Liquid jet delivery method featuring S100A1 gene therapy in the rodent model following acute myocardial infarction.

The S100A1 gene is a promising target enhancing contractility and survival post myocardial infarction (MI). Achieving sufficient gene delivery within safety limits is a major translational problem. This proof of concept study evaluates viral mediated S100A1 overexpression featuring a novel liquid jet delivery (LJ) method. Twenty-four rats after successful MI were divided into three groups (n = 8 ea.): saline control (SA); ssAAV9.S100A1 (SS) delivery; and scAAV9.S100A1 (SC) delivery (both 1.2 × 10¹¹ viral particles). For each post MI rat, the LJ device fired three separate 100 µl injections into the myocardium. Following 10 weeks, all rats were evaluated with echocardiography, quantitative PCR (qPCR) and overall S100A1 and CD38 immune protein. At 10 weeks all groups demonstrated a functional decline from baseline, but the S100A1 therapy groups displayed preserved left ventricular function with significantly higher ejection fraction %; SS group (60 ± 3) and SC group (57 ± 4) versus saline (46 ± 3), P < 0.05. Heart qPCR testing showed robust S100A1 in the SS (10,147 ± 3993) and SC (35,155 ± 5808) copies per 100 ng DNA, while off-target liver detection was lower in both SS (40 ± 40), SC (34,841 ± 3164), respectively. Cardiac S100A1 protein expression was (4.3 ± 0.2) and (6.1 ± 0.3) fold higher than controls in the SS and SC groups, respectively, P < 0.05.

Meiotic spindle checkpoints for assessment of aneuploid oocytes.

The spindle assembly checkpoint suspends cell cycle progression if improperly aligned chromosomes are detected at metaphase. Evolutionarily conserved kinetochore-associated proteins are believed to be key elements of this regulatory pathway. A breakdown in checkpoint function could bring about genomic instability, which may be responsible for the prevalence of aneuploidy in oocytes of older women. Maternal aging remains the overwhelming factor in the etiology of human aneuploidy in assisted reproduction. Defects in cell cycle checkpoint genes may play a role in its development. The existence of such monitoring mechanisms in oocytes has long been controversial. Studies providing evidence in support of and against their existence are reviewed.

Gender determination by multiplex PCR amplification of alphoid repeat sequences from single cells.

Alphoid repeat sequences on the X and Y chromosomes were successfully and consistently coamplified to determine the sex of single cells. Buccal cells served as a convenient model for single blastomeres obtained from preimplantation embryos through in vitro fertilization. The potential use of this technique for preimplantation genetic diagnosis is discussed.

Detection of human leukocyte antigen class I messenger ribonucleic acid transcripts in human spermatozoa via reverse transcription-polymerase chain reaction.

OBJECTIVE: To determine by reverse transcription-polymerase chain reaction (PCR) whether human leukocyte antigen (HLA) class I messenger RNA (mRNA) is present in mature human spermatozoa. DESIGN: Mature human spermatozoa was isolated from donor semen using a swim-up technique. Total RNA was extracted via guanidinium isothiocyanate-cesium chloride ultracentrifugation. By the method previously validated in our laboratory, reverse transcription-PCR was performed using primers specific for HLA class I transcripts. Positive control cells included a choriocarcinoma cell line (JEG) and human fetal tissue. Transformed peripheral blood lymphocytes (PBL) were used as a negative control for somatic cellular contamination. RESULTS: Human spermatozoa were positive for HLA class I (-G and -B) mRNA by reverse transcription-PCR, consistent with the positive controls. We did not detect any mRNA for beta-actin, retinoblastoma (RB), CD4, or kappa light chain genes in the sperm complementary DNA samples, verifying that the class I mRNA detected was not due to somatic cellular contamination of the purified sperm samples. CONCLUSION: These experiments provide the first evidence that mRNA for HLA class I molecules are present in mature human spermatozoa. The physiological role of these transcripts is unknown at present. Further experiments characterizing the expression of HLA class I (-G and -B) mRNA in oocytes and preimplantation embryos are in progress.

Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development.

BACKGROUND: Appropriate gene expression is vital for the regulation of developmental processes. Despite this fact there is a remarkable paucity of information concerning gene activity during preimplantation development. METHODS: We employed reverse transcription and real-time fluorescent PCR to quantify the expression of nine genes (BRCA1, BRCA2, ATM, TP53, RB1, MAD2, BUB1, APC and beta-actin) in oocytes and embryos. A full characterization of all genes was achieved in 42 embryos and four oocytes. The genes analysed have a variety of important cellular functions. RESULTS: Oocytes displayed relatively high levels of mRNA transcripts, while 2-3-cell embryos were seen to contain very little mRNA from any of the genes examined. Recovery of expression levels was not seen until the 4-cell stage or later, with the presumptive activation of the embryonic genome. Some genes displayed sharp increases in expression in embryos composed of 4-8 cells, but, for most, maximum expression was not achieved until the blastocyst stage. CONCLUSIONS: Our data show that it is possible to define characteristic gene expression profiles for each stage of human preimplantation development. The identification of genes active at defined preimplantation phases may provide clues to the cellular pathways utilized at specific stages of development. Expression of genes that function in DNA repair pathways indicate that DNA damage may be common at the cleavage stage. We suggest that specific patterns of gene expression may be indicative of embryo implantation potential.

Association between spindle assembly checkpoint expression and maternal age in human oocytes.

The spindle assembly checkpoint modulates the timing of anaphase initiation in response to the improper alignment of chromosomes at the metaphase plate. If defects are detected, a signal is transduced to halt further progression of the cell cycle until correct bipolar attachment to the spindle is achieved. The mitotic arrest deficient (MAD2) and budding uninhibited by benomyl (BUB1) genes encode conserved kinetochore-associated proteins believed to be components of the checkpoint regulatory pathway. A failure in this surveillance system could lead to genomic instability that may underlie the increased incidence of aneuploidy in the gametes of older women. To explore this possibility, the concentrations of these transcripts in human oocytes at various stages of maturation were determined by real-time rapid cycle fluorescent reverse transcription-polymerase chain reaction (RT-PCR). The results obtained following quantitative analysis suggest that these messages degrade as oocytes age. Potentially, this may impair checkpoint function in older oocytes and may be a contributing factor in age-related aneuploidy.

Quantification of human ooplasmic mitochondria.

It is likely that there is an association between the fitness of mitochondria and their ability to support normal cellular function. Oocytes are greatly enriched in the number of mitochondria as they support essential developmental processes such as oocyte maturation and embryonic development, while their replication is deferred until gastrulation. The mitochondrial DNA (mtDNA) copy number in 87 human oocytes from 29 patients was evaluated after DNA extraction and real-time quantitative polymerase chain reaction (PCR). The average mtDNA copy number was 795,000 (+/- 243,000) in metaphase II oocytes. mtDNA content varied considerably between oocytes, even within the same patient. No relationship was found between mtDNA copy number and maternal age. The findings suggest that mtDNA replication is fully accomplished by the germinal vesicle stage in the fully developed oocyte.

Quantification of mRNA in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR.

Deciphering the complex series of regulatory events that occur during early development depends partly on the ability to accurately quantify stage-specific mRNA species. However, the paucity of biological material coupled with the lack of sensitivity and/or reproducibility of the currently available quantitative methods had been severe limitations on single cell analysis. Rapid cycle DNA amplification is a highly sensitive technique for amplification of specific DNA sequences. With the addition of fluorescence probes, it is possible to monitor the log-linear phase of amplification during which the most useful quantitative data is obtained. Unknown concentrations are extrapolated from standards co-amplified producing a standard curve. Furthermore, micro volume capabilities allow for the analysis of minute samples. Consequently, this approach is ideally suited to the needs of the clinical IVF laboratory. Rapid fluorescence monitored cycling was used to examine expression levels of the housekeeping genes beta-actin and hypoxanthine guanine phosphorlbosyltransferase in individual murine/human oocytes and/or embryos. Results obtained compared favourably with those attained by others and followed the predicted temporal patterns of expression. Once informative reproductive molecular markers are identified by micro-array analysis, minimally invasive techniques can be developed to biopsy cytoplasm and/or polar bodies for clinical evaluation using rapid fluorescence monitored reverse transcription-polymerase chain reaction methods.

Outcome comparison of in vitro fertilization treatment with highly purified subcutaneous follicle-stimulating hormone (Fertinex, a urofollitropin) versus intramuscular menotropins.

OBJECTIVE: The aim of this study was to investigate various outcome measures of stimulation with highly purified subcutaneous follicle-stimulating hormone (Fertinex, a urofollitropin) compared with first- and second-generation urinary human menopausal gonadotropin standards (Pergonal, Metrodin). STUDY DESIGN: Retrospective analysis was restricted to our most efficient in vitro fertilization age group (23-34 years). Data from Institute for Assisted Reproduction in vitro fertilization cycles 1 through 11 with Pergonal, Metrodin, or both were tabulated for hormonal values, oocyte quality, and embryo outcome as baseline data. Patients in cycles 12 through 13 were treated with Fertinex and Pergonal or Fertinex alone and then reviewed for the same parameters. RESULTS: Two hundred thirty-eight in vitro fertilization records with embryo transfer were analyzed. Clinical pregnancy rates per embryo transfer in an optimal age group were similar despite use of first- through third-generation urinary gonadotropin preparations: Pergonal and Metrodin, 67%; Metrodin, 64%; Fertinex and Pergonal, 62%; and Fertinex, 54%. There were no discernible differences in hormonal response, oocyte recovery, or embryonic growth. CONCLUSION: Administered subcutaneously, the third-generation urinary gonadotropin preparation Fertinex is effective in in vitro fertilization treatment in young women.

Use of videocinematography to assess morphological qualities of conventionally cultured and cocultured embryos.

Videocinematography and image analysis procedures were utilized to evaluate the effect of conventional and coculture methodologies on morphological parameters in human embryos derived from in-vitro fertilization (IVF). Following 24-30 h of in-vitro development, cocultured embryos had more acceptable morphological features and less fragmentation present than embryos cultured in medium alone. Cocultured embryos were more advanced at the time of replacement when compared with conventionally cultured embryos. Zona pellucida variation (> or = 20%) also occurred more frequently in cocultured embryos. The morphological characteristic most enhanced after coculture was blastomere expansion. Patients who became pregnant across both culture treatments had a higher proportion of morphologically normal embryos replaced than patients who failed to achieve an ongoing pregnancy. Clinical pregnancy rate for patients following coculture was 49%, which was greater (P < 0.05) than the 29% detected for patients with embryos in the conventional culture group.

Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR.

Rapid cycle DNA amplification is a refinement of the polymerase chain reaction (PCR) method that permits increased product specificity while reducing amplification time by an order of magnitude. Combined with the use of micro volume capillaries, minute samples can be examined by this technique. Thus, this approach is ideally suited to the analysis of gene expression in individual cells. As the current understanding of early developmental processes is still rudimentary, further characterization of transcription in single oocytes and embryos may provide additional insight into the molecular mechanisms directing these events. In this study, we examined the suitability of fluorescence monitored reverse transcription (RT)-PCR for the study of gene expression during oogenesis and embryogenesis using transcripts of the housekeeping gene, beta-actin, as an experimental model. Product accumulation was monitored by either the double-stranded DNA dye SYBR Green I or sequence-dependent hybridization of reporter molecules called molecular beacons. Dyes bind generically and are economical to use. However, both specific and non-specific products are labelled. Hybridization probes permit very specific and sensitive target recognition but they can be costly to manufacture. Once molecular markers indicative of optimal development are identified, this technology could be used in a clinical in-vitro fertilization laboratory as a diagnostic tool.

Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR.

Oocytes, in general, are greatly enriched in mitochondria to support higher rates of macromolecular synthesis and critical physiological processes characteristic of early development. An inability of these organelles to amplify and/or to accumulate ATP has been linked to developmental abnormality or arrest. The number of mitochondrial genomes present in mature mouse and human metaphase II oocytes was estimated by fluorescent rapid cycle DNA amplification, which is a highly sensitive technique ideally suited to quantitative mitochondrial DNA (mtDNA) analysis in individual cells. A considerable degree of variability was observed between individual samples. An overall average of 1.59 x 10(5) and 3.14 x 10(5) mtDNA molecules were detected per mouse and human oocyte, respectively. Furthermore, the mtDNA copy number was examined in polar bodies and contrasted with the concentration in their corresponding oocytes. In addition, the density of mtDNA in a cytoplasmic sample was estimated in an attempt to determine the approximate number of mitochondria transferred during clinical cytoplasmic donation procedures as well as to develop a clinical tool for the assessment and selection of oocytes during in vitro fertilisation procedures. However, no correlation was identified between the mtDNA concentration in either polar bodies or cytoplasmic samples and their corresponding oocyte.

Blastocyst development in co-culture: development and morphological aspects.

A retrospective study was undertaken to determine if initial culture conditions and embryo quality had an effect on subsequent blastocyst development in co-culture for cryopreservation. The apparent effects of freeze-thawing on blastocysts at the ultrastructure level were also observed. On day 3 of culture, embryos were categorized into two groups based on their morphological attributes. Results suggest that the initial culture environment of embryos up to day 3 (5- to 8-cell stage) did not affect the subsequent rate of blastocyst formation in co-culture. However, the initial embryo quality had an impact on blastocyst formation and quality. On day 5.5, 90% (60/67) of the optimal quality embryos (six to eight blastomeres with minimal or no fragmentation on day 3) had attained the blastocyst stage, which was greater (P < 0.01) than the 55% (31/56) observed with the sub-optimal embryos (five to eight blastomeres with 30-50% fragmentation on day 3). Furthermore, 66% (44/67) of embryos initially graded as optimal were suitable for cryopreservation, which was greater (P < 0.01) than attained with embryos of lesser quality (22/56; 39%). At the ultrastructural level, the polarized distribution of plasma membrane microvilli was retained, as was the integrity of the nuclear membrane following thawing.

Beneficial aspects of co-culture with assisted hatching when applied to multiple-failure in-vitro fertilization patients.

A study was conducted on patients who had attempted and failed previous in-vitro fertilization (IVF) procedures an average of 3.8 times following the application of assisted hatching with conventional culture systems. The aim of this investigation was to determine if addition of co-culture methodologies could reduce embryonic abnormalities and thus improve the prognosis for pregnancy. The study population consisted of 123 patients, subdivided into three patient categories. Previous IVF results from conventional culture were used to evaluate any potential benefits derived from the present co-culture application. Following co-culture, the rate of blastomere development was increased and the rate of fragmentation decreased. An increased rate of blastomere development was most noticeable in the patients aged < or = 39 years with no male factor as well as the intracytoplasmic sperm injection (ICSI) subgroup. Similarly, the rate of fragmentation was significantly reduced in the aforementioned subgroups. The most pronounced impact of co-culture was on pregnancy and implantation rates. The overall clinical and ongoing pregnancy rates were 38% (47/123) and 36% (44/123) respectively. The corresponding implantation rate was 17% (72/ 412) as shown by embryonic cardiac activity. The ongoing pregnancy rates in the < or = 39 years no male factor, > or = 40 years no male factor and ICSI no age limit patient subgroups were 41% (21/51), 30% (8/27) and 33% (15/45) respectively. The results indicate that addition of co-culture to the IVF procedure for poor-prognosis patients may be advisable.