1 Department of Clinical Science, Division of Obstetrics and Gynaecology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, Sweden and 2 Institute of Cell and Molecular Biology, University of Edinburgh, Darwin Building, Mayfield Road, Edinburgh EH9, UK
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Abstract |
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Key words: follicular fluid/human/IVF/oocyte/ovarian follicle
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Introduction |
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The aim of this study was to determine whether immature follicles could be obtained from follicular aspirates obtained in our IVF programme, and if these follicles could survive in vitro.
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Materials and methods |
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Identification of follicles
The first aspirate from each ovary was collected in order to obtain follicles from the ovarian cortex caught by the aspiration needle when the ovary was punctured initially. Aspirates with high concentrations of blood were avoided. After the oocytecoronacumulus complexes were removed as part of the clinical treatment of the patient, the remainder of the follicular aspirates was collected into sterilized centrifuge tubes and centrifuged at 200 g for 10 min. The supernatants were removed.
For the first 41 patients, the pellets were resuspended and examined directly. For the following 45 patients, steps were taken to remove erythrocytes as follows: 27 ml of distilled water was added to each tube, which was shaken vigorously and left for 2030 s. Then 3 ml of 10xPBS (phosphate-buffered saline) were added to each tube to restore normal osmolarity. After centrifugation at 200 g for 3 min, the supernatants were removed. The pellets were resuspended and examined.
All the pellets were dissolved in 0.5 ml pre-equilibrated -MEM (Invitrogen Inc., Scotland, UK) and examined both under a dissection microscope at x2070 magnification and under an inverted microscope at x100400 magnification.
In most cases, it was easy to distinguish the follicles from other structures according to the size, shape, brightness and arrangement of the cells. When any doubt existed, the distinguishing procedures were performed.
Distinguishing between follicles and granulosa cell masses
Under the inverted/dissection microscope, some granulosa cell masses resembled follicles. Two methods were used to check whether these cell aggregates contained oocytes. Firstly, granulosa cell aggregates were cultured on 96-well plates (Nunclon; Nunc, Denmark) individually at 37°C in 5% CO2 in the air. Each well contained 200 µl culture medium. Half of the medium was changed every second day. The culture medium consisted of -MEM supplemented with 10% inactivated human serum collected from patients undergoing stimulation for fertility treatment.
The second method involved removing the granulosa cells enzymatically and mechanically. The granulosa cell masses were incubated in 80 IU/ml hyaluronidase (HYASE-10x; Vitrolife, Göteborg, Sweden) for 20 s. After incubation, the granulosa cells were separated using 27 G needles.
Culture of immature follicles
Three follicles (primordial and/or primary) from two patients were cultured individually in the same way as for the granulosa cells.
Histology examination
A small piece of tissue collected from the aspirates was fixed in Bouin's fixative. After fixation, the tissue was dehydrated and embedded in paraffin. The tissue was cut into serial sections of 4 µm and stained with haematoxylin and eosin (H&E) and then examined under light microscope.
Statistical analysis
The 2-test was utilized for analysis of differences in the fertilization rate, pregnancy rate and percentage of mature oocytes between two groups. Fisher's exact test was used when the value was
5. Student's t-test was used to compare age differences in the patients and the number of the oocytes retrieved between two groups. P < 0.05 was considered significant.
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Results |
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Granulosa cell masses that resembled follicles were cultured and they tended to adhere to the bottom of the plate to form a single layer instead of round aggregates during culturing. They degenerated gradually. The number of granulosa cells decreased and the number of vacuoles increased in the culture (Figure 2). After 4 days culture, the granulosa cells had degenerated completely. No oocytes were visible within these cell aggregates over the culture time.
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All three follicles cultured (from two patients) degenerated within 2 days.
A small thin piece of tissue was found that contained vaginal epithelium resembling cluster of follicles under the inverted microscope. After fixation and routine H&E stain, these `follicles' were clearly vaginal epithelium as revealed by light microscope (Figure 3).
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Discussion |
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We found that some components in follicular aspirates appeared to be follicle-like structures when examined under an inverted microscope. Further analysis revealed these structures to be predominantly granulosa cell aggregates. Vaginal epithelia contained in a piece of tissue could also be erroneously identified as follicles if histological analysis was not carried out.
Although the number of cultured follicles was small in the present study, it still implied that it was difficult to culture primordial and primary follicles individually. We have demonstrated in an earlier study that isolated primordial or primary follicles, and those which are surrounded only by a small amount of stromal tissue, do not survive and grow as well as follicles cultured within ovarian tissue slices (Hovatta et al., 1999).
Individual squamous cells were found in the aspirates from four patients, and this was consistent with the report by Artley et al. (Artley et al., 1993). They found squamous cells in cytological examination of fluid obtained from transvaginal aspiration of simple ovarian cysts, suggesting that squamous cells were carried from the vagina into the follicles by the aspiration needle as it passed through the vaginal wall. That is also the explanation for why a piece of tissue consisting of vaginal epithelium was found in follicular aspirate.
In conclusion, our data show that follicular aspirates obtained during oocyte retrieval in IVF units cannot be used as an efficient source of human follicles. Some structures found in the aspirate may be erroneously regarded as follicles.
Note added in proof
After the submission of our manuscript, Moskovtsev et al. reported similarly to us that only a few follicles could be found in follicular aspirates (Moskovtsev et al., 2002).
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Acknowledgements |
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Notes |
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References |
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Hovatta, O., Wright, C., Krausz, T., Hardy, K. and Winston, R.M. (1999) Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation. Hum. Reprod., 14, 25192524.
Lass, A., Silye, R., Abrams, D.C., Krausz, T., Hovatta, O., Margara, R. and Winston, R.M. (1997) Follicular density in ovarian biopsy of infertile women: a novel method to assess ovarian reserve. Hum. Reprod., 12, 10281031.[ISI][Medline]
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Submitted on June 10, 2002; accepted on August 15, 2002.