Orthotopic and heterotopic autografts of frozen–thawed ovarian cortex in sheep

Y. Aubard1,4, P. Piver1, Y. Cognié2, V. Fermeaux3, N. Poulin2 and M.A. Driancourt2

1 Department of Obstetrics and Gynaecology, Service de Gynécologie–Obstétrique, CHU Dupuytren, Avenue Martin Luther-King, 87000 Limoges, 2 INRA, Nouzilly and 3 Department of Pathology, CHU Limoges, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Freezing ovarian cortex is a new option to preserve the fertility of young patients undergoing cancer treatment or in women facing premature menopause. However, the best way to use this banked tissue remains unclear. The function of heterotopic and orthotopic autografts of frozen–thawed ovarian cortex of sheep was compared in the present study. Fresh and frozen–thawed fragments of ovarian cortex were autografted on the uterine horn of six ewes (orthotopic grafts) and under the skin of the belly in nine ewes (heterotopic grafts). In both orthotopic and heterotopic grafts, the resumption of follicular growth and ovulation was monitored. In orthotopically grafted ewes, fertility was recorded. Oocytes from both types of grafts were collected, matured and fertilized in vitro. In both fresh and frozen–thawed grafts follicular growth resumed normally; preantral and antral follicles were first detectable 4 and 10 weeks respectively following grafting but only 5% of the primordial follicles appeared to have survived. This confirms that grafting procedures are more deleterious for follicle survival than cryopreservation. Although ovulation resumed in most ewes, none of the ewes grafted orthotopically became pregnant at a synchronized mating. Seven months following grafting, oocytes could be collected from heterotopic and orthotopic grafts, matured and some of them fertilized, but none developed to the blastocyst stage. Heterotopic grafting may be an alternative to orthotopic grafting to preserve fertility provided follicle survival in the grafts is markedly improved.

Key words: follicles/freezing/ovarian function/ovarian graft/sheep


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemotherapeutic drugs and radiotherapy for the treatment of leukaemias and lymphomas have detrimental effects on the population of ovarian follicles which often result in premature menopause (Marmor, 1995Go). Because more efficient treatments have increased the chances of full remission, maintaining the fertility of such patients may be an important goal (Gosden and Aubard, 1996Go; Donnez and Bassil, 1998Go; Newton, 1998Go). In addition, developing strategies to manage reproduction in women facing premature menopause would also be useful.

Over 30 years ago, Parrott demonstrated that frozen ovarian fragments could restore fertility of mice when re-engrafted (Parrott, 1960Go). These data were extended later to other species: rat (Aubard et al., 1998Go) and sheep (Gosden et al., 1994bGo). This latter model is more relevant to human physiology since sheep, like women, are monovular. However, the pioneering studies of Gosden demonstrated that sheep, following autografting of frozen–thawed ovarian cortex fragments, although fertile, displayed a clear reduction in fertility (Gosden et al., 1994bGo). Only 2/6 ewes maintained with fertile rams for 2 months conceived. This is much lower than expected, assuming a fertility per cycle of 60% in untreated ewes, and three cycles being used by the rams. Hence the first aim of this study was to explore possible reasons for the impaired fertility of orthotopically autografted ewes. More specifically, the questions addressed were: (i) is the timing of resumption of ovulation variable between ewes? (ii) is cyclicity regular in autografted ewes? (iii) what is the ability of the oocytes of large follicles to resume meiosis or fertilize? These features were studied in six ewes autografted with ovarian fragments placed at an orthotopic site (uterus).

An alternative to orthotopic grafting is the use of other sites (heterotopic grafting). This may be valuable if these sites provide a better survival rate of the ovarian follicles [e.g. the kidney capsule of mice (Carroll et al., 1990Go)] and if the implantation sites provide an easy access to oocytes used later for in-vitro fertilization (IVF). Hence the second aim of this study was to explore the potential of a subcutaneous (s.c.) site to replace the grafts. Again, resumption of follicular growth and ovulation, number and duration of cycles and quality of the oocytes collected were assessed in nine ewes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental design
Orthotopic grafting
Six mature (2–4 years old) Ile de France ewes were separated into fresh (n = 2) or frozen–thawed groups (n = 4). In all ewes, one ovary was removed by laparotomy under general anaesthesia induced by barbiturates (i.v. injection of 0.5 g sodium thiopentone, Specia®; Rhone Poulenc Rorer, Montrouge, France) and maintained by halothane in air. The ovarian cortex (mean thickness = 1 mm) was peeled away from the medulla. For the ewes of the frozen–thawed group, four fragments (each of about 1 cm2) were placed in 1.5 ml plastic cryotubes containing Liebovitz medium (Sigma, St Louis, France) and 10% dimethyl sulphoxide (DMSO, Sigma) added. Following a 1h equilibration phase on ice at 5°C, the tubes were frozen as follows. From 5°C to –9°C, the cooling rate was 2°C/min. At –9°C, a manual seeding was done. From –9°C to –40°C, the cooling rate was 0.3°C/min, and from –40°C to –140°C the cooling was at 10°C/min. The tubes were then stored in liquid nitrogen. This protocol is similar to that used by Gosden in his earlier sheep work (Gosden et al., 1994bGo). For the two ewes which received fresh grafts, ovarian cortex fragments were prepared as described above, and immediately grafted using four stitches of Prolene 5/0® (Ethicon, Neuilly/Seine, France) on the ipsilateral uterine horn. The contralateral ovary was ablated. One week later, all frozen ovarian fragments were thawed by immersion in a water bath maintained at 25°C, rinsed twice in pure Liebovitz medium and replaced in the four ewes of the frozen–thawed group using the same procedure as for the ewes of the fresh group. The contralateral ovary of these four ewes was ablated at this time.

Heterotopic grafting
Nine mature (2–4 years old) Ile de France ewes were spayed under general anaesthesia (see above). Ovarian cortex fragments were prepared (see above) to generate three fragments per ovary. The three ewes of the fresh group immediately received all fragments which were sutured (two stitches of prolene 5/0) s.c. under the skin of the belly, on each side of the linea alba about 15 cm apart. For the six ewes receiving frozen–thawed fragments, freezing and thawing was done as described for the ewes grafted orthotopically and grafting was done as described for the fresh heterotopically grafted ewes.

The design of the experiment is presented in Figure 1Go. Three of the fragments were devoted to the study of resumption of follicular growth; this was achieved as follows. Fragments of ovarian cortex from ewes in subgroup 1 (one fresh, two frozen–thawed ewes) were recovered at 1, 4 and 7 weeks following grafting (one fragment each time). Ewes of subgroup 2 (another ewe with fresh and two ewes with frozen–thawed autografts) provided fragments at 4, 7 and 10 weeks following grafting. Finally, ewes of subgroup 3 (one and two ewes respectively with fresh and frozen–thawed autografts) provided fragments at 7, 10 and 13 weeks. The three fragments remaining in each ewe were devoted to the study of cyclic ovarian activity and oocyte quality (see below).



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Figure 1. Diagrammatic presentation of the design of the experiment assessing the function of heterotopic grafts (f = ewes receiving fresh grafts; F/T = ewes receiving frozen/thawed grafts; W = weeks).

 
Histological analysis of the fragments
Immediately following recovery, all grafts were fixed by immersion in Bouin's Liquid (C.H.U. Limoges France, and prepared as paraffin-embedded blocks. All blocks were sectioned at 10 µm and one section in five was mounted and stained with haematoxylin and eosin.

Using the nucleus as a marker (to avoid counting follicles twice), all follicles were counted, measured and ranked in size categories. They were checked for atresia by searching for pycnotic bodies within the granulosa layer or along the border of the antrum (Driancourt et al., 1985Go). Atretic follicles were those exhibiting more than five pycnotic nuclei in the section studied. This is a widely accepted criterion to detect atresia in follicles >500 µm in diameter. In contrast, in smaller follicles pycnotic bodies are very rare (Driancourt et al., 1985Go). Owing to the sampling ratio used, numbers of primordial (<40 µm in diameter) and primary (40–50 µm in diameter) follicles were adjusted using the Abercrombie correction (Abercrombie, 1946Go).

Monitoring of the resumption of cyclic ovarian activity
Resumption of ovulation was monitored by twice weekly semi-quantitative assays of progesterone in blood (Terqui and Thimoniev, 1974). As in an earlier study (Gosden et al., 1994bGo), 3 months were required for ovulation to occur; bleeds were started 3 months following grafting and continued on a twice weekly basis until the end of the experiment. Samples were immediately assayed and samples containing high (>1 ng/ml) versus low (<0.5 ng/ml) concentrations identified relative to reference samples. Normal cycling sheep show alternating periods of high and low concentrations lasting 14 days (4–5 samples) and 3–4 days (one or two samples).

Monitoring of the fertility and of the quality of the oocytes
Fertility was assessed in the six ewes with orthotopic grafts. Six months following grafting, the cycles of these ewes were synchronized by the vaginal insertion of progestagen-impregnated sponges (Chronogest®; Intervet, France) for 14 days. At sponge removal, 600 IU of pregnant mare's serum gonadotrophin (PMSG) were injected. All ewes were checked for oestrus twice daily and mated with a fertile ram. Pregnancy was checked by progesterone assay (Terqui et al., 1974Go), performed 16 days following mating.

In a final experiment performed 7 months after grafting, the abilities of the oocytes of the ewes in the orthotopic and heterotopic groups to resume meiosis, fertilize and develop were assessed. All ewes had their cycles synchronized by the insertion (for 14 days) of progestagen-impregnated sponges. At sponge removal, 1250 IU of PMSG were injected and all ewes killed 24 h later. All follicles >2 mm were dissected from the ovaries, their oocytes obtained and submitted to in-vitro maturation and in-vitro fertilization as previously described (Cognie et al., 1998Go). Oocytes were matured in vitro for 24 h in 0.5 ml TCM 199 (M 199®; Sigma, St Quentin Fallavier, France) supplemented with 10% sheep follicular fluid and 100 ng of follicle-stimulating hormone (FSH)/ml. In-vitro matured oocytes were fertilized in vitro using frozen–thawed semen (Cognie et al., 1991Go). Briefly, after separation of the motile spermatozoa using a Percoll buffered gradient and centrifugation at 500 g for 20 min, 106 spermatozoa/ml were incubated with oocytes in Brackett's defined medium buffered with HEPES and containing 20% heat-inactivated sheep serum at 38.5°C for 17 h. Embryo culture was conducted in synthetic oviductal fluid (Cognie et al., 1998Go).

Statistical analysis
For presentation and analysis of the data, follicles were classified as primordial (<40 µm in diameter), primary (40–50 µm diameter), preantral (50–250 µm), antral (over 250 µm in diameter). Follicle numbers and size of the largest follicle were analysed by one way ANOVA. Results are presented as means ± SEM.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resumption of follicular growth (heterotopic grafts)
All grafts were recovered and all of them contained at least some primordial follicles. There was no significant difference in follicle numbers between fresh and frozen grafts. For example, at 7 weeks following grafting, the three fresh grafts contained 356 ± 145, 40 ± 24 and 2.7 ± 0.9 primordial, primary and preantral follicles while the six frozen–thawed grafts contained 265 ± 201, 30 ± 15, 1.3 ± 1.5 primordial, primary and preantral follicles respectively. The size of the largest follicle was not significantly different between the fresh (73.3 ± 5.9 µm) and frozen–thawed (75.0 ± 12.7) groups. Hence fresh and frozen grafts were combined for further analysis. No atresia was detectable in either group (fresh or frozen). No data on the fresh versus frozen–thawed groups are presented for other stages because meaningful estimates for the fresh group could not be estimated (n = 1 at 1 and 13 weeks, n = 2 at 4 and 10 weeks).

The number of follicles in specific size groups at specific times following grafting (Table IGo) showed that follicular growth resumed between 1 and 4 weeks following grafting. Only primordial follicles could be detected 1 week following grafting while primary follicles were first detected at 4 weeks, preantral follicles at 7 weeks (in 5/9 ewes), antral follicles at 10 weeks (in 4/6 ewes) and large ovulatory/ovulated (4–8 mm) follicles at 13 weeks (in 2/3 ewes). ANOVA did not reveal any effect of the time following grafting on the number of primordial follicles (Table IGo). In contrast, the number of primary (P = 0.04), preantral (P = 0.07) and antral (P < 0.01) follicles increased as the time following grafting increased. The numbers of primary follicles were maximal at 7 weeks and stayed high thereafter. The numbers of preantral follicles were maximal at 10 and 13 weeks as was the number of antral follicles. Interestingly, atresia was never observed within the antral follicles >0.5 mm in diameter, as all of them were devoid of pycnotic bodies.


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Table I. Numbers of primordial, primary, preantral and antral follicles in heterotopic grafts at specific times following grafting (means ± SEM)
 
These changes in follicle numbers produced significant changes in the size of the largest follicle (P < 0.01). At 1, 4, 7, 10 and 13 weeks, the largest follicle diameter was 38 ± 1, 66 ± 21, 74 ± 11, 1008 ± 799 and 4213 ± 2041 µm respectively. It is concluded that, in this heterotopic model, follicular growth from the primordial stage can be achieved in 13 weeks.

Resumption of ovulation (orthotopic and heterotopic grafts)
Amongst the ewes with orthotopic grafts, 4/6 resumed ovulation (1/2 fresh, 3/4 frozen). The mean time lag between grafting and first ovulation was 131.7 ± 12.0 days (range 104–156). Three ewes only had one period of high progesterone, averaging 4.3 ± 1.2 days. One ewe (from the frozen group) had three periods of high progesterone (of 2, 9 and 10 days). In this ewe, extended periods of low progesterone (6–10 days) occurred between the periods of high progesterone.

Amongst the ewes with heterotopic grafts 5/9 resumed ovulation (1/3 fresh, 4/6 frozen). 121 ± 8.1 (range 101–139) days elapsed between grafting and first ovulation. Most of the periods of high progesterone were short (2–6 days), and only one was of normal duration (14 days). Only two ewes (both in the frozen group) displayed repeated periods of high progesterone.

In-vivo monitoring of fertility and of oocyte quality (orthotopic grafts)
When injected with PMSG 6 months following grafting, all ewes with orthotopic grafts came to oestrus and were mated. This was despite the fact that only two of them had shown evidence of high progesterone in the weeks preceding synchronization. However, none of them was pregnant when progesterone concentrations were measured 16 days following mating. When the ewes were slaughtered (see below), examination of the ovaries demonstrated that adhesions had developed in three ewes and that in all the ewes the ovaries were covered by a thin layer of tissue.

In-vitro monitoring of oocyte quality (orthotopic and heterotopic grafts)
Stimulation of the ovaries of the ewes with orthotopic grafts with a high amount of PMSG (1250 IU) generated 1.5 ± 0.2 and 2.0 ± 0.4 pre-ovulatory follicles (4–8 mm) in the ewes autografted with fresh and frozen grafts respectively. Smaller follicles (3–4 mm in diameter) were also present (0.5 per ewe) in the fresh group. Four oocytes were recovered in each of the fresh and frozen groups respectively; of these, one and two respectively fertilized but arrested at the 4-cell stage.

When the ovaries of the ewes bearing heterotopic grafts were stimulated by PMSG, 2/3 ewes in the fresh group and 5/6 ewes in the frozen group developed medium and large follicles; 1.0 ± 0 and 1.7 ± 0.9 pre-ovulatory follicles were dissected in the ewes autografted with fresh and frozen grafts respectively. Smaller follicles were also present in both groups (fresh: 2.0 ± 1.2, frozen 1.0 ± 0.4). A total of 5 and 10 oocytes were collected in the fresh and frozen groups respectively. Of these 2/5 and 8/10 had the ability to resume meiosis up to the metaphase II stage and 1/5 and 2/10 fertilized and were blocked at the 4-cell stage.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present results extend the data reported previously (Gosden et al., 1994aGo) (i) by showing that heterotopic grafting provides identical results as orthotopic grafting in terms of resumption of follicular growth and (ii) by analysing the causes of the reduced fertility observed following orthotopic and heterotopic grafting.

Heterotopic grafting is an interesting alternative to orthotopic grafting. If proved to be adequate, the site selected (skin of the belly) would only require local anaesthesia to handle the grafts and possibly also achieve oocyte collection for IVF. Assessing this alternative to orthotopic grafting (which yields only very limited fertility) is particularly interesting since the IVF procedure for subcutaneously placed grafts should bypass the problems related to ovulation and oocyte collection by fimbria following orthotopic grafting. A number of interesting conclusions were obtained in this model. Firstly, the timing of resumption of follicular growth was established. Preantral follicles of about 80 µm in diameter required 7 weeks to develop from primordial follicles, whereas antral follicles (mostly 250–350 µm in diameter) were present 10 weeks following grafting, and ovulatory follicles present 13 weeks following grafting. Hence, all follicular growth proceeded in about 3 months, a time much shorter than the time calculated for intact adult ewes (Cahill and Mauleon, 1980Go) but close to what has been reported to be required for orthotopic ovarian grafts (Gosden et al., 1994bGo) and fetal sheep ovaries (McNatty et al., 1995Go). Since, as in sheep, follicular growth requires several months in humans (Gougeon, 1996Go), it is likely that the time lag to obtain ovulatory follicles from grafted frozen–thawed human ovarian fragments would be also reduced. Secondly, the follicular population and atresia were checked in the grafts at specific times following grafting. This demonstrated that follicle numbers were markedly reduced. This is in good agreement with earlier studies (Gunasena et al., 1997aGo; Baird et al., 1999Go) reporting a reduction of litter size or an earlier exhaustion of the reserve of primordial follicles. Between 120 and 500 primordial follicles could be counted in a third of an ovary. Although the initial population present in the ovary could not be established owing to the design used, Ile de France ewes display on average 56000 primordial follicles per ovary (Cahill et al., 1979Go). This would indicate that no more than 5% of the follicles survived the freezing/grafting procedures. Such a rate of survival is markedly lower than the 30–50% reported following grafting to the kidney capsule of monkey ovarian cortex fragment (Candy et al., 1995Go, 1997Go) or sheep ovarian fragments (Baird et al., 1999Go). As a consequence, the numbers of preantral and antral follicles were 20–30 fold lower than those found in normal ovaries (Cahill et al., 1979Go). Interestingly, follicular growth was very efficient since hardly any atretic follicles could be detected in heterotopic grafts while over 50% of the follicles >0.5 mm in diameter are atretic in normal ovaries (Cahill et al., 1979Go; Driancourt et al., 1985Go). The reduced follicular population also resulted in a low response to exogenous gonadotrophins to induce ovulation. The dose used in the present study (1250 IU) only induced growth of 1–3 pre-ovulatory follicles while it would be 5–10 fold more efficient in intact ewes (Bindon et al., 1986Go). Thirdly, using both procedures (orthotopic or heterotopic grafting), similar results were obtained using fresh or frozen–thawed grafts. This strongly suggests that the key factor responsible for follicle survival is the post-grafting ischaemia and not damage of germ cells by freezing and thawing. Such a claim is in agreement with earlier reports (Gosden et al., 1994bGo; Candy et al., 1997Go; Gunasena et al., 1997bGo).

Part of the present study was devoted to the analysis of the limited fertility of the ewes with grafted ovaries. Specific questions which were addressed were: (i) are the oocytes present in the follicles growing from the grafts of a high enough quality to resume meiosis, fertilize and develop to the blastocyst stage following in-vitro maturation (IVM), fertilization (IVF) and culture (IVC)? For this purpose, oocytes were collected from superovulated ewes with orthotopic and heteretopic grafts and submitted to IVM/IVF/IVC. A proportion of these could resume meiosis and fertilize but none of them developed to the morula/blastocyst stage. These results compared to the 25–35% blastocyst stage oocytes routinely obtained in the IVF laboratory for sheep (Cognie et al., 1991Go) may indicate that the oocytes may be somewhat deficient in their cytoplasmic maturation. Whether this reflects an inadequate follicular maturation or a low quality of the oocytes which survived freezing/thawing/grafting requires further investigation; (ii) are the corpora lutea developing following ovulation fully functional? This would be identified in ewes with orthotopic ovaries by alternating periods of 14 days of high and 3 days of low progesterone and in ewes with heterotopic ovaries by persistent corpora lutea (ovaries are away from the luteolytic signal produced by the uterus). These patterns were never identified. The majority of the ewes only displayed one period of high progesterone which was often shorter than normal (2–6 days). Such short luteal phases are not uncommon in intact sheep at specific physiological periods and are believed to be caused by inadequate follicular growth and/or inadequate uterine function (Garverick and Smith, 1986Go). In addition, when several episodes of high progesterone concentration were recorded, their detection was at irregular intervals, a finding in agreement with the irregular pattern of vaginal cornification noted in severe combined immune deficient mice xenografted with sheep ovarian cortex (Gosden et al., 1994aGo). Measurement of oestradiol concentrations in plasma or in follicular fluid would be useful to confirm whether follicular maturation is altered in these ovaries. It was also quite puzzling to see that ewes with heterotopic ovaries had spontaneous luteolysis because they were expected to have maintained corpora lutea. For example, ewes with ovaries autografted to the neck (Goding et al., 1967Go) do not ovulate owing to the persistence of corpora lutea. In addition, the few ewes which managed to show repeated periods of high progesterone had extended follicular phases, a situation reminiscent of the premenopausal period in humans.

In conclusion, the present study demonstrated that heterotopic grafting followed by in-vitro fertilization of the oocyte collected may be an alternative to orthotopic grafting to offer a chance of having progeny to young cancer patients treated by chemotherapy and/or radiotherapy (Donnez and Bassil, 1998Go; Newton, 1998Go). A prerequisite before offering this option is to improve follicle survival in grafts and/or restrict its access to young patients. Recent results (Newton et al., 1998Go) suggest that reducing the duration of exposure to DMSO and achieving this at 4°C may minimize follicle loss. Reducing size of the grafts (Krohn, 1977Go) or using pretreatment of the grafts with compounds such as vascular endothelial growth factor or fibroblast growth factor which stimulate vascularization (Redmer and Reynolds, 1996Go) should be considered. In addition, administration of antioxidants following grafting may also be helpful (Nugent et al., 1998Go). Knowledge of the relationship between the number of primordial follicles in a graft and its duration of function will also be needed to establish how to manage the different grafts of each patient. Another most important prerequisite is also to show that grafting does not transmit cancer to the recipient. Since this may happen for specific cancer types (Shaw et al., 1996Go), the grafting strategy should not be offered to all patients.


    Acknowledgments
 
The authors are grateful to the staff of the surgery in Nouzilly for their efficient help and to the staff of the sheep shed for expert care of the animals.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 23, 1998; accepted on April 9, 1999.