Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM

Abstract

Di‐(2‐ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine‐disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time‐lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP‐induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP‐exposed oocytes. VB addition during IVM limited DEHP‐induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.

Assessing equine embryo developmental competency by time-lapse image analysis

“…To thebest of our knowledge, the present study is the first to closely examine equine preimplantation development to the blastocyst stage via TLM with oocytes obtained by OPU from mares of various breeds (both draft and non-draft) that are commonly used as oocyte donors and surrogates for embryo transfer. Marzano et al (2019) also determined that the duration of the second cell cycle, or the time between the first and second mitosis, as reported here, was, 17 h in controls, whereas the time between second and third mitotic division was, 6 h, both of which had large standard deviations. In the present study we demonstrated that the time intervals between the first and second, as well as the second and third, divisions in those equine embryos that formed blastocysts was, 7-10 h and, 1-3 h respectively, with significantly less variation and closer to that observed in human embryos (Wong et al 2010;Chen et al 2013).…”

“…Time-lapse image analysis of human oocyte maturation, fertilisation and preimplantation embryogenesis was initially reported several years ago (Payne et al 1997;Hardarson et al 2002) but only recently used to evaluate embryos from other mammalian species (Pribenszky et al 2010;Sugimura et al 2010Sugimura et al , 2012Weinerman et al 2016). Using abattoirderived equine ovaries from draft mares, Marzano et al (2019) showed differences in the time of second polar body extrusion and duration of the second cell cycle between control and di-(2ethylhexyl)phthalate (DEHP)-treated oocytes. To thebest of our knowledge, the present study is the first to closely examine equine preimplantation development to the blastocyst stage via TLM with oocytes obtained by OPU from mares of various breeds (both draft and non-draft) that are commonly used as oocyte donors and surrogates for embryo transfer.…”

“…Although there are several reports of TLM in other mammalian species besides human (Pribenszky et al 2010;Sugimura et al 2010Sugimura et al , 2012Weinerman et al 2016), only recently was TLM used to evaluate equine preimplantation development to the blastocyst stage (Marzano et al 2019). However, the primary focus of that study was on the effects of a specific toxic chemical present in ovarian follicular fluid and IVM media on embryo morphokinetics.…”

“…However, the primary focus of that study was on the effects of a specific toxic chemical present in ovarian follicular fluid and IVM media on embryo morphokinetics. In addition, the authors used oocytes from abattoir obtained ovaries, only those from draft breeds and the time of intracytoplasmic sperm injection (ICSI) for assessment of certain imaging parameters (Marzano et al 2019). This can have confounding effects on the measurement of other overlapping cell cycle-related characteristics, as shown previously by a report that implemented a biological start point instead of a procedure such as ICSI (Liu et al 2014a).…”

Effect of environmental contamination on female and male gametes – A lesson from bovines

“…A level of ~20 nM MEHP was detected in the follicular fluid aspirated from the DEHP-treated cows ( Kalo et al., 2015 ). Similarly, a level of ~10 nM DEHP was recorded in equine follicular fluid ( Marzano et al., 2019 ). Non-DEHP-originated metabolites such as monoethyl phthalate and monobutyl phthalate were also detected in the follicular fluid, presumably of environmental origin ( Kalo et al., 2015 ).…”