DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease

Molecular Diagnostics in Preimplantation Genetic Diagnosis

“…The second step involves the diagnostic test itself, which can be performed in a region of the IVF laboratory, an adjacent laboratory equipped to perform molecular analyses or in a completely separate dedicated molecular genetics laboratory equipped to process single cell sam- Various (gender determination to exclude hemizygotes) Nested PCR, agarose gel (ϩ/Ϫ X/Y) X-linked disorders 6 Various (gender determination to exclude hemizygotes) Nested PCR, heteroduplexing Cystic fibrosis 22,38,44,75 Tay-Sachs disease 111 3 bp deletion (⌬F508) 4 bp insertion Nested PCR, allele-specific amplification RhD blood typing 3 Myotonic dystrophy 127 ϩ/Ϫ RhD gene determines Rh status Expansion of (CTG)n trinucleotide repeat Nested PCR, restriction enzyme Cystic fibrosis, 23 Beta thalassemia, 83 Marfan syndrome, 107 Epidermolysis Bullosa, 100 Lesch-Nyhan syndrome, 101 Sickle cell anemia, 102 Fanconi's anemia, 103 Ornithine transcarbamylase deficiency, 104 Spinal muscular atrophy [108][109][110] Various point mutations Deletion. Distinguish between gene and pseudogene Nested PCR, restriction enzyme (2 mutations in 1 fragment)…”

“…3 Subsequently, PCR protocols for preimplantation gender determination were refined to include primer sets which simultaneously amplify sequences common to both sex chromosomes (for example single copy genes such as ZFX/ZFY, 4 AMELX/AMELY, 5 ) and repetitive sequences such as DXZ1 and DYZ1. 6,7 Sequences common to the sex chromosomes are identical at the site of primer annealing but differ internally in terms of size or include minor polymorphisms. Despite these technical improvements, the majority of embryo sexing is now accomplished using fluorescent in situ hybridization (FISH) which is less prone to contamination and can also provide the copy number for each chromosome tested thereby potentially avoiding the transfer of common chromosome abnormalities such as triploidy or X-monosomy.…”

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa

“…The production of offspring using sex-sorted spermatozoa in conjunction with in vitro fertilisation (IVF) and embryo transfer (ET) has been reported in cattle (Cran et al 1993, 1995, Fry et al 2004, pigs (Rath et al 1997, 1999, Abeydeera et al 1998, Johnson et al 2000, humans (Levinson et al 1995, Fugger et al 1998, Fugger 1999) and sheep (intra-cytoplasmic sperm injection; Catt et al 1996). Improvements in sorting efficiency (Rens et al 1998, 1999) and cryopreservation methods (Schenk et al 1999) for sex-sorted spermatozoa facilitated the production of offspring after low-dose artificial insemination (AI) in cattle , sheep (Hollinshead et al 2002a), horses (Lindsey et al 2002) and elk (Schenk & DeGrofft 2003).…”

Artificial Insemination in Veterinary Science

“…Since then, DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species (Garner, 2006;Garner et al, 1983;Lu et al, 2010;Pinkel et al, 1982), and offspring have been produced from sexed sperm of at least seven species, including rabbits (Johnson et al, 1989), humans (Levinson et al, 1995), cattle (Cran et al, 1993), horses (Buchanan et al, 2000), sheep (Catt et al, 1996), dogs (Meyers et al, 2008), cats , elk (Schenk & DeGrofft, 2003), buffalo (Presicce et al, 2005) and dolphins (O'Brien & Robeck, 2006). The first offspring born with flow cytometrically sex sorted spermatozoa was in rabbits after surgical AI into the oviduct (Johnson et al, 1989).…”