Stained-free interferometric phase microscopy correlation with DNA 1

Transcription

1 Stained-free interferometric phase microscopy correlation with DNA 1 fragmentation stain in human spermatozoa 2 3 Itay Barnea 1, Lidor Karako 1, Simcha K. Mirsky 1, Mattan Levi 1, Michal Balberg 1,2 4 Natan T. Shaked 1,* 5 6 Affiliations: 7 1- Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel Faculty of Electrical Engineering, Holon Institute of technology, Holon, Israel * Corresponding author: Prof. Natan T. Shaked, PhD. Department of Biomedical 11 Engineering, Tel Aviv University, Tel Aviv, Israel. nshaked@tau.ac.il

2 Abstract 18 Acridine orange (AO) staining is one of the methods used to diagnose the DNA 19 fragmentation status in sperm cells. Interferometric phase microscopy (IPM) is a an 20 optical imaging method based on digital holographic microscopy that provides 21 quantitative morphological and refractive index imaging of cells in vitro without the 22 need for staining. We have imaged sperm cells using stain-free IPM in order to 23 estimate different cellular parameters, such as acrosome dry mass and size, in addition 24 to an embryologist evaluation according to the WHO-2010 criteria. Following this, 25 the same sperm cells were stained by AO, imaged using a fluorescence confocal 26 microscope and assessed by the AO-emitted color, forming five DNA fragmentation 27 groups. These DNA fragmentation groups were correlated with the embryologist- 28 based classification and the IPM-based morphological parameters. Our results 29 indicate on significant differences in IPM-based parameters between groups with 30 different fragmentation levels. Specifically, the size of the acrosome, as measured 31 from stain-free IPM, is a good predictor for the presence of intact DNA. Based on the 32 validation with AO, we conclude that stain-free IPM images analyzed digitally may 33 assist in selecting sperm cells with intact DNA prior to intracytoplasmic sperm 34 injection (ICSI). This information may potentially increase percentage of successful 35 pregnancies Keywords: Digital holographic microscopy; Quantitative phase imaging; Label-free 38 imaging; Sperm analysis; Fertility. 39 2

3 1. Introduction 40 DNA fragmentation in sperm, has long been associated with an impaired ability for 41 spontaneous pregnancy [1], as well as with difficulties in achieving live births after 42 artificial reproductive technology (ART) procedure. It has been shown that couples 43 undergoing conventional in vitro fertilization (IVF) and intracytoplasmic sperm 44 injection (ICSI) where the sperm donor is diagnosed with high percentage of DNA 45 fragmented sperms experience low pregnancy rates, abnormal embryo development 46 and increase miscarriages [2,3]. A meta-analysis summarizing results of over treatment cycles in 41 independent reports has shown that men with higher sperm 48 DNA fragmentation, exhibit a fold increase in the likelihood for failure to 49 achieve clinical pregnancy [4]. DNA fragmentation is not only an important factor in 50 the failure of ICSI cycles involving men with fertility problems, but also those 51 involving men with normal sperm. Reports have shown that in normal semen samples 52 between 20% 30% of sperm cells have fragmented DNA [5]. Moreover, as fertility 53 treatments are becoming more frequent in men in their forties and fifties, the quality 54 of sperm decreases compared to the sperm of younger men, as it has been shown that 55 DNA fragmentation increases with age [6]. 56 There are several methods for the evaluation of DNA fragmentation. These include: 57 the transferase dutp nick end labeling (TUNEL) test [7], which evaluates DNA 58 fragmentation by staining the terminal end of nucleic acids; the Comet test [8], which 59 evaluates DNA strand breaks by electrophoresis of the cell content; and the sperm 60 chromatin dispersion assay (SCD), which evaluate DNA fragmentation by the 61 presence or absence of halo around the cell head [9]. In the present work, we choose 62 to use the acridine orange (AO) method [10], which differentiates between double 63 stranded (dsdna) and single-stranded DNA (ssdna) or RNA. In this method, 64 3

4 dsdna emits green fluorescence and single-stranded DNA or RNA emits red 65 fluorescence, and fluorescent microscopy or flow cytometry is used to evaluate the 66 different colors. 67 The fact that most of the methods for the evaluation of DNA fragmentation listed 68 above require killing of the cells renders them irrelevant for the assessment of the 69 DNA integrity of the individual live sperm cells used in ICSI [11]. Thus, today, the 70 selection of sperm cells for ICSI is performed using the limited capabilities of 71 relatively low-magnification bright-field microscopy (BFM), usually with total magnification. This tool enables the embryologist to select motile sperm on the 73 basis of basic morphological characteristics, such as the sperm head size [12]. 74 Another recent method for sperm selection is intracytoplasmic morphologically 75 selected sperm injection (IMSI). In this method the sperm is imaged using mostly 76 digital magnifications of 6300 or higher, where the cells are imaged without staining 77 using differential interference contrast (DIC) [13]. Due to the higher magnification 78 and enhanced contrast of the IMSI systems, the embryologist can observe 79 morphological characteristics such as cytoplasmic vacuoles. Evidence suggests that 80 these vacuoles are associated with defected chromatin packaging and DNA 81 fragmentations [14,15]. 82 The benefits of IMSI in comparison to ICSI are controversial: one meta-analysis has 83 shown that the use of IMSI significantly improves the percentage of high-quality 84 embryos, implantation and pregnancy rates, and significantly reduces miscarriage 85 rates in comparison to ICSI [16]. In contrast, the critics note that there are not enough 86 randomized trials to confirm the benefits of IMSI, thus this method should be 87 4

5 employed only after the failure of several ICSI cycles and confirmation of male-factor 88 infertility [17]. 89 Interferometric phase microscopy (IPM) is a stain-free imaging technique also known 90 as digital holographic microscopy (DHM), or quantitative phase microscopy (QPM), 91 which can capture both the amplitude and phase profiles of the light that passes 92 through transparent biological cells. IPM provides a quantitative measurement of the 93 optical path delay (OPD) at each point in the sample. OPD is defined as the difference 94 between the refractive index of the sample and that of the surrounding medium 95 multiplied by the sample thickness [18-20] (see Equation 1). Thus, OPD has positive 96 correlation both with the thickness and with the density on the sperm cell. The 97 resulting OPD map is a visualization of a quantified contrast based on the thickness 98 and density at all points of the cell, in contrast to DIC, the basis of IMSI, which 99 records OPD gradient and thus provides contrast only near the cell and organelle 100 edges. Past work done in our group showed agreement between the label-free IPM 101 images and stained cells [11] The particular IPM module used in this work is the τ 102 interferometer, which is a compact external module that can be positioned at the exit 103 of a microscope and thus makes IPM much more accessible to fertility clinics [21]. 104 The quantitative phase measured is defined by φ(x, y) = 2π/λ OPD(x, y), where λ 105 is the illumination wavelength and OPD is defined as follows: 106 OPD(x, y) = [n c (x, y) n m ] h c (x, y), (1) 107 where OPD(x, y) is the OPD at point (x, y), n c (x, y) is the integral refractive index 108 of the sperm cells at point (x, y), n m is the refractive index of the medium and 109 h c (x, y) is the thickness of the cell at point (x, y)

6 111 Our group has demonstrated the agreement between the information obtained by 112 stain-free IPM of fixed unstained sperm cells and the information obtained by BFM of 113 stained sperm cells [11], in connection with the measurement of key morphological 114 parameters using the WHO guidelines. The fact that the IPM and BFM results are in 115 agreement enabled us to assess the OPD maps in accordance to the WHO criteria [22], but without the use of staining. 117 In this work, we compare the information obtained by morphological assessment of 118 stain-free IPM images of sperm cells to categorization of DNA fragmentation by AO 119 staining of the same cells. The ultimate aim is to determine whether stain-free IPM 120 can be used to indicate DNA integrity of the individual sperm cells used in ICSI

7 2. Methods Sample preparation 124 The study was approved by the institutional ethics committee of Tel Aviv University. 125 All sperm donor signed a written Informed consent form. 126 Semen samples were obtained from six childless sperm donors in their twenties. After 127 ejaculation, the semen was liquefied at room temperature for 30 minutes and then 128 spermatozoa were isolated using the PureCeption Bi-layer kit (Origio, Målov, 129 Denmark) in accordance with manufacturer instructions. The upper phases were 130 discarded and the pellet was re-suspended in 5 ml of modified human tubal fluid 131 (HTF) medium (Irvine Scientific, CA, USA) and centrifuged again at 500 g for minutes. Next, the supernatant was discarded and the pelleted sperm cells were 133 resuspended in 0.1 ml HTF. Sperm cells are motile and dynamic. Since the same cells 134 were measured by two microscopy systems (IPM and later, after staining, in BFM), 135 the sperm cells were fixed, which kept their morphology unchanged between the 136 imaging cycles. This is done using drop by drop addition of 10 ml of 3:1 methanol to 137 acetic acid solution. After 5 minutes at room temperature, the cells were centrifuged 138 at 800 g for 5 minutes, the supernatant was discarded, and the pellet was re- 139 suspended in 0.2 ml of fixative solution. 140 We used a 60 mm 20 mm #1 cover slip with 9 laser-engraved tables, each table 141 containing 64 squares of 80 μm 80 μm each (Fig. 1(a)). The slides were rinsed with 142 methanol before use, 10 μl of the fixed sperm cell solution was placed on the 143 engraved grid and kept in a fume hood overnight to ensure the evaporation of the 144 fixative solution. A single slide contained cells from a single donor

8 2.2 IPM imaging and analysis of sperm cells 146 The fixed sperm cells were imaged using 63, 1.4 numerical aperture, oil-immersion 147 objective. The illumination source was a supercontinuum fiber-laser source (SC400-4, 148 Fianium), connected to a computer-controlled acousto-optical tunable filter (SC 149 AOTF, Fianium), tuned to a central wavelength of 633 nm and a full-width-at-half 150 maximum bandwidth of 6.7 nm. We used the IPM system designed by our group and 151 previously described in [11,21]. The camera in the output of the IPM system captures 152 an interferogram that is digitally processed into an OPD map of the sample by digital 153 spatial filtering and phase unwrapping algorithm [21]; a stain-free quantitative image 154 of the sperm representing a cell average refractive index of the cell and thickness at 155 all spatial points Embryologist evaluation 157 The stain-free OPD images of each cell were evaluated by an expert embryologist 158 (M.L.) in accordance with the WHO 2010 guidelines [22]. The embryologist 159 evaluated and individually graded each sperm cell by head shape, acrosome size, 160 cytoplasmic vacuole and cytoplasmic droplet presence, and midpiece form Computer assisted evaluation of sperm cell morphological parameters 162 The isolation and evaluation of sperm cells was performed using a Matlab algorithm 163 that our group had previously designed for this purpose, and was fully presented in 164 Ref. [23]. 165 The output of this procedure was the OPD map of the cell head, nucleus and 166 acrosome, in addition to many parameters that can be calculated based on the OPD, 167 such as dry mass, mean anterior-posterior difference, head, acrosome, and nucleus 168 8

9 areas, and head OPD variance. The OPD variance has a strong connection to the 169 presence or lack of a nucleus, and acrosome area is significant as it is the basis for one 170 of the WHO criteria. Nucleus area is significant as a smaller nucleus may indicate a 171 well-packed nucleus with less DNA fragmentation. The mean anterior-posterior 172 difference was calculated by dividing the isolated head OPD image into two equal 173 halves lengthwise, the anterior and posterior halves of the head. Following this, the 174 mean OPD values of these halves were calculated and the mean posterior OPD was 175 subtracted from the mean anterior OPD in order to get the final mean anterior- 176 posterior difference Dry mass calculation 178 The dry mass of the cell corresponds primarily to the protein concentration [24]. It 179 can be determined from the OPD using the following parameters. In Ref. [25], we 180 determined that the average dry mass of a sperm head was DM=7.51± gram, 181 and the average refractive index of a sperm head was n c = 1.515± This was 182 achieved by comparing the OPD maps with the exact height measurements of the 183 same cells using an atomic force microscope (AFM). Based on Equation 2, we can 184 extract the volume (V) of the cells by: 185 V = Area Thickness = Area OPD n c n m. (2) 186 From the AFM measurements obtained in Ref. [25], we can also obtain the average 187 volume of the cells and calculate the average concentration of dry mass density in 188 sperm cells; = M/V= gram/nm 3 = gram/liter. This value was then 189 used to calculate the dry mass of each cell or its compartment by multiplying the 190 volume calculated by Equation 2 by

10 DM = Area OPD ρ. (3) 192 Acridine orange staining: 193 The fixed sperm cells, previously imaged by IPM, were then stained by AO using the 194 Tejada s method [26]. In short, the slides were exposed to AO (0.19 mg/ml, ph 2.5) 195 for 5 minutes. Staining solution was prepared daily from a stock solution consisting of mg AO (Sigma-Aldrich, Rehovot, Israel) in 1 L of deionized water, and stored in 197 the dark at 4 C. To prepare the staining solution, 10 ml of the stock solution was 198 added to 40 ml of 0.1 M citric acid and 2.5 ml of 0.3 M Na 2 HPO 4 7H 2 O (all from 199 Sigma-Aldrich, Rehovot, Israel). No loss of sperm cells was detected after staining 200 with AO. 201 After staining, the slides were rinsed in a stream of deionized water for 5 minutes, air 202 dried and imaged within two hours by a confocal fluorescence microscope (Zeiss 203 LSM 510-META). Each engraved table of 8 8 squares was imaged using a 25, numerical aperture microscope objective. The cells were excited using light at 205 wavelengths nm, and emission was filtered between nm for the 206 red and nm for green. Each arbitrary fluorescent cell was blindly evaluated 207 by the experimentalist (L.K.) in a color scale of 1 (red) to 5 (green), and the color was 208 incorporated into the database obtained by the algorithm and the embryologist Statistical analysis 210 For each parameter, determined either by the algorithm (e.g. cell area, dry mass) or 211 the embryologist (e.g. head shape), the sperm cells were divided into groups 212 according to their AO colors. For the quantitative parameters, determined by the

11 algorithm, the mean of each of the parameters was calculated and presented in a 214 column chart with error bars representing the standard error. 215 For the embryologist-determined information (binary classification), we calculated 216 the percentage of normal cells, as determined by the embryologist in this particular 217 parameter in each of the AO groups. In this experiment the significance of the 218 differences between the groups was established by the χ 2 test. 219 In computer assisted evaluation of sperm cell morphological parameters, the 220 differences between groups was examined using analysis of variance (ANOVA), 221 complimented by Tukey's multiple comparisons test for comparison between the 222 different groups. The differences between groups were considered statistically 223 significant if the probability, p, for the associated value was smaller than Slope 224 analysis was conducted wherever the changes in a particular parameter were 225 consistent over 4 fragmentation groups. The slope was calculated by linear regression. 226 The significance of the direction of the slope is shown by the 95% confidence 227 interval. All calculations were performed using IBM SPSS STATISTICS The statistical analysis was generated by IBM SPSS STATISTICS The 229 charts were generated by the GraphPad Prism v.7 software

12 3. Results 232 Sperm cells were isolated and fixed on a gridded slide. Figure 1(a) shows a 233 fluorescent image of the fixed sperm cells after staining with AO, superimposed with 234 BFM image. Before the AO staining, the cells, immobilized on the grid, were imaged 235 using IPM (see example in Figs. 1(b) and (c)) and analyzed by the algorithm as well 236 as by the embryologist. 237 Out of 1699 cells and cell fragments that were identified by the computer, 1336 cells 238 were confirmed as sperm cells by an embryologist, and only these cells were used in 239 the statistical analysis. The number of sperm cells from each donor is 139, 98, 106, , 254 and 387 cells. 241 (A) (B) nm (C) nm 15 μm 2 μm (D) (E) 3 μm 15 μm Fig. 1. Imaging of cells by stain-free IPM and evaluation of DNA fragmentation. 243 (A) Human sperm cells were fixed to a gridded slide and stained with AO (BFM and 244 fluorescent images are superimposed), which allowed us to find the same cells on two 245 different microscopy systems. (B) Each square in the grid was captured by IPM and 246 presented to an embryologist for assessment (C). Color bar represents OPD valus. (D) 247 The cells were stained with AO and captures by fluorescent confocal microscope. (E) 248 Fragmentation groups: An experimentalist allocated a color at a scale of 1 (red) to (green), and the color was incorporated into the data obtained by computerized 250 analysis of the IPM image and the embryologist evaluation

13 After the cells were imaged using IPM, the same slides were stained by AO and 252 imaged using the confocal fluorescence microscope (see example in Fig. 1(e)). We 253 then determined the color of each cell, with the color scale being from 1 5, with red 254 being 1 and green being 5 (see scale in Fig. 1(f)). Because the overall color of the cell 255 is determined by the sum of the green and red colors, we can regard the color of the 256 cells as a scale that reflects the proportion between dsdna to ssdna (single stranded 257 or fragmented DNA) and RNA, as well the overall amount of nucleic acids. The 258 number (and the percentage) of cells in each color group, from the most fragmented 259 to the least fragmented was group 1: 84 cell (6.3%), group 2: 152 cells (11.4%), 260 group 3: 349 cells (26.1%), group 4: 624 cells (46.7%) and 5 group: 127 cells (9.5%). 261 Figure 2 shows examples of the same sperm cells, while imaged with label-free 262 qualitative Zernike s phase contrast microscopy (left), label-free quantitative IPM 263 (center), and BFM after AO staining (right), where the colors in the latter indicate the 264 level of fragmentation in respect to Fig. 1(e). As can be seen, a simple visual 265 morphological evaluation cannot predict the level of fragmentation without staining, 266 and computational analysis has to be performed on the IPM images to detect the 267 fragmentation group

14 Accepted to Journal of Biophotonics, 2018 Wiley nm Zernike s 600 AO Group IPM nm 600 AO Group IPM Zernike s nm 600 AO Group IPM Zernike s nm Zernike s IPM AO Group 4 nm Zernike s Zernike s IPM AO Group Fig. 2. Examples of sperm cells imaged with Zernike s phase contrast (left), IPM (center), and BFM after AO staining, and categorized by fragmentation groups. Color bar represents OPD values. White scale bars represent 3 μm on the sample

15 3.1 Distribution of DNA fragmentation level in comparison to WHO parameters 273 According to the WHO 2010 guidelines [22] for the morphological evaluation of 274 smeared and stained semen, there are 5 criteria by which a sperm is determined as 275 having "normal morphology". In order for a semen sample to be considered normal, at 276 least 4% of the sperm cells must have a normal morphology. The criteria are: an 277 acrosome that composes of 40% to 70% of the sperm head area, typical head shape, 278 no excessively large external cytoplasmic droplets, no more than 2 small cytoplasmic 279 vacuoles (<20% of head area) and only in the acrosome area, and a straight and 280 smooth midpiece. Since we have already shown that stain-free IPM and stain-based 281 BFM yield comparable results in sperm analysis [11], these parameters were 282 evaluated by an embryologist examining the stain-free IPM images, and the fraction 283 of cells that were found to be normal in each criterion, respectively, were classified 284 according to the color group, in order to determine the number of cells at each of the 285 five different levels of DNA fragmentation (Fig. 3) Fig. 3. Evaluation of sperm morphology using WHO criteria for the different 288 DNA fragmentation groups. In each fragmentation group, the percentages of cells

16 with "normal" morphology in the categories of: (A) head morphology, (B) acrosome 290 size, (C) midpiece, (D) cytoplasmic vacuoles, and (E) cytoplasmic droplets. χ 2 test 291 shows a statistically significant difference in the distribution of normal cells between 292 different fragmentation groups. Statistically significant difference was found only in 293 "acrosome size" category (p<0.001). 294 As can be seen in Fig. 3, the percentage of cells presenting normal morphology in the 296 categories "acrosome size" rises gradually from group 3 to group 5, the differences in 297 the frequency of normal cells in these categories is statistically significant (p<0.001) According to all other additional criteria evaluated Head morphology, midpiece, 299 cytoplasmic vacuoles and cytoplasmic droplets, it was found that the differences 300 between the percentage of normal cells in each color group are not statistically 301 significant Distribution of DNA fragmentation levels in comparison to IPM analysis 303 2D (Fig. 4) and 3D (Fig. 5) criteria were calculated based on the isolated cell OPD 304 maps using our algorithm Fig. 4. Evaluation of morphologic parameters in different DNA fragmentation 307 groups. In each fragmentation group, the mean projection area of the: (A) sperm 308 head, (B) the nucleus and (C) the acrosome is presented. The values were 309 automatically calculated from the IPM images. The error bars represent standard error 310 of the mean value. ANOVA test was used to determine statistically significant 311 differences (* p < 0.05, *** p < 0.001)

17 314 Fig. 5. Evaluation of OPD-related parameters in different DNA fragmentation 315 groups. The mean dry mass of the, (A) the acrosome expressed in Pico-gram. (B) The 316 mean OPD in nanometer. (C) The variance of the OPD in nanometer. (D) The 317 anterior-posterior difference, representing the difference in OPD between the rear half 318 and the front half of the cell. The error bars represent standard error of the mean 319 value. ANOVA test was used to determine statistically significant differences (* 320 p<0.05, *** p<0.01, *** p<0.001) In exanimating the different morphologic parameters, we observed that overall, head 323 area (Fig. 4A) increased gradually from groups 3 to 5, the nucleus area (Fig. 4B) 324 decreased from group 1 to 4 at a slope of µm 2 per group (95% confidence 325 interval (CI) to ), and acrosome area (Fig. 4C) increased from group to 4 with the decreased fraction of fragmented DNA. 327 The examination of selected dry mass and OPD dependent parameters shows an 328 increase of acrosome dry mass (Fig. 5A) with the increase of un-fragmented DNA 329 from group 1 to 4 with slope of pg per group (95% CI to )

18 The mean OPD representing the mean thickness of the cells (Fig. 5B) declines from 331 group 3 to Many other criteria are useful to distinguish between the different fragmentation 333 groups. We chose to present the OPD variance (Fig. 5C) and the mean anterior- 334 posterior (Fig. 5D). OPD variance is obtained by calculating the statistical variance of 335 the OPD values on the entire cell area. OPD variance is highest in groups 3 and 4. The 336 mean anterior-posterior difference (Fig. 5D) is a metric developed by us and 337 compares the average OPD (Fig. 5B) of the anterior and posterior (Fig. 5D) halves of 338 the sperm head, it shows a gradual increase from group 1 to 4 with slope of (95% CI to 6.197). This trend is reversed in group 5, emphasizing the 340 difference of these cells from group To summarizing the results, we conclude that sperm cells in the most fragmented 342 groups (groups 1 and 2) are characterized by a large nuclear area (Fig. 4B) and small 343 acrosomes (Fig. 4C). The medium fragmented groups (groups 3 and 4) are 344 characterized by a small and compressed nucleus (Fig. 4B). Group 4 is distinguished 345 from group 3 by its larger acrosomes (Fig. 4C) and lower mean OPD (Fig. 5B). Group is characterized by the largest head area (Fig. 4A) and a large acrosome (Fig. 5A) 347 similar to that of cells in group 4. However, cells in group 5 have a smaller mean 348 OPD (Fig. 5B) than cells in group 4. Overall, we determine that there is no single 349 criteria that can distinguish between the different fragmentation groups, thus only a 350 combination of the different criteria can reliably predict the fragmentation status Discussion and Conclusions 352 In this paper, we used AO staining as a verification method to IPM. There are several 353 direct methods to test DNA fragmentation in sperm (reviewed in Ref. [27]). The two

19 most prevalent methods in the clinical setting are sperm chromatin structure assay 355 (SCSA) and the TUNEL assay. For the routine diagnosis of patients, these methods 356 are used with flow cytometry. SCSA uses AO staining [28], resulting in DNA 357 fragmentation index (DFI), which corresponds to the presence of sperm cells that 358 show high levels of red staining. The current threshold for abnormal DFI is 25% of 359 sperm cells. SCSA also identifies a subpopulation of cells that emits high level of 360 green light, which possesses high DNA stainability (HDS), but this population has no 361 effect on the DFI and thus it is not used in clinical decisions. In this work, we have 362 chosen to use Tejada s method [26], rather than an SCSA test. In this case, the emitted 363 fluorescence from the AO-stained sperm cells enabled us to divide the cells into different groups, including group #5 that we attribute as the HDS group, which is 365 characterized by dsdna but with impered DNA packing [29]. 366 Although the different DNA fragmentation assays are efficient in diagnosing patients 367 with high percentage of defective sperm cells, they are not applicable in the selection 368 of sperm for ICSI. An indirect indication for the DNA fragmentation is the lack of 369 acrosome reaction [30] or morphological deformities in sperm head such as 370 globozoospermia (a condition characterized by abnormal sperm morphology, 371 including small or absent acrosome) [31]. Indeed, our results indicate that 372 fragmentation groups 3 and 4 showed a larger acrosome compared to groups 1 and The correlation between DNA fragmentation and acrosomal deficiencies is explained 374 by defects during the late spermatogenesis. During that stage in sperm development, 375 somatic cell histones are replaced by protamines: proteins necessary for the 376 condensed DNA packaging as well as the protection from DNA damage. 377 Concurrently, during spermatogenesis, the acrosome is formed. Thus, sperm cells that 378 underwent defected spermiogenesis are likely to present both defected DNA and

20 defected acrosomes [31-33]. The apparent link between defected DNA and defected 380 acrosomes may have an evolutionary reason: to prevent the useless fertilization of an 381 egg by a defective sperm. However, in the context of ICSI, the acrosome has no role 382 in penetrating the ovule during fertilization, thus this evolutionary mechanism is 383 neutralized. Moreover, current ICSI practice is often conducted under relatively weak 384 magnification such as 20. Under this magnification, the embryologist responsible for 385 selecting the sperm to be used in the fertilization can evaluate only observable 386 characters of the sperm cells such as motility or major defects in morphology. 387 In our results, we demonstrate no statistical link between midpiece morphology and 388 DNA fragmentation. The midpiece is the organelle responsible for cell motility, an 389 abnormal midpiece is correlated with impaired motility and reduced fertilization rate 390 during IVF [34]. In general, it has been long shown that sperm motility is correlated 391 to DNA fragmentation [35]; however, DNA fragmentation was reported as a better 392 predictor for conception than progressive morphology in the context of ICSI [36], 393 making the selection process prone to error. 394 In our results, we did not observe statistically significant differences in the prevalence 395 of cytoplasmic vacuoles in sperm cells with different DNA fragmentation levels. 396 Cytoplasmic vacuoles in sperm cells are subtle morphological nuclear malformations 397 caused by DNA condensation defects in the nucleus [33], and is one of the structures 398 that can easily be observed using IMSI [34]. In that method, sperm cells are examined 399 under a high-magnification (at least 6000x, most of which is a digital magnification, 400 which is convenient for view by the observer on a screen) DIC microscope. Using 401 IMSI, several cellular characteristics, which cannot be observed using a regular ICSI 402 microscope, are distinguished. These include the presence of cytoplasmic vesicles and 403 the head detailed shape. The acrosome can also be viewed in this method; however,

21 its exact size and mass cannot be quantified using DIC microscopy. In any case, the 405 clinical significance of cytoplasmic vesicles is a topic of debate in the clinical 406 community. In some reports, it has been shown that there is a positive correlation 407 between fractions of sperm with cytoplasmic vacuoles and those with DNA 408 fragmentation [35,36], while different conclusions are drawn by other investigators 409 that claim cytoplasmic vacuoles do not correlate with a significant difference in DNA 410 fragmentation or with aneuploidy [14,37]. Overall, IMSI was found by meta-analysis 411 to increase implantation and pregnancy rates as well as to decrease the chances of 412 miscarriage in couples that had at least one failed ICSI attempt [16]. 413 The moderate improvement in fertilization and pregnancy rates using the IMSI 414 method suggests that improved sperm visualization can improve overall results. Thus, 415 we expect that the use of IPM, which is a fully quantitative imaging method for sperm 416 evaluation, will further improve the selection of fertile sperm cells when staining is 417 not possible. In our results, we did not observe statistically significant differences in 418 the prevalence of cytoplasmic vacuoles in sperm cells with different DNA 419 fragmentation levels; however, the low number of cytoplasmic vacuoles observed this 420 cohort of donors is sufficient to draw conclusions on the validity of cytoplasmic 421 vacuoles as a marker for DNA fragmentation. 422 In this work, we demonstrate a trend-correlation between various parameters that can 423 be accurately quantified only by IPM and the ratio of ssdna to dsdna in sperm cells 424 as observed under fluorescent microscopy after staining the same cells with AO. 425 Unlike other works utilizing AO [26], we did not use a single color as a threshold 426 between sperm cells with fragmented or un-fragmented DNA. The benefit in dividing 427 the cells to five different groups by color is that it enables us to examine the 428 morphologic characteristics of each group separately

22 After an embryologist examined the OPD maps of the different groups using the 430 various WHO criteria: head morphology, acrosome size, cytoplasmic vacuoles, 431 midpiece integrity and cytoplasmic droplets, we observed a gradual increase in the 432 percentage of "normal" cells from group 3 to 5 in the category of acrosome size. All 433 other morphological criteria, the distribution of normal head morphology; midpiece 434 integrity and cytoplasmic vacuoles, did not present clear changes in their occurrence 435 between the different fragmentation groups. 436 Following the digital examination of the spermatozoa OPD maps and their 437 comparison to the different fragmentation groups, we observed differences in the 438 values of parameters that can only be calculated by IPM, such as the mean OPD, the 439 size of the acrosome or the mean posterior anterior difference, amongst the different 440 groups. In examining the cells by IPM, we observed that sperm cells in group 5, the 441 cells that emit bright green fluorescence, are distinct from all other cell groups. These 442 cells present larger head area and nucleus compared to groups 3 and 4 with lower 443 density. We presume that this population of cells are HDS, a population of sperm cells 444 with defected DNA arrangement [38]. Interestingly, an embryologist examination of 445 group 5 sperm cells shows a small increase in the percentage of normal cells in the 446 categories of head morphology and acrosome size, in comparison with fragmentation 447 groups 3 and 4. Further research will determine the clinical significance of the 448 difference between fragmentation groups and its effect on ICSI outcomes. Although 449 HDS cells are a clearly defined population of cells [28], to the best of our knowledge 450 no attempt has ever been performed to use these cells ether as a diagnostic tool or as a 451 population to avoid in the selection of sperm in ICSI. 452 Although thousands of cells were analyzed, possible limitations of this work are the 453 relatively small number of donors (six) and the fact that the donors are not known to

23 have fertility problems, making them unrepresentative of men in need of ARTs. 455 However, note that even in healthy donors, only 4% of the cells are defined as normal. 456 Therefore, our sample had enough abnormal sperm cells for the analysis performed. 457 In addition, in order to image the same cells with two microscopy systems (IPM and 458 confocal fluorescence microscopy), we fixed the cells. However, IPM can also be 459 performed for live unfixed cells, as we demonstrated previously [39]. Since the 460 fixation procedure did not visually change the cell morphology, we hypothesize that 461 IPM imaging of live cells in medium will yield similar results in detecting DNA 462 fragmentation. 463 We conclude that IPM imaging can produce a detailed and quantitative morphological 464 map of sperm cells. Computerized analysis of these images produces a set to 465 quantitative measurements for morphological parameters. A combination of these 466 parameters with an associated classifier might be able to automatically predict the 467 DNA fragmentation levels of individual living cells without staining. If the IPM 468 method is to be utilized in the selection process for ICSI, it might improve the 469 selection of sperm and thus increase the odds for successful fertilization. 470 List of References D. P. Evenson, Z. Darzynkiewicz, M. R. Melamed. Relation of mammalian 473 sperm chromatin heterogeneity to fertility. Science 1980, 210(4474), L. Robinson, I. D. Gallos, S. J. Conner, M. Rajkhowa, D. Miller, S. Lewis, J. 475 Kirkman-Brown, A. Coomarasamy. The effect of sperm DNA fragmentation on 476 miscarriage rates: a systematic review and meta-analysis. Hum Reprod 2012, (10), R. Henkel, E. Kierspel, M. Hajimohammad, T. Stalf, C. Hoogendijk, C. Mehnert, 479 C. Hoogendijk. DNA fragmentation of spermatozoa and assisted reproduction 480 technology. Reprod Biomed Online 2003, 7(4), L. Simon, A. Zini, A. Dyachenko, A. Ciampi, D. T. Carrell. A systematic review 482 and meta-analysis to determine the effect of sperm DNA damage on in vitro

24 fertilization and intracytoplasmic sperm injection outcome. Asian J Androl 2016, , K. R. Chohan, J. T. Griffin JT, D. T. Carrell. Evaluation of chromatin integrity in 486 human sperm using acridine orange staining with different fixatives and after 487 cryopreservation. Andrologia 2004, 36(5), N. P. Singh, C. H. Muller, R. E. Berger. Effects of age on DNA double-strand 489 breaks and apoptosis in human sperm. Fertil Steril 2003, 80(6), A. Ahmadi, S. C. Ng. Fertilizing ability of DNA-damaged spermatozoa. J Exp 491 Zool. 1999, 284(6), G. Barroso, M. Morshedi, S. Oehninger. Analysis of DNA fragmentation, plasma 493 membrane translocation of phosphatidylserine and oxidative stress in human 494 spermatozoa. Hum Reprod 2000, 15(6), J. L. Fernández, L. Muriel, M. T. Rivero, V. Goyanes, R. Vazquez, J. G. Alvarez. 496 The sperm chromatin dispersion test: a simple method for the determination of 497 sperm DNA fragmentation. J Androl 2013, 24(1), K. L. Larson, C. J. DeJonge, A. M. Barnes, L. K. Jost, D. P. Evenson. Sperm 499 chromatin structure assay parameters as predictors of failed pregnancy following 500 assisted reproductive techniques. Hum Reprod 2000, 15(8), M. Haifler, P. Girshovitz, G. Band, G. Dardikman, I. Madjar, N. T. Shaked. 502 Interferometric phase microscopy for label-free morphological evaluation of 503 sperm cells. Fertil Steril 2015, 104(1), M. Simopoulou, L. Gkoles, P. Bakas, P. Giannelou, T. Kalampokas, K. Pantos, 505 M. Koutsilieris. Improving ICSI: A review from the spermatozoon perspective. 506 Syst Biol Reprod Med 2016, 62(6), B. Bartoov, A. Berkovitz, F. Eltes. Selection of spermatozoa with normal nuclei 508 to improve the pregnancy rate with intracytoplasmic sperm injection. N Engl J 509 Med 2001, 345(14), F. Boitrelle, F. Ferfouri, J. M. Petit, D. Segretain, C. Tourain, M. Bergere, M. 511 Bailly, F. Vialard, M. Albert, J. Selva. Large human sperm vacuoles observed in 512 motile spermatozoa under high magnification: nuclear thumbprints linked to 513 failure of chromatin condensation. Hum Reprod 2011, 26(7), N. G. Cassuto, A. Hazout, I. Hammoud, R. Balet, D. Bouret, Y. Barak Y, S. 515 Jellada, J. M. Ploucharta, J. Selva, C. Yazbeckde. Correlation between DNA 516 defect and sperm-head morphology. Reprod Biomed Online 2012, 24(2), A. S. Setti, D. P. A. F. Braga, R. C. S. Figueira, A. Iaconelli, E. Borges. 518 Intracytoplasmic morphologically selected sperm injection results in improved 519 clinical outcomes in couples with previous ICSI failures or male factor infertility: 520 a meta-analysis. Eur J Obstet Gynecol Reprod Biol 2014, 183, F. Boitrelle, B. Guthauser, L. Alter, M. Bailly, M. Bergere, R. Wainer, F. 522 Vialard, M. Albert, J. Selva, High-magnification selection of spermatozoa prior 523 to oocyte injection: confirmed and potential indications. Reprod Biomed Online , 28(1), P. Girshovitz, N. T. Shaked. Generalized cell morphological parameters based on 526 interferometric phase microscopy and their application to cell life cycle 527 characterization. Biomed Opt Express 2012, 3(8), G. Popescu. Quantitative phase imaging of cells and tissues: McGraw Hill 529 Professional;

25 20. K. Lee, K. Kim, J. Jung, J. Heo, S. Cho, S. Lee, G. Chang, Y. Jo, H. Park, Y. 531 Park. Quantitative phase imaging techniques for the study of cell 532 pathophysiology: from principles to applications. Sensors 2013, 13(4), P. Girshovitz, N. T. Shaked. Compact and portable low-coherence interferometer 534 with off-axis geometry for quantitative phase microscopy and nanoscopy. Opt 535 Express 2013, 21(5), WHO laboratory manual for the examination and processing of human semen. 537 World Health Organization S. K. Mirsky, I. Barnea, M. Levi, H. Greenspan, N. T. Shaked. Automated 539 analysis of individual sperm cells using stain-free interferometric phase 540 microscopy and machine learning. Cytometry A 2017, 91(9), G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. 542 Feld, K. Badizadegan. Optical imaging of cell mass and growth dynamics. 543 American Journal of Physiology-Cell Physiology 2008, 295(2), C M. Balberg, M. Levi, K. Kalinowski, I. Barnea, S. K. Mirsky, N. T. Shaked. 545 Localized measurements of physical parameters within human sperm cells 546 obtained with wide-field interferometry. J Biophotonics 2017, 10, R. I. Tejada, J. C. Mitchell, A. Norman, J. J. Marik, S. Friedman. A test for the 548 practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil 549 Steril 1984, 42(1), A. Agarwal, A. Majzoub, S. C. Esteves, E. Ko, R. Ramasamy, A. Zini. Clinical 551 utility of sperm DNA fragmentation testing: practice recommendations based on 552 clinical scenarios. Transl Androl Urol 2016, 5(6), D. P. Evenson. Sperm Chromatin Structure Assay (SCSA). In: Methods in 554 molecular biology (Clifton, NJ) 2013, D. Domínguez-Fandos, M. I. Camejo, J. L. Ballescà, R. Oliva. Human sperm 556 DNA fragmentation: Correlation of TUNEL results as assessed by flow 557 cytometry and optical microscopy. Cytometry A 2007, 71(12), B. Ozmen, G. S. Caglar, F. Koster, B. Schopper, K. Diedrich, S. Al-Hasani. 559 Relationship between sperm DNA damage, induced acrosome reaction and 560 viability in ICSI patients. Reprod Biomed Online 2007, 15(2), S. Brahem, M. Mehdi, H. Elghezal, A. Saad. Analysis of sperm aneuploidies and 562 DNA fragmentation in patients with globozoospermia or with abnormal 563 acrosomes. Urology 2011, 77(6), F. G. Iranpour. The effects of protamine deficiency on ultrastructure of human 565 sperm nucleus. Adv Biomed Res 2014, 3(1), A. Perdrix, A. Travers, M. H. Chelli, D. Escalier, J. L. Do Rego, J.P. Milazzo N. 567 Mousset-Siméon, B. Macé, N. Rives. Assessment of acrosome and nuclear 568 abnormalities in human spermatozoa with large vacuoles. Hum Reprod 2011, (1), A. Berkovitz, F. Eltes F, Yaari S, Katz N, Barr I, Fishman A, B. Bartoov. The 571 morphological normalcy of the sperm nucleus and pregnancy rate of 572 intracytoplasmic injection with morphologically selected sperm. Hum Reprod , 20(1), J. B. A. Oliveira, F. C. Massaro, R. L. R. Baruffi, A. L. Mauri, C. G. Petersen, L. 575 F. I. Silva, D. Vagnini, J. G. Franco. Correlation between semen analysis by

26 motile sperm organelle morphology examination and sperm DNA damage. Fertil 577 Steril 2010, 94(5), A. Garolla, D. Fortini, M. Menegazzo, L. De Toni, V. Nicoletti, A. Moretti, R. 579 Selice, B. Engl, C. Foresta. High-power microscopy for selecting spermatozoa 580 for ICSI by physiological status. Reprod Biomed Online 2008, 17(5), A. Fortunato, R. Boni, R. Leo, G. Nacchia, F. Liguori, S. Casale, P. Bonassisa, E. 582 Tosti. Vacuoles in sperm head are not associated with head morphology, DNA 583 damage and reproductive success. Reprod Biomed Online 2016, 32(2), D. P. Evenson, L. K. Jost, M. Corzett, R. Balhorn. Characteristics of human 585 sperm chromatin structure following an episode of influenza and high fever: a 586 case study. J Androl 2000, 21(5), P. Jacob Eravuchira, S. K. Mirsky, I. Barnea, M. Levi, M. Balberg, N. T. Shaked. 588 Individual sperm selection by microfluidics integrated with interferometric phase 589 microscopy. Methods 2018, 136,

27 Acknowledgements 594 This research was supported by Momentum fund from Ramot at Tel Aviv University Graphical Abstract 597 We imaged sperm cells using stain-free interferometric phase microscopy, a 598 quantitative phase imaging method. Next, the same sperm cells were stained by 599 acridine orange (AO), a DNA fragmentation indicator, and imaged using a 600 fluorescence confocal microscope. Our results indicate on significant differences in 601 IPM-based parameters between the AO fragmentation groups. We conclude that stain- 602 free IPM may assist in selecting sperm cells with intact DNA, potentially increasing 603 percentage of successful pregnancies