Early Stages Of Implantation Research Articles

Expression pattern of PRM2, HSP90 and WNT5A in male partners of couples experiencing idiopathic recurrent miscarriages

Recurrent pregnancy loss affects approximately 3–5% of couples attempting to have a child. It is defined as the occurrence of two or more consecutive pregnancy failures before 20 weeks of gestation. In about 40% of cases, the cause remains unknown and are defined as idiopathic recurrent miscarriages (iRM) (Stephenson and Kutteh 2007). However, most of these diagnostics tests are performed in the female partner, with the male’s contribution remaining relatively underexplored (only paternal karyotyping is done). Paternal factor may be the underlying aetiology in RM (Lalancette et al. 2008) and molecular abnormalities in sperm may have an adverse effect on embryonic development. Certain portion of sperm genome remains transcriptionally active and codes for transcripts which are of paramount developmental importance (Krawetz 2005; Gil-Villa et al. 2010). Among these, the role of long-lived sperm RNA which are not translated is controversial. Sperm possess a remarkable set of long-lived messenger RNAs (mRNAs) that have been hypothesized to be required for embryogenesis. Some of these spermatozoal mRNAs are also found in zygote, indicating that these transcripts may be functionally critical during embryonic development (Ostermeier et al. 2004). The delivery of certain sperm transcripts to the oocyte, which could translate to proteins, may be critical for the early stages of embryogenesis and/or implantation. In the vast population of mRNA, transcripts of winglesstype MMTV integration site family, member 5A (WNT5A), heat shock protein 90 (HSP90) and protamine 2 (PRM2) are present in human mature spermatozoa as examined by microarray and serial analysis gene expression (SAGE) technology (Wykes et al. 2000; Zhao et al. 2006). These genes

Preimplantation Human Blastocyst‐Endometrial Interactions: The Role of Inflammatory Mediators

Immune factors such as cytokines, chemokines, and growth factors are known to play important roles in the preimplantation interactions and communication between the blastocyst and receptive endometrium. This crucial dialog occurs during the stages when the blastocyst is in the uterine cavity immediately preceding implantation and the establishment of pregnancy. Human preimplantation processes are difficult to study due to restrictions on tissue availability. This review focuses on the expression and role of immune factors in human blastocyst-endometrial dialog during the very early stages of implantation. It highlights the importance of immune regulators and the need to develop new models to study human implantation.

Finite element modelling of implant designs and cortical bone thickness on stress distribution in maxillary type IV bone

The aims of this study were to examine the effect of implant neck design and cortical bone thickness using 3D finite element analysis and to analyse the stability of clinical evidence based on micromotion and principal stress. Four commercial dental implants for a type IV bone and maxillary segments were created. Various parameters were considered, including the osseointegration condition, loading direction and cortical bone thickness. Micromotion and principal stresses were used to evaluate the failure of osseointegration and bone overloading, respectively. It was found that the maximum stress of the peri-implant bone decreased as cortical bone thickness increased. The micromotion level in full osseointegration is less than that in non-osseointegration and it also decreases as cortical bone thickness increases. The cortical bone thickness should be measured before surgery to help select a proper implant. In the early stage of implantation, the horizontal loading component induces stress concentration in bone around the implant neck more easily than does the vertical loading component, and this may result in crestal bone loss.

Uterine Development and Fertility Are Dependent on Gene Dosage of the Nuclear Receptor Coregulator REA

Although the effectiveness of nuclear hormone-receptor complexes is known to depend on coregulator partner proteins, relatively little is known about the roles of coregulators in uterine development and early stages of pregnancy and implantation. Because conventional genetic deletion of the coregulator, repressor of estrogen receptor activity (REA), was embryonic lethal, we here study REA conditional knockout mice generated by cre-loxP recombination, in which REA function was abrogated only in progesterone receptor-expressing tissues, to define the roles of REA in postembryonic stages and in a tissue-specific manner. We find that REA has gene dose-dependent activity impacting uterine development and fertility. Conditional homozygous mutant (REA(d/d)) mice developed to adulthood and showed normal ovarian function, but females were infertile with severely compromised uterine development and function characterized by cell cycle arrest, apoptosis, and altered adenogenesis (endometrial gland morphogenesis), resulting in failure of implantation and decidualization. By contrast, mice heterozygous for REA (REA(f/d)) had a very different phenotype, with estradiol treatment resulting in hyperstimulated, large uteri showing increased proliferation of luminal epithelial cells, and enhanced fluid imbibition associated with altered regulation of aquaporins. These REA(f/d) female mice showed a subfertility phenotype with reduced numbers and sizes of litters. These findings highlight that uterine development and regulation of estrogen receptor activities show a bimodal dependence on the gene dosage of REA. Optimal uterine development and functional activities require the normal gene dosage of REA, with partial or complete deletion resulting in hyperresponsiveness or underresponsiveness to hormone and subfertility or infertility, respectively.

Hemostatic gelatin sponge is a superior matrix to matrigel for establishment of LNCaP human prostate cancer in nude mice

Matrigels, solubilized basement membrane preparations, are often used to support tumor development in animal models. However, tumors formed by a mixture of tumor cells and Matrigel may vary significantly. The purpose of this study was to compare tumor development and growth of LNCaP human prostate cancer cells mixed with Matrigel or in gelatin sponges. LNCaP cells were mixed with Matrigel or absorbed into VETSPON, a gelatin sponge, and inoculated into the subcutis of nude mice. Tumor incidence and growth rate were determined. Gene expression and cell growth and survival in tumor lesions were evaluated by immunohistochemistry (IHC), immunoblotting, and RT-PCR. All mice (12/12) inoculated with LNCaP cells in VETSPON produced tumors, compared to 70% (19/27) of mice injected with the cells with Matrigel. Tumor volume also varied less with VETSPON implants. No significant differences were observed in gene expression, cell growth, apoptosis, and microvessel density in tumors established from the two types of implants. However, in samples collected on days 1 and 4, more cells in Matrigel implants than those in VETSPON implants were stained positive for cleaved-caspase 3 and -PARP1. Expression of VEGF-A, HIF-1α, and Bcl-2 was elevated in the early VETSPON implants. These data indicate that VETSPON promotes tumor cell survival at the early stage of implantation and suggest that the gelatin sponge is superior to Matrigel in supporting development and progression of human prostate cancer in nude mice. This model should be useful for preclinical studies in nude mice using LNCaP cells.

The effect of adhesive strength of hydroxyapatite coating on the stability of hydroxyapatite-coated prostheses in vivo at the early stage of implantation

IntroductionWith the increase in joint revision surgery after arthroplasty, defects of hydroxyapatite (HA)-coated prostheses have been observed increasingly often. These defects adversely affect the prosthetic stability in vivo. This study has analyzed the potential effect of the adhesive strength of HA coating on the stability of HA-coated prostheses in vivo after its implantation.Material and methodsSixty experimental rabbits were divided into HA- and Ti-coated groups. HA-coated prostheses were implanted into the bilateral epicondyle of rabbits femurs. Ti-coated prostheses were implanted as control. At different time points(4, 9, and 15 weeks) after implantation, bone tissue samples were fetched out respectively for histomorphometric analysis. Push-out testing was used to detect the ultimate shear strength at the bone-prosthesis interface. Scanning electron microscope (SEM) observation and energy-dispersive X-ray spectroscopy (EDX) analysis were used to observe the changes in surface composition of the prostheses after the ultimate shear strength testing. The coating adhesive strength of two kinds of coatings were also examined by scratch testing.ResultsHydroxyapatite coating has an obvious advantage in facilitating osteogenesis and its plays a critical role in the stability of prostheses. However, the ultimate shear strength of HA-coated prostheses is much lower than that of Ti-coated implants (p < 0.01). Further study has demonstrated that the stability of HA-coated prostheses in vivo is affected by the relatively low adhesive strength between coating and substrate.ConclusionsObvious advantage in facilitating osteogenesis around HA-coated prostheses is not the only factor that determines the stability of prostheses in vivo.

Genotoxic and Biological Evaluation of a Nano Silica Cross Linked Composite Specifically Used for Intra-Vas Device

The Context: Aims: To evaluate the biocompatibility and in vitro genotoxicity of a non-copper nano silica polymer modified composite for filtering-type intra-vas devices. Settings and Design: Academic research laboratory, Huazhong University of Science and Technology. Prospective experimental study. Methods and Material: Non-copper nano silica polymer modified composite rods were implanted into the back muscle of rabbits for biocompatibility evaluation. Comet assay was applied to the determination of DNA damage, while, Mutagenic activity was tested by means of Ames test using Salmonella typhimurium TA98 and TA 100 tester strains with and without metabolic activation. Statistical analysis used: qualitative and quantitative data were tested using the Chi-square test and Student’s test. Results: Only mild inflammatory reaction was observed in the surrounding tissues of the implanted nano-silica modified polymer composite in the early implantation stage, which was similar to that of the sham-operated group. The inflammatory reaction was completely disappeared after 12 weeks. No significant DNA damage (P > 0.05) were tested on the nano-silica modified polymer composite in Comet assay. In Ames test, the extracts from non-copper composite did not exert mutagenic effect on the bacterial. Conclusions: The non-copper nano silica modified composite did not exhibit in vitro genotoxicity and obvious inflammation in tissue, it would be a safe biomaterial for further clinical trial.

Comparative Transcriptome Analysis of Human Trophectoderm and Embryonic Stem Cell-Derived Trophoblasts Reveal Key Participants in Early Implantation1

The implantation process begins with attachment of the trophectoderm (TE) of the blastocyst to the maternal endometrial epithelium. Herein we have investigated the transcriptome of mural TE cells from 13 human blastocysts and compared these with those of human embryonic stem cell (hESC)-derived-TE (hESC(troph)). The transcriptomes of hESC(troph) at Days 8, 10, and 12 had the greatest consistency with TE. Among genes coding for secreted proteins of the TE of human blastocysts and of hESC(troph) are several molecules known to be involved in the implantation process, as well as novel ones, such as CXCL12, HBEGF, inhibin A, DKK3, WNT5A, and follistatin. The similarities between the two lineages underscore some of the known mechanisms and offer discovery of new mechanisms and players in the process of the very early stages of human implantation. We propose that the hESC(troph) is a viable functional model of human trophoblasts to study trophoblast-endometrial interactions. Furthermore, the data derived herein offer the promise of novel diagnostics and therapeutics aimed at practical challenges in human infertility and pregnancy disorders associated with abnormal embryonic implantation.

In vitro and in vivo evaluations on osteogenesis and biodegradability of a β‐tricalcium phosphate coated magnesium alloy

Magnesium (Mg) alloys have been demonstrated to be potential orthopedic implants due to their biodegradability in vivo. To enhance its corrosion resistance and improve its osteogenesis, β-tricalcium phosphate (β-TCP) was coated on a Mg alloy (Mg-3AI-1Zn) by phosphating process. In vitro, the human osteosarcoma cell line (SaOS-2) showed significantly good adherence and proliferation on the surface of the β-TCP coated Mg alloy after 24-h incubation. The growth factor bone morphogenetic protein 2 (BMP-2) was highly expressed in SaOS-2 cultured with the β-TCP coated Mg alloy by Western blot analysis (p < 0.05). In vivo, the newborn bone at the implant/bone interface was formed at week 1 and matured at week 4 postimplantation. Villous tissue was found at the implant/bone interface at week 12 postimplantation. The contents of phosphorus and calcium on the surface of the β-TCP coated Mg alloy were decreased at week 4 and week 12 postimplantation, respectively. Immunohistochemical analysis of the experiment results demonstrated that the β-TCP coated Mg alloy implants provided a high BMP-2 expression during the first 4 weeks postimplantation. Compared with the naked Mg alloy which was degraded for 33% in vivo, only 17% of the β-TCP coated Mg alloy was degraded at week 12 postimplantation (p < 0.05). The in vitro cell tests showed that the β-TCP coating provided the Mg alloy with a significantly better surface cytocompatibility, and in vivo results also confirmed that the β-TCP coating exhibited greatly improved osteoconductivity and osteogenesis in the early 12 weeks postoperation period. Moreover, in vivo experiment demonstrated that the β-TCP coating layer could slow down the degradation of the naked Mg alloy at the early stage of implantation.

Tetracycline Double Labelling Tracing Investigation for &lt;i&gt;In Vivo&lt;/i&gt; Osteogenetic Course of PDLLA/HA Composite

The PDLLA(DL-Poly lactic acids)/HA(Hydroxyapatite) composite and pure PDLLA control were implanted in bone for test. Using fluorescent pictures of tetracycline double label and advanced image analysis system, the trend of bone-formation rate for the implanting region of PDLLA/HA composite was obtained via morphometry measurement of the slices labeled with tetracycline. The investigation and calculation results show that, the formation rate of new bone is stable and high (reaches 2.044μm/day) in the early stage of implantation for the PDLLA/HA composite, demonstrating the advantageous capability of bone formation for the composite. The formation rate of new bone seems to decrease continuously with time, and then the bone formation and bone decomposition tend towards equilibration. Namely, the dynamic balance of bony tissue’s metabolism is maintained.

On model of angiogenesis and the mechanism in porous silk fibroin films

The purpose of this paper is to explore the mechanism of the angiogenesis modes in the biomaterials implanted in vivo. By means of experimental observation and analysis of the capillary growing state in the porous silk fibroin film implanted into rats, we intended to develop a modeling expression on the growth mode of the capillaries in the biomaterials. Additionally, we proposed the response model of endothelial cells (ECs) resulting from vascular endothelial growth factor's concentrations at different stages after the implantation. With the implantation experiment, it was identified that angiogenesis developed in the way of capillary sprouting at the early stage of implantation and of intussusception at the late stage. Based on the response model of ECs, experimental results are explained.

Endometrial LGR7 expression during menstrual cycle

In a prospective observational study, 50 healthy patients aged 18-39 years, with regular ovulatory cycle and normal hormone levels, underwent endometrial biopsy in the proliferative and secretory phase of the menstrual cycle for semiquantitative reverse-transcription polymerase chain reaction analysis of mRNA for LGR7, the classic relaxin receptor. LGR7 is constitutively expressed in human endometrium, and an increased LGR7 immunostaining is demonstrated in the secretory phase, confirming the involvement of relaxin in the physiology of endometrium and suggesting its role in implantation.

Effect of nerve growth factor on the early phase of osseointegration around oral implants

To observe the early bone integration of oral implants after injection of exogenous nerve growth factor (NGF) and investigate the effects of NGF on peri-implant osseointegration. Twelve New Zealand white rabbits were used in this study to establish bi-mandible implant model. Then local injection of 1 µg NGF was given on the right side of the mandible as experimental group and normal saline only was injected on the left side as control group once a day for seven days. The rabbits were respectively sacrificed at 2, 4 and 8 weeks after surgery. The implant-bone grinding samples were prepared and stained by toluidine blue for general observation, X-ray, histology and bone histomorphometry analysis. The density of the new bone around implants at 2 and 4 weeks was lower than normal bone. Compared with the control group, the quantity of new bone and bone-implant contact ratio significantly increased in the experimental group. At 8 weeks, the new bone density in both groups was similar to the normal bone. In the experimental group, the haversian system was observed. Bone contact ratio was significantly different between experimental and control group at 2 and 4 weeks, but similar at 8 weeks.[control group at 2 weeks (26.67 ± 3.88)%, 4 weeks (52.59 ± 5.07)% and 8 weeks (97.33 ± 6.75)%, experimental group at 2 weeks (42.24 ± 6.67)%, 4 weeks (72.25 ± 6.30)% and 8 weeks (99.15 ± 4.68)%]. Applying exogenous NGF in the early phase could accelerate the formation and maturation of trabecular bone around the implants and shorten the period of osseointegration. Nerve growth factor could promote osseointegration in the early stage of oral implantation.

Watching Endometriosis Grow? A New Animal Model Using Ovine Eyes

Study Objective: To use the immunologically-privileged site of the anterior chamber of sheep eyes to observe the early stages of implantation and growth of human endometrium through the transparent cornea.

Osseointegration of titanium implants with a roughened surface containing hydride ion in a rabbit model

The objective of this study was to investigate the effect of hydride ion on bone formation and bone-bonding strength at the early stage of implantation. Implants and plates were treated in 2 ways: test implant (implant surface with hydride ion) and control implant (implant surface without hydride ion). Crystal structure of plate surfaces was analyzed by low angle x-ray diffractometry (XRD). Sixty implants (30 test implants and 30 control implants) were inserted into tibias of 10 rabbits. At 2 and 8 weeks postimplantation, tibias were retrieved and prepared for removal torque tests (RTQ) and histomorphometric evaluation, respectively. TiH(2) diffractions appeared on the XRD pattern of the test surface whereas TiH(2) did not appear on the control surface. The test implants showed 51.28% and 64.63% greater RTQ values than did the control implants at 2 and 8 weeks (P = .010, .011, respectively). Histomorphometric evaluation demonstrated the test implants significantly increased bone-implant contact and peri-implant bone formation at 2 and 8 weeks. These results suggest the presence of hydride ion in the implant surface may improve bone integration with implant surfaces at the early stage of implantation.

Biological evaluation of a novel copper-containing composite for contraception

To investigate the biocompatibility of a novel copper-containing composite to provide preclinical data for clinical application of intrauterine device (IUD) or intra-vas device (IVD). Prospective experimental study. Good laboratory practices laboratories. Twenty healthy adult mice (SPF grade Kunming white mice, animal code SCXK 2003-0005). Cytotoxicity tests in vitro were conducted to evaluate the influence of the materials on the morphology, growth, and proliferation of cultured L929 mouse fibroblasts. Acute systemic toxicity tests were conducted to investigate the acute systemic toxic reaction with mice, and then the materials were implanted into the spinal muscle of rabbits (n = 15). The rabbits were sacrificed for pathologic examination at 1, 4, and 12 weeks after surgery. Evaluation of cytotoxicity by MTT assay, cytotoxicity test by direct contact assay, acute systemic toxicity test, and material implantation test. The cytotoxicity grade of the copper-containing composite was 0-1, suggesting that the material was free of cytotoxicity; no acute systemic toxicity was found in any mice; mild inflammatory reaction was observed in the surrounding tissues of the implanted material in the early implantation stage, which was similar to that of the sham-operated sides. Twelve weeks after implantation, the inflammatory reaction was completely disappeared in the implanted tissue, similarly to the sham-operated sides. The fibrosis membrane surrounding the material became stable gradually over time. The copper-containing composite has excellent biocompatibility, which is feasible and safe for the clinical application as a novel contraceptive material.

A sensitive polymerase chain reaction-based method for detection and quantification of metastasis in human xenograft mouse models

Tumor cell dissemination to distant organs accounts for the majority of cancer related deaths. Analysis of the stepwise process of metastasis formation and progression might provide novel therapeutic strategies for the treatment of disseminated cancer. However, studies with both biological and therapeutic endpoints would require highly sensitive and specific methods for precise quantification of the metastatic tumor burden in vivo. We have developed a quantitative real-time PCR-based assay for the detection and quantification of human tumor cells disseminated in mouse organs. The method relies on the parallel amplification of unique, species-specific, conserved and non-transcribed sequences in the mouse and human genomes. We tested the method in xenograft models to assess the metastatic potential of various cancer cell lines, the impact of injection modality and cell type on organ distribution, and the early stages of metastasis implantation and progression. With this method, we observed clear quantitative differences among colon cancer cell lines in terms of metastasis formation in the lung, consistent with the different in vitro growth properties. The mode of cell implantation and cell intrinsic properties strongly affected the metastatic pattern of prostate and breast cancer cell lines in mouse organs. The qPCR assay accurately determined the malignant cell burden even at early stages of metastasis progression in the lung. We describe a very sensitive assay for the highly reproducible detection and accurate quantification of human metastatic cells in mouse tissues and demonstrate its broad applicability to various experimental settings.

Early stages of implantation as revealed by an in vitro model

Our limited understanding of the processes underlying steroid hormonal control of human endometrial receptivity is largely due to the lack of a relevant model system. To overcome scarcity of material, we have developed a model in which mouse embryos attach to human Ishikawa cells, which express functional steroid hormone receptors. Blastocysts flushed from day 4 pregnant superovulated mice were transferred to confluent Ishikawa cell monolayers. After 48 h of co-culture, 85% of the blastocysts had attached loosely, but only 40% attached stably to the epithelial cell surface. In contrast, 95% of the embryos attached stably to tissue culture plastic. Thus, weak attachment of a majority of the embryos was followed by stronger adhesion of a smaller proportion. Seventeen percent of the transferred blastocysts modified the epithelial cell surface with loss of MUC1 at the attachment site, extending variably to adjacent epithelial cells. Initially, stable attachment occurred without disruption to the integrity of the epithelial monolayer, but at later stages after the embryo had spread laterally, displacement of subjacent cells was observed. A modest increase in stable attachment, but no changes to MUC1 clearance, was observed after assisted hatching. After 24 h priming of Ishikawa cells by 17beta-oestradiol (OE(2)) followed by 72-h incubation with medroxyprogesterone acetate and OE(2), stable attachment increased from 40 to 70%. Initial attachment is efficient either in the presence or in the absence of hormone; steroid treatment increased the incidence of stable attachment. Implantation failure is predicted to occur in this model when embryos fail to progress from initial to stable attachment.

Effects of gonadotrophin releasing hormone analogues on human endometrial stromal cells and embryo invasion in vitro

Gonadotrophin releasing hormone (GnRH) analogues are widely used in IVF programmes as a method of suppressing the luteinizing hormone (LH) surge prior to ovarian stimulation, but their roles outside the pituitary remain relatively unknown. A 2002 Cochrane review (Al-Inany et al. Gonadotrophin-releasing hormone antagonists for assisted conception. Cochrane Database Syst Rev 2006;3:CD001750) described lower pregnancy rates in women administered with GnRH antagonist, compared with those using an agonist, as part of an IVF programme, despite the fact that GnRH antagonist is a more effective repressor of LH. This study aimed to analyse the in-vitro effects of GnRH analogues on the decidualizing endometrium, blastocyst invasion and GnRH receptor expression in fertile women. We analysed the in-vitro decidualization capacity of endometrial stromal cells, derived from fertile women during the implantation window, in the presence of GnRH analogues. The influence of GnRH analogues on GnRH receptor expression and blastocyst invasion was assessed by in-vitro assays of biomedical marker secretion, immunoblots and blastocyst attachment to the stromal extracellular matrix. We demonstrate that, at the concentrations and time periods used, GnRH analogues did not significantly influence the extent of decidualization of endometrial stromal cells. In addition, no adverse effect of GnRH analogues was seen on human blastocyst invasion. We suggest that GnRH analogues affect neither the capacity of the endometrium to support invasion nor the invasive potential of the blastocyst in the early stages of implantation.

Histological and Mechanical Evaluation of the in vivo Bone-bonding Ability on the K2TinO2n+1/β-Ti Alloy as a Novel Bioactive Material

AbstractThe purpose of this study was to histologically and mechanically appraise the in vivo bone-bonding abilities of K2TinO2n+1 coated and uncoated Ti-15Mo-3Nb (TMN) implants. According to GB/T16886.6－1997 biological evaluation of medical devices Part 6:Tests for local effects after implantation, the two types of implants were implanted into the proximal metaphyses of Chinese white rabbits’ femurs for 12, 26 and 52 weeks and investigated by pushing out test, scanning electron microscopy (SEM) attached to an energy-dispersive X-ray micro-analyzer (EDX) and light microscopy. The bone-bonding abilities of the K2TinO2n+1 biocoating /Ti-15Mo-3Nb (KBT) gradient biomaterial implants were higher than those of T implants at different periods of implantation. The K2TinO2n+1 biocoating (KB) could stimulate new bone rapid formation at the early stages of implantation. And the implants with the biocoating eventually bonded to bone directly, with no intervening soft tissue layer, that was an osseocoalescence. However, the type of bone-bonding between TMN titanium alloy implants and bone was a simple osseocoaptation. The more excellent bone-bonding ability of the KBT implants should be attributed to the superficial characteristics, the bioactivity of low potassium titanate and biostability of high potassium titanate.