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Hum. Reprod. Advance Access published May 24, 2013

Human Reproduction, Vol.00, No.0 pp. 1–9, 2013

doi:10.1093/humrep/deq098

Hum. Reprod. Advance Access published May 24, 2013 Human Reproduction, Vol.00, No.0 pp. 1–9, 2013 doi:10.1093/humrep/deq098

ORIGINAL ARTICLE Infertility

Hum. Reprod. Advance Access published May 24, 2013 Human Reproduction, Vol.00, No.0 pp. 1–9, 2013 doi:10.1093/humrep/deq098

Y chromosome microdeletions, spe

rm

 

DNA fragmentation and sperm oxidative stress as causes of recurr

ent

spontaneous abortion of unknown etiology

ı

ı

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J. Bellver 1, * , M. Meseguer 1 , L. Muriel 2 , S. Garc´a-Herrero 1 , M.A.M. Barreto 1 , A.L. Garda 3 , J. Remoh´ 1 , A. Pellicer 1 ,

ı

ı

1

Correspondence address. Tel: +34-963050900; Fax: +34-963050999; E-mail:

and N. Garrido

jbellver@ivi es

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1 Instituto Valenciano de Infertilidad (IVI), University of Valencia, Plaza de la Polic´a Local, 3, 46015 Valencia, Spain 2 IVI Vigo, Plaza Francisco Ferna´ndez del Riego, 7, 36203 Vigo, Spain 3 IVI Murcia, Navegante Mac´as del Poyo, 5, 30007 Murcia, Spain

from

* Submitted on December 4, 2012; resubmitted on February 24, 2013; accepted on March 23, 2013
*
Submitted on December 4, 2012; resubmitted on February 24, 2013; accepted on March 23, 2013
A oxidation and
methods: A prospective cohort study was carried out in a private university-affiliated setting. Three groups, each
comprised of 30 males,

background: The aim of the present study was to evaluate the implication of male factor, in terms of sperm DN

fragmentation, and Y chromosome microdeletions in recurrent spontaneous abortion (RSA) of unknown origin in a strictly sel ected cohort.

were compared. The first was formed by healthy and fertile sperm donors (SD) with normal sperm parameters (control gro up), the second by men presenting severe oligozoospermia (SO) without RSA history, and the third by men from couples who had experien ced idiopathic RSA. Frequency of Y chromosome microdeletions and mean sperm DNA fragmentation and oxidation were determined.

results: Y chromosome microdeletions were not detected in any of the males enrolled in the study. Moreover, sperm

DNA oxidation measurements were not demonstrated to be relevant to RSA. Interestingly, sperm DNA fragmentation was higher in the SO group than in the RSA and the SD groups, and also higher in the RSA group compared with the SD group, but lacked an adequate predicti ve power to be employed as a discriminative test of RSA condition. conclusions: Sperm DNA features and Y chromosome microdeletions do not seem to be related to RSA of unknown

Hum. Reprod. Advance Access published May 24, 2013 Human Reproduction, Vol.00, No.0 pp. 1–9, 2013 doi:10.1093/humrep/deq098
Hum. Reprod. Advance Access published May 24, 2013 Human Reproduction, Vol.00, No.0 pp. 1–9, 2013 doi:10.1093/humrep/deq098

origin. Other molecular features of sperm should be studied to determine their possible influence on RSA. Clinicaltrials.gov reference: nCT00447395. Key words: recurrent spontaneous abortion / semen / Y chromosome microdeletions / sperm DNA fragmentation / sperm o xidative stress

Introduction

Recurrent spontaneous abortion (RSA), defined as 3 clinic

2005), some inherited thrombophilias, such as Factor V Leiden and pro- thrombin G20210A gene mutation (Rey et al., 2003; Kovalevsk y et al.,

2004; Krabbendam et al., 2005), congenital or acquired

uterine

anomalies (Devi Wold et al., 2006), endocrine, autoimmune or

alloim-

mune disturbances (Arredondo and Noble, 2006; Christiansen

et al.,

2006) and perhaps unhealthy lifestyle habits (smoking, obesity,

psycho-

logical stress; Lashen et al., 2004; Pandey et al., 2005). Howev

er, most of

these conditions are related to the woman, with the male’s cont

ribution

remaining relatively underexplored.

The alteration of sperm parameters evaluated by classic

criteria

(concentration, motility, morphology; World Health Organi

al preg-

 

nancy losses before the fetus

has reached viability

(Rai an

 

d Regan, 2006), is a frustrating condition that affects 1% of couples of ch

 

ildbear-

ing age (Porter and Scott, 2005). Although some causes h

ave been identified, others remain mere speculation. Thus, 50% of

couples are still classified as having unexplained RSA since the u nderlying mechanism(s) is never found (Porter and Scott, 2005). Known causes include paternal or ‘de novo’ chromosomal aber

couples are still classified as having unexplained RSA since the u nderlying mechanism(s) is never found
couples are still classified as having unexplained RSA since the u nderlying mechanism(s) is never found
couples are still classified as having unexplained RSA since the u nderlying mechanism(s) is never found

rations

(Rai and Regan, 2006), antiphospholipid syndrome (Empson

et

al.,

zation,

& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Introduction Recurrent spontaneous abortion (RSA), defined as 3 clinic 2005 ), some inherited thrombophilias, such as

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1999 ) is not clearly associated with the risk of either sp xfordjo Classic parameters of

1999)

is not clearly associated

with

the

risk

of

either sp

of either sp xfordjo Classic parameters of sperm evaluation cannot be emplo urnals.o rg/ Dow

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Classic parameters of sperm evaluation cannot be emplo

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oradic or recurrent abortion

(Carrell

et

al.,

2003b;

Bhattacharya

yed

to

identify

structural

enpreiss

alterations

in

the

sperm

chromatin

(Er

nloaded

from

 

, 2008), although some of these sperm deficiencies could represent the

mani-

  • al.,

et

2006;

  • 9; Cohen-

Erenpreiss et al., 2008;

Avendano et al.,

200

festation of an underlying related cause. This is the case for e

mbryos

festation of an underlying related cause. This is the case for e mbryos Bacrie et al.,
festation of an underlying related cause. This is the case for e mbryos Bacrie et al.,

Bacrie

et al.,

lated to

2009).

Sperm DNA

festation of an underlying related cause. This is the case for e mbryos Bacrie et al.,
festation of an underlying related cause. This is the case for e mbryos Bacrie et al.,

male

subfertility,

oductive

sporadic

 

fragmentation has been re

with chromosomally abnormalities originating in meiotic segr

abortion

and

 

poorer

repr

egation errors in the spermatozoa of men with severe oligoasthen

egation errors in the spermatozoa of men with severe oligoasthen

oterato-

outcome, especially after assisted conception technologies (

Larson-

Larson-

zoospermia (Bernardini et al., 2005; Pang et al., 2005). In fa

zoospermia ( Bernardini et al., 2005 ; Pang et al., 2005 ). In fa Cook et
zoospermia ( Bernardini et al., 2005 ; Pang et al., 2005 ). In fa Cook et

Cook et

al., 2003;

Lin

et

al.,

Borini et

al.,

2006;

Lewis et al., 2008;

ct, some authors have proposed performing fluorescent in situ hybri

ct, some authors have proposed performing fluorescent in situ hybri 2008 ; Zini et al., 2008
ct, some authors have proposed performing fluorescent in situ hybri 2008 ; Zini et al., 2008

2008; Zini et al., 2008), but with contradictory results (Collin

s et al.,

dization analysis in severely altered sperm samples to detect cases

dization analysis in severely altered sperm samples to detect cases 2008 ; Nicopoullos et al., 2008
dization analysis in severely altered sperm samples to detect cases 2008 ; Nicopoullos et al., 2008

2008;

Nicopoullos et al., 2008;

addition,

Tavalaee

et

al.,

2009).

In

that may benefit from preimplantation genetic screening, thus reduc

that may benefit from preimplantation genetic screening, thus reduc different methods her data of analysis have
that may benefit from preimplantation genetic screening, thus reduc different methods her data of analysis have

different methods

her data

of analysis have been employed to gat

ing the abortion rate (Rubio et al., 2001). Nevertheless, there is no co

[Comet Assay, Sperm Chromatin Structure Assay (SCSA),

TUNEL

 

nsensus in the current medical literature about this topic (Harper et al.,

2008).

Assay; Practice Committee of American Society for Repro

ductive

Medicine, 2008; Zini et al., 2008], and scarce information is c

In addition, many reported cases of male partners of RSA subjects show normal parameters in their ejaculates (Al-Hassan et al.,

2005).

urrently

implication

  • about

available

their

2003a).

in

RSA

(Carrell

et

al.,

Moreover, as sperm physiology is ascertained, an increasing n umber

Similarly, some authors have reported increased DNA

da

of molecular factors

are implicated in male fertility (Garrid

mage

in

 
sperm caused by oxidative stress in infertile men ( Mesegu

sperm

caused by oxidative stress in infertile men (Mesegu

er et al.,

er

et

al.,

 
 

o et al.,

o et al., 2008 ; Aguilar et al., 2009 ), but nothing is known about this

2008; Aguilar et al., 2009), but nothing is known about this in

2008a, b), such as oxidative stress-related molecules

relation

 

(Meseguer et al., 2004), DNA oxidation and fragmentation (Aguilar et al.,

( Meseguer et al., 2004 ), DNA oxidation and fragmentation ( Aguilar et al., 2009 )

2009) and mRNA expression profiles (Garrido et al., 2008a, b), but their implication in RSA has received little or no attention.

to RSA. Two reports have also described a notably higher prevalenc e of Y chromosome microdeletions in the male partners of coupl

 

es

with

RSA (Dewan et al., 2006; Karaer et al., 2008), but this has not been confirmed (Kaare et al., 2008; Lu et al., 2008). The present study was designed to prospectively evalu ate the relationship of Y chromosome microdeletions, sperm DNA frag

The control group included 30 healthy

SD of

18–35 years of

age,

all

Caucasian, with normal karyotype, no family history of diseases,

normal

sperm analysis result (more than 80 million total motile sper matozoa per ejaculate) and previous term pregnancies without complications

men-

con-

tation and sperm DNA oxidation caused by oxidative stress wit h RSA

and to compare their values among healthy and fertile sperm donors (SD) with normal sperm parameters, men with severe oligozo

osper-

mia (SO) and men from couples with RSA.

ceived with their sperm samples. The oligozoospermic group (SO) consisted of 30 Caucasian men with . 1 year infertility, , 45 years of age, absence of autoimmune or endo- crine disorders, normal karyotype, no history of RSA, normal genital examination, normal FSH, LH, testosterone and prolactin values and at least two sperm analysis results showing SO ( 5 million sperm

atozoa/

Materials and Methods

Study design

This is a prospective case–control study,

in which

men

were

enrolled between 1 January 2010 and 1 February 2012. Subjects were assigned to one of the three groups according to the following inclusion crit eria.

ml, but . 1 million of total sperm per ejaculate in order to make f easible the sperm tests). These men were considered to represent cases of severe idiopathic oligozoospermia. No assisted reproduction treat ment had been performed for these males at the time they entered in th e study.

The RSA group was composed

ucasian

of the male partners of 30 Ca

couples

who

rimester

had

previously

experienced

3

clinical

first

t

(5–14 weeks) spontaneous abortions, with normal karyotypes o

f

both

male and female and no autoimmune or endocrine disorders.

These couples had not attempted assisted reproduction treatments at the time

they were accepted for the study. The female partners of these s ubjects were , 38 years old, presented normal ovarian function, a normal uterus confirmed by vaginal ultrasound and/or hysterosalpingog raphy/ hysteroscopy and absence of acquired or inherited thrombophilia

(anti-

phospholipid syndrome, activated protein C fasting

resistance, serum

homocystine,

protein

C,

protein

S,

antithrombin

III,

proth

rombin G20210A gene mutation, factor V Leiden). All nder 45

the men

were

u

years old and previous sperm r only a

analysis had given

a

normal

o

mildly altered result (.

10 million/ml, . 35% A + B motile sperm

atozoa, . 10% of normal spermatozoa following Kruger’s strict criteria). To ensure the reliability of the results obtained, subjects compl eted a clinical questionnaire about previous consumption of alcohol and/or drugs, recent episodes of fever, exposure to gonadotoxins (e.g. during chemotherapy or radiotherapy), pesticide or heavy metals (professi onally) and vitamin intake in the 3 months prior to the enrolment in th e study. Andrological examinations were performed to rule out the prese nce of varicoceles, testicular torsion, traumatisms and other alterations in the

genital tract, such as genital tract inflammation nfections

or recurrent i Applied cturer’s

Science,

Indianapolis,

Ind.)

according

to

the

manufa

(Viloria et al., 2009). None of the men presented any of these con instructions. Screening for Yq microdeletions was carried out in co

ditions. The study was approved by the Institutional Research Board and Ethics

ntrols

and patients using polymerase chain reaction (PCR) techniques by a

mplify-

Committee. The study purposes and procedures were carefully expl ing 20 different sequence-tagged sites (STSs) on the long arm o

ained,

f

the

Y

and the informed consent was obtained from those willing to partic chromosome, corresponding to four AZF loci spread over inter

ipate.

The study

eference

was also

NCT00447395.

registered in

Clinicaltrials.gov, with

the

Y chromosome microdeletions: isolation of DNA and sequence-tagged site analysis

vals 5

r and

6. These included

SY14 from

SRY, SY81, SY84s

and

SY

86 from AZFa; SY182 from KALY, SY121, SYPR3, SY124, SY127, SY128,

SY130,

SY133 and SY134 from AZFb; SY145 and SY152 from proximal AZFc/

d locus; SY242, SY208, SY254 and SY255 from AZFc; and SY157 (

genital tract, such as genital tract inflammation nfections or recurrent i Applied cturer’s Science, Indianapolis, Ind.)

Genomic DNA was extracted from the peripheral blood leukocytes

hetero-

using the MagNA Pure Compact instrument (Roche Diagnostics Corpor ation) and the MagNA Pure Compact Nucleic Acid Isolation I method Roche Male factor and idiopathic recurrent miscarriage

(

chromatic distal Yq region).

Multiplex PCR was performed using the Y chromosome AZF A

nalysis System (Promega, USA) under the PCR conditions recommended by the

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manufacturer. For analysis, the microdeletions PCR products were run by electrophoresis on a 2% agarose gel impregnated with ethidium bro mide for visualization under UV light. Failure of amplification for a given STS was con- firmed twice. DNA from a fertile male and water served as negative Y chromo- some microdeletion controls, whereas DNA from man with a Y chro mosome microdeletion served as a positive control for multiplex reactions.

Sperm analysis

Semen parameters of every ejaculate were evaluated by two inde pendent observers. After 10 min liquefaction of the semen at 378C and 5 % CO2, the samples were examined for concentration and motility in a Mackler chamber (Sefi Laboratories, Tel Aviv, Israel), according to th e WHO guidelines (12). Results were only accepted when the d ifferences between two observers were less than 5%. Two semen aliquots were taken from each semen sample to be pro- cessed for flow cytometry and sperm chromatin dispersion (SCD ) tests. The samples were directly frozen by immersion in liquid nitrogen (SCD) or in cold ethanol 70% (for flow cytometry experiments). These pr otocols have been validated previously and have been demonstrated to be safe in regard to the maintenance of sperm characteristics, allowing the an alysis of all the samples in the same experiment. The total number of spermatozoa collected for the experiments r anged between 1 and 10 million/ml in order to provide a sufficient quan tity for the analysis after centrifugation for 10 min at 400g and eliminatio n of the

supernatant.

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No ejaculate displayed significant (. 1 million/ml) levels of le

ukocytes

(a known and relevant source of ROS), as demonstrated by Quic

k Panop-

from

tic (QCA, Barcelona, Spain) staining of semen smears. The se

nsitivity of

the test is 10 000 leukocytes/ml.

DNA fragmentation (SCD)

Eppendorf tubes with gelled aliquots of low-melting point agar

ose were

placed for 5 min in a water bath at 90–1008C to fuse the

agarose, and sub-

sequently in another water bath at 378C. After 5 min incubation at

378C for

temperature equilibration, 60 ml of the diluted semen sample were

added to

the Eppendorf tube and mixed with the fused agarose. 20

ml of the

semen-agarose mix were pipetted onto slides precoated with

agarose and

covered with a 22 × 22 mm coverslip. The slides were placed

on a cold

plate in the refrigerator (48C) for 5 min to allow the agarose to p

roduce a

microgel with the sperm cells embedded within. The coverslips we

re gently

removed and the slides immediately immersed horizontally in an a

cid solution,

previously prepared by mixing 80 ml of HCl from an Eppendorf t

ube with

10 ml of distilled water and incubated for 7 min. The slides were h

orizontally

immersed in 10 ml of the lysing solution for 25 min. After 5 min w

ashing in a

tray with abundant distilled water, the slides were dehydrated in i

ncreasing

concentrations of ethanol (70–90–100%) for 2 min each and then air-dried. For bright-field microscopy in the improved SCD test (Halo spermw

kit), slides were horizontally covered with a mix of Wright’s staini ng sol- ution (Merck, Germany) and phosphate buffer solution (Merck,

Germany)

(1:1)

for

5–10 min

with

continuous

airflow.

Slid

es were briefly washed in tap water and allowed to dry. Strong staining is p referred to easily visualize the periphery of the dispersed DNA loop halos. For this study, a minimum of 500 spermatozoa per sample were scored under the × 100 objective of the microscope.

DNA oxidation (8-oxoguanine DNA sperm measurement by flow cytometry)

The OxyDNA Assay (OxyDNA assay kit, Calbiochem, Barcelona, S

pain) is based on the direct binding of a fluorescent probe to 8-oxoguanine moieties in the DNA of fixed cells (Aguilar et al., 2009). Fluorescence can then be

quantified using flow cytometry (the OxyDNA Assay is perform

ed

and

observed directly in the cells). Sperm cells were washed twice in PBS and fixed-permeabilized with ice-cold ethanol 70% at 2 208C. Ethanol- treated cells were pelleted at 230 × g for 5 min, and the supernata

nts were

removed and pellets were washed twice with PBS. Sperm cells wer e resus- pended in 3 ml of Wash Solution [Tris-buffered Saline/TWEE Nw 20 Detergent (TBST), containing Thimerosal], and centrifuged at 2 30g for 5 min, after which the supernatant was removed and non-specific binding sites were blocked with 50 ml freshly prepared Blocking Solution for 1 h at 378C. Then 3 ml of Wash Solution was added and washes were repeated twice, and then incubated overnight with 100 ml FITC-Conjugate at 48C. Samples were washed twice (Wash Solution) and resuspended in 2 ml PBS. Cells were maintained in the dark on ice until their resuspe nsion in FACS fluid and were read in a flow cytometer with FITC filters. Flow cytometry analysis was performed on an Epics Elite Fl ow Cyt- ometer (Coulter Cytometry, Hialeah, FL, USA) using an argon- ion laser

tuned

at

488 nm

and

15 mW.

Debris

was excluded by the a

nalysis of

 

the

scatter

properties. At

least 10 000

events per

sample wer

e stored in list-mode files.

Data were expressed as percentage

of

stai

ned cells (compared with negative controls) and fluorescence intensity m easured as fluorescence arbitrary units.

Statistical analysis

Statistical analysis was performed using the Statistical Package f

or Social Science version 17.0 (SPSS Inc., Chicago, IL, USA). Categor ical data were expressed as number and percentage, and numerical data as means with 95% confidence intervals (95% CI). When a quantitativ e analy- sis of the data was performed, groups were compared by analysis of var- iance, with Bonferroni and Scheffe’s post hoc analysis if more than two groups were compared and a Student’s t-test when only two groups were compared. Crosstabs and chi-square tests were applied for the quali- tative analysis of the data. The predictive power of sperm DNA ox idation and fragmentation to forecast miscarriage were determined by receiver operator curve (ROC) analysis. Significance was defined as P , 0

.05.

Results

Demographics

The baseline comparisons of the three groups are presented in T able I. In brief, age was significantly lower among SD males (ranging from

million/ml in sperm concentration, from 0.09 to 7.42 million in total motile sperm cells and from 3 to 53% in A + B forms), as ex pected considering the inclusion criteria. When sperm quality of the SD males was compared with that of the RSA group (concentration ranging from 11 to 110 million/ml), a sig- nificant difference was observed in sperm concentration, but not in other parameters. However, in both SD and RSA, sperm

20 to 34 years) than among men in the SO (range 26–44 years) and RSA (range
20
to
34 years)
than
among men
in
the
SO (range 26–44
years)
and RSA (range 30–42 years) groups. Sperm quality was also
signifi-
cantly higher in the first group, in which the values of sperm c
oncen-
tration (ranging from
29
to
250 million/ml), sperm motility (r
anging
from 29% to 67% of A + B forms) and the total number of
concen-
motile
sperm cells (ranging from 80 to 278 million) were superior, partic
ularly
in comparison with the selected group of SO (ranging from 0.3
to 5
4
Table I Demographics of the study population.
Control donors
Severe oligospermia
Recurrent miscarriage
P-value
.....................................

tration was still normal (. 20 million/ml). The percentages of smokers were statistically comparable in the three groups, and in both males and females, thus ruling out any confounding influence of this parameter on the results. Nevert heless,

Bellver et al.

........................................................................................................................................................

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Age (years) 26.50 (24.58–28.52) a,b Presence of Y chromosome microdeletions 34.00 (32.38–35.32) a 0/30 (0%) 35.24
Age (years)
26.50 (24.58–28.52) a,b
Presence of Y chromosome microdeletions
34.00 (32.38–35.32) a
0/30 (0%)
35.24 (33.84–36.64) b
0/30 (0%)
Smoker (yes/total and %)
3/30 [10% (0–20.74)]
ns
0.15 (0–0.4) a,b
4.02 (3.22–4.83)
10/30 [33.33% (16.56–50.20)]
, 0.001
0/30 (0%) ns
7/30 [23.33%
(8.28–38.46)]
Mean number of cigarettes per day
Ejaculate volume (ml)
Sperm concentration (million/ml)
5.21 (2.06–8.35) a
4.38 (3.56–5.19)
74.70 (55.13–94.28) a,b 3.15 (2.04–4.25) a,c
% of
A + B sperm
Total motile sperm (million)
112.79) b
46.62 (42.86–50.38) a
119.49 (92.76–146.218) a
, 0.001
26.38 (20.66–32.10) a,b
83.91 (55.04–
Female
Age (years)
Smoker (Yes/Total and %)
Mean number of cigarettes per day
Mean number of miscarriages per patient
34.67 (33.61–35.72)
13/30 (43.33 (25.60–61.06))8/30 (26.67 (10.84–42.50))
3.60 (1.50–5.70)
3.76 (3.39–
4.14)
Mean week at pregnancy termination
7.42 (6.93–7.41)
Data are expressed as means or proportions with 95% confidence intervals. Superscript letters (a, b, and c) denote statistical significance between groups within th
e same row sharing the
superindex, and ns denotes non-significant differences. P-values of , 0.05 were considered statistically significant. A + B sperm are defined as sperm cells with
progressive motility either
rapid and straight (A) or less rapid or presenting changing direction (B). Normal ranges are considered above 50% of A + B forms.

from

a trend toward a lower mean number of cigarettes smoked per day

The baseline comparisons of the three groups are presented in T able I . In brief,

was observed

in

the SD

group

(ranging

from

0

to

3 cigarettes/day)

in comparison with

the

SO

(ranging

from

0

to 20 cigarettes/day)

and RSA (ranging from 0 to 30 cigarettes/day) groups.

The baseline comparisons of the three groups are presented in T able I . In brief,

The mean number and mean gestational age of miscarriages are also

presented

in

Table

I.

The number of pregnancy

losses among

The baseline comparisons of the three groups are presented in T able I . In brief,

the

couples of the RSA group ranged from 3 to 7, whereas the mean gestational week in which pregnancy was lost ranged from 5 to 14.

Male’s genetic contribution to RSA

We were unable to find any Y chromosome microdeletions in any of the males included in the SD, SO or RSA groups. Regarding sperm DNA quality, two parameters were assessed:

sperm DNA fragmentation and sperm DNA oxidation. The results can be found in Figs 1 and
sperm DNA fragmentation and sperm DNA oxidation. The results
can be found in Figs 1 and 2.
In short, the mean number of sperm cells with fragmented DNA was
24.06% (95% CI 20.87–27.23) in the SD group, in comparison with
46.01% (95% CI 40.40–51.62) in the SO group and 33.48%
(95% CI
28.24–38.72) in the RSA group, with statistically significant
differences
between the SD and RSA and between the SD and SO groups.
Figure 1 Sperm DNA fragmentation analysis results depending
on
the origin of the sperm sample. The horizontal lines represent
the
medians, the box width the interquartile range, the lower li
The results of
e three
oxidation in th
ne
of
the box the first quartile, the upper line of the box the third qua
groups were comparable in terms of percentage of oxidize
rtile,
and the whiskers are maximum and minimum.
d
with values of 32.14% (95% CI 24.62–39.65) in the S
D
group,
The predictive value of sperm DNA fragmentation and DNA
24.01% (95% CI 16.85–31.17) in the SO group and 32.43
% (95%
CI 24.62–40.24) in the RSA group.
oxi-
dation for forecasting recurrent miscarriage are shown in Fig.
byguest
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oxidation staining intensity was compare 3. The
low values of the areas under the curve indicate the absence of
onMarc
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mean sperm cell DNA oxidation was 87.25 (95% CI 61.16–
a pre-
dictive power of sperm DNA fragmentation or oxidation anal
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relative fluorescence units (RFU) in the SD group,
58
87. ysis in
spermatozoa with respect to RSA.
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(95% CI 63.69–111.48) in the SO group and 68.04 RFU
(95% CI
If only males with non-severe sperm alterations (i.e. mem
bers of
nloaded
from
53.55–82.53)
een
groups were not significant.
betw the SD and RSA groups) are considered,
sperm
the capacity of the
Male factor and idiopathic recurrent miscarriage
5
affected by this condition is not advisable since their alterat
ions are
not currently demonstrated to present a causative role.
DNA oxidative damage is indicative of a number of conditions
invol-
ving the generation of oxygen-free radicals. This damage could
be the
primary source of DNA fragmentation (Aguilar et al., 2009). In
fact, a
defective antioxidant system activity could be related to an imp
airment
of chromatin packaging (Tarozzi et al., 2009). However, this is
not the
only the mechanism of DNA fragmentation (Erenpreiss et al.
,
2006;
Practice Committee of American Society for Reproductive Med
icine,
2008). This is why we considered DNA fragmentation and DN
A oxi-
dation independently because, despite the association tha
t
exists
between the two, they are not synonymous.
We analyzed DNA oxidation by ruling out all the external f
actors
that have been related to increased free radical damage
(see the

merican

‘Materials and Methods’ section; Practice Committee of A

Society for Reproductive Medicine, 2008; Viloria et al., 2009

). Simi- larly, a smoking habit, which is also associated with increas ed DNA damage in
).
Simi-
larly,
a smoking habit, which
is also associated with increas
ed DNA
damage in animal models and humans (Kunzle et al., 2003; Pas
qualotto
et al., 2008), was identified in a similar proportion in the thre
e study
groups. Despite the fact that almost 27% of women include
d
in the
RSA group were smokers (Table I),
the mean number of
ci
garettes
smoked per day was low (3.2). Still, the number of
cigarettes
smoked daily
in the RSA and the SO groups was significantl
y higher
than
in
the SD
group
and this may contribute to the higher
rate
of
DNA
fragmentation. However, a smoking has not been
clearly
related
to
unexplained
recurrent
miscarriage
in
recent
studies
(Zhang
et
al.,
2010). The correlation between male age an
d
sperm
DNA fragmentation is controversial, with some reports show
ing
an
increase in
sperm
DNA
damage with age (Plastira et a
l.,
2007;
Vagnini
et
al.,
2007)
and
others
finding
no
relationship
(Winkle
et
al., 2009),
even after
40 years.
In
our study,
we aimed
to avoid
this possible confounding factor by limiting the age of the study
popu-
lation to 45 years old, although the SD group was significantly y
ounger

Figure 2 Sperm DNA oxidation analysis results depending on the origin of the sample. Results expressed as the percentage of oxi dized cells (positively stained, top) and mean staining levels (bottom). The horizontal lines represent the medians, the box width the interq

uar-

tile range, the lower line of the box the first quartile, the upper l

ine of

the box the third quartile, and the whiskers are maximu

  • m and minimum. The dots outside the box are abnormally high

values. Asterisks here denote extreme values, but not statistical differ ences among groups.

optimal area under the curve should be

. 0.8).

Discussion

In the present study, Y chromosome microdeletions and sper

m DNA fragmentation and oxidation RSA of

did

not

prove to

be related

to

unknown origin, at least in men with normal or mildly altere d sperm samples. Therefore, the assessment of these parameter

s

in

men

DNA

fragmentation and oxidation measurements to discri

minate

between males from couples with or without recurrent misc arriages

can be seen in Fig. 4. Only the percentage of oxidized cells pr esented a mild capacity to adequately predict RSA occurrence, though it was

not

sufficient

for

it

to

be

applied

as

a

diagnostic tool

(

where an

 

compared with the SD and the RSA groups. This also may hav e con- tributed to the higher DNA fragmentation rates in the latt er two groups.

Increased levels of impairment to DNA integrity in severe olig

osper-

mic males have been reported previously in the literature an

d could explain the poor results described in assisted reproduction treat ments by some authors (Meseguer et al., 2008). However, in a recent meta-analysis of 13 studies involving infertile couples, despite a small but statistically significant association between sperm DNA i

According to our results, sperm DNA

compared with the SD and the RSA groups. This also may hav e con- tributed to

DNA fragmentation and oxidation, two of the main symptoms of r eactive

oxygen species attack (Garrido et al., 2004; Aguilar et al., 2009), is

not related to recurrent idiopathic spontaneous abortions. Alth ntegrity

ough statistical differences were confirmed among the groups, th ese do not point to an increase in sperm DNA fragmentation or oxi dation

test results and pregnancy outcome in IVF and ICSI cycles , sperm DNA integrity did not prove to be predictive of pregnancy out

practice

usefulness in the routine

in men from RSA couples with respect to the other groups of pat (Collins

ients. Interestingly, the group with the highest sperm DNA fragmen tation

et al., 2008). The influence of sperm DNA damage on sporadic pregnanc y loss

was that formed by males yielding severely oligospermic s has been mainly evaluated after assisted reproductive tec

amples.

6

Bellver et al.

byguest

onMarc

h17,201

6http://h

umrep.o

xfordjo

urnals.o

rg/Dow

nloaded

from

Figure 3 ROC curve analysis of the predictive value of sperm DNA fragmentation and oxidation to forecast RSA.

Though controversial, a recent systematic review and meta- analysis of 11 studies concluded that the risk of pregnancy loss after IVF a nd ICSI was 2.5 higher in men with DNA damage, but differed depend ing on

the assay (SCSA or TUNEL) employed (Zini et al., 2008). Ho

wever, only two previous reports have considered couples with unexp lained

RSA. In both studies (Carrell et al., 2003a; Qiu et al., 2008), D NA frag-

Similar levels of DNA age of

oxidation, in terms of both the percent

cells

with

oxidized

DNA

and

mean

intensity

of

oxidation

mentation

was significantly higher

in

the

RSA group

tha

     

,

were

 

n

among

 

found

in

all

the three groups

 

assessed

(SD,

SO

and

mal

donors of

known fertility and

men

from

the

general po

es from

 
 

pulation. Classic parameters of sperm quality correlated with sperm DN

 

RSA). Only the percentage of oxidized

cells ex

A frag-

   

predict

RSA

occurrence

(

 

mentation in one of the studies (Qiu et al., 2008), but not in t

     

he other

   

power does

 

not appear to

be suffici

gmen-

(Carrell et al., 2003b). Our results are similar, since sperm sa

mples of

   
 

diagnostic

 

tool

(where

an

optim

the RSA group were normal or only mildly altered, and DNA fra

al area under the curve should be . 0.8).

One of the most significant findings of this work is that no

 

tation

in

the

same group

ng fertile

donor controls. However, when we assessed the clinical pr edictive

 

chromosome microdeletions

value of this finding by nt due to

found between the groups. In fact, no presence of Y letions

 

microde

the overlap

of

values

 

the 90 subjects included.

These res

groups

   

ults are

byguest

onMarc

h17,201

6http://h

(Fig.

4).

The

findings were the

   

mic

men

in complete contrast to what was initially published by Dewa n et al.

were taken into account in the ROC curves (Fig. 3). T

despite the

fact that DNA

 

17 males (82%) from RSA

umrep.o

herefore,

xfordjo

urnals.o

 

or

more of

the

four segm

rg/Dow

nloaded

ased

in

 

the sperm of men with unexplained RSA, the clinical value o

ents of

from

with severely impaired spermat

f sperm

DNA integrity analysis as a predictive tool is negligible. Simil

arly, the causative role of DNA fragmentation in RSA cannot be demons

the proximal AZFc region studied, whereas this percentage dro pped

men

with

proven

fertility.

R

trated.

To the best of our knowledge, this is the first study in which sperm

 

ecently, Karaer et al. (2008) also detected Y chromosome microdeleti ons in

 

oxidative stress

has been

 

assessed in the AZFb an

 

one

or

more

of

the four

STSs

hic

RSA.

d AZFd

 

Male factor and idiopathic recurrent miscarriage

regions

in 16% of men from couples with RSA and repor

ted no case among fertile men. However, the percentage of microde

letions

detected differed hugely between these two studies (82 versus

16%)

and the regions affected were mostly different.

7

Figure 4 ROC curve analysis of the predictive value of sperm DNA fragmentation and oxidation to forecast RSA considering t hose males with

normal sperm parameters.

 
 

byguest

onMarc

In our work, the increased sample size should have allowed chromosome microdeletions in our SO group, since these alter

h17,201

clarifi-

clarifi-

ations

 

6http://h

cation of the considerable differences reported previously by

have been

described in higher proportions

in

men

with

v

umrep.o

Dewan

ery low

xfordjo

et al. (2006). At least, some Y chromosome microdeletions sperm counts

addition,

(, 1 million/ml;

Martinez

et

al.,

2000).

In

urnals.o

rg/Dow

nloaded

should have been observed given

that

we have

considered simil

despite the strict inclusion criteria employed, the sample si

from

ar popu-

 

ze con-

lations. In addition, we assessed 20 Y-linked STSs belonging

sidered in the study (90 men) may also represent a limit

to four

ation for

AZF

loci regions (AZFa,

AZFb, AZFc and

AZFd)

rather

th the results obtained.

 

an only

Similarly to our study, other recent reports have not iden

four STSs

in

one

or

two AZF

regions (Dewan et al.,

2006 tified Y

;

Karaer

 

chromosome microdeletions in male partners of RSA couples.

et al., 2008).

Kaare

Furthermore, Dewan et al. (2006) argue that their patien et al. (2008) evaluated 40 men from couples with at least thr

ts were

ee mis-

selected from a tertiary referral center that receives a dispro

carriages and analyzed 33 STS loci spanning the whole Y chrom

portion-

 

osome

ate number of RSA patients and that these patients were likely plus the 4 STS loci used by Dewan et al. (2006). No microdelet

to have

ion was

failed evaluations and treatments prior to referral, thus ske

found in any case. A study of a Chinese population by Lu et al.

wing the

(2008)

results. In our study, patients were selected following strict i also failed to detect any case of Y chromosome microdeletions

nclusion

 

in 26

criteria.

The

couples

described

as

infertile

in

the Dew

chorionic

villous samples of

abortive male embryos or in

5

an

et

al.

1 blood

 

report exhibited similar conditions of sperm quality as those samples of men whose wives had experienced RSA. They

in our

 

studied

 

study, with

the exception of

those males presenting

azoo 12 STSs in the AZFa, AZFb and AZFc regions of Yq11.2.

spermia,

 

Differences in the presence of Y chromosome microdeletions

who were not included in our population due to the impossibi could

lity of performing the described DNA fragmentation and oxidatio n tests

with

their

samples.

This

fact

may

partly

explain

the

ack of Y

8

aspects

currently

of

the

analysis.

Nevertheless, no clear evidence

be due

to variations

in the study populations (race and e

thnicity),

 

subject selection criteria, the STSs considered and method

l ological

 
 

Bellver et al.

exists

regarding

the

relationship

between

idiopathic

RSA

and

Y

chromosome microdeletions.

To conclude, there seems to be no clinical use for the asse

ssment

of Y chromosome microdeletions and DNA fragmentation an

d oxi-

dation when dealing with couples with idiopathic RSA, at least

when

the sperm samples of the male partner are not severely i

mpaired.

Until such a clinical use is demonstrated, this assessment

can only

serve research purposes. Given the continuous description of

sperm

molecular factors related to male infertility, further resea

rch into

RSA using the newly discovered molecules as markers should

be con-

ducted. Recent advances in microarray technology applied to

sperm

will undoubtedly constitute powerful tools in the characteriza

tion of

these factors.

Authors’ roles

J.B. designed the study, selected

the

patients and wrote th

e paper;

 

M.M. helped in the design of the study and performed the DN

A oxi-

 

dation

studies;

L.M.

performed

the

DNA

fragmentation

studies;

 

S.G.-H.

collaborated

in

the

DNA

oxidation

studies

and

in

the

writing of the paper; M.A.M.B. included many of the patients a

nalyzed

 

and helped

in

the design

of

the

paper; A.L.G.

performe

d

the

Y

outcome of ovum donation cycles. Fertil Steril 2009; June 1 9 [Epub ahead of print].

Al-Hassan S, Hellani A, Al-Shahrani A, Al-Deery M, Jaroudi K, Coskun S. Sperm chromosomal abnormalities in patients with une xplained recurrent abortions. Arch Androl 2005;51:69–76. Arredondo F, Noble LS. Endocrinology of recurrent pregnancy loss. Semin Reprod Med 2006;24:33–39. Avendano C, Franchi A, Duran H, Oehninger S. DNA fragmenta tion of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome. Fertil Steril 2009; M arch 30 [Epub ahead of print].

Bernardini

urini

PL,

LM,

Calogero

AE,

Bottazzi

C,

Lanteri

S,

Vent

Burrello N, De Palma A, Conte N, Ragni N. Low total normal motile count values are associated with increased sperm disomy and d iploidy rates in infertile patients. Int J Androl 2005;28:328–336. Bhattacharya SM. Association of various sperm paramet

ers

with

unexplained repeated early pregnancy loss—which is most imp ortant? Int Urol Nephrol 2008;40:391–395.

chromosome microdeletion analysis; J.R. ı helped in the ı desi

gn and

monitored the study; A.P. helped in the statistical approach and

mon-

itored the study and the paper; N.G. designed the study, perf

ormed

the statistical analysis, coordinate the sperm and blood anal

ysis and

helped in the writing of the paper.

Acknowledgements

The authors would like to acknowledge the Andrology lab tech

nicians

Yolanda Marquez, Carolina Rico, Paloma Rodr´guez, Alicia M

enc´as,

Alicia Zurilla, y Vicente Centelles for their help in the samples

manage-

ment and storage.

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