The Correlation between Malondialdehyde Concentration Level ...

The Correlation between Malondialdehyde Concentration Level ...

The Correlation between Malondialdehyde Concentration Level in Seminal Fluid Analysis and Asthenozoospermia among Infertile Males Faculty of Medicine, Universitas Indonesia Debora Roselita Karo Sekali Kuala Lumpur, 10 October 2016 1 Curriculum vitae Personal Information Formal education Debora Roselita Karo Sekali 2016 present : Honours research, Bachelor

in Medical Science, Monash University 2013 2016 : Bachelor in Medicine, Universitas Indonesia 17 Kings Court, Oakleigh East, Victoria +61410357778 [email protected] [email protected] Research Clinical biochemistry / The Correlation between Malondialdehyde Concentration and Asthenozoospermia Physiology / Understanding Mechanism of Failure in Labor Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 2

The journey to the waiting egg 4000 Miles Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 3 Whats the issue? Infertility (WHO) Inability to conceive after one year regular unprotected sexual intercourse Problem in male and/ or female Is it really a problem

in developing countries? Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 4 Global Prevalence of Infertility WHO 2014 1 in 4 couples in developing countries is infertile Aitken RJ etal. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 1999. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 5 Infertility in Indonesia 20.00%

Fertile couples Infertile couples 80.00% Barten J. Screening for infertility in Indonesia. Results of examination of 863 infertile couples. NCBI. 1978 Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 6 Semen Analysis: Sperm Quality Total numbers 80% male infertility impaired sperm motility (Asthenozoospermia) Vitality and motility

10% of cases of infertile couples is unexplainable Morphology Barten J. Screening for infertility in Indonesia. Results of examination of 863 infertile couples. NCBI. 1978. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 8 Balance or Imbalance Pathological Roles: - Lipid peroxidation Physiological Roles: - Capacitation Acrosome reaction

Hyperactivation Sperm-oocyte binding - DNA damage Antioxidant Defense ROS - Apoptosis Agarwal etal. Effect of Oxidative Stress on Male Reproduction. World J Mens Health. 2014. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 9 Low Sperm Motility Asthenozoospermia WHO 1999: less than 50% are motile, OR less than 25% of

progressive motility (grade a) within 60 minutes of ejaculation Increase in ROS can be one factor of impaired sperm motility Disrupts cytoskeletal proteins + Lipid peroxidation + Malondialdehyde Intracellular ATP Aitken RJ etal. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 1999. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 11 I. PUFA II. Lipid free radical III. Lipid peroxyl radical IV. Lipid peroxyl radical Unstable

V. Malondialdehyde Grotto etal. Importance of the lipid peroxidation biomarkers and methodological aspects FORmalondialdehyde quantification. 2009. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 12 Male Infertility Semen Analysis Asthenozoospermia (motile less than 50%) Biochemical aspect (Not routine, discriminatory ranges not clearly defined) Malondialdehyde (indicator of toxic lipid peroxide) Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 14

Problem Identification The controversies and lack of studies regarding malondialdehyde concentration in seminal plasma and asthenozoospermia Research Question Is there any significant difference in the seminal plasma malondialdehyde concentration between normozoospermic and asthenozoospermic infertile males? Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 15 Hypothesis There is a significant difference in seminal plasma malondialdehyde concentration between normozoospermic and asthenozoospermic infertile males. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 16 Research Objectives General Objective

To understand about oxidative stress and its contributory factor to male infertility To provide basic information in order to choose the best prevention and management related to oxidative stress Specific Objective To evaluate the relationship between MDA and sperm parameters Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 17 Conflicting Statements MDA contributes in low sperm quality MDA has no relationship with sperm motility Naher etal:

Suleiman etal: infertile group has significantly higher concentration of malondialdehyde (MDA) in seminal fluid analysis, an indicator of toxic lipid peroxides Tavilani etal: MDA contributes in various impairments of sperm cells including sperm count, morphology, and motility There is no correlation between MDA and asthenozoospermia Jong etal: Smoking as a source of free radical

contributes in the increase of MDA but does not correlate to infertility Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 20 Research Design and Subjects Research Design Observational-analytical study Comparative case-control design Subject involved Control group : 20 Normozoospermic males Case group : 16 Asthenozoospermic males Both groups are infertile Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 24 Inclusion Criteria

Male 20-50 years old Sexual abstinence for 2-7 days New patients and have not had therapy before. Exclusion Criteria Smoke >20 cigarettes a day Hot water spa in 2 weeks before examination High fever >39oC before examination Drink alcohol more than 8 units per week. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 27

Research Methods Selection of Subjects and Collection of Samples 1. (Done by Staff in RSIA Sayyidah Andrology Laboratory): Requesting informed consent for new patients Semen samples of the patients were collected by masturbation after 4 days of sexual abstinence and examined after liquefaction for 30 min at 37 C. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 29 Research Methods Semen Analysis

2. (Done by Staff in RSIA Sayyidah Andrology Laboratory): Volume, pH, concentration, and motility were evaluated according to WHO guidelines 2010 Determine normozoospermia or asthenozoospermia Centrifuge semen for 20 min at 1600 rpm and get the seminal fluid Sample is stored in -70oc in RSIA Sayyidah Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 30 Parameter Reference value based on Reference value based on WHO 1999

WHO 2010 Volume 2.0 mL or more 1.4 - 1.7 mL Ph 7.2 or more 7.2 or more Sperm concentration 20 x 106 spermatozoa/mL or

12 16 x106 spermatozoa/Ml more Total sperm number Motility 40 x 106 spermatozoa per 33 46 x 106 spermatozoa ejaculate or more per ejaculate 50% or more motile (grades a Total motility (PR+NP): 38 - + b) or 25% or more with

42% progressive motility (grade a) Progressive motility (PR): 31 - within 60 minutes of 34% ejaculation Vitality (live 50% or more live, i.e., spermatozoa) excluding dye

55 63 % Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 31 Research Methods Measurement of Lipid Peroxide (Done in Lab of Biochemistry) Thiobarbituric acid method 3. Preparation of MDA standard by TEP TCA for precipitate protein TBA attach to MDA and read the absorbance Statistical Analysis SPSS for Windows version 20.0

4. Descriptive statistical test Saphiro-Wilk Mann-Whitney U Test Graph pad prisms Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 32 Total Samples (Period June 2015 May 2016) Samples 20 Normozoospermia 14

Asthenozoospermia Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 36 Age distribution Groups Total number Age (years) (mean + SD) p value Control (Normozoospermia)

20 34.1 + 6 0.62 (not significant) Case (Asthenozoospermia) 14 33.1 + 5 Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 37 Semen volume

Seminal plasma volume Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 38 Distribution of Sperm Concentration Groups Median (106/mL) (Range) p value Normozoospermia (n= 20) 1.5 (0.9 4) 0.65

(not significant) Asthenozoospermia (n=14) 1.8 (0.4 4.4) Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 39 Result p value = 0.488 Significantly correlated Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 41 Analysis Parameter

Normo (n = 20) Astheno (n = 14) * p value * Age (years) 34.1 + 6 33.1 + 5 p > 0.05 Semen volume (mL) 2.7 + 1 2.9 + 1.2 p > 0.05

Seminal fluid volume (mL) 1 + 0.5 1 + 0.4 p > 0.05 Sperm concentration 1.7 + 0.8 1.9 + 1.1 P > 0.05 MDA concentration (nmol/mL) 1.1 + 0.5

1.7 + 1.2 p < 0.05 * Result are presented as mean + SD Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 42 INITIAL HYPOTHESIS There is association between concentration of malondialdehyde in seminal fluid and asthenozoospermia RESULT OBTAINED There is correlation between MDA Concentration in Seminal Fluid Analysis and Asthenozoospermia. Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 43

Author Comparison with Other Similar Studies Seminal plasma MDA concentration (nmol/mL) P-value Method of measurement Normozoospermia Asthenozoospermia Sekali (2016 present study) 1.1 + 0.5 * (Infertile normozoospermics) 1.7 + 1.2 *

Significant (p = 0.048) TBA with spectrophotometry Zeba-Un-Naher (2011) Median = 1.88 (0.5 5.37) (Fertile males) Median = 3.17 (1.2 6.21) P<0.001 TBA with

spectrophotometry Colagar (2009) 0.65 + 0.17 0.94 + 0.28 P<0.001 TBA with spectrophotometry Tavilani (2005) 1.2 + 0.3 * (normal healthy men) 1.35 + 0.42 *

P<0.01 TBA method with spectrofluorometry 6.62 + 0.65 * Not significant TBA with spectrophotometry Suleiman (1996) 6.32 + 0.82 * * Result are presented as mean + SD Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 44

Discussion 1. MDA correlates to POOR SPERM QUALITY including low motility. Supported by Colagar et al and Tavilani et al Lipid peroxidation affect the membrane transport activity and the surviving of sperm Lipid peroxidation and decreasing antioxidant capacity lead to low motility, morphology, and sperm count Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 45 Discussion 2. PUFA is very susceptible to lipid peroxide and can be one of indicator of low sperm quality by damaging the sperm plasma membrane Supported by by Vriese et al and Agarwal et al: MDA is mainly attacking PUFA in sperm plasma membrane MDA correlates with the sperm motility because peroxidative damage occur in the sperm membrane and axonemal proteins which cause the permanent impairment in sperm motility

Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 46 Discussion 3. There might be no significant correlation between semen volume, sperm concentration, and seminal plasma volume Not supported by Pouramir et al stated that there is positive correlation between MDA and semen volume HOWEVER, it still needs higher samples to prove this finding and the control samples should be from fertile subjects Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 47 Limitation 1. Selection of infertile males as the control group 2. Collection of only one seminal plasma sample for each subject 3. Prolonged storage time of the samples may decrease the concentration of MDA 4. Failure to collect 20 samples for each group

Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 48 Recommendation 1. Future research with the same topic should: choose healthy, fertile men as the control group. two or more samples for each subject should be taken. pay attention to the duration of storage 2. Research regarding the MDA in spermatozoa 3. Research regarding MDA and motility in bigger population size Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 49 Acknowledgement Professor dr. Mohamad Sadikin, D. Sc. Faculty of Medicine, Universitas Indonesia, Staff in Dept. of Biochemistry and Molecular Biology,

Dr. H. Indra G. Mansur, Sp.And. Andrologists in RSIA Sayyidah and Budhi Jaya Staff in Dept. of Biology in Faculty of Medicine, Universitas Indonesia Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 50 THANK YOU Faculty of Medicine, Universitas Indonesia. Debora Roselita Karo Sekali 51

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