Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.


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Antibodies
Antibodies used "The datasets generated in this study are available in the Gene Expression Omnibus Accession Code GSE152675 or by contacting the corresponding authors." For our mouse studies, the sample size was determined based on a power analysis calculation that took into account various paraments, such as effect size, standard deviation, type 1 error and direction of effect. Because of the genetic nature of our animal studies, and the limitations based on sex and age, we also factored an expected attrition of animals in our breeding schemes.
For the experiments other than those involving mice, a minimum sample size of three was utilized to ensure that findings were consistent across a variety of technical and biological conditions. If findings across the samples were found to be consistent, and that the experimental assumptions were held constant, this was determined to be sufficient sample size. This approach was necessary in the experiments requiring specimens from human donors, where the availability of samples was extremely limited.
For pregnancy studies, only data from all mice deemed to be at the correct stage of pregancy were utilized (based on serum levels of progesterone), or based on the number of blastocysts recovered after flushing of the uterus. Those samples from mice that were deemed not to be at the correct stage of pregnancy were excluded from analysis.
To verify the reproducibility of our studies, experiments were repeated in samples from various subjects and performed on different dates, often by different investigators. Obtaining mice of the same age, sex, and desired genotypes that are at the specific time of pregnancy, required various rounds of mating and timed-mating. Therefore, to obtain enough mice of the correct genotype and stage of pregnancy, the experiments were carried out over the course of at least 6 months, with various rounds of mouse breeding, genotyping, timed mating, dissection and verification of pregnancy. Once the required cohort of mice was obtained, the desired endpoints were assessed and replicated at minimum 3 times. These attempted replicates were successful, given that the techniques assessed are well-established in our laboratory and routinely performed by all labmembers. If unsuccessful attempts were obtained, this was attributed to the absence of a pregnancy, or an incorrectly staged animal.
Samples and organisms were separated into specific groups based on 1) genotype, 2) pregnancy status, 3) or estrous cycles. Because studies were investigating early pregnancy, only female mice were utilized.
Because the analysis was being performed on grouped mice according to genotype, and the differences in gene expression and histology were based on these genetic differences and pregnancy states, blinding was not performed