1 Department of Reproductive Science and Medicine, Division of Paediatrics, Obstetrics and Gynaecology and 2 Division of Pathology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK
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Abstract |
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Key words: follicles/oocyte/organ culture/ovary
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Introduction |
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In the mouse, live offspring have been born from oocytes matured from primordial follicles in vitro (Eppig and O'Brien, 1996). Isolated mouse follicles can be cultured, and there are data regarding the hormones and growth factors required in their growth (Cortvrindt et al., 1998
). Less is known about human ovarian follicles in vitro. Pre-antral human follicles have been isolated enzymatically, and they could be cultured for a few days (Roy and Treacy, 1993
). Mechanically isolated pre-antral (100400 µm) follicles have been cultured to early antral stages (Abir et al., 1997
). The ability to isolate successfully primordial and primary human follicles enzymatically has been studied in vitro (Oktay et al., 1997
), but it has not been possible to establish long-term cultures of these isolated human follicles (Abir et al., 1997
). We have been able to culture human primordial and primary follicles within slices of ovarian tissue, in which growth was initiated and then developed for 3 weeks up to secondary and occasionally to early antral follicles (Hovatta et al., 1997
).
In this study we have compared the growth and development of enzymatically and mechanically partially isolated human primordial and primary follicles with that of follicles within tissue slices, in organ culture.
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Materials and methods |
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The specimens were placed in HEPES-buffered culture medium (MEM, Gibco BRL, Life Technologies, Paisley, UK) and transported to the laboratory. They were sliced (0.31 mm in thickness) and placed in organ culture as described earlier (Hovatta et al., 1997). The tissue was cultured on 24-well plates (Nunclon, Roskilde, Denmark) in Millicell CM inserts (12 mm diameter, 0.4 µm pore size; Millipore, Bedford, MA, USA). The inserts were pre-coated with extracellular matrix (Matrigel; Becton Dickinson, MA, USA), 100 µl per insert, at a dilution of 1:3 with serum-free culture medium. The same batch of Matrigel was used in these experiments. The pieces of tissue were placed in the Matrigel using a pipette. Culture medium 500 µl had been pipetted to the well outside the insert, and three drops into the insert. Culture medium 150 µl was changed every second day, and three drops of medium were added to the inserts. The tissue was cultured in a humidified incubator at 37°C in 5% CO2 in air.
The culture medium was Earle's balanced salt solution (Gibco) supplemented (5%) with inactivated human serum, which was obtained from women undergoing pituitary desensitization for in-vitro fertilization. Follicle stimulating hormone (FSH, Metrodin; Serono, Welwyn Garden City, UK), 0.33 IU/ml, pyruvate (Sigma, St Louis, MO, USA), 0.47 mmol/l, and antibiotics (50 IU penicillin G/ml, 50 IU streptomycin sulphate/ml, 0.125 g amphotericin B/ml, antibiotic antimycotic solution, Gibco) were added to the medium.
After 69 days in culture, half of the tissue from 17 patients was removed from the inserts and placed in medium containing collagenase (Collagenase II; Sigma) at 1 mg/ml (11 patients, mean age 35, range 3038 years) or 0.5 mg/ml (three patients, mean age 34, range 2541 years, one of whom was represented by two samples see below), and incubated at 37°C for 1 or 2h, or from three patients (mean age 38.6, range 3740 years), 0.25 mg/ml overnight at 4°C followed by 23 h at room temperature. All the follicles that could be identified, irrespective of their developmental stages, were then partially isolated from the stromal tissue under a stereo microscope, using 27G needles. Care was always taken to leave some stroma around them.
From four patients (mean age 33, range 2539 years), including one patient with a large biopsy, part of which was also used for collagenase treatment at 0.5 mg/ml, half of the tissue was removed from culture after 8 days, and cut into thin strips. An attempt was made to cut follicles or clusters of follicles out of the stroma without enzyme incubation. It was possible to see some follicles through the dense stroma under a stereo microscope, but cutting was difficult.
The partially isolated follicles or clusters of follicles were placed in new inserts, similar to those used in the first step of culture. The remaining tissues were kept in their original inserts. The cultures were maintained for up to 4 weeks. They were photographed using an inverted microscope every second or third day. Samples of the cultured tissue were taken weekly for histological examination.
For histology, the tissue was fixed in Bouin's solution. After fixation the partially isolated follicles were embedded in 2% gelatine under a stereo microscope. They were dehydrated and embedded in paraffin within the gelatine blocks. The tissue was cut into 2 µm serial sections and stained with haematoxylin and eosin. The numbers of follicles in the samples and per high power field (HPF; x400) were counted, taking care that all the follicles in the tissue were counted, and that each follicle was counted only once (Hovatta et al., 1997; Lass et al., 1997
). Their developmental stages were recorded. Eosinophilia of the ooplasm, contraction and clumping of the chromatin material and wrinkling of the nuclear membrane of the oocyte were regarded as signs of atresia (Gougeon, 1986
).
The 2 test and Fisher's exact text were used for statistical analyses.
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Results |
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After ~2 weeks in culture, some oocytes began to extrude from the partially isolated follicles. This was frequently seen in enzymatically isolated follicles, but also occurred after 23 weeks in mechanically isolated and non-isolated follicles. It was not possible to quantify the proportion of oocytes extruding, because in the culture it was not possible to see if follicles within the tissue had an oocyte or not, and by histology many follicles without oocytes had already disappeared by atresia. As seen in histological preparations, the follicles extruding the oocyte were usually at the secondary stage by the time of extrusion (Figure 2). The oocytes were 2080 µm in diameter, the zona was very thin or absent and they were never surrounded by granulosa cells. Polar bodies and meiotic chromosomes could sometimes be seen in these oocytes, with a minimum diameter of ~40 µm, after they had come out into the gel matrix. The presence of mitotic figures in the granulosa cells throughout the culture period (Figure 3
) showed that the follicles were continuously growing. At the end of the culture period many follicles had reached the secondary stage (Figure 4
).
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As seen in the histological sections, during the first 79 days in the first culture (all non-isolated), a majority of the primary follicles had started to develop (Figure 5, Table I
). In the control tissue before culture, 73% of the follicles were primordial. After the first step of culture in slices, non- isolated, only 5% were primordial. This difference was significant (P < 0.001). At 47 days after the isolation step non-isolated (37%, P = 0.006) and mechanically isolated (47%, P = 0.002) follicles had reached respectively secondary or tertiary stages significantly more often than enzymatically isolated ones (15%) (Table I
), but 1 week later the proportion was similar to that in the other groups. The numbers of tertiary follicles were small, and no statistical analyses could be carried out on them alone.
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Discussion |
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Tertiary follicles were only occasionally seen in these cultures, and they all showed some signs of atresia. In the future, culture conditions will have to be improved to allow the follicles within small slices to develop to early antral stages. It may be best to take the oocytegranulosa cell complexes out of cultured early antral follicles as soon as possible, for further culture.
Partial isolation appears to be better than complete isolation, as completely or almost completely isolated human ovarian follicles can be cultured for only short periods (Roy and Treacy 1993; Oktay et al., 1997
). Ovarian stroma apparently interacts with the follicles. It may also mechanically support the structure of the follicles. Folliclefollicle interaction may also play a role, because the follicles remained in clusters after partial isolation.
Some oocytes were extruded from the follicles after various periods in culture. This has also occurred in cultures of mouse follicles (personal communication, R.Cortvrindt, Free University, Brussels, Belgium), and was thought to result from sub-optimal culture conditions. Growth and development of oocytes and follicles demand intact integrity of the oocyte, the granulosa cells and the other components of the follicle (Gougeon, 1996). Because human follicles are often located in small clusters it is also possible that there is interaction between the co-cultured follicles. This does not, however, explain the differences between the survival of non-isolated and partially isolated follicles, because all cultures contained clusters and individual follicles. Mechanically isolated follicles had some more stroma around them than enzymatically isolated ones, but it appeared not to save them from atresia. It was not possible to distinguish at the moment of isolation between primordial and primary follicles, but all the follicles seen were taken into culture. Some selection of one of the developmental stages could have been possible. If selection has occurred it might have favoured more advanced developmental stages among the enzymatically isolated follicles.
The total numbers of follicles were not high in the biopsies of these patients. A younger patient population would be better for this type of study, but it was difficult enough to obtain these ovarian tissue biopsies. The ages of the patients do not explain the differences between the non-isolated and isolated follicle cultures, because the ages of the tissue donors were similar among them. The possible yield and growth of follicles obtained from follicular aspirates of patients undergoing clinical in-vitro fertilization treatment (Wu et al., 1998) remains to be shown.
Because oocytes which are extruded are usually small and have a thin or absent zona pellucida, it is extremely important to improve these cultures further. Meiotic chromosomes, seen in some immature oocytes in our cultures, have also been described in cultures of human fetal ovarian tissue (Zhang et al., 1995). They appear to be abnormal. To achieve fertilization and embryonic development, larger and more mature oocytes will probably be needed. It is possible that these oocytes have been extruded from follicles which were more advanced at the moment of biopsy. Advanced stages were, however, very rare in the biopsied ovarian cortical tissue. According to our histological findings, the oocytes were usually extruded from secondary follicles.
After collagenase treatment, the oocytes tended to extrude earlier than after mechanical isolation or after culture within slices. Enzymes probably compromise the integrity of the follicles, as has been observed in mouse and bovine follicles (Telfer, 1996). It may also be one reason why human ovarian follicles survive for only a few days after enzymatic isolation (Roy and Treacy, 1993
). Mechanically isolated follicles also showed better development than enzymatically isolated ones, as more advanced developmental stages were found among them following culture. The density of follicles was highest after enzymatic isolation, which means that mechanical isolation was not as successful as the enzymatic one, or that more mechanically isolated follicles underwent atresia and disappeared. Because mechanical isolation proved difficult and very time consuming, the optimal culture method would be to cut the ovarian tissue into small slices, which means that many pieces of stroma only will also be cultured, because it is not possible to see if tissue contains follicles using transmitted light microscopy. In our study the effect of a smaller dose, 0.25 mg/ml of collagenase, remained inconclusive because there were no follicles in the control tissue of the patients concerned.
With our method, it is possible to culture human primordial follicles. After ~4 weeks, many of them will have reached secondary stage. Initiation of growth, based on the observation that the majority of follicles are at primary and secondary stages after 1 week in culture, has already been shown in our earlier study (Hovatta et al., 1997), and it has also been shown in cultured bovine (Wandji et al., 1996
) and baboon (Wandji et al., 1997
) follicles. To reach late pre-antral or early antral stages with full size or almost full size oocytes capable of fertilization and embryonic development in human tissues would probably require several weeks longer in culture. The growth period of follicles within the human ovary is ~12 weeks (Gougeon et al., 1986), and a similar time would be expected in vitro. Research is now required regarding factors regulating early follicular growth in experimental animals and in humans. As regards the optimal culture medium, alpha-MEM might be better than Earle's medium, and the possible deleterious effects of gonadotrophin-releasing hormone in the serum obtained during down-regulation might be avoided by using serum-free medium (Wright et al., 1999
). This information could be used to improve the culture conditions to obtain mature oocytes for clinical use all the way from primordial follicles.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 12, 1998; accepted on July 5, 1999.