6 ART & Reproductive Genetics Dept and PERITOX Laboratory, France
Submission: August 17, 2018; Published: August 29, 2018
*Corresponding author: Moncef Benkhalifa, ART & Reproductive Genetics Dept and PERITOX Laboratory, University Hospital & School of Medicine. Picardie University Jules Verne, Amiens, France, Tel: 0033677867390; Email :email@example.com
How to cite this article: Hazout A, Montjean D, Cassuto NG, Belloc S, Dalleac A, Tesarik J, Benkhalifa M. Free Circulating Nucleic Acids and Infertility. J Gynecol Women’s Health. 2018: 11(3): 555812. DOI: 10.19080/JGWH.2018.11.555812
Background: Human infertility is due to deficiencies in sperm quality in more than half of the cases. Several authors insist on a better exploration of the sperm but little is known about the quality of oocytes and especially about their competence at the crucial moment of transcription. Low ovarian reserve and sperm DNA damage are supposed to be the main culprits in assisted reproductive technology (ART) failures. The presence of excessive cell-free nucleic acids, mainly cell-free DNA (cfDNA), in the serum of men or women has attracted the attention of many practitioners in cases of the so-called unexplained infertility or embryo implantation failures.
Methods: Several and relevant studies are recalled as well as those of our work preceding an european patent granted in 2013 and titled: « cell freee DNA as a therapeutic target for female infertility and diagnostic marker ». The mean cfDNA value in the group of fertile women was 49,2ng/μl versus 98,5ng/μl in the infertile group, which was statistically significant. The mean cfDNA value in the group of fertile men was 60,6ng/μl and 83,34ng/μl in the infertile group which was no statistically significant difference. Comparing cfDNA in the plasma and Follicular Fluid of 42 patients we demonstrated that mean cfDNA value in the FF was less than plasma CFDNA value in the corresponding patients.
Conclusion: Although the excess of cfDNA in the etiology of infertility is not clearly established, our experience and that of other authors suggest that excess cfDNA, might be a marker of oxidative stress or apoptotic and/or necrotic processes deleterious ART outcomes. This review takes stock of the diagnostic, prognostic and therapeutic potentialities, already known and demonstrated in human reproduction
The cause of infertility can be determined in most cases. The known male factors include hormonal defects, genital infections, with or without genital tract obstruction, cryptorchidism, varicocele and spernm DNA fragmentation or chromatin decondensation, whereas female infertility is mainly due to a low oocyte quality. However, the occurrence of unexplained infertility varies between 0% and 6%, according to different authors .
Taking into consideration abnormal results of the evaluation of ovarian function, hysteroscopy, hysterosalpingography, laparoscopy and comprehensive sperm evaluation including testing of DNA and chromatin integrity, the occurrence of unexplained infertility is reduced dramatically. We also know
the deleterious effects of different life-style and environmental factors (smoking, excess caffeine intake, or alcohol and drug abuse). Numerous defects which lead to problems with implantation are unknown and constitute another area of unexplained infertility. Integrins, LIF; G-CSF; GH or other growth factors may affect the percentage of patients with unexplained infertility. But this was not clearly identified.
The presence of circulating cell-free DNA in human plasma was reported in 1948 by Mendel and Metais . Cell-free circulating DNA (cfDNA) has been studied in a wide range of physiological and pathological conditions, including inflammatory disorders, oxidative stress and malignancy . It is present in normalhealthy individuals at low concentrations (< 50ng/ml). Although
the precise mechanism of DNA release into the blood remains
uncertain, it probably derives from a combination of apoptosis,
necrosis and active release from cells (Figure 1).
The clearance of cfDNA from the bloodstream occurs rapidly:
fetal DNA disappeared from the blood of mothers after delivery
with a half life time of 16.3 minutes . It is known that cfDNA
is sensitive to plasma nucleases (for example DNase 1), but
renal and hepatic clearance are also involved in the elimination
of cfDNA. Cultured cells have been shown to release double
stranded DNA into the media, and cfDNA might be incorporated
into cells . However, this hypothesis remains to be proven.
Circulating DNA can be isolated from both plasma and serum
. Recently, It was shown that less than 10% of the 6-fold
higher serum DNA levels were due to contamination by other
sources (i.e. release from leucocytes during the separation of
serum) [6,7]. Cell-free circulating DNA harbors the potential of
a useful biomarker. DNA levels and fragmentation patterns offer
interesting possibilities for diagnostic and prognostic purposes.
Here, we present literature data as well as our recent findings
on cfDNA in infertile female and male patients with regard to
the choice of diagnostic methods, prognostic information and
From the primordial follicles until the ovulation, the follicles
are submitted to a lot of growth factors participating in the
regulation of follicle growth, dominance and oocyte maturation,
and the list of currently known factors is not exhaustive (Table
As in the male, female gametes production comprise mitosis,
meiosis and oocyte maturation. They are named oogonia during
this process, and they stop mitosis once they have reached their
first meiotic division. At that time-point they become primary
oocytes. As they enter meiosis, the primary oocytes become
surrounded by ovarian mesenchymal cells to form primordial
From puberty, a few primordial follicles begin to grow every
day. The follicle first grows quickly. Most of this growth occurs
in the primary oocyte. This is the moment of a major protein
synthesis. Zona pellucida is constituted with gap junctions
between adjacent granulosa cells and between the granulosa
cells and the oocyte.
Cells of the ovarian stroma condense on the membrana
propria to form the theca, which is vascularized. FSH and LH
receptors develop; if not, follicules undergo atresia. FSH and
LH convert the preantral follicles to antral follicles (Graafian
follicles). The theca divides into two layers: the theca interna,
glandular and highly vascular, surrounded by the theca externa,
a fibrous capsule. Fluid starts to appear between the granulosa
cells, creating follicular fluid within the follicular antrum.
The follicle makes androgens and oestrogens. The principal
androgens are androstenedione and testosterone; the principal
oestrogen is Estradiol 17ß. Only the theca interna has LH
receptors. The granulosa cells receive these androgens and
aromatize them to oestrogens. The combination of oestrogen
and FSH causes LH receptors to develop on the granulosa cells.
a) The oocyte, whose first meiotic division has been
interrupted in prophase, resumes meiosis and undergoes
metaphase and anaphase. After the separation of the first polar
body, the oocyte enters rapidly the second meiotic division which
becomes arrested again at the metaphase stage until fertilization.
On the same time, cytoplasmic maturation occurs.
b) The follicle itself matures.
The granulosa cells no longer converts androgen to estrogen
but instead synthetizes progesterone. LH also stimulates this
progesterone synthesis. To be fertilizable the oocyte has to
acquire a real competence during follicular development; Oocyte
competence is defined as the intrinsic ability to complete meiotic
maturation, undergo fertilization, start embryonic development,
and establish a successful pregnancy.
Moreover it is well known that pregnancy rates (PR)
in women over 35 years of age are significantly lower, both
naturally and with assisted reproduction. With the exception of
growth hormone treatment during ovarian stimulation there is
currently no known intervention to improve pregnancy outcome
of elderly women .
Aging and age-related pathologies are frequently associated
with loss of mitochondrial function mainly due to the
accumulation of mtDNA mutations and deletions. In oocytes, low
levels of mitochondrial oxidative phosphorylation may occur for
up to 40 years before follicle maturation and ovulation, further
increasing the risk for mtDNA mutations.
One of the more frequent mtDNA deletions is the ‘‘common
deletion’’of 4977 base pairs, almost a third of the whole mtDNA
genome. This deletion was shown to have a high prevalence in
unfertilized oocytes and oocytes from elderly women  (Figure
To produce, maintain and transport spermatozoa and seminal
plasma to discharge sperm within the female reproductive tract
during sexual intercourse. To produce male sexual hormones
responsible for maintening the male reproductive function.
The testes secrete large amounts of androgens principally
testosterone, but they also secrete small amounts of estrogens.
In the seminiferous tubules, spermatozoa are formed from
the primitive germ cells. Then, they acquire maturation in the
epididymus and, crossing the vas deferens, there are ready to
be ejaculate. Each spermatozoon has its proper morphology
suggesting structure variations and DNA decays according to the
particularity of the head shape, basis and nucleus abnormalities
Epigenetic alterations should be considered among the
idiopathic male infertility etiologies because hyper- and
hypo-methylation of imprinted and non-imprinted genes in
spermatozoa have been associated with oligo-, astheno-, and/or
cf DNA has been detected in human semen . Interestingly,
its concentration in semen is much higher than in other body
fluids. As reported by Chou et al. , cf-DNA concentration in
semen is related with sperm parameters linked to normal sperm
function such as velocity or morphology. cf-DNA level has been
shown to be higher in seminal plasma of azoospermic than in
that of normozoospermic patients .
Wu et al.  showed the existence of a correlation
between methylation of specific promoters in cell-free seminal
DNA and sperm physiopathology. In their study, methylation
of seminal plasma cf-DNA was shown to be higher in the hypo
spermatogenesis group than in other groups with spermatogenic
defects and normozoospermic patients
As expected, methylation profiles of female and male
gametes are quite different. This parental inequality is known as
genomic imprinting. At the moment of gonads colonization, germ
cells are subject to a massive wave of demethylation, allowing
a discount to zero methylation profiles, in a way similar to the
reboot function of a computer. This phase of reprogramming
is a prerequisite to the acquisition of sex-specific methylation
So, at birth, DNA of male germ cells is already methylated.
Then, remethylation of oocyte DNA occurs in post-pubertal
females. During each ovulatory cycle, the oocyte cohort engaged
towards ovulation acquires female-type methylation patterns.
Consequently, men appear to be more likely to develop
methylation disorders in their sperm when exposed to toxic
products, (neuroendocrine disruption during fetal life . In
contrast, similar effects in women could only occur during adult
life (Figure 3).
The presence of cell-free nucleic acids in human plasma and
their importance as candidate biomarkers were recognized
in the early 1990s after the study published by Sorenson et al.
. They include cell-free DNAs (cf-DNAs) and cell-free RNAs
(cf-RNAs), which comprises messenger RNAs (mRNAs) and
three major small non coding RNAs: microRNAs (miRNAs), piwiinteracting
RNAs (piRNAs) and small interfering RNAs (siRNAs).
Cf-DNAs circulate in the bloodstream following their release
from apoptotic and/or necrotic cells . Circulating cf-DNA
is a non-invasive source of material that can be used to collect
genetic and epigenetic information on these cells . Cf-RNAs
have been detected in many biological fluids. Like cf-DNA, they
can be released by dying cells or actively secreted by living cells.
They also may represent promising sources of material
for assessing the gene expression profile of cells and tissues.
Moreover, it has been demonstrated that extracellular small non
coding RNAs may function as signaling molecules in cell-cell
Infertility is defined as the inability to achieve a clinical
pregnancy after one year of regular and unprotected sexual
intercourse. Worldwide, it affects nearly 15% of couples in age
to procreate and trying to conceive . Male-related causes are
involved in 59% of cases. One third is due to known female and
male causes, whereas the cause cannot be idenfied clearly in the
rest. It can be hypothesized that exposure of infertile women
to risks factors, such as intrafollicular cells apoptosis, oxidative
stress with the generation of immuno-inflammatory factors,
cytoplasmic and mitochondrial DNA damages may provide
increased cfDNA in blood of exposed subjects. The fact that
cfDNA can be obtained without invasive or painful procedures
makes it particularly suitable for studies on infertile women and
cfDNA is present in healthy subjects at concentrations
between 0 and 100ng/ml of blood with an average of 30ng/ml
. Assuming that the DNA content of a normal cell amounts
to 6.6pg, these values represent an average of 0-15,000 genome
equivalents per ml of blood with an average of 5000 genomes per
ml. Most of this DNA is double-stranded and is apparently in the
form of nucleoprotein complex.
Clearance of cfDNA from the bloodstream seems to be rapid.
cfDNA is sensitive to plasma endonucleases.
The biological mechanisms of release of free DNA in blood
are not fully understood. Two main mechanisms have been
proposed: apoptosis/necrosis, or release of intact cells into the
bloodstream and their subsequent lysis. It is common to detect
large, quasi-genome size DNA fragments. Mutant DNA fragments
may originate from necrotic cells that have been engulfed by
macrophages which then release partially digested DNA.
Deoxyribonuclease (DNase) is an enzyme that catalyzes
hydrolytic cleavage of phosphodiester linkages in the DNA. Some
DNases cleave only double-stranded DNA, others are specific for
single-stranded molecules, and sothers are in business to both.
The DNase I preferentially cleaves single-stranded DNA, doublestranded
DNA, and chromatin. DNases II, alpha and beta has the
same function. Recombinant human DNase I is a more frequently
DNase I is produced by recombinant means in an organism
with less endogenous RNase greatly facilitating purification of
an enzyme with no RNase. Dornase alfa: Inhalation solution
(Pulmozyme®: Roche Laboratory) is a sterile, highly purified
solution of recombinant human DNaseI.
The protein is produced by genetically engineered Chinese
Hamster Ovary (CHO) cells containing DNA encoding for the
native human protein, deoxyribonuclease I (DNase). The purified
glycoprotein contains 260 amino acids with a molecular weight
of 37,000 daltons. This DNase is easily obtained with Escherichia
Coli but since the recent introduction of glycosylation system
into Yeast, this last microorganism is more attractive.
Animal studies (rats and non human primates) show a
low percentage of dornase systemic absorption (< 15%) after
inhalation. In human DNase administred to patients as an
inhaled aerosol also shows low systemic exposure. These latter
studies have shown that, following intravenous administration,
DNase was rapidly cleared from the serum. Human intravenous
studies suggest an elimination half life from serum of 3-4 hours.
There was not systemic toxicicity.
The patented invention of Bartoov et al.  was based
on the unexpected finding that high levels of cf DNA present
in men’s blood circulation are associated with subfertility and
that administration of exogenous cfDNA reduces sperm quality
and causes sub-fertility. Moreover they have have found that
providing sub-fertile males with a DNase may improve semen
quality and fertility potential. Czamansky-Cohen et al. ,
in a prospective study, examined the cfDNA concentrations
during ovarian stimulation and the relationship between cfDNA
concentration and pregnancy rates in women undergoing IVFembryo
transfer. Thirty seven women underwent IVF treatment.
cfDNA concentrations were measured by a direct fluorescence
assay, pregnancy rates were identified by plasma β human
chorionic gonadotrophin (HCG) concentrations and verified by
vaginal ultrasound to determine gestational sac and fetal heart
On the day of βHCG test in patients undergoing IVF-ET,
plasma cfDNA concentrations were statistically significantly
higher among women who did not conceive in comparison to
those who conceived. The authors concluded than plasma cfDNA
may reflect the presence of factors which interfere with embryo
The patented invention of Hazout et al.  after several
preliminary studies in serum of healthy and infertile women
and males, found a statistically significant difference in cfDNA
concentrations between fertile and infertile women. The authors
demonstrated a beneficial effect, in terms of ongoing pregnancies
and live births, of the treatment with exogenous DNase, in women
with more than two (2 to 8) implantation failures after previous
transfers of good embryos (more than 50%).
In contrast, Hazout and al. did not find, in a preliminary
study, a significant difference in cfDNA concentration between
two groups of fertile and infertile males. The aim of this latter
study was triple. First, to identify, in unexplained infertile
women and men (unexplained infertility since at least two years
with a history of more than 4 IVF/ICSI with embryos available
for transfer and no pregnancy, the level of cfDNA compared to
fertile women (AMH > 2ng/ml and/or a history of pregnancy).
Secondly, to verify the efficacy of the injection of one vial of
alpha Dornase on the blood level of cfDNA in infertile women.
Third, to treat at least 10 infertile women with a high level of
cfDNA (more than 50ng/ml) twice a day, for the last seven days
of luteal phase in a preceding cycle before ovarian stimulation
for ART and to follow up these women in terms of IVF/ICSI
results and outcomes.
A total of 161 men < 50 were included: 73 fertile ones and
88 infertile ones. The authors first tried to prove the concept
of an association of male infertility with high levels of cfDNA,
assuming the fact that DNA fragmentation might contribute to
In a second phase cfDNA quantity in plasma was measured
in 94 fertile women (AMH>2ng/ml) and in 96 infertile women of
less than 37 years of age. A genomic study was made to verify the
origin of the cfDNA particularly in infertile women (Figure 1) All
the genome was concerned.
When samples were available, cfDNA quantification was
also performed in follicular fluid from both spontaneous and
stimulated cycles in corresponding infertile women. A total of
37 follicular fluid samples were included in this study: most
of the patients were stimulated for ART using a long agonist
protocol or antagonist protocols with recombinant FSH or HMG.
The ovulation was triggered with recombinant or urinary HCG,
36 hours before oocyte retrieval. Each follicular fluid aspirated
frome one, two or three dominant follicles was isolated and
centrifuged before storage. Then cfDNA content was evaluated
using the same method that used for plasma/serum (Figure 2).
DNase activity was first determined using Immunometric
Enzyme Immunoassay kit (Orgentec) 100μl of calibrators (using
separate pipette tips), controls and prediluted patient samples
were loaded into the wells of a microplate. The microplate was
closed (parafilm, sealing foil or lid) and incubated for 60 minutes
at 37 °C. The contents of the microwells were discarded and
washed 3 times with 300μl of wash solution. 100μl of enzyme
conjugate solution was added into each well and incubated for
15 minutes at room temperature. The contents of the microwells
were discarded and washed 3 times with 300μl of wash solution.
100μl of TMB substrate solution were dispensed into each
well and incubated for 15 minutes at room temperature. 100μl
of stop solution was added to each well of the modules and
left untouched for 5 minutes. The optical density was read, no
longer than after 30 minutes after of incubation, at 450nm and
the results were calculated with the use of standard absorption
samples (provided with the kit).
Plasma cfDNA was isolated using high pure PCR
template preparation kit (Roche) following manufacturer’s
recommendation: Elution buffer was diluted to a 20%
solution by using ddH20 and prewarmed at 70 °C. Samples
were centrifuged at 16,000g for 5min, 400μl of plasma were
transfered to a 2ml Eppendorf tube avoiding cellular debris.
400μl of binding buffer and 40μl of reconstituted proteinase K
were mixed to the samples. After a brief vortex, the tubes were
incubated for 10min at 70 °C. After incubation 200μl of 100%
isopropanol was mixed with the samples that were subsequenly
transferred to the upper reservoir of a high pure filter collection
tube provided in the kit. The column was centrifuged at 8,000g
for 1min at room temperature. The flow-through and collection
tubes were discarded and the filter was combined to a new
collection tube. This loading step was repeated until the entire
sample had been loaded to the filter. 500μl of inhibitor removal
buffer were added to the upper reservoir and centrifuged for
1min at 8,000g at room temperature. The flow-through and
collection tubes were discarded and the filter as combined to
a new collection tube. The tubes were washed twice by adding
500μl of wash buffer to the upper reservoir and centrifuged for
1min at room temperature. Columns were dried by centrifuging
at maximum speed (approximately 13,000g) for 10s, transferred
to a new 1.5ml Effendorf tube and warmed for 5min at 70 °C
in an incubator. 100μl of pre-warmed 20% elution buffer was
carefully added to the filter. The tube and filter were placed in
the incubator at 70 °C and shook at low speed (approximately
400rpm) for 5min. DNA samples were eluted from the columns
by centrifuging at 8,000g for 5min and subsequently stored at
4 °C before being used or frozen at -70 °C for long term storage.
PCR amplification: The master mix used to amplify JmJC2 and
DXS1285 loci contained 200mM of each dNTP, 1X Taq polymerase
buffer, 2μM of each primer sets, 1.5mM MgCl2, and 0,5U of
Biotaq™ DNA polymerase (Bioline) in a 15μl reaction volume.
The sequences of primers used to amplify JmJC2 (marker of Y
chromosome) and DXS1285 (marker of X chromosome) are
5’-GAACTGACTGTAGAGAAGG-3’, respectively, with a 60 °C
Blood samples were collected in EDTA-containing vacutainer
tubes. They were centrifuged (3,400rpm for 15 minutes) for
plasma isolation. Before cfDNA quantification, plasma and
follicular fluid sample were centrifuged at 3,400g for 20min.
Samples have to be transparent with no red blood cells. Indeed
cfDNA quantification can be altered in couloured coloured
samples. Standard DNA solution was diluted to 20, 50, 100 and
500ng/ml in 166μl to draw the standard curve. 166μl of 1N HCLO4
(Perchloric acid) and 664μl of diphenylamine were added to each
166μl of plasma or follicular fluid supernatant samples. Samples
were incubated at 37 °C for 20h, subsequently centrifuged at
15,000g for 10 minutes. 300μl of the supernatant was transferred
to a 96-well plate and measures in spectrophotometer (Tecan,
Genios) at 600nm.
The mean cfDNA value in the group of fertile men was 60,6ng/
μl and 83,34ng/μl in the infertile group, a difference which was
not statistically significant. The mean cfDNA value in the group
of fertile women was 49,2ng/μl versus 98,5ng/μl in the infertile
group, which was statistically significant (Figure 4).
Comparing cfDNA in the plasma and Follicular Fluid (FF)
of 42 patients we demonstrated that mean cfDNA value in the
FF was lower than plasma cfDNA value in the corresponding
patients. As to the cfDNA, in plasma and FF, levels with regard
to the outcome of 32 FIV/ICSI patients either after ovarian
stimulation or spontaneous cycles (Figure 5&6) we found that:
a) the patients number 5, 9, 15, 17, 19, 24, were pregnant
b) The majority (6/6) had low levels of CFDNA in the
plasma and FF (less than 60ng/microliter)
c) the patients 11, 16, 21 deplored miscarriages (for
probably male reasons?) with normal plasma and FF CFDNA
levels (< 50)
d) the patient 12 had a normal plasma CFDNA level (46,)
but a very high FF CFDNA level (242,7ng/μl) with only one
oocyte from three punctured follicles and no fertilization.
e) six patients were monitored and punctured on
spontaneous cycles after several standard FIV/ICSI failures:
patients number: 1,2,4,15 ,22,23; with respectively 72,5;
27,4; 11,3; 43,5; 30,3;8,5ng/μl in the plasma and 72; 27;11;
53,6; 30,3; 163,1ng/l in the FF.
Patients 1 and 4 had no oocyte (1 with high levels in the
plasma and FF, the other with normal level). Patient 15 was
pregnant and delivered (with normal levels in the plasma and
FF) (Figure 7).
We selected 30 patients with several FIV/ICSI failures but 27
with a high levels of cfDNA in their plasma and 3 with normal
levels (less than 50ng/μl) and then we decided to treat 10
patients among the two groups with the highest cfDNA plasma
levels (Table 1).
All these patients were young women (less than 37) infertile
since 4 years and/or had an history of several ART failures
without obvious explanation. All of them were treated with one
intramuscular injection, twice a day, of DNAse I (5000IU/day) for
seven days in the late luteal phase of the cycle preceding ovarian
stimulation and IVF/ICSI (Table 2). From four patients treated in
Algeria 3 were pregnant and delivered. From 6 patients treated
in Tunisia 3 were pregnant (2 live births and one abortion).
Based on a few studies exploring the relationship of cfDNA
and outcome of ART in infertile couples, and in the light of our
experience, we suggest that there are more failures in assisted
reproduction when the level of blood cfDNA is in excess in
women and / or men.
Boissière A. et al.  showed that little has been clarified
about circulating nucleic acids in spermatogenesis, and in male
infertility and provided a complete summary of the consistent
data on circulating nucleic acids and intracellular miRNAs in
Traver S et al.  demonstrated that follicular fluid cfDNA
level was an independent and significant predictive factor
for pregnancy outcome (adjusted odds ratio=0.69 [0.5;0.96],
p=0.03). In multivariate analysis, the Receiving Operator Curve
(ROC) analysis showed that the performance of FF cfDNA in
predicting clinical pregnancy reached 0.73 (0.66-0.87) with
88% specificity and 60% sensitivity. The authors concluded
that CfDNA might constitute a promising biomarker of follicular
micro-environment quality which could be used to predict
IVF prognosis and to enhance female infertility management.
However, the main limitation of this study was that all FF samples
from the same patient were pooled.
A previous study did not find a statistically significant
difference in cfDNA concentrations comparing women who
conceived in IVF and those who did not. However, in this latter
study the determination of cfDNA was performed one week after
embryo transfer. Czamanski-Cohen et al.  demonstrated an
increase of plasma cfDNA associated with low pregnancy rates
among women undergoing IVF-Embryo transfer.
In our study all the pregnant women had a decrease of cfDNA
after DNase therapy. The only abortion is probably due to the bad
quality of one testicular sperm from an obstructive azoospermic
man. In the non-pregnant group the fall of cfDNA was moderate
suggesting that the dose of DNase I must to be increased in
certain cases with very high levels of cfDNA.
Our clinical results are encouraging and raise a question of
the real DNase I mode of action. The decrease in apoptotic factors
in the plasma, may, promote a better oocyte competence, but also
a better endometrial receptivity. All women treated deplored
more than 4 years of unexplained infertility or many embryo
transfers, at early stage of development, without implantation.
Liu and Li (2010) looked at the relationship between apoptosis in
granulosa cells and IVF–embryo transfer success and speculated
that oxidative stress in granulosa cells had an effect on IVFembryo
transfer failure, and also connected the higher apoptotic
rate to lower oocyte quality.
Díaz-Fontdevila et al.  examined the apoptotic rate of
cumulus cells in relation to infertility diagnosis and spermatozoa
exposure and found that cumulus cells of women with a diagnosis
of endometriosis and those exposed to spermatozoa had higher
rates of apoptosis. Protein p53 was involved in initiation of
apoptosis Choisi-Rossi et al.  and implantation failure .
Given the large number of failures in ART, our new and
effective approach seems to be highly promising.
Cell-free DNA has also been detected in human semen.
Interestingly, its concentration in semen is much higher than
in other body fluids. As reported by Chou et al. seminal cfDNA
(scf-DNA) is associated to sperm parameters linked to normal
sperm function such as velocity or morphology. Sperm cf-DNA
(scfDNA) level has been shown to be higher in seminal plasma of
azoospermic than normozoospermic patients.
These observations suggest that scf-DNA could be used
in a search for biomarkers of sperm quality. Otherwise, it has
been hypothesized that epigenetic alterations could cause male
infertility. The presence of epigenetic information has been
shown on seminal cfDNA  This epigenetic information should
reflect testicular epigenetic aberrations as semen is a mixture
of secretions from the two testes, epididymes, seminal vesicles,
bulbo-urethral glands and prostate. Indeed, Wu et al. 
showed also the existence of a correlation between methylation
of specific promoters (such as CCNA1 and DMRT1) in cell-free
DNA seminal and sperm physiopathology. In that study, the cf-
DNA methylation of these promoters was shown to be higher
in the hypospermatogenesis group than in other groups with
spermatogenic defects and normozoospermic patients.
These findings indicate that seminal cfDNA contains the
epigenetic information of the male genital tract and could be
a novel, non-invasive biomarker to detect spermatogenesis
abnormalities. The mice mutants for the process of germinal
methylation are sterile in both sexes; this demonstrates
the essential character of methylation of gametes for the
reproduction. The absence of methylation induces complete
arrest of spermatogenesis well upstream . The females
suffer from systematic spontaneous abortions of embryos from
fertilization of their oocytes devoid of methylation. So while the
methylation of DNA has an immediate function for the integrity
of male gametes, it does not really matter for the oocyte, but
conditions the viability of the future embryos.
Embryos lacking maternal methylation suffer from
expression disorders of genes essential for development in
utero; hence the systematic miscarriage occurs at the moment
when the embryo becomes dependent of maternal supply via the
So, oocytes and spermatozoa ensure the continuity of the
species by delivering to the next generation a genetic capital
and a reversible epigenetic inheritance. DNA methylation, in
particular, confers intrinsic properties of stability and heritability
allowing its transmission from parental gametes to the embryo,
« Methylation helps to give life and can take it away. In reality,
without methylation, there would be no life at all [29-36].
Excess free nucleic acids in the serum of infertile couples
seems to be a marker of their decreased ability to conceive even
if mechanical causes are excluded. The methylation status of
these free DNA fragments adds an additional, non-invasive factor
of predictability. This need to be confirmed in larger studies as
much as therapeutic solutions are available to change the fate of
male and female gametes.