WHO 6TH EDITION UPDATE FOR SEMEN ANALYSIS

SEMEN ANALYSIS, ANDROLOGY, EMBRYOLOGY, CLINICAL EMBRYOLOGY, IVF

1. SEMEN ANALYSIS: WHO 6 TH EDITION UPDATE DR. DEEPTHI REPALLE IVF LAB DIRECTOR MOHAK IVF SAIMS INDORE

2. Introduction

3. Introduction

4. Introduction

5. Introduction Basic Examination

6. Basic examination Preparations-pre-examination procedures Patients information - Clear written and spoken instructions -Abstinence- 2-7 days Sample collection -Date and time -Clinic/home collection Sample reception -Collection difficulty -Complete collection Initial sample handling

7. Basic examination Time vs. examinations

8. Basic examination Macroscopic examination Appearance: cream/grey opalescent Yellowish Red-brown Liquefaction: 30-60 min Viscosity: slightly viscous Odour: strong odour of urine, putrefaction can be of clinical importance Ph: ≥ 7.2

9. Basic examination Microscopic examination Phase contrast optics Under low magnification (100x) Sperm aggregation Sperm agglutination Insufficient liquefaction Under high magnification (200x/400x) Sperm motility Dilution required Presence of other cells

10. Non-specific aggregation of spermatozoa

11. Sperm agglutination

12. Sperm motility At least 200 sperms counted in all categories. A four-category system for grading motility is recommended. Rapidly progressive ( 25 μm /s) – spermatozoa moving actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of at least 25 μm (or ½ tail length) in one second .

13. Sperm motility Slowly progressive (5 to < 25 μm /s) – spermatozoa moving actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of 5 to < 25 μm (or at least one head length to less than ½ tail length) in one second. Non-progressive (< 5 μm /s) – all other patterns of active tail movements with an absence of progression – i.e. swimming in small circles, the flagellar force displacing the head less than 5 μm (one head length), from the starting point to the end point. Immotile – no active tail movements.

14. Sperm motility

15. Sperm vitality Sperm vitality, as estimated by assessing the membrane integrity of the cells, can be determined routinely on all ejaculates, but is not necessary when at least 40% of spermatozoa are motile . In samples with poor motility, the vitality test is important to discriminate between immotile dead sperm and immotile live sperm.

16. Sperm vitality Eosin-nigrosin

17. Sperm Vitality Hypo Osmotic Swelling test

18. Sperm count The haemocytometer with improved neubauer ruling

19. Sperm count

20. Sperm count Sperm concentration per ml Total number of spermatozoa per ejaculate Formula, C=(N/n) x (1/v) x dilution factor.

21. Sperm Morphology The practical evaluation of human sperm morphology comprises the following steps: Preparing a smear of ejaculate on a slide Air-drying, fixing and staining the slide Mounting the slide with a coverslip if the slide is to be kept for a long time Examining the slide with brightfield optics at ×1000 magnification with oil immersion Assessing approximately 200 sperms.

22. Sperm Morphology

23. Sperm Morphology

24. Sperm Morphology

25. Sperm Morphology Papanicolaou plate Description

26. Sperm Morphology

27. Terminologies

28. Terminologies

29. Reference values

30. Introduction

31. Extended analysis

32. Extended analysis Indices of multiple sperm defects Sperm DNA fragmentation Genetic and genomic tests Tests related to immunology and immunological methods Assessments of interleukins Assessments of immature germ cells Testing for antibody coating of spermatozoa Biochemical assays for accessory sex glands

33. Extended analysis 1. Indices of multiple sperm defects The teratozoospermia index (TZI) The multiple anomalies index (MAI) The sperm deformity index (SDI)

34. Indices of multiple sperm defects

35. Sperm DNA fragmentation Sperm DNA fragmentation ( sDF ) is one of the most common disturbances affecting the genetic material in the form of single or double strand breaks. sDF may be triggered by different processes, including the defective packaging of the DNA during spermatogenesis, and processes of cell death and oxidative stress which may be associated with several pathological and environmental conditions.

36. Sperm DNA fragmentation The techniques of the various SDF tests ( TUNEL, Comet assays , Sperm Chromatin Dispersion test , and Acridine Orange flow cytometry ) have been described and notes on the clinical interpretation of these assays have been included. The editors have also provided suggested thresholds for clinical decision-making. Comet assay should not be used in clinical practice because of an important degree of inter-laboratory variation.

37. Sperm DNA fragmentation The application of sperm DNA tests in clinical practice remains controversial. This controversy stems in part from the modest predictive value of SDF tests in reproduction and the multitude of available tests with variable thresholds and inter-lab variability.

38. Genetic and genomic tests Clinically, there is growing awareness that chromosomal anomalies (numerical, structural, [including microdeletions and microduplications ]) and gene mutations underlie a diverse spectrum of male infertility that underlie many of the anomalies seen in a semen analysis. Sperm aneuploidy test FISH

39. Tests related to immunology and immunological methods Assessments of leukocytes in semen - Staining cellular peroxidase using ortho-toluidine - Panleukocyte (CD45) immunocytochemical staining

40. Tests related to immunology and immunological methods The consensus threshold value of 1.0×10 6 cells/ml for peroxidase -positive cells implies a higher concentration of total leukocytes, since not all leukocytes are peroxidase -positive granulocytes.

41. Assessments of interleukins Markers of male genital tract inflamation . Evaluation of chemokines and cytokines in semen may be required by andrologists and urologists to deepen the diagnostic procedure of infertile males affected by MGT inflammatory states.

42. Assessments of immature germ cells Germ cells include round spermatids and spermatocytes, but rarely spermatogonia. They can be detected in stained semen smears but may be difficult to distinguish from inflammatory cells when the cells are degenerating. The WHO laboratory manual for the examination of human semen and sperm cervical mucus interaction, fourth edition stated that > 6 million immature germ cells/ml was abnormal. This is no longer provided as a reference, as no firm evidence base could be found for this cut-off value.

43. Testing for antibody coating of the spermatozoa If spermatozoa demonstrate agglutination (i.e. motile spermatozoa stick to each other head to head, tail to tail or in a mixed way), the presence of sperm antibodies is one possible cause to be investigated. Sperm antibodies can be present without sperm agglutination; equally, agglutination can be caused by factors others than sperm antibodies. The mere presence of sperm antibodies is insufficient for a diagnosis of sperm autoimmunity.

44. Testing for antibody coating of the spermatozoa Tests for antibodies on spermatozoa (“direct tests”): Two direct tests are described here: the mixed antiglobulin reaction (MAR) test and the immunobead (IB) test. Tests for ASAB in sperm-free fluids, i.e. seminal plasma, blood serum and solubilized cervical mucus (“indirect” tests).

45. Biochemical assays for accessory sex glands Secretory capacity of the prostate . - zinc, citric acid or acid phosphatase in semen gives a reliable measure of prostate gland secretion Secretory capacity of the seminal vesicles . - Fructose in semen reflects the secretory function of the seminal vesicles. Secretory capacity of the epididymis . - L- carnitine , GPC and neutral α- glucosidase are epididymal markers used clinically.

46. Introduction

47. Advanced examination

48. Advanced examination These tests are generally for research purposes. Seminal oxidative stress and reactive oxygen species testing Assessment of acrosome reaction Assessment of sperm chromatin Transmembrane ion flux and transport in semen Computer aided sperm analysis (CASA)

49. Seminal oxidative stress and reactive oxygen species testing Reactive oxygen species (ROS) produced by leukocytes underlie their deleterious effects when present at high level in semen. Luminol Oxidation-reduction potential Total antioxidant capacity

50. Assessment of the acrosome reaction

51. Assessment of the acrosome reaction

52. Assessment of the 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. Calcium influx is believed to be an initiating event in the normal acrosome reaction. In cases of teratozoospermia and oligozoospermia, some patients may have otherwise normal results of ejaculate examination, but spermatozoa may display alterations in the acrosomal structure or in the ability to respond to stimuli of acrosome reaction

53. Assessment of sperm chromatin The stability of the sperm chromatin structure is of fundamental importance for embryo development and quality, probably due to protection of and rapid availability of the paternal genome. Aniline blue assessment Chromomycin A3 assessment

54. Transmembrane ion flux and transport in sperm Diagnostic tools to better understand male factor infertility and disorders of the male reproductive organs. Electrophysiology and kinetic Ca2+ fluorimetry to assess the function of CatSper Electrophysiological and fluorimetric methods to study the function of K+ channels Methods to detect (mal)-function of ion transporters and exchangers

55. Computer-aided sperm analysis ( CASA ) Using CASA to assess sperm motility CASA terminology VCL, velocity along the curvilinear path ( μm /s) VSL, velocity along the straight-line path ( μm /s) VAP, velocity along the average path ( μm /s) ALH, the amplitude of the lateral displacement of the head ( μm ) MAD, mean angular displacement (degrees) Automated systems may be useful for obtaining additional research data (including on morphological subpopulations of spermatozoa, plasma membrane integrity, sperm energy index) and for QC systems, but more research is needed to show their benefits for clinical purposes.

56. Significance The reference ranges described in the 5 th edition should be abandoned as they are of limited value in differentiating fertile and infertile man. Who reference ranges did not adequately reflect fertility dynamics of the male partner. 5 th percentile is commonly used as a statistical approach to determine cut-off norms in medical tests.

57. 5 th edition vs. 6 th edition

58. Decision limits Reference ranges were replaced by the decision limits in the 6 th edition. Normal Borderline Pathological

59. Decision limits Characteristics units Normal Borderline Pathological Volume ML 2-6 1.5-1.9 <1.5 Sperm concentration Million/ML ≥20 10-20 <10 Total sperm count Million/ejaculate ≥80 20-79 <20 Motility % of motile ≥60 40-59 <40 % of progressive ≥50 35-49 <35 % rapid progressive ≥25 Morphology % typical forms ≥14 4-13 <4 Vitality % of live ≥60 40-59 <40

60. Conclusions The 6th edition is a step forward in our understanding of the complex subject of male infertility through evaluation of the human ejaculate.  Several objections to the 5th edition have been addressed in the 6th edition, though areas of controversy remain. For the clinician, the most notable change is the introduction of “decision limits” rather than reference ranges, and the endorsement of SDF as an extended seminal test that can be ordered in certain clinical situations.

61. Conclusions For the laboratory personnel, the manual has been streamlined to facilitate a step-by-step examination with several old tests being abandoned, while several new tests have been introduced. The need for the extended analysis has been explained in detailed. Advanced and emerging technologies are included for research purposes.

62. HISTORY FORM

63. Thank you