Sperm Function Tests Beyond the Semen Analysis

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1 Sperm Function Tests Beyond the Semen Analysis Edmund Sabanegh, Jr., M.D. Chairman, Department of Urology Glickman Urological and Kidney Institute Cleveland Clinic

2 Routine Semen Analysis: Gap in Knowledge?

3 Evolution of WHO semen standards Parameters Volume (ml) ND Sperm count (10 6 /ml) Total sperm count (10 6 ) ND Motility (%) Progressive motility 2 25% 25% (a) 25% (a) Vitality (%) ND Morphology (%) >14* Leukocyte count (10 6 /ml) < 4.7 < 1.0 < 1.0 < 1.0 *Strict (Tygerberg) criterion

4 Evolution of WHO semen standards Parameters Volume (ml) ND Sperm count (10 6 /ml) Total sperm count (10 6 ) ND Motility (%) Progressive motility 2 25% 25% (a) 25% (a) 32% (a+b) Vitality (%) ND Morphology (%) >14* 4* Leukocyte count (10 6 /ml) < 4.7 < 1.0 < 1.0 < 1.0 < 1.0 *Strict (Tygerberg) criterion

5 Overlap Ranges of Sperm Characteristics Overlap range (cut off values) % of Overlap Lower Higher fertile infertile Count (million) Motility (%) Morphology (%) WHO Strict criteria Nallella et al, Fertil Steril 85:629-34, 2006

6 Overlap Ranges of Sperm Characteristics Overlap range (cut off values) % of Overlap Lower Higher fertile infertile Count (million) Motility (%) Morphology (%) Not helpful WHO Strict criteria Nallella et al, Fertil Steril 85:629-34, 2006

7 Overlap Ranges of Sperm Characteristics Overlap range (cut off values) % of Overlap Lower Higher fertile infertile Count (million) More helpful Motility (%) Morphology (%) WHO Strict criteria Nallella et al, Fertil Steril 85:629-34, 2006

8 Overlap Ranges of Sperm Characteristics Overlap range (cut off values) % of Overlap Lower Higher fertile infertile Count (million) Motility (%) Morphology (%) WHO Strict criteria Most helpful Nallella et al, Fertil Steril 85:629-34, 2006

9 Variability in Sperm Concentration WHO 4 th Edition, 1999

10 Count and Conception Time to conception correlates with total sperm numbers and sperm concentration Zinaman (2000)- 200 couples off contraception for 12 months Longitudinal Investigation of Fertility and Environment (LIFE) Study (2014)- 473 couples off contraception for 12 months -Select morphology (head width) and sperm count were associated with shorter time to pregnancy -Overall strict morphology and kinetic parameters were not associated with improved time to conception

11 Routine Semen Analysis: Gap in Knowledge Lacks information on sub cellular / molecular changes in spermatozoa Need for tests to evaluate/stratify interventions

12 Limitations of Current Semen Testing Poor prediction of fertility since 50% of infertile men have normal semen parameters High inter and intra observer variability Morphology in particular is poorly correlated with outcomes The dilemma Men with abnormal semen analyses can be fertile and infertile men can have normal parameters

13 Ideal Sperm Function Test 1. Specific diagnosis of sperm dysfunction 2. Prediction of fertilization/pregnancy rate 3. Suggest targeted therapy to reverse infertility

14 Sperm DNA Sperm chromatin tightly packed due to cross linkages Sperm DNA fragmentation (DFI)- first described in 1993 Correlates with poor semen parameters, leukocytospermia, elevated oxidative stress Associated with unexplained infertility, recurrent miscarriage Sperm DNA integrity and morphology- poor correlation (Avendano, 2010) DNA sperm integrity correlates with reproductive success Elevated DFI- lower spontaneous pregnancy and assisted reproduction success rates

15 Causes of Impaired DNA Integrity Age replication errors Idiopathic Exposure to xenobiotics (Lifestyle, meds, occupational) DNA Damage Exposure to radiofrequency radiation Exposure to scrotal heat stress Defects of spermatogenesis associated with poor or aberrant protamination Assisted conception IVF ICSI Normal embryogenesis Normal DNA repair Abnormal Abnormal development Failed implantation Miscarriage Abnormalities in offspring?

16 Sperm DNA Assessment Sperm karyotyping Sperm FISH analysis Sperm DNA fragmentation tests

17 Methodology Fluorescent labeling of specific chromosome (5+probes) automated image analysis Sperm FISH Analysis Aneuploidy detection noted in 7% of normal men but much higher rates with oligospermic men and those with reciprocal or Robertsonian translocations

18 Sperm FISH Analysis Ramasamy, Fert Steril, Dec 2014

19 Sperm FISH Analysis Widely accepted indications -Men with normal semen analysisunexplained infertility -Partners with recurrent pregnancy loss -Repeated IVF failure -Screening tool to counsel couples regarding potential outcomes from reproductive options Ramasamy, Fert Steril, Dec 2014

20 Sperm DNA Testing COMET Assay TUNEL Assay Sperm with fragmented DNA Sperm with intact DNA The use of the acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA C.F. Martins1,2, M.N. Dode1, S.N. Báo2 and R. Rumpf1; Gen Mol Research, 2007

21 Sperm DNA Fragmentation and Pregnancy Natural Conception 7 times more likely to achieve pregnancy if DFI <30% (n = 362, P = ) Evenson, 2006; Greco, 2005

22 Sperm DNA Fragmentation and Pregnancy Natural Conception 7 times more likely to achieve pregnancy if DFI <30% (n = 362, P = ) Intrauterine insemination 7.3 times more likely to achieve pregnancy if DFI was <30% (n = 518, P = ) Evenson, 2006; Greco, 2005

23 Sperm DNA Fragmentation and Pregnancy Natural Conception 7 times more likely to achieve pregnancy if DFI <30% (n = 362, P = ) Intrauterine insemination Bottom line- DFI>30% and reproductive failure-consider 7.3 times more likely to achieve pregnancy if DFI was <30% (n = 518, P = ) ICSI and possible testicular sperm extraction (lower DNA IVF/ICSI fragmentation in testis vs ejaculated sperm) Infertile couples - 2 times more likely to become pregnant if DFI was <30% (n = 381, P = 0.03) Controversial since paternal genome not expressed until four-cell embryo Varicocele repair prior to ICSI may improve outcomes (Esteves, 2010) Evenson, 2006; Greco, 2005

24 DNA Fragmentation and IVF/ICSI Success Meta-analysis of IVF and ICSI outcomes IVF- High DFI- lower live birth rate ICSI- High DFI- no difference in live birth rates Higher spontaneous abortion rates in IVF and ICSI with high DFI Osman,2015

25 Outcome of Pregnancies with Elevated Sperm DNA Fragmentation High DFI associated with oligospermia and advanced paternal age No effect on intrauterine growth, postnatal physicial development or mental status of offspring of high DFI sperm Elevated sperm dna fragmentation occurs regularly and the machinery of a healthy oocyte might come to the rescue Palermo, Fert Steril, 2014

26 Indications for Sperm DNA Testing Unexplained infertility Recurrent pregnancy loss Prediction of ART outcome Assessment of genetic integrity Post-chemotherapy Advanced paternal age

27 Sperm DNA Challenges Lack of standardization of assays Significant intra-individual variation Technology does not allow individual sperm assessment for ICSI and sperm separation methods are not consistently effective in separating good DNA sperm population from compromised Predictive value for ART may improve if study DFI of motile portion of ejaculate (Palermo, 2014) ASRM (2013) and AUA Best Practice (2011) guidelines- not enough data to recommend routine sperm DNA testing

28 Oxidative Stress What is it : When oxidants outnumber antioxidants Reactive oxygen species (ROS) Generated by seminal leukocytes and sperm React with lipids, proteins and nucleic acids Neutrophils Macrophage Physiological Role: Induces acrosome reaction Mediates activation and capacitation Needed for membrane fluidity Spermatozoa are susceptible to oxidative stress: High concentration of polyunsaturated fatty acids Little capacity for membrane repair Limited cytoplasmic defensive enzymes

29 Clinical Implications of ROS Potential common pathway of injury- cryptorchidism, infection, aging, environmental exposures, cancer (Ko, 2014) Varicocele Increased ROS levels in serum, testes, semen (Sabanegh, 2009) Varicocelectomy increases seminal antioxidant levels and improves bulk semen parameters (Mostafa, 2001) Tobacco Smoking associated with poorer sperm quality (Kunzle, 2003; Raghed, 2009) Infertile smokers- higher levels of seminal oxidative stress than infertile nonsmokers (Saleh, 2002) Smoking cessation- improve semen quality, lowers ROS levels

30 Antioxidant therapy Scavenger: vitamins E and C, lycopene, carnitine, etc. Fertility outcomes Vit E+C supplementation- decreased semen ROS (DeFoor, 2004) Vit C ( mg/day)- increased sperm counts in infertile males (Dawson, 1987) Randomized crossover study of Vit E (600IU q day)-improved zona binding

31 Emerging Technologies Microarray Analyzes transcriptome of cells- comparison at different stages of spermatogenesis Proteomics Seminal fluid has >1000 different proteins and at least 20 have had altered expression in infertile men (Pilch,2006; Pixton, 2004) Genetics and Epigenetics Spermatozoal epigenetic disorders (Boissonnas, 2013; Gannon, 2014; Klaver, 2014) RNA defects (De Mateo, 2014; Jodar, 2013)

32 Proteomics and Male Infertility Study Subjects Sample size Source Technique Main finding Reference Varicocele Varicocele Control (n = 20) (n=20) Spermatozoa 2 DE; MALDI-TOF MS 15 proteins; 4 reduced in varicocele group; 11 overexpressed ; HSP60 and HSP 90 upregulated in varicocele group Chan et al Varicocele Pre-varicocelectomy Post-varicocelectomy (n = 18) Seminal plasma LC/MS 316 proteins; 53 in pre- and 38 in post varicocelectomy; 68 in both groups Camargo et al Oxidative stress Controls Infertile men ROS + and ROS - ( n = 20) ( n = 32) spermatozoa 2-DIGE; LC-MS 1,343 spots in ROS and 1,265 spots in ROS+ samples. 31 spots differentially expressed; 6 spots increased and 25 decreased in ROS- samples. Hamada et al Oxidative stress Controls Infertile men ROS + and ROS - ( n = 20) ( n = 32) Seminal fluid LC-MS 14 proteins; 7 common and 7 unique in both ROS+ and ROS grp. Sharma et al Idiopathic asthenozoospermia Donors Patients (n = 30) (n= 30) 2DE; Tandem mass spectrometry; MALDI-TOF 33 DEP; 15 unique proteins Shen et al Sperm motility proteins Fertile men Asthenozoospermic men (n = 8) (n = 8) spermatozoa 2 DE; MALDI-TOF 14 spots; 10 proteins; Energy metabolism proteins Zhao et al NOA Control NOA Post vasectomy (n =5) (n =5) (n = 5) Seminal plasma Spectral counts 2048 proteins in NOA; Batruch et al Asthenozoospermia Controls Asthenozoospermic men (n = 10) ( n= 20) Spermatozoa 2 DE; 17 protein spots; 8 decreased; 9 increased in asthenozoospermic men Martınez-Heredia et al Asthenozoospermia Controls Asthenozoospermic men (n = 20) (n = 38) Seminal plasma SDS PAGE; LC- MS/MS 741 proteins; 45 upregulated; 56 downregulateddj-11 downregulated in asthenozoospermic gp. Wang et al DE = 2 dimensional gel electrophoresis; MALDI-TOF = matrix assisted laser; LC-MS liquid chromatography mass spectrometry; ROS = reactive oxygen species; 2-DIGE = 2 dimensional in gel electrophoresis

33 Complexity of Proteomics Sharma, 2015

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