Sperm Protamine and Chromatin Integrity: As Biomarkers of Sperm Quality and Assisted Conception Outcome

Abstract

Background: Subfertility affects one out of every seven couples worldwide. After a year, about 25% of couples had no children despite the unprotected sexual activity, 15% got medical advice, and fewer than 5% were still unable to conceive despite their best efforts. In over half of the cases, the underlying etiology is attributed solely to men. Standard sperm analysis is the most essential laboratory investigation for men. Semen analysis, frequently indicates oligozoospermia (low sperm count), asthenozoospermia (poor sperm motility), or morphologically aberrant spermatozoa. In contrast, oligoasthenoteratozoospermia is the condition in which all of these anomalies coexist. The World Health Organization's (WHO) 2010 standard semen analysis, which is used to evaluate treatment options and the chance of spontaneous conception, considers sperm count, motility, and morphology. The use of assisted reproductive technology (ART) to treat subfertility has dramatically increased in recent years. The introduction of these novel procedures, particularly intra-cytoplasmic sperm injection (ICSI), has resulted in a demand for more effective male fertility potential investigation tools. Most of these novel diagnostic approaches focus on the male gamete's genetic integrity. The growing concern about the possible transmission of genetic diseases through ICSI, where natural control mechanisms during spermatocyte interaction are bypassed, potentially resulting in cytogenetic aberration, foetal deformities, and postnatal anomalies in babies born through ICSI. Even though ART has reached its maximal level of efficacy, the "take-home baby" rate has remained stable for several years. One of the explanations could be a lack of male diagnosis and treatment. DNA damage can be caused by abnormal chromatin folding during spermatogenesis, and defective apoptosis just before ejaculation causes abnormal ROS production in the ejaculate, and extra-testicular factors such as age, body mass index, lifestyle, hormonal factors, hyperthermia, and in-vitro semen handling. Spermatozoa preparation technique and storage temperature post preparation affect sperm quality, chromatin integrity, ability to fertilize the oocyte, post-fertilization-embryo cleavage rate, embryo quality, blastocyst formation, and implantation rate. Aim: The study aims to explore the role of protamines in the condensation of sperm chromatin and the impact of abnormal protamine expression on chromatin integrity. 1 General Abstract Additionally, the research aims to assess the effectiveness of utilizing protamine and chromatin integrity as predictors of assisted conception outcomes, including fertilization rates, embryo development, and pregnancy rates. Objective: Current study was designed for the thorough analysis of the molecular and biochemical markers for male subfertility 1. Compare semen parameters, sperm viability, oxidative stress markers, and reproductive hormone levels in fertile and subfertile men. 2. To estimate sperm DNA fragmentation and chromatin damage in fertile and subfertile men, and to assess the clinical value of sperm chromatin structure assay (SCSA), sperm chromatin dispersion assay (SCD), toluidine blue (TB) and acridine orange testing (AOT). 3. To evaluate the impact of high DNA fragmentation and chromatin decondensation on the outcome of standard IVF and ICSI. 4. To evaluate the impact of sperm preparation methods on sperm DNA fragmentation, chromatin condensation, and IVF, ICSI and PGT outcomes. Materials and methods: A total of 753 couples were recruited for the study 146 fertile/control (without any history of fertility problems), and 607 male factor /subfertile (with a history of subfertility) were recruited. A couple of details history and relevant information were collected from each subject with informed consent. Semen and blood from the male partner were obtained for further analysis. Each semen sample was further split into aliquots: 1). standard semen analysis, vitality, reactive oxygen species, sperm DNA damage, and chromatin abnormalities, 2). sperm processing to inseminate oocytes through ICSI/IVF/PGT. According to WHO 2010 standards, sperm counts, motility, and morphology were examined. Sperm vitality was tested utilizing the hypo osmatic test (HOS) and eosin nigrosin stain test. Reactive oxygen species (ROS), thiobarbituric acid-reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) levels were analyzed through a spectrophotometer. Male and female partner serum follicular stimulation hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) levels were 2 General Abstract determined, in men's testosterone (T) levels and female’s estradiol (E2), progesterone (P4) and anti-mullerian hormone levels (AMH) were measured, through spectrophotometer. DNA fragmentation index (DFI) in spermatozoa was analyzed by sperm chromatin dispersion (SCD), sperm chromatin structure assay (SCSA), and acridine orange testing (AO). Chromomycin A3 (CMA3+) chromatin maturity index (CMI) and toluidine blue (TB+) staining to measure sperm chromatin condensation (maturity) in a male subject. In an experiment, the sperm chromatin integrity in the categories of fertile and subfertile patients was measured in two BMI groups (normal weight and overweight). The potential effects of a father's high BMI on fertilisation, embryo quality, the overall live birth rate, and birth weight were also examined, as well as the correlation matrix. Another aspect of the investigation involved measuring the chromatin integrity of sperm from viable and subfertile men that had been cryopreserved and prepared using different techniques such as swim-up (S-UP), density gradient centrifugation (DGC) and magnetic activated cell sorting (MACS). In a different experiment, the success of assisted conception was examined between two groups of sperm DNA damage (20% and >20%). Sperm, protamine, or chromatin condensation (30 and > 30%), as well as female features and hormonal parameters that affect ART results, were compared across the board in the last set of studies. The impact of sperm preparation and storage methods as well as sperm chromatin integrity on the success of assisted conception was also evaluated. To test for the statistically significant differences between the two groups and across all of them, ANOVA with post hoc tests (such as Tukey) was performed. To assess differences in means, a spearman correlation analysis was used between the various parameters. For those outcomes that were connected to one or more examined parameters, prediction models were built. To look for continuous outcome variables, a logistic regression model was used. The statistical package for social sciences (SPSS) 20 IBM was used for all statistical analyses. The Hosmer-Lemeshow goodness-of-fit test was used to determine the model's dependability. A p-value of <0.05 was considered to be statistically significant. Results: Several parameters including male and female age, BMI subfertility duration, socioeconomic status, the weight of the child and in men semen volume, PH, WBC/HPF, 3 General Abstract liquefication time (min) were comparable between the groups, while reduced sperm concentration x106 (p=0.002), progressive motility (p<0.01) and sperm morphology (p <0.001) were elevated in MMF and SMF subfertile couples as compared to control group. CASA parameters VSL, VCL, VAP, and ALH were reduced in MMF and SMF. The sperm membrane integrity (HOS) percentage of fertile subjects measured through HOS was 86.50±0.96%, whereas in MMF was 69.55±0.73%, similarly, SMF had membrane integrity of 63.07±1.21%. Sperm vitality (eosin) in subfertile men was 71.74±1.26 and in MMF and SMF were 48.85±1.18 and 49.69±1.25 respectively. There was a significant difference in SOD (P<0.001) and POD (P<0.001) levels in a fertile male group compared to fertile control. SMF patients had significant (P<0.001) lower levels of SOD, POD, CAT, and higher levels of ROS and TBRAS compared to MMF and fertile male patients. A strong correlation was found between oxidative stress (ROS) with BMI, impaired semen parameters, and HOS. Comparable hormonal (FSH, LH, prolactin) levels (p>0.05) while a significant decrease in serum testosterone concentration was found in MMF and SMF male patients compared to fertile control subjects. Sperm DNA fragmentation and chromatin damage of fertile men was significantly (p<0.05) lesser than in subfertile men categories i.e., moderate male factor (MMF) and severe male factor (SMF), sperm DNA damage correlated with subnormal semen parameters concentration, motility, morphology, sperm velocity. There are raised ROS levels in semen of subfertile men compared to fertile men and a positive correlation between oxidative stress markers and sperm DNA damage in fertile and subfertile men was observed. Sperm chromatin integrity by CMA3+ and TB+ showed a significant (p<0.05) increase in chromatin decondensation in SMF and MMF subfertile men. An increase in sperm DNA fragmentation above 20 % value of SCSA, SCD, and AO affects the men's fertility outcome in MMF and SMF men. Three methods to determine sperm DNA fragmentation were SCSA, SCD and AO and SCD had the same prognostic value as SCSA to evaluate sperm DNA fragmentation. The intra-assay variation in DFI, measured by SCSA was more closed values measured using the SCD technique than AO. Higher than 20% DNA fragmentation and higher than 30 % chromatin decondensation affect the fertilization rate and cleavage rate but had no effect on pregnancy and live birth rate after 4 General Abstract standard IVF and ICSI. DNA fragmentation and chromatin maturity can predict fertilization, cleavage, and but not aneuploidy and live birth rates in ART. Paternal BMI correlates significantly (p<0.05) negatively with semen parameters (concentration, motility, morphology, and vitality), DNA fragmentation, and chromatin maturity. The analysis of the percentage of spermatozoa with chromatin maturity (CMA3+) and chromatin integrity (TB+) was reduced significantly in overweight men (p<0.01) compared with a reference group. Increase in paternal BMI correlate with the increase in sperm chromatin damage (SCD r= 0.282, TB+ r= 0.144, p<0.05), immaturity (CMA3+, r=0.79, p<0.05) and oxidative stress (ROS) (r=0.282, p<0.001). Peri-fertilization effects were an increase in oocytes fertilization in couples with overweight men (FR =67%) compared with normal-weight men (FR=74%), similarly, paternal overweight correlates with poor fertilization(r=-0.187, p<0.01), after multiple regression paternal weight remain predictor of successful fertilization. During the developmental stage, the number of embryo in cleavage was higher in normal-weight men, while day 3 (D3) embryos, percent good quality embryo D3, and blastocyst formation rate were comparable between the groups. A negative correlation was found between implantation rates and paternal BMI (–r=0.110, p<0.01). The paternal overweight group (2952.14±511.64gm) had increased neonatal birth weight (within normal range) when compared with the reference group (2577.24±324.94gm, p<0.001) following assisted reproductive technology (ART). The cumulative live birth rate (CLBR) was higher (p<0.05) than normal weight men compared to the paternal overweight group. In CLBR per embryo transfer and per FR used was the difference between groups statistically significant (p<0.05). We found paternal overweight BMI > 24.5 kg/m2 had a reduced fertilization rate with an OR of 1.98(CI 95% 1.323-2.967, p=0.001). After controlling for several potential confounders, the multiple linear regression model revealed a positive relationship between paternal BMI with fertilisation rate and CLBR (weight within normal range). The present study demonstrated the impact of paternal overweight on male reproductive health, as these patients with overweight had a higher percentage of immature sperm (CMA3+) with impaired chromatin integrity (SCD, TB+) in their semen and had decreased fertilization rate, CLBR following assisted reproductive treatments. The present study supports that paternal overweight should be regarded as one of the predictors for fertilization, CLBR and useful for counseling, to 5 General Abstract consider body mass index not only in women but also for men, in couples opting for ART treatment, and warrant a poor reproductive outcome in overweight men. Male patients with old age (>40) had a higher percentage of immature sperm (CMA3+) with impaired chromatin integrity (SCD, TB+) in their semen. Female characteristics were comparable between all groups. Female age and BMI kg/m2 significantly negatively correlated with the number of the oocyte, fertilization rate, cleavage rate and pregnancy rate. DGC-MACS technique along with the classic sperm preparation (DGC, SU, DGC-SU) methods, significantly (p<0.05) improved semen parameters. DGC-MACS sperm preparation methods, then other methods of sperm preparation (DGC, SU, DGC-SU) and significantly (p<0.05) better yield mature sperm concentration with intact sperm chromatin integrity. Paternal overweight impacts fertilization, embryo quality, live birth rate, and birth weight. In four semen preparation groups i.e., DGC, SU, DGC-SU, and DGC-MACS, in male subjects with teratozoospermia and men with increased (≥ 20%) and normal value (< 20%) SDF a threshold value determine, all four preparation techniques had significant (p<0.05) improved number of spermatozoa with and after DGC-MACS a significant (p<0.05) improvement in mature and intact DNA, condensed chromatin, viability. DGC MACS sperm preparation techniques had significant (p<0.05) ICSI cycles success in improving the percentage of fertilization, cleavage rate, pregnancy rate and non-significant improvement in the live birth rate after DGC-MACS Conclusion: Fertility is linked to sperm quality, and standard sperm analysis is insufficient for predicting male subfertility. Testing for sperm DNA integrity and chromatin condensation are helpful prognostic tools for IVF/ICSI patients. Abnormal sperm morphology can reduce fertilizations and increase failed implantation, and oxidative stress and morphology assessment may be helpful for treating male subfertility. Paternal overweight and age are factors contributing to the global drop in male fecundity, and less than 40 years of age and normal weight prognostic for better ART success in terms of fertilization. Sperm preparation method for better quality sperm separation and post-24-hour quality sperm isolation was combining the density gradient centrifugation with MACS. DGC MACS preparation technique is a safe and cost-effective method to improve assisted 6 General Abstract reproduction outcomes. Further studies are necessary to validate these findings and clarify the issue of male subfertility. Testing for sperm DNA integrity and sperm chromatin condensation are prognostic tools for IVF/ICSI patients, and sperm quality parameters are one of the factors contributing to male fertility. Male age and BMI measurement should be considered in couples opting for ART treatment, and younger age and weight loss before undergoing in-vitro fertilization procedures can improve quality of sperm and increase fertilization rate. Further studies and meta-analyses are necessary to validate these findings and clarify the issue of male subfertility.