Optimizing IVF by controlling for both embryonic aneuploidy and endometrial receptivity using genetic testing

Successful implantation following in vitro fertilization (IVF) requires the transfer of a good quality and competent blastocyst to a receptive endometrium. Since the introduction of IVF, emphasis has been placed mainly on embryo development and quality, and this has guided decisionmaking regarding what day to perform an embryo transfer and how many embryos to transfer. IVF lab techniques to improve culture conditions contributed to an improvement in embryo quality and implantation rates. In recent years, an increase in Preimplantation Genetic Testing for Aneuploidy (PGT-A) involving transfer of euploid embryos has further enhanced these rates. Despite the transfer of good quality euploid blastocysts however, implantation failure still occurs in approximately one third of PGT-A cycles. In order to optimize implantation rates, it is necessary to focus both on the embryonic contribution, and on the role of the endometrium. In this regard, the most likely cause of implantation failure is alteration or desynchrony of endometrial receptivity. Until recently, there has been no objective and reliable diagnostic test to assess endometrial receptivity accurately and provide reproducible results with a clear clinical directive. This, combined with the widely accepted assumption that all women possessed a consistent window of implantation (WOI), meant that meaningful assessment of endometrial receptivity was often omitted from the standard IVF work-up. However, the implementation of personalized medicine to IVF has expanded our understanding of endometrial receptivity and introduced the concept of a personalized WOI (pWOI). A prime example is Endometrial Receptivity Analysis (ERA), a robust molecular test that deciphers the gene expression pattern of an endometrial biopsy sample and employs a bioinformatic predictor that accurately diagnoses the phase of receptivity, providing a recommendation for a personalized embryo transfer (pET).

The overall aim of this thesis is to answer the question: can implantation following IVF be optimized when controlling for both embryonic factor with PGT-A and endometrial factor with ERA? To address this question, the work described here investigates the impact of controlling for embryo aneuploidy and endometrial receptivity, both independently and together, and their effects on clinical outcomes following IVF. This thesis comprises of seven specific aims: a) to provide a preliminary assessment of aneuploidy rates between the polar, mid and mural trophectoderm; b) to establish whether there are improved pregnancy rates following endometrial receptivity analysis and personalized embryo transfer in patients with previous failed implantation after euploid embryo transfer; c) to ask the question of whether the clinical outcomes are different in day-five versus day-six single embryo transfer when endometrial factor is controlled; d) to evaluate clinical outcomes associated with personalized embryo transfers

guided by transition phase results: to ask whether small shifts can lead to big outcomes; e) to ask, through a single case report, what is the narrowest window of implantation by examining the evolution of the endometrial phases via transcriptomic profiling of biopsies taken at various hours of progesterone exposure; f) to provide a novel reanalysis of published data presented pertaining to inter-cycle consistency versus test compliance in endometrial receptivity analysis testing; and g) to assess the prevalence of a displaced WOI in gestational carriers and the clinical utility of applying ERA.

This thesis was largely successful in the fulfillment of these aims in that, the research presented herein provided novel insight into the basic understanding of chromosomal aneuploidy in embryos and endometrial receptivity, along with the clinical impact of controlling for each of these factors, both independently and collectively. My work determined a) a difference in aneuploidy rates between biopsies taken from the mural versus the polar trophectoderm, possibly suggesting an underlying mechanism to embryo implantation and further considerations when studying mosaicism within preimplantation embryos. b) When controlling for both

embryonic aneuploidy and endometrial receptivity, significantly improved implantation rates were achieved in patients with a previous failed euploid transfer, demonstrating the necessity of proper embryo-endometrial synchrony to successful implantation. c) when properly controlling for endometrial receptivity with ERA, similar clinical outcomes between day-five versus day-six blastocysts were observed, suggesting equal viability of blastocysts regardless of blastulation timing. This contributes to our basic understanding of embryo development and potential, and it allows other factors of embryo viability to be prioritized when selecting the best embryo for transfer. d) novel understanding into the WOI was achieved, demonstrating that small shifts of only 12 hours lead to improved outcomes, along with e) showing the existence of an extremely narrow WOI (lasting less than 12 hours). Both these observations clearly epitomize how unique the WOI is in each woman, along with the advantages that precision medicine has to offer in terms of improving clinical IVF outcomes by providing a personalized recommendation for transfer timing based on a woman’s unique genetic profiling. f) It is also demonstrated the consistency of receptivity timing from cycle to cycle, increasing our confidence in transcriptomic analysis for endometrial dating and embryo transfer planning. g) Endometrial transcriptomic studies in gestational carriers (GC) (i.e., women with proven fertility) at time of expected receptivity revealed an unexpectedly high rate of non-receptivity at the standard transfer time in hormone replacement therapy (HRT) cycles, suggesting that receptivity may be commonly altered in this population outside of natural conception cycles. Following the clinical outcomes in GCs when applying a pET, especially when also controlling for embryo aneuploidy, exceptionally high rates of implantation were achieved, suggesting the significance of applying both these technologies, even in a “fertile” population of patients, and again undoubtedly demonstrating the clinical relevance of controlling for both embryo aneuploidy and endometrial receptivity to optimize IVF outcomes.