SPERM FUNCTION TEST IN EMBRYOLOGY .pptx

SPERM FUNCTION TESTS USED in embryology, IVF for assessing the fertilization capacity of sperm

1. SPERM FUNCTION TEST DR. DEEPTHI REPALLE IVF LAB DIRECTOR MOHAK IVF, SAIMS

2. Normal functions of the sperm Fertilization

3. SPERM OOCYTE INTERATION

4. HOWEVER… The sperms should be sufficient in number and have normal structure for fertilizing the eggs Sperms should have intact and functioning membrane to survive the vaginal and uterine environment Normal functioning of the biochemical markers and proteins Motility and mitochondrial function to cross the reproductive barrier Sperm should able to survive from stress Proper Acrosome functioning to fertilize the oocyte Transfer the proper genetic material to the oocyte during the process of fertilization

5. Sperm function tests? What are sperm function tests Sperm count Total sperm number Sperm motility Progressive motility These tests detect function of a certain part of spermatozoa and give insights on the events in fertilization of the oocytes

6. Who needed these tests? Patients with normal semen analysis parameters may not need these specialized test immediately but, may be later Certain patients need some specialized test to understand the functioning of their sperm better like Borderline semen parameters Pathological semen parameters Previous fertilization failures/TFF Prolonged Unexplained infertility/unexplained male infertility

7. SPERM FUNCTION TESTS

8. What these tests specify Understanding the human sperm and fertilization biochemistry Information about important measures like kinematics of sperm capacitation, hyperactivation, The ability of sperm to bind zona-pellucida, The ability to undergo acrosome reaction, Decondensation of the chromatin to form male pronucleus

9. SPERM FUNCTION TESTS

10. Biochemical tests Each secretion has a characteristic marker to detect presence, absence, dysfunction or infection of specific glands of male reproductive system. Each specific substance is contributing to sperm function, but how is still unsure S.no Secretions from Specific Markers Functions 1 Prostate gland Acid phosphatase, Citric acid, Zinc and Magnesium Zinc –protecting and stabilizing condensed sperm chromatin 2 Seminal Vesicles Fructose and Prostaglandins Fructose-energy source for sperm for anaerobic metabolism 3 Epididymis L-Carnitine, alpha-glucosidase and glycerophosphocholine

11. Biochemical tests Fructose test Patients with azoospermia and in low volume samples (<1 ml)-seminal obstruction or atresia or ejaculatory duct obstruction. Reduced fructose leads to reduced motility . Men with high sperm and low motility due to relative deficiency of fructose. Concentration of fructose in semen ranges from 63-500 mg/dl(3.5-28 mmol/l). Seliwanoff’s test for ketose sugars or qualitative assessment of fructose by colorimetric test.

12. SPERM FUNCTION TESTS

13. Leucocyte detection tests There are different methods for assessing leukocytes in semen, including identification of round cells, immunohistochemical staining using monoclonal antibodies, the Endtz test, the peroxidase test, and flow cytometry. The WHO also recommends the peroxidase test , although this test distinguishes only granulocytes and no other WBC types. The gold standard for assessment of WBCs in semen is immunohistochemical staining using monoclonal antibodies against specific WBC subpopulations, but this method is expensive, time-consuming, and not standardized.

14. Leucocyte detection tests The World Health Organization (WHO) defines leukocytospermia as >1 × 10 6  WBCs/mL in a semen sample. Leukocytospermia is considered an inflammatory disease. In most cases, the inflammatory syndrome is secondary to a urogenital bacterial disorder. However, other conditions may also lead to leukocytospermia, including viral infections, varicocele, smoking, or trauma such as spinal cord injury. Studies have evaluated the use of antibiotics, anti-inflammatory agents, and antioxidants as treatment; however, results have been varied, and controversy remains.

15. Leucocyte detection tests Peroxidase test

16. Leucocyte detection tests Endtz test

17. Reactive oxygen species(ROS) Spermatozoa require oxygen for metabolism but the deleterious metabolites produced such as ROS can modify cell function/damage. Seminal plasma naturally contains different antioxidants that help to protect spermatozoa against such oxidants. Seminal plasma oxidative stress develops as a result of an imbalance between ROS generating and scavenging activities. Spermatozoa are more likely susceptible to ROS-induced damage compared to other cells as they have larger quantities of polyunsaturated fatty acids(DHA) in the membrane, and their cytoplasm contains a low concentration of scavenging enzymes.

18. Reactive oxygen species(ROS)

19. Reactive oxygen species (ROS) Ros in semen produced by Immature sperms Leukocytes Epithelial cells ROS can be estimated by using different methods Chemiluminescence using luminol as a substrate -Normal range <0.2 × 10 6 cpm per 20 million spermatozoa -Abnormal values >0.2 × 10 6 cpm per 20 million spermatozoa Semi-quantitative assays using oxidative indicators ROS is inversely correlated with normal sperm function and directly correlated to male infertility

20. Reactive oxygen species (ROS)

21. Reactive oxygen species (ROS)

22. SPERM FUNCTION TESTS

23. Sperm morphology The classification system evolved by Kruger et al. and subsequently accepted by WHO give very low threshold for accepting normal morphology and very few samples show more than 25% normal spermatozoa with most in the range of 4–30% in fertile population. According to strict accepted criteria considering normal spermatozoan are: Normal sperm head is considered to be 3–5 μ in length and 2–3 μ in width with perfect oval shape Mid-piece is about 1 μ in diameter with straight and regular outline. It must be aligned to the longitudinal axis of the head and should be 7–8 μ in length The tail must be slender, uncoiled and at least 45 μ in length.

24. Sperm morphology Staining methods The morphology is evaluated by using air dried smears prepared from whole semen on glass slides and stained using Diff-Quick or PAP method. PAP Diff quick

25. Sperm morphology

26. Sperm morphology

27. Sperm morphology

28. Sperm morphology

29. Sperm morphology

30. SPERM FUNCTION TESTS

31. Sperm vitality It represents the sperm membrane functionality when it enters the female tract. There are different test for sperm vitality HOST test (Hypo Osmotic Swelling Test) Eosin nigrosin test Eosin test

32. Sperm vitality HOST TEST

33. Sperm vitality Eosin-nigrosin test

34. SPERM FUNCTION TESTS

35. Mitochondrial activity test Mitochondria is necessary for motility of the sperm The energy required for the flagellar activity is derived from ATP produced in the mitochondria concentrated in the mid piece of spermatozoa. The long journey of spermatozoa through the female tract requires an optimal quantity of ATP. The mitochondrial oxido -reductive enzyme apparatus can be tested using indicators like nitro blue tetrazolium, which product blue insoluble pigment in and around mid-piece. Evaluation can be carried out in smears showing stained mid-piece from good sperms to poor staining of nonmotile or poor mitochondrial activity sperms.

36. Mitochondrial activity test

37. SPERM FUNCTION TESTS

38. Acrosome reaction tests CAPACITATION Capacitation can be defined as time dependent, reversible changes sperm undergo especially in the female genital tract, which lead to the ability of sperm to acrosome react. The changes occur with loss of extrinsic proteins, including acrosome stabilizing factors along with the loss of membrane cholesterol. These intricate steps results in sperms having more fluid and pliable membranes. During capacitation sperm motility changes to the characteristic pattern, and induction of AR becomes possible. The rapid spiraling pattern of the sperm movement and subsequently hyperactivated sperm motility is commonly associated with sperm capacitation.

39. CAPACITATION

40. Acrosome reaction tests ACROSOME REACTION The integrity of the acrosome structure and the ability to undergo acrosomal exocytosis are necessary for normal fertility. The acrosome reaction is a process that in vivo occurs in the proximity of the oocyte, and which must take place before the spermatozoon can penetrate the oocyte vestments and fuse with the oocyte. Several stimuli are known to induce acrosome reaction. Among these, zona pellucida Proteins and progesterone are considered possible physiological inducers of acrosome reaction in view of their elevated concentration in the proximity of the oocyte. Other stimuli, such as calcium ionophores, will induce the acrosome reaction, but the results are not or less related to those obtained from the zona pellucida-induced acrosome reaction

41. ACROSOME REACTION

42. Acrosome reaction tests Acrosome reactions can be detected by variety of methods like Labeling with fluorescent lectins, Monoclonal antibodies to specific proteins, Histochemical staining or by binding with antibody-bound beads. Flow cytometry also can be used to detect acrosome reacted sperm using specific fluorochrome staining.

43. Acrosome reaction tests

44. Sperm penetration assay .To assess the capacitation and acrosome reaction also known as “ZONA –FREE HAMSTER ASSAY” Zona-free hamster oocyte following the sperm penetration assay. Positive penetration is demonstrated by completely decondensed sperm heads; the tail is still visible (white arrows). All other sperm are seen attached to the surface only and are considered negative.

45. Sperm penetration test

46. Sperm zona binding tests

47. HEMI-ZONA ASSAY The zona pellucida is isolated and divided in half. one half is incubated with fertile donor sperm (positive control) and the other half is incubated with patient sperm. the ratio of fertile to donor binding is measured.

48. Hyaluronan binding assay To test the sperm capacitation in vitro, HBA is the best method

49. SPERM FUNCTION TESTS

50. Sperm DNA fragmentation tests The integrity of paternal genome delivered by the spermatozoa is of paramount importance in the initiation of viable pregnancy. The fragmented DNA delivered by the sperm is incompatible with normal embryonic development. DNA damage can occur due to various reasons DNA damage can be tested by various tests

51. Anti sperm antibodies (ASAB) ASAB in females due to repeated exposure to sperms, ASAB can be present in the cervical mucus (IgA type) or can be circulating in the blood and other body fluids (IgG type). ASAB effect the sperm oocyte interaction and fertilization To what extent the antibodies can affect fertility of sperms is still questionable

52. Anti sperm antibodies (ASAB) There are Various tests to detect the antibodies 1. Immunobead test (IB)- no commercial IB tests are available 2. Mixed antiglobulin reaction (MAR)- commercially available 3. Elisa estimation of antibodies Results of IB/MAR 10-39% of sperms reacted - suspected 40% of sperm reacted- high probability More than 50% reaction is significant cause of subfertility

53. MAR TEST Mar + ve Mar - ve

54. IMMUNOBEAD TEST

55. Sperm function tests Do we need them in this era? Many feel and believe that sperm function testing is now irrelevant due to advances in IVF, especially ICSI technique. ICSI has definitely given hope to couples who previously had virtually no chance of conception with their own gametes. Whether these tests are really needed in the era of ICSI!!! Success of ICSI had led to the cessation of research in the area of sperm function and defects.

56. Importance !!! Because sperm contribute 50% of the genetic material For better understanding of basic and applied biology of subfertility For couples without severe sperm defects we can provide better sperm function tests to ensure normal fertilization without the use of ICSI. It is essential to identify the pathology and treat the male partner which may allow the couples to improve their fertility potential and improve the ART results and also helps in conceiving naturally too. With the rapid growth in ART especially ICSI male partner is overlooked which is responsible for the half of the infertility cases

57. Conclusion The complex nature of molecular interactions between oocyte and sperm leading to fertilization needs further clarification Since there is more than one sperm function involved, no single test of a single function will have perfect sensitivity. Therefore a batteries of tests are required for diagnosis. Tests with high sensitivity, high predictive values and low false positive rates are desirable. Sperm function test should not be routine investigations as they are complex, expensive, not rigorously tested, do not always provide clinically useful information and typically do not effect treatment

58. Thanks