Human Reproduction, Vol. 16, No. 3, 417-422,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Development of antral follicles in human cryopreserved ovarian tissue following xenografting
1 Reproductive Biology Unit, Royal Women's Hospital and 2 Melbourne IVF, Melbourne, Australia
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This study reports the first gross morphological and histological evidence of antral follicle development in human ovarian tissue following cryopreservation. Human ovarian tissue was cryopreserved using propanediol and sucrose and grafted under the renal capsule of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Follicles at all stages of development were observed in the grafted tissue whereas, prior to grafting, only primary and primordial follicles were present. Antral follicles were rarely observed on grafts examined <20 weeks after grafting either non-frozen tissue (fresh, 1/7) or cryopreserved tissue (0/11). In contrast, development of at least one antral follicle was evident on the majority of sites
20 weeks after grafting (fresh 7/9, cryopreserved 18/24). Antral follicle diameters ranged from 0.1 to 5.0 mm. Histological examination of these antral follicles indicated normal follicular morphology, i.e. antral cavities encapsulated by concentric layers of theca and granulosa cells. Pedicles containing germinal vesicle oocytes were observed protruding from the granulosa cell layers. The development and morphology of the cryopreserved and fresh tissue following grafting was similar.
Key words: antral follicle/cryopreservation/human/ovary/transplantation
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Introduction |
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Improvements in cancer therapy have resulted in a higher incidence of long-term survival from many cancers and cure rates of ~70% of adolescent lymphomas and leukaemias (Boring et al., 1994
). As a consequence of these cytotoxic treatments, it is estimated that a third of young women will be rendered sterile (Meirow, 1999). In many who regain ovarian function, premature menopause is imminent (Byrne et al., 1992). The successful cryopreservation of human mature (Porcu et al., 1998) and immature (Tucker et al., 1998) oocytes resulting in live births, and reports of offspring derived from cryopreserved animal ovarian tissue (Parrott, 1960; Gosden et al., 1994; Gunasena et al., 1997; Sztein et al., 1998; Candy et al., 2000; Shaw et al., 2000), has led to patients who are at risk of loss of ovarian function requesting oocyte or ovarian tissue cryopreservation as a means of preserving their fertility. Despite the recent increase in human ovarian tissue banking (Bahadur and Steele, 1996), there has been little progress in establishing whether the follicles within this tissue are viable and capable of function following cryopreservation. Histological examination following cryopreservation of human ovarian tissue has shown retention of morphological integrity in follicles cryopreserved in the presence of dimethylsulphoxide (DMSO) (Hovatta et al., 1996) or 1,2-propanediol (Gook et al., 1999). Neither of the above studies, however, could assess the preservation of follicle function. Although in-vitro studies have suggested some retention of functional viability following cryopreservation (Oktay et al., 1997), the extent of development is very limited (Hovatta et al., 1997) and difficult to assess.
Recent studies in other species have indicated that grafting of cryopreserved tissue may have more potential. Follicular development to the antral stage has been observed following autologous grafting of cryopreserved tissue in sheep (Gosden et al., 1994; Salle et al., 1998; Aubard et al., 1999; Salle et al., 1999), and in mouse (Harp et al., 1994; Candy et al., 1997) and following heterologous grafting of cryopreserved marmoset (Candy et al., 1995) and elephant (Gunasena et al., 1998) tissue. Live births have also been reported following autologous transfer of cryopreserved ovarian tissue (Parrott, 1960; Gosden et al., 1994; Gunasena et al., 1997; Sztein et al., 1998; Candy et al., 2000; Shaw et al., 2000).
There is, however, a lack of evidence of functional development of cryopreserved human ovarian tissue. Antral follicles have developed within fresh (non-frozen) human ovarian tissue following grafting into severe combined immunodeficient (SCID) mice (Oktay et al., 1998; Weissman et al., 1999). However, in human cryopreserved ovarian tissue only initiation of mitosis within primary follicles has been observed following xenografting (Oktay et al., 2000). Clinical ultrasound evidence and rising oestradiol concentrations suggested the development of a single follicle in a patient following autologous grafting of a whole ovary which had previously been cryopreserved (Oktay and Karlikaya, 2000). The aim of the present study was to assess functional preservation of follicles within cryopreserved human ovarian tissue grafted into immunodeficient mice.
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Materials and methods |
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Ovarian tissue was donated from two patients at risk of loss of fertility: a 29 year old with breast cancer (control fresh tissue), and an 18 year old with acute myeloid leukaemia (frozen tissue). This study was approved by the Hospital Ethics Committee. Collection, preparation and cryopreservation of the ovarian tissue has been previously described (Gook et al., 1999
). Thin slices of ovarian cortex (~4x2x0.5 mm) were cryopreserved immediately (frozen tissue) or cultured overnight and grafted the following day for the control fresh tissue. Nine or ten pieces of tissue (equivalent in size to grafted pieces) from each patient were fixed in 4% paraformaldehyde (ProSciTech, Thuringowa, Queensland, Australia) at the time of grafting for the fresh tissue and before cryopreservation for the frozen tissue. The number of each type of follicle present was assessed histologically in each piece of tissue and mean values were obtained.
Cryopreservation
Slices were rinsed briefly in phosphate-buffered saline (PBS; Trace, Clayton, Victoria, Australia) and then dehydrated in 1.5 mol/l PROH (BDH, Kilsyth, Victoria, Australia) and 0.1 mol/l sucrose (BDH) for 90 min at room temperature. All freezing and thawing solutions contained 10 mg/ml human serum albumin (Albumex; CSL, Camperfield, Victoria, Australia) in PBS. Slices were frozen using a slow freezing rate (2°C/min from room temperature to –8°C, at which temperature ice nucleation was induced manually followed by further reduction of temperature to –30°C at the rate of 0.3°C/min and finally to –150°C at 50°C/min) and subsequently stored in liquid nitrogen. The slices were thawed a month later using the rapid thaw procedure previously described (Gook et al., 1999).
Xenografting
Fresh and cryopreserved tissue pieces were grafted into female SCID mice >20 weeks old (Animal Resources Centre, Canningvale, WA, Australia). Mice were anaesthetized with methoxyflurane (Medical Developments Australia; Springvale, Victoria, Australia) in air. Slices were cut into smaller pieces, a single piece (~0.5x1x0.5 mm) of ovarian cortex was placed under the capsule of each kidney in all animals and mice were oophorectomized bilaterally. In the initial study, mice were killed at 14–18 weeks post grafting and kidneys examined for the presence of the grafted tissue. Blood was collected immediately prior to killing by placing a small glass capillary tube in the ocular vein, allowed to clot and the serum separated by centrifugation. Oestradiol was measured using a radioimmunoassay (Coat-A-Count; Diagnostic Products Corporation, Los Angeles, CA, USA). In the later study, i.p. injections of gonadotrophin (1 IU recombinant FSH: Gonal-F; gift from Serono, Australia) or saline were started on day 7 post grafting and given every second day until the completion of the study. Kidneys were removed at 20, 24, 30 or 36 weeks post grafting; follicular development appeared similar at each of these intervals and, due to the low number of animals, results were pooled for the assessment of 20 weeks post grafting. Kidneys were initially examined for follicular development under the dissecting microscope (gross morphology) and subsequently fixed in 4% paraformaldehdye for histology. Prior to fixation, follicle diameters were measured using an ocular micrometer and verified subsequently by estimation of maximum follicular diameters in histological sections. Fixed tissue was paraffin wax-embedded and thick (3 µm) sections cut and stained with haematoxylin-eosin. To overcome repeated counting of the same follicle, follicles were only counted when the germinal vesicle nucleus was detected. Only one mouse (of 29) died from postoperative trauma. Otherwise, all animals were generally healthy.
Follicular assessment
Follicles were classified into six types based on the classification of Gougeon (1986): (i) primordial follicle: containing an oocyte surrounded by single layer of flattened, or a mixture of flattened and cuboidal, pre-granulosa cells (Figure 1D left); (ii) the primary follicle is characterized by a single layer of cuboidal pre-granulosa cells (Figure 1D centre); (iii) in the proliferating primary follicle a partial second layer of cuboidal pre-granulosa are present (Figure 1D right); (iv) the secondary follicle contains at least two complete layers of granulosa cells (Figure 1F); (v) the early antral contains multiple layers of granulosa cells and a small antral cavity (Figure 1G); (vi) in the antral follicle the cavity occupies most of the total follicular volume (>0.4 mm diameter, Figure 1A).
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Gross morphology
Although the majority of grafts, regardless of whether fresh or cryopreserved tissue was grafted, could be identified on the surface of the kidneys <20 weeks after the grafting (fresh, 7/8 grafts recovered; cryopreserved, 11/14 grafts recovered; Table I
), only one site contained a small antral follicle (0.2 mm in diameter). In contrast, more antral follicles were observed in both fresh and frozen grafts 20 weeks after grafting. On gross morphological examination, antral follicles were observed in the majority of fresh (7/9 grafts) and cryopreserved (total 18/24 grafts) pieces of ovarian tissue following grafting. Follicular development was similar in both types of tissue (mean follicular diameter 1.6 mm fresh versus 2.0 mm cryopreserved) and irrespective of gonadotrophin injections (mean diameter 1.5 mm saline versus 2.2 mm FSH). However, the small numbers of follicles analysed meant that no valid statistical comparisons could be made. At least one follicle 2–3 mm in diameter on one graft and small follicles 0.6–1 mm on the other graft were observed in each mouse. The maximum follicular diameter observed in cryopreserved tissue was 5 mm (two grafts) and in fresh tissue 4.3 mm (one graft). One of these follicles which developed in the cryopreserved tissue is shown in Figure 2. Well-developed surface vascularization to the follicles was apparent on a number of grafts (Figure 2; arrow) and in some cases the ovarian tissue had also fused to the liver cortex (l).
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Oestradiol was not detected (assay sensitivity 74 pmol/l) in all animals in which the grafts had developed for <20 weeks and in those in which small antral (
2 mm) follicles had developed following >20 weeks. In animals with larger antral follicles oestradiol ranged between 180 and 1400 pmol/l.
Histological examination
Histology confirmed the presence of antral follicles (Figure 1A
) with fluid-filled antral cavities (a) encapsulated by numerous layers of mural granulosa cells (Figure 1B; g) and theca cells (t). Germinal vesicle stage oocytes on cumulus cell pedicles (Figure 1A and C; o) were detected within these antral follicles. Earlier stage follicles (f) were also present within the tissue (primordial, Figure 1E; primary, Figure 1D centre; proliferating primary, Figure 1B; secondary; Figure 1D right and F; early antral, Figure 1G). The morphological appearance of the antral follicles, other follicles and the overall appearance of the grafted tissue was similar in fresh and cryopreserved tissue.
Sections through the fresh and cryopreserved grafted ovarian tissue (
) revealed that the surface consisted of either cellular remnants/adipose tissue (Figure 1D; #) which abutted healthy stromal cells, or a layer of elongated stromal cells on the surface of the follicles (Figure 1A, E and G). Apart from the surface and some sporadic small areas (Figure 1F; #), the majority of stromal cells appeared intact and densely packed (Figure 1; ). Capillaries (c) were evident on the surface (Figure 1D) and within the ovarian tissue. At the renal interface, either normal (Figure 1E and F) or elongated (Figure 1D and G) stromal cells abutted glomerular cells.
The follicular profile (Table II) of grafted tissue indicated that multiple follicles were present in both the fresh (mean total = 27 per graft) and cryopreserved (saline, 10; FSH, 41) tissue after grafting. No comparison can be made between the profile of the fresh and the cryopreserved grafted tissue, due to tissue from different donors being used. However, cryopreservation in combination with grafting does not appear to have dramatically reduced follicle numbers. The presence of only quiescent follicles (primordial stage) in the tissue before grafting is in contrast to the presence of follicles at all stages of development after grafting.
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Discussion |
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The absence of developing follicles in human ovarian tissue prior to grafting, together with the extended time required for detection of antral follicles in the grafts suggest that antral follicle growth has been initiated after grafting in this study. The absence of follicles more advanced than the primary stage in pre-grafted tissue is consistent with our previous observations (Gook et al., 1999
), and those of others (Hovatta et al., 1996; Oktay et al., 1997) that proliferating or later stage follicles are rarely observed in human ovarian tissue following cryopreservation. This contrasts with grafting studies of marmoset cryopreserved ovarian tissue (Candy et al., 1995) which demonstrated the presence of early antral stage follicles prior to grafting. The development of antral follicles within 2–3 weeks of grafting in the above study suggests that the grafts contained growing follicles which survived cryopreservation. These results are in contrast to the longer times required for antral follicular development in grafted cryopreserved ovarian tissue from other species (Gunasena et al., 1998; Aubard et al., 1999).
The normal morphological appearance of the follicles and surrounding ovarian tissue after grafting confirms the previously reported good morphological preservation using PROH as a cryoprotectant (Gook et al., 1999). Evidence for the suitability of PROH as a cryoprotectant for human ovarian tissue has been limited to observations of post-thaw follicles (Hovatta et al., 1996) and subsequent demonstration of their viability based on live/dead staining following culture (Hovatta et al., 1997) or xenografting (Newton et al., 1996). The subsequent growth and development of multiple follicles, from multiple slices of cryopreserved tissue, to the antral stage in the present study demonstrates clearly that the cryopreservation method used can preserve follicular developmental potential and suggests that preservation is relatively uniform between slices. This latter point may be an important issue in relation to tissue/organ cryopreservation and has been discussed in more detail previously (Gook et al., 1999). Although primordial and primary follicles have been observed in xenografts of human cryopreserved ovarian tissue (Newton et al., 1996; Oktay et al., 2000), and initiation of growth has been inferred in ~12% of follicles based on staining for proliferating cell nuclear antigen (Oktay et al., 2000), the present study is the first report of morphological evidence of antral follicles and an associated rise in oestradiol. Similarly, follicle growth within pieces of human cryopreserved ovarian tissue in vitro has been limited to development to the proliferating or secondary stage (Hovatta et al., 1997). However, clinical ultrasound and endocrine evidence of a single antral follicle following autologous grafting of a reconstituted entire ovary which had been cryopreserved in pieces, using PROH, has recently been reported (Oktay and Karlikaya, 2000).
Antral follicle development has been reported following autografting of cryopreserved tissue in the sheep (Gosden et al., 1994; Salle et al., 1998, 1999; Aubard et al., 1999) and the mouse (Harp et al., 1994; Cox et al., 1996) and live births reported (Parrott, 1960; Gosden et al., 1994; Gunasena et al., 1997; Sztein et al., 1998; Candy et al., 2000; Shaw et al., 2000). Similarly, antral follicles were present in grafts of marmoset cryopreserved ovarian tissue (Candy et al., 1995) grafted under the kidney capsule and elephant ovarian tissue (Gunasena et al., 1998) grafted on to the ovarian bursa of nude mice. The ovarian tissue in all of these studies was cryopreserved using DMSO. Although PROH was used in the present study, the limited data available to date does not permit valid comparison of different cryoprotectant regimens for ovarian tissue.
Although the follicular sizes (antral diameter) reported in the present study are similar to those previously reported following grafting of fresh human ovarian tissue either under the kidney capsule (2.5–5 mm; Oktay et al., 1998) or s.c. (6 mm; Weissman et al., 1999), development to the antral stage was seen earlier in the other studies (17 and 14 weeks respectively). However, FSH was administered during the shorter observation periods in both the above studies. The similarity in maximum size observed suggests that this may relate to a restriction imposed by the site or the host animal. However, this interpretation may be an oversimplification given that similar sized follicles have developed in elephant ovarian xenografts to the ovarian bursa in mice (Gunasena et al., 1998) and in autografts (Gosden et al., 1994; Aubard et al., 1999) in sheep.
Follicular development in mice which did not receive exogenous FSH, in the present study, indicates that the circulating concentrations of FSH in oophorectomized mice are sufficient for follicular development to the antral stage. At present, the small sample size makes it difficult to establish any effect of exogenous FSH, which has previously been suggested to improve development in fresh grafted tissue (Oktay et al., 1998; Weissman et al., 1999). Whether the differences observed in the present and the above studies are attributable to host strain differences which may influence endogenous gonadotrophin concentrations (a distinct possibility in the study of Oktay et al., 1998) is unclear.
In conclusion, this has been the first study to confirm normal growth and development of human follicles to the antral stage following cryopreservation. Although preliminary, the development of antral follicles in tissue from two patients and within almost all of the grafts after 20 weeks suggests that development following grafting is reproducible. Similarly, the high rate of functional preservation of follicles following cryopreservation suggests that cryopreservation of ovarian tissue has real potential for clinical application.
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Acknowledgments |
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We thank Angela Nelson for her expert care of the animals and the Department of Microbiology, University of Melbourne for the use of their animal facility. We also thank Clyde Riley for sectioning of histological blocks, Janell Archer for the hormone assay and general laboratory assistance and Serono Australia for the gift of gonadotrophin.
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Notes |
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3 To whom correspondence should be addressed at: Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Australia. E-mail: gookd{at}cryptic.rch.unimelb.edu.au
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Submitted on September 5, 2000; accepted on November 24, 2000.
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J. Liu, J. Van der Elst, R. Van den Broecke, and M. Dhont Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries Hum. Reprod., March 1, 2002; 17(3): 605 - 611. [Abstract] [Full Text] [PDF]
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