DNA template amplification genetic studies on archival human preimplantation biopsied microtubed cell samples.

Biopsied cell samples can remain in storage following an initial, unequivocally successful preimplantation genetic diagnosis (PGD). The fidelity of the DNA template for polymerase chain reaction (PCR) amplification of microtubed human preimplantation embryonic cells stored at -70 degrees C for extended periods of time (6 months to 4 years) has been assessed. PCR protocols and specific nested primer sets for the beta-globin, ZP3 and CA repeat motif successfully used for previous human embryo PGD research were employed for these studies. The results show that the DNA template of microtubed biopsied blastomere and mural trophectoderm cell samples stored for periods of up to 4 years may be successfully amplified by PCR. Specific gene sequences were able to be analysed at the 1-2 or 3-5 biopsied cell level with a 71-100% success rate. Analysis of DNA fragments amplified from the CA dinucleotide repeat locus showed that in 8/9 samples both alleles were amplified at the cellular level. No DNA contamination was detected in the stored microtubed samples, or in the experimental controls.

Archival fixed and analysed preimplantation human embryonic cells: a DNA resource for retrospective PCR analysis at the cellular level.

Biopsies of human embryonic cell preparations previously analysed by cytogenetic and/or fluorescent in-situ hybridization (FISH) chromosome probes provide a unique reference DNA resource for the archival preimplantation genetic diagnosis (PGD) of the transferred embryo. DNA polymerase chain reaction (PCR) may be utilized on these fixed cell preparations to verify equivocal FISH/PGD results. Retrospective PCR screens of the genotype of biopsied embryonic cell(s) may be of benefit in the case of a suspected genetic mutation. Currently, carrier detection or linkage analysis is often not possible because of early death of the fetus, or of patients with a lethal disease. Alternatively, fixed/stained 'failed fertilized' oocytes provide a resource to extend genetic analysis to infertile patients. A successful research is described which minimizes loss of individual analysed fixed/stained oocytes, metaphase chromosomes, and embryonic cell samples. Initial DNA amplification takes place in situ using a modified PCR protocol. Comparative cellular studies using primer sets previously used for PGD analyses show that 65% of the preparations amplified unequivocally using the modified protocol and primers for a CA repeat motif gene sequence, in comparison with 81% using the original PCR protocol. With further refinement and optimization, the methods outlined have the potential to retrospectively screen archival fixed chromosomes, gametes, and embryonic cells for clinical application, and enable the further study of the fixed human preimplantation embryo at the morphological, cell and molecular level.

Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected 'failed-fertilized' human oocytes.

This study was undertaken to establish baseline data on the chromosomal status of 'failed-fertilized' oocytes derived from in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) procedures. A cytogenetic analysis was undertaken on 162 IVF and 51 ICSI oocytes. In all, 82.1% (133/162) of the IVF and 78.4% (40/51) of the ICSI oocytes had metaphase II (MII) plates, of which 50.4% of the IVF and 47.5% of the ICSI oocytes were analysed further. Chromosomes of the G-group (21-22) were identified with the majority of the anomalies. No overall significant difference in the aneuploidy rate was found for the IVF (37.3%) of ICSI (31.6%) oocytes, or with maternal age. However, chromosome anomalies, e.g. diploidy, fragmented and broken chromatids, single sperm and oocyte chromatids, were found in oocytes from IVF patients aged > 36 years and in the ICSI oocytes throughout the maternal age range (31-38 years). The status of the polar body chromatin indicated that there was no overall significant difference in the maturation of the IVF and ICSI oocytes. Evidence of successful sperm delivery was found in 72.5% (37/51) of the ICSI failed-fertilized oocytes. In this group there was a significant increase in the incidence of premature chromosome condensation: 19.6% (10/51) contained sperm chromosomes, 7.8% (4/51) had swollen sperm heads, and the remaining 45.0% had condensed sperm heads. The presence of both sperm and MII oocyte chromosomes was found in 19.6% (10/51) of the ICSI and 8.6% (14/162) of the IVF failed-fertilized oocytes. Specific fluorescent in-situ hybridization DNA probes were used to re-analyse the chromosomes of karyotyped 'failed-fertilized' IVF oocytes and, for the first time, applied to the karyotyped chromosomes of failed-fertilized ICSI oocytes. The hybridization efficiency was 86-95% for the centromere probe and 100% for probes 21 and 18.

Disruption of murine Hexa gene leads to enzymatic deficiency and to neuronal lysosomal storage, similar to that observed in Tay-Sachs disease.

Tay-Sachs disease is an autosomal recessive lysosomal storage disease caused by beta-hexosaminidase A deficiency and leads to death in early childhood. The disease results from mutations in the HEXA gene, which codes for the alpha chain of beta-hexosaminidase. The castastrophic neurodegenerative progression of the disease is thought to be a consequence of massive neuronal accumulation of GM2 ganglioside and related glycolipids in the brain and nervous system of the patients. Fuller understanding of the pathogenesis and the development of therapeutic procedures have both suffered from the lack of an animal model. We have used gene targeting in embryonic stem (ES) cells to disrupt the mouse Hexa gene. Mice homozygous for the disrupted allele mimic several biochemical and histological features of human Tay-Sachs disease. Hexa-/- mice displayed a total deficiency of beta-hexosaminidase A activity, and membranous cytoplasmic inclusions typical of GM2 gangliosidoses were found in the cytoplasm of their neurons. However, while the number of storage neurons increased with age, it remained low compared with that found in human, and no apparent motor or behavioral disorders could be observed. This suggests that the presence of beta-hexosaminidase A is not an absolute requirement of ganglioside degradation in mice. These mice should help us to understand several aspects of the disease as well as the physiological functions of hexosaminidase in mice. They should also provide a valuable animal model in which to test new forms of therapy, and in particular gene delivery into the central nervous system.

A retrospective analysis of the in-vitro development of 'spare' human in-vitro fertilization preimplantation embryos using 'in-house' prepared medium and 'Medi-Cult' commercial medium.

In-house prepared medium was used routinely in our in-vitro fertilization (IVF) facility prior to the introduction of the commercial 'Medi-Cult' products. A comparative study of the in-vitro development of embryos cultured in two [T6 and Earle's balanced salt solution (EBSS)] human-inactivated serum (HIS)-supplemented media from days 0 to 5 showed that 44.7% (46/103) of the embryos developed to the blastocyst stage in the T6 medium compared with 22.3% (23/103) in EBSS. Following the introduction of the commercial Medi-Cult IVF M2 medium, which is used routinely to culture fertilized eggs from days 0 to 2, new baseline data were required for the in-vitro development of 'spare' embryos from days 2 to 5. When Medi-Cult M3 medium was used, 35.6% (37/104) of the 'spare' day 2 embryos achieved the blastocyst stage. However, if morphologically similar (four normal nucleated blastomeres with no fragmentation) day 2 embryos were selected, an increase in the blastocyst rate to 50.0% (33/66) was achieved. This compared favourably with the 45.0% blastocyst rate (published in the Medi-Cult literature) for M2/M3 medium cultured human embryos. A small series of experiments with T6 + HIS medium and human serum albumin (HSA)-supplemented Ham's F-10, MCDB 302 and M3 media was undertaken to identify a suitable medium which could be used for the culture of M2 medium day 2 embryos. Results show that M2 medium cultured embryos placed in Ham's F-10 medium supplemented with 10 mg/ml HSA gave an acceptable 37.8% (14/45) blastocyst rate. Therefore, this medium could be substituted for M3 medium in an emergency. A total of 483 IVF embryos donated by patients, which were surplus to the therapeutic IVF programme, were used for these studies over a period of 30 months. Late day 2 IVF spare embryos were assigned an embryo score based on a high-power phase-contrast microscopic examination prior to being placed in culture. The embryo score provides an effective in-vitro parameter with which embryos from different patients can be compared. The cleavage and development of individual embryos were monitored on days 2 to 5. In some cases, the continuing normal development and viability of the day 5 cultured embryo were assessed by monitoring the hatching, attachment and outgrowth of the cavitated blastocyst.

Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos.

Human embryos have been biopsied at either the cleavage or the blastocyst stage of development. One to two blastomeres were removed from cleavage-stage embryos and 2-6 cells from blastocysts. The biopsy specimens were subjected to gene amplification by the polymerase chain reaction (PCR) and a comparison made of amplification efficiencies of two unique target sequences, one located within the beta-globin gene and containing the sickle-cell locus and the other a polymorphic dinucleotide repeat. When the cleavage-stage biopsy sample consisted of an intact blastomere with a clearly discernible nucleus, an amplification efficiency of 89% was achieved for each target locus. This was similar to that achieved with cleavage-stage biopsy samples consisting of two blastomeres or with blastocyst biopsy samples consisting of 2-3 trophectoderm cells. When biopsy samples consisted of four or more trophectoderm cells, both target loci were amplified in all samples tested. When the biopsy sample was heterozygous at the dinucleotide repeat locus and the biopsy consisted of one or more intact cells with a clearly discernible nucleus, both alleles were amplified in > 80% of biopsy samples. When four or more trophectoderm cells were used for the PCR, both alleles were amplified in all heterozygous samples. Target sequences were never amplified from biopsy samples which lysed prior to transfer into the reaction tube. Analysis of DNA fragments amplified from the dinucleotide repeat locus indicated that in most cases faithful amplification of biopsy DNA template had taken place.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic diagnosis using polymerase chain reaction and fluorescent in-situ hybridization analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos.

Cultured human preimplantation embryos have been used to develop methods which allow preimplantation genetic diagnosis (PGD) analyses by polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) on biopsied blastomeres and trophectoderm cells from the same embryo. An experimental design is described and experiments undertaken, which demonstrate the feasibility of extending biopsy and PGD procedures currently in use. We have shown that dual-stage biopsies are possible, and that the PCR and FISH analyses of the biopsied cell samples are effective. One to two blastomeres were biopsied from an 8- to 10-cell embryo and processed for the simultaneous PCR amplification of a beta-globin and a cytosine adenine (CA) repeat sequence, or a Y chromosome sequence. FISH procedures were also used to detect the presence of Y chromosome markers. The biopsied cleavage-stage embryo can be cultured to the blastocyst stage, where the serial biopsy of three to five mural trophectoderm cells provides two further cell samples. These can be used to repeat and/or undertake additional PGD analyses. The biopsied blastocyst is either used to confirm earlier diagnoses, or placed in culture for a further 4-24 h. Maintenance of a blastocoele cavity, hatching and formation of an outgrowth demonstrates continuing viability following the dual-stage biopsy procedures. The PCR DNA amplification procedures are effective at the cellular level for both biopsied blastomeres and mural trophectoderm cells. The FISH techniques have shown a definitive Y signal in 50% (one out of two) and 100% (two out of two) of the biopsied blastomeres and 72% (two out of three, four out of five and 7 out of 10) for the trophectoderm cell nuclei. Preliminary experiments have demonstrated that the FISH preparations can be re-amplified to improve the signal, and dual fluorescent procedures using the X and Y probes are effective. A retrospective PCR analysis has also been undertaken on preparations of biopsied cells which were previously used for PGD analysis by FISH.

Biopsy of the human blastocyst and polymerase chain reaction (PCR) amplification of the beta-globin gene and a dinucleotide repeat motif from 2-6 trophectoderm cells.

Cultured human blastocysts have been biopsied on day 5-6 post insemination and 2-6 extra-embryonic cells from the trophectoderm were removed and their DNA subjected to specific amplification by the polymerase chain reaction (PCR). Simultaneous amplification of part of the beta-globin gene and a dinucleotide repeat sequence has been achieved in a high percentage of cases when using the DNA from both trophectoderm cell biopsies and biopsied blastocysts as template for the PCR. A similar success rate was achieved when serial biopsies were taken from the same blastocyst, thus allowing one cell sample to be held in reserve for use should equivocal results be obtained. Over the entire experimental period (5 months), no contamination was experienced with biopsy or PCR procedures. Following biopsy of the trophectoderm cells all blastocysts had reformed a blastocoele cavity within 3-4 h of the biopsy procedure. Those blastocysts remaining in culture after this time showed a high incidence (78-83%) of hatching and outgrowth.

Preimplantation diagnosis of genetic disease.

Preimplantation diagnosis offers couples at high risk of transmitting a genetic disease the opportunity of prenatal diagnosis before the establishment of pregnancy. The successful application of embryo biopsy techniques, and of the polymerase chain reaction for the amplification of DNA, as well as fluorescent in-situ hybridization methods have allowed a small number of IVF units to identify the sex and/or genetic defect in human preimplantation embryos. The development of current methods being applied are reviewed. We suggest that there is a need for constant evaluation of the technique and for improvement of the present methods to ensure reproducibility and accuracy of the diagnostic procedures.

Criteria that optimize the potential of murine embryonic stem cells for in vitro and in vivo developmental studies.

Cultured mouse embryonic stem (ES) cells are used for both in vitro and in vivo studies. The uncommitted pluripotent cells provide a model system with which to study cellular differentiation and development; they can also be used as vectors to carry specific mutations into the mouse genome by homologous recombination. To ensure successful integration into the germ line, competent totipotent diploid ES cell lines are selected using a cell injection bioassay that is both time consuming and technically demanding. The prolonged in vitro culture of rapidly dividing ES cells can lead to accumulated changes and chromosomal abnormalities that will compromise the biological function and abrogate germ line transmission of chimeric mice carrying novel genetic mutations. Such in vitro conditions will vary between individual laboratories; for example, differences in the serums used for maintenance. Using a number of different criteria we attempt in this paper to define the parameters that we found to be key factors for optimization of the biological potential of established ES cell lines. The successful integration into the germ line is dependant on acquiring or deriving a competent totipotent mouse ES diploid cell line. In this paper parameters and criteria are defined which we found to be key factors for the optimization of the biological potential of established ES cell lines.

Preimplantation diagnosis of a human beta-globin transgene in biopsied trophectoderm cells and blastomeres of the mouse embryo.

The preimplantation diagnosis of a HbSA-globin transgene in biopsied trophectoderm cells and blastomeres in embryos using a transgenic mouse model for the trait of human sickle-cell anaemia has been undertaken. A sensitive procedure was developed for the amplification of the human beta-globin gene sequence flanking the sickle mutation. Polymerase chain reaction (PCR) assays were undertaken on one to five biopsied trophectoderm cells and isolated blastomeres of the preimplantation mouse embryo. After biopsy the blastocysts were cultured whilst the cells were analysed for the presence of the transgene, and a high proportion (82-91%) were viable as assessed by the presence of a blastocoele cavity within a 5-h period. The majority of the biopsied cultured blastocysts were frozen and used to confirm the diagnosis; 90 biopsied cultured blastocysts were transferred to pseudopregnant recipients and 34% established pregnancy. Material from day 13.5 post-coitum fetuses was also used to confirm the original diagnosis. The time (4-5 h) required to carry out the analysis obviates a need for extended culture or cryopreservation of the biopsied embryo. In individual experiments under optimal conditions, the presence of the transgene in biopsied cells was detected with 100% accuracy, and the PCR analysis was sensitive at the 1-cell level. The overall success rate of diagnosis and confirmation of the presence or absence of the human beta-globin sequence in the biopsied embryo was 70%. Over the entire experimental period (14 months) DNA contamination from a variety of sources did occasionally occur; the methods used to overcome this problem are discussed.

Localisation of the beta crystallin Cryb gene to mouse Chromosome 2B-->C1 by in situ hybridisation.

The beta crystallin gene Cryb (betaA3/A1) has been assigned to mouse Chromosome 2 region B-->Cl by in situ hybridisation to metaphase chromosomes from mouse foetal liver and bone marrow preparations of Rb(2.17)4H mice using a murine cDNA (pMbeta23Crl) probe.

Immunochemical comparison of the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Expression of the major intrinsic protein (MIP) of eye-lens fibre cell membranes was compared in normal (DBA), cataractous (CAT, LOP, NCT) and chimaeric (CBA-LOP) mice at different stages of development using immunofluorescence microscopy and immunoblotting techniques. MIP of apparent molecular mass 26 kDa was detected in extracts of adult DBA, LOP and CBA-LOP lenses, but only low molecular mass (less than 26 kDa) immunoreactive proteins were detected in similar extracts from adult CAT and NCT lenses. The corresponding MIP distribution patterns confirmed the highly organised fibre-cell histology in embryonic DBA and adult CBA-LOP lenses and also highlighted the severe fibre-cell degeneration in the LOP lens. In contrast, however, no immunoreactive MIP was detected in situ in embryonic CAT and NCT lenses. These results suggest that a structural alteration of MIP occurs during embryonic lens development in the cataractous CAT (dominant) and NCT (recessive) mutant mice.

Sex chimaerism, fertility and sex determination in the mouse.

Adult intraspecific mouse chimaeras, derived by introducing male embryonal stem cells into unsexed host blastocysts, were examined to determine whether gonadal sex was correlated with the sex chromosome composition of particular cell lineages. The fertility of XX in equilibrium XY and XY in equilibrium XY male chimaeras was also compared. The distribution of XX and XY cells in 34 XX in equilibrium XY ovaries, testes and ovotestes was determined by in situ hybridisation using a Y-chromosome-specific probe. Both XX and XY cells were found in all gonadal somatic tissues but Sertoli cells were predominantly XY and granulosa cells predominantly XX. The sex chromosome composition of the tunica albuginea and testicular surface epithelium could not, in general, be fully resolved, owing to diminished hybridisation efficiency in these tissues, but the ovarian surface epithelium (which like the testicular surface epithelium derives from the coelomic epithelium) was predominantly XX. These findings show that the claim that Sertoli cells were exclusively XY, on which some previous models of gonadal sex determination were based, was incorrect, and indicate instead that in the mechanism of Sertoli cell determination there is a step in which XX cells can be recruited. However, it remains to be established whether the sex chromosome constitution of the coelomic epithelium lineage plays a causal role in gonadal sex determination. Male chimaeras with XX in equilibrium XY testes were either sterile or less fertile than chimaeras with testes composed entirely of XY cells. This impaired fertility was associated with the loss of XY germ cells in atrophic seminiferous tubules. Since this progressive lesion was correlated with a high proportion of XX Leydig cells, we suggest that XX Leydig cells are functionally defective, and unable to support spermatogenesis.

Restriction fragment length polymorphisms associated with the gene for the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Cloned cDNAs coding for eye-lens fibre cell-membrane proteins, MIP and MP70, were used to detect restriction fragment length polymorphisms (RFLPs) in genomic DNA from inbred mice with autosomally inherited cataracts. Whereas distinct RFLPs associated with the MIP gene were identified in the Cba Cat and Nct mutants, no such genetic variation was associated with the MP70 gene. RFLPs associated with the mouse MIP gene may provide informative DNA markers in gene linkage studies of murine hereditary cataracts.

Hypoxanthine phosphoribosyl transferase deficiency, haematopoiesis and fertility in the mouse.

We have looked for effects of deficiency in hypoxanthine phosphoribosyl transferase (HPRT) in the mouse comparable to non-behavioural consequences of HPRT-deficiency in humans. HPRT-deficient humans show abnormalities in haematopoiesis and, in heterozygotes, there is strong selection in haematopoietic tissues against HPRT-deficient cells arising as a result of X-chromosome inactivation. We have examined two situations in mice in which HPRT- and HPRT+ cells occur in the same individual. First, in chimaeras resulting from the injection of HPRT- embryonal stem cells into HPRT+ blastocysts the fate of HPRT- and HPRT+ cell populations was monitored by their expression of different isozymes of glucose phosphate isomerase and also, in those chimaeras that resulted from injecting the male ES cells into female blastocysts, by in situ hybridisation using a Y-chromosome-specific repetitive DNA probe. There was a small statistically significant selection against the HPRT- population in haematopoietic tissues in both XX in equilibrium with XY and XY in equilibrium with XY chimaeras. Second, in female mice doubly heterozygous for HPRT-deficiency and for an electrophoretic variant of the X-linked enzyme phosphoglycerate kinase, there was a similar small statistically significant selection against the HPRT- population in haematopoietic tissues. While further work is required to establish whether this selection is a consequence of the HPRT mutation, it is clear that any selection against cells in the haematopoietic system as a consequence of HPRT-deficiency is at most small compared with the effect seen in humans. In HPRT-deficient human males surviving beyond the normal age of puberty, there is testicular atrophy. However, we find no effect of HPRT-deficiency on the fertility of either male or female mice. Thus, as with effects on behaviour, the consequences of HPRT-deficiency for haematopoiesis and testis development in the mouse are at most small compared with those in the human. We conclude that the reason for the difference in effects between the two species lies in a difference in purine-related intermediary metabolism per se, rather than in its interaction with brain amine biochemistry.

In-vitro studies on 'spare' human preimplantation embryos in culture.

Methods previously used for the biopsy of preimplantation mouse embryos have been applied to individual 'spare' human embryos. Early cleavage-stage human embryos have been cultured and individual blastomeres removed following zonae thinning or drilling. Embryos have also been cultured to the blastocyst stage for the biopsy of three to five trophectoderm cells. Both the biopsied embryo and the biopsied cells have been allowed to develop and/or grow in vitro.

The shiverer mouse mutation shi/shi: rescue and preimplantation detection.

The shiverer mouse mutation has been used as a model in this series of experiments. Germ-line therapy of this disorder has been demonstrated by producing mice transgenic for the wild-type gene for myelin basic protein (MBP). The mutation has also been diagnosed in preimplantation mouse blastocysts.

Rapid preimplantation detection of mutant (shiverer) and normal alleles of the mouse myelin basic protein gene allowing selective implantation and birth of live young.

As a model for the detection of human genetic disease in preimplantation embryos, we describe a method in which trophectoderm biopsy samples from viable mouse blastocysts are simultaneously analyzed for the presence of a normal or mutant allele of the myelin basic protein gene by the polymerase chain reaction. The biopsied embryos are kept in culture during analysis of biopsied material and later reintroduced to a foster mother. Prenatal diagnosis can be completed in less than 7 hr. The identity of either amplification product was proved conclusively by direct sequence analysis of amplified products. Ninety-six percent of recovered blastocysts survived biopsy, as judged by re-formation of a blastocyst cavity in culture. Fifty-nine percent of the biopsied embryos established pregnancy by day 6.5, compared to 88% of unmanipulated controls. This approach can be applied to preimplantation diagnosis of human genetic diseases by using extraembryonic cells from blastocysts obtained after in vitro fertilization or uterine lavage. It will make possible the elimination of a mutant allele from a family in a single generation.