Technical aspects of laparoscopic ovarian autograft in ewes after cryopreservation by slow-cooling protocol.

Iatrogenic ovarian failure and infertility are long term-term side effects of anticancerous gonadotoxic treatments in children or women of reproductive year. Ovarian cortex cryopreservation can be a solution to preserve immature germinal cells before gonadotoxic treatment, for later transplantation. The aim of our study was to prove the efficiency of a laparoscopic technique for orthotopic graft after a slow-freezing/thawing protocol, and to evaluate the effect of ovarian cryopreservation and autograft on the primordial follicle survival rate. Experimental surgical study was performed on 6- to 12-month-old ewes. The study was approved by the ethic committee of the Lyon-veterinary-school. The left ovary was removed by laparoscopy and cut in half, and medulla was excised. In group 1 (n = 6), autograft was performed immediately on the right ovary, and in group 2 (n = 6), graft was performed after a slow-freezing/thawing protocol. The second hemi-ovary served as an ungrafted control fragment. A polypropylene/polyglactin mesh was included between graft and base to separate the two structures, to help histological analysis. The mean graft performance time was 71 +/- 8 min in the first group and 57 +/- 10 min in the second. Freezing did not affect the number of primordial follicles. In the ungraft control fragments, the global anomaly rate (cytoplasm plus nuclear anomaly) increased after freezing (p < 0.05). Others results did not reach signification. Pelvic adhesion occurred only once. The post-graft primordial follicle survival rate was 5.1 +/- 2.8% in the non-frozen group vs. 6.3 +/- 2.3% after freezing/thawing. Kruskal-Wallis and Wilkoxon non-parametric tests were used for statistical analysis. Laparoscopy seems to be a well-adapted technique for ovarian tissue orthotopic autograft. The main follicle loss occurs before graft revascularization. Our orthotopic graft's procedure has to be improved to obtain a better graft's neovascularization, and to have a better long-term post-graft primordial follicle survival rate.

Better preservation of endothelial function and decreased activation of the fetal renin-angiotensin pathway with the use of pulsatile flow during experimental fetal bypass.

OBJECTIVE: Pulsatile flow was shown to overcome the progressive rise in peripheral and placental vascular resistances observed during steady-flow bypass, this rise being counteracted by inhibition of nitric oxide synthase. This study quantifies the release of endothelial vasoactive substances during a 60-minute in utero model of fetal bypass. METHODS: Fetuses were randomly allocated into 1 of 2 groups (steady flow, n = 8, or pulsatile flow, n = 13) and subjected to bypass through central cannulation and perfusion with either a centrifugal or pulsatile (125 beats x min(-1)) blood pump. RESULTS: Lactate concentration was high, starting at fetal exteriorization and increasing during fetal preparation in the 2 groups. Once bypass was established, the rise was significant only in the steady-flow group. Plasma nitric oxide metabolites, similar before bypass, reached higher levels during pulsatile flow at the end of bypass (99+/-9 vs. 82+/-23 micromol x L(-1); P =.037). Levels of urinary nitric oxide metabolites were significantly higher in the pulsatile-flow than in the steady-flow group (764+/-143 vs. 508+/-240 micromol x L(-1); P =.005). Plasma cyclic guanosine monophosphate levels increased after 30 minutes of bypass in the pulsatile-flow group (25+/-18 vs. 12+/-8 pmol x mL(-1); P =.004), and urinary cyclic guanosine monophosphate excretion was higher in the pulsatile-flow group (517+/-450 vs. 118+/-78 pmol x mL(-1); P =.024). Plasma endothelin-1 levels increased in the 2 groups and were higher in the steady-flow group at 30 minutes (27+/-5 vs. 23+/-2 pg x mL(-1); P =.04) and 60 minutes of bypass (39+/-7 vs 32 +/- 6 pg x mL(-1); P =.04). Plasma renin concentration increased significantly during bypass only in the steady-flow group (26+/-10 vs. 57+/-42 in ng A1 x mL(-1) x h(-1); P =.04). CONCLUSIONS: Improved placental and peripheral perfusion during fetal pulsatile-flow bypass may be mediated by preservation of fetal/maternal endothelial nitric oxide biosynthetic mechanisms and/or decreased activation of the fetal renin-angiotensin pathway.

Effects of various flow types on maternal hemodynamics during fetal bypass: is there nitric oxide release during pulsatile perfusion?

OBJECTIVE: This study investigates the role of various flow conditions on maternal hemodynamics during fetal cardiopulmonary bypass. METHODS: Normothermic fetal bypass was conducted under pulsatile, or steady flow, for a 60-minute period. Fetal lamb preparations were randomly assigned to 1 of the 3 groups: steady flow (n=7), pulsatile flow (n=7), or pulsatile blocked flow bypass (n=7), where fetuses were perfused with Nomega-nitro-L-arginine after the first 30 minutes of pulsatile flow to assess the potential role of endothelial autacoids. RESULTS: Maternal oximetry and pressures remained unchanged throughout the procedure. Under fetal pulsatile flow, maternal cardiac output increased after 20 minutes of bypass and remained significantly higher than under steady flow at minute 30 (8.8+/-0.7 L x min(-1) vs 5.9+/-0.5 L x min(-1), P=.02). Maternal cardiac output in the pulsatile group also remained higher than in both steady and pulsatile blocked flow groups, reaching respectively 8.7+/-0.9 L x min(-1) vs 5.8+/-0.4 L x min(-1) (P=.02) and 5.9+/-0.3 L min(-1) (P=.01) at minute 60. Maternal systemic vascular resistances were significantly lower under pulsatile than under steady flow after 30 minutes and until the end of bypass (respectively, 9.1+/-0.6 IU vs 12.7+/-1.1 IU, P=.02 and 8.9+/-0.5 IU vs 12.9+/-1.2 IU, P=.01). Infusion of Nomega-nitro-L-arginine was followed by an increase in systemic vascular resistances from 9.3+/-0.7 IU, similar to that of the pulsatile group, to 13.5+/-1 IU at 60 minutes, similar to that of the steady flow group. CONCLUSIONS: Maternal hemodynamic changes observed under fetal pulsatile flow are counteracted after infusion of Nomega-nitro-L-arginine, suggesting nitric oxide release from the fetoplacental unit under pulsatile fetal flow conditions.

The hemodynamic effects of hypoxemia in anesthetized pigs: a comparison between right heart catheter and echocardiography.

UNLABELLED: During hypoxemia, hypoxic pulmonary vasoconstriction and tachycardia are often observed in association with increases in pulmonary artery pressure and cardiac output. Nevertheless, the hemodynamic consequences of hypoxemia have never been evaluated by echocardiography and simultaneously compared with invasive hemodynamic variables. Fourteen open-chest, anesthetized piglets (weight 29-36 kg) were submitted to progressive hypoxemia and reoxygenation. Usual invasive hemodynamic variables were obtained from peripheral and central heart catheters. Direct epicardial echocardiography was used to measure right and left ventricular areas on a short-axis view at mid-papillary level. The mean pulmonary artery pressure (MPAP) increased with pulmonary vascular resistance in a dose-related manner as the fraction of inspired oxygen (FIO2) declined from 0.5 to 0.12. The MPAP correlated with right ventricular end-diastolic area (RVEDA) only at FIO2 0.08. There was a 49% reduction in left ventricular end systolic wall stress (LVESWS) between FIO2 0.5 and 0.08. Left ventricular ejection fraction area (LVEFA) increased by 33% above baseline and correlated with the decrease in LVESWS. No correlation was observed between left ventricular end-diastolic area and pulmonary artery occlusion pressure or left atrial pressure and between cardiac output and LVEFA. Systemic vascular resistance underestimates the magnitude of changes in LVESWS but overestimates the afterload compared with LVESWS. This study demonstrates that, for the lowest FIO2 (0.08), changes in MPAP correlated with changes in RVEDA but not in pulmonary vascular resistance. Moreover, LVESWS decreases significantly in a dose-related manner under progressive hypoxemia and normalizes immediately after reoxygenation. This study also shows that, under hypoxemic conditions, echocardiography enhances understanding of hemodynamic changes compared with right heart catheterization alone. IMPLICATIONS: Acute hypoxemia in pigs is responsible for pulmonary vasoconstriction-induced pulmonary hypertension (which is restricted by the right ventricular failure), as well as a PaO2-dependent decrease in left ventricular afterload. These changes are better displayed by echocardiography than by right heart catheter.

Experimental right ventricle to pulmonary artery discontinuity: outcome of polyurethane valved conduits.

OBJECTIVE: The ideal substitute for the treatment of ventricle-pulmonary artery discontinuity remains a topic of controversy, because of calcifications and degeneration of biologic substitutes leading to subsequent reoperations. Because polyurethane valves used in ventricular assist devices show a satisfactory biocompatibility, the aim of this study was to evaluate a valved conduit composed of a Dacron graft incorporating a trileaflet 25 mm polyurethane valve. METHODS: The conduit was implanted between the right ventricle and the main pulmonary artery in adult sheep, with ligation of the proximal pulmonary artery. The animals received no medications. Serial hemodynamic data were collected at the time of implantation and at postoperative intervals of 6 and 12 months. RESULTS: The peak pressure gradient across the valve increased significantly between implantation (0.17 +/- 5.6 mm Hg) and 6 months after operation (7.3 +/- 3 mm Hg, p = 0.0007) and remained stable thereafter (6.7 +/- 3 mm Hg at 12 months), whereas the cardiac output remained unchanged (4.6 +/- 0.6 L/min at implantation, 4 +/- 0.6 L/min at 6 months, and 3.9 +/- 1.1 L/min at 12 months). At the completion of the study, valve samples were processed and vapor coated with carbon for microscopic examination. There was one instance of nonadherent thrombus formation inside a cusp but no structural failures. The other valves were free of calcium deposits and no significant amounts of phosphorus could be detected by scanning electron microscopy and energy dispersive spectrometry. CONCLUSIONS: These data demonstrate the good hemodynamic performance, low thrombogenicity, and acceptable durability of the polyurethane valves implanted in the right side of the heart in a chronic sheep model.

Flow-induced release of endothelium-derived relaxing factor during pulsatile bypass: experimental study in the fetal lamb.

OBJECTIVE: This study was initiated to test the hypothesis that fetal hemodynamic changes observed under pulsatile flow bypass might be related to the release of endothelium-derived relaxing factor through oscillating shear stress. METHODS: Normothermic bypass was instituted in utero in 21 preterm fetal lambs for a 1-hour period through the right atrium and main pulmonary artery. Ultrasonic flowmeters were positioned around the descending aorta and the umbilical artery. The circuit consisted of an oxygenator and a pump set to either continuous flow (n = 7) or pulsatile flow (n = 7) and adjusted to maintain a fetal main arterial pressure of 50 mm Hg. In seven other animals, endothelium-derived relaxing factor was blocked by a continuous infusion of N omega-nitro-L-arginine after 30 minutes of pulsatile flow. RESULTS: During the first 30 minutes of bypass, pump flows were significantly lower in the continuous-flow group than in the pulsatile-flow or blocked-flow groups (respectively, 612 +/- 144, 907 +/- 153 and 987 +/- 228 ml/min), with similar changes in aortic and umbilical flows. Systemic vascular resistances were significantly lower in the pulsatile-flow and blocked-flow groups than in the continuous-flow group (550 +/- 106 vs 821 +/- 212 dynes/sec/cm-5). However, after blockade of endothelium-derived relaxing factor, resistances increased gradually in the blocked-flow group to reach the level of that of the continuous-flow group at the end of bypass (943 +/- 77 vs 556 +/- 143 dynes/sec/cm-5 in the pulsatile-flow group). CONCLUSIONS: Blockade of endothelium-derived relaxing factor after 30 minutes of pulsatile flow returns fetal hemodynamics to continuous flow conditions. The specific inhibitor of endothelium-derived relaxing factor used in this experiment suggests that nitric oxide may be released by fetal endothelium during pulsatile bypass.

[Effects of active synchronized prolonged coronary perfusion in the animal]. / Effets de la perfusion coronaire active synchronisée prolongée chez l'animal.

The efficacy of a system of active diastolic synchronised coronary perfusion was studied during prolonged balloon angioplasty in 8 sheep. In the first part of the study (group 1) including 5 animals, the aim was to study the effects of high and constant flow (48 ml/min) for 90 minutes perfusion on haemolysis, the arterial wall and the perfused myocardium. The second part of the study (group 2), including 3 animals, assessed whether flow adapted to the extent of the vascular bed perfused (24 to 40 ml/min) could protect the myocardium for an interval of 60 minutes. In group 1, after 90 minutes of perfusion (48 ml/min), there was no haemolysis, or jet lesion of the arterial wall distal to the catheter tip. On the other hand, the creatinine phosphokinase levels increased at the 60th minute (188 vs 119 i.u./l for controls) and at the 90th minute (238 vs 119 i.u./l; p < 0.05). Moreover, the perfused myocardium was the site of histological lesions. These observations showed myocardial changes due to the "overflow phenomenon". In group 2, the flow rate was adapted to each animal, increasing progressively until disappearance of electrocardiographic signs of ischaemia (ST elevation) and maintained for 60 minutes. No signs of haemolysis, jet lesions or myocardial changes were observed, with absence of creatinine phosphokinase elevation and histological abnormalities. These preliminary results show that the system investigated allowed myocardial protection after arterial occlusion for an interval of 60 minutes.

[Hemodynamics of right circulation in ewes: normal values]. / Hémodynamique de la circulation droite chez la brebis: valeurs normales.

The right cardiac pressure was measured on 24 anesthetized adult ewes (Halothane) with a Swan Ganz catheter. After a review of the catheterization technique, the results (mean +/- standard deviation) in mmHg were: right auricle 17 +/- 5, right ventricle 30 +/- 6 (systolic) and 12 +/- 6 (telediastolic), pulmonary artery 29 +/- 6 (systolic), 13 +/- 6 (diastolic) and 20 +/- 6 (mean), capillary pressure 17 +/- 7. We observed slight variations in the pressure curve morphology compared to those found for man and the pressures were 5-10 mmHg higher than what is observed in man and slightly higher than those observed in dogs. These results also demonstrate a great variation between animals.

Pulsatility improves hemodynamics during fetal bypass. Experimental comparative study of pulsatile versus steady flow.

BACKGROUND: The main advantage of pulsatile flow compared with steady flow during cardiopulmonary bypass is to prevent a rise in systemic vascular resistances. We hypothesized that pulsatile flow could overcome the progressive rise in peripheral and placental vascular resistances observed during fetal bypass and leading to progressive irreversible hypoxemia. METHODS AND RESULTS: A study was undertaken in 17 fetal lambs (110 to 140 days of gestation). Fetal bypass was established for a 30-minute period through right atrial and main pulmonary artery cannulation. The circuit had no oxygenator. Flow was delivered by a standard roller pump for the continuous study (group 1, n = 9) or by a centrifugal pulsatile pump for the pulsatile study (group 2, n = 8). Oxymetric and hemodynamic parameters, along with organ blood flow determined by radiolabeled microspheres counting, were recorded before (T1) and after 10 minutes (T2) and 30 minutes (T3) of bypass. SaO2 and PaO2 were significantly higher in group 2 than in group 1 at T2 but thereafter deteriorated similarly in both groups, whereas PCO2 remained unchanged. Pump flow in group 2 was significantly higher than in group 1 at T2 and T3 (957.6 +/- 49 and 1104 +/- 152 versus 437.6 +/- 23 and 467.8 +/- 43 mL/min, respectively). Systemic vascular resistances during pulsatile bypass were also significantly lower than in group 1 at T2 (402 +/- 12 versus 930 +/- 79 dynes/sec/cm-5) and T3 (374 +/- 60 versus 1017 +/- 192 dynes/sec/cm-5). At T2 and T3, all individual blood flows except the brain but including the placenta were statistically higher in group 2 than in group 1. Placental vascular resistances gradually increased during bypass in group 1 to reach 2.9 +/- 0.2 mm Hg.mL-1.min-1.kg-1 at T3 and remained approximately stable in group 2 during 30 minutes of pulsatile bypass, varying from 0.35 +/- 0.02 to 1.26 +/- 0.14 from T2 to T3 (P < .01). CONCLUSIONS: The data suggest that pulsatile flow for 30 minutes of bypass in a fetal lamb preparation temporarily prevents the progressive hypoxemia observed under steady-flow bypass. Pulsatile flow allows higher pump flow through a significant decrease in systemic vascular resistances. Individual organ blood flow, including placenta, was significantly higher under pulsatile bypass. With technical improvements in the design of pulsatile devices adapted to more physiological beat rates, pulsatility may become a valuable adjunct to overcome placental dysfunction observed during experimental fetal cardiac surgery.