Analysis of post-warming degeneration & apoptosis following porcine ovarian tissue vitrification using the ohio-cryo device

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1 Analysis of post-warming degeneration & apoptosis following porcine ovarian tissue vitrification using the ohio-cryo device e-poster: 363 Congress: ESHRE 2008 Type: Scientific poster Topic: ART, laboratory: cryopreservation of gonads Authors: MeSH: A. Kader, C. Biscotti, T. Falcone; Cleveland/US Oocytes [A ] Cryopreservation [E ] Infertility, Female [C ] Fertility [G ] Keywords: Cryopreservation, Ovary, Vitrification, Ovarian tissue Any information contained in this pdf file is automatically generated from digital material submitted to e-poster by third parties in the form of scientific presentations. References to any names, marks, products, or services of third parties or hypertext links to third-party sites or information are provided solely as a convenience to you and do not in any way constitute or imply ESHRE s endorsement, sponsorship or recommendation of the third party, information, product, or service. ESHRE is not responsible for the content of these pages and does not make any representations regarding the content or accuracy of material in this file. As per copyright regulations, any unauthorised use of the material or parts thereof as well as commercial reproduction or multiple distribution by any traditional or electronically based reproduction/publication method is strictly prohibited. You agree to defend, indemnify, and hold ESHRE harmless from and against any and all claims, damages, costs, and expenses, including attorneys fees, arising from or related to your use of these pages. Please note: Links to movies, ppt slideshows and any other multimedia files are not available in the pdf version of presentations.

2 1. Purpose Ovarian tissue cryobanking for cancer patients is now offered in many reproductive centers. The ovarian tissue is mainly intended for future transplantation for fertility preservation purposes. This is of highest importance in cancer patients who are planned to recieve gonadotoxic treatment.(1,2) Ovarian tissue is currently conventionally preserved by slow freezing methods. Vitrification at its current standards is now being anticipated to at least match if not excel in oocytes' cryopreservation.(3,4) Optimum vitrification should enhance the post-thaw viability of the specimens by minimizing or eliminating ice crystals formation. This is especialy important with oocyte cryopreservation. Also, it is more convenient and cost effective as it can be executed in much shorter time than slow freezing, with no need to expensive cryoplanner devices. However, to achieve this, specimens should be rapidly and effectively contacted and cleared from vitrification solution series, which have high concentrations of cryoprotectants. Also, a rapid cooling and heating rates should be achieved. The vitrification of any whole organ would be expected to come at the expense of slowing the heat transfer to the organ cores to be only equal to the organ temperature conduction. The Ohio-Cryo is a novel device that allows the vitrification of any kind of processed tissue or a big load of cells in a closed system (US provisional patent 61/073,392). Ohio-Cryo device: Exploded view The device allows rapid handling of tissue with a proper control on contact time with different types of media, rapid and even heat transfer & the vitrification in a minimal volume of media.

3 The device at its current conception was designed to accomodate up to one processed hemiovary per device. This seems a mandatory requirement for fulfilling transplantation purposes. Currently, successful vitrification of ovarian tissue was mostly achieved with mice ovaries in cryotops.(5) However, using tiny loading devices may not be applicable for human ovarian tissue vitrification as it will require an excessively large number of loading devices. Human ovarian tissue was successfully vitrified with solid surface vitrification.(6) However, the method may not be considered as a closed method, nor does it handle the tissue contact with the media, and may not be practical for highly fragmented, better equilibrated specimen as compared to the ohio cryo handling the ovarian sand. The purpose of our study is to provide some preliminary evaluation on the use of the Ohio Cryo vitrification system, in vitrifying tiny ovarian tissue fragments. Evaluation parameters were focused on the apoptotic index and the histological degenerative changes. The use of the device was compared with the fresh tissue, tissue damaged by direct contact with liquid nitrogen with no cryoprotection and the spontaneously induced apoptosis by prolonged hypoxia. 2. Methods and Materials Seven porcine ovaries from 4 different pigs were collected immediately post-mortem. After removal of the medulla, the ovarian cortex was carefully sliced into about 0.5 mm thick slices, then swiftly chopped at 0.5 mm intervals in 2 perpendicular planes using Mcwillian tissue chopper to make tiny ovarian cubes of almost mm, or what we can refer to as the "Ovarian sand". From each ovary and after fragmentation, a sample was formaline fixed as a fresh control. Another sample was incubated in leibovitz media in a 4 degree C refrigerator for 5 days to spontaneously degenerate, then formaline fixed.(7) Another sample was directly plunged into liquid nitrogen to induce cryo-damage. The remaining sample was vitrified using the Ohio-Cryo. After warming, tissues were incubated for 2 hours in culture media at 37 degree C then fixed. All samples were paraffin embedded & cut at 5um thickness. For each sample, one slide was stained with H& E for histological assessment of degenerative changes. Degenerative changes were judged according to Wood s criteria with slight modification (7). In each section, each follicle was given a score between 0 to 2. Given scores were 0 for no degeneration, 1 for slight degenerative changes & 2 for severe degenerative. An adjacent section was assessed for apoptosis using TUNEL. In a defined visual field, all granulosa cell were given a score as 0 for absent Tunel signal, 1 for mild, 2 for moderate & 3 for intense signal. Ten different fields were randomly chosen in a given slide (8) The Following animations explain the use of the Ohio-Cryo to vitrify the Ovarian sand: [Assembly of the Filtration Member] 1- Assembly of the filtration member [Vitrification] 2- Vitrification [Warming] 3- Warming 3. Results The histologic degeneration scores for primordial follicles was 0.15± 0.16for fresh samples, 0.17±0.23for vitrified samples, 0.56± 0.55for the cryo-induced damage samples & 1.75± 0.08for the degenerating samples. The degenerative scores for preantral follicles were 0.47± 0.4for fresh samples, 0.36± 0.27for vitrified samples, 1.00± 0.34for the cryo-induced damage samples & 1.52± 0.23for the degenerating samples. As for the TUNEL, the apoptotic index was 0.23± 0.08for the fresh samples, 0.32± 0.05for the vitrified samples, 1.01± 0.34for the cryo-induced damage samples & 1.34± 0.59for the degenerating samples.

4 TUNEL & Histological assessment There was no significant difference between vitrification & fresh tissue, with a pvalue of 0.14 for TUNEL, 0.82 & 0.64 for the degenerative scores of primordial & preantral follicles respectively.these indices were all significantly higher than fresh & vitrified tissues when cryo-damage or spontaneous degeneration were applied. Compared to fresh tissue, TUNEL showed a significant increase in the apoptotic index with a pvalue of for cryo-damaged samples & for degenerating samples. Histologic degeneration assessment score showed an increase in primordial follicles degeneration compared to fresh samples, with with a pvalue of 0.22 (NS) for cryo-induced damage & a pvalue <0.001 for spontaneously degenerating tissue. Preantral follicles showed an increase in the same index that was significant for cryo-damage (p = 0.043) & spontaneous degeneration ( p=0.015) as compared to fresh control. 4. Conclusion The vitrification of ovarian tissue in the Ohio-Cryo did not induce significant degenerative changes nor increased the apoptotic index in the ovarian tissue as evidenced by H&E

5 examination and TUNEL assay. With the swiftness of its handling a relatively large volume of processed ovarian tissue for vitrification in a closed system, it may provide a suitable solution for a reliable ovarian tissue vitrification. The device together with the processing technique provide a unique approach that is not currently being proposed in the industry. The ovarian sand created is most suitable for rapid and even contact equilibration and removal of high cryoprotectants concentrations, if compared to larger unprocessed or less processed specimens. Current closed loading devices are either limited in volume so unable to accomodate a similar tissue volume or large with no specific adjustments to compensate for an anticipated compromise in vitrification outcome by low endogenous heat transfer. The device also allows direct contact of the specimen to a rapidly cooling solid surface in an even fashion. With the porcine ovarian volume similarity to human ovary, we would expect to accomodate one human ovary in at least 2 Ohio-Cryo devices. 5. References 1- Donnez J, Squifflet J, Van Eyck AS, Demylle D, Jadoul P, Van Langendonckt A et al. Restoration of ovarian function in orthotopically transplanted cryopreserved ovarian tissue: a pilot experience. Reprod Biomed Online 2008;16: Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005;353: Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A et al. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed Online 2008;16: Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a meta-analysis. Fertil Steril 2006;86: Kagawa N, Kuwayama M, Nakata K, Vajta G, Silber S, Manabe N et al. Production of the first offspring from oocytes derived from fresh and cryopreserved pre-antral follicles of adult mice. Reprod Biomed Online 2007;14: Huang L, Mo Y, Wang W, Li Y, Zhang Q, Yang D. Cryopreservation of human ovarian tissue by solid-surface vitrification. Eur J Obstet Gynecol Reprod Biol Wood TC, Montali RJ, Wildt DE. Follicle-oocyte atresia and temporal taphonomy in cold-stored domestic cat ovaries. Mol Reprod Dev 1997;46: Hussein MR, Bedaiwy MA, Falcone T. Analysis of apoptotic cell death, Bcl-2, and p53 protein expression in freshly fixed and cryopreserved ovarian tissue after exposure to warm ischemia. Fertil Steril 2006;85 Suppl 1:

6 6. Mediafiles Assembly of the Filtration Member A disposable filter is brought to the front of the device and held in place by an O ring the comes in place with a special O ring applicator. Ohio-Cryo device: Exploded view

7 TUNEL & Histological assessment Vitrification First in Equilibration media is brought into contact with the tissue. The filtration device is then used to remove it. The vitrfication media is then added, and after 60 seconds, the filtration device is placed and the vitrification media is removed. The device is capped, and brought to the liquid nitrogen.

8 Warming The device is brought out of liquid nitrogen, then immediately brought to contact a water bath at 37 C. After 15 seconds, the device is uncapped, the filtration device is removed and thawing media is added. After 1 minute, the filtration device is removed, dilution media is added. After 5 minutes, the filtration device removes the media and the tissue is brought in contact with the washing media for at least 10 minutes.

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