Reproductive Toxicology 61 (2016) 142–150

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Reproductive Toxicology
journal homepage: www.elsevier.com/locate/reprotox

Follicular fluid and urinary concentrations of phthalate metabolites

among infertile women and associations with in vitro fertilization
parameters
Yao-Yao Du a , Yue-Li Fang a , Yi-Xin Wang b,c , Qiang Zeng b,c , Na Guo a , Hua Zhao a ,
Yu-Feng Li a,∗
a
Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China
b
Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, Hubei, PR China
c
Key Laboratory of Environment and Health, Ministry of Education & Ministry of Environmental Protection, and State Key Laboratory of Environmental
health (incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Evidence from toxicological studies has demonstrated that phthalates can lead to reduced fertility
Received 23 September 2015 through effects on folliculogenesis, oocyte maturation and embryonic development, but human data
Received in revised form 27 March 2016 are limited. Concentrations of eight phthalate metabolites in 110 follicular fluid (FF) and urine samples
Accepted 7 April 2016
collected from 112 women attending an infertility clinic in Wuhan, China were quantified, and corre-
Available online 8 April 2016
lations between paired matrices were explored. Associations between metabolite concentrations and
in vitro fertilization (IVF) parameters were evaluated with multivariable models. Six metabolites were
Keywords:
detected in >72.73% of the FF samples. MEHP and MBP were the dominant metabolites with a median
Phthalates
Follicular fluid
level of 2.80 and 2.05 ng/mL, respectively. Significant correlations between the two matrices, urine and
IVF parameters FF, were found for MEP (rs = 0.44), and MBP (rs = 0.22). FF and urinary metabolite concentrations were
Female reproduction not associated with any IVF parameters. However, given the prevalence of phthalates exposure, further
Endocrine disruptors work is needed to elucidate the potential hazard on female reproduction.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction products, or as excipients, such as coatings of medications and

Phthalates are a class of man-made industrial chemicals which
have been ubiquitously applied to manufacture polyvinyl chloride
products, such as construction materials, food packaging and chil-
dren’s toys, mainly based on their ability to impart flexibility. They
are also commonly used as solvents in cosmetics and personal care
Abbreviations: AFC, antral follicle count; BMI, body mass index; DEHP,
di(2-ethylhexyl) phthalate; EDCs, endocrine-disrupting chemicals; E2 , estradiol;
FF, follicular fluid; FSH, follicle-stimulating hormone; HCG, human chorionic
gonadotropin; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; LOD,
the limits of detection; MBP, monobutyl phthalate; MBzP, monobenzyl phtha-
late; MEP, monoethyl phthalate; MEHP, mono(2-ethylhexyl) phthalate; MEHHP,
mono(2-ethyl-5-hydroxyhexyl) phthalate; MEOHP, mono(2-ethyl-5-oxohexyl)
phthalate; MMP, monomethyl phthalate; MOP, mono-n-octyl phthalate; %MEHP,
the percentage of DEHP metabolites excreted as MEHP; 2PN, two pronuclei.
∗ Corresponding author at: Reproductive Medicine Center, Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Technology, Wuhan, Hubei,
PR China.
E-mail addresses: yufengli64@163.com, yufengli64@tjh.tjmu.edu.cn (Y.-F. Li).
dietary supplements [1–3]. Owing to the extensive use of phtha-
lates, as well as their non-covalent conjugation with the products,
the general population has been pervasively exposed to these com-
pounds [1]. Once the phthalates enter the human body via various
routes, primarily through ingestion, they can interfere with the
endocrine systems, thus so called “endocrine-disrupting chemi-
cals (EDCs)” [1,4]. To date, detectable levels of phthalates and their
metabolites have been found in a wide range of biological fluids
including human urine, serum, semen, amniotic fluid, umbilical
cord blood and breast milk [5–8].
Prevalent exposure to phthalates has aroused growing pub-
lic health concern based on their endocrine-disrupting potency.
The adverse effects of phthalates on male fertility have been
well documented and extensively studied[9]. In females, on the
other hand, the influence of phthalates on ovarian and repro-
ductive functions remains not well understood. However, there
is accumulated evidence from experimental animal studies sug-
gesting that phthalates exert reproductive toxicity by targeting
the ovary [10,11]. Exposure to phthalates has been demonstrated

http://dx.doi.org/10.1016/j.reprotox.2016.04.005
0890-6238/© 2016 Elsevier Inc. All rights reserved.
 Y.-Y. Du et al. / Reproductive Toxicology 61 (2016) 142–150
to disrupt folliculogenesis, steroidogenesis, oocyte maturation and
embryonic development [11–13], thus lead to reduced fertility. The
effect of di(2-ethylhexyl) phthalate (DEHP) [prototype of mono(2-
ethylhexyl) phthalate (MEHP)] on ovarian follicles and oocyte
cellular structures was evidenced by depleted primordial folli-
cle pool, increased atresia of antral follicles and observed spindle
abnormalities which may result in aneuploidy, miscarriages and
congenital defects [14,15]. A significant decrease of estradiol (E2 )
production in a dose related pattern was observed in primary
cultured granulosa cells after exposure to MEHP [16,17]. Labora-
tory studies using in vitro maturation systems showed that MEHP
impeded the progression of oocyte meiosis and inhibited develop-
mental potential of embryos in different animal models [13,18].
Investigations regarding reproductive toxicity of phthalates on
females conducted to date have been through animal or in vitro
studies, whether they can execute comparable effects in humans
remains to be elucidated, especially, when the high dosages used
in laboratory experiments were considered. The hitherto published
epidemiological literature focusing on phthalates exposure and
female fertility is scarce. Moreover, most previous studies mea-
sured urinary concentrations of phthalate metabolites as internal
exposure biomarkers. Although urinary metabolites are reliable
indicators of individual exposure, they may not effectively reflect
the actual exposure status of the ovary. Follicular fluid (FF), the
micro-environment that oocyte and its surrounding somatic cells
directly contact with, is critical for oocyte health [19]. Any con-
tamination with endocrine disruptors may affect developmental
competence of the oocyte. Therefore, FF may serve as a more suit-
able and biological relevant matrix to assess ovarian phthalate
exposure. Additionally, studies examining other EDCs in FF sam-
ples obtained from women undergoing IVF have demonstrated that
exposure to environmental chemicals can result in detectable con-
centrations in FF [20,21]. Some of these EDCs found in FF were even
suggested to associate with adverse reproductive outcomes includ-
ing elevated risk of fertilization and implantation failure [22,23].
Findings from the above studies underscored the need to identify
whether phthalates would present in the FF and may cause similar
effects observed in experimental animal studies.
To test our hypothesis, we first aimed to quantify eight
phthalate metabolites, including monomethyl phthalate (MMP),
monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP),
MEHP, mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP),
mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), monobenzyl
phthalate (MBzP) and mono-n-octyl phthalate (MOP), in FF and
urine samples from women undergoing in vitro fertilization (IVF).
These metabolites were chosen to determine because their parent
compounds, i.e., dimethyl phthalate (DMP), diethyl phthalate
(DEP), di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP),
di(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DOP),
were among the most commercially significant phthalates with
large volumes of production and widespread uses [24]. The US
Environmental Protection Agency (USEPA) has also identified
the six chemicals as priority pollutants. In order to investigate
whether it was possible to predict concentrations of metabolites
in the FF based on urinary measurements, we went further by
studying the correlations between levels in the two matrices.
Finally, the associations of metabolite concentrations with IVF
parameters, comprising peak E2 levels and oocyte yielding, both
reflecting ovarian response to hyperstimulation [25,26], as well as
early reproductive outcomes, such as the number of matured and
fertilized oocytes, and Day 3 embryo quality, were explored. These
early reproductive end-points which could be only observed in
an IVF laboratory reflect stages of reproduction that are critical to
form a fully competent embryo and predict pregnancy outcomes
[27,28].
143
2. Materials and method
2.1. Study population and data collection
In this prospective cohort study, 112 women seeking infertility
treatment at the Reproductive Medicine Center of Tongji Hospi-
tal, Wuhan, China, between July, 2014 and August, 2014 were
recruited. Among couples attending the infertility clinic, the female
partners aged 20–45 years, diagnosed with infertility (not being
pregnant without protected intercourse for more than one year),
and with indications for IVF or ICSI, were eligible for the study.
Patients who received oocyte donation (n = 3) or used cryo-thawed
oocytes (n = 2) were not included in the study. During the studying
period, the long gonadotropin-releasing hormone (GnRH) agonist
and GnRH antagonist protocols were the main stimulation regi-
mens used in the clinic, which accounted for nearly 90% of the
treatment cycles. The clinical pregnancy rates per transfer cycle at
the clinic maintained at around 55%. All enrolled patients were fol-
lowed up until they withdrew from the study because of achieving
a live birth or discontinuing the treatment due to other reasons.
At the time of recruitment, each participant signed an informed
consent and completed a questionnaire collecting information
about demographics, life-style, and medical and reproductive his-
tories. Their clinical data on antral follicle count (AFC), serum peak
E2 concentrations, and IVF outcomes, etc. were obtained from elec-
tronic medical charts. AFC was determined through transvaginal
ultrasound on the third day of a menstrual cycle as a routine
infertility evaluation at the clinic. The study was approved by the
Institutional Review Board of Tongji Hospital.
2.2. Ovarian stimulation
Patients underwent either a long GnRH agonist or a GnRH antag-
onist protocol, according to their ovarian response. A long GnRH
agonist regimen was offered to patients with a normal ovarian
response, and pituitary suppression was achieved by daily injection
of triptorelin acetate (Decapeptyl; Ferring) initiating from the mid-
luteal phase prior to the stimulation cycle. While among women
with a diminished ovarian reserve or a known poor response, a
flexible GnRH antagonist protocol was conducted by administrat-
ing 0.25 mg Cetrorelix Acetate (Cetrotide; Merck-Serono) when at
least one follicle reached 14 mm. Recombinant follicle-stimulating
hormone (FSH) (Gonal-F, Serono or Puregon, MSD) was given to
induce ovarian stimulation starting with 150 IU/d and adjusted
based on the ovarian response, as previously described [29]. Ovula-
tion was triggered by Recombinant human chorionic gonadotropin
(HCG) (250 mg; Ovidrel; Serono) when a mean diameter of 18 mm
was reached by at least two leading follicles. Ovum pick-up was
performed transvaginally 34–36 h after trigger with HCG.
2.3. IVF procedures
Details on the IVF procedure and embryo culture have been
previously described [30]. Briefly, the oocytes were evaluated for
maturity 2–4 h after ovum pick-up, as evidenced by extruding the
first polar body, and fertilized either by conventional insemina-
tion or intracytoplasmic sperm injection (ICSI). Fertilization was
checked 16–18 h after insemination, and normal fertilization was
defined as the appearance of two pronuclei (2PN). The count of
2PN relative to the amount of retrieved oocytes was referred to
as fertilization rate. Embryo consisted of seven to nine cells with-
out multinucleation and had less than 20% fragments on day 3 after
oocyte pick-up were considered as a good-quality embryo [31]. The
percentage of optimal embryos among the total number of cleav-
age embryos was calculated as good-quality embryo rate. Usually,
fewer than two embryos of good quality were chosen for transfer
144 Y.-Y. Du et al. / Reproductive Toxicology 61 (2016) 142–150
on day 3, with excess embryos being cryopreserved or continuously
cultured to the blastocyst stage. On day 5 or 6, the number of blas-
tocyst formation was noted and divided by the amount of embryos
cultured to blastocyst stage, referred to as blastocyst formation rate.
2.4. Sample collection and hormone measurement
Blood serum on day 3 of the preceding menstrual cycle, together
with serum samples on the day of HCG administration were col-
lected for measurement of basal FSH and peak E2 concentrations,
respectively. Details of hormone measurement could be found in
a previous study [29]. On the day of oocyte retrieval, 110 FF and
110 spot urine samples were obtained from 112 subjects. Among
them, 108 were paired samples. All specimens were collected with
sterile polypropylene containers. Pooled FF from follicles >17 mm
verified by ultrasound was collected. And FF contaminated with
blood or flushing medium was discarded. Following ovum pick-up,
the samples were centrifuged immediately at 1500 revolution/min,
4 ◦ C for 10 min. After the treatment, the supernatant was collected
and aliquoted into 2 mL specimens. Both FF and urine samples were
stored at −80 ◦ C until phthalate metabolites analysis. To adjust for
urine dilution, urinary creatinine concentrations were determined
with an automated clinical chemistry analyzer at the Tongji hospi-
tal clinical laboratory.
2.5. Phthalate metabolites assessment
Concentrations of eight phthalate metabolites, including MMP,
MEP, MEHHP, MBP, MEOHP, MBzP, MEHP and MOP, were quanti-
fied according to previously described methods [32],with minor
modifications. Briefly, 200 ␮L of phosphoric acid was added to
1 mL of FF specimens to terminate hydrolysis, while urine sam-
ples were spared this step due to lack of hydrolytic enzymes.
After being spiked with 80 ␮L of internal standards, both FF
and urine were incubated with 10 ␮L ␤-glucuronidase (Roche,
Mannheim, Germany) at 37 ◦ C for 90 min. Following the enzy-
matic deconjugation, the phthalate monoester compounds were
extracted using solid-phase extraction (SPE) cartridges (Oasis HLB,
Waters Co., Milford, MA). Dried extracts were then analyzed with
high-performance liquid chromatography and tandem mass spec-
trometry (6460LC–MS, Agilent Technologies Co., Santa Clara, CA).
Apart from the study specimens, one blank and two quality con-
trols were run along with each batch of samples. The blank control
contained 1-mL water was used to assess the contaminations dur-
ing sample processing and analysis. Two follicular fluid samples
from unknown subjects spiked with 0.5 and 5 ng/mL of the target
phthalates, respectively, coupled with two urine samples (con-
tained 5 and 50 ng/mL of the target phthalates), were used as quality
controls to determine the accuracy and precision of the method
by calculating the recovery. The linearity correlation coefficient
(R2 ) of calibration curves was >0.99, with a linear range of 0.2–10
and 0.5–200 ng/mL for FF and urine samples, respectively. The lim-
its of detection (LOD) of target metabolites ranged from 0.01 to
0.04 ng/mL, and the recovery for FF metabolites was 86.12-99.26%,
and 88.06-100.93% for urine. According to previous studies [33,34],
the proportion of DEHP detected as the bioactive monoester in
the specimens was calculated by converting the concentrations of
MEHP, MEHHP and MEOHP into nanomoles per milliliter. MEHP
was further divided by the sum of the three metabolites, and mul-
tiplied by 100. The computed value was expressed as %MEHP to
reflect the body burden of MEHP.
2.6. Statistical analyses
The demographic and clinical characteristics, cycle outcomes
and the distributions of metabolite concentrations were summa-
rized with descriptive statistical analyses. Basic characteristics
were presented as mean ± SD (range) or median (IQR) or number
(%), where appropriate. Geometric means, medians and selected
percentiles were calculated to describe the distributions of follic-
√
ular fluid and urinary phthalate metabolites. A value of LOD/ 2
was assigned to samples with concentration below the LOD. To
adjust for urine dilution, concentrations of urinary metabolites
were divided by creatinine and presented as microgram per gram
creatinine, before the analyses. Due to the relatively low detection
frequencies (<50%) of MBzP in the FF specimens and MOP in both
matrices, they were excluded from subsequent analyses. Spearman
correlation analysis was conducted to explore the interrelation-
ships of concentrations between paired matrices. The potential
associations of metabolite levels with patient and cycle character-
istics, including age, BMI and indicators of embryo development,
were assessed by bivariate correlation analyses.
Multiple linear regression analyses were performed on both nat-
ural logarithm transformed concentrations (continuous) as well
as categorized data (untransformed) to evaluate the associations
between FF and urinary concentrations of phthalates and IVF
parameters. Serum peak E2 levels, the number of retrieved oocytes,
mature and normally fertilized oocyte count, and the amount
of blastocyst formed were square root transformed to normal-
ize their distribution, while the number of good-quality embryos
and good-quality embryo rate, with normal distribution, were
kept untransformed. For fertilization and blastocyst formation rate
which were non-normally distributed and could be expressed as a
proportion, namely, 2PN-zygotes among the number of retrieved
oocytes and the amount of blastocyst formed relative to cultured
embryo count, logistic regression was conducted as described by
Rabe-Hesketh and Everit [35]. According to the distribution of
the study population, metabolite concentrations (untransformed)
were divided into tertiles, modeled as an ordinal categorical vari-
able, and assigned with an integer value of 1, 2 and 3, respectively.
Tests for trend were performed by entering the assigned values as a
continuous variable to explore the potential linear dose–response
relationships between tertiles of concentrations and the parame-
ters of interest.
Covariates with either biological relevance or statistical con-
siderations were selected to enter the regression models. Given
their associations with both phthalate exposure and IVF outcomes
reported in the literature [36–38], age and BMI were forced into
regression models regardless of their effect on phthalate parame-
ter estimate. Covariates including Day 3 serum FSH, AFC, duration
of infertility, stimulation protocol (long GnRH agonist versus GnRH
antagonist protocol) and insemination technique (IVF versus ICSI),
whose enter caused >10% change in effect estimates for metabo-
lite regression coefficients were considered as confounders and
retained in the multivariate models. To maintain consistency,
models assessing a group of indicators representing similar out-
comes, such as ovarian response or embryonic development, were
adjusted for the same set of covariates. To control the type I error
rate induced by multiple comparisons, the false discovery rate
(FDR) correction was conducted to adjust the P-values [39]. All tests
with a P-value <0.05 were considered statistically significant. Data
were analyzed with either the Predictive Analytics Suite Worksta-
tion (PASW) version 22.0 (IBM Corporation, Armonk, NY) or Stata
11 (Stata Corp LP, TX, USA).
3. Results
3.1. Characteristics of study population
The patients included in the present study were on average
31 ± 4.4 years old with an average BMI of 22 ± 2.5 kg/m2 , and most
 Y.-Y. Du et al. / Reproductive Toxicology 61 (2016) 142–150 145

Table 1 late metabolites were highly detected in the FF (percent detectable

Demographics and clinical characteristics of the subjects (N = 112).
ranging from 72.73% to 100%), except for MBzP and MOP with a
Characteristic Data detection frequency of 43.64% and 14.55%, respectively. The high-
Age (years) 31 ± 4.4 (21–45) est amount in the FF was found for MEHP with a median level
BMI (kg/m2 ) 22 ± 2.5 (16.4–29.3) of 2.80 ng/mL, followed by MBP (median 2.05 ng/mL). The other
metabolites had much lower concentrations with median levels
Race
Han 107 (95.5) close to their respective LODs (ranging from < LOD to 0.65 ng/mL).
Other 5 (4.5) The majority of urinary metabolites were detectable in >77.27% of
Smoking
participants, with the exception of MOP (16.63%). Generally, the
Never smoked 112 (100%) urine samples had higher percentages of detectable concentra-
Ever smoked 0 tions than that in the FF. Exposure levels of urine varied widely
Gravidity by metabolite, with MBP having the highest median concentration
Yes 53 (47.3%) among the analytes (median 102.30 ␮g/g Cr) which was on aver-
Natural pregnancy 46 (86.8%) age 9-fold higher than the following MMP (median 12.51 ␮g/g Cr)
Assisted pregnancy 7 (13.2%) and MEP (median 12.17 ␮g/g Cr). In the FF samples, the concen-
No 59 (52.7%)
Duration of infertility (years) 4 (2–6)
trations of metabolites were generally two orders of magnitude
lower compared to the corresponding compounds in the urine.
IVF/ICSI treatment indication
Spearman correlation coefficients between levels of metabolites in
Tubal or pelvic factor infertility 66 (58.9)
Ovulation disorders 9 (8.0) paired FF and urine samples are shown in Table 3. Statistically sig-
Diminished ovarian reserve 7 (6.3) nificant correlations between levels in both matrices were found
Endometriosis 6 (5.4) for MEP (rs = 0.44, p < 0.01), and MBP (rs = 0.22, p < 0.05), with mod-
Uterine disorders 1 (0.9) erate to weak correlations. Nevertheless, for the other metabolites,
Male factor 22 (19.6)
no apparent correlations were observed.
Unexplained 1 (0.9)
Day 3 FSH (IU/L) 6.73 (5.64–8.16)
AFC 14 (9-19)
Peak E2 (pg/ml) 3923 (2495–5847)
3.3. Phthalate metabolite exposure and IVF parameters

Stimulation protocol
There were no associations between ln-transformed FF concen-
Long GnRH agonist protocol 95 (84.8)
GnRH-antagonist protocol 17 (15.2)
trations of phthalate metabolites in relation to the reproductive
outcomes as well as urinary results after correction for multiple
Oocyte insemination technique
comparisons (Supplementary Table S1–Table S4). The associations
IVF 80 (72.7)
ICSI 30 (27.3) of phthalate metabolite concentrations in FF with ovarian response
Oocytes retrieveda 13 (8-19) and oocyte maturation in multivariable models are presented in
Mature (MII) oocytes 11 (7-16) Table 4. Both peak serum E2 level and oocyte yield, indicators of
Maturation rate (%) 93.3 (83.8–100) ovarian response, did not correlate with any metabolites in the FF
Normal fertilized (2PN) oocytes 7 (4–11)
Fertilization rate (%) 60 (50–75)
after adjusting for age, BMI, AFC and stimulation protocol. Similarly,
Day 3 good-quality embryos 5 (2–7) the number of M II oocytes showed no associations to increasing
Good-quality embryo rate (%) 50 (33.3–63.6) metabolite concentrations. Table 5 presents the concentrations of
Blastocyst formationb 3 (1–7) FF metabolites relative to parameters of oocyte and embryo devel-
Blastocyst formation rate (%) 71.4 (50–90.9)
opment, including 2PN oocyte counts, the number of good-quality
Data are mean ± SD (range) or median (IQR) or number (%). embryos and blastocyst formed, and exhibits no evidence for any
a
Patients had oocyte retrieval procedures, n = 110. dose–response trend. No statistically significant dose-response
b
Patients had embryos cultivated to the blastocyst stage, n = 97.
patterns between phthalate metabolite concentrations and the fer-
tilization rate, the percent of good-quality embryos and the rate of
of them are Han (95.5%). None of the patients were current smok- blastocyst formation were observed after adjustment for multiple
ers or alcohol users. A total of 53 women (47.3%) had a history of testing (Table 6). To explore dose-response relationships between
pregnancy, and 86.8% got pregnant naturally. More than half of the considered parameters and the percent of DEHP excreted as the
participants underwent ART procedures because of tubal or pelvic bioactive toxicant, %MEHP were also included in the multivari-
infertility, and 19.6% due to male factor infertility. Other patient and able regression models and modeled into tertiles. However, none
cycle characteristics are presented in Table 1. Of the 112 patients, of the parameters showed any associations to %MEHP. With regard
only two women had oocyte retrieval procedures cancelled because to urinary results, there were no correlations between metabolite
of poor response to ovarian stimulation. Seven patients with less concentrations and measured reproductive outcomes in adjusted
than three good-quality cleavage embryos chose for transfer on models with correction for multiple testing (Supplementary Table
day 3 after fertilization. Six women had all day-3 embryos cry- S5–Table S7).
opreserved owing to risk of ovarian hyperstimulation syndrome
or serum progesterone elevation, thus resulting thirteen patients 4. Discussion
being excluded from the blastocyst stage analyses.
In this prospective cohort study of 112 women recruited from
3.2. Distribution of phthalate metabolite concentrations an infertility clinic, we found that the intra-ovarian environment
of the study population has been pervasively exposed to phtha-
Among the 112 participants, 108 women contributed two sam- lates despite the low concentrations. Moreover, no statistically
ples comprising one FF and one urine sample. The remaining significant associations between concentrations of FF and uri-
four participants each contributed a single sample of either kind. nary phthalate metabolites and IVF parameters, including ovarian
Thus, a total of 220 samples were subjected to metabolite concen- response and early IVF outcomes, were observed.
tration assessment. Table 2 presents the distributions of FF and Knowledge about the EDCs exposure status of the ovary is lim-
creatinine-adjusted urinary phthalate metabolites. Most of phtha- ited and of concern for reproductive risk assessment. In the current
146 Y.-Y. Du et al. / Reproductive Toxicology 61 (2016) 142–150

Table 2
Distribution of follicular fluid and urinary phthalate metabolitesa (n = 110).

Metabolites %>LOD GM Median Min Selected percentiles Max

25% 75%

MMP FF 100 0.83 0.65 0.10 0.32 1.34 133

Urine 77.27 4.31 12.51 <LOD 0.53 37.50 16444
MEP FF 96.36 0.15 0.11 0.01 0.07 0.23 11.96
Urine 100 14.80 12.17 0.12 7.26 25.79 548
MEHHP FF 100 0.18 0.15 0.02 0.09 0.04 6.68
Urine 100 10.31 9.88 1.59 6.42 14.56 286
MBP FF 88.18 1.59 2.05 <LOD 0.66 6.41 415
Urine 100 138 102.30 11.66 54.91 176 2324
MEOHP FF 72.73 0.03 0.02 <LOD 0.01 0.25 1.54
Urine 100 6.02 6.46 0.23 3.88 9.26 98.93
MBzP FF 43.64 0.03 <LOD <LOD <LOD 0.16 6.83
Urine 100 0.87 0.72 0.23 0.52 1.28 13.02
MEHP FF 93.64 1.90 2.80 <LOD 0.54 8.06 239
Urine 99.09 4.64 4.57 <LOD 2.68 9.33 113
MOP FF 14.55 0.04 <LOD <LOD <LOD <LOD 4.10
Urine 16.63 0.03 <LOD <LOD <LOD <LOD 0.61
%MEHP FF – 71.29 91.01 4.57 67.31 97.52 99.95
Urine – 21.71 23.59 1.40 16.51 30.80 68.45

%>LOD: Phthalate metabolites above the limits of detection. FF: follicular fluid. GM: geometric mean. Min: minimum. Max: maximum.
%MEHP: the percentage of DEHP metabolites excreted as MEHP.
a
Urinary phthalate metabolite concentrations were creatinine adjusted (␮g/g Cr).

Table 3
Spearman correlation coefficients of phthalate metabolites between paired follicular fluid and urine samplesa (n = 108).

Metabolites MMP MEP MEHHP MBP MEOHP MEHP

rs 0.13 0.44** 0.05 0.22* 0.12 0.11

a
Urinary phthalate metabolite concentrations were creatinine adjusted (␮g/g Cr).
**
P-value < 0.01, two-tailed.
*
P-value < 0.05, two-tailed.

study, for the first time, we determined levels of eight phthalate
metabolites in 110 FF samples in a Chinese population to investi-
gate whether phthalates can reach the intra-ovarian environment
and serve as a biologically relevant marker of exposure. Our results
suggested that phthalate metabolites measured in the FF could be
used for exposure assessment because they were detectable in the
majority of the samples, except for MBzP and MOP. We are aware
of only one other publication [40] with data available on phtha-
late levels in FF. In contrast to our findings, MBzP and MEOHP
were not detected in any of the specimens. This may be attributed
to the small sample size (N = 5) of the previous study. However,
the low concentrations found in our study were comparable to
theirs in which a mean level of all metabolites ranged from 1.19 to
9.34 ng/mL. Additionally, our observation showed that MEHP and
MBP were the dominant metabolites in the FF, similar with their
findings of MEHP showing the highest amount.
Since access to FF is difficult which could be practically obtained
through an IVF setting, we measured concentrations of phthalate
metabolites in paired FF and urine samples to find out if levels
in the FF could be assessed based on measurement in urine. The
urinary concentrations analyzed here were in the ranges of pre-
vious studies in other regions of China with different population
groups [41–43], as well as a study of Japanese women [44]. Our
data were comparable to a recent Chinese study evaluating urinary
concentrations of metabolites in women with clinical pregnancy
loss [43]. The highest amount of MBP measured in our study was
in agreement with the later one. When comparing with data from
American and Puerto Rican women of childbearing age [45–47],
women in the present study had similar distribution for most of the
metabolites, except concentrations of MBP were consistently high,
while MEP exhibiting approximately 3 to 8-fold lower. Discrepan-
cies in the distribution of phthalates may be partially explained
by different exposure profiles related to lifestyle, cultural prac-
tices, governmental legislation and socio–economic factors, such
as long-term exposure to certain phthalate-tainted food, and dif-
ferent frequencies in cosmetics and personal care products use.
Serving as an internal exposure biomarker, the measurement of
urinary phthalate metabolites represents exposure from all routes.
This may be the reason why the concentrations of urinary metabo-
lites were extraordinarily high compared to their counterparts in
the FF. Additionally, when assessing the agreement of metabolite
concentrations between paired urine and FF samples, only weak
to moderate correlations were observed for MBP and MEP. The
reason for the lack of correlations is unclear, but it may reflect
difference in the metabolic clearance of phthalates between the
two matrices. It has been suggested that when administrated with
DEHP orally, the metabolites in serum reached their highest levels
approximately 2 h after exposure and declined rapidly afterward.
While concentrations in urine were still rising and achieved peak
levels 4 h after dosing [48]. Thus, given the fact that FF is a filtrate
of thecal capillary blood, the agreement between levels in FF and
urine samples collected at the same time may not be observed. As
a result, urinary phthalate metabolite measurements may not ade-
quately reflect phthalate metabolite measurements in the follicular
environment, and therefore may not be appropriate for predicting
metabolite levels in FF.
To our knowledge, this is the first epidemiological study to
investigate the associations between FF levels of phthalate metabo-
lites and IVF parameters. It is essential to relate our results to
experimental findings, since there is only a paucity of human data.
Laboratory studies have established that phthalates, especially
DEHP, di-n-butyl phthalate (DBP) and their respective monoesters,
adversely influence ovarian function through effect on oocyte,
granulosa cells and follicles at different stages, resulting in compro-
mised oocyte development, suppressed E2 production, accelerated
primordial follicle pool depletion and antral follicle atresia. How-
 Y.-Y. Du et al. / Reproductive Toxicology 61 (2016) 142–150 147

Table 4
Associations between tertiles of phthalate metabolite concentrations in follicular fluid and peak E2 , number of oocytes retrieved and MII oocytes in multivariable models.

Metabolitetertilesa Peak E2 d Number of oocytes retrievedd Number of MII oocytesd

␤ (95%CI) ␤ (95%CI) ␤ (95%CI)

MMP
1b ( < 0.41) Ref Ref Ref
2 (0.41–0.92) −0.85 (−8.53, 6.83) −0.02 (−0.41, 0.36) −0.11 (−0.45, 0.24)
3 (0.93–133) −0.33 (−8.16, 7.49) 0.20 (−0.20, 0.59) 0.17 (−0.18, 0.52)
Test for trendc 0.93 0.33 0.36
MEP
1b ( < 0.088) Ref Ref Ref
2 (0.088–0.156) −5.37 (−13.37, 2.64) 0.24 (−0.16, 0.64) 0.29 (90.07, 0.65)
3 (0.157–11.96) −1.84 (−9.61, 5.94) 0.38 (−0.01, 0.77) 0.32 (−0.03, 0.67)
Test for trendc 0.66 0.06 0.08
MEHHP
1b ( < 0.11) Ref Ref Ref
2 (0.11–0.212) −2.59 (−10.25, 5.07) −0.27 (−0.65, 0.12) −0.20 (−0.55, 0.15)
3 (0.213–6.68) 0.50 (−7.28, 8.29) −0.12 (−0.51, 0.27) −0.05 (−0.40, 0.31)
Test for trendc 0.91 0.54 0.79
MBP
1b ( < 1.12) Ref Ref Ref
2 (1.12–4.13) 2.51 (−5.41, 10.42) 0.00 (−0.40, 0.41) −0.04 (−0.41, 0.32)
3 (4.14–415) −0.28 (−8.05, 7.49) −0.04 (−0.44, 0.35) 90.10 (−0.45, 0.26)
Test for trendc 0.92 0.83 0.85
MEOHP
1b ( < 0.0124) Ref Ref Ref
2 (0.0124–0.290) 6.09 (−1.50, 13.68) 0.06 (−0.36, 0.45) 0.14 (−0.22, 0.49)
3 (0.291–1.54) −1.98 (−9.67, 5.70) −0.05 (−0.45, 0.35) 0.04 (−0.32, 0.40)
Test for trendc 0.62 0.77 0.85
MEHP
1b ( < 1.08) Ref Ref Ref
2 (1.08–5.24) 3.37 (−4.54, 11.27) 0.15 (−0.25, 0.55) 0.14 (−0.22, 0.50)
3 (5.25–239) 5.52 (−2.38, 13.42) 0.22 (−0.18, 0.62) 0.21 (−0.16, 0.57)
Test for trendc 0.17 0.28 0.26
%MEHP
1b ( < 80.28) Ref Ref Ref
2 (80.28–96.35) −0.54 (−8.21, 7.12) 0.04 (−0.35, 0.43) 0.01 (−0.35, 0.37)
3 (96.36–99.95) 4.76 (−3.07, 12.59) 0.09 (−0.32, 0.49) 0.04 (−0.33, 0.40)
Test for trendc 0.23 0.67 0.84

Adjusted for age, BMI, AFC and stimulation protocol.

a
Phthalate metabolite concentrations were categorized into tertiles.
b
Reference category.
c
P-values for trend.
d
Values were square root transformed.

ever, in contrast to the animal data, no associations between FF
metabolite concentrations and the considered parameters were
observed in the present study. Evidence from animal and in vitro
studies have indicated that much higher levels of phthalates were
required to induce reproductive toxicity than those detected in
our study. Using in vitro cultured human luteinized granulosa
cells, Reinsberg et al. (2009) reported that the minimal concen-
trations of MEHP required to inhibit E2 production by 50% was
three orders of magnitude higher than the median level we mea-
sured [17]. Similarly, exposing oocyte to 50 ␮m MEHP (identical
to 14 ␮g/mL after conversion) could adversely affect its nuclear
and cytoplasm maturation, contributing to reduced developmental
competence of oocytes, decreased cleavage rate of embryos and the
inability to develop to the blastocyst stage [13,49]. A recent study
demonstrated that administration of MEHP to cultured ovaries
and antral follicles at concentrations ranging from 0.2–20 ␮g/mL
and 0.1–10 ␮g/mL, respectively, led to accelerated initial recruit-
ment and declined levels of testosterone, estrone and estradiol [50].
Therefore, the absence of associations may be partly attributed to
the low exposure concentrations compared to the minimum dosage
reported to inducing ovarian toxicity.
Given the fact that there are a finite number of oocytes and
oocyte development occurs from early fetal life through to adult-
hood, the supporting microenvironment (e.g. follicular fluid) poses
a potential route of exposure to various EDCs [34]. It has been noted
that DEHP caused increased antral follicle atresia in adulthood fol-
lowing fetal and neonatal exposure in an animal model [51]. Moyer
et al. (2012) demonstrated that female rodent offspring presented
early reproductive senescence arising from in utero exposure to
MEHP [52]. With regard to human studies, prenatal exposure to
MEP led to decreased serum anti-Müllerian hormone (AMH) levels
and declined incidence of polycystic ovarian morphology (PCO) in
adolescence, suggesting a latent effect of phthalates on the ovary
[53]. DEHP was shown to have an adverse impact on puberty devel-
opment, manifested by negative correlations between reduced
uterus volume among adolescent girls and maternal urinary lev-
els of MEHP and total DEHP [54]. Thus, exposing to phthalates
at critical developmental stages may pose detrimental effects on
female reproduction later in life and make the exposure difficult
to quantify. On the other hand, phthalates measured in FF only
reflect a relatively short period of exposure, because the fluid does
not generate until follicles grow into the antral phase. Therefore,
based on our observation, FF may be a feasible matrix for assess-
ing ovarian exposure to phthalates, but it may not adequately
provide informative results when studying reproductive param-
eters.
With regard to urinary concentrations of phthalates in rela-
tion to interested outcomes, a recent study by Gaskins/Hauser
et al. (2015) found evidence for metabolites of DEHP in associ-
ation with decreased oocyte yield and amount of M II oocytes
[46] in contrast to our negative findings. Their much larger
sample size with sufficient power of test as compared to us,
148 Y.-Y. Du et al. / Reproductive Toxicology 61 (2016) 142–150

Table 5
Associations between tertiles of phthalate metabolite concentrations in follicular fluid and number of 2PN oocytes, good-quality embryos and blastocyst formation in
multivariable models.

Metabolitetertilesa Number of 2PN oocytesd Number of good-qualityembryos Number of blastocyst formationd

␤ (95%CI) ␤ (95%CI) ␤ (95%CI)

MMP
1b (< 0.41) Ref Ref Ref
2 (0.41–0.92) −0.13 (−0.47, 0.20) −0.01 (−1.34, 1.33) 0.32 (−0.15, 0.79)
3 (0.93–133) 0.25 (−0.09, 0.59) −0.22 (−1.57, 1.13) 0.42 (−0.07, 0.90)
Test for trendc 0.18 0.76 0.09

MEP
1b (< 0.088) Ref Ref Ref
2 (0.088–0.156) 0.19 (−0.17, 0.55) 0.46 (90.94, 1.86) 0.11 (−0.41, 0.62)
3 (0.157–11.96) 0.33 (−0.02, 0.67) 0.66 (−0.70, 2.02) 0.34 (−0.15, 0.84)
Test for trendc 0.06 0.34 0.16

MEHHP
1b (< 0.11) Ref Ref Ref
2 (0.11–0.212) −0.19 (−0.53, 0.15) −0.74 (−2.07, 0.59) −0.43 (−0.90, 0.05)
3 (0.213–6.68) −0.05 (−0.39, 0.30) −0.77 (−2.11, 0.58) 0.06 (−0.41, 0.54)
Test for trendc 0.79 0.26 0.80

MBP
1b (< 1.12) Ref Ref Ref
2 (1.12–4.13) −0.10 (−0.46, 0.26) −0.27 (−1.65, 1.12) −0.22 (−0.73, 0.29)
3 (4.14–415) −0.03 (−0.38, 0.32) −0.06 (−1.41, 1.29) −0.09 (−0.58, 0.40)
Test for trendc 0.87 0.95 0.76

MEOHP
1b (< 0.0124) Ref Ref Ref
2 (0.0124–0.290) 0.19 (−0.16, 0.54) 0.39 (−0.97, 1.74) 0.13 (−0.37, 0.63)
3 (0.291–1.54) 0.14 (−0.21, 0.49) −0.27 (−1.64, 1.10) −0.02 (−0.52, 0.48)
Test for trendc 0.44 0.69 0.92

MEHP
1b (< 1.08) Ref Ref Ref
2 (1.08–5.24) −0.06 (−0.41, 0.30) −0.60 (−1.98, 0.78) 0.10 (−0.41, 0.61)
3 (5.25–239) 0.09 (−0.27, 0.45) 0.07 (−1.32, 1.45) 0.16 (−0.34, 0.66)
Test for trendc 0.60 0.89 0.53

%MEHP
1b (< 80.28) Ref Ref Ref
2 (80.28–96.35) −0.06 (−0.41, 0.29) −0.03 (−1.37, 1.32) 90.16 (−0.66, 0.34)
3 (96.36k99.95) −0.10 (−0.45, 0.26) −0.42(−1.79, 0.96) −0.13 (90.63, 0.36)
Test for trendc 0.58 0.54 0.61

Adjusted for age, BMI, AFC, stimulation protocol and oocyte insemination technique (IVF/ICSI).
a
Phthalate metabolite concentrations were categorized into tertiles.
b
Reference category.
c
P-values for trend.
d
Values were square root transformed.

may partially explain the different results between the two to determine the contamination status of FF and measure the IVF
studies. Other possible reasons may rest with differences in end-points.
potential confounding (e.g., exposure to mixed chemicals), and
analytical methods (e.g., our consideration for chance finding).
5. Conclusion
Thus, additional research is needed to investigate the validity
of measuring urinary metabolites with respect to IVF out-
In conclusion, our preliminary data indicated that most phtha-
comes.
late metabolites measured in the study were highly detected in the
There are some limitations of the present study. One major limi-
ovarian FF of women undergoing IVF. However, the levels of phtha-
tation is our small sample size which may not have adequate power
lates are orders of magnitude lower than those shown to induce
to detect significant associations. Another limitation is that the
ovarian toxicity in animal studies. It has to be noted that although
study population was recruited from an infertility clinic which pre-
we did not observe any associations between phthalate metabolite
cluded extrapolation of our results to the general population. The
concentrations and IVF parameters, we cannot exclude their pos-
single spot urine and single measurement of FF used for exposure
sible influences on female reproduction which could be detected
assessment may cause exposure misclassifications owing to their
from a larger sample analysis. Thus, further study with a larger
relatively short biological half-life. However, it has been reported
cohort would be warranted to verify the findings of the present
that when modeled into categories based on exposure levels, a sin-
study.
gle measure of spot urine is possible to represent the exposure
extent over several months [55,56]. Finally, exposure to mixtures
of environmental pollutants with diverse toxicity may influence Conflict of interest
the interpretation of our data. Despite the aforementioned limi-
tations, this study is strengthened by our prospective study design The authors declare that there are no competing financial inter-
and the application of an IVF setting which provided us an approach ests.
 Y.-Y. Du et al. / Reproductive Toxicology 61 (2016) 142–150 149

Table 6
Associations between tertiles of phthalate metabolite concentrations in follicular fluid and fertilization rate, good-quality embryos rate and blastocyst formation rate in
multivariable models.

Metabolitetertilesa Fertilization rated Good-quality embryo ratee Blastocyst formation ratef

OR (95%CI) ␤ (95%CI) OR (95%CI)

MMP
1b (< 0.41) Ref Ref Ref
2 (0.41–0.92) 0.86 (0.67, 1.11) 0.01 (−0.09, 0.12) 1.49 (0.99, 2.24)
3 (0.93–133) 1.16 (0.89, 1.49) −0.03 (−0.13, 0.08) 1.58 (1.03, 2.44)
Test for trendc 0.46 0.66 0.14

MEP
1b (< 0.088) Ref Ref Ref
2 (0.088–0.156) 1.11 (0.85, 1.45) 0.07 (−0.04, 0.18) 1.00 (0.66, 1.53)
3 (0.157–11.96) 1.25 (0.95, 1.63) 0.03 (−0.08, 0.13) 1.83 (1.18, 2.85)
Test for trendc 0.35 0.66 0.07

MEHHP
1b (< 0.11) Ref Ref Ref
2 (0.11–0.212) 1.06 (0.82, 1.38) −0.01 (−0.12, 0.10) 1.20 (0.91, 1.59)
3 (0.213–6.68) 1.11 (0.86, 1.43) −0.11 (−0.22, 0.00) 1.29 (0.97, 1.70)
Test for trendc 0.50 0.35 0.63

MBP
1b (< 1.12) Ref Ref Ref
2 (1.12–4.13) 0.77 (0.60, 1.00) 0.06 (−0.05, 0.17) 0.81 (0.53, 1.22)
3 (4.14–415) 0.98 (0.76, 1.27) −0.04 (−0.15, 0.07) 0.90 (0.60, 1.37)
Test for trendc 0.95 0.63 0.63

MEOHP
1b (< 0.0124) Ref Ref Ref
2 (0.0124–0.290) 1.07 (0.82, 1.39) 0.01 (−0.10, 0.11) 1.18 (0.78, 1.79)
3 (0.291–1.54) 1.20(0.93, 1.55) −0.06 (−0.17, 0.05) 1.23 (0.81, 1.86)
Test for trendc 0.35 0.63 0.46

MEHP
1b (< 1.08) Ref Ref Ref
2 (1.08–5.24) 0.80 (0.61, 1.05) −0.06 (−0.17, 0.05) 0.89 (0.57, 1.40)
3 (5.25–239) 0.86 (0.66, 1.13) −0.09 (−0.20, 0.02) 0.79 (0.52, 1.22)
Test for trendc 0.50 0.39 0.46

%MEHP
1b (< 80.28) Ref Ref Ref
2 (80.28–96.35) 0.78 (0.60, 1.01) 0.02 (−0.09, 0.13) 0.90 (0.58, 1.40)
3 (96.36–99.95) 0.71 (0.55, 0.92) −0.05 (−0.16, 0.06) 0.76 (0.50, 1.16)
Test for trendc 0.07 0.63 0.44
a
Phthalate metabolite concentrations were categorized into tertiles.
b
Reference category.
c
FDR adjusted P-values for trend.
d
Adjusted for age and BMI.
e
Adjusted for age, BMI, AFC, duration of infertility and oocyte insemination technique (IVF/ICSI).
f
Adjusted for age, BMI, FSH and duration of infertility.

Transparency document References

The Transparency document associated with this article can be
found in the online version.
Acknowledgements
We sincerely thank the nurses and technicians in the Reproduc-
tive Medicine Center of Tongji Hospital in Wuhan for clinical data
and sample collection. This study was supported by the National
Natural Science Foundation of China (grant 81571508), the Natural
Science Foundation of Hubei Province (grant 2014CFA069) and the
Self-dependent Innovation Research Funding of Huazhong Univer-
sity of Science and Technology (grant 2014KXYQ017).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.reprotox.2016.
04.005.
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