2

Male Factor

Section Contents 3 Evaluation and Diagnosis of 4 Hormonal Management of Male Infertility 5 of Male Infertility 6 Surgical Management of Male Infertility 7 Genetics and Male Infertility

3

Evaluation and Diagnosis of Male Infertility Sandro C Esteves, Alaa Hamada, Ashok Agarwal

to the andrological armamentarium. Today, it is possible Chapter Contents to correctly classify some cases which were previously ♦♦ Definition and Epidemiology of Male Infertility believed to be idiopathic. The initial evaluation of com- ♦♦ Pathophysiology, Etiology and Classification of Male mon infertility complaint comprises the meticulous his- Infertility tory taking, as well as conducting a thorough physical ♦♦ Probabilities of Conception for Fertile and Infertile examination along with proper laboratory and imaging Couples studies as needed. ♦♦ Goals and the Proper Timing for Fertility Evaluation ♦♦ Approaching the Subfertile Male DEFINITION AND EPIDEMIOLOGY OF MALE INFERTILITY

The infertility is defined as failure of the couples to INTRODUCTION conceive after 12 months of unprotected regular inter- course.2 Infertility is broadly classified into primary t is well known that the motivation to have children infertility, when the male partner has no previous history Iand the formation of a new family unit are essential of fertility, and secondary infertility, when there was components of the individual instinct for existence and previous man’s history of successful impregnation of well-being. Fertility problems may represent a stressful a woman. Subfertility refers to a reduced but not unat- situation to the individual’s life with important nega- tainable potential to achieve pregnancy, while sterility tive psychological consequences.1 The experienced cli- is denoted by permanent inability to induce or achieve nician should realize and comprehend the burdensome pregnancy.3 Fecundity, on the other hand, indicates the bearings and frustrated mood of the infertile individual. capability of a given male or female to produce live Most adults are prompted to discuss their sexual issues births. and behaviors if the interview is conducted in a respect- Epidemiology studies indicate that 13–15% of couples ful, confidential, professional and nonjudgmental way. face difficulties to conceive.4 Approximately 8% of men In the last decades, extraordinary advances have in reproductive age seek medical assistance for fertility- been achieved in the field of male infertility. Significant related problems.5 Isolated male factor infertility, and its progress has been made regarding both diagnostic and combination with female problems accounts for more treatment techniques. Novel tests have been incorporated than 50% of the couples’ inability to conceive.6 The male Section 2 Male Factor Infertility

factor is purely responsible for 20% of infertile couples, PATHOPHYSIOLOGY, ETIOLOGY AND 6 while it is contributory in another 30–40% of infertility. CLASSIFICATION OF MALE INFERTILITY The World Health Organization defines male factor infertility as the presence of abnormalities in the The male reproductive system consists of the testes, analysis, or the presence of sexual or ejaculatory dys- accessory glands and the ductal system for sperm trans- function.7 However, modern has revealed port. The seminal ducts constitute the pathways for sperm that normal semen testing does not guarantee fecundity, transport; and it includes the efferent ductules and rete and unraveled the predictive power of normal semen to testis, , vas deferens, ejaculatory ducts and induce natural pregnancy is about 60%.8 The introduc- urethra. The accessory sex glands include the seminal ves- tion of novel sperm function testing is revolutionizing icles, and bulbourethral glands (Figures 1 and 2). the criteria of diagnosis and definitions of male infertility. The testes perform two important functions: sper- Recent reports indicate that sperm counts of men matogenesis and steroidogenesis. Daily sperm produc- from certain regions of the world have significantly tion is about 40 million and declines progressively with deteriorated. Remarkable differences in sperm pro- aging. Recent data suggests that the duration of sper- duction have been detected among men from different matogenesis is less than 60 days instead of 70 ± 4 days countries in Europe and the United States of America. as previously thought for over 40 years.21 As such, the Differences are also seen in individuals of different sperm production of an individual actually represents regions within the same country. Moreover, there is a the result of influencing biological, physical and occu- noticeable epidemiologic increase in the incidence of pational factors, which acted within the past 2 months and urogenital anomalies.9-12 These from . Steroidogenesis and hormone produc- observations cause public concerns particularly with tion is another essential testicular function. The hormo- regard to the implication of environmental factors nal milieu that regulates spermatogenesis depends on and modern life habits.13-15 Some authors claim that complex coordinated interaction between the testes, the buildup in the prevalence of infertility among men pituitary gland and hypothalamus that form the hypo- may be attributed to increased prevalence of obesity.16-18 thalamic-pituitary-gonadal (HPG) axis (Figure 3). Others factors may also be implicated in the apparent The storage and maturation of spermatozoa occur rising prevalence of male infertility problems such as in the epididymis. However, the maturation process is exposure to cell phones radiation19 and certain endo- exclusively completed within the female reproductive crine disruptors.20 tract. Spermatozoa transit throughout the epididymis for

Figure 1 Schematic illustration of the male reproductive organs 30 Chapter 3 Evaluation and Diagnosis of Male Infertility

transport and sperm function constitute the essential causes of male infertility. The fourth category is represented by “infertility of unknown origin”, a condition in which male infertility has aroused spontaneously, or from an obscure or unknown cause. This category accounts for 37–58% of the cases, and it is divided into two major subtypes: idi- opathic and unexplained male infertility.22-24 Idiopathic male infertility is the cause of about 30% of male infer- tility, and it is characterized by unexplained reduction in semen quality with no previous history associated with fertility problems, and having normal findings on physi- cal examination and endocrine laboratory testing.23 The term “unexplained male infertility”, on the other hand, is reserved for infertile men with infertility of unknown ori- gin with normal semen profile, and in whom female infer- tility factors have been ruled out. This category accounts for 6–27% of male infertility, and it strongly depends on how exhaustive evaluation of the patient is done.22 Table 1 depicts the etiologic classification of male infertility. Figure 2 Schematic representation of the testis and its The most commonly observed abnormality in related structures infertile men seeking evaluation is varicocele followed by obstruction and . Table 2 shows the about 12 days. The epididymides are in continuity with distribution of the causes of male infertility in tertiary the vasa deferentia, which in turn join the emerging care infertility center. It should be noted, however, that ducts from the to form the ejaculatory the frequency distribution of primary infertility causes ducts. They enter the prostate, a gland responsible for is different from secondary infertility causes in which production of a fluid enriched with zinc, citric acid, acid varicocele accounts for 70–80% of the cases, followed by phosphatase and proteases that contributes to liquefac- infection of genital tract.25-27 tion and accounts for approximately 0.5 ml of the ejacu- late. The seminal vesicles produce an alkaline fluid with prostaglandins and fructose that composes 1.5–2.0 ml of PROBABILITIES OF CONCEPTION FOR the seminal fluid. Both seminal vesicles and vas defer- FERTILE AND INFERTILE COUPLES ens have a common embryologic origin. As such, when congenital bilateral absence of vas deferens (CBAVD) is Epidemiology studies indicate that the chance of a cou- diagnosed, it is often associated with seminal vesicles ple to achieve conception is 80–90% after 1 year of regu- hypoplasia/agenesis. This is an important aspect in the lar well-timed intercourse while the monthly chance for differential diagnosis of . In CBAVD, dimin- conception is 20–25%.27 The optimum fertility age for ished or no fructose can be found in the seminal fluid and, conception in both sexes is 24 years. Henceforth, the fer- hence, volume is low (< 1 ml). Under normal conditions, tility potential declines, particularly for female partner spermatozoa are stored in the epididymides rather than after the age of 31 years.28-30 the seminal vesicles. At the time of ejaculation, ductal and On the other hand, the spontaneous pregnancy rate epididymal muscle contractions under sympathetic stim- for infertile couples, due to idiopathic reasons, notice- ulation conduct the spermatozoa towards the prostatic ably depends on the result of . For non- urethra where they join fluids excreted by the prostate, azoospermic men, the pregnancy rate is 1–3% per month, and form the semen. Periurethral muscle contraction is 23% within 2 years and 10% within another two addi- responsible for expelling semen out. Interference in any tional years.31 However, the cumulative pregnancy rate of these steps may lead to male infertility. Herein, the (PR) for infertile couples whose male partner have nor- pathophysiological mechanism involved is dependent on mal semen analyses ranges from 50%–80% over a 3-year which organ or regulatory system is afflicted. period as a function of female age, and from 30%–80% Male infertility causes may range from simple, revers- as a function of infertility duration.32 A cumulative PR ible and correctable causes to uncorrectable ones. Such of 60% may be achieved within 2 years for this category conditions may be inherited or acquired. The factors of patient.33 Infertility periods longer than 3 years are affecting male fertility status can be divided into four associated with very low PR of 1–3% particularly if the 31 major categories: disorders in sperm production, sperm female partner is aged 35 years or older.34 Section 2 Male Factor Infertility

Figure 3 Schematic representation of the components of the hypothalamic-pituitary-testicular axis and the endocrine regulation of spermatogenesis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are secreted by the pituitary gland in response to hypothalamic gonadotropin releasing hormone (GnRH). While FSH acts directly on the germinal epithelium, LH stimulates secretion of testosterone by the Leydig cells. Testosterone stimulates sperm production and also feeds back the hypothalamus and pituitary to regulate GnRH secretion. FSH stimulates Sertoli cells to support spermatogenesis and to secrete inhibin B that negatively feedback FSH secretion

GOALS AND THE PROPER TIMING FOR any risk factor for infertility is present, such as history of FERTILITY EVALUATION infertility with another partner, cryptorchidism, and advanced female age either more than or equal to Generally, the proper timing for an infertile couple to 35 years.6 seek medical advice is after 12 months of regular inter- Advanced paternal age is not only associated with 32 course. However, immediate attention must be given if a decline in the fecundity potential but also with Chapter 3 Evaluation and Diagnosis of Male Infertility

Etiologic classification of male Contd... Table 1 infertility d. Ejaculation 1. Disorders affecting sperm production • Anejaculation • i. Hypothalamic and pituitary causes a. Lack of gonadotropin-releasing hormone secretion iii. Functional obstruction • Kallmann’s syndrome iv. Coital factors • Isolated hypogonadotropic hypogonadism a. Impotence • Midline facial defects b. Infrequent coitus • Congenital syndromes such as Prader-Willi c. Improper coital techniques syndrome, Laurence-Moon-Biedl syndrome b. Lack of luteinizing hormone (LH) secretion • Fertile eunuch syndrome 3. Disorders affecting sperm function • Isolated LH deficiency i. Immune infertility c. Isolated follicle-stimulating hormone deficiency ii. Reactive oxygen species and oxidative stress d. Pituitary disease iii. Sperm DNA damage • Tumors iv. Fertilization defects • Infiltrative disorders a. Zona pellucida binding • Infarction b. Hyperactivation • Radiation c. Acrosome reaction • Hyperprolactinemia d. Fusogenic ability • Endocrinopathies-induced alterations in v. Immotile cilia syndrome pituitary gland responsiveness, such as thyroid dysfunction, glucocorticoid excess, 4. Infertility of unknown origin exogenous androgens or estrogens i. Idiopathic ii. Testicular causes ii. Unexplained a. Vascular causes: torsion, varicocele b. Infection: , orchitis c. Genetic and congenital causes • Congenital causes — Cryptorchidism Distribution of diagnostic — categories of couples seeking — Polyorchidism Table 2 — Microorchidism infertility evaluation in a male • Genetic causes infertility clinic — Chromosomal aneuploidy and structural Category Numbers Percentage defects — Gene defects Varicocele 629 21.9 — Y chromosome microdeletion Infectious 72 2.5 d. Medications Hormonal 54 1.9 e. Castration, testicular trauma Ejaculatory dysfunction 28 1.0 f. Systemic diseases Systemic diseases 11 0.4 g. Environmental factors Idiopathic 289 10.0 2. Disorders affecting sperm transport Normal/Female factor 492 17.1 Immunologic 54 1.9 i. Congenital Obstruction 359 12.5 a. Bilateral absence of vas deferens b. Unilateral absence of the vas Cancer 11 0.4 c. Prostatic/ejaculatory duct cysts Cryptorchidism 342 11.9 Genetic 189 6.6 ii. Acquired a. Iatrogenic (surgical procedures) Testicular failure 345 11.9 b. Infection Total 2,875 c. Trauma Source: ANDROFERT, Campinas, São Paulo, Brazil

Contd... 33 Section 2 Male Factor Infertility

increased incidence of miscarriage and inheritance of Clinical Male Infertility History genetic diseases such as Down’s syndrome as well as Outline (Adapted from Esteves SC, autism and schizophrenia.35-40 The yearly decline in Miyaoka R, Agarwal A. An update on Table 3 sperm parameters has been estimated to be 0.03 ml the clinical assessment of the infertile for volume and 0.7% for motility.41 The probability of male. CLINICS 2011;66(4):691-700, having abnormal sperm counts increases with age. It with permission from the Editor) is 5% at age 30 years, 15% at age 50 years and 50% by Infertility History age 80 years.41 As such, the impact of paternal age on • Age of partners, time attempting to conceive male fertility cannot be underestimated. Current rec- • Contraceptive methods/duration ommendations for immediate evaluation include men • Previous pregnancy (actual partner/other partner) with advanced paternal age, i.e. more than 45 years, • Previous treatments because their fertility potential may be compromised. • Treatments/evaluation of female partner The essential goals of male evaluation for infertil- ity encompass the detection of both correctable and Sexual History uncorrectable causes. The uncorrectable male infertility • Potency, libido, lubricant use can be managed by using assisted conception or donor • Ejaculation, timed intercourse, frequency of masturbation insemination. The identification of genetic and/or chro- mosomal abnormalities are crucial for proper coun- Childhood and Development seling not only regarding the chances of conception • Cryptorchidism, hernia, testicular trauma but also for the health of offspring. Evaluation of males • , infection (e.g. mumps) complaining of infertility may also identify underlying • Sexual development, puberty onset health-threatening medical such as endo- Personal History crinopathies and malignancies.42 • Systemic diseases (diabetes, cirrhosis, hypertension) • Sexually transmitted diseases, tuberculosis, viral infections

APPROACHING THE SUBFERTILE MALE Previous • Orchidopexy, herniorrhaphy, orchiectomy (testicular The basic evaluation of men with infertility com- cancer, torsion) plaint includes a detailed history, a thorough physical • Retroperitoneal and pelvic examination and at least two semen analyses. Additional • Other inguinal, scrotal and perineal surgery laboratory and imaging testing may be ordered accord- • Bariatric surgery, bladder neck surgery, transurethral ing to the initial findings. The role of the urologist in resection of the prostate the management of the subfertile male cannot be under Gonadotoxic Exposure estimated. Whenever possible, he/she is responsible for diagnosing, counseling and treating the existing cause. • Pesticides, alcohol, cocaine, marijuana abuse When no specific treatment is available, the urologist is • Medication (chemotherapeutic agents, cimetidine, sulfasalazine, nitrofurantoin, allopurinol, colchicine, still responsible for referring the patient to a specialized thiazide, β-blockers and α-blockers, calcium blockers, assisted reproductive technology (ART) center, or for ) extracting the male gametes from the or epidi- • Organic solvents, heavy metals dymides as a member of the ART center’s multiprofes- • Anabolic steroids, tobacco use sional team. • High temperatures, electromagnetic energy History • Radiation (therapeutic, nuclear power plant workers),etc. The ’s tasks during the medical interview are Family History to address the patient’s concerns, and to conduct scien- • Cystic fibrosis, endocrine and genetic diseases tific, courteous and nonjudgmental interview. A com- • Infertility in the family prehensive medical history encompasses all aspects of the male’s development from pregnancy to adult Current Health Status life. In Table 3, a comprehensive questionnaire for • Respiratory infection, anosmia interviewing a male patient with infertility complaint • Galactorrhea, visual disturbances is provided. • Obesity 34 Chapter 3 Evaluation and Diagnosis of Male Infertility

Descriptive Elements declines. Consequently, it is also advisable to abstain Male age, duration of infertility, primary vs. second- from ejaculation, not longer than 5 days, when attempt- ary infertility, as well as age and fertility status of the ing to conceive. Woman with regular menstrual cycle can female partner have prognostic and diagnostic signifi- estimate the fertile period by using basal body tempera- cance. Advanced male or female age and longstanding ture and calendar calculations. However, these methods history of infertility are negative factors for fecundity. are both inaccurate and unreliable. The best methods to The secondary infertility carries a better prognosis than prospectively identify the entire fertile window for the the primary infertility. The secondary male infertility is purpose to achieve pregnancy would be either the fer- often associated with correctable causes such as vari- tility charting of vaginal discharge, or the elevation in cocele, infection or ejaculatory problems. A history of urinary estrogen or luteinizing hormone (LH) surge.51 early miscarriages, or occurrence of fetal genetic abnor- The former identifies the changes in vaginal discharge malities may suggest a male factor contribution.43,44 that correspond with high ovarian hormones and can The female partner’s history of having success- roughly estimate the fertile window. The latter method ful pregnancies and live births renders male infertility identifies the rises in urinary estradiol 3-glucuronide more likely. Female age is the most important single (E3G) and urine LH surge, and can prospectively pre- variable influencing the outcome in assisted reproduc- dict the 3–6 days fertile window before ovulation and tion.45 Nevertheless, it is now commonplace to see cou- the day of ovulation.51 ples delaying family planning due to professional and Nonetheless, the best advice for women with economic reasons. This delay mainly jeopardizes the unknown fertile window is to have intercourse at a fre- female fertilizing potential. There is a 50% reduction in quency of two to three times a week during the whole fecundity in woman aged 35 years compared to those menstrual cycle. Unfortunately, some couples find aged 25 years.45 it more stressful and intricate to adhere to frequent schedule of intercourse than to timely assured fruitful Sexual and Coital Aspects intercourse. Although sexual and coital factors account for only 5% A detailed sexual history is also a valuable tool to of infertility causes, it is mandatory to analyze these fac- identify problems. A decreased tors, as they may be the sole cause of the problem.46 It is libido may be caused by complex interaction of social, preferable to discuss these issues with both partners at emotional, psychological and hormonal factors. The the same interview, as this will help to disclose the mis- decreased libido may also be the only sign of hypogo­ apprehension undertaken by the partners regarding the nadism, which results in (ED) and physiology of fertilization. decrease in the sexual drive. Although erectile dysfunc- The clinician should address the concept of fertility tion may be attributed to psychogenic problems, endo- window, and the importance of frequent and well-timed crinopathies such as hypogonadism, diabetes, hypothy- intercourse. Surprisingly, many couples have infrequent roidism and hyperprolactinemia may also be involved. intercourse due to life stressors and irregular work- The incidence of ED increases with age; it is 52% in men ing hours, and are unaware of the importance of timed of age between 40–70 years old, and about 2–27% in men intercourse. The fertility window is defined as the 6-day less than 40 years old.53,54 period within the menstrual cycle when intercourse Though the recommendation to increase the fre- most likely results in a conception. It is estimated to quency of intercourse in the fertile window is evidence- range from 5 days before ovulation to the day of ovu- based, no specific coital technique is praised. Whatever lation for normogonadotropic women.47-49 The highest the position is, the ejaculated sperm in the proximal probability of a successful pregnancy can be anticipated vagina can find the way to the oviduct within five min- if intercourse occurs 1 to 2 days prior to ovulation.48,49 utes.55 Consequent to increased coital frequency, vagi- This phenomenon is based on short duration of human nal dryness may direct the women to use vaginal lubri- ovum longevity and survival that is estimated to be fewer cants. However, commercially available water-based than 16 hours.50,51 On the other hand, human sperm can lubricants are toxic to sperm. These lubricants may survive for up to 6 days in well-estrogenized cervical either induce poor sperm motility or chromatin dam- mucus.52 This relatively protracted longevity and survival age and, correspondingly, loss of fertilizing potential creates an adequate sperm reservoir for fertilization. of sperm.56 Among the lubricants available in the mar- Therefore, it is advisable for couples to have inter- ket, hydroxyethyl cellulose-based lubricant was shown course every other day during the 6 days fertile win- to have the lowest damaging effects on sperm motility dow. Sperm counts are at their maximum after 5 days and chromatin integrity.57 Natural lubricants, such as of ejaculatory abstinence. After that, sperm motility saliva, vegetable oils (canola, vegetable, olive, safflower 35 Section 2 Male Factor Infertility

and peanut), as well as glycerin and petroleum jelly The childhood history of congenital and acquired cause mild decrease in sperm motility.56,58 genitourinary disorders should also be taken into account. Cryptorchidism is the most common genitou- Ejaculatory Disorders rinary disorder and affects about 1–3% of term infants. Natural fertilization requires successful deposition of However, its prevalence among infertile men is 9.4%.71-73 semen into the proximal vagina. Therefore, ejaculatory If cryptorchidism is treated by orchidopexy at an early dysfunction can interfere with this process and may age, there will be higher chance of maintenance sper- render a male infertile. The patient should be asked matogenesis.74 Though men with unilaterally repaired about volume of the ejaculate during coitus and mas- cryptorchidism have variable semen parameters; their turbation. Low semen volume may be attributed to paternity rate seems to be similar to that of general pop- either congenital or acquired obstructive lesion of the ulation.75 However, in those with bilaterally repaired genital tract, male hypogonadism or due to retrograde cryptorchidism a 35–53% decrease in the paternity rates ejaculation. Anejaculation, on the other hand, is a com- (compared to the general population) is observed, with plete absence of antegrade and retrograde ejaculation high incidence of disordered semen parameters such as and is caused by defective seminal emission from semi- oligozoospermia (31%) and azoospermia (42%).76 Other nal vesicles, prostate and the ejaculatory ducts into the rare urogenital anomalies such as microorchidism, poly­­ posterior urethra. This condition is usually associated orchidism, vanishing testis, prune belly syndromes, with intact orgasm, and its etiology is related to neu- posterior urethral valve and bladder exstrophy are well rogenic or drug induced effect.59 Anejaculation should known causes of impairment of male fertility potential. be differentiated from anorgasmia, which is a psy- Inquiries should also include questions about acci- chological disorder characterized by failure to attain dental and surgical trauma to the testes during child- orgasm and is usually associated with anejaculation.60 hood as well as about pediatric hernia repair, which is The prevalence of premature ejaculation among sub- associated with 0.8–2% risk of injury to the inguinal or fertile males is reported to be around 50%.61 However, retroperitoneal vas deferens.77 Unilateral vas injury may premature ejaculation seems to be related to the stress remain unnoticed in the presence of normally function- imposed by the infertility condition rather than to be a ing contralateral testis and contralateral patent seminal primary cause. duct. Bilateral pediatric hernia repair, particularly with the use of mesh, forms the highest risk of clinically evi- Pediatric and Developmental Contribution dent vas injury and male infertility.78 History of testicu- The questionnaire about childhood and pubertal devel- lar torsion, whether treated by orchidopexy or orchiec- opment includes prenatal exposure to certain dietary, tomy can lead to abnormal semen analysis in 30–40% of pharmaceutical and environmental factors that may the affected patients.79 lead to congenital urogenital anomalies, disordered Now, mumps orchitis during childhood is rare hormonal milieu and even altered semen parameters. because of the widely conducted vaccination program. In utero exposure to diethylstilbestrol, a medication Prepubertal mumps orchitis has not been found to that has been banned from the market several years affect testicular function.80,81 However, recent reports ago, leads to appearance of epididymal cyst, cryptor- have raised concern about an increased incidence of chidism and altered semen quality in the male off- postpubertal mumps orchitis after the age of 15 years.82 spring.62 History of parental infertility or the mother Mumps of postpubertal onset is associated with orchi- being subjected to any infertility medical treatment may tis in 25% of cases,83 and in approximately 10–30% of be associated with a decline in the semen parameters cases, bilateral testicular involvement is documented.84 and reproductive potential in the male offspring.63,64 Mumps orchitis can lead to subfertility in 13% and It has been also assumed that maternal exposure to 30–87% of patients with unilateral and bilateral testicu- environmental endocrine disruptors, which are chemi- lar involvement, respectively.84,85 cals present in a variety of pesticides, plant constituents The age of puberty onset is of paramount importance and plastic industry, could induce infertility in adult as it reflects the proper interaction and the integrity of sons. Several compounds have estrogenic activity, as the HPG axis. Most males reach pubertal development such, they are called xenoestrogens, and they have the at age 11–12 years.86 Constitutional delay of puberty is ability to impair testicular development and sperm the most common cause for such condition and is usu- production.65,66 Maternal cigarette smoking and alcohol ally confirmed by positive family history, delayed bone consumption have been also linked to altered semen age and low or normal gonadotropins.86 Nevertheless, quality in the male progeny.67-70 this delay may be attributed to primary testicular failure 36 Chapter 3 Evaluation and Diagnosis of Male Infertility such as , partial androgen resist- scrotal vas deferens, epididymal tubules and testicular ance or other acquired causes, or secondary to hypotha- arteries precipitating vasal and epididymal obstruction lamic or pituitary dysfunction. There are many acquired as well as testicular . Considering all these fer- and congenital causes of hypothalamic and pituitary tility related surgical complications may help to find dysfunction such as Kallmann’s syndrome, Prader-Willi the cause, direct specific diagnostic investigations and syndrome, Laurence-Moon-Biedl syndrome, idiopathic thereby restore fertility potentials. hypogonadotropic hypogonadism and pituitary tumors. Conversely, precocious puberty, which is charac- Medical Aspects terized by appearance of secondary sex features before Medical history of urologic and systemic diseases in 9 years of age, is either caused by premature activation infertile men may be related to the infertility problem. of HPG axis (true precocious puberty) or excessive sex Recurrent attacks of urinary tract, sexually transmitted steroid production (incomplete precocious puberty). infections (STI) and mixed accessory gland infections True precocious puberty may cause testicular enlarge- have all been linked to male infertility. Infected urine ment and normal spermatogenesis so early that a boy is associated with and constitutes a risk factor for geni- at age of 4 years can father children.86,87 However, boys tal tract infections. The STI are commonly caused by with incomplete or pseudoprecocious puberty may , trachomatis, Ureaplasma have pubic hair and penile enlargement, but no testicu- urealyticum, human immunodeficiency virus (HIV) and lar enlargement or production of sperm.87 Such condi- other less common microorganisms. Urethral stricture, tion is often caused by adrenogenital syndrome, a form epididymo-orchitis and postorchitis decline in testicu- of congenital adrenal hyperplasia, or Leydig cell hyper- lar function are well recognized complications of STI.90 plasia or tumors. In pseudoprecocious puberty, there Ureaplasma urealyticum can have deleterious effects on is excessive production of androgens, which suppress sperm motility and DNA condensation leading to poor gonadotropins production. Therefore, there is a lack of fertilization potentials.91 Moreover, recent reports have stimulatory factor of testicular development and sperm shown that Chlamydia trachomatis infection is often production despite the virilized effects of androgens. detected in infertile men. Chlamydia trachomatis may Most men may not memorize the exact timing of attach to the spermatozoa and causes impairment in their puberty unless there is a marked delay or prema- sperm motility, and DNA integrity as well as acrosome ture onset. While some of these conditions may respond reaction (AR) insufficiency.92,93 Mixed accessory gland to hormonal manipulation, others are resistant to hor- infection (MAGI), which is a syndrome characterized by monal and more aggressive such as a chronic inflammatory process that involves the pros- sperm retrieval and assisted reproductive techniques tate, seminal vesicles or epididymides, is also caused may be needed. by the same organisms causing STI such as gonococci, Chlamydia and gram-negative bacteria. This syndrome Surgical Aspects is found in about 10% of infertile men, and it is char- Certain surgical procedures in the vicinity of male acterized by leukocytospermia, increased reactive oxy- reproductive organs may inadvertently injure these gen species and high concentrations of inflammatory organs. Surgery in the pelvis, retroperitoneum, scrotum cytokines.94,95 Symptoms suggestive of MAGI, such as and groin may cause impairment in the male reproduc- urethral discharge, and/or pus or blood associated tive potential. Ejaculatory and erectile dysfunctions are with the ejaculate, should be taken in consideration. known complications of retroperitoneal lymph node Human immunodeficiency virus is one of the STI dissection, deep radical pelvic surgery, bladder neck that impairs male fertility by multiple mechanisms. reconstruction surgery and spinal surgery. Transurethral Orchitis, hypogonadism, enhanced oxidative stress, resection of the prostate and bladder neck incision are oligozoospermia and even azoospermia have been often complicated by retrograde ejaculation. Bilateral described in association with HIV infection.96,97 open or laparoscopic adult repair, par- Some men with autosomal dominant polycystic ticularly with use of knitted mesh, may result in bilateral kidney disease may have low semen volume, oligozoo- vas deferens injury and azoospermia. The prevalence of spermia, and even impaired fertili- such condition is estimated to be 0.3%.88 However, the zation potential.98 unilateral injury may go unnoticed if the contralateral Systemic medical diseases, such as renal insufficiency, testis function and vas deferens are normal. Moreover, liver cirrhosis and endocrinopathies such as thyroid inguinal herniorrhaphy may contribute to testicular dysfunction, Cushing syndrome and diabetes, are all atrophy in 5% of cases.89 On the other hand, scrotal associated with gonadal dysfunction and disordered surgery for and epididymal cyst may injure spermatogenesis.99-102 Men with a history of galactorrhea, 37 Section 2 Male Factor Infertility

headache and impaired vision may have prolactinoma. 3. Inflicting erectile and ejaculatory dysfunction such Gastrointestinal diseases, such as celiac disease, inflam- as beta-blockers and alpha-blockers, or matory bowel diseases and other malabsorption syn- 4. Mitigating libido such as psychotherapeutic medi- dromes, are associated with , hypo­ cations and recreational drugs.113 Table 4 presents a gonadism and impaired semen quality.103,104 comprehensive list of medications and drugs related Some respiratory diseases and male infertility may to fertility impairment. have common genetic origin. More specifically, some syndromes may cause chronic nasal polyps and sino­ Inherited and Familial Aspects pulmonary infections and sperm transport problems Genetics play essential role in male subfertility. such as Young’s syndrome that is associated with ob- Therefore, exploring the personal history of certain struction of the epididymides and vasa deferentia due inherited diseases that may affect the reproductive to inspissated secretions; cystic fibrosis which is as- function as well as the family history of involuntary sociated with congenital bilateral absence of the vas male childlessness are essential components of male deferens; and ciliary dyskinesia syndromes that are fertility evaluation. The retrieved information from characterized by immotile sperm. Other respiratory such enquiries may shed a light on the genetic etiol- diseases may affect the fertility potential through cen- ogy of male infertility. trally induced hypogonadotropic hypogonadism state Personal history of cystic fibrosis, Kallmann’s syn- such as sarcoidosis and obstructive sleep apnea.105,106 drome, Klinefelter’s syndrome, myotonia dystrophica, Furthermore, chronic obstructive airway disease may Kartagener’s syndrome, or adult onset polycystic kid- cause pubertal delay and hypogonadism either through ney disease may explain the etiology of childlessness hypoxia, or corticosteroid-induced hypogonadotropic in infertile men. History of recurrent miscarriages may hypogonadism.107 Men with active pulmonary tubercu- also indicate male chromosomal abnormalities or DNA losis may also have gonadal hormonal dysfunction at- integrity defects. tributed to central mechanism.108 The queries about family history must disclose the Male infertility is also common in sickle cell ane- fertility status of men in the family pedigree of three mia, presumably, due to intratesticular .109,110 generations that include first-degree relatives such Prolonged fever may cause reversible impairment in as father, brothers as well as paternal and maternal semen quality.111 Malignancy and its treatment, such uncles. Some studies detected familial clustering of as radiotherapy and chemotherapy, may also be a risk cases of male subfertility due to known genetic, or even factor for male infertility due to transient or permanent due to undiscovered genetic etiology. A single study alterations in sperm production and, less commonly, has shown that 11.8% of 621 couples have involuntary androgen insufficiency. Testicular cancer, Hodgkin’s childlessness in at least one first-degree and second- disease and acute leukemia are the most common degree relatives on the husband side.114 Another study malignancies in young adult patients. Men with testicu- demonstrated­ that men with impaired semen quality, lar cancer may be oligozoospermic in 50% and azoosper- reported higher incidence of subfertility among their mic in 10–15% of cases before receiving any treatment.112 brothers than that reported by male relatives of normo- Some specific clinical features have genetic relations zoospermic infertile men.115 with male infertility such as anosmia (Kallmann’s syn- Moreover, the husband’s family history of multiple drome), deafness (deafness-infertility syndrome), early births defects also raises the concern about chromosom- cataract (myotonia dystrophica), bronze discoloration al disorders. Husband’s parental consanguinity is also of the skin (hemochromatosis), muscle weakness (myo- considered a risk factor for genetic male infertility. tonia dystrophica, mitochondrial disease) and certain birth marks or scaling skin (X-linked ichthyosis). Environmental Factors and Occupational Exposure The worldwide rising incidence of male infertility has Use of Medication been recently attributed to environmental toxicants. Drugs and medications may undermine male reproduc- Exposure to these harmful factors must be assessed such tive function in various ways. Personal use of drugs and as excessive heat from saunas and hot tubs, use of pesti- medications must be thoroughly elucidated. There are cide and organic solvents, radiation from X-rays or from four mechanisms explaining their impact on male fertility: excessive use of cell phone and heavy metals intoxica- 1. By acting as direct gonadotoxins such as chemother- tion such as lead or cadmium. apeutic agents, Certain occupations constitute a hazard for male 2. Through alterations in the hormonal control of tes- reproductive health. The foundry and steel work- 38 ticular function such as exogenous anabolic steroids, ers, specialized welders, and bakers reported to have Chapter 3 Evaluation and Diagnosis of Male Infertility

Table 4 Effects of medication on male fertility Altered Decreased Erectile Fertilization Medication Gonadotoxic HPG axis libido dysfunction potential Recreational/illicit drugs Alcohol + + + + – Cigarette smoking + – – – – Marijuana + – – – – – + + – – Cocaine – – – + – Antihypertensive drugs Thiazide diuretics – – – + – Spironolactone – + + + – Beta-blockers – – + + – Calcium channel blockers – – – – + Alpha-adrenergic blockers – – – + – Psychotherapeutic drugs Antipsychotics – + + + – Tricyclic antidepressant – + + + – MAOIs – – – + – Phenothiazine – + – – – Lithium – – + + – Chemotherapeutic drug Alkylating agents + – – – – Antimetabolites + – – – – Vinca alkaloids + – – – – Hormones Anabolic steroids + + – + – Testosterone + – – – – Antiandrogens – + + – – Progesterone derivatives – – + + – Estrogens – + + + – Antibiotics Nitrofurantoin + + – – – Erythromycin + – – – – Tetracycline – – – – + Gentamicin + – – – – Miscellaneous medications Cimetidine + + – – – Allopurinol – – – – +

HPG: hypothalamic-pituitary-gonadal axis; MAOIs: monoamine oxidase inhibitors;(+) means that the medication has an effect; (–) means that the medication has no effect heat-induced decline in semen quality. Sedentary Cigarette smoking history should be verified, and the occupations have claimed to increase scrotal tempera- patient should be encouraged to quit smoking. As smok- ture and affect the testicular function. Other occupa- ers have higher incidence of testicular atrophy, altered tions, including exposure to organic solvents, such as semen parameters and sperm function, as well as sperm painters, printers, dyes manufacturers are also consid- with oxidative DNA damage and chromosomal aneu- ered hazardous to male reproductive health. ploidy.116 Alcohol, on the other hand, should be taken in 39 Section 2 Male Factor Infertility

moderation because alcohol may suppress testosterone and intracranial tumors. The combination of galactorrhea, production and consequently impair spermatogenesis.117 visual field defects and infertility is suggestive of prolac- Lastly, obesity and malnutrition must be considered tinoma. Thyroid dysfunction is accompanied by relative as both can have detrimental effect on male reproduc- androgen deficiency and impairment of spermatogenesis. tive function. Such conditions may result in deficiency Chest is examined for the presence of gynecomas- of vitamins and micronutrients which impairs the highly tia and galactorrhea. The signs of chronic sinopulmo- active biological process of spermatogenesis. nary infections, in association with male infertility, may reveal underlying genetic syndromes such as cystic Female Factor Infertility fibrosis, Young’s syndrome and Kartagener’s syndrome. A comprehensive female evaluation by the gynecolo- Abdomen is meticulously inspected and palpated gist is an essential step in the integral evaluation of the to detect signs of chronic liver diseases, such as liver infertile couple. enlargement, and splenomegaly or signs of polycystic kidney diseases. Physical Examination Genitalia General Aspects The genital examination, including digital rectal Initially, the physical examination must be performed examination, should be carried out. The inguinal and in a warm room and the patient is asked to undress and genital regions must be carefully inspected, in order stand with his arms outstretched. This position enables to identify scars from previous surgical interventions, the physician to measure the patient’s height, weight such as hydrocele correction, inguinal hernia repa and arm span as well as to observe abnormal body irs and surgery for undescended . They may hair distribution, abnormal body habitus, muscular account for damage to the testicle blood supply and to hypotonic posture, fat distribution and skin pigmenta- the vas deferens. The genital examination may reveal tion. Physical signs of hypogonadism depend on age of the presence of a hypospadiac urethral meatus, patho- onset in relation to puberty. Prepubertal hypogonadism logic curvature of the phallus or active sexually trans- onset is characterized by eunuchoidism (arm span exceeds mitted disease. These factors may ultimately result in body height by more than 5 cm) due to delayed closure misplacement of spermatozoa inside the vaginal vault of the epiphyseal plates in long bones with sparse facial, following ejaculation. pubic and axillary hair distribution pattern. In contrast, Testis volume can be estimated with the aid of an postpubertal hypogonadism (adult onset) may not affect or can be measured by using a pachymeter the anthropometric measurements; however, decline in (Figure 4A). A normal sized adult testicle should have a the muscle bulk and bone mass with loss of beard and length of 4 cm and a width of 2.5 cm resulting in a volume pubic hair, and thinning of facial skin may be visible. around 20 ml. They should present with firm consist- Both conditions affect skin color and texture, due to ency. Eighty-five percent of the testicular parenchyma is poor development of androgen-dependent sebaceous involved with spermatogenesis. There is no lower limit gland, resulting in dryness, wrinkling and pallor of the for testicular volume to exclude the presence of sperma- skin. Although testicular and penile size may not be tozoa. As such, testicle size cannot be relied on as a clini- affected to a great extent in adult onset, hypogonadism, cal marker to preclude a trial of sperm retrieval.118 preadolescent hypogonadism usually culminates in Bilateral testicular atrophy may be caused by pri- infantile gonads and penis. mary or secondary testicular failure. When serum tes- Although rare, particular attention should be paid tosterone is low, seminal fluid is often of small volume to the diagnosis of specific inherited syndromes that as well. Endocrine workup helps to distinguish both negatively impact the male reproductive potential. conditions. High follicle-stimulating hormone (FSH) Such conditions include Prader-Willi syndrome, which levels accompanied by normal or low testosterone levels is characterized by obesity, hypogonadism, short stat- imply primary testicular failure. These patients ure, skin hypopigmentation and muscle hypotonia, and should be offered genetic evaluation for chromosomal Laurence-Moon-Biedl syndrome featured by obesity, abnormalities and Y chromosome microdeletions. The mental retardation, and retinitis pigmentosa. combined low serum FSH and testosterone levels sug- The physical examination of head and neck includes gest hypogonadotropic hypogonadism, particularly, if testing for smell, visual field defects, midline craniofa- bilateral atrophic testicles are present. In this scenario, cial abnormalities and thyroid enlargement. Anosmia is serum LH is often low. These men should undergo crani- one of the presenting features of Kallmann’s syndrome. al imaging and serum prolactin measurement to exclude 40 Blurred vision and visual field loss are signs of pituitary pituitary gland disease as in Kallmann’s syndrome.119 Chapter 3 Evaluation and Diagnosis of Male Infertility

A B Figures 4A and B Useful tools for the physical examination of the subfertile male. (A) Photograph of the Prader Orchidometer. It is used to measure the volume of the testicles and consists of a chain of 12 numbered beads of increasing size from 1—25 ml. The beads are compared with the testicles of the patient and the volume is read off the bead, which matches most closely in size. Prepubertal sizes are 1—3 ml, pubertal sizes are 4—12 ml and adult sizes are 15—25 ml; (B) Schematic illustration depicting the use of the 9 MHz pencil-probe Doppler stethoscope for varicocele examination. The patient is examined in the upright position and the conducting gel is applied at the upper aspect of scrotum. A venous “rush” may be listened during Valsalva maneuver indicating blood reflux (Reprint with permission from Esteves SC, Miyaoka R, Agarwal A. An update on the clinical assessment of the infertile male. CLINICS 2011;66(4):691-700)

The epididymides have to be evaluated according specificity of approximately 12270%. Varicoceles diag- to their size and consistency as well. The obstructed nosed by this method are termed “clinical” and may epididymis is augmented and ingurgitated (soft). A be graded according to the size. Valsalva maneuver healthy epididymis free of trauma, infection or obstruc- may reveal differences in blood volume in each cord. tion should be firm. Partial regression of an epididymis Internal spermatic veins and cremasteric veins are filled may represent a scenario of CBAVD. The vasa are eas- up, whenever the patient stands. In varicocele patients, ily palpable inside the posterior aspect of the spermatic a venous dilation exists and may be enhanced during cord as a distinct, firm, round, “spaghetti-like” struc- Valsalva maneuver. Large varicoceles (grade III) are ture. Unilateral or bilateral congenital absence of the vas varicose veins seen through the scrotal skin (Figure 5). results in oligozoospermia or azoospermia, respectively. Moderate varicoceles (grade II) and small-sized varico- Narrowing areas of the vasa deferentia may represent celes (grade I) are dilated veins palpable without and an infection or traumatic sequelae. with the aid of the Valsalva maneuver, respectively.123 Absence of the vasa deferentia or vasal agenesis is a No standardized diagnostic method has been defined clinical diagnosis and does not depend on any comple- for the identification of varicocele.124 mentary imaging study. However, 25% of men with uni- Lastly, digital rectal examination should assess the lateral vasal agenesis and about 10% of those with CBAVD pelvic muscle tone in cases of suspected erectile dys- also have unilateral renal agenesis and should undergo function and detect any palpable masses including pro- an abdominal ultrasound to identify this condition.120 static cysts that may obstruct the ejaculatory ducts. Each has to be inspected to assess volume and consistency and to detect existing lipomas Investigation or varicocele. This initial workup consists of semen fluid analysis. Varicocele may be identified by physical examina- Additional testing may be needed depending on the tion with the patient standing in a warm room.121 A results of semen analysis or the information obtained by physical examination alone provides a sensitivity and history and physical examination. 41 Section 2 Male Factor Infertility

variability may exist within the same individual. The interval between the semen analyses is arbitrary and is generally recommended to be 1–2 weeks. Ejaculatory abstinence should be a minimum of 2 days to a maxi- mum of 7 days, ideally 2–3 days.127 Longer abstinence periods lead to higher ejaculatory volume and increased spermatozoa quantity, but motility is usually decreased. The specimen is generally collected by masturbation inside a sterile recipient with a wide opening in order to avoid spillage outside the container that otherwise can be misinterpreted as . The collection should be preferentially done in a proper collection room and no lubricant should be used. If collected at home, the specimen should be brought to the laboratory within 30 minutes, and should be kept close to the body in an effort to maintain physiological temperature dur- ing transportation. The specimen must be identified and allowed to liquefy for 30–60 minutes before analysis is Figure 5 Photograph of a large left grade III varicocele undertaken. Routine semen analysis should include: that can be seen through the scrotal skin (Reprinted with • Physical characteristics of semen, including liquefac- permission from Esteves SC, Miyaoka R, Agarwal A. An update tion, viscosity, pH, color and odor on the clinical assessment of the infertile male. CLINICS • Specimen volume 2011;66(4):691-700) • Sperm count • Sperm motility and progression Seminal Fluid Analysis • Sperm morphology Semen is basically composed of spermatozoa and secre- • Leukocyte quantification tory fluid of the accessory sex glands. The semen analys­ • Fructose detection in cases where no spermatozoa is is is considered as the cornerstone of the laboratory found especially if the total volume is less than 1 ml. evaluation, although it is not a sperm functional test.125 The criteria used for normality according to the World It provides information of the functional status of the Health Organization (WHO) have been recently germ inative cells, epididymides and accessory sexual updated,127 as shown in Table 5. Approximately 2,000 glands. Semen analysis is of great value on the male men from eight countries, whose partners had a time-to- initial investigation and its results are often taken as a pregnancy of either less than or equal to 12 months, were surrogate measure of male fecundity and pregnancy chosen as individuals to provide reference distributions risk. Reference intervals for values of semen parameters for semen parameters. One-sided lower reference limits from a fertile population may provide data, on which (the fifth centile) were generated and have been proposed prognosis of fertility or diagnosis of infertility can be to be considered the cutoff limits for normality (Table 5). extrapolated. Therefore, it is recommended that evalu- Apart from the total sperm number per ejaculate, the ation should be undertaken in a specialized andrology limits of these newly released reference values are lower laboratory and should be analyzed by well-trained tech- than the previously presented ones128-130 but are in agree- nicians under rigorous quality control standards.126 ment with recent observations on the seminal profile of Nonetheless, the prognostic value of semen com- fertile men.131-133 ponents such as sperm number, motility and morpho­ The morphometric description of spermatozoa, logy, as surrogate markers of male fertility, is confound- according to the strict criteria described by Kruger ed in several ways; the fertility potential of a man is et al.134 was definitely incorporated in the new WHO influenced by sexual activity, the function of accessory guidelines. The low proportions of normal spermato- sex glands and other defined, as well as yet unrecog- zoa, as defined by those selected in endocervical mucus, nized conditions. Routine semen analysis itself has inevitably produce very low reference limits for a fer- its own limitations, and does not account for putative tile population. With this method, similar low values sperm dysfunctions such as immature chromatin or a of 3–5% normal forms have been found by others to be fragmented DNA. Results from at least two, preferably optimal cutoff values to discriminate fertile from infertile three separate semen analyses must be obtained before men, whose spermatozoa were used for in vitro fertiliza- 42 a definitive conclusion can be drawn, as wide biological tion,135 intrauterine insemination136 and in spontaneous Chapter 3 Evaluation and Diagnosis of Male Infertility

Cutoff values for semen parameters as published in consecutive World Health Organisation (WHO) manuals (Adapted from Esteves SC, Miyaoka R, Agarwal A. An update on the Table 5 clinical assessment of the infertile male. CLINICS 2011;66(4):691-700, with permission from the Editor) Semen parameters WHO, 1992 WHO, 1999 WHO, 2010* Volume ≥ 2 ml ≥ 2 ml 1.5 ml Sperm concentration/ml ≥ 20 x 106/ml ≥ 20 x 106/ml 15 x 106/ml Total sperm concentration ≥ 40 x 106 ≥ 40 x 106 39 x 106 Total motility (% motile) ≥ 50% ≥ 50% 40% Progressive motility† ≥ 25% (grade a) ≥ 25% (grade a) 32% (a + b) Vitality (% alive) ≥ 75% ≥ 75% 58% Morphology ≥ 30%‡ 14%§ 4%″ Leukocyte count < 1.0 x 106/ml < 1.0 x 106/ml < 1.0 x 106/ml

*Lower reference limit obtained from the lower fifth centile value † Grade a = rapid progressive motility (> 25 μm/s); grade b = slow/sluggish progressive motility (5–25 μm/s); Normal = 50% motility (grades a + b) or 25% progressive motility (grade a) within 60 min of ejaculation ‡ Arbitrary value § No actual value given, but multicenter studies refer to > 14% (strict criteria) for in vitro fertilization ″Normal shaped spermatozoa according to Tygerberg (Kruger) strict criteria pregnancies.137 Interpretation of the reference ranges as it is a simple and low-cost option to detect the pres- requires an understanding that they merely provide ence of peroxidase within neutrophils.139 a description of the semen characteristics of recent fa- In azoospermic patients, diagnosis must be con- thers. The reference limits should not be overinterpret- firmed by lack of any spermatozoa on centrifuged semi- ed to distinguish fertile from infertile men accurately, nal fluid on two separate occasions at high powered but they do represent semen characteristics associated microscopic field evaluation. The WHO recommends with a couple’s achieving pregnancy within 12 months centrifugation for 15 minutes at 3000g or greater.119 of unprotected sexual intercourse; as such, the limits Azoospermia with low ejaculate volume (< 1.0 ml) not provide only a standardized guide regarding a man’s related to hypogonadism or CBAVD can be caused by fertility. None of these values were able to solely dis- ejaculatory dysfunction, although the most common tinguish fertile and infertile men, although morphology cause is ejaculatory duct obstruction (EDO). When sus- was suggested to be the most important. The coexistence pected, EDO can be confirmed by assessing seminal pH of more than one altered seminal parameter significantly and fructose as normal seminal vesicle secretions are increases the risk for infertility.131 A man’s semen char- alkaline and contain fructose. acteristics need to be interpreted in conjunction with his clinical information. The reference limits provided by the Endocrine Evaluation WHO manual are from semen samples initiating natural Endocrine evaluation should be performed in the fol- conceptions. As such, they may indicate whether a man lowing scenarios: would need infertility treatment, but they should not be • Sperm concentration less than 10 million/ml used to determine the nature of that treatment. • Erectile dysfunction Leukocyte count should also be added to the rou- • Hypospermia (semen volume < 1ml) tine semen analysis. Leukocytospermia (> 1 million leu- • of endocrinopathies or kocytes/ml of semen) incidence in infertile men vary hypogonadism. between 3% and 23% and has been correlated with clini- The minimal evaluation includes serum FSH and total cal and subclinical genital infections, elevated levels testosterone. They reflect germ cells and Leydig cells of reactive oxygen species, antisperm antibodies and status respectively. If testosterone level is low, a second deficient sperm function.138 Neutrophils predominate analysis will be recommended along with free testoster- among the inflammatory cells and may be both identi- one, LH, estradiol and prolactin measurements. Isolated fied and quantified through different methods and col- FSH elevation is usually indicative of severe germ cell oring techniques. The Endtz test is one of the most used damage. Elevated FSH and LH levels associated with 43 Section 2 Male Factor Infertility

low-normal or below-normal testosterone levels impli- culture and Giemsa band staining (G band karyotyping). cate diffuse testicular failure which may have a con- Genetic mutations and Y chromosome microdeletions genital (e.g. Klinefelter syndrome) or acquired cause. assessments are also performed by analysis of peripher- Concomitant low levels of FSH and LH may implicate al blood sampling. DNA is amplified using polymerase hypogonadotropic hypogonadism. This may also be chain reaction technique. Table 6 summarizes the indica- congenital, or secondary to pituitary diseases. In these tions and recommended tests for genetic evaluation. cases, a complete workup of pituitary function is rec- Chromosomal abnormalities can be found in about ommended including serum measurement of adreno- 6% of infertile men and its prevalence inversely corre- corticotropic hormone, thyroid-stimulating hormone, lates with sperm count. Azoospermic men can present growth hormone and brain magnetic resonance.140 chromosomal alterations in as much as 16% of cases.144 Gonadotropin values within normal range suggest Sex chromosomal aneuploidy (Klinefelter syndrome; extraductal obstruction in azoospermic subjects. 47,XXY) is the most frequent chromosomal disorder However, patients with sperm maturation arrest and present in infertile men and is generally associated with 10% of those diagnosed with Sertoli-cell-only syndrome hypotrophic or atrophic testicles, elevated serum FSH may present with nonelevated hormonal measurements. levels and azoospermia, although spermatogenesis can Serum estradiol measurement should be done in obese be differently affected in patients with a mosaic karyo- patients and in those presenting with gynecomastia or type (46,XY/47,XXY). hypogonadism. Infertile patients, in whom testosterone The mutation of the cystic fibrosis gene located on to estradiol ratio is less than 10, can harbor significant the long arm of chromosome 7 is the most commonly but reversible seminal alterations.141 Vaucher et al.142 found genetic mutation. According to the extension of suggested that hyperestrogenism secondary to a higher the mutation, cystic fibrosis can be manifested at its full conversion of testosterone into estradiol in Klinefelter clinical presentation (an autosomal recessive poten- syndrome patients inhibits testosterone production via tially fatal disease) or in a mild form, where congenital a negative feedback pathway and may indicate overex- bilateral absence of the vasa deferentia exists and pression of aromatase CYP19 in the testis at a molecu- affects approximately 1.3% of infertile men. Cystic fibro- lar level. As such, there would be scientific rationale sis transmembrane conductance regulator (CFTR) gene for use of aromatase inhibitors in Klinefelter syndrome mutations compromise the development of Wolffian patients.141 ducts derived structures (efferent ducts, epididymides In azoospermic men with normal ejaculate volume, and vasa deferentia) and may even be implicated in a FSH serum level greater than twice the upper normal seminal vesicles hypoplasia or agenesis, and unilateral limit is reliably diagnostic of dysfunctional spermato- renal agenesis. Approximately 80% of men presenting genesis and, when found, diagnostic testicular biopsy with CBVAD have a CFTR mutation. As the diagnostic is usually unnecessary although no consensus exists in methods routinely used are not 100% sensitive, a man this matter.119 If FSH level is normal, a unilateral biopsy with CBAVD should be assumed to harbor a CFTR on the larger testis may be recommended as there is no mutation. Genetic testing should be offered to female guarantee of normal spermatogenesis. partners to exclude the possibility of these being car- Hyperprolactinemia is a rare cause of infertility in riers (approximately 4% risk) before using their hus- healthy men and is more commonly related to erectile band sperm for assisted conception. Genetic coun- dysfunction and pituitary diseases. These men may seling should be offered after genetic testing.119 Recent present micropituitary or macropituitary prolactin- data suggest that azoospermic men with idiopathic secreting adenomas. Serum prolactin must be deter- obstruction and those presenting the triad of chronic mined in infertile men with a complaint of concomitant sinusitis, bronchiectasis and obstructive azoospermia sexual dysfunction or in those who have clinical or labo- (Young’s Syndrome) have an elevated risk for CFTR ratory evidence of pituitary disease. Although hormonal mutation.144 alterations may be present in approximately 10% of The long and short arms of Y chromosome are men who undergo investigation, clinically significant respectively related to spermatogenesis and testicle dysfunctions affect less than 2% (Table 2).143 development. The Y chromosome region related to in- fertility is named azoospermia factor (AZF) locus. The Genetic Evaluation AZF locus can present complete or partial microscopic Genetic factors commonly associated with male infertility deletions, isolated or in combination, and in nonover- include chromosomal aberrations, genetic mutations and lapping subregions called AZFa, AZFb, AZFc and AZFd Y chromosome microdeletions. Chromosomal aberra- (Figure 6). These subregions contain multiple genes con- 44 tions are assessed through peripheral blood lymphocyte trolling different steps of spermatogenesis. Chapter 3 Evaluation and Diagnosis of Male Infertility

Indications for genetic testing in male infertility (Adapted from Esteves SC, Miyaoka Table 6 R, Agarwal A. An update on the clinical assessment of the infertile male. CLINICS 2011;66(4):691-700, with permission from the Editor) Indications Recommended tests Men with infertility of unknown etiology and sperm Y chromosome microdeletion and G band karyotype concentration less than 10 million/ml who are candidates for assisted reproductive technology (ART) Nonobstructive azoospermia in a male considering Y chromosome microdeletion and G band karyotype testicular sperm retrieval for ART Azoospermic or oligozoospermic men with absence CFTR gene mutation of at least one vas deferens at physical examination Azoospermic men with signs of normal spermatogenesis CFTR gene mutation (e.g. obstructive azoospermia of unknown origin) History of recurrent miscarriage or personal/familiar G band karyotype history of genetic syndromes CFTR: cystic fibrosis transmembrane conductance regulator

Figure 6 Illustration of the Y chromosome in humans and the regions involved in fertility and infertility. Interstitial or terminal deletions that include AZFa usually produce the severe phenotype of Sertoli-cell-only syndrome. Interstitial or terminal deletions that include AZFb and/or AZFb+c (hereafter, designated AZFb/c) are mediated by recombination between palindromic repeats, either P5/proximal-P1, P5/distal-P1, or P4/distal-P1. These deletions are associated to the maturation arrest phenotype and azoospermia. Interstitial or terminal deletions that include AZFc only are mediated by recombination between the b2/b4 palindromic repeats and result in a variable phenotype, ranging from azoospermia and Sertoli-cell-only syndrome to severe or mild oligozoospermia. (Adapted from Repping S, Skaletsky H, Lange J, et al. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet. 2002;71(4):906-22. With permission from Elsevier)

The most common Y chromosome deletion in infertile 1.7%.145 Detection of Y chromosome microdeletions pro- men is the one affecting the deleted in azoospermia (DAZ) vides predictive information on the success of obtaining gene located in AZFc region. Severe oligozoospermia or spermatozoa from the testicle for intracytoplasmic sperm azoospermia can be seen in such cases. Y chromosome injection (ICSI). AZFa and AZFb microdeletions present microdeletions can be found in 6% of men presenting with as azoospermia and are associated with germ cell aplasia severe oligozoospermia (< 1 million/ml) and 15% of those and maturation arrest, respectively. In such cases, sperm with azoospermia.144 For sperm counts between 1 million/ retrieval attempt is not indicated because the chances of ml and 5 million/ml, the detection rate drops down to finding testicular sperm are unlikely.146,147 In case of AZFc 45 Section 2 Male Factor Infertility

microdeletion, sperm can be retrieved in approximately use for subclinical varicocele diagnosis is controver- 71% of patients.145 Clinical pregnancy rates after ICSI are sial, as several studies demonstrated no clinical ben- virtually the same as for idiopathic nonobstructive azoo- efit from surgical treatment in this situation.122 When spermic patients who can have their sperm retrieved as there is doubtful physical examination such as in obese well. patients or difficulty to assess the contralateral side of Genetic testing also provides information for genetic a clinically detectable varicocele then ultrasonography counseling, as sons of men with Y chromosome micro- is useful, as correction of a subclinical varicocele con- deletion will inherit the abnormality and may also be comitant to a clinical contralateral one might be justified infertile.148,149 in this scenario. The commonly accepted color-Doppler The prevalence of structural abnormalities in the ultrasonography criterion for varicocele (maximum autosomes, such as inversions and translocations, is also vein diameter of 3 mm or greater) has a sensitivity of higher in infertile men than in the general population. about 50% and specificity of 90% compared to physi- Gross karyotypic abnormalities are related to an elevated cal examination.153 A pencil-probe Doppler (9 MHz) risk for miscarriages and for having children with both stethoscope is an inexpensive tool that may aid in the chromosomal and congenital defects. As such, men with diagnosis of the varicocele. The patient is examined in the nonobstructive azoospermia (NOA) or severe oligozoo- upright position, and a venous “rush” representing blood spermia should be karyotyped before their sperm are reflux is heard with or without the Valsalva maneuver used for ICSI.119 (Figure 4B). Although simple and easily performed in the office, Hirsh et al. demonstrated that more than 50% Imaging of men without clinical varicoceles exhibited a Valsalva Transrectal, scrotal and renal ultrasonography: The indica- maneuver Doppler-positive reflux.154 None of these tions for transrectal ultrasonography (TRUS) include: adjunctive diagnostic methods can differentiate between • Low semen volume (< 1.5 ml) clinical and subclinical varicoceles. The significance of • Abnormal digital rectal examination a positive test result using any of these adjuvant tech- • Ejaculatory disorders (anejaculation, , niques in infertile men remains uncertain. painful ejaculation) Urinary tract ultrasonography is indicated to evalu- It allows for evaluation of the distal extraductal sys- ate renal status in patients diagnosed with CBAVD. tem (seminal vesicles and ejaculatory ducts). Ejaculatory Renal agenesis may be present in 10% of patients with duct obstruction­ (EDO) EDO may be congenital or CBAVD and 25% of those with unilateral absence of vas acquired, and present either as complete or partial deferens.120 obstruction. The characteristic feature of EDO in TRUS is the enlargement of the seminal vesicles; however Magnetic resonance imaging: Use of magnetic resonance visualization of cysts at the level of ejaculatory ducts imaging (MRI) in infertility investigation has gained may coexist.150 When CBAVD is diagnosed, TRUS can importance in the recent years. Situations such as vari- reveal abnormalities at the level of seminal vesicles such cocele, EDO, seminal vesicle agenesis and undescend- as hypoplasia or agenesis. A recent study has suggest- ed testis are male infertility related conditions and can ed that combination of scrotal and TRUS may not only be incidentally seen.155 Pelvic MRI helps to clarify in distinguish obstructive from NOA but also determine detail pictorial changes initially seen at TRUS (Figure 7). the etiologic classification of obstructive azoospermia Moreover, MRI has traditionally been used to exclude (OA). In this condition, ultrasonographic anatomic ab- cranial pathologies manifested by hormonal changes normalities are more commonly seen than in NOA pa- including low testosterone levels, low serum LH and tients (92.2% vs 2.8%, p < 0.001). Sensitivity, specificity FSH values, and high prolactin level. and accuracy of combined assessment in discriminating There is evidence of the optimized usefulness of between OA and NOA were 95.3%, 97.2% and 96.0%, pituitary MRI in men with hypogonadism when pro- respectively.151 Seminal vesicle aspiration and seminal lactin levels are greater than twice the normal range vesiculography may be performed under TRUS guid- or when there are worrisome symptoms suggesting ance and may help to establish the diagnosis of EDO.152 intracranial abnormality (headache, visual disturbanc- In azoospermic men, finding of a large number of sperm es, diffuse metabolic derangements and other).156 In in the seminal vesicle aspirate, strongly suggests EDO. general, pituitary abnormalities can be identified in 25% Concomitant seminal vesiculography can determine the of hypogonadal men. Of these, however, empty sella site of obstruction. and pituitary nonfunctional microadenomas require no The indication for scrotal ultrasonography is to specific treatment and make one wonder about the cost- 46 evaluate palpable nodules or testicular masses. Its effectiveness of their diagnosis. Chapter 3 Evaluation and Diagnosis of Male Infertility

obstruction and rules out testicle or epididymal obstruc- tion. Currently, isolated diagnostic vasography is not recommended, but may be performed concomitantly with surgical approach for obstruction resolution. Sperm Function Laboratory Testing In 10–20% of infertile couples who undergo basic investigation, all diagnostic workup will yield normal results and couples will be etiologically classified as hav- ing unexplained infertility. Additional tests have been developed in order to identify functional disorders and other sperm abnormalities, which are not addressed in conventional semen analysis. Some of them are only uti- lized as research tools (computer-assisted semen analy- sis, acrosome reaction, oxidative stress evaluation using chemiluminescence, hamster egg sperm penetration test, hemizona assay).158 Others, such as antisperm antibodies and sperm DNA fragmentation have been implemented Figure 7 Magnetic resonance imaging showing enlarged in clinical practice. The clinical indications of these high- seminal vesicles with calculosis (Reprint with permission from ly specialized tests are unexplained male infertility or Esteves SC, Miyaoka R, Agarwal A. An update on the clinical idiopathic male infertility with recurrent failure on mul- assessment of the infertile male. CLINICS 2011;66(4):691-700) tiple in vitro fertilization (IVF)/ICSI cycles. A brief over- view of sperm function test are provided below. Nuclear magnetic resonance spectroscopy has been recently proposed as a possible tool to identify meta- Antisperm antibodies: Risk factors for antisperm antibod- bolic signatures associated with various histological ies (ASA) formation include genital infections, testicular states in infertile men. Based on ex vivo analysis of tes- trauma or surgical biopsy, cryptorchidism, testicu- ticular biopsy specimens, concentrations of 19 tissue lar damage secondary to excessive heat exposure and metabolites were acquired and then reassessed in men obst­ruction of extratesticular ductal system (e.g. vasec- with a diagnosis of NOA. A singular pattern could be tomy). Antisperm antibodies can alter sperm motility determined for two testis histological states: normal and and sperm-oocyte interaction. The immunobeads test Sertoli-cell-only syndrome. Proliferating germ cells are is commonly used for antibodies detection and consists related to high phospholipid synthesis and with elevat- in using human anti-immunoglobulin antibody-coated ed phosphocholine. Normal spermatogenesis spectro- polyacrylamide beads that are capable to detect IgA and scopic pattern presents high peaks of phosphocoline as IgG, the two most clinically important immunoglobu- opposed to Sertoli-cell-only syndrome. Further research lin subtypes associated to immunological infertility.159 in this area may aid in the identification of a distinct It precisely detects antibodies in serum and seminal metabolic signature for sperm existence sites, regardless plasma or bound to sperm surface. The rates of sperm of testis histopathology.157 bound to beads above 50% are considered clinically sig- nificant and may cause dysfunctional motility, vitality Vasography: It is indicated in selected cases of obstruc- and capability for egg interaction.127 The detection of tive azoospermia. It can also be performed when there is antibodies in the serum and seminal plasma are of lim- reason to suspect of unilateral obstruction (e.g. previous ited value as antibodies to sperm are the most likely to surgical intervention) in severe oligozoospermic men induce functional alterations. with a hypotrophic contralateral testis. Vasography and seminal vesiculography are usually undertaken Sperm DNA fragmentation testing: Sperm DNA fragmen- by scrotal or transrectal routes, but can also be per- tation seems to be one of the most important causes of formed transurethrally or perineally. The contrast solu- reduced fertility potential.160 Advanced paternal age, tion flows through vas deferens, seminal vesicles and inadequate dietary intake, drug abuse, pesticide ejaculatory duct delineating the anatomy and allowing environmental exposure, tobacco use, varicocele, medi- for identification of obstructive segments. The presence cal disease, scrotal hyperthermia, air pollution, genital of sperm on microscopic analysis of the fluid recovered ­ or infectious diseases can be cited as possi- from vas deferens and seminal vesicles indicates distal ble causes; some of which are reversible. Fragmentation 47 Section 2 Male Factor Infertility

can be secondary to internal factors such as apoptosis such as vitamin C and taurine, chain-breaking antioxi- and oxidative stress (a pathophysiological mechanism dants such as alpha-tocopherol and ROS-metabolizing secondary to a high concentration of free radicals), or enzymes such as superoxide dismutase and those of external factors such as the presence of leukocytes. the glutathione cycle, but very little catalase. Imbalance Assessment of sperm DNA integrity is indicated in the between the oxidants load and natural antioxidant following situations: defense system is called oxidative stress or oxygen • To investigate infertility in men presenting normal paradox. semen analysis as determined by conventional methods The most often used methods for detecting ROS in an • Cases of recurrent spontaneous abortion andrology setting are divided into two major categories: • To aid determining the most appropriate reproduc- 1. Direct methods such as chemiluminescence and flow tive assisted technology when necessary. cytometry Abnormally fragmented sperm DNA can be found 2. Indirect methods such as the colorimetric one. in 5% of infertile men with normal semen analyses and Chemiluminescence uses probes such as lumi- 25% of infertile men with abnormal semen analyses, but nol to detect ROS.167,168 Luminol can penetrate the cell is rarely seen in fertile men.160 Assays to evaluate sperm and react with intracellular reactive oxygen species; in chromatin/DNA intergrity can be divided in three addition, extracellular ROS exhibit chemiluminescence groups: (a) sperm chromatin structural probes using nu- when mixed with an appropriate oxidizing agent. This clear dyes (e.g. microscopic acridine orange test (AOT), property is due to the formation of unstable endoper- sperm chromatin structural assay (SCSA), aniline blue oxide, which ultimately breaks down with the release test (AB), chromomycin-A3 (CMA3) and toluidine blue of light. The emitted light photons are converted to an (TB), (b) tests for direct assessment of sperm DNA frag- electrical signal and measured by luminometer,169 with mentation (e.g. terminal deoxynucleotidyl transferase ROS generation being measured as counted photons per mediated dUTP nick end labeling assay (tunel) and minute (cpm). The normal range is < 0.2 X 106cpm per single-cell gel electrophoresis assay (COMET), and 20 million spermatozoa.170 Intracellular ROS can also be (c) sperm nuclear matrix assays (e.g. sperm chromatin measured by flow cytometry using different fluorescent dispersion test). TUNEL technique seems to be one of probes such as 2’,7’-dichlorofluorescin-diacetate and the most adequate as it offers the possibility to precisely hydroethidine that react with ROS to emit a red fluores- identify all existing endogenous breaks in sperm DNA. cence.171 The colorimetric technique is also widely used It combines both enzymatic and immunohistochemical for indirectly quantifying ROS. It is based on the princi- techniques for direct observation of DNA fragmenta- ple of spectrophotometry and measures lipid peroxide tion using a fluorescence microscope or flow cytometry. end products, mainly malondialdehyde, lipid hydrop- Elevated sperm DNA fragmentation rates significantly eroxides and isoprostanes.172 diminish the chances for natural or assisted pregnancy.161 Sperm-zona pellucida binding: Sperm binding to the Reactive oxygen species: It has been shown that 40–88% of zona pellucida (ZP) is attributed to the presence of nonselected infertile patients have high levels of semi- complimentary binding sites or receptors on the sur- nal reactive oxygen species (ROS).162 Moreover, 11% of face of both gametes; Human ZP3 (hZP3) is believed normozoospermic infertile men have higher ROS lev- to be the primary ZP receptor for capacitated acro- els and reduced total antioxidant capacity (TAC) levels some-intact sperm binding.173,174 Sperm binding to ZP3 compared to normospermic fertile counterpart.163 ROS induces a signal transduction cascade within the sper- in low levels have a physiological role for capacitation, matozoon, involving multiple proteins that lead to the hyperactivation and AR.164,165 ROS also exert their effect AR. Defective ZP-binding sperm is present in approxi- on sperm-oocyte interaction. However, the physiologi- mately 15% and 25% of subfertile men with normal cal ROS levels are still undetermined. and abnormal semen analyzes, respectively.175-177 High ROS levels in semen that often originate from Two tests of sperm binding to the human zona immature spermatozoa and seminal leukocytes can which have been described: exert pathological impact on sperm.166 These free radi- 1. The hemizona assay cals have considerable reactivity and the ability to react 2. The sperm-zona binding ratio test. with and modify the structure of many different kinds In the former, a single zona is bisected and each of biomolecules including proteins, lipids and nucleic zona half is incubated with control and tested sperm acids. suspensions. In the latter, a complete zona is incubated A natural antioxidant defense system offers pro- with equal numbers of motile spermatozoa from con- 48 tection against ROS. It consists of radical scavengers trol and test populations, each labeled with a different Chapter 3 Evaluation and Diagnosis of Male Infertility fluorescent dye. In each case, the number of spermatozoa Sperm penetration testing: The ability of the equatorial from each population bound per whole or half zona is region of the acrosome-reacted human sperm to fuse counted and the number of test sperm expressed as a with the vitelline membrane of the oocyte is tested by ratio of that of the control. using the sperm penetration assay (SPA) also known as the zona-free hamster oocyte penetration test. Acrosome reaction: The AR is defined as the process of Although this test does not assess sperm-ZP binding fusion of sperm plasma membrane with outer acro- it measures the ’s ability to undergo somal membrane leading to release of exocytotic capacitation, AR, fusion and penetration through the proteolytic enzymes (acrosine and hyaluronidase) oolemma, and decondensation within the cytoplasm in response to sperm-ZP binding. ZP3 is considered of an oocyte. The ZP is removed from a hamster the natural stimulus for the AR, which leads to the oocyte, which is then incubated with human sperma- proteolytic dissolution of the ZP. Artificial stimuli tozoa. In its original description, sperm penetration used in vitro to challenge the AR are calcium iono- ability is scored by calculating the percentage of ova phore A23187 and progesterone. Defective AR of that are penetrated; normal sperm are able to pene- clinical significance are: (i) AR prematurity, which trate 10–30% of hamster ova.182 Recent refinement of is defined as high level of spontaneous AR (> 20% of this test includes the incubation of sperm in a more spermatozoa exhibiting spontaneous AR), and (ii) AR potent capacitating media that allow the majority of insufficiency, which is defined as poor responsive- ova to be penetrated; scores are obtained by calculat- ness to AR stimulants (when < 15% of spermatozoa ing the number of subfertile sperm that penetrate each responded to the ionophore A23187 challenge).178 ovum compared to fertile controls. Several studies Both conditions are associated with poor fertilization have evaluated the ability of the SPA to predict success capacity on conventional IVF treatment. Under nor- or failure of IVF. Normal SPA results are predictive mal conditions, > 15% AR in response to ionophore of fertilization by conventional IVF.183 Nonetheless, treatment is expected.179 Various techniques are used semen samples which fail to fertilize hamster ova usu- for visualizing the human sperm acrosome, includ- ally are unable to fertilize human ova.182 Although the ing lectins, monoclonal antibodies or the triple stain. SPA is considered a research tool, it may be of clinical Replicate (minimum two) slides must be scored for value for men with unexplained infertility with poor each determination, with at least 100 spermatozoa fertilization rate on IVF. counted per slide. The frequency of spermatozoa exhibiting defective Postejaculatory Urine Examination ZP induced AR (ZPIAR) is high in subfertile men with Postejaculatory urine (PEU) examination inspects for idiopathic oligozoospermia (65%) and severe teratozoo- the presence of sperm to diagnose retrograde ejacula- spermia (62%, strict normal sperm morphology ≤ 5%), tion. It helps to differentiate from other causes of hypo- but low 25% in normozoospermic subfertile men.176 spermia (volume < 1.5 ml) such as specimen collection Sperm from men with unexplained infertility may be issues, hypogonadism, EDO and congenital bilateral able to bind to the zone but unable to induce the AR, absence of the vas deferens. ultimately resulting in reduced sperm-ZP penetration and failure of fertilization. Patients with this condition Testis Biopsy usually have a long duration of unexplained infertility Testis biopsy is indicated in selected cases of azoo- and normal semen analysis, and have zero or low rates spermia or severe oligozoospermia to distinguish of fertilization with standard IVF.180 The diagnostic fea- obstructive from nonobstructive cases. Histopathology ture is that very low proportions of sperm undergo AR results may reveal normal spermatogenesis, hypo- after binding to the ZP. However, those patients achieve spermatogenesis, germ cell maturation arrest, germ high fertilization and pregnancy rates with ICSI.181 cell aplasia (Sertoli-cell-only syndrome), tubular scle- Although the exact mechanisms of defective AR are rosis or a combination of these conditions. Biopsy can unknown, defective ZPIAR is more likely to be related be performed by percutaneous or open approaches. to major structural defects of the sperm head, such as Intraoperative imprint technique may yield informa- small or abnormal acrosomes, or associated abnormali- tion regarding testicular cytology including the pres- ties in the overlying plasma membrane in severe terato- ence of spermatogenesis or Sertoli-cell-only syndrome; zoospermic subfertile men. In normozoospermic men, it however, this method is limited to provide a spe- has been shown that the seminal zinc concentration as cific and detailed morphological assessment. In cases a decapacitating factor was significantly higher in men biopsies are obtained for diagnostic purposes only, the with defective ZPIAR. authors’ technical choice is to perform the procedure 49 Section 2 Male Factor Infertility

either percutaneously or using the “window” open specialized centers to allow for immediate fresh sam- technique without testis delivery from the scrotum. ple examination and sperm cryopreservation for future Specimens should be placed in an appropriate fixative ICSI, and avoiding the need of repeated procedures. solution such as Bouin’s fixative, Zenker’s fixative or glutaraldehyde; formalin should not be used. In cases Algorithms for Evaluation and Diagnosis of NOA, histology results provide important prognos- A detailed medical history, physical examination, tic information on the chances to retrieve spermatozoa semen analyses and complementary tests, as appropriate, to be used in ART.184 However, as spermatogenesis is are the key to correctly diagnose and to define the bet- often limited and focal in NOA patients who harbor ter treatment strategies. In Figures 8A and B, we present sperm production, it is recommended that biopsy is algorithms summarizing what should be considered undertaken preferentially in assisted reproduction when assessing the infertile male.185

A

Contd...

50 Chapter 3 Evaluation and Diagnosis of Male Infertility

B

Figures 8A and B Algorithms for the workup of the infertile male. (A) Algorithm to be considered on initial evaluation; (B) Algorithm for the male patient presenting with azoospermia on semen repeated analyzes (Adapted from Esteves SC, Miyaoka R, Agarwal A. An update on the clinical assessment of the infertile male. CLINICS 2011;66(4):691-700, with permission from the Editor)

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