Prevention of post-surgical adhesion formation using aspirin in a rodent model: a preliminary report

Pelvic adhesions are one of the major factors which significantlyand adversely affect surgery outcome due to intra-and postoperativemorbidity and reduce future female fertility. Using a rodentmodel, we evaluated the efficacy of aspirin, a non-steroidalanti-inflammatory drug, in the prevention of adhesion formation.A total of 72 female Wistar rats received a standardized primarytraumatic lesion to the right uterine horn. They were randomlydivided into eight groups: group I (control) had no treatmentand group II received a single pre-operative 0.70 mg aspirin.All the succeeding groups (III-VIII) received aspirin in dosesof 0.35, 0.70, or 1.40 mg every 6 h for either 48 or 96 h inaddition to the pre-operative aspirin (0.70 mg). All animalswere killed 4 weeks later and adhesions were assessed usinga modified adhesion scoring scale. The lowest adhesion scorewas found in the group treated with 0.35 mg of aspirin for 96h, and the highest was found among the groups treated with either0.70 or 1.40 mg for 48–96 h respectively (P < 0.05).These results are in line with the hypothesis that administrationof a low dose of aspirin selectively inhibits the productionof thromboxane A₂, whereas basal prostacyclin biosynthesis ispreserved. This phenomenon might contribute to reducing postoperativeadhesion formation in a rat model. Thus, future studies intothe prevention of adhesion formation may require the additionaluse of a non-steroidal anti-inflammatory drug, for which aspirindeserves further attention, before extrapolation into humantherapy.     

Intraperitoneal levonorgestrel-releasing intrauterine device following uterine perforation: the role of progestins in adhesion formation

BACKGROUND: Intrauterine contraception is a widely used, highly effective means of birth control. Uterine perforation is a serious, albeit rare, complication of intrauterine device (IUD) use. Although uterine perforation by levonorgestrel-releasing (20 micro g/day) intrauterine system (LNG-IUS) has already been reported, the peritoneal adhesion potential of this IUD is unknown. METHODS: The medical files of all patients diagnosed with an intra-peritoneal IUD between the years 1990-2002 at Hadassah Medical Center were reviewed. Histopathological study of peritoneal adhesion tissue adjacent to levonorgestrel medicated IUD was conducted in one case. RESULTS: Eight cases of dislocated IUDs were found. Four cases used LNG-IUS and four other cases used copper-IUD. Laparoscopy for IUD removal disclosed mild local peritoneal adhesions between omentum and pelvic organs in all cases. No difference was noted in the appearance of the peritoneum in the presence of either a copper-IUD or LNG-IUS. Histological examination of peritoneal tissue encasing the levonorgestrel-intrauterine system revealed loose connective tissue with aggregates of submesothelial cells with a pseudo-decidual change. Immunohistochemical staining for progesterone receptor was negative. CONCLUSIONS: The peritoneal adhesions potential of LNG-IUS is low, similar to that of the copper-bearing IUD.     

The impact of peritoneal trauma on intra-abdominal fibrinolytic activity, adhesion formation and early embryonic development in a rabbit longitudinal model

Intra-abdominal adhesions may interfere with fertility following gynaecological surgery and injury to the peritoneum plays a central role in the pathogenesis. Tissue plasminogen activator and its antagonists play a pivotal role in the intra-abdominal balance between fibrinolysis and adhesion formation. This process may be cycle-dependent in women. In order to establish the impact of the fibrinolytic activity on adhesion formation after a standardized trauma, a rabbit longitudinal model was developed, which allowed the study of possible differences between the periods before and after ovulation. The influence of extra-genital adhesions on early embryonic development was investigated. No cycle-dependent changes in fibrinolytic activity of the peritoneal fluid (PF) or of the serum could be demonstrated. No correlation was found between post-operative adhesion formation and the fibrinolytic activity during surgery. Three weeks after surgery, a significant increase in fibrinolytic activity of the PF was observed. The rank order of sampling is suggested to account for these differences. Extra-genital adhesions did not markedly influence ovulation, ovum pick-up and fertilization in this hormonally controlled rabbit model.     

Cross-linked hyaluronate hydrogel prevents adhesion formation and reformation in mouse uterine horn model

The aim of this study was to evaluate the efficacy of cross-linked hyaluronate hydrogel (HA gel) as an adjuvant for postoperative adhesion prevention, in a mouse uterine horn model. In experiment 1 uterine horns were abrased with iodine. HA gel was applied to the injured surface before closure in the treatment group. In experiment 2, after injuring the uterine horns, three stitches were placed at equal distances around the uterine horns to appose the injured medial surfaces of the two horns during healing. HA gel was inserted between the uterine horns in the treatment group. In experiment 3 prevention of adhesion reformation was assessed. After lysis of adhesions that were induced as in experiment 2, HA gel was introduced between the serosal surfaces of apposing uterine horns. Untreated animals served as controls in each experiment. Statistical analysis was carried out using Student's t-test. The adhesion score was significantly lower in the HA gel group on the 14th day compared with controls in all the experiments: in experiment 1, 0.3 +/- 0.4 versus 1.7 +/- 1.2; in experiment 2, 0.9 +/- 1.0 versus 2.6 +/- 0.5; and in experiment 3, 1.5 +/- 0.9 versus 2.2 +/- 0.6 respectively. Cross-linked HA gel significantly reduced de-novo adhesions (P< 0.03) and adhesion reformation (P < 0.03).     

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.     

Evaluation of a sprayable polyethylene glycol adhesion barrier in a porcine efficacy model.

BACKGROUND: The formation of adhesions following pelvic surgery remains one of the leading causes of infertility, small bowel obstruction and re-operation for pelvic pain. A novel hydrophilic polyethylene glycol based adhesion barrier (SprayGel) is formed by simultaneously spraying two liquid precursors onto surgical sites. The liquids polymerize to form a gel that effectively coats and adheres to tissue. After about 5 days, the hydrogel layer is absorbed and subsequently undergoes renal clearance. It is believed that the presence of such a barrier would inhibit the formation of adhesions following surgical insult. METHODS: A porcine adhesion model was developed wherein bilateral uterine horn transection and re-anastomosis, along with peritoneal side wall excision was performed via laparotomy. In each animal (n = 10, including the pilot study) one pelvic side wall was treated with adhesion barrier, while the contralateral side remained untreated. RESULTS: At second look laparoscopy, 90% of the untreated sites had adhesions, compared with 30% of the treated sites (P = 0.006). Also observed were statistically significant reductions in the adhesion extent (P = 0.029) and adhesion severity scores (P = 0.023) at the treated sites. However, if the pilot study was excluded (n = 8) the differences obtained were no longer significant. CONCLUSIONS: Polyethylene glycol (SprayGel) merits further investigation as an effective barrier to the formation of post-operative adhesions in this porcine model.     

Severe pelvic adhesions following attempted ultrasound-guided drainage of bilateral ovarian endometriomas: case report.

A 38 year old woman was thought to have bilateral polycystic ovaries and an absence of pelvic or abdominal adhesions at diagnostic laparoscopy. Post-operative transvaginal ultrasound noted bilateral homogeneous hypo-echoic masses with low-level echoes consistent with endometriomas. Transvaginal aspiration was unsuccessful due to the viscosity of the cyst contents. At second laparoscopy severe adhesions with complete cul-de-sac obliteration were noted. This case raises concern about pelvic adhesions following transvaginal drainage of ovarian endometriomas.     

Induced regeneration of endometrium following curettage for abortion: a comparative study

A significant increase in endometrial thickness and volume wasobserved in 30 patients given oestrogen and progestin supplementationfollowing curettage for first trimester abortions, comparedwith 30 women who received no treatment. This indicates an enhancedregeneration of the endometrium following treatment. The abilityto induce this response, creating a space between the intra-cavitysurface area a short time after abortion, may theoreticallybe suggested as preventative treatment to reduce the risk ofintrauterine adhesions.     

The SCID mouse: an experimental model for endometriosis

The purpose of this study was to validate the suitability of the severe combined immunodeficient (SCID) mouse as an experimental model for endometriosis, by defining the morphological and histological features of induced endometrial implants, and characterizing specific biochemical properties of these implants. Human secretory endometrial tissues were injected into the peritoneal cavity of SCID/SCID CB17 mature female mice. Successful peritoneal implantation was observed in 55 of 57 (96.5%) SCID mice and consisted of circumscribed elevated nodules. Haematoxylin-eosin staining of implanting lesions demonstrated the presence of endometrial glandular tissue in a mixed background of stromal and inflammatory cells. When progesterone was administered to mice, epithelial glands underwent well-defined secretory changes. Immunohistochemical analysis using polyclonal human pan-cytokeratin antibodies demonstrated selective positive staining in the glandular epithelium of the human implants with none in the surrounding stroma. In-situ hybridization analysis using complement component 3 cDNA radiolabelled riboprobes yielded significantly more intense signals in glands compared to stroma. As human endometrial implants in SCID mice were shown to retain specific histological, functional and biochemical properties, we conclude that the SCID mouse is an attractive animal model for the study of endometriosis.     

Cutaneous injury following acute UV-B radiation in a mouse model: a pilot histological study

Abstract

Epidemiological and clinical studies have implicated solar ultraviolet (UV) radiation, especially the UV-B radiation (290–320 nm wavelengths) as the principal agent that elicits sunburn, transient inflammation, melanoma and non-melanoma cancer, and premature skin aging. The common form of skin damage described as ‘photoaging’ is caused by repeated sun exposures; it can induce epithelial deoxyribonucleic acid (DNA) damage, and alter the structure and function of dermal connective tissue and extracellular matrix. The present study was aimed to elucidate the effects of a single exposure of different doses of UV-B radiation on the dorsal skin in the hairless mouse. The histopathologic sequels of such exposures were analyzed by using common staining methods, immunohistochemistry, and histomorphometry. Single UV-B exposures seem to elicit dose-graded histological changes some of which have been described in experiments with chronic exposure in this species.     

The fertilization and development of mouse oocytes following cortical granule discharge in the presence of a protease inhibitor.

The effect of leupeptin, a serine protease inhibitor, on the fertilization and development potential of oocytes stimulated to undergo cortical granule exocytosis has been investigated. An in-vitro bioassay system was used in which mouse oocytes were exposed to calcium ionophore, A23187, in the presence and absence of leupeptin, before their fertilization and development to the blastocyst stage was assessed. We have demonstrated that the presence of leupeptin in the incubation medium, at concentrations of 1 micrograms/ml and 10 micrograms/ml during the first 10 min of cortical granule exocytosis, reversed the ionophore-induced decrease in the capacity of oocytes to fertilize and develop to blastocysts. The induction of exocytosis of cortical granules by calcium ionophore was confirmed using fluorescence microscopy. Using this technique, we also confirmed that leupeptin did not inhibit ionophore-induced cortical granule exocytosis, thus supporting the contention that leupeptin acted upon released cortical exudate. It was concluded that leupeptin acted by inhibiting proteases released into the perivitelline space during the early stages of cortical granule exocytosis. Based on these results it was proposed that leupeptin could be used to prevent premature loss of fertility of human oocytes which are inadvertently activated under in-vitro conditions.     

Infertility: Investigation of impaired ovarian function as a contributing factor to infertility in women with pelvic adhesions

A prospective study was performed to determine whether a relationshipexists between infertility as a result of pelvic adhesions,and raised follicle stimulating hormone (FSH). A total of 66patients, who had a diagnostic laparoscopy and dye performedas part of their infertility work-up, had an early cycle serumFSH drawn one to two cycles after this procedure. Patients weredivided into two groups, one with pelvic adhesions as a resultof surgery, infection or endometriosis, and a control groupconsisting of those with a normal pelvis at laparoscopy or withminimal/mild endometriosis without adhesions. In the adhesiongroup, a correlation was noted between American Fertility Society(AFS) adhesion score and basal FSH concentration, although allFSH values for this group were within the normal range. Thisstudy suggests a correlation between pelvic adhesions and ovariancompromise, but a larger study involving more severe cases ofadhesions is required to determine whether this finding is ofclinical importance.     

The correlation of adhesions and peritoneal fluid cytokine concentrations: a pilot study

BACKGROUND: Intra-abdominal adhesion formation and reformation after surgery is a significant cause of morbidity. The greatest problem after the surgical removal of adhesions is their reformation. We examined the concentrations of interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-alpha in the peritoneal fluid throughout the 48 h post-operative period following adhesiolysis, and correlated the results to the extent of adhesion reformation. METHODS: Peritoneal fluid, collected from eight patients following laparoscopy and again at 12, 36 and 48 h after surgery, was analysed using enzyme-linked immunosorbent assay (IL-1 and IL-6) and bioassay (TNF-alpha). At 48 h, a second look laparoscopy was performed to inspect the pelvis for adhesion formation/reformation. RESULTS: Three patients had adhesion reformation >10% at 48 h after surgery. The mean adhesion score 48 h after adhesiolysis was 5 (range 0-17). The mean reduction in adhesion score was 88% (range 83-100%). Newly formed adhesions were filmy, relatively soft and avascular in nature. Adhesion reformation of >10% was associated with (i) high concentrations of IL-6 at 12 h (P < 0.01) and (ii) high concentrations of IL-1 at 48 h (P < 0.001). CONCLUSIONS: Results from this preliminary study suggest that future treatment strategies for adhesion prevention could be aimed at the control of cellular mediators in the peritoneal fluid during the initial adhesion formation period.     

Morphological appearance of the cryopreserved mouse blastocyst as a tool to identify the type of cryoinjury

BACKGROUND: If it were possible to deduce the mechanism of injury in cryopreserved embryos by their appearance, it would help to optimize cryopreservation protocols. METHODS: Mouse blastocysts were treated so that they were damaged by the six types of cryoinjuries listed below, and their appearance was observed at recovery in sucrose solution and a modified phosphate-buffered saline (PB1), and after culture for 1 and 24 h. RESULTS: (i) Intracellular ice: the embryos shrank normally in sucrose solution, but swelled in PB1 and collapsed after culture. (ii) Chemical toxicity of the cryoprotectant: the embryos looked normal in sucrose solution and PB1. After 1 h of culture, however, the blastomeres showed decompaction and degenerated thereafter. If the toxicity was extremely high, embryos looked nearly normal in PB1, but the surface of the cytoplasm was wrinkled as if they were "fixed". (iii) Osmotic swelling: the embryos looked normal in PB1, but after culture they shrank. (iv) Osmotic shrinkage: the embryos swelled in PB1, and then collapsed. (v) Fracture damage: the zona pellucida of the embryos was dissected. (vi) Extracellular ice: the zona of the embryos was elongated. CONCLUSIONS: It was often possible to deduce the type of injury that had occurred in cryopreserved embryos from their appearance at recovery and during subsequent culture. This may help to improve cryopreservation protocols for embryos of many species, including man.     

Reduction of postoperative adhesions with an auto-crosslinked hyaluronan gel in gynaecological laparoscopic surgery: a blinded, controlled, randomized, multicentre study

BACKGROUND: Following myomectomy, postoperative adhesions occur in many patients with adverse effects on fertility. This study investigated the applicability, safety and efficacy of an auto-crosslinked hyaluronan gel in preventing adhesion formation after laparoscopic myomectomy. METHODS: Fifty-two patients aged 22-42 years, undergoing surgery at four centres, were randomly allocated to receive either the gel or no adhesion prevention. The incidence and severity of postoperative adhesions were assessed laparoscopically after 12-14 weeks in a blinded, scored fashion. The primary efficacy variable was the presence/absence of postoperative adhesions at second-look. RESULTS: A nonsignificantly higher proportion of patients receiving the gel were free from adhesions (13 of 21; 62%) compared with control patients (9 of 22; 41%), with a statistically significant difference between the severity of uterine adhesions at baseline and at second-look (0.3 +/- 0.9 versus 0.8 +/- 1.0, P < 0.05). In subjects undergoing myomectomy without concomitant surgery, the proportion of adhesion-free patients was 8 of 12 (67%) and 4 of 11 (36%) (not significant) in the gel and control groups, respectively, with a significant difference in the mean severity scores (P < 0.05). In subjects without uterine adhesions before myomectomy, 12 of 18 (67%) and 8 of 20 (40%) patients in the gel and control groups, respectively were adhesion-free (not significant), with a significant difference in the severity of uterine adhesions (P = 0.05). CONCLUSIONS: Our results suggest that the auto-crosslinked hyaluronan gel may have a favourable safety profile and efficacious antiadhesive action following laparoscopic myomectomy.     

Can the mouse embryo provide a good model for the study of abnormal cellular development seen in human embryos?

Mouse 2-cell embryos arrested in development, either due to the effect of in vitro culture conditions ('2-cell block') or after exposure to the protein synthesis inhibitor anisomycin, were examined to determine the effect of the level of protein synthesis on development. The rate of protein synthesis was found directly to reflect the developmental potential of the embryos. Embryos cultured in the highest dose of the drug failed to divide and had the lowest rate of protein synthesis over the period of investigation, whereas untreated viable 2-cell embryos in the control group had the highest rate of protein synthesis and developed normally. Measurement of the nuclear DNA showed that both arrested and non-arrested embryonic cells completed DNA replication. Furthermore, drug-arrested embryos, like embryos which 'block' in culture, remained morphologically intact when left in culture. Disruption of the nuclear integrity and formation of micronuclei, as is frequently observed in arrested human embryos, was not seen in mouse embryos. These results indicate that developmentally arrested mouse embryos may not be a good model for studying cellular dysfunction in early human development. Experimentation using human material is required to address directly the problem of abnormal human development.     

Impact of hyperglycemia on early embryo development and embryopathy: in vitro experiments using a mouse model

BACKGROUND: The aim of this study is to model the processes of early embryopathy seen in human pregnancy complicated by maternal hyperglycemia secondary to maternal diabetes using a mouse embryo culture system. METHODS: Female mice were superovulated and mated in pairs. Two-cell embryos were harvested from the oviducts and cultured in vitro in KSOM medium (synthetic oviductal medium enriched with potassium) supplemented with 0.2, 5.56, 15.56 or 25.56 mM d-glucose. Cell proliferation, differentiation and apoptosis were assessed. Experiments were performed in constant, embryos exposed to a particular concentration of glucose (0.2, 5.56, 15.56 or 25.56 mM) from harvest to either Day 5 post fertilization (pf) or Day 8 pf, and fluctuating, embryos exposed to alternate high 25.56 mM and normal 5.56 mM concentrations of glucose between harvest and Day 5 pf, glycemic culture. RESULTS: Expected levels of blastocyst formation and hatching were seen at 0.2 and 5.56 mM concentrations of glucose but both were impaired at higher concentrations (chi(2), P < 0.005; P < 0.001). Total cell numbers (P < 0.002) and cell allocation to the inner cell mass (P < 0.01) were reduced, but with no evidence of enhanced apoptosis in the hyperglycemic cultures. Variation in hyperglycemic exposure of the embryos on Days 2, 3 and 4 showed no adverse effects of hyperglycemia up to 24 h, but 48 and 72 h exposures were equally embryopathic (P < 0.01). CONCLUSIONS: Hyperglycemic exposure for >24 h is toxic to early embryo development. These findings may explain the lower than expected implantation rates and higher than expected rates of congenital abnormality and early pregnancy loss seen in patients with diabetes, particularly those with poor diabetic control.     

Developmental competence of mouse embryos following zona drilling using a non-contact holmium: yttrium scandian gallium garnet (Ho: YSGG) laser system

The purpose of this study was to assess the efficacy of theholmium: yttrium scandian gallium garnet (Ho:YSGG) laser, operatingin a pipette-free, non-contact mode, to assist hatching andsustain normal embryonic development. Two-cell mouse embryoswere recovered and assigned to laser-assisted hatching (LAH)treatment or control human tubal fluid (HTF) culture with orwithout serum (HTF-s, HTF-o) or with late serum supplementation(HTF-o/s). The basic experimental apparatus for LAH consistedof a stationary 2.1 µm Ho: YSGG laser beam directed througha mechanical shutter into an input port of a Zeiss Axiomat invertedmicroscope. Fewer (P < 0.05) embryos developed to the blastocyststage in the HTF-s group (81%) than in the LAH (90%), HTF-o(94%) and HTF-o/s (92%) groups. The level of hatching was significantlyincreased (P < 0.01) after the LAH treatment (57%) comparedto HTF-o/s (32%), HTF-s (18%) or HTF-o (5%). Implantation rateswere not significantly impaired following the LAH treatment(21%). These data demonstrate that LAH using the Ho: YSGG laseris a simple, accurate and effective procedure for assisted hatching.