Background The aim of this experimental study was to determine the effect of mild hyperthermia on tumor response and angioneogenesis in an isolated limb perfusion model with a human melanoma xenograft.
Background
The aim of this experimental study was to determine the effect of mild hyperthermia on tumor response and angioneogenesis in an isolated limb perfusion model with a human melanoma xenograft.Methods A human melanoma xenograft was implanted into the hindlimbs of 30 athymic nude rats. The animals were randomized into five groups: group I: control, group II: sham group, group III: external hyperthermia with a tissue temperature of 41.5°C for 30 minutes without ILP, group IV: normothermic ILP (tissue temperature 37°C for 30 minutes, group V: hyperthermic ILP (tissue temperature 41.5°C for 30 minutes). Tumor response was evaluated by tumor size determination and immunohistochemical analysis 6 weeks postoperatively. Tissue sections were investigated for expression of CD34 and basic fibroblast growth factor (bFGF).
Methods
A human melanoma xenograft was implanted into the hindlimbs of 30 athymic nude rats. The animals were randomized into five groups: group I: control, group II: sham group, group III: external hyperthermia with a tissue temperature of 41.5°C for 30 minutes without ILP, group IV: normothermic ILP (tissue temperature 37°C for 30 minutes, group V: hyperthermic ILP (tissue temperature 41.5°C for 30 minutes). Tumor response was evaluated by tumor size determination and immunohistochemical analysis 6 weeks postoperatively. Tissue sections were investigated for expression of CD34 and basic fibroblast growth factor (bFGF).Results Average tumor volumes of the controls (I) increased from 105 mm 3  to 1388 mm 3 . In the sham operated group (II) tumor volumes were significantly larger than in group I. Tumor volumes in group IV were significantly smaller than in group I and lowest in group V. There were no significant differences in size between group I and group III after six weeks. In group III and IV each, 5 animals showed tumor progression and one had a partial tumor response. In group V only 2 animals showed tumor progression. Immunhistochemical analysis of the tissue sections demonstrated that angioneogenesis was more pronounced in group II than in group I and less pronounced in group IV and V compared with group I.
Results
Average tumor volumes of the controls (I) increased from 105 mm 3  to 1388 mm 3 . In the sham operated group (II) tumor volumes were significantly larger than in group I. Tumor volumes in group IV were significantly smaller than in group I and lowest in group V. There were no significant differences in size between group I and group III after six weeks. In group III and IV each, 5 animals showed tumor progression and one had a partial tumor response. In group V only 2 animals showed tumor progression. Immunhistochemical analysis of the tissue sections demonstrated that angioneogenesis was more pronounced in group II than in group I and less pronounced in group IV and V compared with group I.Conclusions Our results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF. External hyperthermia of 41.5°C tissue temperature for 30 minutes only has no impact on tumor growth and angioneogenesis in vivo.
Conclusions
Our results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF.External hyperthermia of 41.5°C tissue temperature for 30 minutes only has no impact on tumor growth and angioneogenesis in vivo.

Background Hyperthermic isolated limb perfusion is an established treatment for multiple, locoregional intransit metastases of malignant melanoma and soft tissue sarcomas of the extremities. It was introduced 1957 by Creech and Krementz [ 1 ]. The procedure allows a high locoregional concentration of cytostatics with few systemic side effects [ 2 ]. Isolated limb perfusion achieves local tumor control in a high percentage of cases. Complication rate of perfusion is acceptably low. A surviving model for isolated perfused rat hindlimbs was reported by Wu et al. [ 3 ] based on the perfusion model of Nagel et al.[ 4 ]. Angioneogenesis has been recognized for many years to play a central role in the growth of primary tumors and the formation of metastases in general [ 5 , 6 ]. Melanomas express basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) in their dermal nevocytes and in the stroma. bFGF promotes angiogenesis  in vivo  [ 7 ] and  in vitro  [ 8 ]. Antisense targeting of bFGF/FGFR-1 in malignant melanomas blocks intratumoral angioneogenesis [ 9 ]. Hyperthermia inhibits angioneogenesis. Some of the antineoplastic effects of hyperthermia are caused by ischemia due to obstruction or destruction of the tumor vessels. Eikesdal et al. described by hyperthermic temperatures disrupture of 25–50 % of the vasculature in malignant tumors. The heated tumors (44°C) had a blood flow reduction of 40–60 % after 24 hours [ 10 ]. In vitro  data indicate that capillary endothelials cells of malignant neoplasms are more thermosensitive than endothelial cells of normal tissues. The vascularization of tumors can be significantly damaged at temperatures which may alter but do not damage the vasculature of normal tissue [ 11 ]. The extent of the proliferation of vessels is also inversely related to temperature [ 12 ]. The critical temperature at which a direct vascular damage occurs is between 42.7 and 43.7 degrees C [ 13 ]. Hyperthermia increases the effects of angioneogenesis inhibitors on tumor growth and tumor angioneogenesis [ 14 ]. The aim of this experimental study was to determine the impact of hyperthermia on tumor response and angioneogenesis in an isolated limb perfusion model.
Background
Hyperthermic isolated limb perfusion is an established treatment for multiple, locoregional intransit metastases of malignant melanoma and soft tissue sarcomas of the extremities. It was introduced 1957 by Creech and Krementz [ 1 ]. The procedure allows a high locoregional concentration of cytostatics with few systemic side effects [ 2 ]. Isolated limb perfusion achieves local tumor control in a high percentage of cases. Complication rate of perfusion is acceptably low. A surviving model for isolated perfused rat hindlimbs was reported by Wu et al. [ 3 ] based on the perfusion model of Nagel et al.[ 4 ].Angioneogenesis has been recognized for many years to play a central role in the growth of primary tumors and the formation of metastases in general [ 5 , 6 ]. Melanomas express basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) in their dermal nevocytes and in the stroma. bFGF promotes angiogenesis  in vivo  [ 7 ] and  in vitro  [ 8 ]. Antisense targeting of bFGF/FGFR-1 in malignant melanomas blocks intratumoral angioneogenesis [ 9 ].in vivoin vitroHyperthermia inhibits angioneogenesis. Some of the antineoplastic effects of hyperthermia are caused by ischemia due to obstruction or destruction of the tumor vessels. Eikesdal et al. described by hyperthermic temperatures disrupture of 25–50 % of the vasculature in malignant tumors. The heated tumors (44°C) had a blood flow reduction of 40–60 % after 24 hours [ 10 ].In vitro  data indicate that capillary endothelials cells of malignant neoplasms are more thermosensitive than endothelial cells of normal tissues. The vascularization of tumors can be significantly damaged at temperatures which may alter but do not damage the vasculature of normal tissue [ 11 ]. The extent of the proliferation of vessels is also inversely related to temperature [ 12 ]. The critical temperature at which a direct vascular damage occurs is between 42.7 and 43.7 degrees C [ 13 ].In vitroHyperthermia increases the effects of angioneogenesis inhibitors on tumor growth and tumor angioneogenesis [ 14 ]. The aim of this experimental study was to determine the impact of hyperthermia on tumor response and angioneogenesis in an isolated limb perfusion model.Methods Thirty inbred male nude athymic rats (Rowett rnu/rnu) weighting 200 to 280 g were used in this study. Animals were kept separately during the experiment with 12 hours of light per day. They were fed a standard laboratory diet and tap water  ad libidum . Maintenance and care of all experimental animals were carried out according to the guidelines of the local responsible Animal Protection Commission and carried out in compliance with national guidelines (National Institute of Health for Use of Laboratory Animals). A solid sample (2 × 2 × 2 mm) of a human melanoma xenograft (SK-MEL-3), derived from a lymph node melanoma metastasis, was implanted into the hindlimbs of 30 nude rats. The largest width and the maximum tumor diameter perpendicular to the width were measured with a micrometer. Only tumors with a diameter greater than > 12 mm in the largest diameter were included in the study. The details of the perfusion system have been described previously [ 4 ]. The equipment consisted of a miniature oxygenator, a heat exchanger and a roller pump. Venous blood of the limb was oxygenated in the oxygenator (99.15% O 2 ; 0.85% CO 2 ) and warmed in the heat exchanger. The warmed arterialized perfusate is driven by a roller pump with two synchronously running pump-heads on a single axis for the arterial and venous lines (Masterflex ® ). The outer diameters of the tubes corresponded with the diameter of the femoral vessels of nude rats weighting about 250 g (arterial 0.7 mm; venous 1.0 mm). Rat hindlimbs were perfused with ringer's solution and sodium heparin (25 IU/100 μl saline) at 37 or 41.5 degrees C for 30 minutes with a flow rate of 4 ml/min. External hyperthermia was applied by an infrared lamp positioned in a fixed distance to the tumor. For temperature measurement during limb perfusion, a nickel-chrom-nickel thermocouple of 0.6 mm in diameter (Standard Integrated Thermocouple Thermocoax, Phillips, Hamburg, Germany) was placed at the macroscopic tumor margin. The thermocouple was calibrated before use in a high-precision water bath. Baseline temperature was recorded for 5 minutes before treatment. Temperature was continuously measured during application. In group I (control) and group II (sham operated) temperature measurement was not performed. The animals were randomized into a control and four study groups of six animals each: group I: control (no therapy), group II: sham group (skin incision without ILP), group III: external hyperthermia with a tissue temperature of 41.5°C for 30 minutes without ILP, group IV: normothermic ILP (Ringer's solution as perfusate, tissue temperature 37°C for 30 minutes, group V: hyperthermic ILP (tissue temperature 41.5°C for 30 minutes). ILP was subsequently performed with the above-mentioned parameters. Antineoplastic agents were not applied. The rats were anaesthetized with Ketamin (Ketanest ® , 80 mg/kg i.m.; Pearl-Davis, Berlin, Germany) and Xylazin (Rompun ® , 10 mg/kg i.m.; Bayer, Leverkusen, Germany), and fixed in a supine position. For perfusion, the animals underwent a 3-cm incision of the groin. The left femoral artery and vein were isolated using an operation microscope. A vascular clip was placed across the artery and a silicon catheter (outer diameter 0.7 mm) was introduced via a transverse arteriotomy. The femoral vein was cannulated with a silicon catheter of 1 mm outer diameter (Fig.  1 ). Tourniquets were applied around the left hindlimb to ensure isolation. Rat hindlimbs were perfused at a flow rate of 4 ml/h, for 30 min. After treatment, the silicon catheters were removed, and the vein and the artery were sutured (8/0 Prolene), to restitute normal blood flow. After the isolated limb perfusion the wound was closed in two layers. Total operation time rated about 90 min. During the first 5 days after operation the rats were weighed daily, and once a week there-after. The animals were kept under standardized conditions and were killed by an overdose of anesthetic and cervical dislocation 6 weeks after treatment. The greatest tumor width and the maximum diameter perpendicular to the width were macroscopically measured with a micrometer. Lesion volume was calculated using the formula v = 4 × π × a × b 2/3, in which a and b are the radii of the measured axes. The tumor was then completely sectioned. Each tumor was divided into two parts. One part was fixed in 4 per cent formalin solution for 5 days, embedded in paraffin and stained with hematoxylin and eosin (H&E). The other part was fixed in 0.9 % saline solution, frozen and stored at -80°C until use. Vascular density and tumor morphology were evaluated by immunohistochemistry. Tumor tissue was tested for expression of basic fibroblast growth factor (bFGF) and CD34. Tissue specimens were fixed in 10 % formaldehyde and embedded in paraffin according to routine protocols. Sections 4 μm thick were deparaffinized and rehydrated using graded ethanols following routine protocols. For antigen retrieval all sections were microwave treated for 20 minutes in target retrieval solution (for bFGF; DAKO, Glostrup, Denmark) or 10 mM citrate buffer (for CD34) at 700 W. The sections were incubated overnight with primary mouse anti-bFGF (1:50; Becton Dickinson, Heidelberg, Germany) and anti-CD34 antibody (1:100; Immunotech, Hamburg, Germany). After rinsing in Tris buffer detection was carried out with a biotinylated secondary rabbit anti-mouse antibody diluted 1:50 (DAKO, Glostrup, Denmark) for 30 minutes at room temperature. The sections were washed again as above in Tris buffer and then incubated with streptavidin-biotin-alkaline-phosphatase (10 μl each solution A+B (Sigma, Deisenhofen, Germany) in 10 ml Tris). Immunohistochemical staining was visualized with Fast Red (2 mg Naphtol-As-Mx-phosphate, 10 μl 1 M Levamisol and 10 mg Fast-Red (Sigma) in 0.2 ml N, N-dimethylformamide, (Merck, Darmstadt, Germany) and 9.8 ml 0.1 M Tris-HCl buffer at pH8.6). The sections were rinsed in H 2 0 and counterstained with hemalaun. Negative controls without primary antibody were run for each sample. Tumor response was graded according to the system described by de Wilt et al. [ 15 ]. Progressive disease (PD) was defined as a tumor size > 125 % of the size at the time of treatment, in tumor sizes of 100 ± 25 % no change (NC) was stated. Response was graded as partial (PR) if tumor sizes was between 10 and 75 % and complete (CR) for tumor size < 10 % of its original diameter. In areas of intense neovascularized spots microvessels were counted in a 100× field. A microvessel was defined as a lumen surrounded by a rim of endothelial cells highlighted by immunostaining with anti-CD34 antibodys or anti-bFGF antibodys. Five separate intense neovascularized areas were assessed, and the mean was calculated as microvessel density of each tumor. The score was assessed by two independent observers. Data were analyzed using SPSS/PC+ statistical software. The mean and range of tumor and lesion volumes were calculated for each group. For comparison of tumor volume and microvessel density between the diffent groups a non-parametric test was used (Kruskal-Wallis). A p value < 0.05 was considered to be significant.
Methods
Thirty inbred male nude athymic rats (Rowett rnu/rnu) weighting 200 to 280 g were used in this study. Animals were kept separately during the experiment with 12 hours of light per day. They were fed a standard laboratory diet and tap water  ad libidum . Maintenance and care of all experimental animals were carried out according to the guidelines of the local responsible Animal Protection Commission and carried out in compliance with national guidelines (National Institute of Health for Use of Laboratory Animals).ad libidumA solid sample (2 × 2 × 2 mm) of a human melanoma xenograft (SK-MEL-3), derived from a lymph node melanoma metastasis, was implanted into the hindlimbs of 30 nude rats. The largest width and the maximum tumor diameter perpendicular to the width were measured with a micrometer. Only tumors with a diameter greater than > 12 mm in the largest diameter were included in the study.The details of the perfusion system have been described previously [ 4 ]. The equipment consisted of a miniature oxygenator, a heat exchanger and a roller pump. Venous blood of the limb was oxygenated in the oxygenator (99.15% O 2 ; 0.85% CO 2 ) and warmed in the heat exchanger. The warmed arterialized perfusate is driven by a roller pump with two synchronously running pump-heads on a single axis for the arterial and venous lines (Masterflex ® ). The outer diameters of the tubes corresponded with the diameter of the femoral vessels of nude rats weighting about 250 g (arterial 0.7 mm; venous 1.0 mm).Rat hindlimbs were perfused with ringer's solution and sodium heparin (25 IU/100 μl saline) at 37 or 41.5 degrees C for 30 minutes with a flow rate of 4 ml/min. External hyperthermia was applied by an infrared lamp positioned in a fixed distance to the tumor.For temperature measurement during limb perfusion, a nickel-chrom-nickel thermocouple of 0.6 mm in diameter (Standard Integrated Thermocouple Thermocoax, Phillips, Hamburg, Germany) was placed at the macroscopic tumor margin. The thermocouple was calibrated before use in a high-precision water bath. Baseline temperature was recorded for 5 minutes before treatment. Temperature was continuously measured during application. In group I (control) and group II (sham operated) temperature measurement was not performed.The animals were randomized into a control and four study groups of six animals each: group I: control (no therapy), group II: sham group (skin incision without ILP), group III: external hyperthermia with a tissue temperature of 41.5°C for 30 minutes without ILP, group IV: normothermic ILP (Ringer's solution as perfusate, tissue temperature 37°C for 30 minutes, group V: hyperthermic ILP (tissue temperature 41.5°C for 30 minutes). ILP was subsequently performed with the above-mentioned parameters. Antineoplastic agents were not applied.The rats were anaesthetized with Ketamin (Ketanest ® , 80 mg/kg i.m.; Pearl-Davis, Berlin, Germany) and Xylazin (Rompun ® , 10 mg/kg i.m.; Bayer, Leverkusen, Germany), and fixed in a supine position. For perfusion, the animals underwent a 3-cm incision of the groin. The left femoral artery and vein were isolated using an operation microscope. A vascular clip was placed across the artery and a silicon catheter (outer diameter 0.7 mm) was introduced via a transverse arteriotomy. The femoral vein was cannulated with a silicon catheter of 1 mm outer diameter (Fig.  1 ). Tourniquets were applied around the left hindlimb to ensure isolation. Rat hindlimbs were perfused at a flow rate of 4 ml/h, for 30 min. After treatment, the silicon catheters were removed, and the vein and the artery were sutured (8/0 Prolene), to restitute normal blood flow. After the isolated limb perfusion the wound was closed in two layers. Total operation time rated about 90 min.During the first 5 days after operation the rats were weighed daily, and once a week there-after. The animals were kept under standardized conditions and were killed by an overdose of anesthetic and cervical dislocation 6 weeks after treatment. The greatest tumor width and the maximum diameter perpendicular to the width were macroscopically measured with a micrometer. Lesion volume was calculated using the formula v = 4 × π × a × b 2/3, in which a and b are the radii of the measured axes. The tumor was then completely sectioned. Each tumor was divided into two parts. One part was fixed in 4 per cent formalin solution for 5 days, embedded in paraffin and stained with hematoxylin and eosin (H&E). The other part was fixed in 0.9 % saline solution, frozen and stored at -80°C until use.Vascular density and tumor morphology were evaluated by immunohistochemistry. Tumor tissue was tested for expression of basic fibroblast growth factor (bFGF) and CD34.Tissue specimens were fixed in 10 % formaldehyde and embedded in paraffin according to routine protocols. Sections 4 μm thick were deparaffinized and rehydrated using graded ethanols following routine protocols. For antigen retrieval all sections were microwave treated for 20 minutes in target retrieval solution (for bFGF; DAKO, Glostrup, Denmark) or 10 mM citrate buffer (for CD34) at 700 W. The sections were incubated overnight with primary mouse anti-bFGF (1:50; Becton Dickinson, Heidelberg, Germany) and anti-CD34 antibody (1:100; Immunotech, Hamburg, Germany). After rinsing in Tris buffer detection was carried out with a biotinylated secondary rabbit anti-mouse antibody diluted 1:50 (DAKO, Glostrup, Denmark) for 30 minutes at room temperature. The sections were washed again as above in Tris buffer and then incubated with streptavidin-biotin-alkaline-phosphatase (10 μl each solution A+B (Sigma, Deisenhofen, Germany) in 10 ml Tris). Immunohistochemical staining was visualized with Fast Red (2 mg Naphtol-As-Mx-phosphate, 10 μl 1 M Levamisol and 10 mg Fast-Red (Sigma) in 0.2 ml N, N-dimethylformamide, (Merck, Darmstadt, Germany) and 9.8 ml 0.1 M Tris-HCl buffer at pH8.6). The sections were rinsed in H 2 0 and counterstained with hemalaun. Negative controls without primary antibody were run for each sample.Tumor response was graded according to the system described by de Wilt et al. [ 15 ]. Progressive disease (PD) was defined as a tumor size > 125 % of the size at the time of treatment, in tumor sizes of 100 ± 25 % no change (NC) was stated. Response was graded as partial (PR) if tumor sizes was between 10 and 75 % and complete (CR) for tumor size < 10 % of its original diameter.In areas of intense neovascularized spots microvessels were counted in a 100× field. A microvessel was defined as a lumen surrounded by a rim of endothelial cells highlighted by immunostaining with anti-CD34 antibodys or anti-bFGF antibodys. Five separate intense neovascularized areas were assessed, and the mean was calculated as microvessel density of each tumor. The score was assessed by two independent observers.Data were analyzed using SPSS/PC+ statistical software. The mean and range of tumor and lesion volumes were calculated for each group. For comparison of tumor volume and microvessel density between the diffent groups a non-parametric test was used (Kruskal-Wallis). A p value < 0.05 was considered to be significant.Results During intervention, the required limb tissue temperature was reached within 8–12 minutes in group IV and 10–15 minutes in group III. Stable temperatures were then maintained for a further 30 minutes with a mean of 41.4°C ± 0.5. To reach limb tissue temperature the perfusion fluid maintained 43°C ± 0.4. Tumor and lesion Volumes Before treatment, the mean (s.e.m) volume of the treated tumors in groups I, II, III, IV and V was 105 mm 3  (4.3), 98 mm 3  (5.7), 108 mm 3  (3.4), 95 mm 3  (5.0) and 107 (4.1) mm 3  respectively, with no significant difference between the groups. Average tumor volume of the controls (I) increased to 1388 mm 3  (101) during six weeks. In the sham operated group (II) tumor volume was significantly larger than in group I (2350 mm 3  (198.6),  P  = 0.021). Tumor volume in group IV was significantly smaller (1009 mm 3  (122.5)) than in group I and lowest in group V 405 mm 3  (103.6) ( P = 0.036 and  P = 0.021, respectively.). There were no significant differences in size between group I and group III (1135 mm 3  (99)) after six weeks ( P  > 0.05)(Fig.  2 ). Body weight After the perfusion, body weight decreased in all groups during the first 5 days with a maximum of 8 %. In the following weeks, body weight mounted up to 130 % of the preoperative value. In the last week of the experiment, the weight almost reached a plateau. No significant differences between the five experimental groups were found. Histology In the H&E staining the untreated melanoma (group I) presented as a subcutanous unilocular nodule of moderate differentiation. The tumor showed mainly tubular structures with vacuole like lumen formation and was infiltrated with fibrous septa. The spontaneous rate of necrosis was estimated to be 20 %. Numerous mitoses were detected. Tumor morphology did not change with ageing or tumor size. Histology was similar in group II. In group V the tumors showed signs of irreversible cell damage after treatment. Tumor cells displayed clear shrinkage and partial loss of cell contact. Thromboses of the larger adjacent vessels were found on the tumor-skin border. Macrophage infiltration was present in all groups, but was more numerous pronounced in group V. In group III and IV there were less signs of irreversible cell damage after treatment than in group V. Immunhistochemical analysis of the histological sections for bFGF demonstrated that microvessel density of group I (Fig  3 ) ranged from 18 to 23 with a mean value of 20. Angioneogenesis was more pronounced in group II (Fig  4 ) than in group I (p = 0.003) and less pronounced in group IV and V (Fig  5 ) compared with group I (p = 0.023, p = 0.001). There were no significant differences in the expression of angiogenetic markers between group I and group III (p >0.05). Immunhistochemical analysis of the histological sections for CD34 showed that microvessel density of group I ranged from 17 to 23 with a mean value of 19. Angioneogenesis was more pronounced in group II than in group I (p = 0,001) and less pronounced in group IV and V compared with group I (p = 0.023, p = 0.017). There were no significant differences in the expression of angiogenetic markers between group I and group III (p > 0.05). Strong expression of bFGF and CD34 was found in the cytoplasm of intimal endothelial and medial smooth muscle cells. Expression of bFGF and CD34 was stronger at the invasion front than in the centre of tumors. Summarized data of the expression intensity of bFGF and CD34 in the tumors are given in table  1 . Tumor remission In group I and II tumor progression was observed in all animals macro- and microscopically. In group III and IV 5 animals showed tumor progression and one had a partial tumor response. In group V, 2 animals showed tumor progression, 2 had a complete remission. One had a partial tumor response and one had no change in tumor response (table  2 ).
Results
During intervention, the required limb tissue temperature was reached within 8–12 minutes in group IV and 10–15 minutes in group III. Stable temperatures were then maintained for a further 30 minutes with a mean of 41.4°C ± 0.5. To reach limb tissue temperature the perfusion fluid maintained 43°C ± 0.4.Tumor and lesion Volumes Before treatment, the mean (s.e.m) volume of the treated tumors in groups I, II, III, IV and V was 105 mm 3  (4.3), 98 mm 3  (5.7), 108 mm 3  (3.4), 95 mm 3  (5.0) and 107 (4.1) mm 3  respectively, with no significant difference between the groups. Average tumor volume of the controls (I) increased to 1388 mm 3  (101) during six weeks. In the sham operated group (II) tumor volume was significantly larger than in group I (2350 mm 3  (198.6),  P  = 0.021). Tumor volume in group IV was significantly smaller (1009 mm 3  (122.5)) than in group I and lowest in group V 405 mm 3  (103.6) ( P = 0.036 and  P = 0.021, respectively.). There were no significant differences in size between group I and group III (1135 mm 3  (99)) after six weeks ( P  > 0.05)(Fig.  2 ).
Tumor and lesion Volumes
Before treatment, the mean (s.e.m) volume of the treated tumors in groups I, II, III, IV and V was 105 mm 3  (4.3), 98 mm 3  (5.7), 108 mm 3  (3.4), 95 mm 3  (5.0) and 107 (4.1) mm 3  respectively, with no significant difference between the groups. Average tumor volume of the controls (I) increased to 1388 mm 3  (101) during six weeks. In the sham operated group (II) tumor volume was significantly larger than in group I (2350 mm 3  (198.6),  P  = 0.021). Tumor volume in group IV was significantly smaller (1009 mm 3  (122.5)) than in group I and lowest in group V 405 mm 3  (103.6) ( P = 0.036 and  P = 0.021, respectively.). There were no significant differences in size between group I and group III (1135 mm 3  (99)) after six weeks ( P  > 0.05)(Fig.  2 ).PPPPBody weight After the perfusion, body weight decreased in all groups during the first 5 days with a maximum of 8 %. In the following weeks, body weight mounted up to 130 % of the preoperative value. In the last week of the experiment, the weight almost reached a plateau. No significant differences between the five experimental groups were found.
Body weight
After the perfusion, body weight decreased in all groups during the first 5 days with a maximum of 8 %. In the following weeks, body weight mounted up to 130 % of the preoperative value. In the last week of the experiment, the weight almost reached a plateau. No significant differences between the five experimental groups were found.Histology In the H&E staining the untreated melanoma (group I) presented as a subcutanous unilocular nodule of moderate differentiation. The tumor showed mainly tubular structures with vacuole like lumen formation and was infiltrated with fibrous septa. The spontaneous rate of necrosis was estimated to be 20 %. Numerous mitoses were detected. Tumor morphology did not change with ageing or tumor size. Histology was similar in group II. In group V the tumors showed signs of irreversible cell damage after treatment. Tumor cells displayed clear shrinkage and partial loss of cell contact. Thromboses of the larger adjacent vessels were found on the tumor-skin border. Macrophage infiltration was present in all groups, but was more numerous pronounced in group V. In group III and IV there were less signs of irreversible cell damage after treatment than in group V. Immunhistochemical analysis of the histological sections for bFGF demonstrated that microvessel density of group I (Fig  3 ) ranged from 18 to 23 with a mean value of 20. Angioneogenesis was more pronounced in group II (Fig  4 ) than in group I (p = 0.003) and less pronounced in group IV and V (Fig  5 ) compared with group I (p = 0.023, p = 0.001). There were no significant differences in the expression of angiogenetic markers between group I and group III (p >0.05). Immunhistochemical analysis of the histological sections for CD34 showed that microvessel density of group I ranged from 17 to 23 with a mean value of 19. Angioneogenesis was more pronounced in group II than in group I (p = 0,001) and less pronounced in group IV and V compared with group I (p = 0.023, p = 0.017). There were no significant differences in the expression of angiogenetic markers between group I and group III (p > 0.05). Strong expression of bFGF and CD34 was found in the cytoplasm of intimal endothelial and medial smooth muscle cells. Expression of bFGF and CD34 was stronger at the invasion front than in the centre of tumors. Summarized data of the expression intensity of bFGF and CD34 in the tumors are given in table  1 .
Histology
In the H&E staining the untreated melanoma (group I) presented as a subcutanous unilocular nodule of moderate differentiation. The tumor showed mainly tubular structures with vacuole like lumen formation and was infiltrated with fibrous septa. The spontaneous rate of necrosis was estimated to be 20 %. Numerous mitoses were detected. Tumor morphology did not change with ageing or tumor size. Histology was similar in group II.In group V the tumors showed signs of irreversible cell damage after treatment. Tumor cells displayed clear shrinkage and partial loss of cell contact. Thromboses of the larger adjacent vessels were found on the tumor-skin border. Macrophage infiltration was present in all groups, but was more numerous pronounced in group V. In group III and IV there were less signs of irreversible cell damage after treatment than in group V.Immunhistochemical analysis of the histological sections for bFGF demonstrated that microvessel density of group I (Fig  3 ) ranged from 18 to 23 with a mean value of 20. Angioneogenesis was more pronounced in group II (Fig  4 ) than in group I (p = 0.003) and less pronounced in group IV and V (Fig  5 ) compared with group I (p = 0.023, p = 0.001). There were no significant differences in the expression of angiogenetic markers between group I and group III (p >0.05).Immunhistochemical analysis of the histological sections for CD34 showed that microvessel density of group I ranged from 17 to 23 with a mean value of 19. Angioneogenesis was more pronounced in group II than in group I (p = 0,001) and less pronounced in group IV and V compared with group I (p = 0.023, p = 0.017). There were no significant differences in the expression of angiogenetic markers between group I and group III (p > 0.05).Strong expression of bFGF and CD34 was found in the cytoplasm of intimal endothelial and medial smooth muscle cells. Expression of bFGF and CD34 was stronger at the invasion front than in the centre of tumors.Summarized data of the expression intensity of bFGF and CD34 in the tumors are given in table  1 .Tumor remission In group I and II tumor progression was observed in all animals macro- and microscopically. In group III and IV 5 animals showed tumor progression and one had a partial tumor response. In group V, 2 animals showed tumor progression, 2 had a complete remission. One had a partial tumor response and one had no change in tumor response (table  2 ).
Tumor remission
In group I and II tumor progression was observed in all animals macro- and microscopically. In group III and IV 5 animals showed tumor progression and one had a partial tumor response. In group V, 2 animals showed tumor progression, 2 had a complete remission. One had a partial tumor response and one had no change in tumor response (table  2 ).Discussion A phenomenon that has been described by numerous authors is the rapid increase of tumor growth following surgical manipulation [ 16 , 17 ]. This was also observed in our sham-operated group of animals (group II) in which tumors grew more rapidly than in the control group (group I). This implies that resection is associated with an accelerated growth in residual tumors. It is assumed that the surgical manipulation induces liberation of growth factors that in addition to their effect on healing also have a stimulating effect on tumor proliferation. This hypothesis is supported by the results of our immunohistochemical investigations, which revealed an enhanced expression of fibroblast growth factor (bFGF) in group II. In an intraperitoneal tumor model, EGGERMONT et al. showed that the surgical intervention of laparotomy induces an increase of carcinomatosis in the entire peritoneal cavity. At the same time, the immunotherapeutic effect of interleukin 2 and natural killer cells is significantly reduced. It must therefore be speculated that surgical trauma induces a temporary immunosuppressive effect that influences tumor growth [ 16 , 18 , 19 ]. In our experiments, group V (hyperthermic perfusion) showed a significant slowing of tumor growth in comparison with the control group. This was also confirmed by the immunohistochemical investigations that showed a reduction in the expression of relevant markers of the vascular endothelium. Indeed, two of the six animals occurred a complete remission of their tumor. The observation that two animals in this group experienced tumor progression can probably be explained by the duration of the intervention, since factors affecting the kinetics of cell death during hyperthermia are not only the maximum temperature induced, but also the duration of the elevated temperature. In an overview report BHUYAN noted that, as a function of various tumor cell types, a temperature of 43°C must be applied between 30 and 150 minutes to irreversibly damage the tumor cells. At a temperature of 45°C, the application time required varied between 13 and 85 minutes [ 20 ]. In our experiments, the application time was only 30 minutes at a temperature of 41.5°C. Nevertheless, the relatively short application time also had an appreciable damaging effect on the metastatic vessels. Although the temperature in group III (external hyperthermia) was also 41.5°C applied for 30 minutes, no significant changes in the growth pattern of the tumor could be observed. Furthermore, the histological and immunohistochemical evaluations showed a mild hyperthermic damage at best. A comparison of treatment groups III and V revealed no differences in terms of duration of treatment and induced tissue temperatures. In contrast to group III, however, hyperthermia in group V has been mediated directly by the vascular system. In normal tissues subjected to warming, blood perfusion increases reactively via dilatation of the vessels together with an increase in vessel wall permeability. A decisive factor is that the tumor cells themselves, as well as the endothelial cells of tumor vessels are more thermosensitive than normal tissue [ 16 , 21 , 22 ]. Other authors have also shown that hyperthermia is capable of destroying vascular architecture, so that perfusion of the tumor is reduced. The sensitivity of various tumors, however, varies considerably [ 14 ]. ILP and other vasculotoxic effects like TNFalpha in combination with melphalan or doxorubicin increase the uptake of these drugs of three to six times [ 23 ]. Heat transfer between the vascular system and the tissue is determined in particular by the nature of the blood vessels. Important factors are the number, length, and diameter of the vessels, and also the velocity of blood flow [ 24 ]. CREZEE described the relationship between tumor perfusion and heating of the tumor tissue under systemic hyperthermia. He could show that the induction of heat in the tumor depends on the density of the blood vessels within the tumor itself. The more vessels in the tumor exist, the easier it is to heat the lesion via the vascular system. Heattransfer is particularly pronounced with large-calibre blood vessels or high flow velocity, and its rate is all the greater the higher the temperature gradient between the tumor tissue and the blood [ 25 , 26 ]. On the other hand, tumor vasculature carries away the externally applied heat (group III) which is then no longer available to destroy the tumor cells. In this case, the temperature gradient decreases from the tumor to the vascular system. Furthermore, in the presence of hyperthermia, microcirculation in the tumor tissue is more affected than in normal tissue [ 27 ]. In addition, manipulation to the vascular system, and thus oxygenation of the tumor, also appears to play an important role. Although the body's own oxygenated blood is added to the perfusate, the dilution effect together with the clamping time, leads to a relative reduction in perfusion. It is known that hypoxia, in conjunction with a decrease in the pH and energy status of the cell, enhances thermosensitivity [ 28 - 30 ], and this increase correlates with the size of the tumor [ 31 ]. Our results have also confirmed that surgical manipulation of the vascular system and perfusion have an influence on the tumor. Overall, the effect of the intravascular application of hyperthermia appears to be mediated by a combination of hypoxia and heat.
Discussion
A phenomenon that has been described by numerous authors is the rapid increase of tumor growth following surgical manipulation [ 16 , 17 ].This was also observed in our sham-operated group of animals (group II) in which tumors grew more rapidly than in the control group (group I). This implies that resection is associated with an accelerated growth in residual tumors. It is assumed that the surgical manipulation induces liberation of growth factors that in addition to their effect on healing also have a stimulating effect on tumor proliferation.This hypothesis is supported by the results of our immunohistochemical investigations, which revealed an enhanced expression of fibroblast growth factor (bFGF) in group II. In an intraperitoneal tumor model, EGGERMONT et al. showed that the surgical intervention of laparotomy induces an increase of carcinomatosis in the entire peritoneal cavity. At the same time, the immunotherapeutic effect of interleukin 2 and natural killer cells is significantly reduced. It must therefore be speculated that surgical trauma induces a temporary immunosuppressive effect that influences tumor growth [ 16 , 18 , 19 ].In our experiments, group V (hyperthermic perfusion) showed a significant slowing of tumor growth in comparison with the control group. This was also confirmed by the immunohistochemical investigations that showed a reduction in the expression of relevant markers of the vascular endothelium. Indeed, two of the six animals occurred a complete remission of their tumor. The observation that two animals in this group experienced tumor progression can probably be explained by the duration of the intervention, since factors affecting the kinetics of cell death during hyperthermia are not only the maximum temperature induced, but also the duration of the elevated temperature.In an overview report BHUYAN noted that, as a function of various tumor cell types, a temperature of 43°C must be applied between 30 and 150 minutes to irreversibly damage the tumor cells. At a temperature of 45°C, the application time required varied between 13 and 85 minutes [ 20 ]. In our experiments, the application time was only 30 minutes at a temperature of 41.5°C. Nevertheless, the relatively short application time also had an appreciable damaging effect on the metastatic vessels.Although the temperature in group III (external hyperthermia) was also 41.5°C applied for 30 minutes, no significant changes in the growth pattern of the tumor could be observed. Furthermore, the histological and immunohistochemical evaluations showed a mild hyperthermic damage at best. A comparison of treatment groups III and V revealed no differences in terms of duration of treatment and induced tissue temperatures.In contrast to group III, however, hyperthermia in group V has been mediated directly by the vascular system. In normal tissues subjected to warming, blood perfusion increases reactively via dilatation of the vessels together with an increase in vessel wall permeability. A decisive factor is that the tumor cells themselves, as well as the endothelial cells of tumor vessels are more thermosensitive than normal tissue [ 16 , 21 , 22 ]. Other authors have also shown that hyperthermia is capable of destroying vascular architecture, so that perfusion of the tumor is reduced. The sensitivity of various tumors, however, varies considerably [ 14 ]. ILP and other vasculotoxic effects like TNFalpha in combination with melphalan or doxorubicin increase the uptake of these drugs of three to six times [ 23 ].Heat transfer between the vascular system and the tissue is determined in particular by the nature of the blood vessels. Important factors are the number, length, and diameter of the vessels, and also the velocity of blood flow [ 24 ]. CREZEE described the relationship between tumor perfusion and heating of the tumor tissue under systemic hyperthermia. He could show that the induction of heat in the tumor depends on the density of the blood vessels within the tumor itself. The more vessels in the tumor exist, the easier it is to heat the lesion via the vascular system. Heattransfer is particularly pronounced with large-calibre blood vessels or high flow velocity, and its rate is all the greater the higher the temperature gradient between the tumor tissue and the blood [ 25 , 26 ].On the other hand, tumor vasculature carries away the externally applied heat (group III) which is then no longer available to destroy the tumor cells. In this case, the temperature gradient decreases from the tumor to the vascular system.Furthermore, in the presence of hyperthermia, microcirculation in the tumor tissue is more affected than in normal tissue [ 27 ]. In addition, manipulation to the vascular system, and thus oxygenation of the tumor, also appears to play an important role. Although the body's own oxygenated blood is added to the perfusate, the dilution effect together with the clamping time, leads to a relative reduction in perfusion. It is known that hypoxia, in conjunction with a decrease in the pH and energy status of the cell, enhances thermosensitivity [ 28 - 30 ], and this increase correlates with the size of the tumor [ 31 ].Our results have also confirmed that surgical manipulation of the vascular system and perfusion have an influence on the tumor. Overall, the effect of the intravascular application of hyperthermia appears to be mediated by a combination of hypoxia and heat.Conclusions Our results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF. External hyperthermia of 41.5°C tissue temperature for 30 minutes has no impact on tumor growth and angioneogenesis in vivo. Hyperthermic isolated limb perfusion effectively suppresses tumor angioneogenesis respectively tumor growth.
Conclusions
Our results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF. External hyperthermia of 41.5°C tissue temperature for 30 minutes has no impact on tumor growth and angioneogenesis in vivo. Hyperthermic isolated limb perfusion effectively suppresses tumor angioneogenesis respectively tumor growth.Competing interests None declared.
Competing interests
None declared.Authors' contributions JP carried out the treatment and drafted the manuscript. MM carried out the treatment. CS carried out the immunohistochemical studies. JG participated in the design of the study. AD carried out the histological studies. WH participiated in the design of the study. TM participiated in the design and coordination of the study. All authors read and approved the final manuscript.
Authors' contributions
JP carried out the treatment and drafted the manuscript.MM carried out the treatment.CS carried out the immunohistochemical studies.JG participated in the design of the study.AD carried out the histological studies.WH participiated in the design of the study.TM participiated in the design and coordination of the study.All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background Postpartum haemorrhage remains an important cause of maternal death despite treatment with conventional therapy. Uncontrolled studies and one randomised comparison with conventional oxytocics have reported dramatic effects with high-dose misoprostol, usually given rectally, for treatment of postpartum haemorrhage, but this has not been evaluated in a placebo-controlled trial.
Background
Postpartum haemorrhage remains an important cause of maternal death despite treatment with conventional therapy. Uncontrolled studies and one randomised comparison with conventional oxytocics have reported dramatic effects with high-dose misoprostol, usually given rectally, for treatment of postpartum haemorrhage, but this has not been evaluated in a placebo-controlled trial.Methods The study was conducted at East London Hospital Complex, Tembisa and Chris Hani Baragwanath Hospitals, South Africa. Routine active management of the third stage of labour was practised. Women with more than usual postpartum bleeding thought to be related to inadequate uterine contraction were invited to participate, and to sign informed consent. All routine treatment was given from a special 'Postpartum Haemorrhage Trolley'. In addition, participants who consented were enrolled by drawing the next in a series of randomised treatment packs containing either misoprostol 5 × 200 μg or similar placebo, which were given 1 orally, 2 sublingually and 2 rectally.
Methods
The study was conducted at East London Hospital Complex, Tembisa and Chris Hani Baragwanath Hospitals, South Africa. Routine active management of the third stage of labour was practised. Women with more than usual postpartum bleeding thought to be related to inadequate uterine contraction were invited to participate, and to sign informed consent. All routine treatment was given from a special 'Postpartum Haemorrhage Trolley'. In addition, participants who consented were enrolled by drawing the next in a series of randomised treatment packs containing either misoprostol 5 × 200 μg or similar placebo, which were given 1 orally, 2 sublingually and 2 rectally.Results With misoprostol there was a trend to reduced blood loss ≥500 ml in 1 hour after enrolment measured in a flat plastic 'fracture bedpan', the primary outcome (6/117 vs 11/120, relative risk 0.56; 95% confidence interval 0.21 to 1.46). There was no difference in mean blood loss or haemoglobin level on day 1 after birth < 6 g/dl or blood transfusion. Side-effects were increased, namely shivering (63/116 vs 30/118; 2.14, 1.50 to 3.04) and pyrexia > 38.5°C (11/114 vs 2/118; 5.69, 1.29 to 25). In the misoprostol group 3 women underwent hysterectomy of whom 1 died, and there were 2 further maternal deaths.
Results
With misoprostol there was a trend to reduced blood loss ≥500 ml in 1 hour after enrolment measured in a flat plastic 'fracture bedpan', the primary outcome (6/117 vs 11/120, relative risk 0.56; 95% confidence interval 0.21 to 1.46). There was no difference in mean blood loss or haemoglobin level on day 1 after birth < 6 g/dl or blood transfusion. Side-effects were increased, namely shivering (63/116 vs 30/118; 2.14, 1.50 to 3.04) and pyrexia > 38.5°C (11/114 vs 2/118; 5.69, 1.29 to 25). In the misoprostol group 3 women underwent hysterectomy of whom 1 died, and there were 2 further maternal deaths.Conclusions Because of a lower than expected incidence of the primary outcome in the placebo group, the study was underpowered. We could not confirm the dramatic effect of misoprostol reported in several unblinded studies, but the results do not exclude a clinically important effect. Larger studies are needed to assess substantive outcomes and risks before misoprostol enters routine use.
Conclusions
Because of a lower than expected incidence of the primary outcome in the placebo group, the study was underpowered. We could not confirm the dramatic effect of misoprostol reported in several unblinded studies, but the results do not exclude a clinically important effect. Larger studies are needed to assess substantive outcomes and risks before misoprostol enters routine use.

Background Excessive bleeding from the genital tract after birth, or postpartum haemorrhage (PPH) is the major cause of maternal deaths in many low-income countries. The global estimate is 125,000 deaths per year [ 1 ]. In South Africa, 240 of 2,445 maternal deaths reported between 1999 and 2001 were due to postpartum haemorrhage, the third most common cause [ 2 ]. In a community-based study in Senegal, estimates of maternal mortality ratio in three regions ranged from 436 to 852 per 100,000 live births. Two-thirds were due to direct obstetric causes, the commonest being haemorrhage [ 3 ]. In the United Kingdom, the risk of maternal death from haemorrhage is about 1 in 100 000 births [ 4 ]. The potential to save mothers' lives with medical interventions for haemorrhage is thus considerable. There is good evidence that a policy of active management of the third stage of labour [ 5 ], and one component of active management, the routine administration of uterotonics (drugs to contract the uerus) such as oxytocin [ 6 ] or oxytocin and ergometrine [ 7 ], are effective in reducing the risk of postpartum haemorrhage. When postpartum haemorrhage occurs, a number of medical and surgical interventions are used to control the bleeding [ 8 ]. A crucial aspect of both prevention and treatment of postpartum haemorrhage is uterotonic therapy. The most commonly used agents are injectable oxytocin and/or ergometrine. Misoprostol is a methyl ester (a synthetic analogue) of natural prostaglandin E1. It is marketed and registered for use in the prevention and treatment of peptic ulcer disease caused by prostanglandin synthetase inhibitors. Administered orally or sublingually, peak plasma concentrations of misoprostolic acid are achieved in less than 30 minutes [ 9 ]. It is a thermostable drug [ 9 ] and is relatively inexpensive. Administered orally or vaginally, it is an effective agent for the induction of abortion [ 10 ] and of labour [ 11 ]. Misoprostol for routine prevention of postpartum haemorrhage Oral and rectal misoprostol have been used for routine management of the third stage of labour (after the birth). Several small trials have given conflicting results. The main side-effects have been shivering and pyrexia, which are dose-dependent [ 12 ]. In the large WHO Collaborative Trial of Misoprostol in the Management of the Third Stage of Labour [ 13 ] and a systematic review of the topic [ 14 ], blood loss >1,000 ml and use of additional oxytocics (the primary outcomes in the WHO trial) were more frequent with misoprostol than with injectable oxytocics, indicating that injectable oxytocics should remain the drug of choice for routine prophylaxis. On the other hand, blood transfusion was used less frequently in the misoprostol group. This may have been a chance occurrence (it was not a primary outcome for the trial). Secondly, there could be a synergistic pharmacological effect between misoprostol and conventional uterotonics, which were given to most women with increased blood loss. Many women in the misoprostol group thus received misoprostol as well as conventional uterotonics, whereas those in the oxytocin group received only conventional oxytocics. Thirdly, we have suggested in the report of a pharmacokinetic study linked to the WHO trial that the longer time to peak levels of misoprostol (20–30 minutes) than syntocinon (3 minutes) may account for more early blood loss with misoprostol [ 15 ]. This does not exclude the possibility that misoprostol may have an effect on more persistent bleeding. Physiological studies have also shown a more rapid onset of uterine contractions following syntometrine than misoprostol after delivery [ 16 ]. Misoprostol has been widely recommended for the prevention of postpartum haemorrhage when other methods are not available [ 17 ]. Misoprostol for treatment of postpartum haemorrhage Apart from the prophylactic use of misoprostol in the third stage of labour, the therapeutic use of misoprostol for the  treatment  of postpartum haemorrhage has been promoted on the basis of unblinded studies. In a systematic review [ 18 ] we located 6 uncontrolled reports (41 women) [ 19 - 24 ] and one small, unblinded randomised trial [ 25 ]. This method has entered clinical use, particularly in developing countries, without systematic research to document the optimal route and dosage, effectiveness or risks of this treatment. The urgent need for randomised trials of this new intervention has been emphasised [ 26 ]. If effective it could have a major impact on maternal mortality, particulary in under-resourced countries. If not, its use should be discouraged because of the dangers of side-effects, and the risks associated with widespread introduction of misoprostol into health systems in which it might be used for labour induction in inappropriate doses, with the risk of fetal compromise and uterine rupture [ 27 , 28 ]. Route and dosage of misoprostol The most common regimens reported for the treatment of postpartum haemorrhage are 800 [ 23 ] or 1,000 μg [ 19 , 21 ] rectally. We reviewed the literature on the pharmacokinetics of misoprostol administered by various routes [ 18 ]. The oral route has the most rapid uptake, but the shortest duration. The rectal route has slow uptake but prolonged duration. The buccal or sublingual route has rapid uptake, prolonged duration and greatest total bioavailability. In order to improve the rapidity of onset of action and the overall bioavailability, we modified the reported practice of using 1,000 μg rectally, by administering 200 μg orally, 400 μg sublingually and 400 μg rectally. Objective The objective of this study was to determine the side-effects and effectiveness of high-dose misoprostol for the treatment of postpartum haemorrhage by means of a double-blind, placebo-controlled, randomised clinical trial. The hypothesis was that measured blood loss of 500 ml or more in the hour after enrolment would be significantly less frequent in the misoprostol than in the placebo group.
Background
Excessive bleeding from the genital tract after birth, or postpartum haemorrhage (PPH) is the major cause of maternal deaths in many low-income countries. The global estimate is 125,000 deaths per year [ 1 ]. In South Africa, 240 of 2,445 maternal deaths reported between 1999 and 2001 were due to postpartum haemorrhage, the third most common cause [ 2 ]. In a community-based study in Senegal, estimates of maternal mortality ratio in three regions ranged from 436 to 852 per 100,000 live births. Two-thirds were due to direct obstetric causes, the commonest being haemorrhage [ 3 ]. In the United Kingdom, the risk of maternal death from haemorrhage is about 1 in 100 000 births [ 4 ]. The potential to save mothers' lives with medical interventions for haemorrhage is thus considerable.There is good evidence that a policy of active management of the third stage of labour [ 5 ], and one component of active management, the routine administration of uterotonics (drugs to contract the uerus) such as oxytocin [ 6 ] or oxytocin and ergometrine [ 7 ], are effective in reducing the risk of postpartum haemorrhage.When postpartum haemorrhage occurs, a number of medical and surgical interventions are used to control the bleeding [ 8 ]. A crucial aspect of both prevention and treatment of postpartum haemorrhage is uterotonic therapy. The most commonly used agents are injectable oxytocin and/or ergometrine.Misoprostol is a methyl ester (a synthetic analogue) of natural prostaglandin E1. It is marketed and registered for use in the prevention and treatment of peptic ulcer disease caused by prostanglandin synthetase inhibitors. Administered orally or sublingually, peak plasma concentrations of misoprostolic acid are achieved in less than 30 minutes [ 9 ]. It is a thermostable drug [ 9 ] and is relatively inexpensive.Administered orally or vaginally, it is an effective agent for the induction of abortion [ 10 ] and of labour [ 11 ].Misoprostol for routine prevention of postpartum haemorrhage Oral and rectal misoprostol have been used for routine management of the third stage of labour (after the birth). Several small trials have given conflicting results. The main side-effects have been shivering and pyrexia, which are dose-dependent [ 12 ]. In the large WHO Collaborative Trial of Misoprostol in the Management of the Third Stage of Labour [ 13 ] and a systematic review of the topic [ 14 ], blood loss >1,000 ml and use of additional oxytocics (the primary outcomes in the WHO trial) were more frequent with misoprostol than with injectable oxytocics, indicating that injectable oxytocics should remain the drug of choice for routine prophylaxis. On the other hand, blood transfusion was used less frequently in the misoprostol group. This may have been a chance occurrence (it was not a primary outcome for the trial). Secondly, there could be a synergistic pharmacological effect between misoprostol and conventional uterotonics, which were given to most women with increased blood loss. Many women in the misoprostol group thus received misoprostol as well as conventional uterotonics, whereas those in the oxytocin group received only conventional oxytocics. Thirdly, we have suggested in the report of a pharmacokinetic study linked to the WHO trial that the longer time to peak levels of misoprostol (20–30 minutes) than syntocinon (3 minutes) may account for more early blood loss with misoprostol [ 15 ]. This does not exclude the possibility that misoprostol may have an effect on more persistent bleeding. Physiological studies have also shown a more rapid onset of uterine contractions following syntometrine than misoprostol after delivery [ 16 ]. Misoprostol has been widely recommended for the prevention of postpartum haemorrhage when other methods are not available [ 17 ].
Misoprostol for routine prevention of postpartum haemorrhage
Oral and rectal misoprostol have been used for routine management of the third stage of labour (after the birth). Several small trials have given conflicting results. The main side-effects have been shivering and pyrexia, which are dose-dependent [ 12 ]. In the large WHO Collaborative Trial of Misoprostol in the Management of the Third Stage of Labour [ 13 ] and a systematic review of the topic [ 14 ], blood loss >1,000 ml and use of additional oxytocics (the primary outcomes in the WHO trial) were more frequent with misoprostol than with injectable oxytocics, indicating that injectable oxytocics should remain the drug of choice for routine prophylaxis. On the other hand, blood transfusion was used less frequently in the misoprostol group. This may have been a chance occurrence (it was not a primary outcome for the trial). Secondly, there could be a synergistic pharmacological effect between misoprostol and conventional uterotonics, which were given to most women with increased blood loss. Many women in the misoprostol group thus received misoprostol as well as conventional uterotonics, whereas those in the oxytocin group received only conventional oxytocics. Thirdly, we have suggested in the report of a pharmacokinetic study linked to the WHO trial that the longer time to peak levels of misoprostol (20–30 minutes) than syntocinon (3 minutes) may account for more early blood loss with misoprostol [ 15 ]. This does not exclude the possibility that misoprostol may have an effect on more persistent bleeding. Physiological studies have also shown a more rapid onset of uterine contractions following syntometrine than misoprostol after delivery [ 16 ].Misoprostol has been widely recommended for the prevention of postpartum haemorrhage when other methods are not available [ 17 ].Misoprostol for treatment of postpartum haemorrhage Apart from the prophylactic use of misoprostol in the third stage of labour, the therapeutic use of misoprostol for the  treatment  of postpartum haemorrhage has been promoted on the basis of unblinded studies. In a systematic review [ 18 ] we located 6 uncontrolled reports (41 women) [ 19 - 24 ] and one small, unblinded randomised trial [ 25 ]. This method has entered clinical use, particularly in developing countries, without systematic research to document the optimal route and dosage, effectiveness or risks of this treatment. The urgent need for randomised trials of this new intervention has been emphasised [ 26 ]. If effective it could have a major impact on maternal mortality, particulary in under-resourced countries. If not, its use should be discouraged because of the dangers of side-effects, and the risks associated with widespread introduction of misoprostol into health systems in which it might be used for labour induction in inappropriate doses, with the risk of fetal compromise and uterine rupture [ 27 , 28 ].
Misoprostol for treatment of postpartum haemorrhage
Apart from the prophylactic use of misoprostol in the third stage of labour, the therapeutic use of misoprostol for the  treatment  of postpartum haemorrhage has been promoted on the basis of unblinded studies. In a systematic review [ 18 ] we located 6 uncontrolled reports (41 women) [ 19 - 24 ] and one small, unblinded randomised trial [ 25 ]. This method has entered clinical use, particularly in developing countries, without systematic research to document the optimal route and dosage, effectiveness or risks of this treatment. The urgent need for randomised trials of this new intervention has been emphasised [ 26 ]. If effective it could have a major impact on maternal mortality, particulary in under-resourced countries. If not, its use should be discouraged because of the dangers of side-effects, and the risks associated with widespread introduction of misoprostol into health systems in which it might be used for labour induction in inappropriate doses, with the risk of fetal compromise and uterine rupture [ 27 , 28 ].Route and dosage of misoprostol The most common regimens reported for the treatment of postpartum haemorrhage are 800 [ 23 ] or 1,000 μg [ 19 , 21 ] rectally. We reviewed the literature on the pharmacokinetics of misoprostol administered by various routes [ 18 ]. The oral route has the most rapid uptake, but the shortest duration. The rectal route has slow uptake but prolonged duration. The buccal or sublingual route has rapid uptake, prolonged duration and greatest total bioavailability. In order to improve the rapidity of onset of action and the overall bioavailability, we modified the reported practice of using 1,000 μg rectally, by administering 200 μg orally, 400 μg sublingually and 400 μg rectally.
Route and dosage of misoprostol
The most common regimens reported for the treatment of postpartum haemorrhage are 800 [ 23 ] or 1,000 μg [ 19 , 21 ] rectally. We reviewed the literature on the pharmacokinetics of misoprostol administered by various routes [ 18 ]. The oral route has the most rapid uptake, but the shortest duration. The rectal route has slow uptake but prolonged duration. The buccal or sublingual route has rapid uptake, prolonged duration and greatest total bioavailability. In order to improve the rapidity of onset of action and the overall bioavailability, we modified the reported practice of using 1,000 μg rectally, by administering 200 μg orally, 400 μg sublingually and 400 μg rectally.Objective The objective of this study was to determine the side-effects and effectiveness of high-dose misoprostol for the treatment of postpartum haemorrhage by means of a double-blind, placebo-controlled, randomised clinical trial. The hypothesis was that measured blood loss of 500 ml or more in the hour after enrolment would be significantly less frequent in the misoprostol than in the placebo group.
Objective
The objective of this study was to determine the side-effects and effectiveness of high-dose misoprostol for the treatment of postpartum haemorrhage by means of a double-blind, placebo-controlled, randomised clinical trial. The hypothesis was that measured blood loss of 500 ml or more in the hour after enrolment would be significantly less frequent in the misoprostol than in the placebo group.Methods The study was conducted at the East London Hospital Complex (Frere Maternity and Cecilia Makiwane Hospitals, Eastern Cape) and Tembisa and Chris Hani Baragwanath Hospitals, Gauteng, South Africa, between Jan 2002 and Dec 2003. All are busy referral hospitals. Treatment packs were prepared independently, ordered in computer-generated random sequence and numbered consecutively. The packs contained either misoprostol 5 × 200 μg or inactive placebo. The treatment sequence was kept sealed, and the code broken only after complete entry and checking of all trial data. A confidential interim analysis, blind to which group was which, was performed by AMG after the first 100 enrolments. Evidence of clear benefit or harm for either group would have prompted stopping the trial, which was not the case. Women in labour were given basic information about the trial in the form of notices in the labour rooms (Table 2 [see  additional file 1
]). Management of labour 3 rd  stage was routine active management with oxytocin 10 units or syntometrine one ampoule soon after the birth. Women aged 18 or more with bleeding judged by the clinician to be more than expected at least 10 minutes after delivery, and thought to be due to uterine atony and requiring additional uterotonic therapy, were given all the routine treatment for PPH (intravenous infusion, uterotonics, etc) from a special 'PPH Trolley' kept in the labour ward. The uterotonics used were oxytocin by intravenous infusion, and/or oxytocin/ergometrine, at the discretion of the attending clinician. Prostaglandin preparations such as sulprostone were not routinely available in the labour wards, and were not part of routine management of postpartum haemorrhage in the participating units. Once all emergency treatment was instituted, and if the women were in a position to give fully informed consent, they were given detailed information about the trial and asked whether they wished to participate. Information about the trial was given verbally and in writing in English or Xhosa (Table 3 [see  additional file 2
]). Those who gave written consent were enrolled in the trial. Baseline data were documented. The next treatment pack containing either misoprostol 5 × 200 μg or inactive placebo was drawn and the number recorded. In addition to routine management, misoprostol or placebo (an inactive base) were given, 1 orally, 2 buccally/sublingually and 2 rectally, and the time recorded. A low-profile plastic 'fracture bedpan' was placed under the woman's buttocks. In previous studies we have shown this to be an efficient method of collecting ongoing blood loss with very little spillage [ 29 ]. Any small swabs soaked with blood were dropped into the bedpan. After 1 hour, the blood collected in the bedpan was measured in a graduated measuring jug, the temperature was measured sublingually with a mercury thermometer, and any shivering was subjectively recorded as 'mild' (not causing any distress), 'moderate' or 'severe'. All other management was by hospital staff according to the hospital routine for the management of postpartum haemorrhage. After 24 hours, blood was collected for haemoglobin estimation and the hospital records checked for use of additional uterotonics and any other complications such as blood transfusion. The primary outcome measures were specified prior to commencing the study: (1) measured blood loss ≥500 ml in 1 hour after enrolment; (2) mean measured blood loss in 1 hour after enrolment; (3) haemoglobin level day 1 after birth <6 g/dl or blood transfusion; (4) Side-effects (pyrexia 38.5°C or more, moderate or severe shivering 1 hour after enrolment). Secondary outcome measures were: (1) blood loss ≥1,000 ml in 1 hour after enrolment; (2) blood transfusion; (3) haemoglobin level 1 day after birth <8 g/dl or blood transfusion; (4) additional uterotonic given after enrolment; (5) manual removal of the placenta; (6) evacuation of retained products of conception; (7) hysterectomy; (8) maternal death. Sample size calculation In the WHO misoprostol trial control group (routine syntocinon), significant postpartum haemorrhage (blood loss >1000 ml) occurred in 2.8% of women. Of these 25% lost >1,500 ml. To reduce blood loss >500 ml after enrolment from 25% to 10%, would require 112 per group (α = 95%, β = 80%). Data were collected on a data form, entered onto an excel spreadsheet, checked for accuracy, then analysed using Epi-info 2002 (United States Department of Health and Human Services, Centres for Diseases Control and Prevention, Epidemiology Program Office) and Review Manager (RevMan) [Computer program] version 4.2 for Windows. Oxford, England: The Cochrane Collaboration, 2003. Results were expressed as relative risks or mean difference with 95% confidence intervals. Analysis was by intention to treat. Ethical considerations All women enrolled in the trial received all the conventional management available for postpartum haemorrhage, in addition to the trial medication (misoprostol or placebo). Rapid conventional therapy was facilitated by the ready availability of the 'PPH trolley' with all the necessary materials. This trolley was also available for women not enrolled in the trial. Participation was limited to women who were in a position to give fully informed consent. Ethical approval was given by the Committee for Research on Human Subjects, University of the Witwatersrand, and the Ethics Committee, East London Hospital Complex. The trial complied with the Helsinki Declaration for research on human subjects.
Methods
The study was conducted at the East London Hospital Complex (Frere Maternity and Cecilia Makiwane Hospitals, Eastern Cape) and Tembisa and Chris Hani Baragwanath Hospitals, Gauteng, South Africa, between Jan 2002 and Dec 2003. All are busy referral hospitals.Treatment packs were prepared independently, ordered in computer-generated random sequence and numbered consecutively. The packs contained either misoprostol 5 × 200 μg or inactive placebo.The treatment sequence was kept sealed, and the code broken only after complete entry and checking of all trial data.A confidential interim analysis, blind to which group was which, was performed by AMG after the first 100 enrolments. Evidence of clear benefit or harm for either group would have prompted stopping the trial, which was not the case.Women in labour were given basic information about the trial in the form of notices in the labour rooms (Table 2 [see  additional file 1
]). Management of labour 3 rd  stage was routine active management with oxytocin 10 units or syntometrine one ampoule soon after the birth. Women aged 18 or more with bleeding judged by the clinician to be more than expected at least 10 minutes after delivery, and thought to be due to uterine atony and requiring additional uterotonic therapy, were given all the routine treatment for PPH (intravenous infusion, uterotonics, etc) from a special 'PPH Trolley' kept in the labour ward. The uterotonics used were oxytocin by intravenous infusion, and/or oxytocin/ergometrine, at the discretion of the attending clinician. Prostaglandin preparations such as sulprostone were not routinely available in the labour wards, and were not part of routine management of postpartum haemorrhage in the participating units.Once all emergency treatment was instituted, and if the women were in a position to give fully informed consent, they were given detailed information about the trial and asked whether they wished to participate. Information about the trial was given verbally and in writing in English or Xhosa (Table 3 [see  additional file 2
]). Those who gave written consent were enrolled in the trial. Baseline data were documented. The next treatment pack containing either misoprostol 5 × 200 μg or inactive placebo was drawn and the number recorded. In addition to routine management, misoprostol or placebo (an inactive base) were given, 1 orally, 2 buccally/sublingually and 2 rectally, and the time recorded.A low-profile plastic 'fracture bedpan' was placed under the woman's buttocks. In previous studies we have shown this to be an efficient method of collecting ongoing blood loss with very little spillage [ 29 ]. Any small swabs soaked with blood were dropped into the bedpan. After 1 hour, the blood collected in the bedpan was measured in a graduated measuring jug, the temperature was measured sublingually with a mercury thermometer, and any shivering was subjectively recorded as 'mild' (not causing any distress), 'moderate' or 'severe'.All other management was by hospital staff according to the hospital routine for the management of postpartum haemorrhage.After 24 hours, blood was collected for haemoglobin estimation and the hospital records checked for use of additional uterotonics and any other complications such as blood transfusion.The primary outcome measures were specified prior to commencing the study: (1) measured blood loss ≥500 ml in 1 hour after enrolment; (2) mean measured blood loss in 1 hour after enrolment; (3) haemoglobin level day 1 after birth <6 g/dl or blood transfusion; (4) Side-effects (pyrexia 38.5°C or more, moderate or severe shivering 1 hour after enrolment).Secondary outcome measures were: (1) blood loss ≥1,000 ml in 1 hour after enrolment; (2) blood transfusion; (3) haemoglobin level 1 day after birth <8 g/dl or blood transfusion; (4) additional uterotonic given after enrolment; (5) manual removal of the placenta; (6) evacuation of retained products of conception; (7) hysterectomy; (8) maternal death.Sample size calculation In the WHO misoprostol trial control group (routine syntocinon), significant postpartum haemorrhage (blood loss >1000 ml) occurred in 2.8% of women. Of these 25% lost >1,500 ml. To reduce blood loss >500 ml after enrolment from 25% to 10%, would require 112 per group (α = 95%, β = 80%). Data were collected on a data form, entered onto an excel spreadsheet, checked for accuracy, then analysed using Epi-info 2002 (United States Department of Health and Human Services, Centres for Diseases Control and Prevention, Epidemiology Program Office) and Review Manager (RevMan) [Computer program] version 4.2 for Windows. Oxford, England: The Cochrane Collaboration, 2003. Results were expressed as relative risks or mean difference with 95% confidence intervals. Analysis was by intention to treat.
Sample size calculation
In the WHO misoprostol trial control group (routine syntocinon), significant postpartum haemorrhage (blood loss >1000 ml) occurred in 2.8% of women. Of these 25% lost >1,500 ml. To reduce blood loss >500 ml after enrolment from 25% to 10%, would require 112 per group (α = 95%, β = 80%).Data were collected on a data form, entered onto an excel spreadsheet, checked for accuracy, then analysed using Epi-info 2002 (United States Department of Health and Human Services, Centres for Diseases Control and Prevention, Epidemiology Program Office) and Review Manager (RevMan) [Computer program] version 4.2 for Windows. Oxford, England: The Cochrane Collaboration, 2003. Results were expressed as relative risks or mean difference with 95% confidence intervals. Analysis was by intention to treat.Ethical considerations All women enrolled in the trial received all the conventional management available for postpartum haemorrhage, in addition to the trial medication (misoprostol or placebo). Rapid conventional therapy was facilitated by the ready availability of the 'PPH trolley' with all the necessary materials. This trolley was also available for women not enrolled in the trial. Participation was limited to women who were in a position to give fully informed consent. Ethical approval was given by the Committee for Research on Human Subjects, University of the Witwatersrand, and the Ethics Committee, East London Hospital Complex. The trial complied with the Helsinki Declaration for research on human subjects.
Ethical considerations
All women enrolled in the trial received all the conventional management available for postpartum haemorrhage, in addition to the trial medication (misoprostol or placebo). Rapid conventional therapy was facilitated by the ready availability of the 'PPH trolley' with all the necessary materials. This trolley was also available for women not enrolled in the trial. Participation was limited to women who were in a position to give fully informed consent. Ethical approval was given by the Committee for Research on Human Subjects, University of the Witwatersrand, and the Ethics Committee, East London Hospital Complex. The trial complied with the Helsinki Declaration for research on human subjects.Results Altogether 244 women were enrolled in the trial. The pack numbers for 6 women were incompletely filled in on the data sheets. The group allocation of these women was therefore unknown, and they could not be included in the analysis. Of these 6 women, the highest measured blood loss after enrolment was 220 ml; none had pyrexia >37.5°C, one had moderate shivering, one had a blood transfusion and none had day 1 haemoglobin level below 8 g/dl or other complications. There was no reason to expect that these exclusions occurred in any selective way which would introduce bias into the final samples, nor could their results have materially altered the conclusions of the study. All the remaining 238 women were included in the analyses. The baseline characteristics are shown in table  1 . As would be expected in a randomised trial, the groups were well matched. Parity was not recorded at all hospitals, but was similar between groups where recorded (misoprostol mean 1.61, SD 1.14; placebo 1.75, SD 1.26). Table 1 Comparison of outcomes between women who received misoprostol or placebo, expressed as proportions (percent) or mean values [standard deviation]. Differences are expressed as relative risks or mean differences, with 95% confidence intervals. Misoprostol (117) Placebo (121) Baseline characteristics n n Age (years) 116 27.1 [6.0] 119 26.2 [6.2] Ergometrine before enrolment 106 36 (34%) 104 34 (33%) Oxytocin ≥20 U before enrolment 116 82 (71%) 117 78 (67%) Primary outcomes: RR/ MD 95% Conf. interval Blood loss ≥500 ml* 117 6 (5.1%) 120 11 (9.2%) 0.56 0.21 to 1.46 Mean blood loss* [SD] (ml) 117 168 [163] 120 176 [173] -8 -51 to 35 24 hour Haemoglobin <6 g/dL or blood transfusion 110 20 (18.2%) 116 17 (14.7%) 1.24 0.69 to 2.24 Pyrexia at 1 hour ≥38, 5°C 114 11 (9.6%) 118 2 (1.7%) 5.69 1.29 to 25 Shivering at 1 hour ≥ moderate 116 63 (54%) 118 30 (25%) 2.14 1.50 to 3.04 Secondary outcomes: Blood loss ≥ 1,000 ml* 117 1 (0.85%) 120 0 (0%) Blood transfusion 115 19 (17%) 119 15 (13%) 1.31 0.70 to 2.45 24 hour haemoglobin <8 g/dL or blood transfusion 110 43 (39%) 116 37 (32%) 1.23 0.86 to 1.75 Additional uterotonic after enrolment 111 63 (57%) 112 63 (56%) 1.01 0.80 to 1.27 Manual removal of the placenta 117 1 (0.85%) 121 4 (3.3%) 0.26 0.03 to 2.28 Evacuation of retained products of conception 117 2 (1.7%) 121 0 (0%) Hysterectomy** 117 3 (2.6%) 121 0 (0%) Maternal death** 117 3 (2.6%) 121 0 (0%) RR = relative risk; MD = mean difference; Conf = confidence; SD = standard deviation *Measured, within 1 hour after enrolment ** One woman died after hysterectomy and is counted in both outcomes The outcomes are shown in table  1 . In the placebo group, the primary outcome (measured blood loss within 1 hour after enrolment of 500 ml or more) was less frequent (9.2%) than the 25% prediction on which the sample size calculation was based. This may have been because the criteria for enrolment were loosely defined as 'more than expected blood loss', resulting in the enrolment of women with less severe blood loss than anticipated. The fact that enrolment was limited to women who were able to received detailed information about the study, may have caused selection of less severe cases. Although the intention was to enrol women who had not responded to conventional therapy, the time taken for counselling of the women and obtaining informed consent may have resulted in some women responding to the primary treatment, and therefore the low rate of ongoing blood loss in the placebo group. The study was thus underpowered to detect a statistically significant reduction in the primary outcome. With misoprostol there was a trend to reduced blood loss ≥500 ml in 1 hour after enrolment (6/117 vs 11/120, relative risk 0.56; 95% confidence interval 0.21 to 1.46). Other proxy estimations of blood loss showed no significant differences between the groups. The well-known side-effects of misoprostol, pyrexia and shivering, were significantly more frequent in the misoprostol than the placebo group. Serious morbidity or mortality occurred in 5 women, all from the misoprostol group. Two women who continued to bleed despite conservative therapy underwent abdominal hysterectomy and recovered well. There were 3 maternal deaths: Case 1 A 22-year old woman with one previous pregnancy developed postpartum haemorrhage after vaginal delivery, managed with a 40-unit oxytocin infusion. She was enrolled and received the trial medication 85 minutes after delivery. Thereafter she received a second 40-unit oxytocin drip, oxytocin/ergometrine (5 units/0.5 mg) and intravenous cyclokapron. In the hour after randomisation, measured blood loss was 125 ml. Subsequently bleeding continued and a sub-total hysterectomy was performed. Coagulopathy developed, bleeding continued through an abdominal drain, and she died 2 days after delivery despite re-laparotomy and multiple blood product transfusions. Case 2 A 32-year-old woman, para 2, gravida 4, delivered normally after labour was induced with misoprostol, 25 μg vaginally and 4 doses of 50 μg orally. Before enrolment she received oxytocin10 units, ergometrine 0.5 mg and a 20-unit oxytocin infusion. She was enrolled and received the trial medication 140 minutes after delivery. After enrolment she received a further 40-unit oxytocin infusion. Measured blood loss in the hour after enrolment was 380 ml. Bleeding continued and a blood transfusion was commenced. Six and a half hours after delivery she suffered a cardiac arrest and was resuscitated. Examination in theatre found the uterus to be empty and intact. The main source of bleeding appeared to be a torn cervix, which was sutured. At 7.5 hours after delivery she suffered a second cardiac arrest and could not be resuscitated. The estimated total blood loss was 3,000 ml. Case 3 A woman with one previous delivery by caesarean section, developed postpartum haemorrhage of about 500 ml after a vaginal delivery. She was enrolled 85 minutes after delivery. Measured blood loss over the next hour was 425 ml, after which she developed hypotension and cardiorespiratory arrest. As no post-mortem examination was performed, the possibility of internal bleeding from a dehisced caesarean section scar could neither be confirmed nor excluded. Unresponsive uterine atony was therefore not confirmed as the main cause of any of the deaths.
Results
Altogether 244 women were enrolled in the trial. The pack numbers for 6 women were incompletely filled in on the data sheets. The group allocation of these women was therefore unknown, and they could not be included in the analysis. Of these 6 women, the highest measured blood loss after enrolment was 220 ml; none had pyrexia >37.5°C, one had moderate shivering, one had a blood transfusion and none had day 1 haemoglobin level below 8 g/dl or other complications. There was no reason to expect that these exclusions occurred in any selective way which would introduce bias into the final samples, nor could their results have materially altered the conclusions of the study. All the remaining 238 women were included in the analyses.The baseline characteristics are shown in table  1 . As would be expected in a randomised trial, the groups were well matched. Parity was not recorded at all hospitals, but was similar between groups where recorded (misoprostol mean 1.61, SD 1.14; placebo 1.75, SD 1.26).Comparison of outcomes between women who received misoprostol or placebo, expressed as proportions (percent) or mean values [standard deviation]. Differences are expressed as relative risks or mean differences, with 95% confidence intervals.RR = relative risk; MD = mean difference; Conf = confidence; SD = standard deviation *Measured, within 1 hour after enrolment ** One woman died after hysterectomy and is counted in both outcomesThe outcomes are shown in table  1 . In the placebo group, the primary outcome (measured blood loss within 1 hour after enrolment of 500 ml or more) was less frequent (9.2%) than the 25% prediction on which the sample size calculation was based. This may have been because the criteria for enrolment were loosely defined as 'more than expected blood loss', resulting in the enrolment of women with less severe blood loss than anticipated. The fact that enrolment was limited to women who were able to received detailed information about the study, may have caused selection of less severe cases. Although the intention was to enrol women who had not responded to conventional therapy, the time taken for counselling of the women and obtaining informed consent may have resulted in some women responding to the primary treatment, and therefore the low rate of ongoing blood loss in the placebo group. The study was thus underpowered to detect a statistically significant reduction in the primary outcome. With misoprostol there was a trend to reduced blood loss ≥500 ml in 1 hour after enrolment (6/117 vs 11/120, relative risk 0.56; 95% confidence interval 0.21 to 1.46).Other proxy estimations of blood loss showed no significant differences between the groups. The well-known side-effects of misoprostol, pyrexia and shivering, were significantly more frequent in the misoprostol than the placebo group.Serious morbidity or mortality occurred in 5 women, all from the misoprostol group. Two women who continued to bleed despite conservative therapy underwent abdominal hysterectomy and recovered well. There were 3 maternal deaths:Case 1 A 22-year old woman with one previous pregnancy developed postpartum haemorrhage after vaginal delivery, managed with a 40-unit oxytocin infusion. She was enrolled and received the trial medication 85 minutes after delivery. Thereafter she received a second 40-unit oxytocin drip, oxytocin/ergometrine (5 units/0.5 mg) and intravenous cyclokapron. In the hour after randomisation, measured blood loss was 125 ml. Subsequently bleeding continued and a sub-total hysterectomy was performed. Coagulopathy developed, bleeding continued through an abdominal drain, and she died 2 days after delivery despite re-laparotomy and multiple blood product transfusions.
Case 1
A 22-year old woman with one previous pregnancy developed postpartum haemorrhage after vaginal delivery, managed with a 40-unit oxytocin infusion. She was enrolled and received the trial medication 85 minutes after delivery. Thereafter she received a second 40-unit oxytocin drip, oxytocin/ergometrine (5 units/0.5 mg) and intravenous cyclokapron. In the hour after randomisation, measured blood loss was 125 ml. Subsequently bleeding continued and a sub-total hysterectomy was performed. Coagulopathy developed, bleeding continued through an abdominal drain, and she died 2 days after delivery despite re-laparotomy and multiple blood product transfusions.Case 2 A 32-year-old woman, para 2, gravida 4, delivered normally after labour was induced with misoprostol, 25 μg vaginally and 4 doses of 50 μg orally. Before enrolment she received oxytocin10 units, ergometrine 0.5 mg and a 20-unit oxytocin infusion. She was enrolled and received the trial medication 140 minutes after delivery. After enrolment she received a further 40-unit oxytocin infusion. Measured blood loss in the hour after enrolment was 380 ml. Bleeding continued and a blood transfusion was commenced. Six and a half hours after delivery she suffered a cardiac arrest and was resuscitated. Examination in theatre found the uterus to be empty and intact. The main source of bleeding appeared to be a torn cervix, which was sutured. At 7.5 hours after delivery she suffered a second cardiac arrest and could not be resuscitated. The estimated total blood loss was 3,000 ml.
Case 2
A 32-year-old woman, para 2, gravida 4, delivered normally after labour was induced with misoprostol, 25 μg vaginally and 4 doses of 50 μg orally. Before enrolment she received oxytocin10 units, ergometrine 0.5 mg and a 20-unit oxytocin infusion. She was enrolled and received the trial medication 140 minutes after delivery. After enrolment she received a further 40-unit oxytocin infusion. Measured blood loss in the hour after enrolment was 380 ml. Bleeding continued and a blood transfusion was commenced. Six and a half hours after delivery she suffered a cardiac arrest and was resuscitated. Examination in theatre found the uterus to be empty and intact. The main source of bleeding appeared to be a torn cervix, which was sutured. At 7.5 hours after delivery she suffered a second cardiac arrest and could not be resuscitated. The estimated total blood loss was 3,000 ml.Case 3 A woman with one previous delivery by caesarean section, developed postpartum haemorrhage of about 500 ml after a vaginal delivery. She was enrolled 85 minutes after delivery. Measured blood loss over the next hour was 425 ml, after which she developed hypotension and cardiorespiratory arrest. As no post-mortem examination was performed, the possibility of internal bleeding from a dehisced caesarean section scar could neither be confirmed nor excluded. Unresponsive uterine atony was therefore not confirmed as the main cause of any of the deaths.
Case 3
A woman with one previous delivery by caesarean section, developed postpartum haemorrhage of about 500 ml after a vaginal delivery. She was enrolled 85 minutes after delivery. Measured blood loss over the next hour was 425 ml, after which she developed hypotension and cardiorespiratory arrest. As no post-mortem examination was performed, the possibility of internal bleeding from a dehisced caesarean section scar could neither be confirmed nor excluded.Unresponsive uterine atony was therefore not confirmed as the main cause of any of the deaths.Discussion Pyrexia and shivering were common side-effects of misoprostol, as found in previous studies. Three women, all in the misoprostol group, had severe pyrexia >40°C. Previous studies of high-dose misoprostol for the treatment of postpartum haemorrhage have mostly used the rectal route, and none has had sufficient numbers to be likely to detect rare adverse outcomes such as severe pyrexia. These side-effects may be related to the rapid absorption of misoprostol given orally, and the rapid absorption and high bioavailability when given sublingually. Because of these serious side-effects, we would recommend that for future trials the dose be reduced. Several previous unblinded studies have reported dramatic effects of misoprostol when used to treat postpartum haemorrhage. In 6 observational studies [ 19 - 24 ], 41 women with postpartum haemorrhage were treated with misoprostol 1,000 μg rectally (32 women), 200 μg rectally (5 women), 200 μg orally 2-hourly (2 women) or 800 μg intrauterine (2 women). In all but 2 of the women (who received 1,000 μg rectally), a prompt response to the treatment was reported. In a single blind randomised trial [ 25 ] subjective prompt cessation of bleeding was reported in 30/32 women who received misoprostol 800 μg rectally compared with only 21/32 who received oxytocin 5 units plus ergometrine 0.5 mg intramuscularly.
Discussion
Pyrexia and shivering were common side-effects of misoprostol, as found in previous studies. Three women, all in the misoprostol group, had severe pyrexia >40°C. Previous studies of high-dose misoprostol for the treatment of postpartum haemorrhage have mostly used the rectal route, and none has had sufficient numbers to be likely to detect rare adverse outcomes such as severe pyrexia. These side-effects may be related to the rapid absorption of misoprostol given orally, and the rapid absorption and high bioavailability when given sublingually. Because of these serious side-effects, we would recommend that for future trials the dose be reduced.Several previous unblinded studies have reported dramatic effects of misoprostol when used to treat postpartum haemorrhage. In 6 observational studies [ 19 - 24 ], 41 women with postpartum haemorrhage were treated with misoprostol 1,000 μg rectally (32 women), 200 μg rectally (5 women), 200 μg orally 2-hourly (2 women) or 800 μg intrauterine (2 women). In all but 2 of the women (who received 1,000 μg rectally), a prompt response to the treatment was reported. In a single blind randomised trial [ 25 ] subjective prompt cessation of bleeding was reported in 30/32 women who received misoprostol 800 μg rectally compared with only 21/32 who received oxytocin 5 units plus ergometrine 0.5 mg intramuscularly.Conclusions In our double blind trial we have been unable to confirm the dramatic effects of misoprostol reported previously in unblinded studies. Our results, however, are consistent with anything from a large beneficial effect to a smaller adverse effect (relative risk of additional blood loss of 500 ml or more anywhere between a reduction of 79% and an increase of 46% with misoprostol). It is important that this possible benefivial effect be assessed by sufficiently powered randomised trials before the treatment enters routine clinical use.
Conclusions
In our double blind trial we have been unable to confirm the dramatic effects of misoprostol reported previously in unblinded studies. Our results, however, are consistent with anything from a large beneficial effect to a smaller adverse effect (relative risk of additional blood loss of 500 ml or more anywhere between a reduction of 79% and an increase of 46% with misoprostol). It is important that this possible benefivial effect be assessed by sufficiently powered randomised trials before the treatment enters routine clinical use.List of abbreviations PPH: postpartum haemorrhage; SD: standard deviation
List of abbreviations
PPH: postpartum haemorrhage; SD: standard deviationCompeting interests None known.
Competing interests
None known.Authors' contributions GJH prepared the first draft of the protocol and the final manuscript, and oversaw the clinical work CN assisted with the design of the protocol and data collection sheet, initiated enrolment at two sites, supervised some of the data collection and contributed to the manuscript SF, ZM, LM, MS and ZJ commented on the protocol, conducted the clinical procedures and collected data. SF entered and collated the data BM undertook the literature search and commented on the protocol. GW contributed to the protocol development AMG contributed to the protocol development and undertook the blinded interim analysis All authors read and approved the final manuscript
Authors' contributions
GJH prepared the first draft of the protocol and the final manuscript, and oversaw the clinical workCN assisted with the design of the protocol and data collection sheet, initiated enrolment at two sites, supervised some of the data collection and contributed to the manuscriptSF, ZM, LM, MS and ZJ commented on the protocol, conducted the clinical procedures and collected data.SF entered and collated the dataBM undertook the literature search and commented on the protocol.GW contributed to the protocol developmentAMG contributed to the protocol development and undertook the blinded interim analysisAll authors read and approved the final manuscriptAdditional files Additional file 1 - Table 2. Notice informing women about the trial, MS Word file: HofmeyrFile1.doc Additional file 2- Table 3: Information and consent form. MS Word file: HofmeyrFile2.doc
Additional files
Additional file 1 - Table 2. Notice informing women about the trial, MS Word file: HofmeyrFile1.docAdditional file 2- Table 3: Information and consent form. MS Word file: HofmeyrFile2.docPre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:Supplementary Material Additional File 1 Table 2. Notice informing women about the trial Click here for file Additional File 2 Table 3: Information and consent form Click here for file
Supplementary Material
Additional File 1
Table 2. Notice informing women about the trialClick here for fileAdditional File 2
Table 3: Information and consent formClick here for file