Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

STUDY QUESTION: Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? SUMMARY ANSWER: No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 µg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. WHAT IS KNOWN ALREADY: Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. STUDY DESIGN, SIZE, DURATION: This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 µg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. MAIN RESULTS AND THE ROLE OF CHANCE: Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. LARGE SCALE DATA: Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. LIMITATIONS, REASONS FOR CAUTION: This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. WIDER IMPLICATIONS OF THE FINDINGS: Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 µg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare.

Architecture of the myocardium in computed tomography.

To determine whether current concepts of myocardial organization were in fact the result of artifacts introduced by the dissection technique, a method comprising air injection of the coronary arteries (which gently separated the myocardial fibers) and computerized tomography was devised. Viewing the tomographs cinematographically confirmed the concept that the heart is formed by a double looping of myocardial fiber bands was correct. Examination of abnormal hearts, eg, one with Duchenne muscular dystrophy, shows that pathologic tissue disarrangements could also be identified.

Morphological hazards of deuterium oxide as a cardioplegic agent.

To determine the cardio-protective effect of heavy water on the ischemic myocardium, a thoracotomy was performed on 18 mongrel dogs. The animals were connected to the extracorporeal circulation in a standardized experimental procedure. Following total cardiopulmonary bypass, 2,000 ml of a standard cardioplegic solution (LK 352) was infused at the aortic root of 10 dogs, which served as controls (group I), and the same solution containing 20% of 99.8% deuterium oxide was given at the aortic root of the remaining animals (group II). At the end of 60 minutes of ischemia, 1,000 ml of the solutions was again administered at the aortic root of the corresponding animals. Myocardial biopsies were taken from the apex of the left ventricle of each dog before cardiopulmonary bypass, immediately after the infusion of the cardioplegic solutions, following 90 minutes of ischemia, and after 30 minutes of reperfusion, and studied ultrastructurally. Whereas the ultrastructure of the myocardium of group I was well preserved at the end of the ischemic period, deuterium-oxide-treated hearts showed extensive focal and global myofilamentolysis and lysis of whole myocytes. Structural damage to glycogen, nuclear chromatin dispersal, severe intracellular edema and complete rupture of the intercalated discs were characteristic findings. At the end of ischemia, all the hearts of group I could be resuscitated. During the ischemia, all the hearts of group II developed into stone hearts. Biochemical studies on a second series showed a higher ATP depletion and a significantly higher lactate accumulation in group II than in group I.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of non-coronary collateral blood supply on the electively arrested heart during ischemia and reperfusion.

To study the influence of non-coronary collateral blood circulation (NCCBC) on the integrity of the ischemic myocardium a right-sided thoracotomy was performed on 15 anesthetized dogs. Following a total cardiopulmonary bypass (CPB), ventricular fibrillation was induced, during which 2,000 ml calcium-free cardioplegic solution LK 352 was given at the aortic root over an 8-10 min period. Precautions were taken to prevent retrograde blood flow into the coronary system via the coronary sinus. After 90 min of ischemia, ten of the dog hearts were reperfused with systemic blood for the next 30 min. Transmural biopsies were taken from the apex of the left ventricle at the following intervals: (1) before CPB, (2) immediately after the infusion of LK 352, (3) following 90 min of ischemia, (4) after 5 min, (5) after 15 min, and finally (6) after 30 min of reperfusion and were then studied ultrastructurally. The presence of NCCBC was documented by the observation of erythrocyte-filled blood vessels in the biopsies corresponding to nos. 2 and 3 of the above. To assess the degree of ischemic injury and the extent of myocardial recovery during reperfusion, a scoring system based on a semiquantitative assessment of the characteristic morphological changes was used. The average result of the separately assessed subendo- and subepicardial layers represented the score, which was plotted on the ischemic injury and the recovery scale, thus making a direct comparison of the hearts possible. All the hearts generously supplied with blood via extracoronary routes during ischemia showed minimal and reversible ischemic injuries. They recovered more quickly and more completely following reperfusion than those hearts without NCCBC. From these results we conclude that despite its warming-up effect on the myocardium and its tendency to wash out the cardioplegic solution, the NCCBC generally protects the myocardium from serious ischemic injuries and shortens the period of recuperation during the reperfusion.

[Catheter tip force probe: a paraclinical development for per- and postoperative monitoring in heart surgery]. / Katheterspitzenkraftaufnehmer: Eine kliniknahe Entwicklung zur per- und postoperativen Uberwachung in der Herzchirurgie.

Local intramyocardial "one-point" force measurement is well adapted to the myocardial architecture. The dense three-dimensional muscle network does not permit direct measurement of longitudinal tension in a particular bundle of muscle fibers. However, measurements on the skeletal muscle have shown changes in tendon stress to be strictly proportional to changes in local force measured simultaneously at different sites on the gastrocnemial muscle. In the heart in situ the response to inotropic and pre- and afterloads changes is extremely sensitive. However, in comparison to left ventricular pressure, local force measurements at different sites and in different ventricular layers show distinct force gradients and phase dislocation, indicating a wide range of inhomogeneities in ventricular dynamics. The method is suitable for clinical application in cardiac surgery: the probe may be implanted directly on the exposed heart or during ventricular catheterization using a pointed catheter tip force probe.

Coronary rheothermia: a quasi non-invasive method for controlling bypass patency.

A method based on the principle of thermodilution was developed for a quasi non-invasive permeability control of aortocoronary bypass. An epivascularly-attached thermistor records the cooling of the bypass wall when, following the intravenous injection of 5 to 10 ml of a NaCl solution at 4 degrees C, a bolus of cooled blood passes through the bypass. During cardiosurgical intervention, the thermistor is attached to the venous bridge by one or 2 sutures. The efferent cable goes through the thorax wall and is coupled to a subcutaneously implanted telemetric amplifier unit. The influence of the vessel wall on the perivascular temperature signal, as compared to the intravascular one, was studied in acute and chronic animal experiments. In acute experiments the perivascular peak of temperature was found to be lower than the intravascular one. Continuous measurements over 9 days showed variations in the perivascular signals which must have been due to changes in the thermal capacity of the tissue coupled to the thermistor as well as to changes in resistance caused by a variable extent of scarred area and by the varying water content of the wound bed. These variables will continue to keep rheothermia within the limitations of a method with primarily binary results (= bypass: open or closed). Given stable coupling conditions after full development of the scar around the thermistor, the signal falsification by the then constantly coupled tissue capacity becomes calculable such as to obtain semiquantitative results which, theoretically should vary predominantly with cardiac output.