Evidence of cancer therapy-induced chronic inflammation in the ovary across multiple species: A potential cause of persistent tissue damage and follicle depletion.

We first evaluated ovarian function and immunopathology evidence in human specimens. Although patient ages were comparable between the control and treatment group, serum AMH concentrations were lower (P 1 year than those of the control patients ( Fig. 1A ). In contrast to AMH, serum MCP1 concentrations increased (P < 0.05) in patients of the treatment group compared with those of the control patients ( Fig. 1B ). IL6 was measurable in serum samples from all patients in the treatment group, whereas it was not detectable (<3.1 pg/ml) in any of the control samples ( Fig. 1B ). The ovarian cortex of the control patients had normal histology with compact stroma and limited blood vessels ( Fig. 1C ). Primordial, primary and secondary follicles resided in the stroma with well-assembled extracellular matrix ( Fig. 1D ; Fig. 2A , 2C and 2E insets). In patients of the treatment group, the ovarian cortex appeared to be loosely organized with a considerable amount of blood vessels, even in area close to the ovarian surface ( Fig. 1E ). Follicles were rarely observed, and the stroma extracellular matrix was disorganized and appeared fibrotic containing a number of hyalinized blood vessels with thick walls ( Fig. 1F ). There were sparse CD68/CD163+, CD4+ ( Fig. 2A ), CD3+ ( Fig. 2C ), or MPO+ ( Fig. 2E ) cells within the ovarian cortex of the control patients. In contrast, clusters of CD68/CD163+ macrophages were evident in the ovarian cortex of patients in the treatment group ( Fig. 2B ). Similar staining patterns were also observed for CD4+ lymphocytes ( Fig. 2B ), CD3+ T lymphocytes ( Fig. 2D ), and MPO+ neutrophils ( Fig. 2F ). Given that ovarian physiology are similar between humans and nonhuman primates, rhesus macaques were utilized for ovarian irradiation to mimic pelvic, abdominal, or spinal irradiation in patients. Although animal ages were comparable between the control and treatment group ( Fig. 3A ), serum AMH concentrations at 8–9 months post-surgery were lower (P < 0.05) in animals that received radiation treatment than those of the control animals ( Fig. 3A ). In contrast to AMH, serum MCP1 concentrations increased (P < 0.05) in animals of the treatment group compared with those of the control group ( Fig. 3B ). IL6 was measurable in serum samples from all animals of the treatment group, whereas it was not detectable (<3.1 pg/ml) in any of the control samples ( Fig. 3B ). In ovaries of the control animals, CD68+ macrophages were primarily present in the corpus luteum at 10–11 months post-surgery ( Fig. 3C ), because immune cells participate in the corpus luteum development and function ( Pate and Keyes, 2001 ). There were sparse macrophages in the ovarian cortex with primordial, primary and secondary follicles observed ( Fig. 3C and 3D ). In contrast, a number of macrophages were evident in the ovarian cortex of animals in the treatment group ( Fig. 3E ). Follicles were sparse in number or absent. There were a number of cysts surrounded by macrophages, and macrophages were also observed inside the cystic cavity ( Fig. 3F ). CD68 staining was absent in negative control sections containing the corpus luteum ( Supplemental figure 1A ). Chemotherapy- and radiotherapy-induced damage of growing follicles can be assessed by serum AMH levels. However, AMH levels do not always reflect the actual status of primordial follicles, i.e., ovarian reserve, because primordial follicles do not produce AMH ( Xu et al., 2016 ). In order to directly determine the alterations of follicles, a relatively low dose of chemotherapy agent was administered to mice with ovaries collected at different time points post-treatment. The numbers of primordial follicles were comparable between the control and CPA-treated animals at 1–2 and 3–4 weeks post-injection ( Supplemental figure 2A ). The number of primordial follicles was lower (P < 0.05) in CPA-treated animals than that of the control group at 5–6 and 7–8 weeks post-injection (5–6 weeks: 824 ± 189 versus 2044 ± 153; 7–8 weeks: 890 ± 172 versus 1851 ± 372) ( Supplemental figure 2A ). There were no differences in the numbers of primary or secondary follicles between animals in the control and treatment group at any assessed time point ( Supplemental figure 2B and 2C ). In ovaries of the control animals, CD68+ macrophages were primarily present in the corpus luteum at all assessed time points ( Fig. 4A ). There were sparse numbers of macrophages in the ovarian cortex with primordial, primary and secondary follicles observed ( Fig. 4B ). In contrast, a number of macrophages were evident in the ovarian cortex from animals at all assessed time points post-CPA injection ( Fig. 4C and 4E ). There were macrophages surrounding primordial, primary, secondary and antral follicles at 3–4 weeks post-injection ( Fig. 4D ). Macrophages were observed inside the granulosa cell layers of secondary and antral follicles, as well as inside the antral cavity of antral follicles, at 7–8 weeks post-injection ( Fig. 4F ). CD68 staining was absent in negative control sections containing the corpus luteum ( Supplemental figure 1B ).

Blood and ovarian tissue were obtained from the Oregon Ovarian Cancer Registry and Tissue Repository at the Oregon Health & Science University (OHSU). Reproductive age women without experiencing menopause (30–47 years old) were recruited ( Table 1 ). Control patients (n = 7) did not receive cancer therapy, whereas patients in the treatment group (n = 7) had chemotherapy and/or radiotherapy completed 1–11 years prior to sample collection. Patients who provided ovarian tissue (n = 4/group) underwent oophorectomy or hysterectomy (including removal of ovaries) due to benign or malignant gynecologic conditions ( Table 1 ). Samples were collected with informed consent. Following tissue collection from a patient, one tissue section from the sample was dedicated for ovarian pathology examination. Only sample without ovarian pathology was utilized for the present study. The experimental protocol was approved by the Institutional Review Board (#921) at OHSU ( Xu et al., 2021 ). Serum levels of anti-Müllerian hormone (AMH), an indicator of follicle growth ( Xu et al., 2016 ), were measured to determine ovarian function. An AMH ELISA kit (AnshLabs) was used for the assay by the Endocrine Technologies Core at the Oregon National Primate Research Center (ONPRC), OHSU ( Xu et al., 2016 ). To assess the systemic inflammation, serum samples were assayed for proinflammatory cytokine. Monocyte chemoattractant protein 1 (MCP1) and IL6 were measured using Human CCL2/MCP-1 and IL-6 Quantikine ELISA Kits (DCP00 and D6050; R&D Systems), respectively. Ovarian tissue was fixed in 4% paraformaldehyde-PBS and embedded in paraffin ( Xu et al., 2021 ). Five micrometer sections were cut and stained with hematoxylin and eosin for morphology evaluation by the Integrated Pathology Core at ONPRC, OHSU. To assess inflammatory cell infiltration, ovarian sections were analyzed by immunohistochemistry, as previously described ( Sereti et al., 2014 ). Sections were incubated at 4 °C overnight with mouse anti-human CD68 (1:400; CM033C, KP1; Biocare), mouse anti-human CD163 (1:500; CM353A; Biocare), rabbit anti-human CD4 (1:1000; ab183685; Abcam), rabbit anti-human CD3 (1:100; RM9107S0; Fisher Scientific), and rabbit anti-human myeloperoxidase (MPO) (1:1,000; A0398; Agilent) antibodies to detect tissue macrophages, T lymphocytes, and neutrophils, respectively. Sections were processed using the Mouse Polink-1 AP and Rabbit Polink-1 HRP staining systems (GBI Labs), developed with Warp Red (Biocare) or ImmPACT DAB (Vector Laboratories), counterstained with CAT-hematoxylin (Biocare), and imaged using an Aperio AT2 scanner (Leica Biosystems). The general care of rhesus macaques ( Macaca mulatta ) were provided by the Division of Comparative Medicine at ONPRC, OHSU. Animals were pair-caged in a temperature-controlled, light-regulated (12 h light/12 h dark) room. Diet consisted of monkey chow supplemented with fresh fruit or vegetables and water ad libitum . Animals were treated according to the National Institutes of Health (NIH)’s Guide for the Care and Use of Laboratory Animals. The protocol was approved by the ONPRC Institutional Animal Care and Use Committee (IACUC) ( Zelinski et al., 2011 ). Reproductive age female animals (8–10 years old; n = 6) exhibiting regular menstrual cycles were assigned randomly to 2 groups (n = 3/group). The control animals underwent a sham surgery, whereas the treatment group animals received a single dose of ovarian irradiation, as previously described ( Zelinski et al., 2011 ). Externalized ovaries were exposed to X-ray-based radiation for 13 min to generate 1,500 rads of absorbed dose (15 Gy), and then returned to the peritoneal cavity. Blood samples and ovaries were collected at 8–9 and 10–11 months post-surgery, respectively. Serum levels of AMH, MCP1 and IL6 were measured, as described above ( section 2.1.1. ) to determine ovarian function and systemic inflammation post-treatment. Ovaries were fixed and sectioned for immunohistochemistry, as described above ( section 2.1.2. ), to assess inflammatory cell infiltration ( Blankenship and Enders, 1997 ). Ovarian sections were incubated at 4 °C overnight with mouse anti-human CD68 (1:200; sc-20060; Santa Cruz Biotechnology) antibody to detect tissue macrophages. Sections with an isotype control antibody served as negative controls. Sections were then incubated with biotinylated anti-mouse secondary antibody and processed using a VECTASTAIN Elite ABC Kit (PK-6102; Vector Laboratories). Following incubation with 3,3΄-diaminobenzidine, sections were counterstained with hematoxylin and imaged using a BX40 microscope and a DP72 camera (Olympus Corporation of the Americas). The general care of ICR mice (CD-1; Envigo) were provided by the Comparative Medicine at the University of Nebraska Medical Center (UNMC). Animals were housed in a temperature-controlled, light-regulated (14 h light/10 h dark) room and provided with food and water ad libitum . Animals were treated according to the NIH’s Guide for the Care and Use of Laboratory Animals. The protocol was approved by the UNMC IACUC. Sexually mature female animals (6 weeks old; n = 32) exhibiting normal estrous cycles were assigned randomly to 2 groups (n = 16/group). The control animals received an intraperitoneal injection of PBS (vehicle), whereas the treatment group animals were injected with a single dose of 150 mg/kg cyclophosphamide (CPA) (PHR1401; MilliporeSigma), as previously described ( Luan et al., 2019 ). Ovaries were collected at 1–2, 3–4, 5–6 and 7–8 weeks post-injection (n = 4/group/time point). Ovaries were fixed and serially sectioned for histology and immunohistochemistry analysis, as described above ( section 2.1.2. ), to assess ovarian reserve and inflammatory cell infiltration. Every 10th section was stained with hematoxylin and eosin. Primordial and preantral (primary and secondary) follicles were counted by microscopy ( Luan et al., 2019 ). Ovarian sections were also incubated at 4 °C overnight with rabbit anti-mouse CD68 antibody (1:100; ab125212; abcam) to detect tissue macrophages, as described above ( section 2.2.2. ). Statistical analysis was performed using a SAS software (SAS Institute). The two-sample t-test was used to analyze differences between the control and treatment group. Differences were considered significant at P < 0.05 and values are presented as mean ± SEM.

We investigated inflammation status and ovarian function after chemotherapy and radiotherapy. For the first time, chronic inflammatory activities, including increased systemic proinflammatory cytokine levels and ovarian inflammatory cell infiltration, were identified in women and animal models. The chronic inflammation induced by chemotherapy agent and/or radiation appeared to be associated with ongoing ovarian tissue damage, follicle depletion, and functional decline. Patients in this study resumed menstrual cycles >1 year after the completion of chemotherapy and/or radiotherapy. Although their serum AMH did not recover to levels observed in the control patients, it indicated the presence of surviving follicles post-treatment that could grow. Data are consistent with a previous study in patients with Hodgkin lymphoma showing that serum AMH decreased during chemotherapy, which recovered after treatment ( Anderson et al., 2018 ). Notably, AMH levels fully recovered in patients receiving an ABVD or AVD regimen, but not in those receiving a BEACOPP regimen. BEACOPP may have greater toxicity to damage more follicles. In addition, BEACOPP, but not ABVD, causes chronic inflammation, as indicated in osteonecrosis development ( Albano et al., 2017 ). If the persistent inflammation is not resolved, the process may result in progressive ovarian tissue damage and follicle loss. Indeed, the evidence of inflammation was found in patients of the treatment group in the present study, including increased systemic levels of pro-inflammatory cytokines, as well as hyalinized blood vessels and inflammatory cell infiltration in the ovary. Correspondingly, ovarian tissue injury was observed as extracellular matrix breakdown ( Sonbol, 2018 ). A limitation of the present study is that the systemic inflammation status of patients is unknown prior to chemotherapy and/or radiotherapy. Because patients medical conditions may potentially affect inflammatory activity, longitudinal studies are warranted in the future to further assess chronic inflammation-associated ovarian function decline in patients with data collected before and after chemotherapy or radiotherapy. Similar to findings in patients, serum AMH was detectable 8–9 months post-irradiation in rhesus macaques in the present study, though it did not recover to levels observed in the control animals. Inflammation was identified in animals of the treatment group, including increased systemic levels of pro-inflammatory cytokines and macrophage infiltration in the ovary. Additionally, cysts associated with macrophages were observed, which could have originated from follicles that underwent atresia. A previous study in rhesus macaques demonstrated that administration of sphingosine-1-phosphate mimetic, FTY720, protected follicles from radiation insult ( Zelinski et al., 2011 ). Animals maintained menstrual cycles over a year after ovarian irradiation. FTY720 attenuates inflammation via regulating the trafficking and activity of immune cells ( Obinata and Hla, 2012 ). The protective effects of FTY720 may be, as least in part, due to its anti-inflammatory action. However, one-time FTY720 administration was not sufficient to prevent chronic inflammatory reactions, which resulted in fibrosis in the ovary of rhesus macaques receiving ovarian irradiation ( Amargant et al., 2021 ). Nevertheless, nonhuman primates offer a unique model for well-controlled studies to investigate chronic inflammation induced by chemotherapy agents or radiation, as well as the long-term effects of inflammatory activities on ovarian tissue conditions and reproductive potential. Consistent to observations in the patient and rhesus macaque ovaries, macrophage infiltration into the ovarian cortex was evident in mice up to 8 weeks post-chemotherapy agent treatment. The decline of primordial follicle numbers became statistically significant 5–6 weeks post-treatment, indicating a progressive follicle loss after clearance of the chemotherapy agent. Although the alteration of preantral follicle numbers was not identified during the present study interval, follicle viability could be negatively impacted by chronic inflammation, as indicated by macrophage invasion into follicles at 7–8 weeks post-treatment. Different from menstrual cycles in humans and rhesus macaques, the estrus cycle in mice only lasts 4–5 days. In the present study, mice were not treated for estrus synchronization because the numbers of early-stage follicles in the mouse ovary do not change during the estrus cycle ( Visser et al., 2007 ). Chemotherapy agent-induced inflammation in the ovary, which is associated ovarian damage, has been studied previously in mice within relatively short intervals post-treatment. One week after a single injection of CPA and busulfan, systemic inflammation was identified, including increased proinflammatory cytokines, e.g., IL6, and decreased anti-inflammatory cytokines in the blood ( Luo et al., 2017 ). Follicular expression of a leukocyte chemoattractive cytokine, stromal cell-derived factor 1, and its receptor was increased, which could lead to follicular atresia. Ovarian vasculature alterations, including hyalinization, endothelial damage, and neovascularization, were observed 2 weeks after a single injection of CPA ( Pascuali et al., 2018 ). Furthermore, anti-inflammatory agents were shown to protect the ovary from CPA-induced damage. For example, co-administration of tocotrienol limited neovascularization and inflammatory cell infiltration over 30 days ( Saleh et al., 2015 ). The mouse model provides opportunities to study inflammation process and ovarian reserve alterations in response to chemotherapy agents and radiation over time, and the underlying mechanisms. The present multi-species research suggests that the ovary could be subject to additional tissue damage due to chronic inflammation triggered by chemotherapy agents or radiation. Inflammatory cell recruitment and activation resulted from the direct acute ovarian injury may lead to excessive proinflammatory cytokine production, which further increases inflammatory cell accumulation in the ovary ( Kim et al., 2014 ; Wang et al., 2010 ). Follicle depletion continues after the completion of chemotherapy and radiotherapy as a result of persistent follicular atresia and stromal/vascular degeneration. Future studies need to further delineate the mechanism of immune activation in the ovary and associated tissue damage. Furthermore, the impact of chronic inflammation on oocytes requires additional investigation, including oocyte developmental competence and genetic integrity. Interventions that dampen the overactivated inflammatory response may protect the ovary to maintain follicle viability and facilitate the continued follicular development in young female patients, and therefore support the ovarian endocrine function and reproductive competence.

The increase of cancer incidence and survival rates in women prior to or during reproductive age draws attention to the long-term side effects of cancer treatment ( Miller et al., 2019 ). Chemotherapy and radiotherapy are known for cytotoxicity that damages the ovary. Ovarian follicle depletion causes primary ovarian insufficiency, which results in early menopause and infertility ( Wallace et al., 2005 ). Impaired ovarian endocrine function is also associated with an increased incidence of osteoporosis and cardiovascular mortality ( Shuster et al., 2010 ). Thus, it is important to understand mechanisms causing the follicle number decline by chemotherapy and radiotherapy, so that interventions may be developed for ovarian protection to improve the quality of life in young female patients. Chemotherapy agents and radiation generate direct deleterious effects on cells, and therefore current ovarian protection strategies predominantly focus on preventing ovarian damage at the time of cancer therapy ( Spears et al., 2019 ). Notably, interrupted ovarian function resulting from the acute injury could resume spontaneously in young patients, as indicated by ovarian hormone production and menses post-treatment, suggesting the presence of primordial follicles that survive cancer therapy ( Jacobson et al., 2015 ; Peigné and Decanter, 2014 ). However, the recovery can take more than six months, a time interval longer than what is estimated for human follicular development from the primordial stage ( Gougeon, 2010 ). In addition, the numbers of growing follicles could remain low for years, as indicated by reduced ovarian hormone production, without full recovery to the levels prior to cancer therapy ( Jacobson et al., 2015 ). It appears that, besides acute injuries, additional mechanisms leading to chronic effects are involved to prevent follicular development and/or induce persistent follicle loss post-treatment. In non-cancerous tissues, cells injured by chemotherapy agents or radiation release proinflammatory cytokines to recruit and activate immune cells that produce additional proinflammatory cytokines ( Kim et al., 2014 ; Wang et al., 2010 ). Chemotherapy agents or radiation can also directly promote proinflammatory cytokine production by immune cells ( Najafi et al., 2018 ; Wang et al., 2012 ). Excessive proinflammatory cytokines lead to damage of the surrounding tissue, which could persist after the completion of cancer treatment, resulting in a state of persistent immune activation and chronic inflammation ( Najafi et al., 2018 ; Wang et al., 2012 ). The inflammatory response induced by chemotherapy agents has been observed in the mouse ovary, including inflammatory cell infiltration and increased expression of proinflammatory cytokines, which was associated with diminished ovarian function ( Luo et al., 2017 ; Saleh et al., 2015 ). Although only acute inflammation was evaluated, these studies provide the basis for investigating long-term effects of cancer treatment on inflammatory activity in the ovary. Therefore, we performed a comprehensive study to examine systemic and local inflammation induced by chemotherapy agents and radiation in human specimens and pre-clinical animal models. The association between chronic inflammation and ovarian tissue damage and follicle depletion was also assessed.

Images include rhesus macaque ovarian section (A) and mouse ovarian section (B). CL, corpus luteum. Scale bar = 100 μm for panel A and 200 μm for panel B. Plots include numbers of primordial follicles (A), primary follicles (B), secondary follicles (C), and antral follicles (D) in ovaries of the control mice and mice at 1–2, 3–4, 5–6 and 7–8 weeks post-cyclophosphamide injection (treatment group). Data are presented as mean ± SEM. *, significant difference at P < 0.05.