Genetic follow-up of male offspring born by ICSI, using a multiplex fluorescent PCR-based test for Yq deletions

Abstract

De-novo deletions involving AZFa, b, c and d are one of the most common chromosomal aberrations in man resulting in defective spermatogenesis and male infertility. Currently, Yq deletion screening involves either single or multiplex PCR using Y-specific sequence tagged site markers and the subsequent analysis of the amplification products on ethidium bromide-stained agarose gels. To improve the practicality of routine and high throughput Yq testing, we have developed a more sensitive multiplex fluorescent (FL)-PCR screening system using genomic DNA extracted from cheek buccal cells as a readily available PCR template. For genetic follow-up studies of ICSI-conceived children, we also developed a DNA fingerprinting system based on the co-amplification of four highly polymorphic markers to validate family samples and detect any potential extraneous DNA contamination that could cause a misdiagnosis. Multiplex FL-PCR analysis of buccal cell DNA from two infertile men who conceived three sons by ICSI demonstrated that their Yq deletions were vertically transmitted. Fine mapping with additional Yq markers revealed identical deletion endpoints involving the loss of AZFdc sequences. This firstly indicates that the extent of the Yq deletion was unchanged on ICSI transmission and secondly supports the view that AZFdc deletions may arise by a common de-novo event. Analysis of paternal, maternal and sibling DNA fingerprints showed the co-inheritance of parental alleles by each male child and confirmed the expected relationship between each family member. The application of these new FL-PCR based screening tests in genetic follow-up studies will assist in confirming transmission of specific genetic defects to male offspring conceived by ICSI and provide a basis for genetic counselling and potential treatment options as these boys approach sexual maturity.

Intracytoplasmic Sperm Injection Effects in Infertile azh Mutant Mice1

“…It was reported that some chromosomal anomalies known to cause infertility were transmitted from fathers to sons via ICSI [11][12][13]. When these children become adults, some of them likely will have the same types of infertility that their parents had, leaving them with ICSI as their only possibility for reproduction.…”

Evolution of intracytoplasmic sperm injection: From initial challenges to wider applications

“… 75 , 76 , 77 , 78 , 79 Furthermore, it has been reported that AZF deletions can expand or cause new de novo deletions during the genetic transmission process via ICSI. 80 , 81 , 82 , 83 , 84 ICSI has become a widely used technique, despite the unresolved issue of potential transmission of AZF microdeletions to subsequent generations. A solution to this issue has yet to be found.…”

Deletion of b1/b3 shows risk for expanse of Yq microdeletion in male offspring

“…To date, there has been 1 published report of a pregnancy achieved via ICSI using sperm from a man with a complete AZFb+c deletion. [ 10 ] Other studies showed that it was possible to sporadically detect spermatozoa in these men, [ 11 , 12 ] indicating that the meiotic process was not completely prevented. However, during genetic counselling of the present patient, ICSI after TESE or m-TESE were not firstly recommended and other options, such as donor insemination or adoption should be considered.…”