1. health and fitness
2. disease
3. infertility

Republic of Iraq
Ministry of Higher Education
And Scientific Research
University of Baghdad
College of Science
Effect of Gonadotropins on Sperm
Characters of infertile patients in vitro Using
SMART Culture Media
A thesis
Submitted to the College of Science, University of Baghdad, in Partial
fulfillment of the Requirements for the Degree of Master of Science in
Biology / Zoology
By
Rasha Makki Mohammed Ali
B.Sc. in Biology (2011)
University of Baghdad
Supervised by
Dr. Sabah N. Alwachi
Dr. Muhammad-Baqir M-R.
Fakhrildin
Professor
1435/Rabee Alawal
Professor
2014/ January
‫َحمهِ الرحِيم‬
‫ِسمِهللا الر‬
‫ب‬
‫ِ‬
‫ِه‬
‫َم‬
‫ِوسَان‬
‫َا اإل‬
‫ْى‬
‫َق‬
‫وا خَل‬
‫إَّ‬
‫ِ‬
‫ِيً‬
‫َل‬
‫وبْت‬
‫َة أ‬
‫وطْف‬
‫مشَاج َّ‬
‫ُّ‬
‫َْ‬
‫ًا‬
‫ِيع‬
‫َاي سَم‬
‫ْى‬
‫َل‬
‫َجَع‬
‫ف‬
‫َاي‬
‫يى‬
‫ًا إ‬
‫ِير‬
‫بص‬
‫َِّ‬
‫ٌَ‬
‫دْ‬
‫َ‬
‫وا َ‬
‫ًا‬
‫ِر‬
‫ما شَاك‬
‫َإ‬
‫السَّبِيل‬
‫َِّ‬
‫ًا‬
‫َفور‬
‫ما ك‬
‫َإ‬
‫و‬
‫َِّ‬
‫َظيم‬
‫َ هللا العلي الع‬
‫دق‬
‫ص‬
‫ََ‬
‫سورة اإلنسان‬
‫اآلية ‪ 2‬و‪3‬‬
Supervisor Declaration
We declare that this thesis was prepared under our supervision at the
Department of Biology/College of Science/ University of Baghdad, as a partial
fulfillment of the requirements for the degree of Master of Science (M.Sc) in
Zoology.
Name: Dr.Sabah N. Alwachi
Name: Dr. Muhammad-Baqir
Title : Professor
M-R. Fakhrildin
Title :
/ / 2014
Professor
/ / 2014
In view of the available recommendations, I forward this thesis for debate by
the examining committee.
Dr. Sabah N. Alwachi
Professor
Head of Department of Biology
College of Science
University of Baghdad
/ / 2014
Committee's Certification
We are the examining committee, certify that we have read this thesis entitled "
Effect of Gonadotropins Addition to SMART Culture Medium on Human
Sperm Parameters and Chromatin Structure Assay during in vitro Sperm
Activation" and have examined the student Rasha makei mohammed ali in it's
content and that in our opinion it is adequate for Degree of Master of Science
(M.Sc.) in Zoology.
Dr. Waleed H. Yousif
Chairman (Professor)
/ / 2014
Dr. Noori M. Luiabi
Member (Assist.Professor)
/
/ 2014
Dr. Sabah N. Alwachi
Dr. Jabaar H. Yanzeel
Member (Assist.Professor)
/
/ 2014
Dr. Muhammad-Baqir
M-R. Fakhrildin
Professor
Professor
/
/ 2014
/
/ 2014
Approved by the College of Committee of graduate studies.
Prof. Dr. Saleh M. Ali
Dean of College of Science
University of Baghdad
/
/ 2014
Dedication
To…
those who inspire and support me
My mother, father, sisters, brother and
fiancé
…..
To all teachers and colleagues for
their support.
 First of all, my praise is to my God "Allah" for giving the persistence to
accomplish my study and helping in all steps of my life.
 I would like to express my grateful thanks and gratitude to my supervisors Prof.
Dr. Muhammad Baqir M-R Fakhrildin and Prof. Dr. Sabah N. Alwachi for
suggesting this work plan, their patience, continuous support, guidance,
encouragement and advice throughout the whole work.
 I would like also express my sincere gratitude & appreciation to the department
of Biology, Collage of science in university Baghdad and the her staff for their
support and cooperation.
 I owe a special love and thanks to my loving family especially my mother for
their great help and support, also, my thanks and appreciation to my fiancé for his
support.
 I also, express my thanks and respect to Dr. Jabbar, brother hussein faleh, my
friends and all colleagues and people I forget to mention their names for their
support.
Finally, to all who wish me a success, my great thanks.
Rasha
Abstract
Semen analysis is considered one of the important examination to diagnoses
male infertility, which involve macroscopic and microscopic examination.
Gonadotropins are glycoprotein secreted by gonadotropes of the anterior lobe of
pituitary gland, which
include
the
follicle-stimulating
hormone
(FSH),
and luteinizing hormone (LH). These hormones are central to the complex
endocrine system that regulates growth, sexual development, and reproductive
function.
This study was conducted to investigate the effect Gonadotropins (FSH &LH)
either each alone or in combination, supplied to culture medium on human sperm
parameters and sperm DNA structure during in vitro sperm activation.
Ninety subjects employed in this study, with age ranges (22-54) years. The
subjects were divided into three major groups according to supplementation of
SMART medium with either FSH or LH or in combination. Semen specimen was
collected from each subject, then semen sample were analysed and DNA
fragmentation assay pre- and post- in vitro sperm activation. For sperm activation,
semen sample was divided into three aliquots and centrifuged at 2500 rpm for 6
minutes, Then, each one of three groups (FSH, LH and Gn) were prepared
including G1 (control group; SMART medium only), G2 (SMART medium
enriched with 0.25 IU hormones) and G3 (SMART medium enriched with 0.5 IU
hormone).
The results showed, that the sperm motility percentage, progressive motility,
normal sperm morphology and sperm DNA fragmentation index (DFI) were
significantly (P<0.05) enhanced for both treated groups compared with control
group (post-sperm activation in vitro) and pre-activation group. Using 0.5IU
I
hormones (FSH, LH, and Gn) within SMART medium (G3) showed a significant
(P<0.05) enhancement in sperm parameters post-sperm activation in vitro as
compared to G2 group (0.25 IU hormones). A significant (P<0.05) in Post-sperm
activation was shown in vitro, in the sperm DNA fragmentation index (DFI) when
using SMART medium enrich with 0.5 IU hormones as compared to 0.25 IU.
It was also found that the addition of high dose of hormone (FSH) to the
culture medium enhances the sperm motility (%) and high concentration of Gn
enhanced the DNA fragmentation.
From the results it can be concluded that the addition of gonadotropins
hormones to the culture media enhanced the sperm parameters especially the high
concentration during in vitro sperm activation, and the high concentration enhance
the DNA fragmentation.
II
List of Contents
Subject
Abstract
List of Contents
List of abbreviations
List of image
List of tables
List of figures
Chapter One: Introduction & Review of Literature
1.1. Introduction
1.2. Literature review
1.2.1. Semen Analysis S.A. Overview
1.2.2. Macroscopic Examination
1.2.2.1 Semen volume
1.2.2.2. Semen Liquefaction Time
1.2.2.3 Semen viscosity
1.2.2.4 Semen PH
1.2.3. Microscopic Examination
1.2.3.1 Sperm motility and grad activity
1.2.3.2 Sperm concentration
1.2.3.3 Sperm morphology
1.2.3.4. Sperm agglutination
1.2.3.5. Round cell count
1.2.4. Correlation between SA and rates of Fertilization and
pregnancy
1.2.5. Gonadotropins , Gn Overview
1.2.5.1. Secretion of Gn
1.2.5.2. Biological action of Gn
1.2.5.3. Role of Gn in Assisted reproductive technology
1.2.6. culture medium CM, Overview
1.2.6.1. Basic and special component of CM
1.2.6.2. Biomedical importance of CM
1.2.6.3. In vitro sperm activation and CM
1.2.6.4. Role of ISA in ART
Page
I
III
V
VI
VII
VIII
1-25
1
3
3
4
4
4
5
5
6
6
8
9
10
11
11
12
13
15
17
17
18
19
20
21
III
List of Contents
1.2.6.5 correlation between ISA and rates of Fertilization
and pregnancy
1.2.7. Sperm chromatin structure assay SCSA, Overview
1.2.7.1. Techniques of HSCSA
1.2.7.2. Factors affecting HSCSA
1.2.7.3. Correlation between HSCSA and ART
Chapter Two: Materials and Methods
2.1 Subjects
2.2 Material and equipments
2.3 Preparation of gonadotropins Gn
2.4 Preparation of culture medium
2.5 Semen analysis
2.5.1 Macroscopic examination
2.5.1.1 Semen Liquefaction time
2.5.1.2 Semen PH
2.5.1.3 Semen viscosity
2.5.2 Microscopic examination
2.5.2.1 Sperm motility and grad activity
2.5.2.2 Sperm concentration
2.5.2.3 Sperm morphology
2.5.2.4 Sperm agglutination
2.5.2.5 Round cell count
2.6. Assessment of DNA Fragmentation index DFi
2.7. Experimental design
2.8. Statically analysis
Chapter Three: The Results
3.1.Follicle stimulating hormone
3.2. Luteinizing hormone
3.3.Gonadotropins
4.4.Compartive study among hormone
Chapter Four: Discussion
Conclusion and Recommendation
References
21
22
24
25
26
27-37
27
27
27
28
29
31
31
31
31
31
32
33
33
34
34
34
35
37
38-63
38
44
50
57
63-68
69
70-97
IV
List of Abbreviations
AO
Acridine orange
AMP
Adenosine mono phosphate
ART
Assisted reproductive technologies
ASA
Anti-sperm antibody
CG
Chorionic gonadotropin
CM
Culture medium
DNA
Deoxyribonucleic acid
DFI
DNA fragmentation index
ER
Endoplasmic reticulum
FSH
Follicle-stimulating hormone
Gn
Gonadotropins
GnRH
Gonadotropin releasing hormone
HPF
High power field
HSA
Human serum albumin
HSCSA
Human sperm chromatin structure assay
ICSI
Intracytoplasmic sperm injection
IUI
Intrauterine insemination
IVSA
IVF
in-vitro sperm activation
in-vitro fertilization
LH
Luteinizing hormone
NP
Non-progressive
PH
Power hydrogen
PR
Progressive motility
ROS
Reactive oxygen species
SA
Semen analysis
SATs
Semen analysis techniques
SMART
Simple media of ART
TSH
Thyroid stimulating hormone
WHO
World health organization
V
List of images
No. of
tables
(3-1)
No. of
Page
sperm head under magnification power of (x100) of oil 56
Title
immersion displaying green fluorescence as normal with intact
DNA.
(3-2)
Sperms head under (x40) HPF displaying normal and
56
abnormal sperm.
VI
List of Tables
No. of
tables
2-1
Title
Equipment and tools utilized in the present study.
No. of
Page
28
2-2
The chemicals used in the present study.
28
2-3
Material and amount preparation culture media
29
2-4
Normal reference limits for semen characteristics according to
30
WHO criteria (2010):
3-1
Percentage of sperm motility pre- and post- activation using
41
SMART medium enriched with two concentrations
of FSH.
3-2
Percentage of sperm motility pre- and post-activation using
46
SMART medium enriched with two concentrations
of LH.
3-3
Percentage of sperm motility pre- and post- activation using
52
SMART medium enriched with two concentrations
of Gonadotropins.
3-4
Assessment of sperm parameters among groups of pre-
59
activation.
3-5
Assessment of sperm parameters when comparing among
60
control groups of post- activation.
3-6
Assessment of sperm parameters among low concentration of
61
FSH, LH and Gn groups of post- activation.
3-7
Assessment of sperm parameters among high concentration of
62
FSH, LH and Gn groups of post- activation.
VII
List of Figures
No. of
Figures
1-1
No.
of
Page
16
Title
Hypothalamic-pituitary-gonadal axis in mammals (Gilbert
2010) (Adapted from Scott F. Gilbert 2010).
2-1
Experimental design
36
3-1
Sperm concentration pre- and post-activation using SMART
40
medium enriched with two concentrations of FSH.
3-2
42
Percentage of sperm morphology pre- and post- activation
using SMART medium enriched with two concentrations of
FSH.
3-3
43
Percentage of sperm DNA fragmentation pre- and postactivation using SMART medium enriched with two
concentrations of FSH.
3-4
Effect Percentage of sperm concentration pre- and post-
45
activation using SMART medium enriched supplied with two
concentrations of LH.
3-5
Percentage of normal sperm morphology pre- and postactivation
using
SMART
medium
enriched
with
48
two
concentrations of LH.
3-6
Percentage of sperm DNA fragmentation pre- and postactivation
using
SMART
medium
enriched
with
49
two
concentrations of LH.
VII
3-7
Percentage of sperm concentration pre- and post-activation
51
using SMART medium enriched with two concentrations of
gonadotropins hormone.
3-8
Percentage of normal sperm morphology activation using
54
SMART medium enriched with two concentrations of
gonadotropins hormone.
3-9
Percentage of sperm DNA fragmentation pre- and postactivation
using
SMART
medium
enriched
with
55
two
concentrations of gonadotropins hormone.
VIII
Chapter one
Introduction
And
Review of literature
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Chapter One
Introduction & Review of Literature
1.1. Introduction
The general definition of infertility is a lesser capacity to conceive
than the mean capacity of the general population (ESHRE, 2000).
Primary infertility is the term used in reproductive medicine for a couple
who failed to achieve a pregnancy for one year of marriage and who was
never pregnant before, while secondary infertility is the term applied to
couple who meet criteria for primary infertility but at some time in the
past have been pregnant (Lunenfeld and Steirteghem, 2004).
Male infertile patients are often classified as oligozoospermic,
asthenozoospermic, or teratozoospermic on the basis of concentration,
motility, and morphology or any of these combination (Agarwal et al.,
2003). Semen analysis is the first tool a medical practitioner uses to
assess the male factor in an infertility workup (WHO, 1999;Agarwal and
Sharma, 2007). Semen analysis is routinely used to predict fertility, the
standard measurements of sperm concentration, percentage motility and
morphology may not reveal sperm defects affecting the integrity of the
male genome. It is clear that abnormalities in the male genome
characterized by damaged Deoxyribonucleic acid (DNA) may be
indication of male subfertility regardless of the routine semen parameters
(Aitken and Krausz, 2001).
There are several methods to determine sperm DNA damage
including, for instance, the sperm chromatin structure assay (SCSA)
(Rybar et al., 2004), terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL) (Martins et al., 2007), comet assay (Fraser and
Strzezek, 2004), and acridine orange (AO) staining (Thuwanut et al.,
2008).
1
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
The synthesis and secretion of the gonadotropic hormones involves
coordination of signal transduction, gene expression, protein translation,
post translation folding and modification and finally secretion (Bousfield
and Dias, 2011). The principal physiological functions of LH receptor are
found in its actions on Leydig cells of the testes to secrete testosterone
(Ascoli et al., 2002, Menon et al., 2004).Follicle stimulating hormone
(FSH) primarily stimulates the growth and development of spermatogenic
tissue (Odedl and Swerdloff, 1968). Therefore, the aims of the study
were:
1- To investigate the effect of (FSH, LH and Gn) supplied to culture
medium on sperm parameters and DNA structure during in vitro
activation.
2- To compare effect three supplementation (FSH, LH and Gn) on
sperm parameters and sperm DNA structure.
2
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
1.2. Review of literature
1.2.1. Semen analysis (SA), Overview:
Semen analysis (SA) is considered to be a diagnostic cornerstone
when evaluating male fertility. Analysis of semen can give us information
about problems in the genital organs of the male; and also can be used to
focus on the continued infertility investigation (NAFA and ESHRESIGA, 2002).
The most basic element of SA, was reports to be the functioning of
the testicular machinery for sperm production and the fluid volume
contributed by the accessory glands (Lingappa and Farey, 2000). The
production and packaging from spermatogonia to spermatozoa in the
testes is critically important because without that functional process there
are no cells and no fertility (Niederberger , 2011). The activation of
motility of epididymal spermatozoa would not take place unless the
spermatozoa were mixed with male accessory gland secretions at the time
of ejaculation, or diluted into a buffer solution containing activating
factor (Morton et al., 1974; Si, 1993).
Analysis of an ejaculate, obtained with masturbation, should be
at the laboratory 30 minutes after ejaculation. Also, special rooms should
be provided for sample collection. Before sample collection, the WHO
manual recommends a maximum interval of abstinence between 2- 7
days, but the interval should be “as constant as possible”. Standardization
of "abstinence time” of 3-4 days is strongly advised (NAFA and ESHRESIGA, 2002). The first portion of the ejaculate, about 5% of it, is made up
of secretion from the Cowper (bulb urethral) and Littre gland. The second
portion derives from the prostate and contributes from 15% to 30% of the
ejaculate. There follow small contributions of the ampulla and epididymis
and, finally, of the seminal vesicle, which contribute the remainder, and
3
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
majority, of the ejaculate (Owen and Katz, 2005). The specimens were
placed in an incubator at 37 ˚C for 30 minutes to allow liquefaction
(NAFA and ESHRE-SIGA, 2002).
1.2.2. Macroscopic examination
The liquefied semen is carefully mixed for few second, and then the
specimen was examined in detail by macroscopic and microscopic
examination within one hour of collection (Baltimore and Alabama,
2004). Macroscopic examination included semen volume, acidity (pH),
liquefaction time, viscosity, colour and odour (Agostini and Lucas,2003).
1.2.2.1. Semen volume
Semen volume normally is in the range between 1.5 - 5 mL and
regarded as an essential part of any semen analysis (WHO, 2010). Semen
volume are in positive relationship with time since last ejaculation and
the dependence of prostate and seminal vesicle fluid secretion on
androgen exposure (Ng et al., 2004). The semen volume was recorded as
hypovolmic if the volume was less than 1mL or hypervolmic if the
volume was more than 6mL (Comhaire et al., 1995).
1.2.2.2. Semen liquefaction time
The freshly ejaculated specimen is a coagulum that should liquefy in
30 minutes, lysozyme and α- amylase from the prostate liquefy the
coagulum, which is produced from the seminal vesicles (Alexander,
1982). The major structural component of human semen coagulum has
been originating from seminal vesicle secretion protein, known as
semenogelin (Yoshida et al., 2003). Proteinase secreted by the prostate is
responsible for semen liquefaction. Several proteases, including prostate4
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
specific antigen and plasminogen activators, play a role in semen
liquefaction. Abnormal liquefaction may be caused by prostatic
abnormalities, e.g. prostatitis (Saunders, 1998).
1.2.2.3. Semen viscosity
After liquefaction, semen viscosity is measured and should not show
evidence of stranding (Turek, 2000). The viscosity, also called
consistency, of the liquefied sample can be estimated by gentle aspiration
into a 5-mL pipette and than allowing the semen to drop by gravity and
observing the length of the thread formed (Rrumbullaku and Agostini,
2003). If the droplets formed threads that were more than 2 cm in length
the sample was considered to express increased viscosity (WHO, 1999).
Increased viscosity may affect sperm motility (WHO, 1992). Abnormally
high viscosity often associated with the presence of anti-sperm antibodies
(ASA) and high percentage of particulate debris (Moulik et al., 1989).
Increased consistency may be related to prostate dysfunction resulting
from chronic inflammation. High viscosity, combined with poor sperm
motility, can lead to a marked decrease in fertilization capacity, due to
problems with sperm release into the cervical mucus (Moulik et al., 1989;
Mortimer, 1997).
1.2.2.4. Semen pH
Normal semen reference pH range is 7.2 to 7.8 (WHO, 1999;
Brunzel, 2004). Variations in volume and low pH may be due to
congenital abnormalities of the genital tract or obstruction. A pH of 8.0
or greater can be associated with infection of the prostate, seminal vesicle
or epididymis (Fink , 2006). Acidic secretion of the prostate and alkaline
secretions of the seminal vesicles determines the pH. The spermatozoa
5
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
can be affected by change in pH. If the pH exceeds 8.0, infection should
be suspected. In acute infection (prostat, seminal vesicles or epididymis)
the seminal pH will be greater than 8.0 when measured soon after
liquefaction. In case of obstruction of the ejaculatory ducts or when only
prostatic fluid are secreted the pH is usually less than 7.0 (Agostini and
Lucas, 2003; Gilbert, 2006; Johnson, 2006). Abnormal pH may be
recorded in cases of incomplete ejaculation and decrease acidic pH(<6.5)
is found in cases of agenesis (or occlusion) of the seminal vesicles
(Agostini and Lucas, 2003).
1.2.3.1. Microscopic Examination
Microscopic examination is to assess sperm concentration, total
sperm count, sperm motility and grading motility, sperm morphology,
sperm vitality, sperm agglutination and round cells count. Semen analysis
includes the examination of spermatozoa, other cells present in semen
and seminal fluid (Agostini and Lucas, 2003).
1.2.3.1. Sperm motility and grade activity
Motility refers to the number (in percent) of sperm that have flagellar
motion (Agostini and Lucas, 2003). The motility should be evaluated
after the semen had liquefied and within 1hour and ideally within the first
30 minutes of collection. Sample that remained viscous, were liquefied by
mechanical pipetting with a large-bore disposable pipette (Doris, 2000;
Chia, 2001). Sperm motility is essential for normal fertilization and is
currently the most common parameter of "sperm quality", acting as an
indirect measure of metabolic activity and sperm viability (Berlinguer,
2009).
6
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A major change in the new WHO (2010) manual is in the evaluation
and categorisation of sperm motility. It is now recommended that
spermatozoa should be categorized as progressively motile, nonprogressively motile and immotile (instead of grade a, b, c or d).
Technicians often found this method difficult to define the forward
progression so accurately without bias (Cooper and Yeung, 2006).
Abandoning the distinction between fast- and slow-progressing
spermatozoa may well be regarded as a backward step ( Bjo¨ rndahl,
2010; Eliasson, 2010). The experience indicates that the technician‟s
ability to distinguish between fast- (previously grade a) and slow(previously grade b) moving spermatozoa is poor, making internal and
external quality control difficult. The decision to reject categorizing
progressive spermatozoa into fast and slow in the WHO (2010) manual
was also based on the inability of technicians to gauge velocities
accurately and the manual suggests that if velocities need to be known, a
computer assisted sperm analyser system should be employed
(Handelsman and Cooper, 2010).
The WHO manual (2010) recommends the use of a simple system for
grading motility which distinguishes spermatozoa with progressive or
non-progressive motility from those that are immotile. The motility of
each spermatozoon is graded as follows:
Progressive motility (PR): spermatozoa moving actively, either linearly
or in a large circle, regardless of speed;
Non-progressive motility (NP): all other patterns of motility with an
absence of progression, i.e., swimming in small circles, the flagellar
Force hardly displacing the head, or when only a flagellar beat can be
observed;
Immotility (IM): no movement.
The percentage of progressively motile sperm is associated with
7
Chapter one
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higher pregnancy rates (Jouannet et al., 1988; Larson et al., 2000 and
Zinaman et al., 2000).
Sperm motility depends greatly on the energetic status of the cell.
Since the mitochondria of the sperm mid piece generate energy to support
movements as a good indicator of functional sperm impairment (Pena et
al., 2003). Also, the sperm DNA fragmentation affects sperm motility and
fertilization rates (Huang et al., 2005).
Sperm motility gives a measure of the integrity of the sperm
axoneme and tail structures as well as the metabolic machinery of the
mitochondria, while sperm morphology is a surrogate measure of the
integrity of DNA packaging and the quality of spermatogenesis (Pacey,
2006).
1.2.3.2. Sperm concentration
The sperm concentration is basic parameters for assessing male
fertility, and there have been many calls for global standardization of this
test (Lu et al., 2007).
The terms „total sperm number‟ and „sperm
concentration‟ describe different concepts. Sperm concentration refers to
the number of spermatozoa per unit volume of semen and is a function of
the number of spermatozoa emitted and the volume of fluid diluting them.
However, total sperm number refers to the total number of spermatozoa
in the entire ejaculate and is obtained by multiplying the sperm
concentration by the semen volume (WHO, 2010).
The total sperm number per ejaculate is recommended as a parameter
that provides information on testicular capacity to produce spermatozoa
(Duran et al., 2002). The total number of spermatozoa per ejaculate
reflects the spermatogenesis and is related to the time of sexual
abstinence before collection. In normal situation spermatogenesis is
considered to be a constant process over time and therefore the total
8
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number of sperm per ejaculate should increase with abstinence time
(Agostini and Lucas, 2003).
The finding of no sperm in the ejaculate (called azoospermia)
suggests either an absence of sperm production or obstruction to sperm
outflow (Rrumbullaku and Agostini, 2003). For normal ejaculates, when
the male tract is unobstructed and the abstinence time short, the total
number of spermatozoa in the ejaculate is correlated with testicular
volume and thus is a measure of the capability of the testes to produce
spermatozoa (Andersen et al., 2000) .
1.2.3.3. Sperm morphology
Assessment of sperm morphology as a parameter of semen analysis
is one of the most important steps in the evaluation of male partner in
infertile couples (Cipak et al., 2009). The head should be 4–5 mm in
length and 2.5–3 mm in width. The total length-to-width ratio should be
1.5–1.75. Additionally, there should be a well-defined acrosomal region
comprising 40–70% of the head area (Menkveld et al., 1990; WHO,
2010). The mid-piece should be slender, less than 1 mm in width, about
one and a half times the length of the head, and attached axially to the
head. The tail should be straight, uniform, and thinner than the mid-piece,
uncoiled and approximately 45 mm long (Menkveld et al., 1990; Cipak et
al., 2009). Physical sperm aberrations may occur during the production of
sperm or during storage in the epididymus. The increased number of
immature spermatozoa may be due to epididymal dysfunction or is a
consequence of frequent ejaculations (Rrumbullaku and Agostini, 2003).
Whatever the cause of abnormal sperm morphology, the sperm head
defects may be markers for other sperm defects that significantly impair
fertility. Sperm nucleus defects have been associated with infertility. A
common consequence of total teratozoospermia is the failure of
9
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
fertilization. Abnormal sperm head reflects abnormality in
spermatogenesis (Demir et al., 1997). Even if sperm morphology is done
correctly and with care, with strict application of the guidelines as
outlined in the WHO manual (2010), the sperm morphology
measurements are still have a very important role to play in the clinical
evaluation of male fertility potential (Menkveld et al., 2011).
1.2.3.4. Sperm agglutination
Agglutination specifically refers to motile spermatozoa sticking to
each other, head to head, tail to tail, or in a mixed way. The motility is
often vigorous with a frantic shaking motion, but sometime the
spermatozoa are so agglutinated that their motion can be limited (WHO,
2010).The increase in the percentage of sperm agglutination can
negatively correlated with sperm motility and grade of activity and
associated with decrease in fertilizing ability of sperm (Zavos et al.,
1998). The implication of anti-sperm antibody (ASA) in clinical
infertility and the ability of ASA to affect sperm function by causing
sperm agglutination and/or immobilization are increased. The anti-sperm
monoclonal antibody were confirmed to agglutinate human spermatozoa,
inhibit sperm penetration of cervical mucus, and inhibit sperm-zone
pellucid binding (Diekman et al., 1997; Yakirevich and Noat, 2000).
1.2.3.5. Round cell count
The round cells observed samples could be either of spermatogenic
origin or varying types of cell non-spermatogenic origin such as epithelial
cell, as well as some leukocytes, are usually present in every semen
sample. In the routine semen analysis, using the Papanicolaou technique
as staining method, the differentiation of these so called round cells into
11
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either spermatogenic cells or leukocytes is sometimes difficult.
Degenerated spermatids having two or more pyknotic nuclei can easily be
falsely diagnosed as polymorphonuclear leukocytes (WHO, 1987, 1992,
1999). The presence of increased number of leukocytes may, however, be
associated with an inflammatory reaction of the male genital tract (Zalata
et al., 1995) and the possibility that the presence of the leukocytes in the
semen interferes with the fertilizing ability of the spermatozoa cannot be
excluded (Sukcharoen et al., 1995).
1.2.4. Correlation between SA and rates of Fertilization and
pregnancy
Few studies performed to date have not produced consistent
relationships between SA results and pregnancy rates, in either subfertile
or general populations. For example, in a study on 1367 subfertile
couples of pregnancy rate, together with duration of infertility (Baker,
2001).
Some of the factors which could be involved in the abnormality of
sperm motility and morphology are changes in seminal plasma osmolarity
(Cohen et al., 1985). In addition to this, the presence of human anti-sperm
anti-bodies in the seminal plasma (autoimmunity) (Hargreave and Eiton
1982),or in the cervical or uterine fluids of the women, could play a
significant role in male infertility (Carson et al.,1988).
1.2.5. Gonadotropins (Gn), Overview:
Reproductive function in mammals is regulated by the pituitary
gonadotropins luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) (Burger et al., 2004), which provide the one-two punch
that drives gamete and gonadal hormone production. In females, FSH
11
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stimulates follicular growth and maturation while LH triggers ovulation
and luteinization (Fevold et al., 1931). It has been demonstrated that LH
binds specifically to Leydig cells, where it stimulates cyclic adenosine
monophosphate (CAMP) accumulation and the conversion of cholesterol
to pregnenolone leading to increased formation of testosterone, the major
testicular steroid product (Dufau et al., 1977). FSH binds to Sertoli cells
and spermatogonia within the seminiferous tubules
(Orth and
Christensen, 1978). Binding of FSH to Sertoli cells is followed by cyclic
AMP accumulation, protein kinase activation, and androgen binding
protein production (Means et al., 1976). FSH also stimulates the
conversion of testosterone to oestradiol by Sertoli cells (Dorrington and
Armstrong, 1974).
The hypothalamic-pituitary-gonadal axis is already functional during
fetal life: In the human fetus, LH and FSH are detectable in pituitary
tissue at the fifth week of gestation (Siler-Khodr et al., 1974). Plasma
gonadotropin levels gradually increase, until they reach maximum levels
at 20 weeks of gestational age. Thereafter, the plasma levels decrease to
very low levels at term, probably due to the development of the negative
feedback mechanism by sex steroids and maternal steroids (Gluckman et
al, 1983).
During puberty, the first hormonal phenomenon is an increase of
serum LH during the night, followed by a pulsatile secretion during the
day, with a distinct sleep-wake pattern ( Wu et al., 1991; Apter et al.,
1993). In boys, the nocturnal rise in LH levels is associated with
nocturnal testosterone secretion, which occurs about 60-90 minutes after
the first high-amplitude LH pulse of the night (Boyar et al., 1974). In
girls, the rise in serum oestradiol occurs the next morning (Boyar et al.,
1976; Goji, 1993).With the progression of puberty the LH secretion
12
Chapter one
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‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
gradually increases in pulse amplitude (Wennink et al., 1988; Wu et al.,
1991; Dunkel et al., 1992; Apter et al., 1993).
FSH is memder of the glycoprotein hormone family, which also
includes thyroid stimulating hormone (TSH), LH, and chorionic
gonadotropin (CG)( Bousfield et al.,1994). Of these members, FSH, LH
and CG are gonadotropins. While TSH is a metabolic hormone( Pierce
and Parsons, 1981).Glycoprotein hormones are heterodimers produced by
the anterior pituitary gland or placenta. They are composed of an α and a
β subunit of which the α subunit is common to all four hormones (Liao
and Pierce, 1971). The β subunit is different for each glycoprotein
hormone and it determines the function of each hormone (Oefner et al.,
1992).
1.2.5.1. Secretion of Gonadotropins
The gonadotropins play a central role in a highly regulated system
known as the hypothalamic pituitary gonadal (HPG) axis as shown in
figure (1-1). Hypothalamic pituitary gonadal axis is a critical part in the
regulation and development of a number of organ systems such as the
reproductive system (Shnaishah, 2011). The spermatogenesis process
and all other aspects of male reproductive function depend on the
presence of reproductive hormones produced by the hypothalamus,
anterior pituitary and testes (Tilbrook and Clarke, 2001).
The hypothalamus produces the decapeptide hormone gonadotropinreleasing hormone (GnRH) into the hypophysial portal circulation. GnRH
is not diluted in the systemic circulation before it reaches the target cells
making it a rapid and efficient signal from the brain (Knobil and Neill,
1998). GnRH binds its G protein-coupled receptor located on
gonadotrope cells in the anterior lobe of the pituitary gland and, in
response to hormone binding, the anterior pituitary synthesizes and
13
Chapter one
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‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
releases the gonadotropins, luteinizing hormone (LH) and follicle
stimulating hormone (FSH) into the peripheral circulation (Lingappa and
Farey, 2000). The gonadotropins then affect their target organs (the
gonads) where specific LH and FSH receptors are expressed (Shnaishah,
2011). The gonadotropins LH and FSH work together to regulate
functions of ovary and testes including gametogenesis and steridogenesis
(Conn et al., 1987).
The biosynthesis of the gonadotropin subunits can be considered in
three steps. Translation involving the cytoplasm and the endoplasmic
reticulum (ER), glycosylation and assembly taking place in the ER,
glycan remodeling, hormone packaging and secretion taking place in the
Golgi and secretogranins. Both subunits are glycosylated, and the timing
of glycosylation relative to translation and transfer to the ER. (Hoshina
and Boime, 1982).
There were many previous studies that focused on the mechanisms
controlling the differential secretion of FSH and LH. It is well established
in mammals that GnH synthesis and release are regulated by multiple
factors including GnRH, sex steroids and gonadal peptides such as activin
and inhibin (Amano et al., 1995).
The gonadal sex hormones from both females and males exert
negative feedback at the level of hypothalamus affecting GnRH secretion
and, at the level of the pituitary, affecting gonadotropin secretion. Thus
this feedback loop helps regulate the levels of LH, FSH and the sex
steroids tightly in the body (Shnaishah, 2011).
1.2.5.2. Biological action of Gonadotropins
Luteinizing hormone, FSH and testosterone are the prime regulators,
which control spermatogenesis. However, androgens are indispensable
for initiation and maintenance of spermatogenesis, although testosterone
14
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
feedback on both gonadotrophic hormones, an additional feedback loop
exists between the testes and the brain for FSH. Inhibin, activin and
follistatin are involved in this regulatory system. While inhibin function
to suppress FSH secretion (Gupta, 2005). FSH stimulates the proliferation
of the spermatogonia and formation of the primary spermatocytes. While
androgens are involved in bringing about the meiosis division of the
primary spermatocyte and their final conversion into the spermatide. FSH
is also influences the secretion of Sertoli cells, secretion of androgen
binding protein (ABP) is under the control of FSH. However, LH controls
the synthesis of androgens by the Leydig cell clusters (Negi, 2009). FSH
and T act through the Sertoli cell since the receptors for those hormone
are located on these cells and not on the germ cells (Verhoeven et al.,
2007). FSH stimulates the production of androgen binding protein by
Sertoli cell. ABP is essential to concentration testosterone in levels high
enough to initiate and maintain spermatogenesis, which can be 20-50
times high than the concentration found in blood. The hormone inhibin
acts to decrease the levels of FSH (Pareek et al., 2007).
15
‫‪introduction & Review of Literature‬‬
‫‪Chapter one‬‬
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
‫‪GnRH‬‬
‫‪GnRH‬‬
‫‪Figure (1-1): Hypothalamic-pituitary-gonadal axis in mammals‬‬
‫‪(Gilbert 2010) (Adapted from Scott F. Gilbert 2010).‬‬
‫‪16‬‬
Chapter one
introduction & Review of Literature
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1.2.5.3. Role of Gonadotropins in Assisted reproductive
technology
The most studies of gonadotropin utilization focus on the total
amount of medication administered per cycle, specifically evaluated live
birth outcomes in relation to the time needed to achieve follicular
development appropriate for oocyte retrieval (Min et al., 2004). Doubling
the dose of gonadotroins dose not enhance the pregnancy rate, but
adverse outcomes such as ovarian hyperstimulation syndrome (OHSS) as
well as multiple pregnancy rate are increased (Cantineau et al., 2007).
The study reported that the length of stimulation did not affect
clinical pregnancy outcomes following ART (Martin et al., 2006). A
number of parameters may influence both the duration of gonadotropin
stimulation and the likelihood of success, and thus, constitute potential
confounders. For example, a meta-analysis of data from 3,865 women
demonstrated that the use of gonadotropin-releasing hormone (GnRH)
antagonists shortened the ovarian response time and was associated with
diminished chance for clinical pregnancy (Al-Inany et al., 2006).
Similarly, obesity is associated with prolonged stimulation phase
(Fedorcsak et al., 2004), and reduced pregnancy rates (Maheshwari et al.,
2007).
1.2.6. Culture medium (CM), Overview
During sperm preparation for ARTs, defined culture medium (CM)
was used and sometimes enriched with protein source and/or sperm
stimulator (Rowell and Braude, 2003). Culture media are isotonic with
semen to prevent any osmotic shock to spermatozoa and developing
embryos during in vitro manipulation steps, it provides optimal buffering
capacity, and maintains the pH within physiological levels to ensure
17
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
sperm survival (Ranch, 2005). Improvement of sperm motility and grade
activity was obtained as a result of special basic components of CM
(Schlegel and Girardi, 1997).
Culture media show to reduce human sperm DNA fragmentation
resulting from oxidative stress, that happened during processing dy
antioxidant activity of its constituent like ascorbic acid, N-acetylcholine,
tocopherol, taurine and hypotaurine (Baumber et al., 2003).
1.2.6.1. Basic and special component of Culture media
The CM used for ARTs should contain protein source and buffers to
promote sperm capacitation and hyperactivation (Baker et al., 2000). The
acidity (pH) of CM is maintained by bicarbonate and CO2 buffer system.
Sodium chloride that is one of the content of the medium plays an
important role in regulation of toxicity of the medium and in turn
preserves the sperm membrane and promotes full sperm function (Lim et
al., 1985). In addition to other types of carbohydrates like pyruvate,
lactate are present in the composition of the medium, which are the
primary nutrient for sperm and source of energy. These material give the
sperm more than one source of energy and make their motility more
improved (young, 1992; Ranch, 2005).
Human serum albumin (HSA) as protein source, and play a major
role in physiology and metabolism of spermatozoa (King and Killian,
1994). Albumin is protein present in the blood serum in humans and it
comprises nearly half of the blood serum protein (Draves, 1998). It has
been necessary to include some kind of proteins in the CM to support
sperm capacitation and/or fertilizing ability (Bavister et al., 2003).
The function of albumin in CM include limited buffering, the binding
of various compounds including steroids and potentially toxic trace
elements and capacitation of spermatozoa. Albumin was available as
18
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
powder or sterile solution (Reiss ,1998). Two essential components of the
SMART medium are serum albumin and bicarbonate is believe to
facilitated the efflux of cholesterol from the sperm plasma membrane by
acting as an acceptor for the lipid (Visconti et al., 2002). Whereas entry
of the bicarbonate ion into spermatozoa has been shown to be involve an
increase in intracellular pH during capacitation (Zeng et al., 1996).
1.2.6.2. Biomedical importance of Culture medium
Bicarbonate plays a major role in the activation of sperm cells
(Boatman and Robbins, 1991; Suzuki et al., 1994; Shi and Roldan, 1995;
Visconti et al., 1995b). The effects of calcium and bicarbonate on
activation of live sperm motility supposedly occur through stimulation of
intracellular cAMP synthesis from ATP via activation of sperm adenylate
cyclase (Morton et al., 1974; Si and Okuno, 1993; Morton and Albali
1973and Okamura et al.,1985).
Human serum albumin used in culture media acts as a powerful
antioxidant that prevents oxidative stress-induced damage (Sikka, 2004).
That means the antioxidant effect of albumin play an important role in
preserving sperms damage and makes its motility more easily.
Furthermore, albumin is considered as nutrition medium for sperms that
supplied them, those proteins in form of albumin which is found in high
concentration in seminal plasma that makes up about one third of the
protein content of semen. Sperm motility appeared to be more negatively
influenced when the medium lacked protein (Owen and Katz, 2005).
1.2.6.3. In vitro sperm activation and Culture medium
The use of in vitro culture media increases sperm motility. The
reason is that the seminal fluid with high viscosity obstructs sperm
19
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
progressive motility so that the uses of in vitro media with aqueous nature
lead to decrease the viscosity of the seminal fluid and as a result sperms
move more freely (Makler et al., 1984). In addition to the high viscosity,
the seminal fluid contains substituent‟s which obstruct sperm forward
progressive motility, as antisperm antibodies, bacteria, leukocytes, and
damaging secretion from the seminal vesicles, thus the use of there in
vitro media decrease the damage occurring by these substituent‟s (Makler
and Jacobi, 1981). Also, when spermatozoa are free of seminal plasma
within culture medium have capacity to achieve capcitation as a results of
removal of both decapacitation factor and acrostatin “arcsine inhibitor”
which prevents oocytes fertilization (Chen et al., 1989).
One of the key events in sperm capacitation is the activation of
adenylate cyclase by high levels of bicarbonate that are present in vitro
fertilization media, and proposed to be locally enriched in upper parts of
the female genital tract (i.e. in the lumen of the oviduct), but virtually
absent in epididymal and seminal plasma (Harrison, 1996). Increased
cAMP levels activate cAMP-dependent PKAs and indirectly induce
protein tyrosine phosphorylation by a yet unknown signaling pathway.
Bicarbonate also induces PKA-dependent changes in the lipid
architecture of the sperm plasma membrane (Harrison and Miller, 2000),
due to phospholipid scrambling (Gadella and Harrison, 2000). In vitro
studies showed that vitamins E and C are major chain–breaking
antioxidants in sperm membranes and appears to have a dose dependent
protective effect (Agarwal and Prabakaran, 2005).
1.2.6.4. Role of In vitro sperm activation in Assisted
reproductive technology
The aim of sperm preparation for ARTs including artificial
insemination (AI) is to maximize the chances of fertilization (Baker et al.,
21
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
2000). Basically the culture media used for ARTs are modification of
balanced salt solution (Sepalla,1985), and it is apparent that spermatozoa
of mammalian species including human can acquire the ability to fertilize
after a short incubation in defined culture media (Ravnik et al.,1992).
Sperm processing and isolation of highly motile spermatozoa from the
whole semen specimen have been tried with variable success prior to its
use for ARTs (Cruz et al., 1986).
The presence of steroids and gonadotropin hormones in serum of the
stimulated females which are used for sperm activation in vitro in the
studies may be responsible for improved sperm motility. The addition of
pasteurized plasma protein solution to the culture media provides safe and
optimal condition for sperm activation and culturing embryo for ART
(Gerritdina et al., 1992).
1.2.6.5. Correlation between ISA and rates of Fertilization
and pregnancy
In vitro sperms activation and IUI will increase the pregnancy by 4%
to 9% per cycle. This may be due to increase the number of oocytes
released, better timing of insemination or correcting subtle ovulation
defect (Ho et al., 1989). In vitro activation of sperm triggers diverse
signaling pathways such as cAMP dependent protein kinase (PKA) and
induced protein tyrosine phosphorylation (Visconti et al., 1995b) and
leads ultimately to the generation of sperm cells with high binding
affinity for the zona pellucida. The sperm activation processes are
collectively termed capacitation (Yanagimachi, 1994).
The first in vitro fertilization (IVF) cases, including that of Louise
Brown, were performed to treat tubal infertility, the increasing number of
men showing poor semen quality prompted the development of wide
array of different laboratory techniques focusing on the selection and
21
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
enrichment of motile and functionally competent spermatozoa from the
ejaculate. First sperm separation methods available only comprised male
germ cell (Ewards et al., 1969; Ewards et al., 1980).
In the light of the influence of the fertilizing spermatozoon not only
on early but also on late embryonic development, selection of the best
sperm from heterogeneous sperm sample would impact positively on the
outcomes of human ARTs. Accurate identification of normal healthy
spermatozoa is of special importance during ICSI, in which a sperm cell
is deliberately injected into the mature oocyte by the technician by
passing all natural barriers. There is great concern about the risk of using
sperm with chromosomal transmission and/ or damage DNA what can
lead to in advertently transmission of genetic diseases to the offspring.
Therefore, improvements of the available sperm selection techniques and
/or development of new methods for precise sperm selection are highly
desirable (Brown et al., 1995).
1.2.7. Sperm chromatin structure assay (SCSA), Overview
The introduction of the sperm chromatin structure assay (SCSA),
first described in 1980, enabled the level of DNA breaks to be quantified
by means of the DNA fragmentation index (DFI) using a flow-cytometric
technique (Evenson et al., 1999). Abnormalities at the level of the sperm
nucleus with implications on reproductive outcome include DNA strand
breaks, numerical and structural chromosomal abnormalities, Y
chromosome microdeletions and alterations in the epigenetic regulation
of the paternal genome. Recently, there has been a focus on the analysis
of
sperm
DNA
damage,
as
an
indicator
of
sperm
quality.
The most common types of identified sperm DNA damage are:
(i) single and double DNA strand breaks; (ii) the chemical modification
of a base by, for example, oxidation or alkylation; (iii)inter- or intrastrand
22
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
crosslinkage; and (iv) DNA–protein crosslinks (Aitken and Iuliis, 2007;
2010).
The damaged of DNA in the single sperm that fertilizes a female
oocyte can have a dramatic negative effect on the embryo development
(Evenson, 1997; 1999a, b). The integrity of mammalian sperm DNA is of
vital importance for the paternal genetic contribution to a normal
offspring and the chromatin status of the sperm is important for
successful embryo development (Bedford et al., 1973; Evenson et al.,
1980).
The sperm chromatin structure assay (SCSA) enables fast
identification of the DNA fragmentation index (DFI, percentage of cells
showing denatured DNA) and percentage of cells with high DNA
stainability (HDS, cells with defective chromatin condensation) in sperm
samples using flow cytometry. By comparison with threshold values (30
DFI and 15% of HDS cells), the results of SCSA can be used as a
predictor of the pregnancy success (Larson-Cook et al., 2003; Virro et al.,
2004; Kennedy et al., 2011). Various hypotheses have been proposed
as the molecular mechanism of sperm DNA damage. The most important
ones are abnormal chromatin packaging, oxidative stress and apoptosis
(Sakkas et al., 1999).
1.2.7.1. Techniques of SCSA
A number of methods for analyzing sperm DNA have been
developed and, in brief, the most common tests are as follows:
1. The sperm chromatin structure assay (SCSA) which uses flow
cytometry to measure the intensity of acridine orange (AO) fluorescence
when it binds to native and fragmented DNA. The percentage of DNA
fragmentation is referred to as the DNA fragmentation index or DFI
23
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
(Evenson and Jost, 1994). The method uses AO, which gives green
fluorescence with native DNA and red fluorescence with single-stranded
DNA. The proportion of red fluorescence to total fluorescence in each
sperm cell is measured by flow cytometry. The percentage of cells
exhibiting a high proportion of red fluorescence was originally known as
COMPat (Cells Outside the Main Population), but is now represented by
the DNA fragmentation index (Ballachey et al., 1987; Evenson et al.,
1999).
2. The sperm chromatin dispersion (SCD or Halosperm) test uses either
fluorescence or bright-field microscopy to evaluate fragmented DNA as
reflected by the presence, in size, or absence of a halo surrounding the
sperm head (Fernandez et al., 2003).
3. Other methods for DNA fragmentation assessment include terminal
deoxynucleotidyl transferase-mediated-dUTP nick end labeling (TUNEL)
assay (Gorczyca
et al.,1993) and the single cell gel electrophoresis
(COMET) assay (Hughes et al.1997). Much-used method of measuring
sperm DNA fragmentation is the terminal TdT-mediated dUTP nick-end
labeling (TUNEL) assay using flow cytometry or fluorescence
microscopy. The TUNEL assay identifies DNA breaks by labelling 3′OH
termini and is a measure of existing DNA damage, whereas the SCSA
measures single-stranded DNA after acid treatment and therefore includes
potential DNA damage. (Evenson et al., 1999; Spano et al., 2000).
1.2.7.2. Factors affecting SCSA
Zinc and copper are trace elements, which play an important role in
the stability of sperm cells chromatin by stabilization of the free thiol
group. The prostate gland secretion is rich with zinc, so that the sperm
chromatin is protected when mixed with seminal plasma during
ejaculation. A lack of zinc leads to increased susceptibility of the sperm
24
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
chromatins to in situ denaturation (Blazak and Overstreet, 1982;
Rodriguez et al., 1985). Some therapeutically used chemicals
(Shalet,1980; Evenson et al., 1999), environmental pollution stress
(Wyrobek et al., 1997; Lemasters et al., 1999; Perreault et al., 2000;
Selevan et al., 2000), cigarette smoking (Spano et al., 1998) and cancer
diseases (Evenson and Melamed, 1983; Evenson et al., 1984; Fossa et al.,
1997).
Poor semen quality has been associated with an increase in the
proportion of sperm with DNA fragmentation (Saleh et al., 2003;
Bochenek et al., 2001). Certainly, the sperm chromatin structure assay
(SCSA) has been recognized as an independent measure of the sperm
quality that may have higher diagnostic and prognostic capabilities than
standard sperm parameters for both in vivo and in vitro fertilization
(Agrawal and Said, 2003).
Chemotherapeutic drugs such as fludarabine, cyclophosphamide and
busolphane can cause testicular damage as manifested by reduced
testicular volume, oligozoospermia, elevated FSH and LH and lower
testosterone concentrations (Chatterjee et al.,2000).
1.2.7.3. Correlation between SCSA and ART
The SCSA has a potential to contribute to more efficient use of in
vitro assisted reproduction techniques (ART) (Evenson and Wixon,
2006a; Bungum et al., 2007).
Semen samples that contain high levels of DNA damage are often
associated with decreased fertilization rates and/or embryo cleavage after
IVF and intra cytoplasmic sperm injection (ICSI) and may be linked to
early embryo death. Although the most normal appearing and motile
25
Chapter one
introduction & Review of Literature
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
spermatozoa are selected during ART, there is always a chance that
sperm containing varying degrees of DNA damage may be used. The
cause of infertility in infertile men with normal semen parameters could
be related to abnormal sperm DNA (Alvarez, 2003).
26
Chapter two
Materials
And
methods
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Chapter Two
Materials and Methods
2.1. Subjects
This study was continued from October, 2012 until April, 2013. All
subjects were involved in this study during their attendance at High
Institute for Infertility Diagnosis and Assisted Reproductive Technologies
/ Al- Nahrain University.
Ninety semen samples were divided to three groups randomly: First
group (FSH) involved thirty males of age mean (30.09±1.39) years with
history of infertility mean was (5.45±0.84) years. Second group(LH)
involves thirty subjects with age mean (34.57±12) year with history of
infertility mean was (4.35±0.48) year. However, third group (Gn)
involves males with age mean (35.25±1.93) year with history of infertility
mean was (6.3±0.71) years. Subjects were instructed to collect a semen
sample by masturbation for all specimens, seminal fluid analysis was
done according to criteria WHO (2010).
2.2 Material and equipments
The material and equipments used in the study are listed in table (21) and (2-2) respectively.
2.3. Preparation of gonadotropins
To prepare a low concentration of FSH and LH 2.5 IU was dissolved
in 10 mL of SMART medium and for high concentration of hormones, 5
IU was added to 10 mL of SMART medium.
Also, addition 2.5 IU FSH and 2.5 IU LH 10 mL of SMART medium
to prepared low concentration of gonadotropins hormone. High
72
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
concentration of gonadotropins hormone added 5 IU from each FSH and
LH to 10 mL of SMART medium.
2.4. Preparation of culture medium
Simple medium for ART (SMART) was prepared according to
(Fakhrildin and Flayyih, 2009) by dissolving materials listed in table (23). The pH value was adjusted at 7.3-7.4. Then, The culture medium was
filtered using (Millipore) filters and stored at 5˚C, before using
encountered to ultra violet light.
Table (2-1): Equipment and tools utilized in the present study.
Equipment and tools
Centrifuge
Fluorescent microscope
Incubator
Light microscope
Micropipette
Millipore filter 0.20 µm
Pasteur pipette (150)
15ml Polystyrene conical tube
Microscope Slides and cover slides
Laminar air flow
Company /Origin
EBA20 Hettich, Germany
BEL Photonic, Italy
Termaks, Norway
Por-Way.Hb.Japan
Slamid, Germany
Sartorius, Germany
Volac John Poulten LTD,England
Falcon, USA
Marienfeld, Garmany
Lab Companion, Germany
Table (2-2): The chemicals used in the present study.
Chemicals
Sodium chloride
Acridine orange stain
Follicle-stimulating hormone
Luteinizing hormone
Calcium chloride-hydrate
Citric acid
Distilled water
Company /Origin
GCC.UK
Sigma,Deisenhofen,Germany
Merck KG, USA
Merck KG, USA
BDH, England
Panreac, Spain
Samara,Iraq
72
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Glacial acetic acid
Human serum albumin
Hydrated sodium phosphate
Hydrochloride acid 0.1N
Magnesium chloride
Methanol
Potassium chloride
Sodium bicarbonate
Sodium hydroxide 0.1N
Sodium pyruvate
Scharlau, Spain
Life Global, USA
BDH, England
BDH, England
HIMEDIA, India
Ajax, Austria
BDH, England
Panreac, Spain
BDH, England
PROLABO, Paris
Table (2-3): Chemicals and amount preparation of SMART.
Chemicals
Bicarbonate
Na-lactate
NaCL
CaCL2
KCL
Na-pyruvate
Human Serum Albumin (HSA)
Penicillin
Streptomycin
Distilled Water
Phenol Red
The amounts
29mΜ
3.2g
6.0g
0.27g
0.4g
0.01g
5%
100IU/mL
100µg/mL
500mL
0.5g
2.5. Semen analysis
Each sample of seminal fluid was collected after 3-5 days of sexually
abstinence directly into a clean, dry and sterile disposable Petri-dish by
masturbation in a private and quite room adjacent to the semen analysis
laboratory. Each Petri-dish was labeled with the person name, age,
abstinence period and time of sample collection. The specimens were
72
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
incubated at 37ºC for 30 minutes to allow liquefaction(NAFA and
ESHRE, 2002). The standard criteria of WHO (2010) was used to record
parameters of the seminal fluid analysis table (2-4).
Table (2-4): Normal reference limits for semen characteristics
according to WHO criteria (2010).
Patient name:
Patient age:
day of abstinence:
Time of collection:
Lab. References No.
Examination date:
Time of examination:
File No.
Macroscopic Examination
Normal Value
Volume
Color
Liquefaction time
Viscosity
pH
1.5 mL
Grey-opalescent
≤ 60 minute
< 2 cm.
≥ 7.2
Microscopic Examination
Normal Value
Sperm concentration ( million/mL )
15 m/mL
Total motility (PR+NP, %)
Progressive motility (PR, %)
Total sperm number (million/ejaculate)
Sperm morphology (normal forms, %)
40 %
32 %
39 m/ejaculate
30 % *
Agglutination (%)
Round cells(WBCs+Germ cells)
<10%
<5 cells\HPF
Others: RBCs+Epithelial cells)
NIL\HPF
*Using WHO 1999
03
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
2.5.1. Macroscopic examination
2.5.1.1. Semen liquefaction time
A normal semen sample liquefies within 60 minutes at room
temperature , although this occurs within a period of
less than 30
minutes. In some cases, semen samples may not liquify probably due to
poor prostate activity or gel particles or mucous streaks (NAFA and
ESHRE, 2002). The sample must be well mixed in the same container
before microscopic examination where incomplete mixing is probably a
major contributor to errors in determining sperm concentration (WHO,
2010).
2.5.1.2. Semen pH
The acidity was measured by pH paper (Agostini and Lucas, 2003).
The pH was measured at a regular time within one hour after ejaculation.
A drop of semen was spread evenly on pH paper. Within 30 seconds, the
color of the impregnated zone became uniform and was compared with
the calibration strip to read the pH. The normal pH should be 7.2 or more
(WHO, 2010).
2.5.1.3. Semen viscosity
Viscosity was measured by drawing part of semen drowns by pasture
pipette and let escape slowly from its mouth fast the sample runs out of
pipette. If the droplets form threads that are more than 2 cm long, note the
increased viscosity should be noted on the sample (NAFA, 2002).
2.5.2. Microscopic examination
A drop of 10μL of liquefied and thoroughly mixed semen was taken
by Eppendorff automatic pipette or pasteur pipette mounted between
warm slide and covered with a standard cover slip (22×22) mm. The
03
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
sample was examined under light microscope at magnification of 40X
objectives. Specimen was assessed for the following five parameters:
2.5.2.1. Sperm motility and grade activity
The prepared slide was examined to determine the percentage of
sperm motility. The number of motile spermatozoa in the ten randomly
selected fields was counted away from cover slip edge. At least one
hundred spermatozoa were counted. The normal sperm progressive
motility was calculated by taking the mean number of forward
progressive motile spermatozoa (grades A+B); which should be ≥32% of
the total sperm count, both were taken within 60 minutes of collection
(WHO, 2010). Therefore, the estimation of percentage of sperm motility
and grade activity, were calculated according to following formula
(normal values):
Grade (A+B): Progressive motility (5 – 25 or more μm/sec).
Grade (C): Non progressive motility (<5 μm/sec).
Grade (D): Immotile.
 Sperm motility (%) =Progressive(%)+Non progressive(%)
Semen sample with less than normal progressive motility percentage
was considered as an asthenozoospermic (WHO, 2010).
Grade activity (%) = Number of sperms in specific motility / Total
number of sperms × 100
07
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
2.5.2.2. Sperm concentration
Sperm concentration per milliliter (mL) was reported from the mean
number of spermatozoa in 10 random microscopically fields of the slide
and multiplying the mean number by a factor of one million (Hinting,
1989). Each spermatozoa per field correspond to the concentration of 1
million sperm/mL(NAFA, 2002). Total sperm count was obtained by
multiplying sperm concentration by semen volume. Semen sample with
concentration of less than 15 million/mL was considered
as an
oligozoospermic semen sample (WHO, 2010).
Sperm concentration (million/mL) =No. of sperms×multiplication
factor(1 million)
2.5.2.3. Sperm morphology
The examination of morphologically normal sperm was performed
by using the same prepared slides for sperm motility. Normal
spermatozoon has an oval shaped head with a pale anterior part
(acrosome 40-70% of the head area) and a darker posterior region. The
length to width ratio of the head should be 1.50 to 1.75. Only one tail
should be attached in a symmetrically situated fosse in the base of the
head. Semen sample with less than 30% of normal sperm morphology
was classified as teratozoospermic (WHO, 2010). At least 100
spermatozoa were counted and percent normal sperm morphology (%) was
calculated according to the following formula:
00
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Normal sperm morphology (%) = No. of normal sperms / Total
number of sperms × 100
2.5.2.4. Sperm agglutination
Agglutination of spermatozoa means that motile spermatozoa stick to
each other head to head, tail to tail or in a mixed way. e.g., head to tail.
The adherence either of immotile spermatozoa to each other or of motile
spermatozoa to mucous threads. Cells other than spermatozoa, or debris is
considered to be nonspecific aggregation rather than agglutination and
should be recorded as such (WHO, 2010). For estimation of percentage of
sperm agglutination, the following formula is used:
Agglutinated sperm (%) = No. of agglutinated sperms /Total
number of sperms × 100
2.5.2.5. Round cell count
The number of round cells in the semen samples was estimated by
counting their mean number in 10 random microscopic fields and
multiplied by a factor of 1 million. The number of round cells value was
counted using high power field (HPF) method. The semen sample with
<5 round cells/ HPF was considered normal (WHO, 1999; 2010).
2.6. Assessment of DNA Fragmentation index (DFi)
Smears were prepared from each semen sample on the slide and
allowed to air dry for about 20 minutes. Then, the slides were fixed in
Carnoy's solution for at least 3 hours to overnight at 4˚C. After that, the
slides were removed from the fixer and allowed to air-dry from a few
03
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
minutes before staining. All solution should be prepared at room
temperature under dim light and pH of the stain was adjusted to 2.5. The
stock solution was stored in the dark at 4˚C, and the AO stain working
solution should be prepared daily. After being placed on a slide holder, a
2-3 mL of the stain was spread over each slide for 5 minutes. They were
gently rinsed in a stream of distilled water. The slides were allowed to
dry, mounted and a 22x50 mm glass cover slip was placed it. Slides were
read on the same day of staining with a (40X) on a fluorescence
microscope, which was equipped with an excitation filter of 460-490 nm
and barrier filter of 520 nm.
The nuclei of 300 spermatozoa from each sample were examined and
scored as fluorescing green, yellow or red. Spermatozoa displaying green
fluorescence were recorded as normal, whereas sperm heads displaying
yellow-red fluorescence were considered as abnormal.
The principle of this test is that AO has been used to label nucleic
acid in solution and intact cells. AO intercalates with the double-strand
DNA as monomer, whereas it binds to single-stranded DNA as an
aggregate. With fluorescent microscope, first state fluoresces green while
in the other fluoresces red or yellow (Tejada et al., 1984).
2.7. Experimental design
The design of the experiments is shown in Figure (2-1).
03
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
90 Semen sample
Semen analysis
Human sperm
chromatin structure
assay
Macroexamin
ation
30 semen sample
30 semen sample
Centrifugation, 2500rpm for 5
min
FSH
0.25 IU
FSH
0.5 IU
30 semen sample
Centrifugation, 2500rpm for 5
Centrifugation, 2500rpm for 5
min
min
Removal supernatant &add
1mL CM
CM
Microexamin
ation
Removal supernatant &add 1mL
CM
CM
LH
0.5 IU
LH
o.25 IU
Removal supernatant &add
1mL CM
CM
GnH
0.25
IU
Incubation for 30 minutes
Assessment of sperm
parameters
Human sperm
chromatin
structure assay
Figure (2-1): Experimental design of the study.
03
GnH
0.5
IU
Chapter Two
Materials and Methods
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
3.8. Statistical analysis
The data were statistically analyzed using SPSS/PC software
(version 18) (SPSS, Chicago).Sperm parameters were analyzed using
complete randomized design (CRD) of one way (ANOVA).
The mathematical model was
Yij = µ + Ti + eij.
Where
Yij= dependent variables (sperm parameters).
µ= overall mean.
Ti= effect of treatments with the hormones (FSH, LH and
gonadotropine).
eij= error term.
Differences among means were computed using the Duncan multiple
ranges test (Duncan, 1955).
02
Chapter Three
Results
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Chapter Three
Results
Ninety semen samples were shared in this study and divided into
three studies involving: 1- Follicle stimulating hormone study, 2Luteinizing hormone study and lastly 3- Gonadotropins study. The range
age for males was (22-54) years.
3.1. Follicle stimulating hormone (FSH)
In this work, thirty infertile males were involved with the mean age
(30.09±1.39) years with range (22-44) years. The mean duration of
infertility of them was (5.45±0.84) years with range of (2-8) years.
Figure (3-1) shows the effect of SMART medium either alone or
supplied one of two concentrations of FSH on sperm concentration after
in vitro sperm activation using centrifugation technique (ISACT). There
is a significant (P>0.05) decrease between sperm concentration pre- and
post-activation. However, non significant (P<0.05) differences are found
among groups of control, low and high FSH concentration.
The results of sperm motility percentages, progressive, non
progressive and immotile sperm motility
pre- and post-ISACT were
shown in table (3-1). The percentage of sperm motility revealed
significant (P<0.05) increase between pre- and post-activation. Also, a
significant (P<0.05) increase were observed between control group and
both treated groups. While, non significant (P>0.05) differences were
found between both treated groups post-ISACT. This reveals that the high
concentration of FSH group have the best effect on sperm motility (%) as
compared with other groups.
83
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
On the other hand, non significant (P>0.05) differences in the
progressive sperm motility (%) was appear between pre-activation group
and control group. While significant (P<0.05) increase in the progressive
sperm motility (%) was noticed between pre-activation group and both
treated group .
Table (3-1) shows a significant (P<0.05) differences in the
precentage of non progressive motility between pre-activation group and
post- activation group. In contrast, non significant (P>0.05) differences
were assessed among groups of control, low and high concentrations of
FSH post-ISACT. The percentage of normal sperm morphology appeared
to have a significant (P<0.05) differences between pre-activation group
and post- activation groups. However, non significant differences
(P>0.05) were obtained for percentage of normal sperm morphology
among all groups post-activation as presented in figure (3-2).
Also, figure (3-3) shows that the percentages of DFI have non
significant (P>0.05) differences in pre-activation group and postactivation group. Also, non significant (P>0.05) differences were noticed
among post-activation groups, and low FSH group showed low DFI (%)
when compare with other groups.
83
Chapter Three
Results
Sperm concentration( millions/mL)
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
45.73 a
50
45
40
35
30
21.13 b
19.40 b
25
23.23 b
20
15
10
5
0
pre- activation
(control)
(0.25 IU of FSH)
(0.5 IU of FSH)
Post-activation
Figure (3-1): Sperm concentration pre- and post-activation using SMART
medium enriched with two concentrations of FSH.




Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means ±SEM.
04
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Table (3-1): Percentage of sperm motility pre- and post- activation using
SMART medium enriched with two concentrations of FSH.
Preactivation
group
G1:Control
G2:Low
concentration
G3:High
concentration
57.800 c
±2.54
77.967 b
±3.34
87.533 a
±3.04
88.133 a
±2.93
30.467 b
±2.60
35.067 b
±3.26
48.767 a
±4.04
51.367 a
±4.31
Nonprogressive
sperm motility
27.333 b
44.233 a
±2.98
±2.98±2.10
38.633 a
±3.07
37.033 a
±2.99
Immotile sperm
41.967 a
±2.55
20.367 b
±3.39
12.467 c
±3.04
11.833 c
±2.93
Sperm motility
Parameters
Sperm motility (%)
Sperm Progressive
grade sperm motility
activity
(%)




±2.58
Post-activation
groups
Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the mean ±SEM.
04
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Normal sperm morphology (%)
60
49.50 a
50.10 a
45.70 a
50
40
30.23 b
30
20
10
0
pre- activation
(control)
(0.25 IU of FSH)
(0.5 IU of FSH)
Post-activation
Figure (3-2): Percentage of sperm morphology pre- and post- activation
using SMART medium enriched with two concentrations of
FSH.




Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means±SEM .
04
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
sperm DNA fragmentation (%)
30
25.42 a
25
20.59 a
19.18 a
18.23 a
20
15
10
5
0
pre- activation
(control)
(0.25 IU of FSH)
(0.5 IU of FSH)
Post-activation
Figure (3-3): Percentage of sperm DNA fragmentation pre- and postactivation using SMART medium enriched with two
concentrations of FSH.




Number of infertile patients =30
Means with similar superscripts within each columns are none significantly different (P>0.05).
Means with different superscripts within each columns are none significantly different (P>0.05).
Data are the means ±SEM.
08
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
3.2. Luteinizing hormone (LH)
The second study consist 30 semen samples for ISA using SMART
medium enriched with two concentrations of luteinizing hormone (LH).
The mean age of infertile males was (34.57±12) years with a range (2339) years. The mean duration of infertility was (4.35±0.48) years with
range of (2-10) years.
Significant (P<0.05) decrease were found in the sperm concentration
by using SMART medium with two concentrations of LH when
compared with pre-activation group. However, non significant (P>0.05)
differences were assessed for sperm concentration among all groups postactivation as presented in figure (3-4).
The percentages of sperm motility and sperm grade activity were
presented in table (3-2). Significant (P<0.05) increase were observed in
the sperm motility (%) and progressive sperm motility(%) between preand post- activation. Also, significant (P<0.05) increase were noticed
between control group and both treated groups post-activation. However,
non significant (P>0.05) differences are observed between low LH
concentration group and high LH concentration group.
On the other hand, non significant (P>0.05) differences in the nonprogressive motile sperm (%) were noticed between pre- and postactivation groups. Similarity, non significant (P>0.05) differences were
assessed among all treated groups. In contrast, immotile sperm (%)
observed significant (P<0.05) decrease between pre- and post-activation
groups. Also, among post-activation groups a significant (P<0.05)
differences were noticed between control group and both treated groups.
While, non significant (P>0.05) differences were appeared between the
groups of low and high concentration of LH (Table 3-2).
00
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Sperm concentration (millions/mL)
45
40.13 a
40
35
30
19.47 b
25
17.97 b
15.50 b
20
15
10
5
0
pre- activation
(control)
(0.25 IU of LH)
(0.5 IU of LH)
Post-activation
Figure (3-4): Sperm concentration pre- and post- activation using
SMART medium enriched with two concentrations of LH.




Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means ±SEM.
04
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Table (3-2): Percentage of sperm motility pre- and post-activation using
SMART medium enriched with two concentrations of LH.
Sperm motility
Parameters
sperm motility (%)
Sperm
grade
Progressive
activity sperm motility
(%).
Non-progressive
sperm motility
Immotile sperm




Preactivation
group
Post-activation
groups
G1:Control
G2:Low
concentration
G3:High
concentration
62.433 c
±1.86
77.667 b
±2.48
87.700 a
±3.06
87.400 a
±3.46
31.767 c
±1.94
39.700 b
3.26
52.800 a
±3.60
52.100 a
±4.19
30.667 a
±1.13
38.633 a
±2.91
34.900 a
±2.65
35.967 a
±3.43
37.567 a
±1.86
22.000 b
±2.43
12.300 c
±3.06
12.567 c
±3.46
Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means ±SEM.
04
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
The percentages of normal sperm morphology for control and treated
groups were found to be with a significant (P>0.05) increase when
compared to pre- activation group. Also, significant (P<0.05) increase
were found in percentage of normal sperm morphology for high LH
concentration group as compared to control group. Furthermore, nonsignificant (P>0.05) differences were observed for low LH concentration
group as compared to high LH concentration group and control group
(Figure 3-5).
Figure (3-6) shows the percentages of sperm DNA fragmentation
pre-and post-activation groups. Non significant (P>0.05) differences were
noticed among all pre- and post-activation groups. However, the lower
percentage of DNA fragmentation were observed when using high LH
concentration within SMART medium.
04
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Normal sperm morphology (%)
60
48.10 ab
43.50 b
50
40
50.40 a
30.40 c
30
20
10
0
pre- activation
(control)
(0.25 IU of LH)
(0.5 IU of LH)
Post-activation
Figure (3-5): Percentage of sperm morphology pre- and post- activation
using SMART medium enriched with two concentrations of
LH.




Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means ±SEM.
03
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Sperm DNA fragmentation (%)
50
45
37.07 a
40
30.27 a
35
25.50 a
30
22.47 a
25
20
15
10
5
0
pre- activation
(control)
(0.25 IU of LH)
(0.5 IU of LH)
Post-activation
Figure (3-6): Percentage of sperm DNA fragmentation pre- and postactivation using SMART medium enriched with two
concentrations of LH.



Number of infertile patients =30
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means ±SEM.
03
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
3.3. Gonadotropins (Gn)
In the third study, SMART medium was used and enriched with low
concentration 0.25 IU and high concentration 0.5 IU of gonadotropins
(both LH and FSH). This study involved 30 semen samples for infertile
males with the mean age of (35.25±1.93) years with range (22-54) years.
The mean duration of infertility was (6.3±0.71) years with range of (2-14)
years.
The results of sperm concentration using ISACT were presented in
the figure (3-7). Significant (P<0.05) decrease was assessed in the sperm
concentration between pre-activation group and post-activation group. In
contrast, non significant (P>0.05) differences were noticed in the sperm
concentration among all control and both treated groups post- activation.
Table (3-3) shows the percentages of sperm motility and sperm
grade activity. Significant (P<0.05) increase were observed in the
percentages of sperm motility and non progressive motility between preactivation group and post-activation groups. While, non significant
(P>0.05) differences were assessed among post- activation groups.
However, the group of high gonadotropins concentration showed the
highest percentages for both parameters when compared to other two
groups of post-activation.
For progressive sperm motility (%), a significant (P<0.05) increase
were assessed between pre-activation group and both treated groups postactivation. However, non significant (P>0.05) differences were appeared
among control and treated groups. Also, non significant (P>0.05)
differences were observed between pre-activation group and control
group post-activation (Table 3-3). From the same table, , immotile
44
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
42.10 a
Sperm concentration (millions/mL)
45
40
35
30
23.57 b
25
20.17 b
18.40 b
20
15
10
5
0
pre- activation
(control)
(0.25 IU of Gn)
(0.5 IU of Gn)
Post-activation
Figure (3-7): sperm concentration pre- and post-activation using
SMART medium enriched with two concentrations of
gonadotropins.




Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means ±SEM.
44
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Table (3-3): Percentage of sperm motility pre- and post- activation using
SMART medium enriched with two concentrations of
gonadotropins.
Sperm motility
Parameters
sperm motility (%)
Sperm Progressive
grade sperm motility
activity
(%).
Non progressive
sperm motility
Immotile sperm




Preactivation
group
Post- activation
groups
G1:Control
58.467 b
±1.90
79.167 a
±3.78
82.533 a
±3.97
84.233 a
±3.71
29.667 b
±1.67
34.767ab
±3.58
39.900 a
±4.80
40.733 a
±4.63
28.800 b
±1.24
44.067 a
±3.46
42.800 a
±3.60
43.467 a
±3.56
41.700 a
±1.92
20.500 b
±3.64
17.467 b
±3.97
15.767 b
±3.71
G2:Low
G3:High
concentration concentration
Number of infertile patients =30
Means with similar superscripts within each columns are non significantly different (P>0.05).
Means with different superscripts within each columns are significantly different (P<0.05).
Data are the means ±SEM.
44
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
sperm (%) showed a significant (P<0.05) decrease between pre-activation
group and post-activation groups.
While, non significant (P>0.05)
differences were assessed among all treated groups .
The percentages of normal sperm morphology were presented in
figure (3-8). Significant (P<0.05) increase were appeared between preactivation group and post-activation groups. On the other hand, non
significant (P>0.05) differences were observed among control and treated
groups post activation. However, high concentration group has the best
percentage for normal sperm morphology as compared to other groups
post-activation.
Figure (3-9) shows effect of gonadotropins supplement within
SMART medium on sperm DNA fragmentation. Significant (P<0.05)
decrease were observed between pre-activation group and post-activation
groups. Whereas, non significant (P<0.05) differences were noticed
between low gonadotropins concentration and both control and groups
with high gonadotropins concentration group. On the other hand, high
gonadotropins concentration group has the best percentage for sperm
DNA fragmentation when compared to the other groups of pre- and postactivation.
A normal sperms headscan be seen in Image (3-1) with intact DNA.
However, Image (3-2) shows sperms displaying green, yellow , and
orange as fragmentation DNA of sperms heads.
48
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
46.03 a
47.33 a
Normal sperm morphology (%)
50
39.83 a
45
40
35
30.30 b
30
25
20
15
10
5
0
pre- activation
(control)
(0.25 IU of Gn)
( 0.5 IU of Gn)
Post-activation
Figure (3-8): Percentage of sperm morphology activation using SMART
medium enriched with two concentrations of gonadotropins.




Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means ±SEM.
40
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Sperm DNA fragmentation (%)
40
36.70 a
35
25.93 b
30
22.44 bc
25
17.81 c
20
15
10
5
0
pre- activation
(control)
(0.25 IU of Gn)
(0.5 IU of Gn)
Post-activation
Figure (3-9): Percentage of sperm DNA fragmentation pre- and post- activation
using SMART medium enriched with two concentrations of
gonadotropins.




Number of infertile patients =30
Means with different superscripts within each columns are significantly different (P<0.05).
Means with similar superscripts within each columns are non significantly different (P>0.05).
Data are the means ±SEM.
44
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Image (3-1): sperm head under magnification power of (1000) of oil
immersion displaying green fluorescence as normal with intact DNA.
Image (3-2): Sperms head under (x40) HPF displaying intact and
abnormal fragmentation DNA of human sperm.
44
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
4.4. Comparative study among hormones
The result of semen analysis of the first, second and third for preactivation groups were presented in table (3-4). It was showed nonsignificant (P>0.05) differences in the all sperm parameters among first,
second and third, except sperm DNA fragmentation it was significantly
(P<0.05) decrease for first group when compared with second and third
groups while, non significant (P>0.05) differences were observed
between second and third groups (Table 3-4).
Table (3-5) shows the effect of SMART medium on all semen
parameters after in vitro sperm activation using centrifugation technique.
Non significant (P>0.05) differences in all sperm parameters were
observed excepted round cell count and DNA fragmentation for FSH
significant (P>0.05) differences compared with other two groups. In
contrast, non significant (P>0.05) differences between LH AND Gn
(Table 3-5).
Effect of low concentration 0.25 IU of FSH, LH and Gn
supplemented to SMART medium on sperm parameters were presented
in table (3-6). Non significant (P>0.05) differences among all sperm
parameters were assessed except progressive sperm motility (%) and
DNA fragmentation (%) . On the other hand, significant (P<0.05)
increase were noticed in progressive motility between Gn group and LH
group. Also, presented of sperm DNA fragmentation were observed
significant (P>0.05) differences between LH group and other treated
groups. Whereas, non significant (P>0.05) differences were reported
between FSH group and Gn group.
Table (3-7) shows the result of all sperm parameters after addition of
high concentration (0.5 IU) from FSH, LH and Gn enriched to SMART
44
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
medium. In general, it was noticed
all sperm parameters have non
significant (P>0.05) differences between all treated groups except DNA
fragmentation. While, significant (P<0.05) decrease were observed in the
sperm DNA fragmentation between LH group and other two groups. In
contrast, non significant (P>0.05) differences were assessed between
FSH and Gn.
43
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Table (3-4): Assessment of sperm parameters among groups of preactivation.
Pre-activation
Sperm Parameters.
First
group
Second
group
Third
group
47.733 a
±3.54
40.133 a
±2.74
42.100 a
±3.58
Progressive
motility
30.467 a
±2.60
31.767 a
±1.94
29.667 a
±1.67
Non Progressive
motility
27.333 a
±2.10
30.667 a
±1.13
28.800 a
±1.24
Immotile
sperm
41.967 a
±2.55
37.567 a
±1.86
41.700 a
±1.92
Normal sperm morphology
(%).
32.233 a
±2.05
33.400 a
±1.09
31.300 a
±1.43
Sperm agglutination (%)
2.333 a
±1.55
2.900 a
±1.71
0.733 a
±0.40
Round cell count (HPF)
9.433 a
±1.32
8.600 a
±1.27
7.967 a
±0.87
DNA fragmentation (%)
25.420 b
±2.95
33.067 a
±2.34
36.703 a
±2.53
Sperm concentration
Millions/ml.
Sperm
grade
activity (%).




Number of infertile patients =90
Means with similar superscripts within each row are non significantly different (P>0.05).
Meanswith different superscripts within each row are significantly different (P<0.05).
Data are the means ±SEM.
43
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Table (3-5): Assessment of sperm parameters when comparing among
control groups of post- activation.
Treatment
Sperm Parameters.
FSH
LH
gonadotropins
13.400 a
±1.5
13.467 a
±1.21
11.400 a
±1.30
35.067 a
±3.26
39.700 a
±3.26
34.767 a
±3.58
Non Progressive 44.233 a
±2.98
motility
38.633 a
±2.91
44.067 a
±3.46
20.367 a
±3.39
22.000 a
±2.43
20.500 a
±3.64
Normal sperm morphology
(%).
45.700 a
±2.66
43.500 a
±2.17
40.833 a
±2.38
Sperm agglutination (%)
0.000 a
±0.00
0.000 a
±0.00
0.000 a
±0.00
Round cell count (HPF)
0.000 b
±0.00
0.100 a
±0.10
0.200 a
±0.15
DNA fragmentation (%)
19.183 b
±2.35
29.267 a
±2.39
25.927 a
±216
Sperm concentration
Millions/ml.
Sperm
grade
activity
(%).
Progressive
motility
Immotile
sperm (%)




Number of infertile patients =90
Means with similar superscripts within each row are non-significantly different (P>0.05).
Meanswith different superscripts within each row are significantly different (P<0.05).
Data are the means ±SEM.
44
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Table (3-6): Assessment of sperm parameters among low concentration
of FSH, LH and Gn groups of post- activation.
Treatment
Sperm Parameters.
FSH
LH
gonadotropins
13.133 a
±1.42
11.500 a
±1.13
12.567 a
±1.53
48.767ab
±4.04
52.800 a
±3.60
39.900 b
±4.80
Non Progressive
motility
38.633 a
±3.07
34.900 a
±2.65
42.800 a
±3.60
Immotile
sperm (%)
12.467 a
±3.04
12.300 a
±3.06
17.467 a
±3.97
Normal sperm morphology
(%).
49.500 a
±2.60
48.100 a
±2.45
44.033 a
±2.54
Sperm agglutination (%)
0.000 a
±0.00
Round cell count (HPF)
0.000 a
±0.00
0.000 a
±0.00
0.000 a
±0.00
DNA fragmentation (%)
18.232 b
±2.83
31.500 a
±3.12
22.443 b
±2.29
Sperm concentration
Millions/ml.
Sperm
grade
activity
(%).




Progressive
motility
0.000 a
±0.00
0.000 a
±0.00
Number of infertile patients =90
Means with similar superscripts within each row are non-significantly different (P>0.05).
Meanswith different superscripts within each row are significantly different (P<0.05).
Data are the means ±SEM.
44
Chapter Three
Results
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Table (3-7): Assessment of sperm parameters among high concentration
of FSH, LH and Gn groups of post- activation.
Treatment
Sperm Parameters.
FSH
LH
gonadotropins
13.233 a
±1.46
12.967 a
±1.29
12.167 a
±1.40
51.367 a
±4.31
52.100 a
±4.19
40.733 a
±4.63
37.033 a
±2.99
35.967 a
±3.43
43.467 a
±3.56
11.833 a
±2.93
12.567 a
±3.46
15.767 a
±3.71
Normal sperm morphology
(%)
50.100 a
±2.88
50.400 a
±2.80
45.333 a
±2.37
Sperm agglutination (%)
0.000 a
±0.00
0.000 a
±0.00
0.000 a
±0.00
Round cell count (HPF)
0.000 a
±0.00
0.000 a
±0.00
0.000 a
±0.00
DNA fragmentation (%)
20.587 b
±3.91
29.467 a
±3.33
17.810 b
±1.59
Sperm concentration
Millions/ml.
Sperm
grade
activity
(%).
Progressive
motility
Non Progressive
motility
Immotile
sperm (%)




Number of infertile patients =90
Means with similar superscripts within each row are non-significantly different (P>0.05).
Meanswith different superscripts within each row are significantly different (P<0.05).
Data are the means ±SEM.
44
Chapter four
Discussion
Chapter Four
Discussion
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Chapter four
Discussion
In vitro sperm activation is very important step in the laboratory
technique that plays an important role in determination the outcome of
assisted reproductive technologies like IUI. In addition, the sperm
preparation technique used, culture media and dependent according to
properties of the semen parameters (Vande vort, 2004).
In the present study, sperm centrifiugation was selected as a method
for in vitro sperm activation depending of the results of the Shaaban
(2007) for different purposes involving removal of effects for
morphologically abnormal spermatozoa, immature sperm cells, epithelial
cells, and lastly seminal leukocytes. Consequently , ROS and sperm
damage are reduced. For the same peroid, is to collect normal
morphologically normal spermatozoa with normal sperm physiology
(Shajer, 2013).
From the results of this study, sperm concentration for all groups
post-activation was significantly reduced (P<0.05) compared to preactivation groups. Similar results was presented by Shaaban (2007). As
mentioned previously, centrifiugation swim-up technique was used in the
present work to remove most sperm with low motility and immotile
sperm, with abnormal morphology, and agglutinated spermatozoa
(Harrison,1976). In addition to remove round cells and epithelial cell
(WHO,1999), and reduced bacterial infection, these results here
considered normal during in vitro activation of human spermatozoa
(Harrison,1976).
63
Chapter Four
Discussion
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
The presence of calcium in the incubation medium is important
because extracellular calcium acts as limitless reservoir of the ion (Beduaddo et al., 2007). Furthermore, Calcium plays a fundamental role in all
the modification of sperm cell properties occurring after the ejaculation,
such as motility, capacitation and the acrosome reaction (Baldi et al.,
1991;Yanagimachi, 1994 ). Therefor, it is not surprising that spermatozoa
maintain calcium homeostasis through the regulation of several types of
calcium channels (Jimenez-Gonzalez et al., 2006; Harper and Publicover,
2005).
In the present study, percentage of sperm motility and progressive
sperm motilty were significantly increased in FSH treated-groups postactivation as compared to the control group and pre-activation group.
Similar results were obtained Arienti et al.(2010). Sperm motility was
enhanced as a result of several factors including migration of sperm from
seminal plasma into culture medium (Ismail and Al-Zaidi, 1998),
contents of SMART medium (Fakhrildin and Flayyih, 2010), generation
of ATP (Litarru and Tiano 2007), improvement of sperm physiology and
metabolism (Mancini et al.,2005).
Follicle stimulating hormone as an agent able to regulate the activity
of ejaculated spermatozoa besides its well known activity on sperm
maturation in the testes and favor the onest of the acrosome reaction
(Arienti et al., 2010). In sperm, the channel is localized primarily to the
tail's principal piece, not the head or mid piece (Ren et al.,2001).
Calcium ion (Ca2+) and cyclic nucleotides control sperm motility
(Hyne and Garbers, 1979; Darszon,1999) and several voltage-dependent
Ca2+-channel (CaV) messenger RNAs and cyclicnucleotide-gated (CNG)
proteins have been detected in sperm cell precursors (Weyand et al.,1994;
Wiesner et al.,1998; Serrano et al.,1999). However, human FSH
64
Chapter Four
Discussion
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
increases [Ca²+] in ovarian (Walker and Cheng, 2005) and in Sertoli cells
(Sharma et al., 1994). FSH is involved in the calcium ion movement of
sperm plasma membrane (McKinney et al., 1994) and induced sperm tail
protein phosphorylation (Lambert et al., 1992; Bajpai et al., 2003) with a
subsequent increase in sperm motility (Tesarik et al., 1994).
The cAMP was demonstrated to play an important role in both sperm
capacitation and the associated protein tyrosine phosphorylation in the
human (Leclerc et al., 1996). Moreover, the intracellular level of cAMP
increaseed through the activation of cAMP protein kinase (Tesarik et al.,
1992; Ho et al., 2002).
In the current study, sperm motility (%) and progressive sperm
motilty (%) were significantly increased for LH and Gn treated groups
post-activation as compared to the control group and pre-activation.
Really, enhancement sperm parameters may be considered as normal
response for sperm physiology after the removal of seminal plasma, pus
cells and agglutinated spermatozoa using sperm preparation techniques.
Furthermore, it was reported that only the active motile sperms will
swim-up to the upper layer of culture medium in vitro human sperm
activation (Mortimer, 2000; Henkel and Schill, 2003).
The results were obtained using a high concentration of the hormone
FSH, LH, or both significantly enhanced in the progressive motility of
sperm compared with the results obtained use low concentration. Perhaps
the reason for that, the high concentration of hormone which help to
move the sperm higher concentration of hormones moves sperm that
seemed quite immotile sperm when microscopic examination exceeded
the progressive motility of sperm. The low concentration for each of the
used hormones can affect the movement speed of motile sperms without
having little impact in moving the immotile sperm.
65
Chapter Four
Discussion
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
when the post-activation groups were compared to per-activation
groups, the normal sperm morphology was significantly enhanced for the
three groups of post-activation. However, high concentration of FSH, LH
and Gn groups produce the best improvement for sperm morphology (%)
as compared to other groups post-activation. In the present study, sperm
activation was done using
centrifugation technique which lead to
precipitate most cells, pus cells and debris within seminal plasma (WHO,
1999). Consequently , most spermatozoa with normal morphology and
progressive motility swim-up into upper layer of culture medium postactivation (Ismail and Al-Zaidi, 1998). Therefore, this procedure will
select live motile sperm with mature condense chromatin. Since, there is
correlation between sperm condensation and morphology, thus this
procedure, also selects sperm with normal morphology (Henkel et
al.,1994).
In the present study, the fragmentation of sperm DNA was
considered an important procedure for male fertility and infertility
diagnosis (Bungum, 2011). SCSA, first described by Evenson et
al.(1980) is show to be an independent marker of fertility in vivo and may
also help in selection of the most effective ART treatment in each in
individual couple (Bungum et al., 2007). Moreover, poor semen quality
has been associated with an increase in the proportion of sperm with
DNA fragmentation (Irvine et al., 2000).
Sperm DNA fragmentation was signifcantly improved for post
activation groups as compared to pre activation group. However, best
improvement for sperm DNA fragmentation was achieved for SMART
medium enriched with high concentration for LH and Gn as compared to
the control group and low concentration for LH and Gn. While, , best
improvement for sperm DNA fragmentation was achieved for SMART
66
Chapter Four
Discussion
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
medium enriched with Low concentration for FSH as compared to the
controle group and high concentration.
Sperm DNA fragmentation was effect by several factors including
oxidative stress (Sharma et al., 2004), in vitro sperm activation tehnique
(Tesarik et al., 2004), presence of sperm with abnormal morphology
(Tang et al., 2010), increment of round cell count (Fariello et al., 2009),
elevation ROS concentration (Altman et al.,1995) and content of SMART
medium (Fakhrildiin and Flayyih, 2010).
Oxidative stress (OS) caused by an imbalance between the
antioxidant ability in seminal plasma and the production of reactive
oxygen species (ROS) leading to the formation of oxidative products such
as 8OHdG is the mechanism that probably most often lies behind sperm
DNA defects. The sperm cell membrane is easily attacked by ROS with
further detrimental effects on nuclear membranes as well as on sperm
DNA (Aitken and Krausz, 2001).
Certainly, DNA damage in sperm can be due to unrepaired DNA
breaks during spermatogenetic chromatin remodeling and packaging or
abortive apoptosis during spermatogenesis. Among other suggested
causes are the effect of endogenous endonucleases and caspases,
exposure to a variety of genotoxic agents because of therapeutic reasons
or because of occupational or environmental exposures, and finally, the
action of oxidative sperm DNA damage (Sakkas and Alvarez, 2010).
In the present study, percentage of sperm agglutination and round
cell concentration, were reduced significantly in all FSH, LH and Gn
when using centrifugation technique for in vitro activation as mentioned
by other study (Younglai et al., 2001; Inaudi et al., 2002). It was noticed
that, the centrifuge technique have efficacy in elimination of agglutinated
sperm and round cell. Moreover, these result can be explained by the
67
Chapter Four
Discussion
‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
effect of centrifugation step in which remove the debris, bacteria and
other cell (effect centrifuge power) (Shaaban, 2007).
Additionally, no significant differences for sperm concentration,
sperm motility (%) and sperm morphology among FSH, LH, and Gn. I
believe these hormone work the same way to in vitro sperm activation.
68
Conclusions
And
Recommendations
Conclusions
From the result of the present study, it was concluded that:
 No differences were found among FSH, LH and Gn in their outcome of
normal sperm morphology, sperm agglutination and concentration of round
cells.
 The high concentration of FSH significantly enhances sperm motility and
high concentration from Gn showed significant enhanced to DNA
 Sperm DNA damage significantly contributes to the growing number of
infertility cases.
Recommendations
It is recommended to follow these suggestions:
 Study the effect of gonadotropin supplementation to culture medium on
sperm mitochondrial apoptosis.
 Study the effect of gonadotropin supplementation to culture medium on
percentage of IVF and early embryonic development in mice as a model for
human.
 Study the effect of gonadotropin supplementation to culture medium on
pregnancy rate for infertile women undergoing IUI.
69
References
References
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Katz, D.F.; Osorio, A.M.; Sublet, V. and Evenson, D.P. (1997).
Assessment of reproductive disorders and birth defects in communities near
hazardous chemical sites, III. Guidelines for field studies of male
reproductive disorders. Reprod. Toxicol. 11:243-259.
 Yanagimachi, R. (1994). Fertility of mammalian spermatozoa:it is
development and relativity. Zygote. 2:371-372.
 Yoshida, K.; Yoshike, T.; Sato, S.T. and Iwamoto, T. (2003).
Quantification of seminal plasma motility inhibitor/semenogelin in human
seminal plasma. J. Androl. 24:987-998.
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70
‫الخالصة‬
‫ٌؼذ ححهٍم انسبئم انًُٕي يٍ أْى انفحٕطبث نخشخٍض انؼقى انزكشي ٔانزي ٌخؼًٍ انفحض انؼٍبًَ‬
‫ٔانًضٓشي‪ْ .‬شيَٕبث انقُذ ًْ ْشيَٕبث األحًبع االيٍٍُّ انخً حفشص يٍ قبم خالٌب انقُذ انًضبص يٍ انفض‬
‫األيبيً نهغذِ انُخبيٍت ٔانخً حشًم انٓشيٌٕ انًحفض نهضشٌب )‪ٔ (FSH‬انٓشيٌٕ انًهٕحٍ (‪ٔ.)LH‬حؼذ ْزِ‬
‫انٓشيَٕبث ػُظشا يشكضٌب فً َظبو انغذد انظًبء انًؼقذ ٔانخً حُظى انًُٕ انطبٍؼً ٔانخطٕس انضُسً‬
‫ٔٔظٍفت ٔانخكبرش‪.‬‬
‫أصشٌج ْزِ انذساست نبحذ حأرٍش ْشيَٕبث انقُذ )‪ )LH(ٔ )FSH‬كم ػهى حذِ أ يضخًؼت بؼذ حضٍٓضْب‬
‫ببنٕسؾ أنضسػً ػهى يؼبٌٍش انُطف انبششٌت ٔحشكٍب انحًغ انُٕٔي انُطف خالل انخُشٍؾ خبسس انضسى‬
‫انحً‪.‬‬
‫حسؼٌٕ شخظب شبسكٕا فً ْزِ انذساست‪ٔ ,‬كبٌ يخٕسؾ أػًبسْى (‪ )45-22‬سُّ‪ .‬قسًج إنى رالد‬
‫يضبيٍغ سئٍسّ اػخًبدا ػهى يكًالث انٕسؾ سًبسث يغ أيب ْشيٌٕ انًحفض نهضشٌب أ انٓشيٌٕ انًهٕحٍ أٔ‬
‫كهًٍٓب يؼب‪ .‬صًؼج ػٍُبث انسبئم انًُٕي يٍ األشخبص ٔاصشي فحض انسبئم انًُٕي ٔفحض حشظً أنذَب‬
‫قبم ٔبؼذ حُشٍؾ انُطف خبسس انضسى‪ .‬قسًج ػٍُت انسبئم انًُٕي إنى رالد أقسبو يخسبٌٔت أصشٌج ػًهٍت‬
‫انطشد انًشكضي(بسشػت ‪ 2400‬دٔسِ‪/‬أنذقٍقّ ٔنًذة ‪ 6‬دقبٌق)‪ .‬أػذث كم يٍ انًضبيٍغ انزالرت‬
‫(‪ )Gn,FSH,LH‬كًب ٌهً‪ :‬أنًضًٕػّ األٔنى (‪ )G1‬يضًٕػت انسٍطشة ٔححخٕي انٕسؾ انضسػً سًبسث‬
‫فقؾ‪ ,‬انًضًٕػت انزبٍَّ(‪ٔ )G2‬ححخٕي انٕسؾ انضسػً سًبسث اغًُ ‪ٔ0.24‬حذِ دٔنٍت يٍ انٓشيٌٕ‪,‬‬
‫ٔانًضًٕػت انزبنزّ (‪ٔ ) G3‬ححخٕي انٕسؾ انضسػً سًبسث اغًُ ‪ٔ%0.4‬حذة دٔنٍت يٍ انٓشيٌٕ‪.‬‬
‫أظٓشث انُخبئش حظٕل اسحفبع يؼُٕي (‪ )P< 0.05‬فً انُسب انًئٌٕت نحشكت انُطف ٔانحشكت انخقذيٍت‬
‫نهُطف ٔانًظٓش انخبسصً نهُطف ٔدنٍم حشظً دَب انُطف نكال يضًٕػخٍٍ ببنًقبسَت يغ يضًٕػت انسٍطشة‬
‫بؼذ اصشاء حقٍُت حُشٍؾ انُطف خبسس انضسى‪ .‬كزانك أظٓشث انُخبئش ٔصٕد اسحفبع يؼُٕي (‪ )P<0.05‬فً‬
‫يؼبٌش انُطف ػُذ إػبفت انخشكٍض ‪ٔ 0.4‬حذِ دٔنٍّ يٍ انٓشيٌٕ (‪ )Gn,FSH,LH‬نهٕسؾ سًبسث فً‬
‫أنًضًٕػّ انزبنزت يقبسَت يغ انًضًٕػت انزبٍَت‪ٔ 0.24‬حذة دٔنٍت‪ٔ.‬اظٓش اسحفبع يؼُٕي(‪ )P<0.05‬فً دنٍم‬
‫حشظً دَب انُطف ػُذ إػبفت ‪ٔ 0.4‬حذة دٔنٍت يٍ انٓشيٌٕ نهٕسؾ أنضسػً يقبسَت يغ انخشكٍض ‪ٔ 0.24‬حذة‬
‫دٔنٍت‪.‬‬
‫فؼال ػٍ رانك فقذ ظٓش أٌ انخشكٍض انؼبنً يٍ انٓشيٌٕ انًحفض نهضشٌب ٌحسٍ يٍ انُسبت انًئٌٕت‬
‫(‪ )%‬نهحشكت يقبسَّ يغ انخشكٍض انٕاؽئ‪ٔ ,‬اٌ انخشكٍض انؼبنً يٍ ْشيَٕبث انقُذ ٌؼضص حشظً أنذَب‪.‬‬
‫ٔيٍ خالل انُخبئش ًٌكٍ االسخُخبس ببٌ إػبفت ْشيَٕبث انقُذ نهٕسؾ أنضسػً ٌحسٍ يٍ يؼبٌٍش انُطف‬
‫ٔإػبفت انخشكٍض انؼبنً ػُذ انخُشٍؾ خبسس انضسى انحً ‪ٔ ,‬اٌ انخشكٍض انؼبنً ٌحسٍ َسبت حشظً أنذَب‪.‬‬
‫جوهىريت العراق‬
‫وزارة التعلين العالي والبحث العلوي‬
‫جاهعت بغداد – كليت العلىم‬
‫تأثير إضافت هريوَاث انقُذ عهى خصائص انُطف نًرضى‬
‫انعقى خارج انجسى انحي باستخذاو انوسط أنزرعي سًارث‬
‫رسالت‬
‫هقدهت الى كليت العلىم‪ ,‬جاهعت بغداد‪ ,‬وهي جسء هي هتطلباث ًيل درجت الواجستير‬
‫في علىم الحياة – علن الحيىاى‬
‫يٍ قبم‬
‫رشا يكي يحًذ عهي‬
‫بكانوريوس عهوو حياة (‪)2011‬‬
‫جايعت بغذاد‬
‫بأشراف‬
‫د‪ .‬صباح َاصر انعهوجي‬
‫د‪ .‬يحًذ باقر يحًذ رشاد‬
‫فخر انذيٍ‬
‫أستار‬
‫ربيع األول‪1435/‬‬
‫أستار‬
‫كاَوٌ انثاَي‪2014 /‬‬