Hasan Acar

Repeated in vitro Fertilization Failure and Its Relation with Thrombophilia

Background: The objective of this study was to determine the prevalence of some thrombophilic factors and its relation to in vitro fertilization (IVF)-embryo transfer failure in women who had had three or more previously failed IVF-embryo transfer cycles. Methods: The study group included 51 consecutive women with three or more previously failed IVF-embryo transfer cycles (group 1). The control group included 50 women who conceived spontaneously with at least one uneventful pregnancy and no previous history of miscarriage. All women were tested for the presence of factor V Leiden, prothrombin (G20210A), and methylenetetrahydrofolate reductase (C677T) mutations. Results: A similar prevalence of factor V Leiden, prothrombin, and methylenetetrahydrofolate reductase mutations was found in both groups. At least one inherited thrombophilic factor was detected in 62.7% of women with repeated IVF failure and in 53.9% of women in group 2. No association between repeated IVF failure and these thrombophilic factors was found statistically. Conclusion: These data suggest that factor V Leiden, methylenetetrahydrofolate reductase and prothrombin gene mutation do not have a significant role in IVF-embryo transfer implantation failure.

Incidence of Chromosome 8, 10, X and Y Aneuploidies in Sperm Nucleus of Infertile Men Detected by FISH

We studied the frequency of aneuploidy in sperm nuclei of six infertile men with abnormal semen profile and normal karyotype, using fluorescence in situ hybridization (FISH) with DNA probes for chromosomes 8, 10, X and Y. The control group consisted of four healthy fertile men with normal karyotype and semen profiles. The purpose of this study was to determine whether there are differences between infertile male donors and control donors for: (1) the incidence of sex chromosome aneuploidy, and (2) the number of disomies for chromosomes 8, and 10 cosegregating with chromosomes X and Y. FISH analysis showed no significant differences of sex ratios of the sperm nuclei in and between infertile and control groups. The most significant abnormalities in the infertile group were clusters of sperm nuclei bearing XY and XYY. In addition, the incidence of disomic sperm nuclei for chromosomes 8 and 10 consegregating with sex chromosomes was not significantly different beween the patient and control groups, nor within them. However, the total frequency of aneuploid sperm nuclei was significantly different beween the infertile group and the control group. We observed a significant excess of sperm nuclei bearing chromosome 10 along with disomy for chromosome Y (10YY). In conclusion, our results from FISH analysis demonstrate a significantly increased frequency of aneuploidy for the sex chromosomes in sperm nuclei from infertile men. Therefore it may be concluded that infertility is a risk factor for sex chromosome aneuploidy in sperm nuclei.

Meiotic segregation analysis of reciprocal translocations both in sperms and blastomeres

Balanced chromosomal rearrangements could lead to unbalanced segregation gametes during meiosis. In this study, sperm flourescence in situ hybridization (FISH) analysis of meiotic segregation products of four reciprocal translocations; 46,XY,t(7;10)(q21;q22), 46,XY,t(15;17)(q11;p12), 46,XY,t(6;13)(p21.1;q32), and 46,XY,t(1;13)(q24;q10) are presented. In three out of these four cases with t(15;17), t(6;13), and t(1;13) additional blastomere FISH analyses are also provided. Multi‐color FISH analysis was applied using diverse probe combinations specific for translocated chromosome segments. The average frequency of sperm nuclei bearing unbalanced products for t(7;10), t(15;17), t(6;13), and t(1;13) were 48.7%, 59.5%, 60.5%, and 62.9%, respectively. Frequencies of blastomeres comprising unbalanced products in cases with t(15;17), t(6;13), and t(1;13) were 80% (12 of 15), 60% (3 of 5), and 50% (2 of 4), respectively. Chi‐square test analysis showed significant differences in the meiotic segregation patterns due to the distribution and numbers of the chiasmatas that could depend on the size of the translocated segments (P < 0.001). In conclusion, FISH analysis of sperm and blastomere for reciprocal translocation carriers effectively estimates the approximate risk of unbalanced products and this result might ensure valuable genetic counseling.

Evaluation of HLA-A, -B, -Cw, and -DRB1 alleles frequency in Turkish patients with nasal polyposis.

Objective To evaluate whether there is a relationship between HLA-A, -B, -Cw, and -DRB1 alleles and developing nasal polyposis (NP). Study Design Data from 66 patients with NP were compared with data from 100 healthy randomly selected controls. Asthma, ASA (acetylsalicylic acid) triad, polyp score, and previous sinonasal surgery were also recorded. Subjects and Methods Genotyping of the HLA-A, -B, -Cw, and -DRB1 alleles were performed with polymerase chain reaction (PCR) with the sequence-specific primer (SSP) method. Data were analyzed by using a Pearson χ 2 test. Results The HLA-B*07 and -Cw*12 alleles were found to be significantly higher in the NP patients compared with the control group, whereas the HLA-B*57 and HLA-Cw*04 alleles were significantly lower (P < 0.05). The HLA-A*24, HLA-Cw*12, and HLA-DRB1*04 alleles were determined to be significantly higher in the NP patients with asthma and ASA triad (P < 0.05). Conclusions Our results show that some of the HLA alleles seem to be associated with the genetic susceptibility to develop NP in the Turkish population.

Evaluation of chromosome 8 and 11 aneuploidies in washings and biopsy materials of bladder transitional cell carcinoma

We compared chromosome 8 and 11 aneuploidies on bladder biopsy tumor tissues and bladder washing samples of transitional cell carcinoma (TCC) and their relationship to tumor malignancy. Interphase fluorescence in situ hybridization (FISH) was applied to nuclei of washing material and biopsy samples of 17 patients with TCC. Incidence of cells having aneuploidy was clearly nonrandom from patient to patient. There was no significant difference in the incidence of aneuploid frequency for chromosomes 8 and 11 between biopsies of bladder tumors and bladder washing samples (P > 0.05). For chromosome 8, incidence of disomic cells (having two signals) in grade III tumors was significantly lower than in grade II tumors of both washing samples (P = 0.004) and biopsy materials (P = 0.005), indicating a high frequency of aneuploidy. The incidence of nuclei with four or more than four signals of chromosome 8 was significantly higher in grade III tumors than in grade II tumors in washing samples (P = 0.031 and 0.003, respectively). Similarly, in biopsy material, the incidence of nuclei with more than four signals of chromosome 8 was significantly higher in grade III tumors than in grade II tumors (P = 0.004). For chromosome 11, in both washing samples and biopsy materials, the incidence of disomic cells (having two signals) in grade III tumors was significantly lower than that detected in grade II tumors (P = 0.031 and 0.014, respectively), indicating a high frequency of aneuploidy. In biopsy materials, the incidence of nuclei with three or four signals was significantly higher than that in grade II tumors (P = 0.014 and 0.012, respectively). These findings suggest that FISH analysis of bladder washing samples can be effectively detected as genetic changes of bladder tumors. It might predict genetic progression of these tumors, which might be related to tumor stage, because higher stages of tumors showed a higher incidence of aneuploidies of chromosomes 8 and 11.

Philadelphia chromosome in chronic myelogenous leukemia: confirmation of cytogenetic diagnosis in Ph positive and negative cases by fluorescence in situ hybridization.

In this study, patients with Philadelphia (Ph) negative and positive chronic myelogenous leukemia (CML) were analyzed by unicolor- and dual color- (DC) fluorescence in situ hybridization (FISH) using abl and bcr cosmid probes. Unicolor- and DC-FISH analysis revealed a BCR-ABL fusion in a Ph-negative patient. DC-FISH to interphase nuclei revealed the BCR-ABL fusion, even though no metaphases were available for metaphase-FISH and conventional cytogenetic analysis. In the remaining cases, the findings of FISH were in agreement with the cytogenetic results.

Identification of variant translocations in chronic myeloid leukemia by fluorescence in situ hybridization

We studied two cases of chronic myeloid leukemia (CML) having variant complex translocations detected by trypsin G-banding and fluorescence in situ hybridization (FISH). Application of dual color- (DC-) FISH using abl and bor cosmid probes permitted us to detect the bor-abl fusion event on both interphase nuclei and metaphase spread. Furthermore, FISH using combinatorial hybridization (centromeric-library and library-library probes) demonstrated the content and the position of the translocations in CML patients with variant (complex type) Ph-positive rearrangements. FISH analysis appears to be superior than conventional cytogenetic analysis.

Chromosome 8 Aneuploidy in Acquired Cholesteatoma

Objective To investigate the incidence of chromosome 8 aneuploidy in acquired cholesteatoma. Material and Methods Cholesteatoma tissue and postauricular skin as a control were surgically obtained from 12 patients with acquired cholesteatoma. Fluorescence in situ hybridization (FISH) analysis using a chromosome 8-specific α-satellite DNA probe was performed on the interphase nuclei. Two hundred cells were analyzed for each of the samples. Results Chromosome 8 aneuploidy was found in 9/12 patients whereas a normal cell structure with 2 signals was observed in the remaining 3 patients. In samples with chromosome 8 aneuploidy, the mean proportion of aneuploidy was 25.6%, including monosomy (3.2%), trisomy (16.1%), tetrasomy (4.9%) and more than tetrasomy (1.4%). The number of aneuploid cells in recurrent cases was more than that in non-recurrent cases. Conclusion A numerical aberration of chromosome 8 was found in patients with acquired cholesteatoma. Our results support the hypothesis that chromosome 8 may be a prognostic factor for cholesteatoma and an indicator in the follow-up of patients with cholesteatoma.

Evaluation of c-MYC status in primary acquired cholesteatoma by using fluorescence in situ hybridization technique.

Objective: The object of study was to investigate the status of c-MYC oncogene in primary acquired cholesteatoma. Study design: Descriptive study. Methods Cholesteatoma samples were obtained from 15 patients with primary acquired cholesteatoma during surgical operation. Fluorescence in situ hybridization with a mixed DNA probe, which is specific for c-MYC located on 8q24 and chromosome 8 specific-alpha-satellite DNA probe (dual color), was used on the interphase nuclei. Results: Copy number of c-MYC oncogene and aneuploidy of chromosome 8 were 21.2% ± 14.4% and 21.7% ± 14.8%, respectively. There was no significant difference between copy number of c-MYC and frequency of chromosome 8 aneuploidy (p > 0.05). Ten of 15 cases showed different percentage of c-MYC and chromosome 8 aneuploidy, whereas 5 (33.3%) of 15 cases showed a normal distribution of c-MYC and chromosome 8 signals. Conclusion: The copy number of c-MYC in 10 of 15 cases was found to be high as observed for chromosome 8 aneuploidy in primary acquired cholesteatoma. These findings suggest that the ability of hyperproliferation of primary acquired cholesteatoma might have been related to c-MYC copy number by deregulating c-MYC expression.

Investigation of Developmental Toxicity and Teratogenicity of Macrolide Antibiotics in Cultured Rat Embryos

Macrolides are considered to be one of the safest anti‐infective groups in clinical use, with severe adverse reactions being rare. However, there are limited data about their embryotoxicity and teratogenicity. We aimed to investigate and compare the effects of these agents on embryonic growth and development. Rat embryos were cultured in vitro for 48 h in rat serum. Whole rat serum was used as a culture medium for the control group while different concentrations of spiramycin and azithromycin (1.25–6.25 μg/ml), and clarithromycin (2.5–30 μg/ml) were added to rat serum for the experimental groups. Dose‐dependent effects of macrolides on embryonic developmental parameters were compared using morphological methods. Embryos were evaluated for the presence of any malformations. After morphological examination of the embryos, total DNA was extracted from the cells using standard procedures to determine fragmentation of nuclear DNA of embryonic cells. When compared with the control embryos, the macrolides significantly decreased all growth and developmental parameters dose dependently. While clarithromycin was found to cause more developmental toxicity than spiramycin and azithromycin, azitromycin was determined to have more teratogenicity potential. Compared with controls, there was no difference regarding the fragmentation of nuclear DNA of all the agents used. According to these results, when the toxic and teratogenic potential of the used agents compared, because of the lower toxic and teratogenic effects observed with spiramycin, this agent may be preferred for parturients.