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
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Abstract |
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Key words: cryopreservation/follicle/human/ovarian tissue/xenografting
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Introduction |
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In the human, preservation of morphological integrity and growth and development of follicles within cryopreserved ovarian tissue have been reported (Gook et al., 2001, 2003
; Van den Broecke et al., 2001a
,b
; Kim et al., 2002
). 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, 2000
; Radford et al., 2001
). 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, 1996
), follicle densities assessed from sampling a small area of ovarian cortex at the time of cryopreservation in cancer patients (Poirot et al., 2002
) 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., 2004
). 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., 1999). 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., 2000
; Gook et al., 2001
; Van den Broecke et al., 2001a
; Van den Broecke et al., 2001b
; Kim et al., 2002
) 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., 2001
; Van den Broecke et al., 2001a
, b
) and these follicles can undergo a normal response to a luteinizing stimulus (Kim et al., 2002
; Gook et al., 2003
). 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 frozenthawed 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.
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Materials and methods |
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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., 2001
, 2003
). 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., 1999
; Gook et al., 2001
), 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., 1998
; Gook et al., 2001
) 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., 2001
), 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 3036 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.
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Results |
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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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Discussion |
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The normal human ovary contains predominantly primordial follicles with a low proportion (<1%) of secondary or more advanced follicles (Gook et al., 1999; Wright et al., 1999
; Qu et al., 2000
; Schmidt et al., 2003a
). These observations together with the time required for antral follicle development following xenografting (Gook et al., 2001
) 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., 2003
). 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., 2001
; Gook et al., 2004
) or small cubes of human ovarian tissue (Hovatta et al., 1997
, 1999
; Wright et al., 1999
). 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., 1999
; Abir et al., 2001
; Gook et al., 2004
), stressing the limited potential of this approach. Addition to the culture medium of factors such as insulin, transferrin and selenium (Wright et al., 1999
), growth differentiation factor-9 (Hreinsson et al., 2002
), and a cyclic guanosine monophosphate analogue (Scott et al., 2004
) 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, 1996
; O'Brien et al., 2003
) 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., 1994; Faddy and Gosden, 1995
), 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, 2004) and studies have shown large variations in follicle density between and within ovaries when multiple samples are examined (Kohl et al., 2000
; Schmidt et al., 2003a
) or a lack of homogeneous follicle distribution even within a single sample (Lass et al., 1997
; Qu et al., 2000
; Poirot et al., 2002
). 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., 2001, 2003
). 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., 2003
; 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, 2000
; Callejo et al., 2001
; Radford et al., 2001
; Kim et al., 2003
; Oktay et al., 2004
; Schmidt et al., 2004
) 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 freezerapid 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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Submitted on July 22, 2004; accepted on September 10, 2004.