Analysis of global gene expression following mouse blastocyst cryopreservation.

BACKGROUND: The aim of this study was to examine the effect of the cryopreservation procedure (slow freezing or vitrification) and cryoprotectants (1,2-propanediol or dimethylsulphoxide) on mouse blastocyst gene expression. METHODS: Cultured mouse blastocysts were cryopreserved with different protocols. Following thawing/warming, total RNA from re-expanded blastocysts was isolated, amplified and then analyzed using mouse whole-genome microarrays. RESULTS: Compared with non-cryopresevered control blastocysts, gene expression was only significantly altered by slow freezing. Slow freezing affected the expression of 115 genes (P < 0.05). Of these, 100 genes exhibited down-regulation and 15 genes were up-regulated. Gene ontology revealed that the majority of these genes are involved in protein metabolism, transcription, cell organization, signal transduction, intracellular transport, macromolecule biosynthesis and development. Neither of the vitrification treatment groups showed statistically different gene expression from the non-cryopreserved control embryos. Hierarchical cluster analysis, did however, reveal that vitrification using 1,2-propanediol could result in a gene expression profile closest to that of non-cryopreserved blastocysts. CONCLUSIONS: Investigating the effects of cryopreservation on cellular biology, such as gene expression, is fundamental to improving techniques and protocols. This study demonstrates that of the cryopreservation regimens employed, slow freezing induced the most changes in gene expression compared with controls.

A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation.

BACKGROUND: The aim of this study was to compare two methods of cryopreservation for the cleavage-stage human embryo: slow freezing and vitrification. METHODS: A total of 466 Day 3 embryos, donated with consent, underwent cryopreservation by either slow freezing in straws or vitrification using the cryoloop. The vitrification procedure did not include dimethyl sulfoxide, but rather employed ethylene glycol and 1,2-propanediol as the cryoprotectants. Survival, embryonic metabolism and subsequent development to the blastocyst were used to determine the efficacy of the two procedures. RESULTS: Significantly, more embryos survived the vitrification procedure (222/234, 94.8%) than slow freezing (206/232, 88.7%; P < 0.05). Consistent with this observation, pyruvate uptake was significantly greater in the vitrification group, reflecting a higher metabolic rate. Development to the blastocyst was also higher following vitrification (134/222, 60.3%) than following freezing (106/206, 49.5%; P < 0.05). In a separate cohort of 73 patients who had their supernumerary embryos cyropreserved with vitrification, the resulting implantation rate and clinical pregnancy rate were 30 and 49%, respectively. CONCLUSIONS: Analysis of metabolism revealed that vitrification had less impact on the metabolic rate of the embryo than freezing, which was reflected in higher survival rate and subsequent development in vitro. Excellent pregnancy outcomes followed the warming and transfer of vitrified cleavage-stage embryos. These data provide further evidence that vitrification imparts less trauma to cells and is, therefore, a more effective means of cryopreserving the human embryo than conventional slow freezing. Clinicaltrials.gov identifier: NCT00608010.

Maintenance of the meiotic spindle during vitrification in human and mouse oocytes.

Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37 degrees C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21-22 degrees C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37 degrees C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37 degrees C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37 degrees C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37 degrees C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.

1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology.

BACKGROUND: The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. METHODS: Intracellular calcium of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using 'nominally calcium free' media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.

The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLCzeta.

When sperm activate eggs at fertilization the signal for activation involves increases in the intracellular free Ca2+ concentration. In mammals the Ca2+ changes at fertilization consist of intracellular Ca2+ oscillations that are driven by the generation of inositol 1,4,5-trisphosphate (InsP3). It is not established how sperm trigger the increases in InsP3 and Ca2+ at fertilization. One theory suggests that sperm initiate signals to activate the egg by introducing a specific factor into the egg cytoplasm after membrane fusion. This theory has been mainly based upon the observation that injecting a cytosolic sperm protein factor into eggs can trigger the same pattern of Ca2+ oscillations induced by the sperm. We have recently shown that this soluble sperm factor protein is a novel form of phospholipase C (PLC), and it is referred to as PLCzeta(zeta). We describe the evidence that led to the identification of PLCzeta and discuss the issues relating to its potential role in fertilization.

Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes.

Fusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca(2+), leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca(2+) oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCzeta) has been identified in mice. Mouse PLCzeta triggers Ca(2+) oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCzeta homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCzeta protein sequences indicates a shorter X-Y linker region in human PLCzeta and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCzeta elicits Ca(2+) oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCzeta elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCzeta from monkeys and mice. These results are consistent with the proposal that sperm PLCzeta is the molecular trigger for egg activation during fertilization and that the role and activity of PLCzeta is highly conserved across mammalian species.

The role of calcium in ABA-induced gene expression and stomatal movements.

There is much interest in the transduction pathways by which abscisic acid (ABA) regulates stomatal movements (ABA-turgor signalling) and by which this phytohormone regulates the pattern of gene expression in plant cells (ABA-nuclear signalling). A number of second messengers have been identified in both the ABA-turgor and ABA-nuclear signalling pathways. A major challenge is to understand the architecture of ABA-signalling pathways and to determine how the ABA signal is coupled to the appropriate response. We have investigated whether separate Ca2+-dependent and -independent ABA-signalling pathways are present in guard cells. Our data suggest that increases in [Ca2+]i are a common component of the guard cell ABA-turgor and ABA-nuclear signalling pathways. The effects of Ca2+ antagonists on ABA-induced stomatal closure and the ABA-responsive CDeT6-19 gene promoter suggest that Ca2+ is involved in both ABA-turgor signalling and ABA-nuclear signalling in guard cells. However, the sensitivity of these pathways to alterations in the external calcium concentration differ, suggesting that the ABA-nuclear and ABA-turgor signalling pathways are not completely convergent. Our data suggest that whilst Ca2+-independent signalling elements are present in the guard cell, they do not form a completely separate Ca2+-independent ABA-signalling pathway.

Usefulness of serum carboxy-terminal propeptide of procollagen type I in assessment of the cardioreparative ability of antihypertensive treatment in hypertensive patients.

BACKGROUND: We investigated whether serum concentration of carboxy-terminal propeptide of procollagen type I (PIP), a marker of collagen type I synthesis, can be used to assess the ability of antihypertensive treatment to regress myocardial fibrosis in hypertensive patients. METHODS AND RESULTS: The study was performed in 37 patients with essential hypertension and hypertensive heart disease. After randomization, 21 patients were assigned to losartan and 16 patients to amlodipine treatment. At baseline and after 12 months, right septal endomyocardial biopsies were performed to quantify collagen volume fraction (CVF) on picrosirius red-stained sections with an automated image-analysis system. Serum PIP was measured by specific radioimmunoassay. Nineteen patients in the losartan group and 11 in the amlodipine group finished the study. Time-course changes in blood pressure during treatment were similar in the 2 groups of patients. In losartan-treated patients, CVF decreased from 5.65+/-2.03% to 3.96+/-1.46% (P<0.01) and PIP from 127+/-30 to 99+/-26 microgram/L (P<0.01). Neither CVF or PIP changed significantly in amlodipine-treated patients. CVF was directly correlated with PIP (r=0.44, P<0.001) in all hypertensives before and after treatment. CONCLUSIONS: These findings suggest that the ability of antihypertensive treatment to regress fibrosis in hypertensives with biopsy-proven myocardial fibrosis is independent of its antihypertensive efficacy. Our data also suggest that blockade of the angiotensin II type 1 receptor is associated with inhibition of collagen type I synthesis and regression of myocardial fibrosis in hypertensives. Thus, determination of serum PIP may be useful to assess the cardioreparative properties of antihypertensive treatment in hypertensives.

Calcium and mitosis.

Calcium is thought to be involved in regulating mitotic transitions. The basis for this view is set out. Recent data from experiments on sea urchin embryos is discussed. The relative simplicity of the embryonic cell cycle and the relative ease with which cell physiology can be done in sea urchin embryos has allowed the clear demonstration that the phosphoinositide-calcium-calmodulin signalling pathway is required for and regulates mitosis entry and anaphase onset. The relevance of the sea urchin work to mitosis in other cell types is briefly discussed.

Serum carboxy-terminal propeptide of procollagen type I is a marker of myocardial fibrosis in hypertensive heart disease.

BACKGROUND: This study was designed to investigate whether the serum concentration of the carboxy-terminal propeptide of procollagen type I (PIP), a marker of collagen type I synthesis, is related to myocardial fibrosis in hypertensive patients. METHODS AND RESULTS: The study was performed in 26 patients with essential hypertension in which ischemic cardiomyopathy was excluded after a complete medical workup. Right septal endomyocardial biopsies were performed in hypertensive patients to quantify collagen content. Collagen volume fraction (CVF) was determined on picrosirius red-stained sections with an automated image analysis system. The serum concentration of PIP was measured by specific radioimmunoassay. Compared with normotensives, both serum PIP and CVF were increased (P<0.001) in hypertensives. A direct correlation was found between CVF and serum PIP (r=0.471, P<0.02) in all hypertensives. Histological analysis revealed the presence of 2 subgroups of patients: 8 with severe fibrosis and 18 with nonsevere fibrosis. Serum PIP was higher (P<0.05) in patients with severe fibrosis than in patients with nonsevere fibrosis. Using receiver operating characteristic curves, we observed that a cutoff of 127 microg/L for PIP provided 78% specificity and 75% sensitivity for predicting severe fibrosis with a relative risk of 4.80 (95% CI, 1.19 to 19.30). CONCLUSIONS: These results show a strong correlation between myocardial collagen content and the serum concentration of PIP in essential hypertension. Although preliminary, these findings suggest that the determination of PIP may be an easy and reliable method for the screening and diagnosis of severe myocardial fibrosis associated with arterial hypertension.

[Coronary revascularization surgery with arterial grafts]. / Cirugía de revascularización coronaria con injertos arteriales.

Coronary artery revascularization surgery is well established procedure throughout the world. The benefits are based on the blood perfusion through the implanted grafts distal to the coronary occlusions and continue as long as the grafts remains patent. The immediate success of this procedure is related to the surgical technique and the anatomical characteristics of the grafted coronary arteries. However, the long term results are mainly dependent on the type of grafts used. The modern era of myocardial revascularization started in the sixties with the use of saphenous vein grafts by Johnson and Favaloro. Alternative vascular conduits to the saphenous vein have been tried, and the internal mammary artery has become the first choice. In recent years, various publications have demonstrated the supremacy of the internal mammary artery over the saphenous vein when used as a single, bilateral, sequential of free graft. In order to obtain full myocardial revascularization, the use of alternatives to the internal mammary artery is required. The gastroepiploic artery, the inferior epigastric artery and the radial artery have been used as alternatives to the saphenous vein with the hope of obtaining long term results similar to the internal mammary artery.

[Percutaneous mitral valvuloplasty. Immediate results with the 30 mm diameter dilatation balloon]. / Valvuloplastia mitral percutánea. Resultados inmediatos con balón de dilatación de 30 mm de diámetro.

Percutaneous balloon dilatation of the mitral valve is a promising alternative to surgical treatment for patients with mitral stenosis. We analysed echocardiographic and haemodynamic results in 62 consecutive patients who underwent mitral dilatation with a big and one chamber balloon (30 mm diameter). The relationship between the dilatation balloon area and body surface was 4.2 (in 98% of patients it was bigger than 3.5). In the whole series we get significant improvements in the haemodynamic parameters and mitral areas (0.91 cm2 versus 1.78 cm2)(p < 0.005). According to the echocardiographic score, patients were divided in two groups. Good results (mitral area > 1.5 cm2, and final, valve area 50% greater than the initial area), were obtained in 93% of patients with a score of 8 or less, and in 45% of patients with a score bigger than 8. Seventy-five percent of patients with bad results (mitral area < 1.5 cm2) were in atrial fibrillation versus 37% of patients where we get a good results. Mortality of procedure was nulle, and two patients required surgery because of procedure failure for mitral insufficiency. We can conclude: percutaneous mitral valvuloplasty with single balloon is valid alternative, and good results can be expected if there is a good relationship between the area of balloon and patient's body-surface. The echocardiographic score and presence of atrial fibrillation can predict immediate results of this technique.

[Ultrasonic decalcification of the aortic valve. Initial experience]. / Decalcificación ultrasónica de la válvula aórtica. Experiencia inicial.

We report our initial experience in 2 patients with degenerative calcific aortic stenosis who underwent ultrasonic debridement of the aortic valve. Compared with preoperative studies, doppler echocardiographic and hemodynamic evaluation before hospital discharge revealed a reduction in the mean aortic valve pressure gradient (80 and 65 mmHg to less than 10 mmHg). There was no change in aortic regurgitation grade. Follow-up doppler echocardiographic evaluation at four and six months showed no changes in gradient or regurgitation in the comparison to the postoperative data. Long-term results will show the convenience to attempt or not ultrasonic salvage of the native aortic valve in severe calcific stenosis.

[Percutaneous cardiopulmonary bypass in angioplasty and valvuloplasty. The technical considerations]. / Bypass cardiopulmonar percutáneo en la angioplastia y la valvuloplastia. Consideraciones técnicas.

To reduce problems during percutaneous transluminal angioplasties and valvuloplasties in the catheterization laboratory, percutaneous cardiopulmonary bypass was used in 6 patients (three aortic, two mitral and one coronary). Percutaneous 21 F (venous) and 17 F (arterial) cannulas were placed through the femoral vessels. Mean flows of 3 l/min were achieved with a roller pump, with a mean blood pressure of 60 mmHg. Bypass time ranged between 22 and 55 minutes (mean 39). Light intravenous sedations and spontaneous breathing was maintained in all cases. Maximum inflation time of balloons were 4 minutes for the aortic valvuloplasty patients and 50 seconds for the mitral. Two patients developed groin hematomas, and one of them required surgical exploration. Percutaneous bypass is a simple and easily achieved technique to ensure safety in the high-risk patient.

In situ right gastroepiploic artery. A conduit for coronary revascularization.

The internal mammary artery is widely recognized as the graft of choice for coronary artery bypass grafting at present. Alternative conduits have been investigated in order to find other adequate long-term grafts. The right gastroepiploic artery has been recently used as a graft to bypass distal coronary vessels. From November 1989 to June 1990, we have implanted this artery in 46 cases. Pedicled grafts were implanted in 20 patients to the main right coronary artery, in 21 patients we grafted the right distal branches, in 3 patients the left anterior descending, and in 2 the circumflex branches. Mean grafts per patient were 3 in this series, with a mean of 2.2 arterial grafts per patient. One patient died in the early postoperative period. The remaining patients had an uncomplicated postoperative evolution. Thirteen patients underwent graft and coronary angiography. Direct or indirect graft patency was confirmed in all cases. The final important issue concerning the long-term patency of this graft will be solved in the future, but short-term patency rates of the right gastroepiploic artery can be anticipated when proper techniques are used.

[Myocardial revascularization with the gastroepiploic artery. The initial experience]. / Revascularización miocárdica con arteria gastroepiploica. Experiencia inicial.

The internal mammary artery is widely recognized as the graft of choice for coronary artery bypass grafting at present. Alternative conduits have been investigated in order to find other adequate long-term grafts. The right gastroepiploic artery has been recently used as a graft to bypass distal coronary vessels. From November 1989 to April 1990 we have implanted this artery in 33 cases (32 in situ and one free grafts). The pedicled grafts were implanted in 17 patients in the main right coronary artery, in 13 we grafted the right distal branches, in one the left anterior descending, and the circumflex branch in one. The free graft was implanted in the first diagonal. Mean grafts per patient was of three in this series, with a mean of 2 arterial grafts per patient. One patient died in the early postoperative period. The remaining patients had an uncomplicated postoperative period. Thirteen patients underwent graft and coronary angiography. Direct or indirect graft patency was confirmed in all cases. The final important issue concerning the long-term patency of this graft will be solved in the future, but short-term patency rates of the right gastroepiploic artery can be anticipated when proper techniques are used.

Calcified constrictive pericarditis. Ultrasonic debridement.

A case of complete debridement of a calcified pericardium in a patient with constrictive pericarditis is reported. The use of an ultrasonic surgical aspirator facilitated disintegration of calcium without damaging the adherent epicardium and myocardium.

Noninvasive determination of pulmonary arterial systolic pressure by continuous wave Doppler.

We evaluated the accuracy of continuous wave Doppler for estimating pulmonary arterial systolic pressure in patients with tricuspid regurgitation. Of 44 patients with a variety of cardiac disorders, 39 (89%) had Doppler-detected tricuspid regurgitation. Adequate spectral profiles of the flow signals were obtained in 34 of them (87%), representing 77% of the entire group. Continuous wave Doppler ultrasound was used to measure the maximum velocity of the regurgitant jet, and by applying the modified Bernoulli equation, the systolic pressure gradient between the right ventricle and the right atrium was calculated. Pulmonary arterial systolic pressure was estimated by adding the transtricuspid gradient to the mean right atrial pressure, and correlated well with catheterization values (r = 0.96). The correlation coefficient was not significantly modified if mean right atrial pressures were excluded in the calculations (r = 0.91). Continuous wave Doppler constitutes a sensitive method for the detection of tricuspid regurgitation. The method using the tricuspid gradient provides an accurate estimation of pulmonary arterial systolic pressure. Combined with other available methods (pulsed wave Doppler), this noninvasive technique can yield information comparable with that obtained at catheterization.