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Summary: Background: In vitro embryo production (IVEP) is increasingly popular but data assessing the outcome of transferred embryos are scarce. Objectives: To determine the likelihood of pregnancy and embryonic loss after transfer of frozen‐thawed IVP embryos and identify factors influencing success. Study design: Retrospective clinical study. Methods: Blastocysts (n = 261) were produced from immature oocytes of Warmblood mares (n = 116) by Intracytoplasmic Sperm Injection (ICSI) and in vitro culture, and cryopreserved. Thawed IVP embryos were transferred into recipient mares on day 4, 5 or 6 after ovulation. The influence of donor mare (age, reproductive history), recipient mare (age, reproductive status, management; in‐house vs. outpatient, day post‐ovulation), embryo (interval from ICSI to blastocyst formation) and management factors (season when ovum pickup was performed, year and method of transfer) on likelihood of pregnancy and embryonic loss was examined, and the developmental stage of the IVP embryo at the time of transfer was estimated. Results: The percentage of mares pregnant 7–10, 23 and 37 days after transfer was 56% (147/261), 49% (129/261), and 48% (124/261), respectively. Development of IVP embryos after transfer equated to day 5 or 6 in vivo embryos. With the exception of year of transfer, none of the factors had an impact on the likelihood of pregnancy or embryonic loss. Nevertheless, the likelihood of pregnancy tended to be lower for IVP embryos from infertile mares or when embryos were transferred into recipient mares on day 6 after ovulation rather than on day 4 or 5. Finally, the diameter of the embryonic vesicle 7 days post transfer was lower for pregnancies that were lost compared to those that were maintained. Main limitations: Small sample size in some of the donor and recipient mare categories. Conclusions: Cryopreserved IVP embryos should be transferred into recipient mares on day 4 or 5 after ovulation and a slower rate of post transfer vesicle expansion indicates a higher risk of subsequent embryonic loss The Summary is available in Portuguese ‐ see Supporting Information [ABSTRACT FROM AUTHOR]

Summary: Background: Advanced mare age is associated with declining fertility and an increased risk of early pregnancy loss. Compromised oocyte quality is probably the primary reason for reduced fertility, but the defects predisposing to embryonic death are unknown. In women, advanced age predisposes to chromosome segregation errors during meiosis, which lead to embryonic aneuploidy and a heightened risk of miscarriage. Objectives: To evaluate the effect of advanced mare age on chromosome alignment and meiotic spindle morphology in in vitro‐matured (IVM) oocytes. Study design: Morphometric and morphological analysis. Methods: To investigate differences in spindle organisation and chromosome alignment between young and old mares, oocytes collected from slaughtered mares were divided into two groups depending on mare age (young, ≤14 years and old, ≥16 years), IVM and stained to visualise chromatin and alpha‐tubulin. Spindle morphology, morphometry and chromosome (mis)alignment were evaluated by confocal microscopy and 3D image analysis. Results: Oocytes from old mares showed a higher incidence of chromosome misalignment (47.4% vs. 4.5%; P<0.001) and a thicker metaphase plate (mean ± s.d.: 5.8 ± 1.0 μm vs. 4.9 ± 0.9 μm; P = 0.04) than oocytes from young mares. Although no differences in spindle morphometry were detected between old and young mares, an increased major spindle axis length was associated with chromosome misalignment (mean ± s.d.: 25.3 ± 6.1 μm vs. 20.8 ± 3.3 μm; P = 0.01) irrespective of age. Main limitations: The oocytes were IVM and may not exactly reflect chromosome misalignment in vivo. Conclusions: Advanced mare age predisposes to chromosome misalignment on the metaphase II spindle of IVM oocytes. The compromised ability to correctly align chromosomes presumably predisposes to aneuploidy in resulting embryos and thereby contributes to the age‐related decline in fertility and increased incidence of early pregnancy loss. The Summary is available in Portuguese – see Supporting Information [ABSTRACT FROM AUTHOR]

The in vitro production of embryos by ovum pickup (OPU) and intracytoplasmic sperm injection (ICSI) is gaining popularity among horse breeders and veterinarians. Various collection media are available for flushing follicles during OPU. The objective of this study was to determine whether the type of flushing media used to aspirate follicles and collect oocytes influences the outcome of a commercial equine OPU-ICSI program. Two commercial embryo flushing media (EFM1 and EFM2) supplemented with heparin were compared with a flushing media designed specifically for the collection of oocytes (oocyte flushing media [OFM]) on the outcome of OPU-ICSI parameters in 234 Warmblood mares. The OPU-ICSI performed in mares using one of the EFM1 resulted in a lower (P < .05) blastocyst rate and blastocysts per OPU-ICSI session (11.9 ± 13.2%, 0.88 ± 1.3) than the OFM (19.2 ± 15.2%, 1.24 ± 1.2). Unlike the EFM2 solution, the heparin used to prepare the EFM1 contained preservatives including benzyl alcohol, a component known to alter the oocyte membrane, which might have been responsible for the lower developmental competence of oocytes collected with EFM1. In conclusion, exposure of oocytes (<1.5 hours) to one of the flushing medium tested in this study affected negatively the outcome of the OPU-ICSI commercial program when compared with flushing media designed for collection of equine oocytes. Care should be taken when choosing the components of the flushing media used to collect oocytes. Further research should be carried out to confirm the potential negative effect of the preservatives used in multidose heparin vials., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Deletions in the short arm of chromosome 1 (1p36) and MYCN amplification are common in neuroblastoma. Previously we showed evidence of at least two different neuroblastoma tumor-suppressor loci on 1p. One is associated with MYCN single-copy tumors and maps distal on 1p36.3. A second, more proximal locus maps to 1p36.1 and is deleted in about 90% of neuroblastomas with MYCN amplification. The cell line UHG-NP has the smallest 1p36 deletion of all neuroblastoma cell lines with MYCN amplifications. We assume that the more proximal locus maps within this deletion, close to its proximal border. Here we present the exact localization of the 1p deletion breakpoint of UHG-NP. A 600-kb PAC contig spanning the breakpoint was analyzed for genes and aberrations. Two more neuroblastoma-associated aberrations were mapped within 150 kb of the UHG-NP breakpoint. Within the contig, we identified nine genes expressed in neuroblastoma cells. One of these genes, AML2, maps 200 kb distal to the UHG-NP breakpoint but is expressed only rarely in neuroblastoma and showed no mutations., (Copyright 2001 Wiley-Liss, Inc.)

Neuroblastoma is an embryonal tumor originating from neural crest-derived cells. Here we present the serendipitous cloning of amplified sequences of chromosome 2p15 in neuroblastoma cell line IMR32. The amplified region was analyzed for oncogene activation using a SAGE (serial analysis of gene expression) library of IMR32. SAGE permits a quantitative analysis of all transcripts of a tissue or cell line. The expression of genes and ESTs mapping within a 30-cR region covering the amplicon was compared to 4 additional SAGE libraries of neuroblastomas and 12 SAGE libraries of other tissues in the CGAP databases. The IMR32 SAGE database revealed increased expression of the MEIS1 oncogene, whereas other SAGE libraries showed little or no MEIS1 expression. MEIS1 turned out to be highly amplified and overexpressed in IMR32. Analysis of 24 neuroblastoma cell lines and 22 tumors showed high-level expression in about 25% of the cases. The MEIS1 homeobox protein forms a complex with the HOXA9 and PBX proteins that are implicated in human leukemia. MEIS1 is a target of retroviral insertion in murine leukemia. This is the first report of a MEIS1 amplification and high expression levels in human cancer and the first time that identification of a candidate target of amplification is facilitated by high-throughput mRNA expression profiling., (Copyright 2001 Academic Press.)

Common genetic aberrations of neuroblastoma are deletions of the short arm of chromosome 1 (1p36) and MYCN amplification. Our deletion analysis of 25 tumor cell lines and 171 tumors strongly suggests that 1p harbors several tumor suppressor loci. Distinct loci are involved in MYCN single-copy versus MYCN-amplified neuroblastoma. Deletions in MYCN single-copy tumors have a shortest region of overlap (SRO) of 20 cM at 1p36.3. MYCN-amplified tumors have large deletions with an SRO of about 60 cM, from 1p36.1 to the telomere. This SRO is defined by D1S7 (1p36.1), which was the most distal locus retained. Therefore, a suppressor gene associated with MYCN-amplified tumors probably maps within a few megabases distal of D1S7. In order to map this locus, we further refined this SRO. We mapped the breakpoint of the MYCN-amplified neuroblastoma with the smallest 1p deletion between 56.6 and 57.2 cM from 1pter. Pulsed-field gel electrophoresis and radiation hybrid mapping were used to construct a 5-Mb physical map of this region. The map includes the region from 82.73 till 92.89 cR from 1pter. About half of it was isolated in P1 and PAC clones. The region harbors the genes FGR, SLC9A1, HMG17, EXTL1, AML2, RH, OP18, four ESTs, and a newly identified gene with a transcript size of approximately 7 Kb. Several of the mapped genes have a putative role in cell growth, differentiation, and morphogenesis. Genes Chromosomes Cancer 27:143-152, 2000., (Copyright 2000 Wiley-Liss, Inc.)

Class I HLA expression is low in neuroblastoma tumours and cell lines. We have recently mapped a modifier of methylation for HLA-C (MEMO-1) to chromosomal bands 1p35-36.1, a region deleted in many neuroblastomas. Hypomethylation of HLA-C is strongly correlated with allelic loss of the MEMO-1 locus. Here, we show that loss of MEMO-1 is associated with hypomethylation of both the 5' and 3' regions of class I HLA loci. We next investigated the relationship between methylation and expression of class I HLA in 28 cell lines of neuroectodermal tumours. Cell lines with hypermethylated HLA-C and HLA-A loci have relatively high expression, while most cell lines with hypomethylated loci have no or a reduced expression. It was reported earlier that high expression of c- or N-myc can suppress class I HLA expression. Remarkably, also N-myc amplification in neuroblastomas is associated with allelic loss of 1p35-36. Therefore, we have analysed the relationships between allelic loss of the MEMO-1 locus, class I HLA methylation and expression, and N-myc amplification and expression. This study shows a tight inter-relationship between these phenomena. Our data suggest a model in which hypomethylation of class I HLA due to loss of the MEMO-1 locus and high N-myc expression could collaborate in the down-regulation of class I HLA expression.

Neuroblastoma is characterized by deletions of the short arm of chromosome 1 (1p) and amplification of the N-myc oncogene. We have made somatic cell hybrids of two human neuroblastoma cell lines, one with and one without N-myc expression and amplification. The expression of the amplified N-myc gene is completely switched off in the hybrids. This suggests that N-myc expression results from loss of a repressor function. As N-myc amplification is associated with loss of heterozygosity (LOH) of 1p36, we analysed 1p deletions in 16 neuroblastoma cell lines. The seven cell lines without N-myc amplification have no deletions or relatively small deletions, with an SRO on 1p36.23-33. This suggests that a tumor suppressor gene maps in this region. All nine cell lines with N-myc amplification have larger deletions, with an SRO from 1p35-36.1 to the telomere. This suggests that a second tumor suppressor gene which is associated with N-myc amplification maps more proximally. Fine mapping of 1p36 deletions in the two cell lines of the fusion experiment suggests that the distal locus is not a repressor of N-myc expression, but the more proximal locus could be a candidate for this function.