Aneuploidy rate and Stemness in Low-level Mosaic Human Embryonic Stem Cells in the Presence/Absence of Bortezomib, Paclitaxel and Lapatinib.

Human embryonic stem cells (hESCs) are predisposed to aneuploidy through continual passages. Some reports indicate more sensitivity of aneuploid hESCs cells to anticancer drugs. The present study was designed to investigate the cytotoxicity of three anticancer drugs (including bortezomib, paclitaxel and lapatinib) and their effect on aneuploidy rate in hESCs. To create a low-level mosaic cell line, normal hESCs (80%) and trisomic hESCs for chromosomes 12 and 17 (20%) were mixed. The effect of the 3 mentioned anticancer drugs on the chromosomal status was assessed by metaphase spread analysis after selection of the nontoxic conditions. Expression of pluripotency genes was analyzed and an alkaline phosphatase test was performed to assess pluripotency preservation. Our data showed that treatment with bortezomib, paclitaxel and lapatinib was nontoxic at 0.01, 0.01, and 0.2µM concentrations, respectively. Alkaline phosphatase and pluripotency gene expression analyses revealed maintenance of pluripotency following treatment with above-noted nontoxic concentrations. Aneuploid cells were dominant in treated and control groups with a minimum abundance of 70%, with no significant differences between groups. Drug treatments had no negative effect on pluripotency. Insensitivity of aneuploid cells in treatment groups could be related to the specific characteristics of each cell line in response to the drug and the proliferative superiority of cells with trisomies 12 and 17.

HOX cluster and their cofactors showed an altered expression pattern in eutopic and ectopic endometriosis tissues.

Endometriosis is major gynecological disease that affects over 10% of women worldwide and 30%-50% of these women have pelvic pain, abnormal uterine bleeding and infertility. The cause of endometriosis is unknown and there is no definite cure mainly because of our limited knowledge about its pathophysiology at the cellular and molecular levels. Therefore, demystifying the molecular mechanisms that underlie endometriosis is essential to develop advanced therapies for this disease. In this regard, HOX genes are remarkable because of their critical role in endometrial development and receptivity during implantation, which is attributed to their ability to mediate some of the sex steroid functions during the reproductive period. Access to the expression profiles of these genes would provide the necessary information to uncover new genes for endometriosis and assist with disease diagnosis and treatment. In this study we demonstrate an altered expression pattern for the HOX clusters (A-D) and their cofactors in both eutopic and ectopic conditions compared to control tissue biopsies. Remarkably, most of the intensive changes occurred in eutopic samples from endometriosis patients compared to control tissue biopsies. Pathway analysis revealed the involvement of differentially expressed genes in cancer that correlate with an association between endometriosis and cancer. Our results suggest critical roles for the HOX cluster and their cofactors in endometriosis pathophysiology.

Lapatinib Decreases the Preimplantation Aneuploidy Rate of in vitro Fertilized Mouse Embryos without Affecting Completion of Preimplantation Development.

One of the major reasons for implantation failure and spontaneous abortion is a high incidence of preimplantation chromosomal aneuploidy. Lapatinib simultaneously inhibits EGFR and HER2, leading to apoptosis. We hypothesized a higher sensitivity for aneuploid cells in preimplantation embryos to lapatinib based on reports of aneuploid cell lines being sensitive to some anticancer drugs. Late 2-cell mouse embryos were treated with lapatinib after determining a nontoxic dose. Morphologies were recorded 24, 48, and 60 hours later. The effect of lapatinib on the aneuploidy rate was evaluated by studying blastocyst cells using FISH. Although the rate of development to 8-cell and morula stage was higher in the control group (p < 0.05), there was no difference in development to the blastocyst stage at the same studied intervals between lapatinib-treated and control groups (p = 0.924). The mean number of cells in morula and blastocyst stages were not different between the groups (p = 0.331 and p = 0.175, respectively). The frequency of aneuploid cells and diploid embryos was, respectively, significantly lower and higher in lapatinib-treated embryos, (p < 0.001). Since lapatinib treatment reduced the aneuploidy rate without impact on the development of mouse preimplantation embryos to the blastocyst stage and number of total cells, lapatinib seems useful for prevention of preimplantation aneuploidy in in vitro fertilization.

Chromosomal instability reducing effect of paclitaxel and lapatinib in mouse embryonic stem cells with chromosomal abnormality.

Chromosomal abnormalities, as a frequent phenomenon in cultured embryonic stem cells (ESCs), is a major obstacle in the ESC application in regenerative medicine. Recent studies showed that aneuploid embryonic stem cells of humans and mice are more vulnerable to anticancer drugs, compared with normal cells. The aim of the current study was to evaluate effects of three anticancer drugs, paclitaxel, lapatinib and bortezomib, on mouse embryonic stem cells (mESCs) as a suitable and available model. To assess in vitro cell toxicity, two mESC lines were treated with the aforementioned drugs. Using G-band karyotyping and micronucleus assay, the effect of anticancer drugs in terms of reduction of chromosomal instability in the mESCs was evaluated in control and treatment groups. Also, apoptosis rate of both groups was estimated by FITC-Annexin V/Propidium Iodide (PI) double staining. In addition, the effect of these three drugs in maintaining the pluripotency was assessed through alkaline phosphatase assay and quantification of the expression of three key pluripotency genes, Nanog, Pou5f1 and Sox-2 was performed using Real Time PCR. The rate of numerical abnormalities after treatment with paclitaxel and lapatinib was lower than the control group. The expression level of pluripotency genes exhibited no significant difference between control and treatment groups. Administration of paclitaxel and lapatinib to the mESCs culture at an appropriate dose and in a timely manner could decrease chromosome stability without affecting pluripotency.

Noninvasive sexing of human preimplantation embryos using RT-PCR in the spent culture media: A proof-of-concept study.

Preimplantation genetic testing (PGT) routinely requires biopsy which is an invasive approach. The aim of this study was to examine a noninvasive approach for sexing of preimplantation embryos using polymerase chain reaction (PCR)/reverse transcriptase-PCR (RT-PCR) based on the presence of SRY DNA/RNA in the spent culture medium. Two groups were evaluated: in group 1, 40 embryos of routine PGT volunteers were cultured individually after biopsy and in group 2, 31 embryos were cultured individually until Day-5 post-fertilization. Each group was further divided into three subgroups: RNA extraction (RE), nucleic acid (NA) and DNase treated (DT). In the NA and DT subgroups, cDNA synthesis was performed directly on culture medium with or without DNase treatment in DT and NA subgroups, respectively. The results of sexing based on the PCR/RT-PCR in the culture medium, were compared with the results of sexing by fluorescence in situ hybridization (FISH) technique. In group 1, all samples were correctly diagnosed. In group 2, five female samples were misdiagnosed. Test's sensitivity, specificity and accuracy were 100 %, 94.44 % and 96.88 %, in RE, 100 %, 81.82 % and 93.55 % in DT and 100 %, 71.43 % and 85.71 % in NA, respectively. Preimplantation sexing without embryo biopsy, in the spent embryo culture media using RNA amplification based methods including RE and DT seem to be more reliable while nucleic acid based method, NA, led to the highest misdiagnoses probably due to DNA contamination. Since all male samples were correctly diagnosed in all subgroups of this preliminary study, preimplantation noninvasive sexing on culture medium seems feasible, however all sources of nucleic acid contamination must be carefully avoided.

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator.

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.

Causes for hospitalization and death in Iranian patients with ß-thalassemia major.

There are limited studies that have focused on the causes for hospitalization as an indicator of morbidity in patients with ß-thalassemia major (BTM). A cross-sectional study was conducted to determine the main causes for hospitalization and death in hospitalized BTM patients in a referral hospital in Shiraz, southern Iran. During a 5-year period, 555 BTM patients were admitted to the hospital, of which 390 (67.7%) were 10 to 20 years of age. The most frequent causes for hospitalization were splenectomy (23%), heart failure (22.6%), liver biopsy (22.2%), uncontrolled diabetes (10.9%), arrhythmia (7.2%), cholecystectomy (3.8%), hypoparathyroidism (2.1%), and sepsis (2%). Of the hospitalized patients, 65 (11%), with a mean age of 16.1 ± 4.2 years, died. The most common causes of death were cardiomyopathy (72.3%), infections (17%), malignancies (3.1%), and cerebrovascular accidents (3.1%). Survival of our patients was less than in developed countries and cardiac complications were the most common cause of mortality and morbidity in these patients. Regarding the key role of iron chelation in prevention of different complications in BTM, correction of iron chelation regimen should be well considered.

Frequency of cystathionine beta-synthase 844INS68 polymorphism in Southern Iran.

Iranian population with an Indo-European origin is one of the oldest populations in the world. Historical evidence suggests the close similarity in the origin of Iranian, European and north Indian population. However, there are few anthropological and genetic evidences on this subject. This study, which is the first report from Iran, was performed to investigate the genetic origin of Iranian population using a polymorphism in Cystathionine beta synthase (CBS) gene known as 844INS68bp in this respect, genomic DNA was extracted from the whole blood of 480 healthy normal blood donors referred to Fars Blood Transfusion Center, using a salting out method. The fragment containing 844INS68bp was amplified, the normal fragment was 174 bp and the fragment containing the insertion was 242 bp in length. Results indicated that 418 (87.08%) out of 480 individuals had a normal (N/N) genotype, 59 (12.29%) individuals were heterozygote (N/I) and 3 (0.63%) had homozygote a mutated genotype (I/I). The total frequency of 844INS68bp allele was found 6.8% which is similar to with the reported in White Caucasians. Comparison of the genotype of this study with the polymorphism in other populations revealed that Southern Iranian population has a great similarity with other Caucasians populations' especially South Italy and North America while differed from East Asian and African populations. These results are in agreement with the result of other studied polymorphisms. Therefore, despite the great admixture of Iranian population with the neighboring non-Caucasian populations during the time, Iranian population still share a genetic background with other Caucasian populations.