Use of aniline blue to assess chromatin condensation in morphologically normal spermatozoa in normal and infertile men*

Hofmann, Norbert; Hilscher, B.

doi:10.1093/oxfordjournals.humrep.a137472

Cited by 95 publications

(62 citation statements)

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“…Similarly, the peak intensity of the 785 cm À1 band varies between 0.45 and 0.74, indicating a var- (Figure 3g), but a few cells have peak ratios that are closer to that of the abnormal cells. This observation is consistent with previous dye binding studies of human sperm considered to have a normal head shape morphology [16]. This finding is extremely important.…”

Section: Discussionsupporting

confidence: 93%

“…Currently clinicians selecting sperm for ICSI and other assisted fertilization methods using single sperm cells pick the sperm to be introduced into the eggs based primarily on their morphology, and in some cases a combination of morphology and motility. The Raman results, as well as the dye binding studies reported earlier [16], indicate that the population of sperm with normal head shapes is clearly heterogeneous in composition, and they may also be variable in their functionality. In summary, we have provided Raman-spectroscopic evidence obtained from analyses of individual cells that indicates that DNA packaging in human sperm cells with abnormal shape is different than in sperm with normal shape.…”

Section: Discussionsupporting

confidence: 54%

“…Sperm head morphology has been established by the World Health Organization (WHO) as the most important criterion to assess the fertility of males. There is, however, evidence and growing concern that not all sperm with normal shape are fully functional and may contain DNA that is improperly packaged [15][16][17]. Fertilization events involving abnormal sperm could have adverse effects on early embryo development or lead to genetic disease.…”

Section: Biophotonicsmentioning

confidence: 99%

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Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells

Huser

Orme

Hollars

et al. 2009

Journal of Biophotonics

108

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Healthy human males produce sperm cells of which about 25–40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro‐Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA‐packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in‐vitro fertilization. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 54%

Section: Biophotonicsmentioning

confidence: 99%

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Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells

Huser

Orme

Hollars

et al. 2009

Journal of Biophotonics

108

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“…The potential value of adding these techniques to standard infertility evaluations lies in their ability to identify abnormal chromatin condensation in morphologically normal and abnormal spermatozoa (Dadoune et al, 1988;Hofmann and Hilscher, 1991). Aniline blue selectively stains lysine-rich histones (Terquem and Dadoune, 1983) and has been used as a clinical assessment of sperm chromatin condensation and associated infertility (Auger et al, 1990).…”

Section: Assessment Of Sperm Chromatin Structure In Fertility Evaluatmentioning

confidence: 99%

Sperm Chromatin Structure Assay: Its Clinical Use for Detecting Sperm DNA Fragmentation in Male Infertility and Comparisons With Other Techniques

Evenson

Larson

Jost

2002

Journal of Andrology

904

768

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The Sperm Chromatin Structure Assay (SCSA) was first described in the December 5, 1980 issue of Science (Evenson et al, 1980a). The data described in that article showed a significant difference between proven fertile and subfertility or infertility in men and bulls and the susceptibility to denaturation of their sperm nuclear DNA. A subsequent study (Evenson et al, 1984) described SCSA data obtained from men with testicular cancer. The data were heterogeneous, and begged the question, what was SCSA measuring? Great effort has been spent over the past 15 years on animal model systems, mostly related to dose-response toxicology experiments and large animal fertility trials. The results from these studies showed that SCSA was highly dose-responsive to toxicants, highly repeatable, and provided meaningful biological information on sperm nuclear DNA defects. The extensive data from nonhumans (Evenson et al, 1985(Evenson et al, , 1989a(Evenson et al, ,b, 1993a(Evenson et al, ,b,c, 1994Ballachey et al, 1987Ballachey et al, , 1988Evenson and Jost, 1993; Sailer et al, 1995a,b;Evenson, 1999a), and human toxicology and fertility studies (Evenson et al, 1978(Evenson et al, , 1980a(Evenson et al, ,b, 1984(Evenson et al, , 1991Evenson and Melamed, 1983;Evenson, 1999b;Larson et al, 1999Larson et al, , 2000Larson et al, , 2001 provide This work was supported in part by grant R827019 from the US Environmental Protection Agency, grants EPS-9720642 and OSR-9452894 from the National Science Foundation, and by SCSA Diagnostics, Inc. This is South Dakota Agricultural Experiment Station publication 3273 of the journal series.Correspondence to: Donald P. Evenson, Olson Biochemistry Laboratories, ASC 136, South Dakota State University, Brookings, SD 57006 (e-mail: donald evenson@sdstate.edu).Received for publication September 20, 2001; accepted for publication September 20, 2001. compelling evidence that SCSA will be useful in clinical human semen analysis (Figure 1).After reviewing current infertility center sites on the World Wide Web and approximately a dozen lay-oriented books on infertility and assisted reproductive technology (ART), it was amazing that not a single reference contained any information about the negative influence of fragmented sperm nuclear DNA on successful pregnancy outcome. Furthermore, few physicians are aware of scientific literature on this subject. Currently, if a spermatozoon has some motility as seen under a light microscope, and reasonable morphology, then many ART clinics assume that the sperm DNA must be fine. However, a number of cases have been documented in which couples have had multiple failed attempts at intracytoplasmic sperm injection (ICSI), even using donor eggs, and we found that the fragmentation of sperm DNA was above our threshold for less than 1% probability of achieving a successful pregnancy. Spermatozoa with defective DNA can fertilize an oocyte, produce high-quality early stage embryos, and then, in relationship to the extent of DNA damage, fail in producing a successful term ...

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“…However, this effect was comparable in all current settings, when corrected for neutrally charged sperm. Multiple studies have reported an association between sperm chromatin compaction and abnormal sperm head morphology [61][62][63] and acrosome abnormalities [64]. Although the compaction of the nuclear chromatin and DNA integrity are two interdependent parameters [65,66], some studies have reported an association between them [67,68].…”

Section: Discussionmentioning

confidence: 99%

Optimization of microelectrophoresis to select highly negatively charged sperm

Lee¹,

Murphy²,

Aston³

et al. 2016

J Assist Reprod Genet

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Purpose The sperm membrane undergoes extensive surface remodeling as it matures in the epididymis. During this process, the sperm is encapsulated in an extensive glycocalyx layer, which provides the membrane with its characteristic negative electrostatic charge. In this study, we develop a method of microelectrophoresis and standardize the protocol to isolate sperm with high negative membrane charge. Methods Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate prior to and following density gradient centrifugation (DGC), and evaluated for sperm DNA damage, and histone retention. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an ICSI needle and directly analyzed for DNA damage. Results When raw semen was analyzed using microelectrophoresis, 94 % were NCS. In contrast, DGC completely or partially stripped the negative membrane charge from sperm resulting PCS and neutrally charged sperm, while the charged sperm populations are increased with an increase in electrophoretic current. Following DGC, high sperm DNA damage and abnormal histone retention were inversely correlated with percentage NCS and directly correlated with percentage PCS. NCS exhibited significantly lower DNA damage when compared with control (P < 0.05) and PCS (P < 0.05). When the charged sperm population was corrected for neutrally charged sperm, sperm DNA damage was strongly associated with NCS at a lower electrophoretic current. Conclusion The results suggest that selection of NCS at lower current may be an important biomarker to select healthy sperm for assisted reproductive treatment.

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Use of aniline blue to assess chromatin condensation in morphologically normal spermatozoa in normal and infertile men*

Cited by 95 publications

References 0 publications

Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells

Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells

Sperm Chromatin Structure Assay: Its Clinical Use for Detecting Sperm DNA Fragmentation in Male Infertility and Comparisons With Other Techniques

Optimization of microelectrophoresis to select highly negatively charged sperm

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