Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting

Debra A. Gook1,2,3,4, D.H. Edgar1,2,3, J. Borg1,2, J. Archer1,2 and J.C. McBain1,2

1 Reproductive Services, Royal Women's Hospital, 132 Grattan Street, Carlton, Victoria 3053, 2 Melbourne IVF, 320 Victoria Parade, East Melbourne, Victoria 3002 and 3 Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Australia

4 To whom correspondence should be addressed at: Reproductive Services, Royal Women's Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. Email: debra.gook{at}rwh.org.au


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Although ovarian tissue cryopreservation for women at risk of losing ovarian function is offered by many clinics, there is a lack of evidence relating to the developmental potential of the stored tissue and, therefore, its clinical potential. This study was designed to use xenografting of cryopreserved tissue from multiple patients to assess the reproducibility of preservating developmental potential, the variation in developing follicle profile and the relationship between pre-freeze histology and post-thaw development. METHODS: Using previously published methods, cryopreserved ovarian cortex from nine patients was thawed and grafted under the kidney capsules of immunodeficient mice. Development of follicles was assessed after 26 weeks and compared to histology prior to freezing. RESULTS: Multiple growing follicles including antral stages were observed in multiple grafts of tissue from all patients. Metaphase II oocytes (n=9) were observed in follicles in grafts from five patients. There was no relationship between pre-freeze histology and developing follicle profile in xenografts. CONCLUSIONS: The propanediol freezing method used in this study is capable of reproducibly preserving the developmental potential of human ovarian follicles. The developing follicle profile after cryopreservation cannot be accurately predicted from pre-freeze histology. Xenografting provides a powerful tool for assessing the potential of human cryopreserved ovarian tissue.

Key words: cryopreservation/follicle/human/ovarian tissue/xenografting


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been known for some time that many young women are likely to suffer ovarian failure as a consequence of the cytotoxic treatments used to eradicate their cancer. The possibility of circumventing this loss of fertility by cryopreserving ovarian tissue prior to treatment appears to be theoretically plausible, but this approach has yet to result in a pregnancy. However, despite this lack of proof of principle, there is a general acceptance that cryopreservation will preserve the developmental potential of follicles within the ovarian tissue and, hence, many groups are cryopreserving ovarian tissue for future clinical use (Bahadur and Steele, 1996Go; Meirow, 2000Go; Oktay and Karlikaya, 2000Go; Callejo et al., 2001Go; Gelety et al., 2001Go; Radford et al., 2001Go; Poirot et al., 2002; Demeestere et al., 2003Go; Schmidt et al., 2003aGo; Oktay et al., 2004Go). Preservation of reproductive potential has, however, been demonstrated in other species with numerous live young produced following autologous transplantation of cryopreserved tissue in both rodents (Parrott, 1960Go; Gunasena et al., 1997Go; Sztein et al., 1998Go; Candy et al., 2000Go; Shaw et al., 2000Go) and sheep (Gosden et al., 1994Go; Salle et al., 2002Go).

In the human, preservation of morphological integrity and growth and development of follicles within cryopreserved ovarian tissue have been reported (Gook et al., 2001Go, 2003Go; Van den Broecke et al., 2001aGo,bGo; Kim et al., 2002Go). In initial clinical studies, in which the whole ovary had been cryopreserved and returned to the patient, resumption of cycling has been reported in two patients who had previously been rendered menopausal as a result of chemotherapy (Oktay and Karlikaya, 2000Go; Radford et al., 2001Go). Although these encouraging results suggest that there has been some preservation of follicular function, the short-lived resumption of cycling also suggests either that a paucity of follicles were present or that the developmental potential of follicles within the tissue may have been limited. In contrast to the ovarian tissue used in animal studies, which was generally obtained from juvenile animals and exhibited uniform follicle density, the ovarian tissue stored for patients is likely to be more variable with respect to follicle density, distribution and fibrous content, parameters which may be unknown at the time of cryopreservation but may impact on the cryopreservation outcome. In addition, although it is well established that there is a negative correlation between primordial follicle density and age (Faddy and Gosden, 1996Go), follicle densities assessed from sampling a small area of ovarian cortex at the time of cryopreservation in cancer patients (Poirot et al., 2002Go) identified numerous young patients with unexpectedly low follicle densities, further suggesting that inter-patient variation may be a major factor in determining the likelihood of successful application of this approach. Uncertainty related to follicular density together with the apparently low developmental potential of frozen tissue following autografting suggests that restoration of fertility by cryopreservation of ovarian tissue may be over-optimistic. Even in a case study in which autografting of the cryopreserved tissue resulted in the development of multiple (~20) follicles and the formation and transfer of a 4-cell stage embryo (with no resultant pregnancy), many of the follicles were found to contain immature or atretic oocytes (Oktay et al., 2004Go). In the absence of diagnostic information on developmental potential, it is not only difficult to provide a prognostic assessment of the value of the stored tissue for an individual patient but it is also impossible to differentiate between factors related to follicle viability and those related to grafting techniques as a likely reason for failure to establish prolonged ovarian function.

Although an accurate estimate of the survival rate of cryopreserved follicles is problematic due to the inadequacy of information on the number of follicles present in a piece of tissue prior to freezing, histological evaluation of the number of damaged follicles can provide an indication of the impact of cryopreservation (Gook et al., 1999Go). This type of assessment, however, is not able to confirm the ability of morphologically intact follicles to initiate and maintain growth and development. A diagnostic test which has the ability to confirm the presence of follicles, indicate survival of cryopreserved follicles, determine the developmental potential of these follicles and assess the normality of the follicle and oocyte would be a valuable tool in the application of ovarian tissue cryopreservation. Previous studies suggest that xenografting may be an appropriate technique for this purpose. The presence of multiple growing follicles within small pieces of thawed ovarian tissue following xenografting (Oktay et al., 2000Go; Gook et al., 2001Go; Van den Broecke et al., 2001aGo; Van den Broecke et al., 2001bGo; Kim et al., 2002Go) demonstrates that these follicles have survived cryopreservation and are capable of initiating development. Follicular growth from the primordial to the antral stage has also been observed within cryopreserved human ovarian tissue following xenografting (Gook et al., 2001Go; Van den Broecke et al., 2001aGo, bGo) and these follicles can undergo a normal response to a luteinizing stimulus (Kim et al., 2002Go; Gook et al., 2003Go). Although the above studies have demonstrated the potential of xenografting as a diagnostic tool for assessing the developmental competence of cryopreserved ovarian follicles, it is difficult to extrapolate its role to general applicability since tissue from very few patients has been assessed and the reproducibility of antral follicle development remains to be established.

The aims of the present study were to: (i) use xenografting to establish the reproducibility of preservation of follicular developmental competence in frozen–thawed ovarian tissue from a range of patients; (ii) assess the value of xenografting in the assessment of the relative developmental potential of stored tissue from multiple patients; and (iii) examine the relationship between pre-freeze histology and follicular development following xenografting of thawed tissue.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Patients
The tissue used in this study was from nine women aged between 18 and 31 years with various forms of malignant disease (details in Table I). All women had requested cryopreservation of ovarian tissue prior to gonadotoxic treatment or bone marrow transplant and consented to a sample of frozen tissue being thawed for diagnostic research purposes. All of the leukaemia patients had previously received chemotherapy. This study was approved by the Royal Women's Hospital Research and Ethics Committee and Animal Ethics.


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Table I. Follicle densities in fresh ovarian cortex from nine patients

 
Ovarian tissue cryopreservation
A piece of ovarian tissue, which generally consisted of less than one-quarter of the ovarian surface, was removed under laparoscopy and transferred immediately to warm (37°C) HEPES-buffered Ham's F-10 medium (Ham's/HEPES; Trace Scientific Ltd, Australia) containing human serum albumin (4 mg HSA/ml; Albumex; CSL, Australia). A small sample (fresh cortex) was removed for routine histopathology and follicle counts prior to cryopreservation. Under the dissecting microscope, all medullary tissue was removed and the cortex was reduced to ~1 mm thickness. Tissue was then cut into small slices ~4 x 2 mm. Slicing was carried out in Ham's/HEPES and slices were periodically moved onto tissue sieves in Ham's F-12 medium (Trace Scientific Ltd) before being placed in a 5% CO2 incubator awaiting completion of slicing. Sieves containing tissue slices were then rinsed briefly in phosphate-buffered saline containing 10 mg HSA per ml (base medium) followed by dehydration in 1.5 mol/l propanediol with 0.1 mol/l sucrose in base medium at room temperature for 90 min (Gook et al., 1999Go). Slices were subsequently transferred to 2 ml cryogenic vials (Nunc, Denmark) containing 1 ml of dehydration solution and loaded into a Planer K10 automated freezing machine. Tissue was frozen using a slow rate of cooling with a manual seed as previously described (Gook et al., 1999Go). Following a minimum storage period of 6 months in liquid nitrogen vapour, a vial containing a small number of slices was thawed for diagnostic evaluation. The rapid thaw procedure has also been previously described (Gook et al., 1999Go).

Xenografting and histological assessment
Following thawing, slices were transferred to Ham's/HEPES and cut into smaller pieces (~0.5 x 0.5 x 1 mm). The procedure for xenografting of cryopreserved ovarian tissue has been described previously (Gook et al., 2001Go, 2003Go). Briefly, a single piece of ovarian tissue was placed under the capsule of each kidney in severe combined immunodeficient (SCID) female mice. Mice were bilaterally oophorectomized at the time of xenografting. Although endogenous gonadotrophin is sufficient to support follicular growth (Weissman et al., 1999Go; Gook et al., 2001Go), to maximize follicular development mice were given an i.p. injection of gonadotrophin (1 IU recombinant FSH; Gonal F: a gift from Serono; and Puregon: a gift from Organon) (Oktay et al., 1998Go; Gook et al., 2001Go) starting on day 7 post grafting, and continued every second day until the completion of the study. Similarly, to provide sufficient time for consistent antral follicular development, as previously shown (Gook et al., 2001Go), the study was completed at a minimum of 27 weeks following grafting, at which time mice were given an i.p. injection of hCG (20 IU, Profasi; Serono). Kidneys were removed 30–36 h after the hCG administration and fixed in 4% paraformaldehyde (ProSciTech, Australia). Antral follicle diameters were measured using an ocular micrometer both prior to fixation and by assessment of the maximum diameter on histological section. Fixed tissue was processed and embedded in paraffin wax. Serial sections (3 µm) were cut and stained with haematoxylin and eosin. Only cavities containing an oocyte were classified as antral follicles. In smaller follicles, to overcome the error associated with counting a follicle more than once, follicles were only counted when the germinal vesicle within the oocyte was visible.

Statistical analysis
Statistical analysis was performed using Spearman's correlation (r) for non-parametric data.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pre-freeze follicular content
A baseline follicular profile was established for tissue from each patient prior to cryopreservation. A minimum of 10 sections of fresh cortex from each patient were examined for the presence of primordial and primary follicles. The mean number of follicles per mm3 was calculated for each patient and the results are shown in Table I. No secondary or antral follicles were observed in tissue from any of the patients. The follicle densities in the fresh cortex indicated that reasonable numbers of primordial and primary follicles were present in tissue from all patients prior to cryopreservation, although there was a large (~100-fold) variation in estimated follicle densities between patients (83–8717 per mm3). Although the estimated follicle densities were higher in the younger (18 years old) patients (P<0.05, r=–0.8), the sample size does not allow any definitive conclusion to be drawn. Similarly, it was not possible to correlate disease diagnosis with the estimate of follicle reserve.

Developing follicles in xenografts of cryopreserved tissue
Table II shows the number of growing follicles which developed within cryopreserved tissue from each patient following xenografting. A small number of primordial and primary follicles (less than one per graft) were also present within this tissue, but only follicles which had clear evidence of granulosa cell proliferation (i.e. initiation of a second layer: Figure 1A; or multiple layers of granulosa cells: Figure 1C and D) were classified as developing follicles. Growth and development of multiple follicles was observed in cryopreserved tissue from all nine patients following thawing and xenografting. However, the pattern and extent of follicular development varied markedly between patients. In tissue from some patients, large numbers of growing follicles were present (almost 10 per graft; patients B and F) whilst in others the total number of developing follicles was relatively low (less than two developing follicles per graft; patients D, E and G. There was no clear relationship (P>0.05, r=0.45) between the follicle density in the fresh tissue and the number of developing follicles/graft after cryopreservation. For example, the tissue which generated the highest number of developing follicles/graft (F) had a similar pre-freeze follicular density to the tissue which generated the lowest number/graft (G).


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Table II. Developing follicle profiles in xenografts of cryopreserved ovarian cortex from nine patients

 


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Figure 1. Follicles which have developed within ovarian tissue from six patients following cryopreservation and xenografting. (A) Histological section through ovarian tissue from patient C adjacent to the mouse kidney (k). Three proliferating follicles (arrows) and a secondary follicle(s) containing a germinal vesicle stage oocyte are present. Scale bar = 100 µm. Magnification: x10. (B) A small antral follicle (<2 mm diameter) in tissue from patient H, situated on the kidney surface. Scale bar = 1 mm. Magnification: x0.63. (C) A histological section through an antral follicle in tissue from patient D, showing an antral cavity (a) containing some blood (darker area within the cavity) and an oocyte (arrow) which has initiated germinal vesicle breakdown. Magnification: x10. (D) A section through tissue from patient F showing multiple antral follicles within a single graft. Three distinct antral cavities (a) are evident, each containing an oocyte within the cumulus pedicle. A secondary follicle (s) is also present. Magnification: x5. (E) A large antral follicle (6 mm diameter) which has developed in tissue from patient J. The follicle wall appears to be particularly thin in the herniated area suggestive of the point of rupture. A halo (arrow) can be visualized through the follicle wall; this is likely to be the cumulus-oocyte complex. Magnification: x0.63. (F) A mature oocyte is present within this follicle which had developed in tissue from patient E. Histological sections of this 4 mm follicle detected metaphase II chromosomes (arrow) within this oocyte and, in a subsequent section, the polar body containing chromatin was observed in the perivitelline space (insert). Magnification: x10.

 
Antral follicles in xenografts of cryopreserved tissue
An antral cavity was observed in 35% of all developing follicles (158/448; Table II) and 63% of all grafts (57/90; Table II) contained at least one antral follicle. Antral follicles were observed in xenografts of cryopreserved tissue from all nine patients with tissue from only one patient (C) failing to generate an antral follicle with a diameter of >2 mm. The total number of antral follicles per graft ranged from 0.5 to 2.8 and the large (>2 mm) antral follicle frequency ranged from 0 to 1.8 per graft. Examples of antral follicles are shown in Figure 1B–. Again, there was no clear relationship between pre-freeze follicular content and antral follicle profile following cryopreservation and xenografting. Oocytes were observed in all 58 large antral follicles with a germinal vesicle present in only 10. The other 48 oocytes (83%) had all undergone germinal vesicle breakdown (GVBD; Figure 1C) and nine metaphase II (MII) oocytes (Figure 1F) were observed. At least one MII oocyte was observed in xenografted cryopreserved tissue from five of the nine patients.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ultimate feasibility of the clinical application of ovarian tissue cryopreservation will be dependent on a number of factors, but the first prerequisite for success is the validation of a cryopreservation method which is consistently capable of not only preserving structural integrity but also maintaining full developmental potential of primordial follicles within stored tissue. Our previous studies using the cryopreservation procedure outlined in this study have preserved both structural integrity (Gook et al., 1999Go, 2000Go; Gook and Edgar, 2003Go) and developmental potential of primordial follicles within human ovarian tissue (Gook et al., 2001Go). These cryopreserved primordial follicles are capable of development to the antral stage and in response to a luteinizing stimulus will undergo luteinization and rupture together with final oocyte maturation (Gook et al., 2003Go). Although encouraging, these studies involved a small amount of stored tissue from only three patients. The observation of large numbers of growing follicles in multiple xenografts of cryopreserved tissue from all nine patients in the present study has confirmed the reproducibility of this method both within the stored tissue from a single patient and between tissue from multiple patients. The general applicability and robust nature of the method is further emphasized by the range of ages and medical indications and also by the fact that the tissue was stored at various times over a period of >2 years. The clinical application of this procedure has yet to be extensively evaluated; however, heterotopic transplantation of tissue cryopreserved using the cryoprotectant propanediol has resulted in development of a large antral follicle (Oktay and Karlikaya, 2000Go). Two other cryoprotectants have been used to cryopreserve human ovarian tissue: dimethylsulphoxide (Gosden et al., 1994Go) and ethylene glycol (Lightman et al., 2001Go; Schmidt et al., 2003bGo, 2004Go). Ovarian tissue cryopreserved using these alternative procedures has been autografted and successful follicle development observed within patients (Callejo et al., 2001Go; Radford et al., 2001Go; Kim et al., 2003Go; Oktay et al., 2004Go; Schmidt et al., 2004Go).

The normal human ovary contains predominantly primordial follicles with a low proportion (<1%) of secondary or more advanced follicles (Gook et al., 1999Go; Wright et al., 1999Go; Qu et al., 2000Go; Schmidt et al., 2003aGo). These observations together with the time required for antral follicle development following xenografting (Gook et al., 2001Go) suggest that the developing and antral follicles reported in the present study have grown subsequent to xenografting. The large number of developing follicles observed on multiple grafts in this study confirms our previous observation that xenografting of human ovarian tissue provides an excellent environment for assessment of follicular developmental potential up to luteinized antral stages, including final oocyte maturation (Gook et al., 2003Go). This is further emphasized by the observation of nine metaphase II oocytes in the present study, although the cytoplasmic maturity and competence of these oocytes for further development is unknown. In contrast to xenografting, extremely limited growth has been observed following in vitro culture of either isolated primordial follicles (Abir et al., 2001Go; Gook et al., 2004Go) or small cubes of human ovarian tissue (Hovatta et al., 1997Go, 1999Go; Wright et al., 1999Go). Generally, the culture period (~2 weeks) is very limited relative to that required for development following xenografting (>26 weeks). Although very little development would be expected from the quiescent primordial stage over this time, difficulties arise in maintaining follicle structure and viability even within this short culture period (Hovatta et al., 1999Go; Abir et al., 2001Go; Gook et al., 2004Go), stressing the limited potential of this approach. Addition to the culture medium of factors such as insulin, transferrin and selenium (Wright et al., 1999Go), growth differentiation factor-9 (Hreinsson et al., 2002Go), and a cyclic guanosine monophosphate analogue (Scott et al., 2004Go) increases the proportion of follicles exiting the resting stage, but at present the requirements for sustaining viability and development to subsequent stages remain elusive. The complexity required within a culture system to achieve consistent growth of human primordial follicles to the large antral stage and to induce oocyte maturation may be difficult to achieve in practice. However, mice have been born following multi-step culture and maturation of primordial follicles from neonate ovaries (Eppig and O'Brien, 1996Go; O'Brien et al., 2003Go) albeit with a relatively low success rate.

In contrast to animal studies, in which tissue is usually obtained from post-natal animals with a uniform distribution of ovarian follicles, the follicle density and distribution may be expected to be much more variable in human ovarian tissue prior to cryopreservation. In addition to the well-established inverse correlation between age and ovarian follicular density (Gougeon et al., 1994Go; Faddy and Gosden, 1995Go), the present study confirms that variability exists even within a relatively tight range of patient ages and also demonstrates that there is no apparent relationship with the number of developing follicles observed after xenografting.

However, the accuracy of estimating follicle density in the human ovary based on a single biopsy/sample has been questioned (Lass, 2004Go) and studies have shown large variations in follicle density between and within ovaries when multiple samples are examined (Kohl et al., 2000Go; Schmidt et al., 2003aGo) or a lack of homogeneous follicle distribution even within a single sample (Lass et al., 1997Go; Qu et al., 2000Go; Poirot et al., 2002Go). Examination of sections to determine follicle densities in the nine patients reported in the present study and similar assessment of ovarian tissue from >200 patients having ovarian tissue cryopreservation in our laboratory (unpublished) supports the conclusion that primordial follicles tend to be located in clusters and not evenly distributed throughout the tissue. Although little prognostic value can, therefore, be placed on the estimated follicle density, it is still reassuring to observe numerous primordial follicles within a sample of the tissue which has been frozen.

The limitations of histology as a prognostic tool in ovarian cryopreservation is emphasized in the present study by the discrepancy between estimated follicular density prior to cryopreservation and the density of developing follicles after thawing and xenografting. This discrepancy may arise from inaccuracy in estimating follicular density due to the factors described above, variations in the intrinsic developmental potential of the primordial follicles or variations in the ability of the follicles to survive cryopreservation. However, all three of the above considerations, or combinations thereof, are circumvented by the use of xenografting as a prognostic tool. Evidence obtained from xenografting a small piece of cryopreserved tissue can confirm not only the presence of follicles after thawing but also their potential for extended development and maturation (Gook et al., 2001Go, 2003Go). The value of xenografting in assessing the post-thaw developmental potential of stored ovarian tissue is further emphasized by the observation that antral follicles in such grafts can respond to a luteinizing signal and the oocytes within can undergo final meiotic maturation (Gook et al., 2003Go; present study). Such evidence of normal function after cryopreservation suggests that the parallel stored tissue should be capable of generating mature oocytes for clinical use under appropriate conditions. Although the clinical and xenografting conditions will differ with respect to site, gonadotrophin concentrations etc., the crude assessment using xenografting provides limited prognostic indication of potential success from the grafted tissue. In contrast, in the clinical studies to date, no prognostic information regarding the cryopreserved tissue was available prior to autografting (Oktay and Karlikaya, 2000Go; Callejo et al., 2001Go; Radford et al., 2001Go; Kim et al., 2003Go; Oktay et al., 2004Go; Schmidt et al., 2004Go) and it is therefore difficult to ascertain the reason(s) for temporary resumption of cycling and poor oocyte quality. Xenografting of the cryopreserved tissue can also potentially identify problems associated with cryopreservation and/or follicular development, such as observed in tissue from patient B in which no mature oocytes were detected in eight of the large antral follicles (all of which were >3.5 mm in diameter). There was also a suggestion of follicle growth retardation in tissue from patients A and C. It is worthy of comment that the primordial follicles present in the ovaries of these three patients had been exposed to chemotherapy.

The present study demonstrates the value of xenografting in establishing the robust nature of a cryopreservation procedure that can, therefore, be applied clinically with confidence. The preservation of developmental potential in tissue from all patients examined indicates that the propanediol slow freeze–rapid thaw procedure is applicable to ovarian tissue from a wide range of patients with a range of diseases and ovarian follicular densities. This study also confirms our previous observations on a small number of tissue samples, that follicles cryopreserved at the primordial stage are capable of growth and development to the periovulatory stage including final oocyte maturation. Finally, we have also established that the prognostic information gleaned from xenografting, in contrast to the information obtained from diagnostic histology, can provide a more realistic assessment of the developmental potential of stored tissue on which to base future clinical management.


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 Results
 Discussion
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Submitted on July 22, 2004; accepted on September 10, 2004.