FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

STUDY QUESTION: Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. WHAT IS KNOWN ALREADY: During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. STUDY DESIGN, SIZE, DURATION: This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. WIDER IMPLICATIONS OF THE FINDINGS: FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Human ovarian gross morphology and subanatomy across puberty: insights from tissue donated during fertility preservation.

Objective: To study ovarian gross morphologic and subanatomic features across pubertal development. Design: Prospective cohort study. Setting: An academic medical center with specimens collected from 2018-2022. Patients: Tissue was obtained from prepubertal and postpubertal participants (0.19-22.96 years) undergoing ovarian tissue cryopreservation before treatment that put them at a significantly or high increased risk of developing premature ovarian insufficiency. Most participants (64%) had not received chemotherapy at tissue collection. Interventions: None. Main Outcome Measures: Ovaries procured for fertility preservation were weighed and measured. Ovarian tissue fragments released during processing, biopsies used for pathology, and hormone panels were analyzed for gross morphology, subanatomic features, and reproductive hormones. Graphical analysis of best-fit lines determined age at maximum growth velocity. Results: Prepubertal ovaries were significantly (1.4-fold and 2.4-fold) smaller than postpubertal ovaries by length and width and 5.7-fold lighter on average. Length, width, and weight grew in a sigmoidal pattern with age. Prepubertal ovaries were less likely to display a defined corticomedullary junction (53% vs. 77% in postpubertal specimens), less likely to have a tunica albuginea (22% vs. 93% in postpubertal specimens), contained significantly more (9.8-fold) primordial follicles, and contained primordial follicles at significantly deeper depths (2.9-fold) when compared with postpubertal ovaries. Conclusions: Ovarian tissue cryopreservation is a resource to study human ovarian biology and pubertal development. Maximum growth velocity occurs late within the pubertal transition (Tanner 3+) after changes in subanatomic features. This ovarian morphology model adds to foundational knowledge of human ovarian development and supports ongoing transcriptomics research.

Workflow for Management of Gonadal Neoplasm in 2 Patients with Differences of Sex Development Enrolled in an Experimental Gonadal Tissue Cryopreservation Protocol.

OBJECTIVE: To outline our experimental gonadal tissue cryopreservation (GTC) protocol that does not disrupt the standard of care in medically-indicated gonadectomy for patients with differences of sex development, including highlighting the multidisciplinary collaborative protocol for when neoplasm is discovered in these cases. METHODS: Two patients with complete gonadal dysgenesis who were undergoing medically-indicated prophylactic bilateral gonadectomy elected to pursue GTC. Both were found to have germ cell neoplasia in situ on initial pathologic analysis, requiring recall of the gonadal tissue, which had been cryopreserved. RESULTS: Cryopreserved gonadal tissue was successfully thawed and transferred to pathology for complete analysis. No germ cells were identified in either patient nor were found to have malignancy, so further treatment beyond gonadectomy was not indicated. Pathologic information was communicated to each family, including that long-term GTC was no longer possible. CONCLUSION: Organizational planning and coordination between the clinical care teams, GTC laboratory, and pathology were key to handling these cases with neoplasia. Processes that anticipated the possibility of discovering neoplasia within tissue sent to pathology and the potential need to recall GTC tissue to complete staging included (1) documenting the orientation and anatomical position of tissue processed for GTC, (2) defining parameters in which tissue will be recalled, (3) efficiently thawing and transferring GTC tissue to pathology, and (4) coordinating release of pathology results with verbal communication from the clinician to provide context. GTC is desired by many families and at the time of gonadectomy and is (1) feasible for patients with DSD, and (2) did not inhibit patient care in 2 patients with GCNIS.

Efficacy of Video-based Education in Improving Understanding of Pediatric Fertility Preservation.

Eighty percent of children diagnosed with cancer in childhood survive into adulthood. Fertility preservation (FP) is an important consideration, and procedures are available to reduce the risk of infertility following gonadotoxic therapies. Discussing FP options eases decision-making and minimizes regret; however, poor comprehension of these topics remains a challenge. This study evaluates if video-based educational tools increase understanding of FP options among pediatric patients and families. Videos were first tested among participants not at risk of infertility to ensure objective utility and optimize quality. In part 1, parents of pediatric surgical patients were randomized to view 2 publicly available educational videos on FP in differing orders. Each group completed pre-surveys and post-surveys assessing the comprehension and perception of video quality. In part 2, the parent and patient participants completed a comprehension assessment before and after viewing our institution-specific educational video, designed based on participant feedback from part 1. Part 1 results demonstrated a significant increase in participant knowledge and perceived understanding after viewing the videos ( P <0.001), regardless of order. In part 2, the post-test comprehension scores were significantly improved for all participants and all subgroups, P <0.01. Results suggest that video-based educational tools may help to reduce barriers to FP in pediatric oncology.

Pediatric, adolescent, and young adult breast and reproductive tumors.

Tumors of the breast and reproductive organs that occur in children, adolescents, and young adults (AYA) have different biological features and can present special challenges. Although prognosis for these tumors is generally favorable, the long-term effects of treatment can be debilitating. Treatments are often multimodal and may include surgery as well as chemotherapy and/or radiation, which can cause considerable distress and anxiety related to loss of femininity or masculinity, concern over future fertility, or sexual dysfunction. Thus, tumors of the reproductive organs in pediatric/AYA patients require special consideration of the treatment effects beyond the intended oncologic outcome. Multidisciplinary teams should be involved in their care and address issues of fertility, sexual dysfunction, and psychosexual concerns before treatment begins. This review addresses histology, risk factors, prognosis, staging and treatment of gynecologic, breast and testicular cancers in pediatric and AYA patients.

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: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.

Fertility Considerations in Pediatric and Adolescent Patients Undergoing Cancer Therapy.

Survivors of pediatric cancer are at increased risk for infertility and premature hormonal failure. Surgeons caring for children with cancer have an important role to play in understanding this risk, as well as advocating for and performing appropriate fertility preservation procedures. Fertility preservation options in males and females vary by pubertal status and include nonexperimental (oocyte harvest, ovarian tissue cryopreservation, sperm cryopreservation) and experimental (testicular tissue cryopreservation) options. This review summarizes the basics of risk assessment and fertility preservation options and explores unique considerations in pediatric fertility preservation.

Feasibility of laparoscopic ovarian tissue cryopreservation after open abdominopelvic tumor surgery.

BACKGROUND: Laparoscopic oophorectomy with tissue cryopreservation (OTC) for fertility preservation is usually performed prior to therapy. When fertility preservation is considered after prior open abdominopelvic tumor surgery there may be a perceived barrier to laparoscopic OTC. This study evaluates the feasibility of OTC with a laparoscopic approach after open surgery. METHODS: This is a single institution retrospective study from 2011 to 2019. RESULTS: Planned laparoscopic OTC was performed after open surgery in 17 of 113 patients. Median age was 4.2 years. The most common diagnoses were Wilms Tumor (35%) and neuroblastoma (35%). The most common procedures were nephrectomy (41%) and exploratory laparotomy with biopsy (35%). The median amount of time between open surgery and OTC was 29 days. Sixteen (94%) had a laparoscopic OTC. Regardless of operative technique, patients resumed therapy a median of 3 days after OTC. CONCLUSIONS: Prior abdominopelvic surgery should not be a barrier to OTC. Laparoscopic OTC is feasible after a variety of open oncologic operations, regardless of time-interval between the procedures and without incurring a significant delay in resuming oncologic therapy.

Participation of Pediatric Surgery Training Programs in Fertility Preservation Initiatives.

Purpose: The significant reduction in childhood cancer mortality has allowed for greater emphasis on survivorship issues, including infertility. This study evaluated the participation of pediatric surgery training programs in fertility preservation (FP) and exposure of fellows to adnexal cases. Materials and Methods: A survey was distributed to pediatric surgery fellowship program directors in the United States and Canada through email. Questions focused on FP participation, operative cases, FP program limitations, and fellow completion of adnexal cases. Results: Survey participation was 49% (28/57). Overall, 43% (12/28) of training programs report participation in FP initiatives. Of those who participated, the most common procedures performed were testicular tissue biopsy (58%) and testicular sperm extraction (42%) in males, and surgical transposition of the ovaries (83%) and laparoscopic oophorectomy (67%) in females. The greatest cited limitations on participation were that FP was another department's responsibility (50%) and lack of multidisciplinary team (31%). Notably, lack of operative experience in benign ovarian and testicular procedures (0%) was not a limitation. All programs, regardless of participation in FP, noted that their fellows performed benign and malignant adnexal cases. Conclusion: Less than half of pediatric surgery training programs participate in FP initiatives, despite adequate advanced minimally invasive training of fellows to perform these procedures.

Establishing an Institutional Gonadal Tissue Cryopreservation Protocol for Patients with Differences of Sex Development.

PURPOSE: Many individuals with differences of sex development experience subfertility. We describe a novel gonadal tissue cryopreservation protocol for those individuals. MATERIALS AND METHODS: Before 2018 patients with differences of sex development electing gonadal tissue cryopreservation were enrolled in a cancer focused protocol. Thereafter, our hospital developed a protocol specifically for these patients undergoing gonadectomy due to neoplasia risk. Protocol development steps and procedures are reported. A retrospective chart review was conducted for patient characteristics and cryopreservation choices. RESULTS: During the institutional review board approval process there were multidisciplinary meetings with stakeholders. The main concerns discussed included preoperative counseling, pathological evaluation and final tissue disposition. Detailed multidisciplinary preoperative counseling is provided regarding potential gonadal tissue cryopreservation for patients undergoing gonadectomy. For enrolled patients the gonad is bisected after removal, with half being sent to pathology and half being processed for cryopreservation. If neoplasia is noted, cryopreserved tissue is recalled for further pathological analysis. Postoperative counseling is performed after pathology results are available, and the final gonadal tissue cryopreservation decision is made. During the study period 7 patients with 5 diagnoses and a median age of 10.99 years (IQR 1.29 to 14.84) elected to attempt gonadal tissue cryopreservation. Of the patients 4 (57%) had germ cells and elected to store tissue. CONCLUSIONS: Gonadal tissue cryopreservation at the time of gonadectomy is feasible for patients with differences of sex development at risk for gonadal neoplasia. The protocol described represents a template for institutions wishing to offer gonadal tissue cryopreservation to patients electing gonadectomy. More than half of patients thus far have cryopreserved gonadal tissue.

Ovarian tissue health after laparoscopic unilateral oophorectomy: A porcine model for establishing optimized fertility preservation techniques in children.

BACKGROUND: The only pre-treatment fertility preservation option for prepubertal girls, who are at risk for infertility due to their diagnosis or treatment, is surgical removal of ovarian tissue for cryopreservation (OTC). We investigated ovarian tissue health following isolation with an ultrasonic advanced energy device (UAED), that has a previously reported thermal spread of ≤2 cm. METHODS: The ovaries of eight Yucatan minipigs were isolated by laparoscopy (1) close dissection with the UAED located up to 2 mm away from the ovarian capsule, (2) far dissection with the UAED located >2 cm away, or by (3) laparotomy for control ovaries using cold scissors. Ovarian cortex tissues were cultured for 4 days to assess tissue health. RESULTS: Ovarian cortex tissue isolated using a UAED produced an altered metabolic ratio in both the far and close dissection compared to control (p < 0.001). There was an increase in folliculogenesis in the control samples over samples isolated with far and close dissection (p < 0.0001), and a reduction in estradiol production in experimental groups (p < 0.0001). CONCLUSIONS: This model defines differences in ovarian tissue health among different isolation techniques. Ongoing work will further define the standard of care surgical technique for OTC.

Gonadal Tissue Cryopreservation for Children with Differences of Sex Development.

INTRODUCTION: Infertility is common for individuals with differences of sex development (DSD) and is a significant concern to these individuals. Fertility potential in many DSD conditions is poorly understood. Gonadal tissue cryopreservation (GTC) for fertility preservation (FP) is offered to children with cancer undergoing gonadotoxic therapy. Our team sought to expand the field of FP by offering and evaluating the success of GTC for individuals with DSD. MATERIALS AND METHODS: GTC was offered to patients with DSD undergoing prophylactic gonadectomy, after extensive multidisciplinary counseling. For those who elected to attempt GTC, data were retrospectively abstracted, including: DSD diagnosis, age at gonadectomy, indication for gonadectomy, pathology results, and final decision about long-term gonadal tissue storage. RESULTS: Ten patients were enrolled to attempt GTC, with a mean age of 11.5 years (range 1-18). Five of the 10 patients had germ cells (GCs) present. Diagnoses (age at gonadectomy) for patients with GCs included ovotesticular DSD (13 months), mixed gonadal dysgenesis (17 months), partial gonadal dysgenesis (3 years), partial androgen insensitivity syndrome (11 years), and mixed gonadal dysgenesis (12 years). Four of the 5 subjects with GCs elected for GTC. One opted against GTC, citing immature gametes that did not match gender identity. CONCLUSION: GTC at the time of gonadectomy for patients with DSD is feasible. In many patients, GCs are present. While questions remain about the timing of gonadectomy, quality of GCs, and future success for use of the tissue based on technological advancement, GTC represents a novel approach to experimental FP for individuals with DSD.

Ethical challenges in regionalization of pediatric surgical care.

PURPOSE OF REVIEW: Rapid advancement in the field of pediatric surgery has resulted in unprecedented opportunities to advance the care of children. However, assuring that the highest quality, most appropriate care is available to all pediatric patients remains challenging. Regionalization of care has been proposed as a means of obtaining this critical goal. In this review, we discuss the ethical challenges associated with regionalization of pediatric surgical care. RECENT FINDINGS: Regionalization of pediatric surgical care is associated with improved patient outcomes. Over half of pediatric surgeons favor regionalization of care. Despite these findings, regionalization of care may be associated with inequitable distribution of resources, financial and emotional burdens for patients and families, and surgeon dissatisfaction and technical decline. SUMMARY: Regionalization of pediatric surgical care is fraught with complex ethical issues. Development of a system of pediatric surgery networks may offer a solution to these challenges.

Gonadal Tissue Cryopreservation for a Girl With Partial Androgen Insensitivity Syndrome.

Individuals with differences/disorders of sex development (DSD) have increased rates of infertility. For children and youth undergoing prophylactic gonadectomy for malignancy risk, our institution offers gonadal tissue cryopreservation, an experimental technique to preserve fertility cryopotential. An 11-year-old girl with partial androgen insensitivity syndrome presented for evaluation for fertility preservation in the setting of a planned bilateral gonadectomy at an outside institution. At presentation, the patient had begun puberty with an elevated serum androgen level and was experiencing undesired virilization. She expressed a strong female gender identity, an understanding of the various treatment options, and a preference for gonadectomy to prevent further virilization. After thorough counseling with the patient and family in our institution's multidisciplinary DSD clinic, she underwent bilateral gonadectomy with gonadal tissue cryopreservation. Her gonadal pathologic examination demonstrated well-developed peripubertal testes, with present, albeit decreased, numbers of spermatogonial germ cells, decreased Leydig cells, and nonspecific degenerative changes. The patient and her family chose to maintain the cryopreserved tissue for the patient's potential future use. To the best of our knowledge, the present case is the first reported case of gonadal tissue cryopreservation in a patient with partial androgen insensitivity syndrome. Storage of gonadal tissue is a feasible method of germ cell preservation in patients with DSD undergoing gonadectomy, although further research advances are required to facilitate development of this tissue into mature gametes capable of biological fertility.

Testicular wedge biopsy for fertility preservation in children at significant risk for azoospermia after gonadotoxic therapy.

BACKGROUND/PURPOSE: Testicular tissue cryopreservation (TTC) provides an experimental option for fertility preservation for male children at significant risk for azoospermia owing to high-risk gonadotoxic treatments. METHODS: A single institution, retrospective review of TTC cases from 2015 to 2017. Children at significant risk for azoospermia were eligible for study inclusion. A unilateral wedge biopsy of the testis was performed for TTC. RESULTS: TTC was performed in 23 patients. Average age was 10 years old (5 months to 18 years). Diagnoses included solid tumor (74%, n = 17), hematologic malignancy (17%, n = 4), and benign hematologic disease (13%, n = 3). Six patients had TTC at the time of disease relapse. Nine patients were referred for TTC prior to stem cell transplantation. The majority (70%, n = 16) of patients had an additional procedure at the time of TTC. One patient developed postoperative scrotal cellulitis that was treated with antibiotics. The majority of patients (96%, n = 22) had normal testicular tissue with the presence of germ cells on histopathological analysis. Median time to start of medical therapy was seven days with no unanticipated treatment delays. CONCLUSIONS: Testicular wedge biopsy for TTC can be performed safely, coordinated with other necessary procedures, and does not delay the start of treatment. TTC remains an experimental option for fertility preservation for children, as no spermatogenic recovery or pregnancies from cryopreserved testicular tissues have been reported to date. LEVEL OF EVIDENCE: IV.

Laparoscopic unilateral oophorectomy for ovarian tissue cryopreservation in children.

BACKGROUND/PURPOSE: Many survivors of childhood cancer will experience premature gonadal insufficiency or infertility as a consequence of their medical treatments. Ovarian tissue cryopreservation (OTC) remains an experimental means of fertility preservation with few reports focused on the surgical technique and postoperative outcomes for OTC in children. METHODS: This is a single institution, retrospective review of OTC cases from January 2011 to December 2017. Children were eligible for OTC if they had a greater than 80% risk of premature ovarian insufficiency or infertility owing to their anticipated gonadotoxic medical treatment. RESULTS: OTC was performed in 64 patients. Median age was 12 years old (range: 5 months-23 years). Nearly half (48%) of the patients were premenarchal. Laparoscopic unilateral oophorectomy was performed in 84% of patients. There were no surgical complications. In 76% of patients, OTC was performed in conjunction with an ancillary procedure. The majority (96%) of patients were discharged within 24 hours. Median time from operation to medical therapy was six days, with no unanticipated treatments delays attributable to OTC. CONCLUSIONS: Laparoscopic unilateral oophorectomy for OTC can be performed safely, in combination with other ancillary procedures, as an outpatient procedure without delaying medical therapy for children facing a fertility-threatening diagnosis or treatment. LEVEL OF EVIDENCE: IV.

Development of a Pediatric Fertility Preservation Program: A Report From the Pediatric Initiative Network of the Oncofertility Consortium.

Infertility is known to decrease quality of life among adults. In some cases, infertility is caused by medical conditions and/or treatments prescribed in childhood, and using methods to protect or preserve fertility may expand future reproductive possibilities. Structured programs to offer counseling about infertility risk and fertility preservation options are essential in the care of pediatric patients facing fertility-threatening conditions or treatments, yet multiple barriers to program development exist. This report was developed from the institutional experiences of members of the Pediatric Initiative Network of the Oncofertility Consortium, with the intent of providing guidance for health care providers aiming to establish programs at institutions lacking pediatric fertility preservation services. The mechanics of building a fertility preservation program are discussed, including essential team members, target populations, fertility preservation options (both established and experimental), survivorship issues, research opportunities, and ethical considerations. Common barriers to program development and utilization, including low referral rates and financial concerns, are also discussed, and recommendations made for overcoming such barriers.