Correlations of follicular fluid oxidative stress
biomarkers and enzyme activities with embryo
morphology parameters during in vitro fertilization
Victor Y. Fujimoto, M.D.,aMichael S. Bloom, Ph.D.,bHeather G. Huddleston, M.D.,aWendy B. Shelley, M.D.,a
Andrew J. Ocque, M.S.,cand Richard W. Browne, Ph.D.c
aDepartment of Obstetrics, Gynecology and Reproductive Sciences, University of California at San Francisco, San Francisco,
California;bDepartments of Environmental Health Sciences and Epidemiology and Biostatistics, University at Albany, State
University of New York, Rensselaer, New York; and
University at Buffalo, State University of New York, Buffalo, New York
cDepartment of Biotechnical and Clinical Laboratory Sciences,
Objective: To measure antioxidant enzyme activities and lipid peroxidation levels within follicular fluid (FF) and
evaluate correlations with early embryo quality.
Design: Individual FF samples were obtained prospectively on the day of oocyte collection and assessed for lipid
peroxidation as specific positional isomers of hydroperoxy and hydroxy fatty acids by high-performance liquid
chromatography and antioxidant enzyme activities by automated kinetic enzyme assays. Spearman rank
correlation coefficients, adjusted for age and day of transfer, were used to assess associations between
antioxidant enzymes and lipid peroxidation products and embryo quality using a 1 follicle-1 oocyte/embryo
approach. Post hoc power analysis was conducted to help interpret null results.
Setting: A university clinic.
Patient(s): Thirty-nine women undergoing IVF.
Main Outcome Measure(s): Embryo cell number and embryo fragmentation score (EFS) at transfer.
Result(s): No significant correlations between lipid peroxidation derivatives or antioxidant enzyme activities and
embryo quality were obtained. Post hoc power analysis indicated possible undetected associations between EFS
and 13-hydroxy octadecatrienoic acid and 13-hydroperoxy octadecadieneoic acid.
Conclusion(s): Our preliminary dataset suggests that most lipid peroxidation products and antioxidant enzyme
However, further consideration of associations between EFS and 13-hydroxy octadecatrienoic acid and
13-hydroperoxy octadecadieneoic acid is warranted. (Fertil Steril?2011;96:1357–61. ?2011 by American
Society for Reproductive Medicine.)
Key Words: Oxidative stress, follicular fluid, embryo quality, lipid peroxidation, antioxidant enzymes
Free radicals and reactive oxygen species (ROS) are generated as
part of normal cellular metabolism and by exogenous sources. Ini-
tially studied solely as products of pathological processes, ROS
and oxidatively modified molecules generated within tissues and
cells are increasingly implicated as necessary reduction-oxidation
signaling molecules (1, 2). Oxidative stress (OS) refers to an
overproduction of ROS and/or a deficit or depletion of the
scavenging capacity of antioxidant defense systems leading to
biologic macromolecule oxidative damage.
ity potential (3–5), debate continues as to whether mammalian
oocyte potential is adversely influenced by OS within the follicle.
There is evidence that OS causes deleterious effects in mammalian
oocytes in vitro (6–9), but the relevance of these studies is
questionable in light of equivocal conclusions reached in a limited
set of clinical studies (10–18). Several factors have prevented
a clear understanding of the relationship between the oocyte and
follicular OS in vivo. First, few studies have been published
tracking individual follicles and allowing detailed correlations
between follicular fluid (FF) measurements and oocyte/embryo
outcomes. Additionally, in studies in which individual follicles and
tracked oocytes were used (14, 16), the set of OS markers
measured was limited and fraught with methodological concerns.
Finally, there is a lack of consistency governing studies used to
assess the oxidative state of pooled follicles because of the use of
a broad array of different OS biomarkers and the various oocyte
end points assessed.
In this preliminary study, we evaluated the relevance of a highly
validated panel of antioxidant enzyme activities and specific lipid
peroxidation end products by correlational analyses with early em-
bryo morphology parameters, embryo fragmentation, and embryo
cell number (ECN), using a 1 follicle-1 oocyte/embryo design.
MATERIALS AND METHODS
Patients and Samples
All patients were recruited into the study with informed consent approved by
the University of California at San Francisco Center for Human Research.
Patient recruitment and clinical protocols were previouslydescribed in detail
Received August 10, 2011; revised September 1, 2011; accepted
September 16, 2011; published online October 18, 2011.
V.Y.F. has nothing to disclose. M.S.B. has nothing to disclose. H.G.H. has
nothing to disclose. W.B.S. has nothing to disclose. A.J.O. has nothing
to disclose. R.W.B. has nothing to disclose.
Reprint requests: Victor Y. Fujimoto, M.D., 2356 Sutter Street, 7th Floor,
Suite J707, San Francisco, CA 94115-0916 (E-mail: fujimotov@obgyn.
Fertility and Sterility?Vol. 96, No. 6, December 2011
Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
(19). The study protocol was approved by the university’s committee on hu-
man research. A total of 39 patients underwent gonadotropin stimulation per
clinical protocols and oocyte retrieval 36 hours after hCG injection. Individ-
oocyte retrieval were immediately processed and frozen at ?80?C until anal-
ysis. Follicles not yielding oocytes were excluded from data analysis. Indi-
vidual embryos from FF study samples were tracked using ECN, on either
tation score (EFS). EFS was defined quantitatively from 1 to 5 as follows:
grade 1, 0% fragmentation; grade 2, 1% to 10% fragmentation; grade 3,
11% to 25% fragmentation; grade 4, 26% to 50% fragmentation; and grade
5, R51% fragmentation.
To evaluate the relation between OS and embryo morphology, we employed
a panel of oxidative stress biomarkers. FF and serum activities of various an-
tioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase
(GPX), glutathione reductase (GR), and glutathione-S-transferase (GST),
were measured by kinetic enzyme assays on the Cobas Fara II automated
chemistry analyzer (Hoffmann-La Roche) (20, 21). Lipid peroxidation was
measured by high-performance liquid chromatography analysis of hydroper-
oxy and hydroxy linolenic acid and linoleic acid oxidation products:
noic acid (13-HPOTE), 13-and 9-hydroxyoctadecadieneoic acid (13-HODE
and 9-HODE), and 13- and 9-hydroperoxy octadecadieneoic acid (13-
HPODE and 9-HPODE) (22, 23). In particular, the lipid peroxidation
products 9- and 13-HODE have been demonstrated to be excellent markers
of lipid peroxidation (24). These assays have been extremely well character-
ized and validated by our group (25).
tions for biomarkers of antioxidant activity and oxidative damage. Distribu-
tions for antioxidant enzymes and lipid peroxidation byproducts were
skewed and were not normalized using data transformations, necessitating
the use of nonparametric tests. Differences for antioxidant enzyme activities
measured in serum and FF specimens were assessed by the Wilcoxon
signed-rank test. Spearman rank correlation coefficients were used to assess
associations among and between antioxidant enzyme activities and lipid per-
oxidation byproducts. Spearman rank correlation coefficients were also used
to assess associations among antioxidant enzymes, lipid peroxidation by-
products, and ECN adjusted for age and day of ET and for EFS adjusted
for only age. Statistical significance was defined as P<.10 for a 2-tailed
testconsistentwiththepreliminarynatureofthisstudy. Nocorrection for po-
tential inflation of type 1 error due to the conduct of multiple statistical tests
was incorporated. Post hoc power analysis was conducted to aid in the inter-
pretation of null study results (26). Sample sizes required to detect statisti-
cally significant partial correlation coefficients equal to those observed
by 9.8% to accommodate our use of the Spearman correlation coefficient,
which is nonparametric (27). All statistical analysis was conducted using
SAS 9.2 (SAS Institute, Inc.).
Of 39 total embryos, 26% were assessed on day 2 and 74% were as-
sessed on day 3 (Table 1). Serum GPX exhibited the highest median
activity (627.76 IU) relative to other enzymes. GR (22.86 IU) dem-
onstrated the second highest activity, followed by SOD (6.81 IU)
the median GPX values exceeded those of the others (482.38 IU);
however, GST demonstrated the second highest median activity
level (34.37 IU), followed by GR (17.58 IU) and SOD (8.58 IU). Se-
rum GPX and GR activities exceeded FF activities for all partici-
pants, with a median difference of 106.67 IU for GPX (95%
confidence interval [CI], 70.51–150.84 IU; P<.0001) and 4.13 IU
for GR (95% CI, 2.64–6.63 IU; P<.0001). In contrast, greater me-
dian activity was detected in FF for GST (35.37 IU; 95% CI,
18.97–47.19 IU; P<.0001) and SOD (2.29 IU; 95% CI, 0.45–3.67
IU; P¼.019) relative to serum. Both SOD and GR activities
exhibited statistically significant correlations between serum and
FF compartments (r ¼ 0.45; 95% CI, 0.15–0.67 IU; P¼.004 and
r ¼ 0.47; 95% CI, 0.17–0.68 IU; P¼.003, respectively).
Serum samples for lipid peroxide analysis were not available
(19, 28). FF lipid peroxide levels for 28 participants with sufficient
sample volumes encompassed median values ranging from 17 to 85
nmol/L. 9-HODE was the predominant regioisomeric peroxidation
product followed by the other hydroxy products 13-HOTE and 13-
HODE. Hydroperoxy product levels were lower than those of the
corresponding hydroxy products among each hydroxy/hydroperoxy
pair. Patient age was significantly associated with increased FF
GPX, FF GR, and FF GST activities (r ¼ 0.48, P¼.002; r ¼ 0.48,
P¼.002; and r ¼ 0.26, P¼.104, respectively), decreased 13-HOTE
levels (r ¼ ?0.53, P¼.004), but increased levels of 13-HODE
activities and lipid peroxide levels in FF did not demonstrate any
consistent patterns of positive or negative associations. GPX was
negatively correlated with both 13-HOTE (r ¼ ?0.34, P<.10) and
13-HPODE (r ¼ ?0.33, P¼.104) with borderline statistical signifi-
cance. GST was negatively correlated with 9-HPODE (r ¼ ?0.33,
P<.10), and SOD was negatively associated with 9-HODE (r ¼
?0.44, P<.05). However, GR was significantly, positively corre-
lated with 13-HODE (r ¼ 0.47, P<.05).
In Table 2, adjusted associations between FF analytes and ECN
and EFS are described. After adjustments for day of ETand patient
age, there were no statistically significant associations between
GPX, GR, GST, and SOD levels or concentrations of lipid peroxides
and ECN. Similarly, there were no statistically significant associa-
tions between any of these enzyme activities or lipid peroxides
and EFS adjusted for age. For the majority of analytes measured,
post hoc power analysis suggested that at a minimum, our sample
size required an approximate 3.3-fold increase to detect a statisti-
cally significant association with 80% power (i.e., 13-HPODE and
ECN), and at a maximum, an increase of more than 1,740-fold
(i.e., SOD and EFS), as noted in Table 2. For 13-HPODE and
13-HOTE, only approximately 2.4-fold and 2.7-fold increases in
the current sample size, respectively, were required for sufficient
power to detect statistically significant positive and inverse associa-
tions with EFS, respectively.
cussed in the literature (29, 30), this is the first prospective study, to
our knowledge, to correlate specific regioisomeric lipid peroxide
species and multiple antioxidant enzyme activities, measured in
single follicle specimens, with distinct early embryo end points. As
previously reported, our data set considered ECN separately from
EFS because the biologic mechanisms responsible for these clinical
observations may differ (19, 28). Other studies have defined
embryo quality based on a combination of morphologic features
that included cell cleavage rate and embryo fragmentation as
a single comprehensive end point and used nonspecific markers of
oxidative damage as summary measurements of total lipid
peroxidation or as the ‘‘peroxidizability’’ of FF (13, 14, 17, 18).
Using these highly specific analytic tools, we did not find
a significant relationship between FF OS and early embryo
Fujimoto et al.
Follicular oxidative stress and embryos
Vol. 96, No. 6, December 2011
outcomes. Our data set confirmed the positive maternal age
association with most of the OS biomarkers (17) but did not indicate
any correlations between OS biomarkers and embryo quality.
Several lines of evidence suggest the importance of OS in oocyte
development and presumably early embryonic development. Stud-
ies have identified glutathione metabolism to be important for oo-
cyte health (9, 31, 32). Although we did not measure glutathione
levels, our GPX, GST, and GR activities did not suggest a critical
role for glutathione metabolism influencing embryo quality
similar to previous reports (11). We identified GST activities to be
local production of GST by granulosa cells (33). Carbone et al. (10)
found increased SOD and reduced GST activities in older women;
however, no differences in GPX or GR activities were seen. In con-
trast, our study found positive correlations between patient age and
FF GST, GPX, and GR activities. Our observations would suggest
a compensatory increase in the glutathione recycling pathway
with age within the ovarian follicle. The lack of correlations with
embryo morphology parameters in our study differed from findings
reported by Das et al. (18), in which ROS in FF was negatively as-
sociated with embryo quality. Elevated ROS levels measured by
a chemiluminescence assay using luminol were seen in FF contain-
ing embryos of poorer morphology at the 4-cell stage (18). A subse-
using the same assay (34).
Similar to our findings, Jozwik et al. (12) found no associations
between lipid hydroperoxide concentrations and reproductive end
points including fertilization and pregnancy rates. Their results
were substantiated by the use of multiple lipid peroxidation markers
in addition to the performance of methodological studies which
demonstrated that there were no artifacts introduced due to
ex vivo peroxidation associated with the methods used. Embryo
morphology was not an outcome variable in their study. One study
found a positive association between FF thiobarbituric acid reactive
substance and FF cotinine levels, suggestive of an important impact
for cigarette smoking on OS (35). However, no association between
FF thiobarbituric acid reactive substance and fertilization rates ap-
pears to exist (12). A recent study demonstrated lower lipid perox-
idation levels and higher reduced-glutathione levels in IVF
patients treated with multivitamin and mineral supplementation
compared with control women, suggesting an effect of various vita-
mins and essential minerals including vitamins A, C, and E on re-
ducing FF OS (36). Although the antioxidant vitamin E inversely
correlates with embryo fragmentation, this observation does not ex-
ist independent of FF high-density lipoprotein (HDL) cholesterol
levels, suggesting that the effect cannot be completely attributable
to the antioxidant potential of vitamin E (28).
Another measure of OS reported in the literature is the thermo-
chemiluminescence assay (17). Using pooled FF, positive correla-
tions were made between thermochemiluminescence curve slope
(indicating increased oxidizability of FF) and number of cleaved
and high-quality embryos (17). The authors interpreted these results
to surmise that some level of OS may indicate a healthy, metaboli-
cally active follicle. Although this is a reasonable hypothesis, it is
Distribution of clinical variables and laboratory analytes measured in FF specimens collected from study participants.
Factor Mean, nSD, % Minimum 25th percentile50th percentile 75th percentileMaximum
Day of ET
Serum enzyme activity, IU (n ¼ 38)
FF enzyme activity, IU (n ¼ 39)
FF lipid peroxides, nmol/L (n ¼ 28)
37.475.12 23.23 35.3938.4840.87 43.71
Note: Maximum indicates the highest observed value, and minimum indicates the lowest observed value. 13-HOTE ¼ 13-hydroxy octadecatrienoic acid;
13-HPOTE ¼ 13-hydroperoxy octadecatrienoic acid; 13-HODE ¼ 13-hydroxy octadecadieneoic acid; 9-HODE ¼ 9-hydroxy octadecadieneoic acid;
13-HPODE ¼ 13-hydroperoxy octadecadieneoic acid; 9-HPODE ¼ 9-hydroperoxy octadecadieneoic acid; FF ¼ follicular fluid.
bP<.05 for difference between serum and FF concentrations.
Fujimoto. Follicular oxidative stress and embryos. Fertil Steril 2011.
Fertility and Sterility?
also possible that the thermochemiluminescence assay was simply
measuring the increased concentration of HDL-associated lipids
(specifically oxidizable polyunsaturated fatty acids), which have
been shown to increase with mammalian follicle maturity (37, 38)
and correlate negatively with embryo fragmentation in humans
(19). In contrast to these prior studies, our data set did not demon-
strate any associations between several preformed lipid peroxide
products in FF and embryo morphology during IVF. Although our
post hoc power analysis indicated that 13-HPODE and 13-HOTE
may ultimately prove to be associated with embryo morphology pa-
rameters in a larger study, the associations may not be related to OS.
Linoleic acid (the precursor of 13-HPODE) can be converted by
prostaglandin endoperoxide synthase to its 9- and 13-hydroperoxy
metabolites (39). Similar reactions have been demonstrated for the
precursor of 13-HOTE, linolenic acid (40). Given that prostaglan-
dins have been shown to play an important role in the ovulation pro-
cess and significant quantities have been found in FF (41) these
observations may be more reflective of follicular eicosanoid metab-
olism rather than OS. Furthermore, the higher GST, an intracellular
antioxidant protein, activities in FF suggestive of local production
within the follicle in our study may be protective against oocyte de-
velopmentalcompromiseasadirect resultofOS. Thus,itis possible
that the antioxidative protective capacity present in the follicle pre-
vents any measurable change in these biomarkers that would impact
downstream clinical outcomes.
It is important to acknowledge that the oxidative state within the
follicle is governed by the interplay between numerous proteins,
lipids, and other small molecules that act to regulate electron trans-
by their association with embryo fragmentation (19). Embryo frag-
mentation may be directly related to the HDL particle constitution
as evidenced by FF phospholipid:cholesterol ratios (42), and in-
volves intrafollicular lipid transport (43). Although various lipo-
philic micronutrients with antioxidant activities within HDL
particles such as b-carotene and b-cryptoxanthine also correlate
retinoic acid receptor agonists with the ability to activate retinoic
acid receptor/retinoid X receptor heterodimers with subsequent
pleiotropic effects on lipid transport may be more relevant (44).
The multiplicity of functions, including antioxidant properties asso-
ciated with HDL, makes it difficult to assess the effect of OS in the
relations between lipid peroxidation products and embryo fragmen-
tation in this data set lends further support to the hypothesis that
intrafollicular lipid transport rather than OS is important to blasto-
mere membrane integrity and the development of early-cleavage–
stage embryo fragmentation.
the measurements of multiple lipid peroxidation products and anti-
oxidant enzyme activities, and  the prospective nature of our
study, which incorporates temporality by design (i.e., exposure pre-
cedes outcome). Whereas the majority of other human studies pub-
lished measured ROS via surrogate ‘‘oxidizability’’ markers or
antioxidants as ‘‘total antioxidant capacity’’ measures, we used mul-
tiple direct measurements of primary lipid peroxidation products to
provide a more specific picture of the level of oxidation occurring in
were not used. Additionally, F2-isoprostanes were not measured,
and this lipid peroxidation product may represent the most sensitive
marker of OS currently available (45). With this limitation, it must
be noted that the one-follicle design significantly restricts the
amount of sample available for analysis. Another limitation is
clearly the sample size, which prevented assessment of other rele-
vant oocyte outcomes, such as oocyte maturation and fertilization.
Given the limitations of sample size, questions remain regardingpo-
we set a fairly liberal ‘‘threshold’’ for statistical significance of
P<.10 in an effort to accommodate the sample size, and moreover,
we didnotadjustfor inflationoftype 1errors. The clinicalrelevance
of FF OS biomarkers on oocyte development must be questioned in
light of this study.
In conclusion, our study results add to the controversy sur-
rounding the role of follicular ROS on oocyte potential as mea-
sured by cleavage-stage embryo parameters, specifically cleavage
and fragmentation rates during IVF. Although the lack of associa-
tions between OS biomarkers and antioxidant enzyme activities
and embryo morphology suggests a largely protective FF environ-
ment against OS, our findings do not necessarily imply that ROS
plays no role in oocyte competence. In fact, post hoc power anal-
ysis raises the possibility for existing, yet undetected, positive and
inverse associations between EFS and 13-HOTE and 13-HPODE,
respectively; however, such associations might reflect eicosanoid
metabolism rather than OS. Extensive inflations in required sample
sizes undermine the tenability for associations among ECN, EFS,
and other analytes measured in this study. Scientific curiosity to
establish a role for OS on oocyte developmental competence
begs further studies to clarify any relationships between more sen-
sitive OS biomarkers and other end points of oocyte/embryo
Spearman rank correlations between embryo morphology
parameters and laboratory analytes measured in FF
specimens collected from study participants.
Enzyme activity (n ¼ 39)
Lipid peroxides (n ¼ 28)
Note: 13-HOTE ¼ 13-hydroxy octadecatrienoic acid; 13-HPOTE ¼ 13-
hydroperoxy octadecatrienoic acid; 13-HODE ¼ 13-hydroxy octa-
decadieneoic acid; 9-HODE ¼ 9-hydroxy octadecadieneoic acid;
13-HPODE ¼ 13-hydroperoxy octadecadieneoic acid; 9-HPODE ¼
9-hydroperoxy octadecadieneoic acid; FF ¼ follicular fluid.
aAdjusted for day of ET and patient age.
bSample size necessary for detection of the observed correlation at
a ¼ 0.10 and 80% power.
cAdjusted for patient age.
Fujimoto. Follicular oxidative stress and embryos. Fertil Steril 2011.
Fujimoto et al.
Follicular oxidative stress and embryos
Vol. 96, No. 6, December 2011
Acknowledgments: We acknowledge Julia Sandler and Giulia Conti for their
contributions to the consenting and collection of samples detailed in this
manuscript. We acknowledge the members of the UCSF IVF laboratory
for their contributions to the processing of the samples. We declare that no
external funding sources were used to conduct this study. Only discretionary
funds from Drs. Fujimoto and Browne were used to conduct this study.
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