P-331 Unlocking metformin’s potential: exploring its impact on inflammation and oxidative pathways in an endometriosis mouse model
Authors: Neto Cerqueira A  C , Botelho M , Rodrigues A , Lamas S , Araújo B , Guimarães J  T , Gouveia A , Almeida H , Neves D 
Published: July 2024

Abstract

Study question
Could endometriosis-associated pro-oxidative and pro-inflammatory condition be mitigated by metformin?

Summary answer
Metformin treatment tends to reduce fibrosis in ectopic tissues, while up-regulating anti-oxidative response enzymes.

What is known already
Endometriosis is a pro-inflammatory and pro-oxidative disease, which causes pain and infertility. Hormonal treatments used to control the disease must be stopped when a woman attempts to conceive, meaning new therapies are necessary. We had shown that endometriosis reduces fertility in a mouse model, which metformin treatment led to its reversal.
Metformin, in addition to reducing blood glucose, reduces inflammation and systemic oxidation. It has also been shown to mitigate the growth of endometrial implants in the animal model. The objective of this work is to evaluate the intervention of metformin in the inflammatory and oxidative response pathways associated with endometriosis.

Study design, size, duration
Female mice were randomly divided into groups: 1-Endometriosis (n = 24); 2-Sham (n = 12); 3-Endometriosis with metformin (n = 21); 4-Sham with metformin (n = 16). Endometriosis was surgically induced by heterologous transplantation of endometrium from a donor into recipients of the same strain. Metformin (0,5mg/mL) was administered orally for 3 months. Half of the mice in each group were mated for the fertility study. Tissues analysed are described with “Eu” for eutopic and “Ec” for ectopic tissues, respectively.

Participants/materials, setting, methods
The implants were confirmed and monitored by ultrasound and serum glucose, estradiol and lipoprotein values were quantified by enzymatic-colorimetric methods. Fibrosis in eutopic and ectopic tissues was quantified after computer-assisted analysis in Masson’s Trichrome images. Dual-labelling immunofluorescence was performed for the proteins F4/80 and Galectin-3 (gal-3) and Western Blot (WB) was performed to quantify their expression and also NFkB, HO-1 and GPX-1, both in eutopic and ectopic tissues.

Main results and the role of chance
No differences were found in serum analysis between groups, apart from a decrease in total cholesterol and an increase in estradiol in pregnant compared to non-mated mice.
Masson’s Trichrome stained sections revealed increased stromal fibrosis in both eutopic and ectopic tissues of non-mated Endometriosis groups compared to Sham groups(EuEnd vs. EuSham p = 0.018; EcEnd vs. EuSham p = 0.022; EuEnd vs. EuShamMet p = 0.0008; EcEnd vs EuShamMet p = 0,001). Metformin treatment showed a decreasing trend in fibrosis both in non-mated and pregnant, particularly in ectopic tissue, although statistical significance was not reached.
Both eutopic and ectopic endometrium presented positive cells for f4/80 and gal-3, with some co-localization, in all groups. Metformin treatment led to the increase of the inflammation markers in the tissues of mice with ENDO: F4/80 was increased in eutopic tissue(p = 0.0002), while gal-3 (p = 0.004) and NF-kB (p = 0.03) augmented in ectopic tissue of non-mated. Pregnancy condition, on the other hand had a tendency for the decrease of these proteins. Metformin increased the expression of antioxidant defense enzymes HO-1 (p = 0,005) and GPX-1 (p = 0,0001) in the ectopic endometrium of non-mated mice.
Metformin has shown to act in the up-regulation of anti-oxidative defenses that intervene in the control of endometriosis progression, but this effect was not evident during pregnancy.

Limitations, reasons for caution
Caution is necessary when extrapolating animal model data to humans due to species-specific differences in endometriosis patterns. Further studies, assessing pro-oxidative species, total antioxidant capacity, and potential anti-inflammatory roles of studied molecules, are essential to clarify the unexpected results in inflammatory pathways.

Wider implications of the findings
These findings highlight metformin, in a subtherapeutic dose, as a novel and safe approach to alleviate oxidative stress in endometriosis, offering a promising pharmacological solution for improving endometriosis-associated infertility, once we also had shown the reversal of infertility associated with endometriosis through metformin treatment.

Trial registration number
not applicable