Extracorporeal perfusion of the human uterus as an experimental model in gynaecology and reproductive medicine

O. Richter1,5, E. Wardelmann2, F. Dombrowski2, C. Schneider3, R. Kiel1, K. Wilhelm4, J. Schmolling1, M. Kupka1, H. van der Ven1 and D. Krebs1

1 Department of Obstetrics and Gynaecology, 2 Department of Pathology, 3 Department of Cardio-Thoracic Surgery and 4 Department of Radiology, University of Bonn, Faculty of Medicine, Sigmund-Freud-Str. 25, 53105 Bonn, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental perfusion of various organs has primarily been used in transplantation medicine to study the physiology, pathophysiology and metabolism of tissues and cells. The purpose of this study was to establish an experimental model for the extracorporeal perfusion of the human uterus with recirculation of a modified, oxygenated Krebs–Henselait solution, in comparison with a non-recirculating perfusion system. With consent of the patients we obtained 25 uteri after standard hysterectomy. We performed an isovolumetric exchange of the perfusion medium at different intervals from 1 to 6 h and examined pH, pO2, pCO2, lactate, lactate dehydrogenase and creatine kinase by taking arterial and venous samples every hour for 24 h. We found the perfusions to be adequate when maintaining flow rates at 15–35 ml/min and at pressures ranging from 70 to 130 mmHg. Isovolumetric exchange of the perfusate every 3–4 h was the maximum interval to keep pH, the arterio-venous gradients of pO2 and pCO2, and the other biochemical parameters in physiological ranges. Examination by light and electron microscopy showed well-preserved features of myometrial and endometrial tissue. However, a 6 h exchanging interval led to increasing hypoxic and cytolytic parameters during the whole perfusion period. X-ray studies using digital subtraction angiography and perfusion studies with methylene blue demonstrated the homogeneous distribution of the perfusion fluid throughout the entire organ.

Key words: extracorporeal perfusion/human uterus/physiological recirculation system


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental perfusion models of various organs are a valuable research tool in transplantation medicine and have primarily been used to study the physiology, pathophysiology, and metabolism of tissues and cells (Kamada et al., 1980Go; van der Wjik et al., 1980Go; Toledo-Pereyra et al., 1982). Extracorporeal perfusion of the human uterus, in particular, offers a new experimental approach to studies of the myometrium, the endometrium and the uterine vasculature. As demonstrated in other organs such as heart, liver and kidney, it can be expected that uterine perfusion with oxygenated media through the uterine arteries could maintain tissue viability and responsiveness to hormones for prolonged periods of time (Iwasaki et al., 1991Go; Marinelli et al., 1991Go; Bresticker et al., 1992Go; Ohura et al., 1995Go; Balden et al., 1997Go). First investigations have shown that physiological organ function of an extracorporeal perfused human uterus for longer periods following hysterectomy depends on a sufficient preservation of cell integrity (Bulletti et al., 1986Go). Insufficient perfusion followed by ischaemia causes cell damage in the perfused organ and consecutive increase of hypoxic and cytolytic parameters.

The purpose of the present study was to establish a system for extracorporeal perfusion of a human uterus with recirculation of the perfusate and regeneration by interval exchange to guarantee tissue vitality of the perfused organ for up to 24 h.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 25 uteri obtained by abdominal or vaginal hysterectomy for benign reasons was studied. Ethical approval by the Ethik-Kommission of the University of Bonn was given under the number 146/97. Every patient signed an information sheet and gave signed consent to the investigation. Patients with previous multiple abdominal surgeries, pelvic endometriosis, pelvic inflammatory disease, large fibroids, adenomyosis (>500 g) or malignant diseases were excluded. The age of each patient and the stage of the cycle was established by anamnestic means; in case of doubt hormone serum plasma concentrations were investigated.

The surgical specimens were carefully prepared, avoiding traction and laceration in order to get vascular stumps suitable for catheterization. Organ weights were always measured immediately before and after perfusion. After cannulation of both uterine arteries with 14 G bulb-headed cannulas, the uterus was flushed bilaterally with a modified heparinized Krebs–Henseleit bicarbonate buffer at 37°C to remove blood products and cell detritus caused by the operation procedures. The composition of the perfusion medium was as follows: pH 7.40, NaCl 6.89 g/l, KCl 0.37 g/l, MgSO4•7H2O 0.25 g/l, CaCl2•2H2O 0.37 g/l, KH2PO4 0.14 g/l, NaHCO3 2.35 g/l, D(+)-glucose 1.50 g/l, saccharose 0.70 g/l, glutatione 0.005 g/l, 1,4-dithiothreitol 0.10 g/l, 100 IE insulin bolus at the beginning of perfusion, refobacin 40 mg/l perfusate, heparin 150 IU/ml. Following this ‘flush-perfusion’ the uterus was connected with the recirculating perfusion system and placed in a cabinet (baby incubator, Dräger, Köln, Germany) at 37°C with humidity values of ~97% on wire mesh above a cylindrical teflon receptacle for perfusate collection under sterile conditions. Oxygenation of the perfusion medium was accomplished by a membrane oxygenator (Jostra, Hirrlingen, Germany) flushed with 95% O2 and 5% CO2 and monitored by gas flowmeters. The perfusate was moved through silicone rubber tubing by separate roller pumps (Storz, Tuttlingen, Germany) monitoring flow and pressure rates throughout the perfusion period.

The recirculation was achieved by pumping the perfusion medium from the teflon receptacle back to the perfusate reservoir with another separate roller pump. In order to coordinate the effluate volume and the recirculation flow rate, a regulating element triggered the recirculation roller pump by measuring the filling level of the perfusate in the collecting receptacle. For the description of the complete perfusion system see Figure 1Go.



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Figure 1. Schematic of the perfusion system. (1) Reservoir for perfusion buffer. (2) Membrane oxygenator. (3) Gas mixture with O2, CO2 and flowmeters. (4) Gas flowmeter. (5) Filter with bubble trap. (6, 16, 17) Roller pumps with pressure transducers and flowmeters. (7) Thermostatic heating element. (8) Temperature transducers. (9) Reservoir for hormone solution. (10) Temperature- and humidity-controlled perfusion chamber. (11) Thermometer. (12) Hygrometer. (13) Arterial catheters with sampling ports. (14) Venous catheters with sampling ports. (15) Reservoir to collect perfusate. (16) Recirculating pump. (17) Pump for perfusion with different hormonal perfusion media. (18) Regulation element for autoregulation between recirculation flow rate and effluate volume.

 
Arterial and venous samples of perfusion medium were taken every 60 min with syringes for measurements of pH, oxygen partial pressure (pO2), carbon dioxide partial pressure (pCO2), lactate, lactate dehydrogenase (LDH) and creatine kinase (CK).

Samples of pH, pO2 and pCO2 were analysed with an ABL 505 Blood Gas Analyzer (Radiometer, Copenhagen, Denmark); all other biochemical parameters were measured using standard procedures (Vitros-Analyzer, Johnson & Johnson, Rochester, NY, USA).

Several myometrium and endometrium biopsies for light and electron microscopy were taken from the corpus uteri and the cervix uteri with a biopsy needle at the beginning, during (at 6, 12 and 18 h of perfusion) and at the end of perfusion.

For light microscopy the samples were fixed in 4% paraformaldehyde and then embedded in paraffin blocks. From these specimens several sections of 4 µm thickness were stained with haematoxylin and eosin (HE). For the electron microscopical investigations samples were fixed in 2.2% glutaraldehyde and PBS buffer. Cubes of about 1 mm3 in size were cut from each specimen and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and were examined with a Philips CM 10 electron microscope (Eindhoven, The Netherlands).

To evaluate the best interval for regeneration of the perfusate in this recirculating perfusion model, isovolumetric exchange of the perfusion medium after 1 (group 1), 2 (group 2), 4 (group 3) and 6 (group 4) h was compared to a non-recirculating system (group 0). In each group five uterine perfusions were performed. After each experiment we performed X-ray studies with opaque contrast medium using digital subtraction angiography (DSA) and perfused each uterus with methylene blue to show the distribution of the perfusion medium throughout the organ. For statistical evaluation we used the Kruskal–Wallis one-way analysis of variance for non-parametric data.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The age of the hysterectomized patients was 28–56 years (mean 42 ± 8.6 years), all of them in a pre-menopausal status. In 16 cases a hormone treatment was finished 2–6 weeks prior to the operation; nine patients did not have any hormone therapy.

Perfusions were considered to be appropriate when constant flow rates of 15–35 ml/min through each artery could be maintained at pressure rates ranging from 70 to 130 mmHg. The venous effluate was collected in a cylindrical teflon reservoir from which the perfusate was recirculated by a separate roller pump to the reservoir for the perfusion buffer (Figure 1Go).

The biochemical parameters were initially elevated in all groups due to preparation and cannulation time. Within the first hours of perfusion, arterio-venous gradients of pH, pO2, and pCO2 decreased significantly in groups 0, 1, 2 and 3 and remained stable in the further course of perfusion as a sign of physiological oxygen consumption of the perfused organ (Figure 2a–cGo).





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Figure 2. (a) Arterio-venous gradients of pH in groups 1, 2, 3 and 4 (i.e. after 1, 2, 4 and 6 h respectively) during perfusion compared to group 0 (a non-recirculating system). (b) Arterio-venous gradients of oxygen partial pressure (pO2) in each group during perfusion. (c) Arterio-venous gradients of carbon dioxide partial pressure (pCO2) in each group during perfusion.

 
The concentrations of lactate, LDH and CK are shown in Figure 3a–cGo. The initial hypoxia in all groups leading to formation of lactic acid at the beginning of perfusion was corrected in groups 0, 1, 2 and 3 as the perfusion proceeded with oxygenated buffer (Figure 3aGo). Similarly, increased LDH and CK levels as indicators for cytolytic tissue processes occurring during the preparation and cannulation period subsided significantly in groups 0, 1, 2 and 3 with continuation of the experiment (Figure 3b,cGo). In contrast, all parameters in group 4 showed an adverse development due to the longer perfusate exchanging period (Figures 2 and 3GoGo). These differences between groups 4 and 0, 1, 2 and 3 were significant ({alpha} = 0.5; P < 0.0001).





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Figure 3. (a) Concentrations of lactic acid in each group during perfusion. (b) Concentrations of lactic dehydrogenase (LDH) in each group during perfusion. (c) Concentrations of creatine kinase in each group during perfusion.

 
Furthermore, examination by light and electron microscopy showed well-preserved intracellular structures in myometrium and endometrium without evidence of intracellular oedema in comparison to the control tissue taken from corpus and cervix immediately after hysterectomy in groups 0, 1, 2 and 3, but not in group 4 (Figure 4a–gGo). There was no significant oedema formation evaluated by comparison of the organ weights before and after the perfusion (Table IGo).










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Figure 4. Light and electron micrographs of myometrial and endometrial tissues. (a) Well-preserved corpus epithelium, group 3, interval-exchange after 4 h. Haematoxylin and eosin (HE); scale bar = 50 µm. (b) Control biopsy as a example for corpus tissue taken immediately after removal of the uterus. HE; scale bar = 50 µm. (c) Autolytic corpus tissue, group 4, interval-exchange after 6 h. HE; scale bar = 50 µm. (d) Intact, mucinous cervix epithelium, group 4, interval-exchange after 4 h. HE; scale bar = 50 µm. (e) Control biopsy as an example for cervix tissue taken immediately after removal of the uterus. HE; scale bar = 50 µm. (f) Dissociation of the cervix epithelium due to autolysis, group 4, interval-exchange after 6 h. HE; scale bar = 50 µm. (g) Electron micrograph shows endometrial epithelial cells in the proliferating phase of the cycle taken immediately after removal of the uterus. The fine chromatin structure and microvilli demonstrate well-preserved epithelium; scale bar = 3.3 µm. (h) Specimen from the same uterus, group 3, interval exchange after 4 h. The electron micrograph shows intact organelles of endometrial epithelial cells, i.e. mitochondria, rough endoplasmic reticulum, centrioles (arrow head), intercellular junctions; scale bar = 1.8 µm.

 

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Table I. Numbers of the perfused uteri and their correponding organ weights in grams before and after the perfusion experiment
 
In group 4 the light microscopic examination of endometrial and myometrial samples taken after 24 h showed severe regressive changes and partial necrosis with predominantly no longer recognizable tissue structures (Figure 4c,fGo). Similiar results were obtained by electron microscopical study of the specimen. Up to a perfusate exchange interval of 4 h the intracellular and intercellular tissue features did not significantly change in comparison to those seen in fresh samples (Figure 4g,hGo). Examination of the tissue samples of group 4, however, showed irreversible damages e.g. degranulation of rough endoplasmatic reticulum with disaggregation of free polyribosomes and clarified mitochondrial matrix. Moreover, frank cavitation with peripherally placed, disorientated, and disintegrated cristae was observed.

The injected X-ray contrast fluid through both arteries showed good distribution of the perfusate throughout the myometrium. The uterus is well supplied with arcuate and radial arteries crossing vertically and horizontally to form a network of small anastomoses which results in perfusion of the entire organ (Figure 5a,bGo). Application of methylene blue demonstrated a homogeneous distribution of the perfusion medium throughout the corpus uteri including the endometrium (not shown).




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Figure 5. Sequences from perfusion study of whole uterus with X-ray material imaged by digital subtraction angiography. (a) Early phase, after 5 s; (b) late phase, after 15 s. Scale bar = 1.5 cm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous investigations emphasized the feasibility of perfusion models for experimental examinations in the human placenta, ovary and uterus (Bulletti et al., 1986Go, 1993Go; Page, 1991Go; Brannstrom and Flaherty, 1995Go; Schmolling et al., 1997Go). The experimental perfusion system of the human uterus described by Bulletti et al. (1987a, 1988a) consisted of an open perfusion circuit, i.e. without recirculation of the perfusate, with excessive demand of perfusate volume, especially in the midterm and longterm perfusion situation. In the present study we developed an extracorporeal perfusion system of the human uterus with recirculation of the perfusion medium allowing a reduction to about a quarter of the necessary perfusate volume compared with the previous model (Bulletti et al., 1987a, 1988aGo).

In our modified experimental model, perfusions under physiological flow and pressure conditions with modified oxygenated Krebs–Ringer bicarbonate buffer could be maintained at 37°C for the entire perfusion period at a steady rate of oxygen consumption, low output of lactate, LDH and CK activity as markers for hypoxia and cytolytic processes and preservation of tissue integrity. In contrast to current clinical methods for extracorporeal preservation of organs by hypothermia, our purpose was to obtain an experimental model suitable for metabolic studies. Cooling causes modifications of physiochemical functions and metabolic processes during prolonged hypothermic storages: in particular, temperatures close to freezing inactivate the sodium–potassium cell pump with consecutive loss of cellular potassium and magnesium and a corresponding sodium influx into the cell with cellular swelling.

In this study it was our intention to simulate the in-vivo situation as close as possible. Therefore, human specimens were used and uterine perfusion was performed at body temperature with the organ placed in an automatically thermo- and humidity-controlled incubator.

With the use of autoregulated perfusion pumps, with adjustable minimum and maximum levels for perfusion flow and perfusion pressure rates according to physiological conditions, microvascular damages such as capillary destruction could be avoided.

In a perfusion system with recirculation of the perfusion medium, the composition of the perfusate is of particular importance. From the biological point of view the use of undiluted heparinized blood appears to be preferable to synthetic media. However, in-vitro perfusions using blood can cause problems in mid-term and long-term perfusion experiments regarding its physiological instability, the probable interaction with experimental pharmacological substances and the large volume required for perfusion.

Currently various preservation solutions, for example Euro–Collins solution, UW–Belzer solution, Bretschneider HTK solution and Krebs–Henseleit bicarbonate buffer solution are used experimentally and clinically for cold storage preservation of organs in transplantation medicine or investigations in oncology (Marinelli et al., 1991Go; Bresticker et al., 1992Go; Collins and Wicomb, 1992Go; Erhard et al., 1994Go; Blech et al., 1997Go; Collins, 1997Go). Based on the experiences of Bulletti et al. (1988b, 1993) we used a modified Krebs–Henseleit bicarbonate buffer with saccharose, glutathione, 1,4-dithiothreitol, insulin and refobacin and demonstrated that exchanging the perfusate every 4 h seems to be sufficient to maintain organ architecture and tissue vitality under optimal conditions of oxygenation and nutrition, thus avoiding infection problems.

Under these circumstances the necessary volume of the expensive perfusion medium could be reduced significantly.

Furthermore, observing uterine tissue fragments by light and electron microscopy, which show good preservation of the endometrial and myometrial intracellular structures as well as the absence of inter- and intracellular oedema up to 24 h of perfusion time, confirm the results of our investigations. Performing X-ray studies with the DSA technique and perfusions with methylene blue demonstrated the homogeneous distribution of the perfusion medium throughout the entire organ.

By avoiding the degeneration of the organ by thrombus formation, problems with organ and perfusate temperature (hypothermia), increased capillary resistance or capillary destruction, unphysiological perfusion flow and perfusion pressure, insufficient gas exchange and relatively activated anaerobic metabolism and bacterial infection, we could maintain the viability and function of the organ.

Previous investigations by Bulletti et al. (1988a,b, 1993a,b, 1997) have shown the feasibility of the extracorporeal perfusion of the human uterus for various purposes. In conclusion, our model for the extracorporeal perfusion of the human uterus with recirculation of the perfusate by isovolumetric interval exchange every 4 h represents a reproducible experimental system which offers, especially in mid-term and long-term perfusion experiments, a physiological and economical scientific approach for further observations on the endometrium and myometrium in reproductive medicine.


    Acknowledgments
 
This research is supported by the Deutsche Forschungsgemeinschaft (DFG), Gz RI 958/1-1 and the BONFOR-Forschungskommission, Gz 103/17.


    Notes
 
5 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 14, 1999; accepted on March 1, 2000.