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ABSTRACT
The purpose of this study will be to analyze Chlamydia and determine the effects of DNA fragmentation on male fertility. Chlamydia is caused by an obligate intracellular bacterium, Chlamydia trachomatis (CT). Chlamydia trachomatis infection is sexually transmitted and most common in sexually active partners. 50% of men who are infected by the bacterium show no signs of the bacterium. The infection has a shelf-life of up to four years, and when couples are infected, they have a much impact on sperm quality. The threat the CT poses on male infertility remains controversial since it is not yet proven. Men act as carriers of Chlamydia infection and its transmission to female cause urinary tract inflammation. This infection can cause damage to sperm DNA and acute epididymitis. Sperm analysis in today’s laboratory procedures is not proved enough about the reproductive outcome and, thus, the necessity for upgraded tests. DNA fragmentation tests are available, but most of the laboratories do not support its use. Sperm DNA fragmentation (SDF) has been used in evaluating male fertility though its clinical indication is not clear. SDF test provides recommendations and acts as a reference for fertility specialists to enhance its improvement. This test is often in patients with abnormal to required sperm grams and is necessary for choosing the most appropriate assisted reproductive technique for those unable to bear children. High SDF is related to repeated loss of pregnancy. Samples of 200 patients who have a history of subfertility, both with normal and abnormal sperm grams, will be examined to test whether CT is present. An ELISA test will also be conducted on the patients’ seminal plasma to check for antibodies against CT. To determine the effect CT infections, have on DNA fragmentation, 50-CT infected patient cases and 25-CT negative tests will be analysed by the Sperm Chromatin Structure Assay (SCSA) using flow cytometry analysis. SCSA test will provide statistical data of exceptional quality.
Keywords: Sperm DNA fragmentation, Chlamydia (Chlamydia trachomatis), male infertility, reproductive assisted technique
INTRODUCTION
1.1 Background Information
Chlamydia trachomatis (CT) is one of the most frequently sexually transmitted infection (STI) in human beings and occurs more in partners who are sexually active. Annually, roughly about 100 million upcoming cases of CT are treated in the world. The spread of CT is highly favored because it is asymptomatic, with an infection rate of about 50% men and 90% women. The research of CT is currently focused on females abandoning males despite the statistics that its frequency of infection is similar to both (Jennifer et al. 2017).
Clinicians and researchers in the discipline of reproductive medicine have raised a concern about the spread of STIs. Research shows that almost about 15% of infertility in men is associated with infection of the genital tract. The part played by CT infections towards infertility in males is disputable. Some researches relate CT to inferior semen quality. In contrast, others indicate that CT is characterized by a reduction in sperm motility and concentration, a drop in the number of sperms ejaculated, and interference with the semen pH (Hanen et al. 2014). Infection with CT causes epididymitis, urethritis, and prostatitis in men. Infertility may be caused by the swelling of the epididymis due to the obstruction of the sperm tract (Band et al. 2018).
Sperm DNA fragmentation (SDF) is caused by aging, diseases, lifestyle-related actions, and body infections. It poses a danger towards reproduction in humans and the health of the infants. SDF is induced by three main factors; apoptosis, oxidative stress, and defects in the maturation of sperm chromatin. Apoptosis and sperm chromatin impairment affects testis and causes breaks in the DNA, resulting in dead spermatozoa. Oxidative stress induces SDF after the ejaculation of sperms (Monica et al. 2019).
SDF has become essential in clinics in the essence of its outcome in assisted reproductive technology (Luca et al. 2019). In most semen analysis, DNA quality in sperm cells is not assessed because of sophisticated and complex technologies for DNA evaluation. The standard technology used is sperm chromatin structure assay (SCSA), used in evaluating the damage in sperm DNA (Fernandez et al. 2015). Research has shown that people presenting high levels of SDF are linked to the unsuccessful production of children than individuals expressing low rates of SDF (Fernandez et al. 2015).
Figure 1: Factors impacting the level of SDF present in a semen sample
2152650126365Fertilization Strategy
Fertilization Strategy
4676775230314529718002284095114300022936204743450150304510477501522095292290542672021621753808095Oocyte DNA Repair Capacity
0Oocyte DNA Repair Capacity
right2722245Iatrogenic Sperm Damage
00Iatrogenic Sperm Damage
4381502722245Patient Characteristics
0Patient Characteristics
right1893570SDF and Reproductive Outcome
SDF and Reproductive Outcome
342900798195Techniques for Sperm Selection
0Techniques for Sperm Selection
3752850798195Values for SDF Obtained on Neat or Selected Samples
0Values for SDF Obtained on Neat or Selected Samples
Source: Fernandez et al. 2015
Methods and Materials
Patients consented to their participation in the study particularly the use of their semen. Samples of male ejaculate were obtained from 200 males aged between 20 and 45. The participants had abnormal to normal sperm counts. The samples were tested for CT using a urethral smear test -u A commercial kit that uses direct immunofluorescence manufactured by Trinity Biotech was used. 64 samples returned positive whereas 136 returned negative. A random sampling method was used to select 50 positive samples and 25 negative samples that would be the control group for the study. Some of the participants who tested positive volunteered information on how long they had been infected with the disease and this was noted down. The participants were required to abstain from sexual activity 48 hours before the commencement of the study. On the day of semen collection, the men masturbated into vials that were appropriately numbered numerically (black ink for positive cases and blue ink for negative cases). Semen analysis was carried out using the standard procedure prescribed by the World Health Organization (WHO). Equal parts from each vial were analyzed for DNA fragmentation and the result was recorded appropriately on the sticker. Seminal plasma was also examined for antibodies using an enzyme-linked immunosorbent assay (ELISA) test.
The first step in the SCSA test was diluting an aliquot of semen from each sample to about 10 million. The diluting agent used was a pH 1.2 buffer of phosphate and salt (PBS). This treatment was allowed to hold for about 35 seconds (It is expected that fragments of DNA with 2 strands break (denature) while those with whole double strands remain unchanged.) The sperm cells were then stained with an acridine orange dye which produces contrasting stains in fragmented and intact DNA (red and green respectively). Flow cytometry analysis was then carried out for the stained samples in which blue light excitation of each sample was carried out and the sample observed for color changes (green to red). The flow cytometry allowed the measurement of the extent of sperm chromatin damage by quantifying the extent of metachromatic shifts from green to red in the cytogram intensity patterns. Denatured DNA was observed as the DNA fluorescent Intensity (DFI) or the ratio of denatured DNA to the total DNA present in a sample. The SCSA Diagnostics software was used to analyze the flow cytometry data and a DFI histogram modeled to present the percentage of denatured DNA strands per sample. The differences between samples from participants who tested positive for CT and those who tested negative for CT were averaged and statistically tested for reliability and validity. Also, the extent of DNA damage as revealed by the SCSA was compared across positive samples to observe whether long-term sufferers had a higher prevalence of fragmented DNA. SCSA has been used successfully to identify sperm with fragmented DNA from those sperm with whole DNA. The test reveals fragmented DNA as a plot with dots. The more the dots the higher the prevalence of SDF.
Results
The target specific test conducted on the samples from the participants indicate the presence of IgG antibody in the 50 samples initially marked for infection. The test was conducted before the study could commence to countercheck the accuracy of infectuion. On the same note, both IgG and IgA antibodies were tested for better interpretation of the results. After conducting the SCSA test for each sample the results for the control group ranged from 3-24 percent which suggest good fertility. Most of the samples from the control group (92%) or 23 showed a DNA Fragmentation Index (DFI) of less than 15 percent which indicated excellent fertility. Only two recorded a score of more than 15 percent but less than 25 which shows a fair potential to achieve a term pregnancy naturally. The DFI scores of the study population was categorized on the basis of how long a patient has know of the infection.
Although the time of infection might have varied only two patients were above the 50 percent DFI score. The DNA fragmentation in the study group was about 5 times higher than in the control group. In relative terms, DNA integrity is compromised in the sperm cell of individuals infected with CT. In comparative terms, the patients showed a negative correlation between sperm composition and sperm DNA fragmentation as compared to the control group.
References
Bryan, E. R., Kollipara, A., Trim, L. K., Armitage, C. W., Carey, A. J., Mihalas, B., … & Beagley, K. W. (2019). Hematogenous dissemination of Chlamydia muridarum from the urethra in macrophages causes testicular infection and sperm DNA damage. Biology of Reproduction, 101(4), 748-759.
Dehghan Marvast, L., Talebi, A. R., Ghasemzadeh, J., Hosseini, A., & Pacey, A. A. (2018). Effects of Chlamydia trachomatis infection on sperm chromatin condensation and DNA integrity. Andrologia, 50(3), e12918.Gallegos, G., Ramos, B., Santiso, R., Goyanes, V., Gosálvez, J., & Fernández, J. L. (2008). Sperm DNA fragmentation in infertile men with genitourinary infection by Chlamydia trachomatis and Mycoplasma. Fertility and sterility, 90(2), 328-334.Moazenchi, M., Totonchi, M., Salman Yazdi, R., Hratian, K., Mohseni Meybodi, M. A., Ahmadi Panah, M., … & Mohseni Meybodi, A. (2018). The impact of Chlamydia trachomatis infection on sperm parameters and male fertility: A comprehensive study. International journal of STD & AIDS, 29(5), 466-473.Samplaski, M. K., Domes, T., & Jarvi, K. A. (2014). Chlamydial infection and its role in male infertility. Advances in Andrology, 2014.Sellami, H., Gdoura, R., Mabrouk, I., Frikha‐Gargouri, O., Keskes, L., Mallek, Z., … & Hammami, A. (2011). A proposed mouse model to study male infertility provoked by genital serovar E, Chlamydia trachomatis. Journal of andrology, 32(1), 86-94.Suarez, J. P., Sanchez, L. R., Salazar, F. C., Saka, H. A., Molina, R., Tissera, A., … & Motrich, R. D. (2017). Chlamydia trachomatis neither exerts deleterious effects on spermatozoa nor impairs male fertility. Scientific reports, 7(1), 1-14.
