EVALUATION OF INFERTILITY
A. Campana, A. de Agostini, P. Bischof, E. Tawfik and A. Mastrorilli
Infertility and Gynecologic Endocrinology Clinic,
Department of Obstetrics and Gynecology,
University Cantonal Hospital, 1211 Geneva 14, Switzerland
The standard medical definition of infertility is the inability of a
couple to conceive after 12 months of intercourse without the use of contraception
(U.S. Congress, Office of Technology Assessment, 1988). The most important
goal of the fertility investigation is to identify the cause(s) of infertility
and to prescribe adequate therapy. This is not the only task of the physician,
however, as s/he should also provide accurate information to the couples
and often correct misinformation gained from friends or family. The physician
has also to provide emotional support for the couple during the period of
investigation and treatment.
The work reported here proposes a standardized and comprehensive approach
to the infertility investigation. For this reason we adopted the idea of
presenting the subject in the form of serial flow charts in well-organized
The infertile couple
The couple must be considered as a single unit as each partner contributes
a share to the infertility potential of the couple. The approach to infertility
requires an efficient and complete initial evaluation. Many couples have
more than one contributory cause which should be identified early in the
evaluation. The main framework for evaluation of the couple is described
in short in flow chart (FC) FC 1. Evaluation must
begin with a detailed history and a complete physical examination of both
partners. History-taking must be directed to complete all the items related
to infertility. A pre-morbid history should be analyzed in detail concerning
the disease, the investigations done, and the treatment. The history and/or
physical examination may direct the evaluation in a particular direction,
but other pertinent fertility factors should not be overlooked.
Diagnosis of female infertility
History-taking and physical examination
Female partner history-taking is described in FC 2
and FC 3 (Campana et al., 1985; Rowe and Farley,
1988; Sciarra, 1992; Speroff et al., 1989; Yen and Jaffe, 1991). The items
of the history were organized so as to cover the most common, and the most
important causes of female partner infertility. The physician should investigate
all the items in order to obtain a valuable impression about the possible
cause(s) of the infertility problem. Any positive history should be analyzed
in detail. The general condition of the female partner should be assessed
by a thorough general physical examination. Pelvic examination is of course
the cornerstone in infertility evaluation. Any suspected lesions should
be investigated thoroughly while keeping always in mind its relation to
the infertility problem (FC 4).
After completion of the initial steps of evaluation (history and physical
examination), a plan should be adopted by the physician to assess the factors
which are necessary to achieve a successful pregnancy. This is accomplished
by the assessment of the menstrual and ovulatory status, the tuboperitoneal
factor, and uterine and cervical factors (FC 5).
Assessment of menstrual and ovulatory status
The different types of menstrual behavior are described in
FC 6. In the presence of regular spontaneous cycles,
the questions to be answered by basal body temperature (BBT) measurement,
endovaginal ultrasound and midluteal serum progesterone evaluations, are
whether the patient is ovulating or not, in the presence of ovulation has
follicular rupture occurred, has it been at the proper time, and has the
luteal phase been adequate? Moreover the status of the endometrium should
also be evaluated (FC 7) (Rowe and Farley, 1988;
Sciarra, 1992; Speroff et al., 1989). Oligomenorrhea which is a cyclic menstrual
bleeding occurring at intervals greater than 35 days, but less than 6 months,
is managed according to the length of the cycle (FC 6).
Patients presenting with primary amenorrhea are subdivided according
to the appearance of the external genitalia into patients with ambiguous
external genitalia and those with normal female external genitalia. The
latter group of patients is further subdivided into four subgroups according
to the presence or absence of the uterus as well as breast development (FC
8) (Mishell et al., 1991).
Patients with primary amenorrhea, intact uterus and absent breast development
should be subjected to serum follicle-stimulating hormone (FSH) measurement.
Accordingly, the patients are classified into either hypogonadotropic hypogonadism
or hypergonadotropic hypogonadism. In the latter group of patients a karyotype
is indicated. If blood pressure is elevated, the likely diagnosis is 17a-hydroxylase
deficiency with 46,XX karyotype. If blood pressure is normal, the results
of karyotyping will be either 46,XX indicating pure gonadal dysgenesis (or
ovarian lesions), 46,XY indicating pure gonadal dysgenesis, or the karyotype
will be abnormal. Where the karyotype is abnormal, one should look for signs
of hirsutism. If hirsutism is present, mixed gonadal dysgenesis is expected.
However, if hirsutism is not present, the case would be 45,X Turner’s syndrome,
mosaicism or 46,XX with abnormal X chromosome (FC 9)
(Mishell et al., 1991).
Patients with primary amenorrhea, absent uterus and developed breasts
should also be subjected to karyotyping. Congenital absence of the uterus
is diagnosed when the karyotype is 46,XX while androgen insensitivity is
the diagnosis when the karyotype is 46,XY (FC 10)
(Mishell et al., 1991).
Patients with primary amenorrhea, absent uterus and absent breast development
all belong to the category of male pseudohermaphroditism with 46,XY karyotype
In patients with primary amenorrhea, intact uterus and well-developed
breasts, one should inquire about the presence of cyclic pelvic pain. Abnormalities
of the müllerian ducts and related embryonic structures should be suspected
in the presence of this type of pain. In the absence of cyclic pain, the
patients should be classified to hypergonadotropic or normo-hypogonadotropic
hypogonadism according to serum FSH levels. Accordingly, the patients are
investigated as in FC 11 (Mishell et al., 1991;
Speroff et al., 1989).
Patients with primary amenorrhea and ambiguous external genitalia should
be karyotyped. 46,XX karyotype combined with elevated androgens and 17-hydroxyprogesterone
(17-OHP) indicates the presence of congenital adrenal hyperplasia. If the
androgens and 17-OHP levels are normal the diagnosis is either elevated
androgen in the maternal blood or 46,XX true hermaphroditism. Patients with
Y containing abnormal karyotype may represent XX/XY true hermaphroditism
or mixed gonadal dysgenesis. Patients with XY karyotype are classified into
three classes according to the presence or absence of the gonads. If the
gonads are present on both sides and the uterus is present, the diagnosis
is true hermaphroditism. If the uterus is absent and there is a rudimentary
wolffian duct, incomplete androgen insensitivity is the likely diagnosis.
If the uterus is absent and the wolffian duct is present, but lacking the
prostate, the diagnosis is 5a-reductase deficiency. If the uterus is absent
and the wolffian duct is completely present, abnormal androgen synthesis
is the diagnosis. If the gonads are present on one side only, the diagnosis
is mixed gonadal dysgenesis. The third class represents those with gonadal
streaks on both sides. If the uterus is absent, this represents testicular
regression syndrome, while if present the diagnosis is late onset agonadism.
If both müllerian and wolffian ducts are present in a rudimentary form the
diagnosis is testicular dysgenesis (FC 12) (Mishell
et al., 1991; Speroff et al., 1989; Yen and Jaffe, 1991).
In patients with secondary amenorrhea, one should start by excluding
a history of hypophysectomy, bilateral oophorectomy, hysterectomy, irradiation
castration and chemotherapy. This should be followed by exclusion of pregnancy,
then the patients are classified into amenorrhea with suggestive history,
amenorrhea with suggestive symptoms and signs and non suggestive amenorrhea
(FC 13) (Mishell et al., 1991; Speroff et al., 1989).
Amenorrhea accompanied by galactorrhea should be first investigated by
measuring prolactin serum levels (FC 13) followed
by the investigations showed in FC 11.
In cases of secondary amenorrhea with recurring cyclic pain and a history
of cervical operation and/or the presence of a cervical mass by pelvic examination,
endovaginal ultrasonography should be done. The presence of hematometra
will confirm the diagnosis of obstructive amenorrhea (FC
History of drug intake with known impact on ovulation and/or the endometrium
may be the cause of amenorrhea. Amenorrhea following curettage or endometritis
arouses the suspicion of Asherman’s syndrome. Amenorrhea following severe
postpartum hemorrhage and failure of lactation is suggestive of Sheehan’s
syndrome. If amenorrhea follows tuberculosis, schistosomiasis or endometrial
irradiation one should suspect endometrial destruction. One should not forget
psychogenic factors, nutritional factors and exercise which are frequent
causes of secondary amenorrhea (FC 14) (Mishell
et al., 1991; Sciarra, 1992; Speroff et al., 1989; Yen and Jaffe, 1991).
In secondary amenorrhea accompanied by hot flushes, karyotyping is essential.
An abnormal karyotype may represent Turner’s syndrome, mosaic Turner’s syndrome
with XO/XX or 46,XX with structural abnormality of the X chromosome. If
the patient is 46,XX a complete set of investigations to exclude the presence
of an autoimmune process is essential. Serum luteinizing hormone (LH), FSH
and estradiol levels should be measured together with endovaginal ultrasound
to differentiate between idiopathic premature menopause and resistant ovary
syndrome (FC 15) (Mishell et al., 1991; Sciarra,
1992; Speroff et al., 1989; Yen and Jaffe, 1991).
Cases of secondary amenorrhea accompanied with hirsutism are investigated
by first excluding the intake of drugs of known androgenic effect. If Cushing’s
syndrome is suspected from the symptoms or the signs, it should be diagnosed
or excluded according to the plan described in FC 16.
However, if Cushing’s syndrome is excluded or is not suspected, serum testosterone
and dehydroepiandrosterone sulfate (DHEAS) levels should be measured (Wilson
and Foster, 1992).
When testosterone levels are <2 ng/ml, polycystic ovary syndrome should
be suspected and should be confirmed by endovaginal ultrasound and its peculiar
hormonal profile. If testosterone levels are >2 ng/ml, endovaginal ultrasound
is indicated to diagnose or exclude the presence of ovarian tumors. When
ultrasonography fails to reveal any ovarian mass, adrenal CAT-scan and ovarian
venous catheterization are indicated.
Serum levels of DHEAS <7 m g/ml, require measurements
of serum 17-OHP levels. If 17-OHP levels are <3 ng/ml, this rules out any
adrenal problem, whereas levels higher than 8 ng/ml point to late onset
adrenal hyperplasia. Serum levels of 17-OHP between 3 and 8 ng/ml require
performing an adrenocorticotropic hormone (ACTH) stimulation test to rule
out or diagnose adrenal hyperplasia. If DHEAS levels are higher than 7
m g/ml, dexamethasone suppression test (2 mg
qid for 5 days) is recommended. At the end of the test, DHEAS should be
measured again. If suppression was achieved, adrenal hyperplasia is suspected
and should be investigated by 17-OHP measurement. If suppression was not
the result, adrenal tumors should be suspected and CAT scan of the adrenals
is recommended (FC 17) (Wilson and Foster, 1992).
Cases of secondary amenorrhea with no suggestive history, signs or symptoms
are investigated as indicated in FC 11.
Patients with abnormal uterine bleeding are candidate for BBT measurement
and endovaginal ultrasonography to exclude or diagnose the presence of organic
lesions. In the absence of organic lesions, the diagnosis is dysfunctional
uterine bleeding of either ovulatory or nonovulatory nature as determined
by BBT charts and the cyclical occurrence of abnormal bleeding (FC
18) (Sciarra, 1992; Speroff et al., 1989).
Assessment of tuboperitoneal factor
Assessment of the tuboperitoneal factor requires investigation of the
patients by hysterosalpingography (HSG) which gives information about tubal
patency, tubal dilatation, presence of diverticulosis, and configuration
of the mucosal folds, but it lacks the precise diagnosis of the presence
or absence of adnexal adhesions and peritoneal endometriotic lesions. Latter
lesions can be easily diagnosed by laparoscopy. Laparoscopy is performed
at the end of the infertility work-up, or immediately after HSG in cases
of abnormal tubal findings (FC 19) (Mishell et al.,
1991; Rowe and Farley, 1988; Sciarra, 1992).
Assessment of uterine factor
Assessment of the uterine factor is done routinely by the use of the
hysterogram and endovaginal ultrasonography. When congenital uterine anomalies
or uterine fibroid are suspected, confirmation is sought by laparoscopy
and hysteroscopy. Hysteroscopy alone should be used to confirm and treat
intrauterine adhesions. If endometrial polyps, submucous fibroids, or endometrial
hyperplasia are suspected, confirmation and treatment should be done by
hysteroscopy and endometrial biopsy (FC 20) (Mishell
et al., 1991; Rowe and Farley, 1988; Sciarra, 1992).
Assessment of cervical factor
Evaluation of the cervical mucus and the postcoital test are essential
to assess the cervical factor. Providing that the partner’s semen parameters
are normal, and vaginal intercourse resulted in complete intravaginal ejaculation,
the interpretation of the results should be as described in
FC 21. Persistently abnormal cervical mucus tested
at the right time is either due to the presence of congenital cervical anomalies
(e.g. lack of endocervical glands, absence of hormonal receptors, and so
on) or due to destruction of the endocervical mucosa by previous cervical
operations. Normal cervical mucus and persistently poor results of the postcoital
test require cervical mucus culture to exclude cervicitis and measurement
of endocervical pH to exclude hyperacidity. Sperm-cervical mucus contact
(SCMC) test is also needed to evaluate a poor postcoital test. An abnormal
SCMC test indicates the presence of anti-sperm antibodies (FC
21) (Campana et al., 1987).
Diagnosis of male infertility
We have classified the history into several well-identified items, to
assist in understanding possible cause-effect relationships. For simplicity
and for the purpose of quick revision of historical factors, one should
refer to the flow charts FC 22 and
Age and other background information considered to be relevant to male fertility
such as occupation, ethnic group, religion, and so on, should be recorded.
Determining whether infertility is primary or secondary is the first
step in the fertility history. This is accomplished by asking the patient
about previous marriages and outcomes. Primary male infertility is the case
when the man has never impregnated a woman. Secondary male infertility applies
when the man has at some time impregnated a woman, even if the woman is
the partner in the presenting couple. In the case of secondary infertility,
one should determine the time elapsed since impregnation. Whether a case
of primary or secondary infertility, one should estimate the duration of
infertility of the present couple. Inquiring about previous work-ups and/or
treatment for infertility is important and should be discussed in detail.
The patient should be asked about the existence of a family history
of infertility, spontaneous abortion, stillbirth, and congenital disease.
Diethylstilbestrol (DES) exposure in utero may be a cause of male infertility
(Speroff et al., 1989).
Diseases can influence fertility either directly or indirectly. Diabetes
mellitus affects fertility through vascular, neurological and metabolic
alteration. Tuberculosis may destroy the urogenital tract. Fever exceeding
38°C can affect spermatogenesis for the succeeding 6 months. Hypogonadism,
gynecomastia, and testicular atrophy are commonly observed in men with hepatic
cirrhosis (Yen and Jaffe, 1991). Chronic renal failure is associated with
hypogonadism and hyperprolactinemia (Yen and Jaffe, 1991). Spermatogenic
arrest has been observed in adrenogenital syndrome (Martin-du Pan and Campana,
1993). Cystic fibrosis is a known factor in male infertility. Bronchiectasis
may be a part of the immotile cilia syndrome or Young’s syndrome. Neurological
diseases such as paraplegia, spina bifida, neuropathies, and congenital
abnormalities of autonomic innervation may affect sexual activity. Irradiation
of the genital area is a risk factor and is dose-dependent: permanent sterility
is observed for single dose field with 600 to 800 rads (Martin-du Pan and
Campana, 1993). Imetidine, spironolactone, nitrofurantoin, sulfasalazine,
and cytotoxic drugs depress sperm density and quality (Speroff et al., 1989).
Tranquillizers, depressant drugs, and some antihypertensive drugs may cause
impotence (Labby, 1982).
Age at puberty (normal, precocious or delayed) should be registered.
Any pathology that possibly causes testicular damage should be inquired
about, with details concerning the site, the treatment given, complications
due to treatment, and evolution of the pathology. A history of cryptorchidism
may lead to testicular atrophy. Testicular injury may initiate anti-sperm
antibody formation or testicular damage especially if it is accompanied
by scrotal bleeding or hematuria. Testicular torsion leads to testicular
damage if not treated within six hours. Mumps orchitis may be the cause
of testicular atrophy. Varicocele can impair testicular function (Yen and
History of sexually transmitted diseases (STD)
A positive history of STD is suggestive of damage and/or obstruction
of the genital tract. Data should be collected about the number of episodes,
the time passed since the most recent episode, and treatment during the
most recent episode. One should also inquire about the causative organism.
Surgical history related to infertility
Testicular and scrotal operations, e. g., orchiectomy, orchiopexy, testicular
detorsion, repair of inguinal hernias, varicocelectomy and hydrocelectomy
may be followed by decreased fertility. Operations of the epididymis and
vas deferentia such as epididymovasostomy and vasovasostomy may lead to
anti-sperm antibody formation or genital tract obstruction. Prostatectomy
is followed by impotence of variable degree and retrograde ejaculation.
Urinary cystectomy, bladder neck operations, repair of urethral strictures,
and repair of a hypospadias can all affect fertility.
The subject should be questioned about environmental and/or occupational
exposure to hazardous factors, such as radiation, chemicals, drugs, heat,
and traumas. Some personal habits can seriously affect men’s fertility,
such as wearing tight pants, taking excessively hot baths, and sauna (Martin-du
Pan and Campana, 1993). Cigarette smoking alters sperm motility and count
(Kulikauskas et al., 1985). Alcohol consumption depresses sperm motility
and count and, if used in high concentration, leads to impotence (Labby,
Daily or more frequent intercourse may depress the sperm count below
normal levels. Abstinence for seven days or more may decrease motility due
to increased proportions of older sperms (Speroff et al., 1989). For most
couples, coitus every 36 hours around the time of ovulation will give the
optimal chance for pregnancy. Intercourse is considered adequate if it occurs
more than twice per month, but if the frequency is twice or less per month
it is considered inadequate and may be an etiologic factor of infertility
and a possible sign of sexual dysfunction (Speroff et al., 1989).
Absence of libido or decreased libido is not only a cause of decreased
coital frequency, but may also be a sign of an endocrine or a psychological
disease. Dyspareunia may underlie either an organic or a psychological problem.
Impotence is defined as the inability to achieve or sustain an erection
for a sufficient duration to have coitus and achieve an orgasm (Yen and
Jaffe, 1991). If coital erection is inadequate, inquiry about early morning
erection and masturbatory erection should be made. If both occur normally,
the diagnosis of primary impotence is excluded and coital impotence is attributed
in most cases to a psychological factor. In primary impotence, erection
cannot be achieved in any circumstance. It may be due to hypogonadism with
androgen failure, pelvic vascular disease, or due to a systemic disease,
such as diabetic neuropathy, anemia, tuberculosis, and cancers (Labby, 1982).
Secondary impotence is due to psychological factors in 90% of cases, and
is characterized by failure of erection in the case of coitus only. Sometimes
the man is impotent only when he consumes alcohol or with the use of some
antihypertensive drugs or central nervous system (CNS) depressants (Labby,
Ejaculation is considered adequate if it occurs intravaginally with emission
of semen from the external urethral meatus. It is considered inadequate
in the following conditions: a) extravaginal ejaculation that may be due
to ejaculation praecox or extreme hypospadias; b) retrograde ejaculation
into the bladder; and c) anejaculation, i. e., failure of emission that
may be of organic or psychological origin (Rowe et al., 1993). In the last
case, if nocturnal and/or masturbatory ejaculation occurs normally, this
indicates a psychological factor. Previous experience of painful ejaculation
may also be a factor behind anejaculation.
Height, arm span, and the upper to lower body ratio should be measured
to exclude eunuchoidism. Gait should be looked at, as it may indicate the
presence of a neurological disease. Body weight, blood pressure and a general
physical examination help assess the general health, and will disclose any
chronic debilitating disease. If there is a history of neuropathy, endocrinopathy,
cardiovascular, respiratory, renal or hepatic disease, or a clinical suspicion
is aroused during the physical examination, the suspected disorder should
be investigated thoroughly. Secondary sexual characteristics including hair
distribution, body configuration, pubertal stage, and evidence of gynecomastia
should be looked for carefully (FC 24).
The penis should be examined for size, circumcision, the presence of
surgical or traumatic scars, and induration plaques of Peyronie’s disease
that may cause deformation of the erect penis, thus hindering vaginal intercourse.
If hypospadias is present, one should look for whether the urethral meatus
is near the corona of the glans, or on the proximal part of the penile shaft.
Delivery of semen could still occur intravaginally if the opening is in
the distal part of the shaft. Semen will be delivered outside the vagina
if the opening is proximal or lies in the perineal region. Any discharge
from the urethra should be cultured. Non-tender induration of the urethra
suggests a stricture. Tenderness and induration may indicate periurethritis
Each scrotum should be examined individually for presence of the testis,
its size, the presence of a hydrocele, masses, nodules, and tenderness.
The average size of the adult testis is 5x3 cm, equivalent to a volume of
25 ml. The lower size limit for a mature testis is about 4x2.5 cm, corresponding
to a volume of approximately 15 ml. Smaller than normal or very soft testes
in an adult indicate impaired germinal tissue mass, due either to primary
testicular failure or to hypothalamic-pituitary insufficiency. When determining
the size of the testis, the patient should be examined in the recumbent
position to avoid the risk of syncope. The scrotal skin is stretched over
the testicle, the contours of which are isolated from the epididymis. The
testis is palpated between the thumb and first two fingers. The volume of
each testicle is compared with the corresponding ovoid of the Prader orchiometer
(Hammond, 1987; Rowe et al., 1993).
A varicocele can only be detected with the patient standing; if present
it should be graded according to the following criteria: Grade III: if the
distended venous plexus visibly bulges through the scrotal skin; Grade II:
if there is intrascrotal venous distension which is easily palpable but
not visible; and Grade I: distension is only palpable while the patient
is performing a Valsalva maneuver. Subclinical varicocele should be recorded
if no distension can be detected, but there is any abnormal finding during
scrotal thermography and/or doppler echography (Rowe et al., 1993).
The epididymis should be examined while the patient is standing. The
epididymis is examined between the thumb and forefinger along its entire
course. A normal one is difficult to palpate, however if the epididymis
is palpable it should have a regular outline, a soft consistency, and should
not be tender. Nodular induration may suggest tuberculosis. Any swelling
and tenderness may indicate epididymitis (Hammond, 1987; Rowe et al., 1993).
The vas deferens should be palpated along its entire course below the
inguinal canal. A normal one should be palpable, but not thickened (Hammond,
1987; Rowe et al., 1993).
Inguinal examination can be performed while the patient is standing,
or in recumbent position. Any palpable or visible abnormalities should be
recorded in detail. If the testes are not palpable in the scrotum, an attempt
to palpate them in the inguinal region should be made (Hammond, 1987; Rowe
et al., 1993).
Rectal examination is carried out with the patient in the knee-elbow
position. Palpation should be performed from the cranial to the caudal part
and from lateral to medial. The prostate is normally soft, regular and not
tender upon slight pressure. The central groove should be easily identified
in a normal prostate. The seminal vesicles are not palpable if normal (Hammond,
1987; Rowe et al., 1993).
After completion of the history and physical exam, some patients can
be diagnosed directly as having sexual and/or ejaculatory dysfunction. Sexual
dysfunction applies to those patients with inadequate erection and/or inadequate
frequency of intercourse. Ejaculatory dysfunction applies when there is
normal intercourse, but followed either by anejaculation or ejaculation
occurring outside the vagina. Retrograde ejaculation is suspected in patients
with normal intercourse and orgasm, but no emission of semen. To be sure
of the diagnosis, a post-orgasm urine sample should be analyzed for the
presence of spermatozoa.
In the case of patients who are not diagnosed as having sexual or ejaculatory
dysfunction, one should advance to the next step in the laboratory work
Laboratory diagnostic methods of male infertility
Laboratory methods available for diagnosing male infertility include
semen analysis and examination of the interactions between sperm and cervical
mucus. We will review the various parameters analyzed and discuss their
interpretation and significance. The reader should refer to original sources
for detailed description of technical procedures.
Definitions concerning semen variables are given in
Table 1 (WHO, 1992).
Semen analysis (SA) comprises a set of descriptive measurements of spermatozoa
and seminal fluid parameters that help to estimate semen quality. Normal
values of semen parameters issued by WHO in 1992 are generally used as reference
values (Table 2). Ideally
each laboratory should set its own normal values reflecting the specific
population analyzed, but this is limited on practical grounds by the availability
of semen from men of proven fertility who have recently achieved impregnation.
It should be emphasized that semen is an exception among biological fluids
as its parameters display very wide intra- and inter-individual variations.
Therefore SA should be repeated to take intra-individual variations over
time into account in order to confirm abnormal parameters. Sperm parameters
assessed in SA include the number of sperm cells, and the viability, motility,
and morphology of the sperm population.
Ejaculate volume, sperm concentration, total sperm count, and viability
The major component of the ejaculate volume is made up of secretions
from the accessory glands (seminal vesicles and prostate). Consequently,
the ejaculate volume is not directly related to spermatogenesis, and the
sperm concentration (spermatozoa/ml) varies according to the ejaculate volume.
The total number of spermatozoa per ejaculate reflects spermatogenesis and
is related to the duration of sexual abstinence before collection. As the
ejaculate volume is related to the secretory function of the seminal vesicles
and prostate, decreased ejaculate volume reflects impaired accessory gland
function. Vital staining of the spermatozoa allows quantitation of the fraction
of living cells independently of their motility.
Sperm motility can be assessed either by manual counting or by using
a computer-assisted semen analysis (CASA) system. Motility is assessed at
the time of semen liquefaction, and after 1 and 3 hours to detect asthenozoospermia.
Manual counting classifies sperm cells into several categories (immotile,
locally motile, non-linear and linear motile) relying on qualitative subjective
criteria of selection. Many infertility centers now use CASA systems for
objective measurement of sperm motion, and positive correlations have been
found between motion parameters such as the amplitude of lateral head displacement,
curvilinear velocity, linearity and straight-line velocity, and fertilization
rates in vitro (Barlow et al., 1991; Liu et al., 1991). The threshold levels
for these motion characteristics have yet to be determined before they can
be used clinically in a prospective way (Oehninger et al., 1992). Moreover,
data obtained by different groups are often difficult to compare due to
the lack of consensus about parameter setting, as well as technical differences
between different systems (Davis and Boyers, 1992).
The evaluation of sperm morphology is performed after Papanicolaou or
similar staining and consists of detailed examination of 100 sperm cells
as well as other cells present in the ejaculate, including leukocytes and
immature sperm cells. Sperm cells represent an unique population in which
up to 50% of the cells can have morphological defects in normal fertile
individuals. These defects affect the head, midpiece, or the tail of the
sperm cell. The percentage of sperm with normal morphology is recorded,
as well as individual abnormalities to detect predominant abnormalities
that suggest genetic defects affecting spermatogenesis. The rare cases of
monomorphic teratozoospermia as well as severe asthenozoospermia can be
subjected to electron microscopic (EM) analysis to detect specific defects
at the ultrastructural level, particularly in the flagella, where abnormal
microtubule assembly can be found such as in the immotile cilia syndrome
(see Zamboni, 1992, for review). Recently Kruger and colleagues have reported
that the use of stricter morphology criteria than those recommended by WHO
gave better predictive value in in vitro fertilization. They report normal
in vitro fertilization rates for cases with >14% normal sperm morphology
(Kruger et al., 1988; Menkveld et al., 1990). There is still controversy
about these numbers and their significance in in vivo fertilization is unknown
(Check, 1992). The evaluation of sperm morphology also includes identification
of other cell types present in semen, such as immature sperm cells and leukocytes.
The presence of immature germ cells in semen indicates spermatogenic dysfunction
at the testicular level whereas leukocytes in concentrations exceeding 1x106/ml
indicate inflammatory conditions possibly related to infection. Direct measurement
of infectious contamination is obtained from bacteriological cultures of
both aerobic and anaerobic germs. In normal conditions, semen is not sterile
but rather colonized at low levels by a variety of germs. Recent studies
have shown that bacterial colonization does not have a negative impact on
sperm-cervical mucus interaction (Chandra and Gray, 1991; Eggert-Kruse et
al., 1992). Distinction between immature germ cells and leukocytes can be
difficult and specific leukocyte staining such as peroxidase staining or
anti-leukocyte monoclonal antibodies can be used to clarify the origin of
round cells in semen (Fisch and Lipshultz, 1992).
The presence of anti-sperm antibodies in semen can alter the fertilizing
ability of sperm. Being haploid, sperm cells display different surface antigens
than their diploid counterparts, and are immunogenic. In normal circumstances,
they are protected from the man’s immune system by a basal membrane constituting
the blood-testis barrier. When this barrier is ruptured, sperm cells induce
the synthesis of anti-sperm antibodies. Antibodies adsorbed on the sperm
surface can be detected by immunological assays using secondary Ig class-directed
antibodies coupled to beads. The percentage of sperm adhering to the beads
reflects in a semi-quantitative manner the presence of anti-sperm antibodies.
Currently available tests include IgG and IgA detection in semen and in
serum (Fisch and Lipshultz, 1992). However there is a poor correlation between
the presence of anti-sperm antibodies in serum and in semen, and serum anti-sperm
antibodies have been shown not to influence fertility prognosis (Eggert-Kruse
et al., 1989a). Sperm-bound antibodies have been found to impair sperm function
only when the degree of antibody binding is very high (>50%) (Barratt et
al., 1992). Accordingly, detection of anti-sperm antibodies in semen should
be preferred, and the presence of anti-sperm antibodies, detected by the
anti-IgG test can now be confirmed by the recently commercialized anti-IgA
Biochemical analysis of secretory components from the prostate, the
seminal vesicle and the epididymis in the semen gives information about
the functional state of these organs. Markers include fructose as a seminal
vesicle marker, zinc or acid phosphatase as prostate markers and carnitine
as an epididymis marker (von der Kammer et al., 1992; Wetterauer, 1986).
Recently Huszar and coworkers have reported that sperm creatine kinase,
a key metabolic enzyme, inversely correlates with fertilizing potential
and could be used as a biochemical marker of sperm fertilizing capacity
(Huszar et al., 1990; Huszar et al., 1992).
Sperm-cervical mucus interaction
Evaluation of sperm-cervical mucus interaction is based on functional
assays and includes the postcoital test, the sperm-cervical mucus contact
test, and the in vitro sperm-cervical mucus penetration assay.
The postcoital test entails analysis of the cervical mucus a few hours
after intercourse. It reflects the physiological situation in vivo and assesses
both the quality of cervical mucus and the penetration ability of sperm.
The quality of cervical mucus varies during the cycle and is favorably influenced
by estrogens, becoming more abundant and fluid at the time of ovulation.
Therefore postcoital tests are scheduled just before ovulation as determined
by cervical mucus changes, or more accurately by follicular sizing by ultrasonography.
The number of motile sperm per high-power microscopic field is recorded,
and, according to WHO guidelines, the test is considered positive when 10
or more motile sperm are found per field. Cervical mucus evaluation (including
volume, consistency, ferning, spinnbarkeit, cellularity and pH) is important
for the interpretation of postcoital test results with respect to sperm
function. A decreased number of sperm in cervical mucus when the cervical
mucus score is low reflects inadequate mucus rather than impaired sperm
function. Cervical mucus is colonized by sperm that are stored for several
hours in cervical crypts. Sperm cells then gradually migrate through the
cervix. Consequently sperm cells are present in cervical mucus constantly
for at least 12h following intercourse, and the timing of postcoital test
(6-12h after intercourse) allows testing of the viability of sperm in this
environment (Kremer and Jager, 1988). Abnormal penetration of cervical mucus
by sperm has been associated with the presence of immobilizing anti-sperm
antibodies (Kremer and Jager, 1992; Pretorius and Franken, 1989). Repeatedly
abnormal postcoital tests with normal cervical mucus associated with normal
sperm concentration and motility should be investigated by additional tests
such as anti-sperm antibody detection in semen, sperm-cervical mucus contact
test, and in vitro cervical mucus penetration assay. The postcoital test
has been used for several decades, but is reported to be inaccurate and
to lack a consensus regarding normal values and methodology (Griffith and
Grimes, 1990; Markham, 1991). Part of the problem may be due to the heterogeneous
nature of cervical mucus that prevents quantitative determination of sperm
concentration. The postcoital test nevertheless remains an inexpensive and
non invasive procedure that gives information about the occurrence of ejaculation,
and the ability of sperm cells to function within the cervical environment
(Markham, 1991). A new approach to the objective assessment of the postcoital
test has been recently described. Cervical mucus is examined after incubation
with bromelains. This procedure allows quantitative measurement of sperm
count in the liquefied cervical mucus in a Neubauer chamber (Campana et
Sperm-cervical mucus contact test
The sperm-cervical mucus contact test consists of mixing semen and cervical
mucus in vitro and measuring the appearance of immobilized " shaking " motile
sperm. This is interpreted as the adherence of antibody-coated sperm cells
to cervical mucus (Kremer and Jager, 1988) and has been shown to correlate
with semen anti-sperm antibodies and pregnancy rates (Franken et al., 1988).
This test can be performed in parallel with donor semen or donor cervical
mucus, and therefore makes it possible to discriminate between a male and
female origin of the sperm immobilizing factor.
In vitro cervical mucus penetration test
The third test of sperm-cervical mucus interactions involves the penetration
of cervical mucus by sperm in vitro. The mucus is placed in a capillary
tube, one end of the tube is dipped in semen and penetration and motility
of sperm in the mucus column is measured. It can be performed with homologous
or donor cervical mucus (Kremer and Jager, 1988). Using cervical mucus standardized
by estrogen treatment, this test has been shown to have good predictive
value of fertility (Eggert-Kruse et al., 1989b). Alternatively, this test
can be performed with commercial mid-cycle bovine cervical mucus or hyaluronic
acid gels. The use of alternative material to human cervical mucus has practical
advantages (availability, reproducibility), but may be less informative
than human material. A better correlation of fertility and sperm penetration
was reported for standardized human than bovine cervical mucus, and sperm
motility was found to decline more rapidly in the latter (Eggert-Kruse et
al., 1989b). Moreover, the distance of sperm penetration into bovine cervical
mucus was found to correlate with in vitro fertilization, but was not accurate
enough for prediction (Morrow et al., 1992). Sperm penetration into hyaluronate
polymers seems to yield better correlations with motility, and with sperm
fusion with hamster oocytes than bovine cervical mucus (Aitken et al., 1992),
but direct correlation with fertility has not yet been shown. Consequently,
whenever possible, human cervical mucus should be used, and for specific
male factor detection, estrogen standardized donor mucus should be preferred.
Sperm functional assays
Sperm functional assays have been developed in an attempt to find a good
predictive test of male fertility. We will discuss the hemizona assay, the
hamster egg penetration assay, and the sperm hypo-osmotic swelling assay.
The hemizona assay (HZA) is the most recently developed sperm functional
assay. It measures the binding of capacitated sperm to isolated human zona
pellucida. Human oocytes are bisected by micromanipulation, thus allowing
for an internally controlled comparison of sperm binding to matching hemizonae
surfaces in the patient relative to a fertile control (Burkman et al., 1988).
The two matched hemizona of the human oocytes have the advantage of providing
functionally equal surfaces, allowing a controlled comparison of sperm binding,
and therefore limiting the amounts of oocytes used. Ethically this assay
is acceptable, as the microsurgical bisection of the oocyte prevents any
inadvertent fertilization. The HZA has been found to be predictive of IVF
outcome with positive and negative predictive values of 83% and 95% respectively
(Oehninger et al., 1989). The major problem with this assay is the limited
availability of human oocytes. Eventually, it could be replaced by a standardized
kit in which recombinant human zona sperm receptors mimic the natural functional
hemizonae used now.
Sperm penetration into zona-free hamster oocytes
The zona-free hamster oocyte sperm penetration assay is a heterologous
bioassay, originally developed to test capacitation, acrosome reaction,
fusion, and sperm chromatin decondensation (Campana et al., 1983). Cross-species
fertilization is made possible by removing the zona pellucida of hamster
oocytes. Using the original test conditions, the limiting step is the low
incidence of spontaneous acrosome reaction in human sperm populations incubated
in vitro, and therefore it has been described as measuring the ability of
sperm to undergo acrosome reaction, rather than the overall fertilization
process (Tesarik and Testart, 1989). Optimized procedures have been introduced
recently that decrease the number of false positives, making it more specific,
and giving good predictive value of IVF (Fisch and Lipshultz, 1992).
Hypo-osmotic swelling of sperm flagella
The hypo-osmotic swelling test (HOS) measures sperm membrane integrity
by its ability to swell when exposed to hypo-osmotic media, and has been
claimed as relevant to fertilizing ability. The biological significance
of this test is unclear and its validity is still controversial. Some authors
have described subnormal HOS scores in patients with low IVF fertilization
rates, but normal semen analysis (Check et al., 1992), whereas other authors
have found that HOS is equivalent to viability staining and correlates with
semen analysis data but not with the hamster oocyte penetration test: they
conclude that HOS does not add relevant information to that obtained from
semen analysis about the fertilizing potential of semen (Van den Saffele
et al., 1992).
Azoospermia and other sperm abnormalities
In all azoospermic patients (FC 26), serum follicle-stimulating
hormone (FSH) should be determined. For patients with elevated FSH levels,
karyotyping is indicated. If the FSH levels are normal, it is necessary
to measure luteinizing hormone (LH) and testosterone levels for differential
diagnosis. High serum levels of LH and testosterone are diagnostic of androgen
resistance. If both LH and testosterone serum levels are normal, the diagnosis
is either obstructive azoospermia or testicular failure depending on history,
and the results of scrotal exploration, vasography, and testicular biopsy.
Azoospermic patients with hypogonadotropic hypogonadism are candidates for
GnRH testing, measurement of serum prolactin, and X-ray evaluation of the
sella turcica to differentiate between a hypothalamic or pituitary origin.
Oligozoospermic patients (FC 27) may need post-orgasm
urine analysis to exclude partial retrograde ejaculation. Apart from that,
the condition may be due to partial genital tract obstruction, testicular
failure, varicoceles, or it may be idiopathic. Severe asthenozoospermic
patients require EM study of sperm to isolate structural tail anomalies.
An immunological work-up to exclude autoimmunization and/or seminal fluid
culture to exclude genital tract infection may be required (FC
27). In cases of teratozoospermia, one should start first by excluding
the presence of monomorphic genetic syndromes like globozoospermia, microcephaly,
and short-tail sperm. The diagnosis then depends on the type of abnormal
morphology. In the presence of abnormal heads, testicular failure of whatever
etiology should be suspected. Abnormal tails require assessment of the male
accessory sex glands for the presence of infection (FC
Male accessory gland infection is diagnosed if the patient has an abnormal
sperm and fulfills at least two of the following criteria:
- History and physical signs: A history of urinary tract infection,
epididymitis, STD, or during the physical examination there is a thickened
or tender epididymis, thickened vas deferens, and/or abnormal rectal
- Prostatic fluid: Abnormal prostatic expression fluid and/or abnormal
urine after prostatic massage.
- Ejaculate signs: More than 1 million WBC/ml in the ejaculate; culture
with significant growth of pathogenic bacteria; abnormal appearance
and/or viscosity and/or pH and/or abnormal biochemistry of the seminal
To diagnose infection, a combination of 2 items from 1 to 3, or two factors
from item 3 should be present. To obtain prostatic expression fluid, a prostatic
massage per rectum should be done. The fluid expressed from the external
urethral meatus is collected on a glass slide, and is either examined wet
in phase contrast or smeared and stained with Giemsa. Normal prostatic expression
fluid contains fewer than 5 WBC/high-power field (HPF). In abnormal fluid,
more than 10 WBC are present per HPF, typically in mucus streaks and together
with degenerated prostatic cells. The presence of 5 to 10 WBC/HPF without
mucus streaks is of doubtful significance. If no prostatic expression fluid
is obtained after prostatic massage, the patient is asked to void urine
and the first void of approximately 10 ml of urine is used for bacteriological
and cytological analysis. The presence of more than 5 WBC or RBC in the
urinary sediment is considered abnormal (Rowe et al., 1993).
Abbreviations used in flow charts
Basal body temperature
Computerized axial tomography
Mixed antiglobulin reaction (MAR) test
Magnetic resonance imaging
Pelvic inflammatory disease
Sperm-cervical mucus contact test
Sexually transmitted disease
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