Sperm Chromatin Structure Assay Parameters Research Articles

Impact of Age and Fertility Status on the Consistency of Repeat Measurements of Sperm DNA Damage: A Single-center, Prospective, Dual Visit Study

ObjectiveTo examine the relationship between abnormal routine semen parameters and DNA damage associated with advanced paternal age and infertility by controlling for common biases contested in the current literature. Materials & MethodsProspective study using 151 semen samples collected from men aged 18-80 at 2 visits with 1-3 months intervals. Samples were collected from both infertile and general population controls. Conventional semen parameters were measured including volume, concentration and motility. Sperm DNA damage was measured using the %DNA Fragmentation Index (%DFI) and High DNA stainability (%HDS) using Sperm Chromatin Structure Assay (SCSA). Patients were then classified according to %DFI as normal (<18), intermediate (18-27), or high (>27). ResultsSignificant correlation between all sperm parameters was seen between both visits regardless of age. DFI had the highest correlation between both visits (R2 = 0.77). Progressive motility, total motility and %DFI were significantly affected in men ≥50 years old when compared to men <35 and men 35-49 years old (P <.001). Forty-eight percent of men with intermediate %DFI changed category on their second visit, whereas men with high and low %DFI changed category in 15% and 9%, respectively. ConclusionSperm and SCSA parameters do not change significantly between 2 visits at 1-3 months intervals in the total population and after subgrouping. Men of advanced age have poorer sperm parameters and more DNA damage. Men with initially normal or elevated %DFI are unlikely to change DNA damage category. Older men are more likely to have sperm parameters and DNA damage vary on repeat semen analysis compared to younger men.

Sperm DNA integrity is critically impacted by male age but does not influence outcomes of artificial insemination by husband in the Chinese infertile couples.

The sperm chromatin structure assay (SCSA) is crucial for assessing male fertility. However, the predictive value of the SCSA parameters, including the DNA fragment indices (DFI) and the percentages of high DNA stainability (HDS), for outcomes of artificial insemination by husband (AIH) remains controversial. This study aims to evaluate the correlations between SCSA parameters and male aging as well as other routine semen parameters, and explore their prognostic powers on AIH outcomes of the Chinese infertile couples. A total of 809 AIH cycles were retrospectively analyzed. The results showed that DFI in the age groups < 35 years were significantly lower than that in the age groups ≥ 35 years (P < 0.001). Meanwhile, there was no statistical difference in HDS between the age groups (P = 0.063). DFI and HDS are negatively correlated with most routine semen parameters (all P < 0.05). The chi-square and generalized linear model tests indicated that neither DFI nor HDS influenced the clinical pregnancy rate of AIH. In summary, this study found that aging is a critical factor leading to increased sperm DFI but not HDS. DFI and HDS are negatively correlated with most semen parameters but do not significantly influence AIH outcomes.

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

In Memoriam: Marcello Spanò: 1954 – 2017

While conversing with his research team at ENEA in Rome, Marcello Spanò suffered a cardiac arrest, and died at the hospital without regaining consciousness a few days later — on January 19, 2017. The meaningless death of Marcello at the age of 62 is a terrible loss for his family, his research team and collaborators at ENEA in Rome, and all his colleagues and friends in a large international network of researchers. Marcello figured out at an early stage that a laboratory method to measure DNA damage in spermatozoa, the so-called Sperm Chromatin Structure Assay (SCSA), had a great potential in research as well as in clinical practice. He established a collaboration with US scientist Professor Don Evenson, who invented this assay and got it to work at his Lab at ENEA in Rome. Subsequently, Marcello's lab has played a major role in numerous epidemiological and experimental studies throughout Europe, addressing toxic effects on male reproductive function by chemicals in our environment. Of major importance, it turned out that the SCSA parameter called DFI (DNA Fragmentation Index) is an independent predictor of male fertilizing potential and therefore has become an important part of diagnostics in infertility make-up. During the past few years, Marcello and his team have worked hard to develop and implement techniques to look into epigenetic changes in the human male genome in spermatozoa. He was brutally taken away just when this work was hoped to provide new important insight into male reproductive function and its susceptibility to environmental chemicals. Marcello was not only a great scientist but also a humanist with profound interest in literature, travel, and ancient as well as modern history and politics. His ability to communicate with other people and to create social relationships was remarkable, perhaps because he was an excellent listener. In human relations, understanding was much more important than judging, the progress and performance of the group more important than that of the individual. Marcello insisted on scientific integrity and high standards and never was tempted to harvest publicity from premature or preliminary research results. Marcello was a great friend and fan of Scandinavia, which he showed, not only by scientific collaboration but also by helping colleagues at Lund University with setting up and running SCSA. He also learned to speak and read Swedish by taking lessons at the Swedish Church in Rome. Many of his e-mails and SMSs sent to his Nordic friends were written in — close to perfect — Swedish. Although research was an important part of his life, he also appreciated his leisure time, which he spent meeting family, friends, reading, traveling, and aquarelle painting. Marcello was a very unique person — as researcher, colleague, and friend. His early death is a tremendous loss to science, colleagues at ENEA and his large international collaborative research network. We will miss Marcello's ever supporting back-up, his optimistic and humoristic approach to life and partnerships, his patience and understanding, a real friend one could always rely on.

Chromatin integrity of ram spermatozoa. Relationships to annual fluctuations of scrotal surface temperature and temperature-humidity index

The objective of the present study was to explore the potential relationships of ovine sperm chromatin integrity, quantified using the sperm chromatin structure assay (SCSA), to the heat load of the scrotum and the discomfort felt by the animals because of fluctuations of microclimatic factors at different time periods before ejaculation. Ejaculates were collected once per week from five Chios rams and four East Friesian rams for 12 months and stored in liquid nitrogen. Frozen-thawed semen samples were analyzed using the SCSA, to determine the DNA fragmentation index (DFI) and the percentage of cells outside the main sperm population (%DFI) in each one of the samples. Scrotal surface temperature (SST) of each ram was measured using an infrared thermometer on a daily basis. Ambient air temperature and relative humidity were recorded at hourly intervals throughout the experimental period and temperature-humidity index (THI) was used to assess the discomfort felt by the rams. Mean values of SST (SSTmean) and THI (THImean) were computed for eight different time periods (up to 61 days) preceding each ejaculation day (Day 0). A linear mixed-effect model analysis was performed to describe the relation of SCSA parameters to collection month, SSTmean, and THImean of different time periods before ejaculation. The results of the statistical analysis revealed a relation of %DFI to the SSTmean of the last 12 days preceding ejaculation, namely the period that resembled the phase of epididymal maturation. On the contrary, the variation of DFI was most adequately described by the linear mixed-effect model applied for Days 54 to 48 before ejaculation, which resembled the phase of spermatogonial mitoses. The effect of collection month was significant for DFI and %DFI, with semen samples collected in September and February exhibiting the lowest DFI values; a less profound seasonal pattern was detected for %DFI. The effect of THImean on DFI and %DFI was proven nonsignificant in regard to all time periods. In conclusion, a relation of SCSA parameters to SSTmean of different periods before ejaculation was shown in the present study, implying an effect of scrotal microenvironment on intratesticular and epididymal sperm population. In contrast, we failed to detect any effect of microclimate-induced discomfort felt by the animals on the chromatin integrity of frozen-thawed ram spermatozoa.

The Relationship Between Post‐Thaw Sperm DNA Integrity and Non‐Return Rate Among Norwegian Cross‐Bred Rams

With the aim of investigating the relationship between sperm DNA integrity and non-return rate (NRR) among Norwegian cross-bred rams, semen from 15 individuals was examined by flow cytometry. Sperm Chromatin Structure Assay (SCSA) quantifies the proportion of spermatozoa with denatured DNA after in situ acid treatment, and the four parameters % DFI, % HDS, MEAN DFI and SD DFI are all different measures of DNA denaturation and maturation. Field fertility, reported as NRR 25 days after insemination was based on all inseminations from a large-scale breeding programme and supplied by the Norwegian Association of Sheep and Goat Farmers. From each ram, four straws from four different weeks of the breeding season were analysed, and the associations between 25-day NRR and the mean of the four SCSA parameters were tested using a logistic regression model. The results revealed no association between fertility and % DFI or % HDS, while SD DFI and MEAN DFI showed a significant negative association with NRR. Further, the SCSA values varied significantly between ejaculates within ram among some of the rams in the study. However, no significant association was seen between these intra-individual differences in sperm DNA integrity and NRR. In conclusion, this study suggests an association between sperm DNA integrity and NRR for rams. However, further research must be conducted to confirm these findings and determine whether sperm DNA assessments can be applied to predict ram fertility.

Influence of microsurgical varicocelectomy on human sperm mitochondrial DNA copy number: a pilot study

There is good evidence to show that varicocele repair can improve conventional sperm parameters, as well as, sperm DNA integrity, in infertile men with a clinical varicocele. To examine the effect of varicocelectomy on sperm quality, specifically, sperm nuclear chromatin integrity and sperm mitochondrial DNA (mtDNA) copy number. A prospective study done between March 2007 and January 2008. We evaluated a consecutive series of infertile men (n = 14) presenting to Ovo clinic with one year or more history of infertility, a clinically palpable varicocele and poor motility (<25 % rapid progressive and <50 % progressive). Microsurgical sub-inguinal varicocelectomy. Conventional sperm parameters, sperm mtDNA copy number (by real time PCR) and sperm chromatin structure assay (SCSA) parameters (%DFI,% HDS) before and 4 months after microsurgical varicocelectomy. Sperm concentration and SCSA parameters (%DFI and %HDS) improved significantly after surgery (P < 0.05). Sperm mitochondrial DNA copy number decreased significantly after surgery (27 ± 30 to 9 ± 6 copies per sperm, respectively, P = 0.032). There was a significant negative correlation between mitochondrial DNA copy number and sperm motility (r = - 0.71, P = 0.002). These findings support the concept that correction of a varicocele can improve spermatogenesis and sperm function, as mitochondrial DNA copy number has been suggested to reflect the efficiency of spermatogenesis and has been inversely related to sperm motility.

2019 EFFECT OF MICROSURGICAL VARICOCELECTOMY ON HUMAN SPERM DNA INTEGRITY AND CHROMATIN COMPACTION

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20112019 EFFECT OF MICROSURGICAL VARICOCELECTOMY ON HUMAN SPERM DNA INTEGRITY AND CHROMATIN COMPACTION Naif Alhathal, Maria San Gabriel, and Armand Zini Naif AlhathalNaif Alhathal Montreal, Canada More articles by this author , Maria San GabrielMaria San Gabriel Montreal, Canada More articles by this author , and Armand ZiniArmand Zini Montreal, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2247AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Human sperm DNA damage may adversely affect reproductive outcomes, and the spermatozoa of infertile men possess substantially more DNA damage than that of fertile men. Further to our previous study on the effect of varicocelectomy on sperm DNA integrity (DFI= DNA fragmentation index) using sperm chromatin structure assay (SCSA), the objective of this study was to further assess human sperm chromatin integrity before and after varicocelectomy by evaluating aniline blue staining (AB) in these samples. METHODS We evaluated 20 men who underwent microsurgical varicocelectomy for clinical varicocele and 6 controls (sperm donors) at our institution. We examined standard semen parameters, sperm DNA integrity (by SCSA) and chromatin integrity (by aniline blue stain) before and after varicocelectomy and for the control group. RESULTS The percentage of sperm with positive AB staining (histone retention) decreased significantly after surgery (from 13.5 to 5.4%). The %DFI (DNA fragmentation index) also decreased significantly (as previously shown). However, there were no significant relationship between AB staining and %DFI, motility or concentration. The only notable relationship was between AB staining and HDS (high DNA stainability–an index of chromatin compaction) post varicocelectomy. Furthermore, the percentage of sperm with positive AB staining was significantly less in the control compared to varicocele patients before surgery (2.5% vs 13.5%). The %DFI also was significantly less in the control group (7% vs 20%). CONCLUSIONS The data show that varicocelectomy is associated with a consistent improvement in sperm DNA integrity and chromatin compaction using two different assays of sperm chromatin integrity (SCSA, Aniline Blue). However, the poor relationship between SCSA parameters (e.g. %DFI) and AB staining suggests that these assays measure different aspects of sperm function. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e807 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Naif Alhathal Montreal, Canada More articles by this author Maria San Gabriel Montreal, Canada More articles by this author Armand Zini Montreal, Canada More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Effect of microsurgical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial

There is evidence from retrospective studies that varicocelectomy can improve sperm DNA damage in infertile men with a clinical varicocele. The objective of this prospective study was to examine further the effect of varicocelectomy on sperm chromatin and DNA integrity. We evaluated a consecutive series of infertile men (n = 25) who underwent microsurgical varicocelectomy for treatment of clinical varicocele. We examined conventional sperm parameters and sperm chromatin structure assay parameters (percentage DFI--DNA fragmentation index and percentage HDS--high DNA stainability, an index of chromatin compaction) before and 4 and 6 months after microsurgical varicocelectomy. Sperm DNA integrity improved significantly after surgery (percentage DFI decreased from 18 ± 11% before surgery to 10 ± 5%, and 7 ± 3%, at 4 and 6 months after surgery respectively). Sperm chromatin compaction also improved significantly after surgery (percentage HDS decreased from 11 ± 7% before surgery to 8 ± 6%, and 7 ± 5%, at 4 and 6 months after surgery, respectively). Sperm concentration and progressive motility improved after surgery, although the differences were not statistically significant when compared with that before surgery. The data show that varicocelectomy is associated with an improvement in sperm DNA integrity and chromatin compaction. These findings support the concept that correction of a varicocele can improve spermatogenesis, particularly spermiogenesis (the stage in spermatogenesis where compaction and stability of the sperm DNA and chromatin occur).

Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction

The objective of this study was to determine if a relationship exists between sperm parameters, measured by sperm chromatin structure assay (SCSA), and spontaneous abortion and multiple births in couples undergoing assisted reproduction treatment. Retrospective analysis of infertility treatment outcomes and occurrence of spontaneous abortion and multiple births was conducted in 233 couples who underwent treatment by intracytoplasmic sperm injection or intrauterine insemination at the Sher Institute for Reproductive Medicine, between 2001 and 2004. Sperm samples used for treatments were analysed for sperm concentration, sperm motility and two different parameters of SCSA (DNA fragmentation index, DFI, and high DNA stainability, HDS). Pregnancy, spontaneous abortion and multiple birth rates were recorded for all couples. A statistically significant relationship ( P < 0.001) was observed between DFI and spontaneous abortion. However, the correlation between HDS and spontaneous abortion was not statistically significant. Significantly lower levels of DFI were observed in men from couples having triplet pregnancies compared with those in the spontaneous abortion group ( P ⩽ 0.05). It is concluded that the parameters of SCSA correlate significantly with spontaneous abortion and multiple birth and may provide guidance for clinical decision making (number of embryos per transfer) and management of spontaneous abortion-prone cases. The goal of this study was to determine if a relationship exists between the sperm chromatin structure assay (SCSA) and spontaneous abortion and multiple births in couples treated via assisted reproductive technologies such as intra-cytoplasmic sperm injection or intrauterine insemination. SCSA is a test used by fertility clinics to determine if the sperm chromosomes or DNA are properly packaged in the sperm head and are therefore protected from damage that could occur during travel in the female reproductive system. A retrospective analysis of the results of SCSA, infertility treatment outcomes and the occurrence of spontaneous abortions and multiple births was conducted for 233 couples attending an infertility clinic. A statistically significant correlation was found between one of the SCSA parameters and spontaneous abortion. In addition, lower levels of this SCSA parameter were observed in men from couples having triplet pregnancies. We conclude that the parameters of SCSA correlate significantly with spontaneous abortions and multiple births and may provide guidance for clinical decision making, such as number of embryos per transfer or intensive management of spontaneous abortion-prone cases.

Sperm chromatin structural integrity in normospermic boars is not related to semen storage and fertility after routine AI

Standard semen parameters are limited in their capacity to distinguish subfertile boars and to assess storage influences on liquid preserved boar semen. The evaluation of sperm chromatin structural integrity could have potential as a diagnostic tool in AI practice. This study assessed whether the determination of sperm DNA integrity adds a useful diagnostic tool for selection of boar ejaculates in routine AI procedure and assessment of storage effects in diluted semen. Special emphasis was laid on the standard spermatological characterization of semen samples in parallel with the determination of the DNA fragmentation index (DFI) through the sperm chromatin structure assay (SCSA). Six hundred ninety two (692) ejaculates from 79 Piétrain boars in an AI center were analyzed for motility, morphology and DFI over a period of 24 weeks. 95.5% of the semen samples had a DFI < 5% with low distribution of variation for DFI due to boar and ejaculate (< 5%). 61.3% of ejaculates with DFI > 5% showed values below thresholds for sperm motility or morphology. Based on field data from 13,239 inseminations, fertility of boars with temporarily elevated DFI was not impaired (P > 0.05). 24 randomly selected diluted semen samples did not show a significant increase of DFI during 168 h storage (P > 0.05). In a further experiment, 42 diluted semen samples from 14 normospermic boars were assessed for motility, membrane integrity (PI, FITC-PNA) and SCSA parameters. Three single ejaculates showed an increase of DFI at 120 and 168 h storage time. This was accompanied by a pronounced loss of both motility and membrane integrity. In conclusion, the evaluation of sperm chromatin structural integrity by the SCSA has only limited value for identifying sperm deficiencies in normospermic fresh or stored boar semen. Temporarily elevated DFIs seem not to be indicative of subfertility in normospermic boars.

Effects of in vitro exposure to natural levels of zearalenone and its derivatives on chromatin structure stability in equine spermatozoa

The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of the DNA fragmentation index (DFI), such as the mean ( X ¯ - DFI ), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, α-zearalenol, and β-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.

Boar Fertility and Sperm Chromatin Structure Status: A Retrospective Report

Little information exists about boar sperm chromatin quality and fertility within a commercial setting. The objective of this report is to provide information about boar sperm chromatin integrity and its relationship to fertility. The sperm chromatin structure assay (SCSA) was used retrospectively to characterize sperm from 18 sexually mature boars having fertility information. Boar fertility was defined by farrow rate (FR) and average total number of pigs born (ANB) per litter of gilts and sows mated to individual boars. Fertility data was compiled for 1867 matings across the 18 boars. The SCSA uses flow cytometry to evaluate the structural integrity of sperm nuclear DNA. The SCSA parameters measured in this retrospective analysis were the percentage DNA fragmentation index (%DFI) and standard deviation of the DNA fragmentation index (SD DFI). The %DFI and SD DFI showed the following significant negative correlations with FR and ANB; %DFI vs FR, r = -0.55, P < .01; SD DFI vs FR, r = -0.67, P < .002; %DFI vs ANB, r = -0.54, P < .01; and SD DFI vs ANB, r = -0.54, P < .02. Although more information is required to better understand the relationship between DFI and boar fertility, this report suggests that the SCSA assay may be an important assay for identification of boars having potential for lowered fertility.

228 BOAR FERTILITY AND SPERM CHROMATIN STRUCTURE ASSAY DEFINED SPERM DNA FRAGMENTATION

The sperm chromatin structure assay (SCSA) was used retrospectively to characterize sperm from 18 sexually mature boars having fertility information. Boar fertility was defined by farrowing rate (FR) and average total number of pigs born (ANB) per litter of gilts and sows mated to individual boars. The SCSA uses flow cytometry to evaluate the structural integrity of sperm nuclear DNA. In brief, aliquots of frozen/thawed semen were treated for 30 s with a pH 1.2 buffer that denatures sperm DNA at the sites of DNA strand breaks. The samples are then stained with acridine orange (AO) that intercalates into native, double-stranded DNA and fluoresces green under the flow cytometer laser beam. Broken and denatured single-stranded DNA associates with AO and then collapses, causing a metachromatic shift from green to red fluorescence. Five thousand sperm are measured per sample at rates of ~200 s–1, thus giving the data strong statistical robustness. This fact coupled with the precision of the AO/DNA biochemistry provides a CV ~2% between replicate samples. The SCSA parameters measured in this study were the %DNA fragmentation index (%DFI; percentage of sperm with fragmented DNA) and standard deviation of DFI (SD DFI). The %DFI and SD DFI showed the following significant negative correlations with FR and ANB; %DFI v. FR r = –0.55, P &lt; 0.01, SD DFI v. FR r = –0.67, P &lt; 0.002, %DFI v. ANB r = –0.54, P &lt; 0.01, and SD DFI v. ANB r = –0.54, P &lt; 0.02. A discrimination analysis was used to distinguish between the boar groups above and below 6% DFI. The variables most useful to discriminate between 2 groups were SD DFI, FR, and ANB. The average squared canonical correlation for SD DFI was 0.82, P &lt; 0.0001. The average squared canonical correlation for FR and ANB was 0.45, P &lt; 0.003 and 0.54, P &lt; 0.001, respectively. Odds ratios for %DFI and SD DFI were 1.5 times (P = 0.0003, CI 1.22, 1.94) and 2.5 times (P = 0.0001, CI 1.87, 3.32), respectively.

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear ( Ursus arctos) spermatozoa

The Cantabrian brown bear ( Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry by the computer-assisted sperm morphology assessment (CASMA) system. Morphometry was analyzed before and after freezing–thawing in order to evaluate the effects of cryopreservation on sperm heads. Each spermatozoon was measured for four primary parameters (length, L; width, W; area, A; perimeter, P) and derived parameters (ellipticity: L/ W, circularity: 4 πA/ P 2, elongation: ( L − W)/( L + W), regularity: πLW/4 A). All the derived parameters significantly differed between bears. Likewise, cryopreservation affected head morphometry by reducing its size. Clustering based on morphometric parameters separated three subpopulations, one of them being significantly more influenced by the cryopreservation process. We obtained high correlations between head morphometry and SCSA parameters: standard deviation of DNA fragmentation index (SD-DFI) was correlated with perimeter and area ( r = 0.75 and r = 0.62, respectively) and DFIm and DFIt (moderate and total DNA fragmentation index) were correlated with perimeter ( r = 0.65 and r = 0.67, respectively). Nevertheless, classification of males according to SCSA or head morphometry did not completely agree so the two assays might explain male variability differently. We conclude that cryopreservation affected morphometry at least in a subset of spermatozoa. These results might improve future application of sperm banking techniques in this species.

Sperm head morphology is related to high deoxyribonucleic acid stainability assessed by sperm chromatin structure assay

To examine the relationship between sperm strict morphology and sperm chromatin integrity. Prospective study. Infertility clinic. Eighty-seven consecutive semen samples from non-azoospermic men presenting for infertility evaluation and 6 samples from fertile donors. Assessment of standard semen parameters and sperm chromatin structure assay (SCSA) parameters (%DFI [DNA fragmentation index] and %HDS [high DNA stainability]). Evaluation of %HDS and %DFI after treatment with dithiothreitol (a thiol-reducing agent used to decondense sperm nuclei) was also undertaken. Relationship between sperm strict morphology defects and SCSA parameters (%DFI and %HDS). We observed significant relationships between the percentage of normal sperm forms and both %HDS (r = -0.40) and sperm motility (r = 0.32). We also found significant relationships between sperm head defects and both %HDS (r = 0.40) and sperm concentration (r = -0.39). Sperm tail, midpiece, and neck defects were not significantly related to the SCSA parameters. Treatment of spermatozoa with dithiothreitol (to induce decondensation) resulted in a substantial increase in %HDS but no measurable change in %DFI. The observed relationship between sperm head defects and %HDS suggests that sperm head abnormalities may, in part, be due to incomplete sperm chromatin condensation.

Reply: Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive of the outcome of ART

Reply: Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive of the outcome of ART

Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART

The sperm chromatin structure assay (SCSA) parameter DNA fragmentation index (DFI) has been shown to predict in vivo and in vitro fertility. So far most SCSA studies have been based on SCSA analysis performed on neat semen. The aim of this study is to assess whether SCSA analysis of sperm prepared by density gradient centrifugation (DGC) could add more information in regard to the prediction of treatment outcome. The study included 510 assisted reproductive technique (ART) cycles. SCSA was performed in neat semen and post DGC. SCSA results were expressed in terms of DFI and high DNA stainability (HDS) cell fractions. The outcome parameter was clinical pregnancy (CP). Scatter-plot diagrams demonstrated that for DGC samples, no DFI cut-off values could be set for in vivo or in vitro fertility. In intrauterine insemination, IVF and ICSI groups the mean difference (95% CI) in DFI post DGC between those who achieved CP and those who did not was 0.2% (-1.7 to 2.0%), 0.4% (-1.9 to 2.8%) and 1.3% (-3.1 to 5.9%), respectively, none of these being statistically significant. The corresponding differences for HDS were 0.1% (-1.3 to 1.5%), 0.1% (-0.7 to 0.9%) and 0.6% (-1.6 to 2.7%), respectively (all P-values >0.6). SCSA performed in semen prepared by DGC cannot predict the outcome of ART.

Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive of the outcome of ART

BACKGROUND The sperm chromatin structure assay (SCSA) parameter DNA fragmentation index (DFI) has been shown to predict in vivo and in vitro fertility. So far most SCSA studies have been based on SCSA analysis performed on neat semen. The aim of this study is to assess whether SCSA analysis of sperm prepared by density gradient centrifugation (DGC) could add more information in regard to the prediction of treatment outcome. METHODS The study included 510 assisted reproductive technique (ART) cycles. SCSA was performed in neat semen and post DGC. SCSA results were expressed in terms of DFI and high DNA stainability (HDS) cell fractions. The outcome parameter was clinical pregnancy (CP). RESULTS Scatter-plot diagrams demonstrated that for DGC samples, no DFI cut-off values could be set for in vivo or in vitro fertility. In intrauterine insemination, IVF and ICSI groups the mean difference (95% CI) in DFI post DGC between those who achieved CP and those who did not was 0.2% (-1.7 to 2.0%), 0.4% (-1.9 to 2.8%) and 1.3% (-3.1 to 5.9%), respectively, none of these being statistically significant. The corresponding differences for HDS were 0.1% (-1.3 to 1.5%), 0.1% (-0.7 to 0.9%) and 0.6% (-1.6 to 2.7%), respectively (all P-values >0.6). CONCLUSIONS SCSA performed in semen prepared by DGC cannot predict the outcome of ART.

Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates

To investigate the relationship between sperm chromatin structure assay (SCSA) parameters, DNA fragmentation index (DFI) and high DNA stainability (HDS), and outcomes of IVF and intracytoplasmic sperm injection (ICSI). Retrospective review and prospective study. Academic human reproduction laboratory. Two hundred twenty-three couples undergoing conventional IVF (n = 137) and ICSI (n = 86). Testing with SCSA on a semen aliquot taken from ejaculate used for assisted reproductive technology (ART). Conventional semen parameters, DFI, HDS, outcomes of IVF and ICSI. There were no significant differences in IVF and ICSI fertilization rate, good embryo rate, and pregnancy rate (PR) between high, moderate, and low DFI or HDS groups. Men with HDS >15% had significantly higher IVF abortion rates. There was a statistically insignificant trend toward an increased abortion rate in the high DFI (>27%) group. The DFI correlated negatively with sperm motility, and HDS correlated negatively with sperm morphology and concentration. Neither DFI nor HDS scores can provide independent information about embryo quality, fertilization, and PRs for infertility patients undergoing ART. Sperm DNA fragmentation probably affects sperm motility. The relationship between HDS and IVF abortion rates provides preliminary evidence that ICSI may be indicated for men with HDS >15%. The potential adverse effect of sperm DNA damage on the quality of postimplantation embryo and spontaneous abortion should be a concern.