fragmentation stain in human spermatozoa 2

Accepted to Journal of Biophotonics, ©2018 Wiley

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Stained-free interferometric phase microscopy correlation with DNA 1

fragmentation stain in human spermatozoa 2

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Itay Barnea1, Lidor Karako

1, Simcha K. Mirsky

1, Mattan Levi

1, Michal Balberg

1,2 4

Natan T. Shaked1,*

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Affiliations: 7

1- Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel. 8

2- Faculty of Electrical Engineering, Holon Institute of technology, Holon, Israel. 9

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* Corresponding author: Prof. Natan T. Shaked, PhD. Department of Biomedical 11

Engineering, Tel Aviv University, Tel Aviv, Israel. Email: [email protected] 12

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https://plus.google.com/u/0/102867044623249178524?prsrc=4


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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. 36

37

Keywords: Digital holographic microscopy; Quantitative phase imaging; Label-free 38

imaging; Sperm analysis; Fertility. 39


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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 8000 47

treatment cycles in 41 independent reports has shown that men with higher sperm 48

DNA fragmentation, exhibit a 1.9-2.3 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


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 200-400× 72

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


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) = [𝑛𝑐(𝑥, 𝑦) − 𝑛𝑚] × ℎ𝑐(𝑥, 𝑦) , (1) 107

where 𝑂𝑃𝐷(𝑥, 𝑦) is the OPD at point (𝑥, 𝑦), 𝑛𝑐(𝑥, 𝑦) is the integral refractive index 108

of the sperm cells at point (𝑥, 𝑦), nm is the refractive index of the medium and 109

ℎ𝑐(𝑥, 𝑦) is the thickness of the cell at point (𝑥, 𝑦). 110


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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-2010 116

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. 121

122


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2. Methods 123

2.1 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 5 132

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. 145


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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. 156

2.3 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. 161

2.4 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


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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. 177

2.5 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±1.03∙10-12

gram, 181

and the average refractive index of a sperm head was nc= 1.515±0.046. 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

𝑛𝑐−𝑛𝑚 . (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=1.013∙10-21

gram/nm3=1.013∙10

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 . 191


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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 196

1 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 Na2HPO4 7H2O (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×, 1.4 204

numerical aperture microscope objective. The cells were excited using light at 205

wavelengths 477 – 488 nm, and emission was filtered between 572 – 668 nm for the 206

red and 505 – 550 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. 209

2.6 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 213


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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 χ2 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 0.05. 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 24.0.0.1. 228

The statistical analysis was generated by IBM SPSS STATISTICS 24.0.0.1. The 229

charts were generated by the GraphPad Prism v.7 software. 230

231


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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, 240

298, 254 and 387 cells. 241

242

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 5 249 (green), and the color was incorporated into the data obtained by computerized 250

analysis of the IPM image and the embryologist evaluation. 251

(A)

(E)(D)

(C)(B)

15 μm

2 μm

nm

nm

15 μm

3 μm


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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. 268


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269

Fig. 2. Examples of sperm cells imaged with Zernike’s phase contrast (left), IPM 270 (center), and BFM after AO staining, and categorized by fragmentation groups. 271 Color bar represents OPD values. White scale bars represent 3 μm on the sample. 272

AO Group 1Zernike’s600

500

400

300

200

100

600

500

400

300

200

100

AO Group 2IPMZernike’s

600

500

400

300

200

100

Zernike’s IPM AO Group 5

600

500

400

300

200

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IPMZernike’s AO Group 4

AO Group 3600

500

400

300

200

100

IPM

IPMZernike’s

Zernike’s

nm

nm

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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). 286

287 Fig. 3. Evaluation of sperm morphology using WHO criteria for the different 288 DNA fragmentation groups. In each fragmentation group, the percentages of cells 289


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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

295

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). 298

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. 302

3.2 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. 305

306 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). 312

313


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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). 321

322

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 –0.09725 µm2 per group (95% confidence 325

interval (CI) –0.1520 to –0.04248), and acrosome area (Fig. 4C) increased from group 326

1 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 0.07505 pg per group (95% CI 0.02898 to 0.1211). 330


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The mean OPD representing the mean thickness of the cells (Fig. 5B) declines from 331

group 3 to 5. 332

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 3.282 339

(95% CI 0.3677 to 6.197). This trend is reversed in group 5, emphasizing the 340

difference of these cells from group 4. 341

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 346

5 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. 351

4. 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 354


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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 5 364

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 2. 373

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 379


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, 404


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. 429


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 454


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

471

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591

592

593


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Acknowledgements 594

This research was supported by Momentum fund from Ramot at Tel Aviv University. 595

596

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. 604

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