Chapter

Male Infertility and Assisted Reproduction

13 Nigel Pereira, Queenie V. Neri, Tyler Cozzubbo, Stephanie Cheung,

Zev Rosenwaks and Gianpiero D. Palermo

Introduction Epidemiology
In most medical conditions, the diagnosis and treat- Infertility is commonly defined as the failure to con-
ment are directly linked, and successful treatment ceive after one year of unprotected intercourse. It
hinges on the functional relationship between a patient is thought to affect approximately 15% of repro-
and medical provider. Infertility, in contrast, involves ductive age couples worldwide [1], with up to 50%
a couple whose general and reproductive health is of cases having some degree of male factor infer-
evaluated by multiple medical providers in paral- tility [3]. Population-based estimates in the United
lel, with treatment focused on the two partners [1]. States suggest that more than 1.1 million men sought
Successful treatment not only entails an orchestrated fertility care in 2002 and that there were 131–172
collaboration between the couple and providers, but infertility-related physician visits per 100,000 insured
also requires congruity of the two partners. Thus, men between 1994 and 2006 [3]. Recent reports have
apart from being a medical condition, infertility often revealed temporal and geospatial variation in the
evolves into a social condition, which encompasses prevalence of male factor infertility [3]. Specifically,
several psychosocial stressors [2]. While the diagno- its prevalence in the United States is highest in New
sis, medical treatment and psychosocial management Mexico (56.4%) and lowest in Mississippi (24.2%). The
of infertility have evolved rapidly over the past four aforementioned distribution of male factor infertility
decades, some difficulties still persist. These difficul- is generally multifactorial [3].
ties are especially apparent in the field of male infer-
tility, where we perennially strive to discover novel
mechanisms underlying the etiology of male infertility, The Spermatozoon [4]
as well as propose accurate diagnoses and treatments The antiquated perception of the spermatozoon as a
of male reproductive dysfunction [1]. In this chapter, delivery device for the male genome has been replaced
we review the epidemiology and diagnostic workup of by more recent findings on the cell’s complex role in
male infertility based on various facets of sperm pro- oocyte fertilization. The general structure of the sper-
duction, genetics and environmental factors. We high- matozoon includes the head and flagellum, which are
light various therapeutic strategies, including sperm both enclosed by a regionally differentiated plasma
retrieval and assisted reproductive techniques, which membrane. The head is mostly occupied by the nucleus
are frequently utilized to help couples conceive. We and is covered by the caplike acrosome, which is
also present the clinical outcomes associated with the derived from the Golgi complex. The acrosome con-
aforementioned approaches and appraise their safety. tains several hydrolytic enzymes that are involved in
Finally, we describe the most recent attempts pertain- the acrosomal reaction, a physiological event essential
ing to and future directions for the treatment of male for oocyte fertilization and subsequent embryo devel-
infertility. opment. The flagellum or tail of the spermatozoon

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.

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 Chapter 13: Male Infertility and Assisted Reproduction

consists of three regions – the midpiece, which is cen- Table 13.1 Reference values for semen analysis per WHO
standards
trally located and generally defined by an aggregated
sheath of mitochondria surrounding the centrosome, Reference value (fifth
the principal piece and the end piece. percentile with 95%
Parameter confidence intervals)

Screening the Male Patient and Volume (mL) 1.5 (1.4–1.7)

Concentration (106 per mL) 15 (12–16)
Semen Analysis Total motility (%) 40 (38–42)

Screening the Male Patient Progressive motility (%) 32 (31–34)

Morphology (normal forms %) 4 (3.0–4.0)
The initial screening of an infertile man [5] should
involve a thorough developmental, medical, surgical,
family, social and sexual history, as well as a metic-
try. Presence of varicoceles should be noted by exam-
ulous physical examination. The developmental his-
ining the patient in both the supine and standing
tory should include a review of hypospadias, cryp-
position. A rectal examination can reveal large cysts,
torchidism, midline defects, hypogonadism and con-
prostate masses or infection, or dilated seminal vesi-
genital infections. Any maternal exposure to diethyl-
cles, and should be performed routinely.
stilbesterol should also be noted. Medical problems
such as unexplained fevers, diabetes, hypertension,
cystic fibrosis or malignancy must be reviewed and Semen Analysis
noted. Given the potential adverse effects that many Evaluation of the ejaculate as a whole has been utilized
medications can have on male fertility, all medica- for over 60 years now, and it continues to be a useful
tions used by a patient, including dosage and route clinical and research tool to investigate the male part-
of administration, should be noted in detail. Surgical ner’s fertility status. In 2010, the World Health Organi-
procedures such as herniorrhaphy, orchidopexy and zation (WHO) published lower reference limits for tra-
retroperitoneal, bladder, pelvic or prostate surgery can ditional semen parameters [6]. Of note, raw data from
impair fertility and should therefore be reviewed. Any about 400–1,900 semen samples, from recent fathers
family history, particularly paternal history of mid- in eight countries spanning three different continents,
line defects, hypogonadism or infertility, should be were used to generate the following reference values in
elicited. The social history should comprise a thor- Table 13.1.
ough review of alcohol consumption, as well as use As evident in Table 13.1, a semen analysis provides
of anabolic steroids, recreational drugs and tobacco useful information regarding viability, production and
products. Furthermore, any occupational exposure to motility of spermatozoa, as well as the patency of the
chronic heat, ionizing radiation, pesticides, herbicides male genital tract. In general, spermatozoa account for
and industrial solvents should be investigated. Elicit- ⬍10% of the total semen, while the remainder of the
ing history of any pregnancies with the current or pre- ejaculate consists of products secreted by the semi-
vious partners, ejaculatory or erectile dysfunction, the nal vesicles (55%), prostate (25%) and bulbouretheral
use of spermicidal lubricants and incorrect patterns of gland (10%) [4]. Assessment of the volume and con-
timing intercourse generally comprises the sexual his- sistency of the ejaculate can offer insight into the con-
tory. dition of the accessory glands. While quantifying the
The physical examination includes a detailed number and motility of spermatozoa is perhaps intu-
assessment of body habitus, specifically obesity or itive, assessment of sperm morphology is more com-
gynecomastia. Genital examination generally involves plex, owing to the variability in criteria utilized to eval-
evaluation of the phallus and testes. For the former, uate their shape and size [4].
any evidence of chordee, plaques, venereal lesions or Human spermatogenesis is completed in 60–80
hypospadias should be noted. For the testes, the size, days, and therefore an individual’s semen analy-
volume, consistency and contours should be assessed. sis reflects biological activity occurring 2–3 months
The epididymides, vas deferens and spermatic cords before [4]. Given the inherent biological fluctuations
should be palpated for nodularity, fullness or asymme- between semen samples, a minimum of two samples

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 Chapter 13: Male Infertility and Assisted Reproduction

should be examined. Ideally, semen samples should be sperm becomes highly condensed and its histones
produced after 2–3 days of sexual abstinence, prefer- are replaced by protamines [12, 13]. This complex
ably without lubricants, and kept at body tempera- organization of DNA and protein into a structure
ture if transport is required [6]. If an individual’s his- called chromatin is highly regulated and different from
tory suggests recent insults to spermatogenesis such as that in somatic cells [12, 13]. During the later stages
medical illness, testicular injury or chemical or toxin of spermiogenesis, breakage of a sizable amount of
exposure, then semen analysis should be expanded single- or double-stranded DNA occurs to allow tight
over several months. Although there can be consid- chromatin compaction and, under ideal conditions,
erable biological variation in semen analyses, men only those spermatozoa with fully repaired chromatin
whose semen contains ⬎48 × 106 sperm per mL are would reach the ejaculate [14]. The integrity of the
deemed fertile [7], while those with ⬍10 × 106 sperm sperm genome is important for embryo development,
per mL are considered subfertile, especially when the specifically blastocyst development and early implan-
specimen contains many immotile sperm and the few tation, and it is thought that DNA breakage may con-
motile sperm have abnormal morphology [4, 7]. It tribute to infertility in a way that is not revealed by
must be noted that a normal semen analysis does simple morphological evaluation of spermatozoa [14].
not guarantee fertility [6] and does not provide func- Thus, tests (biomarkers) of sperm DNA integrity may
tional information about the sperm; that is, a semen be incorporated into the clinical assessment of male
analysis does not predict whether spermatozoa can infertility patients [15].
undergo capacitation or acrosome reaction or fertilize While several studies use the terms DNA integrity
an oocyte [8]. A semen analysis, therefore, correlates and chromatin integrity interchangeably, most tests
with fertility, but does not prove an individual’s fertil- (biomarkers) measure only specific parameters of
ity potential [8]. chromatin [9, 15]. It is postulated that sperm DNA
integrity is closely associated with sperm quality,
male fertility potential and pregnancy outcomes [12].
Other Markers of Male Infertility Specifically, an abnormal DNA fragmentation index
The need for new male infertility biomarkers largely (DFI, %) is thought to have an inverse relationship
arises from the challenges in translating in vivo sper- with male fertility success [16], and if pregnancy
matogenic function into fertility success using semen does occur, then such pregnancies are thought be
analyses [9]. For a long time, a testicular biopsy was at risk for miscarriage [17]. Some of the available
considered the cornerstone in the evaluation of vari- methods for detecting sperm DNA integrity include
ous forms of male infertility [10]. However, its inva- sperm chromatin structure assay (SCSA), terminal
siveness can pose undue risk for the health of the testes deoxynucleotidyl transferase (TdT) dUTP Nick-End
[9]. Furthermore, it provides only a small sample of tis- Labelling assay, TUNEL assay and comet assay, which
sue, and its histology is often unable to reveal the actual will be discussed below [9]. However, it is important
cause of infertility [10]. Thus, evaluation of sperm to note that these assays are consumptive; in other
and sperm-derived biomarkers has been proposed as words, they require permanent fixation of the sperm,
an alternative for evaluating reproductive success [9]. which renders them unsuitable for clinical practice
These biomarkers aim to highlight spermatic function, [18]. Thus, in current clinical practice, there is limited
specifically the fertilization capacity of sperm. Ideally, ability to select sperm with varying degrees of DNA
these biomarkers would aid in the diagnosis of sperm damage for immediate use, assisted reproduction or
dysfunction, would predict fertilization or pregnancy cryopreservation [9].
rates and would indicate suitable therapies for sperm
dysfunction [9].
The packaging of DNA in sperm and its integrity SCSA
have important fertility-related implications. In gen- Variants of SCSA have been commercially avail-
eral, the sperm’s DNA is bundled very densely, owing able for close to 30 years now. It relies on the
to the action of testis-specific serine kinase 6 (TSSK6) metachromatic properties of acridine orange, which
prior to sperm’s transit to the oocyte [11]. During changes colour from green to red when associ-
spermatogenesis and spermiogenesis, the DNA in the ated with single-stranded DNA or RNA [18]. The

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 Chapter 13: Male Infertility and Assisted Reproduction

results of the assay are generally expressed as (␣t) tions [9]. The assay derives its name from the gel elec-
in three ways: red fluorescence/(red + green) fluo- trophoresis pattern of DNA fragments, which splay out
rescence, red fluorescence/total green florescence, and in the shape of a comet and associated tail [9]. Specif-
red fluorescence/COMP␣t. COMP␣t is the number ically, intact DNA remains in place as the comet head,
of sperm outside of the normal population and com- while damaged and fragmented DNA is smaller and
monly represents the DFI [9]. As acridine orange asso- forms the comet tail. Similarly to the TUNEL assay,
ciates poorly with condensed DNA, most SCSA proto- the comet assay lacks a reference assay and relies on
cols require denaturing of DNA with low pH or high the microscopic observation of a few hundred sperm
temperature to promote thorough penetration of DNA [9, 22]. Thus, its cutoffs vary by institution. At least
by acridine orange [18]. The assay also requires flow one study has suggested that higher DNA damage as
cytometry and the use of a reference for successful cal- measured by the comet assay is predictive of failure
culation of COMP␣t [18]. While cutoff values vary by of embryo development after intracytoplasmic sperm
institution, most data indicate that a DFI ⬎27% may injection [22].
be associated with a reduced probability of pregnancy
with assisted reproductive techniques [19].
Chromosomal Markers and FISH
TUNEL Assay In general, autosomal trisomies (93% of trisomy 18,
95% of trisomy 21 and 100% of trisomy 16) originate
The TUNEL assay was first used in somatic cells
in the maternal line, whereas sex chromosomal aneu-
and then subsequently applied to spermatozoa [20].
ploidies are more frequently of paternal origin (50% of
The assay involves transfer of a fluorescence-labeled
47,XXY, 100% of 47,XYY and 70–80% of 45,X) [4,24].
nucleotide to the 3 -hydroxyl group of damaged DNA
While meiotic errors that lead to foetal aneuploidy
strands using the activity of deoxynucleotidyl trans-
occur in both the male and the female gametes, the fre-
ferase [9, 20]. The fluorescence intensity of each sper-
quency of these errors is lower in spermatozoa (9%)
matozoon is then evaluated and a designation of ‘dam-
than in oocytes (20%) [4, 24]. Although fluorescent
aged’ (fluorescence) or ‘undamaged’ (no fluorescence)
in situ hybridization (FISH) has increased the ability
is given [9]. A laboratory technician uses a fluorescent-
to detect chromosomal abnormalities, indications for
light microscope or flow cytometer to report the num-
FISH on sperm are not well established currently [9].
ber of TUNEL positive sperm. In the absence of a refer-
Furthermore, with FISH, only selected regions of inter-
ence assay, most laboratories develop their own proto-
est can be visualized and any estimate of aneuploidy
cols for the TUNEL assay. In addition, a single techni-
from this procedure refers only to the chromosomes
cian is frequently required to perform fluorescent-light
analyzed [4].
microscopy or flow cytometry to ensure consistent
interpretation of the TUNEL assay results [9]. Thus,
given differences in protocols between institutions, Sperm RNA
TUNEL result cutoffs are often institution-specific. In
The recent discovery of RNA in spermatozoa has
general, higher fractions of TUNEL-positive sperm
raised several interesting questions regarding its role
are seen with increasing male age [20]. However, sev-
in male fertility [25, 26]. Analysis of sperm RNA tran-
eral other individual and environmental factors can
scripts reflects prior events in spermatogenesis as well
increase the percentage of TUNEL-positive sperm
as highlighting potential factors that may be critical
[20]. Within the realm of assisted reproduction, a
to fertilization and embryo development [25, 26]. In
higher percentage of TUNEL-positive sperm has been
addition to mRNA, human sperm has been found to
associated with reduced pregnancy rates [21].
carry small noncoding RNAs (sncRNAs). The distri-
bution of sncRNAs in ejaculated specimens is as fol-
Comet Assay lows: 65% repeat-associated small RNAs, 17% Piwi-
The comet assay is used frequently in somatic cells interacting piRNAs, 11% quiescent RNAs, and 7%
to measure single- and double-strand breaks [22, 23]. micro RNAs [26]. Such a complex population of sncR-
When used to assess sperm, the assay involves mix- NAs suggests a role in post-fertilization development,
ing sperm with liquefied agarose gel, followed by elec- making them an emerging biomarker of male infertil-
trophoresis under either alkaline or neutral pH condi- ity [26].

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 Chapter 13: Male Infertility and Assisted Reproduction

Genetic and Epigenetic Basis of have shown that methylation patterns in the testes are
diminished eightfold relative to somatic tissue [31].
Male Infertility Whether this hypomethylation is present in mature
Male germ cell development begins in early embryo- spermatozoa, or whether it represents an epigenetic
genesis, but mature spermatozoa first appear only at process to prepare spermatozoa for increased tran-
puberty [1]. Genetic disorders can disrupt this male- scription following pronuclear development, is still not
specific cell differentiation and maturation at the chro- known [1].
mosomal or molecular DNA level. Genes involved in It is important to note that our comprehen-
spermatogenesis may be expressed functionally in the sion of genetic and epigenetic aspects of human
germ line, during the development of male gonads or spermatogenesis is still poor, and mostly deduced
in testicular somatic cells, but those expressed specifi- from animal studies [1]. Such extrapolation should
cally in the germ line are assumed to be the most rel- be treated with caution due to the myriad altered
evant to regulation of germ cell maturation [1]. For spermatogenesis/spermiogenesis states seen in
example, the RBM, SPGY and DAZ genes are known humans.
to regulate male fertility, and disruption of these genes
may lead to infertility or sterility [27]. There are other
recessive and dominant mutations in somatic cells Scope of Assisted Reproduction
that may indirectly induce infertility as a consequence Following the first successful birth in 1978 using
of other problems, as seen for instance in men with assisted reproductive technologies (ART), their use to
Kartagener’s syndrome, cystic fibrosis or myotonic overcome infertility has increased steadily [32]. An
dystrophy [1]. estimated 456 ART clinics in the United States per-
Chromosomal abnormalities account for approx- formed 157,635 ART procedures in 2012, and these
imately 5% of all male infertility cases and 15% of procedures resulted in 51,261 live deliveries and 65,151
infertility in azoospermic males [9]. Aneuploidy lead- infants [32]. In 2012, ART contributed to 1.5% of all
ing to male infertility may involve the sex chromo- infants born in the United States [32]. ART procedures
somes, for example an additional X-chromosome in consist of several steps over a two-week period, begin-
Klinefelter’s syndrome, or the autosome, for example ning with drug-induced ovarian stimulation, progress-
trisomy 21 [1]. Structural chromosome abnormalities ing to oocyte retrieval and fertilization with sperm in
such as small deletions, inversions, or translocations the laboratory and ultimately leading to embryo trans-
can lead to male infertility and may involve both sex fer [32]. In general, ART includes treatments such as
and autosomal chromosomes [1]. In fact, deletions in IVF, gamete intrafallopian transfer (GIFT) and zygote
the Yq region can be associated with azoospermia. intrafallopian transfer (ZIFT), with IVF accounting for
Specifically, in the region designated AZF (azoosper- approximately 99% of all ART procedures. ART, how-
mia factor), three loci (AZFa, AZFb, AZFc) associated ever, does not include treatments such as intrauterine
with nonobstructive azoospermia have been identified insemination, in which only sperm is handled, or ovu-
[1, 28]. Chromosomal rearrangements like reciprocal lation induction, which involves stimulating oocyte
translocations can also give rise to abnormal meiotic production [32].
chromosome pairing, thus disrupting spermatogene- After the establishment of IVF, it soon became clear
sis [1, 28]. that as many as 40% of the inseminated in vitro cycles
During spermatogenesis, sperm chromatin under- were affected by fertilization failure or by extremely
goes dramatic reorganization, including protamine low fertilization [33]. This was particularly problem-
replacement of histones, histone modifications and atic in patients with marginal semen characteristics
DNA methylation [1, 11]. Modifications of the N- and poor spermatozoa [33]. Specifically, diminished
terminal region of histones confer an epigenetic reg- sperm motility and/or poor morphology presented a
ulatory mechanism of gene expression [29]. Generally, complex obstacle for spermatozoa to penetrate the
methylation of the histones is associated with silenc- zona pellucida (ZP), a thick glycoprotein layer sur-
ing of the gene, while acetylation is associated with rounding the oocyte [34]. Traditional means of over-
transcription [30]. Methylation is carried out by DNA coming such hurdles were limited and dealt primarily
methyltransferases, which transfer a methyl group to with increasing sperm concentration and at enhanc-
deoxycytosines found in CpG islands [30]. Studies ing their selection [1]. Embryologists often increased

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 Chapter 13: Male Infertility and Assisted Reproduction

sperm concentration within the inseminating suspen- Intracytoplasmic Sperm Injection

sion, or if extremely few spermatozoa with impaired
Intracytoplasmic sperm injection (ICSI) is a proce-
motility were available, then a lesser volume of the
dure that involves the injection of a single spermato-
insemination medium was used to facilitate com-
zoon directly into the cytoplasm of an oocyte. ICSI
mingling of the two gametes [1]. Simple procedures
bypasses both the zona pellucida barrier and sperm
included the swim-up method, which selected only
defects in the male gamete that compromise its abil-
highly motile cells in the upper fraction [1]. Other
ity to fertilize [1]. Documented ICSI trials on mam-
methods utilized multilayer density gradients to select
malian gametes date as far back as 1966 [44]. How-
highly motile and, in general, morphologically normal
ever, initial efforts with human gametes yielded a
spermatozoa that also exhibited higher penetration
high incidence of oocyte damage and an unsatisfac-
capacity [35]. While these selection procedures opti-
tory level of embryo implantation [45]. The early use
mized the ability to treat IVF patients with moderately
of ICSI required some adjustments to identify the
compromised semen characteristics, often described
best method for piercing the membrane and identi-
by moderate oligoasthenospermia, they fell short in
fying the best location within the ooplasm in which
dealing with more severe issues of sperm dysfunction
to release the spermatozoon [37]. However, following
such as severe oligoasthenospermia or teratospermia
these adjustments and success in conceiving pregnan-
[1, 36, 37].
cies [43], it soon became apparent that ICSI was capa-
The focus then shifted to efforts to assist the sper-
ble of fertilizing nearly every mature oocyte injected
matozoon in penetrating the ZP. Early attempts involv-
[36, 37]. Moreover, the ability to pinpoint the differ-
ing complete removal of the ZP resulted in polyspermy
ent steps of pronuclei appearance and to monitor the
or impaired embryonic development [1, 38]. These
observation of the first embryonic cleavage without
were followed by attempts to overcome the zona as
the obstructive layer of cumulus cells would facili-
a barrier either by softening it enzymatically with
tate tracking these critical steps in early embryonic
trypsin or pronase [39] or by penetrating it chemically
development [37].
via localized or pinpoint exposure to acidified Tyrode’s
solution prior to sperm exposure [40]. The latter tech-
nique became known as zona-drilling (ZD), but the Popularity of ICSI
low-pH solution involved proved to possibly damage The implementation of micromanipulation techniques
the oocyte [40]. A variant of ZD opened a fissure in in the past 20 years has made it possible to over-
the ZP via mechanical means, called partial zona dis- come male gamete production deficiencies and fer-
section (PZD), and utilized a smaller opening than tilization defects to allow infertile male partners to
did ZD, thus mitigating the rate of polyspermy [41]. reproduce at rates that previously would have been
Still, a large obstacle persisted in the form of sperm deemed unachievable [37]. In a cross-sectional sur-
cells that required further assistance to properly inter- vey of ART procedures performed in 55 countries
act with oolemma [1]. This procedure was replaced by during 2007, the International Committee for Mon-
a more refined technique in which spermatozoa were itoring Assisted Reproductive Technologies reported
brought through the ZP with a pipette and deposited that 65.2% (400,617 of 614,540) of all cycles utilized
beneath the zona into the perivitelline space – sub- ICSI [46]. However, there was considerable variation
zonal insemination (SUZI) [1, 42]. This provided some in ICSI rates, ranging from 49.1% in Asia to 97.8%
success in patients with more impaired sperm motility in the Middle East [46]. In another recent publica-
while controlling the incidence of polyspermy, but still tion analyzing trends in ICSI use between 1996 and
yielded relatively low fertilization rates. This was pri- 2012 in the United States, ICSI use increased from
marily because spermatozoa still needed to undergo 36.4% in 1996 to 76.2% in 2012 [47]. At our centre
a complete acrosome reaction, a necessary precursor (Figure 13.1), there has been a steady and progres-
of fusion with the oolemma [1, 42]. These preliminary sive increase in ICSI prevalence, starting at 32.2% in
efforts to artificially assist sperm penetration soon 1993, rising to 48.8% in 1995 and reaching 73.6% by
became obsolete with the introduction of a microsur- 2002 [37]. Since 1993, ICSI has been used in 29,998
gical method for insertion of spermatozoa directly into cycles compared with 13,454 cycles with conventional
the oocyte [42, 43]. insemination. ICSI has yielded reproductive outcome

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 Chapter 13: Male Infertility and Assisted Reproduction

IVF extremely low sperm count, impaired motility and

100 ICSI poor morphology cannot be helped with IVF alone
90 26.4
[37]. In such cases, ICSI appears as a superior modality
Proportion of ART (%)

80 51.2 in achieving pregnancy. One of the crowning achieve-

70 67.8
60
ments of ICSI is perhaps its role in helping many
50
azoospermic men achieve pregnancy. Until advanced
40 73.6 micromanipulation techniques were available, such
30 48.8
couples relied on donor sperm or adoption [1]. A com-
20 32.2 plete absence of spermatozoa in the ejaculate occurs in
10 1% of all men and approximately 10–15% of all infer-
0 tile men [1]. The diagnosis is further subcategorized
1993 1995 2002
into obstructive azoospermia (OA) and nonobstruc-
Figure 13.1 Prevalence of ICSI cases at our centre in 1993, 1995 tive azoospermia (NOA) [51]. OA can result from con-
and 2002. genital absence of the vas deferens (CBAVD), trauma,
infection, vasectomy or failed vasectomy reversal [1,
comparable to those of conventional IVF, but is also 51]. In general, any form of male infertility due to
capable of consistently overcoming unforeseen sperm obstruction can be treated by ICSI with spermato-
cell dysfunction [37]. The overall fertilization rates zoa microsurgically recovered from either epididymal
(1993–2015) after ICSI and conventional IVF were aspirations, including microscopic (MESA), percuta-
74.4% (161,842/217,449) and 60.6% (74,741/123,316), neous (PESA) and fine-needle aspirations (FNA) [52,
respectively. However, with standard IVF, the two- 53] or testicular aspiration (TESE) [54, 55]. NOA usu-
pronucleated (2PN) formation rate is calculated over ally occurs due to a failure of spermatogenesis and
the total number of oocytes retrieved, so once cor- requires surgical intervention to obtain spermatozoa
rected for ICSI, the fertilization rate is comparable for subsequent injection [1]. TESE and the now refined
between the two insemination methods (58.5% ICSI micro-TESE retrieve seminiferous tubules for search,
vs. 60.6% IVF). The clinical pregnancy rate as defined with the latter achieving a higher probability of sperm
by the presence of a foetal heartbeat on ultrasound was retrieval and maintaining greater anatomical integrity
45.7% (12,066/26,429) for ICSI compared with 40.0% of the testicle [56–58]. It is also important to note that
(4,473/11,189) for IVF. Thus far 16,511 babies have when no motile spermatozoa can be retrieved from
been born from the two ART procedures, of which the epididymis due to epididymal fibrosis or for other
10,199 were conceived with ICSI. reasons, the use of testicular spermatozoa is indicated
There has been an increase in ICSI rates over the [36].
past two decades; however, the use of ICSI for patients Although ICSI requires only a spermatozoon with
with borderline or even normal semen characteristics a functional genome, oocyte activating factor and cen-
has also increased, without clear evidence of a bene- trosome for the fertilization of the oocyte, indications
fit over conventional insemination [48, 49] from using for ICSI are not restricted to low sperm count; they
ICSI. In fact, the American Society for Reproductive also include morphologically impaired spermatozoa
Medicine and the Society for Assisted Reproductive and impaired sperm kinetics [37]. In these scenarios,
Technology confirm that there is insufficient evidence high fertilization and pregnancy rates can be achieved
to support the routine use of ICSI in patients without when a viable spermatozoon is injected, independent
male factor infertility [50]. Thus, it is important to dis- of its characteristics [59]. In addition to these pri-
cuss the indications for ICSI. mary indicators, ICSI is the preferred option for male
patients experiencing various sperm-related problems
that include ejaculatory dysfunction and retrograde
Indications for ICSI ejaculation, as well as complications stemming from
While IVF has become a well-established treatment paraplegia [1]. Conditions linked to ultralow ejaculate
for most types of infertility, including tubal disease, volumes include retrograde ejaculation, lack of emis-
endometriosis, unexplained infertility and even some sion, ejaculatory duct obstruction, hypogonadism and
mild forms of male factor, some couples with an CBAVD. In such patients, high-speed centrifugation of

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 Chapter 13: Male Infertility and Assisted Reproduction

the semen sample and examination of the pellet under Table 13.2 Indications for ICSI
oil at 400× magnification with an inverted microscope Male factor Non-male-factor
is carried out to identify sperm [37].
Ejaculated spermatozoa Prior failed fertilization with IVF
ICSI has also been used in other non-male-factor Oligozoospermia Oocyte dysmorphism
settings. For example, ICSI can be used successfully Asthenozoospermia Low number of oocytes
in patients with complete fertilization failure (CFF) Teratozoospermia Low oocyte maturity
Antisperm antibodies Cryopreserved oocytes
after conventional IVF [39]. It has been hypothesized Fertility preservation In vitro maturation (IVM)
that ICSI might be a better method of fertilization Ejaculatory disorders Preimplantation genetic
than conventional IVF for patients with poor-quality screening
Acrosomeless spermatozoa HIV and hepatitis C discordant
oocytes, as determined by morphologic assessment couples
[60]. The advantage of using ICSI may be twofold in Cryptozoospermia Restrictive legislation
these situations: ICSI confers the ability to confirm Surgically retrieved
that the retrieved oocytes are indeed mature following Epididymal
Obstructive azoospermia
cumulus removal and specifically enhances chances Congenital bilateral
of fertilization by direct sperm injection [37]. ICSI is absence of the vas
commonly used in poor responders with the idea of deferens
Young syndrome
improving fertilization rates in the few oocytes that Failed vasoepididymostomy
are available for fertilization [37, 61]. Another possi- Failed vasovasostomy
ble benefit for ICSI is the prevention of polyspermia. Testicular spermatozoa
In fact, the reported incidence of triploidy in human Necrozoospermia
embryos after conventional IVF ranges from 2 to 10%, All indications for
epididymal sperm
with dispermy being the most common cause [62]. A Nonobstructive
retrospective analysis of 95 couples with ⬎20% inci- azoospermia
dence of 3PN zygotes in their initial conventional IVF
cycles followed by the use of ICSI in a subsequent cycle niques for men with obstructive and nonobstructive
showed that after ICSI, the rate of normally fertilized azoospermia, respectively. The optimal sperm retrieval
zygotes (2PN) was enhanced (65 vs. 34.1%), with a neg- technique depends upon the etiology of azoospermia,
ligible occurrence of 3PN (5.0 vs. 33.9%) [63]. There the technical capabilities of the embryology laboratory,
was no difference in cleavage and quality of embryos and the skill set and preferences of the clinician per-
derived from normal zygotes by the two insemina- forming the sperm retrieval procedure.
tion methods [63]. Thus, ICSI generated a higher num-
ber of diploid zygotes without compromising embryo
development. Table 13.2 summarizes the indications
Clinical Results with ICSI
for ICSI. Between September 1993 and June 2015, we per-
formed 29,998 ICSI cycles. Of these, approximately
91% (n = 27,284) of all ICSI cycles were per-
Sperm Retrieval Methods formed using ejaculated spermatozoa and the remain-
Technical refinements in sperm retrieval methods in der involved specimens that were surgically retrieved
conjunction with ICSI have enabled biological pater- from the epididymis or testis at our centre. In cycles
nity in azoospermic men who were previously con- utilizing ejaculated spermatozoa, a total of 224,247
sidered untreatable [64]. In general, a sperm retrieval MII were oocytes injected, resulting in a survival rate
technique that minimizes trauma to the reproductive of 97.3%. Of those that survived, 75.1% oocytes were
tract and yields the highest-quality sperm in sufficient fertilized normally, with 1PN and 3PN in only 2.4%
quantity for immediate and later use is desirable [64]. and 3.5% oocytes, respectively. No fertilization was
Surgical techniques for sperm retrieval can vary by noted in 16.3% oocytes (Figure 13.2).
anatomical target (epididymis vs. testis) and whether Table 13.5 summarizes the fertilization and
or not the procedure is assisted by intraoperative clinical pregnancy rates in ICSI cycles using ejac-
optical magnification (conventional vs. microsurgical) ulated, epididymal and testicular spermatozoa.
[64]. Tables 13.3 and 13.4 summarize the technical Figure 13.3 compares the fertilization rates between
aspects and spermatozoa yield of sperm retrieval tech- fresh and frozen ejaculated, epididymal and testicular

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 Chapter 13: Male Infertility and Assisted Reproduction

Table 13.3 Sperm retrieval techniques and corresponding yield in men with obstructive azoospermia [65–68]

Technique Yield Sperm retrieval rate Anaesthesia

Percutaneous epididymal sperm Thousands to millions of sperm 80–100% Local with or without sedation
aspiration (PESA) Cryopreservation for later use
possible in most cases
Testicular fine-needle aspiration Hundreds of thousands to millions 52–100% Local with or without sedation
of sperm
Cryopreservation for later use
possible in some cases
Testicular percutaneous biopsy Hundreds of thousands to millions 82–100% Local with or without sedation
of sperm
Cryopreservation for later use
possible in some cases
Microsurgical epididymal sperm Average of 15–95 × 106 total sperm 95–100% General, regional plus sedation,
aspiration (MESA) Cryopreservation for later use or local plus sedation
possible
Testicular sperm extraction Hundreds of thousands to millions 100% General, regional plus sedation,
(TESE) of sperm or local plus sedation
Cryopreservation for later use
possible in some cases
Microsurgical testicular sperm Hundreds of thousands to millions 100% General, regional plus sedation,
extraction (micro-TESE) of sperm or local plus sedation
Cryopreservation for later use
possible in some cases

spermatozoa. When the three different sperm sources Our centre also treats severely oligozoospermic
were examined, encompassing all maternal ages, the men with a concentration of spermatozoa of ⬍1 ×
ejaculated cohort displayed the highest fertilization 106 /mL [36]. Outcomes of ICSI cycles in these men
rates despite having older women (P ⬍ 0.001). Epi- are highlighted in Table 13.6. If the initial semen speci-
didymal spermatozoa achieved a somewhat lower men examination showed no spermatozoa, then high-
fertilization rate but attained the highest clinical speed centrifugation is used. In 311 cycles, after high-
pregnancies, as defined by the presence of at least one speed centrifugation, a mean density of 0.60 ± 1.1 ×
foetal heartbeat. Cycles using testicular spermatozoa 106 /mL and a motility of 39.2 ± 34% were reached. In
had the lowest fertilization rates in spite of having this cohort, a fertilization rate of 59.7% (1,881/3,150)
the youngest women (P ⬍ 0.001). The pregnancy and a clinical pregnancy rate of 37.6% (117/311) were
rates were somewhat lower compared with those in achieved [37].
the other groups. It must be noted that this analysis In cases of NOA, the degree of spermatogenic fail-
is purely academic, because the surgically retrieved ure often varies, and consequently, sufficient sperma-
spermatozoa address different clinical indications. tozoa for ICSI can be identified in only 40–60% of

Table 13.4 Sperm retrieval techniques and corresponding yield in men with nonobstructive azoospermia [65–68]

Technique Yield Sperm retrieval rate Anaesthesia

Testicular fine-needle aspiration ⬍10 to thousands of sperm 17–59% Local with or without sedation
Cryopreservation for later
use possible in some cases
Testicular sperm extraction (TESE) ⬍10 to thousands of sperm 17–70% General, regional plus sedation,
Cryopreservation for later or local plus sedation
use possible in some cases
Microsurgical testicular sperm ⬍10 to thousands of sperm 33–77% General (preferred), regional plus
extraction (micro-TESE) Cryopreservation for later sedation, or local plus sedation
use possible in some cases

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 Chapter 13: Male Infertility and Assisted Reproduction

3PN
MII Injected MII Injected 1PN 3.5%
n = 224,247 n = 224,247 2.4%

Lysed
2.7%

No fert
16.3

2PN
Surviving oocytes 75.1%
97.3%

Figure 13.2 Fertilization characteristics of ICSI cycles performed between September 1993 and June 2015 at our centre.

all patients. It is often necessary to search the biopsy profiles and numbers of oocytes retrieved. The fertil-
for an extended period of time in cases where only ization rate was 44.0% in the search group and 57.1%
a few sperm cells are present. In one study [36], we in the control group (P = 0.002), and live birth rates
investigated whether an extended search for sperma- were 34.3% and 46.8%, respectively (P ⬍ 0.001). Fer-
tozoa in NOA patients has an effect on ICSI outcome. tilization and pregnancy rates were plotted according
The average search time for routine TESE cases was to the length of time spent on each search (30 min–1
no more than 30 min (control), mostly in relation to h, 1–2 h, 2–3 h, ⬎3 h). The fertilization rates for these
the oocyte cohort. The extensive searches, often car- four groups were 54.2, 46.3, 28.0, and 25.4%, respec-
ried out by several embryologists, were divided into tively (R2 = 0.9315; P ⬍ 0.001). A progressive decrease
four groups based on time – 30 min to 1 h, 1–2 h, 2– in pregnancy rate with lengthening search times was
3 h, and ⬎3 h – and compared with clinical outcome. also observed. Specifically, the clinical pregnancy rates
A total of 739 NOA men who underwent 1,087 ICSI were 44.1, 37.8, 31.8 and 23.8%, respectively. Simi-
cycles were included in this study. The mean ages of the larly, the live birth rates were 32.4, 23.5, 18.2 and
female and male patients were 37.2 and 35.4, respec- 9.5%, respectively. Pregnancy loss rates were compa-
tively. Of the 1,087 cycles included in this study, 225 rable between all the extended search groups and con-
(26.1%) required an extended search. The length of trol. Thus, it appears that the length of time required to
sperm search ranged from 30 min to as long as 10 h extensively search a testicular tissue sample and to per-
with a mean of 82 min. The average number of embry- form ICSI on all the oocyte cohorts is inversely related
ologists involved in the searches was 4 ± 2. Pentoxi- to fertilization and pregnancy outcomes. In spite
fylline was used in almost all of the extended search of the time-dependent clinical performance, search-
cycles and in about 57% of the control cycles. The con- ing for precious spermatozoa is still warranted even
trol and the extended search groups had similar patient after several hours. Although labour-intensive and

Table 13.5 ICSI outcomes using ejaculated, epididymal and testicular spermatozoa

Parameter Ejaculated Epididymal Testicular

Maternal age (years) 37.9 ± 5 35.2 ± 5 33.3 ± 6
Cycles 27,284 1,083 1,631
Fertilization rate 168,411/224,247 (75.1%) 7,326/10,314 (71.0%) 8,466/16,188(52.3%)
Clinical pregnancy 12,469 (45.7%) 550 (50.8%) 661 (40.5%)

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 Chapter 13: Male Infertility and Assisted Reproduction

Fresh Figure 13.3 Fertilization rates using

fresh and frozen ejaculated, epididymal
Frozen and testicular spermatozoa between
75.0% September 1993 and June 2015 at our
148,019/197,342 centre.
Ejaculated 75.8%
20,392/26,905

2,602/3,610 72.1%

MESA 4,724/6,704 70.5%

6,314/12,008 52.6%

TESE 2,152/4,180 51.5%

0 10 20 30 40 50 60 70 80 90
Fertilization rate (%)

time-consuming, this procedure still grants many cou- ICSI has also been proposed as a method for rein-
ples the opportunity to conceive [36]. seminating oocytes that failed to fertilize following
conventional IVF [68–71]. Although reinsemination
When Not to Use ICSI of unfertilized oocytes has been performed with ICSI
15 to 18 h after initial insemination, normal fertil-
ICSI requires technical skills that conventional insem-
ization rates after rescue ICSI remain relatively low,
ination does not. In general, it needs to be performed
and the generated embryos achieve low pregnancy
in a highly regulated laboratory environment. It is per-
rates [72]. It is thought that oocyte quality diminishes
formed out of the laminar flow hood, on a heated
during the 24 h after retrieval, and although some
stage outside of the incubator, and requires enzy-
oocytes may still be fertilized, embryos derived from
matic/mechanical removal of the cumulus oophorus
rescue ICSI procedures often arrest at early stages of
[37]. In fact, early ICSI adopters indirectly improved
development [68–72]. Furthermore, high rates of poly-
their pregnancy rates because of the required adjust-
ploidy are reported in embryos fertilized by rescue
ments to more stringent laboratory conditions [37].
ICSI [72, 73], and the proportion of abnormally fer-
Most importantly, ICSI must be performed in an expe-
tilized oocytes seems to be a function of the length of
dited fashion [37]. Thus, ICSI should not be performed
time the oocyte is in culture following the initial failed
when a regulated laboratory environment is not
fertilization assessment [71].
available.

Table 13.6 ICSI outcomes in men with severe oligospermia

(⬍1 × 106/mL of spermatozoa)
Safety of ICSI
ICSI’s safety has often been criticized because the fer-
Parameter Value tilizing spermatozoon neither binds to the zona pel-
Cycles 1,820 lucida nor fuses with the oolemma [74, 75]. Bypass-
Mean concentration (106 per mL ± SD) 0.3 ± 0.3 ing these physiological steps, together with the arbi-
Mean motility (% ± SD) 19.1 ± 24.0 trary selection of the spermatozoon, has been reason
Mean morphology (% ± SD) 0.9 ± 1
for concern [74, 75]. Thus far, ICSI offspring undergo-
ing adolescence and beyond have provided sufficient
Fertilization 11,082/17,360 (63.8%)
information to reassure these qualms. Follow-up stud-
Clinical pregnancy 748 (41.1%) ies of ICSI children, beginning in the mid-1990s, have

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 Chapter 13: Male Infertility and Assisted Reproduction

revealed an incidence of malformations within the found in the IQ assessment between ICSI and natu-
expected range for the general population of New York rally conceived children. No differences were found
state [76]. Another series investigating the outcomes between ICSI and control children in regard to general
of neonates generated by different assisted conception health, such as chronic illnesses or physical develop-
procedures, ICSI versus IVF with conventional fertil- ment. Thus, ICSI and IVF appeared to exert a negative
ization, provided further confirmation of the expected effect on the wellbeing of offspring mainly because of
rate of malformation [77]. In one study evaluating the the association with multiple gestations [74].
medical and developmental state of 1-year-old chil-
dren born after ICSI or IVF as well as after natural con-
ceptions, the authors found that most 1-year-old ICSI Conclusions
children were healthy and were developing normally, Infertility is a common condition, and problems in
as measured by the Bayley Scales of Infant Develop- the male partner are one of the common causes. The
ment [78]. However, about 17% displayed an increase information generated by conventional semen analysis
in learning difficulties compared with those conceived has historically classified patients into categories lack-
by IVF or naturally. A later report dismissed this con- ing knowledge of causality and leaving conventional
cern in 2-year-old ICSI toddlers [79]. In a different therapy somewhat empirical. However, a better under-
follow-up of 10-year-old children, it was found that standing of spermatogenesis and its genetic control
ICSI children and their naturally conceived counter- has in recent years quite rapidly improved our knowl-
parts had similar motor skills and IQ [80]. edge regarding the epidemiology of male reproduc-
Our centre has compared the pregnancy outcomes tion. ICSI remains the most effective means of treat-
and the developmental wellbeing of children con- ing couples with male factor infertility and previous
ceived from 12,866 ICSI cycles with those of chil- fertilization failures. However, assisted reproduction
dren from naturally conceived singleton pregnancies techniques such as IVF and ICSI do not address the
[74]. From a total of 3,277 couples delivering 5,891 underlying cause for infertility, potentially increasing
neonates, the incidences of low birth weight and ges- the risk of transmitting both identified and concealed
tational length were comparable with those for the genetic anomalies [37]. Thus, basic research is needed
naturally conceived counterparts, after controlling for to elucidate the biological mechanisms underlying the
maternal age. Rates of malformation in ICSI offspring genetic and epigenetic effects of ART. Although there is
ranged from 3.5 to 6.2%, compared with 6.5% in still very little known about the long-term health con-
the natural conception group. In the ICSI group the ditions of both infertile men and their offspring, recent
major malformations included two neonates with a data suggest that infertility may serve as a proxy for
cardiac disease (ventricular septal defect and severe general medical ill health [81], with infertile or sub-
tricuspid regurgitation), one with talipes, and one fertile men possibly having increased mortality rates
with trisomy 7 mosaicism. Among the naturally con- [82]. The adverse outcomes in offspring conceived by
ceived pregnancies, there were two neonates with car- IVF or ICSI are generally due to the occurrence of
diac defects (ventricular septal defect and patent fora- high-order pregnancies. Thus, single embryo transfers
men ovale/atrial septal defect), one with encephalopa- are paramount in reducing such adverse outcomes.
thy, one with polydactyly, and one severe midshaft Although perinatal outcomes such as prematurity,
hypospadias with penile angulation. At 3 years of age low birth weight, perinatal mortality and increased
(n = 811), the proportion of children at risk for devel- incidence of malformations have been linked to the
opmental delays was 10.4% in ICSI and 10.7% in IVF techniques of IVF and ICSI, infertility itself seems
singletons. However, high-order gestations were char- to be the larger issue that leads to negative clinical
acterized by 19.4% of the children having compro- outcomes.
mised development. To study the long-term effect of
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