Raman micro-spectroscopy analysis of different sperm regions: a species comparison.

STUDY QUESTION: Is Raman micro-spectroscopy a valid approach to assess the biochemical hallmarks of sperm regions (head, midpiece and tail) in four different species? SUMMARY ANSWER: Non-invasive Raman micro-spectroscopy provides spectral patterns enabling the biochemical characterization of the three sperm regions in the four species, revealing however high similarities for each region among species. WHAT IS KNOWN ALREADY: Raman micro-spectroscopy has been described as an innovative method to assess sperm features having the potential to be used as a non-invasive selection tool. However, except for nuclear DNA, the identification and assignment of spectral bands in Raman-profiles to the different sperm regions is scarce and controversial. STUDY DESIGN SIZE, DURATION: Raman spectra from head, midpiece and tail of four different species were obtained. Sperm samples were collected and smeared on microscope slides. Air dried samples were subjected to Raman analysis using previously standardized procedures. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm samples from (i) two donors attending the infertility clinic at the Centre of Reproductive Medicine and Andrology; (ii) two C57BL/6 -TgN (ACTbEGFP) 1Osb adult mice; (iii) two adult Cynomolgus monkeys (Macaca fascicularis) and (iv) two sea urchins (Arbacia punctulata) were used to characterize and compare their spectral profiles. Differences and similarities were confirmed by principal component analysis (PCA). MAIN RESULTS AND THE ROLE OF CHANCE: Several novel region-specific peaks were identified. The three regions could be differentiated by distinctive Raman patterns irrespective of the species. However, regardless of the specie, their main spectral pattern remains mostly unchanged. These results were corroborated by the PCA analysis and suggest that the basic constituents of spermatozoa are biochemically similar among species. LIMITATIONS REASONS FOR CAUTION: Further research should be performed in live sperm to validate the detected spectral bands and their use as markers of distinctive regions. WIDER IMPLICATIONS OF THE FINDINGS: Raman peaks that have never been described in the sperm cell were detected. Particularly important are those that are unique to the midpiece as they might be a reference to the identification of sperm mitochondria, whose function is highly correlated with that of sperm. In the future, Raman micro-spectroscopy has the potential to be applied in assessment of male fertility. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by BMBF project 'Sperm Ident' (FKZ:13N13024) and the DAAD-CRUP bilateral exchange program (AI A06/16-57213087). S.A. is a recipient of a fellowship from the Portuguese foundation for science and technology (FCT-SFRH/BPD/110160/2015) and R.DC. is a recipient of a DAAD PhD stipend (91590556). There is no competing interest.

In situ visualization of damaged DNA in human sperm by Raman microspectroscopy.

BACKGROUND: Beyond determining the percentage of damaged sperm, current methods of DNA assessment are of limited clinical utility as they render the sample unusable. We evaluated Raman microspectroscopy, a laser-based non-invasive technique that provides detailed chemical 'fingerprints' of cells and which potentially could be used for nuclear DNA-based sperm selection. METHODS: Eight healthy donors provided ejaculates. After system optimization, a minimum of 200 air-dried sperm/sample/donor, prior to/and after UVB irradiation, were assessed by two observers. Spectra were analysed by Principal Component, Spectral Angle and Wavelet Analyses. RESULTS: Spectra provided a chemical map delineating each sperm head region. Principal Component Analysis showed clear separation between spectra from UV-irradiated and untreated samples whilst averaged data identified two regions of interest (1040 and 1400 cm(-1)). Local spectral analysis around the DNA PO(4) backbone peak (1042 cm(-1)), showed that changes in this region were indicative of DNA damage. Wavelet decomposition confirmed both the 1042 cm(-1) shift and a second UVB susceptible region (1400-1600 cm(-1)) corresponding to protein-DNA interactions. No difference was found between observer measurements. CONCLUSIONS: Raman microspectroscopy can provide accurate and reproducible assessment of sperm DNA structure and the sites and location of damage.

The forces generated within the musculature of the left ventricular wall.

OBJECTIVES: To test the hypothesis that two populations of myocardial fibres-fibres aligned parallel to the surfaces of the wall and an additional population of fibres that extend obliquely through the wall-when working in concert produce a dualistic, self stabilising arrangement. METHODS: Assessment of tensile forces in the walls of seven porcine hearts by using needle probes. Ventricular diameter was measured with microsonometry and the intracavitary pressure through a fluid filled catheter. Positive inotropism was induced by dopamine, and negative inotropism by thiopental. The preload was raised by volume load and lowered by withdrawal of blood. Afterload was increased by inflation of a balloon in the aortic root. The anatomical orientation of the fibres was established subsequently in histological sections. RESULTS: The forces in the fibres parallel to the surface decreased 20-35% during systolic shrinkage of the ventricle, during negative inotropism, and during ventricular unloading. They increased 10-30% on positive inotropic stimulation and with augmentation in preload and afterload. The forces in the oblique transmural fibres increased 8-65% during systole, on positive inotropic medication, with an increase in afterload and during ventricular shrinkage, and decreased 36% on negative inotropic medication. There was a delay of up to 147 ms in the drop in activity during relaxation in the oblique transmural fibres. CONCLUSION: Although the two populations of myocardial fibres are densely interwoven, it is possible to distinguish their functions with force probes. The delayed drop in force during relaxation in obliquely oriented fibres indicates that they are hindered in their shortening to an extent that parallels any increase in mural thickness. The transmural fibres, therefore, contribute to stiffening of the ventricular wall and hence to confining ventricular compliance.

A critical evaluation of results of partial left ventriculectomy.

BACKGROUND: Because of the variation in the surgical procedures designed to reduce ventricular radius, along with differences in hospital care, it is difficult to disentangle the factors that may contribute to the success or failure of the partial left ventriculectomy. METHODS AND RESULTS: We undertook partial left ventriculectomy in 18 patients, 10 suffering from idiopathic dilated cardiomyopathy and 8 from ischemic heart disease. We assessed the amount of reduction in wall stress, the systolic thickening of the ventricular wall, and the extent of connective tissue in the excised segment of the wall. Of the overall group, six patients died, three from infarction, one of stroke, one with asystole, and one with ventricular fibrillation. The mean decrease in measured mesh tension was 40% (p < 0.001). Most patients exhibited improvements postoperatively in terms of the systolic thickening of the posterior and superior free walls of the left ventricle. In those in whom the events could be monitored, life-threatening arrhythmias posed complications in three of four patients with ischemic heart disease, and in two of six patients suffering from idiopathic dilated cardiomyopathy. In one patient, death was associated with a transmural alignment of fibrous tissue. CONCLUSIONS: Our measured reductions in myocardial mesh tension were in keeping with the anticipated theoretical reduction in wall stress expected from partial ventriculectomy. The basic concept underscoring surgical maneuvers to reduce ventricular radius, therefore, is sound. A potential trap is the resection of the marginal artery. Critical myofibrosis was a rare complication. Arrhythmias, which are common, can successfully be treated by implantation of antitachycardic and defibrillatory devices.