Laboratory of In vitro Fertilization, Department of Gynaecology, Hospital of Saint Luc, University of Louvain (UCL), B-1200 Brussels, Belgium
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Abstract |
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Key words: cryopreservation/epidermal growth factor/ovarian tissue/primordial follicle/receptor
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Introduction |
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Epidermal growth factor (EGF) is a 53 amino acid growth factor that shows mitogenic effects in a variety of mesodermal and ectodermal tissues, and is involved in regulating cell proliferation in mammals (Fisher and Lakshmanan, 1990). In females, EGF has been found to stimulate the secretion of several hormones in trophoblast cells of the placenta during pregnancy (Miyazawa, 1992
; Qu and Thomas, 1995
). EGF plays a role in oocyte maturation in vitro (Gomez et al., 1993
; Singh et al., 1997
) and also stimulates the proliferation of granulosa cells in vivo and in vitro (Feng et al., 1987
; Roy and Greenwald, 1990
). Transforming growth factor-alpha (TGF-
), with 50 amino acid residues, has a 3540% homology with EGF (Fisher and Lakshmanan, 1990
). EGF and TGF-
both bind to a single EGF receptor, a transmembrane glycoprotein, on the surface of cells. The EGF receptor is present in the ovary and corpus luteum (Ayyagari and Khan-Dawood, 1987
; Feng et al., 1987
) and in endometrium (Imai et al., 1995
). There is evidence that EGF binds to thecal cells, atretic follicles and luteal cells. EGF and its receptor are expressed in the oocytes of primary and preantral follicles of human ovarian tissue (Maruo et al., 1993
; Tamura et al., 1995
). A recent report revealed the expression of TGF-
and EGF in human primordial and primary follicles (Reeka et al., 1998
), though studies of the presence of EGF receptor in human primordial follicles are limited in number. Although the EGF receptor has been found in the primordial follicles of human fetuses (Bennett et al., 1996
), its presence in primordial follicles of human adult ovarian tissue remains to be determined.
The storage of ovarian tissue at low temperature is an attractive concept for in-vitro fertilization (IVF) and ovarian tissue transplantation. It has been demonstrated that cryopreservation of ovarian tissue does not substantially damage follicles in sheep (Gosden et al., 1994), primates (Candy et al., 1995
) and humans (Hovatta et al., 1996
; Oktay et al., 1998a
). After freezing, primate and human ovarian tissues have been successfully grafted into immunodeficient animals (Candy et al., 1995
; Newton et al., 1996
; Oktay et al., 1998b
). Normal follicles developed in cryopreserved marmoset and human ovarian tissues grafted into mice (Candy et al., 1995
; Newton et al., 1996
). In addition, small pieces of ovarian tissue rapidly became revascularized under the kidney capsules of host mice, while human primordial follicles grew to early antral stages in xenografts in severe combined immunodeficient (SCID) mice (Oktay et al., 1998b
).
The freezing of matured oocytes for clinical use is still in debate because of the adverse effects of cooling and cryoprotectants which may damage the microtubular system and meiotic spindle and induce polyploidy, raising questions about the safety of this methodology (Pickering and Johnson, 1987; Tucker et al., 1996
; Oktay et al., 1998a
). Nevertheless, immature oocytes are less likely to be injured by cooling and cryoprotectants because they are small, not well developed, with few organelles, no zona pellucida, and are relatively metabolically quiescent and undifferentiated. Therefore, ovarian tissue containing immature oocytes in primordial follicles may be better suited to cryopreservation and grafting. Advances in developing new technologies for the growth of immature mammalian oocytes open up the possibility of using immature human oocytes from very early follicular stages to full maturity in vivo and in vitro (Eppig and O'Brien, 1996
; Wandji et al., 1997
; Oktay et al., 1998a
). The maturation of human oocytes in primordial and primary follicles in vitro or in vivo, in combination with the cryopreservation of ovarian tissues, may benefit many infertile patients, provide follicles for young cancer patients undergoing chemo- or radiotherapy and for prematurely menopausal women, and revolutionize IVF (Nayudu, 1994
; Gosden and Rutherford, 1997
; Donnez and Bassil, 1998
).
We attempted to set up a bank of human ovarian tissue for the IVF programme in our research unit. To improve the technology for the cryopreservation of ovarian tissue and the growth of oocytes from early follicles in vitro and in vivo, we studied: (i) the distribution of follicles in ovarian tissue samples from patients; (ii) the influence of cryopreservation on the morphology and number of surviving follicles in ovarian tissue; (iii) the expression of EGF receptors in primordial follicles in ovarian tissue; and (iv) the possible effect of freezing on the immunoreactivity of the EGF receptor in the follicles of frozen ovarian tissue.
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Materials and methods |
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Ovarian tissue
After biopsy, ovarian tissue was immediately transported to the laboratory in Leibovitz L-15 medium supplemented with glutamax (Gibco, Paisley, UK) at 4°C. Ovarian tissue from 14 patients was dissected into small pieces (1 mm3). Some pieces of tissue were fixed in Bouin's solution, and immediately underwent histological study; these were assigned to the fresh fragment group. The other pieces of tissue were processed using a programmable freezer and stored in liquid nitrogen as the frozen fragment group. Ovarian tissue from another 10 patients was cut in two different ways. First, ovarian tissue was dissected in the same way as described above. Second, ovarian tissue was cut into strips (1x1x46 mm). A small piece was cut from the same strip, fixed in Bouin's solution, studied histologically, and used as the fresh control fragment. The remaining strip was frozen and stored in liquid nitrogen, and assigned to the frozen strip group. After various periods of time, frozen fragments and strips were thawed and fixed in Bouin's solution for further histological studies.
Freezing
Ovarian tissue samples were frozen using a modification of a published method (Gosden et al., 1994). Briefly, Leibovitz medium containing 2% human albumin (Red Cross, Brussels, Belgium) and 10% dimethylsulphoxide (DMSO) (Sigma, St Louis, MO, USA) was dispensed into (1 ml/vial) cryogenic vials (Simport, Quebec, Canada) precooled on ice. Ovarian tissue fragments (five to six pieces) or strips (one to two pieces) were suspended in the cryoprotective medium, and were equilibrated in the medium for 30 min on ice before cooling was initiated. The cryotubes were cooled in a programmable freezer (Kryo 10, Series III, Planer, Sunbury on Thames, UK) using the following programmes: (i) cooled from 0°C to 8°C at 2°C/min; (ii) seeded manually by touching the cryotubes with forceps prechilled in liquid nitrogen; (iii) cooled to 40°C at 0.3°C/min; (iv) cooled to 150°C at 30°C/min; and (v) transferred to liquid nitrogen (196°C) immediately for storage.
Thawing
The cryogenic vials were thawed at room temperature for 2 min, and immersed in a water bath at 37°C for another 2 min. The ovarian tissues were immediately transferred from the vials to tissue culture dishes (Becton Dickinson, Meylan Cedex, France) in Leibovitz medium, and subsequently washed three times with fresh medium to remove cryoprotectant before further processing.
Histological study
Fixed fresh and frozen samples of ovarian tissue, fragments or strips were dehydrated with ethanol, cleared with toluene and embedded in paraffin. A continuous series of ovarian tissue sections of 6 µm thickness was prepared from each ovarian fragment and mounted on glass slides. The odd slides were stained with haematoxylin and eosin for morphological study and follicle counting, and the even slides were immunohistochemically stained later and not used for counting follicles. Areas of tissue sections of different shapes were viewed under the microscope incorporating a micrometer and calculated individually according to geometrical formulae. Morphological changes associated with the death of follicles were observed microscopically in the sections from each paraffin tissue block, including increased eosinophilia, shrinkage of the cytoplasm, and karyolysis, pyknosis and karyorrhexis of the nucleus. The number of follicles was counted in individual tissue sections. If one follicle was present in several continuous sections, it was only counted in the first section in order to avoid repetition. Unfortunately, no computerized image analysis system was available in our laboratory. Follicles were classified according to the stages of development as follows (Gougeon, 1991, 1996
): (i) primordial follicle: one layer of flattened cells around the oocyte; (ii) primary follicle: one layer of cuboidal cells around the oocyte; (iii) secondary follicle: more than two layers of cuboidal cells around the oocyte.
Immunohistochemical study
Immunohistochemical staining was performed using an avidinbiotinimmunoperoxidase system. The sections were deparaffinized with toluene, rehydrated in ethanol and quenched in 3% hydrogen peroxide for 30 min at 37°C to block the activity of endogenous peroxidase. After washing in water and Tris-buffered solution (TBS; TrisHCl 50 mmol/l, NaCl 150 mmol/l, pH 7.6), normal goat serum (Dako, Glostrup, Denmark) was applied for 1 h at 37°C to minimize non-specific binding. The antibody against human EGF receptor (Oncogene Research Products, Cambridge, MA, USA) was used as the primary antibody at a dilution of 1:50 and added to the sections for overnight incubation in a sealed humidified chamber at 4°C. After washing with TBS, biotinylated secondary antibody (1:250) (Boehringer Mannheim, Brussels, Belgium) was applied, and the specimens were incubated for 30 min at room temperature. Streptomycinavidinperoxidase conjugate (Boehringer Mannheim) was added for 30 min at room temperature. The chromogenic reaction was developed by incubation with a freshly prepared solution of 3,3'-diaminobenzidine (Dako). The sections were counterstained with Mayer's haematoxylin (Merck, Darmstadt, Germany), mounted with a coverslip, and evaluated using microscopy. Optimal working dilutions were determined by serial titration. Control procedures were undertaken to assure the specificity of the immunological reaction. Two negative controls were included simultaneously by replacing the primary antibodies with: (i) phosphate-buffered saline (PBS); and (ii) rabbit IgG (Dako) at the same dilution for the specific primary antibody. In some experiments, the antibody was preabsorbed with EGF receptor peptide (Oncogene Research Products), resulting in the absence of staining for EGF receptor in ovarian tissue sections. Sections of normal human endometrium of the uterus, known to contain EGF receptor (Imai et al., 1995), served as the positive controls.
Statistical analysis
Means between individual groups were analysed by the Wilcoxon signed rank test. The correlation between follicle number and patients' age was analysed by linear regression. A P-value < 0.05 was considered statistically significant.
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Results |
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Age distribution of follicles in ovarian tissue
A negative correlation was observed between the number of follicles in ovarian tissue and the age of patients. Figure 3 shows the age distribution of follicle numbers in fresh and frozen ovarian tissue fragments in patients aged 2141 years. The number of follicles in ovarian tissue declined with the increasing age of the patients in a linear regression (r = 0.485, P = 0.016 in fresh ovarian tissue fragments, and r = 0.515, P = 0.01 in frozen ovarian tissue fragments).
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Discussion |
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Cryopreservation of human ovarian tissue has been extensively studied previously. In addition to an early study (Grischenko et al., 1987) in which cryopreserved human ovarian tissue, when transplanted subcutaneously in women, restored their menstrual cycles, it was also reported that cryopreserved human ovarian tissue could be successfully transplanted beneath the kidney capsule of immunodeficient mice (Newton et al., 1996
). A histological study (Hovatta et al., 1996
) showed that, after cryopreservation, the surviving follicles appeared morphologically normal in frozenthawed human ovarian tissue in comparison with those from fresh ovarian tissue. Approximately two-thirds of the follicles were able to survive freezethawing, as measured by histological examination after frozen human ovarian tissue has been cultured in vitro for 15 days (Hovatta et al., 1997
).
A cryoprotectant is essential for minimizing ice formation and the survival of ovarian tissue after freezing and thawing. Cryopreservation using ethylene glycol or DMSO resulted in the survival of 84% or 74% of the follicles respectively, compared with only 44% when propylene glycol was used, and 10% with glycerol, in frozenthawed human ovarian tissue grafted into the renal capsule of SCID mice (Newton et al., 1996). The extent of follicular survival was presumed to be in part related to the speed of cryoprotectant permeation into ovarian tissue specimens (Candy et al., 1997
). A quantitative study using NMR spectroscopy (Newton et al., 1998
) revealed that at 4°C, ethylene glycol and DMSO permeated human ovarian tissue more efficiently than either propylene glycol or glycerol. Recently, it was reported (Oktay et al., 1998b
) that human primordial follicles could develop to antral-secretory stages in ovarian tissue xenografts in SCID/hpg mice as measured by morphological examination and proliferating cell nuclear antigen (PCNA) immunostaining. Administration of follicle stimulating hormone (FSH) was required to promote the growth of follicles beyond the two-layer stage in the mice. These encouraging results raise hopes for the clinical use of ovarian tissue banking, although at present we have not reached the goal of applying cryopreserved human ovarian tissue to the treatment of patients (Moomjy and Rosenwaks, 1998
; Newton, 1998
; Weissman et al., 1999
). However, the usefulness of ovarian tissue autografting may be limited in patients with ovarian failure caused by endocrine- or immunology-related problems, or in those in whom the ovarian tissue is contaminated with viable cancer cells.
In studies of tissue cryopreservation and transplantation, human ovarian tissue has mainly been collected by biopsy from patients undergoing laparoscopy in clinics. Usually, only one sample is obtained at biopsy. Whether this ovarian tissue sample contains a sufficient number of primordial follicles is vital for the success of autografting or allografting of ovarian tissue in vivo and the maturation of oocytes in vitro. In the present study, we investigated the distribution of follicles in fragments of human ovarian tissue, obtained by biopsy from 24 women, before and after cryopreservation. Our results showed that the number of follicles in ovarian tissue samples differed from patient to patient, ranging from 0 to 64 follicles/mm3. This implies great variation in follicular distribution in ovarian tissue between individual patients. We also observed that even in the same patient, the number of follicles varied to a great extent between tissue fragments. These results suggest that follicles are unevenly scattered in the cortex of the ovary and that the number of follicles counted in one ovarian tissue fragment may not represent the number found in another. Although it is not yet known how many follicles will be needed for grafting or growth of follicles in vitro, it is proposed that gynaecologists collect multiple samples from different parts of the ovary at biopsy in order to reduce the risk of the lack of follicles in obtained ovarian tissue fragments.
The inverse relationship between the age of women and their rate of fertility is well established (Menken et al., 1986). It has been reported that the number of primordial follicles in the ovaries declines from more than 250 000 at menarche to hundreds or thousands at the end of reproductive life (Baker, 1963
). At the age of 51 years (the median age of menopause in Western society), only 1000 follicles remain in the ovaries (Faddy et al., 1992
). In our study, we also observed a negative correlation between follicular number and the age of the study subjects. Follicular density in ovarian tissue declined with patient age between 21 and 41 years. Our observation is consistent with the findings of others (Lass et al., 1997
), who reported a negative correlation between follicular density and ovarian volume, and the age of women.
In the present study, we found that primordial follicles were distributed predominantly in the outer cortical region of the ovary, accounting for 78.682.6% of total follicles. By contrast, primary follicles were sparsely scattered, and secondary follicles could only be found occasionally. This observation indicates that the number of primordial follicles is much greater than primary and secondary follicles in the ovaries. Primordial follicles should therefore be considered as a major potential source of oocytes for future use in IVF programmes in clinics after maturation in vitro or in vivo.
The viability of ovarian tissue after cryopreservation is crucial in the transplantation of ovarian tissue or in-vitro maturation of oocytes. Viability is influenced by many factors, including the method of freezing, the choice of cryoprotectant, and the time lapse between the sample being taken in the operating theatre and the tissue being processed and frozen in the laboratory. In the present study, we used DMSO as a cryoprotectant for freezing ovarian tissue, as previously described. We did not observe any significant change in the morphology of the cortex and medulla of ovarian tissue before and after cryopreservation. Neither was there any decrease in the number of morphologically normal follicles.
In cryopreservation, tissue samples are generally cut into small fragments (about 1 mm3) to allow the cryoprotectant to penetrate to the centre of the tissue mass. However, such a method for ovarian tissue processing is time-consuming and may influence the viability of follicles after freezing. It is also difficult in routine clinical practice to freeze ovarian tissue, especially with large ovarian samples taken by ovariectomy. To avoid cell death, fresh ovarian tissue should be processed not more than 1 h after the samples have been obtained in the operating theatre. In some experiments of this study, ovarian tissue was cut into strips (1x1x46 mm) for cryopreservation. After freezing both ovarian tissue strips and fragments, no significant differences were found between the two sample types in terms of morphological changes and follicular numbers. This suggests that ovarian tissues may be cryopreserved in the form of strips instead of fragments. Compared with ovarian tissue fragments, tissue strips have the advantage of easier handling for storage in the hospital's ovarian bank, with a better chance of having sufficient follicles either for autografting in vivo or for maturation in vitro.
In humans, primordial follicles are an early form of ovarian follicle. As yet, the mechanism involved in the development of primordial follicles to preantral follicles has not been clarified. It has been found that primary imprinting during oocyte growth has a crucial effect on both the expression and repression of maternal alleles during embyrogenesis (Obata et al., 1998). EGF is a growth factor known for its numerous functions in the regulation of cell transformation and differentiation (Fisher and Lakshmanan, 1990
). In women, EGF plays an important role in regulating the secretion of hormones in trophoblast cells in the placenta during pregnancy (Miyazawa, 1992
; Qu and Thomas, 1995
). In the ovary, EGF may also regulate the function of follicular development (Das et al., 1991
; Lonergan et al., 1996
; Goud et al., 1998
). It has been shown that EGF altered the growth of granulosa cells in rats (Bendell and Dorrington, 1990
), and inhibited the action of FSH to augment aromatase activity in human granulosa cells in vitro (Steikampf et al., 1988
). In addition, EGF or TGF-
stimulated the proliferation of porcine granulosa cells (Leal et al., 1990
), while EGF, either alone or in combination with insulin-like growth factor I, stimulated cumulus expansion in bovine oocytes matured in vitro (Lorenzo et al., 1994
). A recent report (Goud et al., 1998
) showed that addition of EGF to maturation medium, and maintenance of the cumulus during culture, improved the nuclear and cytoplasmic maturation of human oocytes in vitro. Furthermore, EGF exposure stimulated the growth of human preantral follicles and induced the expression of TGF-ß type II receptor in almost all follicular cells in the culture, implying a role for EGF in the regulation of preantral folliculogenesis (Roy and Kole, 1998
)
There is increasing evidence that the EGF receptor is present in human ovaries (Maruo et al., 1993; Tamura et al., 1995
; Bennett et al., 1996
; Reeka et al., 1998
). In two of these studies (Maruo et al., 1993
; Tamura et al., 1995
), the immunolocalization of EGF and its receptor in human ovaries during follicular growth and regression was investigated. These authors found immunoreactivity for EGF and EGF receptor in the oocytes of primary and preantral follicles. More recently, it was reported (Reeka et al., 1998
) that both TGF-
and EGF were expressed in the oocytes of primordial and primary follicles. The staining intensity for TGF-
and EGF decreased in the preantral follicle and finally disappeared. By contrast, EGF receptor expression was observed in the granulosa cells of antral follicles. EGF and its receptors were identified by immunohistochemical staining in the ovarian tissue of human fetuses aged 1224 weeks (Bennett et al., 1996
). Expression of EGF receptor was localized in the oocytes, suggesting the presence of EGF receptor in the primordial follicles of fetal ovaries. In the present study, we observed that immunoreactivity for the EGF receptor was present in primordial follicles in human adult ovarian tissue. The strong immunohistochemical staining for EGF receptor was localized in the oocyte, while weak staining was also present in some surrounding pregranulosa cells. In a later study, we also observed the expression of EGF receptor in primary and secondary follicles. Our results corroborate the previous findings of others (Bennett et al., 1996
), and suggest that the EGF receptor can be expressed in the follicles from very early stages of folliculogenesis. Following a later report on the expression of TGF-
and EGF in primordial follicles (Reeka et al., 1998
), these results further suggest that the EGF receptor may be a mediator for the action of EGF/TGF-
in the regulation of the growth of oocytes in primordial follicles. The observations of this study are not consistent with an earlier report (Maruo et al., 1993
) which noted the lack of EGF receptors in primordial follicles. The reason for such a discrepancy is unknown, but one possible explanation is that different techniques were used, for example the fixatives for ovarian tissue staining.
This study was one part of our research project on human ovarian tissue banking and the in-vitro maturation of oocytes in early follicles. The presence of EGF receptor in follicles suggests that EGF or TGF- may be involved in regulating the growth of follicles in human ovarian tissue from very early developmental stages. We assume that EGF or TGF-
may be used in the culture of early follicles from frozen ovarian tissue to promote the growth of immature oocytes in vitro. To exclude the possibility that freezethawing would alter or damage the EGF receptors in ovarian tissue after cryopreservation, we compared the immunoreactivity of the EGF receptor between frozenthawed and fresh ovarian tissues and, indeed, identified no such effect.
In conclusion, the EGF receptor may be expressed in the primordial follicles of human adult ovarian tissue. It would, therefore, be worthwhile in the future to study the effect of EGF/TGF- and their receptors on the in-vitro growth of oocytes in primordial follicles from frozen ovarian tissue.
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Acknowledgments |
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Notes |
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References |
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Submitted on August 11, 1999; accepted on November 1, 1999.