Aboubakr Elnashar Benha University, Egypt

Evaluation of male

infertility

ABOUBAKR ELNASHAR

CAUSES Pretesticular:

Hypothalamic pituitary disease (Secondary

hypogonadism): 1-2%

Testicular:

Primary hypogonadism: 10-15%

Post-testicular defects

disorders of sperm transport: 10 to 20 %

Idiopathic:

Seminiferous tubule dysfunction: 60-80%

including microdeletions of the Y chromosome

ABOUBAKR ELNASHAR

CAUSES OF MALE INFERTILITY

I. Hypothalamicpituitary disorders

(GnRH; LH and FSH deficiency)

Congenital disorders

Congenital GnRH deficiency: Kallmann syndrome

Hemochromatosis

Multiorgan genetic disorders: PraderWilli syndrome,

Laurence Moon Beidl syndrome, familial cerebellar ataxia

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Acquired disorders

Pituitary and hypothalamic tumors: macroadenoma,craniopharyngioma

Infiltrative disorders: sarcoidosis, histiocytosis,

tuberculosis, fungal infections

Trauma, postsurgery, postirradiation

Vascular: infarction, aneurysm

Hormonal: hyperprolactinemia, androgen excess, estrogen

excess, cortisol excess

Drugs: opioids and psychotropic drugs, GnRH agonists or

antagonists

Systemic disorders: Chronic illnesses, Nutritional

deficiencies, Obesity

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II. Primary gonadal disorders

Congenital disorders

Klinefelter's syndrome (XXY) and its variants

(XXY/XY; XXXY)

Cryptorchidism

Myotonic dystrophy

Functional prepubertal castrate syndrome:

congenital anorchia

Varicocele

Androgen insensitivity syndromes

5 alphareductase deficiency

Y chromosome deletions

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Acquired disorders

Viral orchitis: mumps, echovirus, arbovirus

Granulomatous orchitis: leprosy, tuberculosis

Epididymoorchitis: gonorrhea, chlamydia

Drugs: alkylating agents, alcohol, marijuana, antiandrogens,

ketoconazole, spironolactone, histamine2 receptor antagonists

Ionizing radiation

Environmental toxins: dibromochloropropane, carbon disulfide,

cadmium, lead, mercury,environmental estrogens and phytoestrogens

Hyperthermia

Immunologic disorders: polyglandular autoimmune disease

Trauma

Torsion

Castration

Systemic illness: renal failure, hepatic cirrhosis, cancer,

sickle cell disease, amyloidosis, vasculitis, celiac disease ABOUBAKR ELNASHAR

III. Disorders of sperm transport

Epididymal dysfunction: drugs, infection

Abnormalities of the vas deferens: congenital

absence, Young's syndrome, infection, vasectomy

Ejaculatory dysfunction: spinal cord disease,

autonomic dysfunction, premature ejaculation

IV. Unexplained male factor infertility

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EVALUATION

History

Examination

Investigation

1. Conventional Semen analyses

2. Specialized Semen analysis

3. Endocrine testing

4. Genetic tests

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A. History

Focuses on causes of infertility.

Personal:

Age, occupation, special habits

Present:

Type of infertility, duration

Sexual:

Frequency, erection, ejaculation, dysparunia, habits.

libido

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Past:

Medical:

Chronic medical illness

Infections: mumps orchitis, sinopulmonary symptoms,

STI, and GUI (prostatitis)

Surgical:

inguinal and scrotal areas such as vasectomy,

orchiectomy, and herniorrhaphy

Trauma

Developmental:

testicular descent, pubertal development, loss of body

hair, or decrease in shaving frequency

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Drugs and environmental exposures:

alcohol, radiation therapy, anabolic steroids,

cytotoxic chemotherapy, drugs that cause

hyperprolactinemia

exposure to toxic chemicals (e.g. pesticides,

hormonal disrupters)

School performance

determine if he has a history of learning disabilities

suggestive of Klinefelter's syndrome

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PHYSICAL EXAMINATION

General:

Evidence of androgen deficiency

depend upon the age of onset.

during early gestation: ambiguous genitalia

late gestation: micropenis

Childhood: delayed pubertal development

Adulthood: decreased sexual function, infertility,

loss of secondary sex characteristics.

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General appearance

Eunuchoidal proportions (upper/lower body ratio

<1 with an arm span 5 cm >standing height):

androgen deficiency antedating puberty.

increased body fat and decreased muscle mass:

current androgen deficiency.

Skin

Loss of pubic, axillary, and facial hair,

decreased oiliness of the skin, and

fine facial wrinkling: long-standing androgen

deficiency.

Breasts

Gynecomastia suggests a decreased androgen to

estrogen ratio.

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External genitalia

Tanner stage

Phallus and testes

Scrotum

Absence of the vas

Epididymal thickening

Varicocele

Hernia

•Varicocele

should be confirmed with the man standing and

performing a Valsalva maneuver.

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Examination for Varicocele:

distension of the pampiniform venous plexus in the

spermatic cord.

3 grades:

1. Palpable during Valsalva s maneuvers.

2.Palpable without Valsalva s maneuver.

3. Visible distension

Varicocele assessment are not correlated with

pregnancy (ESHRE, 2009)

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Testes:

Decreased volume of the seminiferous tubules can

be detected by measuring testicular size by Prader

orchidometer or calipers.

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I. STANDARD SEMEN ANALYSIS

A. Macroscopic

1. Delayed liquefaction

2. Increased viscosity

3. Semen volume

4. pH

B. Microscopy

1. Agglutination

2. Concentration

3. Motility

4. Morphology

5. Round cells

6. Leukocytes

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Semen analysis: WHO, 2010

:

:

Lower reference limit Parameter

1.5 ml Volume

7.2 pH

15 million/ml Concentration

39 million/ejaculate Total sperm number

40% or PR: 32%

Total motility: (PR+NP)

58% live spermatozoa Vitality

4% (strict criteria). Normal forms

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Other threshold values

Peroxidase-positive leukocytes (106 per ml): <1.0

Mixed Antiglobulin Reaction (MAR) test (motile

spermatozoa with bound particles, %): <50

Immunobead test (motile spermatozoa with bound

beads, %) <50

Seminal fructose (ųmol/ejaculate): ≥13

Seminal neutral glucosidase (mU/ejaculate): ≥20 Seminal zinc (ųmol/ejaculate): ≥2.4

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Collection:

After 2-7 d of sexual abstinence

at the doctor's office

Masturbation

If this is not possible:

condoms without chemical additives

delivered to the laboratory within 1 h

At least 2 samples collected 1-2 w apart & not more

than 3 months apart. marked variation of sperm production within one individual

Any systemic disease during sperm generation time (72 days for spermatogenesis & 14 days for transport through the

epididymis & vas): ±negative impact.

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A. Macroscopic

1. Delayed liquefaction

liquifaction after 1 h

Due to:

chronic prostatitis

seminal vesiculitis

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2. Increased viscosity= Hyperviscosity

length of the thread that forms on withdrawal of

a glass rod >2cm

Due to:

chronic prostatitis

seminal vesiculitis

Kartagner s syndrome : interfere with the semen analysis, in particular, evaluation of sperm motility. Treatment in the laboratory 1. passing the sample via a large gauge needle 2. diluting with a physiological solution 3. enzyme digestion before testing for sperm parameters

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3. pH < 7

+Azoospermia Due to:

Dysgenesis of vas deferens,

seminal vesicles

epididymis

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4. Semen volume Mean: 3.7 mL

lower limit: 1.5 mL

low volume+ azoospermia or severe

oligozoospermia

Genital tract obstruction

Congenital absence of vas deferens: diagnosed

by physical examination and low semen pH

Ejaculatory duct obstruction: diagnosed by dilated

seminal vesicles on transrectal US

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Low semen volume+ normal sperm concentration

1. Semen collection problems: loss of a portion of

the ejaculate

Repeat semen sample collection after emptying

the bladder

2. Partial retrograde ejaculation

neuropathic disorders, including urogenital tract

surgery, sympathetic denervation, and diabetes

low semen volume+ low sperm concentration

Androgen deficiency: Endocrine assessment

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B. Microscopic

1. Agglutination

Stick of motile spermatozoa to each other.

≥10%: suggestive but not conclusive of

immunological infertility.

Confirmed by:

tests for sperm surface antibodies.

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2. Sperm concentration

Lower limit: 15 million/mL (95% CI 12-16)

However, some men with sperm counts considered to be low can be fertile, while others above the lower limit of normal can be subfertile and, for the purposes of fertilization in vitro, 10 million/mL or even less can be satisfactory

ABOUBAKR ELNASHAR

If only a few spermatozoa/HPF

Sensitivity of detecting spermatozoa can be

increased by labeling the spermatozoa with a

fluorescent nuclei stain

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If only a few spermatozoa/HPF

Sensitivity of detecting spermatozoa can be

increased by labeling the spermatozoa with a

fluorescent nuclei stain and then counting the

spermatozoa using a deep chamber. The sensitivity is reduced to 2000 spermatozoa per mL ejaculate

If no spermatozoa are seen:

Centrifuge

whole pellet should be smeared on a slide and

examined for the presence of spermatozoa before

the diagnosis of azoospermia

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1. Adequate motile sperm in the pellet: ICSI with

ejaculated spermatozoa.

2. Few spermatozoa in the ejaculate:

spermatogenesis in a few seminiferous tubules:

microdissection Testicular Sperm Extraction

(TESE) and the testicular spermatozoa used for

ICSI

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3. Sperm motility

Progressive

non-progressive

immotile

At least 40%of spermatozoa should be motile

At least 32% should have progressive motility.

If sperm motility is poor: sperm vitality should be

assessed by

supravital stains or

hypoosmotic swelling test: determine whether

the majority of immotile spermatozoa are dead

The distinction between living, non-moving

sperm, and dead sperm influences the type of

ART ABOUBAKR ELNASHAR

4. Sperm morphology

Previously based mainly on

shape

Now also include:

Length

Width

width ratio

area occupied by the acrosome

neck and tail defects (“strict” criteria)

Strict criteria:

good predictive value in terms of fertilization in

IVF.

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5. Round cells

not > 5 million/ml.

Due to:

1. Leukocytes

2. Immature germ cells: usually indicate disorders

of spermatogenesis.

3. Degenerating epithelial cells.

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6. Leukocytes

Mainly polymorphonuclear leukocytes

frequently present in the seminal fluid.

Assessment by:

peroxidase stain

The peroxidase positive cells are counted using

the hemocytometer

Not ≥: one million/mL.

Increased WBC± :

genital infection/inflammation ±:

poor semen quality release of reactive oxygen

species from the leukocytes.

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Prediction of fertility

The likelihood of infertility

increased with decreases in any of the 3

parameters: M, NM, C

Normal morphology had the greatest

discriminatory power.

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II. SPECIALIZED SEMEN ANALYSIS

Not routinely performed

used to determine the cause of male infertility

1. Sperm autoantibodies

2. Semen biochemistry (semen fructose)

3. Semen culture

4. Sperm cervical mucus interaction tests 5. Sperm function tests

Computer aided sperm analysis

Acrosome reaction Zona free hamster oocyte penetration test Human zona pellucida binding test Sperm reactive oxygen species generation Sperm chromatin/DNA assays

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1. Sperm autoantibodies 4 to 8%of subfertile men.

The presence of agglutination in the initial semen analysis

suggests sperm autoimmunity; this should be confirmed by

the

Mixed antiglobulin reaction (MAR)

Immunobead test, both of which detect sperm

surface antibodies.

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2. Semen biochemistry

Rarely useful in clinical practice. Fructose

marker of seminal vesicle function.

Low or non-detectable:

congenital absence of the vas deferens and

seminal vesicles or

ejaculatory duct obstruction

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3. Semen culture

Indicated: semen samples contain inflammatory

cells Results: usually not diagnostic.

Precautions during sample collection to prevent

skin contamination.

The yield of semen culture may be improved by

performing a prostatic massage before sample

collection.

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4. Sperm-cervical mucus interaction

identifies whether the problem is in the sperm or in

the cervical mucus

The postcoital test :

female partner is in the preovulatory phase of the cycle. The number and motility of sperm in the cervical mucus is assessed 9 to 24 h after SI The in vitro tests

the slide or the capillary tests

performed on sperm and cervical mucus from the infertile couple together with donor semen and cervical mucus. These so-called "crossed tests"

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5. Sperm function tests

Routine:

Impractical and costly

Selective

when the standard semen analysis is normal or near

normal

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Computer-aided sperm analysis: CASA

Assess:

1. sperm concentration

2. morphology.

3. Motility: Quantitative measurement = sperm

kinematics

sperm velocity (curvilinear, straight line, average path)

Amplitude of lateral displacement

other derived functions.

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Useful in:

identifying men with unexplained infertility, predicting

in vivo and in vitro fertilizing capacity, and in

toxicology studies.

Accuracy depend upon:

technology

analytic conditions, and

technical training of the operators.

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Sperm reactive oxygen species

Generation of reactive oxygen species may be a cause of sperm dysfunction and a predictor of fertilization in vitro. Reactive oxygen species lead to lipid peroxidation of the sperm membrane and are also deleterious to sperm motility. This is still regarded as a research test and is not often used for diagnosis of a specific sperm defect.

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ASSESSMENT OF SDF

Test Principle Method

TUNEL

ISNT

Incorporation of probes

at the site of damage

Direct

SCSA

SCD

Comet

Susceptibility of DBs to

denature in acid

solution

Indirect

Aniline blue

Toluidine blue

Incorporation of probes

to nuclear proteins

Chromatin

incorporation

(Feijo and Esteves, 2014)

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Normal= 10

Fragmented= 4

DFI= 4X100/10+4

=28.5%

normal

normal

normal

normal

normal

normal

normal

normal

normal

fragmented

fragmented

fragmented

fragmented

normal

≥30: male infertility

15-30: RM.

≤15: Excellent to Good fertility potential

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ABOUBAKR ELNASHAR

There is insufficient evidence to recommend the

routine use of SDF testing in evaluation and

treatment of infertile couple level C

?????????

For diagnostic test

1. Results must be reproducible

2. Applicable to a given patient

3. Change management of patient

IV. ENDOCRINE TESTS

1. Serum testosterone (T)

Morning T

In men with borderline values:

Repeat

FT

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2. Serum LH and FSH

Indication:

T is low

Interpretation:

high FSH and LH: primary hypogonadism

low or normal: secondary hypogonadism.

low LH + low sperm counts +well-androgenized:

exogenous anabolic or androgenic steroid abuse.

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3. Prolactin

Indication:

low T

normal to low LH

4. Inhibin low serum inhibin concentrations may be an even more sensitive test of primary testicular dysfunction than high serum FSH concentrations, provided the assay is specific for inhibin B

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IV. GENETIC TESTS

ICSI:

Men with severe oligozoospermia and

azoospermia to father children

Genetic risks:

1. Cystic fibrosis conductance regulator (CFTR)

gene,

2. Somatic and sex chromosome abnormalities

3. Microdeletions of the Y chromosome

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OBSTRUCTIVE AZOOSPERMIA

Azospermia+Normal testicular volumes + Normal

FSH, and LH and T

1. Bilateral congenital absence of the vas:

physical examination

low fructose level in the semen.

2. Ejaculatory duct obstruction

Transrectal US:

dilated seminal vesicles.

Patients with obstructive azoospermia: urologist

specialized in infertility for further evaluation and tt.

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