Loss of fertility is a major concern for female reproductive-age cancer survivors, since a common side-effect of conventional cytotoxic cancer therapies is permanent damage to the ovary. While immunotherapies are increasingly becoming a standard of care for many cancers—including in the curative setting—their impacts on ovarian function and fertility are unknown. We evaluated the effect of immune checkpoint inhibitors blocking programmed cell death protein ligand 1 and cytotoxic T lymphocyte-associated antigen 4 on the ovary using tumor-bearing and tumor-free mouse models. We find that immune checkpoint inhibition increases immune cell infiltration and tumor necrosis factor-α expression within the ovary, diminishes the ovarian follicular reserve and impairs the ability of oocytes to mature and ovulate. These data demonstrate that immune checkpoint inhibitors have the potential to impair both immediate and future fertility, and studies in women should be prioritized. Additionally, fertility preservation should be strongly considered for women receiving these immunotherapies, and preventative strategies should be investigated in future studies.
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Previously published microarray data that were re-analyzed here are available under accession code GSE38666. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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The authors acknowledge the technical support of the Monash Animal Research Platform, Monash Histology Platform, Monash FlowCore Platform, Monash Micro Imaging Facility, Peter MacCallum Cancer Centre Animal Research Platform and Peter MacCallum Cancer Centres Flow Facility. We thank P. Bouillet and WEHI Bioservices for the provision of large numbers of BID-deficient mice. We also thank H. Thorne, E. Niedermayr, all the kConFab research nurses and staff, the heads and staff of the Family Cancer Clinics, the National Breast Cancer Foundation of Australia (NBCF), Cancer Australia and the National Institutes of Health (USA) for their contributions to this resource, and the many families who contribute to kConFab. Fig. 6h and the Supplementary Fig. 1 were created using BioRender.com. This work was made possible through Victorian State Government Operational Infrastructure Support and the Australian Government National Health and Medical Research Council (NHMRC) IRIISS. This work was supported by NBCF funding grant no. IIRS-22-092. A.L.W. is supported by DECRA funding grant no. DE21010037 from the Australian Research Council (ARC). L.R.A. is supported by an Australian Government Research Training Program Scholarship and a Monash Graduate Excellence Scholarship. K.-A.P. is an NHMRC Leadership Fellow. S.L. is supported by the NBCF. A.S. is supported by an NHMRC Program Grant no. 1113133, NHMRC Fellowship no. 1116937 and NHMRC Investigator Grant no. 2007887. K.J.H. is supported by an ARC Future Fellowship grant no. FT190100265.
The authors declare no competing interests.
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Extended Data Fig. 1 Flow cytometry gating strategy for tumor-bearing mice.
Gating strategy used to identify ovarian immune cell populations of tumor-bearing mice 4 d after the following final immunotherapy treatment.
Extended Data Fig. 2 Immune checkpoint molecules are expressed in both mouse and human ovary.
CD69 and PD-1 expression in the ovary of healthy WT non-tumor bearing mice was assessed by flow cytometry. (A) The proportions of ovarian CD69+CD4+ and CD69+CD8+ of total effector memory T cells and (B) PD-1+CD4+ and PD-1+CD8+ T cells were quantified in healthy, tumor-free WT mice (n = 3 animals/group). Data are presented as mean ± s.e.m. (C) Log2 expression of PDCD1, CD8A, CD3D, CD274 and CTLA4 in normal human ovary epithelia from n = 12 women and ovarian cancer epithelia from n = 18 women. Data are presented as mean (centre) ± s.e.m. (bounds) of box plots and minima and maxima of whiskers; two-tailed unpaired t-test. (D) Representative immunohistochemical localisation of (i) CD4+ T cells, (ii) CD8+ T cells, (iii) PD-1 and (iv) PD-L1 in human ovarian cortex from n = 4 healthy pre-menopausal women. Arrows indicate ovarian follicles, bars=50 μm.
Extended Data Fig. 3 Ovarian and splenic immune cell numbers in tumor-free mice, 21 days post-final treatment, and flow cytometry gating strategy.
Numbers of ovarian (A) CD4+ T cells, (B) CD8+ T cells and (C) macrophages; and splenic immune cells, including (D) CD4+ T cells, (E) CD8+ T cells and (F) macrophages were analysed by flow cytometry in mice 21 d after the final indicated treatment. Data are presented as mean ± s.e.m.; n = 5 animals/group. (G) Gating strategy used to identify mouse ovarian and splenic immune cell populations 24 h or 21 d following immunotherapy treatment.
Extended Data Fig. 4 Estrous cycling is disrupted in immune checkpoint inhibitor-treated tumor-free mice.
Estrous cycling was monitored by vaginal cytology for a 14-day period following the final indicated treatment in the 21 d cohort of animals administered with 10 mg/kg anti-PD-L1, anti-CTLA-4 or control antibody (IgG). Plots depict the estrous cycles of each individual animal. Vertical axes represent the phase of estrous cycle (P, proestrus; E, estrus; M, metestrus; D, diestrus). Red highlights disrupted phases of the cycle in some anti-PD-L1 or anti-CTLA4 animals; n = 5 animals/group.
Extended Data Fig. 5 Ovarian follicles and ovulation are disrupted even with lower-dose immune checkpoint inhibitor treatment.
Adult female C57BL6/J mice received 5 mg/kg anti-PD-L1, anti-CTLA-4 or control antibody (IgG) on d 1, 4 and 7. Ovaries were collected either 24 h or 14 d later (n = 6/group). At both time points, total numbers of (A) primordial, transitional and primary follicles; Kruskal-Wallis test: *p = 0.0453; one-way ANOVA: ***p = 0.0008, (B) secondary and antral follicles, **p = 0.001, ****p < 0.0001, **p = 0.0017, *p = 0.0204, *p = 0.0429 and (C) corpora lutea were quantified; one-way ANOVA: *p = 0.0454; Kruskal-Wallis test: *p = 0.0353. All data are presented as mean ± s.e.m.
Extended Data Fig. 6 Primordial follicles remain significantly depleted 100 days post-treatment, even after local ovarian cytokine production has returned to control levels.
Adult female C57BL6/J mice received 10 mg/kg anti-PD-L1, anti-CTLA-4 or control antibody (IgG) on d 1, 4 and 7. Ovaries were collected 100 d later (n = 6 animals/group). Total numbers of (A) primordial follicles were quantified. (B) Cytokine mRNA levels in the ovary 100 d after the final indicated treatment were analysed by RT-qPCR (IgG n = 5, anti-PD-L1 n = 4, anti-CTLA-4 n = 6 animals). Data are presented as mean ± s.e.m.; one-way ANOVA; **p = 0.0039, ***p = 0.0003.
Extended Data Fig. 7 Ovarian and splenic immune cell numbers in tumor-free mice, 24 hours post-final treatment.
Numbers of ovarian (A) CD4+ T cells, (B) CD8+ T cells and (C) macrophages; and splenic immune cells, including (D) CD4+ T cells, (E) CD8+ T cells and (F) macrophages were analysed by flow cytometry in mice 24 h after the final indicated treatment. Data are presented as mean ± s.e.m.; one-way ANOVA; *p < 0.05; n = 5 animals/group.
Extended Data Fig. 8 Estrous cycling is not disrupted following immune checkpoint blockade in Bid-/- mice.
(A) Estrous cycling was monitored by vaginal cytology for a 15-day period following the final indicated treatment in the 21-day cohort of Bid-/- animals that had been administered with 10 mg/kg anti-PD-L1 (n = 4) or control antibody (n = 5). Plots depict the estrous cycles of each individual animal. Vertical axes represent the phase of estrous cycle (P, proestrus; E, estrus; M, metestrus; D, diestrus). (B) Compilation of primordial follicle numbers from wild-type and genetic knockout mouse models.
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Winship, A.L., Alesi, L.R., Sant, S. et al. Checkpoint inhibitor immunotherapy diminishes oocyte number and quality in mice. Nat Cancer 3, 1–13 (2022). https://doi.org/10.1038/s43018-022-00413-x
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