Vol.2, No.9, 1010-1017 (2010) Health
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Beneficial effect of reduced oxygen concentration with
transfer of blastocysts in IVF patients older than 40
years old
Javier I. García*, Soledad Sepúlveda, Luis Noriega-Hoces
Laboratory of Assisted Reproduction, Concebir Clinic, Lima, Peru; *Corresponding Author: jgarciaf@hotmail.com
Received 6 February 2010; revised 1 March 2010; accepted 5 March 2010.
The aim of the present study was to determine
the impact of oxygen concentration on implan-
tation, pregnancy and delivery rates in IVF pa-
tients older than 40 year old with transfer of
blastocysts. Included were 558 women aged
23-45 years old undergoing IVF/ICSI procedures
whose embryos were cultured at blastocyst
stage under two different oxygen environments
(a bi-gas system: 5.6% CO2 in air and a tri-gas
system: 5.6% CO2, 5% de O2 and 89.4% N2). The
main outcome measures of this study are im-
plantation, pregnancy and delivery rates. Im-
plantation, pregnancy and delivery rates are
found to be reduced in women older than 40
years old. The implantation and pregnancy rates
are significantly higher in women older than 40
years old from the 5% of O2 group, in compari-
son to the 20% group (25.00% versus 2.70% and
41.38% versus 5.56%; P < 0.05). The deliveries
rates were 13.79% and 5.56% in the 5% and 20%
oxygen groups respectively (P: NS). The birth-
weight was similar in both study groups (P: NS).
Gestational age was significantly longer in wo-
men from the 5% of O2 group, in comparison to
the 20% (36.87 versus 35.87 weeks, P < 0.05).
Results indicated that the embryonic culture
with 5% of oxygen and transfer of blastocysts in
women older than 40 years old improve the re-
sults in the in Vitro fertilization/intracytoplasmic
injection procedures (IVF/ICSI).
Keywords: ART; Blastocyst; IVF; ICSI; Oxygen
Embryos from several mammal species, including hu-
man, were exposed in vivo to low oxygen concentrations,
ranging from 2 to 8% observed in atmospheric air [1-3].
This probably corresponds to an adaptation mechanism,
as it is proven that higher oxygen concentrations may be
harmful to the embryo [4] by generating reactive oxygen
species (ROS) [5-7].
In Vitro fertilization studies (IVF) in mice [8,9]; cattle
[5]; sheep [10]; rabbits [11]; hamsters [12]; rats [13];
cows [14] and pigs [15] have demonstrated that when
cultured in oxygen concentrations of 5% present a higher
viability and a better development to the blastocyst stage.
However, a pioneer study in human embryos showed
that cultures in Vitro in atmospheric concentration (20%)
or reduced (5%) resulted in similar fecundation and
preimplantational embryo development processes [16].
Therefore, in several laboratories of assisted reproduc-
tion, the culture of human embryos using oxygen con-
centration of 20% [17] is now a common practice.
Furthermore, a study in which the effect of oxygen
over the 2nd and 3rd day of human embryo development
was evaluated was unable to find any differences in the
pregnancy and implantation rates when 5% or 20% of O2
was used [17]. This absence of differences in the results
obtained might be because the beneficial effect of low
O2 concentrations happen during the late stages of pre-
implantational embryo development (day 4-6) [18]; how-
ever, the addition of antioxidants to the culture media
had as a result better rates of implantation and pregnancy
when embryos cultured in 5% of O2 are transferred in
the 2nd and 3rd day [5].
The importance of the transfer in blastocyst stage and
the concentration of oxygen have been recently recog-
nized [19,20] These studies reported significant in-
creases in the pregnancy and implantation rates when
transfers were done in blastocyst stage and when the
cultures were made with 5% of O2 compared to the cul-
ture effect in conditions of 20% of O2.
Physiologically, the uterus provides a nutritional en-
vironment different from than in the fallopian tubes.
Therefore, embryo transfer in the cleavage stage would
J. I. García et al. / HEALTH 2 (2010) 1010-1017
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cause homeostatic stress of the embryo and a reduction
in its implantatory potential [21]. Consequently, the tr-
ansfer in blastocyst stage would allow a better synchro-
nization with rhythm of uterine contractions and the em-
bryo [22,23].
However, there are contradictory results in studies
where the human embryos cultured in reduced (5%) or
atmospheric oxygen concentrations (20%) are compared.
Thus, no improvements in terms of pregnancy and im-
plantation rates were observed if the transfer was per-
formed in the 3rd day of the development in terms of
pregnancy and implantation rates [17,20,24]. Similar
results have been observed if transfer was done into
blastocyst stage (day 5) [24-27].
The maternal age is an important factor to be taken
into account in studies on. In fact, there has been a de-
crease in the women’s fertility from 35 years old [28,29],
being this reduction significant from the 40 year olds
and over, in women attending a processes of assisted
reproduction [30,31].
The Latin American Registry of Assisted Reproduc-
tion (REDLARA) reported in 2006 a clinical pregnancy
rate of 39.6% in patients 34 years old, 32.8% in pa-
tients from 35 to 39 years old and 18.6% in women 40
years old respectively [32]. In older women there is
commonly a reduction in the ovarian follicular reserve
and a greater prevalence of chromosomal alterations in
the oocyte, which lead to a significant reduction in the
implantation rates [33,34] and high rates of miscarriages
In the studies comparing the effect of different oxygen
concentrations in the embryo cultures, it has not been
taken into consideration the maternal age impact when
blastocysts are transferred in the programs of assisted
reproduction [19,25,27,37].
In a recent publication Kovačič et al. [26] did not find
improvements in the implantation rates in older women
over 40 years of age whose embryos were cultured with
oxygen at 5% as compared cultures at 20% of O2 and
embryo transfer in the 3rd day. It is possible that effects
of low oxygen concentration may be observed if em-
bryos are transferred in the 5th or 6th days.
We hypothesize that reducing the percentage of oxy-
gen to 5% in the embryo culture systems would have a
much more beneficial effect than the usage of oxygen at
The objective of this study was to evaluate in an
IVF/ICSI program, the relationship between the preg-
nancy and the implantation rates with maternal age
whose embryos were cultured in 5% of O2, compared to
those women whose embryos were cultured at 20% of
O2. In addition, the results of pregnancies were assessed.
2.1. Patients
This is a retrospective non randomized study based on
secondary analysis of data obtained from 558 cycles of
IVF and ICSI at the Laboratories of Assisted Reproduc-
tion of Pranor Group (Lima, Peru) between January
2007 and June 2009. This study was approved by the
Institutional Review Board (IRB) at the Concebir Clinic
(Lima, Peru).
The study group were those gametes and embryos
cultured at 37˚C in an atmosphere of 5.6% CO2, 5% of
O2 and 89.4% N2 (341 cycles); and a control group of
those gametes and embryos cultured at 37 and an at-
mosphere of 5.6% CO2 in air (20% O2) (217 cycles). The
same kind of incubators (Thermo Scientific, USA) was
used for the bi-gas and tri-gas systems.
2.2. Ovarian Stimulation and Oocyte
The patients were submitted to a controlled ovarian
stimulation with Leuprolide Acetate (Lupron®, Abbott
Laboratories) or Ganirelix (Orgalutran®, Organon) in
combination with Recombinant FSH (Puregon®, Or-
ganon Laboratories) or HMG (Humegon®, Organon
Laboratories) according to the established protocols. The
follicular growth was monitored by ultrasound and the
ovulation was induced by applying Human Chorionic
Gonadotropin (hCG) (Ovidrel® 250 ug, Serono Labora-
tories or Pregnyl® 10,000 UI, Organon Laboratories).
The follicular aspiration was made 34 to 36 hours after
giving the hCG. The insemination or ICSI procedure was
made-5 hours after the oocyte recovery.
2.3. Semen Samples
The semen samples were obtained by masturbation of
every patient’s male in aseptic conditions. After the liq-
uefaction process, the motile spermatozoa were recov-
ered from the seminal plasma by centrifugation through
Isolate gradients of 45% and 95% (Irvine Scientific,
USA) for 10 minutes at 300 × g. the recovered sper-
matozoa were washed in Sperm Washing Media (Irvine
Scientific, USA). In oligospermic samples the sper-
matozoa were washed in Sperm Washing Media and
then placed in 10 µL drops of HTF-Hepes + 10% SSS
for the ICSI.
2.4. Fertilization and Embryo Culture
In each one of the evaluated groups, the embryo culture
media and mineral oil were prepared and used according
to the specifications of the company. The CO2 concen-
tration in the incubators was of 5.6% and resulting pH
J. I. García et al. / HEALTH 2 (2010) 1010-1017
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was approximately 7.30 in all the culture media.
The aspired oocytes were washed in a HTF-Hepes
medium (IVFonline, Guelph, ON, Canada) supplement-
ed with 10% vol/vol of SSS (Irvine Scientific, USA) and
cultured in a 200 µL drop of HTF medium + 10% SSS
under mineral oil at 37˚C for 5 hours before the insemi-
nation or ICSI procedure.
The insemination was made with 50,000-100,000 mo-
tile spermatozoa in 200 µL drop of HTF medium + 10%
SSS, where from 1 to 5 oocytes were placed. In the cases
of ICSI, the oocytes in metaphase II were injected in
every patient by using methods previously described
(38). After the insemination or ICSI in 0 day, all the oo-
cytes were cultured up to the evaluation of the fertiliza-
tion at 37˚C.
The fertilization was evaluated 16-18 hours post in-
semination or ICSI by the presence of two pronuclei and
two polar bodies (day 1). The zygotes with two pronu-
clei were cultured individually, under mineral oil, in 10
µL drops of Global medium (IVFonline, Guelph, ON,
Canada) supplemented with 10% vol/vol of SSS (Irvine
Scientific, USA) from day 1 to day 3. On the 3rd day,
the embryos were changed to 10 µL drops of fresh
Global medium + 10% SSS and cultured 2 or 3 days
more up to the transfer day in blastocyst stage. Therefore,
the transfer was made in 5 or 6 days.
2.5. Embryo Transfer
The embryos were transferred in blastocyst stage, being
the average of 1.96 and 2 embryos transferred in the
group of 5% and 20% of O2 respectively (P < 0.05
among the evaluated groups). In the 5% of O2 group, 16
patients received 1 embryo, 324 received 2 embryos and
1 patient received 3 embryos. In the 20% of O2 group, 4
patients received 1 embryo, 209 patients received 2 em-
bryos and 4 patients received 3 embryos (Table 1).
The embryos that were not transferred were cryopre-
served or eliminated according to their morphology. The
embryo transfer was made with a Frydman Ultrasoft
catheter (CCD Laboratoire, Paris, France) that was pre-
viously washed with a culture medium. The catheter was
completely filled with culture medium and the embryos
filled in the last 10 µL of the catheter medium. All the
transfers were made according to the methods previously
described by Mansour [39].
The biochemical pregnancy was determined approxi-
mately 12 to 14 days after the embryo transfer by meas-
uring the Human Chorionic Gonadotropin beta subunit
(hCG-b) in blood. The clinical pregnancy was deter-
mined by the presence of the gestational sac and the
heart beat which were evaluated by ultrasound at the
21st and 28th days post transfer respectively.
2.6. Statistical Analysis
Data were statistically analyzed using the χ2 test and
Student’s t-test as appropriate and differences were con-
sidered to be significant at P < 0.05. All statistical analy-
sis was carried out using the statistic package Stata 10
(StataCorp, College Station, TX).
In this study, the cycles were organized in 3 segments
according to the age of the patient: < 35 years old, 35-39
years old and 40 years old. The normal fertilization
rate was calculated from the number of zygotes with two
pronuclei of IVF and ICSI divided by the number of
mature oocytes inseminated by 100. The rate of implan-
tation was calculated dividing the number of gestational
sacs observed by ultrasound at the 21st day post transfer
divided by the total number of embryos transferred by
100. The rate of clinic pregnancy was calculated from
the number of patients with at least one gestational sac
divided by the total embryo transfers by 100. The abor-
tion rate was defined as the number of pregnancies with
total loss of the gestational sacs before the 20 weeks of
gestation between the numbers of pregnancies by 100.
A total of 558 cycles in which the embryos were cultured
under two different O2 environments were evaluated;
embryos of 341 and 217 cycles were cultured in 5% and
20% of O2 respectively. The age of the patients was
similar in both evaluated groups (P: NS). The fertiliza-
tion rate was similar in the 5% of O2 group versus the
20% in each age group evaluated in this study (Table 2).
Table 1. Characteristics of the two study groups whose em-
bryos were cultured in 5% or 20% of O2.
5% O2 20% O2
Cycles 341 217
Age (y)
Range 25-45 23-45
Mean ± SE 34.47 ± 0.20 34.33 ± 0.26
Tubal Factor 37 (11) 16 (7)
Other female 161 (47) 95 (44)
Male Factor 48 (14) 29 (13)
Multiple Factor 88 (26) 72 (33)
Unexplained 7 (2) 5 (3)
Procedure Class a
Standard IVF 194 (57) 96 (44)
ICSI 147 (43) 121 (56)
Data are Mean ± Standard Error; aValues in parentheses are percentages of
the total number of patients; P: NS.
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Table 2. Implantation rate, pregnancy rate, abortion rate and birth rate by age group in cycles with embryos cultured in at-
mosphere of 5% and 20% of oxygen.
< 35 years 35-39 years 40 years
5% O2 20% O2 5% O2 20% O2 5% O2 20% O2
Cycles 169 108 143 91 29 18
Fertilization rate (%) 83.03 79.78 84.04 78.16 83.52 81.60
Transferred embryos 1.98 ± 0.01 1.99 ± 0.02 1.93 ± 0.02b 2.00 ± 0.02 1.93 ± 0.05a 2.06 ± 0.01
Implantation rate (%) 34.63 35.81 26.09 26.92 25.00a 2.70
Clinical Pregnancy rate (%) 50.89 51.85 42.66 42.86 41.38a 5.56
Abortion rate (%) 5.92 7.41 7.69 10.99 17.24c 00.00
Birth per transfer rate (%) 42.59 44.44 32.12 31.87 13.79 5.56d
Ongoing pregnancy 7 0 6 0 3 0
Data are Mean ± Standard Error; aP < 0.05 compared to the average in patients 40 years old from the 20% O2 group; bP < 0.05 compared to
the average in patients of 35-39 years old from the 20% O2 group; cP < 0.05 compared to the average in patients of < 35 years old from the
5% O2 group; dP < 0.05 compared to the average in patients of < 35 years old from the 5% and 20% O2 group.
The patients 40 years old from 5% group of O2 had
implantation and pregnancy rates significantly higher
compared to those patients from 20% of O2 group (P <
0.05). Furthermore, these patients older than 40 years
old from the 5% of O2 group received a significantly less
number of embryos transferred, compared to the patients
from 20% group (P < 0.05). The group of patients < 35
years old and of 35-39 years old had similar implanta-
tion and pregnancy rates (P: NS).
Women 40 years old from 5% of O2 group had a
higher abortion rate compared to women < 35 years old
from the same study group (P < 0.05). However, in the
group of patients whose embryos were cultured in 20%
of O2, the older women ( 40 years old) had a lower
delivery rates in comparison to women < 35 years old in
both evaluated groups (5% and 20% of O2) (P < 0.05). In
women 40 years old from 20% of O2 group there was
only 1 pregnancy out of 18 transfers, which resulted in a
healthy born baby. In the 5% of O2 group there were 12
pregnancies from which 5 women had an abortion before
the 20th week of gestation, 4 had a normal delivery and
3 pregnancies have a normal development. The delivery
rate was similar in women older than 40 years of age in
the 5% of O2 group compared to the 20% of O2 group
(13.79% vs. 5.56%; P > 0.05).
The pregnancy rates according to the kind of proce-
dure of IVF or ICSI were similar among both procedures
in the evaluated groups (5% and 20% of O2) in women <
35 years old, 35-39 years old and older than 40 years old.
Less pregnancies were achieved in women 40 years
old when their embryos were cultured in a 20% of O2
atmosphere, independently from the kind of procedure of
IVF or ICSI, in comparison to the group of women < 35
years old and of 35-39 years old (P < 0.05) (data not
The percentage of embryos that reached the blastocyst
stage in relation to the total number of fertilized oocytes
is show in Table 3. There were no differences in the
embryonic blastulation rate among patients from the 5%
and 20% of O2 group; these percentages were equally
similar in relation to the age of the patients. Furthermore,
there was no difference in the pregnancy rates according
to the kind of controlled ovarian stimulation in both
evaluated groups in this study (Table 4).
The data about the gestational age at delivery and
birth weight from both groups evaluated in this study is
shown in Table 5. There were 87 deliveries in the 5% of
O2 group and 78 deliveries in the 20% of O2 group.
However, it was only possible to register information
from 39 and 60 in each group respectively. Gestational
age was higher in women whose embryos were cultured
in reduced concentrations of oxygen, in comparison to
those women whose embryos were cultured in atmos-
pheric concentrations of oxygen (P < 0.05) (Table 5).
Table 3. Blastulation rate according to age in both study groups.
5% O2 20% O2 P
Cycles 341 217
No. of embryos (2PN)2281 1612
< 35 years old 39.82% 37.59% 0.487
35-39 years old 36.11% 39.69% 0.348
40 years old 34.01% 31.37% 0.756
Total 37.97% 37.97% 1.000
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Table 4. Pregnancy rate according to the protocol of ovarian
stimulation with agonist or antagonist from the GnRH (GnRHa
–GnRHant) and the recombinant FSH (rFSH) or human
menopausal gonadotropin (HMG) in the study groups.
Stimulation Protocol 5% O2 20% O2
GnRHa + rFSH 60.00% 36.84%
GnRHa + HMG 50.00% 40.00%
GnRHa + rFSH + HMG 45.45% 53.33%
GnRHant + rFSH 50.43% 41.67%
GnRHant + HMG 55.00% 50.00%
GnRHant + rFSH + HMG 48.21% 60.87%
rFSH 27.27% 43.75%
HMG 57.14% 40.00%
rFSH + HMG 35.71% 38.89%
Table 5. Gestational age and birth weight in the study groups.
5% O2 20% O2 P
Biochemical pregnancy 5 4
Clinical pregnancy 159 96
Total delivery 87 78
Deliveries with recorded data 39 60 < 0.05
Gestational age
(weeks) (Mean ± SE)a 36.87 ± 0.30 35.87 ± 0.37
Bithweight of newborns
(gr.) (Mean ± SE)a 2816.26 ± 92.88 2752.96 ± 69.05NS
aData are Mean ± Standard Error
Although human embryos can develop successfully in
atmospheric concentrations of oxygen (20%), some au-
thors have suggested that low oxygen concentrations
(5%) resemble the physiological conditions of the uterus
effectively, and thereby improve the quality, viability
and embryo morphology [40,41].
An important result of this study was to find that cul-
turing embryos at reduced concentrations of O2 (5%) is
beneficial to patients 40 years old who perform as-
sisted reproductive procedures with their own oocyte;
these are the ones who achieve significantly higher im-
plantation and pregnancy rates compared to those pa-
tients of similar age whose embryos were cultured under
atmospheric oxygen concentrations (20%) (25.00% ver-
sus 2.70%; 41.38% versus 5.56%, respectively, P < 0.05).
The implantation and pregnancy rates observed in women
> 40 years old were similar to those seen in women < 35
years old and 35-39 years old (P:NS).
Meintjes et al. (20) embryos cultured in a 5% O2 en -
vironment consistently resulted in higher rates of live
birth implantation and live births when compared with
rates among women whose embryos were cultured in an
atmospheric O2 environment.
Furthermore, Nanassy et al. [27] cultured human em-
bryos under oxygen atmospheric conditions (20%) until
the 3rd day and then cultured the embryos in 5% and
20% of O2 from the 3rd to the 5th day of development
without finding beneficial effects of 5% of O2 in the ad-
vanced stages of preimplantational embryo development.
These results suggest that the beneficial effect of hy-
poxia on embryonic development would be along all
stages of in vitro cultures even from the oocyte before
fertilization up to the blastocyst stage [42], which had
previously been observed in mice [8,43] cattle [44], rab-
bits [45] and pigs [46].
Culturing embryos in 20% of O2, Karagenc [7] showed
damage mainly in the embryonic inner cell mass (ICM).
Similarly, Rhesus monkey embryo cultured in vitro in
20% of O2 showed the ICM morphologically disorgan-
ized, diffuse, with few vacuolated cells, unlike the blas-
tocysts with large and compact ICM cultured in low
concentrations of O2 [47].
Rho et al. [48] culturing bovine embryos has shown
that low concentrations of oxygen produce higher rates
of cleavage and blastocyst stages compared to embryos
cultured in 20% of O2. Also in mice there is a better de-
velopment to blastocyst stage, bigger number and size of
ICM, and gene expression profile similar to those ob-
served in embryo in vivo [49].
The discrepancies between the data obtained in ani-
mals and humans could be explained based on the dif-
ferences in the embryo physiology of each species and a
variety of culture conditions and embryo transfer in the
laboratory [47]. Beneficial effects of culture in 5% of O2
have been demonstrated in animals where embryo trans-
fers routinely occur in blastocyst stage [7,50].
Dumoulin et al. [18], Pabon et al. [8], Quinn and Har-
low [43] suggested that the beneficial effect of O2 in
physiological concentrations should be observed in cul-
tures extended to blastocyst, which are nowadays com-
mon in assisted reproduction laboratories.
Several studies report increases in pregnancy and im-
plantation in blastocyst transfers compared to embryo
transfers on the 3rd day [51] and others report significant
increases only in implantation rates [52], which consid-
ering the results of this study, would have beneficial ef-
fects in those patients over 40 years old.
The culture up to the blastocyst would allow to choose
J. I. García et al. / HEALTH 2 (2010) 1010-1017
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in a more “natural” way embryos with greater potential
for development and implantation, however, this selec-
tion would also depend on the O2 percentage in the sys-
tems in which the embryos are cultured [47] and the oo-
cyte origin associated to the patient’s age [53,54].
There are many authors who have showed the rela-
tionship between chromosomal abnormalities, maternal
age and embryo morphology [35,55-59]. Munné et al.
[54] performed genetic diagnosis for 9 chromosomes
[13,15-18,21,22, X, Y) in > 6000 embryos and found
that women < 35 years old with good quality embryos
had 44% of euploid embryos and that this percentage
decreased to 21% in patients 41 years old. Beside, in
patients with poor morphology embryos only 30% and
12% were euploid embryos in the group of women > 35
years old and 41 years old respectively. It has also been
shown that chromosomal abnormalities in human oo-
cytes are common and that these aneuploidies are closely
related to maternal age, exceeding 60% in women over
40 years old [60].
Since older women have a higher incidence of oocytes
and embryos with aneuploidy [54,60] but with similar
rates of embryonic blastulation than young women ( 35
years old, 35-39 years old; see Table 3) as it has been
demonstrated in this study, we should expect similar
pregnancy and implantation rates independently from the
oxygen concentration under which cultures in vitro are
performed, but in this study we found that low concen-
trations of oxygen (5%) are achieved significantly higher
pregnancy and implantation rates in patients 40 years
old, compared to the results in conditions of atmospheric
concentrations of oxygen.
These results might be caused by high concentrations
of oxygen that would affect the embryonic inner cell
mass [7], an effect that would be even more noticeable
because of the high incidence of aneuploidy observed in
oocytes and embryos in older women.
In this study there were 87 births in the 5% of O2
group and 78 births in the group of 20%, being able to
obtain information of their results, in terms of gesta-
tional age and birthweight, in 39 and 60 cases respec-
Within the data obtained, a significantly higher gesta-
tional age was observed in the group of patients whose
embryos were cultured under 5% O2 compared to the
20% of O2 group (36.87 ± 0.30 vs. 35.87 ± 0.37; P <
0.05) which could be a consequence of preimplantational
embryo development in more physiological concentra-
tions of oxygen to which the embryos were subjected
during in vitro culture, but we believe that further studies
are needed to determinate the possible relationship be-
tween the culture in hypoxic conditions and obstetric
characteristics of pregnancies.
The authors thank to Gustavo F. Gonzales M.D, Ph.D. for his valuable
help and his review of this manuscript.
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