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. 2022 May 9;32(2):10409.
doi: 10.4081/ejtm.2022.10409.

Effects of N-acetylcysteine and metformin treatment on the stereopathological characteristics of uterus and ovary

Bahare Rafiee  1 , Saied Karbalay-Doust  2 , Seyed Mohammad Bagher Tabei  3 , Negar Azarpira  4 , Sanaz Alaee  5 , Parvin Lohrasbi  6 , Soghra Bahmanpour  7
Affiliations

Effects of N-acetylcysteine and metformin treatment on the stereopathological characteristics of uterus and ovary

Bahare Rafiee et al. Eur J Transl Myol. .

Abstract

In this study, the stereo-pathological effect of metformin and N-acetyl cysteine is evaluated on the uterus and ovary of polycystic ovary syndrome (PCOS) mice. 96 mature females (8-weekold, weight of 20-30 gr) BALB/c mice were classified into 6 groups including the control group (n= 16), letrozole-induced PCOS group (n=16), PCOS + metformin (n=16), PCOS+NAC (n=16) and a separate control group for NAC (n=16). Another PCOS group was maintained for a month to make sure that features remain till the end of the study. Testosterone level, vaginal cytology and stereological evaluations were assessed. Vaginal cytology in letrozole-receiving mice showed a diestrus phase continuity. Testosterone level, body weight, uterine weight, endometrial volume, myometrial volume, gland volume, stromal volume, epithelial volume, vessel volume, daughter and conglomerate glands, endometrial thickness, and myometrial thickness exhibited an increasing trend in the uterus of PCOS mice. While normal gland and vessel length decreased in the PCOS group. Ovarian volume, corticomedullary volume, primary follicles, secondary follicles, and ovarian cysts were increased in PCOS ovaries. While corpus luteum, primordial, graafian, and atretic follicles showed a decline in the PCOS group. NAC and metformin, however, managed to restore the condition to normal. Given the prevalence of PCOS and its impact on fertility, the use of noninvasive methods is of crucial significance. NAC can control and treat pathological parameters and help as a harmless drug in the treatment of women with PCOS.

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Polycystic ovary syndrome (PCOS) is a common hormonal disorder in reproductive age. According to Rotterdam criteria, this syndrome can be diagnosed by two of the three symptoms of clinical/biochemical hyperandrogenism, ovarian dysfunction, and the presence of 12 or more follicles of 2-10 mm in the ovary. Although the number of oocytes retrieved in PCOS is higher than the others, most of them are not mature, declining the pregnancy rate. Oocyte, embryo quality, and pregnancy outcomes are weaker in PCOS patients due to changes in the oocyte and follicular fluid microenvironment. Pathological variations of ovaries and uterus in PCOS are the most important causes of ART failure. Uterine exposure with high levels of free insulin, growth factor-׀, and androgens, increases endometrial proliferative activity, and endometrial thickness. The incidence of micropolip is also associated with endometritis (plasma cells infiltration into endometrial stroma), endometrial stromal edema, and peri-glandular hyperemia. In the ovary, the presence of cystic follicles with a thin layer of granulosa cells and the absence of corpus luteum (indicative of anovulation status) is a common finding. One of the most basic treatment lines of PCOS is metformin. Metformin is a biguanide used to treat hyperglycemia, reduce insulin resistance, and inhibit hepatic gluconeogenesis. It also reduces total and free blood androgen, corrects ovarian dysfunction, and increases the quality of oocyte and embryos by reducing ROS and apoptosis levels. Metformin consumption in PCOS patients can decline premature and pre-antral follicles. Metformin consumption can also enhance follicular development, improve the percentage of corpus luteum, and decrease cystic follicles, hence, facilitating ovulation and fertility rate. Metformin also increments endometrial receptivity markers in PCOS patients. Metformin is, however, associated with gastrointestinal side effects. Another treatment that may help reduce PCOS complications and ultimately increase the chances of fertility is N-Acetyl cysteine (NAC). NAC is a derivative of the amino acid cysteine with antioxidant effects. Its anti-apoptotic properties prevent ovarian ischemia. NAC consumption is associated with a decrease in testosterone. Therefore, NAC can have positive effects on ovulation in PCOS patients. NAC can effectively strengthen pregnancy and ovulation rates. It is safe with a high lethal dose threshold, resulting in no side effects. However, the underlying mechanism of how NAC administration protects the utero-ovarian function (that reverse histopathological characteristics in PCOS) still needs further exploration. In this regard, the present study is aimed to evaluate the stereo pathological effects of metformin and N-acetyl cysteine on uterus and ovary in letrozole-induced PCOS mice to find probable mechanisms.

Figures

Figure 1.
Figure 1.
Anatomy, histology, and methods of quantification of the uterus. Anatomy of the uterus in a rat (a). Removal of uterine horn (b). The uterine horn was cut into 8– 12 slabs according to its length (c). Uniform isotropic random sections were obtained from the uterine horn by the orientator method (d).
Figure 2.
Figure 2.
Three layers of the wall uterus (endometrium, myometrium, and perimetrium) (a) and endometrial component (epithelium, gland, stroma, and blood vessels) are indicated on the histological section (b). The point-counting method was used to estimate the volume of the uterine wall layers and endometrial component (c and d).,
Figure 3.
Figure 3.
Methods of quantification of the uterus. The conglomerate (Co), cystic(C), daughter (D) and normal (N) morphological types of endometrial glands are indicated on the histological section (a). The optical disector method was applied to estimate the number of the endometrial glands (b). Estimation of the length of the blood vessels by unbiased counting frame (c). Estimation of the endometrial and myometrial thickness by orthogonal intercept method(d).,
Figure 4.
Figure 4.
The cylindrical paraffin block was cut using isotropic uniform random (IUR) sections. The histological section of the ovary was stained with Hematoxylin and Eosin (H&E) (b). The point-counting method was used to estimate the volume of the cortex, medulla, ovarian cysts, and corpus luteum (c). The optical dissector technique was applied to estimate the number of the follicles (d).
Figure 5.
Figure 5.
Shows uterus in a: control, b: metformin, c: NAC and d: PCOS group. Endometrial and myometrial thickness, epithelial height and gland density increased in PCOS. Endo= Endometrium. Myo= Myometrium. N=Normal. C= Conglomerate. Cy= Cystic. Epi=Epithelium.
Figure 6.
Figure 6.
Shows ovary in a: control, b: metformin, c: NAC and d: PCOS group. Corpus luteom, primordial follicles and grafiaan follicles decreased in PCOS. CL=Corpus luteom.
Figure 7.
Figure 7.
The level of serum testosterone, uterine weight, uterine volume. Data are the mean ± SEM. Statistical analyses were performed by ANOVA followed by Tukey’s test for multiple comparisons. Means without a common letter are significantly different (P<0.05).
Figure 8.
Figure 8.
The endometrial volume, myometrial volume, gland volume, stromal volume, epithelial volume, endometrial blood vessel volume, endometrial thickness, myometrial thickness, endometrial vessel length. Data are the mean ± SEM. Statistical analyses were performed by ANOVA followed by Tukey’s test for multiple comparisons. Means without a common letter are significantly different (P<0.05).
Figure 9.
Figure 9.
Ovarian volume,cortical volume, medullary volume. Data are the mean ± SEM. Statistical analyses were performed by ANOVA followed by Tukey’s test for multiple comparisons. Means without a common letter are significantly different (P<0.05).
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