Lasers in Medical Science

https://doi.org/10.1007/s10103-020-03042-x

REVIEW ARTICLE

Photobiomodulation therapy for male infertility

Luisa Zupin 1 & Lorella Pascolo 1 & Stefania Luppi 1 & Giulia Ottaviani 2 & Sergio Crovella 1,2 & Giuseppe Ricci 1,2

Received: 31 March 2020 / Accepted: 17 May 2020

# Springer-Verlag London Ltd., part of Springer Nature 2020

Abstract
Male infertility is a worldwide critical condition that affects about the 7.5% of males in Europe leading to an increment of the
couples referring to reproductive medicine units to achieve pregnancy. Moreover, in the recent years, an increased number of
patients have required to freeze their gametes in order to preserve their fertility. Photobiomodulation (PBM) therapy is a potential
treatment that has been used for different clinical application basically aimed at biostimulating cells and tissues. Here, we report a
deep overview of the published studies, focusing on PBM mechanism of action, with the aim of expanding the knowledge in the
field of laser light for a rational utilization of irradiation in the clinical practice. In the field of reproductive science, PBM was
employed to increment spermatozoa’s metabolism, motility, and viability, due to its beneficial action on mitochondria, leading to
an activation of the mitochondrial respiratory chain and to the ATP production. This treatment can be particularly useful to avoid
the use of chemicals in the spermatozoa culture medium as well as to promote the spermatozoa survival and movement especially
after thawing or in largely immotile sperm samples.

Keywords Photobiomodulation therapy . Male infertility . Spermatozoa

Male infertility effort arranges to defeat infertility, a resolutive cure is still

Male infertility is a clinical issue with a global burden that has
been increasing in the recent years. The modern medicine is
trying to challenge this problem through the exploiting of
assisted reproductive techniques [1], but, despite the great
* Luisa Zupin
luisa.zupin@burlo.trieste.it
lacking for most of the cases; moreover, a high proportion of
infertile men presents a diagnosis of idiopathic infertility
where no cause is defined [1].
Male infertility affects approximately 35% of the couples
with a diagnosis of infertility, i.e., “the inability of a sexually
active, non-contracepting couple to achieve spontaneous preg-
nancy in one year” accordingly with the definition by World
Health Organization [2].

Lorella Pascolo Etiology

lorella.pascolo@burlo.trieste.it
Stefania Luppi The etiology of this condition is due to different factors both
stefania.luppi@burlo.trieste.it congenital and acquired.
Giulia Ottaviani Obstruction of ejaculatory ducts or epididymis, cryptorchi-
gottaviani@units.it dism (the inability of testes to descend in the scrotum), testic-
ular torsion, varicocele (dilatation of scrotal veins), testicular
Sergio Crovella
sergio.crovella@burlo.trieste.it trauma, hormonal imbalance, and autoimmunity (with pro-
duction of anti-sperm antibodies) can decrease the fertility of
Giuseppe Ricci
giuseppe.ricci@burlo.trieste.it
an individual [3].
Genetic factors can also play a role, especially the chromo-
1
Institute for Maternal and Child Health, IRCCS Burlo Garofolo, via some abnormalities. Severe impaired sperm production is fre-
dell’Istria 65/1, 34137 Trieste, Italy quently associated with higher frequency of numerical and
2
Department of Medicine, Surgery and Health Sciences, University of structural chromosomopathies, possibly resulting in an in-
Trieste, 34149 Trieste, Italy creased risk of chromosomal anomalies in the embryo.

Individuals with low spermatozoa count have a higher risk of
aneuploidies, for example, the Klinefelter syndrome (47,
XXY) and translocations, such as the Robertsonian’s translo-
cation [4]. The impairment of spermatogenesis can be also
caused by deletion or microdeletions on the long arm of Y
chromosome, in the 3 azoospermia factor (AZF) regions:
AZFa, AZFb, and AZFc [4].
Noteworthy, the infections of the urogenital tract can neg-
atively impact on fertility; urethritis, prostatitis, epididymitis,
and orchitis could damage the male reproductive tissues
resulting in poor sperm quality with decreased viability, mo-
tility, and concentration until the obstruction of seminal tract
in the most severe cases [5, 6]. Chronic and not treated infec-
tions are the most harmful conditions for fertility status.
Pathogens can directly damage testis, accessory gland, and
urethra; moreover, bacteria can act through adhesion leading
to the agglutination of sperm. Furthermore, the presence of
leucocytes is associated to deterioration of sperm count and
motility probably due to the production of reactive oxygen
species (ROS); to inflammation, with the secretion of pro-
inflammatory molecules; and to phagocytic process of macro-
phages and neutrophils [7].
Finally, cancer field holds an important position in this
scenario: testicular cancer and germ cell tumors produce direct
gonadotoxic consequences, disrupting the male reproductive
organs; other malignancy can induce hormonal alteration and
production of metabolites toxic for spermatozoa; moreover,
the chemotherapy, targeting proliferating cells, and radiother-
apy can affect spermatogenesis. In these cases, prevention
strategy can be undertaken, preserving the fertility through
the cryopreservation of the spermatozoa or of testicular tissues
[3].
Intriguingly, the scientific community around the world
reported a negative trend for concentration and quality of
spermatozoa during the past years until now, possibly suggest-
ing that environmental chemicals and pollution could alter the
spermatogenesis [3].
Diagnosis and treatments
The semen analysis is still the cornerstone in diagnosis of male
infertility, and it should be carried out following the World
Health Organization guidelines [8] with the macroscopic and
microscopic analysis. The lower reference limit for sperm
concentration is settled at 15 × 106 spermatozoa per ml, for
progressive and total motility at 32% and 40%, respectively,
for normal forms at 4% [8]. In about the 90% of the patients,
the infertility is due to low sperm counts and/or quality; nev-
ertheless, in about 50% of the cases, the infertility remains
undiagnosed and defined as idiopathic [3, 8]; in such cases,
under- or over-expression of some seminal proteins has been
also reported [9].
Lasers Med Sci
The treatments of infertility are exploited accordingly with
the primary cause of infertility, accounting pharmacological
treatments, surgery, or microsurgery [1]; nevertheless, in most
cases of male infertility, the assisted reproductive techniques
represent the only effective treatment [1, 8].
Spermatozoa are prepared separating them from the semi-
nal plasma with simply washing, swim-up, or discontinuous
density gradient to recover the motile spermatozoa and to
eliminate debris, non-sperm, dead cells, epithelial cells, and
leukocytes [10]. Three to six cycles of intrauterine
insemination (IUI) may be considered a first-line treatment
in case of mild to moderate oligoasthenoteratozoospermia (at
least 1 × 106 progressive motile sperm recovered after prepa-
ration) and female partner with normal fertility status [8]. In
couples with repeated IUI failure or sperm parameters less
than IUI threshold levels, conventional IVF or intra-
cytoplasmatic sperm injection (ICSI) may be proposed [8].
Photobiomodulation therapy
Photobiomodulation (PBM) therapy, previously known as
low level laser therapy, is the application of laser light able
to elicit cells and tissues biostimulation, pursuing photochem-
ical and photophysical events and avoiding heating or thermal
effects [11]. PBM exploits generally the wavelengths in the
range from the visible to the near-infrared (600–1100 nm)
[11].
Mechanism of action
It is widely accepted that the mitochondria are the intracellular
targets of PBM at the red and near-infrared wavelengths; more
specifically, the cytochrome C oxidase (Cox) absorbs the laser
light [12]. Cox is the final enzyme of the mitochondrial respi-
ratory chain constituting the complex IV, and it is responsible
of the electron transfer from the cytochrome C to the dioxygen
molecule [12]. The other components of mitochondrial respi-
ratory chain are poorly stimulated by laser light, and this dif-
ference can be due to the peculiar Cu2+ metal centers of the
Cox, CuA, and CuB (and their redox state) whose action and
absorption spectra highlight four peaks at 620, 680, 760, and
820 nm [12].
PBM is able to increment the availability of electrons for
the Cox’s catalytic centers, incrementing O2 uptake, electron
transfer, and proton pumping of Cox. Therefore, PBM induces
a transitory increment of mitochondrial membrane potential
(MMP) that triggers adenosine triphosphate (ATP) production
[13]. Concurrently, oxidative phosphorylation can release a
small quantity of electrons, that, when accepted by oxygen,
produce non-harmful low level of reactive oxygen species
(ROS), particularly superoxide anion (O2−) that can activate
different intracellular pathways [13]. The increment of ROS
Lasers Med Sci
activates redox sensitive transcription factors, as the nuclear
factor kappa-light-chain-enhancer of activated B cells (NF-
kB) able to modulate the transcription and production of dif-
ferent cytokines and cell survival molecules, creating a cross-
talk between mitochondria and nucleus [13]. On the other
hand, when the cells are in a condition of oxidative stress,
PBM is able to decrement it [14].
Nitric oxide (NO) is also incremented after PBM, probably
due to its dissociation from the metal centers of Cox [13] or to
the NO synthesis by Cox acting as a nitrite reductase [13].
These variations lead to the activation of the mitochondrial
respiratory enzymes, to the increment of mitochondrial activ-
ity and cellular metabolism [11, 13].
Notably, these fluctuations of secondary messengers after
PBM therapy point out the biphasic dose response that follows
the Arndt-Schulz law: an upper and lower threshold of energy
and power exist, outside which the light delivered is too weak
and too strong to present a beneficial effect. Huang et al. hy-
pothesized that this effect could be due to generation of ROS
and NO, very low and with minimal effect in the left side of
the curve, low but beneficial to activate intracellular pathways
in the peak, and too high and dangerous in the right side of the
curve [14].
PBM influences also the calcium ion influx from extracel-
lular environment, probably due to the cytosolic alkalization
that facilitates the opening of plasma membrane’s ion chan-
nels or to a direct action on transient receptor (TRP) ion chan-
nel that can be considered alternative photoreceptors of laser
light [13, 15]. Moreover, the oxidative stress can also modu-
late the calcium mobilization from intracellular stores as the
endoplasmic reticulum and the mitochondria [15].
Moreover, another irradiation parameter that should be tak-
en into account is the irradiation mode, in continuous wave
(CW) or in pulsed modality. Some studies actually found dif-
ferent cellular response towards the two, showing a greater
effect when the irradiation was delivered in frequency. Thus,
a new hypothesis arises, regarding the PBM impact on inter-
facial water layers (2–3 H2O monolayers) attached to the cel-
lular surfaces that could lead to change in density with volume
expansion and to reduction in viscosity. These variations lead
to the transmembrane uptake of molecules, including nutrients
with a metabolic beneficial influence and to a biostimulator
effect on ATP synthase, especially in stressful oxidative con-
dition when ROS increment viscosity and inhibit the activity
of the enzyme [16].
Photobiomodulation therapy
on spermatozoa
Different studies reported the beneficial effect of laser light on
spermatozoa both in humans and animals models, favoring
viability and motility of the cells, possibly improving the
outcome of in vitro insemination [17–19]. PBM effect is
linked to photon absorption by mitochondria that is essential
for energy generation. In spermatozoa, these organelles are
located in the midpiece and are fundamental for the tail move-
ment; furthermore, motility is crucial for their fertilization ca-
pacity [20]. In humans, studies employing PBM mainly re-
ported the assessment of motility, but few functional assays
were also conducted [17, 19, 21]. The understanding of how
PBM actually works is fundamental for the translational ap-
plication of PBM on humans in the clinical practice. The stud-
ies are essentially limited to the in vitro experimentation; nev-
ertheless, PBM has the potentiality for a rapid translation in
the clinics, due to its versatility and affordability, avoiding the
use of chemicals or reagents (Fig. 1).
Methodology
A revision of the published articles was conducted on the
following literature databases: Pubmed, Scopus, Embase,
Web of Science, Ovid MEDLINE, and Scholar online data-
base, using the keywords “spermatozoa,” “male infertility”
combined with a range of synonyms of laser therapy, “laser
therapy,” “low level laser therapy,” “photobiomodulation
therapy,” “photobiomodulation,” and “photobiostimulation.”
The reference lists of retrieved articles and textbooks were
also considered. Only English articles conducted on mamma-
lian spermatozoa in vitro were evaluated, with particular focus
on experiments involving human semen for its medical trans-
lational application.
Photobiomodulation therapy on mammalian
spermatozoa
Several studies were conducted in vitro on spermatozoa from
different mammalian species, through the employment of dif-
ferent wavelength (mostly the red one), demonstrating a ben-
eficial effect on vitality and motility by influencing mitochon-
drial activity.
Irradiation by 655-nm wavelength raised the motility in
dog sperm [22], together with a decrement of L-lactate pro-
duction and a boost in functional capacity [23]. In rabbit sper-
matozoa samples, stored at 15 °C, irradiation with wavelength
at 633 nm prior to storage significantly improved cellular mo-
tility, viability, integrity of acrosome, energetic charge, and
Cox activity [24].
PBM at 532 nm was able to increment the spermatozoa
motility in buffalo [25]. In ram sperm, PBM at 633 nm after
thawing strengthened movement and vitality without
impacting DNA integrity. Moreover, augmentation of ATP
production and Cox activity were detected [26]. In bovine
semen, PBM at 660 nm, executed prior to cryopreservation,
enhanced motility, viability, and acrosome integrity after
thawing [27]; likewise, PBM at 633 nm, in this case executed

Fig. 1 Schematic representation of PBM effect on spermatozoa
after thawing of frozen semen, augmented the spermatozoa
movement and mitochondrial membrane potential, possibly
connected to a mitochondrial stimulation [28]. In boars,
PBM at 633 nm maintained the sperm viability and the acro-
some integrity; meanwhile, the not irradiated samples showed
a rapid and progressive decrement of viable cells and of acro-
some integrity. Motility parameters decreased after 10 min
from irradiation, but from this time point, they were main-
tained until 90 min; instead in not treated specimens, they
continued to decrease over time. Mitochondrial membrane
potential displayed an increment after irradiation that was held
until 90 min. Finally, PBM incremented the number of the
in vitro capacitated spermatozoa, resulting in a high rates of
live births [29]. Intriguingly, in bull sperm cells, PBM at
633 nm showed an opposite effect and induced acrosome
reaction but significantly decrease the percentage of dead
sperm at 90 min from irradiation [30]. In tilapia and ram
sperm, PBM set at 660 nm drove sperm motility and ROS
yield, succeeding in higher embryo viability [31].
To unravel cellular mechanisms, some studies were fo-
cused on the PBM impact on the calcium waves. The 633-
and 780-nm laser irradiation influenced the permeability of
the plasma membrane for the calcium ions; specifically, a
transient increment of calcium influx was registered in bull
sperm [32]. The same authors subsequently suggested that
the PBM at 633 nm affected mainly the mitochondria and
specifically the photosensitizers, such as the cytochromes.
Indeed, the 633-nm laser light at low energy and power
Lasers Med Sci
accelerated the mitochondria calcium uptake in permeabilized
bull sperm, whereas high dose inhibited it; moreover, the cal-
cium efflux from mitochondria was not affected by PBM [33].
Further studies indicated that higher wavelengths, as 780 nm,
could act in a different way, possibly both increasing the plas-
ma membrane permeability to calcium, by producing confor-
mational changes of calcium binding proteins and inhibiting
the mitochondrial calcium uptake [34].
The increment of intracellular calcium level after irradia-
tion at 630 nm was also observed in mouse spermatozoa. In
this study, a biphasic influx was detected: the first step, im-
mediately after the treatment, may be caused by a direct action
on plasma membrane thiols, producing a protein conforma-
tional change and favoring a permeability change. The second
step is believed to be due to the activation of voltage-
dependent calcium channel through signal transduction path-
ways involving mitochondria. Moreover, an increment of hy-
drogen peroxide (an oxygen reactive specie) after PBM was
detected. These changes accounted for an increment of the
fertilization capacity of the spermatozoa [35].
In bovine spermatozoa, visible light (400–800 nm)
incremented the NO level under stressful condition (i.e., cells
maintained for a long period in NKM buffer) immediately
after illumination, and this level was maintained until 3 h
post-treatment, with a greater effect with blue (400–500 nm)
than red (600–800 nm) light. This immediate increase could
be caused by photolysis of nitrosothiol; meanwhile, the main-
tenance of the effect should be due to the induction of activity
Lasers Med Sci
of nitric oxide synthase (NOS) by light-induced ROS or by
light directly, since NOS contains chromophores such as fla-
vins and heme [36].
Photobiomodulation therapy on human spermatozoa
The first article published on PubMed reporting the use of
laser on human spermatozoa dates back to the 1984, when
Sato et al. [37] treated semen from normal and infertile sub-
jects with a 647-nm device. The dosage of 4, 8, and 32 J/cm2
incremented by 5–9% the total sperm motility without affect-
ing the sperm velocity, possibly suggesting that the laser light
stimulated non motile live spermatozoa but had no effect on
moving spermatozoa.
In 1989, Lenzi et al. irradiated spermatozoa from healthy
fertile donors [38]. The researchers used an infrared laser de-
vice, employing 4 different protocols (5 mW combined with
2 Hz or 2000 Hz; 30 mW combined with 2 Hz or 2000 Hz).
After 4 h, a higher percentage of spermatozoa with total and
progressive motility was detected, compared to not irradiated
samples, particularly with the setting 30 mW–2000 Hz (from
about 40% of sperm with progressive motility in controls to
over 60%). Moreover, the sperm, 10 h after irradiation,
showed higher velocity, linearity, amplitude of lateral head
displacement, and lower tail beat frequency. The ATP mea-
surement displayed a continuous decrement probably due to a
faster consumption and an improved capacity of exploiting
energetic resources. In addition, spermatozoa concentrations
and sperm progressive motility after swim-up were signifi-
cantly higher in the aliquots irradiated than those in controls.
Therefore, Lenzi et al. suggested a probable energetic modu-
lation effect of PBM on normal spermatozoa.
Some years later, Singer and Colleagues [39] applying a
wavelength of 940 nm, combined with 25 mW power output,
20 mW/cm2 irradiance, 2 cm2 spot size, 4 min, determined an
increment of motility grades of normal and pathological sper-
matozoa immediately after irradiation and also 30 min later.
Same results were observed when the normal spermatozoa
were treated after washing. The morphology, vitality, fructose
level, quantitative and qualitative pattern of proteins, and pro-
portion of acrosome reacted sperm did not change after illu-
mination. Interestingly, the researchers reported a decrement
of viscosity in two samples. The device employed was not a
laser instrument; therefore, Singer et al. [39] postulated that
coherence is not essential to achieve the biostimulatory effect,
although they did not obtain an increment of motile sperma-
tozoa but only of the motility grade.
Soffer et al. [40], using a low energy 630-nm He-Ne laser
device, investigated the effects of sperm irradiation on a
sperm-zona-free hamster egg penetration model. Irradiated
and non-irradiated spermatozoa from infertile men were incu-
bated for 3 h with fresh zona-free hamster eggs. A significant
increase in the sperm penetration capacity after irradiation was
observed only in the poor sperm subgroup.
In 2011, Firestone et al. [41] irradiated normospermic,
asthenospermic, or oligoasthenospermic semen samples
(905 nm, 50 mW/cm2, 1.5 J/cm2, 30 s). At 30 min post expo-
sure, an increment in percentage of motile sperm (+ 17%) was
detected, and when samples were categorized according to
WHO classification [2], this observation was particularly ev-
ident in asthenospermic/oligospermic samples (+ 83.5%)
compared to asthenospermic (+ 11.8%) and normospermic
(+ 5.5%) samples. These results were accompanied by an in-
crement in the straight-line velocity and in the linearity of the
motion. Nevertheless, the effect was not maintained over time,
since at 24 h no difference was observed, nor a change in
amplitude of lateral head displacement, average path velocity,
or curvilinear velocity was identified. The researchers showed
a greater influence of PBM especially in semen samples with
poor mobility, possibly suggesting a low mitochondrial activ-
ity and ATP production (fundamental for the movement) in
patients with asthenospermia. Firestone et al. [41] proposed
PBM as useful tool for intrauterine insemination and IVF with
embryo transfer or to determining which immotile cell is alive.
Shahar et al. [42], by applying a light source (400–800 nm,
40 mW/cm2, 3 min) on capacitated spermatozoa, showed an
increment of hyperactivated motility (HAM, characteristic of
capacitation) over time (180 min), without effect on total mo-
tility. An increment of ROS and the involvement of superox-
ide anion were proved; moreover, the live microscopic analy-
sis showed an enhanced ROS-dependent fluorescence in the
sperm midpiece, the mitochondria’s localization. Light
incremented adenylyl cyclase/cAMP/PKA-dependent HAM,
inducing also the Src phosphorylation/activation. The authors
showed also an increment of intracellular calcium, probably
with the involvement of voltage-dependent Ca2+ channel
(VDCCC). All these mediators are involved in HAM that in
turn is fundamental in the capacitation process [42].
Salma Yazdi et al. [43] treated spermatozoa from patients
with asthenospermia with a GaAlAs laser (830 nm, 100 mW,
0.67 cm2, combined with 4, 6, or 10 J/cm2). They reported that
the motility of spermatozoa in the untreated samples de-
creased over time, while in those irradiated, it was maintained
or increased, and the same results were observed when the
samples were subdivided in grade A (rapid progressive), grade
B (slow progressive), and A + B. These findings were
achieved especially with 4 and 6 J/cm2 fluence, while 10 J/
cm2 fluence seemed to have a slightly inhibitory effect by
passing of time (60 min). No differences were detected be-
tween irradiated and not irradiated samples using the hypo-
osmotic swelling test to evaluate the functional capacity.
Salma Yazdi et al. [43] proposed the use of PBM to maintain
the sperm motility for artificial insemination.
Ban Frangez et al. [44] in the same year, using a light-
emitting diode (LED) device, tested 4 different PBM

protocols: (1) 850 nm, 2.16 mW/cm2; (2) 625, 660 and
850 nm, 3.92 mW/cm2; (3) 470 nm, 5.06 mW/cm2; and (4)
470, 625, 660 nm, 8.23 mW/cm2 (3 min of treatment). After
LED treatment, with all the 4 protocols, sperm cells presented
a faster motility, with increment of the percentage of rapidly
and slow progressive spermatozoa with respect to the immo-
tile fraction, possibly suggesting that the LED PBM effect is
independent from the wavelength used.
Fekrazad et al. [45] determined an increment of total and
progressive motility and a decrement of immotile spermato-
zoa and of spermatozoa with movement without progression
employing 635- and 830-nm wavelength combined with
200 mW and 4 J/cm2. Moreover, they observed an increment
of curvy linear velocity measured using computer-assisted
sperm analysis (CASA). The greater improvement was ob-
tained with the near-infrared wavelength with respect to the
red one.
Salama and Collaborators [46] tested a 636.6-nm LED in-
strument (power output 1.3 W), combined with 0.496, 1.241,
and 2.482 J/cm2 on normal and asthenospermic semen, and
found an increment of the number of sperm with progressive
motility and a decrement of immotile cells 2, 5, and 10 min
after irradiation. The researchers identified these variations
pooling the normal and asthenospermic semen together, while
by separating the two groups, greater changes were detectable
in the normal semen. The PBM experiments were also con-
ducted in cells maintained in the native state, as well as when
sperm was washed and resuspended in the medium: PBM was
able to act on both conditions, but its influence was greater in
washed spermatozoa with respect to those in their native sta-
tus. No change after irradiation was measured for creatine
kinase, membrane integrity, and head chromatin
condensation.
Sommer et al. [47], using a 670-nm LED source and
728 mW/cm2, observed that the spermatozoa motility was
maintained until 60 min at 6.4 J/cm2 fluence; meanwhile, a
decrement of motility was detected at 19.2 J/cm2 and in the
not irradiated samples.
Preece et al. [48] used a 633-nm laser (5.66 mW/cm2,
35 min) in frozen spermatozoa, after thawing. PBM impacted
on curvilinear velocity, incremented it of 17–47%, within
35 min from irradiation.
Gabel et al. [49] experimented two devices on one fresh
and two frozen semen. Specifically, they employed a 104
LED cluster (56 × 660nm, 10 mW, and 48 × 850 nm,
30 mW) with 2 W total power set at 39.5 mW/cm2, 25, 50,
and 75 s (frozen sample) and 50, 100, 200, and 400 s (fresh
sample), and a GaAlAs laser beam at 810 nm with 200 mW
power output set at 90 mW/cm2, 10, 20, and 40 s (frozen
sample) and 15, 20, and 30 s (fresh sample). In frozen sample,
both LED and laser settings incremented the sperm motility
index (SMI) 30 min after irradiation, LED with 75 s of expo-
sure, laser with 10, 20, and 40 s. On the fresh sample, LED
Lasers Med Sci
improved the SMI and total functional sperm count (TFSC) 15
and 30 min after irradiation with 50 and 100 s of exposure
time. The 200 s of treatment was effective only at 15 min,
while the 400 s protocol was detrimental at all time intervals.
The laser protocols at 15 and 20 s incremented the SMI and
TFSC at 15, 30, and 60 min; meanwhile, 30 s of exposure was
harmful. The researchers concluded that PBM could be useful
for intrauterine insemination to increment the probability of
conception or for in vitro insemination of oocytes to increase
the fertilization’s chance.
Espey et al. [50] using a pulsed laser-probe with a wave-
length of 655 nm, 25 mW/cm2 output-power, and an impulse
duration of 200 ns investigated the optimal laser radiation
dose and treatment duration in normozoospermic and
asthenozoospermic subjects. They found that the laser energy
dose of 4 and 6 J/cm2 induced the greatest effect on sperm
motility and velocity parameters in asthenozoospermic group.
In the normozoospermic group, only minor statistically signif-
icant changes for motility or velocity were detected.
Finally, Highland et al. [51] exposed for 15 min semen
from normal and infertile individuals to a near-infrared source
(750–1100 nm). After irradiation, the viability and function-
ality of spermatozoa decreased, as well as the apoptotic cells
incremented. Furthermore, the oxidative stress was enhanced,
while the toroid and nuclear integrity decremented posttreat-
ment. Notwithstanding, the aim of this work was to observe
the harmful effect of near-infrared wavelength and not to per-
form a PBM; indeed, the exposure time is quite long with
respect to those normally used in PBM (15 min versus sec-
onds), and no other parameters were reported.
The principle results are reported in Table 1.
Impact of photobiomodulation therapy on human
spermatozoa DNA
In reproductive technologies is of paramount importance the
safety of the gametes, especially regarding the DNA, that will
be transmitted to the future generations. Since PBM therapy
promotes the motility of the spermatozoa, it may result helpful
for the cells; nevertheless, the assessment of the safeness of
this technique, especially on the DNA, is warranted.
Gabel and Harrison [52] evaluated the effects of PBM on
sperm chromosomal structure and DNA integrity.
Spermatozoa were exposed to a continuous output sources at
a fixed distance and varying doses using a GaAlAr diode laser
at 810 nm, 200 mw, and 500 mW power, with fluences from
100 J (29.5 J/cm2) to 500 J (147.5 J/cm2), corresponding to a
dose range > 100 times the maximal therapeutic dose already
tested in previous studies. No negative effects on chromosom-
al structure and DNA integrity were observed, even at highest
dose, evaluated as DNA fragmentation index (DFI) measured
by the sperm chromatin structure assay (SCSA) on a fluores-
cence activated cell sorter.
Lasers Med Sci

Table 1 Summary of the effects of photobiomodulation therapy on human sperm

Photobiomodulation treatment Effects on spermatozoa Reference

647 nm • Incremented by 5–9% the total sperm motility in spermatozoa from normal and Sato et al. [37]
4, 8, 32 J/cm2 infertile subjects
Infrared • Higher percentage of spermatozoa with total and progressive, higher velocity, Lenzi et al. [38].
5 mW, 2 Hz or 2000 Hz linearity, amplitude of lateral head displacement and lower tail beat frequency,
30 mW, 2 Hz or 2000 Hz lower ATP in spermatozoa from healthy fertile donors
• Higher spermatozoa concentrations and sperm progressive motility after swim-up
940 nm • Increment of motility grades of normal and pathological spermatozoa Singer et al. [39]
25 mW power output, 20 mW/cm2 • Increment of motility grades in normal spermatozoa treated after washing
irradiance, 2 cm2 spot size, 4 min
630 nm • Increment in the sperm penetration capacity (sperm-zona-free hamster egg Soffer et al. [40]
penetration model) in poor-quality spermatozoa
905 nm • Increment in percentage of motile sperm (+ 17%): asthenospermic/oligospermic Firestone et al. [41]
50 mW/cm2, 1.5 J/cm2, 30 s (+ 83.5%), asthenospermic (+ 11.8%), and normospermic (+ 5.5%) samples
400–800 nm • Increment of hyperactivated motility, of ROS, of intracellular calcium on Shahar et al. [42]
40 mW/cm2, 3 min capacitated spermatozoa
• Increment of adenylyl cyclase/cAMP/PKA-dependent hyperactivated motility,
inducing also the Src phosphorylation/activation
830 nm • Maintenance/increment of motility in spermatozoa from patients with Salman Yazdi et al. [43]
100 mW, 0.67 cm2 asthenospermia: greater effect with 4, 6 J/cm2, slightly inhibitory effect with
4, 6, or 10 J/cm2 10 J/cm2
850 nm, • Increment of motility Ban Frangez et al. [44]
2.16 mW/cm2 • Increment of the percentage of rapidly and slow progressive spermatozoa respect to
625, 660, and 850 nm the immotile fraction
3.92 mW/cm2
470 nm, 5.06 mW/cm2
470, 625, 660 nm
8.23 mW/cm2
3 min
635 and 830 nm • Increment of total and progressive motility and a decrement of immotile Fekrazad et al. [45]
200 mW and 4 J/cm2 spermatozoa and of spermatozoa with movement without progression
• Increment of curvy linear velocity measured
636.6 nm • Increment of the number of sperm with progressive motility and a decrement of Salama et al. [46]
1.3 W immotile cells pooled from normal and asthenospermic semen: greater changes in
0.496, 1.241, and 2.482 J/cm2 normal semen
• Increment of the number of sperm with progressive motility and a decrement of
immotile in native semen and after washing greater changes in normal semen:
greater effect in native status
670 nm • Maintenance of motility at 6.4 J/cm2 fluence Sommer et al. [47]
728 mW/cm2, 6.4 J/cm2, 19.2 J/cm2 • Decrement of motility at 19.2 J/cm2
633-nm laser • In frozen spermatozoa, after thawing. PBM impacted on curvilinear velocity, Preece et al. [48]
5.66 mW/cm2, 35 min incremented it of 17–47%, within 35 min from irradiation
660 nm and 850 nm • Increment of sperm motility index (SMI): 660 + 850 nm PBM with 75 s of Gabel et al. [49]
2 W, 39.5 mW/cm2 exposure, 810 nm with 10, 20, and 40 s in frozen sample
25, 50, 75, 100, 200, and 400 s • Increment of SMI and total functional sperm count (TFSC) with 50, 100, and 200 s
810 nm of exposure time (660 + 850 nm PBM), and with 15 and 20 s (810 nm PBM) in
200 mW, 90 mW/cm2 spermatozoa from fresh sample
10, 15 20, 30, and 40 s
655 nm • Increment of motility and velocity with 4 and 6 J/cm2 in spermatozoa from Espey et al. [50]
25 mW/cm2, 4 and 6 J/cm2, impulse asthenozoospermic patients, minor effect in sperm normozoospermic individuals
duration of 200 ns

Firestone et al. [41] utilizing the 905-nm wavelength
(50 mW/cm2, 1.5 J/cm2, 30 s) showed no DNA damage
assessed by DFI using acridine orange staining in flow
cytometry.
Yazdi et al. [43] treated spermatozoa with a GaAlAs laser
(830 nm, 100 mW, 0.67 cm2, 10 J/cm2). Three hours from
irradiation, the percentage of DFI, performing the chromatin
dispersion test, slightly increased in the treated group, al-
though this difference did not reach the statistical significance.
Preece et al. [48] used a 633-nm laser (31 mW/cm2, 30 min
of treatment) in frozen spermatozoa, after thawing, and they
did not identify DNA double strand break using the γH2AX
marker and DNA oxidative damaging measured as the forma-
tion of the 8-hydroxydeoxyguanosine.

Gabel et al. [49] tested two sources, a LED (660 and
850 nm, 39.5 J/cm2, 400 s) and a 810-nm laser (90 mW/
cm2, 1000 s) on fresh semen. The DFI was determined with
SCSA on a fluorescence activated cell sorter, and no differ-
ence was detected between the not irradiated and irradiated
samples, although at very high dose, 33 times the optimal
parameters.
Espey et al. [50] using a pulsed laser-probe with a wave-
length of 655 nm (25 mW/cm2, 200 ns) found that DNA
fragmentation level, analyzed by ligation-mediated real-time
polymerase chain reaction (RT PCR) and by halo assay in
combination with CASA software, was not significantly dif-
ferent in irradiated sperm compared to the control group.
Conclusions
PBM is a technique that presents a translational potential to
treat spermatozoa characterized by poor quality and motility
[21, 53]. To date, a small number of studies were conducted in
this field; moreover, a limitation of the works carried out so far
is the variety of protocol parameters exploited, thus rendering
difficult a direct comparison between the different studies.
Moreover, the results did not reveal a universal uniformity;
in most of the cases, PBM worked well on spermatozoa from
oligoasthenozoospermic men, but in some other articles, the
efficacious results were obtained with normal spermatozoa,
and finally other researchers pooled together normal and ab-
normal spermatozoa. The understanding of this discrepancy is
hard to explain with the actual knowledge; nevertheless, it is
well-known that PBM should be more effective when the cells
are under stressed or abnormal conditions with respect to the
healthy status [11, 13, 14]. Considering that normal sperma-
tozoa are not exposed to any types of lights, it can be specu-
lated that also normal spermatozoa can be sensible to exoge-
nous bright stimulus. Another interestingly issue is the differ-
ence regarding motility, since some works showed an incre-
ment of it, and other observed only a maintenance of the initial
motility that in not treated samples tend to decrease over time.
These differences could be the outcomes of the different PBM
parameters and laser devices employed in the works but also
of the different characteristic of the samples that should be
taken into account. It is important to consider that in some
works, the effect of PBM is not particularly evident and the
impact on the final motility is quite moderate.
For these reasons, the understanding of PBM mechanism
of action on sperm cells should be useful to optimize the
irradiation condition in order to obtain the maximum
effectiveness.
Nevertheless, some milestones were previously achieved
disclosing the molecular effects of PBM especially in studies
conducted on mammals.
Lasers Med Sci
First, the increment of mitochondrial activity with the pro-
duction of ATP [26, 29] is a key process for spermatozoa;
apart from the utilization of this molecule for cellular energetic
reaction, it is essential to supply energy to flagellar dynein
ATPase, required for the motility of the tails, both for progres-
sive motility in the ejaculated semen and for hyperactivated
motility at the site of fertilization [54].
Second, the changes in the intracellular calcium level
could be the key cell response for spermatozoa function-
ality. Calcium intracellular homeostasis is modulated by
mitochondria, since this organelle can function as calcium
stores; on the other hand, calcium promotes the mitochon-
drial electron transport and ATP production. These two
aspects impact on axonemes leading to regulation of pro-
cess like capacitation, hyperactivation, chemotaxis, and
acrosome reaction [54]. Other elements than calcium
seem to play a role in sperm functionality; in fact, we
recently demonstrated by X-ray fluorescence analysis that
the selection of good quality sperm is associated with
reduction of magnesium and copper concentrations [55]:
intriguingly, magnesium is involved in stability of ATP
and other nucleotides; meanwhile, copper is a cofactor in
antioxidant enzymes and metal centers of Cox [56]. Our
undergoing studies are aimed to check how PBM affects
the intracellular levels of these two divalent elements.
Third, a boost of ROS, including increment of NO, after
PBM was reported; ROS (and NO) act as double-edged sword
in spermatozoa, and too high levels are detrimental, while low
amounts are necessary to activate intracellular pathways
resulting in capacitation and acrosome reaction [54].
Taken together, these findings jointly with the increased
motility shown in human spermatozoa highlighted the bene-
ficial effect of PBM. Moreover, the data denoted that PBM is
safe and it does not impact on the DNA structure, assessed
with different assays including SCSA, acridine orange, chro-
matin dispersion test, γH2AX, 8-hydroxydeoxyguanosine,
ligation-mediated RT PCR, and halo assay indicating that dif-
ferent aspects of DNA status were investigated. The mainte-
nance of the DNA integrity after PBM is a fundamental point
to employ the irradiated spermatozoa for the IVF in the clin-
ical practice.
Amplifying the knowledge in this field is advisable,
since a deep comprehension would be the starting point
forward a rational utilization of the laser instruments and
for the optimization of the protocols in the clinical practice.
PBM is a fast, cheap, noninvasive technique, and we deem
that it could replace the use of chemicals to attain the mo-
bilization of immotile spermatozoa for ICSI selection and
can improve the chance of fertilization in the in vitro in-
semination or help the recovery of cryopreserved gametes,
often deteriorated in motility and viability after thawing, or
o f s u r g i c a l l y r e t r i e v e d te s t i c u l a r o r e p i d i d y m a l
spermatozoa.
Lasers Med Sci
Funding information This research was funded by Institute for Maternal
and Child Health, IRCCS Burlo Garofolo, grant number 5mille15D1 and
RC15/17.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
1. Jungwirth A, Giwercman A, Tournaye H et al (2012) European
Association of Urology guidelines on male infertility: the 2012
update. Eur Urol 62:324–332. https://doi.org/10.1016/j.eururo.
2012.04.048
2. WHO (2010) WHO manual for the standardized investigation, di-
agnosis and management of the infertile male. In: WHO. https://
www.who.int/reproductivehealth/publications/infertility/
0521774748/en/. Accessed 28 Nov 2019
3. Naz M, Kamal M (2017) Classification, causes, diagnosis and treat-
ment of male infertility: a review. Orient Pharm Exp Med 17:89–
109. https://doi.org/10.1007/s13596-017-0269-7
4. O’Flynn O’Brien KL, Varghese AC, Agarwal A (2010) The genetic
causes of male factor infertility: a review. Fertil Steril 93:1–12.
https://doi.org/10.1016/j.fertnstert.2009.10.045
5. Comar M, Zanotta N, Croci E et al (2012) Association between the
JC polyomavirus infection and male infertility. PLoS One 7:
e42880. https://doi.org/10.1371/journal.pone.0042880
6. La Vignera S, Condorelli RA, Vicari E et al (2014) Microbiological
investigation in male infertility: a practical overview. J Med
Microbiol 63:1–14. https://doi.org/10.1099/jmm.0.062968-0
7. Fraczek M, Kurpisz M (2015) Mechanisms of the harmful effects of
bacterial semen infection on ejaculated human spermatozoa: poten-
tial inflammatory markers in semen. Folia Histochem Cytobiol 53:
201–217. https://doi.org/10.5603/fhc.a2015.0019
8. World Health Organization (2010) WHO Laboratory Manual for
the Examination and Processing of Human Semen, 5th ed.
9. Giacomini E, Ura B, Giolo E et al (2015) Comparative analysis of
the seminal plasma proteomes of oligoasthenozoospermic and nor-
mozoospermic men. Reprod BioMed Online 30:522–531. https://
doi.org/10.1016/j.rbmo.2015.01.010
10. Ricci G, Granzotto M, Luppi S et al (2015) Effect of seminal leu-
kocytes on in vitro fertilization and intracytoplasmic sperm injec-
tion outcomes. Fertil Steril 104:87–93. https://doi.org/10.1016/j.
fertnstert.2015.04.007
11. Tsai S-R, Hamblin MR (2017) Biological effects and medical ap-
plications of infrared radiation. J Photochem Photobiol B Biol 170:
197–207. https://doi.org/10.1016/j.jphotobiol.2017.04.014
12. Karu T (1999) Primary and secondary mechanisms of action of
visible to near-IR radiation on cells. J Photochem Photobiol B
Biol 49:1–17. https://doi.org/10.1016/S1011-1344(98)00219-X
13. de Freitas LF, Hamblin MR (2016) Proposed Mechanisms of
Photobiomodulation or Low-Level Light Therapy. IEEE J Sel
Top Quantum Electron:22. https://doi.org/10.1109/JSTQE.2016.
2561201
14. Huang Y-Y, Sharma SK, Carroll J, Hamblin MR (2011) Biphasic
dose response in low level light therapy - an update. Dose Response
9:602–618. https://doi.org/10.2203/dose-response.11-009.Hamblin
15. Amaroli A, Ferrando S, Benedicenti S (2019) Photobiomodulation
affects key cellular pathways of all life-forms: considerations on old
and new laser light targets and the calcium issue. Photochem
Photobiol 95:455–459. https://doi.org/10.1111/php.13032
16. Sommer AP (2019) Revisiting the photon/cell interaction mecha-
nism in low-level light therapy. Photobiomodul Photomed Laser
Surg 37:336–341. https://doi.org/10.1089/photob.2018.4606
17. Vladimirovich Moskvin S, Ivanovich Apolikhin O (2018)
Effectiveness of low level laser therapy for treating male infertility.
Biomedicine (Taipei) 8:7. https://doi.org/10.1051/bmdcn/
2018080207
18. Lone S, Mohanty T, Kumaresan A, Bhakat M (2017) Laser irradi-
ation effects and its possible mechanisms of action on spermatozoa
functions in domestic animals. Asian Pac J Reprod 6:97–103.
https://doi.org/10.12980/apjr.6.20170301
19. Borhani S, Yazdi RS (2018) Clinical applications of low-level laser
therapy in reproductive medicine; A Literature Review. https://doi.
org/10.20944/preprints201804.0086.v1
20. Eisenbach M, Giojalas LC (2006) Sperm guidance in mammals —
an unpaved road to the egg. Nat Rev Mol Cell Biol 7:276–285.
https://doi.org/10.1038/nrm1893
21. Karu TI (2012) Lasers in infertility treatment: irradiation of oocytes
and spermatozoa. Photomed Laser Surg 30:239–241. https://doi.
org/10.1089/pho.2012.9888
22. Corral-Baqués MI, Rivera MM, Rigau T et al (2009) The effect of
low-level laser irradiation on dog spermatozoa motility is depen-
dent on laser output power. Lasers Med Sci 24:703–713. https://doi.
org/10.1007/s10103-008-0606-7
23. Corral-Baqués MI, Rigau T, Rivera M et al (2005) Effect of 655-nm
diode laser on dog sperm motility. Lasers Med Sci 20:28–34.
https://doi.org/10.1007/s10103-005-0332-3
24. Iaffaldano N, Rosato MP, Paventi G et al (2010) The irradiation of
rabbit sperm cells with He–Ne laser prevents their in vitro liquid
storage dependent damage. Anim Reprod Sci 119:123–129. https://
doi.org/10.1016/j.anireprosci.2009.10.005
25. Abdel-Salam Z, Dessouki SHM, Abdel-Salam SAM et al (2011)
Green laser irradiation effects on buffalo semen. Theriogenology
75:988–994. https://doi.org/10.1016/j.theriogenology.2010.11.005
26. Iaffaldano N, Paventi G, Pizzuto R et al (2016) Helium-neon laser
irradiation of cryopreserved ram sperm enhances cytochrome c ox-
idase activity and ATP levels improving semen quality.
Theriogenology 86:778–784. https://doi.org/10.1016/j.
theriogenology.2016.02.031
27. Fernandes GHC, de Tarso Camillo de Carvalho P, Serra AJ et al
(2015) The effect of low-level laser irradiation on sperm motility,
and integrity of the plasma membrane and acrosome in cryopre-
served bovine sperm. PLoS One 10:e0121487. https://doi.org/10.
1371/journal.pone.0121487
28. Siqueira AFP, Maria FS, Mendes CM et al (2016) Effects of
photobiomodulation therapy (PBMT) on bovine sperm function.
Lasers Med Sci 31:1245–1250. https://doi.org/10.1007/s10103-
016-1966-z
29. Yeste M, Codony F, Estrada E et al (2016) Specific LED-based red
light photo-stimulation procedures improve overall sperm function
and reproductive performance of boar ejaculates. Sci Rep:6. https://
doi.org/10.1038/srep22569
30. Ocaña-Quero JM, Gomez-Villamandos R, Moreno-Millan M,
Santisteban-Valenzuela JM (1997) Biological effects of helium-
neon (He-Ne) laser irradiation on acrosome reaction in bull sperm
cells. J Photochem Photobiol B Biol 40:294–298. https://doi.org/
10.1016/s1011-1344(97)00072-9
31. Zan-Bar T, Bartoov B, Segal R et al (2005) Influence of visible light
and ultraviolet irradiation on motility and fertility of mammalian
and fish sperm. Photomed Laser Surg 23:549–555. https://doi.org/
10.1089/pho.2005.23.549
32. Lubart R, Friedmann H, Levinshal T et al (1992) Effect of light on
calcium transport in bull sperm cells. J Photochem Photobiol B Biol
15:337–341. https://doi.org/10.1016/1011-1344(92)85139-l
33. Breitbart H, Levinshal T, Cohen N et al (1996) Changes in calcium
transport in mammalian sperm mitochondria and plasma membrane

irradiated at 633 nm (HeNe laser). J Photochem Photobiol B Biol
34:117–121. https://doi.org/10.1016/1011-1344(95)07281-0
34. Lubart R, Friedmann H, Sinyakov M et al (1997) Changes in cal-
cium transport in mammalian sperm mitochondria and plasma
membranes caused by 780 nm irradiation. Lasers Surg Med 21:
493–499. https://doi.org/10.1002/(sici)1096-9101(1997)21:
5<493::aid-lsm12>3.0.co;2-a
35. Cohen N, Lubart R, Rubinstein S, Breitbart H (1998) Light irradi-
ation of mouse spermatozoa: stimulation of in vitro fertilization and
calcium signals. Photochem Photobiol 68:407–413
36. Ankri R, Friedman H, Savion N et al (2009) Visible light induces
no formation in sperm and endothelial cells. Lasers Surg Med 42:
348–352. https://doi.org/10.1002/lsm.20849
37. Sato H, Landthaler M, Haina D, Schill WB (1984) The effects of
laser light on sperm motility and velocity in vitro. Andrologia 16:
23–25. https://doi.org/10.1111/j.1439-0272.1984.tb00229.x
38. Lenzi A, Claroni F, Gandini L et al (1989) Laser radiation and
motility patterns of human sperm. Arch Androl 23:229–234.
https://doi.org/10.3109/01485018908986845
39. Singer R, Sagiv M, Barnet M et al (1991) Low energy narrow band
non-coherent infrared illumination of human semen and isolated
sperm. Andrologia 23:181–184. https://doi.org/10.1111/j.1439-
0272.1991.tb02532.x
40. Soffer Y, Lubart R, Breitbart H (2000) L’irradiation des
spermatozoïdes au Laser HeNe à faible énergie chez la souris et
chez l’homme. Andrologie 10:417–426. https://doi.org/10.1007/
BF03034498
41. Firestone RS, Esfandiari N, Moskovtsev SI et al (2012) The effects
of low-level laser light exposure on sperm motion characteristics
and DNA damage. J Androl 33:469–473. https://doi.org/10.2164/
jandrol.111.013458
42. Shahar S, Wiser A, Ickowicz D et al (2011) Light-mediated activa-
tion reveals a key role for protein kinase a and sarcoma protein
kinase in the development of sperm hyper-activated motility.
Hum Reprod 26:2274–2282. https://doi.org/10.1093/humrep/
der232
43. Salman Yazdi R, Bakhshi S, Jannat Alipoor F et al (2014) Effect of
830-nm diode laser irradiation on human sperm motility. Lasers
Med Sci 29:97–104. https://doi.org/10.1007/s10103-013-1276-7
44. Ban Frangez H, Frangez I, Verdenik I et al (2015)
Photobiomodulation with light-emitting diodes improves sperm
motility in men with asthenozoospermia. Lasers Med Sci 30:
235–240. https://doi.org/10.1007/s10103-014-1653-x
45. Fekrazad E, Keyhan H, Fekrazad R, Tajik A (2014) Effect of diode
lasers on human sperm motility. Acad Res Int 5(5):21–25
46. Salama N, El-Sawy M (2015) Light-emitting diode exposure en-
hances sperm motility in men with and without asthenospermia:
Lasers Med Sci
preliminary results. Arch Ital Urol Androl 87:14. https://doi.org/
10.4081/aiua.2015.1.14
47. Sommer AP, Jaganathan S, Maduro MR et al (2016) Genesis on
diamonds II: contact with diamond enhances human sperm perfor-
mance by 300%. Ann Transl Med 4:407–407. https://doi.org/10.
21037/atm.2016.08.18
48. Preece D, Chow KW, Gomez-Godinez V et al (2017) Red light
improves spermatozoa motility and does not induce oxidative
DNA damage. Sci Rep:7. https://doi.org/10.1038/srep46480
49. Gabel CP, Carroll J, Harrison K (2018) Sperm motility is enhanced
by low level laser and light emitting diode photobiomodulation
with a dose-dependent response and differential effects in fresh
and frozen samples. Laser Ther 27:131–136. https://doi.org/10.
5978/islsm.18-OR-13
50. Espey BT, Kielwein K, Liebenthron J et al (2017) Effects of low-
level-laser-therapy LLLT on human spermatozoa and its future per-
spective for reproductive medicine. J Reprod Med Endocrinol 5:
221
51. Highland H, Rajput N, Sharma R, George L-B (2018) Differential
sensitivity of the human sperm cell to near infrared radiation. J
Photochem Photobiol B Biol 183:119–126. https://doi.org/10.
1016/j.jphotobiol.2018.04.027
52. Gabel P, Harrison K (2009) Sperm DNA integrity is not damaged
by specific low level laser therapy megadose exposure. Lasers Surg
Med 41(supplementary 21):65–66. https://doi.org/10.1002/lsm.
20783
53. Abdel-Salam Z, Harith MA (2015) Laser researches on livestock
semen and oocytes: a brief review. J Adv Res 6:311–317. https://
doi.org/10.1016/j.jare.2014.11.006
54. Piomboni P, Focarelli R, Stendardi A et al (2012) The role of mi-
tochondria in energy production for human sperm motility: mito-
chondria functionality in human spermatozoa. Int J Androl 35:109–
124. https://doi.org/10.1111/j.1365-2605.2011.01218.x
55. Pascolo L, Zupin L, Gianoncelli A et al (2019) XRF analyses reveal
that capacitation procedures produce changes in magnesium and
copper levels in human sperm. Nucl Instrum Methods Phys Res
Sect B 459:120–124. https://doi.org/10.1016/j.nimb.2019.09.005
56. Schmid TE, Grant PG, Marchetti F et al (2013) Elemental compo-
sition of human semen is associated with motility and genomic
sperm defects among older men. Hum Reprod 28:274–282.
https://doi.org/10.1093/humrep/des321
Publisher’s note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.