Expert Review of Molecular Diagnostics

ISSN: 1473-7159 (Print) 1744-8352 (Online) Journal homepage: https://www.tandfonline.com/loi/iero20

Contemporary genetics-based diagnostics of male

infertility

Alberto Ferlin, Savina Dipresa, Andrea Delbarba, Filippo Maffezzoni, Teresa

Porcelli, Carlo Cappelli & Carlo Foresta

To cite this article: Alberto Ferlin, Savina Dipresa, Andrea Delbarba, Filippo Maffezzoni,
Teresa Porcelli, Carlo Cappelli & Carlo Foresta (2019) Contemporary genetics-based
diagnostics of male infertility, Expert Review of Molecular Diagnostics, 19:7, 623-633, DOI:
10.1080/14737159.2019.1633917

To link to this article: https://doi.org/10.1080/14737159.2019.1633917

Accepted author version posted online: 19

Jun 2019.
Published online: 24 Jun 2019.

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EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
2019, VOL. 19, NO. 7, 623–633
https://doi.org/10.1080/14737159.2019.1633917

REVIEW

Contemporary genetics-based diagnostics of male infertility

Alberto Ferlin a, Savina Dipresab, Andrea Delbarbac, Filippo Maffezzonid, Teresa Porcellie, Carlo Cappellia
and Carlo Forestab
a
Department of Clinical and Experimental Sciences, Unit of Endocrinology and Metabolism, University of Brescia, Brescia, Italy; bDepartment of
Medicine, Unit of Andrology and Reproductive Medicine, University of Padova, Padova, Italy; cUnit of Endocrinology and Metabolism, Department
of Medicine, ASST Spedali Civili Brescia, Brescia, Italy; dDepartment of Molecular and Translational Medicine, University of Brescia, Brescia, Italy;
e
Endocrinology, Montichiari Hospital, ASST Spedali Civili Brescia, Montichiari, Italy

ABSTRACT ARTICLE HISTORY

Introduction Thousands of genes are implicated in spermatogenesis, testicular development and Received 7 May 2019
endocrine regulation of testicular function. The genetic contribution to male infertility is therefore Accepted 17 June 2019
considerable, and basic and clinical research in the last years found a number of genes that could KEYWORDS
potentially be used in clinical practice. Research has also been pushed by new technologies for genetic azoospermia; genetics;
analysis. However, genetic analyses currently recommended in standard clinical practice are still Klinefelter; male infertility;
relatively few. Y microdeletion
Areas covered We review the genetic causes of male infertility, distinguishing those already approved
for routine clinical application from those that are still not supported by adequate clinical studies or
those responsible for very rare cause of male infertility. Genetic causes of male infertility vary from
chromosomal abnormalities to copy number variations (CNVs), to single-gene mutations.
Expert opinion Clinically, the most important aspect is related to the correct identification of subjects
to be tested and the right application of genetic tests based on clear clinical data. A correct application
of available genetic tests in the different forms of male infertility allows receiving a better and defined
diagnosis, has an important role in clinical decision (treatment, prognosis), and allows appropriate
genetic counseling especially in cases that should undergo assisted reproduction techniques.

1. Introduction Using these few genetic tests, identifiable genetic abnorm-

alities are documented in 10–15% of the most severe forms of
The prevalence of infertility in Western countries is estimated
male infertility (azoospermia), with the prevalence of genetic
in about 15% considering infertile couples those unable to
anomalies inversely related to sperm concentration [10].
conceive after one year of unprotected intercourses. A male
However, a large proportion (30%-60%) of infertile males
factor is responsible, alone or in combination with female
does not receive a clear diagnosis. In these cases, generally
factors, in about half of the cases [1]. Male infertility represents
reported as idiopathic infertility, there is a strong suspicion of
one of the clearest examples of complex phenotype with
genetic factors yet to be discovered, and research in this field
substantial genetic basis. Several risk factors and causes
will probably reduce the proportion of unexplained infertility
might affect male fertility, including lifestyles, endocrine dis-
in the next years. Nevertheless, studies on genetics of male
eases, testicular trauma, cryptorchidism, varicocele, genitour-
infertility are intrinsically difficult and complicated, because
inary infections, iatrogenic causes and surgical therapies,
this condition is heterogeneous, it has multiple causes, it can
metabolic diseases, but genetic defects account for a large
be classified in different ways (clinical, semen analysis, father-
proportion of cases [1].
hood) and it results from intricate gene–environment interac-
Indeed, thousands of genes are implicated in spermatogen-
tions that are difficult to discriminate. But we are currently in
esis, testicular development and endocrine regulation of tes-
the midst of an era of rapidly advancing molecular technolo-
ticular function [2–11]. The genetic contribution to male
gies that are exponentially increasing our ability to identify
infertility is therefore considerable and basic and clinical
genetic variants responsible for male infertility, such as for
research in the last years found a number of genes that
other complex diseases [2,3,5–11].
could potentially be used in clinical practice. Research has
A correct application of available genetic tests in the dif-
also been pushed by new technologies for genetic analysis,
ferent forms of male infertility allows receiving a better and
such as whole genome association studies, exome sequen-
defined diagnosis, has important role in clinical decision (treat-
cing, and next-generation sequencing using panel of candi-
ment, prognosis), and allows appropriate genetic counselling
date genes [8,10]. However, genetic analyses currently
especially in cases that should undergo assisted reproduction
recommended in standard clinical practice are still relatively
few [10–17].

CONTACT Alberto Ferlin alberto.ferlin@unibs.it Department of Clinical and Experimental Sciences, Unit of Endocrinology and Metabolism, University of
Brescia, Brescia, Italy
© 2019 Informa UK Limited, trading as Taylor & Francis Group
624 A. FERLIN ET AL.
Article highlights
● The genetic contribution to male infertility is high and research in last
years found a number of genes that could potentially be used in
clinical practice. However, genetic tests currently recommended in
routine clinical practice are still relatively few.
● Genetic causes of male infertility vary from chromosomal abnormal-
ities to copy number variations (CNVs), to single-gene mutations.
● Azoospermia and severe oligozoospermia (<10 million total sperm/
ejaculate) caused by primary testicular damage should be tested for
karyotype anomalies and Y chromosome long arm deletions.
● Hypogonadotropic hypogonadism should be tested for candidate
genes by a panel including multiple genes.
● Congenital bilateral or unilateral absence of vas deferens should be
tested for CFTR gene mutations.
● Azoospermia and severe oligozoospermia with signs of androgen
insensitivity (high LH, high/normal testosterone) should be tested
for androgen receptor gene mutations.
● Studies suggested that idiopathic forms also with moderate oligo-
zoospermia could be tested for the gr/gr deletions in the
Y chromosome.
● Rare causes of male infertility (spermatocytic arrest, isolated idio-
pathic complete asthenozoospermia, globozoospermia, macroce-
phaly, history of cryptorchidism) could be tested for specific gene
mutations (TEX11, dynein genes DNAI1, DNAH5, DNAH11, DPY19L2,
AURKC, NR5A1, INSL3, and RXFP2, respectively).
● Pharmacogenetic tests for FSH treatment of male infertility are pro-
mising but not yet applicable routinely on clinical practice.
● A correct application of available genetic tests in the different forms
of male infertility allows receiving a better and defined diagnosis, has
an important role in clinical decision (treatment, prognosis), and
allows appropriate genetic counseling especially in cases that should
undergo assisted reproduction techniques.
techniques, because the risk of the couple to transmit its
genetic characteristics could be assessed.
The most important aspect in the clinical setting is related
to the correct identification of subjects to be tested and the
right application of genetic tests based on clear clinical data.
Genetic causes of male infertility vary from chromosomal
abnormalities to copy number variations (CNVs), to single-
gene mutations. In this review, we examine the genetic tests
currently suggested in the diagnostic work-up of male inferti-
lity and try to expand current guidelines and reviews (refs.
10–17) with genetic tests that overcame basic and clinical
studies to probably be included in clinical practice.
2. Chromosomal abnormalities
Chromosomal abnormalities are found in about 5% of men
with severe oligospermia (usually defined as a sperm concen-
tration less than 5 million/milliliter or less than 10 million/
ejaculate), with an increase to about 15% in non-obstructive
azoospermic males [10,11,18]. Therefore, chromosomal analy-
sis, i.e., karyotype, is not suggested in unselected infertile men,
but rather limited to men with clear clinical signs of primary
testicular failure. However, although the prevalence of karyo-
type anomalies is directly related to the severity of spermato-
genic impairment, also men with moderate oligozoospermia
or even normozoospermia might be carriers of chromosomal
disorders [19–21]. Therefore, it has been suggested that even
these patients should undergo karyotype when assisted repro-
duction techniques are planned to overcome the couple infer-
tility [12].
Chromosomal abnormalities include both numerical and
structural aberrations of chromosomes, that might involve
the sex chromosomes and the autosomes and that can be
homogeneous or in mosaicisms.
2.1. Aneuploidies of sex chromosomes
Aneuploidies are defined by an abnormal number of chromo-
somes with respect to the euploid state (46,XY or 46,XX). The
most frequent aneuploidies detected in infertile men involve
the sex chromosomes, examples include the Klinefelter syn-
drome (at least one extra X chromosome), mixed gonadal
dysgenesis (45,X/46,XY), and the 46,XX male syndrome.
Klinefelter syndrome represents the most frequent karyo-
type abnormality detected in infertile subjects, with
a prevalence of 10–12% in non-obstructive azoospermic
males, and 0.5–1.0% in severely oligozoospermic men
[18,22]. The genetic characteristic is the extra X-chromosome:
approximately 80%-90% of Klinefelter patients men have a 47,
XXY karyotype, whereas the remaining 10%-20% includes
mosaicisms of two different genetic lines such as 47,XXY/46,
XY, or 47,iXq,Y (in which iXq refers to an isochromosome
where the X chromosome is structurally abnormal with the
chromosomal arms being mirrors of the ‘q’ long arm of the
X chromosome rather than a normal long (q) and short (p)
arm), and higher number of X chromosomes (48,XXXY, etc.)
[23,24]. Klinefelter syndrome is due to non-disjunction of the
X chromosomes during maternal or paternal meiosis I or II,
and less frequently (3%) to nondisjunction during early embry-
ogenesis in the fertilized egg [23].
Phenotypically, patients with Klinefelter syndrome demon-
strate substantial variability in the expression and severity of
classic and non-specific features [22–24]. The common feature
includes primary testicular failure starting from mid-puberty,
and infertility with testicular hypotrophy and hypergonadotro-
pic hypogonadism is the classic phenotype. Testicular dysfunc-
tion might lead also to reduced production of testosterone;
therefore, most clinical signs seem attributable to androgen
deficiency (gynecomastia, eunuchoid aspect, propensity
toward obesity, sparse body hair, osteoporosis, reduced
libido). However, many genetic aspects related to the extra
X chromosome (genes escaping from X inactivation, androgen
receptor gene function, CNVs) could contribute to the varia-
bility, heterogeneity, and severity of phenotype [23].
Testicles of men with KS typically demonstrate progressive
hyalinization, fibrosis, and degeneration of germ cells and
Sertoli cells most often resulting in Sertoli cell only (SCO)
syndrome. Therefore, most patients with Klinefelter syndrome
are azoospermic (about 90%), and rarely severely oligozoos-
permic or few sperm in the ejaculate are found. However, the
chance of sperm retrieval by testicular sperm extraction (TESE)
is quite good (45–50%) [25,26] and therefore intracytoplasmic
sperm injection (ICSI) is a reliable opportunity for these
patients to overcome infertility. Genetic counseling in these
cases is fundamental, as a higher incidence of sperm chromo-
somal alterations has been found in Klinefelter patients [27].
The karyotype 47,XYY is the second most frequent aneu-
ploidy of the sex chromosomes with an incidence of 0.1% in
infertile men. The cause of this aneuploidy is non-disjunction

at meiosis II during spermatogenesis (85%) or postzygotic
mitotic error (15%) [28,29]. Most patients are not diagnosed
until later in life due to a typically normal phenotype, and the
47,XYY karyotype is frequently a casual finding [28,30,31].
Fertility is variable, with semen analyses ranging from azoos-
permia to normal sperm counts [30,32], and testosterone
levels are generally normal [31]. Although some studies by
fluorescent in situ hybridization (FISH) showed an increase in
aneuploidy in sperm samples, an increased risk of aneuploidy
in the offspring has not been documented [29,32].
A 46,XX karyotype might be rarely found in infertile
patients with male phenotype [10], both with and without
detection of the SRY gene (in these cases testes formation is
guaranteed by another gene, autosomal or X-linked, involved
in sex determination). 46,XX male subjects are invariably
azoospermic with testicular atrophy and low testosterone
levels (therefore resembling the Klinefelter patients) and the
chance of sperm retrieval by TESE is virtually zero [33].
Men with 45,X/46,XY Mixed gonadal dysgenesis may pre-
sent at birth with ambiguous genitalia or as adults with infer-
tility, gonadal failure and/or short stature [34]. Phenotype is
variable and gonads may develop into testicles or undifferen-
tiated streaks, and may be located in the scrotum, intra-
abdominally, or along the path of descent in the inguinal
canal. Individuals with bilateral scrotal testicles typically pre-
sent as a male with short stature and gonadal failure, quite
invariably azoospermia and very rare chance of sperm retrieval
by TESE [35]. About half of them have primary hypogonadism
with need for testosterone replacement [36]. This syndrome
might present also cardiac and renal malformations, gonado-
blastoma, and germ cell tumors.
2.2. Structural aberrations of the Y chromosome
Further alterations of the sex chromosomes include structural
aberrations of the Y chromosome, such as deletions, rings,
isochromosomes, inversions, and translocations. Among
these, the most frequent alterations are translocations
between the Y chromosome and autosomal chromosomes
[10]. The frequency of Y chromosome aberrations in the gen-
eral population is 0.02%, but the frequency increases to 0.3%
in azoospermic and severely oligozoospermic patients [18].
Deletions of genetic material from the Y chromosome
including genes necessary for spermatogenesis might be
detected by karyotype when including most of the long arm
[18] or might result in a microdeletion within the Yq that
cannot be detected on karyotype and are the cause of up to
10% of men with non-obstructive azoospermia. These are
further discussed below.
2.3. Structural aberrations of autosomes
Autosomes aberrations detected by karyotype analysis include
translocations, inversions, rings, isochromosomes and other
structural abnormalities such as extra satellite marker chromo-
some and clinical syndromes including partial deletions or
duplications (if larger than 10Mb).
Chromosomal translocations can affect male fertility and
pregnancy outcome [4,10–12,15,37,38] and, in particular,
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 625
males with reciprocal translocations have a high rate of
unbalanced spermatozoa due to meiotic segregation errors
[39,40]. Therefore, genetic counseling and analysis of chro-
mosomal constitution in sperm or pre-implantation genetic
diagnosis is suggested in order to assess the risk of trans-
mission of unbalanced forms. From a quantitative point of
view, translocations may be balanced (no loss of quantity of
DNA), or unbalanced (with loss of DNA). Reciprocal translo-
cations involve an exchange of genetic material between
two or more chromosomes, they are the most common
chromosomal structural anomalies in humans and are 10
times more common among infertile men [4,10–
12,15,37,38]. The most frequent translocations in infertile
patients are robertsonian translocations or centric fusions
[4,10–12,15,37,38]. Robertsonian translocations occur with
an incidence of 0.9% of men with severe male factor inferti-
lity [35]. These occur among acrocentric chromosomes
(chromosomes 13, 14, 15, 21, and 22): the long arms of
two chromosomes fuse resulting in loss of genetic material
of the chromosomal short arms (final complement of 45
chromosomes). The most common robertsonian transloca-
tions detected in infertile men include t(13q;14q) and t
(14q;21q). As men with reciprocal translocations, men with
robertsonian translocations are generally phenotypically nor-
mal, but spermatogenesis might be impaired and a high rate
of unbalanced sperm might be found [41].
Autosomal inversions can be considered intrachromosomal
reciprocal translocations without loss of genetic material.
Inversions relevant to male infertility include that of chromo-
some 9. This inversion is found in 3%-5% of men with inferti-
lity and is associated with variable phenotype ranging from
normospermia, oligospermia, azoospermia, and asthenosper-
mia [42,43].
In conclusion, karyotype analysis should be performed in
non-obstructive azoospermic and severely oligozoospermic
men with primary testicular failure, as suggested by testicular
hypotrophy and high plasma levels of FSH (Tables 1 and 2),
and allows identification of a variety of chromosomal altera-
tions (aneuploidies of sex chromosome, structural alterations
of the Y chromosome, structural alterations of autosomes).
Karyotype might also be suggested in all cases of infertility
(including normozoospermia) before assisted reproduction
techniques, after repeated assisted reproduction techniques
failure and repeated abortion [12]. In these latter cases, struc-
tural aberrations of the autosomes might be found allowing
better counseling of the infertile couple.
3. Y chromosome microdeletions
Genetic alterations of the Y chromosome are found with high
frequency in infertile men [1,44–46]. A number of studies have
been conducted in infertile men after the identification of
microdeletions (not detectable by karyotype analysis, but
only with molecular diagnostics) in the Y chromosome long
arm (Yq), removing factors important for spermatogenesis.
These regions of the Yq euchromatin are called azoospermia
factors (AZF) because initially found in azoospermic men. It is
now widely accepted that Y chromosome microdeletions are
clinically important because they are associated not only with
626 A. FERLIN ET AL.

Table 1. Current diagnostics genetic tests suggested for male infertility.

Genetic test Indications
Karyotype Azoospermia and severe
oligozoospermia (<10 million
sperm) caused by primary

(<39 million total sperm/ejaculate)

testicular damage
Repeated abortion
Known chromosomal diseases

Oligozoospermia
Microdeletions of the Y chromosome Azoospermia and severe
long arm (AZFa, AZFb, AZFc) oligozoospermia (<10 million
sperm) caused by primary

N
N
testicular damage
Y chromosome gr/gr deletions Oligozoospermia (<39 million sperm)

CAVD – CFTR mutations

Candidate genes for Kallmann syndrome and normosmic
hypogonadotropic hypogonadism hypogonadotropic hypogonadism
(panel with multiple genes)°
CFTR Congenital absence of vas deferens

gr/gr deletions

Androgen receptor (AR)
gr/gr deletions
TESTICULAR
(bilateral/unilateral)
Azoospermia and severe
oligozoospermia (<10 million

CFTR
sperm) with signs of androgen
insensitivity
NR5A1* Azoospermia and severe

↓FSH, LH, T

Genetic HH
oligozoospermia (<10 million

HH genes
screening
(panel)
sperm) mainly with history of

↓
Table 2. Genetic tests in clinical practice for quantitative spermatogenic defects, based on clinical data to correctly identify subjects to be screened.
cryptorchidism

PRE-
INSL3 and RXFP2* History of cryptorchidism
TEX11* Idiopathic azoospermia
(spermatocytic arrest)

INSENSITIVITY (documented bilateral absence of

Dynein genes (DNAI1, DNAH5, Isolated idiopathic complete
DNAH11) asthenozoospermia
DPY19L2 Globozoospermia

AR mutations CAVD – CFTR mutations

AURKC Macrocephaly
Azoospermia and severe oligozoospermia (<10 million total sperm/ejaculate)

°See ref. 80 for a list of genes

N
N
*Not yet in clinical practice. POST-TESTICULAR

vas deferens)
non-obstructive azoospermia but also with severe oligozoos-

CFTR
permia [1,10,11,14,16,18,44–46].
Microdeletions in the Yq indeed represent the most frequent
ANDROGEN

molecular genetic cause of severe infertility, being detected in

↑/N T
↑LH
↓

10–15% of non-obstructive azoospermic and in 5–10% of

severely oligozoospermic patients [1,10,11,14,16,18,44–46].
AR

Most deletions are de novo and are likely to arise during meiosis
of the gametes from the patient’s father. Although the genetic
pathways and mechanisms of spermatogenic impairment are
Translocations, inversions and other autosomal structural
still largely unknown, three regions AZFa, b and c from proximal
to distal Yq, have been identified [47], but different classifications
have also been proposed based on the mechanism leading to
microdeletions [48–50]. Each of these regions may be deleted
Y chr. microdeletions (AZFa, AZFb, AZFc)

independently or in combination. The six classic forms of AZF

deletions and their corresponding phenotype, in order of
↑FSH
↓

decreasing severity include: AZFabc (Sertoli Cell Only syndrome),

NR5A1 mutations (see table 1)
TEX11 mutations (see Table 1)

AZFa (Sertoli Cell Only syndrome), AZFbc (Sertoli Cell Only syn-
chromosomal aberrations
Y chr. structural aberrations

drome/maturation arrest), AZFb (maturation arrest), AZFc (ran-

ging from severe oligospermia to azoospermia), and partial AZFc
Klinefelter syndrome
NR5A1 (see table 1)
TEX11 (see Table 1)

(from normal spermatogenesis to azoospermia) [45].

Y chr. microdel.

The most frequent microdeletion involves the AZFc

TESTICULAR

46,XX male

region (about 60–70% of the deletions) and produces the

Karyotype

loss of several genes, among whom the DAZ gene is the

major responsible for the phenotype. Different degrees of
spermatogenetic alterations might be found, but, in general,
CLASSIFICATION
SEMEN PHENOTYPE

OLIGOZOOSPERMIA
TESTIS VOLUME

the most part of patients with this alteration have sperm in

GENETIC TESTS
absence of vas
(idiopathic or
documented

the ejaculate or in the testes [45]. The AZFc region spans 4.5
INFERTILITY
HORMONES

DIAGNOSIS
unilateral

deferens)

Mb of euchromatin and contains five protein-encoding

genes shown to be implicated in spermatogenesis: BPY2,
CDY, DAZ, CSPG4LY, and GOLGAZLY [51]. The DAZ family
 EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 627

consists of four genes encoding RNA-binding proteins oligospermia with primary testicular failure, as suggested by
expressed exclusively in germ cells [52]. AZFc deletions are testicular hypotrophy and high plasma levels of FSH (Tables 1
the most common Y-microdeletions because this region is and 2). Deletions of these regions are identified by molecular
composed of repeated regions organized in palindromic techniques, especially PCR, following specific guidelines
sequences (amplicons), which are particularly susceptible to [46,50].
deletions through a mechanism called nonallelic homolo-
gous recombination [53]. Therefore, microdeletions might
be also named based on the repeated regions involving in 4. CNVs (copy number variations) on sex
recombination and causing the loss of genetic material. The chromosomes and autosomes
most frequent deletions occur through recombination
New genetic tools, such as SNP array, CGH array and NGS,
between ampliconic regions b2/b4 (classic AZFc deletion),
allowed in recent years the identification of a number of rare
b1/b3, b2/b3, and gr/gr resulting in the deletion of different
autosomal deletions, X-linked CNVs and Y-linked deletions and
genes including multiple copies of the DAZ genes. gr/gr
duplications that might be implicated in increasing the risk of
deletions represent a particular form of ‘partial AZFc dele-
male infertility [2,3,5–11,17].
tions’ [54], removing only part of the DAZ gene family, and
Among them, deletions in 9p chromosome including the
are described below because they might be classified
DMRT1 gene is particularly attractive as genetic cause of male
among CNVs. Up to 70% of men with AZFc deletions have
infertility, even if with a very low frequency. DMRT1 is a testis-
sperm in the ejaculate, typically less than 2 million sperms
specific transcriptional regulator required for testicular differ-
per milliliter [45]. In men with azoospermia and AZFc dele-
entiation mapping on chromosome 9p24.3, a region involved
tions, TESE can be used to harvest sperm from the testicle in
in XY gonadal dysgenesis [59]. Smaller deletions [60] and
50%-60% of cases [10,45,46,55].
mutations [61] of this gene have been identified in patients
Complete deletions of AZFa are rare, accounting for only
with non-obstructive azoospermia without XY gonadal
3% of Y-microdeletions. They carry the poorest prognosis with
dysgenesis.
azoospermia in all men [46,56]. Histology typically demon-
Other studies provide strong evidence for the involvement of
strates Sertoli Cell Only syndrome, with virtually no chance
X-chromosome CNVs in spermatogenic impairment. In particular,
to find sperm at TESE. The AZFa region harbors two protein-
the group of Krausz reported by X chromosome-specific CGH
coding genes, USP9Y and DBY [56], with the latter playing the
array recurrent deletions on Xq. The authors found that two CNVs
major role in spermatogenesis [56]. Partial AZFa deletions
(CNV64 and CNV69) represent risk factors for spermatogenic
have also been reported rarely, removing only DBY or USP9Y
impairment, whereas another CNV (CNV67), containing the
genes, with less severe phenotypes and testicular histology
gene MAGEA9, is present exclusively in patients [62].
demonstrating hypospermatogenesis [56,57].
Rearrangements other than classic Yq microdeletions,
The AZFb region contains seven protein-encoding genes
namely CNVs in the AZFc region referred to as gr/gr deletion
experimentally shown to be implicated in spermatogenesis.
based on the repeated sequences involved in the recombina-
These include EIF1AY, RPS4Y2, and SMCY located in the
tion have been associated with an increased risk for sperma-
X-degenerate euchromatin, and HSFY, XKRY, PRY, and RBMY
togenic impairment [63,64]. The deletion removes half of the
located in the ampliconic regions [58]. AZFb deletions account
AZFc region gene content, and therefore two of the four
for 15% of Yq microdeletions [46]. Invariably, complete dele-
copies of the DAZ gene [54,64]. gr/gr deletions higher the
tions result in azoospermia and Sertoli Cell Only syndrome or
risk of oligozoospermia (about 2–4 higher risk) and the num-
early maturation arrest of spermatogenesis [45,46,58].
ber and quality of studies performed in last years suggest that
Combinations of deletions in various AZF regions may
genetic test for detecting this CNV is indicated in men with
occur. Combined AZFb e AZFc deletions are the most com-
idiopathic oligozoospermia (Tables 1 and 2), in which the
mon because the two regions overlap and share 1.5 Mb [10].
frequency of gr/gr deletion is 3–4% (compared with
This combination of deletions accounts for approximately 13%
0.4–1.4% in men with normozoospermia) [1,10,11,46,65]. As
of Y-microdeletions. Patient phenotypes demonstrate Sertoli
for complete AZFc deletion, gr/gr deletions will be obligatorily
cell only syndrome or spermatogenic arrest, and sperm retrie-
transmitted to male offspring and reports exist describing the
val by TESE is uniformly unsuccessful [46].
potential extension of gr/gr deletion to complete AZFc dele-
As said, fertility potential is present in some patients with
tion in the next generation [66].
Yq microdeletions, especially those with AZFc deletions. In
these cases, the Y-microdeletion is obligatorily passed to
male offspring, who will have consequent impaired spermato-
5. X-linked gene mutations
genesis. Therefore, appropriate genetic counseling should be
offered to couple undergoing assisted reproductive techni- Although the number of X-linked genes expressed in the testis
ques [4,18]. Furthermore, progressive decreases in sperm pro- and with potential role in spermatogenesis is very high [67],
duction have been reported in men with AZFc deletion, and from a clinical point of view in the setting of infertile males the
therefore preventive cryopreservation of sperm in men with main conditions associated with the X chromosome are repre-
detected deletion, and early descendants of AZFc deletion sented by mutations in the KAL1 gene causing the Kallmann
carriers is suggested for fertility preservation [10]. syndrome, mutations in the androgen receptor (AR) gene
In summary, analysis of Yq microdeletions is recommended causing varying degrees of androgen insensitivity, and muta-
in patients with non-obstructive azoospermia and severe tions in TEX11 gene responsible for spermatogenic arrest.
628 A. FERLIN ET AL.
Kallmann syndrome has a frequency of 1:10.000 and is
characterized by hypogonadotropic hypogonadism (HH) (low
serum level of testosterone, LH and FSH) and anosmia or
hyposmia [68,69]. Beyond HH and midline defects such as
cleft palate, other clinical features associated with Kallmann
syndrome include tall stature, cryptorchidism, unilateral renal
agenesis, and neurogenic deafness [68,69]. The most frequent
genetic alteration responsible for this syndrome is a mutation
in the X-linked gene KAL1 (14% of familial cases and 11% of
sporadic cases), encoding the protein anosmin-1, which is
a cell adhesion protein of the extracellular matrix. During
embryogenesis, anosmin-1 is required for the organized
migration of both olfactory axons and GnRH neurons from
the olfactory placode through the cribriform plate and into
the preoptic area of the hypothalamus. As such, defects result
in anosmia and GnRH deficiency. GnRH deficiency results in
subsequent lack of pulsatile LH that is required for normal
gonadal function. Recent advance in this field, however, iden-
tified many other genes involved in HH mapping on chromo-
somes other than X chromosome (see below).
Mutations in the AR gene have been described in 2–3% of
azoospermic and severely oligozoospermic men and might be
suggested by high/normal testosterone and LH levels [70,71].
Mutations in the AR gene cause a variety of defects known as
androgen insensitivity syndrome, with phenotypes ranging from
female appearance of external genital at birth in the complete
forms, to various kinds of genital abnormalities in the partial
forms and to isolated male infertility in milder forms [70,71].
More than 1000 mutations have been described in this gene;
therefore, complete sequencing is necessary for diagnosis.
TEX11 (Testis-expressed 11) is located on chromosome
Xq13.2 and it encodes for a protein that contributes to repair-
ing double-strand DNA breaks and regulates recombination
and homologous chromosome synapses [72,73]. It is exclu-
sively expressed in spermatocytes and spermatids and hemi-
zygous loss encompassing a part of TEX11 and TEX11
mutations have been described in men with azoospermia
and maturation arrest at testis histology [72,73].
6. Autosomal gene mutations
Other genetic analyses that should be considered in the infer-
tile male are mutation screening in autosomal genes, selected
depending on clinical suspicion.
CFTR gene mutations are responsible for cystic fibrosis (CF),
an autosomal recessive disease due to the presence of severe
mutations in both copies of the gene, which encodes a salt
homeostasis anion channel in epithelial cells. Mutations in this
gene are very frequent in the general population (1/25 subject is
a carrier in the western countries) [74]. Men carrying a mild form
of the CFTR gene mutation are generally phenotypically normal
but might present azoospermia due to congenital bilateral
absence of the vas deferens (CBAVD) (obstructive azoospermia)
[75]. The clinical picture is therefore different from the forms of
primary testicular failure described above, as testicular volumes
and reproductive hormones plasma levels are normal.
Furthermore, unilateral absence of vas deferens (CUAVD) might
develop as a consequence of mild CFTR mutation and these
cases might therefore present with mild oligozoospermia and
even normozoospermia [12]. CBAVD has a prevalence of 1%
among infertile patients and 25% among men with obstructive
azoospermia [75]. Among men presenting with CBAVD, 78%
carry at least one identifiable CFTR mutation, and 46% carry
two mutations (compound heterozygotes) [75]. Azoospermic
men with CBAVD could have fatherhood by assisted reproduc-
tive techniques using sperm obtained through percutaneous
epididymal sperm aspiration (PESA), microsurgical epididymal
sperm aspiration (MESA), testicular sperm aspiration (TESA) or
TESE. Appropriate genetic counseling, genetic screening in the
female partner and consideration of preimplantation genetic
testing is recommended in these cases [12].
Screening for CFTR gene mutations is therefore recom-
mended in infertile men with bilateral or unilateral absence of
vas deferens (Tables 1 and 2) and suspicion for incomplete
obstruction of the genital tract in oligozoospermic men [16].
CFTR gene mutation screening should be performed as the
targeted variant panel that includes causing variants based on
the geographic and ethnic origin of the patient as the first level
test. Ideally, this panel should include the 30–50 most frequent
mutations able to detect >80% of carriers. If clinical suspicion is
high, but this first step found no mutations or one mutation,
Sanger sequencing of exons, 5ʹ flanking regions and intron-exon
boundaries or NGS of the entire gene could be considered
as second step analysis. Anyway, given the high prevalence of
CFTR mutations in the general population, for genetic counseling
dealing with the risk of transmission, it is probably better to
analyze the female partner after the first step analysis, because
the cost of complete gene sequencing is much higher and time
consuming than targeted variant panel analysis. Similarly,
screening for CFTR gene mutations is frequently recommended
by international guidelines before assisted reproduction techni-
ques on both partners to have an informed discussion regarding
the risk of CF in the offspring [12]. Importantly, CFTR analysis
should always include the 5T allele, a variant typically associated
with CBAVD, and TG(n) repeat polymorphism, which influences
its penetrance and clinical effect.
NR5A1 is a gene coding for a transcriptional activator with
essential role in sexual differentiation, development of primary
steroidogenic tissues and regulation of the AMH/Muellerian
inhibiting substance gene [76]. Mutations in NR5A1 have been
initially associated with primary adrenal insufficiency and 46,
XY gonadal dysgenesis [77] but more recently with less severe
phenotypes, including severe forms of male infertility with
primary testicular damage, especially when associated with
a history of cryptorchidism [78].
Genes other than the X-linked gene KAL1 are known to be
involved in hypogonadotropic hypogonadism (HH). In particu-
lar, from a genetic view, Kallmann syndrome and isolated,
normosmic, HH show overlap and some cases of HH not
always follow the rules of Mendelian inheritance, as it can be
caused by digenic or oligogenic transmission [79]. HH is
genetically heterogeneous and, despite the identification of
many novel candidate genes (such as FGFR1, FGF8, HS6ST1,
SOX10, PROK2, PROKR2, TAC3, TACR3, KISS1, KISS1R, GNRH1,
GNRHR), the cause remains unknown in many cases [80].
Indeed, in the clinical suspect of HH, a comprehensive genetic

Table 3. Genetic tests in clinical practice for qualitative sperm defects.
Complete
SEMEN PHENOTYPE asthenozoospermia
GENETIC TESTS Dynein genes screening
(DNAI1, DNAH5, DNAH11)
DIAGNOSIS Genetic asthenozoospermia
screening of a panel of 20–30 genes by NGS is suggested
[10,80]. Even in this way 30–40% of cases still remain without
an identifiable genetic cause.
Other genetic tests that might have clinical value are
related to particular forms of male infertility. INSL3 (insulin-
like factor 3) gene and RXFP2 (relaxin family peptide receptor
2, the receptor for INSL3) gene mutations could be considered
in patients with a history of cryptorchidism [81]. INSL3 is
a member of the relaxin-like hormone family produced by
the Leydig cells and is a major determinant for the transab-
dominal phase of testicular descent [82]. Beside the role in
testicular descent, INSL3 seems to cause endocrine and para-
crine actions in adults, and deficiency of this hormone might
represent a sign of functional hypogonadism [82,83]. Although
mutations in INSL3 and its receptor RXFP2 represent a cause
or risk factor for cryptorchidism, their role in infertile men
without a history of cryptorchidism is still unknown.
Genetic tests should also be considered in infertile men
with specific qualitative sperm defects, such as asthenozoos-
permia, globozoospermia and macrocephaly (Tables 1 and 3).
Mutations in dynein genes cause primary ciliary dyskinesia
(PCD) [84–86]. In particular, three genes are mainly involved:
DNAI1 (axonemal dynein, intermediate chain 1), DNAH5 (axo-
nemal dynein, heavy chain 5) and DNAH11 (axonemal dynein,
heavy chain 11). Axonemal ultrastructural defects of sperm,
including absent or shortened arms of dyneins, can be found
in >50% of PCD patients. Interestingly, dynein genes muta-
tions have been associated with isolated nonsyndromic asthe-
nozoospermia in 8% of cases [87]. Many other genes might be
associated with asthenozoospermia [7], but large studies are
lacking to suggest the ideal panel of genes to be included in
the diagnostic process.
Globozoospermia is a rare autosomal recessive condition
characterized by sperm with a round head and no acrosome,
and it is found in approximately 0.1% of infertile men. Without
an acrosome, the abnormal sperm is unable to undergo acro-
some reaction and get through the outer membrane of an egg
cell. Many different gene mutations have been associated with
mouse and human globozoospermia, but approximately 70%
of the cases are caused by defects in the DPY19L2 gene
(mostly complete deletion of the gene, rarely intragenic dele-
tions and point mutations) involved in the development of the
acrosome and elongation of the sperm head [88]. Genetic
analysis of infertile globozoospermic patients identified causa-
tive mutations also in PICK1, SPATA16, and ZPBP genes [89].
Macrocephaly is a rare condition (<1% of infertile men)
characterized by large-headed sperm. Mutations in AURKC
gene, with a role in meiotic chromosomal segregation, are
the only validated genetic causes of sperm macrocephaly
[90]. About 90% of men with macrozoospermia exhibit
a homozygous deletion of cytosine in exon 3 of the AURKC
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 629
Globozoospermia Macrocephaly
DPY19L2 AURKC
Genetic globozoospermia Genetic macrocephaly
gene (c.144delC). This mutation results in a truncated protein
with consequent alteration in meiotic divisions and formation
of polyploid or tetraploid sperm in cases of alteration in both
meiotic divisions. Geographical differences exist, as this muta-
tion is particularly prevalent in North Africa. Other than
genetic test for this gene, patients with macrocephaly should
receive appropriate genetic counseling, as assisted reproduc-
tion techniques are not advised because of the abnormal
chromosomal constitution of sperm.
Ongoing studies with new genetic tools, especially NGS, of
large cohorts of idiopathic infertile males would undoubtedly
raise the number of genes implicated in the different pheno-
types (quantitative spermatogenic defects, hormonal regula-
tion of spermatogenesis, qualitative sperm defects, obstructive
forms) and our diagnostic possibilities. New genetic tools,
however, will also increase the number of variations of
unknown significance. A recent systematic review [8] already
identified 521 gene–disease relationships, of which only 18%
(92 gene–disease relationships involving 78 genes) have been
at least moderately linked to human male infertility
phenotypes.
7. Pharmacogenetics applied to FSH treatment in
idiopathic male infertility
The currently most promising approach to treat male infertility
is to stimulate spermatogenesis by FSH treatment. However, it
is also clear from many studies published so far that FSH
treatment is not indicated for all infertile men, and also in
selected patients, the effects are not easily predictable [16,91].
One of the most challenging aspects in this field is, therefore,
the identification of the ‘perfect’ patients, i.e. the patient with
the highest probability to respond to treatment.
A personalized treatment regimen which takes into account
clinical and genetic factors controlling spermatogenesis might
resemble the most promising approach to this aim [92] as
discussed below.
Polymorphisms in the FSHR gene have been demonstrated
to influence the expression and/or sensitivity of the receptor for
the hormone and polymorphisms in the FSHB (coding for the β-
subunit of FSH) gene is responsible for the constitutive produc-
tion of FSH [92]. As a consequence, FSH plasma levels and the
reproductive parameters are associated with the individual gen-
otype of these genes both in men and women [92]. The two
most common SNPs in the coding region of FSHR occur at
nucleotides 919 and 2039 in exon 10, in which A⁄G transitions
cause amino acid exchange from threonine (Thr) to alanine (Ala)
at codon 307 and from asparagine (Asn) to serine (Ser) at codon
680, respectively [93]. There is a linkage between these poly-
morphic sites resulting in two major, almost equally common
630 A. FERLIN ET AL.
allelic variants in the Caucasian population, Thr307-Asn680 (TN)
and Ala307-Ser680 (AS), producing two distinct receptor iso-
forms [94], leading to three genotypes (TN/TN, TN/AS and AS/
AS). In the FSHB gene, the most common studied SNP is a G/T
polymorphism located in the promoter of the gene (−211 bp
from the mRNA transcription start site) [95,96]. The combination
of G/T SNP can lead therefore to three genotypes: homozygous
TT and GG and heterozygous GT.
Based on these findings, four studies have been published
so far dealing with the hypothesis that the different genetic
polymorphisms in FSHR and FSHB genes could influence the
response to exogenous FSH administration [97–100]. In parti-
cular, two studies [98,99] analyzed FSHR gene polymorphisms,
one the FSHB gene [100] and one both genes [97]. Therefore,
too few and non-homogeneous data have been published to
draw a conclusion on the clinical utility of evaluating these
polymorphisms in the protocol of FSH treatment, also because
these studies found some discrepancies.
In general, however, a pharmacogenetic approach to FSH
treatment is very intriguing and promising. Once more data
will be available, the knowledge of the individual genotype
could be combined with the other clinical data to obtain
a comprehensive view of the predictors to FSH treatment,
therefore allowing a better selection and characterization of
potential responders and non-responders patients.
Furthermore, FSH treatment scheme (dosage, duration) could
be personalized to maximize the response. Patients with the
less favorable genotypes could theoretically need higher
doses or longer therapy, whereas patients with the most
responsive genotypes could benefit also from lower doses
and/or standard duration of three months. Clearly, such
a pharmacogenetic approach could convert FSH treatment in
idiopathic infertile men from empiric into rational therapy.
8. Conclusion
The body of literature dealing with ‘old’, classic genetic causes
of male infertility (such as chromosomal aberrations,
Y chromosome microdeletions and CFTR gene mutations)
and novel genetic factors associated with spermatogenic
impairment (SNPs, CNVs, gene mutations) better identifiable
by new genetic technologies (SNP array, CGH array, NGS) is
sufficient to suggest a limited number of genetic tests for the
different conditions underlying male infertility:
● Azoospermia and severe oligozoospermia (<10 million
total sperm/ejaculate) caused by primary testicular
damage should be tested for karyotype anomalies and
Y chromosome long arm deletions.
● Congenital bilateral or unilateral absence of vas deferens
should be tested for CFTR gene mutations.
● Azoospermia and severe oligozoospermia with signs of
androgen insensitivity (high LH, high/normal testoster-
one) should be tested for AR gene mutations.
● Idiopathic forms also with moderate oligozoospermia
could be tested for the gr/gr deletions in the
Y chromosome.
● Hypogonadotropic hypogonadism should be tested for
candidate genes by a panel including multiple genes.
● Particular causes of male infertility (spermatocytic arrest,
isolated idiopathic complete asthenozoospermia, globo-
zoospermia, macrocephaly, history of cryptorchidism)
could be tested for specific gene mutations (TEX11;
dynein genes DNAI1, DNAH5, DNAH11; DPY19L2;
AURKC; NR5A1, INSL3, and RXFP2, respectively).
Pharmacogenetic tests for FSH treatment of male infertility are
promising but not yet applicable routinely on clinical practice.
9. Expert opinion
Advances in genetic and genomic tools, as well as most
detailed clinical selection of infertile men to be included in
genetic association studies allowed in the recent past the
discovery of new genetic causes and genetic risk factors that
hopefully in a near future could be routinely applied in the
clinical practice. Application of clinical data to identify sub-
classes of infertile men to be screened would probably prompt
the development of panel of genes and/or genetic tests to be
applied in the diagnostic workup of these men. This, in turn,
will reduce the proportion of infertile men currently diagnosed
as idiopathic.
In the meanwhile, we already have indications for correct
application of the right genetic test to the right infertile man.
Although these genetic analyses are relatively few, their role in
the diagnostic process is fundamental and allows the identifi-
cation of a large proportion of genetic cases: 10–15% of non-
obstructive azoospermia, 5–10% of oligozoospermia, 70–80%
of obstructive forms, 60–70% of HH cases, 80–90% of cases of
globozoospermia and macrocephaly.
Importantly, as the final aim of the clinical approach to
infertility is to achieve a pregnancy and the birth of
a healthy baby, genetic tests should always be coupled with
appropriate genetic counseling, whether it is dealing with
natural conception, application of assisted reproductive tech-
niques, repeated abortion or preimplantation genetic testing.
Clinically, the most important aspect is related to the
correct identification of subjects to be tested and the right
application of genetic tests based on clear clinical data.
A correct application of available genetic tests in the differ-
ent forms of male infertility allows receiving a better and
defined diagnosis and has an important role in clinical deci-
sion (treatment, prognosis). Genetic tests should always be
included with the other clinical data of the patient, from
history and physical examination to highlight other risk
factors for infertility and family data, to semen analysis
(considering quantitative sperm alterations, qualitative
defects of sperm and seminal fluid alterations), hormonal
determination and imaging studies. Subsequent treatment
and prognosis for the couple fertility might benefit from the
identification of genetic factors. As examples, 1. different
AZF region deletions in men with azoospermia are asso-
ciated with different probabilities of sperm recovery at
TESE; 2. a man with azoospermia or severe oligozoospermia
with a left varicocele and AZF deletions or chromosomal
aberrations might not benefit from varicocelectomy; 3. iden-
tification of Klinefelter syndrome in an azoospermic man

allows to predict sperm recovery at TESE in 40–50% of cases
and it is of great importance for the general health of this
patient; 4. identification of a genetic cause in a patient with
oligozoospermia and normal gonadotropin levels suggests
not to proceed with hormonal treatment (such as FSH
therapy).
Furthermore, a correct application of available genetic
tests and identification of genetic factors involved in male
infertility allows appropriate genetic counseling especially in
cases that should undergo assisted reproduction techni-
ques, both determining the risk for the child of inheriting
a monogenic mutation or establishing the risk of unba-
lanced sperm in cases of chromosomal aberrations. This is
also important in the context of pre-implantation genetic
testing and prenatal testing to allow the couple a more
informed decision.
Given the rapid evolution in the field and the development
of high throughput technologies for genetic and genomic
analysis, it is plausible that targeted gene panels including
tens of genes will be used for genetic diagnostics of male
infertility in the near future. In this way specific subgroups of
conditions (such as idiopathic primary testicular damage,
hypogonadotropic hypogonadism, qualitative sperm defects,
cryptorchidism, pharmacogenetics) would be simultaneously
analyzed for multiple genes, reducing the costs and improving
the diagnostic accuracy and precision.
Funding
This paper was not funded.
Declaration of interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
Reviewers Disclosure
Peer reviewers on this manuscript have no relevant financial relationships
or otherwise to disclose.
ORCID
Alberto Ferlin http://orcid.org/0000-0001-5817-8141
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