Single-nucleotide polymorphism microarray-based preimplantation genetic diagnosis is likely to improve the clinical outcome for translocation carriers.

STUDY QUESTION: Is preimplantation genetic diagnosis (PGD) for translocation carriers more effective when done with a single-nucleotide polymorphism (SNP) array using trophectoderm (TE) biopsy and frozen embryo transfer (FET) compared with traditional PGD based on fluorescence in situ hybridization (FISH-PGD) using blastomere biopsy and fresh embryo transfer? SUMMARY ANSWER: The procedure using the SNP array combined with TE biopsy and FET significantly improves the clinical pregnancy rate for translocation carriers. The miscarriage rate also slightly decreases. WHAT IS KNOWN ALREADY: FISH-PGD has been widely used in translocation carriers but the clinical outcomes have not been ideal. SNP arrays can detect both chromosome segmental imbalances and aneuploidy, and may overcome the limitations of FISH in PGD for translocation carriers. STUDY DESIGN, SIZE AND DURATION: This was a retrospective study of 575 couples with chromosomal translocations, including 169 couples treated by SNP-PGD between October 2011 and August 2012, and 406 couples treated by FISH-PGD between January 2005 and October 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was set in an IVF center at the Reproductive and Genetic Hospital of CITIC-Xiangya, China. In total, 169 couples underwent SNP analysis, including 52 Robertsonian translocation carriers and 117 carriers of reciprocal translocations. Blastocysts (n = 773) were biopsied and FET was carried out on the balanced embryos. Four hundred and six couples underwent FISH-PGD, including 149 Robertsonian translocation carriers and 257 reciprocal translocation carriers. In total, 3968 embryos were biopsied and balanced embryos were transferred fresh. The SNP-PGD results and clinical outcomes were compared with those of FISH-PGD. MAIN RESULTS AND THE ROLE OF CHANCE: Reliable SNP-PGD results were obtained for 717 out of 773 (92.8%) biopsied blastocysts. The proportions of normal/balanced embryos, embryos with translocation-related and translocation-unrelated abnormalities, the median number of embryos per patient, the ongoing pregnancy rate per embryo transfer and the miscarriage rate were 58, 23, 19, 2, 69 and 12%, respectively, for Robertsonian translocation carriers and 36, 52, 12, 1, 74 and 11%, respectively, in reciprocal translocation carriers. Reliable FISH-PGD results were obtained for 3452 out of 3968 (87.0%) biopsied embryos. The proportions of normal/balanced embryos, unbalanced embryos, the median number of embryos per patient, the ongoing pregnancy rate per transfer and the miscarriage were 36, 64, 3, 38 and 17%, respectively, for Robertsonian translocation carriers and 20, 80, 1, 39 and 16%, respectively, for reciprocal translocation carriers. Thus, SNP-PGD achieved a higher pregnancy rate but a lower miscarriage rate than FISH-PGD. There were no significant differences in maternal age, basal endocrine level and the average number of retrieved oocytes and good-quality D3 embryos in the SNP-PGD group compared with the FISH-PGD group. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study with the two groups treated in different periods; therefore, there is a chance of sample bias and a possibility that the results were influenced by other factors that changed over time. Furthermore, the two treatment protocols differ in several respects and we cannot say which makes the greatest contribution to the difference in success. Complete pregnancy outcomes of SNP-PGD have not been obtained as some embryos have not been transferred yet. We cannot exclude differences between the final data and the data in the present manuscript. WIDER IMPLICATIONS OF THE FINDINGS: The adoption of SNP-PGD combined with TE biopsy and FET may significantly improve the clinical pregnancy rate, and decrease the miscarriage rate after PGD for translocation carriers.

Cyclic regulation of LPA3 in human endometrium.

BACKGROUND: Lysophosphatidic acid (LPA) belongs to the group of lipid messengers, which plays a pivotal role in the establishment of implantation via its cellular receptor, LPA3. The aims of the present study were to characterize LPA3 mRNA and protein in human endometrium during the normal human menstrual cycle. METHODS: Forty-three normally cycling volunteers without reproductive disorders were randomized to undergo endometrial sampling on a specific cycle day. Samples were assessed for relative LPA3 mRNA expression using real-time PCR and for LPA3 protein using immunohistochemistry and western blot. RESULTS: The expression of LPA3 mRNA significantly increased during the early and late secretory phase compared with other menstrual phases. LPA3 protein was localized to the epithelial and stromal cells and expression levels followed the same pattern as for LPA3 mRNA. CONCLUSION: In the normal human menstrual cycle, LPA3 mRNA and protein expression also change, indicating that this gene may be related to the function of the endometrium.

Concomitant EEG, lactate, and phosphorus changes by 1H and 31P NMR spectroscopy during repeated brief cerebral ischemia.

Pilots of high-performance aircraft are subject to transient loss of consciousness due to cerebral ischemia resulting from sudden high gravitational stress. To assess the effects of gravitational stress-induced blackout on cerebral metabolism and electrical function, we developed an animal model in which global cerebral ischemia is produced repeatedly at short intervals. Rats were prepared by ligation of subclavian and external carotid arteries and the right carotid artery was cannulated bidirectionally to measure circle of Willis and systemic pressures. Ischemia was induced by inflation of an occluder about the left carotid artery. Interleaved 31P and 1H NMR spectra were acquired on a 4.7-T Biospec system simultaneously with EEG recordings. We report results from 20 experiments of 30-min duration in which rats were subject to 30 1-min ischemia:reflow cycles of 10I:50R, 20I:40R, 30I:30R, and 40I:20R [numbers are seconds of ischemia (I) and reflow (R) during each 1-min cycle]. During ischemia the graded delivery of the ischemic insult permitted direct correlations between 2- to 5- and 7- to 20-Hz EEG activity and progressive changes in pH, lactate, ATP, phosphocreatine (PCr) and Pi. The best correlations were found between EEG activity and pH and PCr; correlation coefficients ranged from 0.93 to 0.95. A loss of EEG activity was observed without significant sustained energy loss in all but the most severe cycle.

Evaluation of lactate production and clearance kinetics by 1H NMR in a model of brief repetitive cerebral ischemia.

Pilots of high-performance aircraft are subject to repeated transient cerebral ischemia during high-gravitational stress maneuvers. Previously we have demonstrated that repeated episodes of transient cerebral ischemia and reflow are cumulative and lactate accumulations appear to be exponential. To evaluate the metabolic events determining the kinetics of lactate accumulation, and therefore the rates of substrate utilization, we have used in vivo 1H nuclear magnetic resonance with a 5-s time resolution to measure lactate production and clearance. The individual rates for each animal were then used to predict the accumulation of lactate in the same animal during 30 episodes of ischemia and reflow. Lactate accumulation was modeled as the balance between a zero-order production process during the ischemic period and a first-order clearance process. The predicted lactate accumulation showed excellent agreement with the observed time course, validating the predictive power of the simple model used. The highly reproducible nature of this model and its accuracy in predicting lactate accumulation should enable more accurate studies of the deleterious effects of lactate accumulation in cerebral ischemia by providing a highly reproducible means for generating a specific level of lactate accumulation.

Immunolocalization of FGF-1 and receptors in human renal allograft vasculopathy associated with chronic rejection.

Despite recognition of chronic vasculo-occlusive disease in solid organ transplantation, the exact pathophysiologic events resulting in neointima formation remain to be elucidated. Since acidic fibroblast growth factor (FGF-1) is an established modulator of vascular cell function, we examined the expression of this growth factor and its high affinity receptors in both relevant renal transplant controls (n = 5) and tissue from patients (n = 19) who underwent nephrectomy following graft loss secondary to chronic rejection. In situ hybridization and immunohistochemical studies demonstrated minimal vascular expression and distribution of FGF-1 and FGF high affinity receptors in the normal human kidney. In contrast, vascular lesions in kidney allografts experiencing chronic rejection demonstrated the exaggerated appearance of FGF-1 ligand and receptors. Immunoreactive FGF-1 readily was detected in medial smooth muscle cells and focal areas of intimal hyperplasia, particularly in association with the presence of inflammatory infiltrate. Enhanced staining for FGF-1 mRNA primarily was associated with the appearance of resident inflammatory cells. Medial smooth muscle cells of hyperplastic vascular structures demonstrated the greatest immunoappearance of FGF receptors-however, diffuse immunostaining also was observed in areas of intimal hyperplasia. The enhanced appearance of both FGF-1 and FGF receptors in the vascular wall suggests that this polypeptide mitogen may serve as an important mediator of growth responses associated with neointima development and angiogenesis during chronic rejection of human renal allografts.

Immunolocalization of FGF-1 and receptors in glomerular lesions associated with chronic human renal allograft rejection.

Glomerular lesions are considered one of the more detrimental pathologic changes associated with chronic rejection of renal allografts. To elucidate potential pathophysiologic mechanisms associated with transplant glomerulopathy, we examined the expression of acidic fibroblast growth factor (FGF-1) and its high-affinity receptors (FGFR) in both relevant renal transplant controls (n=5) and tissue from patients (n=19) who underwent nephrectomy following graft loss secondary to chronic rejection. In situ immunohistochemical analyses demonstrated minimal staining and distribution of FGFR and FGF-1, which was localized to the mesangial matrix in glomeruli from normal human kidneys. In situ hybridization failed to detect the presence of FGF-1 mRNA in control tissue. In contrast, each stage of the developing glomerular lesion associated with chronic rejection demonstrated the exaggerated appearance of FGF-1 protein in visceral and parietal epithelial cells. Intense staining for FGF-1 protein did not correlate with the increased appearance of FGF-1 mRNA, which was restricted to circulating inflammatory cells. Glomeruli in kidneys with findings of chronic rejection also exhibited increased immunodetection of both FGFR and PCNA in mesangial and epithelial cells. Immunogold labeling of chronically rejected visceral epithelial cells revealed both cytoplasmic and nuclear/localization of FGF-1, thereby establishing mitogenic potential of the growth factor. The enhanced appearance of both biologically active FGF-1 and FGFR suggests that this polypeptide may serve as an important mediator of growth responses associated with glomerular lesion development during chronic rejection.

Immunolocalization of acidic fibroblast growth factor and receptors in the tubulointerstitial compartment of chronically rejected human renal allografts.

Tubular damage and loss associated with interstitial inflammation and fibrosis may be the most important determinants in chronic renal allograft rejection. To elucidate potential pathophysiologic mechanisms associated with tubulointerstitial lesions, we examined the expression of a fibrogenic cytokine, acidic fibroblast growth factor (FGF-1) and its high-affinity receptors, in both relevant renal transplant controls (n=5) and tissue from patients (n=19) who underwent nephrectomy after graft loss, secondary to chronic rejection. In situ hybridization and immunohistochemical analyses demonstrated minimal expression of FGF-1 mRNA and protein in the tubulointerstitial compartment of the normal human kidney. In contrast, tubulointerstitial lesions in kidney allografts experiencing chronic rejection demonstrated the exaggerated appearance of both FGF-1 protein and mRNA in resident inflammatory and tubular epithelial cells. Patterns of staining were consistent throughout tubular compartments and did not appear to be localized to any particular region. The tubulointerstitium in kidneys with findings of chronic rejection also exhibited increased immunodetection of proliferating cell nuclear antigen in the tubular epithelium, inflammatory cell infiltrate, and neovascular structures. The enhanced appearance of FGF-1 and readily detectable fibroblast growth factor receptors suggests that this polypeptide mitogen may serve as an important mediator of growth and repair responses, associated with development of angiogenesis and tubulointerstitial lesions during chronic rejection of human renal allografts.

Using SNP array to identify aneuploidy and segmental imbalance in translocation carriers.

Translocation is one of the more common structural rearrangements of chromosomes, with a prevalence of 0.2%. The two most common types of chromosomal translocations, Robertsonian and reciprocal, usually result in no obvious phenotypic abnormalities when balanced. However, these are still associated with reproductive risks, such as infertility, spontaneous abortion and the delivery of babies with mental retardation or developmental delay. In recent years, array-based whole-genome amplification (WGA) technologies, including microarray comparative genomic hybridization (array CGH; aCGH) and single-nucleotide polymorphism (SNP) micro-arrays, have enabled the screening of every chromosome for whole-chromosome aneuploidy and segmental imbalance. These techniques have been shown to have clinical application for translocation carriers. Promising studies have indicated that array-based PGD of translocation carriers can lead to transfer pregnancy rates of 45-70% [2]. In addition to genetic testing techniques, the embryo biopsy stage (polar body, cleavage embryo or blastocyst) and the mode of embryo transfer (fresh or frozen embryos) can affect the outcome of PGD. It is now generally recommended that blastomere biopsy should be replaced by blastocyst biopsy to avoid a high mosaic rate and biopsy-related damage to cleavage-stage embryos, which might affect embryo development. However, more clinical data are required to confirm that the technique of SNP array-based PGD (SNP-PGD) combined with trophectoderm (TE) biopsy and frozen embryo transfer (FET) is superior to traditional FISH-PGD combined with Day 3 (D3) blastomere biopsy and fresh embryo transfer.