The effect of follicular fluid selenium concentration on oocyte maturation in women with polycystic ovary syndrome undergoing in vitro fertilization/Intracytoplasmic sperm injection: A cross-sectional study

Abstract Background A high level of free radicals and oxidative substances in women with polycystic ovary syndrome (PCOS) can affect the ovaries through oxidative stress. Antioxidants such as selenium, a vital trace element in human health, can improve the prognosis of PCOS by reducing oxidative stress. Objective This study was performed due to the lack of comprehensive information about selenium concentration in follicular fluid and its effect on the oocyte count and quality in infertile women with PCOS. Materials and Methods In this cross-sectional study, 78 women with PCOS referred to Umm-al-Banin Infertility Clinic Center, Ganjavian Hospital, Dezful, Iran for in-vitro fertilization from March to November 2019 were enrolled. After ovarian stimulation with the antagonist protocol, the oocytes were retrieved under transvaginal ultrasound in in-vitro fertilization/intracytoplasmic sperm injection cycles, and selenium concentrations were measured in the follicular fluid using an atomic absorption method by spectrophotometer device. Oocyte count and morphology were evaluated using inverted optical microscopy. Results There were no significant differences between follicular fluid selenium concentrations in terms of the total number of oocytes and immature oocytes in the metaphase I and germinal vesicle stages. However, a significantly reduced number of metaphase II oocytes was observed at selenium levels < 40 μg/dL (p = 0.001). Conclusion Based on our results, low levels of follicular selenium concentration in infertile women with PCOS can reduce the quality and potency of oocyte maturation.


Introduction
Polycystic ovary syndrome (PCOS), which has metabolic and reproductive outcomes (1), is one of the most common endocrine disorders affecting approximately 7-10% of women of reproductive age worldwide (2,3). Hyperandrogenemia, high body mass index (BMI), insulin resistance, and other conditions related to PCOS can lead to augmented inflammation and oxidative stress (4).
Human follicle development involves several intraovarian and endocrine activities that initiate an alternation in the intrafollicular microenvironment for optimum conditions for oocyte competency (5).
Assessing the quality and morphology of oocytes is the basis of assisted reproductive techniques (6). Although women with PCOS undergoing in vitro fertilization (IVF) have more oocytes and less need for stimulation with gonadotropins, their oocytes are less capable of growth and are unable to complete meiosis and fertilization, and form a normal embryo (7). However, the genetic and environmental aspects of the complexity of PCOS and how PCOS affects the oocyte development are not yet fully understood (8). Follicular fluid (FF) is the net product of the shift of plasma ingredients to follicles and the secretory action of theca cells (5), as well as the blood-follicle fence of the granulosa cells, which plays vital role in oocyte development and maturation. In fact, the development and viability of oocytes are affected by certain compositions of FF (6) that can reflect any alterations in ovarian cell secretory processes and changes in the plasma components due to pathological conditions. Thus, FF improves the oocyte developmental capacity and ovulation and also acts as a medium for the connection between follicular cells and oocytes through follicular growth (9). Studies have shown higher levels of reactive oxygen species (ROS) in the FF of women with PCOS and endometriosis (5, 10). Coskun and colleagues found a positive association between a decrease in oxidative stress and an increase in oocyte maturation in infertile women with PCOS. They concluded that antioxidants could improve the prognosis of PCOS by decreasing oxidative stress (11).
Selenium is an essential element in the human body that, as an effective antioxidant in selenoproteins, supports redox activities (12,13   To minimize any probable interfering, blood-free FF samples were used for the measurements using photometric assay. To precipitate blood cells and remove cellular components, 1 ml of the harvested FF was centrifuged at 3,000 rpm for 10 min. At the final stage, only the supernatant of the blood-free samples was loaded into cryovials and stored at -70°C until assayed (19).

Statistical analysis
All of the statistical analyses were performed using the Statistical Package for the Social Sciences software (SPSS version 22.0 for Windows; SPSS Inc., Chicago, USA). The results were expressed as mean ± SD. The differences between the mean oocyte quantities were assessed by one-way ANOVA and LSD tests. P < 0.05 were considered statistically significant.

Results
In total, 741 oocytes from 78 women with PCOS were collected. The mean ± SD of the total retrieved oocytes and of the MII, MI, and GV oocytes were estimated as 9.61 ± 3.26, 7.68 ± 4.12, 2.87 ± 1.04, and 1.33 ± 0.87, respectively.
The total number of oocytes was significantly higher in women aged < 40 yr than in older women (p = 0.011), and MII oocytes was significantly higher in women aged < 35 yr than in older women (p = 0.008). In women aged ≥ 40 yr, the total number of oocytes and MI oocytes was lower than in women < 40 yr and the number of GV oocytes was significantly higher in 35-yr-old women than in younger ones (Table I) (Table I).
A duration of < 5 of infertility years was associated with a significantly higher in number of immature GV oocytes compared with longer infertility duration. However, in women with an infertility history of 5-10 yr, a significantly smaller total number of oocytes was observed than in other women. Women with an infertility duration of > 10 yr showed a significantly lower number of MII mature oocytes than women with shorter infertility duration. However, no significant relationship was found between the number of immature MI oocytes and the infertility duration ( Table I).
The mean count of MII oocytes was significantly lower in the women with an FF selenium concentration < 40 µg/ml than in the women with higher concentrations (p = 0.001). However, no significant association was observed between the selenium concentration in FF and the count of the total oocyte, the MI oocytes or the GV oocytes (p = 0.98, 0.13, and 0.22, respectively).
Although the highest total oocyte count was seen at selenium concentrations > 60 µg/ml, the difference in the total oocyte count was not significant. The number of MII oocytes in concentrations < 40 µg/ml was significantly lower than in higher concentrations (Table II).   Another study showed that increasing maternal age is associated with oocyte membrane abnormalities which occur due to decreases in fertility (31

Conclusion
According to the present study, low levels of follicular selenium concentration in infertile women with PCOS are associated with the number, maturity, and quality of oocytes taken from their ovaries. Therefore, it can be concluded that by reducing the FF selenium concentration, the oocyte quantity and maturation are likely to decrease.