Serdar Coskun

Clinical application of multiple displacement amplification in preimplantation genetic diagnosis

Multiple displacement amplification (MDA) is a technique used in the amplification of very small amounts of DNA. MDA is reported to yield large quantities of high-quality DNA. The applicability of MDA to single cells was recently demonstrated as a potential technique for preimplantation genetic diagnosis (PGD). This paper shows the first clinical application of MDA in PGD. Two cycles of PGD were performed in two diseases, resulting in two pregnancies. All the diagnoses given on blastomeres were confirmed on the non-transferred whole embryos. The blastomere diagnosis was coupled with short tandem repeat (STR) analysis (16 loci) in all cycles. Allelic drop-out (ADO) assessment and amplification efficiency were evaluated on 40 single lymphocytes derived from parents of each disease. ADO and amplification failure were 10.3 and 2.2% for beta-thalassaemia and 17.9 and 2.2% for cystic fibrosis respectively. HLA matching for A, B and DR was performed successfully on single cell for the beta-thalassaemia family using similar methods to genomic DNA. The PGD protocol used in all diseases consists of MDA amplification, followed by a standard polymerase chain reaction protocol. Although HLA matching was not applied to embryos, its feasibility was shown on single cell DNA amplified by MDA. Altogether, these data show the simplicity and reliability of performing PGD in combination with HLA matching and STR analysis using MDA.

The correlation between endometrial thickness and outcome of in vitro fertilization and embryo transfer (IVF-ET) outcome

BackgroundTo evaluate the relationship between endometrial thickness on day of human chorionic gonadotrophin administration (hCG) and pregnancy outcome in a large number of consecutive in vitro fertilization and embryo transfer (IVF-ET) cycles.MethodsA retrospective cohort study including all patients who had IVF-ET from January 2003–December 2005 conducted at a tertiary center.ResultsA total of 2464 cycles were analysed. Pregnancy rate (PR) was 35.8%. PR increased linearly (r = 0.864) from 29.4% among patients with a lining of less than or equal to 6 mm, to 44.4% among patients with a lining of greater than or equal to 17 mm. ROC showed that endometrial thickness is not a good predictor of PR, so a definite cut-off value could not be established (AUC = 0.55).ConclusionThere is a positive linear relationship between the endometrial thickness measured on the day of hCG injection and PR, and is independent of other variables. Hence aiming for a thicker endometrium should be considered.

Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro.

Purpose:Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization.Methods:Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping.Results:Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results.Conclusion:Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.Genet Med 16 11, 838–845.

Y chromosome microdeletions in infertile men with idiopathic oligo- or azoospermia

About 30–40% of male infertility is due to unknown reasons. Genetic contributions to the disruption of spermatogenesis are suggested and amongst the genetic factors studied, Y chromosome microdeletions represent the most common one. Screening for microdeletions in AZFa, b and c region of Y chromosome showed a big variation among different studies. The purpose of this study was to investigate the prevalence of such deletions in Saudi men. A total of 257 patients with idiopathic oligo- or azoospermia were screened for Y chromosome microdeletions by 19 markers in AZF region. Ten (3.9%) patients had chromosomal rearrangements, six of them showed sex chromosome abnormalities and four patients had apparently balanced autosomal rearrengements. Eight of the remaining 247 patients (3.2%) with a normal karyotype and no known causes of impaired spermatogenesis had Y chromosome microdeletions. Among these, six patients had deletions in AZFc region, one case had a deletion in AZFb and another had both AZFa and AZFc deletions. In conclusion, our study shows that Y chromosome microdeletions are low in our population. We also report for the first time a case with unique point deletions of AZFa and AZFc regions. The lower frequency of deletions in our study suggest that other genetic, epigenetic, nutritional and local factors may be responsible for idiopathic oligo- or azoospermia in the Saudi population.

Effect of Human Chorionic Gonadotropin on Cytokine Production from Human Endometrial Cells in Vitro

PROBLEM: To examine whether human chorionic gonadotropin (hCG) is involved in the regulation of interleukin (IL)‐6, tumor necrosis factor (TNF)‐α, and leukemia inhibitory factor (LIF) secretion from cultured human endometrial cells.

TLE6 mutation causes the earliest known human embryonic lethality

BackgroundEmbryonic lethality is a recognized phenotypic expression of individual gene mutations in model organisms. However, identifying embryonic lethal genes in humans is challenging, especially when the phenotype is manifested at the preimplantation stage.ResultsIn an ongoing effort to exploit the highly consanguineous nature of the Saudi population to catalog recessively acting embryonic lethal genes in humans, we have identified two families with a female-limited infertility phenotype. Using autozygosity mapping and whole exome sequencing, we map this phenotype to a single mutation in TLE6, a maternal effect gene that encodes a member of the subcortical maternal complex in mammalian oocytes. Consistent with the published phenotype of mouse Tle6 mutants, embryos from female patients who are homozygous for the TLE6 mutation fail to undergo early cleavage, with resulting sterility. The human mutation abrogates TLE6 phosphorylation, a step that is reported to be critical for the PKA-mediated progression of oocyte meiosis II. Furthermore, the TLE6 mutation impairs its binding to components of the subcortical maternal complex.ConclusionIn this first report of a human defect in a member of the subcortical maternal subcritical maternal complex, we show that the TLE6 mutation is gender-specific and leads to the earliest known human embryonic lethality phenotype.

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.

Effects of Reducing Insemination Time in Human In Vitro Fertilization and Embryo Development by Using Sibling Oocytes

Purpose:Recent studies showed a beneficial effect of reducing the time of sperm–oocyte interaction on fertilization, division, and implantation rates of the oocytes obtained from randomized patients. In the present study, the effects of reduced insemination time on fertilization and embryo development were evaluated by using sibling oocytes from the same patient.Methods:A total of 464 oocytes from 36 patients was randomly allocated to be inseminated for either 1 hr (reduced) or 18 hr (regular).Results:Fertilization rates were not significantly different between reduced (135/229; 59%) and regular (150/235; 64%) groups. Cleavage rates and embryo quality were similar in both groups. A total of 135 embryos (73 from the reduced and 62 from the regular group) was transferred to 36 patients. Thirty-four embryos implanted in 18 patients (25.2% implantation and 50.0% pregnancy rates).Conclusions:Fertilization, cleavage, and embryo development from 1-hr insemination is comparable, not superior, to those from an 18-hr insemination time, which is commonly used in in vitro fertilization programs. These data suggest that reduced insemination time can be used during in vitro fertilization to avoid unnecessarily longer exposure to spermatozoa.

Genome-wide gene expression profiling and mutation analysis of Saudi patients with Canavan disease

Purpose: Canavan disease, caused by a deficiency of aspartoacylase, is one of the most common cerebral degenerative diseases of infancy. The aims of this study were to identify the mutations associated with Canavan disease in Saudi Arabia and to identify differentially expressed genes likely to contribute to the development of this disease.Methods: Polymerase chain reaction, long polymerase chain reaction, multiplex ligation-dependent probe amplification, sequencing, array comparative genomic hybridization (aCGH), and global gene expression profiling were used to determine putative mutations and likely gene signatures in cultured fibroblasts of patients from Saudi Arabia.Results: One novel and one known large deletion and two previously known mutations (IVS4 + 1G>T and G27R) were identified. Compared with controls, 1440 genes were significantly modulated in Canavan patients (absolute fold change [FC] ≥4). Genome-wide gene expression profiling results indicated that some genes, involved in apoptosis, muscle contraction and development, mitochondrial oxidation, inflammation and glutamate, and aspartate metabolism, were significantly dysregulated.Conclusions: Our findings indicate that the presence of muscle weakness and hypotonia in patients may be associated with the dysregulated gene activities of cell motility, muscle contraction and development, actin binding, and cytoskeletal-related activities. Overall, these observations are in accordance with previous studies performed in a knockout mouse model.

Intracytoplasmic sperm injection and conventional in vitro fertilization are complementary techniques in management of unexplained infertility.

AbstractPurpose: To evaluate the role of ICSI in unexplained infertility. Methods: In 125 cycles with six or more oocytes retrieved per cycle, sibling oocytes were randomly allocated to IVF or ICSI (group A). In 74 cycles with less than six oocytes retrieved per cycle, cycles were allocated to IVF or ICSI (group B). Results: In group A, ICSI fertilization rate of 61% per allocated oocyte was higher than IVF fertilization rate of 51.6% (P < 0.001). Complete fertilization failure occurred in 19.2 and 0.8% of cycles in IVF and ICSI, respectively (P < 0.001). In group B, fertilization rate in IVF cycles was 53.3% as compared to 60.7% per allocated oocyte in the ICSI cycles (P = 0.29). Complete fertilization failure was higher (P = 0.02) in conventional IVF (34.3%) than ICSI cycles (10.3%). Conclusions: Allocation of sibling oocytes to IVF and ICSI in the first cycle minimizes risk of fertilization failure. For patients with limited number of oocytes, ICSI technique is recommended.