Advanced Paternal Age and Sperm DNA Fragmentation: A ...

INTRODUCTION

The average age at which couples first reproduce has increased significantly in recent decades, with the mean age now at around 30 years in many coun-tries [1-3]. Since the 1980s, United States birth rates have increased 40% for men 35 to 49 years old and have subsequently decreased 20% for men less than 30 years old [2]. Increased life expectancy, modern societal

expectations pressures, and advanced age of marriage has resulted in the tendency for couples to delay par-enthood. The increased accessibility to assisted repro-ductive technology (ART) has increased the chance of older couples to conceive children, hence increasing the average paternal age at first childbirth. While increas-ing maternal age is well established as a risk factor for adverse reproductive outcome and offspring fitness, the influence of paternal age on sperm parameters and

Received: Dec 4, 2020 Revised: Feb 15, 2021 Accepted: Mar 4, 2021 Published online Apr 16, 2021Correspondence to: Daniel C. Gonzalez https://orcid.org/0000-0003-1387-7904Department of Urology, Miller School of Medicine, University of Miami, 1150 NW 14th St, #1551, Miami, FL 33136, USA.Tel: +1-305-243-7200, Fax: +1-305-243-6090, E-mail: [email protected]

Copyright © 2022 Korean Society for Sexual Medicine and Andrology

Advanced Paternal Age and Sperm DNA Fragmentation: A Systematic Review

Daniel C. Gonzalez , Jesse Ory , Ruben Blachman-Braun , Sirpi Nackeeran , Jordan C. Best , Ranjith RamasamyDepartment of Urology, Miller School of Medicine, University of Miami, Miami, FL, USA

Purpose:Purpose: Male ageing is often associated with defective sperm DNA remodeling mechanisms that result in poorly packaged chromatin and a decreased ability to repair DNA strand breaks. However, the impact of advanced paternal age on DNA frag-mentation remains inconclusive. The aim of the present systematic review was to investigate the impact of advancing pater-nal age (APA) on DNA fragmentation.Materials and Methods:Materials and Methods: We conducted a thorough search of listed publications in Scopus, PubMed, and EMBASE, in accor-dance with the PRISMA guidelines.Results:Results: We identified 3,120 articles, of which nineteen were selected for qualitative analysis, resulting in a sample of 40,668 men. Of the 19 articles evaluating the impact of APA on DFI% (DNA fragmentation Index) included, 4 were on Normozoo-spermic and subfertile men, 3 on normozoospermic, Oligoasthenoteratozoospermic and Teratozoospermic, 6 on fertile and infertile men, 4 on just infertile men, and 2 evaluated a general population. Seventeen of the ninrnteen studies demonstrated APA’s effect and impact on DFI%.Conclusions:Conclusions: Although there was no universal definition for APA, the present review suggests that older age is associated with increased DFI. In elderly men with normal semen parameters, further studies should be performed to assess the clinical im-plications of DFI, as a conventional semen analysis can often fail to detect an etiology for infertility.

Keywords:Keywords: Aging; DNA fragmentation; Paternal age; Sperm parameters

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Original Article

pISSN: 2287-4208 / eISSN: 2287-4690World J Mens Health 2022 Jan 40(1): 104-115https://doi.org/10.5534/wjmh.200195

Male reproductive health and infertility

mailto:[email protected]

https://orcid.org/0000-0002-7913-3513

https://orcid.org/0000-0002-9176-4334

https://orcid.org/0000-0001-9700-0805

https://orcid.org/0000-0002-6950-2645

https://orcid.org/0000-0002-2364-6627

https://orcid.org/0000-0003-1387-7904

Daniel C. Gonzalez, et al: APA and Sperm DFI: A Systematic Review

105www.wjmh.org

fecundity is unclear [4,5].There’s a preponderance of evidence reporting an

age-related decline in semen volume, motility, and proportion of morphologically normal sperm [3,6-13]. Additionally, compared with fertile men, infertile men exhibit poor sperm chromatin integrity and in vivo fertilizing capability. One study which examined 277 normozoospermic men identified a significantly higher DNA fragmentation Index (DFI) percentage in older (>40 years) men compared to that of younger men [14]. Conversely, Winkle et al [15], found no significant as-sociations with male age, DNA fragmentation, and semen parameters. The mechanisms responsible for age-dependent patterns of DNA fragmentation are not fully understood, but oxidative stress and ineffi-cient apoptosis are thought to be important contribu-tors [16,17].

One intrinsic difficulty with attempting to summa-rize data for advanced paternal age (APA) is that there is no clearly accepted universal definition of APA. Given that the current population mean for paternal age is 27, the most frequently utilized criterion for APA is more than 40 years of age [18]. While the gen-

eral consensus is that APA tends to be associated with a decline in semen quality, as well as an increase in DNA fragmentation [10,16], the suggested data appears to be mixed. Further complicating the study is the lack of a universally accepted definition for an abnormal DNA fragmentation threshold, and standard assays. Therefore, we carried out a systematic review to better elucidate the effect of APA on DNA fragmentation by evaluating both age and DNA fragmentation as con-tinuous variables, rather than using cut-offs. Utilizing data from 19 articles (40,668 subjects) we conducted a systematic review to summarize the impact of increas-ing paternal age on DNA fragmentation.

MATERIALS AND METHODS

1. MethodsA prospective systematic literature review, inclusion

and exclusion criteria, and outcome measurement were prepared a priori according to the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines (Supplement File) [19]. This systematic review was ac-cepted in the International Prospective Register of

Inclu

ded

Elig

ibili

tyS

cre

enin

gId

entification

Search results combined,duplicates removed

(n=2,061)

Records screened(n=2,061)

Full-text articles assessedfor eligibility

(n=126)

Studies included inquantitative synthesis

(n=19)

Records excluded(n=1,935)

Full-text articles excluded,with reasons

Review paper (n=26)Data not provided/

insufficient statisticaldetail (n=49)

Wrong patientpopulation (n=24)

(n=6)(n=105)

Non-English orSpanish language(Portuguese andRussian)

Literature searchDatabases

PumMed (n=146), Scopus (n=2,531),Embase (n=439)

(n=3,116)

Additional records identifiedthrough other sources

(n=4)

Fig. 1. Flow diagram of search and selec-tion strategy in a systematic review on the impact of advancing paternal age on sperm DNA fragmentation Index.

https://doi.org/10.5534/wjmh.200195

106 www.wjmh.org

Systematic Reviews (CRD42020191371) before the com-mencement of the study, which ensured the transpar-ency of the review process and originality of this study.

2. Data sources and search strategyA systematic search of Scopus, PubMed, and Embase

electronic databases was conducted to identify the relevant studies from inception up until March 2020. We conducted the search by using the search string in [All field] setting: “((((((age OR aging)) AND ((sperm OR semen))) AND ((male))) AND ((fertility OR fertile))) AND ((DFI OR DNA fragmentation))”. Additional bib-liography lists of retrieved original articles and review papers were manually searched for additional relevant references. We utilized the preferred reporting items for systematic review and meta-analysis checklist (PRISMA) while conducting this study (Fig. 1).

3. Study selection: inclusion and exclusion criteria

Inclusion criteria were original research articles in English and Spanish language addressing the relation-ship between paternal age, semen parameters, and DNA fragmentation. The search was restricted to stud-ies in humans. Considering the type of participants, studies that assessed DNA fragmentation in fertile men with normal semen analysis in addition to men with a diagnosis of infertility or subfertility were con-sidered, from oligozoospermia (when total sperm count was less than 15 million per mL) to severe oligospermia (when total sperm count was below 5 million per mL). Men who had an underlying varicocele or conditions such as diabetes and obesity were included. This analy-sis included prospective or retrospective comparative studies which examined the association between age and DNA fragmentation as measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL), the sperm chromatin structure assay (SCSA), the single-cell gel electrophoresis (Comet) assay, and the sperm chroma-tin dispersion (SCD) test.

Azoospermic men were not considered eligible and reports including testicular DNA fragmentation were excluded. Articles that were not in English or Spanish language were excluded. Due to the influence of the abstinence period on DNA fragmentation, we excluded articles that did not clearly state the abstinence period. Men who underwent interventions (e.g., chemotherapy,

radiotherapy, antioxidants, etc.) were excluded from the analysis. Men who were diagnosed with a malignancy and performed a semen analysis before treatment were excluded. Additionally, case reports, editorials, review articles, articles for which the full text could not be found, animal experimental studies, and articles for which the data were not extractable were excluded. Lastly, articles studying the influence of factors such as senescence, environmental pollutants, and cryptor-chidism were excluded.

4. Data extractionTwo researchers (D.G and J.B) performed the sys-

tematic review and independently extracted data from all selected articles. The opinion of a third observer (J.O) was sought to gain consensus, in the event of any dis-cordance on selecting studies. Extracted data included on study design, publication year, population character-istics, inclusion and exclusion criteria, population, DNA fragmentation assay, main outcomes and conclusions, adjusted results, and statistical methods. The primary outcome of the study was DNA fragmentation evalu-ation, and volume, concentration, motility, progressive motility, and vitality were considered secondary out-comes.

5. Quality assessmentEach study was scored for their relevance and meth-

odological quality by using the QUADAS 2 (Quality Assessment of Diagnostic Accuracy Studies 2) checklist [20]. Furthermore the following characteristics of the studies were taken into consideration: study population and DNA fragmentation assay.

RESULTS

1. Eligible studies of systematic reviewThe systematic search retrieved a total of 3,120 ar-

ticles: 3,116 were identified utilizing the search strategy and four additional articles were identified by manu-ally searching relevant references. After removing duplicates, we were left with 2,061 potentially relevant articles (Fig. 1). The screening for study inclusion was performed in two stages: titles and abstracts were screened in the first stage, and full manuscripts of the articles identified as relevant in the initial screening were retrieved and read in detail for the second stage. After first stage screening, 1,935 articles were excluded,


107www.wjmh.org

Tabl

e 1.

Cha

ract

erist

ics o

f stu

dies

incl

uded

into

syst

emat

ic re

view

Refe

renc

e(a

utho

r, ye

ar,

natio

nalit

y)

Popu

latio

n ch

arac

teris

ticSa

mpl

e si

zeAg

e ra

nge

(y)

Sper

m D

FI %

Met

hod

used

Volu

me

(mL)

Conc

entr

atio

n (×

106 /m

L)%

Mot

ility

Prog

ress

ive

mot

ility

(%)

Vita

lity

(%)

p-va

lue

Cola

sant

e et

al,

2019

, Bra

zil

[21]

NO

RMO

and

su

bfer

tile

3,12

45

IQR

(2–1

0)TU

NEL

3±1

60±1

077

5<3

5r=

-0.1

7a-

--

--

p<0.

001

1,11

036

–40

r=-0

.16a

--

--

-p<

0.00

11,

239

>41

r=-0

.18a

--

--

-p<

0.00

1M

osko

vtse

v et

al

, 200

6, U

SA

[22]

NO

RMO

and

su

bfer

tile

1,12

5SC

SA-

-57

<30

15.2

±8.4

a3.

2±1.

655

.5±4

9.2

35.3

±13.

7a-

76.7

±7.9

aa p<

0.00

1whe

n co

mpa

red

to >

4538

630

–34

19.4

±12.

1a3.

3±1.

557

.6±5

7.4

33.5

±17.

1a-

71.7

±11.

5a

406

35–4

020

.1±1

0.9a

3.2±

1.5

60.7

±59.

433

.4±1

6.7a

-71

.5±1

1.9a

187

40–4

426

.4±1

6.0a

2.9±

1.4

61.9

±62.

129

.8±1

7.7

-65

.5±1

6.8

89

>45

32.0

±17.

12.

9±1.

865

.5±6

5.7

24.9

±15.

3-

62.5

±14.

6Br

ahem

et a

l, 20

11, T

unisi

a [2

3]

NO

RMO

and

su

bfer

tile

190

TUN

EL10

20–2

9 N

ORM

O10

.5±1

.42.

8±0.

995

.7±2

2.9

53.7

±4.4

--

NS

r=-0

.094

p=0.

516

1630

–39

NO

RMO

10.3

±4.3

2.8±

0.9

93.0

±29.

451

.3±6

.8-

-14

40–4

9 N

ORM

O9.

9±4.

02.

4±0.

3b10

0±46

.248

.1±5

.1-

-10

50–7

0 N

ORM

O9.

0±5.

62.

0±0.

1b99

.1±3

1.3

47.5

±3.5

--

1120

–29

Su

bfer

tile

26.2

±13.

43.

2±1.

662

.0±4

0.9

26.3

±17.

0-

-N

Sr=

0.08

p>0.

0579

30–3

9

Subf

ertil

e27

.6±1

5.8

3.3±

1.5

76.0

±59.

829

.0±1

5.8

--

4040

–49

Su

bfer

tile

30.4

±16.

82.

0±1.

3b97

.9±8

6.1

25.5

±14.

2-

-

1050

–70

Su

bfer

tile

31.6

±18.

02.

4±1.

198

.1±6

1.3

22.0

±17.

4-

-

Guo

et a

l, 20

20,

Chin

a [2

4]N

ORM

O a

nd

subf

ertil

e65

4SC

D71

<30

NO

RMO

13.0

±7.4

-75

.7±4

2.9

68.5

±11.

661

.7±1

0.9

-p=

0.00

6D

FI &

age

for

NO

RMO

8330

–35

NO

RMO

14.0

±8.6

81.0

±51.

363

.3±1

5.5

56.6

±14.

3-

71>3

5 N

ORM

O17

.5±1

0.2a

-82

.2±4

4.3

62.5

±12.

6a55

.6±1

1.7

-13

5<3

0 Su

bfer

tile

14.5

±11.

458

.3±5

1.4

52.8

±21.

646

.9±2

0.0

-p=

0.00

1D

FI &

age

for

subf

ertil

e12

430

–35

Su

bfer

tile

16.0

±11.

0-

58.6

±46.

547

.0±2

1.2

41.0

±19.

8a-

170

>35

Subf

ertil

e19

.8±1

3.9a

-65

.5±6

4.2

45.7

±20.

5a39

.5±1

8.8a

-


108 www.wjmh.org

Tabl

e 1.

Con

tinue

d 1

Refe

renc

e(a

utho

r, ye

ar,

natio

nalit

y)

Popu

latio

n ch

arac

teris

ticSa

mpl

e si

zeAg

e ra

nge

(y)

Sper

m D

FI %

Met

hod

used

Volu

me

(mL)

Conc

entr

atio

n (×

106 /m

L)%

Mot

ility

Prog

ress

ive

mot

ility

(%)

Vita

lity

(%)

p-va

lue

Pete

rsen

et a

l, 20

18, B

razi

l [2

5]

Fert

ile &

in

fert

ile

2,17

8TU

NEL

2.7±

1.4

74.6

±61.

663

.7±1

5.7

56.9

±16.

265

.0±1

4.5

852

<35

14.7

±8.3

a-

--

--

r=0.

10p=

0.00

21,

014

36–4

415

.9±8

.7-

--

--

312

>45

16.2

±8.4

a-

--

--

Kaar

ouch

et

al, 2

018,

M

oroc

co [2

6]

Fert

ile &

in

fert

ile83

TUN

EL42

<40

252.

6±1.

124

.3±2

.933

-60

p<0.

0541

>40

41b

2.4±

1.5

18.4

±2.6

26-

55Co

hen-

Bacr

ie

et a

l, 20

09,

Fran

ce [2

7]

Fert

ile &

in

fert

ile1,

653

TUN

EL68

836

.6±6

.10–

20-

-32

.6±4

9.6

32.3

±20.

774

.3±1

3.0

p=0.

001

463

37.1

±6.4

20–3

0-

-30

.0±5

2.0

28.6

±18.

471

.5±1

2.5

286

38.2

±6.8

30–4

0-

-23

.7±4

0.4a

25.3

±16.

868

.9±1

2.8

216

39.3

±9.6

>40

--

16.6

±28.

5a21

.3±1

6.9

62.3

±14.

8Ev

enso

n et

al,

2020

, USA

[2

8]

Fert

ile &

in

fert

ile25

,262

SCSA

--

-

2,00

020

–29

12.6

±9.7

--

--

-14

,000

30–3

915

.2±1

1.3

--

--

-8,

000

40–4

919

.5±1

4.0

--

--

-1,

000

50–5

926

.8±1

7.4

--

--

-26

260

–80

39.3

±21.

7-

--

--

Anto

noul

i et

al,

2019

, Ge

rman

y [2

9]

Fert

ile &

in

fert

ile15

043

.4±5

.5r=

0.23

b

p=0.

046

SCD

49.5

r=0.

01p>

0.05

54.9

+19.

2a

r=-0

.29

p=0.

012

32.6

±17.

3b-

Blac

hman

-Br

aun

et a

l, 20

20, U

SA

[30]

Fert

ile a

nd

infe

rtile

55

037

.7±6

.512

.7 [7

.8–2

0]SC

SA3.

0±1.

443

.8±3

6.3

47.7

9±10

.5p≤

0.00

1D

FI &

age

38<

3011

.9±7

.93.

3±1.

334

.5±3

3.7

46.3

±19.

8-

-34

830

–40

13.8

±9.4

3.2±

1.5

44.4

±41.

649

.2±1

8.3

--

145

40–5

021

.2±1

6.5a

2.6±

1.2

46.9

±38.

443

.9±1

9.6

--

19>5

029

.5±1

6a2.

4±1.

337

.5±3

039

.3±1

9.9

--

Alsh

ahra

ni e

t al

, 201

4, U

SA

[31]

Infe

rtile

839

19.9

± 1

5.3

TUN

EL3.

1 ±

1.5

-44

.7 ±

19.

7-

-69

<30

16.7

± 1

1.2

3.4

± 1.

542

.0±5

0.5

44.3

± 1

4.4

--

p<0.

005

w

hen

>40

com

pare

d to

al

l gro

ups

298

31–4

019

.1 ±

14.

63

± 1.

436

.6±3

9.7

45.2

± 1

9.5

--

367

<40

18.7

± 1

4. 1

3.1

± 1.

742

.6±4

1.5

45.0

± 1

8.6

--

105

>40

24.4

± 1

8.5b

3.1

± 1.

743

.8±5

2.3

43.5

± 2

2.9

--


109www.wjmh.org

Tabl

e 1.

Con

tinue

d 2

Refe

renc

e(a

utho

r, ye

ar,

natio

nalit

y)

Popu

latio

n ch

arac

teris

ticSa

mpl

e si

zeAg

e ra

nge

(y)

Sper

m D

FI %

Met

hod

used

Volu

me

(mL)

Conc

entr

atio

n (×

106 /m

L)%

Mot

ility

Prog

ress

ive

mot

ility

(%)

Vita

lity

(%)

p-va

lue

Nijs

et a

l, 20

11,

Belg

ium

[32]

Infe

rtile

278

TUN

ELN

S13

5<3

422

.4±1

1.8

40.7

±35.

852

.5±1

8.8

--

r=-0

.006

p=0.

9596

35–3

923

.35±

12.3

40.7

±34.

250

.2±1

6.9

--

r=0.

027

p=0.

7947

>40

24.5

±12.

641

.5±3

1.7

57.3

8±13

.0-

-r=

-0.1

49p=

0.32

Vagn

ini e

t al,

2007

, Bra

zil

[33]

Infe

rtile

508

TUN

EL18

6<3

515

.7±1

0.7b

--

--

-p=

0.03

414

036

–39

18.2

±11.

3b-

--

--

p=0.

022

182

>40

18.3

±11.

0-

--

--

p=0.

93Lu

et a

l, 20

18,

Chin

a [3

4]In

fert

ile1,

010

SCSA

45.3

9±19

.2r=

0.11

5p<

0.00

1a

OR:

0.8

65(9

5% C

I,

0.78

5–0.

954)

p=0.

004

r=0.

145

p<0.

001a

OR:

0.5

48(9

5% C

I, 0.

39–0

.77)

p=0.

001

r=-0

.115

p<0.

001a

OR:

0.9

78(9

5% C

I,

0.96

2–0.

994)

p=0.

008

r=-0

.487

p<0.

001

OR:

0.9

85

(95%

CI,

0.

907–

1.60

9)p=

0.71

3

32.2

9±12

.9-

Pino

et a

l, 20

20, C

hile

, [3

5]

Gene

ral p

ublic

2,67

8SC

D11

921

–30

OR:

1p=

0.02

9 D

FI &

m

en o

ver 5

01,

579

31–4

0O

R: 1

.243

(9

5% C

I, 0.

364–

4.24

0);

p=0.

728

OR:

0.8

21

(95%

CI,

0.

464–

1.45

0);

p=49

7

OR:

0.9

87(9

5% C

I,

0.57

6–1.

690)

;

-O

R: 3

.241

b (9

5% C

I, 1.

175–

8.94

0);

p=0.

023

-

852

41–5

0O

R: 1

.38

(95%

CI,

0.39

–4.8

2);

p=0.

606

OR:

1.3

32

(95%

CI,

0.74

9–2.

369)

; p=

0.32

8

OR:

1.1

88

(95%

CI,

0.

685–

2.06

0)

-O

R: 5

.243

a (9

5% C

I, 1.

892–

14.5

26);

p=0.

001

-

128

>50

OR:

4.5

8b

(95%

CI,

1.

167–

17.9

9)

OR:

2.2

0(9

5% C

I, 1.

11–4

.34)

; p=

0.02

2

OR:

1.18

8a

(95%

CI,

0.

685–

2.06

0)

-O

R: 1

1.91

1a (9

5% C

I, 4.

045–

35.0

73);

p<0.

0001

-


110 www.wjmh.org

Tabl

e 1.

Con

tinue

d 3

Refe

renc

e(a

utho

r, ye

ar,

natio

nalit

y)

Popu

latio

n ch

arac

teris

ticSa

mpl

e si

zeAg

e ra

nge

(y)

Sper

m D

FI %

Met

hod

used

Volu

me

(mL)

Conc

entr

atio

n (×

106 /m

L)%

Mot

ility

Prog

ress

ive

mot

ility

(%)

Vita

lity

(%)

p-va

lue

Wyr

obek

et a

l, 20

06, U

SA

[36]

Gene

ral p

ublic

88Co

met

SCSA

1920

–29

12.9

±7.7

--

--

-r=

0.72

p<0.

001

2030

–39

16.3

±9.6

--

--

-16

40–4

923

.2±1

4.9a

--

--

-17

50–5

935

.4±1

8.6a

--

--

-16

60–8

049

.6±1

7.3a

--

--

-D

as e

t al,

2013

, Ca

nada

[37]

NO

RMO

and

O

AT27

7SC

SA10

7<4

0 N

ORM

O12

±8-

95±6

8-

61±1

4-

p=0.

008

41>4

0 N

ORM

O17

±13a

-99

±58

-58

±17

-97

<40

OAT

12±1

0-

35±3

7-

30±1

6-

p=0.

003

32>4

0 O

AT20

±18a

-33

±37

-24

±14

-Pl

astir

a et

al,

2007

, Gre

ece

[38]

NO

RMO

and

O

AT11

0TU

NEL

2624

–34

NO

RMO

6.5±

1.9

3.3±

0.7

51.0

±22.

456

.5±4

.4-

-r=

-0.1

05p=

0.47

223

35–5

4 N

ORM

O5.

9±1.

73.

4±0.

842

.0±1

4.0

54.7

±4.1

a-

-r=

-0.2

06

p=0.

155

3024

–34

OAT

26.3

±5.3

a3.

2±1.

03.

3±2.

323

.9±9

.6-

-r=

0.55

8p<

0.00

131

35–5

4 O

AT33

.7±6

.7b

2.6±

1.0a

5.0±

2.7

19.2

±8.0

--

r=-0

.294

p=0.

022

Rosia

k-Gi

ll et

al,

2019

, Po

land

[39]

NO

RMO

and

te

rato

ozo-

sper

mic

336

TUN

EL11

6<4

0 N

ORM

O12

.7±9

.43.

8±1.

768

.0±5

0.5

52.4

±12.

9-

-p=

0.00

544

>40

NO

RMO

17.6

±10.

4a3.

3±1.

8a72

.4±4

6.3

56.2

±13.

6-

-13

2<4

0 Te

rato

ozo-

sper

mic

13.9

±10.

93.

3±1.

532

.6±3

2.7

30.3

±18.

5-

-p=

0.00

10

44>4

0 Te

rato

ozo-

sper

mic

21.8

±15.

1a2.

9±1.

6a38

.6±4

2.1

31.6

±20.

4-

-

Valu

es a

re p

rese

nted

as n

umbe

r onl

y, m

ean±

stan

dard

dev

iatio

n, o

r med

ian

[inte

rqua

rtile

rang

e].

NO

RMO

: nor

moz

oosp

erm

ia, I

QR:

inte

rqua

rtile

rang

e, T

UNEL

: ter

min

al d

eoxy

nucl

eotid

yl tr

ansf

eras

e-m

edia

ted

deox

yurid

ine

trip

hosp

hate

nic

k en

d la

belli

ng, S

CSA:

sper

m c

hrom

atin

stru

ctur

e as

say,

NS:

not

sig

nific

ant,

SCD

: spe

rm c

hrom

atin

disp

ersio

n te

st, D

FI: D

NA

fragm

enta

tion

Inde

x, O

R: o

dds

ratio

, CI:

conf

iden

ce in

terv

al, C

omet

: the

sin

gle

cell-

gel e

lect

roph

ores

is as

say,

OAT

: Olig

oast

he-

note

rato

zoos

perm

ic.

a p<0.

001,

b p<0.

05.


111www.wjmh.org

and 126 articles were identified to assess the full text for eligibility. After this second stage of screening, 105 articles were excluded for reasons shown in (Supple-ment Table).

2. Characteristics of included studies and comparison of outcomes

The main characteristics of the present systematic review included 19 studies as summarized in Table 1. There were four studies that examined the impact of APA on DNA fragmentation between Normozoo-spermic and subfertile males [21-24]. Three of the four studies showed a significant difference (p<0.01) in favor of APA increasing DNA fragmentation, even among normozoospermic males [21,22,24]. All six stud-ies examining DNA fragmentation between fertile and infertile males with proven primary or secondary infertility, which reported a significant difference in favor of increasing DNA fragmentation with APA [25-30]. Four studies examined males with proven primary or secondary infertility [31-34]. Two studies examined the effect of APA on DNA fragmentation within a general population of healthy males [35,36]. Two stud-ies examined DNA fragmentation between normozoo-spermic and oligoasthenoteratozoospermic males [37,38] and one study examined teratozoospermic males [39]. Six of the nineteen studies showed a significant dif-ference (p<0.05) in favor of APA decreasing sperm motility [22,24,27,29,34,35]. Moreover, in this review

three studies examined and noted the incidence of varicoceles, which there was no demonstrated effect of varicocele presence on DNA fragmentation, irre-spective of APA [25,31,33]. Five studies showed a sig-nificant difference in favor of APA decreasing semen volume [23,34,35,38,39] and two studies demonstrating APA decreasing concentration [34,38]. Out of the 19 studies, 2 failed to show an association with APA and DNA fragmentation [23,32].

Fig. 2 and Table 2 show the scores on overall risk of bias and concerns regarding applicability in this systematic review according to QUADAS 2. For about half of the studies the patient population examined a mix of infertile and fertile patients and hence was judged to be at “high risk” of bias for QUADAS 2 do-main “patient selection”. Studies were at high risk of applicability concerns in domain “reference standard” when the patient’s age category threshold is not com-parable to the thresholds of other studies. Overall, the domain “index test” was considered to be at “low risk” because there were 10 studies that utilized TUNEL assay [21,23,25-27,31-33,38,39], 6 studies utilizing SCSA [22,28,30,34,35,37], 1 study using Comet assay [36], and 2 studies that utilized the SCD to quantify DNA frag-mentation [24,29].

DISCUSSION

The effect of paternal age on semen quality and

Patientselection

100

80

60

40

20

%

0Index test Reference

standardFlow

and timing

Unclear riskHigh riskLow risk

Patientselection

100

80

60

40

20

%

0Index test Reference

standard

Unclear riskHigh riskLow risk

A B

Fig. 2. Overall risk of bias in systematic review. This figure illustrates the overall risk of bias in the systematic review. (A) Proportion of studies with low, high, or unclear risk of bias (%). (B) Proportion of studies with low, high, or unclear concerns regarding applicability (%).The vertical axis rep-resents the number of studies included. The color of the bars represents the risk of bias.


112 www.wjmh.org

DNA fragmentation remains controversial. We hypoth-esized that APA would be associated with an increase in DNA fragmentation. We performed a systematic review comparing DNA fragmentation in different age groups among normozoospermic, subfertile, and infer-tile men. Our review included data on 40,668 subjects extracted from nineteen available published articles. In the majority of the articles assessed, (17/19) APA was associated with significant increase in DNA fragmen-tation. Conversely, two articles demonstrated that APA did not influence DNA fragmentation [23,32]. The over-all quality of papers demonstrating an association were overall higher than the papers showing no association. Overall, it appears that the majority of articles utiliz-ing SCSA and SCD assays reliably showed an associa-tion with APA and increased DNA fragmentation.

The implications of this systematic review are impor-tant because the average age of men having children has increased [2]. DNA fragmentation is not a part of a standard infertility workup and in the presence of a normal semen analysis, often no further workup is done [40,41]. However, if DNA fragmentation rates

truly do increase as men age, perhaps clinicians should increase their threshold to consider this test in the old-er men with infertility. In addition, couples pursuing in vitro fertilization (IVF) are typically in an older age bracket, and there are several interventions which can improve DNA fragmentation and may improve IVF outcomes in men with high DNA fragmentation [42,43].

In this review, both studies that did not find an ef-fect of APA on DNA fragmentation utilized the TU-NEL assay. Several assays are currently available to assess DNA fragmentation, and these assays can be broadly categorized into two types. The first category includes assays where DNA fragmentation is quanti-fied directly by incorporating probes at the site of dam-age, which detect actual DNA strand breaks. TUNEL, in situ nick translation (ISNT), and Comet assay belong to this category. Conversely, the second category in-cludes assays such as SCD test and SCSA, which utilize the property of fragmented DNA to aid denaturation under certain conditions [44]. SCD is based on the abil-ity of intact DNA deprived of chromatin proteins to loop around the lysed and acid treated sperm nuclear

Table 2. Study characteristics according to QUADAS II recommendations to report the risk of bias for patient selection and the concerns for appli-cability of data collected in manuscripts eligible for the systematic review

Reference (author, year)Risk of Bias Applicability concerns

Patient selection

Index testReference standard

Flow and timing

Patient selection

Index testReference standard

Colasante et al, 2019 [21] Low Low High Low Low Unclear HighMoskovtsev et al, 2006 [22] Low Low Low Low Low Low LowBrahem et al, 2011 [23] Low Low Low Low Low Low LowGuo et al, 2020 [24] Low Low Low Low Low Low LowPetersen et al, 2018 [25] Unclear Low Low Unclear Unclear Unclear LowKaarouch et al, 2018 [26] Unclear High Low Low Unclear Low LowCohen-Bacrie et al, 2009 [27] Low Low High Low Low Low HighEvenson et al, 2020 [28] Unclear Low Low Low Unclear Low LowAntonouli et al, 2019 [29] Unclear Low High Low Unclear Low HighBlachman-Braun et al, 2020 [30] Low Low Low Low Low Low LowAlshahrani et al, 2014 [31] High Low Low Low Low Low LowNijs et al, 2011 [32] High Low Low Low Low Low LowVagnini et al, 2007 [33] High Low Low Low High Low LowLu et al, 2018 [34] High Low Unclear Low High Low LowPino et al, 2020 [35] Low Low Unclear Low High Low LowWyrobek et al, 2006 [36] Low High/lowa Low Low Low High/lowa LowDas et al, 2013 [37] High High High Low Low High HighPlastira et al, 2007 [38] High High High Low Low High HighRosiak-Gill et al, 2019 [39] High High High Low Low High Low

QUADAS: Quality Assessment of Diagnostic Accuracy Studies.aHigh risk for Comet (the single cell gel electrophoresis assay), low risk for sperm chromatin structure assay (SCSA).


113www.wjmh.org

membrane carcass, thus indirectly measuring the sus-ceptibility of DNA to denaturation. Probe incorpora-tion in TUNEL depends on the amount of chromatin that is partially freed from the proteins protecting the DNA, thus it is possible that existing breaks are not detected due to chromatin compaction.

With all systematic reviews, there are limitations that should be taken into consideration. An important weakness of most studies relating to DNA fragmenta-tion and paternal age is that the patient populations are highly selective (i.e., infertile men). The vast ma-jority of studies were retrospective, and therefore the possibility of confounding variables influencing the results cannot be ruled out. It is important to mention that when investigating a paternal age effect on DNA fragmentation, there may be a residual confounding by the presence of varicoceles [45]. Factors such as infertility duration, varicocele, and environmental fac-tors were not reported in several studies. Despite these limitations, our review included data from >40,000 males, and to our knowledge, represents the first for-mal attempt to thoroughly assess the available data on effects of age in males attending an infertility clinic. Similarly reported by Johnson et al. [10], this review found that only two studies demonstrated the effect of APA on declining sperm concentration, while a major-ity of studies (6/19) demonstrated APA decreasing se-men volume. This review and Johnson et al.’s review [10] supports the association of APA with DNA fragmen-tation. This review is unique in that we recorded the method of measurement, and were able to determine how direct vs indirect assays supported the association of APA and DNA fragmentation. After systematically collecting the information of published articles, a meta-analysis was not amenable given the heterogeneity of the reports as there was not a not consistent cut-off point defining APA among authors. Although there is no validated cut-off points to define APA based on DNA fragmentation data with fertile and infertile men as well in those with normal and abnormal semen parameters, several authors favor for using >40 years as a cut-off point to refer to APA [14,18,26,31,39]. This definition still needs to be further validated in the con-text of DNA fragmentation, such an analysis requires making many assumptions about the variation in both age structure and traits across different populations. Despite the limitation of the present study and need of future prospective clinical trials that help validate our

observations, we believe that analyzing DNA fragmen-tation in men with APA starting around the age of 40 years can provide an additional tool to set expectations and counsel couples seeking fertility.

CONCLUSIONS

This study suggests a trend to support the effect of APA on DNA fragmentation. As sperm quality is a pivotal factor in fertility potential and ART outcomes, physicians should consider assessing DNA fragmen-tation in men around the age of 40 years. Given the significant methodological weakness and design of the included studies, future prospective studies are required to investigate the effects of aging on infertile men with normal semen parameters, as a conventional semen analysis can often fail to detect an underlying etiology for infertility.

ACKNOWLEDGEMENTS

The authors thank Dr. Manuel Molina, University of Miami School of Medicine, for his technical assistance for this study.

Conflict of Interest

The authors have nothing to disclose.

Author Contribution

Conceptualization: DCG, RR. Data curation: DCG, RR, JCB, JO. Formal analysis: DCG, RR, JCB, RBB, SN. Funding acqui-sition: None. Investigation: DCG, SN. Methodology: DCG, RR, RBB. Project administration: RR, RBB. Resources: RR. Software: RBB, SN. Supervision: DCG, RR, JCB. Validation: DCG, RBB, JO, RR. Visualization: DCG, RR, RBB, JCB. Writing – original draft: DCG, RR, JO, JCB. Writing – review & editing: JCB, DCG, JO, RR.

Supplementary Materials

Supplementary materials can be found via https://doi.org/10.5534/wjmh.200195.

REFERENCES

1. Humm KC, Sakkas D. Role of increased male age in IVF and


114 www.wjmh.org

egg donation: is sperm DNA fragmentation responsible? Fer-til Steril 2013;99:30-6.

2. Martin JA, Hamilton BE, Ventura SJ, Osterman MJ, Kirmeyer S, Mathews TJ, et al. Births: final data for 2009. Natl Vital Stat Rep 2011;60:1-70.

3. Kühnert B, Nieschlag E. Reproductive functions of the ageing male. Hum Reprod Update 2004;10:327-39.

4. Belloc S, Cohen-Bacrie P, Benkhalifa M, Cohen-Bacrie M, De Mouzon J, Hazout A, et al. Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemi-nation. Reprod Biomed Online 2008;17:392-7.

5. Maheshwari A, Hamilton M, Bhattacharya S. Effect of female age on the diagnostic categories of infertility. Hum Reprod 2008;23:538-42.

6. Auger J, Kunstmann JM, Czyglik F, Jouannet P. Decline in semen quality among fertile men in Paris during the past 20 years. N Engl J Med 1995;332:281-5.

7. Fisch H, Goluboff ET, Olson JH, Feldshuh J, Broder SJ, Barad DH. Semen analyses in 1,283 men from the United States over a 25-year period: no decline in quality. Fertil Steril 1996;65:1009-14.

8. Berling S, Wölner-Hanssen P. No evidence of deteriorating semen quality among men in infertile relationships during the last decade: a study of males from Southern Sweden. Hum Reprod 1997;12:1002-5.

9. Li Y, Lin H, Ma M, Li L, Cai M, Zhou N, et al. Semen qual-ity of 1346 healthy men, results from the Chongqing area of southwest China. Hum Reprod 2009;24:459-69.

10. Johnson SL, Dunleavy J, Gemmell NJ, Nakagawa S. Consistent age-dependent declines in human semen quality: a systematic review and meta-analysis. Ageing Res Rev 2015;19:22-33.

11. Levitas E, Lunenfeld E, Weisz N, Friger M, Potashnik G. Re-lationship between age and semen parameters in men with normal sperm concentration: analysis of 6022 semen samples. Andrologia 2007;39:45-50.

12. Schwartz D, Mayaux MJ, Spira A, Moscato ML, Jouannet P, Czyglik F, et al. Semen characteristics as a function of age in 833 fertile men. Fertil Steril 1983;39:530-5.

13. Rolf C, Behre HM, Nieschlag E. Reproductive parameters of older compared to younger men of infertile couples. Int J An-drol 1996;19:135-42.

14. Das M, Al-Hathal N, San-Gabriel M, Phillips S, Kadoch IJ, Bissonnette F, et al. Infertile normozoospermic men with advanced paternal age have a high prevalence of sperm DNA damage. Hum Reprod 2012;27(Suppl 2):ii121-50.

15. Winkle T, Rosenbusch B, Gagsteiger F, Paiss T, Zoller N. The correlation between male age, sperm quality and sperm DNA fragmentation in 320 men attending a fertility center. J Assist

Reprod Genet 2009;26:41-6.16. Singh NP, Muller CH, Berger RE. Effects of age on DNA dou-

ble-strand breaks and apoptosis in human sperm. Fertil Steril 2003;80:1420-30.

17. Aitken RJ, Baker MA, Sawyer D. Oxidative stress in the male germ line and its role in the aetiology of male infertility and genetic disease. Reprod Biomed Online 2003;7:65-70.

18. Toriello HV, Meck JM; Professional Practice and Guidelines Committee. Statement on guidance for genetic counseling in advanced paternal age. Genet Med 2008;10:457-60.

19. Moher D, Liberati A, Tetzlaff J, Altman DG; PRISMA Group. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. BMJ 2009;339:b2535.

20. Whiting PF, Rutjes AW, Westwood ME, Mallett S, Deeks JJ, Reitsma JB, et al. QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy studies. Ann Intern Med 2011;155:529-36.

21. Colasante A, Minasi MG, Scarselli F, Casciani V, Zazzaro V, Ruberti A, et al. The aging male: relationship between male age, sperm quality and sperm DNA damage in an unselected population of 3124 men attending the fertility centre for the first time. Arch Ital Urol Androl 2019;90:254-9.

22. Moskovtsev SI, Willis J, Mullen JB. Age-related decline in sperm deoxyribonucleic acid integrity in patients evaluated for male infertility. Fertil Steril 2006;85:496-9.

23. Brahem S, Mehdi M, Elghezal H, Saad A. The effects of male aging on semen quality, sperm DNA fragmentation and chro-mosomal abnormalities in an infertile population. J Assist Reprod Genet 2011;28:425-32.

24. Guo LY, Zhou H, Liu M, Li Q, Sun XF. Male age is more criti-cal to sperm DNA integrity than routine semen parameters in Chinese infertile males. Andrologia 2020;52:e13449.

25. Petersen CG, Mauri AL, Vagnini LD, Renzi A, Petersen B, Mattila M, et al. The effects of male age on sperm DNA dam-age: an evaluation of 2,178 semen samples. JBRA Assist Re-prod 2018;22:323-30.

26. Kaarouch I, Bouamoud N, Madkour A, Louanjli N, Saadani B, Assou S, et al. Paternal age: negative impact on sperm ge-nome decays and IVF outcomes after 40 years. Mol Reprod Dev 2018;85:271-80.

27. Cohen-Bacrie P, Belloc S, Ménézo YJ, Clement P, Hamidi J, Benkhalifa M. Correlation between DNA damage and sperm parameters: a prospective study of 1,633 patients. Fertil Steril 2009;91:1801-5.

28. Evenson DP, Djira G, Kasperson K, Christianson J. Rela-tionships between the age of 25,445 men attending infertil-ity clinics and sperm chromatin structure assay (SCSA®) defined sperm DNA and chromatin integrity. Fertil Steril


115www.wjmh.org

2020;114:311-20.29. Antonouli S, Papatheodorou A, Panagiotidis Y, Petousis S,

Prapas N, Nottola SA, et al. The impact of sperm DNA frag-mentation on ICSI outcome in cases of donated oocytes. Arch Gynecol Obstet 2019;300:207-15.

30. Blachman-Braun R, Best JC, Sandoval V, Lokeshwar SD, Patel P, Kohn T, et al. Sperm DNA fragmentation index and high DNA stainability do not influence pregnancy success after intracytoplasmic sperm injection. F S Rep 2020;1:233-8.

31. Alshahrani S, Agarwal A, Assidi M, Abuzenadah AM, Du-rairajanayagam D, Ayaz A, et al. Infertile men older than 40 years are at higher risk of sperm DNA damage. Reprod Biol Endocrinol 2014;12:103.

32. Nijs M, De Jonge C, Cox A, Janssen M, Bosmans E, Ombelet W. Correlation between male age, WHO sperm parameters, DNA fragmentation, chromatin packaging and outcome in assisted reproduction technology. Andrologia 2011;43:174-9.

33. Vagnini L, Baruffi RL, Mauri AL, Petersen CG, Massaro FC, Pontes A, et al. The effects of male age on sperm DNA damage in an infertile population. Reprod Biomed Online 2007;15:514-9.

34. Lu JC, Jing J, Chen L, Ge YF, Feng RX, Liang YJ, et al. Analy-sis of human sperm DNA fragmentation index (DFI) related factors: a report of 1010 subfertile men in China. Reprod Biol Endocrinol 2018;16:23.

35. Pino V, Sanz A, Valdés N, Crosby J, Mackenna A. The effects of aging on semen parameters and sperm DNA fragmenta-tion. JBRA Assist Reprod 2020;24:82-6.

36. Wyrobek AJ, Eskenazi B, Young S, Arnheim N, Tiemann-Boege I, Jabs EW, et al. Advancing age has differential effects on DNA damage, chromatin integrity, gene muta-tions, and aneuploidies in sperm. Proc Natl Acad Sci U S A 2006;103:9601-6.

37. Das M, Al-Hathal N, San-Gabriel M, Phillips S, Kadoch IJ, Bissonnette F, et al. High prevalence of isolated sperm DNA

damage in infertile men with advanced paternal age. J Assist Reprod Genet 2013;30:843-8.

38. Plastira K, Msaouel P, Angelopoulou R, Zanioti K, Plastiras A, Pothos A, et al. The effects of age on DNA fragmentation, chromatin packaging and conventional semen parameters in spermatozoa of oligoasthenoteratozoospermic patients. J As-sist Reprod Genet 2007;24:437-43.

39. Rosiak-Gill A, Gill K, Jakubik J, Fraczek M, Patorski L, Gac-zarzewicz D, et al. Age-related changes in human sperm DNA integrity. Aging (Albany NY) 2019;11:5399-411.

40. Jarow J, Sigman M, Kolettis PN, Lipshultz LR, Dale McClure R, Nangia AK, et al. The optimal evaluation of the infertile male: best practice statement reviewed and validity confirmed 2011 [Internet]. Linthicum (MD): American Urological Associa-tion; c2011 [cited 2020 Dec 21]. Available from: https://www.auanet.org/education/guidelines/male-infertility-d.cfm.

41. Agarwal A, Majzoub A, Esteves SC, Ko E, Ramasamy R, Zini A. Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios. Transl Androl Urol 2016;5:935-50.

42. Telli O, Sarici H, Kabar M, Ozgur BC, Resorlu B, Bozkurt S. Does varicocelectomy affect DNA fragmentation in infertile patients? Indian J Urol 2015;31:116-9.

43. Esteves SC, Roque M, Garrido N. Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis. Asian J Androl 2018;20:1-8.

44. Zini A, Libman J. Sperm DNA damage: clinical significance in the era of assisted reproduction. CMAJ 2006;175:495-500.

45. Enciso M, Muriel L, Fernández JL, Goyanes V, Segrelles E, Marcos M, et al. Infertile men with varicocele show a high relative proportion of sperm cells with intense nuclear dam-age level, evidenced by the sperm chromatin dispersion test. J Androl 2006;27:106-11.

Advanced Paternal Age and Sperm DNA Fragmentation: A ...

Documents

Transcript of Advanced Paternal Age and Sperm DNA Fragmentation: A ...