Exploring the effect of platelet-rich plasma on vascularization and survival of follicles in xenotransplanted human ovarian tissue.

RESEARCH QUESTION: Do platelet-rich plasma (PRP) products, specifically human platelet lysate (hPL) and umbilical cord plasma, enhance vascularization and follicular survival in human ovarian tissue transplanted to immunodeficient mice? DESIGN: Human ovarian tissue was transplanted to subcutaneous pockets in nude mice, followed by daily injections for 6 days of PRP or saline at the transplantation sites. After a grafting period of 3 and 6 days, vascularization was assessed using CD-31 quantification, and gene expression of angiogenic markers (VEGF/Vegf) together with apoptosis-related genes (BAX/BCL-2), oxidative stress markers (HMOX-1/Hmox-1) and pro-inflammatory markers (Il-1ß/Il-6/Tnf-α) was quantitively analysed. Follicle density was analysed in the grafts after 4 weeks. Additionally, a pilot study was conducted exploring the suitability of ultrasound scanning for assessing survival and vascularization in ovarian tissue xenografted to mice. RESULTS: Although there was a significant increase in the CD-31 area from day 3 to day 6 post-grafting, there were no significant differences between the hPL and control groups. Gene expression analysis revealed significant down-regulation of VEGF from day 3 to day 6 for both the hPL and control groups, and significant up-regulation of BAX/BCL-2 in the hPL group compared with the controls. The follicle density showed no significant differences in the hPL group and UCP groups compared with the controls. Furthermore, ultrasound biomicroscopy provided valuable insights into graft morphology, necrotic areas and blood flow, suggesting its potential as a monitoring tool. CONCLUSIONS: Despite the angiogenic properties of PRP, this study was unable to demonstrate a significant impact of hPL on vascularization or of hPL and UCP on follicular survival in xenotransplanted human ovarian tissue.

Survey on the implementation status and reproductive outcomes of oocyte and ovarian tissue cryopreservation in Japan: Historical comparison with nationwide surveys.

PURPOSE: To clarify the reproductive outcomes of fertility preservation (FP) treatment. METHODS: We conducted a mailed-in questionnaire survey at institutions certified by the Japan Society of Obstetrics and Gynecology to investigate the number of oocyte cryopreservations (OC) and ovarian tissue cryopreservations (OTC) performed from December 2016 to the end of 2020. And, we conducted a detailed investigation of cases in which frozen specimens were used during the investigation period, and made historical comparisons with previous nationwide studies. RESULTS: Responses were received from 114 out of 150 facilities (response rate: 76.0%) for OC and 43 out of 51 for OTC (response rate: 84.3%). Breast cancer was the most common disease among patients whose FP specimens were used. During the study period, 1237 OCs and 198 OTCs were performed. In addition, 57 cycles of embryo transfer (ET) using cryopreserved oocytes and 12 cases of ovarian tissue transplantation (OTT) were performed. The mean age of patients who underwent ET using cryopreserved oocytes was 34.8 (±5.8) years, with a median age of 36 years. The pregnancy rate per ET using cryopreserved oocytes was 26.3% and the live birth rate (LBR) was 17.5%. Further, the LBR per patient was 43.3%, and the pregnancy rate following OTTs was 33.3%. Also, controlled ovarian stimulation using the random start method or the combination of aromatase inhibitors had no effect on pregnancy outcome. CONCLUSION: Implementation of both OCs and OTCs have markedly increased over time in Japan, with comparable reproductive outcomes as other reports.

Ovarian tissue cryopreservation for fertility preservation before hematopoietic stem cell transplantation in patients with sickle cell disease: safety, ovarian function follow-up, and results of ovarian tissue transplantation.

PURPOSE: To describe the experience of performing ovarian tissue cryopreservation (OTC) before hematopoietic stem cell transplantation (HSCT), among girls/women with severe sickle cell disease (SCD)(SS or S/ß0-thalassemia) who are, besides the usual surgical risk, at risk of SCD-related complications during the fertility preservation procedure for improving their counseling and management. METHODS: This retrospective study included 75 patients (girls/women) with SCD who have had OTC before myeloablative conditioning regimen (MAC) for HSCT. Characteristics of patients and data on OTC, ovarian status follow-up, and results of ovarian tissue transplantation (OTT) were collected in medical records. RESULTS: At OTC, the median (IQR 25-75; range) age of the patients was 9.6 (6.9-14.1; 3.6-28.3) years, 56/75 were prepubertal, and no SCD or surgery-related complications occurred. The median follow-up post-HSCT was > 9 years. At the last follow-up, among prepubertal patients at HSCT, 26/56 were ≥ 15 years old and presented with a premature ovarian insufficiency (POI), except 2, including the patient who had received an OTT to induce puberty. Eight were 13-15 years old and presented for POI. The remaining 22 patients were under 13. Among the 19 patients who were menarche at HSCT, 2 died 6 months post-HSCT and we do not have ovarian function follow-up for the other 2 patients. All the remaining patients (n = 15) had POI. Five patients had OTT. All had a return of ovarian function. One patient gave birth to a healthy baby. CONCLUSION: OTC is a safe fertility preservation technique and could be offered before MAC independent of the patient's age.

A retrospective study of ovarian tissue cryopreservation in female patients with hematological diseases for fertility preservation.

PURPOSES: To investigate the effect and safety of ovarian tissue cryopreservation (OTC) for fertility preservation in female patients with hematological diseases. METHODS: We designed a retrospective study. The clinical data of patients with hematological diseases undergoing OTC admitted to Peking University People's Hospital from April 2017 to January 2023 were analyzed and summarized. RESULTS: A total of 24 patients were included in the study, including 19 patients with malignant hematological diseases and 5 patients with non-malignant hematological diseases. The former included 14 patients with acute leukemia, 1 patient with chronic leukemia, and 4 patients with myelodysplastic syndrome, while the latter 5 patients were aplastic anemia (AA). 16 patients had received chemotherapy before OTC. The average age of 24 patients was 22.80 ± 6.81 years. The average anti-Mullerian hormone (AMH) was 1.97 ± 2.12 ng/mL, and the average follicle-stimulating hormone (FSH) was 7.01 ± 4.24 IU/L in examination before OTC. FSH was greater than 10.0 IU/L in 4 cases. The pre-OTC laboratory tests showed that the average white blood cell (WBC) count was (3.33 ± 1.35) × 109/L, the average hemoglobin was 91.42 ± 22.84 g/L, and the average platelet was (147.38 ± 114.46) × 109/L. After injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF), blood transfusion, and iron supplementation in pre-OTC treatment, the average WBC count was (4.91 ± 3.07) × 109/L, the average hemoglobin was 98.67 ± 15.43 g/L, and the average platelet was (156.38 ± 103.22) × 109/L. Of the 24 patients, 22 underwent laparoscopic bilateral partial oophorectomy and oophoroplasty, and 2 underwent laparoscopic unilateral oophorectomy. The average duration of OTC was 59.54 ± 17.58 min, and the average blood loss was 32.1 ± 41.6 mL. The maximum blood loss was 200 mL. There was no significant difference in WBC count and hemoglobin concentration after OTC compared to pre-OTC period. Only the platelet count after OTC surgery was significantly different from that before surgery ([134.54 ± 80.84 vs. 156.38 ± 103.22] × 109/L, p < 0.05). None of the 24 patients had serious complications after OTC. 2 patients had mild infection symptoms, but both recovered well. 23 patients underwent hematopoietic stem cell transplantation (HSCT) after OTC. The median and interquartile range from OTC to the pretreatment of HSCT was 33 (57) days, and the median and interquartile range from OTC to HSCT was 41 (57) days. Seven of them began pretreatment of HSCT within 20 days and began HSCT within 30 days after OTC. All patients were followed up. Of the 23 patients who underwent HSCT after surgery, 22 presented with amenorrhea and 1 with scanty menstrual episodes. Seven patients underwent hormone replacement therapy (HRT) after HSCT. A patient with AA underwent ovarian tissue transplantation (OTT) 3 years after HSCT and resumed regular menstruation 6 months after OTT. CONCLUSIONS: Ovarian tissue cryopreservation has a promising future in fertility protection in patients with hematological diseases. However, patients with hematological malignancies often have received gonadotoxic therapy before OTC, which may be accompanied by myelosuppression while patients with non-malignant hematological diseases often present with severe hemocytopenia. So perioperative complete blood count of patients should be paid attention to. There was no significant difference in the WBC count and hemoglobin concentration in patients with hematological diseases before and after OTC surgery, and the platelet count decreased slightly within the normal range. Infection is the most common post-OTC complication, and HSCT pretreatment can be accepted as early as the 10th day after OTC. OTC has no adverse effects on patients with hematological diseases and does not delay HSCT treatment. For young patients with hematological diseases, OTC is an effective method of fertility preservation.

Impact of first chemotherapy exposure on follicle activation and survival in human cryopreserved ovarian tissue.

STUDY QUESTION: Does chemotherapy exposure prior to ovarian tissue cryopreservation (OTC) impact the signaling pathways governing follicle activation and survival for prepubertal and postpubertal patients? SUMMARY ANSWER: Chemotherapy exposure prior OTC increases follicle apoptosis rates but not follicular activation, although the PI3K/AKT/mTOR and Hippo signaling pathways were modified in the cortex. WHAT IS KNOWN ALREADY: OTC is currently the only available fertility preservation procedure for children and for patients who have already started their treatment. While previous studies have not observed harmful impacts of first chemotherapy exposure on OTC outcomes, the consequences of treatment on follicle activation and survival need to be further investigated. To address this question, we evaluated signaling pathway modifications induced by chemotherapy exposure according to pubertal status. STUDY DESIGN, SIZE, DURATION: Cryopreserved ovarian tissues from postpubertal (12-29 years old, n = 8) and prepubertal (3-10 years old, n = 8) cancer patients donated for research were thawed and cultured for 24 h. Analyses of the survival of the follicles and stroma, and of the PI3K/AKT/mTOR and Hippo signaling pathways, were conducted at thawing and after culture. Ovarian fragments exposed to chemotherapy before collection were compared to non-exposed controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological investigations were performed to assess the distribution of the follicles, stroma fibrosis, vessel integrity, and apoptosis levels. It included follicular counting, collagen staining, immunostaining on CD31 and gH2AX, as well as TUNEL staining. To explore follicle activation in the different groups, the PI3K/AKT/mTOR and Hippo signaling pathways were investigated by gene expression analyses of isolated follicles and protein analyses on whole fragments through western blots and immunostaining. MAIN RESULTS AND THE ROLE OF CHANCE: We first assessed the impact of a first exposure to chemotherapy on the collagen density and vessels in ovarian tissues at thawing and after culture. While no differences in collagen density were observed according to age or previous treatment, the vascularization area (CD31+) was significantly lower in tissue from previously exposed patients compared to non-treated ones. Apoptosis analyses (TUNEL) revealed an acute deleterious impact on follicle survival after chemotherapy exposure without affecting the follicular density. Surprisingly, leukemic patients had a significantly higher percentage of gH2AX-positive follicles, indicating a DNA damage response, compared to the other patients. The proportion of activated follicles appeared to decrease following exposure to chemotherapy, suggesting that it at least did not increase activation process. Stable KIT LIGAND gene and protein expression and cKIT protein levels were observed among the groups, confirming the absence of activation. Analysis of the PI3K pathway did not reveal a difference in the AKT phosphorylation level between the groups, but pRPS6 was significantly higher in tissue from patients previously exposed to chemotherapy compared to that from non-exposed patients. Finally, protein and gene analyses on Hippo pathway signaling showed a higher LATS1 protein level in the cortex after chemotherapy exposure. LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of the human fragments, and the limited number of patients included in the cohort have to be considered as important study limitations. Moreover, this study did not explore the long-term consequences of chemotherapy on follicular development. Therefore, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: These results underscore the deleterious effect of previous chemotherapeutic treatment on follicle survival. Although follicular density was not reduced, these data suggested that exposure to chemotherapy impacts follicular apoptosis and the DNA damage response. Chemotherapy-induced activation was not observed despite the impact on mTOR and Hippo signaling pathways in the whole cortex. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by an Excellence of Science (EOS) Grant (ID: 30443682) and was supported by Fonds Erasme. I.D. and M.-M.D. are associate researchers at Fonds National de la Recherche Scientifique de Belgique (FNRS). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Assessing the impact of transplant site on ovarian tissue transplantation: a single-arm meta-analysis.

BACKGROUND: Survival rates of young women undergoing cancer treatment have substantially improved, with a focus on post-treatment quality of life. Ovarian tissue transplantation (OTT) is a viable option to preserve fertility; however, there is no consensus on the optimal transplantation site. Most studies on OTT are nonrandomized controlled trials with limited sample sizes and uncontrolled statistical analyses, leaving the question of which transplant site yields the highest chance of achieving a live birth unanswered. OBJECTIVE: This meta-analysis aimed to assess the effect of different ovarian transplant sites on postoperative reproductive outcomes. METHODS: We adhered to the PRISMA Reporting Items for Systematic Reviews and Meta-Analyses recommendations. Systematic searches were conducted in PubMed, Embase, Web of Science, and the Cochrane Library from inception to September 17, 2023. The inclusion criteria were as follows: (1) women who underwent OTT with a desire for future childbirth, and (2) reports of specific transplant sites and corresponding pregnancy outcomes. The exclusion criteria included the inability to isolate or extract relevant outcome data, case reports, non-original or duplicate data, and articles not written in English. RESULTS: Twelve studies (201 women) were included in the meta-analysis of cumulative live birth rates (CLBR) after OTT. The CLBR, which encompasses both spontaneous pregnancies and those achieved through assisted reproductive technology (ART) following OTT to the ovarian site, was 21% (95% CI: 6-40, I2: 52.81%, random effect). For transplantation to the pelvic site, the live birth rate was 30% (95% CI: 20-40, I2: 0.00%, fixed effect). Combining transplantation to both the pelvic and ovarian sites resulted in a live birth rate of 23% (95% CI: 11-36, I2: 0.00%, fixed effect). Notably, heterotopic OTT yielded a live birth rate of 3% (95% CI: 0-17, I2: 0.00%, fixed effect). CONCLUSION: Pregnancy outcomes were not significantly different after orthotopic ovarian transplantation, and pregnancy and live birth rates after orthotopic OTT were significantly higher than those after ectopic transplantation. REGISTRATION NUMBER: INPLASY202390008.

BNIP3-mediated autophagy via the mTOR/ULK1 pathway induces primordial follicle loss after ovarian tissue transplantation.

PURPOSE: To explore the underlying mechanism of primordial follicle loss in the early period following ovarian tissue transplantation (OTT). METHODS: BNIP3 was selected through bioinformatic protocols, as the hub gene related to autophagy during OTT. BNIP3 and autophagy in mice ovarian grafts and in hypoxia-mimicking KGN cells were detected using immunohistochemistry, transmission electron microscopy (TEM), western blotting, qPCR, and fluorescence staining. The regulatory role played by BNIP3 overexpression and the silencing of KGN cells in autophagy via the mTOR/ULK1 pathway was investigated. RESULTS: Ultrastructure examination showed that autophagic vacuoles increased after mice ovarian auto-transplantation. The BNIP3 and autophagy-related proteins (Beclin-1, LC3B, and SQSTM1/p62) in mice ovarian granulosa cells of primordial follicle from ovarian grafts were altered compared with the control. Administration of an autophagy inhibitor in mice decreased the depletion of primordial follicles. In vitro experiments indicated that BNIP3 and autophagy activity were upregulated in KGN cells treated with cobalt chloride (CoCl2). The overexpression of BNIP3 activated autophagy, whereas the silencing of BNIP3 suppressed it and reversed the autophagy induced by CoCl2 in KGN cells. Western blotting analysis showed the inhibition of mTOR and activation of ULK1 in KGN cells treated with CoCl2 and in the overexpression of BNIP3, and the opposite results following BNIP3 silencing. The activation of mTOR reversed the autophagy induced by BNIP3 overexpression. CONCLUSIONS: BNIP3-induced autophagy is crucial in primordial follicle loss during OTT procedure, and BNIP3 is a potential therapeutic target for primordial follicle loss after OTT.

Summary of the ISFP congress, Brussels, 10-12 November, 2022.

The 7th International Congress of the ISFP was held in Brussels in November 2022. Hundreds of attendees from all over the world had the rare opportunity to hear the most distinguished leaders discuss and debate the latest advances in the field. Participants were also able to attend workshops under the guidance of skilled practitioners. Numerous topics were considered, including a recap on fertility preservation approaches in cancer and benign pathologies and a section on male factor infertility. Other aspects covered were in vitro maturation and poor responders, the impact of chemotherapy on the ovary, and future perspectives. Participants had the chance to listen to a symposium on fertility preservation techniques, and finally, a keynote lecture on fertility preservation in gynecological cancers brought this prominent and highly influential event to a close.

Investigation of the optimal culture time for warmed bovine ovarian tissues before transplantation†.

Ovarian tissue cryopreservation by vitrification is an effective technique, but there are still many unresolved issues related to the procedure. The aim of this study was to investigate the optimal culture time of postwarmed ovarian tissues and their viability before ovarian tissue transplantation. The bovine ovarian tissues were used to evaluate the effect of postwarming culture periods (0, 0.25, 0.5, 1, 2, 5, and 24 h) in the levels of residual cryoprotectant, LDH release, ROS generation, gene and protein abundance, and follicle viability and its mitochondrial membrane potential. Residual cryoprotectant concentration decreased significantly after 1 h of culture. The warmed ovarian tissues that underwent between 0 and 2 h of culture time showed similar LDH and ROS levels compared with fresh nonfrozen tissues. The anti-Mullerian hormone transcript abundance did not differ in any of the groups. No increase in the relative transcript abundance and protein level of Caspase 3 and Cleaved-Caspase 3, respectively, in the first 2 h of culture after warming. On the other hand, an increased protein level of double stranded DNA breaks (gamma-H2AX) was observed in postwarmed tissues disregarding the length of culture time, and a temporary reduction in pan-AKT was detected in postwarming tissues between 0 and 0.25 h of culture time. Prolonged culture time lowered the percentage of viable follicles in warmed tissues, but it did not seem to affect the follicular mitochondrial membrane potential. In conclusion, 1-2 h of culture time would be optimal for vitrified-warmed tissues before transplantation.

Evaluation of an alternative heterotopic transplantation model for ovarian tissue to test pharmaceuticals improvements for fertility restoration.

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.

Strategies to safely use cryopreserved ovarian tissue to restore fertility after cancer: a systematic review.

Ovarian tissue cryopreservation and subsequent autotransplantation is a successful technique for fertility preservation in oncological patients. However, there are concerns regarding safety, as the graft may contain malignant cells that could lead to the reintroduction of cancer. To circumvent this problem several experimental strategies are being pursued. This systematic review was conducted to provide an overview of the strategies aiming to safely use cryopreserved human ovarian tissue to restore fertility after cancer. Thirty-one studies were included, covering five different experimental strategies: (i) in-vitro maturation of oocytes, (ii) constructing an artificial ovary as a scaffold for reseeding pre-antral follicles, (iii) purging strategies aimed at the eradication of contaminating malignant cells, (iv) maturation of oocytes by xenotransplantation, and (v) stem cell-based oogenesis. These strategies to circumvent the reintroduction of cancer cells through ovarian tissue autotransplantation are being developed, but so far have not reached the stage of clinical trials. Further research is required to establish their risks and effectiveness while the ethical aspects associated with these strategies also need to be discussed. Despite the fact that these experimental procedures are still under development, they might provide safe fertility restoration options for oncological patients in the future.

Learning Curve of Surgeons Performing Laparoscopic Ovarian Tissue Transplantation in Women with Premature Ovarian Insufficiency: A Statistical Process Control Analysis.

STUDY OBJECTIVE: To analyze patient safety in laparoscopic ovarian tissue transplantation surgery by tracking the rate of postoperative complications and the learning curves of the surgeons by statistical process control analysis. DESIGN: A retrospective study. SETTING: A university-affiliated hospital. PATIENTS: A total of 100 patients with premature ovarian insufficiency who underwent ovarian tissue cryopreservation by vitrification and then autologous transplantation of frozen-thawed ovarian tissues with in vitro activation. INTERVENTIONS: Ovarian tissue cryopreservation, in vitro activation, and transplantation. MEASUREMENTS AND MAIN RESULTS: We assessed the surgery complications, differences in total surgery time, transplantation time, and transplantation time per ovarian sheet in operations performed by 3 experienced laparoscopic surgeons. Surgeon A performed 80 operations; surgeon B, 29 operations; and surgeon C, 20 operations. Complications occurred in 1.55% of the procedures. Although all 3 surgeons' performance never fell below the unacceptable failure limit, only surgeon A became competent after 66 cases. CONCLUSION: The laparoscopic ovarian tissue transplantation surgery was generally safe given that the postoperative complications were infrequent (1.55%). Although the performance of all 3 surgeons was acceptable, only surgeon A attained the level of competency after 66 cases. The transplantation method may not be the key factor for reducing surgery time in this surgery. An efficient ovarian tissue transplantation team is more important in reducing the surgery time than the surgeon's surgical technique alone.

Failed ovarian cortex transplant but successful ovulation induction in a cancer survivor with premature ovarian failure: A paradox.

Better diagnostic and treatment modalities for malignancies occurring in childhood and young age have increased the overall survival of the affected young girls and boys, but this has come at a cost of developing premature ovarian failure in girls and azoospermia in boys because of the gonadotoxicity of chemotherapy and radiotherapy. Thus, young girls and women of reproductive age who are at risk of ovarian failure due to cancer treatment must be mandatorily offered fertility preservation in any form such as cryopreservation of mature metaphase II oocytes after ovarian stimulation with gonadotropins and oocyte recovery and cryopreservation of embryos if the young female has a partner. However, these modalities may require the postponement of chemotherapy. Thus, in such instances, cryopreservation of ovarian tissue remains the only option. Herein, we report a case of a nulligravid young female patient who had stage IV Hodgkin's lymphoma and had to be taken up for immediate fertility preservation owing to the advanced stage of the disease. Therefore, cryopreservation of ovarian tissue was performed. The uniqueness of the case is that after remission of the disease was achieved with chemotherapy, transplantation of the ovarian tissue was carried out which was not successful, but the desire for motherhood prompted to carry out an ovulation induction in the remnant native ovary with a severely compromised ovarian reserve, and she conceived.

N-acetylcysteine protects ovarian follicles from ischemia-reperfusion injury in xenotransplanted human ovarian tissue.

STUDY QUESTION: Can antioxidant treatment with N-acetylcysteine (NAC) protect ovarian follicles from ischemia-reperfusion injury in xenotransplanted human ovarian tissue? SUMMARY ANSWER: Daily administration of NAC for 7-12 days post-transplantation reduced ischemia-reperfusion injury and increased follicle survival in human ovarian xenografts by upregulating the antioxidant defense system and exerting anti-inflammatory and antiapoptotic effects. WHAT IS KNOWN ALREADY: Freezing of human ovarian tissue is performed with high follicular survival rates but up to 70% of follicles appear to be lost due to hypoxia and ischemia-reperfusion injury during ovarian tissue transplantation (OTT). NAC has been demonstrated to possess antioxidant and antiapoptotic properties, and studies in rodents have shown that intraperitoneal administration of NAC reduces ischemia-reperfusion injury and increases follicle survival in autotransplanted murine ovaries. STUDY DESIGN, SIZE, DURATION: Pieces of frozen-thawed human ovarian tissue from 28 women aged 23-36 years were transplanted to immunodeficient mice in short- and long-term xenograft studies or cultured in vitro. Three short-term xenograft studies (1-week duration) were performed, in which saline or 150 mg/kg NAC was administered for 7 days post-transplantation (n = 12 patients per group). Two long-term xenograft studies (4 weeks of duration) were performed. In one of these studies, saline or 150 mg/kg NAC was administered for 12 days (n = 12 patients per group), while in the other study 50, 150 or 300 mg/kg NAC was administered for 7 days (n = 8 patients per group). In addition, human ovarian tissue (n = 12 pieces from three patients per group) was cultured with increasing concentrations of NAC (0, 5, 25 and 75 mM) for 4 days in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated ovarian tissue was obtained from women who had undergone ovarian tissue cryopreservation for fertility preservation at the University Hospital of Copenhagen. Cortical tissue pieces (5 × 5 × 1 mm) were transplanted subcutaneously to immunodeficient mice and NAC or saline was injected intraperitoneally. Grafts were retrieved after 1 or 4 weeks and follicle density was assessed. Gene expression analysis of antioxidant defense markers (superoxide dismutase; Sod1/SOD1, heme oxygenase-1; Hmox1/HMOX1, catalase; Cat/CAT), proinflammatory cytokines (tumor necrosis factor-alpha; Tnf-α, interleukin-1-beta; Il1-ß, interleukin 6; Il6), apoptotic factors (B-cell lymphoma 2; Bcl2/BCL2, Bcl-2-associated X protein; Bax/BAX) and angiogenic factors (vascular endothelial growth factor A; Vegfa/VEGFA, angiopoietin-like 4; Angptl4/ANGPTL4) was performed in 1-week-old human ovarian xenografts and in cultured human ovarian tissue. Grafts retrieved after 4 weeks were histologically processed and analyzed for vascularization by CD31 immunohistochemical staining, fibrosis by Masson's Trichrome staining and apoptosis by immunofluorescence using cleaved caspase-3. MAIN RESULTS AND THE ROLE OF CHANCE: After 1-week grafting, the relative expression of Sod1, Hmox1 and Cat was significantly higher in the group receiving 150 mg/kg NAC (NAC150-treated group) compared to controls (P = 0.04, P = 0.03, and P = 0.01, respectively), whereas the expression levels of Tnf-α, Il1-ß and Il6 were reduced. The Bax/Bcl2 ratio was also significantly reduced in the NAC150-treated group (P < 0.005). In vitro, the relative gene expression of SOD1, HMOX1 and CAT increased significantly in the human ovarian tissue with increasing concentrations of NAC (P < 0.001 for all genes). However, the expression of VEGFA and ANGPTL4 as well as the BAX/BCL2 ratio decreased significantly with increasing concentrations of NAC (P < 0.02, P < 0.001 and P < 0.001, respectively). After 4-week grafting, fibrosis measured by collagen content was similar in the NAC150-treated group compared to controls (control: 56.6% ± 2.2; NAC150: 57.6% ± 1.8), whereas a statistically significant reduction in the CD31-positive vessel area was found (control: 0.69% ± 0.08; NAC150: 0.51% ± 0.07; P < 0.02). Furthermore, a reduced immunoreactivity of cleaved caspase-3 was observed in follicles of the NAC150-treated xenografts compared to controls. Follicle density (follicles/mm3, mean ± SD) was higher in the NAC150-treated group compared to the control group in the 1-week xenografts (control: 19.5 ± 26.3; NAC150: 34.2 ± 53.5) and 4-week xenografts (control: 9.3 ± 11.0; NAC150: 14.4 ± 15.0). Overall, a 2-fold increase in follicle density was observed in the NAC150-group after 1-week grafting where fold changes in follicle density were calculated in relation to grafts from the same patient. Around a 5-fold increase in follicle density was observed in the NAC150 and NAC300 groups after 4-week grafting. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Follicle density in the human ovarian cortex is highly heterogeneous and can vary 100-fold between cortex pieces from the same woman. A high variability in follicle density within and between treatment groups and patients was found in the current study. Thus, solid conclusions cannot be made. While intraperitoneal injections of NAC appeared to reduce ischemia-reperfusion injury in human ovarian xenografts, different administration routes should be investigated in order to optimize NAC for potential clinical use. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to demonstrate the antioxidant, anti-inflammatory and antiapoptotic properties of NAC in xenotransplanted human ovarian tissue. Therefore, NAC appears to be a promising candidate for protecting ovarian follicles from ischemia-reperfusion injury. This provides the initial steps toward clinical application of NAC, which could potentially reduce the loss of ovarian follicles following OTT. STUDY FUNDING/COMPETING INTEREST(S): We are grateful to the Danish Childhood Cancer Foundation, Hørslev Foundation, Aase and Einar Danielsen's Foundation (grant number: 10-001999), Dagmar Marshalls Foundation, Else and Mogens Wedell-Wedellsborgs Foundation, Knud and Edith Eriksens Mindefond, and Fabrikant Einar Willumsens Mindelegat for funding this study. None of the authors have any competing interests to declare.

Ovarian tissue damage after grafting: systematic review of strategies to improve follicle outcomes.

Frozen-thawed human ovarian tissue endures large-scale follicle loss in the early post-grafting period, characterized by hypoxia lasting around 7 days. Tissue revascularization occurs progressively through new vessel invasion from the host and neoangiogenesis from the graft. Such reoxygenation kinetics lead to further potential damage caused by oxidative stress. The aim of the present manuscript is to provide a systematic review of proangiogenic growth factors, hormones and various antioxidants administered in the event of ovarian tissue transplantation to protect the follicle pool from depletion by boosting revascularization or decreasing oxidative stress. Although almost all investigated studies revealed an advantage in terms of revascularization and reduction in oxidative stress, far fewer demonstrated a positive impact on follicle survival. As the cascade of events driven by ischaemia after transplantation is a complex process involving numerous players, it appears that acting on specific molecular mechanisms, such as concentrations of proangiogenic growth factors, is not enough to significantly mitigate tissue damage. Strategies exploiting the activated tissue response to ischaemia for tissue healing and remodelling purposes, such as the use of antiapoptotic drugs and adult stem cells, are also discussed in the present review, since they yielded promising results in terms of follicle pool protection.

High cryo-resistance of SARS-CoV-2 virus: Increased risk of re-contamination at transplantation of cryopreserved ovarian tissue after COVID-19 pandemic.

Cryopreservation and re-transplantation of ovarian tissue after anticancer treatment is important medical technology. Today, during a pandemic, the risk of contamination of transplanted cells with SARS-CoV-2 virus is extremely high. Data about cryo-resistance (virulence and/or infectivity) of SARS-CoV-2 are limited. Analysis and systematization of literature data allow us to draw the following conclusions: 1) The cytoplasmic membrane of somatic cell, like envelope of corona viruses, consists of lipid bilayer and this membrane, like envelope of corona virus, contains membrane proteins. Thus, we can consider the cytoplasmic membrane of an ordinary somatic cell as a model of the envelope membrane of SARS-CoV-2. It is expected that the response of the virus to cryopreservation is similar to that of a somatic cell. SARS-CoV-2 is more poor-water and more protein-rich than somatic cell, and this virus is much more cryo-resistant. 2) The exposure of somatic cells at low positive temperatures increases a viability of these cells. The safety of the virus is also in direct proportion to the decrease in temperature: the positive effect of low temperatures on SARS-CoV-2 virus has been experimentally proven. 3) Resistance of SARS-CoV-2 to cryoprotectant-free cryopreservation is extremely high. The high viability rate of SARS-CoV-2 after freezing-drying confirms its high cryo-resistance. 4) The risk of SARS-CoV-2 infection after transplantation of cryopreserved ovarian tissues that have been contaminated with this virus, increases significantly. Our own experimental data on the increase in the viability of cancer cells after cryopreservation allow us to formulate a hypothesis about increasing of viability (virulence and/or infectivity) of SARS-CoV-2 virus after cryopreservation.

Disease-inducing potential of two leukemic cell lines in a xenografting model.

PURPOSE: Cryopreserved ovarian tissue transplant restores ovarian function in young cancer patients after gonadotoxic treatment. However, leukemia is associated with increased risk of malignant cell transmission. We aimed to assess the tumor-inducing potential of two different leukemic cell lines when xenografted to immunodeficient mice. METHODS: Fifty-four female immunodeficient mice were grafted with either 100, 200, 500, 1000, and 10,000 chronic myeloid leukemia in blast crisis (BV-173) cells or relapsed acute lymphoblastic leukemia (RCH-ACV) cells, embedded inside a fibrin scaffold along with 50,000 human ovarian stromal cells. Two mice per cell line received the fibrin matrix without leukemic cells as negative controls. Clinical signs of disease were monitored for 20 weeks. Grafts, liver tissue, and masses were collected for macroscopic analysis and gene expression of BCR-ABL1 and E2A-PBX fusion transcripts present in BV-173 and RCH-ACV respectively. RESULTS: BV-173 cells: Mice grafted with 100, 200, or 500 cells showed no sign of disease after and were negative for BCR-ABL1 expression. Three of the 5 animals grafted with 1000 cells and all mice with 10,000 cells developed disease and showed BCR-ABL1-positive expression. RCH-ACV cells: Two out of 4 mice grafted with 100 cells developed disease and were E2A-PBX1-positive. All the animals grafted with higher cell doses showed signs of disease and all but one were E2A-PBX1-positive. CONCLUSION: The present work proves that the disease-inducing potential of BV-173 and RCH-ACV leukemic cells xenografted to SCID mouse peritoneum differs between cell lines, depending on cell number, type, status, and cytogenetic disease profile when ovarian tissue is harvested.

Efficacy of assisted reproductive technology after ovarian tissue transplantation in a cohort of 11 patients with or without associated infertility factors.

PURPOSE: IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor reproductive outcomes. The aim of this study was to evaluate the efficacy of ovarian tissue transplantation (OTT) followed by assisted reproductive technology (ART) in women with or without associated infertility factors. METHODS: This is a prospective cohort study with retrospective data collection including eleven women, four of whom having an infertility factor (IF), who had undergone OTT in one university center between 2005 and 2017, followed by ART in six in vitro fertilization (IVF) centers. RESULTS: In total, 25 of the 85 cycles initiated (29%) were canceled, resulting in 60 oocyte retrievals. Ninety-five oocytes were retrieved: 36 were abnormal or immature, 29/39 fertilized (74%) after ICSI and 13/20 (65%) after IVF. Thirty-five embryos were transferred in seven patients (5/7 patients without IF and 2/4 patients with IF). After ART, one patient with IF experienced two pregnancies, one resulting in a live birth. For all patients, pregnancy rates and live birth rates were 7.4% and 3.7% per embryo transfer, respectively. Nine pregnancies and four live births occurred after spontaneous conception in five patients without IF, none in the infertility group. CONCLUSION: This study confirms that IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor outcomes. However, the chances of natural conception are high in women without IF. Patients with IF, without the possibility of spontaneous pregnancy, should be informed of poor reproductive outcomes after OTT followed by ART. TRIAL REGISTRATION NUMBER: NCT02184806.

Adipose tissue-derived stem cells protect the primordial follicle pool from both direct follicle death and abnormal activation after ovarian tissue transplantation.

PURPOSE: To investigate whether adipose tissue-derived stem cells (ASCs) protect the primordial follicle pool, not only by decreasing direct follicle loss but also by modulating follicle activation pathways. METHODS: Twenty nude mice were grafted with frozen-thawed human ovarian tissue from 5 patients. Ten mice underwent standard ovarian tissue transplantation (OT group), while the remaining ten were transplanted with ASCs and ovarian tissue (2-step/ASCs+OT group). Ovarian grafts were retrieved on days 3 (n = 5) and 10 (n = 5). Analyses included histology (follicle count and classification), immunohistochemistry (c-caspase-3 for apoptosis and LC3B for autophagy), and immunofluorescence (FOXO1 for PI3K/Akt activation and YAP for Hippo pathway disruption). Subcellular localization was determined in primordial follicles on high-resolution images using structured illumination microscopy. RESULTS: The ASCs+OT group showed significantly higher follicle density than the OT group (p = 0.01). Significantly increased follicle atresia (p < 0.001) and apoptosis (p = 0.001) were observed only in the OT group. In primordial follicles, there was a significant shift in FOXO1 to a cytoplasmic localization in the OT group on days 3 (p = 0.01) and 10 (p = 0.03), indicating follicle activation, while the ASCs+OT group and non-grafted controls maintained nuclear localization, indicating quiescence. Hippo pathway disruption was encountered in primordial follicles irrespective of transplantation, with nuclear YAP localized in their granulosa cells. CONCLUSION: We demonstrate that ASCs exert positive effects on the ovarian reserve, not only by protecting primordial follicles from direct death but also by maintaining their quiescence through modulation of the PI3K/Akt pathway.

The Effectiveness of Anti-Apoptotic Agents to Preserve Primordial Follicles and Prevent Tissue Damage during Ovarian Tissue Cryopreservation and Xenotransplantation.

The purpose of this study is to investigate the effectiveness of sphingosine-1-phosphate (S1P) and Z-VAD-FMK (Z-VAD) as anti-apoptotic agents to preserve ovarian function and prevent tissue damage during ovarian tissue cryopreservation and transplantation. This study consisted of two steps, in vitro and in vivo. In the first step, human ovarian tissues were cryopreserved using slow-freezing media alone, S1P, or Z-VAD (control, S1P, Z-VAD group); based on the outcomes in these groups, Z-VAD was selected for subsequent xenotransplantation. In the second step, human frozen/thawed ovarian tissues were grafted into fifty mice divided into three groups: slow-freezing/thawing and transplantation without an anti-apoptotic agent (Trans-control) and xenotransplantation with or without Z-VAD injection (Trans-Z-VAD-positive and Trams-Z-VAD-negative groups, respectively). In the first step, the Z-VAD group had a significantly higher primordial follicular count than the S1P (p = 0.005) and control groups (p = 0.04). Transplanted ovarian tissues were obtained 4 weeks after transplantation (second step). Angiogenesis was significantly increased in the Z-VAD-negative (p = 0.03) and -positive (p = 0.04) groups compared to the control group. This study demonstrated that slow-freezing and transplantation with Z-VAD is an effective method for preserving primordial follicle counts, decreasing double-strand DNA breaks, and increasing angiogenesis in a mouse model. Further molecular and clinical studies are needed to confirm these results.