Sea Level High Altitude
THE EFFECT OF HIGH ALTITUDE ON CELL PROLIFERATION, APOPTOSIS AND FUSION IN THE HUMAN PLACENTA
Camilla E. St. A. Jackson, Tereza Cindrova-‐Davies, Graham J. Burton Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge
1×107
0
Sea Level High Altitude
10
5
0
0.0
There were no significant differences in: • placental weight (p=0.405); • placental volume (p=0.405); • birthweight (p=0.417) or • placental index (p=0.191) at sea level and high al(tude.
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5.0×1010
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Sea Level High Altitude
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Sea Level High Altitude
Total number of STB in placenta
2.0×1010
Altitude
CONCLUSIONS
• High-‐al(tude (3 100m) does not cause a significant difference in the propor(ons of CTB undergoing prolifera(on, apoptosis or fusion in normal term placentae from non-‐na(ve women. • Prolifera(on and apoptosis occur in CTB, not STB. • Fusion of CTB to STB is a rare event in term placentae. • Previous studies have shown a significant decrease in placental weight at high al(tude6. CTB prolifera(on increases during pregnancy but reaches a plateau at term7. Changes in prolifera(on, apoptosis and fusion may occur earlier in gesta(on and may not be obvious at term.
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Graph of the Total number of STB in the placenta at sea level and high altitude
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1.0×108
Graph of the Total number of CTB in the placenta at sea level and high altitude
Altitude
Graph of the ratio of STB to CTB at sea level and high altitude
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Altitude
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Sea Level High Altitude
Altitude
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Total number of CTB in placenta
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Graph showing the proportion of CTB staining positively with a-HERVFRD-1 at sea level and high altitude
Le ve l
0.06
Proportion of +ve CTB nuclei (a-HERVFRD-1)
Altitude
Graph showing the proportion of CTB staining positively with Cleaved Caspase-3 at sea level and high altitude
2.0×108
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H Altitude
Graph of mean numerical density of STB (cm-3) at sea level and high altitude
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Mean Numerical Density STB (cm-3)
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Mean Numerical Density CTB (cm-3)
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3×107
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Graph of mean numerical density of CTB (cm-3) at sea level and high altitude
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Sea Level High Altitude
!
• There were no significant differences in: • mean numerical densi(es of CTB (p=0.873) or STB (p=0.683); • total number of nuclei (CTB p=0.873; STB p=0.524); • STB:CTB (p=0.542) at sea level and high al(tude.
lti
High Altitude
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E
!
Graph showing the proportion of CTB staining positively with PCNA at sea level and high altitude
Le ve l
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Proportion of +ve CTB nuclei (PCNA)
Sea Level
A h ig H
Figure 1: Diagram of unbiased coun(ng frame for ‘physical disector’. Solid lines = forbidden to count, DoUed lines = allowed to count. Cell 1 is not counted because it is on the forbidden line. Cell 2 is not counted since it is present in both. The 3rd cell (green) is counted because it is in the coun(ng frame and only present in the reference sec(on. (Fogarty et al., 20115)
Graph showing the proportion of CTB staining positively with Ki67 at sea level and high altitude
0.5
D
!
• There were no significant differences between the propor(ons of CTB undergoing: • prolifera(on (Ki67 p=0.683; PCNA p=0.683); • apoptosis (CCasp-‐3 p=0.905) or; • fusion (a-‐HERV-‐FRD-‐1 p=0.905) at sea level and high al(tude. • No STB stained posi(vely for Ki67 or Cleaved Caspase-‐3.
Se a
• Ten healthy, term placentae were collected from uncomplicated pregnancies at sea level (London and Cambridge) and high al(tude (Leadville, Colorado; 3 100m) with informed wriUen maternal consent. • Placental samples were preserved in paraffin wax. • The experimenter was blinded to the placental origins throughout un(l decoding for sta(s(cal analysis. • Sequen(al 7µm sec(ons from 10 placentae were stained using immunohistochemistry for markers of: • Prolifera(on (Ki67; PCNA) • Apoptosis (Cleaved Caspase-‐3) • Fusion (a-‐HERV-‐FRD-‐1) • The numbers of CTB and STB nuclei staining posi(vely and nega(vely for each an(body in 100 coun(ng frames (60μm X 60μm X 7μm) were counted for each placental sample manually using the unbiased physical disector technique (see Fig. 1) (4 000 frames in total). • All sta(s(cal analysis was completed using the Mann-‐Whitney U test on GraphPad Prism 6 sodware (GraphPad Sodware Inc., USA).
!
Le ve l
METHODS
C
B
Se a
To inves(gate the effect of high al(tude on trophoblas(c cell: • Prolifera(on • Apoptosis • Fusion for the first (me in normal term human placentae from non-‐na(ve women.
A
Proportion of +ve CTB nuclei (Ki67)
AIM
Figures 2 A – E: Sea level and high al(tude placental samples stained with: 2A Ki67; 2B PCNA; 2C Cleaved Caspase 3; 2D a-‐HERV-‐FRD-‐1; and 2E Nega(ve Control
Proportion of +ve CTB nuclei (CCasp3)
• High al(tude pregnancy is a natural model for the effects of chronic mild hypobaric hypoxia on placental and fetal growth. • Hypoxia is an important cause of complica(ons of pregnancy e.g. intrauterine growth restric(on1. • Human birthweight declines by 102g/1000m ascended independent of other IUGR risk factors2. • Hypoxia increases cytotrophoblast (CTB) prolifera(on and apoptosis and decreases fusion in vitro. However, controversy exists over its effects in vivo 3, 4. • High al(tude causes mild endoplasmic re(culum stress and so might be expected to reduce CTB prolifera(on3. • However, increased CTB numbers have been reported in high al(tude placentae4. This may be explained by increased CTB prolifera(on, decreased apoptosis or decreased fusion. • For the first (me, this study inves(gated the effect of high al(tude on cell prolifera(on, apoptosis and fusion in normal term placentae from non-‐na(ve women living at sea level (London and Cambridge) and high al(tude (Leadville, Colorado; 3 100m).
RESULTS
STB:CTB
INTRODUCTION
Altitude
REFERENCES 1. Zamudio, S. The placenta at high al(tude. High Alt. Med. Biol. 4, 171–191 (2003). 2. Moore, L.G., Brodeur, P., Chumbe, O., D’Brot, J., Hofmeister, S., and Monge, C. (1986). Maternal hypoxic ven(latory response, ven(la(on, and infant birth weight at 4,300 m. J. Appl. Physiol. Bethesda Md 1985 60, 1401–1406. 3. Yung, H. W.,Cox, M.,Tissot van Patot, M. & Burton, G. J. Evidence of endoplasmic re(culum stress and protein synthesis inhibi(on in the placenta of non-‐na(ve women at high al(tude.Faseb J. Off. Publ. Fed. Am. Soc. Exp. Biol. 26,1970–1981 (2012). 4. Ali, K. Z. Stereological study of the effect of al(tude on the trophoblast cell popula(ons of human term placental villi. placenta 18, 447–450 (1997). 5. Fogarty, N.M.E., Mayhew, T.M., Ferguson-‐Smith, A.C., and Burton, G.J. (2011). A quan(ta(ve analysis of transcrip(onally ac(ve syncy(otrophoblast nuclei across human gesta(on. J. Anat. 219, 601–610. 6. Van Patot, M.C.T., Valdez, M., Becky, V., Cindrova-‐Davies, T., Johns, J., Zwerdling, L., Jauniaux, E., and Burton, G.J. (2009). Impact of pregnancy at high al(tude on placental morphology in non-‐na(ve women with and without preeclampsia. Placenta 30, 523–528. 7. Simpson, R.A., Mayhew, T.M., and Barnes, P.R. (1992). From 13 weeks to term, the trophoblast of human placenta grows by the con(nuous recruitment of new prolifera(ve units: a study of nuclear number using the disector. Placenta 13, 501–512.
Camilla E. St. A. Jackson, Tereza Cindrova-‐Davies, Graham J. Burton Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge
1×107
0
Sea Level High Altitude
10
5
0
0.0
There were no significant differences in: • placental weight (p=0.405); • placental volume (p=0.405); • birthweight (p=0.417) or • placental index (p=0.191) at sea level and high al(tude.
ig
h
A
Se a
lti
tu
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Le ve l
tu lti A h
H
H
de tu lti A
A h H Altitude
0.0
h
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de
Le ve l
0.0
5.0×1010
ig
5.0×109
1.0×1011
H
1.0×1010
Sea Level High Altitude
Le ve l
1.5×1010
1.5×1011
Se a
Sea Level High Altitude
Total number of STB in placenta
2.0×1010
Altitude
CONCLUSIONS
• High-‐al(tude (3 100m) does not cause a significant difference in the propor(ons of CTB undergoing prolifera(on, apoptosis or fusion in normal term placentae from non-‐na(ve women. • Prolifera(on and apoptosis occur in CTB, not STB. • Fusion of CTB to STB is a rare event in term placentae. • Previous studies have shown a significant decrease in placental weight at high al(tude6. CTB prolifera(on increases during pregnancy but reaches a plateau at term7. Changes in prolifera(on, apoptosis and fusion may occur earlier in gesta(on and may not be obvious at term.
lti
tu
de
5.0×107
Graph of the Total number of STB in the placenta at sea level and high altitude
ig
ig
15
1.0×108
Graph of the Total number of CTB in the placenta at sea level and high altitude
Altitude
Graph of the ratio of STB to CTB at sea level and high altitude
1.5×108
Altitude
H
Altitude
Sea Level High Altitude
Altitude
Se a
A
A h ig H
Total number of CTB in placenta
-0.02
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Sea Level High Altitude
Graph showing the proportion of CTB staining positively with a-HERVFRD-1 at sea level and high altitude
Le ve l
0.06
Proportion of +ve CTB nuclei (a-HERVFRD-1)
Altitude
Graph showing the proportion of CTB staining positively with Cleaved Caspase-3 at sea level and high altitude
2.0×108
ig
ig H
H Altitude
Graph of mean numerical density of STB (cm-3) at sea level and high altitude
de
Le ve l
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A
Se a
lti ig
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Se a
2×107
Mean Numerical Density STB (cm-3)
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Sea Level High Altitude
Se a
0.0
0.2
Mean Numerical Density CTB (cm-3)
0.1
0.4
3×107
de
0.2
0.6
Graph of mean numerical density of CTB (cm-3) at sea level and high altitude
tu
0.3
Sea Level High Altitude
!
• There were no significant differences in: • mean numerical densi(es of CTB (p=0.873) or STB (p=0.683); • total number of nuclei (CTB p=0.873; STB p=0.524); • STB:CTB (p=0.542) at sea level and high al(tude.
lti
High Altitude
0.8
E
!
Graph showing the proportion of CTB staining positively with PCNA at sea level and high altitude
Le ve l
0.4
Proportion of +ve CTB nuclei (PCNA)
Sea Level
A h ig H
Figure 1: Diagram of unbiased coun(ng frame for ‘physical disector’. Solid lines = forbidden to count, DoUed lines = allowed to count. Cell 1 is not counted because it is on the forbidden line. Cell 2 is not counted since it is present in both. The 3rd cell (green) is counted because it is in the coun(ng frame and only present in the reference sec(on. (Fogarty et al., 20115)
Graph showing the proportion of CTB staining positively with Ki67 at sea level and high altitude
0.5
D
!
• There were no significant differences between the propor(ons of CTB undergoing: • prolifera(on (Ki67 p=0.683; PCNA p=0.683); • apoptosis (CCasp-‐3 p=0.905) or; • fusion (a-‐HERV-‐FRD-‐1 p=0.905) at sea level and high al(tude. • No STB stained posi(vely for Ki67 or Cleaved Caspase-‐3.
Se a
• Ten healthy, term placentae were collected from uncomplicated pregnancies at sea level (London and Cambridge) and high al(tude (Leadville, Colorado; 3 100m) with informed wriUen maternal consent. • Placental samples were preserved in paraffin wax. • The experimenter was blinded to the placental origins throughout un(l decoding for sta(s(cal analysis. • Sequen(al 7µm sec(ons from 10 placentae were stained using immunohistochemistry for markers of: • Prolifera(on (Ki67; PCNA) • Apoptosis (Cleaved Caspase-‐3) • Fusion (a-‐HERV-‐FRD-‐1) • The numbers of CTB and STB nuclei staining posi(vely and nega(vely for each an(body in 100 coun(ng frames (60μm X 60μm X 7μm) were counted for each placental sample manually using the unbiased physical disector technique (see Fig. 1) (4 000 frames in total). • All sta(s(cal analysis was completed using the Mann-‐Whitney U test on GraphPad Prism 6 sodware (GraphPad Sodware Inc., USA).
!
Le ve l
METHODS
C
B
Se a
To inves(gate the effect of high al(tude on trophoblas(c cell: • Prolifera(on • Apoptosis • Fusion for the first (me in normal term human placentae from non-‐na(ve women.
A
Proportion of +ve CTB nuclei (Ki67)
AIM
Figures 2 A – E: Sea level and high al(tude placental samples stained with: 2A Ki67; 2B PCNA; 2C Cleaved Caspase 3; 2D a-‐HERV-‐FRD-‐1; and 2E Nega(ve Control
Proportion of +ve CTB nuclei (CCasp3)
• High al(tude pregnancy is a natural model for the effects of chronic mild hypobaric hypoxia on placental and fetal growth. • Hypoxia is an important cause of complica(ons of pregnancy e.g. intrauterine growth restric(on1. • Human birthweight declines by 102g/1000m ascended independent of other IUGR risk factors2. • Hypoxia increases cytotrophoblast (CTB) prolifera(on and apoptosis and decreases fusion in vitro. However, controversy exists over its effects in vivo 3, 4. • High al(tude causes mild endoplasmic re(culum stress and so might be expected to reduce CTB prolifera(on3. • However, increased CTB numbers have been reported in high al(tude placentae4. This may be explained by increased CTB prolifera(on, decreased apoptosis or decreased fusion. • For the first (me, this study inves(gated the effect of high al(tude on cell prolifera(on, apoptosis and fusion in normal term placentae from non-‐na(ve women living at sea level (London and Cambridge) and high al(tude (Leadville, Colorado; 3 100m).
RESULTS
STB:CTB
INTRODUCTION
Altitude
REFERENCES 1. Zamudio, S. The placenta at high al(tude. High Alt. Med. Biol. 4, 171–191 (2003). 2. Moore, L.G., Brodeur, P., Chumbe, O., D’Brot, J., Hofmeister, S., and Monge, C. (1986). Maternal hypoxic ven(latory response, ven(la(on, and infant birth weight at 4,300 m. J. Appl. Physiol. Bethesda Md 1985 60, 1401–1406. 3. Yung, H. W.,Cox, M.,Tissot van Patot, M. & Burton, G. J. Evidence of endoplasmic re(culum stress and protein synthesis inhibi(on in the placenta of non-‐na(ve women at high al(tude.Faseb J. Off. Publ. Fed. Am. Soc. Exp. Biol. 26,1970–1981 (2012). 4. Ali, K. Z. Stereological study of the effect of al(tude on the trophoblast cell popula(ons of human term placental villi. placenta 18, 447–450 (1997). 5. Fogarty, N.M.E., Mayhew, T.M., Ferguson-‐Smith, A.C., and Burton, G.J. (2011). A quan(ta(ve analysis of transcrip(onally ac(ve syncy(otrophoblast nuclei across human gesta(on. J. Anat. 219, 601–610. 6. Van Patot, M.C.T., Valdez, M., Becky, V., Cindrova-‐Davies, T., Johns, J., Zwerdling, L., Jauniaux, E., and Burton, G.J. (2009). Impact of pregnancy at high al(tude on placental morphology in non-‐na(ve women with and without preeclampsia. Placenta 30, 523–528. 7. Simpson, R.A., Mayhew, T.M., and Barnes, P.R. (1992). From 13 weeks to term, the trophoblast of human placenta grows by the con(nuous recruitment of new prolifera(ve units: a study of nuclear number using the disector. Placenta 13, 501–512.
