ICSI treatments: A prospective single-center study

ICSI treatments: A prospective single-center study

Ecotoxicology and Environmental Safety 188 (2020) 109884 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal ho...

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Ecotoxicology and Environmental Safety 188 (2020) 109884

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

The associations of urinary phthalate metabolites with the intermediate and pregnancy outcomes of women receiving IVF/ICSI treatments: A prospective single-center study

T

Taoran Denga,1, Yaoyao Dua,1, Yixin Wangb,c, Xuemei Tenga, Xiang Huaa, Xiaoqiong Yuana, Yangcheng Yaoa, Na Guoa,∗∗, Yufeng Lia,∗ a

Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095, Jiefang Avenue, Wuhan, Hubei, PR China 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, State Key Laboratory of Environmental Health (incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Phthalate metabolite In vitro fertilization Fertilization Early embryo development Pregnancy outcome

Background: Phthalate exposure was reported to induce defects in ovarian function, and further influence embryo development and pregnancy outcomes. However, the data about the associations of phthalates with intermediate and pregnancy outcomes of in vitro fertilization (IVF) cycles are scarce in the Chinese population. Methods: A total of 663 women receiving IVF/intracytoplasmic sperm injection (ICSI) treatments in our center were enrolled in this analysis. They provided one urine sample on the day of oocyte retrieval. We measured urinary concentrations of eight phthalate metabolites. Generalized linear models were used to analyze the associations of urinary phthalate metabolites with ovarian response, fertilization, early embryo development, and pregnancy outcomes. Results: Among all the phthalate metabolites, mono-n-butyl phthalate (MBP) had the highest urinary concentration with a median level of 101.51 μg/g creatinine (Cr). MBP concentration was inversely associated with normal fertilization odds (overall P-trend < 0.01). There was a significant correlation of monoethyl phthalate (MEP) with decreased odds of normal fertilization in medium-concentration group compared to low-concentration group (overall P-trend = 0.02). No significant associations of metabolite concentrations with the odds of good-quality embryos on day 3 or blastocyst formation were found. Monomethyl phthalate (MMP) and MEP in medium-concentration group reduced 22.4% (95% CI: 0.64–0.94, overall P-trend = 0.04) and 21.9% (95% CI: 0.64–0.95, overall P-trend = 0.05) of the odds to gain good-quality blastocyst compared to low-concentration group. The eight phthalate metabolites were not correlated to clinical pregnancy rate, live birth rate, or early miscarriage rate. There was no significant association of di (2-ethylhexyl) phthalate (DEHP) metabolites observed with any clinical outcomes in the total population. After excluding male infertility, mono (2-ethyl-5hydroxyhexyl) phthalate (MEHHP) in medium-concentration group turned to be associated with a higher number of retrieved oocytes (overall P-trend = 0.04), whereas mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) in medium-concentration group was associated with a lower odds of normal fertilization compared to low-concentration group (overall P-trend = 0.02). Conclusions: Urinary MBP concentration was much higher compared to other phthalate metabolites in this

Abbreviations: IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; LIFE, the Longitudinal Investigation of Fertility and the Environment study; EARTH, the Environment and Reproductive Health study; SEEDS, the Sperm Environmental Epigenetics and Developments Study; FF, follicular fluid; BMI, body mass index; FSH, follicle stimulating hormone; AFC, antral follicle count; MMP, monomethyl phthalate; MEP, monoethyl phthalate; MBP, mono-n-butyl phthalate; DEHP, di (2ethylhexyl) phthalate; MEHP, mono (2-ethylhexyl) phthalate; MEHHP, mono (2-ethyl-5-hydroxyhexyl) phthalate; MEOHP, mono(2-ethyl-5-oxohexyl) phthalate; MBzP, monobenzyl phthalate; MOP, mono-n-octyl phthalate; LOD, limits of detection; 2PN, two pronuclei; Cr, creatinine; EGA, embryonic genome activation; PPAR, peroxisome proliferator-activated receptor; GM, geometric mean; IQR, interquartile range; RR, relative risk; OR, odds ratio; T1, the lowest tertile; T2, the medium tertile; T3, the highest tertile ∗ Corresponding author. No.1095 Jiefang Avenue, Wuhan, 430030, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (N. Guo), [email protected] (Y. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ecoenv.2019.109884 Received 25 April 2019; Received in revised form 15 October 2019; Accepted 26 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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cohort of Chinese IVF/ICSI women, and also higher than it was reported by studies in other countries. MBP showed adverse impacts on fertilization. MMP and MEP could affect blastocyst quality, but not embryo quality on day 3. DEHP metabolites didn’t show consistent reproductive toxicities as demonstrated in previous studies.

1. Introduction

2. Materials and methods

Phthalates, as the most commonly used plasticizer worldwide, are predominantly applied in polyvinyl chloride products and as solvents or matrices, including food packaging, children toys, personal care products, cosmetics, painting materials, coatings for oral medications (Qureshi et al., 2016). Due to their non-covalent bond to plastics, phthalates can leach from these products into the environment, and are absorbed by human body via inhalation, ingestion, and dermal contact (Bui et al., 2016). Exposure to phthalates is ubiquitous for the general population. Phthalates and their metabolites are highly detected in various kinds of human body fluid specimens ((CDC), 2013; Calafat et al., 2004; Du et al., 2016; Frederiksen et al., 2014; Saravanabhavan et al., 2013). In the report of The National Health and Nutrition Examination Survey (NHANES), seven phthalate metabolites could be detected in more than 75% of urine samples obtained from the US population, and higher levels were observed in females than in males with similar age (Silva et al., 2004). Likewise, most phthalate metabolites were found to be of detectable concentrations in other populations worldwide, including Chinese reproductive aged women (Du et al., 2016). Phthalates have been proved to be an endocrine disruptor and linked to adverse reproductive outcomes. Animal experiments have demonstrated that phthalate exposure can induce defects in spermatogenesis, folliculogenesis, steroidogenesis, ovulation, luteal transition, and embryo development (Hannon and Flaws, 2015; Chu et al., 2013a, 2013b). Those reproductive toxicities urged attention to evaluating the effects of phthalates on human fertility. The Longitudinal Investigation of Fertility and the Environment (LIFE) study assessed couple fecundity defined as time to pregnancy among the general preconception population. Paternal exposure to certain phthalates was associated with decreased fecundity (Buck Louis et al., 2016), whereas no association was observed between maternal phthalate exposure and couple fecundity (Buck Louis et al., 2014). Nevertheless, when restricting the participants to those seeking infertility care, certain phthalate metabolites from both female and male partners were found to be correlated with decreased clinical pregnancy rate (CPR) and live birth rate (LBR) (Dodge et al., 2015; Hauser et al., 2016; Al-Saleh et al., 2019). With an in vitro fertilization (IVF) setting, recent work has provided insights into the effects of environmental exposure on human oocyte and early embryo development. However, they have yielded controversial results. Moreover, the data about the associations of phthalates with intermediate and pregnancy outcomes of IVF cycles are scarce in the Chinese population. We previously examined the concentrations of eight phthalate metabolites in 110 pairs of urine and follicular fluid (FF) samples, but no significant associations with intermediate IVF parameters were observed (Du et al., 2016). It was probably because the small sample size had limited power to detect informative results. Therefore, in this study, we enlarged the sample size of participants who received IVF/ intracytoplasmic sperm injection (ICSI) treatments, and reassessed the relationships of urinary phthalate metabolites with ovarian response, fertilization and embryo development. Furthermore, the correlations between phthalate exposure and pregnancy outcomes were evaluated as well.

2.1. Study population The prospective single-center study enrolled 677 participants who received IVF or ICSI treatment during 2014–2016 at Reproductive Medicine Center of Tongji Hospital in Wuhan, China. Women with chromosome abnormalities (n = 10) were ineligible for the study. Four participants who had missing data on the urinary creatinine (Cr) level or phthalate metabolite concentrations were excluded from the cohort. Finally, a total of 663 participants were enrolled and followed up until the delivery or discontinuation of the treatment due to other reasons. Prior to sample collection, all participants signed a written informed consent for participation, and completed a questionnaire regarding demographics, medical histories, reproductive histories, and life-style under the guidance of an investigator. Institutional review board approval was obtained from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The clinical information about intermediate parameters and following pregnancy outcomes of IVF/ICSI cycles was retrieved from electronic medical records. 2.2. Sample collection On the oocyte pick-up (OPU) day, urine samples were collected from female participants with sterile polypropylene containers. All specimens were stored at −80 °C until urinary creatinine analysis and phthalate metabolites measurement. 2.3. Urinary PAEs metabolites measurement Eight phthalate metabolites (mono-n-butyl phthalate (MBP), monoethyl phthalate (MEP), monomethyl phthalate (MMP), mono (2ethylhexyl) phthalate (MEHP), mono (2-ethyl-5-oxohexyl) phthalate (MEOHP), mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), monon-octyl phthalate (MOP), and monobenzyl phthalate (MBzP)) were quantified according to the previously described methods (You et al., 2015; Du et al., 2016). Briefly, after 1mL urine sample being mixed with 3.0mL of ammonium acetate and 80μL of internal standards, urine was incubated with 10μL of β-glucuronidase at 37 °C for 90min. Subsequently, 2mL of formic acid was used to terminate the enzymatic deconjugation. The phthalate metabolites were extracted by solid-phase extraction cartridges (Oasis HLB, Waters Co., Milford, MA). Dried extracts were evaluated with high-performance liquid chromatography and tandem mass spectrometry (6460LC-MS, Agilent Technologies Co., Santa Clara, CA). Each batch of samples includes one blank control, two quality controls, and six standards (0, 1, 10, 50, 100, 200 ng/mL). The blank control (1mL water) was used to detect the presence of contamination during sample processing and analysis. In order to determine the accuracy and precision of the method, two urine samples from a random participant were respectively mixed with 5 and 50 ng/mL of the target phthalate monoesters and used as quality controls by calculating the recovery. The recovery for urine phthalate metabolites was 88.06%–100.93%. The linearity correlation coefficient of calibration curves was > 0.99, with a linear range of 0.5–200 ng/mL for urine samples. The limits of detection (LOD) ranged from 0.01 μg/L to 0.04 μg/L. Urinary creatinine concentrations were measured to adjust for urine dilution. 2

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2.4. IVF/ICSI procedures and embryo quality assessment

Table 1 Demographic and clinical characteristics of all the participants (N = 663).

Details on IVF/ICSI procedures were described previously (Zhu et al., 2013). For ICSI, Mature oocytes (MII oocytes) were characterized with the extrusion of the first polar body after removing the cumulus cells following OPU. For IVF, the total count of mature oocytes was determined at the fertilization check, by summing the number of oocytes presenting at least one pronucleus and those without a pronucleus but extruding a polar body. Normal fertilization was evidenced by the presence of two pronuclei (2PN). The odds of 2PN oocytes developing from MII oocytes was evaluated. At the cleavage stage, embryos were evaluated morphologically according to the Veeck system, based on blastomere multinucleation, cell number, symmetry and amount of fragmentation. Embryos on day 3 which were without multinucleation, consisted of at least six cells and had less than 20% fragments were considered as good-quality embryos. We assessed the odds of good-quality embryos among all cleaved embryos which developed from 2PN oocytes. At the blastocyst stage, embryos were evaluated using Gardner system, based on the expansion of blastocoel cavity (1–6), the number and cohesiveness of inner cell mass and trophectoderm (A-C). Blastocysts that graded ≥3BB on day 5 or 6 were classified as good-quality blastocysts. The odds of blastocyst/ good-quality blastocyst developed from embryos continuously cultured to the blastocyst stage was investigated. Clinical pregnancy was identified when observing a gestational sac with fetal heart activity by ultrasound examination 28 days after embryo transfer. Early miscarriage was defined as the pregnancy loss before gestational week 12. The delivery of live newborns defined live birth. 2.5. Statistical analyses Demographic and clinical characteristics of participants were described using mean ± SD or percentages. To adjust for urine dilution, phthalate metabolite levels were divided by urinary creatinine concentrations. Phthalate metabolites distributions in urine samples were described with the geometric mean (GM), median value and percentiles. Undetectable concentrations were assigned a value of LOD divided by the square root of two. The concentrations of phthalate metabolites were classified into low-, medium-, and high-level groups according to tertiles. The associations of different phthalate metabolite concentrations with oocyte/ embryo quality and pregnancy outcomes were evaluated via multivariable generalized linear models. The median urinary phthalate metabolite concentration in each tertile was calculated as a continuous variable to perform a test of linear trend. When assessing the count data (the number of retrieved oocytes), Poisson distribution and log link function were applied in the models. Oocyte maturity, fertilization, blastocyst formation and good quality statuses for each oocyte or embryo were fitted as binary outcomes. Binomial distribution and logit link function were applied in the models when analyzing fertilization and oocyte/embryo quality. The log-binominal regression model was used to evaluate the associations between phthalate metabolites and pregnancy outcomes due to their high prevalence (Barros and Hirakata, 2003). Estimates of the odds ratio (OR) for fertilization and oocyte/ embryo quality were obtained for each tertile compared to the lowest tertile. Estimates of the relative risk (RR) for retrieved oocyte count and pregnancy outcomes were obtained for each tertile compared to the lowest tertile. RR and OR < 1 respectively indicate the decreased prevalence risk and odds of the target events with one tertile increase in urinary metabolite concentration. Potential covariates were chosen according to previous literature and consisted of age, body mass index (BMI), ethnicity, infertility cause, smoking status, treatment protocol type, insemination technique (IVF and ICSI), overall IVF attempts, and year of treatment (Hauser et al., 2016). Variables included in the final model were statistically

Characteristic

Data

Age (years) < 30 30–39 ≥40 Ethnicity Han Others Smoking status Non-smoker Smoker BMI < 18.5 18.5- < 24 24- < 28 ≥28 Infertility causes Male factor Female factor Mix factors Unexplained COH protocol Long GnRHa Prolonged GnRHa Antagonist Others Overall IVF attempts 1 2 ≥3 Retrieved oocytesa MII oocytes Insemination techniquea IVF ICSI Normally fertilized embryos Cleaved embryos Good-quality embryos on day 3 Embryos cultured past day 3b Blastocyst formation Good-quality blastocysts Fresh cycles with embryo transfer Clinical pregnancy Live birth Miscarriagec Spontaneous abortion before GW 12 Ectopic pregnancy Miscarriage after GW 12

31.3 ± 5.2 279 (42.1%) 332 (50.1%) 52 (7.8%) 636 (95.9%) 27 (4.1%) 660 (99.5%) 3 (0.5%) 21.9 ± 2.7 57 (8.6%) 467 (70.4%) 120 (18.1%) 19 (2.9%) 94 (14.2%) 474 (71.5%) 84 (12.7%) 11 (1.7%) 334 (50.4%) 101 (15.2%) 164 (24.7%) 64 (9.7%) 491 (74.1%) 124 (18.7%) 48 (7.2%) 11.8 ± 7.0 10.7 ± 6.3 458 (69.1%) 197 (29.7%) 7.09 ± 4.77 8.61 ± 5.67 5.09 ± 3.91 5.57 ± 5.04 4.55 ± 4.00 1.57 ± 2.01 328 180 (54.9%) 159 (48.5%) 23 (12.8%) 11 (6.1%) 5 (2.8%) 7 (3.9%)

Data were described as mean ± SD or N (%). GW, gestational week. a Eight participants had oocyte retrieval cancelled. b One hundred and thirty one participants had no embryos continuously cultured to the blastocyst stage. c Two participants have 2 embryos transferred and implanted, but only one child was live born for each women.

significant predictors (P < 0.15) of the outcomes or with vital biological relevance, such as age and BMI (Rittenberg et al., 2011; Bhattacharya et al., 2013). Year of treatment was also forced into the models to avoid measurement bias. After the adjusted models were specified, target phthalate metabolite concentration levels additionally entered the models. Data were analyzed with SPSS 19.0 version software. Results with P < 0.05 were considered statistically significant. 3. Results Demographics of 663 participants are shown in Table 1. The participants enrolled in this study were of an average age of 31.3 ± 5.2 years old, and an average BMI of 21.9 ± 2.7 kg/m2. Most of the participants were Han (95.9%) and non-smokers (99.5%). Four hundred and seventy four couples (71.5%) had a diagnosis for female factor alone, 94 couples (14.2%) had male factor alone, and 84 couples 3

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subgroup analyses focusing on participants diagnosed with female factors or unexplained infertility (Supplementary Tables 1–3). Except for some new findings about di (2-ethylhexyl) phthalate (DEHP) metabolites and MEP, similar results were observed in the subpopulation. In brief, MBP concentration remained inversely associated with the odds of normal fertilization. MEHHP in the medium tertile was associated with a higher number of retrieved oocytes comparing with the lowest tertile (adjusted RR = 1.08, 95% CI: 1.01–1.16). On the other hand, MEOHP in the medium tertile was correlated with lower normal fertilization odds compared to the lowest tertile (adjusted OR = 0.85, 95% CI: 0.74–0.99) (Supplementary Table 1). There were still no associations found between MEHP (the primary metabolite of DEHP) concentration and intermediate or pregnancy outcomes. In addition, MEP in the medium tertile reduced 27% (95% CI: 0.58–0.89) and 31% (95% CI: 0.54–0.87) of the odds for total and good-quality blastocyst formation compared to the lowest tertile (Supplementary Table 2).

(12.7%) had both. Other data about cycle characteristics and clinical outcomes are also listed in Table 1. Eight participants had OPU cancelled because of poor ovarian response. Seventeen women had no oocyte normally fertilized. Seventy-nine women with fewer than three good-quality embryos had chosen for transfer on day 3. One hundred and six women had all embryos transferred and/or cryopreserved on day 3. Hence, a total of 131 participants had no embryos continuously cultured to the blastocyst stage. There were 356 fresh cycles with embryo transfer. Those without embryo transfer were due to lack of transferrable embryos, serum progesterone elevation, or high risk of ovarian hyperstimulation syndrome. When assessing pregnancy outcomes, we excluded 28 women who were diagnosed with uterus malformations or endometriosis, and a total of 328 fresh cycles were evaluated in the final analysis (Supplementary Fig. 1). One hundred and eighty participants had clinical pregnancy, and 88.3% of them (N = 159) had live births. There were 23 women who had a miscarriage. The urinary distributions of phthalate metabolites are presented in Table 2. Most of phthalate metabolites were highly detected in the urine samples (ranging from 94.42% to 100%), except for MOP with a 29.56% detection rate. Among all the phthalate metabolites, MBP had the highest urinary concentration with a median level of 101.51 μg/g Cr. The urinary levels of MMP, MEP, MEHP, MEOHP, and MEHHP were relatively low (median: 9.49, 9.62, 8.84 11.70, and 8.29 μg/g Cr, respectively), while MBzP and MOP had much lower concentrations (median: 0.09 and 0.02 μg/g Cr, respectively), with 75% of the samples less than 1 μg/g Cr. Considering the low detection frequencies of MOP, participants could not be divided into three equal tertiles according to MOP concentration. Hence, MOP was excluded from the subsequent analyses for assessing the associations with IVF/ICSI intermediate clinical outcomes. Table 3 demonstrates the associations of urinary phthalate metabolite concentrations with ovarian response and fertilization. The counts of retrieved oocytes were significantly higher in the highest tertile compared with the lowest tertile for MEP (adjusted RR = 1.08, 95% CI: 1.03–1.15). However, there was a significant association of MEP with decreased odds of normal fertilization for the medium tertile compared to the lowest tertile (adjusted OR = 0.86, 95% CI: 0.76–0.97). MBP concentration was inversely associated with normal fertilization odds. Fig. 1 shows the associations of phthalate exposure with embryo development. None of phthalate metabolites were found to be significant associated with the odds of good-quality embryos on day 3 or blastocyst formation. MMP and MEP in the medium tertile respectively reduced 22.4% (95% CI: 0.64–0.94) and 21.9% (95% CI: 0.64–0.95) of the odds to gain good-quality blastocyst compared to the lowest tertile. The associations of phthalate exposure with pregnancy outcomes were also analyzed in this study (Fig. 2). These eight phthalate metabolites were not correlated to CPR, LBR, or early miscarriage rate. To exclude potential confounding by male infertility, we did

4. Discussion In this large prospective single-center study, we investigated the associations between urinary concentrations of eight phthalate metabolites and IVF/ICSI clinical outcomes reflecting ovarian response, fertilization, early embryo development, pregnancy and birth outcomes. There was an inverse dose-dependent association between MBP and normal fertilization odds. Urinary MEP in medium tertile was correlated with lower odds of normal fertilization and good-quality blastocyst formation. Medium level of MMP was correlated to decreased odds of good-quality blastocyst formation. DEHP metabolites (MEHP, MEHHP and MEOHP) showed none or inconsistent reproductive toxicities in the total population or among participants excluded male infertility. Research on IVF outcomes revealed the environmental impacts on ovarian response and fertilization. Among different populations, varied phthalate metabolites were found to have significant adverse effects. The Environment and Reproductive Health (EARTH) study prospective cohort study included 256 U.S. women who underwent IVF or ICSI to investigate the associations of maternal phthalate exposure with reproductive outcomes (Hauser et al., 2016). They found higher urinary concentrations of MEHP, MEOHP, MEHHP, and ΣDEHP were correlated with reduced counts of retrieved oocytes and mature oocytes, but not associated with fertilization rate. However, a recent study including 136 Israeli women found that urinary levels of MEOHP, MEHHP, ΣDEHP were negatively associated with the number of fertilized oocytes (Machtinger et al., 2018). Additionally, among low molecular weight phthalate metabolites, higher MBP and MEP concentrations were correlated with fewer total, mature, and fertilized oocytes. Similarly, we observed higher levels of MEP and MBP were associated with poorer fertilization. MBP is the most abundant phthalate monoester found in the urine samples of Chinese population from newborns to reproductive-aged people (He et al., 2019; Al-Saleh et al., 2019; Wang

Table 2 The distributions of urinary phthalate metabolites. Phthalatemetabolite

MMP MEP MBP MEHP MEHHP MEOHP MBzP MOP

LOD (μg/L)

0.03 0.02 0.01 0.02 0.01 0.01 0.01 0.04

> LOD (%)

94.42 100 100 98.94 100 100 96.53 29.56

Creatine-adjusted (μg/g Cr)

a

Unadjusted (μg/L)

a

GM

Median (IQR)

GM

Median (IQR)

8.02 11.36 93.78 8.15 12.65 8.60 0.11 0.04

9.49 (4.03, 24.13) 9.62 (4.53, 23.27) 101.51 (50.79, 164.58) 8.84 (3.85, 16.77) 11.70 (7.47, 18.87) 8.29 (4.94, 13.59) 0.09 (0.04, 0.33) 0.02 (< LOD, 0.10)

10.81 15.51 135.03 11.13 17.28 11.74 0.15 0.05

14.50 (5.82, 34.43) 14.35 (6.26, 33.99) 159.34 (62.77, 31.67) 12.27 (4.96, 26.42) 17.57 (9.68, 29.88) 12.04 (5.98, 21.79) 0.14 (0.04, 0.72) 0.03 (0.03, 0.09)

LOD, limit of detection; GM, geometric mean; IQR, interquartile range. a For samples < LOD, a value is assigned LOD/√2. 4

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Table 3 The associations of urinary phthalate metabolite concentrations with ovarian response and fertilization (N = 663). Phthalate metabolite (μg/g Cr) MMP T1 (< 5.56) T2 (5.56- < 17.44) T3 (≥17.44) P-trend MEP T1 (< 5.81) T2 (5.81- < 17.09) T3 (≥17.09) P-trend MBP T1 (< 67.71) T2 (67.71- < 136.11) T3 (≥136.11) P-trend MEHP T1 (< 5.08) T2 (5.08- < 13.69) T3 (≥13.69) P-trend MEHHP T1 (< 8.68) T2 (8.68- < 15.43) T3 (≥15.43) P-trend MEOHP T1 (< 5.94) T2 (5.94- < 11.36) T3 (≥11.36) P-trend MBzP T1 (< 0.049) T2 (0.049- < 0.21) T3 (≥0.21) P-trend

Mature oocytes b Adjusted OR (95% CI)

Normal fertilization c Adjusted OR (95% CI)

Ref 1.01 (0.96, 1.07) 1.03 (0.97, 1.09) 0.59

Ref 1.11 (0.93, 1.33) 0.97 (0.81, 1.17) 0.32

Ref 0.93 (0.82, 1.06) 0.96 (0.85, 1.09) 0.54

Ref 1.03 (0.97, 1.09) 1.08 (1.03, 1.15)* 0.01*

Ref 0.97 (0.81, 1.17) 0.96 (0.80, 1.15) 0.91

Ref 0.86 (0.76, 0.97)* 0.99 (0.88, 1.12) 0.02*

Ref 0.95 (0.90, 1.01) 1.02 (0.96, 1.08) 0.07

Ref 1.07 (0.89, 1.29) 1.10 (0.91, 1.33) 0.61

Ref 0.81 (0.71, 0.92)* 0.81 (0.71, 0.99)* < 0.01**

Ref 0.99 (0.93, 1.04) 0.99 (0.93, 1.04) 0.86

Ref 0.97 (0.81, 1.16) 0.95 (0.79, 1.15) 0.89

Ref 1.05 (0.93, 1.19) 1.07 (0.94, 1.21) 0.59

Ref 1.01 (0.96, 1.07) 1.00 (0.94, 1.02) 0.83

Ref 0.89 (0.74, 1.06) 1.02 (0.85, 1.23) 0.24

Ref 0.94 (0.83, 1.06) 1.03 (0.91, 1.16) 0.35

Ref 0.98 (0.93, 1.04) 0.98 (0.92, 1.04) 0.79

Ref 1.15 (0.95, 1.38) 1.03 (0.85, 1.24) 0.30

Ref 0.88 (0.78, 0.99)* 0.99 (0.86, 1.13) 0.07

Ref 0.99 (0.93, 1.04) 0.96 (0.89, 1.02) 0.42

Ref 1.10 (0.92, 1,31) 1.25 (0.99, 1.57) 0.16

Ref 0.97 (0.86, 1.10) 0.84 (0.72, 0.97)* 0.06

Number of retrieved oocytes Adjusted RR (95% CI)

a

*P < 0.05. **P < 0.01. a Adjusted for age (continuous), BMI (continuous), year of treatment, treatment protocol, and overall IVF/ICSI attempts. b Adjusted for age (continuous), BMI (continuous), year of treatment, infertility cause, and overall IVF/ICSI attempts. c Adjusted for age (continuous), BMI (continuous), year of treatment, and insemination technique.

proportion of overweight or obese participants were higher in the EARTH study (30% vs 21%). The SEEDS study reported the odds of good-quality blastocysts were inversely associated with paternal urinary MBP, MBzP and MMP, but not maternal phthalate exposure. Their combined analysis of paternal phthalate exposure level might attribute to the differently maternal results. Why does maternal phthalate exposure affect blastocyst quality, but not embryo quality on day 3? Phthalate toxic mechanisms, such like inducing oxidative stress and cell apoptosis, occur from folliculogenesis to embryo development (Wang et al., 2012; Chu et al., 2013a, 2013b). Embryo damages are accumulating during this cycle. Molecular changes associated with maternal phthalate exposure might be too subtle to be detected morphologically at the cleavage stage, but later manifested at the morphological level during the blastocyst stage (Wu et al., 2017a). Another potential mechanism for this phenomenon was the regulation of embryonic genome activation (EGA) on day 3. EGA facilitates a transition from maternal to embryonic control at four-to eight-cell stage (Latham and Schultz, 2001). Female phthalate exposure may not show significant adverse impacts on the cleavage stage. However, after EGA, the alterations of paternal genome caused by phthalate exposure (Wang et al., 2016; Jurewicz et al., 2013) would begin to reveal their effects on embryo genome, and subsequently show disruptions in embryo development. The unmeasured male phthalate exposure might affect our results. Notably, many significant results were found in the medium concentrations of phthalate metabolites, but not in the high concentrations when compared to the low exposure level. The nonmonotonic dose-

et al., 2015; Ding et al., 2019). Moreover, recent studies reported MBP and its original form dibutyl phthalate (DBP) were dominant over other phthalate exposure levels in both indoor dust and surface water in South China, showing its high environmental occurrence (Gao et al., 2019a, 2019b). In Chinese IVF population, MBP concentration was not only much higher than other metabolite concentrations, but also one order of magnitude higher than in Israeli women and U.S. women (159.34 vs. 17.4, 12.6 μg/L). The prominent exposure of MBP might result in the consistently female reproductive toxicity. The effects of phthalate metabolites on early embryo development were discordant in different studies. The Israeli study reported that urinary concentrations of DEHP metabolites were inversely associated with the number of top quality embryos on day 3 in IVF cycles (Machtinger et al., 2018). On the other hand, two U.S. research teams, the EARTH team and the Sperm Environmental Epigenetics and Developments Study (SEEDS) (enrolling 50 couples), found no significant adverse impacts of female phthalate exposure on the embryo quality (Wu et al., 2017a; Hauser et al., 2016). In this study, female participant inclusion criteria were similar to the EARTH and SEEDS studies, whereas we found maternal MMP and MEP levels inversely associated with blastocyst quality, without affecting embryo quality on day 3. Limited sample sizes, as well as different patient characteristics (age, BMI, infertility diagnosis and treatment protocols) and exposure levels might contribute to the inconsistent results among these studies. The mean age of our population was much younger than that of women in the EARTH study (31.3 vs 35.3 years). BMI of our participants was also lower than U.S. women (21.9 ± 2.7 vs 24.1 ± 4.3), and the 5

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Fig. 1. The associations of urinary phthalate metabolite concentrations with the odds of good-quality embryos on day 3, and total/good-quality blastocyst formation (N = 663) a. Adjusted for age (continuous), BMI (continuous), year of treatment, infertility cause, treatment protocol, insemination technique, and overall IVF/ICSI attempts. b. Adjusted for age (continuous), BMI (continuous), year of treatment, infertility cause, treatment protocol, and insemination technique. c. A total of 532 participants had embryos continuously cultured to the blastocyst stage. *P < 0.05. The P value is for the overall trend. 6

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Fig. 2. The associations of urinary phthalate metabolite concentrations with the rates of clinical pregnancy (N = 328), live birth (N = 328), and early miscarriage (N = 180) a. Adjusted for age (continuous), BMI (continuous), year of treatment, insemination technique, and overall IVF/ICSI attempts. b. Adjusted for age (continuous), BMI (continuous), year of treatment, and overall IVF/ICSI attempts. c. Adjusted for age (continuous), BMI (continuous), and year of treatment. *P < 0.05. The P value is for the overall trend. 7

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phthalates on pregnancy outcomes. On the other hand, it should also be noted that the single measurement of urinary metabolite concentrations on the day of OPU might misclassify the exposure levels during critical windows for long-term clinical outcomes. The intermediate IVF/ICSI outcomes might reflect more accurate effects of preconception phthalate exposure compared with pregnancy outcomes. Our strengths are the till-now largest sample size and systematic exploration into the clinical outcomes among Chinese women receiving assisted reproductive technology treatments. Although we had improved the statistical power than those in the previous studies, the sample size is still far from being sufficiently effective to achieve an optimal power. Hence, the recruitment of the prospective cohort with participants receiving IVF is undergoing now. Another limitation of this study is the single measure of urine specimens. Longer-chained phthalates metabolites (such as MEHP and MBzP) showed a poorer temporal stability compared to shorter-chained phthalates (such as MBP and MEP) (Johns et al., 2015). Although it is suggested that a single sample may adequately represent one’s exposure level over several months, measurements of multiple urine samples would serve to further reduce exposure misclassification, especially for longer-chained metabolites due to their low reproducibility over time (Johns et al., 2015). The third limitation of this study is lacking the male partner characteristics and phthalate exposure assessment. The concentrations of paternal phthalate metabolites have been proved to be associated with sperm quality, fertilization, embryo development, implantation, and pregnancy outcomes (Dodge et al., 2015; Wang et al., 2015; Wu et al., 2017a, 2017b). The SEEDS team used couples-level models and showed an even more important role of male exposure in embryo quality compared to the female exposure. Al-saleh also emphasized the importance of a couplebased approach in assessing fertility outcomes (Al-Saleh et al., 2019). Therefore, multiple measurements and couple-level exposure study would be the future trend. In addition, we wonder if there are other types of specimens that can be measured to better present the phthalate exposure in the innerenvironment. Although we previously noticed that phthalate metabolite concentrations were generally two orders of magnitude higher in urine samples compared to those in FF (Du et al., 2016), FF is still an important candidate type of specimen because it is the direct exposure environment for oocyte development. Our pilot study reported no association between phthalates in FF and intermediate IVF outcomes, which is possibly due to limited power of the small sample size (Du et al., 2016). There may be new findings in the study of a larger sample size.

responses such as inverted U-shape curves are commonly observed in the field of endocrine disruption chemicals (EDCs) (Gore et al., 2015). So far, there have been some other studies which reported non-dosedependent trends on obesity development induced by MEHP, perfluorooctanoic acid and bisphenol A (Angle et al., 2013; Hao et al., 2012; Hines et al., 2009). We speculated that it could be correlated with receptor occupancy. Phthalate and their metabolites bind to and activate peroxisome proliferator-activated receptor (PPAR) to start the inhibition of E2 synthesis (Sarath Josh et al., 2014; Reinsberg et al., 2009). It is possible that low-dose phthalate activates PPAR activation in a high efficient way, while high-dose leads to degradation of PPAR. This might explain the influences of lower degree caused by high-level exposure than medium-level exposure of phthalates. Although both animal research and certain epidemiologic studies showed that DEHP exposure could significantly affect oocyte and early embryo quality, we found no associations between maternal DEHP metabolites with oocyte parameters and intermediate IVF/ICSI outcomes. Even after participants with male infertility were excluded, DEHP metabolites still showed non-significant or inconsistent associations with IVF/ICSI outcomes. Apart from the varied patient characteristics, urinary phthalate metabolite levels in our study were adjusted using creatinine concentration instead of specific gravity. Hence, the concentrations reported in the EARTH study might not be directly comparable to our study, and the potential influences of different adjusting methods on analytical results were unknown. Compared to our cohort and U.S. women (14.35 μg/L, 52.2 μg/L), Israeli and Saudi Arabian women had much higher urinary MEP concentrations (150.5 μg/L, 333 μg/L) (Al-Saleh et al., 2019; Hauser et al., 2016; Machtinger et al., 2018). Such high MEP level was associated with poorer oocyte quality, as well as higher risks of failed clinical pregnancy and miscarriage. In the present study, the MEP concentration demonstrated positive associations with the count of total oocytes, but an inverse association with the odds of normal fertilization and good-quality blastocyst formation. High exposure level of certain phthalate metabolite might show more consistent effects on reproductive outcomes. However, new data of more participants were still needed to elucidate, confirm and interpret the associations between urinary MEP concentration and reproductive outcomes among Chinese women undergoing IVF/ICSI. Several studies evaluated the effects of phthalate exposure on pregnancy outcomes. Mu et al. and Yi et al. examined the levels of the same five phthalate metabolites among couples who conceived naturally and underwent pregnancy loss. Interestingly, they respectively found MEP, MiBP, MBP and MMP, MEHP were correlated to increasing risk of miscarriage (Mu et al., 2015; Yi et al., 2016). The inconsistent results might due to different adjusting methods for phthalate concentrations. When restricting the participants to those seeking infertility care, the EARTH studies reported the concentrations of certain phthalate metabolites from both paternal and maternal sides were inversely correlated with odds of clinical pregnancy and live birth, and elevated risk ratios of miscarriage (Dodge et al., 2015; Hauser et al., 2016; Messerlian et al., 2016). Similarly, a recent Saudi Arabian study included 599 couples undergoing IVF and showed that maternal MEP and MEHP concentrations were associated with higher risk of failed clinical pregnancy and live birth (Al-Saleh et al., 2019). However, the Israeli study found none of urinary phthalate metabolites were associated with pregnancy outcomes among women undergoing IVF because of male factors or unexplained infertility (Machtinger et al., 2018). These findings suggested that couples with female infertility factors might be a more vulnerable subgroup to environmental chemicals. Nevertheless, although the current study also included infertile women, no associations were observed between phthalate exposure and pregnancy outcomes. Al-saleh et al. pointed out the enhancement of maternal adverse effects when jointly adjusted phthalate metabolite levels in the male partner (Al-Saleh et al., 2019). The lack of paternal exposure in our study might conceal the maternal influences of

5. Conclusions In this cohort of Chinese women receiving IVF/ICSI treatments, urinary MBP concentration was much higher compared to other phthalate metabolites, and also higher than it was reported by studies in other countries. MBP showed adverse impacts on fertilization. MMP and MEP could affect blastocyst quality, but not embryo quality on day 3. DEHP metabolites didn’t show consistent reproductive toxicities as demonstrated in previous studies. Funding This work was supported by the grants from National Natural Science Foundation of China [No. 81571508; No. 81771654]. Declaration of competing statement None. Acknowledgement The authors thank all the doctors, nurses, and embryologists in the 8

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Reproductive Medicine Center of Tongji Hospital for their help in collecting samples and data.

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