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DNA fragmentation in spermatozoa: Implications for failed fertilization in intracytoplasmic sperm injection
Stephanie Lopes
A thesis submitted in conformity with the requirements for the degree of Masters of Science
Institute of Medical Science University of Toronto
O Copyright by Stephanie Lopes, 1997
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DNA fragmentation in sperrnatozoa: Implications for failed fertilization in intracytoplasmic spem injection.
Masters of Science, 1997 Stephanie Lopes Institute of Medical Science University of Toronto
Objectives: To correlate the percentage of sperrn with darnaged DNA with semen
analysis parameters and with fertilization rates in ICSI. To evaluate the effect of
exogenously generated reactive oxygen species (ROS) on the integrity of the DNA of
human spermatozoa and the effectiveness of pretreatment with antioxidants to reduce
DNA darnage.
Design: Semen samples were collected fiom men in the ICSI program at a tertiary-
care centre. DNA fragmentation was deterrnined using the method of TUNEL.
Reactive oxygen species was generated in washed sperm sarnples in an attempt to
cause DNA fragmentation. Antioxidants were added to 17 sarnples to determine if
DNA damage could be prevented.
Results: A negative correlation was found between the percentage of DNA
fragmentation and motility and morphology of the ejaculated sperm. A significant
negative association was also found between the percentage of sperm with DNA
fragmentation and fertilization rate, When the unfertilized oocytes were analyzed, we
determined that almost 25% contained sperm with fragmented DNA and 25% of the
sample group demonstrated fragmented oocyte DNA. Lastly, we established that
CAUgGl lVU3 AUD LU C;~USC dIIlIU3L LUUIIUIU mC;rE;asc III UIYA uamage wnicri can De
prevented by the addition of antioxidants.
Conclusions: Since poor quality semen sarnples are the indication for ICSI, Our
results suggest that there is a greater likelihood that some sperm selected for injection
despite appearing normal, contained fragmented DNA.
Abstract Table of Figures Table of Contents: Tables List of Abbreviations Acknowledgements
1. Background
1.1 Spermatogenesis 1.2 Male Factor Infettility 1.3 Intracytoplasmic Spem Injection ( ICSI) 1.4 Apoptosis 1.5 The TUNEL assay 1.6 Reactive Oxygen Species
2. Objectives
3. Materials and Methods
3.1 Sperm samples used for ICSI 3.2 The TUNEL method 3.3 TUNEL on unfertilized, injected oocytes 3.4 Generation of reactive oxygen species 3.5 Supplernentation with antioxidants
4. Resutts
S. Discussion
6. Conclusions
7. Future Research
8. References
TABLE OF CONTENTS: Figures
Figure 1 : Human spermatozoa after swim-up analyzed for fragmented DNA
Figure 2: Correlation between DNA fragmentation rate and fertilization
Figure 3a & 3b: Human oocytes analyzed for fragmented oocyte DNA
Figure 3c & 3d: Human oocytes analyzed for fiagmented spem DNA
Figure 3e: Correlation of materna1 age with DNA fragmentation of oocytes
Figure 4: Kinetics of reactive oxygen species generation in the xanthine oxidase systern
Figure 5: Protective effect of reduced-glutathione on DNA of spermatozoa
Figure 6: Protective effect of hypotaurine on DNA of human spermatozoa
Figure 7: Effect of catalase on DNA of human spermatozoa
Figure 8: Protective effect of N-acetylcysteine on human spermatozoa
Figure 9: Protective effect of glutathione + hypotaurine on sperrnatozoa 44
Table 1 : The Spearman rank order correlation between sperm DNA fragmentation and fertilization and embryo cleavage rates
Table 2: Association between fertilization rate in ICSI and sperm DNA fragmentation of < 25% or > 25% using the TUNEL assay.
Table 3: DNA fragmentation of unfertilized oocytes from patients undergoi ng KSI
Table 4: Sperm chromatin patterns in unfertilized oocytes afier ICSI
ART
FSH
GSSG
GSH
HEPES
HTF
KSI
IVF
LW
LPO
MI1
MHTF
PCD
ROS
TUNEL
X
XO
_ - - - - - - _ - . -
Assisted reproductive technology
FoIlicle stimulating hormone
Glutathione peroxidase
Glutathione reductase
N-2-hydroxyethylpiperazine-N'-2-ethanesdphonic acid
Human tuba1 fluid
Intracytoplasmic sperm injection
In vitro fertilization
Luteinizing hormone
Lipid peroxidation
Metaphase-II oocytes
Modified human tubal fluid
Programmed cell death
Reactive oxygen species
Terminal transferase mediated d-UTP nick-end labeling
Xanthine
Xanthine oxidase
1 am most indebted to my supervisor Bob Casper, for his guidance, patience and being my role model. 1 would like to express my gratitude to the members of my program cornmittee, Sue Varmuza and Ted Brown, for their time, constructive comments and assistance.
A special thank you to my colleague, Andrea Junsicova, who always offered her help and support despite her busy schedule and to everyone at T.C.A.R.T., in particular the lab, who without them this project would not have been possible. Their expertise in this area has contri buted significantly to the scientifrc quality of this thesis.
1 thank my parents for their support in al1 my academic endeavours. I would also like to thank my fiancé Matthew for his patience, support, understanding and love.
Spermatogenesis is a cornplex, highly ordered process of ce11 division and
differentiation by which spermatogonial stem cells give nse to mature spematozoa.
Spermatogenesis takes places in the seminiferous tubules of the testis which produce
approximately 120 million spermatozoa daily in the normal male (Amann and Howards,
1980). Maintenance of a high production rate requires the coordination of cellular
division of spermatogonia to replenish stem ce11 reserves and to undergo further
differentiation into spermatocytes, meiotic divisions of spermatocytes to produce
spermatids containing a haploid number of chromosomes and differentiation of
spermatids into mature spematozoa (Matsumoto, 1996).
At puberty, when the testis is stirnulated by follicle-stimulating hormone (FSH)
and luteinizing hormone (LH) produced by the anterior pituitary gland, spematogonia
undergo mitosis and differentiate into a specialized cell. The more primitive
spermatogonia are referred to as Type A and the products of their division are both Type
A and the more differentiated Type B cells. Type A cells are necessary to maintain a
reserve ceIl population. Type B ceIls undergo furiher divisions before they enter into a
phase of DNA dupIication without a subsequent mitotic division, thereby transforming
them into primary spermatocytes. It is the primary spermatocyte which undergoes the
first meiotic (reduction) division. This separates the pairs of chromosomes so that only
one of each pair passes into each of the two daughter cells, secondary spermatocytes.
These then undergo a second meiotic division to produce haploid spermatids.
A1 lnls stage, sperm are si111 wnsiucrcu Irr irI iaLuic; ariu I I I U ~ L uc r i a i i 3 i u i i i i G u I I I L U u
spermatozoon through the process of spermiogenesis, before becoming capable of
fertilizing an oocyte. Maturation involves growth of a tail, reduction of the cytoplasm
and formation of the acrosome, a membrane-enclosed cap containing enzymes thought to
be necessary for penetration of the oocyte vestments. In addition, condensation of the
nucleus occurs as the histones in the DNA of the sperm chromosomes are replaced with
protamine (Balhorn et al, 1988). The protamination of the chromatin when complete,
leads to the formation of disulphide bonds and extremely tight coiling of the DNA which
tends to protect it from chernical or physical denaturation (Balhorn et al., 1982).
The time for a complete cycle of spermatogenesis from the division of a
spermatogonium until the spermatid is about to be released to the epididymis, takes about
74 days (Heller and Clermont, 1963). As a result of the long duration of
spermatogenesis and an additional epididymal transit time of approximately 12 days in
humans, an insult to the testes that may result in arrested development of germ cells will
not be manifested by reduced spermatozost in the ejacutate, until months later.
Disorders of sperm production are not uncornmon because of the long time and
the cornplex series of events which are required for sperm maturation. This process is
thought to be highly sensitive to disruption by a number influences such as environmental
toxins, irradiation and excessive heat. In addition, the male reproductive system seems to
be tolerant of abnormal sperm which rnay be produced and which are not usually selected
out. Therefore it is not unusual to observe morphologie sperm abnormalities within a
fertile semen sample during routine semen analysis. It is also likely that morphologically
is this premise which forms the basis of the research described in this thesis.
1.2 Male Factor Infertilitv:
Infertility, defined as the inability to produce a pregnancy within a 1 year penod
of sexual intercourse without contraceptive rneasures, affects approximately 15% of
married couples (Menning, 1980). It is estimated that in 40-50% of infertile couples, the
man is infertile which in the general population equaIs about 5-10% of married men
(Brugo-Omedo et al, 1990). Male infertility is a heterogenous group of disorders.
Recognizable causes account for onIy 30-50% of the cases and there is emerging
evidence to suggest that a genetic basis for male infertility may exist in many men
currently classified as idiopathic.
Currently, disorders for which there are logical or effective treatments are
diagnosed in only a small proportion of men seen for infertility and include genital tract
obstruction, varicocele, homonal disorders and sperm autoimmunity. Although these
disorders are treatable, pregnancies are often difficult to achieve through natural
intercourse. Thus assisted reproductive technology (ART) has become important in the
management of such patients.
Another group of men seen for infertility is found to have reduced semen quality
on the basis of normal values determined by the World Health Organization (1992).
According to WHO guidelines, a semen sample is considered abnonnal if; sperm
concentration is less than 20 million/ml, less than 40% of sperm are motile andor more
than 30% demonstrate abnormal morphology. The outcome for pregnancy in this group
Due to the lack of effective treatments to enhance semen quality, failure to conceive afier
empinc or simple therapies such as techniques of spem selection and concentration for
intrauterine insemination, often leads to in vitro fertilization (IVF).
IVF involves the placement of spem and egg together in vitro in order to
facili tate fertilization. Although IVF is suitable to treat infertility due to fernale factor,
the use of abnormal/poor quality sperm from subfertile males for this procedure produced
less than satisfactory results. It has been show that even when several samples of
sperm from oligozoospermic (low spem count) men are combined to result in an overall
normal concentration, and are placed directly with oocytes in culture, they do not fertilize
at the same rates as sperm from othetwise normal men (Acosta et al, 1988). Secondly,
adequate numbers of sperm cannot be obtained fiom al1 men to allow insemination of
oocytes with the usual numbers of gametes necessary for successfbl IVF, i.e. 50,000 to
100,000 sperndoocyte.
In addition to low spem concentration, other problems leading to failure of
fertilization in IVF include absence or deficiency of acrosomal enzymes, failure of the
acrosome reaction to occur, poor spem rnotility, or morphologically abnormal spenn,
leading to consequent failure of spenn to penetrate the zona petlucida of the oocyte.
Furthemore for men who are azoospermic (no spem present in the ejaculate), IVF offers
little help. To overcome these problems in subfertile males, a new technique known as
intracytopiasmic sperm injection (KSI) was developed.
Direct injection of a single sperm into the cytoplasm of an oocyte during IVF had
been tied for several years without success. It was not until the dernonstration of
fertilization and live births as a result of a number of technologie advances by Palermo et
al. in 1992, that ICSI developed a widespread clinical application. Since that time, KSI
has been performed extensively to treat patients with severe male factor infertility.
ICSI is an assisted reproductive technique in which a single sperm is
microinjected into the ooplasm. The development of KSI offered a breakthrough for
male factor patients since poor sperm parameters such as low concentration, low motility
or a large proportion of sperm with abnormal morphology appeared to be irrelevant to the
success of this procedure. With ICSI, fertilization and pregnancy rates appear to be
independent of sperm quality (Cohen et al, 1992; Van Steirtegham et al, 1993) which is
the opposite of what has been dernonstrated for both IUi and IVF (Acosta et al, 1988).
Nagy et al., (1995) reviewed the effect of spermatozoal factors on results of ICSI
in 966 cycles. Despite the absence of morphologically normal sperm or of highly motile
sperm in a semen preparation, pregnancy can still be achieved. Men are classified as
asthenozoospennic if they demonstrate ~ 4 0 % motility in a semen sample. Even if a
patient has poorly motile sperrn, pregnancy can still be achieved as long as one motile
sperm can be selected to injected each oocyte. The only semen abnomality that had a
significantly adverse effect on fertilization and prebmancy rates with lCSI was the
complete absence of motile sperrn. When no sperm motility was observed, it appeared
that sperm viability may be decreased as well. The use of the hypo-osmotic swelling test
has been described to select viable sperm for KSI from samples with complete absence
between live, non-motile sperm and dead spem when selecting for ICSL This technique
has led to a marked improvement in fertilization and prepancy rates for couples with
male infertility secondary to complete asthenozoospermia. The basis for this test is
when live spem are placed in a hypo-osmotic solution (that is non-toxichon-hamifbl to
sperm), the solution enters the spem head causing a slight increase in volume. The
result is a change in plasma membrane tension of the spem resulting in coiling of the
sperm tail. When dead sperm are placed in the same solution, fluid enters the sperm
easily, but unlike live sperm, the solution also freely moves out of the head as well.
Therefore the ce11 volume does not change and tail coiling is not observed. The lab
technologist will then select sperm with coiled tails indicating viability, for injection into
the oocyte.
The application of ICSI has allowed treatrnent of couples who until very recently
were considered sterile and untreatable. Men with bilateral congenital absence of the vas
deferens and other unreconstructable obstructions of the male reproductive tract are good
candidates for KSI. In these men, microsurgical retrieval with or without
cryopreservation of spenn is possible despite the fact that the sperm are immature.
Aspiration of sperm from the epididymis or testis can also provide spem for ICSI cycles
(Palermo et al., 1996a).
The ICSI procedure has recently created a controversy regarding the use of sperm
from abnormal samples to achieve fertilization, as physiological selection processes are
bypassed by the injection (Curnmins and Jequier, 1995). In particular, there is concern
that sex chromosome abnormalities may be more frequent in ICSI pregnancies (In't Veld,
numbers of chromosomal abnormalities when compared with the general population
(FIVNAT, 1995).
Although the rate of chromosornal abnormalities does not appear to increase with
the use of ICSI, there is new concem that a genetic defect in some men could cause
infertility. In the past decade, there has been mounting evidence that genes on the long
a m of the Y-chromosome (Yq) play a critical role in spermatogenesis (Burgoyne, 1987).
Evidence from Y autosomal translocations along with information fiom patients with
deletions of the long a m of the Y-chromosome, suggest that al1 of the euchromatic
portions of the Y-chromosome must be present to achieve normal spermatogenesis
(Nagafuchi et al., 1993). Deletion mapping of the Y-chromosome in males with
macroscopic deletions of the Y are consistent with the proposa1 that the azoospermia
çene is located on Yq deletion interval 6. Men with macroscopic deletions in this reçion
are azoospermie. In the past, the presence of infertility would have prevented further
propagation of this genetic defect. However with the use of ICSI and sperm retrieved by
testicular biopsy, there is concern that men with microdeletions on the Y-chromosome
resulting in azoospermia, can now pass this defect on to their offspnng. Studies should
be done on the resultant offspnng of ICSI to detemine if defects of the Y-chromosome
are passed on to the next generation, in order to adequately judge if ICSI tmly produces
normal, fertile oflspring.
Even though the rnost normal-appearing, motile spermatozoa are selected for
injection in the ICSI procedure, the quality of semen sample fiom which the sperrn is
chosen must be taken into consideration as well. The ICSI fertilization rate in general,
each mature oocyte (Palenno et al, 1995; Payne and Matthews, 1995; Svalander et al,
1995). A possible explanation for this lower than expected fertilization rate could be that
spem seiected from semen of male-factor patients may have defects in their DNA. Such
abnormalities as loosely packaged chrornatin and damaged DNA have already been
observed in poor quality semen samples (Evenson et al, 1980; Foresta et al. 1992; Sailer
et al, 1995). We have recently established that spem used for IVF contained an average
DNA fragmentation rate of 3.13% and that the percentage of spem with DNA damage
correlated negatively with fertilization rates (Sun et al, 1997). We hypothesize that spem
selected for ICSI will also demonstrate DNA fragmentation, likely at a higher rate due to
the poorer quality of sample obtained with respect to semen analysis parameters.
1.4 Apoutosis:
Although DNA fragmentation has already been established to be present in spem
used for IVF, it is not known if this phenornenon represents apoptosis. As discussed
above, spermatogenesis is a dynamic process of germ ceIl proliferation and
differentiation from spermatogonia to sperrnatozoa. In the mammalian testis, germ cells
clonally expand through many rounds of mitosis before undergoing differentiation and
maturation steps that result in spermatozoa. This clona1 expansion is excessive, requiring
that a mechanism exist to match the number of germ cells with the supportive capacity of
the surrounding environment. Overproliferation of early germ cells is reduced by
selective apoptosis of their progeny (Bartke, 1995; Billig et al, 1995).
regulate the homeostasis, development and differentiation of many multicellular
organisms (Raff, 19%). Upon induction by an appropriate trigger, the ce11 activates, or
altematively represses suppressor gene products responsible for the suicida1 mechanism.
The most common series of morphological changes which result in ce11 death is referred
to as apoptosis (Kerr et al. 1972). It is important to note however, that not every
apoptotic characteristic is evident in each cell. At first, the ce11 shrinks and becomes
denser and chromatin becomes pyknotic and denseiy packed within the nuclear
membrane. This is followed by extensive condensation of cytoplasm in the remnants of
the cell. The ce11 membrane convolutes and the cytoplasm is shifted into extracellular
membrane-covered processes (cytoplasmic blebbing). Subsequently, the ce11 breaks up
into several membrane-bound buds of variable size (apoptotic bodies) In addition,
endogenous nucleases digest DNA into fragments of approximately 180 base pairs called
01 igonucleosomes. In many cases, DNA fragmentation occurs early in apoptosis, often
appearing several hours before the appearance of decreased ceil viability (Jurisicova et
al, 1995). PCD is under genetic control and can be initiated by an interna1 clock, by
DNA damage, by ceIl cycle arrest, or by extracellular agents such as hormones,
cytokines, killer cells and a variety of chemical physical, and viral agents (Majno and
Joris, 1995).
Testicular gemi ce11 apoptosis occurs normafly and continuously throughout life.
In addition, testicular germ ce11 loss is known to result from toxic chemical exposure,
radiation and alteration of hormonal support (Blanchard et al., 1996; Meistrich, 1993;
Sinha et al., 1995). In many of these situations, germ cells are known to undergo
when the testicular environment cannot support spematogenesis. However, the elements
that control this process have yet to be identified.
Due to the spem DNA being already condensed and since there is little
cytoplasm is present in the germ ceIl to cause blebbing and formation of apoptotic
bodies, morphologie changes associated with apoptosis cannot be identified. Thus, from
the results of TUNEL alone, it cannot be distinguished whether or not DNA
fragmentation in spermatozoa is a feature of apoptosis or simply necrosis. In contrast to
PCD, necrosis is often a consequence of cellular injury, toxic insult or changes in pH.
Cells undergoing this phenornenon demonstrate swelling resulting in widespread
disrupt ion of membranes, DNA is randomly degraded (Schwartzman and Cidlowski ,
1993), and often necrotic injury affects adjacent cells. We speculate that DNA
fragmentation in human spermatozoa, is more likely due to apoptosis than to necrosis
since it is known that spermatids can follow a specific 'suicide' pathway when in excess.
Several PCD proteins have been identified in the testes such as Fas (Lee et al., 1997).
indicating again that an apoptotic pathway exists in this system. However, we have been
unable to detect a typical Iadder pattern associated with apoptosis that represents
oligonucleosome fragmentation in human spermatozoa. This may be due to the fact that
the amount of fragmented DNA obtained is not in sufficient amounts to produce visible
banding patterns on a gel. On the other hand, it is not known if spenn DNA is capable of
forming oligonucleosomes (i.e. 180 bp). Thus if spem are incapable of fragmenting into
these specific formations, this may explain why we do not observe the characteristic
ladder pattern of apoptosis in these cells.
As mentioned above, we have previously reported DNA fiamentation in spem
used for IVF, using a teminal deoxynucleotidyl transferase-mediated dUTP nick-end
labelin!: technique (TUNEL) (Jurisicova et al., 1996; Sun et al.. 1997). The TUNEL
assay utilizes exogenous TdT to label 3'OH ends of DNA strand breaks characteristic of
apoptotic cells (Figure 1). Multiple copies of a selected deoxyribonucleotide can
potentially be added to each 3'OH end of DNA by TdT. In comparison, normal resting
or DNA synthesizing (S-phase) cells also have 3' ends, however they are capped by
telomeres and are thus inaccessibIe to enzymes. Although TUNEL specifically detects
3'OH ends, only DNA with strand breaks readily expose their 3' ends, therefore labeling
indicates cells containing nicked DNA. DifTerent deoxyribonucleotides have been
utilized for incorporation, most charactenstically with different modifications to allow
subsequent detection. In this study, we utilize a variation on the TUNEL assay by
incorporating d-UTP labeled with biotin and subsequent detection with avidin.
The TUNEL technique as been used in various cells and is successful because it
does not require a DNA template as in other nick-translation assays. Secondly it is a
hiçhly sensitive method which allows detection of nicks in individual cells. Lastly, the
assay requires the use of only very small volumes of reagent and is quick to perform.
One disadvantage of this technique is that although TUNEL demonstrates
evidence of DNA breaks, quantification of the number of nicks present is not possible.
However, this is not a problem in our present study since we are prirnarily observing if
sperm contain fragnented DNA or not. It would be useful to determine the number of
DNA nicks in future studies perhaps by limiting 3'OH additions to a single nucleotide.
triphosphate. Therefore each label would indicate one nick. Secondly, hi& background
signals can sometimes be obtained fiom this assay depending on the celUtissue used and
length of incubation. This could present a problem if celts appeared labeled but are
actually artifact staining. Lastly, it is important to note that although this technique
demonstrates evidence for DNA breaks, we cannot per se state that this is an indication
of apoptosis based on this one result. Other morphological changes characteristic of
apoptosis would have to be looked at as well.
Figure 1. Diagram of how the TUNEL assay works.
+ Terminal Transfease + x-dNtP ( m)
~t thiç time, the etiology of sperm DNA fragmentation seen in infertile men is
unclear. Over the past decade, studies have demonstrated that peroxidative damage to the
sperm plasma membrane by reactive oxygen species (ROS) may impair spem function,
leading to the onset of male infertility (Aitken and Clarkson, 1987; Iwasaki and Gagnon,
1992). ROS includes free radicals which are active oxidizing agents. Spermatozoa are
highly susceptible to oxidative damage due to an abundance of polyunsaturated fatty
acids within the plasma membrane (Jones et al, 1979) and a low concentration of
scavenging enzymes within the cytoplasm. However, the production of ROS by sperm is
a normal physiologic process required for the occurrence of capacitation and the
acrosome reaction (de Lamirande and Gagnon, 1993a; de Lamirande et al, 1993b).
Capacitation is a requisite maturation process which sperm must undergo in order to
fertilize successfully. This process takes place in tbe fernale tract (although it can be
induced in vitro) and appears to involve a destabilization of the cell membrane of the
sperm. This takes about 6-7 h in humans and permits the sperm to release the contents of
the acrosome. The release of enzymes is termed the acrosome reaction and is necessary
to enable sperm to penetrate the vestments surrounding the oocyte. (Saling, 19%)
It has been demonstrated that semen sarnples from infertiie men have increased
levels of ROS suggesting that deficient sperm may produce excessive ROS (Iwasaki and
Gapon, 1992). High levels of ROS present in seminal plasma have been associated.
with poor morphology, motiiity and low spem count (Mazzilli et al, 1994; Aitken et
al, 1989). Furthemore, i t has been reported that a key factor in the production of ROS
by damaged or deficient sperm may be the spem preparation technique. Spem washing
higher ROS production (Agarwal et al. 1994).
Spematozoa are very suceptible to ROS attack resulting in a decrease in spem
motility, sperm viability and an increase in midpiece morphology defects, with
deletenous effects on spem capacitation and the acrosome reaction. Lipid peroxidation
(LPO) of the sperm membrane is considered to be the key mechanism of this ROS-
induced sperm darnage (Alvarez et al, 1987) leading to infertility. Besides membrane
effects, LPO can also damage DNA and proteins either through oxidation of DNA bases
(primarily guanine) or through covalent binding to malondialdehyde (an end product of
LPO), resulting in strand breaks and cross-linking (Sikka et al, 1995). Furthemore, ROS
has been implicated in the cause of DNA fragmentation in somatic cells (Buttke and
Sandstrom, 1994; Ratan et al., 1994). From the above mentioned studies, we hypothesize
that ROS will have a similar effect on the DNA of human spennatozoa.
To counteract the harrnful effects of ROS, there are a number of biological
aritioxidants presently known which scavenge, dispose andor suppress the formation of
free radicals. Free radical scavengers are necessary in order to maintain a balance, i.e.
allow just enough ROS to cause capacitation, but not more. The cytoplasm of the hurnan
sperm is extremely limited in terms of its volume and does not possess a significant
amount of antioxidants. In contrast, seminal plasma contains superoxide dismutase
(SOD), catalase and reduced glutathione, which al1 have a significant role in scavenging
ROS. However, once seminal plasma is washed away during sperm preparation for ART,
spenn are virtually unprotected from oxidative damage.
removing ROS. SOD spontaneously dismutates 0; to fonn O2 and H20z, whereas
catalase converts H,02, to 0, and H,O very effectively. Glutathione has a similar effect
on H20,. in addition to neutralizing OH-. Several chemical antioxidants are available that
are also known to attack ROS and LPO. In ow study we propose to pretreat spermatozoa
wi th; ( i ) N-acetylc ysteine (a precursor of glutathione which scavenges H202), (ii )
hypotaurine (an antioxidant which rernoves cytotoxic aldehydes), (iii)reduced glutathione
and (iv) catalase (both are antioxidants present in sperm), prior to ROS çeneration in an
attempt to prevent DNA damage.
i ) To correlate the percentage of sperm containing damaged DNA with semen analysis
parameters and with fertilization rates in ICSI.
i i ) To assess the presence of fiagmented DNA derived fiom sperm or oocyte
chromosomes in oocytes injected during the ICSI procedure which fail to
demonstrate pronuciear formation, i.e. apparently unfertilized oocytes.
iii) To evaluate the effect of exogenously generated reactive oxyçen species (ROS) on
the integrity of the DNA of human sperrnatozoa and the effectiveness of pretreatment
with antioxidants to reduce DNA damage.
3.1 ICSI semen sam~fes:
Sample Collection
A total of 150 semen samples were collected fiom couples undergoing ICSl at the
Toronto Center for Advanced Reproductive Technology. Wn'tten consent for use of the
sperm for research was obtained fiom patients according to guidelines established for
research on human subjects by the University of Toronto.
Semen Pre~aration
Semen samples were collected after at least 48 h of abstinence. After
approximately 30 min of liquefaction at room temperature, both routine semen analysis
and swim-up assessrnent were performed using standard techniques (Jaffe and Oates,
1994). Semen analysis is used to detennine sperm concentration, motility and
rnorphology of a sample. The percentage of motility is calculated by counting motile and
immotile sperm in five randomly selected fields containing at least 100 cells using phase
contrast microscopy. To determine concentration, the fresh sample is diluted 1 : 10 with
human tubal fluid (HTF) (Irvine Scientific, Santa Ana, CA) supplemented with 5%
hurnan semm albumin (HSA) (Irvine Scientific, Santa Ana, CA). The chamber of a
Neubouer hematocytometer is loaded with the diluted sample. Five red blood ceIl areas
are counted using 20X magnification. The total number of sperm counted in these five
areas is divided by 20 (depth of hematocytorneter) and multiplied by 10 (dilution factor)
.- - -
are counted and divided into categories such as normal, immature, macrocephalic,
microcephalic, midpiece defects and amorphous. The number of normal spem is
expressed as a percentage of the total sperm counted.
For swim-up, the sample was diluted with HTF supplemented with 5% HSA. The
diluted semen was wasbed twice by centrifugation for 10 min at 220 x g; the final pellet
was resuspended in approximately 200 pl of medium and layered gently under 1 ml of
fresh medium supplemented with 5% HSA. The motile spermatozoa were allowed to
swim up for 1 h into the overlaying medium at 37OC in a 5% CO2 incubator. The
supernatant, containing swim-up sperm was aspùated and the final number of motile
sperm and concentration was evaluated using a Neubaur hemocytometer.
3.2 TUNEL Assav
Nicked DNA in the sperm recovered by swim-up was measured using a
modification of the method of terminal deoxynucleotidyl transferase (TdT)- mediated
dUTP-biotin end-labeling (TUNEL) which has been previously described (Jurisicova et
al., 1996; Sun et al., 1997). Immediately after the ICSI procedure was perforrned, the
remaining sperm were fixed with 1 % formaldehyde (BDW Inc., Toronto, ON, Canada)
for 10 min at room temperature. A slide was prepared of the fixed sperm and allowed to
air dry. Afier washing in PBS (pH 7.4), 100 pl prewash buffer containing single strength
One-Phor-Al1 buffer (100 mM Trisacetate, 100 mM magnesiurn acetate, 500 m M
potassium acetate; Pharmacia LiU3 Biotech, Picataway, NJ) and 0.1 % Triton x- 100
-
minutes at room temperature.
The prewash was washed off using PBS and 50 pl of TdT bufSer containing 3 p M
biotin-16-dUTP (Boehnnger Mannheim, Laval, PQ, Canada), 12 p M dATP (Phamacia
LKB Biotech), 0.1% Triton X-100, and 10 U of TdT enzyme (Phamacia LKB Biotech)
was added to the slide and allowed to incubate at 37OC for 60 min. Afier two washes
with PBS, the fixed, pemeabilized sperm were treated with 100 pl of staining buffer
consisting of 0.1 % Triton x- 100 and 1 % Streptavidiflexas red anti-biotin (Calbiochem-
Novabiochem Corporation, La Jolla, CA) and incubated at 4°C in the dark for 30 min.
The stained cells were washed in PBS. Slides wcre placed in a staining jar containing 50
ml PBS and 20 pl 4,6-diamidino-2-phenyhdole (DAPI) for 5 min to counter-stain al1
chromatin prior to analysis.
For negative controls, the enzyme terminal transferase was omitted from the
reaction mixture. For positive controls, the samples were pretreated with O. 1 IU DNAse 1
(Phamacia LKB Biotech) for 30 min at room temperature and then labeled with TUNEL.
Analvsis
Using a fluorescent microscope, sperm stained with DAPI were first rnanually
counted under UV iight. This number represented total sperm in the field. Red
fluorescence labeling was detected using a 640 nrn filter on the same field. The number
of sperm demonstrating red fluorescence was expressed as a percent of total sperm in the
field to give an average (I SEM) nicked DNA rate. This assessrnent was perfomed on
10 fields per sample in which 100-500 cells were counted.
Sigmastat (Jandel Corporation, San Raphael. CA) was used for data analysis.
Since the data was not nonnally distributed, we used the Spearman rank order correlation
to determine the correlation of DNA fragmentation with fertilization and embryo
cleavage rate. Multiple linear regession analysis was used to determine if a significant
influence of semen analysis parameters on DNA fragmentation existed. A p value of <
0.05 was considered significant.
3.3 Unfertilized, iniected oocvtes:
Patients
The study was performed on unfertilized oocytes from 55 patients undergoing
ICSI over a time period of 9 rnonths. The mean age (It SEM) of the women was 34.7 f
3.4 years (range 25-42). Written consent for use of unfertilized oocytes for research was
obtained from patients according to guidelines established for research on human
subjects by the University of Toronto
Oocvte Retrievai
Ovulation induction for assisted conception procedures was achieved with human
menopausal gonadotrophin (HMG). During the 9 month period of this study, different
sources of this hormone was used; Pergonal and Metrodin HP (Serono), Humegon
(Organon). Cycles were monitored using a combination of ultrasound and serum
estradiol estimation. Human chorionic gonadotrophin (Serono) was administered at 34-
guidance.
Preparation of oocvtes for ICST
Following oocyte retneval, the cumulus cells were removed by exposing the
cumulus corona-oocyte complex to hyaluronidase (3 Nlml) (Sigma, St. Louis, MO) in
modifed human tubal fluid (MHTF) (Irvine Scientific, Santa Ana, CA). MHTF difiers
from HTF as it contains N-2-hydroxyethylpiperazine-N'-2-ethanesuphonc acid (HEPES)
which is used successfully as a buffer in media for human oocyte collection and embryo
handling. Each oocyte was assessed for maturity. Those with a first polar body present
(MI) were selected for KSI.
Preparation of spermatozoa for lCSl
Spermatozoa was prepared on the day of oocyte retrieval as described in semen
preparation section above. 1 0% pol yviny 1 pyrrolidone (PVP) was used for the injection
procedure.
The ICSI procedure
All microinjection procedures were carried out on the 37°C heated stage of an
inverted microscope (magnification x200 or x400). Prior to injection, individual
morphologically normal motile sperm were selected from the spermlPVP droplet and
immobilized. The sperm is immobilized by touching the tail with the intracytoplasrnic
spem injection pipette. Immobilized sperrn perform better than spermatozoa that are
injected while still motile. This irnrnobilization process may induce changes in the
al., 1996b). The injection of spem into the mature oocytes was perfomed by trained
IVF lab technologists. The oocyte is held in place by gentle suction on the holding
pipette. The first polar body is positioned at the 6 or 12 o'clock position. With the
spem positioned close to the tip and the edge of the pipette and the oolernma in focus,
the intracytoplasmic sperm injection pipette was forced gently horizontally fiom the 3
07clock position deep into the ooplasm. Following injection, oocytes were transferred
into 100 pl droplet of HTF medium supplemented with 5% human serum albumin in a
plastic 60 x 15 mm petri dish, covered with mineral oil and incubated in a humidified 5%
COz environment at 37°C.
Assessrnent of fertilization:
Cultured human oocytes were assessed for the presence of pronuclei 16- 18 h after
ICSI. Fertilization was considered normal when there were two distinct pronuclei and
two polar bodies present. Only those oocytes that had failed fertilization were selected
for this study.
Detection of DNA fragmentation
The zona peliucida of each oocyte was reinoved by exposure to 20 pl of acidic
tyrode's solution under minera1 oil (pH 2.0) (Sigma, MO) for 20 sec. Oocytes were then
fixed with 4% paraformaldehyde in HTF medium for 10 minutes, placed on a slide and
allowed to air dry. Nicked DNA was measured using a modification of the method of
terminal deoxynucleotidyl transferase (TdT)- mediated dUTP-biotin end-labeling
penneabilizing agent Tween-20 (Sigma) in place of Triton X-100. Tween-20 was used
as more aggressive permeabilization was required of the oocyte membrane in order for
TdT to enter the oocyte and then the sperrn head within the oocyte.
Oocytes pretreated with DNase 1 served as positive controls, and oocytes in which
the TdT enzyme was omitted, as negative controls. In addition, 20 unfertiiized oocytes
from IVF cycles were anaiyzed using the same method to control against possible
chrornatin damage caused by the ICSI procedure itself These oocytes were treated in an
identical manner as the ICSI oocytes with the exception that the IVF oocytes did not
undergo the injection procedure. The mean age of patients from the IVF group was 32.8
years and therefore was comparable to the ICSI patients.
Statistical Anaiysis
Since the DNA fragmentation vs. materna1 age data was not normally distributed,
the Mann-Whitney U-test was used to determine if there was a significant difference
between the sample groups. To detect a difference between DNA damage of udertilized
oocytes from IVF cycles and those from KSI, the x2 was used. A difference of p < 0.05
was considered significant.
Sample Collection
A total of 47 semen samples were collected fiom men undergoing semen analysis
in the andrology clinic at The Toronto General Hospital, General Division, prior to their
IVF cycle. These men were undergoing analysis ensure that sperm concentration,
motiiity, and morphology were within normal parameters for IVF. AI1 samples selected
for this study demonstrated normal semen analysis parameters according to the WHO
criteria (2992). Age was not taken into consideration, although al1 men were under the
age of 43 years. Written consent for use of the spenn for research was obtained from
patients according to guidelines established for research on human subjects by the
University of Toronto.
Semen Preparation
Semen preparation was similar as to the technique described above text in semen
preparation for ICSI spem.
Generation of reactive oxvgen species
Reactive oxygen species were generated using the xanthine-xanthine oxidase
(X/XO) system described by McCord and Fridovich ( 1 968). Stock solutions of xanthine
oxidase (Sigma, St. Louis, MO) (50 mu) and xanthine (Signa, St. Louis, MO) (200 mM)
were prepared in human tuba1 fluid (HTF) (Irvine Scientific, Santa Ana, CA) and added
to 30 samples immediately after swim up. At timed intervals of 15 min, 30 min, I hr and
fomaldehyde on a slide and allowed to air dry.
The concentration of ROS produced by this system was assessed by a
chemiluminescence assay (Shekarriz et al, 1995) using luminol (5-amino-2,Sdihydro-
1,4-phthalazinedione) (Sigma, St. Louis, MO) dissolved in water (5mM). Luminol
deoxygenates in the presence of 0;- and H202, giving off a signal which can be detected
by chemiluminescence. For analysis, 20 yL of lurninol solution was added to each spem
aliquot. Cherniluminescence was measured using a Berthold luminometer (Lumat LB
950 1, Wallace Inc., Gaithersburg, MD) immediateIy before the addition of XKO and at
time intervals of 15 min, 30 min, 1 hr and 2 hrs after X/XO addition. ROS production
was expressed as counted photons x IO5 (cpm) in 20 sec. One aliquot was used to
measure the background luminescence for each specimen before adding luminol. The
background reading was subtracted fiom the actual test value to obtain the ROS level.
3.5 Antioxidant pretreatment
The second part of this study involved the pretreatment of swim up samples with
antioxidants. 17 specimens were divided into 5 aliquots. The sperm concentration was
adjusted in each aliquot with HTF solution so that a concentration of 2 milIion/ml was
present in each sarnple. Antioxidants [N-acetylcysteine (.O1 mM), catalase (500 U/ml),
reduced glutathione (10 mM) and hypotaurine (10 mM)] were freshly prepared in HTF
and added to the swim-up sample 10 minutes prior to X/XO addition; the same procedure
was repeated as above to prepare slides for the TUNEL assay.
DNA fragmentation in the sperm recovered afier exposure to ROS and/or
antioxidants, was measured using the TUNEL method and analyzed using fluorescent
m icroscopy as described above.
Approximately 20 pL of spem fiom each swim up sample was removed and
fixed before xanthine oxidase or antioxidants were adrninistered. These were control
slides for cornparison with the treated slides.
Statistical Evaluation
Since the data were not normally distributed, we used the two way analysis of
variance to detect a significant difference between antioxidant treatment groups and the
sample goup with no antioxidant treatrnent. A p value of less than .O5 was considered
sipificant.
ICSI semen samples:
A total of 150 male partners from the KSI program provided spem samples
processed for TUNEL. The mean (A SEM) DNA fragmentation rate for this sample
group was 14.5 1.5% and ranged from 0.5 - 75%. An example of the degree of red
fluorescence obtained with the technique is shown in Figure 2. The negative control
sperm sample in which terminal transferase was omitted, demonstrated 0% red
fluorescence. The positive control spem sample which was pretreated with DNase 1,
showed 97% labeled sperm. Using the Spearman rank correlation coefficient, a negative
correlation was found between the percentage of sperm with DNA fragmentation after
swim-up and the ICSl fertilization rate (r = -0.23; p = 0.01 17; n = 13 1; Table 1 and 2).
In contrast, there was no correlation between percentage of sperm with DNA
fragmentation and the embryo cleavage rate. Embryo deavage rate was defined as the
number of embryos that have divided at 48 hrs out of the total nurnber of oocytes that
dernonstrated fertilization at 24 hrs.
To detect an influence of each or a combination of the three semen analysis
parameters on DNA fragmentation, multiple linear regession analysis was used. We
observed that DNA fragmentation could be predicted by the cornbination of motility of
the ejaculated sperm and percentage normal morphology in the raw semen samples prior
to swim-up (p = 0.002). Sperm concentration on the other hand, did not appear to
demonstrate a significant influence on DNA fragmentation (p>0.05).
Table 1: The Spearman rank order correlation between spem DNA fragmentation and the fertilization and embryo cleavage rates with KSI.
- -
Variable 11 Coefficient p value*
Fertilization rate 131 - 0.23 0.01 17
Embryo cleavage rate 129 0.00 NS
- - . . . - - -- - - -
* NS, not significant
Table 2: Association between fertilization rate in ICSI and sperm DNA fragmentation of (25% or > 25% assessed using the TUNEL assay. n = 13 1
FERTlLIZATiON DNA FRAGMENTATION RATE (%) < 25% >25 %
- ---- - -- -- , .,.,..m... . .'yV. . ' . C . C . . k \ ' U U. C Y . 4 .. ... ' -r , , .YU . ". . . Y , . L V U L. L . ' .. 1 ... .., .. ., point: at unlabeled normal sperni head with intact DNA.
A$, spcrrnritozoa froin patient undersoing TCST. A) Sperm stained with a DNA dye (DAPi) B) The satnc: t i dd at'ter I'UNEl.. using biotinylated nucleotidcs and Streptav
red conj ug t c . din Texas-
C,D, spermatozou from positive control sample. C ) Sperin staincd with a DNA dye (DAPI) D) The same tield aller pretreatment with DNast: I prior to TUNEL. 97% DNA
fragrne~tatim is evidcnt.
Figure 2: The percentage of DNA fragmentation observed in 131 ICSI spem sampies according to fertilization rate.
Honmnml bar indiaics median of DNA l i a p u t a i i o n in rach p u p . Kecmglc mdicstcs 95% limiu about thc me<ii;in. Errot b u s Qmonsmie range or DNA fngmenlalion values m euh group.
The clinical results obtained dunng the period of study are surnmarized in Table
3. A total of 102 oocytes that failed to show rnorphological evidence of normal
fertilization 1 6-1 8 h afier spenn injection were anal yzed (1.8Ypatient). Unfertilized
oocytes were fixed from 55 cycles of ICSI and assessed using the TLJNEL assay.
Fluorescence labeling of fragnented oocyte DNA was observed as bnght red staining of
the chromosomes (Fig. 3B) of almost 25% of oocytes in the sample. niere was a
significant increase in the incidence of DNA fragmentation in oocytes from patients
greater than 35 years old (p = 0.01) (Fig. 3E). This age group criteria was selected as
previous cytogenetic studies have demonstrated higher rates of aneuploidy in women
who are in their late 30s (Kajii and Ferrier, 1978; Zenzes et al., 1992). In addition, of
those oocytes that had a fim polar body present which could be analyzed following
fixation and staining procedures, 68.4% (60/88) contained fragmented DNA. Not al1
oocytes had a polar body present due to the removal of the zona pellucida.
Of the oocytes subjected to micromanipulation, a proportion did not show any
presence of spermatozoa inside, f 7 out of 102 (1 7.3%). This is consistent with studies by
Flaherty et al (1995) and Sakkas et ul (1996), who reported that 19% and 16%
respectively, of their oocytes examined had ejected the spermatozoa afier KSI. In the
remainder of the oocytes assessed, 30.6% contained sperm that remained condensed
while 39% presented sperm either decondensing or decondensed, visible by DAPI
staining (Table 4). Of the sample of oocytes, almost 25% demonstrated fragmented
spem DNA (Fig. 3D). In addition, we also observed that condensed sperm had a greater
proportion of DNA nicks in cornparison to decondensed sperm.
demonstrated 0% red fluorescence. Positive control samples which were pretreated with
DNase 1, showed almost 100% labeling of both oocyte and sperm DNA. Furthemore,
when 20 unfertilized oocytes fiom IVF cycles were analyzed using the same technique,
no significant difference was evident (p = 0.35) between the proportion of unfertilized
KSI oocytes with fragrnented DNA and those fiom IVF. This finding indicates that
since the TVF oocytes were treated in an identical manner as the ICSI oocytes with the
exception of injection of sperm, that the ICSI procedure does not damage oocyte
chromatin.
A) 0oc;te stained wiih a DNA dye (DAPl) kom0 yciir old patient. 1%) The same oocyte aRer 'l7UNEL, drmonstrating fragmented oocyte DNA.
Arrows point at labeied chromatin indicating fragrnented DNA. S - sperin head. C ) Oocyte slained witli ri DNA dye. (DAPI) from 27 year old patient. D) The same oocyte afer TUNEI,, deinonstrating liagmented sperin DNA.
Figure 3E: Mean percentage of oocytes with fragmented DNA from 24 phents == 35 years old compared to patients 3 1 > 35 yean old p < -05.
<35YRS >35 YRS
AGE (W.)
Table 3. DNA fragmentation of unfertilized oocytes fiom patients undergoing ICSI during the study.
No. of patients:
No. of cycles:
Mean age + SEM (years): 34.7 f 3.4 Range: 25 - 42
No. of oocytes: 102
Fragrnented oocyte DNA: 25 (24.4%)
Fragmented sperm DNA: 26 (25.8%)
" spennatozoa visible
Table 4. Spenn chromatin patterns in unfertilized oocytes afier intracytoplasmic sperm
injection (ICSI).
No. of oocytes:
Oocytes with sperrn present:
Sperrnatozoa;
condensed
decondensed*
1 Oz
85 (83.3 %)
Intact spenn DNA Fragmented sperm DNA
* partially or completely decondensed
A total of 47 sperm samples were obtained from males undergoing routine semen
analysis. ROS was generated exogenously in these samples, then spematozoa were
analyzed for DNA fragmentation using the TUNEL method. Before treamient, the
percentage of spem with fragrnented DNA was less than 4% in the majority of simples
but ranged from 0% to 16%. A mean (* SEM) basal level of ROS in the samples was
measured to be 2.85 x IO5 4.3 cpm which represented the amount of free radicals
present in the swimup sample before treatment. ROS was then generated in 30 samples
creating an average ROS concentration of up to 28.8 x IO5 * 8.9 cpm which decreased
over time (Fig. 4). Using the Wilcoxon rank sum test, a significant fourfold increase in
the DNA fragmentation rate between time O and 2 hrs was observed afier spenn were
exposed to ROS for up to 2 hrs (p = 0.000 1). The remaining 17 of the original 47 samples
were divided into 5 aliquots each containing approximately 2 million spedrnl. Each
sample was pretreated with one of four antioxidants or a combination of these prior to
XB(0 generation of ROS (Figs. 5-9). The concentrations used were obtained fiom
previous studies which have used antioxidant supplementation to improve sperm quality
(Sikka et al., 1995). Using a two-way analysis of variance, the most effective prevention
of DNA fragmentation was the addition of a combination of two antioxidants, reduced
glutathione and hypotaurine (p = .0001). The addition of N-acetylcysteine (p = 0.04),
hypotaurine (p = 0.0 1 ), and reduced glutathione (p = .O0 1) were observed to also have a
significant protective effect on DNA fragmentation, although not as effective as the
combination group. In contrast, the pretreatment of samples with catalase (p 0.05)
demonstrated no significant effect with respect to DNA damage.
1 1 I 1
O 15min 30 min 1 hr 2 hrs
TIME OF EXPOSURE TO ROS
Figure 5: Protective effect of rduced-glutathione on DNA of human spermatozoa. n = 17
TIME OF EXPOSURE TO ROS
4 glutathione +- contml
* A signi ficant difference berneen the antioxidant treated group and the non-rreated control group was evident. p = 0.01. Error bars indicate the standard error of the mean.
Figure 6: Protecave effect of hypotaurine on DNA of human spematozoa. n = 17
O 15 min 30 min 1 hr 2 h n
TiME OF EXPOSURE TO ROS
* A significant difference between the antioxidant treated goup and the non-treated controi group was evident, p = 0.01. Emr bars indicate the standard error of the m a n .
Figure 7: Effect of cacalase on DNA of human spematozoa n = 17
18
16 -
14 - I
12 - I O -
2 -
O 1 1 1 1 I I
O 15 min 30 min 1 hr 2hrs
TIME OF EXPOSURE TO ROS
- &lase + control I
t A significant difference between the antioxidant treated group and the non-ueated control g o u p was not evidenf p > 0.05. Error bars indicate the standard error of the mean.
Figure 8: Protective effect of N-acety lcy steine on DNA of human spennatozoa. n = 17
TIME OF EXPOSURE TO ROS
O
O 15 min 30 min 1 hr 2hm
* A significant difference between the antioxidant treated group and the non-treated conuol group was evident. p = 0.04. Error bars indicate the standard error of the mean.
Figure 9: Protective effect of reduced-glutathione t hypotaurine on DNA of human spermatozoa. n = 17
O
O 15 min 30 min 1 hr 2 hrs
T b E OF EXPOSURE TO ROS
* A significant difference between the antioxidant treated group and the non-treated control group was evident, p = 0.001. Error bars indicate the standard emor of the mean.
ICSI is now accepted as an appropnate treatrnent for male factor infertility
patients and results in high fertiliuition and pregnancy rates (Palemo et al., 1995).
Favorable results are achieved even from semen samples with severe oligo-
asthenoteratozoospermia defined by WHO Criteria (1992) as samples containing: 0 0
million/ml spem concentration, <40% motile sperm and <30% normal sperrn. However,
the importance of sperm contribution to fertilization, embryo development and fetal
viability must be considered when performing intracytopIasmic injection of spermatozoa
from subfertile males, especially those with a high percentage of abnomal spermatozoa.
The results of the present study demonstrated that spermatozoa fiom patients with
abnormal semen parameters, have increased levels of nicked DNA and that the more
sperm containing nicked DNA in the sample, the lower the fertilization rate. In addition,
when injected, unfertil ized oocytes were examined, almost 25% of unfertilized oocytes
after ICSI, were injected with sperm containing fragmented DNA. These observations
are likely to be of importance to the ICSl procedure since it is assumed that
morphologically normal, motiIe sperm selected for injection are in fact normal. This
study suggests that sperm for injection may often be selected from a spem population
with a relatively high incidence of fiagmented DNA. Our results indicate that a
significantly greater proportion of intact, condensed spem were observed to have
damaged DNA, compared to those with decondensed sperm. It is possible that a high
level of abnorrnalities in the chromatin of a spematozoon selected for ICSl may impede
the initiation or completion of decondensation, thereby leading to a failure of
fertilization (Sakkas et al., 19%).
primarily due to the replacement of histones by protamine and increased disulfide bond
formation (Balhorn et al, 1988). Therefore, sperm DNA is nomally highly resistant to
physical or chernical denaturation. Using the acndine orange staining test, it was
observed that fewer sperm with double-stranded DNA were present in men who had poor
fertilization in N F compared to those with apparently normal fertilization (Tejada et al.,
1984). This finding suggests that since acridine orange stains protamines, the fewer
protamines present, the less disulphide bonds formed within the DNA. It is believed that
chromatin packaging anomalies in human spermatozoa arke because of defects in the
sperm nuclear condensation mechanisms such as faulty protamine deposition during
spematogenesis in some patients (Balhorn et al., 1988; Belokopytova et al., 1993;
Manicardi et al., 1995). This hypothesis is supporîed by the increased aniline blue
staining of sperm observed in infertile men, indicating the persistence of histones
(Foresta et al., 1992). These abnormalities may be associated with increased DNA
instability and sensitivity to denaturing stress. Griveau ci uZ. (1992) also found that
asthenozoospermic men displayed a high percentage of spermatozoa with abnomal
nuclei and demonstrated that their sperm chromatin decondensed slowly and
incompletely compared to norrnozoospermic men. Abnomal chromatin may indicate
faulty condensation of the chromatin therefore decondensation will also be affected if the
DNA was packaged properly.
The present study utilized the specific activity of TdT to incorporate biotinylated
deoxyniridine to 3'-OH ends of DNA in order to detect DNA frapentation. The signal
was amplified by streptavidin-Texas red conjugate. Sperm with normal DNA in which
those with frapented DNA (multiple chromatin 3'-OH ends) fluoresced bnghtly. This
technique has previously been used along with DAPl staining of chromatin, to confirm
the occurrence of DNA fragmentation associated with apoptosis in fiagmented human
embryos (Jurisicova et al., 1996) and to demonstrate low levels of spem DNA
fragmentation in normal appearing semen samples used for IVF (Sun et al., 1997).
The reason for spem DNA fragmentation is unclear at present. Gorczyka et al
(1993) proposed that the presence of endogenous nicks in ejaculated human spem is
characteristic of programmed ce11 death as seen in apoptosis of somatic cells. In this
context, apoptosis may lead to functionat elimination of possibly defective germ cells
from the genetic pool. Apoptosis could be triggered by a vanety of factors within the
testes such as hormonal changes, physical trauma and chemical insult (Blanchard et al.,
1996; Meistrich, 1993; Sinha et al., 1995).
Another study suggests that the appearance of DNA nicks during the late
spermatid stage when histones are replaced by protamines, may facilitate the packaging
of DNA into a very small volume during spermiogeneisis (McPherson and Longo, 1993).
McPherson and Longo state that the rernoval of histones creates torsional strain on the
DNA strucnire, therefore endonucleases such as topoisomerase 11, must create nicks in
the DNA to relieve this stress. However, if DNA fragmentation was a natural phenomena
and simply a step in DNA packaging, we would expect to see nicking in every spem cell,
which is not the case in this study.
A third speculation is that proteîn replacement and chromatin re-amangement are
related to nick translation sensitivity (McPherson and Longo, 1992). On this basis, spem
For esample, there may be a defect in histone rernoval, therefore not as many protamines
are able to bind to the DNA. The outcome would be less condensed nuclear material in
the spem since protamines are necessary to form disulphide bonds which aid in the tight
packaging of chromatin. Poorly condensed DNA would be susceptible to various
denaturing stresses such as heat or irradiation, thereby possibly resulting in fragmented
DNA.
Alternatively, it is known that DNA fragmentation in somatic cells can be caused
by reactive oxygen species (ROS) (Buttke and Sandstrom, 1994; Ratan et al., 1994).
Several pathologie processes such as vancocele and infection with pyospermia rnay
increase the level of ROS (Lenzi et al., 1994). Therefore, increased concentrations of
ROS, particularly in the presence of reduced protamination and disulphide bond
formation, may be associated with poor semen quality and spem DNA fragmentation.
Finally, another potential cause of sperm DNA fragmentation might be exposure
to environmental toxins. The relationship between declining semen quality and
environmental pollutants is a matter of considerable interest in both the scientific and lay
literature. Currently there is little evidence to support this theory. However, in a
previous study from the Casper laboratory. a significant increase was found in the
percentage of spem with fragmented DNA in the group of men who smoked compared
to nonsmokers (Sun et al., 1997). This observation may provide some support for an
adverse effect of environmental toxins on spem DNA. As mentioned above, if çpem
DNA is packaged loosely, environmental pollutants could cause denaturation and
perhaps DNA nicking as well.
Although spem chromatin appears to be an important deciding factor in the
outcome of ICSI, our results indicate that nomality of oocyte DNA plays an equal role.
In this snidy, bright fluorescence labeling was detected in the cytoplasm of almost 25%
of the oocytes indicating nicked DNA. DNA fragmentation often represents the initial
step in apoptosis which refers to rnorphologic appearance of cells undergoing
programmed ceIl death. Apoptotic oocytes are not viable and hence will not fertilize as
demonstrated by our results. The first polar body on the other hand, was observed to be
labeled almost 70% of the time which was an expected result. The polar body is
evidently programmed to die and thus always undergoes degeneration, even if
fertilization occurs.
As in the case of spermatozoa, the cause of DNA damage in oocytes is unclear
although it seems likely from our study that age may be a factor. The relationship
between matemal age and fertility has long been recognized. Age-dependent loss of
reproductive capacity is observed in women in their late thirties who tend to have lower
prepancy rates and higher early abortion rates. In addition, chromosornal abnonnatities
of the fetus are detected more frequently in this older age group. Very little data is
available on human oocyte DNA. However Fujino et cil. (1996) reported that the rate of
DNA fragmentation detected by TUNEL, was significantly higher for oocytes from aged
mice. The age group criteria in Our study was selected since previous cytogenetic studies
have demonstrated higher rates of aneuploidy in women who are in their late 30s (Kajii
and Ferrier, 1978; Zenzes et al.. 1992). In our study, oocytes from women over 35 years
old, demonstrated a higher DNA frapentation rate than patients younger than 35 years.
fertilization at the time of ovulation.
Hman oocytes remain arrested in prophase 1 before birth until gonadotrophins
trigger resumption of meiosis. This implies a long duration of the first meiotic division,
allowing for cumulative exposure to extemal insults. One toxin in particular which has
been snidied extensively, is the affect of cigarette smoke on human oocytes. These
accumulated effects appear to induce alterations in cytoplasmic organization by the
presence of higher proportions of diploid oocytes in smokers compared with non-smokers
(Zenzes et al, 1995). Although we did not note the cigarette smoke exposure status of
patients from our study, according to Zenzes et al.(1995), it seems likely that smoking
may be hazardous to the viability and function of developing oocytes, particularly on
oocyte chromatin and the resulting embryo.
Another speculation of the cause of DNA fragmention in oocytes is thought to be
indirect exposure to UV irradiation. UV activates lipid peroxidation as well as fiee
radical generation (Bouquet et al, 1993), both of which we eluded to previously as being
potential causes of DNA fragmentation. DNA strand breaks although not produced by
UV irradiation per se, may occur as the result of failed nucleotide excision repair shut
down caused by ROS exposure. The final outcome could lead to chromosomal DNA
becoming incapacitated, therefore preventing the oocyte from taking part in subsequent
development. Furthemore, when bovine oocytes were exposed to UV irradiation, DNA
repair was detected only in immature germinal vesicle stage oocytes but was limited in
metaphase-1 and metaphase41 stage oocytes (Bradshaw et al, 1995). Hence, mature MI-
mechanisms, contibuting to the failure to fertilize.
Similarly to Zenzes et al. (1992), we observed that multiple oocytes of a patient
tend to be sirnilar with respect to chromatin constitution. In particular, 2 patients who
participated in more than one ICSI cycle each during the study penod and had poor
fertilization outcornes, dernonstrated almost equal proportions of fiagrnented oocyte
DNA from each cycle. This nonrandomness in chromosome normality or abnormality
(Le. fragmented DNA) of multiple oocytes indicates that some ICSI patients produce
either repeated chromosomally normal conceptions or repeated abnonnal conceptions.
The latter group of patients may thus be at an increased risk for recurrent failure in the
ICSI procedure.
ROS and its effect on DNA of human soerrnatozoa
Depending on the nature and concentration of the particular ROS involved, ROS
can have beneficial or detrimental effects on sperm function (de Lamirande and Gagnon,
1995). Free radicals are necessary for maintaining hyperactivation and the ability of
sperm to undergo the acrosome reaction (de Lamirande and Gagnon, 1993a; de
Lamirande et al., 1993b). Under normal physiologie conditions, seminal plasma which
contains a high degree of antioxidant activity, does not contain sperm-generated ROS.
However, as many as 25% of semen samples fiom infertile men have been demonstrated
to have increased levels of ROS (Iwasaki and Gagnon, 1992) suggesting that deficient
sperm may produce excessive ROS. Free radicals have been hypothesized to play a
causative role in the etiology of defective sperm function through peroxidation of the
High levels of ROS were correlated with decreased concentrations of motile sperm
while conversely, greater spem motility was observed in samples with lesser amounts of
detectable ROS. This obsentation implies that elevated incidence of ROS formation may
be associated with certain types of male infertility.
In the present study, 1 demonstrate that fiee radicals, such as 0;' and H,02
created exogenously by the X/XO system, can cause DNA damage in hurnan sperm when
exposed for time periods consistent with clinical sperm preparation techniques for ICSI
or IVF. An ROS concentration of up to 28 x 10' cpm was measured by luminescence
which is comparable to the reported mean ROS value of 34.3 x IO5 cpm produced by
sperm during routine sperm preparation techniques (Shekarriz et al, 1995). As mentioned
above, sperm nuclear chromatin is highly condensed primarily due to the replacement of
histones by protamine and increased disulfide bond formation. Therefore, sperrn DNA is
normally highly resistant to physical or chernical denaturation. However, in subfertile
men, defects in chromatin condensation have been show to result in increased DNA
instability and sensitivity to denaturing stress (Balhom et al., 1988; Manicardi et al.,
1995). We have confirmed this finding in the early part of the study by demonstrating
that s p e m of poor quality used for KSI, contained higher amounts of fragmented DNA
than sperm frorn men in the IVF program or from normal fertile males. We can speculate
that sperm from poor quality samples not only have the capabiiity to produce high levels
of ROS, but also are much more susceptible to DNA damage caused by oxidative stress.
It has been reported that a key factor in the production of ROS by damaged or
deficient sperm, may be the spem preparation technique. Sperm washing and swim up is
---- ----- - . -
production (Aganval et al., 1994). This finding is of particular relevance during
preparation of spenn for KSI where poor quality sperm is characteristic of samples
selected for this procedure. Sperm washing procedures could trigger increased ROS
production which then accumulate in the sarnple. The outcome is prolonged exposure of
sperm to ROS afier swimup during the time period when oocytes are prepared for
injection. Our results indicate that sperm exposed to ROS for greater than 1 hr have an
increased DNA fragmentation rate.
To counteract the damaging effects of ROS, a variety of antioxidant systems are
present within the serninal plasma including, superoxide dismutase (SOD), catalase,
GSSG (glutathione peroxidase) and GSH (glutathione reductase) (Alvarez et al., 1987).
However, once seminal plasma is washed away during sperm preparation for assisted
reproduction techniques, spenn are virtually unprotected. In addition, the cytoplasm of
the human sperm is extrernely limited in tems of its volume and does not possess a
significant amount of antioxidants. Cellular damage arises when the equilibrium
between the amount of ROS produced and that scavenged by antioxidants is disturbed,
and this imbalance has been s h o w to correlate with idiopathic infertility (Sharma and
Aganval, 1996).
We demonstrate that a combination of reduced glutathione and hypotaurine,
which are considered "suicide antioxidants", was most protective against DNA
fragmentation. In addition to having the ability to neutralize O/, reduced glutathione is
also a substrate for GSSG which rnetabolizes H202 and OH-. Hypotaunne on the other
hand, is able to react directly with cytotoxic aldehydes . produced during lipid
al. ( 1996) have also demonstrated the effectiveness of glutathione in combination with
hypotaurine on sperm motility.
Catalase, which scavenges H,02, was the least effective with respect to preventing
sperm DNA damage suggesting that although H202 may be an important initiator of lipid
peroxidation, that 0;' has a more direct effect on chromatin. Previous studies have
indicated the use of antioxidants to irnprove sperm function (Krausz et al., 1994; Lenzi et
al., 1994). Our results suggest an additional therapeutic reason for supplementation of
media with free radical scavengers.
In summary, we have demonstrated that a negative correlation exists between the
percentage of sperm with DNA fragmentation afier swim up and fertilization rate in KSI.
We established that samples with a high percentage of DNA fragmentation were less
likely to fertilize in ICSI compared to samples which had little or no DNA frapentation.
In cases of severe male factor infertility we suggest that a significant proportion of spem
injected into oocytes may contain fiagmented DNA. This suggestion might explain the
inability of most clinics to achieve a mean fertilization rate of more than 65% with ICSI.
We also detemined that both spem motility and morphology have an influence on DNA
fragmentation in sperm, although spenn concentration does not.
Secondly, concems have been expressed that micromanipulation may produce
adverse genetic consequence for children bom as a results of this procedure. However,
this study demonstrates that bypassing of extra-oocyte bamers to fertilization still results
in a seiection process related to DNA integnty at the time of fertiiization. Oocytes
injected with sperm containing normal or slightly abnormal chromatin may fertilize
successfully, while an increased amount of abnormal chromatin in sperm will most likely
prevent decondensation and thus fertilization. In addition, we observed that oocyte
chromatin damage may also play a role in the failure to fertilize and that matemal age
influences the rate of DNA fragmentation.
Lastly, we have determined that exogenous ROS generation causes an increase in
DNA fragmentation in human sperm after swim-up. An increase in oxidative damage to
the spem membrane, intracellular proteins and DNA is associated with alterations in
signal transduction mechanisms that can affect fertility (Sikka et al, 1995). Poor quality
ROS during routine sperm preparation and our results indicate that sperrn are prone to
DNA damage when exposed to ROS for at least lhr. DNA damage in tum could lead to
the failure of fertilization if a spem containing fragmented DNA is selected for injection.
Furthermore, we determined that the administration of antioxidants prevented
excess DNA nicking caused by exogenous ROS. This suggests another advantage for
antioxidant supplernentation of media which has been discussed in other studies.
This current project has provided us with many answers to the integnty of DNA
in human spermatozoa and how damaged DNA may affect fertilization. However, it has
also brought up new questions of which the answers remain unknown
During this study, testicular biopsy samples fiom azoospemic men were also
treated with the TUNEL assay. We observed that almost 55% of round spematids in the
samples contained fragmented DNA. Round spennatids are currently being investigated
for use in ICSI, although to date the fertilization rate is very poor (less than 25%) and
only three pregnancies have been reported. It would be worthwhile to cany on this study
on round spematids which we speculate do not fertilize possibly due to fragmented
DNA. Pretreatment of testicular biopsies with antioxidants in an effort to reduce DNA
damage, wou1d also be an important area to look at.
There have been many debates in the recent media regarding the decline in sperrn
quality in the male population and its link to environmental pollutants. Although we
have established that ROS can cause DNA fragmentation, it would be worthwhile to
determine the effect of other toxins such as dioxin and nicotine, on sperm DNA as well.
Lastly, we have yet to determine if DNA fragmentation in sperm is a feature of
the early stages of apoptosis. A suitable method would have to be developed to obtain
enough fragmented DNA. Currently, we cannot observe a distinct ladder pattern during
gel electrophoresis which would indicate apoptosis, however we suspect that there may
simply not be enough DNA to demonstrate this pattern. On the other hand, other
techniques such as annexin V binding rnay be utilized for sperm to determine early
apoptosis.
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