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INFERTILITY: DIAGNOSIS & EVALUATION

See 2015 CUA Azoospermia Guideline Notes

Diagnosis and Evaluation

General princimples
- 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 that may underlie the infertility or associated medical comorbidities that require medical attention
    - Male infertility may occasionally be the presenting manifestation of an underlying life-threatening condition such as testicular cancer or pituitary tumours
  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.
  - 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).
- In couples with failed assisted reproductive technology cycles or recurrent pregnancy losses (RPL) (two or more losses), evaluation of the male should be considered.
- Timing
  - Infertility should be evaluated after 6 months of attempted conception when the female partner is over 35 years of age
UrologySchool.com Summary
- 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.§

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 virilisation
- 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
  - 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):
        
        Appendix testis
        
        Prostatic utricle
      - Male structures derived from urogenital sinus:
        
        Prostate
        
        Bulbourethral glands
  - Bilateral absence
    - Consider investigation for CF gene mutation
      - Congenital bilateral absence of the vas deferens (CBAVD) is associated with CF gene mutations in 85% of patients.
        
        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

Serum

Initial screen (6):
1. Total testosterone
2. SHBG
3. Albumin (to calculate bioavailable testosterone)
4. LH
5. FSH
6. Estradiol
- Prolactin, if clinically indicated.

Total testosterone
- ≥300 ng/dL considered adequate (164 ng/dL bioavailable)
LH
- Determine if hypoandrogenism testicular (primary) or pituitary (secondary) in nature
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
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.

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
    - Presence of abnormal semen parameters suggests the presence of a male factor in an infertile couple, encouraging physicians to consider further evaluation of the male and management to enhance male reproductive function

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
  - Alpha blockers
  - Spinal cord injury
  - Diabetes
  - Multiple sclerosis
- Hypervolemia (>5 mL), dilutes sperm therefore interferes with reproduction.
  - IUI can be used to concentrate sperm
Concentration <15 million/mL

Visual assessment under microscopy

Total number <39 million/ejaculate
- Most important semen analysis parameter
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

Progressive motility <32%
- Progressive motility: moving actively, linearly or in large circle, regardless of speed
- Nonprogressive motility: all other patterns with absence of progression
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, environemental 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”.
Vitality <58%

Metabolically active living cells
Necrospermia: reduced vitality
Cannot evaluate in complete asthenospermia since assay relies on association of moving particles proximal to motile sperm

Therefore, to rule out necrospermia when motility is 0%, consider vital stain
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
- In immotile cilia syndrome is conrimed, patients can subsequently be counselled to undergo IVF

Viscosity, coloration, pH

In spinal cord injury, a brown hue can be noted in the semen
pH should be 7.2-7.8 (alkaline due to seminal vesicle contribution)

Secondary semen analyses:

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

Leukocytes

Often present in the semen of infertile men. Should be < 1 million/mL
Often indicate functional damage from DNA fragmentation to sperm membrane lipid peroxidation from reactive oxygen species released from the leukocytes; seldom due to antisperm antibodies or documentable infection.
Leukocytes can occur with UTIs, but unless urine is in the semen, this is an unlikely source. Sexual transmitted infections can also lead to leukocytes in semen and this does need to be ruled-out, however, even PCR DNA testing for sexual transmitted pathogens are often negative verifying the situation is most commonly idiopathic
Semen WBC staining is indicated only if increased numbers of round cells are noted in the semen
Leukocytes and immature germ cells are not differentiable with light microscopy. A stain such as Papanicolaou allows them to be distinguished

DNA fragmentation

Direct measures of sperm DNA fragmentation include the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and the comet assay at neutral pH.
AUA: Currently, the tests have inadequate sensitivity and specificity to be of value as screening tests for pregnancy by intercourse
- The literature on outcomes associated with abnormally high sperm DNA fragmentation levels is inconsistent
  - Some investigators hypothesize that abnormally high levels of sperm DNA fragmentation are associated with abnormal reproductive outcomes, including lower fertilization, pregnancy, live birth rates, and higher rates of miscarriage.

At this time, the WHO categorizes sperm DNA integrity tests as "research procedures".

Currently no effective therapy to correct an abnormal DNA fragmentation result

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

Genetic testing

Karyotype

Indicated in all men with azoospermia and severe oligospermia (<5 million sperm/mL)

Y Chromosome Microdeletion

A region in the long arm of the Y chromosome critical to formation of sperm is known as AZF – azoospermia factor
AZFa (also known as AZF1) or AZFb microdeletion: generally result in absence of spermatogenesis.
- Common phenotypic manifestations of deletions in AZFa region are azoospermia and Sertoli cell-only syndrome
- Genes in the AZFb region have been found to support the growth and maturity of sperm and are critical for efficient progression of spermatogenesis. Common phenotypic manifestations of deletions in this region are spermatogenic arrest and azoospermia
AZFc microdeletion: may result in spermatogenic impairment but not necessarily absence of spermatogenesis
- Genes in the AZFc region have a diverse role, but overall, they are essential to complete spermatogenesis. AZFc deletions have been associated with drastic reduction in sperm count, and there are subsets of men with AZFc microdeletions that experience progressive declines in their sperm count.
Originally, the AZFb and AZFc genes were identified and thought to be separate regions. They were later found to be overlapping and are now referred to as AZF2.
Should be done in azoospermia before surgical sperm extraction to counsel likelihood of retrieval

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

References

Wein AJ, Kavoussi LR, Partin AW, Peters CA (eds): CAMPBELL-WALSH UROLOGY, ed 11. Philadelphia, Elsevier, 2015, chap 24