Cryopreservation of human ovarian tissue

European Journal of Obstetrics & Gynecology and Reproductive Biology 113S (2004) S41–S44

Cryopreservation of human ovarian tissue Debra A. Gooka,b,*, D.H. Edgara,b, C. Sterna,b a

Reproductive Services, Royal Women’s Hospital, 132 Grattan Street, Carlton, Vic. 3053, Australia b Melbourne IVF, Melbourne, Vic., Australia

Abstract Approaches to the cryopreservation of human ovarian tissue are typically characterised by the use of slow freezing/rapid thawing methods using dimethyl sulfoxide or 1,2-propanediol (PROH) as cryoprotectants. This paper reviews current experience with these procedures. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Cryopreservation; Follicle; Human; Oocyte; Ovary

1. Introduction Early detection and aggressive chemotherapy and radiotherapy treatments are improving long term survival rates for many young women with cancer. As a consequence of these cytotoxic treatments their reproductive future is either short lived or eradicated. For young single women with cancer, cryopreservation of mature oocytes offers the best potential for achieving a pregnancy using their own gametes [1,2]. Unfortunately, the urgency to commence cytotoxic treatment often prohibits cryopreservation of mature oocytes. Conversely, cryopreservation of ovarian tissue eliminates the delay necessary to obtain mature oocytes but the subsequent potential for establishing pregnancy is unknown. Although cryopreservation of ovarian tissue is an attractive alternative and commonly used for patients with these conditions, little is published on the efficacy of protocols. Cryopreservation of ovarian tissue is more complex than that of gametes or embryos, requiring preservation of multiple cell types which vary in volume and water permeability. Essentially, ovarian tissue cryopreservation is more similar to organ cryopreservation than to that of gametes. The majority of follicles within the ovarian cortex are at the quiescent primordial stage (91% [3]), consisting of an oocyte (approximately one-third the size of a mature oocyte) surrounded by a single layer of flattened pre-granulosa cells. These follicles in the human are situated a millimetre below the cortical epithelium, embedded in a dense cortex of stromal cells and collagen bundles. Depending on the milieu * Corresponding author. Tel.: þ61-3-9344-2143; fax: þ61-3-9349-1387. E-mail address: [email protected] (D.A. Gook).

which will be used for subsequent growth and development of these follicles, i.e. in vitro culture or transplantation, it may also be necessary to preserve the integrity of the surrounding cortex components. At present the two cryoprotectants predominantly used to cryopreserve human ovarian tissue are—(a) dimethyl sulfoxide (DMSO) and (b) 1,2-propanediol (PROH). 1.1. Cryopreservation using DMSO The DMSO procedure was initially reported by Gosden et al. [4] for the cryopreservation of sheep ovaries and subsequent autologous grafting, which resulted in the birth of a lamb. This procedure (Table 1) or slight modifications of the procedure (Table 2) have been used to cryopreserve human ovarian tissue. Newton et al. [5] established the presence of at least one primordial follicle per slice of ovarian tissue which had been cryopreserved with DMSO and xenografted into mice. Intact pre-granulosa and stromal cell membranes were evident within primordial follicles isolated from tissue cryopreserved in DMSO [6] and initiation of development to the primary stage following xenografting was detected using PCNA [7]. The results achieved using the DMSO procedure appear inconsistent with Kim et al. [8] reporting an absence of follicles within fibrotic tissue after xenograft. Nisolle et al. [9] also reported that almost 70% of the cryopreserved tissue was fibrotic after xenografting compared to 40% in non-frozen tissue. The development of a large antral follicle, detected on ultrasound following autografting of cryopreserved ovarian tissue [10], is the best evidence to date of the successful preservation of follicular function with the DMSO procedure.

0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2003.11.009

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D.A. Gook et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 113S (2004) S41–S44

Table 1 Standard DMSO Cryopreservation Procedure [4] Preparation Blocks of tissue cut 1 mm thick Dehydration Load immediately into vials containing 1.5 M DMSO þ10% serum in Leibovitz L-15 medium Rotate on ice (4 8C) for 15 min Load into automated freezer, starting at 0 8C Slow cooling Rate

2 8C/min to 7 8C 0.3 8C/min to 40 8C 10 8C/min to 140 8C

Manual seed Store in liquid nitrogen

Rapid thaw 2 min in air, 2 min in 22 8C water bath Wash 3 with medium

Table 2 Modifications of the DMSO procedurea and assessment of tissue Dehydration solution

Dehydration

Cooling rate

Assessment

[10] [8] [7] [19]

1.5 M 1.5 M 1.5 M 4.2 M

4 8C, 4 8C, 4 8C, 22 8C,

Slow seed 9 8C Slow seed 9 8C Slow seed 7 8C Ultrarapid

Autograft, 20 mm follicle Xenograft, No follicles Xenograft, Primary folls Fetal tissue cultured, MII oocytes

a

Procedure in Table 1.

DMSO, DMSO, DMSO, DMSO,

2.5% albumin 2.5% albumin 0.1 M sucrose 0.35 M sucrose

30 min 30 min 30 min 7 min

1.2. Cryopreservation using the cryoprotectant PROH Although some success has been achieved with the DMSO procedure, we have concentrated solely on the use of PROH, which we had previously validated as safe for cryopreservation of human oocytes [11–14]. Numerous regimens using the cryoprotectant PROH have been evaluated (Table 3) to establish critical parameters within the

cryopreservation procedure which impact on not only cellular survival but also organelle normality. Parameters examined were dehydration duration, stepwise addition of cryoprotectants, cryoprotectant concentrations and rate of freezing. Morphological evaluation of all cell types within the ovarian tissue, i.e. stromal cells, collagen bundles, pregranulosa cells and the oocyte, was assessed using both light and electron microscopy [3]. Lysis of pre-granulosa cells

Table 3 Proportions of intact follicular cells following various PROH cryopreservation regimens Dehydration Solution 4.5 M 3.0 M 1.5 M 1.5 M 1.5 M 1.5 M 1.5 M 1.5 M 1.5 M 1.5 M

PROH, PROH, PROH, PROH, PROH, PROH, PROH, PROH, PROH, PROH,

b

% Intact pre-granulosa cells

% Intact cytoplasmic normal oocytes

10 10 30 30 10 30 60 30 60 90

Ultra rapid Ultra rapid Intermediate Slow Slow Slow Slow Slow Slow Slow

<5 <5 8 55 14 10 43 46 61 74

0 2 21 56 12 0 0 44 50 85 Antral follicles following xenografting

4 8C, 30 min –

Slow Slow

17 mm follicle following autografting [18] Fetal ovary, some secondary follicles following xenografting [20]

Time 0.2 M 0.2 M 0.1 M 0.1 M 0.2 M 0.2 M 0.2 M 0.1 M 0.1 M 0.1 M

a

sucrose sucrosea sucroseb sucroseb sucrosea sucrosea sucrosea sucrose sucrose sucrose

Other modifications 1.5 M PROH, 10% serum 1.5 M PROH, 0.1 M sucrose 20% SSS a

Rate of cooling

Two step dehydration, first step 1.5 M PROH þ 0:1 M Sucrose for 10 min. Two step dehydration, first step 1.5 M PROH for 10 min.

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Fig. 1. Histological section of primordial follicles from human ovarian tissue cryopreserved using; (a) dehydration in 4.5 M PROH þ 0:2 M sucrose followed by an ultra rapid freezing rate showing lysis of the pre-granulosa cells and oocyte, together with shrinkage of the germinal vesicle; (b) long dehydration (90 min) in 1.5 M PROH þ 0:1 M sucrose followed by a slow freezing rate, showing intact and normal appearance of both pre-granulosa cells and oocyte (magnification 100). (c and d) Electron micrographs of the cytoplasm adjacent to the GV within oocytes from cryopreserved ovarian tissue (magnification 10,000). (c) Abnormal cytoplasm in which the mitochondria are absent, replaced by vesicles containing electron dense material. Numerous areas devoid of organelles are also apparent. (d) Normal mitochondria and cytoplasm.

and oocytes was common with fast cooling rates (Fig. 1a) compared to slow rate (Fig. 1b). Nuclear ghosts were often the only components remaining within pre-granulosa cells. Large areas devoid of organelles and mitochondria without internal cristae were frequently observed within oocytes (Fig. 1c). To assist in quantifying this damage, the relative proportion of the oocyte cytoplasmic area occupied by vacuoles in non-frozen oocytes was estimated to be <10%. Oocytes within cryopreserved tissue which exceeded this level of vacuolation were deemed to be above the normal level of vacuolation. Similarly, relative normality of mitochondrial morphology was assessed for each oocyte in cryopreserved compared to non-frozen tissue (Fig. 1d). Very few regimens examined preserved a reasonable proportion (50%) of both pre-granulosa cells and ‘‘normal oocytes’’. These regimens have been previously reported and discussed in more detail by Gook et al. [3,15], who concluded that 90 min dehydration in 1.5 M PROH þ 0:1 M sucrose followed by slow freezing resulted in the highest proportion of

intact pre-granulosa cells and ‘‘normal ‘‘oocytes. This regimen also preserved the highest proportion of follicles with all of the pre-granulosa cells intact and the lowest proportion with no intact granulosa cells [16]. Due to the low number of pre-granulosa cells within a primordial follicle (10) the loss of 1 or 2 cells as a result of cryopreservation may be sufficient to compromise the subsequent development of the follicle. Evidence of preservation of function using this cryopreservation regimen (Table 4) has recently been established by follicular growth to the antral stage following xenografting of cryopreserved human ovarian tissue into immunodeficient mice [17]. The development of numerous antral follicles using this regimen, and the growth of a 17 mm follicle following autologous transplantation of tissue which had been cryopreserved using a modified PROH cryopreservation regimen [18] indicate that the first hurdle towards clinical ovarian tissue storage (i.e. a successful cryopreservation protocol) may have been overcome.

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Table 4 Standard PROH cryopreservation procedure [3] Preparation Cortex cut into thin slices (2 mm  4 mm  1 mm) Dehydration Load into cell sieves in wells containing 1.5 M PROH þ 0:1 M sucrose in phosphate buffered saline with 10 mg/ml human serum albumin Room temperature for 90 min Load into vials containing 1 ml of above solution Load into automated freezer, starting at 18 8C Slow cooling Rate

2 8C/min to 7 8C 0.3 8C/min to 30 8C 50 8C/min to 150 8C

Manual seed Store in liquid nitrogen

Rapid thaw 2–3 min 37 8C water bath Gradual dilution of cryoprotectant (similar to embryo thaw procedure)

2. Condensation Current protocols used for cryopreservation of human ovarian tissue are reviewed with emphasis on possible approaches to evaluation of successful preservation of primordial follicles.

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