An investigation into the mechanisms of syncytial nuclear aggregate formation

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Abstract

The outer surface of the human placenta, the syncytiotrophoblast, results from the fusion of many cytotrophoblast cells such that many nuclei are contained in this layer. It is possible for these nuclei to cluster forming syncytial nuclear aggregates (SNAs). SNAs have been linked to pathology with increased numbers and earlier formation of SNAs in preeclampsia and fetal growth restriction (FGR). SNAs can be grouped into subtypes including bridges, knots and sprouts, dependent on morphology and attachment to surrounding placental villi. Little is known about SNA formation, but the pyknotic appearance of nuclei within SNAs has led to development of a hypothesis that SNAs are the terminal point of nuclear turnover in the syncytiotrophoblast. Some cytoskeletal proteins have been associated with SNAs indicating their potential involvement in SNA formation. This project aimed to uncover differences between SNA subtypes, whether the degenerate nuclear morphology represents apoptosis and to understand which mechanisms drive nuclear collection into SNAs.Experimental approaches included a review of an electron micrograph archive and application of immunohistochemical techniques to ex vivo placental tissue. A long-term explant model was developed to examine SNA development in vitro; these experiments were further explored using an isolated primary cytotrophoblast model.Nuclei within SNAs were more frequently pyknotic and less frequently eukaryotic than nuclei dispersed in the syncytiotrophoblast. However, few SNAs were positive for the cytokeratin-M30 neoepitope, a caspase dependent breakdown product of cytokeratin-18 and no subtype of SNA showed greater M30 staining than general areas of syncytiotrophoblast. There were increased syncytial knots and decreased syncytial bridges in placentas from women with preeclampsia compared to controls and FGR. While cytoskeletal proteins are seen surrounding SNAs, inhibition of actin and tubulin had no effect on SNA turnover or stability. Very limited nuclear movement was recorded from in vitro culture indicating that syncytiotrophoblast nuclei move far less than had been expected.These data suggest that cell death was not prominent within SNAs but different prevalence of subtypes were present in preeclampsia indicating that SNAs might represent larger changes in placenta structure. As nuclei moved less and SNAs were more static than expected it is suggests that SNAs are more stable than previously thought. Overall, the hypothesis that SNAs are highly active in preeclampsia is questioned and new hypotheses of the role of SNAs are considered in the light of these experimental findings, including whether they form by chance and represent changes in cell turnover of the syncytiotrophoblast.

Layman's Abstract

A correctly functioning placenta is essential for a healthy pregnancy. When things go wrong with placental development it can be detrimental for the health of both mother and fetus. The outer layer of the placenta is adjacent to the maternal circulation and is the primary site for gas and nutrient exchange. This layer is made by fusion of many cells so there are no cell boundaries and many nuclei. Clusters of nuclei can form in the outer surface of the placenta; in many pregnancy diseases there is an increase in nuclear cluster number. This project aimed to examine nuclear clusters in more detail. Currently, we do not know how or by what mechanism these nuclear clusters form or what their function is, but nuclei in these clusters had signs of degeneration. Also, structural proteins which control cell shape and cell movement can be seen surrounding nuclear clusters. Many authors suggested that nuclear clusters were formed by a particular type of cell death called apoptosis and nuclei were collected together by structural proteins.My project examined these nuclear clusters in placentas after birth. Staining for markers of apoptosis revealed little evidence of this process in nuclear clusters; nuclear clusters were actually no more likely to show apoptosis than other areas of the outer placental layer. Thus, these nuclear clusters do not seem to be the end point of apoptotic cell death.Although the majority of pregnancies are healthy, pregnancy diseases such as preeclampsia and fetal growth restriction affect around 10% of pregnancies in the UK. Preeclampsia is noted by high blood pressure and proteinuria and fetal growth restriction is where the baby fails to reach its growth potential. Comparing nuclear cluster types in healthy, preeclamptic and fetal growth restricted placentas showed no differences between normal and fetal growth restricted samples but significant differences in preeclampsia. In preeclampsia there were fewer connecting nuclear cluster and more unconnected nuclear clusters which indicated there were fewer connections between placental villi compared to healthy placentas. This suggests that the microscopic structure of preeclamptic placentas is less well supported, potentially making these placentas more vulnerable to damage.Finally, I used two models to replicate nuclear clusters in order to learn how they form and what is changed in disease. Using cultured placental tissue and isolated placental cells I was able to show that nuclear clusters were very stable and there was less nuclear movement than had been anticipated, which means that structural proteins may not be responsible for moving nuclei around the outer layer of the placenta.Overall, there are changes in nuclear clusters during pregnancy and in disease but this work has shown that these are much more stable than previously anticipated. Therefore, previous suggestions that nuclear clusters were the end point for degenerate nuclei in the placenta have been questioned. Furthermore, I developed new hypotheses for nuclear cluster formation, including whether they form by chance or are symptomatic of other events, such as ageing, that occur in the placenta throughout pregnancy.

Keyword(s)

Cytoskeleton; Cytotrophoblast; Fetal Growth Restriction; Placenta; Preeclampsia; Pyknotic; Syncytial Bridge; Syncytial Knot; Syncytial Nuclear Aggregate; Syncytiotrophoblast

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