Aquaporin-dependent excessive intrauterine fluid accumulation is a major contributor in hyper-estrogen induced aberrant embryo implantation

In mammalian species, the window of implantation is defined as a limited period when implantation-competent blastocyst can interact with the receptive endometrium¹. Besides the acquisition of blastocyst competency and uterine receptivity, initiation of embryo implantation also involves a timely reabsorption of intrauterine fluids, which facilitates the apposition of embryo with the uterine lining². These processes are coordinated by preimplantation ovarian estrogen (E2) and progesterone (P4): a small E2 surge imposed on a P4-primed environment initiates implantation¹, where the level of E2 and ratio of P4/E2 must be kept within an optimal range^3,4. To date, numerous genes regulating uterine receptivity and blastocyst implantation have been identified using transgenic mouse models⁵. However, genes controlling uterine fluid homeostasis are much less explored, and their pathophysiological significance in embryo implantation remains poorly understood. In the present study, combining genetic and pharmacological approaches, we demonstrate that an excess of intrauterine fluids caused by increased expression of two water channel genes, aquaporin-5 (Aqp5) and -8 (Aqp8), is a major contributor to abnormal implantation in pregnant mice treated with supraphysiological doses of E2. We also showed that P4 administration neutralized E2-induced Aqp5/8 overexpression, preventing excessive intrauterine fluid accumulation and improving implantation.

To explore the underlying mechanism for the substantial increase in luminal fluid volume, we performed microarray analyses of E2- and vehicle-treated uteri and searched for potential genes responsible for the drastic intrauterine fluid increase. Among the possible candidates, two water channel genes, Aqp5 and Aqp 8, showed a simultaneous increase in expression levels. As aquaporin-mediated fluid transport is involved in a wide range of physiological and pathological conditions¹¹, and previous studies have shown that the expression of aquaporins is dynamically regulated in the uterus during pregnancy^12,13,14, we next examined the uterine expression levels of aquaporin family members in day-4 uteri (untreated, treated with E2 or ER antagonist ICI 182780) by RT-PCR analysis and confirmed the drastic increase in the expression levels of Aqp5 and Aqp8 after E2 treatment (Figure 1J). A weak expression of Aqp5 was observed in the absence of E2 treatment, which was inhibited by ICI treatment (Figure 1J). Similar results were obtained using real-time PCR analysis (Figure 1K and 1L). The expression levels of Aqp5/8 showed a gradual increase, which coincides with the increase in the volume of intrauterine fluids after E2 treatment (Supplementary information, Figure S1G-S1I). We next performed immunofluorescence analysis for Aqp5 and Aqp8 proteins (antibody specificity was confirmed in positive tissues, Supplementary information, Figure S1J and S1K) in day-4 uteri and observed an enhanced staining of Aqp5 and Aqp8 after E2 treatment (Figure 1M). Notably, both Aqp5 and Aqp8 proteins are specifically localized on the apical surface, the secretion side of the uterine glandular epithelium (Figure 1M), suggesting an active role in E2-induced luminal fluid secretion.

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