Infection during pregnancy increases the risk of negative outcomes during childbirth, and prenatal mycoplasma infection is associated with various adverse pregnancy outcomes such as preterm delivery, spontaneous abortion, and premature rupture of membranes [[1], [2]],. Azithromycin is a macrolide antibiotic that inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit and interfering the bacterial transpeptidation. Azithromycin is currently considered to be the most effective medication for treating mycoplasma infection during pregnancy [3,4]. In recent years, increasing attention has been given to the potential toxicity of prenatal azithromycin exposure (PAzE). A population-based cohort study showed that macrolide antibiotics, including azithromycin, used in early pregnancy (4–13 week's gestation) were associated with an increased risk of malformations, particularly cardiovascular malformations [5]. In addition, macrolides used at any stage of pregnancy were associated with an increased risk of genital malformations. Studies have also demonstrated that azithromycin treatment during pregnancy is related to fetal liver developmental toxicity, maxillary malformations, and neural tube defects [[2], [6]],. These results suggest that PAzE has potential developmental toxicity.

The hippocampus is an important part of the limbic system in humans and vertebrates and is responsible for regulating emotions, learning and memory [7]. Hippocampal lesions may lead to emotional disorder and dysmnesia, neurobehavioral abnormalities, and seizures [[8], [9], [10]]. During the development and maturation of hippocampal neurons, synapses formed by connections between different neurons are the structural foundation for determining the early postnatal function of the brain. In addition, hyperactivated glial cells (including microglia and astrocytes, etc.) would cause neuroinflammation [11]. During nervous system development, hippocampal neuroinflammation and impairment of synaptic plasticity could result in cognitive deficiency in the offspring [12]. Previous studies have demonstrated that exposure to a variety of xenobiotics during pregnancy, such as caffeine [13], dexamethasone [14], and nicotine [15], could result in hippocampal developmental toxicity in the offspring. Exposure to antibiotics of fetus or in the first two years after birth is associated with an increased risk of psychiatric disorders later in childhood [16]. Therefore, intrauterine fetal hippocampal development is susceptible to adverse maternal environmental factors.

Mycoplasma infection during pregnancy can be effectively treated with azithromycin and is widely used in clinical practice. However, the potential developmental toxicity of azithromycin on the hippocampus during pregnancy remains unclear. This study established PAzE mice models at different doses, courses, and time based on the actual clinical medication to investigate the hippocampal morphology and a variety of nerve cells, including neurons, astrocytes and microglia damage under different models. The “toxic window” effect of PAzE-induced hippocampal developmental toxicity was explored, providing an experimental and theoretical basis for the rational usage of azithromycin during pregnancy. Furthermore, the study assessed the risk of long-term diseases related to fetal hippocampal developmental toxicity.