Review

Oxidative Stress as an Underlying

Mechanism of Bacteria-Inflicted
Damage to Male Gametes

Eva Tvrdá, Filip Benko and Michal Ďuračka

Special Issue
Review Papers in Oxygen
Edited by
Prof. Dr. John T. Hancock and Prof. Dr. César Augusto Correia de Sequeira

https://doi.org/10.3390/oxygen2040036
Review
Oxidative Stress as an Underlying Mechanism of
Bacteria-Inflicted Damage to Male Gametes
Eva Tvrdá 1, * , Filip Benko 2 and Michal Ďuračka 3

1 Institute of Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in
Nitra, Tr. A. Hlinku 2, 94976 Nitra, Slovakia
2 Institute of Applied Biology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in
Nitra, Tr. A. Hlinku 2, 94976 Nitra, Slovakia
3 AgroBioTech Research Centre, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 94976 Nitra, Slovakia
* Correspondence: evina.tvrda@gmail.com; Tel.: +421-37-641-4918

Abstract: Bacterial infestation of the male reproductive system with subsequent effects of bacteria
on the structural integrity and functional activity of male gametes has become a significant factor in
the etiology of male reproductive dysfunction. Bacteria may affect male fertility either by directly
interacting with structures critical for sperm survival or indirectly by triggering a local immune
response, leukocytospermia or reactive oxygen species (ROS) overproduction followed by oxidative
stress development. This review aims to provide an overview of the currently available knowledge
on bacteriospermia-associated sperm damage with a special emphasis on oxidative mechanisms
underlying sperm deterioration caused by bacterial action. At the same time, we strive to summarize
readily available alternatives to prevent or counteract alterations to spermatozoa caused by bacterial
colonization of semen or by oxidative stress as an accompanying phenomenon of bacteriospermia.

Citation: Tvrdá, E.; Benko, F.; Keywords: bacteriospermia; infection; sperm quality; oxidative stress; reactive oxygen species;
Ďuračka, M. Oxidative Stress as an subfertility; bacterial contamination
Underlying Mechanism of
Bacteria-Inflicted Damage to Male
Gametes. Oxygen 2022, 2, 547–569.
https://doi.org/10.3390/ 1. Introduction
oxygen2040036
A global challenge of today’s era lies in the understanding, prevention, and manage-
Academic Editors: John T. Hancock ment of ever-increasing male reproductive dysfunction. Particularly in mammals, male sub-
and César Augusto Correia de or infertility is a complex and multifactorial condition that may be caused by a multitude
Sequeira of factors, ranging from genetic causes and systemic ailments to environmental exposure or
Received: 7 October 2022
nutrition [1]. Within this vast array of etiologies, bacteriospermia—the presence of bacteria
Accepted: 3 November 2022
in semen—is thought to be the causative agent in around 15% of all cases of subfertile
Published: 6 November 2022
males [1,2]. In clinical practice, bacteriospermia is acknowledged when the bacterial count
exceeds 1.000 colony-forming units (CFU)/mL of semen [3]. The primary reason for bac-
Publisher’s Note: MDPI stays neutral
terial colonization of ejaculates lies in an acute or chronic infection of the urogenital tract
with regard to jurisdictional claims in
including diseases such as urethritis, prostatitis, epididymitis or orchitis, subsequently
published maps and institutional affil-
compromising proper spermatogenesis and sperm maturation [4]. Alternatively, bacteria
iations.
may invade the reproductive system from urinary tract infection [5], by hematogenous dis-
semination [6] or by sexual intercourse [7]. In certain cases, bacteriospermia may be caused
by the overgrowth of natural microflora present in the urogenital tract even in clinically
Copyright: © 2022 by the authors.
healthy subjects [8,9]. Semen may be also infested by bacteria from external sources, such
Licensee MDPI, Basel, Switzerland. as skin, urine, feces [10] as well as laboratory tools, equipment or media needed for semen
This article is an open access article collection and/or processing for assisted reproduction techniques (ARTs) [11].
distributed under the terms and Bacteria present in semen may affect male gametes through adhesion [12] and aggluti-
conditions of the Creative Commons nation events, which will ultimately result in sperm immobilization and inability to reach
Attribution (CC BY) license (https:// and fertilize the oocyte [13]. The released bacterial toxins may have direct cytotoxic effects
creativecommons.org/licenses/by/ on the male gamete, leading to increased sperm cell death [14]. Furthermore, bacterial
4.0/). infection may trigger persistent inflammation and leukocytic infiltration accompanied by

Oxygen 2022, 2, 547–569. https://doi.org/10.3390/oxygen2040036 https://www.mdpi.com/journal/oxygen

Oxygen 2022, 2 548

the release of pro-inflammatory cytokines that will contribute to permanent damage of

structures vital for sperm survival [9,15].
While the mechanisms of which bacteria may cause alterations to male reproductive
cells are diverse, a convincing body of evidence indicates a notable involvement of reac-
tive oxygen species (ROS) in the phenomena associated with bacteriospermia. As such,
understanding the role of oxidative processes underlying bacteria-inflicted damage to
spermatozoa may assist in the development of adequate intervention strategies to mitigate
bacteriospermia-associated male subfertility.

2. Sources of ROS in Bacteriospermia

The involvement of ROS in male reproduction with beneficial as well as adverse
effects has become indisputable in modern andrology. Carefully controlled physiologi-
cal ROS levels play a crucial role in signaling pathways involved in sperm production
and maturation, as well as during capacitation, hyperactivation, acrosome reaction and
sperm-egg fusion. Nevertheless, ROS accumulation has been previously acknowledged
to act either as a primary cause or an accompanying phenomenon in a vast array of male
reproductive pathophysiologies [16–19]. Correspondingly, earlier reports on bacteriosper-
mia have frequently observed increased oxidative damage resulting from a shift in the
prooxidant–antioxidant milieu in favor of prooxidants, favoring further ROS release and
action [20–23].
By and large, three sources of ROS are most relevant to bacteriospermia: (1) immature,
damaged or dead spermatozoa, (2) bacterial metabolism, and (3) activated leukocytes.

2.1. Spermatozoa
The process of sperm maturation involves a series of events that take place during
the sperm transition from the seminiferous epithelium to the epididymis. These processes
include structural remodeling, male germ cell differentiation, acquisition of the ability
to move and to accomplish acrosome reaction, all of which are dependent on changes in
the architecture and fluidity of the plasma membrane [24]. In morphologically normal
spermatozoa, cytoplasmic deposits located in the midpiece are extruded to allow for
subsequent cell elongation and nuclear condensation to occur during spermiogenesis [25]
(Figure 1a).
If sperm maturation is not carried out properly, spermatozoa will retain the cytoplasm
in the form of proximal or distal droplets, containing large amounts of cytoplasmic enzymes
such as lactate dehydrogenase, superoxide dismutase (SOD) and glucose-6-phosphate de-
hydrogenase [24–26], all of which have been shown to be directly involved in the cellular
oxidative balance reviewed by [27] (Figure 1b). In particular, glucose-6-phosphate dehy-
drogenase has been repeatedly associated with an increased risk for peroxidative damage,
since it produces nicotinamide adenine dinucleotide phosphate (NADPH). Subsequently,
ROS is generated from NADPH via the intramembrane-located NADPH oxidase 5 (NOX
5) [28], a prime enzyme responsible for ROS production in the acrosomal, equatorial or post-
acrosomal regions of morphologically abnormal spermatozoa [29]. In addition, significant
associations have been reported between ROS production and the stage of sperm devel-
opment, with particularly high ROS levels being observed in immature spermatozoa [30].
Correspondingly, increased ROS production has been reported in patients suffering from
teratozoospermia [31] or in subjects with a high occurrence of cytoplasmic droplets [25,26].
Oxygen 2022, 2 549

Figure 1. Sources of ROS in normal (a), immature (b) or dead (c) spermatozoa.

On the other hand, it has−

been suggested that apart from immature or morphologically
abnormal sperm, increased amounts of damaged or dead male gametes may represent a
threat to still viable and functional spermatozoa. The risks associated with the presence of
dying spermatozoa
− lie in an increased release of intracellular content into their surround-
ings, containing large amounts of ROS, which may then attack the polyunsaturated fatty
acids (PUFAs) located in the membrane of viable male gametes. Such compromised cells
will then exhibit phosphatidylserine externalization, loss of membrane fluidity and an
increased occurrence of DNA breaks, all of which will ultimately result in the activation
of apoptotic or even necrotic cell death (Figure 1c). In this sense, cell necrosis represents
a larger risk for the loss of viable spermatozoa, since any disturbance to intracellular
homeostasis is succeeded by the rupture of membranous structures and a subsequent con-
tamination of the extracellular space with toxic metabolites, sustaining the chain reaction
of damage to other functional male gametes [32]. Moreover, an increased occurrence of
dead spermatozoa has been suggested to trigger the immune system and to contribute to
the formation of sperm granulomas. These abnormal structures consist of spermatozoa,
macrophages, and other immune cells, eventually developing into a mass attached to the
vas deferens, causing an obstruction and raising the intraluminal pressure [33].
(a) Mitochondria are the primary source of ROS in structurally and functionally normal
spermatozoa. Superoxide (O2 •− ) is created by electron leakage within NADH dehy-
drogenase (complex I) and ubiquinol cytochrome C oxidoreductase (complex III). The
radical is quickly scavenged by superoxide dismutase (SOD) to generate hydrogen
peroxide (H2 O2 ). Alternatively, O2 •− and H2 O2 are generated by the NADPH-oxidase
isoform 5 (NOX5) located in the plasma membrane and are activated through NADPH
and calcium ions (Ca2+ ). Modified from [29]. CI—complex I, CII—complex II (succi-
Oxygen 2022, 2 550

nate dehydrogenase), CIII—complex III, CIV—complex IV (cytochrome C oxidase),

CV—complex V (ATP synthase).
(b) Immature or abnormal spermatozoa present with cytoplasmic droplets containing
glucose-6-phosphate dehydrogenase (G6PD), which produces NADPH serving as a
substrate for the creation of O2 •− by NADPH oxidase systems (NADPH Ox). SOD
present in the droplets catalyzes the conversion of O2 •− to H2 O2 . In the meantime, lac-
tate dehydrogenase
− produces the reduced form of nicotinamide adenine dinucleotide
−
(NADH), which then proceeds to be oxidized by the NADH dehydrogenase (complex
I) in the mitochondria. Modified from [28].
(c) In necrotic spermatozoa, the intracellular content including ROS will be released into the
environment, subsequently contaminating the surrounding cells with toxic metabolites.
Created with BioRender.com (supplementary: Confirmation of Publication and Licens-
ing Rights) (accessed on 28 October 2022).

2.2. Bacteria
The extent of ROS production by bacterial action is largely affected by the bacterial load,
diversity, and metabolism of bacteria representing the seminal microflora [34]. In aerobic
bacteria, more than 90% of cellular oxygen will be consumed by oxidative phosphorylation
to produce ATP and water. Similarly to eukaryotic cells, a small portion of molecular
oxygen captures 0.1%–1% of electrons present in the transport chain, continually forming
superoxide radical (O2 •− ) by NADPH − oxidase, which is the primary source of ROS in
bacteria [35] (Figure 2a).

Figure 2. Sources of ROS in aerobic bacteria (a) and leukocytes (b).

Oxygen 2022, 2 551

While obligate anaerobes are not equipped to perform aerobic respiration, it has been
speculated that these can deploy low-potential electron transfer pathways to produce
ROS [36]. O2 •− can be generated abiotically from the reaction of O2 with ferrous ion-like
metal ions [37,38]. Correspondingly to higher organisms, hydrogen peroxide (H2 O2 ) in
bacteria is generated as part of the dismutation of O2 •− catalyzed by SOD [38] (Figure 2a).
These two ROS are prevalent reactive intermediates produced by the bacterial cell, and the
presence of antioxidant enzymes (such as SOD, catalase, thioxiredoxin or thiol peroxidase)
in several bacterial species fortifies the evidence that bacteria themselves are capable of
producing, regulating and sustaining certain levels of free radicals [39].
Additionally, O2 •− as well as H2 O2 have been observed to be actively released by
numerous potentially uropathogenic bacteria [40,41]. As suggested by Fraczek et al. [22],
additional ROS may subsequently cause significant damage to the sperm membranes,
evidenced by increased malondialdehyde (MDA) levels in spermatozoa subjected to
in vitro culture in the presence of Campylobacter ureolyticus, Staphylococcus haemolyticus, and
Escherichia coli. Similar experiments of in vitro bacteriospermia designed by Ďuračka et al. [41]
also revealed that selected staphylococci contributed to increased ROS levels in a sperm-
bacteria co-culture, leading to decreased sperm motility, mitochondrial membrane potential
and DNA integrity.

2.3. Leukocytes
Under physiological conditions, the primary source of ROS in semen is polymorphonu-
clear neutrophilic granulocytes, which are activated by an ongoing inflammatory process.
The primary role of such peroxidase-positive leukocytes is to dispose of the pathogen
by an increased release of ROS into the source of inflammation [42]. In this case, high
amounts of O2 •− and H2 O2 are produced by a series of reactions referred to as respiratory
burst. This phenomenon is mediated by the NADPH-oxidase complex, which is rapidly
assembled following neutrophil activation. As discussed previously, NADPH oxidase
produces O2 •− similarly to NOX5 found in immature spermatozoa [28,29,43]. Subsequent
dismutation catalyzed by SOD takes place to create H2 O2 . Both ROS may then be released
individually, or these may interact with products of other microbicidal systems to generate
ozone, a hydroxyl radical or singlet oxygen [43] (Figure 2b). While the primary role of
respiratory burst lies in an additional line of protection to the cells against a pathogen,
excessive infiltration and inappropriate activation of leukocytes may result in the release of
excessive ROS and a subsequent deterioration of sperm function, as repeatedly observed in
subjects suffering from leukocytospermia [9,10,44,45].
(a) O2 •− is created by NADPH oxidase localized on the bacterial plasma membrane.
The molecule is further processed by SOD to generate H2 O2 . Additional ROS may
be generated by the Fenton and Haber–Weiss reaction catalyzed by iron. Modified
from [37].
(b) Respiratory burst is activated by the NADPH-oxidase complex, which will produce
O2 •− . Subsequently, SOD will dismutate the radical to generate H2 O2 .
Created with BioRender.com (supplementary: Confirmation of Publication and Licens-
ing Rights) (accessed on 28 October 2022).

3. Adhesion and Agglutination Events

The ability of bacteria to adhere to the cell surface is a crucial prerequisite for sub-
sequent bacterial colonization of ejaculate since adhesion events may lead to alterations
to the sperm morphology and motion behavior [46]. Furthermore, bacterial species at-
tached to the sperm structures may release signaling molecules to attract other bacteria to
form agglutinating complexes that will completely immobilize male gametes [47]. Further
development of agglutination structures may then stimulate the release of extracellular
polymeric substances and initiate biofilm formation [46,48]. Finally, successful bacterial
adherence may stimulate the secretion of bacterial endotoxins with cytotoxic effects on
sperm functional activity and fertilization ability [49].
Oxygen 2022, 2 552

3.1. Bacterial Adhesion

Bacteria accomplish adhesion events with polymeric fibers called “fimbriae” or “pilli”
that serve to establish contact among the cellular surfaces and serve as virulence factors
able to mediate the formation of bacterial aggregates by the recognition of receptors located
on the host cell [49,50]. In the case of G− bacteria, their pilli possess a high affinity to
mannose receptors found in the sperm membrane [47,51]. Two major types of pilli have
been studied in uropathogenic G− bacteria, namely Type 1 fimbrinae with a high affinity
to mannose located in the sperm head [47] and a mannose-resistant P fimbriae, which
recognizes a-D-galpl-4-9-D-galp (gal gal) located predominantly in the sperm tail [52].
With respect to G+ bacteria (particularly Lactobacillus and Corynebacterium), the most
common fimbrinae involved in bacterial colonization is SpaCBA with a versatile ability
to bind to host cells, mucin, and mucous collagen. It has been revealed that bacterial
aggregation enabled by this pillus only affects sperm motility without any impact on the
morphology or vitality of male reproductive cells [46,47].
Several factors play a role in bacterial adherence, particularly the physical properties
of the bacterial cell wall, area of contact, charge distribution or hydrophobicity [46]. While
the exact molecular pathways driving the adhesion events are not completely understood
yet, pilot studies indicate at least a partial involvement of oxidative mechanisms in enabling
proper bacterial anchoring into the sperm membrane.
Integrins are transmembrane receptors that are intricately involved in important cell
functions, such as cell adhesion or migration by regulating direct cell-to-cell associations
or their interplay with extracellular matrix (ECM) proteins [53]. In addition to somatic
cells, integrins have also been reported on the sperm surface and are known to play an
important role in sperm membrane remodeling, organization of sperm protein complexes
and sperm-oocyte fusion [54]. Nevertheless, activation of integrins by their specific ligands
opens a transmembrane link for the transmission of molecular signals or mechanical forces
across the plasma membrane in both directions. Several integrin receptors may be exploited
by pathogenic agents to enable first contact with the host cell [53]. Furthermore, a growing
list of recent reports demonstrates that integrin engagement by ECM proteins activates
several intracellular cascades accompanied by induced ROS production promoting tight
cell adhesion and cytoskeleton organization [55].
Although the underlying molecular mechanism of integrin-initiated ROS production
remains to be elucidated, it has been hypothesized that the interplay between integrins
and ECM proteins triggers ROS production by promoting changes in the mitochondrial
redox function and activation of several oxidoreductases including NADPH-oxidases,
5-lipoxygenase and cyclooxygenase-2 [56–58]. The GTPase Rac1 activated by integrin
engagement is essential for the stimulation of all ROS-producing systems [56]. Conversely,
there is evidence that ROS can also affect integrin-mediated inside-out signaling by in-
ducing conformational changes required for the activation of integrins [58]. According
to Taddei et al. [59] an immediate cell-to-cell interaction carried out by integrins induces
structural changes to adjacent adhesion molecules that are accompanied by mitochondrial
ROS in the early stage of adhesion while 5-lipoxygenase-derived ROS peak during later
stages of cell attachment. Furthermore, Chiarugi et al. [57] proposed that modulation of
integrin signaling and cell adhesion by ROS is partially mediated by upregulation of focal
adhesion kinase through oxidative inhibition of low molecular weight protein tyrosine
phosphatases. Since ROS are capable of interacting with a wide array of biomolecules,
their impact on cellular signaling events during cell adhesion depends on the site of
production [56] and on the modification of redox-sensitive molecules. As such, target
molecules may include phosphatases and kinases, receptors, transcription factors, actin
and actin-associated proteins [56,58–60].
Finally, a recent report by Strempel et al. [61] has unraveled that H2 O2 and hypochlorite
that are frequently used as disinfecting agents may induce adaptive signaling pathways
in Pseudomonas aeruginosa, particularly by increasing intracellular levels of the second
messenger c-di-GMP and diguanylate cyclase. This enzyme is pivotal in mediating the
Oxygen 2022, 2 553

motility and interactions of Pseudomonas aeruginosa with macrophages, and plays major
roles in the biofilm formation typical of late stages of bacterial adhesion and aggregation. As
such, it may be worth to assume that even extracellular oxidative agents traditionally used
to counteract bacterial growth may in fact contribute to the initiation of bacterial adaptive
mechanisms, which may further aggravate bacterial contamination of biological samples.

3.2. Sperm Agglutination

Sperm agglutination regarded as a direct result of bacterial adherence to the cell
surface is defined as a phenomenon when previously motile spermatozoa stick to each
other. Such compromised male gametes may be connected to each other either in a head-
to-head, tail-to-tail, or mid-piece-to-tail pattern. Nonspecific agglutination may also occur
by the adherence of either nonmotile sperm to each other or of motile sperm to cell debris,
mucus, or other cell types [62]. The type of agglutination is by and large predetermined
by the type of fimbrinae responsible for the initial bacterial adherence: while head-to-
head agglutination is primarily caused by Type 1 fimbrinae, P-fimbriae are responsible
for tail-to-tail agglutination. If the sample is infested by different bacterial types, a mixed
agglutination may occur [63,64].
Events associated with sperm immobilization and an inherent high agglutination
capacity of bacteria will ultimately lead to the formation of a complex structure called a
biofilm. The unique architecture of biofilm provides a more favorable environment for
bacteria to grow and reproduce, by creating niches occupied by bacterial cells that are
covered by layers of extracellular polymeric molecules preventing the entry of antibacterial
remedies [64,65]. The bacterial load within the biofilm is usually very high, promoting the
spread of drug resistance phenotypes [65].
Two molecules released by bacteria play a pivotal role in the promotion of sperm
agglutination and immobilization—the sperm agglutinating factor (SAF) and the sperm
immobilizing factor (SIF). SAFs have been primarily detected in Escherichia coli [66] or
staphylococci [67,68], and are able to block sperm motility, cause morphological abnormal-
ities and act as spermicides at higher concentrations. The motility inhibition lies in the
ability of SAFs to inhibit Mg2+ -dependent ATPase as well as to trigger membrane receptors
responsible for the initiation of apoptosis [66–68].
SIFs are molecules previously isolated from Escherichia coli [69] and Staphylococcus aureus [70],
which directly immobilize male gametes without causing their agglutination. These
molecules also have a specific receptor on the sperm membrane, and besides inhibiting ATP
synthesis, also promote a premature acrosome reaction by causing an ionic imbalance in the
sperm cell [69]. Furthermore, it has been revealed that Staphylococcus aureus-derived SIFs
may have a direct cytotoxic effect on the male reproductive cell [70]. Besides Escherichia coli
and staphylococci, sperm agglutination or immobilization have also been reported in the
presence of Chlamydia trachomatis [71], Mycoplasma [72] or Trichomonas vaginalis [73].
Agglutinated sperm present with a high proportion of morphological and acrosomal
abnormalities. Since both SAFs and SIFs target primarily ATPase, sperm agglutination
and/or immobilization may lead to a disruption in the mitochondrial activity necessary
for sperm motion [62]. As suggested by Wang et al. [74], sperm agglutination favors thiol-
rich, oxidative-stressed, and apoptotic or necrotic spermatozoa, which themselves become
additional sources of ROS. Furthermore, copper-induced lipid peroxidation (LPO) has been
implicated in the loss of cholesterol from the membranous structures with a subsequent
membrane destabilization in agglutinated spermatozoa [75]. Consequently, rupture of the
plasma and mitochondrial membranes of spermatozoa engulfed by bacterial aggregates
may result in the release of cytochrome C and ROS, leading to cell death and a decreased
semen quality [14].

4. Bacterial Toxins
In addition to detrimental effects induced by physical contact between male gametes
and bacteria, damage to spermatozoa may be caused by an array of molecules that are being
Oxygen 2022, 2 554

synthesized and released by bacteria, including lipopolysaccharide (LPS) and hemolysins

(Figure 3).

Figure 3. Damage to the sperm cell caused by (a) hemolysins or (b) lipopolysaccharide (LPS).

4.1. Lipopolysaccharide
LPS is a major component of the outer membrane of G−− bacteria [76]. The molecule
is defined as a prototypical endotoxin, which triggers a significant immune response [77].
During bacteriospermia, LPS is primarily recognized by the Toll-like receptor 4 (TLR4) in
the acrosomal and tail regions of spermatozoa [78], leading to the activation of nuclear
κ to commence the transcription of downstream inflammatory factors [79]. LPS has
factor-κB
been consistently linked to reprotoxicity [78,80,81] by affecting the expression patterns of
pro-apoptotic genes [78,82], and by decreasing the levels of cyclic adenosine monophos-
phate (cAMP), Ca2+ , and the intensity of protein phosphorylation [80]. LPS is also a very
potent prooxidant, exposure to which leads to increased levels of mitochondrial ROS and
occurrence of cell death with a concomitant decrease in sperm motility [78,83]. He et al. [78]
observed that LPS induces mitochondrial transcription factor A (TFAM) translocation,
which activates mitochondrial DNA (mtDNA) replication and subsequent activation of the
mitochondrial oxidative phosphorylation system (OXPHOS) in boar sperm. As a result,
ROS production in the mitochondria is elevated, which leads to oxidative stress (OS),
mitochondrial membrane LPO, and a decrease in sperm motility and viability [84]. In this
sense, the activation of TLR4 and mitochondrial translocation of TFAM are considered to
act as prime mechanisms underlying LPS action [78,85].
Oxygen 2022, 2 555

4.2. Hemolysins
Hemolysins are unique pore-forming toxins that cause alterations to the membrane
integrity and thus contribute to sperm immobilization. Escherichia coli is the sole producer
of α-hemolysin, which forms pores in the host cell membrane, ultimately leading to cellular
lysis [86]. The molecule is receptor-independent, since it presents with a versatility to
permeabilize lipid bilayers of different architectures and compositions, and subsequently
disturbs the intracellular osmotic pressure by forming voltage-dependent ion channels [87].
As such, α-hemolysin is only released if the bacterium adheres to the sperm surface [46].
With respect to spermatozoa, hemolytic Escherichia coli immobilizes spermatozoa more
quickly and effectively when compared with nonhemolytic strains. An important side
effect of hemolysin toxicity lies in the accumulation of lipoxygenase products, increased
release of ROS (particularly O2 •− ) and reactive nitrogen species [87]. This disturbance of
the oxidative milieu is accompanied by a decline in the antioxidant capacity and sperm
mitochondrial membrane potential initiated by cellular rupture [88].
β-hemolysin is a toxin produced by Enterococcus that presents with the same mecha-
nism of action as α-hemolysin and that exerts its toxic effects primarily on the sperm head,
neck, and the middle segment of the tail. The head seems to be the primary target of the
toxin, since a premature acrosome reaction and an increased release of hydrolytic enzymes
are typical characteristics of urogenital infections caused by enterococci [89,90].
Finally, bacteriospermia is often accompanied by elevated levels of enzymes that are
not per se toxic to male gametes but serve to create an optimal environment for the bacterial
colonization of semen to proceed. These include but are not limited to proteases [91],
lipases [92], coagulase or coagulation factors [93].
(a) Since hemolysins are receptor-independent, a direct contact between the bacterium
and the sperm cell is needed for their secretion. Once hemolysins are released, these
will form pores in the plasma membrane, leading to the loss of its semipermeability
and subsequent alterations to the intracellular osmotic balance. Hemolysins will
additionally promote ROS overproduction, leading to peroxidation of the membrane
lipids followed by membrane disintegration.
(b) Once LPS is released, it will be recognized by the Toll-like receptor 4 (TRL4) located
on the sperm surface. Its activation and a subsequent release of ROS will then trigger
nuclear factor-κB (NF-κB) to promote inflammation, as well as caspases to initiate
apoptosis. Meanwhile, LPS will activate the mitochondrial oxidative phosphorylation
system, leading to increased ROS production by the mitochondria, peroxidation
of mitochondrial lipids and a subsequent mitochondrial rupture. Initiation of the
apoptotic process as well as mitochondrial dysfunction will then result in the loss of
sperm motility.
Created with BioRender.com (supplementary: Confirmation of Publication and Licens-
ing Rights) (accessed on 28 October 2022).

5. Leukocytospermia
Semen is a heterogeneous biological fluid containing an array of cells other than sper-
matozoa. Several components representing molecular immunity are found in ejaculates,
such as immunoglobulins, chemokines, cytokines, or growth factors [94]. Furthermore,
almost every semen specimen will contain white blood cells including granulocytes, lym-
phocytes, or macrophages, which represent the principal defense mechanism against
foreign organisms [95]. Supraphysiological amounts of leukocytes is a condition defined as
leukocytospermia, which according to the World Health Organization (WHO), is acknowl-
edged if the concentration of peroxidase-positive white blood cells exceeds 1 × 106 /mL of
semen [96]. This cut-off value is considered a potential indicator of an ongoing and often
asymptomatic male genital infection [95], particularly if a bladder infection or urethritis
have been previously excluded [97].
Symptomatic as well as “silent” urogenital infections accompanied by increased levels
of leukocytes are frequently observed in clinical practice [98]. Nevertheless, a conclusive
Oxygen 2022, 2 556

interconnection among leukocytospermia and sub-fertility needs to be reinforced since

some reports observed a direct correlation between increased concentrations of leukocytes
and alterations to the sperm motion, morphology, or chromatin stability [99], while others
excluded any impact of leukocytes on the fertilization potential of spermatozoa, particularly
in the case of ARTs [100,101]. Despite this controversy, it is acknowledged that the presence
of leukocytes in semen, regardless of their final concentration, is associated with ROS
overproduction that may be of negative influence on certain sperm quality parameters [96].
Different mechanisms of leukocyte-inflicted damage to male gametes are known, out
of which three may be considered as pre-dominant: (1) spermiophagy, (2) secretion of pro-
inflammatory cytokines and (3) extracellular traps (ETs). Alterations to the sperm structure
or function may occur individually or simultaneously, during isolated leukocytospermia or
by leukocytospermia diagnosed concurrently with bacteriospermia [102].
Spermiophagy occurs when white blood cells, inherently programmed to detect and
dispose of damaged or dead sperm, become overactivated and thus surround, engulf, and
destroy even healthy male gametes [98,102,103]. The engulfment of spermatozoa by the
cytoplasm of phagocytic cells is preceded by a direct contact of both cells and adhesion of
leukocytes to the sperm head, midpiece, and flagellum, which is further reinforced by the
secretion of proinflammatory cytokines [102,103].
According to Fraczek and Kurpisz [102], cytokines act within a network, where the
toxicity of one cytokine may be modulated in the presence of other immune molecules.
Among the most common pro-inflammatory cytokines, tumor necrosis factor (TNF) α
released during inflammation and/or infection is most frequently reported to induce
sperm cell death or to contribute to the loss of DNA integrity [104,105]. The cytotoxic
behavior of TNF α may be further mediated via ROS or nitric oxide [106] that is inversely
correlated with sperm motility. Within a vast and heterogenous group of proinflammatory
interleukins (ILs), IL-1b, IL-6, IL-8, IL-12, and IL-18 have been reported to be involved
in inflammation-inflicted damage to spermatozoa in response to bacterial infestation of
semen and a compromised motion behavior [18,105,107]. Similarly to TNF α, increased
levels of ILs have been observed in subfertile subjects or in semen samples with ROS
overload [8,42,100].
A recently observed immune response to the presence of bacteria is the creation of ETs
by activated white blood cells. ETs represent complex 3D web-like scaffolds of DNA strands
embedded with histones and other antimicrobial molecules, such as myeloperoxidase,
lactoferrin, elastase, bactericidal permeability-increasing protein or cathepsin G [108,109].
Physical contact between a leukocyte and a sperm cell leads to fast activation of the white
blood cell, releasing ET structures that will then engulf the male gamete, causing its
immobilization. This phenomenon is aggravated by phagocytosis, degranulation, and
cytokine release [110,111]. Recent studies have observed that ROS are intricately involved
in most reaction cascades promoting the release of ETs. Evidence gathered from a variety
of perspectives has shown that ET-osis involves ROS generation by NADPH oxidase. This
event occurs either directly via molecular signaling that enables ET formation and release,
or indirectly by affecting other factors that modulate the process. Oxidative mechanisms
are likely to act as important players in the regulatory network that determines whether
the process of ET release and activation will be beneficial or detrimental [112].

6. Oxidative Stress and its Impact on Sperm Function

Regardless of the causes for ROS overproduction, oxidative stress has become one
of the leading contributors to alterations in the sperm architecture or functional behavior.
Oxidative stress is defined as a phenomenon when oxidants “overpower” antioxidants,
when fast chain reactions of peroxidation develop and when these processes exhibit patho-
logical effects on the cell [113]. The impact of OS is proportional to the intensity of these
events since the cell is capable of overcoming minor perturbations and returning to its
original state.
Oxygen 2022, 2 557

The complex and intricate cellular structure of male gametes predisposes them to
be particularly vulnerable to OS, since their plasma membranes are characterized by
large quantities of PUFAs, while containing only minor concentrations of antioxidant
molecules in their intracellular compartments [17]. Increased ROS generation has been
associated with a decline in sperm motility [18] either through the ability of H2 O2 to
diffuse across the membranes and inhibit the activity of several enzymes crucial for the
sperm movement [114] or through inhibition of phosphorylation of axonemal proteins and
subsequent sperm immobilization [115]. Furthermore, the motility loss is highly correlated
with LPO, suggesting that oxidation of lipid structures is a major cause for alterations in
the sperm motion behavior [116]. LPO of the sperm membrane is considered to be the
key mechanism of ROS-induced sperm damage leading to subfertility [116], since PUFAs
involved in the maintenance of membrane fluidity and transmembrane communication
are most susceptible to oxidative insults [96]. Sperm LPO is a self-propagating process
during which alkoxyl and peroxyl lipid radicals are formed, and subsequently act on
other lipids in the membrane until all of them have undergone peroxidative changes [19].
These domino reactions will ultimately contribute to oxidative disintegration of DNA and
proteins through lipoperoxides [19,116].
Within the molecule of DNA, bases and phosphodiester backbones are prone to
oxidative insults. While sperm DNA protects itself against ROS by its specific compact
organization and by antioxidants in the seminal plasma, spermatozoa lack any DNA repair
mechanisms and rely on the oocyte to accomplish any repair following fertilization [117].
Out of different types of DNA damage caused by ROS, deletions, base modifications, DNA
cross-links, occurrence of base-free sites, and chromosomal rearrangements are among the
most commonly found in infertile males [118]. OS has also been linked to high frequencies
of single- and double-strand DNA breaks and gene mutations that have been reported
to occur, especially during sperm production and maturation [117,118]. Furthermore,
mutations in mtDNA, which may also be affected by ROS, may compromise mitochondrial
energy metabolism and sperm motility in vivo [119].
Oxidative insults to proteins may lead to in site-specific amino acid modifications,
changes to the electric charge, disintegration of the peptide chain, and alterations to the
susceptibility to proteolysis. Sulphur-containing amino acids are ought to be extraordinarily
susceptible to changes in the oxidative balance. Such disturbance of the structure and
function of sperm proteins may ultimately result in a decreased sperm ability to penetrate
the zona pellucida and to fuse with the ovum [120].
Finally, ROS may trigger a chain of reactions that will ultimately lead to apoptosis.
Under normal circumstances, programmed cell death is a natural process that helps to
dispose of abnormal germ cells. Nevertheless, high ROS levels may disrupt the inner and
outer mitochondrial membrane, accompanied by cytochrome C release and activation of
caspases responsible for the promotion of apoptosis [121]. In the case of bacteriospermia,
previous reports hypothesize that the principal pro-apoptotic mechanism lies in the activity
of bacterial endotoxins, such as LPS or porins to interact with Toll-like receptors 2 and
4 located on the sperm surface [122]. Exposure of male gametes to these toxins may
lead to ROS overproduction and subsequent mitochondrial depolarization followed by
activation of caspase 3-mediated cell death [123]. Furthermore, the immune system may
play a significant role in sperm apoptosis through the cytokine network, since it has been
observed that IL-1b, IL-6, IL-8 or IL-18 could activate the Fas/Fas ligand complex on the
sperm membrane, and subsequently initiate caspase 8-driven apoptotic machinery [14].

7. Clinical Effects of Bacteriospermia on Semen Quality

The effects of bacterial presence in semen on the resulting sperm structural integrity
and functional activity are complex and multivariable as previously reported in several
studies on humans [3,8,10,102,124,125] as well as domestic animals [23,90,126–131]. By and
large, these reports agree that bacteriospermia may lead to the loss of sperm membrane
stability [23,102,126,127,132], acrosome rupture and morphological alterations to the sperm
Oxygen 2022, 2 558

head, mid-piece, and tail [23,48,102,127,133], and mitochondrial dysfunction accompanied

by ATP depletion [23,126,127,133], all of which will ultimately result in the loss of sperm
motility and a subsequent inability of spermatozoa to reach and fertilize the oocyte. A
frequently observed phenomenon associated with the presence of bacteria in semen is
sperm agglutination, DNA disintegration and early onset of cell death [14,41,126,127]. Fur-
thermore, the presence of bacteria in semen has been frequently associated with the onset
of leukocytospermia and increased levels of cytokines with cytotoxic properties [3,10,102].
Finally, preliminary reports have indicated that bacterial activity could modify the biochem-
ical or physicochemical properties of semen, thus creating a less favorable environment
for sperm survival [134,135]. Table 1 provides a summary of the most prominent effects of
uropathogens on male gametes across currently available original papers published on the topic.

Table 1. Leading consensual in vivo and in vitro effects of the most frequent uropathogenic bacteria
on semen quality and/or sperm structural integrity and functional activity.

Bacterium In Vivo Observations In Vitro Observations

↓ sperm count [8,9,23,126,136,137]
↓ sperm motility [8–10,23,126,136–138]
↓ sperm viability [8,9,23,126,136]
↓ sperm morphology [9,136] ↓ sperm motility and viability [20,21,133,139]
↓ acrosome integrity [8,23,126] ↓ sperm morphology [139]
Escherichia coli
↑ DNA damage [8,9,23] ↓ acrosome integrity [140]
↓ chromatin integrity [8] ↑ caspase 3 and 7 activity [20]
↑ leukocytospermia [8,9,23,44,126,137]
↑ concentration of cytokines [8,23,126]
↓ concentration of antibacterial proteins [8,126]
↓ sperm count, motility, viability and acrosome
integrity [126] ↓ sperm motility [20,21,141]
↓ mitochondrial activity [126] ↓ sperm viability [20,21]
Klebsiella pneumoniae
↑ DNA damage [8,9,126] ↓ sperm morphology [21]
↑ leukocytospermia and cytokine levels [126] ↑ caspase 3 and 7 activity [20]
↓ concentration of antibacterial proteins [126]
↓ sperm motility [143]
↓ sperm count, motility and morphology [128,142]
Pseudomonas aeruginosa ↓ sperm viability [20]
↓ sperm viability [122,128]
↑ caspase 3 and 7 activity [20]
↓ sperm count, motility and acrosome integrity [8,9]
↓ sperm viability [8,9]
↓ sperm morphology [9] ↓ sperm motility and viability [20,90]
↓ mitochondrial activity [8,9,126] ↓ acrosome integrity [90]
Enterococcus spp. ↑ sperm apoptosis and phospholipid scrambling [9] ↓ mitochondrial activity [90]
↑ DNA damage [8,9,126] ↑ caspase 3 and 7 activity [20]
↓ chromatin integrity [8,86,126] ↓ chromatin integrity [90]
↑ leukocytospermia and cytokine levels [8,9,126]
↓ concentration of antibacterial proteins [8,126]
↓ sperm count and motility [8,9,23,127]
↓ sperm viability [8,9,127]
↓ sperm morphology [9]
↓ acrosome integrity [8,23,127] ↓ sperm motility and viability [41,133,139]
↓ mitochondrial activity [8,9,23,127] ↓ sperm morphology [139]
Staphylococcus spp.
↑ sperm apoptosis and phospholipid scrambling [9] ↓ acrosome integrity [41]
↓ chromatin integrity [8,23,127] ↓ mitochondrial activity [41,133]
↑ leukocytospermia [8,9,23,127]
↑ concentration of cytokines [8,23,127]
↓ concentration of antibacterial proteins [8]
Oxygen 2022, 2 559

Table 1. Cont.

Bacterium In Vivo Observations In Vitro Observations

↓ sperm count [9,137]
↓ sperm motility and viability [9]
↓ cell morphology [9,137]
Campylobacter ureolyticus ↓ mitochondrial activity [9] N/A
↑ sperm apoptosis, DNA damage and phospholipid
scrambling [9]
↑ leukocytospermia [9,137]
↓ sperm count and motility [9,144,145]
↓ sperm viability [9]
↓ sperm morphology [9,144]
Ureaplasma urealyticum ↓ mitochondrial activity [9] ↓ sperm motility and viability [133]
↑ sperm apoptosis, DNA damage and phospholipid
scrambling [9]
↑ leukocytospermia [9]
↓ sperm count and motility [9,146–148]
↓ sperm morphology [9,146,148]
↓ sperm viability [9,146]
↑ sperm apoptosis, DNA damage and phospholipid
Mycoplasma spp. ↓ sperm motility and morphology [149]
scrambling [9]
↓ chromatin integrity [148]
↑ leukocytospermia [9,147]
↑ concentration of cytokines [147]
↓ sperm count, motility and morphology
[148,150,151]
Chlamydia trachomatis N/A
↓ sperm viability [151]
↓ chromatin integrity [148,150]

Recent studies on bacteriospermia have also provided a solid body of evidence, in-
dicating a significant involvement of OS in bacteria-inflicted damage to male gametes.
Interactions among bacteria and spermatozoa have been shown to trigger ROS overgenera-
tion and subsequent oxidative insults, particularly to the membranous structures of the
sperm cell, leading to the loss of sperm functionality in vivo or in vitro. Changes observed
in the oxidative profile of semen and/or spermatozoa affected by the presence of bacteria
are summarized in Table 2.

Table 2. Leading consensual in vivo and in vitro effects of the most frequent uropathogenic bacteria
on oxidative characteristics of semen and/or spermatozoa.

Bacterium In Vivo Observations In Vitro Observations

↑ intracellular ROS production
↑ intracellular ROS
[23,126]
production [21,22]
↑ superoxide production [9]
Escherichia coli ↑ mitochondrial ROS
↓ antioxidant capacity [8,23,126]
production [20]
↑ LPO [8,9,23,126]
↑ LPO [22]
↑ protein oxidation [23,126]
↑ intracellular ROS production [126] ↑ mitochondrial ROS
Klebsiella pneumoniae ↓ antioxidant capacity [126] production [20]
↑ LPO and protein oxidation [126] ↑ LPO [141]
↑ mitochondrial ROS
Pseudomonas aeruginosa N/A production [20]
↑ LPO [20]
Oxygen 2022, 2 560

Table 2. Cont.

Bacterium In Vivo Observations In Vitro Observations

↑ intracellular ROS production [126]
↑ intracellular ROS
↑ superoxide production [9]
production [90]
Enterococcus spp. ↓ antioxidant capacity [8,126]
↑ mitochondrial ROS
↑ LPO [8,9,126]
production [20]
↑ protein oxidation [126]
↑ superoxide production [9]
↑ intracellular ROS
↓ antioxidant capacity [8,23,127]
Staphylococcus spp. production [22,41]
↑ LPO [8,9,23,127]
↑ LPO [22]
↑ protein oxidation [23,127]
↑ intracellular ROS
↑ superoxide production [9]
Campylobacter ureolyticus production [22]
↑ LPO [9,137]
↑ LPO [22]
↑ superoxide production [9]
Ureaplasma urealyticum N/A
↑ LPO [9]
↑ superoxide production [9]
Mycoplasma spp. N/A
↑ LPO [9]

8. Management of Bacteriospermia in Practice: Strategies, Options and Alternatives

8.1. Antibiotics
Current management of bacteriospermia both in vivo as well as in vitro is by and large
dependent upon antibiotics. The primary mechanism of action of these substances is based
on inducing cell death by interfering with primary structural targets or by corrupting target-
specific processes within the respective bacterial cell. Nevertheless, it is now well known
that these target-specific interactions trigger stress responses that induce redox-related
physiological alterations resulting in the formation ROS, which will further contribute to
bacterial damage and/or death [152].
If bacteriospermia is caused by chronic or acute bacterial infections, these will be
generally treated using a broad spectrum of antibiotics, among which ciprofloxacin, nor-
floxacin, and ofloxacin are most recommended in clinical practice [153]. In the meantime,
supplementation of streptomycin, penicillin, spectinomycin, and lincomycin to semen ex-
tenders and cryopreservation media is required by law to avoid any potential transmission
of disease among recipients [154].
Despite an indisputable contribution of traditional antibiotics to the prevention of
bacteria-inflicted damage to male gametes, several reports have indicated that the in-
herently toxic properties of antibiotics could at least partially affect the male gametes
themselves [155]. It has been recently reported that ciprofloxacin causes a rapid in vitro
disintegration of sperm DNA [156], while gentamycin and ofloxacin administered in vivo
may affect sperm motility and the extent of testicular apoptosis [157]. In this sense, there
is a need to precisely define effective doses and eventual toxicity of antibiotics to male
gametes in order to avoid any adverse effects.
Another aspect that must be taken into consideration lies in often-irrational antibiotic
overuse in practice, which may endanger antibiotic susceptibility and lead to increased
multidrug resistance in a wider range of bacterial species. A recent study has unrav-
eled that more than 56% of all bacterial species found in boar semen were resistant to
gentamycin, while only every fifth isolate was susceptible to gentamycin, lincomycin, peni-
cillin, and neomycin [158]. In the meantime, Pseudomonas aeruginosa and Proteus mirabilis
found in boar semen were resistant to spectinomycin, lincomycin, florfenicol, and strepto-
mycin [159]. An alarming resistance of all isolates retrieved from bovine semen to penicillin
has been reported, while the majority also presented with resistance patterns to tylosin
and lincomycin [160]. With respect to humans, vancomycin and rifampicin were inef-
fective against G+ bacteria detected in Iraqi patients with fertility issues [161]. A similar
Oxygen 2022, 2 561

study on infertile men unraveled the presence of Klebsiella pneumoniae, Escherichia coli, and
Staphylococcus epidermidis with multidrug resistance patterns [162]. Furthermore, 90% of
bacterial isolates found in in vitro fertilization (IVF) culture dishes were resistant to at least
one of the antibiotics commonly used in ARTs [163], complementing a recent report on
increased resistance of staphylococci in ART clinics [164].

8.2. Physical Removal of Bacteria

Since different concerns have arisen from an improper use of antibiotics in human
or veterinarian andrology, several efforts have been made to develop procedures that
would help with the physical removal of bacteria prior to further semen processing. Semen
washing with an extra swim-up step has been reported to be more efficient in limiting the
occurrence of potentially pathogenic bacteria in semen when compared to antibiotics [165].
Previous studies have also emphasized the advantages of density gradient centrifugation
in removing bacteria and viruses from semen. Besides improving the microbial status of
ejaculates, density gradient centrifugation also eliminates leukocytes or dead spermato-
zoa, significantly improving the quality of neat or thawed ejaculates even of suboptimal
quality [41,90,165–167]. In addition, sperm filters have been developed for semen pro-
cessing, causing less damage to the plasma membrane in comparison to centrifugation
techniques [168]. Magnetic-activated cell-sorting (MACS), which identifies and eliminates
apoptotic cells from semen using annexin V-conjugated superparamagnetic microbeads,
could also become a suitable technique to reduce the proportion of damaged or dead
spermatozoa prior to ARTs [169].
A new line of antimicrobial defense is represented by nanoparticles. Recent reports
on iron oxide nanoparticles [170] or silver-carbon nanoparticles [171] applied to semen
reveal that their use did not have any impact on sperm motility, viability, morphology or
DNA integrity, while being effective in the elimination of several uropathogens including
Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa.

8.3. Alternative Antibacterial Supplements

A better prevention or management of bacteriospermia could also be accomplished by com-
plementing conservative prescription medications with traditional remedies. Ethnopharmacolog-
ical herbs such as copperleaf (Acalypha wilkesiana) [172], white weed (Ageratum conyzoides) [173],
clove basil (Ocimum gratissimum) [174], pheasant-berry (Phylantus discoideus) [175], or
Guinea pepper (Aframomum melegueta) [176] have been used for centuries now as remedies
for male urogenital infections caused by pathogens such as Staphylococcus aureus, Escherichia
coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecium and Pseudomonas aeruginosa,
all of which may additionally compromise semen quality. Herbal extracts and essential oils
prepared from the above-mentioned plants may be used as floral beverages or functional
foods with minimal known side effects on health. Furthermore, their use might help in
reducing dependence on antibiotics and minimizing antibiotic resistance [177].
Furthermore, oxidative stress resulting from bacterial infestation of semen may be alleviated
by products from Ajwain (Trachyspermum copticum), juniper (Juniperus communis), Gokshur
(Tribulus terrestris), holy basil (Ocimum sanctum) or Ceylon cinnamon (Cinnamomum verum),
which present with significant antioxidant properties [178]. Preliminary studies have
indicated that administration of these remedies in the form of essential oils or extracts
improved male steroidogenesis, which was accompanied by increased sperm motility,
viability and DNA integrity [179,180].
Finally, the use of plant extracts [181] or pure natural biomolecules [90] as supplements
to semen extenders could be yet another strategy to stabilize the oxidative milieu of in vitro
processed spermatozoa while at the same time acting as antibacterial agents. A complex
assessment of the biological activity of Schisandra chinensis revealed that the Omija extract
was effective against Streptococcus pneumoniae and Enterococcus faecalis, while acting as
a motion-promoting and metabolism-enhancing supplement to bovine sperm in vitro
culture [182]. In the meantime, it was observed that marigold (Calendula officinalis) extract
Oxygen 2022, 2 562

exhibited antibacterial activity against Staphylococcus aureus and Enterococcus faecalis [183] and
at the same time provided significant antioxidant protection to extended bull semen [184].
In their experiments, Eini et al. [185] observed that tea tree (Melaleuca alternifolia) and
rosemary (Rosmarinus officinalis) essential oils were capable of exerting similar effects to
ampicillin on swine artificial insemination doses deprived of spermatozoa and spiked with
Escherichia coli. In the meantime, moringa (Moringa oleifera) and ginger (Zingiber officinale)
extracts supplemented with chilled banana shrimp spermatophores were able to maintain
a higher percentage of viable sperm while inhibiting growth of pathogenic Vibrio and
Pseudomonas [186]. With respect to pure biomolecules, a pivotal study by Duracka et al. [90]
revealed that resveratrol, quercetin and curcumin provided protection to rabbit sperm
structural integrity and functional activity during in vitro induced bacteriospermia by
Enterococcus faecalis while exhibiting partial antibacterial activity against the bacterium.
While the preliminary results on alternative remedies against bacteriospermia collected
from currently available literature are undoubtedly promising, precise and detail-oriented
toxicological studies of these herbs, extracts and biomolecules are essential to elucidate their
suitability for effective in vivo or in vitro management of male reproductive dysfunction
caused by bacterial infection.

9. Conclusions
An increasing body of evidence strongly indicates that the presence and/or activity
of bacteria in semen may exhibit detrimental effects on the structure and function of male
gametes, rendering them to be ineffective in accomplishing their primary role of fertilizing
the ovum. While the exact molecular machinery underlying bacteria-inflicted damage
to spermatozoa is still not fully understood, it is feasible to state that oxidative mecha-
nisms play a pivotal role in the etiology of bacteriospermia. Further studies unraveling
the involvement of reactive oxygen species in bacterial infestation of ejaculates and conse-
quences resulting from this condition will be needed to enable the development of new
techniques to diagnose bacteriospermia in a more effective manner, followed by suitable
strategies to manage the after-effects of bacterial action and/or oxidative damage to male
reproductive cells.

Author Contributions: Conceptualization, E.T.; methodology, E.T. and M.Ď.; software, E.T.; valida-
tion, E.T. and F.B.; formal analysis, E.T., F.B. and M.Ď.; resources, E.T.; data curation, E.T., F.B. and
M.Ď.; writing—original draft preparation, E.T., F.B. and M.Ď.; writing—review and editing, E.T., F.B.
and M.Ď.; visualization, F.B.; supervision, E.T.; project administration, E.T.; funding acquisition, E.T.
All authors have read and agreed to the published version of the manuscript.
Funding: This publication was supported by the projects VEGA 1/0239/20, APVV-21-0095, KEGA
008SPU-4/2021 and by the Operational program Integrated Infrastructure within the project: Demand-
driven research for sustainable and innovative food, Drive4SIFood 313011V336, cofinanced by the
European Regional Development Fund.
Acknowledgments: We wish to thank the CeRA Team of Excellence for their support.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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