Increased Sperm DNA Fragmentation Research Articles

COMPARISON OF SPERM DNA FRAGMENTATION(DFI) AMONG NON-SMORKERS, REGULAR SMORKERS, ELECTRONIC-CIGARETTE(E-CIGARETTE) SMORKERS AND REGULAR/E-CIGARETTE SMORKERS

The integrity of sperm DNA is important for fertilization and development of healthy offspring and is cited as one of the leading causes of male infertility. Among them, increased sperm DNA fragmentation is one of the causes of infertility that affects changes in sperm integrity. Toxins from smoking are known to potentially affect sperm development and function and negatively affect semen parameters. Recently, the use of e-cigarettes is increasing. This new phenomenon is due to the fact that e-cigarettes are considered a safer alternative to regular cigarettes.

IMPACT OF MICROFLUID SPERM SORTING ON EMBRYO PLOIDY RATES IN COUPLES WITH HIGH SPERM DNA FRAGMENTATION?

Increased sperm DNA fragmentation (SDF) is one of the leading causes of male infertility. Conventional methods for sperm preparation have induced questioning of sperm recovery rates. The microfluidic sperm sorting (MSS) technique selects highly motile sperm with lower levels of SDF compared to density-gradient centrifugation (DGC) technique. This study aimed to compare MSS to DGC technique in couples with repeated implantation failure (RIF) and high SDF referred to a PGT-A (preimplantation genetic testing for aneuploidies) cycle.

Sperm as a Carrier of Genome Instability in Relation to Paternal Lifestyle and Nutritional Conditions

Endogenous and exogenous factors can severely affect the integrity of genetic information by inducing DNA damage and impairing genome stability. The extent to which men with and without subfertility are exposed to several adverse lifestyle factors and the impact on sperm DNA fragmentation (SDF), sperm chromatin maturity (condensation and decondensation), stability (hypo- and hypercondensation) and sperm aneuploidy are assessed in this study. Standardized assays employing flow cytometry were used to detect genome instability in 556 samples. Semen parameters deteriorated with age, BMI, increased physical activity and smoking. Age and BMI were associated with increased SDF. Increased BMI was associated with increased hypocondensed chromatin and decreased decondensed chromatin. Increase in age also caused an increase in sex chromosome aneuploidy in sperms. Surprisingly, alcohol abuse reduced chromatin hypercondensation and drug abuse reduced SDF. Although genome instability was more pronounced in the subfertile population as compared to the fertile group, the proportion of men with at least one lifestyle risk factor was the same in both the fertile and subfertile groups. While one in three benefited from nutritional supplementation, one in five showed an increase in SDF after supplementation. Whilst the message of ‘no smoking, no alcohol, no drugs, but a healthy diet’ should be offered as good health advice, we are a long way from concluding that nutritional supplementation would be beneficial for male fertility.

P-070 Comparison of Microfluidic Sperm Sorting (MFSS) versus Physiological Intracytoplasmic Sperm Injection (PICSI) versus Density Gradient versus Swim Up in high DNA fragmentation index sperm samples

Abstract Study question To evaluate the effectiveness of using Microfluidic Sperm Sorting (MFSS) over other technique in patient with high DNA fragmentation index (DFI) sperm samples? Summary answer Microfluidic is not only correlated with better DNA integrity but also with the better reproductive outcome. What is known already DNA damage is unrecognisable in living sperm prior to insemination and an increased sperm DNA fragmentation index has been associated with lower fertilization rates, impaired embryo development and reduced pregnancy rates. Standard semen processing techniques are associated with centrifugation, which may induce reactive oxygen species and DNA damage. In strategies to minimize sperm DNA fragmentation, Physiological ICSI can relatively reduce sperm DNA fragmentation by 67.9% (Parmegiani et al., 2010) while new technique Microfluidic sperm sorter technique also demonstrate sperm selection with significantly reduced DNA damage Study design, size, duration A prospective randomised study was conducted from 1st January 2019 to 1st December 2021. Four hundred patients were randomised by computer generated list and divided into 4 groups. Group A (n = 100) , in which sperm were processed by microfluidic sperm sorter (MFSS) while in group B (n = 100), sperm were selected by Physiological Intracytoplasmic Sperm Injection (PICSI), Group C (n = 100), sperm were processed by density gradient and Group D (n = 100), sperm were processed by swim-up technique. Participants/materials, setting, methods The study period included all normozoospermic patients with high DNA fragmentation index (>25%) while oligospermic, asthenozoospermic samples, patients with poor ovarian reserve and advanced age were excluded from the study. All A grade embryos were vitrified and transferred in frozen embryo replacement cycle. All the 3 groups were compared on the basis of fertilisation rate, day 3 grade A embryo development rate, clinical pregnancy rate and miscarriage rate. Main results and the role of chance Cycle characteristics (female age, length of stimulation, gonadotrophin dose, number of oocytes and number of transferred embryos) were similar among all 4 groups. Among 4 groups, There was a statistically significant increase observed in Group A (Microfluidic Sperm Sorting), day 3 grade A embryo development rate (60% vs. 42% vs. 38% vs. 40%, p-0.016) and clinical pregnancy rate (62% vs. 46% vs. 41% vs 43%, p-0.049), while no statistical significant difference observed in fertilisation rate (82% vs. 78% vs. 76% vs. 78%, p-0.80) comparing group B, group C and group D. But significantly higher miscarriage rate (12% vs. 11% vs. 25%, 12%) observed in density gradient (Group C) technique. Limitations, reasons for caution Larger randomised control studies are needed to strengthen these results. Wider implications of the findings We have demonstrated that sperm sorted by microfluidic helps in selection of sperm with better DNA integrity over Physiological ICSI, Density Gradient and Swim-up techniues. Using it in routine practice can help in reducing the negative effect of reactive oxygen species and thus improve pregnancy rate and live birth rate. Trial registration number MCDH/2019/35

P-068 Oxidative stress and DNA fragmentation of spermatozoa in patients with cancer

Abstract Study question Does cancer increase sperm DNA fragmentation (sDF) level by increasing oxidative stress in human spermatozoa? Summary answer Cancer increases both sDF and oxidative stress, but further studies are necessary to understand whether higher levels of ROS are responsible for sperm DNA damage. What is known already In recent years, it has been emerging that not only oncological therapies but also cancer itself can induce abnormal spermatogenesis. In addition, several Authors reported that occurrence of malignancy provokes also increases of sperm DNA damage, although such finding was not confirmed by others and the possible mechanisms responsible for such damage are presently unknown Study design, size, duration This was a prospective observational study conducted from 2018 to today, conducted in 102 patients affected by cancer and in 66 control subjects. Participants/materials, setting, methods We recruited 102 patients affected by cancer and 66 male partners of infertile couples as control subjects, in the Andrology Clinic of University of Florence. Control subjects were normozoospermic with absence of leukocytospermia, semen viscosity, smoking habit and recent antibiotic therapies. In the recruited men, we evaluated standard semen parameters, sperm DNA Fragmentation with SCD (Sperm Chromatin Dispersion) Test and oxidative stress as percentage of viable spermatozoa with MitoSOX™ Red labeling on total viable spermatozoa. Main results and the role of chance We found poorer standard semen parameters (sperm motility, concentration and number) in cancer patients (both testicular and hematological ones) with respect to control group, whereas no differences were observed between the two types of cancer. Testicular, but not hematological patients, were younger than control subjects. No difference was seen in the other tested characteristics (sperm morphology, abstinence, semen volume and pH, BMI). Regarding sDF, we found higher median values [IQR] in cancer patients (total: 22.25[17.00-25.95], n = 68; hematological: 23.00[20.13-26.38], n = 28; testicular: 21.13[16.13-25.73], n = 40) vs control subjects (12.50[8.25-14.75], n = 53); p < 0.05, test U di Mann-Whitney. In addition, the amount of sperm oxidative stress was dramatically higher in patients with cancer (total:38.92[24.90-58.87], n = 79; hematological: 38.85[24.98-50.77], n = 34; testicular: 38.92[20.59-63.59], n = 45) vs control subjects (11.50[8.38-17.20], n = 62); p < 0.05, test U di Mann-Whitney. We also studied the occurrence of a correlation between levels of sDF and oxidative stress. We found a sharp correlation when both cancer patients and control subjects were analysed (Spearman coefficient = 0.62, p < 0.001, n = 103), but such correlation was completely lost when only cancer patients were considered (Spearman coefficient = 0.10, p > 0.05, n = 50). This finding suggests that mechanisms different from ROS attack to DNA could explain the increase of sDF levels in cancer patients. Limitations, reasons for caution The study did not investigate, because of scarce availability of semen samples from cancer patients, other possible mechanisms (i.e apoptosis, defects in sperm chromatin maturation, failure in DNA system repair) which could cause the observed increase of sperm DNA damage in such patients. Wider implications of the findings Cancer patients show high levels of both sDF and oxidative stress. This finding rises concern, as cancer patients cryopreserve semen for using it with Assisted Reproductive Tecnhologies and both parameters represent a threat for natural and assisted reproduction. In addition, emerging evidence suggest that oxidative stress may alter sperm epigenome. Trial registration number not applicable

Relationship between spermatogenesis, DNA fragmentation index, and teratozoospermia index

Infertility has proven a global medical issue, with 15-20 percent of married worldwide experiencing difficulties in conceiving. One of the causes of male infertility is sperm morphology abnormalities, or teratozoospermia, which is often associated with sperm DNA damage. Teratozoospermia can increase sperm DNA fragmentation through several mechanisms, including abortive apoptosis and induce ROS formation. Unfortunately, many forms of sperm flaws, including genetic defects, could not be validated by routine semen examination. In the Sixth Edition, WHO introduced a more thorough sperm morphology examination, named the teratozoospermia index (TZI), in which every aberrant sperm is examined for up to four defects. Analyzing spermatozoa abnormalities in more detail can provide an overview of the function of the male reproductive organs, particularly the testes and epididymis. However, using this method is not entirely easy due to subnormal variations between cells so that quality control may be needed when using TZI for sperm morphology evaluation.

Mixture analysis on associations between semen quality and sperm DNA integrity and occupational exposure to polycyclic aromatic hydrocarbons

The objective of this study was to assess relationships between exposure to PAHs at occupational levels and outcomes of human semen quality and sperm DNA integrity. Personal breathing zone air samples were collected to quantify exposure of 16 targeted PAHs to coke-oven workers at a steel company in southern Taiwan. Semen quality, including concentration, motility, morphology, and viability, were assessed. Sperm DNA fragmentation, 8-oxodGuo, bulky PAH adducts, and benzo[a]pyrene diol epoxide-DNA adducts served as biomarkers for assessment of sperm DNA integrity. The Bayesian Kernel Machine Regression modeling was employed to estimate mixture effects of the PAH mixture on the outcomes of semen quality and sperm DNA integrity and to identify individual compounds of PAH mixtures associated with the mixture effects. Exposure to the PAH mixture was inversely associated with sperm viability, while benzo(b)fluoranthene (B[b]F) was identified as the main predictor for sperm viability. Exposure to the PAH mixture also exhibited a positive trend with sperm DNA fragmentation. B[b]F and benzo(a)anthracene (B[a]A) were identified as individual PAH compounds associated with increased sperm DNA fragmentation.

Assessment of the Impact Induced by Different Incubation Time, Storage Time, Storage Medium and Thawing Methods on Sperm DNA Fragmentation Assay: A Before-After Study.

The sperm DNA fragmentation has been considered an important index in the field of male infertility. Our study aims to evaluate the impact of different factors, including incubation time, storage time, storage medium and method of thawing, on DNA fragmentation of semen samples. This study was designed as a before-after study in five experiments. Experiment 1 was conducted to assess the effect of storage time in liquid nitrogen on 15 semen samples. In experiment 2, DNA fragmentation was performed on 10 semen samples with different incubation times before freezing. In experiments 3, 4, two different storage media and thawing methods were applied respectively in two separate groups, each containing 30 samples and the DNA fragmentation index (DFI) was measured using the sperm chromatin structure assay method. Data were analysed using Stata version 11. There was a significant increase in sperm DNA fragmentation of samples stored in liquid nitrogen for 1 month. This increase occurred in the first 2 weeks. Furthermore, our results showed a significant increase in the DFI after 120 min of incubation at room temperature (RT) and also thawing in RT separately. It is better to use fresh samples to measure DNA fragmentation up to 2 h after ejaculation to achieve more accurate results. Furthermore, if sperm freezing is inevitable, the use of a water bath (37°C) to thaw will be the most appropriate option, as it can lead to less DNA damage.

Bisphenol A and Male Infertility: Role of Oxidative Stress.

Bisphenol A (BPA) is an endocrine-disrupting chemical that is capable of mimicking, antagonizing, and interfering with the normal biological functioning of the endocrine system. BPA is used in diverse industries, hence its vast sources of exposure. Although the half-life of BPA is relatively short (<24hours), studies have reported its detection in the urine of different populations. It, therefore, became important to investigate its effect on general health, including male reproductive health. The adverse effects of BPA on male fertility have been evaluated and reported from both in vivo and in vitro studies. Up to date, reports from randomized controlled trials remain controversial, as some revealed decreased sperm quality, sperm concentration, and total sperm count, while others reported that no adverse effect was seen after exposure. Findings from animal model studies and in vitro experiments have shown that exposure to BPA led to a reduction in sperm quality and increased sperm DNA fragmentation, and some even revealed altered expression of the gene that encodes gonadotropin-releasing hormone. This shows that BPA not only may adversely affect male fertility by acting as an endocrine disruptor but also can potentially impact male fertility via its possible contribution to oxidative stress. Therefore, this book chapter aims to identify and elucidate the effect of BPA exposure on male fertility, and to as well illustrate the mechanisms through which this occurs, while emphasizing the role of oxidative stress as a potential pathway.

Evaluating the therapeutic effect and toxicity of theophylline in infertile men with asthenoteratozoospermia: a double-blind, randomized clinical trial study

Theophylline as a cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor (cAMP-PDEI) elevates cAMP levels. We aimed to evaluate the therapeutic effect and toxicity of theophylline on the sperm parameters, oxidative stress (OS), and inflammation in asthenoteratozoospermic men. Sixty asthenoteratozoospermic patients were divided into groups of placebo and theophylline (200 mg/day). After 3 months of oral treatment, sperm parameters, viability, and DNA fragmentation were analyzed by the CASA system, eosin nigrosin staining, sperm DNA fragmentation kit, respectively. The seminal plasma level of reactive oxygen species (ROS) of neat semen samples, malondialdehyde (MDA), total antioxidant capacity (TAC), tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) was assessed. Data were analyzed statistically using the independent samples t-test and the paired t-test and the means were considered significantly different at p < 0.05. Sperm motility, viability, and the number of sperms with normal morphology and the seminal plasma level of TAC and IL-10 and also sperm DNA fragmentation increased significantly in the theophylline group compared to the placebo. The MDA, TNF-α, and ROS levels decreased significantly in the theophylline group compared to the placebo. Theophylline improved sperm parameters, reduced OS and inflammation, but also created genotoxicity and increased sperm DNA fragmentation. Therefore, to benefit from the desired effects of theophylline and inhibit the toxicity of it in the treatment of men with asthenoteratozoospermia, it is suggested to be used simultaneously with another antioxidant to protect sperm DNA from fragmentation.

Evaluation of Sperm Parameters and DNA Integrity Following Different Incubation Times in PVP Medium.

Polyvinylpyrrolidone (PVP) is a chemical material used in intracytoplasmic sperm injection (ICSI) program. The aim of this study was to investigate the ideal time that sperm can be safely incubated in PVP with less structure and DNA damage. Thirty-one Oligoasthenoteratospermia (OAT) samples were used. Sperm samples were prepared by discontinuous density-gradients method and incubated in 10% PVP at different time intervals (0, 5, 10, 15, 20, and 30 min). The effect of PVP was assessed on sperm DNA fragmentation and viability via SCD assay and Eosin-nigrosin staining respectively. Data showed there was a significant increase in sperm DNA fragmentation at 10 min compared to 0 min. The viability rate also significantly reduced at 10 min compared to 0 min. As a result, sperm samples could be incubated with PVP for less than 10 min. While prolonged incubation may significantly damage the sperm DNA integrity and viability.

Low-dose Methotrexate Therapy Does Not Affect Semen Parameters and Sperm DNA.

Methotrexate is widely used in inflammatory diseases during the patients' reproductive years. The effect on male fertility and sperm DNA integrity is largely unknown. We evaluated sperm DNA integrity and basic semen parameters according to the World Health Organization (WHO) in male patients with inflammatory diseases treated with methotrexate. Semen samples from 14 patients on low-dose maintenance methotrexate were compared with samples from 40 healthy volunteers. Further, 5 patients delivered samples on and off methotrexate therapy for paired comparison. Sperm DNA fragmentation index (DFI), concentration, motility, and morphology were evaluated. Blood sex hormones and methotrexate levels were measured in blood and semen. DNA fragmentation index in methotrexate-treated patients was comparable with that in healthy volunteers (DFI, 11.5 vs 15.0; P = .06), and DFI did not change significantly on and off methotrexate in the paired samples (DFI, 12.0 vs 14.0; P = 0.35). Sperm concentration, motility, and morphology did not differ between men treated with methotrexate and healthy volunteers. Sperm progressive motility increased off therapy compared with on therapy (65.0% vs 45.0%, P = .04), but all fluctuations in progressive motility were within the WHO reference interval. All methotrexate polyglutamates1-5 were detected in blood, but only methotrexate polyglutamate1 in semen. Serum testosterone was unaffected by methotrexate therapy. Patients treated with low-dose methotrexate have a sperm quality comparable with that of healthy volunteers, and methotrexate treatment does not increase sperm DNA fragmentation. This study does not support cryopreservation of semen before treatment initiation nor a 3-month methotrexate-free interval prior to conception.

Astaxanthin Induces the Expression of CatSper1 Gene and Protects Sperms in Toxicity Induced by Cadmium in Mice

Cadmium (Cd) as a heavy metal damages testis and decreases fertility, however, antioxidants can improve sperm parameters and decrease male infertility. In this study we investigated the effect of astaxanthin (AST) on sperm parameters, expression levels of CatSper1 and CatSper2 genes in presence of Cd in mice. Thirty adults' mice were divided into 4 groups, sham group received olive oil and saline (olive oil is the solvent of AST and saline is the solvent of Cd), Cd group received 1 mg/kg Cdcl2, a group received 10 mg/kg AST and 1 mg/kg Cdcl2 and a group received 10 mg/kg AST. The treatments were done intraperitoneally for 14 days. After 14 days sperm parameters were analyzed. Malondialdehyde level, catalase enzyme activity, the alteration of CatSper1 and CatSper2 genes expression were measured in testis. Results showed that Sperm count, viability, CatSper1 gene expression and catalase activity significantly decreased by Cd compared to sham group. Cd significantly increased sperm DNA fragmentation (SDF), abnormal sperm morphology and malondialdehyd level compared to sham group. AST significantly increased sperm count, viability and CatSper1 gene expression and decreased SDF and abnormal sperm in comparison with Cd group. AST protected testis and decreased oxidative stress induced by Cd. Our findings indicated that AST could protect sperm DNA, enhanced CatSper1 gene expression and sperm quality in presence of Cd. No significant differences were found in CatSper2 expression among treatments. Therefore, AST as a strong antioxidant can help to protect the potential of fertility against Cd toxicity.

P–012 A new sperm preparation technique for ICSI

Abstract Study question Is there any difference in this technique(swim-in) according to clinical results in ICSI cycles? Summary answer Fertilization rates are significantly better with this new technique(swim-in). There is no difference in pregnancy and ongoing pregnancy rates between swim-in and DGS techniques. What is known already Sperm separation is crucial in assisted reproductive technologies, based on different principles like migration, filtration, or density gradient centrifugation techniques. However, there are some studies that centrifugation steps and using gradient solutions may increase sperm DNA fragmentation. On the other hand, multiple contacts to different plastic surfaces such as pipettes, plastic tubes, etc., and also pipetting the ejaculate in sperm preparation may have detrimental effects. The ideal sperm separation technique should be non-invasive, not time-consuming, easy, and cost-effective, not cause sperm damage or non-physiological alterations of the separated sperm cells, eliminate dead spermatozoa and other cells, including leukocytes. Study design, size, duration This is a prospective randomized study between 01/02/2019...01/01/2021. Sperms were prepared either with this new technique that we call “swim-in”(n = 359) or with density gradient centrifugation(n = 404) before microinjection (ICSI). Fertilization rates, clinical pregnancy rates, and on-going pregnancy rates were compared between groups. Sperm motility is less than%20 patients are not included in the study. t-test and Chi-square test used. p &amp;lt; 0.05 significant. Participants/materials, setting, methods For this new centrifugation free technique(swim-in), a Braun injector, 21G green needle, and sperm washing medium are used. First, we pulled a 0.5 ml sperm wash medium in a Braun injector. After sperm liquefaction, we put Braun injector with the needle in the sperm cap perpendicularly and waited 30 minutes at 37-degree Celcius. Sperms swam through the needle to the injector used for ICSI. For DGC sperms were prepared according to the 2015 WHO manual. Main results and the role of chance: We try to make a centrifugation-free, gentle, cost-effective, and easy sperm preparation technique and called it a swim-in technique. Groups were comparable according to mean age, collected oocyte numbers, and oocyte maturation ratios. Fertilization rates are%73,2(1786/2438) vs%66,9(2167/3237) respectively for swim-in and DGC groups. Clinical pregnancy rates are%45,1 vs%42,3 respectively for swim-in and DGC groups. On-going pregnancy rates are%41,5 vs%39,6 for swim-in and DGC respectively. There is a significant difference between the two techniques according to fertilization rates but no difference according to clinical pregnancy and ongoing pregnancy rates. Limitations, reasons for caution Sperms can move by their own motility from ejaculate to the sperm washing medium for that reason with severe sperm problem patients are not suitable for this technique. Wider implications of the findings: This technique is centrifugation free so there are no detrimental g-forces. The needle used may mimic the physiological environment like Uterine Tube. Alternative sperm washing mediums like follicular fluid or chemical attractor progesterone rich medium can be used.DNA fragmentation rates and live birth rates should be established for future aspect. Trial registration number Not applicable

P–271 Should intracytoplasmic sperm injection (ICSI) of delayed mature oocytes become a routine practice in the IVF Laboratory?

Abstract Study question Do delayed mature oocytes result in similar euploid blastocyst rates as their immediate mature sibling oocytes? Summary answer Once a blastocyst is obtained, delayed mature oocytes have similar euploid rates compared to immediate mature oocytes. What is known already Intracytoplasmic sperm injection (ICSI) of metaphase II oocytes few hours post oocyte retrieval is standard practice in IVF laboratories. Immature metaphase I (MI) and prophase I (GV) oocytes are usually discarded. Immature oocytes may mature overnight, after which ICSI can be performed. Studies demonstrated lower fertilization and blastulation rates for these delayed mature oocytes. However, live births have been reported from blastocysts transferred. The evidence available is not compelling, since most of the studies had either low sample size, no preimplantation genetic testing for aneuploidies (PGT-A), or the outcome was not compared to sibling MII oocytes at time of denudation. Study design, size, duration A single-center retrospective sibling oocyte study was performed between January 2019 and December 2020 at ART Fertility clinics Abu Dhabi, UAE. A total of 345 PGT-A cycles, with at least one delayed mature oocyte inseminated by ICSI, were included: 2506 immediate mature oocytes and 669 delayed mature oocytes. Participants/materials, setting, methods Following controlled ovarian stimulation, MII oocytes at the time of denudation were inseminated by ICSI/IVF (immediate mature). Immature oocytes (MI/GV) were cultured for 16–24 hours in fertilization medium and injected the next day if matured (delayed mature). Trophectoderm biopsy was performed on day 5/6/7 and samples were subjected to Next Generation Sequencing to screen the ploidy state of the blastocyst. Main results and the role of chance The 345 controlled ovarian stimulation cycles resulted in the insemination of 2506 MII oocytes on the day of oocyte retrieval (Day0) and 669 delayed mature oocytes on day 1. Normal fertilization rate was significantly higher in the immediate mature oocytes compared to delayed mature oocytes (68% vs 56%, p &amp;lt; 0.0001). Similarly, the usable blastocyst rate was significantly higher in immediate mature oocytes (59% vs 19%, p &amp;lt; 0.0001). On day 5 of development, a significantly higher-good quality blastocyst formation rate was obtained from immediate mature oocytes (65% vs 27%, p &amp;lt; 0.0001). The rate of good quality blastocyst on the day of biopsy was significantly higher in the immediate mature oocytes group (76% vs 62%, p &amp;lt; 0.015). Fisher’s Exact Test was performed to compare the euploid rate of blastocysts biopsied on day 5/6/7 originating from immediate mature oocytes or sibling delayed mature oocytes. The euploid potential of blastocyst biopsied showed no significant difference between the two groups (p = 0.388). Limitations, reasons for caution The timing of MI/GV oocytes transition to MII stage was not recorded since the incubation was done in a benchtop incubator. Furthermore, the same sperm sample was used to inseminate immediate and delayed mature oocytes, which might contribute to the compromised embryo development due to increased sperm DNA fragmentation. Wider implications of the findings: Insemination of delayed mature oocytes by ICSI, should be considered as a tool to increase patients’ chances of obtaining a euploid embryo. Especially in cases where low yield of euploid embryos is expected. Trial registration number Not applicable

P–084 Microfluidic Sperm Sorting (MFSS) technique versus Physiological Intracytoplasmic Sperm Injection (PICSI) technique in high DNA fragmentation index sperm samples

Abstract Study question To evaluate the effectiveness of using Microfluidic Sperm Sorting (MFSS) technique and Physiological Intracytoplasmic Sperm Injection (PICSI) technique in patient with high DNA fragmentation index (DFI) sperm samples. Summary answer Sperm selected by microfluidic sorting are associated with significant increase in day 3 grade A embryo development rate, clinical pregnancy rate over PICSI. What is known already DNA damage is unrecognisable in living sperm prior to insemination and an increased sperm DNA fragmentation index has been associated with lower fertilization rates, impaired embryo development and reduced pregnancy rates. Standard semen processing techniques are associated with centrifugation, which may induce reactive oxygen species and DNA damage. In strategies to minimize sperm DNA fragmentation, Physiological ICSI can relatively reduce sperm DNA fragmentation by 67.9% (Parmegiani et al., 2010) while new technique Microfluidic sperm sorter technique also demonstrate sperm selection with significantly reduced DNA damage. Study design, size, duration A prospective randomised study was conducted from 1st August 2019 to 31st December 2020. Two hundred patients were randomised by computer generated list and divided into 2 groups. Group A (n = 100) , in which sperm were processed by microfluidic sperm sorter (MFSS) while in group B (n = 100), sperm were selected by Physiological Intracytoplasmic Sperm Injection (PICSI) technique and morphologically normal motile sperm were injected by Intracytoplasmic sperm injection (ICSI) technique in all mature oocytes. Participants/materials, setting, methods The study period included all normozoospermic patients with high DNA fragmentation index (&amp;gt;25% ) while oligospermic, asthenozoospermic samples, patients with poor ovarian reserve and advanced age were excluded from the study. All A grade embryos were vitrified and transferred in frozen embryo replacement cycle. Both groups were compared on the basis of fertilisation rate, day 3 grade A embryo development rate , clinical pregnancy rate and miscarriage rate. Main results and the role of chance Cycle characteristics (female age, length of stimulation, gonadotrophin dose, number of oocytes and number of transferred embryos) were similar in both groups. Between the 2 groups, There was a significant increase observed in day 3 grade A embryo development rate (60% vs. 42%, p–0.016) and clinical pregnancy rate (62% vs. 46%, p–0.049), while no statistical significant difference observed in fertilisation rate (82% vs. 78%, p–0.80) and miscarriage rate ( 12% vs. 11%, p- 1). Limitations, reasons for caution: Larger randomised control studies are needed to strengthen these results. Wider implications of the findings: We have demonstrated that sperm sorted by microfluidic helps in selection of sperm with better DNA integrity over Physiological ICSI. Using it in routine practice can help in reducing the negative effect of reactive oxygen species and thus improve pregnancy rate and live birth rate. Trial registration number MCDH/2019/31

Chronic Prostatitis/Chronic Pelvic Pain Syndrome Leads to Impaired Semen Parameters, Increased Sperm DNA Fragmentation and Unfavorable Changes of Sperm Protamine mRNA Ratio.

Background: Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a frequent disease affecting men of every age and accounting for a great number of consultations at urology departments. Previous studies suggested a negative impact of CP/CPPS on fertility. As increasing attention has been attributed to additional aspects, such as sperm DNA integrity and sperm protein alterations, besides the WHO standard semen analysis when assessing male fertility, in this prospective study, we aimed to further characterize the fertility status in CP/CPPS patients with a focus on these parameters. Methods: Sperm DNA fragmentation measured by sperm chromatin structure assay (SCSA) and protamine 1 to protamine 2 mRNA ratio assessed by RT-qPCR were analyzed along with conventional ejaculate parameters and inflammatory markers in 41 CP/CPPS patients and 22 healthy volunteers. Results: We found significant differences between the groups concerning multiple conventional ejaculate parameters. A significant increase in sperm DNA fragmentation was shown in CP/CPPS patients with association to other sperm parameters. The majority of CP/CPPS patients exhibited protamine mRNA ratios out of the range of regular fertility. Conclusions: This is a pioneering study with a strong practical orientation revealing that CP/CPPS leads to increased sperm DNA damage and changes in sperm protamine levels, emphasizing an unfavorable impact of CP/CPPS on fertility.

Sperm DNA fragmentation and total antiradical activity of sperm in men with exaggerated anamnesis

Various intracellular and extracellular stimuli can promote excessive accumulation of reactive oxygen species and lead to detrimental effects on sperm function, in particular, damage to sperm DNA, thus often resulting into infertility. The aim of the study was to assess the degree of sperm DNA fragmentation by flow cytometry and to study total antiradical activity of sperm in men with infertility and chronic prostatitis using spectrophotometric technique. A total of 75 men were examined. The comparison group consisted of 45 men with chronic prostatitis beyond exacerbation. The observation group included 30 men with chronic prostatitis in remission state. We have found that in the subjects during the period of acute inflammation, pathological fragmentation of sperm DNA was revealed, being statistically significant (p = 0.004). It was registered 1.7 times more often than in the examined men during the remission of chronic prostatitis. Increased fragmentation of the sperm DNA was noted in 50% of men from comparison group. In the patients from observation group, the degree of sperm DNA fragmentation over reference ranges was observed in 80% of the subjects. In men diagnosed with chronic prostatitis at the exacerbation stage, there is a statistically significant (p 0.05) relationship between sperm DNA fragmentation and development of acute inflammatory process. It was shown that in the subjects from observation group, the mean value of antiradical sperm activity did not show statistically significant differences (p = 0.378) compared with results found in men from the comparison group. The presented results suggest a higher degree of sperm DNA fragmentation in the examined patients with chronic prostatitis at the stage of decompensation, which may point to excessively increased production of free radicals during the acute inflammatory process. Obviously, exacerbation of chronic prostatitis contributes to imbalance in the redox homeostatic system in men, thus creating prerequisites for excess of reactive oxygen species and significant fragmentation of sperm DNA. The identified changes, i.e., an increased sperm DNA fragmentation levels and a deficiency of total antiradical activity of sperm in men with chronic urogenital diseases, may play a pathogenetic role in occurrence and further development of infertility. The degree of sperm DNA fragmentation and markers of redox processes may serve as informative activity indices of the inflammatory process.

The effect of sperm DNA fragmentation on intracytoplasmic sperm injection outcome.

Our study objective was to assess the effect of various sperm DNA fragmentation levels on clinical intracytoplasmic sperm injection outcome. This retrospective study included 392 patients who underwent ICSI and performed sperm DNA fragmentation testing before the procedure. Based on sperm DNA fragmentation cut-off values, the patients were differentiated into 3 groups as <20%, 20%-30% and >30%. According to the female status, patients were differentiated into favourable group (n=259) with female age <35years and anti-Mullerian hormone level ≥7.1pmol/L; and unfavourable group (n=133) with female age ≥35years and anti-Mullerian hormone level ≤7.1pmol/L. The patient's medical records were reviewed, and patient's demographic, laboratory data including semen analysis, sperm DNA fragmentation determined by means of sperm chromatin dispersion, hormonal profile and data regarding intracytoplasmic sperm injection cycle were collected. This cohort reported that the clinical reproductive outcomes of intracytoplasmic sperm injection showed no statistical significance with increase sperm DNA fragmentation levels. In sperm DNA fragmentation above 30%, favourable females had significantly higher clinical pregnancy rate and live birth rate than unfavourable females, while fertilisation rate and miscarriage rate showed no significance between the subgroups. High sperm DNA fragmentation is linked to poor semen parameters.

Paternal Fenitrothion Exposures in Rats Causes Sperm DNA Fragmentation in F0 and Histomorphometric Changes in Selected Organs of F1 Generation.

The adverse effects of maternal pesticides exposure on the progeny is very well established. However, the impact of paternal exposure to pesticides such as Fenitrothion (FNT) on the histomorphometry of progeny’s organs in unexposed mothers are much less well studied. Therefore, this study aims to evaluate the effects of paternal FNT exposure on the sperm quality of the parent rat and its effects on the histomorphometry of the progeny’s organs. Randomly, male Sprague Dawley rats (n = 24) categorized as F0 were distributed equally into three groups namely Control, FNT-10, and FNT-20. Control received 1 mL/kg corn oil while FNT-10 and FNT-20 received 10 mg/kg and 20 mg/kg of FNT, respectively, via oral force feeding for 28 consecutive days. At the end of the study, male rats were mated with unexposed female rats and the male rats were sacrificed to obtain sperm for sperm characterization and DNA damage evaluation. Meanwhile, the rats’ progeny (F1) namely pControl, pFNT-10, and pFNT-20 were left to grow until postnatal day 70 before being sacrificed to obtain the matured organs for histology and morphometric analysis. Our results showed that both doses of FNT reduced sperm quality and caused DNA fragmentation in F0 rats compared with the control group (p < 0.05). The number of Leydig cells as well as the diameter of the seminiferous tubules and glomerulus of the pFNT-20 group had significantly decreased (p < 0.05) compared with the pControl group. The Bowman’s space of the pFNT-20 group had significantly increased (p < 0.05) compared with the pFNT-10 and pControl groups. Therefore, paternal exposure to FNT reduced the sperm quality and increased sperm DNA fragmentation in F0 male Sprague Dawley rats and altered the histology and morphometry of the selected organs in the F1 progeny.