Female fertility preservation Ariel Revel, MD 2012 arielr2@hadassah.org.il The problem • Loss of fertility is a major concern of cancer patients. • Cancer therapy increases oocyte apotosis – 99% of BMT with TBI pts will develop permanent premature ovarian failure! Fertility Preservation: An issue less than half of affected cancer patients are told about Terry had run 5,373 km and was forced to stop running after 143 days on 1/9/1980 at Thunder Bay, Ontario Osteosarcoma •most frequent primary bone malignancy •affects children & young adults (10-25) • accounts 5% of all childhood cancers. •Common in bones around the knee •But can appear in any bone region. Treatment 1. Surgery 2. radiation therapy 3. Chemotherapy Terry Fox Run Alkylating agents The run is the single largest single-day cancer fundraiser in the world. Ifosfamide The fertility specialist The biological clock • • • • • • Women are born with about 2 million eggs in their ovaries. By the time they reach puberty, 90% of these eggs have died Age 20 , only about 50,000 of the original 2 million eggs remain. Steady decline until age of 35-37, at which point rate of loss speeds up dramatically. increasingly fewer eggs available for fertilization beyond 35. Effect of length of exposure on ovarian xenograts High risk Intermediate risk Low/no risk OR = 4 Cyclophosphamide Doxorubicin Methotrexate Busulfan Cisplatin Melphalan Bleomycin Carboplatin OR= 1,8 5-Fluorouracil Chlorambucil Actinomycin-D Dacarbazine Mercaptopurine Procarbazine Vincristine Effect of dosage Ifosfamide Thiotepa Nitrogen mustard Meirow, 1999 AMH Reference ranges Ovarian Fertility Potential pmol/L Optimal Fertility 28.6 pmol/L 48.5 pmol/L Satisfactory Fertility 15.7 pmol/L 28.6 pmol/L Low Fertility 2.2 pmol/L 15.7 pmol/L Very 0.0 pmol/L Low/Undetect 2.2 pmol/L able Antral follicle count •early Graafian follicles •2–10mm •round to oval •echolucent-fluid–filled •cohort of follicles awaiting further recruitment and stimulation by gonadotropins. Oktem, 2007 apoptosis Ovarian reserve Chemo-therapy Bcl-2 S1-P + BAX Caspase-2 Ceramide Apoptotic Oocytes David Hachey Reviewed in: Oocyte Apoptosis: Like Sand through an Hourglass. Yutaka Morita and Jonathan L. Tilly Developmental Biology 1999 Anti apoptosis TECHNIQUES OF FERTILITY PRESERVATION Fertility Preservation Options GnRHa ?  Oophoropexy  Cryopreservation   Egg  Embryo  Ovarian cortex GnRHa for fertility protection Evidence based medicine Ovarian transposition and oophporopexy Sella, T. et al. Am. J. Roentgenol. 2005;184:1602-1610 Cryo Preservation Techniques Embryo Freezing M II Freezing OTCP IVM The IVF revolution- from Louise Brown to fertility preservation •Woldwide 4.5 millions children born through IVF •20% of the children born through cryopreservation procedure IVF method- ovulation induction Stimulatory Phase GnRH-agonist Menses Gonadotropins 1 2 3 ……………………………………………………… hCG ~10 OPU ~12 IVF/culture ~14 – 16 Egg freezing Embryos cryopreservation GnRH-antagonist Menses Gonadotropins 1 2 3 ……………………………………………………… Tamoxifen, Letrozole GnRH-antagonist hCG OPU ~10 ~12 IVF/culture ~14 – 16 Egg freezing Embryos cryopreservation Rationale for use of Tamoxifen or Letrozole • Slow freezing • Vitrification The problem with M-II oocytes Cryopreservation Microtubule system chain Chromosomes Slow freezing 90-120 min. Vitrification Vitrification 1 sec. - 0.3 C/min - 50.000 C/min Oocyte Vitrification PROH sucrose water • Most patients cannot delay their cancer treatment • The only potential option in prepubertal girls • Menstrual cycle independent Banking ovarian cortex Sheep Human Biopsies Unilateral oophorectomy Ovarian cryopreservation protocol  Consultation  Unilateral Laparoscopic oophorectomy  transfer ovary immediately to IVF lab  Oocytes from tissue  all antral follicles observed on the ovarian surface are aspirated  19 gauge needle  Follicle contents immediately flushed into culture dishes ○ HTF-hepes medium supplemented with 10% synthetic serum  searched for cumulus-oocyte complexes.      Following the dissection of the ovarian tissue, the remaining media dishes were searched as well. Cortex removal Preparation of slivers Absorbing cryoprotectant ovarian cortex cryopreserved according to appropriate protocols (Gosden, 2000) Cryopreservation of ovarian cortical tissue The patient is in full remission Thawing the tissue Transplantation (Ortho/heterotopic) Follicles Isolation (IVG/IVM/IVF) Indication and patient survival (1996-2010) 30 25 Achsentitel 20 15 In red=deceased 10 5 0 Surgery: Laparoscopic oophorectomy Preop surgery postop • Discuss with anasthesiology • Combine other procedure?? • 20-30 min • General anasthesia • Quick recovery (hours) • Chemo can start next day Single port laparoscopic oophorectomy Oocyte collection: Aspiration Searching in the medium Revel, Fertil Steril 2003 Ovarian transplantation Risk of metastasis General Experience Hadassah Experience (NA & LH) Ischemia Risks of ovarian metastasis • Hodgkin’s disease – no ovarian metastases – >10 HD pts transplanted • No relapse • Sarcoma – Low risk of Ovarian metastases • Metastatic Breast cancer – Possible ovarian metastases • Non Hodgkin’s Lymphoma – Possible ovarian metastases Techniques to exclude ovarian metastasis • Imaging – sonography, CT • Laboratory – Immunohistochemistry – PCR • to exclude single malignant cells • xenotransplantation of small pieces of frozen/ • thawed ovarian tissue in SCID Mice • in cases in which ovaries are at risk to contain malignant cells, sophisticated techniques are required to exclude ovarian metastasis. Orthotopic re-transplantation of ovarian tissue Ovarian orthotopic transplantation st Nasser Arin 1 Hadassah patient Ariel Revel Hadassah, July 2006- Sept 2010 NA • Born 1982, Thalassemia beta Major • Conservative treatment • 2001 (age 19) – Laparoscopic Oophorectomy • ovarian cryopreservation – 6 oocytes (4 GV; 2 GVBD) Oocytes thawed IVM attempted No maturation – Successful BMT from sister • Ovarian failure – Amenorrhea – Elevated FSH – Ovarian cortex (at transplantation) – no follicles found Thawing Flushing out cryoprotectant RIGHT RIGHT MICRO ORGANS: Universal Biological “constant” Rational: Epithelial cells are always epithelium supported by a stroma connective tissue or stroma Revel et al, 2011 Micro-Organs (MOs) Organ fragments of microscopic thickness that preserve the organ micro-architecture and are of such dimensions that no cell is more than 150 microns away from a source of nutrients. Revel et al, 2011 Hadassah ov –transplant sonographic followup 5 month 3 month 7 months 7 months Hadassah ovarian –transplantation Oocytes from IVF Human micro-organs proliferate in-vitro for at least 30 days Revel et al, 2011 Rat ovary micro organ transplant Revel et al, 2011 Cutting ovarian cortex with a specially designed apparatus into 350 micrometer thick MOs A B C D E F Revel et al, 2011 Regular vs microorgan ovarian cortex Revel et al, 2011 Hormonal levels before and after ovarian transplantations FSH 35 Estradiol 2195 30 25 1700 20 1090 15 10 570 5 70 70 70 70 0 before oophorectomy after oophorectomy before transplant after transplant 1 before transplant after transplant 2 before transplant after transplant 3 1 2 3 NA followup Table 1: patient ovarian transplantations and IVF cycle results date 25 July 2006 20.10. 06 28.12. 06 28.2.07 15 April 2008 22.10. 08 30.11. 08 6.1.09 28.1.09 30 March 2009 14.7.09 6.8.09 6.10.09 13.1.10 number of aspirated ooc ytes right left 0 1 0 2 1 PB GVBD GV degenerative 2 1 1 day of fertilized embryo transfer ET ovarian transplantation- laparotomy 2 1 2 1 day 3 day 2 Embryo 1 Embryo 2 beta hCG 4C c 2C c 6C c negative negative ovarian transplantation- laparoscopy 1 1 1 1 0 2 2 1 2 1 1 1 1 1 0 0 day 2 day 3 2C c 5C c negative negative ovarian transplantation- laparotomy – microorgans to right neo-ovary 0 0 0 0 0 1 0 2 1 2 0 0 1 2 day 2 2C a 2C b positive Collaboration with the Tel Aviv medical center Fouad Azem, MD Dalit Ben Yossef, PhD Ami Amit, MD Joseph B. Lessing, MD LH, 28 yo ELAL air hostess • 2000 – May- Hodgkin’s lymphoma 3a – June -ovarian tissue cryopreservation – December – completed 6 ABVD and GnRHa LH, cont’ • 2005 – July- PET CT-relapse of Hodgkin’s lymphoma – Aug – chemotherapy DVIP – December – autologous BMT • 2006 – POF • 2010 – June- Micro-organ ovarian transplantation » Revel- Mitrani team Ovarian cortex Avascular transplantation Whole ovary transplantation The major problem with ovarian cortex is the loss of follicles by apoptosis Whole organ transplantation could be the solution Revel Arav Elami Supported by the Israeli Academy of Sciences MRI of transplanted sheep ovary Ariel REVEL, MD IVF unit, Department of Obstetrics and Gynecology Hadassah Hebrew University Hospital Oocyte retrieval following autotransplantation of intact sheep ovary Revel, 2004, Arav, 2005 Arav A, Gavish Z, Elami A, Revel A, Gosden RG, Patrizio P. A Six-Year Record of Ovarian Function after Orthotopic Vascular Transplantation of Whole Cryopreserved Sheep Ovaries RMB on line 20(1): 48-52, 2010 Revel, 2004, Arav, 2005 Egg donation What if no cryopreservation was performed? Gregor Mendel - Collaboration Reprofit, Brno, Czech Republic Prof. MUDr. Ladislav Pilka, DrSc. Scientific Dir- Reprofit International Hadassah Reprofit results • • • • • More than 300 patients treated Pregnancy rate (bHCG +ve ) 55% Ongoing pregnancy rate 49% Implantation rate 29% Multiple pregnancy rate 40% – 30% twins + 10% triplets Fertility preservation in Israel • Permitted – Cancer Pts • Embryo, oocyte and ovary cryop • Ovarian transplantation – For healthy patients • Oocyte cryopreservation age 30-41 • Funding – For cancer patients • In the health basket since 2011 Conclusions Cancer treatment causes infertility  This can be measured Fertility consequences should be discussed with patients and parents  prior to chemotherapy Fertility preservation should be offered  Various options to suit individual needs should be available here The future of fertility preservation • • • • • Less gonadotoxic treatment protocols Ovarian apoptosis inhibitors Oocyte maturation and vitrification Whole ovary? Testicular cryopreservation Mentorship Team work Hadassah • MD – – – – • Collaborations Neri Laufer Alex Simon Benny Reubinof Yoel Shufaro • Aby Lewin • Mohamed Fatum • Assaf Ben Meir              –     Naomi Rabinovitch Moriah Koler Hanna Achache   Nurses • • • • –  Michal Bloch Einat Zivi Doron Kabiri Erez Berkovitch  Nitshia Geva Zurit Malamud Miri Heringman Hagit Gugenheim  •  • • • •  Einat Aizenman Meital Leibovich Yael Siminovsky Eli Girsh Zehava Guatta-Rangini – – Anat Safran Anat Porat-Katz   Amir Elami Ed Mitrani Israel vlodavsky Yuval Dor Eli Keshet Amir Arav Amir Bor Diti Natan Saar Yavin Israeli fertility preservation SIG  IVF lab Dina Ben Yehuda/Hematology Tamar Peretz/Oncology Reuven Or/BMT Michael Weintraub/Pediatric Hemato – Oncology Pathology/ Diana Prus Hebrew university  Fertility preservation  Hadassah Treating units Sheba/ Dror Meirow & Hila Raanani Rambam/Zeev Blumenfeld Tel Aviv/ Fouad Azem Rabin/ Avi Benaroush International Society for fertility preservation