Study Protocol: Effect of Lifestyle Modification on Sperm DNA Fragmentation Index

ABSTRACT

Background:

Infertility is a growing concern, affecting 8%–12% of reproductive-age couples worldwide. Male infertility accounts for 20%–30% of cases and contributes to 50% of fertility problems. Semen analysis is the traditional method used to assess male fertility, but it may not adequately reflect sperm DNA integrity, which plays a crucial role in successful conception. Increased oxidative stress (OS) and lifestyle factors like obesity, smoking, and alcohol consumption are known to damage sperm DNA, leading to infertility. Sperm DNA fragmentation (SDF) has emerged as a critical parameter for assessing male fertility potential. This study aims to evaluate the impact of lifestyle modifications on sperm DNA fragmentation in men with infertility.

Objective:

The objectives of this study are to identify male partners exhibiting abnormal semen parameters, with a focus on assessing the impact of lifestyle modifications—such as quitting smoking, reducing alcohol intake, and increasing physical fitness—on sperm DNA fragmentation. Additionally, the study aims to evaluate the role of sperm DNA integrity in the diagnosis of infertility, as sperm DNA fragmentation is increasingly recognized as a critical factor influencing fertility outcomes. By examining these factors, the study seeks to enhance understanding of how lifestyle changes can potentially improve male fertility through the reduction of DNA fragmentation.

Materials and Methods:

This prospective study will be conducted at Sawangi, Maharashtra, over 6 months. The study will include male patients with sperm abnormalities and high DNA fragmentation enrolled at an IVF center. Semen analysis, DNA fragmentation testing (using the sperm chromatin dispersion test), and a detailed lifestyle questionnaire will be conducted. Participants will receive advice on lifestyle changes, including cessation of smoking and alcohol consumption and encouragement of physical fitness. Post-modification sperm DNA fragmentation levels will be reassessed.

Expected Results:

A reduction in sperm DNA fragmentation index (DFI) following lifestyle modifications results in improved sperm quality and fertility outcomes.

Study Implications:

This study will offer valuable insights into the potential benefits of lifestyle changes on male fertility. It will highlight the importance of sperm DNA integrity and provide a diagnostic tool for male infertility.

INTRODUCTION

When a clinical pregnancy cannot be established after 12 months of regular, unprotected sexual activity, it has been defined as infertility. Around the world, between 8% and 12% of couples who are of reproductive age are thought to be affected by infertility. However, males account for 20%–30% of infertility and contribute to 50% of it.[1] The traditional method of Semen analysis is used now to assess infertile males, even though it cannot accurately forecast the effectiveness of ART (assisted reproductive technology) or the potential for male fertility. Average semen analysis is present in 15% of infertile people. Assessments of sperm quantity, motility, and shape, however, might not accurately represent sperm DNA integrity issues.[2] Sperm function tests, however, have become less popular in most andrology laboratories with the development of intracytoplasmic sperm injection (ICSI).[3] A wide range of variables might affect male fertility, and one reason for the downward trend might be environmental, occupational, and lifestyle variables that contribute to the decline in sperm quality. The following are the lifestyle factors impacting male infertility: obesity, alcohol intake, smoking, psychological stress, and advanced paternal age.[4] Redox imbalance, DNA damage, and decreased sperm motility in Reactive oxygen species (ROS) are raised, and antioxidant defense is diminished in sperm. Due to their cell membranes’ high concentrations of unsaturated fatty acids, spermatozoa are especially susceptible to the negative consequences of ROS. Lipid peroxidation is facilitated by reactive oxygen species, which increases the intracellular oxidative burden. Lipid peroxidation, membrane permeability loss due to loss of membrane integrity, structural damage to DNA, reduced sperm motility, and apoptosis are the subsequent processes. Increased oxidative stress (OS) in the male reproductive system has been linked to several intrinsic and extrinsic variables.[5] ROS are produced in excess compared to the follicular fluid’s size to detoxify them, creating oxidative stress inside the ovarian fluid surrounding the growing egg. Because of harm to the egg’s DNA, alterations in the follicular milieu, and a lower supply of severe oxidative stress, antioxidants in the follicular fluid have been linked to reduced oocyte quality and decreased fertilization rates. Ultimately, this can result in a decline in the pregnancy rate.[6] ROS, a hazardous oxidant, are increased by smoking in seminal plasma. This imbalance between antioxidants and oxidants causes oxidative stress. Sperm DNA may become harmed because of oxidative stress. Additionally, research has demonstrated that developing DNA addicts linked to cigarette smoking may also result in sperm DNA damage. This could impact the developing embryo and the sperm epigenome and genome.[7]

The integrity of sperm DNA has drawn interest in the last 30 years due to research on novel methods of evaluating sperm abnormalities. The sperm DNA fragmentation (SDF) assay, which assesses the sperm’s suitability as a DNA package carrier, was more remarkable than the parameters considered in the earlier semen assay.[8] Since 1999, studies have shown a noticeable rise in demonstrating a connection between SDF and male infertility. Based on a recent scientific metric analysis, the primary topics of SDF research over the preceding 20 years have been lifestyle factors and asthenozoospermia, varicocele, and Increased (SDF) Stages have a connection with illnesses such as varicocele, advanced paternal age, male accessory gland infection, chronic sickness, cancer, exposure to environmental contaminants, lifestyle choices, and infertility in males. Furthermore, some research has shown that lower SDF negatively affects conceiving rates. Evidence indicates that DNA damage in spermatozoon can affect children’s welfare and health.[2] DNA damage, such as denaturation and fragmentation, which might negatively impact embryo development and fertilization, can cause infertility. There is a greater likelihood that the progeny of spermatozoa with DNA damage will have genetic disorders. Lower sperm DNA integrity is observed in infertile males, and men with typical semen test results may have more significant DNA damaged than fertile males with SDF. SDF has been added to semen analysis processes due to its utility as a separate index for assessing the quality of semen. Although ICSI has increased in popularity globally, instances of successful fertilization following ICSI have increased. Poor sperm DNA or semen analysis results have raised concerns about the clinical utility of SDF testing. We now live in an era where ICS has numerous advantages over natural selection. When employing ART, the choice of sperm with damaged DNA might have unfavorable effects such as increased miscarriage rates, decreased pregnancy success rates, congenital disabilities, abnormalities in chromosomes, and other genetic or congenital diseases in the progeny. Therefore, it’s critical to correctly grasp and oversee sperm nuclear DNA integrity in the era of ART.[9] This study determines the assessment of sperm DNA fragmentation index with lifestyle modification.

The objective of the study

1. To identify male partners with abnormal semen parameters.

2. Study of sperm DNA fragmentation in males with lifestyle modification.

3. To assess the integrity of genetic material within the sperm cells.

4. Understanding the level of DNA infertility helps diagnose related to infertility.

Hypothesis

Adopting a healthier lifestyle is expected to reduce the sperm DNA fragmentation index in males while improving fertility outcomes and sperm quality.

MATERIALS AND METHODS

The study will be conducted at Sawangi, Maharashtra, for the duration of 6 months

Study population – Patients with lifestyle modification and increased DNA fragmentation enrolled at IVF Sawangi (M), fitting into inclusion and exclusion criteria [Table 1].

Table 1.

Inclusion and exclusion criteria

Inclusion	Exclusion
Patients having infertility issues.	The age of patients is more than 50 years.
Patients with sperm abnormalities.	Patients who are not willing to give samples.
Patients with poor semen parameters	Patients suffering from infectious disease.

Methodology

Patients visiting the infertility center for assistance with fertility issues undergo a thorough evaluation to ensure they meet the necessary health standards for further testing. Initially, their reports are verified for HIV and hepatitis to maintain safety protocols. Semen analysis is then conducted to assess sperm count and morphology, evaluating overall sperm quality. A DNA fragmentation test follows to determine the integrity of sperm DNA, identifying patients with high DNA fragmentation for potential inclusion in the study. A comprehensive history is taken, including general and physical examination, dietary habits, occupational background, and lifestyle practices, to evaluate the overall health status of the patients. Lifestyle modifications form a significant part of the study, with patients being strongly advised to discontinue alcohol intake and quit smoking to improve long-term health and fertility outcomes. Patients are also encouraged to adopt a fitness routine with the guidance of a professional and to prioritize physical exercise, which positively impacts reproductive health. Additionally, the importance of staying adequately hydrated is emphasized to support optimal reproductive function, with these lifestyle adjustments contributing to overall well-being and enhancing fertility outcomes.

Methods of sperm DNA fragmentation index

Assessing human sperm DNA fragmentation through the Sperm Chromatin Dispersion (SCD) test is a crucial step in evaluating male fertility. Sperm DNA integrity is recognized as an essential factor for successful reproductive outcomes, making DNA fragmentation testing a valuable tool. Compared to routine semen analysis, sperm DNA fragmentation tests offer superior diagnostic and prognostic potential. While various methods exist for identifying DNA fragmentation, the SCD test stands out as a simple, reliable, and repeatable technique. This method allows accurate quantification of DNA fragmentation using conventional bright-field microscopy, providing critical insights into sperm quality and its impact on fertility.

Kit contents

• Pre-coated slide 10 Nos

• Agarose 12 Nos

• Solution A

• Solution B

• Solution C

• Solution D.

Sample size calculation

$$n=\frac{(\sigma 21+\sigma 22/K){(Z1-\alpha /2+Z1-\beta )}^2}{{\Delta }^2}$$

Were,

Mean implantation rate in study group = 0 = 0.148

Mean implantation rate in control group = 0.04

σ1 = SD of implantation rate in study group = 0.23

σ2 = SD of implantation rate in control group = 0.13

For detecting mean difference of 0.108 i.e., ∆ = 0.148-0.04 = 0.108

$$n=\frac{(0.23*0.23+0.13*0.13){(1.96+084)}^2}{(0.108*0.108)}$$

= 50 patients required in each of the total sub-groups.

Analysis

This study examines the impact of lifestyle modifications on sperm DNA fragmentation in male infertility patients. A total of 100 patients with lifestyle-related sperm abnormalities were enrolled and underwent semen analysis and DNA fragmentation tests. After lifestyle interventions, including alcohol cessation, smoking cessation, and exercise encouragement, statistical analysis using SPSS or R was conducted to compare sperm DNA fragmentation before and after the modifications. These lifestyle changes are expected to significantly reduce DNA fragmentation, improve fertility outcomes, and provide insights into which factors most influence sperm DNA integrity.

EXPECTED RESULTS

Reduction in sperm DNA fragmentation index (DFI) following lifestyle modification, improving sperm quality and fertility outcomes.

DISCUSSION

According to Ashok Agarwal et al.[3] 2023, D.N.A. from sperm plays a crucial role in human reproduction. They give sufficient data to conclude that SDF testing is a helpful diagnostic tool for assessing male infertility. They said the last 20 years of study helped to understand the function of SDF testing and its indication. Along with Siemen’s analysis, SDF should be considered, while evaluating male factor fertility.

According to Alahamar Ahmed T 2019, Reproductive mechanisms, including enhanced ROS capacitation and the acrosome reaction, require reactive oxygen species. Lid to oxidative stress status, which causes sperm lipid membrane peroxidation, sperm DNA damage and high risk of genetic disease in offspring. Research for oxidative stress and different diagnostic and treatment methods has been established. There needs to be more consensus in choosing OS stress, the kind and length of antioxidant treatment, and identifying the patient population to be targeted. To increase the reproductive potential and lower neonatal risk for genetic disorders and cancerous tumors, further study is warranted to overcome these limitations.[5]

Valeria Maria Iommiello et al.[9] 2015 said that Sperm DNA fragmentation and high concentration of round cells is related to OS. It also noted that assessing OS and damaged sperm DNA would significantly improve semen analysis profile and serve as a tool for determining male infertility, particularly in idiopathic origin.

According to Akira Komiya et al.[10] 2023 they said Comorbid medical conditions and modified lifestyles affect men trying to conceive. The individual’s medical condition and lifestyle should be considered to provide appropriate advice during semen analysis. In 99% of patients, minimal one of the medical or lifestyle variables that impact fertility was present.

Peter Humaidan et al.[11] 2022 reported changes in lifestyle and regimen of antioxidant therapy that decreased the DFI of patients for 3 months. Then, they compared the healthy group that gave two semen samples in 14 days without any lifestyle changes, and then they saw no difference OR. Lifestyle changes and antioxidant interventions may improve fertility by improving DFI.

According to Sandro C Esteves et al.[12] 2020 research on the influence of sperm DNA fragmentation on the development of sperm DNA fragmentation, 41 suggestions were created using the evidence gathered 13 of these suggestions deal with the technical component of sperm DNA fragmentation test, and the remaining 28 deal with clinical treatment and management of sperm DNA fragmentation test.

Study implications

This study explores the impact of lifestyle modifications on sperm DNA integrity, emphasizing healthy habits like quitting smoking, reducing alcohol, and improving fitness to enhance male fertility. By using the SCD test highlights advanced diagnostics for accurate infertility assessment.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.