Correlation of Uterine and Umbilical Arteries Doppler With Placental Pathology in IUGR
Condition: Intrauterine Growth Restriction
MeSH terms: Fetal Growth Retardation
Study type: Observational

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
      (e.g. 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, i)central 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 3.5-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
      systolic/end-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
      (e.g. 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, i)central 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 (1.5 × 1.5 × 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 6.0) for VEGF. The sections
      were incubated at room temperature for one hour with EP1176Y rabbit polyclonal antibodies
      reactive with VEGF (1:100; 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 3,3-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 (0.09 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)
    

Intervention name(s): Doppler velocimetry
Intervention type(s): Radiation
Intervention description(s): Doppler studies were performed within the last week before delivery using a 3.5-Mhz transducer, 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.
Observational model: Case-Only
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        1. Group 1 ( control group) :

             Twenty five uneventful pregnancies with appropriate for gestational age fetuses are
             selected as control.

          2. Group 2:

        Fifty women with intrauterine growth restricted fetuses were included in the study.
        Gestational age was based on the precisely dated last menstrual period and
        ultra-sonographic examination of crown-rump length in the first trimester. Caesarean
        sections were performed on clinical grounds
      
Biospecimen description: 
      The general shapes of placentas were assessed and weightedby trimming the membranes and
      umbilical cord. The diameters and thickness of placentas were noted cord insertion site. The
      maternal surface at a distance of 1-2 cm in bread loaf manner and examined for the pale
      areas. Whole thickness villous tissue blocks were obtained from three zones ;central,
      peripheral and intermediate zones

      Placental bed biopsies were obtained at Caesarean sections .. 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 . For studying these criteria 8 random microscopic fields
      were chosen and 100 villi were counted in each field