Natural Selection of Human Embryos: Decidualizing Endometrial Stromal Cells Serve as Sensors of Embryo Quality upon Implantation

Background

Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown.

Methodology/Principal Findings

We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo.

Conclusions

Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies.     

Proteomic Insights into Endometrial Receptivity and Embryo‐Endometrial Epithelium Interaction for Implantation Reveal Critical Determinants of Fertility

In vitro fertilization has overcome infertility issues for many couples. However, achieving implantation of a viable embryo into the maternal endometrium remains a limiting step in optimizing pregnancy success. The molecular mechanisms which characterize the transient state of endometrial receptivity, critical in enabling embryo‐endometrial interactions, and proteins which underpin adhesion at the implantation interface, are limited in humans despite these temporally regulated processes fundamental to life. Hence, failure of implantation remains the “final frontier” in infertility. A human coculture model is utilized utilizing spheroids of a trophectoderm (trophoblast stem) cell line, derived from pre‐implantation human embryos, and primary human endometrial epithelial cells, to functionally identify “fertile” versus “infertile” endometrial epithelium based on adhesion between these cell types. Quantitative proteomics identified proteins associated with human endometrial epithelial receptivity (“epithelial receptome”) and trophectoderm adhesion (“adhesome”). As validation, key “epithelial receptome” proteins (MAGT‐1/CDA/LGMN/KYNU/PC4) localized to the epithelium of receptive phase (mid‐secretory) endometrium obtained from fertile, normally cycling women but is largely absent from non‐receptive (proliferative) phase tissues. Factors involved in embryo‐epithelium interaction in successive temporal stages of endometrial receptivity and implantation are demonstrated and potential targets for improving fertility are provided, enhancing potential to become pregnant either naturally or in a clinical setting.     

Molecular aspects of implantation failure

Despite expanding global experience with advanced reproductive technologies, the majority of IVF attempts do not result in a successful pregnancy, foremost as a result of implantation failure. The process of embryo implantation, a remarkably dynamic and precisely controlled molecular and cellular event, appears inefficient in humans and is poorly understood. However, insights gained from clinical implantation failure, early pregnancy loss, and emerging techologies that enable molecular interrogation of endometrial–embryo interactions are unravelling this major limiting step in human reproduction. We review current molecular concepts thought to underlie implantation failure, consider the contribution of embryonic and endometrial factors, and discuss the clinical value of putative markers of impaired endometrial receptivity. Finally we highlight the nature of the dialogue between the maternal endometrium and the implanting embryo and discuss the concept of natural embryo selection. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.     

A proteomic atlas of ligand–receptor interactions at the ovine maternal–fetal interface reveals the role of histone lactylation in uterine remodeling

Well-orchestrated maternal–fetal cross talk occurs via secreted ligands, interacting receptors, and coupled intracellular pathways between the conceptus and endometrium and is essential for successful embryo implantation. However, previous studies mostly focus on either the conceptus or the endometrium in isolation. The lack of integrated analysis impedes our understanding of early maternal–fetal cross talk. Herein, focusing on ligand–receptor complexes and coupled pathways at the maternal–fetal interface in sheep, we provide the first comprehensive proteomic map of ligand–receptor pathway cascades essential for embryo implantation. We demonstrate that these cascades are associated with cell adhesion and invasion, redox homeostasis, and the immune response. Candidate interactions and their physiological roles were further validated by functional experiments. We reveal the physical interaction of albumin and claudin 4 and their roles in facilitating embryo attachment to endometrium. We also demonstrate a novel function of enhanced conceptus glycolysis in remodeling uterine receptivity by inducing endometrial histone lactylation, a newly identified histone modification. Results from in vitro and in vivo models supported the essential role of lactate in inducing endometrial H3K18 lactylation and in regulating redox homeostasis and apoptotic balance to ensure successful implantation. By reconstructing a map of potential ligand–receptor pathway cascades at the maternal–fetal interface, our study presents new concepts for understanding molecular and cellular mechanisms that fine-tune conceptus–endometrium cross talk during implantation. This provides more direct and accurate insights for developing potential clinical intervention strategies to improve pregnancy outcomes following both natural and assisted conception.     

A model for implantation: coculture of blastocysts and uterine endometrium in mice

One of the limitations in embryo implantation research is the lack of an available in vitro model that faithfully replicates embryo-uterine interactions. In previous studies, embryos were cultured on a monolayer of either uterine epithelial cells or extracellular matrix substratum on which embryos could adhere and outgrow. However, these models failed to display embryonic invasion, primarily because of the shortage of critical structural and molecular supports that are available in vivo. In the present study, we used intact mouse uterine endometrium collected on Day 4 of pregnancy and placed in contact with blastocysts to initiate coculture experiments in a defined medium at the air-liquid interface. The culture medium was composed of Ham F-12/Dulbecco modified Eagle medium (1:1), 30% fetal calf serum, 63.5 nmol/L of progesterone, 7.14 nmol/L of estradiol-17beta, 100 mug/ml of insulin, and 20 ng/ml of epidermal growth factor, whereas the incubation condition was mixed air of 50% oxygen, 5% carbon dioxide, and 45% nitrogen with a humidity of greater than 90% at 37 degrees C. Our observations from 24 h of culture clearly demonstrated that embryos were capable of attachment to the uterine endometrium and displayed partial invasion into the endometrial stroma. Interestingly, no outgrowth of trophoblasts on the surface of uterine endometrium was seen, but embryos exhibited a pole-specific attachment. Overall, this model is capable of demonstrating a true invasion of embryo within the endometrial stroma and may be suitable in studies related to early embryo implantation.     

Analysis of HLA-G in maternal plasma, follicular fluid, and preimplantation embryos reveal an asymmetric pattern of expression

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.     

Omics insights into extracellular vesicles in embryo implantation and their therapeutic utility

Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance. Omics technologies including mass spectrometry have been instrumental in dissecting EV cargo that regulate these processes as well as molecular changes in embryo and endometrium to facilitate implantation. This has also led to discovery of potential cargo in EVs in human uterine fluid (UF) and embryo spent media (ESM) of diagnostic and therapeutic value in implantation success, fertility, and pregnancy outcome. This review discusses the contribution of EVs in functional hallmarks of embryo implantation, and how the integration of various omics technologies is enabling design of EV-based diagnostic and therapeutic platforms in reproductive medicine.     

Role of progesterone on peri-implantation stage endometrium-embryo interaction in the primate

Progesterone secretion during the luteal phase influences oviductal and endometrial functions which are essential for embryo viability and implantation in a number of species including primates. Luteal phase estrogen is not essential for progesterone-dependent endometrial receptivity towards implantation and pregnancy in the rhesus monkey and in the human. However, synchronous development of embryo and endometrium is an essential prerequisite for evolutive implantation. Progesterone helps to maintain synchronous development of preimplantation embryo through its action on maternal uterus. The anti-nidatory action of mifepristone, a potent progesterone receptor modulator (PRM) with pronounced antiprogestagenic activity, is known to be associated with desynchronization of endometrium along with repression of glandular secretory differentiation and vascular maturation. Thus, it is likely that early luteal phase administration of mifepristone affects paracrine action of the secretory stage endometrium on the preimplantation stage embryo, and thereby inhibits embryonic development and viability. We shall examine this hypothesis using the rhesus monkey as a primate model.     

Expression of plasminogen activators in preimplantation rat embryos developed <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. The invasion of trophoblast cells into the endometrium during the implantation process can be blocked by inhibitors of serine proteases, illustrating the role of these enzymes in the invasion process. As in vitro developing embryos resulted in lower implantation rate than those developed in vivo we assume that a reduced PAs activity may lead to it. There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. The purpose of this study was to assess the expression of urokinase type (uPA) and tissue type (tPA) plasminogen activators in in vivo and in vitro preimplantation development in rat embryos using immunofluorescence confocal microscopy and computerized image analysis.     

Differential expression of genes in the endometrium at implantation: upregulation of a novel member of the E2 class of ubiquitin-conjugating enzymes

The process of embryo attachment and implantation is accompanied by dramatic cellular and functional changes in the endometrium, the control and mechanisms of which are not clearly understood. The cDNA cloning of differentially expressed genes, specifically at implantation sites in the rabbit endometrium, was used to identify genes controlling functional and remodeling changes. Tissue from the endometrium of Day 6(3/4) (preimplantation) and Day 8 (implantation initiation) pregnant rabbits was used to screen for differentially expressed genes by combined cDNA subtraction/suppressive hybridization. Twenty-nine differentially expressed genes were identified encoding protein modification enzymes, signaling proteins, structural proteins, and enzymes. One of these is a novel member of the E2 ubiquitin-conjugating enzyme family we have designated UBCi (i for implantation), which displayed dramatic nucleotide and deduced amino acid sequence conservation between rabbits, humans, and mice. In situ hybridization indicated UBCi expression exclusively in the luminal epithelium of the endometrium while glandular epithelium, trophoblast, and myometrium were negative. Expression was specific for epithelial cells at implantation sites and was not detected in non-implant-site endometrium. UBCi mRNA was detected in both the mesometrial and antimesometrial epithelial cells of the implantation sites, sites undergoing both differentiation and/or apoptosis. These results identify a group of differentially expressed genes in the endometrium including UBCi and provide new focal targets for studying processes controlling cellular remodeling during implantation. The important roles of ubiquitination in controlling the activities and turnover of key signaling proteins suggest potential roles in controlling critical aspects of implantation.     

Constitutive proteins of the oocytes and early embryos of rats]

5 protein fractions were identified and their relative mobility was determined in the rat oocytes and cleaving embryos by means of vertical capillary microdisc-electrophoresis in 7.5% polyacrilamide gel (PAA-gel). The same fractions were identified in the cleaving embryos devoid of zona pellucida. A conclusion was drawn that these proteins were present in the oocyte cytoplasm and kept in the cleaving embryos until the stage of implantation. 4 groups of proteins with different anodic mobility were identified in the isolated zona pellucida by means of microdisc-electrophoresis in 7.5% PAA-gel added with 1% sodium dodecylsulfate (SDS). The molecular weight of low molecular weight proteins of oocytes and preimplantation embryos was determined by means of disc-electrophoresis in 14% PAA-gel with 1% SDS. The zona pellucida of one embryo contained, according to the data of capillary spectrophotometry, 5 ng of protein.     

Significant differences between mouse and human trophinins are revealed by their expression patterns and targeted disruption of mouse trophinin gene.

Trophinin has been identified as a membrane protein mediating apical cell adhesion between two human cell lines: trophoblastic HT-H cells, and endometrial epithelial SNG-M cells. Expression patterns of trophinin in humans suggested its involvement in embryo implantation and early placental development. The mouse trophinin gene maps to the distal part of the X chromosome and corresponds to human chromosome Xp11.21-22, the locus where the human trophinin gene maps. Western blot analysis indicates that the molecular weight of mouse trophinin is 110 kDa, which is consistent with the calculated value of 107 kDa. Positive signals for trophinin proteins were detected in preimplantation mouse embryos at the morula and blastocyst stages. Implanting blastocysts do not show detectable levels of trophinin protein, demonstrating that trophinin is not involved in blastocyst adhesion to the uterus in the mouse. Mouse embryo strongly expressed trophinin in the epiblast 1 day after implantation. Trophinin protein was not found in the mouse uteri and placenta after 5.5 days postcoitus (dpc). Targeted disruption of the trophinin gene in the mouse showed a partial embryonic lethality in a 129/SvJ background, but the cause of this lethality remains undetermined. The present study indicates significant differences between mouse and human trophinins in their expression patterns, and it suggests that trophinin is not involved in embryo implantation and placental development in the mouse.     

PP2Cdelta (Ppm1d, WIP1), an endogenous inhibitor of p38 MAPK, is regulated along with Trp53 and Cdkn2a following p38 MAPK inhibition during mouse preimplantation development

Preimplantation embryos utilize mitogen-activated protein kinase signaling (MAPK) pathways to relay signals from the external environment to prepare appropriate responses and adaptations to a changing milieu. It is therefore important to investigate how MAPK pathways are regulated during preimplantation development. This study was conducted to investigate whether PP2Cdelta (Ppm1d, WIP1) is expressed during mouse preimplantation development and to determine the influences of p38 MAPK inhibition on expression of Trp53 (p53), Ppm1d, (WIP1), and Cdkn2a (p16) during mouse preimplantation development. Our results indicate that Trp53, Ppm1d, and Cdkn2a mRNAs and TRP53 and PP2Cdelta proteins are expressed throughout mouse preimplantation development. Treatment of 2-cell embryos with SB220025 (potent inhibitor of p38 MAPK alpha/beta/MAPK 14/11) significantly increased Trp53, Ppm1d and Cdkn2a and Mapk14 mRNA levels at 12 and 24 hr. Treatment of 8-cell embryos with SB220025 for 12 hr increased Trp53, Ppm1d, and Cdkn2a mRNA levels, but not Mapk14 mRNA levels. Treatment of 8-cell embryos for 24 hr increased Trp53, and Ppm1d mRNA levels, but decreased Cdkn2a and Mapk14 mRNA levels. Therefore, blockade of p38 MAPK activity is associated with embryo stage specific influences on Trp53, Ppm1d, Cdkn2a, and Mapk14 expression during mouse preimplantation development. These results define downstream targets of p38 MAPK during preimplantation development and indicate that the p38 MAPK pathway regulates Trp53, Ppm1d, and Cdkn2a expression. This study increases our understanding of the mechanisms controlling preimplantation development and of the interactions between preimplantation embryos and their culture environments.