Percentage Of Aneuploidy Research Articles

DNA aneuploidy identifies a subset of Luminal subtype breast carcinoma patients with worse clinical outcome.

In breast carcinoma (BC), the relationship between the ploidy pattern and molecular subtyping is still unknown. We aim to investigate the prognostic impact of DNA ploidy within molecular subtypes of a large cohort of BC patients. The series involved 520 BC patients with no neoadjuvant therapy and a median follow-up of 115.5 months. Immunohistochemical assessment of hormonal receptors, ERBB2 (HER2) status and Ki67 proliferative activity was the basis of the surrogate molecular subtyping. Ploidy was evaluated by DNA flow cytometry in fresh/frozen tumour samples. Kaplan-Meier (K-M) survival estimation was used for prognostic statistical analysis. Luminal A subtype had the lowest prevalence of disease recurrences (23.6%) and deaths from disease (18.3%), while Luminal B (42.2%/37.9%), Triple-negative (46.4%/40.6%), and HER2-positive (55.9%/50.0%) subtypes had the highest. The Triple-positive subtype shows an intermediate/low frequency of adverse events (29.4% of disease recurrences and 17.6% of deaths from disease). Luminal A tumours were mostly diploid (55.3%), while Triple-negative and HER2-positive tumours showed a high incidence of aneuploidy (82.6% and 88.2%, respectively). Luminal B and Triple-positive tumours had an intermediate percentage of aneuploidy (63.8% and 70.6%, respectively). K-M survival curves showed that DNA aneuploidy is significantly associated with shorter disease-free survival and overall survival in Luminal A and Luminal B molecular subtypes. DNA aneuploidy identifies a subset of Luminal BC patients with worse clinical outcome, potentially eligible for more aggressive adjuvant therapy.

Polymorphic Rearrangements of Human Chromosome 9 and Male Infertility: New Evidence and Impact on Spermatogenesis.

Chromosomal polymorphisms are structural variations in chromosomes that define the genomic variance of a species. These alterations are recurrent in the general population, and some of them appear to be more recurrent in the infertile population. Human chromosome 9 is highly heteromorphic, and how its rearrangement affects male fertility remains to be fully investigated. In this study, we aimed to investigate the association between the polymorphic rearrangements of chromosome 9 and male infertility via an Italian cohort of male infertile patients. Cytogenetic analysis was carried out, along with Y microdeletion screening, semen analysis, fluorescence in situ hybridization, and TUNEL assays using spermatic cells. Chromosome 9 rearrangements were observed in six patients: three of them showed a pericentric inversion, while the others showed a polymorphic heterochromatin variant 9qh. Of these, four patients exhibited oligozoospermia associated with teratozoospermia, along with a percentage of aneuploidy in the sperm of above 9%, in particular, an increase in XY disomy. Additionally, high values for sperm DNA fragmentation (≥30%) were observed in two patients. None of them had microdeletions to the AZF loci on chromosome Y. Our results suggest that polymorphic rearrangements of chromosome 9 might be associated with abnormalities in sperm quality due to incorrect spermatogenesis regulation.

P-519 Relationship between the quality control parameters in PGT-A results by NGS with the chromosomal mosaicism diagnosis in trophoectoderm biopsies

Abstract Study question Are quality control (QC) parameters in NGS results related to mosaicism rate in trophoectoderm samples? Summary answer A high number of filtered reads in PGT-A results correlates with a higher mosaicism rate. But the overall noise does not influence the mosaicism rate. What is known already The implementation of NGS have led to increased reporting of mosaicism. There is intense research on the biological mechanisms behind mosaicism, but also on the technical factors that might be influencing in its diagnosis. Low quality samples and DNA amplification artefacts lead to an increase in overall noise after sample analysis. The Derivative Log Ratio (DLR) quality control parameter in sequencing results measures the overall noise present in a sample. High DLR values indicate noisy profiles. Additionally, insufficient numbers of sequencing reads will result in noisy profiles and false copy number assignments. Study design, size, duration A retrospective analysis of PGT-A results, corresponding to trophoectoderm biopsies from embryos on day 5, 6 or 7 of development, was performed (February 2017- November 2021). After the quality analysis, 5245 samples that reached the optimum QC measures were included (samples with DLR < 0.4 and number of filtered reads > 250.000). Participants/materials, setting, methods PGT-A was carried out with the VeriSeq kit (Illumina), after genome amplification with the Picoplex technique. Sequencing data was analyzed with BlueFuse Multi software (Illumina). Data analysis was performed using SPSSv20.0. An embryo was designated as mosaic when the percentage of aneuploidy for at least one chromosome was between 25 and 50%. For this study, we considered euploid-aneuploid and aneuploid-aneuploid mosaic embryos. Main results and the role of chance Considering the 5245 samples included in the analysis, the number of embryos carrying chromosomal mosaicism was 960, which corresponds to 18.3%. To analyze whether the percentage of mosaic embryos varied according to the DRL, we used a logistic regression analysis, introducing maternal and paternal age, embryo quality, and biopsy day as confounding variables, and categorized the DLR using as a point cut the median (0.19). There were no significant differences in the percentage of embryos carrying mosaicism between the group of higher DLR (≥0.19) compared to the group of lower DLR (<0.19) (18.7% vs 18.0%; p = 0.278). On the other hand, to study the relationship between mosaicism and the number of filtered reads obtained in the sequencing analysis, we performed the same analysis using the median of filtered reads (556.688) as cut point. The samples with a number of filtered reads higher than the median had a higher rate of mosaicism (19.3% vs 17.4%, p = 0.041). Moreover, we analyse the data according to the type of mosaicism (segmental or whole chromosome) and we did not observe any difference neither for the DLR nor for the reads. Limitations, reasons for caution The main limitation of this study compared with other studies is the different criteria in the mosaicism diagnosis between the different laboratories. Moreover, we are not able to identify if worse QC are due to issues with the biopsy procedure, sample quality, DNA amplification or library preparation. Wider implications of the findings The overall noise in PGT-A results doesn't seem to influence the mosaicism rate. However, increased mosaicism rate is observed in samples with high number of filtered reads. This finding suggests that too many cells may lead to amplification saturation and underestimation of the relative quantity of DNA. Trial registration number Not applicable

P–532 Embryo quality needs to be considered as a main criterion when selecting mosaic embryos for transfer

Abstract Study question Should we consider embryo quality as one of the most important criteria to follow when transferring a mosaic embryo? Summary answer Embryo quality is an implantation biomarker both for euploid and mosaic embryos, and also a determinant for selecting the most eligible mosaic for transfer. What is known already Several studies show the benefit of transferring mosaic embryos when there are no euploid embryos to transfer, and they still result in ongoing pregnancies and what is more important is that they result in healthy babies. Studies and guidelines suggest prioritizing mosaic embryos based on maternal age, chromosomes impacted, percentage of aneuploidy, number of chromosomes involved, type of mosaic (simple vs complex, segmental vs complete, monosomy vs trisomy) but embryo quality is never part of these criteria. Studies claim that mosaic implantation rate is lower than euploid embryos, but they never show if both populations are comparable in terms of quality. Study design, size, duration This is a retrospective observational study performed in a private centre between February 2018 and January 2020. The study includes the data analysis of 96 euploid blastocysts and 14 low risk mosaic blastocysts (defining low risk regarding chromosome syndromes and less than 50% level mosaicism). All transferred in single embryo transfer (SET) to 105 patients after PGT-A (mean maternal age 38,9 years). The SET factor enables us to track the implantation outcome of all embryos. Participants/materials, setting, methods PGT-A with NGS technology was offered to patients of advanced maternal age and/or with repeated IVF failures. Trophectoderm biopsies were performed on day 5 and/or day 6 embryos, with laser assistance. Blastocyst morphology was scored in 3 groups: A: excellent (AA, AB, BA), B: good (BB), C: average and poor-quality embryos (BC, CB, CC). (Gardner-Schoolcraft classification) Low risk mosaic embryo transfer was offered to patients with no euploid embryos to transfer. Main results and the role of chance We found no significant differences between both populations (euploid and mosaic embryos) in terms of embryo quality (Chi^2 p-value =0,0975) so we were able to compare the overall implantation of similar quality populations. Despite euploid implantation being higher as described in most studies, no statistical differences (Chi^2 p-value = 0,4344) were found in terms of implantation rates between mosaic (57,0%) and euploid (67,6%) blastocysts during the same period. There are no differences between the mean age of both groups (39,7 vs 38,8 years, respectively). The implantation rates for euploid blastocysts were 79,5% (n = 39), 62,7% (n = 51) and 33,3% (n = 6) in the A, B and C blastocyst quality groups, respectively, showing significative differences among the three groups. The implantation rates of low-risk mosaic blastocysts were 100% (n = 3), 62,5% (n = 8) and 0,0% (n = 3) in the A, B and C blastocyst morphology groups, respectively, showing also still significant differences among the three groups despite the small population. (Chi^2 p-values according to implantation: Euploid =0,0434; Mosaic=0,0419) We have also compared the three quality categories between both populations showing no significative differences (Chi^2 p-values according to quality: A = 0,4344; B = 0,9894; C = 0,2568), concluding that same quality embryos behave the same way despite being euploid or mosaic. Limitations, reasons for caution The study is limited by its retrospective nature and the low number of mosaic embryos transferred as they are the last option for transfer. Additionally, it is common to transfer more than one mosaic embryo to increase the chances of pregnancy, therefore losing implantation track. Wider implications of the findings: Embryo quality has always been a strong biomarker predictable for implantation and this is also true for mosaic embryos as well. It is a simple concept, but we cannot compare implantation potential of euploid embryos with mosaic embryos without describing both populations in terms of quality. Trial registration number Not applicable

Mesenchymal Stem/Stromal Cell Engulfment by Breast Cancer Cells Generates a Hybrid Cancer Cell Population with Dormancy and Chemoresistance

IntroductionDrug‐resistant dormant tumor cells are a major causes of breast cancer relapse and metastasis. It has been reported that in the tumor microenvironment breast cancer cells (BCC) can engulf mesenchymal stem/stromal cells (MSCs) and acquire dormancy. We previously documented the presence of BCC that engulf MSCs in clinical samples of breast cancer metastasis and showed that MSC engulfment leads to a more aggressive breast cancer phenotype, with an upregulated gene signature consisting of MSR1, WNT5A, ELMO1, IL1RL2, ZPLD1 and SIRPB1. However, the mechanisms of MSC engulfment, and the phenotypic features of hybrid breast cancer cells are still unclear. Here, we tested the hypothesis that MSC engulfment generates hybrid cancer cells exhibit a multinucleated phenotype and increased survival advantage, dormancy, and chemoresistance, and that the genes we identified may mediate MSC engulfment.MethodMSC labeled with DS‐RED and MDA‐MB‐231 labeled with GFP (GFP‐231) were co‐cultured in microfluidic cell pairing devices, and treated with antibodies against MSR1, WNT5A, ELMO1, IL1RL2, ZPLD1 and SIRPB1 or IgG control to quantify the percentage of MSC engulfing on‐chip. The conditioned media was subjected to a cytokine array. Live Imaging Microscopy was used to characterize the hybrid cancer cells in co‐cultures compared the GFP‐231. In Co‐cultures of DS‐RED‐MSC with GFP‐231 stably transduced with shWNT5A, shMSR1 or lentivirus control, and treated with Doxorubicin or untreated, we analyzed the percentage of DS‐RED+/GFP+ hybrid population, Ki‐67 low cells in G1, multinucleated cells (N4+), and the percentage of apoptotic cells using annexin V/PI.ResultsNeutralizing antibodies or shRNAs against WNT5A and MSR1 significantly reduced the percentage of MSC engulfment by MDA‐MB‐231 cells, and changed the cytokine profile in the conditioned medium of co‐cultures. In co‐cultures with DS‐RED‐MSCs, DS‐RED+/GFP+ hybrid cells had a higher percentage of Ki‐67 low in G1 and higher percentage of aneuploidy compared to GFP‐231+ cells, which were reversed by WNT5A and MSR1 shRNA downregulation. Doxorubicin treatment increased the percentage of DS‐RED+/GFP+ hybrids cells and decreased the percentage of apoptosis in the co‐cultures when compared to untreated controls.ConclusionsMSC engulfment by BCCs results in a hybrid multinucleated cell population with increased percentage of aneuploidy than BCC controls and a dormant phenotype characterized by higher of Ki‐67 low in G1 compared to controls. Hybrid cancer cells have a distinct cytokine profile than control BCCs with increased levels of pro‐metastatic proteins including thrombosponding‐1, UPAR, and VEGF. Hybrid cancer cells show increased resistance to Doxorubicin compared to control BCCs, which can be rescued by downregulation of WNT5A and MRS1.Support or Funding InformationRole of EZH2 Breast Cancer Progression – R01 to C. Kleer. Role of CCN6 (WISP3) in the progression and metastasis – R01 to C. Kleer‐ AIRC Investigator Grant (IG#18602) to S. Andò‐PRIN2015#2015B7M39T to S. Andò

Can cytoplasmic donation rescue aged oocytes?

BackgroundThe pregnancy and delivery rates following assisted reproductive technology (ART) start to decrease and that the miscarriage rate increases rapidly from 35 years old. The miscarriage rate exceeds 50% at 43 years old. The number of aneuploid fetuses in miscarriages increases according to female age, reaching more than 90% when women are over 40 years old.MethodsDifferent cytoplasmic donation technologies used to rescue aged oocytes with high percentage of aneuploidy were analyzed, and their efficacy compared.Main findings (Results)Germinal vesicle transfer (GVT) might be superior to spindle chromosome transfer (ST) theoretically from the point of higher capability of rescuing the disjunction at meiosis I which cannot be helped by ST. However, actually, in vitro maturation (IVM) of oocyte after GVT has not yet been totally completed. ST among other nuclear donations showed the higher possibility to rescue them, due to the fact it does not require in vitro maturation and it has an ethical advantage over pronuclear transfer (PNT) which requires the destruction of an embryo.ConclusionSpindle chromosome transfer has the potential to rescue aged oocytes to some extent, but we have to continue the basic study further to establish the clinical application of cytoplasmic donation to rescue aged oocytes.

A novel algorithm for determining the level of mosaicism in preimplantation genetic screening (PGS) with next-generation sequencing (NGS)

Introduction: Filtering PCR-artifacts, normalization and aberrations calling are common tasks implemented in any software designed for analyzing NGS data. However, most of the algorithms have been developed for post-natal diagnosis, where NGS has recently become a useful but not fully compatible approach for PGS. Previous methods based on whole-genome sequencing (WGS) require high-depth coverage on the whole genome scale and are not cost-efficient. Moreover, they do not determine precisely the level of mosaicism, because this is very uncommon in post-natal diagnosis. In order to accurately identify genome-wide aneuploidies by NGS, we implemented an algorithm that can identify the percentage of aneuploidy for embryo-biopsies. This algorithm is able to improve confidence-value and reduce the risk of misdiagnosis. Less reads are required enabling high-level multiplexing.

Aging of bone marrow– and umbilical cord–derived mesenchymal stromal cells during expansion

Background aimsMesenchymal stromal cells (MSCs) are used as experimental immunotherapy. Extensive culture expansion is necessary to obtain clinically relevant cell numbers, although the impact on MSCs stability and function is unclear. This study investigated the effects of long-term in vitro expansion on the stability and function of MSCs. MethodsHuman bone marrow–derived (bmMSCs) and umbilical cord–derived (ucMSCs) MSCs were in vitro expanded. During expansion, their proliferative capacity was examined. At passages 4, 8 and 12, analyses were performed to investigate the ploidy, metabolic stability, telomere length and immunophenotype. In addition, their potential to suppress lymphocyte proliferation and susceptibility to natural killer cell lysis was examined. ResultsBmMSCs and ucMSCs showed decreasing proliferative capacity over time, while their telomere lengths and mitochondrial activity remained stable. Percentage of aneuploidy in cultures was unchanged after expansion. Furthermore, expression of MSC markers and markers associated with stress or aging remained unchanged. Reduced capacity to suppress CD4 and CD8 T-cell proliferation was observed for passage 8 and 12 bmMSCs and ucMSCs. Finally, susceptibility of bmMSCs and ucMSCs to NK-cell lysis remained stable. ConclusionsWe showed that after long-term expansion, phenotype of bmMSCs and ucMSCs remains stable and cells exhibit similar immunogenic properties compared with lower passage cells. However, immunosuppressive properties of MSCs are reduced. These findings reveal the consequences of application of higher passage MSCs in the clinic, which will help increase the yield of therapeutic MSCs but may interfere with their efficacy.

Insights on the association between somatic aneuploidy and ostreid herpesvirus 1 detection in the oysters Crassostrea gigas , C. angulata and their F1 hybrids

Cytogenetic abnormalities associated with viral infections, including from viruses of the Herpesvirales order, have been reported in vertebrate species. Ostreid herpesvirus 1 (OsHV-1) has been detected worldwide during mortality outbreaks of the Pacific oyster Crassostrea gigas. On the other hand, a high proportion of aneuploid cells in somatic tissues have been observed in C. gigas. In this study, we analysed the putative association between aneuploidy levels and the detection of OsHV-1 in gills of C. gigas, the Portuguese oyster C. angulata and their F1 hybrids cultured in Ria Formosa (Portugal). OsHV-1 was detected by PCR in 5.4% of the total of oysters analysed (n = 111) namely in 11.1%, 8.0% and 1.7% of C. gigas, C. angulata and F1 hybrid respectively. Sequencing analysis of a viral fragment amplified with the C2/C6 primer pair revealed a high similarity with the OsHV-1 reference type. Moreover, in situ hybridization confirmed the presence of OsHV-1 in gill tissue. Oysters where OsHV-1 was detected had a significantly higher mean percentage of aneuploid cells (25%) than the ones where the virus was not detected (18%). However, the overall low percentage of positive samples contrasted with the high mean percentage of aneuploidy observed, with 50% of the oysters analysed showing a percentage of aneuploid cells between 20% and 30%. We hypothesize that somatic aneuploidy may adversely affect oysters making them more prone to OsHV-1 infection, but the virus is unlikely to be the cause of somatic aneuploidy.

Apoptosis mediated by phosphatidylserine externalization in the elimination of aneuploid germ cells during human spermatogenesis.

It has been described that aneuploidies trigger cell cycle checkpoints leading to apoptosis. The aim of this study was to assess the relationship between the presence of chromosomal abnormalities and apoptosis in germ cells and in Sertoli cells. Fourteen diagnostic testicular biopsies from infertile patients were processed following a sequential methodology, which included enzymatic disaggregation, apoptotic staining, cell sorting, cell fixation, and fluorescent in situ hybridization analysis. The chromosome constitution of germ cells (interphase pre-meiotic germ cells, meiotic figures, post-reductional germ cells, and spermatozoa) and Sertoli cells was evaluated in non-sorted and flow-sorted cell populations (apoptotic and viable). The mean percentage of aneuploidy was compared between the three fractions in each cell type using a Kruskal-Wallis test. If significant results were obtained, a two-by-two Chi-squared test was performed. There were significant differences between the apoptotic fraction and the viable and non-sorted fractions in the pre-meiotic germ cells (p<0.01). In the remaining cell types, no association between the presence of aneuploidy and apoptotic processes was observed, even in the case of post-reductional germ cells in which we detected the highest rates of aneuploidy regardless of the fraction analyzed. From our data, it can be inferred that most of the aneuploid post-reductional germ cells are efficiently removed from the testicular epithelium without differentiating into spermatozoa. Our results suggest that the elimination of aneuploid testicular epithelial cells is triggered by different mechanisms. Accordingly, the cellular elimination of aneuploid germ cells beyond the blood-testis barrier does not involve phosphatidylserine externalization.

The relationship between blastocyst grade, time of blastocyst formation, maternal age and chromosomal aneuploidy

To evaluate the correlation between blastocyst grade, time of blastocyst formation, maternal age and chromosomal aneuploidy. Retrospective analysis. 377 patients who underwent IVF at our center from Jan 2013 to Dec 2013 were included. A total of 1403 blastocysts underwent trophectoderm biopsy and comparative genomic hybridization (CGH). The association of morphologic score, time of blastocyst formation, maternal age and the rate of aneuploidy were analyzed. Embryos that progressed to blastocysts on day5 were less likely to be aneuploid than day6 blastocysts (Table1 A). A surprisingly high percentage of grade AA blastocysts were aneuploid. More than half of the grade AB and BB blastocysts were aneuploid (Table 1B). The aneuploidy rate increased significantly with maternal age (Table 1C). Our data suggest a correlation between aneuploidy and the time of blastocyst formation, blastocyst quality and maternal age. A significant proportion of grade AA blastocysts were aneuploid suggesting morphologic analysis alone is insufficient to ensure transfer of normal embryos. Younger age groups had a high percentage of aneuploidy suggesting that PGS testing may be considered even in younger patients. Very few D6 blastocysts from patients >40 years were euploid. PGS-testing should be strongly considered in D6 embryos in this age group.Tabled 1THE ASSOCIATION BETWEEN TIME OF BLASTOCYST FORMATION, EMBRYO GRADE, MATERNAL AGE AND RATE OF ANEUPLOIDYTable1 (A) Association between time of blastocyst formation and aneuploidyAneuploidy Rate (%)Age GroupsD5 BlastocystD6 Blastocystp valuedonor29 (n=217)38 (n=88)0.17<3524 (n=229)46 (n=128)0.00*35-3737 (n=139)53 (n=100)0.02*38-4055 (n=160)68 (n=124)0.04*41-4276 (n=80)84 (n=45)0.36>4276 (n=42)93 (n=51)0.04*Table1 (B) Association between embryo grade and aneuploidyAneuploidy Rate (%)p valueAAAB/BABBAA vs AB/BAAA vs BBAB/BA vs BBD5 Blastocyst31 (n=527)55 (n=197)58 (n=118)0.00*0.00*0.64D6 Blatocyst42 (n=177)58 (n=118)69 (n=224)0.120.00*0.04*Table1 (C) Association between maternal age and aneuploidyAge Groupsdonor<3535-3738-4041-42>42Aneuploidy Rate (%)323344617985p valuedonor vs <35 p=0.87; donor vs 35-37 p=0.00*; donor vs 38-40 p=0.00*; donor vs 41-42 p=0.00*; donor vs >42 p=0.00*; <35 vs 35-37 p=0.00*, <35 vs 38-40 p=0.00*; <35 vs 41-42 p=0.00*; <35 vs >42 p=0.00*; 35-37 vs 38-40 p=0.00*; 35-37 vs 41-42 p=0.00*; 35-37 vs >42 p=0.00*; 38-40 vs 41-42 p=0.00*; 38-40 vs >42 p=0.00*; 41-42 vs >42 p=0.29* p<0.05 statistical significant difference Open table in a new tab

Comparison of clinical staging of benign and malignant ovarian tissues with DNA flow cytometry

The prevention, diagnosis and treatment of ovarian cancer are major issues. The outcome of patients with advanced ovarian cancer is poor despite aggressive therapy including surgery, combination drug chemotherapy and radiation treatments. From the literature, the direct correlation between DNA ploidy and survival is greatly enhanced using high resolution DNA measurements, which results in a coefficient of variation (CV) range between 1%-2% (1.42 ± 0.19, n=66) for trout red blood cells (TRBC) and 2%-3% (2.18 ± 0.46 SD, n=22) for tonsil nuclei derived from formalin-fixed, paraffin-embedded tissues (deparaffinated). DNA nuclear determinations from 50 ovarian cancer and 21 benign patients is presented. This was accomplished by measuring the DNA content of nuclei simultaneously isolated from deparaffinated tissues, stained with the fluorescent DNA specific dye, 4’, 6-diamidino-1-phenylindole (DAPI) and analyzed on a high resolution flow cytometer. A high percentage of aneuploidy (92.0%) was determined from the ovarian cancer patients, especially of the aneuploid DNA histogram types, such as hypodiploid, multiploid and hypertetraploid (64.0%), which have shown poor prognosis in a variety of cancers including ovarian. Furthermore, aneuploidy was detected in 23.8% of the benign patients. DNA flow cytometry may complement pathological assessment of ovarian cancer to better determine malignancy, thus justifying a closer follow-up with more specialized approaches to treatment in clinical trials that involve new therapies including the use of chemically defined, natural products, which may help offset the overall poor prognosis seen in ovarian cancer.

Optimizing IVF outcomes for women with diminished oocyte reserve

Women with diminished oocyte reserve do not have very poor oocyte quality similar to women of advanced reproductive age. The very poor pregnancy rates found in many studies of IVF-ET in this population seems to be related to the use of high-dosage FSH stimulation. The presence of significant elevated FSH levels leave many women more prone to FSH receptor downregulation. The best ‘hypothesis’ to fit the poor success rate found with high FSH stimulation is that FSH receptor downregulation leads to an insufficient production of a factor needed to prevent non-disjunction of chromosomes, leading to the creation of embryos with a high percentage of aneuploidy. Mild stimulation, to a reasonable degree obviates the problem with FSH receptors and results in the production of live healthy babies.

DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer

ObjectiveThe present study was undertaken to analyze the impact of epigenetic alterations with a main focus on nuclear area, aneuploidy, hyperploidy, and proliferation in 70 ovarian cancer specimens. MethodsMorphometric changes and somatic chromosomal ploidy status were assessed by Feulgen spectrophotometry. DNA-hypomethylation of LINE1 repeats was analyzed by means of MethyLight PCR, and methylation levels of satellite 2 (Sat2) and satellite alpha (Satα) DNA sequences in chromosome 1 were measured by Southern blot analysis. These parameters were analyzed with regard to correlations as well as to recurrence and survival. ResultsWe identified a significant association between LINE1 DNA-hypomethylation and patient age (p=0.029). Furthermore, LINE1 DNA-hypomethylation was positively correlated with the nuclear area (r=0.47; p<0.001) and the proliferation index (r=0.36; p<0.001). Univariate survival analysis showed that the nuclear area and LINE1 DNA-hypomethylation were prognostic factors for overall (p=0.015 and =0.006, respectively) and progression-free survival (p=0.020 and p=0.001 respectively), the percentage of aneuploidy only for overall survival (p=0.031). Subgroup survival analyses revealed that the prognostic value of these factors is strictly confined to mucinous cancers. In serous cancers no prognostic value could be pointed out for any analyzed parameter. Multivariate analysis of the entire cohort showed that the percentage of hyperploidy was an independent prognostic parameter for overall survival (p=0.003) and LINE1 DNA-hypomethylation for progression-free survival (p=0.03). In mucinous cancers nuclear area and LINE1 DNA-hypomethylation were found to be independent predictors of progression-free and overall survival. ConclusionsIn this study we identified the correlations between early cancer-associated genome DNA-hypomethylation, nuclear morphometric changes, somatic chromosomal ploidy status and the proliferation index. Prognostic relevance of nuclear area and LINE1 DNA-hypomethylation was revealed exclusively in mucinous ovarian cancers.

Prognostic factors correlation between the cell cycle phases and apoptosis in basal cell carcinoma

Basal cell carcinoma (BCC) is the most common skin malignancy, are found in various forms depending on their clinical and biological behavior. The objective of study was analyzed the phases of the cell cycle and correlations between BCC of low and high risk of recurrence and correlation prognostic factors. The quantity of content DNA in tissues of normal skin, showed small amount of cells in apoptosis and mostly in phase quiescent and rare aneuploidy cells. In BCC, apoptosis was higher in the BCC at high risk than low risk, probably due to their high rates of cell proliferation, and present of aneuploidy cells, when compared to the average percentage of aneuploidy. The DNA content from cells of normal skin shows that the majority is in the quiescent phase; compatible with tissues that are is refreshing. There is presence of apoptosis in the epidermis by probable normal process of differentiation. The aneuploidy in BCC showed a direct correlation with the degree of tumor aggressiveness.

45,X/46,XX mosaicism below 30% of aneuploidy: clinical implications in adult women from a reproductive medicine unit

Turner's syndrome (TS) is well known, but prognosis for 45,X/46,XX mosaicism below 30% of aneuploidy has not been established. We evaluated differences in clinical features and biological parameters between patients with numerical sex chromosome mosaicism diagnosed incidentally and control women. Retrospective observational study of clinical features and biological parameters. Standard endocrinological and gynecological examination was done and early-follicular-phase blood values were collected from the medical records of women aged 21-43, who were referred to our ward from 1996 to 2006 because of infertility and were karyotyped. Seventy-one women with sex chromosome mosaicism (45,X/46,XX) ranging from 4 to 28% were assigned a chromosomally normal woman (46,XX) matched according to age (n=71). In group 45,X/46,XX, 8% or more of aneuploidy accounted for a smaller height compared to controls (P=0.01). Body mass index was increased from 6% of aneuploidy (P=0.02) and was positively correlated to the percentage of 45,X cells (P=0.0001); menarche occurred earlier from 10% of aneuploidy (P=0.01) and was inversely correlated to the percentage of 45,X cells (P=0.045). No difference was found between the groups for FSH, LH, estradiol, inhibin B, and TSH values. Spontaneous abortions were more frequent in case of mosaicism (P=0.01), and recurrence was positively correlated to the percentage of aneuploidy (P=0.008). Sex chromosome mosaicism is responsible for clinical changes from 6% of aneuploidy, corresponding to the main phenotypical features of TS.

Aneuploidy Rates in IVF Patients ≤34 Years Old Based on the Indication for Preimplantation Genetic Diagnosis Aneuploidy Screening (PGD-AS)

Background: Studies on the rate of aneuploidy in IVF patients have shown varied results, ranging from 50% to 90% of the embryos. Despite this compelling data, controversy remains regarding which patients, if any, benefit from PGD-AS.Objective: We compared the rates of aneuploidy in young IVF patients based on the indication for PGD-AS to further elucidate which patient groups benefit most from this technique.Design: Cohort case report.Materials and Methods: All patients with female age ≤34 years undergoing PGD-AS at the ART Reproductive Center from January 2007 through November 2007 were divided into categories based on their indication for PGD-AS: recurrent pregnancy loss (RPL), recurrent implantation failure (RIF), gender determination (GD), and egg donor recipient cycles (ED).Tabled 1Age (n = 46)≤34 yrsGD (n = 17)49.6 (59/119)RIF (n = 4)40 (14/35)RPL (n = 12)27.9 (29/104)ED (n = 13)51.2 (80/156) Open table in a new tab Tabled 1Age (n = 46)≤34 yrsGD vs. RIF.342GD vs. RPL.001GD vs. ED.597ED vs. RIF.297ED vs. RPL.001RIF vs. RPL.207 Open table in a new tab There was no significant difference in aneuploidy rates in GD patients compared with patients who underwent PGD-AS for ED. Women with a history of RPL had a significantly higher rate of aneuploidy than patients who requested PGD-AS for GD or ED. There was no significant difference in aneuploidy rates between patients in the ROF and RPL groups.Conclusions: The nonsignificant difference between the RIF and RPL groups may be biased by the small sample sizes of both groups. In women ≤34 years old with RIF, the percentage of aneuploidy is the same as that for the GD and ED groups. Women ≤34 years of age with RPL showed a significantly higher rate of aneuploidy compared with those who had PGD-AS for GD and ED cycles, suggesting that this subset of patients may benefit from PGD-AS. Aneuploidy screening remains the only way to determine whether an embryo, regardless of its morphology, possesses a balanced set of chromosomes and is therefore capable of initiating a sustainable pregnancy. Background: Studies on the rate of aneuploidy in IVF patients have shown varied results, ranging from 50% to 90% of the embryos. Despite this compelling data, controversy remains regarding which patients, if any, benefit from PGD-AS. Objective: We compared the rates of aneuploidy in young IVF patients based on the indication for PGD-AS to further elucidate which patient groups benefit most from this technique. Design: Cohort case report. Materials and Methods: All patients with female age ≤34 years undergoing PGD-AS at the ART Reproductive Center from January 2007 through November 2007 were divided into categories based on their indication for PGD-AS: recurrent pregnancy loss (RPL), recurrent implantation failure (RIF), gender determination (GD), and egg donor recipient cycles (ED). There was no significant difference in aneuploidy rates in GD patients compared with patients who underwent PGD-AS for ED. Women with a history of RPL had a significantly higher rate of aneuploidy than patients who requested PGD-AS for GD or ED. There was no significant difference in aneuploidy rates between patients in the ROF and RPL groups. Conclusions: The nonsignificant difference between the RIF and RPL groups may be biased by the small sample sizes of both groups. In women ≤34 years old with RIF, the percentage of aneuploidy is the same as that for the GD and ED groups. Women ≤34 years of age with RPL showed a significantly higher rate of aneuploidy compared with those who had PGD-AS for GD and ED cycles, suggesting that this subset of patients may benefit from PGD-AS. Aneuploidy screening remains the only way to determine whether an embryo, regardless of its morphology, possesses a balanced set of chromosomes and is therefore capable of initiating a sustainable pregnancy.

Aneuploidy and Confined Chromosomal Mosaicism in the Developing Human Brain

BackgroundUnderstanding the mechanisms underlying generation of neuronal variability and complexity remains the central challenge for neuroscience. Structural variation in the neuronal genome is likely to be one important mechanism for neuronal diversity and brain diseases. Large-scale genomic variations due to loss or gain of whole chromosomes (aneuploidy) have been described in cells of the normal and diseased human brain, which are generated from neural stem cells during intrauterine period of life. However, the incidence of aneuploidy in the developing human brain and its impact on the brain development and function are obscure.Methodology/Principal FindingsTo address genomic variation during development we surveyed aneuploidy/polyploidy in the human fetal tissues by advanced molecular-cytogenetic techniques at the single-cell level. Here we show that the human developing brain has mosaic nature, being composed of euploid and aneuploid neural cells. Studying over 600,000 neural cells, we have determined the average aneuploidy frequency as 1.25–1.45% per chromosome, with the overall percentage of aneuploidy tending to approach 30–35%. Furthermore, we found that mosaic aneuploidy can be exclusively confined to the brain.Conclusions/SignificanceOur data indicates aneuploidization to be an additional pathological mechanism for neuronal genome diversification. These findings highlight the involvement of aneuploidy in the human brain development and suggest an unexpected link between developmental chromosomal instability, intercellural/intertissular genome diversity and human brain diseases.

Prognostic significance of DNA cytometry in combination with AgNOR investigation

Conclusion. While most results concerning DNA and nucleolar organizer region (AgNOR) parameters fit with previous studies, the percentage of aneuploidy looks like a promising prognostic parameter. The observed intratumoral heterogeneity could represent a possible source of conflicting and inconsistent results. Objectives. The aims of our study were to determine the prognostic relevance of different DNA and AgNOR parameters in laryngeal squamous cell carcinoma (SCC) and compare these findings with established prognostic factors including tumor stage and grade, as well as the detection of possible intratumoral heterogeneity. Materials and methods. Sections from 62 laryngeal SCCs were analyzed for DNA content, DNA index, S-phase, percentage of aneuploidy, and AgNOR. Of 62 samples, 31 morphologically similar tumor samples were analyzed for the same parameters in three different tumor areas defined as tumor center, invasive tumor margin, and transformation margin between tumor and normal-appearing mucosa. Results. Our study showed that DNA and AgNOR parameters correlated with T stage, lymph node involvement, and histologic grade regardless of tumor areas. Significant correlation was found between mean number of AgNOR per nucleus and percentage of aneuploidy. Clinical stage and percentage of aneuploidy correlated with survival (p<0.02). Heterogeneity DNA study revealed aneuploidy in central portions of 90% of tumors, while in margins aneuploidy was demonstrated in about half of the patients.

4CHROMOSOMAL STABILITY OF AFRICAN WILD CAT (FELIS SILVESTRIS LIBICA) SOMATIC CELLS AND CLONED EMBRYOS

Nuclear transfer (NT) procedures are generally inefficient and chromosomal abnormalities have been suggested as one causative factor. Embryos derived by somatic cell NT show a high incidence of aneuploidy, which seems to be reflective of the donor cell line with high chromosomal abnormalities (Bureau et al., 2003, Theriogenology 59, 239). Also, aneuploidies in some cell lines increase progressively with the number of passages (Denning et al., 2001, Cloning 3, 221–231). Therefore, the aim of the present study was to analyze the chromosomal stability of donor cells at different passages as well as that of reconstructed embryos derived from these cells. A primary culture of African wild cat (AWC) fibroblast cells was established from a skin sample and cultured until cells stopped dividing at passage 9. Chromosome numbers were determined in cells using a karyotyping technique (Iwasaki et al., 1992 J Exp Zool 261, 79–85) at passages 1 and 3 through 9. At passages 1, 3, 4 and 9, NT of AWC fibroblast cells into domestic cat cytoplasts was done (Gomez et al., 2003, Biol Reprod 69, 1032–1041). Reconstructed blastocysts were treated with colcemid (0.28μgmL−1) and fixed for karyotyping to evaluate chromosome numbers in the blastomeres. Blastomere metaphases were often highly contracted or overlapping, so that only 1 to 7 sets of metaphase chromosomes per embryo were spread sufficiently to allow determination of ploidy. No attempt to produce NT embryos using cells at passages 5 through 8 was done because the percentages of aneuploidies of these passages were not significantly different (P&amp;gt;0.05). Data were analyzed by chi-square test (P&amp;lt;0.05). As shown in the Table, the percentage of aneuploidy in the somatic cells increased progressively with duration of culture. Furthermore, the percentage of chromosomal abnormalities in reconstructed embryos was similar to that of the cells from which they were derived. Accordingly, for future NT, we propose to use donor cells at early passages when the percentage of cells with chromosomal abnormalities is still relatively low. Research was funded partially by the John &amp;amp; Shirley Davies Foundation. Chromosomal abnormalities in AWC cells and cloned embryos