Comparative Effects of Metformin and Dienogest on PI3K/AKT Signaling in Experimental Endometriosis

Technical Summary: Comparative Effects of Metformin and Dienogest on PI3K/AKT Signaling in Experimental Endometriosis

Problem Statement
Endometriosis is an estrogen-dependent disorder affecting 6–10% of women of reproductive age, characterized by ectopic endometrial growth, chronic inflammation, and metabolic alterations. While conventional treatments involve surgical excision and hormonal suppression, these approaches often yield suboptimal outcomes with risks of recurrence and adverse effects. The PI3K/AKT signaling pathway, which regulates cell growth, survival, metabolism, and angiogenesis, is known to be aberrantly activated in endometriotic tissues. However, the specific molecular mechanisms by which pharmacological agents modulate this pathway to induce lesion regression remain under-explored. This study aims to evaluate and compare the therapeutic efficacy of Metformin and Dienogest on the PI3K/AKT pathway in a rat model of induced endometriosis.

Methodology
The study utilized a surgically induced endometriosis model in 32 adult female Sprague Dawley rats. Endometriosis was created by suturing autologous uterine horn explants to the omentum. Twenty-one days post-surgery, the rats were randomized into four groups (n=8 per group):

1. Control: No surgery, no treatment.
2. Endometriosis: Surgery only, no treatment.
3. Endometriosis + Dienogest: Surgery plus oral Dienogest (0.3 mg/kg/day) for 28 days.
4. Endometriosis + Metformin: Surgery plus oral Metformin (100 mg/kg/day) for 28 days.

Treatments commenced on day 21 and concluded on day 49. Rats were sacrificed during the diestrus phase. The study employed the following analytical techniques:

• Macroscopic Assessment: Measurement of endometrial lesion surface area (length × width × height).
• Biochemical Analysis: Serum levels of estradiol (E2), progesterone, and Vascular Endothelial Growth Factor (VEGF) were quantified via ELISA.
• Histopathology: Tissues were stained with Hematoxylin and Eosin (H&E) to evaluate inflammatory infiltration, tissue degeneration, and structural integrity.
• Molecular Analysis: Expression of PI3K, p-PI3K, AKT, and p-AKT was assessed in both endometriotic lesions and uterine tissue using Immunohistochemistry (IHC) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

Key Results

• Lesion Regression: Metformin treatment resulted in the most significant reduction in lesion size (3.5 ± 2.36) compared to both the untreated Endometriosis group (177.8 ± 214.01) and the Dienogest group (103 ± 143.58), with a statistically significant difference (p = 0.002). While the Dienogest group showed a reduction in lesion size compared to the untreated group, this specific difference did not attain statistical significance.
• Hormonal and Biochemical Profiles: Serum estradiol (E2) levels were significantly higher in the Metformin group compared to all other groups (p = 0.05). VEGF levels showed no significant variation between groups, though the highest mean was observed in the Metformin group. Progesterone levels were highest in the Metformin group but differences were not statistically significant.
• Histopathological Findings: Both treatment groups exhibited reduced inflammatory infiltration and tissue degeneration compared to the untreated Endometriosis group. The Metformin and Dienogest groups showed smaller ectopic tissue areas with restricted stromal and inflammatory cells.
• Molecular Expression (RT-PCR):
  • In endometriosis lesions, the Dienogest group showed significant upregulation of PI3K (1.88-fold) and p-PI3K (2.96-fold) compared to the untreated group. In the Metformin group, although PI3K, AKT, and p-PI3K expression levels appeared higher than in the untreated Endometriosis group, no statistically significant difference was detected between these two groups.
  • In uterine tissue, the Endometriosis group showed a significant decrease in p-PI3K expression compared to controls. The Dienogest group showed a significant decrease in PI3K expression. Notably, the Metformin group exhibited a statistically significant increase in AKT expression (73.86-fold) in uterine tissue compared to the control group (p = 0.05).
• Molecular Expression (IHC): Immunohistochemical analysis revealed differential protein regulation. In lesions, AKT expression was higher in the Dienogest group compared to both the untreated and Metformin groups. In uterine tissue, PI3K expression was highest in the control group, while p-AKT expression was lower in both treatment groups compared to the control and untreated groups.

Significance and Claims
The authors conclude that both Metformin and Dienogest are effective agents for inducing the regression of endometriotic implants in a rat model. However, the study highlights distinct mechanisms of action:

• Metformin demonstrated superior efficacy at macroscopic, microscopic, and molecular levels, achieving the most significant lesion reduction. Interestingly, this regression occurred despite significantly elevated serum E2 levels, suggesting that Metformin's anti-endometriotic activity is not mediated through systemic estrogen suppression but rather through local mechanisms, potentially involving anti-inflammatory and antiproliferative properties.
• Dienogest also promoted lesion regression and reduced inflammatory markers, consistent with its known progestin-mediated effects, though its impact on lesion size was less pronounced than Metformin in this specific model.
• Pathway Modulation: The study found that histopathological improvements did not correlate with a uniform decrease in PI3K/AKT pathway expression in the lesions. In fact, some pathway components were upregulated in treatment groups. This suggests that the therapeutic benefits of these drugs may not be solely explained by the suppression of the PI3K/AKT pathway in endometriotic foci, or that the pathway's role is complex and context-dependent.

The paper asserts that Metformin's comprehensive effects across multiple biological levels underscore its therapeutic potential. However, the authors maintain a modest stance, noting that further research is essential to fully unravel the specific molecular mechanisms and to explore clinical applications, particularly regarding fertility outcomes and pain relief, which were not evaluated in this experimental model.