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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