During the critical period of development, adverse environmental exposure, such as stress, toxins, or an unhealthy diet, will affect the growth trajectory before birth, significantly impact health, and increase disease susceptibility in offspring (Han et al., 2018). Maternal factors play important roles in offspring development and postnatal life in adulthood. In mice, exposure to maternal obesity before and during pregnancy can cause early embryonic developmental delay and fetal developmental delay (Jungheim et al., 2010; Luzzo et al., 2012; McPherson et al., 2015; Sasson et al., 2015; Wu et al., 2015). It has been reported that maternal iron limitation mediated by hepcidin during pregnancy causes embryonic anemia, tissue iron deficiency, and weight loss (Sangkhae et al., 2020). Offspring growth restriction and impaired metabolism in adulthood result from hyperglycemia exposure before and during pregnancy (Bianco and Josefson, 2019). The transgenerational inheritance of epiphenotypes in mammals could be caused by disruptions to gut microbiota, DNA methylation in oocytes, and other pathways (Chen et al., 2016; Li et al., 2016; Consitt et al., 2018; Han et al., 2018; Zhang et al., 2018). Maternal obesity during pregnancy can cause cognitive and social behavior dysfunction in offspring (Liu et al., 2021). These deficiencies derive from changes in the structure of the gut microbiota and alterations in metabolites in both the mother and offspring (Liu et al., 2021).

5-Hydroxytryptamine (5-HT), also known as serotonin, is a neurotransmitter that is widely distributed in the central nervous system and gastrointestinal tract, where it also functions as a hormone and a growth factor. In humans, most 5-HT is found in peripheral tissues, of which approximately 90% is synthesized and mainly distributed in intestinal enterochromaffin cells (Kato, 2013). Notably, previous evidence has shown that 5-HT is detected in reproductive tissues and plays an essential role in cell proliferation, follicular development, embryo development, and fetal growth (Tanaka et al., 1993; Edwards, 1995; Il'kova et al., 2004; Amireault and Dube, 2005; Kim et al., 2010; Oberlander, 2012; Amireault et al., 2013). 5-HT is involved in the pathophysiology of many mental illnesses and is the target of many drug therapies, such as selective serotonin reuptake inhibitor (SSRI) drugs widely used in depression and anxiety (Brummelte et al., 2017). In humans, ingestion of a combination of two or more serotonin (5-HT)-enhancing drugs and overdose of a single drug can cause serious adverse reactions, such as serotonin syndrome (Haberzettl et al., 2015). Serotonin syndrome is a potentially life-threatening drug-induced condition caused by too much serotonin in the brain synapses (Foong et al., 2018). The incidence of serotonin syndrome has gradually increased worldwide, which has mostly resulted from the use of antidepressants, such as monoamine oxidase inhibitors, selective serotonin reuptake inhibitors, and serotonin-norepinephrine reuptake inhibitors (Wang et al., 2016b; Bartlett, 2017). Serotonin syndrome usually manifests as a triad of autonomic dysfunction, neuromuscular excitement, and altered mental status, although not all of these symptoms consistently exist in all patients with the disorder (Boyer and Shannon, 2005). However, there is no evidence of whether a mother suffering from serotonin syndrome will impact the development of the fetus.

As a place for the exchange of nutrients and metabolic waste between the mother and the fetus, the placenta plays an important role in maintaining pregnancy by secreting a variety of hormones, growth factors, and cytokines (Cross, 1998; Rossant and Cross, 2001). Once the development and function of the placenta are impaired, the nutrients, hormones, and growth factors required for embryonic and fetal development cannot be guaranteed (Rossant and Cross, 2001; Regnault et al., 2002). It is easier to transfer toxic maternal substances to the fetus through the placental barrier, which will inevitably damage the growth and development of the embryo and fetus (Dimasuay et al., 2016). In addition, the placenta is an important barrier for fetal immune protection during pregnancy and can protect the fetus from maternal immune system attack (Regnault et al., 2002). Prior evidence has shown that placental developmental disorders can hinder the normal growth of the fetus, causing miscarriage, fetal growth restriction, premature delivery, and stillbirth (Rock et al., 2020). During pregnancy, placental inflammation and impaired function caused by maternal metabolic endotoxemia lead to an adverse early-life environment, which is connected to metabolic dysfunction in offspring (Elahi et al., 2009). It has been reported that maternal circulating microRNAs (miRNAs) can cross the placenta and affect the phenotype of offspring (Nelson and Nadeau, 2010; Sun et al., 2020). Cote found that there were the same 5-HT levels in both tph1^+/− (tryptophan hydroxylase and the rate-limiting enzyme in 5-HT synthesis) female mice and wild-type females, but in tph1^−/− females, it dropped to 3–15% of the level of wild-type females (Cote et al., 2003). Interestingly, the offspring derived from heterozygous mothers have been exposed to a normal 5-HT level, no matter whether their genotype is tph1^−/− or tph1^+/−(Francine et al., 2006). Therefore, maternal 5-HT levels are essential for the normal development of fetuses.

However, it is unclear whether the suffering of mothers from serotonin syndrome before pregnancy can affect the development of offspring. Therefore, we used intraperitoneal injection to change prepregnancy 5-HT levels to explore the effects of maternal prepregnancy 5-HT exposure on the mother, placenta, and fetus. This study provides new insights into the relationships among 5-HT, mother, and fetus and develops new directions for the effects of 5-HT on intergenerational inheritance.

It has been claimed that the environment experienced by the parents affects the offspring who have never experienced the environment (Dietz et al., 2011; Seong et al., 2011; Rando, 2012). For example, exposure to environmental challenges, such as stress, toxins, or an unhealthy diet, can affect the development of the parents (Han et al., 2018). The changes could then be passed on to offspring and could make them more susceptible to illness (Han et al., 2018). Therefore, the change in maternal developmental level is closely related to the developmental ability of the offspring. When the mother takes an overdose of antidepressants, it can even cause 5-HT syndrome, resulting in an increase in 5-HT in the body (Bartlett, 2017). However, whether the increase in 5-HT and even 5-HT syndrome in mothers affects the development of offspring has not yet been studied. Thus, we aimed to understand the potential effects of maternal 5-HT levels on the mother and fetus. In the present study, we first found that the offspring of mothers injected with 5-HT had lower body weight at 2 weeks, which indicates that the mother having 5-HT syndrome before pregnancy may affect the development of the offspring. Previous research mainly focused on obesity, depression, and other diseases of the mother during pregnancy that would affect the development of offspring. Therefore, the result that the prepregnancy changes in the mother affected the development of the offspring in this study is enough to attract more research attention.

In the gastrointestinal tract, 5-HT is a key signaling molecule involved in intestinal secretion, sensation, and peristalsis (Bulbring and Crema, 1958; Gershon, 1999; Shajib and Khan, 2015). It has been demonstrated that 5-HT is involved in mediating interdigestive contractions of the small intestine in rats (Pineiro-Carrero et al., 1991). 5-HT3 and 5-HT4 receptors play essential roles in regulating intestinal interdigestive contractions (Taniguchi et al., 2008). In the present study, we found that intraperitoneal injection of 5-HT could prevent the increase in maternal weight and reduce hormone levels before and during pregnancy in female mice, which was consistent with a previous study in rats (Edwards, 1995). Simansky reported that neural 5-HT could inhibit food intake and tends to reduce body weight gain and even increase energy expenditure (Simansky, 1996). Therefore, we speculate that 5-HT injection affects the contraction and peristalsis of the intestines, and reduces nutrient intake; on the other hand, 5-HT injection inhibits the appetite of mice and reduces food intake, which ultimately causes slower weight gain. We also noticed that there was a reduction in placental weight caused by 5-HT exposure. When the development of the placenta is impaired, the nutrients, hormones, and growth factors that were necessary for the development of the embryo and fetus cannot be passed from the mother, contributing to the abnormal growth and development of offspring. This may explain the increasing fetal absorption rate in the 5-HT group, as well as the reduced body weight of offspring.

Ovarian hormones are known to perform important roles related to various neurotransmitters, such as 5-HT, the noradrenergic system, and glutamate (Paredes et al., 2019). For example, sex hormones affect the synthesis, reuptake, and degradation of the serotoninergic system. Moreover, previous studies have reported that estrogens could regulate the serotonergic system to protect against 5-HT-related diseases. However, in turn, estrogen synthesis was affected to restore homeostasis by 5-HT (Hudon Thibeault et al., 2019). In the present study, we found that 5-HT exposure could reduce maternal estrogen and progesterone levels, which was likely based on the negative feedback effect of 5-HT on hormone synthesis. Low concentrations of estrone-sulfate and estradiol-17β were found in cows that gave birth to stillborn calves, which emphasizes the importance of maintaining proper estrogen concentrations during pregnancy (Kindahl et al., 2004; Nguyen et al., 2012). In primate pregnancy, estrogen deprivation leads to insulin resistance in offspring (Maniu et al., 2016).

5-HT has an essential role in the regulation of embryo development, but the explicit effects remain controversial (Chebotar et al., 1995; Sakharova et al., 1997; Vesela et al., 2003; Il'kova et al., 2004). Our results showed that maternal prepregnancy 5-HT exposure did not affect the cleavage and blastocyst rate in preimplantation embryos. However, much evidence has shown that maternal changes in the environment, nutrition, and endocrine stress during pregnancy can interfere with epigenetic modifications, which further affects gene expression and the growth and development of offspring (Marco et al., 2016; Wang et al., 2016a). For instance, epigenetic changes have been observed in the offspring of the Cohen strain of diabetic rats and human placenta (Ornoy et al., 2015; Petropoulos et al., 2015). In mice, maternal high-fat intake altered mRNA m6A modifications in adipose and skeletal muscle tissues in offspring (Li et al., 2016). The effects of these epigenetic changes may appear in the pro-implantation stage and after birth, rather than in the preimplantation embryo. Further exploration is needed to determine whether maternal 5-HT exposure contributes to the changes in epigenetic modification.

A previous study confirmed that both 5-HT and estrogen are important regulators of the immune system (Shajib and Khan, 2015). In the 1980s, 5-HT was identified as an immunomodulator because of its ability to stimulate or inhibit inflammation (Cloez-Tayarani et al., 2003). In mice, 5-HT administration increased the release of proinflammatory cytokines in macrophages in dextran sodium sulfate (DSS)-induced colitis (Khan and Ghia, 2010). 5-HT can promote the recruitment of mast cells, eosinophils, DCs, and neutrophils in acute inflammation (Boehme et al., 2004). In addition, there is a strong connection between estrogen levels and inflammation (Au et al., 2016). Women who are ovariectomized as well as those with natural menopause exhibit systemic inflammation (Cioffi et al., 2002; Abu-Taha et al., 2009). In the present study, we found that maternal prepregnancy 5-HT exposure caused inflammation in the placenta and increased the expression of proinflammatory markers, which might result from changes in 5-HT and estrogen levels. Therefore, placental inflammation may be directly caused by maternal 5-HT exposure, or 5-HT may indirectly promote inflammation by reducing estrogen levels. Further study should be performed examining the underlying mechanism of this phenomenon.

Maternal inflammation during pregnancy can affect the development of the fetus by interrupting murine intestinal development, influencing placental function, and increasing the risk of neurodevelopmental disorders (Romero et al., 2007; Elgin et al., 2019). In the present study, prepregnancy 5-HT exposure caused inflammation in the placenta and reduced the weights of 2-week-old fetuses. The placenta is an important communication organ between the mother and fetus. Inflammation of the placenta may affect nutrient transfer from the mother to the fetus, which further leads to the abnormal development of the fetus. It ultimately impacts offspring development and causes growth retardation and slow weight gain in offspring.

In summary, our research showed that maternal prepregnancy 5-HT exposure could affect offspring development by reducing maternal body weight, suppressing estrogen and progesterone secretion, and causing placental inflammation. This study provides a basis for the relationship between 5-HT and intergenerational inheritance and opens up new perspectives for future research directions.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by The Animal Care and Use Committee of Jilin University.

BT designed and coordinated the study. YH performed the embryo culture and ELISA and wrote the manuscript. YH, MZ, and JD collected tissues and weighed. LL, JD, and HC performed the qPCR. SZ, YZ, XA, and QL provided the technical assistance and contributed to the data analysis. ZL and XZ revised the manuscript. All authors contributed to the article and approved the submitted version.

The study was funded by the National Natural Science Foundation of China (no. 31472093; 31872434; and 32172803), the National Key R&D Program of China (no. 2017YFA0104400), and the Scientific Research Project of Education Department of Jilin Province (JJKH20211136KJ).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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