Background

Biomedical science has changed a lot since new technologies have come out. Now, it is where many different areas of science and engineering meet to find solutions to problems that people are facing. Innovative uses of technology in healthcare can improve patient safety, clinical effectiveness, and patient monitoring, as well as help accelerate and lower the cost of develo** and testing medical therapies. Some of the new and innovative technologies that are changing the healthcare system are the carbodiimide and N-hydroxy succinimide cross-linking system [1,2,3] thin film hydration techniques [4], visible light photocatalysts [5], sonochemical approach [6], hydrothermal methods [7] photocatalytic compounds with band gap [8], high-performance photocatalytic process [9] and facile combustion routes [10].

Single-nucleotide polymorphisms (SNPs) are the alteration in the single base-pair sequence of the deoxyribonucleic acid (DNA) when it takes place at a high frequency of the human genome also termed as modern units of genetic variation [11]. These genetic variations are identified by cutting-edge research technique Genome-Wide Association Studies (GWASs) through comprehensive sequencing and analysis of the complete genome of thousands of SNPs at once, allowing for the identification of common SNPs that are linked to certain diseases [12]. Focusing on SNPs throughout the entire genome is an exciting new direction in the study of complex, common illnesses in which several genes contribute in identifying the genes to a person's risk, pharmacological reactions, susceptibility to contaminants and environmental factors; in prognosis and diagnosis of illness or trait [13].

Women of reproductive age (12–45 years) are disproportionately affected by polycystic ovary syndrome (PCOS), which has a varying prevalence rate across different ethnic groups (ranging from 2.2% to 26% worldwide) responsible for almost 40% of female infertility in India [14]. The clinical presentation of PCOS is more genetically variable and can have an impact on a wide range of organs including the ovary, pituitary, hypothalamus, pancreas, liver, adrenal glands, etc. Negative body image, compromised physical and mental health, and a lower quality of life are all serious concerns for women who suffer from PCOS [15]. At least 70 candidate genes have been identified as potentially contributing to the diseases of the syndrome, although the exact aetiology is still unclear [16]. PCOS symptoms include absent or irregular periods, anovulation, male-pattern hair growth, acne, acanthosis nigricans, obesity with central or distinct abdominal obesity, etc. Impaired glucose tolerance (IGT), atherogenic dyslipidaemia (AD), type 2 diabetes mellitus, hepatic steatosis, cardiovascular problems such as hypertension, and metabolic complications, as well as endometrial cancer, are all more common in these individuals [17].

During ovulation, the oocyte is accompanied by the cumulus granulosa cells (CGCs), immediately surrounding the oocyte, supporting the oocyte by sharing nutrients and a safe, caring environment. These cells emit hyaluronic acid, stabilises the produced proteins; and collectively hel** the oocyte to resume meiotic division, initiating the maturation process and facilitating the formation of adult cumulus-oocyte-complexes [17]. The mural granulosa cells (MGCs) line the antrum of develo** follicles surrounding the fluid-filled section of the follicle and in close proximity to the basal lamina [18]. The MGCs differentiate into luteal cells and remain in the ovary after ovulation called corpus luteum, whereas CGCs transport the egg to the oviduct. Many reports and studies have shown that the quality of oocyte production and follicular maturation are both affected by the state of these cells [19]. There are several genes present within the follicles that are differently expressed between these two divisions of granulosa cells: MGCs and CGCs [20].

Since then, CGCs and MGCs have been important in a wide range of cellular processes, such as signal transduction, making extracellular matrix, folliculogenesis, ovulation, and fertilisation. For the improvement of knowledge, many methods are used. One of these is the modern GWAS [18]. Therefore, the primary goal of this study was to look into the genome-wide association of the SNP in order to find out more about SNPs that are linked to PCOS. If these SNPs are found and proven, they could help us know and understand more about the molecular mechanism and genetic diversity of PCOS. The secondary goal of this research was to find out how different cumulus cells and mural cells are genetically and what role they play in the development of PCOS. This was carried out by looking at SNPs and gene expressions in granulosa cells with SNP arrays.

Methods

Study Participants and centre

Twenty-four women with PCOS and twenty-four control women of the same ethnicity, age range (25–37 years), and body mass index (BMI) were enrolled in the trial, which took place between January 2020 and September 2021 at Morpheus Prasad International IVF Centre and Indira IVF Fertility Centre in Dehradun. Subjects were categorised based on clinical manifestations and diagnostics, without regard to genotype. The cells' DNA was taken and combined into a pool (n = 6), and then millions of SNPs were found. This GWA study was conducted at the TERI School of Advanced Studies, New Delhi, India.

Criterion of inclusion

The age range of the women in the research was from 25 to 37 years old. According to the Rotterdam Criteria agreement, PCOS is diagnosed when two out of three of the following conditions are present: hyperandrogenism; oligoanovulation; and polycystic ovaries on ultrasound examination (less than 12 follicles measuring 2–9 mm in diameter and/or an ovarian volume & increased ovarian volume, i.e., > 10 cm3 in a single ovary). Women without any gynaecological problems serving as a control group are those who choose to undergo the in vitro fertilisation/Intracytoplasmic sperm injection (IVF/ICSI) process for infertility caused by male factors (husband or male partner). Women in the control group had normal ovarian morphology as determined by ultrasonography and had androgen levels within the reference range (0.4–3.5 nmol/L) throughout the follicular phase of their menstrual cycles.

Criterion of exclusion

A diagnosis of androgen-producing tumours, a lack of the enzyme 21-hydroxylase, non-classical adrenal hyperplasia, hyperprolactinemia, active thyroid disease, or Cushing syndrome would rule out participation. Medications likely to affect carbohydrate metabolism or endocrine parameters (oral contraceptives, anti-hypertensives, lipid-lowering, and anti-inflammatory drugs) for at least three months prior to entering the study were also exclusion criteria for both women with PCOS and control women. BMIs below 18 kg/m2 or above 45 kg/m2 were also excluded. Participants who did not sign an informed consent form were also excluded from the research.

Bioethics

All participants provided written informed consent, and the study was approved by the University Research and Ethics Committee (UREC) (DITU/UREC/2019/07/2) on July, 9th 2019. All data collected were anonymous and identity of individuals will remain confidential.

Separation of CGCs from follicular fluid

As part of IVF/ICSI, routine short or long-term gonadotropin-releasing hormone agonist injection protocols were used to stimulate the ovaries in a controlled way. After about 36 h, the follicular fluid was taken and the oocytes were taken while the patient was under a light anaesthetic. Each person's follicular fluid was taken from 3–5 follicles and put into a 15 ml polypropylene centrifuge tube with a conical bottom. The fluid was then put into Petri dishes so it could be looked at under a microscope to find cumulus oocyte complexes. After the multiple washes, both the CGCs and the pipette tips dropped off the dish. The CGCs were then mixed together and washed with 1X Phosphate Buffer Saline. Six patients' CGCs were pooled together for further study [21].

Isolation of MGCs

The rest of the follicular fluid from the previous step was put back into the tubes of the centrifuge, which contained the granulosa cells. The tubes were then spun at 800 × g for 10 min at room temperature. The supernatant was discarded, and the remaining cells at the bottom of the centrifuged tube were re-suspended with a small amount of phosphate buffer saline. This suspension was then carefully layered (to avoid mixing at this stage) over the same amount of 50% (v/v) PercollTM and centrifuged at 500 × g for 20 min at room temperature [76]. To identify the similarities and differences between variants we used both hg19 and hg38 because hg19 is a unified representation of several genomes and hg38 is a representation of alternative sequences in line with previous study [77]. The genes expressed in the nervous system (2.13 and 4.09 in hg-19 and hg-38, respectively) and the reproductive system have the highest matching scores (2.44 and 3.98 in hg-19 and hg-38, respectively). This shows that granulosa cells have the genes needed for these systems substantiate the fact that they contribute in pathogenesis of PCOS through hyperandrogenism, obesity and insulin resistance [78].

Insulin resistance is known to be linked to PCOS through Protein Kinase AMP-Activated Catalytic Subunit Alpha 2 (PRKAA2), which is also found in granulosa cells. Our study also found Matrix Metallopeptidase 9 (MMP9) is linked to a higher risk of cardiovascular disease, which is also a serious problem that can happen with PCOS in line with earlier study [79]. Since haptoglobin (HP) is responsible for anti-oxidant and anti-inflammatory properties, its levels may be lower in people with PCOS supporting the fact that it has significant pro-inflammatory role in diabetes and metabolic syndrome [80]. Through GWA studies of SNPs, the results of this study showed the presence of PRKAA2, MMP9, and HP. This shows that granulosa cells are expressing these genes, which may lead to PCOS and complications.

The results of this study show that the granulosa cells have genes that make HP, which binds to haemoglobin (Hb) and causes haemolysis when macrophages recognise this. It shows that the HP gene in granulosa cells is linked to PCOS problems due to insulin resistance mentioned in previous study [81]. The most found genes Ceramide Kinase (CERK), MMP9, Protein Phosphatase 1 Regulatory Subunit 16B (PPP1R16B), Phospholipase C Beta 1 (PLCB1), Pantothenate Kinase 2 (PANK2), Piezo Type Mechanosensitive Ion Channel Component 2(PIEZO2), THO Complex Subunit 1 (THOC1), Component of Oligomeric Golgi Complex 4 (COG4), RNA Polymerase II Subunit C (POLR2C), Nuclear Pore Complex Interacting Protein Family Member B3 (NPIPB3), PPFIA Binding Protein 2 (PPFIBP2), Neuron Navigator 2 (NAV2), Nuclear Protein, Coactivator of Histone Transcription (NPAT), Glutamate Ionotropic Receptor Kainate Type Subunit 4 (GRIK4), Heterogeneous Nuclear Ribonucleoprotein C (HNRNPC), Rab Geranylgeranyltransferase Subunit Alpha (RABGGTA), F-Box And Leucine Rich Repeat Protein 16 (FBXL16), Meteorin, Glial Cell Differentiation Regulator (METRN), Coiled-Coil Domain Containing 78 (CCDC78), Tryptase Alpha/Beta 1 (TPSAB1), Deoxyribonuclease 1 (DNASE1), etc.) are mostly expressed in the brain, ovary, and testis. These supports the fact that there is a variation in expression of individual genes in organs [82]. Genes shared by epithelial cells, hearts, ovaries, and kidneys ArfGAP With Coiled-Coil, Ankyrin Repeat and PH Domains 3 (ACAP3), G Protein Subunit Beta 1 (GNB1), Retention In Endoplasmic Reticulum Sorting Receptor 1 (RER1), Multiple EGF Like Domains 6 (MEGF6), Rho Guanine Nucleotide Exchange Factor 10 Like (ARHGEF10L), Platelet Activating Factor Acetylhydrolase 2 (PAFAH2), Small Nuclear Ribonucleoprotein U5 Subunit 40 (SNRNP40), Zinc Finger CCCH-Type Containing 12A (ZC3H12A), Spermatogenesis Associated (6SPATA6), Solute Carrier Family 1 Member 7 (SLC1A7), Ras Association Domain Family Member 7 (RASSF7), Troponin I2, Fast Skeletal Type (TNNI2), PPFIBP2, NAV2, GRIK4, HNRNPC, CERK, Suppressor Of Glucose, Autophagy Associated 1 (SOGA1), and RABGGTA) are involved in neuromuscular development, neurotransmitter reuptake, positive regulation of miRNA catabolic process, immune response-activating signal transduction, vagus nerve development, etc., as mentioned in previous study [83]. It is possible that these genes, which are found in granulosa cells, are involved in the altered physiological activities that occur with PCOS.

We found six genes that are common between hg19 and hg38 by comparing the SNP data from the two genomes. These genes include AT-Rich Interaction Domain 4B(ARID4B), COMT, HP, MMP9, NID2 and Mucin 5AC, Oligomeric Mucus/Gel-Forming(MUC5AC). ARID4B of granulosa cells, which is expressed in the testis, bone marrow, bladder, and adrenal glands, among other places, and is responsible for the overexpression of testosterone from sertoli cells, may be the cause of hyperandrogenism [84].This links to the development of hyperandrogenism commonly seen in PCOS. Placental, adrenal, and ovarian Catechol-O-methyltransferase (COMT) expression is first implicated in symptoms of PCOS such as fibromyalgia, chronic fatigue, and mild inflammation. As per the literature and results of present study these genes were also found in granulosa cells, which indicates that a significant role may be played by the genes of granulosa cells [85]. The PCOS-related behaviours of diabetic nephropathy, coronary artery disease, and inflammatory illness were all discovered in granulosa cells, indicating that HP may have a role in the aetiology of PCOS similar to the fact produced in earlier study they were found strongly associated with obesity and glucose tolerance [42]. The MMP9 gene has a role in the remodelling of tissue, reproduction, embryonic development, and the local proteolysis of the extracellular matrix, as well as in the differentiation of macrophages and the migration of leukocytes. These study outcomes validate the role of this gene in reduction in scar formation and promote neovascularization [86]. PCOS is a syndrome in which there is a disruption in the normal functions of the immune system, the development of embryos, reproduction, and the remodelling of several different tissues, organs, and systems. This provides some insight into the potential function of these genes in granulosa cells. The expression of Mucin 5AC, Oligomeric Mucus/Gel-Forming (MUC5AC) in the stomach has been connected to biliary tract sickness, itchy-dry eye disease, Sjogren's syndrome, cystic fibrosis, and other disorders implicated with PCOS [87]. In our study MUC5AC expression found in CGCs and MGCs. The fact that NID2, which is linked to cell-adhesion, cardiovascular disease, polycystic ovarian syndrome, gastric cancer, and other diseases and conditions, is expressed in these granulosa cells is an indication that MGCs and CGCs are involved in PCOS pathogenesis through expression of this gene. Earlier study correlated other diseases and conditions, such as nasopharyngeal, esophageal, and oral carcinoma linking to NID2 methylation [88].

The Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREBBP) gene plays a vital role in the interactive signalling process, as shown by the findings of studies on the interactions between proteins. It might be because of its essential function in the cellular phosphorylation signalling system, which influences processes such as cell differentiation, proliferation, growth, and cyclic regulation as mentioned in previous study [89]. If these steps are changed in PCOS, it could be because the expression of genes in granulosa cells has changed. GNB1, which we found, acts as a modulator or transducer in several transmembrane signalling pathways that control cell growth and differentiation, transport activities, protein synthesis, and other processes affected by PCOS. This is in line with the outcome of previous study explaining its importance in clinical etiopathogenesis of mutation [90]. Another gene of Kinesin family member 2C (KIF2C) is involved in chromosomal segregation during mitosis reflected in previous study [91] was also present in granulosa cells which is one of the most common complications in PCOS. In this GWA study, we found that Collagen alpha-1(XVIII) chain (COL18A1), which is involved in extracellular matrix architecture, Phospholipase-C pathway, collagen chain trimerization, ERK signalling, etc., similar to hepatotoxicity induced by drugs presented by previous study [92] affects the signalling process in PCOS, showing the role of this gene in the signal transduction pathway in skeletal muscle, which causes a change in glucose uptake as mentioned in earlier study [93].

Heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) is an oncogenic gene that is found in a wide range of tissues and organs, such as bone marrow, brain, ovary, bladder, testis, thyroid, endometrium, etc. These results are supported by one of the study carried out on hnRNP family [94, 95]. It is involved in protein metabolism, signalling through RhoGTPases, and the Rho Related BTB Domain-GTPase (RHOBTB-GTPase) Cycle. It was also found in MGCs and CGCs. Because of this gene, the cyst might continue to recur even after surgery as reported in earlier study [96, 97].

Conclusion

We have compiled a list of the characteristics of a large number of genes that are present in MGCs and CGCs, as well as their direct and indirect connections to PCOS. Based on the results, we can propose that the MGCs and CGCs express genes PRKAA2, MMP9, HP, ARID4B, COMT, MUC5AC, NID2, CREBBP, GNB1, KIF2C, COL18A1, and HNRNPC mostly connected to the clinical symptoms of PCOS, such as insulin resistance, cardiovascular disease, low-grade inflammation, hyperandrogenism, fibromyalgia, chronic fatigue, and itchy- dry eyes. We do suggest, though, that more detailed studies be done with a larger number of patients from different hospitals, regions, and ethnic groups. This would give us more information from which to reach a conclusion on the role of CGCs and MGCs in pathogenesis of PCOS.