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Serdar Coskun

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Chorionic Gonadotropin↗

Flow cytometric ploidy analysis of testicular biopsies from sperm-negative wet preparations.

BACKGROUND: The use of testicular sperm in assisted reproduction depends on the availability of sperm in wet preparations. It is not always possible to recover sperm from the testis, even with previous sperm-positive histopathological findings. The purpose of this study was to evaluate the sperm-negative wet preparation search results with flow cytometric ploidy analysis and histopathological examination. METHODS: Two pieces of testicular tissue were obtained from azoospermic patients to investigate the spermatogenic status of the testis, and to determine the presence of sperm through a wet preparation. The testicular tissue was shredded and then vortexed; the cellular suspension was then processed for a wet preparation sperm search, while the residual tissue was exposed to enzymatic digestion for flow cytometric ploidy analysis. RESULTS: A total of 38 patients had sperm-negative wet preparation results. Of those, six (16%) were shown to have haploid cells after flow cytometric analysis. Histopathological examination showed three samples with maturation arrest at the spermatid stage, and the other three at the spermatocyte stage. CONCLUSIONS: Flow cytometric ploidy analysis can be used to verify the results of a wet preparation sperm search when no sperm were detected. Flow cytometric ploidy analysis can also reveal the presence of spermatids when no sperm are available.

Biopsy↗

Pregnancy after preimplantation genetic diagnosis for Ataxia Telangiectasia.

Ataxia Telangiectasia (AT) is an autosomal recessive disorder with an incidence estimated at 1 in 40 000 to 1 in 100 000 live births. More than 100 different somatic and germ-line mutations have been identified in the AT gene, the majority of which cause premature protein truncation. The immense size of the AT gene (66 exons) complicates the detection of mutations. A Saudi family with three affected children suffering from AT consulted our IVF centre for preimplantation genetic diagnosis (PGD). Despite advanced maternal age and unknown mutation, the family was screened for AT mutations. A large deletion in the gene was found to be responsible for the phenotype of AT. The mutation detection permitted us to perform PGD on AT for the first time. Single cell PCR consisted of amplifying one of the deleted exons, exon 19. Homozygous affected embryos show an absence of the exon, while in heterozygous or normal embryos the exon is amplified successfully. After ICSI, three embryos were suitable for embryo biopsy. After biopsy only one embryo showed exon amplification and was transferred. A singleton pregnancy ensued and prenatal diagnosis confirmed the presence of exon 19. This report demonstrates that PGD is feasible despite advanced maternal age and poor response to follicle stimulation.

Adolescent↗

Nucleolar precursor body distribution in pronuclei is correlated to chromosomal abnormalities in embryos.

In-vitro generated human embryos have low implantation rates and high chromosomal abnormalities. Embryos are mostly selected on the basis of microscopic morphological examination. The relationship between pronuclear morphology and chromosomal abnormalities was investigated in this study. Zygotes were scored according to pronuclear morphology on day 1. Excess embryos that were not transferred or cryopreserved on day 3 were fixed. Chromosomes 13, 18, 21, X and Y were analysed by fluorescence in-situ hybridization (FISH). A total of 125 embryos were analysed; 58 (46%) were abnormal, 32 (26%) were mosaic and 35 (28%) were normal. Results were analysed according to different pronuclear morphology. Zygotes with polarized pattern had a significantly lower incidence of chromosome abnormality than those with a non-polarized pattern. The presence of cytoplasmic halo, the size of each pronucleus and the number of nucleolar precursor body had no significant effect on chromosomal abnormalities. In conclusion, embryos generated from zygotes with polarized pattern have fewer chromosomal abnormalities compared with other patterns. A simple microscopic examination during fertilization confirmation would be useful to select embryos with fewer chromosomal abnormalities, preferably in combination with other observations shown to correlate with chromosomal abnormalities.

Adult↗