Infertility: Diagnosis and Evaluation

See 2020 AUA/ASRM Guidelines on Infertility

See 2015 CUA Azoospermia Guideline Notes

General principles

• Goals of evaluation are to identify (5):★
  1. Potentially correctable conditions for treatment to allow for conception through intercourse or through techniques, such as IUI or IVF, when those approaches would otherwise not be possible
  2. Irreversible conditions that are amenable to ART using the sperm of the male partner
  3. irreversible conditions that are not amenable to the above, and for which donor insemination or adoption are possible options
  4. Life- or health-threatening conditions (e.g. testicular cancer or pituitary tumours) that may underlie the infertility or associated medical comorbidities that require medical attention
  5. Genetic abnormalities or lifestyle and age factors that may affect the health of the male patient or of offspring particularly if ART are to be employed
• For initial infertility evaluation, both male and female partners should undergo concurrent assessment.
  • Maternal age is the strongest predictor of fertility outcome in couples undergoing therapy.
  • Many couples have more than one fertility issue present.
    • For the female partner, tests are indicated to evaluate ovarian reserve, ovulatory function, tubal structures as well as assessment of the uterine cavity.
    • For the male partner, good clinical practice to obtain a reproductive history, perform a physical examination and basic diagnostic tests of reproductive function (Appendix I).
• Men with abnormal SAs and/or abnormal reproductive history, including physical examination and selected laboratory and radiologic assessment should be evaluated by specialists in male reproduction
• Counsel infertile men or men with abnormal semen parameters of the health risks associated with abnormal sperm production.
  • 1-6% of men have undiagnosed medical diseases at the time of an infertility evaluation
  • Abnormal semen parameters associated with increased risk of
    • Testicular cancer
    • Mortality
• In couples with failed assisted reproductive technology cycles or recurrent pregnancy losses (RPL) (two or more losses), evaluation of the male should be considered.
  • In this clinical setting, the male partner should be evaluated by a male reproductive expert and consideration given to evaluation of sperm DNA fragmentation and karyotype testing of the male.
    • Even with a "normal" SA, a sperm that appears morphologically and functionally normal may not be chromosomally normal or may have a high level of DNA fragmentation.
  • Some experts would also consider sperm aneuploidy testing
    • This test is not universally available for all centers.
• Timing
  • Infertility should be evaluated after 6 months of attempted conception when the female partner is over 35 years of age★

UrologySchool.com Summary

2020 AUA Guidelines

• Recommended
  • History and physical exam
  • Labs
    • Semen analysis
      • At least two SAs obtained a month apart are important to consider, especially if the first SA has abnormal parameters
        • Semen parameters are highly variable biological measures and may vary substantially from test to test
• Optional
  • Labs
    • Testosterone, FSH, if indicated (see below)
    • Genetic testing, if indicated
      • Karyotype
      • Y-microsome deletion
      • Cystic fibrosis

History and physical exam

History

• Evaluation of men with secondary infertility should include a focus on conditions or exposures that have developed or occurred after initiation of the earlier pregnancy(ies).§
• Risk factors
  • Can be categorized into (TICS - see Infertility: Epidemiology and Etiology Chapter Notes):
    1. Toxins (medications, chemotherapy, radiation, social habits)
    2. Infections of the GU tract
    3. Childhood (congenital causes (recall DUNKY-XX), hydrocele or hernia surgery, trosion, cryptorchidism)
    4. Sexual history (lubricants)
• Infertility history
  • Duration of infertility
  • Whether the infertility is primary or secondary
  • Any treatments to date
  • Libido, sexual function, sexual activity
• General health of the male (diabetes, respiratory issues, recent illnesses)
  • Medical comorbidities (e.g., hypertension, hyperlipidemia, obesity, diabetes) that can contribute to infertility.
  • If there has been a recent serious medical illness or injury or evidence of a recent reproductive tract infection, semen testing should be repeated at least 3 months following recovery from the illness
• Surgery of the reproductive tract: testis cancer, undescended testis, hydrocelectomies, spermatocelectomies, varicocelectomies, vasectomies
  • Vasectomy reversal may represent a more cost-effective option compared to IVF in couples with adequate ovarian function§
• Proven or suspected GU infections/inflammation: sexually transmitted infections, epididymo-orchitis, mumps orchitis
• Medications and therapies which might have an adverse impact on spermatogenesis
  • See Infertility: Epidemiology and Etiology Chapter Notes
  • If there has been exposure to any gonadotoxic agents, these medications should be stopped and semen should be retested in 3-6 months
• Environmental exposures (pesticides, excessive heat on the testicles)
• Lifestyle (cigarette smoking, recreational drugs, alcohol use, stress, anabolic steroid usel)
• History of any genetic abnormalities in the patient or the family

Physical exam

• Body habitus
  • Obese men have
    • Elevated estradiol
      • Mechanism: adipose cells have aromatase which metabolizes testosterone to estradiol
    • Reduced serum testosterone and reduced SHBG (therefore, more bioavailable testosterone component of total testosterone)
• State of virilization
• Abdominal examination
  • Scars indicative of previous inguinal surgery or treatment of undescended testis
• Scrotal examination
  • Size and consistency of the testis; size correlates well with sperm production
    • Long axis length <4.6cm associated with impaired spermatogenesis
    • Volume <20mL considered low
    • Obstructive azoospermia is suspected if the physical examination reveals testes of normal size, fully descended into the scrotum and bilaterally indurated epididymides with or without absence of the vas deferens
    • When the testes are atrophied and soft, especially in the presence of FSH greater than 7.6 IU/L, the results are suggestive of spermatogenic failure rather than obstructive azoospermia.
  • Epididymis (engorgement may suggest obstruction)
• Phallus (meatal displacement)
• Prostate and seminal vesicles
• Vas deferens
  • Unilateral absence
    • Suggests complete lack of Wolffian (duct development on that side, including renal agenesis.
      • The absent vas should raise a red flag for possible ipsilateral renal agenesis because the ureteral bud and vas are both derived from the wolffian duct
      • Recall, male structures derived from Wolffian ducts:
        • Body and tail of epididymis (note efferent ductules and head of epididymis from mesonephric tubules)
        • Vas deferens
        • Seminal vesicles
          • Distally, the wolffian ducts join the urogenital sinus by about 30 days gestation, where they develop into the seminal vesicles
        • Ejaculatory duct
        • Appendix epididymis
      • Male structures derived from Müllerian duct (2):
        1. Appendix testis
        2. Prostatic utricle
      • Male structures derived from urogenital sinus:
        1. Prostate
        2. Bulbourethral glands
  • Bilateral absence
    • Consider investigation for CF gene mutation
      • Mutations in the CFTR gene are present in up to 80% of men with congenital bilateral absence of the vas deferens (CBAVD), 20% of men with CUAVD and 21% of men with idiopathic epididymal obstruction★
        • The most common CFTR mutation is ΔF508, which is severe
        • ≈7% of brothers of patients with CBAVD will have also vasal agenesis
        • No association between CBAVD and Y microdeletions.
      • If the male partner is being tested for CFTR, such is in CBAVD, both patient and female partner should be tested for CFTR to determine risk of cystic fibrosis in offspring (CUA Azoospermia Guidelines).
        • Only a portion of CFTR mutations are detected by routine testing.
          • A male with CBAVD should be assumted to be a CFTR carrier despite a negative CFTR gene test and the female partner still needs to be tested prior to any assisted reproductive techniques.
    • Semen is almost always of low volume and acidic in patients with CBAVD due to hypoplasia or absence of the seminal vesicles, which provide alkalinity
  • In men with congenital bilateral or unilateral absence of the vas deferens who are not carriers of cystic fibrosis mutations, abdominal US to assess for renal agenesis is indicated since these men have a higher chance of having absence of one of their kidneys
    • 26% of males with unilateral congenital absence of the vas deferens and 11% of males with CBAVD had an absent ipsilateral kidney;
      • Most of the bilateral CAVD patients with an absent ipsilateral kidney are in patients with no identifiable CF gene mutation.
• Varicoceles
  • Large varicoceles are associated with greater preoperative impairment in semen quality than are small varicoceles
  • Varicocele treatment may be more cost effective than assisted-reproductive therapy or can lower the intensity of treatment§
  • See Varicocelectomy Chapter Notes

Laboratory

Semen analysis

• Results cannot precisely distinguish fertile from infertile men except in cases of azoospermia and some types of teratozoospermia (e.g., complete globozoospermia), necrozoospermia, or complete asthenozoospermia

WHO semen parameter percentiles

• Derived from men whose partners became pregnant (fertile population) within 1 year of discontinuation of contraceptives.
• The 5th percentile is used to describe infertility cut-offs for 7 semen parameters
  • Values falling above or below the lower limit do not by themselves predict either fertility or infertility
    • There are males who have abnormal semen parameters, yet they have contributed to a prior successful pregnancy through natural conception
      • Individual sperm parameters diagnostic of infertility (4):
        1. Azoospermia: defined as absence of sperm in the ejaculate, distinguished from aspermia (absence of antegrade ejaculate; dry ejaculate) and RE (where semen with sperm are released into the prostatic urethra but travel backward (retrograde) into the bladder)
        2. Some types of teratozoospermia (e.g., complete globozoospermia)
        3. Necrozoospermia
        4. Complete asthenozoospermia
    • The odd ratio for infertility increases as the number of abnormal parameters increases.
    • Presence of abnormal semen parameters suggests the presence of a male factor in an infertile couple which should prompt further evaluation of the male

1. Volume <1.5mL
   • See 2015 CUA Azooospermia Guideline Notes
   • Causes:
     • Obstruction or hypoplasia (severe androgen deficiency, CBAVD) of the prostate and seminal vesicles
     • Retrograde ejaculation
       • Can be present in men with various neuropathies (e.g., diabetes, spinal cord injury, after RPLND)
     • Alpha blockers
     • Multiple sclerosis
   • Hypervolemia (>5 mL), dilutes sperm therefore interferes with reproduction.
     • IUI can be used to concentrate sperm
2. Concentration <15 million/mL
   • Visual assessment under microscopy
3. Total number <39 million/ejaculate
   • Most important semen analysis parameter
   • When a semen analysis shows azoospermia, the laboratory should then centrifuge the ejaculate and re-suspend the pellet in a small volume of seminal plasma and examine under wet mount microscopy for the presence of rare sperm. If no sperm are present, a second SA should be performed at least one to two weeks later. If the sample is azoospermic, then another pellet analysis should be performed.★
4. Total motility <40%
   • Visual assessment under microscopy
   • Asthenospermia is when total motility <40% or progressive motility <32%
     • Causes of asthenospermia§
       1. Sperm structural defects
       2. Prolonged abstinence periods
       3. Genital tract infection
       4. Anti-sperm antibodies
       5. Partial ductal obstruction
       6. Varicocele
5. Progressive motility <32%
   • Progressive motility: moving actively, linearly or in large circle, regardless of speed
   • Nonprogressive motility: all other patterns with absence of progression
6. Normal forms <4%
   • Teratozoospermia: normal forms <4%
   • Globospermia: failure of acrosomal head to form small, round heads.
     • Without the acrosome, fertilization with sperm in the natural setting or with incubational in vitro fertilization will not be successful. Intracytoplasmic sperm injection is required.
       • Ejaculated sperm is available in this patient, and therefore surgical sperm extraction is unnecessary.
   • Higher rates of aneuploidy with many abnormal forms.
     • Conditions associated with aneuploidy:
       • Aging, cytotoxic agents, environmental exposures (smoking)
     • Consider aneuploidy in patients with recurrent pregnancy losses
     • intracytoplasmic sperm injection with biological gametes is not recommended with sperm associated with macrocephaly and multiple tails due to the high rate of aneuploidy
   • A variety of abnormal forms rules out excludes rare genetic conditions such as failure of formation of the acrosomal cap.
   • Strict morphology is not a consistent predictor of fertility, and for this reason, the AUA states that strict morphology “should not be used in isolation to make prognostic or therapeutic decisions
7. Vitality <58%
   1. Metabolically active living cells
   2. Necrospermia: reduced vitality
      1. Cannot evaluate in complete asthenospermia since assay relies on association of moving particles proximal to motile sperm
      2. Therefore, to rule out necrospermia when motility is 0%, consider vital stain
      3. In the complete absence of motility and necrospermia have been ruled out, electron microscopy can identify if ultrastructural tail defects are present in the immotile cilia syndrome
      4. In immotile cilia syndrome is confirmed, patients can subsequently be counselled to undergo IVF
8. Viscosity, coloration, pH
   1. In spinal cord injury, a brown hue can be noted in the semen
   2. pH should be 7.2-7.8 (alkaline due to seminal vesicle contribution)
      1. A low volume, acidic pH, azoospermic ejaculate can be indicative of obstruction in the genital tract.

Secondary semen analyses

• DNA fragmentation
  • Negatively associated with pregnancy rates and positively associated with miscarriages
  • Direct measures of sperm DNA fragmentation include (2):
    • Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
    • Comet assay
  • Not recommended in the initial evaluation of the infertile couple★
    • May be useful in couples undergoing IVF with repeated IVF failure
      • For male partners with high sperm DNA fragmentation, a clinician may counsel them that there is a possible association with infertility and compromised outcome after ART
  • Currently no effective therapy to correct an abnormal DNA fragmentation result
  • Decreased abstinence may be an intervention to limit sperm DNA damage.[1]
  • If high sperm DNA fragmentation, consider using surgically obtained sperm in addition to ICSI
    • In a prospective cohort study of over 100 couples with high DNA fragmentation, testicular sperm yielded substantially higher live birth rates than ejaculated sperm.[2]
• Semen WBC staining
  • Increased levels of round cells in the semen may result from
    1. Presence of elevated levels of white blood cells in the semen (pyospermia)
       • White blood cells in the semen may result from infection or inflammation in the proximal or distal male genital tract.
    2. Spermatogenic problem where immature germ cells (spermatocytes and/or round spermatids) are present in the ejaculate
       • No evidence that elevated levels of immature sperm in the semen is deleterious to fertility, and they may be present in semen of infertile men and fertile men with high sperm counts.
    3. Idiopathic
       • Most common cause
  • Leukocytes and immature germ cells are not differentiable with light microscopy
    • Important to know whether men with elevated levels of round cells in the semen have immature germ cells (a condition that cannot be treated) or an infectious or inflammatory etiology
    • Papanicolaou staining may be used
    • Immunocytochemical staining provides more information to aid in distinguishing between inflammation and those subtypes involved in fighting off infection
  • Indications★
    • Increased round cells on SA (>1million/mL)
      • Upper limit of normal as <1 million white blood cells/mL of semen
  • Pyospermia should be evaluated for the presence of infection★
    • Chronic prostatitis due to bacterial infection may require long courses of antibiotic treatment, and some cases of elevated levels of white blood cells may result from chronic nonbacterial prostatitis.
    • Sexual transmitted infections can also lead to leukocytes in semen and this does need to be ruled-out
    • Leukocytes can occur with UTIs, but unless urine is in the semen, this is an unlikely source.
    • Inflammation may be medically treated with anti-inflammatory drugs.
• Anti-sperm antibodies
  • Varying clinical significance depending on degree of binding
  • Associated with history of vasectomy, testis trauma, orchitis, cryptorchidism, testis cancer, varicocele
    • Vasectomy disrupts the blood-testis barrier, resulting in detectable levels of serum antisperm antibodies in 60% to 80% of men.
  • Consider if semen analysis demonstrates sperm agglutination or reduced sperm motility
• Sperm penetration assay most closely models incubational in vitro fertilization
• Sperm culture: limited seminal concentrations of the majority of bacteria including E. Coli have minimal or no effects on sperm motility in vivo

Serum

Total testosterone and FSH

• Not recommended as a primary first-line test in the evaluation of male infertility★
• Indications (6):★
  1. Azoospermia
  2. Oligozoospermic (particularly if sperm concentration) <10 million/mL)
  3. Impaired libido
  4. Erectile dysfunction
  5. Atrophic testes
  6. Evidence of hormonal abnormality on physical evaluation
• Total testosterone
  • Should be defined based upon a blood sample drawn in the morning, since levels drop during the day
  • ≥300 ng/dL considered adequate (164 ng/dL bioavailable)
  • If the fasting morning total testosterone level is low (<300 ng/dL), a repeat measurement of total and free testosterone (or bioavailable testosterone) as well as determination of serum LH, estradiol, and prolactin levels should be obtained.
    • The relationship of testosterone, LH, FSH, and prolactin helps to identify the clinical condition.
• FSH
  • Indirect assessment of germ cell mass
    • If normal testicle size and FSH <7.6 IU/L --> consider obstructive azoospermia
    • If reduced testicle size and FSH >7.6 IU/L --> consider spermatogenic dysfunction

Other

• LH
  • Determine if hypoandrogenism testicular (primary) or pituitary (secondary) in nature
• Estradiol
  • Ratio of total T:E < 10:1 indicates reproductive dysfunction
• Prolactin
  • Abolishes GnRH pulsatility, suppresses testosterone production
  • Pituitary disease (hyperplasia, adenoma, tumours), including prolactinoma, is associated with visual field changes, headache, and erectile dysfunction
  • Obtain prolactin if clinically indicated. Consider MRI if very elevated.
    • Prolactin is a labile assay. Before continuing with further diagnostic assessment or therapy, moderately elevated assay results should first be confirmed with a second test.

	Severely impaired spermatogenesis	Obstructive azoospermia	Hypogonadotropic hypogonadism
LH	Increased or no change	No change	Decreased
FSH	Increased	No change	Decreased
Testosterone	Decreased or no change	No change	Decreased

Post-ejaculate Urine Analysis

• Used to diagnose retrograde ejaculation
• Viable sperm from urine or any location within the male reproductive tract can be used with ART to achieve a pregnancy.

Genetic Testing

Options

1. Karyotype
2. Y Chromosome Microdeletion
3. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutation carrier testing

Indications

• Karyotype and Y chromosome microdeletion
  • Primary infertility and azoospermia or severe oligozoospermia (<5 million sperm/mL) with (3):
    1. Elevated FSH OR
    2. Testicular atrophy OR
    3. Presumed diagnosis of impaired sperm production as the cause of azoospermia
• Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutation carrier testing
  1. Vasal agenesis/abnormalities
  2. Idiopathic obstructive azoospermia
  3. If the female partner is a carrier (to consider for preimplantation diagnosis)

Karyotype

• Evaluates the number and structure of chromosomes in order to detect abnormalities
• Karyotype abnormalities (e.g. Klinefelter syndrome) are the most common known genetic abnormalities that cause male infertility.

Y Chromosome Microdeletion Analysis

• Y chromosome microdeletions are the second most common known genetic cause of infertility in the male
• Should be done in azoospermia before surgical sperm extraction to counsel likelihood of retrieval
  • Men with complete deletions of AZFa and/or AZFb should not undergo TESE for ART. ★
  • Men with deletions of AZFc and smaller partial deletions of AZFa and/or AZFb should be counseled that sperm may or may not be found with TESE.★

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutation carrier testing

• Including assessment of the 5T allele
  • In addition to classic mutations, the 5-thymidine (5T) variant of the polythymidine tract in the splice site of intron 8 (which regulates exon 9 splicing efficiency) is also commonly found in men with obstructive azoospermia due to CFTR abnormalities.
• For men who harbor a CFTR mutation, genetic evaluation of the female partner should be recommended.
  • In cases where the male patient has a mutation in the CFTR gene and the partner is also a carrier, then there is a risk of an affected offspring (25% if both partners are carriers, and up to 50% if the male has mutations in both alleles with a female partner who is a carrier)

Imaging

• Scrotal Ultrasound: testicular size, varicoceles
• TRUS: suggestive of ejaculatory duct obstruction if AP diameter of seminal vesicle >1.5cm
• Vasography: patency of vas.
• MRI brain: evaluate for functioning and non-functioning pituitary tumors in secondary hypogonadotropic hypogonadism with a elevated or normal serum prolactin level
  • 2018 AUA Testosterone Deficiency Guidelines: total testosterone <150 ng/dL in combination with a low or low/normal LH should undergo a pituitary MRI regardless of prolactin levels, as non-secreting adenomas may be identified.

Testicular biopsy

• Indications (must have all):
  1. Azoospermic
  2. Testis of normal size and consistency
  3. Palpable vasa deferentia
  4. Normal serum follicle-stimulating hormone (FSH) levels
  5. Negative serum anti-sperm antibody assay
  6. Biopsy will distinguish obstructive from nonobstructive azoospermia
• In men with congenital absence of vasa and normal serum FSH levels, biopsy is not needed since these patient’s azoospermia is likely obstructive rather than testicular in cause
• Spermatogenesis may be highly focal in men with azoospermia so a random biopsy may miss areas of sperm production

Special Scenarios

Recurrent pregnancy loss

• Defined as two or more failed pregnancies
  • Distinct from infertility
• Most miscarriages are related to abnormalities within the fetus itself★
• Causes of recurrent miscarriages (6)
  1. Genetic causes (e.g., chromosomal translocations)
  2. Anatomic abnormalities of the female uterus (e.g., septum, submucosal fibroids, adhesions)
  3. Infections
  4. Hematologic and immunologic disorders of the female partner
  5. Female partner endocrine issues (e.g., thyroid and diabetes)
  6. Male factor issues*#*Most common identified etiologic issues in males: karyotypic abnormalities and sperm DNA fragmentation.

References

• Wein AJ, Kavoussi LR, Partin AW, Peters CA (eds): CAMPBELL-WALSH UROLOGY, ed 11. Philadelphia, Elsevier, 2015, chap 24
• Schlegel, Peter N., et al. "Diagnosis and treatment of infertility in men: AUA/ASRM guideline part I." The Journal of urology 205.1 (2021): 36-43.