Human Reproduction, Vol. 18, No. 6, 1158-1164,
June 2003
© 2003 European Society of Human Reproduction and Embryology
Density and distribution of primordial follicles in single pieces of cortex from 21 patients and in individual pieces of cortex from three entire human ovaries
1 Laboratory of Reproductive Biology, section 5712, 2 The Fertility Clinic, section 4071 and 3 Department of Pediatrics, section 4064, University Hospital of Copenhagen, Rigshospitalet, DK-2100 Copenhagen, Denmark
4 To whom correspondence should be adressed at: The Fertility Clinic, section 4071, The Juliane Marie Centre for Women, Children and Reproduction, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. e-mail: rh12735{at}rh.dk
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Abstract |
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BACKGROUND: At the time of cryopreservation of ovarian tissue for fertility preservation a small biopsy of ovarian cortex is usually taken for histological evaluation of the follicular reserve. The purpose of this study was to evaluate the distribution and density of primordial follicles in single pieces of cortex from individual patients and in pieces of cortex comprising entire ovaries, all prepared for cryopreservation. METHODS: Cortical biopsies from 21 patients and the whole cortex of one ovary were evaluated histologically prior to cryopreservation. In addition, the cortex of two whole ovaries was cryopreserved before histological evaluation. The volume of each cortical fragment was measured, all follicles counted and the follicular density calculated. RESULTS: In individual pieces of cortex follicular density showed a significant inverse linear correlation with age. The follicular density per cortical fragment prepared from each of the three entire ovaries varied from 1.8 to 166, 0.007 to 140 and 0.04 to 4.48 follicles/mm3 cortical tissue. CONCLUSION: The density of primordial follicles varied more than two orders of magnitude in cortical fragments from each of the three ovaries. Primordial follicles were very unevenly distributed throughout the cortex of these ovaries, although a significant linear correlation between age and follicular density was found.
Key words: cryopreservation/density/ovary/primordial follicles
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Introduction |
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Chemotherapy and/or radiation therapy as treatment for serious diseases may, as a side effect, deplete the ovarian follicular reserve, causing infertility and early menopause. Cryo preservation of ovarian tissue has now become an option for fertility preservation and maintenance of menstrual cycles in such women. After thawing, the ovarian tissue can be reimplanted and follicular development and ovarian steroid production may be re-established (Oktay et al., 2001
; Radford et al., 2001). Although no children have yet been born after reimplantation of thawed ovarian tissue, many hospitals cryopreserve human ovarian tissue.
In conjunction with preparation of ovarian tissue for cryopreservation, a small piece of the ovarian cortex is normally removed and prepared for histology in order to evaluate the number and density of primordial follicles (Meirow et al., 1999; Radford et al., 2001; Poirot et al., 2002). However, there is only limited information on the follicular density of ovarian cortical biopsies (Lass et al., 1997; Kohl et al., 2000; Qu et al., 2000; Poirot et al., 2002), and in particular there is a paucity of information as to whether a small biopsy will represent the follicular density of the entire cortex, or whether the follicular density varies throughout the ovarian cortex. Recent studies have indicated that follicles are not homogeneously distributed within the ovarian cortex, as evaluated in one random sample of cortical tissue (Poirot et al., 2002), between different locations of the biopsy in one ovary (Qu et al., 2000) or from the left and the right ovary in five women (Kohl et al., 2000). To our knowledge no studies have so far assessed the distribution pattern of follicles in pieces of cortex from an entire human ovary.
The purpose of the present study was to determine the density of primordial follicles in individual pieces of cortex from patients participating in our cryopreservation programme. Furthermore, the number of primordial follicles in the entire cortex of an ovary was assessed using the total number of cortical pieces from each of three ovaries prepared for cryopreservation in order to evaluate the distribution and density of primordial follicles within a whole ovary.
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Materials and methods |
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Single ovarian biopsy analysis
Twenty-one cortical biopsies were included in the study. A total of 17 patients with a malignant disease had one of their ovaries cryopreserved. Each had an ovarian cortical biopsy taken for histological examination before freezing. In addition, four patients who underwent open pelvic surgery for cervical cancer agreed to donate one ovarian biopsy to the study. The patient characteristics are given in Table I. Six of the 21 patients had already received chemotherapy prior to cryopreservation according to the following schedule. Patient 1 (patient numbers refer to Table I) received a total of 240 mg/m2 topoisomerase II inhibitor, 3 g cyclophosphamide and 45 mg metotrexate. Patient 2 is identical to case 2 from ‘Whole ovarian cortex analysis’. Before cryopreservation, one fresh cortical piece was taken for histological evaluation. Patient 3 received seven + three series of topoisomerase II inhibitor until remission, but no alkylating agents. Patient 4 received a total of 146.9 mg adriamycin, 17.5 mg vincristin, 545 mg Cytosar, 297 mg VP-16, 3.2 g cytarabine, 887.4 mg etoposide, 3.6 g cyclophosphamide, 48 mg methotrexate intraspinally and 60 820 IU asperaginase. Patient 5 received nine series of ABVD/COPP (doxurubicin, bleomycin, vinblastine, darcarbacine/cyclophosphamide, oncovin, procarbazine, prednisone). Patient 6 received a total of 960 mg etoposide and 5.1 g cyclophosphamide.
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Whole ovarian cortex analysis
An entire ovary was obtained from each of three patients. All ovaries were prepared for cryopreservation as described below. The ovarian cortex from cases 1 and 2 was actually cryopreserved, while the cortex from case 3 was prepared for histology immediately after oophorectomy.
Case 1: a 12.5-year-old girl was referred to our department in March 2000 for cryopreservation of ovarian tissue. Nine months earlier she had been diagnosed with acute myeloid leukaemia (AML). She received a total of 30 mg/m2 mitoxantrone, 225 mg/m2 doxorubicin and large doses of antimetabolites, but no alkylating agents. In February 2000 she was re-admitted because of relapse, and bone marrow transplantation (BMT) was planned following whole body irradiation and large doses of alkylating agents. At the time of referral the patient had not yet entered menarche, but she did show breast development. Before BMT, her left ovary was laparoscopically removed for cryopreservation. The patient died 3 months later.
Case 2: a 10-year-old girl was referred in May 2001 for cryopreservation of ovarian tissue. Prior to this she was diagnosed with acute lymphoblastic leukaemia (ALL) for which she received a total of 12 mg/m2 plant alkaloids, 80 mg/m2 doxorubicin and small doses of antimetabolites, but no alkylating agents. Because of relapse BMT was planned following whole body irradiation and large doses of alkylating agents. Prior to this treatment, she had one of her ovaries removed laparoscopically for cryopreservation. The patient died before receiving any further treatment.
Case 3: a 29-year-old woman, with one child, was referred for oophorectomy in August 2001. Two years earlier she was diagnosed with an estrogen receptor-positive breast cancer of the right breast. She had a right-sided mastectomy and was shortly thereafter referred for cryopreservation of ovarian tissue (right ovary) in October 1999. Immediately thereafter she received radiation therapy to the breast as well as chemotherapy. She received a total of 9.4 g of cyclophosphamide. In addition, she was treated with the anti-estrogen medication tamoxifen. The patient maintained menstrual cycles throughout the treatment period. Despite the treatment she developed bone metastases in the spring of 2001, and was consequently referred for laparoscopic oophorectomy of the remaining ovary (the left). She donated the ovary to the study. The cortex of the ovary was isolated immediately after retrieval and cut into fragments exactly as though cryopreservation was going to be performed. The tissue was directly processed for histology instead of being cryopreserved.
Cryopreservation of ovarian tissue
Immediately after recovery of the ovary the cortex was isolated into 1–2 mm of thickness and cut into 5 x 5 mm fragments. The fragments were collected without taking into consideration the order of their placement in the ovary. During the isolation procedure the tissue was rinsed several times in an isotonic saline solution. The pieces were transferred to 30 ml of 0.1 mol/l sucrose and 1.5 mol/l ethyleneglycol in phosphate-buffered saline, and equilibrated for 30 min at 1°C on a tilting table. The pieces of cortex were stored in 1.8 ml cryovials (Nunc A/S, Roskilde, Denmark), each containing 1 ml of cryoprotectant, and cryopreserved using a programmable Planer freezer (Planner K10, Planner Ltd, UK). The following programme was used: 2°C/min to –9°C, 5 min of soaking, then manual seeding for ice crystal nucleation induction, 0.3°C/min to –40°C, 10°C/min to –140°C, at which temperature the samples were plunged into liquid nitrogen at –196°C (Newton, 1996). Samples were thawed rapidly in a 37°C water bath. Each fragment was fixed in Bouin’s solution before being transferred to a 70% ethanol solution. All fragments were processed for paraffin embedding and sliced into 30-µm thick sections, mounted on glass slides and stained with periodic acid Schiff reagents and Mayer’s haematoxylin. The follicles were classified as follows: primordial follicle (an oocyte surrounded by a single layer of flattened granulosa cells), primary follicle (single layer of cuboidal granulosa cells) and secondary follicle (more than one layer of cuboidal granulosa cells). All follicles were counted, but structures that had collapsed to a degree to which they could no longer with certainty be recognized as a follicle were excluded.
Calculation of follicular density and volume of cortical pieces
In order to calculate the total number of follicles we developed a mathematical model as described below.
Each fragment was assessed, and all primordial follicles counted in every second section. The mean diameter (i.e. the perpendicular measurements of the largest cross section between basement membrane) of a primordial follicle was measured to be 44 µm (SEM 0.9 µm, n = 20). Since the diameter of the primordial follicle exceeds the thickness of the individual sections, a correction factor, 
, was calculated in order to account for the probability of counting the same follicle two or three times:
where t is the actual thickness of slice and d the diameter of the primordial follicle (i.e. 44 µm).
The total number of follicles was calculated using the following general formula:
in which ni is the number of follicles in section i, and N is the total number of sections, 
is a correction factor used to account for the possibility of only a fraction of sections being counted on every slide (i.e. is 2 in our study, expressing that every second section has been counted).
The density of follicles in a piece of cortex was calculated using the following formula:
in which Ai expresses the area of section I and ti expresses the height of section i.
The volume of each piece was calculated based on measurements of the height and area of every fourth section using a computer-assisted stereological system (C.A.S.T.-Grid; Olympus DK A/S, Albertslund, Denmark).
The research programme on cryopreservation of ovarian tissue prior to treatment of a malignant disease was approved by the Ethical Committee of Copenhagen and Frederiksberg. Before entering the programme each patient was informed in writing as well as orally, and gave her written consent. Parents signed on behalf of the under-aged girls. In the cases where ovarian tissue was donated, patients gave their written consent after oral and written information.
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Results |
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Single ovarian biopsy analysis
The follicular density of the 17 patients enrolled in our cryopreservation programme and the four patients donating ovarian biopsies is shown in Figure 1. As can be seen, the density of cortical tissue varied from 1.1 to 190.6 follicles/mm3 between the patients, and all of the cortical pieces contained primordial follicles. Despite the random nature by which the piece of cortex destined for histology was collected from all of the cortical pieces of the ovary prepared for cryopreservation, there was a significant inverse correlation between follicular density and age (r = –0.48, P < 0.001).
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Follicular density of ovarian cortex from the six patients that had already received chemotherapy showed no correlation with age, was non-homogeneous and the sample size too small to allow for a comparison with those patients who were not treated with chemotherapy prior to cryopreservation.
Whole ovarian cortex analysis
The volume of the isolated ovaries was 3, 4 and 8 ml for cases 1, 2 and 3, respectively. The volume, number of follicles, and density of each cortical fragment is shown in Table II. A total of 14 pieces of cortex were isolated from the ovary of case 1. The cortex had a total volume of 421.4 mm3 and contained a total of 22 310 primordial follicles. From case 2, a total of 15 cortical fragments were isolated from one ovary, with a volume of 237.8 mm3 and 15 888 primordial follicles. The ovary of case 3 resulted in 19 pieces of cortex comprising a total volume of 926.2 mm3 and 1011 primordial follicles. There was a wide variety in the number of primordial follicles between each fragment of the same ovary, ranging from 58 to 5861 in case 1, from 2 to 2497 in case 2 and from 1.5 to 130 in case 3. Also, the follicular density varied markedly within the ovary, ranging from 1.8 to 166.1 follicles/mm3 in case 1, from 0.007 to 140 follicles/mm3 in case 2 and from 0.04 to 4.48 follicles/mm3 in case 3. In the case of the second ovary the follicular density in cortical fragments varied by a factor of 20 000.
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Table III shows the mean, SEM and range of volume, number of primordial follicles, and density in each of the three ovaries.
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The most predominant type of follicle in the tissue was the primordial follicle. Primary oocytes and secondary oocytes were scarce, especially in the ovaries of cases 1 and 2. The distribution of primordial, primary and secondary follicles is listed in Table IV. The variability of the density of follicles within cortical fragments from one ovary is illustrated in Figure 2.
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In all three ovaries the primordial follicles tended to lie in clusters in the fragments rather than being evenly distributed throughout the tissue.
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Discussion |
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The present study is, to our knowledge, the first to evaluate the follicular density in the entire cortex of human ovaries. The results showed that follicles in the human ovarian cortex as prepared for cryopreservation were unevenly distributed, and, as a consequence, there was a huge variation in follicular density. For each of the three ovaries evaluated in toto, the difference in density of primordial follicles between pieces of cortex was greater than two orders of magnitude from fragment to fragment within the same ovary. In the most extreme case two follicles were found in one fragment and >2000 in another fragment of the same ovary, resulting in a follicular density that varied by a factor of >20 000. Thereby the present results confirm and extend those of Qu et al. (2000)
, who reported that the follicular density in ovarian cortex from a biopsy in each of two patients varied greatly between the fresh and the cryopreserved part. An uneven follicular distribution was also observed by Poirot et al. (2002), who found no primordial follicles in samples of ovarian cortex from three young women having received no previous chemotherapy. Collectively, there is now good evidence to suggest that the follicular density in the human ovarian cortex is very uneven and suggests that at the time of re-implantation of tissue multiple pieces of ovarian cortex should ideally be replaced. In addition, this study suggests that a single small biopsy of ovarian cortex should not be used to evaluate the total number of follicles present in an ovary. However, we do acknowledge that a small biopsy may offer some diagnostic value, especially in cases where it contains a large number of follicles.
The follicular density in ovarian cortical biopsies from the 21 women receiving cryopreservation showed a significant inverse correlation with age, although a large variation in follicular density was obvious. This confirms and extends earlier observations showing an almost linear decay curve of primordial follicles in relation to age (Faddy, 2000; Qu et al., 2000). These findings support the diagnostic value of a single ovarian biopsy, but in cases where no or only a few follicles are present in a biopsy, a random biopsy may not be representative of the follicular reserve of that particular ovary. Also, in conjunction with ovarian cryopreservation, histological evaluation of a small biopsy regarding the follicular reserve should not stand alone, but should also include other data. Age, menstrual cycles, transvaginal sonography of the ovaries including measurements of volume (Lass and Brinsden, 1999), antral follicle count (Bukman and Heinemann, 2001; Bancsi et al., 2002) and Dopplerflow (Sladkevicius and Campbell, 2000), previous exposure to chemotherapy or irradiation, and hormonal status including levels of FSH, inhibin B and estradiol on days 2–5 of the menstrual cycle (Bukman and Heinemann, 2001), may provide valuable information. Different stimulation and challenge tests have been developed in the hope of finding a good diagnostic tool for the clinician when predicting the ovarian reserve of a woman. The clomiphene citrate challenge test, the GnRH agonist stimulation test and the exogenous FSH ovarian reserve test all seem to offer some potential in predicting the outcome of different assisted reproductive technology programmes, but none is optimal, and further studies are called for (Broekmans et al., 1998; Bukman and Heinemann, 2001).
Clearly, further non-invasive methods for better evaluation of follicular reserve are needed, not least when counselling the woman on her future potential benefits of having her ovary cryopreserved. This is especially the case in women who have already received some antineoplastic treatment or in older women with a diminished ovarian reserve. When harvesting ovarian tissue for cryopreservation, some clinics choose to remove small ovarian biopsies only, rather than the entire ovary (Meirow et al., 1999; Meirow and Nugent, 2001). However, if a biopsy with only a limited number of follicles is obtained, this may deprive the woman of a future fertility option. In order to avoid this situation we suggest, in cases with two intact ovaries, the removal of one entire ovary as already proposed by Donnez and Bassil (1998).
The present study did not attempt to assess the survival of follicles in relation to the cryopreservation procedure. Clearly, not all follicles survived the procedure as evaluated from the histological appearance. However, the cryopreserved ovarian tissue was fixed for histology immediately after thawing and we found no evidence to suggest that any follicles had collapsed to a degree to which they were not recognizable as such. This observation justifies comparison of the frequency by which different types of follicles appear in the three ovaries, although two of the ovaries were used with, and one without, cryopreservation. A recent study addressed the changes associated with early atresia of primordial and primary follicles in the human ovary (de Bruin et al., 2002). Based on ultrastructural changes they found that most follicles in ovarian biopsies were of good quality, with little sign of atresia, and demonstrated that early signs of atresia were characterized by changes in mitochondrial and nuclear membranes as well as changes in the smooth endoplasmatic reticulum of the oocyte. The material in the present study consists of two groups of ovarian cortex, one prepared for histology immediately after recovery and one where cryopreservation actually took place. The rate of atresia differs between these two groups, and we find the above-mentioned parameters difficult to assess at the light microscopic level using 30 µm thick sections. We acknowledge that studies to evaluate the rate of atresia in human primordial follicles, and especially before and after cryopreservation, are needed, but do not find the present material optimal for that purpose.
As expected, the primordial follicle was the most predominant type of follicle. In the cortex of the adult ovary (case 3) 94% of the follicles were primordial, 5.3% primary and 0.7% secondary. This distribution is in accordance with the studies by Gook et al., who studied frozen thawed tissue from ovarian biopsies (Gook et al., 1999). In cortical fragments of ovaries from the prepubertal girls the frequency by which primordial follicles appeared was increased to 98.2–99.7% of all the follicles. This is in accordance with the early work of Block, who described that the number of growing follicles increases with age (Block, 1952).
Although the present study provides information on the follicular density for three whole ovaries, we acknowledge that they do not represent the normal, human ovary. All three patients had received an ovarian ablative treatment prior to cryopreservation, and the total number of follicles is less than would be expected for their age, namely 
400 000–300 000 in girls aged 10–15 years, and 50 000 in the 30-year-old woman (Faddy, 2000). The results obtained in this study in relation to the distribution and the density of follicles are likely to apply to the normal ovary as well; however, only evaluations of normal ovaries will prove this. Human ovarian tissue for scientific purposes is scarce, and entire ovaries even more so, and this naturally limits the study of the healthy human ovary. On the other hand, many of the patients eligible for cryopreservation of ovarian tissue have already received some sort of antineoplastic treatment prior to cryopreservation, which makes our study representative of the ovarian morphology of this particular group of patients.
In conclusion, the present study shows that the density of primordial follicles exhibits great variation within individual pieces of cortex representing entire human ovaries. Despite this variation in follicular density, random samples from 21 patients show the expected significant inverse correlation between density and age. It is suggested that in connection with cryopreservation of ovarian tissue for fertility preservation removal of a whole ovary may benefit the woman more than the removal of a small biopsy, and that a small ovarian biopsy may not reflect the fertility potential of the woman from whom it was removed.
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Acknowledgements |
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The authors wish to thank laboratory technicians Inga Husum and Tiny Roed for their excellent help in processing the tissue for cryopreservation and histology. We also wish to thank Dr Peter Frandsen Jakobsen for his help in developing the mathematical models for estimating follicular numbers and density. Finally, we wish to thank the Danish Medical Research Council (grant number 11-062/00) and the Hovedstadens Sygehusfællesskab Research Fund for their financial support.
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C.B. Lambalk, C.H. de Koning, A. Flett, Y. van Kasteren, R. Gosden, and R. Homburg Assessment of ovarian reserve: Ovarian biopsy is not a valid method for the prediction of ovarian reserve Hum. Reprod., May 1, 2004; 19(5): 1055 - 1059. [Abstract] [Full Text] [PDF]
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A. Lass Assessment of ovarian reserve: Is there still a role for ovarian biopsy in the light of new data? Hum. Reprod., March 1, 2004; 19(3): 467 - 469. [Abstract] [Full Text] [PDF]
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F. I. Sharara and R. T. Scott Assessment of ovarian reserve. Is there still a role for ovarian biopsy?: First do no harm! Hum. Reprod., March 1, 2004; 19(3): 470 - 471. [Abstract] [Full Text] [PDF]
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K.L.T. Schmidt, E. Ernst, A.G. Byskov, A. Nyboe Andersen, and C. Yding Andersen Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation Hum. Reprod., December 1, 2003; 18(12): 2654 - 2659. [Abstract] [Full Text] [PDF]
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