Putative Window Of Implantation Research Articles

The use of propensity score matching to assess the benefit of the endometrial receptivity analysis in frozen embryo transfers

To study the impact of the endometrial receptivity analysis (ERA) on live birth rates in frozen embryo transfer (FET) cycles. Retrospective cohort study. A single, large, university-affiliated infertility practice. Autologous FET cycles between January 1, 2014, and June 30, 2019, were reviewed. Multiple covariates that impact outcomes were used for propensity score matching; 133 ERA patients were matched to 353 non-ERA patients. Patients were assigned to the ERA group if they had an ERA during treatment and underwent at least one "personalized" FET based on the ERA recommendations. No interventions administered. Live birth rates per cycle in the FET cycle after ERA compared with that of matched non-ERA patients. The live birth rates for the ERA group, 49.62%, and the matched non-ERA group, 54.96%, (odds ratio 0.8074; 95% confidence interval, 0.5424-1.2018) were not significantly different, nor was a difference seen in subanalyses based on prior number of FETs or receptivity status. The ERA identifies a patient's putative window of implantation with the goal of improving synchrony with the embryo, thereby achieving higher live birth rates. This study used propensity score matching to control for multiple covariates in a heterogenous group of patients to compare live birth rates. There was no difference in the live birth rate in patients who underwent the ERA compared with that of those who did not.

Inflammosome in the human endometrium: further step in the evaluation of the “maternal side”

To investigate the expression of inflammosome components (NALP-3, associated speck-like protein containing a CARD [ASC]) and their activation (caspase-1, interleukin [IL]-1β, and IL-18 secretion) in the human endometrium from fertile and women with history of recurrent pregnancy loss (RPL). Experimental study. University hospital. Ten fertile women (control group [CTR]) and 30 women with RPL. None. Endometrial samples were collected by hysteroscopy during the putative window of implantation and evaluated for chronic endometrial inflammation by hystopathological analysis. Inflammosome expression was analysed by immunohystochemical staining (27 RPL and 10 CTR women). The expression of NALP-3 and ASC protein was quantified by Western blot (30 RPL and 10 CTR women). Caspase-1 activation and IL-1β and IL-18 secretion was quantified by ELISA (30 RPL and 10 CTR women). We observed a significantly increased expression of inflammasome NALP-3 and ASC protein, an increased activation of caspase-1, and increased levels of IL-1β and IL-18 in RPL endometrium compared with CTR. Abnormal activation of endometrial innate immunity by means of inflammosome, stimulated by pathogen- or damage-associated molecular patterns, may represent an additional mechanism, currently not investigated, negatively interfering with endometrial receptivity. More studies are required [1] to identify the primary trigger of endometrial inflammosome activation and its clinical impact in the occurrence of RPL; and [2] to validate the inflammosome components as a novel family of endometrial biomarkers and promising therapeutic targets in RPL.

Role of endometrial concentrations of heavy metals (cadmium, lead, mercury and arsenic) in the aetiology of unexplained infertility

ObjectiveTo determine the role of endometrial concentrations of heavy metals (cadmium, lead, mercury and arsenic) in the aetiology of unexplained infertility. Study designThirty-three women with unexplained infertility and 32 fertile women were recruited. Endometrial biopsies were collected during the putative window of implantation (cycle days 20–24). The concentrations of cadmium, lead, mercury and arsenic were measured in endometrial biopsy specimens using atomic absorption spectrometry. ResultsCadmium was detected in 91% (30/33) of women with unexplained infertility, compared with 34% (11/32) of fertile women. The median endometrial cadmium concentration was 19.58 (interquartile range 1.46–30.23)μg/l in women with unexplained infertility, compared with 0.00 (interquartile range 0.00–0.40)μg/l in fertile women. Lead was detected in 15% (5/33) of women with unexplained infertility and 3% (1/32) of fertile women. Mercury and arsenic were not detected in any endometrial samples from either group. ConclusionA significant difference in endometrial cadmium concentration was found between women with unexplained infertility and fertile women. This suggests that cadmium may be a contributing factor in the aetiology of unexplained infertility.

Expression of VLA1, VLA2 and VLA3 Integrin Molecules in Uterine Endometrium of Infertile Women with Unexplained Aetiology in Ahwaz-Iran.

Recent attention has focused on the expression of integrin molecules within the endometrium, and their relation to infertility. The present prospective study was undertaken to determine whether the endometrium of women with unexplained infertility differs in the expression of very late activation antigens (VLA) from the endometrium of normal fertile women. Thirty samples of endometrial biopsies from hysterectomies with nonendometrial pathology and 28 endometrial samples by uterine curetting from infertile women in secretary phase at implantation time were collected, stained with three monoclonal antibodies against beta1 integrin subunits including VLA-1 to VLA-3 by immunohistochemical technique and then assessed semiquantitively by microscope. Chi-Square test was used to compare the expression of VLA antigens on epithelial cells, stromal cells, lymphocytes and vessels within endometrial tissues between two groups. The results showed that most VLA integrins were present in fertile and infertile endometrium tissues. There were similarities and differences in the expression of VLA molecules in different compartments. VLA-2, VLA-3 expression on endometrial compartments showed an unaltered pattern of staining during the putative window of implantation in either fertile or infertile women with no significant differences (P-value> 0.5). VLA-1 expression on endometrial compartments changed in fertile and unexplained infertile women, the differences were related to the presence or lack of the molecules on epithelial and stromal cells respectively. Differences may explain causes of unexplained infertility, and suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation and early placental development which requires more investigations.

Estrogen and Progesterone Response Elements in Genes Relevant for Human Embryo Implantation Identified by Microarray/Bioinformatics

The process of embryo implantation is a condition of equilibrium in the up- and down-regulation of diverse endometrial genes/proteins under control of steroid hormones and other local factors. The objective of this study was to identify putative estrogen and progesterone response elements (ERE and PRE) in the 5’-flanking regions of a subset of genes found to be differentially expressed in two clinical scenarios: (1) during the transition of the pre-receptive (early luteal phase, day 16) to the receptive endometrium (mid-luteal phase, day 21) in natural cycles; and (2) during the putative window of implantation (day 21) of gonadotropin-stimulated controlled ovarian hyperstimulation (COH) cycle of oocyte donors in GnRH agonist or antagonist cycles compared to temporally-matched natural cycles. Prospective, randomized and blinded clinical research study. RNA was extracted from endometrial specimens; for global gene expression profiling cRNA was generated and hybridized to Affymetrix Hu95A microarrays. Statistical analysis of the genomic data was performed using pair wise multiple group comparison (SAM software). To validate microarray findings we quantified the expression of selected genes using semiquantitative RT-PCR. We retrieved a 3500 bp-long sequence encompassing 3000 bp upstream and 500 bp downstream of the transcriptional start site of each gene. The search for putative EREs was carried out with ERE finder software with a range of sensitivity threshold from 0.75 to 0.87. The presence of potential EREs was also investigated using the MATCH program using nucleotide distribution matrix for ER with core similarity set to the stringency levels optimal to reduce false positives. The search for putative PREs was conducted using a pattern-based method implemented in the GeneQuest module of Lasergene software. (1) Candidate ERE sequences were found in the 5’-flanking regions of 10 genes among 12 genes analyzed that were differentially expressed when comparing the receptive versus the pre-receptive endometrium; 8 of such genes (CD55, SERPING1, ID4, GATA2,MAPK3K5, CIR, MSX1 and GATA 2) had multiple ERE-like sequences. Seven genes had putative PREs. (2) Linear discriminant analysis identified 4 genes (OPTN, SNX7, PCOLN3, and COX 17) significantly up regulated in COH versus natural cycles with maximal potential functional significance. For these genes, the presence of ERE-like sequences was significantly higher than an average for the human genome. Of major interest was the discovery of an identical ERE-like sequence (TGGCCA-NNN-TGGTCT) in the promoter regions of extended homology of these genes. PREs were identified in two genes. This study identified a number of genes that may have functional significance for the opening and maintenance of the window of receptivity. Although induction of expression by steroid hormones has been previously detected for some of these genes, the mechanism of their transcriptional activation remains unknown. It is reasonable, however, to assume that in the endometrium, transcription of the implantation-related genes is directly regulated by nuclear estrogen and/or progesterone receptors. Our results demonstrated that most of these genes depict putative EREs and PREs. The identification of a unique pattern of ERE-like sequence in a subset of genes upregulated during COH cycle provides strong support for their functional significance.

Genomic and proteomic analyses of the endometrium during the window of implantation following laser capture microdissection: Comparison of natural and controlled ovarian hyperstimulation (COH) cycles

The process of embryo implantation is a condition of equilibrium in the up- and down-regulation of diverse endometrial genes/proteins under control of steroid hormones and other local factors. The objective of this study was to compare endometrial gene and protein expression during the window of implantation of natural and COH cycles following tissue compartmentalization using laser capture microdissection (LCM). Prospective, randomized and blinded research study. Endometrial biopsies were obtained during the putative window of implantation (day 21) from (a) COH cycles of oocyte donors (n = 10); and (b) natural cycle controls (n = 5). Oocyte donors were stimulated either with a GnRH agonist (long protocol) and recombinant FSH (rFSH) or with rFSH and a GnRH antagonist. For global gene expression profiling, cRNA was generated and hybridized to Affymetrix Hu95A microarrays. Prior to proteomic analysis, LCM was used to isolate specific endometrial cell types, i.e. epithelial and stromal cells. Proteomics was performed to the compartmentalized endometrium using surface enhanced laser desorption/ionization (SELDI)-time of flight spectrometry (TOF) technology. Protein lysates were loaded robotically on an IMAC 3 chip pretreated with CuSO4. Each resulting protein peak was determined and its intensity normalized for total ion current. Statistical analysis of the genomic data was performed using pair wise multiple group comparison (SAM software), while statistical analysis of the proteomic data was based on the Student’s t test and CART analysis using Ciphergen’s Biomarker Patterns Software. SAM analysis reveled that the endometrium of COH cycles differed in 18 genes in comparison to the timely matched natural cycles. Optineurin and procollagen type 3 endopeptidase were genes up regulated in the COH group, which were validated by real time RT-PCR. SELDI spectra revealed similar protein fingerprinting in the dissected endometrial epithelium in natural versus COH cycles stimulated with a GnRH antagonist. On the other hand, microdissected epithelium from GnRH agonist cycles depicted a different fingerprinting compared to natural and GnRH antagonist cycles. Furthermore, microdissected stromal compartments differed in the protein fingerprinting in the three studied groups. Several proteins, with masses ranging from 2.5 to 17 kD were differentially expressed among the three groups. Importantly, two unique 11 kD and 15 kD protein peaks were observed in the GnRH agonist cycles. Minimal differences in gene expression were found when comparing COH versus natural cycles. SELDI fingerprinting revealed a similar protein expression pattern in the epithelium of natural and GnRH antagonist cycles. However, differences were observed in GnRH agonist cycles. The stromal compartment demonstrated divergent protein fingerprinting amongst groups. These results provide a basic background on global gene and protein expression analysis of the endometrium during the window of implantation under natural and stimulated conditions.

Fluctuations of fatty acid amide hydrolase and anandamide levels during the human ovulatory cycle

Implantation is possible within a defined period of the menstrual cycle, referred to as the ‘implantation window’. It is during this critical period that proper dialog can be established between the blastocyst and a receptive endometrium. If for any reason this dialog is not established or is altered, the embryo is aborted. The factors responsible for the interaction between the embryo and the mother at the moment of implantation remain poorly understood. Recent studies indicate that endocannabinoids may contribute to the development of an adequate milieu at the implantation site. Here we show that the levels of anandamide and of its degrading enzyme, the fatty acid amide hydrolase, in peripheral lymphocytes undergo specific variations during the various phases of the human ovulatory cycle. In particular, we found the highest levels of fatty acid amide hydrolase activity and protein content, paralleled by the lowest anandamide concentrations, in the period that temporally coincides with the putative window of implantation in humans. On the other hand, the anandamide-synthesizing phospholipase D, the anandamide membrane transporter and the anandamide-binding cannabinoid receptors of lymphocytes did not change during the menstrual cycle. This study indicates that high fatty acid amide hydrolase activity and lowanandamide levels may be among the factors that co-operate in the success of implantation. This would add to our understanding of the pathophysiological and therapeutic implications of the endocannabinoid system in human fertility.

Expression of αvβ3 and α4β1 integrins throughout the putative window of implantation in a cohort of healthy fertile women

Background. Blastocyst implantation is a dynamic process requiring a specific cascade of cellular interactions and endometrial changes. The aim of this prospective observational study was to investigate the endometrial expression of αVβ3 and α4β1 integrins throughout the window of implantation in healthy fertile women.Materials and methods. Forty fertile women (mean age 29.7 ± 6.2 years) were recruited for this study. All underwent two endometrial biopsies in a single menstrual cycle. The first biopsy was performed on postovulatory day 6, while the second was taken on postovulatory day 8. Histological dating and immunohistochemical assessments of αVβ3 and α4β1 integrins were analyzed. Oestradiol and progesterone serum concentrations were also measured at the time of biopsy.Results. Second endometrial biopsy could not be performed in two of the 40 women (5%) because their menses had started in advance. No inflammatory or reactive phenomena were observed after biopsy. All the samples showed an in‐phase endometrium. αVβ3 integrin expression was found in 21 out of the 38 first biopsies and in all 38 second biopsies (55.2% vs. 100%, p < 0.001). α4β1 integrin expression was not significantly different between first and second biopsies (68.4% and 76.3%, NS). Oestradiol and progesterone serum concentrations were similar throughout the implantation window. Twelve of the 38 women (31.6%) conceived within 12 months of completing the study. All these had significantly higher αVβ3 integrin expressions in mid‐luteal phase biopsies compared with those who did not conceive.Conclusions. These immunohistochemical findings demonstrate that both αVβ3 and α4β1 integrins have a spatial and temporal expression throughout the implantation window in the endometrium of fertile women. Moreover, the shortage of αVβ3 integrin expression during the mid‐luteal phase results in a detrimental effect for blastocyst implantation.

Expression of α v β 3 and α 4 β 1 integrins throughout the putative window of implantation in a cohort of healthy fertile women

Blastocyst implantation is a dynamic process requiring a specific cascade of cellular interactions and endometrial changes. The aim of this prospective observational study was to investigate the endometrial expression of alpha(v)beta3 and alpha4beta1 integrins throughout the window of implantation in healthy fertile women. Forty fertile women (mean age 29.7 +/- 6.2 years) were recruited for this study. All underwent two endometrial biopsies in a single menstrual cycle. The first biopsy was performed on postovulatory day 6, while the second was taken on postovulatory day 8. Histological dating and immunohistochemical assessments of alpha(v)beta3 and alpha4beta1 integrins were analyzed. Oestradiol and progesterone serum concentrations were also measured at the time of biopsy. Second endometrial biopsy could not be performed in two of the 40 women (5%) because their menses had started in advance. No inflammatory or reactive phenomena were observed after biopsy. All the samples showed an in-phase endometrium. alpha(v)beta3 integrin expression was found in 21 out of the 38 first biopsies and in all 38 second biopsies (55.2% vs. 100%, p < 0.001). alpha4beta1 integrin expression was not significantly different between first and second biopsies (68.4% and 76.3%, NS). Oestradiol and progesterone serum concentrations were similar throughout the implantation window. Twelve of the 38 women (31.6%) conceived within 12 months of completing the study. All these had significantly higher alpha(v)beta3 integrin expressions in mid-luteal phase biopsies compared with those who did not conceive. These immunohistochemical findings demonstrate that both alpha(v)beta3 and alpha4beta1 integrins have a spatial and temporal expression throughout the implantation window in the endometrium of fertile women. Moreover, the shortage of alpha(v)beta3 integrin expression during the mid-luteal phase results in a detrimental effect for blastocyst implantation.

A prospective evaluation of the effect of salpingectomy on endometrial receptivity in cases of women with communicating hydrosalpinges.

We aimed to assess whether salpingectomy in women with communicating hydrosalpinges influenced endometrial receptivity. The inclusion criteria were: women with communicating hydrosalpinges, absence of other confounding infertility factors and aged <40 years. Patients were scheduled for laparoscopy during the putative window of implantation (cycle days 19-21). In patients in whom salpingectomy was decided upon due to the severity of tubal disease (n = 10), an intra-operative endometrial biopsy was performed. Post-treatment endometrial sampling was done between day 19-21 of the fourth consecutive cycle. Pre-treatment and post-treatment samples were assessed by both conventional histologic criteria and alpha(v)beta3 integrin immunostaining, where histological score (HSCORE) was used for quantification. Despite normal histological maturation assessed by conventional criteria, 8/10 hydrosalpinx cases yielded an epithelial HSCORE of <0.7, which was below the accepted threshold. Following salpingectomy, luminal endometrial epithelium demonstrated a significantly increased alpha(v)beta3 integrin expression (Wilcoxon's signed rank test, P = 0.017). Although the mean HSCORE for glandular epithelia improved, it failed to reach statistical significance. Ultrasound visible hydrosalpinges (n = 5) and non-visible cases (n = 5) were also compared. However, neither the pre-treatment integrin expression, nor the postoperative improvement were significantly different between these groups. We conclude that the surgical treatment of communicating hydrosalpinges may improve endometrial receptivity as assessed by alpha(v)beta3 integrin expression. Women with hydrosalpinges may undergo endometrial evaluation by the molecular markers of implantation, such as alpha(v)beta3 integrin. This evaluation may be decisive in determining the optimal management of cases, and may also be used to assess the efficacy of the treatment. The expression of the implantation markers should be correlated with implantation and clinical pregnancy rates in IVF-embryo transfer programs.

The effect of salpingectomy on endometrial receptivity in women with communicating hydrosalpinges.

Objective: To assess the affect of surgical treatment of communicating hydrosalpinges on endometrial receptivity. Design: Prospective pre-post study. Materials/Methods: The women less than 40 years of age with documented communicating hydrosalpinges and lacking other confounding infertility factors were recruited. The consented patients were assigned to laparoscopy during the putative window of implantation (cycle days 19–21). The patients in whom salpingectomy was decided (n = 10) due to the severity of tubal disease, an intraoperative and a post-treatment endometrial biopsy in the 4th consecutive cycle were employed. Pretreatment and posttreatment endometrial samples were evaluated according to the conventional histologic criteria, and also stained for an integrin marker of endometrial receptivity (αvβ3). Histological score (HSCORE) was used for the evaluation of epithelial αvβ3 immunostaining. Results: Despite normal histological maturation assessed by conventional criteria, 8 out of 10 hydrosalpinx cases yielded an epithelial HSCORE <0.7, which was below the accepted cut-off. Following salpingectomy, luminal endometrial epithelium demonstrated significantly increased αvβ3 expression (Wilcoxon signed rank test, p = 0.017). Although the mean HSCORE for glandular epithelia improved, no statistical significance yet documented (p = 0.192). Ultrasound visible hydrosalpinges (n = 5) and nonvisible cases (n = 5) were also compared, but neither preoperative integrin expression, nor postoperative improvement was not statistically different between these groups. Conclusions: The surgical treatment of communicating hydrosalpinges improves endometrial receptivity assessed by immunofluorescent αvβ3 integrin staining. The initial impaired integrin expression and later improvement following the surgical intervention is independent to the conventional endometrial maturation criteria. Supported by: Hacettepe Hospital Research Fund.

Messenger ribonucleic acid encoding interferon-inducible guanylate binding protein 1 is induced in human endometrium within the putative window of implantation.

The putative window of embryo implantation in the human opens between days 19--24 of the menstrual cycle. During this period, the endometrium undergoes distinctive structural and functional changes orchestrated by steroid hormones, growth factors, and cytokines to attain a receptive phase in which it acquires the ability to implant the developing embryo. A major challenge in the study of human reproduction is to identify the molecular signals that participate in the establishment of this critical receptive phase in the context of the natural cycle. Toward this goal, we analyzed human endometrial biopsies at various days of the menstrual cycle by employing messenger RNA (mRNA) differential display technique. We isolated several complementary DNAs representing genes that are either up- or down-regulated within the putative window of implantation. We identified one of these genes as that encoding interferon (IFN)-inducible guanylate-binding protein 1 (or GBP1), which possesses GTPase activity. Analysis of endometrial biopsies by Northern blotting and RT-PCR demonstrated that GBP1 mRNA is specifically induced at the midsecretory phase of the menstrual cycle. In situ hybridization analysis revealed that GBP1 mRNA expression is localized in the glandular epithelial cells as well as in the stroma in the immediate vicinity of the glands. We observed that treatment of human endometrial adenocarcinoma cell, Ishikawa, with IFN-gamma or IFN-alpha markedly induced the expression of GBP1 mRNA. IFN-gamma was, however, a more potent inducer of GBP1 than IFN-alpha. Consistent with this finding, the temporal profile of GBP1 expression during the menstrual cycle resembled that of IFN-gamma mRNA more closely than that of IFN-alpha, predicting a regulatory role of IFN-gamma in GBP1 expression in midsecretory human endometrium. Although the precise function of GBP1 in the receptive human uterus remains unclear, its unique expression overlapping the putative window of implantation suggests that it might serve as a useful marker of uterine receptivity in the human.

Messenger Ribonucleic Acid Encoding Interferon-Inducible Guanylate Binding Protein 1 Is Induced in Human Endometrium within the Putative Window of Implantation

Messenger Ribonucleic Acid Encoding Interferon-Inducible Guanylate Binding Protein 1 Is Induced in Human Endometrium within the Putative Window of Implantation

Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation.

The human endometrium acquires the ability to implant the developing embryo within a specific time window that is thought to open between days 19-24 of the secretory phase of the menstrual cycle. During this period the endometrium undergoes pronounced structural and functional changes induced by the ovarian steroids, estrogen and progesterone, that prepare it to be receptive to invasion by the embryo. The identification of reliable biochemical markers to assess this critical receptive phase in the context of the natural cycle remains one of the major challenges in the study of human reproduction. Our previous studies in a rat model system demonstrated that the expression of calcitonin, a peptide hormone involved in calcium homeostasis, is transiently induced by progesterone in the glandular epithelium at the onset of implantation. Attenuation of calcitonin synthesis in the uterus during the preimplantation phase by administration of calcitonin antisense oligodeoxynucleotides severely impairs implantation of rat embryos, suggesting that this peptide hormone plays a critical role in uterine receptivity. To investigate whether calcitonin is also expressed in the human endometrium during implantation, we monitored the spatio-temporal expression of calcitonin on various days of the menstrual cycle. Our studies employing RT-PCR showed that calcitonin messenger ribonucleic acid is expressed in human endometrium during the postovulatory midsecretory phase (days 17-25) of the menstrual cycle, with maximal expression occurring between days 19-21. Very little calcitonin expression was detected in the endometrium in either the preovulatory proliferative (days 5-14) or the late secretory (days 26-28) phase. In situ hybridization and immunocytochemical analyses localized the calcitonin expression predominantly in the glandular epithelial cells of the endometrium. Our studies further showed that calcitonin expression in the human endometrium is under progesterone regulation. Treatment of women with an antiprogestin, mifepristone (RU-486), drastically reduced calcitonin expression in the endometrium. Collectively, these findings reveal that progesterone-induced expression of calcitonin in the secretory endometrium temporally coincides with the putative window of implantation in the human.