Human Blastocyst Implantation Research Articles

An in vitro Model for Human Blastocyst Implantation

An in vitro Model for Human Blastocyst Implantation

Insights into the mechanism of CXCL12-mediated signaling in trophoblast functions and placental angiogenesis.

The chemokine CXCL12 and its receptor CXCR4 are important signaling components required for human blastocyst implantation and the progression of pregnancy. Growing evidence indicates that the CXCL12/CXCR4 axis can regulate trophoblast function and uterine spiral artery remodeling, which plays a fundamental role in placentation and fetal outcome. The orphan receptor CXCR7 is also believed to partly regulate the function of the CXCL12/CXCR4 axis. Additionally, the CXCL12/CXCR4/CXCR7 axis can enhance the cross-talk between trophoblasts and decidual cells such as uterine natural killer cells and decidual stromal cells which are involved in regulation of trophoblast differentiation and invasion and placental angiogenesis. In addition, recent studies proved that CXCL12 expression is elevated in the placenta and mid-trimester amniotic fluid of pregnant women with preeclampsia, implying that dysregulation of CXCL12 plays a role in the pathogenesis of preeclampsia. Further understanding of the regulatory mechanisms of CXCL12-mediated signaling in trophoblast functions and placental angiogenesis may help to design novel therapeutic approaches for pregnancy-associated diseases.

Progesterone and its downstream molecules as blastocyst implantation essential factors.

This review is to update the previous review (Am J Reprod Immunol, 63, 2010 and 413) on the research on blastocyst implantation essential factors (BIEFs). Focus of the current review is on progesterone and its downstream molecules in the process of blastocyst implantation. To understand the process of implantation, we need to know where and when the BIEFs are expressed and what they do. Progress in this research area is rapid, and its update is indeed necessary. The basic concept of BIEFs is that they have dual functions, one physiological and the other immunological (J Reprod Dev, 58, 2012 and 196). As we are still exploring the mechanism of implantation, available data are incomplete and human data are few. Thus, I will use information obtained through research on animal models, in vitro studies, cell lines, and some human studies where available. The ultimate goal of the review is to understand human blastocyst implantation.

Multi-level and multi-scale integrative approach to the understanding of human blastocyst implantation

Implantation is a complex process which results in fixation of zona pellucida free blastocyst to the maternal uterine endometrium. In the human, it involves progesterone mediated preparation of endometrium, age- and stage-matched development of pre-implantation embryo, and interaction between embryo and endometrium. In the present essay, we present the case to explain why there is a necessity of undertaking multi-level, multi-scale integrative approach to deconstruct the succession process of endometrial development to the climax of implantation.

Polyethylene glycated leukemia inhibitory factor antagonist inhibits human blastocyst implantation and triggers apoptosis by down-regulating embryonic AKT

To study the effect of polyethylene glycated leukemia inhibitory factor (LIF) antagonist (PEGLA) in the human blastocyst viability and implantation process. In vitro study. University hospital and research laboratory. Endometrial biopsy samples from fertile donors (n = 20), and surplus, frozen, good-quality human embryos obtained from an in vitro fertilization (IVF) clinic that survived thawing (n = 51). Timed human endometrial biopsy on the day of luteinizing hormone peak + 4 days (LH + 4). Human embryo attachment rate, embryo quality, and expression of AKT and caspase-3. PEGLA significantly reduced the embryo attachment rate to the endometrial construct. It decreased both mRNA and protein for LIF in the endometrial construct. Inhibition of embryonic LIF triggered apoptosis. Analysis of these blastocysts by immunofluorescence and real-time polymerase chain reaction showed a down-regulation in AKT activation and an increase in caspase-3 activation compared with the control group of blastocysts. The LIF inhibitor PEGLA could be a potential nonsteroidal fertility-regulating agent in humans. It acts on endometrial epithelial cells by down-regulating endometrial epithelial LIF. Inhibition of blastocyst LIF decreased its cell survival factor p-AKT and increased apoptosis (cleaved caspase-3). This highlights that embryonic LIF is vital for human embryo implantation.

Impact of cleavage-stage embryo biopsy in view of PGD on human blastocyst implantation: a prospective cohort of single embryo transfers

Human embryo biopsy is performed for preimplantation genetic diagnosis (PGD). The impact of 1- or 2-cell removal at cleavage-stage on future embryo development and implantation capacity is highly debated. In order to explore this issue further, a cohort of Day 5 single embryo transfers was analysed prospectively for embryological and clinical outcome. All transferred embryos resulted from 8-cell embryos on Day 3, from which subsequently either one cell (group I, n = 182) or two cells (group II, n = 259) were removed, or on which no invasion by means of embryo biopsy was performed (group III, control group, n = 702). RESULTS Blastocyst formation was significantly better in group III compared with group II, and similar to group I. Group I and group II did not differ in Day 3 nor in Day 5 embryo development. The overall live birth rate was significantly higher in group I (37.4%, CI 29.0-47.4%) than in group II (22.4%, CI 17.0-28.9%), and comparable to the reference ICSI population (35.0%, CI 30.8-39.7%). The clinical outcome of 1-cell biopsy was significantly better than that of 2-cell biopsy, even when adjusted for availability of genetically transferable embryos.

The implantation sites of early gestational sacs after embryo transfer and natural conception

OBJECTIVE: The implantation site can be estimated from the location of the early gestational sacs (GS) by transvaginal ultrasound. The objective of this study was to investigate the most frequent implantation site and to compare the implantation sites of pregnancies after embryo transfer (ET) and natural conceived pregnancies.DESIGN: Prospective study.MATERIALS AND METHODS: A total of 426 singleton pregnancies (244 of IVF-ET, 181 of natural conception) were analysed. In all embryo transfers, the catheter was introduced into the endometrial cavity guided by abdominal ultrasound, with the catheter tip placed at the upper point of the endometrial cavity. Gestational sacs (GS) were evaluated with two-dimensional transvaginal ultrasound between the 4th and the 6th weeks of gestation. The uterine cavity was divided into four parts (fundal, upper, middle and lower regions) and the upper and middle region was subdivided into lateral, middle anterior and middle posterior areas, making a total of 8 areas.RESULTS: Of the 425 singleton pregnancies, 76%(323/425) of the GS were detected in the fundal and upper region, 21.2%(90/425) in the middle region and 2.8%(12/425) in the lower region. The upper posterior area was the most frequent implantation site in both IVF-ET and natural conception (24.3%, 23.8%). There was no statistical difference in the implantation sites between IVF-ET and natural conception. And also there was no statistical difference in the miscarriage rate of each region.CONCLUSIONS: When embryos were transferred to the upper area of the uterine cavity, the implantation sites of IVF-ET pregnancies were not different from that of naturally conceived pregnancies. The endometrium suitable for human blastocyst implantation was at the upper area of the uterine cavity, especially upper posterior area. OBJECTIVE: The implantation site can be estimated from the location of the early gestational sacs (GS) by transvaginal ultrasound. The objective of this study was to investigate the most frequent implantation site and to compare the implantation sites of pregnancies after embryo transfer (ET) and natural conceived pregnancies. DESIGN: Prospective study. MATERIALS AND METHODS: A total of 426 singleton pregnancies (244 of IVF-ET, 181 of natural conception) were analysed. In all embryo transfers, the catheter was introduced into the endometrial cavity guided by abdominal ultrasound, with the catheter tip placed at the upper point of the endometrial cavity. Gestational sacs (GS) were evaluated with two-dimensional transvaginal ultrasound between the 4th and the 6th weeks of gestation. The uterine cavity was divided into four parts (fundal, upper, middle and lower regions) and the upper and middle region was subdivided into lateral, middle anterior and middle posterior areas, making a total of 8 areas. RESULTS: Of the 425 singleton pregnancies, 76%(323/425) of the GS were detected in the fundal and upper region, 21.2%(90/425) in the middle region and 2.8%(12/425) in the lower region. The upper posterior area was the most frequent implantation site in both IVF-ET and natural conception (24.3%, 23.8%). There was no statistical difference in the implantation sites between IVF-ET and natural conception. And also there was no statistical difference in the miscarriage rate of each region. CONCLUSIONS: When embryos were transferred to the upper area of the uterine cavity, the implantation sites of IVF-ET pregnancies were not different from that of naturally conceived pregnancies. The endometrium suitable for human blastocyst implantation was at the upper area of the uterine cavity, especially upper posterior area.

Possible involvement of crosstalk cell-adhesion mechanism by endometrial CD26/dipeptidyl peptidase IV and embryonal fibronectin in human blastocyst implantation

When human blastocysts hatch through the zona pellucida, gaining the ability to adhere to the endometrium, crosstalk between the embryo and the uterus may represent a successful outcome of their synchronized development and differentiation. CD26/dipeptidyl peptidase IV is known as a marker molecule of the implantation phase endometrium. To study the role of CD26 in implantation, 35 human hatched blastocysts were prepared by enzymatic treatment of expanded blastocysts that had been grown on schedule from frozen-thawed surplus embryos at the 2- or 4-cell stage. The blastocysts were placed on CD26-overexpressing or mock-transfected control monolayer cell cultures. The CD26-overexpression caused significantly higher blastocyst adhesion rate (53.3% versus 25.0%, P < 0.05) and significantly larger outgrowth area of trophectoderm (1.7-fold, P < 0.05). The second part of the present study was to show the expression of fibronectin, a CD26 ligand, in human preimplantation embryos, using the same donated resources. Fibronectin mRNA was detected by RT-PCR from the single hatched blastocyst (2/2) and from the single early blastocyst (3/6) but not from the single morula (0/5) samples. An indirect immunofluorescence technique verified the localization of fibronectin on the surface of the blastocyst. These results indicate that the adhesion mechanism by endometrial CD26 and embryonal fibronectin may be involved in human blastocyst implantation.

Coexpression of pinopodes and leukemia inhibitory factor, as well as its receptor, in human endometrium

Objective To determine cell-type–specific expression of leukemia inhibitory factor (LIF) and LIF receptor (LIFR) proteins relative to formation of pinopodes in human endometrial samples. Design Prospective clinical study. Setting Hospital-based unit for reproductive health and university-affiliated reproductive research laboratories. Patient(s) Twenty-six healthy fertile women with normal menstrual cycles. Main outcome measure(s) Routine blood and urine samples were obtained, and vaginal ultrasonography and endometrial biopsy were performed. Pinopode formation and expression of LIF and LIFR were examined in endometrial samples. Result(s) Samples obtained during LH days 6 through 9 had pinopodes at different developmental stages. Both surface and glandular epithelial cells expressed maximal levels of LIF and LIFR protein, in biopsy samples showed fully developed pinopodes. Immunostaining of LIF was more intense in the glandular epithelium, whereas immunostaining of LIFR was most intense in the surface epithelium. Before and after the appearance of pinopodes, LIF and LIFR immunostaining was less intense or faint. Stromal endometrial cells showed faint LIF accumulation. Conclusion(s) The simultaneous positive spatial and temporal expression of pinopodes and LIF and LIFR proteins in endometrial samples from healthy women suggests that both molecular and structural cell changes are important in the initiation of human blastocyst implantation.

Determination of blastocyst implantation site in spontaneous pregnancies using three-dimensional transvaginal ultrasound.

Three-dimensional transvaginal ultrasound was used to determine the location of early gestational sacs (E-GSs) and, as a result, the physiological implantation sites of human blastocysts. We examined 138 patients who were found by three-dimensional transvaginal ultrasound to have a GS with an inner diameter of 3 to 6 mm (E-GS). The uterine cavity was divided into three parts: upper, middle, and lower regions. The upper region was subdivided into the right, middle, and left areas, and the middle region was subdivided into the right and left areas. Thus, overall 6 areas were designed and the frequency of the E-GS detection in each area was evaluated. Of the 138 patients, 123 (89.1%) had E-GSs detected in the upper region, which was found to be the most frequent region. When the frequency of E-GS detection among the upper three areas was compared, the right and left upper areas had a higher frequency than the middle upper area. As to the miscarriage rate, patients with E-GSs detected in the upper region had a significantly lower rate than those in the middle and lower regions (p<0.05). The endometrium suitable for human blastocyst implantation under physiological conditions is at the uterine fundus, especially near the uterotubal junction.

In vitro models of human blastocyst implantation

This paper reviews different in vitro models used for the study of blastocyst implantation in animals and the human. Furthermore, results from human blastocyst–endometrial interactions in vitro, investigated by scanning electron microscopy (SEM), light microscopy (LM) and transmission electron microscopy (TEM), are presented. SEM demonstrates the preference of human blastocysts to adhere to pinopode-presenting areas on endometrial cell cultures. LM and TEM show that the first morphological sign of cell contact, defined as junction formation, is present at the apical-to-lateral border of endometrial epithelial cells, whereas trophoblast attachment to apical endometrial epithelial plasma membranes was not observed. More advanced stages illustrate that the human blastocyst penetrates the epithelial lining by the intrusive penetration mechanism.

In Vitro Studies of Human Blastocyst Implantationa

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