SARS-CoV-2 and Implantation Window: Gene Expression Mapping of Human Endometrium and Preimplantation Embryo

Understanding whether SARS-CoV-2 could infect cells and tissues handled during ART is crucial for risk mitigation, especially during the implantation window when either endometrial biopsies are often practiced for endometrial receptivity assessment or embryo transfer is performed. To address this question, this review analyzed current knowledge of the field and retrospectively examined the gene expression profiles of SARS-CoV-2-associated receptors and proteases in a cohort of ART candidates using our previous Affymetrix microarray data. Human endometrial tissue under natural and controlled ovarian stimulation cycles and preimplantation embryos were analyzed. A focus was particularly drawn on the renin-angiotensin system, which plays a prominent role in the virus infection, and we compared the gene expression levels of receptors and proteases related to SARS-CoV-2 infection in the samples. High prevalence of genes related to the ACE2 pathway during both cycle phases and mainly during the mid-secretory phase for ACE2 were reported. The impact of COS protocols on endometrial gene expression profile of SARS-CoV-2-associated receptors and proteases is minimal, suggesting no additional potential risks during stimulated ART procedure. In blastocysts, ACE2, BSG, CTSL, CTSA and FURIN were detectable in the entire cohort at high expression level. Specimens from female genital tract should be considered as potential targets for SARS-CoV-2, especially during the implantation window.

Hypothyroidism Affects Uterine Function via the Modulation of Prostaglandin Signaling

Thyroid hormones control the functions of almost all body systems. Reproductive dysfunctions, such as abnormal sexual development, infertility, or irregularities in the reproductive cycle, might be associated with thyroid disorders. Uterine receptivity is the period when the uterus is receptive to the implantation of an embryo. During the receptivity period (implantation window), a newly formed blastocyst is incorporated into the uterine epithelium. Prostaglandins are well-known primary mediators of pathological conditions such as inflammation and cancer but are also essential for the physiology of female reproduction. The aim of this study was to evaluate the possible relationship between hypothyroidism and changes in the prostaglandin signaling pathways in the uterus and in the process of uterine receptivity in a rat model. The results show that hypothyroidism impaired uterine receptivity by decreasing the level of E2 as well as decreasing the expression of the uterine-receptivity factors homeobox A10 and osteopontin. Moreover, hypothyroidism caused changes in the expression of elements of the prostaglandin E2, F2α, and I2 signaling pathways and changed the levels of those prostaglandins in the uterine tissue. The results suggest that the mechanisms by which hypothyroidism affects female reproductive abnormalities might involve the prostaglandin signaling pathway, resulting in a subsequent reduction in uterine receptivity.

Expression and significance of miR-30d-5p and SOCS1 in patients with recurrent implantation failure during implantation window

Background Poor endometrial receptivity is a major factor that leads to recurrent implantation failure. However, the traditional method cannot accurately evaluate endometrial receptivity. Various studies have indicated that microRNAs (miRNAs) are involved in multiple processes of embryo implantation, but the role of miRNAs in endometrial receptivity in patients with recurrent implantation failure (RIF) remains elusive. In the present study, we investigated the presence of pinopodes and the roles of miR-30d-5p, suppressor of cytokine signalling 1 (SOCS1) and the leukaemia inhibitory factor (LIF) pathway in women with a history of RIF during the implantation window. Methods Endometrial tissue samples were collected between January 2018 to June 2019 from two groups of women who underwent in vitro fertilisation and embryo transfer (IVF-ET) or frozen ET. The RIF group included 20 women who underwent ≥ 3 ETs, including a total of ≥ 4 good-quality embryos, without pregnancy, whereas the control group included 10 women who had given birth at least once in the past year. An endometrial biopsy was performed during the implantation window (LH + 7). The development of pinopodes in the endometrial biopsy samples from all groups was evaluated using scanning electron microscopy (SEM). Quantitative reverse transcription-polymerase chain reaction and western blotting were used to investigate the expression levels of miR-30d-5p, SOCS1, and the LIF pathway. Results The presence of developed pinopodes decreased in patients with RIF on LH + 7. The expression level of miR-30d-5p decreased in the endometria during the implantation window of patients with RIF, whereas the mRNA and protein levels of SOCS1 were significantly higher in the RIF group than in the control group. Furthermore, a negative correlation was observed between the expression of miR-30d-5p and SOCS1 (r2 = 0.8362). In addition, a significant decrease in LIF and p-STAT3 expression was observed during the implantation window in patients with RIF. Conclusions MiR-30d-5p and SOCS1 may be potential biomarkers for endometrial receptivity. Changes in pinopode development and abnormal expression of miR-30d-5p, SOCS1 and LIF pathway in the endometrium could be the reasons for implantation failure.

Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterised endometrial assembloids, consisting of gland-like organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by eliminating the emergence of senescent decidual cells. In co-culture experiments, accelerated decidualization resulted in entrapment of collapsed human blastocysts in a robust, static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings suggest that decidual senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

O-199 Proof of concept: implantation window must be wider than proposed. Report of seven twins after asynchronous double embryo transfer

Study question Can simultaneous transfer of two embryos that were cryopreserved at different stages (D3 and Blastocyst) be appropriate to enhance success in women with more than three failed embryo transfers? Summary answer Double asynchronous embryo transfer offered excellent results in RIF. Unexpectedly high twin rate suggests that embryo-endometrium synchrony is overemphasized. Implantation window must be wider. What is known already Transcriptomic signature of the endometrium has been investigated in the last few years trying to understand the best moment for embryo implantation. Nevertheless, the optimal period has not been well established yet in humans. Simultaneous transfer of two human embryos at different developmental stages (D3 and Blastocyst) on Day 4 was proposed to help couples who have had RIF. Study design, size, duration Observational case-control study. From April 2016 to January 2021, we offered double asynchronous embryo transfer only after Recurrent Implantation Failure (RIF). Two requirements were necessary: 1) Double embryo transfer was acceptable by the couple due to poor reproductive outcome. 2) Availability of two embryos cryopreserved at different stage (D3 and Blastocyst). Results were compared with good prognosis patients (all patients under 35 years in that period who had elected to transfer two day 3 cryopreserved embryos). Participants/materials, setting, methods Forty-five patients accepted to participate in the study. Results were compared with all patients (237) under 35 years where two D3 thawed embryos were transferred. All cases received same protocol (oral estradiol 6mg/d or vaginal estradiol 4mg/d until ultrasound showed endometrial growth) LH, P4 and E2 were monitored in all patients to detect spontaneous LH surge. All cases received transvaginal micronized progesterone 800 mg/d. Embryo transfers were ultrasound guided and Wallace Embryosure catheter was employed. Main results and the role of chance Limitations, reasons for caution Multiple pregnancy rate was unacceptably high. Therefore, it should not be suggested for good prognosis couples where single embryo transfer is clearly advidsed. Our main limitation was the combination of D3 embryos with blastocysts. The retrospective design make the results to be considered as a proof of concept. Wider implications of the findings Double asynchronic embryo transfer can offer new insights in the understanding of human implantation. The concept of implantation window is clearly challenged. Aiming to the center of the window is fine, but we still dońt know how wide is that center. Trial registration number not applicable

P–392 Clinical outcomes of endometrium receptivity analysis(ERA) testing in patients with repeated IVF failures

Study question Is ERA testing different between RIF patients with control group? Summary answer In RIF patients, there were more chances of non-receptive endometrium. ERA testing may be helpful for the patients with repeated IVF failure. What is known already: The endometrium receptivity analysis testing might have the ability to detect the implantation window. In repeat implantation failure patients, detecting of precisely implantation window may have some benefits. Study design, size, duration This was a single-center retrospective observational study. Two hundred and forty-nine patients who underwent ERA testing following frozen-thawed embryo transfer in our center were including in this study between January 2019 and May 2020. Participants/materials, setting, methods 181 patients having unexplained repeated IVF failure (RIF group, at least tow implantation failure) and 68 patients having no experience with embryo transfer (Control group) who underwent ERA testing were including in this study. Both of Patients having a receptive (R) ERA and having a non-receptive (NR) ERA underwent a personalized embryo transfer (pET) on ERA. ERA results and clinical outcomes compared between RIF group and control group were analyzed by Chi-square test. Main results and the role of chance The proportion of R/NR results were 33:35 for the RIF group and 118:63 for the Control group, demonstrating the displacement of the window of implantation in patients with RIF. Our results revealed an endometrial factor in 51% RIF patients, which was significantly greater than the Control group 34.8% (P = 0.02). Among the patients with NR ERA result, there are not significantly difference in clinical pregnancy rate in the RIF group compared with control group (57.1%. vs. 61.9%). The clinical pregnancy rate of the patients with receptive ERA result also is comparable in both group (70.3% vs. 66.7%). Limitations, reasons for caution This is a retrospective, single center study with limited case number. There were may some bias with ERA testing errors. Wider implications of the findings: In RIF patients, there were more chances of non-receptive endometrium. ERA testing may be helpful for the patients with repeated IVF failure. Larger randomized studies are required to validate these results. Trial registration number 18MMHISO70e

P–361 Endometrium optical coherence tomography (OCT) and histomorphometry in implantation window and their relationships with reproductive failure and implantation outcome

Study question How do endometrium OCT image characteristics during peri-implantation window correlate with histomorphometry and associate with implantation outcomes in women with reproductive failure? Summary answer Endometrium OCT intensity correlated with stromal cell density and gland size. Endometrium with recurrent implantation failure had low OCT intensity but reversed in successful implantation. What is known already OCT is a non-invasive imaging technique using low energy near-infrared light to capture micrometer-scale resolution images from optical scattering media. An image produced by OCT resembles tissue architecture observed in histology, so OCT imaging has been regarded as “optical biopsy”. Our previous findings demonstrated OCT is better than ultrasound to identify endometrial pathology. We also showed association of OCT signal with microvessel density in peri-implantation endometrium. However, other histomorphometry were not evaluated. It is still unclear whether endometrium OCT image characteristics are different in reproductive failure and can predict implantation outcomes. Study design, size, duration This was a prospective study conducted at teaching hospital of The Chinese University of Hong Kong from Jan 2018 to Dec 2019. 46 infertile women with or without recurrent miscarriage (RM) and implantation failure (RIF) were recruited in this study. Endometrium OCT imaging and subsequent biopsy were performed on the seventh day after luteal hormone surge (LH + 7) in natural cycle prior to the consecutive natural conception or embryo transfer (ET) cycle. Participants/materials, setting, methods At least 5 systematic random endometrium OCT images from uterine fundus, body and lower segment of each subject were included for intensity analysis by two independent observers. OCT intensity of each image was classified as low, moderate, high based on optical range and then average OCT intensity in each uterine region was calculated for group comparison. Endometrium glandular epithelial, stromal, endothelial, uNK cells were defined by standard H&E and specific immunostaining for histomorphometry and correlation. Main results and the role of chance OCT intensity significantly correlated with endometrial cell and gland parameters regardless classifications of reproductive failure and implantation outcome. Higher OCT intensity indicated higher stromal cell density, gland to stromal (G/S) ratio and average gland area, but fewer microvessel and uNK cells. None of the endometrium histomorphometry were significantly different among different reproductive failure types and implantation outcomes, suggesting it may not be sensitive enough to detect the abnormal histological features. However, OCT intensity was significantly lower in the uterine fundus and body of RIF group than in that of infertile and RM groups. There was no significant difference of OCT intensity in the lower part of the endometrium among three groups. It indicates that OCT intensity is a sensitive marker to differentiate endometrium with RIF from the endometrium with other conditions and also endometrium with RIF is characterized with less stromal cells and smaller glands. Compared with infertile group with unsuccessful implantation, OCT intensity was higher in all three parts of the uterus from the infertile group with successful implantation, but the results were not statistically different. The results further implied that endometrial cells and gland size may potentially contribute to the endometrium receptivity for implantation. Limitations, reasons for caution Current endometrium OCT imaging depth is within 3mm, change beyond this thickness is inaccessible but still the most important layer for implantation. This is a pilot and small study with lack of normal fertile control. Endometrium OCT imaging in the same natural conception or ET cycle will be more accurate. Wider implications of the findings: OCT imaging could be used as a potential noninvasive modality to evaluate peri-implantation window endometrium. It enables real-time and in-situ visualization of endometrium structure and pathology with no additional biopsy risk and examination delay. Larger clinical trials are needed to confirm its clinical applications and utilities. Trial registration number CREC 2016.160

P–297 Endometrium receptivity test in patients with failed embryo implantation

Study question Studying the receptivity of the endometrium for improving the synchronicity between the embryo and the condition of the endometrium. Summary answer Determination of the level of endometrial receptivity with PGT may significantly increase the effectiveness of IVF in patients with recurrent implantation failure What is known already Modern approach to the treatment of patients with recurrent implantation failure, using assisted reproductive technologies imply preimplantation genetic testing (PGT) of embryos for chromosomal abnormalities. However, in some cases, even with the transfer of a genetically complete embryo, pregnancy does not occur. A possible reason is a violation of embryo implantation due to a shift in the period of the “implantation window” . The ERA (Endometrium Receptivity Assay) is a personalized molecular genetic test specifically designed to analyze the level of endometrium receptivity for determine the period of the “implantation window”. Study design, size, duration ERA test is carried out for patients with idiopathic infertility, in cases where repeated transfer of a genetically complete embryo does not lead to implantation and pregnancy: at least three attempts in the case of women under 37 years old and two attempts - for older women (without pathological morphological changes in the endometrium). The test was performed in a cycle with hormone replacement therapy (HRT) or a natural cycle. Participants/materials, setting, methods Were investigated the endometrial Pipell biopsy specimen in a cycle of hormone replacement therapy (HRT) or a natural cycle. Endometrial examination was implemented using the Endometrium Receptivity Assay, the analysis of mRNA of 238 genes was carried out, which showed a difference in the level of expression when analyzing samples of prereceptive, receptive and postreceptive endometrium. Main results and the role of chance The results of the studies of the level of endometrial receptivity revealed a shift in the period of the “implantation window” in 28% of patients. The results obtained make it possible to carry out the transfer of embryos in an individual order for each patient, taking into account the data on the level of receptivity of the endometrium, which makes it possible to implement the so-called Personalized Embryo Transfer (pET) principle. In 72% of cases, the study showed the “receptive” status of the endometrium. The result means that the period of the “implantation window” for a particular type of cycle falls on the moment of biopsy and this period of time is the most favorable for implantation of the embryo. For patients from this group, embryo transfer is carried out at the next repetition of the cycle option selected for the study of receptivity, while the blastocyst transfer occurs on the same day of the cycle when the endometrial biopsy was performed. Limitations, reasons for caution The ERA test is unique technique. Wider implications of the findings: Using the ERA test allow to determine the state of maturity of the endometrium. Unlike other methods for receptivity of the endometrium, the ERA test allows not only to accurately determine the period of the “implantation window”, but also to reliably predict its displacement to an earlier or later time. Trial registration number Not applicable

Screening of Variants in the Transcript Profile of Eutopic Endometrium from Infertile Women with Endometriosis during the Implantation Window

Objective Abnormalities in the eutopic endometrium of women with endometriosis may be related to disease-associated infertility. Although previous RNA-sequencing analysis did not show differential expression in endometrial transcripts of endometriosis patients, other molecular alterations could impact protein synthesis and endometrial receptivity. Our aim was to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis and controls during the implantation window. Methods Data from RNA-Sequencing of endometrial biopsies collected during the implantation window from 17 patients (6 infertile women with endometriosis, 6 infertile controls, 5 fertile controls) were analyzed for variant discovery and identification of functional mutations. A targeted study of the alterations found was performed to understand the data into disease's context. Results None of the variants identified was common to other samples within the same group, and no mutation was repeated among patients with endometriosis, infertile and fertile controls. In the endometriosis group, nine predicted deleterious mutations were identified, but only one was previously associated to a clinical condition with no endometrial impact. When crossing the mutated genes with the descriptors endometriosis and/or endometrium, the gene CMKLR1 was associated either with inflammatory response in endometriosis or with endometrial processes for pregnancy establishment. Conclusion Despite no pattern of mutation having been found, we ponder the small sample size and the analysis on RNA-sequencing data. Considering the purpose of the study of screening and the importance of the CMKLR1 gene on endometrial modulation, it could be a candidate gene for powered further studies evaluating mutations in eutopic endometria from endometriosis patients.