Transplantation of the Uterus in the Male Rat : Transplantation

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Original Basic Science

Transplantation of the Uterus in the Male Rat

Yang, Liu MD1; Wang, Tong MD1; Chen, Lin MD1; Li, Xia PhD2; Song, Yajuan MD1; Yu, Zhou MD, PhD1; Song, Baoqiang MD, PhD1

Author Information
Transplantation 107(10):p 2168-2178, October 2023. | DOI: 10.1097/TP.0000000000004599
  • Open

Abstract

Background. 

Uterus transplantation (UTx) is one of the potential methods to cure absolute uterine factor infertility of transgender. However, this mostly comes with many technological challenges.

Methods. 

Left inguinal UTx was performed in 13 castrated male rats. End-to-end anastomosis of donor common iliac vessels to recipient femoral vessels was used for transsexual UTx. Sampling was performed on day 30 after transplantation. Grafts were used to analyze the histological changes. TUNEL assay was applied to stain the apoptotic cells. Immunological rejection was judged by flow cytometry.

Results. 

Six uteri, 4 ovaries, and 4 upper vaginas were found at day 30 posttransplantation. Similar histological changes to proestrus, estrus, and diestrus of female rats were examined in the transplanted uteri. The histological changes of transplanted vaginas showed similarity to proestrus, estrus, and metestrus of the female rats. Follicles of different stages and corpus luteum with distinct morphological appearances were also observed. The TUNEL assay revealed a higher apoptosis of granulosa cells in transplanted ovaries compared with normal ovaries.

Conclusions. 

A rat model of transsexual unilateral inguinal uterine transplantation in castrated rats was established, which will provide a reference for bilateral transsexual UTx in animals and genetically 46 XY individuals who wish to become real women through transsexual UTx.

INTRODUCTION

Transsexual women or genetically XY women face the same situation that they cannot conceive and deliver their children, which may play an integral role in the expression and consolidation of female identity.1 The prevalence of male-to-female transsexualism is between 1 of 45 000 and 1 of 10 000, and the incidence has a trend to aggravate.2 Traditionally, gender remodeling changes their appearance and make them look more like their preferred gender. However, the function to be a true woman remained unsolved. Not only that, when the Imperial College London investigated the reproductive aspirations of 182 transgender women >16 y of age, the desire of these transgender women to have genetically related children was the same as the cisgender women; 94% of them had the desire to experience gestation, and 88% of them wanted to experience menstruation to enhance perceptions of their femininity. More importantly, more and more of the male-to-female transgender people would rather freeze sperm when uterus transplantation (UTx) becomes a realistic treatment option for transgender women, and 99% of them recognize that UTx can consolidate the happiness and bring hope in the future.3 However, as things stand, adoption or surrogacy may be the only existing ways to have their children. In terms of surrogacy, both commercial and altruistic surrogacy have ethical drawbacks, such as the commodification of children and instrumentalizated women.4 So, surrogacy cannot be legally supported in China. Similarly, adoption is also accompanied by many potential ethical and social issues. Therefore, UTx is a more suitable way to solve the infertility problem of women whose karyotype is 46 XY.

Up to now, >80 human UTx operations have been performed globally, resulting in >40 live births.5 Of the UTx, approximately two-thirds of the UTx procedures have been performed using uteri obtained from living donors, and the success rate (78%) is slightly higher than those obtained from deceased donors (64%). Twenty-four live births have now been reported in the media following UTx.6 Of them, 17 live births are recorded from living donor procedures, and 2 live births are reported from dead donor procedures. A majority of the live births come from their relatives, but the different genetic background still can normally deliver.7 This implies that recipients who received a transplanted uterus of different genetic background still harbor the fertility potential, and the present immunosuppressive regimen for UTx is safe to support UTx. Moreover, a survey from Turkey investigated the uterine donation wish of 31 female-to-male transgender people, and 30 of them (96.7%) had a positive attitude.8 This suggests a sufficient uterus supply for uterine transplantation. However, all these just occurred in cisgender female recipients; the possibility of heterosexual uterine transplantation in transgender females is still unknown.

In the United Kingdom, the Equality Act (2010) affords transgender people explicit protection from discrimination related to their gender reassignment.9 Hence, male-to-female transgender women are legally entitled to the same treatment as women assigned female at birth. Many scientists thought that UTx in transgender women may be ethically and legally permissible. However, they also stated that the transsexual UTx surgery and treatment strategy deserved further research.9–12 Actually, the first, and so far only, transsexual UTx attempt may have been performed in 1931 in Germany, as reported in media.13 Thereafter, no human transsexual UTx was reported.

Animal-based research for more than a decade formed the fundamentals for the successful introduction of human UTx in women. The present study is the first study to address UTx in a male animal model, namely, the castrated male rat.

MATERIALS AND METHODS

Animals

Male (7 wk, n = 13, 200–250 g) and female (6 wk, n = 13, 150–200g) Sprague-Dawley (SD) rats purchased from the Fourth Military Medical University were used as the organ recipients or donors. The rats were housed in a specific pathogen-free condition with humidity (60 ± 5%) and temperature (22 ± 2 °C)-controlled rooms under 12-h light/12-h dark cycles and were provided free access to food and water. All animal procedures were approved by the Animals Ethics Committee of the Fourth Military Medical University (IACUC-20190821).

Anesthesia Protocol

The experimental rats were anesthetized with 2% isoflurane in air (500 mL/min) and O2 (500 mL/min), followed with an intraperitoneal injection of 1% (10 mg/Kg) pentobarbital sodium immediately. The individual rat was then placed in a supine position on the operating table, and their limbs were secured with gauze.

Donor Surgery

The surgical procedure of donor preparation was similar to the study of Racho et al.14 Briefly, the female rats were used to achieve the left ovary, fallopian tube, uterine body, uterine horn, cervix, and upper vagina. The left common iliac vessels were selected as vascular grafts, and the separated vessels ranged from the left common iliac artery and vein to the left superior vesical artery and vein (Figure 1A). The left uterine artery and utero-ovarian vein were ligated to the branch of the renal blood vessels. The ureter and the accompanying vessels proximate to the bladder were ligated and cauterized. Symphysis pubis was opened by scissor carefully. The left horn of the uterus was dissected free from the underlying tissue, and the right uterine horn was ligated and excised at a site just cranial to its branching from the common uterine cavity. The vaginal vessels and vessels supplying the caudal portion of the cervix were separated and cauterized. The bladder, ureter, rectum, and upper vagina were separated. The upper vagina of about 0.7-cm length was kept for transplantation. Finally, the other blood vessels in the pelvis were ligated, and the whole grafts were kept in the donor until perfusion finished.

F1
FIGURE 1.:
The procedure of left inguinal uterus transplantation (UTx) in castrated male rats. A, Donor preparation: The left common iliac artery (LCIA) and left common iliac vein (LCIV) were used as the donor-anastomosed vessels. The left superior vesical artery and vein derived from the left common illac artery and vein supplied the left uterus with bloods. B, The left uterus, ovary, and upper vagina after perfusion. C, Recipient preparation: The left femoral artery (LFA) and left femoral vein (LFV) were used as the anastomosis vessels of receptors. D, Recanalization after vascular anastomosis was completed. E, The transplanted left uterine grafts in the groin after hyperemia. F, The closed inguinal incision and vaginal ostomy. AS, anastomotic site; LO, left ovary; LU, left uterus; UV, upper vagina.

The perfusion was performed in vivo. The left common iliac artery and vein were transected, and a catheter was inserted into the left common iliac artery. About 10 mL perfusion fluids composed of acetate Ringer solution (25 mol/L) and 0.4% heparin sodium were used to flush the graft until the tissue was blanched. The left uterus, ovary, and vagina along with common iliac vessels were procured (Figure 1B). The donor was euthanized finally.

Recipient Surgery

The male rats used as recipients were castrated before UTx. A small incision was made in the midline of the scrotum. The testes were squeezed out, and the spermatic cord blood vessels were ligated. Then, the testes were removed, and the scrotal skin incision was sutured. After castration, uterine transplantation including the left uterus, ovary, and upper vagina was performed. An incision was made in the left groin; then, the inguinal fat pad was removed to make room for the transplants. The left femoral vessels were identified (Figure 1C). The distal branches were ligated and dissected from the inguinal ligament until the branching of the saphenous vessels. Vascular clamps were placed proximally to the anastomosis sites. The grafts were placed parallel to the abdominal wall. Then, the left common iliac vessels of donor and femoral vessels of recipient were anastomosed in an end-to-end fashion with interrupted sutures (Figure 1D). After documenting vascular patency, the clamps on the blood vessels were removed. The grafts became reddish from pale (Figure 1E). Finally, the graft was fixed to the left abdominal wall with 6-0 silk. To secure the excretion of vaginal discharges, a cruciate incision was made in the inguinal skin, which was also used to fix the upper vagina. Finally, the inguinal incision was closed with 5-0 suture using continuous sutures (Figure 1F).

Grafts Follow-up

Empirical immunosuppression protocol was used to prevent immune rejection until biopsy.15 Cyclosporin A ([CsA] 10 mg/Kg, once a day and sustained for 30 d) was administered through intraperitoneal injection. Subcutaneous injection of heparin (400 IU/Kg) was also performed for 2 d to prevent thrombosis. Penicillin (400 IU/Kg) was used as antibiotic prophylaxis, and the antibiotic injection was sustained for 3 d. Tissue sampling was performed on day 30 after the operation.

Flow Cytometry

Blood samples from 4 recipient rats were taken after UTx, and the blood of normal, healthy male rats (n = 4) was used as control. Briefly, 100 μL whole bloods were added to 2 mL red cell lysing reagents. The mixture was incubated for 15 min at room temperature, following by centrifugation with 500g ×5 min at 4 °C. After washing with PBS, single-cell suspensions of peripheral blood cells were immunostained with fluorescence-conjugated antibodies (CD3, Cat 201403; CD4, Cat 201509; CD8, Cat 200607; CD45, Cat 202220; Biolegend) for 30 min at 4 °C in the dark environment. Isotype-matched antibodies corresponding to each specific antibody were used as negative controls. Finally, the cells were used for flow cytometry with an ACCURI C6 instrument.

HE Staining

A total of 13 UTx were performed. Of them, 6 recipients survived to day 30 after the surgery and were killed for sampling. Two recipients died on the 6th and 14th d after surgery, respectively. The remaining 5 recipients died because of postoperative graft thrombosis. The samples biopsied from the 6 survived recipients were fixed in 10% formaldehyde, dehydrated, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin and examined under the light microscopy.

TUNEL Assay

Four UTx ovaries and 4 normal ovaries were applied for TUNEL assay to analyze the apoptotic ovarian granulosa cells. The procedure was conducted using a TUNEL kit (Roche Applied Science, Indianapolis, IN), according to the protocol of the manufacturer.

Statistical Analysis

The results were expressed as median (range). The difference between the groups was analyzed with a Mann-Whitney U test or Kruskal-Wallis test. All analyses were performed with GraphPad Prism 6 (GraphPad, Inc) software. A value of P < 0.05 was considered to be statistically significant.

RESULTS

Operation Time

A total of 13 heterosexual UTx procedures were recorded. The time of the procedure was shown in Table 1. The whole procedure of the operation took about 145.15 ± 33.80 min, in which the acquisition of the donor grafts was about 44.77 ± 8.26 min and the recipient acquisition time was about 55.46 ± 9.05 min. The graft perfusion time was about 9.23 ± 4.11 min. The total vascular anastomosis time was 36.46 ± 8.57 min, in which the arterial anastomosis time was 17.15 ± 4.62 min, whereas the venous anastomosis time was 19.31 ± 5.23 min.

TABLE 1. - Operation time of the surgical procedures
Surgical procedure Time, minutes
Recipient operation 55.46 ± 9.05
Donor operation 44.77 ± 8.26
Perfusion 9.23 ± 4.11
Venous anastomosis 19.31 ± 5.23
Arterial anastomosis 17.15 ± 4.62
Total anastomosis 36.46 ± 8.57
Operation time 145.15 ± 33.80

Macroscopic Morphology of Transplants

Of the 13 heterosexual UTx rats, 7 of them died from vascular embolism and were excluded from further research. The remaining 6 animals were subjected to further investigations. During the process of biopsy at day 30 after UTx, 6 uteri, 4 ovaries, and 4 upper vaginas were found. The anastomotic vessels could also be examined. Besides, the grafts were surrounded by fibrous connective tissue. The vagina was connected to the surrounding tissue and appeared pink (Figure 2A). No obvious swelling was observed. The uterus was connected to the abdominal wall and showed a pink color (Figure 2B). Vascular patency and the anastomosed site were also found (Figure 2C). The ovary was indistinguishable from the connective tissue (Figure 2D).

F2
FIGURE 2.:
Macroscopic appearances of the grafts on day 30 after transplantation. A, The stoma was pink in color, and the anteroposterior vaginal walls were adjacent to the skin of the groin. B, Uterus adhered to abdominal wall with a good blood supply. C, Anastomosed blood vessels showing smooth blood flow. D, Uterus and ovary surrounded by fibrous connective tissue. AS, anastomotic site; LU, left uterus; UV, upper vagina.

Immunologic Changes of the Recipients

As shown in the results of flow cytometry (Table 2), the median percentage of CD3+ T lymphocytes in the blood from the UTx group was 9.6% and was less than that from the control group (38.3%; P < 0.05). In contrast to the control group, the median percentages of CD4+ T lymphocytes in the UTx group showed an upward trend after surgery, but no significant difference was found. The median percentages of CD8+ T lymphocytes in the UTx group were 43.1%, 51.8%, 60.6%, 59.4%, and 54.7% at days 6, 12, 18, 24, and 30 postoperation; compared with the median percentage of CD8+ T lymphocytes in the control group (30.2%), significant differences were found at days 18, 24, and 30 (P < 0.05). No significant differences were found in the median percentage ratio of CD4+/CD8+ T lymphocytes and the median percentages of CD45+ T lymphocytes between the control group and UTx group.

TABLE 2. - Phenotypic analysis of T lymphocytes in the recipients
Factor Control Day 6 Day 12 Day 18 Day 24 Day 30
n Median (range) (%) n Median (range) (%) n Median (range) (%) n Median (range) (%) n Median (range) (%) n Median (range) (%)
CD3 4 38.3 (32.2–47.4) 4 51.2 (27.0–59.7) 4 35.7 (15.3–41.9) 4 14.3 (7.3–24.3) 4 11.1 (10.6–26.6) 4 9.6 (3.7–19.5)a
CD4 4 37.4 (33.6–55.5) 4 46.5 (33.0–66.2) 4 56.4 (47.6–60.9) 4 59.0 (23.6–63.2) 4 61.1 (56.8–70.3) 4 54.4 (39.3–71.8)
CD8 4 30.2 (20.2–42.0) 4 43.1 (25.3–48.5) 4 51.8 (46.8–54.2) 4 60.6 (41.7–66.6)a 4 59.4 (48.7–67.3)a 4 54.7(50.6–61.9)a
CD4/CD8 4 1.3 (1.1–2.0) 4 1.2 (0.8–1.7) 4 1.1 (0.9–1.3) 4 1.0 (0.4–1.4) 4 1.1 (0.9–1.2) 4 1.0 (0.8–1.2)
CD45 4 95.6 (94.6–96.7) 4 98.4 (97.9–98.8) 4 92.9 (86.3–98.0) 4 90.6 (80.3–96.7) 4 98.0 (96.8–99.7) 4 99.1 (93.8–99.9)
aIndicated P < 0.05, compared with the control group.

Histological Analysis of Uteri

Grossly, the transplanted uteri retained complete structures composed of endometrium, myometrium, perimetrium, and glands. Dilated blood vessels can be found in all of the transplanted uteri. The histopathological changes of uteruses from UTx1 were similar to proestrus of female rats (Figure 3A); the endometrial epithelium was cuboidal to tall columnar and accompanied by mitotic activity. The endometrial stroma was infiltrated by a few leukocytes (Figure 3B). The uterine endometrial glands displayed dilation, and mitotic activity was also observed in the glandular cells (Figure 3C). The sample from UTx5 was similar to estrus (Figure 3D), and the uterine endometrial epithelium showed notable necrosis and severe leukocyte infiltration (Figure 3E). Besides, dilated glands were also examined. No mitotic activity or apoptosis appeared in the gland cells (Figure 3F). Regarding to the uterine samples from UTx2, UTx3, UTx4, and UTx6, they showed the histological changes similar to diestrus of female rats (Figure 3G), and the endometrial epithelium was reduced in size to lower columnar (Figure 3H). No characteristics of mitosis or apoptosis were found in the gland cells (Figure 3I).

F3
FIGURE 3.:
Histological features of the transplanted uteri. A, The whole morphology of UTx1. B, The endometrium was lined by cuboidal to columnar epithelium. Mitosis (yellow arrow) and infiltrated leukocytes (black arrows) were present in the transplanted uterus (UTx1). C, Uterine endometrial gland dilation and mitotic activity (yellow arrow, UTx1) were observed in the glandular cells. D, Image from the uterus of UTx5. E, The cells lining the endometrial lumen presented notable necrosis and infiltrated with lot of neutrophils (black arrows, UTx5). F, Dilated glands were examined (UTx5). G, The histological appearance of uterus of UTx4. H, Uterine endometrium lined by low columnar epithelium (UTx4). I, The endometrial gland cells showed no characteristics of mitotic activity or apoptosis (UTx4). UTx, uterus transplantation.

Histological Analysis of Vaginas

Four vaginas were found in the transplants. All the vaginas were composed of the mucosal layer, muscular layer, and fibrous layer, but distinct histopathological characteristics were found in the vaginal samples. In the specimen from UTx4 (Figure 4A), stratum germinativum, stratum granulosum, stratum corneum, and stratum mucification constituted the vaginal epithelium, and dilated blood vessels and mild edema can be seen in the vaginal stroma (Figure 4B). In vaginal samples from UTx1 and UTx5 (Figure 4C), similar histological changes were examined. Numerous dilated blood vessels were seen in the stroma. Variable numbers of leukocytes were present in them. Both attached and detached cornified epithelia were found, and the attached cornified epithelium covered most of the vaginas (Figure 4D). While in the vagina from UTx6 (Figure 4E), the stratum granulosum and stratum corneum were lost. Meanwhile, polymorphonuclear inflammatory cells were present within the degenerating stratum germinativum (Figure 4F).

F4
FIGURE 4.:
Histological characteristics of the transplanted vaginas. A, The gross appearance of vagina from UTx4. B, The vagina was composed of stratum germinativum (SGerm), stratum granulosum (SG), stratum corneum (SC), and stratum mucification (SM). C, Both attached and detached cornified epithelia were examined underneath the vagina from UTx1. D, Inflammatory cells (black arrows) infiltrated in the vaginal stroma. E, In the vagina from UTx6, the stratum corneum and stratum granulosum were lost. F, Polymorphonuclear inflammatory cell (black arrow) present in the vaginal epithelium. UTx, uterus transplantation.

Histological Analysis of Ovaries

In total, 4 transplanted ovaries were recovered. Grossly, the transplanted ovaries were enclosed by a large amount of fibrous tissue; the border between ovary and fibrous tissues was clear. The ovarian cortex and medulla were difficult to distinguish (Figure 5A). No ovulation was found. Follicles of different stages with normal or abnormal histological appearances were unevenly distributed. The normal primordial, primary, secondary, and tertiary follicles were shown in Figure 5B–E. The abnormal primordial, primary, secondary, and tertiary follicles were shown in Figure 5F–I. Besides, corpus luteum with different histological characteristics can be found. Some of the corpus luteum was accompanied by fibrous tissue proliferation in the central cavity, and cells with eosinophilic cytoplasm can be seen (Figure 5J and K). Interestingly, cells with basophilic cytoplasm can also be found in the corpus luteum (Figure 5L). Histologically, the majority of the follicles were abnormal or atretic. The atresia rates of primordial follicles, primary follicles, secondary follicles, tertiary follicles, and graafian follicles were 50% to 100%, 75% to 100%, 92% to 100%, 90% to 100%, and 100%, respectively (Table 3). The TUNEL assay further revealed a higher apoptosis of granulosa cells in the transplanted ovaries than in the normal ovaries (Figure 6A–F).

TABLE 3. - Quantification of the atretic follicles in the transplanted ovaries
Ovary Primordial follicles Primary follicles Secondary follicles Tertiary follicles Graafian follicles
Total Atresia Total Atresia Total Atresia Total Atresia Total Atresia
UTx1 2 1 (50%) 8 6 (75%) 12 11 (92%) 4 4 (100%) 2 2 (100%)
UTx2 12 10 (83%) 3 3 (100%) 3 3 (100%) 26 25 (96%) 1 1 (100%)
UTx4 4 3 (75%) 3 3 (100%) 7 7 (100%) 3 3 (100%)
UTx6 2 2 (100%) 3 3 (100%) 3 3 (100%) 10 9 (90%)
UTx, uterus transplantation.

F5
FIGURE 5.:
Histological analysis of transplanted ovaries. A, The transplanted ovary was surrounded by fibrous tissue. Follicles of different stages were found in the transplants. The normal primordial (B, black arrow), primary (C), secondary (D), and tertiary follicles (E) can be found, respectively. Abnormal primordial (F, black arrow), primary (G), secondary (H), and tertiary follicles (I) could also be observed. Corpus luteum with different histological characteristics were found in the transplanted ovaries. Some of the cells in the corpus luteum harbored eosinophilic cytoplasm. Fibrous tissue proliferation could also be found (J and K). L, Cells with basophilic cytoplasm can be observed in the corpus luteum.
F6
FIGURE 6.:
TUNEL staining affirmed the apoptosis of granulosa cells in both normal (A–C) and transplanted ovaries (D–F). The green fluorescence indicated the apoptotic cells (A and D). The nucleus was stained with DAPI (B and E). The merged images (C and F). The median numbers of apoptotic cells in the normal ovaries and transplanted ovaries were 10 (4, 28) and 82 (7, 387), respectively. **P < 0.01.

DISCUSSION

UTx is one of the most commonly used methods to solve the problem for absolute uterine factor infertility,16 and the female pelvic cavity is a candidate position for UTx. However, male individuals have a narrow pelvis, and combined ovario-utero-vaginal transplantation requires more space. Moreover, intraperitoneal uterine transplantation may also lead to infertility and chronic pain.17 Besides, in order to restore the original position of the vagina, the transplanted vagina should pass through the space between the rectum and bladder; this makes UTx surgery more complex and harder to perform. Then, we thought that ectopic UTx may help solve these problems using castrated male rats. Previously, the groin is a commonly used place for ectopic UTx. Jiga et al18 reported uterine transplantation in the groin of the same sex, using end-to-end anastomosis of donor abdominal aorta to recipient common femoral artery and donor inferior vena cava to recipient common femoral vein. They also found that transplanting the uterus into the groin made in vivo observation and repeated biopsies easier. In addition, reducing the influence of testosterone on grafts through orchieotectomy may contribute to graft survival and functional maintenance. Besides, bilateral UTx takes more time and is hard to perform. Therefore a unilateral inguinal transsexual UTx model in castrated rats using end-to-end anastomosis of donor common iliac vessels to recipient femoral vessels was established, 13 UTx surgeries were performed in total. Of these recipient rats, 6 animals were sacrificed for biopsy on day 30 after UTx; 2 recipients died on the 6th and 14th d after surgery, respectively. The remaining 5 recipients died because of postoperative graft thrombosis. During biopsy and sampling, we found that the grafts survived well in the recipient castrated rats, except that the grafts were surrounded by fibrous connective tissues. This suggests that the unilateral inguinal UTx model is a suitable alternative to study transsexual UTx.

During the process of biopsy and sampling after UTx, we monitored the immune rejection status of grafts. Macroscopically, the grafts were ruddy in color, soft in texture, and enclosed by fibrous tissues. No visible swelling and necrosis were seen. Histologically, inflammatory cells infiltration could be found in the transplants. Immunologically, a decreased median percentage of CD3+ lymphocytes in the blood was found after UTx, whereas the percentages of CD4+ cells kept relatively stable. The percentages of CD8+ lymphocytes cells increased after UTx. No significant difference was found in the ratio of CD4+/CD8+ lymphocytes and the percentages of blood CD45+ lymphocytes before and after UTx. The results of gross observation and flow cytometry detection demonstrated that the immune rejection was effectively controlled. This may be attributed to the use of the transsexual UTx model of SD female rats to castrated SD male rats. Some scientists believe that practicing UTx with a syngeneic model permits avoiding immune reaction19; the immunosuppressant may be not necessary. However, to ensure the survival and function maintenance of transplanted uteri, the application of immunosuppressant may be needed: first, previous study had confirmed that grade 1 acute rejection did occur after syngeneic transplantation.20 Monique et al21 found that the transplanted kidneys exhibited morphologic evidence of rejection in syngeneic SD to SD models. This suggested that the application of cyclophilin A may help reduce the occurrence of immune rejection. Cyclophilin A is an immunosuppressant widely used in rats and patients planning to conceive. Previous reports have proved that it had no severe reproductive toxicity.22 Wranning et al23 found that CsA delayed uterine graft rejection in the mild and long course. Csencsits et al24 also verified that the CD4+/CD8+ T-cell ratios were negatively correlated with the graft survival. Thus, CsA could effectively suppress the immune rejection according to the results of immunological molecules. Based on the complexity of human body, a more effective immunosuppressive regimen for heterosexual uterine transplantation should be established before transsexual UTx.

Similar histopathological changes corresponding to different stages of the female rat reproductive cycle were examined in the specimens. In the uterine samples from UTx1, cuboidal to tall columnar endometrial epithelium, dilated endometrial glands, and a few inflammatory infiltrations were found. Mitotic activity was examined in the endometrial epithelium and gland cells. These histological changes correspond to the morphological characteristics of proestrus of female rats. In the uterine samples from UTx5, cellular degeneration/necrosis occurred in the lining epithelium, and severe leukocyte infiltrations were found. However, no apparent apoptosis was noticed in the gland cells, which is different from the histological changes of estrus of normal female rat uterus. Regarding the samples from UTx2, UTx3, UTx4, and UTx6, which showed histological characteristics similar to diestrus of normal female rats, the endometrial epithelium was reduced in size to lower columnar. However, no mitosis and apoptosis were recorded in the gland cells.25,26 In summary, the transplanted uteri from the recipient male rats showed some of the histological changes of proestrus, estrus, and diestrus of female rats. However, whether a transplanted uterus in the castrated male recipient can experience the completed reproductive cycle of female rats deserves further investigations.

With regard to the transplanted vaginas, the vaginal epithelium from UTx4 was composed of stratum germinativum, stratum granulosum, stratum corneum, and stratum mucification, and dilated blood vessels and mild edema could be seen in the stroma, and these were similar to the histological changes of proestrus of female rats. Regarding the vaginal samples from UTx1 and UTx5, they showed some of the classic changes of estrus, for both the attached and detached cornified epithelia were examined, and a varying number of leukocytes were found. In terms of the vaginal specimen from UTx6, absent stratum granulosum and stratum corneum were found, and polymorphonuclear inflammatory cells appeared within the degenerating stratum germinativum, and these showed similar histological changes to metestrus of female rats. Surprisingly, synchronous histological changes of vaginas and uteruses corresponding to a specific stage of reproductive cycle were observed in only UTx5. To be specific, the vaginal histological changes of UTx1, UTx5, UTx4, and UTx6 were similar to estrus, estrus, proestrus, and metestrus, whereas the histological changes of uteri from UTx1, UTx5, UTx4, and UTx6 were similar to proestrus, estrus, diestrus, and diestrus, manifesting that the vaginal changes match the uterine changes in UTx5.

With regard to the transplanted ovaries, follicles of different stages could be found, but the atresia rate was high; this may attribute to the irregular regulation of hormones in the castrated male rats. In addition, the development of follicles is also controlled by the hypothalamic-pituitary-gonadal axis, but we do not know whether and how the difference in hypothalamic-pituitary-gonadal axis between the male and female rats will influence the follicles in the transplanted ovaries. In a normal ovary, except for a few follicles with normal structure, the majority of follicles were at the quiescent stage; the remaining ones showed a trend of apoptosis or atresia.27 The results of TUNEL assay further revealed an increased number of apoptotic granulosa cells. In the transplanted ovaries, cells with either eosinophilic cytoplasm or basophilic cytoplasm were present in the corpus luteum, and these represent the different features of corpus luteum being in distinct periods of reproductive cycle.28

Based on these results, we deduce that after transsexual UTx, the effects of combined ovarian transplantation on the survival and functional maintenance of transplanted uterus are limited, and hormone replacement therapy may be a better alternative method. Besides, transsexual UTx without ovaries will simplify the operation process and reduce the difficulty of the surgery, which may also be helpful to the success of transsexual UTx.

Regarding this study, 2 limitations need to be acknowledged. First, the study was performed with SD rats of different sex, which may not be able to effectively reflect the influence of immune rejection on the recipient and grafts during allogeneic uterine transplantation. The use of immunosuppressants may not be necessary for uterine transplantation using syngeneic animals. Second, we only established a unilateral inguinal UTx model in castrated male rats to test the possibility of transsexual UTx in a relatively short time, and this cannot evaluate the possibility of bilateral uterine transplantation in the male pelvis, especially for long-time monitoring.

CONCLUSIONS

A unilateral inguinal transsexual UTx model was established using castrated male rats; the transplanted uteruses and vaginas survived well in the recipients. Synchronous histological changes of uteruses and vaginas corresponding to a specific stage of reproductive cycle were rarely observed in the same recipients. Follicles of different stages with either normal or abnormal histological appearances were examined. These results may provide a reference for bilateral transsexual UTx in animals and genetically 46 XY individuals who wish to become real women through transsexual UTx.

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