Research Laboratory on Human Reproduction, Université Libre de Bruxelles (ULB), Brussels, Belgium.
Department of Biomedical Research, HUB-Hopital Erasme, Université Libre de Bruxelles, Brussels, Belgium.
Hum Reprod. 2023 Mar 1;38(3):408-420. doi: 10.1093/humrep/dead013.
Does chemotherapy exposure prior to ovarian tissue cryopreservation (OTC) impact the signaling pathways governing follicle activation and survival for prepubertal and postpubertal patients?
Chemotherapy exposure prior OTC increases follicle apoptosis rates but not follicular activation, although the PI3K/AKT/mTOR and Hippo signaling pathways were modified in the cortex.
OTC is currently the only available fertility preservation procedure for children and for patients who have already started their treatment. While previous studies have not observed harmful impacts of first chemotherapy exposure on OTC outcomes, the consequences of treatment on follicle activation and survival need to be further investigated. To address this question, we evaluated signaling pathway modifications induced by chemotherapy exposure according to pubertal status.
STUDY DESIGN, SIZE, DURATION: Cryopreserved ovarian tissues from postpubertal (12-29 years old, n = 8) and prepubertal (3-10 years old, n = 8) cancer patients donated for research were thawed and cultured for 24 h. Analyses of the survival of the follicles and stroma, and of the PI3K/AKT/mTOR and Hippo signaling pathways, were conducted at thawing and after culture. Ovarian fragments exposed to chemotherapy before collection were compared to non-exposed controls.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological investigations were performed to assess the distribution of the follicles, stroma fibrosis, vessel integrity, and apoptosis levels. It included follicular counting, collagen staining, immunostaining on CD31 and gH2AX, as well as TUNEL staining. To explore follicle activation in the different groups, the PI3K/AKT/mTOR and Hippo signaling pathways were investigated by gene expression analyses of isolated follicles and protein analyses on whole fragments through western blots and immunostaining.
We first assessed the impact of a first exposure to chemotherapy on the collagen density and vessels in ovarian tissues at thawing and after culture. While no differences in collagen density were observed according to age or previous treatment, the vascularization area (CD31+) was significantly lower in tissue from previously exposed patients compared to non-treated ones. Apoptosis analyses (TUNEL) revealed an acute deleterious impact on follicle survival after chemotherapy exposure without affecting the follicular density. Surprisingly, leukemic patients had a significantly higher percentage of gH2AX-positive follicles, indicating a DNA damage response, compared to the other patients. The proportion of activated follicles appeared to decrease following exposure to chemotherapy, suggesting that it at least did not increase activation process. Stable KIT LIGAND gene and protein expression and cKIT protein levels were observed among the groups, confirming the absence of activation. Analysis of the PI3K pathway did not reveal a difference in the AKT phosphorylation level between the groups, but pRPS6 was significantly higher in tissue from patients previously exposed to chemotherapy compared to that from non-exposed patients. Finally, protein and gene analyses on Hippo pathway signaling showed a higher LATS1 protein level in the cortex after chemotherapy exposure.
LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of the human fragments, and the limited number of patients included in the cohort have to be considered as important study limitations. Moreover, this study did not explore the long-term consequences of chemotherapy on follicular development. Therefore, the results should be interpreted with caution.
These results underscore the deleterious effect of previous chemotherapeutic treatment on follicle survival. Although follicular density was not reduced, these data suggested that exposure to chemotherapy impacts follicular apoptosis and the DNA damage response. Chemotherapy-induced activation was not observed despite the impact on mTOR and Hippo signaling pathways in the whole cortex.
STUDY FUNDING/COMPETING INTEREST(S): This work was funded by an Excellence of Science (EOS) Grant (ID: 30443682) and was supported by Fonds Erasme. I.D. and M.-M.D. are associate researchers at Fonds National de la Recherche Scientifique de Belgique (FNRS). There are no competing interests.
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卵巢组织冷冻保存（OTC）前的化疗暴露是否会影响调控卵泡激活和存活的信号通路，无论是在青春期前还是青春期后的患者？
OTC 前的化疗暴露增加了卵泡凋亡率，但不影响卵泡激活，尽管 PI3K/AKT/mTOR 和 Hippo 信号通路在皮质中发生了改变。
OTC 目前是儿童和已经开始治疗的患者唯一可用的生育力保存程序。虽然以前的研究没有观察到第一次化疗暴露对 OTC 结果的有害影响，但需要进一步研究治疗对卵泡激活和存活的影响。为了解决这个问题，我们根据青春期状态评估了化疗暴露引起的信号通路改变。
研究设计、规模、持续时间：来自青春期后（12-29 岁，n=8）和青春期前（3-10 岁，n=8）癌症患者的捐赠用于研究的冷冻卵巢组织被解冻并培养 24 小时。在解冻和培养后，对卵泡和基质的存活、PI3K/AKT/mTOR 和 Hippo 信号通路进行分析。将暴露于收集前化疗的卵巢片段与未暴露的对照组进行比较。
参与者/材料、设置、方法：进行组织学研究以评估卵泡、基质纤维化、血管完整性和凋亡水平的分布。它包括卵泡计数、胶原染色、CD31 和 gH2AX 的免疫染色以及 TUNEL 染色。为了探讨不同组中卵泡的激活，通过分离卵泡的基因表达分析和整个片段的 Western blot 和免疫染色对 PI3K/AKT/mTOR 和 Hippo 信号通路进行了研究。
我们首先评估了第一次接触化疗对解冻和培养后卵巢组织中胶原密度和血管的影响。虽然根据年龄或以前的治疗，胶原密度没有差异，但与未治疗的患者相比，以前暴露于化疗的患者组织中的血管化区域（CD31+）明显较低。凋亡分析（TUNEL）显示化疗暴露后对卵泡存活有急性有害影响，但不影响卵泡密度。令人惊讶的是，与其他患者相比，白血病患者的 gH2AX 阳性卵泡比例明显更高，表明存在 DNA 损伤反应。化疗暴露后，激活卵泡的比例似乎减少，表明它至少没有增加激活过程。各组中均观察到稳定的 KIT LIGAND 基因和蛋白表达以及 cKIT 蛋白水平，证实了没有激活。PI3K 通路分析显示各组之间 AKT 磷酸化水平没有差异，但与未暴露于化疗的患者相比，以前暴露于化疗的患者的 pRPS6 明显更高。最后，对 Hippo 通路信号的蛋白和基因分析显示，化疗暴露后皮质中的 LATS1 蛋白水平升高。
局限性、谨慎的原因：人类片段的异质性以及队列中患者数量有限是重要的研究局限性。此外，本研究并未探讨化疗对卵泡发育的长期影响。因此，结果应谨慎解释。
这些结果强调了先前化疗治疗对卵泡存活的有害影响。尽管卵泡密度没有降低，但这些数据表明，化疗暴露会影响卵泡凋亡和 DNA 损伤反应。尽管对 mTOR 和 Hippo 信号通路在整个皮质中产生影响，但未观察到化疗诱导的激活。
研究资金/利益冲突：这项工作得到了卓越科学（EOS）赠款（ID：30443682）的资助，并得到了 Erasme 基金会的支持。I.D. 和 M.-M.D. 是比利时国家科学研究基金会（FNRS）的副研究员。没有利益冲突。
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