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Extended adverse effects of cyclophosphamide on mouse ovarian function

Abstract

Purpose

Most patients with cancer undergo multiple administrations of anticancer drugs during treatment, resulting in chronic impairment of their reproductive health. As improved treatment options increase cancer survival, it has become increasingly important to address fertility issues in cancer survivors. In this study, we examined the pathophysiological effects of multiple exposures to cyclophosphamide (Cy) on the ovaries of mice and their underlying molecular mechanism.

Methods

Female C57BL/6 mice were intraperitoneally injected with 100 mg/kg Cy six times over 2 weeks; 4 weeks later, the mice were sacrificed and their ovaries, sera, and oocytes were collected for histological observation, measurement of anti-Müllerian hormone levels, and assessment of oocyte quantity and quality in response to hormonal stimulation. Gene expression changes in Cy-treated ovaries were examined by microarray and bioinformatics analyses.

Results

After repeated Cy exposure, the anti-Müllerian hormone level was decreased, and follicle loss and impairments in the quality of oocyte were irreversible. The expression levels of genes involved in folliculogenesis, oogenesis, and zona pellucida glycoprotein transcription displayed sustained alterations in Cy-exposed ovaries even after 4 weeks.

Conclusion

The adverse effects of Cy on ovarian function and oocytes remained even after chemotherapy was complete. Therefore, strategies to prevent ovarian damage or restore ovarian function after treatment are required to safeguard the fertility of young cancer survivors.

Peer Review reports

Background

Most patients with ovarian cancer are administered multiple rounds of chemotherapy, the off-target toxicities of which can result in dangerous side effects that must be addressed [1]. Anticancer agents have complex mechanisms of action and their effects depend on their drug type, dose, and therapeutic duration [2]. During cancer treatment, the same drugs are administered every 2–3 weeks for more than four cycles; this repeated exposure can severely affect the quality of life of patients [3]. In female survivors, concerns include early-onset menopause and treatment-related infertility [4].

The need for female survivors to perform family planning is increasing. Care providers recommend that women wait from 6 months to 2 years after finishing chemotherapy before becoming pregnant to avoid adverse effects on the infant [5]. The long-term effects of repeated exposure to anticancer drugs remain unclear; however, most animal studies have used single or short-term exposures to investigate adverse effects on ovarian function and their mechanisms. Reports on the mechanisms of chronic ovarian dysfunction after repeated cancer treatment are lacking.

Cyclophosphamide (Cy) is a widely used alkylating agent that is toxic to both cancer cells and reproductive cells [6, 7]. Cy exposure directly and indirectly leads to apoptosis by inducing DNA damage, suppressing proliferation, and causing mitochondrial dysfunction, resulting in diminished ovarian reserves [8,9,10,11]. A previous study showed that potent regulatory factors can persist or prevent acute ovarian toxicity induced by short-term Cy treatment [12]. Studies are needed to understand the molecular mechanism and changes in gene expression under chronic impaired conditions following rigorous Cy treatment. This may help in the prevention of the extended toxic effects of Cy treatment. To address the chronic effects of repeated Cy treatment, we evaluated ovarian function 4 weeks after the cessation of Cy exposure and investigated the molecular mechanisms underlying chronic ovarian damage.

Methods

Mice

Experimental animal protocols were approved by the Institutional Animal Care and Use Committee at Korea Institute of Oriental Medicine (19–019, Daejeon, Korea). Eight-week-old female C57BL/6 mice (18–20 g) were obtained from Narabiotech (Pyeongtaek, Korea) and housed under specific pathogen-free conditions. Animals were randomly divided into two groups and administered intraperitoneal injections of saline without (n = 12) or with 100 mg/kg Cy (Sigma-Aldrich, St. Louis, USA) six times over 2 weeks (n = 12). The mice were sacrificed 4 weeks after the final Cy injection. This timeframe was selected because it provides sufficient time for newly recruited primordial follicles to complete the preantral period [13]. Blood was collected from the inferior vena cava of mice anesthetized with 1.2% avertin (0.6 mL/mouse, Sigma-Aldrich). Each mouse was euthanized by cervical dislocation to collect ovaries and oocytes after blood collection. The ovaries were removed, weighed, and immediately fixed in 4% paraformaldehyde (Biosesang, Seongnam, Korea).

Hormonal assessment by enzyme-linked immunosorbent assay (ELISA)

Sera separated from the blood samples were frozen at − 70 °C until analysis. The concentration of anti-Müllerian hormone (AMH) was measured by ELISA (MyBiosource, San Diego, CA, USA) in triplicate according to a standard protocol and the manufacturers’ instructions. The inter-assay coefficient of variation was < 10% and sensitivity was 0.19 ng/mL.

Histological assessment of ovarian follicles

The whole ovaries were serially sectioned to 5-μm thickness and stained with hematoxylin and eosin. Primordial, primary, secondary, and preovulatory follicles with visible oocytes were counted in every fifth stained section to avoid counting the same follicle twice. The follicle stage was classified as previously described [14, 15]: primordial follicles had a single flat layer of granulosa cells surrounding the oocyte, primary follicles had a single cuboidal granulosa cell layer, secondary follicles had at least two granulosa cell layers and a theca cell layer, and preovulatory follicles had a complete antrum and theca cell layer.

Assessment of oocyte quality

Cyclophosphamide- or saline-injected mice were superovulated via intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (Prospec, Rehovot, Israel), followed by 5 IU human chorionic gonadotropin (hCG, Prospec) at 48 h later. Oocytes were collected 18 h post-hCG injection in preincubated Human Tubal Fluid medium (Irvine scientific, CA, USA). Oocytes were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) for 10 min. Oocytes were blocked in phosphate-buffered saline containing 3% bovine serum albumin (Genedepot, Katy, TX, USA), and then incubated with rabbit anti-α-tubulin antibody (1:200, Cell Signaling Technologies, Danvers, MA, USA). Oocytes were mounted with VECTASHIELD Antifade Mounting Medium with 4′6’-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) to visualize the chromosomes, and observed by fluorescence microscopy (Olympus BX51, Tokyo, Japan). Oocytes with well-organized, bipolar spindles and chromosomes that were tightly aligned at the metaphase plate were scored as normal. Oocyte quality was also evaluated by measuring morphometrical parameters, including the complete oocyte, ooplasm, and perivitelline space (PVS) using NIS-elements BR 4.60.00 software (Nikon, Tokyo, Japan) [16].

Microarray analysis

Ovaries from Cy- or saline-injected mice were collected, and total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purity and integrity of the extracted RNA were evaluated using a NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All samples were of high purity (optical density (OD)260/OD280 > 2.00) and integrity (RNA integrity number > 7.0). Hybridization on GeneChip Mouse Gene 2.0 ST arrays (Affymetrix) was controlled using GeneChip Command Console Software (AGCC, Affymetrix, Santa Clara, CA, USA). We used Affymetrix Expression Console 1.4 Software for basic data extraction (CEL files) and quality control metrics. A fold change value > 2.0 and a p-value < 0.05 were used as thresholds to identify differentially expressed genes (DEGs). Functional annotation of the DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery version 6.8 (https://david.ncifcrf.gov/hom.jsp). Gene ontology (GO) analysis was performed to identify potential functions of DEGs in the biological process, molecular function, and cellular component categories [17].

Statistical analyses

Data are presented as means ± standard deviation (SD). The statistical significance of differences between the two groups was determined by Student’s t-test using GraphPad Prism, version 8.4.0 (GraphPad, Inc., La Jolla, CA, USA). P < 0.05 was considered as statistically significant.

Results

Impaired physiological conditions endure after cy exposure

Mice were monitored throughout the study and sacrificed to collect ovaries and blood 4 weeks after Cy treatment. The treated mice had significantly lower body weights than control mice (Fig. 1a). A significant loss of body weight has been associated with negative therapeutic responses [18]. We also measured AMH levels in the serum of Cy- and saline-injected mice (Fig. 1b). The AMH level was significantly decreased in Cy-injected mice, suggesting a decline in the number of growing follicles (p < 0.01).

Fig. 1
figure1

Body weights and serum AMH levels 4 weeks after Cy exposure Mice were injected with Cy (n = 5) or saline (n = 5) for 2 weeks, and then housed for 4 weeks before the mice and ovaries were weighed, and serum AMH levels were measured. a Mouse body weights in individual control and Cy-injected mice. b Serum AMH levels in each group. Data represent the mean ± standard deviation. Statistical analysis was performed by Student’s t-test

Follicle loss after cessation of cy exposure

To investigate the side effects of rigorous Cy exposure on the ovaries, we performed histological analysis of isolated ovaries 4 weeks after Cy treatment. Entire follicles were damaged, and the number of follicles at all stages was significantly decreased (Fig. 2a, p < 0.05). The proportions of primordial and preovulatory follicles were significantly decreased and increased, respectively (Fig. 2b, p < 0.01). The decrease in primordial follicles may have been due to a combination of damage and growth activation. Although surviving granulosa cells in the growing follicles secreted AMH, its level was low, and only a small number of growing follicles survived and were activated within one menstrual cycle.

Fig. 2
figure2

Histological observation of ovarian follicles 4 weeks after Cy exposure Mice were injected with Cy (n = 5) or saline (n = 5) for 2 weeks, and then housed for 4 weeks before histological observation of the ovaries. a Number of ovarian follicles in control and Cy-injected mice. b Proportions of primordial, primary, and secondary and preovulatory follicles in each group. Data represent the mean ± standard deviation. Statistical analysis was performed by Student’s t-test

Cy-induced impairment of oocyte quantity and quality is irreversible

To investigate the effects of rigorous Cy exposure on oocytes, mice were hormonally superovulated. Oocytes were collected from the oviducts 18 h post-hCG, and their quantity and quality were assessed.

As expected, the total number of retrieved oocytes and number that matured to metaphase II (MII) were both significantly decreased (Fig. 3a and b, p < 0.05). The MII oocytes of Cy-injected mice displayed increased chromosomal abnormalities and spindle misalignments compared to control oocytes (Fig. 3c, p < 0.001).

Fig. 3
figure3

Quality of retrieved oocytes 4 weeks after Cy exposure Mice were injected with Cy (n = 4) or saline (n = 4) for 2 weeks, then housed for 4 weeks. The mice were superovulated and (a) total and (b) MII retrieved oocytes were quantified. The oocytes were immunostained for α-tubulin to assess spindle and chromosome alignment (c). Data represent the mean ± standard deviation. Statistical analysis was performed by Student’s t-test

Analysis of the morphology of Cy-treated oocytes revealed that the surrounding zona pellucida (ZP) was loosely compacted and less uniformly shaped than that in control oocytes (Fig. 4a). The area of the ooplasm was decreased in Cy-treated and control oocytes with a significantly increased PVS (Fig. 4b, p < 0.0001). Abnormal PVS morphology is a negative indicator of an oocyte’s developmental potential and has been linked to lower fertilization rates [19, 20]. These results indicate that even after chemotherapy ends, residual Cy metabolites or surviving follicles with damaged granulosa cells can impair oocyte viability and quality in response to superovulation.

Fig. 4
figure4

Retrieved oocytes 4 weeks after Cy exposure Mice were injected with Cy (n = 4) or saline (n = 4) for 2 weeks, and then housed for 4 weeks. a Mice were superovulated and the retrieved oocytes were observed. b Oocyte, ooplasm, and PVS were compared between the control and Cy-treated groups. Data represent the mean ± standard deviation. Statistical analysis was performed by Student’s t-test

Gene expression is continuously altered in response to repeated cy exposure

We next performed microarray experiments to analyze and compare the RNA expression patterns in ovaries from Cy- or saline-injected mice. Hierarchical clustering analysis revealed marked differences among the two mouse groups (Fig. 5a). Of the 41,345 genes detected, 46 were significantly different between the two groups (Fig. 5b). Of these, seven genes (15.2%) were upregulated and 39 genes (84.8%) were downregulated in Cy-injected mice compared with control mice (Tables 1 and 2). Interestingly, the expression profiles of genes associated with fertilization and ovarian follicle development, such as ZP glycoprotein 2 and 3 (Zp2 and Zp3); solute carrier family 18, member 2 (Slc18a2); WEE1 homolog 2 (Wee2); NLR family, pyrin domain containing 5 (Nlrp5) and 2′-5′-oligoadenylate synthetase 1d and 1e (Oas1d and Oas1e), were changed. GO analysis revealed that in molecular functions, the DEGs were enriched in protein binding, acrosin binding, and 2′5′-oligoadenylate synthetase activity (Table 3). In biological process, enriched GO terms included ovarian follicle development, oogenesis, binding of sperm to ZP, and immune response (Table 3). For cellular components, GO analysis revealed that the DEGs were enriched in GO terms such as cytoplasm, secretory granule, extracellular region, and matrix (Table 3).

Fig. 5
figure5

Hierarchical clustering and analysis of DEGs Ovaries from mice injected with Cy (n = 3) or saline (n = 3) were used for transcriptomic analysis. Microarrays were performed to compare ovarian gene expression 4 weeks after Cy exposure to that in control mice. a Hierarchical clustering among microRNA expression profiles. b Significantly upregulated and downregulated genes are shown as green and red dots, respectively

Table 1 Upregulated genes in Cy-exposed mice
Table 2 Downregulated genes in Cy-exposed mice
Table 3 Functional annotation of differentially expressed genes

Discussion

Currently, young women diagnosed with cancer have a greater chance of long-term survival than ever before. However, successful survivorship includes maintaining a high quality of life after cancer diagnosis and treatment [21], and lifesaving treatments such as chemotherapy, radiation, and surgery can impact survivors by impairing their reproductive and endocrine health. Patients exposed to several months of rigorous chemotherapy can suffer from infertility or premature ovarian failure. In addition, patients who retain fertility after cancer therapy have increased risk factors for fetal and maternal complications during subsequent pregnancies [22]. As fertility issues in cancer survivorship have become increasingly important, additional studies are needed to evaluate these effects. Most studies examining cancer therapy-related infertility have used mouse models induced by single or short-term exposure to anticancer agents, which is typically not performed in the clinic [3]. To consider the pathology of chronic ovarian impairment in young cancer survivors, we repeatedly administered Cy and analyzed physiological conditions at 4 weeks after treatment completion.

Cy exposure leads to deterioration of oocyte quality [23, 24]. Koike et al. reported a decrease in the number of retrieved oocytes, whereas the rates of fertilization and blastocyst development were similar compared to those in controls at 2 weeks after single 400 mg/kg Cy administration in mice [25]. However, the mouse model following single exposure of Cy did not reflect the clinical situation regarding the extent of damage to the follicle and oocyte quality in oocytes and embryos after rigorous Cy treatment.

Interestingly, both follicles and oocytes were susceptible to Cy-induced damage even after the 4 weeks had passed, allowing sufficient time for the generation of new preovulatory follicles [13]. Only a small number of oocytes were retrieved, which had phenotypic indications of low fertilization potential. This leads to poor reproductive outcomes such as a high risk of non-viable fetuses and malformation at 4 weeks after Cy exposure [26].

There is limited information regarding the effects of Cy in the ovaries. Cy is thought to act as a direct ovotoxin that destroys dormant primordial follicles and activates quiescent primordial follicles by inducing apoptosis in pregranulosa cells and oocytes [27]. Cy exposure also generates increased reactive oxygen species in oocytes, resulting in mitochondrial dysfunction and disrupting the meiotic spindle [23, 28].

To examine whether the adverse effects of rigorous chemotherapy persisted after cancer treatment ends, we performed microarray and bioinformatic analyses on ovaries 4 weeks after Cy exposure. In the microarray data, seven genes were found to be upregulated. Of them, Zbtb1 is a member of the Krüeppel C2H2-type zinc-finger protein family and regulated by the PI3K/PTEN/AKT pathway, which has a critical role in regulating dormancy and initial primordial follicle activation [29]. However, exposure to Cy disturbs this balance by destroying growing follicles or activating the PI3K/PTEN/Akt pathway, causing reservoir “burnout” [30, 31].

A total of 39 downregulated DEGs associated with folliculogenesis and oogenesis are involved in the prolonged effects of repeated Cy treatment. WEE2, one of the oocyte-specific kinase, is responsible for the follicular development, oocyte meiotic regulation, and fertilization in humans and mice [32, 33]. Reduced WEE2 levels induce fertilization failure and abnormal blastocyst formation [34]. Slc18a2 is highly expressed in granulosa cells of growing follicles and its downregulation indicates that granulosa cells were damaged by Cy exposure [35]. Additionally, expression of OAS1D, which regulates the translational regulator of newborn ovary homeobox gene (Nobox), was decreased, leading to rapid follicle loss after birth [36]. Taken together, altered gene expression continuously impaired ovarian follicle development even after Cy exposure was complete.

The DEGs also included oocyte-specific genes associated with fertilization, including Zp1, 2, and 3, which are critical for proper organization of the ZP surrounding oocytes and showed decreased expression 4 weeks after Cy exposure. Consistent with the microarray data, morphological observation revealed a loosely compacted ZP and enlarged PVS, which can affect fertility [37].

The microarray data indicate that genetic regulation in ovaries remains impaired 4 weeks after repeated Cy exposure. We found that impaired follicular growth correlated with oocyte abnormalities caused by rigorous Cy treatment. However, in our study, we did not determine whether these abnormal oocytes are directly caused by Cy and/or indirectly through other cells such as granulosa cells, and assessment of the fertilization potential of Cy-damaged oocytes requires further studies. In recent decades, several studies have been conducted to develop strategies for protecting fertility from the effects of chemotherapeutic drugs, but they have been performed during chemotherapy in animal models [38]. Our results suggest that, for young cancer survivors, persistent treatment is required to prevent chronic damage to the ovaries after chemotherapy ends.

Conclusion

We found that ovarian cell damage induced by repeated Cy treatment continuously alters the expression of genes associated with fertility and has persistent effects on ovarian function, resulting in diminished ovarian reserves even after the completion of chemotherapy. Studies to prevent chronic damage to the ovaries and/or restore their function are required to ensure fertility preservation in cancer survivors.

Availability of data and materials

All data generated and analyzed during this study are included in this article. The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

AMH:

Anti-Müllerian hormone

Cy:

Cyclophosphamide

DEGs:

Differentially expressed genes

GO:

Gene ontology

hCG:

Human chorionic gonadotropin

KEGG:

Kyoto Encyclopedia of Genes and Genomes

Nobox :

Newborn ovary homeobox gene

Nlrp5 :

NLR family, pyrin domain containing 5

Oas1d :

2′-5′-oligoadenylate synthetase 1d

Oas1e :

2′-5′-oligoadenylate synthetase 1e

PVS:

Perivitelline space

PMSG:

Pregnant mare’s serum gonadotropin

Slc18a2 :

Solute carrier family 18, member 2

Wee2 :

WEE1 homolog 2

References

  1. 1.

    Lin A, Giuliano CJ, Palladino A, John KM, Abramowicz C, Yuan ML, Sausville EL, Lukow DA, Liu L, Chait AR, Galluzzo ZC, Tucker C, Sheltzer JM. Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. Sci Transl Med. 2019;11(509):eaaw8412. https://doi.org/10.1126/scitranslmed.aaw8412.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Wang Y, Qi X, Li D, Zhu T, Mo X, Li J. Anticancer efficacy and absorption, distribution, metabolism, and toxicity studies of aspergiolide a in early drug development. Drug Des Devel Ther. 2014;8:1965–77. https://doi.org/10.2147/dddt.S64989.

    Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Hulvat MC, Jeruss JS. Maintaining fertility in young women with breast cancer. Curr Treat Options in Oncol. 2009;10(5–6):308–17. https://doi.org/10.1007/s11864-010-0116-2.

    Article  Google Scholar 

  4. 4.

    Letourneau JM, Ebbel EE, Katz PP, Oktay KH, McCulloch CE, Ai WZ, Chien AJ, Melisko ME, Cedars MI, Rosen MP. Acute ovarian failure underestimates age-specific reproductive impairment for young women undergoing chemotherapy for cancer. Cancer. 2012;118(7):1933–9. https://doi.org/10.1002/cncr.26403.

    Article  PubMed  Google Scholar 

  5. 5.

    Lawrenz B, Banys M, Henes M, Neunhoeffer E, Grischke EM, Fehm T. Pregnancy after breast cancer: case report and review of the literature. Arch Gynecol Obstet. 2011;283(4):837–43.

    PubMed  Google Scholar 

  6. 6.

    Handolias D, Quinn M, Foo S, Mileshkin L, Grant P, Dutu G, Rischin D. Oral cyclophosphamide in recurrent ovarian cancer. Asia Pac J Clin Oncol. 2016;12(1):e154–60. https://doi.org/10.1111/ajco.12074.

    Article  PubMed  Google Scholar 

  7. 7.

    Madden JA, Hoyer PB, Devine PJ, Keating AF. Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity. Toxicol Appl Pharmacol. 2014;277(1):1–7. https://doi.org/10.1016/j.taap.2014.03.006.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Ludovini V, Antognelli C, Rulli A, Foglietta J, Pistola L, Eliana R, Floriani I, Nocentini G, Tofanetti FR, Piattoni S, Minenza E, Talesa VN, Sidoni A, Tonato M, Crino L, Gori S. Influence of chemotherapeutic drug-related gene polymorphisms on toxicity and survival of early breast cancer patients receiving adjuvant chemotherapy. BMC Cancer. 2017;17(1):502. https://doi.org/10.1186/s12885-017-3483-2.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Madden JA, Keating AF. Ovarian xenobiotic biotransformation enzymes are altered during phosphoramide mustard-induced ovotoxicity. Toxicol Sci. 2014;141(2):441–52. https://doi.org/10.1093/toxsci/kfu146.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Himelstein-Braw R, Peters H, Faber M. Morphological study of the ovaries of leukaemic children. Br J Cancer. 1978;38(1):82–7. https://doi.org/10.1038/bjc.1978.166.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Shah MA, Schwartz GK. Cell cycle-mediated drug resistance: an emerging concept in cancer therapy. Clin Cancer Res. 2001;7(8):2168–81.

    CAS  PubMed  Google Scholar 

  12. 12.

    Kalich-Philosoph L, Roness H, Carmely A, Fishel-Bartal M, Ligumsky H, Paglin S, Wolf I, Kanety H, Sredni B, Meirow D. Cyclophosphamide triggers follicle activation and "burnout"; AS101 prevents follicle loss and preserves fertility. Sci Transl Med. 2013;5(185):185ra62. https://doi.org/10.1126/scitranslmed.3005402.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    McGee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocr Rev. 2000;21(2):200–14. https://doi.org/10.1210/edrv.21.2.0394.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Pedersen T, Peters H. Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil. 1968;17(3):555–7.

    CAS  PubMed  Google Scholar 

  15. 15.

    Myers M, Britt KL, Wreford NG, Ebling FJ, Kerr JB. Methods for quantifying follicular numbers within the mouse ovary. Reproduction. 2004;127(5):569–80. https://doi.org/10.1530/rep.1.00095.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Balaban B, Urman B. Effect of oocyte morphology on embryo development and implantation. Reprod BioMed Online. 2006;12(5):608–15. https://doi.org/10.1016/s1472-6483(10)61187-x.

    Article  PubMed  Google Scholar 

  17. 17.

    Ren YM, Duan YH, Sun YB, Yang T, Tian MQ. Bioinformatics analysis of differentially expressed genes in rotator cuff tear patients using microarray data. J Orthop Surg Res. 2018;13(1):284. https://doi.org/10.1186/s13018-018-0989-5.

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Gadducci A, Cosio S, Fanucchi A, Genazzani AR. Malnutrition and cachexia in ovarian cancer patients: pathophysiology and management. Anticancer Res. 2001;21(4b):2941–7.

    CAS  PubMed  Google Scholar 

  19. 19.

    Figueira R, de Almeida Ferreira Braga DP, Semião-Francisco L, Madaschi C, Iaconelli A Jr, Borges E Jr. Metaphase II human oocyte morphology: contributing factors and effects on fertilization potential and embryo developmental ability in ICSI cycles. Fertil Steril. 2010;94(3):1115–7. https://doi.org/10.1016/j.fertnstert.2009.11.039.

    Article  Google Scholar 

  20. 20.

    Setti AS, Figueira RC, Braga DP, Colturato SS, Iaconelli A Jr, Borges E Jr. Relationship between oocyte abnormal morphology and intracytoplasmic sperm injection outcomes: a meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2011;159(2):364–70. https://doi.org/10.1016/j.ejogrb.2011.07.031.

    Article  PubMed  Google Scholar 

  21. 21.

    Waimey KE, Smith BM, Confino R, Jeruss JS, Pavone ME. Understanding fertility in young female cancer patients. J Women's Health (Larchmt). 2015;24(10):812–8. https://doi.org/10.1089/jwh.2015.5194.

    Article  Google Scholar 

  22. 22.

    Kort JD, Eisenberg ML, Millheiser LS, Westphal LM. Fertility issues in cancer survivorship. CA Cancer J Clin. 2014;64(2):118–34. https://doi.org/10.3322/caac.21205.

    Article  PubMed  Google Scholar 

  23. 23.

    Jeelani R, Khan SN, Shaeib F, Kohan-Ghadr HR, Aldhaheri SR, Najafi T, Thakur M, Morris R, Abu-Soud HM. Cyclophosphamide and acrolein induced oxidative stress leading to deterioration of metaphase II mouse oocyte quality. Free Radic Biol Med. 2017;110:11–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Nguyen QNZN, Liew SH, Findlay JK, Hickey M, Hutt KJ. Cisplatin- and cyclophosphamide-induced primordial follicle depletion is caused by direct damage to oocytes. Mol Hum Reprod. 2019;25(8):433–44. https://doi.org/10.1093/molehr/gaz020.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Koike MKA, Kido K, Goto K, Kumasako Y, Nagaki M, Otsu E, Araki Y, Araki Y, Kawabe F, Kai Y, Utsunomiya T. Effects of cyclophosphamide administration on the in vitro fertilization of mice. Reprod Med Biol. 2018;17(3):262–7. https://doi.org/10.1002/rmb2.12099.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Meirow DEM, Lewis H, Nugent D, Gosden RG. Administration of cyclophosphamide at different stages of follicular maturation in mice: effects on reproductive performance and fetal malformations. Hum Reprod. 2001;16(4):632–7. https://doi.org/10.1093/humrep/16.4.632.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Kim SY, Cho GJ, Davis JS. Consequences of chemotherapeutic agents on primordial follicles and future clinical applications. Obstet Gynecol Sci. 2019;62(6):382–90. https://doi.org/10.5468/ogs.2019.62.6.382.

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Zhang M, ShiYang X, Zhang Y, Miao Y, Chen Y, Cui Z, Xiong B. Coenzyme Q10 ameliorates the quality of postovulatory aged oocytes by suppressing DNA damage and apoptosis. Free Radic Biol Med. 2019;143:84–94. https://doi.org/10.1016/j.freeradbiomed.2019.08.002.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Zhou WSH, Zhang D, Dong J, Cheng W, Wang L, Teng Y, Yu Z. PTEN signaling is required for the maintenance of spermatogonial stem cells in mouse, by regulating the expressions of PLZF and UTF1. Cell Biosci. 2015;5:42. https://doi.org/10.1186/s13578-015-0034-x.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Roness HGZ, Cohen Y, Meirow D. Ovarian follicle burnout: a universal phenomenon? Cell Cycle. 2013;12(20):3245–6. https://doi.org/10.4161/cc.26358.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Luan YEM, Woodruff TK, Kim SY. Inhibitors of apoptosis protect the ovarian reserve from cyclophosphamide. J Endocrinol. 2019;240(2):243–56. https://doi.org/10.1530/joe-18-0370.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Bonnet A, Servin B, Mulsant P, Mandon-Pepin B. Spatio-temporal gene expression profiling during in vivo early ovarian folliculogenesis: integrated transcriptomic study and molecular signature of early follicular growth. PLoS One. 2015;10(11):e0141482. https://doi.org/10.1371/journal.pone.0141482.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Bonnet A, Cabau C, Bouchez O, Sarry J, Marsaud N, Foissac S, Woloszyn F, Mulsant P, Mandon-Pepin B. An overview of gene expression dynamics during early ovarian folliculogenesis: specificity of follicular compartments and bi-directional dialog. BMC Genomics. 2013;14:904. https://doi.org/10.1186/1471-2164-14-904.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Sang Q, Li B, Kuang Y, Wang X, Zhang Z, Chen B, Wu L, Lyu Q, Fu Y, Yan Z, Mao X, Xu Y, Mu J, Li Q, Jin L, He L, Wang L. Homozygous mutations in WEE2 cause fertilization failure and female infertility. Am J Hum Genet. 2018;102(4):649–57. https://doi.org/10.1016/j.ajhg.2018.02.015.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Gallardo TD, John GB, Shirley L, Contreras CM, Akbay EA, Haynie JM, Ward SE, Shidler MJ, Castrillon DH. Genomewide discovery and classification of candidate ovarian fertility genes in the mouse. Genetics. 2007;177(1):179–94. https://doi.org/10.1534/genetics.107.074823.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Rajkovic A, Pangas SA, Ballow D, Suzumori N, Matzuk MM. NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression. Science. 2004;305(5687):1157–9. https://doi.org/10.1126/science.1099755.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Gosden R, Lee B. Portrait of an oocyte: our obscure origin. J Clin Invest. 2010;120(4):973–83. https://doi.org/10.1172/jci41294.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Spears N, Lopes F, Stefansdottir A, Rossi V, De Felici M, Anderson RA, Klinger FG. Ovarian damage from chemotherapy and current approaches to its protection. Hum Reprod Update. 2019;25(6):673–93.

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

Not applicable.

Funding

This study was financially supported by a project grant from the Korea Institute of Oriental Medicine (KSN2013240). The study funder had no further role in the study design, data collection, analyses, interpretation of results, writing of the article, or the decision to submit it for publication.

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All authors have read and approved the manuscript. JK conceived the study, performed experiments, and wrote and revised the manuscript. SY performed experiments, revised the manuscript, and is responsible for correspondence.

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Correspondence to Sooseong You.

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All the experiments and analyses were conducted in accordance with the relevant guidelines and regulations. Experimental animal protocols were approved by the Institutional Animal Care and Use Committee at Korea Institute of Oriental Medicine (19–019, Daejeon, Korea). No permission was necessary to collect the specimens in our study.

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I understand that the text and any pictures published in the article will be freely available on the internet and may be seen by the general public. I have been offered the opportunity to read the manuscript.

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Kim, J., You, S. Extended adverse effects of cyclophosphamide on mouse ovarian function. BMC Pharmacol Toxicol 22, 3 (2021). https://doi.org/10.1186/s40360-020-00468-5

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Keywords

  • Cyclophosphamide
  • Chronic side effect
  • Ovarian dysfunction
  • Oocyte
  • Bioinformatics analysis
  • Mice