Comparative effects of the antioxidant glutathione with metformin and Diane-35 on hormonal, metabolic, and inflammatory indicators in a DHEA-induced PCOS rat model

Figures & data

Figure 1. Interventions in study groups.

PCOS; Polycystic Ovary Syndrome, DHEA; Dehidroepiandrosteron, sc; subcutaneous, o; orally, ip; intraperitoneal

Figure 2. Serum concentrations of IL-6, insulin, testosterone, hs-CRP, MDA and SHBG in study groups.

PCOS; Polycystic Ovary Syndrome, DHEA; Dehidroepiandrosteron, GSH; glutathione, IL-6; interleukin-6, hs-CRP; high-sensitive C-reactive protein, MDA; Malondialdehyde; SHBG; Sex hormone binding globulin

Table 1. Serum IL-6, insulin, testosterone, hs-CRP, MDA and SHBG levels in study groups.

Variables	Group 1 Control (n = 5)	Group 2 PCOS (n = 5)	Group 3 Diane 35 (n = 5)	Group 4 Metformin (n = 5)	Group 5 Glutathione (n = 5)	p value
IL-6 (ng/L)	–	32.16 ± 6.97	31.69 ± 8.37	10.5 ± 4.02**, ¥¥	16.13 ± 1.13**,¥¥	<0.0001
Insulin (mIU/L)	–	39.33 ± 18	35.79 ± 16.72	10.49 ± 4.71*, ¥	13.59 ± 2.84*, ¥	0.0002
Testosterone (ng/L)	1887 ± 750	1949 ± 689.6	1837 ± 798.6	551.9 ± 236.8**,¥	555.1 ± 352.5**,¥¥	<0.0001
hs-CRP (ng/L)	–	5.74 ± 2.13	4.97 ± 1.98	1.83 ± 0.79*, ¥	1.86 ± 0.97*, ¥	0.0002
MDA (nmol/mL)	–	12.03 ± 0.66	10.62 ± 0.89	3.88 ± 1.45**, ¥¥	2.41 ± 1.04**, ¥¥	<0.0001
SHBG (ng/mL)	–	8824 ± 2023	6768 ± 3321	2736 ± 1538*	2664 ± 1565*,¥	0.0016

PCOS; Polycystic Ovary Syndrome, IL-6; Interleukin-6, hs-CRP; High-sensitive C-Reactive Protein, MDA; Malondialdehyde; SHBG; Sex hormone binding globulin. All values are expressed as mean ± SD. One way ANOVA test was used for intergroup comparisons. Tukey Post Hoc test was used for multiple comparisons. p < 0.05 was considered statistically significant.

* ^,** Comparison with PCOS group; p < 0.05 and p < 0.001, respectively.

^{¥ , ¥¥} Comparison with Diane 35 group; p < 0.05 and p < 0.001, respectively.

Figure 3. Comparative follicle assessment in study groups.

PCOS; Polycystic Ovary Syndrome, DHEA; Dehidroepiandrosteron, GSH; glutathione, IL-6; interleukin-6, hs-CRP; high-sensitive C-reactive protein, MDA; Malondialdehyde; SHBG; Sex hormone binding globulin. a) Number of primordial, primary, secondary and antral follicles in study groups. *p < 0.05, **p < 0.01, ***p < 0.001, compared to DHEA (PCOS) group. +p < 0.05, ++p < 0.01, +++p < 0.001, compared to Control group. b) Number or atretic and cystic follicles. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared to DHEA (PCOS) group. +p < 0.05, ++++p < 0.0001, compared to Control group.

Figure 4. Photomicrographs of the representative ovary cross-sections in all experimental groups. Control group showed normal ovarian structures with several healthy follicles (arrow) at different developmental stages and corpus luteum (CL). DHEA (PCOS) group showed multiple cystic follicles (*) with a thin layer of flattened granulosa cells and a few healty follicles and CL. In the DHEA + 4,5 mg/kg Diane and DHEA + 300 mg/kg Metformin groups, number of cystsic follicles were decreased with the presence of growing healthy follicles. DHEA + 100 mg/kg GSH group showed a marked reduction in the of cystsic follicles with the presence of growing healthy follicles which were almost comparable with Control group. Healthy follicle (arrow), corpus luteum (CL), cystic follicle (*). Hematoxylin and eosin staining, Bar: 1000 µm in lower magnification and 500 µm in high magnification.

Table 2. Comparison of the primordial, primary, secondary, antral, atretic and cystic follicle counts in study groups.

Variables	Group 1 Control (n = 5)	Group 2 PCOS (n = 5)	Group 3 Diane 35 (n = 5)	Group 4 Metformin (n = 5)	Group 5 Glutathione (n = 5)	p value
Primordial follicle counts	130.4 ± 9.6	120.8 ± 7.5	126 ± 12.41	132.4 ± 19.83	138.2 ± 19.07	0.4862
Primary follicle counts	44.2 ± 8.9	67.75 ± 7.63	56.4 ± 6.87	45.8 ± 11.61	36 ± 7.51	0.0003*
Secondary follicle counts	20.2 ± 5.1	29.5 ± 3.41	19 ± 3.67	16.4 ± 5.77	20.2 ± 2.58	0.0039*
Antral follicle counts	13.6 ± 2.9	5.25 ± 1.5	5 ± 1.87	9 ± 3.08	11.6 ± 1.81	<0.0001*
Atretic follicle counts	2.8 ± 1.3	8.5 ± 1.29	5.6 ± 1.81	4 ± 1.00	4.4 ± 1.14	<0.0001*
Cystic follicle counts	0.2 ± 04	5.75 ± 1.7	2.2 ± 1.3	1.2 ± 0.83	0.8 ± 0.83	<0.0001*

All values are expressed as mean ± SD. One way ANOVA was used for comparisons between study groups.

* p < 0.05 significant difference in comparison of all groups.

Tukey Post Hoc Analyses.

Primordial follicle counts; Control vs DHEA (PCOS) p = 0.8; Control vs Diane-35 p = 0.9; Control vs Metformin p = 0.9; Control vs Glutathione p = 0.9; DHEA (PCOS) vs Diane-35 p = 0.9; DHEA (PCOS) vs Metformin p = 0.7; DHEA (PCOS) vs Glutathione p = 0.3; Diane-35 vs Metformin p = 0.9; Diane-35 vs Glutathione p = 0.6; Metformin vs Glutathione p = 0.9.

Primary follicle counts; Control vs DHEA (PCOS) p = 0.005; Control vs Diane-35 p = 0.2; Control vs Metformin p = 0.9; Control vs Glutathione p = 0.5; DHEA (PCOS) vs Diane-35 p = 0.2; DHEA (PCOS) vs Metformin p < 0.01; DHEA (PCOS) vs Glutathione p < 0.01; Diane-35 vs Metformin p = 0.2; Diane-35 vs Glutathione p < 0.01; Metformin vs Glutathione p = 0.3.

Secondary follicle counts; Control vs DHEA (PCOS) p = 0.04; Control vs Diane-35 p = 0.9; Control vs Metformin p = 0.5; Control vs Glutathione p = 0.9; DHEA (PCOS) vs Diane-35 p < 0.01; DHEA (PCOS) vs Metformin p < 0.01; DHEA (PCOS) vs Glutathione p < 0.05; Diane-35 vs Metformin p = 0.7; Diane-35 vs Glutathione p = 0.9; Metformin vs Glutathione p = 0.4.

Antral follicle counts; Control vs DHEA (PCOS) p = 0.0004; Control vs Diane-35 p = 0.0001; Control vs Metformin p = 0.04; Control vs Glutathione p = 0.6; DHEA (PCOS) vs Diane-35 p = 0.9; DHEA (PCOS) vs Metformin p = 0.09; DHEA (PCOS) vs Glutathione p < 0.01; Diane-35 vs Metformin p < 0.05; Diane-35 vs Glutathione p < 0.01; Metformin vs Glutathione p = 0.2.

Atretic follicle counts; Control vs DHEA (PCOS) p < 0.0001; Control vs Diane-35 p = 0.02; Control vs Metformin p = 0.6; Control vs Glutathione p = 0.3; DHEA (PCOS) vs Diane-35 p < 0.05; DHEA (PCOS) vs Metformin p < 0.01; DHEA (PCOS) vs Glutathione p < 0.01; Diane-35 vs Metformin p = 0.2; Diane-35 vs Glutathione p = 0.5; Metformin vs Glutathione p = 0.9.

Cystic follicle counts; Control vs DHEA (PCOS) p < 0.0001; Control vs Diane-35 p = 0.05; Control vs Metformin p = 0.6; Control vs Glutathione p = 0.8; DHEA (PCOS) vs Diane-35 p < 0.01; DHEA (PCOS) vs Metformin p < 0.01; DHEA (PCOS) vs Glutathione p < 0.01; Diane-35 vs Metformin p = 0.5; Diane-35 vs Glutathione p = 0.2; Metformin vs Glutathione p = 0.9.

Data availability statement

The data that support the findings of this study are available from the corresponding author, Pınar Özcan, upon reasonable request.