Sexual and Reproductive Health

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 1 year [1749]. Primary infertility refers to couples that have never had a child and cannot achieve pregnancy after at least 12 consecutive months having sex 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). Recurrent pregnancy loss is distinct from infertility and is defined as two or more failed pregnancies [1750,1751].

11.2. Epidemiology/aetiology/pathophysiology/risk factors

11.2.1. Introduction

About 15% of couples do not achieve pregnancy within 1 year and seek medical treatment for infertility. One in eight couples encounter problems when attempting to conceive a first child and one in six when attempting to conceive a subsequent child [1752]. In 50% of involuntarily childless couples, a male-infertility-associated factor is found, usually together with abnormal semen parameters [1749]. 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 [1749]:

• congenital or acquired urogenital abnormalities;
• gonadotoxic exposure (e.g., radiotherapy or chemotherapy);
• malignancies;
• urogenital tract infections;
• increased scrotal temperature (e.g., as a consequence of varicocele);
• endocrine disturbances;
• genetic abnormalities;
• iatrogenic factors (e.g., previous scrotal surgery);
• immunological factors.

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 11.3.2). 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. 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 [1753].

Advanced paternal age has emerged as one of the main risk factors associated with the progressive increase in the prevalence of male factor infertility [1754-1761]. 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 [1762,1763]. This should include the age and ovarian reserve of the female partner, since these parameters might determine decision-making in terms of timing and therapeutic strategies (e.g., assisted reproductive technology [ART] vs. surgical intervention) [1754-1757]. Table 52 summarises the main male-infertility-associated factors.

Table 52: Male infertility causes and associated factors and percentage of distribution in 10,469 patients 

11.2.2. Recommendations on epidemiology and aetiology

11.3. Diagnostic work-up

Focused evaluation of male patients must always be undertaken and 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 [1765-1767], and hormonal evaluation [1768]. Other investigations (e.g., genetic analysis and imaging) may be required depending on the clinical features and semen parameters.

11.3.1. Medical/reproductive history and physical examination

11.3.1.1. 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 sexually transmitted infections), history of testicular surgery and exclude any potential known gonadotoxins [1769].

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.

11.3.1.2. Physical examination

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 [1770]; orchidometry may over-estimate testicular volume when compared with US assessment [1771]. 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) [1770-1772]. The mean Prader’s orchidometer-derived testis volume reported in the European general population is 20.0 ± 5.0 mL [1770], whereas in infertile patients it is 18.0 ± 5.0 mL [1770,1773-1775]. The presence of the vas deferens, fullness of epididymis and presence of a varicocele should be always 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

A comprehensive andrological examination is always indicated in every infertile couple, both if semen analysis shows abnormalities, and even in the case of normal sperm parameters as compared with reference values [1776]. Important treatment decisions are based on the results of semen analysis and most studies evaluate semen parameters as a surrogate outcome for male fertility. However, semen analysis cannot precisely distinguish fertile from infertile men [1777]; therefore, it is essential that the complete laboratory work-up is standardised according to reference values (Table 53). There is consensus that modern semen analysis must follow these guidelines. Ejaculate analysis has been standardised by the WHO and disseminated by publication of the updated version of the WHO Laboratory Manual for the Examination and Processing of Human Semen. The 6^th edition the WHO Manual for the Examination and Processing of Human Semen [1767] has been published on July 2021 and reports some differences compared to the previous edition (5^th) [1778]. Therefore, it is possible that the worldwide implementation in the everyday clinical practice of the newly-released version could be gradual.

The 6^th edition of the WHO Manual is more like a technical guideline rather than a clinical guideline. Accordingly, it comprises three sections: i) semen examination; ii) sperm preparation and cryopreservation; and, iii) quality assessment and quality control.

Overall, the procedures for semen examination are divided into three chapters:

• Basic examinations, which contains fewer investigations than the previous edition that should be performed by every laboratory, based on step-wise procedures and evidence based techniques.
• Extended analyses, which are performed by choice of the laboratory or by special request from the clinicians.
• Advanced examinations, that are classified as focused on very specialized as well as mainly research methods and other emerging technologies.

Although only preliminary findings have been published in the real-life setting, a number of relevant differences have been identified between 6^th and 5^th editions [1779].

Basic examination:

• 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 very high. In this edition, 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 back 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.

Moreover, vitality test should not be performed in all samples and only if few motile sperm are found.

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.

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 and sperm chromatin structure and stability, computer-assisted sperm analysis.

Reference ranges and reference limits

In the 5^th edition, the distribution of values from approximately 1,800 men who have contributed to a natural conception within 12 months of trying was presented and the lower fifth percentile of this distribution has been considered as a true cutoff limit for normal vs. abnormal sperm parameters [1765].

The 6^th edition highlights that distribution of data from reference men do not represent limits between fertile and subfertile individuals [1780]. Indeed, in the latest edition of the WHO Manual, the data presented in the 5^th edition have been further evaluated and complemented with data from around 3,500 men in 12 countries [1776]. Of note, the distributions do not differ much from the compilation of 2010. Table 53 reports the lower reference limits for semen characteristics according to the 2010 and 2021 versions of the WHO Manual.

According to the new WHO Manual, the lower fifth percentile of data from men in the reference population (Table 53) does not represent a limit between fertile and infertile men. For a general prediction of live birth in vivo as well as in vitro, a multiparametric interpretation of the entire men’s and partner’s reproductive potential are needed.

It has also become clear from studies that more complex testing than semen analysis may be required in everyday clinical practice, particularly in men 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, in these patients there is evidence that sperm DNA may be damaged, thus resulting in pregnancy failure [1753,1781,1782] (see below).

Table 53: Lower reference limits (5th centiles and their 95% CIs) for semen characteristics

CIs = confidence intervals; MAR = mixed antiglobulin reaction; NP = non-progressive; PR = progressive (a+bmotility).
* Distribution of data from the population is presented with one-sided intervals (extremes of the reference population data). The lower 5th percentile represents the level under which only results from 5% of the men 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. According to WHO reference criteria 5^th edition, it is important to differentiate between the following [1778]:

• oligozoospermia: < 15 million spermatozoa/mL;
• asthenozoospermia: < 32% progressive motile spermatozoa;
• teratozoospermia: < 4% normal forms.

None of the individual sperm parameters (e.g., concentration, morphology and motility), are diagnostic per se of infertility. According to the WHO reference criteria 6th edn., this subdivision is not reported, although the EAU Guidelines panel considers this further segregation still clinically relevant in the everyday clinical practice.

Often, all three anomalies occur simultaneously, which is defined as oligo-astheno-terato-zoospermia (OAT) syndrome. As in azoospermia (namely, the complete absence of spermatozoa in semen), in severe cases of oligozoospermia (spermatozoa < 5 million/mL) [1783], there is an increased incidence of obstruction of the male genital tract and genetic abnormalities. In those cases, a more comprehensive assessment of the hormonal profile may be helpful to further and more accurately differentially diagnose among pathological conditions.

In azoospermia, the semen analysis may present with normal ejaculate volume and azoospermia after centrifugation. A recommended method is semen centrifugation at 3,000 g 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 [1780]. 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.

11.3.3. Measurement of sperm DNA Fragmentation Index (DFI)

Semen analysis is a descriptive evaluation and may be unable to discriminate between the sperm of fertile and infertile men. Therefore, it is now apparent that sperm DNA damage may occur in men with infertility. DNA fragmentation, or the accumulation of single- and double-strand DNA breaks, is a common property of sperm, and an increase in the level of sperm DNA fragmentation has been shown to reduce the chances of natural conception. Although no studies have unequivocally and directly tested the impact of sperm DNA damage on the clinical management of infertile couples, sperm DNA damage is more common in infertile men and has been identified as a major contributor to male infertility, as well as poorer outcomes following ART [1784,1785], including impaired embryo development [1784], miscarriage, recurrent pregnancy loss [1781,1782,1786], and birth defects [1784]. Sperm DNA damage can be increased by several factors including hormonal anomalies, varicocele, chronic infection and lifestyle factors (e.g., smoking) [1785].

Several assays have been described to measure sperm DNA damage. 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 [1785,1787]. Of those, 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 DNA fragmentation assessment. Sperm chromatin structure assay is still the most widely studied and one of the most commonly used techniques to detect DNA damage [1788,1789]. In SCSA, the number of cells with DNA damage is indicated by the DNA fragmentation index (DFI) [1790], whereas the proportion of immature sperm with defects in the histone-to-protamine transition is indicated by high DNA stainability [1791]. 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) [1789]. Furthermore, DFI values > 50% on SCSA are associated with poorer outcomes from in vitro fertilisation (IVF). More recently, 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 [1785].

Testicular sperm is reported to have lower levels of sperm DFI when compared to ejaculated sperm [1792]. Couples with elevated DNA fragmentation may benefit from combination of testicular sperm extraction (TESE) and ICSI, an approach called TESE-ICSI, which may not overcome infertility when applied to an unselected population of infertile men with untested DFI values [1789,1792]. However, further evidence is needed to support this practice in the routine clinical setting [1792].

11.3.4. Hormonal determinations

In men with testicular deficiency, hypergonadotropic hypogonadism (also called primary hypogonadism) is usually present, with high levels of FSH and LH, with or without low levels of testosterone. Generally, the levels of FSH negatively correlate with the number of spermatogonia [1793]. 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 [1793]. However, for patients undergoing TESE, FSH levels do not accurately predict the presence of spermatogenesis, as men with maturation arrest on histology can have both normal FSH and testicular volume [1794,1795]. Furthermore men with non-obstructive azoospermia (NOA) and high levels of FSH may still harbour focal areas of spermatogenesis at the time of TESE or microdissection TESE (mTESE) [1795,1796].

11.3.5. Genetic testing

All urologists working in andrology must have an understanding of the genetic abnormalities most commonly associated with infertility, so that they can provide correct advice to couples seeking fertility treatment. Men with low sperm counts can still be offered a reasonable chance of paternity, using IVF, ICSI and sperm extraction from the testes in cases of azoospermia. However, the spermatozoa of infertile men show an increased rate of aneuploidy, structural chromosomal abnormalities, and DNA damage, carrying the risk of passing genetic abnormalities to the next generation. Current routine clinical practice is based on the screening of genomic DNA from peripheral blood samples. However, screening of chromosomal anomalies in spermatozoa (sperm aneuploidy) and preimplantation genetic testing are also feasible and indicated in selected cases (e.g., recurrent miscarriage) [1797-1803].

11.3.5.1. 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 men, the incidence of chromosomal abnormalities was 5.8% [1804]. 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 [1804]. The frequency of chromosomal abnormalities increases as testicular deficiency becomes more severe. Patients with sperm count < 5 million/mL already show a 10-fold higher incidence (4%) of mainly autosomal structural abnormalities compared with the general population [1805,1806]. Men with NOA are at highest risk, especially for sex chromosomal anomalies (e.g., Klinefelter syndrome) [1807,1808].

Based on the frequencies of chromosomal aberrations in patients with different sperm concentration, karyotype analysis is currently indicated in men with azoospermia or oligozoospermia (spermatozoa < 10 million/mL) [1806]. This broad selection criterion has been recently externally validated, with the finding that the suggested threshold has a low sensitivity, specificity, and discrimination (80%, 37%, and 59%, respectively) [1809]. In this context, a novel nomogram, with a 2% probability cut-off, which allows for a more careful detection of karyotype alterations has been developed [1809]. Notwithstanding, the clinical value of spermatozoa < 10 million/mL remains a valid threshold until further studies, evaluating the cost-effectiveness, in which costs of adverse events due to chromosomal abnormalities (e.g., miscarriages and children with congenital anomalies) are performed [1810]. If there is a family history of recurrent spontaneous abortions, malformations or mental retardation, karyotype analysis should be requested, regardless of the sperm concentration.

11.3.5.1.1. Sex chromosome abnormalities (Klinefelter syndrome and variants )

Klinefelter syndrome is the most common sex chromosomal abnormality [1811]. Adult men with Klinefelter syndrome usually have small firm testes along with features of primary hypogonadism. The phenotype is the final result of a combination between genetic, hormonal and age-related factors [15]. 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. Leydig cell function is also commonly impaired in men with Klinefelter syndrome and thus testosterone deficiency is more frequently observed than in the general population [1812], although rarely observed during the peri-pubertal period, which usually occurs in a normal manner [15,1813]. Rarely, more pronounced signs and symptoms of hypogonadism can be present, along with congenital abnormalities including heart and renal problems [1814].

The presence of germ cells and sperm production are variable in men with Klinefelter syndrome and are more frequently observed in mosaicism, 46,XY/47,XXY. Based on sperm fluorescence in situ hybridisation (FISH) studies showing an increased frequency of sex chromosomal abnormalities and increased incidence of autosomal aneuploidy (disomy for chromosomes 13, 18 and 21), concerns have been raised about the chromosomal normality of the embryos generated through ICSI [1815]. The production of 24,XY sperm has been reported in 0.9% and 7.0% of men with Klinefelter mosaicism [1816,1817] and in 1.36-25% of men with somatic karyotype 47,XXY [1818-1821]. In patients with azoospermia, TESE or mTESE are therapeutic options as spermatozoa can be recovered in up to 50% of cases [1822,1823]. Although the data are not unique [1823], there is growing evidence that TESE or mTESE yields higher sperm recovery rates when performed at a younger age [1807,1824].

Numerous healthy children have been born using ICSI without pre-implantation genetic diagnosis (PGD) although the conception of one 47,XXY foetus has been reported [1811]. Although data published so far have not reported any difference in the prevalence of aneuploidy in children conceived using ICSI in Klinefelter syndrome compared to the general population, men with Klinefelter syndrome undergoing fertility treatments should be counselled regarding the potential genetic abnormalities in their offspring.

Regular medical follow-up of men with Klinefelter syndrome is recommended as testosterone therapy may be considered if testosterone levels are in the hypogonadal range when fertility issues have been addressed [18]. Since this syndrome is associated with several general health problems, appropriate medical follow-up is therefore advised [16,1825,1826]. In particular, men with Klinefelter syndrome are at higher risk of metabolic and cardiovascular diseases, including venous thromboembolism (VTE). Therefore, men with Klinefelter syndrome should be made aware of this risk, particularly when starting testosterone therapy [1827]. In addition, a higher risk of haematological malignancies has been reported in men with Klinefelter syndrome [16].

Testicular sperm extraction in peri-pubertal or pre-pubertal boys with Klinefelter syndrome aiming at cryopreservation of testicular spermatogonial stem cells is still considered experimental and should only be performed within a research setting [1828]. The same applies to sperm retrieval in older boys who have not considered their fertility potential [1829].

11.3.5.1.2. 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. As with Klinefelter syndrome, sperm FISH analysis provides a more accurate risk estimation of affected offspring. However, the use of this genetic test is largely limited by the availability of laboratories able to perform this analysis [1830]. When IVF/ICSI is carried out for men with translocations, PGD or amniocentesis should be performed [1831,1832].

11.3.5.2. Cystic fibrosis gene mutations

Cystic fibrosis (CF) is an autosomal-recessive disorder [1833]. 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 [1834]. Congenital bilateral absence of the vas deferens is a rare reason of male factor infertility, which is found 1% of infertile men and in up to 6% of men with obstructive azoospermia [1835]. Clinical diagnosis of absent vasa is easy to miss and all men with azoospermia should be carefully examined to exclude CBAVD, particularly those with a semen volume < 1.0 mL and acidic pH < 7.0 [1836-1838]. In patients with CBAVD-only or CF, TESA, epididymal sperm aspiration (micro or percutaneous; MESA and PESA respectively) or TESE 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 compared with CF patients [1834].

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 [1839,1840]. Given the functional relevance of a DNA variant (the 5T allele) in a non-coding region of CFTR [1841], it is now considered a mild CFTR mutation rather than a polymorphism and it should be analysed in each CBAVD patient. As more mutations are defined and tested for, almost all men with CBAVD will probably be found to have mutations. It is not practical to test for all known mutations, because many have a low prevalence in a particular population. Routine testing is usually restricted to the most common mutations in a particular community through the analysis of a mutation panel. Men with CBAVD often have mild clinical stigmata of CF (e.g., history of chest infections). When a man has CBAVD, it is important to test also his partner for CF mutations. If the female partner is found to be a carrier of CFTR mutations, the couple must consider carefully whether to proceed with ICSI using the man’s sperm, as the risk of having a child with CF or CBAVD will be 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% [1842].

11.3.5.2.1. 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 [1843]. Consequently, in these subjects CFTR mutation screening is not indicated. Men with CUAVD are usually fertile, and the condition is most commonly encountered as an incidental finding in the vasectomy clinic. Cystic fibrosis transmembrane conductance regulator gene mutation screening is indicated in men with unilateral absence of the vas deferens with normal kidneys. The prevalence of renal anomalies is rare in patients who have CBAVD and CFTR mutations [1844]. 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 [1845].

11.3.5.3. Y microdeletions – partial and complete

Microdeletions on the Y-chromosome are termed AZFa, AZFb and AZFc deletions [1846]. 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 [1847]. In each AZF region, there are several spermatogenesis candidate genes [1848]. Deletions occur en bloc (i.e., removing more than one gene), it is not possible to determine the role of a single AZF gene from the AZF deletion phenotype and it is unclear if they all participate in spermatogenesis. Gene-specific deletions, which remove a single gene, have been reported only in the AZFa region and concern the USP9Y gene. These studies have suggested that USP9Y is most likely to be a “fine tuner” of sperm production, and its specific screening is not advised [1849]. It has been observed that a number of commercial laboratories can use a limited number of primer sets over the AZF a, b and c regions in their Y chromosome microdeletion assay. This can eventually miss smaller microdeletions and clinicians should be aware of this in the work-up of patients scheduled for testicular
surgery [1850,1851].

11.3.5.3.1. Clinical implications of Y microdeletions

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

• They are not found in normozoospermic men, proving there is a clear cut cause-and-effect relationship between Y-deletions and spermatogenic failure [1852].
• The highest frequency of Y-deletions is found in azoospermic men (8-12%), followed by oligozoospermic (3-7%) men [1853,1854].
• Deletions are extremely rare with a sperm concentration > 5 million/mL (~0.7%) [1855].
• 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%) [1856].
• Complete deletion of the AZFa region is associated with severe testicular phenotype (Sertoli cell only syndrome), 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 at the time of TESE and sperm is not found in these patients. Therefore, TESE should not be attempted in these patients [1857,1858].
• Deletions of the AZFc region causes a variable phenotype ranging from azoospermia to oligozoospermia.
• Sperm can be found in 50-75% of men with AZFc microdeletions [1857-1859].
• Men with AZFc microdeletions who are oligo-zoospermic 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 [1855,1860].

The specificity and genotype/phenotype correlation reported above means that Y-deletion analysis has both a diagnostic and prognostic value for testicular sperm retrieval [1860].

11.3.5.3.1.1. 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 recent meta-analysis assessing the prevalence of microdeletions on the Y chromosome in oligo-zoospermic men in 37 European and North American studies (n1398042195=139804219512,492 oligo-zoospermic men) showed that the majority of microdeletions occurred in men with sperm concentrations < 1 million sperm/mL, with < 1% identified in men with > 1 million sperm/mL [1855]. 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 efforts 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 [1861].

11.3.5.3.1.2. Genetic counselling for AZF deletions

After conception, any Y-deletions are transmitted to the male offspring, and genetic counselling is therefore mandatory. In most cases, father and son will have the same microdeletion [1861], but occasionally the son may have a more extensive deletion [1862]. The extent of spermatogenic failure (still in the range of azoo-/oligo-zoospermia) cannot be predicted entirely in the son, due to the different genetic background and the presence or absence of environmental factors with potential toxicity on reproductive function. A significant proportion of spermatozoa from men with complete AZFc deletion are nullisomic for sex chromosomes [1863,1864], indicating a potential risk for any offspring to develop 45,X0 Turner’s syndrome and other phenotypic anomalies associated with sex chromosome mosaicism, including ambiguous genitalia [1865]. Despite this theoretical risk, babies born from fathers affected by Yq microdeletions are phenotypically normal [1860,1861]. This could be due to the reduced implantation rate and a likely higher risk of spontaneous abortion of embryos bearing a 45,X0 karyotype.

11.3.5.3.1.3. Y-chromosome: ‘gr/gr’ deletion

A new type of Yq deletion, known as the gr/gr deletion, has been described in the AZFc region [1866]. This deletion removes half of the gene content of the AZFc region, affecting the dosage of multicopy genes mapping inside this region. This type of deletion confers a 2.5 to 8-fold increased risk for oligozoospermia [1861,1867-1869]. The frequency of gr/gr deletion in oligozoospermic patients is ~5% [1870].

According to four meta-analyses, gr/gr deletion is a significant risk factor for impaired sperm production [1868-1870]. It is worth noting that both the frequency of gr/gr deletion and its phenotypic expression vary among different ethnic groups, depending on the Y-chromosome background. For example, in some Y haplo-groups, the deletion is fixed and appears to have no negative effect on spermatogenesis. Consequently, the routine screening for gr/gr deletion is still a debated issue, especially in those laboratories serving diverse ethnic and geographic populations. A large multi-centre study has shown that gr/gr deletion is a potential risk factor for testicular germ cell tumours (GCT) [1841]. However, these data need confirmation in an ethnically and geographically matched case-control study setting. For genetic counselling it is worth noting that partial AZFc deletions, gr/gr and b2/b3, may predispose to complete AZFc deletion in the next generation [1871].

11.3.5.3.1.4. Autosomal defects with severe phenotypic abnormalities and infertility

Several inherited disorders are associated with severe or considerable generalised abnormalities and infertility (e.g., Prader-Willi syndrome [1872], Bardet-Biedl syndrome [1873], Noonan’s syndrome, Myotonic dystrophy, dominant polycystic kidney disease [1874,1875], and 5 α-reductase deficiency etc., [1876-1879]) Pre-implantation genetic screening (PGS) may be necessary in order to improve the ART outcomes among men with autosomal chromosomal defects [1880,1881].

11.3.5.4. Sperm chromosomal abnormalities

Sperm can be examined for their chromosomal constitution using FISH both in men with normal karyotype and with anomalies. Aneuploidy in sperm, particularly sex chromosome aneuploidy, is associated with severe damage to spermatogenesis [1804,1882-1884] and with translocations and may lead to recurrent pregnancy loss or recurrent implantation failure [1885]. In a large retrospective series, couples with normal sperm FISH had similar outcomes from IVF and ICSI on PGS. However, couples with abnormal FISH had better clinical outcomes after PGS, suggesting a potential contribution of sperm to aneuploidic abnormalities in the embryo [1886]. In men with sperm aneuploidy, PGS combined with IVF and ICSI can increase chances of live
births [1799].

11.3.5.5. Measurement of oxidative stress

Oxidative stress is considered to be central in male infertility by affecting sperm quality, function, as well as the integrity of sperm [1887]. Oxidative stress may lead to sperm DNA damage and poorer DNA integrity, which are associated with poor embryo development, miscarriage and infertility [1888,1889]. 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 [1890]. However, these data have not been supported by RCTs. Furthermore, there are no standardised testing methods for ROS and the duration of antioxidant treatments. Although ROS can be measured by various assays (e.g., chemiluminescence), routine measurement of ROS testing should remain experimental until these tests are validated in RCTs [1891].

11.3.5.6. Outcomes from assisted reproductive technology and long-term health implications to the male and offspring

It is estimated that > 4 million babies have been born with ART since the first baby was conceived by IVF in 1978 [1892]. As the number of couples undergoing ART has increased [1893,1894], safety concerns related to ART have been raised. Assisted reproductive technology-conceived offspring have poorer prenatal outcomes, such as lower birth weight, lower gestational age, premature delivery, and higher hospital admissions compared with naturally conceived offspring [1895,1896]. However, the exact mechanisms resulting in these complications remain obscure. Birth defects have also been associated with children conceived via ART [1897-1899]. Meta-analyses have shown a 30-40% increase in major malformations linked with ART [1900-1902]. However, debate continues as to whether the increased risk of birth defects are related to parental age, ART or the intrinsic defects in spermatogenesis in infertile men [1903-1908].

As for the long-term outcomes, post-natal growth patterns are mostly not associated with ART [1897,1909,1910]. However, a number of studies have shown that ART children are taller [1911,1912]. This may be important as there is evidence showing that rapid weight gain during early childhood is linked with higher blood pressure levels in children conceived via ART [1913]. It is also suggested that ART-conceived children have similar childhood illnesses and hospital services rates as compared with naturally conceived children [1914-1916]. Some studies have shown an increased risk of retinoblastoma [1917] and hepatoblastoma in children after ART. However, these studies have been challenged with other studies that have not supported these findings [1918]. The current evidence for cancer risk in children conceived with ART is inadequate and further studies are warranted [1919,1920]. Finally, several epigenetic alterations seem to be caused by ART, which might be the molecular basis to some complex traits and diseases [1921].

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) [1771]. In clinical practice, Prader’s orchidometer-derived testicular volume is considered a reliable surrogate of US-measured testicular volume, easier to perform and cost-effective [1770]. 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, where the epididymis is large in comparison to the total testicular volume [1770,1771]). Ultrasound patterns of testicular inhomogeneity [1922,1923] is usually associated with ageing, although it has also been reported in association with testicular atrophy and fibrosis [1771]. At present, a diagnostic testicular biopsy is not recommended when testicular inhomogeneity is detected [1922,1923].

11.3.6.1. Scrotal US

Scrotal US is widely used in everyday clinical practice in patients with oligo-zoospermia or azoospermia, as infertility has been found to be an additional risk factor for testicular cancer [1924,1925]. It can be used in the diagnosis of several diseases causing infertility including testicular neoplasms and varicocele.

11.3.6.1.1. Testicular neoplasms

In one study, men with infertility had an increased risk of testicular cancer (hazard ratio [HR] 3.3). When infertility was refined according to individual semen parameters, oligo-zoospermic men had an increased risk of cancer compared with fertile control subjects (HR 11.9) [1926]. In a recent systematic review infertile men with testicular microcalcification (TM) were found to have a ~18-fold higher prevalence of testicular cancer [1927]. However, the utility of US as a routine screening tool in men with infertility to detect testicular cancer remains a matter of debate [1924,1925].

One issue in undertaking routine screening for testicular neoplasms in this cohort of patients is the risk of overdiagnosis and the increased detection of indeterminate lesions of the testis. These testicular lesions are often detected during the diagnostic work-up of infertile men and 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 definitely distinguish benign from malignant testicular masses is currently not available. However, in a study with 81 patients with a lesion size < 10 mm, on histology showed that 56 (69%) were benign lesions, although one-third were malignant. All lesions < 5 mm in diameter were benign [1928]. Available data suggest that the smaller the lesion, the less likely that it is malignant [1929], and lesions < 5 mm could be monitored, as they have a low probability of malignancy.

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) [1771].

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 sonographic abnormality; a hypoechoic lesion was found in 20 patients (14%), hyperechoic lesions were seen in 10 patients (7%); and, a heterogeneous appearance of the testicular parenchyma was seen in 19 patients (13%). Of 18 evaluable patients, 11 had lesions < 5 mm; 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. The authors concluded that men with severe infertility who have incidental testicular lesions, negative tumour markers and lesions < 5 mm may be observed with serial scrotal US examinations and enlarging lesions or those of greater dimension can be considered for histological biopsy [1930].

Other studies have suggested that if a testicular lesion is hyperechoic and non-vascular on colour Doppler US and associated with negative tumour markers, the likelihood of malignancy is low and consideration can be given to regular testicular surveillance, as an alternative to radical surgery. In contrast, hypoechoic and vascular lesions are more likely to be malignant [1931-1935]. However, most lesions cannot be characterised by US (indeterminate), and histology remains the only certain diagnostic tool. A multidisciplinary team discussion (MDT), 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 remains controversial, although several authors have proposed its value in the intra-operative diagnosis of indeterminate testicular lesions [1936]. Although the default treatment after patient counselling and MDT 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 or is hypogonadal. In men who have severe abnormalities in semen parameters (e.g., azoospermia), a concurrent mTESE can also be performed at the time of diagnostic biopsy.

In summary, if an indeterminate lesion is detected incidentally on US in an infertile man, MDT discussion is highly recommended. Based upon the current literature, lesions < 5mm in size are likely to be benign and serial US and self-examination can be performed. However, men with larger sized lesions (> 5mm), which are hypoechoic or demonstrate vascularity, may be considered for open US-guided testicular biopsy, testis sparing surgery with tumour enucleation for frozen section examination or radical orchidectomy. Therefore, in making a definitive treatment decision for surveillance vs. intervention, consideration should be given to the size of the lesion, echogenicity, vascularity and previous history (e.g., cryptorchidism, previous history of GCT). If intervention is to be undertaken in men with severe hypospermatogenesis (e.g., azoospermia), then a simultaneous TESE can be undertaken, along with sperm banking.

11.3.6.1.2. Varicocele

At present, 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 [1771]. Definitive evidence of reflux and venous diameter may be utilised in the decision to treat (see Sections 11.4.3.1 and 11.4.3.2).

11.3.6.1.3. Other

Scrotal US is able to 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 [1937,1938].

11.3.6.2. Transrectal US

For patients with a low seminal volume, acidic pH and severe oligozoospermia or azoospermia, in whom obstruction is suspected, scrotal and transrectal US are of clinical value in detecting CBAVD and presence or absence of the epididymis and/or seminal vesicles (SV) (e.g., abnormalities/agenesis). Likewise, transrectal US (TRUS) has an important role in assessing obstructive azoospermia (OA) secondary to CBAVD or anomalies related to the obstruction of the ejaculatory ducts, such as ejaculatory duct cysts, seminal vesicular dilatation or hypoplasia/atrophy, although retrograde ejaculation should be excluded as a differential diagnosis [1771,1939].

11.3.7. Recommendations for the diagnostic work-up of male infertility

11.4. Special Conditions and Relevant Clinical Entities

11.4.1. Cryptorchidism

Cryptorchidism is the most common congenital abnormality of the male genitalia; at 1 year of age nearly 1% of all full-term male infants have cryptorchidism [1940]. 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.

11.4.1.1. 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 then it is termed congenital while diagnosis of acquired cryptorchidism refers to men that have been previously noted to have testes situated within the scrotum. Cryptorchidism is classified as bilateral or unilateral and according to 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 [1941,1942]. However, testicular volume and hormonal function are reduced in adults treated for congenital bilateral cryptorchidism compared to unilateral cryptorchidism [1943].

11.4.1.1.1. 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 [1944]. Cryptorchidism has also been linked with maternal gestational smoking [1945] and premature birth [1946].

11.4.1.1.2. Pathophysiological effects in maldescended testes

11.4.1.1.2.1. 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 [1947]. During the second year, the number of germ cells declines. Early treatment is therefore recommended (surgery should be performed within the subsequent year) to conserve spermatogenesis and hormone production, as well as to decrease the risk for tumours [1948]. Surgical treatment is the most effective. Meta-analyses on the use of medical treatment with GnRH and hCG have demonstrated poor success rates [1949,1950]. It has been reported that hCG treatment may be harmful to future spermatogenesis; therefore, the Nordic Consensus Statement on treatment of undescended testes does not recommend it use on a routine basis [1951]. See also the EAU Guidelines on Paediatric Urology [1952].

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) [1941,1953]. This implies that unilateral cryptorchidism may affect the contralateral testis and patients and parents should be counselled appropriately.

11.4.1.1.2.2. Relationship with fertility

Semen parameters are often impaired in men with a history of cryptorchidism [1954]. Early surgical treatment may have a positive effect on subsequent fertility [1955]. In men with a history of unilateral cryptorchidism, paternity is almost equal (89.7%) to that in men without cryptorchidism (93.7%). In men with bilateral cryptorchidism, oligozoospermia can be found in 31% and azoospermia in 42%. In cases of bilateral cryptorchidism, the rate of paternity falls to 35-53% [1956]. 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 [1957].

11.4.1.1.2.3. Germ cell tumours

As a component of TDS, cryptorchidism is a risk factor for testicular cancer and is associated with testicular microcalcifications and intratubular germ cell neoplasia in situ (GCNIS), formerly known as carcinoma in situ (CIS) of the testes. In 5-10% of testicular cancers, there is a history of cryptorchidism [1958]. The risk of a 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 [1940]. Orchidopexy performed before the onset of puberty has been reported to decrease the risk of testicular cancer [1959]. However, there is evidence to suggest that even men who undergo early orchidopexy still harbour a higher risk of testicular cancer than men without cryptorchidism [1960]. 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 [1961]. There is also observational study data suggesting that cryptorchidism may be a risk factor for worsening clinical stage of seminoma but this needs to be substantiated with future prospective studies [1962].

11.4.1.2. Disease management

11.4.1.2.1. Hormonal treatment

Human chorionic gonadotropin or GnRH is not recommended for the treatment of cryptorchidism in adulthood. Although some studies have recommended the use of hormonal stimulation as an adjunct to orchidopexy to improve fertility preservation, there is a lack of long-term data and concerns regarding impairment to spermatogenesis with the use of these drugs [1963].

11.4.1.2.2. Surgical treatment

In adolescence, removal of an intra-abdominal testis (with a normal contralateral testis) can be recommended, because of the risk of malignancy [1964]. 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 [1965] and regular testicular self-examination is not an option in these patients. In patients with unilateral undescended testis (UDT) and impaired testicular function on the contralateral testis as demonstrated by biochemical hypogonadism and/or impaired sperm production (infertility), an orchidopexy may be offered to preserve androgen production and fertility. However, based on Panel consensus multiple biopsies of the UDT 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 [1966]. Vascular damage is the most severe complication of orchidopexy and can cause testicular atrophy in 1-2% of cases. In men with non-palpable testes, the post-operative atrophy rate was 12% in cases with long vascular pedicles that enabled scrotal positioning. Post-operative atrophy in staged orchidopexy has been reported in up to 40% of patients [1967]. 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, < 12 mL testicular volume, poor spermatogenesis [1968]).

11.4.1.3. 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 [1969]. 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., in Denmark and Norway) to 2/100,000 (e.g., in Finland and the Baltic countries). Generally, seminomas and non-seminomas are preceded by GCNIS, and untreated GCNIS eventually progresses to invasive cancer [1970-1972]. There has been a general decline in male reproductive health and an increase in testicular cancer in western countries [1973,1974]. In almost all countries with reliable cancer registries, the incidence of testicular cancer has increased [1860,1975]. 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. As detailed above, the adverse sequelae of TDS include cryptorchidism hypospadias, infertility and an increased risk of testicular cancer [1944]. Endocrine disrupting chemicals have also been associated with sexual dysfunction [1976] and abnormal semen parameters [1977]. These cancers arise from premalignant gonocytes or GCNIS [1978]. Testicular microcalcification, seen on US, can be associated with TGCT and GCNIS of the testes [1927,1979,1980].

11.4.2.1. Testicular germ cell cancer and reproductive function

Sperm cryopreservation is considered standard practice in patients with cancer overall, and not only in those with testicular cancer [1981,1982]. As such, it is important to stress that all men 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, radiotherapy or retroperitoneal surgery).

Men with TGCT have decreased semen quality, even before cancer treatment. Azoospermia has been observed in 5–8% of men with TGCT [1983] and oligospermia in 50% [1984]. Given that the average 10-year survival rate for testicular cancer is 98% and it is the most common cancer in men of reproductive potential, it is mandatory to include counselling regarding fertility preservation prior to any gonadotoxic treatment [1984,1985]. Semen analysis and cryopreservation are therefore recommended prior to any gonadotoxic cancer treatment and all patients should be offered cryopreservation of ejaculated sperm or sperm extracted surgically (e.g., c/mTESE) if shown to be azoospermic or severely oligozoospermic. Given that a significant number of men with testicular cancer at the time of first presentation have severe semen abnormalities (i.e., severe oligozoospermia/azoospermia) even prior to any treatment [1978], it is recommended that men should undergo sperm cryopreservation prior to orchidectomy. As mentioned above, in those who are either azoospermic or severely oligo-zoospermic this will allow an opportunity to perform TESE prior to further potential gonadotoxic/ablative surgery [1984]. The use of cryopreservation has been demonstrated to be the most cost effective strategy for fertility preservation in patients undergoing potential gonadotoxic treatments [1986,1987]. In cases of azoospermia, testicular sperm may be recovered to safeguard patient’s fertility (onco-TESE) potential. The surgical principles in onco-TESE do not differ from the technique of mTESE for men with infertility (e.g., NOA) [1988,1989]. In this context, referral to a urologist adept in microsurgery is desirable with facilities for sperm cryopreservation.

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 eventually benefit from fertility-preserving procedures (e.g., onco-mTESE at the time of orchidectomy). Therefore, counselling about fertility preservation is a priority and needs to be broached earlier in men with testicular cancer [1984]. There are controversial arguments that performing cryopreservation prior to orchidectomy may delay subsequent treatment and have an adverse impact on survival. In this context, orchidectomy should not be unduly delayed if there are no facilities for cryopreservation or there is a potential delay in treatment.

Treatment of TGCT can result in additional impairment of semen quality [1990] and increased sperm aneuploidy up to two years following gonadotoxic therapy [1991]. Chemotherapy is also associated with DNA damage and an increased DNA fragmentation rate [1992]. However, sperm aneuploidy levels often decline to pre-treatment levels 18-24 months after treatment [1991]. 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 radiotherapy [1993].

In addition to spermatogenic failure, patients with TGCT have Leydig cell dysfunction, even in the contralateral testis [1994]. The risk of hypogonadism may therefore be increased in men treated for TGCT. 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. Men who have had TGCT and have low normal androgen levels should be advised that they may be at increased risk of developing hypogonadism, as a result of an age-related decrease in testosterone production and could potentially develop MetS; there are no current long-term data supporting this. 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 [1995]. 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 [1970]. The risk of low libido and ED is also increased in TGCT patients [1996]. Patients treated for TGCT are also at increased risk of CVD [1992]. Therefore, patients may require a multi-disciplinary 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 selective oestrogen receptor modulators (SERMs; e.g., clomiphene) or gonadotrophin analogues (e.g., hCG), although these are off-label treatments in this particular clinical setting.

11.4.2.2. Testicular microcalcification (TM)

Microcalcification inside the testicular parenchyma can be found in 0.6-9% of men referred for testicular US [1997,1998]. 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, male pseudohermaphroditism and varicocele [1945]. The incidence reported seems to be higher with high-frequency US machines [1999]. 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% [2000-2002]. A recent 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) [1927].

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 [2003]. However, TM can also occur in benign testicular conditions and the microcalcification itself is not malignant. Therefore, 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. However, clinicians and patients should be reassured that testicular cancer does not develop in most men with asymptomatic TM [1980]. Available data indicate that only men in whom TM is found by US, and who have an increased risk of TGCT, should be offered testicular biopsy to exclude GCNIS. 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 [1974,1979]. The normal mean testicular volume is estimated to be 12-30 mL and < 12 mL is considered small [1997]. Patients with a history of TGCT and TM in the contralateral testis and sub-fertile patients have been demonstrated to have an increased risk of GCNIS [1980], while there are only a few studies showing a further increase in GCNIS with TM in the context of cryptorchidism [1974,1998,2004]. A useful algorithm has been proposed [1974] to stratify 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. With the lack of availability of large cohort studies, these recommendations must be treated with caution given the risk of overtreatment (i.e., biopsy) in these patients.

Decastro et al., [2005] suggested that testicular cancer would not develop in most men with TM (98.4%) during a 5-year follow-up. As such, an extensive screening programme would only benefit men at significant risk. In this context it would be 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 [1945]. As testicular atrophy and infertility have an association with testicular cancer, some authors recommend biopsy or follow-up US if TM is seen [1974]. However, most patients who are azoospermic will be undergoing therapeutic biopsy (i.e., with the specific purpose of sperm retrieval) and therefore a definitive diagnosis can be made and 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.

11.4.2.3. 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:

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

11.4.3.1. Classification

The following classification of varicocele [1749] 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.

Overall, the prevalence of varicocele in one study was 48%. Of 224 patients, 104 had unilateral and 120 had bilateral varicocele: 62 (13.30%) were grade 3; 99 (21.10%) were grade 2; and 63 (13.60%) were grade 1 [2006]. Worsening semen parameters are associated with a higher grade of varicocele and age [2007,2008].

11.4.3.2. 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 [1749,2009]. A maximum venous diameter of > 3 mm in the upright position and during the Valsalva manoeuvre and venous reflux with a duration > 2 seconds correlate with the presence of a clinically significant varicocele [2010,2011]. 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 [2012]. Patients with isolated, clinical right varicocele should be examined further for abdominal, retroperitoneal and congenital pathology and anomalies.

11.4.3.3. Basic considerations

11.4.3.3.1. 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 [1749,2007,2013,2014]. 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% [1749,2014].

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 [2014].

A meta-analysis showed that improvements in semen parameters are usually observed after surgical correction in men with abnormal parameters [2015]. Varicocelectomy can also reverse sperm DNA damage and improve OS levels [2013,2014].

11.4.3.3.2. 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 [2013,2016-2019]. Pain resolution after varicocelectomy occurs in 48-90% of patients [2020]. A recent systematic review has shown greater improvement in higher-grade varicoceles and this should be taken into account during patient counselling [2021].

In RCTs, varicocele repair in men with a subclinical varicocele was ineffective at increasing the chances of spontaneous pregnancy [2022]. 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 [2023]. 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 [2023]. 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 [2023]. 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) [2024]. Significantly improved post-operative semen parameters where reported in treated patients compared to controls with regards to sperm concentration (SMD 1.73; 95% CI 1.12 to 2.34; p<0.001), total sperm count (SMD 1.89; 95% CI 0.56 to 3.22; p < 0.05), progressive sperm motility (SMD 3.30; 95% CI 2.16 to 4.43; p < 0.01), total sperm motility (SMD 0.88; 95% CI 0.03 to 1.73; p=0.04) and normal sperm morphology (SMD 1.67; 95% CI 0.87 to 2.47; p < 0.05) [2024].

A Cochrane review from 2012 concluded that there is evidence to suggest that treatment of a varicocele in men from couples with otherwise unexplained subfertility may improve a couple’s chance of spontaneous pregnancy [2025]. Similarly, a Cochrane review from 2021 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 to 2.26) [2026]. 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 [2019,2025]. Average time to improvement in semen parameters is up to two spermatogenic cycles [2027,2028] with spontaneous pregnancy occurring between 6 and 12 months after varicocelectomy [2029,2030]. A further meta-analysis has reported that varicocelectomy may improve outcomes following ART in oligozoospermic men with an OR of 1.69 (95% CI: 0.95-3.02) [2031].

11.4.3.3.3. 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 [2032]. 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 [2033,2034].

More novel considerations for varicocelectomy in patients with NOA, hypogonadism and DNA damage are described below:

Varicocelectomy and NOA

Several 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 post-operatively with an increase in ensuing natural or assisted pregnancies [2035]. There were further beneficial effects on sperm retrieval rates (SRRs) and ICSI outcomes. 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, 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 post-operative ejaculate. These findings indicate that varicocelectomy in patients with NOA and clinical varicocele is associated with improved SRR, and overall, 44% of the treated men have sperm in the ejaculate and may avoid sperm retrieval. 1397256787However, the quality of evidence available 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 [2017]. This must necessarily take into consideration the infertile couple together, especially considering the time needed for a possible SRR and the baseline characteristics of the female partner (i.e., age, medical history, anti-Müllerian hormone (AMH) levels = good ovarian reserve, etc.).

Varicocelectomy and hypogonadism

Evidence also suggests that men with clinical varicoceles who are hypogonadal may benefit from varicocele intervention. One meta-analysis studied the efficacy of varicocele intervention by comparing the pre-operative and post-operative serum testosterone of 712 men. The combined analysis of seven studies demonstrated that the mean post-operative serum testosterone improved by 34.3 ng/dL (95% CI: 22.57-46.04, p < 0.00001,
I² = 0%) compared with their pre-operative levels. An analysis of surgery vs. untreated control results showed that mean testosterone among hypogonadic patients increased by 105.65 ng/dL (95% CI: 77.99-133.32 ng/dL), favouring varicocelectomy [2036]. However, 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 in this setting. Although, varicocelectomy may be offered to hypogonadal men with clinically significant varicoceles, patients must be advised that the full benefits of treatment in this setting must be further evaluated with prospective RCTs.

11.4.3.3.4. Varicocelectomy for assisted reproductive technology and raised DNA fragmentation

Varicocelectomy can improve sperm DNA integrity, with a combined mean difference of -3.37% to - 5.46% (95% CI: 2.65% to -4.09% and 95% CI: -4.79 to -6.13, respectively) [2032,2037]. A systematic review and meta-analysis analysed data from 1,070 infertile men with clinical varicocele and showed that varicocelectomy was associated with reduced post-operative sperm DNA fragmentation rates (weighted mean difference 7.23%; 95% CI: 8.86 to 5.59) [2038]. 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 pre-operative mean fragmentation index > 20% than that in studies with sperm deoxyribonucleic acid fragmentation (SDF) < 20%, suggesting that varicocelectomy might be more beneficial in men with elevated baseline SDF values [2038]. The magnitude of the effect size increased as a function of preoperative SDF levels (coefficient: 0.23; 95%CI: 0.07 to 0.39).

Varicocelectomy can improve sperm DNA integrity, with a mean difference of -3.37% (95% CI: -2.65% to -4.09%) [2032]. There is now increasing evidence that varicocele treatment may improve DNA fragmentation and outcomes from ART [2031,2032]. As a consequence, more recently it has been suggested that the indications for varicocele intervention should be expanded to include men with raised DNA fragmentation. 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 [2039], and exclusion of other causes of raised DNA fragmentation [2032,2040].The dilemma remains as to whether varicocele treatment is indicated in men with raised DNA fragmentation 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 [2031]. 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).

11.4.3.4. Disease management

Several treatments are available for varicocele (Table 54).

Impact on pregnancy rate and semen parameters

Current evidence indicates that microsurgical varicocelectomy is the most effective among the different varicocelectomy techniques [2032,2041]. 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 to 1.36) [2026]. 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 [2023]. However, the most recent Cochrane review showed inconclusive results about the effect of surgical ves. radiological treatment on pregnancy rates and varicocele recurrence [2026]. 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 [2026,2042,2043]; however, this procedure, requires microsurgical training. The various other techniques are still considered viable options, although recurrences and hydrocele formation appear to be higher [2043].

Radiological techniques (sclerotherapy and embolisation) are minimally invasive approaches for varicocele treatment. Although higher recurrence rates have been reported compared to microscopic varicocelectomy [1754], a meta-analysis showed that the incidence of varicocele recurrence was similar after surgical ligation and sclero-embolisation [2044]. In terms of complications, a meta-analysis of twelve studies comparing 738 cases of surgical ligation vs. 647 cases of sclero-embolisation, showed that overall complications rate did not differ significantly between the groups (OR 1.48; 95% CI 0.86–2.57, p = 0.16) [2044]. The incidence of post-operative hydrocele is significantly higher after surgical ligation than sclero-embolisation, but radiological techniques are associated with higher incidence of post-operative orchiepidydimitys [2044].

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 [2045-2047].

Table 54: Recurrence and complication rates associated with treatments for varicocele

11.4.3.5. Summary of evidence and recommendations for varicocele

11.4.4. Male accessory gland infections and infertility

11.4.4.1. Introduction

Infection of the male urogenital tract is a potentially curable cause of male infertility [2059-2061]. The WHO considers urethritis, prostatitis, orchitis and epididymitis to be male accessory gland infections (MAGIs) [2059]. The effect of symptomatic or asymptomatic infections on sperm quality is contradictory [2062]. A systematic review of the relationship between sexually transmitted infections, 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 sexually transmitted infections and male infertility due to the limited quality of reported data [2063].

11.4.4.2. Diagnostic evaluation

11.4.4.2.1. Semen analysis

Semen analysis (see Section 11.3.2) clarifies whether the prostate is involved as part of a generalised MAGI and provides information regarding sperm quality. Leukocyte analysis allows differentiation between inflammatory and non-inflammatory chronic pelvic pain syndrome (CP/CPPS) (NIH IIa vs. NIH 3b National Institutes of Health classification for CP/CPPS).

11.4.4.2.2. Microbiological findings

After exclusion of UTI (including urethritis), > 10⁶ peroxidase-positive white blood-cells (WBCs) per millilitre of ejaculate indicate an inflammatory process. In these cases, a semen culture or polymerase chain reaction (PCR) analysis should be performed for common urinary tract pathogens. A concentration of > 10³ CFU/mL urinary tract pathogens in the ejaculate is indicative of significant bacteriospermia [2064]. 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 [2065]. The ideal diagnostic test for isolating C. trachomatis in semen has not yet been established [2066], but the most accurate method is PCR [2067-2069].

Historical data show that Ureaplasma urealyticum is pathogenic only in high concentrations (> 10³ CFU/mL ejaculate). Fewer than 10% of samples analysed for Ureaplasma exceeded this concentration [2070]. Normal colonisation of the urethra hampers the significance of mycoplasma-associated urogenital infections, using samples such as the ejaculate [2071].

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) [2072].

The prevalence of human papilloma virus (HPV) in the semen ranges from 2 to 31% in the general population and is higher in men with unexplained infertility (10-35.7%) [2073,2074]. Recent systematic reviews have reported an association between male infertility, poorer pregnancy outcomes and semen HPV positivity [2075-2077]. However, data still needs 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 men may be associated with lower sperm quality compared to that in HSV-negative infertile men [2062]. However, it is unclear if anti-viral therapy improves fertility rates in these men.

11.4.4.2.3. White blood cells

The clinical significance of an increased concentration of leukocytes in the ejaculate is controversial [2078]. 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 [2079]. According to the WHO classification, leukocytospermia is defined as > 10⁶ WBCs/mL. Only two studies have analysed alterations of WBCs in the ejaculate of patients with proven prostatitis [2080,2081]. Both studies found more leukocytes in men with prostatitis compared to those without inflammation (CPPS, type NIH 3b). Furthermore, leukocytospermia should be further confirmed by performing a peroxidase test on the semen. There is currently no evidence that treatment of leukocytospermia alone without evidence of infective organisms improves conception rates [2082].

11.4.4.2.4. Sperm quality

The deleterious effects of chronic prostatitis (CP/CPPS) on sperm density, motility and morphology have been demonstrated in a recent systematic review based on case-controlled studies [2083]. Both C. trachomatis and Ureoplasma spp. can cause decreased sperm density, motility, altered morphology and increased DNA damage. Data from a recent retrospective cross-sectional study showed that U. urealyticum was the most frequent single pathogen in semen of asymptomatic infertile men; a positive semen culture was both univariably (p < 0.001) and multi-variably (p = 0.04) associated with lower sperm concentration [2084]. Human papilloma virus can also induce changes in sperm density, motility and DNA damage [2073,2074]. Mycoplasma spp. can cause decreased motility and development of antisperm antibodies [2062].

11.4.4.2.5. Seminal plasma alterations

Seminal plasma elastase is a biochemical indicator of polymorphonuclear lymphocyte activity in the ejaculate [2061,2085,2086]. 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 [2087-2089].

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 [2090]. However, elevated cytokine levels do not depend on the number of leukocytes in expressed prostatic secretion [2091].

11.4.4.2.6. 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; the seminal plasma concentrations of these factors are usually altered during infection and inflammation. However, they are not recommended as diagnostic markers for MAGIs [2092].

11.4.4.2.7. 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. However, the levels of ROS in infertile patients with asymptomatic C. trachomatis and M. hominis in the semen are low, making it difficult to draw any firm conclusions [2093]. Chronic urogenital infections are also associated with increased leukocyte numbers [2094]. However, their biological significance in prostatitis remains unclear [2061].

11.4.4.2.8. Disease management

Treatment of CP/CPPS is usually targeted at relieving symptoms [2095,2096]. The indications and aims of therapy are:

• reduction or eradication of micro-organisms in prostatic secretions and semen;
• normalisation of inflammatory (e.g., leukocytes) and secretory parameters;
• improvement of sperm parameters associated with fertility impairment [2097].

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 [2097], there is no evidence that treatment of CP/CPPS increases the probability of natural conception [2061,2098].

Asymptomatic presence of C. trachomatis and M. hominis in the semen can be correlated with impaired sperm quality, which recovers after antibiotic treatment. However further research is required to confirm these findings [2093].

11.4.4.3. Epididymitis

Inflammation of the epididymis causes unilateral pain and swelling, usually with acute onset. Among sexually active men aged < 35 years, epididymitis is most often caused by C. trachomatis or N. gonorrhoea [2099,2100]. Sexually transmitted epididymitis is usually accompanied by urethritis. Non-sexually transmitted epididymitis is associated with UTIs and occurs more often in men aged > 35 years [2101].

11.4.4.3.1. Diagnostic evaluation

11.4.4.3.1.1. Ejaculate analysis

Ejaculate analysis according to WHO Laboratory Manual for the Examination and Processing of Human Semen (6^th edition) criteria, may indicate persistent inflammatory activity. Transient reductions in sperm counts and progressive motility can be observed [2099,2102,2103]. 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).

11.4.4.3.1.2. 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 told to also refer their sexual partners for evaluation and treatment [2104].

11.4.4.4. Summary of evidence and recommendation for male accessory gland infections

11.5. Non-Invasive Male Infertility Management

11.5.1. Idiopathic male infertility and oligo-astheno-terato-zoospermia

Oligo-astheno-teratozoospermia (OAT) is a clinical condition with a reduced number of spermatozoa in the ejaculate, which is also characterised by reduced sperm motility and morphology; often referred to as OAT syndrome (OATS). Several conditions can cause OATS, although the aetiology may be unknown in a significant number of cases [104,1925].

11.5.2. Empirical treatments

11.5.2.1. Lifestyle

Studies suggest that environmental and lifestyle factors may contribute to idiopathic infertility acting additively on a susceptible genetic background [104,1925]. Hence, lifestyle improvement can have a positive effect on sperm parameters (see below).

11.5.2.1.1. Weight loss

Few authors have investigated the role of weight loss on male fertility outcomes. Non-controlled studies have suggested that weight loss can result in improved sperm parameters [104,2105,2106]. However, 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 morbidly obese men [2107]. Data on ART outcomes are lacking. However, it is important to recognise that weight loss can improve obesity-related secondary hypogonadism, which may result in better outcomes in couples seeking medical care for infertility, and is important for the general health of the male partner [2105,2107].

11.5.2.1.2. Physical activity

Regular physical activity is recommended by the WHO in order to prevent and reduced the risk of several long-term chronic diseases [2108]. A recent meta-analysis has documented that moderate-intensity (20–40 metabolic equivalents [METs]/week) or even high-intensity (40–80 METs-h/week) recreational physical activity can result in better semen parameters [2109]. In addition, similar to what is observed from weight loss, improvements in hormonal profile have also been reported [2105].

11.5.2.1.3. Smoking

Epidemiological data indicates that about one in three men of reproductive age smokes, with the highest prevalence observed in Europe among all the WHO regions [2110]. Data derived from a large meta-analysis of 20 studies with 5,865 participants clearly show a negative association between smoking and sperm parameters [2110]. Experimental studies performed in rats have shown that nicotine has a dose-dependent deleterious effect on sperm, which can be improved by nicotine cessation [2111]. Data in men are lacking and only one case report has indicated an improvement of sperm parameters after 3 months of a smoking cessation programme [2112]. Similar data have been reported in a recent non-controlled study, which showed a possible benefit on ART after the male partner stopped smoking [2113].

11.5.2.1.4. Alcohol consumption

Data derived from a recent 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 [2114]. Similar to what has been reported for weight loss; however, heavy chronic alcohol consumption (defined as > 2 drinks/day [2115]) can reduce testosterone levels, which can be restored by alcohol cessation [2116].

11.5.2.2. Antioxidant treatment

Inflammation is a positive reaction of the human body to overcome potential noxious stimuli. However, chronic inflammation can induce several negative biochemical and metabolic effects that contribute to the development of several medical conditions. Oxidative stress is considered to be 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 DNA fragmentation [2117]. Accordingly, seminal levels of ROS have been negatively associated with ART outcomes [2118]. Despite this, evidence for the role of antioxidant therapy in male infertility is still conflicting. A Cochrane systematic review and meta-analysis including 34 RCTs and 2,876 couples using various antioxidant compounds, it was concluded that antioxidant therapy had a positive impact on live-birth and pregnancy rates in sub-fertile couples undergoing ART cycles [2119]. Similar results were also reported in the most recent meta-analysis including 61 studies with 6,264 infertile men, aged 18-65 years [2120]. More recently, the Males, Antioxidants, and Infertility (MOXI) trial found that antioxidants did not improve semen parameters or DNA integrity compared to placebo among infertile men with male factor infertility. Moreover, cumulative live-birth rate did not differ at 6 months between the antioxidant and placebo groups (15% vs. 24%) [2121]. However, all the aforementioned studies also recognised important limitations: data were derived from low-quality RCTs with serious risk of bias due to poor methods of reporting randomisation; failure to report on the clinical outcomes including live-birth and clinical pregnancy rates; high attrition rates; and imprecision due to often low event rates and small overall sample sizes [2120]. No clear conclusions were possible regarding the specific antioxidants to use or and/or therapeutic regimes for improving sperm parameters and pregnancy rate [2120].

11.5.2.3. Selective oestrogen receptor modulators

Selective oestrogen receptor modulators (SERMs) have been advocated as a possible empirical treatment in male idiopathic infertility. The proposed mechanism of action is based on the activity of these compounds to block oestrogen receptors at the level of the hypothalamus, which results in stimulation of GnRH secretion leading to an increase in pituitary gonadotropin release. The latter effect, by stimulating spermatogenesis, represents the rational basis for SERM administration to patients with reduced sperm count [2122]. In an initial meta-analysis including 11 RCTs, in which only 5 were placebo-controlled, it was concluded that SERMs were not associated with an increased pregnancy rate in the 459 patients analysed [2123]. In a subsequent Cochrane review published 1 year later, these findings were confirmed in a larger number of studies (n = 10 and 738 men), although positive effects on hormonal parameters were documented. More recently, Chua et al., meta-analysed data derived from 11 RCTs and showed that SERMs were associated with a significantly increased pregnancy rate [2124]. Additionally, a significant improvement in sperm and hormonal parameters was detected. Similar results were confirmed in the latest updated meta-analysis of 16 studies [2122]. However, it should be recognised that the quality of the papers included was low and only a few studies were placebo-controlled. In conclusion, although some positive results relating to the use of SERMs in men with idiopathic infertility have been reported, no conclusive recommendations can be drawn due to poor quality of the available evidence. Furthermore, complications from the use of SERMs were under-reported.

11.5.2.4. Aromatase inhibitors

Aromatase, a cytochrome p450 enzyme, is present in the testes, prostate, brain, bone, and adipose tissue of men; 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, aromatase inhibitors (AIs) may decrease oestrogen production by reversibly inhibiting cytochrome p450 isoenzymes 2A6 and 2C19 of the aromatase enzyme complex inhibiting the negative feedback of oestrogen on the hypothalamus resulting in stronger GnRH pulses that stimulate the pituitary to increase production of FSH [2125-2128]. Aromatase activity has been associated with male infertility characterised by testicular dysfunction with low serum testosterone and/or testosterone to oestradiol ratio. In this context, AIs have been reported to increase endogenous testosterone production and improve spermatogenesis in the setting of infertility as an off-label option for treatment [2129]. Either steroidal (testolactone) and non-steroidal (anastrozole and letrozole) AIs significantly improve hormonal and semen parameters in infertile men, with a safe tolerability profile, although prospective RCTs are necessary to better define the efficacy of these medications in this clinical setting [2127,2129].

11.5.3. Hormonal therapy

11.5.3.1. Gonadotrophins

Follicle Stimulating Hormone is primarily involved in the initiation of spermatogenesis and testicular growth during puberty. The role of FSH post puberty has not been clearly defined. Luteinising hormone stimulates testosterone production in the testes, but due to its short half-life, it is not suitable for clinical use. Human Chorionic Gonadotrophin acts in a similar manner to LH and can be used pharmacologically to stimulate testosterone release in men with failure of their hypothalamic-pituitary-gonadal axis. Human Chorionic Gonadotrophin can adequately stimulate spermatogenesis in men who have developed hypopituitarism after normal puberty. Therefore, the treatment of men with secondary hypogonadism depends on whether or not they developed hypothalamic-pituitary failure before or after puberty [5].

11.5.3.2. Secondary hypogonadism

11.5.3.2.1. Pre-Pubertal-Onset

Congenital causes resulting in low gonadotropin production are associated with testicular size < 4 mL and/or cryptorchidism. Testes size of < 4 mL occurs when they have not been exposed to any gonadotropins at all. These conditions require combination therapy with both hCG and FSH with subcutaneous administration or GnRH by pulsed delivery using a subcutaneous pump [2130]. However, GnRH treatment requires a pulsatile secretion using specific devices for either intravenous or subcutaneous administration, which may limit patient compliance. Moreover, GnRH therapy should be limited to subjects with a residual pituitary gonadotropic activity [5].

As for the type of gonadotropin treatment, it is usual to commence hCG first and titrate the dose to achieve testosterone levels within the normal physiological range. However, FSH can be given first or in combination with hCG [141]. Human Chorionic Gonadotrophin is given twice weekly and in patients with congenital secondary hypogonadism in high dose, commencing at 1,000 IU twice weekly. Testosterone levels can be assayed every 2 weeks with dose increases until ideally mid-range testosterone is achieved. Dose increases can be to 2,000, 3,000, 4,000 and 5,000 IU two or three times a week, until normal testosterone levels are achieved [2131-2134]. Failure to achieve normal testosterone status at the high dose would indicate that primary testicular failure is present; probably as a result of cryptorchidism or failure of testicular development. Human Chorionic Gonadotrophin is also used to stimulate testicular descent into the scrotum in individuals with cryptorchidism. Once the hCG dose giving a normal level testosterone is established with the implication that intra-testicular testosterone has occurred, FSH 75-150 IU three times per week subcutaneously should be commenced. Usually the higher 150 IU dose three times weekly is needed to be successful in men with testicular volume < 4mL. The trophic response of the testes to FSH is variable in these patients and it may range from no effect to achieving testicular sizes of 12-15 mL [2135]. A trophic response is usually an indication of an increase in spermatogenesis. The production of new spermatogenesis may be evident after 3 months of FSH therapy, but could occur even after 18 months of treatment [2133-2135]. A low baseline sperm concentration does not indicate a poor response to gonadotropin therapy [2136]. Semen analysis can be assessed at 3-monthly intervals. These patients can be fertile with low sperm counts < 20 million/mL as there is a high proportion of motile sperm. Follicle-stimulating hormone therapy prior to GnRH is also effective in stimulating testicular growth and fertility in men with congenital hypogonadotropic hypogonadism (HH) [2137]. A larger initial testicular volume is the best prognostic factor for induction of successful spermatogenesis [2138].

11.5.3.2.2. Post-Pubertal Onset Secondary

If secondary hypogonadism develops after puberty, hCG alone is usually required first to stimulate spermatogenesis. Doses of subcutaneous hCG required may be lower than those used in individuals with pre-pubertal onset; therefore, a starting dose of 250 IU twice weekly is suggested, and if normal testosterone levels are reached, hCG doses may be increased up to 2,000 IU twice weekly as for pre-pubertal onset. Again, semen analysis should be performed every 3 months to assess response, unless conception has taken place. If there is a failure of stimulation of spermatogenesis, then FSH can be added (75 IU three times per week, increasing to 150 IU three times per week if indicated). Similarly, combination therapy with FSH and hCG can be administered from the beginning of treatment, promoting better outcomes in men with HH [141]. No difference in outcomes were observed when urinary-derived, highly purified FSH was compared to recombinant FSH [141].

Greater baseline testicular volume is a good prognostic indicator for response to gonadotrophin treatment [2138]. Data had suggested that previous testosterone therapy can have a negative impact on gonadotropin treatment outcomes in men with HH [2138]. However, this observation has been subsequently 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 [141].

In the presence of hyperprolactinaemia, causing suppression of gonadotrophins resulting in sub-fertility the 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.

11.5.3.3. 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 [105,2139].

11.5.3.4. Idiopathic Male Factor Infertility

There is some evidence that FSH treatment increases sperm parameters in idiopathic oligozoospermic men with FSH levels within the normal range (generally 1.5 – 8 mIU/mL) [2140]. It has also been reported that FSH may improve sperm DNA fragmentation rates as well as ameliorating AMH and inhibin levels [2141-2144]. High-dose FSH therapy is more effective in achieving a testicular response than lower doses are [2145]. 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. However, no significant difference among groups was observed when ICSI or IUI were considered [2146]. In a more recent 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 [2147]. A further study showed that in azoospermic men undergoing TESE-ICSI there were improved SRRs and higher pregnancy and fertilisation rates in men treated with FSH compared to untreated men [2148]. In men with NOA, combination hCG/FSH therapy was shown to increase SRR in only one study [2149]. Human chorionic gonadotrophin alone prior to TESE in NOA has not been found to have any benefit on SRRs [2150]. 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.

11.5.3.5. Anabolic Steroid Abuse

Oligospermia or azoospermia as a result of anabolic abuse should be treated initially 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. If after this interval the condition persists, then hCG without or in combination with FSH as an alternative to clomiphene can be used to try and stimulate spermatogenesis [2151].

11.5.3.6. Recommendations for treatment of male infertility with hormonal therapy

11.6. Invasive Male Infertility Management

11.6.1. Obstructive azoospermia

Obstructive azoospermia (OA) is the absence of spermatozoa in the sediment of a centrifuged sample of ejaculate due to obstruction [2059]. Obstructive azoospermia is less common than NOA and occurs in 20-40% of men with azoospermia [2152,2153]. Men with OA usually have normal FSH, testes of normal size and epididymal enlargement [2154]. Of clinical relevance, men with late maturation arrest may present with normal gonadotropins and testicular size and may be only distinguished from those with OA at the time of surgical exploration. The vas deferens may be absent bilaterally (CBAVD) or unilaterally (CUAVD). Obstruction in primary infertile men is more frequently present at the epididymal level.

11.6.1.1. Classification of obstructive azoospermia

11.6.1.1.1. Intratesticular obstruction

Intratesticular obstruction occurs in 15% of men with OA [2155]. Congenital forms are less common than acquired forms (post-inflammatory or post-traumatic) (Table 55).

11.6.1.1.2. Epididymal obstruction

Epididymal obstruction is the most common cause of OA, affecting 30-67% of azoospermic men [2155-2158]. Congenital epididymal obstruction usually manifests as CBAVD, which is associated with at least one mutation of the CF gene in 82% of cases [2158]. Other congenital forms of epididymal obstruction include chronic sinu-pulmonary infections (Young’s syndrome) [2159]. Acquired secondary to acute (e.g., gonococcal) and subclinical forms (e.g., Chlamydial) epididymitis are most commonly due to infections [2160,2161]. Other causes may be trauma or surgical intervention [2162,2163] (Table 55).

11.6.1.1.3. Vas deferens obstruction

Vas deferens obstruction is the most common cause of acquired obstruction following vasectomy [2160] (Table 55). Approximately 2-6% of these men request vasectomy reversal (see 2019 EAU Guidelines on Male Infertility). Vasal obstruction may also occur after hernia repair [2164,2165]. The most common congenital vasal obstruction is CBAVD, often accompanied by CF. Unilateral agenesis or a partial defect is associated with contralateral seminal duct anomalies or renal agenesis in 80% and 26% of cases, respectively [1844].

11.6.1.1.4. Ejaculatory duct obstruction

Ejaculatory duct obstruction is found in 1-5% of cases of OA and is classified as cystic or post-inflammatory or calculi of one or both ejaculatory ducts [1984,2166] (Table 55). Cystic obstructions are usually congenital (i.e., Müllerian duct cyst or urogenital sinus/ejaculatory duct cysts) and are typically midline. In urogenital sinus abnormalities, one or both ejaculatory ducts empty into the cyst [2167], while in Müllerian duct anomalies, the ejaculatory ducts are laterally displaced and compressed by the cyst [2168]. Paramedian or lateral intraprostatic cysts are rare [2169]. Post-inflammatory obstructions of the ejaculatory duct are usually secondary to urethra-prostatitis [2170]. Congenital or acquired complete obstructions of the ejaculatory ducts are commonly associated with low seminal volume, decreased or absent seminal fructose, and acidic pH. The seminal vesicles (anterior-posterior diameter > 15 mm) and ejaculatory duct (> 2.3 mm in width) are usually dilated [2166,2170-2172].

11.6.1.1.4.1. Functional obstruction of the distal seminal ducts

Functional obstruction of the distal seminal ducts might be attributed to local neurogenic dysfunction [2173]. This abnormality is often associated with urodynamic dysfunction. Impaired sperm transport can be observed as idiopathic or due to spinal cord injury, multiple sclerosis, retroperitoneal lymph node dissection, pelvic surgery, SSRIs, α-blockers and typical antipsychotic medications [2174].

Table 55: Causes of obstruction of the genitourinary system

11.6.1.2. Diagnostic evaluation

11.6.1.2.1. Clinical history

Clinical history-taking should follow the investigation and diagnostic evaluation of infertile men (See Section 10.3). Risk factors for obstruction include prior surgery, iatrogenic injury during inguinal herniorrhaphy, orchidopexy or hydrocelectomy.

11.6.1.2.2. Clinical examination

Clinical examination should follow the guidelines for the diagnostic evaluation of infertile men. Obstructive azoospermia is indicated by at least one testis with a volume > 15 mL, although a smaller volume may be found in some patients with:

• obstructive azoospermia and concomitant partial testicular failure;
• enlarged and dilated epididymis;
• nodules in the epididymis or vas deferens;
• absence or partial atresia of the vas deferens.

11.6.1.2.3. Semen analysis

Azoospermia means the inability to detect spermatozoa after centrifugation at ×400 magnification. At least two semen analyses must be carried out [2175,2176] (see Section 10.3). When semen volume is low, 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.

11.6.1.2.4. 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 [2177].

11.6.1.2.5. Genetic testing

Inability to palpate one or both sides of the vas deferens should raise concern for a CFTR mutation. Any patient with unilateral or bilateral absence of the vas deferens or seminal vesicle agenesis should be offered CFTR testing [2178].

11.6.1.2.6. Testicular biopsy

Testicular biopsy must be combined with TESE for cryopreservation. Although studies suggest that a diagnostic or isolated testicular biopsy [2179] is the most important prognostic predictor of spermatogenesis and sperm retrieval, the Panel recommends not to perform testis biopsies (including fine needle aspiration [FNA]) without performing simultaneously a therapeutic sperm retrieval, as this will require a further invasive procedure after biopsy. Furthermore, even patients with extremes of spermatogenic failure (e.g., Sertoli Cell Only syndrome [SCOS]) may harbour focal areas of spermatogenesis [2180,2181].

11.6.1.3. Disease management

Sperm retrieval

11.6.1.3.1. Intratesticular obstruction

Only TESE allows sperm retrieval in these patients and is therefore recommended.

11.6.1.3.2. Epididymal obstruction

Microsurgical epididymal sperm aspiration (MESA) or percutaneous epididymal sperm aspiration (PESA) [2182] is indicated in men with CBAVD. Testicular sperm extraction and percutaneous techniques, such as testicular sperm aspiration (TESA), are also options [2183]. 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 fertilisation, pregnancy, or miscarriage rates [2184]. Usually, one MESA procedure provides sufficient material for several ICSI cycles [2185] and it produces high pregnancy and fertilisation rates [2186]. In patients with OA due to acquired epididymal obstruction and with a female partner with good ovarian reserve, microsurgical epididymovasostomy (EV) is recommended [2187]. Epididymovasostomy can be performed with different techniques such as end-to-site and intussusception [2188].

Anatomical recanalisation following surgery may require 3-18 months. A recent 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% [2189]. Before microsurgery, and in all cases in which recanalisation is impossible, epididymal spermatozoa should be aspirated intra-operatively by MESA and cryopreserved to be used for subsequent ICSI procedures [2170]. Patency rates range between 65% and 85% and cumulative pregnancy rates between 21% and 44% [2163,2190]. Recanalisation success rates may be adversely affected by pre-operative and intra-operative findings. Robot-assisted EV has similar success rates but larger studies are needed [2191].

11.6.1.3.3. 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 [2163,2190]. The average time to patency is 1.7-4.3 months and late failures are uncommon (0-12%) [2189]. Robot-assisted vasovasostomy has similar success rates, and larger studies, including cost-benefit analysis, are needed to establish its benefits over standard microsurgical procedures [2191].

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 [2192-2194]. Simultaneous sperm retrieval may be performed for future cryopreservation and use for ICSI; likewise, patients should be counselled appropriately.

11.6.1.3.4. 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 [2195] can be used for cryopreservation for future ICSI. Prostate cancer patients who express an interest in future fertility should be counselled for cryopreservation [2196,2197].

11.6.1.3.5. 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 [2166,2170]. 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 [2166,2170].

Intra-operative TRUS makes this procedure safer. If distal seminal tract evaluation is carried out at the time of the procedure, installation of methylene blue dye into the seminal vesicles (chromotubation) can help to confirm intra-operative opening of the ducts. Pregnancy rates after TURED are 20-25% [1984,2166,2198]. Complications following TURED include epididymitis, UTI, gross haematuria, haematospermia, azoospermia (in cases with partial distal ejaculatory duct obstruction) and urine reflux into the ejaculatory ducts and seminal vesicles [2166].

Alternative therapies for EDO include, seminal vesiculoscopy to remove debris or calculi and balloon dilation and laser incision for calcification on TRUS [2199]. The alternatives to TURED are MESA, PESA, TESE, proximal vas deferens sperm aspiration and seminal vesicle-ultrasonically guided aspiration.

11.6.1.4. Summary of evidence and recommendations for obstructive azoospermia

11.6.2. Non-obstructive azoospermia

Non-obstructive azoospermia (NOA) is defined as the absence of sperm at the semen analysis after centrifugation, with usually a normal ejaculate volume. This finding should be confirmed at least at two consecutives semen analyses [1780]. 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.

11.6.2.1. Investigation of non-obstructive azoospermia

The diagnosis of NOA is based on the evidence of two consecutive semen analyses confirming azoospermia. Causes of OA should be ruled out. Patients with NOA should undergo a comprehensive assessment to identify genetically transmissible conditions, potential treatable causes of azoospermia, and potential health-relevant co-morbidity (e.g., testicular cancer and hypogonadism [of any type]). A detailed medical history (e.g., history of cryptorchidism, previous gonadotoxic treatments for cancer, etc.) and socio-demographic characteristics [2200], along with a comprehensive physical examination should be performed in every patient to detect conditions potentially leading to azoospermia, while ruling out co-morbidity frequently associated with azoospermia. Non-obstructive azoospermia can be the first sign of pituitary or GCTs of the testis [2201-2203]. Patients with NOA have been shown to be at increased risk of being diagnosed with cancer [2204]. Moreover, other systemic conditions such as MetS, T2DM, osteoporosis and CVDs have been more frequently observed in patients with NOA compared to normozoospermic men [2205-2207]. Azoospermic men are at higher risk of mortality [2208,2209]. Therefore, investigation of infertile men provides an opportunity for long-term risk stratification for other co-morbid conditions [2210].

Genetic tests should be performed in patients with NOA to detect genetic abnormalities. As discussed (see Section 11.3), patients should undergo karyotype analysis [1805,1806], along with a screening of Y-chromosome micro-deletions [1855,2211] and of the gene coding for CFTR in order to exclude concomitant mutations, and to rule out CBAVD [1839,1840]. Genetic counselling for eventual transmissible and health-relevant genetic conditions should be provided to couples.

All patients should undergo a complete hormonal investigation to exclude concomitant hypogonadism, which has been found in about 30% of patients with NOA [356,2212,2213]. A correct definition of the type of the associated hypogonadism (i.e., hypogonadotropic hypogonadism vs. hypergonadotropic vs. compensated hypogonadism) is relevant to differentiate diagnostic and therapeutic approaches to the patient [2214].

Scrotal US may show signs of testicular dysgenesis (e.g., non-homogeneous testicular architecture and/or microcalcifications) and testicular tumours. Testicular volume may be a predictor of spermatogenic function [1771] 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 [2215]; however, to date, data are inconsistent to suggest a routine role of testicular Doppler evaluation before TESE. In a recent multicentre study including 806 men submitted to mTESE, the size of seminiferous tubules assessed with pre-operative US was significantly associated with sperm retrieval outcomes, with a sensitivity and specificity of 76.7% and 80.7% for a cut-off point of 250 μm, respectively [2216].

11.6.2.2. Surgery for non-obstructive azoospermia

Surgical treatment for NOA is mostly 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. Techniques and indications for surgical sperm retrieval in patients with NOA are discussed below. Any surgical approach aimed at sperm retrieval must be considered not a routine and simple biopsy; in this context, performing a diagnostic biopsy before surgery (any type) unless dedicated to ART protocols is currently considered inappropriate.

11.6.2.3. 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 [2217,2218]. Numerous predictive factors for positive sperm retrieval have been investigated, although no definitive factors have been demonstrated to predict sperm retrieval [2218].

Historically, there is a good correlation between the histology found at testicular biopsy and the likelihood of finding mature sperm cells during testicular sperm retrieval [2179,2219,2220]. The presence of hypospermatogenesis at testicular biopsy showed good accuracy in predicting positive sperm retrieval after either single or multiple conventional TESE or mTESE compared with maturation arrest pattern or SCOS [2179,2219,2220]. However, formal diagnostic biopsy is not recommended in this clinical setting for the reasons outlined above.

Hormonal levels, including FSH, LH, inhibin B and AMH have been variably correlated with sperm retrieval outcomes at surgery, and data from retrospective series are still controversial [2148,2221-2226]. Similarly, conflicting results have been published regarding testicular volume as a predictor of positive sperm retrieval [2148,2179,2224]. Therefore, no clinical variable may be currently considered as a reliable predictor for positive sperm retrieval throughout ART patient work-up [2218].

In case of complete AZFa and AZFb microdeletions, the likelihood of sperm retrieval is zero and therefore TESE procedures are contraindicated [1860]. Conversely, patients with Klinefelter syndrome [1823] and a history of undescended testes have been shown to have higher chance of finding sperm at surgery [2224].

Historically, surgical techniques for retrieving sperm in men with NOA include testicular sperm aspiration (TESA), single or multiple conventional TESE (cTESE) and mTESE.

Fine needle aspiration mapping

Fine needle aspiration (FNA) mapping technique has been proposed as a prognostic procedure aimed to select patients with NOA for TESE and ICSI [2227]. The procedure is performed under local anaesthesia in the office and percutaneous aspiration is performed with 23G needle in multiple sites, ranging from 4 to 18 [2227]. The retrieved tissue is sent for cytological and histological evaluation to provide information on the presence of mature sperm and on testicular histological pattern. Given that focal spermatogenesis may occur within the testes of patients with NOA, FNA mapping may provide information on the sites with the higher probability of retrieving sperm, thus serving as a guide for further sperm retrieval surgery in the context of ART procedures (e.g., ICSI). Turek et al. have shown that a higher number of aspiration sites may increase the chance of finding sperm [2228,2229]. The extent and type of subsequent sperm retrieval procedure can be tailored according to the FNA mapping results: TESA or TESE could be suggested in case of multiple positive sites for sperm, while a more precise and potentially more-invasive technique, such as mTESE, could be considered for patients with few positive sites at FNA [2227]. However, no RCTs have compared the diagnostic yield from FNA and mTESE. A positive FNA requires a secondary therapeutic 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 men who have undergone FNA mapping. Therefore, FNA mapping cannot be recommended as a primary therapeutic intervention in men with NOA until further RCTs are undertaken.

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 [2230-2233]. Data have shown that using larger needles (18-21G) with multiple passes could yield a higher chance of positive sperm retrieval [2233]. Complications after TESA are uncommon and mainly include minor bleeding with scrotal haematoma and post-operative
pain [2233].

As a less-invasive and less-costly procedure TESA has been proposed as a possible first-line approach before sending patients for a more-invasive procedure [2233]. To date no RCTs have compared SRRs from TESA, cTESE and mTESE. 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 sperm retrieval as compared with TESA [2218]. Given the low success rates compared with TESE, TESA is no longer recommended in men with NOA.

Conventional and microTESE

In patients with NOA, a testicular sperm extraction procedure is required to retrieve sperm that can be utilised in ARTs. Testicular sperm extraction was first performed through a single or multiple open biopsy of the testicle (conventional TESE [cTESE]). Conventional TESE requires a scrotal incision and open biopsy of the testes [2234]. Reported SRRs in single-arm studies are about 50% [2217]. Observational studies have demonstrated that multiple biopsies yield a higher chance of sperm retrieval [2217,2235].

In 1999, Schlegel pioneered the use of a micro testicular extraction of sperm (mTESE) approach, which utilised an operative optical microscope to inspect seminiferous tubules at a magnification of 20-25x and extract those tubules which were larger, dilated and opaque as these were more likely to harbor sperm [2234]. The rationale of this technique is to increase the probability of retrieving sperm with a lower amount of tissue sampled and a subsequent lower risk of complications. A meta-analysis that pooled data analysis of case-control studies comparing cTESE with mTESE showed a lower unadjusted SRR of 35% (95% CI: 30-40) for cTESE and 52% for mTESE [2218]. A more recent meta-analysis comparing cTESE and mTESE in patients with NOA showed a mean SRR of 47% (95% CI: 45;49%). No differences were observed when mTESE was compared with cTESE (46 [range 43-49] % for cTESE vs. 46 [range 42-49] % for mTESE, respectively) [2236].

Meta-regression analysis demonstrated that the SRR per cycle was independent of age and hormonal parameters at enrolment. However, the SRR increased as a function of testicular volume. Retrieved sperms resulted in a live-birth rate of up to 28% per ICSI cycle [1912]. The difference in surgical sperm retrieval outcomes between the two meta-analyses may be explained by the data studied [2218] only one analysed case control studies whilst Corona et al. [1912] also included the single randomised controlled trial), but it is important to note that all the studies comparing cTESE and mTESE have shown that the latter is superior in retrieving sperm.

The probability of finding vital sperm at TESE varies also according to testicular histology: data from non-randomised studies comparing cTESE with mTESE have shown a higher chance of sperm retrieval with mTESE only for patients with a histological diagnosis of SCOS [2237]. In such cases, results ranged from 22.5 to 41% and from 6.3 to 29% for mTESE vs. cTESE, respectively [2237]. Conversely, no difference between the two techniques has been found when comparing patients with a histology suggestive of maturation arrest [2237]. A single study showed a small advantage of mTESE when hypospermatogenesis was found [2238]. In light of these findings, some authors have advocated that cTESE could be the technique of choice in patients with a histological finding of maturation arrest or hypo-spermatogenesis [2218,2237].

In a study assessing the role of salvage mTESE after a previously failed cTESE or TESA, sperm were successfully retrieved in 46.5% of cases [1857]. In studies reporting sperm retrieval by micro-TESE for men who had failed percutaneous testicular sperm aspiration or non-microsurgical testicular sperm extraction, the SRR was 39.1% (range 18.4-57.1%) [2239,2240]. Similarly, a variable SRR has been reported for salvage mTESE after a previously failed mTESE (ranging from 18.4% to 42.8%) [2241,2242].

Conventional TESE has been associated with a higher rate of complications compared with other techniques [2217]. A total of 51.7% of patients have been found with intratesticular haematoma at scrotal US 3 months after surgery, with testicular fibrosis observed in up to 30% of patients at 6-months’ assessment [2243].

A recent meta-analysis investigated the risk of hypogonadism after TESE due to testicular atrophy [2244]; patients with NOA experienced a mean 2.7 nmol/L decrease in total testosterone 6 months after cTESE, which recovered to baseline within 18-26 months. Lower rates of complications have been observed with mTESE compared to cTESE, both in terms of haematoma and fibrosis [2237]. Both procedures have shown a recovery of baseline testosterone levels after long-term follow-up [2238,2245].

Follow-up after TESE

When compared with cTESE, mTESE has been reported to have fewer post-operative complications and negative effects on testicular function. In a recent meta-analysis analysing the complications of TESE, men with Klinefelter syndrome and NOA had the largest decrease in total testosterone levels 6 months after TESE (mean decrease of 4.1 and 2.7 nmol/L, respectively), which recovered to baseline levels 26 and 18 months after TESE, respectively [2244,2245]. Therefore, it would be reasonable to provide long-term endocrinological follow-up after TESE (any type) to detect hypogonadism, particularly for patients with Klinefelter syndrome. Testosterone measurement could be offered in asymptomatic men at 18 months post-TESE or in those men who become symptomatic for hypogonadism after surgery [2246]. Temporary discontinuation of treatment may reveal the expected recovery of testosterone secretion and revise the decision for testosterone therapy [2247]. Human chorionic gonadotropin or selective oestrogen receptors modulators (SERMs) administration could be considered in highly selected, hypogonadal patients who have not completed their fertility attempts to increase intratesticular testosterone concentration and manage the hypogonadal symptoms [2245].

The main limitation to contemporary literature is the paucity of randomised controlled studies comparing cTESE and mTESE. Although no difference in SSR was observed between cTESE/mTESE techniques in patients with NOA in the latest and most comprehensive meta-analysis [2236], it is important to note that in all the individual trials comparing cTESE and mTESE the latter was superior in retrieving sperm. Furthermore, the current data suggests that mTESE has less complications than cTESE and therefore the consensus opinion of the guidelines panel is that mTESE is the optimum approach for surgical sperm retrieval procedures. However, this is based on low-quality evidence and larger RCTs comparing SSR, risks and costs between the two techniques are urgently needed.

Hormonal therapy prior to surgical sperm retrieval approaches

Stimulating spermatogenesis by optimising intratesticular testosterone (ITT) has been proposed to increase the chance of sperm retrieval at the time of surgery in men with NOA. Similarly, increasing FSH serum levels could stimulate spermatogenesis. There is evidence that treatment with hCG can lead to an increase in ITT [2143] and Leydig cells within the testes [2248]. It has been shown in azoospermic patients with elevated gonadotropins levels that administration of hCG and/or FSH can lead to a so-called “gonadotropins reset”, with a reduction in FSH plasma concentrations and improvement in Sertoli cells function [2249]. Similarly, clomiphene citrate may increase pituitary secretion by blocking feedback inhibition of oestradiol, thus inducing an increase in FSH and LH in patients with NOA [2250]. While azoospermic patients with secondary hypogonadism should be treated accordingly to stimulate sperm production [356], no RCT has shown a benefit of hormonal treatment to enhance the chances of sperm retrieval among patients with idiopathic NOA [2247]. In a large multicentre case-control study, 496 patients with idiopathic NOA treated with a combination of clomiphene, hCG and human menopausal gonadotropin according to hormonal profile, were compared with 116 controls subjected to mTESE without receiving any pre-operative treatment [2149]. A total of 11% of treated patients had sperm in the ejaculate at the end of treatment; of the remaining patients, 57% had positive sperm retrieval at mTESE as compared with 33% in the control group. Likewise, in a small case-control study including 50 men with idiopathic NOA, of whom 25 were treated with recombinant FSH before mTESE, there was observed a 24% SRR compared with 12% in the control group [2148]. Conversely, Gul et al. [2150] failed to find any advantage of pre-operative treatment with hCG compared with no treatment, in 34 idiopathic NOA patients candidates for mTESE. A recent meta-analysis has suggested that hormone stimulation in men prior to TESE does not improve sperm retrieval rates in hypergonadotropic hypogonadal patients [2251]; however, the included studies had moderate or severe risk of bias and randomised studies are needed to confirm these findings.

Hormonal therapy has also been proposed to increase the chance of sperm retrieval at salvage surgery after previously failed cTESE or mTESE. Retrospective data have shown that treatment with hCG and recombinant FSH could lead to a 10-15% SRR at salvage mTESE [2143,2252]. In a small case-control study 28 NOA patients were treated with hCG with or without FSH for 4-5 months before salvage mTESE and compared with 20 controls subjected to salvage surgery [2253]. Sperm retrieval rate was 21% in the treated group compared with 0% in the control group. The histological finding of hypo-spermatogenesis emerged as a predictor of sperm retrieval at salvage surgery after hormonal treatment [2253]. Further prospective trials are needed to elucidate the effect of hormonal treatment before salvage surgery in NOA patients, with a previously failed cTESE or mTESE. However, patients should be counselled that the evidence for the role of hormone stimulation prior to sperm retrieval surgery in men with idiopathic NOA is limited [2254]. Currently, it is not recommended in routine practice.

11.6.2.4. Recommendations for Non-Obstructive Azoospermia

11.7. Assisted Reproductive Technologies

11.7.1. Types of assisted reproductive technology

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 [2255,2256].

Once couples have been prepared for treatment, the following are the steps that make up an ART cycle:

1. Pharmacological stimulation of growth of multiple ovarian follicles, while at the same time other medications is given to suppress the natural menstrual cycle and down-regulate the pituitary gland.
2. Careful monitoring at intervals to assess the growth of the follicles.
3. Ovulation triggering: when the follicles have reached an appropriate size, a drug is administered to bring about final maturation of the eggs.
4. Egg collection (usually with a trans-vaginal US probe to guide the pickup) and, in some cases of male infertility, sperm retrieval.
5. Fertilisation process, which is usually completed by IVF or ICSI.
6. Laboratory procedures follow for embryo culture: culture media, oxygen concentration, co-culture, assisted hatching etc.
7. The embryos are placed into the uterus. Issues of importance here include endometrial preparation, the best timing for embryo transfer, how many embryos to transfer, what type of catheter to use, the use of US guidance, need for bed rest etc.
8. Luteal phase support, for which several hormonal options are available.

Fertility treatments are complex and each cycle consists of several steps. If one of the steps is incorrectly applied, conception may not occur [2255].

Several ART techniques are available:

11.7.1.1. Intra-uterine insemination (IUI)

Intra-uterine insemination is an infertility treatment that involves the placement of the prepared sperm into the uterine cavity timed around ovulation. This can be done in combination with ovarian stimulation or in a natural cycle. The aim of the stimulation cycle is to increase the number of follicles available for fertilisation and to enhance the accurate timing of insemination in comparison to the natural cycle IUI [2257-2259].

Intra-uterine insemination is generally, though not exclusively, used when there is at least one patent fallopian tube with normal sperm parameters and regular ovulatory cycles (unstimulated cycles) and when the female partner is aged < 40 years.

The global pregnancy rate (PR) and delivery rate (DR) for each IUI cycle with the partner’s sperm are 12.0% and 8.0%, respectively. Using donor sperm, the resultant PR and DR per cycle are 17.0% and 12.3%, respectively [2260]. The rates of successful treatment cycles for patients decrease with increase in age, and the birth rates across all age groups have remained broadly stable over time. The highest birth rates have been reported in patients younger than 38 years (14% in patients aged < 35 years and 12% in those aged 35-37 years). The rates of successful treatment are low for patients older than 42 years. The multiple pregnancy rate (MPR) for IUI is ~8% [2258]. Intra-uterine insemination is not recommended in couples with unexplained infertility, male factor infertility and mild endometriosis, unless the couples have religious, cultural or social objections to proceed with IVF [2261].

Intra-uterine insemination with ovarian stimulation is a safer, cheaper, more patient-friendly and non-inferior alternative to IVF in the management of couples with unexplained and mild male factor infertility [2257,2258]. A recent RCT showed lower multiple pregnancy rates and comparable live-birth rates in patients treated with IUI with hormonal stimulation when compared to women undergoing IVF with single embryo transfer [2262]. Additionally, IUI is a more cost-effective treatment than IVF for couples with unexplained or mild male subfertility [2263].

11.7.1.2. In vitro fertilisation (IVF)

Involves using controlled ovarian hyperstimulation to recruit multiple oocytes during each cycle from the female partner. Follicular development is monitored ultrasonically, and ova are harvested before ovulation with the use of US-guided needle aspiration. The recovered oocytes are mixed with processed semen to perform IVF. The developing embryos are incubated for 2-3 days in culture and then placed trans-cervically into the uterus.

The rapid refinement of embryo cryopreservation methods has resulted in better perinatal outcomes of frozen-thawed embryo transfer (FET) and makes it a viable alternative to fresh embryo transfer (ET) [2264,2265]. Frozen-thawed embryo transfer seems to be associated with lower risk of gestational complications than fresh ET. Individual approaches remain appropriate to balance the options of FET or fresh ET at present [2266].

Generally, only 20%-30% of transferred embryos result in clinical pregnancies. The global PR and DR per aspiration for non-donor IVF is 24.0% and 17.6%, respectively [2260].

According to the NICE guidelines, IVF treatment is appropriate in cases of unexplained infertility for women who have not conceived after 2 years of regular unprotected sexual intercourse [2267].

11.7.1.3. Intracytoplasmic sperm injection

Intracytoplasmic sperm injection is a procedure through which a single sperm is injected directly into an egg using a glass micropipette.

The difference between ICSI and IVF is the method used to achieve fertilisation. In conventional IVF, oocytes are incubated with sperm in a Petri dish, and the male gamete fertilises the oocyte naturally. In ICSI, the cumulus–oocyte complexes go through a denudation process in which the cumulus oophorus and corona radiata cells are removed mechanically or by an enzymatic process. This step is essential to enable microscopic evaluation of the oocyte regarding its maturity stage, as ICSI is performed only in metaphase II oocytes [2268]. A thin and delicate glass micropipette (injection needle) is used to immobilise and pick up morphologically normal sperm selected for injection. A single spermatozoon is aspirated by its tail into the injection needle, which is inserted through the zona pellucida into the oocyte cytoplasm. The spermatozoon is released at a cytoplasmic site sufficiently distant from the first polar body. During this process, the oocyte is held still by a glass micropipette [2268].

With this technique the oocyte can be fertilised independently of the morphology and/or motility of the spermatozoon injected.

Intracytoplasmic sperm injection is currently the most commonly used ART, accounting for 70–80% of the cycles performed [2269].

The procedure was first used in cases of fertilisation failure after standard IVF or when an inadequate number of sperm cells was available. The consistency of fertilisation independent of the functional quality of the spermatozoa has extended the application of ICSI to immature spermatozoa retrieved surgically from the epididymis and testis [2270]. Intracytoplasmic sperm injection is the natural treatment for couples with severe male factor infertility and is also used for a number of non-male factor indications (Table 56) [2271].

The need to denude the oocyte allows assessment of the nuclear maturity of the oocyte. Intracytoplasmic sperm injection is also preferred in conjunction with pre-implantation genetic diagnosis and has recently been used to treat HIV discordant couples, in whom there is a pressing need to minimise exposure of the oocyte to a large number of spermatozoa [2270].

The global PR and DR per aspiration for ICSI is 26.2% and 19.0%, respectively [2260]. For all ages and with all the different sperm types used, fertilisation after ICSI is at approximately 70%-80% and it ensures a clinical pregnancy rate of up to 45% [2269,2270].

Existing evidence does not support ICSI in preference over IVF in the general non-male factor ART population; however, in couples with unexplained infertility, ICSI is associated with lower fertilisation failure rates than
IVF [2271].

Overall, pregnancy outcomes from ICSI are comparable between epididymal and testicular sperm and also between fresh and frozen–thawed epididymal sperm in men with OA [2272]. However, these results are from studies of low evidence [2271].

Sperm injection outcomes with fresh or frozen–thawed testicular sperm have been compared in men with NOA. In a meta-analysis of 11 studies and 574 ICSI cycles, no significant difference was observed between fresh and frozen–thawed testicular sperm with regards to fertilisation rate (RR: 0.97, 95% CI: 0.92–1.02) and clinical pregnancy rates (RR: 1.00, 95% CI: 0.75–1.33) [2273]. However, no meta-analysis was performed on data regarding implantation, miscarriage, and low-birth rates.

11.7.1.4. Testicular sperm in men with raised DNA fragmentation in ejaculated sperm

The use of testicular sperm for ICSI is associated with possibly improved outcomes compared with ejaculated sperm in men with high sperm DNA fragmentation [1792,2271]. Men with unexplained infertility with raised DNA fragmentation 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 have not yet been confirmed in large scale randomised studies [2274].

In terms of a practical approach, urologists may offer the use of testicular sperm in patients with high DNA fragmentation. However, patients should be counselled regarding the low levels of evidence for this (i.e., non-randomised studies). Furthermore, testicular sperm should only be used in this setting once the common causes of oxidative stress have been excluded including varicoceles, modifications of dietary/lifestyle factors and treatment of accessory gland infections.

Table 56: Fertilisation methods for male factor and non-male factor infertility (adapted from )

ICSI = intracytoplasmic sperm injection; IVF = in vitro fertilisation; OAT = oligo-asthenoter-atozoospermia

Intracytoplasmic sperm injection is carried out using viable sperm populations. Several semen processing techniques have been developed to select the optimal sperm fraction for ICSI. Density gradient centrifugation (DGC) and the swim-up procedures have been used as standards for semen preparation for ICSI for more than two decades [2276]. However, these traditional sperm selection techniques are unable to select sperm fractions with optimal DNA integrity and functional characteristics. Advanced sperm selection techniques have been introduced to optimise the selection of high-quality sperm for ICSI [2277]. These selection methods are based on sperm surface charge (electrophoresis and zeta potential), apoptosis (magnetic-activated sperm cell sorting (MACS) and glass wool), membrane maturity (hyaluronic acid binding), or ultra-morphological sperm assessment [2278].

11.7.1.5. Intra-cytoplasmic morphologically selected sperm injection

Intra-cytoplasmic morphologically selected sperm injection (IMSI) was first introduced in 2002 as a modification of the ICSI technique [2279]. This technique increases the magnification of sperm to > 6,000 times; the purpose of which is to perform the motile sperm organelle morphology examination (MSOME), a method used to select spermatozoa that have the choicest morphology in couples with the most severe male factor. Bartoov et al. showed that, for patients with a history of ICSI failure, addition of IMSI resulted in a 60% pregnancy rate, compared with a 30% rate for patients not using IMSI [2280]. The pregnancy rate following IVF-IMSI was significantly higher and the miscarriage rate significantly lower, than for the routine IVF-ICSI procedure (60.0% vs. 25.0%, and 14% vs. 40%, respectively) [2281]. However, the most recently updated Cochrane review neither supported nor refuted the clinical use of IMSI [2282].

11.7.1.6. Physiological ICSI (PICSI) technique: a selection based on membrane maturity of sperm

Human oocytes are surrounded by hyaluronic acid (HA), which acts as a natural selector. Only mature sperm that express receptors specific to HA can reach the oocytes and fertilise them. Those sperm have normal shapes, low DNA fragmentation rates, and low frequency of chromosomal aneuploidy [2283]. Several studies have attempted to verify whether sperm selection based on HA binding affects IVF outcomes. A meta-analysis included six prospective randomised studies and one retrospective study, all of which used a PICSI sperm-selection dish (a plastic culture dish with microdots of HA hydro gel on its inner surface) or the Sperm Slow method (a viscous medium containing HA). No improvements in fertilisation and pregnancy rates were recorded, although embryo quality was superior in PICSI compared with conventional ICSI [2283]. A recent large-sample multicentre randomised trial provided conclusive evidence against the use of PICSI in ART (PICSI live-birth rate vs. ICSI: OR: 1.12, 95% CI: 0.95–1.34) [2284]. A time-lapse study found no difference in embryo development dynamics in oocytes fertilised via HA-ICSI vs. conventional ICSI [2285].

11.7.1.7. Magnetic-activated cell sorting

Magnetic-activated cell sorting (MACS) is an advanced sperm-selection technique used to isolate sperm that do not show signs of apoptosis and, therefore, are presumed to have a lower rate of DNA damage [2277]. Use of MACS after density gradient centrifugation (DGC) has been found to improve sperm morphology and decrease DNA fragmentation and apoptotic markers, but it reduces motility of the selected sperm [2277,2278]. Magnetic-activated cell sorting failed to improve ICSI outcomes compared with DGC or swim-up, although a slightly higher pregnancy rate (RR: 1.5, 95% CI: 1.14–1.98) was observed in MACS patients relative to the control group [2286]. No difference in implantation or miscarriage rate was noted (RR: 1.03, 95% CI: 0.8–1.31 and RR: 2, 95% CI: 0.19–20.9, respectively).

Finally, another RCT performed on infants conceived via ovum-donation IVF cycles did not report any differences in terms of obstetrical and perinatal outcomes between pregnancies or babies conceived with sperm selected via MACS or swim-up [2287].

11.7.2. Safety

The most significant risk of pre-implantation ART treatment is the ovarian hyperstimulation syndrome, a potentially life-threatening condition resulting from excessive ovarian stimulation during ART techniques, ranging from 0.6% to 5% in ART cycles [2288].

Other problems include the risk of multiple pregnancies due to the transfer of more than one embryo and the associated risks to mother and baby, including multiple and preterm birth. The most prevalent maternal complications include pre-eclampsia, gestational diabetes, placenta previa, placental abruption, postpartum haemorrhage, and preterm labour and delivery [2222,2289,2290]. The risks of foetal demise during the third trimester, perinatal mortality, preterm birth, and low birth weight increase with the number of foetuses in the pregnancy. The foetal consequences of preterm birth (cerebral palsy, retinopathy, and broncho-pulmonary dysplasia) and foetal growth restriction (polycythaemia, hypoglycaemia, and necrotising enterocolitis) are significant [2291].

The average number of embryos transferred in fresh non-donor IVF and ICSI cycles in 2011 was 1.91, compared with 2.09 in 2008, 2.00 in 2009, and 1.95 in 2010, reflecting a continuing decrease from previous years. The average number of embryos transferred in frozen ET cycles decreased from 1.72 in 2008 to 1.65 in 2009 to 1.60 in 2010 and to 1.59 in 2011 [2292].

The global multiple birth rate for fresh cycle transfer has decreased from 21.5% in 2010 to 20.5% in 2011 and for frozen ET cycles from 12.0% to 11.5% [2260].

In 2011, the rate of early pregnancy loss was 20.1% after fresh ET, compared with 25.4% after frozen ET. Both rates showed wide regional variation [2260]. The multiple birth rates after fresh non-donor ET were 19.6% (twins) and 0.9% (triplets and higher-order births); for frozen ET non-donor cycles, twin and triplet and higher-order birth rates were 11.1% and 0.4%, respectively [2260].

Rates of premature delivery and perinatal mortality were lower for frozen ETs than for fresh ETs. The global preterm DR after non-donor fresh ET was 19.1%, and after frozen ET was 13.1%. The perinatal mortality rate per 1,000 births after non-donor fresh ET was 16.3 and after frozen ET was 8.6.

In terms of potential adverse effect, ICSI-conceived offspring has a greater neonatal morbidity, obstetric complications and congenital malformations, compared with spontaneous conception [2289,2293,2294]. Additionally, epigenetic disorders and impaired neurodevelopment have been observed in infants born using ICSI compared with naturally conceived children [2271]. Among singleton infants born at 37 weeks of gestation or later, those following IVF had a risk of low birth weight that was 2.6 times (95% CI: 2.4–2.7) greater than in the general population (absolute risk of low birth weight with spontaneous vs. resulting from IVF was 2.5% vs. 6.5%) [1896]. Singleton infants after IVF were 39% more likely (adjusted RR: 1.39, 95% CI: 1.21–1.59) to have a non-chromosomal birth defect (particularly gastrointestinal and musculoskeletal) compared with all other singleton births. No single ART procedure (e.g., ICSI, fresh, or frozen ETs) was found to substantially increase the risk of birth defects.

Analyses from the Massachusetts Outcome Study of ART reported a 50% increase (adjusted prevalence ratio of 1.5, 95% CI: 1.3–1.6) in birth defects in infants after IVF vs. spontaneous pregnancy, and a 30% increase (adjusted prevalence ratio of 1.3, 95% CI: 1.1–1.5) in birth defects in infants after subfertility vs. spontaneous pregnancy [2295-2297]. No difference in risk of cancer was found between ART-conceived children and those spontaneously conceived [2298].

Health differences between ICSI and IVF conceptions have not been comprehensively assessed and results are contradictory. Some authors found a significantly reduced risk of birth defects in IVF compared to ICSI conceived infants [1899], while two meta-analyses demonstrated no difference in risk of congenital malformations between IVF and ICSI conception [1902,2299]. Data about ICSI- and IVF-conceived adolescents or young adults are scarce but it seems that there is no difference in outcomes between the two techniques. Further research into health outcomes in adolescence and adulthood is required before conclusions can be drawn about the long-term safety of ICSI compared to IVF [2300].

11.8. Psychosocial aspects in men’s infertility

Male infertility impacts men’s psychological well-being in different ways. It results in emotional distress and challenges men’s sense of identity. Factors such as personality style, sociocultural background, and treatment specificities (e.g., repeated cycles, treatment side-effects), may determine men’s adjustment to infertility [2301]. The effects may be particularly worst in socially isolated men, with an avoidant coping style [2302]. Infertility-associated distress and psychiatric morbidity in men are further related to the male and mixed factor and increases after the clinical diagnosis [2303]. In this regard, special attention has been given to men’s psychological adaptation after the failure of medically assisted reproduction treatments. While the risk factors for emotional maladjustment encompass difficulties in couples’ communication or avoidance/religious coping style from the female partner, the protective factors include seeking information, reframe infertility by assigning it a positive meaning, having social and spouse support, and talk openly about the infertility issue [2304]. Is worth noting that a failed treatment often results in a prolonged grief response, requiring post-treatment psychological support [2305]. The literature supports the relevance of addressing men’s psychological needs, as a means to reduce the impact of infertility treatments across all of its stages. 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 [2301]. Furthermore, there should be a deeper focus on preventive policies; it has been recognised that men, similar to women, want to become parents. Yet, they have very limited knowledge about infertility related risk factors, including a lack of awareness on the age-related decline in fertility, and tend to overestimate the chance of spontaneous conception [2306,2307].