Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

SPERMATOZOAL COMPONENTS AND THEIR

EFFECT ON REPRODUCTIVE OUTCOME

Various components of the fertilizing spermato-

zoon have been studied for their impact on the

reproductive process. Anomalies of either the sperm

cytoplasmic membrane or cytoskeleton are most

likely to affect the postfertilization processes. Indeed,

centriolar abnormalities have largely been shown to

impede fertilization,

22,23

although an effect on later

stages cannot be excluded. The mitochondria are

believed to be maternally inherited, although some

argue that this inheritance could be skewed by micro-

manipulation techniques such as those utilized in

assisted reproductive technologies.

24,25

The nucleus

therefore remains as the greatest contributor to the

potential success of a reproductive outcome.

SPERM NUCLEAR DETERMINANTS

IMPACTING ON REPRODUCTIVE

OUTCOME

A multitude of acquired spermatozoal nuclear fac-

tors may affect reproductive outcome. These include

numerical abnormalities in spermatozoal chromo-

some content, Y chromosome microdeletions,

alterations in the epigenetic regulation of paternal

genome, and, finally, non-specific DNA strand

breaks (reviewed in reference 16).

The impact of aneuploidy of paternal origin and

Y-chromosome microdeletions are clear and have

both been extensively investigated.

Animal studies reveal that the male germ cell has

a role in epigenetic inheritance,

and suggest that

epigenetic marks, especially DNA methylation, are

unstable, and may be altered by culture condi-

tions.

26,27

Assisted reproductive technologies (ART)

rely on manipulation and culture of gametes and

embryos at times when epigenetic programs are

being acquired and modified. An initial study of

samples from 92 children conceived using ICSI found

no evidence of altered methylation at 15q11-13, the

locus linked to the pathogenesis of the imprinting

disorders Angelman and Prader-Willi syndromes.

However, six studies have recently reported two

imprinting disorders, Beckwith-Wiedemann syn-

drome

29–32

and Angelman syndrome

33,34

in associa-

tion with the use of ART. The report by Halliday et al

was the first case–control study to suggest that a

child with Beckwith-Wiedemann syndrome had a

probability of having been conceived using IVF

that is 18 times greater than for a child without the

syndrome. Although these data are worrying, they

may also be exposing particular genetic and devel-

opmental traits in infertile couples that influence

imprinting.

A number of studies have examined imprinting

in spermatozoa of men with abnormal semen

parameters. An initial study by Manning et al

used

polymerase chain reaction (PCR)-based techniques

to analyze DNA extracted from spermatozoa of men

with normal semen analysis (n ⫽ 30) and from men

with medium (n ⫽ 30, 5–20 million spermatozoa/

mL) and high grade semen pathology (n ⫽ 30, ⬍5

million spermatozoa/mL) undergoing ICSI. They

failed to detect a difference in methylation status

between the groups; however, this does not confirm

that differences may not exist. More recently, Marques

et al

studied two oppositely imprinted genes in

spermatozoal DNA from normozoospermic and

oligozoospermic patients. In the mesodermal specific

transcript gene (MEST), bisulfite genomic sequenc-

ing showed that maternal imprinting was correctly

erased in all 123 patients. However, methylation of

the H19 gene did not change in any of 27 normo-

zoospermic individuals (0%) compared with methyl-

ation changes in eight moderate (17%, p ⫽ 0.026)

and 15 severe (30%, p ⫽ 0.002) oligozoospermic

patients. Their findings suggest an association

between abnormal genomic imprinting and hypo-

spermatogenesis, and that spermatozoa from oligo-

zoospermic patients carry a raised risk of transmitting

imprinting errors.

Testicular sperm extraction and intracytoplas-

mic sperm injection (TESE-ICSI) is a frequently

used therapeutic option in azoospermic males.

Manning et al

used hemi-nested methylation-

specific PCR for the target region 15q11-13 to ana-

lyze imprinting at the single-cell level in cells from

different stages of spermatogenesis. They analyzed

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SPERM DNA AND EMBRYO DEVELOPMENT

spermatozoa, elongated spermatids, and round

spermatids, and found completed establishment of

the correct paternal imprint in these three develop-

mental stages. However, there was a high rate of

amplification failure in round spermatids in this

study, raising a factor of uncertainty. It should be

emphasized that the mechanism that establishes

paternal epigenetic marks which contribute to the

future genome are still major issues that remain to

be explored.

NUCLEAR DNA STRAND BREAKS IN

SPERMATOZOA

The presence of nuclear DNA strand breaks in ejac-

ulated spermatozoa as an aspect of sperm quality

has been examined more closely in the past decade.

Their presence was initially reported in the early

1990s,

39–41

and their origin and subsequent impact

is still not completely understood.

21,42,43

The complex process of spermatogenesis involves

an intricate series of mitoses and a meiotic division

followed by marked changes in cell structure. In

addition to proliferation and differentiation, the out-

come of spermatogenesis is affected by the extent of

germ cell death. During spermatogenesis, germ cell

death occurs normally and continuously, and is

estimated to result in the loss of up to 75% of the

potential number of spermatozoa.

It is believed

that nuclear DNA strand breaks may arise due to the

failure or inadequacy of the DNA policing and/or

processing mechanisms that take place during sper-

matogenesis. The three main mechanisms responsi-

ble for the quality of DNA in the spermatozoa are

DNA repair, apoptosis, and chromatin remodeling.

All of these steps have been implicated in the

generation of DNA strand breaks in the ejaculated

spermatozoa.

DNA REPAIR

A failure in effective DNA repair in human testicu-

lar cells is reflected in an accumulation of DNA

damage adducts such as 8-oxoG and benzo(a)pyrene

in human sperm

15,45–47

and by the plethora of papers

now showing DNA damage in ejaculated sperm.

Although some of these defects are clearly environ-

mentally related, with smoking as a good (or bad)

example,

48,49

many are thought to be intrinsic to the

patients’ clinical makeup; whether they are related

to a failure in the patients’ DNA repair mechanisms

is as yet unknown.

APOPTOSIS

Apoptosis represents an additional mechanism for

removing cells with DNA lesions, and operates in

relation to DNA repair. Spontaneous apoptosis does

occur in all cell types of the testis during normal

spermatogenesis in man,

and testicular cells are

very sensitive to apoptotic stimuli such as high-dose

chemotherapy.

It is likely that the total level of

DNA lesions is of decisive importance for the fate of

the spermatogenic cell.

On the other hand, there is evidence that

benzo(a)pyrene (B(a)P) adducts can indeed accu-

mulate without eliciting apoptosis in spermatogenic

cells, since ejaculated sperm from smokers contain

more B(a)P adducts than from non-smokers.

Furthermore, such adducts derived from the father

do not prevent fertilization and are persistent even

at the blastocyst level,

also indicating that they are

not always completely repaired in the fertilized

oocyte. A recent publication

showed that assisted

fertilization was 40% less likely to produce a live off-

spring if the father was a smoker, compared with if

he was a non-smoker.

Four possible functional roles have been proposed

for apoptosis during normal spermatogenesis.

(1) Maintenance of an optimal germ cell/Sertoli

cell ratio. It has been established that each

Sertoli cell can support only a finite number of

germ cells throughout their development into

spermatozoa. Therefore, supraoptimal num-

bers of spermatogonia may undergo apoptosis

to maintain an optimal ratio.

(2) Elimination of abnormal germ cells. There may

be a selective process in which abnormal germ

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HUMAN PREIMPLANTATION EMBRYO SELECTION

cells, especially chromosomally abnormal germ

cells, are eliminated from the population by

apoptosis.

(3) Formation of the blood–testis barrier by tight

junctions between Sertoli cells. Elimination of

excessive germ cells is necessary for this process.

Suppression of germ cell apoptosis by inactiva-

tion of the apoptosis-promoting factor bax pre-

vents the formation of these tight junctions.

(4) Creation of a prepubertal apoptotic wave. A

massive wave of germ cell apoptosis normally

takes place as mammalian species approach

puberty. This wave serves as a regulator of the

ratio between germinal cells in various stages

and Sertoli cells, and facilitates the establish-

ment of mature spermatogenesis. There is evi-

dence that preventing this wave of apoptosis by

expression of apoptosis-inhibitory proteins,

such as Bcl-xL or Bcl-2, results in highly

abnormal adult spermatogenesis accompanied

by sterility.

Whereas apoptosis appears to be a component of

normal spermatogenesis, one puzzling occurrence

in ejaculated human spermatozoa is the presence of

apoptotic marker proteins. In the human, we origi-

nally found that men with abnormal sperm param-

eters display higher levels of the apoptotic protein

Fas on their ejaculated spermatozoa.

The presence

of Fas on ejaculated spermatozoa correlated strongly

with a decreased sperm concentration and sperm

with abnormal morphology. More recently, we and

others also found that other apoptotic markers such

as Bcl-x, p53, caspase, and Annexin V are also pres-

ent on ejaculated human spermatozoa and show

distinct relationships with abnormal semen param-

eters.

58–61

Double labeling experiments have shown

that ejaculated human sperm expressing apoptotic

marker proteins can also display chromatin damage

and show signs of immaturity, such as cytoplasmic

retention.

Interestingly, exclusion of sperm dis-

playing these apoptotic markers can also limit the

number of sperm possessing DNA strand breaks,

but this is not exclusive.

Said et al

have shown

that annexin V-negative sperm displayed superior

quality in terms of high motility, low caspase 3 acti-

vation, MMP integrity, and small extent of DNA

fragmentation. In the same study it was shown that

these sperm also had a higher fertilization potential.

DNA REMODELING

DNA strand breaks detected in male germ cells

may also be due to factors unrelated to apoptosis.

Indeed, McPherson and Longo

64,65

demonstrated

the presence of endogenous DNA strand breaks in

elongating rat spermatids, when chromatin structure

and nucleoproteins are modified. They proposed

that the presence of endogenous nicks in ejacu-

lated spermatozoa might be indicative of incom-

plete maturation during spermiogenesis. They also

postulated

64,65

that chromatin packaging might

involve endogenous nuclease activity in order to

create and ligate nicks during the replacement of

histones by protamines. The transient presence of

DNA breaks has been reported in both mouse and

human.

66–68

THE IMPACT OF SPERM NUCLEAR DNA

STRAND BREAKS ON REPRODUCTIVE

OUTCOME

An increased presence of DNA strand breaks in

ejaculated spermatozoa could affect fertilization,

blastocyst development, and pregnancy rates.

Investigation of the possible association between

DNA strand breaks in spermatozoa and fertilization

rates in patients undergoing ART have in general

found no strong correlation between DNA integrity

of ejaculated spermatozoa and IVF and ICSI fertil-

ization rates.

69–72

In contrast to these reports, a neg-

ative correlation between sperm DNA strand breaks

and IVF and ICSI fertilization rates has been reported

using the terminal deoxynucleotidyl transferase

mediated dUTP nick end labeling (TUNEL) assay.

45,73

A recent meta-analysis examining eight articles using

the TUNEL and sperm chromatin structure assay

(SCSA) found that sperm DNA damage did not affect

either IVF or ICSI fertilization.

Unfortunately, many

of the human studies examine sperm populations and

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SPERM DNA AND EMBRYO DEVELOPMENT

are unable to examine individual spermatozoa which

actually fertilize the egg, as in animal studies.

Activation of embryonic genome expression occurs

at the 4–8-cell stage in human embryos,

suggest-

ing that the paternal genome may not have an influ-

ence on development until that stage. Therefore, an

elevated incidence of DNA strand breaks in sperma-

tozoa may not have an impact on early embryo

development. Indeed, a number of studies have

failed to find an association between sperm DNA

damage and early embryo development.

45,76

However, evaluating the outcomes of ART cycles

that involved egg sharing, we did observe a paternal

effect on embryo development prior to embryonic

genome activation.

During egg-sharing cycles, the

eggs from one woman are shared between herself

and a recipient, and spermatozoa from two different

partners are used to fertilize the eggs. When com-

paring embryo development between egg-sharing

pairs, we found that approximately 30% of patients

showed a difference in mean embryo score of ⱖ5 in

all embryo development, and 14% showed a differ-

ence in the quality of embryos available for trans-

fer on day 2 and 3. While sperm DNA integrity was

not evaluated in this study, its results indicate that

a spermatozoal component may impede embryo

development as early as the 4–8-cell stage in

human.

A number of early studies suggested a paternal

effect on blastocyst development in relation to sperm

quality

and when comparing IVF and ICSI.

interesting observation in a number of these reports

was that embryo quality was similar until day 2–3

and then the differences in blastocyst development

were observed in the day 3 to day 5 stage.

79,80

This

again indicates that a paternal effect may manifest

itself postembryonic genome activation. The possi-

ble consequences of fertilisation with sperm pos-

sessing abnormal DNA are shown in Figure 25.1.

Whether the extent of nuclear DNA damage in

ejaculated spermatozoa affects blastocyst develop-

ment after IVF and ICSI has also been examined. A

negative correlation using both the TUNEL assay to

evaluate spermatozoa processed for IVF

and SCSA

to evaluate unprocessed spermatozoa

has been

observed. In our study we found that when the cut-

off score for sperm DNA damage (as measured by

TUNEL) was ⬎50%, four out of eight patients had

no blastocysts developing in vitro. In contrast, only

two out of the 41 remaining patients did develop

blastocysts, all of whom had sperm DNA damage of

⬍50%. In the study by Virro et al,

males with ⱖ30%

DNA fragmentation index (DFI) had low blasto-

cyst rates (⬍30%) and no ongoing pregnancies.

These findings illustrate not only the importance of

sperm DNA integrity and its association with the

embryo’s ability to develop postembryonic genome



(a) No DNA Repair

Fertilisation

Failure

Abnormal

offspring

Normal

offspring

DNA Repair

Fertilisation

Partial DNA Repair

Fertilisation

Developmental failure

post - embryonic

genome activation

Development

and offspring

(b) (c)

(d)

Figure 25.1 Consequences of fertilization with sperm contain-

ing damaged DNA (red). (A) The spermatozoon may fail to

fertilize. (B) The spermatozoon containing damaged DNA may

be repaired upon fertilization and a normal blastocyst develops

with no paternal abnormalities (green). (C) The spermatozoon

containing damaged DNA may be partially repaired upon fertil-

ization and a normal blastocyst develops with paternal abnor-

malities (red). (D) The spermatozoon containing damaged

DNA may fertilize, however, the embryo fragments post-

embryonic genome activation and fails to form a blastocyst.

HPE_Chapter25.qxp 7/14/2007 5:52 PM Page 329

HUMAN PREIMPLANTATION EMBRYO SELECTION

activation, but also indicate that it is not an all-or-

none phenomenon, as sperm that can support

embryo development are present in the population.

Pregnancy rates after IVF are also reduced in

couples who have higher percentages of spermato-

zoa with DNA strand breaks detected by in situ nick

translation.

Similarly, there is strong evidence for

a relationship between sperm nuclear DNA integrity,

as assessed with the SCSA, and fertility after normal

intercourse

83,84

or ART.

Interestingly, Evenson et al

found cases in which

the classical criteria (concentration, motility, and

morphology) were within the normal ranges, but

the SCSA values were poor and not compatible with

good fertility after intercourse. Based on these results,

Evenson has proposed that SCSA parameters may

be independent predictors of reproductive out-

come, beyond World Health Organization (WHO)

parameters. On the other hand, in a recent study,

Gardner et al did not find a difference in the implan-

tation and pregnancy rates between two groups that

had 16% vs 40% fragmentation rates detected by

SCSA.

This result contradicts previous findings

using this technique.

The SCSA measures a number of parameters:

the DNA fragmentation index (DFI), that is, the

sperm fraction with detectable denaturable single-

stranded DNA, mainly due to DNA breaks; and the

highly DNA stainable cells (HDSs), the sperm frac-

tion showing increased double-stranded DNA acces-

sibility to acridine orange, mainly because of defects

in the histone-to-protamine transition process. As

these parameters are not correlated to each other,

they represent independent aberrations of the human

mature male gamete in the ejaculate. DFI has been

shown to influence normally initiated pregnancy:

83,84

increasing levels of DFI (⬎30%), independent of

WHO standard semen parameters, were associated

with a decreased probability of fathering a child.

Initially, SCSA was thought to be a powerful new

tool to predict ART outcome. An initial small pilot

study (24 men) showed that when DFI was over

27%, no pregnancies could occur with IVF or ICSI.

A number of subsequent studies reinforced this

finding. Larson-Cook et al

examined 89 couples

undergoing IVF/ICSI; the endpoint was clinical

pregnancy as assessed by serum human chorionic

gonadotropin (hCG) and ultrasonographic detection

of a gestational sac. They showed that all patients

who achieved a pregnancy had a DFI under 27%;

however, on the other hand HDS was not correlated

to pregnancy. Saleh et al

evaluated 19 couples

undergoing IUI, ten couples undergoing IVF, and

four couples undergoing ICSI. In this study, levels of

DFI (but not of HDS) were negatively correlated

with biochemical pregnancy. The highest DFI value

in biological fathers was 28%. Although the findings

were quite consistent, some discrepancies emerged

from these two studies: sperm concentration, per-

centage motility, and percentage morphology were

significantly lower in patients who failed to initiate a

clinical pregnancy in the Saleh et al

study but not

in the study by Larson-Cook et al.

The fertilization

rate was related to DFI in the Saleh et al

study but

not in the Larson-Cook et al

investigation. It is

noteworthy that Benchaib et al

also showed that

higher (⬎10%) sperm DNA fragmentation levels

(this time evaluated by TUNEL assay on the dis-

continuous gradient centrifugation selected sperm)

were a negative predictor of pregnancy via ICSI (but

not via IVF) and no pregnancies occurred if DNA

fragmentation was over 20%. This study assessed

the clinical pregnancy rate in a cohort of 50 IVF

patients and 54 ICSI patients by positive serum hCG

and ultrasound detection of a fetal heartbeat. The

enthusiasm generated by these original studies

regarding the existence of an upper DNA fragmen-

tation threshold above which no pregnancy can be

obtained after ART, has cooled down as more inves-

tigations have been published. Firstly, Gandini et al,

in a study involving 34 couples (12 IVF and 22

ICSI), did not note any difference between patients

initiating pregnancies or not, and, above all, they

reported healthy full-term pregnancies even with

high levels of DFI (up to 66.3%). Pregnancy rates

were 25% for IVF and 40.9% for ICSI. HDS did not

correlate with pregnancy or live birth rate, and no

association was found between the SCSA parame-

ters and the fertility rate. Secondly, Bungum et al

investigated 306 consecutive couples undergoing

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SPERM DNA AND EMBRYO DEVELOPMENT

ART (131 IUI, 109 IVF, and 66 ICSI), taking into

account biochemical pregnancy (positive serum

hCG), clinical pregnancy (intrauterine gestational

sac with a heartbeat 3 weeks after a positive hCG

test), and delivery. The delivery rate was 15.3% after

IUI, 28.4% after IVF, and 37.9% after ICSI. They

reported that the chance of pregnancy/delivery after

IUI was significantly higher in the group with DFI

under 27% (and HDS under 10%): only one delivery

resulted among the 23 men with a DFI over 27%.

The combination of DFI and HDS gave a higher

predictive value regarding the outcome of IUI. On

the other hand, no statistically significant difference

in the outcome after IVF/ICSI was noted by dividing

patients according to a DFI level of 27%. However,

the results after ICSI were significantly better than

those after IVF: for example, as far as the group with

DFI over 27% was concerned, comparing ICSI with

IVF performances the authors reported higher

clinical pregnancy (52.9% vs 22.2%), implantation

(37.5% vs 19.4%), and delivery (47.1% vs 22.2%)

rates. In addition, by restricting the analysis to IVF

patients only, the group with a low DFI level under

27% consistently showed better clinical pregnancy

(36.6% vs 22.2%), implantation (33.3% vs 19.4%),

and delivery (29.7% vs 22.2%) rates as compared

with the group of men with DFI over 27%. A more

recent analysis by the same group

assessed the pre-

vious 988 cycles of IUI, IVF, and ICSI showed that

for IUI, the odds ratios (ORs) for biochemical, clin-

ical pregnancy, and delivery were significantly lower

for couples with DFI ⬎30% as compared with those

with DFI ⱕ30%. No statistical difference between

the outcomes of ICSI versus IVF in the group with

DFI ⱕ30% was seen. In the DFI ⬎30% group, the

results of ICSI were significantly better than those of

IVF. Virro et al

studied 249 couples undergoing

IVF/ICSI and noted that men with DFI under 33%

had a significantly greater chance of initiating a

clinical pregnancy (positive hCG), lower rate of

spontaneous abortions, and an increased rate of

ongoing pregnancies at 12 weeks (47% vs 28%).

HDS and standard WHO parameters were not

related to pregnancy outcomes. Finally, a recent

meta-analysis by Li et al

showed that sperm DNA

damage, as assessed by the TUNEL assay, signifi-

cantly decreases only the chance of IVF clinical

pregnancy, but not that of ICSI clinical pregnancy.

In addition they revealed that sperm DNA damage,

when assessed by the SCSA, had no significant effect

on the chance of clinical pregnancy after IVF or

ICSI treatment. All of these studies demonstrate

that high levels of DNA damage are still compatible

with pregnancy and delivery after IVF/ICSI.

The increasing number of publications in this

field indicates that the relevance of sperm nuclear

DNA tests is not completely black and white.

From

the ever-increasing wealth of data collected so far

about the SCSA and TUNEL techniques and their

predictive power in ART, we can speculate that an

increased fraction of sperm showing DNA damage

is certainly a negative trait that reduces the chance

of fathering a child; however, a magic number or

percentage of DNA damaged sperm in the population

that is not compatible with pregnancy is far from

being established. In addition, the studies of Bungum

et al

89,90

indicate that the predictive power of the

SCSA seems to lose its strength from natural con-

ception to ICSI, passing through IUI and IVF. The

contradictory results for ICSI can also be explained

on the basis that SCSA parameters refer to the DNA

status of a whole sperm population, but the selec-

tion of just one single good-looking sperm for ICSI

is biased by the intervention of a human operator

on the basis of his own criteria. We know that SCSA

parameters are only weakly associated with WHO

standard semen parameters. The technician who

performs ICSI attempts to select a sperm with normal

morphology, in order to reduce the risk of introduc-

ing a sperm with DNA breaks. The selection of sperm

from a subpopulation with no significant DNA

damage, as assessed by the SCSA, cannot be excluded.

This indicates that the sample tested by the sperm

DNA testing techniques should also reflect the way

in which they will be used. Therefore, while assess-

ing DNA damage levels in the unprocessed semen

sample may be predictive of natural fertility and IUI

outcome, it may be more diagnostically relevant to

use the prepared sample for assessment in order to

predict IVF or ICSI outcome.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

NOVEL TECHNIQUES TO REMOVE

SPERMATOZOA WITH

DAMAGED DNA

Cleansing the ejaculate of spermatozoa with abnor-

mal DNA would therefore overcome the above

problems. A number of studies have now examined

the possibility of improving the efficiency of prepa-

ration techniques in order to eliminate spermatozoa

with nuclear anomalies. The simplest of these is a

method that is routinely used in most ART labora-

tories, i.e. density gradient centrifugation. This may

explain why there is a better correlation between

sperm DNA tests when the final prepared sperm

sample is used for testing. A number of studies,

including our own, have shown that using sperma-

tozoa prepared with a density gradient centrifuga-

tion technique significantly improves the quality

of spermatozoa in the preparation. These studies

indicate that density gradient centrifugation can

enrich the sperm population by separating out

those with nicked DNA and with poorly condensed

chromatin.

70,92–94

A number of novel techniques have now been

proposed that may assist in limiting the chance of

selecting an abnormal spermatozoon prior to ICSI.

Bartoov et al

reported that they were able to achieve

a pregnancy rate of 58% in a small group of patients

who had previously failed at least five consecutive

routine cycles of IVF and ICSI, by selecting sperma-

tozoa under high magnification. Aitken’s group

96,97

report a novel electrophoretic sperm isolation tech-

nique for preparing functional human spermatozoa

free of significant DNA damage. Briefly, the separa-

tion system consists of a cassette comprising two

chambers. Semen is introduced into one chamber

and a current applied that within seconds leads to a

purified suspension of spermatozoa collecting on

the other side of the chamber. Suspensions generated

by the electrophoretic separation technique contain

motile, viable, morphologically normal spermatozoa

that exhibit lower levels of DNA damage. Another

recently reported technique is one which sorts sper-

matozoa that possess the apoptotic marker protein

annexin V. Said et al

used a magnetic cell sorting

technique to sort spermatozoa on the basis of

annexin V labeling, producing two sperm fractions:

an annexin V-negative (non-apoptotic) and annexin

V-positive (apoptotic).

They postulate that this

technique may benefit the success of assisted repro-

duction techniques. A final selection technique has

recently been reported by Huszar et al. They had

previously reported that sperm that are able to bind

to hyaluronic acid (HA) are mature and have com-

pleted the spermiogenetic process of sperm plasma

membrane remodeling, cytoplasmic extrusion, and

nuclear histone-protamine replacement.

Testing

this technique has shown that the HA bound sperm

have lower rates of cytoplasmic, nuclear, and chro-

mosomal abnormalities.

98,99

They concluded that

using this method to select sperm for ICSI might

reduce the potential genetic complications and

adverse public health effects of ICSI.

CONCLUSION

There is accumulating evidence linking sperm

nuclear DNA anomalies to poor reproductive out-

come in relation to ART (Table 25.1). The concern

as to the significance of this relationship is definitely

greater in relation to ICSI. The tests currently

available only provide an inkling of the impact of

sperm nuclear DNA abnormalities on outcomes.

More research is needed to improve our current

knowledge in relation to the DNA anomalies in



Table 25.1 Selected studies indicating whether

abnormalities in sperm DNA impact on reproductive

outcome in animals and human

Animal Human

Ye s N o Yes N o

Fertilization — 100–102 73 74

Embryo 103–105 — 81,82 —

development

Miscarriages 102,106 — 83,84,107 —

and pregnancy

Future 7,106 — ? —

generations

HPE_Chapter25.qxp 7/14/2007 5:52 PM Page 332

SPERM DNA AND EMBRYO DEVELOPMENT

spermatozoa, how to detect them more accurately

and how they may relate to failed reproductive out-

comes. Additionally, more standardized, large-scale

trials are needed to assess the predictive value of

sperm DNA fragmentation techniques as useful

pregnancy predictors in ART.

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