Human Reproduction, Vol. 14, No. 10, 2519-2524,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation
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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Ovarian cortical tissue, donated by 20 women aged 25–43 years during gynaecological laparoscopies or laparotomies, was first cultured for 7–9 days as tissue slices, 0.1–0.3 mm in thickness, in extracellular matrix, to initiate the growth of the primordial and primary follicles. It was then divided into two parts, one of which was cultured further as slices, and the other one used for enzymatic (collagenase at 1, 0.5 or 0.25 mg/ml; 17 patients) or mechanical (four patients) partial isolation of the follicles. The tissue slices and the partially isolated follicles were cultured for a further 1–3 weeks in the matrix. After ~2 weeks in culture, some oocytes began to extrude from the follicles, which were usually at the secondary stage. They were small, 20–80 µm in diameter, and had a thin or absent zona. Polar bodies and meiotic chromosomes could be seen in these naked oocytes. This premature extrusion probably resulted from sub-optimal culture conditions. It occurred sooner in follicles that had been partially isolated using collagenase. Histologically, larger numbers of oocytes were observed in non-isolated slice cultures than in the partially isolated cultures. Initiation of growth of the follicles occurred during the first 7–9 days in culture within slices. In non-isolated slices and following mechanical partial isolation there were significantly more secondary follicles after 11–18 days in culture than following isolation with collagenase. The proportion of atretic follicles increased during all cultures, and it was significantly higher after partial isolation. Because partial isolation did not improve the survival or development of the follicles the optimal method for human ovarian follicles could be to culture them non-isolated within small tissue slices.
Key words: follicles/oocyte/organ culture/ovary
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Human oocytes from small antral follicles can already be matured, fertilized in vitro and even give rise to live births following transfer (Cha et al., 1991
; Barnes et al., 1995; Cha and Chian, 1998; Mikkelsen et al., 1998). In ovarian cortical biopsies, the numbers of primordial follicles are significantly higher than those at later stages (Lass et al., 1997). If biopsied cortical tissue is to be used, especially after cryopreservation, a method for in-vitro culture of primordial and primary follicles would be useful. Autografting or transplantation (Hovatta et al., 1998; Oktay et al., 1998) would be another option, but autografting carries a risk of transmission of a malignant disease (Gosden et al., 1997; Shaw and Trounson 1997).
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 (100–400 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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Ovarian cortical tissue pieces about 5x5x5 mm were obtained during gynaecological laparoscopies or laparotomies by biopsy from 20 patients who were treated for infertility. The mean age of the women was 35 (range 25–41) years. They all gave their informed consent. The study had been approved by the Research Ethics Committee of the Imperial College School of Medicine at Hammersmith Hospital. They were not using any hormones by the time of the operation. They did not have polycystic ovaries. The stage in the menstrual cycle at which these women underwent their operations could not be regulated.
The specimens were placed in HEPES-buffered culture medium (MEM, Gibco BRL, Life Technologies, Paisley, UK) and transported to the laboratory. They were sliced (0.3–1 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 6–9 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 30–38 years) or 0.5 mg/ml (three patients, mean age 34, range 25–41 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 37–40 years), 0.25 mg/ml overnight at 4°C followed by 2–3 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 25–39 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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Follicles were cultured throughout the whole 4 week period of this study, both within tissue slices and in partial isolation. Growth of the partially isolated follicles was observed by means of an inverted microscope (Figure 1
).
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In histology, 0–85 (mean 10) follicles were counted in the control pieces taken from the biopsies before cultures. In four control samples, no follicles could be seen, but these patients had two, seven, 15 and 45 follicles in the cultured tissue.
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 2–3 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 20–80 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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Partial isolation was easiest after incubation in the highest concentration (1 mg/ml) of collagenase. Longer-term incubation in a lower concentration (0.25 mg/ml) of collagenase did not prove successful, as no follicles were found. However, no non-isolated follicles were found in the control histological samples from these patients, aged 37, 39 and 40 years, either.
As seen in the histological sections, during the first 7–9 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 4–7 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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Although similar amounts of tissue were used for follicle isolation and for further culture in tissue slices, the total numbers of follicles observed were clearly higher in tissue slices than they were after isolation (Table II
), which reflected both the difficulty of isolation of follicles from the dense stroma, and the disappearance of the partially isolated follicles through atresia. The density of the follicles per HPF was highest among the enzymatically isolated follicles (Table II). The yields of follicles after enzymatic and mechanical isolation were similar, except in the case of overnight incubation in collagenase at 0.25 mg/ml, after which no follicles were found. In all groups, more than half of the follicles seen were located in clusters of two to ten follicles. In this study, the follicles within clusters appeared to develop at equal speed to solitary ones.
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The proportion of atretic follicles increased over the culture period in all groups, and 11–21 days after isolation there were significantly (P < 0.001) more atretic follicles after both enzymatical and mechanical isolation than there were among the non-isolated follicles within slices (Table I
, Figure 6).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The survival of ovarian follicles was significantly better among follicles cultured within tissue slices than it was among partially isolated follicles. Although we did not measure the exact amounts of tissue used for partial isolation and that continued as cultured slices, the amounts were approximately the same. The differences in the numbers of follicles observed after both enzymatic and mechanical isolation and after culture within slices were very clear, with more follicles being observed in slices. Hence, partial isolation does not appear to have any advantage over small tissue slices for the culture of human ovarian primordial, primary or secondary follicles.
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 oocyte–granulosa 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. Follicle–follicle 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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We thank Mr Raul Margara and Mr Geoffrey Trew for taking the ovarian biopsies.
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Notes |
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3 To whom correspondence should be addressed at:Department of Obstetrics and Gynaecology, Karolinska Institutet, Huddinge Hospital, SE-14 1 86 Huddinge, Sweden
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Submitted on November 12, 1998; accepted on July 5, 1999.
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J. G. Hreinsson, M. Otala, M. Fridstrom, B. Borgstrom, C. Rasmussen, M. Lundqvist, T. Tuuri, N. Simberg, M. Mikkola, L. Dunkel, et al. Follicles Are Found in the Ovaries of Adolescent Girls with Turner's Syndrome J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3618 - 3623. [Abstract] [Full Text] [PDF]
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I. Demeestere, A. Delbaere, C. Gervy, M. Van den Bergh, F. Devreker, and Y. Englert Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice Hum. Reprod., August 1, 2002; 17(8): 2152 - 2159. [Abstract] [Full Text] [PDF]
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S. I Moskovtsev, J. T Griffin, C.M. Peterson, and D. T Carrell Primordial and pre-antral follicles are not commonly observed in IVF aspirates Hum. Reprod., July 1, 2002; 17(7): 1783 - 1787. [Abstract] [Full Text] [PDF]
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O. Hovatta, V. Soderstrom-Anttila, T. Foudila, L. Tuomivaara, K. Juntunen, A. Tiitinen, and K. Aittomaki Pregnancies after oocyte donation in women with ovarian failure caused by an inactivating mutation in the follicle stimulating hormone receptor Hum. Reprod., January 1, 2002; 17(1): 124 - 127. [Abstract] [Full Text] [PDF]
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J. Liu, J. Van der Elst, R. Van den Broecke, and M. Dhont Live Offspring by In Vitro Fertilization of Oocytes from Cryopreserved Primordial Mouse Follicles after Sequential In Vivo Transplantation and In Vitro Maturation Biol Reprod, January 1, 2001; 64(1): 171 - 178. [Abstract] [Full Text]
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H. Louhio, O. Hovatta, J. Sjoberg, and T. Tuuri The effects of insulin, and insulin-like growth factors I and II on human ovarian follicles in long-term culture Mol. Hum. Reprod., August 1, 2000; 6(8): 694 - 698. [Abstract] [Full Text] [PDF]
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J. M.Sztein, M. J.O'Brien, J. S.Farley, L. E.Mobraaten, and J. J.Eppig Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term Hum. Reprod., March 1, 2000; 15(3): 567 - 571. [Abstract] [Full Text] [PDF]
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