Newborns’ overall health and lifetime development are greatly influenced by their mode of delivery which influences their gut flora and has an effect on their short- and long-term well-being. The immune system, mental health issues, and metabolic diseases have all been associated with abnormalities gut microbiota, highlighting the critical role that gut microbiota plays in determining one’s quality oflife. The gut microbiota functions as an extension of the host genome, containing 50-100 times more genes than the host and providing vital enzymes that the host is unable to manufacture. Many disorders can result from any changes in this microbial mix, known as dysbiosis.

The mode of delivery along with other factors like feeding schedule have a significant impact on an infant’s gut microbiome health.Delivery methods such as vaginal and caesarean sections are crucial in determining the microbiological makeup. Because lactobacilli are so prevalent in the vagina, newborns delivered vaginally have higher than average quantities of these bacteria. On the other hand, babies born by caesarean section have lower microbe counts from the bacteroides, genus.

The mode of delivery related differences in the composition of the gut microbiota is closely related to the establishment and development of the neonates immune system. Research has indicated that the gut microbiota plays a significant role in controlling the immune system, and it has been linked to allergic and hypersensitive reactions via affecting the expression of the genes including FOXP3 CTLA4, GATA3, IKZF2, IFNG, IL10RA, and TGFB1 gene.

So, this work deliberately focuses on important genes-CTLA4, FOXP3, GATA3, IKZF2, IFNG, IL10RA, and TGFB1-that are known to affect immune regulation and response. Immunological homeostasis is maintained by CTLA4, a crucial regulator of T-cell activation that sheds light on the fine balance between tolerance and immunological response. The project’s focal point, FOXP3, serves as a pivotal player in the development of the immune system and is particularly linked to regulatory T cells (Treg). It provides insight into the complex interplay that occurs between the gut microbiota and immune regulation. The greater influence of gut microbiota on immune cell development is shown by GATA3, a crucial component in T-cell differentiation. The transcription factor IKZF2’s participation provides insight into how gut microbiota may affect the genetic machinery responsible for immune cell specification. The foundation of immune responses, IFNG, sheds light on the complex interplay between gut microbiota and the maturation of efficient immune defence systems. The anti-inflammatory cytokine IL-10 receptor, or IL10RA, provides insight into possible regulatory pathways by which the gut microbiota may affect immune responses and the finely balanced relationship between inflammation and tolerance. The investigation focuses on how gut microbiota may influence signalling cascades essential for immune systemdevelopment is furthered by the discovery of

TGFB1, a crucial regulator of immune cell activity. We will also be looking into the central focus of our study FOXP3 gene expression in T cells by flowcytometric measurement and quantification. This will be performed by using extracellular and intracellular fluorochrome markers to identify the cell populations showing increased FOXP3 gene expression which will provide further insights into the immune status of the infant. So, this work aims to decipher the complex genetic patterns influencing immunological development in neonates by synthesising these genetic aspects with the mode of delivery. Understanding the complex interactions among the host genome, gut microbiota, and immune system helps advance our understanding of the factors that influence health in the early years of life.

Study Design: A Prospective Cohort study

Sample Size: 40 Newborn babies will be included in the study.

Group A: 20 healthy infants delivered through caesarean section delivery.

Group B: 20 healthy infants delivered through normal vaginal delivery.

Inclusion & Exclusion Criteria :

Inclusion Criteria: All consenting individuals born in BLDE (DU)’s Shri B M Patil Medical Collage, Hospital & Research Centre who are breast fed will be included in the study.

Exclusion Criteria:

Those neonates whose mothers not willing to give consent.

Deliveries of unknown gestational age

Babies with known health conditions that may interfere with the gut microbiota and immune functions of the infant will be excluded from the study.

Who are not breast fed born to mothers with known health conditions with known infectious diseases, congenital anomalies, metabolic diseases.

Written Consent: Written informed consent will be obtained from all the participants before enrolment.

Study Protocol:

• Gene Expression Studies:

Extracting RNA:

5ml of blood samples will be collected in EDTA tubes from infant delivered after the 36 weeks gestation, after 03 months and 06 months of age.

To extract RNA, use TRIzol.

Analysis of the purity and concentration of RNA.

Specific primers will be designed for FOXP3, CTLA4, GATA3, IKZF2, IFNG, IL10RA, and TGFB1. Use FOXP3, CTLA4, GATA3, IKZF2, IFNG, IL10RA, and TGFB1 primers and CDNA will be synthesised to do PCR.

Electrophoresis of gels:

Examination of the PCR results will be done by using an agarose gel.

Relative expression of FOXP3, CTLA4, GATA3, IKZF2, IFNG, IL10RA, and TGFB1 will be done by aPCR.

Statistical Analysis:

Statistical analysis will be done in SPSS software.

In gene expression plots, fold change is plotted, but statistical analysis will be made on threshold cycle (ACt) values. The p values<0.05 will be considered significant.

Sequencing data analysis

Multivariate ANOVA (PERMANOVA) will be used to evaluate group differences based on UniFrac distance matrices. Differences were considered significant at P<0.05.