Seminar

Male infertility
Ashok Agarwal, Saradha Baskaran*, Neel Parekh*, Chak-Lam Cho, Ralf Henkel, Sarah Vij, Mohamed Arafa, Manesh Kumar Panner Selvam,
Rupin Shah

It is estimated that infertility affects 8–12% of couples globally, with a male factor being a primary or contributing Lancet 2021; 397: 319–33
cause in approximately 50% of couples. Causes of male subfertility vary highly, but can be related to congenital, Published Online
acquired, or idiopathic factors that impair spermatogenesis. Many health conditions can affect male fertility, which December 10, 2020
https://doi.org/10.1016/
underscores the need for a thorough evaluation of patients to identify treatable or reversible lifestyle factors or medical
S0140-6736(20)32667-2
conditions. Although semen analysis remains the cornerstone for evaluating male infertility, advanced diagnostic
*Authors contributed equally
tests to investigate sperm quality and function have been developed to improve diagnosis and management. The use
American Center for
of assisted reproductive techniques has also substantially improved the ability of couples with infertility to have Reproductive Medicine
biological children. This Seminar aims to provide a comprehensive overview of the assessment and management of (Prof A Agarwal PhD,
men with infertility, along with current controversies and future endeavours. S Baskaran PhD,
Prof R Henkel PhD,
M K Panner Selvam PhD) and
Introduction The cause of infertility lies solely with the man in Department of Urology
WHO defines infertility as the inability to conceive 20–30% of cases and a male cause is contributory in a (N Parekh MD, S Vij MD),
after at least 12 months of regular, unprotected sexual further 20%.16,17 In 1992, a large meta-ana­lysis by Carlsen Cleveland Clinic, Cleveland, OH,
USA; SH Ho Urology Center,
intercourse.1 Infertility is a major health problem world­ and colleagues confirmed that sperm counts had declined
Department of Surgery,
wide and is estimated to affect 8–12% of couples in the by 50% during a 60-year period.18 Subsequently, numerous Chinese University of
reproductive age group.2 A Global Burden of Disease studies have shown similar declines glo­bally,19,20 although Hong Kong, Hong Kong
survey reported that between 1990 and 2017, the age- some studies have disputed this claim.21,22 A systematic (C-L Cho FRCSEd[Urol]);
Department of Medical
standardised prevalence of infer­tility increased annually review by Levine and colleagues23 reported that sperm
Bioscience, University of
by 0·370% in women and by 0·291% in men.3 counts decreased by 50–60% between 1973 and 2011. Western Cape, Bellville,
Infertility causes substantial psychological and social The causes of male subfertility are wide ranging and South Africa (Prof R Henkel);
distress,4,5 and imposes a considerable economic burden poorly understood in most cases.24–26 Although various Department of Metabolism,
Digestion and Reproduction,
on patients and health-care systems.6 Early diagnosis and diagnostic tests are available, their interpretation is
Imperial College London,
appropriate management can mitigate these factors. In imprecise and often subjective.27 Intracytoplasmic sperm London, UK (Prof R Henkel);
a prospective study of 384 419 Danish men, Glazer and injection has made it possible to achieve pregnancy with Male Infertility Unit, Urology
colleagues7 reported a higher risk of mortality among men very poor semen quality—eg, in cases of azoospermia Department, Hamad Medical
Corporation, Doha, Qatar
with male factor infertility than among men who were for which surgically retrieved testicular sperm are used.28
(M Arafa MD); Andrology
fertile. Ventimiglia and colleagues8 showed that impaired Exciting new therapies using stem cells and in-vitro Department, Cairo University,
male reproductive health (including poorer semen para­ sperm maturation are still experimental. This Seminar Cairo, Egypt (M Arafa); and
meters and lower testosterone levels) was associated with aims to review our current understanding of these issues Department of Urology,
Lilavati Hospital and Research
a higher Charlson Comorbidity Index, which is a proxy of Center, Mumbai, India
decreased general health status.9 Severe male infertility is (R Shah MCh[Urology])
also associated with a greater incidence of cancer.10 Thus, Search strategy and selection criteria
Correspondence to:
early detection of male subfertility offers the opportunity We searched Scopus and PubMed for relevant articles on male Prof Ashok Agarwal, American
for identification and correction of medical conditions infertility using the search term “male infertility” in
Center for Reproductive
Medicine, Cleveland Clinic,
affecting not only fertility, but also general health and combination with the search terms “epidemiology”, “etiology”, Cleveland, OH 44195, USA
wellbeing.11 “pathophysiology”, “investigations”, “azoospermia”, agarwaa@ccf.org
There is increasing evidence that paternal health at “oligoasthenoteratozoospermia”, “asthenozoospermia”,
the time of conception can affect the offspring’s meta­ “varicocele”, “genetic abnormalities”, “cryptorchidism”,
bolic health and reproductive potential, through transge­ “testicular cancer”, “obstruction”, “hypogonadism”, “ejaculatory
nerational transmission of epigenetic modifications.12 dysfunction”, “idiopathic”, “risk factors”, “diagnosis”, “clinical
Thus, obesity or diabetes13 might contribute not only to evaluation”, “sperm DNA fragmentation index”, “reactive
male subfertility, but can also com­promise the health of oxygen species”, “genetic testing”, “imaging”, “management”,
future progeny. A study of 744 men with infertility revealed “treatment”, “antioxidant therapy”, “varicocelectomy”, “ART”,
that 15·4% of men who met the criteria suggestive of or “omics”. We selected articles mostly published in the past
prediabetes were at increased risk of hypogonadism, 5 years and highly cited older publications. We also reviewed
higher sperm DNA fragmentation, and non-obstructive the reference list of the retrieved articles and selected articles
azoospermia.14 Men who are oligozoospermic are more that discussed male infertility, were published within the last
likely to have metabolic syn­drome than men who are 5 years, and were not retrieved in the initial search. Highly
normozoospermic.15 Therefore, it is important to look referenced reviews and book chapters are cited to provide
beyond a semen analysis, and to view male infertility as a readers with more information and references than this
condition connected to and promoting a state of impaired Seminar can accommodate.
metabolism.

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and provide practice guidelines for individualising and defects. Among acquired factors, varicocele is the most
optimising the management of men with subfertility. common and correctable cause of infertility in men,
with a prevalence of 40%.30,32–34 About 30–50% of male
Causes infer­tility cases are idiopathic, with no discernible cause
A multitude of causes and risk factors contribute to the or contributory female infertility.35,36 Male oxidative stress
increasing incidence of male infertility,29,30 which can be infertility involves altered semen characteristics and
stratified as congenital, acquired, and idiopathic (panel 1). oxidative stress, and affects about 37 million men with
The primary known genetic causes of male infertility idiopathic male infertility.37 Environ­ mental or occu­
are congenital bilateral absence of the vas deferens pational exposure to toxic chemicals38 and various lifestyle
associated with cystic fibrosis gene mutations, Kallmann factors (eg, smoking,39,40 alcohol consumption,41 recre­
syndrome,31 chromosomal abnormalities leading to ational drug use,42–44 obesity,45,46 and psychological stress47)
deterioration of testicular function, and Y chromosome are all potential risk factors for male infertility.48
microdeletions resulting in isolated spermatogenic
Evaluation
Infertility evaluation and treatment is recom­mended for
Panel 1: Causes and risk factors of male infertility couples who do not conceive naturally after at least
Congenital factors 12 months of regular, unprotected sexual intercourse,49,50
• Anorchia or after 6 months for couples in which the female partner
• Congenital absence of vas deferens is older than 35 years. Evaluation and treatment before
• Cryptorchidism 12 months might be considered on the basis of medical
• Y chromosome microdeletions history and physical examination, and men who have
• Chromosomal or genetic abnormalities concerns about their future fertility can also be evaluated.
• Klinefelter syndrome and its variants (47,XXY; The American Society for Reproductive Medicine
46,XY/47,XXY mosaicism) (ASRM) and the European Association of Urology (EAU)
• Kallmann syndrome both recommend an initial evaluation consisting of a
• Robertsonian translocation reproductive history and at least one semen analysis,30,49
• Mild androgen insensitivity syndrome although the American Urological Association (AUA)
• Genetic endocrinopathy insists on two semen analyses.50 If the initial evaluation
• Congenital obstruction shows abnormal results, refer­ ral to a reproductive
specialist is recommended for a thorough evaluation that
Acquired factors includes a physical examination and taking a complete
• Varicocele medical history. Depending on the results, further
• Testicular trauma andrological assess­ ments and procedures might be
• Testicular torsion recommended.
• Germ cell tumours
• Acquired hypogonadotropic hypogonadism Medical history
• Recurrent urogenital infections (prostatitis, Successful diagnosis of male infertility can be challenging,
prostatovesciculitis) because the process of conception involves multiple organs
• Postinflammatory conditions (epididymitis, mumps and requires the evaluation of two individuals. The initial
orchitis) step in evaluating infertility is obtaining a thorough history
• Urogenital tract obstruction (panel 2). Infertility can be classified as either primary
• Exogenous factors (eg, chemotherapy, medications, (ie, no previous fertility) or secondary (ie, previously fertile,
radiation, heat) currently infertile).1 Although this distinction can narrow
• Systemic diseases (live cirrhosis, renal failure) differential diagnosis, men classified with primary or
• Anti-sperm antibodies secondary infertility should be assessed in the same way.50
• Surgeries that can comprise vascularisation of the testis Various childhood conditions (eg, cryptorchidism,
• Sexual dysfunction (erectile or ejaculatory dysfunction) postpubertal mumps orchitis, and testicular torsion or
Idiopathic risk factors trauma) can result in testicular atrophy or decreased
• Smoking semen quality.51–53 Infections of the male urogenital tract
• Alcohol (prostatitis, urethritis, epididymitis, and orchitis) can
• Recreational drugs contribute to male infertility.30 The prevalence of male
• Obesity urogenital tract infection was reported to be as high as
• Psychological stress 35% in a study of more than 4000 men with infertility.54
• Advanced paternal age A cross-sectional study of 1689 men revealed that 20% of
• Dietary factors men with primary infertility had asymptomatic semen
• Environmental or occupational exposure to toxins infections, which were associated with impaired sperm
concentrations.55 Prostatitis, a common urogenital

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disease caused by Escherichia coli, can have detrimental Semen analysis

effects on various sperm parameters.56 Among sexually WHO recommends conventional semen analysis as
active men younger than 35 years, Chlamydia trachomatis the first step in the evaluation of male fertility potential.
and Neisseria gonorrhoeae are the most common path­ The WHO Laboratory Manual for the Examination
ogens to cause epididymitis. E coli is the predominant and Processing of Human Semen and Sperm–Cervical Mucus
pathogen found in men older than 35 years who have Interaction has been published since 1980,69–72 with the
infertility. Although semen analysis is not recom­
mended in acute cases of epididymitis or prostatitis,
men with chronic epididymitis or prostatitis might Panel 2: Important attributes of history taking in the
present with leukocytospermia (>1 × 10⁶ white blood evaluation of men with infertility
cells per mL), which is a sign of inflammation30 and can
be confirmed by peroxidase test in semen.57 Infertility history
Lifestyle factors such as smoking, alcohol consumption, • Duration of infertility
recreational drug use (eg, cocaine, opioid narcotics, • Previous pregnancies and outcomes (primary vs
cannabis, and anabolic steroids), and obesity are also secondary infertility)
relevant to male infertility.40–42 A large meta-analysis • Partner’s fertility history
involving 5865 men from 20 studies showed deterioration • Previous fertility investigation and treatment
of semen quality in moderate and heavy smokers.40 Sexual history
Similarly, a meta-analysis of 15 cross-sectional studies • Libido
revealed the negative asso­ ciation between alcohol con­ • Erectile dysfunction
sump­ tion and sperm param­ eters.41 Cannabis, the most • Ejaculatory dysfunction
frequently used recreational drug, negatively effects • Type of lubricants
male fertility by inhibiting the hypothalamic–pituitary– • Frequency and timing of coitus
gonadal axis, spermatogenesis, and sperm function.58 The • Sexually transmitted disease
association between obesity and male infertility has been
widely investigated as the global prevalence of obesity Medical history
continues to rise.12 Obesity-induced endocrine alterations • Cryptorchidism
that result in peripheral conversion of testosterone to • Timing of puberty
oestrogen have been linked with reduced sperm con­ • Anosmia
centrations.59 Among the subsets of obesity, metabolically • History of testicular torsion
unhealthy obesity (ie, with metabolic abnormalities such • History of testicular trauma
as diabetes, hypertension, dyslipidaemia, insulin resis­ • Diabetes
tance) is known to be a risk factor for erectile dysfunction, • Neurological conditions (spinal cord injury, multiple
and the combination of erectile dysfunction and meta­ sclerosis)
bolically healthy obesity (ie, without evidence of metabolic • Infections (urinary infections, epididymitis or prostatitis,
and cardiovascular disease) in men represents an early tuberculosis, mumps orchitis, recent febrile illness)
marker for future adverse metabolic consequences.60 • Renal disease
The couple’s sexual practices, including the timing of • Cancer
coitus and erectile and ejaculatory function, should be Surgical history
assessed. Ovulation tracking methods should be used to • Orchidopexy
ensure that couples are timing intercourse effectively. • Retroperitoneal or pelvic surgery
Inter­course is recommended every 48 h around the time • Herniorrhaphy
of ovulation, to maximise the chance of fertilisation.61 • Vasectomy
The most common sexual disorders that affect men with • Bladder neck or prostatic surgery
infertility are hypoactive sexual desire and an absence
of sexual satisfaction (pleasure, positive feeling, and Gonadotoxin exposures
orgasm).62 One in six men with infertility has erectile • Medications (endocrine modulators, antihypertensives,
dysfunction, or premature ejaculation, or both.63 The antibiotics, antipsychotics)
psychological effects of sexual dysfunction and male • Environmental (pesticides, heavy metals)
infertility can be a substantial barrier to successful • Chemotherapy or radiotherapy
fecundity, and should be screened for during clinical • Lifestyle (obesity, tobacco, vaping, recreational drugs,
evaluation. Also, many couples use vaginal lubricants, anabolic steroids)
but these can be spermicidal.64,65 Vegetable oil, raw egg Family history
white, and fertility-friendly lubricants (eg, Pre-Seed, ING • Infertility
Fertility, Spokane, WA, USA) have the least spermicidal • Cystic fibrosis
effects, but couples should still be made aware to use • Androgen receptor deficiency
them in moderation.66–68

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WHO manual WHO manual WHO manual WHO manual WHO manual
1st edn (1980)69 2nd edn (1987)70 3rd edn (1992)71 4th edn (1999)72 5th edn (2010)57
Volume ND ≥2·0 mL ≥2·0 mL ≥2·0 mL ≥1·5 mL
Sperm concentration 20–200 × 10⁶/mL* ≥20 × 10⁶/mL ≥20 × 10⁶/mL ≥20 × 10⁶/mL ≥15 × 10⁶/mL
Total sperm count ≥40 × 10⁶/mL ≥40 × 10⁶/mL ≥40 × 10⁶/mL ≥40 × 10⁶/mL ≥39 × 10⁶/mL
Sperm motility (% progressive) ≥60% ≥50% ≥50% ≥50% ≥32%
Sperm vitality (%) ND ≥50% ≥75% ≥75% ≥58%
Sperm morphology (% normal) ≥80·5%† ≥50% ≥30%‡ ≥15%§ ≥4%

Data extracted from the WHO manuals. ND=not defined. *Probably based on MacLeod’s work.73 †Mean of fertile population. ‡Arbitrary value. §Value not defined but strict
criteria and in-vitro fertilisation data suggest a 14% cutoff value.

Table: The evolution of normal values for semen parameters from 1980 to 2010 across the first five editions of the WHO Laboratory Manual for the Examination
and Processing of Human Semen and Sperm–Cervical Mucus Interaction

most recent manual released in 2010.57 The recom­ semen analysis. Results of Agarwal and colleagues’
mended cutoff values for semen parameters have evolved prospective study85 of semen analysis show that this
dramatically over the years (table), yet nomenclature device is a reliable diagnostic tool, providing clinically
related to semen quality has remained unchanged acceptable results, as defined by WHO 5th edition
(panel 3). The lower reference limits depicted in the guidelines.
latest edition of the WHO manual57 are derived from Home-based collection of semen samples is another
the statistical analysis of the semen parameters of advancement in semen analysis.86 Technologies that
1953 fertile men from around the world.74 However, support being able to test sperm at home provide a
these reference limits have been criticised for not potential solution for men who feel uncom­ fortable
considering the female factor, high biological variation about providing a semen specimen in an unfamiliar
among individuals, and the absence of data from environment.87,88 Home-based sperm testing systems are
representative ethnic groups.75–77 Consequently, standard mainly based on antibody reactions, micro­fluidics, or
semen analysis has limited accuracy for determining smartphone technology. The accuracy of these devices
male fertility potential or predicting reproductive for determining sperm concentration ranges from
success. In fact, interpreting semen analysis using 95% to 98%, making them a practical and affordable way
WHO 2010 reference values resulted in samples being to do preliminary screening for male infertility.89
considered normal that would have been considered
abnormal if using the 1999 manual.78 Ombelet and Physical examination
colleagues used receiver operating characteristic curve Physical examination is a key part of evaluating male
analysis to determine the diagnostic potential and cutoff infertility, and should include an assessment of body
values for single and combined sperm parameters.79 habitus, secondary sexual characteristics, and genitalia.
Their prospective study revealed that single sperm An eunuchoid body habitus, decreased body hair
parameters were of little clinical value for differentiating compared with Tanner stage V, obesity, or gynae­ co­
­
men who were fertile from men with subfertility, and mastia might be seen in patients with endocrinopathies
showed it was important to use a combination of sperm (eg, low serum testosterone, Klinefelter syndrome,
parameters to predict a man’s fertility status.79 Another hyperprolactinaemia).90,91
problem with standard semen analysis is that not all The genital examination should begin with the phallus,
laboratories comply strictly with the WHO manual carefully assessing for penile curvature, plaques, epi­
methods. Less than 60% of laboratories in the USA spadias, or hypospadias, all of which can impair semen
complied with WHO guidelines, and less than 5% in deposition in the vaginal vault. The testicles should be
the UK.80,81 It is of paramount importance that all examined for presence, size, and consistency. Testicular
laboratories follow the WHO manual guidelines strictly, size should be assessed using a Prader orchidometer
to provide reliable and comparable results. or callipers (normal volume 20 mL or 4 × 3 cm).92 Scrotal
Several semi-automated and fully automated computer- ultrasonography can be useful when the patient’s body
assisted sperm analysis systems have been introduced. habitus or scrotal anatomy (hydrocele, dilated epididymis,
Despite their shortcomings for evaluating sperm mor­ or inguinal testis) might render testicular measurement
phology accurately,82,83 computer-assisted sperm analysis by Prader orchidometer unreliable.93 A testicular mass
systems are widely used in many andrology and in-vitro should be ruled out, because men with infertility are at
fertilisation clinics that strictly adhere to quality control increased risk of testicular neoplasm.94 The epididymides
protocols to quantify semen parameters accurately.84 Sys­ should be palpated to assess for enlargement that might
tems such as the LensHooke (Bonraybio Co, Taichung indicate distal obstruction. A hypoplastic epididymis
City, Taiwan) incorporate artificial intelligence to simplify with either unilateral or bilateral non-palpable vas

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deferens is consistent with vasal agenesis and can be

associated with genetic or renal abnormalities.
The spermatic cords should be assessed in the supine
Panel 3: Nomenclature related to semen quality
and standing positions, allowing for the detection of
a varicocele. Varicoceles are graded by size: grade 1 is Aspermia
palpable only by Valsalva manoeuvre, grade 2 is palpable No semen (no ejaculation or retrograde ejaculation)
without Valsalva manoeuvre, and grade 3 is visible at
Asthenozoospermia
rest.95 Although digital rectal exami­nation is not routinely
Percentage of progressively motile spermatozoa below the lower reference limit
done in young men with subfertility, it is indicated in
men with low ejaculate volume. The prostate should Asthenoteratozoospermia
be assessed for size and consistency. A midline cyst or Percentages of both progressively motile and morphologically normal spermatozoa
prominent seminal vesi­cles might indicate ejaculatory below the lower reference limits
duct obstruction.92
Azoospermia
No spermatozoa in the ejaculate (given as the limit of quantification for the assessment
Hormonal evaluation
method used)
Hormonal evaluation is an important tool in the
management of male infertility. Many clinicians consider Cryptozoospermia
hormonal assessment to be part of the routine inves­ Spermatozoa absent from fresh preparations but seen in a centrifuged pellet
tigation for every male patient with infertility,96 although
international societies recommend limiting use to Haemospermia (haematospermia)
particular groups of patients, including men with a Presence of erythrocytes in the ejaculate
sperm concentration below 10 × 10⁶/mL or impaired Leukospermia (leukocytospermia, pyospermia)
sexual function, or if endocrinopathy is suspected.49,50 Presence of leucocytes in the ejaculate greater than the threshold value
The recommended basic hormonal evaluation should
include analysis of follicle-stimulating hormone and total Necrozoospermia
testosterone (panel 4). If total testosterone concentration Low percentage of live, and high percentage of immotile, spermatozoa in the ejaculate
is found to be low, a more thorough endocrine evaluation Normozoospermia
is recommended. This process includes repetition of Total number (or concentration, depending on outcome reported)* of spermatozoa,
total testosterone and addition of luteinising hormone and percentages of progressively motile and morphologically normal spermatozoa, equal
assay to differentiate primary from secondary hypo­ to or greater than the lower reference limits
gonadism. Prolactin analysis is also recommended in
such cases.49,50 The validity of the ASRM guidelines Oligoasthenozoospermia
for hormonal evaluation of male infertility has been chal­ Total number (or concentration, depending on outcome reported)* of spermatozoa,
lenged for predicting hypogonadism.97 A retro­spective and percentage of progressively motile spermatozoa, less than the lower reference
study by Ventimiglia and colleagues97 revealed that the limits
guidelines had a low predictive value, with 58% overall Oligoasthenoteratozoospermia
accuracy, 75% sensiti­vity, and 39% specificity. There is Total number (or concentration, depending on outcome reported)* of spermatozoa,
no general consensus on the lower cutoff value for and percentages of both progressively motile and morphologically normal spermatozoa,
testosterone concentrations. The ASRM adopts the value less than the lower reference limits
of less than 300 ng/dL as a cutoff for diagnosing
hypogonadism, and the EAU recommends 230 ng/dL Oligoteratozoospermia
(8 nmol/L).98,99 Total number (or concentration, depending on outcome reported)* of spermatozoa,
Measuring total testosterone concentration alone could and percentage of morphologically normal spermatozoa, less than the lower reference
be insufficient in cases in which sex hormone-binding limits
globulin is increased (eg, in men older than 75 years, Oligozoospermia
thyroid disease, or diabetes). In these cases, measurement Total number (or concentration, depending on outcome reported)* of spermatozoa less
of free testos­terone is recommended. Although reverse than the lower reference limit
equilibrium dialysis is the gold standard for measuring
free testosterone, it is expensive and technically chal­ Teratozoospermia
lenging. Using calculated free testosterone can be a Percentage of morphologically normal spermatozoa less than the lower reference limit
more clinically accurate method in assessing men with The suffix spermia refers to the ejaculate and zoospermia refers to the spermatozoa.
hypogonadal symptoms.99,100 Therefore, the following terms should not be used: asthenospermia,
Although the role of prolactin in female fertility is well asthenoteratospermia, cryptospermia, oligoasthenospermia, oligoteratospermia,
established, its role in male infertility is not clear, oligospermia, teratospermia.
although mild elevations are not important. Severe
Adapted from WHO 5th edn, 2010.57 *Preference should always be given to the total number, as this parameter takes precedence
hyperprolactinaemia might be associated with lower over concentration.
total testosterone concentrations, thereby affecting

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defects. Karyotype anomalies are the most common

Panel 4: Clinical interpretations of hormonal assessments type of genetic defect, with a prevalence of 12–15% in
in men with infertility azoospermia, 5% in severe oligozoospermia, and less
Hypogonadotropic hypogonadism than 1% in normal semen.110–112 The most common
Decreased concentrations of follicle-stimulating hormone, karyotype defect is Klinefelter syndrome (also known
luteinising hormone, and testosterone as 47,XXY), followed by translocations, inver­sions, and
deletions. Different professional societies agree on
Testicular failure (oligoasthenoteratozoospermia or recom­ mending karyotype analysis for men with
non-obstructive azoospermia) azoospermia or severe oligozoospermia (sperm count
Increased concentrations of follicle-stimulating hormone and <5 × 10⁶/mL).113–115 However, the EAU extended their
luteinising hormone, and decreased or normal guideline recom­ mendations to include men with a
concentrations of testosterone sperm count of less than 10 × 106/mL.30,35 The EAU also
Inconclusive: normal spermatogenesis or defective recommends obtaining karyotype if there is a family
spermatogenesis history of recurrent spontaneous abortions, malfor­
Normal concentrations of follicle-stimulating hormone, mations, or intellectual disability,30,35 regardless of the
luteinising hormone, and testosterone sperm concentration.30 This recommendation35 was
retrospectively validated in a cohort study of 1168 men,
Hyperprolactinaemia which found that the suggested threshold had moderate
Increased concentrations of prolactin, and normal or sensitivity (80%), but low specificity (37%) and dis­crimi­
decreased concentrations of testosterone nation (59%).116 Therefore, use of the EAU guidelines
primarily on the basis of sperm count might lead to
unnecessary use of karyotype analysis, which is an
spermatogenesis and male sexual function.101 Hyperpro­ expensive and laborious test.
lactinaemia is caused by prolactinomas in 40% of cases.102 Y chromosome microdeletion analysis is indicated
Follicle-stimulating hormone is usually negatively for patients with azoospermia or oligozoospermia and
associated with spermatogenesis, so increased follicle- a sperm count of less than 5 × 10⁶/mL.117 A meta-analysis
stimulating hormone would be seen in cases of by Kohn and colleagues showed that the majority of
defective spermatogenesis with absent or diminished Y chromosome microdeletions occur in men with sperm
spermatogonia.103,104 However, in some cases of sper­ counts of less than 1 × 10⁶/mL.118 The latest EAU guide­
matogenic arrest at the level of spermatocyte or sper­ lines recommend Y chromosome microdeletion testing
matid, concentrations of follicle-stimulating hormone, if sperm concentrations are less than 5 × 10⁶/mL, and
luteinising hormone, and testosterone might be normal, make such testing mandatory for sperm con­centrations
which limits the predictive value of endocrine evaluation of less than 1 × 10⁶/mL.30 Y chromosome microdeletion
in men with non-obstructive azoospermia. affects azoospermia factor a, b, or c in the long arm of
the Y chromosome. Although sperm can be retrieved
Genetic testing from the testes of men with azoospermia factor c
Genetic abnormalities related to male infertility affect deletions, azoospermia factor a or b deletions carry a
about 15% of men with infertility.105 A recent systematic very poor prognosis and sperm retrieval is not advised in
review and clinical validity assessment of male infertility such cases. Importantly, Y chromosome microdeletions
genes revealed a total of 78 genes linked to 92 male infer­ can be transmitted to male offspring, so counselling
tility phenotypes.106 Several genes and gene mutations couples is recommended before intracytoplasmic sperm
related to spermatogenesis have been discovered.26,107 injection.119,120
Men with genetic abnormalities usually show defective Most patients with cystic fibrosis have congenital
spermatogenesis, resulting in severe oligozoospermia or bilateral absence of the vas deferens and about two-
azoospermia and increased aneuploidy.108 Genetic muta­ thirds of men with this condition have CFTR mutations
tions in embryos might lead to repeated intracytoplasmic without any other cystic fibrosis manifestations.121,122 For
sperm injection failure, recurrent miscarriage, or vertical men with structural abnor­malities of the vas deferens, it
transmission of paternal genetic defects. Therefore, iden­ is recommended that both partners be tested for CFTR
tifying genetic defects is crucial for diagnostic purposes mutations containing a minimal panel of common point
and proper counselling before intracytoplasmic sperm mutations and the 5T allele.30
injection. Vertical trans­mission of genetic defects can be
prevented through preimplantation genetic testing and Imaging
transfer of genetically healthy embryos.109 Genetic testing Full evaluation of a man with infertility can involve
is also important for predicting the success of sperm imaging in some circumstances. Scrotal ultraso­nography
retrieval.109 is a preferred imaging modality because of its non-
Karyotyping (also known as chromosomal analysis) invasive nature, safety, and low cost. It provides details
detects numerical chromosomal defects, or structural about testicular size and volume, testicular echogenicity

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and blood flow, varicocele presence, and epididymal Specialised tests

anatomy. Because scrotal ultrasonography is not indi­ Conventional semen parameters do not detect defects
cated for the diagnosis of subclinical varicocele,123 associated with functional aspects of spermatozoa,127 so
ultrasonography can be avoided in men with a normal sperm function tests have been developed to augment
physical examination result. Patients in whom proximal semen analysis (figure 1). The clinical importance of the
genital tract obstruction is suspected (on the basis of sperm function tests came to light after the emergence
history, physical examination, and semen analysis) need of in-vitro fertilisation and intracytoplasmic sperm
to have transrectal ultrasound to evaluate for seminal injection.129–131 In conventional in vitro fertilisation, defec­
vesicle dilation, midline prostatic cyst, and ejaculatory tive sperm–zona interaction is the main reason for
duct dilation.93,124 Transrectal ultrasound can be used in fertilisation failure. However, in the current era of
combination with seminal vesicle aspiration to more intracytoplasmic sperm injection, hemizona or acrosome
accurately diagnose ejaculatory duct obstruction.124 If function assays are no longer used in clinical practice,
more detailed imaging of the genitourinary tract is because the penetrating capability of sperm is bypassed
required, MRI can be done. In men with infertility, by intracytoplasmic sperm injection. There­fore, greater
hypogonadism, and elevated prolactin, cranial MRI can emphasis is placed on the assessment of sperm chromatin
diagnose a pituitary pathology (most commonly pro­ quality using sperm DNA fragmentation testing.132–134
lactinoma) as an underlying cause of hyperprolactinaemia Sperm DNA fragmentation assays potentially provide a
and hypogonadism.125 Vasography is an invasive imaging more comprehensive assessment of the overall fertility
modality to confirm patency or delineate an obstruction status than conventional semen parameters.135 Currently,
of the vas deferens or ejaculatory duct,126 and is usually terminal deoxynucleotidyl transferase-mediated dUTP
done only as part of definitive reconstructive surgery. nick-end labelling, sperm chromatin structure assay,
In many cases, physical exami­ nation alone allows a and sperm chromatin dispersion are among the most
specialist in male infertility to make a diagnosis, but commonly used sperm DNA fragmentation assays.136
the aforementioned imaging methods can be used for Although test protocols and cutoff values have substan­
inconclusive cases, or intraoperatively during recon­ tially improved precision and decreased variations for the
structive microsurgery.92 sperm DNA fragmentation test, the absence of strict

Basic Advanced
Sperm zona pellucida
binding
Sperm DNA fragmentation

Hemizona assay
SCD, TUNEL, SCSA, comet
assay
ROS
ROS

Reactive oxygen species

Acrosome reaction

Chemiluminescence
Macroscopic Microscopic (luminometer) ORP
FITC-PSA staining
(MiOXSYS)

Volume Concentration Motility

Mitochondrial function
pH Morphology Agglutination

Mitochondrial membrane
Appearance Round cells Viability potential

Viscosity

Figure 1: Laboratory evaluation for male infertility

Standard semen analysis comprises the analysis of macroscopic and microscopic parameters. An advanced sperm function test comprises the determination of ROS,
sperm DNA fragmentation, acrosome reaction, and MMP using different techniques. FITC-PSA=fluorescein isothiocyanate-labelled Pisum sativum agglutinin.
MiOXSYS=male infertility oxidative system. MMP=mitochondrial membrane potential. ORP=oxidation-reduction potential. ROS=reactive oxygen species. SCD=sperm
chromatin dispersion test. SCSA=sperm chromatin structure assay. TUNEL=terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. Adapted from
Agarwal and colleagues,128 by permission of the Korean Society for Sexual Medicine and Andrology.

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 Seminar

intro­duced to measure global oxidative stress in semen

Panel 5: Clinical indications for sperm DNA fragmentation testing samples using the Male Infertility Oxidative System,
Clinical varicocele which is a quick and simple test.145 The potential clinical
• Sperm DNA fragmentation testing is recommended in patients with grade 2 or 3 value of the oxi­ dation-reduction potential assay was
varicocele with normal conventional semen parameters reported in a multicentre study that established a cutoff
• Sperm DNA fragmentation testing is recommended in patients with grade 1 value of 1·34 mV/10⁶ sperm per mL to differentiate
varicocele with borderline or abnormal conventional semen parameters men with normal and abnormal semen parameters.146
Although seminal oxidative stress can be determined
Unexplained infertility or intrauterine insemination failure or recurrent pregnancy by various assays, the EAU guidelines recommend
loss that routine testing of reactive oxygen species should
• Sperm DNA fragmentation testing should be offered to couples with infertility and remain experimental until these tests are validated in
recurrent pregnancy loss, or before intrauterine insemination randomised controlled trials (RCTs).30
• Early in vitro fertilisation or intracytoplasmic sperm injection might be an alternative
treatment for couples with infertility and recurrent pregnancy loss or failed Management
intrauterine insemination Azoospermia
In vitro fertilisation failure, or intracytoplasmic sperm injection failure, or both The causes of azoospermia can be classified as
• Sperm DNA fragmentation testing is indicated in patients with recurrent failure of pretesticular, testicular, or post-testicular. Pretesticular
assisted reproduction causes of azoospermia include endocrine abnormali­
• The use of testicular sperm rather than ejaculated sperm might be beneficial in men ties involving the hypothalamic–pituitary–gonadal axis.
with oligozoospermia, high sperm DNA fragmentation, and recurrent in vitro Although congenital and acquired hypogonado­ tro­
pic
fertilisation failure hypogonadism is rare, it is one of the few medi­
cally treatable causes of male infertility. Common notable
Borderline abnormal (or normal) semen parameters with risk factor causes of hypogonadotropic hypogonadism include
• Sperm DNA fragmentation testing should be offered to patients who have a modifiable Kallmann syndrome and exogenous androgen excess.
lifestyle-risk factor for male infertility Hypogonadotropic hypogonadism is characterised by
low concentrations of serum follicle-stimulating hormone
and testosterone during the hormonal evalu­ ation of
standardisation and clear threshold values deter its men who are azoospermic. The combination of human
wider application.137 Hence, although emerging evidence chorionic gonadotropin and human meno­pausal gonado­
supports the role of sperm DNA fragmentation in tropin is commonly used in clinical practice as a sub­
reproductive outcomes (whether natural or via assisted stitute for luteinising hormone and follicle-sti­mu­lating
reproductive techniques),49 routine use of sperm DNA hormone respectively, to induce fertility in patients
fragmen­tation testing is not recommended by the AUA with hypo­gonadotropic hypogonadism. Successful preg­
or ASRM.30,49,50 In 2017, a publication on clinical practice nancies were reported for female partners of 16–57% of
guidelines consolidated the available data on sperm DNA men with congenital hypogonadotropic hypogonadism
fragmentation testing and provided recom­ mendations after treament.147
in four specific clinical scenarios138 (panel 5). The EAU Once a pretesticular cause has been ruled out, azoo­
guidelines recom­ mend sperm DNA fragmen­ tation spermic men are categorised as having either obstructive
testing in couples with recurrent pregnancy loss, or in azoospermia or non-obstructive azoospermia (figure 2).
men with unexplained infertility.30 A DNA fragmentation Testicular biopsy is no longer recommended to make a
index of more than 30% by sperm chromatin structure diagnosis. Generally, a cutoff value of 7·6 mIU/mL for
assay is associated with a lower incidence of pregnancy follicle-stimulating hormone and a testicular long axis
via natural conception or intrauterine insemination.138 of 4·6 cm are used to differentiate obstructive azoo­­sper­
Measuring seminal oxidative stress could be another mia from non-obstructive azoospermia.148 Patients with
means of sperm functional assessment, considering obstructive azoospermia have several options, including
the close and potentially causal relationship between epididymal or testicular sperm retrieval for intracytoplasmic
sperm DNA fragmentation and reactive oxygen species. sperm injection or surgical reconstruc­tion.149
Excessive amounts of reactive oxygen species, if not Impaired spermatogenesis as a result of primary
counterbalanced by antioxidants, lead to oxidative stress testicular failure is the most common cause of non-
and result in protein, lipid, and DNA damage.139,140 obstructive azoospermia. Although successful testicular
Direct measurement of reactive oxygen species in sperm retrieval from men with obstructive azoospermia is
semen using chemiluminescent or fluorescent tech­ highly likely, success rates in men with non-obstructive
niques can have prognostic value in the evaluation azoospermia are substantially lower.150–152 Although sperm
of the male fertility potential,141–143 with a cutoff value of production in non-obstructive azoospermia is often
less than 102·2 RLU/s/10⁶ sperm per mL to distinguish inadequate to reach the ejaculate, the finding of hetero­
between men who are fertile and men with infertility.144 geneous patchy spermatogenesis on testicular biopsy, and
Seminal oxidation-reduction potential is a novel concept demonstrable sperm within the testes in 60% of men with

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 Seminar

Azoospermia
Karyotype and Y chromosome Vas absent
microdeletion and hormone CFTR gene mutation
evaluation
Vas present
Sperm retrieval and ICSI

Normal volume Low volume

Small testes Normal testes Post ejaculate urine

analysis

Positive Negative

FSH low FSH high Normal FSH and bulky Retrograde ejaculation Transrectal ultrasound
epididymis, with or
without testicular biopsy Abnormal Normal

Post-orgasmic urine and

ICSI
Hypogonadotropic Primary testicular failure Obstructive azoospermia Seminal vesicle aspiration Failure of emission
hypogonadism
Positive Negative

Luteinising hormone or Sperm retrieval and ICSI Scrotal exploration with Ejaculatory duct Vasal or epididymal Sperm retrieval and ICSI
FSH replacement with or without obstruction obstruction
or without sperm vasoepididymostomy
retrieval and ICSI
Transurethral resection Scrotal exploration with
of the ejaculatory ducts or without vasovasotomy,
vasoepididymostomy,
or sperm retrieval and ICSI

Figure 2: Classification of azoospermia

FSH=follicle-stimulating hormone. ICSI=intracytoplasmic sperm injection. Adapted from Agarwal and colleagues,128 by permission of the Korean Society for Sexual Medicine and Andrology.

non-obstructive azoo­ spermia, provide the rationale for considered before counselling patients about a particular
sperm retrieval in the management of non-obstructive sperm retrieval technique, as there is no clear recom­
azoospermia.150,151 Although testicular sperm aspiration mendation about which tech­nique to use.30 There has been
can be done percutaneously using local anaesthetic, low considerable debate about the role of varicocele repair
sperm retrieval renders the procedure uncommon, except in patients with non-obstructive azoospermia, because
when used in conjunction with testicular mapping.153 surgical sperm retrieval rates and outcomes for intra­
Micro­dissection testicular sperm extraction might be more cytoplasmic sperm injection have yet to be defined.156
efficient than conventional testicular sperm extraction Despite advances in reproductive medicine, sperm
(surgical sperm retrieval 52% vs 35%), on the basis of a retrieval is not successful in about 50% of men with
meta-analysis of data from 15 case-controlled studies.154 non-obstructive azoospermia, leaving these men with the
Importantly, microdissection testicular sperm extraction option of donor sperm insemination or adoption.
gets a larger quantity of sperm with less testicular tissue
removed and has the lowest com­ plication rates.151,154 Varicocele
However, a subsequent meta-analysis showed no differ­ Varicoceles are dilations of the veins of the pampiniform
ence in sperm retrieval or livebirth outcomes between plexus that drain blood from the testicles, and are present
microdissection and con­ventional testicular sperm extrac­ in 15% of healthy men and 25% of men with abnormal
tion in men with non-obstructive azoospermia.152 Similar semen analysis.30 The mechanism by which varicoceles
findings were noted in patients with Klinefelter syndrome, affect testicular function is likely to be multifactorial, but
for whom surgical sperm retrieval and livebirth outcomes the most commonly accepted theory includes a relative
were compared after either conventional or microdis­ stasis of venous blood in the pampiniform plexus, which
section testicular sperm extraction.155 Further well designed increases testicular temperature and results in elevated
RCTs are needed to clarify which technique is more reactive oxygen species.157
efficient. Several variables (eg, surgical skill, testicular The indications and surgical approach for varicocele
histology, cost, and risk of complications) should be repair have been a matter of controversy. In men with

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clinical varicoceles and abnormal semen analysis, vari­ and reducing oxidative stress in men with infertility.169
cocele repair can significantly improve semen para­ A 2019 Cochrane review that meta-analysed 61 RCTs
meters.30,33,158 Current guidelines do not recommend in 6264 men with subfertility treated with a combination
varicocelectomy in men with infertility who have a of antioxidants, reported low-quality evidence suggesting
normal semen analysis, or in men with a subclinical improvement in the proportion of clinical pregnancies
varicocele. However, varicocele repair is recommended and livebirths with antioxidant supplementation.170 The
for men with infertility who have clinical varicocele, review recognised important limitations, including low-
abnormal semen parameters, and unexplained infertility quality RCTs with serious risk of bias owing to poor
with a female partner who has healthy hormone levels reporting of randomisation methods, failure to report on
indicating good egg counts.30 A systematic review and clinical outcomes (eg, livebirths, clinical pregnancies),
meta-analysis concluded that varicocele repair improved high attrition rates, and imprecision owing to an often
livebirth outcomes following assisted reproductive tech­ low number of events and small overall sample sizes.170
nology procedures, even if semen parameters did not Further large-scale RCTs reporting clinically relevant
improve.159 outcomes are therefore necessary before an optimal
Surgical repair is the primary treatment approach for antioxidant regimen can be recommended.
varicocele, and radiological percutaneous embolisation
is a viable alternative.160 Varicocelectomy can be done Role of assisted reproductive technology
through retroperitoneal, laparoscopic, or robot-assisted The use of assisted reproductive technology has substan­
laparoscopic, microsurgical inguinal, or subinguinal tially improved the ability of couples with infertility to
approaches.161 There is no substantial difference in the have biological children. For intrauterine insemination,
success rates between the different surgical approaches, progressively motile sperm are separated from the semen
but microsurgical subinguinal varicocelectomy has been and inseminated directly into the uterine cavity during
considered the gold standard on the basis of a lower the time of ovulation. In cases of more severe male factor
risk of varicocele recurrence (0·4%) or postoperative infertility, conventional in vitro fertilisation or intra­
hydrocele forma­tion (0·44%) than other approaches.30,32 cytoplasmic sperm injection can be used. Despite the
Varicocelectomy can improve semen parameters and success of these techniques, some couples still have poor
reduce oxidative stress, potentially sparing couples from outcomes, which might result from the poor quality of
costly assisted reproductive technology procedures.32 the egg, or sperm, or both. Lee and colleagues showed
that intracytoplasmic sperm injection cycles using sperm
Idiopathic male infertility from men with severe oligoasthenoteratozoospermia and
In men with idiopathic infertility, despite completing non-obstruc­ tive azoospermia had worse outcomes in
diagnostic investigations, the cause of altered semen terms of embryo implantation and clinical pregnancy
parameters cannot be identified.35 Current treatment of than for men who were normozoospermic.171 This finding
idiopathic male infertility consists of assisted reproductive shows the importance of the paternal contribution, and
technology or empirical medical therapy, which includes the need to select the best sperm before intracytoplasmic
lifestyle improvement and hormonal or non-hormonal sperm injection. Optimal management of couples with
therapy. Lifestyle modifications (eg, weight loss, physical infertility should involve correction of sperm defects,
activity, and cessation of smoking) are important non- even for couples destined for assisted reproductive
invasive measures,30 and have been linked to improved technology. The use of testicular derived sperm is of
sperm parameters.162–165 growing importance, because testicular sperm can have
The mainstays of hormonal empirical medical therapy lower amounts of sperm DNA fragmentation compared
are selective oestrogen receptor modulators and aroma­ with ejaculated sperm.172 As such, testicular sperm
tase inhibitors. Selective oestrogen receptor modulators extraction–intracytoplasmic sperm injection can be used
(specifically clomiphene citrate) have been used off-label in men who are not azoospermic but have elevated sperm
to improve semen parameters, but there are too few DNA fragmentation and have had previous failed intra­
high-quality RCTs to prove its efficacy conclusively.166,167 cytoplasmic sperm injection cycles. However, additional
Exogenous testosterone should not be used for male evidence is needed to support this practice in the routine
infertility treatment because it inhibits spermatogenesis.168 clinical setting.
The role of oxidative stress as a cause of male infertility
is supported by elevated seminal oxidation-reduction Future therapies and challenges
potential in 80% of men with infertility.37 Because Advancements in the current era of omics technologies
oxidative stress is potentially reversible, this provides an facilitate the diagnosis and management of male
opportunity for treatment. As a result, oral antioxidants infertility at genetic, molecular, and cellular levels.
are the most commonly adopted empirical medical Next-generation sequencing technologies, such as
therapy. Although there is heterogeneity across studies disease-targeted sequencing, whole exome and genome
in the literature, a systematic review showed the efficacy sequencing, and epigenetic analysis of sperm, are pro­
of antioxidant therapy in improving semen parameters mising techniques in genetic testing.173 Next-generation

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 Seminar

sequencing technologies have enabled identification of characteristics possessed by spermatogonial stem cells
novel candidate genes associated with male infertility have opened up new perspectives in the therapeutics
conditions such as azoospermia,174 oligozoospermia,175 of male infertility.199,200 Autografting cryobanked sper­
and idiopathic male infertility.107 Discoveries on the matogonial tissue was proposed as a new strategy of
role of small RNAs and microRNAs in epigenetic fertility preservation for paediatric patients who have
regula­tions,176 and their involvement in spermatogenesis undergone gonadotoxic therapy.201 However, several
and epididymal sperm maturation, have expanded barriers, including ethical issues and the risk of trans­
current understanding of these pro­ cesses.177–180 Meta­ mitting genetic insults to the offspring during in vitro
bolic fingerprinting of seminal plasma is another culture of stem cells, must be overcome before stem cell
promising area of research, especially in cases of therapy can be used for the management and treatment
idiopathic male infertility.181 A study published in 2019 of male infertility.
found that reactive oxygen species-induced epigenetic Contributors
alterations of sperm DNA and seminal metabolic All authors wrote this Seminar and read and approved the final
profile were correlated with semen quality in men with manuscript.
infertility who were normozoospermic.182 Declaration of interests
The paradigm shift to proteomic research of male We declare no competing interests.
reproduction has revealed several pro­teins as biomarkers Acknowledgments
that are associated with various causes of male infertility, We thank Joseph Terry and Mary Reagan, Center for Medical Art and
Photography, Cleveland Clinic, for their assistance in preparing the
such as oxidative stress-mediated sperm dysfunc­tion,183,184 figures.
varicocele,176,185,186 asthenozoospermia,187,188 globozoosper­
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