Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro

Abstract

STUDY QUESTION What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? SUMMARY ANSWER Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. WHAT IS KNOWN ALREADY Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. STUDY DESIGN, SIZE, DURATION Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21–41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 μM in separate experiments, using biopsies from seven women (aged 27–41) and six women (aged 21–34), respectively. Biopsies from four women (aged 28–38) were used to investigate the effect of rapamycin or AMH only. PARTICIPANTS/MATERIALS, SETTING, METHODS Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. MAIN RESULTS AND THE ROLE OF CHANCE Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. LIMITATIONS, REASONS FOR CAUTION These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. TRIAL REGISTRATION NUMBER N/A.

AMH protects the ovary from doxorubicin by regulating cell fate and the response to DNA damage

“…The Ccng1/Mdm2/p53 axis has emerged as a strategic target for new precision molecular and genetic cancer therapies as well as chemo-sensitization 41 . While AMH has been proposed to contribute to the retention of the ovarian reserve following chemotherapy by preventing excessive activation of primordial follicles 11,14,42,43 , its potential salutary effects on growing follicles and other cell types of the ovary, and its involvement in the response to DNA-damage have not been thoroughly explored.…”

Gynotoxic Effects of Chemotherapy and Potential Protective Mechanisms

“…Given its low toxicity, high specificity against tumors expressing a specific receptor, and ability to inhibit the proliferation of drug-resistant tumor cells, recombinant AMH appears to be an interesting candidate to support the effects of cancer drugs [ 216 ], as well as to limit their deleterious effects on the ovaries [ 217 ]. On the other hand, several scientific articles have reported the possible use of AMH as a potential new method to limit the decline of PMFs during chemotherapy [ 6 , 168 , 169 , 170 , 207 ].…”

“…According to the study’s authors, intraovarian administration of AMH prior to cyclophosphamide may be a relatively non-invasive method of limiting the adverse effects of the chemotherapeutic agent [ 170 ]. In tests on ovarian cortex biopsies taken from healthy women, AMH was confirmed to attenuate cyclophosphamide-induced effects, including impaired PMFs and follicle transition health, as well as increased PI3K signaling [ 207 ].…”