11. MALE INFERTILITY

11.1. Definition and classification

Infertility is defined by the inability of a sexually active, non-contraceptive couple to achieve spontaneous pregnancy within 12 months [1608]. Primary infertility refers to couples that have never had a child and cannot achieve pregnancy after at least 12 consecutive months of sexual intercourse without using birth control methods. Secondary infertility refers to infertile couples who have been able to achieve pregnancy at least once before (with the same or different sexual partner).

In 30-40% of cases, no male-associated factor is found to explain the underlying impairment of sperm parameters and historically was referred to as idiopathic male infertility. These men present with no previous history of diseases affecting fertility and have normal findings on physical examination and endocrine, genetic and biochemical laboratory testing, although semen analysis may reveal pathological findings (see Section 10.3.2). It is now believed that idiopathic male infertility may be associated with several previously unidentified pathological factors, which include but are not limited to endocrine disruption as a result of environmental pollution, generation of reactive oxygen species (ROS)/sperm DNA damage, or genetic and epigenetic abnormalities [1609]. Unexplained male infertility is defined as infertility of unknown origin with normal sperm parameters and partner evaluation. Between 20 and 30% of couples will have unexplained infertility.

11.2. Epidemiology, aetiology, pathophysiology and risk factors

11.2.1. Introduction

About 15% of couples do not achieve pregnancy within 12 consecutive months and seek medical treatment for infertility [1610]. One in eight couples encounter problems when attempting to conceive a first child and one in six when attempting to conceive a subsequent child [1611]. In 50% of involuntarily childless couples, a male-infertility-associated factor is found, usually together with abnormal semen parameters [1608]. For this reason, in all infertile couples, the male should undergo medical evaluation by a urologist trained in male reproduction.

Male fertility can be impaired as a result of many different conditions, including [1608]:

• congenital or acquired urogenital abnormalities
• genetic abnormalities
• malignancies
• urogenital tract infections
• varicocele
• increased scrotal temperature (e.g. because of varicocele)
• endocrine disturbances
• immunological factors
• iatrogenic factors (e.g. previous scrotal surgery)
• gonadotoxic exposure (e.g. RT or chemotherapy).

Advanced paternal age has emerged as an additional risk factor associated with the progressive increase in prevalence of male factor infertility [1612-1619].

Likewise, advanced maternal age must be considered in the management of every infertile couple, and in the subsequent decisions in the diagnostic and therapeutic strategy of the male partner [1620,1621]. This should include the age and ovarian reserve of the female partner, as these parameters may guide decision-making on timing and therapeutic strategies (e.g. assisted reproductive technology [ART] vs. surgical intervention) [1612-1615]. Earlier evaluation is still a matter of debate in couples with female partners older than 35 years who have not conceived for six months, as ovarian reserve may fall [1622-1624]. Table 11.1 summarises the main male-infertility-associated factors.

Table 11.1: Male infertility causes and associated factors and percentage of distribution in 10,469 patients

11.2.2. Summary of evidence and recommendations on epidemiology and aetiology of male infertility

11.3. Diagnostic work-up

Important treatment decisions are based on the results of semen analysis, and most studies indicate semen parameters are a surrogate outcome for male fertility. A semen analysis per se cannot distinguish fertile from infertile men [1625].

The Panel concludes that a comprehensive andrological examination is always indicated in infertile couples, both if semen analysis shows abnormalities and in men with normal sperm parameters as compared with reference values [1626-1628]. Infertile men should be properly counselled and followed-up, considering their higher risk of developing malignant and non-malignant comorbid conditions later in life [1629,1630].

Focused evaluation of male patients should include a medical and reproductive history; physical examination; semen analysis – with strict adherence to World Health Organization (WHO) reference values for human semen characteristics [1631,1632]; and hormonal evaluation [1633]. Other investigations (e.g. genetic analysis and imaging) may be required depending on the clinical features and semen parameters.

11.3.1. Medical history, reproductive history and physical examination

11.3.1.a. Medical and reproductive history

Medical history should evaluate any risk factors and behavioural patterns that could affect the male partner’s fertility, such as lifestyle, family history (including testicular cancer), comorbidities (including systemic diseases: e.g. hypertension, diabetes mellitus, obesity, MetS, testicular cancer, etc.), genito-urinary infections (including STIs), history of testicular surgery, and exclude any potential known gonadotoxic medication or recreational drugs [1633].

Typical findings from the history of a patient with infertility include:

• cryptorchidism (uni- or bilateral)
• testicular torsion and trauma
• genitourinary infections
• exposure to environmental toxins
• gonadotoxic medications (anabolic drugs, chemotherapeutic agents, etc.)
• exposure to radiation or cytotoxic agents.

11.3.1.b. Physical examination

A focused physical examination is compulsory in the evaluation of every infertile male, including presence of secondary sexual characteristics. The size, texture and consistency of the testes must be evaluated. In clinical practice, testicular volume is assessed by Prader’s orchidometer [4]; orchidometry may over-estimate testicular volume compared to US assessment [1634]. There are no uniform reference values in terms of Prader’s orchidometer-derived testicular volume, due to differences in the populations studied (e.g. geographic area, nourishment, ethnicity and environmental factors) [4,1634,1635]. The mean Prader’s orchidometer-derived testis volume reported in the European general population is 20.0 ± 5.0mL [4], whereas in infertile patients it is 18.0 ± 5.0mL [4,1636-1638]. The presence of the vas deferens, fullness of epididymis and presence of a varicocele should always be determined. Likewise, palpable abnormalities of the testis, epididymis and vas deferens should be evaluated. Other physical alterations, such as abnormalities of the penis (e.g. phimosis, short frenulum, fibrotic nodules, epispadias, hypospadias, etc.), abnormal body hair distribution and gynecomastia, should also be evaluated.

Typical findings from the physical examination of a patient with characteristics suggestive for testicular deficiency include:

• abnormal secondary sexual characteristics
• abnormal testicular volume and/or consistency
• testicular masses (potentially suggestive of cancer)
• absence of testes (uni-bilaterally)
• gynaecomastia
• varicocele.

11.3.2. Semen analysis

The 6^th edition the WHO Manual for the Examination and Processing of Human Semen [1632] was published in July 2021 and comprises of three sections: i) semen examination; ii) sperm preparation and cryopreservation; and, iii) quality assessment and quality control.

Procedures for semen examination are divided:

• Basic examinations that should be performed by every laboratory, based on standardised procedures and evidence-based techniques.
• Extended analyses, which are performed by choice of the laboratory or by special request from the clinicians.
• Advanced examinations.

Basic examination [1631]

• Assessment of sperm numbers: the laboratory should not stop assessing the number of sperm at low concentrations (2 million/mL), as suggested in the 5^th edition, but report lower concentrations, noting that the errors associated with counting a small number of spermatozoa may be high. It is recognised that the total sperm numbers per ejaculate (sperm output) have more diagnostic value than sperm concentration; therefore, semen volume must be measured accurately.
• Assessment of sperm motility: the categorisation of sperm motility has reverted to fast progressively motile, slow progressively motile, non-progressively motile and immotile (grade a, b, c or d) because presence (or absence) of rapid progressive spermatozoa is recognised to be clinically important.
• Assessment of sperm morphology: the 6^th edition has recommended the Tygerberg strict criteria by sperm-adapted Papanicolaou staining.
• Assessment of vitality should not be performed in all samples, only if more than 60% of spermatozoa are immotile.

Extended examinations

This Chapter contains procedures to detect leukocytes and markers of genital tract inflammation, sperm antibodies, indices of multiple sperm defects, sequence of ejaculation, methods to detect sperm aneuploidy, semen biochemistry and sperm DNA fragmentation (SDF).

Reference ranges and limits

The lower fifth percentile of the distribution of semen analysis values from approximately 3,500 males in 12 countries who have contributed to a natural conception within 12 months of trying does not represent a limit between fertile and infertile males. For a general prediction of live birth in vivo as well as in vitro, a multiparametric interpretation of the entire male’s and partner’s reproductive potential are needed. Reference values for semen parameters are represented in Table 11.2 [1626].

More complex testing than classic semen analysis may be required in everyday clinical practice, particularly in males belonging to couples with recurrent pregnancy loss from natural conception or ART and in men with unexplained male infertility. Although definitive conclusions cannot be drawn given the heterogeneity of the studies, increased sperm DNA damage is associated with pregnancy failure [1609,1639,1640].

Table 11.2: Lower reference limits (fifth centiles and their 95% CIs) for semen characteristics

CIs = confidence intervals; MAR = mixed antiglobulin reaction; NP = non-progressive; PR = progressive (a + b motility).
* Distribution of data from the population is presented with one-sided intervals (extremes of the reference population data). The lower fifth percentile represents the level under which only results from 5% of the males in the reference population were found.

If semen analysis is normal according to WHO criteria, a single test is sufficient. If the results are abnormal on at least two tests, further andrological investigation is indicated.

None of the individual sperm parameters (e.g. concentration, morphology and motility), are diagnostic per se of infertility. According to WHO 5^th edition reference criteria, it is important to differentiate between the following [1641]:

• oligozoospermia: < 16 million sperm/mL;
• asthenozoospermia: < 32% progressive motile sperm; and
• teratozoospermia: < 4% normal forms.

According to the WHO 6^th edition, reference criteria for this subdivision is not reported, although the Panel considers this further segregation still clinically relevant in everyday clinical practice.

Often, all three anomalies occur simultaneously, which is defined as oligo-astheno-terato-zoospermia syndrome. As in azoospermia (the complete absence of spermatozoa in semen), in severe cases of oligozoospermia (spermatozoa < 5 million/mL) [1642], there is an increased incidence of obstruction of the male genital tract and genetic abnormalities. In case of azoospermia, full andrological investigation should be warranted to classify obstructive azoospermia (OA) versus non-obstructive azoospermia (NOA). A recommended method to diagnose absolute azoospermia versus cryptozoospermia is semen centrifugation at 3,000g for 15 minutes and a thorough microscopic examination by phase contrast optics at ×200 magnification of the pellet. All samples can be stained and re-examined microscopically [1632]. This is to ensure that small quantities of sperm are detected, which may be potentially used for intra-cytoplasmic sperm injection (ICSI); therefore, removing the need for surgical intervention.

Advanced examinations
Obsolete tests, such as the human oocyte and human zona pellucida binding and the hamster oocyte penetration tests, have been completely removed. Research tests include assessment of ROS and oxidative stress, membrane ion channels, acrosome reaction, sperm chromatin structure and stability, and computer-assisted sperm analysis.

Measurement of oxidative stress
Oxidative stress is considered central in male infertility by affecting sperm quality, function, and sperm integrity [1643]. Oxidative stress may lead to sperm DNA damage and poorer DNA integrity, which are associated with poor embryo development, miscarriage and infertility [1644,1645]. Spermatozoa are vulnerable to oxidative stress and have limited capacity to repair damaged DNA. Oxidative stress is generally associated with poor lifestyle (e.g. smoking) and environmental exposure, and, therefore, antioxidant regimens and lifestyle interventions may reduce the risk of DNA fragmentation and improve sperm quality [1646]. These data have not been supported by RCTs. Although ROS can be measured by various assays (e.g. chemiluminescence), no standardised testing methods for ROS are available and routine measurement of ROS testing should remain experimental until these tests are validated in RCTs [1647].

11.3.3. Measurement of Sperm DNA Fragmentation Index

Sperm DNA fragmentation, or the accumulation of single- and double-strand DNA breaks, occur in sperm, and an increase in the level of SDF has been shown to reduce the chances of natural conception [1648]. Although no studies have unequivocally and directly tested the impact of SDF on the clinical management of infertile couples, SDF is more common in infertile men and has been identified as a major contributor to male infertility, as well as poorer outcomes following ART [1649,1650], including impaired embryo development [1649], miscarriage, recurrent pregnancy loss [1639,1640,1651], and birth defects [1649]. Sperm DNA fragmentation can be increased by several factors including hormonal anomalies, varicocele, chronic infection and lifestyle factors (e.g. smoking) [1650,1652]. Repeated ejaculation separated by a three-hour time interval has been reported to significantly decrease SDF [1653]. In a cohort study of 10,000 semen samples, SDF was also increased in men in older age groups [1654].

Several assays have been described to measure SDF. It has been suggested that current methods for assessing sperm DNA integrity still do not reliably predict treatment outcomes from ART and there is controversy whether to recommend them routinely for clinical use [1650,1655,1656]. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) and the alkaline comet test (COMET) directly measure DNA damage. Conversely, sperm chromatin structure assay (SCSA) and sperm chromatic dispersion test (SCD) are indirect tools for SDF assessment. The SCSA is still the most widely studied and one of the most used techniques to detect SDF [1657,1658]. In SCSA, the number of cells with DNA damage is indicated by the DNA fragmentation index (DFI) [1659], whereas the proportion of immature sperm with defects in the histone-to-protamine transition is indicated by high DNA stainability [1660]. It is suggested that a threshold DFI of 25%, as measured with SCSA, is associated with reduced pregnancy rates via natural conception or intra-uterine insemination (IUI) [1658]. Furthermore, DFI values > 50% on SCSA are associated with poorer outcomes from IVF. The mean COMET score and scores for proportions of sperm with high or low DNA damage have been shown to be of value in diagnosing male infertility and providing additional discriminatory information for the prediction of both IVF and ICSI live births [1650].

Raised SDF in pregnancy loss or spontaneous abortion
Recurrent pregnancy loss or repeated spontaneous abortion is not uniformly defined in the literature. In a meta-analysis SDF assessed by seven SCSA studies, nine SCD studies and eight TUNEL studies was associated with recurrent pregnancy loss. Although the meta-analysis was limited by the small numbers of participants in each trial and heterogeneity in definition and inclusion criteria, clinicians should be aware of this association and opportunity to assess a possible correctable biomarker for paternal contribution to pregnancy loss [1639].

Testicular sperm in men with raised SDF in ejaculated sperm
Testicular sperm is reported to have lower levels of SDF compared to ejaculated sperm [1661]. The use of testicular sperm for ICSI is associated with potentially improved outcomes as compared with ejaculated sperm in men with high SDF [1661,1662]. Males with unexplained infertility with raised SDF may be considered for TESE after failure of ART, although they should be counselled that live-birth rates are under reported in the literature and patients must weigh up the risks of performing an invasive procedure in a potentially normozoospermic or unexplained condition. The advantages of the use of testicular sperm in men with cryptozoospermia or oligozoospermia have not yet been confirmed in large scale randomised studies [1663,1664]. A meta-analysis has suggested that TESE-ICSI may improve the outcomes from ART but there is significant heterogeneity of data and RCTs are needed to validate the use of TESE in men with raised SDF [1665].

In terms of a practical approach, urologists may offer the use of testicular sperm in patients with high SDF on a case-by-case basis and after a full discussion including reproductive specialists. Patients should be informed of the low levels of evidence for this. Testicular sperm should only be used in this setting once the common associations of SDF have been excluded, including varicoceles, modifications of dietary/lifestyle factors, treatment of accessory gland infections, and reduction of abstinence time. Additionally, any confounding female factors must also be taken into consideration before using TESE in this setting.

11.3.4. Hormonal determinations

In males with testicular deficiency, hypergonadotropic hypogonadism (primary hypogonadism) is usually present, with high levels of FSH and LH, with or without low testosterone. Generally, the levels of FSH negatively correlate with the number of spermatogonia [1666]. When spermatogonia are absent or markedly diminished, FSH level is usually elevated; when the number of spermatogonia is normal, but maturation arrest exists at the spermatocyte or spermatid level, FSH level is usually within the normal range [1666]. However, for patients undergoing TESE, FSH levels do not accurately predict the presence of spermatogenesis, as males with maturation arrest on histology can have both normal FSH and testicular volume [1667,1668]. Furthermore, males with NOA and high levels of FSH may still harbour focal areas of spermatogenesis at the time of TESE or microdissection TESE (mTESE) [1668,1669]. Despite the need for current findings to be confirmed, growing data suggest that lower preoperative serum anti-Müllerian hormone (AMH) levels are associated with a higher likelihood of positive sperm retrieval in men undergoing mTESE [1670,1671].

11.3.5. Genetic testing

All urologists working in andrology must understand the genetic abnormalities most commonly associated with infertility, so that they can provide correct advice to couples seeking fertility treatment. Current routine clinical practice in genetic testing is based on the screening of genomic DNA from peripheral blood samples. Screening of chromosomal anomalies in spermatozoa (sperm aneuploidy) and preimplantation genetic testing are also feasible and indicated in selected cases (e.g. recurrent miscarriage) [1672-1678].

11.3.5.a. Chromosomal abnormalities

Chromosomal abnormalities can be numerical (e.g. trisomy) or structural (e.g. inversions or translocations). In a survey of pooled data from 11 publications, including 9,766 infertile males, the incidence of chromosomal abnormalities was 5.8% [1679]. Of these, sex chromosome abnormalities accounted for 4.2% and autosomal abnormalities for 1.5%. In comparison, the incidence of abnormalities was 0.38% in pooled data from three series, with a total of 94,465 new-born male infants, of whom 131 (0.14%) had sex chromosomal abnormalities and 232 (0.25%) autosomal abnormalities [1679]. The frequency of chromosomal abnormalities increases as testicular deficiency becomes more severe. Patients with sperm count < 5 million/mL already show a ten-fold higher incidence (4%) of mainly autosomal structural abnormalities compared to the general population [1680,1681]. Males with NOA are at highest risk, especially for sex chromosomal anomalies (e.g. Klinefelter syndrome) [1682,1683].

Based on the frequencies of chromosomal aberrations in patients with different sperm concentration, karyotype analysis is currently indicated in males with azoospermia or oligozoospermia (spermatozoa < 10 million/mL) [1681]. The clinical value of spermatozoa < 10 million/mL remains a valid threshold until further studies evaluating the cost-effectiveness, including the costs of TRAEs due to chromosomal abnormalities (e.g. miscarriages and congenital anomalies), are conducted [1684].

11.3.5.a.1. Sex chromosome abnormalities (Klinefelter syndrome and variants [47, XXY; 46, XY/47, XX mosaicism])

Klinefelter syndrome is the most common sex chromosomal abnormality [1685]. Adult males with Klinefelter syndrome usually have small firm testes along with features of primary hypogonadism. The phenotype is the result of a combination between genetic, hormonal and age-related factors [12]. The phenotype varies from that of a normally virilised male to one with the stigmata of androgen deficiency. In most cases, infertility and reduced testicular volume are the only clinical features that can be detected, although neuro-psychological features, learning or language processing disabilities are often present. Leydig cell function is also commonly impaired in males with Klinefelter syndrome and thus testosterone deficiency is more frequently observed than in the general population [1686], although rarely observed during the peri-pubertal period which usually occurs in a normal manner [12,1687]. Rarely, more pronounced signs and symptoms of hypogonadism can be present, along with congenital abnormalities including heart and renal problems [1688].

The presence of germ cells and sperm production are variable in males with Klinefelter syndrome and are more frequently observed in mosaicism 46, XY/47, XXY. In patients with azoospermia, TESE or mTESE are therapeutic options, as spermatozoa can be recovered in up to 50% of cases [1689,1690]. Although the data are not unique [1690], there is some evidence that TESE or mTESE yields higher sperm recovery rates when performed at a younger age, but an age threshold cannot be provided [1682,1691].

Klinefelter syndrome is associated with several general health problems and appropriate medical follow-up is advised [13,1692,1693]. Testosterone therapy may be considered if testosterone levels are in the hypogonadal range when fertility issues have been addressed [15]. Males with Klinefelter syndrome are at higher risk of metabolic and CVD, including venous thromboembolism and diabetes, particularly when starting testosterone therapy [1694]. A higher risk of haematological malignancies has been reported in males with Klinefelter syndrome [13].

Testicular sperm extraction in peri-pubertal or prepubertal boys with Klinefelter syndrome for the purpose of cryopreservation of testicular spermatogonial stem cells is currently considered experimental and should only be undertaken within a research setting [1695]. The same applies to sperm retrieval in older boys who have not yet considered their future fertility potential [1696].

11.3.5.a.2. Autosomal abnormalities

Genetic counselling should be offered to all couples seeking fertility treatment (including IVF/ICSI) when the male partner has an autosomal karyotype abnormality. The most common autosomal karyotype abnormalities are Robertsonian translocations, reciprocal translocations, paracentric inversions, and marker chromosomes. It is important to look for these structural chromosomal anomalies because there is an increased associated risk of aneuploidy or unbalanced chromosomal complements in the foetus. When IVF/ICSI is carried out for males with translocations, preimplantation genetic testing or amniocentesis should be performed [1697,1698].

11.3.5.b. Cystic fibrosis gene mutations

Cystic fibrosis (CF) is an autosomal-recessive disorder [1699]. It is the most common genetic disease of Caucasians; 4% are carriers of gene mutations involving the CF transmembrane conductance regulator (CFTR) gene located on chromosome 7p. It encodes a membrane protein that functions as an ion channel and influences the formation of the ejaculatory duct, seminal vesicle, vas deferens and distal two-thirds of the epididymis. Approximately 2,000 CFTR mutations have been identified, and any CFTR alteration may lead to congenital bilateral absence of the vas deferens (CBAVD). However, only those with homozygous mutations exhibit CF disease [1700]. Congenital bilateral absence of the vas deferens is a rare reason of male factor infertility, which is found in 1% of infertile males and in up to 6% of males with OA [1701]. Clinical diagnosis of absent vasa is easy to miss and all males with azoospermia should be carefully examined to exclude CBAVD, particularly those semen volume < 1.0mL and acidic pH < 7.0 [1702-1704]. In patients with CBAVD-only or CF, epididymal sperm aspiration (microsurgical [MESA] or percutaneous [PESA]), TESA, or TESE in combination with ICSI, can be used to achieve pregnancy. However, higher sperm quality, easier sperm retrieval, and better ICSI outcomes are associated with CBAVD-only patients as compared with CF patients [1700].

The most frequently found mutations are F508, R117H and W1282X (according to their traditional definitions), but their frequency and the presence of other mutations largely depend on the ethnicity of the patient [1705,1706]. Given the functional relevance of a DNA variant (the 5T allele) in a non-coding region of CFTR [1707], it is now considered a mild CFTR-mutation rather than a polymorphism, and it should be analysed in each CBAVD patient. Males with CBAVD often have mild clinical stigmata of CF (e.g. history of chest infections). When a male has CBAVD, it is important to test their partner for CF mutations. If the female partner is found to be a carrier of CFTR mutations, the couple must carefully consider whether to proceed with ICSI, as the risk of having a child with CF or CBAVD is 50%, depending on the type of mutations carried by the parents. If the female partner is negative for known mutations, the risk of being a carrier of unknown mutations is ~0.4% [1708].

11.3.5.b.1. Unilateral or bilateral absence/abnormality of the vas and renal anomalies

Congenital unilateral absence of the vas deferens (CUAVD) is usually associated with ipsilateral absence of the kidney and probably has a different genetic causation [1709]. Cystic fibrosis transmembrane conductance regulator gene mutation screening is indicated in males with unilateral absence of the vas deferens with normal kidneys. The prevalence of renal anomalies is rare for patients who have CBAVD and CFTR mutations [1710]. Abdominal US should be undertaken both in unilateral and bilateral absence of vas deferens without CFTR mutations. Findings may range from CUAVD with ipsilateral absence of the kidney, to bilateral vessel and renal abnormalities, such as pelvic kidney [1711].

11.3.5.c. Y microdeletions – partial and complete

Microdeletions on the Y-chromosome are termed AZFa, AZFb and AZFc deletions [1712]. Clinically relevant deletions remove partially, or in most cases completely, one or more of the AZF regions, and are the most frequent molecular genetic cause of severe oligozoospermia and azoospermia [1713]. In each AZF region, there are several spermatogenesis candidate genes [1714].

11.3.5.c.1. Clinical implications of Y microdeletions

The clinical significance of Yq microdeletions can be summarised as follows:

• They are not found in normozoospermic males, proving there is a clear cut cause-and-effect relationship between Y-deletions and spermatogenic failure [1715].
• The highest frequency of Y-deletions is found in azoospermic males (8-12%), followed by oligozoospermic (3-7%) males [1716,1717].
• Deletions are extremely rare with a sperm concentration > 5 million/mL (~0.7%) [1718].
• AZFc deletions are most common (65-70%), followed by Y-deletions of the AZFb and AZFb+c or AZFa+b+c regions (25-30%). AZFa region deletions are rare (5%) [1719].
• Complete deletion of the AZFa region is associated with severe testicular phenotype (Sertoli cell-only syndrome [SCOS]), while complete deletions of the AZFb region is associated with spermatogenic arrest.
• Complete deletions that include the AZFa and AZFb regions are of poor prognostic significance for retrieving sperm with TESE. Therefore, TESE should not be attempted in these patients [1720,1721].
• Deletions of the AZFc region causes a variable phenotype ranging from azoospermia to oligozoospermia.
• Testicular sperm can be found in 50-75% of men with AZFc microdeletions [1720-1722].
• Males with AZFc microdeletions who are oligo-azoospermic or in whom sperm is found at the time of TESE must be counselled that any male offspring will inherit the deletion.
• Classical (complete) AZF deletions do not confer a risk for cryptorchidism or testicular cancer [1718,1723].
• A meta-analysis indicated that the fertilisation rate but not live birth rate or male birth rate after ICSI in males with Y chromosome microdeletions was significantly decreased, compared to ICSI controls [1724]. The quality of the included studies was limited by sample size, but counselling of patients may be refined.

The specificity and genotype/phenotype correlation reported above means that Y-deletion analysis has both a diagnostic and prognostic value for testicular sperm retrieval [1723].

11.3.5.c.1.a. Testing for Y microdeletions

Historically, indications for AZF deletion screening are based on sperm count and include azoospermia and severe oligozoospermia (spermatozoa count < 5 million/mL). A meta-analysis assessing the prevalence of microdeletions on the Y chromosome in oligo-zoospermic males in 37 European and North American studies (n = 12,492 oligo-zoospermic males) showed that the majority of microdeletions occurred in males with sperm concentrations ≤ 1 million sperm/mL, with < 1% identified in males with > 1 million sperm/mL [1718]. In this context, while an absolute threshold for clinical testing cannot be universally given, patients may be offered testing if sperm counts are < 5 million sperm/mL but must be tested if ≤1 million sperm/mL.

With the contribution of the European Academy of Andrology (EAA) Guidelines and the European Molecular Genetics Quality Network external quality control programme (http://www.emqn.org/emqn/), Yq testing has become more reliable in different routine genetic laboratories. The EAA Guidelines provide a set of primers capable of detecting > 95% of clinically relevant deletions [1725].

11.3.5.d. Exome sequencing

The diagnostic yield of exome sequencing in males with NOA is increasing rapidly, revealing monogenic causes of male infertility, genetic variants that may impact the function of genes implicated in male factor infertility, and candidate genes [1726,1727]. The diagnostic yield in patients with presumed idiopathic spermatogenic failure may also increase significantly in time [1728]. Case control series of exome sequencing in males with asthenoteratozoospermia add significant genetic variants that may add to panels for ciliopathy/sperm flagellum phenotype. Catalogues of human genes associated with specific sperm morphology and dysfunction characteristics underline the increase in knowledge and the importance of genotyping for specific severe male factor infertility conditions [1729,1730]. These findings suggest that the introduction of exome sequencing in NOA will improve diagnosis and possible management of patients, although validation in cohorts with different geographical and ethnic backgrounds are needed before recommendations can be made for standard clinical.

In addition, SNPs in the genetic makeup of the methylenetetrahyudrofolate reductase gene have been identified as a potential genetic vulnerability factor to male factor infertility in predominantly Asian population cohorts [1731]. The possible protective role of SNPs associated with oxidative stress pathway genes and seminal plasma antioxidant capacity are under investigation [1732]. A meta-analysis reported on the possible role of XRCC1 gene polymorphism as a risk factor for male factor infertility, although gene-environment interaction could not be evaluated [1733].

11.3.6. Imaging in infertile men

In addition to physical examination, a scrotal US may be helpful in: (i) measuring testicular volume; (ii) assessing testicular anatomy and structure in terms of US patterns, thus detecting signs of testicular dysgenesis often related to impaired spermatogenesis (e.g. non-homogeneous testicular architecture and microcalcifications) and testicular tumours; and, (iii) finding indirect signs of obstruction (e.g. dilatation of rete testis, enlarged epididymis with cystic lesions, or absent vas deferens) [1634]. In clinical practice, Prader’s orchidometer-derived testicular volume is considered a reliable surrogate of US-measured testicular volume, as it is easier to perform and cost-effective [4]. Nevertheless, scrotal US has a relevant role in testicular volume assessment when Prader’s orchidometer is unreliable (e.g. large hydrocele, inguinal testis, epididymal enlargement/fibrosis, thickened scrotal skin, small testis, or where the epididymis is large in comparison to the total testicular volume [4,1634]). Ultrasound patterns of testicular inhomogeneity [1734,1735] is usually associated with ageing, although it has also been reported in association with testicular atrophy and fibrosis [1634]. When testicular inhomogeneity is detected, a diagnostic testicular biopsy is not recommended [1734,1735].

11.3.6.a. Scrotal ultrasound

Scrotal US is widely used in everyday clinical practice in patients with oligozoospermia or azoospermia, as infertility is an additional risk factor for testicular cancer [1736,1737]. It can be used in the diagnosis of several diseases causing infertility, including OA (see section 11.4) and testicular neoplasms, and to provide specific radiological metrics, such as venous diameter and reflux for the diagnosis of varicoceles.

11.3.6.a.1. Testicular neoplasms

Males with infertility had an increased risk of testicular cancer compared to fertile controls (pooled OR = 1.91; 95% CI: 1.52–2.42) [1738]. When infertility was refined according to individual semen parameters, oligozoospermic males had an increased risk of cancer compared with fertile control subjects (HR: 11.9) [1739]. In a systematic review, infertile men with testicular microcalcification (TM) were found to have a ~18-fold higher prevalence of testicular cancer [1740]. The utility of US as a routine screening tool in men with infertility to detect testicular cancer remains a matter of debate [1736,1737]. These testicular lesions are difficult to characterise as being benign or malignant based only upon US criteria, including size, vascularity and echogenicity.

A dichotomous cut-off of certainty in terms of lesion size that may distinguish benign from malignant testicular masses is currently not available. A systematic review and meta-analysis were conducted by the EAU Testicular Cancer Guidelines Panel and this Panel to determine which scrotal US or MRI characteristics can predict benign or malignant disease in pre- or post-pubertal males with indeterminate testicular masses [1741]. Benign and malignant masses were classified using the reported reference test: that is, histopathology, or 12 months progression-free radiological surveillance. A total of 32 studies were identified, including 1,692 masses of which 28 studies and 1,550 masses reported scrotal US features, and four studies and 142 masses reported MRI features. Meta-analysis of different scrotal US (B-mode) values in post-pubertal males demonstrated that a size of ≤ 0.5cm had a significantly lower OR of malignancy compared to masses of > 0.5cm (p < 0.001). Comparison of masses of 0.6-1.0cm and masses of > 1.5cm also demonstrated a significantly lower OR of malignancy (p = 0.04). There was no significant difference between masses of 0.6-1.0cm and 1.1-1.5cm. Scrotal US in post-pubertal men also had a significantly lower OR of malignancy for heterogenous masses compared to homogenous masses (p = 0.04), hyperechogenic versus hypoechogenic masses (p < 0.01), normal versus increased enhancement (p < 0.01), and peripheral versus central vascularity (p < 0.01), respectively. There were limited data on pre-pubertal scrotal US, pre-pubertal MRI and post-pubertal MRI [1741].

Small hypoechoic/hyperechoic areas may be diagnosed as intra-testicular cysts, focal Leydig cell hyperplasia, fibrosis and focal testicular inhomogeneity after previous pathological conditions. Hence, they require careful periodic US assessment and follow-up, especially if additional risk factors for malignancy are present (i.e. infertility, bilateral TM, history of cryptorchidism, testicular atrophy, inhomogeneous parenchyma, history of testicular tumour, history of/contralateral tumour) [1634].

In the case of interval growth of a lesion and/or the presence of additional risk factors for malignancy, testicular biopsy/surgery may be considered, although the evidence for adopting such a management policy is limited. In 145 men referred for azoospermia who underwent US before testicular biopsy, 49 (34%) had a focal US abnormality; a hypoechoic lesion was found in 20 patients (14%); hyperechoic lesions were seen in ten patients (7%); and a heterogeneous appearance of the testicular parenchyma was seen in 19 patients (13%). Of 18 evaluable patients, 11 had lesions < 5mm, all of which were confirmed to be benign. All other patients with hyperechoic or heterogeneous areas on US with subsequent tissue diagnoses were found to have benign lesions. Men with severe infertility who have incidental testicular lesions, negative tumour markers, and lesions < 5mm may be observed with serial scrotal US, while enlarging or larger lesions can be considered for histological biopsy [1742]. Other studies have suggested that if a testicular lesion is hyperechoic and non-vascular on colour Doppler US and is associated with negative tumour markers, the likelihood of malignancy is low and regular testicular surveillance can be considered as an alternative to radical surgery. In contrast, hypoechoic and vascular lesions are more likely to be malignant [1743-1747]. Most lesions cannot be characterised by US (indeterminate), and histology remains the only certain diagnostic tool. A multidisciplinary team discussion, including invasive diagnostic modalities, should therefore be considered in these patients.

The role of US-guided intra-operative frozen section analysis in the diagnosis of testicular cancer in indeterminate lesions can be considered, and several authors have proposed its value in the intra-operative diagnosis of indeterminate testicular lesions [1748]. Although the default treatment after patient counselling and multidisciplinary team discussion may be radical orchidectomy, an US-guided biopsy with intra-operative frozen section analysis may be offered as an alternative to radical orchidectomy and potentially obviate the need for removal of the testis in a patient seeking fertility treatment. In men with azoospermia, a concurrent TESE with sperm banking can also be performed at the time of surgical intervention.

11.3.6.a.2. Varicocele

The clinical management of varicocele is still mainly based on physical examination; nevertheless, scrotal colour Doppler US is useful in assessing venous reflux and diameter when palpation is unreliable and/or in detecting recurrence/persistence after surgery [1634]. Definitive evidence of reflux and venous diameter may be utilised in the decision to treat (see Section 11.4.3.1 and 11.4.3.2).

11.3.6.a.3. Other

Scrotal US can detect changes in the proximal part of the seminal tract due to obstruction. Especially for CBAVD patients, scrotal US is a favourable option to detect the abnormal appearance of the epididymis. Given that, three types of epididymal findings are described in CBAVD patients: tubular ectasia (honeycomb appearance), meshwork pattern, and complete or partial absence of the epididymis [1749,1750].

11.3.6.b. Transrectal ultrasound

For patients with a low seminal volume, acidic pH and severe oligozoospermia or azoospermia in whom obstruction is suspected, scrotal US and TRUS are of clinical value in detecting CBAVD and presence or absence of the epididymis and/or seminal vesicles (e.g. abnormalities/agenesis). Likewise, TRUS has an important role in assessing OA secondary to CBAVD or anomalies related to the obstruction of the ejaculatory ducts, such as ejaculatory duct cysts, seminal vesicle dilatation or hypoplasia/atrophy, although RE should be excluded as a differential diagnosis [1634,1751].

11.3.7. Summary of evidence and recommendations for the diagnostic work-up of male infertilityhormonal determinations

11.4. Special conditions and relevant clinical entities

11.4.1. Cryptorchidism

Cryptorchidism is the most common congenital abnormality of the male genitalia; at one year of age nearly 1% of all full-term male infants have cryptorchidism [1752]. Approximately 30% of undescended testes are non-palpable and may be located within the abdominal cavity. These Guidelines will only deal with management of cryptorchidism in adults.

11.4.1.a. Classification

The classification of cryptorchidism is based on the duration of the condition and the anatomical position of the testes. If the undescended testis has been identified from birth, it is termed congenital, while diagnosis of acquired cryptorchidism refers to men in whom testes were situated within the scrotum. Cryptorchidism is categorised as bilateral or unilateral and by the location of the testes (inguinal, intra-abdominal or ectopic).

Studies have shown that treatment of congenital and acquired cryptorchidism results in similar hormonal profiles, semen analysis and testicular volumes [1753,1754]. However, testicular volume and hormonal function are reduced in adults treated for congenital bilateral cryptorchidism compared to unilateral cryptorchidism [1755].

11.4.1.a.1. Aetiology and pathophysiology

It has been postulated that cryptorchidism may be a part of the so-called testicular dysgenesis syndrome (TDS), which is a developmental disorder of the gonads caused by environmental and/or genetic influences early in pregnancy, including exposure to endocrine disrupting chemicals. Besides cryptorchidism, TDS includes hypospadias, reduced fertility, increased risk of malignancy, and Leydig/Sertoli cell dysfunction [1756]. Cryptorchidism has also been linked with maternal gestational smoking [1757] and premature birth [1758].

11.4.1.a.2. Pathophysiological effects in maldescended testes

11.4.1.a.2.a. Degeneration of germ cells

The degeneration of germ cells in maldescended testes is apparent even after the first year of life, and varies depending on the position of the testes [1759]. During the second year, the number of germ cells declines further. Treatment between the age of six to 18 months is therefore recommended to conserve spermatogonial stem cells, safe guard future spermatogenesis and hormone production, and decrease the risk of tumours [1760]. Surgical treatment is the most effective. Meta-analyses on the use of medical treatment with GnRH and hCG have demonstrated poor success rates [1761,1762]. It has been reported that hCG treatment may be harmful to future spermatogenesis [1763]. The EAU Guidelines on Paediatric Urology do not recommend endocrine treatment to achieve testicular descent on a routine basis, but endocrine treatment with GnRH analogues in boys with bilateral undescended testis is recommended [1764].

There is increasing evidence to suggest that, in unilateral undescended testis, the contralateral normal descended testis may also have structural abnormalities, including smaller volume, softer consistency and reduced markers of future fertility potential (spermatogonia/tubule ratio and dark spermatogonia) [1753,1765]. This implies that unilateral cryptorchidism may affect the contralateral testis and patients and parents should be counselled appropriately.

11.4.1.a.2.b. Relationship with fertility

Semen parameters are often impaired in men with a history of cryptorchidism [1766]. Early surgical treatment may have a positive effect on subsequent fertility [1767]. In males with a history of unilateral cryptorchidism, paternity is almost equal (89.7%) to that in males without cryptorchidism (93.7%). Outcome studies for untreated bilateral undescended testes revealed that 100% are oligospermic and 75% azoospermic. Among those successfully treated for bilateral undescended testes, 75% still remain oligospermic and 42% azoospermic [1768]. It is also important to screen for hypogonadism, as this is a potential long-term sequelae of cryptorchidism and could contribute to impaired fertility and potential problems, such as testosterone deficiency and MetS [1769].

11.4.1.a.2.c. Germ cell tumours

As a component of TDS, cryptorchidism is a risk factor for testicular cancer and is associated with TM and intratubular germ cell neoplasia in situ (GCNIS), formerly known as carcinoma in situ of the testes. In 5-10% of testicular cancers, there is a history of cryptorchidism [1770]. The risk of a germ cell tumour (GCT) is 3.6-7.4 times higher than in the general population and 2-6% of men with a history of cryptorchidism will develop a testicular tumour [1752]. Orchidopexy performed before the onset of puberty has been reported to decrease the risk of testicular cancer [1771]. However, there is evidence to suggest that even men who undergo early orchidopexy still harbour a higher risk of testicular cancer than males without cryptorchidism [1772]. Therefore, all men with a history of cryptorchidism should be warned that they are at increased risk of developing testicular cancer and should perform regular testicular self-examination [1773].

11.4.1.b. Disease management

11.4.1.b.1. Hormonal treatment

Human chorionic gonadotropin or GnRH is not recommended for the treatment of cryptorchidism in adulthood.

11.4.1.b.2. Surgical treatment

In adolescence, removal of an intra-abdominal testis (with a normal contralateral testis) can be recommended, because of the risk of malignancy [1774]. In adults with a palpable undescended testis and a normal functioning contralateral testis (i.e. biochemically eugonadal), an orchidectomy may be offered, as there is evidence that the undescended testis confers a higher risk of GCNIS and future development of a GCT [1775] and regular testicular self-examination is not an option in these patients. In patients with unilateral undescended testis and impaired testicular function on the contralateral testis demonstrated by biochemical hypogonadism and/or impaired sperm production (infertility), an orchidopexy may be offered to preserve androgen production and fertility. Based on Panel consensus, multiple biopsies of the unilateral undescended testis are recommended at the time of orchidopexy to exclude intra-testicular GCNIS as a prognostic indicator of future development of GCT. As indicated above, the correction of bilateral cryptorchidism, even in adulthood, can lead to sperm production in previously azoospermic men and therefore may be considered in these patients or in patients who place a high value on fertility preservation [1776]. Vascular damage is the most severe complication of orchidopexy and can cause testicular atrophy in 1-2% of cases. In males with non-palpable testes, the postoperative atrophy rate was 12% in cases with long vascular pedicles that enabled scrotal positioning. Postoperative atrophy in staged orchidopexy has been reported in up to 40% of patients [1777]. At the time of orchidectomy in the treatment of GCT, biopsy of the contralateral testis should be offered to patients at high risk for GCNIS (i.e. history of cryptorchidism, < 12mL testicular volume, poor spermatogenesis) [1778].

11.4.1.c. Summary of evidence recommendations for cryptorchidism

11.4.2. Germ cell malignancy and male infertility

Testicular germ cell tumour (TGCT) is the most common malignancy in Caucasian men aged 15-40 years, and affects approximately 1% of sub-fertile men [1779]. The lifetime risk of TGCT varies among ethnic groups and countries. The highest annual incidence of TGCT occurs in Caucasians and varies from 10/100,000 (e.g. Denmark and Norway) to 2/100,000 (e.g. Finland and Baltic countries). Generally, seminomas and non-seminomas are preceded by GCNIS, and untreated GCNIS eventually progresses to invasive cancer [1780-1782]. There has been a general decline in male reproductive health and an increase in testicular cancer in Western countries [1783,1784]. In almost all countries with reliable cancer registries, the incidence of testicular cancer has increased [1723,1785]. This has been postulated to be related to TDS, which is a developmental disorder of the testes caused by environmental and/or genetic influences in pregnancy. Endocrine disrupting chemicals have also been associated with sexual dysfunction [1786] and abnormal semen parameters [1787]. These cancers arise from premalignant gonocytes or GCNIS [1788]. Testicular microcalcification, seen on US, can be associated with TGCT and GCNIS of the testes [1740,1789,1790].

11.4.2.a. Testicular germ cell cancer and reproductive function

All males with cancer must be offered sperm cryopreservation prior to the therapeutic use of gonadotoxic agents or ablative surgery that may impair spermatogenesis or ejaculation (i.e. chemotherapy, RT or retroperitoneal surgery) [1791,1792].

Males with TGCT have decreased semen quality, even before cancer treatment. Azoospermia has been observed in 24% of males with TGCT [1793] and oligospermia in 50% [1794]. Given that the average ten-year survival rate for testicular cancer is 98% and it is the most common cancer in males of reproductive potential, it is mandatory to include counselling regarding fertility preservation prior to any gonadotoxic treatment [1794,1795]. All patients should be offered ejaculated semen preservation as the most cost-effective strategy for fertility preservation, or sperm extracted surgically (e.g. c/mTESE). Treatment for TGCT, including orchidectomy owing to the risk of a non-functioning remaining testicle, may have a negative impact on reproductive function [1793]. If a male is shown to be azoospermic or severely oligozoospermic, sperm cryopreservation is recommended before to orchidectomy to allow the option of concomitant TESE before any further potential gonadotoxic/ablative surgery [1794]. The surgical principles in onco-TESE do not differ from the technique of TESE for men with infertility (e.g. NOA) [1796,1797]. In this context, it is recommended to organise cryopreservation care delivery networks that enable referral to a urologist adept in TESE.

Rates of under-utilisation of semen analysis and sperm cryopreservation have been reported to be high; resulting in the failure to identify azoospermic or severely oligozoospermic patients at diagnosis who may benefit from advanced fertility-preserving procedures, such as onco-TESE. The argument that performing cryopreservation prior to orchidectomy may delay subsequent treatment is not supported by contemporary clinical practice; adverse impact on survival has not been investigated. Orchidectomy should not be unduly delayed if there are no facilities for cryopreservation or there is a potential delay in treatment.

Since chemotherapy and RT are teratogenic, contraception must be used during treatment and for at least six months after completion [1798]. Both chemotherapy and RT can impair fertility. Long-term infertility is rare after RT and dose-cumulative-dependent with chemotherapy. Treatment of TGCT can result in additional impairment of semen quality [1799] and increased sperm aneuploidy up to two years following gonadotoxic therapy [1800]. Spermatogenesis usually recovers one to four years after chemotherapy [82]. Chemotherapy is also associated with DNA damage and an increased SDF rate [1801]. However, sperm aneuploidy levels often decline to pre-treatment levels 18-24 months after treatment [1800]. Several studies reviewing the offspring of cancer survivors have not shown a significant increased risk of genetic abnormalities in the context of previous chemotherapy and RT [1802].

In addition to spermatogenic failure, patients with TGCT have Leydig cell dysfunction, even in the contralateral testis [1803]. The measurement of pre-treatment levels of testosterone, SHBG, LH and oestradiol may help to stratify those patients at increased risk of hypogonadism and provide a baseline for post-treatment hypogonadism. The risk of hypogonadism may be increased in men treated for TGCT. Likewise, the risk of hypogonadism is increased in the survivors of testicular cancer and serum testosterone levels should be evaluated during the management of these patients [1804]. However, this risk is greatest at 6-12 months post-treatment and suggests that there may be some improvement in Leydig cell function after treatment. Therefore, it is reasonable to delay initiation of testosterone therapy until the patient shows continuous signs or symptoms of testosterone deficiency [1780]. The risk of low libido and ED is also increased in TGCT patients [1805]. Patients treated for TGCT are also at increased risk of CVD [1801]. Therefore, patients may require a multidisciplinary therapy approach and, in this context, survivorship programmes incorporating a holistic view of patients considering psychological, medical and social needs could be beneficial. In patients who place a high value on fertility potential, the use of testosterone therapy in men with symptoms suggestive for TDS needs to be balanced with worsening spermatogenesis. In these patients, consideration can be given to the use of SERMS (e.g. clomiphene) or gonadotrophin analogues (e.g. hCG), although these are off-label treatments in this clinical setting.

11.4.2.b. Testicular microcalcification

Microcalcification inside the testicular parenchyma can be found in 0.6-9% of men referred for testicular US [1806,1807]. Although the true incidence of TM in the general population is unknown, it is most probably rare. Ultrasound findings of TM have been seen in men with TGCT, cryptorchidism, infertility, testicular torsion and atrophy, Klinefelter syndrome, hypogonadism, disorders of sex development, and varicocele [1757]. The incidence reported seems to be higher with high-frequency US machines [1808]. The relationship between TM and infertility is unclear, but may relate to testicular dysgenesis, with degenerate cells being sloughed inside an obstructed seminiferous tubule and failure of the Sertoli cells to phagocytose the debris. Subsequently, calcification with hydroxyapatite occurs. Testicular microcalcification is found in testes at risk of malignant development, with a reported incidence of TM in men with TGCT of 6-46% [1809-1811]. A systematic review and meta-analysis of case-control studies indicated that the presence of TM is associated with a ~18-fold higher odds ratio for testicular cancer in infertile men (pooled OR: 18.11; 95% CI: 8.09; 40.55; p < 0.0001) [1740].

Testicular microcalcification should therefore be considered pre-malignant in this setting and patients counselled accordingly. Testicular biopsies from men with TM have found a higher prevalence of GCNIS, especially in those with bilateral microcalcifications [1812]. However, TM can also occur in benign testicular conditions and the microcalcification itself is not malignant. The association of TM and TGCT is controversial and the challenge is to identify those men at risk of harbouring GCNIS and future risk of TGCT. Further investigation of the association between TM and GCNIS requires testicular biopsies in large series of men without signs of TGCT with or without risk factors for TGCT. Clinicians and patients should be reassured that testicular cancer does not develop in most men with asymptomatic TM [1790]. Men potentially at high-risk of harbouring or developing GCNIS include those with infertility, atrophic testes, undescended testes, history of TGCT, and contralateral TM, and it has been suggested that men with these risk factors could be offered testicular biopsy [1784,1789]. Patients with a history of TGCT and TM in the contralateral testis, as well as subfertile patients, have an increased risk of GCNIS [1790], although only a few studies suggest a further increase in GCNIS when TM occurs in the context of cryptorchidism [1784,1807,1813]. A useful algorithm has been proposed [1784] to stratifying those patients at increased risk of GCNIS who may benefit from testicular biopsy. However, when undertaking a biopsy in this setting, the full risks and complications of adopting this strategy must be explained to the patient.

Decastro et al., [1814] suggested that testicular cancer would not develop in most men with TM (98.4%) during a five-year follow-up. As such, an extensive screening programme would only benefit men at significant risk. It is prudent to advise patients with TM and risk factors for testicular cancer to at least undergo regular testicular examination. It has been suggested that these patients could also be offered annual physical examination by a urologist and US follow-up, although follow-up protocols may be difficult to implement in this invariably young cohort of patients [1757]. As testicular atrophy and infertility have an association with testicular cancer, some authors recommend biopsy or follow-up US if TM is seen [1784]. However, most patients who are azoospermic will be undergoing therapeutic biopsy (i.e. for the specific purpose of sperm retrieval), allowing a definitive diagnosis to be made. There is a lack of evidence demonstrating a higher prevalence of testicular cancer in patients with both TM and testicular atrophy. In patients with incidental TM, the risk of GCNIS is low and a logical approach is to instruct patients to perform regular testicular self-examination.

11.4.2.c. Summary of evidence and recommendations for germ cell malignancy and testicular microcalcification

11.4.3. Varicocele

Varicocele is a common congenital abnormality, that may be associated with the following andrological conditions:

• failure of ipsilateral testicular growth and development
• male sub-fertility
• symptoms of pain and discomfort
• hypogonadism.

11.4.3.a. Classification

The following classification of varicocele [1608] is useful in clinical practice:

• Subclinical: not palpable or visible at rest or during Valsalva manoeuvre but can be shown by special tests (Doppler US).
• Grade 1: palpable during Valsalva manoeuvre.
• Grade 2: palpable at rest.
• Grade 3: visible and palpable at rest.

11.4.3.b. Diagnostic evaluation

The diagnosis of varicocele is made by physical examination and scrotal Doppler US is indicated if physical examination is inconclusive or semen analysis remains unsatisfactory after varicocele repair to identify persistent and recurrent varicocele [1608,1815]. A maximum venous diameter of > 3mm in the upright position and during the Valsalva manoeuvre and venous reflux with a duration > two seconds correlate with the presence of a clinically significant varicocele [1816,1817]. To calculate testicular volume, Lambert’s formula (V = L x W x H x 0.71) should be used, as it correlates well with testicular function in patients with infertility and/or varicocele [1818]. Patients with isolated, clinical right varicocele should be examined further for abdominal, retroperitoneal and congenital pathology and anomalies.

11.4.3.c. Basic considerations

11.4.3.c.1. Varicocele and fertility

Varicocele is present in almost 15% of the normal male population, in 25% of men with abnormal semen analysis and in 35-40% of men presenting with infertility [1608,1819-1821]. The incidence of varicocele among men with primary infertility is estimated at 35–44%, whereas the incidence in men with secondary infertility is 45–81% [1608,1820,1821]. Worsening semen parameters are associated with a higher grade of varicocele and age [1820,1822].

The exact association between reduced male fertility and varicocele is unknown. Increased scrotal temperature, hypoxia and reflux of toxic metabolites can cause testicular dysfunction and infertility due to increased overall survival and DNA damage [1819,1821].

11.4.3.c.2. Varicocelectomy

Varicocele repair has been a subject of debate for several decades. A meta-analysis of RCTs and observational studies in men with only clinical varicoceles has shown that surgical varicocelectomy significantly improves semen parameters in men with abnormal semen parameters, including men with NOA with hypo-spermatogenesis or late maturation (spermatid) arrest on testicular pathology [1819,1823-1826]. A meta-analysis showed that improvements in semen parameters are usually observed after surgical correction in men with abnormal semen parameters [1827-1829]. Varicocelectomy can also reverse sperm DNA damage and improve OS levels [1819,1821]. Pain resolution after varicocelectomy occurs in 48-90% of patients [1830]. A systematic review has shown greater improvement in higher-grade varicoceles, and this should be considered during patient counselling [1831].

In RCTs, varicocele repair in men with a subclinical varicocele was ineffective at increasing the chances of spontaneous pregnancy [1832]. Also, in randomised studies that included mainly men with normal semen parameters, no benefit was found to favour treatment over observation. This was also reported in a systematic review and meta-analysis including prospective randomised and non-randomised studies [1833]. In studies including patients with abnormal semen parameters pregnancy rates (OR: 1.29; 95% CI: 1.00–1.65; p = 0.04) and total sperm count (mean difference: 12.34 million/mL; 95% CI: 3.49–21.18; p = 0.006) were significantly improved by varicocele treatment compared with observation. A benefit for varicocele treatment was not found for sperm progressive motility and normal sperm morphology [1833]. When pre- versus post-treatment values were considered in the varicocele treatment arm, only a benefit in terms of sperm count, progressive motility, and normal morphology was found [1833]. Another systematic review and meta-analysis evaluated the change in conventional semen parameters after varicocele repair (n = 1,426) compared to untreated controls (n = 996) [1834]. Significantly improved postoperative semen parameters where reported in treated patients compared to controls with regards to sperm concentration (SMD 1.73; 95% CI: 1.12–2.34; p < 0.001); total sperm count (SMD 1.89; 95% CI: 0.56–3.22; p < 0.05); progressive sperm motility (SMD 3.30; 95% CI: 2.16–4.43; p < 0.01); total sperm motility (SMD 0.88; 95% CI: 0.03–1.73; p=0.04) and normal sperm morphology (SMD 1.67; 95% CI: 0.87–2.47; p < 0.05) [1834].

A 2012 Cochrane review concluded that treatment of a varicocele in men from couples with otherwise unexplained subfertility may improve the chance of spontaneous pregnancy [1835]. Similarly, a 2021 Cochrane review including 5,384 participants showed that varicocele treatment may improve pregnancy rates compared to delayed or no treatment (RR: 1.55; 95% CI: 1.06–2.26) [1836]. Two meta-analyses of RCTs comparing treatment to observation in men with a clinical varicocele, oligozoospermia and otherwise unexplained infertility, favoured treatment, with a combined OR of 2.39–4.15 (95% CI: 1.56–3.66) and (95% CI: 2.31–7.45), respectively [1826,1835]. Average time to improvement in semen parameters is up to two spermatogenic cycles [1837,1838] with spontaneous pregnancy occurring between six and 12 months after varicocelectomy [1839,1840]. A further meta-analysis reported that varicocelectomy may improve outcomes following ART in oligozoospermic men with an OR of 1.69 (95% CI: 0.95–3.02) [1841].

11.4.3.c.3. Prophylactic varicocelectomy

In adolescents with a varicocele, there is a significant risk of over-treatment because most adolescents with a varicocele have no problem achieving pregnancy later in life [1842]. Prophylactic treatment is only advised in case of documented testicular growth deterioration confirmed by serial clinical or Doppler US examinations and/or abnormal semen analysis [1843,1844].

Varicocelectomy and non-obstructive azoospermia
Several non-randomised studies have suggested that varicocelectomy may lead to sperm appearing in the ejaculate in men with azoospermia. In one such study, microsurgical varicocelectomy in men with NOA led to sperm in the ejaculate postoperatively with an increase in ensuing natural or assisted pregnancies [1845]. Meta-analyses have further corroborated these findings; 468 patients diagnosed with NOA and varicocele underwent surgical varicocele repair or percutaneous embolisation. In patients who underwent varicocelectomy, sperm retrieval rates (SRRs) increased compared to those without varicocele repair (OR: 2.65; 95% CI: 1.69–4.14; p < 0.001). In 43.9% of the patients (range: 20.8–55.0%), sperm were found in postoperative ejaculate. These findings indicate that varicocelectomy in patients with NOA and clinical varicocele is associated with improved SRR, and sperm retrieval may be avoided when sperm reappear in the ejaculate following varicocelectomy. 1397256787The quality of available evidence is low and the risks and benefits of varicocele repair must be discussed fully with the patient with NOA and a clinically significant varicocele prior to embarking upon treatment intervention [1824,1846]. The current understanding of the underlying genetic defects of NOA must be considered when interpreting contemporary literature.

Varicocelectomy and hypogonadism
Evidence also suggests that men with clinical varicoceles and hypogonadism may benefit from varicocele intervention. One meta-analysis studied the efficacy of varicocele intervention by comparing the preoperative and postoperative serum testosterone of 712 men. The combined analysis of seven studies demonstrated that the mean postoperative serum testosterone improved by 34.3ng/dL (95% CI: 22.57–46.04; p < 0.00001; I² = 0%) compared with their preoperative levels. An analysis of surgery versus untreated control results showed that mean testosterone among patients with hypogonadism increased by 105.65ng/dL (95% CI: 77.99–133.32ng/dL), favouring varicocelectomy [1847]. In this setting, results must be treated with caution and adequate cost-benefit analysis must be undertaken to determine the risks and benefits of surgical intervention over testosterone therapy. Although varicocelectomy may be offered to men with hypogonadism with clinically significant varicoceles, patients must be advised that the full benefits of treatment in this setting must be further evaluated with prospective RCTs.

11.4.3.c.4. Varicocelectomy for assisted reproductive technology and raised sperm DNA fragmentation

Varicocelectomy can improve sperm DNA integrity [1842,1848-1850]. A systematic review and meta-analysis analysed data from 1,070 infertile men with clinical varicocele and showed that varicocelectomy was associated with reduced postoperative SDF rates (weighted mean difference 7.23%; 95% CI: 8.86–5.59) [1851]. Improvement of DNA integrity was independent from the assay used (SCSA vs. TUNEL vs. SCD) and the surgical technique performed. The estimated weighted mean difference was greater in studies with preoperative mean SDF ≥ 20% than that in studies with SDF < 20%, suggesting that varicocelectomy might be more beneficial in men with elevated baseline SDF values [1851]. The magnitude of the effect size increased as a function of preoperative SDF levels (coefficient: 0.23; 95% CI: 0.07–0.39).

There is increasing evidence that varicocele treatment may improve SDF and outcomes from ART [1841,1842]. Consequently, it has been suggested that the indications for varicocele intervention should be expanded to include men with raised SDF. If a patient has failed ART (e.g. failure of implantation, embryogenesis or recurrent pregnancy loss), there is an argument that if DNA damage is raised, consideration could be given to varicocele intervention after extensive counselling [1852] and exclusion of other causes of raised SDF [1842,1853]. The dilemma remains as to whether varicocele treatment is indicated in men with raised SDF and normal semen parameters. This decision would need a full and open discussion with the infertile couple, taking into consideration the female partners ovarian reserve and the surgical risks and potential delays in ART associated with varicocele intervention.

In a meta-analysis of non-azoospermic infertile men with clinical varicocele by Estevez et al., four retrospective studies were included of men undergoing ICSI, and included 870 cycles (438 subjected to ICSI with prior varicocelectomy, and 432 without prior varicocelectomy). There was a significant increase in the clinical pregnancy rates (OR: 1.59; 95% CI: 1.19–2.12; I2 = 25%) and live birth rates (OR: 2.17; 95% CI: 1.55–3.06; I2 = 0%) in the varicocelectomy group compared to the group subjected to ICSI without previous varicocelectomy. A further study evaluated the effects of varicocele repair and its impact on pregnancy and live birth rates in infertile couples undergoing ART in male partners with oligo-azoospermia or azoospermia and a varicocele [1841]. In 1,241 patients, a meta-analysis demonstrated that varicocelectomy improved live birth rates for the oligospermic (OR = 1.699) men and combined oligo-azoospermic/azoospermic groups (OR = 1.761). Pregnancy rates were higher in the azoospermic group (OR = 2.336) and combined oligo-azoospermic/azoospermic groups (OR: 1.760). Live birth rates were higher for patients undergoing IUI after intervention (OR: 8.360).

11.4.3.d. Disease management

Several treatments are available for varicocele (Table 11.3).

Impact on pregnancy rate and semen parameters
Current evidence indicates that microsurgical varicocelectomy is the most effective among the different varicocelectomy techniques [1842,1854]. A Cochrane review reported that microsurgical subinguinal varicocelectomy probably improves pregnancy rates slightly more compared to other surgical treatments (RR: 1.18; 95% CI: 1.02–1.36) [1836]. A subgroup analysis from a systematic review of prospective randomised and non-randomised studies reported that surgical approach (including all possible surgical techniques) significantly improved pregnancy rates and sperm concentration as compared with controls, while the same was not demonstrated for radiological treatment [1833]. However, a Cochrane review showed inconclusive results about the effect of surgical versus radiological treatment on pregnancy rates and varicocele recurrence [1836]. There are no large prospective RCTs comparing the efficacy of the various interventions for varicocele.

Complications
Microsurgical repair results in fewer complications and lower recurrence rates compared to the other techniques [1836,1855,1856]; however, this procedure requires microsurgical training. The various other techniques are still considered viable options, although recurrences and hydrocele formation appear to be higher [1856].

Radiological techniques (sclerotherapy and embolisation) are minimally invasive approaches for varicocele treatment. Although higher recurrence rates have been reported compared to microscopic varicocelectomy [1841], a meta-analysis showed that the incidence of varicocele recurrence was similar after surgical ligation and sclero-embolisation [1857]. In terms of complications, a meta-analysis of 12 studies comparing 738 cases of surgical ligation versus 647 cases of sclero-embolisation, showed that the overall complication rate did not differ significantly between the groups (OR: 1.48; 95% CI: 0.86–2.57; p = 0.16) [1857]. The incidence of postoperative hydrocele is significantly higher after surgical ligation than sclero-embolisation, but radiological techniques are associated with higher incidence of postoperative orchiepidydimitys [1857].

Robot-assisted varicocelectomy has a similar success rate compared to the microscopic varicocelectomy technique, although larger prospective randomised studies are needed to establish the most effective method [1858-1860].

Table 11.3: Recurrence and complication rates associated with treatments for varicocele

11.4.3.e. Summary of evidence and recommendations for varicocele

11.4.4. Male accessory gland infections and infertility

11.4.4.a. Introduction

Infection of the male urogenital tract is a potentially curable cause of male infertility [1872-1874]. The WHO considers urethritis, prostatitis, orchitis and epididymitis to be male accessory gland infections [1872]. The effect of symptomatic or asymptomatic infections on sperm quality is contradictory [1875]. A systematic review of the relationship between STIs, such as those caused by Chlamydia trachomatis, genital mycoplasmas, Neisseria gonorrhoeae, Trichomonas vaginalis and viruses, and infertility was unable to draw a strong association between STIs and male infertility due to the limited quality of reported data [1876].

11.4.4.b. Diagnostic evaluation

11.4.4.b.1. Semen analysis

Semen analysis (see Section 11.3.2) clarifies whether the prostate is involved as part of a generalised male accessory gland infection and provides information regarding sperm quality.

11.4.4.b.2. Microbiological findings

After exclusion of urinary tract infection (including urethritis), > 10⁶ peroxidase-positive white blood cells per millilitre of ejaculate indicate an inflammatory process. Semen culture or polymerase chain reaction analysis should be performed for common urinary tract pathogens in all suspected cases of genitourinary tract infections. A concentration of > 10³ colony forming units per millilitre urinary tract pathogens in the ejaculate is indicative of significant bacteriospermia [1877]. The sampling should be delivered the same day to the laboratory because the sampling time can influence the rate of positive micro-organisms in semen and the frequency of isolation of different strains [1878]. The ideal diagnostic test for isolating C. trachomatis in semen has not yet been established [1879], but the most accurate method is polymerase chain reaction [1880-1882].

Historical data show that Ureaplasma urealyticum is pathogenic only in high concentrations (> 103 colony forming units per millilitre ejaculate). Fewer than 10% of samples analysed for Ureaplasma exceeded this concentration [1883]. Normal colonisation of the urethra hampers the significance of mycoplasma-associated urogenital infections, using samples such as the ejaculate [1884].

A meta-analysis indicated that Ureaplasma parvum and Mycoplasma genitalium were not associated with male infertility, but a significant relationship existed between U. urealyticum (OR: 3.03; 95% CI: 1.02–8.99) and Mycoplasma hominis (OR: 2.8; 95% CI: 0.93– 3.64) [1885]. For these reasons, the treatment is not always recommended.

Human papillomavirus (HPV) is commonly found in semen samples, with a reported prevalence of 20% in infertile populations and 8% in men from the general population [1886]. Systematic reviews have reported an association between male infertility, poorer pregnancy outcomes and semen HPV positivity [1886-1889]. However, data still need to be prospectively validated to clearly define the clinical impact of HPV-infection in semen. Additionally, seminal presence of Herpes simplex virus (HSV)-2 in infertile males may be associated with lower sperm quality compared to that in HSV-negative infertile males [1875]. However, it is unclear if anti-viral therapy improves fertility rates in these males.

11.4.4.b.3. White blood cells

The clinical significance of an increased concentration of leukocytes in the ejaculate is controversial [1890]. Although leukocytospermia is a sign of inflammation, it is not necessarily associated with bacterial or viral infections and therefore cannot be considered a reliable indicator [1891]. According to the WHO classification, leukocytospermia is defined as > 10⁶ white blood cells per millilitre. Only two studies have analysed alterations of white blood cells in the ejaculate of patients with proven prostatitis [1892,1893]. Both studies found more leukocytes in males with prostatitis compared to those without inflammation (CPPS, type NIH 3b). Leukocytospermia should be further confirmed by performing a peroxidase test on the semen. A meta-analysis of case-control studies showed that leukocytospermia in males seeking consultation for couple subfertility was not associated with reduced fertility after ART and altered semen quality in males without symptoms of genital tract infections [1894]. There is currently no evidence that treatment of leukocytospermia alone without evidence of infective organisms improves conception rates [1895].

11.4.4.b.4. Sperm quality

The deleterious effects of CP/CPPS on sperm density, motility and morphology have been demonstrated in a systematic review based on case-controlled studies [1896]. Both C. trachomatis and Ureoplasma spp. can cause decreased sperm density, motility, altered morphology and increased DNA damage. Data from a retrospective cross-sectional study showed that U. urealyticum was the most frequent single pathogen in semen of asymptomatic infertile males; a positive semen culture was both univariably (p < 0.001) and multi-variably (p = 0.04) associated with lower sperm concentration [1897]. Human papillomavirus is associated with changes in semen density, sperm motility and sperm DNA damage [1898,1899]. Mycoplasma spp. can cause decreased motility and development of anti-sperm antibodies [1875].

11.4.4.b.5. Seminal plasma alterations

Seminal plasma elastase is a biochemical indicator of polymorphonuclear lymphocyte activity in the ejaculate [1874,1900,1901]. Various cytokines are involved in inflammation and can influence sperm function. Several studies have investigated the association between interleukin (IL) concentration, leukocytes, and sperm function through different pathways, but no correlations have been found [1902-1904]. The prostate is the main site of origin of IL-6 and IL-8 in the seminal plasma. Cytokines, especially IL-6, play an important role in the male accessory gland inflammatory process [1905]. However, elevated cytokine levels do not depend on the number of leukocytes in expressed prostatic secretion [1906].

11.4.4.b.6. Glandular secretory dysfunction

The secretory function of the prostate gland can be evaluated by measuring seminal plasma pH, citric acid, or γ-glutamine transpeptidase levels, although these parameters are no longer evaluated in numerous laboratories; the seminal plasma concentrations of these factors are usually altered during infection and inflammation. They are not recommended as diagnostic markers for male accessory gland infections [1907].

11.4.4.b.7. Reactive oxygen species

Reactive oxygen species may be increased in infertile patients with asymptomatic C. trachomatis and M. hominis infection, with subsequent decrease in ROS upon antibiotic treatment. Reactive oxygen species levels in infertile patients with asymptomatic C. trachomatis and M. hominis in the semen are low, making it difficult to draw any firm conclusions [1908]. Chronic urogenital infections are also associated with increased leukocyte numbers [1909], but their biological significance in prostatitis remains unclear [1874].

11.4.4.b.8. Disease management

Only antibiotic therapy of chronic bacterial prostatitis (NIH II according to the classification) has provided symptomatic relief, eradication of micro-organisms, and a decrease in cellular and humoral inflammatory parameters in urogenital secretions. Although antibiotics might improve sperm quality [1910], there is no evidence that treatment of CP/CPPS increases the probability of natural conception [1874,1911].

Asymptomatic presence of C. trachomatis and M. hominis in the semen can be correlated with impaired sperm quality, which recovers after antibiotic treatment. Further research is required to confirm these findings [1908].

11.4.4.c. Epididymitis

Inflammation of the epididymis causes unilateral pain and swelling, usually with acute onset. Among sexually active males aged < 35 years, epididymitis is most often caused by C. trachomatis or N. gonorrhoea [1912,1913]. Sexually transmitted epididymitis is usually accompanied by urethritis. Non-sexually transmitted epididymitis is associated with urinary tract infections and occurs more often in males aged > 35 years [1914].

11.4.4.c.1. Diagnostic evaluation

11.4.4.c.1.a. Ejaculate analysis

According to the WHO Laboratory Manual for the Examination and Processing of Human Semen (6th edn) criteria, ejaculate analysis may indicate persistent inflammatory activity. Transient reductions in sperm counts and progressive sperm motility can be observed [1912,1915,1916]. Semen culture might help to identify pathogenic micro-organisms. Development of stenosis of the epididymal ducts, reduction of sperm count, and azoospermia are more important potential sequelae to consider in the follow-up of bilateral epididymitis (see Section 11.3.2).

11.4.4.c.1.b. Disease management

Treatment of epididymitis results in:

• microbiological cure of infection
• improvement of clinical signs and symptoms
• prevention of potential testicular damage
• prevention of transmission
• decrease of potential complications (e.g. infertility or chronic pain).

Patients with epididymitis known or suspected to be caused by N. gonorrhoeae or C. trachomatis must be informed to also refer their sexual partners for evaluation and treatment [1917].

11.4.4.d. Summary of evidence and recommendation for male accessory gland infections

11.5. Non-invasive male infertility management

11.5.1. Empirical treatments

11.5.1.a. Lifestyle

Environmental and lifestyle factors may contribute to male infertility, acting additively on a susceptible genetic background [90,1737]. Hence, lifestyle improvement can have a positive effect on sperm parameters.

This includes:

• Weight loss: non-controlled studies have suggested that weight loss can result in improved sperm parameters [90,1918,1919]. Data derived from RCTs are more conflicting. A meta-analysis of 28 cohort studies and 1,022 patients documented that bariatric surgery did not improve sperm quality and function in males that are morbidly obese [1920]. Data on ART outcomes are lacking. Weight loss can improve obesity-related secondary hypogonadism, which may result in better outcomes in couples seeking medical care for infertility [1918,1920].
• Physical activity: a meta-analysis reported that moderate-intensity (20–40 metabolic equivalents per week) or even high-intensity (40–80 metabolic equivalents hours per week) recreational physical activity can result in better semen parameters [1921]. Physical activity might improve hormonal profile [1918].
• Smoking: data derived from a large meta-analysis of 20 studies with 5,865 participants showed a negative association between smoking and sperm parameters [1922].
• Alcohol consumption: Data derived from a meta-analysis including 15 cross-sectional studies and 16,395 men suggested that moderate alcohol does not adversely affect semen parameters, whereas high alcohol intake can have a detrimental effect on male fertility [1923]. Heavy chronic alcohol consumption (defined as > two drinks/day [1837]) can reduce testosterone levels [1924].

11.5.1.b. Antioxidant treatment

Oxidative stress is one of the most important contributing factors in the pathogenesis of idiopathic infertility. Reactive oxygen species, the final products of OS, can impair sperm function acting at several levels, including plasma membrane lipid peroxidation, which can affect sperm motility, the acrosome reaction and chromatin maturation leading to increased SDF [1925]. Accordingly, seminal levels of ROS have been negatively associated with ART outcomes [1926]. Evidence for the role of antioxidant therapy in male infertility is still conflicting. An Cochrane systematic review and meta-analysis including 34 RCTs and 2,876 couples using various antioxidant compounds concluded that antioxidant therapy had a positive impact on live-birth and pregnancy rates in sub-fertile couples undergoing ART cycles [1927]. Similar results were also reported in a meta-analysis including 61 studies with 6,264 infertile males, aged 18-65 years [1928]. The quality of the reported studies is poor. The Males, Antioxidants, and Infertility (MOXI) trial found that antioxidants did not improve semen parameters or DNA integrity compared to placebo among infertile males with male factor infertility. Cumulative live-birth rates did not differ at six months between the antioxidant and placebo groups (15% vs. 24%) [1929]. No clear conclusions were possible regarding the specific antioxidants to use or and/or therapeutic regimes for improving sperm parameters and pregnancy rate [1928].

11.5.1.c. Probiotic treatment

Prebiotic/probiotic supplementation may influence hormone secretion; facilitating the scavenging of free radicals, and improving the microenvironment of the prostate and indirectly enhance sperm function. A RCT including 56 males with idiopathic infertility treated with a prebiotic/probiotic compound versus placebo showed a significant increase in sperm parameters in the treated group (i.e. sperm concentration, motility and normal morphology) as well as an increase in DNA integrity [1930]. A RCT of 78 infertile males who underwent varicocelectomy with or without postoperative probiotic supplementation found a significant early (three months) increase of sperm parameters (sperm concentration and normal morphology) in favour of the probiotic group versus placebo [1931]. Further high-powered RCTs are warranted to further investigate the use of prebiotic/probiotic supplementation in the context of male infertility.

11.5.1.d. Selective oestrogen receptor modulators

Selective oestrogen receptor modulators block oestrogen receptors at the level of the hypothalamus, which results in stimulation of GnRH secretion, leading to an increase in pituitary gonadotropin release and stimulation of spermatogenesis [1932]. Meta-analysed data derived from 11 RCTs showed that SERMs significantly increased pregnancy rate, sperm and hormonal parameters [1933]. Similar results were confirmed in a meta-analysis of 16 studies [1932]. Previous systematic reviews failed to find any association between SERMs and pregnancy rate [1934]. The quality of the papers considered was low, only a few studies were placebo controlled and complications from the use of SERMs were under-reported. In conclusion, although some positive results on the use of SERMs in males with idiopathic infertility have been reported, no conclusive recommendations can be drawn due to poor quality of the available evidence.

11.5.1.e. Aromatase inhibitors

Aromatase, a cytochrome p450 enzyme, is present in the testes, prostate, brain, bone, and adipose tissue of males; it converts testosterone and androstenedione to oestradiol and oestrone, respectively. Oestradiol negatively feeds back on the hypothalamus and pituitary to reduce gonadotropic secretions, ultimately affecting spermatogenesis. In this context, AIs may reduce oestrogen production by reversibly inhibiting cytochrome p450 isoenzymes 2A6 and 2C19 within the aromatase enzyme complex. This inhibition reduces oestrogen-mediated negative feedback on the hypothalamus, resulting in stronger GnRH pulses that stimulate the pituitary to increase production of FSH [1935-1938]. Aromatase activity has been associated with male infertility characterised by testicular dysfunction with low serum testosterone and/or testosterone to oestradiol ratio. As an off-label treatment option, AIs have been reported to increase endogenous testosterone production and improve spermatogenesis in the setting of infertility [1939]. Both steroidal (testolactone) and non-steroidal (anastrozole and letrozole) AIs significantly improve hormonal and semen parameters in infertile males, with a safe tolerability profile, although prospective RCTs are necessary to better define the efficacy of these medications in this clinical setting [1937,1939].

11.5.2. Summary of evidence and recommendation for non-invasive male infertility management

11.5.3. Hormonal therapy

11.5.3.a. Secondary hypogonadism

A brief discussion on pre-pubertal onset can be found in Appendix 7, online supplementary evidence.

Post-pubertal onset: hCG alone is usually required first to stimulate spermatogenesis. A starting dose of 250 IU hCG twice weekly is suggested, and if normal testosterone levels are reached, hCG doses may be increased up to 2,000 IU twice weekly. Semen analysis should be performed every three months to assess response, unless conception has taken place. If there is a failure of stimulation of spermatogenesis, FSH can be added (75 IU three times per week, increasing to 150 IU three times per week, if indicated). Combination therapy with FSH and hCG can be administered from the start of treatment, promoting better outcomes in males with hypogonadotropic hypogonadism [133]. No difference in outcomes were observed when urinary-derived, highly purified FSH was compared to rFSH [133].

Greater baseline testicular volume is a good prognostic indicator for response to gonadotrophin treatment, while previous testosterone therapy can have a negative impact on gonadotropin treatment outcomes in males with hypogonadotropic hypogonadism [1940]. This observation was refuted by a meta-analysis that did not confirm a real negative role of testosterone therapy in terms of future fertility in this specific setting [133].

11.5.3.a.1. Secondary hypogonadism due to hyperprolactinemia

In the presence of hyperprolactinaemia, which suppresses gonadotrophins and leads to subfertility, treatment (independent of aetiology, including a pituitary adenoma) is dopamine agonist therapy or withdrawal of the drug that causes the condition. Dopamine agonists used include bromocriptine, cabergoline and quinagolide.

11.5.3.b. Primary hypogonadism

There is no substantial evidence that gonadotrophin therapy has any beneficial effect in the presence of classical testicular failure. Likewise, there are no data to support the use of other hormonal treatments (including SERMs or AIs) in the case of primary hypogonadism to improve spermatogenesis [91,1941].

11.5.3.c. Idiopathic male factor infertility

There is some evidence that FSH treatment increases sperm parameters in idiopathic oligozoospermic males with FSH levels within the normal range (generally 1.5–8mIU/mL) [1942]. In a meta-analysis including 12 trials with 924 patients, FSH treatment resulted in an improvement of both semen parameter and SDF [1943]. It has also been reported that FSH, along with improvement of SDF rates, may ameliorate AMH and inhibin B levels [1944-1947]. High-dose FSH therapy is more effective in achieving a testicular response than lower doses [1948]. A Cochrane review including six RCTs with 456 participants, different treatment protocols and follow-up periods, concluded that FSH treatment resulted in higher live-birth and pregnancy rates compared with placebo or no treatment. No significant difference among groups was observed when ICSI or IUI were considered [1949]. In a meta-analysis including 15 trials with > 1,200 patients, similar findings after FSH treatment were observed in terms of both spontaneous pregnancies and pregnancies after ART [1950]. A further study showed that in azoospermic males undergoing TESE-ICSI, there were improved SRRs and higher pregnancy and fertilisation rates in males treated with FSH compared to untreated males [1951]. In males with NOA, combination hCG/FSH therapy was shown to increase SRR in only one study [1952]. Human chorionic gonadotrophin alone prior to TESE in NOA has not been found to have any benefit on SRRs [1953]. Overall, the evidence for the use of hormone therapy prior to SSR is limited and treatment should be confined to clinical trials and not used routinely in clinical practice.

11.5.3.d. Anabolic steroid abuse

Oligospermia or azoospermia caused by anabolic abuse should initially be treated by withdrawal of the anabolic steroid. There is no common indication for treating this disorder; the management is based on case reports and clinical experience. Usually, adequate sperm numbers and quality will improve over a six to twelve-month period from cessation. If after this interval the condition persists, then hCG without or in combination with FSH as an alternative to clomiphene can be used to stimulate spermatogenesis [1954].

11.5.3.e. Summary of evidence and recommendations for treatment of male infertility with hormonal therapy

11.6. Invasive male infertility management

11.6.1. Obstructive azoospermia

Obstructive azoospermia is the absence of spermatozoa in the sediment of a centrifuged sample of ejaculate due to obstruction [1872]. Obstructive azoospermia occurs in 20-40% of males with azoospermia [1955,1956] and it is characterised by normal FSH values, testes of normal size, and epididymal enlargement [1957]. The most common causes of OA are reported in Table 11.4.

Table 11.4: Causes of obstruction of the genitourinary system

CBAVD = congenital bilateral absence of the vas deferens; CUAVD = Congenital unilateral absence of the vas deferens; MESA = microsurgical epididymal sperm aspiration.

11.6.1.a. Diagnostic evaluation

Clinical history-taking should follow the investigation and diagnostic evaluation of infertile males (See Section 11.3). Risk factors for obstruction include prior surgery, iatrogenic injury during inguinal herniorrhaphy, orchidopexy or hydrocelectomy.

11.6.1.a.1. Clinical examination

Clinical examination should follow the guidelines for the diagnostic evaluation of infertile males. Obstructive azoospermia is indicated by at least one testis with a volume > 15mL, although a smaller volume may be found in some patients with OA and concomitant partial testicular failure. Other finding might include:

• enlarged and dilated epididymis
• nodules in the epididymis or vas deferens
• absence or partial atresia of the vas deferens.

When semen volume is low or absent, a search must be made for spermatozoa in urine after ejaculation. Absence of spermatozoa and immature germ cells in the semen pellet suggest complete seminal duct obstruction.

11.6.1.a.2. Hormone levels

Hormones, including FSH and inhibin-B, should be normal but do not exclude other causes of testicular azoospermia (e.g. NOA). Although inhibin‐B concentration is a good index of Sertoli cell integrity reflecting closely the state of spermatogenesis, its diagnostic value is no better than that of FSH and its use in clinical practice has not been widely advocated [1958].

11.6.1.a.3. Genetic testing

In any patient with unilateral or bilateral absence of the vas deferens or seminal vesicle agenesis, CFTR gene testing should be performed [1959].

11.6.1.a.4. Testicular biopsy

Testis biopsies (including fine-needle aspiration [FNA]) without performing simultaneously a therapeutic sperm retrieval are not recommended, as this will require a subsequent invasive procedure. Furthermore, even patients with extremes of spermatogenic failure (e.g. SCOS) may harbour focal areas of spermatogenesis [1960,1961].

11.6.1.b. Disease management

11.6.1.b.1. Sperm retrieval

Intratesticular obstruction
Only testicular sperm retrieval can extract sperm in these patients and is therefore recommended.

Epididymal obstruction
Microsurgical epididymal sperm aspiration or PESA [1962] is indicated in males with CBAVD. Testicular sperm extraction and percutaneous TESA, are also options [1963]. The source of sperm used for ICSI in cases of OA and the aetiology of the obstruction do not affect the outcome in terms of pregnancy or miscarriage rates [1964,1965]. Usually, one MESA procedure provides sufficient material for a number of ICSI cycles [1966] and it produces high pregnancy and fertilisation rates [1967]. Overall, pregnancy outcomes from ICSI in males with OA are comparable between epididymal and testicular sperm and also between fresh and frozen–thawed epididymal sperm [1968]. However, these results are from studies of low evidence.

In patients with OA due to acquired epididymal obstruction and with a female partner with good ovarian reserve, microsurgical epididymovasostomy (EV) is recommended [1969]. Epididymovasostomy can be performed with different techniques such as end-to-site and intussusception [1970]. Anatomical recanalisation following surgery may require 3-18 months. A systematic review indicated that the time to patency in EV varies between 2.8 to 6.6 months. Reports of late failure are heterogeneous and vary between 1 and 50% [1971]. At the time of microsurgery, and in all cases in which reconstruction is impossible, sperm retrieval should be performed intra-operatively by MESA/TESE and cryopreserved to be used for subsequent ICSI procedures [1972].

Patency rates range between 63% and 85% and cumulative natural pregnancy rates between 21% and 45% [1973-1975]. Better patency rates might be achieved by performing bilateral EV and by targeting the caudal or corpus part of the epididymis when possible [1975,1976]. Recanalisation success rates may be adversely affected by preoperative and intra-operative findings. Robot-assisted EV has similar success rates, but larger studies are needed [1977].

Vas deferens obstruction after vasectomy
Vas deferens obstruction after vasectomy requires microsurgical vasectomy reversal. The mean post-procedural patency and pregnancy rates weighted by sample size were 90-97% and 52-73%, respectively [1973,1974]. The average time to patency is 1.7-4.3 months and late failures are uncommon (0-12%) [1971]. Robot-assisted vasovasostomy has similar success rates, and larger studies, including cost-benefit analysis, are needed to establish its benefits over standard microsurgical procedures [1977].

The absence of spermatozoa in the intra-operative vas deferens fluid suggests the presence of a secondary epididymal obstruction, especially if the seminal fluid of the proximal vas deferens has a thick “toothpaste” appearance; in this case microsurgical EV may be indicated [1978-1980]. Simultaneous sperm retrieval may be performed for future cryopreservation and use for ICSI; likewise, patients should be counselled appropriately.

Vas deferens obstruction at the inguinal level
It is usually impossible to correct large bilateral vas deferens defects, resulting from involuntary excision of the vasa deferentia during hernia surgery in early childhood or previous orchidopexy. In these cases, TESE/MESA/PESA or proximal vas deferens sperm aspiration [1981] can be used for cryopreservation for future ICSI.

Ejaculatory duct obstruction
The treatment of ejaculatory duct obstruction (EDO) depends on its aetiology. Transurethral resection of the ejaculatory ducts (TURED) can be used in post-inflammatory obstruction and cystic obstruction [1972,1982]. Resection may remove part of the verumontanum. In cases of obstruction due to a midline intraprostatic cyst, incision, unroofing or aspiration of the cyst is required [1972,1982].

• Pregnancy rates after TURED are 20-25% [1794,1982,1983]. Complications following TURED include epididymitis, urinary tract infection, gross haematuria, haematospermia, azoospermia (in cases with partial distal ejaculatory duct obstruction) and urine reflux into the ejaculatory ducts and seminal vesicles [1982].
• Alternative therapies for EDO include, seminal vesiculoscopy to remove debris or calculi and balloon dilation and laser incision for calcification on TRUS [1984]. The alternatives to TURED are MESA, PESA, TESA, TESE, proximal vas deferens sperm aspiration and seminal vesicle-ultrasonically guided aspiration.

11.6.1.c. Summary of evidence and recommendations for obstructive azoospermia

11.6.2. Non-obstructive azoospermia

Non-obstructive azoospermia is defined as the absence of sperm in the ejaculate after centrifugation. Ejaculate volume is usually normal. Azoospermia should be confirmed by at least two consecutives semen analyses [1632]. The severe deficit in spermatogenesis observed in NOA patients is often a consequence of primary testicular dysfunction or may be related to a dysfunction of the hypothalamus-pituitary-gonadal (HPG) axis.

11.6.2.a. Investigation of non-obstructive azoospermia

Clinical history-taking and clinical examination should follow the investigation and diagnostic evaluation of infertile males (See Section 11.3). Non-obstructive azoospermia can be the first sign of pituitary or TCGT [1985-1987]. Patients with NOA have been shown to be at increased risk of long-term chronic non-communicable diseases (e.g. cardio-metabolic diseases, cancer) and mortality [1988-1993]. Therefore, investigation of infertile males provides an opportunity for long-term risk stratification for other comorbid conditions [1994]. A complete hormonal investigation and scrotal US are important in the diagnostic work-up of NOA males [1995,1996].

Concomitant hypogonadism has been found in about 30% of patients with NOA [303,1995,1996]. Biochemical evaluation should be performed to differentiate the types of hypogonadism (i.e. hypogonadotropic hypogonadism vs. hypergonadotropic vs. compensated hypogonadism) as this will determine different therapeutic strategies to treat the hypogonadal male [1997].

Scrotal US may show signs of testicular dysgenesis (e.g. non-homogeneous testicular architecture and/or microcalcifications) and rare testicular tumours can be identified. Testicular volume may be a predictor of spermatogenic function [1634] and is usually, but not invariably, low in patients with NOA. Some authors have advocated that testicular perfusion detected at US Doppler assessment can predict surgical sperm retrieval at TESE and guide testicular biopsies [1998]; however, to date, data are inconsistent to support a routine role of testicular Doppler evaluation before TESE in order to predict sperm retrieval outcome.

As discussed (see Section 11.3), patients should undergo karyotype analysis [1892,1893], along with a screening of Y-chromosome micro-deletions [1718,1999]. In patients with clinical suspicion of CBAVD assessment of mutations in the gene coding for CFTR is also to be recommended [1705,1706]. Genetic counselling for eventual transmissible and health-relevant genetic conditions should be provided to couples.

11.6.2.b. Surgery for non-obstructive azoospermia

Surgical treatment for NOA is aimed at retrieval of vital sperm directly from the testes (either uni- or bilaterally). This treatment is normally part of ART protocols, including IVF cycles via ICSI. Testicular biopsy before TESE is not recommended.

11.6.2.c. Indications and techniques of sperm retrieval

Spermatogenesis within the testes may be focal, which means that spermatozoa can usually be found in small and isolated foci. With a wide variability among cohorts and techniques, positive SRRs have been reported in up to 50% of patients with NOA [2000,2001]. Numerous predictive factors for positive SSR have been investigated (see below), although no definitive factors have been demonstrated to predict SSR [2001].

• Histology: The presence of hypospermatogenesis at testicular biopsy showed good accuracy in predicting positive sperm retrieval after TESE compared with maturation arrest pattern or SCOS [2002-2004].
• Hormonal levels: FSH, LH, inhibin B and AMH have been variably correlated with sperm retrieval outcomes, but data from retrospective series are controversial [1670,1951,2005-2009].
• Testicular volume has been inconsistently found to be a predictor of positive SSR [1951,2002,2008].

In case of complete AZFa and AZFb microdeletions, the likelihood of sperm retrieval is almost zero and therefore TESE procedures are contraindicated [1723]. Conversely, patients with Klinefelter syndrome [1690] and a history of undescended testes have been shown to have higher chance of finding sperm at surgery [1690,2008,2010,2011]. Studies investigating the use of machine learning algorithms to predict sperm retrieval in males with NOA are emerging and might prove a useful tool in the future [1669,2012].

Fine-needle aspiration mapping
Fine-needle aspiration mapping technique has been proposed as a prognostic procedure aimed to select patients with NOA for TESE and ICSI [2013]. The retrieved tissue is sent for cytological and histological evaluation to provide information on the presence of mature sperm and on testicular histological pattern. Fine-needle aspiration mapping may provide information on sites with higher probability of sperm retrieval, serving as a guide for further sperm retrieval surgery in the context of ART procedures (e.g. ICSI). A positive FNA requires a secondary therapeutic invasive surgical approach, which may increase the risk of testicular damage and, without appropriate cost-benefit analysis, is not justifiable. No studies have evaluated the salvage rate of mTESE in males who have undergone FNA mapping. Until further RCTs are undertaken, FNA mapping is not recommended as a primary therapeutic procedure in males with NOA.

Testicular sperm aspiration
Testicular sperm aspiration is a minimally invasive, office-based procedure in which testicular tissue is retrieved with a biopsy needle under local anaesthesia. Reported SRRs with TESA range from 11 to 60% according to patient profile and surgical techniques [2014-2017]. Complications after TESA are uncommon and mainly include minor bleeding with scrotal haematoma and postoperative pain [2017]. A meta-analysis including data from case-control studies reported that TESE was two times (95% CI: 1.8–2.2) more likely to result in successful SSR as compared with TESA [2001]. Given the low success rates compared with TESE, TESA is no longer recommended in males with NOA.

Conventional and microdissection testicular sperm extraction
Conventional TESE requires a scrotal incision and open biopsy of the testes [2018]. Reported SRRs in single-arm studies are about 50% [2000]. Observational studies have demonstrated that multiple biopsies yield a higher chance of sperm retrieval [2000,2019]. Conventional TESE has been associated with a higher rate of complications compared with other techniques [2000]. A total of 51.7% of patients had intratesticular haematomas at scrotal US three months after surgery, with testicular fibrosis observed in up to 30% of patients at six-months’ assessment [2020]. The functional clinical significance of this in the longer term remains unclear.

Microdissection
TESE is performed using an operative optical microscope to inspect seminiferous tubules at 20-25x magnification. This enables identification and extraction of larger, dilated and more opaque tubules, which are more likely to harbour sperm [2018]. The rationale is to increase the probability of retrieving sperm with a lower amount of tissue sampled and a subsequent lower risk of complications. Lower rates of complications have been observed with mTESE compared to conventional TESE, both in terms of haematoma and fibrosis [2021]. Both procedures have shown a recovery of baseline testosterone levels after long-term follow-up [2022,2023]. It is reasonable to provide long-term endocrinological follow-up after TESE (any type) to detect hypogonadism.

A meta-analysis that pooled data of case-control studies comparing conventional TESE with mTESE showed a lower unadjusted SRR of 35% (95% CI: 30–40) for TESE and 52% (95% CI: 47–58) for mTESE [2001]. A meta-analysis comparing conventional TESE and mTESE in patients with NOA showed a mean overall SRR of 47% (95% CI: 45–49%). No differences were observed when mTESE was compared with conventional TESE (46 [range 43–49] % for conventional TESE vs. 46 [range 42–49] % for mTESE, respectively) [2010]. Meta-regression analysis demonstrated that the SRR per cycle was independent of age and hormonal parameters at enrolment. The SRR increased as a function of testicular volume. Retrieved sperms resulted in a live-birth rate of up to 28% per ICSI cycle [1999]. The difference in surgical sperm retrieval outcomes between the two meta-analyses may be explained by the data studied – one analysed only case control studies [2001] whilst the other also included the single RCT [1999]; however, all studies directly comparing conventional TESE and mTESE have shown that the latter is superior in retrieving sperm.

Studies showed a higher chance of sperm retrieval with mTESE only for patients with a histological diagnosis of SCOS [2021]. Results ranged from 22.5 to 41% and from 6.3 to 29% for mTESE versus conventional TESE, respectively [2021]. No difference between the two techniques was found when comparing patients with a histology suggestive of maturation arrest [2021]. A single study showed a small advantage of mTESE when hypospermatogenesis was found [2023].

In a study assessing the role of salvage mTESE after a previously failed conventional TESE or TESA, sperm were successfully retrieved in 46.5% of cases [2024]. In studies reporting SSR by mTESE for males who had failed TESA or conventional TESE, the SRR was 39.1% (range 18.4–57.1%) [2025,2026]. Hypospermatogenesis might be a predictor for successful salvage mTESE after a failed conventional TESE, but this is based on retrospective studies with a limited number of patients [2027]. Similarly, a variable SRR has been reported for salvage mTESE after a previously failed mTESE (ranging from 18.4% to 42.8%) [2028,2029].

A meta-analysis investigated the risk of hypogonadism after TESE due to testicular atrophy [2030]; patients with NOA experienced a mean 2.7nmol/L decrease in total testosterone six months after conventional TESE, which recovered to baseline within 18-26 months. Lower rates of complications have been observed with mTESE compared to conventional TESE, both in terms of haematoma and fibrosis [2021]. Both procedures have shown a recovery of baseline testosterone levels after long-term follow-up [2022,2023].

Only one RCT has investigated surgical sperm retrieval in 100 males with NOA, and mTESE was compared to a modified TESA using a large biopsy needle (18G) with multiple needle passes [2031]. The SRR was significantly higher in mTESE compared to multiple needle-pass TESA (43% vs. 22%; rate difference -0.21; 95% Cl: -0.39–0.03; p = 0.02). A salvage mTESE was offered to males who failed multiple needle-pass TESA and the combined SRR from the two procedures was 29%. This was not significantly different from the SRR achieved by mTESE, but the study was not powered for this comparison. Taken together, this study shows that the SRR from mTESE is superior to TESA.

Most available studies on surgical sperm retrieval in males with NOA are limited by their observational designs, heterogeneity in NOA diagnostic criteria, surgical techniques, and reporting of outcomes. However, the main limitation to contemporary literature is the paucity of randomised controlled studies comparing conventional TESE and mTESE. Although no difference in SSR was observed between conventional TESE and mTESE techniques in patients with NOA in the latest and most comprehensive meta-analysis [2010], it is important to note that in all the individual trials comparing conventional TESE and mTESE, the latter was superior in retrieving sperm. Furthermore, the current data suggests that mTESE has less complications than conventional TESE and therefore the consensus opinion of the Panel is that mTESE is the optimum approach for surgical sperm retrieval procedures. This is based on low-quality evidence, and larger RCTs comparing SSR, risks and costs between the two techniques are urgently needed. Such trials should report the specifics of the surgical technique, the procedure followed by the embryologists, clinical outcomes including quantity and quality of retrieved sperm, and detailed ART outcomes including live-birth rates [2032].

Hormonal therapy prior to surgical sperm retrieval approaches
Stimulating spermatogenesis by optimising intratesticular testosterone has been proposed to increase the chance of SSR in males with NOA. Similarly, increasing FSH serum levels could stimulate spermatogenesis. To this aim, several treatment options are available, including hCG and/or FSH [1946,2033,2034] or SERMs [2035], but a standardised protocol is lacking.

No RCT has shown a benefit of hormonal treatment to enhance the chances of sperm retrieval among patients with idiopathic NOA [2036]. A meta-analysis has suggested that hormone stimulation prior to TESE might improve SRR in eugonadal but not in patients with hypergonadotropic hypogonadism [2037]. The included studies had moderate or severe risk of bias and randomised studies are needed to confirm these findings.

Hormonal therapy has been proposed to increase the chance of sperm retrieval at salvage surgery after previously failed conventional TESE or mTESE. Only small retrospective studies with conflicting results have been conducted [1946,2037-2039]. The histological finding of hypo-spermatogenesis emerged as a predictor of sperm retrieval at salvage surgery after hormonal treatment [2039]. Patients should be counselled that the evidence for the role of hormone stimulation prior to sperm retrieval surgery in males with idiopathic NOA is limited [2040]. Currently, it is not recommended in routine practice.

11.6.2.d. Recommendations for non-obstructive azoospermia

11.7. Assisted reproductive technologies

Assisted reproductive technology consists of procedures that involve the in vitro handling of both human oocytes and sperm, or of embryos, with the objective of establishing pregnancy. A limited summary of ARTs, including a discussion on safety, can be found in Appendix 7 online supplementary evidence.

11.8. Psychosocial aspects in men’s infertility

Male infertility affects psychological wellbeing, leading to emotional distress and challenges to men’s sense of identity. A failed treatment often results in a prolonged grief response, requiring psychological support post-treatment [2041]. The mental health expert is thus regarded as part of the infertility intervention team, acting in all intervention stages, using strategies that may range from psycho-education techniques to more comprehensive psycho-therapeutic approaches [2042]. There should be a deeper focus on preventive policies. It has been recognised that men, like women, may wish to become parents. However, they often have limited knowledge of infertility-related risk factors, including poor awareness of age-related declines in fertility, and tend to overestimate the likelihood of spontaneous conception [2043,2044].