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1. Semen analysis

2. Semen analysis- Introduction Test to evaluate fertility potential in a male. Reflects efficiency of spermatogenesis, integrity of post testicular structures necessary for sperm transport and ejaculation. As per WHO reference values, the majority of fertile men are expected to have their parameters within the range of the 50th percentile.  The results may correlate with “ fertility ,”this the assay is not a direct measure of fertility. It provides no insights into the functional potential of the spermatozoon to fertilize an ovum or to undergo the subsequent maturation processes required to achieve fertilization.

3. Semen analysis- Introduction Seminal analysis needs to be complemented with sperm functional assay , which indirectly measures the ability of one spermatozoon to deliver the correct complement of chromosomes to an ovum.

4. Semen analysis- Introduction Fertility is a “couple-concept”. Severe alterations of semen parameters (much below the fifth percentile) can be compatible with the induction of pregnancy, provided that the female partner’s fertility status is optimal. Vice versa, exceptionally good sperm count does not always imply natural conception.

5. Specimen Requirements and Procedure Semen sample is collected by self-masturbation near the laboratory or at home.  Recommended to be collected after a minimum of 3 days and a maximum of 7 days of abstinence. Clear instructions regarding the collection of the semen sample must be provided before collection to ensure all fractions of the ejaculate are collected and complete. Any loss of the sample must be reported to the laboratory during submission.

6. Specimen Requirements and Procedure Sample should be collected in a clean, wide-mouthed container that is nontoxic to spermatozoa. Container should be kept at ambient temperature between 20 deg C and 37 deg C. Latex condoms should not be used for sample collection. They have chemical agents that can affect sperm viability and motility. Non toxic condoms to be used for sample collection during intercourse. Semen should be delivered to the laboratory within 1 hour of its collection As semen samples can show substantial variation, a minimum of 2 properly collected and transported samples, ideally collected over 2 spermatogenic cycles, should be examined at 37°C. Safety guidelines must be strictly adhered to at the laboratory while handling semen samples.

7. Semen Analysis: General Concepts Semen evaluation is divided into two parts: macroscopic and microscopic analysis.

8. Macroscopic evaluation refers to the chemical and physical parameters of the ejaculate. 1. Liquefaction   of the semen needs 15–30 min (> 2 hrs abnormal), and this process is regulated by the prostatic secretion, which is rich in citric acid acting in synergy with proteolytic enzymes (lysozyme, α- amylase, and β- glucuronidase) and prostatic specific antigen (PSA), a trypsin-like protease, that cleaves the semenogelin proteins.  2. Viscosity   is homogeneous stickiness of semen sample. A normally liquefied sample will have an opalescent appearance, with a cream/grey color . A transparent ejaculate might indicate an extreme reduction in the number of spermatozoa. While a highly concentrated specimen will be opaque. High viscosity may indicate prostatitis.

9. Macroscopic evaluation 3. Volume - The normal volume of ejaculate after 2-7 days of sexual abstinence is about 2-6 mL. secretions of the accessory glands (about 90%) whereas the contribution of the epididymis and bulbourethral glands are minimal. Prostate and seminal vesicles are target organs of androgens; therefore, severe androgen deficiency is associated with lower semen volume. Hypospermia :  <0.5 mL of semen (Cause- Improper collection, hypogonadism, retrograde ejaculation, obstruction of lower urinary tract may yield low volume) Hyperspermia :  >6 mL of semen ejaculated (prolonged abstinence or excessive secretion from the accessory sex glands).

10. Macroscopic evaluation 4. pH 7.2-8.2 prostate produces an acidic fluid, while seminal vesicles produce an alkaline fluid leading to the typical neutral  pH. Changes are usually due to inflammation of the prostate or seminal vesicles.

11. Microscopic evaluation Sperm number is a quantitative marker of spermatogenesis, whereas sperm motility, vitality, and morphology are qualitative parameters. 1. Sperm concentration/ number is influenced by accessory gland activity and does not represent a direct measure of testicular sperm output. It is done using a phase contrast microscope by volumetric dilution and hemocytometry and is reported as millions of sperm per mL.   The fifth percentile reference value for TSC is >39 millions of spermatozoa per ml. Conditions of abnormal sperm count. Azoospermia: absence of spermatozoa in the ejaculate and in the pellet after centrifugation. Cryptozoospermia : absence of spermatozoa in the ejaculate but present in the pellet after centrifugation. Oligozoospermia : sperm concentration or TSC lower than the fifth percentile value (< 20 million per ml in severe Oligospermia)

12. 2. Sperm motility   is classified based on the direction and velocity of sperm movement into the following categories: progressive motility (rapid and slow) is calculated by the speed at which sperm moves with flagellar movement in a given volume as a percentage (range 0%-100%) by counting 200 sperms (  >25 μ m/s at 37°C and >20 μ m/s at 20°C) non-progressive sperm movement  (<5 μ m/s) immotile spermatozoa  Asthenozoospermia is reduction in sperm motility total motility <42% and progressive motility <30% can be due to congenital or acquired factors.

13. 3. Sperm Vitality test is indicated in case the percentage of immotile sperm is higher than 40% to distinguish vital and dead cells. The extreme reduction in  sperm vitality  is known as necrozoospermia . 4. Sperm morphology WHO method classifies abnormally shaped sperm into specific categories based on specific head, tail, and midpiece abnormalities. Normal - the 50th percentile of reference values would correspond to only 14% of typical or normal forms.

14. Types of Sperm morphological defects: Head defects:  Large, small, tapered, pyriform, round, amorphous, vacuolated (>20% of the head area occupied by unstained vacuolar areas) heads with small acrosomal area (<40% of head area), double heads, any combination of these. Neck and midpiece defects:  Bent neck; asymmetrical insertion of midpiece into head; thick, irregular midpiece; abnormally thin midpiece; any combination of these. Tail defects:  Short, multiple, hairpin, broken, bent, kinked, coiled tails, or any combination of these. Cytoplasmic droplets:  Greater than one-third of the area of a normal sperm head.

15. Teratozoospermia is <4% normal forms. It includes head, midpiece, tail, and cytoplasmic residue defects different spermatozoa may present different types of abnormalities. These mixed morphological defects are usually related to defective spermatogenesis or epididymal inflammation. 

16. Leukocytospermia Leukocytospermia is defined as the presence of more than 1 × 10 6  leukocytes per ml of semen.  The presence of a high number of WBCs and a reduced ejaculated volume has been proposed as an indicator for infections and inflammations of the genitourinary tract. However, the association of leukocytospermia with fertility potential is controversial. Infection of the male reproductive tract may directly or indirectly cause infertility. Pyospermia is a laboratory finding categorized as the abnormal presence of leukocytes in human ejaculate and may also indicate genital tract inflammation

17. Agglutination is the tendency of motile spermatozoa to form clumps maybe due to presence of  anti-sperm antibodies  (ASA)  ASA have been found in several pathological conditions which may lead to the interruption of the blood-testis barrier, such as testicular torsion, testicular carcinoma, and orchitis. Immunological cause of infertility confirmed by mixed antiglobulin reaction test (MAR test) and immunobead-binding assay (for IgA, IgG, and IgMs ) A positive finding of >50% of motile sperm with attached beads is considered to be clinically significant.

18. Summary of the main semen alterations and related suspected clinical conditions

19. BIOCHEMICAL ASSESSMENT OF SEMINAL PLASMA, PROSTATE, EPIDIDYMIS, AND SEMINAL VESICLES clinically relevant in patients with hyperviscous semen and to understand genital fluid interactions during the semen coagulation-liquefaction process. Decreased levels of zinc, citric acid, and glucosidase may indicate either seminal vesicle, prostate dysfunction, or prostatic duct obstruction.

20. Other tests TESTS OF SPERM CAPACITATION TESTS OF HEMIZONA AND ZONA PELLUCIDA BINDING SPERM PENETRATION ASSAY OR SPERM CAPACITATION INDEX  TESTS OF SPERM DNA DAMAGE ASSESSMENT OF REACTIVE OXYGEN SPECIES

21. From Semen Analysis to Diagnosis Semen analysis, together with medical history and physical exam, represents the first step of the diagnostic work-up of infertile men.  If result of semen analysis is suggestive for specific forms of reproductive impairments. It will confirmed by subsequent laboratory and instrumental exams. e.g., additional semen microbiological examinations in case of suspected infections or inflammations transrectal ultrasound exam in case of suspected distal obstruction genetic exams in case of monomorphic teratozoospermia , etc

22. Azoospermia/ Cryptozoospermia Azoospermia is absence of spermatozoa in the ejaculate and in the pellet after centrifugation. Affect about 1% of the general male population It is incompatible with natural pregnancy.  Diagnosis of azoospermia must be confirmed in at least two semen analysis (possibly after the whole spermatogenic cycle) because it could be a temporary disorder, or it could alternate with cryptozoospermia . differentiate between azoospermia and cryptozoospermia .

23. Importance in distinguishing between these two conditions refers to different in vitro fertilization options, i.e., in azoospermia testicular sperm extraction (TESE) must precede ICSI. Azoospermia can be divided into forms : obstructive (OA) – -spermatogenesis is unaffected absence of spermatozoa in the ejaculate is due to bilateral distal or proximal obstruction of the urogenital tract. 2. non-obstructive (NOA) – Sertoli cell-only syndrome maturation arrest at different stages of germ cell maturation hypospermatogenesis . 

24. When no spermatozoa is present in the ejaculate, it is important to evaluate the sediment after centrifugation, as their presence is indicative of cryptozoospermia  . When azoospermia is confirmed by examining the sediment, three parameters are useful to distinguish between OA and NOA: semen volume, pH, and the presence of immature germ cells.  For example: an acidic pH indicates the absence of the seminal vesicles secretions, which together with the absence of spermatozoa could strongly suggest congenital bilateral absence of the vas deferens (CBAVD) associated to the agenesis of seminal vesicles. Reduced semen volume with normal pH , can be seen in case of severe hypoandrogenism When semen volume and pH are normal, the analysis of the pellet smears for the presence of spermatogenetic cells is useful. If they are present, a maturation arrest can be suspected. while if they are absent, a proximal obstruction can be the cause.

25. Asthenozoospermia   Asthenozoospermia is the most frequent sperm defect observed in infertile men, with variable degrees of severity. In case of total sperm immotility , it is mandatory to distinguish between immotile live and dead sperm ( necrozoospermia ). A large proportion of live but immotile cells, as seen in structural defects in the flagellum. Complete asthenozoospermia is due to a high percentage of dead cells can be associated with an epididymal pathology Alterations of motility and morphology classified as asthenoteratozoospermia .

26. Value and Limits of the Semen Analysis Routine semen analysis is a valuable diagnostic test although it has its own limitations. It remain fundamental step to explore aetiology behind defects. While azoospermia undoubtedly causes infertility. The presence of triple defects—reduced sperm number, motility, and morphology—also increases the likelihood of a male factor responsible for couple infertility. It is not possible to predict fertility using parameters from either partner alone, unless there is azoospermia in the man or premature ovarian failure in the woman. To further analyse extended examinations including a series of test as per WHO should be done. However, thier clinical utility remains still controversial 

27. Semen analysis is performed in specialized laboratories strictly following the WHO guidelines and participating at external quality control programs.   Clinicians should also be aware of the fact that semen parameters are susceptible of variations due to pre-analytical, analytical factors, together with intraindividual biological variability. Therefore, when semen alterations are observed, a second evaluation is mandatory.

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