Pretreatment with melatonin improves ovarian tissue cryopreservation for transplantation.

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Marcos Eiji Shiroma, Luciana Lamarão Damous, Fernanda Pereira Cotrim, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-93401/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2021 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Melatonin has anti-inflammatory and antioxidative actions at the mitochondrial level. This indole may protect the ovarian graft during the cryopreservation process. Therefore, our study aimed to determine whether melatonin pretreatment improves rat ovarian graft quality. Methods: Twenty female rats were allocated to two study groups of ten animals each: 1) control group: ovaries cryopreserved using the standard protocol; and 2) melatonin group: ovaries cryopreserved in a medium with melatonin. Following 24-h freezing, whole ovaries underwent autologous and avascular transplants with retroperitoneal placement. After postoperative (PO) day 15, daily vaginal smears were obtained for estrous cycle characterization. Between PO days 30 and 35, animals were euthanized and ovarian grafts were recovered for histological and immunohistochemical (Ki-67, cleaved caspase-3, TUNEL, von Willebrand factor, estrogen, and progesterone receptors) analysis. The ovaries of the three remaining rats from each group were studied immediately after thawing to assess cryopreservation effects. The ANOVA and the Tukey tests were used, and the rejection level of the null hypothesis was set at 0.05 or 5% (p<0.05). Results: Melatonin promoted faster restart of the estrous cycle and increased the expression of mature follicles, collagen type I, von Willebrand factor, Ki-67, and cleaved caspase-3 on corpora lutea and estrogen receptors in the ovaries. There was a reduction in apoptosis by TUNEL on follicles, corpora lutea, and collagen type III. Conclusion: Melatonin may promote the quality of ovarian grafts through the evaluated parameters. Reproductive function enhancement could be further studied. Endocrinology & Metabolism Sexual & Reproductive Medicine melatonin ovary transplantation fertility preservation cryopreservation tissue preservation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Recent advances in the treatment of oncological diseases have resulted in the significantly improved survival of young cancer patients [ 1 – 3 ], with a cure rate of over 90% [ 4 ]. In the USA, yearly estimates run as high as 60,000 new cancer cases in females younger than 40 years of age; of these, 4,000 are prepubertal children and adolescents [ 5 ]. However, both chemotherapy and radiotherapy may impair the reproductive future of these patients [ 1 , 3 , 4 – 7 ]. Premature ovarian failure is estimated to occur in up to 68% of the women treated with alkylating agents for breast cancer during menacme; in 38–57% of those aggressively treated with cytotoxic chemotherapy and radiotherapy for lymphomas; and in over 90% of the patients undergoing a conditioning regimen for bone marrow transplantation [ 1 – 4 ]. Radiotherapy is also known to destroy the follicle reserve [ 1 , 4 , 5 ]. Fertility preservation is a major cause of concern in young women. Preservation of the fertility of women is a challenge, likewise the offer of the best chances of maternity to patients at risk of premature, induced, or iatrogenic menopause [ 3 , 4 ]. Such an offer may be made not only to patients with malignant diseases, but also to patients with sickle cell anemia and thalassemia, who may require a bone marrow transplant, patients with lupus, or even lesions leading to bilateral oophorectomy, such as recurring cysts [ 1 – 4 ], all entailing reproductive failure. These are further reasons for improving the technique. The options for maintaining reproductive capacity include oocyte, embryo, and ovarian tissue freezing [ 3 – 5 ]. Ovarian tissue cryopreservation is the only technique still deemed experimental. It allows the storing of a large quantity of primordial and primary follicles quickly and at any phase of the menstrual cycle [ 1 , 6 ]. It is the only option for preserving fertility in prepubertal girls [ 1 , 2 , 4 , 5 , 7 ]. In cryopreserved ovarian tissue transplantation, one of the major difficulties to surmount is the improvement in the vascular bed of the receptor area [ 4 – 6 ], given the potential occurrence of ischemic lesions over the time it takes for graft revascularization [ 1 , 2 , 6 , 7 ]. This lesion results in fibrosis and apoptosis, which affect the follicle survival rate and graft lifespan, both key in fertility restoration [ 1 , 7 ]. Besides, follicle activation in the graft speeds up after transplantation, leading to discrepancies in cell maturation in the granulosa and oocyte development, possibly due to hypoxic stress and lack of anti-Mullerian hormone in the graft [ 4 ]. Finally, the freezing–thawing process induces structural and morphological changes, especially in the theca cell layer [ 4 ]. The main reasons for the ovarian graft’s limited quality are inflammatory processes and oxidative stress, which damage the cells and their mitochondria. These issues are the target of melatonin action [ 8 ]. Treatments such as ischemic preconditioning [ 9 ] and cell therapy with stem cells [ 10 ] have been studied by our group, with moderate benefits. Therefore, additional investigations are needed to improve the ovarian transplantation technique. One hypothesis for the impaired outcome is the presence of factors capable of interfering in tissue response, chief among them is a large number of free radicals derived from the procedure [ 1 ]. In this experimental research scenario, new techniques have been proposed, the most prominent of which is melatonin administration for reducing oxidative stress. Melatonin acts as a free radical scavenger with vast antioxidant power and antiapoptotic function [ 6 , 11 – 13 ]. Melatonin action diminishes free radical-induced lesions in several diseases, including cancer, and it also inhibits the mitochondrial apoptotic pathway as it reduces BCL2 expression and cleaved caspase-3 activity [ 6 , 14 ]. The antioxidant action of melatonin is more potent than that of vitamin C or E, as even its metabolites act as free radical scavengers, in a process called the cascade effect [ 12 , 13 ]. Besides acting directly against free radicals, melatonin activates antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase [ 12 , 13 , 14 ]. The surplus free radicals interact with lipids, proteins, and nucleic acids, causing the loss of membrane integrity, functional and structural changes in proteins, and nucleic acid lesions [ 13 , 15 ]. Melatonin action is also recognized in diverse biological functions, such as circadian cycle control and anticancer action and those related to the reproductive system (ovarian activity, pregnancy, and delivery)[ 11 , 13 ] and neuroendocrinology, cardiology, and neuroimmunology [ 15 ]. It is a hydrophilic and lipophilic molecule [ 11 , 13 , 15 ], easily diffusing in various subcellular compartments, such as membranes, cytoplasm, nucleus, and mitochondria [ 11 , 12 , 13 ]. There is evidence of functional enhancement in human thawed sperm quality when melatonin is applied to the cryopreservation medium [ 16 ]. Therefore, melatonin may favor cell survival. However, it is not clear if this propriety may occur during the cryopreservation of tissue fragments such as ovarian grafts. Thus, this study aimed to evaluate rat ovarian grafts with melatonin added to the culture medium prior to cryopreservation. Materials And Methods The study was approved by the local Ethics Committee on the Use of Animals (CEUA-FMUSP 024/15). The animal group consisted of 26 adult female Wistar rats ( Rattus norvegicus albinus ) aged 3 months and weighing approximately 250 g each. The animals were kept under proper conditions of temperature and feeding as well as under a controlled light/dark cycle of 12/12 h. Only animals with three regular estrous cycles were included. The animals were allocated to two study groups (n = 13 each): control and melatonin. In the control group, slow cryopreservation was performed according to the standard protocol with the M2 culture medium, dimethyl sulfoxide (DMSO) [ 17 ], and ethyl alcohol vehicle, whereas in the melatonin group, melatonin (Sigma Aldrich, Saint Louis, MO, USA) was added to the medium at a concentration of 10 − 7 M [ 12 ]. Three animals from each group underwent graft analysis immediately after thawing from cryopreservation to verify the potential differences between groups induced up to the freezing-thawing process itself. In both groups, the ovaries underwent slow cryopreservation and were kept in liquid nitrogen (N 2 ) for 24 h. Thawing took place at room temperature (25 °C). The ovaries were implanted in the retroperitoneum of their respective donors, one on each side of the aorta, without anastomosis, and fixed by means of a simple stitch with an unabsorbable thread (nylon 4 − 0). Estrous cycle control At the beginning of the experiment, vaginal smears were obtained daily, always at the same time (8–10 h AM), to characterize the estrous cycle using the Shorr-Harris technique. Only animals with regular estrous cycles of 4–5 days were used; diestrus was the standard phase for the surgical procedures (oophorectomy for cryopreservation and euthanasia). Daily (8–10 h AM) vaginal smear collection resumed from postoperative (PO) days 15 to 30; then euthanasia was performed as the animals entered the diestrus phase (PO day 30–35). Protocol for anesthesia After being weighed, the animals were anesthetized with xylazine (15 mg/kg) and ketamine (60 mg/kg) administered via intraperitoneal injection. Oophorectomy protocol After a median longitudinal opening of the abdominopelvic cavity, the ovaries were removed bilaterally and washed with saline solution (0.9% NaCl). The procedure was performed between 9 and 10 h of AM. Cryopreservation and thawing protocol The ovaries were placed in 1.2-mL cryotubes with 1 mL of 1.4 M DMSO as a cryoprotectant and the M2 medium with or without melatonin added to the medium, depending on the study group, and kept at room temperature for 5 min. Slow-freezing was performed using the CL-8800 temperature controller and the Cryogenesis software, and then controlling the freezer from 25–10 o C at 1 o C/min and to -7 o C at 0.5 o C/min, keeping the temperature for 5 min. Next, the temperature was lowered to -55 o C at 0.5 o C/min. At this point, the ovaries were transferred into liquid N 2 at -196 o C and kept for 24 h [ 17 ]. The cryotubes were thawed at room temperature until all the ice melted (15–20 min). The tissue was then transferred to 5 mL of TL-HEPES at room temperature for 10 min, while it was gently agitated to promote DMSO efflux. The tissue was kept in TL-HEPES at 37 o C until transplantation [ 17 ]. Collection and analysis of the material The ovarian grafts were recovered and cut in half for analysis. The rats were euthanized with a lethal dose of the previously employed anesthetics. Imaging and measurements were performed with a computer system comprised of a light microscope (Carl Zeiss) adapted to a high-resolution camera (Axio Cam MRC, Carl Zeiss) and a color video monitor. Measurements were taken with an image analysis software program (AxionVision REL 4.6, Carl Zeiss). Counting was always performed using four fields per animal. The analyses comprised: 1) Estrous cycle With the animal immobilized, the vaginal smear was first obtained using a swab impregnated with saline solution and then placed on a standard slide for subsequent staining using the Shorr-Harris technique [ 18 ]. The slides were subsequently analyzed under a light microscope at 10 × and 40 × magnification. The phases of the estrous cycle were determined according to the proportion of cells observed in the smears as follows: 1) proestrus: predominance of nucleated epithelial cells; 2) estrus: predominance of non-nucleated keratinized cells; 3) diestrus: equal proportion of leukocytes and nucleated keratinized epithelial cells [ 18 ]. 2) Histology To assess follicle development, the ovarian follicles were counted and then categorized into developing follicles, regardless of their stage and atretic follicles. The former were classified according to the degree of maturation as follows: immature follicles (including primordial, primary, and secondary), mature follicles (with a single voluminous antrum), and corpora lutea. For counting purposes, the ovarian follicles comprised of both viable and atretic follicles as well as both normal and degenerating corpora lutea [ 19 ]. Blood vessel count was performed in a 100-square-micrometer area in five randomly selected fields and analyzed by two independent observers. For evaluation of fibrosis, the slides were stained with picrosirius red, and measurements were taken in eight fields per animal in the ovarian stroma, with a magnification of 400 × times, and the results were expressed as a percentage of the positive area (unit/mm 2 ). The evaluation of the slides was conducted at our Medical Investigation Laboratory (LIM-58). For the quantification of the parameters evaluated, the images were captured using a high-resolution camera (AxioCam-MCR, Carl Zeiss) adapted to a light microscope (Axiolab, Carl Zeiss) adjusted to the 40 × objective lens [ 17 ]. 3) Immunohistochemistry Slides with sections of the ovarian grafts were stained using immunohistochemistry to measure the von Willebrand factor (AB6994, 1:100, Abcam Inc., Cambridge, MA, USA), Ki67 (M724001-2, 1:100, Dako North America Inc., Carpinteria, CA, USA), cleaved caspase-3 (SANT-SC-1226, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling, Roche, Indianapolis, IN, USA). Hormonal receptor expression for estrogen (E1644, 1:50, Springe Bioscience Corporation, Pleasanton, CA, USA) and progesterone (AB51896, 1:30, Abcam Inc., Cambridge, MA, USA) was also studied. All preparations were performed according to the manufacturer’s instructions. Microscopy images were obtained using a computer program (Leica DM2500), and quantifications were performed with the LeicaQWin V3 program. The red-brown coloration of the cytoplasm and nucleus of granulosa cells and antral follicles (for apoptosis and Ki-67) or stroma (for fibrosis and expression of endothelial cells) was considered as positive expression and any other color, as negative. Hormone receptors were evaluated in both the stroma and follicular cells. The analysis was performed in eight different fields per animal at 400 × magnification, and the results were expressed as a percentage of the positive area (unit/mm 2 ). The interpretation was performed by two independent and blinded investigators. Statistical analysis The data from each group of animals were analyzed according to the type of variables. The ANOVA and the Tukey tests were used, and the rejection level of the null hypothesis was set at 0.05 or 5% (p < 0.05). Results Evaluation after freezing–thawing Three animals from each group were analyzed immediately after the freezing-thawing process to verify the effects of melatonin specifically related to the cryopreservation process. Histological assessment of immature follicles, mature follicles, corpora lutea, and blood vessels showed no significant difference between the control and melatonin (Table 1 ). Table 1 Histological analysis Control Melatonin p Immature Follicles 10.33 ± 2.90 4.66 ± 0.88 0.13 Mature Follicles 11.00 ± 3.51 5.33 ± 1.20 0.20 Corpora Lutea 10.00 ± 3.46 10.33 ± 4.70 0.95 Blood vessels 4.66 ± 1.45 2.66 ± 1.76 0.43 Histological analysis comparing the use of melatonin added to cryopreservation medium or not, in rat ovarian slide, freezing-thawing group (n = 3 each group) Immunohistochemical assessment of the frozen-thawed group showed no significant difference in cleaved caspase-3, and TUNEL activities between the control and melatonin groups (Table 2 ). Table 2 Immunohistochemical analysis Control Melatonin p Caspase Stroma 1.61 ± 0.32 1.95 ± 0.33 0.48 Caspase Follicles 3.39 ± 0.75 2.37 ± 0.33 0.18 Tunel Stroma 0.05 ± 0.01 0.21 ± 0.09 0.05 Tunel Follicles 0.04 ± 0.01 0.07 ± 0.01 0.26 Immunohistochemical analysis comparing the use of melatonin added to cryopreservation medium or not, in rat ovarian slide, freezing-thawing group (n = 3 each group) Evaluation After Transplantation Ten animals in the melatonin and control groups were analyzed after autologous transplantation. 1) Estrous cycle evaluation For all animals in both groups, there was a characterization of the estrus phase of the estrous cycle, demonstrating ovarian hormonal activity. The melatonin group presented faster resumption of the estrous cycle after transplantation (16.22 ± 0.50 days) compared to the control group (20.75 days ± 1.89, p = 0.0017), indicating an enhancement in functional activity. 2) Morphology and morphometry The melatonin group had an increase in mature follicle count compared to the control group, but no significant difference was observed in immature follicles or corpora lutea expression. With regard to immature follicles, we did not find any significant difference between the groups, even on the primordial follicular account. Blood vessel count did not differ significantly between groups. The melatonin group showed enhancement and reduction in collagen type I and type III expression, respectively, compared to the control group (Table 3 , Fig. 1 ). Table 3 Histological analysis Control Melatonin p Immature Follicles 7.20 ± 3.23 6.00 ± 2.55 0.66 Mature Follicles * 3.01 ± 0.91 8.75 ± 2.02 0.04 Corpora Lutea 7.86 ± 1.71 5.22 ± 1.05 0.34 Collagen type I* 5.75 ± 0.52 8.52 ± 1.04 0.03 Collagen type III* 3.53 ± 0.28 1.49 ± 0.15 < 0.001 Collagen type I and III ratio 1.62 5.71 Blood vessels 20.13 ± 3.66 18.63 ± 3.65 0.99 Ovarian follicle density, collagen and blood vessels (mean ± standard deviation) comparing the use of melatonin added to cryopreservation medium or not, in rat ovarian slide, autologous cryopreserved grafts after 30th day of transplantation (n = 10 each group); *p < 0.05 T test 3) Immunohistochemistry: The melatonin group presented a significant increase in endothelial cells (vWF), cellular proliferation (Ki67), and estrogen receptors both on the follicles and corpora lutea, and apoptosis in the corpora lutea using the cleaved caspase-3 assay compared to that in the control group. There was a significant reduction in apoptosis using TUNEL on follicles and corpora lutea. There were no significant differences in apoptosis using the cleaved caspase-3 assay on follicles and progesterone receptors on follicles or corpora lutea between both groups (Table 4 , Figs. 2 – 7 ). Table 4 Immunohistochemical analysis Control Melatonin p Cleaved caspase 3 F 11.30 ± 1.87 13.70 ± 1.79 0.38 Cleaved caspase 3 CL 5.88 ± 0.82 24.50 ± 2.06 < 0.001* Tunel F 0.40 ± 0.14 0.04 ± 0.02 < 0.001* Tunel CL 1.58 ± 0.23 0.10 ± 0.03 < 0.001* Ki67 F 1.46 ± 0.29 4.09 ± 0.55 0.002* Ki67 CL 1.67 ± 0.33 5.27 ± 0.54 < 0.001* Estrogen receptor F 2.41 ± 0.93 5.37 ± 0.73 0.02* Estrogen receptor CL 2.25 ± 0.38 6.43 ± 0.85 < 0.001* Progesterone receptor F 4.26 ± 0.53 4.65 ± 1.13 0.77 Progesterone receptor CL 16.62 ± 1.07 13.47 ± 1.72 0.14 von Willebrand factor 1.69 ± 0.28 3.19 ± 0.38 0.003* Immunohistochemical analysis comparing the use of melatonin added to cryopreservation medium or not, in rat ovarian slide, autologous cryopreserved grafts 30 days after transplantation (n = 10 each group); * p < 0.05; F = follicle; CL = corpora lutea Discussion Some studies have suggested the beneficial effects of melatonin on ischemic-reperfusion injury [ 11 ]. In the ovary, there are references to two specific receptors for melatonin (MT1 and MT2) [ 20 ] of the three identified so far, and they act via cyclic AMP reduction [ 21 , 22 , 23 ]. The same receptors have been identified in human ovarian follicles [ 21 ]. They act on ovarian function, modulating steroidogenesis, thereby notably increasing progesterone synthesis [ 23 ] and that of its receptor. They also modulate the formation of corpora lutea, indicating optimization of the ovulatory function [ 22 ]. Changes in serum hormone levels are related to ovulatory disorders in both rats and humans [ 21 ]. Melatonin-deprived rats have a persistent anovulatory cycle, premature vaginal opening, ovarian hypertrophy, and increased vaginal cell cornification. Moreover, melatonin replacement reverses these effects. The hormone is found in ever higher concentrations in the ovarian follicle as it develops, reaching maximum concentration, when compared to plasma in the preovulatory follicle [ 11 , 13 , 15 ]. The balance between free radicals and antioxidants in the ovarian follicle appears to be fundamental for the proper functioning of oocytes and granulosa cells [ 11 , 13 , 15 ]. Moreover, melatonin reduction also decreases embryo implantation [ 21 ]. These are well-known properties of melatonin in the reproductive system. However, the action of melatonin on free radicals and antioxidants to reduce the damage to the ovarian graft is a new direction for applying this indolamine. Our study found that previous melatonin treatment on the graft was beneficial during the cryopreservation and transplantation of the rat ovarian graft. The first study on the effect of melatonin on rat ovarian autotransplantation reported the indirect benefits of the intraperitoneal administration of the substance [ 24 ]. The authors described low levels of malondialdehyde as well as high superoxide dismutase and glutathione peroxidase levels with low ovarian necrosis in melatonin-treated rats. Another study with animal xenotransplant treated with melatonin via the oral route and vitamin E showed improvement in ovarian graft survival as evidenced by follicle count, the apoptosis index, and VEGF and PCNA expression [ 6 ]. None of these previous studies investigated the use of melatonin in the medium used for cryopreserving ovarian grafts. Our results suggest that melatonin pretreatment in the cryopreservation medium also presents beneficial effects on ovarian graft after transplantation: a) precocious resumption of the estrous cycle; b) a higher number of mature follicles; c) enhancement in tissue proliferation; and d) reduction in apoptosis. The animals subjected to subsequent ovarian transplantation showed that the melatonin group demonstrated faster recovery of regular estrous cycles compared to the control. This finding suggests that melatonin could have enabled better conditions for ovulatory and hormonal activity recovery. Histological analysis showed that the melatonin group presented more mature follicles than the control group, indicating that the indolamine promoted enhancement in the graft function. In contrast, corpora lutea counting did not show any significant difference between groups. In rats, the estrous cycle can present corpora lutea formed by previous cycles, thus limiting quantitative comparison, which may explain this result. Blood vessel quantification did not differ between groups, and the melatonin group showed an enhancement in the von Willebrand factor expression, compared to the control. Previous studies, although not involving transplantation, have also reported such diverse effects of melatonin on blood vessel proliferation [ 22 , 25 ]. Melatonin may have a modulatory influence on vessels in different biological scenarios [ 26 ]. Estrogen receptor expression was significantly higher in the melatonin group than in the control group. Indolamines can directly activate this hormone receptor activity or indirectly through the enhancement in follicular cell proliferation, which in turn synthesizes more estrogen. This hormone is the main representative activity of ovulatory follicles [ 27 ], thereby inferring that melatonin can enhance graft hormonal performance. The present study is the first to assess progesterone receptors in transplanted rats and found no significant difference between the groups. An extended period of analysis may suggest that the application of melatonin to the cryopreservation medium could enhance progesterone receptor expression. Melatonin can modulate the apoptosis process [ 28 ]. From the present study the melatonin group showed an increase in cleaved caspase-3 on corpora lutea and no difference in follicles. For follicles, melatonin increased follicle development which could be related to diminish cleaved caspase-3. For corpora lutea, the natural degradation process after ovulation might be related to the enhancement in cleaved caspase-3. This study is the first to analyze cleaved caspase-3 on ovarian rat autotransplantation treated with melatonin and maybe further studies might confirm how melatonin modulates cleaved caspase-3 activity in this scenario. DNA fragmentation analysis using the TUNEL assay is the final result of cell degeneration either through apoptosis cascade or not [ 29 ]. The present study revealed that TUNEL quantification was significantly reduced in the melatonin group, both in follicles and corpora lutea, compared to the control group. This finding relates to the most consolidated biochemical property of melatonin investigated in various tissues reported in the literature: the oxidative free radical scavenger, which could reduce DNA degradation, independently of the apoptotic pathway. Among experimental ovary transplantation studies, the first research to investigate the effect of melatonin also indicated a reduction in TUNEL [ 24 ]. This research revealed that the melatonin group showed enhancement in type I collagen and reduction in type III collagen compared to the control. Previous studies have related collagen expression to tissue response against hypoxia. Fibroblast activity in tissue cultures have correlated an increase in type I collagen [ 30 ] and a reduction in type III collagen [ 31 ] with better oxygenation. This result corroborates other reports of the present study, indicating that melatonin promoted better functional and follicular activity in the ovarian graft. The ovulation process is indeed related to the production and lysis of collagen in a dynamic process that ultimately produces ovarian follicle rupture and oocyte liberation [ 32 ]. Conclusion Melatonin may promote a better quality of ovarian grafts by evaluating histological and protein expression parameters. Future research can further assess the potential enhancement provided by melatonin in graft reproductive performance. List Of Abbreviations PO postoperative DMSO dimethyl sulfoxide vWF von Willebrand factor TUNEL MT VEGF PCNA terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling receptors for melatonin vascular endothelial growth factor proliferating cell nuclear antigen Declarations Ethics approval The study was approved by the University of Sao Paulo Faculty of Medicine Ethics Committee on the Use of Animals (CEUA-FMUSP 024/15). Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES). Authors' contributions Marcos Eiji Shiroma, Luciana Lamarão Damous and José Maria Soares-Jr contibuted to the study conception and design. Marcos Eiji Shiroma and Luciana Lamarão Damous made acquisition of data. Marcos Eiji Shiroma, Luciana Lamarão Damous and José Maria Soares-Jr analyzed the data. Edmund Chada Baracat, Russel Joseph Reiter, José Cipolla-Neto, Fernanda Pereira Cotrim and Cristiane Lima Roa contributed revising the work. All authors read and approved the final manuscript. Acknowledgments Authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) for the support. 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Hemadi M , Saki G, Shokri S, Ghasemi Follicular dynamics in neonate vitrified ovarian grafts after host treatment with melatonin. Folia Morphol (Warsz). 2011;70:18-23. Mirza-Aghazadeh-Attari M, Reiter RJ, Rikhtegar R, Jalili J, Hajalioghli P, Mihanfar A, Majidinia M, Yousefi B. Melatonin: An atypical hormone with major functions in the regulation of angiogenesis. IUBMB Life. 2020;72:1560-84 Drummond AE, Fuller Ovarian actions of estrogen receptor-β: an update. Semin Reprod Med. 2012;30:32-8 Rodriguez C, Martín V, Herrera F, García-Santos G, Rodriguez-Blanco J, Casado-Zapico S, Sánchez-Sánchez AM, Suárez S, Puente-Moncada N, Anítua MJ, Antolín I. Mechanisms involved in the pro-apoptotic effect of melatonin in cancer cells. Int J Mol Sci. 2013;14:6597-613. Negoescu A, Guillermet C, Lorimier P, Brambilla E, Labat-Moleur F. Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity. Biomed Pharmacother. 1998;52:252-8. Hara-Saito Y, Kato H, Saito N, Shiomi A, Uenoyama A, Takagi R, Izumi K. Distinct differences in hypoxic responses between human oral mucosa and skin fibroblasts in a 3D collagen matrix. In Vitro Cell Dev Biol –Animal.2020;56:452-79. Wang X, Lin L,Chai X, Wu Y, Li Y, Liu X. Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblastos. Int J Mol Med. 2020;45:175-85. Lofman C, Zackrisson U, Mikuni M, Block M, Janson PO, Brannstrom M. A method for longitudinal microscopic in vivo examinations of morphology, vascularity, and motility in the ovary and the oviduct of the rat. J Soc Gynecol Investig. 2002;6:379-85.Top of Form Cite Share Download PDF Status: Published Journal Publication published 03 Feb, 2021 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted Editorial decision: Major revision 06 Nov, 2020 Review # 1 received at journal 01 Nov, 2020 Review # 2 received at journal 25 Oct, 2020 Reviewer # 2 agreed at journal 22 Oct, 2020 Reviewer # 1 agreed at journal 21 Oct, 2020 Reviewers invited by journal 08 Oct, 2020 Editor assigned by journal 07 Oct, 2020 First submitted to journal 06 Oct, 2020 Submission checks completed at journal 06 Oct, 2020 Editor invited by journal 06 Oct, 2020 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-93401","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":3560254,"identity":"d38dc425-d342-45a4-9eb6-1a57e93bdbec","order_by":0,"name":"Marcos Eiji Shiroma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYHACNoYEEMXeACQMLEjRwnMApEWCSC1gIAHWSIQW3fb2Zw8e7rGJ5p/5/OqGHwUSDPzt3Ql4tZidOWNukPAsLXfG7Zyymz1Ah0mcObsBv5YbOWwSCQcO5zbczkm7wQPUYiCRS0DL/efPwFrm3zyTdvMPUVpuMJiBtWy4wX7sNnG2nMkBaUnL3Xgmh+22jIEED2G/HD/+TPLHAZvceUDGzTd/bOT423vxa0ECPAZgkljlIMD+gBTVo2AUjIJRMIIAABxwTa/MnqYNAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0318-9489","institution":"Universidade de Sao Paulo Faculdade de Medicina Hospital das Clinicas","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Marcos","middleName":"Eiji","lastName":"Shiroma","suffix":""},{"id":3560255,"identity":"a2804df6-7bb3-4ae8-a92d-a91a1cff0142","order_by":1,"name":"Luciana Lamarão Damous","email":"","orcid":"","institution":"Universidade de Sao Paulo Faculdade de Medicina","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luciana","middleName":"Lamarão","lastName":"Damous","suffix":""},{"id":3560256,"identity":"0aadae12-f8af-4e6c-b4f1-7bdf77e26cd8","order_by":2,"name":"Fernanda Pereira Cotrim","email":"","orcid":"","institution":"Universidade de Sao Paulo Faculdade de Medicina","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fernanda","middleName":"Pereira","lastName":"Cotrim","suffix":""},{"id":3560257,"identity":"9a56d142-ae3c-4895-9da6-81511796bbe1","order_by":3,"name":"Cristiane Lima Roa","email":"","orcid":"","institution":"Universidade de Sao Paulo Faculdade de Medicina","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cristiane","middleName":"Lima","lastName":"Roa","suffix":""},{"id":3560258,"identity":"4914c1f7-505e-4ea8-bb54-245097294614","order_by":4,"name":"José Cipolla-Neto","email":"","orcid":"","institution":"Universidade de Sao Paulo Instituto de Ciencias Biomedicas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"José","middleName":"","lastName":"Cipolla-Neto","suffix":""},{"id":3560259,"identity":"c11c7372-ff93-45f4-af7c-200ceb5f5521","order_by":5,"name":"Russel Joseph Reiter","email":"","orcid":"","institution":"University of Texas UTHSCSA: The University of Texas Health Science Center at San Antonio","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Russel","middleName":"Joseph","lastName":"Reiter","suffix":""},{"id":3560260,"identity":"3c7c6829-0312-4fef-8640-448737ff6173","order_by":6,"name":"Edmund Chada Baracat","email":"","orcid":"","institution":"Universidade de Sao Paulo Faculdade de Medicina","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Edmund","middleName":"Chada","lastName":"Baracat","suffix":""},{"id":3560261,"identity":"1d9f37e3-85bb-4530-bd76-8487d48a0edf","order_by":7,"name":"José Maria Soares","email":"","orcid":"","institution":"Universidade de Sao Paulo Faculdade de Medicina","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"José","middleName":"Maria","lastName":"Soares","suffix":""}],"badges":[],"createdAt":"2020-10-15 20:14:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-93401/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-93401/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12958-021-00705-4","type":"published","date":"2021-02-03T15:02:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3071193,"identity":"99417cf2-3633-49eb-b31b-3cf837276c72","added_by":"auto","created_at":"2020-10-19 22:15:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":122228,"visible":true,"origin":"","legend":"Picrosirius stain analysis of types I and III collagen comparing the use of melatonin added to cryopreservation medium or not, in ovarian rat autologous cryopreserved grafts 30 days after transplantation (n=10 each group); A control, type I collagen, 400x; B melatonin, type I collagen, 400x; C control, type III collagen, 400x; D melatonin, type III collagen, 400x","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-93401/v1/b864aa33bed4645d4e10bd82.jpg"},{"id":3071194,"identity":"f525f289-8a16-4c31-a0ea-5f4d53e3c8d2","added_by":"auto","created_at":"2020-10-19 22:15:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":161341,"visible":true,"origin":"","legend":"Immunohistochemical analysis of cleaved caspase 3 comparing the use of melatonin added to cryopreservation medium or not, in ovarian rat autologous cryopreserved grafts 30 days after transplantation (n=10 each group); A control, follicle, 400x; B melatonin, follicle, 400x; C control, corpus luteum, 400x; D melatonin, corpus luteum, 400x; E negative control, 100x","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-93401/v1/c671732621eda70f7ffccba8.jpg"},{"id":3071195,"identity":"95c47523-295b-47d0-8ad1-448605a01458","added_by":"auto","created_at":"2020-10-19 22:15:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139461,"visible":true,"origin":"","legend":"Immunohistochemical analysis of TUNEL comparing the use of melatonin added to cryopreservation medium or not, in ovarian rat autologous cryopreserved grafts 30 days after transplantation (n=10 each group); A control, follicle, 400x; B melatonin, follicle, 400x; C control, corpus luteum, 400x; D melatonin, corpus luteum, 400x; E negative control, 100x","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-93401/v1/ea7111c217aecec860b5eaeb.jpg"},{"id":3071196,"identity":"aa851e3b-12bf-4dc6-b98f-176a30db8b35","added_by":"auto","created_at":"2020-10-19 22:15:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":144687,"visible":true,"origin":"","legend":"Immunohistochemical analysis of Ki-67 comparing the use of melatonin added to cryopreservation medium or not, in ovarian rat autologous cryopreserved grafts 30 days after transplantation (n=10 each group); A control, follicle, 400x; B melatonin, follicle, 400x; C control, corpus luteum, 400x; D melatonin, corpus luteum, 400x; E negative control, 100x","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-93401/v1/1a287cb0af5757c1c2296753.jpg"},{"id":3071197,"identity":"7ee6e85e-458c-4d59-94bf-66923b3ab710","added_by":"auto","created_at":"2020-10-19 22:15:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":141749,"visible":true,"origin":"","legend":"Immunohistochemical analysis of estrogen receptor comparing the use of melatonin added to cryopreservation medium or not, in ovarian rat autologous cryopreserved grafts 30 days after transplantation (n=10 each group); A control, follicle, 400x; B melatonin, follicle, 400x; C control, corpus luteum, 400x; D melatonin, corpus luteum, 400x; E negative control, 100x","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-93401/v1/f5d3cc113e35330d2c22eb43.jpg"},{"id":3071198,"identity":"e8d89394-f22c-47c6-a753-2de81de896b5","added_by":"auto","created_at":"2020-10-19 22:15:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":183993,"visible":true,"origin":"","legend":"Immunohistochemical analysis of progesterone receptor comparing the use of melatonin added to cryopreservation medium or not, in ovarian rat autologous cryopreserved grafts 30 days after transplantation (n=10 each group); A control, corpus luteum, 400x; B melatonin, corpus luteum, 400x; C control, follicle, 400x; D melatonin, follicle, 400x; E negative control, 100x","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-93401/v1/4a807dc5ef27d8f8edb93ec8.jpg"},{"id":3071199,"identity":"29cb84aa-41dc-4476-a815-831ae6e60992","added_by":"auto","created_at":"2020-10-19 22:15:21","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87939,"visible":true,"origin":"","legend":"Immunohistochemical analysis of von Willebrand factor comparing the use of melatonin added to cryopreservation medium or not, in ovarian rat autologous cryopreserved grafts 30 days after transplantation (n=10 each group); A control, 400x; B melatonin, 400x; C negative control, 100x","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-93401/v1/2131d64999ba06610e3fb75e.jpg"},{"id":13605151,"identity":"6a9a24bb-7c3f-45d3-a453-be1b261b5e4d","added_by":"auto","created_at":"2021-09-17 06:02:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":937963,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-93401/v1/a08dca7e-25d0-43ff-ba15-14b230766b01.pdf"}],"financialInterests":"","formattedTitle":"Pretreatment with melatonin improves ovarian tissue cryopreservation for transplantation.","fulltext":[{"header":"Background","content":"\u003cp\u003eRecent advances in the treatment of oncological diseases have resulted in the significantly improved survival of young cancer patients [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e], with a cure rate of over 90% [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the USA, yearly estimates run as high as 60,000 new cancer cases in females younger than 40\u0026nbsp;years of age; of these, 4,000 are prepubertal children and adolescents [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, both chemotherapy and radiotherapy may impair the reproductive future of these patients [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Premature ovarian failure is estimated to occur in up to 68% of the women treated with alkylating agents for breast cancer during menacme; in 38\u0026ndash;57% of those aggressively treated with cytotoxic chemotherapy and radiotherapy for lymphomas; and in over 90% of the patients undergoing a conditioning regimen for bone marrow transplantation [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. Radiotherapy is also known to destroy the follicle reserve [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Fertility preservation is a major cause of concern in young women.\u003c/p\u003e\n\u003cp\u003ePreservation of the fertility of women is a challenge, likewise the offer of the best chances of maternity to patients at risk of premature, induced, or iatrogenic menopause [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. Such an offer may be made not only to patients with malignant diseases, but also to patients with sickle cell anemia and thalassemia, who may require a bone marrow transplant, patients with lupus, or even lesions leading to bilateral oophorectomy, such as recurring cysts [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e], all entailing reproductive failure. These are further reasons for improving the technique.\u003c/p\u003e\n\u003cp\u003eThe options for maintaining reproductive capacity include oocyte, embryo, and ovarian tissue freezing [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Ovarian tissue cryopreservation is the only technique still deemed experimental. It allows the storing of a large quantity of primordial and primary follicles quickly and at any phase of the menstrual cycle [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is the only option for preserving fertility in prepubertal girls [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. In cryopreserved ovarian tissue transplantation, one of the major difficulties to surmount is the improvement in the vascular bed of the receptor area [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e], given the potential occurrence of ischemic lesions over the time it takes for graft revascularization [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. This lesion results in fibrosis and apoptosis, which affect the follicle survival rate and graft lifespan, both key in fertility restoration [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Besides, follicle activation in the graft speeds up after transplantation, leading to discrepancies in cell maturation in the granulosa and oocyte development, possibly due to hypoxic stress and lack of anti-Mullerian hormone in the graft [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. Finally, the freezing\u0026ndash;thawing process induces structural and morphological changes, especially in the theca cell layer [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. The main reasons for the ovarian graft\u0026rsquo;s limited quality are inflammatory processes and oxidative stress, which damage the cells and their mitochondria. These issues are the target of melatonin action [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eTreatments such as ischemic preconditioning [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e] and cell therapy with stem cells [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e] have been studied by our group, with moderate benefits. Therefore, additional investigations are needed to improve the ovarian transplantation technique. One hypothesis for the impaired outcome is the presence of factors capable of interfering in tissue response, chief among them is a large number of free radicals derived from the procedure [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. In this experimental research scenario, new techniques have been proposed, the most prominent of which is melatonin administration for reducing oxidative stress. Melatonin acts as a free radical scavenger with vast antioxidant power and antiapoptotic function [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Melatonin action diminishes free radical-induced lesions in several diseases, including cancer, and it also inhibits the mitochondrial apoptotic pathway as it reduces BCL2 expression and cleaved caspase-3 activity [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. The antioxidant action of melatonin is more potent than that of vitamin C or E, as even its metabolites act as free radical scavengers, in a process called the cascade effect [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Besides acting directly against free radicals, melatonin activates antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. The surplus free radicals interact with lipids, proteins, and nucleic acids, causing the loss of membrane integrity, functional and structural changes in proteins, and nucleic acid lesions [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eMelatonin action is also recognized in diverse biological functions, such as circadian cycle control and anticancer action and those related to the reproductive system (ovarian activity, pregnancy, and delivery)[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] and neuroendocrinology, cardiology, and neuroimmunology [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is a hydrophilic and lipophilic molecule [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e], easily diffusing in various subcellular compartments, such as membranes, cytoplasm, nucleus, and mitochondria [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. There is evidence of functional enhancement in human thawed sperm quality when melatonin is applied to the cryopreservation medium [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, melatonin may favor cell survival. However, it is not clear if this propriety may occur during the cryopreservation of tissue fragments such as ovarian grafts. Thus, this study aimed to evaluate rat ovarian grafts with melatonin added to the culture medium prior to cryopreservation.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eThe study was approved by the local Ethics Committee on the Use of Animals (CEUA-FMUSP 024/15). The animal group consisted of 26 adult female Wistar rats (\u003cem\u003eRattus norvegicus albinus\u003c/em\u003e) aged 3 months and weighing approximately 250\u0026nbsp;g each. The animals were kept under proper conditions of temperature and feeding as well as under a controlled light/dark cycle of 12/12\u0026nbsp;h. Only animals with three regular estrous cycles were included.\u003c/p\u003e\n\u003cp\u003eThe animals were allocated to two study groups (n\u0026thinsp;=\u0026thinsp;13 each): control and melatonin. In the control group, slow cryopreservation was performed according to the standard protocol with the M2 culture medium, dimethyl sulfoxide (DMSO) [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e], and ethyl alcohol vehicle, whereas in the melatonin group, melatonin (Sigma Aldrich, Saint Louis, MO, USA) was added to the medium at a concentration of 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Three animals from each group underwent graft analysis immediately after thawing from cryopreservation to verify the potential differences between groups induced up to the freezing-thawing process itself.\u003c/p\u003e\n\u003cp\u003eIn both groups, the ovaries underwent slow cryopreservation and were kept in liquid nitrogen (N\u003csub\u003e2\u003c/sub\u003e) for 24\u0026nbsp;h. Thawing took place at room temperature (25\u0026nbsp;\u0026deg;C). The ovaries were implanted in the retroperitoneum of their respective donors, one on each side of the aorta, without anastomosis, and fixed by means of a simple stitch with an unabsorbable thread (nylon 4\u0026thinsp;\u0026minus;\u0026thinsp;0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstrous cycle control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the beginning of the experiment, vaginal smears were obtained daily, always at the same time (8\u0026ndash;10\u0026nbsp;h AM), to characterize the estrous cycle using the Shorr-Harris technique. Only animals with regular estrous cycles of 4\u0026ndash;5 days were used; diestrus was the standard phase for the surgical procedures (oophorectomy for cryopreservation and euthanasia).\u003c/p\u003e\n\u003cp\u003eDaily (8\u0026ndash;10\u0026nbsp;h AM) vaginal smear collection resumed from postoperative (PO) days 15 to 30; then euthanasia was performed as the animals entered the diestrus phase (PO day 30\u0026ndash;35).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtocol for anesthesia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter being weighed, the animals were anesthetized with xylazine (15\u0026nbsp;mg/kg) and ketamine (60\u0026nbsp;mg/kg) administered via intraperitoneal injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOophorectomy protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter a median longitudinal opening of the abdominopelvic cavity, the ovaries were removed bilaterally and washed with saline solution (0.9% NaCl). The procedure was performed between 9 and 10\u0026nbsp;h of AM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryopreservation and thawing protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ovaries were placed in 1.2-mL cryotubes with 1\u0026nbsp;mL of 1.4\u0026nbsp;M DMSO as a cryoprotectant and the M2 medium with or without melatonin added to the medium, depending on the study group, and kept at room temperature for 5\u0026nbsp;min. Slow-freezing was performed using the CL-8800 temperature controller and the Cryogenesis software, and then controlling the freezer from 25\u0026ndash;10\u003csup\u003eo\u003c/sup\u003eC at 1\u003csup\u003eo\u003c/sup\u003eC/min and to -7\u003csup\u003eo\u003c/sup\u003eC at 0.5\u003csup\u003eo\u003c/sup\u003eC/min, keeping the temperature for 5\u0026nbsp;min. Next, the temperature was lowered to -55\u003csup\u003eo\u003c/sup\u003eC at 0.5\u003csup\u003eo\u003c/sup\u003eC/min. At this point, the ovaries were transferred into liquid N\u003csub\u003e2\u003c/sub\u003e at -196\u003csup\u003eo\u003c/sup\u003eC and kept for 24\u0026nbsp;h [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe cryotubes were thawed at room temperature until all the ice melted (15\u0026ndash;20\u0026nbsp;min). The tissue was then transferred to 5\u0026nbsp;mL of TL-HEPES at room temperature for 10\u0026nbsp;min, while it was gently agitated to promote DMSO efflux. The tissue was kept in TL-HEPES at 37\u003csup\u003eo\u003c/sup\u003eC until transplantation [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection and analysis of the material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ovarian grafts were recovered and cut in half for analysis. The rats were euthanized with a lethal dose of the previously employed anesthetics. Imaging and measurements were performed with a computer system comprised of a light microscope (Carl Zeiss) adapted to a high-resolution camera (Axio Cam MRC, Carl Zeiss) and a color video monitor. Measurements were taken with an image analysis software program (AxionVision REL 4.6, Carl Zeiss). Counting was always performed using four fields per animal.\u003c/p\u003e\n\u003cp\u003eThe analyses comprised:\u003c/p\u003e\n\u003cp\u003e1) Estrous cycle\u003c/p\u003e\n\u003cp\u003eWith the animal immobilized, the vaginal smear was first obtained using a swab impregnated with saline solution and then placed on a standard slide for subsequent staining using the Shorr-Harris technique [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The slides were subsequently analyzed under a light microscope at 10\u0026thinsp;\u0026times;\u0026thinsp;and 40\u0026thinsp;\u0026times;\u0026thinsp;magnification. The phases of the estrous cycle were determined according to the proportion of cells observed in the smears as follows: 1) proestrus: predominance of nucleated epithelial cells; 2) estrus: predominance of non-nucleated keratinized cells; 3) diestrus: equal proportion of leukocytes and nucleated keratinized epithelial cells [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e2) Histology\u003c/p\u003e\n\u003cp\u003eTo assess follicle development, the ovarian follicles were counted and then categorized into developing follicles, regardless of their stage and atretic follicles. The former were classified according to the degree of maturation as follows: immature follicles (including primordial, primary, and secondary), mature follicles (with a single voluminous antrum), and corpora lutea. For counting purposes, the ovarian follicles comprised of both viable and atretic follicles as well as both normal and degenerating corpora lutea [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBlood vessel count was performed in a 100-square-micrometer area in five randomly selected fields and analyzed by two independent observers.\u003c/p\u003e\n\u003cp\u003eFor evaluation of fibrosis, the slides were stained with picrosirius red, and measurements were taken in eight fields per animal in the ovarian stroma, with a magnification of 400\u0026thinsp;\u0026times;\u0026thinsp;times, and the results were expressed as a percentage of the positive area (unit/mm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eThe evaluation of the slides was conducted at our Medical Investigation Laboratory (LIM-58). For the quantification of the parameters evaluated, the images were captured using a high-resolution camera (AxioCam-MCR, Carl Zeiss) adapted to a light microscope (Axiolab, Carl Zeiss) adjusted to the 40\u0026thinsp;\u0026times;\u0026thinsp;objective lens [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e3) Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eSlides with sections of the ovarian grafts were stained using immunohistochemistry to measure the von Willebrand factor (AB6994, 1:100, Abcam Inc., Cambridge, MA, USA), Ki67 (M724001-2, 1:100, Dako North America Inc., Carpinteria, CA, USA), cleaved caspase-3 (SANT-SC-1226, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling, Roche, Indianapolis, IN, USA). Hormonal receptor expression for estrogen (E1644, 1:50, Springe Bioscience Corporation, Pleasanton, CA, USA) and progesterone (AB51896, 1:30, Abcam Inc., Cambridge, MA, USA) was also studied. All preparations were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003eMicroscopy images were obtained using a computer program (Leica DM2500), and quantifications were performed with the LeicaQWin V3 program. The red-brown coloration of the cytoplasm and nucleus of granulosa cells and antral follicles (for apoptosis and Ki-67) or stroma (for fibrosis and expression of endothelial cells) was considered as positive expression and any other color, as negative. Hormone receptors were evaluated in both the stroma and follicular cells. The analysis was performed in eight different fields per animal at 400\u0026thinsp;\u0026times;\u0026thinsp;magnification, and the results were expressed as a percentage of the positive area (unit/mm\u003csup\u003e2\u003c/sup\u003e). The interpretation was performed by two independent and blinded investigators.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003cp\u003eThe data from each group of animals were analyzed according to the type of variables. The ANOVA and the Tukey tests were used, and the rejection level of the null hypothesis was set at 0.05 or 5% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation after freezing\u0026ndash;thawing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree animals from each group were analyzed immediately after the freezing-thawing process to verify the effects of melatonin specifically related to the cryopreservation process. Histological assessment of immature follicles, mature follicles, corpora lutea, and blood vessels showed no significant difference between the control and melatonin (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eHistological analysis\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMelatonin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ep\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImmature Follicles\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMature Follicles\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e5.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.20\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCorpora Lutea\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.95\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBlood vessels\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.43\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eHistological analysis comparing the use of melatonin added to cryopreservation medium or not, in rat ovarian slide, freezing-thawing group (n\u0026thinsp;=\u0026thinsp;3 each group)\u003c/p\u003e\n\u003cp\u003eImmunohistochemical assessment of the frozen-thawed group showed no significant difference in cleaved caspase-3, and TUNEL activities between the control and melatonin groups (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eImmunohistochemical analysis\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMelatonin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ep\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCaspase Stroma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.48\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCaspase Follicles\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.18\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTunel Stroma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTunel Follicles\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.26\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eImmunohistochemical analysis comparing the use of melatonin added to cryopreservation medium or not, in rat ovarian slide, freezing-thawing group (n\u0026thinsp;=\u0026thinsp;3 each group)\u003c/p\u003e\n\u003c/div\u003e\u003cp\u003e\u003cstrong\u003eEvaluation After Transplantation\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTen animals in the melatonin and control groups were analyzed after autologous transplantation.\u003c/p\u003e\n\u003cp\u003e1) Estrous cycle evaluation\u003c/p\u003e\n\u003cp\u003eFor all animals in both groups, there was a characterization of the estrus phase of the estrous cycle, demonstrating ovarian hormonal activity. The melatonin group presented faster resumption of the estrous cycle after transplantation (16.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 days) compared to the control group (20.75 days\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89, p\u0026thinsp;=\u0026thinsp;0.0017), indicating an enhancement in functional activity.\u003c/p\u003e\n\u003cp\u003e2) Morphology and morphometry\u003c/p\u003e\n\u003cp\u003eThe melatonin group had an increase in mature follicle count compared to the control group, but no significant difference was observed in immature follicles or corpora lutea expression. With regard to immature follicles, we did not find any significant difference between the groups, even on the primordial follicular account.\u003c/p\u003e\n\u003cp\u003eBlood vessel count did not differ significantly between groups. The melatonin group showed enhancement and reduction in collagen type I and type III expression, respectively, compared to the control group (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eHistological analysis\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMelatonin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ep\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImmature Follicles\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.66\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMature Follicles *\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.04\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCorpora Lutea\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.34\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCollagen type I*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCollagen type III*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCollagen type I and III ratio\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBlood vessels\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18.63\u0026thinsp;\u0026plusmn;\u0026thinsp;3.65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.99\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eOvarian follicle density, collagen and blood vessels (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation) comparing the use of melatonin added to cryopreservation medium or not, in rat ovarian slide, autologous cryopreserved grafts after 30th day of transplantation (n\u0026thinsp;=\u0026thinsp;10 each group); *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u0026nbsp;T test\u003c/p\u003e\n\u003cp\u003e3) Immunohistochemistry:\u003c/p\u003e\n\u003cp\u003eThe melatonin group presented a significant increase in endothelial cells (vWF), cellular proliferation (Ki67), and estrogen receptors both on the follicles and corpora lutea, and apoptosis in the corpora lutea using the cleaved caspase-3 assay compared to that in the control group. There was a significant reduction in apoptosis using TUNEL on follicles and corpora lutea. There were no significant differences in apoptosis using the cleaved caspase-3 assay on follicles and progesterone receptors on follicles or corpora lutea between both groups (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eImmunohistochemical analysis\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMelatonin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ep\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCleaved caspase 3\u0026nbsp;F\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e13.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.38\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCleaved caspase 3 CL\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e24.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTunel F\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTunel CL\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eKi67 F\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.002*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eKi67 CL\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e5.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstrogen receptor F\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e2.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e5.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.02*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstrogen receptor CL\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eProgesterone receptor F\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.77\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eProgesterone receptor CL\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e16.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e13.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.14\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003evon Willebrand factor\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e3.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.003*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eImmunohistochemical analysis comparing the use of melatonin added to cryopreservation medium or not, in rat ovarian slide, autologous cryopreserved grafts 30 days after transplantation (n\u0026thinsp;=\u0026thinsp;10 each group); * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; F\u0026thinsp;=\u0026thinsp;follicle; CL\u0026thinsp;=\u0026thinsp;corpora lutea\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSome studies have suggested the beneficial effects of melatonin on ischemic-reperfusion injury [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. In the ovary, there are references to two specific receptors for melatonin (MT1 and MT2) [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e] of the three identified so far, and they act via cyclic AMP reduction [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The same receptors have been identified in human ovarian follicles [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. They act on ovarian function, modulating steroidogenesis, thereby notably increasing progesterone synthesis [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] and that of its receptor. They also modulate the formation of corpora lutea, indicating optimization of the ovulatory function [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Changes in serum hormone levels are related to ovulatory disorders in both rats and humans [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Melatonin-deprived rats have a persistent anovulatory cycle, premature vaginal opening, ovarian hypertrophy, and increased vaginal cell cornification. Moreover, melatonin replacement reverses these effects. The hormone is found in ever higher concentrations in the ovarian follicle as it develops, reaching maximum concentration, when compared to plasma in the preovulatory follicle [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. The balance between free radicals and antioxidants in the ovarian follicle appears to be fundamental for the proper functioning of oocytes and granulosa cells [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, melatonin reduction also decreases embryo implantation [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. These are well-known properties of melatonin in the reproductive system. However, the action of melatonin on free radicals and antioxidants to reduce the damage to the ovarian graft is a new direction for applying this indolamine. Our study found that previous melatonin treatment on the graft was beneficial during the cryopreservation and transplantation of the rat ovarian graft.\u003c/p\u003e\n\u003cp\u003eThe first study on the effect of melatonin on rat ovarian autotransplantation reported the indirect benefits of the intraperitoneal administration of the substance [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. The authors described low levels of malondialdehyde as well as high superoxide dismutase and glutathione peroxidase levels with low ovarian necrosis in melatonin-treated rats. Another study with animal xenotransplant treated with melatonin via the oral route and vitamin E showed improvement in ovarian graft survival as evidenced by follicle count, the apoptosis index, and VEGF and PCNA expression [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. None of these previous studies investigated the use of melatonin in the medium used for cryopreserving ovarian grafts.\u003c/p\u003e\n\u003cp\u003eOur results suggest that melatonin pretreatment in the cryopreservation medium also presents beneficial effects on ovarian graft after transplantation: a) precocious resumption of the estrous cycle; b) a higher number of mature follicles; c) enhancement in tissue proliferation; and d) reduction in apoptosis.\u003c/p\u003e\n\u003cp\u003eThe animals subjected to subsequent ovarian transplantation showed that the melatonin group demonstrated faster recovery of regular estrous cycles compared to the control. This finding suggests that melatonin could have enabled better conditions for ovulatory and hormonal activity recovery.\u003c/p\u003e\n\u003cp\u003eHistological analysis showed that the melatonin group presented more mature follicles than the control group, indicating that the indolamine promoted enhancement in the graft function. In contrast, corpora lutea counting did not show any significant difference between groups. In rats, the estrous cycle can present corpora lutea formed by previous cycles, thus limiting quantitative comparison, which may explain this result. Blood vessel quantification did not differ between groups, and the melatonin group showed an enhancement in the von Willebrand factor expression, compared to the control. Previous studies, although not involving transplantation, have also reported such diverse effects of melatonin on blood vessel proliferation [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Melatonin may have a modulatory influence on vessels in different biological scenarios [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eEstrogen receptor expression was significantly higher in the melatonin group than in the control group. Indolamines can directly activate this hormone receptor activity or indirectly through the enhancement in follicular cell proliferation, which in turn synthesizes more estrogen. This hormone is the main representative activity of ovulatory follicles [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e], thereby inferring that melatonin can enhance graft hormonal performance.\u003c/p\u003e\n\u003cp\u003eThe present study is the first to assess progesterone receptors in transplanted rats and found no significant difference between the groups. An extended period of analysis may suggest that the application of melatonin to the cryopreservation medium could enhance progesterone receptor expression.\u003c/p\u003e\n\u003cp\u003eMelatonin can modulate the apoptosis process [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. From the present study the melatonin group showed an increase in cleaved caspase-3 on corpora lutea and no difference in follicles. For follicles, melatonin increased follicle development which could be related to diminish cleaved caspase-3. For corpora lutea, the natural degradation process after ovulation might be related to the enhancement in cleaved caspase-3. This study is the first to analyze cleaved caspase-3 on ovarian rat autotransplantation treated with melatonin and maybe further studies might confirm how melatonin modulates cleaved caspase-3 activity in this scenario. DNA fragmentation analysis using the TUNEL assay is the final result of cell degeneration either through apoptosis cascade or not [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. The present study revealed that TUNEL quantification was significantly reduced in the melatonin group, both in follicles and corpora lutea, compared to the control group. This finding relates to the most consolidated biochemical property of melatonin investigated in various tissues reported in the literature: the oxidative free radical scavenger, which could reduce DNA degradation, independently of the apoptotic pathway. Among experimental ovary transplantation studies, the first research to investigate the effect of melatonin also indicated a reduction in TUNEL [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThis research revealed that the melatonin group showed enhancement in type I collagen and reduction in type III collagen compared to the control. Previous studies have related collagen expression to tissue response against hypoxia. Fibroblast activity in tissue cultures have correlated an increase in type I collagen [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] and a reduction in type III collagen [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e] with better oxygenation. This result corroborates other reports of the present study, indicating that melatonin promoted better functional and follicular activity in the ovarian graft. The ovulation process is indeed related to the production and lysis of collagen in a dynamic process that ultimately produces ovarian follicle rupture and oocyte liberation [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMelatonin may promote a better quality of ovarian grafts by evaluating histological and protein expression parameters. Future research can further assess the potential enhancement provided by melatonin in graft reproductive performance.\u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003ctable\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003ePO\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"523\"\u003e\n\u003cp\u003epostoperative\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eDMSO\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"523\"\u003e\n\u003cp\u003edimethyl sulfoxide\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003evWF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"523\"\u003e\n\u003cp\u003evon Willebrand factor\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eTUNEL\u003c/p\u003e\n\u003cp\u003eMT\u003c/p\u003e\n\u003cp\u003eVEGF\u003c/p\u003e\n\u003cp\u003ePCNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"523\"\u003e\n\u003cp\u003eterminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling\u003c/p\u003e\n\u003cp\u003ereceptors for melatonin\u003c/p\u003e\n\u003cp\u003evascular endothelial growth factor\u003c/p\u003e\n\u003cp\u003eproliferating cell nuclear antigen\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the University of Sao Paulo Faculty of Medicine Ethics Committee on the Use of Animals (CEUA-FMUSP 024/15).\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\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMarcos Eiji Shiroma, Luciana Lamar\u0026atilde;o Damous and Jos\u0026eacute; Maria Soares-Jr contibuted to the study conception and design. Marcos Eiji Shiroma and Luciana Lamar\u0026atilde;o Damous made acquisition of data. Marcos Eiji Shiroma, Luciana Lamar\u0026atilde;o Damous and Jos\u0026eacute; Maria Soares-Jr analyzed the data. Edmund Chada Baracat, Russel Joseph Reiter, Jos\u0026eacute; Cipolla-Neto, Fernanda Pereira Cotrim and Cristiane Lima Roa contributed revising the work. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors thank Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) for the support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDemeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Orthotopic and heterotopic ovarian tissue transplantation. Hum Reprod Update. 2009;15: 649-65.\u003c/li\u003e\n\u003cli\u003eAbir R, Fisch B, Jessel S, Felz C, Ben-Haroush A, Orvieto R. 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Transp Proc. 2008;40:861-4.\u003c/li\u003e\n\u003cli\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Damous%20LL%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25889829\"\u003eDamous LL\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Nakamuta%20JS%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25889829\"\u003eNakamuta JS\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20Carvalho%20AE%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25889829\"\u003ede Carvalho AE\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Soares%20JM%20Jr%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25889829\"\u003eSoares-Jr JM\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20Jesus%20Sim%C3%B5es%20M%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25889829\"\u003ede Jesus Sim\u0026otilde;es M\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Krieger%20JE%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25889829\"\u003eKrieger JE\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Baracat%20EC%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25889829\"\u003eBaracat EC\u003c/a\u003e\u003csup\u003e. \u003c/sup\u003eAdipose tissue-derived stem cell therapy in rat cryopreserved ovarian grafts. \u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/25889829\"\u003eStem Cell Res Ther\u003c/a\u003e. 2015;6:57.\u003c/li\u003e\n\u003cli\u003eReiter RJ, Tan DX, Manchester LC, Paredes SD, Mayo JC, Sainz RM. Melatonin and Reproduction Revisited. Biol Reprod. 2009;81:445-56.\u003c/li\u003e\n\u003cli\u003eWang F, Tian XZ, Zhang L, Tan DX, Reiter RJ, Liu GS. Melatonin promotes the in vitro development of pronuclear embryos and increases the efficiency of blastocyst implantation in murine. J Pineal Res. 2013;55:267-74.\u003c/li\u003e\n\u003cli\u003eCruz MHC, Leal CLV, Cruz JF, Tan DX, Reiter RJ. Essential actions of melatonin in protecting the ovary from oxidative damage. Theriogenology. 2014;82:925-32.\u003c/li\u003e\n\u003cli\u003eFerreira CS, Maganhin CC, Sim\u0026otilde;es RS, Gir\u0026atilde;o MJBC, Baracat EC, Soares-Jr JM. Melatonina: modulador de morte celular. Rev Assoc Med Bras. 2010;56:715-8.\u003c/li\u003e\n\u003cli\u003eTamura H, Takasaki A, Taketani T, Tanabe M, Kizuka F, Lee F, Tamura I, Maekawa R, Aasada H, Yamagata Y, Sugino N. The role of melatonin as an antioxidant in the follicle. J Ovarian Res. 2012;5:5.\u003c/li\u003e\n\u003cli\u003eKarimfar MH, Niazvand F, Haghani K, Ghafourian S, Shirazi R, Bakhtiyari S. The protective effects of melatonin against cryopreservation-induced oxidative stress in human sperm. Int J Immunopth Ph. 2015;28:69-76.\u003c/li\u003e\n\u003cli\u003eGuanasena KT, Lakey JRT, Villines PM, Crister ES, Crister JK. Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice. Biol Reprod. 1997;57:226-31.\u003c/li\u003e\n\u003cli\u003eMarcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol. 2002;62:609-14.\u003c/li\u003e\n\u003cli\u003eJunqueira LC, Carneiro J. Histologia b\u0026aacute;sica. 13th ed. Rio de Janeiro: Guanabara Koogan; 2008\u003c/li\u003e\n\u003cli\u003eSoares-Jr JM, Masana MI, Ersahin C, Dubocovich ML. Functional melatonin receptors in rat ovaries at various stages of the estrous cycle. J Pharmacol Exp Ther. 2003;306:694-702.\u003c/li\u003e\n\u003cli\u003eMaganhin CC, Carbonel AAF, Hatty JH, Fuchs LFP, Oliveira-Junior IS, Sim\u0026otilde;es MJ, Sim\u0026otilde;es RS, Baracat EC, Joares-Jr JM. Efeitos da melatonina no sistema genital feminino: breve revis\u0026atilde;o. Rev Assoc Med Bras. 2008;54:267-71.\u003c/li\u003e\n\u003cli\u003eRomeu LRG, Motta ELA, Maganhin CC, Oshima CTF, Fonseca MC, Barrueco KF, Sim\u0026otilde;es RS, Pellegrino R, Baracat EC, Soares-Jr JM. Effects of melatonin on histomorphology and on the expression of steroid receptors, VEGF, and PCNA in ovaries of pinealectomized female rats. Fertil Steril. 2011;95:1379-84.\u003c/li\u003e\n\u003cli\u003eTeixeira CP, Sim\u0026otilde;es RS, Santos MA, Cali\u0026oacute; ML, Soares-Jr JM, Sim\u0026otilde;es MJ, Bertoncini CR, Higa EM, Carbonel AF. Soybean concentrated extract counteracts oxidative stress in the uterus of rats. Climacteric. 2014;17:402-9.\u003c/li\u003e\n\u003cli\u003eSapmaz E, Ayar A, Celik H, Sapmaz T, Kilic N, Yasar MA. Effects of melatonin and oxytetracycline in autologous intraperitoneal ovary transplantation in rats. Neuroendocrinol Lett. 2003;24:350-4.\u003c/li\u003e\n\u003cli\u003eHemadi \u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate\u0026amp;term=Hemadi+M\u0026amp;cauthor_id=21604248\"\u003eM\u003c/a\u003e,\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate\u0026amp;term=Saki+G\u0026amp;cauthor_id=21604248\"\u003e Saki\u003c/a\u003e G, \u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate\u0026amp;term=Shokri+S\u0026amp;cauthor_id=21604248\"\u003eShokri\u003c/a\u003e S,\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate\u0026amp;term=Ghasemi+FM\u0026amp;cauthor_id=21604248\"\u003e Ghasemi\u003c/a\u003e Follicular dynamics in neonate vitrified ovarian grafts after host treatment with melatonin. Folia Morphol (Warsz). 2011;70:18-23.\u003c/li\u003e\n\u003cli\u003eMirza-Aghazadeh-Attari M, Reiter RJ, Rikhtegar R, Jalili J, Hajalioghli P,\u0026nbsp;Mihanfar A, Majidinia M, Yousefi B. Melatonin: An atypical hormone with major functions in the regulation of angiogenesis. IUBMB Life. 2020;72:1560-84\u003c/li\u003e\n\u003cli\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Drummond+AE\u0026amp;cauthor_id=22271292\"\u003eDrummond\u003c/a\u003e AE,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Fuller+PJ\u0026amp;cauthor_id=22271292\"\u003eFuller\u003c/a\u003e Ovarian actions of estrogen receptor-\u0026beta;: an update. Semin Reprod Med. 2012;30:32-8\u003c/li\u003e\n\u003cli\u003eRodriguez C, Mart\u0026iacute;n V, Herrera F, Garc\u0026iacute;a-Santos G, Rodriguez-Blanco J, Casado-Zapico S, S\u0026aacute;nchez-S\u0026aacute;nchez AM, Su\u0026aacute;rez S, Puente-Moncada N, An\u0026iacute;tua MJ, Antol\u0026iacute;n I. Mechanisms involved in the pro-apoptotic effect of melatonin in cancer cells. Int J Mol Sci. 2013;14:6597-613.\u003c/li\u003e\n\u003cli\u003eNegoescu A, Guillermet C, Lorimier P, Brambilla E, Labat-Moleur F. Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity. Biomed Pharmacother. 1998;52:252-8.\u003c/li\u003e\n\u003cli\u003eHara-Saito Y, Kato H,\u0026nbsp;Saito N,\u0026nbsp;Shiomi A,\u0026nbsp;Uenoyama A,\u0026nbsp;Takagi R, Izumi\u0026nbsp;K. Distinct differences in hypoxic responses between human oral mucosa and skin fibroblasts in a 3D collagen matrix. In Vitro Cell Dev Biol \u0026ndash;Animal.2020;56:452-79.\u003c/li\u003e\n\u003cli\u003eWang X, Lin L,Chai X,\u0026nbsp;Wu\u0026nbsp;Y, Li Y,\u0026nbsp;Liu X. Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblastos. Int J Mol Med. 2020;45:175-85.\u003c/li\u003e\n\u003cli\u003eLofman C, Zackrisson U, Mikuni M, Block M, Janson PO, Brannstrom M. A method for longitudinal microscopic in vivo examinations of morphology, vascularity, and motility in the ovary and the oviduct of the rat. J Soc Gynecol Investig. 2002;6:379-85.Top of Form\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"melatonin,ovary, transplantation, fertility preservation, cryopreservation, tissue preservation","lastPublishedDoi":"10.21203/rs.3.rs-93401/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-93401/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Melatonin has anti-inflammatory and antioxidative actions at the mitochondrial level. This indole may protect the ovarian graft during the cryopreservation process. Therefore, our study aimed to determine whether melatonin pretreatment improves rat ovarian graft quality. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Twenty female rats were allocated to two study groups of ten animals each: 1) control group: ovaries cryopreserved using the standard protocol; and 2) melatonin group: ovaries cryopreserved in a medium with melatonin. Following 24-h freezing, whole ovaries underwent autologous and avascular transplants with retroperitoneal placement. After postoperative (PO) day 15, daily vaginal smears were obtained for estrous cycle characterization. Between PO days 30 and 35, animals were euthanized and ovarian grafts were recovered for histological and immunohistochemical (Ki-67, cleaved caspase-3, TUNEL, von Willebrand factor, estrogen, and progesterone receptors) analysis. The ovaries of the three remaining rats from each group were studied immediately after thawing to assess cryopreservation effects. The ANOVA and the Tukey tests were used, and the rejection level of the null hypothesis was set at 0.05 or 5% (p\u0026lt;0.05).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Melatonin promoted faster restart of the estrous cycle and increased the expression of mature follicles, collagen type I, von Willebrand factor, Ki-67, and cleaved caspase-3 on corpora lutea and estrogen receptors in the ovaries. There was a reduction in apoptosis by TUNEL on follicles, corpora lutea, and collagen type III. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Melatonin may promote the quality of ovarian grafts through the evaluated parameters. Reproductive function enhancement could be further studied.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Pretreatment with melatonin improves ovarian tissue cryopreservation for transplantation.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-10-19 22:15:18","doi":"10.21203/rs.3.rs-93401/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-11-07T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-11-02T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-10-25T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-10-22T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-10-21T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-10-08T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-10-07T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-10-06T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-10-06T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-10-06T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10094d11-dd40-455a-ae05-0384ffe35d88","owner":[],"postedDate":"October 19th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":824552,"name":"Endocrinology \u0026 Metabolism"},{"id":824553,"name":"Sexual \u0026 Reproductive Medicine"}],"tags":[],"updatedAt":"2021-02-07T15:03:33+00:00","versionOfRecord":{"articleIdentity":"rs-93401","link":"https://doi.org/10.1186/s12958-021-00705-4","journal":{"identity":"reproductive-biology-and-endocrinology","isVorOnly":false,"title":"Reproductive Biology and Endocrinology"},"publishedOn":"2021-02-03 15:02:04","publishedOnDateReadable":"February 3rd, 2021"},"versionCreatedAt":"2020-10-19 22:15:18","video":"","vorDoi":"10.1186/s12958-021-00705-4","vorDoiUrl":"https://doi.org/10.1186/s12958-021-00705-4","workflowStages":[]},"version":"v1","identity":"rs-93401","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-93401","identity":"rs-93401","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}