Name: surgical approach, challenges, and resolutions for uterus transplantation in rats
Uploaded: 2023-04-14T14:00:00
Description: Watch this Scientific Journal Video about Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats at JoVE.com

This study was supported by the Swiss National Science Foundation (project grant no. 310030_192736). We would like to thank Dr. Frauke Seehusen from the Institute of Veterinary Pathology of the University of Zurich for her histopathological support.

All animal experiments were performed following Swiss Federal Animal Regulations and approved by the Veterinary Office of Zurich (n&#176; 225/2019), assuring human care. Female virgin Lewis rats (body weight of 170-200 g) and female virgin Brown Norway rats (170-200 g) were used as uterus donors/recipients, while male Lewis rats (300-320 g) were used for mating. The rats were aged from 12-15 months. The animals were obtained from commercial sources (see Table of Materials) and were housed in controlled c...

<ol>
	<li>Richards, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterus+transplantation:+state+of+the+art+in+2021.">Uterus transplantation: state of the art in 2021.</a> Journal of Assisted Reproduction and Genetics. 38 (9), 2251-2259 (2021).</li><li>Jones, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Options+for+acquiring+motherhood+in+absolute+uterine+factor+infertility;+adoption,+surrogacy+and+uterine+transplantation.">Options for acquiring motherhood in absolute uterine factor infertility; adoption, surrogacy and uterine transplantation.</a> The Obstetrician &amp; Gynaecologist. 23 (2), 138-147 (2021).</li><li>Brannstrom, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+clinical+trial+of+uterus+transplantation:+surgical+technique+and+outcome.">The first clinical trial of uterus transplantation: surgical technique and outcome.</a> American Journal of Transplantation. 14, 44 (2014).</li><li>Ejzenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Livebirth+after+uterus+transplantation+from+a+deceased+donor+in+a+recipient+with+uterine+infertility.">Livebirth after uterus transplantation from a deceased donor in a recipient with uterine infertility.</a> Lancet. 392 (10165), 2697-2704 (2018).</li><li>Lavoue, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+donor+for+uterus+transplants:+brain-dead+donor+or+living+donor?+A+systematic+review.">Which donor for uterus transplants: brain-dead donor or living donor? A systematic review.</a> Transplantation. 101 (2), 267-273 (2017).</li><li>O'Donovan, L., Williams, N. J., Wilkinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethical+and+policy+issues+raised+by+uterus+transplants.">Ethical and policy issues raised by uterus transplants.</a> British Medical Bulletin. 131 (1), 19-28 (2019).</li><li>Kisu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+outcome+and+rejection+after+allogeneic+uterus+transplantation+in+cynomolgus+macaques.">Long-term outcome and rejection after allogeneic uterus transplantation in cynomolgus macaques.</a> Journal of Clinical Medicine. 8 (10), 1572 (2019).</li><li>Ozkan, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterus+transplantation:+From+animal+models+through+the+first+heart+beating+pregnancy+to+the+first+human+live+birth.">Uterus transplantation: From animal models through the first heart beating pregnancy to the first human live birth.</a> Womens Health. 12 (4), 442-449 (2016).</li><li>Favre-Inhofer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involving+animal+models+in+uterine+transplantation.">Involving animal models in uterine transplantation.</a> Frontiers in Surgery. 9, 830826 (2022).</li><li>El-Akouri, R. 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N., Diaz-Garcia, C., Brannstrom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy+after+syngeneic+uterus+transplantation+and+spontaneous+mating+in+the+rat.">Pregnancy after syngeneic uterus transplantation and spontaneous mating in the rat.</a> Human Reproduction. 26 (3), 553-558 (2011).</li><li>Wranning, C. A., Akhi, S. N., Kurlberg, G., Brannstrom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterus+transplantation+in+the+rat:+Model+development,+surgical+learning+and+morphological+evaluation+of+healing.">Uterus transplantation in the rat: Model development, surgical learning and morphological evaluation of healing.</a> Acta Obstetricia et Gynecologica Scandinavica. 87 (11), 1239-1247 (2008).</li><li>Brannstrom, M., Wranning, C. A., Altchek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+uterus+transplantation.">Experimental uterus transplantation.</a> Human Reproduction Update. 16 (3), 329-345 (2010).</li><li>Diaz-Garcia, C., Akhi, S. N., Wallin, A., Pellicer, A., Brannstrom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+report+on+fertility+after+allogeneic+uterus+transplantation.">First report on fertility after allogeneic uterus transplantation.</a> Acta Obstetricia et Gynecologica Scandinavica. 89 (11), 1491-1494 (2010).</li><li>R, E., Brown, M. J., Karas, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anesthesia and Analgesia in Laboratory Animals. 2nd edn. , (2008).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Current Protocols. , 8 (2006).</li><li>Rutledge, C., Raper, D. M. S., Abla, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+I+do+it:+superficial+temporal+artery-middle+cerebral+artery+bypass+for+flow+augmentation+and+replacement.">How I do it: superficial temporal artery-middle cerebral artery bypass for flow augmentation and replacement.</a> Acta Neurochirurgica. 162 (8), 1847-1851 (2020).</li><li>Kuo, S. C. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple-U+technique:+a+novel+microvascular+anastomosis+technique+that+guarantees+everted+anastomosis+sites+with+solid+intima-to-intima+contact.">The multiple-U technique: a novel microvascular anastomosis technique that guarantees everted anastomosis sites with solid intima-to-intima contact.</a> Plastic and Reconstructive Surgery. 149 (5), 981 (2022).</li><li>Magee, D. J., Manske, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology and Intervention in Musculoskeletal Rehabilitation. 2nd edn. , 25-62 (2016).</li><li>Diaz-Garcia, C., Johannesson, L., Shao, R. J., Bilig, H., Brannstrom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy+after+allogeneic+uterus+transplantation+in+the+rat:+perinatal+outcome+and+growth+trajectory.">Pregnancy after allogeneic uterus transplantation in the rat: perinatal outcome and growth trajectory.</a> Fertility and Sterility. 102 (6), 1545-1552 (2014).</li><li>Canovai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGL-1+as+a+preservation+solution+in+intestinal+transplantation:+a+multicenter+experience.">IGL-1 as a preservation solution in intestinal transplantation: a multicenter experience.</a> Transplant International. 33 (8), 963-965 (2020).</li><li>Habran, M., De Beule, J., Jochmans, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGL-1+preservation+solution+in+kidney+and+pancreas+transplantation:+A+systematic+review.">IGL-1 preservation solution in kidney and pancreas transplantation: A systematic review.</a> PLoS One. 15 (4), 0231019 (2020).</li><li>Mosbah, I. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGL-1+solution+reduces+endoplasmic+reticulum+stress+and+apoptosis+in+rat+liver+transplantation.">IGL-1 solution reduces endoplasmic reticulum stress and apoptosis in rat liver transplantation.</a> Cell Death &amp; Disease. 3 (3), 279 (2012).</li><li>Wiederkehr, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+IGL-1+preservation+solution+in+liver+transplantation.">Use of IGL-1 preservation solution in liver transplantation.</a> Transplantation Proceedings. 46 (6), 1809-1811 (2014).</li><li>Tilney, N. L., Guttmann, R. 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The protocol presented here offers detailed instructions for the surgical approach behind uterus transplantation in rats. The protocol has been optimized to increase the odds of live births following UTx and subsequent mating. The original protocol has been taken over from the Br&#228;nnstr&#246;m group12,13, inspired by the mouse work of Akouri et al.10, and modified based on the authors&#39; experiences over the past years. As such, the ...

Uterine transplantation (UTx) is a novel treatment for absolute uterine factor infertility (AUFI). AUFI results from an absence (congenital or acquired) or malformation of the uterus and affects 3%-5% of women worldwide1. Ethical, legal, or religious reasons rule out adoption or surrogacy for many women who have a desire for motherhood but suffer from AUFI2. For these women, UTx remains the only option to start their own family. UTx has been applied in the clinic, albeit with mixed success; the procedure is technically challenging and requires steady improvement for its clinical establishment.
In 2014, the first transplantation of a uterus from a live donor (LD)-resulting in successful pregnancy-was performed by the pioneering Swedish group of Br&#228;nnstr&#246;m3. The first birth following UTx from a deceased donor (DD) was reported in 2016 in Brazil4. By 2021, more than 80 UTxs have been performed worldwide, however with a success rate of about 50% and with grafts coming from LD for the majority1.
Although not life-saving, UTx is an increasingly popular procedure to fulfill the desires for own progeny. As such, the demand for grafts is mounting, placing DD donation into a future focus. However, DD donation is complicated due to considerably longer cold (and in the case of cardiac death, also warm) ischemic exposures, elevating the risks of graft dysfunction and rejection5,6. Surgical technique, demanding compatibility matching, and associated immunosuppression remain critical issues regarding UTx outcomes7.
To manage the above risks in the clinic, appropriate animal models for the exploration of ischemia and immunosuppression are needed. The most clinically relevant endpoint for animal models remains successful birth; to date, pregnancies following experimental UTx have been achieved in mice, rats, sheep, rabbits, and cynomolgus monkeys8. While larger animals are predestined for acquiring and optimizing surgical techniques, rodents come with the distinct advantage of short gestation periods. Therefore, rodent models are superior regarding practical, financial, and ethical considerations9. However, the main challenge of UTx in mice is the small anatomy, with the highly demanding surgery tied to the low reproducibility of murine UTx10. By contrast, rats are surgically more accessible and retain the advantages of short gestation times. As such, the rat has become the model of choice for UTx9. Wranning et al. introduced the rat model of orthotopic UTx in 2008, and using this model, the first live birth following UTx and natural mating has been reported11,12,13. Subsequent studies have had critical contributions to the implementation of UTx in humans9.
Nonetheless, UTx remains challenging in rats, and only a few groups as of yet have mastered this surgical technique. One relevant obstacle to the spread of rat UTx among researchers is the lack of a precise description of the individual microsurgical steps, the pitfalls, and the according measures for troubleshooting14. This protocol aims to provide a detailed guide for this highly complex microsurgical procedure to facilitate the implementation of this animal model into future research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Angled to Side Scissor 5 mm</td>
			<td>F.S.T</td>
			<td>15008-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Big Paper Clip</td>
			<td>No specific</td>
			<td></td>
			<td>Used as retractor</td>
		</tr>
		<tr>
			<td>Blunt Bend Needle G30</td>
			<td>Unimed S.A.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bupivacain 0.5%</td>
			<td>Sintetica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Temgesic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dosiernadel G25</td>
			<td>H.SIGRIST&#38; PARTNER AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilon 10/0</td>
			<td>Ethicon</td>
			<td>2810G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilon 6/0</td>
			<td>Ethicon</td>
			<td>667H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilon 7/0</td>
			<td>Ethicon</td>
			<td>EH7446H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilon 8/0</td>
			<td>Ethicon</td>
			<td>2808G</td>
			<td></td>
		</tr>
		<tr>
			<td>Femal Brown Norway Rats (150-170 g)</td>
			<td>Janvier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femal Lewis Rats (150-170 g)</td>
			<td>Charles River Deutschland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>F.S.T</td>
			<td>14060-09</td>
			<td>Any other small scissor works too</td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder</td>
			<td>F.S.T</td>
			<td>12500-12</td>
			<td>Any other small needholder works too</td>
		</tr>
		<tr>
			<td>Heparin Natrium 25000 I.E./ 5 mL</td>
			<td>B. Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Institute Georges Lopez Perfusion Solution (IGL)</td>
			<td>Institute Georges Lopez</td>
			<td></td>
			<td>Organ preservation solution&#160;&#160;</td>
		</tr>
		<tr>
			<td>Male Lewis Rats (300-320 g)</td>
			<td>Charles River Deutschland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Serrefines 13 mm</td>
			<td>F.S.T</td>
			<td>18055-04&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Serrefines 16 mm gebogen</td>
			<td>F.S.T</td>
			<td>18055-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Serrefine Clamp Applicator with Lock&#160;&#160;</td>
			<td>F.S.T</td>
			<td>18056-14&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>M&#246;lnlyncke Op Towel</td>
			<td>M&#246;lnlyncke</td>
			<td>800300</td>
			<td>Sterile drape</td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>B.Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Octenisept</td>
			<td>Sch&#252;lke</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paper Tape</td>
			<td>Tesa</td>
			<td></td>
			<td>For fixing the animal</td>
		</tr>
		<tr>
			<td>Philips Avent Schneller Flaschenw&#228;rmer SCF358/02</td>
			<td>Philips</td>
			<td>12824216</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringerfundin</td>
			<td>B.Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun 2%</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine</td>
		</tr>
		<tr>
			<td>Round Handled Needle Holders</td>
			<td>F.S.T</td>
			<td>12075-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Handled Needle Holders</td>
			<td>F.S.T</td>
			<td>12075-12</td>
			<td></td>
		</tr>
		<tr>
			<td>S&#38;T Vessel Dilating Forceps - Angled 45&#176;</td>
			<td>F.S.T</td>
			<td>00276-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Sacryl Naht</td>
			<td>KRUUSE</td>
			<td>152575</td>
			<td></td>
		</tr>
		<tr>
			<td>Scapel No 10</td>
			<td>Swann Morton</td>
			<td>201</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Histo-Container</td>
			<td></td>
			<td></td>
			<td>Any small histo-container works fine-for coldstorage of the graft</td>
		</tr>
		<tr>
			<td>Small Plastik Bags</td>
			<td></td>
			<td></td>
			<td>Any transparant plastic bags are fine</td>
		</tr>
		<tr>
			<td>Steril Cotton swab</td>
			<td>Lohmann-Rauscher</td>
			<td></td>
			<td>Any steril cotton swab is fine</td>
		</tr>
		<tr>
			<td>Sterile Gauze</td>
			<td>Lohmann-Rauscher</td>
			<td></td>
			<td>Any steril gauze is fine</td>
		</tr>
		<tr>
			<td>Straight Scissor 8mm</td>
			<td>F.S.T</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope &#8211; SZX9</td>
			<td>Olympus</td>
			<td>OLY-SZX9-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutter Non Stick GLISS 0.4 mm</td>
			<td>Sutter</td>
			<td>78 01 69 SLS</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Tying Forceps&#160;</td>
			<td>F.S.T</td>
			<td>00272-13</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoLux warming mat</td>
			<td>ThermoLux</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Forceps for Skin</td>
			<td></td>
			<td></td>
			<td>Any tissue forceps are fine</td>
		</tr>
		<tr>
			<td>Vesseldilatator Forceps</td>
			<td>F.S.T</td>
			<td>00125-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl&#160; plus 4/0</td>
			<td>Ethicon</td>
			<td>VCP292H</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical approach, challenges, and resolutions for uterus transplantation in rats

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer from AUFI. These women were deprived of the option to have children until the advent of UTx. The clinical application of UTx was driven by experimental studies in animals, and the first successful UTx was achieved in rats. Given their physiological, immunological, genetic, and reproductive characteristics, rats are a suitable model system for such transplants. In particular, their short gestation period is a clear advantage, as the usual endpoint of experimental UTx is successful pregnancy with live birth. The biggest challenge for rat models remains the small anatomy, which requires advanced microsurgical skills and experience. Although UTx has led to pregnancy in the clinic, the procedure is not established and requires continuous experimental optimization. Here, a detailed protocol is presented, including essential troubleshooting for rat UTx, which is expected to make the entire procedure easier to grasp for those without experience in this type of microsurgery.

Results from two groups of rats are presented. UTx was carried out before (group 1, n = 8) and after (group 2, n = 8) protocol adjustment (Table 1)&#160;to demonstrate the effects of our modifications (please see the Discussion for an explanation of our modifications)12,15,21.
The outcome of rat UTx is associated with three key phases. The first phase is successful re...

Watch this Scientific Journal Video about Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats at JoVE.com

Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats

The present protocol describes all the essential steps for successful uterine transplantation (UTx) in rats. The rat model has proven suitable to promote the clinical implementation of UTx; however, rat UTx is a highly complex procedure requiring careful instructions.

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer ...

Right uterus transplantation belongs to one of the most complex microsurgical procedures mastered by only a few. Our protocol shifts right uterus transplantation from a experimental stage to an applicable methodology. Our protocol leads to a high rate of live births post uterus transplantation.It soon will promote the application of right uterus transplantation for actual research. Patience and stamina are key traits during the whole procedure. After performing a median laparotomy, on an anesthetized rat, harvest the right uterine horn with the common uterine cavity and cervix along with vascular pedicles.Cauterize the wound and ligate the left uterus horn with a 4-0 polygalactin suture. Now sever the left uterine horn adjacent to the branching from the common uterine cavity. Separate the cervical and vaginal portion of the future graft from the rectal attachment.Separate the future graft from the paravaginal and paracervical ligaments. Using diathermy, dissect the vagina around two to three millimeters caudal of the cervix. Locate the uterine artery and veins at their origins.Ligate all vessels caudal of the uterine vessels with 8-0 polyamide place. Place 8-0 polyamide ligatures directly around the right common iliac artery and vein proximal to the aortic and caval bifurcations. Make a 0.5 to one millimeter long incision into the right common iliac artery directly adjacent to the bifurcation.Insert a blunt straight 25 gauge needle into the lumen to flush it out. Secure the needle with a 6-0 polyamide ligature. To enable outflow during flushing, dissect the common iliac vein caudal to the ligature at the right common iliac vein.Using a three milliliter syringe, flush the uterus with cold Ringer's solution, followed by organ preservation solution both supplemented with heparin and xylazine. Cut the common iliac artery caudal of the ligature at the abdominal aortic bifurcation. Then using blunt dissection, sever the common iliac vessels from the aorta vena cava bifurcation at the division of the uterine vessels.And remove the transplant when the uterine tissue turns pale. Place the transplant into the cold organ preservation solution to facilitate back table preparation and storage before transplantation. After placing the graft in the abdominal cavity and positioning the graft vein, place one 10-0 polyamide stay suture into the cranial corner and one loose suture into the caudal corner of the slit on the right common iliac vein.Anastomose one side of the graft vein into the recipient's vein using six to eight loops of a continuous suture. Similarly, anastomose the other side of the vessel starting from the outside. Then using 10-0 polyamide, tie one knot at the cranial stay suture and another knot at the caudal stay suture.Perform arterial anastomosis using 10-0 polyamide interrupted sutures at the right common iliac arteria with eight to 10 loops. To begin graft perfusion, when the sites appear patent, release the vascular clamps on the graft vessels and check the graft for signs of reperfusion. Connect the vaginal cuff of the transplant to the vaginal vault using six to seven 6-0 polygalactin interrupted sutures.Using five to seven polyamide 7-0 interrupted sutures, anastomose the graph uterine horn end to end to the remaining recipient cranial uterine segment. Monitor the rat post operation. Post uterine transplantation, two Group 1 rats displayed signs of pregnancy.However, examination revealed dilated uterus, suggesting that the pups developed normally, but the rats failed to deliver the pups. Histological examination in the fetuses showed that the pups developed up to parturition. Five Group 2 rats displayed pregnancy with three giving birth to pups.The Lewis female gave birth twice. All pups in the first litter survived while only two of the three pups from the second litter survived. The brown Norway female also gave birth twice and all surviving pups displayed normal development.The protocol adaptation improved both direct surgical outcomes and the rate of live births following uterine transplantation. It increased the two-week graft survival rate from 50%in Group 1 to 75%in Group 2. It is important to keep the suture knot at the caudal corner loose for better adjustment and to prevent pursed string effects.Right uterus transplantation is particularly suited to assess pre-transplant approaches that may improve uterus transplantation outcomes such as ex vivo hypodermic organ perfusion or other techniques for the mitigation of organ injury. In general, uterus transplantation in animals has been and is a must to introduce to establish uterus transplantation in the clinic.

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