Talk:Carnegie stage 4

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Nature 443, 376-377 (28 September 2006) | doi:10.1038/443376a; Published online 27 September 2006



Natural selection of human embryos: decidualizing endometrial stromal cells serve as sensors of embryo quality upon implantation

PLoS One. 2010 Apr 21;5(4):e10258.

Teklenburg G, Salker M, Molokhia M, Lavery S, Trew G, Aojanepong T, Mardon HJ, Lokugamage AU, Rai R, Landles C, Roelen BA, Quenby S, Kuijk EW, Kavelaars A, Heijnen CJ, Regan L, Brosens JJ, Macklon NS.

Department of Reproductive Medicine and Gynecology, University Medical Center Utrecht, Utrecht, The Netherlands.

Abstract 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-1beta, -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.

PMID: 20422011

Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss

Salker M, Teklenburg G, Molokhia M, Lavery S, Trew G, Aojanepong T, Mardon HJ, Lokugamage AU, Rai R, Landles C, Roelen BA, Quenby S, Kuijk EW, Kavelaars A, Heijnen CJ, Regan L, Macklon NS, Brosens JJ. PLoS One. 2010 Apr 21;5(4):e10287. PMID: 20422017


Secretory role for human uterodomes (pinopods): secretion of LIF

Mol Hum Reprod. 2005 Aug;11(8):553-9. Epub 2005 Aug 26.

Kabir-Salmani M, Nikzad H, Shiokawa S, Akimoto Y, Iwashita M. Source Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Mitaka, Tokyo, Japan.


The differentiation of human endometrial epithelium is a dynamic event, which occurs throughout the menstrual cycle in preparation for pregnancy. The appearance of uterodomes (pinopods) in this regard was first introduced in rodents with an established pinocytotic function, whereas little evidence was available in humans in this context. This study was undertaken to identify the potential physiological roles of uterodomes in the implantation process. To address this, endometrial biopsies from early, mid- and late luteal phases of the menstrual cycle of 23 fertile female patients with regular menses were used. Scanning and transmission electron microscopies (SEM and TEM) as well as immunofluorescence and immunogold TEM were performed to study the morphological changes and the expression pattern of leukaemia inhibitory factor (LIF) at uterodomes. Our results illustrated a high level of LIF expression in the human uterodomes, which was colocalized with the well-known biochemical markers of exocytosis, including syntaxin-1, 25-kDa synaptosomal protein (SNAP-25) and vesicle-associated membrane protein-2 (VAMP-2). Our morphological and immunocytochemical findings illustrated a secretory function for human uterodomes for the first time. In conclusion, this novel function for uterodomes provides an important clue in detection of their physiological function(s) during the process of the plasma membrane transformation.

PMID 16126773


Ultrastructure of human blastocyst-endometrial interactions in vitro

J Reprod Fertil. 2000 Nov;120(2):337-50.

Bentin-Ley U, Horn T, Sjögren A, Sorensen S, Falck Larsen J, Hamberger L. Source Department of Obstetrics and Gynaecology, Herlev University Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark. Abstract The interactions of seven human blastocysts with cultured endometrial cells were investigated by light microscopy and transmission electron microscopy. Trophoblastic-endometrial contact was observed at the lateral border of endometrial epithelial cells where trophoblast and endometrial epithelial cells shared apical junctional complexes and desmosomes. The first sign of penetration was invasion of a trophoblastic cytoplasmic protrusion between endometrial epithelial cells. In broad contact areas, lateral displacement of endometrial epithelial cells and formation of a peripheral pseudostratified epithelium were observed. When trophoblastic cells were interposed fully among endometrial epithelial cells, they formed a penetration cone and appeared to dislodge endometrial epithelial cells from the stromal compartment. A single penetration cone only was found in each specimen. Endometrial or trophoblastic degeneration was not observed. Formation of multinucleate (>/= three nuclei per cell) trophoblast cells was not observed, but many cells displayed areas with abrupt disappearance of well-defined plasma membranes, which is indicative of syncytium formation. In this study, adhesion and penetration occurred at the same time. The human blastocysts penetrated the endometrial surface epithelium by intrusive penetration. Epithelial penetration was achieved primarily by cellular syncytiotrophoblast-like cells and the first indications of syncytium formation were observed simultaneously with penetration of the epithelium.

PMID 11058449


From blastocyst to placenta: the morphology of implantation in the baboon

Hum Reprod Update. 1997 Nov-Dec;3(6):561-73.

Enders AC, Lantz KC, Peterson PE, Hendrickx AG. Source Department of Cell Biology and Human Anatomy, School of Medicine, and California Regional Primate Research Center, University of California, Davis 95616, USA.


Implantation and placentation in the baboon share many morphological features with other primates, as well as having some specific distinctions. The ability to use deturgescence of the sex skin as a method of timing ovulation and the ease with which the uterine lumen can be flushed have been used to examine morphological aspects of blastocyst differentiation and implantation in this species. Preimplantation blastocysts were obtained by non-surgical flushing of the uterus 6-8 days after ovulation, and implantation sites were excised from uteri removed on days 10-16 of gestation. All tissues were prepared for electron microscopy by aldehyde fixation and plastic embedding. Maturation of trophoblast from the compacted morula stage to the expanded blastocyst stage includes increase in numbers of polyribosomes, changes in conformation of mitochondria, and development of an effective endocytic apparatus. An endodermal layer forms beneath the inner cell mass prior to loss of the zona pellucida, and parietal endodermal cells extend beyond the inner cell mass. Azonal blastocysts have regions of syncytial trophoblast adjacent to the inner cell mass, and they may represent adhesion stages of early implantation. In early postimplantation stages, trophoblast replaces the uterine epithelium and processes of syncytial trophoblast invade dilated superficial maternal vessels. In subsequent lacunar stages there is rapid elevation of the developing conceptus above the uterine surface as the lacunae enlarge. Cytotrophoblast rapidly enters maternal vessels, and arterioles are partially or completely occluded by migrating cytotrophoblast. The early access to controlled maternal blood flow apparently allows trophoblastic lacunae to expand superficially as opposed to more extensive endometrial invasion. PMID 9584946


Studies on the mechanism of embryo implantation

[Article in Japanese]

Nippon Sanka Fujinka Gakkai Zasshi. 1996 Aug;48(8):591-603.

Tominaga T.

Department of Obstetrics and Gynecology, Fukui Medical School. Abstract Implantation is a complex process accomplished by synchronization and interactions between embryo and endometrium by local exchange of signals including a number of cytokines and growth factors and direct cell-cell and cell-matrix contact. However, the research in early events of human implantation is still in its infancy. This presentation comprises the results of our attempts to investigate the mechanisms of human implantation process in its early stage by cell-biological method, including establishment of experimental implantation model in vitro. 1. Human trophoblast of early stage of gestation showed active cell locomotion, active endocytosis, and invasion of endometrial cell monolayer in mixed cultures. Trophoblast invasion was later arrested by transformed endometrial cells similar to decidual cells in vivo. These results appeared to indicate the interactions between trophoblast and endometrial cells in implantation. 2. Coculture system of rabbit preimplantation blastocyst and endometrial epithelium reformed from isolated endometrial epithelial cells on basement membrane matrix (Matrigel) simulated the in vivo rabbit implantation processes. This coculture system may provide a useful experimental implantation model. 3. A human trophoblast cell line was established from chorionic tissues of normal early pregnancy. These cells were cytotrophoblast-like morphology and endocrine functions. They formed the villous structures similar to those in vivo in culture on Matrigel and invasion of Matrigel was observed. These indicated the extracellular matrix may affect the morphology and function of invading trophoblast in implantation site. 4. Human endometrial epithelial single cells were cultured on Matrigel. Reconstruction of gland followed by epithelium formation quite similar to in vivo structures by migration and proliferation of isolated cells was demonstrated. Height of gland was promoted by estrogen and initiation of epithelization was upregulated by platelet-derived growth factors. This system revealed the extracellular matrix regulated morphogenesis of endometrial epithelium in vivo and is an essential substrate in experimental implantation model of endometrial epithelium. 5. Parallel cultures of endometrial epithelial cells on Matrigel were carried out with the IVF. ET patients to evaluate the endometrial morphology at time of ET. Endometrial cultures were initiated in previous cycles on Matrigel and the sera of patients were added to her own cultures from 1st day of IVF treatment cycle. Evaluation of reformed epithelium revealed the apparently unsuitable morphology for implantation in group of patients who eventually failed in pregnancy. This system may provide a useful measures in evaluation of endometrial receptivity and modality of treatment for endometrial aberrations. 6. Cyclic changes of extracellular matrix components in endometrium were investigated. Collagen I, III, IV, V were immunohistochemically estimated. Relative levels of all types of collagen except for collagen V declined at early secretory phase. In rodents, not only collagen but also laminin and fibronectin levels declined at early secretory phase. These changes may facilitate trophoblast invasion of endometrium. Collagen V distributed in myometrial surface was found to consist of subunit (alpha 1)2 alpha 2 and trophoblast growth was inhibited on substrate of alpha 1 subunit. Collagen V in myometrial surface may have a role in blocking trophoblast invasion. 7. HGF (hepatocyte growth factor) mRNA was demonstrated to be expressed during menstruation and secretory phase in endometrium distinctly and its receptor in endometrial epithelial cells and decidual cells. Positive correlation between plasma HGF levels and ultrasonographic thickness of endometrium was observed at late secretory phase. Recombinant HGF promoted proliferation of endometrial epithelial cells and decidual cells and upregulated initiation of endometrial epithelization of Matrigel.

PMID 8808827


Ultrastructure in human implantation: transmission and scanning electron microscopy

Baillieres Clin Obstet Gynaecol. 1991 Mar;5(1):1-14.

Lindenberg S. PMID 1855333


Ultrastructure of the blastocyst and endometrium of the roe deer (Capreolus capreolus) during delayed implantation

J Anat. 1975 Apr;119(Pt 2):369-84.

Aitken RJ.


Transmission electron microscopy of the trophoblast cells during diapause revealed an abundance of electron-dense, membrane-bound granular inclusions and a marked lack of cytoplasmic organelles. The cells also possessed a well-developed covering of branched microvilli, numerous caveolae, micropinocytotic vesicles and a lamina of fine fibrillae. The progressive enlargement of the blastocyst during diapause was correlated with a decline in the height of the trophoblast cells and a reduction in the density of microvilli and caveolae associated with the outer membrane. The granular inclusions also declined in number and electron density during the delay phase, suggesting the progressive utilisation of energy reserves. Embryonic elongation was associated with the disappearance of the granular inclusions, a reduction in the amount of fibrillar material and a dramatic increase in the development of cytoplasmic organelles. During diapause, clear vesicles, apparently derived from the Golgi apparatus, gradually accumulated in the supranuclear region of each gland and non-ciliated duct cell. Embryonic elongation was associated with the sudden release of these vesicles into the glandular lumen and thence into the uterine lumen. Numerous apical protrusions were also observed projecting from the luminal and ductal epithelia at this time, suggesting the formation of an apocrine secretion. Another type of secretion was produced during the early stages of placental attachment by the hypertrophied granular endoplasmic reticulum of the ductal epithelium.

PMID 166066


Electron microscopic studies on the mammalian blastocyst in the pre-implantation stages

Keio J Med. 1974 Jun;23(2):77-94.

Hamada Y.


This work was designed to investigate the fine structures of the rat and the rabbit blastocysts with both transmission and scanning electron microscopes. The cells of the trophoblast form a single layer beneath the intact zona pellucida and the inner cell mass and blastocystic cavity are formed. In the blastocyst on day 5 to 6 of pregnancy, the trophoblast cells were differentiated from the inner cell mass cells. However, the difference in density was not distinct and the distribution and structures of the organelles were almost similar in both cells.

The tight junctional complex between the trophoblast cells and the loose association among the inner cell mass cells were observed. The organelles of the blastocysts were quite specific. In the rabbit blastocyst, as inclusions restricted to the blastocyst stage, crystal-like inclusions were present.

From the standpoint of functional aspects the incorporation of 3H-uridine and 3H-thymidine into rat blastocysts was studied using electron microscopical radioautography. The result showed that incorporated 3H-uridine was found in all regions and 3H-thymidine mostly in nucleus. Moreover, with scanning electron microscope the surface of blastocyst was observed after the zona pellucida was removed with pronase. The surface of zona-free rat blastocyst was wholly covered with dense, fine microvilli and abembryonic proliferation was seen. Each bulging trophoblast cell was bounded by grooves and distinguishable to one another.

PMID 4479880

Recent research on implantation in animals

Proc R Soc Med. 1974 Sep;67(9):927-32.

Finn CA. PMID 4215085

The morphology of blastocyst implantation

J Reprod Fertil. 1974 Jul;39(1):187-94.

Nilsson O. PMID 4605300

Implantation of the rabbit blastocyst: the adhesive phase of implantation

J Anat. 1971 Jul;109(Pt 2):215-27.

Steer HW. PMID 5558231


J Anat. 1968 Jun;103(Pt 1):77-90. The ultrastructure of implantation in the mouse. Potts DM. PMID 5691967