Hananel Holzer, MD Medical Director, MUHC Reproductive Center McGill University Health Center Director, REI Division, Dept. of Obstetrics & Gynecology McGill University McGill University Health Centre

Fertility preservation

1 st live birth 1986 (Chen et al) But: oocytes vulnerable to freezing process intracellular ice formation membrane rupture, abnormal cortical granule reaction, zona hardening, meiotic spindle and cytoskeleton damage : 5 live births No male partner: Oocyte cryopreservation

Using PROH/sucrose slow freezing protocol and introduction of ICSI improved pregnancy rates (Porcu et al 1997) Reported survival rates for mature oocytes (collected from stimulated ovaries) are 50-70% No male partner: Oocyte cryopreservation

Oocyte vitrification Cryoprotectants in high concentration used to induce glasslike state, cell then rapidly frozen avoiding formation of intracellular ice Kuleshova et al, 1999: survival rate 65%, PR/ET 21% Yoon et al, 2003: survival rate 69%, implantation rate 6.4%, PR/ET 21.4% Katayama et al, 2003: survival rate 94%, PR/ET 33%

Oocyte vitrification 165 live births (Chian et al ASRM 2007: McGill Reproductive Center in Canada, Instituto Mexicano de Infertilidad in Mexico and CECOLFES in Colombia) A few thousand babies Vitrification is emerging to be a better technique.

Embryo or oocyte cryopreservation after ovarian stimulation Pregnancies reported are result of fertilization of frozen/thawed oocytes collected after ovarian stimulation However: − −time interval needed for ovarian stimulation 2-6 weeks. − −Starting during menstruation − −Time may not be available for cancer patients − −Recent studies: stimulation luteal phase. (Von Wolff 2009, Sonmezer 2011) − −ovarian stimulation associated with high estrogen levels which may not be safe in cases of hormone sensitive tumors such as breast cancer − −Estrogens may have an indirect mitogenic effect on receptor negative cancers. (Gupta PB 2006)

Embryo or oocyte cryopreservation after ovarian stimulation Aromatase inhibitor + FSH: letrozole started 2 days before FSH administration, then given concomitantly, no increased risk of relapse (Oktay 2005, 2007, Azim 2008) Lower estradiol levels does not totally avoid stimulation Early follicular start of treatment. More studies are needed

Collection of immature oocytes from unstimulated ovaries Pincus, J Exp Med 1935; Edwards, Nature, 1965,1969. Cha et al,1991: Immature oocyte laparotomy for oocyte donation Trounson et al, 1994: IVM: vaginal collection of immature oocytes, for PCO related infertility

Mature oocytes at oocyte collection in IVM cycles 8mm 12mm 14mm

Vitrification of IVM oocytes? Could oocytes collected from unstimulated ovaries, matured in vitro, then vitrified survive thawing and be fertilized? Could transfer of these embryos result in a viable pregnancy ?

No. of patients20 Mean age30.7 ±3.7 No. of mature oocytes retrieved7 No. of immature oocytes retrieved295 Mean oocyte maturation rate (%) No. of oocytes vitrified and thawed215 No. of oocytes survived (mean % + SEM )148 ( ) No. of oocytes fertilized (mean % + SEM)96 ( ) No. of embryos transferred (median; range)64 (3.2; range 1 - 6) No. of implantations (mean % + SEM)5 ( ) No. of clinical pregnancies (%)4 (20.0) No. of live births (%)4 (20) Holzer et al ESHRE 2007 Vitrification of IVM oocytes

Conclusions Vitrification of in-vitro matured oocytes collected from unstimulated ovaries followed by later thawing and fertilization can result in successful pregnancies and live births

Conclusions Preliminary results 20 patients, all with PCO 20% pregnancy rate (vs. 35% in “Fresh IVM” ) Lower implantation rate (10.8% vs. 14.4%) Learning curve?

Conclusions Collection of immature oocytes from unstimulated ovaries followed by IVM and vitrification of mature oocytes could be offered to patients with hormone- sensitive disease and/or when there is not enough time to stimulate ovaries – –no risk of aggravating disease – –no theoretic risk of re-instituting metastatic malignant disease – –does not require same amount of time as that needed for ovarian stimulation, no need to wait for next cycle

When ovarian tissue is being harvested Additional strategy of fertility preservation combines ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue, followed by in vitro maturation (IVM) and vitrification (Huang, Tulandi, Holzer, Tan, Chian Fertil Steril 2007) When surgery performed to remove ovarian tumor or for other therapeutic indications

Fertility preservation in prepubertal children Childhood and adolescence period of emotional and psychological instability; issues of sexuality, including fertility, are of particular importance Depletion of primordial oocytes after gonadotoxic treatment proportional to size of oocyte pool Younger patients have more oocytes; thus gonadal damage could seem to be less severe than in older patients

Fertility preservation in prepubertal children Global incidence of acute ovarian failure in childhood cancer survivors ranges from 6.3 to 12% (Bakker 2004) In Childhood Cancer Survivor Study ;the relative risk for survivors of ever being pregnant was 0.81 (Green DM 2009) Very few treatments benefit younger patients at risk of infertility after treatment

Fertility preservation in prepubertal children; Ovarian tissue cryobanking Primary method of fertility preservation for prepubertal girls One ovary removed from 47 patients aged y (median: 5) Ovarian tissue fragments frozen strong inverse correlation found between age and follicular density none of the cases had visible ovarian tumour components (Poirot 2007) 58 patients years, underwent ovarian tissue cryopreservation. 1 case of ovarian lymphoma infiltration (Jadoul 2010)

Fertility preservation in prepubertal children: Combined approach Unilateral oophorectomy: 19 patients aged 5-20 years (median 15 yrs.)Unilateral oophorectomy: 19 patients aged 5-20 years (median 15 yrs.) Antral follicles aspirated, average no. of oocytes: 9 (0-37)Antral follicles aspirated, average no. of oocytes: 9 (0-37) −GV oocytes – IVM −mature oocytes – frozen Ovarian tissue – cryopreserved in fragmentsOvarian tissue – cryopreserved in fragments (Revel 2008)

Fertility preservation in prepubertal children: Ovarian tissue cryobanking Reimplantation of ovaries cryopreserved before puberty not yet performed in either nonpubertal or pubertal recipients Animal models: puberty and fertility restored (Sauvat 2008) Removal of entire ovary, although strongly advocated by some, may not be recommended for pediatric patients in whom fertility outcome is often uncertain. How much? Based on the risk of the planned treatment and the ovarian volume.

Fertility preservation in pre- pubertal children Fertility preservation should now be considered in children facing cancer treatment with high risk of gonadal toxicity, including high-dose chemotherapy and bilateral irradiation of gonads at toxic doses Multidisciplinary team