Novel strategy with potential to identify developmentally competent IVF blastocysts.

BACKGROUND: Currently there are no markers fully predictive of developmental competence of human IVF embryos. The present study investigated a novel strategy involving blastocyst biopsy and DNA fingerprinting to link developmental competence with gene expression patterns. METHODS: Patient's blastocysts were biopsied to remove 8-20 trophectoderm (TE) cells for molecular analysis prior to transfer. Biopsy samples were amplified and gene expression was evaluated using microarrays. Sibling TE biopsies and cells from resulting offspring were subjected to DNA fingerprinting to identify which blastocyst(s) in the transfer cohort developed to term. RESULTS: Blastocyst biopsy did not appear to impair developmental competence. Comparative microarray analysis of cDNA from pooled 'viable' and 'non-viable' TE samples identified over 7000 transcripts expressed exclusively in 'viable' blastocysts. The most significant of these included transcripts involved in cell adhesion and cell communication, key processes that have been associated with mammalian implantation. DNA fingerprinting of three cohorts of sibling blastocysts identified those blastocyst(s) that produced term pregnancies. CONCLUSIONS: The combination of blastocyst biopsy, microarray gene expression profiling and DNA fingerprinting is a powerful tool to identify diagnostic markers of competence to develop to term. This strategy may be used to develop a rapid diagnostic assay or for refining existing criteria for the selection of the single most viable blastocyst among a cohort developing in vitro.

Gene expression profiling of human oocytes following in vivo or in vitro maturation.

BACKGROUND: Immature human oocytes matured in vitro, particularly those from gonadotrophin stimulated ovaries, are developmentally incompetent when compared with oocytes matured in vivo. This developmental incompetence has been explained as poor oocyte cytoplasmic maturation without any determination of the likely molecular basis of this observation. METHODS: Replicate whole human genome arrays were generated for immature and mature oocytes (matured in vivo and in vitro, prior to exposure to sperm) recovered from women undertaking gonadotrophin treatment for assisted reproduction. RESULTS: More than 2000 genes were identified as expressed at more than 2-fold higher levels in oocytes matured in vitro than those matured in vivo (P < 0.05, range 4.98 x 10(-2) -2.22 x 10(-4)) and 162 of these are expressed at 10-fold or greater levels (P < 0.05, range 4.98 x 10(-2)-1.38 x 10(-3)). Many of these genes are involved in transcription, the cell cycle and its regulation, transport and cellular protein metabolism. CONCLUSIONS: Global gene expression profiling using microarrays and bioinformatics analysis has provided a molecular basis for differences in the developmental competence of oocytes matured in vitro compared with in vivo. The over-abundance of transcripts identified in immature germinal vesicle stage oocytes recovered from gonadotrophin stimulated cycles and matured in vitro is probably due to dysregulation in either gene transcription or post-transcriptional modification of genes. Either mechanism would result in an incorrect temporal utilization of genes which may culminate in developmental incompetence of any embryos derived from these oocytes.

Glucose metabolism of human morula and blastocyst-stage embryos and its relationship to viability after transfer.

The pregnancy rate and implantation rate following blastocyst transfer in the human have been reported to be high; however, it has remained necessary to transfer 2-3 blastocysts to achieve these rates. Morphological criteria are currently used to select blastocysts for transfer and have some limited correlation with ongoing viability. Glucose metabolism of 189 human morula to blastocyst stage embryos was analysed using a non-invasive ultramicrofluorescence technique to determine if this could be used to predict viability. There was a linear trend to increased glucose uptake with progression from the morula to the hatching/hatched blastocyst stage of development, whereas glycolytic activity did not vary. There was no consistent difference in glucose uptake or glycolytic activity for embryos at the various morphological stages on day 5 compared to day 6 in vitro. Glucose uptake and glycolytic activity of the nine embryos positively identified as having implanted following transfer varied and were apparently not different from the values for embryos that failed to implant. In addition, viability was demonstrated to be compatible with high glycolytic activity, with four of nine implanted embryos having a glycolytic activity in the highest 15% of the population of embryos studied. Glucose uptake and glycolytic activity of male and female embryos did not appear to be different. Glucose metabolism cannot be used prospectively to select viable human morula or blastocyst stage embryos for transfer and it is also unlikely to be a useful tool to predict the sex of the embryo.

Prolonged administration of a dithiol antioxidant protects against ventricular remodeling due to ischemia-reperfusion in mice.

The prolonged production of reactive oxygen species due to ischemia-reperfusion (I/R) is a potential cause of the pathological remodeling that frequently precedes heart failure. We tested the ability of a potent dithiol antioxidant, bucillamine, to protect against the long-term consequences of I/R injury in a murine model of myocardial infarction. After transiently occluding the left anterior descending coronary artery for 30 min, saline or bucillamine (10 microg/g body wt) was injected intravenously as a bolus within the first 5 min of reperfusion. The antioxidant treatment continued with daily subcutaneous injections for 4 wk. There were no differences in infarct sizes between bucillamine- and saline-treated animals. After 4 wk of reperfusion, cardiac hypertrophy was decreased by bucillamine treatment (ventricular weight-to-body weight ratios: I/R + saline, 4.5 +/- 0.2 mg/g vs. I/R + bucillamine, 4.2 +/- 0.1 mg/g; means +/- SE; P < 0.05). Additionally, the hearts of bucillamine-treated mice had improved contractile function (echocardiographic measurement of fractional shortening) relative to saline controls: I/R + saline, 32 +/- 3%, versus I/R + bucillamine, 41 +/- 4% (P < 0.05). Finally, I/R-induced injury in the saline-treated mice was accompanied by a fetal pattern of gene expression determined by ribonuclease protection assay that was consistent with pathological cardiac hypertrophy and remodeling [increased atrial natriuretic peptide, beta-myosin heavy chain (MHC), skeletal alpha-actin; decreased sarco(endo)plasmic reticulum Ca2+ ATPase 2a, and alpha-MHC-to-beta-MHC ratio]. These changes in gene expression were significantly attenuated by bucillamine. Therefore, treatment with a dithiol antioxidant for 4 wk after I/R preserved ventricular function and prevented the abnormal pattern of gene expression associated with pathological cardiac remodeling.

Cytokines regulate matrix metalloproteinases and migration in cardiac fibroblasts.

We sought to define the relationship between cytokine stimulated release of matrix metalloproteinases (MMPs) and cell migration using adult rat cardiac fibroblasts. Interleukin-1beta (IL-1beta) increased release of MMP-2, -3, and -9, and TIMP-1, by 3-6-fold, measured by immunoblotting and gel zymography. Tumor necrosis factor-alpha (TNFalpha) augmented IL-1beta stimulated release of MMP-9, but not MMP-2 or -3. Transforming growth factor-beta1 (TGFbeta1) attenuated all the responses to IL-1beta. IL-1beta was also the most robust stimulus of adult rat cardiac fibroblast migration, measured in Boyden chamber assays. The combination of IL-1beta plus TNFalpha substantially enhanced migration, whereas TGFbeta1 strongly inhibited the migratory response to IL-1beta. The pan-selective MMP inhibitor GM 6001 effectively blocked IL-1beta stimulated migration. Pharmacologic inhibitors selective for ERK, JNK, and p38 MAP kinase pathways inhibited the IL-1beta regulation of individual MMPs. Increased MMP activity associated with migration of cardiac fibroblasts may be important determinants of cytokine-directed remodeling of injured myocardium.

Optimization of a microarray based approach for deriving representative gene expression profiles from human oocytes.

The purpose of the present study was to optimize a protocol for deriving reproducible and representative gene expression profiles from very rare research samples of human oocytes using microarrays. Immature oocytes produced as a result of administration of gonadotrophins for the treatment of infertility were donated to research. Linear amplification (L-amp) and exponential amplification (E-amp) were both capable of generating sufficient product for hybridization to the microarrays even from the low amount of template mRNA present in a single human oocyte. Slightly higher numbers of transcripts were detected by microarray following linear rather than E-amp but both techniques generated a product with reliably reproducible sensitivity and fidelity providing oocytes were pooled in minimum numbers of three. The majority of the variance associated with amplification and hybridization to arrays comes from the molecular processing. Slightly greater additional variance is associated with biological differences in immature oocytes from the same or different donors. The findings suggest that representative gene expression profiles can be generated from human oocytes for comparative purposes following L-amp and hybridization to microarray. However, oocytes must be pooled for the starting template for each array and sufficient independent microarray experiments performed to minimize the variance associated with molecular processing.

Developmental effects of sublethal mitochondrial injury in mouse oocytes.

Mitochondrial dysfunction may be acquired or inherited by oocytes without detectable morphological abnormalities. This pathology may account for some examples of unexplained pregnancy loss in women following transfer of morphologically normal in vitro fertilization (IVF) embryos. The present study was intended to determine whether sublethal mitochondrial injury in mouse oocytes before IVF negatively affects pre- and postimplantation development, and to further define the latency of developmental compromise in relation to aberrant mitochondrial metabolism. Mature mouse oocytes were loaded with the mitochondrial fluorophore rhodamine-123 and photosensitized for 20 sec, a duration previously found to permit preimplantation embryo development to the blastocyst stage and so deemed "sublethal." This treatment resulted in some aberrations in cytoplasmic patterning of organelles, but did not inhibit zygote mitochondrial metabolism. Blastocyst development following IVF was not significantly inhibited following sublethal oocyte photosensitization; however, a decrease in trophectoderm cell numbers was observed relative to untreated controls. Following intrauterine transfer, blastocysts derived from sublethally photosensitized oocytes implanted but later aborted at a higher rate, formed fetuses with lower average weights, and, in rare cases, formed abnormal fetuses relative to controls. Photosensitization for more prolonged durations resulted in failed fertilization (2 min) and rapid oocyte degeneration (10 min). Therefore, photosensitization duration and the consequent degree of mitochondrial dysfunction are negatively related to the onset of developmental compromise. Acquired low-level mitochondrial injury is heritable by the resultant embryos and can cause postimplantation developmental compromise that may be relevant to some clinically observed outcomes following human assisted reproduction strategies, including reduced birth weights for gestational age. Future strategies for the detection and prevention of mitochondrial dysfunction may assist in improving outcomes for some clinically infertile women.

Effect of female age on mouse oocyte developmental competence following mitochondrial injury.

Oocytes from aging ovaries contain mitochondria with morphological and genetic flaws. How these flaws relate to phenotypes of oocyte developmental compromise associated with clinical infertility is not well understood. This study was conducted to investigate the role of mitochondria in the developmental compromises observed with female aging using a mouse model of mitochondrial dysfunction. Oocytes obtained from aging (30-40 wk) (C57BL/6J x CBACaH)F1 (B6CBAF1) hybrid female mice were photosensitized with mitochondrial fluorophore rhodamine-123 for variable durations and compared to similarly treated oocytes derived from pubertal mice (4-6 wk). Blastocyst development of normally fertilized oocytes from both age-groups correlated negatively in mathematically unique profiles with irradiation time, with a more sudden decline in development for oocytes from aging mice. Complete inhibition of blastocyst development occurred following a shorter duration of photosensitization for oocytes from aging compared to pubertal animals (60 vs. 90 sec). Prolonged photosensitization resulted in mitochondrial uncoupling and promoted localized generation of reactive oxygen species, mitochondrial permeabilization, and apoptotic phenotypes. Thus, aging oocytes are more developmentally sensitive to mitochondrial damage than pubertal oocytes but undergo similar metabolic and apoptotic responses. These and future findings may encourage further optimization of laboratory-based strategies to minimize mitochondrial injury to oocytes, particularly those from older women, and improve clinical outcomes for women with age-related etiologies of infertility.

Mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro.

Oocyte mitochondrial dysfunction has been proposed as a cause of high levels of developmental retardation and arrest that occur in human preimplantation embryos generated using assisted reproductive technology in the treatment of some causes of female infertility. To investigate this, a model of mitochondrial dysfunction was developed in mouse oocytes using a method of photosensitization of the mitochondrion-specific dye, rhodamine-123. After in vitro fertilization, dye-loaded and photosensitized oocytes showed developmental arrest in proportion to irradiation time. Morphological and metabolic assessments of zygotes indicated an increase in mitochondrial permeability that subsequently resulted in apoptotic degeneration. Development was partially restored by inhibition of mitochondrial permeability transition pore formation by oocyte pretreatment with cyclosporin A. Oocyte mitochondria are therefore physiological regulators of early embryo development and potential sites of pathological insult that may perturb oocyte and subsequent preimplantation embryo viability. These findings have important implications for the treatment of clinically infertile women using assisted reproductive technologies.

Identification and characterisation of known and novel transcripts expressed during the final stages of human oocyte maturation.

The final stages of oocyte maturation, from the germinal vesicle (GV) stage to metaphase II (MII) oocytes, are characterised by a series of dynamic events. These include germinal vesicle break down (GVBD), resumption of meiosis, and nuclear and cytoplasmic maturation to produce MII oocytes ready for fertilisation. To investigate the specific genes transcribed during these stages of oogenesis, we have prepared and analysed amplified cDNA representing the transcribed genes in a series of GV and MII oocytes. Differential display analysis disclosed that the overall gene expression profiles between different samples of GV oocytes are very similar, regardless of their source, while those between the MII oocytes are markedly variable. A comparison of expression profiles in oocytes and somatic (cumulus) cells identified several known genes preferentially-expressed in oocytes (e.g., a zona pellucida gene), as well as five novel sequences. Two of the five novel sequences are homologous to retrotransposon sequences, long terminal repeat (LTR) and long interspersed nuclear element (LINE) 1, and two other sequences show partial homology to known ESTs and genomic sequences. The remaining sequence, which is identical to shorter ESTs isolated from germ cell tumor cDNA libraries, was extended towards its 5' end by PCR, using the original cDNA preparation from which it was isolated as a template. Expression of the resultant 1.1-kb transcript is restricted to the testis and ovary, and its expression correlates with cell pluripotency in that it is expressed in embryonal carcinoma cells, but not in their differentiated derivative cells.