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INFERTILITY

SPERM COUNT y The generalization that total sperm number reflects testicular sperm productivity may not hold for electroejaculates from men with spinal cord injury, those with androgen defi ciency, or for samples collected after prolonged abstinence or partial retrograde ejaculation.

While measurements made on the whole population of ejaculated spermatozoa cannot defi ne the fertilizing capacity of the few that reach the site of fertilization, semen analysis nevertheless provides essential information on the clinical status of an individual. All aspects of semen collection and analysis must be done by properly standardized procedures if the results are to provide valid, useful information. total number of spermatozoa in the ejaculate is correlated with testicular volume, and thus is a measure of the capability of the testes to produce spermatozoa and the patency of the male tract.
Total sperm number refers to the total number of spermatozoa in the entire ejaculate and is obtained by multiplying the sperm concentration by the semen volume.

To reduce sampling errors, a critical number of spermatozoa have to be counted (preferably a total of at least 400, from replicate counts of approximately 200)

It is recommended to calculate and report the total sperm number per ejaculate, as this parameter provides a measure of the capability of the testes to produce spermatozoa and the patency of the male tract. This is obtained by multiplying the sperm concentration by the volume of the whole ejaculate. Procedure: y 1. 2. 3. 4. 5. 6. Examine the well-mixed, undiluted preparation of liquefied semen on a glass slide under a coverslip. Mix semen with diluent and fixative. Load the mixture to the hemocytometer and wait for it to settle in a humid chamber after 10-15 minutes. Count at least 200 spermatozoa per replicate. Calculate the concentration of the spermatozoa per ml. The lower reference limit for sperm concentration is 15 106 spermatozoa per ml (5th centile, 95% CI 1216 106). Calculate the total spermatozoa per ejaculate = sperm concentration x volume of the whole ejaculate.

Lower Reference Limit for Total Sperm Number: 39 106 spermatozoa per ejaculate (5th centile, 95% CI 3346 106)
1. Errors in estimating numbers The precision of the estimate of sperm number depends on the number of spermatozoa counted. Poisson distribution: the standard error (SE) of a count (N) is its square root (_N) and the 95% confi dence interval (CI) for the number of spermatozoa in the volume of semen is approximately N 1.96 _N (or N approximately 2 _N). If 100 spermatozoa are counted, the SE = 10 (_100), and the 95% CI = 80 120(100 20). If 200 spermatozoa are counted, the SE = 14 (_200), and the 95% CI = 172 228 (200 28). If 400 spermatozoa are counted, the SE = 20 (_400) and the 95% CI = 360 440 (400 40). The sampling errors can be conveniently expressed as a percentage of the count = (100(_N/N)). Counting too few spermatozoa will produce an uncertain result, which may have consequences for diagnosis and therapy.This may be unavoidable when spermatozoa are taken for therapeutic purposes and sperm numbers are low. When semen volume is small and fewer spermatozoa are counted than recommended, the precision of the values obtained will be signifi cantly reduced. If fewer than 200 spermatozoa are counted per replicate, report the sampling error When an accurate assessment of low sperm numbers is not required (0 to 4 per 400 HPF or 0 to 16 per 200

HPF) No spermatozoa observed: Mix the sample. If viscous, reduce viscousity

Remove a 1-ml aliquot of semen and centrifuge at 3000g for 15 minutes. Decant supernatant and resuspend the sperm pellet in the remaining approximately 50 ul of seminal plasma. Place one 10-ul aliquot of the pellet on each of two slides under 22 mm 22 mm coverslips. This will create two wet preparations approximately 20 um deep Examine the slides with phase-contrast optics at 200 or 250 magnification Scan the entire coverslip systematically field by field

CRYPTOZOOSPERMIA - presence of spermatozoa in either replicate AZOOSPERMIA absence of spermatozoa in both replicate * The absence of motile spermatozoa from the aliquot examined does not necessarily mean that they are absent from the rest of the sample. MORPHOLOGY y Defective spermatogenesis and some epididymal pathologies are commonly associated with an increased percentage of spermatozoa with abnormal shapes. Morphological defects: increased DNA fragmentation increased incidence of structural chromosomal aberrations immature chromatin aneuploidy Head defects: large or small tapered pyriform round amorphous vacuolated (more than two vacuoles or >20% of the head area occupied by unstained vacuolar areas) vacuoles in the post-acrosomal region small or large acrosomal areas (<40% or >70% of the head area), double heads, or any combination of these Neck and midpiece defects: asymmetrical insertion of the midpiece into the head thick or irregular sharply bent abnormally thin or any combination of these Principal piece defects: short, multiple broken smooth hairpin bends sharply angulated bends of irregular width coiled, or any combination of these If there are many such defects, their prevalence relative to spermatozoa can be determined. C = S (N/400) If, N = number of cells with defects counted in the same number of fields as 400 spermatozoa S = concentration of spermatozoa (106 per ml) C = concentration (C) of the defects (106 per ml)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Aspermia - no semen (no or retrograde ejaculation) Asthenozoospermia - percentage of progressively motile (PR) spermatozoa below the lower reference limit Asthenoteratozoospermia - percentages of both progressively motile (PR) and morphologically normal spermatozoa below the lower reference limits Azoospermia - no spermatozoa in the ejaculate (given as the limit of quantifi cation for the assessment method employed) Cryptozoospermia - spermatozoa absent from fresh preparations but observed in a centrifuged pellet Haemospermia (haematospermia) - presence of erythrocytes in the ejaculate Leukospermia (leukocytospermia, pyospermia) - presence of leukocytes in the ejaculate above the threshold value Necrozoospermia - low percentage of live, and high percentage of immotile, spermatozoa in the ejaculate Normozoospermia - total number (or concentration, depending on outcome reported)* of spermatozoa, and percentages of progressively motile (PR) and morphologically normal spermatozoa, equal to or above the lower reference limits Oligoasthenozoospermia - total number (or concentration, depending on outcome reported)* of spermatozoa, and percentage of progressively motile (PR) spermatozoa, below the lower reference limits Oligoasthenoteratozoospermia - total number (or concentration, depending on outcome reported)* of spermatozoa, and percentages of both progressively motile (PR) and morphologically normal spermatozoa, below the lower reference limits Oligoteratozoospermia - total number (or concentration, depending on outcome reported)* of spermatozoa, and percentage of morphologically normal spermatozoa, below the lower reference limits Oligozoospermia - total number (or concentration, depending on outcome reported)* of spermatozoa below the lower reference limit Teratozoospermia - percentage of morphologically normal spermatozoa below the lower reference limit Lower reference limit 1.5 (1.41.7) 39 (3346) 15 (1216) 40 (3842) 32 (3134) 58 (5563) 4 (3.04.0) > 7.2 <1.0 <50 <50 >2.4 >13 >20

Parameter Semen volume (ml) Total sperm number (106 per ejaculate) Sperm concentration (106 per ml) Total motility (PR + NP, %) Progressive motility (PR, %) Vitality (live spermatozoa, %) Sperm morphology (normal forms, %) Other consensus threshold values pH Peroxidase-positive leukocytes (106 per ml) MAR test (motile spermatozoa with bound particles, %) Immunobead test (motile spermatozoa with bound beads, %) Seminal zinc (_mol/ejaculate) Seminal fructose (_mol/ejaculate) Seminal neutral glucosidase (mU/ejaculate)

* Men whose semen characteristics fall below the lower limits given here are not necessarily infertile; their semen characteristics are below the reference range for recent fathersas are, by definition, those of 5% of the fertile men who provided data used in the calculation of the reference range. CRYOPRESERVATION Fertility preservation Semen may be obtained and stored before a man undergoes a procedure or exposure that might prevent or impair his fertility, such as: vasectomy (in case of a future change in marital situation or desire for more children); treatment with cytotoxic agents or radiotherapy, which is likely to impair spermatogenesis permanently (Meseguer et al., 2006; Schmidt et al., 2004); active duty in a dangerous occupation, e.g. in military forces, in countries where posthumous procreation is acceptable. Infertility treatment Spermatozoa may be stored for treatment of the mans partner by artifi cial insemination by husbands semen (AIH), IUI, IVF or ICSI, in cases of:

severe oligozoospermia or intermittent presence of motile spermatozoa in the semen (as backup for ICSI) (Bourne et al., 1995); treatment of infertility that may not persist, such as surgery for genital tract obstruction or gonadotrophin treatment for hypothalamo-pituitary hypogonadism; the need for special collection, such as assisted ejaculation for patients with spinal cord injury, spermatozoa from retrograde ejaculation in urine, or surgical collection from the genital tract; men who are unable to provide fresh semen on the day of an ART procedure. * For fertility preservation or infertility treatment, enough normal specimens should be stored for 10 or more inseminations, to ensure a good chance of pregnancy. With abnormal semen, pooling of multiple samples for AIH has not been proven to be useful.