Role of NR5A1 Gene Mutations in Disorders of Sex Development: Molecular and Clinical Features.

Disorders/differences of sex development (DSDs) are defined as broad, heterogenous groups of congenital conditions characterized by atypical development of genetic, gonadal, or phenotypic sex accompanied by abnormal development of internal and/or external genitalia. NR5A1 gene mutation is one of the principal genetic alterations implicated in causing DSD. This review outlines the role of NR5A1 gene during the process of gonadal development in humans, provides an overview of the molecular and functional characteristics of NR5A1 gene, and discusses potential clinical phenotypes and additional organ diseases due to NR5A1 mutations. NR5A1 mutations were analyzed in patients with 46,XY DSD and 46,XX DSD both during the neonatal and pubertal periods. Loss of function of the NR5A1 gene causes several different phenotypes, including some associated with disease in additional organs. Clinical phenotypes may vary, even among patients carrying the same NR5A1 variant, indicating that there is no specific genotype-phenotype correlation. Genetic tests are crucial diagnostic tools that should be used early in the diagnostic pathway, as early as the neonatal period, when gonadal dysgenesis is the main manifestation of NR5A1 mutation. NR5A1 gene mutations could be mainly associated with amenorrhea, ovarian failure, hypogonadism, and infertility during puberty. Fertility preservation techniques should be considered as early as possible.

Fertility preservation before and after cancer treatment in children, adolescents, and young adults.

Fertility is a top concern for many survivors of cancer diagnosed as children, adolescents and young adults (CAYA). Fertility preservation (FP) treatments are effective, evidence-based interventions to support their family building goals. Fertility discussions are a part of quality oncology care throughout the cancer care continuum. For nearly 2 decades, clinical guidelines recommend counseling patients about the possibility of infertility promptly at diagnosis and offering FP options and referrals as indicated. Multiple guidelines now recommend post-treatment counseling. Infertility risks differ by cancer treatments and age, rendering risk stratification a central part of FP care. To support FP decision-making, online tools for female risk estimation are available. At diagnosis, females can engage in mature oocyte/embryo cryopreservation, ovarian tissue cryopreservation, ovarian suppression with GnRH agonists, in vitro oocyte maturation, and/or conservative management for gynecologic cancers. Post-treatment, several populations may consider undergoing oocyte/embryo cryopreservation. Male survivors' standard of care FP treatments center on sperm cryopreservation before cancer treatment and do not have the same post-treatment indication for additional gamete cryopreservation. In practice, FP care requires systemized processes to routinely screen for FP needs, bridge oncology referrals to fertility, offer timely fertility consultations and access to FP treatments, and support financial navigation. Sixteen US states passed laws requiring health insurers to provide insurance benefits for FP treatments, but variation among the laws and downstream implementation are barriers to accessing FP treatments. To preserve the reproductive futures of CAYA survivors, research is needed to improve risk stratification, FP options, and delivery of FP care.

Fertility in young-onset colorectal patients with cancer: a review.

Although the overall incidence and mortality of colorectal cancer have declined, diagnosed cases of young-onset colorectal cancer have increased significantly. Concerns about future fertility are second only to concerns about survival and may significantly affect the quality of life of young cancer survivors. Fertility preservation is an important issue in young-onset colorectal patients with cancer undergoing oncotherapy. Here, we discussed the effects of different treatments on fertility, common options for fertility preservation, factors affecting fertility preservation and improvement measures, and the relationship between fertility and pregnancy outcomes in young-onset colorectal patients with cancer.

Characterizing attitudes related to future child-bearing in young women diagnosed with early-stage breast cancer.

PURPOSE: This study characterizes attitudes and decision-making around the desire for future children in young women newly diagnosed with early-stage breast cancer and assesses how clinical factors and perceived risk may impact these attitudes. METHODS: This is a prospective study in women < 45 years with newly diagnosed stage 1-3 breast cancer. Patients completed a REDCap survey on fertility and family-building in the setting of hypothetical risk scenarios. Patient, tumor, and treatment characteristics were collected through surveys and medical record. RESULTS: Of 140 study patients [median age = 41.4 (range 23-45)], 71 (50.7%) were interested in having children. Women interested in future childbearing were younger than those who were not interested (mean = 35.2 [SD = 5.2] vs 40.9 years [3.90], respectively, p < 0.001), and more likely to be childless (81% vs 31%, p < 0.001). 54 women (77.1% of patients interested in future children) underwent/planned to undergo oocyte/embryo cryopreservation before chemotherapy. Interest in future childbearing decreased with increasing hypothetical recurrence risk, however 17% of patients wanted to have children despite a 75-100% hypothetical recurrence risk. 24.3% of patients wanted to conceive < 2 years from diagnosis, and 35% of patients with hormone receptor positive tumors were not willing to complete 5 years of hormone therapy. CONCLUSION: Many young women diagnosed with early-stage breast cancer prioritize childbearing. Interest in having a biologic child was not associated with standard prognostic risk factors. Interest decreased with increasing hypothetical recurrence risk, though some patients remained committed to future childbearing despite near certain hypothetical risk. Individual risk assessment should be included in family-planning discussions throughout the continuum of care as it can influence decision-making.

Microwave-assisted dehydration, long-term storage at non-freezing temperatures, and rehydration of cat germinal vesicles.

Germinal vesicles (GVs) are alternative targets for female fertility preservation due to their availability and high resilience against non-physiological conditions. Preserved GVs can then be transferred to fresh cytoplasts to reconstitute viable oocytes. Here, we describe a GV preservation method that employs non-ionizing microwave radiations imparting energy to water molecules, which results in rapid and homogeneous drying of the sample. Trehalose is added as a xero-protectant before the radiations, enabling isothermal vitrification of the disaccharide sugar during drying. While the technique is still considered experimental, studies have shown that DNA and structural integrity can be effectively maintained in dried/rehydrated GVs. Importantly, the dry-preservation approach allows supra-zero temperature storage of the samples, offering a cost-effective and energy-saving alternative to traditional methods relying on ultra-low freezing temperatures. The protocol outlines a comprehensive procedure involving GV oocyte collection, trehalose loading, microwave drying, storage, and rehydration. The simplicity of the protocol facilitates ease of manipulation, making it an accessible method for researchers. While initially developed for domestic cats, the protocol can be adapted for other species with necessary modifications, considering potential species-specific responses to dehydration stress.

Prediction of complete regression in fertility-sparing patients with endometrial cancer and apical hyperplasia: the GLOBAL model in a large Chinese cohort.

BACKGROUND: Fertility preservation treatment is increasingly essential for patients with apical endometrial hyperplasia (AEH) and early endometrial cancer (EEC) worldwide. Complete regression (CR) is the main endpoint of this treatment. Accurately predicting CR and implementing appropriate interventions during treatment are crucial for these patients. METHODS: We conducted a retrospective study involving 193 patients diagnosed with atypical AEH or EEC, enrolled from January 2012 to March 2022 at our center. We evaluated 24 clinical parameters as candidate predictors and employed LASSO regression to develop a prediction model for CR. Subsequently, a nomogram was constructed to predict CR after the treatment. We evaluated the performance of the nomogram using receiver operator characteristic (ROC) curve and decision curve analysis (DCA) to assess its predictive accuracy. Additionally, we employed cumulative curves to determine the CR rate among patients. RESULTS: Out of the 193 patients, 173 achieved CR after undergoing fertility preservation treatment. We categorized features with similar properties and provided a list of formulas based on their coefficients. The final model, named GLOBAL (including basic information, characteristics, blood pressure, glucose metabolism, lipid metabolism, immunohistochemistry, histological type, and medication), comprised eight variables identified using LASSO regression. A nomogram incorporating these eight risk factors was developed to predict CR. The GLOBAL model exhibited an AUC of 0.907 (95% CI 0.828-0.969). Calibration plots demonstrated a favorable agreement between the predicted probability by the GLOBAL model and actual observations in the cohort. The cumulative curve analysis revealed varying cumulative CR rates among patients in the eight subgroups. Categorized analysis demonstrated significant diversity in the effects of the GLOBAL model on CR among patients with different total points (p < 0.05). CONCLUSION: We have developed and validated a model that significantly enhances the predictive accuracy of CR in AEH and EEC patients seeking fertility preservation treatment.

Laparoscopic-Assisted Vaginal Trachelectomy with Prophylactic Cerclage: A Safe Fertility-Sparing Treatment for Early Stage Cervical Cancer.

BACKGROUND: In recent years fertility-sparing treatments are increasingly developing in patients with early stage cervical cancer.1,2 Among these, trachelectomy represents a milestone with a wide range of surgical approaches,3 evidence of oncological safety, and positive obstetric outcomes.4 PATIENTS AND METHODS: A 26-year-old patient underwent conization for CIN3 with a subsequent diagnosis of squamous cervical cancer stage FIGO IB1. After a negative laparoscopic bilateral pelvic nodes sampling and the radiologic evidence [positron emission tomography-computed tomography (PET-CT) and magnetic resonance imaging (MRI)] of a disease limited to the cervix, the patient was a candidate for trachelectomy according to her fertility-sparing desire. RESULTS: The first laparoscopic time is dedicated to the safe opening of the vesicouterine and rectovaginal spaces until the medial pararectal fossa. Ureters are found and bilateral ureterolysis performed under vision. Colpotomy is then vaginally achieved, and the cervix is closed in a vaginal cuff to avoid tumor spread. Careful dissection of the anterior and posterior septa is carried out until reunification with laparoscopic dissection. Bilateral parametrectomy is performed. Vaginal trachelectomy is finalized with a negative deep margin at the frozen section. In the second laparoscopic time a monofilament polypropylene sling cerclage is bilaterally positioned from posterior to anterior through the broad ligaments and fixed anteriorly on the uterine isthmus to prevent an eventual preterm delivery. CONCLUSION: Laparoscopic-assisted vaginal trachelectomy is a feasible procedure combining the conservative advantages of the vaginal approach and the oncological safety of laparoscopic spaces dissection with possible good obstetric outcomes.

Evaluating testicular tissue for future autotransplantation: focus on cancer cell contamination and presence of spermatogonia in tissue cryobanked for boys diagnosed with a hematological malignancy.

STUDY QUESTION: What is the contamination rate by cancer cells and spermatogonia numbers in immature testicular tissue (ITT) harvested before the start of gonadotoxic therapy in boys with a hematological malignancy? SUMMARY ANSWER: Among our cohort of boys diagnosed with acute lymphoblastic leukemia (ALL) and lymphomas, 39% (n = 11/28) had cancer cells identified in their tissues at the time of diagnosis and all patients appeared to have reduced spermatogonia numbers compared to healthy reference cohorts. WHAT IS KNOWN ALREADY: Young boys affected by a hematological cancer are at risk of contamination of their testes by cancer cells but histological examination is unable to detect the presence of only a few cancer cells, which would preclude autotransplantation of cryobanked ITT for fertility restoration, and more sensitive detection techniques are thus required. Reduced numbers of spermatogonia in ITT in hematological cancer patients have been suggested based on results in a limited number of patients. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 54 pre- and peri-pubertal boys who were diagnosed with a hematological malignancy and who underwent a testicular biopsy for fertility preservation at the time of diagnosis before any gonadotoxic therapy between 2005 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Among the 54 patients eligible in our database, formalin-fixed paraffin-embedded (FFPE) testicular tissue was available for 28 boys diagnosed either with ALL (n = 14) or lymphoma (n = 14) and was used to evaluate malignant cell contamination. Hematoxylin and eosin (H&E) staining was performed for each patient to search for cancer cells in the tissue. Markers specific to each patient's disease were identified at the time of diagnosis on the biopsy of the primary tumor or bone marrow aspiration and an immunohistochemistry (IHC) was performed on the FFPE ITT for each patient to evidence his disease markers. PCR analyses on the FFPE tissue were also conducted when a specific gene rearrangement was available. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age at diagnosis and ITT biopsy of the 28 boys was 7.5 years (age range: 19 months-16 years old). Examination of ITT of the 28 boys on H&E stained sections did not detect malignant cells. Using IHC, we found contamination by cancerous cells using markers specific to the patient's disease in 10 of 28 boys, with a higher rate in patients diagnosed with ALL (57%, n = 8/14) compared with lymphoma (14%, n = 2/14) (P-value < 0.05). PCR showed contamination in three of 15 patients who had specific rearrangements identified on their bone marrow at the time of diagnosis; one of these patients had negative results from the IHC. Compared to age-related reference values of the number of spermatogonia per ST (seminiferous tubule) (Spg/ST) throughout prepuberty of healthy patients from a simulated control cohort, mean spermatogonial numbers appeared to be decreased in all age groups (0-4 years: 1.49 ± 0.54, 4-7 years: 1.08 ± 0.43, 7-11 years: 1.56 ± 0.65, 11-14 years: 3.37, 14-16 years: 5.44 ± 3.14). However, using a cohort independent method based on the Z-score, a decrease in spermatogonia numbers was not confirmed. LIMITATIONS, REASONS FOR CAUTION: The results obtained from the biopsy fragments that were evaluated for contamination by cancer cells may not be representative of the entire cryostored ITT and tumor foci may still be present outside of the biopsy range. WIDER IMPLICATIONS OF THE FINDINGS: ITT from boys diagnosed with a hematological malignancy could bear the risk for cancer cell reseeding in case of autotransplantation of the tissue. Such a high level of cancer cell contamination opens the debate of harvesting the tissue after one or two rounds of chemotherapy. However, as the safety of germ cells can be compromised by gonadotoxic treatments, this strategy warrants for the development of adapted fertility restoration protocols. Finally, the impact of the hematological cancer on spermatogonia numbers should be further explored. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by a grant from the FNRS-Télévie (grant n°. 7.4533.20) and Fondation Contre le Cancer/Foundation Against Cancer (2020-121) for the research project on fertility restoration with testicular tissue from hemato-oncological boys. The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

STUDY QUESTION: What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? SUMMARY ANSWER: Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. WHAT IS KNOWN ALREADY: Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. STUDY DESIGN, SIZE, DURATION: Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 µM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. LIMITATIONS, REASONS FOR CAUTION: These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. TRIAL REGISTRATION NUMBER: N/A.

Experiences and intentions of patients undergoing medically indicated oocyte or embryo cryopreservation: a qualitative study.

STUDY QUESTION: What structural (logistical) and psychological challenges do patients who cryopreserve oocytes or embryos for medical reasons face, including possible barriers to using their frozen materials? SUMMARY ANSWER: The majority of women who underwent oocyte or embryo cryopreservation for medical reasons reported a desire to use their frozen oocytes or embryos but had been impeded by ongoing medical issues, the need for a gestational carrier, or the lack of a partner. WHAT IS KNOWN ALREADY: Current data suggest that many women who have frozen oocytes or embryos for medical indications are concerned about the prospect of infertility and have unique emotional and financial needs that differ from patients with infertility. Further, most patients have not returned to use their cryopreserved materials. STUDY DESIGN, SIZE, DURATION: This is a qualitative interview study of 42 people who cryopreserved between January 2012 and December 2021. Interviews were conducted between March 2021 and March 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants were cisgender women who had undergone oocyte or embryo cryopreservation for medical indications at an academic fertility center. Participants were invited to interview by email if they were younger than 40 years old when their oocytes or embryos were cryopreserved. Interviews were conducted over the internet and transcribed verbatim. Data were analyzed using thematic analysis with the constant comparison method. MAIN RESULTS AND THE ROLE OF CHANCE: Saturation was reached at 42 interviews. The median age of participants was 35 years old (range 28-43) at interview and 31 years old (range 25-39) at cryopreservation. Of the 42 women, 30 had a cancer diagnosis, while 7 had non-cancer chronic medical conditions, and 5 had hereditary cancer susceptibility syndromes. There were 12 women who banked embryos and 30 who banked oocytes. The majority of women indicated a desire to use their cryopreserved materials, but many were unsure about how or when. Four had already used their frozen oocytes or embryos, while another four had conceived without assisted reproduction. The cryopreservation experience was described by the majority as highly emotionally challenging because they felt out of place among couples receiving infertility treatment and, for cancer patients, overwhelmed by the complex decisions to be made in a short time period. Common reported barriers to using frozen materials included ongoing medical issues preventing pregnancy, the need for a gestational carrier, the lack of a partner, and the desire for unassisted conception. Some were glad to have frozen oocytes or embryos to allow more time to meet a partner or if they were considering becoming single parents. LIMITATIONS, REASONS FOR CAUTION: The majority of participants had their oocytes or embryos frozen at a single, urban, academic fertility center, which may limit generalizability. We also could not calculate a response rate because the snowball technique was used to identify additional participants, so did not know the total number of people invited to participate. Like other interview studies, our study may be subject to response bias because those who agreed to participate may have particularly positive or negative views about their experiences. Furthermore, the mean follow-up time since freezing was relatively short (3.3 years, median 2.7 years), which may not have been enough time for some patients to use their frozen materials. WIDER IMPLICATIONS OF THE FINDINGS: Learning about the experiences of patients undergoing medically indicated oocyte and embryo cryopreservation can help clinicians better counsel these patients regarding decisions and hurdles they may encounter. We found that most patients had not returned to use their frozen materials because of ongoing medical issues, the need for a gestational carrier, lack of a partner, or the desire to attempt unassisted reproduction. STUDY FUNDING/COMPETING INTEREST(S): This study did not receive any funding. The authors of this study have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Rapamycin prevents cyclophosphamide-induced ovarian follicular loss and potentially inhibits tumour proliferation in a breast cancer xenograft mouse model.

STUDY QUESTION: To what extent and via what mechanism does the concomitant administration of rapamycin (a follicle activation pathway inhibitor and antitumour agent) and cyclophosphamide (a highly toxic ovarian anticancer agent) prevent cyclophosphamide-induced ovarian reserve loss and inhibit tumour proliferation in a breast cancer xenograft mouse model? SUMMARY ANSWER: Daily concomitant administration of rapamycin and a cyclic regimen of cyclophosphamide, which has sufficient antitumour effects as a single agent, suppressed cyclophosphamide-induced primordial follicle loss by inhibiting primordial follicle activation in a breast cancer xenograft mouse model, suggesting the potential of an additive inhibitory effect against tumour proliferation. WHAT IS KNOWN ALREADY: Cyclophosphamide stimulates primordial follicles by activating the mammalian target of the rapamycin (mTOR) pathway, resulting in the accumulation of primary follicles, most of which undergo apoptosis. Rapamycin, an mTOR inhibitor, regulates primordial follicle activation and exhibits potential inhibitory effects against breast cancer cell proliferation. STUDY DESIGN, SIZE, DURATION: To assess ovarian follicular apoptosis, 3 weeks after administering breast cancer cells, 8-week-old mice were randomized into three treatment groups: control, cyclophosphamide, and cyclophosphamide + rapamycin (Cy + Rap) (n = 5 or 6 mice/group). Mice were treated with rapamycin or vehicle control for 1 week, followed by a single dose of cyclophosphamide or vehicle control. Subsequently, the ovaries were resected 24 h after cyclophosphamide administration (short-term treatment groups). To evaluate follicle abundance and the mTOR pathway in ovaries, as well as the antitumour effects and impact on the mTOR pathway in tumours, 8-week-old xenograft breast cancer transplanted mice were randomized into three treatment groups: vehicle control, Cy, and Cy + Rap (n = 6 or 7 mice/group). Rapamycin (5 mg/kg) or the vehicle was administered daily for 29 days. Cyclophosphamide (120 mg/kg) or the vehicle was administered thrice weekly (long-term treatment groups). The tumour diameter was measured weekly. Seven days after the last cyclophosphamide treatment, the ovaries were harvested, fixed, and sectioned (for follicle counting) or frozen (for further analysis). Similarly, the tumours were resected and fixed or frozen. PARTICIPANTS/MATERIALS, SETTING, METHODS: Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed to examine ovarian follicular apoptosis in the short-term treatment groups. All subsequent experiments were conducted in the long-term treatment groups. Tumour growth was evaluated using the tumour volume index. The tumour volume index indicates the relative volume, compared to the volume 3 weeks after tumour cell injection (at treatment initiation) set to 100%. Tumour cell proliferation was evaluated by Ki-67 immunostaining. Activation of the mTOR pathway in tumours was assessed using the protein extracts from tumours and analysed by western blotting. Haematoxylin and eosin staining of ovaries was used to perform differential follicle counts for primordial, primary, secondary, antral, and atretic follicles. Activation of the mTOR pathway in ovaries was assessed using protein extracts from whole ovaries and analysed by western blotting. Localization of mTOR pathway activation within ovaries was assessed by performing anti-phospho-S6 kinase (downstream of mTOR pathway) immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Ovaries of the short-term treatment groups were resected 24 h after cyclophosphamide administration and subjected to TUNEL staining of apoptotic cells. No TUNEL-positive primordial follicles were detected in the control, Cy, and Cy + Rap groups. Conversely, many granulosa cells of growing follicles were TUNEL positive in the Cy group but negative in the control and Cy + Rap groups. All subsequent experimental results were obtained from the long-term treatment groups. The tumour volume index stabilized at a mean of 160-200% in the Cy group and 130% in the Cy + Rap group throughout the treatment period. In contrast, tumours in the vehicle control group grew continuously with a mean tumour volume index of 600%, significantly greater than that of the two treatment groups. Based on the western blot analysis of tumours, the mTOR pathway was activated in the vehicle control group and downregulated in the Cy + Rap group when compared with the control and Cy groups. Ki-67 immunostaining of tumours showed significant inhibition of cell proliferation in the Cy + Rap group when compared with that in the control and Cy groups. The ovarian follicle count revealed that the Cy group had significantly fewer primordial follicles (P < 0.001) than the control group, whereas the Cy + Rap group had significantly higher number of primordial follicles (P < 0.001, 2.5 times) than the Cy group. The ratio of primary to primordial follicles was twice as high in the Cy group than in the control group; however, no significant difference was observed between the control group and the Cy + Rap group. Western blot analysis of ovaries revealed that the mTOR pathway was activated by cyclophosphamide and inhibited by rapamycin. The phospho-S6 kinase (pS6K)-positive primordial follicle rate was 2.7 times higher in the Cy group than in the control group. However, this effect was suppressed to a level similar to the control group in the Cy + Rap group. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The combinatorial treatment of breast cancer tumours with rapamycin and cyclophosphamide elicited inhibitory effects on cell proliferative potential compared to cyclophosphamide monotherapy. However, no statistically significant additive effect was observed on tumour volume. Thus, the beneficial antitumour effect afforded by rapamycin administration on breast cancer could not be definitively proven. Although rapamycin has ovarian-protective effects, it does not fully counteract the ovarian toxicity of cyclophosphamide. Nevertheless, rapamycin is advantageous as an ovarian protective agent as it can be used in combination with other ovarian protective agents, such as hormonal therapy. Hence, in combination with other agents, mTOR inhibitors may be sufficiently ovario-protective against high-dose and cyclic cyclophosphamide regimens. WIDER IMPLICATIONS OF THE FINDINGS: Compared with a cyclic cyclophosphamide regimen that replicates human clinical practice under breast cancer-bearing conditions, the combination with rapamycin mitigates the ovarian follicle loss of cyclophosphamide without interfering with the anticipated antitumour effects. Hence, rapamycin may represent a new non-invasive treatment option for cyclophosphamide-induced ovarian dysfunction in breast cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This work was not financially supported. The authors declare that they have no conflict of interest.

Eggsurance? A randomized controlled trial of a decision aid for elective egg freezing.

STUDY QUESTION: Does a purpose-designed Decision Aid for women considering elective egg freezing (EEF) impact decisional conflict and other decision-related outcomes? SUMMARY ANSWER: The Decision Aid reduces decisional conflict, prepares women for decision-making, and does not cause distress. WHAT IS ALREADY KNOWN: Elective egg-freezing decisions are complex, with 78% of women reporting high decisional conflict. Decision Aids are used to support complex health decisions. We developed an online Decision Aid for women considering EEF and demonstrated that it was acceptable and useful in Phase 1 testing. STUDY DESIGN, SIZE, DURATION: A single-blind, two-arm parallel group randomized controlled trial was carried out. Target sample size was 286 participants. Randomization was 1:1 to the control (existing website information) or intervention (Decision Aid plus existing website information) group and stratified by Australian state/territory and prior IVF specialist consultation. Participants were recruited between September 2020 and March 2021 with outcomes recorded over 12 months. Data were collected using online surveys and data collection was completed in March 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Females aged ≥18 years, living in Australia, considering EEF, proficient in English, and with internet access were recruited using multiple methods including social media posts, Google advertising, newsletter/noticeboard posts, and fertility clinic promotion. After completing the baseline survey, participants were emailed their allocated website link(s). Follow-up surveys were sent at 6 and 12 months. Primary outcome was decisional conflict (Decisional Conflict Scale). Other outcomes included distress (Depression Anxiety and Stress Scale), knowledge about egg freezing and female age-related infertility (study-specific measure), whether a decision was made, preparedness to decide about egg freezing (Preparation for Decision-Making Scale), informed choice (Multi-Dimensional Measure of Informed Choice), and decision regret (Decision Regret Scale). MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 306 participants (mean age 30 years; SD: 5.2) were randomized (intervention n = 150, control n = 156). Decisional Conflict Scale scores were significantly lower at 12 months (mean score difference: -6.99 [95% CI: -12.96, -1.02], P = 0.022) for the intervention versus control group after adjusting for baseline decisional conflict. At 6 months, the intervention group felt significantly more prepared to decide about EEF than the control (mean score difference: 9.22 [95% CI: 2.35, 16.08], P = 0.009). At 12 months, no group differences were observed in distress (mean score difference: 0.61 [95% CI: -3.72, 4.93], P = 0.783), knowledge (mean score difference: 0.23 [95% CI: -0.21, 0.66], P = 0.309), or whether a decision was made (relative risk: 1.21 [95% CI: 0.90, 1.64], P = 0.212). No group differences were found in informed choice (relative risk: 1.00 [95% CI: 0.81, 1.25], P = 0.983) or decision regret (median score difference: -5.00 [95% CI: -15.30, 5.30], P = 0.337) amongst participants who had decided about EEF by 12 months (intervention n = 48, control n = 45). LIMITATIONS, REASONS FOR CAUTION: Unknown participant uptake and potential sampling bias due to the recruitment methods used and restrictions caused by the coronavirus disease 2019 pandemic. Some outcomes had small sample sizes limiting the inferences made. The use of study-specific or adapted validated measures may impact the reliability of some results. WIDER IMPLICATIONS OF THE FINDINGS: This is the first randomized controlled trial to evaluate a Decision Aid for EEF. The Decision Aid reduced decisional conflict and improved women's preparation for decision making. The tool will be made publicly available and can be tailored for international use. STUDY FUNDING/COMPETING INTEREST(S): The Decision Aid was developed with funding from the Royal Women's Hospital Foundation and McBain Family Trust. The study was funded by a National Health and Medical Research Council (NHMRC) Project Grant APP1163202, awarded to M. Hickey, M. Peate, R.J. Norman, and R. Hart (2019-2021). S.S., M.P., D.K., and S.B. were supported by the NHMRC Project Grant APP1163202 to perform this work. R.H. is Medical Director of Fertility Specialists of Western Australia and National Medical Director of City Fertility. He has received grants from MSD, Merck-Serono, and Ferring Pharmaceuticals unrelated to this study and is a shareholder of CHA-SMG. R.L. is Director of Women's Health Melbourne (Medical Practice), ANZSREI Executive Secretary (Honorary), RANZCOG CREI Subspecialty Committee Member (Honorary), and a Fertility Specialist at Life Fertility Clinic Melbourne and Royal Women's Hospital Public Fertility Service. R.A.A. has received grants from Ferring Pharmaceuticals unrelated to this study. M.H., K.H., and R.J.N. have no conflicts to declare. TRIAL REGISTRATION NUMBER: ACTRN12620001032943. TRIAL REGISTRATION DATE: 11 August 2020. DATE OF FIRST PATIENT'S ENROLMENT: 29 September 2020.

Fertility preservation before cancer treatment: the dilemma of saying 'no' as the price of glory.

A great deal of work has been carried out by professionals in reproductive medicine in order to raise awareness about fertility preservation (FP) techniques, particularly for women, and to ensure that FP is included in the care of young adults treated for cancer or a pathology requiring gonadotoxic treatment. If the importance of the development of our discipline is obvious, our militancy in favour of FP and our emotional projections must not make us forget that medical thinking must be carried out not only on a case-by-case basis, weighing up the benefit-risk balance, but also without losing sight that conceiving a child with one's own gametes is not a vital issue. The cultural importance given to the genetic link with offspring may bias patients' and physicians' decisions, while other ways of achieving parenthood exist, and are often more effective. Systematic information should be provided on the existence of FP techniques, but this should not lead to their systematic implementation, nor should it obscure that early information will also allow patients to begin projecting themselves in alternative options to become parents.

Robotic versus vaginal radical trachelectomy for reproductive-aged patients with early-stage cervical carcinoma: A multi-center cohort study.

BACKGROUND: A randomized non-inferiority trial showed worse survival in women with early-stage cervical cancer treated with radical hysterectomy by minimally invasive approach compared to laparotomy; the impact of surgical approach on survival following radical trachelectomy is unknown. OBJECTIVE: To examine oncologic outcomes in women with early-stage cervical cancer who underwent robotic or vaginal radical trachelectomy at Canadian cancer centers with the highest volumes of radical trachelectomy procedures. STUDY DESIGN: Retrospective multi-centre cohort analysis which includes patients who had surgery between 2006 and 2019. Women with International FIGO 2009 stage IA-IB cervical cancer who underwent radical trachelectomy and lymph node assessment were grouped by surgical approach (vaginal versus robotic surgery). RESULTS: A total of 197 patients were included from 4 regional referral centres. 56 women underwent robotic radical trachelectomy and 141 underwent vaginal radical trachelectomy. All patients had lymph node assessment by a minimally invasive technique. Median age was 32 years, median tumor size was 12 mm, and median depth of invasion was 5 mm. Recurrence-free survival was 97% in both groups at a median follow-up of 57 months. On multivariable analysis, after adjusting for previously chosen confounders (high risk pathologic criteria, tumor size, and LVSI) there was no statistically significant difference in PFS between the 2 groups (HR 2.1, 95%CI 0.3-7.1, p = 0.5). Tumor size larger than 2 cm (HR 9.4, 95%CI 2.8-26, p = 0.003) was the only variable predictive of recurrence. CONCLUSION: Survival outcomes were excellent in both cohorts of patients undergoing robotic vs. vaginal radical trachelectomy. The surgical approach was not significantly associated with risk of recurrence after adjusting for clinically important confounders.

Reducing radicality in fertility-sparing surgery is associated with improved in vitro fertilization outcome in early-stage cervical cancer: A national retrospective study.

OBJECTIVE: Fertility-sparing surgery (FSS) aims to achieve oncological outcomes that are non-inferior to radical treatment while preserving fertility and optimizing reproductive results. This study assesses in vitro fertilization (IVF) outcomes in early-stage cervical cancer survivors following FSS, comparing radical and non-radical approaches. METHODS: This retrospective analysis used data from Hungary's National Health Insurance Fund (2004-2022) on patients who underwent IVF treatment following FSS for early-stage cervical cancer at ten Hungarian fertility clinics. Patients were classified into radical and non-radical surgical groups, with the uterine arteries being spared in the non-radical procedures. RStudio (R software version: 4.2.2) was used for statistical analysis. Student's t-test was used to compare group means, and Fisher's exact test was applied to assess independence and distributions between categorical variables, and to estimate odds. RESULTS: The study analyzed data from 122 IVF treatment cycles involving 36 patients. The non-radical group had a significantly higher live birth rate (83%, 5/6 compared to the radical group (17%, 5/30). Additionally, the non-radical group had a significantly higher implantation rate and cumulative live birth rate per oocyte retrieval (37%, 7/19 and 55%, 6/11 respectively) compared to the radical group (8%, 12/148 and 6%, 5/80 respectively). CONCLUSION: This is the largest study to evaluate IVF outcomes in young cervical cancer survivors who have undergone FSS. The findings suggest that less radical procedures are associated with significantly better IVF outcomes. These results emphasize the importance of considering oncological safety and reproductive outcomes together when choosing FSS for early-stage cervical cancer patients. It also highlights the reproductive benefits of performing less radical surgery.

What reproductive follow-up for adolescent and young women after cancer? A review.

Fertility capacity has been shown to be one of the main concerns of young cancer survivors. Gonadotoxic treatments may lead to both premature ovarian failure and/or infertility. This review aimed to define which, and when, reproductive indicators should be followed-up to help doctors to counsel patients regarding their fertility and ovarian function, and to determine if a second stage of fertility preservation after the end of cancer treatment is clinically relevant. Longitudinal assessment of anti-Müllerian hormone (AMH) concentrations during cancer treatment indicates the degree of follicular depletion, and allows discrimination between low and high gonadotoxic treatments. Sustained low AMH concentrations after treatment, especially in the case of alkylating protocols, may reduce the duration of the conception window significantly, and expose the patient to the risk of premature ovarian failure. It remains unknown whether this may impact further fertility capacity because of the lack of systematic follow-up of adolescent and young adult (AYA) women after chemo-radiotherapy. It appears that dedicated reproductive follow-up of AYA women under cancer treatment is needed to refine fertility preservation strategies, and to determine if low AMH concentrations after treatment impact the chance of pregnancy in this specific survivor population.

Cryopreservation of ovarian tissue for fertility preservation in breast cancer patients: time to stop?

Fertility preservation is currently offered to young women with breast cancer to increase their chances of motherhood after a potentially gonadotoxic treatment. Ovarian stimulation with oocyte vitrification and cryopreservation of ovarian tissue remain the most commonly used methods of choice. Whichever method is preferred is very much dependent on the practice and experience of the clinics, although for breast cancer in particular one method might be superior to the other. Cryopreservation of ovarian tissue is inevitably associated with the iatrogenic reduction of the ovarian reserve of a patient and should only be offered to women with a high risk of premature ovarian insufficiency following treatment. However, for younger breast cancer survivors, pregnancy and delivery rates are reassuringly high, even after chemotherapy. Despite its widespread use, few women come back to make use of their cryopreserved tissue. It is argued here that cryopreservation of ovarian tissue is not an appropriate option for breast cancer patients and discuss the reasons for this opinion.

A compact, high-throughput semi-automated embryo vitrification system based on hydrogel.

RESEARCH QUESTION: What is the efficiency and efficacy of the novel Biorocks semi-automated vitrification system, which is based on a hydrogel? DESIGN: This comparative experimental laboratory study used mouse model and human day 6 blastocysts. Mouse oocytes and embryos were quality assessed post-vitrification. RESULTS: The Biorocks system successfully automated the solution exchanges during the vitrification process, achieving a significantly improved throughput of up to 36 embryos/oocytes per hour. Using hydrogel for cryoprotective agent delivery, 12 vessels could be processed simultaneously, fitting comfortably within an assisted reproductive technology (ART) workstation. In tests involving the cryopreservation of oocytes and embryos, the system yielded outcomes equivalent to the manual Cryotop method. For example, the survival rate for mouse oocytes was 98% with the Biorocks vitrification system (n = 46) and 95% for the manual Cryotop method (n = 39), of which 46% and 41%, respectively, progressed to blastocysts on day 5 after IVF. CC-grade day 6 human blastocysts processed with the Biorocks system (n = 39) were associated with a 92% 2 h re-expansion rate, equivalent to the 90% with Cryotop (n = 30). The cooling/warming rates achieved by the Biorocks system were 31,900°C/minute and 24,700°C/minute, respectively. Oocyte quality was comparable or better post-vitrification for Biorocks than Cryotop. CONCLUSIONS: The Biorocks semi-automated vitrification system offers enhanced throughput without compromising the survival and developmental potential of oocytes and embryos. This innovative system may help to increase the efficiency and standardization of vitrification in ART clinics. Further investigations are needed to confirm its efficacy in a broader clinical context.

Effect of various contraceptives on oocyte yield and maturation in patients undergoing planned oocyte cryopreservation.

RESEARCH QUESTION: Do the various forms of hormonal and non-hormonal contraceptives have any association with ovarian stimulation outcomes, such as oocyte yield and maturation, in patients undergoing planned oocyte cryopreservation (POC)? DESIGN: This retrospective cohort study included all patients who underwent POC cycles between 2011 and 2023. The use of types of contraception before a POC cycle was recorded. The study evaluated the median number of cumulus-oocyte complexes obtained after vaginal oocyte retrieval and the proportion of metaphase II oocytes that underwent vitrification among all the cohorts. RESULTS: A total of 4059 oocyte freezing cycles were included in the analysis. Eight types of contraceptive method were recognized in patients undergoing ovarian stimulation: intrauterine device (IUD), copper (n = 84); IUD, levonorgestrel low dose (<52 mg) (n = 37); IUD, levonorgestrel (n = 192); subdermal etonogestrel implant (n = 14); injectable medroxyprogesterone acetate (n = 11); etonogestrel vaginal ring (n = 142); combined oral contraceptive pills (n = 2349); and norelgestromin transdermal patch (n = 10). The control group included patients not using contraceptives or using barrier or calendar methods (n = 1220). Among all the cohorts the median number of cumulus-oocyte complexes retrieved during oocyte retrieval was comparable (P = 0.054), and a significant difference in oocyte maturity rate with median number of vitrified oocytes was found (P = 0.03, P < 0.001, respectively). After adjusting for confounders a multivariate analysis found no association between the type of contraceptive and proportion of metaphase II oocytes available for cryopreservation. CONCLUSIONS: Among the various forms of contraception, none was shown to have an adverse association with oocyte yield or maturation rate in patients undergoing POC.

Fertility preservation parameters in patients with haematologic malignancy: a systematic review and meta-analysis.

Patients with haematologic malignancies represent one of the most common groups referred for fertility preservation before gonadotoxic oncological treatment. The aim of this systematic review and meta-analysis was to evaluate the effect of haematologic cancer on ovarian reserve and response to ovarian stimulation compared with healthy controls. A total of eight observative studies were included in the final quantitative analysis. Despite a younger age (mean difference -4.17, 95% CI -6.20 to -2.14; P < 0.0001), patients with haematologic malignancy had lower serum anti-Müllerian hormone levels compared with the control group (MD -1.04, 95% CI -1.80 to -0.29; P = 0.007). The marginally higher total recombinant FSH dose (MD 632.32, 95% CI -187.60 to 1452.24; P = 0.13) and significantly lower peak oestradiol serum level (MD -994.05, 95% CI -1962.09 to -26.02; P = 0.04) were demonstrated in the study group compared with the healthy controls. A similar number of retrieved oocytes were achieved in both groups (MD 0.20, 95% CI -0.80 to 1.20; P = 0.69). In conclusion, haematologic malignancies may detrimentally affect ovarian function manifesting in decreased AMH serum levels despite a younger age compared with healthy controls. This effect can be overcome by the application of relevant IVF protocols and stimulation doses to achieve an adequate oocyte yield.