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Department of Obstetrics and Gynecology, Center for Reproductive Biology (D.T.B., B.K.C., L.M.H.), and the Department of Physiology (R.G.G.), University of Edinburgh, Edinburgh, Scotland EH3 9EW, United Kingdom; and the Roslin Institute (Edinburgh) (R.W.), Roslin, Midlothian, Scotland EH 259PS, United Kingdom
Address all correspondence and requests for reprints to: Prof. David T. Baird, Department of Obstetrics and Gynecology, Center for Reproductive Biology, University of Edinburgh, 37 Chalmers Street, Edinburgh, Scotland EH3 9EW, United Kingdom.
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Abstract |
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These results demonstrate that despite a drastic reduction in the total number of primordial follicles, cyclical ovarian function is preserved in sheep after autotransplantation of frozen/thawed ovarian tissue and provide experimental confirmation that such a technique could provide a means of preserving fertility in women undergoing chemo- or radiotherapy for malignant disease.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An alternative approach for large animals is to transplant small pieces of ovarian cortical tissue into which oxygen and nutrients can diffuse before vascularization takes place. We have previously demonstrated in the sheep that primordial follicles in thin strips of ovarian cortex remain viable after freezing to -196 C in cryopreservant (8). In the initial experiment, we demonstrated that viable ovarian tissue was recovered from all six ewes at slaughter, 9 months after transplantation of frozen-thawed ovarian slices to the ovarian pedicle (9). During this experiment one ewe became pregnant and delivered a healthy lamb after transplantation.
These results suggested that it might be possible to restore fertility
in girls and young women whose ovaries were likely to be destroyed as a
result of chemo- or radiotherapy for malignant disease such as
leukemia. However, before offering such an option clinically, it would
be necessary to know how long such grafts might survive so as to
determine whether it was likely that pregnancy could occur. Only a
small portion of total ovarian tissue is transplanted (
5%), and
approximately 50% of oocytes survive the freezing and thawing (10).
Hence, although cyclical ovarian function occurs after
autotransplantation, there is a drastic reduction in the total number
of oocytes present in the grafted ovaries. This situation is analogous
to that which exists clinically during incipient ovarian failure before
spontaneous or radiation-induced menopause when certain minor
abnormalities in ovarian function have been described (2). Although the
cyclical pattern of secretion of estradiol and
progesterone is maintained, characteristically the
concentration of FSH in the early follicular phase of the cycle is
elevated above normal and, in one study, is associated with low levels
of inhibin B (11).
The purpose of the current study was to investigate the endocrine function of the ovaries for a period of 2 yr in sheep after autotransplantation of frozen-thawed ovarian slices.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Plasma was separated within 2 h
of collection and stored at -20 C until analysis.
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Twenty-eight days later, in mid-January 1994, a second laparotomy was performed under general anesthesia when four thawed ovarian slices (two on each side) were anastomosed on to the ovarian pedicle close to the site of ovariectomy as identified by the silk suture. The slices were attached to the ovarian pedicle by two 6/0 prolene sutures (Ethicon Ltd., Edinburgh, UK). Six and 3 days before surgery, 100 µg estradiol benzoate were injected im to increase the vascularity of the uterus and ovarian pedicle.
During anestrus (April to September 1994) the frequency of blood samples was reduced to once per week before being increased to three times per week at the onset of the breeding season (September 1994).
A more detailed assessment of endocrine function was made throughout a complete estrous cycle 1 month before (November 1993) and 11 months after transplantation (November 1994) by collecting blood samples twice daily (0900 and 1600 h) for a period of approximately 20 days from the second estrus after synchronization with progestogen-impregnated sponges and/or cloprostenol (100 µg; Estrumate, Cooper’s Animal Health Ltd., Crewe, UK).
On October 15, 1995, after the onset of estrous behavior in the control ewes, all ewes were injected with 100 µg cloprostenol to induce luteal regression. All ewes showed estrus within 3 days and were given a further injection of cloprostenol on day 8 of the next cycle. Seven days later (approximately day 5 of the cycle), the ewes were anesthetized with thiopentone and 2% halothane, and samples of ovarian venous blood were collected from each side. Careful note of the presence of corpora lutea and follicles and their sizes was made before slaughtering the animals with an overdose of thiopentone. The ovaries were dissected free from the ovarian pedicle, weighed, and fixed in 5% paraform-aldehyde.
Cryopreservation of ovarian tissue
The ovaries were transferred to Leibovitz-L15 medium at room
temperature for preparing cortical strips for cryopreservation. Cortex
that was free of obvious follicles or luteal tissue was cut with a
scalpel to a thickness of approximately 1 mm, avoiding the hilar
area.
The strips intended for autografting were trimmed to remove ragged
edges (5 x 5 mm), and pieces were also set aside for grafting
into immunodeficient SCID mice (see below).
The tissue was cryopreserved using the same protocol as that described by Gosden et al. (9). It was equilibrated for 30 min at 0 C in cryogenic vials containing Leibovitz medium containing 10% bovine calf serum and 1.5 M dimethylsulfoxide. The tubes were transferred to a programmable freezer (Planar Products) and cooled at 2 C/min to 1–7 C for seeding. The second cooling ramp was at 0.3 C/min to -40 C and subsequently at 10 C/min to -140 C. Finally, the tubes were plunged into liquid nitrogen and stored in a dewar for approximately 1 month. The tissue was thawed rapidly by swirling in a water bath at room temperature. It was immediately transferred to fresh medium and washed three more times to remove the cryoprotectant. Four strips were then used for autografting on to the ovarian pedicle.
Further fresh strips were used for assessment of follicle survival in SCID mice. Strips of ovarian tissue were transferred to fresh medium and held at 0 C for 2–3 h before trimming as 1 x 1 x 1-mm blocks. The pieces were allocated randomly to three experimental groups. Each sheep provided four tissue slices for frozen storage and four for fresh grafts.
Group 1
The tissues were fixed overnight in aqueous Bouin’s fluid and
prepared as paraffin wax blocks, serially sectioned at 6 µm, stained
with hematoxylin and eosin, and mounted.
Group 2
The tissues were grafted under the renal capsule of virgin
female SCID mice, aged 8–10 weeks. The procedures were carried out
under strictly aseptic conditions in a laminar flow hood. The tissues
were incubated for 6 h on Milli-cell membranes (Millipore Corp., Bedford, MA) in MEM containing 10% donor calf serum,
gentamicin, and amphotericin B to eliminate any microbial
contamination. The animals were anesthetized with tribromoethanol (0.6
g/kg BW), with the left kidney from each of 32 mice (16 in groups 2 and
3) being exposed by a flank incision for inserting a graft at both
poles. The wounds were closed, and the animals were returned to a
positive pressure isolator, where they were provided with sterile food
and water. They were killed by cervical dislocation 3 weeks later, and
the grafts were retrieved for histology as in group 1.
Group 3
These tissues were cryopreserved using the same protocol as
before. They were stored in liquid nitrogen for 1 week, thawed, washed
thoroughly in fresh Leibovitz medium to remove the cryoprotectant, and
subsequently grafted into SCID mice as before. The timing of tissue
recovery and subsequent histology of the grafts were identical to those
in the other groups. The slides were given code numbers to avoid bias
when counting the total number of primordial follicles per graft.
Hormone assays
Gonadotropin and steroid plasma concentrations were measured in
duplicate using a previously described double antibody RIA. FSH (12),
LH (13), and progesterone were determined in unextracted
jugular venous samples (12). Androstenedione and estradiol were
measured in ovarian venous plasma samples after solvent extraction
(13). The sensitivities of the assay for FSH, LH, progesterone,
androstenedione, and estradiol were 0.3 µg/liter (USDA, oFSH,
SIAFP-RP-2), 0.2 µg/liter (NIDDK, oLH, S23), 380 pmol/liter, 175
pmol/liter, and 50 pmol/liter, respectively. The concentration of
inhibin A in ovarian venous plasma was measured by two-site
enzyme-linked immunosorbent assay described for use in human plasma
samples (14) and modified for use in sheep plasma (15, 16). The
sensitivity of the enzyme-linked immunosorbent assay was 30 ng/liter,
and the intra- and interassay variations in the immunoassays used were
less than 15% in the ED20–80 range.
Statistical analysis
Hormone profile data from all experiments was log transformed
and analyzed by repeated measures ANOVA, with data being partitioned on
the basis of treatment and time (ANOVA). The data from a complete
estrous cycle before and 11 months after autografting was grouped
around the day of behavioral estrus, as there were minor differences in
cycle length between animals. The t test was used for
comparison of ovarian venous blood.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Two ewes became pregnant inadvertently (the ram was incompletely
vasectomized) and delivered healthy twins and triplets in April and
June, respectively. Estrous behavior and cyclical fluctuations in
progesterone were observed in all control ewes in the
autumn of 1995 before slaughter in November.
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and 3). One ewe became pregnant
inadvertently in December 1994 and delivered triplets on May 31, 1995.
There was no difference between control and treated ewes in the length
of the estrous cycle (mean ± SE, 17.0 ± 0.4
vs. 17.3 ± 0.2 days, respectively) or in the number of
detected estruses (excluding those ewes that became pregnant: control,
8 and 9; treatment: mean, 8.4, range, 5–10). In autumn of 1995, all
ewes showed estrus on at least 1 occasion before slaughter in
November.
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).
In the ewes with transplanted ovaries there was a progressive rise in
the concentrations of FSH and LH after ovariectomy in December 1993 to
reach castrate levels by 3 weeks (P < 0.001). After
the injection of estradiol benzoate, the concentration was suppressed
again to levels similar to those in intact ewes (Figs. 2 and 3). After
autotransplantation of ovarian strips, the concentration of FSH rose
progressively to reach a peak value at 14 weeks (P <
0.007). The concentration of FSH then declined steeply
(P < 0.001) to values (4–5 ng/liter) midway between
those in intact (1 µg/liter) and those in castrate animals (8
µg/liter). The values of FSH remained relatively constant before
declining again (P < 0.001) to those found in intact
ewes at the end of the breeding season (March 1995). After 7–8 weeks,
the concentration of FSH increased again (P < 0.001)
to 4–5 µg/liter. In contrast, the concentration of FSH in intact
control ewes remained constant at 1–2 µg/liter throughout the
experimental period (Fig. 4).
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Detailed hormone profile over the estrous cycle
In the control ewes the hormone profiles in November 1993 and 1994
were identical (data not shown). In the treatment group there was no
significant difference in the profile of progesterone
concentration in samples collected before transplantation and that in
samples collected 12 months later i.e. 11 months after
transplantation (Fig. 5). In contrast,
the concentration of FSH was significantly raised (P <
0.001) throughout the luteal phase of the cycle in treated ewes, and
there was an absence of the peaks of FSH on days 1–2 and days 6–8
normally observed in intact ewes. In the follicular phase there was a
marked decline in FSH concentrations (P < 0.0002) in
treated ewes, so that by the day of estrus the levels were not
significantly different from those in the intact ewes. The
concentration of LH during the luteal phase of the cycle was also
raised in the ewes with transplanted ovaries (P <
0.006). There was no significant difference in the magnitude or timing
of the LH surge.
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). There was no significant difference
in the concentrations of estradiol and androstenedione between the
controls and ewes with transplanted ovaries. In contrast, the
concentration of inhibin A was significantly lower in the transplanted
animals than that in the control ewes (mean ± SE,
409 ± 118 vs. 1914 ± 555 pg/ml).
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).
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The average number of follicles per tissue block was slightly less than
200. This was reduced by 65% after grafting the fresh tissue and by
only another 7% after freezing and thawing before grafting (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although none of the primordial oocytes in the grafts in SCID mice showed evidence of development, previous research has demonstrated that preantral follicle development occurs if more time is allowed to elapse (8).
In contrast to controls, no ewes showed evidence of cyclical ovarian function within 3 months of transplantation during the remainder of the breeding season of 1993/1994. In two ewes there was transient elevation of the serum progesterone concentration, indicating the presence of a corpus luteum 60–80 days after transplantation. As few, if any, developing follicles survive cryopreservation and transplantation, these results indicate that the time taken from recruitment of primordial follicle to a large antral follicle is about 80 days. Previous estimates based on the mitotic index of granulosa cells at different stages of development have suggested that 5–6 months are required for folliculogenesis in this species (17, 18). It is possible that the raised levels of gonadotropins observed in these animals accelerated the rate of follicle development compared with that found in intact ewes or that in two ewes some small follicles survived the autotransplantation. The occurrence of anestrus prevented an accurate estimation of the time of reestablishment of ovulation, but clearly it can be as short as 80 days but no longer than 6 months, because cyclical ovarian function was observed in all ewes by the onset of the next breeding season.
The prompt rise in the concentrations of FSH and LH after ovariectomy was probably due to lack of the inhibitory effects of ovarian hormones. After the injection of estradiol benzoate, there was a sharp fall in the concentration of FSH, such that by the time of transplantation it had reached precastration levels. After autotransplantation, the rise in FSH concentration was similar to that observed after castration, indicating that the ovary was secreting minimal amounts of estradiol and inhibin. By mid-May 1994 (4 months after transplantation), there was a significant fall in the concentrations of FSH and LH, probably reflecting the development of large antral follicles in the ovaries. However, the decline in FSH secretion coincided with a period of summer anestrus, which is associated with reduced release of GnRH from the hypothalamus (19).
In the majority of ewes, the concentration of FSH fluctuated from day to day, although the overall mean concentration remained consistently higher than that in control ewes. It is likely that the raised levels of FSH were due to reduced secretion of inhibin A and estradiol, although the secretion of the latter was not different from that in control ewes at the time of slaughter. In sheep, over 90% of estradiol is secreted by large antral follicles greater than 4 mm in diameter (20). In contrast, a significant proportion of inhibin production is derived from small antral follicles (21, 22). The reduced inhibin secretion from transplanted ovaries probably reflects the reduced pool of small antral follicles from which the ovulatory follicles are selected. We have observed similar changes in hemicastrated old ewes (>12 yr), in which the pool of primordial follicles is drastically reduced (23).
Although the concentrations of FSH and LH were raised above normal in the ewes with transplanted ovaries, the pattern of hormones throughout the cycle was similar to normal. The similar concentration of progesterone in the luteal phase suggests that the numbers of corpora lutea (and ovulation rate) were the same before and after transplantation despite the reduced pool of antral follicles. Further support for this hypothesis is derived from the observation that the concentration of FSH declined normally after luteal regression during the follicular phase of the cycle. Direct evidence of the maintenance of ovulation rate was obtained in the four ewes that had ovulated in the few days before slaughter in November 1995 and by the birth of triplets to a ewe that inadvertently became pregnant.
It might be expected that the persistently raised levels of FSH would lead to an increase in the number of large antral follicles and hence an increased ovulation rate. Two factors were probably responsible for maintaining a normal number of ovulatory follicles. Firstly, the total number of follicles in the ovaries was drastically reduced by the transplantation procedure, and hence, although it is likely that a greater proportion of developing follicles avoided atresia, the pool of small antral follicles from which the ovulatory follicles are selected is likely to be limited. Secondly, although the basal level of FSH in the luteal phase was raised, it was suppressed to normal levels during the follicular phase of the cycle. This inhibitory effect of estradiol and inhibin secreted by the dominant follicles ensures that the number of ovulatory follicles is restricted to that which is appropriate for that breed (24).
Although ovarian cyclicity was observed after transplantation, by the time of slaughter at 22 months, there was evidence of abnormalities in all ewes. Only four of the eight ewes had ovulated within 7 days of induction of luteal regression by injection of cloprostenol. The ovaries of all but one ewe had at least one large antral follicle (>7 mm diameter). It is likely in the four ewes in which the ovaries contained no corpora lutea that these large cystic structures (19–24 mm) represent preovulatory follicles that had failed to ovulate. The large follicles (8–9 mm) in the ovaries of three of the ewes that had ovulated may be due to stimulation by the raised levels of FSH of the cohort of follicles (4–6 mm diameter) that developed in the early luteal phase of the sheep estrous cycle. The persistently raised level of LH is likely to have induced luteinization of the theca and disruption of folliculogenesis (25).
In the sheep, the basal secretion of LH is suppressed by a combination of progesterone secreted by the corpus luteum and estradiol derived from large antral follicles. As the concentration of progesterone is normal after transplantation, it must be presumed that the secretion of estradiol is reduced due to a depletion of large antral follicles. The fact that there was no significant difference in LH concentration during the follicular phase between intact and transplanted ewes suggests that the secretion of estradiol from the preovulatory follicles is normal. This would be in keeping with the decline in FSH concentration in transplanted ewes during the follicular phase when estradiol is a more important regulator of FSH than inhibin (22).
Although the number of oocytes in each graft was not measured, estimates can be made based on the number in 1-mm cubes (192). In each ewe, four cortical strips measuring 5 x 5 x 1 mm were autografted, containing approximately 19,200 primordial oocytes (4 x 25 x 192), of which 5376 (28%) survived freezing, thawing, and transplantation. The fact that by the time of slaughter the levels of FSH were near those found in castrate animals together with the presence of cystic ovaries suggests that ovarian failure was imminent. These findings indicate that the grafts have a limited lifespan and that when such techniques are applied clinically, women should be advised to defer autotransplantation until that they wish to become pregnant.
The primary purpose of the present experiment was to determine how long ovarian function could be preserved in ewes after autotransplantation of cryopreserved ovarian slices. The results demonstrate that despite drastic reduction in the pool of oocytes, cyclical ovarian function was observed in all ewes for nearly 2 yr. Despite the raised basal levels of FSH, the cyclical pattern of ovarian hormones consistent with ovulation was preserved. These results have encouraged us to offer cryostorage of ovarian tissue using similar techniques to girls and young women in whom ovarian function may be lost due to chemo- or radiotherapy.
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Acknowledgments |
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Footnotes |
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2 Present address: Division of Agriculture and Horticulture,
University of Nottingham School of Biological Sciences, Sutton
Bonington Campus, Loughborough, Leicestershire, United Kingdom LE12
5RD.
3 Present address: Division of Obstetrics and Gynecology, University
of Leeds, D Floor, Clarendon Wing, Belmont Grove, Leeds, United Kingdom
LS2 9NS.
Received August 3, 1998.
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References |
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-ßA dimer) during the oestrous cycle, after
manipulation of ovarian activity and during pregnancy in the ewe. J
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M. A. Bedaiwy and T. Falcone Ovarian tissue banking for cancer patients: Reduction of post-transplantation ischaemic injury: intact ovary freezing and transplantation Hum. Reprod., June 1, 2004; 19(6): 1242 - 1244. [Abstract] [Full Text] [PDF]
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C.G. Almodin, V.C. Minguetti-Camara, H. Meister, J.O.H.R. Ferreira, R.L. Franco, A.A. Cavalcante, M.R.M. Radaelli, A.S. Bahls, A.F. Moron, and C.G.V. Murta Recovery of fertility after grafting of cryopreserved germinative tissue in female rabbits following radiotherapy Hum. Reprod., June 1, 2004; 19(6): 1287 - 1293. [Abstract] [Full Text] [PDF]
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M. Sonmezer and K. Oktay Fertility preservation in female patients Hum. Reprod. Update, May 1, 2004; 10(3): 251 - 266. [Abstract] [Full Text] [PDF]
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C.P. Schroder, H. Timmer-Bosscha, J.G. Wijchman, L.F.M.H. de Leij, H. Hollema, M.J. Heineman, and E.G.E. de Vries An in vitro model for purging of tumour cells from ovarian tissue Hum. Reprod., May 1, 2004; 19(5): 1069 - 1075. [Abstract] [Full Text] [PDF]
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B. K. Campbell, E. E. Telfer, R. Webb, and D. T. Baird Evidence of a Role for Follicle-Stimulating Hormone in Controlling the Rate of Preantral Follicle Development in Sheep Endocrinology, April 1, 2004; 145(4): 1870 - 1879. [Abstract] [Full Text] [PDF]
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K. Oktay and M. Sonmezer Ovarian tissue banking for cancer patients: Fertility preservation, not just ovarian cryopreservation Hum. Reprod., March 1, 2004; 19(3): 477 - 480. [Abstract] [Full Text] [PDF]
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K.L.T. Schmidt, E. Ernst, A.G. Byskov, A. Nyboe Andersen, and C. Yding Andersen Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation Hum. Reprod., December 1, 2003; 18(12): 2654 - 2659. [Abstract] [Full Text] [PDF]
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S.S. Kim Ovarian tissue banking for cancer patients: To do or not to do? Hum. Reprod., September 1, 2003; 18(9): 1759 - 1761. [Abstract] [Full Text] [PDF]
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D. A. Gook, D.H. Edgar, J. Borg, J. Archer, P.J. Lutjen, and J.C. McBain Oocyte maturation, follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting Hum. Reprod., September 1, 2003; 18(9): 1772 - 1781. [Abstract] [Full Text] [PDF]
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H. Yin, X. Wang, S.S. Kim, H. Chen, S. L. Tan, and R. G. Gosden Transplantation of intact rat gonads using vascular anastomosis: effects of cryopreservation, ischaemia and genotype Hum. Reprod., June 1, 2003; 18(6): 1165 - 1172. [Abstract] [Full Text] [PDF]
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 T. Israely, H. Dafni, D. Granot, N. Nevo, A. Tsafriri, and M. Neeman Vascular Remodeling and Angiogenesis in Ectopic Ovarian Transplants: A Crucial Role of Pericytes and Vascular Smooth Muscle Cells in Maintenance of Ovarian Grafts Biol Reprod, June 1, 2003; 68(6): 2055 - 2064. [Abstract] [Full Text] [PDF]
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 K. Oktay, H. C. M. T. Prinsen, W. R. de Vries, H. P. F. Koppeschaar, and M. G. M. de Sain-van der Velden Evidence for Limiting Ovarian Tissue Harvesting for the Purpose of Transplantation to Women Younger than 40 Years of Age J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1907 - 1908. [Full Text] [PDF]
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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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R.A. Cushman, C.M. Wahl, and J.E. Fortune Bovine ovarian cortical pieces grafted to chick embryonic membranes: A model for studies on the activation of primordial follicles Hum. Reprod., January 1, 2002; 17(1): 48 - 54. [Abstract] [Full Text] [PDF]
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S. S. Kim, J. Radford, M. Harris, J. Varley, A. J. Rutherford, B. Lieberman, S. Shalet, and R. Gosden Ovarian tissue harvested from lymphoma patients to preserve fertility may be safe for autotransplantation Hum. Reprod., October 1, 2001; 16(10): 2056 - 2060. [Abstract] [Full Text] [PDF]
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J. Callejo, C. Salvador, A. Miralles, S. Vilaseca, J. M. Lailla, and J. Balasch Long-Term Ovarian Function Evaluation after Autografting by Implantation with Fresh and Frozen-Thawed Human Ovarian Tissue J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4489 - 4494. [Abstract] [Full Text] [PDF]
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 R Grundy, R G Gosden, M Hewitt, V Larcher, A Leiper, H A Spoudeas, D Walker, and W H B Wallace Personal practice: Fertility preservation for children treated for cancer (1): scientific advances and research dilemmas Arch. Dis. Child., April 1, 2001; 84(4): 355 - 359. [Full Text]
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C.J. Candy, M.J. Wood, and D.G. Whittingham Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries Hum. Reprod., June 1, 2000; 15(6): 1300 - 1304. [Abstract] [Full Text] [PDF]
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 H A Spoudeas, W H B Wallace, and D Walker Is germ cell harvest and storage justified in minors treated for cancer? BMJ, January 29, 2000; 320(7230): 316 - 316. [Full Text]
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 D. C. Linch, R. G. Gosden, T. Tulandi, S.-L. Tan, and S. L. Hancock Hodgkin's Lymphoma: Choice of Therapy and Late Complications Hematology, January 1, 2000; 2000(1): 205 - 221. [Abstract] [Full Text] [PDF]
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Y. Aubard, P. Piver, Y. Cognie, V. Fermeaux, N. Poulin, and M.A. Driancourt Orthotopic and heterotopic autografts of frozen–thawed ovarian cortex in sheep Hum. Reprod., August 1, 1999; 14(8): 2149 - 2154. [Abstract] [Full Text] [PDF]
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) ovariectomy and
autotransplantation of four frozen/thawed ovarian cortical strips.
). The wall of the follicular cyst in the transplanted
ovary is devoid of granulosa cells and ia lined by a thick luteinized
theca externa. Hemotoxylin and eosin staining was used. Magnification,
x20.