Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation

Background: In order to isolate the â??bestâ?? sperm for assisted conception a discontinuous two-step density gradient centrifugation is usually employed. This technique is known to isolate a subpopulation with good motility, morphology and nuclear DNA (nDNA) integrity. As yet its ability to isolate sperm with unfragmented mitochondrial DNA (mtDNA) is unknown. Methods: Semen was obtained from men (n=28) attending our Regional Fertility Centre for infertility investigations. We employed a modified long polymerase chain reaction to study mtDNA and a modified alkaline Comet assay to determine nDNA fragmentation. Results: The high- density fraction displayed significantly more wild type mtDNA (75% of samples) than that of the low- density fraction (25% of samples). In the high-density fraction, there was a higher incidence of single, rather than double or multiple deletions and the deletions were predominantly small scale (0.1-4.0kb). There was a strong correlation between nDNA fragmentation, the number of mtDNA deletions (r=0.7, p

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity

Objective: To investigate effects of cryopreservation on sperm motility and DNA integrity. Design: Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. Setting: A hospital andrology laboratory. Patient(s): Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. Intervention(s): Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. Main Outcome Measure(s): Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. Result(s): Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. Conclusion(s): Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.

Fas and DNA double stranded breaks in human ejaculated sperm

Objective: to determine the incidence of Fas positivity and DNA double stranded breaks (DSB) as indicators of early and late stage apoptosis in ejaculated sperm. Design: Fas positivity was assessed by flow cytometry and DSB by neutral Comet assay Setting: Andrology Laboratory, Royal Maternity Hospita, Belfast Northern Ireland, UK. Patients: 45 infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies Main Outcome measures: Perecentage Fas positive cells, percentage DNA fragmentation, olive tail moments Results: The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozoospermic semen. No Fas positivity was observed in fertile mens’ sperm. DSB were greater in infertile than in fertile mens’ sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage. Conclusion: Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis.

The effects of short and long incubations on DNA fragmentation of testicular

DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4 hr and 24 hr incubations, and after cryopreservation. The effect is intensified by post-thaw incubations. Testicular sperm to be used clinically in ICSI should be injected without delay.

The Effects of Delta–9–Tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana, on human sperm function in vitro.

Objective: to investigate the effects of tetrahydrocannabinol; THC on human sperm function in vitro. Design: laboratory analysis of sperm motility with and without exposure to THC using computer assisted semen analysis (CASA) and acrosome reaction by fluoroscein isothiocyanate labelled peanut agglutinin (FITC-PNA) staining. Setting: An ART unit in a tertiary medical centre. Patients: semen was obtained from 78 men attending the Regional Fertility Centre, Belfast. Interventions: Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45% (the poorer subpopulation) fractions by density centrifugation and incubated with, or without (controls), tetrahydrocannabinol (THC) at concentrations equivalent to therapeutic (0.032�?�¯?�?�­M) and recreational (4.8 and 0.32�?�¯?�?�­M) plasma levels, at 37�?�¯?�?�°C for 3 hours. Main outcome measures: Sperm motility, spontaneous and induced acrosome reactions Results: There was a dose-dependent decrease in percentage progressive motility (-21% at 4.8�??�?�µM, p0.05) in the 90% fraction. The 45% fraction showed a greater decrease in percentage progressive motility (-56% at 4.8�??�?�µM, p=0.011; -23% at 0.32�??�?�µM, p= 0.039; and -28% at 0.032�??�?�µM, p=0.004). A decrease in the straight line velocity; VSL (-10%) and the average path velocity; VAP (-10%) were also observed in the 90% fraction. A significant inhibition (-15% at 4.8�??�?�µM, p=0.04) in spontaneous acrosome reaction was observed in the 90% fraction. The 45% fraction showed a more marked inhibition [-35% (p

Sildenafil citrate improves sperm motility but causes a premature acrosome reaction in vitro

Objective: to determine if sildenafil citrate; a cyclic monophosphate specific type 5 phosphodiesterase inhibitor, influences sperm motility or the acrosome reaction. Design: laboratory analysis of sperm motility after exposure to sildenafil citrate using computer assisted semen analysis (CASA) and acrosome reaction by fluoroscein isothiocyanate labelled peanut agglutinin (FITC-PNA) staining. Setting: An ART unit Patients: 57 male patients Interventions: Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45% (the poorer subpopulation) fractions by density centrifugation and incubated with sildenafil citrate ( 0.67uM) at 37ï?°C for up to 180 minutes. Main outcome measures: both the numbers and velocity of progressively motile sperm were significantly increased by sildenafil citrate between 15 and 135 minutes. Further, samples revealed that these effects were consistent in the 90% and 45% subpopulations of sperm. In both subpopulations, sildenafil also caused a significant increase in the proportion of acrosome reacted sperm - 22.1% compared with 11.8% in the control group of the good quality fraction (p

Reduced sperm yield from testicular biopsies of vasectomized men is due to increased apoptosis

Objective: To compare sperm yields, apoptotic indices, and sperm DNA fragmentation from vasectomized men and fertile men undergoing vasectomy.

Design: Testicular biopsies from vasectomized (n 26) and fertile men (n 46), were milked to calculate sperm/gram and also formalin-?xed to determine the numbers of developing sperm and incidence and intensities of testicular FasL, Fas, Bax, and Bcl-2. Testicular sperm DNA fragmentation was assessed using the alkaline Comet assay.

Setting: An ART unit.

Patient(s): Twenty-six men attending for intracytoplasmic sperm injection (ICSI) and 46 men attending for vasectomies.

Main Outcome Measure(s): Spermatocyte, spermatid and sperm yields, Fas, FasL, and Bax staining.

Result(s): Sperm yields from men vasectomized 5 years previously were markedly reduced compared to fertile men. Increased intensities of FasL and Bax staining were observed in the seminiferous tubules of vasectomy men. FasL positivity (percentage) also increased in Sertoli cells, and both FasL and Fas positivity (percentage) increased in primary spermatocytes and round spermatids of vasectomized men. Sperm DNA fragmentation, an end point marker of apoptosis, increased signi?cantly in vasectomized men compared to fertile men.

Conclusion(s): Reduced sperm yields after vasectomy are associated with increased apoptosis through the Fas–FasL and Bax pathways. Sperm after vasectomy displayed increased DNA fragmentation. (Fertil Steril 2007;87:834–41. ©2007 by American Society for Reproductive Medicine.)

Incidence of Fas positivity and deoxyribonucleic acid double-stranded breaks in human ejaculated sperm.

Objective

To determine the incidence of Fas positivity and DNA double-strand breaks (DSB) as indicators of early- and late-stage apoptosis in ejaculated sperm.

Design

Fas positivity was assessed by flow cytometry and DSB by the neutral Comet assay.

Setting

Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom.

Patient(s) and intervention(s)

Forty-five infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies.

Main outcome measure(s)

Percentage Fas-positive cells, percentage DNA fragmentation, olive tail moment.

Result(s)

The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozospermic semen. No Fas positivity was observed in fertile mens' sperm. Deoxyribonucleic acid fragmentation (DSB) was greater in infertile than in fertile men's sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage.

Conclusion(s)

Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis.

The genetic basis of infertility in men.

Subfertility in men is a heterogeneous syndrome, its pathophysiology remaining unknown in the majority of affected men. A large number of genes and loci are associated with sterility in experimental animals, but the human homologues of most of these genes have not been characterized. A British study suggested that, in a large proportion of men with idiopathic infertility, the disorder is inherited as an autosomal recessive trait; this provocative hypothesis needs confirmation. Because normal germ cell development requires the temporally and spatially co-ordinated expression of a number of gene products at the hypothalamic, pituitary and testicular levels, it is safe to predict that a large number of autosomal, as well as X- and Y-linked, genes will probably be implicated in different subsets of male subfertility.

Necrospermia and chronic spinal cord injury.

OBJECTIVE: To determine whether improvement in quality of semen over 4 consecutive days of electroejaculation in men with chronic spinal cord injury (SCI) was consistent with epididymal necrospermia. DESIGN: Prospective study of a random sample of men with SCI. SETTING: A southeastern Australian SCI management center in collaboration with the specialist andrology service of a university-based department of obstetrics and gynecology in a tertiary referral hospital. PATIENT(S): Nine men with chronic spinal cord injury. INTERVENTION(S): Semen samples were obtained by using electroejaculation, and testicular biopsy samples were obtained by using fine-needle tissue aspiration. MAIN OUTCOME MEASURE(S): Semen analysis was performed according to World Health Organization criteria. Testicular biopsy and electron microscopy were done by using standard techniques. RESULT(S): During up to 4 days of consecutive-day electroejaculation, sperm motility and viability in semen obtained from men with chronic SCI increased by an average of 23% on days 2 and 3. The severity of the degenerative changes and the numbers of spermatozoa affected on day 1 became less marked by day 4. The changes were not present in late spermatids obtained from testicular biopsies. CONCLUSION(S): The asthenospermia of chronic SCI is similar to epididymal necrospermia and can be improved by consecutive-day electroejaculation.