1. Introduction and Aim of work
Human spermatogenesis begins at adolescence and continues
throughout life. This process includes morphologic, cytological and
biological changes, leading to the formation of mature spermatozoa
(Wald et al., 2004). However, the development of spermatids into
spermatozoa, termed spermiogenesis, is characterized by striking
morphological and molecular transformations (Meistrich, 1993). In
elongating and condensing spermatids, major restructuring of the somatic
chromatin takes place in which the histones are first replaced by a group
of arginine and lysine rich proteins called transition proteins (TPs), which
are in turn replaced by protamines (Meistrich, 1989). At that time, the
nucleosomal-type chromatin is transformed into a smooth fiber, and
condensation begins at this stage (Kundu and Roa, 1996).
Male infertility may be caused by several reasons, including
oligozoo-spermia at variable degrees and complete absence of mature
spermatozoa (Wald et al., 2004). Patients with fertility problems have
often been characterized by an increased frequency of producing
spermatozoa with abnormal chromatin (Hughes et al., 1996). Moreover,
chromatin condensation may be altered by various factors, such as a
shortage of zinc or alterations in protamines, which affects the fertilizing
capacity of the spermatozoon (Kramer and Krawetz, 1997).
Infertile cases cannot be evaluated by routine spermatogram,
measuring sperm counts, motility and morphology; hence, it is of great
importance to evaluate the chromatin status when testing the fertilizing
capacity of spermatozoa. However, to date, no single laboratory test can
assess a man’s total fertility as many of these tests used light microscope
observations and suffered from labor intensiveness, intra-observer
variations and low numbers of spermatozoa analyzed, leading in turn to
poor statistical power (Amann, 1989).
These difficulties were approached through the development of
computer-driven instrumentation, most notably the computer-assisted
sperm analysis (CASA) systems for motion analysis, elementary
morphometry and sperm concentration measures.
On the other hand, flow cytometry is a simple rapid procedure that
quantitates DNA content and chromatin condensation for cells present in
human semen. Only 10 minutes were required to measure 5,000 cells per
sample (Evenson and Melamed 1983). Therefore, the computer-
interfaced flow cytometry (FCM) provides a powerful advantage over
1

2. light microscopy techniques in terms of speed, multiple parameters
measured per cell, objectivity, lack of bias in sample selection, and
thousands of cells measured per sample, and thus provides a very high
degree of statistical analysis during evaluation of sperm morphometry
and sperm concentration measures (Shapiro, 1994).
Aim of the work:
This study was intended to evaluate sperm chromatin status of
infertile men, which is of great importance when testing their fertilizing
capacity. We aimed to compare between image cytometry, computer
assisted semen analysis (CASA) and flow cytometry (FCM) methods that
identify different spermatogenic cells and evaluate their chromatin
structure in the semen ejaculate obtained from infertile patients.
These parameters may be of value to evaluate the extent of male
factors in the occurrence of infertility and to predict the outcome of
assisted reproductive techniques (ARTs) as intra uterine insemination
(IUI), in-vitro fertilization (IVF) and intra cytoplasmic sperm injection
(ICSI) particularly with testicular sperm extraction (TESE) in oligo-
spermic patients.
*******
2

3. The Spermatogenic cells and Spermatozoa
The male reproductive system is composed of the testes, genital
ducts, accessory glands and penis (Figure 1). The dual function of the
testis is to produce hormones and spermatozoa. Each testis is surrounded
by a thick capsule of dense connective tissue, the tunica albuginea. The
tunica albuginea is thickened on the posterior surface of the testis to form
the mediastinum testis, from which fibrous septa penetrate the gland,
dividing it into about 250 pyramidal compartments called the testicular
lobules. These septa are incomplete, and there is frequent
intercommunication between the lobules. Each lobule is occupied by one
to four seminiferous tubules enmeshed in a web of loose connective
tissue that is rich in blood and lymphatic vessels, nerves and interstitial
cells, also known as Leydig cells. Seminiferous tubules produce male
reproductive cells, the spermatozoa, whereas interstitial cells secrete
testicular androgens (Junqueira and Carneiro, 2005).
Figure 1: The male genital system. The testis and the epididymis are shown in
different scales than the other parts of the reproductive system. Note the
communication between the testicular lobules (Junqueira and Carneiro, 2005).
Functionally and microscopically, the testis is composed of two
important components, the seminiferous tubules and the intertubular
tissue surrounding the tubules (Snell, 1984).
I. The intertubular tissue: Fibroblasts, macrophages and some mast
cells intermingle with the connective tissue fibers, but the most
conspicuous cells in this intertubular compartment of the testis are Leydig
cells (Fawcett et al., 1973).
3

4. Leydig cells are found singly or in groups, almost invariably
around capillaries for an effective blood supply (Kerr and Dekrester,
1981). Their outline is generally polygonal and their nuclei are oval,
eccentric and slightly irregular. The nuclear chromatin is arranged in the
form of a dense peripheral band attached to the nuclear membrane. The
nucleolus is usually well developed and is close to the nuclear membrane.
Two nucleoli may be present. The cytoplasm is usually abundant,
granular and basophilic (Leeson et al., 1985).
II. The Seminiferous tubules: Each seminiferous tubule is a highly
convoluted tubule about 0.2 mm in diameter and 30 to 70 cm long
(Leeson et al., 1985). The seminiferous tubule is lined by a single layer of
Sertoli cells that are epithelial elements of mesodermal origin. The
complexity of the germinal epithelium itself is attributable to diversity of
germinal elements that pack the spaces between Sertoli cells (Huckins
and Meacham, 1991).
Within the tubules, there are two main types of cells, the Sertoli
cells (sustentacular cells) and the spermatic cells (germ cells) in different
stages of development (Rodriguez-Rigau et al., 1980).
A. The Sertoli cells:
Sertoli first described these cells in 1865, as columnar cells with
cytoplasmic processes extending from the basement membrane to the
lumen of the seminiferous tubule and enveloping the neighboring germ
cells to provide them with physical support (Nursing Cells). Sertoli cells
are the only non-germinal elements within the seminiferous tubules
(Steinbeger A and Steinberger E, 1977).
Sertoli cells are linked by tight junctions along their lateral borders,
dividing the seminiferous tubule into two distinct and separate
physiologic compartments: A basal compartment occupied by
spermatogonia, preleptotene and leptotene spermatocytes, and an
adluminal compartment, occupied by the other stages of the germ cells
population (Hagenäs et al., 1978).
In histological sections under the light microscope Sertoli cells are
usually seen at the periphery of the tubule attached to the basement
membrane. At the fine structural level, these cells are seen to have a pale
ovoid nucleus surrounded by cytoplasmic filaments separating it from the
other cytoplasmic organelles and the large nucleolus often has a tripartite
appearance due to having the so-called chromecentres (Fawcett, 1975).
4

5. Rough endoplasmic reticulum is sparse in the cytoplasm, but
smooth endoplasmic reticulum is abundant. This suggests the secretion of
estrogenic hormone (Kerr and Dekrester, 1981). Lipofuscin granules are
present occasionally. The cytoplasm also contains wavy bundles of fibrils
(Sohval et al., 1971).
Sertoli cells have phagocytic potentialities; they can eliminate the
fragmented cytoplasm following the process of spermiogenesis (Zakaria,
1974).
Figure 2: Part of a seminiferous tubule with its surrounding tissues. The seminiferous
epithelium is formed by two cell populations: the cells of the spermatogenic lineage
and the supporting or Sertoli cells (Junqueira and Carneiro, 2005).
B. The Spermatogenic cells:
The successive order of spermatogenic cells arranged from the
basement membrane to the lumen of seminiferous tubules include
spermatogonia, spermatocytes and spermatids, the exfoliated cells that
become the male gametes or spermatozoa (Gustafson, 1979).
1. Spermatogonia:
The spermatogonia are the stem cells of all other spermatogenic
cells, are situated adjacent to the basement membrane of the seminiferous
tubule. In the human testis four spermatogonial types have been
recognized [A-long (Al), A-dark (Ad), A-pale (Ap) and B] and the
possibility of additional types of A cells has been raised. It is apparent
that type-B spermatogonia are differentiated cells that ultimately produce
5

6. preleptotene spermatocytes. The relationship among the types of type A
spermatogonia is unclear, however, all have been considered for a stem
cell role. The Ad and Ap spermatogonia are numerous while the Al
Spermatogonia occur infrequently (Huckins and Meacham, 1991).
Dark type A spermatogonia (Ad): have spherical or ovoid nucleus.
Their finely granular chromatin is deeply stained and thus looks
homogeneous. One or more clear cavities are present in the chromatin
mass and frequently containing a nucleolus, which is free from adhering
chromatin granules (Clermont, 1963).
Pale type A spermatogonia (Ap): have an ovoid nucleus containing
a uniformly pale gray granular chromatin which gives a ground glass
texture to the nucleus, one or more nucleoli free of chromophilic
chromatin usually present close to the nuclear membrane (Clermont,
1963).
Long type A spermatogonia (Al): are elongated cells with elongated
nucleus and are occasionally mitotically active (Huckins and Meacham,
1991).
Type B spermatogonia (B): have a nucleus containing fine
chromatic granulation, several heavily stained chromatin masses and a
nucleolus detached from the nuclear membrane. In humans, it is difficult
to differentiate these 4 types on biopsy unless Zenker formal fixative is
used (Clermont, 1963).
Initially, it was suggested that Ad spermatogonia were stem cells
that gave rise to Ap spermatogonia (Clermont, 1966). This theory was
subsequently modified to state that Ad spermatogonia were reserve stem
cells that did not normally contribute to spermatogenesis, whereas the Ap
spermatogonia may represent a phase of the cell cycle rather than a
specific cell type (Huckins and Meacham, 1991).
Entry of type B spermatogonia into the prophase of meiosis is
represented by the conversion of those cells into primary spermatocytes,
which divide to form secondary spermatocytes. The latter, after a very
short life span of ± 6 hours in the human, divide to form round
spermatids. These two divisions result in the conversion of the diploid to
the haploid chromosomal complement. Cells passing through the long
phase of the first meiotic division can be subdivided on a cytological
basis into preleptotene, leptotene, zygotene, pachytene and diplotene
stages.
6

7. 2. Spermatocytes:
The spermatocytes undergo meiosis i.e. the two successive
divisions leading to the production of the haploid cells, the spermatids.
The term primary refers to the diploid germ cells during the first meiotic
division, while the secondary refers to the haploid cells during the second
meiotic division (Kerr and Dekrester, 1981).
Primary spermatocytes:
The primary spermatocytes have a diploid number of
chromosomes. They are the largest cells of the spermatogenic cells
(Clermont, 1963). They result from the division of type B spermatogonia,
and then they lose their contact with the basement membrane of the
tubule and begin the long prophase of meiosis, which can be subdivided
into the classical stages of leptotene, zygotene, pachytene, diplotene and
diakinesis. This classification is based on the changes in chromatin
configuration (Kerr and Dekrester, 1981).
Primary spermatocytes have a life span of 24 days in
contradistinction to secondary spermatocytes, which have a short life
span so they are hardly seen in testicular biopsies (Courot et al., 1970).
Preleptotene stage: After arising from B spermatogonia during the
interphase and before entering the long prophase, the spermatocytes are
preleptotene. The preleptotene spermatocyte resembles the B
spermatogonia and the difference is that the nucleus of the preleptotene
spermatocyte is slightly smaller and its chromatin is more deeply stained.
By the end of this interphase, the amount of deoxyribonucleic acid
(DNA) is duplicated. DNA is synthesized only in this stage of
spermatogenesis (Heller and Clermont, 1964).
Leptotene stage: It is the beginning of the meiotic prophase in
which the chromatin crusts or granules resolve into finely beaded
filaments, (chromosomes) and they become clearly filamentous
(Clermont, 1972).
Zygotene stage: The homologous chromosomes are paired. These
paired chromosomes assume the shape of long loops attached by their
extremity on one given area of the nuclear envelop forming the
characteristic (banquet) configuration (Heller and Clermont, 1964).
7

8. Pachytene stage: There is increase in the nuclear volume. Large
spherical nucleolus becomes visible and the chromosomes shorten and
become thicker. They remain in this condition for a long period.
Diplotene stage: This stage is very short and characterized by
maximum size of the nucleus. The chromosomes partially split
longitudinally and the nucleoli split at the end of this stage.
Secondary spermatocytes:
The secondary spermatocytes are characterized by smaller nuclei
than those of late pachytene spermatocytes and contain a pale stained
granular chromatin and several globules of chromophilic chromatin free
in the nucleoplasm or associated with the nucleus envelop. Each cell
enters the second maturation division without duplicating their DNA
resulting in the formation of the haploid spermatids (Courot et al., 1970;
Clermont, 1972).
3. Spermatids:
The spermatids are haploid cells resulting from division of
secondary spermatocytes and no longer capable of division. They are
present near the lumen of the seminiferous tubules and have deeply
stained nuclei. Spermatids undergo a complex series of changes giving
rise to sperms (Clermont, 1963).
By a process, called spermiogenesis the spermatid gives highly
differentiated germ cells called the spermatozoa. No cell division occurs
in this process, which is divided into 12 steps defined by the stages of
development of the positive acrosomic system, these steps were
distributed over four phases (Clermont and Lebland, 1985). The
morphological changes of the spermatid nucleus as seen in ordinary
Hematoxylin and Eosin stained sections are used to identify six
characteristic types of spermatids namely a, b1, b2, c, d1 and d2.
Spermatid a (Sa): The nucleus is spherical, centrally located and
contains pale large chromophilic granules. An empty looking
hemispherical vesicle is occasionally seen at the nuclear surface
corresponding to a distended acrosomic structure.
Spermatid b1 (Sb1): The nucleus is irregular and centrally located. It
shows an increase overall staining while the chromatin appears more
homogenous.
8

9. Spermatid b2 (Sb2): The nucleus is slightly elongated, asymmetric
and contains more deeply stained homogenous chromatin. The nucleus
comes in contact with the cytoplasmic membrane where the head cap
develops. The caudal tube of machete appears at the opposite pole of the
nucleus.
Spermatid c (Sc): The nucleus is elongated and deeply stained. The
apical portion is conical and covered by the unstained head cap while the
caudal extremity is globular. The conical extremity protrudes at the
surface of the elongated cytoplasmic body. The caudal tube surrounds a
clear visible flagellum (Burger et al., 1976).
Figure 3: Top: The principal changes occurring in spermatids during spermiogenesis.
The basic structural feature of the spermatozoon is the head, which consists primarily
of condensed nuclear chromatin. The reduced volume of the nucleus affords the sperm
greater mobility and may protect the genome from damage while in transit to the egg.
The rest of the spermatozoon is structurally arranged to promote motility. Bottom:
The structure of a mature spermatozoon (Junqueira and Carneiro, 2005).
Spermatid d1 (Sd1): The nucleus has undergone further
condensation and flattens dorsoventrally such that in the side view the
apical end is pointed and the caudal end is globular and in the front view
it is paddle-shaped. The flagellum is cleanly visible while the caudal tube
has become indistinct, some eosinophilic granules accumulate around the
flagellum (Fawcett and Bedford, 1979).
Spermatid d2 (Sd2): The nucleus is identical to Sd1 with clear
flagellum and eosinophilic granules. The cell has discarded most of its
cytoplasm called (residual body) while contains basophilic masses. The
9

10. cell now referred to as spermatozoa remains covered with tightly fitting
cytoplasmic membrane, except at the junction of nucleus and flagellum
where a head of cytoplasm is retained (Heller and Clermont, 1964).
4. Mature spermatozoon:
Two features of the spermatozoon make it unique among the cells
of the body. It is flagellated and devoid of cytoplasm (Glover et al.,
1990-b). Under the light microscope the spermatozoon (Figure 3) appear
to be composed of head and tail.
The head (4-5 microns) contains pear shaped nucleus with a
tapered acrosomal end, surrounded by a narrow strip of cytoplasm devoid
of organelles (Nistal and paniagua, 1984).
The tail is subdivided from anterior to posterior into four segments:
1. The neck, which is the area of connection between the head and the
tail.
2. The middle piece (5-7 microns), which contains a sheath of
mitochondria that help motility.
3. The principal piece (45-50 microns), a region whose outer
circumference contains fibrous sheath that aids in support of the tail
region.
4. The end piece (5 microns), which constitute the terminal segment of
the tail (Gustafson, 1979).
*******
10

11. Sperm Chromatin
The condensation of chromatin during spermatogenesis,
epididymal transport and its decondensation at the time of fertilization are
essential for successful fertilization. The development of spermatids into
mature spermatozoa is accomplished by a series of structural and
chemical modifications including a gradual replacement of virtually
lysine-rich histones by transition proteins and then by protamines which
bind more tightly to DNA than do histones and this results in compaction
of chromatin in the sperm nucleus, a process which is termed ‘sperm
chromatin condensation’. Mammalian sperm DNA is the most tightly
packed eukaryotic DNA, being at least six times more highly condensed
than DNA in mitotic chromosomes, which allows the DNA to be
compacted into a small volume (Ward and Coffey, 1991).
Sperm chromatin condensation:
In eutherian mammals, the condensation of sperm chromatin has
two main phases. The first phase, which occurs in the testis, involves the
substitution of somatic histones by testis-specific protamines (Bellvé et
al., 1975; Goldberg et al., 1977).
Protamines are small, only half the size of the core histones they
replace, and are extremely basic. Between 55% and 70% of the amino
acids is arginine. Sperm protamines also contain numerous cysteine
residues, which are used to generate disulfide cross-links between
adjacent protamine molecules during chromatin condensation. Bull sperm
protamine contains 47 amino acids, with 24 arginine and 6 cysteine
residues (Coelingh et al., 1972); and rat sperm protamine consists of 50
amino acids, with 32 arginine and 5 cysteine residues (Marushige Y and
Marushige K, 1975-a). Both protamine molecules are of sufficient length
to fill one turn of DNA, with adjacent protamines locked in place around
DNA by multiple disulfide bridges (Coelingh et al., 1972).
The formation of large numbers of disulfide cross-links between
protamine molecules describes what occurs in the second main phase of
chromatin condensation. These cross-links are formed after the
spermatozoa have exited the caput epididymis and are in route to the
cauda epididymis (Aravindan et al., 1997; Golan et al., 1996).
Spermatozoa that are isolated from the caput epididymis contain
84% of total sulfhydryl (SH) + disulfides (SS) groups in the head region
as thiols; whereas, sperm heads from the cauda epididymis contain only
11

12. 14% of total SH + SS groups as thiols. This difference indicates that
during transit between the two epididymis, almost 1.5 billion disulfide
bonds are formed per individual sperm. Therefore, it is not surprising that
after chromatin condensation, sperms are highly resistant to a variety of
agents such as strong acids, proteases, DNAse, and detergents (Mahi and
Yanagimachi, 1975). The overall effect of chromatin condensation is a
transient inactivation of the male genome (Bedford and Calvin, 1974).
Chromatin condensation is directly related to the capacity of sperm
to fertilize the ovum. For example, spermatozoa from both the caput
epididymis and the proximal corpus epididymis lack the ability to
fertilize; whereas, spermatozoa from the distal corpus epididymis and the
cauda epididymis has this ability (Haidl, 1994; Orgebin-Crist et al.,
1976; Weissenberg et al., 1994).
Sperm chromatin decondensation:
In contrast to spermatogenesis, the process of fertilization requires
that disulfide bonds between protamine molecules be broken. This occurs
before chromatin decondensation, pronucleus formation, and DNA
synthesis (Zirkin et al., 1989; Longo, 1981).
It has been proposed that glutathione, which is present in the egg
cytoplasm, provides the reducing equivalents for the reduction of the
disulfide bonds (Zirkin et al., 1989). The possibility that mitochondria,
located in the middle piece of the spermatozoon, might be involved in
decondensation via a lactate/pyruvate shuttle system (Gallina et al.,
1994) has also been considered. It has also been suggested that chromatin
decondensation is the result of a trypsin-like, acrosomal protease that
causes a proteolytic degradation of sperm protamine (Marushige Y and
Marushige K, 1975-b).
Effect of oxidation on sperm chromatin:
There is no question that the oxidation and reduction of sulfhydryl
groups is critical to sperm chromatin condensation/decondensation.
However, very little is known about the processes, or whether each
utilizes the same mechanisms. The usual recipient for reducing
equivalents is NAD(P)+, which is reduced to NAD(P)H. Unless the
nuclear region contains an unlimited supply of NAD(P)+, it is critical that
NAD(P)H transfer its reducing equivalents to some other molecule
(Burgos et al., 1982; Coronel et al., 1986).
12

13. During the early stages of spermatogenesis, reducing equivalents
can be transferred from the cytoplasm into the mitochondria via shuttle
systems (Burgos et al., 1982; Coronel et al., 1986). However,
spermatozoa lack cytoplasm, and their mitochondria are located in the
middle piece (Monesi, 1972). Without cytoplasm, it is unlikely that
spermatozoa can transfer reducing equivalents from the head region to the
middle piece. The unique structure of spermatozoa, relative to that of a
typical cell, suggests that their pathway for oxidizing NAD(P)H is unique
as well, indicating that endogenous NAD(P)H was the source of the
reducing equivalents (Chapman and Michael, 2003).
Chromatin abnormalities and male infertility:
Human spermatozoa in which the chromatin is not completely
condensed are reported to have a low percentage of fertilization
(Hammadeh et al., 1998). In a recent study, human sperm that were
incompletely condensed failed to fertilize, even after their injection
directly into the ovum (Rosenbusch, 2000).
However, incomplete chromatin condensation is independent of
other causes of infertility, such as abnormalities in sperm morphology
(teratozoo-spermia), low sperm count (oligo-spermia), or poor sperm
motility (asthenozoo-spermia). It has been suggested that incompletely
condensed sperm constitute a significant factor in the assessment of male
fertility (Hammadeh et al., 2001).
Chromosomal abnormalities and male infertility:
Chromosomal abnormalities are common in infertile men
(Egozcue et al., 2000). Both numerical and structural abnormalities may
predispose to severe congenital abnormalities during formation of
gametes (Diemer and Desjardins, 1999).
Meiosis is a double-division process that is preceded by only one
DNA replication event to produce haploid gametes. The defining event in
meiosis is prophase I, during which chromosome pairs locate each other,
become physically connected, and exchange genetic information (Cohen
et al., 2006).
Primordial germ cell development, spermatogonial proliferation
and survival as well as the various stages of meiosis are regulated by a
number of germ cell-specific proteins. These proteins include lactate
dehydrogenate C4, phosphoglycerate kinase 2, cytochrome cT, and the
heat shock protein (HSP) 70-2. Absence of these proteins results in partial
13

14. to complete arrest in meiosis, leading to male infertility (Eddy and
O'Brien, 1998; Venables and Cooke, 2000).
Infertile men with a normal karyotype and low sperm concentration
or certain types of morphologically abnormal spermatozoa have a
significantly increased risk of producing aneuploid spermatozoa,
particularly for the sex chromosomes (Shi and Martin, 2001).
Meiotic disorders are frequent in infertile males, and increase with
severe oligoasthenozoo-spemia and/or high follicle stimulating hormone
concentrations. These patients produce spermatozoa with autosomal and
sex chromosome disomies, and diploid spermatozoa. Their contribution to
recurrent abortion depends on the production of trisomies, monosomies
and of triploids (Egozcue et al., 2000).
The most frequent sperm chromosome anomaly in infertile males is
diploidy, originated either by meiotic mutations or by a compromised
testicular environment. However, in chromosomally normal infertile
males, the rates of chromosome 21 and sex chromosome disomy in
spermatozoa are increased. Higher incidences of trisomy 21 (seldom of
paternal origin) and sex chromosome aneuploidy are also found. XXY
and XYY patients produce increased numbers of XY, XX and YY
spermatozoa, indicating an increased risk of production of XXY, XXX
and XYY individuals (Egozcue et al., 2000).
Klinefelter's syndrome is the most common sex chromosomal
abnormality seen in infertility clinic (Pandiyan and Jequier, 1996). In
this condition, the white cell karyotype shows the presence of a 47,XXY
configuration. This karyotypic anomaly is always associated with primary
testicular disease presented clinically as azoospermia. Testicular size is
always reduced and serum levels of gonadotrophins are rose. The
testicular histology is characterized by almost total atrophy of
seminiferous tubules (Klinefelter et al., 1942).
Genetic abnormalities and male infertility:
About 2,000 genes regulate spermatogenesis. Most of them are
present on somatic chromosomes. However, approximately 30 genes are
located on the Y-chromosome. Autosomal genes regulate the metabolic
processes in spermatogenic cells. Y genes are not essential for general
body function. However, they are vital to male reproductive processes
(Hargreave, 2000).
14

15. Three different spermatogenesis loci have been mapped on the Y
chromosome and named "azoospermia factors" (AZF a, b, and c).
Deletions in these regions remove one or more of the candidate genes
(DAZ, RBMY, USP9Y, and DBY) and cause severe testiculopathy
leading to male infertility (Foresta et al., 2001).
Moreover, Reijo et al. (2000) mentioned that DAZ and DAZL
proteins are present in the nucleus and cytoplasm of fetal gametocytes. In
adults both proteins are abundant in the nucleus of spermatogonia.
However they are transit to the cytoplasm of primary spermatocytes at
meiosis.
Azoospermia factor (AZF) deletions are genomic deletions in the
euchromatic part of the long arm of the human Y chromosome (Yq11)
associated with azoospermia or severe oligozoo-spermia. Consequently, it
can be assumed that these deletions remove Y chromosomal genes
required for spermatogenesis. However, these ‘classical’ or ‘complete’
AZF deletions, AZFa, AZFb and AZFc, represent only a subset of
rearrangements in Yq11 (Vogt, 2005).
About 7% of male factor infertility has been attributed to
submicroscopic deletions of the Y-chromosome (Hargreave, 2000). The
prevalence of Y chromosome microdeletions is 4 % in oligozoo-spermic
patients, 14 % in idiopathic severely oligozoo-spermic men, 11 % in
azoo-spermic men, and 18 % in idiopathic azoo-spermic subjects
(Foresta et al., 2001).
Therefore routine cytogenetic analysis of all infertile male patients
is required but it may be advisable to limit routine Y chromosome
microdeletion screening to patients with severe male factor infertility
(<or=5 x 106/mL) (Quilter et al., 2003).
*******
15

16. Male infertility
Infertility is a reproductive health problem that affects many
couples in the human population. Infertility is defined as the state in
which a couple wanting a child cannot conceive after 12 months of
unprotected intercourse (Mueller and Daling, 1989; Thonneau et al.,
1991).
Infertility is either primary or secondary. In primary infertility no
pregnancy has ever occurred. In secondary infertility however, there has
been a pregnancy, regardless of the outcome. About 67-71% and 29-33%
of patients have primary and secondary infertility, respectively (Mueller
and Daling, 1989; Thonneau et al., 1991; Irvine, 1998).
Infertility affects 15% couples attempting pregnancy and in
40-50% of these cases the male partner has qualitative or quantitative
abnormalities of sperm production (Dada et al., 2003).
A man’s role in conception is considered to be relatively simple
and straightforward, but in fact his reproductive physiology is quite
complicated. It involves secretion of testosterone, communication
between the pituitary gland and testes, and a highly involved process of
sperm production, maturation and delivery.
Male factor infertility is defined as an abnormality in sperm
production, function or delivery that impedes a couple from establishing a
pregnancy. The manufacturing and delivery systems must function
properly in order to produce large quantities of healthy sperm, which is
the basis of effective conception. Therefore, when a couple is having
trouble conceiving it makes sense to not only evaluate the female partner
but also the male. Male infertility has been found to be the major cause of
a couple’s inability to conceive in 50% of childless couples.
Replication errors and DNA fragmentation are two types of DNA
damage that occur in spermatozoa. Spermatozoa of older men have a
history of more cell divisions than those of younger men. Consequently,
spermatozoa of older men might exhibit a higher incidence of mutations
because of replication errors. It has been shown that the occurrence of a
dominant genetic disease might involve a mutation in the father's germ
line and is strongly correlated with paternal age (Crow, 1997). DNA
fragmentation is represented by single and double DNA strand breaks. It
is particularly frequent in the ejaculates of sub fertile men (Irvine et al.,
2000).
16

17. Causes of male infertility:
There are many causes of male infertility including: 42%
varicocele, 14% obstructions, 23% idiopathic and 21% other (including
chromosome abnormalities, infections, undescended testes, systemic
illnesses, environmental causes, social habits and sexual dysfunction).
Varicocele:
Varicocele is the result of retrograde reflux of blood down the
internal spermatic vein causing dilatation of pampiniform plexus
(Wishahi, 1991). Varicocele is present in 19% - 41% of infertile men as
listed in Table 1 (Nagler et al., 1997).
Left-sided varicoceles are 10 times more common than those on the
right side are. It is due to the hemodinamically unfavorable merging of
the left spermatic vein into the left renal vein, while the right spermatic
vein drains directly into the inferior vena cava. In addition, the left
internal spermatic vein is approximately 8 - 10 cm longer in course than
the right internal spermatic vein, leading to a relative increase in the
hydrostatic pressure within. In addition, the relatively increased pressure
in the left spermatic vein is due to compression of the left renal vein
between the superior mesenteric artery and aorta, the so called "nut-
cracker phenomenon" (Coolsaet, 1980). In addition, the left renal vein
shows narrowing of its lumen as it crosses the abdominal aorta, leading to
increase in the pressure in left spermatic vein (Buschi et al., 1980).
Varicocele may be graded clinically according to (Dubin and
Amelar, 1970; Hudson et al., 1986) as follows:
• Grade I: palpable enlargement of veins only with valsalva maneuver.
• Grade II: clearly palpable enlargement of veins with and without
valsalva.
• Grade III: visible enlargement of the veins.
Varicocele may be primary, which is the most common, or
secondary due to compression of renal or internal spermatic veins by a
neoplasm. Secondary varicocele characteristically does not disappear
when the patient attains the supine position (Browse, 1991).
As regards age incidence, varicocele is rare in prepubertal children
(Stewart, 1974) while in adolescents, grade I varicocele was found in
18%, grade II in 12%, and grade III in 5% of populations examined
(Niedzielski et al., 1997). A dramatic increase in the incidence occurs
17

18. with the onset of puberty, which might be due to increased perfusion at
that time, unmasking the underlying abnormalities (Stewart, 1974).
Number Men with
Authors %
of patients varicocele
* Dubin and Amelar, 1970 21294 512 40
* Hendry et al., 1973 166 32 19
* Stewart, 1974 195 48 25
* Johnson, 1975 120 38 32
* Greenberg et al., 1977 425 159 37
* Rodriguez et al., 1978 455 108 24
* Cockett et al., 1984 600 246 41
* Aufjes and Vander vijver, 1985 742 180 24
* Marks et al., 1986 1255 480 38
Table 1: Incidence of varicocele among infertile men (Nagler et al., 1997)
Varicocele appears to affect spermatogenesis regardless of the
fertility status (Saypol, 1981). In infertile patient with varicocele, semen
analysis may show oligozoo-spermia, terato-spermia or asthenozoo-
spermia, or a variable combination of these elements (Dubin and
Amelar, 1970). On the other side, Mahmoud et al., (1983) showed that
there was a significant quantitative increase in the DNA content of
spermatozoa in infertile patient with varicocele. However, in some cases
of infertility with varicocele, semen analysis shows perfectly normal
quantitative parameters, in this condition, sperm function test will be
important.
Possible causes of infertility in men with varicocele:
An increase in testicular temperature: The scrotum has an
important role in maintaining the testes at a temperature lower than the
abdominal temperature (Junqueira and Carneiro, 2003). Lewis and
Harrison (1970) reported an increase in scrotal temperature when
varicocele is associated with abnormal spermatogenesis and no increase
of scrotal temperature when varicocele is associated with normal
spermatogenesis. However, Mieusset et al. (1987) reported an increase in
scrotal temperature in infertile men with no varicocele. Thus, one cannot
rely solely on thermography to diagnose varicocele, but can use it to
confirm the clinical impression (Nagler et al., 1997).
Hypoxia: Venous stasis in varicocele leads to hypoxia and
hypercapnia with an adverse effect on spermatogenesis (Donohue and
18

19. Brown, 1969). Among patients with large varicoceles, it is possible that
the weight of the blood in a varicocele could impede the arterial input
into the testis and thus causes infertility by a simple ischaemic change
(Comhaire et al., 1983).
Antisperm antibodies: Hyperthermia resulting from venous stasis
in case of varicocele may damage the blood testicular barrier with
subsequent formations of anti sperm antibodies (Nistal and Paniagua,
1984). There is a significant increase in the level of antisperm antibodies
in infertile group of patients with varicocele in comparison with other
infertile men with no varicocele (Golomb et al., 1986).
Reflux of renal and adrenal metabolites: Reflux of metabolites of
the kidney and adrenal gland into left spermatic vein cause a harmful
effect of varicocele on testicular function (Turner, 1983). Among the
accused metabolites are prostaglandin E, prostaglandin F (Ito et al.,
1982), prostaglandin F2α , Catecholamines (Cohen et al., 1975) and
serotonin (Cockett et al., 1984). Cockett et al. (1998) suggested that the
poor sperm motility associated with varicocele might be attributed to the
increased serotonin level in seminal plasma.
Changes in hormonal levels: Testosterone deficiency, with its
negative effect on spermatogenesis, has been demonstrated in patients
with varicocele by (Comhaire and Vermeulen, 1975). In 1983 and 1985,
Hudson et al., studied the effect of varicocele on hypothalamic-pituitary
axis and reported an excessive gonadotrophin response to exogenous
GnRH in a group of infertile patients with varicocele, with sperm count
less than one million.
Epididymal factor: It has been suggested that varicocele may
impair epididymal sperm maturation and may cause occult epididymal
obstruction (Glezerman et al., 1976).
Primary testicular diseases:
Primary testicular disease is a primary disorder of the testes and not
secondary to any malfunction of the pituitary or the hypothalamus.
Primary testicular disease causes a disruption of the spermatogenic
process and will be presented clinically as either oligo-spermia or in more
severe cases as azoospermia (Jequier and Holmes, 1993). The main
causes of infertility in men are oligo-spermia, astheno-spermia, teratozoo-
spermia and azoospermia, which account for 20-25% of case (Egozcue et
al., 2000; Hargreave, 2000).
19

20. Although in the majority of men with this condition its etiology
may be unknown, many causes can be identified from the history. One of
the most common identifiable causes is testicular maldescent. It is
present in around 20% of men with primary testicular disease (Jequier
and Holmes, 1993) and may be present in around 5-9% of the infertile
men presenting at the clinic (Mieusset et al., 1995). How testicular
maldescent might damage sperm production is unclear. However, it is
likely to be caused by the increased temperature that is present within the
abdominal cavity. Testicular temperature in the scrotum is around 35 oC
whereas in the intra abdominal temperature is 37-38 oC. It is known that
warming the testis impairs sperm production (Tessler and Krahn, 1966).
Another identifiable cause for primary testicular disease is
testicular torsion, which is frequently associated with testicular
maldescent. When a testis undergoes torsion, the venous return and then
the arterial input are cut off. Consequently, the testis will become
ischaemic. Clinically, it is presented with acute pain. The diagnosis often
confirmed by using color doppler ultrasound, which will demonstrate a
lack of blood flow through the testis, which has undergone torsion
(Yazbeck and Patriquin, 1994).
Trauma to the testis is an important cause of primary testicular
disease, as it is known that nearly 10% of male children have suffered
from significant trauma to the gonads prior to puberty (Finkelhor and
Wolak, 1995). In adults, testicular biopsy is a common form of trauma
(Schlegel and Su, 1997). Injury may cause oedema or intra-testicular
hematoma. Either of these abnormalities will increase intra-testicular
pressure and reduce the venous drainage and then the arterial input to the
testis affecting its blood supply. The consequent ischaemia, if prolonged,
will thus result in reduction in the activity of the spermatogenic
epithelium resulting in infertility (Markey et al., 1995).
Orchitis is another cause of primary testicular disease. The most
famous cause of orchitis as an agent of infertility is the viral disease of
mumps (Jameson, 1981). Clinically, it can be quite difficult to
distinguish an acute epididymo-orchitis from testicular torsion as both are
presented clinically by acute sudden pain. Doppler imaging of the
testicular arterial blood flow will distinguish between the two (Paltiel et
al., 1998).
The use of anticancer therapy and x-irradiation in the treatment of
malignancy. Irradiation has its main effect upon the spermatogonia.
Thus, a change in the sperm count will appear (Rowley et al., 1974).
20

21. Endocrinal diseases:
Acromegaly is an endocrinal disorder affecting 2-4 per million of
the population (Alexander et al., 1980). The disease is caused by over
production of growth hormone by the anterior pituitary gland and is
usually associated with the growth of an acidophil adenoma of the
pituitary. Impotence and loss of libido occur in around one third of men
with this disease (Nabarro, 1987) and this is probably one of the major
causes of infertility in these men. Another cause is reduced sperm
production and motility due to decreased serum testosterone and elevated
prolactin level (Franks et al., 1976).
Hyper-prolactinaemia is another cause of infertility. Excess
secretion of prolactin results in reduced secretion of LH resulting in a
reduction in testosterone secretion. In addition, testosterone response to
human chorionic gonadotrophin (HCG) is impaired, thus hyper-
prolactinemia may present with a low sperm count and infertility
(Merino et al., 1997). Other causes of infertility present in patients with
hyperprolactinaemia include retrograde ejaculation (Ishikawa et al.,
1993) and even epididymal obstruction (Jequier et al., 1979).
Hypothyroidism can cause male infertility through sexual
dysfunction that may be present in as many as 80% of male patients with
severe primary myxedema (Griboff, 1962). This form of thyroid disease
may also affect semen quality (Buitrago and Diez, 1987).
Another endocrinal disorder as Cushing’s disease. In which the
anterior pituitary gland secretes excessive quantities of adreno-cortico-
trophic hormone (ACTH), resulting in excessive production of cortisol by
adrenal gland (Cushing, 1932). Hypercortisolism may occur due to
primary adrenal hyperplasia or as a result to administration of large doses
of corticosteriodes. In male patients with Cushing’s disease, there is
usually reduction of LH and testosterone level in serum. In addition, the
LH and FSH response to gonadotrophin releasing hormone are also
blunted (Luton et al., 1977). Because of these changes, these men show
signs of hypogonadism. Oligo-spermia is frequently encountered
(Gabrilove et al., 1974).
In male patients with uremia, testosterone production is reduced
and spermatogenesis may be impaired (Lim, 1994). There may also be an
element of Leydig cell resistance in these men (Holdsworth et al., 1977).
Diabetes is one of the most common endocrine diseases and will
indeed frequently present in an infertility clinic (Dunsmuir and Holmes,
21

22. 1996). Impotence is a very common complication of diabetes and occurs
in 50% of all diabetics (Sexton and Jarow, 1997). The serum testosterone
in men with diabetic impotence may be reduced (Murray et al., 1987).
Testicular biopsies from impotent diabetic patients show a wide range of
changes that vary from a minimal reduction in spermatogenesis to the
presence of totally hyalinized and non-functional seminiferous tubules
(Cameron et al., 1985).
Men with a condition called Haemochromatosis are frequently
infertile (Tweed and Roland, 1998). Haemochromatosis is a disorder of
iron metabolism. Excessive quantities of iron are laid down in the tissues.
One of these tissues is pancreas. Thus, all the vascular and neurological
complications of diabetes may be seen in patients with haemochromatosis
and disorders of potency and ejaculation that relate to diabetes may occur
in patients with this disorder. Large amounts of iron are deposited in the
cells of anterior pituitary (Peillon and Racadot, 1969) resulting in a
reduced production of both FSH and LH leading to reduction of
testosterone (Stocks and Powell, 1972).
It has been pointed out that some men who are clinically normal
but who have oligozoo-spermia may in fact resistant, as elevated
testosterone levels were noted in a group of them (Aiman and Griffin,
1982).
Drugs and toxins:
Many drugs may have a role in causation of infertility, such as
sulphasaiazine, which is now very common agent, used in treatment of
inflammatory bowel disease, in particular in the control of ulcerative
colitis (Levi et al., 1979). This agent will change the sperm concentration
together with changes in both sperm motility and morphology (Birnie et
al., 1981).
One of the most commonly prescribed chemotherapeutic agents is
cyclophosphamide. The damage done by this agent is also dependent in
daily doses of 3.7 mg/kg. Damage to spermatogenesis always occurs
(Hsu et al., 1979) resulting in oligo-spermia or azoospermia.
Phenytoin, which is anticonvulsant, is known to reduce sperm
count probably by its action in reducing FSH level (Stewart et al., 1976).
Caffeine may also interfere with fertility but its mode of action is
unclear (Gerhard and Runnebaum, 1992).
22

23. Nicotine is also known to have adverse effect on fertility. Cigarette
smoking also has an important relationship with the incidence of erective
failure (Gilbert et al., 1986), this effect is a result of the direct action of
nicotine on tissue but in long term is also likely to act by the induction of
arteriosclerosis causing reduction of the blood flow in the penile artery
(Gilbert et al., 1986). Nicotine may reduce the sperm count and increase
the frequency of morphologically abnormal forms in semen sample
(Gerhard and Runnebaum, 1992).
Smoking marijuana cigarettes cause a reduction in the secretion of
gonadotrophins (Smith and Asch, 1987). Consequently, testosterone level
in the serum will be reduced (Kolodny et al., 1974).
A number of different effects of the opiates (heroin) have been
described. The depression of gonadotrophins secretions will last for 2-3
hours (Wang et al., 1978). This will lead to a reduction in serum
testosterone. The opiates will also stimulate the production of prolactin,
which may interfere with testosterone production (Van Vugt et al., 1979).
Alcohol may induce changes in morphology of spermatozoa (Dixit
et al., 1976) because semen samples from alcoholic men will show a
greatly increased incidence of abnormal forms (Lester and Van Thiel,
1977). Alcohol will also have an effect on reproductive function. The
major endocrine effect of alcohol is on Leydig cell function, which results
in a reduction in testosterone synthesis. Both alcohol and its metabolites
inhibit testicular enzymes involved in testosterone production (Johnston
et al., 1981).
Toxic agents are now becoming an important cause of infertility in
the male. They can act directly on the gonad itself or they may interfere
with the normal function of the pituitary-hypothalamic axis. Cadmium is
well known as testicular toxin, the effect of cadmium on the testis appears
to be manifest in the Sertoli cells and probably cause damage to
spermatogenic epithelium (Suzuki et al., 1978).
Lead has been known to reduce fertility by increasing
abnormalities of the semen analysis and by causing decreased libido
(Lancranjan et al., 1975). Lead may have an action on the FSH
receptors, thus interfering with spermatogenesis (Wiebe et al., 1983).
The ethylene glycols used in the printing industry may also
interfere with spermatogenesis. Indeed, an increased incidence of
oligozoo-spermia was demonstrated in a group of patients whose paint
contained these solvents (Hardin, 1983).
23

24. Flow cytometry
Flow cytometry is the measurement of numerous cell properties
(cytometry) as the cells move in a single file (flow) in a fluid column and
interrupt a beam of laser light. The method allows the quantitative and
qualitative analysis of several properties (multiparameter) of cell
populations from body fluids. (Wood, 1993(.
Flow cytometers function as particle analyzers in all of the
applications such as interrogation of membrane, cytoplasmic, and nuclear
antigens, also cellular constituents, such as organelles, nuclei, DNA,
RNA, chromosomes, cytokines, hormones, and protein content (Radcliff
and Jaroszeski, 1998).
Cells or particles are prepared as single-cell suspension for flow
cytometric analysis. This allows them to flow as single file in a liquid
stream past a laser beam. As the laser strikes the individual cells. First
light scattering occurs that is directly related to structural and
morphological cell features. Second, fluorescence occurs if the cells are
attached to a fluorescent probe. Fluorescent probes are typically
monoclonal antibodies that have been conjugated to fluorochromes; they
can also be fluorescent stains reagents that are not conjugated to
antibodies (Parks and Herzenberg, 1989).
After acquisition of light scattering and fluorescence data for each
particle, the resulting information can be analyzed utilizing a computer
and specific software that are associated with the cytometer (Rose et al.,
1992). Flow cytometry is particularly important for biological
investigations because it allows qualitative and quantitative examination
of whole cells and cellular constituents that have been labeled with a wide
range of commercially available reagents, such as dyes and monoclonal
antibodies (Melamed et al., 1990).
Flow cytometry has become a powerful tool to be used in research
as well as in the clinical investigations because cytometers have the
capability to process thousands of individual particles in a matter of
seconds (Longobardi-Given, 1992).
History of flow cytometry:
Quantitative cytometry has its origins in the 1930s in the
pioneering work of nucleic acid measurements of the cell by Caspersson
(Caspersson, 1930). The need to make measurements of large cell
24

25. populations rapidly and accurately stimulated the development of
instruments that were the forerunners of present day flow cytometers.
Light scattering was used as an indicator of the presence of a particle. A
significant discovery was the report by Coons and Kaplan of the
conjugation of fluorescein to antibodies, which opened the field of
detection of tissue antigens by specific antibodies using fluorescence. The
next important development took advantage of the low electrical
conductivity of a cell in respect to saline solutions. The rise of electrical
impedance as cells suspended in saline passed through an orifice was
used as a measure of cell volume. In the early 1960s, Kamentsky
developed a rapid cell spectrophotometer. Utilizing computer technology
to make accurate statistical analysis of data possible, the instrument
measured cell size (by light scattering at 410 nm) and DNA content (by
absorption at 260 nm) (Lee, 1999(.
The need for multiparameter analysis was satisfied by the
introduction of fluorescent dyes for measurement of total DNA content in
the detection of cancer cells and fluorescent antibodies specific for cell
surface markers in the separation of cell subpopulations. Flow cytometers
have been commercially available since the early 1970's, and their use has
been increasing since then. One of the early commercially available flow
cytometers, the Hemalog (Technicon), found wide applications in
hematology laboratories for differential blood counts. Light scattering
was combined with absorption measurements at different wavelengths
using dyes and chromogenic substrates of enzyme action to identify the
blood cell populations. Eventually, other light sources such as helium;
neon or argon ion lasers were used in the modem flow (Steen, 1990).
Throughout history, few other scientific techniques have involved
the contributions of specialists from so many different backgrounds and
disciplines as flow cytometry. A partial list of the various disciplines
involved in the development of flow cytometry includes biology,
biotechnology, computer science, electrical engineering, laser technology,
mathematics, medicine, molecular biology, organic chemistry, and
physics (Robinson, 1993).
By the mid 1970s, commercial flow cytometers began to appear on
the market. New focus was placed on fluorchrome development, methods
of cell preparation and enhanced electronic data handling capabilities.
Scientists, instrument manufactures, and biochemical industries
perpetuated the development of flow cytometry throughout the 1980s and
early 1990s (Radcliff and Jaroszeski, 1998).
25

26. Design of flow cytometers:
Flow cytometers can be described as four interrelated systems
which are common to all cytometers regardless of the instrument
manufacturer and whether or not the cytometer is designed for analysis or
sorting (Melamed et al., 1990; Longobardi-Given, 1992).
1. The fluidic system that transports particles from a sample through the
instrument for analysis.
2. The illumination system that is used for particle interrogation.
3. The optical and electronic system for direction, collection, and
translation of scattered and fluorescent light signals that result when
particles are illuminated.
4. The data storage and computer control system that interprets
translated light and electrical signals and collates them into
meaningful data for storage and subsequent analysis.
1. Fluidic system:
Flow cytometers involve sophisticated fluidics. As the cell
suspension is carried by the sample delivery system to the flow cell where
it is injected through a small-bore (50-300 µm) injection needle into a
larger diameter, rapidly flowing sheath stream (flow rate of l0 m/sec.).
When conditions are right, sample fluid flows in a central core that does
not mix with the sheath fluid (which is a free particles fluid). This is
termed laminar flow. The fluidics hydro-dynamically focuses the cells
within the sheath fluid into a stream allowing the cells to be in a row of
diameter hovering around diameter of one separate individual cell.
Although it makes measurements on one cell a time, it can process
thousands of cells in a few seconds. Since different cell types can be
distinguished by quantitating structural features, flow cytometry can be
used to count cells of different types in a mixture (Givan, 2001).
The pressure of the sheath fluid against the suspended particles
aligns the particles in a single file fashion. This process is called
hydrodynamic focusing and allows each cell to be interrogated by the
illumination source individually while traveling within the sheath fluid
stream (Ormerod, 1994; Radcliff and Jaroszeski, 1998).
2. Illumination system (Figure 4):
In most flow cytometers, the light source of choice is a laser, which
can provide from milliwatts to watts of light. It can be inexpensive, air-
cooled units or expensive, water-cooled units and it can provide coherent
26

27. light of specific wavelength. Scattered and emitted fluorescent light is
collected by two lenses (one set in front of the light source and one set at
right angles) and by a series of optics, beam splitters and filters, specific
bands of fluorescence can be measured. It can measure physical
characteristics such as cell size, shape and internal complexity. Any cell
component or function that can be detected by a fluorescent compound
can be examined (Murphy, 1996).
The flow cytometers used in clinical laboratories have an argon ion
laser that emits light at 488 nm wavelength (blue to blue-green). For the
fluorochrome to be useful, the fluorescent wavelength must be longer
than the excitation light. The fluorescence emitted by each fluorochrome
is usually detected in a unique fluorescence channel located perpendicular
to the excitation light beam and is detected in the side or 90° scatter
channel. The specificity of detection is controlled by the wavelength
selectivity of optical filters and mirrors. This location was chosen to
minimize the amount of excitation light that is scattered into the
fluorescence photo-detectors (Lenkei et al., 1998).
Figure 4: Illumination, optical and electronics system
27

28. 3. Optical and electronic system (Figure 4):
Light and fluorescence are generated when the focused laser beam
strikes a particle within the sample stream. These light signals are then
quantitated by the optical and electronic system to yield data that is
interpretable by the user.
The optical and electronic system of a typical flow cytometer is
responsible for collecting and quantitating at least five types of
parameters from this scatter light and emitted fluorescence. Two of these
parameters are light scattering properties. Light that is scattered in the
forward direction (in the same direction as the laser beam) is analyzed as
one parameter, and light scattered at 90o relative to the incident beam is
collected as a second parameter (Shapiro, 1994).
The laser light can excite cellular constituents such as cytochromes
to fluorescence causing cells to emit light at longer wavelength, this
property is called auto-fluorescence. Certain dyes (fluorochromes) can
absorb the laser light and emit light at longer wavelengths, this is known
as fluorescence.
Fluorescence is detected using networks of mirrors, optics, and
beam splitters that direct the emitted fluorescent light toward highly
specific optical filters. The filters collect light within the range of wave
lengths associated with each of the three fluorescent channels. Filtered
light is directed toward photo-multiplier tubes (PMTs) for conversion into
electrical signals. The signals are then digitized, which results in a
fluorescent intensity for each analyzed cell or particle (Murphy, 1996).
A. Forward-scatter (FS) sensor and detector:
Figure 5: The forward angle light scatter sensor
The forward angle light scatter (FALS) sensor (Figure 5) detects
the light scattered by a cell in the forward direction near the axis of the
28

29. incident beam (along the same axis that the laser light is traveling) and
detected in the forward scatter channel.
The signal is approximately proportional to cell size and optical
homogeneity of cells (or other particles). FS is particularly useful for
discrimination between cells and debris in different cell types. The FS
channel is a photodiode, which is used for strong signals when saturation
is a potential problem (Zola, 2000).
B. Side-scatter (SS) sensors and detectors:
Figure 6: Analysis of human peripheral blood cells by flow cytometry. Blood cells are
separated on the bases of their size that scatters light in a forward direction (FS), as
well as on the basis of their granularity that scatters light at a 90˚ angle (SS).
Lymphocytes (L) (small and non-granular) occupy the lower left area of the screen.
Polymorpho-nuclear leucocytes (P) (large with many granules) occupy the upper
right. Monocytes (M) are detected in an intermediate position (Lee, 1999).
The collected optics for SS sensor is located perpendicular to the
excitation light beam and is detected in the side or 90° scatter channel.
The intensity of side scatter is proportional to the size, shape and optical
homogeneity of cells (or other particles). Forward scatter tends to be
more sensitive to surface properties of particles (e.g., cell ruffling) than
29

30. side scatter and can be used to distinguish live from dead cells. Side
scatter tends to be more sensitive to inclusions within cells than forward
scatter and can be used to distinguish granulated cells from non-
granulated cells (Figure 6) as a result of differences in cell size and
granularity, light scattering (FS and SS) separates blood cell into three
major populations: lymphocytes, monocytes, and granulocyte (Robinson
et al., 2002).
C. Fluorescence sensor and detector:
Figure 7: The fluorescence detector
In contrast to the light scattered in the forward direction,
fluorescence emissions of dye-labeled cells are lower in intensity. This
necessitates the use of high-sensitivity, low-noise photo-detectors such as
the photo-multiplier tubes (PMTs), to detect the fluorescence emissions
(Figure 7). PMT are more sensitive than photodiode but can be destroyed
by exposure to too much light (Murphy, 1996).
Electrical pulses are very weak and must be amplified.
Amplification is either linear or logarithmic. The linear form of data
presentation more accurately represents differences in fluorochrome
concentrations between cells. In linear amplification, the output signal is
directly proportional to the input signal. Each channel represents the same
increment in signal value. In logarithmic amplification, the output is
proportional to the logarithm of the input pulse. This is important when
cell populations that vary widely in their characteristics, such as in cell
surface markers, are reviewed. Because the logarithmic amplification
compresses a wide input range, populations with similar intensities can
30

31. not be resolved. Immuno-phenotyping is the most common application of
logarithmic amplification (Murphy, 1996).
Fluorescence intensities are typically measured at several different
wavelengths simultaneously for each cell. Fluorescent probes are used to
report the quantities of specific components of the cells. Fluorescent
antibodies are often used to report the densities of specific surface
receptors and thus to distinguish subpopulations of differentiated cell
types, including cells expressing a transgene by making them fluorescent.
The binding of viruses or hormones to surface receptors can be
measured. Intracellular components can also be reported by fluorescent
probes, including total DNA/cell (allowing cell cycle analysis), newly
synthesized DNA, specific nucleotide sequences in DNA or mRNA,
filamentous actin, and any structure for which an antibody is available.
Flow cytometry can also monitor rapid changes in intracellular free
calcium, membrane potential, pH, or free fatty acids (Darzynkiewicz et
al., 2000).
4. Data storage and computer control system:
Flow cytometry is concerned with the measurement of the light
intensity of a cell whether it is scattered laser light or fluorescence
emitted by a fluorochrome. Light is detected by a photo-multiplier tube
(PMT), which converts it via an amplifier to a voltage i.e. electrical
output that is proportional to the original fluorescence intensity. Each
measurement from each detector is referred to as a "parameter". Data are
acquired as a "list" of the values for each "parameter" (variable) for each
"event" (cell). The electrical impulses are analog signals that are
converted to digital signals with converters. The sensor may process
either the brightest signal emitted from the cell (peak-sense-and-hold
processing) or all signals (integrated signal processing), which measures
total cell fluorescence (Lee, 1999).
After light scattering and fluorescence is converted to electrical
signals by the optical and electronics system, the information is converted
into digital data that the computer can interpret. The computer is a very
important part of flow cytometers because it is used to control most
functions of the instrument. The signals generated from cells or particles
are referred to as events and are stored by the computer. Flow cytometry
data files are known as list-mode files, which contain unprocessed data of
all the measured parameters (Rose et al., 1992).
31

32. The number of events acquired for each sample is always
determined before analysis and is usually set-using software designed to
control cytometer operation. A conventional acquisition value is 100.000
events per sample. However, this value may vary and range upward of
events per sample depending on the experimental objective (Melamed et
al., 1990).
Data analysis:
Data analysis is a very critical part of any experiment that utilizes
flow cytometry. Although many of the specialests of operating the flow
cytometer through the computer will be handled by a dedicated or
experienced operator, the beginning user must be aware of several types
of control samples that are critical (Radbruch, 1992).
Data from these control samples serve as reference points for the
information acquired from experimental samples. There are basic types of
control samples.
• Negative-control samples are used to adjust instrument parameters so
that all data appears on scale.
• Positive controls are used to ensure that the antibodies used are
capable of recognizing the antigen of interest.
• Compensation controls are employed when performing multi-
fluorochrome analysis to adjust for spectral overlap (Radcliff and
Jaroszeski, 1998).
To present and to analyze data collected by flow cytometry there
are several software available commercially. From these software the cell
quest. It can be used for data analysis through gating and set a different
quadrant, which identifies each studied population according to its
parameters. It allows different way of data presentation such as
histogram, dot plot, three-dimension plot, etc.
1. Histogram:
It display and analyze the results of an experiment dated, processed
and reduced to one or more histograms. The most commonly used are one
or two parameter histograms. One parameter histogram is the frequency
distribution of one of the collected parameters. It is usually displayed as
the number of counts accumulated for each intensity value (channel
number) of that parameter. Two parameter histograms are a bivariate or
two-dimensional map of the frequency distribution of two of the collected
parameters (Lee, 1999).
32

33. 2. Dot plot:
It is a two dimensional way of showing the number of events
acquired by the flow cytometry, (Figure 6) gives example of how data is
shown using dot plot or histogram. Dot plots are probably the most
common type of two-parameter plots, and they are the easiest to
understand (Robinson, 1993).
Finally, we remember that flow cytometers are very complex
instruments that are composed of four closely related systems: the fluidic
system - the illumination system - the resulting light scattering and
fluorescence - the data storage and computer control system. These four
systems provide a very unique and powerful analytical tool for
researchers and clinicians (Shapiro, 1988; Longobardi-Given, 1992).
*******
33

34. Applications of flow cytometry
Flow cytometry is used for immuno-phenotyping and measuring
DNA content of a variety of specimens including whole blood, bone
marrow, serous cavity fluids, cerebrospinal fluid, urine and solid tissues.
Characteristics that can be measured include cell size, cytoplasmic
complexity, DNA or RNA content (Recktenwald, 1993).
In the past, flow cytometers were found only in larger academic
centers. However, advances in technology now made it possible for
community hospitals to use this methodology (Orfao et al., 1995).
The use of flow cytometry in the clinical laboratory has grown
substantially in the past decade. This was attributable in part to the
development of smaller, less expensive instruments and a continuous
increase in the number of clinical applications (Brown and Wittwer,
2000).
1. Immuno-phenotyping Applications:
The distributed nature of the hematopoietic system markers is
amenable to flow cytometric analysis, mainly surface proteins and
glycoproteins on erythrocytes, leukocytes and platelets (Hartwell, 1998).
The availability of monoclonal antibodies directed against these
surface proteins facilitates flow cytometric analysis of erythrocytes
leukocytes and platelets. Antibodies against intracellular proteins such as
myeloperoxidase and terminal deoxynucleotidyl transferase are also
commercially available and permit analysis of an increasing number of
intracellular markers (Brown and Wittwer, 2000).
A- Erythrocyte analysis:
The quantitative test most frequently used in clinical laboratories
was the Kleihauer-Betke acid-elution test. This test was fraught with
inter-observer and inter-laboratory variability and was tedious and time
consuming (Polesky and Sebring, 1981).
However, the use of flow cytometry for the detection of fetal cells
is much more objective, reproducible, and sensitive than the Kleihauer-
Betke test (Bayliss et al., 1991; Bromilow and Duguid, 1997).
In the blood bank, flow cytometry can be used as a
complementary or replacement test for red cell immunology,
34

35. including RBC-band immunoglobulins and RBC antigens. Also, it can
be used to accurately identify and phenotype the recipient’s red cells and
to assess leukocyte contamination in leukocyte-reduced blood products
(Lombardo et al., 1993; Griffin et al., 1994; Barclay et al., 1998;
Garratty and Arndt, 1999).
B- Platelet analysis:
The analysis of platelets by flow cytometry is becoming more
common in both researches and clinical laboratories. Platelet-associated
immunoglobulin assays by flow cytomerty can be direct or indirect
assays, similar to other platelet-associated immuno-globulin immuno-
assays. In autoimmune thrombocytopenic purpura, free serum
antibodies are not found as frequently as platelet-bound antibodies
(Kokawa et al., 1993; Stockelberg et al., 1996).
C- Leukocyte analysis:
Blood samples are usually collected in tubes containing sodium
heparin, human leucocytes are stable at room temperature and should not
be refrigerated. It is best to process the sample within 6 hours,
lymphocytes generally can be recovered up to 24 hours after sample
collection with minimal effect on yield or cell viability. Whole blood can
be used or different cell fractionation methods are used to enrich the
stained cell population for the desired cell type prior to flow cytometric
analysis. Most commonly, the cells are fractionated with the respect to
their buoyant density gradient via non-continuous density gradient
centrifugation using Ficoli hypaque or percoll. (McFaul, 1990).
With the advent of monoclonal antibodies and a uniform
nomenclature system defining antibody reactivity in terms of clusters of
differentiation (CD), an independent means of characterizing acute
leukemias using cellular antigen expression has evolved (Maslak et al.,
1994).
Comparative studies of cell surface antigen expression between
normal and leukemic cells indicate that most, if not all, leukemias express
phenotypes that are not observed on most normal maturing cells. This
aberrant expression of cellular antigens suggests that leukemias are not
proliferations of cells arrested at one stage of normal maturation, rather
leukemic cells maintain a genetic program that can produce expression of
antigens of any lineage. Nearly all laboratories performing immuno-
fluorescence analysis use different reagents, some of the most widely
used antibodies for leukemia immuno-phenotyping are listed in Table 2 A
35

36. and B (Duque et al., 1990; Terstappen and Loken, 1990; Terstappen et
al., 1990 and 1991).
The ability to analyze multiple cellular characteristics, with new
antibodies and strategies, has substantially enhanced the utility of flow
cytometry in the diagnosis of leukemias and lymphomas (Jennings and
Foon, 1997).
CD10 CD 19 CD 22 CD 3 CD 7 IgM IgG
Early pre-B + + + - - - -
Pre-B + + + - - + -
B - + + - - - +
T - - - + + - -
Table 2 (A): Acute Lymphoblastic Leukemia
M1 M2 M3 M4 M5
DR 70% 91 7 95 92
CD33 70% 71 94 85 92
CD13 76% 85 76 71 45
CD15 22% 55 14 70 75
CD11b 35% 62 15 81 89
CD14 8% 12 2 63 57
Table 2 (B): Acute Myeloid Leukemia
D- Quantification of stem cells:
In the past, stem cell enumeration needed a hematology counter
(Erber et al., 1994). Nowadays, flow cytometry has become the major
technique for the quality control of stem cell-containing products such as
apheresis concentrates, bone marrow or cord blood (Götte, 2001).
Stem cells can be easily identified with flow cytometry due to their
unique characteristics. They demonstrate a medium level of CD34
expression, a low level of CD45 expression and a low forward side
scattered (Jennings and Foon, 1997).
E- Human Immune-deficiency Virus (HIV) monitoring:
It was demonstrated that, during HIV infection the amount of
CD4+ T-helper cells decreases. Therefore, the exact determination of the
numbers of T-helper cells is important for establishing the particular stage
36

37. of HIV infection, and for monitoring the progress of a patient undergoing
treatment (Saag et al., 1996).
In addition, in the course of HIV infection, CD8+ cytotoxic
suppresses T-cells express the activation marker CD38. Quantification of
the amount of CD38 antigen on CD8+ T-cells is possible using the BD
quantibrite system. Early clinical trials have indicated that this may be a
valuable tool for HIV monitoring (Saag et al., 1996).
Recently, a modern four-color analysis system allows the
quantification of CD4+ and CD8+ T-cells in one tube. It provides
automatic gating on lymphocytes and direct absolute counting (Götte,
2001).
F- Other markers:
There are a number of additional phenotypic parameters of
potential biological and clinical interest that are amenable to FCM
investigation, including carcinoembroynic antigen in colon and other
tumors (Bizzaro et al., 1982).
HLA-B27 is clinically relevant to the evaluation of seronegative
spondylo-arthropathies. There is a strong association of HLA-B27 with
ankylosing spondylitis as well as with several other disorders, such as
Reiter’s syndrome, psoriatic arthritis and inflammatory bowel disease. A
flow cytometric HLA-B27 test is much faster than the classical
microcytoxicity test (Jennings and Foon, 1997; Götte, 2001).
2. Cell cycle analysis:
The measurement of the DNA content of cells was one of the first
major applications of flow cytometry and is still one of the biggest
applications in the laboratory today (Albro et al., 1993).
The DNA content of the cell can provide a great deal of
information about the cell cycle, and consequently the effect on the cell
cycle of added stimuli e.g. transferred genes or drug treatment (Duque et
al., 1993).
A- Normal cell cycle:
Howard and Plec (1953) introduced the concept of the cycle in its
current form. When they were studying incorporation of DNA precursors
by autoradiography, they observed that DNA synthesis (S phase) in
37

38. individual cells was discontinuous and occupied a discrete portion of the
cell life and was constant in duration. Mitotic division was seen to occur
after certain period following DNA replication. A distinct phase between
DNA replication and mitosis was also apparent.
The G1 phase represents the gap in time between mitosis and the
start of DNA replication while the G2 represents the gap between the end
of DNA replication and onset of mitosis. It is possible to discriminate
between G1 Vs, S Vs, G2 or M cells (Figure 8) because of the difference
in their DNA content (Rabinovitch, 1993).
Figure 8: The cell cycle (the DNA histogram in the center shows the relative DNA
content of normal cells in various phases of the cell cycle)
Normal tissue predominantly consists of cells, which are in the pre-
synthetic phase (G1) of the cell cycle. They have diploid chromosomes
(2C = 46 chromosomes) and a corresponding content of DNA. In human
cells, this constitutes approximately 7 pg per cell. In tissue, which is not
actively growing, there is only a few cells past the S phase (synthesis)
with an increased DNA and protein content. In the post synthesis phase
(G2), there exists a double set of chromosomes (4C = 92 chromosomes)
and a DNA content of about 14 pg per cell (Zimmermann and Truss,
1979; Ormerod et al., 1998).
38

39. B- Evaluation of DNA Histogram:
Tribukait and his associates (1975) presented the methodological
aspects of single parameter DNA analysis by the flow cytometer in
karolinska' Institute, Sweden.
Since the cell material always contained normal diploid cells
such as leukocytes or normal kidney cells, these were used as an
internal standard and regarded as diploid (2C) (Tribukait, 1984).
Human Ficol-prepared lymphocytes, fixed in ethanol, were used as
external standard (Gustafson, 1982).
The degree of aneuploidy was determined by the DNA index,
which represents the ratio of fluorescence intensity of aneuploid cells
to the diploid cells. The DNA index of a diploid tumor is 1.0,
whereas, aneuploid tumors are designated by progressively higher
indices (Ormerod, 1994; Götte, 2001; Gordon et al., 2003).
3. Determination of Apoptosis:
Apoptosis frequently referred to, as programmed cell death is an
active and physiological mode of cell death, in which the cell itself
designs and executes the program of its own demise and subsequent body
disposal. Early and delayed patterns of apoptosis are present. Many cell
types, particularly cells of hematopoietic origin, undergo apoptosis
rapidly, within few hours following exposure to relatively high
concentration of cytotoxic agents (Arends et al., 1990; Del Bino et al.,
1990 and 1991; Oltvai and Korsmeyer, 1994; Majno and Joris, 1995).
There are a number of caspases in mammalian cells that have been
shown to be involved in the early stages of apoptosis e.g. (caspase 2,
caspase 3, caspase 6, caspase 7, caspase 8, caspase 9 and caspase 10). The
functions of these enzymes are not yet entirely clear but it appears that
after an initial signal to the cell to undergo apoptosis, they may be
responsible for the activation, amplification and execution of the
apoptotic cascade (Cohen and al-Rubeai, 1995).
As cells die or become apoptotic the refractive index of the internal
cytoplasm becomes more similar to that of the extra cellular medium.
This manifests itself as a reduction in forward scatter signal. At the same
time, intracellular changes and invagination of the cytoplasmic membrane
lead to an increase in side (or orthogonal or 90o) scatter (Cohen and al-
Rubeai, 1995).
39

40. A- Apoptosis and DNA analysis:
During apoptosis, calcium and magnesium dependent nucleases are
activated leading to DNA degradation. This means that within the DNA
there are nicks and fragmentation. These can be detected in three ways:
using DNA analysis to look at a sub-G1 peak, using strand break labeling
(TUNEL) to detect broken DNA or using Hoechst binding to detect DNA
conformational changes (Majno and Joris, 1995).
The sub-G1 method (Figure 9) relies on the fact that after DNA
fragmentation, there are small fragments of DNA that are able to be
eluted following washing in either a phosphate buffer solution or a
specific phosphate-citrate buffer. This means that after staining with a
quantitative DNA-binding dye, cells that have lost DNA will take up less
stain and will appear to the left of the G1 peak. The advantage of this
method is that it is very rapid and will detect cumulative apoptosis and is
applicable to all cell types (Cohen and al-Rubeai, 1995; Darzynkiewicz
et al., 1997).
Figure 9: Sub-G1 peak by Propidium Iodide Staining
The cell must have lost enough DNA in order to be seen in the sub-
G1 area. However, if cells enter apoptosis from the S or G2/M phase of
the cell cycle or if there is an aneuploid population undergoing apoptosis,
they may not appear in the sub-G1 peak (Schwartz and Osborne, 1993).
In addition, cells that have lost DNA for any other reason e.g. death by
some other form of oncosis will appear in the sub-G1 region (Nicoletti et
al., 2001).
40

41. B- Apoptosis and cell membrane analysis:
In normal cells, phosphatidyl serine (PS) residues are found in the
inner membrane of the cytoplasmic membrane. During apoptosis, the PS
residues are translocated in the membrane and are externalized. In
General, though not always, this is an early event in apoptosis and is
thought to be a signal to neighboring cells that a cell is ready to be
phagocytosed (Robinson, 1993).
Annexin-V is a specific PS-binding protein that can be used to
detect apoptotic cells. Annexin-V is available conjugated to a number
of different fluorochromes. Early apoptotic cells are annexin positive
but Propidium Iodide (PI) negative (Koopman et al., 1994).
C- Apoptosis and Organelle analysis:
During apoptosis, there is often a collapse in the mitochondrial
membrane potential. This can be detected in a number of ways by flow
cytometry. Two dyes, in particular, are useful, CMXRos and LDs-751.
CMXRos has a chloromethyl group, which accumulates in active
mitochondria, but in cells that are undergoing apoptosis, the
mitochondrial membrane is unable to take up CMXRos leading to a
decrease in fluorescence (Robinson, 1993; Chapman et al., 1995).
4. Andrological applications:
A- Assessment of Spermatogenesis:
Various approaches have been proposed to quantify spermato-
genesis in histologic preparations, using the mean Johnsen score as an
indicator for spermatogenesis. Thus, as with other histomorphological
methods, the shortcoming is the extensive time required for a quantitative
and semi quantitative evaluation of the tubular epithelium. Furthermore,
the patients have to undergo surgery (Hittmair et al., 1992).
Thorud et al. (1980) studied fine needle aspiration biopsy of
human testis using DNA measurements by flow cytometry. They
concluded that flow cytometry might be useful in the evaluation of
spermatogenesis.
B- Assessment of semen:
The standard determination of sperm concentration is by use of a
haemocytometer. It has been shown, however, that there is a high inter-
41

42. personal variability in results obtained by this method (Neuwinger et al.,
1990). Therefore, the application of automated methods might be useful.
The clinical application of flow cytometery as a suitable method
for supplementing the information obtained from the spermiogram of
patients with infertility was proposed by Otto et al. (1979).
The incidence of applying flow cytometric techniques to semen
analysis has gradually increased over the last 10 to 15 years. Although
cost considerations have limited their use primarily to research, more
affordable "bench-top" flow cytometers are being developed. The
advantages flow cytometry offers for sperm evaluation are speed,
accuracy, precision and large sample size. The use of flow cytometry for
sperm analysis is an attempt to address the long-standing problem of the
subjective nature of the manual method commonly used to perform
semen samples analysis. Other sources of laboratory variation arise from
the number of sperm cell analyzed, usually lower for manual techniques
(Davis and Gravance, 1993).
Evenson and Melamed (1983) described flow cytometery as a
simple rapid procedure that quantities DNA content and chromatin
condensation for cells present in human semen. They used in their study a
fresh semen sample (1-6 hours post emission) or frozen samples. They
demonstrated that only 10 minutes were required to measure 5,000 cells
per sample.
Otto and Hettwer (1990) described an improved method for
differential staining and high resolution for flow cytometric measurement
of human semen. They used a mild pretreatment with citric acid
detergent. This provided excellent preservation and good discrimination
of all cells, which are present in normal and pathological semen samples.
However, Hacker-Klom et al. (1999) determined the relative
proportions of the various cell populations, which are mature haploid
spermatozoa, abnormal diploid spermatozoa, cellular fragments,
immature germ cells (haploid round spermatids, diploid S-phase cells and
4C cells) and leukocytes as indicator of infection. They stated that there
are ten DNA histograms of human semen (Figures 10 and 16). The
histogram represent Sub-haploid region <1 cc (debris), 1 cc peak (mature
haploid spermatozoa), 1 c peak (haploid round spermatids), 2 cc peak
(diploid spermatozoa), More than 2 cc level including cells at 2 c
(lymphocytes, spermatogenic primary spermatocytes, etc.).
42

43. Figure 10: Different forms of DNA histogram of human semen
(Hacker-Klom et al., 1999)
a) The peak representing haploid spermatozoa at 1cc is split into two peaks
representing Y and X chromosome bearing spermatozoa.
b) DNA distribution of normospermic man representing ≥90 % mature haploid
spermatozoa at 1cc.
c) Nearly normal DNA histogram with 71 % mature haploid spermatozoa at 1cc.
d) Sever disturbance of spermatogenesis, 67 % only at 1cc.
e) DNA histogram reflecting ≥5 % of diploid spermatozoa at 2cc.
f) DNA histogram showing that the chromatin condensation of 55 % of the haploid
spermatozoa is disturbed, as reflected by the shifting of the DNA histogram to the
left.
g) Histogram showing that >10 % of different immature cell types at 1c, 2c, s and 4c
levels.
h) Histogram showing that >10 % of different immature cell types at 1c, 2c, s and 4c
levels.
i) The DNA histogram reflects a total count of only 3.5 million spermatozoa in the
ejaculate.
j) DNA histogram shows that only cellular debris is present in the ejaculate.
43

44. C- Sex preselection:
Preimplantation genetic diagnosis (PGD) has been practiced since
1968 (Gardner and Edwards, 1968) and embryo biopsy in humans has
been practiced since 1990 to reach a disease free embryo (Handyside et
al., 1990). These methods are the only one used routinely on a
commercial scale.
Preimplantation genetic diagnosis (PGD), involving in-vitro
fertilization (IVF), allows the transfer of unaffected embryos to the
uterus. During this procedure, one or two blastomeres from 4- or 8-cell-
stage embryos are biopsied and analyzed either by polymerase chain
reaction (allowing the sexing of the embryos and the detection of a
specific mutation), or by fluorescence in-situ hybridization (allowing the
sexing of the embryos and the detection of some aneuploidies). PGD
might offer an alternative to pre-natal diagnosis (PND) for couples at risk
of transmitting a genetic defect, and avoid the difficult decision of
whether or not to terminate a pregnancy (Liebaers et al., 1992; Lissens et
al., 1996; Handyside and Delhanty, 1997).
Spanò and Evenson (1993) concluded that various flow cytometric
techniques are already available to identify germ cell subpopulation
undergoing both proliferative and maturative processes. Precise DNA
content measurement allows accurate analysis to determine the proportion
of X and Y chromosome bearing sperm and sorting of this subpopulation
for gender preselection.
Johnson et al. (1993) reported the use of flow cytometry to
separate human X and Y chromosome bearing spermatozoa based on the
2.8% total DNA content difference between human X and Y chromosome
bearing sperm cells. The authors evaluated the efficiency of their
selection by single FISH, using alpha satellite DNA probes for the X or Y
chromosome, in decondensed sperm nuclei. Their results reflected an
average of 82% and 75% success for X-enrichment and Y-enrichment
sorting respectively. Flow cytometric separation of X and Y bearing
sperm cells is a reliable procedure that may be useful for the prevention
of X-linked disorders when used in conjunction with intrauterine
insemination (IUI), in-vitro fertilization (IVF) or intra-cytoplasmic sperm
injection (ICSI) especially with preimplantation genetic diagnosis of X-
linked diseases.
Flow cytometric technology has reached a maturation level that
allows its inclusion in the list of available and routine methods for
44

45. reproductive studies in human and animal populations. However,
although the reported efficiency of the selection procedure is very high,
its use should be followed by prenatal or preimplantational diagnosis to
prevent the birth of children affected by a sex-linked disease (Levinson et
al., 1995).
Johnson and Welch (1999), concluded that sex preselection that is
based on flow cytometric measurement of sperm DNA contents to enable
sorting of X from Y chromosome bearing sperm, has proven reproducible
at various locations and with many species at greater than 90% purity.
Offspring of the pre-determined sex in both domestic animals and human
beings have been born using this technology since its introduction in
1989.
Correspondingly, Seidel (1999) postulated that within males,
spermatozoa were essentially identical phenotypically so that no
convincing phenotypic difference has been detected between X and Y
chromosome bearing spermatozoa. The only consistently successful, non-
destructive approach to sexing spermatozoa was to quantify the DNA
contents in spermatozoa using a fluorescing DNA binding dye, followed
by flow cytometery and cell sorting. Since X-chromosome, bearing
spermatozoa has about 4% more DNA compared with Y-chromosome
bearing spermatozoa, accuracy of sorting can exceed 90% routinely.
D- Prognosis of Cancer:
Evenson et al. (1984) analyzed semen samples from fourteen
patients with testicular cancer, by flow cytometric techniques and by
conventional semen analysis. Samples were obtained post-unilateral
orchidectomy and prior to further treatment. The assessment of
spermatogenesis by flow cytometric study of sperm chromatin appeared
to be a sensitive and valuable.
Giwercman et al. (1988) studied the content of cellular DNA in
ejaculates from eight patients with carcinoma in situ of the testis and
twenty-six controls without evidence of testicular neoplasia by flow
cytometry. An aneuploid cell population with a ploidy value similar to
that found for carcinoma in situ cells was detected in seminal fluid from
four of the eight men with carcinoma in situ but in none of the controls.
One year after orchidectomy and local irradiation in these four men, no
aneuploid cells were found in the semen. These findings showed that a
detectable proportion of malignant germ cells might be released into the
seminal fluid of patients with carcinoma in situ of the testis and analysis
45

46. of seminal fluid might therefore aid in screening for early neoplasia of the
testis.
5. Flow Sorting:
Sorting can be defined as the physical separation of cell or particle
of interest from a heterogeneous population. In general, flow cytometers
use a principle involving the electrostatic deflection of charged
droplets similar to that used in ink-jet printers. Cells are aspirated
from a sample and ejected one by one from a nozzle in a stream of
sheath fluid, which is generally phosphate buffer solution which may
be any ionized fluid (Robinson, 1993).
As the cell intercepts with the laser beam, scattered light and
fluorescence signals are generated and the sort logic boards make a
decision as to whether the cell is to be sorted or not (Ormerod, 1994).
Figure 11: Strand Break Labeling (As the drop containing the cell of interest leaves
the solid fluid stream it will carry a charge, either positive or negative)
The distance between the laser intercept and the break off point is
called the drop delay. If a cell of interest i.e. to be sorted (Figure 11), has
been detected the cytometer waits until that cells has traveled from the
46

47. intercept to the break off point and then charges the stream (Owens and
Loken, 1995).
Therefore, it is possible to sort two separate populations from the
same sample, by applying two different levels of charge to the left or the
right streams. It is actually possible to sort two streams on either side.
Both the Cytomation Moflo and the Becton Dickinson are capable of this
(Shapiro, 1994).
6. New applications:
The number of new applications for flow cytometry in clinical
research is growing every day. The exciting application of flow
cytomtery is the identification of antigen-specific T-cells. The BD fast
immune antigen-specific assay identifies activated T-cells in whole blood
that has been stimulated with the antigen interest, through the activation
marker CD69. Other intracellular cytokines can also be detected
simultaneously. Activated T-cells express a specific pattern of cytokines
depending on the type of response. Th1 cells express IL-2, INF α and
IFN δ, whereas Th2 express IL-4, IL-5 and IL-10. This kind of specific
functional test is of particular interest to the pharmaceutical industry,
which is constantly trying to reduce the time required to test new drugs
(Orfao et al., 1995; Götte, 2001).
*******
47

48. Subjects and Methods
Patients:
In this work sperm chromatin evaluation was performed on 50
men, presenting with infertility of different etiology, in the andrology unit
of Mansoura University hospital, during the period between 11/2006 and
8/2008, their age ranged from 23 to 52 years with a mean age of 33.74 (±
05.41 S.D.) years.
Control:
Another 10 normal fertile men (each has more than one child) were
used as a control, their age ranged from 36 to 44 years with a mean age of
40.90 (± 02.23 S.D.) years.
Each of the sixty cases was routinely investigated to assess the
extent of sperm chromatin changes presented in their semen ejaculate.
Each case was subjected to the following:
• History taking.
• Clinical examination.
• Conventional laboratory investigations.
• Histological examination of semen and image cytometric analysis.
• Computer assisted semen analysis (CASA).
• Flow cytometry of semen and flow cytometric analysis.
According to the percentage of abnormal spermatozoa in the semen
analysis, all examined cases were classified into three groups (Table 3
and figure 12):
Group 1: 10 normal fertile men (with abnormal spermatozoa less
than 40 %) used as a control group. They represent 16.67 % of cases,
with a mean age of 40.90 years (± 02.23 S.D.).
Group 2: 41 infertile cases presented with abnormal spermatozoa
ranging from 40-60 %. They represent 68.33 % of cases, with a mean age
of 34.54 years (± 00.84 S.D.).
48