Ovarian tissue cryopreservation

Ovarian tissue cryopreservation has been developed as a fertility preservation strategy in recent decades. It does not require controlled ovarian stimulation and is therefore indicated in prepubertal patients and those who cannot postpone their oncological treatment or have already started it (1). The American Society for Reproductive Medicine (ASRM) removed the experimental label from this technique in 2019, based on the overall evidence of safety of cryopreserving and further transplanting the ovarian tissue (2). 

Ovarian biopsy samples

Follicles are located inside the ovarian cortex, and thus tissue samples for cryopreservation are collected from the surface of the organ. A biopsy can be taken during any gynecological procedure, by laparoscopy or laparotomy, and may be composed of one or several cortical fragments, or even a whole ovary, depending on the surgical indications and the risk of premature ovarian failure (POF) after the treatment. However, if no extensive surgery is needed (often the case with pelvic malignancies), biopsies should be obtained by simple laparoscopy performed under general anesthesia. Palmer forceps are inserted through one of the 5-mm trocars placed in the iliac fossa, and are used to grasp the ovary and cut a fragment from its surface. A cortical biopsy can also be easily obtained with laparoscopic scissors (Figure 1)(3).

The number of biopsies taken varies according to the size of the patient’s ovaries and the estimated risk of POF. Indeed, POF after chemotherapy is dependent on age, drug used and dose given. Biopsy samples are immediately transferred to the laboratory in Leibovitz L-15 medium supplemented with Glutamax® (Invitrogen, Paisley, UK) on ice. To minimize any tissue damage due to ischemia, the samples are transferred within minutes to the laboratory for processing.

The amount of ovarian tissue to be harvested for cryopreservation varies depending on the risk of ovarian insufficiency and existing ovarian volume. Oophorectomy is usually indicated in very young girls due to the small size of the ovaries. Otherwise, one-third of the total surface area is typically recovered by laparoscopy and then prepared for cryopreservation. Biopsy thickness should be around 1 mm, as preparation entails separating the cortex from the remaining medulla in order to preserve only the cortical layer accommodating early-stage follicles. Preference is for this to be performed in a Biosafety Cabinet Class 2 or in a clean room under a laminar flow hood. Cortical fragments of around 5×10 or 3×4 mm are then placed into cryovials containing freezing medium in sterile conditions (4-6). 

Freezing procedure

There are two methods of OTC: slow-freezing and vitrification. The first one involves ovarian fragment cooling at a slow rate in both the initial phase until extracellular ice is formed (seeding) and subsequent stage to at least -30°C, then a swift temperature drop to approximately -140°C. The tissue is exposed to low concentrations of cryoprotectants throughout the whole process, before being stored under liquid nitrogen or in nitrogen vapor (Anderson and Baird, 2019). The second, vitrification, is an ultra-rapid freezing technique that requires high concentrations of cryoprotectant in order to remove intracellular water before cooling begins. Samples are directly plunged into liquid nitrogen to achieve a glass-like state without ice crystal formation. They are then stored in liquid nitrogen for the long term. Although cryopreservation by vitrification is an emerging approach in some departments, slow-freezing is still the most widely used in clinical practice yielding the major number of babies born so far in the world after transplantation (1,7). 

For European countries, the EU Tissues and Cells Directive states that “where tissues or cells are exposed to the environment during processing, without a subsequent microbial inactivation process, an air quality with particle counts and microbial colony counts equivalent to those of Grade A, as defined in the current European Guide to Good Manufacturing Practice (GMP), Annex 1 and Directive 2003/94/EC, is required with a background environment appropriate for the processing of the tissue/cell concerned but at least equivalent to GMP Grade D in terms of particles and microbial counts” (8).

The samples are transferred under laminar flow in sterile conditions to a Petri dish containing a sterilized glass slide and 1-2 ml of L-15 medium. The tissue temperature is kept close to 4°C by placing the dish on top of a glass box containing crushed ice. The ovarian medulla is then separated from the cortex using forceps and surgical scissors, and disposed of. The remaining cortex is cut on the glass slide into strips of 2 x 6-8mm. These strips are transferred into 2-ml cryovials (Simport, Quebec, Canada) containing 800 µl of L-15 medium and stored at 0°C in a cooler box (Nalgene® Labtop cooler, Cat. No. 5116-0032; Nalge Nunc International, Rochester, NY, USA), each of the tubes containing 2-5 strips. Two strips are randomly put aside and immersed in a 37% paraformaldehyde solution for histological examination. Leibovitz medium is then twice replaced with 800 µl of freshly prepared cryoprotective solution containing 88% L-15 medium, 2% human albumin 20% (Red Cross, Brussels, Belgium) and 10% dimethylsulfoxide (Sigma-Aldrich Co., Irvine, UK).

The standard protocol for cryopreservation of human ovarian tissue is slow freezing. Freezing of ovarian tissue is carried out according to the protocol described by Gosden (9). The cryovials are cooled in a programmable freezer (Kryo 10, Series III; Planer, Sunbury-on-Thames, UK) as follows:

• At 0°C, place cryovials inside the freezer, keep stable at 0°C for 15 min
• From 0 to -8°C, cool at a rate of -2°C/min
• Keep stable at -8°C for 8 min for soaking
• Seed manually (induction of ice crystal formation) by grasping the cryovials (for 5-10 s each) with forceps prechilled in liquid nitrogen (Figure 2)
• Keep stable at -8°C for 15 min
• From -8 to -40°C, cool at a rate of -0.3°C/min
• From -40 to -150°C, cool at a rate of -30°C/min

The cryovials are then transferred to liquid nitrogen (-196°C) for storage.

Thawing procedure

For thawing, the cryovials are removed from the liquid nitrogen and left at room temperature for 2 min. Thawing is subsequently completed by immersing the cryovials in a warm (37°C) water bath for 2 min. The tissue samples are then grasped with small forceps and placed in a Petri dish containing L-15 medium, which is replaced three times (5 min each wash) to remove the cryoprotectant completely

Figure 1. (a) and (b) Ovarian tissue biopsy using Palmer forceps

Figure 2 Manual seeding using prechilled forceps

References

1. 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
2. ASRM Practice Committee. Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion. Fertil Steril 2019;112(6):1022–33.
3. Meirow D, Fasouliotis SJ, Nugent D, et al. Laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer. Fertil Steril 1999; 71: 948-51
4. Andersen CY, Mamsen LS, Kristensen SG. FERTILITY PRESERVATION: Freezing of ovarian tissue and clinical opportunities. Reproduction. 2019 Nov;158(5):F27-F34.
5. Gook DA, Edgar DH. Cryopreservation of female reproductive potential. Best Pract Res Clin Obstet Gynaecol. 2019 Feb;55:23-36.
6. Dolmans MM, Donnez J, Cacciottola L. Fertility Preservation: The Challenge of Freezing and Transplanting Ovarian Tissue. Trends Mol Med. 2021 Aug;27(8):777-791.
7. Shapira M, Dolmans MM, Silber S, Meirow D. Evaluation of ovarian tissue transplantation: results from three clinical centers. Fertil Steril 2020;114:388–97.
8. COMMISSION DIRECTIVE 2006/86/EC, implementing Directive 2004/23/EC5. Gosden RG, Baird DT, Wade JC et al. Restoration of fertility to oophorectomised sheep by ovarian autografts stored at -196°C. Hum Reprod 1994; 9: 597-60
9. Gosden RG. Low temperature storage and grafting of human ovarian tissue. Mol Cell Endocrinol. 2000 May 25;163(1-2):125-9.