Establishing a Swine Model to Study Uterus Dynamic Preservation and Transplantation

Summary

A detailed and reproducible swine uterus model is described, from surgical procurement to the initiation of machine perfusion, allowing for the study of uterus preservation in transplantation.

Abstract

To date, uterus transplantation is the only option for women with absolute uterine infertility, such as those with Rokitansky syndrome, to experience pregnancy and give birth. Despite the growing interest in uterus transplantation in recent years, several issues still require further research, including ischemia-reperfusion injury and its impact on graft quality and rejection. Recent literature has highlighted a thrombotic complication rate of up to 20% following uterus transplantation. This type of complication may result from hypoxia-induced endothelial cell damage, often leading to uterine graft rejection. Hypoxia is induced during static cold storage, which remains the gold standard for graft preservation in solid organ transplantation. Recently, dynamic preservation using machine perfusion has been shown to improve the long-term storage of conventional and marginal organs by reducing ischemic and hypoxic injury. In this protocol, we aim to describe every surgical step involved in porcine uterus procurement and dynamic preservation, based on both uterine pedicles, to enable the connection and initiation of the machine perfusion protocol.

Introduction

Uterus transplantation (UTx) has significantly developed over the last ten years, with several teams starting clinical research programs. To date, the main indication of UTx is absolute uterine infertility due to uterine agenesis, including the Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. MRKH syndrome is a congenital disorder with a prevalence of one in 5,000 female live births¹. UTx could potentially address additional causes of infertility, including those resulting from hysterectomy due to malignant disease, postpartum hemorrhage, uterine fibroids, infectious sequelae, and various congenital malformations. This suggests that approximately 1 in 500 women may be eligible for UTx.

The first-ever clinical UTx occurred in 2000 in Saudi Arabia², but vascular complications led to a hysterectomy three months later. Since then, several cases of UTx have been performed, based on both living and deceased donors, resulting in more than 80 live births^3,4. Similar to the realm of solid organ transplantation and vascularized composite allotransplants (VCA), immune rejection is a significant challenge in UTx.⁵ Several factors can lead to graft rejection, including microcirculatory failure and venous stasis, both of which can lead to thrombotic complication. In a recent review studying uterine vascularization in transplantation, Kristek et al. reported up to 15% arterial thrombosis and 5% venous thrombosis⁶. Additionally, cold and warm ischemia are critical factors that must be addressed for successful transplantation, as ischemia-reperfusion injury (IRI) can lead to graft dysfunction and acute rejection^7,8. Myocytes respond to ischemic stress by producing lactate for up to 6 h⁹, after which muscle cell damage is irreversible. The impact of cold ischemia on the myometrium has been documented in clinical studies, and the use of intracellular-like University of Wisconsin solution during static cold storage (SCS) has been shown to improve preservation with better contractile response to prostaglandin and higher ATP concentrations when compared to Ringer's acetate solution¹⁰. However, the impact of warm and cold ischemia remains poorly explored in UTx.

SCS remains the gold standard for VCA preservation, including the uterus, and for most solid organ transplants. However, in recent years, significant advancements in machine perfusion systems and preservation solutions have led to a paradigm shift. There is now strong evidence supporting that dynamic machine perfusion can improve and prolong the preservation of healthy and marginal solid organs^{11,12,13,14,15}. This technique is now commonly used in clinical practice for lung, heart, liver, and kidney transplantation^14,16,17,18. Dynamic organ preservation demonstrated multiple benefits, including minimizing cold ischemia and hypoxia injuries by providing continuous oxygen and nutrient supply, clearing toxic metabolites, and improving graft quality and viability parameters^12,19. Multiple modalities have been developed, ranging from hypothermic to normothermic machine perfusion (with or without oxygen carriers), with several perfusates available, but only a few have been tested on the uterus²⁰. To ensure the substantial contribution of such research perspectives, relevant preclinical surgical models are of crucial importance.

In this work, subnormothermic machine perfusion (SNMP) is used as an oxygenated dynamic organ preservation method at room temperature (around 20 °C) by circulating a perfusate through a roller pump and an oxygenator. A porcine model is employed that is relevant for studies on UTx and preservation due to its similarities with the human reproductive system in terms of anatomy, physiology, and vessel size^21,22. The uterus is procured following circulatory death, providing relevance for donation after cardiac death and suggesting the possibility for a procurement delay after all other relevant solid organs^23,24. In addition, this model facilitates the development of uterus preservation studies within established transplant laboratories focusing on other organs, applying the "3Rs" principles²⁵. The aim is to establish a new preservation model based on uterine pedicles and assess its reliability for dynamic preservation. All the procedure steps are detailed, from the hysterectomy to the preservation, encompassing highlighted key points on using SNMP.

The protocol described below preceded a preliminary experiment based on a single pump and a "Y-tubing" inflow system for both uterine arteries (Supplementary Figure 1). After 4 h-SNMP, the organ gained over 50% of its initial weight. Flow, pressure, resistance, and weight variation are shown in Supplementary Figure 2. A single perfusion system separated into two inflows did not allow modulation of each flow rate to each side's pressure. In this case, SNMP led to substantial edema in half of the organ (Supplementary Figure 3). This system proved unsuitable for the uterine model, partly because it should not be considered a perfectly symmetrical model. Therefore, two systems of machine perfusion were used in this protocol, one for each uterine artery.

Protocol

All animals received humane care following the National Institute of Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC). Overall, 6 female Yucatan minipigs weighing 30-40 kg were used for uterus procurement, with four uteri undergoing SNMP. All animals were heparinized with one full dose (100 IU/kg) before euthanasia. Organ procurement occurred post-mortem with less than 60 min of warm ischemia. Other organs could have been harvested from the same donor for different studies, according to the "3Rs" principles²⁵. See the Table of Materials for details about all reagents and equipment used in the protocol.

1. Preoperative preparation (day before the surgery)

1. Prepare the perfusate solution. For subnormothermic machine perfusion, a Steen+ solution optimized for VCA was used^26,27. One liter of solution per uterus was used, and the composition is detailed in Table 1.
   NOTE: A large quantity of sodium hydroxide is added to the perfusate with the aim of achieving a pH of around 7.5-7.6. This value is expressly high but necessary as the pH will tend to fall as the machine is circulated and oxygenated with a carbogen mixture (95% oxygen; 5% carbon dioxide).
2. Set up the machine perfusion system (Figure 1). Check for leaks and bubbles when perfusate is circulated.

2. Post-mortem uterus procurement

NOTE: To simulate donation after cardiac death and/or post-mortem procurement, the animal should be euthanized according to the local IACUC guidelines. Exsanguination should be preferred to intravenous Pentobarbital injection in order to avoid toxicity that could interfere with the study.

1. Place the euthanized animal in the supine position. Scrub the abdominal area and place sterile drapes.
2. Make a 10 cm median infra umbilical incision with a #20 blade.
3. Open the subcutaneous tissue and aponeurosis with a monopolar electric scalpel.
   NOTE: Attention must be paid to not damage intestines by opening the abdominal cavity.
4. Set aside the small intestine with a surgical gauze and expose the uterus.
   NOTE: The uterine anatomy of the model used is shown in Figure 2A.
5. Proceed similarly for the left and right sides as follows:
   1. Identify the uterine vessels.
      NOTE: The uterine vein is positioned laterally to the uterine artery (Figure 3).
   2. Create an opening into the broad ligament laterally to the uterine vein with right-angle forceps.
   3. Through this opening, insert 2-0 silk tie sutures to ligate the ovarian vessels and release the uterus from the surrounding connective tissue in the broad ligament using cautery.
   4. Ligate the utero-ovarian vessels with 2-0 silk tie sutures and remove the ovary and tube.
   5. Skeletonize uterine vessels and divide them as close to the internal iliac vessels as possible.
      NOTE: Attention must be paid to keeping the pedicle as long as possible to facilitate cannulation and anticipate pedicle retraction after it has been severed.
   6. Repeat steps 2.5.1-2.5.5 on the opposite side.
6. Remove the uterus by cutting through the cervix with a monopolar electric scalpel.
   NOTE: Use a long contact time to ensure proper cervix vessel coagulation, preventing leakage during perfusion.

3. Preparation for perfusion

1. On a side table, dilate both uterine arteries using a microsurgical dilator and insert an angiocatheter. Secure the cannulation with 3-0 silk ties (Figure 2B).
   NOTE: Here, an 18 G angiocatheter was used for all arteries. Care must be taken not to insert the catheter too far to avoid selective cannulation, as the bifurcation is relatively close. The uterine veins are not cannulated, as venous outflow is sufficient to keep the lumen of these vessels open, allowing easy collection. In addition to saving time, traumatic cannulation may lead to vascular damage and potentially affect venous flow.
2. Slowly manually flush both uterine arteries with 20 mL of heparin solution on each side until all the vessels are washed out and the outflows are clear.
   NOTE: Attention must be paid not to flush with high pressure, which can lead to microvascular injuries and perfusion failure.
3. Weigh the uterus.

4. Subnormothermic machine perfusion

NOTE: For the uterus, two independent systems of machine perfusion are required. Each uterine artery is connected to a perfusion system composed of a roller pump, an oxygenator, a bubble trap, and a pressure sensor. The perfusate in a reservoir circulates through silicone tubes connected to the elements listed above before running through the organ via the uterine artery to the uterine vein on each side, where the perfusate exits and is released in the same reservoir.

1. Connect the uterus to the machine perfusion systems by connecting the uterine artery cannulas into the inflow tubing (Figure 1).
2. Using the roller pump, adjust the flow rate to low (2.5-4.0 mL/min) to maintain a constant arterial pressure between 25-35 mmHg.
3. Assess viability parameters at each predefined time point in both inflow and outflow using a 1 mL syringe and analyzing samples with the blood gas system machine [e.g., blood gas metrics (pH, pCO₂, pO₂, lactate, base excess, bicarbonate), glucose, sodium, potassium, calcium, chloride].
   NOTE: In this protocol, the perfusion lasts for 4 h, and samples from the inflow and the outflow are taken every 30 min.
4. Weigh the uterus at the end of perfusion.

Results

During perfusion, the system was connected to a pressure sensor that recorded the pressure during the experiment. The pressure was initially recorded for a uterus-free system, which was subtracted from pressure recordings during uterus perfusion to obtain the real organ pressure. The flow rate was adapted to maintain the pressure within the desired range and was controlled by the roller pump. The resistance was calculated using the formula R = P / Q (R: resistance (mmHg.mL.min^-1); P: pressure (mmHg); Q: flow r...

Discussion

Uterus transplantation, often considered part of VCA, has rapidly developed in the last few years. In parallel, machine perfusion started to be explored in VCA since it demonstrated robust evidence in improving solid organ preservation. Hypothermic and subnormothermic machine perfusion has allowed up to 24 h preservation in swine models of myocutaneous and bone-containing VCA^26,27,28. Since the uterus presents challenges compara...

Materials

Name	Company	Catalog Number	Comments
Affinity Pixie Oxygenation System	Medtronic	BBP241	Oxygenator
Bovin serum albumin	Sigma-Aldrich	A9647	Perfusate component
Calcium chloride dihydrate	Sigma-Aldrich	223506	Perfusate component
Carbon Dioxide Oxygen	Airgas	UN3156	Carbon Dioxide Oxygen mix gas
D-(+)-Glucose monohydrate	Sigma-Aldrich	49159	Perfusate component
Dexamethasone	Sigma-Aldrich	D2915	Perfusate component
Dextran	Thermo scientific	406271000	Perfusate component
Heparin sodium injection	Eugia Pharma	63739-953-25	Perfusate component
Humulin Regular Insulin human	Lilly	0002-8215-01	Perfusate component
Hydrocortisone sodium succinate	Pfizer	0009-0011-03	Perfusate component
Magnesium chloride hexa-hydrate	Sigma-Aldrich	M9272	Perfusate component
MasterFlex L/S	Cole-Parmer	77200-32	Roller pump
Polyethylene glycol 35000	Sigma-Aldrich	25322-68-3	Perfusate component
Potassium chloride	Sigma-Aldrich	7447-40-7	Perfusate component
Pressure Monitor, Portable, PM-P-1	Living Systems Instrumentation	PM-P-1	Pressure sensor
Radnoti Bubble Trap Compliance Chamber	Radnoti	130149	Bubble trap
RAPIDPoint500	Siemens	500	Blood Gas System
Sodium bicarbonate	Sigma-Aldrich	S5761	Perfusate component
Sodium chloride	Sigma-Aldrich	S9888	Perfusate component
Sodium hydroxide	Sigma-Aldrich	72068	Perfusate component
Sodium phosphate monobasique dihydrate	Sigma-Aldrich	71505	Perfusate component
Syringe 1 mL	BD	309659	Sample procurement
Vancomycine hydrochloride	Slate run pharmaceuticals	70436-021-82	Perfusate component