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Effect of paclitaxel on primordial follicular reserve in mice

2001; Elsevier BV; Volume: 76; Issue: 3 Linguagem: Inglês

10.1016/s0015-0282(01)01959-8

1556-5653

Fatih Güçer, Petek Balkanlı-Kaplan, Latife Doğanay, M. Ali Yüce, Ebru Demıralay, N. Cenk Sayın, Turgut Yardım,

Male Breast Health Studies

Premature ovarian failure and infertility are long-term consequences of chemotherapy in young women. Because primordial follicular cells in the ovary cannot regenerate, destruction of these cells results in ovarian dysfunction and premature ovarian failure. Histologic studies of chemotherapy-induced ovarian damage showed loss of primordial follicles and ovarian volume (1Montz F.J Wolff J.A Gambone J.C Gonadal protection and fecundicity rates in cyclophosphamide-treated rats.Cancer Res. 1991; 51: 2124-2126PubMed Google Scholar). Paclitaxel is an anticancer agent widely used in patients with carcinoma of the ovary, breast, and lung. In addition, promising results have been reported in patients with germ cell tumors and non-Hodgkin lymphomas, which occur frequently in young patients. Paclitaxel damages healthy mature oocytes (2Mailhes J.B Carabatsos M.J Young D London S.N Bell M Albertini D.F Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes.Mutat Res. 1999; 423: 79-90Crossref PubMed Scopus (57) Google Scholar) and affects short-term reproductive potential in a dose-dependent manner (3Kai S Kohmura H Hiraiwa E Koizumi S Ishikawa K Kawano S et al.Reproductive and developmental toxicity studies of paclitaxel. (I) Intravenous administration to rats prior to and in the early stages of pregnancy.J Toxicol Sci. 1994; 19: 57-67Crossref PubMed Google Scholar). However, a search of the MEDLINE database of the U.S. National Library of Medicine (1980 to 2000) found no study that evaluated the potential effect of paclitaxel on the primordial follicle reserve in healthy ovaries. The aim of our study was to evaluate the dose-related effects of paclitaxel on primordial follicle reserve in young mice.Forty inbred Balb/c mature female mice aged 5 to 6 weeks were randomly divided into four groups of 10 mice each. With the use of sterile techniques, we administered paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) intraperitoneally in normal saline as a single injection at doses of 2.5, 5.0, or 7.5 mg/kg in three groups. Ten control mice received only sterile saline in similar volumes.The length of the mouse estrous cycle is approximately 5 days. Therefore, to evaluate the toxic effect of paclitaxel in the subsequent cycle, the ovaries were removed 7 days after paclitaxel administration, fixed in 10% formalin, and embedded in paraffin. Serial sections were prepared in 5-μm slices and stained with hematoxylin and eosin (H&E). Although primordial follicle toxicity can be adequately established by counting five random sections (4Smith B.J Plowchalk D.R Sipes I.G Mattison D.R Comparison of random and serial sections in assessment of ovarian toxicity.Reprod Toxicol. 1991; 5: 379-383Crossref PubMed Scopus (55) Google Scholar), for the purpose of the study, one of the investigators (LD) counted the primordial follicles in every fifth section. Primordial follicles were identified by the presence of oocytes encapsulated partially or completely by a single layer of squamous pregranulosa cells without a theca layer.Institutional review board approval was obtained prior to the study. The guidelines for the care and use of the animals approved by the local institution were followed.The differences in the mean number of primordial follicular counts among the study groups were compared by one-way analysis of variance (ANOVA) using SPSS software (version 9.0, Statistical Package for Social Sciences, Chicago, IL). Regression models were used to analyze the relationship between the dose of paclitaxel and the primordial follicular counts. P values <.05 were considered statistically significant.The mean ± standard deviation (SD) number of primordial follicles in the control group was 353.8 ± 94.8. The number of primordial follicles decreased in all groups that received paclitaxel (Fig. 1). The administration of 7.5 mg/kg of paclitaxel resulted in a 36% reduction in the mean number of primordial follicles. A statistically significant decrease in the mean ± SD number of primordial follicles was found between the control group and the group that had received 7.5 mg/kg of paclitaxel as a single dose (353.8 ± 94.8 vs. 227.7 ± 45.8, respectively; P = .001). The differences in the mean ± SD number of primordial follicles between the control group and the 2.5-mg/kg group (353.8 ± 94.8 vs. 313.3 ± 66.9; P = .554) and between the control group and 5.0-mg/kg group (353.8 ± 94.8 vs. 279 ± 42.5; P = .087) were not statistically significant. Linear regression analysis showed a statistically significant inverse relationship between the increasing doses of paclitaxel and the primordial follicular counts (ANOVA, P <.0001).We found an inverse relationship between increasing doses of paclitaxel and primordial follicular counts in mice. Primordial follicular counts decreased at all administered doses of paclitaxel compared with controls. The difference in the mean number of primordial follicular count between control group and group that received 7.5 mg/kg of paclitaxel was statistically significant. We chose paclitaxel dosages of 2.5, 5.0, and 7.5 mg/kg because significant damaging effects of paclitaxel on mature follicles have been observed at these doses (2Mailhes J.B Carabatsos M.J Young D London S.N Bell M Albertini D.F Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes.Mutat Res. 1999; 423: 79-90Crossref PubMed Scopus (57) Google Scholar).In mature oocytes, paclitaxel induces meiotic maturation delay and spindle defects resulting in aneuploid oocytes (2Mailhes J.B Carabatsos M.J Young D London S.N Bell M Albertini D.F Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes.Mutat Res. 1999; 423: 79-90Crossref PubMed Scopus (57) Google Scholar). In addition, mature follicles are more sensitive to chemotherapy-induced damage. The patient's age and the specific chemotherapeutic agent and its dosage are the major prognostic factors for ovarian function and fertility after chemotherapy (5Sanders J.E Hawley J Levy W Gooley T Buckner C.D Deeg H.J et al.Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation.Blood. 1996; 87: 3045-3052PubMed Google Scholar, 6Koyama H Wada T Nishizawa Y Iwanaga T Aoki Y Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer.Cancer. 1977; 39: 1403-1409Crossref PubMed Scopus (284) Google Scholar). The possibility of permanent ovarian failure after chemotherapy is higher in older women than in younger ones (5Sanders J.E Hawley J Levy W Gooley T Buckner C.D Deeg H.J et al.Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation.Blood. 1996; 87: 3045-3052PubMed Google Scholar, 6Koyama H Wada T Nishizawa Y Iwanaga T Aoki Y Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer.Cancer. 1977; 39: 1403-1409Crossref PubMed Scopus (284) Google Scholar). This clinical observation has been ascribed to the age-related diminished primordial follicular reserve of older patients and further depletion of ovarian follicular reserve by chemotherapy (7Meirow D Lewis H Nugent D Epstein M Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide clinical importance and proposed accurate investigative tool.Hum Reprod. 1999; 14: 1903-1907Crossref PubMed Scopus (212) Google Scholar).However, younger patients with apparently normal ovarian function after chemotherapy are at elevated risk for premature ovarian failure in subsequent years. Strong destruction of ovarian follicular units affects ovarian function immediately and leads to permanent ovarian failure (1Montz F.J Wolff J.A Gambone J.C Gonadal protection and fecundicity rates in cyclophosphamide-treated rats.Cancer Res. 1991; 51: 2124-2126PubMed Google Scholar). In one study in rats, 80% or more depletion of ovarian follicular units by chemotherapy resulted in a sharply decreased fertility rate, whereas fertility was much better in rats treated with lower dosages resulting in less primordial follicular damage (1Montz F.J Wolff J.A Gambone J.C Gonadal protection and fecundicity rates in cyclophosphamide-treated rats.Cancer Res. 1991; 51: 2124-2126PubMed Google Scholar). On the other hand, Meirow et al. (7Meirow D Lewis H Nugent D Epstein M Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide clinical importance and proposed accurate investigative tool.Hum Reprod. 1999; 14: 1903-1907Crossref PubMed Scopus (212) Google Scholar) showed that destruction of half of the primordial follicular reserve by chemotherapy in young mice treated with cyclophosphamide did not affect the short-term fertility rate of the follicles exposed to chemotherapy during the primordial follicle stage.Paclitaxel binds and stabilizes cellular microtubules, leading to cell death (8Wang T.H Wang H.S Soong Y.K Paclitaxel-induced cell death. Where the cell cycle and apoptosis come together.Cancer. 2000; 88: 2619-2628Crossref PubMed Scopus (517) Google Scholar). Also, paclitaxel initiates apoptosis through various mechanisms (8Wang T.H Wang H.S Soong Y.K Paclitaxel-induced cell death. Where the cell cycle and apoptosis come together.Cancer. 2000; 88: 2619-2628Crossref PubMed Scopus (517) Google Scholar). The concentration of paclitaxel is important in the mechanisms of the paclitaxel-induced cell death (8Wang T.H Wang H.S Soong Y.K Paclitaxel-induced cell death. Where the cell cycle and apoptosis come together.Cancer. 2000; 88: 2619-2628Crossref PubMed Scopus (517) Google Scholar). However, the mechanisms leading to chemotherapy-induced destruction of primordial follicles are unknown and require further study.Our results are the first to show that the administration of paclitaxel to young mice causes the depletion of primordial follicular reserve in a dose-dependent manner. Should the observed effect of paclitaxel on the primordial follicular reserve in mice also be valid in humans, then the use of paclitaxel may damage the primordial follicular reserve of young women in a similar fashion. To what extent this effect of paclitaxel leads to premature ovarian failure and infertility in young patients requires further study. Premature ovarian failure and infertility are long-term consequences of chemotherapy in young women. Because primordial follicular cells in the ovary cannot regenerate, destruction of these cells results in ovarian dysfunction and premature ovarian failure. Histologic studies of chemotherapy-induced ovarian damage showed loss of primordial follicles and ovarian volume (1Montz F.J Wolff J.A Gambone J.C Gonadal protection and fecundicity rates in cyclophosphamide-treated rats.Cancer Res. 1991; 51: 2124-2126PubMed Google Scholar). Paclitaxel is an anticancer agent widely used in patients with carcinoma of the ovary, breast, and lung. In addition, promising results have been reported in patients with germ cell tumors and non-Hodgkin lymphomas, which occur frequently in young patients. Paclitaxel damages healthy mature oocytes (2Mailhes J.B Carabatsos M.J Young D London S.N Bell M Albertini D.F Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes.Mutat Res. 1999; 423: 79-90Crossref PubMed Scopus (57) Google Scholar) and affects short-term reproductive potential in a dose-dependent manner (3Kai S Kohmura H Hiraiwa E Koizumi S Ishikawa K Kawano S et al.Reproductive and developmental toxicity studies of paclitaxel. (I) Intravenous administration to rats prior to and in the early stages of pregnancy.J Toxicol Sci. 1994; 19: 57-67Crossref PubMed Google Scholar). However, a search of the MEDLINE database of the U.S. National Library of Medicine (1980 to 2000) found no study that evaluated the potential effect of paclitaxel on the primordial follicle reserve in healthy ovaries. The aim of our study was to evaluate the dose-related effects of paclitaxel on primordial follicle reserve in young mice. Forty inbred Balb/c mature female mice aged 5 to 6 weeks were randomly divided into four groups of 10 mice each. With the use of sterile techniques, we administered paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) intraperitoneally in normal saline as a single injection at doses of 2.5, 5.0, or 7.5 mg/kg in three groups. Ten control mice received only sterile saline in similar volumes. The length of the mouse estrous cycle is approximately 5 days. Therefore, to evaluate the toxic effect of paclitaxel in the subsequent cycle, the ovaries were removed 7 days after paclitaxel administration, fixed in 10% formalin, and embedded in paraffin. Serial sections were prepared in 5-μm slices and stained with hematoxylin and eosin (H&E). Although primordial follicle toxicity can be adequately established by counting five random sections (4Smith B.J Plowchalk D.R Sipes I.G Mattison D.R Comparison of random and serial sections in assessment of ovarian toxicity.Reprod Toxicol. 1991; 5: 379-383Crossref PubMed Scopus (55) Google Scholar), for the purpose of the study, one of the investigators (LD) counted the primordial follicles in every fifth section. Primordial follicles were identified by the presence of oocytes encapsulated partially or completely by a single layer of squamous pregranulosa cells without a theca layer. Institutional review board approval was obtained prior to the study. The guidelines for the care and use of the animals approved by the local institution were followed. The differences in the mean number of primordial follicular counts among the study groups were compared by one-way analysis of variance (ANOVA) using SPSS software (version 9.0, Statistical Package for Social Sciences, Chicago, IL). Regression models were used to analyze the relationship between the dose of paclitaxel and the primordial follicular counts. P values <.05 were considered statistically significant. The mean ± standard deviation (SD) number of primordial follicles in the control group was 353.8 ± 94.8. The number of primordial follicles decreased in all groups that received paclitaxel (Fig. 1). The administration of 7.5 mg/kg of paclitaxel resulted in a 36% reduction in the mean number of primordial follicles. A statistically significant decrease in the mean ± SD number of primordial follicles was found between the control group and the group that had received 7.5 mg/kg of paclitaxel as a single dose (353.8 ± 94.8 vs. 227.7 ± 45.8, respectively; P = .001). The differences in the mean ± SD number of primordial follicles between the control group and the 2.5-mg/kg group (353.8 ± 94.8 vs. 313.3 ± 66.9; P = .554) and between the control group and 5.0-mg/kg group (353.8 ± 94.8 vs. 279 ± 42.5; P = .087) were not statistically significant. Linear regression analysis showed a statistically significant inverse relationship between the increasing doses of paclitaxel and the primordial follicular counts (ANOVA, P <.0001). We found an inverse relationship between increasing doses of paclitaxel and primordial follicular counts in mice. Primordial follicular counts decreased at all administered doses of paclitaxel compared with controls. The difference in the mean number of primordial follicular count between control group and group that received 7.5 mg/kg of paclitaxel was statistically significant. We chose paclitaxel dosages of 2.5, 5.0, and 7.5 mg/kg because significant damaging effects of paclitaxel on mature follicles have been observed at these doses (2Mailhes J.B Carabatsos M.J Young D London S.N Bell M Albertini D.F Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes.Mutat Res. 1999; 423: 79-90Crossref PubMed Scopus (57) Google Scholar). In mature oocytes, paclitaxel induces meiotic maturation delay and spindle defects resulting in aneuploid oocytes (2Mailhes J.B Carabatsos M.J Young D London S.N Bell M Albertini D.F Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes.Mutat Res. 1999; 423: 79-90Crossref PubMed Scopus (57) Google Scholar). In addition, mature follicles are more sensitive to chemotherapy-induced damage. The patient's age and the specific chemotherapeutic agent and its dosage are the major prognostic factors for ovarian function and fertility after chemotherapy (5Sanders J.E Hawley J Levy W Gooley T Buckner C.D Deeg H.J et al.Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation.Blood. 1996; 87: 3045-3052PubMed Google Scholar, 6Koyama H Wada T Nishizawa Y Iwanaga T Aoki Y Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer.Cancer. 1977; 39: 1403-1409Crossref PubMed Scopus (284) Google Scholar). The possibility of permanent ovarian failure after chemotherapy is higher in older women than in younger ones (5Sanders J.E Hawley J Levy W Gooley T Buckner C.D Deeg H.J et al.Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation.Blood. 1996; 87: 3045-3052PubMed Google Scholar, 6Koyama H Wada T Nishizawa Y Iwanaga T Aoki Y Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer.Cancer. 1977; 39: 1403-1409Crossref PubMed Scopus (284) Google Scholar). This clinical observation has been ascribed to the age-related diminished primordial follicular reserve of older patients and further depletion of ovarian follicular reserve by chemotherapy (7Meirow D Lewis H Nugent D Epstein M Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide clinical importance and proposed accurate investigative tool.Hum Reprod. 1999; 14: 1903-1907Crossref PubMed Scopus (212) Google Scholar). However, younger patients with apparently normal ovarian function after chemotherapy are at elevated risk for premature ovarian failure in subsequent years. Strong destruction of ovarian follicular units affects ovarian function immediately and leads to permanent ovarian failure (1Montz F.J Wolff J.A Gambone J.C Gonadal protection and fecundicity rates in cyclophosphamide-treated rats.Cancer Res. 1991; 51: 2124-2126PubMed Google Scholar). In one study in rats, 80% or more depletion of ovarian follicular units by chemotherapy resulted in a sharply decreased fertility rate, whereas fertility was much better in rats treated with lower dosages resulting in less primordial follicular damage (1Montz F.J Wolff J.A Gambone J.C Gonadal protection and fecundicity rates in cyclophosphamide-treated rats.Cancer Res. 1991; 51: 2124-2126PubMed Google Scholar). On the other hand, Meirow et al. (7Meirow D Lewis H Nugent D Epstein M Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide clinical importance and proposed accurate investigative tool.Hum Reprod. 1999; 14: 1903-1907Crossref PubMed Scopus (212) Google Scholar) showed that destruction of half of the primordial follicular reserve by chemotherapy in young mice treated with cyclophosphamide did not affect the short-term fertility rate of the follicles exposed to chemotherapy during the primordial follicle stage. Paclitaxel binds and stabilizes cellular microtubules, leading to cell death (8Wang T.H Wang H.S Soong Y.K Paclitaxel-induced cell death. Where the cell cycle and apoptosis come together.Cancer. 2000; 88: 2619-2628Crossref PubMed Scopus (517) Google Scholar). Also, paclitaxel initiates apoptosis through various mechanisms (8Wang T.H Wang H.S Soong Y.K Paclitaxel-induced cell death. Where the cell cycle and apoptosis come together.Cancer. 2000; 88: 2619-2628Crossref PubMed Scopus (517) Google Scholar). The concentration of paclitaxel is important in the mechanisms of the paclitaxel-induced cell death (8Wang T.H Wang H.S Soong Y.K Paclitaxel-induced cell death. Where the cell cycle and apoptosis come together.Cancer. 2000; 88: 2619-2628Crossref PubMed Scopus (517) Google Scholar). However, the mechanisms leading to chemotherapy-induced destruction of primordial follicles are unknown and require further study. Our results are the first to show that the administration of paclitaxel to young mice causes the depletion of primordial follicular reserve in a dose-dependent manner. Should the observed effect of paclitaxel on the primordial follicular reserve in mice also be valid in humans, then the use of paclitaxel may damage the primordial follicular reserve of young women in a similar fashion. To what extent this effect of paclitaxel leads to premature ovarian failure and infertility in young patients requires further study. The authors thank M. Türe, Ph.D., for assistance with statistical analysis of the data.