Difference between revisions of "Trophoblast"

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'''Chorionic villi''' are part of a normal [[pregnancy]].  They are derived from the conceptus, i.e. NOT maternal.  
'''Trophoblast''' is part of a normal [[pregnancy]].  Trophoblasts are derived from the conceptus, i.e. '''not''' maternal.
Part of the trophoblast forms '''chorionic villi'''.


Tumours arising from the trophoblast are dealt with in ''[[gestational trophoblastic disease]]''.
Tumours arising from the trophoblast are dealt with in ''[[gestational trophoblastic disease]]''.


=Trophoblast table=
Based on Shih ''et al.'':<ref name=pmid11192071/>
{| class="wikitable"
|
| '''Cytotrophoblast'''
| '''Syncytiotrophoblast'''
| '''Villous intermediate<br>trophoblast'''
| '''Implantation site<br>intermediate trophoblast'''
| '''Chorionic-type<br>intermediate trophoblast'''
|-
| Nuclear features
| round, small
| multinucleated
| polyhedral
| pleomorphic +/-multinucleation <br>(occasional), +/-nucleoli
| round / polyhedral
|-
| Cell borders
| well-defined
| poorly defined (?)
| well-defined
| moderately-defined
| poorly defined (?)
|-
| Cytoplasm
| minimal, clear to granular
| abundant, vacuolated, basophilic (???)
| abundant & eosinophilic to clear
| abundant & eosinophilic
| abundant & eosinophilic
|-
| Location
| villi - deep to <br>syncytiotrophoblast
| villi - superficial to <br>cytotrophoblast - <br>nearest to maternal blood
| villi
| implanatation site
| chorion
|-
| Function
| stem cell, produces beta-hCG<ref name=pmid12242037>{{Cite journal  | last1 = Kovalevskaya | first1 = G. | last2 = Genbacev | first2 = O. | last3 = Fisher | first3 = SJ. | last4 = Caceres | first4 = E. | last5 = O'Connor | first5 = JF. | title = Trophoblast origin of hCG isoforms: cytotrophoblasts are the primary source of choriocarcinoma-like hCG. | journal = Mol Cell Endocrinol | volume = 194 | issue = 1-2 | pages = 147-55 | month = Aug | year = 2002 | doi =  | PMID = 12242037 }}</ref>
| produces hormones
| ???
| anchor placenta
| ???
|-
| IHC
| beta-hCG -ve (???), p63 +ve<ref name=pmid19188816>{{Cite journal  | last1 = Zhang | first1 = HJ. | last2 = Xue | first2 = WC. | last3 = Siu | first3 = MK. | last4 = Liao | first4 = XY. | last5 = Ngan | first5 = HY. | last6 = Cheung | first6 = AN. | title = P63 expression in gestational trophoblastic disease: correlation with proliferation and apoptotic dynamics. | journal = Int J Gynecol Pathol | volume = 28 | issue = 2 | pages = 172-8 | month = Mar | year = 2009 | doi = 10.1097/PGP.0b013e318189555b | PMID = 19188816 }}</ref>
| beta-hCG +ve
| p63 +ve,<ref name=pmid19188816/> PLAP +ve
| inhibin +ve, hPL +ve, beta-hCG -ve<ref name=pmid1694585>{{cite journal |author=Yeh IT, O'Connor DM, Kurman RJ |title=Intermediate trophoblast: further immunocytochemical characterization |journal=Mod. Pathol. |volume=3 |issue=3 |pages=282–7 |year=1990 |month=May |pmid=1694585 |doi= |url=}}</ref> CD146+,<ref name=pmid10451481>{{Cite journal  | last1 = Shih | first1 = IM. | title = The role of CD146 (Mel-CAM) in biology and pathology. | journal = J Pathol | volume = 189 | issue = 1 | pages = 4-11 | month = Sep | year = 1999 | doi = 10.1002/(SICI)1096-9896(199909)189:14::AID-PATH3323.0.CO;2-P | PMID = 10451481 }}</ref> p63 -ve
| hPL +ve/-ve, p63 +ve, CD146 -ve
|-
| Image
| [http://pathology2.jhu.edu/trophoblast/images/intro_sm.jpg ST (jhu.edu)]<ref name=jhu>URL: [http://pathology2.jhu.edu/trophoblast/introduction.cfm http://pathology2.jhu.edu/trophoblast/introduction.cfm]. Accessed on: 13 August 2011.</ref>
| [http://pathology2.jhu.edu/trophoblast/images/intro_sm.jpg CT (jhu.edu)]<ref name=jhu>URL: [http://pathology2.jhu.edu/trophoblast/introduction.cfm http://pathology2.jhu.edu/trophoblast/introduction.cfm]. Accessed on: 13 August 2011.</ref>
|
| [http://commons.wikimedia.org/wiki/File:Intermediate_trophoblast_-_high_mag.jpg (WC)]
|
|-
| Associated tumour
| [[Choriocarcinoma]]
| Choriocarcinoma
| ?
| [[Placental site trophoblastic tumour]]
| [[Epithelioid trophoblastic tumour]]
|-
|}
hPL = human placental lactogen.
Notes:
*Keratin is positive in all trophoblastic tissue and negative in decidual tissue.<ref name=pmid1694585/>
=Chorionic villi=
==Chorionic villi 101==
==Chorionic villi 101==
*Maternal blood is around villi.
*Maternal blood is around villi.
Line 8: Line 83:


===Basic histology===
===Basic histology===
*Outer layer of villus: syncytiotrophoblasts.
*Syncytiotrophoblasts: outer layer of villus / closer to mother.
*Beneath syncytiotrophoblast (and closer to fetus) cytotrophoblasts.
*Cytotrophoblasts: inner layer of villus / closer to fetus.
*Hofbauer cells: cells in the stroma of the villi, abundant bubbly cytoplasm - villus macrophages.<ref name=pmid21401637>{{Cite journal  | last1 = Tang | first1 = Z. | last2 = Abrahams | first2 = VM. | last3 = Mor | first3 = G. | last4 = Guller | first4 = S. | title = Placental Hofbauer cells and complications of pregnancy. | journal = Ann N Y Acad Sci | volume = 1221 | issue =  | pages = 103-8 | month = Mar | year = 2011 | doi = 10.1111/j.1749-6632.2010.05932.x | PMID = 21401637 }}</ref>
 
====Images====
<gallery>
Image: Chorionic villi - low mag.jpg | Chorionic villi - low mag. (WC)
Image: Chorionic villi - intermed mag.jpg | Chorionic villi - intermed. mag. (WC)
Image: Chorionic villi - high mag.jpg | Chorionic villi - high mag. (WC)
Image: Chorionic villi - very high mag.jpg | Chorionic villi - very high mag. - image has numerous annotations. (WC)
</gallery>
www:
*[http://courseweb.edteched.uottawa.ca/medicine-histology/english/reproduction/Placenta/Fig08Placenta.htm Placental villi - histology (uottawa.ca)].


==Chorionic villi 102==
==Chorionic villi 102==
*Syncytiotrophoblasts (eosinophilic cytoplasm with vacuoles (contain hCG), multiple hyperchromatic nuclei).
===Syncytiotrophoblasts===
*Multiple hyperchromatic nuclei, eosinophilic cytoplasm with vacuoles (contain hCG).
**Large + many irreg. or lobular hyperchromatic nuclei.  
**Large + many irreg. or lobular hyperchromatic nuclei.  
**Eosinophilic vacuolated cytoplasm (contains hCG).
**Eosinophilic vacuolated cytoplasm (contains hCG).
**Closest to mom - covers cytotrophoblast.<ref>[http://upload.wikimedia.org/wikipedia/commons/4/45/Gray37.png http://upload.wikimedia.org/wikipedia/commons/4/45/Gray37.png]</ref>
**Closest to mom - covers cytotrophoblast.<ref>[http://upload.wikimedia.org/wikipedia/commons/4/45/Gray37.png http://upload.wikimedia.org/wikipedia/commons/4/45/Gray37.png]</ref>
*Cytotrophoblasts (polygonal shape, distinct borders, clear cytoplasm, in cords, single nucleus).
 
Note:
*Syncytiotrophoblasts arise from cytotrophoblasts.<ref name=pmid24595308>{{Cite journal  | last1 = Díaz | first1 = P. | last2 = Wood | first2 = AM. | last3 = Sibley | first3 = CP. | last4 = Greenwood | first4 = SL. | title = Intermediate conductance Ca2+-activated K+ channels modulate human placental trophoblast syncytialization. | journal = PLoS One | volume = 9 | issue = 3 | pages = e90961 | month =  | year = 2014 | doi = 10.1371/journal.pone.0090961 | PMID = 24595308 }}</ref>
 
====Images====
<gallery>
Image: Seminoma with syncytiotrophoblasts -- low mag.jpg | Syncytiotrophoblasts in seminoma - low mag.
Image: Seminoma with syncytiotrophoblasts -- intermed mag.jpg | Syncytiotrophoblasts in seminoma - intermed. mag.
Image: Seminoma with syncytiotrophoblasts -- high mag.jpg | Syncytiotrophoblasts in seminoma - high mag.
Image: Seminoma with syncytiotrophoblasts -- very high mag.jpg | Syncytiotrophoblasts in seminoma - very high mag.
</gallery>
 
===Cytotrophoblasts===
*Polygonal shape, distinct borders, clear cytoplasm, in cords, single nucleus.
**Polygonal shaped cells in cords/masses.
**Polygonal shaped cells in cords/masses.
**Distinct cell borders.
**Distinct cell borders.
Line 25: Line 125:
==Age of villi==
==Age of villi==
===Young villi===
===Young villi===
*Do not have [[blood vessel]]s.
*Few [[blood vessel]]s, central location.
*No syncytial knots.
*Few syncytial knots.


===Old villi===
===Old villi===
*Blood vessels present and at the periphery of villus - contain nucleated (fetal) RBCs.
*Blood vessels present and at the periphery of villi.
*Syncytial knots.
*Syncytial knots.


==See also==
==Villi and age - table==
{| class="wikitable"
| '''Gestational age'''
| '''Nucleated [[RBC]]s'''
| '''Cytotrophoblast / <br>syncytiotrophoblast'''
| '''Villi size'''
| '''Vessels'''
| '''Other'''
|-
| First trimester (<13 weeks<ref name=pmid22712058>{{Cite journal  | last1 = Haggerty | first1 = CL. | last2 = Seifert | first2 = ME. | last3 = Tang | first3 = G. | last4 = Olsen | first4 = J. | last5 = Bass | first5 = DC. | last6 = Karumanchi | first6 = SA. | last7 = Ness | first7 = RB. | title = Second trimester anti-angiogenic proteins and preeclampsia. | journal = Pregnancy Hypertens | volume = 2 | issue = 2 | pages = 158-163 | month = Apr | year = 2012 | doi = 10.1016/j.preghy.2012.01.005 | PMID = 22712058 }}</ref>)
| present
| both can be identified
| large
| few; location central
|
|-
| Second trimester (>=13 weeks and <28 weeks<ref name=pmid22146095>{{Cite journal  | last1 = Grewal | first1 = E. | last2 = Kansara | first2 = S. | last3 = Kachhawa | first3 = G. | last4 = Ammini | first4 = AC. | last5 = Kriplani | first5 = A. | last6 = Aggarwal | first6 = N. | last7 = Gupta | first7 = N. | last8 = Khadgawat | first8 = R. | title = Prediction of gestational diabetes mellitus at 24 to 28 weeks of gestation by using first-trimester insulin sensitivity indices in Asian Indian subjects. | journal = Metabolism | volume = 61 | issue = 5 | pages = 715-20 | month = May | year = 2012 | doi = 10.1016/j.metabol.2011.10.009 | PMID = 22146095 }}</ref>)
| rare
| cytotrophoblast difficult to identify; syncytial knots uncommon
| intermediate
| intermediate; location: central/periphery
|
|-
| Third trimester
| absent
| cytotrophoblast indistinct; syncytial knots common
| small
| abundant; location: periphery of villi
| focal calcifications
|}
Notes:
*The villi size is not very helpful in differentiating second and trimester placentas.
 
=Intermediate trophoblast=
*Abbreviated: ''IT''.
===General===
*Thought to be the cell of origin for:<ref name=pmid11192071>{{cite journal |author=Shih IM, Kurman RJ |title=The pathology of intermediate trophoblastic tumors and tumor-like lesions |journal=Int. J. Gynecol. Pathol. |volume=20 |issue=1 |pages=31–47 |year=2001 |month=January |pmid=11192071 |doi= |url=}}</ref>
**[[Exaggerated placental site]] (EPS).
**[[Placental site nodule]] (PSN).
**[[Placental site trophoblastic tumour]] (PSTT).
**[[Epithelioid trophoblastic tumour]] (ETT).
 
===Subtypes===
Three subtypes of IT are recognized:<ref name=pmid11192071>{{cite journal |author=Shih IM, Kurman RJ |title=The pathology of intermediate trophoblastic tumors and tumor-like lesions |journal=Int. J. Gynecol. Pathol. |volume=20 |issue=1 |pages=31–47 |year=2001 |month=January |pmid=11192071 |doi= |url=}}</ref>
#Implanation site IT.
#Villous IT.
#Chorionic-type IT.
 
===Microscopic===
Features:
*Large cells.
*Eosinophilic cytoplasm.
**May be multi-nucleated.
 
====Images====
<gallery>
Image:Intermediate trophoblast - intermed mag.jpg | IT - intermed. mag. (WC/Nephron)
Image:Intermediate trophoblast 2 - intermed mag.jpg | IT - intermed. mag. (WC/Nephron)
Image:Intermediate_trophoblast_-_high_mag.jpg | IT - high mag. (WC/Nephron)
Image:Intermediate_trophoblast_-_lmwk_-_high_mag.jpg | IT - LMWK - high mag. (WC/Nephron)
</gallery>
www:
*[http://pathology2.jhu.edu/trophoblast/introduction.cfm IT - high mag. (jhu.edu)].
 
====IHC====
Features:<ref name=pmid1694585>{{cite journal |author=Yeh IT, O'Connor DM, Kurman RJ |title=Intermediate trophoblast: further immunocytochemical characterization |journal=Mod. Pathol. |volume=3 |issue=3 |pages=282–7 |year=1990 |month=May |pmid=1694585 |doi= |url=}}</ref>
*hPL (human placental lactogen) +ve.
*beta-hCG -ve.
*Keratin +ve.
**[[CK7]] +ve.<ref name=pmid19069710>{{Cite journal  | last1 = Trenkić | first1 = M. | last2 = Basić | first2 = M. | last3 = Milentijević | first3 = M. | last4 = Petrović | first4 = A. | last5 = Zivković | first5 = V. | last6 = Lazarević | first6 = V. | title = Immunohistohemical evidences of pregnancy in uterine curettage tissue by the use of a double immunocytochemical staining technique using cytokeratin 7 and vimentin antibodies. | journal = Vojnosanit Pregl | volume = 65 | issue = 11 | pages = 810-3 | month = Nov | year = 2008 | doi =  | PMID = 19069710 }}
</ref>
*[[EMA]] +ve in 2nd & 3rd trimester.
*CD146 +ve.<ref>URL: [http://www.epitomics.com/diagnostics/product/5618 http://www.epitomics.com/diagnostics/product/5618]. Accessed on: 14 August 2011.</ref>
 
Notes:
*Cytotrophoblast and syncytiotrophoblast - keratin +ve.<ref name=pmid1694585/>
 
=See also=
*[[Endometrium]].
*[[Endometrium]].
*[[Products of conception]].
*[[Products of conception]].
*[[Ectopic pregnancy]].
*[[Ectopic pregnancy]].
*[[Choriocarcinoma]].
*[[Choriocarcinoma]].
*[[Arias-Stella reaction]] - benign atypical changes associated with chorionic tissue.


==References==
=References=
{{reflist|2}}
{{reflist|2}}


[[Category:Gynecologic pathology]]
[[Category:Gynecologic pathology]]
=External links=
*[http://www.med.yale.edu/obgyn/kliman/placenta/articles/AJP%20Commentary.html Uteroplacental Blood Flow: The Story of Decidualization, Menstruation and Trophoblast Invasion (yale.edu)].

Latest revision as of 05:18, 21 May 2016

Trophoblast is part of a normal pregnancy. Trophoblasts are derived from the conceptus, i.e. not maternal. Part of the trophoblast forms chorionic villi.

Tumours arising from the trophoblast are dealt with in gestational trophoblastic disease.

Trophoblast table

Based on Shih et al.:[1]

Cytotrophoblast Syncytiotrophoblast Villous intermediate
trophoblast 		Implantation site
intermediate trophoblast 		Chorionic-type
intermediate trophoblast
Nuclear features round, small multinucleated polyhedral pleomorphic +/-multinucleation
(occasional), +/-nucleoli 		round / polyhedral
Cell borders well-defined poorly defined (?) well-defined moderately-defined poorly defined (?)
Cytoplasm minimal, clear to granular abundant, vacuolated, basophilic (???) abundant & eosinophilic to clear abundant & eosinophilic abundant & eosinophilic
Location villi - deep to
syncytiotrophoblast 		villi - superficial to
cytotrophoblast -
nearest to maternal blood 		villi implanatation site chorion
Function stem cell, produces beta-hCG[2] produces hormones ??? anchor placenta ???
IHC beta-hCG -ve (???), p63 +ve[3] beta-hCG +ve p63 +ve,[3] PLAP +ve inhibin +ve, hPL +ve, beta-hCG -ve[4] CD146+,[5] p63 -ve hPL +ve/-ve, p63 +ve, CD146 -ve
Image ST (jhu.edu)[6] CT (jhu.edu)[6] (WC)
Associated tumour Choriocarcinoma Choriocarcinoma ? Placental site trophoblastic tumour Epithelioid trophoblastic tumour

hPL = human placental lactogen.

Notes:

  • Keratin is positive in all trophoblastic tissue and negative in decidual tissue.[4]

Chorionic villi

Chorionic villi 101

  • Maternal blood is around villi.
  • Fetal blood (nucleated (fetal) RBCs) in the villi.

Basic histology

  • Syncytiotrophoblasts: outer layer of villus / closer to mother.
  • Cytotrophoblasts: inner layer of villus / closer to fetus.
  • Hofbauer cells: cells in the stroma of the villi, abundant bubbly cytoplasm - villus macrophages.[7]

Images

  • Chorionic villi - low mag. (WC)

  • Chorionic villi - intermed. mag. (WC)

  • Chorionic villi - high mag. (WC)

  • Chorionic villi - very high mag. - image has numerous annotations. (WC)

www:

Chorionic villi 102

Syncytiotrophoblasts

  • Multiple hyperchromatic nuclei, eosinophilic cytoplasm with vacuoles (contain hCG).
    • Large + many irreg. or lobular hyperchromatic nuclei.
    • Eosinophilic vacuolated cytoplasm (contains hCG).
    • Closest to mom - covers cytotrophoblast.[8]

Note:

  • Syncytiotrophoblasts arise from cytotrophoblasts.[9]

Images

  • Syncytiotrophoblasts in seminoma - low mag.

  • Syncytiotrophoblasts in seminoma - intermed. mag.

  • Syncytiotrophoblasts in seminoma - high mag.

  • Syncytiotrophoblasts in seminoma - very high mag.

Cytotrophoblasts

  • Polygonal shape, distinct borders, clear cytoplasm, in cords, single nucleus.
    • Polygonal shaped cells in cords/masses.
    • Distinct cell borders.
    • Covered by syncytiobrophoblast[10] - closer to fetus than syncytiotrophoblasts.
    • Clear cytoplasm.
    • Single uniform nucleus.

Age of villi

Young villi

Old villi

  • Blood vessels present and at the periphery of villi.
  • Syncytial knots.

Villi and age - table

Gestational age Nucleated RBCs Cytotrophoblast /
syncytiotrophoblast 		Villi size Vessels Other
First trimester (<13 weeks[11]) present both can be identified large few; location central
Second trimester (>=13 weeks and <28 weeks[12]) rare cytotrophoblast difficult to identify; syncytial knots uncommon intermediate intermediate; location: central/periphery
Third trimester absent cytotrophoblast indistinct; syncytial knots common small abundant; location: periphery of villi focal calcifications

Notes:

  • The villi size is not very helpful in differentiating second and trimester placentas.

Intermediate trophoblast

  • Abbreviated: IT.

General

Subtypes

Three subtypes of IT are recognized:[1]

  1. Implanation site IT.
  2. Villous IT.
  3. Chorionic-type IT.

Microscopic

Features:

  • Large cells.
  • Eosinophilic cytoplasm.
    • May be multi-nucleated.

Images

  • IT - intermed. mag. (WC/Nephron)

  • IT - intermed. mag. (WC/Nephron)

  • IT - high mag. (WC/Nephron)

  • IT - LMWK - high mag. (WC/Nephron)

www:

IHC

Features:[4]

  • hPL (human placental lactogen) +ve.
  • beta-hCG -ve.
  • Keratin +ve.
  • EMA +ve in 2nd & 3rd trimester.
  • CD146 +ve.[14]

Notes:

  • Cytotrophoblast and syncytiotrophoblast - keratin +ve.[4]

See also

References

  1. ↑ 1.0 1.1 1.2 Shih IM, Kurman RJ (January 2001). "The pathology of intermediate trophoblastic tumors and tumor-like lesions". Int. J. Gynecol. Pathol. 20 (1): 31–47. PMID 11192071.
  2. ↑ Kovalevskaya, G.; Genbacev, O.; Fisher, SJ.; Caceres, E.; O'Connor, JF. (Aug 2002). "Trophoblast origin of hCG isoforms: cytotrophoblasts are the primary source of choriocarcinoma-like hCG.". Mol Cell Endocrinol 194 (1-2): 147-55. PMID 12242037.
  3. ↑ 3.0 3.1 Zhang, HJ.; Xue, WC.; Siu, MK.; Liao, XY.; Ngan, HY.; Cheung, AN. (Mar 2009). "P63 expression in gestational trophoblastic disease: correlation with proliferation and apoptotic dynamics.". Int J Gynecol Pathol 28 (2): 172-8. doi:10.1097/PGP.0b013e318189555b. PMID 19188816.
  4. ↑ 4.0 4.1 4.2 4.3 Yeh IT, O'Connor DM, Kurman RJ (May 1990). "Intermediate trophoblast: further immunocytochemical characterization". Mod. Pathol. 3 (3): 282–7. PMID 1694585.
  5. ↑ Shih, IM. (Sep 1999). "The role of CD146 (Mel-CAM) in biology and pathology.". J Pathol 189 (1): 4-11. doi:10.1002/(SICI)1096-9896(199909)189:14::AID-PATH3323.0.CO;2-P. PMID 10451481.
  6. ↑ 6.0 6.1 URL: http://pathology2.jhu.edu/trophoblast/introduction.cfm. Accessed on: 13 August 2011.
  7. ↑ Tang, Z.; Abrahams, VM.; Mor, G.; Guller, S. (Mar 2011). "Placental Hofbauer cells and complications of pregnancy.". Ann N Y Acad Sci 1221: 103-8. doi:10.1111/j.1749-6632.2010.05932.x. PMID 21401637.
  8. ↑ http://upload.wikimedia.org/wikipedia/commons/4/45/Gray37.png
  9. ↑ Díaz, P.; Wood, AM.; Sibley, CP.; Greenwood, SL. (2014). "Intermediate conductance Ca2+-activated K+ channels modulate human placental trophoblast syncytialization.". PLoS One 9 (3): e90961. doi:10.1371/journal.pone.0090961. PMID 24595308.
  10. ↑ http://upload.wikimedia.org/wikipedia/commons/4/45/Gray37.png
  11. ↑ Haggerty, CL.; Seifert, ME.; Tang, G.; Olsen, J.; Bass, DC.; Karumanchi, SA.; Ness, RB. (Apr 2012). "Second trimester anti-angiogenic proteins and preeclampsia.". Pregnancy Hypertens 2 (2): 158-163. doi:10.1016/j.preghy.2012.01.005. PMID 22712058.
  12. ↑ Grewal, E.; Kansara, S.; Kachhawa, G.; Ammini, AC.; Kriplani, A.; Aggarwal, N.; Gupta, N.; Khadgawat, R. (May 2012). "Prediction of gestational diabetes mellitus at 24 to 28 weeks of gestation by using first-trimester insulin sensitivity indices in Asian Indian subjects.". Metabolism 61 (5): 715-20. doi:10.1016/j.metabol.2011.10.009. PMID 22146095.
  13. ↑ Trenkić, M.; Basić, M.; Milentijević, M.; Petrović, A.; Zivković, V.; Lazarević, V. (Nov 2008). "Immunohistohemical evidences of pregnancy in uterine curettage tissue by the use of a double immunocytochemical staining technique using cytokeratin 7 and vimentin antibodies.". Vojnosanit Pregl 65 (11): 810-3. PMID 19069710.
  14. ↑ URL: http://www.epitomics.com/diagnostics/product/5618. Accessed on: 14 August 2011.

External links

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