• Clinical and Experimental Reproductive Medicine
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• v.49 no.1
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• pp.40-48
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• 2022

Objective: As the associations of sperm DNA fragmentation with morphology have not been examined in detail, this study aimed to investigate the relationship between abnormalities of morphological details and DNA integrity in human sperm. Methods: In this cross-sectional study, men from infertile couples were enrolled at Hue Center for Reproductive Endocrinology and Infertility, Vietnam. Conventional semen parameters, including morphological details, were analyzed following the World Health Organization 2010 criteria. Sperm DNA fragmentation was evaluated using a sperm chromatin dispersion assay. The relationships and correlations between semen parameters, sperm morphology, and the type of halosperm and the DNA fragmentation index (DFI) were analyzed. Results: Among 130 men in infertile couples, statistically significant differences were not found in the sperm halo type between the normal and abnormal sperm morphology groups. The percentage of round-head spermatozoa was higher in the DFI >15% group (16.98%±12.50%) than in the DFI ≤15% group (13.13% ±8.82%), higher values for amorphous heads were found in the DFI >15% group, and lower values for tapered heads were observed in the DFI ≤15% group; however, these differences were not statistically significant. Small-halo sperm and the DFI were positively correlated with round-head sperm (r=0.243, p=0.005 and r=0.197, p=0.025, respectively). Conclusion: The rate of general sperm morphological abnormalities in semen analysis was not related to sperm DNA integrity. However, round sperm heads were closely associated with sperm DNA fragmentation.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.2
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• pp.67-75
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• 2019

Objective: Sperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test. Methods: In total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups. Results: There was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability. Conclusion: Conventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.4
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• pp.199-206
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• 2016

Objective: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. Methods: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. Results: The sperm DFI showed a significant correlation (r=-0.347, p< 0.001) with sperm motility and morphology (r=-0.114, p< 0.05) but not with other semen parameters. The DFI ($11.5%{\pm}2.0%$) of semen samples was significantly reduced by DGC ($8.1%{\pm}4.1%$) or MACS alone ($7.4%{\pm}3.9%$) (p< 0.05). The DFI was significantly further reduced by a combination of DGC and MACS ($4.1%{\pm}1.3%$, p< 0.05). Moreover, the combination of DGC and MACS ($1.6%{\pm}1.1%$, p< 0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC ($4.4%{\pm}3.2%$) or MACS alone ($3.4%{\pm}2.2%$). Conclusion: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.3
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• pp.132-140
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• 2017

Objective: Correlations between semen parameters and sperm DNA fragmentation index (DFI) were investigated to identify characteristics of sperm without DNA damage that could be used in selecting sperm for intracytoplasmic sperm injection (ICSI). Pregnancy outcomes were compared to determine whether in vitro fertilization (IVF) or ICSI is a better choice for patients who have sperm with a high-DFI. Methods: Semen analysis was carried out in 388 patients who visited our IVF center for the first time to investigate correlations between sperm DFI and semen parameters. In addition, 1,102 IVF cycles in 867 patients were carried out in the present study; 921 cycles in the low-DFI group (DFI < 30%) and 181 cycles in the high-DFI group ($DFI{\geq}30%$). Both the low- and high-DFI groups were subdivided into IVF and ICSI cycle groups. Results: Sperm DFI showed significant inverse correlations with sperm motility (r = -0.435, p< 0.001) and morphology (r = -0.153, p< 0.05). Sperm DFI also showed significant correlations with rapid motility (r = -0.436, p< 0.001), and the kinetic parameters of average-path velocity (r = -0.403) and linearity (r = -0.412). Although there was no significant difference in the pregnancy rates between IVF (48.6%) and ICSI (44.8%) in the low-DFI group, the pregnancy rate of ICSI cycles (44.8%, p< 0.05) was significantly higher than IVF cycles (25.0%) in the high-DFI group. No significant difference was observed in the abortion rates between the low-DFI (52 of 921, 5.6%) and high-DFI groups (7 of 181, 3.8%). Conclusion: ICSI is a better choice than IVF for improving the pregnancy outcomes of patients who have sperm with a high DFI.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.1
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• pp.14-21
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• 2019

Objective: The usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay. Methods: In this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group). Results: The alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r = 0.70 and r = 0.68, respectively; p< 0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r = 0.77, p< 0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r = 0.59, r = 0.57, and r = 0.72, respectively; p< 0.001). Conclusion: The TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.3
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• pp.185-191
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• 2023

Objective: Although intracytoplasmic sperm injection (ICSI) is a way to deal with in vitro fertilization failure, 3% of couples still experience repeated fertilization failure after attempted ICSI, despite having sperm within normal parameters. These patients are a challenging group whose sperm cannot fertilize the egg during ICSI. Unfortunately, no test can predict the risk of fertilization failure. Phospholipase C zeta (PLCζ) and transition nuclear proteins (TNPs) are essential factors for chromatin packaging during sperm maturation. This study aimed to assess PLCζ1 and TNP1 expression in the sperm of patients with fertilization failure and the correlations among the DNA fragmentation index, PLCζ1 and TNP1 gene and protein expression, and the risk of fertilization failure. Methods: In this study, 12 infertile couples with low fertilization rates (<25%) and complete failure of fertilization in their prior ICSI cycles despite normal sperm parameters were chosen as the case group. Fifteen individuals who underwent ICSI for the first time served as the control group. After sperm analysis and DNA fragmentation assays, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to compare the gene and protein expression of PLCζ and TNP1 in both groups. Results: DNA fragmentation was significantly higher in the fertilization failure group. The qRT-PCR and Western blot results demonstrated significantly lower PLCζ and TNP1 gene and protein expression in these patients than in controls. Conclusion: The present study showed that fertilization failure in normozoospermic men was probably due to deficient DNA packaging and expression of TNP1.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.3
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• pp.245-251
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• 2010

Objective: Human sperm nucleus DNA damage may negatively affect pregnancy outcome, and the spermatozoa of infertile men have more DNA damage than that of fertile men. The aim of this study was to evaluate the effect of microsurgical varicocelectomy on human sperm nucleus DNA integrity. Methods: We reviewed the medical records of 18 subfertile male patients who underwent microsurgical varicocelectomy at our hospital from April 2006 to April 2007. Varicocele was diagnosed by physical examination and Doppler ultrasound. Standard semen analysis was performed in 18 patients before and 4 months after microsurgical varicoceletcomy using a computer assisted semen analyzer. Sperm nucleus DNA integrity was assessed by a single-cell gel electrophoresis (comet assay). Results: No recurrence of varicocele was observed after 4 months later. The DNA fragmentation index improved after varicocelectomy compared with pre-operatively (19.3 versus 13.7%, respectively, p<0.05). Semen analysis parameters (total count, concentration, motile sperm, viability, strict morphology) increased after varicocelectomy, but the difference did not reach statistical significance. Conclusion: Our data suggest that microsurgical varicocelectomy can improve semen analysis parameters and human sperm nucleus DNA integrity in infertile men with varicocele.

• Clinical and Experimental Reproductive Medicine
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• v.48 no.1
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• pp.61-68
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• 2021

Objective: This study was conducted to investigate the relationship of semen parameters in samples used for intracytoplasmic sperm injection (ICSI) with fertilization and pregnancy rates in infertile couples. Methods: In this prospective study of Infertile couples with male factor infertility that had undergone ICSI, fractions of the same semen samples obtained for microinjection (to ensure the best predictability) were evaluated to determine the semen parameters and sperm DNA fragmentation index (DFI) on the day of oocyte recovery. Results: In total, 120 couples completed the study and were subdivided into fertilized (n=87) and non-fertilized couples (n=33). The fertilized couples were further classified into pregnant (n=48) and non-pregnant (n=39) couples. Compared to non-fertilized and non-pregnant couples, fertilized and pregnant couples showed statistically significantly higher sperm viability and percentage of normal sperm morphology, as well as significantly lower sperm DFI values. A receiver operating characteristic curve analysis of data from the 120 ICSI cycles showed that sperm viability, normal sperm morphology percentages, and sperm DFI were significant prognostic indicators of fertilization at cutoff values of 40%, 7%, and 46%, respectively. A sperm DFI of 46% showed sensitivity and specificity of 95% and 90%, respectively, for predicting fertilization, and no clinical pregnancies occurred in couples with a sperm DFI above 46%. Conclusion: Semen parameters from the ICSI day sample, especially sperm viability, normal morphology, and DFI, had an impact on fertilization and pregnancy outcomes in ICSI cycles.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.9
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• pp.1411-1420
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• 2020

Objective: The aim of study was to investigate the effects of season and single layer centrifugation (SLC) before cryopreservation on post-thaw bull sperm quality in Thailand. Methods: Semen was collected from 6 bulls (Bos indicus) in summer, rainy season and winter 2014 through 2016. Semen characteristics, sperm morphology, sperm kinematics, viability, chromatin structure and mitochondrial membrane were evaluated. Meteorological data were available from the local meteorological station; Results: Season had an effect on semen characteristics in the raw ejaculate, with higher proportions of normal spermatozoa and lower abnormalities in winter than in the other two seasons. Sperm kinematics, viability, DNA fragmentation index, and mitochondrial membrane potential were not different between seasons. Sperm samples selected by SLC had greater normal morphology and a lower proportion with bent tails than controls and higher values of progressive motility (PRO), beat cross frequency, linearity, straightness, wobble (WOB), and lower values of slow motility, velocity average path (VAP), velocity curved line, and amplitude of lateral head displacement than controls. In addition, SLC-selection had a favorable effect on PRO, VAP, and WOB that differed among seasons. Conclusion: Our results suggested that these bulls were well adapted to their location, with season having an effect on sperm morphology. Moreover, SLC could be used prior to cryopreservation, regardless of season, to enhance normal morphology and kinematics of bull sperm samples without adversely affecting other parameters of sperm quality. However, there was considerable variation among bulls in DNA fragmentation index, mitochondrial membrane potential and sperm viability. In addition, SLC had a positive effect on sperm morphology and sperm kinematics, which could be expected to influence fertility.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.1
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• pp.53-62
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• 2023

Objective: The aim of this study was to investigate the relationships of serum folate (vitamin B9), cobalamin (vitamin B12) levels and diet with semen parameters (semen standard parameters [SSP] and DNA fragmentation index [DFI]) in infertile men. Methods: Sperm samples were assessed for SSP and DFI (using the sperm chromatin dispersion test). Serum vitamin concentrations were measured with an immuno-electrochemiluminescence assay, and men completed a semi-quantitative food frequency questionnaire (FFQ). Results: Serum folate levels were positively correlated with sperm progressive motility and DFI. A comparison of SSP between two groups of patients according to serum folate concentration (B9 <4.840 ng/mL and B9 ≥4.840 ng/mL) showed significantly higher sperm concentration and sperm progressive motility in the latter group. However, there was no difference between these groups regarding DFI. Interestingly, serum folate levels were significantly higher in patients with a high DFI (using the cut-offs of 30% or 18%). FFQ data showed that the consumption of fruits and egg yolk correlated positively with sperm concentration and sperm motility, respectively. Conclusion: Serum folate levels showed significant associations with sperm concentration and sperm progressive motility. However, the positive association of serum folate with DFI raises the need for careful prescription of folate supplements.