A synopsis of global frontiers in fertility preservation.

Since 2007, the Oncofertility Consortium Annual Conference has brought together a diverse network of individuals from a wide range of backgrounds and professional levels to disseminate emerging basic and clinical research findings in fertility preservation. This network also developed enduring educational materials to accelerate the pace and quality of field-wide scientific communication. Between 2007 and 2019, the Oncofertility Consortium Annual Conference was held as an in-person event in Chicago, IL. The conference attracted approximately 250 attendees each year representing 20 countries around the world. In 2020, however, the COVID-19 pandemic disrupted this paradigm and precluded an in-person meeting. Nevertheless, there remained an undeniable demand for the oncofertility community to convene. To maintain the momentum of the field, the Oncofertility Consortium hosted a day-long virtual meeting on March 5, 2021, with the theme of "Oncofertility Around the Globe" to highlight the diversity of clinical care and translational research that is ongoing around the world in this discipline. This virtual meeting was hosted using the vFairs ® conference platform and allowed over 700 people to participate, many of whom were first-time conference attendees. The agenda featured concurrent sessions from presenters in six continents which provided attendees a complete overview of the field and furthered our mission to create a global community of oncofertility practice. This paper provides a synopsis of talks delivered at this event and highlights the new advances and frontiers in the fields of oncofertility and fertility preservation around the globe from clinical practice and patient-centered efforts to translational research.

Clinical summary guide: reproduction in women with previous abdominopelvic radiotherapy or total body irradiation.

STUDY QUESTION: What is the evidence to guide the management of women who wish to conceive following abdominopelvic radiotherapy (AP RT) or total body irradiation (TBI)? SUMMARY ANSWER: Pregnancy is possible, even following higher doses of post-pubertal uterine radiation exposure; however, it is associated with adverse reproductive sequelae and pregnancies must be managed in a high-risk obstetric unit. WHAT IS KNOWN ALREADY: In addition to primary ovarian insufficiency, female survivors who are treated with AP RT and TBI are at risk of damage to the uterus. This may impact on its function and manifest as adverse reproductive sequelae. STUDY DESIGN SIZE DURATION: A review of the literature was carried out and a multidisciplinary working group provided expert opinion regarding assessment of the uterus and obstetric management. PARTICIPANTS/MATERIALS SETTING METHODS: Reproductive outcomes for postpubertal women with uterine radiation exposure in the form of AP RT or TBI were reviewed. This included Pubmed listed peer-reviewed publications from 1990 to 2019, and limited to English language.. MAIN RESULTS AND THE ROLE OF CHANCE: The prepubertal uterus is much more vulnerable to the effects of radiation than after puberty. Almost all available information about the impact of radiation on the uterus comes from studies of radiation exposure during childhood or adolescence.An uncomplicated pregnancy is possible, even with doses as high as 54 Gy. Therefore, tumour treatment doses alone cannot at present be used to accurately predict uterine damage. LIMITATIONS REASONS FOR CAUTION: Much of the data cannot be readily extrapolated to adult women who have had uterine radiation and the publications concerning adult women treated with AP RT are largely limited to case reports. WIDER IMPLICATIONS OF THE FINDINGS: This analysis offers clinical guidance and assists with patient counselling. It is important to include patients who have undergone AP RT or TBI in prospective studies to provide further evidence regarding uterine function, pregnancy outcomes and correlation of imaging with clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: This study received no funding and there are no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Cisplatin- and cyclophosphamide-induced primordial follicle depletion is caused by direct damage to oocytes.

It is well established that DNA-damaging chemotherapies can cause infertility and ovarian endocrine failure by depleting the ovarian reserve of primordial follicles. Currently, no effective pharmacological therapies exist for the preservation of long-term fertility and ovarian function in female cancer patients, due to a limited understanding of the mechanisms of chemotherapy-induced follicle depletion. This study investigated the cellular targets, molecular mechanisms, and temporal course of ovarian reserve depletion following treatment with commonly used chemotherapeutic drugs. Adult female C57BL/6 mice were injected i.p. with saline, cisplatin (5mg/kg), or cyclophosphamide (300mg/kg); ovaries were harvested after 8 or 24 hours. Follicle counts showed depletion of all follicular stages 24 hours after administration of cisplatin or cyclophosphamide. Eight hours post-treatment, H2A histone family member X (γH2AX) immunofluorescence showed DNA double-stranded breaks at all follicular stages, including within primordial follicle oocytes. This staining was resolving by 24 hours, indicating that primordial follicle oocytes begin to undergo either apoptosis or repair in this timeframe. γH2AX-positive follicles were further examined to identify the specific cell types damaged. In primordial, transitional, and primary follicles, only oocytes sustained DNA damage, whereas in secondary and antral follicles, only somatic cells were affected. TUNEL staining confirmed that apoptosis occurs in these targeted cell types. Whilst multi-drug and multi-dose regimens were not examined, this study conclusively shows that cyclophosphamide and cisplatin cause direct damage to primordial follicle oocytes, which then undergo apoptosis. Therefore, future pharmacological strategies to prevent chemotherapy-induced infertility in females must specifically prevent primordial follicle oocyte death.

The primordial follicle reserve is not renewed after chemical or γ-irradiation mediated depletion.

Reports indicate that germ-line stem cells present in adult mice can rapidly generate new oocytes and contribute to the primordial follicle reserve following conditions of ovotoxic stress. We further investigated the hypothesis that adult mice have the capacity to generate new oocytes by monitoring primordial follicle numbers throughout postnatal life and following depletion of the primordial follicle reserve by exposure to doxorubicin (DXR), trichostatin A (TSA), or whole-body γ-irradiation. We show that primordial follicle number remains stable in adult C57BL/6 mice between the ages of 25 and 100 days. However, within 2 days of treatment with DXR or TSA, primordial follicle numbers had declined to 65 and 51% respectively (P<0.05-0.01 when compared to untreated controls), with no restoration of follicle numbers evident after 7 days for either treatment. Furthermore, ovaries from mice subjected to sterilizing doses of γ-irradiation (0.45 or 4.5 Gy) revealed complete ablation of all primordial follicles 5 days after treatment, with no indication of follicular renewal. We conclude that neo-folliculogenesis does not occur following chemical or γ-irradiation mediated depletion of the primordial follicle reserve.

Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation.

BACKGROUND: The demand for cryopreservation of human oocytes is increasing in assisted reproduction clinics and yet remains an experimental procedure. Surprisingly, little is known about the effects of cryopreservation on spindle-chromosome interactions and the recovery of meiotic spindle functionality. The goal of these studies was to evaluate the process of meiotic spindle reassembly and chromosome alignment in cryopreserved human metaphase II oocytes. METHODS: Unfrozen control oocytes were compared with frozen oocytes fixed at 0, 1, 2 and 3 h after thawing. Oocytes were analysed by confocal microscopy and subjected to 3-dimensional image analysis to evaluate spindle integrity. RESULTS: Freezing resulted in a loss of spindle bipolarity and chromosome alignment. One hour following thawing, most oocytes recovered spindle bipolarity and equatorial chromosomal alignment. However, between 2 and 3 h, a progressive loss of chromosome alignment was observed. Further analysis revealed a positive correlation between spindle length and number of displaced chromosomes following freezing. This time-dependent redistribution of chromosomes involved outward displacement from the equatorial plate and retention at the surface of the meiotic spindle. CONCLUSIONS: Spindle disassembly incurred by cryopreservation is rapidly reversed and is coordinated with chromosome alignment within 1 h but is not sustained at later times.

Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation.

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle. RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 +/- 0.29 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm s(-1) were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.

Kit ligand and c-Kit have diverse roles during mammalian oogenesis and folliculogenesis.

Paracrine signalling between the oocyte and its surrounding somatic cells is fundamental to the processes of oogenesis and folliculogenesis in mammals. The study of animal models has revealed that the interaction of granulosa cell-derived kit ligand (KL) with oocyte and theca cell-derived c-Kit is important for multiple aspects of oocyte and follicle development, including the establishment of primordial germ cells within the ovary, primordial follicle activation, oocyte survival and growth, granulosa cell proliferation, theca cell recruitment and the maintenance of meiotic arrest. Though little is known about the specific roles of KL and c-Kit during human oogenesis, the expression profiles for KL and c-Kit within the human ovary suggest that they are also functionally relevant to female fertility. This review details our current understanding of the roles of KL and c-Kit within the mammalian ovary, with a particular focus on the functional diversity of this receptor-ligand interaction at different stages of oocyte and follicle development.

An investigation into the ultrasonic treatment of polluted solids.

Granular pieces of brick impregnated with copper oxide were used as a model for contaminated soil. Washing this model substrate by passing water across the substrate on an ultrasonically shaken tray irradiation afforded a 40% reduction in copper content. This was compared with only a 6% reduction when the sample was treated under otherwise identical conditions but using a tray shaken conventionally. The majority of the copper was removed as a result of the removal of surface materials which were more heavily contaminated with the copper oxide.