Phthalates and phenols are two classes of potential endocrine disrupting chemicals (EDCs) present in the environment, food, and everyday consumer products (Baccarelli et al. 2000; Diamanti-Kandarakis et al. 2009). These ubiquitous compounds are associated with adverse health outcomes in humans (Bornehag et al. 2004; Feige et al. 2007; Gore et al. 2015; Miodovnik et al. 2011; Philippat et al. 2012). Human exposure to phthalates and phenols occurs through several routes, including ingestion, inhalation, and dermal contact (Hauser and Calafat 2005). The most common sources of exposure to these chemicals include personal care products (cosmetics, shampoos, perfumes), solvents, medical devices (like IV tubing), thermal receipts, and food packaging materials (Carwile et al. 2011; Guo and Kannan 2013; Hauser and Calafat 2005). Previous epidemiologic studies have shown that some phthalates and phenols are associated with adverse female fertility outcomes (Ehrlich et al. 2012; Hauser et al. 2016b; Jukic et al. 2016; Machtinger et al. 2018a; Meeker and Ferguson 2014; Mínguez-Alarcón et al. 2016; Toft et al. 2012).

There is increasing interest in molecular markers, such as microRNAs, and how they might act as an intermediate between environmental exposures like EDCs and the development of diseases and disorders (Casati et al. 2015; Derghal et al. 2016; Fleisch et al. 2012). MicroRNAs are short, non-coding RNA molecules that can post-transcriptionally regulate gene expression (Li et al. 2007; Lim et al. 2005; Sætrom et al. 2007) and can be free-floating or packaged in extracellular vesicles (EV-miRNAs). Extracellular vesicles (exosomes, micro-vesicles, and other membrane-bound vesicles) have been detected in almost every biofluid, including follicular fluid (Weber et al. 2010), and can act as a vehicle carrying proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs) (Raposo and Stoorvogel 2013). These EV-miRNAs are microRNAs packaged in extracellular vesicles and are stable compared to their free-floating counterparts. They are actively released by viable cells and likely represent an active means of communication between cells and tissues (Machtinger et al. 2016b; Yáñez-Mó et al. 2015). EV-miRNAs expression has been associated with concentrations of phenols and phthalate metabolites, with resultant downstream alterations in gene expression in testicular tissue from zebrafish (Lee et al. 2018), in vitro mouse Sertoli cells (Cho et al. 2010), ovarian tissue from rodents (Liu et al. 2018b), hippocampal tissue from rodents (Luu et al. 2017), and human placental cells (Avissar-Whiting et al. 2010; De Felice et al. 2015b; LaRocca et al. 2016; Meruvu et al. 2016). However, to the best of our knowledge, the relationship between EV-miRNAs expression in follicular fluid and ubiquitous environmental chemicals known to have reproductive toxicity is unstudied.

The ovarian follicle not only houses the oocyte itself, but as the follicle matures, it induces cellular differentiation occurs, creating cellular layers of thecal, granulosa, and cumulus cells. Thecal and granulosa cells make up the membrane of the follicle itself, while cumulus cells surround the encapsulated oocyte (Hennet and Combelles 2012; Rimon-Dahari et al. 2016). The granulosa cells that embed the ovarian follicle secret hyaluronan and chondroitin sulfate that generates an osmotic gradient in the follicle. This gradient pulls in fluid derived from the vasculature of the theca cells that surround the granulosa cells in the ovarian follicle (Rodgers and Irving-Rodgers 2010). The follicular fluid, a critical microenvironment for the development of oocytes (Rodgers and Irving-Rodgers 2010; Zuccotti et al. 2011), contains a mixture of proteins, metabolites, ions, plasma components, numerous other molecules, including EV-miRNAs. Thus, the objective of this study was to determine whether urinary concentrations of phenols and phthalate metabolites are correlated with the expression of EV-miRNAs isolated from follicular fluid in women undergoing in vitro fertilization (IVF). The ability to quantify biomarkers of exposure to phthalates and phenols and how they impact EV-miRNAs in follicular fluid may provide insight into the potential influence of EDCs on female reproduction.

In the present study, we identified significant correlations between phenol and phthalate biomarker urinary concentrations and EV-miRNA profiles in follicular fluid. Among these women undergoing IVF, hsa-miR-125b and hsa-miR-15b were positively associated with DEHP, while hsa-miR-106b, and hsa-miR-374a had an inverse association with DEHP (increased DEHP with decreased expression). Concentrations of MBP were positively associated with levels of hsa-miR-24. Hsa-let-7c was positively associated with urinary concentrations of MEOHP, MEHHP, MECPP, and ΣDEHP. Additionally, phthalate alternative MHINCH was positively associated with hsa-miR-19a. Among the phenols examined, increased urinary concentration of ethyl paraben was associated with decreased expression hsa-miR-375. These associations remained significant after FDR adjustment.

To our knowledge, this is the first study to identify correlations between select potential EDCs and EV-miRNA expression in follicular fluid. EV-miRNAs play a role in intra- and inter- cellular communication within the ovarian follicle. Follicular fluid EVs can originate from the oocyte, cumulus or mural granulosa cells in the ovarian follicle. As the field of EVs and reproduction is evolving, the origin of these follicular fluid EVs, their production and their turnover rate remains largely unexplored. Recent studies suggest that EV-miRNAs can impact granulosa cell function and can alter follicular development and oocyte maturation (Di Pietro 2016; Machtinger et al. 2016a) and several of the significant EV-miRNAs that were associated with phthalates and phenols biomarkers in our study play key roles in the ovarian follicle. MiR-375 is expressed in both granulosa cells and oocytes and mediates the effect of genes that regulate follicular growth with downstream proliferation, spread, and apoptosis of cumulus cells (Chen et al. 2017; Liu et al. 2018a). Overexpression of miR-375 blocked the proliferation ability and increased the rate of apoptosis of bovine cumulus cells (Chen et al. 2017; Liu et al. 2018a) and suppressed estradiol production and follicular development in porcine granulosa cells (Yu et al. 2017). In rodents, overexpression of a miR-125b mimic in oocytes and mouse embryonic stem cells could target and block expression of specific genes required for embryo progress beyond the two-cell stage (Kim et al. 2016). In another study, higher levels of miR-24 in bovine culture media was associated with embryos that failed to undergo differentiation. Moreover, when adding more miR-24 to the functional blastocyst containing media, development was significantly altered (Kropp and Khatib 2015). This suggests that over-expression of hsa-miR-375, hsa-miR-24, and hsa-miR-125b in follicular fluid is influenced by environmental exposures and could impact ovarian function through mechanistic pathways yet to be elucidated.

There is growing concern regarding the underlying pathways or mechanisms by which EDCs, such as some phenols and phthalates, impact ovarian reserve, fertility, and pregnancy rates. In rodents, DEHP and its metabolites negatively affect follicular growth and estrogen synthesis (Craig et al. 2014; Gupta et al. 2010). However, in humans, the adverse effects of DEHP on female fertility and pregnancy are not as clear. Epidemiologic studies suggest that higher urinary concentrations of ΣDEHP significantly decreased the antral follicle count among sub-fertile women (Messerlian et al. 2016). Furthermore, an increase in urinary concentrations of DEHP metabolites was associated with a lower number of oocytes retrieved, mature oocytes, fertilized oocytes, and poor embryo quality (Machtinger et al. 2018b) along with lower clinical pregnancy rates and live birth rates following fertility treatment (Hauser et al. 2016a). Higher urinary DEHP metabolite concentrations were also associated with a higher risk of biochemical pregnancy loss (Messerlian et al. 2016). Another study examining the same population as our current study found associations between urinary concentrations of MBP, ΣDEHP, and DEHP metabolites and the number of oocytes (total yield, mature, and/or fertilized) and top quality embryos (Machtinger et al. 2018b). Similarly, in our study, we identified significant associations of EV-miRNAs with urinary concentrations of MBP and ΣDEHP, and individual DEHP metabolites MEHP, MEOHP, MEHHP, and MECPP. The urinary concentration of MHiNCH, a phthalate alternative, was not associated with adverse clinical IVF outcomes (Machtinger et al. 2018b), however, we identified one significant EV-miRNA with MHiNCH in this current study. The agreement between our study and the previous epidemiology studies implies the existence of pathways through which certain phenols and phthalates may affect IVF outcomes. Despite evolving evidence of the negative effects of DEHP on female reproduction, little is known about the possible underlying mechanisms. Our results suggest that exposure to certain phthalates and phenols may affect biological pathways that are associated with follicular development and oocyte maturation by altering the expression of EV-miRNAs in follicular fluid.

We ran KEGG analyses for the eight FDR-adjusted significant EV-miRNAs and identified 17 pathways that were associated with follicular development and oocyte maturation. Additionally, we found five genes that appeared in more than 10 of these KEGG pathways, mapk1, akt3, map2k1, pik3r3, and raf1. MapK1 and map2K1 are mitogen activated protein kinases and are both involved in the meiotic regulation and oocyte maturation, as well as key genes involved in the MAPK signaling pathway (Dupre et al. 2011). Akt3, involved in spindle formation and meiotic maturation (Hoshino and Sato 2008), is also a player in the PI3K-Akt and MAPK pathways. Pik3r3, a gene that is involved in the PI3K-Akt and MAPK pathways, has also been associated with ovarian cancer (Zhang et al. 2007). All five of these genes were identified in the EGFR tyrosine kinase inhibitor, endocrine resistance, ErbB signaling, focal adhesion, FoxO signaling, progesterone-mediated oocyte maturation, prolactin signaling, and PI3K-Akt signaling pathways.

The PI3K-Akt signaling pathway is of great relevance. PI3K-Akt has been associated with recruitment of primordial follicles, a dynamic and tightly controlled process that initiates folliculogenesis (Lopez-Cardona et al. 2017; Sánchez and Smitz 2012), with granulosa proliferation and with ovarian function (Andrade et al. 2017). It is also involved in cell proliferation, apoptosis, DNA repair and protein synthesis. In rodents, exposure to DEHP decreased primordial follicle recruitment by dysregulation of the PI3K signaling pathway (Hannon et al. 2014). In humans, an in vitro study showed that DEHP exposed Hep3B cells induced oxidative stress and oxidative DNA damage (Chen et al. 2013). When a Pl3K-Akt inhibitor was added, reactive oxygen species levels were lower compared to those cells that were exposed to DEHP alone. The oxidative DNA damage persisted, even in the presence of the Pl3K-Akt inhibitor. Additionally, DEHP increased DNA replication rates, but this change was abated by the addition of the Pl3K-Akt inhibitor. The authors concluded that DEHP induced increased cellular proliferation and oxidative damage by activating the Pl3K-Akt pathway (Chen et al. 2013). The Pl3KAkt pathway also activates the mTOR and FoxO pathways, both which are regulated in the oocyte (Makker et al. 2014). In mice, dysregulation of FoxO can lead to stunted oocyte growth and follicle development, as well as luteinization of ruptured follicles (Liu et al. 2007). Deletion of mTOR genes in rodents causes early activation of all primordial follicles and leads to premature ovarian failure (Wang et al. 2014).

While EV-miRNAs have been identified in different tissue types (Ludwig et al. 2016), relatively few studies have examined the associations with phenol and phthalate exposures (Avissar-Whiting et al. 2010; Chou et al. 2017; De Felice et al. 2015a; LaRocca et al. 2016; Liu et al. 2018b; Meruvu et al. 2016; Tilghman et al. 2012). In rodents, exposure to DEHP was associated with increased expression of let-7b, miR-17, miR-181a, and miR-151 (Liu et al. 2018b). In our present study, we did not find similar associations; however, we did observe a positive association between expression miR-151–3p and urinary concentrations of MiBP (p = 0.04). Two studies measured BPA and EV-miRNA in placental tissue and found significant increases in miR-146a (Avissar-Whiting et al. 2010; De Felice et al. 2015a). However, we did not identify a signal with BPA or our parabens. Additionally, an in vitro placental cell line exposed to MEHP increased expression of miR-16 (Meruvu et al. 2016). A recent epidemiology study found decreased expression of free-floating miR-142 in placenta to be associated with urinary phthalate metabolite concentrations (LaRocca et al. 2016). Furthermore, an inverse association was identified between free-floating miR-15a and miR-185 in placenta and urinary phenols (LaRocca et al. 2016). In our current study, we found associations between miR-15a and propyl paraben (p = 0.01), however we did not identify any associations with miR-142 or miR-185. Inconsistencies observed between these prior studies and ours are likely the result of differences in species (human vs rodent) or type of study (in vitro), measurement of microRNAs (free floating vs EV packaged), underlying study populations (USA vs Israel), and exposure type (induced in vitro exposures vs urinary biomarkers capturing environmental exposure). Most of the differences, however, are likely due to differences in the type of tissue used to measure EV-miRNAs. EV-miRNAs are tissue specific and certain EV-miRNAs are present in placental tissues that are not present in follicular fluid (Landgraf et al. 2007).

This study has several limitations. First, our exposure and outcome measurements were not taken in the same tissue type. Although urinary concentrations of phthalates, phenols and DINCH are the gold standard biomarker of exposure, it is unknown how representative urinary concentrations are of concentrations within the follicle. Second, we ran our GO and KEGG pathway analyses on a relatively small number of EV-miRNAs. To minimize that our results may be subject to overfitting of the selected pathways by the software, we set our inclusion of genes to those found by at least six separate miRNA-to-gene prediction softwares. Third, we only quantified EV-miRNA profiles from one follicle and it is unknown whether this represents EV-miRNAs within a woman’s cohort of follicles. Additionally, we present our results as FDR adjustments for the 133 EV-miRNA and not for the FDR adjustments by EV-miRNA and biomarker. Our results, therefore, may be subject to spurious associations and false positives. As it was beyond the scope of our study, we did not collect serum samples in the current studies and could not look adjust to patient hormonal levels as FSH, LH or estradiol. Including only young patients with normal ovarian response, we assume that all of those were in the normal range. We are also limited by the current methods of extracting EV-miRNAs. We can assure we have a pellet enriched in EVs, however, we cannot assume that it is a pure EV fraction. It is possible that miRNAs in the pellet were derived both from necrotic cells, bound to proteins, or packaged in EVs (Endzelins et al. 2017). However, our protocol has been optimized by running thousands of samples from a lab that has a strong experience in large epidemiological studies investigating EV-miRNAs and random samples were checked by Nanosight in order to evaluate EV size distribution. As it was beyond the scope of this study to assess what was the origin of these EV-miRNA in the ovarian follicle, we encourage further studies to test the origin of these EV-miRNA as it may add important information regarding the cell types that are more vulnerable to EDCs. Last, only internal controls were present in replicate reactions and miRNAs were analyzes in a single reaction, as required by the standard protocol of this technique. Addition of technical replicates might help to monitor intra- and inter-assay variability. Despite these limitations, our study has several strengths. This is the first study, to our knowledge, to examine any environmental exposure with EV-miRNA profiles in follicular fluid. In the current study, It is also one of the largest studies to examine EV-miRNAs in follicular fluid, a target sample for IVF outcomes. This study may provide potential mechanistic information which could support previous animal and epidemiological studies on phthalate and phenol exposure and adverse reproductive outcomes such as reduced antral follicle counts, pregnancy loss, and reduced live birth rates.

Our findings suggest that select urinary phenol and phthalate biomarkers are associated with altered expression of eight EV-miRNAs in follicular fluid, hsa-let-7c, hsa-miR-375, hsa-miR-24, hsa-miR-19a, hsa-miR-125b, hsa-miR-106b, hsa-miR-374a, and hsa-miR-15b. Understanding how environmental stimuli might alter the function of EV-miRNAs, especially in follicular fluid, will help elucidate how exposures to these environmental chemicals might affect biological pathways associated with female fertility.