Impact of the cryopreservation technique and vascular bed on ovarian tissue transplantation in cynomolgus monkeys.

PURPOSE: The aim of this study was to determine the best combination in terms of cryopreservation techniques and vascular bed preparation before grafting in order to obtain functional ovarian tissue after transplantation. METHODS: Five cynomolgus monkeys were used. Strips from 10 ovaries were cryopreserved, 5 by vitrification (V), and 5 by slow-freezing (SF). Pieces of fresh ovarian tissue were used for controls. After 1 month, the strips were autografted to two different vascular beds, healed (HB) or freshly decorticated (FDB), constituting four study groups: SF-HB, SF-FDB, V-HB, and V-FDB. These were compared to fresh tissue. After 6 months, the ovaries were removed and several parameters analyzed: follicle quality, stage, density, proliferation, apoptosis, functionality, vascularization, and fibrosis. Mixed effect linear regression models were built to assess the impact of cryopreservation and vascular bed preparation on ovarian tissue viability and functionality. p values were adjusted for multiple testing using the Benjamini-Hochberg method, and q values < 0.20 were considered significant in order to achieve a 20% false discovery rate. RESULTS: Compared to fresh tissue, no difference was observed in the percentage of morphologically normal follicles, while a significant increase was noted in the follicle proliferation rate (41%, q = 0.19), percentage of antral follicles (12%, q = 0.14), and number of vessels per area (3.3 times, q = 0.07) in the V-FDB group. CONCLUSIONS: Vitrification associated with FDB vascular bed preparation is the best combination to obtain functional autografted ovarian tissue. Further studies are nevertheless required, with confirmed pregnancies and live births before introducing the procedure into clinical practice.

Critical reappraisal of embryo quality as a predictive parameter for pregnancy outcome: a pilot study.

AIM OF THE STUDY: Pilot study to analyse the efficacy and embryo morphology using a new human embryo culture medium (GM501) versus the conventional used medium (ISM1). METHODS: Over a four-month period, all patients at the Leuven Institute of Fertility and Embryology (LIFE) were -randomly allocated to have their embryos cultured in either the standard sequential culture medium ISM1 (control) or in a new universal medium (GM501) (study group). Primary outcome parameters were clinical pregnancy and live birth rate. The secondary outcome parameter was the correlation of embryo fragmentation rate with pregnancy outcome. RESULTS: We did not observe any differences between the ISM1 control group and GM501 study group with regard to fertilization, pregnancy, implantation rates, ongoing pregnancy, and babies born. The number of embryos with a minimal fragmentation rate (less than 30%) was significantly higher in the GM501 study group. CONCLUSION: Although a significant higher embryo fragmentation rate was seen in In vitro culture of embryos in GM501, pregnancy outcome results were comparable to those of embryos cultured in ISM1. According to our results the value of embryo morphological criteria as a parameter for pregnancy outcome should be examined and discussed again.

Adhesion formation after laparoscopic surgery: what do we know about the role of the peritoneal environment?

In spite of the approaches that have been proposed to reduce postoperative peritoneal adhesions, they remain a major clinical problem because of the associated intestinal obstruction, chronic pelvic pain, female infertility and difficulties at the time of reoperation. The pathogenesis of the process have been focused almost exclusively on the local events induced by the surgical trauma, and the strategies for adhesion prevention thus focused on barriers to separate surgically denuded areas. The important role of the peritoneal cavity environment only recently became apparent and is not yet incorporated in adhesion reducing strategies. Recent data demonstrate that, in the presence of a direct surgical trauma, the entire peritoneal environment is quantitatively the most important factor in adhesion formation and hence adhesion prevention after both open and laparoscopic surgery. Indeed mesothelial hypoxia (CO2 pneumoperitoneum) or hyperoxia (open surgery), desiccation and surgical manipulation have been identified as factors cumulatively enhancing adhesions--. The clinical implication is especially relevant for laparoscopic surgery because the pneumoperitoneum, being a closed environment, can be easily conditioned. Although human studies are lacking, animal data indicate that peritoneal adhesions can be reduced by over 80% with a good surgical technique, with adequate pneumoperitoneum conditioning as adding 3-4% of oxygen to the CO2 pneumoperitoneum, prevention of desiccation and slight cooling. Adhesion prevention barriers remain additionally effective, although quantitatively less important. The relevance of all these strategies for adhesion prevention still have to be confirmed in humans, but since it seems that the peritoneal environment is quantitatively much more important than the surgical trauma, adhesion prevention research and strategies should be directed more to conditioning the peritoneal cavity than to the use of agents.

Hyperoxia and prevention of adhesion formation: a laparoscopic mouse model for open surgery.

OBJECTIVE: CO(2) pneumoperitoneum with more than 10% oxygen enhances adhesions. As during open surgery the peritoneum is exposed to air (20% oxygen), in this hyperoxia-enhanced adhesion model we evaluated the effect of hypothermia and products with known effectiveness in hypoxia (pure CO(2) pneumoperitoneum) and normoxia (CO(2) pneumoperitoneum plus 3-4% oxygen) models. Results were expected to be important for adhesion prevention in open surgery, and, moreover, similarities and differences between the three models would be important to identify differences in pathways of adhesion formation between laparoscopy and laparotomy. DESIGN: Two experiments were performed in which the effect of hypothermia (32 degrees C), a surfactant (phospholipids), a barrier (Hyalobarrier gel), reactive oxygen species scavengers (superoxide dismutase, SOD, and ascorbic acid, AA), anti-inflammatory agents (dexamethasone and nimesulide), a calcium channel blocker (diltiazem) and recombinant plasminogen activator (r-PA) were evaluated upon adhesions. SETTING: University Hospital. POPULATION: BALB/c mice. METHODS: Hyperoxia-enhanced adhesions were induced by performing laparoscopically bipolar lesions during 60 minutes of CO(2) pneumoperitoneum plus 12% oxygen at 37 degrees C body temperature. MAIN OUTCOME MEASURES: Adhesions were scored after 7 days. RESULTS: In this model, adhesions were reduced by hypothermia (P < 0.02; Wilcoxon), phospholipids (P = 0.03), Hyalobarrier gel (P < 0.004), dexamethasone (P < 0.005) and diltiazem (P < 0.01). A significant but quantitatively borderline effect was seen for AA (P < 0.002) and r-PA (P = 0.0005), whereas SOD and nimesulide did not have any effect. CONCLUSIONS: Hyperoxia-enhanced adhesions were prevented by hypothermia, dexamethasone, phospholipids, Hyalobarrier gel, diltiazem, r-PA and AA. All effects were similar to those in the hypoxia-enhanced adhesion model, suggesting that the underlying mechanisms are similar.

Prevention of adhesion formation in a laparoscopic mouse model should combine local treatment with peritoneal cavity conditioning.

BACKGROUND: Adhesion formation results from a series of local events at the trauma site. This process can be enhanced by factors derived from the peritoneal cavity such as mesothelial cell hypoxia (pneumoperitoneum with pure CO(2)), reactive oxygen species (pneumoperitoneum with more than 4% oxygen), desiccation and mesothelial trauma produced through manipulation. Adhesion prevention, therefore, should combine local treatment while minimizing adverse peritoneal factors through conditioning of the pneumoperitoneum. METHODS: In a laparoscopic mouse model, adhesion induction comprised a mechanical lesion together with a humidified pneumoperitoneum for 60 min with pure CO(2) at 37 degrees C. Adhesion prevention consisted of a combination of treatments known to reduce adhesions, i.e. pneumoperitoneum with CO(2) with the addition of 3-4% O(2), reduction of body temperature (BT) to 32 degrees C and application of antiadhesion products such as anti-inflammatory drugs (dexamethasone, nimesulide), calcium-channel blockers (diltiem), surfactants (phospholipids), barriers (Hyalobarrier gel), reactive oxygen species scavengers (superoxide dismutase and ascorbic acid) and recombinant plasminogen activator. RESULTS: The addition of 3% O(2) to the pneumoperitoneum or a lower BT decreased adhesions by 32% or 48%, respectively (P < 0.05, Wilcoxon), but were without additional effects when combined. In addition, if dexamethasone or Hyalobarrier((R)) gel were administrated, the total reduction was 76% (P = 0.04) or 85% (P < 0.02), respectively. CONCLUSIONS: Combining pneumoperitoneum conditioning together with dexamethasone or a barrier resulted in significant adhesion reduction in a laparoscopic mouse model.

Effect of reactive oxygen species scavengers, antiinflammatory drugs, and calcium-channel blockers on carbon dioxide pneumoperitoneum-enhanced adhesions in a laparoscopic mouse model.

BACKGROUND: Postoperative adhesions are a clinical problem. They can cause female infertility, intestinal obstruction, chronic pelvic pain, and difficulties at the time of reoperation. A variety of approaches described to prevent adhesions have shown variable and inconsistent results. Therefore, this study aimed to evaluate most known substances in a laparoscopic mouse model to obtain quantitative and comprehensive information on adhesion prevention. Specifically, this first study aimed to investigate the effects of reactive oxygen species (ROS) scavengers, antiinflammatory agents, and a calcium-channel blocker on pneumoperitoneum-enhanced adhesions. METHODS: Adhesions were induced during laparoscopy in BALB/c female mice by creation of a bipolar lesion. Carbon dioxide (CO2) pneumoperitoneum was maintained for 60 min using humidified CO2. Six experiments were conducted to evaluate the effects of ROS scavengers (superoxide dismutase [SOD], catalase, melatonin, and ascorbic acid), antiinflammatory agents (dexamethasone, tenoxicam, ibuprofen, parecoxib, nimesulide, anti-tumor necrosis factor [TNF]-alpha), and a calcium-channel blocker (diltiazem). Adhesions were scored after 7 days during laparotomy. RESULTS: Adhesions were reduced by SOD (p < 0.01, proc general linear methods (GLM) of experiments 1 and 2), diltiazem (p = 0.05, Wilcoxon), and dexamethasone (p < 0.03), but not by nonsteroidal antiinflammatory drugs (NSAIDs) nor by anti-TNF-alpha. When all the experiments were grouped for analysis, adhesions also decreased with one and three doses of SOD (p < 0.01 and p < 0.01, respectively) and with one and three doses of ascorbic acid (p < 0.02 and p = 0.05, respectively). CONCLUSIONS: These experiments confirm that SOD, diltiazem, and dexamethasone can decrease adhesion formation. The absence of effect from the other antiinflammatory drugs and anti-TNF-alpha is surprising.

Effect of temperature upon adhesion formation in a laparoscopic mouse model.

BACKGROUND: Pneumoperitoneum can be a cofactor in adhesion formation. Pneumoperitoneum with non-humidified gas causes desiccation in the peritoneal cavity which decreases temperature. The effect of desiccation upon adhesion formation is widely accepted. The specific effect of the associated cooling upon adhesion formation remains unexplored, and was addressed specifically in our laparoscopic mouse model. METHODS: Adhesions were induced during laparoscopy and scored after 7 days during laparotomy. Pneumoperitoneum was performed using CO2 or CO2 with oxygen with or without humidification. Animals were placed at different environmental temperatures to modulate body and intraperitoneal temperature. RESULTS: Anaesthesia, environment with a lower temperature and pneumoperitoneum all independently decrease body temperature. A decrease in body temperature decreases adhesion formation (P=0.004). Therefore, at 37 degrees C, pneumoperitoneum-enhanced adhesion formation is more important than at room temperature (P=0.04). As was observed at room temperature, adhesion formation at 37 degrees C increases with the duration (P=0.01) of pneumoperitoneum and decreases with the addition of 3% of oxygen (P=0.03). CONCLUSIONS: Hypothermia reduces pneumoperitoneum-enhanced adhesion formation, which supports hypoxia as a driving mechanism, since hypothermia decreases the toxic effects of hypoxia and of the ischaemia-reperfusion process. These data could open up new possibilities for adhesion prevention in laparoscopic surgery.

Reactive oxygen species and adhesion formation: clinical implications in adhesion prevention.

Postoperative adhesion formation is a major clinical problem. It has been demonstrated that the pneumoperitoneum used during laparoscopy is a cofactor in adhesion formation. Reactive oxygen species (ROS) are produced in a hyperoxic environment and during the ischaemia/reperfusion process. ROS activity is deleterious for cells, which protect themselves by an antioxidant system known as ROS scavengers. ROS activity can increase by up-regulation of ROS themselves or by down-regulation of ROS scavengers. Recent data also point to a role for ROS in adhesion formation since the administration of ROS scavengers decreases adhesion formation in several animal models. ROS activity increases during both laparotomy and laparoscopy. During laparoscopy, the pneumoperitoneum determines ischaemia at the time of insuflation and reperfusion at the time of deflation. During laparotomy, the environment has a 150 mmHg partial pressure of oxygen (pO(2)), which is much higher than the intracellular pO(2) (5-40 mmHg). This can explain the increase in ROS activity. The aim of this debate is to open a discussion about the importance of ROS activity, besides the known players and mechanisms involved, in adhesion formation and in adhesion prevention.

Inhibition of endothelial cell proliferation and tumor-induced angiogenesis by pentoxifylline.

PURPOSE: In this study we investigated the effect of pentoxifylline (PTX) on tumor-induced neovascularization as well as on different steps involved in the angiogenic process. METHODS: To assess angiogenesis inhibition. we injected intradermally (i.d.) 10 B16-F10 melanoma cells into C57BL/6J mice which were subsequently intraperitoneally (i.p.) inoculated with PTX or saline. On day 7 the number of blood vessels converging to the remnant of injected material was counted and the volumes of incipient tumors were calculated in each case. In vitro growth inhibition by PTX was evaluated in two different cell lines of endothelial origin and in human umbilical vein endothelial cells. Motility assays, as well as zymographic assays carried out to analyze gelatinolytic metalloproteinases and urokinase-type plasminogen activator, were performed in one of the endothelial cell lines. RESULTS: A significant inhibition of tumor-induced angiogenesis was observed in C57B1/6 mice i.p. inoculated with PTX, that paralleled reduced incipient tumor volumes. The endothelial cells derived from different sources were inhibited in a dose-response manner by PTX in vitro. Non-cytotoxic PTX concentrations assayed in one of the endothelial cell lines did not inhibit its in vitro cell motility nor its gelatinase secretion, but its low molecular weight urokinase-type plasminogen activator expression. CONCLUSIONS: Our findings suggest that the inhibitory effect of PTX on tumor angiogenesis is related to antiproliferative action on endothelial cells, as well as to down regulation of u-PA secreted by them.

Differential anthracycline sensitivity in two related human colon carcinoma cell lines expressing similar levels of P-glycoprotein.

Chemosensitivity of the human colon carcinoma HCT-15 cell line to 4'-epidoxorubicin proved to be 100-fold higher than that of its variant HCT-15 EDR. Confocal scanning microscopy showed significant less drug accumulation in HCT-15 EDR. A 2-fold increase in hsp27 expression was found in HCT-15 EDR, with no alteration in hsp70. The expression of the drug exporter Pgp was similar in both cell lines, despite the lower drug accumulation shown by HCT-15 EDR in respect to HCT-15. Other molecules implicated in the acquisition of enhanced chemoresistance or a more active Pgp variant present in HCT-15 EDR, could explain the phenomenon.

Inhibitory effect of Lovastatin on spontaneous metastases derived from a rat lymphoma.

The HMGCoA reductase inhibitor Lovastatin (LOV) has previously shown to abrogate p21ras farnesylation, which is associated with invasive and metastatic abilities in many tumor models. Considering the scarcity of therapeutic resources against metastasis, our objective was to study LOV as an antimetastatic agent on L-TACB rat lymphoma, which as a syngeneic tumor model resembles more closely the situation in human cancer. We also aimed to analyze the effect of LOV on chemoinvasion, motility, metalloproteases secretion, angiogenic capacity, and adhesion to the reconstituted basement membrane Matrigel. Our results showed that LOV caused no effect on cell motility, metalloprotease secretion and neovascularization. Conversely, LOV produced a significant inhibition of invasiveness, which could be a consequence of an impaired cell adhesion to the basement membrane observed. These effects could explain, at least in part, the inhibitory action of LOV on L-TACB rat lymphoma metastases.

Studies on the mechanisms responsible for inhibition of experimental metastasis of B16-F10 murine melanoma by pentoxifylline.

Pentoxifylline (PTX), a methylxanthine derivative widely used as a hemorheological agent in the treatment of peripheral vascular disease, was studied to unveil the mechanisms responsible for its inhibitory action on B16-F10 experimental metastasis. In vitro pretreatment of B16-F10 cells with noncytotoxic concentrations of PTX significantly inhibited their adhesion to reconstituted basement membrane Matrigel(R) and type IV collagen as well as the relative activity of secreted 92 kD metalloproteinase. However, PTX pretreatment of B16-F10 cells did not affect their in vitro invasiveness. Heterotypic organ adhesion assays carried out with B16-F10 cells and suspended organ tissues demonstrated that pretreatment with noncytotoxic concentrations of PTX of both, tumor cells or lung tissue, brought about a dose-dependent inhibition of melanoma cell adhesion to lung. Immunohistochemical studies using antibodies against CD31 adhesion molecule (PECAM-1) revealed that B16-F10 cells adhere to lung endothelial cells. Our results suggest that PTX may exert its inhibitory effect on tumor lodgment, and as a consequence of that on experimental metastases, through an inhibitory action on cell adhesion molecules.

Antiangiogenesis and apoptosis as mediators of concomitant tumor resistance induced by Calu-6, a human lung carcinoma cell line, in nude mice.

Concomitant resistance (CR), the phenomenon by which tumor-bearing hosts are able to inhibit secondary implants of the same tumor at distant sites of the body, has been previously observed by us and others in different murine tumor models. Here, we verified the generation of CR in nude mice by tumors induced by SC inoculation of Calu-6, a human lung carcinoma cell line. Histological analysis of secondary tumors subject to CR did not reveal macrophage infiltration nor cytotoxic signs. Although serum from tumor-bearing mice inhibited in vitro [3H]thymidine uptake by Calu-6 cells, no significant differences in [3H]thymidine labeling index of tumors implanted in the right flank of mice with and without a primary tumor in the left flank were detected. In our model, the presence of a primary tumor hindered remote tumor angiogenesis, as well as serum from tumor-bearing mice inhibited in vitro proliferation of an endothelial cell line derived from a murine hemangioendothelioma. Conversely, an enhancement of the apoptotic index was observed in secondary tumor implants carried out in tumor-bearing mice. The results reported herein show that human tumor cells are capable of inducing CR, and that this phenomenon would be a consequence of an impaired neovascularization as well as an increased programmed cell death at sites distant from the primary tumor.