lian M, Cheah FSH, Chen M, Tan ASC, Prasath EB, et al. FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome. Expert Rev Mol Med 2017; 19: e10.

[15] Handyside AH, Lesko JG, Tarín JJ, Winston RM, Hughes MR. Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N Engl J Med 1992; 327: 905-909.

[16] Meseguer M, Garrido N, Remohí J, Simón C, Pellicer A. Gender selection: Ethical, scientific, legal, and practical issues. J Assist Reprod Genet 2002; 19: 443-446.

[17] Joshi BD, De R, Goyal SP. Utility and applicability of a universal set of primers in identifying the sex of South and Southeast Asian mammals. Zool Stud 2019; 58: e19.

[18] Osman J, Murad NAA, Chin SF, Jamal R. Highly sensitive and reliable human sex determination using multiplex PCR. Asia-Pacific J Mol Med 2016; 4: 1.

[19] Settin A, Elsobky E, Hammad A, Al-Erany A. Rapid sex determination using PCR technique compared to classic cytogenetics. Int J Health Sci 2008; 2: 49-52.

[20] Wu H, Ding C, Shen X, Wang J, Li R, Cai B, et al. Mediumbased noninvasive preimplantation genetic diagnosis for human α-thalassemias-SEA. Medicine 2015; 94: e669.