Differential Gene Expression Analysis of Human Ovarian Follicular Cumulus and Mural Granulosa Cells Under the Influence of Insulin in IVF Ovulatory Women and Polycystic Ovary Syndrome Patients Through Network Analysis

ABSTRACT

Background

Polycystic ovarian syndrome (PCOS) is a commonly occurring reproductive disorder among the reproductive-aged women. Its global occurrence varies based on diagnostic guidelines, ethnicities, and locations of concern. Insulin resistance (IR) is commonly observed around 65–70% of women diagnosed with PCOS, representing a prevalent association. Consequently, the study was designed with an objective of illustrating the effect of insulin on mural and cumulus granulosa cells (GCs) of PCOS patients in comparison to normal ovulating women.

Methodology

This study is a case–control design, wherein a total of 80 participants were recruited meeting criterion of inclusion and exclusion, divided into 8 groups with each group consisting of 10 samples. The process involves the isolation and culturing of mural granulosa cells (MGC) and cumulus granulosa cells (CGC) with and without exposure to insulin. The proteins released by untreated GCs and insulin-treated GCs were extracted, and complex protein mixtures were digested with trypsin, followed by tandem mass spectrometry analysis and data processing using bioinformatics.

Results

We found 595 proteins in both control and PCOS samples, of which 310 were contributed by MGCs and 285 by CGCs. The PCOS MGCs expressed 20%, both the normal MGCs and CGCs have equal representation of 16% by each, whereas the PCOS CGCs proteins contributed 15% of the total of the proteomic expression. However, the poor expression observed with the Insulin exposure, the Insulin treated PCOS CGCs contributes 13%, PCOS MGCs contributes 8%. The normal MGCs upon the Insulin treatment give 8% then and there only 4% of proteins expressed by normal CGCs after Insulin treatment. The Venn analysis widened on their precise expression topographies. The examination of strings exhibited important protein–protein interaction pathways.

Conclusion

This is a pioneering investigation aimed to establish the link between hyperinsulinemia in localized follicular GCs and PCOS mechanisms by comparing them to control group. The examination of various attributes, mechanisms, and traits shown by genes and proteins in individuals with PCOS compared to control populations, alongside the investigation of the dynamics of these genes and proteins following exposure to insulin, holds promise for the formulation of novel hypotheses and strategies in the identification of new biomarkers.

KEYWORDS:

• Gene analysis
• gene ontology
• insulin stress
• protein expression
• tandem mass spectrometry
• venn analysis

Acknowledgments

We also acknowledge thanks to Mr Achal Tiwari, Ms Laxmi Arya, Mr Nitin Kataria, and all other technical and non-technical staff of Indira IVF Fertility Centre, Dehradun for their help and support throughout the study. We also thank Dr Neha Sharma, Application Scientist, ATPC, RCB, Faridabad (Haryana)-India.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization, H. C.; methodology, H. C. P. P.; software, P. P.; validation, P. P., H.C. and R. S.; formal analysis, P.P. H. C.; investigation, P. P.; resources, H.C.; data curation, R.S.; writing – original draft preparation, H.C., P. P.; writing – review and editing, P. P., H.C. and R. S, H. O. P., R. P., M. A. A. O., and N.M.; visualization, H.C., P. P.; supervision, H.C., M R. S, H. O. P., R. P.; project administration, H.C.; R. P.,H. O. P.; funding acquisition, H. C. All authors have read and agreed to the published version of the manuscript.

Data availability statement

Data is available with the first author of the article.

Informed consent Statement

Informed consent was obtained from all subjects involved in the study.

Institutional review board statement

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

List of abbreviations

BMI	=	Body mass index
CGCs	=	Cumulus Granulosa cells
DHEA	=	Dehydroepiandrosterone
GO	=	Gene Ontology
ICSI	=	Intracytoplasmic sperm injection
IVF	=	In vitro fertilization
MGCs	=	Mural Granulosa cells
Normal-MGCs+Insulin	=	Non-PCOS patients Mural Granulosa cells exposed to Insulin
Normal-MGCs	=	Untreated Non-PCOS patients Mural Granulosa cells
Normal-CGCs+Insulin	=	Non-PCOS Cumulus Granulosa cells exposed to Insulin
Normal-CGCs	=	Untreated non-PCOS Cumulus Granulosa cells
OMIM	=	Online Mendelian Inheritance in Man (OMIM®)
PBS	=	Phosphate buffer saline
PCOS	=	Polycystic ovary syndrome
PCOS-MGCs+Insulin	=	PCOS patients Mural Granulosa cells exposed to Insulin
PCOS-MGCs	=	Untreated PCOS patients Mural Granulosa cells
PCOS-CGCs+Insulin	=	PCOS patients Cumulus Granulosa cells exposed to Insulin
PCOS-CGCs	=	Untreated PCOS patients Cumulus Granulosa cells

Additional information

Funding

This research was supported by the Bayer Pharmaceuticals, Germany