Semen analysis What’s new GAB 26/10/04 Ahmed Mahmoud, Frank Comhaire Center for Medical & Urological Andrology UZ Gent www.infertiliteit.net www.andrology.be

1999 2000

Male reproductive tract

Ejaculatory sequence Seminal vesicles 2-5 ml Cowper’s glands & glands of Littré ± 0.2 ml Prostate, ampulla, epididymis (+ spermatozoa) ± 0.5 ml Seminal vesicles 2-5 ml

Aims of semen analysis To discriminate between fertile semen and semen with impaired fertilizing potential To detect causal factors or mechanisms of impaired fertilizing potential. To direct treatment and predict treatment outcome

The semen sample

The semen sample By masturbation (or Special Condom e.g. Male factor Pack) After 2-7 days of sexual abstinence Within one hour of collection If 1st abnormal then 2nd sample Container: clean, dry warm (20-40°c) also during transport Plastic: test for toxicity ‘Motility’ Wide-mouthed

Sperm counting

Reusable chambers

Disposable CC

Sperm concentration Sample liquified, mix Liquefaction problem: use Bromelain (1g/l) Pipette: positive displacement (at least for viscous samples)

Mahmoud et al. Fertil Steril 1997 counted bead concentration (million/mL) Counting chambers (beads) 55 50 45 40 35 30 25 DROP DROP1 Standard Cell Cell Micro 2X JCD Makler Bürker Improved NEUBSPP† count vu vision cell cel Neubauer Range ‡ Cost x 2 Mahmoud et al. Fertil Steril 1997 DEVICE

Mahmoud et al. Fertil Steril 1997 Sperm concentration (million/ml) Counting chambers (sperm) 65 60 +SD § 55 Ok 50 Best 45 Mean § 40 35 -SD § 30 Experience ± Experience +++ 25 Cell Vision Makler Bürker Improved Neubauer NEUSMP† CONC‡ Mahmoud et al. Fertil Steril 1997 DEVICE

Sperm Motility

Sperm motility Manual Automated systems: Difficulties in quality control especially for grades A, B Accepted CV 10 % maximum Automated systems: Fully automated: Expensive, not problem-free Semi-automated (Autosperm)

The AUTOSPERM

Start A B C D E 1 2 3 4 5 Stop Press Button (4)

Sperm morphology (Anton van Leeuwenhoek)

Liberal vs Strict criteria Mahmoud A., Comhaire F. Antwerp 15/10/02

Liberal criteria Abnormal forms Defined Other Spermatozoa Normal Phase contrast x 1000+

Spontaneous pregnancy Grade a motility 42 % Spontaneous pregnancy Comhaire et al. (IJA 1987) Morphology %

Spontaneous pregnancy * * Spontaneous pregnancy Menkveld et al. (HR 2001) * *

Determinants in-vivo * Same criterion as IVF!!! Grade 6>100µm/sec!! C: Comhaire 1988, M: Menkveld 2001 Variable Sens. Spec. Criterion value Grade a % Grade (1-6) C 84 81 M 80 68 42 % 4.5 Morphology WHO C 75 72 M 75 77 30 % Morphology strict 4 %* * Same criterion as IVF!!! Grade 6>100µm/sec!!

Strict criteria & IUI pregnancy rate per cycle 30 25 20 15 10 5 p=NS n=91 n=267 n=53 Pregancy per cycle (%) 0-4%A 5-14% >14% Morphology SC (% normal) Check et al., Arch Androl, 2002

Morphology & IVF Kruger et al. 1988 Female (tubal) factor infertility Normal concentration >20 mil/ml, motility >30% Sperm morphology <14 % normal “strict criteria” Morphology of successful vs. failed IVF

a. Normal b. +/- “slightly amorphous” c. Abnormal Kruger et al. 1988 “severely amorphous” Kruger et al. 1988

Morphology & IVF Kruger et al. 1988 50 Successful R2=0.56 Failed 40 R2=0.36 30 Morphology % normal 20 R2=0.44 10 Normal Strict&WHO “Slightly amorphous” Strict=Abnormal WHO=normal Mophology index =WHO

Morphology & IVF Host et al. 1999 (Acta Obstet Gynecol Scand) 100 couples (50 tubal factor, 50 unexplained) Technician blinded WHO criteria better predictor for fertilization rate than Kruger's criteria (p<0.002)

Morphology CV%: 1 sperm misclassified Method & number Reading 1 % normal Reading 2 % normal CV % Strict 100 14 15 4.88 Strict 200 14.5 2.5 WHO 100 30 31 2.3

CV of different methods of sperm morphology More Liberal Strict 50 50 % * CV is even higher Without transformation 45 American Society Clinical Pathology 40 35 33 % 30 CV % 25 20 21 % 15 10 5 ASCP WHO Strict* * After data transformation Method Keel et al., HR 2000

I quote Rune Eliasson, Androlog mail, 16.09.02 “Classification of sperm morphology according to 'strict criteria' has been accepted by gynecologists and many others to a degree that is totally unfounded. It has become a new paradigm and will take a considerable amount of work and time to get rid of”. N. B. Kruger (1986) morphology is a modification of the methods by Eliasson !! (1971) & MacLeod (1962)

Conclusions (morphology) Use high magnification, good optics (phase contrast x1000+) Define your own normal values Morphology less important in vivo Be liberal, Use liberal criteria

Immunological infertility The role of antisperm antibodies in male infertility

Spermatozoa “loaded” with spermagglutinins stick to the glycoprotein filaments as soon as they come with contact with cervical mucus. Cervical mucus containing spermagglutinins provides the penetrating spermatozoa with the spermagglutinins and afterwards the spermatozoa stick to the glycoprotein filaments.

Diagnosing Immunological Infertility Antibodies in semen: IgG and IgA-class Antibodies in serum: agglutinating, cytotoxic (requiring complement) Current techniques in routine analysis of semen and serum

Detecting Antisperm Antibodies attached to Spermatozoa (direct tests)

Mixed Antiglobulin Reaction (MAR)

Schematic representation of the direct MAR test

Immunobead test

Detecting Antisperm Antibodies in Serum (indirect tests)

Indirect SpermMAR test

Antisperm Antibodies Immunobead SpermMAR Motility rapidly good Preparation time non S. Volume 0.5-2.0 ml 10 µ l Shelf life 1 month 1 year Sensitivity-specificity Better Price ~ X 2

2. Reactive oxygen species & WBCs

Secretory Products of the accessory Sex Glands Refining the Diagnosis of MAGI

Prostate Epididymis Seminal vesicles Citric acid Acid phosphatase Gamma-glutamyl-transferase Zinc-calcium pH (acidic) Liquefaction Seminal vesicles Semen volume pH (alkaline) Coagulum Fructose Prostaglandins Epididymis Alpha-glucosidase L-carnitine Glyceryl phosphoryl choline Antioxidants

Assessing the Function of the Epididymides

Alpha-glucosidase and sperm fertilizing potential Milingos et al. (1996) Eur.J.Obstet.Gynecol.Reprod.Biol 64, 115 In IUI more pregnancies occurred when markers of epidydimal function (Shorr stain & alpha-glucosidase) were normal (OR: 11.1, CI: 2.1-59). Criterion value: 78 mIU/ejaculate

Alpha glucosidase and sperm concentration 120 100 80 Alpha glucosidase (mU/L) 60 40 20 ND AZOOSPERMIA (n=67) OLIGOZOOSPERMIA (n=216) NORMOZOOSPERMIA (n=189) Difference between groups: p<0.05 (ANOVA)

ALPHA-GLUCOSIDASE (mU/L) Alpha glucosidase in azoospermia 30 crit <=13.5 U/L sens: 82 % spec: 70 % 25 20 ALPHA-GLUCOSIDASE (mU/L) 15 10 N D Epididymal (caput) Functional azoospermia Vasectomy (n=27) (n=5) (n=33)

Epididymis and Antioxidants Antioxidants are produced in the same region of the epididymides that produce alpha-glucosidase Semen with low alpha-glucosidase activity presents high oxidative overload

Alpha-glucosidase: correlations Gamma-GT 384 0.62 <0.001 WBCs (mill/ml) Cases > 1 mill/ml 165 -0.30 ROS 104 -0.27

Assessing the Function of the Prostate and/or the Seminal Vesicles

Localizing the affected organ(s) in MAGI

Markers of accessory sex glands in semen Prostatitis Prostato Prostato Epididymitis vesiculitis epididymitis Ejac. volume =  = = Fructose (conc.) =  = = GT/Citric acid    = -glucosidase = =   Effect on treatment: Choice of antibiotic, Use of antioxidants