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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cryopreservation/density/ovary/primordial follicles
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 12 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 Bouins 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 Mayers 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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 25 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 98.299.7% 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 000300 000 in girls aged 1015 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.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Block, E. (1952) Quantitative morphological investigations of the follicular system in women. Acta Anat., 14, 108123.[ISI]
Broekmans, F.J., Scheffer, G.J., Bancsi, L.F.J.M.M., Dorland, M., Blankenstein, M.A. and te Velde, E.R. (1998) Ovarian reserve tests in infertility practice and normal fertile women. Maturitas, 30, 205214.[CrossRef][ISI][Medline]
Bukman, A. and Heinemann, M.J. (2001) Ovarian reserve testing and the use of prognostic models in patients with subfertility. Hum. Reprod. Update, 7, 581590.
deBruin, J.P., Dorland, M., Spek, E.R., Posthuma, G., van Haaften, M., Looman, C.W.N. and te Velde, E.R. (2002) Ultrastructure of the resting ovarian follicle pool in healthy young women. Biol. Reprod., 66, 11511160.
Donnez, J. and Bassil, S. (1998) Indications for cryopreservation of ovarian tissue. Hum. Reprod. Update, 4, 248259.
Faddy, M.J. (2000) Follicle dynamics during ovarian ageing. Mol. Cell. Endocrinol., 163, 4348.[CrossRef][ISI][Medline]
Gook, D.A., Edgar, D.H. and Stern, C. (1999) Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1,2-propanediol. Hum. Reprod., 14, 20612068.
Kohl, J., Dittrich, R., Siebzehnrübl, E. and Wildt, L. (2000) Determination of follicle numbers in human ovarian biopsies a method for estimation of outcome of ovarian cryopreservation. Fertil. Steril., 74, Suppl. 1, P-212.
Lass, A. and Brinsden, P. (1999) The role of ovarian volume in reproductive medicine. Hum. Reprod., 5, 256266.[CrossRef]
Lass, A., Silye, R., Abrams, D.-C., Krausz, T., Hovatta, O., Margara, R. and Winston, R.M.L. (1997) Follicular density in ovarian biopsy of infertile women: a novel method to assess ovarian reserve. Hum. Reprod., 12, 10281031.[CrossRef][ISI][Medline]
Meirow, D. and Nugent, D. (2001) The effects of radiotherapy and chemotherapy on female reproduction. Hum. Reprod. Update, 7, 535543.
Meirow, D., Fasouliotis, S.J., Nugent, D., Schenker, J.G., Gosden, R.G. and Rutherford, A.J. (1999) A laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer. Fertil. Steril., 71, 948951.[CrossRef][ISI][Medline]
Newton, H., Aubard, Y., Rutherford, A., Sharma, V. and Gosden, R. (1996) Low temperature storage and grafting of human ovarian tissue. Hum. Reprod., 11, 14871491.
Oktay, K., Economos, K., Kan, M., Rucinski, J., Veeck, L. and Rosenwaks, Z. (2001) Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm. JAMA, 286, 14901493.
Poirot, C., Vacher-Lavenu, M.-C., Helardot, P., Guibert, J., Brugières, L. and Jouannet, P. (2002) Human ovarian tissue cryopreservation: indications and feasibility. Hum. Reprod., 17, 14471452.
Qu, J., Godin, P.A., Nisolle, M. and Donnez, J. (2000) Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation. Hum. Reprod., 15, 302310.
Radford, J.A., Lieberman, B.A., Brison, D.R., Smith, A.R.B., Critchlow, J.D., Russell, S.A., Watson, A.J., Clayton, J.A., Harris, M., Gosden, R.G. et al. (2001) Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkins lymphoma. Lancet, 357, 11721175.[CrossRef][ISI][Medline]
Sladkevicius, P. and Campbell, S. (2000) Advanced ultrasound examination in the management of subfertility. Curr. Opin. Obstet. Gynecol., 12, 221225.[CrossRef][ISI][Medline]
Submitted on July 7, 2002; resubmitted on December 9, 2002; accepted on February 24, 2003.