Ovary Transplantation, VS Oocyte Freezing

Outline of Talk Ovarian Tissue Cryopreservation Oocyte Cryopreservation Ovary Tissue vs Oocyte Freezing

It All Begins Here The Epiblast

Primordial Germ Cells Primordial Germ Cells - the origin of all germ cell lineage - expressing pluripotent genes - Undergoing genome-wide reprogramming (massive DNA demethylation and conversion of histone modifications) Yamaji et al (2008) Nat.Genet. - When transferred into testis or ovary, PGCs give rise to functional sperm or oocytes.

Oocyte Freezing For Fertility Preservation Versus Ovarian Tissue Freezing The intrinsic fertility of the human oocyte very gradually drops from the late teens to the early 30s. But then at around age 35, there is a dramatic fall in fertility of 10% per year, so that by age 40 only about one percent of oocytes are capable of resulting in a live baby.

Fertility Of The Human Oocyte Related to Age r = 1/(a + exp(b*(t - c)))

COH Vs Natural and Mini IVF

Intrinsic Fertility vs Stimulated Fertility Live Baby per oocyte with no stimulation for under age 37 is 26 %. In index papers with COH (Patrizio and Cobo) it is only 6-7 %.

Six years' experience in ovum donation using vitrified oocytes (Cobo et al 2015) You want 30 frozen oocytes to Have 84 % success. You get 200,000 oocytes to with ovary tissue. 40 frozen oocytes to have a 97 % success. Cobo et al

Age of Menopause in Japanese Women Yasui et al 2012

Age of Menopause in After Unilateral Oophorectomy Yasui et al 2012

World Wide Results Ovarian Tissue Freezing Worldwide there have been over 148 documented and peer reviewed live births from frozen ovary tissue, but the majority have been case reports. Nonetheless based on the tabulated series of young patients (i.e under 35 years old when frozen) in Brussels, Israel, Denmark, and our center, live birth rates vary from 41% to 76%.

Results of Fertility Preservation With Ovarian Tissue Freezing Most are spontaneous pregnancies not requiring IVF. These were young women who had ovary cortex frozen prior to goonadotoxic treatment for cancer or other diseases.

Results of Fertility Preservation With Ovarian Tissue Freezing It is not known yet what the success rate would be for healthy women just wishing to delay childbearing. Patients almost always have return of ovarian function 5 months post transplant with regular menstrual cycling.

Results With Ovary Tissue Freezing AMH rises to very high levels as the FSH declines to normal or near normal. Four to eight months later, the AMH declines to very low levels. Nonetheless, the grafts remain functional for up to 5 years or longer.

Gamete and Embryo Freezing For Fertility Preservation Versus Ovarian Tissue Freezing Patients almost always have return of ovarian function 5 months post transplant with regular menstrual cycling. AMH rises to very high levels at the same time that the FSH declines to normal.

RELATIONSHIP OF FSH AND AMH AFTER OVARY TRANSPLANTATION

Gamete and Embryo Freezing For Fertility Preservation Versus Ovarian Tissue Freezing Four to eight months after the FSH returns to normal, the AMH declines again to very low levels. Nonetheless, the ovary grafts remain functional for up to 5 years or longer despite very low AMH levels.

Ovary Tissue Freezing vs Oocyte Freezing Thus ovary tissue cryopreservation, at least for young cancer patients, is a robust method for preserving a woman s fertility. How does that compare to oocyte cryopreservation for preserving a woman s fertility?

Oocyte Freezing vs Ovarian Tissue Before vitrification the only option for cancer patients from 1996 until 2006 was to freeze ovarian tissue as advocated by Gosden. This was successful because the oocytes in primordial follicles are small (30 microns compared to 140), and arrested in early meiosis with no metaphase spindle.

Oocyte Freezing vs Ovarian Tissue However for mature oocytes retrieved via ovarian stimulation, the slow freeze technique was very ineffective. The water content of the mature oocyte could only be reduced from 70% to 30%, allowing ice crystal formation still to damage the oocyte.

Oocyte Freezing vs Ovarian Tissue The smaller oocyte in the primordial follicle, with no spindle could more easily tolerate the resuced ice crystal formation inside the oocyte with slow freeze. The mature oocyte could not tolerate this. However with vitrification, there would be no ice crystal formation at all to worry about. Therefore the larger mature metaphase II eggs could now be safely cryopreserved with vitrification.

VITRIFICATION VS SLOW FREEZE

Slow Freeze VS Vitrification Vitrification completely avoids any ice crystal formation by using a very dense concentration of cryotprotectant, and an ultra-rapid speed of cooling. Slow freeze relies on partial and gradual removal of water from the cell by encouraging ice crystal formation preferentially on the outside of the cell. The freezing point is higher outside the cell (because of lower osmolality), thus drawing water out of the cell ( reducing water from 70 % to 30 %) as the cell cools.

Slow Freeze Protocol For Embryos 1.5 Molar PROH at Room Temperature 10 Minutes. 1.5 Molar PROH and 0.1 Molar Sucrose <15 Seconds. Load into Straw Between Air Bubbles and Heat Seal. Start at 20 degrees C and drop at -2 degrees C/minute. Hold at -8 degrees C for 10 minutes and Seed. Drop at -.3 degrees C/minute to -30 degrees C. Then Drop -50 degrees C/minute to -150 degrees C. Then Plunge into Liquid Nitrogen.

Thaw Following Embryo Slow Freeze Plunge Straw into 30 degree Water Bath 40 seconds. Cut Heat Sealed End of Straw and Expel (at 20 deg C): Into 1.0 Molar PROH and o.2 M Sucrose 5 minutes. NOTE: ALL FREEZE MEDIA: 20% PROTEIN Transfer into 0.5 Molar PROH & 0.2 M Sucrose 5 min. Transfer to 0.2 Molar Sucrose for 10 minutes. Transfer to Isotonic Hepes 10 min. Place in culture at 37 degrees C.

Vitrification 3min 3min 9min 15sec x 4 Cryotop WS + ES1 + ES2 ES3 (20μl for each drop, at R.T.) VS1 VS2 VS3 VS4 (7.5%EG,7.5%DMSO in ES) (15%EG,15%DMSO,0.5M Suc in VS) Thawing (1M Suc in TS, 0.5M Suc in DS) LN2 TS 1min, 37 DS 3min WS1,2 5min 2 P1 2hrs Before ICSI

Oocyte Freezing vs Ovarian Tissue Cobo s live baby rate per oocyte was 6.5% quite similar to that of Patrizio with fresh oocytes. Nagy in 2009 showed the same good results in the U.S. Cobo computed that for a woman to have a 97% chance of having at least one baby, 40 oocytes needed to be preserved.

Worldwide Well Documented Oocyte Freeze Results # Oocytes Frozen # Oocytes Survived LBR/Oocyte # Total Live Babies Cobo et al. 2015 42,152 37,725 6.5% 2,306 Martin et al. 2010 2,470 X 7.3% 181 1,044 X 5.0% 52 Emanuele et al. 2014 74,418 X 1.8% 1,342 TOTAL 120,084 X 3.2% 3,881

Live Baby Rate Per Oocyte Silber et al. 2017 (Natural) Kato et al. 2018 (Clomid) Cobo et al. 2015 (Frozen COH) Patrizio et al. 2009 (Fresh COH) Lemmen et al. 2016 (Fresh COH) (18%) 40yrs (26%) 35yrs (18.5%) 40yrs (32%) 35yrs (6.5%) 40yrs (5.8%) 40yrs (4.9%) 40yrs

Vitrification For Oocyte or Embryo Freezing Place in 7.5 % DMSO and 7.5 % EG with 20 % Protein : Equilibration Media (ES). All at 23 degrees C. <15 minutes in gradually increasing concentrations. Watch embryo contract, re-expand, and sink. Place in 15.0% DMSO and 15.0 % EG with 0.5 Molar Sucrose, and 20 % Protein, in Hepes (Vitrification Solution-VS) for 60 to 90 seconds (or more). Watch embryo contract, but float. Must help it, and then it sinks wait for it to RE- EXPAND. VS solution is NOT toxic. Wait for re-expansion. Place on tip of Cryotop. Draw off all fluid from microdrop. Dunk cryotop in liquid nitrogen in Styrofoam box, swishing back and forth.

Preparation of each solution

ES Equilibration: shrinks, re-expands, and sinks

VS 1 washing of oocyte/embryo

VS Solution Retrieve floating oocyte/ embryo that floated to the surface of VS1 and contracts and maintain it near the tip of the pipette with minimal VS1 back loaded. Blow the oocyte/ embryo again to the bottom of VS1. Watch it contract and float. Then it re-expands and sinks.

VS2 washing of oocyte/embryo (Step 5-8)

Do not vitrify until oocyte reexpands (usually 60-90 seconds)

Cryoprotectants Are Not Toxic It is only the severity and rapidity of osmotic pressure gradient that makes them appear to be toxic. Poor results are obtained when the oocyte is not left in the VS media long enough for fear of toxicity. Look at the oocyte. Watch it re-expand.

The Bridge technique for Oocyte Freezing

Preparing Embryo With Equilibrum(ES) Solution Before Finally Vitrifiction(VS) and 0.5 M SUCROSE

5 minute

12 minute Watch embryo contract, re-expand, and then sink!

Ovary Transplant Oocyte Clip 1

Thawing of Vitrified Oocyte Thaw (TS) solution is 1.0 molar sucrose. Place oocyte carrier instantly ( within one second after removing from liquid nitrogen) into TS solution at 37 degrees. Oocyte floats. Does not expand or contract. After one minute place in (DS) diluting solution (0.5 molar sucrose) for 3 minutes at room temperature. Oocyte will sink, and shrink. Then it will begin to re-expand. Then wash solution, and culture several hours while oocyte fully re-expands.

Ovary Transplant Oocyte Clip 2

Ovary Transplant Oocyte Clip 3

Disadvantage of Oocyte Freezing For Cancer Cancer patients have little time to preserve their fertility. They certainly need more than one oocyte retrieval. Many will have already undergone initial chemotherapy and have no developing follicles to respond to ovarian stimulation.

Many leukemia patients will have already undergone this initial chemotherapy However, many primordial follicles will have survived this initial chemotherapy, and can be preserved with ovarian tissue freezing. So in general we think ovary tissue freezing is the best approach for cancer patients.

COST For healthy women just wanting to put off child bearing, oocyte freezing may at first seem easier. But we still would want to do at least 3 cycles to give them some measure of assurance. In the United States this could add up to 45,000 dollars or more.

Ovary Freezing vs Oocyte Freezing A simple ovary freeze would be less than 6,000 dollars, and require just one simple procedure and no hormonal stimulation. Furthermore transplanting back ovarian tissue can give them hormonal function even after they might otherwise be in menopause 20 years later.

For Healthy Women: Ovary Freezing vs Oocyte Freezing Many female business executives and physicians have chosen this otherwise less popular approach because it is quick and simple. So each woman will have to decide which method of preserving her fertility is most appropriate for her individual situation.

Ovary Tissue Transplants

Primordial Follicles All Located in Cortex

Follicles Develop Inward Toward the Softer Ovarian Medulla

40 x

PREPARATION OF OVARIAN CORTICAL SLICES

Ovary Transplant Oophorectomy & Vitrification

Ovary Transplant Vitrification Clip 4

ULTRA-THIN SLICE ON METAL GRID: GOOD FOR VITRIFICATION AND RAPID REVASCULARIZATION

Vitrification of Ovarian Tissue ES Solution is the same as for Embryo or Oocyte vitirfication, 15 % cryoprotectant (7.5 % DMSO and 7.5 % EG with 20 % Protein in Hepes. VS Solution however is 40 % cryoprotectant (20 % DMSO and 20 % EG, instead of 15 % DMSO and 15 % EG). Leave tissue slices in ES Solution for 25 minutes. Then place in VS solution until the thin tissue slices fall to the bottom of the centrifuge tube (usually < 15 minutes).

Ovary Transplant Tissue Slices in Test Tube

Vitrification Procedure (Thaw) For thaw, metal strip is immersed directly into 40 ml of 1.0 molar sucrose at 37 degrees C for at least 1 minute. Like with oocyte thaw, at first the tissue slices float. Must be stirred. Then transfer into 0.5 molar sucrose for 5 minutes at room temp. Now they sink. Then wash twice. No ice crystal formation observed.

Fresh Human Tissue Pre-Freeze

HumanTissue Post Vitrification

Post-thaw Survival of Oocytes: Vitrification vs Slow Freeze Ovarian tissues Number Collected (%) of oocytes Surviving Vitrified 8 1122 1000 (89.1%) Fresh 2 358 329 (91.9%) Slow- Freeze 6 821 342 (41.7%)

Ovary Transplant Tissue Quilting Clip 1

Ovary Transplant Transplant Clip 5

FSH Levels (miu/ml) RETURN OF FSH TO NORMAL AFTER FROZEN OVARY GRAFT

Relation of Recovery of FSH to AMH and Over Recruitment

Preg Fresh Transplant: 7/9 (78%) Preg Frozen Transplant : 10/13 (77%) Healthy Babies from Fresh Ovary Transplant: 11, from 7 of 9 transplants. Healthy Babies from Frozen Ovary Transplant: 13, from 10 of 13 transplants (2 from 4 vitrified, and 11 from 9 slow freeze. ). Total: 24 healthy babies

Ovary Tissue Freeze Transplants Date of Transplant Age At Transplantation Age At Freeze Pregnant Live Birth Time Until Pregnancy (Days) Miscarriages Duration of Ovarian Function (Days) 3/6/07 26 24 YES YES 174 675 (Ended) 1/13/09 31 20 YES YES 272 885 (Ended) 6/9/09 29 24 YES 276 1 561 (Ended) 6/17/11 33 20 NO 1155 (Ended) 10/12/12 33 31 YES YES 481 1882 (Still Functioning) 3/29/13 32 25 YES YES 243 794 (Ended) 4/5/13 33 30 YES YES 665 1707 (Still Functioning) 4/12/13 25 18 YES YES 502 1700 (Still YES YES 998 Functioning) YES YES 1578 10/1/13 29 28 NO 1528 (Still Functioning) 10/7/13 39 24 YES YES 1287 1522 (Still Functioning) 7/21/15 28 25 NO 870 (Still Functioning) 8/5/15 32 21 YES YES 343 855 (Still Functioning) 9/18/14 36 20 YES YES 473 1176 (Still YES YES 908 Functioning) Totals 13 Cases 13 Babies 10 Pregnant (77% live baby pregnancy) 1 Miscarriage 4 Vitrification 9 Slow Freeze YES

Benefit of Ovary Tissue Freeze For Cancer Patients vs Eggs Less (no) time to wait before cancer treatment. No problem if chemotherapy has already begun. One tenth the cost. Less troublesome than going through ovarian hyperstimulation. Return of hormone function as well as fertility of eggs.

No problem if chemotherapy has already begun.

Why Do These Grafts Last So Long? Very low antral follicle count. Very low AMH. Only one fourth to one sixth of a single ovary.

TRANSPLANT STILL WORKING (8 YEARS): AGE 46

Leukemia-January 1997: 24 Years Old

Cured and Married October 2012: 41 Years Old Now

1 st Baby Born (11/24/17f)

DOB: 6/11/73, DOF: 7/97, DOT: 10/7/13 (24 year old tissue) Diagnosis: Leukemia 1 LBB (11/24/17f)

Leukemia, Tissue Frozen 15 Years Earlier

First Baby DOB: 5/8/15

Second Baby DOB: 9/15/16

Thrid Baby DOB: 4/21/18

LEUKEMIA CASES SUMMARY Date of Transplant Age At Transplantation Age At Freeze Diagnosis Pregnant Live Birth Time Until Pregnancy (Days) Miscarriages Duration of Ovarian Function (Days) 4/12/13 25 18 Leukemia YES YES 502 1700 (Still YES YES 998 Functioning) YES YES 1578 10/7/13 39 24 Leukemia YES YES 1287 1522 (Still Functioning) 7/21/15 28 25 Leukemia NO 870 (Still Functioning) Totals 3 Cases 4 Babies 4 Pregnancies 0 Miscarriage 2 Became Pregnant

OTF Breast Cancer Patient 35 y.o. And not yet ready to have chldren EVEN IF SHE DID NOT HAVE CANCER SHE WILL BE INFERTILE SOON

AUTO-IMMUNE DISEASE Autologous Ovary Transplant For Multiple Sclerosis

TISSUE PRESSURE Over-recruitment of primordial follicles, followed by depletion. Decreased primordial follicle recruitment with decreasing ovarian reserve. Long duration of transplant function despite very low AMH. Late menopause in multips. AMH decreases in pregnancy. Think about the horse ovary.

Necessity of Oocyte Preservation 36.1 y.o at 1 st transplantation (embryo) Resulting in pregnancy 483 Women 38.3 y.o. at initiation of Tx for 2 nd baby Expected AMH at 38.3 y.o For pregnancy and breast feeding Teramoto 2016

WHAT IS IT THAT LOCKS AND UNLOCKS THE PRIMORDIAL FOLLICLE: TISSUE PRESSURE