Pilot study of isolated early human follicles cultured in collagen gels for 24 hours

R. Abir1,3, P. Roizman2, B. Fisch1, S. Nitke1, E. Okon2, R. Orvieto1 and Z. Ben Rafael1

1 Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, and 2 Department of Pathology, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, Israel


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human ovarian cortex contains mainly primordial and primary follicles. The ability to mature these follicles in vitro could be of great importance for infertility treatments. Fresh and frozen–thawed ovarian tissue was incubated with collagenase and DNase. Follicles with one layer or an incomplete second layer of granulosa cells were then dissected. The follicles were embedded in collagen gels and cultured with Earle's balanced salt solution, 10% fetal calf serum and 0.5 IU/ml follicle stimulating hormone. Increases in the number of granulosa cell layers and in oocyte size were observed in 40 and 38.7% of the follicles from fresh and frozen–thawed tissue respectively, during a 24 h culture period. All the growing follicles were surrounded by cellular outgrowths. Attempts to culture the follicles longer resulted in deterioration of the follicles and oocyte release. Since our study was purely morphological, further growth parameters, e.g. DNA synthesis, should be examined in the future.

Key words: cryopreservation/follicles/in vitro/primary/primordial


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human ovarian cortex contains mostly unilaminar primordial follicles (Hovatta et al., 1996Go). The ability to mature these follicles in vitro may help many infertile women. However, attempts to date have yielded only limited success because the signals that induce transformation of unilaminar follicles into preantral (multilaminar) follicles remain unknown.

Culturing isolated unilaminar follicles enables their direct monitoring during the culture period, which is of special importance considering the poorly populated human ovary (Hovatta et al., 1996Go). Unilaminar follicles isolated from mouse (Torrance et al., 1989Go), bovine (Hulshof et al., 1995Go) and rat (Cain et al., 1995Go) have been grown in culture. Granulosa cells (GCs) from primary porcine follicles proliferated (Morbeck et al., 1993Go), and morphologically normal human primordial follicles have survived (Oktay et al., 1997Go).

Culturing of mouse (Torrance et al., 1989Go), bovine (Hulshof et al., 1995Go) and porcine (Hirao et al., 1994Go) follicles within collagen gels has been shown to provide maximal support to the follicle and to maintain its three-dimensional structure (Torrance et al., 1989Go). We describe a combined enzymic and mechanical isolation method for obtaining unilaminar human follicles and their subsequent culture in collagen gels.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian material
Ovarian material was obtained from 10 women aged 29 ± 11 (mean ± SD) years undergoing gynaecological operations at Rabin Medical Center, Israel.

Cryopreservation and thawing of ovarian tissue
Cryopreservation and thawing of tissue specimens from seven patients was carried out with a combination of 1,2-propanediol (Sigma, St Louis, MO, USA) and sucrose (Sigma), as described in detail elsewhere (Hovatta et al., 1996Go). In the present study the freezing and thawing medium contained synthetic serum substitute (Irvine Scientific, Santa Ana, CA, USA). After thawing, the specimens were transferred to Earle's balanced salt solution (EBSS) (Biological Industries, Beit Haemek, Israel) with 0.4% human serum albumin (HSA) (Irvine Scientific).

Isolation of follicles
Fresh or frozen–thawed ovarian tissue was cut into slices 0.5–1 mm thick and incubated at 37°C for 2 h with collagenase IX (5944 IU/ml, Sigma) and pancreatic deoxyribonuclease IV (180 IU/ml, Sigma) dissolved in EBSS (Biological Industries) and with 0.4% HSA (Irvine Scientific). The tissue was then placed in EBSS (Biological Industries) with 0.4% HSA (Irvine Scientific). EBSS with HSA was used for all isolation and rinsing processes unless stated otherwise. The pH of the solution was maintained at 7.0–7.5, despite the absence of HEPES during dissection. Follicles were dissected under an inverted microscope with 21 gauge needles attached to 1 ml syringes and further aspirated through a fine bore pipette. The follicles were rinsed three times, the last rinse containing the medium used for culture (see below).

Culturing methods
Collagen gel solutions were prepared according to the manufacturer's instructions (Becton Dickinson, NJ, USA). Follicles were placed at the centre of each well (4-well culture plates) (Nunclon, Roskilde, Denmark) and overlaid with 200 µl collagen gel solution at 37°C. The gel setting took 30 min, after which 1 ml of culture medium was poured into every well, and the plates were returned to the incubator for a further 24 h. The culture medium was EBSS (Biological Industries) containing 0.47 mM (Hovatta et al., 1997Go) sodium pyruvate (Sigma), 2 mM/ml L-glutamine (Biological Industries), 10% heat inactivated fetal calf serum (Biological Industries), 0.5 IU/ml (Hovatta et al., 1997Go) highly purified human follicle stimulating hormone (Metrodin HP, a generous gift from Teva Pharmaceutical Industries, Petah Tikva, Israel) and antibiotics (Biological Industries).

Two types of follicles were cultured: unilaminar follicles with one layer of GCs surrounding the oocyte; and follicles with a partially developed second layer of GCs. The follicles and their oocytes were measured with a calibrated eyepiece micrometer at the beginning of the culture (0 h) and after 24 h. The number of GC layers was counted before and after culture. The index for GC layer number was assigned a range of 1–3; with 1, 2, 3 indicating full layers, 0.5 a very partial layer, and 0.7 an almost full layer. In this manner, we were able to follow changes in GC layers of the same follicle. Preliminary experiments showed that secondary follicles having two or more fully developed GC layers did not survive in culture.

Histological preparation
For light microscopy, collagen gels were fixed in Bouin's solution after 1 h (Torrance et al., 1989Go) and 24 h of culture and prepared for paraffin embedding and sectioning. Repeated attempts to prepare 0 h gels for light microscopy resulted in insufficient staining.

For transmission electron microscopy (TEM), some of the 24 h gels were treated with 2972 IU/ml collagenase IX (Sigma) for 30 min. The follicles were recovered, fixed in 3% glutaraldehyde (Sigma) and prepared for TEM. Ultrathin sections were examined with a JEOL (JEM 1010) electron microscope.

Statistical analysis
The data were analysed by analysis of variance (ANOVA) and {chi}2 test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An average of 7.4 follicles per subject from fresh tissue (10 subjects) and 8.9 follicles per subject from frozen–thawed tissue (seven subjects) were isolated and cultured. In all, 30 out of 74 (40%) follicles from fresh tissue and 24 out of 62 (38.7%) follicles from frozen–thawed tissue grew in culture.

Table IGo presents the sizes of the fully isolated follicles and their oocytes and the number of GC layers from fresh and frozen–thawed tissue after 0 and 24 h of culture. The cultured follicles were divided into growing and non-growing groups. Non-growing follicles and their oocytes could not be measured after 24 h because of rapid structural deterioration and oocyte release and degeneration. The largest follicles reached a diameter of 90–100 µm after culture, some having doubled in size. Growing follicles and their oocytes were initially larger than the non-growing ones and their oocytes (P < 0.0001) and had more GC layers (P < 0.009). There was an increase in size of the growing follicles and their oocytes and in the number of GC layers (P < 0.0001) between 0 and 24 h of culture. The morphology of the follicles after isolation and culture is shown in Figures 1–3GoGoGo. The cellular outgrowths seen around growing follicles are shown in Figure 1Go. Culture periods of >24 h resulted in disruption of the follicular structure and oocyte release in previously growing follicles.


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Table I. Sizes of follicles and oocytes and numbers of granulosa cell (GC) layers from fresh and frozen–thawed tissue after 0 and 24 h of culture. Values shown are mean ± SD
 


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Figure 1. Micrograph of a multilaminar follicle after 24 h of culture. Note the multilaminar granulosa cell (GC) layers surrounding the oocyte and the cellular outgrowths (arrows). At 0 h, this follicle was unilaminar and was 45 µm in diameter; after 24 h, the follicle was multilaminar and 90 µm in diameter. Scale bar = 2.9 µm.

 


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Figure 2. Section of a collagen gel containing two follicles that grew from unilaminar to multilaminar stages. One follicle is seen in mid-section; at 0 h, it was 45 µm in diameter and after 24 h, it was 80 µm in diameter. Scale bar = 7.0 µm.

 


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Figure 3. Electron micrograph of an oocyte from a follicle digested after 24 h of culture. The image shows part of a normal oocyte, the nuclear membrane (N), and the nucleolus (NU). Note that the nuclear membrane is surrounded by various cytoplasmic organelles including mitochondria. Follicle diameter was 45 µm at 0 h and 70 µm after 24 h. The follicle had one granulosa cell (GC) layer at 0 h and after 24 h it had a partial third GC layer. Scale bar = 0.9 µm.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first report to show an increase in GC layers and oocyte diameter of small human follicles isolated and cultured in collagen gels. There was no difference in the proportions of growing follicles from fresh (40%) or frozen–thawed (38.7%) tissue. These results are in accordance with other studies that used human tissue (Zhang et al., 1995Go; Hovatta et al., 1997Go; Oktay et al., 1997Go) and showed no difference between follicles from fresh or frozen–thawed tissue.

It is, however, unclear how the follicles and the oocytes could increase in size so rapidly in only 24 h. One possibility is that removal of the stroma layer abolishes growth-inhibiting signals. Alternatively, the increase in size might have been due to fluid absorption. This is, however, very unlikely considering the concomitant increase in the number of GC layers, and in oocyte size, which would have shrunk drastically during histological preparation had there been fluid absorption. Nevertheless, further growth parameters, e.g. bromodeoxyuridine (BrdU) labelling (Hulshof et al., 1995Go), should be examined before this possibility can be ruled out altogether. We also cannot explain why the follicles did not grow beyond 24 h. If basement membrane damage caused by the enzymic treatment restricted growth, why were the follicles not affected during the first 24 h? Moreover, since the non-growing follicles were smaller than the growing ones, it is likely that their lack of growth was due to their size.

Three other studies have shown follicular culture in collagen gels (Torrance et al., 1989Go; Hirao et al., 1994Go; Hulshof et al., 1995Go). In these reports, growth was evaluated mainly by standard light microscopy methods, although growth of bovine follicles was also studied by BrdU labelling (Hulshof et al., 1995Go) and large pig preantral follicles were studied using TEM (Hirao et al., 1994Go). Mouse follicles cultured in collagen gels to preantral stages were shown to grow further under the kidney capsule of immunodeficient mice (Telfer et al., 1990Go), and porcine cumulus-enclosed oocytes aspirated from antral follicles grown in collagen gels were further matured in culture (Hirao et al., 1994Go). Despite the longer culture periods in these studies, fluid absorption was not indicated as a cause of size increase in the collagen gels, which supports our findings.

Unilaminar mouse (Torrance et al., 1989Go), bovine (Hulshof et al., 1995Go) and human follicles reached only preantral stages in collagen gels, though large preantral follicles from pigs grew to antral stages (Hirao et al., 1994Go). Furthermore, the current study showed that isolated human multilaminar follicles with two or more full GC layers did not grow in the collagen gels, while smaller follicles did. Thus, it seems that in-vitro follicular maturation systems are both species- and stage-specific. Evaluation of further growth parameters and improvements in the culture system and medium are required. Our study is, however, a first encouraging step towards successful in-vitro maturation of human oocytes from small follicles.


    Acknowledgments
 
R.A. established the methods described in this paper at Prof. R.M.L. Winston's laboratory at Hammersmith Hospital, London, UK with funding from The Birthgard Foundation. Prof. O.Hovatta (Karolinska Institute, Stockholm, Sweden) introduced R.A. to ovarian cryopreservation methods. The authors are grateful to Dr J.Sulkes and Ms G.Ganzach from Rabin Medical Center for statistical advice and English editing respectively. The study was partially supported by the Israel Cancer Association and the Leo Mintz Fund (Tel Aviv University).


    Notes
 
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, IVF Research Laboratory, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cain, L., Chatterjee, S. and Collins, T.J. (1995) In vitro folliculogenesis of rat preantral follicles. Endocrinology, 136, 3369–3377.[Abstract]

Hirao, Y., Nagai, T., Kubo, M. et al. (1994) In vitro growth of and maturation of pig oocytes. J. Reprod. Fertil., 100, 333–339.[Abstract]

Hovatta, O., Silye, R., Kraustz, T. et al. (1996) Cryopreservation of human ovarian tissue by using dimethylsulphoxide and propandiol–sucrose as cryoprotectants. Hum. Reprod., 11, 1268–1272.[Abstract]

Hovatta, O., Silye, R., Abir, R. et al. (1997) Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture. Hum. Reprod., 12, 1032–1036.[ISI][Medline]

Hulshof, S.C.J., Figueiredo, J.R., Beckers, J.F. et al. (1995) Effects of fetal bovine serum, FSH and 17ß-estradiol on the culture of bovine preantral follicles. Theriogenology, 44, 216–226.

Morbeck, D.E., Flowers, W.L. and Britt, JH. (1993) Response of granulosa cells isolated from primary and secondary follicles to FSH, 8-bromo-cAMP and epidermal growth factor in vitro. J. Reprod. Fertil., 99, 577–584.[Abstract]

Oktay, K., Nugent, D., Newton, H. et al. (1997) Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil. Steril., 67, 481–486.[ISI][Medline]

Telfer, E., Torrance, C. and Gosden, R.G. (1990) Morphological study of cultured preantral ovarian follicles of mice after transplantation under the kidney capsule. J. Reprod. Fertil., 89, 565–571.[Abstract]

Torrance, C., Telfer, E. and Gosden, R.G. (1989) Quantitative study of the development of isolated mouse pre-antral follicles in collagen gel culture. J. Reprod. Fertil., 87, 367–374.[Abstract]

Zhang, J., Liu, J., Xu, K.P. et al. (1995) Extracorporeal development and ultrarapid freezing of human fetal ova. J. Assist. Reprod. Genet., 12, 361–368.[ISI][Medline]

Submitted on May 8, 1998; accepted on January 5, 1999.