ది స్ ఇస్ ఎక్ససర్' ప్టెడ్ ఫ్రామ్ ది బుక్ The Sperm Cell 2న్డ్ ఎడిషన్
Sperm RNA and Its Use as a Clinical Marker
Meritxell Jodar, Ester
Anton and Stephen A. Krawetz
Introduction
Infertility affects 12–30% of
childbearing-age couples [1], and it is
expected that its prevalence will keep rising
[2]. This likely reflects the influence of several lifestyle factors in developed countries,
such as progressive delay in maternal
age, stress, obesity, exposure to
environmental hazards and the use of drugs.
It is suggested that upon presentation to an infertility clinic half of these infertile couples
involve a genetically associatedmale
factor. Nevertheless, a significant proportion
of male infertility (around 25%) remains
diagnosed as idiopathic [3]. The
current standard of practice in assessing male
fertility in the andrology laboratory is usually through semen analysis. This includes a microscopic
analysis in which sperm concentration,
motility and morphology are evaluated
[4]. Although this test is widely used
in clinics to ascertain male reproductive potential, it has been suggested that additional
clinicalmarkers are needed for more
accurate assessments [5, 6]. In this
context, several parameters have been proposed
as suitable fertility biomarker candidates, some ofwhich have already been incorporated in
clinics (e.g. sperm FISH studies, sperm
DNA integrity) [7]. The recent
development of new molecular methodologies
has been driven by genomics, transcriptomics, proteomics and metabolomics. In this chapter, we present a synopsis of the current state of the art of
spermatozoal RNAs, with a glimpse to the
future. Spermatozoal
RNAs Spermatogenesis is a complex biological
process that requires a highly regulated
genetic program to orchestrate a
successful differentiation program. This process occurs over at least 64 days in humans,
beginning as a primordial germ cell
differentiates into a spermatogonium. The
proliferation and differentiation of spermatogonia
into spermatocytes, the progression of spermatocytes
through meiosis to form spermatids, and
final differentiation yield spermatozoa. Dramatic changes in the shape of the round spermatid
and nucleus, as well as the formation of
new organelles, typify the mature sperm.
This is exemplified by the development
of the flagellum and the acrosome, while
most cytoplasm is removed during the final steps of spermatogenesis [8–10].These last steps are
also characterized by a general shutdown
of transcription and translation, as the
genome is compacted by the progressive, albeit
incomplete replacement of themajority of
histones by protamines. Despite the resulting transcriptional blockage and the loss of a large proportion of RNAs during the final stages of
spermatogenesis, a small but complex
population of RNAs is preserved in mature
sperm. Several studies have suggested that the
retention of these transcripts may not be random, but rather reflects residues of spermatogenic
processes or the selection of molecules
that will display their function in
early embryogenesis [11–15]. A single
spermatozoon contains _50–90 fg of long RNA (_200 nt), including coding and noncoding RNAs [16]. This is about 200 times less than
a somatic cell [14]. An extensive and
detailed catalogue of sperm RNAs has
been generated with the recent application
of Next Generation Sequencing (NGS) as summarized
in Figure 4.1, revealing the singularity of
RNAs retained in the sperm [14, 17].The most abundant transcripts in sperm correspond to ribosomal RNAs (rRNAs), but unlike those in somatic
cells, these rRNAs in sperm are
selectively cleaved, most likely to ensure
the translational quiescent state of spermatozoa [18]. A substantial portion of spermcoding
RNAs also The
Sperm Cell, Second Edition, ed. Christopher J. De Jonge and
Christopher L. R. Barratt. Published by Cambridge University Press. _C Cambridge University Press 2017. 59 https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.006 Downloaded from https:/www.cambridge.org/core. Boston University Theology
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Figure 4.1 Characteristics of spermatozoal RNAs. The
spermatozoa contain a complex population of coding and noncoding long RNAs and small noncoding RNAs. Whereas the
majority of coding RNAs are in a biologically fragmented state showing significant
3’ end profile bias (i.e. RN2), a small
percentage of transcripts are intact where all exons are well represented (i.e.
DDX3X). A high percentage of sperm RNAs correspond
to long noncoding RNAs including rRNAs, annotated long noncoding RNAs, natural antisense
transcripts and sperm-specific RNAs
named intronic elements. High levels of specific intronic sperm RNAs are also
observed, while the coding regions of this transcript are absent in sperm (i.e. QRICH1). Several types
of small noncoding RNAs are also observed in sperm, including repeat-associated
small RNAs, piRNAs, transcription start
sites/promoter associated RNAs, miRNAs, small nucleolar RNAs and small nuclear
RNAs. appear fragmented as remnants of
spermatogenesis. However, some
transcripts are maintained intact, with potential
roles in sperm transit through the female
reproductive tract, fertilization and early embryogenesis (Figure 4.1) [17]. A large proportion of human spermatozoal RNAs correspond to noncoding RNAs. Some are
annotated in other cell types as long
intergenic noncoding RNAs (lincRNAs),
transposable elements (LINE1, ERVLMaLR, etc.),
natural antisense transcripts (NAT) and
chromatin-associated (CAR) and small-nuclear
ILF3/NF30-associated (snaR) RNAs. Regulatory roles have been ascribed to these long noncoding
RNAs, functioning both at the
transcriptional and posttranscriptional level.
In addition to this group of known RNAs,
sperm-specific noncoding RNAs are also
observed [14, 17]. Typically they range in size
from 100 to 300 nt and overlap either the coding, intronic or untranslated (UTR) regions of an
otherwise low-expressed or absent
transcript (Figure 4.1).Their specific
retention in all samples sequenced (more than
100 individuals) and the presence of the corresponding full transcript in testes suggests that they
may be part of a separate regulatory
mechanism [17, 19]. Additionally, sperm
contain approximately 0.3 fg of small
noncoding RNAs (sncRNAs) [14] which have
been described as playing an important regulatory role throughout spermatogenesis [20, 21]. Their
presence in human spermatozoa was first
described by Ostermeier et al. [22], and
subsequently, several studies have evaluated
their content in fertile and infertile males. To date, 2,588 mature microRNAs (miRNAs) have
been described in humans (Sanger miRBase
v.21;1)
[23]. It is estimated that they regulate
the expression of up to 60% of the
coding genes [24]. sncRNAs include several
classes of RNA transcripts with sizes ranging from _21 to 30 nucleotides (nt) for miRNAs [25]
and up to _35
nt for piRNAs and tRNA fragments. The bestcharacterized are the miRNAs, which are considered to be highly relevant to regulating important
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that encompass germ cell development, differentiation and proliferation [26]. The study of sperm
sncRNAs has been directed primarily
towards miRNAs as new potential
fertility biomarkers [27]. This is reflected in
the enrichment of categories of processes modulated by miRNAs. The canonical pathway of miRNA
biogenesis is highly regulated through
their transcription, processing, loading
into effective ribonucleoprotein complexes
and turnover [28]. For example, the polymerase
II pri-miRNAs transcripts can be as large
as 3–4 kb, encoding multiple miRNAs [29]. The primiRNAs are then processed by the RNase III Drosha into shorter 41–180 nt (mode 83 nt)
pre-miRNAs [30]. When exported to the
cytosol by exportin 5, they are primed
for processing by the RNase III Dicer, yielding
semicomplementary double-stranded structures. These are unwound through a helicase to yield
two 16– 27 nt (mode 22 nt) mature miRNAs
[30] denoted as -5p and -3p,
respectively, for their strand of origin.
Each mature miRNA is capable of regulating the translation of several target mRNAs by forming imperfect 3’UTR complementary stem–loop structures,
subsequent to their assembly as part of
an Argonaute (AGO) family protein
complex. Another major class of sncRNAs
is the piwiinteracting RNAs
(piRNAs).They have been described as
essential for mammalian spermatogenesis [31],
although their function and biogenesis are still not fully understood. To date, _30,000
piRNAs have been described in humans
(piRNABank2)
[32]. piRNAs can be distinguished from
miRNAs using several criteria. These
include their larger size (24–32 nt), their genesis from single-strand precursors through an
RNase III-independent mechanism, and the
formation of effector complexes with
PIWI proteins (germ-linespecific subfamily
members of AGO) [33, 34].The primary functions
of piRNAs are transposable element silencing
[35], epigenetic programming [36] and posttranscriptional regulation of gene expression [37]. Recent studies have shown the existence of a
turnover mechanism that promotes the
active degradation of the
ribonucleoprotein complex piRNAs-MIWI (mouse
PIWI protein) in the late stages of spermatogenesis through the ubiquitin–proteasome pathway
[38]. This turnover has been proposed to
be part of transposon silencing to avoid
transmitting paternal piRNAs to the
embryo, where they could function in a similar
manner and may impede the zygote-to-embryo transition [39]. Although the depletion of
piRNAs in the later stages of germ-cell
development has been observed [40–42],
elimination may not be complete. Recent
data describe the presence of several piRNA
species in human sperm from fertile individuals [43, 44], along with a population of different
intact mRNA transcripts, in mature sperm
cells [14]. The reduction in piRNA
abundance parallels that observed for the
population of mRNAs as the elongating spermatid
matures and the cytoplasm is expelled [45, 46]. In the human spermatozoa, the major classes
of sncRNAs identified by the first
RNA-seq study [43] included repeat-associated
smallRNAs (65%), piRNAs (_17%),
transcription start sites/promoter-associated
RNAs (_11%), miRNAs (_7%),
small nucleolar RNAs (0.3%) and small
nuclear RNAs (0.1%) [43]. Heterogeneity among
the three sperm samples reflected 20–60%
donor specificity. Interestingly, only 35 miRNAs were consistently present in the three
samples sequenced. This included several
epi-miRNAs (miRNAs that specifically
target effectors of the epigenetic machinery)
and miRNAs with a potential exclusive paternal
origin for early embryonic development (not
detected in mouse oocytes but present in the
zygote). The most abundant miRNA was hsa-miR-34c, which has been described as playing an
important role in spermatogenesis [47]
and early embryogenesis [48], although
species-specific conflicting data have been
reported [49]. Interestingly, most potential targetmRNAs of these ubiquitousmiRNAs have not been detected in sperm [50] supporting their
potential role in translational
suppression by degradation. In direct contrast,
1,137 piRNAs were detected (the most abundant
of which was has-piR-020548), which preferentially target MER, LINE1 and LTR elements. More recently, Pantano et al. [44] used the
same technology to evaluate the sncRNA
in two other normozoospermic individuals.
They observed 182 miRNAs present in both
samples with predicted targets among
sperm-specific genes.The most abundant miRNA
detected by these authors was hsa-miR-1246.
Discordance with respect to this new study and that of Krawetz et al. [43] primarily reflects the
exclusion of miRNA with multiple
alignments to the genome by Krawetz et
al. [43] and the rapid progress in technology.
The similarity between hsa-miR-1246 and U2 small nuclear RNAs is notable. However, the
corresponding immature form, pri-miR-1246,
has been identified in sperm, confirming
its presence. Considering only the
unique aligned miRNAs, hsa-miR-34c was
also found as the most abundant. In their study, Pantano et al. [44] also observed piRNAs as
the most 61 https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.006 Downloaded from https:/www.cambridge.org/core. Boston University Theology
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abundant sncRNAs molecules (some of them processed from pseudogenes), including 408 small RNA clusters containing _1
known piRNA (34 clusters in particular
accounted for 13,509 piRNAs). The preferential
targets predicted for these piRNAs also included LINE1 transposons, concordant with the study
of Krawetz et al. [43]. Discovering Potential Causes of Male Infertility The diagnosis of male infertility is
currently based on the study of seminal
parameters such as sperm concentration, motility
and morphology. To date, a limited number
of causes associated with altered parameters
have been identified [51]. This is exemplified by the general class of chromosomal
abnormalities that are detected in
approximately 5% of patients with
fertility problems. Examples include numerical or structural chromosomal aberrations, as in
Klinefelter syndrome (47,XXY),
structural chromosome reorganizations (e.g.
balanced translocations or inversions) and
Y chromosome deletions. All can have a direct
negative influence on spermatogenesis, thereby affecting sperm production, probably disrupting meiotic pairing. As an adjunct, the study of
ejaculated sperm RNAs has emerged. It
has provided insight into the basicmolecularmechanisms
that regulate production, maturation and
transit of sperm, as well as the pathogenesis
of male infertility. Coding RNAs and Male
Infertility The
initial approach to assessing male fertility by
spermatozoa RNAs relied on RT-PCR for quantification of sperm coding RNAs known to be essential during spermatogenesis. This was followed by the first-generation high-throughput
techniques, i.e. microarrays, which
revealed specific biological pathways affecting
the seminal parameters from the different
subtypes of male infertility [52–55]. The relationship of spermatozoal coding RNAs to humanmale fertility is presented in Table 4.1. The
corresponding summary of the published
data highlighting the potential causes
of different infertility phenotypes associated
with semen parameter alterations follows. Oligozoospermia Oligozoospermia is characterized by a
sperm concentration or total number of
ejaculate sperm below 15 or 39 million
sperm cells, respectively [4], but excluding
the absence of spermatozoa (azoospermia).There are many known causes for oligozoospermia,
including hormonal and chromosomal
disorders, single genetic defects,
testicular and post-testicular factors, such
as varicocele and hypogonadism, and environment
insults [56]. However, even with this multitude of correlative presentations, the primary
causative factor of oligozoospermia
remains to be identified. The observed
reduction of sperm density in unexplained
oligozoospermia suggests that altered spermatogenesis could reflect an altered transcript profile. Gene knockout technology has shown that about 388 genes are critical for murine
spermatogenesis (Mouse Genome
Informatics3).
Down-regulation of some of these genes
has been shown to lead to significantly
decreased RNA levels in infertile men presenting
oligozoospermia. Examples include the DEAD
(Asp-Glu-Ala-Asp) Box Polypeptide 4 (DDX4)
[57], Ubiquitin-Conjugating Enzyme E2B (UBE2B) [58] and some heat-shock proteins (HSPA4,
HSF1and HSF2) [59]. These transcripts
encode proteins that play an essential
role in the early stages of spermatogenesis,
e.g. DDX4, which is key to the differentiation of primordial germ cells and spermatogonia [60] and UBE2B is involved in chromatin
organization of meiotic cells [61].
Microarray studies have shown that the
transcript profiles of oligozoospermic patients
also display a massive down-regulation of
transcripts involved in germ cell development and spermatogenesis [54]. Asthenozoospermia Asthenozoospermia is characterized by
reduced sperm motility or the absence of
motile sperm in greater than 35% of the
spermatozoa examined. Although a high
percentage of oligozoospermic patients
also present low sperm motility, some infertile
patients have a normal sperm count but very poor sperm motility. This can be caused by the
presence of ultrastructural anomalies,
seminal infections and antisperm
antibodies. In addition, different habits
such as smoking, alcohol intake or a poor diet have also been associated with asthenozoospermia
[62–64]. In contrast to oligozoospermia,
RT-PCR studies revealed that
asthenozoospermic patients present alterations
of RNAs associated with sperm maturation
or sperm metabolism in the latter stages of spermatogenesis. For example, some of the transcripts encode for sperm nuclear proteins such as
protamines 62 https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.006 Downloaded from https:/www.cambridge.org/core. Boston University Theology Library,
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Table 4.1 Summary of studies showing
altered spermatozoa transcripts and disrupted pathways associated with altered
seminograms. Male
infertility phenotype GO biological altered (microarray) Transcripts altered (RT-PCR)
References Oligozoospermic Protein–lipid modification mRNA transcription Spermatogenesis and motility Protein targeting and localization, phospholipid metabolism Nucleoside, nucleotide and nucleic acid metabolism DDX4 HSPA4,
HSF1, HSF2 TPD52L3, PRM2, JMJD1A, NIPBL Guo et al., 2007 [57] Ferlin et al., 2010 [59] Montjean et al., 2012 [54] Asthenozoospermic UBE2B PRM1, PRM2
HILS1, TNP1, TNP2 VDAC2 NFE2L2
Yatsenko et al., 2013 [58] Kempisty
et al., 2007 [65] Jedrzejczak et al.,
2007 [66] Liu et al., 2010 [68] Chen et al., 2012 [70] Spermatid development Ubiquinone biosynthesis pathway Metabolic processes Translation
Cell cycle Localization Protein transport Meiosis
DNA repair ANXA2, BRD2, OAZ3,
PRM1, PRM2 Jodar et al., 2012 [53] Bansal
et al., 2015 [52] Oligoasthenozoospermic
CABYR NTRK1 BDNF Shen
et al., 2015 [69] Li et al., 2010 [79] Zheng et al., 2011 [80] Teratozoospermic Ubiquitin–proteasomal
pathway Apoptotic pathway MAP kinase signalling pathway Platts et al., 2007 [76] Oligoteratozoospermic HSPA2 Cedenho et al.,
2006 [78] (PRM1 and PRM2) [65], transition nuclear
proteins (TNP1 and TNP2) and Histone
Linker H1 Domain, Spermatid-Specific 1
(HILS1) [66]. Changes in the expression
of sperm nuclear proteins are associated
with abnormal sperm chromatin condensation and higher DNA damage, which might trigger
apoptosis, inactivating mitochondria and
thereby affecting sperm motility [67].
The expression of other transcripts representative
of processes associated with the regulation
of energy metabolism and sperm motility,
e.g. Voltage-Dependent Anion Channel 2 (VDCA2) located in the mitochondrial outer membrane
[68] and Calcium-Binding
Tyrosine-(Y)-Phosphorylation Regulated
(CABYR) [69], are also detected as altered.
Microarray-based discovery studies have confirmed some of the trends brought forth by RT-PCR.
In this case, disturbances of later
events of germ cell production such as
spermatid development were also observed.
Alterations in the energy production with
perturbations in ubiquinone biosynthesis pathways and in different metabolic processes were
also emphasized [52, 53]. Different antioxidant trials have attempted
to negate the effects of reactive oxygen
species known to contribute to male
infertility. It was hoped that seminal parameters
would improve, reflecting onmale fertility.
Consistent with the hypothesis, nuclear factorNFE2L2 RNA was down-regulated in asthenozoospermic patients. Interestingly, this transcription
factor regulates the expression of
several antioxidant enzymes [70]. This
is consistent with the beneficial effect of
antioxidant intake therapy on sperm motility [71]. Teratozoospermia Teratozoospermia is diagnosed when less
than 4% of sperm have normal morphology
evaluated using Kruger’s strict criteria
[72]. However, sperm morphology has not
been consistently predictive of fecundity,
and its etiology essentially remains unexplored by conventional approaches. The one exception
is 63 https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.006 Downloaded from https:/www.cambridge.org/core. Boston University Theology
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globozoospermia, which is characterized by altered differentiation of the Golgi
apparatus/acrosome. Diseases such as
Hodgkin’s disease and celiac disease, lifestyle
factors and habits may also be contributing
factors [73–75]. A single
microarray-based study has been published
which examined the teratozoospermic sperm RNA profile [76]. As in asthenzoospermia,
teratozoospermic patients also present a
deficiency of spermatocyte and spermatid
transcripts, indicating a disruption of
the later stages of spermatogenesis. Specifically,
the proteasome is broadly downregulated,
likely affecting sperm capacitation, thereby impeding the hyperactive motility of
spermatozoa and the ability to undergo
the acrosome reaction [77]. Combined
Phenotypes The
three seminal parameters, sperm count, motility
and morphology, are usually interrelated and appear coordinated in the individual.
Oligozoospermia is often accompanied by
poor motility (asthenozoospermia) and
abnormal morphology (teratozoospermia), which
leads to even lower fertility. Alterations of
sperm RNAs involved in early spermatogenesis, but not in later events, are observed in these
collective phenotypes. For example, a
transcript encoding heat-shock protein
(HSPA2), essential for the maintenance of
the synaptonemal complex during meiosis,
is down-regulated in oligoteratozoospermic patients [78]. Additionally, a receptor (Neurotrophic
Tyrosine Kinase, Receptor, Type 1;
NTRK1) [79] and a nuclear factor
(Brain-Derived Neurotrophic Factor; BDNF)
[80], involved in the paracrine regulation of spermatogenesis, are down-regulated in sperm from
oligoasthenozoospermic individuals.
These results suggest that alterations
of sperm RNAs resulting in reduced sperm
production are more obvious than these affecting sperm motility and/or morphology. sncRNAs and Male Infertility Few studies have been published describing
the sncRNA content of infertile
individuals. Abu-Halima et al. [81] used
microarrays to analyze the sperm miRNA
content in infertile males with asthenozoospermia (n = 9) and oligoasthenozoospermia (n = 9) and compared the results with those of
normozoospermic individuals (n = 9). They identified 27 down-regulated and 50 up-regulated miRNAs
in spermfromasthenozoospermicpatients
and44downregulated and 42 up-regulated
miRNAs in sperm from oligoasthenozoospermic
patients. Among these differentially expressed
miRNAs, five were selected (hsamiR- 34b∗,
hsa-miR-34b, hsa-miR-34c-5p, hsa-miR- 429
and hsa-miR-122) and evaluated in a larger population of infertilemales [82]. Based on these
results, the authors suggest this panel
of five miRNAs as potential biomarkers
of impaired spermatogenesis in affected individuals. Recently Salas-Huetos et al. used TaqMan
arrays to evaluate the miRNA profiles in
sperm from fertile (n = 10) [83] and infertile individuals with a
single seminal parameter affected:
oligozoospermia (n=10),
teratozoospermia (n = 10) and asthenozoospermia (n=10) [84].The results showed a stable
population of ontologically related
miRNAs corresponding to spermatogenesis and
embryogenesis in sperm from fertile individuals.
In comparison, each group of infertile individuals
presented a differential pattern ofmiRNAs.
These ‘altered’ profiles included 18 differential miRNAs in the oligozoospermic group, 19 in the
teratozoospermic group and 32 in the
asthenozoospermic group. Interestingly,
ontological analysis of predicted targets
of these differential miRNAs showed a direct
relationship with biological processes involved in the specific seminal alterations present in each
population. Interestingly, certain
miRNAs were correlated with specific
demographic parameters such as age (miR-
34b-3p), sperm motility (hsa-miR-629–3p) and sperm concentration (hsa-miR-335–5p, hsa-miR-885–5p
and hsa-miR-152–3p), indicating that
these miRNAs may act as biomarkers for
these attributes. Unfortunately, studies
describing the sperm piRNA cargo in infertile
individuals have yet to be published, and their functional significance potential as biomarkers of
fertility remains to be determined. Integrated Analysis of mRNAs and sncRNAs Altered in Patients with Altered Sperm Parameters
Differential
patterns of mRNAs and miRNAs in patients
presenting altered seminal parameters (oligozoospermia, asthenozoospermia and teratozoospermia) suggests a possible functional relationship between different RNA molecules. It is well
known that miRNAs regulate gene
expression through translational
repression and/or mRNA deadenylation and
degradation (Figure 4.2A). Integrated analysis
is complex because each miRNA can target different 64 https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.006 Downloaded from https:/www.cambridge.org/core. Boston University Theology
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Pathways altered in teratozoospermia and
asthenozoospermia Pathways altered in
oligozoospermia A B 5′ 3′ 5′ 3′UTR Mitosis
Mitosis spermatogonia Spermatogenesis Spermatid
development Sperm motility Sperm capacitation has-miR-139-5p hsa-miR-143-3p hsa-miR-370
hsa-miR-198, hsa-miR-1305, hsa-miR-432-3p PSMC5, PSMD14, GABRB1 TNP1 VDAC2 2n GMPS
n Meiosis Spermiogenesis Sperm function (motility and
capacitation) TRANSLATION
INHIBITION/ mRNA DEGRADATION/ mRNA DEADENYLATION AGO
Protein-coding region AAAA Figure 4.2 Integrated analysis ofmRNAs
and sncRNAs altered in patients with altered seminal parameters. (A) The
mature miRNAs, in conjunction with
Argonaute (AGO) proteins, form a complex able to regulate gene silencing by
translational repression followed by
mRNA deadenylation and degradation. (B) Results of an integrated
analysis of mRNA and miRNA altered in patients presenting altered sperm parameters (oligozoospermia,
asthenozoospermia and teratozoospermia). A correlation of some miRNAs and the
corresponding predicted mRNA targets is
apparent. For example, guanine monophosphate synthase (GMPS), which is
essential during mitosis of spermatogonia,
is post-transcriptionally regulated by hsa-miRNA-139–5p, and both RNAs are
altered in oligozoospermia. Several miRNAs
(hsa-miRNA-143–3p, hsa-miRNA-370, hsa-miR-198, hsa-miR-1305 and
hsa-miR-432–3p) and their corresponding targets Transition nuclear protein 1 (TNP1), Voltage-Dependent Anion
Channel 2 (VDAC2), Proteasome (Prosome, Macropain) 26S Subunit, ATPase, 5
(PSMC5) and Proteasome (Prosome,
Macropain) 26S Subunit, Non-ATPase, 14 (PSMD14) and Gamma-Aminobutyric Acid
(GABA) A Receptor, Beta 1 (GABARB1) are
affected in asthenozoospermia or teratozoospermia. These RNAs have important
roles during spermatid development (TNP1),
sperm motility (VDAC2) and sperm capacitation (PSMC5, PSMD14 and
GABARB1). mRNAs and a single mRNA can be regulated
by different miRNAs. The analysis showed
that more than 50% of predicted targets
from the differentially abundant miRNAs
in one altered sperm parameter were also
potentially affected with other alterations of
seminal parameters. However, 278 predicted targets were unique to oligozoospermia; 2,346 were
unique to asthenozoospermia and 1,531
were unique to teratozoospermia. Only
one known altered transcript in
oligozoospermia (Guanine Monophosphate Synthase
(GMPS)) was predicted to be targeted by a
miRNA known to be altered specifically in oligozoospermic individuals (hsa-miR-139) [54, 83, 84]. GMPS is crucial for purine synthesis.
Alterations 65 https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.006 Downloaded from https:/www.cambridge.org/core. Boston University Theology
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in purine content could alter DNA synthesis and consequently, mitosis in spermatogonia
(Figure 4.2B). Five transcripts
associated with asthenozooospermia (Voltage-Dependent
Anion Channel 2 (VDAC2), Gamma-Aminobutyric
Acid (GABA) A Receptor, Beta 1
(GABARB1), Transition Nuclear Protein 1 (TNP1),
C1D Nuclear Receptor Corepressor (C1D) and
Solute Carrier Family 25 (Mitochondrial Carrier; Phosphate Carrier), Member 3 (SLC25A3)) are
predicted targets of four differentially
expressed miRNAs (hsa-miR-143,
hsa-miR-370 and hsa-mir-432 and has-miR-615)
in asthenozoospermic individuals (Figure
4.2B). hsa-miR-143 regulates both the nuclear
protein TNP1 and C1D, which is involved in the recruitment of RNA to the exosome [52, 66,
83, 84]. Sperm motility is affected by
and reflective of abnormal chromatin
condensation, but it is not known how alterations
of C1D could affect sperm function. Two sperm
channels involved in sperm metabolism and
capacitation (VDCA2 and GABRB1) are predicted to be regulated by two differentially expressed
miRNAs in asthenozoospermic individuals
(hsa-miR-370 and hsa-mir-432,
respectively) [53, 68, 83, 84]. Finally, one
differentially expressed miRNA in asthenozoospermia, hsa-miR-615, regulates a solute mitochondrial carrier (SCL25A3) associated with sperm
motility as identified using a
microarray [52, 83, 84]. Two subunits of
the proteasome (Proteasome 26S Subunit, ATPase,
5 (PSMC5) and Proteasome 26S Subunit, Non-ATPase,
14 (PSMD14)) are highly disrupted in teratozoospermia
and regulated by two altered miRNAs in
teratozoospermic individuals (has-miR-198
and hsa-miR-1305) (Figure 4.2B). Functional analysis of the potential targets of these seven
miRNAs in the specific phenotypes could
elucidate the mechanism by which miRNAs
affect sperm concentration, motility and
morphology. Spermatozoal
RNAs in the Reproductive Clinic Reproductive treatments are typically
recommended to couples unable to
conceive after one year of unprotected intercourse.The
appropriate fertility treatment for each
couple is established after both the female and
the male partner are evaluated. If any severe female or male factor is identified, e.g. an
ovulatory or tubular disorder in females
or a diagnosis of azoospermia or severe
oligoasthenozoospermia in males, assisted
reproductive technologies such as in vitro fertilization (IVF) or intracytoplasmic sperm injection
(ICSI) are recommended. Less invasive
treatments such as the combination of
ovarian hyperstimulation with controlled
timed intercourse (TIC) and intrauterine
insemination (IUI) are typically indicated as the first treatment for couples with mild to moderate
male or female factor and unexplained
infertility [85]. While ART has proven
to be invaluable for couples with severely
compromised semen parameters, the success
of TIC or IUI in infertile patients with normal seminal parameters (normozoospermia) is
unpredictable. After three or four
unsuccessful cycles, idiopathic infertile
couples are usually shunted to ART. Some
studies suggest the primary use ofART, even if seminal parameters are normal, thereby avoiding
failed fertilization [86]. On one hand,
the use of ART for treating some
idiopathic infertile couples might be costeffective when compared to providing IUI followed by ART in those cases that fail [87]. On the
other hand, a successful TIC or IUI
cycle minimizes exposure of the female
partner to invasive treatments such as egg
collection. NGS of spermatozoal
RNAs has revealed a set of sperm RNA
elements that may indicate the best fertility
treatment for idiopathic infertile couples
[19]. RNA-seq can provide a greater resolution than microarrays, as the distribution of
sequencing reads, not the individual
probe, reflects transcription from that
region. Each individual annotated or unannotated transcribed genomic region can be defined as
a sperm RNA element.This strategy
allowed the identification of 648
abundant sperm RNA elements (SREs) that were
essentially at equivalent levels across seven fertile individuals, indicative of a natural
conception. It was observed that
patients expressing all SREs were more
likely to achieve live birth (LB) by TIC/IUI (73%; 22 of 30 individuals) than those with one or
more SREs absent (27%; 3 of 11 males;
two-tailed Fisher’s exact test, P = 0.012).These findings suggested that in those patients lacking at least one of the
SREs, the earlier use of ART would avoid
unsuccessful IUI cycles. Approximately
one-third of the idiopathic infertile males
included in the study (19 of 56 infertile patients) did not present the complete set of SREs,
suggestive of amale factor underlying
the couple’s infertility. In contrast, the
presence of all SREs in the remaining 37 idiopathic infertilemales could indicate the presence of
an unknown female or couple’s factor. About 40% of the male fertility SREs were
located within exonic regions of genes
known to be involved in 66 https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.006 Downloaded from https:/www.cambridge.org/core. Boston University Theology
Library, on 28 May 2017 at 16:50:26, subject to the Cambridge Core terms of
use, available at Chapter
4: Sperm RNA and Its Use as a ClinicalMarker
Table 4.2 Sperm RNA elements (SRE)
required for natural conception
(described by Jodar et al., 2015) located within transcripts altered in different infertile
phenotypes (see Table 4.1). Altered seminogram Transcripts altered (number of containing SRE) Oligozoospermia DDX3X (1) TAF10 (1)
TPT1 (1) Asthenozoospermia OAZ3
(1) TNP1 (2) CANX (3)
BRD2 (7) HSP90AB1 (1) RPS24 (2)
EIF4G2 (2) HNRNPC (1) TBC1D3C (4)
CABYR (2) Teratozoospermia SKP1
(1) ODF2 (9) RERE (4)
spermatogenesis,
spermmotility, fertilization and processes
that precede implantation. As expected, some of the altered transcripts detected by RT-PCR
ormicroarrays associated with alteration
of one seminal parameter (oligozoospermia,
asthenozoospermia and teratozoospermia; see
Section 3.1) contain some of the required
SREs for natural conception (Table 4.2).
New Perspectives
The ability of sperm RNAs to inform male factor status in idiopathic infertile couples, together
with the rapidly decreasing cost of NGS,
suggests that deep sequencing of sperm
RNA could change the counselling approach
for couples seeking reproductive care. The
routine use of this technique in reproductive clinics might also make it possible to evaluate the
quality of the genetic contribution from
the male to the embryo.With the use of
suitable computational tools, it may be
possible to identify genetic variants reflected
in the RNA-seq data [88].This strategy may be useful to evaluate the presence of single-nucleotide
polymorphisms associated with male
infertility, as well as different monogenic
diseases or allelic imbalance. It is
well known that environmental contaminants
and lifestyle factors influence human fertility, which may be reflected to different degrees in the
spermatozoa [89]. These changes can be
transmitted to future generations and
may affect their health and fertility. Several
examples of epigenetic transgenerational
inherence are described in different animal models, e.g. altered metabolism of rodent offspring
due to paternal diet (low protein or
high fat diet) [90, 91] or presence of
cardiac malformations in zebrafish offspring
after paternal exposure to bisphenol A similar to those after direct exposure [92]. Some
authors suggest that transgenerational
epigenetic inherence could reconcile the
adaptation of species to new environments
to which the parents were exposed. This is exemplified in the case of the innate defence
traumatic mechanism against predators
transmitted by olfactory receptors [93].
The concept is controversial, as it
implies transference of hereditary information
from soma to germline. A recent study from Cossetti et al. (2014) proposed that
somatic-cell-derived RNAs can be
transferred to epididymal spermatozoa, likely
through exosomes [94]. Growing evidence points
to spermatozoal RNAs, e.g. miRNAs, as causing
phenotypic variations in the progeny reflective of the father’s life experience [95, 96]. One
example is the observation of a
decreased fear response and the presence
of depressive symptoms in the offspring (F2)
of traumatized male mice [95]. Mice exposed to traumatic early postnatal events showed a dysregulation
of several sperm miRNAs that target
genes involved in DNA, RNA and
epigenetic regulation. The injection of
sperm RNAs from these stressed males into wild
type fertilized oocytes resulted in offspring with similar behavioural disorders, suggesting that sperm
RNAs participate in epigenetic
transgenerational inheritance. Although
F3 also showed similar behavioural disorders,
the population of sperm miRNA from F2 did
not present any alterations. This suggests that
the information provided by altered miRNA in F1 is reversibly rooted in the F2 genome, i.e. by
an epigenetic mechanism, possibly DNA
methylation or chromatin organization.
Interestingly, it was observed that diet-induced
paternal obesity in humans and rodents could
disturb metabolic processes in female offspring
[91, 97]. Obese males also exhibit changes in sperm miRNAs as well as altered sperm DNA
methylation. However, intervention
through a diet and exercise program
during two complete spermatogenic cycles
appears to transmit a normal metabolic profile to female offspring [98]. These findings suggest
that the use of spermatozoal RNAs in
clinics may assist the clinical care of
male infertility and serve as a predictor
of childhood outcomes. All the
studies reviewed in this chapter are based
on the study of sperm RNAs. However, spermatozoa 67 https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.006 Downloaded from https:/www.cambridge.org/core. Boston University Theology
Library, on 28 May 2017 at 16:50:26, subject to the Cambridge Core terms of
use, available at Chapter
4: Sperm RNA and Its Use as a Clinical Marker
account for only 5% of the ejaculate, while the remaining 95% corresponds to secretions from the
epididymis, prostate and seminal
vesicles. These accessory sex glands
release a substantial number of exosomes,
containing a large repertoire of RNAs and proteins, into the seminal fluid [99].The majority of
seminal fluid (_70%
by volume) is produced by the seminal vesicles,
with these secretions being rich in fructose,
a sugar essential for the nutrition of the spermatozoa during the transit to the oocyte. The
secretions fromthe prostate,which
constitute approximately 20% by volume,
contain proteins required for the coagulation
and liquefaction of semen aswell as immune components. These classes of protein typically serve
varying roles in intercellular
interaction and determination of immune
properties. Seminal fluid plays amuch greater
role than simply being a medium to carry the
spermatozoa through the female reproductive tract. New perspectives suggest that the seminal
fluid also provides an optimal
environment for the development and
successful implantation of the preimplantation
embryo, and that its alteration may impact the success of the early pregnancy [100].The integrative
analysis of spermand seminal fluid
transcriptomic and proteomic high-throughput
data has revealed the active transit of seminal
fluid proteins required for sperm maturation
[101], promoting the inclusion of seminal fluid RNAs in future studies ofmale infertility. Some
seminal fluid RNAs appear to provide
additional molecular markers of foci of
spermatogenesis in azoospermic patients thatmay
foreshadow the likelihood of testicular sperm
extraction [102]. Conclusion When a couple first visit a reproductive
clinic, they initiate a well-defined
clinical protocol with the goal of determining
the etiology of their infertility and receive
advice on the treatment options, as well as the success rate and costs. Currently, the evaluation of
the male partner is principally based on
the analysis of seminal parameters in
order to exclude a severe male factor, defined
as the absence or immobility of spermatozoa.
ART is recommended to those patients with a severe male or female factor. The counselling
of 15– 30% of the patients diagnosed
with unexplained infertility [103]
presents a unique challenge. Despite ARTs
having a high success rate in cases of unexplained infertility, they present an increased risk
to the female patient when the use of
less invasive techniques such Deep
sequencing of spermatozoal RNAs and miRNA may predict future health problems in offspring ART ART NO
FEMALE FACTOR TIC/IUI FEMALE FACTOR OR
SEVERE MALE FACTOR COMPLETE SET OF SRE Sperm RNA Elements analysis
Extensive physical and molecular
evaluation Physical and seminal parameters evaluation MILD OR NO MALE FACTOR
INCOMPLETE SET OF NO FEMALE
FACTOR SRE Figure
4.3 Proposed practice for reproductive counselling in couples displaying infertility. Reproductive
counselli
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