A microfluidic sperm-sorting device reduces the proportion of sperm with double-stranded DNA fragmentation.

Sperm DNA fragmentation can be produced in one (ssSDF) or both (dsSDF) DNA strands, linked to difficulties in naturally achieving a pregnancy and recurrent miscarriages, respectively. The techniques more frequently used to select sperm require centrifugation, which may induce sperm DNA fragmentation (SDF). The objective of this study was to assess whether the microfluidic-based device FertileChip® (now ZyMot®ICSI) can diminish the proportion of sperm with dsSDF. First, in a blinded split pilot study, the semen of nine patients diagnosed with ≥60% dsSDF, was divided into three aliquots: not processed, processed with FertileChip®, and processed with swim up. The three aliquots were all analyzed using neutral COMET for the detection of dsSDF, resulting in a reduction of 46% (P < 0.001) with FertileChip® (dsSDF: 34.9%) compared with the ejaculate and the swim up (dsSDF: 65%). Thereafter, the FertileChip® was introduced into clinical practice and a cohort of 163 consecutive ICSI cycles of patients diagnosed with ≥60% dsSDF was analyzed. Fertilization rate was 75.41%. Pregnancy rates after the first embryo transfer were 53.2% (biochemical), 37.8% (clinical), 34% (ongoing) and the live birth rate was 28.8%. Cumulative pregnancy rates after one (65.4% of patients), two (27.6% of patients) or three (6.4% of patients) transfers were 66% (biochemical), 56.4% (clinical), 53.4% (ongoing) and the live birth rate was 42%. The selection of spermatozoa using Fertile Chip® significantly diminishes the percentage of dsSDF, compared with either the fresh ejaculate or after swim up. Its applicability in ICSI cycles of patients with high dsSDF resulted in good laboratory and clinical outcomes.

Sperm telomere length in motile sperm selection techniques: A qFISH approach.

Several studies have associated telomere shortening with alterations in reproductive function. The objective of the present study was to determine telomere length (TL) in spermatozoa selected by either density-gradient centrifugation (DGC) or swim-up. The analysis of TL was performed using quantitative fluorescent in situ hybridisation (qFISH) using PNA probes in combination with a chromatin decompaction protocol in sperm cells. Results of TL were 24.64 ± 5.00 Kb and 24.95 ± 4.60 Kb before and after DGC, respectively, and 19.59 ± 8.02 Kb and 20.22 ± 5.18 Kb before and after swim-up respectively. Sperm selected by DGC or swim-up did not show any significant differences in TL as compared to nonselected sperm (p > .05). Negative correlations between TL and sperm motility (r = -.308; p = .049) and concentration (r = -.353; p = .028) were found. Furthermore, exposure of sperm to increasing concentrations of hydrogen peroxide during incubation resulted in a reduction in TL. These data indicate that oxidative stress may be one of the main factors involved in the reduction of TL in sperm. Preliminary clinical results from patients included in this study indicate that TL was shorter in spermatozoa from couples who never achieved a pregnancy compared to couples who did achieve at least one natural pregnancy (p < .05); however, the clinical utility of this biomarker still needs to be confirmed in further studies.

Nuclear degraded sperm subpopulation is affected by poor chromatin compaction and nuclease activity.

There is an interest in the nuclear degraded sperm subpopulation because, although it is present in a low percentage in all semen samples, patient groups such as varicocele and rearranged genome carriers show high levels of these degraded spermatozoa. This study is designed with two objectives in mind: first, incubations of H2 O2 and nuclease on DTT-treated and untreated samples to show the aetiology of this subpopulation and second, assessment of the correlation between the protamine ratio and nuclear degraded spermatozoa. A very high increase in the nuclear degraded subpopulation has been found with nuclease incubation, and it is even higher when it has been merged with nuclear decompaction using DTT. Alternatively, incubation with H2 O2 with and without DTT did not show such a significant increase in nuclear degraded spermatozoa. The protamine ratio correlated with this subpopulation, showing, in patients, that poor nuclear compaction would turn the sperm susceptible to degradation. Then, the assessment of nuclear degraded spermatozoa might not be only a measure of DNA degradation but also an indicator of chromatin compaction in the spermatozoa. Different patient groups would fit this model for sperm nuclear degradation, such as varicocele patients, who show a high percentage of immature spermatozoa and nuclear degraded spermatozoa, and reorganised genome carriers, where reorganisation might also cause poor chromatin compaction on the sperm nucleus.

Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay.

Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected.

Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men.

Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.

The primary aim of this study was to determine the effect of oral antioxidant treatment (1500 mg of l-Carnitine; 60 mg of vitamin C; 20 mg of coenzyme Q10; 10 mg of vitamin E; 10 mg of zinc; 200 µg of vitamin B9; 50 µg of selenium; 1 µg of vitamin B12) during a time period of 3 months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0 h, 2 h, 6 h, 8 h and 24 h) at 37 °C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P < 0.01). The proportion of DDS was also significantly reduced (P < 0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques.

Varicocele, leucocitospermia y su impacto en la fragmentación del ADN espermático / Varicocele, leukocytospermia and its impact on the spermatic DNA fragmentation

Introducción: El estrés oxidativo en una de las causas que puede explicar la presencia de unos niveles elevados de daño en la molécula de ADN. En 2 cuadros clínicos que afectan a la línea germinal del varón, la leucocitospermia y el varicocele, se reconoce la incidencia de niveles elevados de estrés oxidativo. Objetivos: En el presente trabajo se compararon 2 cuadros clínicos, varicocele y leucocitospermia, con objeto de comprobar si existe una intensidad diferencial de la fragmentación del ADN espermático. Material y métodos: Se incluyeron como controles externos donantes de espermatozoides con fertilidad probada y pacientes con factor masculino no determinado. A diferencia de otros estudios de fragmentación del ADN espermático, en este caso, se consideraron tanto los niveles de fragmentación absolutos (SDF), como la proporción de espermatozoides degradados en el total de fragmentados (índice de degradación [ID]) de acuerdo con la información obtenida tras la aplicación del test Halosperm. Resultados: Se demostró un aumento muy significativo de la fragmentación del ADN espermático en las muestras seminales de pacientes con varicocele y con leucocitospermia. Los pacientes con varicocele mostraron un ID 2 veces mayor que el observado en pacientes con factor masculino no determinado o en pacientes con leucocitospermia, y 6 veces mayor que en los donantes. Discusión: La presencia de unos niveles de estrés oxidativo elevados podría ser una explicación asumible para justificar los altos niveles de daño observado en los espermatozoides de los pacientes tanto con varicocele como con leucocitospermia, pero probablemente estos niveles sean más elevados en el caso del varicocele ya que los niveles de degradación del ADN espermático son superiores a los observados en cuadros de leucocitospermia... (AU)

Introduction: High levels of oxidative stress can explain the presence of high levels of damage in the DNA molecule. The impact of high levels of oxidative stress in 2 clinical circumstances affecting the male germ line has been well established: leukocytospermia and varicocele. Objective: To assess sperm DNA fragmentation in patients diagnosed with leukocytospermic and varicocele. Material and methods: Leukocytospermic and varicocele patients and external controls (donors with proven fertility and patients with undetermined male factor). Unlike in other studies of sperm DNA fragmentation, in this study both the proportion of damaged sperm after using the sperm chromatin dispersion test (Halosperm), and the proportion of degraded sperm in total fragmented (degradation index [DI]) were taken into consideration. Results: A highly significant increase in sperm DNA fragmentation has been observed in semen samples of patients with varicocele and leukocytospermia. Varicocele patients showed a DI twice as high as that observed in patients with undetermined male factor or in patients with leukocytospermia, and 6 times as high as that observed in the donors. Discussion: The presence of high levels of oxidative stress could be an acceptable explanation for the high levels of damage observed in the spermatozoa of varicocele patients or with leukocytospermia; the level of sperm degradation is higher in the case of varicocele than those observed in leukocytospermia. Conclusions: SDF levels in patients with leukocytospermia and varicocele are significantly higher than those observed in donors or men with undetermined male factor. The DI in sperm samples from patients with varicocele is the highest of all the samples studied in this analysis. The routine determination of the DI may have a practical value, by guiding the patient towards the potential diagnosis of varicocele, even when this is subclinical (AU)

Alkaline and neutral Comet assay profiles of sperm DNA damage in clinical groups.

BACKGROUND: The analysis of sperm DNA fragmentation has become a new marker to predict male infertility, and many techniques have been developed. The sperm Comet assay offers the possibility of differentiating single- and double-stranded DNA (ssDNA and dsDNA) breaks, which could have different effects on fertility. The objective of this study was to perform a descriptive characterization of different groups of patients, such as those with asthenoteratozoospermic (ATZ) with or without varicocele, oligoasthenoteratozoospermic (OATZ) or balanced chromosome rearrangements, as compared with fertile donors. The Comet assay was used to investigate sperm samples for ssDNA and dsDNA breaks. METHODS AND RESULTS: The analysis of alkaline and neutral Comet assays in different groups of patients showed different sperm DNA damage profiles. Most fertile donors presented low values for ssDNA and dsDNA fragmentation (low-equivalent Comet profile), which would be the best prognosis for achieving a pregnancy. OATZ, ATZ and ATZ with varicocele presented high percentages of ssDNA and dsDNA fragmentation (high-equivalent Comet assay profile), ATZ with varicocele being associated with the worst prognosis, due to higher levels of DNA fragmentation. Rearranged chromosome carriers display a very high variability and, interestingly, two different profiles were seen: a high-equivalent Comet assay profile, which could be compatible with a bad prognosis, and a non-equivalent Comet assay profile, which has also been found in three fertile donors. CONCLUSIONS: Comet assay profiles, applied to different clinical groups, may be useful for determining prognosis in cases of male infertility.

Dynamics of sperm DNA fragmentation in patients carrying structurally rearranged chromosomes.

This investigation was conducted to assess the baseline level of sperm DNA fragmentation (SDF) in a cohort of patients presenting chromosomal rearrangements (nine reciprocal translocations and two inversions). In a separate experiment, a dynamic analysis to calculate the rate of SDF (rSDF), after a varying period of sperm storage (0 h, 1 h, 4 h, 8 h and 24 h) at 37 °C, was performed. Results were compared with eight fertile donors. Different experimental approaches to assess SDF, such as terminal transferase dUTP nick-end labelling (TUNEL), sperm chromatin structure assay (SCSA) and sperm chromatin dispersion test (SCDt), were used. No differences for the baseline level of SDF were found. Carriers of reorganized genomes showed statistically higher levels of SDF than did control donors (p = 0.025 for TUNEL; p = 0.022 for SCSA; p = 0.014 for SCDt). However, 54.5% (6/11) of the patients presented values similar to those of control donors. There was no significant difference in rSDF (p = 0.34). Nevertheless, the results suggest that a high variability for SDF and rSDF exists in these patients. Routine analysis of SDF and rSDF should be considered in patients presenting rearranged genomes to determine fertility status for assisted reproductive techniques (ART) purposes.

Sperm DNA integrity and meiotic behavior assessment in an infertile male carrier of a 9qh+++ polymorphism.

Although several reports on male infertility suggest a relationship between chromosome 9 polymorphisms and infertility, the effects on the phenotype have not been extensively reported. In this study, an infertile patient was found to carry a 9qh+++ chromosome. The flow cytometric TUNEL assay and SCD test have been applied to characterize sperm DNA integrity. In order to assess its meiotic behaviour, synapsis, recombination, and aneuploidy, analyses have been also performed. Sperm DNA fragmentation (SDF) was 77.81% and 87% for the TUNEL and SCD tests, respectively. Ninety-two percent of pachytene cells analyzed showed meiotic abnormalities. The mean number of MLH1 foci per pachytene in the control group was higher (49) than the mean found in the 9qh+++ patient (38) (P < .0001). In spermatozoa, significant increases of disomy rates were observed for chromosome 18 and for the sex chromosomes (P < .0001). These disturbances could be present in other male carriers of a less marked 9qh+.

Synovial vascular patterns and angiogenic factors expression in synovial tissue and serum of patients with rheumatoid arthritis.

OBJECTIVE: To determine whether subgroups of rheumatoid arthritis (RA) patients classified according to their synovial vascular pattern have a different expression of angiogenic mediators or exhibit distinct clinical or biological characteristics. METHODS: Arthroscopies were performed in 27 patients with RA and synovial samples were obtained. Vascular morphology was classified in three patterns: straight (S), tortuous (T) and mixed (M). Immunostaining was performed with anti-vascular endothelial growth factor (anti-VEGF), anti-vascular endothelial growth factor receptor (VEGFR)-1, anti-VEGFR-2, anti-IL-8 and anti-TGF-beta, and measured by digital image analysis. Serum levels of VEGF, TGF-beta and IL-8, and clinical, radiographic and serological data were also analysed. RESULTS: Eleven (41%) patients had the S pattern, nine (33%) the M pattern and seven (26%) the T pattern. The S and M groups had a higher prevalence of rheumatoid factor positivity and erosive disease, and higher levels of markers of systemic inflammation compared with the T group. Synovial expression of VEGF was higher in the S and T groups compared with the M group, whereas TGF-beta was higher in the T compared with the S and M groups. Distinct synovial distribution of VEGF and TGF-beta between groups was also observed. CONCLUSIONS: This preliminary study suggests that RA patients with the S and M patterns share different clinical, biological and serological characteristics compared with those with the T pattern, which may constitute a group with less severe disease. Differences in the intensity and distribution of synovial expression of VEGF and TGF-beta observed between groups could have pathophysiological relevance. However, larger, prospective multicentre studies would be need to determine the clinical relevance of vascular patterns in RA.

p53 expression in rheumatoid and psoriatic arthritis synovial tissue and association with joint damage.

BACKGROUND: Overexpression and functional mutations of p53 have been found in the synovial tissue (ST) of patients with rheumatoid arthritis (RA), but their clinical significance remains unclear. OBJECTIVE: To analyse p53 expression in the ST of patients with RA and psoriatic arthritis (PsA) and its association with joint damage. METHODS: Synovial biopsy specimens were obtained by arthroscopy in 45 patients (27 RA, 18 PsA). Radiographs of hands, feet, and the joint undergoing arthroscopy were obtained to evaluate the presence of erosive disease. Synovial cell populations were analysed using CD4, CD8, CD138, CD20, and CD68 monoclonal antibodies (mAbs). The p53 protein was determined by immunohistology using DO7 mAb in 34 patients (18 RA, 16 PsA). In 11 patients with early RA, the association between p53 and 1 year progression of radiographic damage was analysed using the Larsen-Scott method. RESULTS: The p53 protein was detected in 16/18 (89%) patients with RA and in 9/16 (56%) patients with PsA, but its expression in RA was significantly higher than in PsA. In RA, p53 expression was significantly associated with erosive disease, and its scores were higher in patients with radiological progression. CD68 expression was also associated with erosions and radiological progression in RA. No association was found between either p53 or CD68 and erosive disease in PsA. CONCLUSIONS: These results suggest that p53 ST overexpression and association with joint damage is characteristic of RA rather than PsA, and that p53 ST expression might be a prognostic marker of joint damage in RA.