Optimizing the place of autologous (heterotopic and orthotopic) ovarian transplantation following vitrification-warming procedure in rabbit

Optimizing the place of autologous (heterotopic and orthotopic) ovarian transplantation following vitrification-warming procedure in rabbit | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimizing the place of autologous (heterotopic and orthotopic) ovarian transplantation following vitrification-warming procedure in rabbit Ensiye Sajadian-Jaghaegh, Abbas Parham, Armin Attaranzadeh, Niloufar Tashakkori, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7212845/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: To maintain survival, notably for endangered species, it is essential to preserve female and male gametes. Ovarian tissue cryopreservation and subsequent re-transplantation can restore both endocrine function and fertility in humans and animals. The aim of this study was to evaluate the optimal transplantation site for ovarian tissue fragments after freezing/thawing for restoring fertility in rabbits. Methods: Rabbits were randomly assigned into 3 experimental groups according to ovarian auto-transplantation sites (n=6), as follows: I) cervical subcutaneous transplanted group (orthotopic transplantation), II) intramuscular transplanted group (orthotopic transplantation) and III) ovarian bursa transplanted group (heterotopic transplantation). After anesthesia, oophorectomy was performed on each rabbit. The ovarian cortex was separated from the medulla and divided into four strips. One strip was saved as a control in 10% formalin, the other three were frozen for two weeks, then one of the frozen ovarian parts, after warming process, was re-implanted into the implantation sites. Biopsies from ovarian strips were taken eight weeks after transplantation. Vaginal cytology and hormonal levels were determined a day before oophorectomy, two weeks after oophorectomy, and 2, 4, 6, and 8 weeks following implantation. Microscopically, the number and area (µm 2 ) of primordial follicles were measured. Results: The number of intact primordial follicles was significantly reduced in the re-implanted ovarian tissues compared with the fresh ovary in all groups. The implanted ovarian tissues in three groups did not show any evident changes in area and number of intact primordial follicle. There was a statistically significant difference in serum hormonal concentrations two weeks after oophorectomy compared to other sampling times in each group but, there were no significant differences among three groups as to the serum hormonal concentrations in implanted ovarian tissues. The present study revealed that the morphological changes of vaginal cells in the vaginal smear after implantation were similar to those before oophorectomy. Conclusions: Cryopreservation and implantation of small pieces of ovarian tissues could restore fertility and the endocrine function of ovarian tissue. Generally, the subcutaneous, ovarian bursa and intramuscular area are all available sites for implantation and have similar rates of acceptance, despite some differences in the details of implantation. This study can be used strategically to preserve the female gametes of endangered species, wild and important domestic animals. Cryopreservation Heterotopic transplantation Orthotopic transplantation Ovarian tissue Rabbit Figures Figure 1 Figure 2 Figure 3 Background Numerous exotic, domestic, and wild animal species have been lost during the last years [ 1 ]. Today, 24 species of mammals and a third of breeding animals have faced the threat of extinction [ 2 ]. It is essential to prevent other animals from facing a similar fate by implementing rescue strategies to guarantee their survival in the future [ 3 ]. The reproductive technologies developed to preserve human oocytes and ovarian tissues in patients with infertility disorders [ 4 ]. These methods can now be used in veterinary medicine for various reasons including preservation of species and the fight to preserve genetic diversity, reduction of polymorphism, inbreeding and inbreeding depression [ 2 ]. Some of these fertility preservation methods include cryopreservation of oocytes, embryos or ovarian tissue [ 5 ]. Cryo-banking of oocytes extracted from follicles or ovarian tissue may be considered as two approaches for fertility preservation [ 6 , 7 ]. The success rate of cryo-banking of oocytes is limited due to high volume-to-surface ratio, chilling injury, alteration in the zona pellucida and cumulus cell interaction [ 7 ]. Embryos cryopreservation has been introduced as a routine method since the 1980s, but this procedure is not suitable for immature females [ 8 ]. Ovarian tissue cryopreservation and re-transplantation is a relatively new technique that has emerged as a promising option for preserving fertility and restoring the endocrine function of the ovary in animals and women facing gonadotoxic treatments or premature ovarian failure [ 9 – 12 ]. The ovarian cortex contains many primordial follicles that could be cryopreserved for future transplantation or in vitro culture. Thus, cryopreservation of whole ovarian tissue or ovarian cortex can preserve female gametes [ 3 ]. Removing and freezing ovarian tissue, then thawing and transplanting it into the patient at desired times allows for the potential restoration of ovarian function, leading to natural conception [ 9 ]. Cryopreserving of ovary may be done using one of three methods: vitrification, slow-freezing and ultra-rapid freezing. In the vitrification procedure, ovarian tissue are subjected to high concentrations of cryo-protective agents to prevent the formation of ice crystals during rapid cooling in liquid nitrogen [ 13 , 14 ]. While the efficacy of this technique has been demonstrated in human studies [ 15 – 20 ], further research is necessary to optimize the procedure and ensure its safety and efficacy. In addition to the freezing-thawing process, the re-implantation site of ovarian tissue is important due to angiogenesis and nature gestation [ 2 ]. The ovarian autograft can be done either orthotopically or heterotopically [ 2 , 21 ]. It was indicated that the main loss of follicles is because of ischemia before revascularization after re-implantation [ 2 ]. Animal models can provide valuable insights into the potential complications and limitations of the procedure, allowing for the refinement of the technique before clinical use. Rabbits are often used as a pre-clinical model for studying ovarian tissue transplantation because of their similar reproductive anatomy and physiology to humans [ 22 ]. In the present study, the feasibility of autologous ovarian transplantation after a vitrification/warming cycle was evaluated in rabbit. The study assessed the viability, follicular density, and hormone levels of ovarian tissue transplanted using both heterotopic and orthotopic methods. The results of this study would offer implications for developing and optimizing this technique for clinical use in medicine and veterinary medicine. Materials and Methods Experimental design Eighteen mature female mixed breed rabbits with a mean weight of 3.2 kg were included in this study and individually caged in the animal center of the faculty of Veterinary Medicine, Ferdowsi University of Mashhad, and maintained under a 12-hour light-dark cycle, at 22–25°C, light off at 6 pm. The rabbits were acclimatized to their housing environment for a week with ad libitum access to food and water. All data used in this study was approved by the Ferdowsi University Animal Ethics Committee (IR.UM.REC.1400.335). The rabbits were randomly assigned into 3 experimental groups (n = 6), as follows: 1) cervical subcutaneous transplanted group (treatment 1), 2) intramuscular (opened gluteal muscle) transplanted group (treatment 2) and 3) ovarian bursa transplanted group (treatment 3). The rabbits were anesthetized with intramuscular injection of a cocktail of ketamine-HCl (50mg kg − 1 , Alfasan, Netherlands) and xylazin (5mg kg − 1 , Alfasan, Netherlands) [ 23 ]. Analgesia and antibacterial prophylaxis were performed using SC meloxicam 2% (0.2 mg kg − 1 , Meloxivet®, Iran, q24h for 2 days after surgery) [ 24 ] and IM cefazolin (100 mg, Cefzolix®, Iran) [ 23 ]. Following preparation of surgical site, bilateral oophorectomy was performed on each rabbit using midline approach [ 23 ]. The cortex of the ovaries was immediately separated from the medulla and divided into four fragments using a scalpel blade. One fragment of the fresh cortex from each rabbit was fixed in 10% formalin (Neutron-chemistry company, Iran) for histological evaluation; another three fragments were frozen into liquid nitrogen (-196̊ C) according to vitrification-warming procedure. Base on the group of study, after warming process, each one of the frozen ovarian strips was re-implanted into the implantation site as auto graft. Eight weeks later re-implanted ovarian strips were harvested from the implantation sites by surgical incision and fixed in formalin 10%. All fixed samples were submitted for histological evaluation [25]. Vitrification- warming procedure For the vitrification procedure, ovarian fragments were placed in two vitrification solutions (All chemicals used in the vitrification and warming processes were achieved from the company of Sigma-Aldrich chemie, Steinheim, Germany). The vitrification solutions were prepared as vitrification solution 1 (VS1) supplemented with 75 ethylene glycol (EG) + 75% dimethylsulphoxide (DMSO) in 10% Ham's tissue culture medium (HAMS) as a handling medium for 25 min at room temperature (25̊C), and vitrification solution 2 (VS2) consisted of 20% EG + 20% DMSO + 0.5 M/L sucrose in 10% 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES 3.5 ml) for 15 min (25°C). The ovarian fragments from each rabbit were placed in separate cryo-vials plunged into liquid nitrogen (-196°C) and stored until thawing [26]. Two weeks after vitrification, the thawing process was done, the cryovials were held at room temperature for 20 min then the ovary fragments were placed in thawing solution (TS1) (1M sucrose and 10% HEPES with HAMS as the base media), TS2 (10% HEPES and 0.5M sucrose), and TS3 (10% HEPES and 0.25M sucrose) for 1, 5, and 10 min (25̊C), respectively [26]. Vaginal cytology observation The vaginal smear was collected using a long-wet cotton swab with taking a thin smear of the vaginal mucosa. The smears were stained by Giemsa and studied under the light microscope at magnification of ×1000 [ 27 ]. The vaginal cells were classified, according to Tsiligianni et al. [ 28 ], as parabasal (rounded or oval cells with a high nuclear–cytoplasmatic ratio), intermediate (rounded, oval (small intermediate) or polygonal (large intermediate) cells with a structurally normal nucleus), superficial (polygonal cells with a pycnotic nucleus) and anuclear (polygonal/cornified epithelium with no nucleus and a clear cytoplasm). Vaginal cytology was performed a day before the start of bilateral oophorectomy, two weeks after bilateral oophorectomy and 2, 4, 6 and 8 weeks following transplantation. Endocrinologic evaluation Whole blood samples were collected from the marginal vein one day before bilateral ovariectomy, two weeks after bilateral oophorectomy and 2, 4, 6, 8 weeks after re-implanting the cortex strips into the implantation sites. Serum of whole blood was separated by centrifuging at 2500 rpm for 10 minutes and preserved at -20°C until endocrinologic evaluation. Serum concentration of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol (E2) were evaluated by the ELISA method [ 29 ]. Histopathological evaluation The histological samples included fresh and implanted ovarian tissues. Tissues were fixed in 10% formalin, and following tissue processing, ovarian samples were embedded in paraffin blocks, sectioned at five µm and stained with H&E [ 25 ]. The area (µm 2 ) and number of morphologically normal primordial follicles (oocytes surrounded by one layer of flattened granulosa cells) [ 30 ] was estimated by light microscopy (BX‑71, Olympus Co., Tokyo, Japan) (10 contiguous microscopic fields over the cortex biopsy at ×400 magnification). In order to avoid recounting of the same follicle, follicles were only analyzed when an oocyte nucleolus was present. Statistical analysis Data were analyzed using SPSS (IBM, SPSS, Inc) version 26. Kolmogorov-Smirnov test was used to evaluate the distribution of data. One-way repeated measure ANOVA or Friedman test on rank was conducted to compare the count and area of morphologically normal primordial follicles, serum LH, FSH and E2 level based on the normality test results. All results were compared pairwise using post hoc test. The Kruskal-Wallis H test or one-way ANOVA (with post hoc tests for pairwise analysis) was employed to compare survival proportion of primordial follicles between groups based on the normality test findings. Results were expressed as the mean ± SD and median (Q1, Q3) and P value < 0.05 was considered as significant level. Results All anesthesia and surgeries were performed smoothly. None of the animals were died or lost during the study; minor complications were recorded at the implantation site. An intact ovarian complex and circulation by revascularization established in implantation sites of rabbits. Of the 18 rabbits, 16 showed restored ovarian function, as evidenced by vaginal cytology, a reproductive endocrine profile and intact primordial follicles after re-implantation. Vaginal cytology Analysis of epithelial cell types within vaginal smears was observed to choose those rabbits with normal estrous cycle before the experiment and to evaluate the recovery of ovarian function after transplantation. Vaginal cytology revealed normal results before ovariectomy in all examined rabbits. The presence of intermediate and superficial cells in vaginal samples were evident in 16 rabbits with restored ovarian function two weeks after re-implanted ovarian surgery (Fig. 1 a-c). Vaginal smears obtained from two rabbits in group 2 and group 3 after implantation showed evidence of absence of ovarian function, by a predominance of parabasal cells with a minor presence of intermediate cells. Endocrinologic evaluation Ovarian hormones were detected in all rabbits. Measured serum levels of FSH, LH and E2 were considered as a normal reproductive hormonal profile before bilateral oophorectomy (Fig. 2 a-c). The level of serum FSH was higher at two weeks after bilateral oophorectomy compared to other sampling times in each group (P 0.05) (Fig. 2 a). Patterns of LH secretion were statistically different among the three groups two weeks after transplantation surgery (P < 0.05). The LH level in treatment group 3 was significantly lower than the other groups. Statistically significant differences in LH level at different sampling dates in each group were recorded (Fig. 2 b). The concentration of E2 showed that there is a significant difference between treatment one versus three and treatment two versus treatment three. Also, the E2 level at two weeks after bilateral oophorectomy was significantly different from sampling dates in each group (P < 0.05) (Fig. 2 c). Histopathological findings The area and the number of primordial follicles were evaluated in histological slides (Fig. 3 a-d). After re-implantation, total primordial follicle count was significantly lower in all groups than controls (P 0.05) (Table 1 ). No significant difference between the fresh and implanted ovary and also between groups was recorded in terms of primordial follicles areas (µm 2 ). There was not any healthy follicle in a histological slide of re-implanted ovary in cervical subcutaneous region in a rabbit and the number of atretic follicles were increased in this slide (Fig. 3 e). Dystrophic calcification of the ovarian cortex in one rabbit of the treatment group 3 was reported (Fig. 3 f). Table 1 Primordial follicular count and area (µm 2 ) of fresh and implanted ovary in treatment groups Fresh After Re- Implantation Count Area (µm 2 ) Count *** Area (µm 2 ) Treatment 1 129 (143, 79) 1257.69 ± 425.68 58.5 (74,0) 1181.20 ± 697.70 Treatment 2 107(151, 67) 1242.02 ± 362.17 56 (58, 34) 1336.36 ± 391.27 Treatment 3 118 (158, 78) 1409.09 ± 510.01 44 ( 58, 0) 1097.50 ± 637.13 Total primordial follicle count was significantly lower in all groups after re- implantation than fresh ovary (*** p < 0.001, ** 0.001 < p < 0.01 and * 0.01 < p < 0.05). No significant difference between the fresh and implanted ovary and among groups was recorded in primordial follicles areas. Discussion The successful cryopreservation of the ovarian cortex for long-term storage is essential in veterinary and human medicine [ 31 , 32 ]. Cryopreservation of the ovarian cortex is crucial in oocytes storage for future breeding plans especially in endangered animals when valuable animals die suddenly or undergo ovariectomy/ovariohysterectomy surgery for medical reasons [ 31 ]. Also, this method is helpful for fertility preservation in women who need immediate chemotherapy [ 33 ]. The vitrification method was used for cryo-banking of ovarian tissues in this study because this method is easy, cheap, and effective in preserving ovarian fragments compared to other freezing methods. Sajadian Jaghargh et al. showed that follicular development to the antral stage was observed in all implanted ovarian strips into gluteal muscle after vitrification/ warming procedure [ 14 ]. Also no significant difference between follicular proportions of the primary and secondary stages in slow-frozen and vitrified tissues immediately after warming were reported [ 11 ]. It remains to be studied which ovarian transplantation site is most effective and practical. The major challenge of ovarian transplantation is ischemia-reperfusion time. Factors influencing this period include the re-anastomosis duration and the vascular condition of the transplantation site [ 21 ]. It was reported that the cervical subcutaneous region, gluteus intermuscular, and back muscle sites were chosen as graft sites for fresh ovarian transplantation because they mimic of ovarian conditions and have suitable angiogenesis [ 21 , 30 ]. Based on these investigations, the present study selected the gluteus intermuscular and cervical subcutaneous regions as transplantation sites (heterotopic transplantation) following a freeze/thaw cycle and compared them to transplantation of the ovary after a freeze/thaw cycle to the ovarian bursa (orthotopic transplantation) in rabbit as an animal model. In this study, grossly, neo-vascularization surrounded the grafted strip of ovarian cortex well in all rabbits. Microscopically, dystrophic calcification of the ovarian cortex which occurs in damaged or necrotic tissue, may primarily be attributed to cryoinjury and subsequent microvascular perfusion of a large mass in one rabbit in treatment group 3 in this study. Additionally, the population of atretic follicles were increased due to the lack of nutrients caused by delayed re-angiogenesis in a histological slide of re-implanted ovary in cervical subcutaneous region (treatment group 1) in a rabbit in this study. It was revealed that there were some limitations on follicular growth and development, such as temperature variation, atmospheric pressure, and mechanical contacts in the subcutaneous region [ 21 ]. The present study investigated the number and area of primordial follicles as histological findings because these follicles constituted the dominant follicular population (more than 90%) in the ovarian cortex [ 1 ]. The small size, ability to grow in hypoxic condition, and relatively inactive metabolism of these follicles could make them more resistant to damage from vitrified-thawed procedures [ 1 , 21 , 25 , 33 ]. The mean count of primordial follicles decreased in the ovarian microvascular anastomosis surgery group (13.99 ± 3.21) versus the control group (18.68 ± 3.86) significantly [ 25 ]. Also, previous studies have reported a high percentage of follicular loss after transplantation [ 21 , 34 ]. In agreement with previous research, it has been shown that there was a significant reduction in the number of primordial follicles eight weeks after the implantation of the vitrified-thawed ovary in the present study. According to this data, the survival rate of primordial follicles has been reported as 38%, 48% and 53% in the treatment group 1, treatment group 2 and treatment group 3, respectively and there was no significant difference between groups. There were various reasons for primordial follicular loss after implantation, such as the lack of nutrients, hypoxia conditions, and the ischemic effects caused by delayed re-angiogenesis (main factor), developmental stages of follicles, and the cryoinjury [ 21 , 30 , 33 , 35 ]. Treatment with some antioxidants and antiapoptotic agents such as vascular endothelial growth factors (VEGF), transforming growth factors (TGF), fibroblast growth factors (FGF), and vitamin E could relieve hypoxic tissue damage by promoting neovascularization [ 35 ]. The reproductive endocrinology can be an accurate assessment of ovarian function. Callejo et al. reported a transient decrease in serum E2 level with an increase in serum FSH level after ovary removal [ 36 ]. It was noticed in this study that two weeks after ovariectomy, the values of FSH and LH were reported as remarkably higher, and E2 was reported as remarkably lower versus the values at other sampling times in study groups. Decreasing E2 may be due to the removal of the ovary as main source of E2 production and increasing the values of FSH and LH may be due to negative feedback with E2 production amount. Changes in FSH and E2 levels showed that heterotrophic allografted and xenografted fresh ovarian transplantation without vascular anastomosis likely had similar characteristics [ 29 ]. A study of ovarian auto-transplantation without a vascular pedicle in rats published that the E2 level produced by the ovary in the sub-peritoneal region was remarkably higher than the subcutaneous region near the inguinal plexus [ 37 ]. The elevation of the serum E2 value (57.5 pg/mL) emphasized the return of ovarian functions 14 days after heterotopic autografted of cryobanked ovarian tissue in a 37 years old woman [ 38 ]. The FSH, LH, E2, and P4 measurements before surgery and one week and six months after ovarian transplantation with vascular anastomosis indicated restored ovarian function [ 25 ]. In agreement with research on ovarian transplantation after vitrification/ warming procedure, endocrine functions of the ovary were restored 2 weeks after transplantation in this study. Future research should optimize cryopreservation and thawing processes to reduce follicular loss. Studies are needed to explore the use of antioxidants and growth factors to improve revascularization and follicular survival post-transplantation [ 33 ]. Additionally, research on long-term outcomes and fertility restoration in larger animal models and eventually in clinical trials with humans will be essential. Conclusion The optimal implantation site has features such as suitable conditions for ovarian tissue survival, development and monitoring of follicles, oocyte harvesting and spontaneous ovulation [ 35 ]. These results statistically indicated that all three implantation sites could preserve a number of primordial follicles and restore the endocrine function of vitrified ovarian tissue. However, we have introduced opened gluteal muscle site as better auto-heterotopic transplantation place for preservation of frozen/thawed ovarian fragments than other two places in rabbits because of less surgery risks, more enable oocyte monitoring and frequent egg retrieval from graft, easier implantation and less damage to other tissues around the transplantation sites. These findings pave the way for further research and potential clinical applications in fertility preservation. Abbreviations VS vitrification solution EG ethylene glycol DMSO dimethylsulphoxide HAMS Ham's tissue culture medium TS thawing solution LH luteinizing hormone FSH follicle-stimulating hormone E2 estradiol Declarations Ethics approval and consent to participate All data used in this study was approved by the Ferdowsi University Animal Ethics Committee (IR.UM.REC.1400.335). Consent for publication Not applicable Availability of data and materials Except the raw data, authors accept sharing of the data after publishing the article. Acknowledgment The authors are gratefully acknowledged the support and guidance provided by the Physiology and Surgery Department of the Veterinary Clinic of Ferdowsi University of Mashhad, Iran throughout the course of this research. Also, the authors extend their thanks to Dr. Marzie Faezi for her constructive discussions and shared expertise that enriched the quality of this work. Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this paper. Competing interests The authors declare no competing interests. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contributions Ensiye Sajadian Jaghaegh: Data Curation, Investigation, Methodology, Resources, Validation, Writing – Original Draft Preparation. Abbas Parham: Methodology, Resources, Writing –Review & Editing. Armin Attaranzadeh: Conceptualization, Resources. Niloufar Tashakkori: Writing – Review & Editing. Ahmad Reza Mohammadnia: Data Curation, Investigation, Methodology, Resources, Formal Analysis, Supervision, Writing –Review & Editing. 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Fertil Steril. 2004;82(4):930–2. 10.1016/j.fertnstert.2004.02.137 . Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":952152,"visible":true,"origin":"","legend":"\u003cp\u003eThe smears of vaginal mucosa were made by Gimsa staining (×1000)\u003c/p\u003e\n\u003cp\u003ea: Intermediated cells in vaginal smear of the rabbit 4 weeks after re- implantation (treatment 1).\u003c/p\u003e\n\u003cp\u003eb: Parabasal cells in vaginal smear of the rabbit 8 weeks after re- implantation (treatment 2).\u003c/p\u003e\n\u003cp\u003ec: Anuclear superficial cells in a smear of vaginal cytology of the rabbit 2 weeks after re implantation (treatment 3).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7212845/v1/be6e5dfcb06b863f385dede0.png"},{"id":91991955,"identity":"8cba5cbc-1586-489e-a51c-93c7264dd635","added_by":"auto","created_at":"2025-09-23 12:55:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":359438,"visible":true,"origin":"","legend":"\u003cp\u003eThe serum level of FSH, LH and E2 at different times of study in treatment groups\u003c/p\u003e\n\u003cp\u003eFigure 2a: The serum level of FSH at different times of study in treatment groups\u003c/p\u003e\n\u003cp\u003eAll groups have a significant difference between time four against time five.\u003c/p\u003e\n\u003cp\u003eThere is also a significant difference between time two with other times. (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ** 0.001\u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and * 0.01\u0026lt; \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05). (Time 1: 2 weeks before bilateral ovariectomy, time 2: 2 weeks after bilateral ovariectomy, time 3: 2 weeks after ovary transplantation, time 4: 4 weeks after ovary transplantation, time 5: 6 weeks after ovary transplantation and time 6: 8 weeks after ovary transplantation).\u003c/p\u003e\n\u003cp\u003eFigure 2b: The serum level of LH at different times of study in treatment groups\u003c/p\u003e\n\u003cp\u003eThere is a significant difference between time 1 versus time 3** and time 4* in all groups. There is also a significant difference in time 2 with times 1, 3 *** and 4, 5 and 6**. There is a statistically significant difference between the treatment 1 group against the treatment 3 group and the treatment 2 group versus the treatment 3 group in time 3 of study. (***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ** 0.001\u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and * 0.01\u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). (Time 1: 2 weeks before bilateral ovariectomy, time 2: 2 weeks after bilateral ovariectomy, time 3: 2 weeks after ovary transplantation, time 4: 4 weeks after ovary transplantation, time 5: 6 weeks after ovary transplantation and time 6: 8 weeks after ovary transplantation).\u003c/p\u003e\n\u003cp\u003eFigure 2c: The serum level of LH at different times of study in treatment groups\u003c/p\u003e\n\u003cp\u003eThere is a significant difference between time 2 with other times. There is also a statistically significant difference between the treatment 1 group against the treatment 3 group and the treatment 2 group versus the treatment 3 group in time 4 of this study. (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ** 0.001\u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and * 0.01\u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). (Time 1: 2 weeks before bilateral ovariectomy, time 2: 2 weeks after bilateral ovariectomy, time 3: 2 weeks after ovary transplantation, time 4: 4 weeks after ovary transplantation, time 5: 6 weeks after ovary transplantation and time 6: 8 weeks after ovary transplantation).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7212845/v1/78e3020b5e2f2d805866ebd5.png"},{"id":91991969,"identity":"9a5c292a-3c5d-4b02-be8e-62d51f281f48","added_by":"auto","created_at":"2025-09-23 12:55:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7081196,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue sections stained with hematoxylin and eosin (H \u0026amp; E)\u003c/p\u003e\n\u003cp\u003ea: Fresh ovarian tissue (black arrow: primordial follicle) (Scale bar: 50 µm).\u003c/p\u003e\n\u003cp\u003eb: Ovarian piece implanted to cervical subcutaneous site (black arrow: primordial follicle) (Scale bar: 50 µm).\u003c/p\u003e\n\u003cp\u003ec: Ovarian piece implanted into opened gluteal muscle site (black arrow: primordial follicle) (Scale bar: 50 µm).\u003c/p\u003e\n\u003cp\u003ed: Ovarian piece implanted to ovarian bursa site (black arrow: primordial follicle, yellow circle: area of primordial follicle) (Scale bar: 50 µm).\u003c/p\u003e\n\u003cp\u003ee: Ovarian piece implanted to cervical subcutaneous site without healthy follicles (black arrow: atrophic follicle) (Scale bar: 50 µm)\u003c/p\u003e\n\u003cp\u003ef: Dystrophic calcification of the ovarian implanted to ovarian bursa site (black arrow: atrophic follicle) (Scale bar: 200 µm).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7212845/v1/2c7f50fa4ecfb2b7b358eec1.png"},{"id":93528362,"identity":"23bc8bba-f0f0-4d38-b035-f0405c5cf349","added_by":"auto","created_at":"2025-10-14 20:24:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8370872,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7212845/v1/b16a4bcd-f495-4277-a0af-b720ba64f877.pdf"},{"id":91991958,"identity":"f5a9036b-0548-4acd-a074-5821699df546","added_by":"auto","created_at":"2025-09-23 12:55:54","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":351886,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-7212845/v1/fbf2522afeb5b05d19bc7689.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimizing the place of autologous (heterotopic and orthotopic) ovarian transplantation following vitrification-warming procedure in rabbit","fulltext":[{"header":"Background","content":"\u003cp\u003eNumerous exotic, domestic, and wild animal species have been lost during the last years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Today, 24 species of mammals and a third of breeding animals have faced the threat of extinction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is essential to prevent other animals from facing a similar fate by implementing rescue strategies to guarantee their survival in the future [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The reproductive technologies developed to preserve human oocytes and ovarian tissues in patients with infertility disorders [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These methods can now be used in veterinary medicine for various reasons including preservation of species and the fight to preserve genetic diversity, reduction of polymorphism, inbreeding and inbreeding depression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Some of these fertility preservation methods include cryopreservation of oocytes, embryos or ovarian tissue [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cryo-banking of oocytes extracted from follicles or ovarian tissue may be considered as two approaches for fertility preservation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The success rate of cryo-banking of oocytes is limited due to high volume-to-surface ratio, chilling injury, alteration in the zona pellucida and cumulus cell interaction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Embryos cryopreservation has been introduced as a routine method since the 1980s, but this procedure is not suitable for immature females [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Ovarian tissue cryopreservation and re-transplantation is a relatively new technique that has emerged as a promising option for preserving fertility and restoring the endocrine function of the ovary in animals and women facing gonadotoxic treatments or premature ovarian failure [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The ovarian cortex contains many primordial follicles that could be cryopreserved for future transplantation or in vitro culture. Thus, cryopreservation of whole ovarian tissue or ovarian cortex can preserve female gametes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Removing and freezing ovarian tissue, then thawing and transplanting it into the patient at desired times allows for the potential restoration of ovarian function, leading to natural conception [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Cryopreserving of ovary may be done using one of three methods: vitrification, slow-freezing and ultra-rapid freezing. In the vitrification procedure, ovarian tissue are subjected to high concentrations of cryo-protective agents to prevent the formation of ice crystals during rapid cooling in liquid nitrogen [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. While the efficacy of this technique has been demonstrated in human studies [\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], further research is necessary to optimize the procedure and ensure its safety and efficacy. In addition to the freezing-thawing process, the re-implantation site of ovarian tissue is important due to angiogenesis and nature gestation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The ovarian autograft can be done either orthotopically or heterotopically [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It was indicated that the main loss of follicles is because of ischemia before revascularization after re-implantation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Animal models can provide valuable insights into the potential complications and limitations of the procedure, allowing for the refinement of the technique before clinical use. Rabbits are often used as a pre-clinical model for studying ovarian tissue transplantation because of their similar reproductive anatomy and physiology to humans [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the present study, the feasibility of autologous ovarian transplantation after a vitrification/warming cycle was evaluated in rabbit. The study assessed the viability, follicular density, and hormone levels of ovarian tissue transplanted using both heterotopic and orthotopic methods. The results of this study would offer implications for developing and optimizing this technique for clinical use in medicine and veterinary medicine.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eExperimental design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEighteen mature female mixed breed rabbits with a mean weight of 3.2 kg were included in this study and individually caged in the animal center of the faculty of Veterinary Medicine, Ferdowsi University of Mashhad, and maintained under a 12-hour light-dark cycle, at 22\u0026ndash;25\u0026deg;C, light off at 6 pm. The rabbits were acclimatized to their housing environment for a week with ad libitum access to food and water. All data used in this study was approved by the Ferdowsi University Animal Ethics Committee (IR.UM.REC.1400.335).\u003c/p\u003e\u003cp\u003eThe rabbits were randomly assigned into 3 experimental groups (n\u0026thinsp;=\u0026thinsp;6), as follows: 1) cervical subcutaneous transplanted group (treatment 1), 2) intramuscular (opened gluteal muscle) transplanted group (treatment 2) and 3) ovarian bursa transplanted group (treatment 3).\u003c/p\u003e\u003cp\u003eThe rabbits were anesthetized with intramuscular injection of a cocktail of ketamine-HCl (50mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Alfasan, Netherlands) and xylazin (5mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Alfasan, Netherlands) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Analgesia and antibacterial prophylaxis were performed using SC meloxicam 2% (0.2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Meloxivet\u0026reg;, Iran, q24h for 2 days after surgery) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and IM cefazolin (100 mg, Cefzolix\u0026reg;, Iran) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFollowing preparation of surgical site, bilateral oophorectomy was performed on each rabbit using midline approach [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The cortex of the ovaries was immediately separated from the medulla and divided into four fragments using a scalpel blade. One fragment of the fresh cortex from each rabbit was fixed in 10% formalin (Neutron-chemistry company, Iran) for histological evaluation; another three fragments were frozen into liquid nitrogen (-196̊ C) according to vitrification-warming procedure. Base on the group of study, after warming process, each one of the frozen ovarian strips was re-implanted into the implantation site as auto graft. Eight weeks later re-implanted ovarian strips were harvested from the implantation sites by surgical incision and fixed in formalin 10%. All fixed samples were submitted for histological evaluation [25].\u003c/p\u003e\u003cp\u003e\u003cb\u003eVitrification- warming procedure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the vitrification procedure, ovarian fragments were placed in two vitrification solutions (All chemicals used in the vitrification and warming processes were achieved from the company of Sigma-Aldrich chemie, Steinheim, Germany). The vitrification solutions were prepared as vitrification solution 1 (VS1) supplemented with 75 ethylene glycol (EG)\u0026thinsp;+\u0026thinsp;75% dimethylsulphoxide (DMSO) in 10% Ham's tissue culture medium (HAMS) as a handling medium for 25 min at room temperature (25̊C), and vitrification solution 2 (VS2) consisted of 20% EG\u0026thinsp;+\u0026thinsp;20% DMSO\u0026thinsp;+\u0026thinsp;0.5 M/L sucrose in 10% 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES 3.5 ml) for 15 min (25\u0026deg;C). The ovarian fragments from each rabbit were placed in separate cryo-vials plunged into liquid nitrogen (-196\u0026deg;C) and stored until thawing [26].\u003c/p\u003e\u003cp\u003eTwo weeks after vitrification, the thawing process was done, the cryovials were held at room temperature for 20 min then the ovary fragments were placed in thawing solution (TS1) (1M sucrose and 10% HEPES with HAMS as the base media), TS2 (10% HEPES and 0.5M sucrose), and TS3 (10% HEPES and 0.25M sucrose) for 1, 5, and 10 min (25̊C), respectively [26].\u003c/p\u003e\u003cp\u003e\u003cb\u003eVaginal cytology observation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe vaginal smear was collected using a long-wet cotton swab with taking a thin smear of the vaginal mucosa. The smears were stained by Giemsa and studied under the light microscope at magnification of \u0026times;1000 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The vaginal cells were classified, according to Tsiligianni et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], as parabasal (rounded or oval cells with a high nuclear\u0026ndash;cytoplasmatic ratio), intermediate (rounded, oval (small intermediate) or polygonal (large intermediate) cells with a structurally normal nucleus), superficial (polygonal cells with a pycnotic nucleus) and anuclear (polygonal/cornified epithelium with no nucleus and a clear cytoplasm).\u003c/p\u003e\u003cp\u003eVaginal cytology was performed a day before the start of bilateral oophorectomy, two weeks after bilateral oophorectomy and 2, 4, 6 and 8 weeks following transplantation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEndocrinologic evaluation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhole blood samples were collected from the marginal vein one day before bilateral ovariectomy, two weeks after bilateral oophorectomy and 2, 4, 6, 8 weeks after re-implanting the cortex strips into the implantation sites. Serum of whole blood was separated by centrifuging at 2500 rpm for 10 minutes and preserved at -20\u0026deg;C until endocrinologic evaluation. Serum concentration of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol (E2) were evaluated by the ELISA method [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistopathological evaluation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe histological samples included fresh and implanted ovarian tissues. Tissues were fixed in 10% formalin, and following tissue processing, ovarian samples were embedded in paraffin blocks, sectioned at five \u0026micro;m and stained with H\u0026amp;E [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The area (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e) and number of morphologically normal primordial follicles (oocytes surrounded by one layer of flattened granulosa cells) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] was estimated by light microscopy (BX‑71, Olympus Co., Tokyo, Japan) (10 contiguous microscopic fields over the cortex biopsy at \u0026times;400 magnification). In order to avoid recounting of the same follicle, follicles were only analyzed when an oocyte nucleolus was present.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using SPSS (IBM, SPSS, Inc) version 26. Kolmogorov-Smirnov test was used to evaluate the distribution of data. One-way repeated measure ANOVA or Friedman test on rank was conducted to compare the count and area of morphologically normal primordial follicles, serum LH, FSH and E2 level based on the normality test results. All results were compared pairwise using post hoc test. The Kruskal-Wallis H test or one-way ANOVA (with post hoc tests for pairwise analysis) was employed to compare survival proportion of primordial follicles between groups based on the normality test findings. Results were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and median (Q1, Q3) and \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as significant level.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAll anesthesia and surgeries were performed smoothly. None of the animals were died or lost during the study; minor complications were recorded at the implantation site. An intact ovarian complex and circulation by revascularization established in implantation sites of rabbits. Of the 18 rabbits, 16 showed restored ovarian function, as evidenced by vaginal cytology, a reproductive endocrine profile and intact primordial follicles after re-implantation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVaginal cytology\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnalysis of epithelial cell types within vaginal smears was observed to choose those rabbits with normal estrous cycle before the experiment and to evaluate the recovery of ovarian function after transplantation. Vaginal cytology revealed normal results before ovariectomy in all examined rabbits. The presence of intermediate and superficial cells in vaginal samples were evident in 16 rabbits with restored ovarian function two weeks after re-implanted ovarian surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c). Vaginal smears obtained from two rabbits in group 2 and group 3 after implantation showed evidence of absence of ovarian function, by a predominance of parabasal cells with a minor presence of intermediate cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEndocrinologic evaluation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOvarian hormones were detected in all rabbits. Measured serum levels of FSH, LH and E2 were considered as a normal reproductive hormonal profile before bilateral oophorectomy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c).\u003c/p\u003e\u003cp\u003eThe level of serum FSH was higher at two weeks after bilateral oophorectomy compared to other sampling times in each group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No difference was recorded among three other groups for this hormone (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003ePatterns of LH secretion were statistically different among the three groups two weeks after transplantation surgery (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The LH level in treatment group 3 was significantly lower than the other groups. Statistically significant differences in LH level at different sampling dates in each group were recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eThe concentration of E2 showed that there is a significant difference between treatment one versus three and treatment two versus treatment three. Also, the E2 level at two weeks after bilateral oophorectomy was significantly different from sampling dates in each group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistopathological findings\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe area and the number of primordial follicles were evaluated in histological slides (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). After re-implantation, total primordial follicle count was significantly lower in all groups than controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The survival rates were 38%, 53% and 48% in treatment 1, 2 and 3, respectively, which were not significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No significant difference between the fresh and implanted ovary and also between groups was recorded in terms of primordial follicles areas (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e). There was not any healthy follicle in a histological slide of re-implanted ovary in cervical subcutaneous region in a rabbit and the number of atretic follicles were increased in this slide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Dystrophic calcification of the ovarian cortex in one rabbit of the treatment group 3 was reported (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimordial follicular count and area (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e) of fresh and implanted ovary in treatment groups\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eAfter Re- Implantation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCount\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eArea (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCount ***\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eArea (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e129 (143, 79)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1257.69\u0026thinsp;\u0026plusmn;\u0026thinsp;425.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.5 (74,0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1181.20\u0026thinsp;\u0026plusmn;\u0026thinsp;697.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e107(151, 67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1242.02\u0026thinsp;\u0026plusmn;\u0026thinsp;362.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56 (58, 34)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1336.36\u0026thinsp;\u0026plusmn;\u0026thinsp;391.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e118 (158, 78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1409.09\u0026thinsp;\u0026plusmn;\u0026thinsp;510.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44 ( 58, 0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1097.50\u0026thinsp;\u0026plusmn;\u0026thinsp;637.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eTotal primordial follicle count was significantly lower in all groups after re- implantation than fresh ovary (***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ** 0.001\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and * 0.01\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNo significant difference between the fresh and implanted ovary and among groups was recorded in primordial follicles areas.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe successful cryopreservation of the ovarian cortex for long-term storage is essential in veterinary and human medicine [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Cryopreservation of the ovarian cortex is crucial in oocytes storage for future breeding plans especially in endangered animals when valuable animals die suddenly or undergo ovariectomy/ovariohysterectomy surgery for medical reasons [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Also, this method is helpful for fertility preservation in women who need immediate chemotherapy [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The vitrification method was used for cryo-banking of ovarian tissues in this study because this method is easy, cheap, and effective in preserving ovarian fragments compared to other freezing methods. Sajadian Jaghargh et al. showed that follicular development to the antral stage was observed in all implanted ovarian strips into gluteal muscle after vitrification/ warming procedure [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Also no significant difference between follicular proportions of the primary and secondary stages in slow-frozen and vitrified tissues immediately after warming were reported [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt remains to be studied which ovarian transplantation site is most effective and practical. The major challenge of ovarian transplantation is ischemia-reperfusion time. Factors influencing this period include the re-anastomosis duration and the vascular condition of the transplantation site [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It was reported that the cervical subcutaneous region, gluteus intermuscular, and back muscle sites were chosen as graft sites for fresh ovarian transplantation because they mimic of ovarian conditions and have suitable angiogenesis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Based on these investigations, the present study selected the gluteus intermuscular and cervical subcutaneous regions as transplantation sites (heterotopic transplantation) following a freeze/thaw cycle and compared them to transplantation of the ovary after a freeze/thaw cycle to the ovarian bursa (orthotopic transplantation) in rabbit as an animal model. In this study, grossly, neo-vascularization surrounded the grafted strip of ovarian cortex well in all rabbits. Microscopically, dystrophic calcification of the ovarian cortex which occurs in damaged or necrotic tissue, may primarily be attributed to cryoinjury and subsequent microvascular perfusion of a large mass in one rabbit in treatment group 3 in this study. Additionally, the population of atretic follicles were increased due to the lack of nutrients caused by delayed re-angiogenesis in a histological slide of re-implanted ovary in cervical subcutaneous region (treatment group 1) in a rabbit in this study. It was revealed that there were some limitations on follicular growth and development, such as temperature variation, atmospheric pressure, and mechanical contacts in the subcutaneous region [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe present study investigated the number and area of primordial follicles as histological findings because these follicles constituted the dominant follicular population (more than 90%) in the ovarian cortex [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The small size, ability to grow in hypoxic condition, and relatively inactive metabolism of these follicles could make them more resistant to damage from vitrified-thawed procedures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The mean count of primordial follicles decreased in the ovarian microvascular anastomosis surgery group (13.99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21) versus the control group (18.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86) significantly [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Also, previous studies have reported a high percentage of follicular loss after transplantation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In agreement with previous research, it has been shown that there was a significant reduction in the number of primordial follicles eight weeks after the implantation of the vitrified-thawed ovary in the present study. According to this data, the survival rate of primordial follicles has been reported as 38%, 48% and 53% in the treatment group 1, treatment group 2 and treatment group 3, respectively and there was no significant difference between groups. There were various reasons for primordial follicular loss after implantation, such as the lack of nutrients, hypoxia conditions, and the ischemic effects caused by delayed re-angiogenesis (main factor), developmental stages of follicles, and the cryoinjury [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Treatment with some antioxidants and antiapoptotic agents such as vascular endothelial growth factors (VEGF), transforming growth factors (TGF), fibroblast growth factors (FGF), and vitamin E could relieve hypoxic tissue damage by promoting neovascularization [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe reproductive endocrinology can be an accurate assessment of ovarian function. Callejo et al. reported a transient decrease in serum E2 level with an increase in serum FSH level after ovary removal [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. It was noticed in this study that two weeks after ovariectomy, the values of FSH and LH were reported as remarkably higher, and E2 was reported as remarkably lower versus the values at other sampling times in study groups. Decreasing E2 may be due to the removal of the ovary as main source of E2 production and increasing the values of FSH and LH may be due to negative feedback with E2 production amount. Changes in FSH and E2 levels showed that heterotrophic allografted and xenografted fresh ovarian transplantation without vascular anastomosis likely had similar characteristics [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A study of ovarian auto-transplantation without a vascular pedicle in rats published that the E2 level produced by the ovary in the sub-peritoneal region was remarkably higher than the subcutaneous region near the inguinal plexus [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The elevation of the serum E2 value (57.5 pg/mL) emphasized the return of ovarian functions 14 days after heterotopic autografted of cryobanked ovarian tissue in a 37 years old woman [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The FSH, LH, E2, and P4 measurements before surgery and one week and six months after ovarian transplantation with vascular anastomosis indicated restored ovarian function [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In agreement with research on ovarian transplantation after vitrification/ warming procedure, endocrine functions of the ovary were restored 2 weeks after transplantation in this study.\u003c/p\u003e\u003cp\u003eFuture research should optimize cryopreservation and thawing processes to reduce follicular loss. Studies are needed to explore the use of antioxidants and growth factors to improve revascularization and follicular survival post-transplantation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, research on long-term outcomes and fertility restoration in larger animal models and eventually in clinical trials with humans will be essential.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe optimal implantation site has features such as suitable conditions for ovarian tissue survival, development and monitoring of follicles, oocyte harvesting and spontaneous ovulation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These results statistically indicated that all three implantation sites could preserve a number of primordial follicles and restore the endocrine function of vitrified ovarian tissue. However, we have introduced opened gluteal muscle site as better auto-heterotopic transplantation place for preservation of frozen/thawed ovarian fragments than other two places in rabbits because of less surgery risks, more enable oocyte monitoring and frequent egg retrieval from graft, easier implantation and less damage to other tissues around the transplantation sites. These findings pave the way for further research and potential clinical applications in fertility preservation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003evitrification solution\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eethylene glycol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edimethylsulphoxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHAMS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHam's tissue culture medium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ethawing solution\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eluteinizing hormone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFSH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efollicle-stimulating hormone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eE2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eestradiol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data used in this study was approved by the Ferdowsi University Animal Ethics Committee (IR.UM.REC.1400.335).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcept the raw data, authors accept sharing of the data after publishing the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are gratefully acknowledged the support and guidance provided by the Physiology and Surgery Department of the Veterinary Clinic of Ferdowsi University of Mashhad, Iran throughout the course of this research. Also, the authors extend their thanks to Dr. Marzie Faezi for her constructive discussions and shared expertise that enriched the quality of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnsiye Sajadian Jaghaegh: Data Curation, Investigation, Methodology, Resources, Validation, Writing – Original Draft Preparation. Abbas Parham: Methodology, Resources, Writing –Review \u0026amp; Editing. Armin Attaranzadeh: Conceptualization, Resources. Niloufar Tashakkori: Writing – Review \u0026amp; Editing. Ahmad Reza Mohammadnia: Data Curation, Investigation, Methodology, Resources, Formal Analysis, Supervision, Writing –Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are gratefully acknowledged the support and guidance provided by the Physiology and Surgery Department of the Veterinary Clinic of Ferdowsi University of Mashhad, Iran throughout the course of this research. Also, the authors extend their thanks to Dr. Marzie Faezi for her constructive discussions and shared expertise that enriched the quality of this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCommin L, Buff S, Rosset E, Galet C, Allard A, Bruyere P, et al. Follicle development in cryopreserved bitch ovarian tissue grafted to immunodeficient mouse. Reprod Fertil Dev. 2012;24(3):461\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDemirci B, Lornage J, Salle B, Th\u0026egrave;rese Poirel M, Fran\u0026ccedil;ois Guerin J, Franck M. The cryopreservation of ovarian tissue: uses and indications in veterinary medicine. Theriogenology. 2003;60(6):999\u0026ndash;1010.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSantos RR, Amorim C, Cecconi S, Fassbender M, Imhof M, Lornage J, et al. Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of endangered species and breeds. Anim Reprod Sci. 2010;122(3\u0026ndash;4):151\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePicton HM. Preservation of female fertility in humans and animal species. 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Fertil Steril. 2004;82(4):930\u0026ndash;2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.fertnstert.2004.02.137\u003c/span\u003e\u003cspan address=\"10.1016/j.fertnstert.2004.02.137\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cryopreservation, Heterotopic transplantation, Orthotopic transplantation, Ovarian tissue, Rabbit","lastPublishedDoi":"10.21203/rs.3.rs-7212845/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7212845/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e To maintain survival, notably for endangered species, it is essential to preserve female and male gametes. Ovarian tissue cryopreservation and subsequent re-transplantation can restore both endocrine function and fertility in humans and animals.\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to evaluate the optimal transplantation site for ovarian tissue fragments after freezing/thawing for restoring fertility in rabbits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Rabbits were randomly assigned into 3 experimental groups according to ovarian auto-transplantation sites (n=6), as follows: I) cervical subcutaneous transplanted group (orthotopic transplantation), II) intramuscular transplanted group (orthotopic transplantation) and III) ovarian bursa transplanted group (heterotopic transplantation). After anesthesia, oophorectomy was performed on each rabbit. The ovarian cortex was separated from the medulla and divided into four strips. One strip was saved as a control in 10% formalin, the other three were frozen for two weeks, then one of the frozen ovarian parts, after warming process, was re-implanted into the implantation sites. Biopsies from ovarian strips were taken eight weeks after transplantation. Vaginal cytology and hormonal levels were determined a day before oophorectomy, two weeks after oophorectomy, and 2, 4, 6, and 8 weeks following implantation. Microscopically, the number and area (µm\u003csup\u003e2\u003c/sup\u003e) of primordial follicles were measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The number of intact primordial follicles was significantly reduced in the re-implanted ovarian tissues compared with the fresh ovary in all groups. The implanted ovarian tissues in three groups did not show any evident changes in area and number of intact primordial follicle. There was a statistically significant difference in serum hormonal concentrations two weeks after oophorectomy compared to other sampling times in each group but, there were no significant differences among three groups as to the serum hormonal concentrations in implanted ovarian tissues. The present study revealed that the morphological changes of vaginal cells in the vaginal smear after implantation were similar to those before oophorectomy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Cryopreservation and implantation of small pieces of ovarian tissues could restore fertility and the endocrine function of ovarian tissue. Generally, the subcutaneous, ovarian bursa and intramuscular area are all available sites for implantation and have similar rates of acceptance, despite some differences in the details of implantation. This study can be used strategically to preserve the female gametes of endangered species, wild and important domestic animals.\u003c/p\u003e","manuscriptTitle":"Optimizing the place of autologous (heterotopic and orthotopic) ovarian transplantation following vitrification-warming procedure in rabbit","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 12:55:49","doi":"10.21203/rs.3.rs-7212845/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bba7cad3-a21c-40e7-9c5b-437185e6d3e4","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-14T20:08:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 12:55:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7212845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7212845","identity":"rs-7212845","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}