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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 5, Iss. 5 — May. 1, 2014
  • pp: 1690–1699
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Confocal Raman micro-spectroscopy for rapid and label-free detection of maleic acid-induced variations in human sperm

Ning Li, Diling Chen, Yan Xu, Songhao Liu, and Heming Zhang  »View Author Affiliations


Biomedical Optics Express, Vol. 5, Issue 5, pp. 1690-1699 (2014)
http://dx.doi.org/10.1364/BOE.5.001690


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Abstract

Confocal Raman microspectroscopy is a valuable analytical tool in biological and medical research, allowing the detection of sample variations without external labels or extensive preparation. To determine whether this method can assess the effect of maleic acid on sperm, we prepared human sperm samples incubated in different concentrations of maleic acid, after which Raman spectra from the various regions of sperm cells were recorded. Following the maleic acid treatment, Raman spectra indicated significant changes. Combined with other means, we found that the structures and chemical compositions of sperm membranes were damaged, and even the sperm DNA was damaged by the incorporation of maleic acid. Thus, this technique can be used for detection and identification of maleic acid-induced changes in human sperm at a molecular level. Although this particular application of Raman microspectroscopy still requires further validation, it has potentially promise as a diagnostic tool for reproductive medicine.

© 2014 Optical Society of America

1. Introduction

Approximately 15 to 20% of couples face the fertility problem worldwide [1

1. Y. Nishimune and H. Tanaka, “Infertility caused by polymorphisms or mutations in spermatogenesis-specific genes,” J. Androl. 27(3), 326–334 (2006). [CrossRef] [PubMed]

]. Even when fertilization is successful, poor-quality embryos, low implantation rates, and high miscarriage rates are still serious problems for some couples [2

2. P. Devroey and A. Van Steirteghem, “A review of ten years experience of ICSI,” Hum. Reprod. Update 10(1), 19–28 (2004). [CrossRef] [PubMed]

5

5. C. Kennedy, P. Ahlering, H. Rodriguez, S. Levy, and P. Sutovsky, “Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction,” Reprod. Biomed. Online 22(3), 272–276 (2011). [CrossRef] [PubMed]

]. In general, male factor infertility is responsible for 40–50% of cases [6

6. V. M. Brugh 3rd and L. I. Lipshultz, “Male factor infertility,” Med. Clin. North Am. 88(2), 367–385 (2004). [CrossRef] [PubMed]

]. Various reasons may result in male infertility including genetic defects, illnesses, injuries, environmental factors, or lifestyle choices [7

7. A. E. Willets, J. M. Corbo, and J. N. Brown, “Clomiphene for the treatment of male infertility,” Reprod. Sci. 20(7), 739–744 (2013). [CrossRef] [PubMed]

]. And semen quality is closely linked to the probability of infertility.

At present, numerous methods are available for the measurement of sperm status and assessment of sperm quality. Computer-aided sperm analysis (CASA) and computer-aided sperm morphometric assessment (CASMA) are employed to evaluate sperm physiological parameters and morphological parameters, respectively, including sperm concentration, motility, ellipticity, and regularity. However, relevant studies suggest that methods only using the above parameters lack sufficient evidence to evaluate sperm’s potential for fertilization [8

8. D. S. Guzick, J. W. Overstreet, P. Factor-Litvak, C. K. Brazil, S. T. Nakajima, C. Coutifaris, S. A. Carson, P. Cisneros, M. P. Steinkampf, J. A. Hill, D. Xu, and D. L. VogelD. S. GuzickJ. W. OverstreetP. Factor-LitvakC. K. BrazilS. T. NakajimaC. CoutifarisS. A. CarsonP. CisnerosM. P. SteinkampfJ. A. HillD. XuD. L. VogelNational Cooperative Reproductive Medicine Network, “Sperm morphology, motility, and concentration in fertile and infertile men,” N. Engl. J. Med. 345(19), 1388–1393 (2001). [CrossRef] [PubMed]

10

10. C. Avendaño, A. Franchi, S. Taylor, M. Morshedi, S. Bocca, and S. Oehninger, “Fragmentation of DNA in morphologically normal human spermatozoa,” Fertil. Steril. 91(4), 1077–1084 (2009). [CrossRef] [PubMed]

]. Other analytical techniques with high spatial resolution, such as optical or electron microscopy, X-ray imaging, or secondary ion-mass spectroscopy, either damage the sample during analysis, or do not provide detailed information about structure and chemical composition simultaneously [11

11. K. Meister, D. A. Schmidt, E. Bründermann, and M. Havenith, “Confocal Raman microspectroscopy as an analytical tool to assess the mitochondrial status in human spermatozoa,” Analyst (Lond.) 135(6), 1370–1374 (2010). [CrossRef] [PubMed]

]. In any case, several methods are currently available for the analysis of sperm nuclear DNA (nDNA) quality, such as sperm chromatin dispersion, the comet assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), and sperm chromatin structure assay [12

12. J. L. Fernández, L. Muriel, V. Goyanes, E. Segrelles, J. Gosálvez, M. Enciso, M. LaFromboise, and C. De Jonge, “Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test,” Fertil. Steril. 84(4), 833–842 (2005). [CrossRef] [PubMed]

15

15. M. Bungum, L. Bungum, and A. Giwercman, “Sperm chromatin structure assay (SCSA): a tool in diagnosis and treatment of infertility,” Asian J. Androl. 13(1), 69–75 (2011). [CrossRef] [PubMed]

]. However, these methods also face a similar problem, in that the processes required may lead to the destruction of the sperm sample. What is required clinically, then, is a reliable, non-invasive, and non-destructive analytical technique that provides precise information on the quality of a sperm while not affecting the integrity of the cell, thereby qualifying it for use in assisted reproductive technology (ART).

Raman spectroscopy has been recognized as a valuable analytical technique by providing chemical fingerprint information of substances without external labels or extensive preparation. This can be used to identify and characterize biomolecules, cells, or tissues. Using a confocal microscope affords three-dimensional spatial resolution, permitting the identification of molecules in organelles [11

11. K. Meister, D. A. Schmidt, E. Bründermann, and M. Havenith, “Confocal Raman microspectroscopy as an analytical tool to assess the mitochondrial status in human spermatozoa,” Analyst (Lond.) 135(6), 1370–1374 (2010). [CrossRef] [PubMed]

,16

16. C. Krafft, T. Knetschke, R. H. W. Funk, and R. Salzer, “Identification of organelles and vesicles in single cells by Raman microspectroscopic mapping,” Vib. Spectrosc. 38(1-2), 85–93 (2005). [CrossRef]

,17

17. C. Matthäus, T. Chernenko, J. A. Newmark, C. M. Warner, and M. Diem, “Label-free detection of mitochondrial distribution in cells by nonresonant Raman microspectroscopy,” Biophys. J. 93(2), 668–673 (2007). [CrossRef] [PubMed]

]. In biomedicine, Raman microspectroscopy has been used as a powerful tool in the discrimination, classification and diagnosis of pathological conditions, such as various malignancies and tumors [18

18. C. M. Krishna, G. D. Sockalingum, J. Kurien, L. Rao, L. Venteo, M. Pluot, M. Manfait, and V. B. Kartha, “Micro-Raman spectroscopy for optical pathology of oral squamous cell carcinoma,” Appl. Spectrosc. 58(9), 1128–1135 (2004). [CrossRef] [PubMed]

20

20. M. S. Vidyasagar, K. Maheedhar, B. M. Vadhiraja, D. J. Fernendes, V. B. Kartha, and C. M. Krishna, “Prediction of radiotherapy response in cervix cancer by Raman spectroscopy: A pilot study,” Biopolymers 89(6), 530–537 (2008). [CrossRef] [PubMed]

]. Being non-invasive, it has also been employed in the evaluation of various living cells without any adverse reaction experienced by the cells themselves [21

21. K. C. Schuster, I. Reese, E. Urlaub, J. R. Gapes, and B. Lendl, “Multidimensional information on the chemical composition of single bacterial cells by confocal Raman microspectroscopy,” Anal. Chem. 72(22), 5529–5534 (2000). [CrossRef] [PubMed]

23

23. A. Downes, R. Mouras, and A. Elfick, “Optical spectroscopy for noninvasive monitoring of stem cell differentiation,” J. Biomed. Biotechnol. 2010, 101864 (2010). [CrossRef] [PubMed]

]. Recent studies have successfully demonstrated the utility of Raman microspectroscopy for investigating the molecular compositions and sub-cellular organelles of human sperm [11

11. K. Meister, D. A. Schmidt, E. Bründermann, and M. Havenith, “Confocal Raman microspectroscopy as an analytical tool to assess the mitochondrial status in human spermatozoa,” Analyst (Lond.) 135(6), 1370–1374 (2010). [CrossRef] [PubMed]

, 24

24. T. Huser, C. A. Orme, C. W. Hollars, M. H. Corzett, and R. Balhorn, “Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells,” J Biophotonics 2(5), 322–332 (2009). [CrossRef] [PubMed]

28

28. Z. Huang, G. Chen, X. Chen, J. Wang, J. Chen, P. Lu, and R. Chen, “Rapid and label-free identification of normal spermatozoa based on image analysis and micro-Raman spectroscopy,” J Biophotonics1–5 (2013).

].

2. Materials and methods

2.1 Sample preparation

The resulting sperm suspension was divided into five aliquots. One aliquot was left untreated, while the remaining four were subjected to the increasing amounts of maleic acid reagent (0.01 M, 0.02 M, 0.04 M, 0.08 M). After 45 minutes of incubation at 37 °C in ambient air with 5% CO2, the sperm were washed with PBS (800 × g, 10 min, 4 °C). Subsequently, the supernatants were discarded and the pellets resuspended in PBS. In addition, each sample was further divided into three aliquots, which were assessed for sperm quality by Raman micro-spectroscopy, flow cytometry, and hypo-osmotic swelling (HOS) testing [33

33. S. Ramu and R. Jeyendran, “The Hypo-osmotic Swelling Test for Evaluation of Sperm Membrane Integrity,” in Spermatogenesis, D. T. Carrell and K. I. Aston, eds. (Humana Press, 2013).

].

2.2 Confocal Raman microspectroscopy

A drop of 15 μL sperm suspensions were smeared onto aluminium slices and left to air-dry. This kind of pure metal has no Raman spectral features and very low background interference. For Raman spectral measurement, a confocal micro-Raman spectroscopy system (Renishaw Invia, UK) with an excitation wavelength of 514.5 nm generated by an Ar+ laser (~10mw) was employed, in which a × 50 objective lens is used to focus the laser beam and to collect the Raman signal. The lateral resolution of the instrument was about 1 μm, and the power of the laser beam measured at the sample was 1.5 mw approximately. Raman spectra were recorded by a Peltier-cooled charge-coupled device (CCD) camera with an integration time of 30 s, and three accumulations. At least five sperm cells were scanned for each sample. Three scanning points were selected for each individual sperm according to anatomical structure, including the acrosome, nucleus, and middle piece.

2.3 Flow cytometry

The flow-cytometric method based on acridine orange was used to assess the degree of sperm DNA damage, as described elsewhere [4

4. D. P. Evenson, L. K. Jost, D. Marshall, M. J. Zinaman, E. Clegg, K. Purvis, P. de Angelis, and O. P. Claussen, “Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic,” Hum. Reprod. 14(4), 1039–1049 (1999). [CrossRef] [PubMed]

]. Briefly, samples were diluted with TNE buffer (0.01 M Tris·Cl, 0.15 M NaCl, 1 mM EDTA, pH 7.4) and transferred into tubes for flow cytometry (Lab test tubes; Beckmann Coulter); 0.4 mL of acid detergent solution (0.08 N HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 1.2) was added, and, after 30 seconds of gentle agitation, 1.2 mL of acridine orange staining solution (6 μg/mL) was added. The samples were kept for 3 min at 4 °C in the dark and then analyzed by flow cytometry (Cytomics FC 500, Beckman Coulter, USA). Fluorescence data were acquired from a total of 5,000 sperm per sample. Excitation by a 488-nm-wavelength light source resulted in the emission of red fluorescence (single-stranded DNA) and green fluorescence (double-stranded DNA). The results were expressed as a DNA fragmentation index (DFI, Flow cytometry software: FCS Express, version 3), which represents the percentage of cells exhibiting increased red fluorescence.

2.4 Data analysis

Standard principal component analysis (PCA) was performed on the data of the Raman spectra from both the control sperm cells and the sperm cells incubated with maleic acid. Computations were performed using the MATLAB (R2012b) software system. In this paper, we chose the first two principal components accounted for more than 75% of the accumulative total contribution for analysis.

3. Results and discussion

3.1 The Raman spectra of sperm

Figure 1(a)
Fig. 1 (A) Bright-field image of sperm cell and (B) averaged Raman spectra correspond to the regions of nucleus (a), middle piece (b), and acrosome (c) in normal human sperm, respectively.
shows a bright-field image of a normal sperm cell; it exhibits a regular outline and a clearly distinguished acrosomal cap. Note that the sperm acrosome outline is clearly visible without any staining treatment. Single point Raman spectra of the nucleus, middle piece and acrosome regions in normal sperm cells are presented in Fig. 1(b).

3.2 Effect on the sperm nucleus region

In order to quantitatively identify how maleic acid treatment influenced the variation in DNA, we selected specific Raman peaks and compared the changes in their spectral intensities. The results are shown in Fig. 3(a). For sperm cells exposed to maleic acid, the Raman intensities corresponding to DNA, such as 787, 1094, 1337 and 1421 cm−1, were reduced in comparison with the control. The peak at 988 cm−1 noticeably shifted to 973 cm−1 (Fig. 4
Fig. 4 Comparison of the spectra of control and treated sperm cells with the different concentrations of maleic acid in the region of 900 cm−1 to 1050 cm−1 from the nucleus region of human sperm..
). It is possible that broken covalent bonds between desoxyribose and phosphodiester or desoxyribose and base, caused by the maleic acid damage, can change the groups and their force-bearing environments, changing their Raman activities [41

41. Y. Xu, Z. Zhou, H. Yang, Y. Xu, and Z. Zhang, “Raman spectroscopic study of microcosmic photodamage of the space structure of DNA sensitized by Yangzhou haematoporphyrin derivative and Photofrin II,” J. Photochem. Photobiol. B 52(1-3), 30–34 (1999). [CrossRef] [PubMed]

]. All of these changes indicate the destruction of the DNA structures.

3.3 Effect on the sperm middle-piece region

The Raman peaks at 1128 cm−1 and 1071 cm−1, belonging to the trans stretching vibrations of C-C skeleton, and 1086 cm−1, belonging to the gauche vibrations of C-C skeleton were chosen for data analysis. These peaks are very sensitive to the change of the conformation of membrane lipids. The longitudinal order-parameters in chains (Strans) [44

44. B. P. Gaber and W. L. Peticolas, “On the quantitative interpretation of biomembrane structure by Raman spectroscopy,” Biochim. Biophys. Acta 465(2), 260–274 (1977). [CrossRef] [PubMed]

] was used to assess the damage to the middle piece membranous lipids. The more content of the trans conformation, the higher order of the vertical chain. Increased content of the trans conformation also results in less liquidity of the middle piece membrane [45

45. N. Li, S. X. Li, Z. Y. Guo, Z. F. Zhuang, R. Li, K. Xiong, S. J. Chen, and S. H. Liu, “Micro-Raman spectroscopy study of the effect of mid-ultraviolet radiation on erythrocyte membrane,” J. Photochem. Photobiol. B 112, 37–42 (2012). [CrossRef] [PubMed]

]. The value of Strans is calculated following Eq. (1). For the solid dipalmitoylphosphatidylcholine (DPPC), the intensity ratio of 1128cm−1/1086 cm−1 was 1.77 [44

44. B. P. Gaber and W. L. Peticolas, “On the quantitative interpretation of biomembrane structure by Raman spectroscopy,” Biochim. Biophys. Acta 465(2), 260–274 (1977). [CrossRef] [PubMed]

]. Our calculated results (Table 1

Table 1. Longitudinal order-parameters in chains Strans and the rate of variation in the middle piece membrane before and after the effects of maleic acid.

table-icon
View This Table
) mean that the percentage of trans conformation increased and gauche conformation decreased in the membrane, as compared to the control sample, and so the liquidity and ionic permeability of the membrane in the middle piece region was reduced.

strans=(I1128/I1086)sample(I1128/I1086)DPPCofsolid=(I1128/I1086)sample1.77
(1)

3.4 Effect on the sperm acrosome region

Next, we discuss the peaks at 1238 and 1303 cm−1, which are assigned to the β-sheet and α-helix of amide III, respectively. The α-helix and the β-sheet are both common secondary structures in proteins and are maintained by hydrogen bonds. Amide III is very sensitive to changes of the main chain’s conformation in membrane proteins [45

45. N. Li, S. X. Li, Z. Y. Guo, Z. F. Zhuang, R. Li, K. Xiong, S. J. Chen, and S. H. Liu, “Micro-Raman spectroscopy study of the effect of mid-ultraviolet radiation on erythrocyte membrane,” J. Photochem. Photobiol. B 112, 37–42 (2012). [CrossRef] [PubMed]

]. Its quantity decreased with increasing maleic acid concentrations as indicated in Fig. 3(c). This suggests the occurrence of denaturation and conformational changes to the acrosome membrane proteins. This change can be identified as the rupture of the peptide and hydrogen bonds in membrane proteins.

Thirty-four Raman spectra of control sperm cells (CSC) and 36 Raman spectra of treated sperm cells (TSC) were used for PCA. All the Raman spectra were from the acrosome region of the sperm cells. The result in Fig. 6
Fig. 6 PC1 vs PC2 plot for the spectra from the acrosome region in the control sperm cells (CSC) and treated sperm cells (TSC) with maleic acid incubation.
shows that although there are similarities, spectra from the control sample are sufficiently distinguishable and significantly different from those obtained after incubation with different concentrations of maleic acid. All of these changes indicate the destruction of the acrosome membrane.

3.5 Evaluation of human sperm damage

To verify the results obtained with Raman microspectroscopy, we performed the HOS test and flow-cytometry. A functional and intact membrane is required for sperm to be fertile. The HOS test can evaluate the integrity of the sperm’s plasma membrane and also serves as a useful indicator of fertility potential [46

46. R. S. Jeyendran, H. H. Van der Ven, M. Perez-Pelaez, B. G. Crabo, and L. J. D. Zaneveld, “Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics,” J. Reprod. Fertil. 70(1), 219–228 (1984). [CrossRef] [PubMed]

48

48. H. N. Sallam, A. Farrag, A.-F. Agameya, Y. El-Garem, and F. Ezzeldin, “The use of the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in patients treated with ICSI: a randomized controlled study,” Hum. Reprod. 20(12), 3435–3440 (2005). [CrossRef] [PubMed]

]. It predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or “swell.” A higher percentage of swollen sperm indicates the presence of sperm having functional and intact plasma membranes [33

33. S. Ramu and R. Jeyendran, “The Hypo-osmotic Swelling Test for Evaluation of Sperm Membrane Integrity,” in Spermatogenesis, D. T. Carrell and K. I. Aston, eds. (Humana Press, 2013).

]. Changes to membranous structure can affect the functional integrity of the sperm’s plasma membrane. The results of HOS test indicate that the sperm membrane has been damaged after the incorporation of maleic acid (Fig. 7
Fig. 7 Evaluation of sperm membrane integrity with HOS test.
). This result is in agreement with the Raman results. Since the membrane is subjected to damage, the intracelluar organelles and molecules will lose its protection.

The flow-cytometric method based on acridine orange was used to assess the degree of sperm DNA damage. The results were expressed as DNA fragmentation index (DFI). Analysis of the DNA fragmentation by flow cytomtry confirmed that increasing levels of sperm DNA damage occurred after the treatments with maleic acid (Fig. 8
Fig. 8 Flow-cytometric assessment of sperm DNA damage showing the different levels of fragmention induced by maleic acid.
). This result is also in agreement with the Raman results.

4. Conclusion

In summary, our study has shown that Raman micro-spectroscopy is capable of assessing the variations and damage at a molecular level in human sperm cells induced by maleic acid treatment. Following the maleic acid treatment, Raman spectra indicated significant changes in different regions of the sperm cells. All of these results suggest destruction and conformational changes in proteins and lipids, and damage to DNA structure, including nDNA and mitochondrial DNA. We can see that maleic acid can affect the quality of sperm. In contrast, maleic acid or its chemically modified compounds exhibit promise as cervical or vaginal contraceptives because it disrupts the structural and functional integrity of sperm via either electrostatic or pH-lowering effects. In the future, non-invasive and label-free Raman micro-spectroscopy could prove to be a promising diagnostic tool with further potential to identify normal sperm, allowing for their routine selection in ART. This method can also be applied to the exploration of the biochemical and molecular mechanisms of human sperm function with the chemical fingerprints we obtained.

Acknowledgments

We gratefully acknowledge the financial support for this work via grants from china national ministry of science and technology plan projects (2011BAI01B02), and Guangdong science and technology plan projects (2012A030100005), and Guangdong Natural Science Foundation Grant (S2013040016159).

References and links

1.

Y. Nishimune and H. Tanaka, “Infertility caused by polymorphisms or mutations in spermatogenesis-specific genes,” J. Androl. 27(3), 326–334 (2006). [CrossRef] [PubMed]

2.

P. Devroey and A. Van Steirteghem, “A review of ten years experience of ICSI,” Hum. Reprod. Update 10(1), 19–28 (2004). [CrossRef] [PubMed]

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Z. Pandian, A. Templeton, G. Serour, and S. Bhattacharya, “Number of embryos for transfer after IVF and ICSI: a Cochrane review,” Hum. Reprod. 20(10), 2681–2687 (2005). [CrossRef] [PubMed]

4.

D. P. Evenson, L. K. Jost, D. Marshall, M. J. Zinaman, E. Clegg, K. Purvis, P. de Angelis, and O. P. Claussen, “Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic,” Hum. Reprod. 14(4), 1039–1049 (1999). [CrossRef] [PubMed]

5.

C. Kennedy, P. Ahlering, H. Rodriguez, S. Levy, and P. Sutovsky, “Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction,” Reprod. Biomed. Online 22(3), 272–276 (2011). [CrossRef] [PubMed]

6.

V. M. Brugh 3rd and L. I. Lipshultz, “Male factor infertility,” Med. Clin. North Am. 88(2), 367–385 (2004). [CrossRef] [PubMed]

7.

A. E. Willets, J. M. Corbo, and J. N. Brown, “Clomiphene for the treatment of male infertility,” Reprod. Sci. 20(7), 739–744 (2013). [CrossRef] [PubMed]

8.

D. S. Guzick, J. W. Overstreet, P. Factor-Litvak, C. K. Brazil, S. T. Nakajima, C. Coutifaris, S. A. Carson, P. Cisneros, M. P. Steinkampf, J. A. Hill, D. Xu, and D. L. VogelD. S. GuzickJ. W. OverstreetP. Factor-LitvakC. K. BrazilS. T. NakajimaC. CoutifarisS. A. CarsonP. CisnerosM. P. SteinkampfJ. A. HillD. XuD. L. VogelNational Cooperative Reproductive Medicine Network, “Sperm morphology, motility, and concentration in fertile and infertile men,” N. Engl. J. Med. 345(19), 1388–1393 (2001). [CrossRef] [PubMed]

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11.

K. Meister, D. A. Schmidt, E. Bründermann, and M. Havenith, “Confocal Raman microspectroscopy as an analytical tool to assess the mitochondrial status in human spermatozoa,” Analyst (Lond.) 135(6), 1370–1374 (2010). [CrossRef] [PubMed]

12.

J. L. Fernández, L. Muriel, V. Goyanes, E. Segrelles, J. Gosálvez, M. Enciso, M. LaFromboise, and C. De Jonge, “Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test,” Fertil. Steril. 84(4), 833–842 (2005). [CrossRef] [PubMed]

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16.

C. Krafft, T. Knetschke, R. H. W. Funk, and R. Salzer, “Identification of organelles and vesicles in single cells by Raman microspectroscopic mapping,” Vib. Spectrosc. 38(1-2), 85–93 (2005). [CrossRef]

17.

C. Matthäus, T. Chernenko, J. A. Newmark, C. M. Warner, and M. Diem, “Label-free detection of mitochondrial distribution in cells by nonresonant Raman microspectroscopy,” Biophys. J. 93(2), 668–673 (2007). [CrossRef] [PubMed]

18.

C. M. Krishna, G. D. Sockalingum, J. Kurien, L. Rao, L. Venteo, M. Pluot, M. Manfait, and V. B. Kartha, “Micro-Raman spectroscopy for optical pathology of oral squamous cell carcinoma,” Appl. Spectrosc. 58(9), 1128–1135 (2004). [CrossRef] [PubMed]

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20.

M. S. Vidyasagar, K. Maheedhar, B. M. Vadhiraja, D. J. Fernendes, V. B. Kartha, and C. M. Krishna, “Prediction of radiotherapy response in cervix cancer by Raman spectroscopy: A pilot study,” Biopolymers 89(6), 530–537 (2008). [CrossRef] [PubMed]

21.

K. C. Schuster, I. Reese, E. Urlaub, J. R. Gapes, and B. Lendl, “Multidimensional information on the chemical composition of single bacterial cells by confocal Raman microspectroscopy,” Anal. Chem. 72(22), 5529–5534 (2000). [CrossRef] [PubMed]

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24.

T. Huser, C. A. Orme, C. W. Hollars, M. H. Corzett, and R. Balhorn, “Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells,” J Biophotonics 2(5), 322–332 (2009). [CrossRef] [PubMed]

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F. Liu, Y. Zhu, Y. Liu, X. Wang, P. Ping, X. Zhu, H. Hu, Z. Li, and L. He, “Real-time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucida,” Fertil. Steril. 99, 684–689.e684 (2013).

28.

Z. Huang, G. Chen, X. Chen, J. Wang, J. Chen, P. Lu, and R. Chen, “Rapid and label-free identification of normal spermatozoa based on image analysis and micro-Raman spectroscopy,” J Biophotonics1–5 (2013).

29.

P. D. Brown-Woodman, E. J. Post, P. Y. Chow, and I. G. White, “Effects of malonic, maleic, citric and caffeic acids on the motility of human sperm and penetration of cervical mucus,” Int. J. Fertil. 30(3), 38–44 (1985). [PubMed]

30.

D. M. Stein and H. Schnieden, “Effect of antidepressant drugs on the in-vitro eggpenetrating ability of golden hamster epididymal spermatozoa,” Reprod. Fertil. 68(1), 227–233 (1983). [CrossRef]

31.

H. Singh, M. S. Jabbal, A. R. Ray, and P. Vasudevan, “Effect of anionic polymeric hydrogels on spermatozoa motility,” Biomaterials 5(5), 307–309 (1984). [CrossRef] [PubMed]

32.

R. Sanchez, E. Toepfer-Petersen, R. J. Aitken, and W. B. Schill, “A new method for evaluation of the acrosome reaction in viable human spermatozoa,” Andrologia 23(3), 197–203 (1991). [CrossRef] [PubMed]

33.

S. Ramu and R. Jeyendran, “The Hypo-osmotic Swelling Test for Evaluation of Sperm Membrane Integrity,” in Spermatogenesis, D. T. Carrell and K. I. Aston, eds. (Humana Press, 2013).

34.

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, and T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990). [CrossRef] [PubMed]

35.

W. L. Peticolas, “Raman spectroscopy of DNA and proteins,” in Methods in Enzymology, S. Kenneth, ed. (Academic Press, 1995).

36.

H. Deng, V. A. Bloomfield, J. M. Benevides, and G. J. Thomas Jr., “Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence,” Biopolymers 50(6), 656–666 (1999). [CrossRef] [PubMed]

37.

J. M. Benevides and G. J. Thomas Jr., “Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D2O solutions and application to B, Z and A-DNA,” Nucleic Acids Res. 11(16), 5747–5761 (1983). [CrossRef] [PubMed]

38.

S. C. Erfurth and W. L. Peticolas, “Melting and premelting phenomenon in DNA by laser Raman scattering,” Biopolymers 14(2), 247–264 (1975). [CrossRef] [PubMed]

39.

H. Deng, V. A. Bloomfield, J. M. Benevides, and G. J. Thomas Jr., “Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy,” Nucleic Acids Res. 28(17), 3379–3385 (2000). [CrossRef] [PubMed]

40.

S. Krimm and J. Bandekar, “Vibrational analysis of peptides, polypeptides, and proteins. V. Normal vibrations of beta-turns,” Biopolymers 19(1), 1–29 (1980). [CrossRef] [PubMed]

41.

Y. Xu, Z. Zhou, H. Yang, Y. Xu, and Z. Zhang, “Raman spectroscopic study of microcosmic photodamage of the space structure of DNA sensitized by Yangzhou haematoporphyrin derivative and Photofrin II,” J. Photochem. Photobiol. B 52(1-3), 30–34 (1999). [CrossRef] [PubMed]

42.

J. Mo, W. Zheng, and Z. Huang, “Fiber-optic Raman probe couples ball lens for depth-selected Raman measurements of epithelial tissue,” Biomed. Opt. Express 1(1), 17–30 (2010). [CrossRef] [PubMed]

43.

Z. Zhuang, N. Li, Z. Guo, M. Zhu, K. Xiong, and S. Chen, “Study of molecule variations in renal tumor based on confocal micro-Raman spectroscopy,” J. Biomed. Opt. 18(3), 031103 (2013). [CrossRef] [PubMed]

44.

B. P. Gaber and W. L. Peticolas, “On the quantitative interpretation of biomembrane structure by Raman spectroscopy,” Biochim. Biophys. Acta 465(2), 260–274 (1977). [CrossRef] [PubMed]

45.

N. Li, S. X. Li, Z. Y. Guo, Z. F. Zhuang, R. Li, K. Xiong, S. J. Chen, and S. H. Liu, “Micro-Raman spectroscopy study of the effect of mid-ultraviolet radiation on erythrocyte membrane,” J. Photochem. Photobiol. B 112, 37–42 (2012). [CrossRef] [PubMed]

46.

R. S. Jeyendran, H. H. Van der Ven, M. Perez-Pelaez, B. G. Crabo, and L. J. D. Zaneveld, “Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics,” J. Reprod. Fertil. 70(1), 219–228 (1984). [CrossRef] [PubMed]

47.

D. Lechniak, A. Kedzierski, and D. Stanislawski, “The use of HOS test to evaluate membrane functionality of boar sperm capacitated in vitro,” Reprod. Domest. Anim. 37(6), 379–380 (2002). [CrossRef] [PubMed]

48.

H. N. Sallam, A. Farrag, A.-F. Agameya, Y. El-Garem, and F. Ezzeldin, “The use of the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in patients treated with ICSI: a randomized controlled study,” Hum. Reprod. 20(12), 3435–3440 (2005). [CrossRef] [PubMed]

OCIS Codes
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(170.1530) Medical optics and biotechnology : Cell analysis
(170.5660) Medical optics and biotechnology : Raman spectroscopy
(300.6450) Spectroscopy : Spectroscopy, Raman

ToC Category:
Cell Studies

History
Original Manuscript: March 14, 2014
Revised Manuscript: April 20, 2014
Manuscript Accepted: April 23, 2014
Published: April 29, 2014

Citation
Ning Li, Diling Chen, Yan Xu, Songhao Liu, and Heming Zhang, "Confocal Raman micro-spectroscopy for rapid and label-free detection of maleic acid-induced variations in human sperm," Biomed. Opt. Express 5, 1690-1699 (2014)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-5-5-1690


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References

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  27. F. Liu, Y. Zhu, Y. Liu, X. Wang, P. Ping, X. Zhu, H. Hu, Z. Li, and L. He, “Real-time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucida,” Fertil. Steril.99, 684–689.e684 (2013).
  28. Z. Huang, G. Chen, X. Chen, J. Wang, J. Chen, P. Lu, and R. Chen, “Rapid and label-free identification of normal spermatozoa based on image analysis and micro-Raman spectroscopy,” J Biophotonics1–5 (2013).
  29. P. D. Brown-Woodman, E. J. Post, P. Y. Chow, and I. G. White, “Effects of malonic, maleic, citric and caffeic acids on the motility of human sperm and penetration of cervical mucus,” Int. J. Fertil.30(3), 38–44 (1985). [PubMed]
  30. D. M. Stein and H. Schnieden, “Effect of antidepressant drugs on the in-vitro eggpenetrating ability of golden hamster epididymal spermatozoa,” Reprod. Fertil.68(1), 227–233 (1983). [CrossRef]
  31. H. Singh, M. S. Jabbal, A. R. Ray, and P. Vasudevan, “Effect of anionic polymeric hydrogels on spermatozoa motility,” Biomaterials5(5), 307–309 (1984). [CrossRef] [PubMed]
  32. R. Sanchez, E. Toepfer-Petersen, R. J. Aitken, and W. B. Schill, “A new method for evaluation of the acrosome reaction in viable human spermatozoa,” Andrologia23(3), 197–203 (1991). [CrossRef] [PubMed]
  33. S. Ramu and R. Jeyendran, “The Hypo-osmotic Swelling Test for Evaluation of Sperm Membrane Integrity,” in Spermatogenesis, D. T. Carrell and K. I. Aston, eds. (Humana Press, 2013).
  34. G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, and T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature347(6290), 301–303 (1990). [CrossRef] [PubMed]
  35. W. L. Peticolas, “Raman spectroscopy of DNA and proteins,” in Methods in Enzymology, S. Kenneth, ed. (Academic Press, 1995).
  36. H. Deng, V. A. Bloomfield, J. M. Benevides, and G. J. Thomas., “Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence,” Biopolymers50(6), 656–666 (1999). [CrossRef] [PubMed]
  37. J. M. Benevides and G. J. Thomas., “Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D2O solutions and application to B, Z and A-DNA,” Nucleic Acids Res.11(16), 5747–5761 (1983). [CrossRef] [PubMed]
  38. S. C. Erfurth and W. L. Peticolas, “Melting and premelting phenomenon in DNA by laser Raman scattering,” Biopolymers14(2), 247–264 (1975). [CrossRef] [PubMed]
  39. H. Deng, V. A. Bloomfield, J. M. Benevides, and G. J. Thomas., “Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy,” Nucleic Acids Res.28(17), 3379–3385 (2000). [CrossRef] [PubMed]
  40. S. Krimm and J. Bandekar, “Vibrational analysis of peptides, polypeptides, and proteins. V. Normal vibrations of beta-turns,” Biopolymers19(1), 1–29 (1980). [CrossRef] [PubMed]
  41. Y. Xu, Z. Zhou, H. Yang, Y. Xu, and Z. Zhang, “Raman spectroscopic study of microcosmic photodamage of the space structure of DNA sensitized by Yangzhou haematoporphyrin derivative and Photofrin II,” J. Photochem. Photobiol. B52(1-3), 30–34 (1999). [CrossRef] [PubMed]
  42. J. Mo, W. Zheng, and Z. Huang, “Fiber-optic Raman probe couples ball lens for depth-selected Raman measurements of epithelial tissue,” Biomed. Opt. Express1(1), 17–30 (2010). [CrossRef] [PubMed]
  43. Z. Zhuang, N. Li, Z. Guo, M. Zhu, K. Xiong, and S. Chen, “Study of molecule variations in renal tumor based on confocal micro-Raman spectroscopy,” J. Biomed. Opt.18(3), 031103 (2013). [CrossRef] [PubMed]
  44. B. P. Gaber and W. L. Peticolas, “On the quantitative interpretation of biomembrane structure by Raman spectroscopy,” Biochim. Biophys. Acta465(2), 260–274 (1977). [CrossRef] [PubMed]
  45. N. Li, S. X. Li, Z. Y. Guo, Z. F. Zhuang, R. Li, K. Xiong, S. J. Chen, and S. H. Liu, “Micro-Raman spectroscopy study of the effect of mid-ultraviolet radiation on erythrocyte membrane,” J. Photochem. Photobiol. B112, 37–42 (2012). [CrossRef] [PubMed]
  46. R. S. Jeyendran, H. H. Van der Ven, M. Perez-Pelaez, B. G. Crabo, and L. J. D. Zaneveld, “Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics,” J. Reprod. Fertil.70(1), 219–228 (1984). [CrossRef] [PubMed]
  47. D. Lechniak, A. Kedzierski, and D. Stanislawski, “The use of HOS test to evaluate membrane functionality of boar sperm capacitated in vitro,” Reprod. Domest. Anim.37(6), 379–380 (2002). [CrossRef] [PubMed]
  48. H. N. Sallam, A. Farrag, A.-F. Agameya, Y. El-Garem, and F. Ezzeldin, “The use of the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in patients treated with ICSI: a randomized controlled study,” Hum. Reprod.20(12), 3435–3440 (2005). [CrossRef] [PubMed]

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