Histomorphometric study for volume proportion of certain parameters in placenta of pregnancy induced hypertension

Assistant Professor, Department of Anatomy, Calcutta National Medical College, Kolkata, West Bengal, India. Professor, Department of Anatomy. MGIMS, Sewagram, Maharashtra, India Professor & Head, Department of Gynaecology and Obstetrics, MGIMS, Sewagram, Maharashtra, India Assistant Professor, Department of Physiology, Calcutta National Medical College, Kolkata, West Bengal, India. Consultant Anaesthesiology, Medica Super Speciality Hospital, Kolkata, West Bengal, India. Professor & Head, Department of Anatomy, MGIMS, Sewagram, Maharashtra, India


Introduction
Placenta is the organ belonging to the developing mammalian conceptus which is never incorporated in the body of foetus but it is vital to the well-being of the baby in the womb and it offers protective, nutritional, respiratory, synthetic and excretory functions. Thus it exists as a vital link between mother and developing fetus 1 .
Placenta is basically chorionic tissue and trophoblasts are the representative cells. Hence trophoblasts are responsible for a successful outcome of pregnancy-a healthy baby and healthy mother, at the completion of intra-uterine life 2,3,4,5,6 .
The outcome of pregnancy depends on many factors. One such factor is the health and wellbeing of mother throughout pregnancy. Any deviation from normal physiology would adversely affect not only the mother and foetus but the other gestational tissues as well 1and 7 .
Rise of blood pressure during pregnancy (gestational hypertension or pregnancy induced hypertension-'PIH') is one of the most common causes of altered physiological process in pregnancy 6 .
The diagnosis of gestational hypertension is made in women whose blood pressure reaches 140/90 mm Hg or greater for the first time during pregnancy 6 . A number of hypotheses have been proposed to explain the aetiology of PIH. According to Sibai (2003) those are 1. Abnormal trophoblastic invasion of uterine vessels. 2. Immunological intolerance between maternal and foeto-placental tissues. 3. Maternal mal-adaptation to cardiovascular or inflammatory changes of normal pregnancy. 4. Dietary deficiencies. 5. Genetic influences 6 .
There are very few scientific works in support of such hypotheses. Since placenta acts as a mirror to the disease process in mother and fetus or both we decided to study gross and microscopic placental structure in PIH.
IJBR (2014) 05 (03) www.ssjournals.com 2.1 The chief study material was placenta Forty (40) placentae were collected from Institutional labour room immediately after term delivery of mothers having uneventful antenatal period as control. Another forty (40) placentae were collected after term delivery of pregnant mothers who had clinically proven hypertension (pregnancy induced hypertension-PIH).

Inclusion criteria for all subjects:
Age of mother between 20 to 38 years 10

Specific inclusion criterion for subjects enrolled as cases (experimental):
Clinically proven hypertension in antenatal (beyond 20 weeks of gestation) or in intranatal period.
Inclusion criteria for Subjects enrolled as controls (normal pregnancy)  Subjects free from any pre existing clinical disorder.  Uneventful antenatal period

Exclusion criteria for Subjects enrolled as experiment:
Hypertension with other gestational disorders, pre-existing hypertension, subjects having still birth, gross congenital anomalies in baby, complicated vaginal delivery, emergency lower segment caesarean section, invasive procedure during gestation.

Preparation for examination of placenta:
The intact sample of placenta was kept for one week in 10% formal saline for fixation and hardening so as to make it suitable for cutting into 2-3 cm pieces for further routine histological processing followed by section cutting, staining, microscopy and microphotography.

Method of light microscopy (Based on Hamilton and Boyd, 1970)
The stained slides of placentae of two groups were examined under binocular light microscope under different magnifications. Light microscopy was performed under different magnifications (X 100 with low power and X 400 in high power) under self illuminated binocular microscope. Hematoxylin and Eosin (H/E) staining was performed routinely while Masson's trichrome as special stain. In each slide all the serial sections were observed under microscope.

Identification under microscope Areas of microscopic calcification:
Amorphous discrete basophilic matter present in villi of term placenta signifies calcification . The eosinophilic homogeneous amorphous or fine lamellar or fibrillary or hyalinised substance seen in H/E stained sections around the villi (perivillous), within the vilus (intra villous), within the basal plate (Nitabuch's stria, many times referred to as maternal floor infarct), adjoining intervillous space and chorionic plate (Langhans stria) or rarely adjoining intervillous space and basal plate (Rohr's stria). It appears red in Masson's trichrome stained sections (Microphotograph No.2& 3).   Five fields of ten sections of each placenta were examined (50 fields of each placenta) for determination of volume proportion of a given parameter. For each placenta the average value of the desired parameter was obtained from these 50 fields and recorded in Table No. I.

Method for Statistical analysis
All the parameters judged for histo-morphometry were subjected to statistical analysis by comparing the data of case (experimental group) versus control and then the difference was assessed for statistical significance using student' t' test.

Histomorphometry
The sections were studied under light microscope with the help of square and linear graticules. The control placentae were showing the mean volume proportion of calcification 0.645 and the figure in PIH placenta was 4.954. The increased calcification in PIH cases was statistically significant (Table No.II).

Discussion
There was evidence of fibrinoid material in control and experimental placental tissue. So we quantified the fibrinoid by taking the volume proportion of fibrinoid in whole placental tissue in both groups and found a significant increase in experimental group (Table No. I and II  The basic hypothesis that the changes occurring in the villi were compensatory to hypoxic condition induced by PIH was supported by an increase in surface area of the terminal villi. Though the weight, volume, diameter and central thickness of experimental group of placentae was reduced and there was much more increased fibrinoid material in such placentae the trophoblastic cell proliferation and increase of villous diameter compensated these to some extent. This allowed greater exchange of materials from intervillous space to foetal blood vessels of the villi through syncytiotrophoblast (ST). Zacutti (1992) opined that mosaicism of the placenta probably led to placental insufficiency and ultimately to foetal growth retardation, thus creating a vicious cycle 11 .
Our gross finding of calcification in placenta was confirmed by microscopy. The calcified areas were more evident in experimental cases. Calcification could be seen in even those placentae where there was no evidence of calcification in gross examination. We found a statistically significant increase of calcification in experimental group of placentae (Table No. II and Graph 2).
On histochemical study Mangal et al (2005) had proved that in PIH cases the placental Alkaline Phosphatase synthesis was increased by syncytiotrophoblast. Presence of this enzyme is clearly implicated in those areas where active calcium deposition has to occur. Hence our finding of increased calcification supports the result of Mangal et al (2005) 12 .

Conclusions
The present study suggests that the gestational hypertension does cause structural and micro-structural changes in placenta which are negatively affecting the mass and surface area of placenta along with microscopic changes reactionary and compensating the hypoxia.