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Deviations from the norm can prevent the establishment of pregnancy or contribute to obstetrical disorders associated with aberrant placentation. Through basic and translational research, our mission is to identify the critical cellular and molecular events that are required for successful implantation and to understand their relationship with the pathologies of early pregnancy. Our objective is therefore to understand the biology of the developing blastocyst, which requires the differentiation of the trophectoderm—its outer epithelium—into invasive trophoblast cells and the maintenance of a cohort of pluripotent embryonic stem cells. We thus focus on the developing blastocyst, trophectoderm, and pluripotent embryonic stem cells and investigate the interactions between the uterine endometrium and implanting blastocyst at the inception of pregnancy. We are evaluating agents that would provide cellular and molecular evidence of successful and pathologic implantation.
Regulation of blastocyst development
Previous work with mouse blastocysts revealed signaling pathways regulated by oxygen, growth factors, and the extracellular matrix; these pathways advance the trophoblast's intrinsic developmental program. Using in vitro approaches with human trophoblast cell lines, we investigate the role of the maternal environment on pre- and peri-implantation development. Survival signaling by heparin-binding EGF-like growth factor (HBEGF) plays a major role in the ability of fetal cells to develop normally during the first trimester, when oxygen levels are extremely low. As development proceeds and oxygen becomes readily available, trophoblast cells lose their ability to induce HBEGF in response to hypoxia and become apoptotic at low oxygen levels. In the coming year, we plan to extend our studies of trophoblast differentiation and examine how the oxygen microenvironment regulates HBEGF production in these cells through intracellular signaling pathways. Our collaborator Carol Brenner has established the rhesus monkey pre-implantation embryo model and works with several rhesus embryonic stem cell lines. We found that some genes regulating cell fate in the blastocyst are expressed differently in this primate than in rodent models. Thus the transcription factor OCT4, which directs embryonic cells to become pluripotent stem cells, is also expressed in trophectoderm cells of late blastocysts. However, CDX2, a gene required for the formation of the placenta, is confined to the trophectoderm, while NANOG, a gene thought to be essential for maintaining pluripotency, is only expressed by the pluripotent inner cell mass, as predicted from mouse studies. As a translational model, the monkey preimplantation embryo is thus proving very useful for understanding the regulation of early development in humans.
Nuclear and cytoplasmic damage of pre-implantation embryos
Fluorescence in situ hybridization analysis of blastomeres from pre-implantation embryos of young rhesus macaque females revealed a high frequency of chromosomal abnormalities that was surprisingly similar to rates demonstrated by in vitro—produced (IVP) human embryos. Using this model, we found that about half the morphologically normal eight-cell embryos contain a mixture of cells that are normal diploid and those that are aneuploid, which could confound efforts to conduct preimplantation genetic diagnosis on single biopsied blastomeres. We are assessing mitochondrial replication and function to determine their impact on cytoplasmic cellular components, as the mitochondrion is central to energy and toxicity management. The proportion of mtDNA deletions in stimulated oocytes and embryos from rhesus macaques is significantly higher than that of mtDNA deletions in immature, unstimulated oocytes derived from ovaries of age-matched monkeys. The findings validate non-human primates as a model for investigating reproductive mechanisms relevant to human infertility. Our studies will have an impact on human in vitro fertilization, where aneuploidy in embryos is common.
Metabolomics of frozen/thawed immature human oocytes
Given that ATP is a principal donor of free energy and phosphate in many intracellular metabolic reactions and signal transduction pathways, we measured the ATP level in oocytes after a freezing/thawing process and compared the level with that in fresh oocytes (human oocytes from a stimulated IVF cycle that are discarded owing to their nuclear immaturity). ATP levels from surviving frozen/thawed oocytes are significantly lower than those in fresh oocytes. The low level results from cryopreservation, but, once the mitochondria begin to function after thawing, the ATP level approaches that of the fresh oocyte. We are continuing to measure the ATP levels in a larger number of oocytes and will extend our study by incubating post-thawed oocytes for either two or three hours to demonstrate the effect of the incubating time after thawing.
Novel approaches for prenatal diagnostics
The evaluation of trophoblast differentiation and survival during human implantation is critical to successful establishment of a pregnancy. We are studying multifunctional growth factors involved in normal trophoblast development and associated pathologies. By collecting fetal trophoblast cells migrating from the placenta to the cervix, information about ongoing pregnancies can be obtained shortly after a woman is found to be pregnant. Using a non-invasive approach identical to the collection of cells for a PAP smear, we are able to identify fetal trophoblast cells beginning as early as the sixth week of pregnancy by labeling them with an antibody against a protein specifically expressed by trophoblast cells. Interestingly, while cervical specimens from normal intrauterine pregnancies contain fetal cells at similar frequencies, samples obtained from ectopic pregnancies or those containing a blighted ovum have significantly fewer trophoblasts. Future prospective studies are planned to explore the predictive value of this test for a variety of pathologies and to develop methods to use cervical trophoblast cells for other kinds of prenatal diagnostic testing.