P-587 effects of erzhi-tiangui granules to NEAT1 and apoptotic factors expression in granulosa cells of women with diminished ovarian reserve and Kidney-deficiency syndrome
Authors: Dong L , Xin X , Xiang S , Yu Y , Yu C , Qiu Y , Wu H , Lian F 
Published: June 2023

Abstract

Study question
To investigate whether erzhi-tiangui granules (ETG) can reduce granulosa cells (GCs) apoptosis levels by regulating NEAT1 expression, thereby improving the cycle outcomes of patients.

Summary answer
ETG significantly improved oocyte quality and decreased apoptotic factor levels in GCs, which may be related to its intervention in NEAT1 expression.

What is known already
The mechanism by which diminished ovarian reserve (DOR) occurs is mainly due to extensive apoptosis of GCs, which leads to follicular atresia and reduced follicle numbers. It has been found that changes in NEAT1 expression in ovarian tissues can exert influence the extent of apoptosis of GCs, which leads to diseases such as POF and PCOS. While ETG, a clinical formula used to improve female fertility, can improve the quality of oocytes and embryos by down-regulating the expression of apoptosis-related effector proteins, thus improving clinical outcomes. Whether ETG improves clinical outcomes in DOR patients via NEAT1 hasn’t been elucidated.

Study design, size, duration
This study was conducted in two separate parts from January 2020 to September 2021. Experiment I included 15 women each with normal ovarian reserve (NOR) and DOR, aiming to investigate the expression differences of NEAT1, key apoptosis proteins (Bcl-2 and Caspase-3). Experiment II, a randomized controlled trial, recruited 70 DOR patients, aimed to investigate if ETG could regulate the expression levels of Bcl-2 and Caspase-3 through NEAT1, thus acting to improve clinical outcomes.

Participants/materials, setting, methods
We detected expression levels of lncRNA NEAT1 and apoptotic proteins Bcl-2 and Caspase-3 of DOR and NOR women using RT-qPCR and Western-blotting, respectively. DOR patients were randomly assigned to ETG and control group, treated with ETG and placebo respectively. Clinical efficacy was confirmed by comparing two groups regarding changes in clinical and laboratory data. We also probed the changes in expression levels of NEAT1, Bcl-2 and Caspase-3 to investigate effects of ETG on GCs’ apoptosis.

Main results and the role of chance
In Experiment I, compared to the NOR group, the expression levels of NEAT1 and Bcl-2 were down-regulated while those of Caspase-3 were up-regulated in the GCs of DOR patients (P &lt; 0.05). In Experiment II, a total of 65 of 70 patients completed the study and were included in the outcome analysis. There were no significant differences between the two groups regarding baseline characteristics. Before treatment, there was no significant difference in the Kidney-deficiency syndrome scores between the two groups. After treatment, the ETG group had significantly lower Kidney-deficiency syndrome scores compared to the control group (P &lt; 0.01). Also, Kidney-deficiency syndrome scores were significantly lower in the ETG group post-treatment compared with that pre-treatment (P &lt; 0.001); there was no significant change in the control group (P &gt; 0.05). There were no significant differences between the two groups in the duration of gonadotrophin stimulation, serum estrogenand progesterone levels on the trigger day, and normal fertilization rate. Compared to the control group, the ETG group had lower gonadotropin dosage and increased number of oocytes, transferable embryos and top embryos obtained (P &lt; 0.05). Furthermore, the expression of NEAT1 was upregulated (P &lt; 0.05), Caspase-3 expression was decreased (P &lt; 0.01) in GCs from the ETG group than those in the control group (P &lt; 0.05).

Limitations, reasons for caution
The small number of observed cases and the lack of long-term efficacy may lead to biased study conclusions. Further studies are also expected in the future to understand the relevant pathway mechanisms.

Wider implications of the findings
In this study, not only to judge the therapeutic effects of ETG by using clinical symptoms and clinical laboratory indexes as observations, but also to explore the molecular mechanism of its treatment of Kidney-deficiency type DOR based on NEAT1, which may be related to affecting the apoptotic process.

Trial registration number
No. 82274573, No. 82174429, No. ZR2021MH255, No. tsqn202103182