ARTICLE IN PRESS

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Q2 Article
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3 bs_bs_query Multi-centre assessment of nitroblue tetrazolium

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5 bs_bs_query reactivity in human semen as a potential marker of

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7 bs_bs_query oxidative stress

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Q1 Jaime Gosálvez a, Lamberto Coppola b, Jose Luis Fernández c, Carmen López-Fernández a,
10 bs_bs_query Alfredo Góngora d, Ricardo Faundez e, John Kim f, Nabil Sayme g, Moises de la Casa h,
11 bs_bs_query Rebeca Santiso c, Keith Harrison i, Ashok Agarwal j, Stephen Johnston k,*,
12 bs_bs_query Sandro C Esteves l
a
13 bs_bs_query Department of Biology, Universidad Autónoma de Madrid, Madrid, 28049, Spain
b
14 bs_bs_query Tecnomed, Centro Medico Biologico, Nardò, Lecce, Italy
c
15 bs_bs_query Genetics Unit, INIBIC-Complejo Hospitalario Universitario A Coruña (CHUAC), As Xubias, 84, 15006 A Coruña, Spain
d
16 bs_bs_query Centro de Fertilidad, Tuxpan 6. Col. Roma Sur. Mexico DF, Mexico
e
17 bs_bs_query Invimed Fertility Clinics, Embryology Laboratories, Center of Biomedical Research, WULS, 36 Rakowiecka Street, 02-532 Warsaw, Poland
f
18 bs_bs_query SJ Science Inc., 972-8, Shin Jung 4 Dong, Yang Chun Gu, Seoul, Republic of Korea
g
19 bs_bs_query Arzt, Frauenarzt (Gynäkologe), Weiterbildungen: Reproduktionsmedizin, Ambulante Operationen, Brühlstr. 19, 30169 Hannover, Germany
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Q3 h
Ginefiv, Madrid, Spain
i
21 bs_bs_query Queensland Fertility Group, 55 Little Edward Street, Spring Hill 4000, Australia
j
22 bs_bs_query Clinical Pathology, Cleveland Clinic Main Campus, Mail Code X11, 9500 Euclid Avenue, Cleveland, OH 44195, USA
k
23 bs_bs_query School of Agriculture and Food Sciences, The University of Queensland, Gatton 4343, Australia
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24 bs_bs_query ANDROFERT, Andrology and Human Reproduction Clinic, Av. Dr. Heitor Penteado, 1464, Campinas, SP 13075-460, Brazil
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27 bs_bs_query Jaime Gosálvez is Professor of Genetics at the Department of Biology, University Autónoma of Madrid. He has
28 bs_bs_query published 345 journal articles and co-edited two books. He has also been principal investigator in 21 national
29 bs_bs_query and international research projects and has participated in five patents.
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31 bs_bs_query KEY MESSAGE

32 bs_bs_query Semen samples with high levels of oxidative stress as determined by a nitroblue tetrazolium assay, have di-
33 bs_bs_query minished sperm DNA longevity after ejaculation. Given that seminal plasma was found to be the primary source
34 bs_bs_query of oxidative stress, rapid separation of this fraction may improve sperm DNA quality in these patients.

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36 bs_bs_query A B S T R A C T
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38 bs_bs_query The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially sur-
39 bs_bs_query veyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]);

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42 bs_bs_query * Corresponding author.

43 bs_bs_query E-mail address: s.johnston1@uq.edu.au (S Johnston).
44 bs_bs_query http://dx.doi.org/10.1016/j.rbmo.2017.01.014
45 bs_bs_query 1472-6483/© 2017 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.

Please cite this article in press as: Jaime Gosálvez, Lamberto Coppola, Jose Luis Fernández, Carmen López-Fernández, Alfredo Góngora, Ricardo Faundez, John Kim, Nabil
Sayme, Moises de la Casa, Rebeca Santiso, Keith Harrison, Ashok Agarwal, Stephen Johnston, Sandro C. Esteves, Multi-centre assessment of nitroblue tetrazolium reactiv-
ity in human semen as a potential marker of oxidative stress, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.01.014
 ARTICLE IN PRESS
2 REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■–■■

L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay
was used to anlysise samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen
samples incubated for 24 h at 37oC showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further char-
acterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the
most significant fraction of •O2− localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan
precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan
and not all leukocytes presented with equivalent production of superoxide anions.
© 2017 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.

(sperm versus seminal plasma), or of the prevalence of positive re-

Introduction
Reactive oxygen species (ROS) are commonly known to negatively affect
somatic or germ cell lines and are a major cause of cellular damage.
Examples of ROS include the superoxide anion (•O2−), hydrogen per-
oxide (H2O2), hydroxyl radical (•OH) and the peroxyl radical (•HO2−);
the former being the most abundant ROS in human semen and the
precursor of other ROS types. The superoxide anion is primarily pro-
duced from mitochondrial electron transport chain complexes I and
III (Chen et al., 2009), and there has been significant interest in using
it as a marker of oxidative damage given its ubiquity and strong chemi-
cal reactivity. All types of ROS can induce cellular damage in biological
molecules such as proteins, lipids and DNA (Agarwal et al., 2014; Du
Plessis et al., 2015; Kohen and Nyska, 2002). Excessive ROS produc-
tion in the male reproductive tract is of concern because it leads to
oxidative stress, which has potential toxic effects on sperm quality
and function. In fact, between 25 and 50% of patients attending in-
fertility clinics have high concentrations of ROS that may be associated
with abnormal sperm motility, membrane integrity and DNA quality
(Aitken, 1995; Ferramosca et al., 2007, 2013). For example, patients
with a varicocele, typically have ROS concentrations higher than those
of other infertile patients; in these patients, the affect of ROS is likely
to be associated with heat stress affecting developing germ cells (Smith
et al., 2006). Nevertheless, the exact prevalence of ROS in the subfertile
male population remains poorly understood (Gharagozloo and Aitken,
2011). Part of the problem stems from the complexity of some tech-
niques that are used to measure ROS, which limits the widespread
utilization of ROS measurements as a routine procedure in the
andrology clinic. Additionally, the plethora of different ROS radicals
being assessed and how they are being assessed makes it challeng-
ing to draw firm conclusions on the real prevalence and significance
of ROS in male infertility.
Various strategies have been used to assess the presence and
effects of ROS (Agarwal et al., 2014; Kohen and Nyska, 2002). The NBT
assay is a technique that has traditionally been used to determine
the production of •O2− in somatic and germ cells (Choi et al., 2006;
Dimitrova et al., 2013; Sharma and Agarwal, 1996). The yellow NBT
molecule is water-soluble, membrane permeable and reduced by •O2−
to a blue formazan deposit (Halliwell and Gutteridge, 1985). The speci-
ficity of this reaction has been demonstrated by the inhibitory effect
of superoxide dismutase (SOD) as this enzyme catalyzes the dis-
mutation of two molecules of •O2− to form one molecule of oxygen
and one molecule of H2O2 (Baehner et al., 1975). Therefore, the levels
of •O2−, H2O2 and •OH are in constant conversion until equilibrium is
established. Although •O2− in semen samples has previously been
evaluated using NBT (Amarasekara et al., 2014; Esfandiari et al., 2003;
Tunc et al., 2010), a clear understanding of the association between
positive reactions to NBT and the different fractions of the ejaculate
actions in the neat ejaculate within a large cohort of men seeking
fertility assistance, is lacking. Moreover, the effect of ROS on sperm
DNA quality deserves more detailed attention, especially in estab-
lishing direct relationships between both concepts.
We conducted a multinational and multicentre cross-sectional study
to determine the prevalence of positive responses to NBT in the ejacu-
lates of a large number of infertile men attending infertility clinics
around the world. The following were studied: the relationship of high
and low NBT reacting ejaculates on initial sperm DNA quality and after
incubation at 37°C for up to 24 h; the relative contribution of seminal
plasma, spermatozoa, or both, to the strength of NBT reaction; and
which primary region of the sperm cell the signal is being gener-
ated from.
Material and methods
Study design
This prospective design was a cross-sectional study that incorpo-
rated fresh ejaculates obtained from 707 men seeking fertility
evaluation in 2014 and 2015 at seven participating clinics in Australia,
Brazil, Germany, Mexico, Poland, South Korea and Spain. All partici-
pants provided informed consent to use their semen samples for the
analysis. The data obtained by individual centres were compiled and
subsequently analysed at the Genetics Unit of the Autónoma Univer-
sity of Madrid. This study was approved by the research committee
or internal clinical board of each participant institution and com-
plied with the standards for the reporting of cross-sectional studies
(STROBE statement, http://strobe-statement.org). Patients were Q4
offered the assay in addition to the standard seminogram and in-
formed that their semen sample would be analysed as part of larger
worldwide multicentre study on seminal oxidative stress. No charged
was levied for the assay and signed consent for participation was
requested.
Participants
Men were included if they were between the ages of 18 and 45 years,
seeking fertility evaluation and consented to donate a semen speci-
men for research. Participants were asked to abstain from ejaculation
for 2–3 days before collection. All participants used a collection room
located in the same facility as the andrology laboratory. Ejaculates
were collected by masturbation into sterile cups. A single ejaculate
was obtained from each individual and all specimens were dis-
carded after assessments. Men were excluded if they reported semen
spillage or loss during collection, had a history of the following, or

Please cite this article in press as: Jaime Gosálvez, Lamberto Coppola, Jose Luis Fernández, Carmen López-Fernández, Alfredo Góngora, Ricardo Faundez, John Kim, Nabil
Sayme, Moises de la Casa, Rebeca Santiso, Keith Harrison, Ashok Agarwal, Stephen Johnston, Sandro C. Esteves, Multi-centre assessment of nitroblue tetrazolium reactiv-
ity in human semen as a potential marker of oxidative stress, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.01.014
 ARTICLE IN PRESS
REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■–■■ 3

46 bs_bs_query both: medical illness, including fever in the past 3 months, evidence semi-quantitative micro-densitometric approaches or image analy- 78
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47 bs_bs_query of clinical or subclinical (leukocyte >1 million/ml of semen) genital sis protocols, results obtained after direct visual observation are not 79
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48 bs_bs_query infection, severe oligozoospermia (<1 million sperm per ml of semen), significantly different (de la Casa et al., 2015); therefore, a visual quali- 80
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49 bs_bs_query azoospermia, or use of gonadotoxic medication. tative classification, which is both less expensive and easier to carry 81
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out, was used here. This was achieved by visual colour comparison 82
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51 bs_bs_query Assessment of •O2− levels in neat semen with a pre-established pantone supplied with the kit. A single expe- 83
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rienced senior technician performed the Oxisperm® test in each 84
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53 bs_bs_query The presence •O2− in the semen specimens was assessed using the centre; the technician in each centre underwent before training as 85
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54 bs_bs_query Oxisperm® kit (Halotech® DNA, Madrid, Spain); this protocol is based provided by Halotech® DNA instructions. Each run was carried out 86
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55 bs_bs_query on the nitroblue tetrazolium (NBT) test that produces a stable with appropriate positive and negative controls. Positive controls in- 87
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56 bs_bs_query colourimetric reaction in a biological sample that may contain •O2−. cluded the use of activated leukocytes from peripheral blood that were 88
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57 bs_bs_query To perform this assay, a tube containing the reagent gel was placed incubated with tetradecanoylphorbol acetate (TPA; Sigma Aldrich, 89
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58 bs_bs_query in a water bath at 90°C for about 5 min to allow the gel to liquefy. Madrid, Spain) at a concentration of 1.6 × 10−7 for 30 min at 37°C. Nega- 90
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59 bs_bs_query After gel equilibration at 37°C, liquefied semen was added accord- tive controls involved use of non-activated leukocytes from peripheral 91
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60 bs_bs_query ing to the kit specifications and the mixture incubated for 45 min at blood. Positive and negative controls were processed in parallel with 92
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61 bs_bs_query 37°C. Depending on the concentration of superoxide anion and the the test specimens. Two trained observers independently scoring 60 93
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62 bs_bs_query cellular content of the various oxido-reductases, a colour precipi- samples twice in a blinded fashion evaluated the reproducibility of 94
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63 bs_bs_query tate that ranged from a light pink to dark purple-black was produced. test scores in one of the participating centres (Brazil). The inter- 95
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64 bs_bs_query The NBT reaction gives rise to solid deposits of formazan on the plasma observer variability was calculated by analysing the readings obtained 96
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65 bs_bs_query membranes, which are also readily detectable by bright field mi- by two observers while the intra-observer variability was analysed by 97
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66 bs_bs_query croscopy. In this study, we only considered three levels of colourimetric calculating the difference between two readings obtained by the same 98
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67 bs_bs_query reaction: L1, low/absence of colour; L2, medium; and L3, high observer. 99
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68 bs_bs_query (Figure 1). Although the colour assessment can be carried out using 100
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Sperm DNA fragmentation assessment 101

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102
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Initial sperm DNA fragmentation (SDF) was assessed 30 min after 103
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ejaculation and was determined from chromatin dispersion pat- 104

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terns using the Halosperm kit (Halotech DNA SL, Madrid, Spain) in 105
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143 samples (25 corresponding to L1, 102 to L2 and 16 to L3). To assess 106
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the dynamic behaviour of SDF, 30 semen samples that stained as L1 107

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or L3 after •O2− assessment were incubated at 37°C for 0, 2, 6 and 108

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24 h and the SDF assessed at each time interval using the Dyn- 109
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Halosperm kit (Halosperm DNA, Madrid, Spain). The morphology of 110

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different sized haloes was visualized by epifluorescence micros- 111

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copy after being stained with the DNA intercalating fluorochrome Gel 112
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Red (Biotium, Hayward, CA, USA) and anti-fading agent Vectashield 113
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(Vector Laboratories, Burlingame, CA, USA). Image visualization and 114

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69 bs_bs_query capture was carried out using a Leica DMLB microscope with 115
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epifluorescence light-emitting diode illumination with a charge- 116

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coupled device (Leica Microsystems, Barcelona, Spain). 117

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Assessment of •O2− levels in semen fractions 119

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120
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In a parallel experiment, each patient’s response to the NBT reac- 121

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tion was assessed in the neat ejaculate, seminal plasma and sperm 122
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fractions. A total of 100 individuals who had a positive reaction to NBT 123
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were included in this analysis. Thirty minutes after ejaculation, semen 124
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samples were centrifuged at 400 g for 6 min to produce a sperm pellet 125
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(spermatozoa) and supernatant (seminal plasma). The supernatant 126

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was directly reacted with NBT by mixing 50 μL of the reactive gel in- 127
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cluded in the kit with 50 μL of seminal plasma and incubated for 45 min 128
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at 37°C; the resulting reaction colour was recorded. The sperm pellet 129
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70 bs_bs_query Figure 1 – Visualization of the three pre-established colour levels was further diluted using phosphate-buffered saline to achieve a final 130
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71 bs_bs_query of reactivity using the Oxisperm® kit to assess •O2− content in and homus sperm concentration of 50 × 106 mL−1 and, from this di- 131
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72 bs_bs_query neat semen samples and visual colour comparison using a lution, 50 μL of the re-suspended sperm pellet was reacted with 50 μL 132
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73 bs_bs_query pre-established pantone. L1, no detectable reactivity or low of the reactive gel following the same protocol as the seminal plasma. 133
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74 bs_bs_query reactivity level; L2, Medium reactivity level; L3, high reactivity Reactivity results were compared in each specimen before and after 134
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75 bs_bs_query level. The reaction was conducted in multi-well microtitre plates. semen fractionation. Specimens were classified on the basis of pres- 135
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76 bs_bs_query Positive and negative controls correspond to leukocytes incubated ence or absence of reactivity as follows: P1, negative response (L1) 136
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77 bs_bs_query with and without tetradecanoylphorbol acetate, respectively. to NBT reactivity in the neat ejaculate and in both semen fractions 137
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Please cite this article in press as: Jaime Gosálvez, Lamberto Coppola, Jose Luis Fernández, Carmen López-Fernández, Alfredo Góngora, Ricardo Faundez, John Kim, Nabil
Sayme, Moises de la Casa, Rebeca Santiso, Keith Harrison, Ashok Agarwal, Stephen Johnston, Sandro C. Esteves, Multi-centre assessment of nitroblue tetrazolium reactiv-
ity in human semen as a potential marker of oxidative stress, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.01.014
 ARTICLE IN PRESS
4 REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■–■■

138 bs_bs_query (frequencies not included in this study); P2, positive response (L2 or
139 bs_bs_query L3) to NBT reactivity in the neat ejaculate and in both semen fractions;
140 bs_bs_query P3, positive response (L2 or L3) in the neat semen and in the seminal
141 bs_bs_query plasma fraction, but negative (L1) in the sperm fraction and P4, posi-
142 bs_bs_query tive response (L2 or L3) to NBT reactivity in the neat specimen and in
143 bs_bs_query the sperm fraction but negative (L1) in the seminal plasma fraction.
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145 bs_bs_query Microscope analysis of •O2− assessment in sperm fractions

146 bs_bs_query

147 bs_bs_query The semen fractions from the ejaculates of 100 individuals were as-
148 bs_bs_query sessed under bright-field microscopy after the completion of the NBT
149 bs_bs_query reaction to determine the location of the positive NBT signals. Once
150 bs_bs_query the reaction was completed in the gel, a smear was produced using
151 bs_bs_query a 20 μL aliquot of sperm suspension on a clean microscope glass slide.
152 bs_bs_query The slides were dried at room temperature and counterstained using
153 bs_bs_query methyl green 2% (Sigma Aldrich, Madrid, Spain). A standard bright
154 bs_bs_query field microscope equipped with a Jenoptik ProgRes CCD (Jenoptik
155 bs_bs_query Optical System, GmbH) was used for image capture; a non-oil-
156 bs_bs_query immersion 63x plan-achromatic objective provided sufficient resolution.
157 bs_bs_query
Figure 2 – Distribution of colour levels (L1–L3) (Figure 1) from all 197 bs_bs_query

158 bs_bs_query Statistical analysis participating centres after assessment of •O2− content, using the 198 bs_bs_query

159 bs_bs_query
Oxisperm® assay, in test neat semen specimens (n = 707). 199 bs_bs_query

160 bs_bs_query The Statistical Package for Social Sciences (SPSS v.11; Chicago, IL, 200 bs_bs_query

161 USA) was used for statistical analysis. P < 0.05 was considered sta-
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medium (L2) level of reactivity and a low proportion with high reac- bs_bs_query

162 tistically significant. Absolute and per cent distributions of the different
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tivity (L3). Nevertheless, significant difference (contingency, chi- bs_bs_query

163 NBT reaction categories were calculated for each participant centre
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square = 66.93; df = 12; Pα 0.05 = 0.000) was found between the values bs_bs_query

164 and a contingency table chi-squared test was used to compare results
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reported by the different clinics for the levels of NBT reactivity bs_bs_query

165 among the different centres. For intra- and inter-observer variabil-
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observed. bs_bs_query

166 bs_bs_query ity, absolute and per cent differences between individual measurements,
206 bs_bs_query

167 bs_bs_query and the final recorded value with the cumulative percentages, were
Influence of superoxide presence on sperm 207 bs_bs_query

168 bs_bs_query calculated. The non-parametric Mann–Whitney U statistical test was

DNA fragmentation 208 bs_bs_query

169 bs_bs_query used to compare initial SDF values between samples from individu-
209 bs_bs_query

170 bs_bs_query als in the two groups whereas the dynamic loss of DNA quality was
To compare the effect on sperm DNA fragmentation (SDF) with the 210 bs_bs_query

171 bs_bs_query assessed using the non-parametric maximum likelihood Kaplan–

different levels of superoxide in the ejaculate, SDF was compared 211 bs_bs_query

172 bs_bs_query Meier estimator. To compare the survival distributions of the sperm
30 min after ejaculation (T0 – static assessment) and after a period 212 bs_bs_query

173 bs_bs_query samples from each group, a Log Rank (Mantel–Cox) test was used
of incubation at 37°C (T0, T2, T6 and T24h – dynamic assessment) 213 bs_bs_query

174 bs_bs_query (SPSS v.16.0 for Windows, SPSS Inc. 233 S. Wacker Drive. 60606 Chicago,
(Figure 3). With static assessment (T0), 143 individuals were in- 214 bs_bs_query

175 bs_bs_query IL). Sperm DNA accumulated survival, in this case was represented
cluded in the analysis (25 corresponding to L1, 102 to L2 and 16 to 215 bs_bs_query

176 as a varying accumulated frequency over time for sperm that were
L3). The mean ± SD values of SDF obtained in the different groups
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216 bs_bs_query

177 not affected by fragmentation at each time period of the incubation.

were 23.3 ± 13.7, 23 0 ± 14.4 and 21.3 ± 10.8 for L1, L2 and L3, re-
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217 bs_bs_query

178 bs_bs_query

spectively; no significant difference was observed between the different 218 bs_bs_query

179 bs_bs_query
groups (Kruskal–Wallis, Chi-Square 0.174; df). With dynamic assess- 219 bs_bs_query

180 bs_bs_query Results ment of SDF, 30 individuals were scored for SDF after incubation of 220 bs_bs_query

181 bs_bs_query
the neat ejaculate at T0, T2, T6 and T24 h at 37°C. Fifteen of initial 221 bs_bs_query

182 bs_bs_query Prevalence of a positive NBT reaction in the neat ejaculate ejaculates were classified as L1 whereas the remainder (n = 15) were 222 bs_bs_query

183 bs_bs_query classified as L3. Our analysis revealed that within this selected semen 223 bs_bs_query

184 bs_bs_query Assessment of the •O2− levels within the same clinic revealed an intra- sample population we could observe no statistical differences in 224 bs_bs_query

185 bs_bs_query class correlation coefficient between observers of 0.94 and an inter- the initial T0 value for sperm DNA (Figure 3A); the mean ± SD SDF 225 bs_bs_query

186 bs_bs_query class correlation coefficient between two readings by the same for the L1 and L3 groups was 17.8 ± 10.5 and 21.1 ± 11.2 (Mann– 226 bs_bs_query

187 bs_bs_query observer of 0.96. The pooled distribution of the different reactivity levels Whitney U = 94.0; Z = −0.768), respectively. After comparing the DNA 227 bs_bs_query

188 bs_bs_query from all clinics is shown in Figure 2. Most patients (n = 538 [76%]) dynamics between the L1 and L3 groups, however, significant differ- 228 bs_bs_query

189 bs_bs_query were categorized as having medium (L2) reactivity. In contrast, only ences in SDF were observed. Individuals characterized as L3 showed 229 bs_bs_query

190 bs_bs_query 4% (n = 28) of the cohort exhibited high reactivity (L3) whereas 20% a faster rate of DNA damage than those categorized in the L1 230 bs_bs_query

191 bs_bs_query (n = 141) showed absence/low reactivity (L1). Overall, 80.1% of the group (Figure 3B) (Kaplan–Meier; Log-Rank; Mantel–Cox: 242.1; df 1; Q5 231 bs_bs_query

192 bs_bs_query studied population showed semen reactivity to the NBT test, indicat- P < 0.001). Differences in SDF dynamics were also observed when 232 bs_bs_query

193 bs_bs_query ing the presence of superoxide anion in the ejaculate. The distribution the different individuals in each NBT reactivity category were 233 bs_bs_query

194 bs_bs_query of reactivity levels by participating centres is shown in Table 1. In compared (L1; Kaplan–Meier; Log–Rank; Mantel–Cox: 116.7; df 14; 234 bs_bs_query

195 bs_bs_query general, a similar trend was observed among centres in the clustering P < 0.001) (Figure 3C) and (L3; Kaplan–Meier; Log–Rank; Mantel–Cox: 235 bs_bs_query

196 bs_bs_query of specimens, and indicated a large proportion of patients with a 148.0; df 14; P < 0.001) (Figure 3D). 236 bs_bs_query

Please cite this article in press as: Jaime Gosálvez, Lamberto Coppola, Jose Luis Fernández, Carmen López-Fernández, Alfredo Góngora, Ricardo Faundez, John Kim, Nabil
Sayme, Moises de la Casa, Rebeca Santiso, Keith Harrison, Ashok Agarwal, Stephen Johnston, Sandro C. Esteves, Multi-centre assessment of nitroblue tetrazolium reactiv-
ity in human semen as a potential marker of oxidative stress, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.01.014
 ARTICLE IN PRESS
REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■–■■ 5

237 bs_bs_query Table 1 – Prevalence of nitroblue tetrazolium reactivity levels (L1–L3)a in neat semen specimens (n = 707) according to centre.
238 bs_bs_query NBT Centre 1 Centre 2 Centre 3 Centre 4 Centre 5 Centre 6 Centre 7
239 bs_bs_query Level 1, n (%) 15 (15) 6 (5.6) 33 (33.3) 41 (24.6) 11 (35.5) 20 (19.4) 16 (16.2)
240 bs_bs_query Level 2, n (%) 83 (83) 102 (94.4) 65 (65.7) 116 (69.4) 20 (64.5) 71 (68.9) 82 (82.8)
241 bs_bs_query Level 3, n (%) 2 (2) 0 (0.0) 1 (1.0) 10 (6.0) 0 (0.0) 12 (11.7) 1 (1.0)
242 bs_bs_query Total, n (%) 100 (100) 108 (100) 99 (100) 167 (100) 31 (100) 103 (100) 99 (100)
243 bs_bs_query
a
See Figure 1.
244 bs_bs_query NBT, nitroblue tetrazolium.
245 bs_bs_query

246 bs_bs_query NBT reactivity in neat and fractioned semen and 10% to P4 categories (Figure 4); once a positive colour reaction 260 bs_bs_query

247 bs_bs_query was confirmed in the neat ejaculate, a positive signal was further iden- 261 bs_bs_query

248 bs_bs_query After assessing 100 individuals in which the neat ejaculate was posi- tified in the sperm and seminal plasma fractions in 32% and 90% of 262 bs_bs_query

249 bs_bs_query tive to NBT, 22% of these samples were assigned to P2, 68% to P3 the cases, respectively. The colour intensity of sperm samples in P4 263 bs_bs_query

250 bs_bs_query

251 bs_bs_query Figure 3 – The effect of L1 and L3 levels of nitroblue tetrazolium reactivity on the static and dynamic values of sperm DNA fragmentation
252 bs_bs_query (SDF). (a) Box-whisker plot showing static values for SDF at T0 (after ejaculation) in L1 (n = 15) and L3 (n = 15) groups created according to
253 bs_bs_query the level of nitroblue tetrazolium reactivity. Dynamic values of sperm DNA fragmentation are expressed as sperm DNA accumulated
254 bs_bs_query survival defined as accumulated frequency over time of those sperm not affected by DNA fragmentation at each time period of the
255 bs_bs_query incubation. Dynamic behaviours have been normalized to the SDF at t0, set as ‘1’ on the Y axis; (b) mean values of sperm DNA dynamics in
256 bs_bs_query Oxisperm®-L1 (n = 15; blue) and Oxisperm®-L3 (n = 15; red) designated spermatozoa, Kaplan–Meier; Log–Rank; Mantel–Cox; 242.1; df 1;
257 bs_bs_query P < 0.001 for L1 versus L3; (c) intra-group variation of sperm DNA dynamics in group L1 (n = 15, Kaplan–Meier; Log–Rank; Mantel–Cox:
258 bs_bs_query 116.7; df 14; P < 0.001 for the comparison between individuals; (d) intra-group variation of sperm DNA dynamics in group L3 (n = 15),
259 bs_bs_query
Q8 Kaplan–Meier; Log–Rank; Mantel–Cox: 148.0; df 14; P < 0.001 for the comparison between individuals.
Please cite this article in press as: Jaime Gosálvez, Lamberto Coppola, Jose Luis Fernández, Carmen López-Fernández, Alfredo Góngora, Ricardo Faundez, John Kim, Nabil
Sayme, Moises de la Casa, Rebeca Santiso, Keith Harrison, Ashok Agarwal, Stephen Johnston, Sandro C. Esteves, Multi-centre assessment of nitroblue tetrazolium reactiv-
ity in human semen as a potential marker of oxidative stress, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.01.014
 ARTICLE IN PRESS
6 REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■–■■

264 bs_bs_query Figure 4 – Colourimetric characterization of different reaction

265 bs_bs_query patterns according to the reactivity to nitroblue tetrazolium (NBT)
266 bs_bs_query in the neat ejaculate, seminal plasma and sperm fractions.
267 bs_bs_query P1 - negative response to NBT assay in the neat ejaculate, seminal
268 bs_bs_query plasma and sperm fractions; P2, positive response (L2 or L3)
269 bs_bs_query (Figure 1) in the neat ejaculate and in both seminal plasma and
270 bs_bs_query sperm fractions; P3, positive reaction in the neat semen and in the
271 bs_bs_query seminal plasma, but negative in the sperm fraction; P4, positive
272 bs_bs_query reaction to NBT in the neat specimen and in the sperm fraction,
273 bs_bs_query but negative in the seminal plasma. PBS, phosphate-buffered
274 bs_bs_query saline.

275 bs_bs_query

276 bs_bs_query was slightly less than those sperm registered in both the neat ejacu-
277 bs_bs_query late and seminal plasma fractions (P2) (Figure 4). None of the patients
278 bs_bs_query presenting with a negative response in the neat ejaculate showed a
279 bs_bs_query positive reaction in the seminal plasma or in sperm fractions.
280 bs_bs_query

281 bs_bs_query Signal localization in NBT reactive sperm fractions

282 bs_bs_query

283 bs_bs_query In those patient ejaculates that showed NBT reactivity in both the neat
284 bs_bs_query specimen and sperm fractions (irrespective of the response in seminal Figure 5 – Visualization of formazan precipitates after reacting the 308 bs_bs_query

285 bs_bs_query plasma), the signal was localized to spermatozoa, leukocytes, or both. sperm pellet with Oxisperm®. (a) Spermatozoa showing absence 309 bs_bs_query

286 bs_bs_query Staining of the sperm cell was preferentially located in the mid- of any precipitate; (b) spermatozoa showing positive signal in the 310 bs_bs_query

287 bs_bs_query piece and post-acrosomal regions, but rarely covered the entire surface mid-piece; (c) spermatozoa showing intense precipitation at the 311 bs_bs_query

288 bs_bs_query of the sperm head (Figure 5B and 5C). The intensity of labelling also post-acrosomal region; (d) overview of a sperm sample that had a 312 bs_bs_query

289 bs_bs_query varied among cells within the same ejaculate (Figure 5E). The pro- negative reaction; (e) overview of a sperm sample that positively 313 bs_bs_query

290 bs_bs_query portion of labelled spermatozoa also varied from one individual to reacted – note spermatozoa with (red arrows) and without 314 bs_bs_query

291 bs_bs_query another; in 10 randomly selected positive staining ejaculates, the pro- precipitates (green arrows) in the head; (f) overview of a sperm 315 bs_bs_query

292 bs_bs_query portion of labeled sperm varied from 2–60% after counting 300 cells. sample in which polymorphonuclear leukocytes leukocytes were 316 bs_bs_query

293 bs_bs_query The NBT staining of spermatozoa was present in all patients who were labelled (red arrows); (g,h) two selected leukocytes showing 317 bs_bs_query

294 bs_bs_query identified as having polymorphonuclear leukocytes (PMN) in the ejacu- positive low (g) and high (h) levels of formazan precipitation. Scale 318 bs_bs_query

295 bs_bs_query late (Figure 5F). Similarly, the labelling intensity of leukocytes also bar: a–c: 2 μm; d–f: 10 μm; g–h: 5 μm. 319 bs_bs_query

296 bs_bs_query varied (compare Figure 5G bottom with Figure 5F top). No patients 320 bs_bs_query

297 with a negative response in the neat semen showed traces of formazan
2003; Tunc et al., 2010). In this survey examining over 700 patients 321
bs_bs_query

bs_bs_query

298 precipitate in the sperm fraction (Figure 5A and 5D).

evaluated across seven different infertility clinics in four continents, 322
bs_bs_query

bs_bs_query

299 bs_bs_query

80% of the studied population showed semen reactivity to NBT, in- 323 bs_bs_query

300 bs_bs_query
dicating the presence of the superoxide anion in the ejaculate. Despite 324 bs_bs_query

301 bs_bs_query Discussion some individual variation in relative proportions of colourimetric cat- 325 bs_bs_query

302 bs_bs_query
egories across the different clinics, most participants evaluated (76%) 326 bs_bs_query

303 bs_bs_query Prevalence of NBT reactivity in the ejaculate presented with a medium level of NBT reactivity. Only one in 25 in- 327 bs_bs_query

304 bs_bs_query dividuals produced ejaculates with NBT high reactivity. 328 bs_bs_query

305 bs_bs_query The NBT reaction provides information about the levels of •O2− in a Significant variation in the prevalence of NBT reactivity levels (L1– 329 bs_bs_query

306 bs_bs_query range of biological samples (including semen) by means of a simple L3) across the seven centres was found, particularly in levels L1 and 330 bs_bs_query

307 bs_bs_query colourimetric reaction (Amarasekara et al., 2014; Esfandiari et al., L2. For example, centres 1, 2 and 7 all reported high levels of L2 and 331 bs_bs_query

Please cite this article in press as: Jaime Gosálvez, Lamberto Coppola, Jose Luis Fernández, Carmen López-Fernández, Alfredo Góngora, Ricardo Faundez, John Kim, Nabil
Sayme, Moises de la Casa, Rebeca Santiso, Keith Harrison, Ashok Agarwal, Stephen Johnston, Sandro C. Esteves, Multi-centre assessment of nitroblue tetrazolium reactiv-
ity in human semen as a potential marker of oxidative stress, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.01.014
 ARTICLE IN PRESS
REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■–■■ 7

332 bs_bs_query may have possibly overestimated the incidence of this category or been processing. Nevertheless, it is important to consider that a negative 392
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333 bs_bs_query unable to differentiate it sufficiently from L1. We attempted to account result to NBT does not preclude these spermatozoa from resulting 393
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334 bs_bs_query for this variability, however, by having samples analysed by two dif- in a higher risk of miscarriage after ICSI, if they also have damaged 394
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335 bs_bs_query ferent observers (inter) in the same centre and twice by the same DNA (Agarwal et al., 2016). The most obvious adverse effect would 395
bs_bs_query

336 bs_bs_query observer (intra) and determined a correlation coefficient of 0.94 and be oxidative damage combined with compromised sperm DNA in- 396
bs_bs_query

337 bs_bs_query 0.96, respectively. It was impossible to compare the same ejaculate tegrity. This phenomenon has already been well described, and having 397
bs_bs_query

338 bs_bs_query between centres, as the NBT reaction typically occurs within 45 min, direct implications to clinical aspects of reproduction, has been vari- 398
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339 bs_bs_query so that the time spent transporting the semen sample between dif- ously addressed (Agarwal et al., 2005; Aitken and Baker, 2006; Cho 399
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340 bs_bs_query ferent laboratories made this comparison impractical. Although some et al., 2016; Esteves et al., 2014, 2015; Evenson et al., 2002; Henkel 400
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341 bs_bs_query of the variability may also be attributed to the technician’s indi- et al., 2005). 401
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342 bs_bs_query vidual perception of the colour change, we found in an earlier Nicotinamide adenine dinucleotide phosphate-dependent oxido- 402
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343 bs_bs_query preliminary study that, although simple visual colour determination reductase, localized at the level of mitochondria within the sperm mid- 403
bs_bs_query

344 bs_bs_query can result in a slightly lower colour discrimination ability compared piece, has been shown to contribute ROS and is a major site of oxidative 404
bs_bs_query

345 bs_bs_query with spectrophotometric and image analysis, these three methods were stress (Marchetti et al., 2002; Sabeti et al., 2016) Background ROS, 405
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346 bs_bs_query all still highly correlated (de la Casa et al., 2015). Despite our best therefore, would normally be expected to be associated with sperm 406
bs_bs_query

347 bs_bs_query efforts to standardize our patient cohorts by excluding those with mitochondria but this level of activity is not usually sufficient to reduce 407
bs_bs_query

348 bs_bs_query medical illness, it is also possible that the reported variation in the NBT resulting in visible formazan precipitates; rather, these precipi- 408
bs_bs_query

349 bs_bs_query distribution of NBT reactivity levels was simply a reflection of the rela- tates only seem to be associated with increased local ROS production 409
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350 bs_bs_query tive difference in patient cohorts at each centre. For example, patients and probably not compensated by antioxidant capacity. In addition to 410
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351 bs_bs_query in Mexico were younger than those attending the Spanish clinic. Such the detrimental effect on nuclear and mitochondrial sperm DNA, a 411
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352 bs_bs_query specific differences associated to NBT reactivity in patient cohorts are high level of •O2− can also compromise ICSI outcomes by nega- 412
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353 bs_bs_query topics for future investigation. tively affecting the oocyte cytoplasm. The role of centrosomes and 413
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354 bs_bs_query centrioles, the post-transcriptional modifications of alpha tubulins, 414

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355 bs_bs_query The cellular contribution to NBT reactivity and modifications to the caboxy-terminal tyrosine amino acid residue 415
bs_bs_query

356 bs_bs_query from alpha tubulin, have several potential implications for the manner 416
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357 bs_bs_query A high level of NBT reactivity in the neat ejaculate of some patients in which the microtubule array is, as a large part of this oxidative stress Q6 417
bs_bs_query

358 bs_bs_query was attributable to spermatozoa, leukocytes, or both. Although an as- is localized at the mid-piece where the mitochondria are located, and 418
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359 bs_bs_query sociation between high levels of NBT reactivity and sperm DNA damage to these organelles may putatively affect the first steps of 419
bs_bs_query

360 bs_bs_query fragmentation was found in this study, this association was also time fertilization by disrupting the normal formation of tubulin filaments 420
bs_bs_query

361 bs_bs_query dependent in that the incidence of sperm SDF in high and low NBT that are necessary for syngamy after sperm penetration. In fact, it 421
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362 bs_bs_query staining ejaculates increased over time; the rate of SDF was also sig- is well known that oxidative stress has the capacity to produce tubulin 422
bs_bs_query

363 bs_bs_query nificantly higher in L3 ejaculates. This finding was not surprising, given depolymerization (Lee et al., 2005), and this effect may be associ- 423
bs_bs_query

364 bs_bs_query the undisputed association between oxidative stress and DNA damage ated with the lack of orthodox chromatin-tubulin assembly after sperm 424
bs_bs_query

365 bs_bs_query in spermatozoa (Wright et al., 2014); however, it is interesting that injection (Gook et al., 1998). 425
bs_bs_query

366 bs_bs_query the baseline level of SDF observed in the neat ejaculate 30 min after Direct mapping of the NBT reaction at the level of the sperm cell 426
bs_bs_query

367 bs_bs_query ejaculation did not seem to be affected by the corresponding level or leukocyte is relatively straightforward using the Oxisperm® tech- 427
bs_bs_query

368 bs_bs_query of •O2− reactivity. This observation suggests that a high level of NBT nique (Figure 4), but the respective contributions of individual sperm 428
bs_bs_query

369 bs_bs_query reaction in the neat ejaculate was not initially and directly associ- cell damage to overall ROS damage is still not clear and deserves 429
bs_bs_query

370 bs_bs_query ated with DNA damage linked to innate sperm production in the patient, further investigation. The differential production of •O2− depending 430
bs_bs_query

371 bs_bs_query but rather is more likely to be associated with prolonged semen ex- on the leukocyte observed within the same patient may also be an 431
bs_bs_query

372 bs_bs_query posure to oxidative stress post ejaculation. A prudent strategy in these important factor at the time of evaluating the quantitative effect of 432
bs_bs_query

373 bs_bs_query patients to reduce DNA damage before insemination would be to sepa- •O2− on other cells. The production of •O2− would not only depend 433
bs_bs_query

374 bs_bs_query rate the spermatozoa rapidly from the seminal plasma or from on the number of PMN observed in the sample but also on the amount 434
bs_bs_query

375 bs_bs_query exposure to leukocytes. of •O2− that each cell is able to produce (Walrand et al., 2003). At 435
bs_bs_query

376 bs_bs_query The fact that some patients presented with a high level of NBT the cellular level it would be important to distinguish between the 436
bs_bs_query

377 bs_bs_query labelling in both the neat ejaculate and spermatoza, whereas in others sources of ROS (PMN leukocytes, sperm or both), as the clinical im- 437
bs_bs_query

378 bs_bs_query the spermatoza were free of such labelling, suggests that sperma- plications of infiltrating leukocytes are different from the pathological 438
bs_bs_query

379 bs_bs_query tozoa affected by the overproduction of superoxide may not be suitable conditions in which sperm are themselves the source of ROS. 439
bs_bs_query

380 bs_bs_query for IVF or intracytoplasmic sperm injection (ICSI). Given that it is still 440
bs_bs_query

381 bs_bs_query currently problematic to detect compromised sperm cells for ICSI, Seminal plasma contribution to NBT reactivity 441
bs_bs_query

382 bs_bs_query the possibility exists that embryologists may unintentionally select 442
bs_bs_query

383 bs_bs_query spermatoza with oxidative damage. Incubation of oocytes with sper- Although the NBT reaction is supposedly based on the generation of 443
bs_bs_query

384 bs_bs_query matozoa originating from semen samples with high NBT reactivity, ROS by sperm and leukocytes, by analysing the distribution of NBT 444
bs_bs_query

385 bs_bs_query and in which sperm DNA fragmentation increases as a function of reactivity in respective sperm and seminal plasma fractions, we ob- 445
bs_bs_query

386 bs_bs_query incubation time, are likely to be sub-optimal and this might help to served that the larger contribution •O2− was derived from the seminal 446
bs_bs_query

387 bs_bs_query explain the poorer assisted reproduction technique outcomes ob- plasma, with only 32% of samples showing sperm staining positive 447
bs_bs_query

388 bs_bs_query served when conventional IVF is used in couples whose male partner to the NBT reaction. Our results suggest that assessing the re- 448
bs_bs_query

389 bs_bs_query has high SDF (Agarwal et al., 2016). Conversely, we might expect that sponses to NBT in both the neat ejaculate and semen fractions 449
bs_bs_query

390 bs_bs_query sperm not staining positive for NBT might have better reproductive (seminal plasma and spermatozoa) may provide an additional quali- 450
bs_bs_query

391 bs_bs_query outcomes, because sperm are microinjected into oocytes shortly after tative framework to discriminate the source of oxidative damage in 451
bs_bs_query

Please cite this article in press as: Jaime Gosálvez, Lamberto Coppola, Jose Luis Fernández, Carmen López-Fernández, Alfredo Góngora, Ricardo Faundez, John Kim, Nabil
Sayme, Moises de la Casa, Rebeca Santiso, Keith Harrison, Ashok Agarwal, Stephen Johnston, Sandro C. Esteves, Multi-centre assessment of nitroblue tetrazolium reactiv-
ity in human semen as a potential marker of oxidative stress, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.01.014
 ARTICLE IN PRESS
8 REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■–■■

452 bs_bs_query individual patients. The location of labelling in sperm primarily in- A R T I C L E I N F O 510 bs_bs_query

453 bs_bs_query volved the mid-piece region, suggested that most of the •O2− was 511 bs_bs_query

454 bs_bs_query linked to mitochondrial activity. The ability to differentiate the source Article history: 512 bs_bs_query

455 bs_bs_query of the oxidative damage, whether sperm, leukocyte and/or seminal Received 26 May 2016 513 bs_bs_query

456 bs_bs_query plasma, could be highly instructive as to the respective causes of these Received in revised form 26 January 2017 514 bs_bs_query

457 bs_bs_query abnormally high levels of ROS, thereby potentially facilitating spe- Accepted 27 January 2017 515 bs_bs_query

458 bs_bs_query cific treatments to reduce oxidative damage. Declaration: While Profs Jaime 516 bs_bs_query

459 bs_bs_query A positive response of the NBT reactant with the seminal plasma Gosálvez and Jose Luis Fernández were 517 bs_bs_query

460 bs_bs_query after ejaculate fractionation and the absence of any discrete precipi- the initial developers of the Sperm 518 bs_bs_query

461 bs_bs_query tate associated to any cell remains difficult to interpret. The seminal Chromatin Dispersion test, the 519 bs_bs_query

462 bs_bs_query plasma contains natural antioxidant such as vitamins C and E, su- Halosperm® assay was developed by 520 bs_bs_query

463 bs_bs_query peroxide dismutase, glutathione, uric acid and free radical scavengers INDAS laboratories (Madrid, Spain); the 521 bs_bs_query

464 bs_bs_query (Henkel et al., 2005; Khosrowbeygi and Zarghami, 2007) but it has also work presented here represents no 522 bs_bs_query

465 bs_bs_query been associated with oxidative stress (Lissak et al., 2004; Palan and conflict of interest for any of the 523 bs_bs_query

466 Naz, 1996). Recently, it has been demonstrated that variations of the authors. 524
525
bs_bs_query
bs_bs_query

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467 bs_bs_query proteome associated with the seminal plasma are also related to levels 526 bs_bs_query

468 bs_bs_query of oxidative stress (Intasqui et al., 2015), such that the seminal plasma Keywords: 527 bs_bs_query

469 bs_bs_query can also be used as an index of ROS imbalance or oxidative stress Nitroblue tetrazolium 528 bs_bs_query

470 bs_bs_query that may be occurring both in the sperm or even in the prostate. Human spermatozoa 529 bs_bs_query

471 bs_bs_query The presence of a positive signal in the seminal plasma in an in- Oxidative stress 530 bs_bs_query

472 bs_bs_query dividual characterized as P2 (neat ejaculate positive, seminal plasma Oxisperm® 531 bs_bs_query

473 bs_bs_query positive, and sperm positive) can be assumed to be caused by excess •O2− generation 532 bs_bs_query

474 bs_bs_query free formazan deposits obtained after the reaction of •O2− emanat-
475 bs_bs_query ing from spermatozoa or PMNs. On the contrary, the reason why those 533 bs_bs_query

476 bs_bs_query individuals characterized as P3 (neat ejaculate positive, seminal plasma

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535
478
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536
479 be contributing to the generation of ROS; these subcellular par-
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480 bs_bs_query ticles are surrounded by lipid membranes so that the oxidative Agarwal, A., Gupta, S., Sharma, S., Sharma, R., 2005. Oxidative stress 537 bs_bs_query

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Please cite this article in press as: Jaime Gosálvez, Lamberto Coppola, Jose Luis Fernández, Carmen López-Fernández, Alfredo Góngora, Ricardo Faundez, John Kim, Nabil
Sayme, Moises de la Casa, Rebeca Santiso, Keith Harrison, Ashok Agarwal, Stephen Johnston, Sandro C. Esteves, Multi-centre assessment of nitroblue tetrazolium reactiv-
ity in human semen as a potential marker of oxidative stress, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.01.014