Objective To reduce the number of multiple pregnancies after IVF/ICSI treatment by an elective single embryo transfer, without significant reduction of pregnancy rate (PR). Design A pilot study. Setting Department of Obstetrics and Gynecology, University Hospital, Olomouc. Methods The patients were devided into the three groups according to number and quality of transferred embryos: elective Single EmbryoTransfer (eSET, n = 14), Single EmbryoTransfer (SET, n = 21), and Double EmbryoTransfer--control group (DET, n = 85). After 3 days of cultivation the embryos were transferred. In all groups the following parameters were compared: age of the patient, number of oocytes obtained, fertilization rate, number of frozen embryos, pregnancy rate (PR), implantation rate (IR), abortions (AB) and the number of multiple pregnancy. Results In eSET group PR and (or IR) was 42.8%, in SET group PR and (or IR) was 19%. There were no monozygotic twins in these groups. In DET group PR was 40.7% and IR 25.9%. Nine of these 33 pregnancies were twins (27%). Because of small number of the patients detailed statistical analyses were not performed. Conclusion An elective transfer of one TOP embryo can reduce the number of multiple pregnancies without significant decrease of pregnancy rate.
Imatinib změnil přistup k lecbě pacientů s chronickou myeloidni
leukemii (CIVIL). Na zakladě vynikajicich výsledků studie IRIS
se stal u nemocných s C M L lekem prvni volby. Nicmeně dat o
ucincich imatinibu na neselektovane populaci pacientů je stale
poskrovnu. Cilem studie bylo popsat ucinnost a toxicitu
imatinibu jako leku prvni volby u neselektovane skupiny
nemocných s CML, kteři byli prospektivně zařazovani do databaze
INFINITY. V databazi jsou evidovani vsichni po sobě
přichazejici nemocni s CML v 1. chronicke fazi, leceni
imatinibcm na dvou velkých hematoonkolo-gických pracovistich v
Praze a Brně. Analyzovana byla data od 105 pacientů (51 mužů a
54 žen), median věku 54 let (20-77). Median sledovani byl k
datu analýzy 16 měsiců (0,3-50). Median průměrne denni davky
imatinibu pro pacienta byl 400 mg. Po 12 měsicich lecby byly
zjistěny nasledujici lecebne odpovědi: kompletni hematologicka
remise 94 %, kompletni cytogeneticka odpověď 70 %, parcialni
cytogeneticka odpověď 14 %, velka molekularni odpověď 42 % se 4
% kompletnich molekularnich odpovědi. Median exprese Bcr-Abl
transkriptu ve 12. měsici lecby byl 0.15 % (0-31 %). Celkem 15
nemocných lecbu hnal mihem během sledovani ukoncilo: 5
podstoupilo alogenni transplantaci krvetvorných buněk, u 7
nemocných byl imatinib nahrazen jiným tyrosinkinazovým
inhibitorem pro lecebne selhani (6 dasatinib, l nilotinib) a u
3 nemocných byl imatinib nahrazen dasatinibem pro zavažnou
nehematologickou toxicitu. Během sledovani bylo zaznamenano
pouze jedno umrti, jehož přicinou vsak nebyla CML, ale
intoxikace alkoholem.
In a 66 years old female patient with acute myeloblastic leukemia (AML) complex chromosomal rearrangements involving 11q23 were diagnosed by G-banding and confirmed by different fluorescence in situ hybridization (FISH) techniques. The amplification of MLL gene differed in various sidelines as shown by locus specific probes for 11q23 and 11q13. Complex karyotype rearrangements involving deletions del(5)(q31) and del(7)(q31) were verified by multicolor fluorescence in situ hybridization (mFISH).
A variant of del(20q), an isochromosome of the long arm with the loss of an interstitial part of 20q, ider(20q), has been reported in patients with myeloid diseases (Li et al, 2004). About 40 cases with this rearrangement have been reported up to 2012 (reviewed by Mullier et al, 2012). Molecular cytogenetic and array techniques have been used for mapping of the deleted region on 20q (Douet-Guilbert et al, 2009). The proximal breakpoints are consistently located in the 20q11.21 band, and the distal breakpoints span from band 20q13.13 to band 20q13.33. A 79-year-old male patient was admitted to our hospital with a diagnosis of refractory anaemia with ring sideroblasts (RARS). His haemoglobin level was reduced to 108 g/l; other blood parameters were normal. A bone-marrow aspirate was typical for RARS and revealed mild hypogranular and strong vacuolated neutrophils. No specific haematological therapy was suggested, other than transfusion-supported treatment. The patient provided his written informed consent for the use of his sample for research purposes. The 46,XY,ider(20)(q10)del(20)(q11q13) karyotype was established in bone marrow cells. Fluorescence in situ hybridization (FISH) with a locus-specific probe, ON MDS 20q- (PTPRT 20q12)/20q11 (KREATECH Diagnostics, Amsterdam, the Netherlands) confirmed the 20q11/20q12 deletion and FISH studies with a subtelomeric probe, Vysis ToTel 20p/20q (Abbott Molecular, Des Plaines, IL, USA), proved the duplication of the subtelomeric 20q region and the deletion of the subtelomeric 20p region. Multicolour FISH banding of chromosome 20 (XCyte 20; MetaSystems, Altlussheim, Germany) showed ider(20)(q10)del(20)(q11.1q13.1). Array comparative genomic hybridization (aCGH, CytoChip Cancer 180K, BlueGnome, Cambridge, UK) was used to detect gene copy number variations. Two duplicated regions, the first ranging from 20p11 to 20q11.21 (bp 25 805 264–30 960 195) and the second from 20q13.2 to 20q13.33 (bp 52 080 063–62 949 120), were detected with two other deletions that included the short arm of chromosome 20 (except 20cen-20p11.2) and the 20q11.21–20q13.2 region (bp 30 971 874–52 045 077) (Fig 1A). This analysis confirmed that ider(20q) was a dicentric chromosome. Metaphase FISH mapping with a set of six bacterial artificial chromosome (BAC) probes (BlueGnome) distributed in 20q11.21 and 20q13.2, with a chromosome-20-specific centromeric probe [SE 20 (D20Z1); KREATECH Diagnostics] was performed. Based on the results (Table 1), we deduced that the region 20q11.21–20q13.2, including parts of the additional sex combs like 1 (ASXL1) and teashirt zinc finger homeobox 2 (TSHZ2) genes, had been deleted and the corresponding RNA levels were analysed to confirm this hypothesis. RNA was isolated from mononuclear cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was prepared (Marková et al, 2009). Four forward primers covering the hypothetically disrupted 5′ end of the ASXL1 gene were designed, together with 15 reverse primers distributed along the whole TSHZ2 gene. Eight multiplex polymerase chain reaction (PCR) reactions were performed using one of the forward primers together with a mixture of reverse primers 1–8 or 9–15. The amplified products were subjected to electrophoresis on 2% agarose gel and the only band amplified (247 bp, resulting from the ASXL1-for2 and TSHZ2-rev1–8 mixed primers) was directly sequenced using the forward primer and the BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems, Branchburg, NJ, USA). Sequencing analysis revealed a fusion between exon 4 of the ASXL1 gene and exon 3 of the TSHZ2 gene. This result was validated with a single PCR using primers ASXL1-for2 (5′-GTCATAGAGGCAGAAGGACT-3′) and TSHZ2-rev5 (5′-AGGAGGAGTTCAATGAGTTC-3′). Using a combination of all the techniques described above, the karyotype was shown to be 46,XY,idic(20)(p11)del(20)(q11.21q13.2), with the fusion gene ASXL1/TSHZ2 (Fig 1). The frequency of ider(20q) is very low, this is the first case we have observed in approximately 1500 patients with myeloid malignancies examined during the last 10 years. The identification depends on the resolution of the methods used. Li et al (2004) used G- and R- banding techniques in combination with FISH to first describe this aberration as ider(20)(q10)del(20)(q11q13). In follow-up studies, the same group revised all previously observed cases by mapping the 20p11.21–20p11.22 region with a BAC/PAC contig and showed that ider(20q) is a dicentric chromosome (Li et al, 2006). We delimited the proximal and distal breakpoints in our patient using aCGH and FISH with sets of BAC probes for the 20q11.21 and 20q13.2 regions respectively, and confirmed the aberrant dicentric chromosome. On the mRNA level, the disruption and partial loss of the ASXL1 (exons 5–12) and TSHZ2 (exons 1 and 2) genes was described. The ASXL1 gene maps to chromosomal region 20q11 and belongs to a family of three paralogues. This gene is one of the most frequently mutated genes in malignant myeloid diseases, and the majority of mutations affect exon 12 (with the consequent loss of the PHD domain), although a few deletions and translocations with fusions to the PAX5 gene have also been reported (reviewed by Gelsi-Boyer et al, 2012). Recent studies have also highlighted the role of the truncation of the PHD domain in malignant progression (Baker et al, 2008). The TSHZ2 gene is located in the 20q13.2 band and its role in aberrant methylation has been demonstrated in breast and prostate cancer, when the TSHZ2 and TSHZ3 genes were shown to be the most important candidates for novel tumour suppressor genes (Yamamoto et al, 2011). The TSHZ2 gene was partially deleted in our patient, so we hypothesized that its expression was downregulated. Similarly, the DNMT3B gene, responsible for DNA methylation, and MYBL2, a tumour suppressor gene, were deleted, which may contribute to the malignant transformation of cells. Several mechanisms accompanying the formation of ider(20q) contribute to the tumourigenesis of myeloid malignancies—the loss of tumour suppressor genes in the deleted regions on the short and long arms of chromosome 20 (Clarke et al, 2013); the overexpression of genes in the duplicated regions (Mackinnon et al, 2010); the aberrant methylation/demethylation of gene promoters (Yamamoto et al, 2011); and the formation of fusion genes. To our knowledge, this is the first report describing ASXL1/TSHZ2 fusion gene. The identification of further cases will be necessary to determine the frequency and significance of the ASXL1/TSHZ2 fusion and its implications for the treatment of patients with this rare but recurrent aberration. J.B. was responsible for the design of the study, the interpretation of the molecular cytogenetic results, and with H.B., for writing the manuscript. I.S., S.R., and S.I. performed the FISH analyses, and participated in the design and interpretation of the experiments. H.B. and K.K. performed the aCGH, and Z.Z. was responsible for the interpretation of the results of these two techniques and participated in the revision of the manuscript. J.M. performed the RNA and PCR analyses. J.S. provided medical care for the patient. K.M. supervised the manuscript preparation. All the authors read and approved the final manuscript. This work was supported by grants UHKT2005 00023736, RVO-VFN64165, GACR P302/12/G157/1, and PRVOUK-P27/LF1/1. All authors disclose that they have no conflicts of interest.
To evaluate morphological parameters of embryos obtained in the process of ICSI.A prospective study.Centre of Assisted Reproduction, Dept. of Obstetrics and Gynecology, Palacký University Medical School, Olomouc.In the present study 1116 embryos developing after ICSI (IntraCytoplasmic Sperm Injection) procedure in the period of 2001-2004 were evaluated. The beginning of the mitotic cleavage was assessed within the interval of 22-27 hours after insemination. The embryos were divided into three groups according to the speed of their division as Early Cleavage (EC) embryos, where two blastomeres were present at the time of assessment, Break Down ProNuclei stage (BDPN) where the pronuclei had already disappeared, and ProNuclei (PN) embryos, where both pronuclei were still present. In these groups the degree of fragmentation was evaluated on day two of cultivation and embryos were divided into four categories as: A--regular blastomeres, without fragmentation, B--irregular blastomeres or fragmentation below 30%, C--fragmentation 30-50%, D--fragmentation above 50%. The speed of further cleavage and average number of blastomeres were evaluated on day two and three of cultivation. Statistical analysis was preformed at the Palacky University Computer Centre. The chi2 test and t-test for independent samples were used.EC embryos were found in 37.4%, BDPN in 33.1% and PN in 29.5%. The degree of fragmentation between evaluated groups of embryos were statistically significant (p = 0.000). EC embryos were less fragmented (p = 0.000), had more blastomeres at the time of evaluation (p = 0.000) and their speed of cleavage was faster (p = 0.000). The cleavage of EC embryos was faster in comparison with the PN group (p = 0.000), but there were no significant differences between the EC and BDPN groups on day two of cultivation. On day three significant differences were found also between the EC and BDPN groups (p = 0.000). The embryonic developmental arrest was found only in PN embryos.The speed of the first cell cleavage is a useful additional criterion for the embryo selection for embryotransfer. EC embryos usually have better morphology and more blastomeres than the BPDN and PN ones.
