Sexing ofin vitro-fertilized preimplantation mouse embryos by the PCR method

Abstract

SummaryThe applicability of the dual PCR method to embryo sexing was examined with the aim of establishing a noninvasive method of preimplantation diagnosis for human genetic disorders. Mouse pre-embryos obtained by in vitro fertilization were studied. The Sry gene sequence and the myogenin sequence were amplified as the Y-specific and internal control sequences, respectively. Amplification of as little as 10 pg of mouse genomic DNA was possible with the dual PCR method, the sensitivity being 10-fold greater than that of the single PCR method. The sex was identified in 100% (24/24) and 96% (23/24) of the pre-embryos tested at the 16-and 4-cell stages, respectively. In addition, the sex of all four single blastomeres dissociated from 4-cell pre-embryos agreed in 76% (16/21) of the specimens tested and 94% (79/84) of dissociated blastomeres could be sexed. The sex of single blastomeres biopsied from pre-embryos at the 8-cell stage could be identified. After transfer of 13 male and 25 female sexed pre-embryos, six viable fetuses were obtained. Histological examination showed that all these fetuses were of the predicted sex.Sexing of biopsied single blastomeres by the dual PCR method was rapid and reliable, suggesting its feasibility for preimplantation diagnosis of in vitro fertilized human pre-embryos.

Methods for Prenatal Sex Determination and Their Importance in Understanding and Prevention of Gender-Related Birth Defects

“…Successful assays are simple, need small amount of tissue, and are accurate during the entire pregnancy. Measuring the activity of X chromosomelinked enzymes [60] or RNA-based PCRs is complicated by the presence of some gene products only at certain developmental stages [61]. However, this problem is not present when the test is based on DNA (see Table 2).…”

Differences in metabolite burden of di(2-ethylhexyl)phthalate in pregnant and postpartum dams and their offspring in relation to drug-metabolizing enzymes in mice

“…Sexing was performed using their tails by analyzing the Sry gene at the Y-chromosome-specific target and the myogenin gene as a control by the dual polymerase chain reaction (PCR) method, according to Yano (1993) and Hayashi et al (2011).…”

Genetic sex determination assays in 53 mammalian species: Literature analysis and guidelines for reporting standardization

“…Genetic methods have the advantage of being less time consuming in certain situations (use of noninvasive samples instead of laborious tracking down of the animal), require smaller samples, are easier to perform, and can be noninvasive (e.g., hair). In the field of embryo sexing, many implant sexing techniques, such as karyotyping (Yano, 1993), are limited by the lack of material, while molecular assays need as little as a single blastomere (Tsai et al., 2011). Measuring the activity of X chromosome‐linked enzymes (Yano, 1993) or RNA‐based PCRs is further complicated by the presence of some gene products only at certain developmental stages (Prantner, Ord, Medvedev, & Gerton, 2016); this problem is not present when the test is based on the DNA.…”

“…In the field of embryo sexing, many implant sexing techniques, such as karyotyping (Yano, 1993), are limited by the lack of material, while molecular assays need as little as a single blastomere (Tsai et al., 2011). Measuring the activity of X chromosome‐linked enzymes (Yano, 1993) or RNA‐based PCRs is further complicated by the presence of some gene products only at certain developmental stages (Prantner, Ord, Medvedev, & Gerton, 2016); this problem is not present when the test is based on the DNA. Furthermore, embryo freezing is not needed (contrary to in situ hybridization), reducing the chance of freezing‐induced damage (Mara et al., 2004).…”