High DNA Stainability Research Articles

Age-related changes in human conventional semen parameters and sperm chromatin structure assay-defined sperm DNA/chromatin integrity

Research questionWhat are the correlations between male age, traditional semen parameters, sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) in a sufficiently large sample size? DesignRetrospective cohort study of 18,441 semen samples, with data divided into seven age groups according to male age: ≤25, 26–30, 31–35, 36–40, 41–45, 46–50 and ≥51 years. ResultsAge was negatively correlated with semen volume, total sperm count, motility and HDS, and positively correlated with sperm concentration and DFI (P < 0.001). After 35 years of age, semen volume and total sperm count began to decline. After 30 years of age, motility and HDS decreased consistently. Sperm concentration and DFI increased from 26–30 years of age. DFI was negatively correlated with sperm concentration, total sperm count, motility and normal morphology (P < 0.001) and positively correlated with semen volume and HDS (P < 0.001). HDS was negatively correlated with all parameters (P < 0.001) except semen volume (r = –0.013, P = 0.074) and DFI (r = 0.124, P < 0.001). Patients aged ≥40 years had higher DFI than those aged <40 years in the entire cohort, in the abnormal semen parameters cohort, and in the normal semen parameters cohort (OR 2.145, 2.042, 1.948, respectively, P < 0.001). The ≥40 years age group had a lower HDS than the <40 years age group in the entire cohort and abnormal semen parameters cohort (OR 0.719, 0.677, respectively, P < 0.001). ConclusionsAgeing is a negative effector of sperm quantity and quality, and routine sperm parameters have weak but significant correlations with sperm DNA/chromatin integrity.

Unravelling how in vitro capacitation alters ram sperm chromatin before and after cryopreservation.

Sperm chromatin structure provides valuable information for the prediction of male fertility and can be altered during different procedures. Previous studies have shown that sperm chromatin condensation decreased during in vitro capacitation. Moreover, cryopreservation can affect sperm DNA integrity and chromatin compaction. This study aimed to investigate dynamic modifications produced in the chromatin structure of ram spermatozoa during in vitro capacitation before and after cryopreservation. Chromatin decondensation (AB+), DNA methylation, DNA fragmentation index (%DFI) and high DNA stainability (HDS) were evaluated in fresh and frozen-thawed ram spermatozoa incubated under capacitating (CAP) conditions at 1, 5, 15, 30, 60, 120, 180 and 240minutes and under non-capacitating (NC) conditions at 0, 15 and 240minutes. Incubation in NC conditions did not induce significant changes in chromatin condensation (P>.05; AB+and HDS). However, incubation of fresh and cryopreserved ram spermatozoa under CAP conditions significantly increased chromatin decondensation (P<.05), reaching the highest percentage of AB+and HDS from 180 to 240minutes in fresh samples and from 5 to 30minutes in cryopreserved samples. Both variables (HDS and AB+) were positively correlated with tyrosine phosphorylation, total motility, progressive motility, curvilinear velocity and amplitude of lateral head displacement, as well as between them under CAP conditions in fresh and cryopreserved spermatozoa. DNA methylation significantly increased in cryopreserved spermatozoa (P<.05), but only after extended incubation under CAP conditions (60-240minutes), while the %DFI, albeit higher in cryopreserved samples, remained constant under CAP and NC conditions in both types of sample (P>.05). Our results suggest that sperm chromatin condensation decreased progressively during in vitro capacitation of ram spermatozoa, while sperm DNA integrity remained intact. Such changes in chromatin condensation appeared faster after sperm cryopreservation.

Analysis of age‐associated alternation of SCSA sperm DNA fragmentation index and semen characteristics of 1790 subfertile males in China

BackgroundIt has been identified that incidence of infertility was about 20% among couples worldwide, about 50% caused by male elements. However, conventional semen laboratory detections could not handle clinical needs, which led to more comprehensive parameters for male fertility evaluation. We aimed to investigate the clinical relationship of age‐linked changes and the sperm chromatin structure assay (SCSA) sperm DNA fragmentation index (DFI), and routine semen characteristics among subfertile Chinese males.Methods1790 clinical semen specimens were enrolled from February 2018 to October 2019. Clinical and laboratory data including routine semen analyses, sperm DFI, and sperm morphology were collected and showed age‐related alterations in semen parameters.ResultsOur results, displayed an increase in sperm DFI with age, were demonstrated in three age‐groups, particularly within the ≥35‐year cohort. There were positive and inverse correlations of sperm DFI with abnormal semen characteristics and with normal morphological parameters, respectively. Furthermore, age, sperm morphology, concentration, and progressive motility, immotile sperm percentage, semen volume, sperm survival, and high acridine orange DNA stainability (indicating immature forms) were found to be independent risk factors affecting sperm DNA integrity. Likewise, men aged ≥35 years had a higher sperm DFI than did normozoospermic men in the overall cohort. Routine semen characteristics, sperm DFI, and morphology tended to alter with age.ConclusionsThe SCSA sperm DFI showed the greatest clinical application in the assessment of male fertility in this study, which should help infertility clinics decide on reproductive options for the treatment of older infertile couples.

DNA fragmentation of sperm: a radical examination of the contribution of oxidative stress and age in 16 945 semen samples.

What is the relationship between sperm DNA fragmentation and oxidative stress (OS) with increasing male age? Sperm DNA fragmentation increases with age and is likely related to both defective spermatogenesis and increasing OS levels. Sperm quality declines with age. The presence of DNA damage in a high fraction of spermatozoa from a raw semen sample is associated with lower male fertility in natural conception and intrauterine insemination. A retrospective cohort study of 16 945 semen samples analysed at a single reference laboratory between January 2010 and December 2018. All males were undergoing an infertility evaluation. The cohort was divided into seven age categories: <30, 30-34, 35-39, 40-44, 45-49, 50 to <54 and ≥55 years. The mean age was 37.6 years (SD 6.8). Sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) were calculated using flow cytometry. OS levels were measured using the oxidative stress adducts (OSA) test, by spectrophotometry. ANOVA with weighted polynomial contrast analysis was used to evaluate trends for DFI, OSA and HDS values across age categories. Mean DFI significantly increased across all age groups (Ptrend < 0.001). OSA was lowest in patients <30 years old (mean 3.6, SD 1.0) and also increased as age increased (Ptrend < 0.001). There was a statistically significant difference between age groups for each of the three parameters (P < 0.001). There was a significant linear trend for DFI, OSA and HDS across the seven age categories (P < 0.001). Among patients with high DFI, there was a decreasing age-dependent trend in the patients observed with high OSA (P < 0.001). This is a retrospective study. All males included in the study were undergoing a work-up for infertility and may not be representative of a fertile population. Additional patient demographics and clinical data were not available. DNA and/or oxidative damage in sperm may be just as important to understand as the chromosomal aberrations that are carried in the oocyte. Further studies are needed to evaluate the effect of advancing paternal age on the male genome and, ultimately, on the health of the offspring. No funding was obtained for this study. V.D. is an employee of Reprosource/Quest Diagnostics. D.S. reports he was a Scientific Advisor to Cooper Surgical. N/A.

SPERM DNA FRAGMENTATION INDEX AND HIGH DNA STAINABILITY DO NOT INFLUENCE PREGNANCY ACHIEVEMENT FOLLOWING ICSI

To evaluate the ability of sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) to influence the chance of achieving pregnancy in couples undergoing intracytoplasmic sperm injection (ICSI) cycles.

Sperm content of TXNDC8 reflects sperm chromatin structure, pregnancy establishment, and incidence of multiple births after ART

ABSTRACT Male germline-specific thioredoxin domain containing 8 (TXNDC8; alias SPTRX3) accumulates indefective human spermatozoa. We assessed the efficiency of two-step semen purification inremoving spermatozoa carrying TXNDC8, and examined the relationship of TXNDC8 with theoutcomes of assisted reproductive therapy (ART), conventional semen parameters, and sperm DNA integrity in sperm chromatin structure assay (SCSA). Semen samples (n = 255) from 91 ART couples were screened in two independent trials, both including a two-step, gradient-and-swim-up separation procedure yielding A-samples (raw semen), B-samples (gradient separated), and C-samples (gradient-and-swim-up). The C-samples were used for intracytoplasmic sperm injection (ICSI) with morphologically selected spermatozoa (IMSSI). Percentage of TXNDC8-positive spermatozoaincreased progressively from A to B/C-samples in both trials. In the first trial (35 couples), the TXNDC8 correlated positively with sperm DNA fragmentation index (%DFI; r = 0.66) measured before separation, and negatively with sperm concentration (r = −0.57) and motility (r = −0.67), also taken before separation. The high DNA stainability index (%HDS) correlated with the percentage of spermatozoa lacking TXNDC8 (r = 0.68). Both SCSA and TXNDC8 parameters showed moderate correlations (r = 0.33–0.66) with blood serum levels of hCG on day 11 (Beta 1) and day13 (Beta 2) after oocyte retrieval. In the second trial (56 couples), fathers of multiplets had a significantly lower percentage of TXNDC8-positive spermatozoa in B-sample (gradient separationonly) compared to men who conceived a singleton pregnancy (p = 0.01) and those who produced no pregnancy (p = 0.02). Those multiplets’ fathers also had a significantly higher sperm concentration while their SCSA parameters did not differ from others. It is concluded that theTXNDC8 levels correlate with SCSA and conventional raw semen parameters, and are predictive of pregnancy outcome and multiple births after ART. Two-step purification does not efficiently remove TXNDC8 carrying spermatozoa. Abbreviations ART– assisted reproductive therapy; DFI- DNA fragmentation index; FC- flow cytometry (FC); hCG: human chorionic gonadotropin; HDS: high DNA stainability index; HEPES- (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); HTF- human tubal fluid; ICSI- intracytoplasmic sperm injection; IgG- immunoglobulin G; IMSSI- ICSI with morphologically selected spermatozoa; IVF- in vitro fertilization; IU-: intrauterine insemination; NGS- normal goat serum; PBS- phosphate buffered saline; PVP- polyvinylpyrrolidone; SAB- spontaneous abortion; SCSA- sperm chromatin structure assay; SPTRX3- spermatid specific thioredoxin 3; SSS- synthetic serum substitute; TRITC- tetramethyl rhodamine isothiocyanate; TX-100- Triton X-100; TXNDC- thioredoxin domain-containing proteins; TXNDC8- thioredoxin domain containing 8; TUNEL- Terminal deoxynucleotidyl transferase dUTP nick end labeling

Sperm DNA fragmentation index and high DNA stainability do not influence pregnancy success after intracytoplasmic sperm injection

ObjectiveTo evaluate the ability of sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) to influence the chance of achieving pregnancy in couples undergoing intracytoplasmic sperm injection (ICSI) cycles.DesignA retrospective study evaluating couples who underwent an ICSI cycle between 2009 and 2018.SettingReproductive center.Patient(s)Consecutive couples who underwent an ICSI cycle and had a semen analysis with subsequent DFI and HDS testing, evaluated using Sperm Chromatin Structure Assay.Intervention(s)Measurement of DFI and HDS prior to ICSI cycle.Main Outcome Measure(s)To determine whether DFI or HDS of sperm was predictive of the number of ICSI cycles until the first clinical intrauterine pregnancy.Result(s)A total of 550 couples who underwent 1,050 ICSI cycles were analyzed. Of those, a total of 330 couples achieved pregnancy. As expected, in couples who achieved pregnancy, females were younger and underwent fewer cycles. Importantly, the DFI and HDS were similar between couples who achieved pregnancy (DFI% 12.9; HDS% 9.3) and couples who did not (DFI% 12.2; HDS% 9.1). A multivariable-adjusted analysis evaluating female age at the first cycle was associated negatively with pregnancy.Conclusion(s)Neither DFI nor HDS at baseline influenced the chances of a couple to achieve pregnancy after ICSI. Increased female age and couples who underwent more ICSI cycles were associated with lower chances of achieving pregnancy.

Could high DNA stainability (HDS) be a valuable indicator of sperm nuclear integrity?

BackgroundThe Sperm Chromatin Structure Assay (SCSA®), in addition to identifying the DNA Fragmentation Index (DFI) also identifies High DNA satiability (HDS), supposed to reflect the nuclear compaction of spermatozoa. However, data on what exactly this parameter reveals, its relevance and usefulness are contradictory. In order to shed light on this situation, spermatozoa of a cohort (N = 397) of infertile men were subjected to the SCSA®, TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling) and CMA3 (Chromomycin A3) tests. In a smaller subcohort (N = 100), aniline blue (AB) and toluidine blue (TB) staining were performed in addition. The objective of this study was thus to answer the question of whether HDS is a relevant and reliable parameter to be taken into account?ResultsHDS does not appear to be a reliable indicator of nuclear immaturity because it shows a weak correlation with the CMA3, AB and TB stains. The low correlation of HDS with sperm DNA fragmentation (TUNEL and SCSA®) and DNA condensation (CMA3, AB and TB) tests suggests that these two parameters could be decoupled. Unlike DFI and TUNEL, HDS has not been shown to correlate with classic clinical situations of male infertility (asthenozoospermia, teratozoospermia or astheno-teratozoospermia).ConclusionHDS correlates poorly with most tests that focus specifically on the level of maturity of the sperm nucleus. To our knowledge, this study is the first to compare SCSA®, TUNEL, AB, TB and CMA3 assays on identical samples. It shows the potency, consistency and limitations of each test and the care that must be taken in their interpretation.

Differences in sperm functionality and intracellular metabolites in Norwegian Red bulls of contrasting fertility

In the dairy breeding industry, prediction of bull fertility in artificial insemination (AI) is important for efficient and economically sustainable production. However, it is challenging to identify bulls with superior fertility applying conventional in vitro sperm assays. In the present study, sperm functionality was investigated to identify a multivariate model that could predict fertility. Two groups of young Norwegian Red bulls were selected, one with inferior fertility (18 bulls) and one with superior fertility (19 bulls) based on non-return rate after 56 days (NR56). Frozen-thawed semen doses were analysed for sperm chromatin integrity, viability, acrosome integrity, motility, and ATP content. A targeted approach was used to study intracellular concentrations of amino acids and trace elements in viable sperm cells. Significant differences between the two groups of bulls were observed, both for sperm functional attributes and intracellular concentrations of metabolites. Pearson correlation analyses indicated a negative relationship between NR56 and chromatin integrity parameters, DNA fragmentation index (DFI) and high DNA stainability (HDS). Several motility parameters correlated positively with NR56. The concentrations of cysteine and glutamic acid in sperm cells correlated negatively with NR56, while the concentrations of aspartic acid, leucine and serine showed a positive NR56-correlation. The sperm intracellular concentrations of the trace elements Fe, Al and Zn, correlated negatively with NR56. Correlations were observed between several sperm parameters and metabolites. Stepwise multiple regression analysis indicated that the best predictor of NR56 was a model containing %DFI, together with the intracellular sperm concentration of aspartic acid, Fe and Zn. This model explained 59% of the variability in NR56.

Concentrated ambient PM2.5exposure affects mice sperm quality and testosterone biosynthesis

BackgroundStudies suggested that PM2.5 exposure could lead to adverse reproductive effects on male animals. However, the underlying mechanism is still not clear. Besides, animals in the majority of previous studies were exposed to PM2.5 through intratracheal instillation which should be improved. In addition, limited amount of research has been conducted in China where the PM2.5 concentration is higher and the PM2.5 components are different. The aim of this work is to explore the effects of concentrated ambient PM2.5 (CAP) on mice sperm quality and testosterone biosynthesis.MethodsA total of 12 male C57BL/6 mice were exposed to filtered air (FA) or CAP for 125 days using the Shanghai Meteorological and Environmental Animal Exposure System. The mice sperm concentration, sperm motility, DNA fragmentation index, high DNA stainability and plasma testosterone were analyzed. Testicular histology and sperm morphology were observed through optical microscope. Testosterone biosynthesis related gene expressions were analyzed using real-time PCR, including cytochrome P450 CHOL side-chain cleavage enzyme (P450scc), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β HSD), 17β-hydroxysteroid dehydrogenase, cytochrome P450 aromatase (P450arom), estrogen receptor (ER), androgen receptor (AR) and follicle stimulating hormone receptor (FSHR).ResultsExposure to CAP resulted in disturbance of various stages of spermatogenesis and significant higher percentage of abnormal sperm (FA vs. CAP: 24.37% vs. 44.83%) in mice testis. CAP exposure significantly decreased sperm concentration (43.00 × 106 vs. 25.33 × 106) and motility (PR: 63.58% vs. 55.15%; PR + NP: 84.00% vs. 77.08%) in epididymis. Plasma testosterone concentration were significantly declined (0.28 ng/ml vs. 0.69 ng/ml) under CAP exposure. Notably, the levels of testosterone biosynthesis related genes, StAR, P450scc, P450arom, ER and FSHR were significantly decreased with CAP exposure.ConclusionConcentrated ambient PM2.5 exposure altered mice sperm concentration, motility and morphology, which might be mediated primarily by the decline in testosterone concentration and testosterone biosynthesis process.

H3K4me2 accompanies chromatin immaturity in human spermatozoa: an epigenetic marker for sperm quality assessment

ABSTRACTChromatin remodeling, including histone post-translational modifications, during spermatogenesis can affect sperm quality and fertility, and epigenetic marks may therefore be useful for clinical evaluations of sperm. Together with histone hyperacetylation, the dimethylation of histone H3 on lysine K4 (H3K4me2) is also required during protamination. Accordingly, we evaluated the utilization of this epigenetic mark for the identification of sperm with decrease quality and immature chromatin. In this study, 99 semen samples, including 22 normozoospermic (N), 63 asthenozoospermic (A), and 14 oligoasthenozoospermic (OA) samples, were comprehensively analyzed with respect to H3K4me2 levels, DNA damage (DNA fragmentation index, DFI), and sperm immaturity (high DNA stainability, %HDS), as determined by a sperm chromatin structure assay using flow cytometry. We detected a significant relationship between H3K4me2 and %HDS (r = 0.47; p < 0.001). Furthermore, we observed negative correlations between H3K4me2 and sperm concentration, motility, and mitochondrial activity (p < 0.05). The increase in immaturity as semen quality decreased (N > A > OA) indicates the importance of chromatin immaturity and histone code deviations in sperm evaluations. Using various approaches, our study elucidated H3K4me2 as a molecular marker of sperm quality with potential use in reproductive medicine.Abbreviations: A: asthenozoospermic; AO: acridine orange; ART: assisted reproductive therapy; BWW: Biggers-Whitten Whittingham; DAPI: 4ʹ,6ʹ -diamidino-2-phenylindole; DFI: DNA fragmentation index; H3K4me2: dimethylation of lysine K4 on histones H3; HDS: high DNA stainability; HRP: horseradish peroxidase; MACS: magnetic-activated cell sorting; N: normospermic; NGS: normal goat serum; OA: oligoasthenozoospermic; PTM: post-translational modification; SCSA: sperm chromatin structure assay; SUTI: sperm ubiquitin tag assay; TBS-T: TBS with 0.5% Tween-20

High DNA stainability in the SCSA® is associated with poor embryo development and lower implantation rate

Objective To determine if the high DNA stainability index (HDS), as measured by the sperm chromatin structure assay (SCSA®), is associated with in vitro embryo development and concurrent assisted reproductive technology (ART) outcomes. Design A retrospective IRB approved, chart review of patients who completed at least one ART cycle (in vitro fertilization or intracytoplasmic sperm injection) and had a male partner in a DNA fragmentation index (DFI) of 25%. Materials and Methods Semen samples for ART patients were analyzed at SCSA® Diagnostics Inc. A list of male partners with a DFI less than 25% and an HDS greater than 25% was compared to those who completed ART cycle(s) with their female partner. We collected data on the number of patients, the number of cycles, quality of Day 3 and Days5/6 embryos, positive serum human chorionic gonadotropin (hCG) results, fetal cardiac activity (FCA) and live births (LB). We stratified the data into four groups by mean HDS. Results 75 IVF cycles had confirmed DFI levels below 25% and HDS levels above 25%. The number of top quality Day 3 and Day 5 embryos decreased when the HDS was above 31% (25%-30%, n=344; 31-35%, n=132; 36-40%, n=79%; 41+%, n=65), however the total percentage of cycles with successful blastocyst formation was similar (25-30% = 33%, 31-35% = 25%; 36-40% = 39%; 41+% = 42%). The total number as well as percentage of positive hCG results after fresh embryo transfer diminished as HDS climbed (25-30%, n = 18 (42%); 31-35% n = 6 (46%); 36-40% n = 4 (44%); 41+% n = 1 (10%). The same trend occurred with FCA (25-30%, n = 18 (42%); 31-35% n = 6 (46%); 36-40% n = 2 (22%); 41+% n = 0 (0%), and LB outcomes (25-30%, n = 14 (33%); 31-35% n = 3 (23%); 36-40% n = 2 (22%); 41+% n = 0 (0%). The implantation rate declined when the HDS was above 36% (25-30% = 30%, 31-35% = 30%; 36-40% = 16%; 41+% = 15%). Conclusions These data suggest that while a sperm with an HDS above 25% can fertilize oocytes, as HDS rises, it may be associated with delayed and/or impaired embryo development in vitro as well as poor live birth outcomes. Disclosures Co-author D. Evenson is the owner and CEO of SCSA® Diagnostics Inc. Funding None

Sperm chromatin structure assay high DNA stainability sperm as a marker of early miscarriage after intracytoplasmic sperm injection

To determine whether high DNA stainability (HDS), as assessed by the sperm chromatin structure assay (SCSA), predicts the risk of early miscarriage after invitro fertilization with intracytoplasmic sperm injection (IVF-ICSI). Retrospective cohort study of consecutive pregnancies after IVF and ICSI treatment. Reproductive medicine center. A total of 1,602 pregnancies after 832 IVF and 770 ICSI treatments. HDS measured using SCSA. Early miscarriage (≤12weeks). The HDS represents the proportion of immature spermatozoa lacking the normal exchange of histone for protamine-complexed DNA, and the outcome parameter was early miscarriage (≤12weeks). For all treatments, the odds ratio (OR) and 95% confidence interval (CI) for early miscarriage was 1.41 (1.07-1.85) if HDS >15% compared with HDS ≤15%. When comparing the two HDS categories, for ICSI, the OR was 1.44 (1.01-2.04) whereas for IVF the results were not statistically significant. There is a small but increased risk of early miscarriage if HDS >15% compared with HDS ≤15%. This increased risk is seen only after ICSI, not after IVF. These findings suggest that HDS can be used as a predictor of an increased risk of miscarriage in ICSI treatments.

Male age is associated with sperm DNA/chromatin integrity

The diagnosis of sperm DNA integrity is increasingly recognized as being crucial to inform the clinical course in infertile couples. An internationally accepted sperm DNA fragmentation assay that determines the proportion of sperm and degree of broken sperm nuclear DNA with recognised clinical thresholds for identifying men at risk of infertility is the Sperm Chromatin Structure Assay (SCSA®). In this study, SCSA® test was utilised to evaluate the relevance of male age on sperm DNA quality in total of 6881 males of Indian origin. Analysis of proportions of DNA fragmentation index (%DFI) and high DNA stainability (%HDS) was performed based on four groups (<35, 35–40, 40–45, and >45 years of age). The impact of increasing male age on %DFI revealed that males >45 years of age had the highest %DFI and lowest %HDS compared to all other age groups (p<.001). This study is the largest population study and first of its kind in India that utilises SCSA® to assess the relevance of %DFI and %HDS to increasing age with potentially important implications for the choice of clinical course based on age and sperm quality of infertile males in India.

Sperm DNA fragmentation (SDF) was most effectively improved by a sperm separation device compared to different gradient and swimup methods

SDF is correlated with poor embryo development, implantation, and ongoing pregnancy rates. Reducing DNA fragmentation index (DFI) in the sample used for insemination may improve IVF outcomes. Prospective study to compare different sperm preparation techniques on the same semen sample to determine which methods most effectively improves DFI and other sperm health biomarkers, oxidative stress adducts (OSA) and high DNA stainability (HDS) index. Thirty samples from semen analysis patients were obtained. Samples sent to ReproSource (Woburn, MA) were blinded by freezing the samples in vials labeled with a random number. An aliquot of 0.3 mL of neat semen was frozen and the remainder of the semen allocated between 5 preparation methods (1-5). A sterile syringe was used to load 0.8 mL of raw semen in ZyMōt Sperm Separation Device (1,DxNow) then layered with G-IVF+ and incubated for 30 minutes. At the end of incubation, 0.3 mL of sperm was removed from the out port and frozen. Approximately 0.8 mL of semen was layered over the Isolate gradient (2,Irvine) and SpermGrad gradient (3,Vitrolife) per package insert and centrifuged for 15 minutes. The samples were reconstituted with GIVF+ and an aliquot of each was frozen. The pellet was re-formed and the tube incubated at an angle to obtain the swim-up (SU) Isolate + SU (4) and Spermgrad + SU (5), for each gradient. After one hour, an aliquot from the top of the top of the tube was frozen. Each blinded sample, 6 per semen sample, was analyzed using the Sperm DNA Fragmentation Assay (acridine orange/flow cytometry SDFA™) and the OSA™ test which directly measures sperm damage from oxidative stress by quantifying the presence of “adducts,” molecules in semen covalently modified by free radicals/reactive oxygen species. Statistical analysis was performed in JMP (SAS 2017). The mean age of men in the study was 37±8.3 years. Neat sperm concentrations ranged from 7 to 104 million/mL with 23.3% oligospermic. The motility of the samples ranged from 15 to 80% motility with an average of 52.2±16.9%. DFI, OSA, and HDS were compared between preparation methods on the same ejaculate using Wilcoxon Rank Sums between each pair with P<0.05 considered significant (See table).Tabled 1Prep Method 1Prep Method 2DFI p-valueOSA p-valueHDS p-valueNeatIsolate0.00520.00020.0011NeatIsolate + SU0.00230.0002<0.0001NeatSpermGrad0.0740.05740.2837NeatSpermGrad + SU0.01840.00240.0002NeatDxNow<0.0001<0.0001<0.0001IsolateIsolate + SU0.67890.83600.0389IsolateSpermGrad0.37500.06560.0326IsolateSpermGrad + SU0.65200.73940.1808IsolateDxNow<0.00010.0657<0.0001SpermGradSpermGrad + SU0.44640.31830.0044SpermGradIsolate + SU0.17610.06560.0005SpermGradDxNow<0.00010.0004<0.0001DxNowIsolate + SU<0.00010.0224<0.0001DxNowSpermGrad + SU<0.00010.0428<0.0001SpermGrad + SUIsolate + SU0.47790.97640.0044DFI, OSA, and HDS were compared between preparation methods on the same ejaculate using Wilcoxon Rank Sums between each preparation method. Open table in a new tab DFI, OSA, and HDS were compared between preparation methods on the same ejaculate using Wilcoxon Rank Sums between each preparation method. Sperm preparation methods have changed very little even with the rapid advances in the IVF laboratory. The sperm separation device is a novel method that effectively reduced DFI (p<0.0001) compared to two different gradients (2,3) and gradients followed by swim-up (4,5). The device also has eliminated centrifugation, a step known to increase oxidative stress. The device reduced OSA levels, a measurement of oxidative stress, and HDS, which is a measurement of immature cells and high histone retention. These are both indicators of sperm health and function. Overall, the quality of the sperm obtained post-processing was improved by the use of the separation device, which may increase the chance of a healthy sperm being used for fertilization.

Characterization of the Sperm Proteome and Reproductive Outcomes with in Vitro, Fertilization after a Reduction in Male Ejaculatory Abstinence Period

Semen samples from men after a short ejaculatory abstinence show improved sperm quality and result in increased pregnancy rates, but the underlying mechanisms remain unclear. Herein, we report that ejaculates from short (1-3 h) compared with long (3-7 days) periods of abstinence showed increases in motile sperm count, sperm vitality, normal sperm morphology, acrosome reaction capacity, total antioxidant capacity, sperm mitochondrial membrane potential, high DNA stainability, and a decrease in the sperm DNA fragmentation index (p, < 0.05). Sperm proteomic analysis showed 322 differentially expressed proteins (minimal fold change of ±1.5 or greater and p, < 0.05), with 224 upregulated and 98 downregulated. These differentially expressed proteins are profoundly involved in specific cellular processes, such as motility and capacitation, oxidative stress, and metabolism. Interestingly, protein trimethyllysine modification was increased, and butyryllysine, propionyllysine, and malonyllysine modifications were decreased in ejaculates from a short versus, long abstinence (p, < 0.05). Finally, the rates of implantation, clinical pregnancy, and live births from in vitro, fertilization treatments were significantly increased in semen samples after a short abstinence. Our study provides preliminary mechanistic insights into improved sperm quality and pregnancy outcomes associated with spermatozoa retrieved after a short ejaculatory abstinence.

Acrosome reaction and chromatin integrity as additional parameters of semen analysis to predict fertilization and blastocyst rates

BackgroundTraditional semen parameters have shown little to none predictive value for fertilization and blastocyst viability for a successful pregnancy. Therefore, the purpose of this study was to explore the usefulness of incorporating the acrosome reaction (AR) and chromatin integrity to conventional semen analysis to individually predict the fertile potential of sperm samples.MethodsA cross-sectional study was conducted in 69 participants undergoing IVF using oocyte donation. Semen samples were collected and evaluated for: AR [spontaneous (sAR) and induced (iAR)] by flow cytometry using anti-CD46-FITC, Acrosome Response to an Ionophore Challenge (ARIC), chromatin integrity by Sperm Chromatin Structure Assay (DNA Fragmentation Index-%DFI and High DNA Stainability-%HDS), WHO semen analysis, fertilization and blastocyst rates.ResultsThe participant age was 40.0 ± 6.1 years (66% were normozoospermic). Sperm morphology, sAR, iAR, and ARIC were associated with the fertilization (β = 3.56, R2 = 0.054; β = − 5.92, R2 = 0.276; β = 1.83, R2 = 0.150; and β = 2.10, R2 = 0.270, respectively, p < 0.05). A logit model was developed to calculate the probability of fertilization (≥ 60%) for each participant, using the sperm morphology and ARIC as independent variables, followed by ROC analysis to determine a cutoff probability of 0.65 (specificity = 80.6%, sensitivity = 63.2%). %DFI was inversely associated with the viable blastocyst rate (β = − 1.77, R2 = 0.057, p = 0.003), by the logit model and ROC analysis, a cutoff probability of 0.70 (specificity = 80.6%, sensitivity = 72.3%) was obtained to predict blastocyst viability (≥ 40%). There was no difference in the results with normozoospermic samples (n = 46).ConclusionsThe incorporation of ARIC and %DFI allowed to obtain predictive models for high fertilization and blastocyst rates in an individualized way, being promising tools to improve the diagnosis of male fertility potential for research or assisted reproduction, even in men with unknown infertility.

Environmental exposure to non-persistent endocrine disrupting chemicals and semen quality: An overview of the current epidemiological evidence

Some of the recent publications have reported a decline in semen quality in the last few decades. This phenomenon is associated with environmental factors, particularly with exposure to endocrine disrupting chemicals (EDCs). The aim of this publication is to critically review the literature on exposure to the following 6 ubiquitous environmental non-persistent EDCs: bisphenol A, triclosan, parabens, synthetic pyrethroids, organophosphate pesticides and phthalates, and on their influence on semen quality measured as sperm concentration, sperm volume, total sperm count, motility, total motile count, morphology, sperm motion, sperm DNA damage (comet extent, tail length, tail distributed moment, percent of DNA located in the tail (tail%), DNA fragmentation index, high DNA stainability, X:Y ratio and aneuploidy. Several electronic databases were systematically searched until 31 August 2016. Studies were qualified for the review if they: linked environmental exposure to non-persistent EDCs to semen quality outcomes, were published in English after 2006 (and, in the case of phthalates, if they were published after 2009) and were conducted in the case of humans. Out of the 970 references, 45 articles were included in the review. This review adds to the body of evidence that exposure to non-persistent EDCs may affect semen quality parameters and decrease semen quality. Int J Occup Med Environ Health 2018;31(4):377–414

Morphological defects, sperm DNA integrity, and protamination of bovine spermatozoa.

The association between sperm morphology characteristics and DNA conformation and integrity is still controversial. In bulls, major morphological sperm abnormalities have been associated with reduced fertility, and morphological assessment is used to provide an indication of potential fertility of the individual. Sperm DNA fragmentation and damage has a negative effect on embryo development and subsequently fertility, with bull spermatozoa generally displaying low levels of DNA damage and tight chromatin. However, sensitive methods for detecting chromatin damage may reveal associations with morphological defects. The objective was to determine whether morphological sperm abnormalities and variables expressing sperm DNA integrity and protamination are correlated in bulls, using the sperm chromatin structure assay (SCSA) and the sperm protamine deficiency assay (SPDA). Electroejaculated samples (n=1009) from two-year-old tropically adapted bulls were split and fixed and submitted to microscopic sperm morphology assessment, and snap-frozen for sperm nuclear integrity assessments by SPDA and SCSA. For SPDA, the variables were defective (MCB) and deprotaminated (HCB), and for SCSA, the variables were DNA fragmentation index (DFI) and high DNA stainability (HDS). HCB correlated with DFI; τKen2 =0.317 and HDS; 0.098, and MCB correlated with DFI; 0.183 (p<0.001). The percentage of morphological normal spermatozoa was correlated negatively to DFI; τKen2 =-0.168, MCB; -0.116 and HCB; -0.137 (p<0.001). HCB and DFI were both positively correlated to head defects, proximal droplets, and spermatogenic immaturity, but not to distal droplets, vacuoles, or diadems. Sperm DNA integrity and protamination, using the SCSA and SPDA, respectively, in bulls show associations with morphological parameters, particularly with head shape abnormalities and indicators of spermatogenic immaturity, including proximal droplets. The vacuoles and diadem defects were not correlated with sperm nuclear integrity, and hence, these are likely physiological features that may not directly affect sperm chromatin configuration.

Exposures to Atmospheric PM10 and PM10-2.5 Affect Male Semen Quality: Results of MARHCS Study.

Studies have shown that the effects of ambient particulate matter (PM) may be related to particle's size. However, results on the relationships between different PM and reproductive health are controversial. To explore the impacts of various PM fractions on male reproductive health, a total of 796 eligible subjects recruited in 2013 baseline investigation. In addition, there were 656 (82.4%) and 568 (71.3%) subjects participated follow-up surveys in 2014 and 2015, respectively. We used multivariable regression analysis and mixed-effect model to investigate the associations between air pollutants PM10, PM10-2.5, and PM2.5 exposures and semen quality, sperm DNA fragmentation and serum reproductive hormones of subjects. In the preliminary regression analysis, PM10, PM10-2.5, and PM2.5 exposure all associated with sperm concentration, morphology, sperm high DNA stainability (HDS), serum estradiol and testosterone levels. However, in mixed models, we only found that PM10 exposure were negatively associated with sperm normal morphology (95% CI: -14.13, -24.47) but positively associated with sperm progressive motility (95% CI: 23.00, 8.49), and PM10-2.5 exposure was inversely associated with sperm concentration (95% CI: -9.06, -27.31) after multiplicity adjustment. Our results provide the evidence that air PM10 and PM10-2.5 exposures, not PM2.5, are risk factors of semen quality.