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Background: The aim of this study was to compare the effectiveness of two different vitrification methods and slow freezing in terms of the recovery of endocrine function, follicular morphology and proliferation, apoptosis of stromal cells, and angiogenesis after heterotopic transplantation of human ovarian tissue., Methods: Ovarian tissue from young women aged 29 to 40 was subjected to two vitrification methods and one slow freezing method. The thawed ovarian tissue was then transplanted into nude mice and divided into three groups (VF1 group, VF2 group, SF group) according to the different freezing methods. Ovarian tissue samples were collected at 4 and 6 weeks post-transplantation. The recovery of ovarian function was evaluated by observing the estrous cycle and measuring estradiol levels using Elisa. Histological evaluation was performed to assess the integrity of ovarian follicles. TUNEL assay was used to detect stromal cell apoptosis, and immunohistochemistry was conducted to evaluate follicular proliferation and tissue angiogenesis., Results: After heterotopic transplantation, mice in the experimental groups exhibited restoration of the estrous cycle. Hormone levels showed an increasing trend in the vitrification groups. At 6 weeks post-transplantation, the VF2 group had significantly higher hormone levels compared to the VF1 group and the slow freezing (SF) group (P < 0.05). At 4 weeks post-transplantation, the proportion of normal follicles was higher in the VF2 group compared to the other two groups (P > 0.05), and at 6 weeks post-transplantation, the VF2 group was significantly higher than the SF group (P < 0.05) and slightly higher than the VF1 group. Immunohistochemistry analysis indicated a higher proportion of proliferating follicles in the vitrification groups compared to the slow freezing group (P > 0.05). CD31 expression was established in all groups at 4 and 6 weeks post-transplantation, with better results in the slow freezing group compared to the vitrification group. TUNEL analysis showed that stromal cell apoptosis was higher in the SF group compared to the vitrification group at 4 weeks post-transplantation (P < 0.05), while there was no significant statistical difference among the groups at 6 weeks post-transplantation., Conclusions: Vitrification showed better results than slow freezing, with the VF2 group performing slightly better than the VF1 group. Considering the lower economic and time costs associated with vitrification, it may be more suitable for ovarian tissue cryopreservation in major research centers in the future., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Ethics Board of Peking University Shenzhen Hospital. Consent for publication: Our manuscript does not contain any individual person’s data in any form. However, three attenders provided written consents for the publication of the study. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Preconditioning before hematopoietic stem cell transplantation (HSCT) severely damages ovarian function, resulting in infertility and premature ovarian insufficiency (POI) in young women and girls. Ovarian function and fertility preservation before HSCT is crucial. In China, many patients miss this opportunity, highlighting the need for ovarian function and fertility preservation after HSCT. Ovarian tissue cryopreservation (OTC) is a standard method for fertility preservation and protecting ovarian function. The objective of this case report is to report a case of OTC performed on an 8-year-old girl after HSCT, and present a review about the necessity and feasibility of ovarian preservation after HSCT., Case: An 8-year-old girl required a second HSCT due to a relapse of dermatomyositis. Before the procedure, she visited our center for OTC. Hormonal assessments showed FSH 1.17 IU/L, LH 0.00 IU/L, E2 < 11.80 pg/ml, and AMH 0.81 ng/ml. Pelvic ultrasound revealed bilateral ovarian sizes of approximately 1.5 × 0.7 × 0.7 cm and 1.6 × 0.9 × 0.7 cm, with 10 and 4 visible follicles, respectively. We proceeded with OTC, surgically retrieving the entire left ovary via laparoscopy. Seven ovarian cortical slices were cryopreserved by slow freezing, with an average of 1079 follicles in per 2 mm diameter cortical tissue slice., Conclusion: Patients who miss fertility preservation before HSCT should consult fertility preservation and gynecological endocrinology experts as early as possible after HSCT and undergo regular follow-up. If clinical evidence indicates residual ovarian function, fertility protection measures should be discussed promptly. OTC should be assessed as a successful option for women after HSCT.

Background: This study aimed to investigate the effects of different volumes of ovarian tissue transplantation on the reproductive endocrine function of rats after oophorectomy., Methods: Female rats were selected to establish a castration model and then underwent different volumes of ovarian tissue transplantation. Group I served as the sham operation group. The transplantation group was divided into five subgroups based on the calculated ratio of ovarian weight to body weight in normal female rats, δ = (2.52 ± 0.17) ×10 -4 : Group II: transplanted ovarian volume was δ; Group III: 0.75δ; Group IV: 0.5δ; Group V: 0.25δ; Group VI: without ovarian transplantation. The post-transplant oestrous cycle recovery was observed, and blood samples were collected every 2 weeks to measure serum hormone levels. Histological evaluation was performed at the end of the observation period., Results: Rats in Group V exhibited disrupted oestrous cycles after transplantation, which were significantly longer than those in Group I. Rats in Groups II, III, and IV showed no cyclic changes. At 6 weeks post-transplantation, rats in Group V had lower E2 and AMH levels and higher FSH levels compared to Group I. The uterine wet weight and the number of normal follicles in Group V were significantly lower than those in Group I, but the number of atretic follicles was higher than in Group I., Conclusion: The larger ovarian tissue transplantation resulted in a faster recovery with a higher survival rate of the uterus and normal follicles, compared to smaller ovarian tissue transplantation.

Ovarian tissue cryopreservation and transplantation is one of the most advanced and promising fertility preservation methods. Prior to any procedure that may lead to a toxic ovarian injury (such as chemotherapy or radiotherapy), a portion of the ovary is removed and cryopreserved. At an appropriate time, after toxic therapy is concluded, the cryopreserved ovarian tissue is then thawed and transplanted back to the patient when conditions permit. This technique can not only preserve female fertility but also restore ovarian endocrine function. However, there is no standardization for ovarian tissue cryopreservation and transplantation in China. In order to promote the standardized development of ovarian tissue cryopreservation technology in the whole country, it is urgent to establish the standard of this technology.

Introduction: Ovarian tissue cryopreservation (OTC) is recommended by scientific societies for women undergoing highly gonadotoxic cancer treatments. Following transplantation, the restoration of ovarian function is typically characterised by the resumption of spontaneous menstruation. Yet, a few studies have looked at the longitudinal hormonal variations following transplantation. This study aims to investigate the fluctuation of gonadotropins and granulosa/theca cells secretions during the interval between ovarian transplantation and the recovery of menstrual function in two young women with no residual ovarian activity., Method: We selected two patients diagnosed with Hodgkin's lymphoma, initially referred for OTC at the ages of 19 and 15, respectively, and who had both undergone two consecutive stem cell transplants due to recurrent disease episodes. Both patients presented with premature ovarian failure and returned at ages 29 and 26, respectively, for ovarian cortex transplantation. Hormonal secretions and menstrual function were closely monitored both prior and in the months following the ovarian transplantation., Results: Menstruation resumed at 7 and 5 months post-transplantation, respectively. FSH and LH levels significantly decreased as early as 1 and 3 months before the first menstruation. As for ovarian hormonal secretion, AMH, measured with an ultra-sensitive assay ("pico AMH"), and Inhibin B were the first to increase, starting 1 month before the resumption of menstruation. Subsequently, AMH levels consistently remained very low throughout the follow-up, as did androgens, which showed a slight increase after the graft but remained at postmenopausal levels., Conclusion: Pico AMH, measured by an ultra-sensitive assay, Inhibin B and estradiol are the first ovarian hormones to be secreted following an ovarian graft, with levels rising 1 month prior the return of menstruation. However, the earliest hormonal indicators of graft success are the significant drops in FSH and LH levels, accompanied by a rise in estradiol levels, which occur 1-3 months before menstruation resumes., Competing Interests: Declarations. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose. IRB ethical approval: Not applicable. Informed consent: The authors affirm that human research participants provided informed consent for publication., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The use of parthenogenetic silkworm (Bombyx mori) strains, which eliminate the problem of recombination, is a useful tool for maintaining transgenic clonal lines. The generation of genetically identical individuals is becoming an important tool in genetic engineering, allowing replication of an existing advantageous trait combination without the mixing that occurs during sexual reproduction. Thus, an animal with a particular genetic modification, such as the ability to produce transgenic proteins, can reproduce more rapidly than by natural mating. One obstacle to the widespread use of parthenogenesis in silkworm genetic engineering is the relatively low efficiency of downstream transgenesis techniques. In this work, we seek to optimize the use of transgenesis in conjunction with the production of parthenogenetic individuals. We found that a very important parameter for the introduction of foreign genes into a parthenogenetic strain is the precise timing of embryo microinjection. Our modification of the original method increased the efficiency of transgene injection as well as the survival rate of injected embryos. We also provide a detailed description of the methodological procedure including a graphical overview of the entire protocol. [ABSTRACT FROM AUTHOR]

The use of parthenogenetic silkworm (Bombyx mori) strains, which eliminate the problem of recombination, is a useful tool for maintaining transgenic clonal lines. The generation of genetically identical individuals is becoming an important tool in genetic engineering, allowing replication of an existing advantageous trait combination without the mixing that occurs during sexual reproduction. Thus, an animal with a particular genetic modification, such as the ability to produce transgenic proteins, can reproduce more rapidly than by natural mating. One obstacle to the widespread use of parthenogenesis in silkworm genetic engineering is the relatively low efficiency of downstream transgenesis techniques. In this work, we seek to optimize the use of transgenesis in conjunction with the production of parthenogenetic individuals. We found that a very important parameter for the introduction of foreign genes into a parthenogenetic strain is the precise timing of embryo microinjection. Our modification of the original method increased the efficiency of transgene injection as well as the survival rate of injected embryos. We also provide a detailed description of the methodological procedure including a graphical overview of the entire protocol.

Ovarian tissue cryopreservation and transplantation (OTCT) offers hope for preserving fertility and endocrine functions in patients undergoing gonadotoxic treatments. Advancements in techniques for the procedure have transformed OTCT from an experimental procedure into a viable option. There is a growing interest in utilizing OTCT to delay menopause and alleviate associated health issues. Menopausal transition affects women globally, leading to symptoms and long- term health risks. OTCT has the potential to restore endocrine functions, reducing menopause-related symptoms while mitigating health consequences such as osteoporosis and cardiovascular diseases. Although the use of OTCT for delaying menopause is not clinically proven, the discussion around shows potential for future utilization. In essence, the remarkable advancements in OTCT have bestowed upon us the ability to safeguard fertility and sustain the delicate endocrine functions of the ovaries. However, it is the tantalizing prospect of utilizing this technique to postpone menopause and alleviate its associated symptoms that truly captivates the imagination. Further research is imperative to substantiate the clinical efficacy of OTCT; nonetheless, its potential in menopausal therapy is both promising and warrants comprehensive exploration. This review highlights advancements and the feasibility of OTCT to postpone menopause as an alternative approach to currently used conventional menopause therapy methods., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Ovarian cryopreservation is a promising technique despite being hindered by damage from freezing and thawing. Melatonin can mitigate this outcome., Objective: This study aimed to evaluate the effect of melatonin on the follicular dynamics of ovarian tissue in a cryopreserved cell culture., Methods: Three-month-old adult female Wistar rats (n = 24) weighing approximately 250 g were oophorectomized and divided into two groups (n = 12): the control group (CG) and the melatonin group (MG). In the CG, slow cryopreservation was performed according to the standard protocol with Medium M2 and dimethyl sulfoxide (DMSO). In MG, melatonin diluted in ethyl alcohol vehicle at a concentration of 0.1 μm was added to the culture medium. In both groups, the ovaries were cryopreserved by slow freezing and kept in liquid nitrogen for 24 h. Subsequently, after thawing, the ovaries were reimplanted in the retroperitoneum, one on each side of the great vessels (inferior vena cava and aorta). After 30 days, the animals were euthanized during the diestrus phase; then, the grafts were removed and processed for histomorphometric and immunohistochemical analyses, whereas the blood was subjected to biochemical analysis. Student's t test was used to assess the difference between the groups., Results: The FSH levels in MG (83.79 ± 32.37) were lower than those in CG (120.52 ± 36.59), p = 0.03. The FSH/AMH ratios were also lower in MG (3.53 ± 1.13) than in CG (6.52 ± 2.85), p = 0.001. The SOD2 immunoexpression was higher in MG than in CG regarding all parameters except for the degenerated follicles (follicular cells and internal thecal cells): CG (16.80 ± 4.80 [13.36-20.24]) and MG (14.91 ± 4.04 [12.01-17.79]) p = 0.351. Statistically, the difference in intact follicles (theca + interstitium) between CG (6.60 ± 2.59 [4.75-8.45]) and MG (9.31 ± 3.09 [7.09-11.51]) was significant (p = 0.049), with a small difference in the expression of regular antral follicles., Conclusions: Melatonin can improve the quality of cryopreserved tissues, as evidenced in this study, and the evaluation of cryopreserved ovarian grafts, as shown in the melatonin group with better hormonal parameters and greater immunohistochemical expression of the SOD2 antioxidant. Thus, damage is reduced during cryopreservation and transplantation is improved., (© 2024. The Author(s).)

Objectives: Recent studies have shown that oxidative stress, inflammation, and apoptosis play substantial roles in the pathogenesis of tissue damage and distant organ damage during ovarian transplantation. Because of their potential antioxidant and anti-inflammatory properties, investigating natural compounds like curcumin and gallic acid is crucial. This study investigated the effects of curcumin, gallic acid, and their combination on liver and kidney tissues in a rat model of ovarian transplantation., Materials and Methods: For this study, we used 42 adult female Sprague-Dawley rats aged 11 to 12 weeks, which we randomly divided into 6 groups. After the transplant procedures, we performed histopathological analysis, biochemical examinations, and statistical analysis., Results: Significant damage was shown in the liver and kidney tissues of the ovarian transplant only and transplant plus corn oil groups compared with the control group, as evidenced by histopathological evaluation, increased BAX/BCL-2 intensity indicating apoptotic activity, and elevated interleukin 6 levels. However, treatment with curcumin, gallic acid, or their combination attenuated these adverse effects, suggesting potential protective effects against transplant-induced injury., Conclusions: Our findings highlight the therapeutic potential of these compounds in mitigating tissue damage and inflammation associated with ovarian transplant.

The idea of utilizing unused oocytes present in the ovaries has been tested in various ways to produce offspring. However, only a limited number of studies succeeded in offspring generation. They include transplantation of ovaries into autologous or allogeneic animals, and acquisition of pups from oocytes obtained by transplanting mouse ovaries into immunodeficient rats. Here we report successful production of rat oocytes by transplanting rat ovaries under the kidney capsule of immunodeficient mice with addition of hormone administration to the mice. In addition, these oocytes were developed by in vitro fertilization, and transplanted into the oviducts of pseudopregnant rats, resulting in successful delivery of pups. The modified gene of the donor rat was confirmed to be correctly inherited to the pups. These results show that xenotransplantation of ovarian tissue makes it possible to leave offspring, beginning a new phase in developmental engineering., (© 2024. The Author(s).)

Ovarian tissue cryopreservation and transplantation (OTCT) has emerged in recent years as a potential method for reversing abnormal endocrine and reproductive functions, particularly in patients receiving gonadotoxic cancer treatments having longer survival rates. From its first rodent experiments to human trials, OTCT has evolved tremendously, opening new windows for further utilization. Since then, significant progress has been achieved in terms of techniques used for surgical removal of the tissue, optimal fragment size, freezing and thawing procedures, and appropriate surgical sites for the subsequent reimplementation of the graft. In addition, various approaches have been proposed to decrease the risk of ischemic injury, which is the leading cause of significant follicle loss during neo-angiogenesis. This review aims to discuss the pros and cons of ovarian and retroperitoneal transplantation sites, highlighting the justifications for the viability and efficacy of different transplantation sites as well as the potential advantages and drawbacks of retroperitoneal or preperitoneal area.

Indoleamine 2,3-dioxygenase (IDO) plays important roles in maternal immune tolerance. Female Sprague Dawley rats (9-11 weeks old) were randomly divided into an autoplastic transplantation group (n = 75) and an allograft transplantation group (n = 300) was further divided into subgroups of ovarian transplantation, allograft ovarian transplantation, allograft ovarian transplantation with cyclosporine A treatment, allograft ovarian transplantation and transfection with IDO-expressing lentiviruses, and allograft ovarian transplantation and transfection with control lentiviruses. IDO was successfully transfected into the transplanted ovarian tissue. The survival rate, success rate of ovarian transplantation, period until estrous cycle restoration, and estrogen levels of rats that received IDO-expressing lentiviruses were significantly different from those of rats that underwent allograft transplantation and with control transfection (all P < 0.05), but not significantly different from those rats that received autoplastic transplantation (all P > 0.05). The number of ovarian follicles in the transplanted ovarian tissue of rats that received IDO-expressing lentiviruses was also significantly higher. The expression level of IDO protein detected by immunohistochemistry and western blotting was especially high in ovaries that had received IDO-containing lentiviruses. Naturally pregnant rats were found in each group postoperatively. These results indicated that IDO-expressing lentiviruses were successfully transfected into transplanted ovarian tissues of rats and that IDO was stably expressed within a certain time. These findings suggest that the expression level of IDO protein is associated with an enhanced success rate of ovarian tissue transplantation and a short restoration period of endocrine function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cancer survival rates in prepubertal girls and young women have risen in recent decades due to increasingly efficient treatments. However, many such treatments are gonadotoxic, causing premature ovarian insufficiency, loss of fertility, and ovarian endocrine function. Implantation of donor ovarian tissue encapsulated in immune-isolating capsules is a promising method to restore physiological endocrine function without immunosuppression or risk of reintroducing cancer cells harbored by the tissue. The success of this approach is largely determined by follicle density in the implanted ovarian tissue, which is analyzed manually from histologic sections and necessitates specialized, time-consuming labor. To address this limitation, we developed a fully automated method to quantify follicle density that does not require additional coding. We first analyzed ovarian tissue from 12 human donors between 16 and 37 years old using semi-automated image processing with manual follicle annotation and then trained artificial intelligence program based on follicle identification and object classification. One operator manually analyzed 102 whole slide images from serial histologic sections. Of those, 77 images were assessed by a second manual operator, followed with an automated method utilizing artificial intelligence. Of the 1181 follicles the control operator counted, the comparison operator counted 1178, and the artificial intelligence counted 927 follicles with 80% of those being correctly identified as follicles. The three-stage artificial intelligence pipeline finished 33% faster than manual annotation. Collectively, this report supports the use of artificial intelligence and automation to select tissue donors and grafts with the greatest follicle density to ensure graft longevity for premature ovarian insufficiency treatment., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Childhood cancer incidence, especially in high-income countries, has led to a focus on preserving fertility in this vulnerable population. The common treatments, such as radiation and certain chemotherapeutic agents, though effective, pose a risk to fertility. For adult women, established techniques like embryo and egg freezing are standard, requiring ovarian stimulation. However, for prepubescent girls, ovarian tissue freezing has become the primary option, eliminating the need for hormonal preparation. This review describes the beginning, evolution, and current situation of the fertility preservation options for this young population. A total of 75 studies were included, covering the steps in the current fertility preservation protocols: (i) ovarian tissue extraction, (ii) the freezing method, and (iii) thawing and transplantation. Cryopreservation and the subsequent transplantation of ovarian tissue have resulted in successful fertility restoration, with over 200 recorded live births, including cases involving ovarian tissue cryopreserved from prepubescent girls. Despite promising results, challenges persist, such as follicular loss during transplantation, which is attributed to ischemic and oxidative damage. Optimizing ovarian tissue-freezing processes and exploring alternatives to transplantation, like in vitro systems for follicles to establish maturation, are essential to mitigating associated risks. Further research is required in fertility preservation techniques to enhance clinical outcomes in the future. Ovarian tissue cryopreservation appears to be a method with specific benefits, indications, and risks, which can be an important tool in terms of preserving fertility in younger women.

Ovarian tissue cryopreservation and transplantation is the only way to preserve fertility for female cancer patients in prepubertal ages and those who cannot delay radiotherapy or chemotherapy. However, the success rate of cryopreservation and transplantation of ovarian tissue is still low at present due to the risk of ischemia and hypoxia of the grafted tissues. Abnormal activation of primordial follicles and ischemia-reperfusion injury after blood supply recovery also cause massive loss of follicles in grafted ovarian tissues. Various studies have explored the use of different drugs to reduce the damage of follicles during freezing and transplantation as well as to extend the duration of endocrine and reproductive function in patients with ovarian transplantation. For example, melatonin, N -acetylcysteine, erythropoietin or other antioxidants have been used to reduce oxidative stress; mesenchymal stem cells derived from different tissues, basic fibroblast growth factor, vascular endothelial growth factor, angiopoietin 2 and gonadotropin have been used to promote revascularization; anti-Müllerian hormone and rapamycin have been used to reduce abnormal activation of primordial follicles. This article reviews the research progress on the main mechanisms of follicle loss after ovarian tissue transplantation, including hypoxia, ischemia-reperfusion injury and associated cell death, and abnormal activation of follicles. The methods for reducing follicle loss in grafted ovarian tissues are further explored to provide a reference for improving the efficiency of ovarian tissue cryopreservation and transplantation.

Purpose: Transplantation of ovarian tissue is a valuable method to rescue mouse strains with fertility problems and to revitalize archived strains. The purpose of this study was to investigate the effect of (i) different sizes of transplanted ovary pieces on reproductive outcome, (ii) use of immunodeficient recipients to overcome the limitation of histocompatibility, and (iii) to compare different protocols for cryopreservation of ovarian tissue. Methods: Halves, quarters, and eights of mouse ovaries were transplanted. Half ovaries from B6 donors were transferred into immunodeficient mice. Halves of ovaries were frozen according to four different protocols, thawed and transferred. Results: Pregnancy rate after transplantation of ovarian tissue was high (90–100%) independent of the transplant size. Although, the average litter size was significantly lower for recipients of quarters and eights (4.4 and 4.6 vs. 6.5), the total number of offspring produced per donor ovary was higher compared with recipients of halves. Pregnancy rate of immunodeficient recipients was 40% (mean 4.7 offspring per litter). All four cryopreservation protocols used were able to preserve functionality of the ovarian tissue. Conclusions: Transplantation of ovarian tissue smaller than halves resulted in reduced litter sizes. The distribution of ovarian tissue of one donor female to 4 or 8 recipients will therefore yield in a higher total number of offspring in a certain time period. The use of immunodeficient recipients is an option for non-histocompatible donors. Cryopreservation of ovarian tissue is generally feasible but the function of frozen-thawed ovary halves after transplantation differs depending on the freezing protocol used. [ABSTRACT FROM AUTHOR]

Study Question: Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles?Summary Answer: We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles.What Is Known Already: Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice.Study Design, Size, Duration: Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells.Participants/materials, Setting, Methods: Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24 hours followed by 24 hours of 1 mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N = 241) and human (N = 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks.Main Results and the Role Of Chance: Incubation in 0.1% SDS for 18-24 hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88-98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%).Large Scale Data: N/A.Limitations, Reasons For Caution: Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells.Wider Implications Of the Findings: This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration.Study Funding/competing Interest(s): This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication. [ABSTRACT FROM AUTHOR]

Objective: To compare the outcomes of orthotopic and heterotopic ovarian tissue transplantation (OTT) techniques., Design: Mixed prospective-retrospective cohort study., Setting: Academic hospital., Patients: A total of 14 recipients of autologous OTT., Interventions: Of the 14 women, 12 who received orthotopic (n = 6) or heterotopic (n = 6) transplants met the inclusion criteria. All orthotopic transplants and one heterotopic ovarian tissue transplant were performed laparoscopically. Although 5 of the 6 remaining heterotopic transplants were performed subcutaneously under local anesthesia or intravenous sedation, one was performed with robotic assistance. With the exception of one recipient who solely desired restoration of endocrine function, all underwent oocyte retrieval either to cryopreserve oocytes and embryos before the graft function ceased or because they could not otherwise conceive (hysterectomy, radiation damage, and heterotopic transplant)., Main Outcome Measures: Primary outcome measures were graft function and longevity, and the number of embryos generated per retrieval., Results: The mean age at ovarian tissue harvesting and transplantation was lower in patients with orthotopic vs. heterotopic transplants, although the proportion of transplanted ovarian cortex was lower in heterotopic transplant cases. All grafts restored ovarian endocrine function. Fertilization rates, the number of embryos generated per retrieval, and the mean number of nonarrested embryos were significantly lower in heterotopic OTT. However, time to function and graft longevity were similar between the groups. Although 4 of the 6 women conceived and delivered 7 children among orthotopic ovarian tissue recipients, one recipient had 3 spontaneous live births after heterotopic OTT, presumably because of the induction of function in the remaining menopausal ovary., Conclusions: It appears that orthotopic OTT results in higher gamete and embryo quality. However, the endocrine function restoration rate and longevity are similar between the 2 approaches. When feasible, orthotopic OTT should be preferred for those who intend to conceive, although a less invasive heterotopic OTT can be performed for those who primarily desire ovarian endocrine function., Competing Interests: Declaration of interests K.H.O. has nothing to disclose. L.M. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: To report the results of cryopreserved ovary tissue transplantation for leukemia and other cancers, in a single US center.Methods: One hundred eight females between age 6 and (median age 24) 35 were referred for possible ovary tissue cryopreservation over a 20-year period, with either slow freeze or vitrification. Thus far 13 patients returned up to 18 years later to have their tissue transplanted back.Results: All 13 patients had return of ovarian function 5 months post transplant with regular menstrual cycling. AMH rose to very high levels as the FSH declined to normal. Four months later, the AMH again declined to very low levels. Nonetheless, the grafts remained functional for up to 5 years or longer. Ten of the 13 (77%) became spontaneously pregnant at least once, resulting in 13 healthy babies. A total of 24 healthy babies have been born 11 from fresh transplanted ovarian tissue and 13 from cryopreserved transplanted ovarian tissue.Conclusions: (1) Ovary tissue cryopreservation is a robust method for preserving a woman's fertility. (2) Cortical tissue pressure may be a key regulator of primordial follicle arrest, recruitment, and ovarian longevity. (3) This is the only such series yet reported in the USA. [ABSTRACT FROM AUTHOR]

Ishigameyoh (gap) was identified as a spontaneous mutant of Bombyx mori with an apterous and sterile phenotype. Phenotypic observations revealed that the gap mutation affected the development of imaginal discs and the primordia of adult organs, including the wings, legs, compound eyes, and reproductive organs. Therefore, Ishigameyoh (gap) is a metamorphosis-defective mutant. We found that oogenesis depends on the gap gene, whereas spermatogenesis is independent. Ovary transplantation experiments suggest a cell-autonomous requirement for the gap gene in the process of oogenesis. The gap mutation has been maintained by crossing +gap gap moths as the u90 strain. However, the u90 strain is difficult to maintain because +gap+gap and +gapgap moths cannot be distinguished phenotypically. For efficacious maintenance, we established a molecular marker that is closely linked to the gap locus. First, we localized the gap locus to chromosome 5 using phenotypic markers. Second, the gap locus was narrowed down to within a 1-Mb region using PCR-based markers. Within this 1-Mb region, we finally established a PCR-based marker that amplified different-sized products from +gap+gap and gap offspring of the u90 strain. This marker allows the distinction of +gap+gap and +gapgap moths with an error rate of <1% and therefore reduces the labor needed to maintain the u90 strain. Moreover, this marker helps to identify +gap and gap individuals before pupation, which is necessary for larvae-based genetic and physiological analyses of the gap gene. [ABSTRACT FROM AUTHOR]

Ovarian tissue autotransplantation is an innovative fertility preservation technique that has provoked ongoing investigations. The purpose of the present study was to assess the safety and reproductive performance of ovarian tissue autotransplantation. This review is conducted according to PRISMA guidelines. Seven studies met the inclusion criteria. A total of 3427 patients underwent ovarian tissue cryopreservation and 205 received an autotransplantation. Tissue retrieval was mainly performed by laparoscopy and only one major complication occurred. Transplantations were predominantly performed by open procedures and data on safety were insufficient. A total of 295 autotransplantations were analyzed, resulting in 104 pregnancies. Sixty-five pregnancies led to live births, while nine were ongoing at that time. A pregnancy rate (PR) of 50.7% and a live-birth rate (LBR) of 32.7% were observed. Natural conception accounted for 46.3% of live births. No birth deficits were recorded. Ovarian tissue autotransplantation seems to be a safe procedure with acceptable pregnancy rates., (This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

INTRODUÇÃO: A insuficiência ovariana decorre de ooforectomia bilateral em diversas afecções do ovário, como neoplasia e sepse, além de quimioterapia, radiação pélvica e fatores relacionados à idade. Suas complicações sistêmicas, como insuficiências hormonais, podem ser de difícil controle. OBJETIVOS: Avaliar aspectos endócrinos e histológicos ovarianos após seu transplante ortotópico, sem anastomose vascular e submetidos à imunossupressão pela ciclosporina. MATERIAL E MÉTODO: Foram utilizadas 12 coelhas da raça Nova Zelândia Branca (n = 6) e Califórnia (n = 6), divididas em dois grupos: grupo 1 (n = 4), de controle, submetido apenas a laparotomia, sem outro procedimento cirúrgico; e grupo 2 (n = 8), submetido a ooforectomia bilateral e transplante ortotópico dos ovários, que foram trocados entre as duas raças de animais. De um lado, foi transplantado o ovário íntegro e, do outro, o ovário fatiado. Não houve anastomose vascular. As coelhas foram imunossuprimidas com ciclosporina (10mg/kg ao dia). No final do período de acompanhamento de seis meses, foram feitas dosagens de estradiol, progesterona, hormônio foliculoestimulante (FSH) e hormônio luteinizante (LH), além de estudo histológico de ovários, tuba uterina e útero. RESULTADOS: Os ovários transplantados estavam rodeados por tecido conjuntivo, bem vascularizados, mostrando folículos em diferentes estádios de desenvolvimento. A histologia tubária e uterina também foi preservada em ambos os grupos. A dosagem hormonal foi normal em todas as coelhas, indicando que, do ponto de vista endócrino, o ovário transplantado estava funcionando. O transplante ovariano homógeno ortotópico, sem pedículo vascular e submetido à imunossupressão pela ciclosporina, foi eficaz para a manutenção de hormônios ovarianos em níveis normais.INTRODUCTION: Ovarian failure may be due to bilateral oophorectomy on several ovarian diseases, such as infection and neoplasm; chemo and radiotherapy as well as factors related to age led to endocrine disturbances followed by systemic complications. OBJECTIVES: To assess endocrine and histological ovarian aspects following allogeneic ovarian transplantation without vascular pedicle and immunosupressed by cyclosporine. MATERIAL AND METHOD: Twelve female New Zealand White and California rabbits were divided into 2 Groups: group 1 (n = 4), sham operation; and group 2 (n = 8), submitted to a laparotomy and ovarian bilateral transplantation between pairs of animals. In one side the ovary was intact and in the other side the ovary was sliced. At the end of the follow-up period, blood samples were studied for estradiol, progesterone, FSH and LH. At the end of the experiment, histological studies of ovaries, uterus and tubes were carried out. RESULTS: The ovary implants were surrounded by connective tissue; they were well vascularized, and presented follicles in different stages of development. Hormonal values were normal in all rabbits. CONCLUSIONS: Allogeneic orthotopic ovarian transplantation without vascular pedicle and immunosupressed by cyclosporine was effective to preserve normal level of ovarian hormone.

Objective: To study the effect of freezing, in vitro culture (IVC) and grafting to chorioallantoic membrane (CAM) on follicle outcomes in human ovarian tissue., Design: An experimental study., Setting: University-based research laboratory., Patients: Fresh and cryopreserved ovarian tissue from 10 patients was donated to research with their consent and institutional review board approval., Interventions: Fresh and frozen-thawed ovarian cortical pieces were in vitro-cultured and compared (fresh-IVC vs FT-IVC). The FT-IVC fragments were then examined against fragments grafted to CAM (FT-CAM). After both IVC and CAM grafting, ovarian cortical pieces (4×2×1 mm 3 ) were analyzed on days 0, 1, and 6., Main Outcome Measures: Follicle analyses included histology (count and classification) and immunohistochemistry (Ki67 [proliferation], caspase-3 [apoptosis], 1A and 1B light chain 3B [autophagy], p-Akt, FOXO1, and p-rpS6 [PI3K activation]). Droplet digital polymerase chain reaction further explored expression of PI3K pathway- and oocyte-related genes in tissue sections., Results: No major differences were detected between fresh-IVC and FT-IVC tissues in any conducted analyses. Although a significant drop was observed in primordial follicle (PF) proportions in the fresh-IVC and FT-IVC groups (d0 vs. d6, P<.002), they held steady in the FT-CAM group (d0 vs. d6, P>.05). The PF rates were also significantly higher in the FT-CAM group than the FT-IVC group on d6 (P=.02). Importantly, avian erythrocytes were already present in 30% of implants from d1. Apoptotic and autophagic follicle rates increased during IVC (P<.008), but remained significantly lower in the FT-CAM group (P<.01), confirming superior follicle preservation in CAM-grafted tissue. Upregulation of the PI3K/FOXO pathway was established in the IVC groups, demonstrating PF activation, whereas significant pathway downregulation was detected in the FT-CAM group (P<.03). The droplet digital polymerase chain reaction tests confirmed oocyte growth during IVC and follicle autophagy in all groups; however, the PI3K pathway appeared to be differentially modulated in tissues and follicles., Conclusions: In vitro culture induces PF depletion with no additional impact of freezing. Grafting to CAM preserves the PF pool by curbing follicle activation, apoptosis, and autophagy, probably thanks to rapid graft revascularization and/or the circulating embryonic antimüllerian hormone. These findings highlight the importance of enhancing neoangiogenesis in ovarian grafts and investigating the potential benefits of administering antimüllerian hormone to prevent PF burnout., (Copyright © 2022 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: To assess the feasibility, effectiveness, and reproductive outcomes of transplantation of tiny cryopreserved ovarian pieces through a pipelle cannula during laparoscopic surgery., Methods: A retrospective study of patients who underwent ovarian tissue transplantation for fertility restoration between 2004 and 2022. The "pipelle group" had their ovarian cortex cut into tiny pieces of ~ 1-2 mm 3 before cryopreservation. The pieces were too small to be handled and transplanted via standard laparoscopic tools. Transplantation was performed using a pipelle cannula during laparoscopic surgery. The "control group" underwent transplants of ovarian cortex pieces 1-2 mm thick, measuring approximately 25-50 mm 2 pieces, using standard procedures., Results: The pipelle group consisted of 4 patients aged 19, 21, 27, and 28 years old at ovarian tissue cryopreservation (OTC). The control group consisted of 14 patients aged 21-30 years old. All pipelle patients restored their endocrine activity, and all of them conceived. FSH levels dropped during the first 3 months following the pipelle transplant. IVF cycle outcomes were similar for both groups. All patients from the pipelle group conceived, resulting in 5 pregnancies and 4 live births (one patient had 2 deliveries, and one additional pregnancy is ongoing), compared to the control group, where 8 patients achieved a total of 20 pregnancies and 18 live births., Conclusion: Pipelle transplantation for tiny cryopreserved ovarian pieces is feasible and effective. This study opens a door for patients who had their ovaries cut into small pieces and may even simplify the procedure in some instances, making ovarian transplant more accessible., Trial Registration: (#6531-19-SMC) [18/09/2019]., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Study Question: What are the pregnancy and live birth rates for ovarian tissue transplantation and which factors are associated with the success rate?, Summary Answer: Pregnancy and live birth rates per transplanted woman are 32.7% and 26.5% and success rate is associated with female age and first versus repeated transplantation., What Is Known Already: Live birth rates after ovarian tissue transplantations have been reported to be between around 24% and 41% per patient. Success rates seem to be negatively associated with increasing female age at the time of tissue cryopreservation and with pelvic radiation. Success rates are apparently not reduced after overnight transportation of ovarian tissue before freezing., Study Design, Size, Duration: Registry analysis of 244 transplantations in 196 women, performed by 26 FertiPROTEKT network centres from 2007 to 2019 with follow-up till December 2020., Participants/materials, Setting, Methods: Orthotopic ovarian tissue transplantations were performed in 196 women, 191 with previous malignant and 5 with previous non-malignant diseases. Size of transplanting centres varied between 1 and 100 transplantations per centre (median: 2). Factors possibly associated with success rate such as female age, first and repeated transplantation, experience of the transplanting centre and overnight transportation of the ovarian tissue before freezing were analysed., Main Results and the Role of Chance: Average age of all 196 transplanted women was 31.3 years (SD 5.2; range 17-44) at the time of cryopreservation of tissue and 35.9 years (SD 4.8; range 23-47) at the time of transplantation. Pregnancy rate was 30.6% (95% CI, 24.2-37.6%) per first transplantation and 32.7% (95% CI, 26.1-39.7%) per patient. Pregnancy rate was higher after first transplantation (30.6% (95% CI, 24.2-37.6%)) compared to second and subsequent transplantations (11.8% (95% CI, 3.3-27.5%)). Live birth rate per first transplantation was 25.0% (95% CI, 19.1-31.7%) and per patient 26.5% (95% CI, 20.5-33.3%). Success rate decreased with increasing age at the time of ovarian tissue freezing. Live birth rate was 28.2% (95% CI, 20.9-36.3%) in women <35 years and 16.7% (95% CI, 7.9-29.3%) in women >35 years. Pregnancy rates after first transplantation were higher in centres who had performed ≥10 transplantations (35.1%) compared to centres with <10 transplantation (25.4%) (P = 0.12). Corresponding live birth rates were 27.0% and 18.6%. Success rates were not different in women with and without overnight transportation of tissue before cryopreservation., Limitations, Reasons for Caution: The data were drawn from a registry analysis. Data such as ovarian reserve and premature ovarian insufficiency were not available for all women. Data might be influenced by different follow-up policies of the centres., Wider Implications of the Findings: The study reveals the high potential of ovarian tissue freezing and transplantation, but only if freezing is performed in younger women. The study suggests focus should be placed on the first and not on repeated transplantations. It also opens the discussion of whether transplantation should rather be performed by experienced centres., Study Funding/competing Interest(s): No funding. No competing interests., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Besides the exponentially increasing number of mouse strains, the rising number of rat strains, due to the establishment of transgenic and coisogenic strains in this species, surpasses the capacity of most animal houses. Cryopreservation of gametes may be a means of solving these problems. Here we describe an easy and fast method for the cryopreservation and transplantation of frozen–thawed ovaries of the rat. Ovaries of the rat inbred strain WKY/Ztm were frozen with dimethylsulphoxide as cryoprotectant and stored at -196°C. Orthotopical transplantation was performed into ovariectomized syngenic recipients. Re-establishment of the reproductive cycle in the recipients was confirmed by vaginal cytology. The morphological integrity of frozen and unfrozen ovaries was compared by histological means after staining with haematoxylin and eosin. The number of litters and offspring was recorded. Reproductive cycle was re-established in all recipients of unfrozen ovaries and in more than 50% of recipients that received frozen–thawed ovaries. One-third of the former and more than 25% of the latter became pregnant and delivered at least one litter. Cyropreservation of ovaries can thus be considered as a reliable method of preserving scientifically and economically important mutant stock, as well as congenic rat strains that are currently not required. [ABSTRACT FROM AUTHOR]

Background and Aim: To examine the effects of intratesticular transplantation on the growth and maturation of young murine ovaries. Methods: Two-week-old ovaries from transgenic mice with enhanced green fluorescent protein expression were transplanted under the testicular capsule of 4-week-old non-transgenic mice. Results: Two months after transplantation all successfully grafted ovaries had survived, based on the presence of bright green fluorescence. The grafts showed various stages of folliculogenesis, including expanded follicles. The neighboring seminiferous tubules had a normal structure and mature sperm in their lumens, indicating active spermatogenesis, and all the recipient males were fertile. There was no evidence of extensive cell migration from the grafted ovaries into the testis. Similar findings were obtained for the grafted ovaries 6 months after surgery, although cell death (as evidenced by yellowish or pale fluorescence) was more frequent. Conclusion: Young murine ovaries can grow and mature autonomously for at least 6 months unaffected by the male hormonal environment. (Reprod Med Biol 2006; 5: 71–77) [ABSTRACT FROM AUTHOR]

Summary We investigated the capacity of young ovaries, transplanted into old ovariectomized CBA mice, to improve remaining life expectancy of the hosts. Donor females were sexually mature 2-month-olds; recipients were prepubertally ovariectomized at 3 weeks and received transplants at 5, 8 or 11 months of age. Relative to ovariectomized control females, life expectancy at 11 months was increased by 60% in 11-month recipient females and by 40% relative to intact control females. Only 20% of the 11-month transplant females died in the 300-day period following ovarian transplantation, whereas nearly 65% of the ovariectomized control females died during this same period. The 11-month-old recipient females resumed oestrus and continued to cycle up to several months beyond the age of control female reproductive senescence. Across the three recipient age groups, transplantation of young ovaries increased life expectancy in proportion to the relative youth of the ovary. Our results relate to recent findings on the gonadal input upon aging in Caenorhabditis elegans and may suggest how the mammalian gonad, including that of humans, could regulate aging and determine longevity. [ABSTRACT FROM AUTHOR]

Background: Ovarian insufficiency is a major concern for long-term cancer survivors. Ovarian tissue cryopreservation for fertility preservation is an emerging technique that has proven successful over the past decade through transplantation of frozen-thawed ovarian tissue. Compared to other established techniques, such as oocyte freezing, ovarian tissue cryopreservation preserves actual organ function and thus the production of sex hormones. Endometriosis in perimenopausal women is rare, however it can be surprising diagnosis in the planned transplantation of cryopreserved ovarian tissue and the already thawed tissue may not be transplanted, so that it has to be refrozen., Results: Ovarian function returned in the patient two months after transplantation, as shown by estrogen production. Ten months after the ovarian tissue transplantation mild stimulation with FSH was initiated in accordance with a low-dose protocol. When ultrasonography revealed a follicle 17 mm in size in the ovarian graft, hCG was added and after follicular puncture one oocyte was obtained. The oocyte could be fertilized by IVF and transferred to the uterus. On day 14 after embryo-transfer, a positive hCG-Level was detected and after an uncomplicated pregnancy a healthy child was delivered., Conclusions: We report the first pregnancy and live birth achieved using transplantation of thawed and refrozen ovarian tissue in a woman treated by chemotherapy and subsequent endometriosis surgery. Refreezing of cryopreserved ovarian tissue is not a hindrance to successful transplantation of ovarian tissue. Against the background of increasing numbers of candidates for transplantation of ovarian tissue is expected that the combination chemotherapy followed by endometriosis will increase., (© 2022. The Author(s).)

Background: Ovarian tissue cryopreservation and transplantation (OTCTP) is currently the main option available to preserve fertility in prepubertal patients undergoing aggressive cancer therapy treatments. However, a major limitation of OTCTP is follicle loss after transplantation. The mouse is a model of choice for studying ovarian function and follicle development after ovarian tissue grafting in vivo. In these mouse models, ovarian tissue or ovaries can be transplanted to different sites. Our aim was to evaluate a new alternative to heterotopic transplantation models that could be useful to test pharmaceutical improvement for ovarian grafts after OTCTP., Methods: Slow frozen murine whole ovaries were transplanted into the mouse ears (between the external ear skin layer and the cartilage). Ovarian transplants were recovered after 3, 14 or 21 days. Grafts were analyzed by immunohistochemistry and follicle density analyses were performed., Results: An increase of ovarian vascularization (CD31 and Dextran-FITC positive staining), as well as cellular proliferation (Ki67 staining) were observed 3 weeks after transplantation in comparison to 3 days. Fibrosis density, evaluated after Van Gieson staining, decreased 3 weeks after transplantation. Furthermore, transplantation of cryopreserved ovaries into ovariectomized mice favored follicle activation compared to transplantation into non-ovariectomized mice., Conclusion: The present study indicates that surgical tissue insertion in the highly vascularized murine ear is an effective model for ovarian grafting. This model could be helpful in research to test pharmaceutical strategies to improve the function and survival of cryopreserved and transplanted ovarian tissue., (© 2022. The Author(s).)

Objective: To report our experience with robot-assisted (RA) autologous cryopreserved ovarian tissue transplantation (ACOTT) with the use of a neovascularizing extracellular matrix scaffold., Design: Case series with meta-analytic update., Setting: Academic., Patient(s): Seven recipients of RA-ACOTT., Intervention(s): Before or shortly after initiating chemotherapy, ovarian tissue was cryopreserved from 7 women, who then underwent RA-ACOTT 9.9 ± 1.8 years (range, 7-12 years) later. Perioperatively, they received transdermal estrogen and low-dose aspirin to enhance graft vascularization. Ovarian cortical pieces were thawed and sutured on an extracellular matrix scaffold, which was then robotically anastomosed to the bivalved remaining ovary in 6 cases and retroperitoneally (heterotopic) to the lower abdomen in 1 case., Main Outcome Measure(s): Ovarian function return, the number of oocytes/embryos, aneuploidy %, live births, and neonatal outcomes were recorded. Graft longevity was compared with the mean from the meta-analytic data., Result(s): Ovarian function returned 13.9 ± 2.7 weeks (11-16.2 weeks) after ACOTT, and oocytes were retrieved in all cases with 12.3 ± 6.9 embryos generated. In contrast to orthotopic, the heterotopic ACOTT demonstrated low embryo quality and an 80% aneuploidy rate. A recipient did not attempt to conceive and 2 needed a surrogate, whereas 4 of 4 delivered 6 healthy children, compared with 115 of 460 (25% pregnancy rate) from the meta-analytic data (n = 79). The mean graft longevity (43.2 ± 23.6/47.4 ± 22.8 months with/without sensitivity analysis) trended longer than the meta-analytic mean (29.4 ± 22.7), even after matching age at cryopreservation., Conclusion(s): In this series, RA-ACOTT resulted in extended graft longevity, with ovarian functions restored in all cases, even when the tissues were cryopreserved after chemotherapy exposure., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Cryopreservation of ovarian tissue followed by transplantation represents a strategy to restore ovarian function and fertility. Stress from cryopreservation-thawing processes can lead to alterations and/or damage to mitochondrial structure and functionality. High resolution respirometry and histological analysis were used to evaluate the effect of cryopreservation and transplantation on ovarian tissue. Four different conditions were performed: Fresh non-transplanted tissue, Fresh transplanted tissue, Cryopreserved non-transplanted tissue and Cryopreserved transplanted tissue. All groups were able to respond to the substrates-uncoupler-inhibitor protocol. We found a dramatic decrease in general oxygen consumption in hemi-ovaries submitted to cryopreservation and/or transplantation. The effect of cryopreservation on mitochondrial metabolism was less intense than effect of transplantation, since the transplantation affected all of the mitochondrial states. A total of 2644 follicles were analyzed. Of these, 2198 were classified as morphologically normal. The percentage of morphologically normal follicles was significantly lower in the Cryopreserved transplanted group when compared to the Cryopreserved non-transplanted group and the Fresh transplanted group (p-value < 0.05). Despite decreased follicular viability and mitochondrial activity, the cryopreservation followed by transplantation of ovarian tissue proved feasible for attempts to restore ovarian function., (© 2021. The Author(s).)

Purpose: This study evaluated the concentrations of hormones resulting from the transplantation of ovarian tissue (OTT) in relation to whether the tissue was frozen at a time close to puberty or above the age of 19 years., Methods: Six girls and adolescents (aged 9-14 years) who underwent ovarian tissue cryopreservation (OTC) were followed after transplantation in adulthood. After OTT, the women were followed via regular blood samples to evaluate the concentrations of FSH, LH, oestradiol and AMH. Twenty-three women undergoing OTT (aged 19-36 years at the time of OTC) were included as a reference group. All of the women had postmenopausal levels of gonadotropins at the time of transplantation., Results: The return of FSH and LH to normal premenopausal concentrations in adult women transplanted with ovarian tissue that was frozen at a time close to puberty was similar to the profiles in women from the reference group. Serum AMH levels were below the detection limit (via the Roche Elecsys assay) in many samples, but four out of six young girls showed measurable concentrations. Oestradiol similarly increased in the first 12 weeks following transplantation, after which it tended to be higher in women having frozen tissue in adulthood., Conclusions: Ovarian tissue that was excised from girls at a time close to puberty, after which it was frozen and transplanted in adulthood, interacts with pituitary tissue in a similar manner to ovarian tissue that is frozen from adult women. Follicles located in the ovarian tissue from young girls are equally sensitive to gonadotropin stimulation as follicles from adult women when exposed to postmenopausal levels of gonadotropins. This result indicates that it is not the ovaries that require maturation to sustain full reproductive potential but rather proper FSH and LH stimulation. Moreover, these results support the continued use of OTC in young women., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Since the survival rates of pediatric patients undergoing cancer treatment or hematopoietic stem cell transplantation (HSCT) have increased rapidly in recent decades, the late effects of treatment are now an important focus of patient care. Access to fertility preservation (FP) procedures as well as their financing differs considerably across Europe. However, some countries in Europe have recently changed the legal basis for financing FP procedures; therefore, the implementation of structures is mandatory to give patients access to FP. In this prospective cohort study, we characterized the process for establishing pediatric fertility counseling, including the development of an in-house standard procedure for recommendations regarding FP with potentially gonadotoxic treatment and valuating data from all FP counseling sessions. All data concerning patient characteristics (pubertal status, disease group) and recommendation of FP measures were prospectively collected and adoption of FP measures analyzed. Prior to the establishment of a structured process for FP in our pediatric oncology and stem cell transplantation center, there was no standardized FP counseling. We demonstrate that with the establishment of an inhouse standard procedure, it is possible to give consistent yet individualized FP counseling to approximately 90% of our patients facing gonadotoxic treatment, counseling over 200 patients between 2017 and 2019. This pilot study could potentially be adapted in other pediatric hematology, oncology, and stem cell transplantation centers to allow a more standardized handling of FP counseling for all patients facing gonadotoxic treatment., (© 2021. The Author(s).)

Objective: To evaluate the use of cryopreserved ovarian tissue in the Danish fertility preservation cohort., Design: Retrospective cohort study., Setting: University hospitals and fertility clinics., Patient(s): Ovarian tissue cryopreservation (OTC) was performed for 1,186 Danish girls and women from 1999-2020, of whom 117 subsequently underwent ovarian tissue transplantation (OTT). Subgroup 1 included 759 patients with a follow-up period of >5 years. Out of these, OTT rates were further analyzed for those patients who were alive and aged >24 years in July 2020 (subgroup 2; n = 554)., Intervention(s): OTC and OTT., Main Outcome Measure(s): OTT, death, donation of tissue., Result(s): In subgroup 1, 14% of the patients had undergone OTT, 18% had died, 9% had donated their tissue for research, and 59% still had their tissue stored. In subgroup 2, 19% had undergone OTT and for most diagnoses the OTT rates ranged from 15% to 22% with benign hematologic diseases having the highest OTT rate (35%). On the basis of the entire cohort, stratified age analysis indicated that women aged ≥30 years at OTC were more likely to return for OTT than women aged 18-29 years at OTC; mean storage times were 3.7 and 3.6 years, respectively. Only 4% of the girls aged <18 years at OTC had undergone OTT., Conclusion(s): The OTT rates depended on the diagnosis, age at OTC, and follow-up time. Specific criteria are needed for reporting and comparing OTT rates. Six out of 10 patients still had their cryopreserved tissue stored and longer follow-up is needed, especially for younger girls., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The huge loss of ovarian follicles after transplantation of frozen/thawed ovarian tissue is considered a major drawback on the efficacy of the procedure. Here we investigate whether Er:YAG laser treatment prior to xenotransplantation can improve re-vascularization and subsequently follicle survival in human ovarian tissue., Methods: A total of 99 frozen/thawed human ovarian cortex pieces were included of which 72 pieces from 12 woman were transplanted to immunodeficient mice. Tissues from each woman were included in both an 8-day and an 8-week duration study and treated with either full-beam laser (L1) or fractionated laser (L2), or served as untreated controls. Vascularization of the ovarian xenografts were evaluated after 8 days by qPCR and murine Cd31 immunohistochemical analysis. Follicle densities were evaluated histologically 8 weeks after xenografting., Results: Gene expression of Vegf/VEGF was upregulated after L1 treatment (p=0.002, p=0.07, respectively), whereas Angpt1, Angpt2, Tnf-α, and Il1-β were significantly downregulated. No change in gene expression was found in Cd31/CD31, ANGPT1, ANGPT2, ANGTPL4, XBP1, or LRG1 after any of the laser treatments. The fraction of Cd31 positive cells were significantly reduced after L1 and L2 treatment (p<0.0001; p=0.0003, respectively), compared to controls. An overall negative effect of laser treatment was detected on follicle density (p=0.03)., Conclusions: Er:YAG laser treatment did not improve re-vascularization or follicle survival in human ovarian xenografts after 8 days and 8 weeks grafting, respectively. However, further studies are needed to fully explore the potential angiogenic effects of controlled tissue damage using different intensities or lasers., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To describe molecular and paracrine signaling changes produced by human bone marrow-derived stem cells (BMDSC) in human ovarian cortex., Design: Experimental study., Setting: University hospital research laboratories., Patient(s): Ovarian cortex from poor responder women (n = 7)., Animals: Immunodeficient NOD/SCID female mice (n = 18)., Intervention(s): Human ovarian cortex strips were xenografted into ovariectomized NOD/SCID female mice. A week later, mice were infused with phosphate-buffered saline, 1 × 10 6 BMDSC, or 3 × 10 5 CD133 + cells via tail vein. Gene expression changes and enriched pathways were assessed by RT 2 Profiler Arrays. Several upregulated genes were validated in individual samples by real-time quantitative PCR, and transcriptomic results were reinforced by a proteomic assessment., Main Outcome Measure(s): Gene expression changes, enriched Kyoto Encyclopedia of Genes and Genomes pathways, and paracrine factors., Result(s): Seventy-four Kyoto Encyclopedia of Genes and Genomes pathways were upregulated, with the PI3K-Akt signaling pathway the most enriched after BMDSC and CD133 treatments. The greatest transcriptomic changes were seen on day 14 in the BMDSC group, affecting the regulation of paracrine factors such as KITLG, THBS1, SERPINF1, and TIMP2. Proteomics data verified changes in FoxO signaling, actin cytoskeleton remodeling, and apoptosis by BMDSC., Conclusion(s): We identified paracrine factors and pathways regulated by BMDSC that may be future targets of treatment for the increasing number of poor responder women. Our findings suggest that BMDSC upregulated soluble factors such as KITLG, THBS1, SERPINF1, and TIMP2 as well as PI3K-Akt signaling and regulation of actin cytoskeleton pathways. The identification of these putative underlying mechanisms informs future experiments aiming to optimizing clinical application of BMDSC., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The occurrence of ice crystallization during ovarian tissue (OT) cryopreservation causes unavoidable cryodamage, and ice recrystallization during the warming is more detrimental than ice crystallization. Here, we investigated that antifreeze protein (AFP) treatment during the warming procedure can improve the bovine OT quality after xenotransplantation (XT). Bovine OTs (n=120) were evenly assigned to four groups: fresh, vitrified-warmed, vitrified-warmed with 10 mg/mL Leucosporidium ice-binding protein (LeIBP, a type of AFP) (LeIBP-10), and vitrified-warmed with 20 mg/mL LeIBP (LeiBP-20). LeIBPs were added to the first warming solution. Twenty pieces of OTs were assigned to each category. The remaining 10 OTs from each category were assigned to the XT-Fresh control, XT-Vitrified-warmed control, XT-LeIBP-10, and XT-LeIBP-20 groups, respectively, and xenotransplanted to 9-week-old ovariectomized nude mice for one week. LeIBP treatment during the warming step increased morphological follicle normality and decreased apoptotic follicle ratios after vitrification-warming and XT. The XT-vitrified-warmed control group showed significantly reduced microvessel density and increased fibrosis when compared to that of the XT-fresh group. Microvessel density and fibrosis were recovered in both LeIBP treated groups. There was no significant difference between the LeIBP-10 and LeIBP-20 groups in all outcomes. AFP treatment during the warming procedure can prevent OT damage, and improve ovarian follicle morphology and apoptosis in both the vitrified-warmed bovine OT and its graft. After confirmation in a human study, AFPs can potentially be applied to human OT cryopreservation to reduce cryodamage and improve the OT quality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kong, Hong, Lee, Youm, Lee, Suh and Kim.)

The feasibility of freezing and thawing ovarian tissue is nowadays widely documented. However, ovarian tissue transplantation (OTT) is happening at a much slower pace, and clinical experience is somewhat limited. In this review, five European centers present their collective experience of transplanting ovarian tissue in 285 women. The focus is on surgical techniques and OTT outcomes, reproductive outcomes, the impact of chemotherapy before ovarian tissue cryopreservation (OTC), the risk of relapse, and endocrine resumption and longevity of transplanted tissue. The risk of relapse due to reimplantation of ovarian tissue appears to be very low according to current data. Recovery of endocrine function is seen in almost all women undergoing transplantation of ovarian tissue, and about one in four gives birth to a healthy child. The efficacy of in vitro fertilization in these patients is not very high, however, and needs to be substantially improved. Radiation to the pelvis, especially with relatively high doses, appears to considerably decrease the likelihood of a successful pregnancy and may be contraindicated. Our results demonstrate that chemotherapy before OTC does not impair the chances of success, depending, of course, on the total dose and type of chemotherapy administered. At this early stage of development of OTT for restoration of fertility, the results are encouraging and demonstrate clear potential. However, the method is far from being fully developed and requires continued research efforts to optimize our approach., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: After ovarian tissue transplantation, ischemia-reperfusion injury and free radicals cause follicle depletion and apoptosis. Therefore, the use of antioxidants to reduce the production of free radicals is an important method to address the consequences of ischemia-reperfusion injury. Resveratrol is a natural active polyphenol compound with anti-inflammatory, antitumor, strong antioxidant and anti-free radical properties. The aim of this study was to investigate whether resveratrol could improve the effect of autologous ovarian transplantation after cryopreserve-thawn mouse ovarian tissue., Methods: Whole-ovary vitrification and autotransplantation models were used to investigate the effects of resveratrol. Six-week-old female mice from the Institute of Cancer Research (ICR) were subjected to vitrification. All ovaries were preserved in liquid nitrogen for 1 week before being thawed. After thawing, ovarian tissues were autotransplanted in the bilateral kidney capsules. Mice (n = 72) were randomly divided into four groups to determine the optimal concentration of resveratrol (experiment I). Treatments were given as follows: saline, 5 mg/kg resveratrol, 15 mg/kg resveratrol and 45 mg/kg resveratrol, which were administered orally for one week. Grafted ovaries were collected for analysis on days 3, 7, and 21 after transplantation. Ovarian follicle morphology was assessed by hematoxylin and eosin staining. Serum FSH and E 2 levels were measured to estimate the transplanted ovarian reserve and endocrine function. Other mice were randomly divided into two groups-saline and 45 mg/kg resveratrol to further evaluate the effect of resveratrol and explore the mechanisms underlying this effect (experiment II). Ovarian follicle apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. Immunohistochemistry, qRT-PCR and western blotting (MDA, SOD, NF-κB, IL-6 and SIRT1) were used to explore the mechanisms of resveratrol. Moreover, oocytes derived from autotransplanted ovaries at 21 days were cultured and fertilized in vitro., Results: The proportions of morphologically normal (G1) follicles at 3, 7 and 21 days were significantly higher in the 45 mg/kg resveratrol group than in the saline group. The TUNEL-stained follicles (%) at 7 days were significantly decreased in the 45 mg/kg resveratrol group compared with the saline group. Western blot analysis revealed that SOD2 and SIRT1 levels were significantly higher in the 45 mg/kg resveratrol group than in the saline group at day 7 and that MDA and NF-κB levels were lower in the saline group on day 3. Likewise, IL-6 was lower in the saline group on day 7. These results are basically consistent with the qRT-PCR results. In addition, the mean number of retrieved oocytes and fertilization and cleavage were significantly increased in the 45 mg/kg resveratrol group compared with the saline group., Conclusions: Administration of resveratrol could improve the quality of cryopreserved mouse ovarian tissue after transplantation and the embryo outcome, through anti-inflammatory and antioxidative mechanisms.

Backgroud: Melatonin has anti-inflammatory and antioxidative actions at the mitochondrial level. This indole-containing molecule may protect ovarian grafts during the process of cryopreservation. Therefore, we aimed to determine whether melatonin pretreatment improves rat ovarian graft quality., Methods: Twenty-six female rats were allocated to two study groups of thirteen animals each: 1) control group: ovaries cryopreserved using the standard protocol; and 2) melatonin group: ovaries cryopreserved in a medium with melatonin. Ten rats of each group were submitted to 24-h freezing, and whole ovaries autologous and avascular transplantation with retroperitoneal placement. After postoperative (PO) day 15, daily vaginal smears were obtained for estrous cycle characterization. Between PO days 30 and 35, the 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) analyses. The ovaries of the three remaining rats from each group were studied immediately after thawing to assess the effects of cryopreservation. ANOVA and Tukey's 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 as compared to control. There was a reduction in apoptosis by TUNEL on follicles, corpora lutea, and collagen type III., Conclusion: Based on the evaluated parameters, melatonin may promote the quality of ovarian grafts. Reproductive function enhancement should be further studied.