Placenta
Volume 31, Issue 3 , Pages 245-248, March 2010

Comparison of Phospholipid Molecular Species between Terminal and Stem Villi of Human Term Placenta by Imaging Mass Spectrometry

  • Y. Kobayashi

      Affiliations

    • Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan
    • First two authors equally contributed to the study.
    • ,
  • T. Hayasaka

      Affiliations

    • Department of Molecular Anatomy, Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan
    • First two authors equally contributed to the study.
    • ,
  • M. Setou

      Affiliations

    • Department of Molecular Anatomy, Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan
    • ,
  • H. Itoh

      Affiliations

    • Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan
    • Corresponding Author InformationCorresponding author. Tel.: +81 53 435 2309; fax: +81 53 435 2308.
    • ,
  • N. Kanayama

      Affiliations

    • Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan

Accepted 29 December 2009. published online 29 January 2010.

Article Outline

Abstract 

Placental villi play pivotal roles in the feto-maternal transportation and phospholipids constitute major part of villous membrane. However, the functional contributions as well as pathological roles of placental phospholipids are yet to be fully clarified, because tissue distribution of phospholipids in the placental villi has not been identified. Recently, 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 identification with highly sensitive mass spectrometry from mixtures of ions generated on tissue surfaces. In the present study, we applied this technology to the molecular identification of phospholipids in the human term placenta and found that sphingomyelin (d18:1/16:0) and phosphatidylcholine (16:0/20:4) were distributed differently between stem and terminal villi. This methodology detected a distinct tissue distribution of phosphatidylcholine (16:0/20:4) of terminal villi, coupling with arachidonic acid (AA), which might be a clue leading to the future investigation of the possible involvement the synthetic cascade of eicosanoids in the physiology as well as pathological development of terminal villi, such as fetal growth restriction and/or fetal hypoxia, since terminal villi plays the central roles for nutrient and oxygen supply from maternal to fetal circulation.

Keywords: Pregnancy, Imaging mass spectrometry, Fetal growth restriction, Terminal villi, Eicosanoids

 

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1. Introduction 

The placenta is the largest organ of fetal origin and pivotal not only for the supply of nutrients enabling proper fetal development but also for both maternal and fetal functions from implantation to delivery, expressing various kinds of bioactive substances, such as leptin, resistin, [1] and olfactory receptors [2]. Phospholipids account for three-fourths of all lipid and 9% of the dry weight of the human placenta [3], being mostly composed of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin [4]. Phospholipids not only constitute the membranous structure of placental cells, but also contribute to specific functions in the placenta. Phospholipid complexes in the human placenta function as carriers of amino acids from maternal to fetal blood [5]. We reported that the injection of microvesicles of phospholipids into pregnant mice caused placental coagulopathy and fetal growth restriction [6]. Various steroid-metabolizing enzymes in the placenta have been found to require phospholipids for activity [7], [8]. Nevertheless, the physiological as well as pathophysiological roles of placental phospholipids have not been well clarified, because their tissue distribution has not been established.

Coordinated development of the fetal villous tree of the human placenta is necessary to maintain appropriate growth and the well-being of the fetus, from the stem villi to large numbers of terminal villi exchanging gas and nutrients [9]. However, a debate persists regarding the relative importance of stem and terminal villous pathology to the pathophysiology of the onset of fetal growth restriction [10]. Percy et al. reported significant differences in the composition of phosphatidylcholine and acylglycerol between the placentas from growth-restricted and normally grown newborns [11]. The development of mass spectrometry (MS) technology has revealed the numerous patterns of structural modifications of phospholipids after initial synthesis [12]. To our knowledge, there has been little information regarding MS analysis of the phospholipids in the human placenta. Therefore, biological significance of structural modifications of phospholipids after initial synthesis has not been well understood. Recent development of imaging mass spectrometry (IMS) technology has enabled the two-dimensional assessment of structural modifications of phospholipids by coupling mass spectra with spatial information simultaneously recorded to obtain expression patterns of various molecules in specimens to be analyzed [12], [13], [14]. In the present study, we speculated that the information of the tissue distribution of phospholipids in the structure of villi by IMS would give us a methodological clue to investigate the biological roles of structural modifications of phospholipids after initial synthesis in the placenta. The reason is because the villous membrane is closely associated with various placental functions, such as gas exchange, nutrient transport, etc., critical to the survive as well as rapid development of the fetus and also because phospholipids are among main components of the villous membrane and plays vital roles in the maintenance of membranous integrity and/or in the modulation of the synthetic cascade of various eicosanoids as donors of arachidonic acid [3], [4].

In IMS, the coupling of a time-of-flight (TOF) mass analyzer with either a matrix-assisted laser desorption ionization (MALDI) or secondary ion mass spectrometry (SIMS) ion source has created a new generation of mass spectrometers suitable for the direct analysis of lipids in mammalian tissues [15], [16]. Further introduction of an electrospray ionization source and intermediate-pressure MALDI combined with a linear ion trap imaging system has improved this technology. MALDI-based IMS can provide clear two-dimensional molecular identification with highly sensitive mass spectrometry from a mixture of ions generated on the tissue surface [17]. Recently, we applied this technology to an analysis [18] of synapse-localized E3 ubiquitin ligase-knockout mice [19].

In this paper, results of the imaging and molecular identification of phospholipids differently distributed in stem and terminal villi of the human placenta at term are presented.

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2. Methods 

2.1. Preparation of placental tissue block 

Placental tissue blocks were obtained from three Japanese pregnant women at uncomplicated elective caesarean section due to histories of cesarean delivery during 37 weeks of gestation at Hamamatsu University Hospital, informed consents after a full explanation of the study. Immediately after delivery, the five tissue blocks of approximately 1 cubic centimeter were obtained from each placenta at least 2 cm inside from placental margin, excluding the insertion of umbilical cord. After removing amnion and decidual tissues bluntly, they were snap frozen in liquid nitrogen. The blocks were stored separately at −80 °C in polystyrene tubes until the assessment.

2.2. Analysis of lipid character in the placental extracts 

Thin-layer chromatography (TLC) as well as MS analysis was carried out by using lipid extract from the placental tissues to confirm the major components of placental phospholipids, as pre-assessments of IMS analysis (data not shown).

2.3. Preparation of samples for lipid analysis 

After 30 min keeping at −20 °C a placental tissue block was mounted onto the specimen disc of a cryostat (CM1950; Leica Microsystems, Wetzler, Germany) using optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA). The OCT compound was not used for embedding the entire tissue, because residual polymer on the sections might have affected the mass spectra. The tissue sections were sliced to a thickness of 5 μm using a cryostat and mounted onto indium tin oxide (ITO)-coated glass slides (Bruker Daltonics, Bremen, Germany). The experiments were carried out repeatedly by using the tissues obtained from three different pregnant women and representative data were reported.

2.4. Matrix application 

A thin matrix layer was applied to the surface using an airbrush with a 0.2 mm nozzle (Procon Boy FWA Platinum, Mr. Hobby, Tokyo, Japan) [20]. A total of 500 μL of 2,5-dihydroxybenzoic acid (2,5-DHB; Bruker Daltonics) solution (50 mg/mL in 70% methanol/0.1% trifluoroacetic acid) was sprayed. During spraying, the distance between the nozzle and the tissue surface was kept at 15 cm.

2.5. Mass spectrometry (MS) 

All imaging results shown in this paper were acquired using a MALDI-TOF/TOF type mass spectrometer (ultraflex II; Bruker Daltonics), equipped with a 355 nm Nd:YAG laser [18]. To install the ITO-coated glass slides in the ionization chamber, we used a special holder (MTP Slide Adapter II; Bruker Daltonics) having concavities. All data were acquired in the positive-ion mode using an external calibration method. The external calibration peptides, human angiotensin II ([M + H]+, m/z 1046.54; Sigma Aldrich, St. Louis, MO) and human bradykinin fragment 1–7 ([M + H]+, m/z 757.40; Sigma Aldrich), were deposited on the ITO-coated slides to minimize mass shift. In the imaging experiment, a total of 200 laser shots per point (a total of 6550 points) were irradiated (1 s/point) and the interval between data points was 50 μm. These mass spectra were acquired from each spot with flexControl 3.0 (Bruker Daltonics) (Fig. 1A).

  • View full-size image.
  • Fig. 1 

    Photos of a section with a white square indicating the area imaged (B), hematoxylin and eosin staining of a nearby section (C) and the averaged mass spectrum from the area (A). Imaging results for m/z 725.6 (D) and m/z 804.7 (E). These peaks were analyzed by direct MS/MS and identified as fragment ions of sphingomyelin (d18:1/16:0) (F) and phosphatidylcholine (16:0/20:4), respectively (G).

2.6. Image reconstruction 

The absolute intensity of mass spectra should be processed using an adequate normalization, because the ionization efficiency of an analyte on tissue sections could vary depending on the matrix-analyte co-crystallization conditions [21]. Ion images were reconstructed from the spectra with flexImaging 2.1 (Bruker Daltonics). The two-dimensional image thus obtained (Fig. 1D and E) was compared to the hematoxylin and eosin (HE) staining of a nearby section (Fig. 1C).

2.7. Identification of phospholipid molecular species 

Several ions that showed high intensities on the mass spectrum averaged by raster scanning were identified as phospholipid molecular species by MS/MS analyses. Serial sections were used for the MS/MS analyses. A MALDI-hybrid quadrupole TOF type mass spectrometer (QSTAR XL; Applied Biosystems/MDS Sciex, Foster City, CA) was also used in the MS/MS analyses. The collisional activation of selected ions was determined using a relative collision energy of 20–35 V with argon as the collision gas. In the product ion spectrum, when the product ions corresponded to the specific neutral loss of trimethylamine [N(CH3)3: 59 dalton (Da)] and/or the cyclophosphane ring (C2H5O4P: 124 Da) from [14], [22], the m/z value was transferred to the Metabolite MS Search (http://www.hmdb.ca/).

2.8. Approval 

The Ethics Committee of the Hamamatsu University School of Medicine approved all the procedures.

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3. Results and discussion 

3.1. Mass spectrum obtained from the placental section 

The time required to complete a raster scanning at intervals of 50 μm in 15 mm2 was less than 3 h. The averaged mass spectrum from a raster scanning was shown in the range of m/z 700–900, in which many ions representing phospholipids were detected (Fig. 1A). Several ions chosen based on their signal intensity in the spectrum were used as precursor ions to be analyzed with MS/MS. In this study, the two ions m/z 725.6 and m/z 804.7 colored in red are discussed.

3.2. Imaging and identification of phospholipids 

We visualized small molecules from sections of human term placentas. Photographs of the human placental section with a white square indicating the area imaged (Fig. 1B) and HE staining of a nearby section (Fig. 1C) are presented. As shown in Fig. 1C, a stem villus, indicated by an arrowhead, was surrounded by numerous terminal villi in the scanned area. The stem villus was identified by the relative size of its area. We decided to analyze two peaks, at m/z 725.6 and m/z 804.7, although the raster scanning detected a large number of peaks (Fig. 1A). The reason is because m/z 725.6 was detected in a stem villus, but not in terminal villi (Fig. 1D), while m/z 804.7 was observed in terminal villi, but not in the stem villus (Fig. 1E).

We performed MS/MS analyses for these peaks and successfully identified the molecular species. Fig. 1F shows the product ion mass spectrum obtained from the precursor ion of m/z 725.6. Three product ions, such as m/z 147,0, m/z 542.5, and m/z 666.5, were detected. The peaks at m/z 542.5 and m/z 666.5 corresponds to the neutral losses (NLs) of 183 Da (headgroup [N(CH3)3C2H5O4P]) and 59 Da (trimethylamine [N(CH3)3]) for the precursor ion at m/z 725.6 which often are detected by MS/MS analyses of sphingomyelin or phosphatidylcholine. The odd number of precursor ion indicated that the molecular species is sphingomyelin. Moreover the peak at m/z 147.0 corresponds to cyclophosphane ring (C2H5O4P) adduct sodium ion. Therefore, we identified the molecular species as a sodium-ion-adducted sphingomyelin (d18:1/16:0) [23]. Sphingomyelin (d18:1/16:0) is distributed in stem villi, but not in terminal villi. Fig. 1G shows the product ion spectrum obtained from the precursor ion of m/z 804.7. The peaks, such as m/z 621.5 and m/z 745.5, and the even number of precursor ion indicated that the molecular species is phosphatidylcholine. The NL of 22 from m/z 621.5 to m/z 599.6 proved that the phosphatidylcholine is adducted with a sodium ion. Therefore, we identified the molecular species at a sodium-ion-adducted phosphatidylcholine (36:4) [14]. Moreover, m/z 313.3, m/z 441.3 and m/z 489.3 correspond to the product ion of [lyso-phosphatidylcholine (16:0) – 183 + H]+ and the neutral losses of (20:4 + trimethylamine) and (16:0 + trimethylamine) from each precursor ion, representatively. Therefore, we identified the peak at m/z 804.7 as [phosphatidylcholine (16:0/20:4) + Na]+. Phosphatidylcholine (16:0/20:4) is present in terminal villi, but not stem villi.

In the term human placenta, terminal villi, especially trophoblast cells, are the main compartment of gas and nutrient transports as well as secretion of placental hormones, while terminal villi play an important role in supplying fetal blood to the numerous terminal villi. However, it is yet to be clarified how phospholipids are involved in the functional differentiation of term and stem villi. In the present study, we revealed the distinct tissue distribution of sphingomyelin (d18:1/16:0) and phosphatidylcholine (16:0/20:4) between stem and terminal villi.

It is noteworthy that phosphatidylcholine (16:0/20:4), coupling with arachidonic acid (AA), was distinctively distributed in terminal villi, because AA is closely associated with synthetic cascades of eicosanoids. Fetal growth restriction in one of serious problems of perinatal/neonatal medicine and morphological as well as functional disorders of terminal villi is regarded as one of critical causative factors, because terminal villi plays the central roles in the nutrient and oxygen transport from maternal to fetal circulation. Abnormal dilatation of the micro-vessels in the terminal villi, chorangiosis, was often reported in growth-restricted fetuses [24] and we recently reported a significant change of placental oxygenation in cases of chorangiosis [25]. The distinct distribution of AA coupling phosphatidylcholine (20:4) in the terminal villi suggests that in the near future placental IMS could clarify the possible involvement of AA associated synthesis of eicosanoids, such as vasodilative prostacyclin and/or vasoconstrictive thromboxane in the development of abnormal vessels in the placenta of fetal growth restriction, leading us to the better understanding of etiology of the abnormal fetal growth.

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4. Summary 

Two-dimensional imaging and molecular identification from tissue surfaces using MALDI-TOF/TOF and MALDI-hybrid quadrupole TOF type mass spectrometers were applied to an analysis of the distribution of phospholipids in the human placenta. This methodology may provide a new clue for the investigation of physiological and pathophysiological changes in villous development.

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PII: S0143-4004(10)00004-4

doi:10.1016/j.placenta.2009.12.026

Placenta
Volume 31, Issue 3 , Pages 245-248, March 2010

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