title: Cryopreservation of in vitro matured oocytes in addition to ovarian tissue freezing for fertility preservation in paediatric female cancer patients before and after cancer therapy.
abstract: OBJECTIVE
  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?
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  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.
  
  
  BACKGROUND
  N/A.
authors: IVF and Infertility Unit, Beilinson Women Hospital, Rabin Medical Center, Petach Tikvah, Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Ramat Aviv, Israel ronita@clalit.org.il ronitabir@gmail.com.
authors: R
authors: R
authors: Abir
authors: Institute of Oncology, Davidoff Center, Rabin Medical Center, Petach Tikvah, Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Ramat Aviv, Israel.
authors: I
authors: I
authors: Ben-Aharon
authors: IVF and Infertility Unit, Beilinson Women Hospital, Rabin Medical Center, Petach Tikvah, Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Ramat Aviv, Israel.
authors: R
authors: R
authors: Garor
authors: Department of Paediatric Hematology Oncology, Schneider Children's Medical Center of Israel, Petach Tikvah, Israel, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors: I
authors: I
authors: Yaniv
authors: Department of Paediatric Hematology Oncology, Schneider Children's Medical Center of Israel, Petach Tikvah, Israel, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors: S
authors: S
authors: Ash
authors: Institute of Oncology, Davidoff Center, Rabin Medical Center, Petach Tikvah, Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Ramat Aviv, Israel.
authors: S M
authors: SM
authors: Stemmer
authors: IVF and Infertility Unit, Beilinson Women Hospital, Rabin Medical Center, Petach Tikvah, Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Ramat Aviv, Israel.
authors: A
authors: A
authors: Ben-Haroush
authors: Department of Paediatric Surgery, Schneider Children's Medical Center of Israel, Petach Tikvah, Israel, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors: E
authors: E
authors: Freud
authors: Department of Paediatric Surgery, Schneider Children's Medical Center of Israel, Petach Tikvah, Israel, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors: D
authors: D
authors: Kravarusic
authors: IVF and Infertility Unit, Beilinson Women Hospital, Rabin Medical Center, Petach Tikvah, Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Ramat Aviv, Israel.
authors: O
authors: O
authors: Sapir
authors: IVF and Infertility Unit, Beilinson Women Hospital, Rabin Medical Center, Petach Tikvah, Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Ramat Aviv, Israel.
authors: B
authors: B
authors: Fisch
mesh_terms: D000293:Adolescent; D000367:Age Factors; D000970:Antineoplastic Agents / Q000009:adverse effects / Q000627:therapeutic use; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D015925:Cryopreservation; D005240:Feasibility Studies; D005260:Female; D059247:Fertility Preservation / Q000009:adverse effects*; D006801:Humans; D059471:In Vitro Oocyte Maturation Techniques; D007557:Israel; D009369:Neoplasms / Q000188:drug therapy / Q000473:pathology*; D009865:Oocytes / Q000187:drug effects / Q000473:pathology*; D010052:Ovariectomy / Q000009:adverse effects; D010053:Ovary / Q000187:drug effects / Q000473:pathology* / Q000601:surgery; D011446:Prospective Studies; D062606:Tertiary Care Centers; D058989:Vitrification
chemical_list: D000970:Antineoplastic Agents
keywords: anti-Mullerian hormone; atretic oocytes; fertility preservation; in vitro maturation; oocyte vitrification; paediatric cancer patients; pre-antral follicles