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What’s in the Mix? Comparing Freezing Media and Protocols for Ovarian Tissue

By Claus Yding Andersen

Background:

Our decision on which cryoprotectant compositions to use for freezing ovarian tissue dates back to the inception of our program at the turn of the last century. At that time, based on available research (1), studies using sheep ovaries indicated that the best results were achieved with the small, low-molecular-weight cryoprotectants DMSO or Ethylene Glycol (EG) at concentrations of 1.5 mol/L. We tested four different cryoprotectant combinations using mouse ovaries (N = 6 per group), incorporating 0.1 mol/L sucrose as a cell non-penetrating agent to reduce extracellular water activity (2). Based on morphological evaluation, the highest survival rates were observed with a solution containing 1.5 M EG, 0.1 M sucrose, and 10 mg/mL human serum albumin (HSA). Subsequently, we assessed the quantitative survival of follicles after slow freezing and thawing, using this cryoprotectant mixture in human ovarian cortex tissue. The evaluation was conducted with the vital stain Neutral Red (3). Our findings demonstrated a 7% loss of non-growing follicles post-hawing, with survival remaining consistent over an 18-year period for tissue stored in liquid nitrogen (3).

Cryoprotectant Preparation and Quality Control

A single batch of 800 mL cryoprotectant medium is prepared in-house, with a shelf life of three months. The solution is formulated using GLP-approved PBS, commercially sourced EG and sucrose, and is sterile-filtered and tested for sterility and endotoxins before use. Both tests must return negative results (endotoxin levels below the assay detection limit). Human serum albumin is added only on the day of use.

Cryoprotectant Composition: 1.5 mol/L Ethylene Glycol; 0.1 mol/L Sucrose; 10 mg/mL HSA

Slow-Freezing Temperature Profile

  • Equilibration: Incubation on ice with rotation (0–2°C for 30 min)
  • Cooling: -2°C/min until reaching -9°C
  • Manual Seeding: Induction of extracellular ice crystal formation using a pre-cooled cotton swab
  • Controlled Cooling: -0.3°C/min until reaching -40°C
  • Rapid Cooling: -10°C/min until reaching -140°C
  • Storage: Transferred to liquid nitrogen (-196°C)

References

  1. Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod. 1996;11:1487
  2. Rosendahl M, Schmidt KT, Ernst E, Rasmussen PE, Loft A, Byskov AG, Andersen AN, Andersen CY. Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique. Reprod Biomed Online. 2011;22:162
  3. Kristensen SG, Liu Q, Mamsen LS, Greve T, Pors SE, Bjørn AB, Ernst E, Macklon KT, Andersen CY. A simple method to quantify follicle survival in cryopreserved human ovarian tissue. Hum Reprod. 2018;33:2276

 

Answer by Debra Gook

Method: 1.5 M propanediol (PROH) and 0.1 M sucrose in Hepes buffered HTF with 10mg/ml human serum albumin with dehydration for 90 minutes at 22°C and slow freeing rate manually seeded at -7°C.
My initial studies developing the slow freezing method using propanediol to dehydrate human oocytes indicated that it was safe for oocytes and therefore likely to be suitable for preserving oocytes within the primordial follicles of the ovarian tissue. Also, that if trace amounts remained after thawing the tissue, which was my major concern in choosing a cryoprotectant for tissue, propanediol would be oxidized to pyruvic and lactic acid.
A number of propanediol and sucrose concentrations and dehydration times were tested together with one and two step addition of cryoprotectants. A painstaking histological evaluation of human tissue showed 91% normal intact oocytes with 94% normal organelles and <10% vacuolation, together with 74% intact granulosa cells however only 45 % of the stromal tissue was intact1. At a similar time, a study by Newton’s showed the rate of cryoprotectant infiltration into tissue was temperature dependent. Her fundamental work showed that at 4°C all cryoprotectants (DMSO, ethylene glycol and PROH) only reach a tissue saturation of 80% in 60 minutes whereas at 37°C PROH was at 99% saturation within 30 minutes while the other cryoprotectants plateau at 80%2. She had also shown the benefit of a non- permeating cryoprotectant; sucrose, which at appropriate concentration resulted in low lactate dehydrogenase levels, (a measure of cell lysis) in tissue after cryopreservation. Newton’s results aligned with our histological study where we had found 90 minutes at 22°C to give the best survival of primordial follicles.
I continued to attempt to improve stromal survival by altering the freezing rates unfortunately to the detriment of the primordial oocyte3, and this work was subsequently abandoned. Electron microscopy of tissue frozen with the above method also showed good preservation of organelles, especially mitochondria within the oocytes and gap junction between granulosa cells and the oocyte3. To evaluate potential function of the follicle in the frozen tissue, I subsequently xenografted small fragment of frozen thawed tissue under kidney capsule in immunodeficient mice. Multiple follicles developed and following hCG some follicles contained MII oocytes4 and ovulated5. The reproducibility of the freezing procedure across multiple patient’s tissue confirmed the method as a robust, consistent method for preserving potential function in a large number of primordial follicles within the small slices of tissue6. This has since been confirmed with our clinical data of frozen thawed tissue grafted to an abdominal site7.
References
  1. Gook D.A., Edgar D.H and Stern C. (1999) Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1,2-propanediol. Hum Reprod 14, 2061-2068
  2. Newton H., Fisher J., Arnold J.R. P., Pegg D.E., Faddy M.J., and Gosden R.G. (1998) Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod 13, 376-380
  3. Gook D.A., Edgar D.H. and Stern C. (2000) The effects of cryopreservation regimens on the morphology of human ovarian tissue. Mol Cell Endrocrin 169, 99-103
  4. Gook D.A., McCully B.A., Edgar D.H. and McBain J.C. (2001) Development of antral follicles in human cryopreserved ovarian tissue following xenografting. Hum Reprod 16, 417-422
  5. Gook D.A., Edgar D.H., Borg J., Archer J, Lutjen P.J. and McBain J. C. (2003) Oocyte maturation, follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting. Hum Reprod 18, 1772-1781.
  6. Gook D.A., Edgar D.H., Borg J., Archer J and McBain J. C. (2005) Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting. Hum Reprod 20, 72-78.
  7. Gook, D., L. Hale, A. Polyakov, T. Manley, G. Rozen, and K. Stern, Experience with transplantation of human cryopreserved ovarian tissue to a sub-peritoneal abdominal site. Hum Reprod, 2021. 36(9), 2473-2483.

 

Answer by Jana Bender-Liebenthron

Material:

Material Custodiol Multipurpose Handling

Medium-Complete

(MHM-C) with

Gentamicin and HSA

 CryoSure-DMSO (at

room temperature)
Distributor Dr. Franz Köhler Chemie, Germany FUJIFILM Irvine

Scientific, US

 WAK-Chemie Medical

GmbH, Germany
Needs Sterile, CE-certified for human/IVF-use Sterile, CE-certified for

human/IVF-use

 Sterile, CE-certified for

human/IVF-use

First, a single batch of 500mL Custodiol medium, certified and specialised for organ transport, which has a shelf life of three months, is sterile aliquoted for later use as a preparation medium. Each batch is microbiologically tested for sterility prior to use. Test must be negative. Secondly, a single batch of 500mL of 1.5M cryopreservation medium with a shelf life of two months is prepared in-house, as there is no commercial media available for cryopreservation of ovarian tissue. The solution is formulated using Multipurpose Handling Medium-Complete (MHM-C) with Gentamicin, HSA (5%) and CryoSure-DMSO (10%). Each batch is microbiologically tested for sterility prior to use. Test must be negative.

Procedure:

Preparation and Cryopreservation of Ovarian Tissue:

  • A separate clean room laboratory with a contamination-free environment and a sterile class II laminar airflow hood, equipped with an integrated cooling plate (provided by FRYKA- Kältetechnik GmbH, Esslingen, Germany), is used for the sterile and cooled preparation of ovarian tissue at 2-6°C.
  • The tissue is prepared by removing the medulla ovarii from the cortex ovarii, leaving only a small portion of the residual stroma. This ensures an optimal starting point for neovascularization of the grafts [2].
  • Depending on size and quality, rectangular strips are cut from the prepared cortex, equilibrated in medium for cryopreservation and then cryopreserved in individual portions for future therapeutic (re-) transplantations [3,4].
  • DMSO is used as our preferred cryoprotectant, dissolved in a complete medium containing a protein additive to achieve the best survival rates of embedded germ cells and intact tissue morphology after cryopreservation with slow freezing and thawing [3,5,6].
  • Ovarian tissue strips (approximately ten strips per patient, about 4x8x1 mm3, adjusted by patient age) are pre-incubated for 25 minutes in 2-6°C pre-cooled cryopreservation medium containing 10% DMSO on an orbital shaker with cooling plate (200 cycles/min):
  • For patients under 25 years of age, the tissue samples are typically smaller, about 5-6x4x1 mm3.
  • For patients over 25 years of age, the tissue samples are typically larger, about 8x4x1 mm3.
  • Small tissue pieces from prepubertal girls were not prepared and frozen as whole tissue pieces.
  • Cryopreservation is performed using a slow freezing protocol, modified from that described by Gosden [7].
  • Tissue strips are transferred into 1.8mL cryo vials and slowly frozen in a computer-controlled freezer (IceCube 14S-A, SY-LAB, Neupurkersdorf, Austria) with automatic seeding.
  • The freezing program starts 40 minutes after pre-incubation in cooled cryopreservation media, including:
  • 25 minutes pre-incubation on an orbital shaker with cooling plate, 200 cycles/min,
  • 5-10 minutes on a cooling block after transfer of the tissue strips into cryo vials
  • and finally 5-10 minutes in a cooling block for transportation of cryo vials with tissue strips to the freezer

Important: Do not interrupt at any time the 2-6°C cooling chain!

  • Freezing program:
  • Starts at 2°C
  • Cooling at a rate of -2°C/min unitil -6°C
  • Automatic seeding begins when the temperature of the medium in the vial reaches – 6°C for 100 seconds in the freezer = induction of extracellular ice crystal formation
  • After successful seeding, the temperature decreases at -0.3°C/min to -40°C
  • This is followed by more rapid cooling at -10°C/min to -140°C
  • Finally, the cryo vials are immersed in liquid nitrogen and transferred to storage boxes under liquid nitrogen.
  • Storage is performed in vapor-phase storage tanks (MVE 1500 series) at -190°C (Chart – MVE BioMedical Industries, Inc., Garfield Heights, USA).

Determination of Ovarian Cortex Biopsies for Potential Testing Before and After Cryopreservation [1,8]: The success of fertility preservation by cryopreservation depends on the reproductive age at the time of cryopreservation of ovarian tissue and the start of oncological therapy. To determine the reproductive age, ovarian reserve is assessed in-house by sonographic determination of AFC (antral follicle count), ovarian size, AMH (anti-Müllerian hormone) concentration, age, and additionally by counting of primordial and primary follicles in normed biopsies of prepared cortical tissue.

  • Standardized two-millimetre biopsies are taken from different cortical areas using a biopsy punch (3×2 mm biopsies for fresh viability testing prior to cryopreservation and after surgical removal and transport, and an additional 3×2 mm biopsies as thawing samples, for a total of 6×2 mm biopsies).
  • Preparation of a solution containing 2 μL Calcein-AM (Sigma-Aldrich), 998 μL pre-warmed DPBS solution (FUJIFILM Irvine Scientific), and 6–8 mg Collagenase type I A (Sigma-Aldrich).
  • 500 μL of this solution is pipetted into one well of a Nunc 4-well plate, and each 3×2 mm biopsy is transferred into the Calcein-AM/DPBS/Collagenase type I A solution.
  • The incubation time is 1.5–2 hours at 5% CO2 and 37°C. After 1 hour, the lysate is carefully homogenized with a 1000 μL pipette, and this step is repeated after 2 hours. The reaction is terminated by removing the samples from the incubator, placing them at room temperature, and storing the specimens in a light-protected environment.
  • A quantitative evaluation of healthy follicles (green fluorescent) is conducted using inverted fluorescence microscopy (10x magnification). The determined value is divided by 3 to obtain an approximate number of follicles per 2 mm biopsy.

The use of DMSO in our ovarian tissue cryopreservation program: Proven success Our choice of cryoprotective agent (CPA) for freezing ovarian tissue goes back to the pioneering work of Markus Montag in my former laboratory in Bonn in the late 1990s. At that time, DMSO (dimethylsulfoxide) was considered to be a non-toxic and very suitable CPA [7]. In particular, it showed low toxicity when used at concentrations of around 5-10% and at appropriately chilled temperatures (4°C) [9].

Another important study from this period showed that DMSO alone, without the need for additional CPAs such as ethylene glycol or additives such as sucrose, achieved the necessary preservation and recovery of ovarian function [10].  Also, a later study of Westphal et al. confirmed that the use of 10% DMSO for cryopreservation of human ovarian tissue allowed over 95% preservation of tissue integrity, including follicles and vascular endothelium [11]. Today, DMSO is still a widely used and established CPA for ovarian tissue [1,12-14]. Finally, if looking back on my 15 years of experience, I still stand by the decision to use DMSO as the CPA of choice for slow freezing ovarian tissue (with minor modifications introduced in 2016 regarding temperature optimisation and a revised choice of the DMSO carrier medium). Our protocols – both freezing and thawing – consistently gives excellent results, with high follicle survival rates and successful fertility restoration after transplantation, leading to healthy live births – predominantly by spontaneous pregnancies [3,6,14-16].

References:

  1. Liebenthron J, Montag M. Cryopreservation and Thawing of Human Ovarian Cortex Tissue Slices. Methods Mol Biol. 2021;2180:485-499. doi: 10.1007/978-1-0716-0783-1_23. PMID: 32797429.
  2. Donnez J, Dolmans MM. Ovarian tissue freezing: current status. Curr Opin Obstet Gynecol. 2015 Jun;27(3):222-30. doi: 10.1097/GCO.0000000000000171. PMID: 25811258.
  3. Liebenthron J, Montag M, Reinsberg J, Köster M, Isachenko V, van der Ven K, van der Ven H, Krüssel JS, von Wolff M. Overnight ovarian tissue transportation for centralized cryobanking: a feasible option. Reprod Biomed Online. 2019 May;38(5):740-749. doi: 10.1016/j.rbmo.2019.01.006. Epub 2019 Jan 19. PMID: 30733076.
  4. Beckmann MW, Lotz L, Toth B, Baston-Büst DM, Fehm T, Frambach T, Germeyer A, Goeckenjan M, Häberlin F, Henes M, Hirchenhain J, Hübner S, Korell M, Krüssel JS, Müller A, Reinsberg J, Schwab R, Seitz S, Sütterlin M, van der Ven H, van der Ven K, Winkler-Crepaz K, Wimberger P, von Wolff M, Liebenthron J, Dittrich R. Concept Paper on the Technique of Cryopreservation, Removal and Transplantation of Ovarian Tissue for Fertility Preservation. Geburtshilfe Frauenheilkd. 2019 Jan;79(1):53-62. doi: 10.1055/a-0664-8619. Epub 2018 Oct 9. PMID: 30686834; PMCID: PMC6336469.
  5. Bastings L, Liebenthron J, Westphal JR, Beerendonk CC, van der Ven H, Meinecke B, Montag M, Braat DD, Peek R. Efficacy of ovarian tissue cryopreservation in a major European center. J Assist Reprod Genet. 2014 Aug;31(8):1003-12. doi: 10.1007/s10815-014-0239-7. Epub 2014 Jun 14. PMID: 24928054; PMCID: PMC4130937.
  6. Lotz L, Bender-Liebenthron J, Dittrich R, Häberle L, Beckmann MW, Germeyer A, Korell M, Sänger N, Kruessel JS, von Wolff M; FertiPROTEKT (Transplantation group). Determinants of transplantation success with cryopreserved ovarian tissue: data from 196 women of the FertiPROTEKT network. Hum Reprod. 2022 Nov 24;37(12):2787-2796. doi: 10.1093/humrep/deac225. PMID: 36272106.
  7. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196 degrees C. Hum Reprod. 1994 Apr;9(4):597-603. doi: 10.1093/oxfordjournals.humrep.a138556. PMID: 8046009.
  8. Liebenthron J, Reinsberg J, van der Ven K, Saenger N, Kruessel JS, von Wolff M. Serum anti-Müllerian hormone concentration and follicle density throughout reproductive life and in different diseases-implications in fertility preservation. Hum Reprod. 2019 Dec 1;34(12):2513-
  9. doi: 10.1093/humrep/dez215. Erratum in: Hum Reprod. 2020 Apr 28;35(4):1009-1010. doi: 10.1093/humrep/deaa058. PMID: 31782794.
  10. Newton H, Fisher J, Arnold JR, Pegg DE, Faddy MJ, Gosden RG. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod. 1998 Feb;13(2):376-80. doi: 10.1093/humrep/13.2.376. PMID: 9557842.
  11. Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, Lass A, Winston RM. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod. 1996 Jun;11(6):1268-72. doi: 10.1093/oxfordjournals.humrep.a019370. PMID: 8671438.
  12. Westphal JR, Gerritse R, Braat DDM, Beerendonk CCM, Peek R. Complete protection against cryodamage of cryopreserved whole bovine and human ovaries using DMSO as a cryoprotectant. J Assist Reprod Genet. 2017 Sep;34(9):1217-1229. doi: 10.1007/s10815-017- 0963-x. Epub 2017 Jun 3. PMID: 28580514; PMCID: PMC5581782.
  13. Meirow D, Roness H, Kristensen SG, Andersen CY. Optimizing outcomes from ovarian tissue cryopreservation and transplantation; activation versus preservation. Hum Reprod. 2015 Nov;30(11):2453-6. doi: 10.1093/humrep/dev210. Epub 2015 Sep 6. PMID: 26345687.
  14. Dolmans MM, Jadoul P, Gilliaux S, Amorim CA, Luyckx V, Squifflet J, Donnez J, Van Langendonckt A. A review of 15 years of ovarian tissue bank activities. J Assist Reprod Genet. 2013 Mar;30(3):305-14. doi: 10.1007/s10815-013-9952-x. Epub 2013 Feb 16. PMID: 23417329; PMCID: PMC3607674.
  15. Ruan X, Du J, Lu D, Duan W, Jin F, Kong W, Wu Y, Dai Y, Yan S, Yin C, Li Y, Cheng J, Jia C, Liu X, Wu Q, Gu M, Ju R, Xu X, Yang Y, Jin J, Korell M, Montag M, Liebenthron J, Mueck AO. First live birth in China after cryopreserved ovarian tissue transplantation to prevent premature ovarian insufficiency. Climacteric. 2022 Aug;25(4):421-424. doi: 10.1080/13697137.2022.2064215. Epub 2022 May 3. PMID: 35504301.
  16. Dolmans MM, von Wolff M, Poirot C, Diaz-Garcia C, Cacciottola L, Boissel N, Liebenthron J, Pellicer A, Donnez J, Andersen CY. Transplantation of cryopreserved ovarian tissue in a series of 285 women: a review of five leading European centers. Fertil Steril. 2021 May;115(5):1102-1115. doi: 10.1016/j.fertnstert.2021.03.008. PMID: 33933173.
  17. Ruan X, Du J, Jin F, Gu M, Cheng J, Li Y, Dai Y, Korell M, Montag M, Liebenthron J, Mueck AO. Second pregnancy in China 5 years after ovarian tissue transplantation to prevent iatrogenic POI. Climacteric. 2025 Apr;28(2):212-216. doi: 10.1080/13697137.2025.2471062. Epub 2025 Mar 10. PMID: 40063149.

Comments

Luciana Cacciottola

Yunior board member
17/06/2025 at 18:54

Thanks to the authors of this scientific challenge to share their protocols. I would like to raise two questions for discussion:

  1. Do we currently have enough published or ongoing comparative studies to support the choice of one cryoprotectant over another in ovarian tissue cryopreservation?
  2. While preserving follicle integrity is clearly a primary goal, how important is stromal preservation for the success of transplantation in terms of graft revascularization and longevity? Is there any clinical or experimental evidence supporting its role?
Claus Yding Andersen

President
02/10/2025 at 12:35

  1. Do we currently have enough published or ongoing comparative studies to support the choice of one cryoprotectant over another in ovarian tissue cryopreservation?

The key problem is selecting an appropriate endpoint for the investigation. Ideally, pregnancy after transplantation would be the primary outcome, but a study powered for this endpoint would require many years to complete, so surrogate endpoints must be used. One possibility is quantitative assessment of follicle survival after thawing (e.g. Neutral Red staining). However, thaw survival per se is high with current protocols (we observe only ~7% loss of viable follicles following freezing/thawing with ethylene glycol, sucrose and hSA as CPA's), so differences between different CPA's are likely to be too small to guide a definitive choice. Small, non-growing human follicles appear relatively resistant to cooling and freezing; the principal challenge is therefore securing follicle survival during the immediate post-transplantation period, when a substantial proportion of follicles are lost.

  1. While preserving follicle integrity is clearly a primary goal, how important is stromal preservation for the success of transplantation in terms of graft revascularization and longevity? Is there any clinical or experimental evidence supporting its role?

That is an important question and several arguments can be made. The majority of follicles that survive freezing/thawing and grafting are non-growing primordial follicles that lack a theca cell layer. When these follicles activate and enter growth, the theca lineage is recruited from local stromal precursors; this recruitment will only occur once the graft has re-established itself within the host. One could therefore argue that, provided stromal recovery is adequate after transplantation, the theca compartment will be available when needed. Conversely, impaired stromal viability caused by the transplantation injury could lead to locally persistent defects in the graft and compromise follicle maturation in some regions. This returns us to the central methodological difficulty: choosing appropriate, feasible study end-points that reflect the clinically relevant biology.

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