Original Article

Vol.
48 (1): 131-156, January - February, 2022

ORIGINAL ARTICLE
doi: 10.1590/S1677-5538.IBJU.2021.0604
The combined effect of lifestyle intervention and antioxidant

therapy on sperm DNA fragmentation and seminal oxidative
stress in IVF patients: a pilot study
_______________________________________________
Peter Humaidan 1, 2, Thor Haahr 1, 2, Betina Boel Povlsen 2, Louise Kofod 2, 3, Rita Jakubcionyte Laursen 2,
Birgit Alsbjerg 1, 2, Helle Olesen Elbaek 2, Sandro C. Esteves 1, 2, 4, 5
1
Department of Clinical Medicine, Aarhus University, Denmark; 2 The Fertility Clinic Skive, Skive Regional
Hospital, Denmark; 3 Department of Obstetrics and Gynecology, Regional Hospital Herning, Denmark;
4
ANDROFERT, Clínica de Andrologia e Reprodução Humana, Campinas, SP, Brasil; 5 Departamento
de Cirurgia, Divisão de Urologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas
(UNICAMP), Campinas, SP, Brasil
ABSTRACT ARTICLE INFO
Purpose: Sperm DNA fragmentation (SDF) and seminal oxidative stress are emerging Peter Humaidan
measurable factors in male factor infertility, which interventions could potentially https://orcid.org/0000-0001-6884-5366
reduce. We evaluated (i) the impact of lifestyle changes combined with oral antioxidant
intake on sperm DNA fragmentation index (DFI) and static oxidation-reduction potential Keywords:
(sORP), and (ii) the correlation between DFI and sORP. DNA Fragmentation;
Materials and Methods: We conducted a prospective study involving 93 infertile males with Reproductive Techniques,
a history of failed IVF/ICSI. Ten healthy male volunteers served as controls. Semen analysis Assisted; Infertility
was carried out according to 2010 WHO manual, whereas seminal sORP was measured using
Int Braz J Urol. 2022; 48: 131-56
the MiOXSYS platform. SDF was assessed by sperm chromatin structure assay. Participants
with DFI >15% underwent a three-month lifestyle intervention program, primarily based on
diet and exercise, combined with oral antioxidant therapy using multivitamins, coenzyme _____________________
Q10, omega-3, and oligo-elements. We assessed changes in semen parameters, DFI, and sORP, Submitted for publication:
and compared DFI results to those of volunteers obtained two weeks apart. Spearman rank August 18, 2021
correlation tests were computed for sORP and DFI results. _____________________
Results: Thirty-eight (40.8%) patients had DFI >15%, of whom 31 participated in the Accepted after revision:
intervention program. A significant decrease in median DFI from 25.8% to 18.0% was seen August 20, 2021
after the intervention (P <0.0001). The mean DFI decrease was 7.2% (95% CI: 4.8-9.5%; _____________________
Published as Ahead of Print:
P <0.0001), whereas it was 0.42% (95%CI; -4.8 to 5.6%) in volunteers (P <0.00001). No
August 30, 2021
differences were observed in sperm parameters and sORP. Based on paired sORP and DFI
data from 86 patients, no correlation was observed between sORP and DFI values (rho=0.03).
Conclusion: A 3-month lifestyle intervention program combined with antioxidant
therapy reduced DFI in infertile men with elevated SDF and a history of failed IVF/ICSI.
A personalized lifestyle and antioxidant intervention could improve fertility of subfertile
couples through a reduction in DFI, albeit controlled trials evaluating reproductive
outcomes are needed before firm conclusions can be made.
Trial registration number and date: clinicaltrials.gov NCT03898752, April 2, 2019.
131
IBJU | LIFESTYLE AND ANTIOXIDANTS ON SPERM FUNCTION IN IVF PATIENTS
INTRODUCTION the automated Male Infertility Oxidative System

(MiOXSYS) (13). This automated method has been
Male infertility accounts for approximate- suggested as a less time-consuming and more effi-
ly 20-30% of infertility cases; however, combined cient way of assessing the complete redox balance
with female factor infertility it contributes to at of all known and unknown oxidants and antioxi-
least 50% of infertility cases (1). Male infertility dants in semen (14). However, until now, results
involves a broad causative spectrum, including are conflicting regarding the correlation between
infections, endocrine disorders, cryptorchidism, the seminal ORP level, routine semen parameters,
genetic conditions, and varicocele, but in up to and DFI (15-17). Moreover, to suggest a clinical
50% of cases, the cause of male infertility remains implementation of ORP measurement and DFI tes-
unknown (2). An important contributor to male ting in daily practice, a potential causal inference
factor infertility is sperm DNA fragmentation, needs to be made.
which is generally not routinely assessed before To further explore this emerging field of
or during infertility treatment (3, 4). male infertility, the objectives of the present stu-
It has been shown that more than 20% of dy were to evaluate the combined impact of a
normozoospermic males have a DNA fragmen- three-month questionnaire-based personal lifes-
tation index (DFI) >27%, resulting in decreased tyle change and oral antioxidant therapy on DFI
chances of conception after intrauterine insemi- and ORP in men with infertility and DFI levels
nation (IUI) (5). Moreover, in vitro fertilization >15%, undergoing medically assisted reproduc-
(IVF) success rates are lower in men with high DFI tion (MAR); moreover, the possible correlation be-
(6, 7). A recent meta-analysis comparing IVF and/ tween DFI and ORP was also investigated.
or intracytoplasmic sperm injection (ICSI) success
rates in men with low and high DFI reported hi- MATERIALS AND METHODS
gher clinical pregnancy rates in favor of the low
DFI group, regardless of fertilization method; for Ethics
IVF, the odds ratio (OR) was 1.92 (95% CI; 1.33- The Central Denmark Region Committees
2.77) and for ICSI, 1.49 (95% CI; 1.11-2.01) (8). on Health Research Ethics approved the conduct of
Notably, a routine semen analysis with se- this study, file number 1-10-72-148-19. The study
men parameters within normal ranges does not was registered at clinicaltrials.gov: NCT03898752.
preclude the presence of elevated DNA fragmen- All participants provided written informed consent.
tation, which has been associated with poor re-
productive outcome after IUI and IVF treatment Participants
(3, 4, 6). In addition, it has been suggested that the In this pilot study, a total of 93 consecu-
use of testicular sperm to be superior as compared tive men with idiopathic or unexplained infertili-
to ejaculated sperm for ICSI in patients with DFI ty undergoing MAR treatment with their partners
levels >29% (9, 10), and that the use of this indi- were prospectively included between November
vidualized approach resulted in increased live bir- 2018 and May 2020. Only patients who had at le-
th rates -especially for patients in whom previous ast two failed IVF/ICSI trials for no apparent rea-
ICSI cycles using ejaculated sperm had failed (3, sons, resulting in either embryo developmental ar-
6). Since testicular sperm aspiration is an invasive rest, implantation failure, biochemical pregnancy,
procedure, other less-invasive interventions that or miscarriage were invited to participate. Before
could potentially reduce DFI levels in high DFI embarking on our IVF program, males underwent
males should be explored. a standard workup, including history, physical exa-
Oxidative stress (OS) is a potentially modi- mination, semen analysis, and serology. Hormone
fiable cause of DFI elevation, seen in male factor testing, ultrasound scan, and genetic testing were
infertility (11, 12). A surrogate measure for the le- applied as appropriate.
vel of seminal OS is the oxidation-reduction po- Participants were non-smokers, including
tential (ORP). The ORP can be quantified by using no intake of cannabis, and were not taking me-
132
dication with known gonadotoxic effects when ORP measurement

entering the IVF program. Moreover, at the first ORP in semen was measured in-house
consultation, all patients were advised by the tre- using the MiOXSYS platform (MiOXSYS, Aytu
ating physician to reduce exposure to pesticides, BioScience Inc, Englewood, CO, USA). MiOXSYS
irradiation, and excess heat in daily life and work. is a qualitative electrochemical test for the detec-
Furthermore, patients with sperm concentrations tion of oxidative stress in fresh or frozen semen
below 5 million/mL in raw semen had a testicular specimens. A 30µL of the semen sample was loa-
ultrasound to exclude malignancies. Genetic testing ded into the MiOXSYS sensor, which was imme-
including karyotype, Y-chromosome microdeletion diately inserted into the analyzer. The analysis
screening, and cystic fibrosis transmembrane con- was completed in approximately three minutes,
ductance regulator (CFTR) gene analysis were per- and the final static oxidation-reduction potential
formed in males with sperm concentrations below (sORP) reading in millivolt (mV) was displayed on
1 million/mL in the raw semen, according to the the MiOXSYS display screen. This value was nor-
recommendation by the Danish Health Authority. malized to the sperm concentration of the sam-
Patients with varicocele, malignancies, genetic cau- ple. The result was reported as mV/106 sperm/mL.
ses of infertility, and those using any medication A cut-off value of 1.36 mV/106 sperm/mL sORP
were excluded from the study. has previously been proposed based on correla-
tions to sperm concentration, motility, and sperm
Healthy volunteers morphology (14). sORP values above the normal
Through direct contact to the local military range imply an imbalance between oxidants and
base a convenience sample consisting of 10 healthy antioxidants, signaling the presence of oxidati-
male conscript volunteers with a mean age of 31 ve stress in the specimen. No positive/negative
years, defined as non-smokers and presumably not control protocols are provided by the manufac-
suffering from any known disease served as con- turer. However, to assess test reproducibility, we
trols. None of the controls had children or fathered conducted paired assessments, using specimens
previous pregnancies. The main aim of this group of ten patients. Variability was analyzed by cal-
was to investigate the biological variability in se- culating the mean difference between the two
men parameters. The controls delivered two semen readings. The mean difference was 0.10 mV/106
samples, respecting a 14-day interval under similar sperm/mL sORP (Standard error: 0.20; P=0.60).
circumstances as the IVF patients, regarding absti-
nence time and examination method in the same DFI analysis
laboratory. These samples were blinded, and hence, Part of the semen sample was shipped to
the DFI laboratory could not identify the male vo- a central laboratory (SPZ lab A/S, Copenhagen,
lunteers, nor did the DFI laboratory know the re- Denmark) for DFI analysis. An aliquot of 0.2mL
sults of the first DFI test. The first measurements of of the semen sample was transferred to a cryo-
sORP and standard semen quality parameters were -vial and diluted with 0.2mL sperm washing me-
also blinded for the laboratory technician who per- dium (G-IVF™ PLUS, Vitrolife) and snap-frozen
formed the analysis of the second test. at -80°C. The samples were shipped in a dry-
-shipper container containing liquid nitrogen
Sampling and semen analysis (-196°C). DFI was determined using the sperm
Patients were instructed to have a mini- chromatin structure assay (SCSA) (19). Results
mum of two days and a maximum of three days were expressed as the percentage of sperm with
abstinence before delivering the semen sample DNA fragmentation (% DFI) using the flow cyto-
on-site. Semen analysis was performed as part of meter software. A minimum of 10,000 events was
diagnostic testing according to WHO 2010 crite- recorded, and all tests were run in parallel with
ria (18), measuring ejaculate volume, sperm con- positive and negative controls.
centration, total sperm number, total motility, and
progressive motility.
133
Questionnaire-based personalized lifestyle in- Antioxidant therapy

tervention All patients with DFI >15% received an-
All males with a DFI >15% were asked to tioxidant therapy, which included a combination
embark on a three-month lifestyle intervention of daily nutraceutical supply of coQ10 100mg
program based on a questionnaire consisting of (OmniQ10 Energy, Biosym A/S), one multivitamin
lifestyle parameters. The patients received indi- (Apovit®) tablet, and 1g Omega-3 (Apovit®) dai-
vidualized recommendations as to lifestyle inter- ly. One multivitamin tablet contained: vitamin A
vention according to their answers to a structured 800ug, vitamin B1 1.1mg, vitamin B2 1.4g, vita-
questionnaire including a total 68 of questions, min B6 1.4mg, vitamin B12 2.5ug, niacin 16mg,
covering among others smoking, alcohol con- pantothenacid 6mg, folic acid 200ug, vitamin C
sumption, caffein intake, diet, exercise, body wei- 80mg, vitamin D 5ug, vitamin E 12mg, magnesium
ght, stress, and work-related exposure (Appendix). 100mg, molybdenum 50ug, iron 14mg, zinc 10mg,
The proposed lifestyle interventions included pri- copper 1mg, manganese 2mg, chromium 40ug, se-
marily general health recommendations for adults lenium 55ug, and iodine 150ug. The choice of the
from the Danish Health Authority (www.sst.dk/ above commercially available multivitamin tablet
da/Viden). In brief, at least 30 minutes of exer- was primarily based on patient convenience as it
cise daily was recommended. Regarding nutrition is widely available in our market and the fact that
and food consumption, a reduction in meat in- the manufacturer’s recommended daily doses was
take (maximum of 500g/week) and, in particular, mostly in accordance to the recommended dietary
caution about red meat, more intake of fruit and allowance. Moreover, published data indicate that
vegetables, and reducing high sugar-containing the above vitamin/mineral combination and dosa-
drinks, including soda and energy drinks were ge provides adequate antioxidant effects (23).
recommended. Moderate alcohol intake <6 units
per week was also recommended. All patients em- Follow-up and outcome
barking on our IVF program were already non- After three months of intervention (lifes-
-smokers by recommendation. We considered any tyle and antioxidant therapy), a new semen sam-
potential work-related exposures and recommen- ple was delivered, respecting the same abstinence
ded, for example, facemasks to reduce airborne time as for the initial semen sample. The treating
exposure when relevant. In case of a subjective physician checked patient compliance with the
feeling of stress, we recommended mindfulness. intervention at the follow-up visit. The primary
Finally, we advised against having the cellphone outcome was the possible effect of life-style in-
in the trouser pocket and the laptop on the lap. tervention and antioxidant therapy on sORP and
The decision to use DFI >15% as cut-off DFI levels. Other outcomes were to investigate a
was based on (i) our preliminary SCSA data, in- possible correlation between sORP and DFI and
dicating that reproductive outcomes of couples compare the effect of the intervention on elevated
undergoing IVF/ICSI are reduced when threshol- DFI in IVF patients to the biological DFI variation
ds exceed 15% (20), and (ii) clinical SCSA data in healthy controls.
on natural pregnancy published elsewhere (21),
revealing that men producing ejaculates with Sample size
adequate routine semen parameters and less than A formal sample size calculation was not
15% DFI had no delay in pregnancy initiation possible as this was a single-arm exploratory pilot
(<4 months), whereas counterparts with 15%- study using for the first time a personalized lifestyle
30% DFI took a longer time to achieve pregnancy intervention in combination with antioxidant
(4-12 months). The decision to use a three-mon- therapy and a DFI cut-off of 15%. However, a post-
th lifestyle intervention period was based on the hoc power analysis was conducted on the basis of
time for spermatogenesis, previously reported to the mean difference in DFI results from specimens
be 64+/-8 days (22). of healthy volunteers obtained two weeks apart
134
and the results of studied patients from before to RESULTS

after intervention.
Lifestyle and antioxidant intervention and im-
Statistical methods pact on DFI, sORP on semen parameters
Baseline data is described with median A total of 38 of 93 patients (40.8%) with
and range. Spearman rank correlation test was a history of failed IVF had DFI >15% (Figu-
computed for sORP and DFI results to have a re-1). Of them, six males declined to participate
nonparametric comparator to the regression in the three-month intervention despite having
model. The linear regression model was based DFI >15% (DFI ranged from 15.4-17.5% in tho-
on the standard assumptions, plots of the re- se six patients). In addition, one male was lost to
siduals, and a leverage plot to investigate ex- follow-up. Thus, a total of 79% (31/38) of patients
treme outliers. In case data did not meet the with DFI >15% participated in the three-month
assumptions on the original scale, we inves- personalized lifestyle intervention, after which a
tigated log-transformed values. We applied a follow-up semen sample was delivered.
chi-square test to evaluate the association be- Table-1 shows the patient’s DFI, sORP, and
tween DFI and sORP as a binary variable using semen parameters, before and after a 3-month
the cut-offs stated. Adjustment for total moti- lifestyle intervention and antioxidant therapy. A
lity was made by multiplying the motility per- significant decrease in median DFI from 25.8% to
centage on the sORP value. For the follow-up 18.0% was seen after the intervention (P <0.0001).
analysis pre/post-intervention, we used paired The mean DFI decrease was 7.2% (95% CI: 4.8-
t-test on the differences after testing distribu- 9.5%; P <0.0001), and its correspondent distri-
tion with quantile-quantile plots and variance bution ranged from -19.7% to 5.3%, i.e., at the
in Bland-Altman plots. If data did not meet the individual level, a high variance was observed as
assumptions on the original scale, log-trans- some males experienced a large decrease in DFI,
formed values were investigated, reporting the whereas others had a slight increase in DFI (Fi-
median ratio between pre/post-intervention gure-2). Notably, a total of 84% (26/31) had a de-
measurements. The estimates are given with the crease in DFI, and 29% (9/31) had a DFI of ≤15%
95% confidence interval (CI) and the minimum at follow-up. In our study, a total of 13 men had
and maximum values when relevant. baseline DFI above 27%; after the intervention,
In order to compare healthy volunteers 69% (9/13) of them had a DFI below 27%. Only
and IVF patients with follow-up data, we used a one patient discontinued the interventional pro-
one-sample t-test on the differences before and gram before completion for unknown reasons. All
after intervention in IVF patients and the mean others complied with the intervention program for
in controls. Sensitivity analyses were also made three months.
according to the 95% CI around the mean in the A total of 28 males were available for sORP
controls. The rationale in these analyses was to analysis before and after lifestyle intervention as
compare biological variation in semen parameters three patients had a semen concentration below 1
to those seen after the intervention. One patient million/mL. According to the manufacturer’s ins-
lost to follow-up was not included in the analysis. tructions, sORP should only be performed in spe-
A total of eight men decided to take another an- cimens with sperm concentration above 1 million/
tioxidant therapy than recommended by the unit mL. No statistically significant sORP reduction
(Punalpin®; N=4) and Inoman®; N=4)). These pa- was seen after lifestyle intervention as given by
tients were included in the primary analysis but the median ratio between pre/post lifestyle inter-
excluded from the sensitivity analysis. Data were vention (sORP values: 0.67; 95% CI: 0.41 to 1.11).
analyzed using STATA IC©, version 16.1, Stata- The distribution of sORP values was large; some
Corp LLC, USA. men experienced an 8-fold increase in sORP post-
135
Figure 1 - Flow diagram showing total patient breakdown.
Table 1 - Semen parameters, sperm DNA fragmentation index (DFI) and seminal static oxidation-reduction potential (sORP)
after 3-month lifestyle intervention and antioxidant therapy in 31 infertile men with DFI>15%.
Baseline Follow-up* Ratio** P-value
Sperm concentration (x106/mL) 12.9 (7.0-23.7) 13.4 (7.3-24.4) 1.03 (0.8-1.4) 0.812
Total motile sperm count (x106/mL) 15.5 (8.1-29.9) 13.7 (7.6-24.7) 0.89 (0.6-1.3) 0.499
%DFI 25.8 (23.0-29.0) 18.0 (15.1-21.6) 0.70 (0.6-0.8) 0.000
sORP (mV/106 sperm/mL) 1.3 (0.7- 2.5) 0.9 (0.4-1.8) 0.67 (0.4-1.1) 0.114
*After 3-month intervention program
Values are median and interquartile range
**Ratio between post-intervention and baseline values
136
Figure 2 - Boxplots showing the difference between sperm DNA fragmentation (DFI) measurements of 10 healthy
controls who delivered two semen specimens within a 14-day interval and 31 IVF patients with baseline DFI >15% who
had lifestyle intervention and antioxidant therapy for 3 months. The boxplot includes the median (horizontal line in the
box), 25-75% interquartile range box (i.e., representing 50% of the data), minimum and maximum values excluding
outliers (whiskers extending outside of the box), and outlier (blue dot). The DFI differences between the groups were
significant (P <0.0001).
-intervention, whereas others had a 20-fold re- Correlation between DFI and sORP at baseline
duction in sORP (range: 0.05 to 8.10). In the sub- A total of 86 patients from the 93 to-
group of patients with sORP >1.36mV/106 sperm/ tal study population was eligible for the corre-
mL (N=12), we observed a significantly lower lation analysis (Table-2). Seven patients with a
sORP in the second measurement than the first sperm concentration in the raw sample less than
measurement (t-test: median ratio 0.34; 95% CI: 1 million/mL were excluded from the correlation
0.15 to 0.77), but the equivalent non-parametric analysis, but not from the intervention part of the
test did not reveal a significant difference. Sensiti- study. The seven patients with <1 million/mL were
vity analysis excluding patients who administered clear outliers, presenting with extremely high
either Punalpin (N=4) or Inoman (N=4) yielded si- sORP measurements assumed to be caused by the
milar results (data not shown). normalization to very low sperm concentration.
The median ratio in sperm concentration Using the sORP cut-off at 1.36 and the DFI cut-off
before and after the intervention was small and not at 15% to evaluate outcome as binary variables,
statistically significant (median ratio 1.03; 95% CI: no correlation between sORP and DFI was seen
0.8 to 1.4). The total motile sperm count was also not (OR=0.81; 95% CI; 0.34 to 1.90, Table-3).
statistically different from before to after interven- As raw data did not meet the assumptions
tion (median ratio 0.89; 95% CI: 0.6 to 1.3). to fit a linear model, a Spearman’s rank correla-
137
Table 2 - Baseline data of 86 infertile men included in the correlation analysis between sperm DNA fragmentation index (DFI)
and seminal static oxidation reduction potential (sORP).
Variable Median (IQR)
Demographics:
Age; years 35 (24-55)
Body Mass Index (BMI); kg/m2 26 (19-42)
Semen analysis:
Volume; mL 2.4 (0.5-8.0)
Concentration; 106/mL 25.0 (1.0-300.0)
Total motile count; 106 32.4 (0.4-556.0)
Sperm function:
DNA Fragmentation Index (DFI); % 14.3 (4.0-74.8)
Seminal Oxidation Reduction Potential (sORP)*; mV/106 sperm/mL 1.30 (0.00-30.50)
*Two samples had sORP values which were below 0.00 at the MIYOXIS display –these were relabeled as 0.00.
IQR: interquartile range
Table 3 - 2x2 table of dichotomized sperm DNA fragmentation index (DFI) and static oxidation-reduction potential (sORP).
Normal DFI: ≤15% (N) High DFI: >15% (N) Total (N)
Normal sORP level: ≤1.36 mV/10 sperm/ 6

24 22 46
mL (N)
High sORP level: >1.36 mV/106 sperm/

23 17 40
mL (N)
Total (N) 47 39 86
The chi-square statistic is 0.24. The P value is 0.62.
tion was used to test the overall hypothesis of mo- Lifestyle intervention compared to biological
notonic correlation between sORP and DFI. Again, variation in DFI
the test did not support any correlation (rho=0.03; In the volunteer group (N=10), the mean
Figure-3A). After log-transformation, data passed difference between the two consecutive DFI me-
the standard assumptions of the linear regression asurements performed 14 days apart was 0.42%
model. However, the 95% CI of the regression coe- (95% CI: -4.8 to 5.6%), indicating no systematic
fficient exceeded 0 (0.05; 95% CI; -0.04 to 0.15), and difference between one DFI measurement to the
consequently, there was no evidence to support an other. Moreover, the DFI difference in volunteers
association between DFI and sORP. A multiple linear was significantly lower than the relatively high
regression model adjusted for age and BMI showed a difference observed after lifestyle and antioxidant
similar regression coefficient of 0.05 (95% CI; -0.04 intervention in IVF patients (P <0.00001; Figu-
to 0.15). As a recent study suggested that the sORP re-2). The sensitivity analyses comparing mean
adjusted for total motility might be a better outcome DFI difference in IVF patients according to the
marker (24), this was also investigated; however, with 95% CI of the mean difference in controls was also
similar results (rho= -0.09; Figure-3B). highly significant (P <0.00001). The differences in
138
Figure 3 - Scatterplot of the correlation between Sperm DNA Fragmentation and Seminal Oxidation Reduction Potential,
excluding patients with sperm concentration below 1 million/mL. A total of 86 patients was included. As raw data did
not meet the assumptions to fit a linear model, a Spearman's rank correlation was run to test the overall hypothesis of
monotonic correlation between sORP and DFI (A), and sORP normalized for motility and DFI (B). The test indicated a very
weak correlation: rho=0.03 and -0.09, respectively.
sORP from the first to the second measurement sperm concentration and total motile sperm count.
after 14 days were not normally distributed and The DFI reduction seen in the IVF patients was sig-
had unequal variance. The median sORP differen- nificantly higher than the DFI biological variation
ce was 0.12 lower at the second measurement af- in the control group of healthy volunteers who
ter 14 days, but ranged from 15.91 lower to 2.95 delivered a semen sample 14 days apart without
higher (P=0.15). lifestyle intervention. Additionally, no significant
A post-hoc power analysis based on the effect of lifestyle intervention and antioxidant tre-
mean DFI results ultimately obtained from the two atment was observed on sORP; however, in the
specimens of volunteers and patients subjected to subgroup of patients with elevated sORP >1.36
the intervention indicated that the power of the (N=12), we observed a significantly lower sORP
studied cohort to have an 80% chance of detec- by parametric testing, albeit this was not signifi-
ting, as significant at the 5% level, a decrease of cant in the nonparametric test. Thus, this finding
approximately 7% in DFI with the interventional warrants further exploration in a larger prospecti-
program was 96.5%. ve trial. Finally, no significant correlation between
sORP and DFI was seen.
DISCUSSION
Interpretation
Main findings The present study was not designed to
This pilot study investigated (i) the effect of evaluate sORP or DFI as individual markers of re-
lifestyle and antioxidant therapy on DFI and ORP in productive health, and thus, the results cannot be
a single-arm intervention trial in male IVF patients used as evidence for one marker being superior
with IVF failure and DFI >15%, (ii) the correlation to the other. In contrast, our interpretation of the
between sORP and DFI, and (iii) lifestyle interven- lack of correlation between sORP and DFI is that
tion compared to biological variation in DFI. these two qualitative seminal markers (at least
We found that a three-month lifestyle when measured as reported herein) should be con-
changes alongside oral antioxidant intake redu- sidered separately, i.e., an elevated sORP does not
ced DFI, whereas no significant effect was seen on necessarily correlate with an elevated DFI and vice
139
versa. Thus, although oxidative stress is widely re- generally or in the group of males with a high
ported to be one of the major causes of elevated baseline DFI >25% (28).
DFI (25, 26), oxidative stress, when measured by The most recent Cochrane meta-analysis
sORP, cannot be directly correlated to DFI. concluded that the current evidence on interven-
In relation to this aspect, a recent retros- tion with antioxidants in subfertile men is incon-
pective cross-sectional study also investigated the clusive due to the low quality of the evidence (29).
correlation between DFI and sORP in male infer- Nevertheless, in the Cochrane meta-analysis, low-
tility patients (N=1068) (24). Similar to our study, -quality evidence according to GRADE anticipa-
the conclusion was that the DFI and sORP should tes increased live birth rates in case of antioxi-
be considered as two separate measurements. Ho- dant intervention; thus, there is a need for more
wever, in contrast to the present study, the authors well-conducted studies in this field. Collectively,
did not exclude a potential correlation between results of the antioxidant intervention remain
DFI and sORP when taking into account sperm equivocal, and interstudy comparison is restricted
motility in the sORP measurements. Neverthe- by differences in baseline characteristics, the in-
less, the baseline characteristics of the patients in tervention per se, and differences in methods to
the aforementioned study differ remarkably from evaluate the outcome. In this aspect, it could be
this study as those patients generally had higher speculated whether regulation should be improved
DFI and sORP levels. Moreover, DFI was measu- in the field of antioxidants, as frequently antioxi-
red differently (i.e., using the Halosperm G2 test dants are considered food supplements -not medi-
kit), and importantly, it could be argued that the cal drugs- leading to inadequate rigor regarding
linear relationship assumed by receiver operating efficacy and safety.
characteristics curves, made in that study, cannot Importantly, not only lifestyle interven-
sufficiently estimate cut-points for optimal sen- tions and antioxidants seem to impact the DFI (30).
sitivity and specificity in exponential sORP data. A meta-analysis evaluating the effect of follicle-
Intervention-based studies for elevated DFI -stimulating hormone (FSH) therapy on DFI in
and sORP in male infertility only recently star- male infertility reported a significant reduction in
ted to emerge. One study investigated the effect the mean DFI after treatment with exogenous FSH
of a three-month administration of an antioxi- (4.24%; 95% CI 0.24-8.25%) (31). Also, varicoce-
dant preparation containing a variety of vitamins le repair has been shown to significantly reduce
and nutrients. The study included a group of pa- DFI in infertile men with palpable varicocele (32,
tients with male infertility of idiopathic (N=119) 33). In these lines, use of testicular sperm for ICSI
or unexplained (N=29) origins (12). That study has been explored in non-azoospermic males with
evaluated the effect of antioxidant therapy on high DFI values, as DFI was shown to be marke-
DFI and sORP, reporting that antioxidant treat- dly reduced in testicular as compared to ejaculated
ment significantly improved both DFI and sORP sperm (9, 34).
in both patients subgroups. This effect was even
more pronounced when evaluating the group of Lifestyle and semen quality
men with high baseline DFI -in that study defined To the best of our knowledge, this is the
as DFI >30% and using the sperm chromatin dis- first study evaluating the effect of lifestyle inter-
persion test. Another recent study included men vention combined with antioxidant therapy on
from a single fertility center based on DFI >16% DFI, ORP and standard semen parameters. Althou-
(N=35), investigating the effect of intervention gh the effect of the present lifestyle intervention
with 1800mg freeze-dried açai pulp per day (27). cannot lead to isolated evidence for any single in-
After at least 74 days of follow-up, a statistically tervention, we point to the fact that the lifestyle
significant and relatively high reduction in DFI interventions used were quite basic and with only
(-17.03±2.51%) was observed. In contrast, a re- minor adjustments to the general health recom-
asonably large and well-conducted RCT reported mendations for adults set by the Danish Health
that antioxidant intervention did not reduce DFI Authority. It could be speculated that most ma-
140
les already follow these simple recommendations, baseline were part of the study. Another limitation
but in fact, a recent large (N=1149) questionnaire- refers to the use of only DFI and ORP as outcome
-based study in subfertile men provided evidence measures. Other indicators of oxidative stress such
against this assumption (35). Interestingly, subfer- as lipid peroxidation, reactive oxygen species as-
tile men were reported to more frequently have sessment, 8-oxoguanine determination could have
lifestyle risks and on top of that, being poorly provided additional information, but these assays
educated on lifestyle factors and their association were not available to us.
with semen quality. A strength of the study is that we selected
The evidence for intervention with physi- patients based on previous IVF failure and baseline
cal exercise is mostly circumstantial. Cross-sec- DFI values shown to affect the probability of preg-
tional studies show that a sedentary lifestyle is nancy potentially. The inclusion of a healthy vo-
associated with low semen quality (36), whereas lunteer group for comparison is also a strength of
self-reported increased physical activity appears the study as random biological variations occurring
to be associated with increased motility (37) and within 14 days do not appear to be the cause of the
total sperm count (38). Cell-phone “trousers” sto- DFI reduction observed after three month’s lifestyle
rage has been associated with increased DFI (39, intervention in the IVF patient group. We did not
40). This effect -among others- could be media- evaluate reproductive outcomes in this pilot study;
ted by radiation damage to the NAD+ isocitrate however, natural pregnancies during the interven-
dehydrogenase enzyme (41). As for diet, a recent tion were reported by two patients. Whether the
large cross-sectional study in young Danish men DFI reduction after an intervention like ours trans-
(N=2935) found that a prudent diet -as recom- lates into improved reproductive outcomes, both
mended in the present study- was associated with naturally and after ART, warrants further research.
higher median sperm count as compared to a more Nevertheless, recent data indicate that the magni-
“western” diet, including high amounts of proces- tude of SDF decrease after other interventions (e.g.,
sed foods, high on fat and sugar (42). microsurgical varicocelectomy) was similar to that
reported in the current study, with a potential posi-
Strengths and limitations tive impact of on reproductive success (32).
The limitations of the present pilot study
are the small sample size, lack of formal a priori CONCLUSIONS
power calculation, and lack of a control group of
male infertility patients with DFI >15%. However, We herein report that a three-month lifesty-
our single-arm trial was primarily designed to ob- le change and antioxidant therapy program signifi-
tain preliminary evidence for the efficacy of the cantly reduced the DFI. This effect was statistically
interventional treatment. Thus, the present findin- significant compared to a healthy control group
gs could be considered pivotal for the design and who delivered two semen samples 14 days apart
power calculation of future trials. In these lines, without any lifestyle intervention. No difference
our post-hoc analysis based on the mean DFI re- in ORP was seen after lifestyle intervention. Mo-
sults obtained from volunteers and treated patients reover, no correlation between sORP measured by
indicated that the number of included subjects MiOXSYS and DFI measured by the SCSA method
was close to that needed to obtain 100% power, was observed. This finding suggests that these two
using type I and type II errors of 0.05 and 0.20, seminal markers should be evaluated separately.
respectively, to detect a decrease of approximately Personalized lifestyle and antioxidant intervention
7% in DFI with the interventional program. may potentially improve the fertility of subferti-
A potential selection bias is the fact that le couples through an improved DFI, albeit larger
patients who declined to participate in the follow- controlled trials evaluating pregnancy and live bir-
-up (N=6) were all close to the DFI threshold. In th rates are needed before firm conclusions can be
total, only six males with a DFI between 15-20% at made.
141
ACKNOWLEDGEMENTS 7. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L,

Erenpreiss J, et al. Sperm DNA integrity assessment in
prediction of assisted reproduction technology outcome.
We thank all staff, including the study nur-
Hum Reprod. 2007; 22:174-9.
se office at The Fertility Clinic, Regional Hospital 8. Simon L, Zini A, Dyachenko A, Ciampi A, Carrell DT. A
Skive, Denmark for their enthusiastic collaboration systematic review and meta-analysis to determine the
during the study period. Moreover, we gratefully effect of sperm DNA damage on in vitro fertilization and
thank the patients who participated in this study. intracytoplasmic sperm injection outcome. Asian J Androl.
2017; 19:80-90.
DATA ACCESSIBILITY STATEMENT 9. Esteves SC. Testicular versus ejaculated sperm should be
used for intracytoplasmic sperm injection (ICSI) in cases
Trial data will be made available on rea- of infertility associated with sperm DNA fragmentation |
sonable request to the corresponding author. Dei- Opinion: Yes. Int Braz J Urol. 2018; 44:667-75.
dentified participant data will be provided after 10. Esteves SC, Roque M, Bradley CK, Garrido N. Reproductive
outcomes of testicular versus ejaculated sperm for
approval by the corresponding author..
intracytoplasmic sperm injection among men with high
levels of DNA fragmentation in semen: systematic review
CONFLICT OF INTEREST and meta-analysis. Fertil Steril. 2017; 108:456-467.e1.
11. Agarwal A, Panner Selvam MK, Arafa M, Okada H, Homa
None declared. S, Killeen A, et al. Multi-center evaluation of oxidation-
reduction potential by the MiOXSYS in males with abnormal
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26. Jeremias JT, Belardin LB, Okada FK, Antoniassi MP, Lassen TH, et al. Is Sedentary Lifestyle Associated With
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47:275-83. 38. Sun B, Messerlian C, Sun ZH, Duan P, Chen HG, Chen YJ, et
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39. Rago R, Salacone P, Caponecchia L, Sebastianelli A, Marcucci 42. Nassan FL, Jensen TK, Priskorn L, Halldorsson TI, Chavarro
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2021: 1-10. Epub ahead of print. _______________________
Correspondence address:
41. Hagras AM, Toraih EA, Fawzy MS. Mobile phones Peter Humaidan, Prof, DMSc
electromagnetic radiation and NAD+-dependent The Fertility Clinic, Skive Regional Hospital and
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E-mail: [email protected]
144
APPENDIX
Baseline questionnaire
A large number of factors, including lifestyle can lead to reduced fertility in men. Recent research within the
field shows that males can improve their fertility by lifestyle intervention.
At the Regional Hospital in Skive, a trial is being carried out, in which we investigate different factors impacting
male fertility. We already have a presumption about some of the important factors, but we want to document
the effect in depth. Thus, your participation in this study is important to move the field ahead.
In the questionnaire, please provide information about your life and lifestyle as accurately as possible. Based
on the information, we will subsequently review your answers and suggest personalized life style changes
wherever possible. The goal is for you to possibly achieve an improvement in fertility prior to embarking on
fertility treatment.
In connection with the study, your sperm quantity will be examined. Moreover, an advanced quality analysis
of the sperm (e.g., DNA fragmentation analysis) will also be performed. As the production of new sperms takes
approximately 3 months, possible changes in quantity and quality will be assessed 3 months after lifestyle
intervention through a repeat sperm analysis.
Identification:
1. Name: ___________________________________________________________________
2. CPR: ____________________________________________________________________
3. Email: ___________________________________________________________________
Information on fertility and infertility
4. Have you achieved pregnancy / children in previous relationships?
Yes
No
If yes, enter the year your last child was born: _____________
5. Have you achieved pregnancy / children in the current relationship?

Yes
No
If yes, enter the year your last child was born: _____________
145
Information about genetic disorders in you or your family
6. Are you a carrier of a hereditary disease or a gene defect?

Yes
No
If yes, state which hereditary disease and / or gene defect: _______________________________________
7. Is there anyone in your family who is a carrier of a hereditary disease or a gene defect?
Yes
No
If so, state which hereditary disease or gene defect and who in the family has this disease:
___________________________________________
Diseases and medication consumption
8. Have you previously been admitted to hospital or psychiatric ward for treatment?
Yes
No
If yes, please state the following:
Year Name disease
Possibly, Additional Information:

_______________________________________________________________
_______________________________________________________________
146
9. Do you take any kind of medicine?

Yes
No
If yes, please state preparation/medicine, dose and against which disorder:

_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
10. Did you take any medication during the last 6 months?
Yes
No
If so, please indicate the preparation/medicine and for which illness:

_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
11. Are there any medications you cannot tolerate?

Yes
No
If yes, please indicate preparation/medicine:

_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
12. Do you have malformations? (for example, were you born with both testicles in your scrotum?). All
types of malformations must be mentioned:
Yes
No
147
Supplementary comments: ____________________________________________

_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
13. Do you have or have you had varicose veins in your scrotum? (pouches, varicocele)
Yes
No
14. If so, have you had surgery for varicose veins in the scrotum?
Yes
No
15. Have your testicles / scrotum at any time been exposed to an impact, injury, inflammation or alike?
Yes
No
Supplementary comments: __________________________________________
16. Do you have any kind of chronic diseases? (eg: diabetes, high blood pressure or similar)
Yes
No
If so, which chronic disease (s): ________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
17. Do you suffer from the following (put x in the correct answer for each disorder)?
Yes
No
Asthma Supplementary comments: ___________________________
Yes
No
Allergy Supplementary comments: ___________________________
Yes
No
Hay fever Supplementary comments: ___________________________
148
18. Do you have problems with upset stomach, abdominal pain or flatulence?
Yes
No
Do you suspect that consumption of specific foods cause these nuisances?:

Yes □ Gluten
Yes □ Milk
Yes □ Eggs
Yes □ What food types? ____________________________________________
_______________________________________________________________
_______________________________________________________________
19. Have you had any type of cancer?

Yes
No
Which type of cancer: ___________________________

Year this cancer was discovered? _________________
20. Did you receive chemotherapy or radiation to treat cancer?

Yes
No
What type of chemotherapy / radiation? : ___________________________________________
21. Have you had any acute illnesses during the last 6 months?
Yes
No
Which disease/illness?: _________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
22. Have you had a fever above 38 ° C for more than 24 hours during the last 6 months?
Yes
No
23. How many days of fever? ______________________________________
149
24. Have you had mumps (parotiditis) as a child?

Yes
No
25. How old were you when you had mumps? ______________
26. Have you been vaccinated against mumps?

Yes
No
Information on general well-being
27. For each statement in the following form, please tick the option which comes closest to how you
have felt during the last 2 weeks?
During the All the time Most of the A little more A little less A little of At no time
last 2 week time than half than half the time
the time the time
I have been
happy and in
good mood
I have
feltcalm and
relaxed
I have felt
active and
energetic
I woke up
fresh and
rested
my everyday
life has been
filledwith
things that
have
interestedme
Information about diet, lifestyle, etc.
28. Is your diet mixed or vegetarian?
Answers include all types of meat (red meat, poultry, seafood, etc.)
□ Vegetarian. I never eat meat.
□ Mixed. I eat meat approx. 3 times a week.
□ Mixed. I eat meat at least 6 times a week.
150
29. Intake of meat from poultry.

I eat poultry about______ times a month.
30. Intake of meat from seafood.

I eat seafood about ______ times per month.
31.How much meat do you eat approximately per meal?

□ I eat 100 grams or less
□ I eat about 250 grams
□ I eat 200 grams or more
32. Do you take a multi-vitamin pill every day or other types of vitamin preparations?
Yes
No
If yes, please indicate which preparation(s) and how long you have been taking it/(them:
Preparation: ____________________ Number of months: ___________________________
33. Do you use dietary supplements - for example with protein?

Yes
No
If yes, state preparation for how long you have been taking supplements (months):
34. Do you prefer light or dark bread?

□ I never eat dark bread
□ I eat mostly light bread.
□ I eat both light and dark bread.
□ I eat mostly dark bread.
□ I never eat light bread.
151
35. How many times a day do you eat vegetables or fruit?

Number of times per day: ____________________
(e.g. an apple, a carrot or other vegetables / fruit corresponding to about 100 grams (e.g. salad, etc.)).
36. How often do you drink coffee?

□ I do not drink coffee
□ I rarely drink coffee
□ I drink coffee once a day
□ I drink coffee several times a day
37. Approximate number of cups of coffee per day: _____________________
38. How often do you drink tea?

□ I do not drink tea
□ I rarely drink tea
□ I drink tea once a day
□ I drink tea several times a day
39. Approximate number of cups of tea per day: ____________________
40. How often do you drink sugary soft drinks such as sodas, juices, energy drinks, juices and the
like?
□ I rarely drink that kind of thing
□ I drink it once a day
□ I drink it several times a day.
41. Approximate number of glasses per day: ___________________
42. How often do you eat chocolate, cakes and sweets?

□ Very often (several times a day)
□ Once a day.
□ Occasionally (3 times a week)
□ Rarely (once a week)
□ Never
43. Your height and weight

Weight ___________ kg
Height ___________ cm
152
44. How many items of alcohol do you drink per week?

________ items of beer.
________ items of wine.
________ items of spirits.
Information for calculating the number of units:
1 regular beer = 1 item

1 strong beer = 1.5 items
1 glass of wine = 1 item
4 cl spirits = 1 item
1 “alco-pop” = 1.5 items (eg Barcardi Breezer)
45. Smoking habits:

□ I have never smoked
□ I smoke about ____ cigarettes (or pipe tobacco) daily.
□ I smoke e-cigarettes.
□ I have stopped smoking.
46. When did you stop smoking?

□ During the last 3 months.
□ 3 to 6 months ago.
□ 6 to 12 months ago.
□ Over 1 year ago.
47. I used to smoke about _________ cigarettes (or pipe tobacco) daily and have smoked for about ___
years.
48. Do you use snus?

□ I have never used snus
□ I have used snus about ______ times a day
□ I have stopped using snus
If you use snus, state which brand: ______ Number of months you have taken it: ______________
49. How many times a week do you exercise? ___________
50. Approximate duration per time ___________ min.
153
51. How big a part of your training is cardio training (swimming, cycling, running, etc.)?
□ Approx. 0% (I only do yoga, strength training, etc.)
□ Approx. 25%
□ Approx. 50%
□ Approx. 75%.
□ 100%.
52. Did you ever used performance-enhancing drugs? (e.g. anabolic steroids)
Yes
No
Specify preparation: ___________________________________
Number of months you took it: __________
53. Do you consider yourself to be in good shape?

□ Yes
□ No.
□ Do not know
54. What type of bath do you take?

□ I always take a shower
□ I occasionally use the bath or spa. (approx. once a month)
□ I often use a bath or spa. (at least once a week).
Work environment
55. What is your position / profession? __________________________________
Which of the following descriptions apply to your work environment?
56. Does your job involve handling toxic substances such as chemicals and pesticides? (e.g.:
hairdresser, farmer or similar)
Yes
No
154
57. Does your work involve a risk of metal intake? (e.g. welders, plumbers and the like)
Yes
No
Supplementary comments: _______________________________________
58. Does your job involve exposure to air pollution (e.g. exhaust fumes) or inhaling dust (for example
when cutting or grinding)?
Yes
No
Supplementary comments: _______________________________________
59. Is your work stressful?

□ Yes, to a greater extent
□ Yes, to a lesser extent
□ Yes, at times
□ No.
60. Does your work take place in a warm environment? (for example: chef, baker and the like).
Yes
No
61. Does your work involve wearing protective clothing/overalls that can increase your body
temperature making you sweat often during light work? Please note whether this outfit also includes
trousers, which can increase the temperature in the groin.
□ Yes, I often sweat at work

□ Yes, the protective clothing also includes trousers
□ No.
62. Do you sit down for more than 4 hours a day? (eg: office work, driver).
Yes
No
If yes, indicate the number of hours per day you sit down: ________________
155
63. Do you often sit with your laptop on your lap?

Yes
No
64. Does your job involve a lot of driving? (for example: salesman, driver, etc).
Yes
No
If yes, indicate the number of hours per day you drive: __________
65. Do you use heat in the car seat?

Yes
No
66. Do you often travel by plane? (e.g.: business man or similar).

Yes
No
If so, how many times a month do you travel by plane?: _______
67. Do you often have your mobile phone in your trouser pocket?
Yes
No
68. Does your work mean that you often eat lunch or dinner in connection with work/business?
□ Yes, we have a lunch club or similar in connection with work

□ Yes, I often eat evening meals in connection with work
□ No, I rarely eat lunch or dinner in connection with work
You have reached the end of the questionnaire

Thank you for your participation
156

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Vol.

48 (1): 131-156, January - February, 2022

The combined effect of lifestyle intervention and antioxidant

ABSTRACT ARTICLE INFO

INTRODUCTION the automated Male Infertility Oxidative System

dication with known gonadotoxic effects when ORP measurement

Questionnaire-based personalized lifestyle in- Antioxidant therapy

and the results of studied patients from before to RESULTS

Figure 1 - Flow diagram showing total patient breakdown.

Baseline Follow-up* Ratio** P-value

%DFI 25.8 (23.0-29.0) 18.0 (15.1-21.6) 0.70 (0.6-0.8) 0.000

Variable Median (IQR)

Body Mass Index (BMI); kg/m2 26 (19-42)

Normal sORP level: ≤1.36 mV/10 sperm/ 6

High sORP level: >1.36 mV/106 sperm/

The chi-square statistic is 0.24. The P value is 0.62.

ACKNOWLEDGEMENTS 7. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L,

Information on fertility and infertility

4. Have you achieved pregnancy / children in previous relationships?

5. Have you achieved pregnancy / children in the current relationship?

Information about genetic disorders in you or your family

6. Are you a carrier of a hereditary disease or a gene defect?

If yes, state which hereditary disease and / or gene defect: _______________________________________

Diseases and medication consumption

If yes, please state the following:

Year Name disease

Possibly, Additional Information:

9. Do you take any kind of medicine?

If yes, please state preparation/medicine, dose and against which disorder:

If so, please indicate the preparation/medicine and for which illness:

11. Are there any medications you cannot tolerate?

If yes, please indicate preparation/medicine:

Supplementary comments: ____________________________________________

Do you suspect that consumption of specific foods cause these nuisances?:

19. Have you had any type of cancer?

Which type of cancer: ___________________________

20. Did you receive chemotherapy or radiation to treat cancer?

23. How many days of fever? ______________________________________

24. Have you had mumps (parotiditis) as a child?

26. Have you been vaccinated against mumps?

Information on general well-being

Information about diet, lifestyle, etc.

28. Is your diet mixed or vegetarian?

29. Intake of meat from poultry.

30. Intake of meat from seafood.

31.How much meat do you eat approximately per meal?

33. Do you use dietary supplements - for example with protein?

34. Do you prefer light or dark bread?

35. How many times a day do you eat vegetables or fruit?

36. How often do you drink coffee?

37. Approximate number of cups of coffee per day: _____________________

38. How often do you drink tea?

39. Approximate number of cups of tea per day: ____________________

41. Approximate number of glasses per day: ___________________

42. How often do you eat chocolate, cakes and sweets?

43. Your height and weight

44. How many items of alcohol do you drink per week?

1 regular beer = 1 item

45. Smoking habits: