Two novel techniques to detect follicles in human ovarian cortical tissue

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Human Reproduction Vol.21, No.7 pp. 1720–1724, 2006


Advance Access publication March 3, 2006.

Two novel techniques to detect follicles in human ovarian cortical tissue R.Soleimani1,4, W.De Vos2, P.Van Oostveldt2, S.Lierman1, R.Van den Broecke3, P.De Sutter1, M.Dhont1 and J.Van der Elst1 1

Infertility Centre, Ghent University Hospital, Ghent, Belgium, 2Department of Molecular Biotechnology and 3Gynecological Oncology, Department of Obstetrics and Gynaecology, Ghent University Hospital, Ghent, Belgium


To whom correspondence should be addressed at: Infertility Centre, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. E-mail: [email protected]

Key words: cryopreservation/follicle visualization/ovarian transplants/rhodamine 123

Introduction Recent successes in transplantation of the ovary and of cryopreserved human ovarian cortical tissue show its importance in restoring fertility in patients with extraneous premature ovarian failure (Donnez et al., 2004; Smitz and Cortvrindt, 2004; Meirow et al., 2005; Silber et al., 2005). Human ovarian cortical tissue contains a pool of primordial follicles, which is the source of future oocytes (Newton et al., 1996; Van den Broecke et al., 2001). Assessment of the number of follicles present in the cryopreserved tissue before transplantation has major prognostic value, although quantification without affecting the safe clinical use of the analysed tissue is a daunting task. Cortvrindt and Smitz (2001) reported that histological counting is reliable but has a long turnaround time; therefore they introduced fluorescent methods using Calcein AM and Picogreen, giving information on follicular density within 1 h, but both methods are cytotoxic and therefore prohibit the clinical use of the analysed tissues (Jonsson et al., 1996). We investigated two novel methods of detecting primordial follicles in ovarian cortical tissue strips before transplantation while allowing re-use of the tissue. One method is based on rhodamine

123 (R123) vital staining in combination with laser scanning confocal microscope (LSCM). R123 is a lipophilic cationic fluorescent dye that directly and selectively accumulates in the inner mitochondrial membrane and stains mitochondria of living cells (Johnson et al., 1981; Chen et al., 1982; Davis et al., 1985; Emaus et al., 1986; Chen, 1988). The second proposed method is simple stereomicroscopic visualization of follicles in ovarian cortical strips using glass-bottom dishes and transillumination. Materials and methods Source of human ovarian tissue Cryopreserved human ovarian tissue was obtained from a consenting 22-year-old female-to-male transsexual and was kept frozen for about 3 years. Cryopreservation had been done with the method described previously by Van den Broecke et al. (2001). The use of this tissue for the current study was approved by the Ghent University Hospital Ethical Committee. Staining of ovarian cortical tissue strips A stock solution of 1 μg/μl R123 (R302, Invitrogen, Molecular Probes, Merelbeke, Belgium) dissolved in methanol was prepared and

1720 © The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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BACKGROUND: Ovarian tissue cryopreservation and transplantation are becoming increasingly important issues for preserving female fertility as shown by recent successes in restoring ovarian activity and even fertility. Primordial follicle content before transplantation is a key issue for success. We investigated two novel methods to detect primordial follicles in human ovarian cortical tissue strips. METHODS: The first method used the fluorescent mitochondrial stain rhodamine 123 in combination with laser scanning confocal microscopy (LSCM). The first method used the fluorescent mitochondrial stain rhodamine 123 (R123) in combination with laser scanning confocal microscopy (LSCM). The second used a simple stereomicroscopic method with glass-bottom dishes for detecting primordial follicles in ovarian cortical tissue strips. Potential toxic effects of R123 and of the exposure to confocal laser were investigated in a mouse ovarian allograft model. RESULTS: Follicles were visible as white spots in thin cortical strips using LSCM in single and fast scanning at low magnification, allowing a fair estimation of the number of primordial follicles present. Using the second method, ovarian follicles were also visible using glass-bottom dishes under the stereomicroscope, although tissue thickness and density were limiting factors of its success. DISCUSSION: Follicles can be visualized in human cortical ovarian strips with R123 in combination with LSCM. Stereomicroscopy using glass-bottom dishes and transmitted illumination is a simple alternative method and has the advantage of allowing further safe clinical use of the analysed tissue.

Two novel techniques to detect follicles in human ovarian cortical tissue strips

kept at –20°C. Staining was done by adding 1 μl of the stock solution to 1 ml of medium before incubation. Vials containing thin human ovarian tissue strips were thawed in a water bath at room temperature. Tissue strips were washed five times with KSOM-Hepes (Potassium Simplex Optimized Medium + Hepes) buffered medium in 60 mm tissue culture dishes (Falcon; BD 35-3002, VWR, Leuven Belgium) to remove the cryoprotectant dimethylsulphoxide (Sigma, D5879, Bornem, Belgium). Tissue strips were cut to small pieces of about 0.5–1 mm2 with surgical blades (NO 24) and were washed again in 5 ml KSOM-Hepes buffered medium. These mini strips were then incubated in 35 mm tissue culture dishes (Falcon; BD 35-3001) containing 2 ml of MCDB 105 (Sigma, M-6395): M199 (Sigma, M-2154) 1 : 1 with 1 ng/ml R123 for 60 min in a 5% CO2 incubator.

Visualization of follicles in human ovarian cortical tissue strips by LSCM Visualization of stained tissue by R123 using glass-bottom dishes was done with a Nikon Eclipse TE300 epifluorescence microscope equipped with a Biorad Radiance 2000 confocal system. R123 was excited with a 514 nm Argon laser and detected with a photomultiplier tube (PMT) through a 590/70 nm HQ BP filter. Average duration of visualization was 5 min. The microscope objectives used were a ×100 plan oil immersion objective with a numerical aperture (NA) of 1.3, a ×40 oil objective (NA = 1.3), a ×10 dry objective (NA = 0.45) and a ×4 dry objective (NA = 0.13). Digital images were acquired with Lasersharp 2000 software under Windows 2000. Scanned tissues were fixed by buffered formaldehyde (10% v/v, Mallinckrodt Baker B.V., Deventer, The Netherlands) overnight at room temperature and processed followed by serial sectioning of the tissues to 4 μm thickness for haematoxylin-eosin (H&E) staining. Localization of follicles detected by LSCM was verified by light microscopy. Mouse ovarian tissue transplantation as a viability assay after exposure to R123 and confocal laser Two 12-day-old (C57BL/6j × CBA/Ca) F1 female hybrid mice, sacrificed by cervical neck dislocation, served as the donor. Left ovaries were removed, cut into half, washed with 2 ml pre-warmed MCDB 105 (Sigma, M-6395): M199 medium (Sigma, M-2154) 1 : 1 and incubated for 60 min in a 35 mm Falcon tissue culture dish with 2 ml of the same medium containing 1 ng/ml R123 (R302, Invitrogen) under an atmosphere of 5%CO2. Hemi-ovaries were evaluated by LSCM as described using low magnification. After exposure to R123 and confocal visualization, mouse ovarian tissues were allografted. This protocol was approved by the Ghent University Hospital Animal Ethical Committee. Two 8-week-old female (C57BL/6j × CBA/Ca) F1 hybrid recipients were anaesthetized by i.p. injection of Natrium pentobarbital 25 mg/kg (Nembutal, CEVA Santé Animale-France) at room temperature. LSCM-examined mouse ovaries were allografted into the back muscle of recipient mice about 15 min after examination. A small dorsal incision was made in the skin and body wall, and fine watchmakers’ forceps were used to make a deep hole (3–5 mm) in the back muscle. Ovarian tissue was inserted using other watchmakers’ forceps, while

Visualization of follicles in human ovarian cortical tissue strips using stereomicroscopy and glass bottom dishes Human ovarian cortical mini strips were thawed and washed as described above. Tissue strips of approximately 0.5 mm (476 ± 176 μm; mean ± SD) (thickness) × 1 mm × 1 mm were then placed in prewarmed KSOM–Hepes medium in a glass-bottom dish (without R123). Visualization was done under transillumination using an Olympus SZ-60 stereomicroscope. Statistical methods Statistic analysis was performed by chi-square test with Graphpad Prism software. P < 0.05 was considered significant.

Results Fluorescence observation of human ovarian tissue strips Using fluorescent microscopy, ovarian tissue stained as a large bright object, although it was not possible to discern the follicles (Figure 1). Visualization of follicles in human ovarian cortical tissue strips by LSCM White spots resembling small follicles were detected in cortical tissue strips by a single and fast scanning at a low magnification (×4) using LSCM (Figure 2). Very bright round spots in a dark grey field of tissue were easy to distinguish and were counted, giving an estimation of the number of follicles in the tissue strips (Table I).

Figure 1. Fluorescent staining of human ovarian fragment with R123 (scale bar 1 mm).


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Conventional fluorescence microscopic observation of human ovarian cortical tissue strips Standard fluorescent images of human ovarian cortical tissue strips were obtained from stained tissue with R123 using glass-bottom dishes (MatTek, Ashland, USA) with a Nikon TE2000-Eclipse epifluorescence microscope equipped with a custom Nikon DS-U1 colour camera. This was done to inspect if it could be useful in visualizing the follicles in ovarian strips.

the first forceps remained in their original location, keeping the hole open. Finally, the body wall and skin were closed. All procedures were performed under aseptic conditions. One week after grafting, ovarian stimulation was started by i.p. injection of 1 IU FSH (Puregon, Organon, Oss, The Netherlands), given every second day for 2 weeks. Finally, two doses of 5 IU FSH were given followed by 5 IU HCG 48 h later. Another 14 h later, all animals were autopsied after cervical dislocation. Skin was removed gently to retrieve the grafted ovarian tissues. Both grafts were removed and fixed in 10% (v/v) formaldehyde overnight at room temperature and processed for paraffin embedding and H&E staining. Histological examination was performed microscopically after serial sectioning to 4 μm thickness. To prevent recounting, a follicle was only counted once at the time that the dark-stained nucleolus was seen. Follicles were classified as primordial (oocytes surrounded by one layer of flattened pregranulosa cells), primary (surrounded by one layer of cuboidal granulosa cells), pre-antral (with two or more layers of granulosa cells without antrum), antral (with an antral cavity), ovulated (MII oocytes found in a cavity formed at the grafting site) and corpora lutea. Age-matched ovarian grafts that had not been exposed to rhodamine 123 and LSCM before transplantation served as controls.

R.Soleimani et al.

Table II. Mouse ovarian follicular development after exposure to R123, confocal visualization of follicles and allotransplantation

B1 B2 Control

Primordial and primary (%)

Pre-antral, antral and ovulatory (%)


212 (85.1) 298 (84.4) 337 (88.2)

37 (14.9) 55 (15.6) 45 (11.8)

249 353 382

B1, B2: Two mice with hemi-ovaries transplanted to the back muscle site. Control: mean percentage of six grafts (non-R123 and LSCM exposed). P-value = 0.2930.

Figure 2. Laser scanning confocal microscopy (LSCM) of human ovarian cortical tissue at a low magnification: Round spots resembling human ovarian follicles are visible. (A) Scale bar 70 μm, (B) scale bar 50 μm.

Strip number

1 2 3 4 5 6

Figure 4. Mouse ovary allografted to the back muscle site (scale bar 200 μm). (a) Grafted ovary fragment, (b) back muscle tissue.

Number of follicles LSCM


9 0 6 3 0 7

8 0 7 3 0 6

Visualization of follicles in human ovarian cortical tissue strips using stereomicroscopy and glass-bottom dishes Ovarian follicles were seen as light bright round spots in a mat field of tissue by simple evaluation of a thin cortical strip of human ovarian tissue under the stereomicroscope in combination with the use of glass-bottom dishes and transmitted illumination. It was easy to detect follicles by adjusting the light intensity with an inbuilt mirror. In a thicker part of the tissue, detection was not so successful because there was insufficient transmission of light (Figure 5). Discussion

Figure 3. Laser scanning confocal microscopy (LSCM) of human primordial follicle with higher magnification. (A) Serial scanning of human primordial follicle, (B) LSCM of human primordial follicle: (a) granulosa cells, (b) cytoplasm, (c) nucleus (scale bar 10 μm), (C) the same follicle after histological examination with haematoxylin– eosin staining (scale bar 12 μm).

When preparing ovarian tissue for transplantation, it would be helpful to have a visualization technique for detecting small follicles that allow the subsequent use of the tissue. For this purpose, we studied both a laser confocal method and a simple stereomicroscopic method. Follicles in human ovarian cortical tissue strips were clearly detected by LSCM combined with exposure to the mitochondrial stain R123. We found that shortterm exposure to R123 and LSCM was not toxic to follicular

Higher magnifications confirmed the classical threedimensional structure of follicles. Nuclei were visualized as dark (absence of fluorescence), cytoplasm stained weakly and granulosa cells were stained bright (Figure 3). Mouse ovarian tissue transplantation as viability assay after exposure to R123 and confocal visualization Both grafted hemi-ovaries were recovered. Follicular development was observed in grafted tissue as seen on histomorphological evaluation of both grafts. There was no significant difference in follicular development in grafts that had been exposed to R123 compared to non-exposed controls (Table II, Figure 4). 1722

Figure 5. Visible human ovarian follicles in a thin piece of cortical tissue using glass-bottom dishes. (A) Low magnification under stereomicroscope (arrows) (scale bar 100 μm). (B) Higher magnification under microscope (scale bar 18 μm).

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Table I. Estimation of follicle content of human cortical tissue strips with laser scanning confocal microscopy (LSCM) and follicle counting after haematoxylin–eosin (H&E) histological staining of the same tissue samples

Two novel techniques to detect follicles in human ovarian cortical tissue strips

of laboratories, it is not possible to use expensive techniques such as LSCM to evaluate the tissues. Also, the use of a fluorescent mitochondrial dye may not be acceptable for human tissue that has to be used subsequently for transplantation. Therefore, it was necessary to establish a simple technique allowing the tissue to be re-used with certainty. Using glass-bottom dishes under the stereomicroscope is a simple method, although this technique requires an experienced operator with sufficient knowledge of intact follicle morphology to detect the follicles by adjustment of the light and the lens. Using glass-bottom dishes prevents scattering of transmitted light during evaluation of the tissue, which is the case when using plastic dishes during evaluation of the tissue. The thickness and density of the tissue can be a limiting parameter. The best thickness seems to be 0.5 mm or less. The great advantage of this method is that no chemical dye has to be used and that the same tissue on which the visualization was done can be used for further experiments. However, the method is restricted to transparent tissues. In contrast, with LSCM whole tissues can be evaluated even if they are not transparent. Though previously described techniques such as Calcein AM and Picogreen (Cortvrindt and Smitz, 2001) are also very good methods that can be used to estimate follicular density in ovarian tissues, their cytotoxic nature prohibits their clinical use. The two methods presented here for the detection of follicle presence in ovarian tissue may help clinicians and researchers to evaluate fresh or cryopreserved ovarian tissue before any experiments such as transplantation or long-term culture. Acknowledgements The authors thank the Department of Pathology, Faculty of Medicine and Health Sciences, Ghent University Hospital for their kind assistance in preparation of histological sections. Also our special thanks go to the Organon Co. for providing us with recombinant FSH (Puregon) used in this study. We also thank MatTek Co. for providing us with glass-bottom culture dishes. We thank Mrs Vera David for her help in taking care of the mice. Particular thanks go to Ms. Elke Heytens for assistance in reading and editing.

References Alonso-Pozos I, Rosales-Torres AM, Avalos-Rodriguez A, Vergara-Onofre M and Rosado-Garcia A (2003) Mechanism of granulosa cell death during follicular atresia depends on follicular size. Theriogenology 60,1071–1108. Bedaiwy MA and Falcone T (2004) Ovarian tissue banking for cancer patients: reduction of post-transplantation ischemic injury: intact ovary freezing and transplantation. Hum Reprod 19,1242–1244. Chen LB (1988) Mitochondrial membrane potential in living cells. Annu Rev Cell Biol 4,155–181. Chen LB, Summerhayes IC, Johnson LV, Walsh ML, Bernal SD and Lampidis TJ (1982) Probing mitochondria in living cells with rhodamine 123. Cold Spring Harb Symp Quant Biol 46,141–155. Cortvrindt RG and Smitz JE (2001) Fluorescent probes allow rapid and precise recording of follicle density and staging in human ovarian cortical biopsy samples. Fertil Steril 75,588–593. Davis S, Weiss MJ, Wong JR, Lampidis TJ and Chen LB (1985) Mitochondrial and plasma membrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinoma-derived MCF7 cells. J Biol Chem 260,13844–13850. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, MartinezMadrid B and van Langendonckt A (2004) Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 16,1405–1410. Emaus RK, Grunwald R and Lemasters JJ (1986) Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria. Spectral and metabolic properties. Biochim Biophys Acta 850,436–448.


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development in mouse ovarian allografts. Stereomicroscopy using fine glass-bottom dishes to visualize primordial follicles in thin sections of human ovarian tissue was revealed as a simple alternative to LSCM for detection of follicles. Cryopreservation and banking of ovarian cortical tissue in view of future preservation of female fertility is a well-known technique (Newton et al., 1996; Newton, 1998; Bedaiwy and Falcone, 2004; Oktay and Sonmezer, 2004). Recent reports on successful ovarian tissue transplantation showed that ovarian activity and even fertility can be restored in patients at risk of losing their ovarian activity (Donnez et al., 2004; Meirow et al., 2005; Oktay and Tilly, 2004; Smitz and Cortvrindt, 2004; Silber et al., 2005). Because an estimation of follicle density is mandatory to choose the right tissue strips with sufficient amount of primordial/early primary follicles, non-harmful visualization of follicles before ovarian tissue transplantation provides a useful tool. As the number and distribution of follicles in the cortical part of the adult women’s ovaries are very variable according to age and physiologic status, absence of any follicle in the ovarian cortical tissue strips selected for transplantation is a potential hazard (Lass et al., 1997). Recording of follicle density can be equally important before cryopreservation or in vitro culture (Cortvrindt and Smitz, 2001). We introduced two methods to inspect ovarian tissues for the presence of follicles before transplantation. R123, a vital mitochondrial stain that can be visualized in cells by means of LSCM, seems to be a good technique for this purpose. It is generally accepted that R123, as a permeant cationic fluorochrome, is taken up specifically by functional, active mitochondria. This means that only follicles surrounded with live granulosa cells will be visible, and it is not possible to detect dead follicles (Alonso-Pozos et al., 2003). As the aim was to have a rapid estimation of the presence of ovarian follicles in tissue strips, only a single scan of the tissue with a minimum energy source of laser beam and small magnification of the lens was done. This was enough to see white spots resembling follicles in a dark field of tissue, allowing us to estimate the number of follicles in the tissue. Higher magnifications and H&E staining confirmed the results. Grafting of mouse ovarian tissue after LSCM–R123 proved that follicles survive and are able to develop even up to the ovulatory stage. Thus, our animal model shows that the combination of LSCM and shortterm R123 exposure is not cytotoxic for mouse ovarian tissue. This is supported by previous unpublished data showing that short-term exposure of mouse zygotes to R123 up to 90 min is not toxic for in vitro blastocyst development. However, if this method of evaluation is to become of practical value, viability of human xenograft after exposure to R123 and LSCM will have to be demonstrated. In addition to the confocal microscopy, we also introduced a very simple stereomicroscopic method to evaluate cryopreserved human ovarian cortical tissue strips after thawing. The basic success of this technique is the use of glass-bottom dishes and varying the light incidence. Both methods presented here for follicle visualization in human ovarian cortical tissue have their own advantages and disadvantages. LSCM needs a well-equipped laboratory but gives a fair estimation of the number of follicles. It is clear that for lots

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Oktay K and Sonmezer M (2004) Ovarian tissue banking for cancer patients: fertility preservation, not just ovarian cryopreservation. Hum Reprod 19,477–480. Oktay K and Tilly J (2004) Live birth after cryopreserved ovarian tissue autotransplantation. Lancet 364,2091–2092. Silber SJ, Lenahan KM, Levine DJ, Pineda JA, Gorman KS, Friez MJ, Crawford EC and Gosden RG (2005) Ovarian transplantation between monozygotic twins discordant for premature ovarian failure. N Engl J Med 353,58–63. Smitz J and Cortvrindt R (2004) First childbirth from transplanted cryopreserved ovarian tissue brings hope for cancer survivors. Lancet 16,1405–1410. Van den Broecke R, Van der Elst J, Liu J, Hovatta O and Dhont M (2001) The female-to-male transsexual patient: a source of human ovarian cortical tissue for experimental use. Hum Reprod 16,145–147. Submitted on October 13, 2005; resubmitted on January 10, 2006, February 2, 2006; accepted on February 3, 2006

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