Fertility Preservation Options Research Articles

Trainee-Led Quality Improvement Project to Improve Fertility Preservation Counseling for Patients With Cancer.

Oncofertility counseling regarding the reproductive risks associated with cancer therapy is essential for quality cancer care. We aimed to increase the rate of oncofertility counseling for patients of reproductive age (18-40 years) with cancer who were initiating systemic therapy at the Johns Hopkins Cancer Center from a baseline rate of 37% (25 of 68, June 2019-January 2020) to 70% by February 2021. We formed an interprofessional, multidisciplinary team as part of the ASCO Quality Training Program. We obtained data from the electronic medical record and verified data with patients by phone. We surveyed patients, oncologists, and fertility specialists to identify barriers. After considering a prioritization matrix, we implemented Plan-Do-Study-Act (PDSA) cycles. We identified the following improvement opportunities: (1) oncologist self-reported lack of knowledge about counseling and local fertility preservation options and (2) lack of a standardized referral mechanism to fertility services. During the first PDSA cycle (February 2020-August 2020, disrupted by COVID-19), we introduced the initiative to increase oncofertility counseling at faculty meetings. From September 2020 to November 2020, we implemented a second PDSA cycle: (1) educating and presenting the initiative at Oncology Grand Rounds, (2) distributing informative pamphlets to oncologists and patients, and (3) implementing an electronic medical record order set. In the third PDSA cycle (December 2020-February 2021), we redesigned the order set to add information (eg, contact information for fertility coordinator) to the patient after-visit summary. Postimplementation (September 2020-February 2021), counseling rates increased from 37% to 81% (38 of 47). We demonstrate how a trainee-led, patient-centered initiative improved oncofertility care. Ongoing work focuses on ensuring sustainability and assessing the quality of counseling.

Equine ovarian tissue xenografting: impacts of cooling, vitrification, and VEGF.

Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans.Lay summaryDue to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.

Effect of Genetic Variation in CYP450 on Gonadal Impairment in a European Cohort of Female Childhood Cancer Survivors, Based on a Candidate Gene Approach: Results from the PanCareLIFE Study.

Simple SummaryChildhood cancer patients receiving treatment containing alkylating agents are at risk of infertility, yet inter-individual variability in treatment-related ovarian damage is observed. Alkylating agents are metabolized by cytochrome P450 (CYP450) enzymes and polymorphisms in these CYP450 enzymes may explain this variability in ovarian damage. This study on genetic variation in CYP450 enzymes of chemotherapy-induced gonadotoxicity, using anti-Müllerian hormone (AMH) levels as a proxy for ovarian reserve, in female childhood cancer survivors (CCSs) may identify patients at risk of infertility. This unique global collaboration of two large CCS studies shows the significant gonadotoxic effect of enzyme CYP3A4*3 and significant protective effect of CYP2B6*2 on gonadal function in CCSs receiving alkylating agents. Genetic variation in CYP3A4 and CYP2B6 have previously been associated with gonadotoxicity after cancer treatment. These findings could guide risk prediction models determining patients at risk of chemotherapy-induced gonadal impairment.Background: Female childhood cancer survivors (CCSs) carry a risk of therapy-related gonadal dysfunction. Alkylating agents (AA) are well-established risk factors, yet inter-individual variability in ovarian function is observed. Polymorphisms in CYP450 enzymes may explain this variability in AA-induced ovarian damage. We aimed to evaluate associations between previously identified genetic polymorphisms in CYP450 enzymes and AA-related ovarian function among adult CCSs. Methods: Anti-Müllerian hormone (AMH) levels served as a proxy for ovarian function in a discovery cohort of adult female CCSs, from the pan-European PanCareLIFE cohort (n = 743; age (years): median 25.8, interquartile range (IQR) 22.1–30.6). Using two additive genetic models in linear and logistic regression, nine genetic variants in three CYP450 enzymes were analyzed in relation to cyclophosphamide equivalent dose (CED) score and their impact on AMH levels. The main model evaluated the effect of the variant on AMH and the interaction model evaluated the modifying effect of the variant on the impact of CED score on log-transformed AMH levels. Results were validated, and meta-analysis performed, using the USA-based St. Jude Lifetime Cohort (n = 391; age (years): median 31.3, IQR 26.6–37.4). Results: CYP3A4*3 was significantly associated with AMH levels in the discovery and replication cohort. Meta-analysis revealed a significant main deleterious effect (Beta (95% CI): −0.706 (−1.11–−0.298), p-value = 7 × 10−4) of CYP3A4*3 (rs4986910) on log-transformed AMH levels. CYP2B6*2 (rs8192709) showed a significant protective interaction effect (Beta (95% CI): 0.527 (0.126–0.928), p-value = 0.01) on log-transformed AMH levels in CCSs receiving more than 8000 mg/m2 CED. Conclusions: Female CCSs CYP3A4*3 carriers had significantly lower AMH levels, and CYP2B6*2 may have a protective effect on AMH levels. Identification of risk-contributing variants may improve individualized counselling regarding the treatment-related risk of infertility and fertility preservation options.

Documentation of Oncofertility Communication in Adolescents and Young Adults with Cancer: A Retrospective Analysis.

Background: Increasing numbers of adolescents and young adults with cancer (AYACs) are surviving long term, highlighting the importance of effective oncofertility communication. We undertook this study to understand documentation of fertility discussions with AYACs, what options are offered, and how this differs for AYACs on treatment compared with those post-treatment. Methods: We reviewed the documentation of fertility discussions with 122 AYACs treated between 2000 and 2020: 72 AYACs on treatment and 50 AYACs at least 3 years post-treatment ("late effects" cohort). Results: Diagnoses were split evenly between hematological and solid tumor diagnoses, and biological sex. Seventy-five percent of patients were diagnosed and treated by the AYAC team and 25% by the pediatric team. Median age at diagnosis was 19 years (range 4-24) for on-treatment patients and 16 years (range 3-25) for late effects patients. Fertility was discussed with 93% of on-treatment patients and 48% of late effects patients. Seventy-nine percent of on-treatment patients and 48% of late effects patients pursued a pre-treatment fertility preservation option. Post-treatment, 84% of late effects patients had a discussion and 57% pursued an option. Only four patients across both cohorts underwent oocyte or ovarian tissue cryopreservation. Those referred to specialist reproductive medicine clinics had more detailed documentation about fertility discussions. Nurse-led late effects clinics had a key role in facilitating post-treatment discussions. Conclusions: It is important to communicate oncofertility options to AYACs repeatedly throughout treatment. Referral to specialist oncofertility services and adequate information for both sexes is important pre-treatment, and can be facilitated post-treatment by a late effects service.

The ethics of offering fertility preservation to pediatric patients: A case-based discussion of barriers for clinicians to consider

Decisions about fertility preservation can be difficult in general but the recent application of preservation techniques to pediatric patients has ushered in a host of new ethical considerations. Fertility preservation (FP) may be considered for all patients who are at risk for infertility due to their medical diagnosis or treatment, including those undergoing gonadotoxic chemotherapy, those with differences of sex development (DSD) undergoing gonadectomy,1-3 and transgender patients undergoing gender affirming surgery. The focus of this paper is to review the ethical issues involved in offering FP to pediatric oncology patients and, to a lesser extent, the new ethical issues that apply to patients with DSD. Some of the techniques and approach to counseling will also apply to transgender individuals, although that is beyond the scope of this work. We aim to discuss several barriers to offering FP and to advise how to counsel families in the setting of rapid changes in this field. Families should be educated about:•Infertility risk assessment and options for FP.•Experimental nature of certain technologies.•Appropriate consent of minor individuals and the concept of assent to treatment.•Financial barriers to storage and use of genetic material.•Time-sensitive nature of decision making.•Ownership of genetic material over the lifetime of a patient.Specific guidance for clinicians regarding some of these points was recently published in an American Academy of Pediatrics Clinical Report,1 and we will illustrate the use of these guidelines in four case presentations.

OUTCOME OF SIMULTANEOUS PREIMPLANTATION GENETIC TESTING (PGT) FOR ANEUPLOIDY AND HEREDITARY BREAST CANCER (PGT-A/M) FOR FERTILITY PRESERVATION OR PREVENTION OF VERTICAL TRANSMISSION

Hereditary Breast Cancer (BC) accounts for 5-10% of BCs. Carriers of mutations in BRCA1/2, CHECK2, ATM, Lynch sy and other oncogenes have higher lifetime risk for BC (65-80%), ovarian (30-45%) and other cancers, with chance of 50% transmission to their offspring. Oocyte and/or embryo cryopreservation is an option for fertility preservation (FP), and when coupled with PGT-A/M for BC mutations, allowing prevention of vertical transmission. Our objective was to evaluate the utilization and outcomes of simultaneous PGT-A/M for BC mutation carriers.

An Educational Package for Oncology Nurses Regarding Fertility Preservation among Female Cancer Patients

affected with cancer. However, chemo-radiotherapy regimens have a negative impact on future fertility. Oocyte cryopreservation is one of the available methods to females to preserve their fertility. Study Aimed to evaluate the effect of educational package for oncology nurses regarding fertility preservation among female cancer patients. Subjects and Method: A quasi-experimental pre and post-test design. Purposive sampling consisted of 61 nurses selected according to the inclusion criteria. The study conducted at oncology center, Mansoura University Hospital. Tools: Self-Administered Structured Questionnaire, Nurses’ knowledge regarding fertility preservation, Nurse’s attitude towards Fertility Preservation and Nurses Satisfaction Scale. Results: There was statistically significant improvement in the total knowledge score regarding fertility preservation from (8.2 ±3.1) pre-intervention to (12.3 ±5.4) post-intervention with highly significant difference (P= 0.001). The total attitude score of the study sample regarding fertility preservation were improved significantly from (26.5 ±4.9) pre-intervention to (40.4 ±4.6) post-intervention with highly significant difference (P= 0.001). Furthermore, the majority of nurses were satisfied, their satisfaction level reach 73.8%. There was significant association between the post-intervention total knowledge and attitude level among the nurses (X2 7.389, p=0.025). Conclusion: Educational package was effective to improve the nurses’ knowledge and attitudes towards fertility preservation as a novel method to sustain future reproductive capacity in newly diagnosed cancer female patients. Recommendations: The importance of considering this novel method to sustain fertility preservation in newly diagnosed cancer female patients as one of the main items in the nursing curriculum and evolve protocols and pathways to empower timely counselling and communication between health care team and the newly diagnosed cancer patients regarding fertility preservation options. Also, the need for a multidisciplinary collaboration between oncology health team and reproductive health care providers to improve awareness and availability of future fertility preservation in newly diagnosed cancer patients.

THE THERAPEUTIC POTENTIAL OF IMMATURE OOCYTES FROM FERTILITY PRESERVATION PROCEDURES AND THEIR IN-VITRO MATURATION AT DIFFERENT AGES

Cryopreservation of ovarian cortical tissue (OTC) followed by auto-transplantation has emerged as a promising fertility preservation option . Immature oocyte can be found in the dissection medium during tissue handling and the ability to mature them in vitro and cryopreserve them as mature oocytes, provides an additional option for fertility preservation. In this work we aim to evaluate the developmental potential and epigenetic function of these premature oocytes, in different age groups.

SPERM CRYOPRESERVATION IN MALE CANCER PATIENTS: UTILIZATION AND SPECIMEN QUALITY

Sperm cryopreservation is an easy and effective fertility preservation option in men before initiating gonadotoxic treatment. However, referral to a fertility specialist, banked sperm disposition, and information on pregnancy outcomes remain limited. The objective of the study was to assess utilization and results of sperm cryopreservation, as well as the barriers to its use.

TESTICULAR TISSUE CRYOPRESERVATION IN A COORDINATED NETWORK OF ACADEMIC CENTERS

Testicular tissue cryopreservation (TTC) is the only fertility preservation option for prepubertal boys who are at risk of infertility due to treatments for cancer, benign diseases requiring BMT, and gender dysphoria. We established a coordinated network of 12 centers in the US that offer TTC with tissue processing and cryopreservation occurring at a centralized laboratory in Pittsburgh. Each patient donates a portion of their tissue to research. We used those tissues to evaluate the impacts of disease, previous medical treatments and duration of cryogenic storage on testicular histology and function.

IVF OUTCOMES IN BRCA CARRIERS WITH AND WITHOUT ADDITION OF LETROZOLE TO STIMULATION

Women with BRCA 1/2 mutations are commonly referred to reproductive endocrinologists to discuss fertility preservation options prior to ovarian and breast cancer risk reducing surgery. Researchers remain divided about whether BRCA carriers might be predisposed to decreased ovarian reserve and accelerated ovarian aging compared to noncarriers, and debate optimal stimulation dosage and protocols.1 The aromatase inhibitor letrozole is commonly used during controlled ovarian stimulation (COH) of breast cancer patients to minimize circulating Estradiol levels, but it is unclear whether its use negatively affects stimulation response, fertilization, and embryo quality.

Fertility preservation strategies for patients with lymphoma: a real-world practice survey among Fondazione Italiana Linfomi centers.

Improvement in the prognosis of lymphomas in recent decades has allowed focus on reducing long-term toxicity of treatment, including infertility. The aim of this study was to assess the fertility preservation knowledge and practices among hematologic centers affiliated with Fondazione Italiana Linfomi (FIL) in Italy. A survey questionnaire was provided to 152 FIL centers between December 2019 and December 2020. Responses from 58 centers (38%) were received. All respondents reported informing patients about treatment-related gonadotoxicity. A minority of patients (10% female, 20% male) refused fertility preservation due to personal reasons. The most common fertility preservation options offered to female patients were mature oocyte cryopreservation (43.1%), ovarian tissue cryopreservation (6.9%), and mature oocyte or ovarian tissue cryopreservation (39.7%). Six centers (10.3%) did not perform any procedures. All centers offered sperm cryopreservation for male patients. Challenges regarding the time intervals between lymphoma diagnosis and fertility consultation (up to 20 days) as well as between consultation and fertility preservation procedure (up to 40 days) were revealed. This survey provides insight into fertility preservation practices among Italian hematologic centers and points out an urgent need to improve close cooperation between hematologists and fertility preservation specialists in order to avoid unacceptable delays in lymphoma treatment.

Adult cancer patients and parents of younger cancer patients have little information about fertility preservation: a survey of knowledge and attitude

BackgroundImpaired fertility is one of the side effects of effective cancer therapy. Saving the potential or storing the material to enable people to have biological children after cancer treatment can be of high importance to many cancer survivors. Therefore, we designed a study to determine knowledge and attitudes to fertility preservation (FP) in adult cancer patients and the parents of patients with cancer. Participants who completed this survey were a convenience sample of 384 parents of cancer patients < 18 years and cancer patients ≥ 18 years from two large referral hospitals. A 25-item self-administered questionnaire measured knowledge and attitudes to FP. Responses were yes/no, or on a 4-point Likert scale (greatly, usually, rarely, never) scored from 1 for never to 4 for greatly.ResultsMost parents and most cancer patients were unaware of the FP methods of embryo cryopreservation (96.3% and 88.4%, respectively) and sperm cryopreservation (97.5% and 89.0%, respectively). Attitudes among cancer patients and parents to use of FP options, based on a 4-point Likert scale, were determined by financial cost, lack of access and information on FP options.ConclusionOf concern in this sample of Iranian adult cancer patients and their parents is that knowledge of the fertility risk associated with cancer therapy and knowledge of FP treatment options was generally poor, particularly among the parents. To enable cancer patients or their parents to make the best decisions about using FP services, oncologists and fertility specialists should discuss FP options during their consultation.

The fertility preservation decision-making and testicular sperm retrieval outcome in older adolescents with nonmosaic Klinefelter syndrome and azoospermia.

This study aims to analyze the fertility preservation decision-making and the sperm retrieval rate (SRR) in older adolescents (age 15-19 years) with nonmosaic Klinefelter syndrome (KS) and azoospermia in a male reproductive clinic, and to determine the accumulated SRR in older adolescents by literature review. Older adolescents with nonmosaic KS and azoospermia referred for hypogonadism and fertility concerns were enrolled. Reproductive counseling and fertility preservation options were offered to patients/parents. The acceptability and the reasons affecting the reproductive decision-making were analyzed. Patients/parents who agreed on fertility preservation received microdissection testicular sperm extraction (mTESE) and cryopreservation. A comprehensive literature review regarding the SRRs in older adolescents with KS was conducted. A total of eight older adolescents were enrolled. After fertility preservation counseling, three patients/parents (37.5%) agreed to receive mTESE, and spermatozoa were successfully retrieved in two. "Lack of interest" and "inconsistent sperm retrieval result" were the main reasons for refusal. A total of 89 older adolescents from nine articles, and ours were collected for SRR analysis. Most of the reports had a limited number of cases, and none of them described the acceptance rate of sperm retrieval in adolescents. Forty-three out of 89 older adolescents (48.3%) had successful sperm retrieval, and there was no significant difference in the SRR between the mTESE and conventional TESE. Successful testicular sperm retrieval in older adolescents with KS is not superior to those reported in adults. Adolescents and their parents should undergo a detailed reproductive consultation process and shared decision-making discussion before considering testicular sperm retrieval.

P–434 Effect of let–7a mimic as a new pharmaco-protective agent against chemotherapy-induced ovarian damage on subsequent follicular development and oocyte quality using mouse ovarian transplantation model

Abstract Study question What is the long-term impact of let–7a-mimic transfection on oocytes development in new-born mice ovaries exposed to chemotherapy in vitro following transplantation in the kidney? Summary answer The let–7a-mimic restoration protects against chemotherapy-induced ovarian apoptosis and preserves subsequent follicular developmental and acquisition of oocyte maturation competence in mouse. What is known already It is well known that cyclophosphamide and its active metabolites (4-hydroperoxycyclophosphamide, 4-HC) cause irreversible ovarian damage and impair future fertility of cancer survivors. Besides the available fertility preservation options, microRNAs/miRNAs appear to be very attractive and novel targets to prevent theses damage. We showed that miRNAs were dysregulated after exposure to 4-HC in postnatal-day–3 (PND3) ovaries, let–7a being the most downregulated among them. By replacing let–7a function, let–7a-mimic was able to protect mouse follicles against 4-HC in vitro. This previous study suggested that it could preserve the reproductive potential after treatment. However, the impact on subsequent oocytes development is unknown Study design, size, duration PND3 ovaries from C57blxCBAF1 hybrid mice were cultured under 3 conditions: control, chemotherapy for 24h (4-HC/20μΜ/24h), chemotherapy for 24h+let–7a-mimic (4-HC/20μΜ/24h+let–7a-mimic). Nine PND3 ovaries were cultured in the different conditions and then transplanted under the kidney’s capsule of C57blxCBAF1 hybrid adult mice for follicular growth/apoptosis evaluation. Then, 21 ovaries (≥7/condition) were used for oocyte maturation assessment after transplantation and ovarian stimulation. All transplanted mice were observed during 21 days before PND3 ovaries collection. Participants/materials, setting, methods: PND3 ovaries were cultured in vitro using inserts under different conditions. A liposome-based system was used to deliver let–7a-mimic into ovaries and QPCR-assays validated its expression levels after transfection. Apoptosis was evaluated by TUNEL Assay while haematoxylin/eosin staining was used for assessing the follicular morphology, stage and count. The oocyte maturation rate was evaluated at day 21 post-transplantation after gonadotropins injection, mechanical eggs collection and in vitro maturation for 24 hours. Main results and the role of chance The apoptosis assessment confirmed that let–7a-mimic transfection reduced the chemotherapy-induced damage in PND3 ovaries in vitro. The number of primordial follicles was significantly reduced (p &amp;lt; 0.05) compared to control after chemotherapy exposure. However, it was increased in chemo24h+let–7a-mimic compared to chemo24h alone while remaining lower than control (p &amp;gt; 0.05). Accordingly, the number of the transitory follicles reflecting follicular activation was significantly higher in chemo24h compared to control (p &amp;lt; 0.05) and chemo24h+let–7a-mimic but for the last one, the result was not significant. Consequently, chemotherapy induces follicle activation while let–7a restoration tends to slow down this effect. To evaluate the long-term effects of chemotherapy and let–7a-mimic transfection, in vitro exposed PND3 ovaries were transplanted under kidney’s capsule in female adult mice. After 21 days, the ovarian reserve was higher in control, but we observed a slight increase of follicular density in the chemo24+let–7a-mimic compared to chemo24h. Similarly, the percentage of damaged/apoptotic cells was higher in all chemotherapy exposed groups compared to control but the impact was lower after let–7a restoration (12,0% and 28,2% in chemo24+let–7a-mimic and chemo24h, respectively). Importantly, the oocyte maturation rate after transplantation was higher in chemo24h+let–7a-mimic compared to chemo24h (40% versus 18%, respectively), suggesting a preservation of oocytes maturation competence. Limitations, reasons for caution The multiple in vitro/in vivo steps may introduce study bias. Moreover, the oocyte competence and live offspring is currently evaluated. The blastocyst formation and embryo development from oocytes fertilized in vitro, are more relevant parameters for oocyte quality assessment. The birth of healthy animals will confirm let–7a-mimic-transfection safety. Wider implications of the findings: Our previous study demonstrated the anti-apoptotic effect of let–7a restoration in mouse ovaries against chemotherapy. In the current study, we demonstrated a long-term beneficial effect of let–7a restoration strategy on follicular development and oocytes maturation capacity. The results open new perspectives in fertility preservation using pharmacological approach. Trial registration number Not applicable

P–456 Impact of first-line chemotherapy prior to ovarian tissue cryopreservation on primordial follicle activation and survival in pre-pubertal and young adult patients

Abstract Study question How does chemotherapeutic regimen administered prior to ovarian tissue cryopreservation affect the signaling pathways regulating ovarian reserve and follicular survival in pre-pubertal and adult patients? Summary answer Chemotherapy induces key signaling processes of follicle activation and increases apoptosis in quiescent follicle. However, damages were different according to the age of the patient. What is known already Therapeutic regimen can damage the ovarian reserve leading to infertility of cancer survivors. Among fertility preservation options, cortical tissue banking appears to be an attractive alternative for patients who cannot delay their treatment or have already started chemotherapy. Though previous studies showed that first-line chemotherapy may induce follicular damages, the impact on follicle activation signaling pathways in human remains poorly understood. Deciphering the signaling modifications under chemotherapy is critical to have a better understanding of the follicle depletion process. Moreover, only few studies on cryopreserved tissue were conducted in children whereas follicle distribution differs compared to post-pubertal women. Study design, size, duration Cryopreserved ovarian tissue from young adult (16–27 years old, n = 6) and pre-pubertal (3–10 years old, n = 6) cancer patients were used as model. Fragments were thawed and cultured for 24 hours after size homogenization (4x2x0.5 mm). Patients who received chemotherapy before ovarian tissue cryopreservation were compared to non-exposed patients. PI3K/AKT/mTOR and Hippo pathways, as well as follicles and stroma survival, were assessed among the different groups at thawing and after culture. Participants/materials, setting, methods The impact of previous chemotherapy exposure on follicle activation, on the PI3K/AKT/mTOR and Hippo pathways was assessed at thawing and after 24 hours of culture by protein analyses (immunostaining and western blot). Histological analyses (follicular counting, immunostaining and TUNEL staining) were performed at the two timepoints to assess follicle distribution, morphology, stroma structure and apoptosis. Main results and the role of chance: The damage of chemotherapeutic regimen prior to cryopreservation was observed specifically on quiescent follicles after thawing by TUNEL staining in both adult and pre-pubertal patients. Surprisingly, apoptosis occurred more specifically in oocytes of pre-pubertal treated tissue while adult treated patients showed granulosa cells death. After culture, apoptosis was observed in the stroma but healthy follicles were observed in all conditions. Atretic follicles were observed similarly in pre-pubertal and adult cortex previously exposed to chemotherapy while not in the unexposed tissue. Protein analyses showed a higher expression of PI3K and Hippo proteins among all groups at thawing compared to cultured groups while difference was observed between pre-pubertal and adult cortex. At thawing, cortical tissues previously exposed to chemotherapy had a higher expression of phosphorylated forms of AKT and RPS6 compared to untreated groups, irrespective to the age. Moreover, immunostainings showed an oocyte-specific localization of p-AKT while p-RPS6 was more pronounced in the granulosa cells, suggesting an early process of follicle activation. Limitations, reasons for caution This study was limited to the evaluation of two major signaling pathways, PI3K/AKT/mTOR and Hippo. Moreover, considering the scarcity and the heterogeneity of our model, the number of patients included in this study is limited and the results should be interpreted with caution. Wider implications of the findings: Our results highlight the involvement of age and previous chemotherapeutic treatment in the regulation of signaling pathways regulating follicular activation, growth, and survival. Besides sustaining the chemotherapy-induced “burn out effect” theory, it opens perspectives to regulate the deleterious impacts of chemotherapy on follicles though molecular control of the altered pathways. Trial registration number Not applicable

P–474 Psychosocial aspects of fertility preservation options for young cancer patients in the Czech Republic: a qualitative study

Abstract Study question What is the real accessibility of fertility preservation techniques and its perception by patients in the Czech Republic? Summary answer Fertility preservation options are not offered on a routine basis and often are not part of a treatment plan. Patients wish to be adequately informed. What is known already Every year, more than 82.000 people in the Czech Republic develop cancer. The incidence of newly diagnosed cancers in individuals in their reproductive age represents 4.4% of all newly diagnosed oncological diseases. Because the prognosis of treatment in this group of patients is very favourable, the priority in treatment is the emphasis on quality of life after surviving. One of the important parameters of quality of life for many people is the ability to reproduce and the possibility of having a genetic bond to their children. Study design, size, duration This qualitative study was conducted in 2019 and relied on explorative in-depth semi-structured interviews. Participants were chosen through occasional sampling. Participants/materials, setting, methods We interviewed 13 cancer patients/survivors in their reproductive age (aged 21 to 36; 7 females and 6 males); 4 close family members of young cancer patients; and 8 experts from relevant professional fields. Data gathered from semi-structured interviews were analysed by interpretative phenomenological analysis. Main results and the role of chance Cancer was perceived as a threat to life and one’s safety. It causes uncertainty and a feeling of loss of control. It also has a negative impact on a self-concept. Loss of fertility was perceived as a “injury of personality”. The possibility of maintaining fertility has increased the subjectively experienced quality of life. They make decisions under great time pressure, in a mentally demanding situation. They are usually in the early stages of coping with the diagnosis. The main factors that influenced the respondents’ decision were the amount and quality of information, psychological stress and time pressure. Patients and their families are interested in being informed about the risks that the disease and its treatment pose to their reproductive health. They want to be able to decide whether to undergo any of the fertility protection techniques. They want to keep their future open. It is therefore desirable that, within oncofertility, we focus on the process of passing on information and supporting decision-making on issues of fertility and its protection. A tool that could be a good informational platform may be so-called decision aid – a tool used to inform patients about available treatments, along with potential benefits, risks and costs. Limitations, reasons for caution As all qualitative data, our findings cannot be generalized. Selection bias could have occurred because it is likely that those interested and open to sharing participated. Wider implications of the findings: Oncofertility treatment is highly relevant and should be offered and discussed with all patients in their reproductive age. Addressing fertility preservation options should be a part of cancer treatment plan of all these patients. Trial registration number 0

P–734 Parenthood among men diagnosed with cancer in childhood and early adulthood – trends over time in a Danish national cohort

Abstract Study question Is the rate of fatherhood among men diagnosed with cancer in childhood and early adulthood different from men without cancer – have differences changed over time? Summary answer Men diagnosed with cancer had significantly reduced rates of fatherhood compared with undiagnosed men. Rates of fatherhood among the cancer survivors increased markedly over time. What is known already The number of children and young adolescents who survive cancer has steadily increased over the past decades, with a current 5-year survival rate of approximately 80%. Consequently, life circumstances after cancer have gained increasing importance, including the desire among survivors to have children and a family. MAR technologies to aid reproduction among cancer survivors have been developed, and fertility preservation is increasingly a topic being discussed before undergoing cancer treatment. But the potential for fertility preservation differs depending on age at diagnosis and type of cancer. Earlier studies have shown decreased fertility rates among survivors of childhood and adolescent cancer. Study design, size, duration This study is a national, register-based cohort study. Men diagnosed with cancer in childhood and early adulthood (&amp;lt;30 years of age) were registered in the Danish Cancer Register in 1978–2016 (n = 15,600). At time of diagnosis, each cancer-diagnosed man was randomly age-matched with 150 undiagnosed men from the background population within the same birth year. The men were followed in medical registers and socio-demographic population registers until death, migration or end of study December 31st, 2017. Participants/materials, setting, methods Fatherhood among the boys and young men diagnosed with cancer was compared with the age-matched comparison group in all statistical analyses. Cancer diagnoses were categorized as central nervous system (CNS), haematological cancers or solid cancers. Also, analyses were stratified by age at diagnosis (0–9, 10–19, 20–29 years) and year of diagnosis (1978–89, 1990–99, 2000–16). Death was incorporated as a competing risk in all analyses. Main results and the role of chance The study population consisted of 15,600 boys and young men diagnosed with cancer between 1978 and 2016 and 1,386,493 men in the age-matched comparison group. Men surviving CNS cancer had the lowest hazard ratio of fatherhood compared with the age-matched comparison group (HR = 0.64, 95% CI 0.57–0.73), followed by survivors of haematological cancers (HR = 0.90, 95% CI 0.82–0.98) while the highest chance of fatherhood was slightly increased among survivors of solid cancers (HR = 1.13, 95% CI 1.10–1.16). The hazard ratio of becoming a father increased over time. From the first decade to the last decade 30 years later, the hazard ratio of becoming a father increased for solid tumours (HR 0.76, 95% CI 0.72–0.80 to HR 1.07, 95% CI 0.96–1.19), haematological tumours (HR 0.60, 95% CI 0.51–0.71 to HR 0.97, 95% CI 0.76–1.23) and CNS tumours (HR 0.47, 95% CI 0.39–0.58 to HR 1.04, 95% CI 0.56–1.93) compared to the age-matched comparison group. Also, men diagnosed with cancer when aged 20–29 years more likely became fathers over time (HR 0.79, 95% CI 0.74–0.84 to HR 1.09, 95% CI 0.98–1.22). Limitations, reasons for caution The study was based on register data, and information was not available about the men’s fertility potential, whether they had a desire to have children and whether it was possible for them to find a partner. Also, information about fertility preservation, e. g. sperm freezing, could have provided additional insights. Wider implications of the findings: Information and education of male patients diagnosed with cancer about fertility preservation options, and chances to create their own family is crucial. Reassuringly, time trends showed more men with a previous cancer diagnosis becoming fathers in recent years than earlier, reflecting that survival and fertility preservation have improved over time. Trial registration number N/A

P–457 Emergency fertility preservation (FP) in female patients- utilisation rate of the stored eggs/embryos and pregnancy outcomes

Abstract Study question The aim of the study was to determine the percentage of patients returning to use their stored eggs/embryos following FP and their pregnancy outcomes. Summary answer The patient utilisation rate for eggs/embryos was 17% with a live birth rate of 59%. What is known already Fertility preservation is considered as a vital issue for individuals in the reproductive stage of life when their future fertility may be compromised. Increased cancer survival rate and advances in assisted reproductive techniques make this an essential service to offer to patients facing life limiting disease or long-term medical conditions.FP is important to improve the quality of life in cancer survivors. Study design, size, duration A retrospective analysis was performed over a period of ten years between January 2010 to December 2020 in our tertiary unit. A total of 75 patients who underwent FP were identified. Participants/materials, setting, methods Infertility database for embryology and andrology (IDEAS) was used for the data collection and analysis. Patient’s age, reasons for fertility preservation, type of benign/cancer condition, protocol used for controlled ovarian stimulation (COS), dose of the gonadotropins, number of eggs collected, number of eggs/embryos cryopreserved, duration between storage and fertility treatment, pregnancy outcomes were included in the analysis. Main results and the role of chance Seventy-five patients underwent FP during the 10-year study period. The mean age was 30 years (range 17–43). Seventy-two patients (96%) underwent treatment for oncological reasons and the rest (4%) were for gender transition and Crohn’s disease. The most common types of malignancies include breast cancer (36%), Hodgkin’s lymphoma (18%) and cervical cancer (15%). Ninety-two percentage of patients underwent COS with an antagonist cycle, with an average of 10.8 eggs collected. Recombinant follicle stimulating hormone (FSH) was used in 92% of the cycles and human menopausal gonadotropin (HMG) was used in 8%. Fifty-eight percentage were given a maximum dose of 300IU of gonadotropin. The mean yield of eggs was higher in patients with breast cancer (12.62) followed by Hodgkin’s (10.5) and cervical cancer (9.6). Majority (60%) had embryo cryopreservation (82% at blastocyst stage and 18% at day 3 cleavage stage) and the rest (40%) had egg cryopreservation. A total of 17% (12) of patients returned for treatment with a livebirth rate of 59% and miscarriage rate of 8%. One third of livebirths were achieved through surrogacy. The average duration between fertility preservation and return for treatment was 2.4 years. Limitations, reasons for caution During the last 5 years, there has been an increase in the number of young women requiring FP in our unit. These women may require a considerable amount of time to complete their oncological treatment before embarking on pregnancy using their stored eggs/embryos. Wider implications of the findings: As cancer survival rate improves, there will be a likely increase in the utilisation rate for follow up treatment among young women who had FP. The overall awareness of the gonadotoxic effect of cancer therapy and available fertility preservation options among both patients and clinicians needs to be increased. Trial registration number NA

P–436 Identification of ovarian cell subpopulations by multicolor flow cytometry and its potential impact on ovarian reconstruction programs

Abstract Study question How could multicolor flow cytometry (MFC) help to identify ovarian subpopulations that could be used for ovarian reconstruction with isolated follicles? Summary answer MFC is useful to identify ovarian cell subpopulations in the ovarian cortex. What is known already Ovarian tissue cryopreservation is a fertility preservation option for women before gonadotoxic chemo- and/or radiotherapy. However, graft of cryopreserved ovarian tissue must be performed with caution in women suffering from malignancies that may metastasize to the ovaries. For this purpose, functional ovarian tissue qualification is essential to identify ovarian cell subpopulations that could be used for ovary reconstruction in combination with isolated follicles. Furthermore, ischemic tissue damage occurring after the graft is currently another important issue to be resolved for successful ovarian reuse. Study design, size, duration We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Then, we used MFC for the identification of different cell subpopulations in the cell suspension thus obtained. Participants/materials, setting, methods Human ovarian tissues from patients undergoing surgery for polycystic ovary syndrome were used in this study. Biopsies of ovarian cortex (fresh or frozen-thawed) were dissociated using an automated dissociation method. We used FVS780 and SYTO13 markers to gate viable ovarian cells by MFC. Variable markers were chosen to differentiate and identify cell subpopulations among the viable ovarian cells. Main results and the role of chance The dissociation yield was on average 1.59 ± 1.58 x 106 and 0.78 ± 0.72 x 106 viable ovarian cells per 100 mg of fresh (n = 17) and frozen-thawed (n = 43) ovarian cortical tissue, respectively. On average, 35.4 ± 13.1% of viable ovarian cells were CD34 + (n = 61, stromal phenotype). Concerning endothelial phenotype, 7.8 ± 5.5% of CD31+ cells (n = 51) and 5.3 ± 3.6% of CD144+ cells (n = 29) were identified among viable ovarian cells. Vimentin marker is found in 25.6 ± 10.8% of viable ovarian cells (n = 23) and CD326 (EpCAM expression) in 0.6 ± 0.8% (n = 16). Finally, pericyte phenotype (CD34-/Vimentin-/CD31-/CD146+/ CD140b+) was identified in 4.6 ± 4.3% of viable ovarian cells (n = 7). Limitations, reasons for caution We do not know how these ovarian cell subpopulations could be a factor associated or not with time for ovarian function recovery in vivo after ovarian tissue graft and the impact of these ovarian cells on the ovarian microenvironment of an artificial ovary. Wider implications of the findings: Functional qualification of ovarian tissue can be performed by MFC. MFC is a promising tool for ovarian cortex qualification before reuse of cryopreserved ovarian tissue. Cell sorting could be used to separate and isolate cell subpopulations and add these cells with isolated follicles in an ovarian reconstruction program. Trial registration number Not applicable