Decrease in Sphingomyelin (d18:1/16:0) in Stem Villi and Phosphatidylcholine (16:0/20:4) in Terminal Villi of Human Term Placentas with Pathohistological Maternal Malperfusion

<div><p>Placental villi play pivotal roles in feto-maternal transportation and phospholipids constitute a major part of the villous membrane. We have been developing and optimizing an imaging system based on a matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometer, which provides clear two-dimensional molecular distribution patterns using highly sensitive mass spectrometry from mixtures of ions generated on tissue surfaces. We recently applied this technology to normal human uncomplicated term placentas and detected the specific distribution of sphingomyelin (SM) (d18:1/16:0) in stem villi and phosphatidylcholine (PC) (16:0/20:4) in terminal villi. In the present study, we applied this technology to nine placentas with maternal or fetal complications, and determined whether a relationship existed between these specific distribution patterns of phospholipid molecules and the six representative pathological findings of placentas, i.e., villitis of unknown etiology (VUE), thrombus, atherosis, chorioamnionitis (CAM), immature terminal villi, and multiple branched terminal villi. In two placentas with the first and second largest total number of positive pathological findings, i.e., five and three positive findings, the specific distribution of SM (d18:1/16:0) in stem villi and PC (16:0/20:4) in terminal villi disappeared. The common pathological findings in these two placentas were atherosis, immature terminal villi, and multiple branched terminal villi, suggesting the possible involvement of the underperfusion of maternal blood into the intervillous space. On the other hand, the number of pathological findings were two or less in the seven other placentas, in which no specific relationships were observed between the differential expression patterns of these two phospholipids in stem and terminal villi and the pathological findings of the placentas; however, the specific distribution pattern of SM (d18:1/16:0) in stem villi disappeared in four placentas, while that of PC (16:0/20:4) in terminal villi was preserved. These results suggested that the absence of the specific distribution of PC (16:0/20:4) in terminal villi, possibly in combination with the absence of SM (d18:1/16:0) in stem villi, was linked to placental morphological changes in response to maternal underperfusion of the placenta.</p></div

Expression profiles of sphingomyelin (d18:1/16:0) in stem villi and phosphatidylcholine (16:0/20:4) in terminal villi by imaging mass spectrometry.

<p>Samples grouped as—showed significant decreases (p<0.05) with an analysis of variance and Tukey’s test from the other group denoted as +.</p><p>Expression profiles of sphingomyelin (d18:1/16:0) in stem villi and phosphatidylcholine (16:0/20:4) in terminal villi by imaging mass spectrometry.</p

Representative averaged mass spectra from entire sections.

<p>Signals collected between <i>m/z</i> 700–900 were shown for sample Nos.2 (A), 8 (B), and 9 (C) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142609#pone.0142609.t003" target="_blank">Table 3</a>. Peaks corresponding to representative phospholipids were labeled. Imaging results for <i>m/z</i> 725.5 and <i>m/z</i> 804.5 were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142609#pone.0142609.g003" target="_blank">Fig 3</a> and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142609#pone.0142609.t003" target="_blank">Table 3</a>. Pathological findings of the placenta were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142609#pone.0142609.t001" target="_blank">Table 1</a>.</p

Ion images for <i>m/z</i> 725.5 (B, F, J), <i>m/z</i> 804.5 (C, G, K), and <i>m/z</i> 734.5 (D, H, L).

<p>A-D; placenta No. 2. E-H; placenta No.8. I-L; placenta No. 9. The peaks of <i>m/z</i> 725.5 corresponding to sphingomyelin (d18:1/16:0) and <i>m/z</i> 804.5 corresponding to phosphatidylcholine (16:0/20:4) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142609#pone.0142609.ref021" target="_blank">21</a>] were visualized. Imaging results for <i>m/z</i> 725.5 and <i>m/z</i> 804.5 associated with pathological findings were summarized in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142609#pone.0142609.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142609#pone.0142609.t003" target="_blank">3</a>. An ion image of <i>m/z</i> 734.5 (D, H, L) was presented as a positive control independent of pathological findings. A, E, I; HE staining of consecutive sections. Pathological findings of the placenta were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142609#pone.0142609.t001" target="_blank">Table 1</a>. The white arrows indicate stem villi. The areas surrounded by white dotted lines correspond to stem villi, in which the specific distribution of <i>m/z</i> 725.5 was observed in (J), but not in (B) or (F). The outside areas of white dotted circles are mostly terminal villi, in which the specific distribution of <i>m/z</i> 804.5 was observed in (G) and (K), but not in (C). +; Preservation of the specific distribution of <i>m/z</i> 734.5 in stem villi (J) or <i>m/z</i> 804.5 in terminal villi (G, K). Red squares also indicate the preservation of the specific distribution of <i>m/z</i> 734.5 in stem villi (J) or <i>m/z</i> 804.5 in terminal villi (G, K). −; Absence of the specific distribution of <i>m/z</i> 734.5 in stem villi (B, F) or <i>m/z</i> 804.5 in terminal villi (C).</p

Pathological findings in placentas enrolled.

<p>VUE; villitis of unknown etiology. CAM; Chorioamnionitis.</p><p>Pathological findings in placentas enrolled.</p

Gas Chromatography-mass spectrometry (GC-MS) analyses show (A) the FAs per testicular weight (mean ± SD; n = 5), and (B) the ratios between each FA per total FAs in the testes of the three developmental male morphotypes.

<p>(A) the FAs which were detected in the testes of the three morphotypes consisted of 14:0, 15:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. In term of relative quantities it was shown that the OC testes contained highest amounts of FAs 16:0, 18:0, 16:1, 18:1, 18:2, 20:1, and contained higher amounts of 14:0, 15:0, and 20:2 when compared with BC while the differences were not significant when compared to SM. Moreover, testes of SM and OC contained higher amounts of FAs 17:0, and 20:5 (EPA) when compared to BC, whereas FAs 20:4 (ARA) and 22:6 (DHA) showed no statistical difference among the testes of the three groups. (B) FA ratios showed that the testes of SM contained higher accumulations of 17:0, 20:1, 20:2, 20:5 (EPA) and 22:6 (DHA) when compared with OC while there were no significant differences between SM and BC. 20:4 = ARA, Arachidonic acid; 20:5 = EPA, Eicosapentaenoic acid; 22:6 = DHA, Docosahexaenoic acid; SM = small male; OC = orange claw male; BC = blue claw male; * = significant difference at P<0.05.</p

IMS showing the intensity and distribution of PCs in the STs of OC males at high magnifications.

<p>Micrographs from H&E-stained sections (a-c) show areas surrounded by white dashed lines corresponding to the same areas that display ion images (d-o). Groups A and B STs containing mostly developing cells, and intertubular area (IT) (d-e, g-h, j-k, m-n), show higher levels of the signal intensity compared with group C STs, which contains only mature spermatozoa (Sz) (surrounded by orange dashed circle) (f, i, l, o). The areas containing Sz have very low signal intensities in all groups. Sc = spermatocytes; St = spermatids; Scale bars = 200 μm; Relative intensity bar shows the intensity level of the ion images.</p

MS/MS analysis showing product ions from the precursor ions at (A) <i>m/z</i> 780.5 and (B) <i>m/z</i> 798.5.

<p>The product ions from the precursor ion at <i>m/z</i> 780.5 represent neutral losses of a PC head group [(CH₃)₃N(CH₂)₂PO₄H] at <i>m/z</i> 597.5 and trimethylamine [(CH₃)₃N] at <i>m/z</i> 721.5. The minor peaks at <i>m/z</i> 465.3 and 441.3 correspond to neutral losses of FAs (16:0 and 18:2) from a peak at <i>m/z</i> 721.5. Therefore, the molecule was assigned as [PC (16:0/18:2) + Na]⁺. The product ions from the precursor ion at <i>m/z</i> 798.5 represent neutral losses of the PC head group [(CH₂)₂PO₄H] at <i>m/z</i> 615.5 and trimethylamine [(CH₃)₃N] at <i>m/z</i> 739.5. The minor peaks at <i>m/z</i> 483.5 and 457.5 correspond to neutral losses of FAs (16:0 and 18:1) from a peak at <i>m/z</i> 739.5. Therefore, the molecule was assigned as [PC (16:0/18:1) + K]⁺.</p

Separation and identification of phosphatidylcholines (PCs) by thin-layer chromatography (TLC) showing bands duplicated bands (upper panels) and histograms of the intensity of PCs (lower panel) in (A) each group of seminiferous tubules (ST), and (B) testes of the three developmental male morphotypes.

<p>Group B STs, containing mostly spermatids and some immature spermatozoa, show significant differences of PCs intensities compared with groups A and C (<i>P</i><0.05; means ± S.D.; n = 5). The data support the IMS results of the PC (16:0/18:1). Moreover, the testes of OC males also contain significantly more PCs than those of SM and BC males (<i>P</i><0.05). SM = small male; OC = orange claw male; BC = blue claw male. Bar = S.D.; * = significant difference at <i>P</i><0.05.</p

HE-stained section of case 1.

<p>(A) Thyroid papillary cancer tissue was localized on the left, and normal thyroid tissue was localized on the right (original magnification 40×). The stromal region was excluded. The ROI was determined from the corresponding HE-staining results. The black boxes indicate the representative region of cancer and normal thyroid tissue. (B) Magnified representative regions of cancer and normal tissue (original magnification 200×). The cancer cells had a high cytoplasmic ratio and displayed nuclear features characteristic of papillary thyroid cancer. Histologic findings of thyroid papillary cancer consisted of columnar thyroidal epithelium set in papillary projection. The normal thyroid tissue is composed of many spherical hollow sacs called thyroid follicles.</p