Competent blastocyst and receptivity endometrium improved clinical pregnancy in fresh embryo transfer cycles: a retrospective cohort study.

BACKGROUND: Embryo quality is usually regarded as a key predictor of successful implantation and clinical pregnancy potential. The identification of embryos that have the capacity to implant and result in a healthy pregnancy is a crucial part of in vitro fertilization (IVF). Usually, morphologically high-quality embryos are chosen for embryo transfer in IVF treatment. The aim of this study was to assess the association between the available blastocyst formation rate and the clinical pregnancy outcome following the first fresh embryo transfer cycle and provide systematic individual treatment to adjust endometrial receptivity for the next transfer cycle. METHODS: This retrospective, single-center study included 512 fresh embryo transfers conducted between 11/2019 and 08/2021, which consisted of 385 cleavage-stage (Day 3) and 127 blastocyst-stage (Day 5) embryo transfers. The two groups were divided into a clinical pregnancy group and a nonclinical pregnancy group for comparison. The association between the available blastocyst formation rate and the clinical pregnancy rate in the Day 3 and Day 5 transfer groups were considered. RESULTS: In the Day 3 group, there were 275 clinical pregnancies, and the clinical pregnancy rate was 71.43%. Although the two pronuclei (2PN) oocyte rate and available embryo rate at Day 3 were significantly higher in the clinical pregnancy group than the nonclinical pregnancy group (P < 0.05), the blastocyst formation rate and the available blastocyst formation rate were not significantly different between the clinical pregnancy group and the nonclinical pregnancy group (P > 0.05). In the Day 5 group, there were 81 clinical pregnancies, and the clinical pregnancy rate was 63.78%. No baseline characteristics showed any obvious differences between the clinical pregnancy group and nonclinical pregnancy group (P > 0.05). The blastocyst formation rate in the nonclinical pregnancy group was higher than that in the clinical pregnancy group, but the difference was not statistically significant (81.06% vs. 77.03%, P = 0.083). Interestingly, the available blastocyst formation rate and the Day 5 available blastocyst formation rate were significantly higher in the nonclinical pregnancy group than the clinical pregnancy group (66.19% vs. 60.79%, P = 0.014; 54.58% vs. 46.98%, P = 0.007). CONCLUSIONS: In fresh cycles, the available blastocyst formation rate was not associated with the clinical pregnancy outcome for Day 3 embryo transfers, and the available blastocyst formation rate was not positively correlated with the clinical pregnancy outcome for Day 5 embryo transfers.

Detection and Classification of Hysteroscopic Images Using Deep Learning.

BACKGROUND: Although hysteroscopy with endometrial biopsy is the gold standard in the diagnosis of endometrial pathology, the gynecologist experience is crucial for a correct diagnosis. Deep learning (DL), as an artificial intelligence method, might help to overcome this limitation. Unfortunately, only preliminary findings are available, with the absence of studies evaluating the performance of DL models in identifying intrauterine lesions and the possible aid related to the inclusion of clinical factors in the model. AIM: To develop a DL model as an automated tool for detecting and classifying endometrial pathologies from hysteroscopic images. METHODS: A monocentric observational retrospective cohort study was performed by reviewing clinical records, electronic databases, and stored videos of hysteroscopies from consecutive patients with pathologically confirmed intrauterine lesions at our Center from January 2021 to May 2021. Retrieved hysteroscopic images were used to build a DL model for the classification and identification of intracavitary uterine lesions with or without the aid of clinical factors. Study outcomes were DL model diagnostic metrics in the classification and identification of intracavitary uterine lesions with and without the aid of clinical factors. RESULTS: We reviewed 1500 images from 266 patients: 186 patients had benign focal lesions, 25 benign diffuse lesions, and 55 preneoplastic/neoplastic lesions. For both the classification and identification tasks, the best performance was achieved with the aid of clinical factors, with an overall precision of 80.11%, recall of 80.11%, specificity of 90.06%, F1 score of 80.11%, and accuracy of 86.74 for the classification task, and overall detection of 85.82%, precision of 93.12%, recall of 91.63%, and an F1 score of 92.37% for the identification task. CONCLUSION: Our DL model achieved a low diagnostic performance in the detection and classification of intracavitary uterine lesions from hysteroscopic images. Although the best diagnostic performance was obtained with the aid of clinical data, such an improvement was slight.

Metformin ameliorates endometrial thickness in a rat model of thin endometrium.

Metformin, a well-established anti-diabetic drug, is also used in managing various other metabolic disorders including polycystic ovarian syndrome (PCOS). There are evidences to show that metformin improves endometrial functions in PCOS women. However, fewer studies have explored the direct effects of metformin on endometrium. Previous in vitro studies have shown that therapeutic serum concentrations of metformin enhance endometrial epithelial cell proliferation. The present study was undertaken to investigate in vivo effects of metformin on endometrial proliferation in a rat model of thin endometrium. Toward this, a rat model of thin endometrium was developed. Metformin (0.1% or 1% w/v) was administrated orally for 15 days in rats with thin endometrium. Oral metformin administration for three consecutive estrous cycles (15 days) in the thin endometrium rat model led to an increase in endometrial thickness compared to sham endometrium. Histological analysis showed a significant increase in the number of endometrial glands (P < 0.05), stromal cells (P < 0.01) and blood vessels (P < 0.01) in metformin-treated (n = 10 in each group) uterine horns compared to sham (saline-treated) uterine horns in rats. The expression of proliferating cell nuclear antigen and vascular epithelial growth factor was found to be upregulated on treatment with 1% metformin-treated group (n = 7). However, pregnancy outcomes in the rats treated with metformin remained unaltered despite the restoration of endometrial thickness. In conclusion, the study demonstrated that metformin ameliorates endometrial thickness in a rat model of thin endometrium by increasing endometrial proliferation and angiogenesis, without restoration of embryo implantation.

Effect of low uterine segment involvement on prognosis of early stage endometrial cancer.

AIM: Our aim is to investigate the effect of uterine lower segment involvement on prognosis of early-stage endometrial cancer cases diagnosed and treated in our clinic. MATERIALS AND METHODS: The file records of 316 cases reviewed retrospectively.Only stage I (a and b, n=209) cases were investigated, because they were more homogeneous group. RESULTS: The lymphovascular invasion rate was found to be higher in patients with stage Ia and uterine lower segment involvement (p < 0.001). Adjuvant treatment requirement was higher in patients with stage Ia and uterine lower segment involvement (p < 0.001). Among stage Ia cases, the recurrence rate between 1 and 3 years was found to be higher in cases with uterine lower segment involvement (p = 0.001). Among the stage Ib cases, lymphovascular invasion was found to be higher in cases with uterine lower segment involvement (p < 0.001). The recurrence rate between 1 and 3 years was found to be higher in stage Ib compared to Ia (p = 0.01). Uterine lower segment involvement was found to be associated with high lymphovascular invasion rate in all stage I cases (p < 0.001). It was determined that the need for adjuvant treatment was higher in cases with uterine lower segment involvement (p < 0.001). It was determined that the probability of recurrence between 1 and 3 years was higher in cases with uterine lower segment involvement (p = 0.007). CONCLUSION: Uterine lower segment involvement is associated with increased lymphovascular invasion even in the early stages. It is an important risk factor for systemic spread such as lymphovascular invasion, myometrial invasion, and lymph node involvement.

Safety and Potential Effect of Intrauterine Infusion of Autologous Adipose Tissue-Derived Regenerative Cells in Patients With Implantation Failure: A Pilot Study.

BACKGROUND: Implantation failure due to thin endometrium has emerged as a major cause of infertility. In this study, we aimed to assess the safety and preliminary efficacy of adipose tissue-derived regenerative cells (ADRCs), a source of adipose-derived stem cells, in infertility patients with implantation failure. METHODS: Five infertile women with implantation failure despite artificial reproductive technology were enrolled in this study and treated with ADRCs via the intrauterine route. The primary outcome was the incidence of adverse events. Additional outcomes were endometrial thickness after ADRC treatment and pregnancy success after embryo transfer. RESULTS: There were no adverse events in any patient. There was no elevation of white blood cell count, C-reactive protein, or D-dimer levels. There was a significant difference in endometrial thickness in the secretory phase before versus after intrauterine transplantation of ADRCs (3.8 ± 1.3 mm versus 8.8 ± 2.8 mm, respectively; p<0.05). A gestational sac and fetal heartbeat were detected on transvaginal ultrasound in two of five patients. CONCLUSION: Intrauterine infusion of autologous ADRCs is a simple and safe procedure that may ameliorate the endometrial microenvironment in infertile women with implantation failure.

Extracellular vesicles secreted by human aneuploid embryos present a distinct transcriptomic profile and upregulate MUC1 transcription in decidualised endometrial stromal cells.

STUDY QUESTION: Do extracellular vesicles (EVs) secreted by aneuploid human embryos possess a unique transcriptomic profile that elicits a relevant transcriptomic response in decidualized primary endometrial stromal cells (dESCs)? SUMMARY ANSWER: Aneuploid embryo-derived EVs contain transcripts of PPM1J, LINC00561, ANKRD34C, and TMED10 with differential abundance from euploid embryo-derived EVs and induce upregulation of MUC1 transcript in dESCs. WHAT IS KNOWN ALREADY: We have previously reported that IVF embryos secrete EVs that can be internalized by ESCs, conceptualizing that successful implantation to the endometrium is facilitated by EVs. Whether these EVs may additionally serve as biomarkers of ploidy status is unknown. STUDY DESIGN SIZE DURATION: Embryos destined for biopsy for preimplantation genetic testing for aneuploidy (PGT-A) were grown under standard conditions. Spent media (30 µl) were collected from euploid (n = 175) and aneuploid (n = 140) embryos at cleavage (Days 1-3) stage and from euploid (n = 187) and aneuploid (n = 142) embryos at blastocyst (Days 3-5) stage. Media samples from n = 35 cleavage-stage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocysts were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. PARTICIPANTS/MATERIALS SETTING METHODS: EVs were isolated from pools of media by differential centrifugation and EV-RNA sequencing was performed following a single-cell approach that circumvents RNA extraction. ESCs were decidualized (estradiol: 10 nM, progesterone: 1 µM, cAMP: 0.5 mM twice every 48 h) and incubated for 24 h with EVs (50 ng/ml). RNA sequencing was performed on ESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc = -5.13, P = 0.011), LINC00561 (log2fc = -7.87, P = 0.010), and ANKRD34C (log2fc = -7.30, P = 0.017) and more abundant in TMED10 (log2fc = 1.63, P = 0.025) compared to EVs of euploid embryos. Decidualization per se induced downregulation of MUC1 (log2fc = -0.54, P = 0.0028) in ESCs as a prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualized ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2fc = 0.85, P = 0.0201). LARGE SCALE DATA: Raw data have been uploaded to GEO (accession number GSE234338). LIMITATIONS REASONS FOR CAUTION: The findings of the study will require validation utilizing a second cohort of EV samples. WIDER IMPLICATIONS OF THE FINDINGS: The discovery that the transcriptomic profile of EVs secreted from aneuploid cleavage stage embryos differs from that of euploid embryos supports the possibility to develop a non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a Marie Sklodowska-Curie Actions fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd. The authors have no conflicts of interest to declare.

The Impact of High Adiposity on Endometrial Progesterone Response and Metallothionein Regulation.

CONTEXT: Obesity is a disease with deleterious effects on the female reproductive tract, including the endometrium. OBJECTIVE: We sought to understand the effects of excess adipose on the benign endometrium. DESIGN: A physiologic in vitro coculture system was developed, consisting of multicellular human endometrial organoids, adipose spheroids, and menstrual cycle hormones. Native human endometrial tissue samples women with and without obesity were also analyzed. SETTING: Academic institution. PATIENTS: Benign endometrial tissues from premenopausal women were obtained following written consent. MAIN OUTCOME MEASURES: Gene expression, protein expression, chromatin binding, and expression of DNA damage and oxidative damage markers were measured. RESULTS: Under high-adiposity conditions, endometrial organoids downregulated endometrial secretory phase genes, suggestive of an altered progesterone response. Progesterone specifically upregulated the metallothionein (MT) gene family in the epithelial cells of endometrial organoids, while high adiposity significantly downregulated the MT genes. Silencing MT genes in endometrial epithelial cells resulted in increased DNA damage, illustrating the protective role of MTs. Native endometrium from women with obesity displayed increased MT expression and oxidative damage in the stroma and not in the epithelium, indicating the cell-specific impact of obesity on MT genes. CONCLUSIONS: Taken together, the in vitro and in vivo systems used here revealed that high adiposity or obesity can alter MT expression by decreasing progesterone response in the epithelial cells and increasing oxidative stress in the stroma.

Obesity attenuates the beneficial effect of an intrauterine infusion of autologous platelet-rich plasma during in vitro fertilization.

OBJECTIVE: To assess how obesity, normal weight (NW) versus overweight/obese (OW/OB), impacts platelet-rich plasma's (PRP) effectiveness during in vitro fertilization and how obesity affects platelets during the menstrual cycle. METHODS: Endometrial mean thickness (EMT), embryo implantation, and clinical pregnancy were assessed using a self-controlled retrospective study that enrolled 59 patients with two failed cycles and treated with autologous PRP (three-dose scheme). The NHANES dataset was used to assess platelet changes during the menstrual cycle, using the mean platelet volume to platelet count ratio (MPR) index. The COSINOR packages for R were used to determine rhythmicity. RESULTS: PRP treatments significantly improved the EMT (2.5 ± 1.4 mm, P<0.001), unaffected by obesity. After the PRP treatment, one patient spontaneously became pregnant; therefore, 58 patients underwent embryo transfer (62 cycles), of which in 39 cycles the embryos implanted (63.9%). This was a significant improvement from their previous cycle (vs. 22.6%, P<0.001). Clinical pregnancy also improved with the PRP treatment over the previous cycle (57.4% vs. 16.1%, P<0.001). When stratified by obesity, there was an appreciable decrease in embryo implantation and clinical pregnancy rates for the OW/OB group; nevertheless, the PRP treatment significantly improved embryo implantation and clinical pregnancy (P<0.05). A rhythm was observed with the MPR index (P<0.05) only for the NW group, suggesting that the platelets normally fluctuate during the menstrual cycle. CONCLUSION: PRP improved embryo implantation and clinical pregnancy rates; however, these beneficial effects were attenuated by obesity. PRP presumptively promoted a change in the uterine environment to mimic the normal findings associated with normal-weight women.

Machine learning to identify endometrial biomarkers predictive of pregnancy success following artificial insemination in dairy cows.

The objective was to identify a set of genes whose transcript abundance is predictive of a cow's ability to become pregnant following artificial insemination (AI). Endometrial epithelial cells from the uterine body were collected for RNA sequencing using the cytobrush method from 193 first-service Holstein cows at estrus prior to AI (day 0). A group of 253 first-service cows not used for cytobrush collection were controls. There was no effect of cytobrush collection on pregnancy outcomes at day 30 or 70 or on pregnancy loss between day 30 and 70. There were 2 upregulated and 214 downregulated genes (FDR < 0.05, absolute fold change >2-fold) for cows pregnant at day 30 versus those that were not pregnant. Functional terms overrepresented in the downregulated genes included those related to immune and inflammatory responses. Machine learning for fertility biomarkers with the R package BORUTA resulted in identification of 57 biomarkers that predicted pregnancy outcome at day 30 with an average accuracy of 77%. Thus, machine learning can identify predictive biomarkers of pregnancy in endometrium with high accuracy. Moreover, sampling of endometrial epithelium using the cytobrush can help understand functional characteristics of the endometrium at AI without compromising cow fertility. Functional characteristics of the genes comprising the set of biomarkers is indicative that a major determinant of cow fertility, at least for first insemination after calving, is immune status of the uterus, which, in turn, is likely to reflect the previous history of uterine disease.

Isolation and characterization of feline endometrial mesenchymal stem cells.

BACKGROUND: Recently, there has been a growing interest in stem cells for human medicine. Limited feline endometrial mesenchymal stem cell (fEM-MSC) research in veterinary medicine necessitates reporting for future feline disease research and therapy. OBJECTIVES: This study aimed to isolate fEM-MSCs from feline endometrial tissues and evaluate their morphology, proliferative ability, differentiation ability, and immunophenotype. METHODS: Feline endometrial tissues were obtained from the ovariohysterectomies of healthy cats and isolated using an enzymatic method. The morphology and proliferative ability of the isolated cells were assessed using a doubling time (DT) assay from passages 3 to 6 (P3 - P6). We measured pluripotency gene expressions of cells in P2 using quantitative real-time polymerase chain reaction (qRT-PCR). To investigate MSC characteristics, a trilineage differentiation assay was conducted in P4, and cells in P4 were immunophenotyped using flow cytometry. RESULTS: fEM-MSCs showed a typical spindle-shaped morphology under a microscope, and the DT was maintained from P3 to P6. fEM-MSCs could differentiate into adipocytes, osteoblasts, and chondrocytes, and expressed three pluripotency markers (OCT4, SOX2, and NANOG) by qRT-PCR. Immunophenotypic analysis showed that the fEM-MSCs were CD14 -, CD34 -, CD45 -, CD9+, and CD44+. CONCLUSIONS: In this study, the feline endometrium was a novel source of MSCs, and to the best of our knowledge, this is the first report on the isolation method and characteristics of fEM-MSCs.

High-intensity focused ultrasound for endometrial ablation in adenomyosis: a clinical study.

Objective: The present study aimed to investigate the clinical efficacy of endometrial ablation with high-intensity focused ultrasound (HIFU) for symptom relief in women with adenomyosis. Methods: Between July 2014 and July 2020, 167 patients with adenomyosis treated at the Zhongshan City People's Hospital were enrolled in this study. Patients were divided into two groups according to patient aspirations: the control group, including patients who only underwent ablation of adenomyosis lesions (group A) and the treatment group, including patients who underwent removal of adenomyosis lesions and endometrial ablation (group B). Results: The reduced dysmenorrhea scores (visual analog scale) and menstrual volume scores (pictorial blood assessment chart) were measured before and after treatment. The scores were obtained by subtracting the postoperative scores from the preoperative scores and were compared to determine whether the symptoms had alleviated. Compared with the menstrual volume of group A, that in group B showed significant improvements. The average relief rates of dysmenorrhea in the two groups also showed significant improvement. However, the scores in group B showed a more significant improvement than those in group A. Conclusion: Therefore, our findings suggest that endometrial ablation using HIFU may be superior to conventional therapy with regard to alleviating the symptoms of increased menstruation in women with adenomyosis.

NK cell receptor profiling of endometrial and decidual NK cells reveals pregnancy-induced adaptations.

Natural killer (NK) cells, with a unique NK cell receptor phenotype, are abundantly present in the non-pregnant (endometrium) and pregnant (decidua) humanuterine mucosa. It is hypothesized that NK cells in the endometrium are precursors for decidual NK cells present during pregnancy. Microenvironmental changes can alter the phenotype of NK cells, but it is unclear whether decidual NK cell precursors in the endometrium alter their NK cell receptor repertoire under the influence of pregnancy. To examine whether decidual NK cell precursors reveal phenotypic modifications upon pregnancy, we immunophenotyped the NK cell receptor repertoire of both endometrial and early-pregnancy decidual NK cells using flow cytometry. We showed that NK cells in pre-pregnancy endometrium have a different phenotypic composition compared to NK cells in early-pregnancy decidua. The frequency of killer-immunoglobulin-like receptor (KIR expressing NK cells, especially KIR2DS1, KIR2DL2L3S2, and KIR2DL2S2 was significantly lower in decidua, while the frequency of NK cells expressing activating receptors NKG2D, NKp30, NKp46, and CD244 was significantly higher compared to endometrium. Furthermore, co-expression patterns showed a lower frequency of NK cells co-expressing KIR3DL1S1 and KIR2DL2L3S2 in decidua. Our results provide new insights into the adaptations in NK cell receptor repertoire composition that NK cells in the uterine mucosa undergo upon pregnancy.

TSG6-Exo@CS/GP Attenuates Endometrium Fibrosis by Inhibiting Macrophage Activation in a Murine IUA Model.

Intrauterine adhesion (IUA) is characterized by the formation of fibrous scar tissue within the uterine cavity, which significantly impacts female reproductive health and even leads to infertility. Unfortunately, severe cases of IUA currently lack effective treatments. This study presents a novel approach that utilizes tumor necrosis factor-(TNF) stimulated gene 6 (TSG6)-modified exosomes (Exos) in conjunction with an injectable thermosensitive hydrogel (CS/GP) to mitigate the occurrence of IUA by reducing endometrium fibrosis in a mouse IUA model. This study demonstrate that TSG6-modified Exos effectively inhibits the activation of inflammatory M1-like macrophages during the initial stages of inflammation and maintains the balance of macrophage phenotypes (M1/M2) during the repair phase. Moreover, TSG6 inhibits the interaction between macrophages and endometrial stromal fibroblasts, thereby preventing the activation of stromal fibroblasts into myofibroblasts. Furthermore, this research indicates that CS/GP facilitates the sustained release of TSG6-modified Exos, leading to a significant reduction in both the manifestations of IUA and the extent of endometrium fibrosis. Collectively, through the successful construction of CS/GP loaded with TSG6-modified Exos, a reduction in the occurrence and progression of IUA is achieved by mitigating endometrium fibrosis. Consequently, this approach holds promise for the treatment of IUA.

Uterine fluid microRNAs in repeated implantation failure.

Recurrent implantation failure (RIF) is a significant obstacle in assisted reproductive procedures, primarily because of compromised receptivity. As such, there is a need for a dependable and accurate clinical test to evaluate endometrial receptiveness, particularly during embryo transfer. MicroRNAs (miRNAs) have diverse functions in the processes of implantation and pregnancy. Dysregulation of miRNAs results in reproductive diseases such as recurrent implantation failure (RIF). The endometrium secretes several microRNAs (miRNAs) during the implantation period, which could potentially indicate whether the endometrium is suitable for in vitro fertilization (IVF). The goal of this review is to examine endometrial miRNAs as noninvasive biomarkers that successfully predict endometrium receptivity in RIF.

De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering.

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.

Endometrial responsiveness to interferon-tau and its association with subsequent reproductive performance in dairy heifers.

Our objectives were to evaluate the endometrial responsiveness of dairy heifers to an intrauterine infusion of recombinant bovine interferon-tau (rbIFN-τ) and to associate endometrial responses to rbIFN-τ with subsequent reproductive performance. In Experiments 1 and 2, cyclic heifers were enrolled in a 5-d CIDR Cosynch program for estrous synchronization, and blood sampling and ultrasonography examinations were performed on d 0, 4, 7, 11, and 14 of the estrous cycle. In Experiment 1, heifers were randomly assigned to receive an intrauterine infusion containing 2 µg of rbIFN-τ (rbIFN-τ = 19) or saline (CTRL = 19) into the uterine horn ipsilateral to the corpus luteum (CL) on d 14 of the estrous cycle. Six hours after the infusion, the infused uterine horn was flushed for sampling of the uterine luminal fluid (ULF) for analyses of composition, and the endometrium was biopsied for transcriptomics. In Experiment 2, 100 heifers received an intrauterine infusion of rbIFN-τ, and the same procedures for uterine sample collection described in Experiment 1 were performed. After the intrauterine test, heifers were enrolled in a breeding program and classified as highly fertile (HF; pregnant at first AI) or subfertile (SF; not pregnant at first AI). Statistical analyses were performed using linear regression models, which included the effects of treatment (Experiment 1: CTRL vs. rbIFN-τ) or fertility group (Experiment 2: HF vs. SF) and block of samples. Intrauterine infusion of rbIFN-τ increased the expression of classical interferon-stimulated genes in the endometrium (e.g., ISG15, MX1, OAS2, IRF9, and USP18), and an antiviral response was predicted to be the main downstream effect of the transcriptome changes. In addition, rbIFN-τ increased the abundance of cholesterol, glycerol, and the overall concentration of oxylipins in the ULF. Analysis of endometrial transcriptome between HF and SF heifers revealed important differences in the expression of proteins associated with cell signaling, metabolism, attachment, and migration, with a large representation of genes encoding extracellular matrix proteins. In general, differently expressed genes were expected to be downregulated by IFN-τ but seemed to fail to be downregulated in SF heifers, resulting in higher expression in SF compared with HF heifers. Subfertile heifers had lower concentrations of glycerol and an altered profile of oxylipins in the ULF, with lower abundance of oxylipins derived from arachidonic acid and dihomo-γ-linolenic acid, and greater abundance of oxylipins derived from linoleic acid. Measurements of ovarian function did not differ between groups and, therefore, did not influence the observed results in uterine biology. In conclusion, the endometrial responsiveness to IFN-τ is variable among individuals and associated with subsequent fertility of heifers, indicating that communication between conceptus and endometrium is critical for the uterine receptivity and survival of pregnancy.

The impact of estradiol supplementation on endometrial thickness and intrauterine insemination outcomes.

The impact of estrogen supplementation during the follicular/proliferative phase on the endometrial lining thickness (EMT) prior to intrauterine insemination (IUI) remains largely unstudied. Our study examined changes in EMT and rates of clinical pregnancy, miscarriage, and live birth for all patients who completed an IUI cycle at Stanford Fertility Center from 2017-2023 (n = 2281 cycles). Cycles with estradiol supplementation (n = 309) were compared to reference cycles without supplementation (n = 1972), with the reference cohort further categorized into cycles with a pre-ovulatory EMT of < 7 mm ("thin-lining", n = 536) and ≥ 7 mm ("normal-lining", n = 1436). The estradiol group had a statistically significant greater change in EMT from baseline to ovulation compared to the thin-lining reference groups (2.4 mm vs 1.9 mm, p < =0.0001). Similar rates of clinical pregnancy and live birth were observed. After adjusting for age, BMI, race/ethnicity, infertility diagnosis, and EMT at trigger, the estradiol cohort had a significantly increased odds of miscarriage versus the entire reference cohort (2.46, 95 % confidence interval [1.18, 5.14], p = 0.02). Thus, although estradiol supplementation had a statistically significant increase in EMT compared to IUI cycles with thin pre-ovulatory EMT (<7 mm), this change did not translate into improved IUI outcomes such as increased rates of clinical pregnancy and live birth or decreased rate of miscarriage. Our study suggests that supplemental estradiol does not appear to improve IUI outcomes.

Bioengineering approaches for the endometrial research and application.

The endometrium undergoes a series of precise monthly changes under the regulation of dynamic levels of ovarian hormones that are characterized by repeated shedding and subsequent regeneration without scarring. This provides the potential for wound healing during endometrial injuries. Bioengineering materials highlight the faithful replication of constitutive cells and the extracellular matrix that simulates the physical and biomechanical properties of the endometrium to a larger extent. Significant progress has been made in this field, and functional endometrial tissue bioengineering allows an in-depth investigation of regulatory factors for endometrial and myometrial defects in vitro and provides highly therapeutic methods to alleviate obstetric and gynecological complications. However, much remains to be learned about the latest progress in the application of bioengineering technologies to the human endometrium. Here, we summarize the existing developments in biomaterials and bioengineering models for endometrial regeneration and improving the female reproductive potential.

Uterine macrophages and NK cells exhibit population and gene-level changes after implantation but maintain pro-invasive properties.

Introduction: Prior to pregnancy, hormonal changes lead to cellular adaptations in the endometrium allowing for embryo implantation. Critical for successful pregnancy establishment, innate immune cells constitute a significant proportion of uterine cells prior to arrival of the embryo and throughout the first trimester in humans and animal models. Abnormal uterine immune cell function during implantation is believed to play a role in multiple adverse pregnancy outcomes. Current work in humans has focused on uterine immune cells present after pregnancy establishment, and limited in vitro models exist to explore unique functions of these cells. Methods: With single-cell RNA-sequencing (scRNAseq), we comprehensively compared the human uterine immune landscape of the endometrium during the window of implantation and the decidua during the first trimester of pregnancy. Results: We uncovered global and cell-type-specific gene signatures for each timepoint. Immune cells in the endometrium prior to implantation expressed genes associated with immune metabolism, division, and activation. In contrast, we observed widespread interferon signaling during the first trimester of pregnancy. We also provide evidence of specific inflammatory pathways enriched in pre- and post-implantation macrophages and natural killer (NK) cells in the uterine lining. Using our novel implantation-on-a-chip (IOC) to model human implantation ex vivo, we demonstrate for the first time that uterine macrophages strongly promote invasion of extravillous trophoblasts (EVTs), a process essential for pregnancy establishment. Pre- and post-implantation uterine macrophages promoted EVT invasion to a similar degree as pre- and post-implantation NK cells on the IOC. Conclusions: This work provides a foundation for further investigation of the individual roles of uterine immune cell subtypes present prior to embryo implantation and during early pregnancy, which will be critical for our understanding of pregnancy complications associated with abnormal trophoblast invasion and placentation.