Jaime Gosálvez

Epigenetics and its role in male infertility

Male infertility is a common and complex problem affecting 1 in 20 men. Despite voluminous research in this field, in many cases, the underlying causes are unknown. Epigenetic factors play an important role in male infertility and these have been studied extensively. Epigenetic modifications control a number of processes within the body, but this review will concentrate on male fertility and the consequences of aberrant epigenetic regulation/modification. Many recent studies have identified altered epigenetic profiles in sperm from men with oligozoospermia and oligoasthenoteratozoospermia. During gametogenesis and germ cell maturation, germ cells undergo extensive epigenetic reprogramming that involves the establishment of sex-specific patterns in the sperm and oocytes. Increasing evidence suggests that genetic and environmental factors can have negative effects on epigenetic processes controlling implantation, placentation and fetal growth. This review provides an overview of the epigenetic processes (histone-to-protamine exchange and epigenetic reprogramming post-fertilization), aberrant epigenetic reprogramming and its association with fertility, possible risks for ART techniques, testicular cancer and the effect of environmental factors on the epigenetic processes.

Types, Causes, Detection and Repair of DNA Fragmentation in Animal and Human Sperm Cells

Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues.

Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species

The dynamic onset of DNA fragmentation in mammalian sperm populations varies widely in different species when the spermatozoa are incubated in vitro at body temperature for several hours, and recent studies have shown that the dynamic rate of DNA fragmentation within a species has considerable predictive value in terms of fertility. The reasons for such variation are unclear, but here we show that differences in protamine sequence and identity could be partially responsible. Sets of 10 normal semen samples from 11 species (ram, goat, boar, white‐tailed deer, rabbit, human, domestic and Spanish fighting bull, horse, donkey, rhinoceros, and koala) were cryopreserved, thawed, diluted in an appropriate extender for each species, and then incubated for 4 hr at 37°C. Semen samples from human infertility patients were also included for comparison with the donors. DNA fragmentation analysis was undertaken immediately after thawing (t0) and after 4 hr (t4) using the Halomax/Halosperm procedure, and the differences in DNA fragmentation between t0 and t4 were examined in the context of the respective protamine genomes. The expression of protamine 2 in a species significantly enhanced the likelihood of sperm DNA fragmentation; greater numbers of cysteine residues in protamine 1 tended to confer increased sperm DNA stability, and there were logical evolutionary relationships between species in terms of their sperm DNA stability. Human spermatozoa from infertility patients exhibited considerably higher DNA instability than the normal semen donors, a difference that could be indirectly attributed to unbalanced protamine 1‐to‐protamine 2 ratios. Mol. Reprod. Dev. 78:951–961, 2011.

Protamine 1 to protamine 2 ratio correlates with dynamic aspects of DNA fragmentation in human sperm

OBJECTIVE To investigate the relationship between the protamine 1 to protamine 2 (P1/P2) ratio and the rate of sperm DNA fragmentation in sperm samples from human males with proven fertility and three different cohorts of male patients. DESIGN P1/P2 ratio was analyzed using acid-urea polyacrylamide acid-urea gels electrophoresis (PAGE). Sperm DNA fragmentation using sperm chromatin dispersion methodology was analyzed after 0, 4, 8, and 24 hours of incubation at 37°C. SETTING University medical school and hospital. PATIENT(S) A total of 32 human males: six with proven fertility, seven carriers of chromosome reorganizations, nine clinical varicocele patients, and ten subclinical varicocele patients. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) P1/P2 ratio, sperm DNA fragmentation (SDF) and the rate of sperm DNA fragmentation (rSDF). RESULT(S) P1/P2 ratio correlated with SDF and rSDF. Statistical differences were detected between fertile controls and patients for the three pathologies studied. rSDF yielded information that differed from baseline SDF. No differences were detected for P1/P2 ratio among patient groups, in reference to the three pathologies studied. CONCLUSION(S) SDF and rSDF correlates with P1/P2 ratio in human sperm, and statistical differences were detected when fertile controls were compared with three different cohorts of patients.

A two-tailed Comet assay for assessing DNA damage in spermatozoa

DNA fragmentation is considered an important parameter of semen quality, and of significant value as a predictor of male fertility. Poor quality chromatin is closely associated with, and highly indicative of, some fertility problems. Many methodologies to assess DNA fragmentation in spermatozoa are available, but they are all unable to differentiate between single-stranded DNA breaks (SSB) and double-stranded DNA breaks (DSB) in the same sperm cell. The two-tailed Comet assay (2T-Comet) protocol overcomes this limitation. A modification of the original Comet assay was developed for the simultaneous evaluation of DNA SSB and DSB in human spermatozoa. The 2T-Comet assay is a fast, sensitive, and reliable procedure for the quantification and characterization of DNA damage in spermatozoa. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology.

A dynamic assessment of sperm DNA fragmentation versus sperm viability in proven fertile human donors

OBJECTIVE To analyze any possible dynamic correlation between sperm DNA fragmentation and sperm viability. DESIGN The rate of viability loss and the rate of increase of the frequency of sperm cells with fragmented DNA were determined at 0, 1.5, 4.5, and 24.0 hours after thawing samples from donors with proven fertility. SETTING Academic biology and reproductive medicine centers. PATIENT(S) Fifteen male donors with proven fertility for a maximum of six births at the reproductive medicine center. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Sperm DNA fragmentation and viability dynamics expressed as logarithmic coefficients of change. RESULT(S) The dynamics of sperm DNA fragmentation and sperm viability adjusted to a logarithmic function with an initial highest velocity that progressively decreases. Nevertheless, the rates were not statistically significantly correlated. CONCLUSION(S) In the short term, dynamic dysfunction of membrane permeability does not result in DNA fragmentation and thus must be considered as independent parameters of sperm quality.

Histological damage in chronic hepatitis C is not related to the extent of infection in the liver.

It has not been completely elucidated whether the liver injury induced by the hepatitis C virus (HCV) is due to direct cytopathic damage or to an immune-mediated response against HCV-infected hepatocytes. In this work, we have determined the percentage of HCV-infected hepatocytes, the histological activity index, and the viremia levels in chronically HCV-infected patients with different grades of liver injury to investigate any possible correlation between them. For that purpose, liver biopsies from 27 patients with HCV chronic hepatitis were analyzed by in situ hybridization. This technique revealed that the percentage of infected hepatocytes ranged from 0.04% to 83.6%. Regarding the viremia levels, HCV RNA concentration ranged from 1.8 x 10(3) to 1.4 x 10(6) genome copies/ml. A significant correlation (r = 0.54; P = 0.003) between the percentage of infected hepatocytes and the viremia levels was found. In contrast, no correlation was observed between the percentage of HCV-infected hepatocytes or the viremia levels and the histological activity index. In conclusion, we have shown that the HCV viremia reflects the extent of the infection in the liver and that the liver injury in chronic HCV infection is not directly related to either the number of infected hepatocytes or the serum HCV RNA concentration.

Sperm deoxyribonucleic acid fragmentation dynamics in fertile donors

OBJECTIVE To study the velocity of sperm DNA fragmentation in frozen-thawed sperm samples from male sperm donors of proved fertility. DESIGN Sperm DNA fragmentation assessment with use of sperm chromatin dispersion methodology after 0, 4, 8, and 24 hours of incubation in IVF medium. SETTING Academic biology and reproductive medicine centers. PATIENT(S) Twenty male fertility donors with proved fertility for a maximum of six births at the reproductive medicine center. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The velocity of sperm DNA fragmentation between two consecutive incubations was scored. Best adjustment of sperm DNA fragmentation index versus incubation time for linear, logarithmic, or exponential function was tested. RESULT(S) Increase of sperm DNA fragmentation through time accounted for a substantial percentage of the overall variation. The highest velocity of sperm DNA fragmentation was observed in the first 4 hours of incubation, decreasing by 50% during the second incubation period and being of the order of 1% in the final experimental period. The tendency to increase in sperm DNA fragmentation is not homogeneous among donors; they may adjust to a logarithmic, linear, or exponential function rendering high values for R(2). CONCLUSION(S) Sperm DNA fragmentation occurs rapidly after thawing, and it is an important cause of the rapid decline of sperm quality. Thus, the use of sperm samples as quickly as possible after thawing is highly recommended in clinical practice. Different sperm DNA fragmentation dynamics among individuals were observed.

Comparison of reproductive outcome in oligozoospermic men with high sperm DNA fragmentation undergoing intracytoplasmic sperm injection with ejaculated and testicular sperm

OBJECTIVE To investigate the effectiveness of intracytoplasmic sperm injection (ICSI) using testicular sperm as a strategy to overcome infertility in men with high sperm DNA fragmentation (SDF). DESIGN Prospective, observational, cohort study. SETTING Private IVF centers. PATIENT(S) A total of 147 couples undergoing IVF-ICSI and day 3 fresh ETs whose male partner has oligozoospermia and high SDF. INTERVENTION(S) Sperm injections were carried out with ejaculated sperm (EJA-ICSI) or testicular sperm (TESTI-ICSI) retrieved by either testicular sperm extraction (TESE) or testicular sperm aspiration (TESA). SDF levels were reassessed on the day of oocyte retrieval in both ejaculated and testicular specimens. MAIN OUTCOME MEASURE(S) Percentage of testicular and ejaculated spermatozoa containing fragmented DNA (%DFI) and clinical pregnancy, miscarriage, and live-birth rates. RESULT(S) The %DFI in testicular sperm was 8.3%, compared with 40.7% in ejaculated sperm. For the TESTI-ICSI group versus the EJA-ICSI group, respectively, the clinical pregnancy rate was 51.9% and 40.2%, the miscarriage rate was 10.0% and 34.3%, and the live-birth rate was 46.7% and 26.4%. CONCLUSION(S) ICSI outcomes were significantly better in the group of men who had testicular sperm used for ICSI compared with those with ejaculated sperm. SDF was significantly lower in testicular specimens compared with ejaculated counterparts. Our results suggest that TESTI-ICSI is an effective option to overcome infertility when applied to selected men with oligozoospermia and high ejaculated SDF levels.

CYTOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF THE IN SITU ENDONUCLEASE DIGESTION OF FIXED TENEBRIO MOLITOR CHROMOSOMES

Cytological and biochemical experiments were undertaken in order to characterize the action of several restriction enzymes on fixed chromosomes of Tenebrio molitor (Coleoptera). EcoRI cuts the satellite DNA of this organism into suunit monomers of 142 bp in naked DNA and acts on fixed chromosomes cleaving and extracting these tandemly repeated sequences present in median centromeric heterochromatin. AluI, in contrast, is unable to attack the satellite sequences but does cut the main band DNA both in naked DNA and in fixed chromosomes. These enzymes therefore permit the in situ localization of satellite DNA or main band DNA in T. molitor. Other enzymes such HinfI or Sau3A do not produce longitudinal differentiation in chromosomes because of the extraction of DNA from satellite and main band DNA regions. In situ hybridization with a satellite DNA probe from T. molitor confirms that the DNA extracted from the chromosomes is the abundant and homogenous highly repeated DNA present in pericentromeric regions. These results plus the analysis of the DNA fractions retained on the slide and solubilized by the action of the restriction enzymes in situ provide evidence that: (a) as an exception to the rule EcoRI (6 bp cutter) is able to produce chromosome banding; (b) the size of the fragments produced by in situ digestion of satellite DNA with EcoRI is not a limiting factor in the extraction; (c) there is a remarkable accord between the action of EcoRI and AluI on naked DNA and on DNA in fixed chromosomes, and (d) the organization of specific chromosome regions seems to be very important in producing longitudinal differentiation on chromosomes.