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Study Question: Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy?, Summary Answer: Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy., What Is Known Already: Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy., Study Design, Size, Duration: A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre., Participants/materials, Setting, Methods: The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy., Main Results and Role of Chance: Ovarian tissue was successfully collected from 78.7% of the 42 patients. Oocytes were obtained from 20 patients before chemotherapy and 13 after chemotherapy. The youngest patients from whom oocytes were retrieved were aged 2 years (two atretic follicles) and 3 years. Of the 395 oocytes collected, ∼30% were atretic (29.6% in the pre-chemotherapy group, 37% in the post-chemotherapy group). One hundred twenty-one oocytes (31%) were matured in vitro and vitrified: 67.8% from patients before chemotherapy, the rest after chemotherapy. Mature oocytes suitable for vitrification were obtained from 16/20 patients before chemotherapy and from 12/13 patients after chemotherapy (maturation rate, 32 and 26.4%, respectively). There were significant correlations of the number of vitrified oocytes with patient age (more matured oocytes with older age) (P = 0.001) and with pre-oophorectomy AMH levels (P = 0.038 pre-chemotherapy group, P = 0.029 post-chemotherapy group). Oocytes suitable for vitrification were obtained both by manual aspiration of antral follicles (45%) and from rinse solutions after dissection. There were significantly more matured oocytes in the pre-chemotherapy group from aspiration than in the post-chemotherapy group after both aspiration (P < 0.033) and retrieval from rinsing fluids (P < 0.044). The number of pre-antral follicles per histological section did not differ in the pre- versus post-chemotherapy. AMH levels dropped by approximately 50% after ovarian removal in both groups, with a significant correlation between pre- and post-oophorectomy levels (P = 0.002 pre-chemotherapy group, P = 0.001 post-chemotherapy group)., Limitations, Reasons for Caution: There were no patients between 5 years and 10 years old in the post-chemotherapy group, which might have affected some results and correlations. Oocytes from patients soon after chemotherapy might be damaged, and caution is advised when using them for fertility-restoration purposes. The viability, development capability and fertilization potential of oocytes from paediatric patients, especially prepubertal and after chemotherapy, are unknown, in particular oocytes recovered from the media after the tissue dissection step., Wider Implications of the Findings: Although more oocytes were collected and matured from chemotherapy-naïve paediatric patients, ovarian tissue and immature oocytes were also retrieved from young girls in whom cancer therapy has already been initiated. Our centre has established a protocol for potential maximal fertility preservation in paediatric female patients with cancer. Vitrified-in vitro-matured oocytes may serve as an important gamete source in paediatric female patients with cancer because the risk of reseeding the disease is avoided. Further studies are needed on the fertility-restoring potential of oocytes from paediatric and prepubertal patients, especially after exposure to chemotherapy., Study Funding/competing Interests: The study was conducted as part of the routine procedures for fertility preservation at our IVF unit. No funding outside of the IVF laboratory was received. Funding for the AMH measurements was obtained by a research grant from the Israel Science Foundation (to B.-A.I., ISF 13-1873). None of the authors have competing interests., Trial Registration Number: N/A., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To investigate the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors, GDNF alpha 1 (GFR-alpha1) and tyrosine kinase receptor for rearranged during transfection (RET), in human ovaries at the protein and mRNA levels., Design: Samples were prepared for immunohistochemical staining for GDNF, GFR-alpha1, and RET and in situ hybridization for the mRNA of GFR-alpha1. The mRNA expression of two GDNF isoforms and two RET isoforms was investigated by reverse transcription polymerase chain reaction., Setting: Infertility unit at a university-affiliated tertiary medical center., Patient(s): Fifteen patients who underwent pregnancy terminations at 21-35 gestational weeks and 28 girls/women aged 5-39 years who underwent ovarian laparoscopies., Intervention(s): None., Main Outcome Measure(s): Laboratory analysis of human ovarian specimens., Result(s): The GDNF protein was detected in oocytes of all samples. Granulosa cells (GCs) stained for GDNF in a portion of fetal samples and in all samples from girls/women. The proteins for GFR-alpha1 and RET were detected in both oocytes and GCs with weak staining for the long RET isoform. GFR-alpha1 mRNA transcripts were detected in oocytes and GCs from all samples. The mRNA transcripts for the two GDNF isoforms and the two RET isoforms were detected in all fetal and adult specimens., Conclusion(s): The presence of the receptors of GDNF in the GCs of human primordial follicles suggests that GDNF may be involved in the regulation of primordial follicular activation., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To compare the development of human fetal follicles from late-gestational-age fetuses frozen-thawed gradually and slowly with dimethylsulfoxide (DMSO) or 1,2 propanediol (PROH) and sucrose after renal grafting into follicle stimulating hormone-treated immunodeficient mice., Design: Controlled histologic study of grafted human fetal ovaries., Setting: Major tertiary care academic center., Patient(s): Eleven women undergoing pregnancy termination at 22 to 33 gestational weeks., Intervention(s): None., Main Outcome Measure(s): Microscopic morphometric analysis and immunohistochemistry for proliferating cell nuclear antigen (PCNA)., Result(s): Only follicles from samples frozen-thawed with PROH developed to secondary and antral stages 4 to 6 months after grafting, with PCNA expression in their granulosa cells. However, the number of surviving/developing follicles per section was very low (4-25 per graft), compared with 71 to 406 follicles in pretransplantation samples. Graft recovery was very high, with similar rates for transplants frozen-thawed with PROH and DMSO. Normal ovarian structure after grafting was identified only in the PROH frozen-thawed grafts. In deteriorated grafts, frozen-thawed with either DMSO or PROH, net-like hollows replaced follicles, whereas tubule-like structures were only identified in DMSO frozen-thawed grafts., Conclusion(s): This is the first report of the development of late-pregnancy-stage human fetal follicles in immunodeficient mice. PROH freezing-thawing supported development and survival better than DMSO. However, the low follicular survival points to the urgent need for efficient methods to enhance vascularization rate and prevent ischemia.

Objective: To investigate the presence of growth hormone (GH) and its receptor (GH-R) in early developing follicles., Design: Immunocytochemical and in situ hybridization study., Setting: Major tertiary care and referral academic centers., Patient(s): Ten ovarian samples from adults/girls aged 6-38 years and from 10 fetuses of women undergoing second and third trimester pregnancy terminations., Intervention(s): None., Main Outcome Measure(s): Immunocytochemistry and in situ hybridization on paraffin sections of human ovaries from fetuses and women/girls., Result(s): The proteins and the mRNA transcripts for GH and GH-R were detected in oocytes, granulosa (GC), and stroma cells from both sources (fetuses and women/girls), with low staining intensity only in a portion of the fetal GC., Conclusion(s): This is the first report of the expression of GH-R in human ovaries from fetuses as well as women/girls and of GH in human fetal ovaries. Be that as it may, further experiments should be conducted to elucidate if indeed GH is involved in the initiation of human primordial follicular growth.