Ignite Creation Date:
2024-05-06 @ 9:49 AM
Last Modification Date:
2024-10-26 @ 12:20 PM
Study NCT ID:
NCT03081754
Status:
COMPLETED
Last Update Posted:
2017-03-16
First Post:
2017-03-10
Brief Title:
Correlation of Uterine and Umbilical Arteries Doppler With Placental Pathology in IUGR
Sponsor:
Ahmed Maged
Organization:
Cairo University
Study Overview
Official Title:
Study of Placental Bed of Growth Restricted Fetuses Correlation of Doppler Velocimetries of Uterine and Umbilical Arteries With Placental Pathology
Status:
COMPLETED
Status Verified Date:
2017-03
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
When indicated a conservative management plan of IUGR was undertaken Doppler studies were performed within the last week before delivery The results of Umbilical artery UA Doppler velocimetry were categorized as normal increased absent and reversed Patients were admitted for close surveillance in the case of worsening of maternal or fetal conditions eg absent or reversed UA blood flow and severe preeclampsia
Tissue samples The general shapes of placentas were assessed The collected placentas were weighed by trimming the membranes and umbilical cord Then the diameters and thickness of placentas were noted The position of insertion of umbilical cord on the fetal surface of placenta was observed Transverse cuts were made through the maternal surface at a distance of 1-2 cm in bread loaf manner and examined for the pale areas All placentas were immersed in 10 formalin overnight and examined on the next day For each placenta blocks containing cord membrane and full thickness of villous tissue were prepared Whole thickness villous tissue blocks were obtained from three zones icentral zone ii peripheral zone and iii intermediate zone between the first two zones so as to include all areas of placenta
Placental bed biopsies were obtained at Caesarean sections with direct visualization of the placental site Biopsies of at least 1cm were taken The specimens were fixed in buffered formalin The tissues were processed and stained with Haematoxlyin and Eosin Microscopic study of placenta was carried out utilizing a set of standard criteria for villous and intervillous lesions
Immunohistochemistry Expression of VEGF and CD34 was analyzed in 75 50 placenta of IUGR and 25 of control placental villous tissues
Immunostaining was performed by the streptavidin-biotin-peroxidase method Evaluation of immunohistochemical staining To determine the MVD the stained placental vasculature Tissue sections were initially screened microscopically at low power 100 to identify the areas of highest vascularization hot spots
Evaluation of immunohistochemical staining of VEGF
Tissue samples The general shapes of placentas were assessed The collected placentas were weighed by trimming the membranes and umbilical cord Then the diameters and thickness of placentas were noted The position of insertion of umbilical cord on the fetal surface of placenta was observed Transverse cuts were made through the maternal surface at a distance of 1-2 cm in bread loaf manner and examined for the pale areas All placentas were immersed in 10 formalin overnight and examined on the next day For each placenta blocks containing cord membrane and full thickness of villous tissue were prepared Whole thickness villous tissue blocks were obtained from three zones icentral zone ii peripheral zone and iii intermediate zone between the first two zones so as to include all areas of placenta
Placental bed biopsies were obtained at Caesarean sections with direct visualization of the placental site Biopsies of at least 1cm were taken The specimens were fixed in buffered formalin The tissues were processed and stained with Haematoxlyin and Eosin Microscopic study of placenta was carried out utilizing a set of standard criteria for villous and intervillous lesions
Immunohistochemistry Expression of VEGF and CD34 was analyzed in 75 50 placenta of IUGR and 25 of control placental villous tissues
Immunostaining was performed by the streptavidin-biotin-peroxidase method Evaluation of immunohistochemical staining To determine the MVD the stained placental vasculature Tissue sections were initially screened microscopically at low power 100 to identify the areas of highest vascularization hot spots
Evaluation of immunohistochemical staining of VEGF
Detailed Description:
When indicated a conservative management plan of IUGR was undertaken according to a defined protocol including antenatal visits ultrasound surveillance The frequency of fetal surveillance was assessed at each visit according to the maternal and fetal conditions
Doppler studies were performed within the last week before delivery using a 35-Mhz transducer color-flow mapping and a 50-Hz high-pass filter all measurements were performed with the mothers in a semi recumbent position Color-flow imaging was used to visualize the ascending branch of the uterine arteries Pulsed Doppler velocimetry was performed with a sample volume of 5 mm
A minimum of three separate recordings was taken for each examination The wave contour of the uterine arteries was studied for the presence of a diastolic notch from which the systolicend-diastolic S D ratio was calculated Abnormal uterine velocimetry was defined as an average of left and right S D ratio and by the bilateral presence of diastolic notching Umbilical artery waveform was measured from free-floating loop of cord during fetal quiescence The pulsatility index PI maximum velocity - minimum velocity mean velocity was calculated and the average of three measurements was used An abnormal umbilical artery PI was defined as standard deviations above the mean for gestational age based reference standards Chitty and Altman 1999
The results of Umbilical artery UA Doppler velocimetry were categorized as normal end-diastolic velocity 90th percentile of our reference curve increased end-diastolic velocity 90th percentile absent and reversed Madazil R et al 2002 Patients were admitted for close surveillance in the case of worsening of maternal or fetal conditions eg absent or reversed UA blood flow and severe preeclampsia Preeclampsia was defined according to standard criteria Arduini D et al 2002
Tissue samples The general shapes of placentas were assessed The collected placentas were weighed by trimming the membranes and umbilical cord Then the diameters and thickness of placentas were noted The position of insertion of umbilical cord on the fetal surface of placenta was observed Transverse cuts were made through the maternal surface at a distance of 1-2 cm in bread loaf manner and examined for the pale areas All placentas were immersed in 10 formalin overnight and examined on the next day For each placenta blocks containing cord membrane and full thickness of villous tissue were prepared Whole thickness villous tissue blocks were obtained from three zones icentral zone ii peripheral zone and iii intermediate zone between the first two zones so as to include all areas of placenta
Placental bed biopsies were obtained at Caesarean sections with direct visualization of the placental site Biopsies of at least 1cm were taken The specimens were fixed in buffered formalin The tissues were processed and stained with Haematoxlyin and Eosin Microscopic study of placenta was carried out utilizing a set of standard criteria for villous and intervillous lesions Kotigwar S et al 2011 For studying these criteria 8 random microscopic fields were chosen and 100 villi were counted in each field and studied for the presence of following criteria
1 Syncytial knots 30 in one field
2 Fibrinoid necrosis 5 in one field
3 Placental infarction 5 in one field Intervillous space
a Chorangiosis b Perivillous fibrinoid deposition 5 in one field c Infarctions d Presence of calcification e Thickened hylinosed blood vessels For statistical purpose such changes are labeled as Abnormal placenta
Immunohistochemistry Expression of VEGF and CD34 was analyzed in 75 50 placenta of IUGR and 25 of control placental villous tissues Samples 15 15 1 cm in diameter taken from the maternal surface of each placenta infarct areas were excluded from the study All tissues were fixed in formalin embedded in paraffin and cut into 5-μm-thick sections which were collected on slides coated with poly-L-lysine After the paraffin was removed the sections were rehydrated
Immunostaining was performed by the streptavidin-biotin-peroxidase method Endogenous peroxidase activity was blocked using 3 hydrogen peroxide Antigen retrieval was carried out in a microwave oven for 15 minutes in 10 nM citrate buffer pH 60 for VEGF The sections were incubated at room temperature for one hour with EP1176Y rabbit polyclonal antibodies reactive with VEGF 1100 Genova Spain CD34 mouse monoclonal antibodies Ventana USA After washing in phosphate-buffered saline with Tween-20 the tissues were incubated with a biotin-conjugated secondary antibody and then with a biotin-streptavidin complex for 30 min at room temperature Reactions were visualized with 33-diaminobenzidine tetrahydrochloride DAB Sections were counterstained with hematoxylin rinsed and mounted
Evaluation of immunohistochemical staining
Evaluation of immunohistochemical staining of CD34
Microvessel Density Determination
The most popular method to study the angiogenic activity in a tissue is to count the number of microvessels per unit area of tissue section known as the microvessel density MVD Hasan J et al 2002 To determine the MVD the stained placental vasculature Tissue sections were initially screened microscopically at low power 100 to identify the areas of highest vascularization hot spots Five high-power 400 fields were then chosen randomly and the number of microvessels in each high-power field 009 mm2 was counted for each sample with the use of an ocular grid MVD for each sample was taken as the mean of the five values obtained Mustafa G et al 2012
Evaluation of immunohistochemical staining of VEGF
The intensity and localization of the staining reaction in chorionic villous stromal cells vascular smooth muscle cells villous vascular endothelial cells cytotrophoblasts syncytiotrophoblasts and extravillous trophoblasts was evaluated Immunoreactivity for antibodies was scored using a semi-quantitative scale for intensity of staining 0 negative no staining 1 weak positive 2 moderately positive 3 strongly positive Barut F et al 2010
Doppler studies were performed within the last week before delivery using a 35-Mhz transducer color-flow mapping and a 50-Hz high-pass filter all measurements were performed with the mothers in a semi recumbent position Color-flow imaging was used to visualize the ascending branch of the uterine arteries Pulsed Doppler velocimetry was performed with a sample volume of 5 mm
A minimum of three separate recordings was taken for each examination The wave contour of the uterine arteries was studied for the presence of a diastolic notch from which the systolicend-diastolic S D ratio was calculated Abnormal uterine velocimetry was defined as an average of left and right S D ratio and by the bilateral presence of diastolic notching Umbilical artery waveform was measured from free-floating loop of cord during fetal quiescence The pulsatility index PI maximum velocity - minimum velocity mean velocity was calculated and the average of three measurements was used An abnormal umbilical artery PI was defined as standard deviations above the mean for gestational age based reference standards Chitty and Altman 1999
The results of Umbilical artery UA Doppler velocimetry were categorized as normal end-diastolic velocity 90th percentile of our reference curve increased end-diastolic velocity 90th percentile absent and reversed Madazil R et al 2002 Patients were admitted for close surveillance in the case of worsening of maternal or fetal conditions eg absent or reversed UA blood flow and severe preeclampsia Preeclampsia was defined according to standard criteria Arduini D et al 2002
Tissue samples The general shapes of placentas were assessed The collected placentas were weighed by trimming the membranes and umbilical cord Then the diameters and thickness of placentas were noted The position of insertion of umbilical cord on the fetal surface of placenta was observed Transverse cuts were made through the maternal surface at a distance of 1-2 cm in bread loaf manner and examined for the pale areas All placentas were immersed in 10 formalin overnight and examined on the next day For each placenta blocks containing cord membrane and full thickness of villous tissue were prepared Whole thickness villous tissue blocks were obtained from three zones icentral zone ii peripheral zone and iii intermediate zone between the first two zones so as to include all areas of placenta
Placental bed biopsies were obtained at Caesarean sections with direct visualization of the placental site Biopsies of at least 1cm were taken The specimens were fixed in buffered formalin The tissues were processed and stained with Haematoxlyin and Eosin Microscopic study of placenta was carried out utilizing a set of standard criteria for villous and intervillous lesions Kotigwar S et al 2011 For studying these criteria 8 random microscopic fields were chosen and 100 villi were counted in each field and studied for the presence of following criteria
1 Syncytial knots 30 in one field
2 Fibrinoid necrosis 5 in one field
3 Placental infarction 5 in one field Intervillous space
a Chorangiosis b Perivillous fibrinoid deposition 5 in one field c Infarctions d Presence of calcification e Thickened hylinosed blood vessels For statistical purpose such changes are labeled as Abnormal placenta
Immunohistochemistry Expression of VEGF and CD34 was analyzed in 75 50 placenta of IUGR and 25 of control placental villous tissues Samples 15 15 1 cm in diameter taken from the maternal surface of each placenta infarct areas were excluded from the study All tissues were fixed in formalin embedded in paraffin and cut into 5-μm-thick sections which were collected on slides coated with poly-L-lysine After the paraffin was removed the sections were rehydrated
Immunostaining was performed by the streptavidin-biotin-peroxidase method Endogenous peroxidase activity was blocked using 3 hydrogen peroxide Antigen retrieval was carried out in a microwave oven for 15 minutes in 10 nM citrate buffer pH 60 for VEGF The sections were incubated at room temperature for one hour with EP1176Y rabbit polyclonal antibodies reactive with VEGF 1100 Genova Spain CD34 mouse monoclonal antibodies Ventana USA After washing in phosphate-buffered saline with Tween-20 the tissues were incubated with a biotin-conjugated secondary antibody and then with a biotin-streptavidin complex for 30 min at room temperature Reactions were visualized with 33-diaminobenzidine tetrahydrochloride DAB Sections were counterstained with hematoxylin rinsed and mounted
Evaluation of immunohistochemical staining
Evaluation of immunohistochemical staining of CD34
Microvessel Density Determination
The most popular method to study the angiogenic activity in a tissue is to count the number of microvessels per unit area of tissue section known as the microvessel density MVD Hasan J et al 2002 To determine the MVD the stained placental vasculature Tissue sections were initially screened microscopically at low power 100 to identify the areas of highest vascularization hot spots Five high-power 400 fields were then chosen randomly and the number of microvessels in each high-power field 009 mm2 was counted for each sample with the use of an ocular grid MVD for each sample was taken as the mean of the five values obtained Mustafa G et al 2012
Evaluation of immunohistochemical staining of VEGF
The intensity and localization of the staining reaction in chorionic villous stromal cells vascular smooth muscle cells villous vascular endothelial cells cytotrophoblasts syncytiotrophoblasts and extravillous trophoblasts was evaluated Immunoreactivity for antibodies was scored using a semi-quantitative scale for intensity of staining 0 negative no staining 1 weak positive 2 moderately positive 3 strongly positive Barut F et al 2010
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None