In vitro growth of secondary follicles from cryopreserved-thawed ovarian cortex.

STUDY QUESTION: Can secondary follicles be obtained from cultured cryopreserved-thawed human ovarian cortical tissue? SUMMARY ANSWER: We obtained high-quality secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue from cis female donors (cOVA), but not from trans masculine donors (tOVA) in the same culture conditions. WHAT IS KNOWN ALREADY: The in vitro growth of oocytes present in unilaminar follicles into metaphase II stage (MII) oocytes has been previously achieved starting from freshly obtained ovarian cortical tissue from adult cis female donors. This involved a multi-step culture protocol and the first step included the transition from unilaminar follicles to multilayered secondary follicles. Given that the ovarian cortex (from both cis female and trans masculine donors) used for fertility preservation is cryopreserved, it is crucial to investigate the potential of unilaminar follicles from cryopreserved-thawed ovarian cortex to grow in culture. STUDY DESIGN, SIZE, DURATION: Cryopreserved-thawed ovarian cortical tissue from adult trans masculine donors (n = 3) and adult cis female donors (n = 3) was used for in vitro culture following the first culture step described in two published culture protocols (7-8 days and 21 days) and compared to freshly isolated ovarian cortex from trans masculine donors (n = 3) and to ovarian cortex prior to culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortical tissue was obtained from adult trans masculine donors undergoing gender-affirming surgery while using testosterone, and from adult cis female donors undergoing oophorectomy for fertility preservation purposes before chemotherapy. The ovarian cortex was fixed either prior (day 0) or after the culture period. Follicular survival, growth, and morphology were assessed through histology and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: We quantified the different stages of follicular development (primordial, primary, secondary, and atretic) after culture and observed an increase in the percentage of secondary follicles as well as an increase in COLIV deposition in the stromal compartment regardless of the culture media used. The quality of the secondary follicles obtained from cOVA was comparable to those prior to culture. However, in the same culture conditions, the secondary follicles from tOVA (fresh and cryo) showed low-quality secondary follicles, containing oocytes with small diameter, granulosa cells that expressed abnormal levels of KRT19 and steroidogenic-marker STAR and lacked ACTA2+ theca cells, when compared to tOVA secondary follicles prior to culture. LIMITATIONS, REASONS FOR CAUTION: The number of different donors used was limited. WIDER IMPLICATIONS OF THE FINDINGS: Our study revealed that cryopreserved-thawed cOVA can be used to generate high-quality secondary follicles after culture and those can now be further tested to evaluate their potential to generate functional MII oocytes that could be used in the clinic. However, using the same culture protocol on tOVA (fresh and cryo) did not yield high-quality secondary follicles, suggesting that either the testosterone treatment affects follicular quality or adapted culture protocols are necessary to obtain high-quality secondary follicles from tOVA. Importantly, caution must be taken when using tOVA to optimize folliculogenesis in vitro. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the European Research Council Consolidator Grant OVOGROWTH (ERC-CoG-2016-725722 to J.S.D.V. and S.M.C.D.S.L.), the Novo Nordisk Foundation (reNEW NNF21CC0073729 to H.C., F.W., J.S.D.V., S.M.C.D.S.L.), and China Scholarship Council (CSC 202008320362 and CSC 202008450034 to H.C. and F.W.), respectively. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Fertility preservation in patients undergoing gonadotoxic treatments: a Canadian Fertility and Andrology Society clinical practice guideline.

The management of young patients with cancer presents several unique challenges. In general, these patients are ill prepared for the diagnosis and the impact on their fertility. With the improved survival for all tumour types and stages, the need for adequate fertility counselling and a multidisciplinary approach in the reproductive care of these patients is paramount. Recent advances in cryopreservation techniques allow for the banking of spermatozoa, oocytes, embryos and ovarian tissue without compromising survival. This Canadian Fertility and Andrology Society (CFAS) guideline outlines the current understanding of social and medical issues associated with oncofertility, and the medical and surgical technologies available to optimize future fertility.

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.

Modeling delay of age at natural menopause with planned tissue cryopreservation and autologous transplantation.

BACKGROUND: Ovarian tissue cryopreservation has been proven to preserve fertility against gonadotoxic treatments. It has not been clear how this procedure would perform if planned for slowing ovarian aging. OBJECTIVE: This study aimed to determine the feasibility of cryopreserving ovarian tissue to extend reproductive life span and delay menopause by autotransplantation near menopause. STUDY DESIGN: Based on the existing biological data on follicle loss rates, a stochastic model of primordial follicle wastage was developed to determine the years of delay in menopause (denoted by D) by ovarian tissue cryopreservation and transplantation near menopause. Our model accounted for (1) age at ovarian tissue harvest (21-40 years), (2) the amount of ovarian cortex harvested, (3) transplantation of harvested tissues in single vs multiple procedures (fractionation), and (4) posttransplant follicle survival (40% [conservative] vs 80% [improved] vs 100% [ideal or hypothetical]). RESULTS: Our model predicted that, for most women aged <40 years, ovarian tissue cryopreservation and transplantation would result in a significant delay in menopause. The advantage is greater if the follicle loss after transplant can be minimized. As an example, the delay in menopause (D) for a woman with a median ovarian reserve who cryopreserves 25% of her ovarian cortex at the age of 25 years and for whom 40% of follicles survive after transplantation would be approximately 11.8 years, but this extends to 15.5 years if the survival is 80%. As another novel finding, spreading the same amount of tissue to repetitive transplants significantly extends the benefit. For example, for the same 25-year-old woman with a median ovarian reserve, 25% cortex removal, and 40% follicle survival, fractionating the transplants to 3 or 6 procedures would result in the corresponding delay in menopause (D) of 23 or 31 years. The same conditions (3 or 6 procedures) would delay menopause as much as 47 years if posttransplant follicle survival is improved to 80% with modern approaches. An interactive Web tool was created to test all variables and the feasibility of ovarian tissue freezing and transplantation to delay ovarian aging (here). CONCLUSION: Our model predicts that with harvesting at earlier adult ages and better transplant techniques, a significant menopause postponement and, potentially, fertile life span extension can be achieved by ovarian tissue cryopreservation and transplantation in healthy women.

Comprehensive fertility preservation can be offered in a timely manner around gonadotoxic therapy in children and adolescents.

BACKGROUND: Treatment for certain childhood cancers and nonmalignant conditions can lead to future infertility and gonadal failure. The risk of treatment delay must be considered when offering fertility preservation (FP) options. We examined the timeline from FP referral to return to treatment (RTT) in pediatric patients who underwent FP due to iatrogenic risk for infertility. METHODS: A retrospective review was performed of patients with FP consultation due to an increased risk of iatrogenic infertility at Ann & Robert H. Lurie Children's Hospital of Chicago from 2018 to 2022. Data on diagnosis, age, treatment characteristics, and procedure were collected. RESULTS: A total of 337 patients (n = 149 with ovaries, n = 188 with testes) had an FP consultation. Of patients with ovaries, 106 (71.1%) underwent ovarian tissue cryopreservation (OTC), 10 (6.7%) completed ovarian stimulation/egg retrieval (OSER), and 33 (22.1%) declined FP. Of the patients with testes, 98 (52.1%) underwent testicular tissue cryopreservation (TTC), 48 (25.5%) completed sperm banking (SB), and 42 (22.3%) declined FP. Median time from referral to FP consultation was short (ovaries: 2 days, range: 0-6; testes: 1 day, range: 0-5). OSER had a significantly longer RTT versus OTC and no FP (52.5 vs.19.5 vs. 12 days, p = .01). SB had a significantly quicker RTT compared to TTC or no FP (9.0 vs. 21.0 vs. 13.5 days; p = .008). For patients who underwent OTC/TTC and those who declined FP, there was no significant difference in time from consultation to treatment. CONCLUSIONS: It is feasible to promptly offer and complete FP with minimal delay to disease-directed treatment.

De novo design of a nanoregulator for the dynamic restoration of ovarian tissue in cryopreservation and transplantation.

The cryopreservation and transplantation of ovarian tissue underscore its paramount importance in safeguarding reproductive capacity and ameliorating reproductive disorders. However, challenges persist in ovarian tissue cryopreservation and transplantation (OTC-T), including the risk of tissue damage and dysfunction. Consequently, there has been a compelling exploration into the realm of nanoregulators to refine and enhance these procedures. This review embarks on a meticulous examination of the intricate anatomical structure of the ovary and its microenvironment, thereby establishing a robust groundwork for the development of nanomodulators. It systematically categorizes nanoregulators and delves deeply into their functions and mechanisms, meticulously tailored for optimizing ovarian tissue cryopreservation and transplantation. Furthermore, the review imparts valuable insights into the practical applications and obstacles encountered in clinical settings associated with OTC-T. Moreover, the review advocates for the utilization of microbially derived nanomodulators as a potent therapeutic intervention in ovarian tissue cryopreservation. The progression of these approaches holds the promise of seamlessly integrating nanoregulators into OTC-T practices, thereby heralding a new era of expansive applications and auspicious prospects in this pivotal domain.

Comparison of liquid nitrogen-free slow freezing protocols toward enabling a practical option for centralized cryobanking of ovarian tissue.

Geographically distributed ovarian tissue cryobanks remain limited due to the high facility and staff costs, and cold transportation to centers is associated with ischemia-induced tissue damage that increases with transport distance. It is ideal to perform the cryopreservation procedure at a tissue removal site or local hospital before shipment to cost-effective centralized cryobanks. However, conventional liquid nitrogen-based freezers are not portable and require expensive infrastructure. To study the possibility of an ovarian tissue cryopreservation network not dependent on liquid nitrogen, we cryopreserved bovine ovarian tissue using three cooling techniques: a controlled rate freezer using liquid nitrogen, a liquid nitrogen-free controlled rate freezer, and liquid nitrogen-free passive cooling. Upon thawing, we evaluated a panel of viability metrics in frozen and fresh groups to examine the potency of the portable liquid nitrogen-free controlled and uncontrolled rate freezers in preserving the ovarian tissue compared to the non-portable conventional controlled rate freezer. We found similar outcomes for reactive oxygen species (ROS), total antioxidant capacity (TAC), follicular morphology, tissue viability, and fibrosis in the controlled rate freezer groups. However, passive slow cooling was associated with the lowest tissue viability, follicle morphology, and TAC, and the highest tissue fibrosis and ROS levels compared to all other groups. A stronger correlation was found between follicle morphology, ovarian tissue viability, and fibrosis with the TAC/ROS ratio compared to ROS and TAC alone. The current study undergirds the possibility of centralized cryobanks using a controlled rate liquid nitrogen-free freezer to prevent ischemia-induced damage during ovarian tissue shipment.

A case of non-Hodgkin lymphoma of the upper gingiva with minimal residual disease detected in cryopreserved ovarian tissue: A case report.

Recently, more than 200 live births following ovarian tissue cryopreservation (OTC) and transplantation in cancer survivors have been reported worldwide. However, cancer survivors with minimal residual disease (MRD) in cryopreserved ovarian tissue are at the risk of relapse through the graft. Here, we report a rare case of a 19-year-old female patient with non-Hodgkin lymphoma who had MRD in the ovary harvested for OTC. The patient was diagnosed with aggressive B-cell lymphoma after gingival biopsy. The 18F-fluoro-2-deoxy-D-glucose positron emission tomography scan performed before OTC showed no viable lesions in either ovary. However, on histological evaluation, we detected infiltration of lymphoma cells in the ovary. Informed consent about MRD is required even if there is no evidence of MRD in the ovary before OTC. Patients whose cryopreserved ovaries have MRD may require the development of alternative assisted reproductive technologies such as in vitro growth or artificial ovary.

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.

Ethical, legal, social, and policy issues of ovarian tissue cryopreservation in prepubertal girls: a critical interpretive review.

PURPOSE: Despite the increasing number of childhood cancer survivors, significant advances in ovarian tissue cryopreservation (OTC) technique and medical societies' recommendations, fertility preservation (FP) and FP discussions are not always offered as a standard of care in the pediatric context. The aim of this literature review is to understand what ethical, legal, social, and policy issues may influence the provision of FP by OTC in prepubertal girls with cancer. METHODS: A critical interpretive review of peer-reviewed papers published between 2000 and January 2023 was conducted, guided by the McDougall's version of the critical interpretive synthesis (Dixon-Woods), to capture recurring concepts, principles, and arguments regarding FP by OTC for prepubertal girls. RESULTS: Of 931 potentially relevant papers, 162 were included in our analysis. Data were grouped into seven thematic categories: (1) risks of the procedure, (2) unique decision-making issues in pediatric oncofertility, (3) counseling, (4) cultural and cost issues, and (5) disposition of cryopreserved reproductive tissue. CONCLUSION: This first literature review focusing on ethical, legal, social, and policy issues surrounding OTC in prepubertal girls highlights concerns in the oncofertility debate. Although OTC is no longer experimental as of December 2019, these issues could limit its availability and the child's future reproductive autonomy. This review concludes that specific actions must be provided to enable the offer of FP, such as supporting families' decision-making in this unique and complex context, and providing pediatric patients universal and full access to free or highly subsidized OTC.

Investigating the impact of vitrification on bovine ovarian tissue morphology, follicle survival, and transcriptomic signature.

PURPOSE: Ovarian tissue cryopreservation is vital for fertility preservation, yet its effect on ovarian tissue follicle survival and transcriptomic signature requires further investigation. This study delves into the effects of vitrification on tissue morphology, function, and transcriptomic changes, helping to find possibilities for vitrification protocol improvements. METHODS: Ovarian cortex from 19 bovine animals were used to conduct pre- and post-vitrification culture followed by histological assessment, immunohistochemistry, and TUNEL assay. Follicles' functionality was assessed for viability and growth within the tissue and in isolated cultures. RNA-sequencing of ovarian tissue was used to explore the transcriptomic alterations caused by vitrification. RESULTS: Follicle density, cell proliferation, and DNA damage in ovarian stroma were unaffected by vitrification. However, vitrified cultured tissue exhibited reduced follicle density of primordial/primary and antral follicles, while freshly cultured tissue manifested reduction of antral follicles. Increased stromal cell proliferation and DNA damage occurred in both groups post-culture. Isolated follicles from vitrified tissue exhibited similar viability to fresh follicles until day 4, after which the survival dropped. RNA-sequencing revealed minor effects of vitrification on transcriptomic signatures, while culture induced significant gene expression changes in both groups. The altered expression of WNT and hormonal regulation pathway genes post-vitrification suggests the molecular targets for vitrification protocol refinement. CONCLUSION: Vitrification minimally affects tissue morphology, follicle density, and transcriptomic signature post-thawing. However, culture revealed notable changes in vitrified tissue samples, including reduced follicle density, decreased isolated follicle survival, and alteration in WNT signalling and ovarian hormonal regulation pathways, highlighted them as possible limitations of the current vitrification protocol.

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.

Ovarian tissue cryopreservation and transplantation as a natural means to delay menopause.

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.

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.

Ovarian tissue cryopreservation and transplantation: a review on reactive oxygen species generation and antioxidant therapy.

Cancer is the leading cause of death worldwide. Fortunately, the survival rate of cancer continues to rise, owing to advances in cancer treatments. However, these treatments are gonadotoxic and cause infertility. Ovarian tissue cryopreservation and transplantation (OTCT) is the most flexible option to preserve fertility in women and children with cancer. However, OTCT is associated with significant follicle loss and an accompanying short lifespan of the grafts. There has been a decade of research in cryopreservation-induced oxidative stress in single cells with significant successes in mitigating this major source of loss of viability. However, despite its success elsewhere and beyond a few promising experiments, little attention has been paid to this key aspect of OTCT-induced damage. As more and more clinical practices adopt OTCT for fertility preservation, it is a critical time to review oxidative stress as a cause of damage and to outline potential ameliorative interventions. Here we give an overview of the application of OTCT for female fertility preservation and existing challenges; clarify the potential contribution of oxidative stress in ovarian follicle loss; and highlight potential ability of antioxidant treatments to mitigate the OTCT-induced injuries that might be of interest to cryobiologists and reproductive clinicians.

Prior exposure to chemotherapy does not reduce the in vitro maturation potential of oocytes obtained from ovarian cortex in cancer patients.

STUDY QUESTION: Does chemotherapy exposure affect IVM potential of immature oocytes retrieved from the ovarian cortex following ovarian tissue cryopreservation (OTC) for fertility preservation? SUMMARY ANSWER: The IVM potential of oocyte retrieved from ovarian cortex following OTC is not affected by prior exposure to chemotherapy but primarily dependent on patient's age, while successful retrieval of immature oocytes from the ovarian tissue is negatively affected by chemotherapy and its timing. WHAT IS KNOWN ALREADY: The potential and feasibility of IVM in premenarche patients was previously demonstrated, in smaller studies. The scarce data that exist on the IVM potential of oocytes retrieved during OTC following chemotherapy support the feasibility of this process, however, this was not previously shown in the premenarche cancer patients population or in larger cohorts. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study evaluating 229 cancer patients aged 1-39 years with attempted retrieval of oocytes from the ovarian tissue and the medium following OTC in a university affiliated fertility preservation unit between 2002 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 172 chemotherapy naïve and 57 chemotherapy exposed patients aged 1-39 years underwent OTC in university affiliated tertiary infertility and IVF center. OTC and IVM outcomes were compared between the chemotherapy naïve and exposed groups. The main outcome measure was mean IVM rate per patient in the chemotherapy naïve and exposed groups, with subgroup analysis of a 1:1 chemotherapy exposed group matched for age at OTC and type of malignancy. We additionally analyzed premenarche and postmenarche patients' outcomes separately and investigated the effect of time from chemotherapy to IVM, malignancy type and chemotherapy regimen on oocyte number and IVM outcomes in the chemotherapy exposed group. MAIN RESULTS AND THE ROLE OF CHANCE: While the number of retrieved oocytes and percentage of patients with at least one oocyte retrieved was higher in the chemotherapy naïve group (8.7 ± 7.9 versus 4.9 ± 5.6 oocytes and 87.2% versus 73.7%, P < 0.001 and P = 0.016, respectively), IVM rate and number of mature oocytes were comparable between the groups (29.0 ± 25.0% versus 28. 9 ± 29.2% and 2.8 ± 3.1 versus 2.2 ± 2.8, P = 0.979 and P = 0.203, respectively). Similar findings were shown in subgroup analyses for premenarche and postmenarche groups. The only parameter found to be independently associated with IVM rate in a multivariable model was menarche status (F = 8.91, P = 0.004). Logistic regression models similarly showed that past chemotherapy exposure is negatively associated with successful retrieval of oocytes while older age and menarche are predictive of successful IVM. An age and the type of malignancy matched (1:1) chemotherapy naïve and exposed groups were created (25 patients in each group). This comparison demonstrated similar IVM rate (35.4 ± 30.1% versus 31.0 ± 25.2%, P = 0.533) and number of matured oocytes (2.7 ± 3.0. versus 3.0 ± 3.9 oocytes, P = 0.772). Type of malignancy and chemotherapy regimen including alkylating agents were not associated with IVM rate. LIMITATIONS, REASONS FOR CAUTION: This study's inherited retrospective design and the long study period carries the possible technological advancement and differences. The chemotherapy exposed group was relatively small and included different age groups. We could only evaluate the potential of the oocytes to reach metaphase II in vitro but not their fertilization potential or clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: IVM is feasible even after chemotherapy broadening the fertility preservation options of cancer patients. The use of IVM for fertility preservation, even after exposure to chemotherapy, should be further studied for optimal postchemotherapy timing safety and for the in vitro matured oocytes potential for fertilization. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study by any of the authors. The authors report that no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Long-term follow-up to assess criteria for ovarian tissue cryopreservation for fertility preservation in young women and girls with cancer.

STUDY QUESTION: Do the Edinburgh Selection Criteria correctly identify female cancer patients under the age of 18 who are at risk of premature ovarian insufficiency (POI) as candidates for ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Patient assessment using these criteria accurately identifies those at risk of POI, who can be offered OTC and future transplantation as a means of fertility preservation. WHAT IS KNOWN ALREADY: Treatment for childhood cancer can have adverse consequences on future fertility; at the time of diagnosis, fertility risk assessment should be undertaken in order to identify patients to whom fertility preservation should be offered. The Edinburgh selection criteria, based on planned cancer treatment and patient health status, are utilized to identify those at high risk and therefore eligible for OTC. However, this procedure is not without risk and there are few data on the efficacy of the procedure in prepubertal patients. As such, long-term follow-up of reproductive outcomes is necessary, to ensure that OTC is being offered appropriately. STUDY DESIGN, SIZE, DURATION: Cohort study encompassing all females diagnosed with cancer under the age of 18 in South East Scotland, from 1 January 1996 to 30 April 2020. Patients were followed up for reproductive outcomes to assess for diagnosis of POI. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 638 eligible patients were identified; patients under the age of 12 or deceased before the age of 12 were excluded from the study, leaving a study population of 431 patients. Electronic records were reviewed for reproductive function, assessed by current menstruation, pregnancy (in the absence of POI diagnosis), reproductive hormone measurements, pubertal progression, or diagnosis of POI. Patients on hormonal contraception (other than for treatment of POI or panhypopituitarism with no history of gonadatoxic treatment) were excluded from analysis (n = 9). Analysis on remaining 422 patients was carried out using the Kaplan-Meier methods, with POI as the defined event, and Cox proportional hazards model. MAIN RESULTS AND THE ROLE OF CHANCE: In the study population of 431 patients, median ages at diagnosis and analysis were 9.8 and 22.2 years, respectively. Reproductive outcomes were unavailable in 142 patients; the assumption was made that these patients did not have POI, but a subanalysis excluding these patients was also performed. Of the 422 patients aged >12 at analysis and not taking hormonal contraception, OTC was offered to 37 patients and successfully performed in 25 patients. Of the 37 patients offered OTC (one at time of relapse), nine (24.3%) developed POI. Of the 386 not offered OTC, 11 (2.9%) developed POI. The probability of developing POI was significantly higher in those offered OTC (hazard ratio [HR] 8.7 [95% CI 3.6-21]; P < 0.0001), even when those patients with unknown outcomes were excluded from the analysis (HR 8.1 [95% CI 3.4-20]; P < 0.001). All patients offered OTC who developed POI did so after treatment for primary disease; in those not offered OTC, five patients (45.5%) developed POI after treatment for disease relapse. LIMITATIONS, REASONS FOR CAUTION: A significant number of patients had unknown reproductive outcomes; many of these patients were engaged in ongoing follow-up but did not have documented reproductive assessment. This may have introduced bias to the analysis and highlights the need for reproductive follow-up as part of routine cancer aftercare. In addition, the relatively young age of the patient population and short duration of follow-up in some cases demonstrates the need for ongoing follow-up of this cohort. WIDER IMPLICATIONS OF THE FINDINGS: The prevalence of POI after childhood cancer is low, but the Edinburgh selection criteria remain a robust tool for selecting those at high risk at the time of diagnosis, to offer OTC appropriately. However, disease relapse necessitating more intensive treatments remains a challenge. This study additionally highlights the importance of routine assessment and documentation of reproductive status in haematology/oncology follow-up. STUDY FUNDING/COMPETING INTEREST(S): K.D. is supported by a CRUK grant (C157/A25193). This work was undertaken in part in the MRC Centre for Reproductive Health, (supported by MRC grant MR/N022556/1). R.A.A. has received consulting fees from Ferring and Roche Diagnostics; payment from Merck and IBSA for educational events; and laboratory materials from Roche Diagnostics. The other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Ovarian tissue cryopreservation can be combined simultaneously with oocyte retrieval after controlled ovarian hyperstimulation.

STUDY QUESTION: Can ovarian tissue cryopreservation (OTC) be performed after controlled ovarian hyperstimulation (COH)? SUMMARY ANSWER: Unilateral oophorectomy after transvaginal oocyte retrieval is feasible on stimulated ovaries during one surgical step. WHAT IS KNOWN ALREADY: In the fertility preservation (FP) field, the timeframe between patient referral and start of curative treatment is limited. Combining oocyte pick-up with ovarian tissue (OT) extraction has been reported to improve FP but COH applied before OT extraction is not currently recommended. STUDY DESIGN, SIZE, DURATION: This retrospective cohort-controlled study involved 58 patients who underwent oocyte cryopreservation immediately followed by OTC between September 2009 and November 2021. The exclusion criteria were a delay between oocyte retrieval and OTC of >24 h (n = 5) and IVM of oocytes obtained ex vivo in the ovarian cortex (n = 2). This FP strategy was performed either after COH (stimulated group, n = 18) or after IVM (unstimulated group, n = 33). PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte retrieval followed by OT extraction on the same day was performed either without previous stimulation or after COH. Adverse effects of surgery and ovarian stimulation, mature oocyte yield and pathology findings of fresh OT were retrospectively analysed. Thawed OTs were analysed prospectively, for vascularization and apoptosis using immunohistochemistry, when patient consent was obtained. MAIN RESULTS AND THE ROLE OF CHANCE: No surgical complication occurred after OTC surgery in either group. In particular, no severe bleeding was associated with COH. The number of mature oocytes obtained increased after COH (median = 8.5 (25% = 5.3-75% = 12.0)) compared to the unstimulated group (2.0 (1.0-5.3), P < 0.001). Neither ovarian follicle density nor cell integrity was affected by COH. Fresh OT analysis showed congestion in half of the stimulated OT which was higher than in the unstimulated OT (3.1%, P < 0.001). COH also increased haemorrhagic suffusion (COH + OTC: 66.7%; IVM + OTC: 18.8%, P = 0.002) and oedema (COH + OTC: 55.6%; IVM + OTC: 9.4%, P < 0.001). After thawing, the pathological findings were similar between both groups. No statistical difference in the number of blood vessels was observed between the groups. The oocyte apoptotic rate in thawed OT was not statistically different between the groups (ratio of positive cleaved caspase-3 staining oocytes/total number of oocytes equal to median 0.50 (0.33-0.85) and 0.45 (0.23-0.58) in unstimulated and stimulated groups respectively, P = 0.720). LIMITATIONS, REASONS FOR CAUTION: The study reports FP from a small number of women following OTC. Follicle density and other pathology findings are an estimate only. WIDER IMPLICATIONS OF THE FINDINGS: Unilateral oophorectomy can be successfully performed after COH with limited bleeding risk and an absence of impact on thawed OT. This approach could be proposed to post pubertal patients when the number of mature oocytes expected is low or when the risk of residual pathology is high. The reduction of surgical steps for cancer patients also has positive implications for introducing this approach into clinical practice. STUDY FUNDING/COMPETING INTEREST(S): This work was made possible through the support of the reproductive department of Antoine-Béclère Hospital and of the pathological department of Bicêtre Hospital (Assistance Publique Hôpitaux de Paris, France). The authors have no conflict of interest to disclose in this study. TRIAL REGISTRATION NUMBER: N/A.

Live birth rate after female fertility preservation for cancer or haematopoietic stem cell transplantation: a systematic review and meta-analysis of the three main techniques; embryo, oocyte and ovarian tissue cryopreservation.

STUDY QUESTION: What are the chances of achieving a live birth after embryo, oocyte and ovarian tissue cryopreservation (OTC) in female cancer survivors? SUMMARY ANSWER: The live birth rates (LBRs) following embryo and oocyte cryopreservation are 41% and 32%, respectively, while for IVF and spontaneous LBR after tissue cryopreservation and transplantation, these rates are 21% and 33%, respectively. WHAT IS KNOWN ALREADY: Currently, fertility preservation (FP) has become a major public health issue as diagnostic and therapeutic progress has made it possible to achieve an 80% survival rate in children, adolescents and young adults with cancer. In the latest ESHRE guidelines, only oocyte and embryo cryopreservation are considered as established options for FP. OTC is still considered to be an innovative method, while it is an acceptable FP technique in the American Society for Reproductive Medicine guidelines. However, given the lack of studies on long-term outcomes after FP, it is still unclear which technique offers the best chance to achieve a live birth. STUDY DESIGN, SIZE, DURATION: We performed a systematic review and meta-analysis of published controlled studies. Searches were conducted from January 2004 to May 2021 in Medline, Embase and the Cochrane Library using the following search terms: cancer, stem cell transplantation, FP, embryo cryopreservation, oocyte vitrification, OTC and reproductive outcome. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 126 full-text articles were preselected from 1436 references based on the title and abstract and assessed via the Newcastle-Ottawa Quality Assessment Scale. The studies were selected, and their data were extracted by two independent reviewers according to the Cochrane methods. A fixed-effect meta-analysis was performed for outcomes with high heterogeneity. MAIN RESULTS AND THE ROLE OF CHANCE: Data from 34 studies were used for this meta-analysis. Regarding cryopreserved embryos, the LBR after IVF was 41% (95% CI: 34-48, I2: 0%, fixed effect). Concerning vitrified oocytes, the LBR was 32% (95% CI: 26-39, I2: 0%, fixed effect). Finally, the LBR after IVF and the spontaneous LBR after ovarian tissue transplantation were 21% (95% CI: 15-26, I2: 0%, fixed-effect) and 33% (95% CI: 25-42, I2: 46.1%, random-effect), respectively. For all outcomes, in the sensitivity analyses, the maximum variation in the estimated percentage was 1%. LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of the literature prevents us from comparing these three techniques. This meta-analysis provides limited data which may help clinicians when counselling patients. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the need for long-term follow-up registries to assess return rates, as well as spontaneous pregnancy rates and birth rates after FP. STUDY FUNDING/COMPETING INTEREST(S): This work was sponsored by an unrestricted grant from GEDEON RICHTER France. The authors have no competing interests to declare. REGISTRATION NUMBER: CRD42021264042.

Effects of needle puncturing on re-vascularization and follicle survival in xenotransplanted human ovarian tissue.

BACKGROUND: Ovarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex. METHODS: Cryopreserved human ovarian cortex pieces (N = 36) from 20 women aged 24-36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150-200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors (Vegfα, Angptl4, Ang1, and Ang2), and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks. RESULTS: A significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Vegfα was consistently higher in the needle group compared to control at all three time points, but not statistically significant. The expression of Ang1 and Ang2 increased significantly from day 3 to day 10 in the control group (p < 0.001, p = 0.0023), however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density (follicles/mm3, mean ± SEM) was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045). CONCLUSIONS: Needle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology.