Uterus transplantation may become the surgical therapeutic modality of choice for uterine factor infertility. However, this procedure still faces technical, therapeutic, and immunologic challenges that limit its success and clinical application. Experimental studies are therefore still needed to address various challenges in the field of uterus transplantation. Among various laboratory animals, small animals are ideal models for the purpose of experimental uterus transplant. However, clinical success in small animal models is not generalizable to clinical application and treatment for uterine factor infertility in humans. Large animal models are necessary because their uterine anatomy and reproductive physiology closely resemble those of humans. In the literature, in general with small or large animal models, the same striking characteristic has been previous regular menstruation. Anesthesia was usually induced through inhalation and/or intraperitoneal injection in small models and intravenous injection in large models. Systemic heparinization was usually performed after preparation of uterus and vessels and before cross-clamping of the vessels. Flushing of the graft was performed through the interior iliac artery or aorta. A grafted segment was frequently selected only from one horn of the uterus. The uterine artery, internal iliac artery, and aorta have been frequently used for arterial revascularization into the recipient's external iliac artery or abdominal aorta. The uterine vein, internal iliac vein, and inferior vena cava have been used for venous drainage into the recipient's inferior vena cava, external iliac vein, or uteroovarian vein. In most models, the native uterus was resected to reconstruct the grafted uterus continuity. Other models have left the native uterus in the recipient's abdomen, and stomas have been used for end of the grafted uterus.
Key words : Animal model, Complications, Uterine factor infertility
Uterus transplantation (UTx) may become the surgical therapeutic modality of choice for uterine factor infertility, allowing women with this condition to carry their own child during the entire pregnancy.1 However, UTx still faces technical, therapeutic, and immunologic challenges that limit its success and clinical application. Experimental studies are therefore needed to address various challenges in the field of UTx. Experimental UTx was first introduced by Eraslan and associates in canines in 1966.2 Several years later, after the first human UTx by Fageeh and associates in 2000, attempts at other experimental UTx models have become more intense.3
Small animals, such as mice and rats, are ideal models for the purpose of experimental UTx as they are well-established, inexpensive, and ethically acceptable. There are various inbred strains of mice and rats available with well-defined histocompatibility properties that make the immune system manipulation feasible. Despite these advantages, however, the model remains somewhat technically difficult to master, especially in the microsurgical portion. Uterus transplant was first described in mice by Brännström and associates4 in 2003 and in rats by Ionac and associates5 in 2002. Several modifications and developments have since been adopted in these models. However, technical complexities and high mortality rates have plagued their use.
The clinical success in small models could not be translated as a permanent treatment for uterine factor infertility in humans. Therefore, large animal models are necessary because their uterine anatomy and reproductive physiology closely resemble those of humans.6 In addition, the ethical principles established by the International Federation of Gynecology and Obstetrics dictate that human UTx should only occur after adequate significant successful trials in appropriate large animal models, including primates.6-9
In this work, we reviewed the most important methods in the literature for UTx in various experimental models and compared the advantages and disadvantages of each so that investigators in this field will be able to better select and standardize the required techniques depending on their aims and objectives of study.
We conducted a search of all Medline-published papers regarding UTx in various experimental models, documenting characteristics of animals, methods of animal care, techniques of procurement, recipient procedures, including methods of arterial and venous reconstructions, reconstruction of the uterus continuity, different methods of uterus exteriorization, and posttransplant care, with particular emphasis on surgical techniques. Where possible, attempts were made to give an overview of the advantages and disadvantages of the different techniques.
In our literature search, although different small models (mice and rats) or large models (sheep, cynomolgus monkey, and baboon) were used, the same striking characteristic was shown: previous regular menstruation,10 with some studies including even previous pregnancy history.11 As noted, housing and feeding of animals should be performed according to institutional practices.12,13 For the purpose of experimental UTx, rodents have become ideal animals14 because they are less expensive and ethically accepted and the genome is better understood.15
Among the technical challenges faced are preoperative hypothermia and electrolyte imbalance, which can result in high mortality. Some technical strategies have been developed to prevent these challenges. For example, partial wrapping of both donor and recipient rats in isolating aluminum foil and surrounding wrapped animals with warm bags16,17 can maintain adequate body temperature to avoid hypothermia. Maintaining water and electrolyte balance is important to avoid early mortality.7 Several research groups have allowed animals free access to food and water without any special limitations,18,19 whereas others kept the animals fasted for 12 to 48 hours20,21 depending on the research aim. Fasted animals should, however, receive a daily volume (2 mL/100 g/24 h) of normal saline via subcutaneous injection in small models22,23 and intravenous injection in large models.24
Regarding anesthesia, outbred Sprague-Dawley rats have been described as ideal rodent models for microsurgical procedures because they tolerate the anesthesia well and are less expensive than other species. Inbred Lewis rats are preferred for physiologic and immunologic studies.25,26 For small animal model anesthesia, accepted routes are inhalation and intraperitoneal injection. For induction, several products have been used. Inhalation products have included ether,27-29 isoflurane, and methoxyflurane.30 Intraperitoneal products include pentobarbital (40 mg/kg),31 3% sodium pentobarbital (0.1 mL/100 g),23,32 and ketamine (100 mg/kg).33-35 An intramuscular injection is painful, and it is not routinely recommended.36,37 However, some groups have used the intramuscular route after primary anesthesia to prolong the anesthesia period during operation.6,38 Young mice and rats do better than older rates because older rats have more fat, making surgical dissection more difficult and increasing respiratory complications.
The accepted routes for large animal model anesthesia include inhalation and intraperitoneal injection. For the purpose of induction, in large animals, the products used are the same as for small animals.30,39-44 However, accepted anesthesia in large models include induction with ketamine (20-22 mg/kg intramuscularly) and xylazine 5% (0.02-0.04 mg/kg intramuscularly) and then intubation with subsequent anesthesia maintained by inhalation of halothane in an oxygen/air mixture or isoflurane (0.5%-1.0%).6,7,11,34,40,45,46
Preoperation antibiotic therapy has been performed by some groups for a few days or immediately before the surgical procedure. Trimethoprim/-sulfamethoxazole (17%/83%) at 15 mg/kg intravenously and metronidazole at 5 mg/kg intravenously44 and dihydrostreptomycin sulfate 0.02 to 0.1 mL/kg intramuscularly18 were used for both small and large animal models.
Systemic heparinization is usually performed after preparation of uterus and vessels and before cross-clamping of the vessels.24,33,34 Heparin has also been frequently added to the flushing solution.41,47,48 No heparinization has also been reported.6 The heparin dose varies from 3000 to 10000 IU.41,47
Flushing of the graft has been performed through the interior iliac artery11,18,24,44 and aorta.49-51 Perfadex (XVIVO Perfusion, Gothenburg, Sweden),51,52 iced lactated Ringer solution,20,24 and normal saline11,33 have been frequently adopted as perfusion solutions. University of Wisconsin solution47 and histidine-tryptophan-ketoglutarate34,44 have also been used. Uterus transplant models have selected the total or have selected one horn of the uterus and the corresponding vessels; the grafted total uterus was frequently selected from the upper vagina to salpinx.6,11,20,33,52 Some models have included only one horn of the uterus.19,20,48 The common iliac artery50,51 or aortic Carrel patch21,49,53,54 in small animal models and uterine artery (UA)11,18,34,55 or interior iliac artery6,20,44 in large animal models have been used for arterial revascularization. The inferior vena cava (IVC)33,53,54 or common iliac vein52 in small models and uterine vein (UV),6,11 internal iliac vein,20 and ovarian vein24,34,55 have been used for venous drainage. The graft is preserved at +4°C in the same solution used for perfusion.
Choosing to resect or to leave the recipient uterus in place has been different in various models, depending on the research aim. In most models, the native uterus has been removed from the recipient's body11,33,44; other models have left the native uterus in the recipient's abdomen.19,27,49
The arterial anastomosis is the key technical step in the entire procedure. Arterial anastomosis for UTx was first described by Eraslan and associates2 and involved dissection of the hypogastric common artery. This method had required fairly extensive retroperitoneal dissection to free sufficient aorta and grafted artery.54 Differences in techniques of arterial reconstruction have been based on the 3 following variables: donor's artery, recipient's artery, and reconstruction technique. The donor's interior iliac artery has been dominantly used as the graft artery.6,40,44 The donor's aorta has also been used by several teams in small animal models (Figure 1),19,27 with the UA used in large animal models (Figure 2).6,18,24 In one research approach, the common iliac artery was selected.48 For the purpose of arterial reconstruction, the recipient's external iliac artery in large models (Figure 3) and recipient's abdominal aorta (Figure 1) in small models have been most frequently used to perform end-to-side anastomosis with the donor's artery.6,11,24,44,48,53 Some investigators have reported the use of the recipient's UA for end-to-end anastomosis.11,33,55 The drawback of this method is the probable increased risk of thrombosis and graft rejection because the diameter of these arteries is narrow.21,41
To obtain short warm ischemia time, many studies perform vessel anastomose unilaterally. Blood flow from the unilateral UA is limited for graft supply. Therefore, for pregnancy, the UA should be bilaterally grafted.50,56 However, the use of only one horn of the uterus with unilateral vessels anastomose has also been described (Figure 4).19,48,53
Regarding suture techniques, the running 10-0 nylon,52 the running 11-0 nylon,6,18 and the running 8-0 Prolene24,44 have been described. During organ transplant, various methods of microvascular anastomosis have been developed and documented. For example, the hand-sewn, simple interrupted technique still remains the criterion standard and is performed by most surgeons.57 Some groups have preferred the simple continuous suture technique (SCST) to reduce the surgery time for both safety and economic purposes.58 However, surgeons require several months of training to achieve the required dexterity for this technique. Despite these advantages, there are still concerns regarding SCST. This technique can, at least in theory, narrow the anastomotic site, limit systolic expansion, and leave a greater amount of suture material in contact with the bloodstream, thus potentially leading to stasis and thrombosis. The continuous locked suture technique has also been used.
This suture technique has combined the advantages of the interrupted suture technique and the SCST. The continuous locked suture technique is as time-saving as the SCST, although leaving less suture material in contact with the bloodstream compared with SCST.59 In small animal models, the rodent cuff technique can also be used between graft external iliac artery or aorta and recipient's aorta to further reduce graft warm ischemia time and to obtain higher success rates. This method is used widely by many researchers.22 Table 1 summarizes the arterial reconstruction techniques.
The first technique of vein anastomosis used for UTx involved the use of the hypogastric common vein; however, this approach required temporally long dissection.2 Venous blood drainage has been most often created by forming an end-to-side anastomosis between the donor's external iliac vein,8,9 internal iliac vein (Figure 3),20,45 deep UV,6,18,34,60 or ovarian vein11,40,46,61 and the recipient's external iliac vein (Figure 3) in large models.8,9,18,21,40 In small models, it is created between the donor's IVC19,27,53 and recipient's IVC (Figure 1),17,20,50,53 which has been performed with 9-0 nylon18,21,55 or 11-0 nylon19,48,51 continuous sutures. Other large animal models have therefore performed drainage of the donor's UV into the recipient's UV, thus providing a more physiologic model.18,34,55 By avoiding the temporary clamping of the IVC, this model reconstitutes the natural perfusion and prevents recipient hemodynamic instability. Some large animal models have involved vein anastomosis through simplified donor ovarian vein and recipient external iliac vein anastomosis because the diameter of the ovarian vein is greater than the UV and there was less risk of vein thrombosis in nonhuman primate experiments.11 Table 2 summarizes vein reconstruction techniques.
Most published experimental models have involved the total uterus and recipient's uterus from upper side of the vagina to salpinx replaced by the donor's organ. Total UTx is more favorable for researchers who want to compare different surgical techniques, immunosuppression, and preservation (Figure 2).11,33,52,55 Other investigators have used only one horn of the uterus, based on the research aims, known as the sham technique (Figure 4). The use of a one-horn graft has been based on 2 motives. It seemed that the unilateral UA cannot supply the total uterus to provide a regular menstruation cycle and resulted in late pregnancy.56 In addition, a unilateral vessel anastomosis reduces warm ischemia time and rate of rejection, allowing an easier surgical technique.8,19,27,48,52
Other groups have placed the grafted uterus in a heterotopic position in the recipient while the cervix was exteriorized and sutured as a stoma to the skin of the abdomen, with the native uterus of the recipient left in place (Figure 5).17,19,20,27,51 This nonphysiologic technique is performed to compare the grafted uterus versus the native uterus to induce a pronounced rejection process.19,20,27 The anastomosis technique for the distal end of the uterus has been generally conducted with an end-to-end anastomosis using continuous sutures with 5-0 Prolene in small models18 and 2-0 polydioxanone in large models.40,44
The onset and progression of rejection have been shown to be the same with all 3 types of allografts studied.14
Some heterotopic models of UTx have also been described in mice. Uterus autotransplants to the cervical region have been technically feasible, with a 100% technical success rate. The procedure results in normal function despite the "tight" location of the graft. However, the subcutaneous space in the neck is limited.49 Table 3 summarizes uterus continuity techniques.
All animals should be observed daily posttransplant. Based on research aims, posttransplant biopsies and further evaluations should respect animal welfare principles in accordance with institutional guidelines on animal care.
After transplant, small animals should be kept warm (eg, under heat lamp).11,49,53 Animals should be individually caged. In small animal models, recipient animals can be injected with a few milliliters of donor blood and/or normal saline.8 Some groups have kept the recipients fasted for the first 12 hours4 or the first 24 hours40 and then provide the animals with 20-mL subcutaneous injection of lactated Ringer during the first 48 hours.40 Large animals are to be observed in accordance with pre- and postoperative care guidelines for children (The Queen Silvia Children's Hospital, Sahlgrenska University Hospital).24
Antibiotics posttransplant have included cefuroxime (40 mg/kg intramuscularly),19 cefazolin sodium (20 mg/d subcutaneously) for 5 days,41 or streptomycin (50 mg subcutaneously)62 in small animal models. Moxifloxacin hydrochloride (400 mg/d orally),34 trimethoprim/sulfamethoxazole (15 mg/kg intramuscularly) plus metronidazole (20 mg/kg/d orally) have been used in large animal models.24,44 After the acute posttransplant phase, free water and food must be made available. After transplant, general status, body weight, limb movement, and intestinal function are generally assessed.63 Biopsy samples have been obtained transvaginally or via a stoma sample of the endometrium, cervix, or uterus.41,55 Some investigators have performed abdominal magnetic resonance imaging.21
Uterus transplant has been improved in the experimental setting after related complications have been investigated and solved using various animal models. Along the way, UTx in small or large animal models have been developed and modified. The preferred technique varies according to the researcher's objectives and the surgeon's experience. For long-term survival studies, more physiologically and anatomically oriented techniques should be performed; for short-term survival studies, faster and simpler techniques, which may not completely mimic the original physiology, can be employed.
Volume : 16
Issue : 2
Pages : 119 - 126
DOI : 10.6002/ect.2017.0187
From the 1Departments of Gynecology and Obstetrics MKK Gelnhausen, Gelnhausen,
Germany; the 2Medicine Kreiskrankenhaus Bergstraße, Heppenheim, Germany; the
3Medical University Ardabil, Ardabil, Iran; and the 4Medical University Zahedan,
Acknowledgements: The authors have no sources of funding for this study and have no conflicts of interest to declare.
Corresponding author: Jalal Arvin, Departments of Gynecology and Obstetrics MKK, Herzbachweg 14, 63571, Gelnhausen, Germany
Phone: +49 60 51/87 2353
Figure 1. Revascularization of the Uterine Graft Using End-to-Side Arterial Anastomosis Between Donor’s Aorta and Recipient’s Abdominal Aorta
Figure 2. Revascularization of Uterine Graft Using End-to-End Arterial Anastomosis Between Donor’s Uterine Artery and Recipient’s Uterine Artery
Figure 3. Revascularization of the Uterine Graft Using End-to-Side Arterial Anastomosis Between Donor’s Internal Iliac Artery and Recipient’s ExternalIliac Artery
Figure 4. Reconstruction of Uterine Continuity Through End-to-End Vagino-Vaginotomy Using One Horn of Uterus as Sham Technique
Figure 5. Reconstruction of Uterine Continuity Through Exteriorization of the Distal Ends of the Graft With Left Native Uterus in Abdomen
Table 1. Different Types of Artery Reconstruction in Uterus Transplant
Table 2. Different Types of Vein Reconstruction in Uterus Transplant
Table 3. Three Different Restoration Techniques of the Uterus Continuity in Uterus Transplant