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Real-time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucida
ORIGINAL ARTICLES: ANDROLOGY
Real-time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucida Feng Liu, B.Sc.,a Yong Zhu, Ph.D.,a Yufei Liu,a Xiaobo Wang,a Ping Ping, M.D., Ph.D.,a Xinyuan Zhu, Ph.D.,b Hongliang Hu, Ph.D.,a Zheng Li, M.D., Ph.D.,a and Lin He, Ph.D.b a
Department of Urology, Renji Hospital, Shanghai Human Sperm Bank, Sperm Development and Genetics Laboratory, Shanghai Institute of Andrology, Shanghai Jiao Tong University School of Medicine; and b Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai, People’s Republic of China
Objective: To determine if Raman microspectroscopy (RMS) can distinguish sperm bound to the human zona pellucida (ZP) from those unbound sperm. Design: Paired experiments to compare Raman scanning features of ZP-bound and unbound sperm. Setting: Public hospital–based clinical assisted reproduction center. Patient(s): Sperm samples from ten fertile donors were used in this study. Intervention(s): None. Main Outcome Measure(s): Sperm-ZP binding, ZP-induced acrosome reaction, and scanning intensity of various regions of sperm. Result(s): The RMS found two slightly low-intensity regions (800–900 and 3,200–4,000 cm1) shifted to high-intensity grade at the acrosome region of the ZP-bound sperm compared with unbound sperm. Moreover, principal component analysis and statistical analysis showed that the RMS can distinguish the ZP-bound sperm from the unbound sperm. Conclusion(s): RMS scanning of single live sperm could be used to distinguish ZP-bound sperm from unbound sperm. Thus, RMS may be a useful tool to detect normal functional sperm and to select sperm for intracytoplasmic sperm injection. (Fertil SterilÒ 2013;99:684–9. Ó2013 by American Society for Reproductive Use your smartphone Medicine.) to scan this QR code Key Words: Raman microspectroscopy, scanning of single live sperm, human zona pellucida– and connect to the bound sperm Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/liuf-raman-micro-spectroscopy-sperm-human-zona-pellucida/
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n assisted reproductive technology (ART) clinics, >50% of infertile couple are routinely treated by intracytoplasmic sperm injection (ICSI) (1). Although ICSI can significantly improve the fertilization rate, a low number of high-quality embryos and low
implantation rates are still major concerns for some couples (2, 3). It is still debatable how to select the best sperm which not only fertilize the oocyte but also ensure the safety of the offspring (4–6). During the fertilization process, either in vivo or under conventional
Received July 3, 2012; revised October 18, 2012; accepted October 21, 2012; published online November 10, 2012. F.L. has nothing to disclose. Y.Z. has nothing to disclose. Y. L. has nothing to disclose. X.W. has nothing to disclose. P.P. has nothing to disclose. X.Z. has nothing to disclose. H.H. has nothing to disclose. Z.L. has nothing to disclose. L.H. has nothing to disclose. F.L. and Y.Z. contributed equally to this work. Supported by the Science and Technology Commission of Shanghai Municipality (grant no. 10JC1409900), National Basic Research Program of China (grant no. 2011CB944504), and the Shanghai Municipal Commission of Population and Family Planning Commission Science and Technology Developing Foundation (grant no. 2012JG07). Reprint requests: Zheng Li, M.D., Ph.D., Department of Urology, Shanghai Institute of Andrology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200001, People’s Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 3, March 1, 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.10.035 684
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in vitro fertilization, a fertile sperm has been biologically selected through sperm-oocyte interaction, in particular a thick layer of zona pellucida (ZP). For a fertile man, the average ejaculation contains 100 106–200 106 motile sperm, but only 14% of motile sperm are capable of binding to the ZP (7, 8). Most sperm bound to the ZP have normal morphology and chromatin DNA (9). Therefore the ZP-bound sperm are likely the favored or fertile population of sperm in any given ejaculate. Therefore, using ZP-bound sperm for ICSI would in theory enhance the outcomes. There are a few reports demonstrating that the use of ZP-bound sperm for ICSI significantly improves the embryo quality, implantation rate, and clinical pregnancy rate compared with conventional ICSI (10). Therefore, it VOL. 99 NO. 3 / MARCH 1, 2013
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FIGURE 1
Mapping of Raman spectral peaks to different regions of sperm, including (A) acrosome, (B) nucleus, (C) equatorial, (D) neck, (E) midpiece, and (F) tail. (G) The combined spectral map. The x-axis presents the continuous wave lengths (cm1) of the spectra, and the y-axis presents the intensity of the spectra, including three grades of intensity peaks: low intensity (50–100 a.u.), medium intensity (100–200 a.u.), and high intensity (>200 a.u.). Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
would advantageous to develop a new approach of sperm selection based on the characteristic of ZP binding (10–12). Recently Raman microspectroscopy (RMS), a laser-based noninvasive technique, was shown to provide a detailed chemical ‘‘fingerprint’’ of cells in various medical fields, such as in the classification of different malignancies (13–15). It has been shown that RMS can detect sperm DNA packaging or DNA structure damage and their mitochondrial state (16–18). In the present study, the RMS scanning technique was used to distinguish ZP-bound sperm from unbound sperm.
MATERIALS AND METHODS Sperm and Oocytes This study was approved by the Renji Hospital Research and Ethics Committee. Both sperm donors and patients (who donated unfertilized oocytes) signed a consent to use their gametes for the purposes of this study. Sperm from 10 fertile donors obtained from Shanghai Sperm Bank were examined (supplemental Table 1). All the men had normal semen analysis parameters according to the current World Health Organization (WHO) standards. The motile sperm were separated by gradient centrifugation into two layers (upper layer 45% and lower layer: 90%) of SpermFilter (Cryo-International, Aarhus, Denmark), The SpermFilter was diluted with Queen’s Advantage Medium HEPES supplemented with 0.5% human serum albumin. VOL. 99 NO. 3 / MARCH 1, 2013
Immature human oocytes were obtained from ICSI patients treated at the ART clinic of the Jinghua Hospital, Shenyang Dongfang Medical Group. All oocytes were stored in 1M ammonium sulfate at 4 C. Before using the oocyte for sperm-ZP binding experiments, the salt-stored oocytes were washed with the HEPES medium to remove the salt as described previously by Liu and Baker (19).
Experiment Design The ZP-binding sperm test was performed according to Liu et al. (10) with some minor modifications. Immature oocytes (three oocytes/each sperm microdroplet) were incubated with motile sperm (1.5 million in 50 mL culture medium under oil) for 2.5 hours at 37 C in ambient air with 5% CO2. After incubation, the oocytes were washed to dislodge the unbound sperm (control group) and record the tightly ZPbound sperm. The ZP-bound sperm were then removed and collected (10). Duplicate sperm smears were made from both ZP-bound and unbound sperm for each sample. One of the slides was used for assessment of the acrosome reaction and another slide was used for RMS scanning as described in detail below. The acrosome reaction was assessed with the use of Pisum sativum agglutinin labeled with fluorescein isothiocyanate (PSA-FITC; Sigma Aldrich). After drying the sperm smear in air, it was fixed with 100% ethanol for 1 hour, and then stained with 30 mg/mL PSA-FITC for 2 hours at 37 C. Slides 685
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FIGURE 2
The grade of intensity shifts at (A) the acrosome region and (B) the equatorial region when treated by ZP-binding assay. The red arrow indicates the sperm treated by ZP binding, and the blue arrow indicates the sperm without ZP-binding treatment. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
were washed with a phosphate-buffered saline solution and 200 or more sperm were scored under a Nikon fluorescent microscope (Supplemental Fig. 1, available online at www.fertstert.org). Liu and Baker (9) reported that the process of removing ZP-bound sperm from the surface of oocytes had no effect of sperm motility, morphology, or DNA damage.
Real-Time Raman Microspectroscopy Both ZP-bound and unbound sperm were scanned with the use of the Senterra R200-L dispersive Raman microscope (Bruker Optik) with a 532-nm laser source, and accumulation times were 2 5 s for a single spectrum. For each sample, 100 ZPbound and 100 unbound sperm were scanned. In total, 16 scanning points were selected for each individual sperm according to the anatomic structure, including the acrosome, nucleus, equatorial region, neck, middle piece, and tail, as illustrated in Supplemental Figure 2 (available online at www.fertstert. org). For the acrosome, equatorial, and nucleus regions, three linear equidistant marks (every 1 mm distance) were set in the cross-section. The other marks were set in the vertical section for neck (1 mark), middle piece (3 linear equidistant marks), and tail (3 linear equidistant marks). Wave number was calibrated automatically by Opus 6 software, and the peak assignments were based on the attributions of recent research (16–18). 686
Standard principal component analysis (PCA) was performed on the data of the Raman peaks from both the ZP-bound and the unbound sperm. Scores from the first two principal components accounted for 80% of the major peaks and clearly separated ZP-bound from unbound sperm.
Statistical Analysis All of the RMS data of the marks between ZP-bound and unbound sperm were analyzed by a Student paired t test with statistical significance defined as a P value of %.05.
RESULTS Results of semen analysis, sperm-ZP binding, and acrosome status of motile sperm isolated by gradient centrifugation and sperm bound to the ZP are presented in Supplemental Table 1 (available online at www.fertstert.org). As expected, all donor sperm had >100 sperm bound/ZP, and the acrosome reaction of the ZP-bound sperm (ZP-induced acrosome reaction [ZPIAR]) was an average of 26% (range 7.8%–43%) in ten fertile men (Supplemental Table 1; Supplemental Fig. 1). By Raman scanning, the shape of spectral peaks varied between different regions of sperm after ignoring the background noise. Three grades of intensity peaks were sorted VOL. 99 NO. 3 / MARCH 1, 2013
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FIGURE 3
Statistical analysis (P<.01; unpaired two-tailed Student t test) results of the two groups (ZP binding in red and ZP nonbinding in blue) showed that the mean spectral intensity of the 13 typical Raman peaks were differentiated at the acrosome, nucleus, and equatorial regions, at which the spectral lengths of (A) 789 cm1, (B) 810 cm1, (C) 838 cm1, (D) 1,092 cm1, (E) 1,100 cm1, (F) 1,357 cm1, (G) 1,421 cm1, (H) 1,441 cm1, (I) 1,482 cm1, (J) 1,505 cm1, (K) 1,575 cm1, (L) 1,582 cm1, and (M) 3,240 cm1, as picked up by Opus 6.0 software. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
into low intensity (50–100 a.u.), medium intensity (100–200 a.u.), and high intensity (>200 a.u.). According to this definition, four regions were identified with low intensity, one with medium intensity, and two with high intensity (Supplemental Table 2, available online at www.fertstert.org; Fig. 1). Furthermore, the grade of intensity shifts at the sperm acrosome in ZP-bound sperm. Two obviously low-intensity regions had wavelengths at 800–900 cm1 and 3,200–4,000 cm1, then shifted to a high intensity grade (Fig. 2A). Although at the equator of a sperm, only one low-intensity region (the wavelength of 3,200–4,000 cm1) shifted to high intensity (Fig. 2B). Except for the intensity that varied between different treatments with sperm, another new single peak (1,000 cm1) was detected at both the acrosome and equatorial regions by RMS scanning. Statistical analysis results showed that the mean spectral intensity was differentiated at 13 typical Raman peaks (Fig. 3) VOL. 99 NO. 3 / MARCH 1, 2013
when the ZP-bound sperm was compared with unbound sperm. The increased intensity of ZP-bound sperm peaks were 789 cm1, 810 cm1, 838 cm1, 1,092 cm1, 1,100 cm1, 1,357 cm1, and 1,421 cm1, whereas the descending intensity of ZP-bound sperm peaks were 1,441 cm1, 1,482 cm1, 1,505 cm1, 1,575 cm1, 1,582 cm1, and 3,240 cm1, as detected by Opus 6.0 software. PCA demonstrated spectral differentiation between the ZP-bound and unbound sperm. Above all, our data showed that the spectra from the ZP-bound sperm were clearly distinguished from those from unbound sperm (Fig. 4). Thus the RMS can distinguish whether sperm are bound or unbound to the human ZP.
DISCUSSION In clinical ICSI, selection of a fertile and healthy sperm is critical, not only to enhance the outcome of an ICSI treatment, 687
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FIGURE 4
Principal component (PC) analysis shows the different spectral data between ZP binding (red) and ZP nonbinding (blue) and distinguished by these methods in three-dimensional coordinates. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
but also to obtain a healthy child. However, there is a lack of effective and noninvasive methods to identify and select a normal fertile sperm for ICSI. The ZP-based selection and the ZPIAR were validated as useful functional tests to predict the outcome of fertilization in vitro (11, 12). Earlier studies have shown that using ZP-bound sperm for ICSI resulted in higher embryo quality, implantation rate, and likely subsequently higher clinical pregnancy rate than conventional ICSI (10). However, the use of human ZP for selection of sperm for ICSI is not practical owing to limited ZP materials. In the present study, our data shows that the RMS technique can distinguish ZP-bound sperm from unbound sperm. Therefore, it is possible to further develop the RMS technique into a simple routine procedure for the selection of the most fertile sperm for ICSI. The acrosome reaction is a prelude for fertilization in mammalian sperm (22). Liu et al. (8) reported that an average of 48% (range 20%–98%) of the ZP-bound sperm in fertile men had undergone an acrosome reaction when they used fresh human ZP. In the present study, we used salt-stored human ZP. The ZPIAR was at an average of 26% (range 7.8%– 43%) in the ten fertile sperm donors (Supplemental Table 1), which was much lower than reported by Liu et al. It is possible that our low ZPIAR may be due to our use of frozen donor sperm, salt-stored ZP, and/or different culture medium. It has been reported that the Raman spectra may be able to detect the ‘‘chemical fingerprints’’ for different regions of the sperm, i.e., the acrosomal cap and the tail (16–18). Most recently, Mallidis et al. (18) found the DNA PO4 backbone peak (1,042 cm1) shifts in this region, which was indicative of DNA damage in sperm. In the present study, the grade of intensity shifts at the acrosome region for ZPbound sperm compared with unbound sperm. A similar difference was also found in the equatorial region. Physiologically, the ZPIAR causes acrosomal exocytosis and releases hyaluronidase and acrosin. Two slightly intense regions (800– 900 cm1 and 3,200–4,000 cm1) were found to be shifted to a corresponding high-intensity grade at the acrosome region. DNA backbones (838 cm1), RNA backbones (810–820 688
cm1), and water bands (8,240 cm1) are found in response to this region (16–18,20,21). The intensity of the nucleic acid backbone increase is due to the preacrosome’s broken membrane, then the nucleic-acid exposed to only the sperm membrane instead of two membranes, including preacrosome and cell membranes. The acrosome reaction also is proven to trigger the opening of ion channels (i.e., Ca2þ, Na2þ) and these positive ions influx with H2O (23–29). These identified chemical bands could also be candidate molecules to uncover the mechanism underlining ZP-bound sperm selection in future. Although the applications for RMS are growing in medicine and biology, the knowledge of the Raman spectral chemical database is modest and limited (30–33). For example, the RMS of the single cell or single sperm contains tens of thousands chemical bands, but only a few hundred are known from the currently available literature. Furthermore, the safety of RMS should be confirmed regarding what type of laser and time course are safer for scanning live sperm. In conclusion, RMS can distinguish ZP-bound sperm from unbound sperm. RMS should be further developed into a diagnostic tool to identify normal functional sperm, allowing for their routine selection for ICSI. In addition, the chemical fingerprints obtained by RMS may be able to explore the biochemical and molecular mechanisms of human sperm function. Acknowledgments: The authors thank the staff of the ART clinics of Jinghua Hospital, Shenyang Dongfang Medical Group, for collecting and supplying immature human oocytes. The authors thank and greatly appreciate Dr. De Yi Liu (Melbourne IVF and University of Melbourne in Australia) and Dr. Philip S. Li (Center for Male Reproductive Medicine and Microsurgery, Cornell Institute for Reproductive Medicine, and Department of Urology, Weill Medical College of Cornell University) for their comments and assistance in preparation of the manuscript.
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SUPPLEMENTAL FIGURE 1
The acrosome reaction was assessed by Pisum sativum agglutinin labeled with fluorescein isothiocyanate: (A) all sperm with intact acrosome in motile sperm isolated by gradient centrifugation; (B) the acrosome-reacted sperm bound to the ZP showing a single fluorescent band at the equatorial segment region. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
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SUPPLEMENTAL FIGURE 2
(A) The marks of Raman scanning corresponding to sperm structure indicated by red Xs. (B) Schematic map of human sperm structure, including acrosome, nucleus, equatorial, neck, midpiece, and tail. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
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SUPPLEMENTAL TABLE 1 Semen analysis results, including volume, concentration, progressive motility (PR), normal sperm morphology, intact acrosome (IA), number of sperm bound/zona pellucida (NSB/ZP), and ZP-induced acrosome reaction (ZPIAR) in ten fertile men. Donor
Volume (mL)
Concentration (106)
PR (%)
Normal morphology (%)
IA (%)
NSP/ZP
ZPIAR (%)
1 2 3 4 5 6 7 8 9 10 Mean
1.5 4 2.5 3 2.5 3.5 4 2.5 1.5 2 2.7
200 182 110 90 120 95 105 150 120 166 133.8
72 70 70 68 75 80 85 78 75 69 74.2
35 30 35 32 38 34 32 30 35 31 33.2
67 95 89 93 96 95 52 66 98 97 85
110 120 104 101 105 112 113 105 110 127 110.7
15 7.8 40.3 25 38.5 10.5 14.3 43.8 32 33.3 26.05
Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
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SUPPLEMENTAL TABLE 2 Four low-intensity regions (50–100 a.u.), one medium-intensity region (100–200 a.u.), and two high-intensity regions (>200 a.u.) were identified in sperm spectral map based on the Raman spectral intensity. Grade type Low intensity
Wavelength range (cmL1) 400–480 810–900
Medium intensity High intensity
2,300–2,400 3,200–3,500 520–540 1,000–1,100 1,375–1,510
Chemical bands Skeletal modes of carbohydrates (starch and glucose) Nucleic acid backbones Unknown Water S-S str Phosphate, CC skeletal, and COC string from glycosidic link Adenine, thymine, guanine, cytosine
Reference Schuster et al. (2000) (29) Deng et al. (1999) (30); Schuster et al. (2000) (29); Maquelin et al. (2002) (20); Uzunbajakava et al. (2003) (31, 32) Harz et al. (2009) (33) Maquelin et al. (2002) (20) Maquelin et al. (2002) (20) Uzunbajakava et al. (2003) (31, 32)
Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
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Real-time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucida Feng Liu, B.Sc.,a Yong Zhu, Ph.D.,a Yufei Liu,a Xiaobo Wang,a Ping Ping, M.D., Ph.D.,a Xinyuan Zhu, Ph.D.,b Hongliang Hu, Ph.D.,a Zheng Li, M.D., Ph.D.,a and Lin He, Ph.D.b a
Department of Urology, Renji Hospital, Shanghai Human Sperm Bank, Sperm Development and Genetics Laboratory, Shanghai Institute of Andrology, Shanghai Jiao Tong University School of Medicine; and b Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai, People’s Republic of China
Objective: To determine if Raman microspectroscopy (RMS) can distinguish sperm bound to the human zona pellucida (ZP) from those unbound sperm. Design: Paired experiments to compare Raman scanning features of ZP-bound and unbound sperm. Setting: Public hospital–based clinical assisted reproduction center. Patient(s): Sperm samples from ten fertile donors were used in this study. Intervention(s): None. Main Outcome Measure(s): Sperm-ZP binding, ZP-induced acrosome reaction, and scanning intensity of various regions of sperm. Result(s): The RMS found two slightly low-intensity regions (800–900 and 3,200–4,000 cm1) shifted to high-intensity grade at the acrosome region of the ZP-bound sperm compared with unbound sperm. Moreover, principal component analysis and statistical analysis showed that the RMS can distinguish the ZP-bound sperm from the unbound sperm. Conclusion(s): RMS scanning of single live sperm could be used to distinguish ZP-bound sperm from unbound sperm. Thus, RMS may be a useful tool to detect normal functional sperm and to select sperm for intracytoplasmic sperm injection. (Fertil SterilÒ 2013;99:684–9. Ó2013 by American Society for Reproductive Use your smartphone Medicine.) to scan this QR code Key Words: Raman microspectroscopy, scanning of single live sperm, human zona pellucida– and connect to the bound sperm Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/liuf-raman-micro-spectroscopy-sperm-human-zona-pellucida/
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n assisted reproductive technology (ART) clinics, >50% of infertile couple are routinely treated by intracytoplasmic sperm injection (ICSI) (1). Although ICSI can significantly improve the fertilization rate, a low number of high-quality embryos and low
implantation rates are still major concerns for some couples (2, 3). It is still debatable how to select the best sperm which not only fertilize the oocyte but also ensure the safety of the offspring (4–6). During the fertilization process, either in vivo or under conventional
Received July 3, 2012; revised October 18, 2012; accepted October 21, 2012; published online November 10, 2012. F.L. has nothing to disclose. Y.Z. has nothing to disclose. Y. L. has nothing to disclose. X.W. has nothing to disclose. P.P. has nothing to disclose. X.Z. has nothing to disclose. H.H. has nothing to disclose. Z.L. has nothing to disclose. L.H. has nothing to disclose. F.L. and Y.Z. contributed equally to this work. Supported by the Science and Technology Commission of Shanghai Municipality (grant no. 10JC1409900), National Basic Research Program of China (grant no. 2011CB944504), and the Shanghai Municipal Commission of Population and Family Planning Commission Science and Technology Developing Foundation (grant no. 2012JG07). Reprint requests: Zheng Li, M.D., Ph.D., Department of Urology, Shanghai Institute of Andrology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200001, People’s Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 3, March 1, 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.10.035 684
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in vitro fertilization, a fertile sperm has been biologically selected through sperm-oocyte interaction, in particular a thick layer of zona pellucida (ZP). For a fertile man, the average ejaculation contains 100 106–200 106 motile sperm, but only 14% of motile sperm are capable of binding to the ZP (7, 8). Most sperm bound to the ZP have normal morphology and chromatin DNA (9). Therefore the ZP-bound sperm are likely the favored or fertile population of sperm in any given ejaculate. Therefore, using ZP-bound sperm for ICSI would in theory enhance the outcomes. There are a few reports demonstrating that the use of ZP-bound sperm for ICSI significantly improves the embryo quality, implantation rate, and clinical pregnancy rate compared with conventional ICSI (10). Therefore, it VOL. 99 NO. 3 / MARCH 1, 2013
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FIGURE 1
Mapping of Raman spectral peaks to different regions of sperm, including (A) acrosome, (B) nucleus, (C) equatorial, (D) neck, (E) midpiece, and (F) tail. (G) The combined spectral map. The x-axis presents the continuous wave lengths (cm1) of the spectra, and the y-axis presents the intensity of the spectra, including three grades of intensity peaks: low intensity (50–100 a.u.), medium intensity (100–200 a.u.), and high intensity (>200 a.u.). Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
would advantageous to develop a new approach of sperm selection based on the characteristic of ZP binding (10–12). Recently Raman microspectroscopy (RMS), a laser-based noninvasive technique, was shown to provide a detailed chemical ‘‘fingerprint’’ of cells in various medical fields, such as in the classification of different malignancies (13–15). It has been shown that RMS can detect sperm DNA packaging or DNA structure damage and their mitochondrial state (16–18). In the present study, the RMS scanning technique was used to distinguish ZP-bound sperm from unbound sperm.
MATERIALS AND METHODS Sperm and Oocytes This study was approved by the Renji Hospital Research and Ethics Committee. Both sperm donors and patients (who donated unfertilized oocytes) signed a consent to use their gametes for the purposes of this study. Sperm from 10 fertile donors obtained from Shanghai Sperm Bank were examined (supplemental Table 1). All the men had normal semen analysis parameters according to the current World Health Organization (WHO) standards. The motile sperm were separated by gradient centrifugation into two layers (upper layer 45% and lower layer: 90%) of SpermFilter (Cryo-International, Aarhus, Denmark), The SpermFilter was diluted with Queen’s Advantage Medium HEPES supplemented with 0.5% human serum albumin. VOL. 99 NO. 3 / MARCH 1, 2013
Immature human oocytes were obtained from ICSI patients treated at the ART clinic of the Jinghua Hospital, Shenyang Dongfang Medical Group. All oocytes were stored in 1M ammonium sulfate at 4 C. Before using the oocyte for sperm-ZP binding experiments, the salt-stored oocytes were washed with the HEPES medium to remove the salt as described previously by Liu and Baker (19).
Experiment Design The ZP-binding sperm test was performed according to Liu et al. (10) with some minor modifications. Immature oocytes (three oocytes/each sperm microdroplet) were incubated with motile sperm (1.5 million in 50 mL culture medium under oil) for 2.5 hours at 37 C in ambient air with 5% CO2. After incubation, the oocytes were washed to dislodge the unbound sperm (control group) and record the tightly ZPbound sperm. The ZP-bound sperm were then removed and collected (10). Duplicate sperm smears were made from both ZP-bound and unbound sperm for each sample. One of the slides was used for assessment of the acrosome reaction and another slide was used for RMS scanning as described in detail below. The acrosome reaction was assessed with the use of Pisum sativum agglutinin labeled with fluorescein isothiocyanate (PSA-FITC; Sigma Aldrich). After drying the sperm smear in air, it was fixed with 100% ethanol for 1 hour, and then stained with 30 mg/mL PSA-FITC for 2 hours at 37 C. Slides 685
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FIGURE 2
The grade of intensity shifts at (A) the acrosome region and (B) the equatorial region when treated by ZP-binding assay. The red arrow indicates the sperm treated by ZP binding, and the blue arrow indicates the sperm without ZP-binding treatment. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
were washed with a phosphate-buffered saline solution and 200 or more sperm were scored under a Nikon fluorescent microscope (Supplemental Fig. 1, available online at www.fertstert.org). Liu and Baker (9) reported that the process of removing ZP-bound sperm from the surface of oocytes had no effect of sperm motility, morphology, or DNA damage.
Real-Time Raman Microspectroscopy Both ZP-bound and unbound sperm were scanned with the use of the Senterra R200-L dispersive Raman microscope (Bruker Optik) with a 532-nm laser source, and accumulation times were 2 5 s for a single spectrum. For each sample, 100 ZPbound and 100 unbound sperm were scanned. In total, 16 scanning points were selected for each individual sperm according to the anatomic structure, including the acrosome, nucleus, equatorial region, neck, middle piece, and tail, as illustrated in Supplemental Figure 2 (available online at www.fertstert. org). For the acrosome, equatorial, and nucleus regions, three linear equidistant marks (every 1 mm distance) were set in the cross-section. The other marks were set in the vertical section for neck (1 mark), middle piece (3 linear equidistant marks), and tail (3 linear equidistant marks). Wave number was calibrated automatically by Opus 6 software, and the peak assignments were based on the attributions of recent research (16–18). 686
Standard principal component analysis (PCA) was performed on the data of the Raman peaks from both the ZP-bound and the unbound sperm. Scores from the first two principal components accounted for 80% of the major peaks and clearly separated ZP-bound from unbound sperm.
Statistical Analysis All of the RMS data of the marks between ZP-bound and unbound sperm were analyzed by a Student paired t test with statistical significance defined as a P value of %.05.
RESULTS Results of semen analysis, sperm-ZP binding, and acrosome status of motile sperm isolated by gradient centrifugation and sperm bound to the ZP are presented in Supplemental Table 1 (available online at www.fertstert.org). As expected, all donor sperm had >100 sperm bound/ZP, and the acrosome reaction of the ZP-bound sperm (ZP-induced acrosome reaction [ZPIAR]) was an average of 26% (range 7.8%–43%) in ten fertile men (Supplemental Table 1; Supplemental Fig. 1). By Raman scanning, the shape of spectral peaks varied between different regions of sperm after ignoring the background noise. Three grades of intensity peaks were sorted VOL. 99 NO. 3 / MARCH 1, 2013
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FIGURE 3
Statistical analysis (P<.01; unpaired two-tailed Student t test) results of the two groups (ZP binding in red and ZP nonbinding in blue) showed that the mean spectral intensity of the 13 typical Raman peaks were differentiated at the acrosome, nucleus, and equatorial regions, at which the spectral lengths of (A) 789 cm1, (B) 810 cm1, (C) 838 cm1, (D) 1,092 cm1, (E) 1,100 cm1, (F) 1,357 cm1, (G) 1,421 cm1, (H) 1,441 cm1, (I) 1,482 cm1, (J) 1,505 cm1, (K) 1,575 cm1, (L) 1,582 cm1, and (M) 3,240 cm1, as picked up by Opus 6.0 software. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
into low intensity (50–100 a.u.), medium intensity (100–200 a.u.), and high intensity (>200 a.u.). According to this definition, four regions were identified with low intensity, one with medium intensity, and two with high intensity (Supplemental Table 2, available online at www.fertstert.org; Fig. 1). Furthermore, the grade of intensity shifts at the sperm acrosome in ZP-bound sperm. Two obviously low-intensity regions had wavelengths at 800–900 cm1 and 3,200–4,000 cm1, then shifted to a high intensity grade (Fig. 2A). Although at the equator of a sperm, only one low-intensity region (the wavelength of 3,200–4,000 cm1) shifted to high intensity (Fig. 2B). Except for the intensity that varied between different treatments with sperm, another new single peak (1,000 cm1) was detected at both the acrosome and equatorial regions by RMS scanning. Statistical analysis results showed that the mean spectral intensity was differentiated at 13 typical Raman peaks (Fig. 3) VOL. 99 NO. 3 / MARCH 1, 2013
when the ZP-bound sperm was compared with unbound sperm. The increased intensity of ZP-bound sperm peaks were 789 cm1, 810 cm1, 838 cm1, 1,092 cm1, 1,100 cm1, 1,357 cm1, and 1,421 cm1, whereas the descending intensity of ZP-bound sperm peaks were 1,441 cm1, 1,482 cm1, 1,505 cm1, 1,575 cm1, 1,582 cm1, and 3,240 cm1, as detected by Opus 6.0 software. PCA demonstrated spectral differentiation between the ZP-bound and unbound sperm. Above all, our data showed that the spectra from the ZP-bound sperm were clearly distinguished from those from unbound sperm (Fig. 4). Thus the RMS can distinguish whether sperm are bound or unbound to the human ZP.
DISCUSSION In clinical ICSI, selection of a fertile and healthy sperm is critical, not only to enhance the outcome of an ICSI treatment, 687
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FIGURE 4
Principal component (PC) analysis shows the different spectral data between ZP binding (red) and ZP nonbinding (blue) and distinguished by these methods in three-dimensional coordinates. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
but also to obtain a healthy child. However, there is a lack of effective and noninvasive methods to identify and select a normal fertile sperm for ICSI. The ZP-based selection and the ZPIAR were validated as useful functional tests to predict the outcome of fertilization in vitro (11, 12). Earlier studies have shown that using ZP-bound sperm for ICSI resulted in higher embryo quality, implantation rate, and likely subsequently higher clinical pregnancy rate than conventional ICSI (10). However, the use of human ZP for selection of sperm for ICSI is not practical owing to limited ZP materials. In the present study, our data shows that the RMS technique can distinguish ZP-bound sperm from unbound sperm. Therefore, it is possible to further develop the RMS technique into a simple routine procedure for the selection of the most fertile sperm for ICSI. The acrosome reaction is a prelude for fertilization in mammalian sperm (22). Liu et al. (8) reported that an average of 48% (range 20%–98%) of the ZP-bound sperm in fertile men had undergone an acrosome reaction when they used fresh human ZP. In the present study, we used salt-stored human ZP. The ZPIAR was at an average of 26% (range 7.8%– 43%) in the ten fertile sperm donors (Supplemental Table 1), which was much lower than reported by Liu et al. It is possible that our low ZPIAR may be due to our use of frozen donor sperm, salt-stored ZP, and/or different culture medium. It has been reported that the Raman spectra may be able to detect the ‘‘chemical fingerprints’’ for different regions of the sperm, i.e., the acrosomal cap and the tail (16–18). Most recently, Mallidis et al. (18) found the DNA PO4 backbone peak (1,042 cm1) shifts in this region, which was indicative of DNA damage in sperm. In the present study, the grade of intensity shifts at the acrosome region for ZPbound sperm compared with unbound sperm. A similar difference was also found in the equatorial region. Physiologically, the ZPIAR causes acrosomal exocytosis and releases hyaluronidase and acrosin. Two slightly intense regions (800– 900 cm1 and 3,200–4,000 cm1) were found to be shifted to a corresponding high-intensity grade at the acrosome region. DNA backbones (838 cm1), RNA backbones (810–820 688
cm1), and water bands (8,240 cm1) are found in response to this region (16–18,20,21). The intensity of the nucleic acid backbone increase is due to the preacrosome’s broken membrane, then the nucleic-acid exposed to only the sperm membrane instead of two membranes, including preacrosome and cell membranes. The acrosome reaction also is proven to trigger the opening of ion channels (i.e., Ca2þ, Na2þ) and these positive ions influx with H2O (23–29). These identified chemical bands could also be candidate molecules to uncover the mechanism underlining ZP-bound sperm selection in future. Although the applications for RMS are growing in medicine and biology, the knowledge of the Raman spectral chemical database is modest and limited (30–33). For example, the RMS of the single cell or single sperm contains tens of thousands chemical bands, but only a few hundred are known from the currently available literature. Furthermore, the safety of RMS should be confirmed regarding what type of laser and time course are safer for scanning live sperm. In conclusion, RMS can distinguish ZP-bound sperm from unbound sperm. RMS should be further developed into a diagnostic tool to identify normal functional sperm, allowing for their routine selection for ICSI. In addition, the chemical fingerprints obtained by RMS may be able to explore the biochemical and molecular mechanisms of human sperm function. Acknowledgments: The authors thank the staff of the ART clinics of Jinghua Hospital, Shenyang Dongfang Medical Group, for collecting and supplying immature human oocytes. The authors thank and greatly appreciate Dr. De Yi Liu (Melbourne IVF and University of Melbourne in Australia) and Dr. Philip S. Li (Center for Male Reproductive Medicine and Microsurgery, Cornell Institute for Reproductive Medicine, and Department of Urology, Weill Medical College of Cornell University) for their comments and assistance in preparation of the manuscript.
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SUPPLEMENTAL FIGURE 1
The acrosome reaction was assessed by Pisum sativum agglutinin labeled with fluorescein isothiocyanate: (A) all sperm with intact acrosome in motile sperm isolated by gradient centrifugation; (B) the acrosome-reacted sperm bound to the ZP showing a single fluorescent band at the equatorial segment region. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
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SUPPLEMENTAL FIGURE 2
(A) The marks of Raman scanning corresponding to sperm structure indicated by red Xs. (B) Schematic map of human sperm structure, including acrosome, nucleus, equatorial, neck, midpiece, and tail. Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
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SUPPLEMENTAL TABLE 1 Semen analysis results, including volume, concentration, progressive motility (PR), normal sperm morphology, intact acrosome (IA), number of sperm bound/zona pellucida (NSB/ZP), and ZP-induced acrosome reaction (ZPIAR) in ten fertile men. Donor
Volume (mL)
Concentration (106)
PR (%)
Normal morphology (%)
IA (%)
NSP/ZP
ZPIAR (%)
1 2 3 4 5 6 7 8 9 10 Mean
1.5 4 2.5 3 2.5 3.5 4 2.5 1.5 2 2.7
200 182 110 90 120 95 105 150 120 166 133.8
72 70 70 68 75 80 85 78 75 69 74.2
35 30 35 32 38 34 32 30 35 31 33.2
67 95 89 93 96 95 52 66 98 97 85
110 120 104 101 105 112 113 105 110 127 110.7
15 7.8 40.3 25 38.5 10.5 14.3 43.8 32 33.3 26.05
Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
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SUPPLEMENTAL TABLE 2 Four low-intensity regions (50–100 a.u.), one medium-intensity region (100–200 a.u.), and two high-intensity regions (>200 a.u.) were identified in sperm spectral map based on the Raman spectral intensity. Grade type Low intensity
Wavelength range (cmL1) 400–480 810–900
Medium intensity High intensity
2,300–2,400 3,200–3,500 520–540 1,000–1,100 1,375–1,510
Chemical bands Skeletal modes of carbohydrates (starch and glucose) Nucleic acid backbones Unknown Water S-S str Phosphate, CC skeletal, and COC string from glycosidic link Adenine, thymine, guanine, cytosine
Reference Schuster et al. (2000) (29) Deng et al. (1999) (30); Schuster et al. (2000) (29); Maquelin et al. (2002) (20); Uzunbajakava et al. (2003) (31, 32) Harz et al. (2009) (33) Maquelin et al. (2002) (20) Maquelin et al. (2002) (20) Uzunbajakava et al. (2003) (31, 32)
Liu. Raman scanning of ZP-bound and unbound sperm. Fertil Steril 2013.
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