Human Reproduction vol.l3 no.2 pp.376-380, 1998

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

H.Newton1•6, J.Fisher, J.R.P.Arnold3, D.E.Pegg4, M.J.Faddy5 and R.G.Gosden1

1Centre for Reproduction, Growth and Development, School of Clinical Medicine, University of Leeds, Clarendon Wing, Leeds General Infirmary, 2School of Chemistry, 3Department of Biology, University of Leeds, Leeds LS2 9NS, 4Medical Cryobiology Unit, Biology Department, The University of York, York, UK and 5Department of Mathematics, The University of Queensland, Brisbane, Queensland, Australia

6To whom correspondence should be addressed

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or per­manent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretic­ally, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an import­ant determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4°C and 37°C. We have also studied the effect of adding different concentrations of the non-penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4°C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37°C all four cryoprotectants penetrated at a faster rate, however con­cern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4°C; this achieved a mean tissue concentra­tion that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.

376

Key words: cryopreservation/follicle/oocyte/ovary

Introduction Human females are born with a finite store of germ cells represented by primordial follicles in the cortex of the ovary. The number of germ cells reaches a peak of approximately 7 million at mid-gestation and thereafter declines throughout life by the natural processes of ovulation and atresia until the follicular stock falls below a threshold of approximately 1000 and fecundity is lost (Faddy and Gosden, 1995). The treatment of cancer by chemo- and radiotherapy has a highly deleterious effect on the ovary causing a severe depletion of the follicular store. If the treatment is particularly aggressive, or the patient is older than 30 years of age, there is a high possibility of permanent sterility (Apperley and Reddy, 1995; Sanders et al., 1996). The improvements in the prognosis of many young cancer patients, in particular paediatrics, have led to an increased impetus to find an efficient method for the prevention of iatrogenic sterility. One strategy is to conserve the follicular population by cryopreserving strips of ovarian cortical tissue until the patient is in full remission. Fertility can then be restored by returning the tissue to the body either as an orthotopic autograft or, more speculatively, by isolating foll­icles from the thawed tissue for growth in culture followed by in-vitro fertilization (IVF) (Oktay et al., 1997). Recent studies have shown that healthy follicles are present in human ovarian tissue after freezing and thawing (Hovatta et al., 1996) and that 3 weeks after grafting tissue into immunodeficient mice 44-84% of the follicular population survive, depending on the cryoprotectant used (Newton et al., 1996). These encouraging results were not, however, based on an optimized technique and further work is required if this procedure is to become a clinical reality.

The first requirement for cryopreservation is to permeate the tissue with a compound that moderates the normally lethal effect of ice formation and the build-up of extracellular salts. Many such cryoprotectives (CPA) are known and their mode of action is quite well understood (Nash, 1962; Pegg and Diaper, 1988). The permeation of isolated cells with such CPAs is relatively fast, but diffusion in multicellular tissue systems is much slower, which means the exposure may have to be prolonged to reach adequate concentrations in the centre of the tissue thereby exposing cells near the surface to the risk of excessive toxicity. Consequently it is important to balance equilibration time against the harmful effects of overexposure to CPA prior to cryopreservation and during its removal. The penetration of CPAs has been studied using radioactive tracers

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(Karow et al., Clark et al., but more recently

proton nuclear magnetic resonance (NMR) analysis (Abraham

et al., 1988) has been used to measure dimethyl sulphoxide

(DMSO) and (EO) in corneal tissue (Taylor

and Wusteman, communication) and

et al., 1994; Wusteman et ccc,,uuuuc presents a proton spectrum for a given

and since the method is with water and

CPA giving distinct spectra, the concentration of CPA within

the tissue over time can be calculated directly, which makes

it to estimate the necessary equilibration time. In the

we have the rate of penetration of

four commonly used CPAs through human ovarian tissue at

both 4oC and 37oC. A second class of CPA does not permeate cells, and

it includes sugars such as sucrose and polymers such as

hydroxyethyl starch. Their mode of action is less clear than

that of penetrating CPAs and their relative effectiveness is generally lower, but many have indicated a beneficial

effect of supplementing the freezing medium with low concen-

trations of these solutes 197 In the present

the release of the intracellular enzyme lactate dehydrogenase

(LDH) following freezing was used to assay cell damage when

sucrose was included in the preservation medium: this approach

was previously used to optimize a method for the cryopreserv­

ation of rabbit renal tissue (Jacobsen et al., 1984).

Materials and methods

Comparison of the rates of penetration o.f four cryoprotective agents NMR spectroscopy

Ovarian tissue was collected from five premenopausal patients aged 32-49 years undergoing total abdominal hysterectomy-oophorectomy or routine gynaecological surgery. The patients gave informed consent to the use of their tissue for research which was approved by the local ethics cmm11ittee of the United Leeds Teaching Hospitals Trust. Slices of ovarian cortex were cut at a thickness of 2 1mn into uniform disks of 4 mm diameter with a biopsy punch (Stiefel, Bucks, UK). The tissue was on ice in Leibovitz L-15 medium and used within 2 h.

EG, DMSO, propylene glycol (PROH) and glycerol (GLY) were used at a concentration of 1.5 M in Leibovitz-Ll5 medium supple­mented with 10% fetal calf semm. 1.5 ml of cryoprotective media were pipetted into 1.8 ml cryovials (Nunc, Kamstn1p, Denmark) for equilibration at either 37°C or 4 oc.

Ovarian biopsies were transfened to individual vials and gently rolled ( l Hz) at either 4 oc or 3TC for 5, 10, 20, 40 or 90 min. Following incubation, the tissue was retrieved and surface moisture was removed with filter paper. The tissue samples were then transferred to 0.5 ml Eppendorf tubes and immersed in 400 ~ll of deuterium oxide (D20, 99.9%; Sigma, London, UK). The tubes were sealed and stored for up to 5 months at -20°C The experiment was repeated four times at each time with single pieces of tissue from different patients.

Samples were thawed at room temperature, and left overnight on a 'Spiramix' to ensure equilibration of water and cryoprotectant between the tissue and the D20. Samples of 350 !Jl of the solution were transferred to 5 nm1 glass NMR tubes (Wilmad Glass Co., Buena, NJ, USA),

All spectra were recorded on a GE Omega 500 spectrometer ( 1 H

Cryopn;servation of ovariaill tissmc

frequency 500 MHz). Prior to the time course

inversion recovery solvents at concentrations those in

Spectra were then acquired over 64 transients with 2500Hz in 16 384 data s. The relaxation between transients was 28 s and a 30 pulse width (3 flS duration) was transient was approximately three times the The data were recorded at 25°C, and were the NMR package FELIX® (version L3, Diego, CA, USA). Prior to Fourier transformation the free induction decays (FIDs) were an line factor of 1 Hz. The area under each standard procedures within

Calibration data for each of the agents was obtained by recording the spectra of L5 l\!1 standard solutions to a cryoprotectant to water ratio. These values were measured as DMSO 0.0235; EG 0.0248; PROH 0.0320 and GLY 0.0300. A

deuterium oxide was also to obtain a value for the background water signal for subtraction from all water The

of cryoprotectant as a fraction of was calculated all the test values for each cryoprotective agent.

Assessment (LDH) assay

Ovarian cortical from a further five 25-50 years old were used in the Tissue was cut into 2 nm13 blocks under a dissecting and randomly Five of the groups were transfened into l ml either (i) Leibovitz-LI5 medium, (ii) 1.5 M DMSO, (iii) !.5 lVI DMS0/0.05 M sucrose, (iv) 1.5 M DMS0/0.1 M :;ucrose or (v)

1.5 M DMS0/0.25 M sucrose, ail of which were same medium. Tbe final group was incubated for medium to act as a fresh control.

The vials were rolled (1 Hz) for 30 min penetration of the agent before programmable freezer 10, Planar, Middx, UK). cooled at 2°C/min to -9°C when were seeded u~cm~cm.1 .

continued at 0.3°C/min to -40oc and then at the min to -l40°C Vials were stored in until

Samples were thawed in a water bath at 20°C

for 1-2 min. Tissue was retrieved, washed in medium and placed in individual microtitre wells 150 ~Ll of sterile PBS for overnight incubation at JrC. The tissue was recovered and the wet weight recorded. The LDH levels in the PBS were measured with a UV assay on a BM/Hitachi 717 apparatus at 3~/oC

(Boehringer Mannheim, East Sussex, UK).

Statistical """'"'"'" Regression lines were determined for the NMR

linear least squares method. The LDH data were of variance and Student's t-tesL

Results

time a mean and sn

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!lLNewhm et

10 20 30 40 50

T~mo {m!nu!es)

DMSO EG PROH GLY

· ·DMSO and EG ~ PROH an<.1 GL Y

70 90

The rate of pcl.JcctauLvu

(EG), (PROH) and ovarian tissue at 4 oc. Plotted lines represent the Lhe form = l~a.exp (~bt) ~ (l~a) exp (~ct)

is time, b and c are the peJrle"lratto over time, and ( 1 ~a) are the vrcmo·rtH)f! process over time. Each represents the mean and SD from four c)<AllHJ.l'.Co.

b and c an: are the "''''''~''''''~" of the process At both 4oc and 37oC the data showed variation between nms with some solutes more variation than others: PROH showed the greatest vmiation and EG the and these differences were

< 0.01 at 4oc and P < 0.01 at The rate of of each of the four ,,.,.,,".'''t"'''h

agents at both 4 ac and 37oC was characterized of diffusion followed a second slower

to the rate b> >c in the above The 70% of

for PROH at this

and after min At the same temper-

ofDMSO was not different 1, broken but this

than that of the other After 30 min DMSO and EG had reached a

79%. At 37oC PROH <

2, broken line). After 30 min EG and GLY had reached 81% whilst PROH had reached almost total The parametGr estimates at each Table l

geuase Our results shmv that the smallest amount

the lactate

unfrozen control tissue released LDR 2. and this increased

i.2

/--i l

T t ' 0.8 ,t· t· I 1 l

0.6.

0.4 ' DMSO EG . PROH GLY

-PAOH 0.2 · · · · · DMSO/EG/Gl Y

10 20 30 40 50 60 70 80 90

Tirne (minutes}

Figure 2. The rate of of sulphoxide (DMSO), ethylene (EO), glycol (PROH) and glycerol (GLY)

human ovarian tissue at 3rC. Plotted lines represent the best fit curve of the form y = 1 ~a.exp ( ~bt) ~ (1 ~a) exp ( ~ct) where y is and t is time. Each point represents the mean and SD from four samples.

25

20

15

!f " .~

~ 10

1.6M DMSO 1.5M DMSO 1.5M DMSO 1.5M DMSO Leibovitz Fresh 0.05M sucrose 0.1 M sucrose 0.25M sucrose

Figure 3. The amount of lactate dehydrogenase (LDH) released from human ovarian tissue in one of five different freezing media: L5 M DMSO, 1.5 M DMS0/0.05 IV! sucrose, 1.5 M DMSO/O.l M sucrose, 1.5 M DMS0/0.25 M sucrose and Leibovitz-L15 medium alone, to fresh unfrozen control tissue; n = 23~24 per group.

Table t Parameter estimates

a

DMSO and EG at 4oc 0.7 (0.04) PROH and GLY at 4oC 0.7 (0.04) DMSO, EG and GLY at 3rC 0.78 (0.03) PROH at 37oC

Values in parentheses are SE.

b

0.28 (0.05) 0.19 (0.03) 0.4 (0.07) 0.27 (0.05)

a = proportion of penetTation process over time. b and c = penetration rates over tiine.

c

0.012 (0.004) 0.006 (0.0039) 0.0048 (0.0027) 0.27 (0.05)

DMSO = dimethyl sulphoxide; EG = ethylene glycol; PROH = propylene glycol; GLY = glycerol.

to a mean value of 20.8 ± 2.4 tissue was frozen~thawed in media without a

< The release decreased signific-when 1.5 M DMSO was

included in the medium The further addition of either 0.05 M and l M sucrose decreased LDH release to I 1.8 ± 1.4 and 9.8 ± 1.2 however,

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0.25 M sucrose caused a greater LDH release of 14.6 :±: 1.5 IU/g than freezing in 1.5 M DMSO alone but none of these differences was even though the group sizes were = 0.14, from of variance).

Discussion

In the present experiment we investigated the rates of penetra­tion of four different cryoprotectants and found that at 4oC

glycol and permeate human ovarian tissue more efficiently than either propylene glycol or glycerol. Thomas et al. have recently reported results for the transport ofDMSO at 0-2°C into 3-5 mm thick porcine and human ovarian tissue slices. It was observed that DMSO reached 45% equilibration in 20 min, which compares with about 76% at that time in the 4oc group in our study. This discrepancy most probably is due to the greater thickness and lower temperature used in their investigation. In our previous

of ovarian cryopreservation we found that glycol and dimethyl sulphoxide supported the survival of 84% and 74% of the follicular population after freeze-thawing, whereas 44% and 10% survived in tissue frozen in propylene glycol and glycerol respectively. Thus it would appear that the extent of follicular survival is at least in part determined the speed of CPA permeation and the final tissue concentration achieved. This theory is supported by the work of Candy et al. who murine ovarian tissue to different cryoprotectants for defined periods of time at room temperature before cryopreservation. Histological examination of the tissue on revealed that the dial follicle population was frozen in but that the numbers increased the longer the tissue was to the CPA. Primordial follicle counts taken from the tissue frozen in DMSO, EG and PROH did not differ significantly and did not increase over time from 5-60 min. These results probably reflect the fact that murine ovarian stroma is less dense than that of the human and at room temperature total equilibration is reached in this tissue in less than 5 min.

The effect of temperature on the speed of is also demonstrated in the cunent where the initial rate of of all four CPAs was found to be more rapid at 3rc than at 4 oc. there was little effect of temper-ature on the tissue concentrations of GLY or EG achieved at 30 min. Propylene however, to exhibit a greater dependence but it must be noted that it also displayed the greatest level of variation between runs. In practice the advantage of more rapid diffusion at 37oC would have to be balanced against the probability of toxicity, and this was not in the present study.

The benefit of adding a such as sucrose or mannitol has been demonstrated in both single cells and embryos et al., One suggested mechanism for the beneficial effect may be that they act as an osmotic buffer against the stresses incurred the cells the addition and removal of penetrating

agents. The results of the present study suggest

CryoiJreservation or ovarian tissue

that the addition of low concentrations of sucrose to the media does not

following slight decrease in the release of LDH the addition of 0.1 M or 0.05 M sucrose and thus it n1ay be n'"""'"m

media as an added

to sterilized hosts the ovarian tissue at the s1te

was first in the was frozen and then thawed in a solution insertion the endocrine function and some gave birth to healthy litters of pups

of lmnn~vt~ments in the for many young

cancer patients have altered this situation and there is now increased interest in tissue as a means of

induced automated freezers and more efficient agents have led to follicular survival rates as has been mouse and marmoset ovarian tissue et These can be

the fact that the with ovarian tissue

to 6/l et The human ovary is more fibrous than that of small

animals and ethical and have tended to the study of follicular survival after and auto-grafting. As an model was chosen because its ovaries are to those of the human. When were returned to the animals gave birth encouraging results from the ovine extend the work to human tissue.

showed that

The

present within the factor to determine follicular survival to be the CPA used

et al., In the absence of any CPA the were found to be minute and sterile

Human ovarian tissue has also been incubated in an in-vitro organ culture for up to 15 after cryopreserv-ation. examination of the tissue shovved

two thirds of the follicles had survived freeze-and extended culture et al.,

loss from ovarian tissue the u-.v6 .• u~"" may be due to the formation of intracellular ice or to the increase in the extracellular salt concentration that

Meryman, CPA cells the amount of ice formed at !.ow temperatures as a

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H.Newton et al.

result of their colligative properties, thus decreasing the external salt concentration. To date, the protocol used for the cryopre­servation of ovarian tissue has been based on the methods used for mature oocytes. Since tissues have the added problems of cellular heterogeneity and the rates of diffusion of CPA in and out (Pegg et al., 1979), it was anticipated that whole ovarian tissue would require changes in technique, including more time for cryoprotectant penetration into the cells and for total washout from the tissue after thawing.

In the present study we have investigated the penetration rates of the four CPAs DMSO, EG, PROH and GLY at both 4oc and 37°C. We decided to use 4oc in order to minimize toxicity and at this temperature we have shown that bathing the tissue for 30 min in a 1.5 M solution of either EG or DMSO produces mean tissue CPA concentrations approaching 80% of that in the bathing medium. In addition we have also found that no statistically significant protection is offered by adding low concentrations of sucrose to the freezing media.

Acknowledgements We gratefully acknowledge the help of Ken Newton with the LDH assays and the Leukaemia Research Fund (London) for supporting this work. J.R.P.A. also acknowledges personal support from the Wellcome Trust.

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Received on July 3, 1997; accepted on October 20, 1997

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