Abstract
Hepatitis C virus (HCV) poses a global health challenge, especially due to its genetic diversity and capacity to infect 71 million people worldwide. This infection is prevalent in Pakistan, with 15% of the population affected, with varying rates across provinces. HCV primarily targets liver cells but can also impact other bodily systems, including the reproductive system. Liver infection, oxidative stress, and inflammation may directly or indirectly affect the production, transport, and functions of reproductive hormones and sperm. This study hypothesized that HCV infection may reduce male fertility in the patients. In this case–control study, 74 HCV-infected men and 12 healthy controls from Rawalpindi and Islamabad were compared for semen quality. Participants provided semen samples following strict guidelines, ensuring the reliability of results. The semen analysis, conducted using manual and computer-assisted techniques, revealed significant (p < .05) reductions in volume, concentration, motility, and morphology among HCV-infected men compared to the control group. In addition, a weak negative correlation between virus load and semen parameters was observed. These findings reveal broader health implications of HCV beyond liver damage, highlighting the need for targeted reproductive health interventions for affected men. Improved fertility preservation options and informed reproductive guidance for HCV-infected men could significantly benefit those undergoing treatment.
Introduction
Many health-related problems faced by human societies worldwide remained unsolved even at the turn of the millennium (Simoni et al., 1997). Hepatitis C virus (HCV) is a significant global health challenge. The HCV genome is highly variable, which adds to its complexity. This virus is classified into nine distinct genotypes, further divided into 30 subtypes (Choo et al., 1991). Genotype 1 is widely distributed worldwide, with subtypes 1a and 1b being more common in the Americas and Europe thansubtype1b in Asia (Bukh et al., 1995). Each year, approximately 1.5 million new cases of HCV are reported (Sy & Jamal, 2006). The global statistics for HCV infections are estimated to be more than 71 million cases representing about 3% of the world’s population (World Health Organization [WHO], 2016).
The prevalence of HCV infection varies by region (Arumugam et al., 2022). Pakistan, the sixth most populous country in the world, has approximately 10 million people affected by HCV. This results in an estimated prevalence of 10% to 15% among the Pakistani population (Ayoub et al., 2018; Jamil et al., 2010). Within the country, the HCV prevalence rates differ among its four provinces: Punjab has a prevalence of 6.7%, Sindh 5%, Balochistan 1.5%, and Khyber Pakhtun khwa is the most affected province, with a prevalence of 6.07% (Khan et al., 2017; Mahmood & Raja, 2017).
Hepatitis C disease presents several signs and symptoms. The virus primarily targets hepatocytes and can also affect the extra hepatic system. The infection may become chronic and pose a significant public health concern (Ryerson et al., 2020). Most hepatitis C infections arise from exposure to blood due to unsafe injection practices, risky medical procedures, unscreened blood transfusions, drug injections, and sexual interactions (Prati, 2006). Consequently, the prevalence of HCV infection is higher among men of reproductive age, particularly in societies like Pakistan, where men are more likely to be the primary earners and are consequently more exposed to these risks.
HCV primarily affects the liver; however, as a systemic infection, can affect other organs and systems, including the reproductive system. HCV infection may disrupt male fertility through various physiological processes (Hofny et al., 2011). The virus may indirectly lower testosterone levels by affecting the balance of hormones released from the hypothalamus and pituitary gland. The liver plays a crucial role in hormone metabolism. It regulates the levels of sex hormone-binding globulin (SHBG), which binds to testosterone and estradiol in the blood, affecting their availability. Liver damage caused by HCV can alter SHBG levels, resulting in reduced bioavailable testosterone, which is vital for sperm production and overall semen quality (Rhyu & Yu, 2021).
Men with HCV may have lower serum testosterone levels, either due to impaired liver function or direct effects of the virus on testicular cells. Low testosterone can lead to reduced spermatogenesis, affecting sperm count, motility, and morphology (Hofny et al., 2011). Chronic HCV infection may lead to the production of reactive oxygen species (ROS) and oxidative stress, which can damage testicular tissue and negatively affect sperm. This oxidative damage can reduce sperm motility, sperm morphology, and lower sperm count. HCV triggers chronic inflammation, releasing pro-inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin (IL)-6) that can disrupt the environment within the testes and epididymis where sperm matures (Medvedev et al., 2016). This inflammatory response can impair the seminiferous tubules leading to defective spermatogenesis. Men with chronic HCV are often at higher risk for metabolic disorders such as insulin resistance and obesity, which can also disrupt the hormonal balance and negatively affect semen parameters (Hasan et al., 2022).
Previous studies have indicated that patients with HCV infection and those undergoing antiviral therapies tend to have worse sperm and reproductive hormone parameters compared to control groups. Most of these studies have primarily focused on the risks of viral transmission and whether the viral genome is present in semen, either through intercourse or assisted reproductive technologies (Taha et al., 2018).
Investigating the effects of HCV on semen parameters can provide insights into the virus’s broader health impacts including male infertility. The hormonal disruption may affect both general male health and fertility. This study could have therapeutic implications, particularly for men undergoing treatment for HCV. This study could inform decisions related to fertility preservation, the timing of conception, or the need for fertility treatments. Limited research has previously been conducted on the reproductive status of HCV-infected men within the Pakistani population. This study aims to evaluate the impact of HCV infection on various semen parameters in Pakistani men.
Methods
Study Design
This study was a prospective case–control study approved by the ethical committee of the university (No. PMAS-AAUR/IEC/318). The study was conducted between February 2021 and December 2023. Informed written consent was obtained from all the participants before the data and semen samples were acquired.
Study Area and Population
This study was conducted in the twin cities of Rawalpindi (35.651° N, 73.0169° E) and Islamabad (33.0430° N, 72.0550° E). Rawalpindi is a historical city with a relatively higher population density than Islamabad, the capital city. Rawalpindi, with a population of 6,118,911 residents, and Islamabad, with 2,363,863 residents, comprise a diverse demographic (https://www.pbs.gov.pk/).
Sampling
A convenient sample comprised of HCV-infected men (n = 74) for at least 6 months served as cases and HCV-negative men (n = 12) as the control group. The semen samples were obtained from patients visiting various hospitals and diagnostic centers in Rawalpindi and Islamabad.
Inclusion and Exclusion Criteria
The cases were adult men aged 18 to 50 years, diagnosed with Hepatitis C infection confirmed by anti-HCV antibodies and/or HCV RNA testing. Men were excluded with co-infections such as human immunodeficiency virus or hepatitis B, a prior history of antiviral therapy for Hepatitis C, individuals with a history of reproductive disorders (e.g., varicocele, mumps, hormonal imbalance, or genetic infertility conditions), or those with lifestyle factors that could affect semen quality, including heavy smoking, alcohol abuse, and drug use. Men currently undergoing fertility treatment, using medications known to influence reproductive hormones, or having any recent severe illness were excluded. In addition, a control group of age-matched healthy men without Hepatitis C or any other chronic infection were included for comparison.
Data and Sample Collection
A predefined self-administered questionnaire on the socio-demographic factors, lifestyle, and medical history along with HCV load was filled out by each participant before providing a single semen sample by masturbation, ejaculating directly into a sterile vial in a private, designated area within the hospital or diagnostic center. The sexual abstinence of 3 to 5 days was ensured.
Semen Analysis
The semen samples were subjected to manual and computer-assisted semen analysis (CASA). The analysis focused on parameters such as concentration, motility, and morphology, using the program settings provided by the manufacturer (Spermolyzer (Computer-Assisted Sperm Analysis) Model IVOS II). The parameters were categorized according to the semen analysis guidelines of the WHO (2001).
Liquefaction, pH, and Semen Volume
After collection, each semen sample is allowed to liquefy at room temperature, and the time of liquefaction was noted by using a glass rod, and time was noted by using a stopwatch. Full liquefaction was indicated when the semen no longer had a gel-like consistency and flowed easily when the container was tilted. Semen volume was noted by reading the graduation on the semen sample collecting container or using a pasture pipette. A digital pH meter was used for determining the pH of semen (HI 2210 pH meter HANNA instrument, USA).
Morphology, Concentration, and Motility of Sperms
An aliquot of the sample was placed on a prewarmed slide and covered with a cover slip for analysis under a phase-contrast microscope. CASA software, integrated with the microscope, measured key semen parameters, including sperm concentration, motility, and morphology (Light Microscope Olympus CH2O, 400×, Japan). CASA evaluated motility by tracking individual sperm cells to distinguish between progressive, nonprogressive, and immotile sperm. CASA assessed sperm morphology by analyzing size, shape, and structure to identify any abnormalities. The system provided detailed data on parameters like sperm velocity, linearity, and amplitude of lateral head displacement. At least three replications were done for each semen sample.
Statistical Analysis
The mean ± S.E from the semen parameters for HCV patients and control were calculated and presented in tables and figures. The parameters in these groups were statistically compared using the Student’s t-test. A one-way analysis of variance followed by the Duncan Multiple Range Test was used to check the variation in the different age groups of the HCV-infected men. Statistical significance was defined at a p value of p = .05.
Results
Table 1 shows the profiles of men infected with HCV. The sample was comprised of Panjabi (43.37%), Pakhtoon (29.41%), and Urdu-speaking (27.70%) men. The patient population consisted of 33.74% from the labor community, 32.53% from private businesses, and 33.73% from government employees. The duration of infection among patients was categorized into three groups: 1 year (33.78%), 2 years (37.83%), and more than 2 years (28.37%). In addition, the majority of patients (90.63%) had no history of HCV infection in their families while 8.43% reported a history of infertility within their families (Table 1).
Assessment of Reproductive Health Status of the HCV-Infected (n = 74) Men
Semen Liquefaction, pH, and Volume
The mean liquefaction time and pH for the HCV-infected and the control group (n = 12).were statistically similar. The semen volume was significantly (t = −2.50; df = 12, p < .05) reduced in the HCV-infected men compared to control men (Table 2).
Semen Parameter (Mean ± SD) of HCV-Infected Patients (N = 74) and Normal Controls (N = 12)
Significantly different at p < .05 (t = −2.50; df = 12).
Sperm Concentration
The comparison of mean sperm concentration between normal and HCV human male patients reveals the sperm concentration was significantly (t = −2.97; df = 15; p < .05) lower in the experimental HCV-infected group (62.75 ± 40.30 million/ml) compared to normal men (121.92 ± 11.59 million/ml) (Figure 1).
Comparison of Sperm Concentration (Million/ml) Between Normal and HCV Human Male Patients Assessed by Computer-Assisted Semen Analysis
Sperm Motility
According to the results, all the mean measures of sperm motility were significantly (p < .05) reduced in HCV-infected men than in normal men (Figure 2). These parameters of HCV-infected and normal men include total sperm motility (61.62% ± 7.26% vs. 70.87% ± 7.46%), progressive motility (34.96% ± 8.87% vs. 44.89% ± 9.29%), rapid progressive motility (5.49% ± 1.88% vs 14.25% ± 3.71%), and slow progressive motility (30.88% ± 5.57% vs 40.86% ± 10.22%).
Sperm Progressive Motility, Rapid Progressive, Slow Progressive Motility, and Immobility Between Normal and HCV Patients
Sperm Morphology
Figure 3 shows the sperm morphology index (%) of HCV-infected and normal men. The sperm morphology index (%) was statistically higher (t = 3.39; df = 13; p < .05) in normal (72.17 ± 5.79) than in HCV-infected men (51.38 ± 7.141).
Sperm Morphology Index (%) Between Normal and HCV Human Male Patients Assessed by Computer-Assisted Semen Analysis
Various Other Parameters
Table 3 shows various parameters of sperm and semen that were found nonsignificantly (p > .05) different between HCV-infected and normal men.
Various Parameters of Sperms in HCV-Infected and Normal Men
Correlation of Semen Parameters With Virus Load, Age, and Duration of Disease
Most of the semen parameters were negatively correlated with the HCV load. This correlation was weak and statistically nonsignificant (p > .05). A similarly correlation was observed between the semen quality parameters and age of the patient (Table 4).
Correlation Coefficient (r) of Semen Quality Parameters of HCV-Infected Men With the Viral Load and Age
Discussion
This study offers detailed insights into effects of HCV infection on male fertility parameters. Our sample consisted of a diverse group of patients, primarily from Panjabi, Pakhtoon, and Urdu-speaking backgrounds, representing individuals in various socio-economic status, including labor, private business, and government employment. The socio-economic and ethnic diversity observed indicated that HCV infection crossed demographic boundaries, was a significant public health concern. Most participants did not report a family history of HCV, suggesting that transmission likely occurred outside of familial or genetic factors. A small subset had a family history of infertility pointing to potential genetic or environmental influences that could worsen reproductive challenges in individuals infected with HCV.
The evaluation of semen liquefaction time and pH showed no significant differences between HCV-infected patients and healthy controls, indicating that these specific characteristics remain unaffected. The semen volume was significantly reduced in HCV-infected men, which may suggest impaired function of the accessory glands, including the seminal vesicles and prostate. These glands maintain adequate ejaculate volume. This finding is consistent with previous research, such as the study by Hofny et al. (2011), which found decreased semen volume in individuals with chronic viral infections may be due to systemic inflammation or direct viral effects on glandular secretion.
The study found a significant decrease in sperm concentration among men infected with HCV compared to the control group, confirming earlier research (Durazzo et al., 2006; Moretti et al., 2008). This reduction in sperm concentration may result from direct viral damage to testicular cells or from an immune-mediated response that disrupts the process of spermatogenesis that could be a concern for a successful conception. In addition, sperm motility was compromised in HCV-infected men. Motility directly impacts the sperm’s ability to navigate the female reproductive tract and achieve fertilization. The decline in motility has been linked to viral-induced mitochondrial dysfunction and inflammatory responses that impair motility mechanisms (Safarinejad et al., 2010). Research by Zignego and Brechot (1999) and Hofer et al. (2010) supports the connection between HCV infection and reduced sperm motility due to oxidative stress and the presence of viral components in seminal plasma.
Sperm morphology was significantly impaired in men infected with HCV. Abnormal morphology is associated with reduced fertilization potential, which further increases the risk of infertility in HCV patients. This alteration in sperm structure may be due to oxidative stress or the immune response to chronic HCV infection, which disrupts normal sperm development and maturation. Hofny et al. (2011) observed similar results, reporting an increase in abnormal sperm forms among men with HCV infection.
Our study found negative correlations between the age of HCV patients and several parameters of semen quality. The HCV RNA viral load showed negative correlations with semen quality, highlighting how levels of viral replication can affect reproductive health. Our findings align with previous research, reinforcing the link between chronic HCV infection and declining semen parameters. For example, Hofny et al. (2011) reported reductions in semen volume, sperm concentration, and motility among patients with chronic HCV. Similarly, studies by Brault et al. (2016) and Machida et al. (2006) indicate that HCV infection may induce oxidative stress, which can impair spermatogenesis and lead to reduced sperm motility.
The findings highlight the negative effects of chronic HCV infection on male reproductive health, specifically regarding sperm concentration, motility, and morphology. These impairments can significantly decrease the chances of successful conception in HCV-infected men. The study found a correlation between the age, viral load, and semen quality. Early diagnosis and treatment of HCV infection may help preserve reproductive potential. This research emphasizes the need for more studies to explore the effects of HCV on spermatogenesis and to investigate potential therapeutic strategies to mitigate these impacts, particularly for individuals of reproductive age. Overall, this study adds to the existing evidence about the harmful effects of viral infections on male fertility and underscores the importance of addressing reproductive health in the clinical management of chronic HCV infection.
This study has several limitations. First, its cross-sectional design restricts the ability to establish limiting the ability to determine causality or assess changes in semen quality over time in HCV-infected men. The study included only 74 HCV-infected men and 12 healthy controls, which limits the ability to generalize the findings to broader populations. A larger sample size would be necessary for stronger conclusions. The participants were recruited from Rawalpindi and Islamabad cities, which may not represent the whole population of Pakistan or the wider global population. Although the semen analysis provides valuable information, it does not fully address other important aspects of male fertility, such as sperm DNA integrity, hormone levels, or functional sperm tests. Moreover, the study does not consider potential confounding factors such as age, lifestyle factors, environmental conditions, or other medical conditions that could influence semen parameters. The study does not account for the genetic diversity of HCV, which may influence the severity of infection and its impact on reproductive health. While the study suggests that liver infection, oxidative stress, and inflammation could affect reproductive health, it does not provide direct evidence or an in-depth study of these mechanisms.
Footnotes
Authors’ Contribution
S.A. conceived this research idea and supervised the study. J.D., S.A., and M.A. conducted the field study by collecting the samples and questionnaire data besides writing the initial manuscript. M.I. and M.J.A. facilitated data analysis and interpretation of the results. M.A. developed the study area map. J.D. wrote the initial manuscript and M.I. edited the final draft.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Ethical Statement
We strictly followed the PMAS-Arid Agriculture University’s Ethical Committee’s protocol and guidelines (reviewed in the 26th meeting held on 6th January 2020, No. PMAS-AAUR/IEC/318) during the conduction of this research study. No animal, in any form, was harmed during this study.
Information on Patient Consent
A written informed consent was acquired from all the men participated in this study.
