1 Reproductive Services, Royal Womens Hospital, Carlton, Victoria, 2 Melbourne IVF, East Melbourne, Victoria and 3 Department of Obstetrics and Gynaecology, Sandringham Hospital, Sandringham, Australia
4 To whom correspondence should be addressed at: Reproductive Services, Royal Womens Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. e-mail: debra.gook{at}rwh.org.au
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Abstract |
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Key words: cryopreservation/human/luteinized follicle/mature oocyte/xenotransplantation
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Introduction |
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Cryopreservation of animal ovarian tissue was initially reported during the 1950s (Deanesly, 1954; Green et al., 1956
; Parkes, 1958
; Parrott, 1960
), and live births were subsequently documented following the autografting of such tissue (Parrott, 1960
; Gosden et al., 1994a
; Gunasena et al., 1997
; Sztein et al., 1998
; Candy et al., 2000
; Shaw et al., 2000
). In particular, the birth of a lamb (Gosden et al., 1994a
) and resumption of cycling for a period of 2 years in ewes (Baird et al., 1999
) following grafting of cryopreserved ovine ovarian tissue suggested that similar success may be possible in the human given the similarity in follicle distribution and density of stromal tissue. Initial attempts to freeze human ovarian strips using the same procedure as that used in the sheep studies (Gosden et al., 1994a
) indicated that some primordial follicles had survived the cryopreservation (Hovatta et al., 1996
; Newton et al., 1996
; Oktay et al., 1997
). Further studies with human tissue have demonstrated initiation of mitosis in some primary follicles following xenografting (Oktay et al., 2000
). However, the reduction in follicle numbers, high rate of fibrosis in the tissue (Kim et al., 2000
; Nisolle et al., 2000
) and histological evidence of follicular damage associated with manipulation of the cryopreservation procedure (Gook et al., 1999
), highlight some fundamental concerns associated with the cryopreservation of human ovarian tissue. Until recently, growth of follicles following xenografting had only been established in non-frozen human ovarian tissue (Oktay et al., 1998
; Weissman et al., 1999
). More recent reports of growth to the antral stage within cryopreserved ovarian tissue following xenografting (Gook et al., 2001
; Van den Broecke et al., 2001a
; Kim et al., 2002
) are encouraging, and indicate preservation of developmental potential in the follicles. Reports of albeit transient resumption of cycling in two patients following auto-transplantation of cryopreserved ovarian tissue (Oktay and Karlikaya, 2000
; Radford et al., 2001
) further support the potential of the technology. In contrast to these encouraging results, there is a lack of fundamental evidence that the primordial follicles cryopreserved within human ovarian tissue can fulfil their full developmental potential and progress to ovulation of mature oocytes.
The aim of the present study was to examine whether periovulatory changes could be induced by administration of hCG in human antral follicles which had developed within xenografted cryopreserved ovarian tissue.
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Materials and methods |
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Cryopreserved ovarian tissue was xenografted into 12 mice, using methods reported previously (Gook et al., 2001). Following thawing, slices were transferred to Hams F12 medium (Trace Scientific Ltd, Noble Park, Australia) with 10 mg HSA/ml, and cut into smaller pieces (
0.5x0.5x1 mm). Female, 16-week-old severely compromised immunodeficient (SCID) mice were anaesthetized with an i.p. injection of 2,2,2-tribromoethanol (0.4 mg/g body weight; Avertin; Sigma-Aldrich). A single piece of ovarian cortex was inserted under the capsule of each kidney, and the mice were oophorectomized bilaterally. An i.p. injection of antibiotic (4 µg/g body weight; Gentamycin; Pharmacia-Upjohn) was given while the animal was anaesthetized. At 7 days after surgery, i.p. injections of gonadotrophin (1 IU recombinant FSH; Gonal F; a gift from Serono, Australia, and Puregon; a gift from Organon, Australia) were commenced and continued every second day until completion of the study. At
27 weeks following grafting, mice received an i.p. injection (20 IU) of hCG (Profasi; Serono). The kidneys were removed at 3036 h after hCG administration, examined initially for gross morphological changes in the appearance of antral follicles, and subsequently fixed in 4% paraformaldehyde (ProSciTech, Thuringowa, Queensland, Australia) for histology. Follicular diameters were measured using an ocular micrometer both prior to fixation and at maximum diameter on histological section. Fixed tissue was processed followed by embedding in paraffin wax, and 3 µm serial sections were cut and stained with haematoxylin and eosin. Due to the possibility of folds occurring within the follicle wall, only cavities containing an identifiable oocyte were classified as antral follicles.
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Results |
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Corpora lutea
On one site (14), a follicle was detected which was morphologically different from all other follicles, and was classified as a recent corpus luteum. The entire cavity was filled with blood (Figure 4A) which precluded the identification of any oocyte. The cavity was surrounded incompletely by a fibrous wall that was thick and contained very few cells, some of which were luteinized (Figure 4B; L). Luteinized cells were also present in the stromal tissue adjacent to the wall. The fibrous wall was completely absent in one region, in which the cavity contents merged with the stromal tissue. Higher resolution examination of the cavity contents (Figure 4B) revealed erythrocytes, polymorphs and macrophages in association with large luteinized cells (some of which contained yellow pigmentation) abutting the wall. Heavily eosin-stained granules were present throughout the remainder of the cavity and also within the large luteinized cells.
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Summary of observed periovulatory changes
All the characteristics reported were more prevalent within those follicles (n = 15) with a larger diameter (2 mm). Thinning of the exterior follicular wall occurred in over half (17/32) of all follicles, and eight follicles (all of which had a diameter
2 mm) had completely ruptured. Blood was observed within nine follicles, seven of which were
2 mm in diameter. Although a mucified cumulus oocytes complex was observed in all 32 follicles, detachment of the complex from the pedicle rarely occurred in the smaller (<2 mm) follicles. In almost all (14/15) of the larger follicles, partial and complete detachment of the complex from the pedicle was observed. Resumption of meiosis had occurred in over half (20/32) of all oocytes. Five were at MII, seven at MI, and GVBD had commenced in eight. Two corpora lutea were also detected.
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Discussion |
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The morphology observed in the present study was consistent with generally accepted periovulatory changes in mammalian ovarian follicles. Difficulties associated with obtaining periovulatory and ovulatory samples have limited reported observations on human tissue. However, in the rodent and rabbit (Burr and Davies, 1951; Blandau, 1967
; Byskov, 1969
; Motta et al., 1971
; Bjersing and Cajander, 1974
) similar observations to those presented in the present studythat is, the destruction of the granulosa and theca cell layers, mainly in the apical regionhave been reported. At
1 h before ovulation in the rabbit, evidence was observed (Blandau, 1967
) of separation and vacuolation of the granulosa and theca cells at the stigma site. Progressive deterioration was evident, resulting in only a few strands of connective tissue remaining just prior to ovulation. The wall finally ruptured, releasing the mucified cumulusoocyte complex, which had dislodged from the pedicle. As in the present study, blood has been observed within the cavity (Burr and Davies, 1951
) or associated with the escaping cumulus oocyte complex (Blandau, 1967
; Motta et al., 1995
). The withdrawal of the corona cell processes, mucification of the cumuluscorona cell complex and nuclear maturation of the oocyte seen in the present study are also consistent with ovulated oocytes (Motta et al., 1995
) and routine observations following follicle aspiration for clinical assisted reproduction (Veeck, 1991
).
Evaluation of the pre-existing follicle population in cryopreserved human ovarian tissue (Gook et al., 1999) and the observed time requirement for antral follicle development following xenografting (Gook et al., 2001
) indicate that these antral follicles developed to the periovulatory stage, following xenografting, from primordial follicles which were present in the cryopreserved tissue. The full range of ovulatory changes in multiple follicles reported in the present study demonstrates that full functional potential has been preservedan important prerequisite for clinical application of this technology.
Histological evidence of luteinization in human cryopreserved ovarian tissue following xenografting (Kim et al., 2002) has also been observed when dimethylsulphoxide (DMSO) was used as the cryoprotectant. The DMSO cryopreservation regimen, which is frequently used for animal and human ovarian tissue, was initially reported for use with sheep ovarian tissue (Gosden et al., 1994a
), and numerous live births have been achieved from autografting of ovarian tissue cryopreserved using this DMSO regimen in animals. However, data relating to the extended development of follicles within human ovarian tissue cryopreserved with DMSO are limited, thereby precluding a conclusive comparison of the relative effectiveness of the two cryoprotectants. Recent evidence of temporary resumption of menses in a single patient following autologous transplantation of pieces of a whole ovary, which had been cryopreserved using the DMSO procedure (Radford et al., 2001
), and the presence of corpora lutea in a subsequent study (Kim et al., 2002
) in which a modification of this procedure was used, suggest that this method can preserve follicular function within the human ovarian cortex. However, in contrast to the minimal follicle loss (7%) observed as a consequence of cryopreservation damage in sheep (Baird et al., 1999
), a significant loss of cellular structure has been reported following DMSO cryopreservation and xenografting of human ovarian tissue (Kim et al., 2000
; 2002; Nisolle et al., 2000
) and has been estimated to result in a 50% reduction in the number of primordial and primary follicles compared with non-frozen tissue (Nisolle et al., 2000
). Degenerative changes in oocytes and granulosa cells have also been observed (Nisolle et al., 2000
; Van den Broecke et al., 2001b
) in human ovarian tissue cryopreserved using DMSO. The follicular development and luteinization observed in the present study, together with the temporary return to menses in a patient receiving ovarian tissue cryopreserved using the PROH procedure (Oktay and Karlikaya, 2000
), indicate that this approach, which has previously been optimized for human ovarian cortex (Gook et al., 1999
; 2000), is consistent with survival and subsequent functional competence. The total number of antral follicles (fewer than 10) previously reported to have developed within human cryopreserved ovarian tissue following autografting or xenografting remains limited (Oktay and Karlikaya, 2000
; Radford et al., 2001
; Van den Broecke et al., 2001a
; Kim et al., 2002
). The number of follicles which developed in the present study (n = 32) and in a previous study (n = 6; Gook et al., 2001
) indicate that preservation of cellular function within follicles is highly reproducible using this cryopreservation procedure.
Interestingly, although the follicles which developed in the present study were approximately one-fifth of the diameter of periovulatory human follicles, they contained mature oocytes of normal size and exhibited the full range of morphological characteristics associated with ovulation. Similar or smaller follicle sizes to those in the present study have been observed following xenografting of human tissue, whether non-frozen (Oktay et al., 1998; Weissman et al., 1999
; Nisolle et al., 2000
; Gook et al., 2001
) or cryopreserved (Kim et al., 2000
; 2002; Nisolle et al., 2000
; Oktay et al., 2000
; Gook et al., 2001
; Van den Broecke et al., 2001a
), suggesting that the inability to achieve normal size does not relate to the cryopreservation. Smaller than normal diameters have also been observed in sheep follicles (Gosden et al., 1994b
; Salle et al., 1998
; 1999; Aubard et al., 1999
; Jeremias et al., 2001
) which developed following autologous transplantation of cryopreserved tissue. This phenomenon has also been reported following autologous (Lee et al., 2002
) and heterologous (Candy et al., 1995
) transplantation of cryopreserved monkey ovarian tissue. These results suggest that restricted follicle size may be a consequence of the transplantation site.
The present report is the first describing mature (MII) oocyte development from cryopreserved human ovarian cortex. The resumption of nuclear maturation in all oocytes within the larger follicles indicates the reproducible nature of the preservation of not only follicular but also oocyte function using the PROH cryopreservation procedure. Oocytes have been observed in antral cavities in previous studies using human cryopreserved ovarian tissue (Kim et al., 2000; Oktay et al., 2000
; Gook et al., 2001
; Van den Broecke et al., 2001a
). In these studies, however, no exogenous stimulus for induction of maturation was administered and oocytes remained at the germinal vesicle stage. The identification of mature oocytes is a crucial step towards the success of human ovarian tissue cryopreservation, and also in the future development of ovarian tissue transplantation. Initial clinical reports using various transplantation techniques have, to date, resulted in the recovery of only one metaphase I oocyte (Oktay et al., 2001
).
In conclusion, the highly reproducible and extensive evidence presented in the present study illustrates clearly that primordial follicles within human ovarian tissue, when cryopreserved using PROH, can subsequently develop to the antral stage and undergo the full range of morphological changes associated with ovulation, luteinization and oocyte maturation. Not only has this confirmed the clinical potential of this approach, but it has also provided a model which has allowed characterization of the process of human ovulation.
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Acknowledgements |
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Submitted on March 21, 2003; accepted on May 20, 2003.