Administration of calcitonin promotes blastocyst implantation in mice by up-regulating integrin β3 expression in endometrial epithelial cells.

STUDY QUESTION: Does exogenous calcitonin improve the efficiency of implantation in mice by increasing uterine receptivity?

SUMMARY ANSWER: The administration of calcitonin could improve the efficiency of implantation by increasing the expression of several receptivity-related genes in endometrial epithelial cells (EECs).

WHAT IS KNOWN ALREADY: Calcitonin is one of the biomarkers of uterine receptivity, which is transiently produced in the uterine epithelia during the period of implantation both in humans and mouse.

STUDY DESIGN, SIZE, DURATION: Hormone-replaced mice were used for in vivo experiments. To evaluate the effect of calcitonin on uterine receptivity, the expression of endometrial genes was analyzed 36 h after i.p. injection of 0.5 IU calcitonin in a treatment group versus saline in the control. To evaluate the effect of calcitonin on implantation efficiency in vivo, two groups received 0.5 IU or 2 IU calcitonin (i.p.) 24 h before embryo transfer, and a control group received saline (i.p.) (n = 18 mice per group). Implantation sites were counted 7 days after embryo transfer. The RL95-2 human endometrial carcinoma cell line was used to study the mechanisms underlying the effect of calcitonin on gene expression in the endometria. Using an in vitro model of endometrium-trophoblast interaction, established with RL95-2 cells and JAR (human choriocarcinoma cell line) trophoblast, endometrial receptivity was evaluated by comparing attachment and outgrowth of JAR spheroids in control and treatment groups.

PARTICIPANTS/MATERIALS, SETTING, METHODS: Uterine receptivity in ovariectomized mice was induced by injection of estradiol and progesterone. Expression of eight genes in murine endometrium and RL95-2 cells was analyzed by real-time RT-PCR, western blot, immunohistochemical analysis, flow cytometry and enzyme-linked immunosorbent assay. We tested the effects of a protein kinase C inhibitor, matrigel and an antibody against integrin αvβ3 using RL95-2 cells and performed attachment and outgrowth assays using the in vitro model of endometrium-trophoblast interaction. Implantation efficiency was evaluated by counting the implantation sites after embryo transfer.

MAIN RESULTS AND THE ROLE OF CHANCE: Calcitonin up-regulated αvβ3 in RL95 cells, which in turn resulted in increased levels of the leukemia inhibitory factor (LIF) and heparin binding-epidermal growth factor (HB-EGF) mRNA (both P < 0.01 versus control) and protein (both P < 0.05 versus control). The attachment and expansion of JAR spheroids was promoted by pretreatment of EECs with calcitonin (P < 0.05 versus control) together with significantly increased expression of αvβ3, LIF and HB-EGF. Moreover, the injection of calcitonin in the preimplantation phase increased the total number of implantation sites in treatment groups (55 in control versus 78 and 85 in 0.5 and 2 IU groups, respectively). Compared with the control group (3.11 ± 2.14), the average number of implantation sites in the 2 IU calcitonin treatment group increased (4.72 ± 1.87, P = 0.022).

LIMITATIONS, REASONS FOR CAUTION: Experiments were performed in mice and human cell lines but not in primary cultures of human endometrial cells.

WIDER IMPLICATIONS OF THE FINDINGS: The findings presented here have important implications, in that calcitonin administration (currently used for treatment of hypercalcemia or osteoporosis) may have clinical benefits in assisted reproduction programs, by facilitating endometrial receptivity and embryo implantation. However, further studies are required to confirm these findings.

STUDY FUNDING/COMPETING INTEREST(S): This work was supported by National Science Foundation of China (No. 81170619). There are no financial or commercial conflicts in this study.