Hum. Reprod. Advance Access published online on February 8, 2008
Human Reproduction, doi:10.1093/humrep/dem413
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Selection of patients before and after anticancer treatment for ovarian cryopreservation
1 Infertility and IVF Unit, Helen Schneider Hospital for Women, Rabin Medical Center, Beilinson Campus, Petach Tikva 49100 and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Department of Pathology, Rabin Medical Center, Beilinson Campus, Petach Tikva 49100 and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
5 Correspondence address. Tel: +972-3-9377618; Fax: +972-3-9240533; E-mail: ronita{at}clalit.org.il
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Abstract |
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BACKGROUND: Although ovarian cryopreservation in patients with cancer should ideally be performed before the initiation of therapy, cryopreservation from such patients often becomes an option only later. The justification for the procedure needs to be elucidated.
METHODS: Eighteen cancer patients before chemotherapy and 23 others after chemotherapy participated in the study. Freshly dissected ovarian samples were prepared for light microscopy to demonstrate follicular numbers and apoptosis, transmission electron microscopy to enhance intracellular changes, and staining with fluorescent markers (calcein AM, rhodamin 123 and ethidium homodimer) to test for viability.
RESULTS: High numbers of preantral follicles were detected in ovaries of patients 
20 years. No antral follicles were detected. All the follicles were viable and not apoptotic. Deterioration in follicular quality was observed after chemotherapy, manifested mainly as an increase in abnormal granulosa cell nuclei (P < 0.05–0.0001) and in oocyte vacuolization (P < 0.0001).
CONCLUSIONS: Our study stresses the importance of prechemotherapy ovarian cryopreservation. However, the large number of viable, non-apoptotic follicles in ovaries of younger patients (age 20 years) indicates that ovarian cryopreservation might be considered after treatment in this age group. Further studies of ovarian samples from women aged 20–30 years are needed to determine the exact age margin wherein postchemotherapy ovarian cryopreservation can be suggested.
Key words: follicles/chemotherapy/transmission electron microscopy/apoptosis/viability
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Introduction |
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As cancer treatment improves, more young patients of reproductive age survive (Abir et al., 1998
; Meirow, 2000; Meirow and Nugent, 2001). However, many experience severe side effects of the radiation and chemotherapy, including follicular depletion and ovarian failure leading to infertility. The level of ovarian damage varies by patient age and cancer type dependent treatment protocol. Studies have shown that the younger the patient, the lower the likelihood of severe ovarian failure (Meirow and Nugent, 2001). In prepubertal girls, the lesser postchemotherapy induced damage may also be partly attributable to the premenarcheal hormonal status (Marcello et al., 1990; Abir et al., 1998).
Regarding the treatment protocol, the integrity of the ovaries is affected by the type of chemotherapeutic agents, the cumulative dose of chemotherapeutic agents, the treatment duration and the total dosage effect (Abir et al., 1998). In general, alkylating agents such as cyclophosphamide are associated with the highest risk of infertility (Meirow and Nugent, 2001), although ovarian failure has also been reported in patients treated with other agents such as cisplatin and vinca alkaloids (Abir et al., 1998; Meirow and Nugent, 2001). Of the multiagent treatment protocols, the adriamycin (doxorubicin), bleomycin, vinblastine and decarbazine (ABVD) combination is considered less cytotoxic than other protocols (Meirow and Nugent, 2001). Furthermore, the pre-bone marrow transplantation (BMT) conditioning protocol of busulfan and cyclophosphamide has been found to lead to high rates of sterilization, whereas the high-dose melfalan seems to be safer.
The ovarian damage induced by radiation depends on the dose and the irradiated zone in relation to ovary location (Abir et al., 1998). Patients treated with total body irradiation before BMT together with high doses of chemotherapeutic agents are very likely to have severe ovarian dysfunction. However, even scattered radiation, not directed specifically to the pelvis or abdomen, can cause ovarian damage. The combination of high levels of abdominal-pelvic radiation and alkylating agents increases the risk of amenorrhea (Abir et al., 1998; Meirow, 2000; Meirow and Nugent, 2001).
Most human ovarian follicles are quiescent primordial follicles (30–50 µm in diameter) with a single flat granulosa cell (GC) layer (Gougeon, 1996). Chemotherapeutic agents affect dividing cells by damaging the spindle apparatus (Meirow, 2000; Meirow and Nugent, 2001), however, the mechanism by which they affect primordial follicles is unknown (Abir et al., 1998; Raz et al., 2002). Researchers assume that chemotherapeutic agents affect primordial follicles by initiating apoptosis (Tilly, 1996). Postchemotherapy intracellular changes can be identified by transmission electron microscopy (TEM) (Marcello et al., 1990; Familiari et al., 1993), also including apoptotic characteristics, such as deletion of single cells, membrane blebbing, cell shrinkage, phagocytosis and compaction of the chromatin into uniformly dense masses (Tilly, 1996). In addition, apoptosis can be identified by in situ DNA-end labeling assays (TUNEL) (Meirow, 2000; Meirow and Nugent, 2001; Abir et al., 2002), because the DNA is cleaved at regularly spaced nucleosomal units. To date, postchemotherapy apoptotic changes have been reported only in mature murine oocytes exposed in vitro to doxorubicin (Perez et al., 1997), and in GCs of human primordial follicles cultured with cisplatin (Meirow, 2000; Meirow and Nugent, 2001).
Follicular viability can be evaluated with fluorescent dyes (Schotanus et al., 1997; Cortvrindt and Smitz, 2001; Hreinsson et al., 2003; Donnez et al., 2004); namely calcein (yellow–green fluorescent signal dependent on ubiquitous esterase activity in viable cells) in combination with rhodamin (red fluorescent signal in active mitochondria) (Schotanus et al., 1997) or in combination with ethidium homodimer (red fluorescent signal in dead cells, wherein the membranes become permeable) (Schotanus et al., 1997; Cortvrindt and Smitz, 2001; Hreinsson et al., 2003; Donnez et al., 2004).
The options for fertility preservation in young female cancer patients are limited (Abir et al., 1998; Meirow, 2000; Meirow and Nugent, 2001). Cryopreservation of ovarian tissue containing primordial follicles is conducted in many medical centers worldwide. The restoration of ovarian function from cryopreserved–thawed ovarian tissue by replantation (Radford et al., 2001; Oktay et al., 2003, 2004; Donnez et al., 2004; Meirow et al., 2005, 2007a; Demeestere et al., 2006; Rosendahl et al., 2006) has so far led to pregnancy in only four patients (Donnez et al., 2004; Meirow et al., 2005; Demeestere et al., 2006; Rosendahl et al., 2006), with two live births (Donnez et al., 2004, Meirow et al., 2005). The development of in vitro culture of primordial follicles, followed by routine IVF would eliminate the risk of retransmission of some cancers by ovarian grafts (Abir et al., 2006). However, to date the culture protocol has been successful only in murine models (O'Brien et al., 2003), and its application in the near future to primordial follicles from cancer survivors seems impractical.
Ovarian cryopreservation in patients with cancer should preferably be performed before initiation of therapy. However, in many patients, treatment cannot be postponed, and in cases of recurrent malignancies, tissue may not have been cryopreserved after the initial diagnosis. Therefore, the justification to preserve ovarian tissue after cancer treatment remains a major dilemma. The aims of the present study were 3-fold: to evaluate the number and quality of follicles in patients before and after chemotherapy; to correlate these results with age and treatment protocols; and to draw initial conclusions regarding the possible merit of postchemotherapy ovarian cryopreservation.
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Materials and Methods |
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Ovarian material and patients
The study protocol was approved by the local Human Investigations Committee. Ovarian tissue was donated by 18 cancer patients before chemotherapy (Table I) aged 8–39 years (mean ± SD, 23 ± 9 years, P < 0.04 compared with age of patients after chemotherapy-Table II), and by 23 different patients after chemotherapy (Table II) aged 5–39 years (mean ± SD, 17 ± 10). The prechemotherapy group included one premenarcheal girl (age 8; Table I, Patient 1), and the postchemotherapy group included seven premenarcheal girls (aged 5–13 years; Table II, Patients 1–6, 9). Eighteen patients were treated with alkylating agents (Table II, Patients 1, 2, 5–11, 13–15, 18–23): 15 with cyclophosphamide (Table II, Patients 1, 5–7, 9–11, 13–15, 18–23), two with carmustine (Table II, Patients 2, 8) and one with cyclophosphamide as well as carmustine (Table II, Patient 6). The other five were treated with protocols devoid of alkylating agents (Table II, Patients 3, 4, 12, 16, 17). All patients underwent ovarian retrieval for fertility preservation (Abir et al., 1998
), except two (Table II, Patients 21 and 23), in whom prophylactic hysterectomy/oophorectomy was performed after treatment for breast cancer.
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All the pubertal patients (Tables I and II) had regular menstruation cycles of 28–32 days before chemotherapy initiation, with first menses at age 12–15 years. Moreover, in all the seven patients with recurrent malignancies (Tables II, Patients 7, 12–14, 16–18) regular menses were documented until the second anticancer treatment cycles; the only exception (Tables II, Patient 19) had recurrent acute myeloid leukemia (AML) every few years from 5 to 25 years of age, with occasional, irregular menses after puberty among the malignancy episodes. The 8-year-old girl with ovarian cancer (Tables I, Patient 1) in the prechemotherapy group had no genetic history of the disease. One 31-year-old postchemotherapy patient had received treatment during pregnancy that resulted in the birth of a healthy boy (Tables II, Patient 20); 18 months later, she gave birth to a healthy baby girl, conceived spontaneously.
The freshly dissected ovarian samples from each patient (Tables I and II) were split into three pieces: for fluorescence staining (uniform-sized samples measuring 1–2 x 1–2 mm) of either frozen sections or isolated follicules (see Follicular isolation and fluorescence microscopy studies) (Abir et al., 2001) and for light microscopy (LM) fixation (uniform-sized samples measuring 1–2 x 1–2 mm to enable accuracy in the follicular counts); and for TEM fixation (samples measuring 0.5–1 x 0.5–1 mm).
Follicular isolation and fluorescence microscopy studies
In the initial experiments, we utilized frozen sections (5 µm in thickness) after fixation of ovarian slices (1 x 1 mm) in 4% paraformaldehyde or after rapid freezing at –70°C. The sections were incubated at 37°C for 45 min with a solution of alpha minimal essential medium (
MEM) (Biological Industries, Beit Ha'emek, Israel) and 5% human serum albumin (HSA) (Irvine Scientific, Santa Ana, CA, USA) combined with: calcein AM (2 µM) (Molecular Probes, Leiden, The Netherlands) with either rhodamin 123 (10 µM) (Sigma, St. Louis, USA), or ethidium homodimer (5 µM) (Molecular Probes) (Schotanus et al., 1997) and rinsed twice with MEM (Biological Industries) and 5% HSA (Irvine Scientific). These attempts did not yield clear signals but rather diffuse red staining and were regarded as positive controls, including positive cellular death signals (ethidium homodimer).
Thereafter, preantral follicles were immediatly isolated for fluorescent labeling, a method previously found successful (Hreinsson et al., 2003; Dolmans et al., 2006). Freshly dissected ovarian tissue was further cut into slices of 0.5–1 x 0.5–1 mm and incubated at 37°C for 2 h with collagenase IX (5944 U/ml, Sigma) and pancreatic deoxyribonuclease (DNase IV-180 U/ml, Sigma) dissolved in MEM (Biological Industries) with 5% HSA (Irvine Scientific) (Abir et al., 2001). The tissue was then placed in organ culture dishes (Becton Dickinson, NJ, USA) containing MEM (Biological Industries) and 5% HSA (Irvine Scientific). Follicles were dissected under an inverted microscope with 21 gauge needles (Becton Dickinson). Fully isolated follicles were further released from the stroma cells by aspirating the follicles through a fine bore pipette.
The isolated follicles were then transferred using fine bore pipettes to four well plate dishes (Nunclon, Roskilde, Denmark) at room temperature. Transfers between wells were conducted very rapidly. The first well-contained MEM (Biological Industries) and 5% HSA (Irvine Scientific) with either calcein AM (2 µM) (Molecular Probes) with rhodamin 123 (10 µM) (Sigma) or calcein AM (2 µM) with ethidium homodimer (5 µM) (Molecular Probes) (Schotanus et al., 1997), to avoid dilution of the actual fluorescent incubation medium during follicular transfer. The isolated follicles were then transferred to a second well containing the same solution of fluorescent stains, and the plates were incubated at 37°C for 45 min. Thereafter, the isolated follicles were transferred to two additional wells and rinsed twice at room temperature with MEM (Biological Industries) and 5% HSA (Irvine Scientific). The labeled follicles were examined under a fluorescent microscope (Olympus IX50, Olympus Optical Corporation, Tokyo, Japan) equipped with a filter for both green and red signals (wave lengths for calcein = 585 nm; rhodamin = 522 nm and ethidium homodimer = 661 nm).
Histological preparation for LM and follicular counts
Fresh ovarian pieces were fixed in Bouin's solution, dehydrated with increasing concentrations of ethanol (Biolab, Jerusalem, Israel) and toluene (Biolab) and prepared for paraffin embedding (Abir et al., 2001). Sections were cut at 5 µm of thickness and stained with haematoxylin (Pioneer Research Chemicals Ltd, Colchester Essex, UK) and eosin (Sigma).
Human cortical follicles were classified according to Gougeon (1996):
- ‘Unilaminar’ follicles with a single GC layer surrounding the oocyte that include mostly the 'primordial' follicles (see definition in Introduction), but also 'primary' follicles with cuboidal GCs.
- ‘Secondary, multilayered’ follicles with at least two cuboidal GC layers surrounding the oocytes.
- ‘Antral’ follicles, multilayered follicles containing a fluid filled cavity.
- ‘Preantral follicles’ are the group of follicles (primordial, primary and secondary) preceding the 'antral' stage.
The number of preantral and antral follicles per paraffin section of every patient was counted in two different levels of the paraffin blocks (with at least 50 µm between layers to avoid counting the same follicle twice) (Abir et al., 2006). Counting and classification was conducted with a computerized image analyser (analySIS, Soft Imaging System, Digital Solutions for Imaging and Microscopy, System GmbH, Munster, Germany).
Terminal deoxynucleotidyl transferase (TdT)-TUNEL apoptosis assay
Unstained sections were placed on OptiPlus positive charged microscope slides (Pioneer Research Chemicals Ltd) for TdT assay (TUNEL) conducted with an ApopTag In Situ Detection Kit (Intergen Company, Purchase, NY, USA) (Abir et al., 2002). Unless otherwise stated, all chemicals used were from this kit, all dilutions were performed with phosphate buffered saline at pH 7.6 (Biological Industries), which also served as the main rinsing solution, and all the incubations were carried out at the room temperature in a humidified chamber.
The slides were first deparaffinized, rehydrated and rinsed. Sections from rat mammary glands 4 days after lactation termination served as positive controls. Additional positive controls were prepared by pretreating our ovarian samples with DNAse I (final concentration 1 µg/ml, specific activity 10 000–1000 U/ml, Sigma) for 10 min. Thereafter, all slides were treated with proteinase K (Sigma) for 15 min and rinsed in distilled water. To block endogenous peroxidase activity, the samples were quenched with 3% H2O2 (Gadot, Binyamina, Israel) for 5 min in the dark and rinsed.
The slides were then preincubated with an equilibrium buffer for up to 1 h, followed by an incubation with working strength TdT in reaction buffer at 37°C in the same chamber for 1 h. The negative controls were incubated with the same volume of distilled water. Rinsing the sections in 5% stop/wash buffer for 10 min terminated the reaction. The samples were transferred to 70% ethanol (Biolab) and kept at –20°C for no longer than 1 week, to allow the assay to be carried out over 2 days instead of one long day.
When the assay was resumed, samples were rinsed, incubated for half an hour with anti-digoxigenin peroxidase, washed and exposed to a diaminobenzidine urea H2O2 solution in distilled water (Sigma Fast tablets, Sigma) for 5 min. The sections were then rinsed with distilled water and counterstained with 0.5% methyl green free of crystal blue (Sigma) for 10 min. Finally, the slides were rinsed with distilled water and dehydrated with 100% n-butanol (Sigma) and toluene (Biolab). The TUNEL procedure was repeated for at least two sections per patient. Presence of brown apoptotic GCs and oocytes was evaluated.
Histological preparation for TEM
For TEM preparation, all the ovary pieces were cut to a size not larger than 0.5–1 x 0.5–1 mm and were then fixed in 3% glutaraldehyde (Sigma) (Abir et al., 2001; Raz et al., 2002). These samples were prepared for TEM using standard methods with the following modifications: an overnight infiltration at room temperature with an araldite resin (Sigma) solution followed by an additional infiltration with a fresh araldite resin (Sigma) mixture at 60°C for 90 min with periodic stirring. Semi-thin (0.5–0.75 µm) plastic sections for LM were prepared and stained with toluidine blue (BDH Chemicals Ltd, Poole, UK). Whenever possible follicles were identified in two different tissue levels with at least 50 µm between layers to avoid re-sectioning the same follicle. After follicles were identified, ultra-thin (1 Å) sections for TEM were prepared and stained with uranil acetate (BDH) and lead citrate (BDH).
The follicles were examined with a JEOL (JEM 1010) electron microscope equipped with a Gatan digital system (Gatan Inc., Abingdon, UK) for the following parameters: changes in the basement membrane (BM), normality of GC and oocyte nuclei, number of vacuoles in GC and oocytes, normality of intracellular organelles (Abir et al., 2001; Raz et al., 2002). The number of vacuoles has been scored as 0 (lack of vacuoles), 1 (up to 3 vacuoles/follicle), 2 (from 4–14 vacuoles/follicle) or 3 (at least 15 vacuoles/follicle), according to a system established previously by our group (Abir et al., 2001; Raz et al., 2002). In addition, the morphological characteristics of apoptosis were examined (Tilly, 1996; Raz et al., 2002).
Statistical analysis
Data were statistically analysed by a chi-square test, Fisher's exact test, unpaired t-test and analysis of variance as required. A value of P < 0.05 was considered significant.
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Results |
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Antral follicles were not detected in LM sections, TEM sections or fluorescent labeled follicles. A total of 228 preantral follicles (mean ± SD, 14 ± 15/section) were identified by LM in prechemotherapy ovarian sections (Table I) and 1264 preantral follicles (mean ± SD, 57 ± 61/section) in postchemotherapy LM ovarian samples (P < 0.006 for the difference in number before and after therapy) (Table II). Single primordial ovarian follicles per field were identified in two prechemotherapy samples from ovarian cancer patients (8 and 35 years of age, Table I, Patients 1 and 17). In the postchemotherapy group, very few preantral follicles were identified in samples from women >30 years of age (Table II). In addition, all the follicles detected in a sample from a 25-year-old woman after 20 years of recurrent AML were totally damaged (Table II, Patient 19). By treatment protocol, 1102 preantral follicles (mean ± SD, 55 ± 59/section) were identified in the ovaries of the 18 patients treated with alkylating agents (Table II, Patients 1, 2, 5–11, 13–15, 18–23) and 355 preantral follicles (mean ± SD, 71 ± 81/section) in the ovaries of the five patients treated with protocols that did not include alkylating agents (Table II, Patients 3, 4, 12, 16, 17). Ovarian follicles from both study groups (Tables I and II) did not show apoptosis (Fig. 1A), while the positive controls were stained for apoptosis (Fig. 1B).
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Fifty-seven preantral follicles from 14 prechemotherapy patients (Fig. 2A) and 73 from 19 postchemotherapy patients (Figs 2B and C) were examined by TEM. Table III presents the parameters associated with an increase in postchemotherapy cellular deterioration, as monitored by a decrease in normal GC nuclei (P < 0.05–0.0001), concomitant with an increase in oocyte vacuolization (P < 0.0001). Significant changes were noted after treatment with alkylating agents, and also after protocols that did not include alkylating agents. Significant intracellular alterations were noted in preantral follicles from all minors (patients less than 18-year old, premenarcheal and menarcheal), although the follicular damage was greater in the group of patients treated with alkylating agents. The percentage of normal BM was lower in the whole subgroup of patients treated with alkylating agents, as well as in the menarcheal minors treated with alkylating agents (but this was not significant). No morphological characteristics of apoptosis were identified in any of the samples tested.
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Calcein staining was noted in 320 isolated preantral follicles from seven prechemotherapy patients and was noted in 750 isolated preantral follicles from 14 postchemotherapy patients, without positive staining for rhodamin or ethidium homodimer (Fig. 3A). Digested stroma cells attached to the follicles stained with ethidium homodimer (Fig. 3B).
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Discussion |
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In the present study, there was no reduction in the number of postchemotherapy ovarian preantral follicles, although there was a significant decrease in follicular quality as indicated by a reduction in normal GC nuclei and a parallel increase in oocyte vacuolization. Although more cellular damage was identified after treatment with alkylating agents, treatment protocols that did not include alkylating agents also induced significant follicular deterioration. These results suggest that it is not the premenarcheal hormonal status that serves as an ovarian protector, but rather young age per se, when large numbers of ovarian follicles are present (Gougeon, 1996
). The ovarian follicles showed lack of apoptosis, and all were viable. However, none stained positively for active mitochondria. It is noteworthy that if samples from the same patients had been available for comparison before and after chemotherapy, the results would have been more accurate, but this option is of course unrealistic. Moreover, as our ethically approved study protocol did not include the collection of blood samples, we did not measure serum markers of estimated follicular reserve such as anti-Mullerian hormone (Broekmans et al., 2006; Lutchman et al., 2007).
Surprisingly, our study does not support earlier research reporting a decrease in ovarian follicles after chemotherapy (Familiari et al., 1993; Abir et al., 1998; Meirow, 2000; Meirow and Nugent, 2001). This discrepancy may be attributable to the young age of most of our patients (20 years), particularly in the postchemotherapy group. It is possible, however, that a reduction in the number of preantral follicles would have been identified if samples from the same patients had been examined before and after chemotherapy.
Marcello et al. (1990) examined ovaries of 10 girls with acute leukemia treated with various protocols that included prednisolone and vincristine in combination with other drugs, although only three premenarcheal girls were exposed to an alkylating agent (cyclophosphamide). In contrast to our research, intracellular ovarian changes, mostly stromal and capillary, were identified only in the three menarcheal patients, and not in the seven premenarcheal girls. Familiari et al. (1993) conducted a TEM study of ovarian samples from four postchemotherapy patients with Hodgkin lymphoma aged 19–31 years. Two were treated with an exclusive ABVD protocol, one with ABVD combined with methotrexate, vincristine, procarbazide and prednisolone (MOPP) and one with MOPP only. Both previous studies (Marcello et al., 1990, Familiari et al., 1993) did not find a difference in ovarian morphology by treatment protocol. The main cellular changes identified by Familiari et al. (1993) after chemotherapy were an increase in oocyte and GC vacuolization and in BM thickness. Although our results regarding postchemotherapy intracellular damage correlate with previous studies (Marcello et al., 1990; Familiari et al., 1993), there are dissimilarities in specific cellular changes that can be explained by the differences in anticancer protocols, the use of fewer alkylating agents in older protocols (Marcello et al., 1990; Familiari et al., 1993), and by the heterogenic malignancies in our study. The present in vivo findings are in line with our previous report (Raz et al., 2002) of a decrease in normal GC nuclei and BM in human primordial follicles cultured with cyclophosphamide. It is noteworthy also that the number of preantral follicles identified here in the TEM sections was lower than those in the LM sections, mainly because the TEM blocks, and therefore the sections, were smaller (to allow proper fixation and infiltration), and also because different ovarian areas were fixed for LM and for TEM.
Perez et al. (1997) exposed mature murine oocytes to doxorubicin for 24 h. They detected several apoptotic characteristics such as oocyte condensation, cellular fragmentation and genomic DNA segregation into multiple apoptotic bodies. Another group (Meirow, 2000; Meirow and Nugent, 2001) cultured human primordial follicles with cisplatin for up to 36 h, after which a TdT assay identified apoptosis in the GCs, as well as detection of GC nuclei swelling and cytokeratin accumulation. The findings of the present study do not confirm postchemotherapy follicular apoptosis, which was not identified either by a TdT assay or morphologically, by TEM. This discrepancy may be explained by differences between in vivo and in vitro (Perez et al., 1997; Meirow, 2000; Meirow and Nugent, 2001) outcomes; the lack of doxorubicin association with significant follicular damage (Perez et al., 1997; Meirow and Nugent, 2001); the possibility that apoptosis in mature oocytes does not reflect the situation in primordial follicles; and by the inclusion of cisplatin (in combination with the alkylating agent, carmustine) only in one of our treatment protocols (Table II, Patient 8). Moreover, we cannot rule out the possibility that apoptotic follicles disappear within hours of initiation of anticancer treatment. However, this seems very unlikely, given our failure to detect any traces of apoptosis even 4 days after onset of chemotherapy (Table II, Patient 10).
We speculate that follicular destruction after chemotherapy occurs probably in response to the changes in the GCs, as reported here as well as previously (Familiari et al., 1993; Meirow, 2000; Meirow and Nugent, 2001), because follicles cannot remain viable without normal physiological interactions between the oocytes and GCs (Gougeon, 1996; Senbon et al., 2003). Therefore, although a large number of follicles were identified in ovaries of girls 20 years old after therapy, it is possible that a portion of them will eventually deteriorate as a result of poor quality, especially of the GCs. Alternatively, depletion of primordial follicles might be a result of chemotherapy induced vascular shut-down of certain ovarian cortical areas, causing local ischemia (Meirow et al., 2007b). This theory is supported by findings in ovaries of 17 patients (mean age ± SD, 30.5 ± 7.9 years) with heterogenic malignancies exposed to alkylating agents: injuries to blood vessels, formation of immature blood vessels without any pattern of organization and proliferation of collagen fibers.
Calcein staining in human ovarian follicles has been identified before (Cortvrindt and Smitz, 2001) and after (Hreinsson et al., 2003; Donnez et al., 2004) cryopreservation-thawing. In contrast to our current findings, two previous studies (Hreinsson et al. 2003; Dolmans et al., 2006) reported low numbers of either dead GCs or dead follicles (Dolmans et al., 2006) after incubation of isolated human follicles with calcein and ethidium homodimer. Their isolation method (Hreinsson et al. 2003; Dolmans et al., 2006), however, differed from ours (Abir et al., 2001), and included centrifugation, filtering and transfer through pipettes (Hreinsson et al., 2003; Dolmans et al., 2006). Our procedure was more delicate and consisted mainly of manual follicular dissection, followed by aspiration through fine bore pipettes (Abir et al., 2001); explaining the lack of dead follicular cells identified by us in these fragile follicles. It seems highly unlikely that our initial rapid follicular collection at room temperature in a culture medium containing the fluorescent stains (before the actual incubation at 37°C) contributed to their viability.
In contrast to the present study also, Schotanus et al. (1997) noted both calcein and rhodamin staining in cultured bovine secondary follicles. The lack of rhodamin staining in our study was probably a consequence of the low metabolic activity of the primordial follicles (Gougeon, 1996). Alternatively, considering that similarly isolated human follicles developed in culture (Abir et al., 2001), the mitochondria may have been temporally affected by the enzymatic isolating process. This discrepancy between the bovine (Schotanus et al., 1997) and the human might also be attributable to differences in follicular class, species, or in in vitro (Schotanus et al., 1997) compared with in vivo conditions.
The two prechemotherapy patients (8 and 35 years of age, Table I, Patients 1 and 17) with ovarian cancer who were included in our study showed almost total depletion of follicles. As ovarian cancer is rare in young patients (Prat et al., 2005; Vanderhyden, 2005), especially children, there are no studies correlating the malignancy itself with a reduction in follicles. Be that as it may, it is known in mice that premature ovarian failure with germ cell loss may lead to ovarian cancer, and in human menopause ovarian cancer may be induced by loss of germ cells as well as elevated gonadotrophin production.
Only three reports described restoration of ovarian function after transplantation of postchemotherapy cryopreserved–thawed ovarian tissue (Radford et al., 2001; Meirow et al., 2005, 2007a, b; Demeestere et al., 2006). In the first, a 32-year-old woman with Hodgkin lymphoma was initially treated with a chemotherapy protocol that included cyclophosphamide combined with cervical and upper abdominal radiotherapy (Radford et al., 2001). Four years later, she underwent three chemotherapy cycles with chlorambucil, vinblastine, procarbazine, prednisolone, etoposide, vincristine and adriamycin, followed by ovarian cryopreservation. The cryopreserved–thawed ovarian tissue was replanted, inducing only one menstrual period. In contrast, a 25-year-old woman with non-Hodgkin lymphoma was initially also treated with a chemotherapy protocol that included cyclophosphamide (Meirow et al., 2005, 2007a). Six months later, she underwent two courses of mesna, isosfamide and mitoxantrone, and her ovaries were cryopreserved. Replantation of the cryopreserved–thawed ovarian tissue resulted in return of her menstrual periods (that resumed even after giving birth); IVF treatment resulted in a live birth, although her endocrine profile indicated a gradual reduction in follicular reserve after delivery (Meirow et al., 2007a). The third patient was a 24-year-old woman with Hodgkin lymphoma who underwent ovarian cryopreservation after one course of ABVD (Demeestere et al., 2006). Replantation of the cryopreserved–thawed ovarian tissue was followed by six documented menstrual cycles, a spontaneous pregnancy which ended in miscarriage after which there was a progressive decrease in ovarian function.
These limited reports (Radford et al., 2001; Meirow et al., 2005, 2007a; Demeestere et al., 2006) cannot provide clear answers to whether there is merit in postchemotherapy ovarian cryopreservation for fertility preservation. In addition, as the patients in the current study were within a large age range with heterogenic malignancies, our conclusions can only be limited. Be that as it may, as our study and previous ones (Marcello et al., 1990; Familiari et al., 1993) showed that ovarian follicles are of better quality before chemotherapy, it is indeed advisable to cryopreserve ovarian tissue before anticancer treatment. However, given that a pregnancy was obtained in a woman whose ovaries were cryopreserved after treatment with ABVD (Demeestere et al., 2006), we suggest the consideration of postchemotherapy ovarian cryopreservation in patients treated with ‘ovary safe' protocols, especially before BMT. The influence of young age (20 years) on the large numbers of postchemotherapy viable non-apoptotic follicles, the poor results obtained after grafting in the 36-year-old woman described by Radford et al. (2001) and the lower number of follicles in the ovaries of women >30 years of age, especially after chemotherapy, indicates that age is the major factor when considering patients for ovarian cryopreservation, especially postchemotherapy. Although our results suggest that postchemotherapy ovarian cryopreservation may be considered at 20 years of age, there might be a possible benefit in ovarian cryopreservation after chemotherapy in patients 20–30 years old, supported by the live birth achieved after transplantation of postchemotherapy ovarian tissue in a 25-year-old patient (Meirow et al., 2005, 2007a). As our study included limited numbers of women between 20 and 30 years old, further studies in this age group are needed to accurately define the age margin within which a high number of ovarian follicles remain after chemotherapy.
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The study was partially supported by grants from Tel Aviv University (R.O., R.A.) and Israel Cancer Association (B.F., R.A.).
The study was partially presented at the annual meeting of the European Society for Human Reproduction and Embryology (ESHRE), Copenhagen, Denmark, 2005.
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Acknowledgements |
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We are greatly indebted to Lihi Dalal, Ortal Arbiov, Reut Suleiman and Tehila Lavi for their assistance in this project as a requirement for their high school matriculation diploma in medicine (L.H., O.A.) and biotechnology (R.S., T.L.). The authors are also grateful to Gloria Ganzach of the Editorial Board of Rabin Medical Center for the English editing.
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3 Present address: IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center 52621, Tel-Hashomer, Israel
4 Present address: infertility and IVF Unit, Brazilai Medical Center, Ashkelon and Ben Gurion University, School of Medicine, Beer Sheva, Israel
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Abir R, Fisch B, Raz Ah, Nitke S, Ben Rafael Z. Preservation of fertility in women undergoing chemotherapy-current approach and future prospects. J Assist Reprod Genet (1998) 15:469–477.[CrossRef][Web of Science][Medline]
Abir R, Fisch B, Nitke S, Okon E, Raz Ah, Ben-Rafael Z. Morphological study of fully and partially isolated early human follicles. Fertil Steril (2001) 75:141–146.[CrossRef][Web of Science][Medline]
Abir R, Orvieto R, Dicker D, Zukerman Z, Barnett M, Fisch B. Preliminary studies of apoptosis in human fetal ovaries. Fertil Steril (2002) 78:259–264.[CrossRef][Web of Science][Medline]
Abir R, Nitke S, Ben-Haroush A, Fisch B. In vitro maturation of human primordial ovarian follicles—clinical significance, progress and methods for growth evaluation. Histol Histopathol (2006) 21:887–898.[Medline]
Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update (2006) 12:685–718.
Cortvrindt RG, Smitz JE. Fluorescent probes allow rapid and precise recording of follicle density and staging in human ovarian cortical samples. Fertil Steril (2001) 75:588–593.[CrossRef][Web of Science][Medline]
Demeestere I, Simon P, Buxant F, Robin V, Fernandez SA, Centner J, Delbaere A, Englert Y. Ovarian function and spontaneous pregnancy after combined heterotopic and orthotopic cryopreserved ovarian tissue transplantation in a patient previously treated with bone marrow transplantation: a case report. Hum Reprod (2006) 21:2010–2014.
Dolmans MM, Michaux N, Camboni A, Martinez-Madrid B, Van Langendonckt A, Nottola S, Donnez J. Evaluation of liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles. Hum Reprod (2006) 21:413–420.
Donnez J, Dolmans MM, Demylle D, Jadoul P, Picard C, Squifflet J, Martinez-Madrid B, Van Langendonckt A. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet (2004) 364:1405–1410.[CrossRef][Web of Science][Medline]
Familiari G, Caggiati A, Nottola SA, Ermini M, Di Benedeto MR, Motta PM. Ultrastructure of human ovarian primordial follicles after combination chemotherapy for Hodgkin's disease. Hum Reprod (1993) 8:2080–2087.
Gougeon A. Regulations of ovarian follicular development in primates: facts and hypotheses. Endocrine Rev (1996) 17:121–154.
Hreinsson J, Zhang P, Swahn ML, Hultenby K, Hovatta O. Cryopreservation of follicles in human ovarian cortical tissue. Comparison of serum and human serum albumin in the cryoprotectant. Hum Reprod (2003) 18:2420–2428.
Lutchman SK, Singh K, Muttukrishna S, Stein RC, McGarrigle HH, Patel A, Parikh B, Groome NP, Davies MC, Chatterjee R. Predicators of ovarian reserve in young women with breast cancer. Br J Cancer (2007) 18:1808–1816.
Marcello MF, Nuciforo G, Romeo R, Di Dino G, Russo O, Palumbo G, Schiliro G. Structural and ultrastructural study of the ovary in childhood leukemia after successful treatment. Cancer (1990) 66:2099–2104.[CrossRef][Web of Science][Medline]
Meirow D. Reproduction post-chemotherapy in young cancer patients. Mol Cell Endocrinol (2000) 169:123–131.[CrossRef][Web of Science][Medline]
Meirow D, Nugent D. The effects of radiotherapy and chemotherapy on female reproduction. Hum Reprod Update (2001) 7:535–543.
Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, Schiff E, Dor J. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med (2005) 353:318–321.
Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Yemini Z, Dor J. Monitoring the ovaries after autotransplantation of cryopreserved ovarian tissue: endocrine studies, in vitro fertilization cycles, and live birth. Fertil Steril (2007) a87:418.e7–418.e15.
Meirow D, Dor J, Kaufman B, Shrim A, Rabnovici J, Schiff E, Raanani H, Levron J, Fridman E. Cortical fibrosis and blood-vessels damage in human ovaries exposed to chemotherapy. Potential mechanisms of ovarian injury. Hum Reprod (2007) b22:1626–1633.
O'Brien MJ, Pendola JK, Eppig JJ. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their development competence. Biol Reprod (2003) 68:1682–1686.
Oktay K, Buyuk E, Rosenwaks Z, Rucinski J. A technique for transplantation of ovarian cortical strips to the forearm. Fertil Steril (2003) 80:193–198.[Web of Science][Medline]
Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, Opsahl M, Rosenwaks Z. Embryo development after heterotopic transplantation of cryopreserved ovarian tissue. Lancet (2004) 363:837–840.[CrossRef][Web of Science][Medline]
Perez GI, Knufdon CM, Leykin L, Korsmeyer SJ, Tilly JL. Apoptosis-associated signaling pathways are required for chemotherapy-mediated female germ cell destruction. Nature Med (1997) 3:1228–1232.[CrossRef][Web of Science][Medline]
Prat J, Ribe A, Gallardo A. Hereditary ovarian cancer. Hum Pathol (2005) 36:861–870.[CrossRef][Medline]
Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russel SA, Watson AJ, Clayton JA, Harris M, Gosden RG, et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma. Lancet (2001) 357:1172–1175.[CrossRef][Web of Science][Medline]
Raz Ah, Fisch B, Okon E, Feldberg D, Nitke S, Raanani H, Abir R. Possible direct cytoxicity effects of cyclophosphamide on cultured human follicles: an electron microscopy study. J Assist Reprod Genet (2002) 19:498–504.
Rosendahl M, Loft A, Byskov AG, Ziebe S, Schmidt KT, Andersen AN, Ottosen C, Andersen CY. Biochemical pregnancy after fertilization of an oocyte aspirated from a heterotopic autotransplant of cryopreserved ovarian tissue: case report. Hum Reprod (2006) 21:2006–2009.
Schotanus K, Hage WJ, Vanderstiehele H, Van den Hurk. Effects of conditioned medium from murine cell lines on the growth of isolated bovine preantral follicles. Theriogenol (1997) 48:471–483.[CrossRef]
Senbon S, Hirao Y, Miyano T. Interactions between the oocyte and surrounding somatic cells in follicular development: lessons from in vitro culture. J Reprod Develop (2003) 49:259–269.[CrossRef]
Tilly J. Apoptosis and ovarian function. Rev Reprod (1996) 1:162–172.[Abstract]
Vanderhyden BC. Loss of ovarian function and risk of ovarian cancer. Cell Tissue Res (2005) 322:117–124.[CrossRef][Medline]
Submitted on June 16, 2007; resubmitted on November 15, 2007; accepted on November 30, 2007.
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