The human embryonic genome is karyotypically complex, with chromosomally abnormal cells preferentially located away from the developing fetus.

STUDY QUESTION: Are chromosome abnormalities detected at Day 3 post-fertilization predominantly retained in structures of the blastocyst other than the inner cell mass (ICM), where chromosomally normal cells are preferentially retained? SUMMARY ANSWER: In human embryos, aneuploid cells are sequestered away from the ICM, partly to the trophectoderm (TE) but more significantly to the blastocoel fluid within the blastocoel cavity (Bc) and to peripheral cells (PCs) surrounding the blastocyst during Day 3 to Day 5 progression. WHAT IS KNOWN ALREADY: A commonly held dogma in all diploid eukaryotes is that two gametes, each with 'n' chromosomes (23 in humans), fuse to form a '2n' zygote (46 in humans); a state that remains in perpetuity for all somatic cell divisions. Human embryos, however, display high levels of chromosomal aneuploidy in early stages that reportedly declines from Day 3 (cleavage stage) to Day 5 (blastocyst) post-fertilization. While this observation may be partly because of aneuploid embryonic arrest before blastulation, it could also be due to embryo 'normalization' to a euploid state during blastulation. If and how this normalization occurs requires further investigation. STUDY DESIGN, SIZE, DURATION: A total of 964 cleavage-stage (Day 3) embryos underwent single-cell biopsy and diagnosis for chromosome constitution. All were maintained in culture, assessing blastulation rate, both for those assessed euploid and aneuploid. Pregnancy rate was assessed for those determined euploid, blastulated and subsequently transferred. For those determined aneuploid and blastulated (174 embryos), ICM (all 174 embryos), TE (all 174), Bc (47 embryos) and PC (38 embryos) were analyzed for chromosome constitution. Specifically, concordance with the original Day 3 diagnosis and determination if any 'normalized' to euploid karyotypes within all four structures was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients (144 couples) were undergoing routine preimplantation genetic testing for aneuploidy in three IVF clinical settings. Cleavage-stage biopsy preceded chromosome analysis by next-generation sequencing. All patients provided informed consent. Additional molecular testing was carried out on blastocyst embryos and was analyzed for up to four embryonic structures (ICM, TE, Bc and PC). MAIN RESULTS AND THE ROLE OF CHANCE: Of 463/964 embryos (48%) diagnosed as euploid at Day 3, 70% blastulated (leading to a 59% pregnancy rate) and 30% degenerated. Conversely, of the 501 (52%) diagnosed as aneuploid, 65% degenerated and 35% (174) blastulated, a highly significant difference (P < 0.0001). Of the 174 that blastulated, the ratio of '(semi)concordant-aneuploid' versus 'normalized-euploid' versus 'other-aneuploid' embryos was, respectively, 39%/57%/3% in the ICM; 49%/48%/3% in the TE; 78%/21%/0% in the PC; and 83%/10%/5% in the Bc. The TE karyotype therefore has a positive predictive value of 86.7% in determining that of the ICM, albeit with marginally higher aneuploid rates of abnormalities (P = .071). Levels of abnormality in Bc/PC were significantly higher (P < 0.0001) versus the ploidy of the ICM and TE and nearly all chromosome abnormalities were (at least partially) concordant with Day 3 diagnoses. LIMITATIONS, REASONS FOR CAUTION: The results only pertain to human IVF embryos so extrapolation to the in vivo situation and to other species is not certain. We acknowledge (rather than lineage-specific survival, as we suggest here) the possibility of other mechanisms, such as lineage-specific movement of cells, during blastulation. Ethical considerations, however, make investigating this mechanism difficult on human embryos. WIDER IMPLICATIONS OF THE FINDINGS: Mosaic human cleavage-stage embryos can differentiate into a euploid ICM where euploid cell populations predominate. Sequestering of aneuploid cells/nuclei to structures no longer involved in fetal development has important implications for preimplantation and prenatal genetic testing. These results also challenge previous fundamental understandings of mitotic fidelity in early human development and indicate a complex and fluid nature of the human embryonic genome. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Organon Pharmaceuticals and Merck Serono by grants to W.G.K. W.G.K. is also an employee of AdvaGenix, who could, potentially, indirectly benefit financially from publication of this manuscript. R.C.M. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133747. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.K.G. provides paid consultancy services for Care Fertility. TRIAL REGISTRATION NUMBER: : N/A.

Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice [corrected].

Overexpression of the gene encoding the beta-amyloid precursor protein (APP) may have a key role in the pathogenesis of both Alzheimer's disease (AD) and Down Syndrome (DS). We have therefore introduced a 650 kilobase (kb) yeast artificial chromosome (YAC) that contains the entire, unrearranged 400 kb human APP gene into mouse embryonic stem (ES) cells by lipid-mediated transfection. ES lines were generated that contain a stably integrated, unrearranged human APP gene. Moreover, we demonstrate germ line transmission of the APP YAC in transgenic mice and expression of human APP mRNA and protein at levels comparable to endogenous APP. This transgenic strategy may prove invaluable for the development of mouse models for AD and DS.

Preimplantation genetic diagnosis and screening.

Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation embryos prior to embryo transfer. PGD is an exciting technology that may improve the likelihood of a successful pregnancy and birth for five distinct patient groups: (1) those with infertility related to recurrent miscarriages or unsuccessful in vitro fertilization (IVF) cycles, (2) those with unexplained infertility, (3) advanced maternal age, (4) severe male factor infertility, and (5) couples at risk for transmitting a hereditary disease to their offspring. PGD is always performed following an IVF cycle where multiple oocytes are retrieved and fertilized. Sophisticated techniques such as multiprobe, multicolor fluorescence in situ hybridization are used to test single cells for structural or numerical chromosome abnormalities, whereas the polymerase chain reaction, linkage analysis, and DNA sequencing are used to analyze single cells for disease-specific DNA mutations. PGD allows one to transfer only those embryos identified as being free of genetic abnormalities, thus potentially increasing the implantation rate and decreasing the miscarriage rate. These technologies identify embryos free of specific genetic abnormalities and may increase the likelihood of achieving the patient's goal: the birth of a healthy infant.

Expression of the human multidrug resistance and glucocerebrosidase cDNAs from adeno-associated vectors: efficient promoter activity of AAV sequences and in vivo delivery via liposomes.

Recombinant adeno-associated viruses (rAAV) are attractive tools for gene therapy. We designed plasmids in which the human multidrug resistance gene (hMDR1) cDNA was placed downstream from portions of the 5' end of AAV including either a 234-bp cassette or the entire AAV p5 promoter. The drug-resistant phenotype conferred by the P-glycoprotein (Pgp) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cells transfected with these plasmids. The 234-bp region alone showed promoter activity similar in strength to that of the entire p5 promoter or the retroviral Harvey murine sarcoma virus long terminal repeat (LTR); this result demonstrates that the 234-bp cassette might be used as a small and efficient promoter in rAAV designed to express large genes approaching the packaging limit of AAV particles. After transfection of AAV-MDR1 vectors, the integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH). In addition, Southern analysis of low-molecular-weight DNA extracted from drug-resistant cells grown under continuous selection pressure indicated the persistence of nonintegrated AAV-MDR1 plasmids. Coordinate expression of Pgp and human glucocerebrosidase (hGC) was observed in drug-selected NIH-3T3 cells transfected with a bicistronic vector in which MDR1 cDNA was linked to hGC cDNA via the encephalomyocarditis internal ribosome entry site sequence. Moreover, following a single intravenous injection of the bicistronic vector complexed to cationic liposomes into recipient mice, delivery of MDR1 and GC cDNAs was achieved in all the organs we tested. Our results demonstrate that the efficiency of liposomes as vehicles for in vitro and in vivo gene delivery, the advantages of AAV-vectors, and the use of MDR1 as a selectable marker might be successfully combined in gene therapy protocols.

Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells.

The adeno-associated virus 2 (AAV)-based vector system has been suggested for its potential use in human gene therapy because the wild-type (wt) AAV genome appears to integrate into the human chromosomal DNA in a site-specific manner. We systematically investigated the integration patterns of the recombinant AAV genomes lacking one or both the viral coding sequences. Four recombinant AAV genomes were constructed containing the genes for resistance to tetracycline (TcR) and the herpesvirus thymidine kinase (TK) promoter-driven gene for resistance to neomycin (neoR; vTc.Neo), the genes for resistance to ampicillin (ApR) and TK-neoR (vAp.Neo), the genes for AAV replication (rep) genes and TK-neoR (vRep.Neo), and the AAV capsid (cap) genes and TK-neoR (vCap.Neo). The integration pattern of each of the recombinant AAV genomes in individual clonal isolates of the human nasopharyngeal carcinoma cell line (KB) analyzed on Southern blots using a neo-specific DNA probe was distinctly different. In addition, in none of the clones examined was the proviral genome covalently linked to the previously described AAV right-junction (Rt.Jn.) human chromosomal DNA fragment, the putative specific-site of integration for the wt AAV genome. Furthermore, whereas a 276-bp DNA fragment could be readily amplified from each of these clones, using a neo-specific primer-pair by polymerase chain reaction (PCR), no amplified DNA product was obtained using the neo- and the Rt.Jn. primer-pair under identical conditions. Fluorescence in situ hybridization (FISH) analyses further revealed the lack of integration of the recombinant AAV into human chromosome 19, even in the presence of a functional rep gene as determined by rescue of the recombinant AAV genome in the presence of adenovirus. These data suggest that the recombinant AAV genomes integrate at sites that are different from that characterized for the wt AAV genome. These studies may have implications in the development of the AAV-based vector system for its potential use in human gene therapy.

Unbalanced translocation, t(18;21), detected by fluorescence in situ hybridization (FISH) in a child with 18q- syndrome and a ring chromosome 21.

We report on an 8-year-old girl with minor anomalies consistent with 18q- syndrome and mild developmental delay. Initially cytogenetics showed a terminal deletion of chromosome 21 with mosaicism for a small ring chromosome 21 as the only apparent karyotypic abnormality: mos 45,XX,-21/46,XX,+r(21) (48%/52%). Further studies including FISH and DNA analysis demonstrated a de novo unbalanced translocation of chromosomes 18 and 21 with the likely breakpoints in 18q23 and 21q21.1. Most of 21q was translocated to the distal long arm of one chromosome 18, and this derivative 18 appeared to lack 18q23-qter. The small ring chromosome 21 [r(21)], present in only 52% of the patient's blood lymphocytes, did not appear to be associated with the abnormal phenotype since all 13 chromosome 21 markers that were examined in genomic DNA were present in 2 copies, and the phenotype of the patient was consistent with the 18q- syndrome. The karyotype was reinterpreted as mos 45,XX,-18,-21,+der(18) t(18;21) (q23;q21.1)/46,XX,-18,-21,+der(18) t(18;21) (q23;q21.1), +r(21) (p13q21.1) (48%/52%). These results demonstrate the power of FISH in conjunction with DNA analysis for examination of chromosome rearrangements that may be misclassified by traditional cytogenetic studies alone.

Cytogenetics of somatic cells and sperm from a 46,XY/45,X mosaic male with moderate oligoasthenoteratozoospermia.

OBJECTIVE: To determine aneuploidy frequencies in sperm from a patient with normal phenotype and 46,XY/45,X mosaicism in somatic cells (peripheral lymphocytes). DESIGN: Case report. SETTING: Infertility clinic and genetics laboratory. PATIENT: A 30-year-old male with primary infertility and moderate oligoasthenoteratozoospermia. INTERVENTION(S): Cytogenetic analysis of somatic cells and determination by fluorescence in situ hybridization of aneuploidy frequencies for the gonosomes (sex chromosomes) and chromosome 18 in sperm from whole and Percoll-separated semen. MAIN OUTCOME MEASURE(S): Somatic and gametic aneuploidy were scored. RESULT(S): Analysis of lymphocyte metaphase cells showed a mosaic 46,XY (90%)/ 45,X (10%) karyotype. Significantly higher frequencies of gonosomal (semen, 1.92% versus 0.70%; Percoll, 1.12% versus 0.46%), and chromosome 18 (semen, 0.89% versus 0.28%; Percoll, 0.26% versus 0.10%) disomy were detected in the sperm of the patient compared with those observed in spermatozoa from a proved fertile control. CONCLUSION(S): Significantly higher frequencies of aneuploid sperm suggest that the patient is at elevated risk of producing offspring with numerical chromosome abnormalities.

Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection.

OBJECTIVE: To determine aneuploidy frequencies in pellet and swim-up semen fractions from 10 infertile men with severe oligoasthenoteratozoospermia (OAT) who were donating sperm for intracytoplasmic sperm injection and to determine whether the swim-up isolation method would successfully separate aneuploid from haploid sperm. DESIGN: Prospective study. SETTING: Infertility clinic and molecular genetics laboratory. PATIENT(S): Ten patients with severe OAT. INTERVENTION(S): Cytogenetic analyses by fluorescence in situ hybridization to determine aneuploidy frequencies for chromosomes 1, 13, 18, 21, X, and Y in sperm from swim-up and pellet fractions. MAIN OUTCOME MEASURE(S): Gametic aneuploidy was scored in sperm fractions separated by the swim-up technique and clinical results after intracytoplasmic sperm injection were tabulated. RESULT(S): In all cases, chromosome aneuploidy levels in patients were significantly greater than in controls. The type and percentage of aneuploid sperm for all patients with OAT found in both swim-up and pellet fractions were not different, with the exception of diploid sperm, which remained in the pellet fraction. After ET, 2 (20%) of 10 couples achieved successful pregnancies. CONCLUSION(S): The data show significantly higher rates of diploidy, autosomal disomy and nullisomy, sex chromosome disomy and nullisomy, and total aneuploidy in sperm from all separated fractions obtained from all patients with OAT versus controls. This patient population with OAT may be at increased risk of producing aneuploid offspring.

ESHRE PGD Consortium data collection V: cycles from January to December 2002 with pregnancy follow-up to October 2003.

The fifth report of the ESHRE PGD Consortium is presented (data collection V). For the first time, the cycle data were collected for one calendar year (2002) in the following October, so that data collection was complete for pregnancies and babies. The data were collected using a Filemaker Pro database and divided into referrals, cycles, pregnancies and babies. There are currently 66 active centres registered with the consortium; however, the data presented here were obtained from 43 centres and included 1603 referrals, 2219 cycles, 485 pregnancies and 382 babies born. The cycle data were divided into preimplantation genetic diagnosis (PGD) for inherited disorders (including chromosome abnormalities, sexing for X-linked disease and monogenic disorders), aneuploidy screening (PGS) and the use of PGD for social sexing. Data collection V is compared with the previous cumulative data collection (I-IV), which comprised 4058 PGD/PGS cycles that reached oocyte retrieval.

Effects of diacylglycerol and inositol trisphosphate on steroidogenesis by ovarian granulosa from pigs.

In addition to increasing cyclic adenosine 3',5'-monophosphate (cAMP) levels, luteinizing hormone (LH) stimulation of granulosa results in phosphoinositide hydrolysis producing inositol trisphosphate (IP3) and diacylglycerol. The roles of these putative second messengers were investigated by measuring production of progesterone and inositol phosphates by granulosa from medium-sized porcine follicles (3-7 mm) after 15 min incubation with or without LH (1 microgram/ml), 5 microM dibutyryl cAMP (dbcAMP), or 5 microM 1-oleoyl,2-acetylglycerol (OAG). Compared to a control rate of 5.4 pmoles/10(7) cells/15 min, LH and dbcAMP stimulated progesterone production to 12.8 and 15.9 pmoles, respectively, and OAG decreased progesterone production to 3.7 pmoles. LH also stimulated inositol phosphate (IP) and bisphosphate (IP2) accumulations by approximately 5-fold and IP3 accumulation by 20-fold. In experiments where granulosa were premeabilized with saponin, LH, dbcAMP, and IP3 stimulated progesterone production from 1.3 pmol in control cells to 5.2, 3.2, and 5.1 pmol, respectively, and OAG decreased progesterone production to 1.0 pmol. LH stimulated accumulation of all inositol phosphates in permeabilized cells, whereas the addition of IP3 only increased IP2 and IP3 accumulations. In granulosa preincubated with 0.9 mM [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, A23187 increased progesterone production from 3.7 to 5.8 pmol. Addition of 1-20 nmoles IP3 to 10(7) granulosa incubated in a Ca2+-free medium increased Ca2+ efflux linearly. These data suggest that IP3 may have a role in regulating steroid production in granulosa by regulating intracellular Ca2+.

Origin of chromosome rearrangements in two long-lived human keratinocyte lines.

We have determined the origin of the extra chromosomal material in the karyotypes of two spontaneously-occurring long-lived human keratinocyte lines, HKC-N2 and HKC-N6. In each case the extra material was derived from the chromosome on which it was located. Possible relationships between the triplication of chromosomal material and the overcoming of cell senescence are discussed.

Nuclear receptor co-repressor gene localizes to 17p11.2, a frequently deleted band in malignant disorders.

The t(8;21) between the AML1 and ETO genes is a commonly seen genetic alteration in acute myeloid leukemia. Recently, we reported that the fusion partner ETO binds to the human nuclear receptor co-repressor (NCOR), a member of the NCOR/SIN3/histone deacetylase complex. This complex mediates transcriptional repression as a result of chromatin remodeling. Here, we used a combination of fluorescence in situ hybridization and hybrid panels to localize the human NCOR gene (NCOR) to chromosome band 17p11.2. The position of human NCOR on 17p11 raises the possibility of deranged transcriptional regulation in malignant disorders associated with deletions of 17p.

Isolation and characterization of an ornithine aminotransferase-related sequence (OATL3) mapping to 10q26.

We used a near full-length human ornithine delta-amino-transferase cDNA, huOAT6, as a probe under low stringency hybridization conditions to identify a new autosomal ornithine delta-aminotransferase-related sequence (OATL3). Cloning and characterization of this sequence reveal it to be a partial nonprocessed pseudogene corresponding to exon 3 and flanking intronic sequences of the ornithine delta-aminotransferase structural gene. Using somatic cell hybrids and fluorescence in situ hybridization, we mapped OATL3 to 10q26, adjacent to the ornithine delta-aminotransferase structural gene locus.

A complex chromosomal rearrangement detected prenatally and studied by fluorescence in situ hybridization.

We report of case of a complex chromosomal rearrangement detected prenatally and studied with traditional banding methods and fluorescence in situ hybridization. The combination of these techniques showed that four chromosomes were involved in the translocation. Nine breakpoints were proposed to explain these results. Some of the findings could only be detected with fluorescence in situ hybridization, demonstrating the usefulness of this technique in characterizing chromosomal abnormalities that would otherwise be difficult to interpret correctly with classical cytogenetics alone.

Assignment of the hypoxia-inducible factor 1alpha gene to a region of conserved synteny on mouse chromosome 12 and human chromosome 14q.

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor that mediates homeostatic responses to hypoxia. HIF-1 is a heterodimer consisting of HIF-1alpha, which is encoded by the HIF1A gene, complexed with HIF-1beta, which is encoded by the ARNT gene. In this paper we report the assignment of Hif1a and HIF1A to mouse chromosome 12 and human chromosome 14, respectively. HIF1A was assigned to human chromosome 14q21-q24 by analysis of somatic cell hybrids and by fluorescence in situ hybridization. Hif1a was localized by interspecific backcross analysis within a region of mouse chromosome 12 encompassing >30 cM that demonstrates conservation of synteny with a region of human chromosome 14 extending from PAX9 at 14q12-q13 to IGHC at 14q32.33.

Duplication of the short arm of the X chromosome in mother and daughter.

An 11-year-old girl with short stature, mental retardation, and mild dysmorphic features was found to have an inverted duplication of most of the short arm of the X chromosome [dic inv dup(X)(qter-->p22.3::p22.3-->cen:)]. Her mother, who is also short and retarded, carries the same duplication. Fluorescence in situ hybridization with an X chromosome library, and with X centromere-specific alpha satellite and telomere probes, was useful in characterizing the duplication. In most females with structurally abnormal X chromosomes, the abnormal chromosome is inactivated. Although the duplicated X was consistently late replicating in the mother, X chromosome inactivation studies in the proband indicated that in 11% of her lymphocytes the duplicated X was active.

Latent adeno-associated virus infection elicits humoral but not cell-mediated immune responses in a nonhuman primate model.

Latent infection with wild-type (wt) adeno-associated virus (AAV) was studied in rhesus macaques, a species that is a natural host for AAV and that has some homology to humans with respect to the preferred locus for wt AAV integration. Each of eight animals was infected with an inoculum of 10(10) IU of wt AAV, administered by either the intranasal, intramuscular, or intravenous route. Two additional animals were infected intranasally with wt AAV and a helper adenovirus (Ad), while one additional animal was inoculated with saline intranasally as a control. There were no detectable clinical or histopathologic responses to wt AAV administration. Molecular analyses, including Southern blot, PCR, and fluorescence in situ hybridization, were performed 21 days after infection. These studies indicated that AAV DNA sequences persisted at the sites of administration, albeit at low copy number, and in peripheral blood mononuclear cells. Site-specific integration into the AAVS1-like locus was observed in a subset of animals. All animals, except those infected by the intranasal route with wt AAV alone, developed a humoral immune response to wt AAV capsid proteins, as evidenced by a >/=fourfold rise in anti-AAV neutralizing titers. However, only animals infected with both wt AAV and Ad developed cell-mediated immune responses to AAV capsid proteins. These findings provide some insights into the nature of anti-AAV immune responses that may be useful in interpreting results of future AAV-based gene transfer studies.