Relationship between sperm telomere length and sperm quality in infertile men

Telomeres, noncoding and repetitive DNA sequences play a significant function in chromatin integrity. Telomere length is age-dependent in somatic cells, while it increases in sperm cell with age. Therefore, we aimed to assess sperm chromatin, leucocyte and sperm telomere length (LTL, STL) in spermatozoon of 38 infertile and 19 fertile men aged between 20 and 50 years. Protamine deficiency (chromomycin A3 test), DNA fragmentation (TUNEL assay), lipid peroxidation (Bodipy probe) and telomere length (quantitative real-time PCR) were assessed. A significant decrease in mean of sperm concentration and motility and a significant increase in means of sperm abnormal morphology, DNA fragmentation, lipid peroxidation and protamine deficiency were observed in infertile compared with fertile men. In addition, the mean of LTL and STL were significantly shorter in infertile men compared with fertile individuals. We observed significant associations between telomere length with sperm concentration, DNA fragmentation and lipid peroxidation. We hypothesised that increased oxidative stress in spermatozoa of infertile men can result in abnormal packaging of chromatin, damage of DNA and shorter sperm telomere length. Together, these anomalies may account for fertility failure in these individuals.     

Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men

Normal chromatin condensation is important for sperm fertilising ability. However, routine semen analysis does not identify defects in sperm chromatin structure. This study aimed to investigate the condensation of chromatin and DNA integrity in spermatozoa of infertile men and deduce the relationship with sperm quality, as measured by conventional semen parameters. Semen analysis was carried out to assess sperm quality according to World Health Organization criteria. The remaining aliquot of each sample was processed for transmission electron microscopy, chromomycin A3 (CMA3) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays. The ultrastructural analysis of spermatozoa from infertile men showed heterogeneity in sperm nuclear morphology. Some spermatozoa displayed a round nucleus with incomplete chromatin condensation. Immunoreactivity with antitransitional protein and antiprotamine antibodies indicated nuclear maturation defects in the spermatozoa of infertile men. Spearman's correlation analysis indicated a positive correlation between the percentages of CMA3- and TUNEL-positive spermatozoa. In addition, these two parameters were negatively correlated with sperm concentration, motility and normal morphology. This study demonstrated that men with morphologically normal spermatozoa of <30% have greater degree of protamine deficiency and DNA damage than men with morphologically normal spermatozoa of >30%. Evaluation of chromatin integrity appears to be a useful tool for assessing male fertility.     

Imprinting analysis in spermatozoa prepared for intracytoplasmic sperm injection (ICSI)

Genetic imprinting is a mechanism of gene regulation by which only one of the parental copies of a gene is expressed. This process is mediated by the methylation of DNA. As spermatozoa represent exclusively the paternal contribution to a future individual, they are expected to carry the paternal imprint only. For intracytoplasmic sperm injection (ICSI), spermatozoa mostly have to be selected from samples with pathological semen parameters. Correct establishment of the paternal imprint in these spermatozoa has not yet been demonstrated. In the present study, imprinting analysis was undertaken using DNA extracted from spermatozoa from men with normal semen analysis (group A: n=30 patients) and from men with an abnormal sperm count (B: n=30 patients with 5--20 million spermatozoa/mL and C: n=30 patients with < or =5 million spermatozoa/mL) from the ICSI program. It was performed using firstly a conventional methylation-specific polymerase-chain-reaction (M-PCR) and secondly a more sensitive modified hemi-nested M-PCR technique. In addition, a single cell PCR was performed on a total of 88 single spermatozoa (collected from nine males) and on 25 leucocytes (control group). With the conventional M-PCR, exclusively paternal imprints were found in all groups. Using the more sensitive hemi-nested M-PCR, additional maternal imprints were found in 63% of the samples in A, 57% in B and 60% in C. In the single cell PCR, exclusively paternal imprints were detected. Because of the very small amount of DNA (3 pg), a complete amplification failure occurred in 43% of spermatozoa. The correct paternal and maternal imprints were found in 56% of the analysed leucocytes (complete amplification failure in the other 44%). In conclusion, ejaculated spermatozoa from males with medium or high-grade semen pathology proved to have the same imprinting status as those from males with normal semen parameters. As the additional maternal imprints were never found at the single cell level, they were classified as contamination by diploid cells such as leucocytes or immature germ cells in the processed and purified semen samples, which can be detected by a more sensitive PCR method in contrast to the conventional standard PCR.     

Correlation between male age, WHO sperm parameters, DNA fragmentation, chromatin packaging and outcome in assisted reproduction technology

In the human, male ageing results in reproductive hormonal and cellular changes that can influence semen quality (volume, motility, concentration and morphology) and ultimately result in a reduced fertilising capacity and a longer 'time to pregnancy' for ageing men as well as an increased risk for miscarriage. This prospective cohort study of 278 patients undergoing a first in vitro fertilisation or intracytoplasmic sperm injection treatment was undertaken to examine whether patient's age was reflected in sperm motility, concentration, morphology as well as in DNA fragmentation (DFI) and immature chromatin (unprocessed nuclear proteins and/or poorly condensed chromatin) as measured by the sperm chromatin structure assay. This study also investigated the possible influence of male age (after correcting for female age) on their fertilising capacity, on obtaining a pregnancy and a healthy baby at home. Logistic regression analysis did not reveal any male age-related influences on sperm parameters like concentration, motility or morphology. No significant male age-related increase in DFI or immature chromatin was demonstrable for these patients. Elevated male age, after correcting for female age, was not related to lower fertilisation rates or significant decreases in the chance for a healthy baby at home.     

Sperm DNA fragmentation in subfertile men: the effect on the outcome of intracytoplasmic sperm injection and correlation with sperm variables

OBJECTIVE

To present the first UK data on sperm DNA fragmentation levels in subfertile men and fertile controls, the correlation with semen variables, and to assess the effect on the outcome of intracytoplasmic sperm injection (ICSI).

PATIENTS, SUBJECTS AND METHODS

In all, 56 subfertile men undergoing ICSI (28 with positive and 28 with a negative outcome for paternity) and 10 control fertile semen donors were recruited. The sperm DNA fragmentation index (DFI) was assessed on raw pre‐preparation samples using the sperm chromatin structure assay. A mean of 5212 sperm were analysed per sample and DFI data are presented by fertility status, ICSI outcome and correlated with semen variables (assessed using World Health Organisation criteria).

RESULTS

Total DFI was significantly higher in subfertile men than in fertile controls (mean and median of 22.8% and 17.0% vs 8.4% and 5.0%; P < 0.001), as was the proportion of both moderate DFI (16.4% and 13.0% vs 6.4% and 4.0%; P = 0.001) and high DFI (6.2% and 6.1 vs 2.0% and 1.0%; P = 0.01). This difference remained significant when the control men were compared only with the subfertile men with successful paternity. There was no significant difference in DFI in the subfertile men when analysed by ICSI outcome (mean and median of 24.5% and 17.0% vs 22.3% and 21.0% for successful and unsuccessful cycles, respectively; P = 0.94). There was a positive statistically significant correlation (r = 0.37; P = 0.02) between the DFI and sperm morphology.

CONCLUSIONS

This study confirms a relationship between male subfertility and sperm DFI; we discuss the correct role for genetic testing of sperm in the evaluation of subfertile men. Although DNA fragmentation data might help to decide a suitable treatment, once it is decided to proceed with ICSI, DFI levels have no effect on the outcome.     

The influence of male age on treatment outcomes and neonatal birthweight following assisted reproduction technology involving intracytoplasmic sperm injection (ICSI) cycles

The aim of this study was to investigate the effect of male age on treatment outcomes and neonatal birthweight following intracytoplasmic sperm injection (ICSI). This study included 2,474 ICSI cycles. Male partners were stratified into 5‐year age categories (up to 25, 26–30, 31–35, 36–40 and 41 and up). Multilevel logistic regression was used to evaluate the relationship between male age and treatment outcomes. After adjusting for confounders, we found no difference in the clinical pregnancy rate. However, we observed that the 31‐ to 35‐year group had a higher odds of live birth than that of the >41‐year group (aOR 1.63, p = .03), and that the risk of abortion in the 31‐ to 35‐year group was lower than that of the reference group (aOR 0.41, p = .02). A total of 754 single‐foetus newborns and 556 twin newborns were analysed. Among the singletons, none of the variables differed among the five groups (p > .05). Among the twins, the infants in the 36‐ to 40‐year group had a lower neonatal birthweight and a higher low‐birthweight rate than those of the other groups (p < .05). Our study indicates that increased paternal age negatively affects the live birth and miscarriage rates. In addition, advanced paternal age may affect the birthweight of twins.     

TUNEL assay: Establishing a sperm DNA fragmentation cut‐off value for Egyptian infertile men

Male factor infertility is responsible for half of all infertility cases. Conventional semen analysis is inadequate to evaluate male fertility. Sperm DNA fragmentation (SDF) test can be done by: direct methods such as Terminal deoxynucleotidyl transferase dUTP Nick‐End Labeling (TUNEL) and Comet assay, or indirect like Sperm Chromatin Structure Assay (SCSA) and Sperm Chromatin Dispersion (SCD). TUNEL assay measures both single‐ and double‐strand breaks and is technically less demanding, while SCSA tests for the susceptibility for nuclear DNA denaturation and samples should be sent to the reference lab. Studies showed that a single cut‐off value does not fit all. Therefore, this study aimed at establishing a cut‐off value to discriminate between fertile and infertile Egyptian men. We enrolled 354 infertile men and 40 proven fertile volunteers.TUNEL assay was performed using Apo‐Direct kit and bench top flow cytometer.The calculated SDF cut‐off value was 20.3% with a sensitivity of 96.6% and specificity of 87.5%, and the overall accuracy of the test was 95.7%. Sperm DNA fragmentation Test using TUNEL assay is valuable tool for male infertility evaluation, and it assists in offering the best treatment options based on it's results.     

New perspectives on male fertility evaluation: Innovative approach for the qualitative analysis of spermatozoa

The identification of idiopathic infertility cases, actually, is impossible. Among new functional tests, developed to improve the male fertility diagnosis, the evaluation of spermatic myo-inositol (MI) level, known as Andrositol^® test (AT), is one of the most interesting, considering its weak economic burden and ease of use. The aim of this study was to evaluate the predictive power of AT and its potential use for a preliminary evaluation of semen samples. To evaluate the predictive power of AT, 87 sperm samples were analysed in comparison with spermiogram and sperm chromatin dispersion (SCD) Test, the gold standard analyses for male fertility evaluation. The application of AT resulted very useful for a preliminary sample evaluation, predicting the absence of DNA fragmentation in case of Low Responder samples precisely, and the presence of DNA fragmentation in case of medium or High Responder samples with abnormal morphology, predicting SCD results with a probability of 80% for Medium Responder sample and of 96.7% for High Responder sample. Considering the predictive power of this method, we could imagine, as preliminary qualitative analysis, its application before SCD test, deepening sperm analysis, improving the daily activities of laboratory operators and maintaining a good reliability of sperm evaluation.     

Sperm DNA integrity and male infertility: a narrative review and guide for the reproductive physicians

Background and ObjectiveConventional semen analysis (SA) remains an essential tool in the initial male fertility evaluation and subsequent follow-up. However, it neither provides information about the functional status of spermatozoa nor addresses disorders such as idiopathic or unexplained infertility (UI). Recently, assessment of sperm DNA fragmentation (SDF) has been proposed as an extended sperm test that may help overcome these inherent limitations of basic SA. In this review, we aim to: (I) discuss the pathophysiological aspects of SDF, including natural repair mechanisms, causes, and impact on reproductive outcomes; (II) explain different assessment tools of SDF, and describe potential therapeutic options to manage infertile men with high SDF; and (III) analyse the strengths, weaknesses, opportunities and threats (SWOT) of current research on the topic.MethodsThis review was constructed from original studies, systematic reviews and meta-analyses that were published over the years up until August 2021, related to the various aspects of SDF.Key Content and FindingsDifferent mechanisms lead to high SDF, including defective chromatin packaging, apoptosis, and seminal oxidative stress. The relevance of sperm DNA integrity to male fertility/infertility has been supported by the frequent observation of high levels of SDF in infertile men, and in association with risk factors for infertility. Additionally, high SDF levels have been inversely correlated with the outcomes of natural pregnancy and assisted reproduction. Terminal deoxynucleotidyl transferase dUTP nick end labelling, sperm chromatin structure assay, sperm chromatin dispersion, and Comet assay are four commonly used assays for measurement of SDF. Addressing lifestyle risks and underlying conditions, antioxidants, hormonal therapy, and advanced sperm selection techniques have all been proposed as potential therapeutic options to lower SDF.ConclusionsThe sum of literature provides evidence of detrimental effects of high SDF on both natural and assisted fertility outcomes. Standardization of the techniques used for assessment of SDF and their incorporation into the work up of infertile couples may have significant implications on the future management of a selected category of infertile men with high SDF.     

Protective effect of alpha glucosyl hesperidin (G‐hesperidin) on chronic vanadium induced testicular toxicity and sperm nuclear DNA damage in male Sprague Dawley rats

The study was conducted to evaluate the vanadium‐induced testicular toxicity and its effect on sperm parameters, sperm nuclear DNA damage and histological alterations in Sprague Dawley rats and to assess the protective effect of G‐hesperidin against this damage. Treatment of rats with vanadium at a dose of 1 mg kg bw^?1 for 90 days resulted in significant reduction in serum testosterone levels, sperm count and motility. Further, a parallel increase in abnormal sperm morphology and adverse histopathological changes in testis was also associated with vanadium administration when compared to normal control. Moreover, sperm chromatin dispersion assay revealed that vanadium induces sperm nuclear DNA fragmentation. A marked increase in testicular malondialdehyde levels and decreased activity of antioxidant enzymes such as superoxide dismutase and catalase indicates vanadium‐induced oxidative stress. Co‐administration of G‐hesperidin at a dose of 25 and 50 mg kg bw^?1 significantly attenuated the sperm parameters and histological changes by restoring the antioxidant levels in rat testis. These results suggested that vanadium exposure caused reduced bioavailability of androgens to the tissue and increased free radical formation, thereby causing structural and functional changes in spermatozoa. G‐hesperidin exhibited antioxidant effect by protecting the rat testis against vanadium‐induced oxidative damage, further ensures antioxidant potential of bioflavonoids.     

Selection of normal spermatozoa with a vacuole‐free head (x6300) improves selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates

Intracytoplasmic morphologically selected sperm injection (IMSI, 6300× magnification with Nomarski contrast) of a normal spermatozoon with a vacuole‐free head could improve the embryo's ability to grow to the blastocyst stage and then implant. However, the most relevant indications for IMSI remain to be determined. To evaluate the potential value of IMSI for patients with a high degree of sperm DNA fragmentation (n = 8), different types of spermatozoa were analysed in terms of DNA fragmentation. Motile normal spermatozoa with a vacuole‐free head selected at 6300× magnification had a significantly lower mean DNA fragmentation rate (4.1 ± 1.1%, n = 191) than all other types of spermatozoa: non‐selected spermatozoa (n = 8000; 26.1 ± 1.5% versus 4.1 ± 1.1%; P < 0.005), motile spermatozoa (n = 444; 20.8 ± 2.7% versus 4.1 ± 1.1%; P < 0.001) and motile, normal spermatozoa selected at 200× magnification (n = 370; 18.7 ± 2.7% versus 4.1 ± 1.1%; P < 0.001) and then motile, morphometrically normal spermatozoa with anterior vacuoles (n = 368; 15.9 ± 2.9% versus 4.1 ± 1.1%; P < 0.05) or posterior vacuoles (n = 402; 22.5 ± 3.6% versus 4.1 ± 1.1%; P < 0.001) selected at 6300× magnification. For patients with high sperm DNA fragmentation rates, selection of normal spermatozoa with a vacuole‐free head (6300×) yields the greatest likelihood of obtaining spermatozoa with non‐fragmented DNA.     

66例男性不育患者采用自然周期IVF/M结合胞浆内单精子注射技术治疗的研究

目的探讨自然周期成熟卵母细胞结合未成熟卵母细胞体外成熟方法(natural cycle in vitro fertilization combined with in vitro maturation of immature oocytes,自然周期IVF/M)用于治疗男性因素引起不育的临床价值。方法66例适应证患者分成3组:少弱精子组、偶见活精子组和无精子症组[行经皮附睾精子抽吸术(PESA)、睾丸精子抽吸术(TESA)或睾丸切开取精术(TESE)],接受72个自然周期IVF/M,比较三组受精率和临床妊娠率。结果64例患者完成70个治疗周期。少弱精子组、偶见活精子组和无精子症组分别占完成周期数的54.3%、38.6%和7.1%,共获得卵母细胞812个,平均(11.3±5.2)个。卵母细胞成熟率为61.2%,正常受精率为82.2%,卵裂率为90.0%(377/419)。共26例患者获得临床妊娠,每取卵周期的妊娠率为36.4%(26/72),每移植周期的妊娠率为37.1%(26/70)。三组受精率分别为84.5%、78.9%和80.0%,三组间差异无统计学意义(P>0.05),临床妊娠率分别为39.5%,29.6%和60.0%,PESA/PESE和TESE组的临床妊娠率(60%)显著高于精液中偶见活精子组(29.6%,P<0.05)。结论自然周期IVF/ M技术对于治疗由于男性因素引起的不育如少弱精子症以及无精子症是一种简便有效的方法。     

流式细胞术检测精子DNA损伤的标准化与质量控制初步研究

目的:对流式细胞术检测精子DNA损伤的标准化和质量控制进行初步研究.方法:随机选取精液样本,观察酸变性时间,吖啶橙(AO)染色时间,精液样本冷藏、冷冻及反复冻融对精子DNA碎片指数(DFI)结果的影响.结果:随着酸变性时间延长,精子DFI逐渐增高,与酸变性30 s相比,酸变性至2 min时DFI即显著增加(P＜0.05...