Perinatal exposure to glyphosate or a glyphosate-based formulation disrupts hormonal and uterine milieu during the receptive state in rats

Highlights

• Glyphosate pure and in formulation increased preimplantation losses in F1 rats.

• Glyphosate pure and in formulation increased serum 17β-estradiol in early pregnancy.

• Glyphosate pure and in formulation disrupted uterine implantation-related genes.

• Glyphosate pure and in formulation induced similar deleterious effects.

Abstract

We investigated the effects of perinatal exposure to a glyphosate-based herbicide (GBH) or glyphosate alone (Gly) on female fertility and the hormonal and uterine milieu during the preimplantation period. F0 pregnant rats orally received a GBH or Gly in a dose of 2 mg of glyphosate/kg/day from gestational day (GD) 9 until weaning. F1 females were evaluated to determine the reproductive performance on GD19; and the sex steroid serum levels, the expression of estrogen receptor alpha (ERα), progesterone receptor (PR) and implantation-related genes on GD5 (preimplantation period). GBH and Gly induced preimplantation losses in F1 rats. GBH and Gly groups exhibited higher 17β-estradiol serum levels, without changes in progesterone. Both compounds increased the uterine ERα protein expression, with no differences at transcript level; and only Gly decreased PR mRNA expression. Also, GBH and Gly downregulated Hoxa10 and Lif genes, with no difference in Muc1 and Areg expression. To conclude, perinatal exposure to a GBH or Gly disrupted critical hormonal and uterine molecular targets during the receptive state, possibly associated with the implantation failures. Overall, similar results were found in GBH- and Gly-exposed rats, suggesting that the active principle might be the main responsible for the deleterious effects.

Introduction

Implantation is the limiting factor to achieve pregnancy for both natural female reproduction and in vitro fertilization (Lessey, 2011). Successful implantation requires the reciprocal interaction between a normal and functional embryo at the blastocyst developmental stage and a receptive uterus (Simón et al., 2000). Approximately two-thirds of embryo implantation failures are associated with an inadequate uterine receptivity, which may contribute to infertility in women (Fatemi and Popovic-Todorovic, 2013). Uterine receptivity refers to the window of limited time in which the uterine endometrium undergoes morphological, biochemical and genetic changes conducive to blastocyst attachment, leading to implantation and subsequent pregnancy establishment (Vasquez and DeMayo, 2013). Synchronized production of the steroid hormones progesterone (Pg) and 17β-estradiol (E2) mediates structural and functional changes in the uterus that enable the implantation process (Zhang et al., 2013). Optimal levels of these hormones are critical in regulating molecular mechanisms for uterine receptivity (Ma et al., 2003; Song et al., 2007). Progesterone and estrogen act primarily through its cognate nuclear receptors, progesterone receptor (PR) and estrogen receptor alpha (ERα), respectively, to trigger transcription pathways compromised in implantation (Vasquez and DeMayo, 2013). This complex process involves the expression of multiple factors (membrane-bound, soluble, and secretory proteins) which are downstream effectors of ER and PR and characterize the transition of the uterus from the nonreceptive to the receptive state (Lessey and Young, 2019). Several hormone-responsive genes including Homeobox A10 (Hoxa10), Leukemia inhibitory factor (Lif), Mucin 1 (Muc1), Amphiregulin (Areg), among others play key roles during the secretory phase in order to sustain endometrial receptivity (Byun et al., 2008; Leach et al., 2012; Lee and DeMayo, 2004). Various environmental chemicals including pesticides, known for their action as endocrine disruptors, have shown to affect female fertility and more specifically, interfere with the implantation process (Meeker et al., 2011; Milesi et al., 2015; Mokhtar et al., 2013).

Among pesticides, glyphosate is the most widely applied in Argentina and worldwide and constitutes the active ingredient of all glyphosate-based herbicides (GBHs) (Benbrook, 2016; Castro Berman et al., 2018). GBHs are used to eliminate weeds in farmlands and urban gardens and to control aquatic flora (Held et al., 2016). Although there are microorganisms in soil and water that can degrade glyphosate (Sviridov et al., 2015), the global intensive use of this herbicide has led to an increased environmental contamination, as it has been detected in several matrixes such as surface waters (Montiel-León et al., 2019; NAIADES, 2018), groundwaters and open-reservoir tank waters (Demonte et al., 2018), rainwaters and soil (Lupi et al., 2019), dust (Mendez et al., 2017) and air (Ravier et al., 2019). Several environmental and biomonitoring studies evidence that individuals are likely to be exposed to glyphosate in their occupational settings (Connolly et al., 2017; Kongtip et al., 2017) but also, through foodstuff (Bai and Ogbourne, 2016; Rodrigues and Ferreira de Souza, 2018) and drinking water (Rendon-von Osten and Dzul-Caamal, 2017) becoming an issue of concern to countryside and urban populations.

Scientific literature exhibits numerous reports of glyphosate and its commercial formulations showing endocrine effects both in in vitro and experimental animal models (Manservisi et al., 2019; Mesnage et al., 2015). In this regard, we found evidence of endocrine disruption of a GBH formulation on the female reproductive tract in rats. We showed that neonatal exposure to a GBH alters uterine morphology and the expression pattern of key proteins for uterine organogenetic differentiation in prepubertal rats (Guerrero Schimpf et al., 2017), and impairs endometrial decidualization at adulthood (Ingaramo et al., 2017, 2016). More recently, we also demonstrated that GBH exposure during early postnatal development increases uterine sensitivity to E2 and induces histomorphological and molecular alterations indicative of endometrial hyperplasia in E2-treated ovariectomized rats (Guerrero Schimpf et al., 2018). However, the Endocrine Disruptor Screening Program lead by the United States Environmental Protection Agency (EPA, 2015) and later, the European Food Safety Authority (EFSA, 2017) resolved that there were not sufficient evidence to support endocrine disrupting properties of glyphosate by evaluating different modes of action (as an estrogen-, androgen-, thyroid-like molecule or as an interfering in the steroidogenesis pathway).

Glyphosate is never used alone, but instead is combined with other chemicals named as “inert ingredients” to constitute GBHs, increasing its adhesion to plant surfaces or facilitating penetration into plant tissues in order to enhance herbicide action (Vandenberg et al., 2017). Despite the fact that the co-formulants are classified as “inert” substances, some evidence indicates GBH formulations are more toxic than pure glyphosate (Defarge et al., 2016; Mesnage et al., 2013). Moreover, some authors reported that even though co-formulants can be more toxic than glyphosate alone, glyphosate but not GBH formulations or co-formulants are able to induce a proliferative response together with ERα activation in human cancer cells (Mesnage et al., 2017). At present, there are no conclusive results indicating whether GBH formulation or glyphosate alone cause the adverse effect observed in different studies.

Previously, we showed that a GBH formulation administrated during gestation and lactation induces subfertility in F1 female rats due to a decrease in embryo implantation (Milesi et al., 2018). In the present work, we sought to assess the effects of perinatal exposure to a GBH or the active ingredient glyphosate (Gly) on female fertility. Moreover, considering the potential of glyphosate as endocrine disruptor, we hypothesized that both GBH and Gly might lead to hormonal and uterine milieu alterations which could disrupt the window of uterine receptivity for embryo implantation. We investigate the effects of perinatal exposure to a low dose of GBH or Gly on: 1) the reproductive performance of female rats on GD19, 2) the serum levels of E2 and Pg and their nuclear receptors in the uterus on GD5 (preimplantation period), and 3) implantation-related gene expression (Hoxa10, Lif, Muc1, Areg,) in the preimplantation uterus.