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BACKGROUND: Assisted Reproductive Technologies (ARTs) have been validated in human and animal to solve reproductive problems such as infertility, aging, genetic selection/amplification and diseases. The persistent gap in ART biomedical applications lies in recapitulating the early stage of ovarian folliculogenesis, thus providing protocols to drive the large reserve of immature follicles towards the gonadotropin-dependent phase. Tissue engineering is becoming a concrete solution to potentially recapitulate ovarian structure, mostly relying on the use of autologous early follicles on natural or synthetic scaffolds. Based on these premises, the present study has been designed to validate the use of the ovarian bioinspired patterned electrospun fibrous scaffolds fabricated with poly(ε-caprolactone) (PCL) for multiple preantral (PA) follicle development. METHODS: PA follicles isolated from lamb ovaries were cultured on PCL scaffold adopting a validated single-follicle protocol (Ctrl) or simulating a multiple-follicle condition by reproducing an artificial ovary engrafted with 5 or 10 PA (AO5PA and AO10PA). The incubations were protracted for 14 and 18 days before assessing scaffold-based microenvironment suitability to assist in vitro folliculogenesis (ivF) and oogenesis at morphological and functional level. RESULTS: The ivF outcomes demonstrated that PCL-scaffolds generate an appropriate biomimetic ovarian microenvironment supporting the transition of multiple PA follicles towards early antral (EA) stage by supporting follicle growth and steroidogenic activation. PCL-multiple bioengineering ivF (AO10PA) performed in long term generated, in addition, the greatest percentage of highly specialized gametes by enhancing meiotic competence, large chromatin remodeling and parthenogenetic developmental competence. CONCLUSIONS: The study showcased the proof of concept for a next-generation ART use of PCL-patterned scaffold aimed to generate transplantable artificial ovary engrafted with autologous early-stage follicles or to advance ivF technologies holding a 3D bioinspired matrix promoting a physiological long-term multiple PA follicle protocol.

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An artificial ovary based on the alginate (ALG) hydrogel has been widely implemented to preserve prepubertal female fertility. However, this platform is not fully capable of successful an ovary microenvironment simulation for follicle development, holding great potential for its improvement. Therefore, this experimental study aimed to evaluate the effect of an amniotic membrane extract (AME) -loaded hydrogel on the mouse preantral follicles in vitro development. In order to have better follicle development, first, the impact of different concentrations of follicle-stimulating hormone (FSH) was evaluated on the mouse preantral follicles encapsulated in ALG. Later, the appropriate dose was adjusted for the follicles encapsulated in the ALG-AME hydrogel. Results demonstrated that 100 mIU/ml FSH showed a significant follicle survival rate compared with 10 mIU/ml FSH (P=0.005). According to MTT assay finding, the rate of weight loss, and rheology evaluations, ALG containing 1 mg/ml AME was identified as an optimal sample of follicle culture instead of other AME concentrations. Follicle diameter significantly increased in the ALG-AME 1 hydrogel compared with the ALG control group without AME (P=0.027). The storage modulus of ALG-AME 1 was 773 Pa and retained the follicle morphology for 13 days. No statistically substantial difference was seen in survival, antrum cavity formation, and competent oocyte in terms of the normal chromosomal arrangement and meiotic spindle rate in comparison with the control group. It can be concluded that ALG-AME 1 could not significantly impact the mouse preantral follicle.

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BACKGROUND: Studies have shown that chemotherapy and radiotherapy can cause premature ovarian failure and loss of fertility in female cancer patients. Ovarian cortex cryopreservation is a good choice to preserve female fertility before cancer treatment. Following the remission of the disease, the thawed ovarian tissue can be transplanted back and restore fertility of the patient. However, there is a risk to reintroduce cancer cells in the body and leads to the recurrence of cancer. Given the low success rate of current in vitro culture techniques for obtaining mature oocytes from primordial follicles, an artificial ovary with primordial follicles may be a good way to solve this problem. METHODS: In the study, we established an artificial ovary model based on the participation of mesenchymal stem cells (MSCs) to evaluate the effect of MSCs on follicular development and oocyte maturation. P2.5 mouse ovaries were digested into single cell suspensions and mixed with bone marrow derived mesenchymal stem cells (BM-MSCs) at a 1:1 ratio. The reconstituted ovarian model was then generated by using phytohemagglutinin. The phenotype and mechanism studies were explored by follicle counting, immunohistochemistry, immunofluorescence, in vitro maturation (IVM), in vitro fertilization (IVF), real-time quantitative polymerase chain reaction (RT-PCR), and Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) assay. RESULTS: Our study found that the addition of BM-MSCs to the reconstituted ovary can enhance the survival of oocytes and promote the growth and development of follicles. After transplanting the reconstituted ovaries under kidney capsules of the recipient mice, we observed normal folliculogenesis and oocyte maturation. Interestingly, we found that BM-MSCs did not contribute to the formation of follicles in ovarian aggregation, nor did they undergo proliferation during follicle growth. Instead, the cells were found to be located around growing follicles in the reconstituted ovary. When theca cells were labeled with CYP17a1, we found some overlapped staining with green fluorescent protein(GFP)-labeled BM-MSCs. The results suggest that BM-MSCs may participate in directing the differentiation of theca layer in the reconstituted ovary. CONCLUSIONS: The presence of BM-MSCs in the artificial ovary was found to promote the survival of ovarian cells, as well as facilitate follicle formation and development. Since the cells didn't proliferate in the reconstituted ovary, this discovery suggests a potential new and safe method for the application of MSCs in clinical fertility preservation by enhancing the success rate of cryo-thawed ovarian tissues after transplantation.

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PURPOSE: To preserve fertility before gonadotoxic therapy, ovarian tissue can be removed, cryopreserved, and transplanted back again after treatment. An alternative is the artificial ovary, in which the ovarian follicles are extracted from the tissue, which reduces the risk of reimplantation of potentially remaining malignant cells. The PTEN inhibitor bpV(HOpic) has been shown to activate human, bovine and alpacas ovarian follicles, and it is therefore considered a promising substance for developing the artificial ovary. The purpose of this study was to examine the impact of different scaffolds and the vanadate derivative bpV(HOpic) on mice follicle survival and hormone secretion over 10 days. METHODS: A comparative analysis was performed, studying the survival rates (SR) of isolated mice follicle in four different groups that differed either in the scaffold (polycaprolactone scaffold versus polyethylene terephthalate membrane) or in the medium-bpV(HOpic) versus control medium. The observation period of the follicles was 10 days. On days 2, 6, and 10, the viability and morphology of the follicles were checked using fluorescence or confocal microscopy. Furthermore, hormone levels of estrogen (pmol/L) and progesterone (nmol/L) were determined. RESULTS: When comparing the SR of follicles among the four groups, it was observed that on day 6, the study groups utilizing the polycaprolactone scaffold with bpV(HOpic) in the medium (SR: 0.48 ± 0.18; p = 0.004) or functionalized in the scaffold (SR: 0.50 ± 0.20; p = 0.003) exhibited significantly higher survival rates compared to the group using only the polyethylene terephthalate membrane (SR: 0). On day 10, a significantly higher survival rate was only noted when comparing the polycaprolactone scaffold with bpV(HOpic) in the medium to the polyethylene terephthalate membrane group (SR: 0.38 ± 0.20 versus 0; p = 0.007). Higher levels of progesterone were only significantly associated with better survival rates in the group with the polycaprolactone scaffold functionalized with bpV(HOpic) (p = 0.017). CONCLUSION: This study demonstrates that three-dimensional polycaprolactone scaffolds improve the survival rates of isolated mice follicles in comparison with a conventional polyethylene terephthalate membrane. The survival rates slightly improve with added bpV(HOpic). Furthermore, higher rates of progesterone were also partly associated with improved survival.

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STUDY QUESTION: Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles in vitro? SUMMARY ANSWER: A feeder layer of OSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. WHAT IS KNOWN ALREADY: In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of OSCs. This co-culture notably enhances follicle survival and growth. STUDY DESIGN SIZE DURATION: Pre-antral follicles were isolated from human frozen-thawed ovarian tissue biopsies and then encapsulated in 1% alginate scaffolds. These embedded preantral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth, and hormone production between the different groups. PARTICIPANTS/MATERIALS SETTING METHODS: Primordial/intermediate and primary follicles were isolated from frozen-thawed ovarian tissue of cancer patients (n = 6). OSCs were then isolated from ovarian tissue of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean ± SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of OSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (P < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 µm compared to 67.3 ± 7.2 µm without it (P = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (P < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; P < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75< x <200 µm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (P = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without OSCs (29.9 ± 4.0 pg/ml). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology, and steroidogenesis in alginate scaffolds with human OSCs. WIDER IMPLICATIONS OF THE FINDINGS: Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human OSCs has shown the potential to overcome this limitation. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., PhD scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

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Recent advances in anticancer treatment have significantly improved the survival rate of young females; unfortunately, in about one third of cancer survivors the risk of ovarian insufficiency and infertility is still quite relevant. As the possibility of becoming a mother after recovery from a juvenile cancer is an important part of the quality of life, several procedures to preserve fertility have been developed: ovarian surgical transposition, induction of ovarian quiescence by gonadotropin-releasing hormone agonists (GnRH-a) treatment, and oocyte and/or ovarian cortical tissue cryopreservation. Ovarian tissue cryostorage and allografting is a valuable technique that applies even to prepubertal girls; however, some patients cannot benefit from it due to the high risk of reintroducing cancer cells during allograft in cases of ovary-metastasizing neoplasias, such as leukemias or NH lymphomas. Innovative techniques are now under investigation, as in the construction of an artificial ovary made of isolated follicles inserted into an artificial matrix scaffold, and the use of stem cells, including ovarian stem cells (OSCs), to obtain neo-folliculogenesis and the development of fertilizable oocytes from the exhausted ovarian tissue. This review synthesizes and discusses these innovative techniques, which potentially represent interesting strategies in oncofertility programs and a new hope for young female cancer survivors.

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Ovarian dysfunction poses significant threats to the health of female individuals. Ovarian failure can lead to infertility due to the lack or inefficient production of fertilizable eggs. In addition, the ovary produces hormones, such as estrogen and progesterone, that play crucial roles not only during pregnancy, but also in maintaining cardiovascular, bone, and cognitive health. Decline in estrogen and progesterone production due to ovarian dysfunction can result in menopausal-associated syndromes and lead to conditions, such as osteoporosis, cardiovascular disease, and Alzheimer's disease. Recent advances in the design of bioengineered three-dimensional (3D) ovarian models, such as ovarian organoids or artificial ovaries, have made it possible to mimic aspects of the cellular heterogeneity and functional characteristics of the ovary in vitro. These novel technologies are emerging as valuable tools for studying ovarian physiology and pathology and may provide alternatives for fertility preservation. Moreover, they may have the potential to restore aspects of ovarian function, improving the quality of life of the (aging) female population. This review focuses on the state of the art of 3D ovarian platforms, including the latest advances modeling female reproduction, female physiology, ovarian cancer, and drug screening.

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STUDY QUESTION: What is the current state-of-the-art methodology assessing decellularized extracellular matrix (dECM)-based artificial ovaries for treating ovarian failure? SUMMARY ANSWER: Preclinical studies have demonstrated that decellularized scaffolds support the growth of ovarian somatic cells and follicles both in vitro and in vivo. WHAT IS KNOWN ALREADY: Artificial ovaries are a promising approach for rescuing ovarian function. Decellularization has been applied in bioengineering female reproductive tract tissues. However, decellularization targeting the ovary lacks a comprehensive and in-depth understanding. STUDY DESIGN SIZE DURATION: PubMed, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials were searched from inception until 20 October 2022 to systematically review all studies in which artificial ovaries were constructed using decellularized extracellular matrix scaffolds. The review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol. PARTICIPANTS/MATERIALS SETTING METHODS: Two authors selected studies independently based on the eligibility criteria. Studies were included if decellularized scaffolds, regardless of their species origin, were seeded with ovarian cells or follicles. Review articles and meeting papers were removed from the search results, as were articles without decellularized scaffolds or recellularization or decellularization protocols, or control groups or ovarian cells. MAIN RESULTS AND THE ROLE OF CHANCE: The search returned a total of 754 publications, and 12 papers were eligible for final analysis. The papers were published between 2015 and 2022 and were most frequently reported as coming from Iran. Detailed information on the decellularization procedure, evaluation method, and preclinical study design was extracted. In particular, we concentrated on the type and duration of detergent reagent, DNA and extracellular matrix detection methods, and the main findings on ovarian function. Decellularized tissues derived from humans and experimental animals were reported. Scaffolds loaded with ovarian cells have produced estrogen and progesterone, though with high variability, and have supported the growth of various follicles. Serious complications have not been reported. LIMITATIONS REASONS FOR CAUTION: A meta-analysis could not be performed. Therefore, only data pooling was conducted. Additionally, the quality of some studies was limited mainly due to incomplete description of methods, which impeded specific data extraction and quality analysis. Several studies that used dECM scaffolds were performed or authored by the same research group with a few modifications, which might have biased our evaluation. WIDER IMPLICATIONS OF THE FINDINGS: Overall, the decellularization-based artificial ovary is a promising but experimental choice for substituting insufficient ovaries. A generic and comparable standard should be established for the decellularization protocols, quality implementation, and cytotoxicity controls. Currently, decellularized materials are far from being clinically applicable to artificial ovaries. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the National Natural Science Foundation of China (Nos. 82001498 and 81701438). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: This systematic review is registered with the International Prospective Register of Systematic Reviews (PROSPERO, ID CRD42022338449).

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Today, fertility preservation is receiving more attention than ever. Cryopreservation, which preserves ovarian tissue to preserve fertility in young women and reduce the risk of infertility, is currently the most widely practiced. Transplantation, however, is less feasible for women with blood-borne leukemia or cancers with a high risk of ovarian metastasis because of the risk of cancer recurrence. In addition to cryopreservation and re-implantation of embryos, in vitro ovarian organ reconstruction techniques have been considered as an alternative strategy for fertility preservation. In vitro culture of oocytes in vitro Culture, female germ cells induction from pluripotent stem cells (PSC) in vitro, artificial ovary construction, and ovaria-related organoids construction have provided new solutions for fertility preservation, which will therefore maximize the potential for all patients undergoing fertility preservation. In this review, we discussed and thought about the latest ovarian organ function reconstruction techniques in vitro to provide new ideas for future ovarian disease research and fertility preservation of patients with cancer and premature ovarian failure.

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BACKGROUND: Artificial ovary (AO) is an alternative approach to provide physiological hormone to post-menopausal women. The therapeutic effects of AO constructed using alginate (ALG) hydrogels are limited by their low angiogenic potential, rigidity, and non-degradability. To address these limitations, biodegradable chitin-based (CTP) hydrogels that promote cell proliferation and vascularization were synthesized, as supportive matrix. METHODS: In vitro, follicles isolated from 10-12-days-old mice were cultured in 2D, ALG hydrogels, and CTP hydrogels. After 12 days of culture, follicle growth, steroid hormone levels, oocyte meiotic competence, and expression of folliculogenesis-related genes were monitored. Additionally, follicles isolated from 10-12-days-old mice were encapsulated in CTP and ALG hydrogels and transplanted into the peritoneal pockets of ovariectomised (OVX) mice. After transplantation, steroid hormone levels, body weight, rectal temperature, and visceral fat of the mice were monitored every two weeks. At 6 and 10 weeks after transplantation, the uterus, vagina, and femur were collected for histological examination. RESULTS: The follicles developed normally in CTP hydrogels under in vitro culture conditions. Additionally, follicular diametre and survival rate, oestrogen production, and expression of folliculogenesis-related genes were significantly higher than those in ALG hydrogels. After one week of transplantation, the numbers of CD34-positive vessels and Ki-67-positive cells in CTP hydrogels were significantly higher than those in ALG hydrogels (P < 0.05), and the follicle recovery rate was significantly higher in CTP hydrogels (28%) than in ALG hydrogels (17.2%) (P < 0.05). After two weeks of transplantation, OVX mice implanted with CTP grafts exhibited normal steroid hormone levels, which were maintained until week eight. After 10 weeks of transplantation, CTP grafts effectively ameliorated bone loss and atrophy of the reproductive organs, as well as prevented the increase in body weight and rectal temperature in OVX mice, which were superior to those elicited by ALG grafts. CONCLUSIONS: Our study is the first to demonstrate that CTP hydrogels support follicles longer than ALG hydrogels in vitro and in vivo. The results highlight the clinical potential of AO constructed using CTP hydrogels in the treatment of menopausal symptoms.

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Fertility restoration in patients that survived a hematological cancer during childhood is a core part of their care pathway. Nonetheless, there might be a risk of contamination of the gonads by cancer cells, especially in patients presenting with leukemia and lymphoma. When only a few cancer cells have reached the gonad, they may not be detected by routine histological examination, and therefore more sensitive techniques are required before being confident of the safety of transplanting cryostored testicular and ovarian tissues or cells back to the patient after recovery. Furthermore, if neoplastic cells are identified in the gonadal tissue, methods to eliminate such cells are urgently awaited as the presence of only a few cancer cells may induce disease relapse in these patients. In this review, contamination rates of human gonadal tissue in the case of leukemia or lymphoma as well as decontamination methods applied to both adult and prepubertal testicular and ovarian tissues are presented. Prepubertal gonads will be the main focus as we aim to show how far we have come in establishing safe approaches to fertility restoration. Advances have been made using animal tissue that is usually artificially contaminated by the addition of cancer cell lines to the gonadal cells or tissue, but these techniques need to be improved and still await development in the case of in vivo cancer cell invasion of tissue.

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INTRODUCTION: The in vitro culture of primordial follicles is the only available option for preserving fertility in prepubertal girls with malignant tumors. The cultivation of primordial follicles in scaffolds as artificial ovaries is a promising approach for this. METHODS: Dissociated follicles were placed into an artificial ovarian scaffold composed of fibrinogen and thrombin. The follicles were cultured in a dish dedicated to live cell imaging and observed for growth using immunofluorescence and development via optical microscopy. The morphology of the follicles in the scaffold was three-dimensionally reconstructed using the Imaris software. Growth and development were also quantified. RESULTS: The morphology of artificial ovaries began to degrade over time. Within approximately 7 days, primordial follicles were activated and grew into secondary follicles. A comparison of optical and confocal microscopy results revealed the superior detection of live cells using confocal microscopy. The three-dimensional reconstruction of the confocal microscopy data enabled the automatic enumeration and evaluation of the overall morphology of many follicles. CONCLUSIONS: The novel artificial ovary-enabled primordial follicles to enter the growth cycle after activation and grow into secondary follicles. The use of a fibrin scaffold as a carrier preserves the developmental potential of primordial germ cells and is a potentially effective method for preserving fertility in prepubertal girls.

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Current assisted reproduction technologies (ART) are insufficient to cover the slice of the population needing to restore fertility, as well as to amplify the reproductive performance of domestic animals or endangered species. The design of dedicated reproductive scaffolds has opened the possibility to better recapitulate the reproductive 3D ovarian environment, thus potentially innovating in vitro folliculogenesis (ivF) techniques. To this aim, the present research has been designed to compare ovine preantral follicles in vitro culture on poly(epsilon-caprolactone) (PCL)-based electrospun scaffolds designed with different topology (Random vs. Patterned fibers) with a previously validated system. The ivF performances were assessed after 14 days under 3D-oil, Two-Step (7 days in 3D-oil and on scaffold), or One-Step PCL protocols (14 days on PCL-scaffold) by assessing morphological and functional outcomes. The results show that Two- and One-Step PCL ivF protocols, when performed on patterned scaffolds, were both able to support follicle growth, antrum formation, and the upregulation of follicle marker genes leading to a greater oocyte meiotic competence than in the 3D-oil system. In conclusion, the One-Step approach could be proposed as a practical and valid strategy to support a synergic follicle-oocyte in vitro development, providing an innovative tool to enhance the availability of matured gametes on an individual basis for ART purposes.

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In recent decades, there has been increasing attention toward the quality of life of breast cancer (BC) survivors. Meeting the growing expectations of fertility preservation and the generation of biological offspring remains a great challenge for these patients. Conventional strategies for fertility preservation such as oocyte and embryo cryopreservation are not suitable for prepubertal cancer patients or in patients who need immediate cancer therapy. Ovarian tissue cryopreservation (OTC) before anticancer therapy and autotransplantation is an alternative option for these specific indications but has a risk of retransplantation malignant cells. An emerging strategy to resolve these issues is by constructing an artificial ovary combined with stem cells, which can support follicle proliferation and ensure sex hormone secretion. This promising technique can meet both demands of improving the quality of life and meanwhile fulfilling their expectation of biological offspring without the risk of cancer recurrence.

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PURPOSE: The aim of this study was to design and fabricate a three-dimensional (3D) printed artificial ovary. METHODS: We first compared the printability of gelatin-methacryloyl (GelMA), alginate and GelMA-alginate bioinks, of which GelMA was selected for further investigation. The swelling properties, degradation kinetics and shape fidelity of GelMA scaffolds were characterized by equilibrium swelling/lyophilization, collagenase processing and micro-computed tomography evaluation. Commercial ovarian tumor cell lines (COV434, KGN, ID8) and primary culture ovarian somatic cells were utilized to perform cell-laden 3D printing, and the results were evaluated by live/dead assays and TUNEL detection. Murine ovarian follicles were seeded in the ovarian scaffold and their diameters were recorded every day. Finally, in vitro maturation was performed, and the ovulated oocytes were collected and observed. RESULTS: Our results indicated that GelMA was suitable for 3D printing fabrication. Its scaffolds performed well in terms of hygroscopicity, degradation kinetics and shape fidelity. The viability of ovarian somatic cells was lower than that of commercial cell lines, suggesting that extrusion-based 3D culture fabrication is not suitable for primary ovarian cells. Nevertheless, the GelMA-based 3D printing system provided an appropriate microenvironment for ovarian follicles, which successfully grew and ovulated in the scaffolds. Metaphase II oocytes were also observed after in vitro maturation. CONCLUSIONS: The GelMA-based 3D printing culture system is a viable alternative option for follicular growth, development and transfer. Accordingly, it shows promise for clinical application in the treatment of female endocrine and reproductive conditions.

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Background: A functional artificial ovary is a promising strategy to recover fertility and restore endocrine function in cancer patients. The aim of this study is to optimize the follicle isolation protocol for cryopreserved human ovarian tissues. Methods: Each of the cryopreserved human ovarian cortex pieces (OCPs) from 10 patients was cut into two equal parts and randomly distributed into two treatment groups. Group 1: OCPs digested with Tumor Dissociation Enzyme (TDE); Group 2: OCPs digested with Liberase Dispase High (DH). The efficiency of both groups were evaluated in terms of yield, viability, morphology, and a short-term in vitro culture (IVC) in alginate scaffolds. Results: The TDE can isolate more primordial follicles and smaller diameter of follicles than Liberase DH. The TDE also enabled the isolation of more bright red follicles, higher percent of viable follicles, more morphologically normal follicles, and lower oxidative stress levels compared with Liberase DH. After eight days of IVC, follicles in the TDE group had a higher growth rate from Day 0 to Day 8, and higher viability on Day 8 than the Liberase DH Group. Conclusion: The TDE can be considered an alternative to Liberase DH, enables the isolation of a higher number of healthy follicles from human OCPs, and improves follicle survival after IVC in contrast to Liberase DH.

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The proportion of cancer patients that survive is increasing because of improvements in cancer therapy. However, some cancer treatments, such as chemo- and radio-therapies, can cause considerable damage to reproductive function. The issue of fertility is paramount for women of childbearing age once they are cured from cancer. For those patients with prepubertal or haematogenous cancer, the possibilities of conventional fertility treatments, such as oocyte or embryo cryopreservation and transplantation, are limited. Moreover, ovarian tissue cryopreservation as an alternative to fertility preservation has limitations, with a risk of re-implanting malignant cells in patients who have recovered from potentially fatal malignant disease. One possible way to restore fertility in these patients is to mimic artificially the function of the natural organ, the ovary, by grafting isolated follicles embedded in a biological scaffold to their native environment. Construction and cryopreservation of an artificial ovary might offer a safer alternative option to restore fertility for those who cannot benefit from traditional fertility preservation techniques. This review considers the protocols for constructing an artificial ovary, summarises advances in the field with potential clinical application, and discusses future trends for cryopreservation of these artificial constructions.

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An artificial ovary is a promising approach for preserving fertility in prepubertal girls and women who cannot undergo current cryopreservation strategies. However, this approach is in its infancy, due to the possible challenges of creating a suitable 3D matrix for encapsulating ovarian follicles and stromal cells. To maintain the ovarian stromal cell viability and proliferation, as a first step towards developing an artificial ovary, in this study, a double network hydrogel with a high water swelling capacity (swelling index 15-19) was developed, based on phenol conjugated chitosan (Cs-Ph) and silk fibroin (SF) through an enzymatic crosslinking method using horseradish peroxidase. The addition of SF (1%) to Cs (1%) decreased the storage modulus (G') from 3500 Pa (Cs1) to 1600 Pa (Cs-SF1), and the hydrogels with a rapid gelation kinetic produced a spatially homogeneous distribution of ovarian cells that demonstrated 167% proliferation after 7 days. This new Cs-SF hydrogel benefits from the toughness and flexibility of SF, and phenolic chemistry could provide the potential microstructure for encapsulating human ovarian stromal cells.

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The ovarian reserve (OR) indicates ovarian function by representing the quantity and quality of ovarian follicles, and it gradually decreases with increasing age. With the prolongation of women's lives, the protection provided by estrogen is lost for decades in postmenopausal women, and the related cardiovascular and cerebrovascular diseases, osteoporosis, and decreased immunity are the main risk factors affecting women's quality of life and longevity. Pharmacologic hormone replacement therapy (PHRT) has been controversial, and the construction of artificial ovary (AO) has attracted increasing attention. The most critical step of AO generation is the establishment of an in vitro culture (IVC) system to support the development of isolated follicles. This article mainly compares the advantages and disadvantages of different polymer biomaterials for use in follicle IVC, provides theoretical support for the development and construction of the follicle IVC system using natural biological materials, and provides a theoretical basis for establishing mature AO technology.

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OBJECTIVE: Ovarian tissue cryopreservation is one of the crucial options for fertility preservation. Transplantation of cryopreserved ovarian tissue was proven to restore ovarian endocrine function in patients with premature ovarian insufficiency. Ovaries from deceased donors potentially serve as an excellent and readily available tissue for the translational and basic research. In this study, we used ovaries obtained from 5 deceased donors aged 18-26 years, to evaluate the number and quality of ovarian follicles isolated before and after cryopreservation. DESIGN: Preclinical. SETTING: Academic biomedical research laboratory. PATIENTS: De-identified deceased human donors. INTERVENTIONS: Slow-freeze cryopreservation and thawing. MAIN OUTCOME MEASURES: Follicle count, follicle density, follicle viability using immunohistochemical staining (TUNEL). RESULTS: The follicle density negatively correlated with age in both cryopreserved/thawed and fresh group. A total of 2803 follicles from fresh and 1608 follicles from cryopreserved tissues were classified and analyzed using Hematoxylin and eosin staining. There was no significant difference in the percent of morphologically normal follicles between two groups. TUNEL assay indicated no higher DNA damage in the follicles and the stroma cells after cryopreservation. Morphologically normal preantral follicles were enzymatically isolated from both fresh and cryopreserved tissue with 88.51 ± 5.93% (mean ± SD) of the isolated follicles confirmed viable using LIVE/DEAD evaluation. CONCLUSIONS: Our results indicate the ovarian tissue from deceased donors maintain high quality after long time extracorporeal circulation and transportation from the hospital to the laboratory. High survival rate of follicles at different developmental stages suggested tolerance to the cryopreservation process. Human ovarian tissues obtained from deceased donors is an ample source tissue and can be applied to promoting research and future clinical applications.

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