Sex Of Preimplantation Embryos Research Articles

Noninvasive sexing of human preimplantation embryos using RT-PCR in the spent culture media: A proof-of-concept study

Preimplantation genetic testing (PGT) routinely requires biopsy which is an invasive approach. The aim of this study was to examine a noninvasive approach for sexing of preimplantation embryos using polymerase chain reaction (PCR)/reverse transcriptase-PCR (RT-PCR) based on the presence of SRY DNA/RNA in the spent culture medium.Two groups were evaluated: in group 1, 40 embryos of routine PGT volunteers were cultured individually after biopsy and in group 2, 31 embryos were cultured individually until Day-5 post-fertilization. Each group was further divided into three subgroups: RNA extraction (RE), nucleic acid (NA) and DNase treated (DT). In the NA and DT subgroups, cDNA synthesis was performed directly on culture medium with or without DNase treatment in DT and NA subgroups, respectively. The results of sexing based on the PCR/RT-PCR in the culture medium, were compared with the results of sexing by fluorescence in situ hybridization (FISH) technique.In group 1, all samples were correctly diagnosed. In group 2, five female samples were misdiagnosed. Test’s sensitivity, specificity and accuracy were 100 %, 94.44 % and 96.88 %, in RE, 100 %, 81.82 % and 93.55 % in DT and 100 %, 71.43 % and 85.71 % in NA, respectively.Preimplantation sexing without embryo biopsy, in the spent embryo culture media using RNA amplification based methods including RE and DT seem to be more reliable while nucleic acid based method, NA, led to the highest misdiagnoses probably due to DNA contamination. Since all male samples were correctly diagnosed in all subgroups of this preliminary study, preimplantation noninvasive sexing on culture medium seems feasible, however all sources of nucleic acid contamination must be carefully avoided.

Non-invasive preimplantationsexingusingRT-PCRon the spent embryo culture media

Introduction Preimplantation sexing is an alternative method for Preimplantation GeneticTesting (PGT) to prevent the birth of children suffering from x-linked genetic disorders.Social sexing for non-medical reasons is also legal for some cases. PGT routinely needsto biopsy of embryonic cells or oocyte polar bodies which are invasive and could lead to implantation failure. SRY gene expression begins in male embryos following genome activation in preimplantation embryos. The aim of this study was sexing of preimplantation embryos without biopsy based on the presence of SRY RNA and DNA in the spent culture medium as a biomarker of sexing of human preimplantation embryos. Materials and Methods In this double-blind study, two groups were evaluated. In the first group, on the third day after the biopsy, the human embryos were cultured individually; we received the spent media of the non-degenerated 5-day embryos those have been underwent sexing by Fluorescent In Situ Hybridization(FISH) method via blastomere biopsy on the third day. In the second group, embryos of ART candidateswere cultivated on the third day individuallyuntil 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 were GAPDH positive and SRY negative considered as females. The results of sexing based on spent culture media was compared with the results of sexing based on FISH. Results For the first group, 12 samples were evaluated, after comparing the results of PCR and FISH, the results for all the samples were concordant.In the second group, we were able to correctly diagnose 12 of the 14 samples. Two embryos with false diagnosis were females that diagnosed as males by direct PCR. Conclusion Preimplantation sexing without embryo biopsy by RT-PCR on the spent embryo culture media 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.

Sex determining of cat embryo and some feline species

Sex identification in mammalian preimplantation embryos is a technique that is used currently for development of the embryo transfer industry for zootechnical animals and is, therefore, a resource for biodiversity preservation. The aim of the present study was to establish a rapid and reliable method for the sexing of preimplantation embryos in domestic cats. Here we describe the use of nested PCR identify Y chromosome-linked markers when starting from small amounts of DNA and test the method for the purpose of sexing different species of wild felids. To evaluate the efficiency of the primers, PCR analysis were performed first in blood samples of sex-known domestic cats. Cat embryos were produced both in vitro and in vivo and the blastocysts were biopsied. A Magnetic Resin System was used to capture a consistent amount of DNA from embryo biopsy and wild felid hairs. The results from nested PCR applied on cat blood that corresponded to the phenotypical sex. Nested PCR was also applied to 37 embryo biopsies and the final result was: 21 males and 16 females. Furthermore, beta-actin was amplified in each sample, as a positive control for DNA presence. Subsequently, nested PCR was performed on blood and hair samples from some wild felines and again the genotyping results and phenotype sex corresponded. The data show that this method is a rapid and repeatable option for sex determination in domestic cat embryos and some wild felids and that a small amount of cells is sufficient to obtain a reliable result. This technique, therefore, affords investigators a new approach that they can insert in the safeguard programmes of felida biodiversity.

Detection of bovine fetal DNA by polymerase chain reaction for prenatal sexing

The ability to determine the sex of embryos prior to transfer to recipients has commercial application in the embryo transfer industry. A number of methods of sex determination have been attempted; however, none of these has been commercially successful to date. The identification of repetitive, male-specific bovine chromosomal DNA fragments enables the use of DNA-probe technology in determining the sex of preimplantation embryos from a small embryonic sample. The identification and isolation of three repetitive, male-specific bovine chromosomal DNA fragments, and the application of these probes in a sensitive and highly accurate embryo sexing assay are reported.

Amplification of X-and Y-chromosome-specific regions from single human blastomeres by polymerase chain reaction for sexing of preimplantation embryos

We describe a polymerase chain reaction (PCR) assay for the simultaneous detection of X and Y chromosomes in human blastomeres. In a first round of PCR, both X- and Y-specific fragments are amplified with primers which are common to both chromosomes and are derived from the X-linked steroid sulphatase gene and the Y-linked pseudogene. In a second round of PCR, fragments specific to each chromosome are generated: both an X and a Y fragment in male embryos and, due to the absence of a Y chromosome, only an X fragment in females. The efficiency and accuracy of this assay are high; it generates no false positive amplification signals and allows sexing in approximately 6 h after embryo biopsy. We therefore believe that it is suitable for gender determination by preimplantation diagnosis for couples at risk for X-linked genetic diseases.

Amplification of the Sry gene allows identification of the sex of mouse preimplantation embryos

The Sry gene is key in determining male testis formation located in the sex-determining region of the Y chromosome during gonadal differentiation. In the present study, the Sry gene, chosen as the Y-specific target sequence, was amplified in order to sex mouse preimplantation embryos rapidly using the polymerase chain reaction (PCR). Meanwhile, the actin gene on the autosome was also amplified and served as a control for both male and female samples. When genomic DNA of male and female origin was amplified with Sry and actin primers, a 504 bp fragment was detected in both samples, while a 227 bp product was observed in only the male DNA. Morula and blastocyst stage embryos (n=135) were typed either as male or female with the exception of 2 samples. The Sry gene fragment was detected in about half of the embryos, which showed a 1:1 male and female ratio (P > 0.05). Based on developmental rates of embryos during morula and blastocyst stages, approximately 65% of faster-developing embryos were diagnosed as male and 63% of slower-developing embryos as female. The products of PCR were confirmed by restriction mapping and indicated that the sex of a preimplantation embryo can be determined accurately by amplifying the Sry gene. Use of this method would allow animal breeders to determine the sex of other mammalian preimplantation embryos before transfer to recipient females.

Diagnosis of sex in preimplantation embryos by fluorescent in situ hybridisation.

The aim of prenatal genetic diagnosis is to give parents the option of terminating affected pregnancies. For most X linked conditions specific diagnosis is not possible and the only option is induced abortion of all male fetuses, of which half would be unaffected. An altemative is offered by preimplantation diagnosis of sex in embryos obtained by in vitro fertilisation and selective transfer of female embryos. The sex of preimplantation embryos has been determined by amplification of a DNA fragment specific to the Y chromosome by the polymerase chain reaction after biopsy of the embryo at the eight cell stage.' Live births of female infants have followed this procedure, testifying to its safety.2 Misdiagnosis is, however, possible because of contamination, failure of amplification, or the sampling of anuclear cytoplasmic fragments.' We report the application of dual colour fluorescent in situ hybridisation for sexing interphase nuclei of three day old human preimplantation embryos.

Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats.

We describe a quick and efficient method of determining the sex of DNA samples from humans, cattle, sheep, and goats. Using universal primers we have amplified 447 or 445 bp fragments from male or female genomic DNA corresponding to the ZFY or ZFX genes. Restriction fragment length polymorphism (RFLP) analysis of the fragments yielded specific banding patterns between the two sexes in these species. Horse, pig and rainbow trout fragments did not show RFLPs with the group of enzymes chosen for the other species. Use of this method would allow animal breeders to determine the sex of preimplantation embryos before transfer to recipient females. Human embryos at risk for sex-linked disorders could be sexed soon after in vitro fertilization (IVF) and options considered before implantation.

The use of male-specific chromosomal DNA fragments to determine the sex of bovine preimplantation embryos

The ability to determine the sex of embryos prior to transfer to recipients has commercial application in the embryo transfer industry. A number of methods of sex determination have been attempted; however, none of these has been commercially successful to date. The identification of repetitive, male-specific bovine chromosomal DNA fragments enables the use of DNA-probe technology in determining the sex of preimplantation embryos from a small embryonic sample. The identification and isolation of three repetitive, male-specific bovine chromosomal DNA fragments, and the application of these probes in a sensitive and highly accurate embryo sexing assay are reported.

Investigation of sperm cytotoxicity as an indicator of ability of antisera to detect male-specific antigen on preimplantation mouse embryos.

H-Y antisera were produced in C57BL/6 female mice by repeated intraperitoneal injections of syngeneic male spleen cells. Epididymal spermatozoa were incubated in the presence of H-Y antisera and guinea-pig serum as a complement source. Levels of ATP remaining after treatment were used to calculate the amount of specific killing. Sera of different cytotoxic titres were used in an indirect immunofluorescent assay with a fluorescein isothiocyanate-conjugated IgG fraction of goat anti-mouse IgG (Fc fragment specific) as second antibody. Embryos were classified as fluorescent or nonfluorescent, transferred to pseudopregnant recipients, and allowed to develop to term. Of 12 sera tested for sperm cytotoxicity, 5 were different from a nonimmunized control serum (P less than 0.05). Percentage specific killing in each of these sera was 7.8 +/- 4.2, 11.7 +/- 3.0, 26.0 +/- 2.2, 27.7 +/- 3.7 and 39.2 +/- 4.8, respectively (mean +/- s.e.m. with three replicates). The 5 sera and an additional one (4.9 +/- 1.3% specific killing) were used in the embryo sexing experiment. The accuracy with which these sera correctly identified sex of preimplantation embryos was 60, 46, 74, 73, 74 and 48%, respectively. Correlation coefficients were 0.86 (P less than 0.05) for specific sperm cytotoxicity and percentage of nonfluorescent embryos that were female and 0.78 (n.s.) for specific sperm cytotoxicity and percentage of fluorescent embryos that were male. Therefore, although the sperm cytotoxicity test is useful for screening antisera for the study of H-Y antigen expression on preimplantation embryos, nonfluorescent embryos are more accurately classified as females than are fluorescent embryos as male.