P–434 Effect of let–7a mimic as a new pharmaco-protective agent against chemotherapy-induced ovarian damage on subsequent follicular development and oocyte quality using mouse ovarian transplantation model
Authors: Alexandri C , Nguyen A , Va. De. Steen G , Demeestere I 
Published: August 2021

Abstract

Study question
What is the long-term impact of let–7a-mimic transfection on oocytes development in new-born mice ovaries exposed to chemotherapy in vitro following transplantation in the kidney?

Summary answer
The let–7a-mimic restoration protects against chemotherapy-induced ovarian apoptosis and preserves subsequent follicular developmental and acquisition of oocyte maturation competence in mouse.

What is known already
It is well known that cyclophosphamide and its active metabolites (4-hydroperoxycyclophosphamide, 4-HC) cause irreversible ovarian damage and impair future fertility of cancer survivors. Besides the available fertility preservation options, microRNAs/miRNAs appear to be very attractive and novel targets to prevent theses damage. We showed that miRNAs were dysregulated after exposure to 4-HC in postnatal-day–3 (PND3) ovaries, let–7a being the most downregulated among them. By replacing let–7a function, let–7a-mimic was able to protect mouse follicles against 4-HC in vitro. This previous study suggested that it could preserve the reproductive potential after treatment. However, the impact on subsequent oocytes development is unknown

Study design, size, duration
PND3 ovaries from C57blxCBAF1 hybrid mice were cultured under 3 conditions: control, chemotherapy for 24h (4-HC/20μΜ/24h), chemotherapy for 24h+let–7a-mimic (4-HC/20μΜ/24h+let–7a-mimic). Nine PND3 ovaries were cultured in the different conditions and then transplanted under the kidney’s capsule of C57blxCBAF1 hybrid adult mice for follicular growth/apoptosis evaluation. Then, 21 ovaries (≥7/condition) were used for oocyte maturation assessment after transplantation and ovarian stimulation. All transplanted mice were observed during 21 days before PND3 ovaries collection. Participants/materials, setting, methods: PND3 ovaries were cultured in vitro using inserts under different conditions. A liposome-based system was used to deliver let–7a-mimic into ovaries and QPCR-assays validated its expression levels after transfection. Apoptosis was evaluated by TUNEL Assay while haematoxylin/eosin staining was used for assessing the follicular morphology, stage and count. The oocyte maturation rate was evaluated at day 21 post-transplantation after gonadotropins injection, mechanical eggs collection and in vitro maturation for 24 hours.

Main results and the role of chance
The apoptosis assessment confirmed that let–7a-mimic transfection reduced the chemotherapy-induced damage in PND3 ovaries in vitro. The number of primordial follicles was significantly reduced (p &lt; 0.05) compared to control after chemotherapy exposure. However, it was increased in chemo24h+let–7a-mimic compared to chemo24h alone while remaining lower than control (p &gt; 0.05). Accordingly, the number of the transitory follicles reflecting follicular activation was significantly higher in chemo24h compared to control (p &lt; 0.05) and chemo24h+let–7a-mimic but for the last one, the result was not significant. Consequently, chemotherapy induces follicle activation while let–7a restoration tends to slow down this effect. To evaluate the long-term effects of chemotherapy and let–7a-mimic transfection, in vitro exposed PND3 ovaries were transplanted under kidney’s capsule in female adult mice. After 21 days, the ovarian reserve was higher in control, but we observed a slight increase of follicular density in the chemo24+let–7a-mimic compared to chemo24h. Similarly, the percentage of damaged/apoptotic cells was higher in all chemotherapy exposed groups compared to control but the impact was lower after let–7a restoration (12,0% and 28,2% in chemo24+let–7a-mimic and chemo24h, respectively). Importantly, the oocyte maturation rate after transplantation was higher in chemo24h+let–7a-mimic compared to chemo24h (40% versus 18%, respectively), suggesting a preservation of oocytes maturation competence.

Limitations, reasons for caution
The multiple in vitro/in vivo steps may introduce study bias. Moreover, the oocyte competence and live offspring is currently evaluated. The blastocyst formation and embryo development from oocytes fertilized in vitro, are more relevant parameters for oocyte quality assessment. The birth of healthy animals will confirm let–7a-mimic-transfection safety.
Wider implications of the findings: Our previous study demonstrated the anti-apoptotic effect of let–7a restoration in mouse ovaries against chemotherapy. In the current study, we demonstrated a long-term beneficial effect of let–7a restoration strategy on follicular development and oocytes maturation capacity. The results open new perspectives in fertility preservation using pharmacological approach.

Trial registration number
Not applicable