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Journal of the Anatomical Society of India

Histochemical Analysis of Placental Alkaline Phosphatase in Hypertensive Disorders complicating Pregnancy

Author(s): Mangal A, Shrivastava P, Gaur U, Jain A, Goyal U, Rath G

Vol. 54, No. 2 (2005-07 - 2005-12)

Mangal A*, Shrivastava [email protected], Gaur U*, Jain A**, Goyal U#, Rath [email protected]
Department of Anatomy* @, Biochemistry** and, Obstetrics and Gynecology#.
Lady Hardinge Medical College and Associated Hospitals* # **, and
Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi.

Abstract: Placental alkaline phosphatase ( PALP) has been reported to be secreted in second and third trimester of pregnancy by placental syncytiotrophoblasts. In this study, attempt has been made to find the difference in localization or intensity of PALP in placenta of normotensive and hypertensive mothers. Forty placentae were collected from normotensive and hypertensive mothers. Fresh tissues from maternal side of placenta were collected and 4 micron thick cryostat sections were cut. The tissues were treated with Naphthol AS-BI phosphate (substrate) followed by Fast red TR for coupling. Sections were mounted on slide with help of glycerine jelly. PALP activity was noticed only in syncytiotrophoblastic membrane in all control and experimental cases. Cytotrophoblast, stroma and capillaries in placental villi did not show any PALP activity and presented a bleached appearance. In all experimental groups, dense localization of PALP was present in the syncytiotrophoblastic membrane as compared to control ones. At higher magnification, at some places PALP was also localized in outer and inner cell membrane of syncytiotrophoblast. This bilaminar arrangement was discernible in the experimental group. The intensity of localization was proportional to the rise in maternal blood pressure. This high intensity of PALP in syncytiotrophoblast of placenta of hypertensive mothers may be due to increased synthesis of this enzyme by syncytiotrophoblast. This could be a compensatory mechanism adapted by hypertensive placentae, to combat the hazardous effect on developing fetuses of these ailing mothers as the fetal outcome is not very distressing.

Key Words: Placenta, Hypertension, Placental Alkaline Phosphatase (PALP).

Introduction:

Human Placental Alkaline Phosphatase (PALP) is synthesized in placenta during pregnancy and is also expressed in many cancer patients. It has a growth factor – like effect on human fetus and mouse embryo fibroblast, She et al (2000). PALP also has a role in cell division in normal and transformed cells, Telfer and Green (1993), Dabare et al (2000). This enzyme is responsible for active transport of phosphate, Lobel et al (1962), transfer of maternal IgG to fetus, Makiya and Stigbrand (1992) and Stefaner et al (1997), absorption of nutrients, Fishman and Ghosh (1967) and phosphorylation and dephosphorylation of other substances, Neri and Korngold (1970), destined for the growth and development of the fetus. Placenta synthesizes PALP during the second and third trimester of pregnancy, which steadily increases in concentration till term. In pregnant women, decreased serum level of PALP may be associated with intra-uterine growth retardation, Rodin et al (1989), premature rupture of membrane, premature labour, Holmgren et al (1979). The normal pattern of increase in PALP activity in placenta is not seen in cases of fetus dying in-utero during the last trimester, Sussman and Bowman (1968). These observations suggest that PALP may play some role in the development of fetus. According to Jeacock et al (1963) and Mckay et al (1958), phosphatase activity in the placenta usually increases during pregnancy. In case of pre-eclampsia, PALP activity has been appreciably increased, Dempsey and Wislocki (1945) whereas according to Jones and Fox (1976), placenta from pre-eclamptic women showed diminished activity of PALP. Therefore an attempt has been made to localise PALP in placenta of mothers with hypertensive disorders immediately after delivery to find out its correlation with growth and development of fetus.

Materials and Methods

Hypertension in pregnancy has been classified into Gestational hypertension, Pre-eclampsia, Eclampsia, Pre-eclampsia superimposed on chronic hypertension and Chronic hypertension, Cunningham et al (2001) (Table I).

Following the above classification, we collected placenta from forty pregnant mothers between 20-25 years of age. Out of which 15 placentae were procured from cases of gestational hypertension, 6 from preeclampsia, 5 from superimposed pre-eclampsia on chronic hypertension, 4 from chronic hypertension and 10 from normotensive uncomplicated pregnancies, which served as control. During the course of this study, we did not get any case of eclampsia. Placenta was collected immediately after delivery from the Department of Obstetrics and Gynaecology, Lady Hardinge Medical College and S K Hospital, New Delhi. Most of the cases from experimental group were delivered by cesarean section, during 36 to 38 weeks of gestation. 0.5 mm thick fresh tissue from the maternal side of placenta was taken and 4 µ thick cryostat sections were cut (Microm International GmbH cryostat HM505). Sections were collected on gelatin coated slides, and then incubated for 7 minutes at room temperature in the incubating solution using Naphthol AS B1 phosphate and Fast Red TR (pH 8) Bancroft et al (1996). After incubation at room temperature, tissues were washed with double distilled water and counterstained in 2% methyl green. These sections were washed again in running tap water, and mounted in glycerine jelly. Then they were examined and photographed with the help of Nikon E 400 light microscope.

Table I – Classification of Hypertensive Disorders complicating pregnancy

Classification of Hypertension Signs and Symptoms
Gestational Hypertension a) B P ³ 140/90mm Hg for first time during pregnancy
  b) No Proteinuriac) BP returns to normal < 12 weeks post partum
Pre-eclampsia a) B P ³ 140/90 mm Hg after 20 weeks of gestation
  b) Proteinuria ³ 300 mg/ 24 hrs or ³ 1+ dipstick
Eclampsia a) Seizures with Pre-eclampsia (no other cause known)
Superimposed Pre-eclampsia a) New – onset proteinureia ³ 300 mg/ 24 hours in hypertensive woman (on chronic hypertension) after 20 weeks gestation
  b) Sudden increase in proteinurea or B P or platelet count < 100,000/ mm ³ in women with hypertension + proteinurea before 20 weeks gestation
Chronic Hypertension a) B P ³ 140/90 mm hg before 20 weeks gestation or before pregnancy or
  Hypertension first diagnosed after 20 weeks gestation and persistent after 12 weeks post partum

The Apgar score and Weight of newborns were also noted down (Table II).

To supplement our study, serum samples were also collected from these patients during their antenatal check up from 26 weeks of gestation onwards at the interval of 2 weeks till term and PALP was measured by thermal inactivation method described by Fishman et al (1972).

Observation:

The cases were grouped and studied according to the classification of hypertensive disorders in pregnancy.

Control: These normotensive mothers delivered babies with a mean weight of 2.75 kg and mean Apgar score of 9. PALP activity was seen in the syncytiotrophoblast under low magnification which was red in colour due to use of Fast Red TR which is the main component of naphthol AS-B1 method for study of alkaline phosphatase activity. Under high magnification the localization was discernible in outer and inner membrane of syncytiotrophoblast. It was continuous in outer membrane whereas it was interrupted in basal membrane. The stroma, blood vessels and cytotrophoblast did not pick up any stain presenting bleached appearance. As the sections were from fresh tissue the RBCs appeared green in colour due to methyl green, which was used for counterstaining. (Fig 1 and 2)

Gestational hypertension: In this group the mean baby weight of the newborns was 2.49 kg. The mean Apgar score of these newborns was 6.5. Like control, PALP was localized in the outer and inner membrane of syncytiotrophoblast, but the intensity was more in the outer membrane of syncytiotrophoblast in majority of cases. (Fig 3)

Table II Consequences of Hypertension on newborns and Placenta

Study groups No of Cases Blood Pressure in mmHg (Mean) Baby Weight in Kg (Mean) APGAR Score (Mean) Intensity of localization of PALP in Placenta
Normotensive cases 10 120/82 2.75 9 +
GestationalHypertension 15 144/ 94 2.49 6.5 ++
Pre- eclampsia 6 150/100 2.35 6 ++
Chronic Hypertension 4 150/104 2.13 6.5 +++
Superimposed Pre-eclampsiaon chronic hypertension 5 160/106 2.1 kg 5 ++++

Photomicrograph of tertiary villi

Fig. 1: Photomicrograph of tertiary villi showing PALP activity in Syncytiotrophoblast (S) of normotensive placenta . X 40

High power view of syncytiotrophoblast

Fig. 2: High power view of syncytiotrophoblast (S) of tertiary villi of normotensive placenta presenting PALP localization in the outer and inner membrane. Appearance of stroma (St) and capillaries is bleached out and red blood cells (R) is green due to counterstain. X 200.

Photomicrograph showing dense localization of PALP

Fig. 3: Photomicrograph showing dense localization of PALP in the outer and inner membrane of syncytiotrophoblast (S) in gestational hypertension. Stroma (St), red blood cells (R), intervillous space (IVS). X 50

Photomicrograph showing similar dense localization of PALP

Fig. 4: Photomicrograph showing similar dense localization of PALP in the outer and inner membrane of syncytiotrophoblast (S) in preeclampsia . Stroma (St), red blood cells (R), intervillous space (IVS). X 500.

Photomicrograph showing moderately high intensity of localization of PALP

Fig. 5: Photomicrograph showing moderately high intensity of localization of PALP in the outer and inner membrane of syncytiotrophoblast (S) in chronic hypertension. Stroma (St), red blood cells (R), intervillous space (IVS). X 500.

Pre-eclampsia: Mean baby weight in six cases in this group was 2.35 kg. The mean Apgar score was 6. The localization of PALP was similar to control and gestational hypertension presenting bilaminar localization. (Fig 4)

Chronic Hypertension: In this group mean baby weight was 2.13 kg and mean Apgar score was 6.5. PALP was localized in both the outer and inner membrane of the syncytiotrophoblast and the intensity was moderately high. (Fig 5).

Superimposed Pre-eclampsia on chronic hypertension: Only five cases were obtained for study in this group in which the mean baby weight was 2.1 kg and mean Apgar score was 5.The intensity of PALP activity in syncytiotrophoblast was very high in comparison to the above groups. In this group, almost in all cases, PALP was localized in outer and inner membrane of syncytiotrophoblast. In most of these cases the PALP was also spilling over the cytoplasm. (Fig 6)

Photomicrograph showing very high intensity of localization of PALP

Fig. 6: Photomicrograph showing very high intensity of localization of PALP in the outer and inner membrane of syncytiotrophoblast (S) with cytoplasmic spilling in superimposed preeclampsia on chronic hypertension. Stroma (St), red blood cells (R), intervillous space (IVS). X 200.

The intensity of localization was recorded by visual perception only, with the help of light microscope having constant magnification in all the cases. Depending upon intensity, grading was done, keeping control as the baseline.

Serum levels of PALP in different groups of hypertensive disorders complicating pregnancy were significantly raised and were almost proportional to the severity of maternal hypertension (Table III).

Discussion:

subject of many studies because this enzyme plays an important role in transport mechanism. Our study corroborates the reported observation concerning PALP in placenta. We have found that PALP was localized in the outer and inner layers of the syncytiotrophoblast, with the intervening area negatively stained in the placenta of the control (normotensive) group Wielenga and Willighagen (1962) and Harris (1989). PALP precipitates were found to be continuously distributed with no interruption on the brush border of the outer membrane of the syncytiotrophoblast whereas on the inner membrane PALP precipitates appeared interrupted, in the placentae of control group, Hulstaert et al (1973). The fetal stroma, fetal blood vessels and cytotrophoblast showed negative straining. However Wachstein et al (1963) found PALP activity in the cytotrophoblastic cells too.

PALP localization in the placentae of hypertensive pregnancies has been extensively studied. Dempsey and Wislocki (1947) worked on eclamptic and preeclamptic placentae. They found increased phosphatase activity in outer and inner layers of syncytiotrophoblast of pre-eclamptic placentae. And in eclamptic placentae very strong phosphatase activity was observed in the two layers of syncytiotrophoblast.

TABLE III Placental Alkaline Phosphatase (PALP) levels in Serum (IU/l)

  Study groups
Period of Gestation in weeks   26-28 28-30 30-32 32-34 34-36 36-38 38-40
1. Normotensive Cases Mean 87.88 107.56 126.24 151.6 182.68 234.52 268.42
  ± S.D. ±18.13 ±23.67 ±27.61 ±38.37 ±46.32 ±52.61 ±58.58
T test 0.001 0.001 0.001 0.001 0.001 0.001 0.001
2. Gestational Hypertension Mean 158.56 179.76 210.04 226.16 259.36 307.56 302.33
  ± S.D. ±59.76 ±63.64 ±64.72 ±69.01 ±79.89 ±96.32 ±91.2
T test 0.001 0.001 0.001 0.001 0.001 0.001 0.001
3. Pre-eclampsia Mean 156.46 178.46 207.20 224.48 255.14 302.66 301.16
  ± S.D. ±55.21 ±60.16 ±61.22 ±65.21 ±72.84 ±92.64 ±90.32
T test 0.001 0.001 0.001 0.001 0.001 0.001 0.001
4. Chronic Hypertension Mean 168.16 190.20 215.20 234.64 264.42 316.12 320.18
  ± S.D. ±53.33 ±56.24 ±60.61 ±63.45 ±67.16 ±84.32 ±86.71
T test 0.01 0.01 0.01 0.01 0.001 0.001 0.001
5. Superimposed Pre-eclampsia Mean 188.24 210.62 236.34 255.44 274.60 330.50 340.22
  ± S.D. ±54.22 ±58.30 ±62.27 ±64.33 ±69.23 ±86.33 ±88.22
T test 0.01 0.01 0.01 0.01 0.01 0.01 0.01

But, Jones and Fox (1976) found normal PALP activity with normal intensity in the outer and inner layers of syncytiotrophoblast in a few pre-eclamptic placentae and in few other pre-eclamptic placentae diminished intensity of PALP in the syncytium was also observed. Jones and Fox (1976) studied only four pre-eclamptic placentae, in contrast to thirty hypertensive placentae of all varieties (Table I) opted by us for better understanding of PALP localization. Though PALP localization was observed in the outer and inner membrane of syncytiotrophoblast of all the study groups, the degree of intensity was variable. It was noticed that increase of PALP activity is directly proportional to maternal blood pressure. It was noticed that placentae collected from superimposed preeclampsia on chronic hypertension showed maximum intensity of PALP in the outer layer of syncytiotrophoblast in comparison to other groups. This may be due to localization in the microvilli, which is not discernible under light microscope. Staining positivity in microvilli and spilling of PALP were also reported in the cytoplasm of syncytiotrophoblast. The increasing hypertension causing increased PALP activity can be explained by placental ischaemia resulting due to maternal hypertension. Alkaline phosphatase of the placenta appears to be moderately resistant to hypoxia. There is considerable increase in lysosomal activity in hypertensive placentae, presumably a response to placental ischaemia which occurs in pre eclampsia and which by altering the tissue pH of trophoblast, stimulate lysosomal activity. This leads to syncytial damage and destruction in pre eclampsia, which releases PALP from vesicles into cytoplasm, Fox and Agrofojo-Blanco (1974).

The other aspect dealt with in this study is the fetal outcome, measured by mean baby weight and mean Apgar score. The increasing severity of hypertension during antenatal period, do show increase in PALP activity but do not appear to have any correlation with fetal outcome as the baby weight and Apgar score are not much altered. Shane and Suzuki (1974) also found no correlation of serum PALP levels with birth weight.

Many workers have worked on the serum placental alkaline phosphatase showing rising levels in the second and third trimesters of pregnancy, which coincides, with the period of calcification of fetal skeleton. PALP is a membrane-bound glycoprotein, which facilitates the mobilization of calcium from the maternal system for the fetal calcification process Barry (1970), Hunter and Herrett (1973) and Wilde and Dakey (1976). McMaster et al (1964) confirms the placental origin of alkaline phosphatase in maternal circulation. Placental phosphatase first appears in the first trimester of pregnancy and increases during gestation to comprise 40-67% of the total serum phosphatase during the third trimester. After delivery the placental isoenzyme decreases but persists as a minor fraction in the serum initially up to 12 weeks postpartum, Sussman and Bowman (1968). Posen et al (1969) found that alkaline phosphatase in fetal circulation share none of the characteristics of placental alkaline phosphates concluding that PALP enters maternal but not fetal circulation. In toxemic pregnancies,Curzenand Morris (1968) show rising values of PALP in serum. These rising values may be linked to placental damage, Lee and Lewis (1963), Bagga et al (1969) Quisley et al (1970),Kapoor and Mehta (1973). In our study also it is noticed that PALP levels in serum are significantly raised. As there is increase in intensity of localization of PALP in placenta with the increase of maternal blood pressure, similar correlation is noticed between rising serum levels of PALP with increasing hypertension. Thus it is concluded that increased placental localization of PALP reflects as increased levels of PALP in serum or vice versa. And these PALP levels in serum and placenta are affected by increasing maternal blood pressure, during pregnancy. The growth and development of fetus is not significantly affected in our study even in situation of severe placental ischaemia i.e.hypertensive disorders complicating pregnancy.This may be due to increased localization of PALP in placenta which might be helping in the increased transport demand between mother and fetus. But this needs to be further investigated.

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