ARTICLE IN PRESS

Relationship of reactive oxygen species levels in day

3 culture media to the outcome of in vitro fertilization/
intracytoplasmic sperm injection cycles
Mohamed A. Bedaiwy, M.D., Ph.D.,a Reda Z. Mahfouz, M.D.,b Jeffrey M. Goldberg, M.D.,b
Rakesh Sharma, Ph.D.,b Tommaso Falcone, M.D.,b Mohamed F. Abdel Hafez, M.S.,b and Ashok Agarwal, Ph.D.b
a
Department of Obstetrics and Gynecology, University Hospitals Case Medical Center, Case Western Reserve University; and
b
Center for Reproductive Medicine, Glickman Urological and Kidney Institute, & Ob-Gyn and Women’s Health Institute,
Cleveland Clinic, Cleveland, Ohio

Objective: To examine the relationship of early human embryonic development parameters to day 3 reactive
oxygen species (D-3 ROS) levels in culture media.
Design: Prospective study.
Setting: Tertiary care hospital.
Patient(s): Patients were undergoing IVF (n ¼ 92; 36 with intracytoplasmic sperm injection [ICSI]).
Intervention(s): The D-3 ROS levels in sample and control of each embryo culture dish were measured by the
chemiluminescence method using a luminol probe.
Main Outcome Measure(s): Embryo quality (days 3 and 5) and pregnancy rates (PR).
Result(s): The D-3 ROS level was significantly lower in pregnant cycles 26.8  13.9  106 cpm (counted photon
per minute) versus nonpregnant cycles 66.4  39.4  106 cpm. This relationship was maintained when the cycles
were stratified to conventional IVF (27.1  14.95 vs. 67.0  39.9  106 cpm) or ICSI (25.6  12.75 vs. 65.5 
39.7  106 cpm). After controlling for all variables, D-3 ROS levels were negatively correlated with blastocyst de-
velopment rate as well as PR. Odds ratio (OR) (95% confidence interval [CI]) of clinical pregnancy corresponding
to a 10  106 cpm increase in D-3 ROS was 0.47 (0.30–0.74) for ICSI and 0.56 (0.37–0.85) for IVF.
Conclusion(s): During extended in vitro culture, ROS generated in culture media by day 3 may be an important
biochemical marker for blastulation. An increase of 10 units in D-3 ROS may decrease the clinical pregnancy
by 41%. (Fertil Steril 2010;-:-–-. 2010 by American Society for Reproductive Medicine.)
Key Words: Reactive oxygen species, fertilization, blastulation, ICSI, IVF, extended embryo culture

The evaluation and selection of embryos for fresh uterine transfer or
cryopreservation is largely based on their morphology. Ideally, se-
lection of normal viable embryos with high implantation potential
would be based on chromosomal integrity with expression of the ap-
propriate developmental genes and normal metabolic function. The
metabolic alterations of developing preimplantation embryos re-
mains poorly understood (1–4). At present, specific early embryonic
molecular markers indicative of optimal development are yet to be
identified (5, 6). We identified reactive oxygen species (ROS) and
total antioxidant capacity in day 1 culture media as possible meta-
bolic markers of in vitro embryonic development (4, 7). Both
markers, at certain levels, were significantly correlated with in vitro
embryonic development parameters, as well as clinical pregnancy.
Oxygen species has also been shown to be involved in the etiol-
ogy of defective embryo development and embryonic fragmenta-
tion. The ROS generation may be from embryo metabolism or
embryo surroundings (8, 9). Although the embryo has several mech-
anisms of defense against ROS (10, 11), rapid embryonic develop-
ment continues to generate more ROS (4). The exact role of
oxygen species in early embryonic development has yet to be fully
identified.
Several media that improve embryo culture to the blastocyst stage
are available. Day 5 embryo transfer at the blastocyst stage permits
full expression of the embryonic genome, which occurs after the
eight-cell stage. Earlier transfer at the cleavage stage may cause un-
expressed genetic defects to go unnoticed (12, 13). High implanta-
tion rates have been reported for blastocyst transfer by several
investigators (14–18).
This study was designed to quantify ROS in the embryo culture
media 3 days after oocyte retrieval (day 3 media) in conventional
IVF and intracytoplasmic sperm injection (ICSI) cycles and to as-
sess the relationship of day 3 ROS (D-3 ROS) levels to embryo qual-
ity on day 3 and 5, and pregnancy rates (PR) for patients undergoing
day 5 embryo transfer.

Received August 2, 2009; revised December 3, 2009; accepted December MATERIALS AND METHODS
8, 2009.
This study was approved by the Institutional Review Board (IRB) of
M.A.B. has nothing to disclose. R.Z.M. has nothing to disclose. J.M.G. has
nothing to disclose. R.S. has nothing to disclose. T.F. has nothing to dis-
the Cleveland Clinic. A total of 92 cycles (56 IVF cycles and 36 ICSI
close. M.F.A.H. has nothing to disclose. A.A. has nothing to disclose. cycles) in 91 patients between 2000 and 2001 were included.
Presented in part at the 63rd Annual Meeting of the American Society for
Reproductive Medicine, October 13–17, 2007, Washington, DC.
Reprint requests: Ashok Agarwal, Ph.D., Director, Center for Reproductive
Controlled Ovarian Stimulation
Medicine, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, All patients underwent pituitary down-regulation with the GnRH
OH 44195 (FAX: 216-445-6049; E-mail: Agarwaa@ccf.org). agonist leuprolide acetate (LA; Lupron; Tap Pharmaceutical,

0015-0282/10/$36.00 Fertility and Sterility Vol. -, No. -, - 2010 1

doi:10.1016/j.fertnstert.2009.12.020 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
 ARTICLE IN PRESS
Deerfield, IL) at a daily SC dose of 10 U (0.5 mg) initiated on cycle
day 21. The dose was subsequently reduced to 5 U (0.25 mg) once
serum E2 was suppressed to 50 pg/mL and continued until the day
hCG was administered. Controlled ovarian stimulation with SC re-
combinant FSH (Gonal F; Serono, Randolph, MA) or Follistim (Or-
ganon, West Orange, NJ) was begun after pituitary down-regulation.
The standard initial dose was 300 IU. Monitoring of the ovarian re-
sponse was performed by serial serum E2 levels and transvaginal
ultrasonograms beginning on the fifth day of stimulation. The
subsequent FSH doses and monitoring frequency were individual-
ized based on patients’ results. Stimulation was continued until at
least two follicles reached a mean diameter of 18–20 mm, at which
time hCG 10,000 IU SC was administered 36 hours before oocyte
recovery.
Oocyte Retrieval
Oocytes were collected by transvaginal ultrasound (TVUS)-guided
needle aspiration of the follicles. This procedure was performed un-
der deep conscious sedation. The retrieved oocytes were rinsed,
graded, and placed in N-2-hydroxyethylpiperazine-N0 -2-ethanesul-
fonic acid (HEPES)-buffered human tubal fluid (HTF) (Irvine Sci-
entific, Santa Ana, CA) at 37 C under 5% CO2, 5% O2, and 90% N2.
Sperm Preparation
After 2 days of abstinence, semen samples were collected from the
male partner by masturbation in a sterile container. The sperm were
prepared by double density gradient centrifugation technique. A sin-
gle 2-mL layer of 90% ISolate (Irvine Scientific) was used for most
specimens. After preparation, sperm were maintained in HTF me-
dium with 5% synthetic serum substitute (SSS; Irvine Scientific)
at room temperature until the time of IVF or ICSI. In conventional
IVF, 150–200  103 sperm were added to each culture dish contain-
ing 4–5 oocytes. For ICSI, the single best looking spermatozoon was
selected for microinjection of the oocytes.
Embryo Culture
After cumulus dissection and washing, the oocytes were placed in 1
mL of HTF supplemented with 6% SSS. Fertilization was confirmed
14–16 hours after insemination or ICSI by the presence of two pro-
nuclei and the extrusion of the second polar body. The fertilized oo-
cytes were cultured in groups of 4–5 oocytes in 1 mL of HTF with
SSS until the early afternoon of day 3. They were then placed in 1
mL of blastocyst media (Irvine Scientific) after a five-drop rinse
in the same medium. A second change to fresh blastocyst medium
was done on the morning of day 5 after embryo evaluation and be-
fore embryo transfers. Embryo transfers were scheduled between
noon and 2:00 PM on day 5.
Embryo Evaluation
On days 3 and 5 of development, the embryos were evaluated with
an Olympus X 70 inverted microscope (Olympus America, Mel-
ville, NY) equipped with Hoffman Modulation Optics (Narishige,
Tokyo) at 600 magnification. Cell number and degree and pattern
of fragmentation were recorded on day 3 of development. The de-
gree of fragmentation was expressed as a percentage and defined
as the embryonic volume occupied by denucleated cytoplasmic
fragments. Development on day 5 was recorded as well. An orga-
nized and distinct inner cell mass (ICM) and a cohesive layer of nu-
merous tightly packed cells in the trophectoderm cells were
considered normal. Highly irregularly arranged ICM or trophecto-
2 Bedaiwy et al. Reactive oxygen species in day 3 embryo culture media
derm cells were considered abnormal. All abnormal embryos on
days 5 and 6 were considered arrested.
ROS Measurement
After evaluating the embryos the morning of day 3, the culture me-
dia in the central well of the culture dish was collected as the test
sample. The media in the outer well of the culture dish served as
the control. Both were immediately centrifuged for 7 minutes at
600  g, and the supernatant was removed. Aliquots of the superna-
tant were prepared for ROS measurement.
The ROS production was measured by the chemiluminescence
assay using luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione;
Sigma, St. Louis, MO) as the probe. Ten microliters of luminol
(5 mM) prepared in dimethyl sulfoxide (Sigma) was added to
400 mL of the day 1 media. The ROS levels were determined by
measuring chemiluminescence with a luminometer (LKB 953,
Wallac, Gaithersburg, MD) in the integrated mode for 15 minutes,
and results were expressed as 106 cpm (counted photon per minute).
Implantation rate The implantation rate was calculated by dividing
the number of gestational sacs on TVUS by the number of embryos
transferred.
Clinical pregnancy rate The clinical pregnancy rate represents the
number of cycles with fetal cardiac activity on ultrasound divided
by the number of cycles initiated.
Statistical Methods
The D-3 ROS levels, PRs, and other studied parameters were ana-
lyzed separately and collectively for the IVF and ICSI groups. De-
scriptive statistics are presented as frequency (percentage) or
mean  SD. Associations with categorical variables were assessed
using Fisher’s exact test or c2 test. Associations with quantitative
and ordinal variables were assessed using logistic regression.
Odds ratios (OR) of pregnancy were reported for quantitative vari-
ables. The association between D-3 ROS levels and PRs was as-
sessed separately for the IVF and ICSI groups. Logistic regression
was used to estimate an OR for clinical pregnancy with respect to
a 10-fold increase in D-3 ROS. Multivariate modeling analysis
was done to examine the effect of other predictive factors on D-3
ROS levels. Differences were considered significant if P%.05.
Analyses were performed using R software version 2.3.1 (19).
RESULTS
Ninety-two patients underwent 93 cycles of assisted reproduction
technology (ART) during the study duration. Of those, 56 were
conventional IVF cycles and 36 were ICSI cycles. Demographic
features of the study population and the indications for ART are
presented in Table 1. Cycle outcome parameters are presented in
Table 2. Correlation between D-3 ROS levels and pregnancy are
presented in Tables 3 and 4.
Comparison of Demographic Variables
Patients’ ages, height, weight, body mass index (BMI), parity, pri-
mary or secondary infertility, and infertility factors were not signifi-
cantly different in pregnant versus nonpregnant overall (Table 1).
There were no statistically significant differences in any of those pa-
rameters between the pregnant and nonpregnant patients within the
IVF or ICSI groups (data not shown).
Vol. -, No. -, - 2010
 ARTICLE IN PRESS

TABLE 1
Demographic features and indications for ART in both IFV/ICSI groups.

Nonpregnant cycles Pregnant cycles

No. % N % P value OR (95% CI) (units)

No. of patients 42 50
No. of cycles 42 51
Age (y), mean  SD 31.7  3.6 32.5  3.5 .2c 1.07 (0.95–1.21) (1)
Weight (kg), mean  SD 70.0  18.5 69.8  13.3 .9c 0.99 (0.87–1.13) (5)
Height (m), mean  SD 1.64  0.07 1.7  0.07 .1c 1.04 (0.98–1.10) (0.01)
BMI, mean  SD 25.85  5.8 25.3  4.9 .6c 0.91 (0.62–1.34) (5)
Parity .07b
Nulliparous 26 63.4 23 46
Multiparous 15 36.6 27 54
Infertility .08b
Primary 24 58.5 22 44
Secondary 17 41.5 28 56
Infertility factors .7a
Male 11 26.2 10 19.6
Tubal 7 16.6 14 27.5
Ovarian 5 11.9 5 9.8
Endometriosis 9 21.4 9 17.6
Unexplained 10 23.9 13 25.5
ART
IVF 26 61.9 30 58.8 .51b
ICSI 16 38.1 21 41.2
Note: Associations with categorical variables assessed using: OR ¼ odds ratios of pregnancy reported for quantitative variables and pertains to a change of x
units, where x is reported in parentheses; 95% CI ¼ 95% confidence interval; No. of cycles ¼ number of cycles for infertility and infertility factors; ART ¼
assisted reproductive technology; ICSI ¼ intracytoplasmic sperm injection; BMI ¼ body mass index.
a
Fisher’s exact test.
b 2
c test.
c
Associations with quantitative and ordinal variables assessed using logistic regression.

Bedaiwy. Reactive oxygen species in day 3 embryo culture media. Fertil Steril 2010.

Comparison of Cycle Outcome
The number of days of FSH administration, total FSH units used, cy-
cle day 3 E2 levels, peak E2 levels, number of mature follicles, number
of retrieved oocytes, fertilization rate, blastocyst rate, and number of
embryos transferred were similar between the pregnant and nonpreg-
nant groups (Table 2). These parameters were also not significantly
different within the IVF or ICSI groups (data not shown). The overall
clinical PR was 51%, with a multiple PR of 45.1%.
Correlation Between D-3 ROS Levels and Cycle Outcome
The pregnant cycle group showed significantly lower D-3 ROS levels
( 106 cpm) and log10 D-3 ROS overall as well as in the IVF and ICSI
groups (Table 3). The D-3 ROS level was negatively correlated with
high (>7) day 3 cell number in IVF and ICSI cycles. A significant neg-
ative correlation was also observed between D-3 ROS levels and
worst fragmentation rate in both ICSI and IVF cycles. A significant
correlation was seen between high D-3 ROS levels and low blastocyst
development rates in conventional IVF and ICSI cycles (Table 4).
Multivariate Modeling Analysis for Predictive Factors of
Clinical Pregnancy
The covariate-adjusted odds ratio (95% confidence interval [CI]) for
a 0.1-U change in Log10 (ROS) was 0.54 (0.39–0.74) (P<.001).
Therefore, the univariable result (OR ¼ 0.52; 95% CI 0.38–0.70;
P<.001) was essentially unchanged, even after adjusting for age,
BMI, diagnosis, total units, oocytes recovered, blastocysts at day
5, and worst observed fragmentation.
DISCUSSION
The ROS may block or retard early embryonic development by
affecting key cellular organelles required for rapid cell division.
The ROS may cause aggregation of cytoskeleton components, con-
densation of the endoplasmic reticulum, loss of membrane fluidity,
and embryo fragmentation (20, 21). Free radicals have many patho-
logical effects including DNA damage. The balance between ROS in
the culture media and the ability of embryo to neutralize them may
affect in vitro embryo development.
This study showed that slow development (<7 cells embryo on
day 3), worst fragmentation, and reduced formation of morpholog-
ically normal blastocysts are associated with increased levels of D-3
ROS. Lower blastocyst development rates have been observed in
ICSI cycles (22). The concern about sperm DNA damage by ROS
is especially relevant during the ICSI procedure (23, 24).
Lower clinical PRs were also associated with higher D-3 ROS
levels in both conventional IVF and ICSI cycles. Day 3 embryos
with reduced blastomere numbers and higher fragmentation due to
the negative association with D-3 ROS would be predicted to de-
velop into blastocysts at a lower rate. Such embryos have been
shown to yield higher PRs after assisted hatching, fragment removal,
and embryo transfer on day 3 rather than by extending culture to day
5 (25, 26).

Fertility and Sterility 3

 ARTICLE IN PRESS

TABLE 2
Comparison of the overall outcome of pregnant versus nonpregnant cycle parameters in the study population.

Nonpregnant cycles Pregnant cycles

Factor (n [ 42) (n [ 51) P valuea OR (95% CI) (units)

Days of stimulation 9.3  1.5 9.1  1.5 .5 0.92 (0.69–1.22) (1)

FSH used (IU) 2,383.8  922.2 2,311.1  885.3 .7 0.99 (0.95–1.04) (100)
Day 3 E2 (pg/mL) 41.1  18.2 36.4  15.9 .2 0.85 (0.65–1.11) (10)
E2 on day of hCG administration (pg/mL) 2,401.3  932.2 2,350.15  960.3 .8 0.99 (0.95–1.04) (100)
Number of mature follicles (>15 mm) on 15.2  6.4 16.7  6.1 .3 1.04 (0.97–1.12) (1)
day of hCG administration
No. of oocytes retrieved 17.1  6.0 17.8  6.1 .6 1.02 (0.95–1.09) (1)
Fertilization rate 67.3  17.3 70.3  15.4 .3
No. of blastocyst 4.5  2.1 4.6  1.6 .4
No. of cryopreserved blastocyst 2.0  2.2 2.3  1.8 .28
No. cryopreserved embryos 1.7  3.3 2.9  3.6 .03
No. of embryos transferred 2.47  0.63 2.35  0.59 .3 0.72 (0.36–1.41) (1)
Total embryos 10.9  3.9 12.2  4.4 .1 1.08 (0.98–1.20) (1)
Normal fertilization 11.1  4.1 12.3  4.4 .1 1.07 (0.97–1.18) (1)
High cell number 5.4  2.1 5.3  1.6 .6
Worst fragmentation 4.2  2.7 4.1  2.1 .7
No. of fetuses, events/trials (%)
1 28 (54.9)
2 21 (41.2)
3 2 (3.9)
Previous gestations
Term 0.28  0.51 0.53  0.77 .08 1.88 (0.93–3.81) (1)
Alive 0.25  0.49 0.53  0.77 .06 2.04 (0.98–4.22) (1)
Blastocysts at day 5 3.48  2.57 4.14  2.09 .17 1.13 (0.95–1.36) (1)
Note: All values mean  SD unless otherwise indicated. OR ¼ odds ratios of pregnancy reported for quantitative variables and pertains to a change of x units,
where x is reported in parentheses; 95% CI ¼ 95% confidence interval.
a
Associations with quantitative and ordinal variables assessed using logistic regression.

Bedaiwy. Reactive oxygen species in day 3 embryo culture media. Fertil Steril 2010.

Actually we have done the multivariate modeling for other predic-
tive factors including the embryo morphology/fragmentation and we
got confirmation of our findings. The univariable result was essen-
tially unchanged, even after adjusting for age, BMI, diagnosis, total
units, oocytes recovered, blastocysts at day 5, and worst observed
fragmentation. Worst fragmentation is defined as the greatest degree
of fragmentation observed among the total embryos.
Reduction of the gametes exposure is the most proper way to
reduce their oxygen species-induced damage. The IVF clinics are
trying to minimize the gametes and embryos exposure time to
conditions that allow high free radical generation. Eliminating expo-
sure of gametes and embryos to air during handling can partially
help. Exogenous ROS is mostly produced by spermatozoa that are
used to inseminate oocytes in IVF. With ICSI where there is only

TABLE 3
Correlation between D-3 ROS levels in pregnant and nonpregnant cycles.

Nonpregnant cycles, Pregnant cycles,

Factor mean ± SD mean ± SD P valuea OR (95% CI) (units)

Overall, n 42 51
Day 3 ROS level ( 106 cpm) 66.4  39.4 26.8  13.9 < .001 0.47 (0.30–0.74) (10)
Log10 (D-3 ROS) level 1.74  0.29 1.36  0.26 < .001 0.59 (0.47–0.74) (0.1)
IVF, n 26 30
Day 3 ROS level ( 106 cpm) 67.00  39.91 27.11  14.95 < .0001 0.47 (0.30–0.74) (10)
Log10 (D-3 ROS) level 1.75  0.26 1.36  0.27 < .001 0.52 (0.36–0.74) (0.1)
ICSI, n 16 20
Day 3 ROS level ( 106 cpm) 65.53  39.79 25.6  12.75 .006 0.56 (0.37–0.85) (10)
Log10 (D-3 ROS) level 1.71  0.34 1.34  0.26 .005 0.66 (0.49–0.88) (0.1)
Note: OR ¼ odds ratios of pregnancy reported for quantitative variables; the odds ratio pertains to a change of x units, where x is reported in parentheses;
95% CI ¼ 95% confidence interval; D-3 ROS ¼ day 3 reactive oxygen species.
a
Associations with quantitative and ordinal variables assessed using logistic regression.

Bedaiwy. Reactive oxygen species in day 3 embryo culture media. Fertil Steril 2010.

4 Bedaiwy et al. Reactive oxygen species in day 3 embryo culture media Vol. -, No. -, - 2010
 ARTICLE IN PRESS
in conventional IVF cycles. Furthermore, other indicators of oxida-
TABLE 4 tive stress, such as lipid peroxides and total antioxidant capacity,
Correlation between D-3 ROS with embryo development could have provided a more comprehensive picture of oxidative
and pregnancy outcome in IVF and ICSI. stress in culture. It is unclear if the relationship between embryo
quality and high day 3 ROS levels in culture media is one of cause
IVF (n [ 56) ICSI (n [ 36)
or effect and is independent of whether single or multiple culture
Factor r P value r P value media is used. Actually we believe that it may be a cause-and-effect
in the same time, as high culture media ROS may compromise the
High (>7) day 3 cell number –0.023 < .001 –0.4 < .001
embryo quality through inducing uncorrectable oxidative stress
Worst fragmentation rate –0.01 < .001 –0.36 < .001
damage. In addition, low embryo quality may produce a high ROS
Blastocyst rate –0.78 < .001 –0.36 < .001
Clinical pregnancy –0.56 < .001 –0.58 < .001 production and hence its ROS level may be used as a predictor for
the embryo quality. In our study because we use the outer well as a neg-
Note: D-3 ROS ¼ day 3 reactive oxygen species; ICSI ¼ intracytoplas- ative control, our findings mainly indicate that the source of ROS is
mic sperm injection. P< .05 was significant.
from the embryos themselves, whatever their quality. However, we
Bedaiwy. Reactive oxygen species in day 3 embryo culture media. Fertil Steril did not isolate or remove low quality embryos to study the effect of
2010. such removal on the ROS levels. Our findings refer to the use of day
3 culture media ROS levels as predictor of embryo quality or clinical
pregnancy outcome in both IVF and ICSI procedures. Another poten-
tial application of our findings is the use of day 3 culture media ROS as
one spermatozoon per oocyte, our findings also show that high ROS
an indicator for culture media quality control and this may be used as
levels can be hazardous.
an additional parameter for media quality testing and assurance.
Most IVF laboratories use 14–16 hours insemination times. Such
In conclusion, increasing levels of ROS generation in day 3
long exposure of oocytes to spermatozoa can increase oxygen spe-
in vitro embryo culture media may have a detrimental effect on the
cies-induced damage (27, 28), and therefore, some laboratories
in vitro embryo growth parameters, as well as clinical PRs in both
have considered shortening this time. Reports show the beneficial
conventional IVF and ICSI cycles. Also, we can improve blastocyst
effects of short coincubation of gametes in IVF (29–31), whereas
development rates in the mouse embryo model by a short coincuba-
other investigators noted the opposite (32, 33).
tion time of the gametes in conventional IVF cycles and supplement-
This study is limited by the inability to evaluate the ROS levels
ing the culture media with antioxidants to help scavenge excessive
of each individual embryo as they were cultured in groups (4–5
production of ROS in the culture media. These measures may be
embryos in each group). However, culturing embryos individually
applied clinically in an attempt to improve ART outcomes.
may deprive them of potentially beneficial embryonic paracrine
functions, including antioxidants (34–36). In addition, we could Acknowledgments: The authors thank Kurt Miller, Ph.D., and Jeffery Ham-
not determine the relative contribution of ROS from the blastomeres, mel, M.S., for their valuable assistance during the preparation of the manu-
or cumulus cell mass in culture, or their potential antioxidant effects script.

REFERENCES
1. Gardner DK. Changes in requirements and utilization
of nutrients during mammalian preimplantation em-
bryo development and their significance in embryo
culture. Theriogenology 1998;49:83–102.
2. Porterfield DM, Trimarchi JR, Keefe DL, Smith PJ.
Characterization of oxygen and calcium fluxes from
early mouse embryos and oocytes. Biol Bull
1998;195:208–9.
3. Trimarchi JR, Liu L, Porterfield DM, Smith PJ,
Keefe DL. Oxidative phosphorylation-dependent
and -independent oxygen consumption by individual
preimplantation mouse embryos. Biol Reprod
2000;62:1866–74.
4. Bedaiwy MA, Falcone T, Mohamed MS, Aleem AA,
Sharma RK, Worley SE, et al. Differential growth of
human embryos in vitro: role of reactive oxygen spe-
cies. FertilSteril 2004;82:593–600.
5. Trimarchi JR, Liu L, Smith PJ, Keefe DL. Noninva-
sive measurement of potassium efflux as an early in-
dicator of cell death in mouse embryos. Biol Reprod
2000;63:851–7.
6. Somers J, Smith C, Donnison M, Wells DN,
Henderson H, McLeay L, et al. Gene expression pro-
filing of individual bovine nuclear transfer blasto-
cysts. Reproduction (Cambridge, England)
2006;131:1073–84.
7. Bedaiwy M, Agarwal A, Said TM, Goldberg JM,
Sharma RK, Worley S, et al. Role of total antioxidant
capacity in the differential growth of human embryos
in vitro. Fertil Steril 2006;86:304–9.
8. Goto Y, Noda Y, Mori T, Nakano M. Increased gen-
eration of reactive oxygen species in embryos cul-
tured in vitro. Free Radical Biol Med 1993;15:
69–75.
9. Booth PJ, Holm P, Callesen H. The effect of oxygen
tension on porcine embryonic development is depen-
dent on embryo type. Theriogenology 2005;63:
2040–52.
10. Lima PF, Oliveira MA, Santos MH, Reichenbach HD,
Weppert M, Paula-Lopes FF, et al. Effect of retinoids
and growth factor on in vitro bovine embryos pro-
duced under chemically defined conditions. Animal
Reprod Sci 2006;95:184–92.
11. Guyader-Joly C, Guerin P, Renard JP, Guillaud J,
Ponchon S, Menezo Y. Precursors of taurine in female
genital tract: effects on developmental capacity of bo-
vine embryo produced in vitro. Amino Acids
1998;15:27–42.
12. Grifo JA, Flisser E, Adler A, McCaffrey C, Krey LC,
Licciardi F, et al. Programmatic implementation of
blastocyst transfer in a university-based in vitro fertil-
ization clinic: maximizing pregnancy rates and mini-
mizing triplet rates. Fertil Steril 2007;88:294–300.
13. Rehman KS, Bukulmez O, Langley M, Carr BR,
Nackley AC, Doody KM, et al. Late stages of embryo
progression are a much better predictor of clinical
pregnancy than early cleavage in intracytoplasmic
sperm injection and in vitro fertilization cycles with
blastocyst-stage transfer. Fertil Steril 2007;87:
1041–52.
14. Hill DL, Surrey MW. Successful outcome after pre-
implantation genetic screening and very delayed em-
bryo transfer. Fertil Steril 2009;91:1602–3.
15. Holte J, Berglund L, Milton K, Garello C,
Gennarelli G, Revelli A, et al. Construction of an ev-
idence-based integrated morphology cleavage em-
bryo score for implantation potential of embryos
scored and transferred on day 2 after oocyte retrieval.
Hum Reprod (Oxford, England) 2007;22:548–57.
16. Katsoff B, Check JH, Fox F, Choe JK, Iacone K. A re-
assessment of comparative pregnancy and implanta-
tion rates following embryo transfer in recipients vs
their infertile donors also trying to conceive in the
background of performing salpingectomy for hydro-
salpinx. Clin Exp Obstet Gynecol 2006;33:143–4.
17. Stokes PJ, Hawkhead JA, Fawthrop RK, Picton HM,
Sharma V, Leese HJ, et al. Metabolism of human em-
bryos following cryopreservation: implications for
the safety and selection of embryos for transfer in
clinical IVF. Hum Reprod (Oxford, England)
2007;22:829–35.
18. Van Voorhis BJ, Dokras A. Delayed blastocyst trans-
fer: is the window shutting? Fertil Steril 2008;89:
31–2.
19. Team RDC, ed. A language and environment for sta-
tistical computing. Vienna, Austria: R Foundation for
Statistical Computing, 2006.
20. Agarwal A, Gupta S, Sharma RK. Role of oxidative
stress in female reproduction. Reprod Biol Endocri-
nol 2005;3:28.

Fertility and Sterility 5


21. Gupta S, Agarwal A, Banerjee J, Alvarez JG. The role
of oxidative stress in spontaneous abortion and recur-
rent pregnancy loss: a systematic review. Obstet Gy-
necol Sur 2007;62:335–47. quiz 53–4.
22. Agarwal A, Said TM. Role of sperm chromatin abnor-
malities and DNA damage in male infertility. Hum
Reprod Update 2003;9:331–45.
23. Dumoulin JC, Coonen E, Bras M, van Wissen LC,
Ignoul-Vanvuchelen R, Bergers-Jansen JM, et al.
Comparison of in-vitro development of embryos orig-
inating from either conventional in-vitro fertilization
or intracytoplasmic sperm injection. Hum Reprod
(Oxford, England) 2000;15:402–9.
24. Menezo Y, Barak Y. Comparison between day-2
embryos obtained either from ICSI or resulting
from short insemination IVF: influence of maternal
age. Hum Reprod (Oxford, England) 2000;15:
1776–80.
25. Alikani M, Cohen J, Tomkin G, Garrisi GJ, Mack C,
Scott RT. Human embryo fragmentation in vitro and
its implications for pregnancy and implantation. Fer-
til Steril 1999;71:836–42.
6 Bedaiwy et al. Reactive oxygen species in day 3 embryo culture media
ARTICLE IN PRESS
26. Keltz MD, Skorupski JC, Bradley K, Stein D. Predictors
of embryo fragmentation and outcome after fragment re-
moval in invitro fertilization. Fertil Steril 2006;86:321–4.
27. Aitken RJ, Irvine DS, Wu FC. Prospective analysis of
sperm–oocyte fusion and reactive oxygen species
generation as criteria for the diagnosis of infertility.
Am J Obstet Gynecol 1991;164:542–51.
28. Nallella KP, Sharma RK, Allamaneni SS, Agarwal A.
Identification of male factor infertility using a novel
semen quality score and reactive oxygen species
levels. Clinics (Sao Paulo, Brazil) 2005;60:317–24.
29. Gianaroli L, Cristina Magli M, Ferraretti AP,
Fiorentino A, Tosti E, Panzella S, et al. Reducing
the time of sperm-oocyte interaction in human
in-vitro fertilization improves the implantation rate.
Hum Reprod (Oxford, England) 1996;11:166–71.
30. Quinn P, Lydic ML, Ho M, Bastuba M, Hendee F,
Brody SA. Confirmation of the beneficial effects of
brief coincubation of gametes in human in vitro fertil-
ization. Fertil Steril 1998;69:399–402.
31. Kattera S, Chen C. Short coincubation of gametes in
in vitro fertilization improves implantation and preg-
nancy rates: a prospective, randomized, controlled
study. Fertil Steril 2003;80:1017–21.
32. Boone WR, Johnson JE. Extending the coincubation
time of gametes improves in vitro fertilization. J As-
sist Reprod Genetics 2001;18:18–20.
33. Gil MA, Ruiz M, Vazquez JM, Roca J, Day BN,
Martinez EA. Effect of short periods of sperm–oocyte
coincubation during in vitro fertilization on embryo
development in pigs. Theriogenology 2004;62:
544–52.
34. Guerin P, El Mouatassim S, Menezo Y. Oxidative
stress and protection against reactive oxygen species
in the pre-implantation embryo and its surroundings.
Hum Reprod Update 2001;7:175–89.
35. Harvey AJ. The role of oxygen in ruminant preim-
plantation embryo development and metabolism.
Ani Reprod Sci 2007;98:113–28.
36. Orsi NM, Leese HJ. Protection against reactive oxy-
gen species during mouse preimplantation embryo
development: role of EDTA, oxygen tension, cata-
lase, superoxide dismutase and pyruvate. Mol Reprod
Dev 2001;59:44–53.
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