Extended adverse effects of cyclophosphamide on mouse ovarian function

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.


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 shortterm 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.

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.

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 /OD 280 > 2.00) and integrity (RNA integrity number > 7.0). Hybridization on Gene-Chip 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.

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).

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.

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).
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.
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).

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 shortterm 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.  Genes were significantly upregulated in Cy-injected mice when they displayed > 2.0-fold expression compared to in the control (p < 0.05) 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]. Genes were significantly downregulated in Cy-injected mice when they displayed > 2.0-fold differences compared to the control (p < 0.05) 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 oocytespecific 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.