Forensic significance of phenotypical transformations … · issues associated both to the...

Rom J Leg Med [25] 131-139 [2017]DOI: 10.4323/rjlm.2017.131© 2017 Romanian Society of Legal Medicine

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FUNDAMENTAL RESEARCH

Forensic significance of phenotypical transformations of villus terminalis in human placenta

Gheorghe S. Drăgoi1,2,*, Elena Pătraşcu3, Ileana Dincă3, Mihaela Meşină Botoran3, Petru Răzvan Melinte3

_________________________________________________________________________________________ Abstract: The authors aim at deciphering the morphogenous synchronic and diachronic synergisms that are present in the epigenesis of villus terminalis in human placenta. The objectives are the spatial distribution of syncytial nuclear aggregates of the trophoblast, of the location and relations of intravillous fibrinoid substance, of the cyto- and histo- architecture of skeleton villus terminalis in the third-semester gestation placenta as well as of the perivillous pseudo-tumoural proliferations in partial hydatidiform mole miscarriage. The microanatomic analysis of villus terminalis structures visualized in histochemial and immuno-histochemical methods made possible the evaluation of morphological variability of phenotypical transformations of the trophoblast and of the villus terminalis centre that synergically contribute to the re-formation of villus terminalis in the normal and/or pathological evolution of gestation with implicatons in the forensic evaluation of pregnacy or miscarriage. Key Words: villus terminalis, syncytial knots, syncytial sprouts, intravillous fibrinoid substance, partial hydatidiform mole.

1) Romanian Academy of Medical Sciences, Branch of Craiova, Craiova, Romania* Corresponding author: E-mail: [email protected]) Doctoral School of University of Medicine and Farmacy of Craiova, Craiova, Romania3) University of Medicine and Farmacy of Craiova, Department of Morphology, Craiova, Romania

INTRODUCTION The evaluation of the phenotypical transformations, of the trophoblast and/or of the conjunctival – vacular stroma in the villus terminalis of the human placenta is difficult in histo-pathological practice because of the morphogenous synergisms that lie at the basis of micro-anatomic transformations. There still is a large number of unclear issues associated both to the determining factors of phenotypical transformations of villus terminalis and their significance in general and/or forensic pathology, namely: What is the contribution of the cyto-trophoblast in determining phenotypical transformations of villus terminalis? What are the correlations between the phenotypical transformations of the trophoblast and the stereo-distribution of the intravillous fibrinoid substance;

Might intravillous fibrinoid substance be the cause or the effect of phenotypical transformations of the trophoblast? What is the contribution of the increased value of the trophoblast apoptosis in the morphogenesis of the syncytial nuclear aggregates? What is the significance of the pathological or/and of the forensic presence of syncytial nuclear aggregates? In what ways do cyto-skeleton of the trophoblast epithelia and the histo-skeleton structure in the villous centre participate in maintaining the architecture of phenotypical transformations? Considering the phenotypical transformations of villus terminalis, in what way could the latter be seen as a marker of the morpho-functional evolution of the human placental system? The authors aim at the temporal and spatial micro-anatomic evaluation as well as at the agreement and disagreement of synchronic and diachronic

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Drăgoi G.S. et al. Forensic significance of phenotypical transformations of villus terminalis in human placenta

synergisms that are found in the structural epigenesis of placental villus terminalis. The objectives of the micro-anatomic study are: the stereodistribution of the syncytial nuclear aggregates in the trophoblast; the analysis of the location and the ratios of syncytial nuclear aggregates; the evaluation of the histo-skeleton architecture of the villous centre; the analysis of the architecture of the epithelial cyto-skeleton of the villous trophoblast and of the phenotypical transformations of chorial villous structures in partial hydatidiform mole;

MATERIAL AND METHODS

The research was carried out on 21 placentae taken from 36-week gestation (six cases, of which 2 after caesarean operation, 3 after natural delivery and one case after pre-eclampsia), from 37-week gestation (5 cases, of which a caesarean operation, 2 after the death of the fetus antepartum, and 2 after natural delivery), from 38-week gestation (7 cases, of which 3 after caesarean operation, 2 after restriction of the intrauterus development of the fetus and 2 after the antepartum death of the fetus) and from 3-month gestation (2 miscarriages from partial hydatidiform mole). The placentae were fixed for 3 days in formalin 5%. Five samples were taken from each placenta (2 from the central zone and 3 from the periphery). Following paraffin embedding, ten serial sections were made from each block. They were used to visualize the microanatomy of the structural elements of villus terminalis: staining in hematoxylin eosine for the histo-topographic analysis of the structures, van Gieson staining for the visualization of picro-fuchsinofile collagen fibres (collagen type XVIII), reduced silver nitrate Gömöri method for reticulin collagen fibres (collagen type III) and immunomarking for cyto-keratin 7, alpha-Actin and desmine. The sections were examined under light microscopy Nikon Eclipse 80i. Microanatomic images were obtained on Nikon DS Fi1 Digital Camera and the soft programme Nis-Element AR 3.20 64 bit.

RESULTS

The microanatomic study was carried out on the stereodistribution of syncytial nuclear aggregates, on the implication of intravillous fibrinoid substance in the process of re-structuring villus terminalis, on the architecture of the histo- and cyto-skeleton of epithelial and mesenchimal structures found in placenta villi as well as on the phenotypical transformations of villous structures in partial hydatidiform mole.

A. Microanatomic analysis of syncytial nuclear aggregates On examining serial sections of placenta fragments after 36 and 37 – week gestations, stained in hematoxylin eosine, magnified x4 and x10, the authors easily identified the structural heterogeneity of villus terminalis induced by the phenotypical transformations of villous trophoblast epithelia. We identified 2 subtypes of syncytial nuclear aggregates: Nodi syncytiale (Syncytial knots) and Gemmae syncytiale (Syncytial sprouts) (Figs 1 and 2). Location, form and structure of these aggregates vary according to the sectioning planes that impose the spatial geometry of villus terminalis. Spatial location was identified both in the equatorial and meridional planes. The geometric form depends on the number of syncytiotrophoblast nuclei included in the section planes. The structure of the nuclear aggregates is variable and influenced by the intensity and frequency of the process of nuclear apoptosis. On magnifier x60, nuclear heterochromatin is dominated by apoptotic nuclei (Figs 1 and 2). In nodi syncytiales, nuclear agregates of the syncytiotrophoblast were identified in unipolar, bipolar, equatorial and polar – equatorial mixed (Fig. 1). In Gemmae syncytiales nuclear aggregates expand in the intravillous space (Fig. 2).

B. Microanatomic analysis of the location and ratios of the intravillous fibrinod substance involved in the re-making of villus terminalis Intravillous fibrinoid substance, through its diachronic stereodistribution as well as through its relations is an important factor in phenotypical transformations that take place in time and space at the level of villous structures (Fig. 3). On examining serial sections x20, x40, stained in hematoxylin eosine,the authors identified the primary location of fibrinoid substance in the immediate neighbourhood of the cytotrophoblast (Figs 3A and 3B). The dissociation of the cytotrophoblast from the syncytiotrophoblast is easily seen through the centripetal expansion of the fibrinoid substance. The cytotrophoblast appears dislocated within the conjunctive stroma of the villous centre (Figs 3C, 3D and 3E). Part of the cytotrophoblastic epithelium, with nuclei in apoptosis, is incorporate in the fibrinoid substance. (Figs 3F, 3G and 3H). Concommitantly, the syncytiotrophoblast undergoes phenotypical transformations within the fibrinoid substance expanded over the entire villous centre area (Figs 3 H and 3I).

C. Microanatomic analysis of the histo- and cyto- skeleton architecture in the epithelial - mesenchymal structures of villus terminalis

The architecture of the histo-skeleton is defined by location and the relations of both picrofuchsinophile

Romanian Journal of Legal Medicine Vol. XXV, No 2(2017)

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and reticulin collagen fibres in centrum villi. On examining serial sections x20, x40, stained by van Giesen method, the authors identified fascicles of collagen fibres (collagen XVIII) at the level of villus terminalis integrated in the network of the villous histo-skeleton. They make up a three-dimensional network in which angio- and vasculo- genesis processes occur due to the presence of myofibroblasts and the extension of the intravillous fibrinoid substance (Figs 4A, 4B and 4C). On examining, reduced siver nitrate Gömöri method, serial sections x10 and x40, the authors noticed the presence of fascicles of reticulin collagen fibres (Colagen III) spatially distributed in the stroma of villus terminalis either under the form of subtrophoblastic and subendothelial basal membranes or as a network within the stroma of the villous centre (Fig. 4 C). At the level of intermediate mature villi we saw the fascicular spiral of reticulin collagen fibres around presinusoid arterioles as a support of the presinusoid arteriolar sphincter. By means of immunochemistry we distinguished the intermediary filaments of cytokeratin (Filamentum cytokeratini), of desmine (Filamentum desmini) and of alpha-actin (Filamentum actini) from the structural elements of the cyto-skeleton. On examining x60 the serial sections for the immunomarking of keratin 7 we noticed alpha-keratin in the cells and syncytial elements of the trophoblast in villus terminalis (Figs 4D, 4E and 4F). On the serial sections immnomarked for alpha-keratin the authors studied the stereodistribution and the relations of alpha-keratin filaments around the blood vessels both in villus terminalis and in the intermediary mature villosities and stem villi (Figs 5G, 5H and 5 I). A similar distribution was seen on the serial sections in the immunomarked desmine filaments (Figs 5J, 5K and 5L).

D. Microanatomic analysis of phenotypical transformations in placental villous structures in partial hydatiditiform mole

In partial hydatidiform mole phenotypical transformations of chorial villosities were evaluated both at the level of the trophoblast and of the stroma in the villous centre (Fig. 5). On examination x 4 and x10 we identified the presence of large chorial villosities, of a vesiculous aspect, with irregular margins (Figs 5A, 5B and 5C). The perivillous trophoblast of pseudotumoural extravillous proliferation generates aisles adjacent to the limiting chorial villosities (Figs 5A, 5B and 5C). The stroma of the villous centre lacks vessels and contains conjunctival tissue and interstitial edema, and relatively frequent Hofbauer cells. We also identified villus terminalis of bilayered trophblast. The syncytiotrophoblast is made up of ovoid, hyperchromatic nuclei, eosinophilic cytoplasm and a syncytial membrane that is equipped with microvillosities oriented towards the intervillous space (Figs 5D, 5E and 5 F). Syncytial nuclear

aggregates, under the form of syncytials sprouts were visualized both on the longitudinal and the transverse sections made in villus terminalis (Figs 5D and 5E). At the periphery of villus terminalis, eosinophilic fibrinoid substance was seen on the hematoxylin eosin – stained perivillous sections (Figs 5J, 5K and 5 L).

DISCUSSION

Human placenta is an anatomic and functional system of variable morphology. This variable character is dictated by the synergic, diachronic and/or synchronic transformations that occur throughout all the stages of gestation. The diverse and complex functions occurring at the level of placental barrier make possible both gestation, on the one hand and the growth of the embryo/fetus, on the other. The results obtained highlight the implication of syncytial nuclear aggregates, of the intravilous fibrinoid substance and extravillous pseudotumour proliferations of the trophoblast from partial hydatidiform mole in the morphogenesis of phenotypical transformations in the structure of placental villi. These aspects rise two essential issues, amply discussed in literature, one connected to the formation of syncytial nuclear aggregates and another to their significance in patomorphogenesis. They are closely associated to the trophoblast turnover that includes 3 morpho-differentiation processes: cytotrophoblast proliferation and differentiation [1]; the syncytial fusion of the cytotrophoblast [2] and the differentiation of the syncytiotrophoblast generating the syncytial nuclear aggregates through apoptosis. Hupertz et al. (2006) [3] believe that the presence of dense heterochromatin nuclei is a sign of apoptosis. However, Coleman et al. (2013) [4] demonstrate that this indicator is not specific to apoptosis. Again, Burton and Jones (2009) [5] and Longtine et al. (2012) [6] noticed the presence of apoptotic changes in normal syncyotrophoblast. Syncytial nuclear aggregates formation mechanism is still obscure [7]. However, the proteins existing in the cyto-skeleton of villous trophoblast epithelia are associated to the syncytial nuclear aggregates formation mechanism. The cyto-keratin filaments tightly envelop syncytial nuclear aggregates and give them high stability [4 and 8]. Microfilaments of alfa-Actin are also part of syncytial nuclear aggregates genesis [9-11]. The significance of the part played by syncytial nuclear aggregates in pathological morphology is also unclear [12, 13]. Nodi syncytiales (Syncytial knots) are equally taken as markers in preeclampsia [14] a sign of overdue pregnancy [8] and associated to oxydizing stress [15]. Extravillous hyperplasia of the syncytiotro-phoblast and ample secretion of gonadotropic hormones are characteristic in hydatid mole. The presence of the embryo is an indicator of partial hydatid mole, unlike in complete hydatid mole in which the embryo is missing.

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Figure 1. Nodi Syncytiales (Syncytial Knots). 36-week old gestation placenta. 1. Syncytiotrophoblast. 2. Sinusoid blood vessels. 3. Ecuatorial location. 4. Polar location. 5. Stasis in sinusoid blood vessels. 6. Secant section through nodi syncytiales in ecuatorial location. 7. Nuclei in apoptosis. 8. Anucleated syncyotrophoblast zone. Arrowhead – vasculo-syncytial membrane. Formalin fixed. Paraffin embedded sections. Hematoxylin Eosin stain. Magnified: x 420 (A-I).


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Figure 2. Gemmae syncytiales (Syncytial sprouts). 37-week old gestation placenta. 1. Gemmae syncytiales in differentiation process. 2. Syncytiotrphoblast nuclei. 3. Cytotrophoblast nuclei. 4. Anucleated syncytiotrophoblast zone. 5. Sinusoid blood vessels. 6. Syncytiotrophoblast nuclei in apoptosis 7. Substantia fibrinoidea perivillosa including nuclei of syncytiotrofoblast in apoptosis. Formalin fixed. Paraffin embedded sections. Hematoxylin Eosin stain. Magnified: x 420 (A-L).

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Figure 3. Structural re-formation of villus terminalis. 38-week old gestation placenta. 1. Cytotrophoblast. 2. Syncytiotrophoblast. 3. Sinusoid blood vessels. 4. Anucleated syncytiotrophoblast zone. 5. Substantia fibrinoidea intra-villosa. 6. Mitosis at the level of a trophoblastic cells. 7. Nodi syncitiales. 8. Binucleated trophoblastic cells. 9. Gemmae syncytiales. 10. Trophoblastic cells included in fibrinoid substance. 11. Villous structures erased. Formalin fixed. Paraffin embedded sections. Hematoxylin Eosin stain. Magnified: x 420 ( A – I).


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Figure 4. Histo – and Cyto – skeleton in placental villosities. 36-week old gestation placenta. 1. Fascicles of collagen fibres XVIII (picrofuxinofile) in stem villi, mature intermediate villi and villus terminalis. 2. Tip XVIII collagen (picrofuxinofil) in villus terminalis 3. Nodi syncytiales. 4. Villus terminalis 5. Transverse-sectioned sinusoid blood vessels. 6. Tip III colagen (reticulin) in centrum villi. 7. Mature intermediate villi. 8-10.Immunomarking of cytokeratin 7 in trophoblast epithelia. 11, 12. Positive reaction to alpha-Actin in mature intermediate villi and in villus terminali. 13-15. Immunomarking for desmine in stem villi, in mature intermediate villi and in villus treminalis. Formalin fixed. Paraffin embedded sections. Van Gieson stain (A, B), Reduced silver nitrate Gömöri method (C), Immunochemistry on paraffin section for the qualitative identification by light microscopy of cytokeratin 7 (D-F), alpha-Actin (G-I) and desemine molecule (J-L). Magnified : x 420 (A-I).

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Figure 5. Partial hydatidiform mole. 1. Chorial avascular villosities affected by interstitial edema and cystic degenerescence. 2. Perivillositary pseudo-tumoural proliferation of trophoblast. 3. Fibrinoid substance within the proliferation aisle of the trophoblast. 4. Syncytiotrophoblast in superficies vill. 5. Gemma syncytiales. 6. Cytotrophoblast in superficies villi. 7. Gemma syncytiales in longitudinal section. 8. Gemma syncytiales in transverse section. 9. Proliferation of trophoblast epithelial cells. 10. Stereodistribution of fibrinoid substance in villus terminalis. Formalin fixed. Paraffin embedded sections. Hematoxilin Eosin stain. Magnified: x 28 (A); x 70 (B;C) ; x 420 (D-L).


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Nevertheless, the diagnosis needs to be endorsed by cytogenetic analyses that will prove that the chromosomes come from the biological father. The phenotypical transformations in the structures of placental barrier are involved in pathological morphology: limitation of the intrauterine growth of the fetus, antepartum death of the fetus, distribution of nodi syncytiales in mother’s blood flow.

CONCLUSIONS

1. The evaluation of morphological variability of placental villi is essential for the understanding of phenotypical transformations that are the basis of a stable balance in the spatial and time development of the functions of the placental barrier. 2. Two structural elements are involved in the turnover of morphogenous synergisms: the trophoblast

and the villous centre that interactively participate in the formation and transformation of the functional structures of the villous system. 3. The structure and stereodistribution of syncytial nuclear aggregates are consequences of phenotypical transformations of syncytiotrophoblast nuclei and the presence of cytokeratin and alpha-actin protein filaments. 4. Total structural re-making of villus terminalis is achieved through synchronous phenotypical transformations processes, the genesis and the centripetal expansion of intravillous fibrinoid substance. 5. Phenotypical transformations of villous structures are important in the medico-legal evaluation of pregnancy and miscarriage.

Conflict of interest. The authors declares that there is no conflict of interest.

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