SIRT1 and antioxidants in infertile females: Exploration of the role of vitamin D.

Infertility is defined as the inability for a couple to conceive after 12 months of regular sexual intercourse [ 1 ]. In the reproductive cycle, follicular granulosa cells form an important association with follicle’s survival that determines ovulation and hence fertility [ 2 ]. The development of follicles in the available follicle pool is dependent on a number of factors like hypoxia, heat stress, and oxidative stress (OS) [ 3 ]. Several toxicants and pesticides modify the defence system producing various lifestyle-related diseases due to OS and reproductive disorders like endometriosis, PCOS, preeclampsia, spontaneous abortion, and unexplained infertility [ 4 , 5 ]. High levels of OS markers are hypothesized to compromise the quality of oocyte and subsequently, reproductive potential in women living with infertility [ 6 ]. Therefore, it is essential that the redox environment in the oocyte is maintained at beneficial levels. VD acts as a membrane antioxidant, protects cell membranes against free radical-induced lipid peroxidation through interface with phospholipid fatty acid side chains and intensifies stabilization of the membrane structure and follicular development [ 7 – 9 ]. VD status is considered to be deficient, inefficient and sufficient on the basis of serum 25-hydroxyvitamin D (25(OH)D) level of less than 20 ng/mL, between 20 to 29.9 ng/ml greater than 30 ng/ml30 ng/mL [ 10 ]. VD levels above 100 ng/ml are labelled hypervitaminosis D [ 11 ]. Deficiency of VD with an increase in OS markers, altered sperm parameters and impaired fertility has already been explored [ 12 ]. VD supplementation in women has been shown to reduce the accumulation of pro-inflammatory advanced glycation end-products (AGEs) and inhibit the formation of reactive oxygen species (ROS) in ovarian tissue [ 8 ]. The estimation in follicular fluid (FF) of women living with infertility has suggested its important role in adjusting steroid hormones with a reduction in OS and hence enhancing chances of fertility [ 13 ]. ( Fig 1 ). SIRT1, silent information regulator 1; ROS, reactive oxygen species; FSH, follicle-stimulating hormone; LH, luteinizing hormone. Silent information regulator 1 (SIRT1) is a histone deacetylase one of the members of the Sirtuins family, and has been shown to maintain the redox environment in the body, via the regulation of mitochondrial OS [ 14 ]. Mitochondrial dysfunction, which may also affect the redox environment in the cell, has been linked to infertility, via the Sirtuins family of proteins [ 15 ]. It works through transcription factor Forkhead box O3 (FOXO3a) and peroxisome-proliferator activator-receptor γ-coactivator 1α (PGC-1α), which is a transcription co-activator. Together, they induce the expression of antioxidant genes and deacetylation of histone proteins [ 16 ]. SIRT1 regulates many antioxidants, including manganese superoxide dismutase (MnSOD) [ 16 ]. It is a mitochondrial antioxidant, playing an essential role in the aerobic respiration of cells [ 6 ]. SIRT1, known to regulate many antioxidant levels, is positively correlated with visfatin, glutathione reductase (GR), adrenaline, and VD. These findings represent the positive influence of elevated Sirtuins on a total antioxidant status and a negative association with cortisol [ 17 ]. Glutathione is a family of enzymes that includes glutathione peroxidase (GPx), glutathione S-transferase (GST), and glutathione reductase (GSH) [ 18 ]. It is an endogenous antioxidant that protects the cell from OS and also preserves other antioxidants such as vitamins C and E; the role of these vitamins has been already documented [ 19 ]. Visfatin is an adipokine released from visceral adipose tissue dominantly [ 20 ]. Superoxide dismutase 1 (SOD1) knockout mice show that SOD1 is required for the development of ovaries and female reproductive function [ 21 ]. Adrenaline is a chemical stressor released from the adrenal medulla as well as cortisol. Cortisol does affect the maternal metabolic adaptions and embryonic development [ 22 ]. Increased levels of SIRT1 are related to decreased levels of cortisol [ 23 ]. Therefore, in this study, we want to estimate the levels of VD, SIRT1 and antioxidants (MnSOD, GR and visfatin) and oxidants (adrenaline & cortisol) in individuals living with infertility and explore the association of VD with SIRT1 expression (levels), antioxidants, and oxidants contributing to infertility in women.

Table 1 reports the baseline characteristics of the participants, in the present study, there were 342 samples; primary type of infertility was higher than the secondary cause. With median age of 22 (19–26) years at the time of marriage, the duration of marriage was 7(4–11) years. Results showed that the highest cause of infertility was due to PCOS followed by endometriosis. PCOS, polycystic ovary syndrome; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone. Table 2 demonstrates the interquartile range of fertility parameters. All the antioxidants and levels of VD are statistically significantly higher in the fertile group; however, adrenaline and cortisol were lower in the fertile group when compared with the group living with infertility. Fig 2 represents the levels of VD and glutathione reductase in fertile controls and women with infertility. (a) Vitamin D; (b) Glutathione Reductase. SIRT1, silent information regulator 1; MnSOD, manganese superoxide dismutase; IQR, interquartile range. Table 3 represents the correlation of VD with the study variables. The absolute magnitude of the coefficient (r) indicating the strength of the linear relationship between VD and MnSOD, SIRT1, Visfatin and the adrenaline was weak. The correlation of VD with Glutathione reductase was moderately strong (r = 0.507, p < 0.01). **Spearman Correlation is significant at the 0.05 level (2-tailed). SOD, superoxide dismutase; SIRT1, silent information regulator 1. Table 4 represents the levels of all study parameters based on VD deficient and sufficient groups. MnSOD levels were significantly high in VD sufficient groups however, adrenaline and cortisol levels were significantly high in groups suffering from VD deficiency. Dependent Variable: Vitamin D Mann-Whitney U test Deficient: 30ng/ml Insufficient: 20.1–29.9ng/ml (no sample was categorized as Vitamin D insufficient None of the study subjects fell in the insufficient group of VD.

Oxidants and antioxidants impact female fertility. The deficiency of VD is associated with a decrease in SIRT1 and other antioxidants, which may deter natural reproductive functions leading to infertility. The study suggests that VD deficiency can result in decreased levels of antioxidants and SIRT1, leading to inflammation, mitochondrial malfunctioning, and apoptosis through the hypothalamic-pituitary-ovarian axis, resulting in poor-quality oocytes and infertility. The study findings further propose that VD supplementation may be a potential therapeutic option for improving female fertility. However, further studies are required to determine the cause-effect relationship of VD deficiency on conception and the involved mechanisms. Overall, this study provides valuable insights into the potential role of VD and antioxidants in female infertility. SIRT1 is a notable target in various disease situations due to the promise of pharmacological and/or natural modulators of SIRT1 activity within the framework of endocrine and immune-related disease models.

This study received extended approval from the Ethical Review Committee (ERC: 2020-0314-14433), Aga Khan University, Karachi, Pakistan. The cross-sectional study was carried out from July 2020 to July 2021 at the Aga Khan University (AKU), Karachi, Pakistan collaborating with the Australian Concept of Infertility Medical Centre (ACIMC), Karachi, Pakistan. Patients were recruited from the infertility clinic (ACIMC) during the clinic visits employing the convenient sampling method. The sample size was estimated using Open-Source Epidemiologic Statistics for Public Health. Observing Pakistan’s infertility rate of 23% and in order to achieve 80% power and detecting an odds ratio of at least two, assuming hypothetical proportion of control with exposure to be 23%, and two-sided confidence (1-alpha) of 5%, the minimum sample size was 320 female participants [ 24 ]. Out of the sample size of 342 women, there were 135 (39.5%) and 207 (60.5%) fertile female subjects. All patients were taken after acquiring written informed consent. Patients were examined for general health checkups, height and weight measurements, and body mass index (BMI) calculations were done. The baseline hormonal profile (follicular stimulating hormone, luteinizing hormone) was obtained and noted from desk records of ACIMC. Fertile women aged 18–40 years old were included in the study from all ethnic backgrounds bearing a child less than three years of age and were in sexual interaction with their male partners for at least the preceding three months. The cases comprised female subjects with primary infertility of more than two years. Women with secondary infertility, including ovarian cysts and endometriosis with ovarian pathologies, who had been using contraceptive pills in the preceding three months were excluded from the study. Women with endocrine disorders like diabetes mellitus and thyroid problems were also excluded. From each subject, 3 ml blood samples were collected in a tube. The collected samples were transported in ice boxes to AKU Multidisciplinary laboratory for storage and further analysis. The cases were previously diagnosed cases of infertility. Serum samples were used for estimation of MnSOD, SIRT1, visfatin, glutathione reductase, VD, adrenaline, and cortisol in fertile and infertile female participants. Estimation of MnSOD . Serum manganese superoxide dismutase was estimated with the commercially available Enzyme Immunoassay kit for Human Mitochondrial Superoxide dismutase (SOD2) ELISA Kit, Cat. No. SG-10189 (Sinogeneclon Co., Ltd., Hangzhou, China) according to the manufacturer’s protocol. The sensitivity of the kit was 3 pg/ml and the detection range of the standard was 15.6–1000 pg/ml with intra and inter-assay coefficient of variation: CV<8% and CV<10% respectively. Estimation of SIRT1 . Serum SIRT1 were analyzed with a commercially available Enzyme Immunoassay kit for Human Sirtuin 1 (SIRT1) ELISA Kit, Cat. No. SG-10458 (Sinogeneclon Co., Ltd., HangZhou, China) according to manufacturer’s protocol. The sensitivity of the kit was 0.1 ng/ml and detection range of the standard was 0.5–18 ng/ml with intra and inter assay coefficient of variation: CV<8% and CV<10% respectively. Estimation of visfatin . Serum visfatin was determined by Human visfatin (VF) ELISA Kit, Cat. No. SG-10381 (Sinogeneclon Co., Ltd., HangZhou,China) according to manufacturer’s protocol. The sensitivity of the kit was 0.3 μg/L and detection range of the standard was 1–20 μg/L with intra and inter assay coefficient of variation: CV<8% and CV<10% respectively. Estimation of glutathione reductase . Serum glutathione reductase was determined by Human Glutathione Reductase (GR) ELISA Kit, Cat. No. SG-00523 (Sinogeneclon Co., Ltd., HangZhou, China) according to manufacturer’s protocol. The sensitivity of the kit was 0.1 pg/ml and detection range of the standard was 18–1000 pg/ml with intra and inter assay coefficient of variation; CV<8% and CV<10% respectively. Estimation of vitamin D . VD was analyzed by a 25(OH) Vitamin D ELISA kit, Cat. No. ab213966 (Abcam, Waltham, MA 02453, USA) according to manufacturer’s protocol. The sensitivity of the kit was 1.98 ng/ml and detection range of the standards was 0.5–1010 ng/ml with intra and inter assay coefficient of variation: CV<4% and CV<15% respectively. Estimation of adrenaline . Serum adrenaline was analyzed with a Human Epinephrine (EPI) ELISA Kit, Cat. No. SG-10545 (Sinogeneclon Co., Ltd., HangZhou, China) according to manufacturer’s instructions. The sensitivity of the kit was 0.25 ng/ml and the detection range of the standards was 7–150 ng/L with intra and inter-assay coefficient of variation: CV<8% and CV<10% respectively. Estimation of cortisol . Serum cortisol was determined using a Cortisol ELISA Kit, Cat. No. DKO001 (DiaMetra, Perugia, Italy) as per manufacturer’s instruction. The assay range of the kit was 10–500 ng/mL. The quantitative variables were divided into two categories: women who were fertile and infertile. The chosen grouping categories were based on causes of infertility including PCOS, male factor, endometriosis, tubal and unexplained infertility. These groups were chosen based on the increasing number of women attending infertility clinic with these causes. Data were analyzed by using IBM SPSS Statistics (RRID: SCR_016479) version 23.0. Counts with percentages have been reported for baseline features of studied samples. To determine the distribution of the variable studied parameters of fertility, it was important for choosing an appropriate statistical method. Thus, the Shapiro-Wilk test was performed and showed that the distribution of all parameters departed significantly from normality (p-value < 0.01). Based on this outcome, non-parametric tests (Mann-Whitney U, Spearman’s correlation) were used, and the median with the interquartile range was used to summarize the variables. Mann-Whitney U test was performed to compare the differences in fertility parameters between the fertile and infertile groups. Spearman’s correlation analysis was performed to study the strength of the association between parameters.

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