Modificari structurale ale interfeei materno-fetale

i evoluia postnatal a copilului

Structural changes of the maternal-foetal interface
and postnatal development of the child

Scientific Coordinator: Prof. Anastasiu Doru Mihai, MD, PhD

PhD Candidate: Ilie Rodica, MD

SUMMARY

LIST OF ABBREVIATIONS
INTRODUCTION
CHAPTER I MATERNAL-FETAL INTERFACE, DATA IN THE LITERATURE

1 Implantation embryo-maternal interaction

1 Early stages of embryonic development

2 Maternal-embryonic dialogue

3 Factors needed for proper implantation

2 Immunological considerations on maternal-foetal interface

1.2.1. Implantation immunology and formation of multiple maternal-foetal interfaces
1.2.2. Foetal trophoblast and HLA expression
1.2.3. Trophoblast immune evasion
1.2.4. Immune cell populations in the MFI
1.2.5. Foetus-specific immune regulation by maternal T cells
1.3 Maternal-foetal interface
1.3.1. Maternal-foetal interface structure
1.3.2. Morphology of structures ensuring maternal-foetal exchanges
4 Maternal-foetal interface homeostasis

CHAPTER II FOETAL HYPOXIA, PLACENTAL DISTRESS AND IUGR

1 Hemodynamic changes in foetal hypoxia

2 Chronic foetal hypoxia and oxidative stress
3 IUGR

CHAPTER III PERSONAL STUDIES

CHAPTER IV MATERIAL AND METHOD

4.1. STUDY MATERIAL SELECTION OF CASES

4.2. METHODS OF HISTOLOGICAL PROCESSING OF PLACENTA

4.2.1. Conventional staining methods
4.2.2. Immunohistochemical methods

4.3. DATA PROCESSING METHODS

CHAPTER V RESULTS

5.1. PIH
5.1.1 Placental lesions in PIH by standard histological stainings
5.1.2 Placental lesions in PIH by immunohistochemical staining
 5.1.3 Clinical data regarding the infants born to PIH mothers

5.2 GDM
5.2.1. Placental lesions in GDM by standard histological stainings
5.2.2. Placental lesions in GDM by immunohistochemical staining
5.2.3. Clinical data regarding the infants born to GDM mothers

5.3 PID

CHAPTER VI DISCUSSIONS

6.1. HISTOLOGICAL PLACENTAL LESIONS BY STANDARD STAININGS

6.1.1. Significance of histological lesions in PIH
6.1.2. Significance of histological lesions in GDM

6.2. PLACENTAL LESIONS BY IMMUNOHISTOCHEMICAL STAINING FOR

MONOCLONAL ANTIBODIES
6.2.1. Placental lesions in PIH by immunohistochemical staining
6.2.2. Placental lesions in GDM by immunohistochemical staining

6.3. NEWBORNS CLINICAL DATA

CHAPTER VII CONCLUSIONS

7.1. HISTOLOGICAL STUDY CONCLUSIONS

7.2. EARLY POSTNATAL EVOLUTION CONSEQUENCES OF MATERNAL-FETAL
INTERFACE DISTRESS
7.3. PROPOSALS FOR SYSTEMATIC PLACENTAL INVESTIGATION BY THE
MEANS OF EXAMINATION PROTOCOLS

REFERENCES

LIST OF ABBREVIATIONS

PIH = Pregnancy-induced hypertension

GDM = Gestational diabetes mellitus
MFI = Maternal-foetal interface
IUGR = Intrauterine growth retardation
PID = Placental insufficiency degree
GA = Gestational age
BW = Birth weight
DM = Diabetes mellitus
SG = Study group
CG = Control group
INTRODUCTION

The first organ to develop during pregnancy is the placenta, a temporary organ connecting the
baby to its mother. It controls metabolic and oxygen exchanges between mother and foetus, it
produces growth factors and hormones and it ensures the transfer of nutrients that support foetal
growth. Pregnancy success means to avoid its rejection by the mothers immune system. Foetal
and maternal cells come into direct contact in the decidua, a highly specialised mucous
membrane playing a key role in foetal tolerance and allowing for the development of maternal-
foetal interface (MFI).

Physiological conversion of spiral arteries and subsequent appropriate placental perfusion are the
essential keys for a successful human pregnancy. Defective placentation with diminished
trophoblast invasion and insufficient spiral artery remodelling leads to unfavourable pregnancy
outcome with such consequences as early pregnancy miscarriage, intrauterine growth
retardation (IUGR) and/or PIH. These complications are the predisposing factors that will
greatly impact on the development of diseases in adulthood. Abnormal placental function
consisting in increased vascular resistance, improper nutrient transport and epigenetically
imprinted genes has a major impact on adaptation of foetal programming.

PERSONAL STUDIES

Work hypothesis and study objectives

The research on placenta should be a priority for the entire medical community, as it is a
valuable diary of human been prenatal development. This organ must be systematically studied
at birth, and then preserved for a long period of time in order to extend its anatomopathological
study in case of unfavourable development of the newborn. Research on placenta is currently an
almost ignored theme. Starting from these premises, the personal research aims at achieving the
following objectives:

1. To define two major groups of maternal conditions with major impact on placentation,
placental development and on foetal development, implicitly: PIH and GDM.

2. To set up, based on inclusion and exclusion criteria, two homogenous groups of newborns
by selecting those at highest risk of both disorders.

3. To harvest placentas for morphopathological study, in both pathological conditions.

4. To form two equal groups of newborns, the control groups (CASE-CONTROL study
design), as well as to collected the corresponding placentas, thus also creating the two control
groups for the histological study;
5. To identify and assess in each group the main MFI specific lesions supporting a correlation
with the perinatal pathology.

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6. To validate the hypothesis that placenta is an important witness of pathological events in
the prenatal period, as certain morphological changes in the MFI become specific markers of
disturbances during pregnancy with postnatal pathological consequences. Morphological
research of placenta has been focused on MFI, a very complex exchange structure belonging
both to the mother and the foetus, the injury and distress of which determines the perinatal
pathology and some aspects of adult pathology.

7. To set up a placental insufficiency degree (PID) score to aid in establishing rapid

correlations (necessary to the neonatologist in case of emergency) between MFI complex of
lesions and postnatal pathological events at the time of birth. This research starts from the
evidence in many studies showing that early hypoxia causes multiple maternal-foetal vascular,
immunological and metabolic impairments, ultimately followed by irreversible structural
changes. All these, reunited into a PID score studied immediately after birth, aim to
retrospectively ascertain the degree of newborns hypoxic damage, followed by prevention
and/or correction of clinical changes before it is too late.

STUDY MATERIAL

The present study has been conducted within the Obstetrics-Gynaecology and Neonatology
Clinics of the County Clinical Emergency Hospital Timisoara following approval by their mana-
gers. It has also been approved by the Ethics Committees of Victor Babes University of Medici-
ne and Pharmacy Timisoara and County Clinical Emergency Hospital Timisoara.
SAMPLES COLLECTION

Study fragments samples for PIH and GDM were collected from the placentas immediately after
birth. The samples were divided into two groups 68 placentas of PIH patients and normoten-
sive pregnant women (controls), respectively, and other 30 placentas provided the fragments
from GDM pregnant women and normal pregnancy expecting mothers (controls).

Five sectional fragments were taken for each case from the following different regions: foetal
site , median section, maternal site, amnion and chorion.

The samples were fixed in 10% buffered formalin for 24-48 hours, and then prepared for their
specific processing by the 3 conventional histological staining types and respectively, the 3
monoclonal antibodies types by immunohistochemical methods.

SELECTION OF CASES

For the babies born to mothers with PIH:

Inclusion criteria were the following: Gestational Age (GA) 28 weeks; Birth Weight
(BW) 1000g and <2500 g; mother with PIH, without complications; singleton
pregnancy; favourable neonatal evolution, with hospital discharge; presence of various
degrees of neonatal hypoxic pathology according to SARNAT grading scale.

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 Exclusion criteria were the following: GA < 28 weeks; BW < 1000 g; twin pregnancy;
death during neonatal period; associated severe pathology (infection, congenital
malformations, perinatal asphyxia); mother with PIH with complications.
A study group comprising 34 babies born to mothers with PIH and an equal corresponding
control group were formed (following the CASE-CONTROL study design). These groups were
defined as Study Group (SG) and Control Group (CG).
For the babies born to mothers with GDM:

Inclusion criteria were the following: GA < 37 weeks; mother with uncomplicated GDM
(cases of DM before pregnancy were excluded); singleton pregnancy; favourable
neonatal development, with hospital discharge; presence of various degrees of neonatal
hypoxic pathology according to SARNAT grading scale.

Exclusion criteria were the following: twin pregnancy; death during neonatal period;
mother with complicated DM; severe pathology in the newborn.
The two groups of newborns from mothers with GDM were formed, as follows: SG comprising
15 newborns and CG, respectively, comprising an equal number of newborns.
Diagnosis of hypoxic neonatal distress has been based on clinical data in the SARNAT Grading
Scale, neurologic examination of the newborn and his/her subsequent development. Based on
these we were able to classify the newborns with hypoxic manifestations in mild, moderate and
severe forms. Fifteen parameters were comparatively analysed for both pathologies, measuring
newborns physical and maturity indicators, as well as their adaptation problems and neonatal
hypoxic pathology.

STUDY METHODS

METHODS OF HISTOLOGICAL PROCESSING OF PLACENTAS

Conventional staining methods

The five sections harvested as study samples were processed by Hematoxylin-Eosin, Massons
Trichrome and Van Gieson staining techniques.

Immunohistochemical methods

We have decided to fix the fragments harvested immediately after birth from 15 placentas of PIH
patients and 7 placentas of GDM patients for 48 hours in 10% buffered formalin. Samples were
prepared for immunohistochemistry procedure after deparaffinization and rehydration.

In order to unmask the CK 7 antigen, we have treated the studied tissue to induce the unmasking
epitope, using antigen unmasking solution (pH 6), for CK 5 30 minutes at 99 degrees C and for
vimentine 5 minutes, at 99 degrees C. After endogenous peroxidase blocking step, samples were
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incubated for 30 minutes at room temperature, as follows: CK 5 (monoclonal mouse, XM26 clone,
RTU, Leica Microsystems), CK 7 (monoclonal mouse, RN7 clone, Leica Microsystems), and
Vimentine (V9 clone, RTU, Dako Carpinteria USA). We have continued with NOVOLINK
Polymer System and we have used Diaminobenzidine as chromogen. Overstaining has been
performed using Lillies modified Hematoxylin. No marker antibody has been added so as to get
the negtive control. The positive reaction was assessed for all markers as being moderate at the
membrane level, and intense at cytoplasmic level, while :1 cm scale in the figure represents 50 m
in-situ.

METHODS OF DATA PROCESSING

Histological and immunohistochemical data processing. We have used optical microscopy for
pathological examinations of the tissues samples by the means of an AmScope microscope, using
x10, x20, x40 and x100 objectives, respectively, depending on the structure to be evidenced.
Images were captured with a 1.3 Megapixels High speed digital camera and then processed on a
computer (equipped with Windows 7 operating system) by the means of ACDSee Pro 5
programme.

Statistical processing of newborns clinical data. The data base was built using an Excel file by
sequential data input from each patients documents (medical record). Statistical study of data
has been performed by the aid of SPSS 17.0 programme. Parametric and nonparametric
methods were used for statistical processing.

The results were considered statistically significant when p<0.05, for a 95% confidence
interval, and strongly statistically significant at p<0.01, for a 99% confidence interval.
RESULTS
Placental histological lesions in PIH by standard histological stainings
The primary purpose of histological examinations was to study MFI structure, lesions of
terminal villosities, spiral arterioles of maternal decidua particularly their tunica intima and
media amnion and chorion, aiming to reveal their lesions, as well as other significant
degenerations.

By comparing placentas in the two groups, those harvested from mothers with PIH and those
from the controls, we have obtained the statistical results of histological lesions in the Figure 1,
as follows:

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 Figure 1. Histological lesions of placenta in PIH

The degenerative lesions group has the strongest histological significance, being 4.54.8 times
more frequent as compared with the control group. Among the subgroups of this lesion type
decrease in number and shrinkage of villi (up to ghost villi), thickening of endothelia of
terminal villi capillaries and fibrinoid necrosis in the spiral arterioles, the differences have
basically no statistical significance.

The proliferative lesions group shows a 3.9 higher peak than the control group, through the
subgroup with basal arterioles smooth muscle hypertrophy; fibrosis due to capillary loss and
hyaline stromal villi rarefaction, and syncytial changes by agglutination, respectively, are 2.3
times lower than in the peak group and 2 times higher than in controls, in which they are absent.

In the circulatory lesions group, the endothelial lesions subgroup is 4.5 times higher than the
controls, but the subgroup of thromboses and/or spiral arterioles infarctions is 2.2 times lower,
while the decidual endothelial atheromatosis subgroup is 3.3 times lower than the peak value,
although absent in controls.
Clinical data of babies born to mothers with PIH
The parameters considered in this study were comparatively analysed between SG and CG.
The presented and graphically displayed data show a major impact of PIH on foetal growth and
development associated with substantial alteration of foetal weight, leading to a mean difference
in this parameter of over 1,000 g between the two groups.
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The chronic suffering degree with intrauterine growth restriction, as well as the preterm birth,
which are significantly more frequent in the SG have also influenced other parameters, such as:
neonatal adaptation (APGAR score), need of resuscitation at birth and need of mechanical
ventilation within first 24 hours of life.
Medical history and clinical data were focused on neonatal hypoxic pathology that had a
significantly higher incidence in the SG vs. the CG.

Placental lesions in GDM by standard histological stainings

The main aim of this histopathological study was to analyse injuries that may contribute to
pathological changes in the MFI in patients with insulin controlled GDM. We have investigated
the following regions: terminal villi, villous capillaries, villous and extravillous
syncytiotrophoblast, trophoblast membranes, Hofbauer cells, amnion, and chorion, aiming to
reveal their lesions, as well as other significant degeneration areas.

By comparing the histology of placentas in the two groups, from mothers with GDM and
controls, respectively, we have obtained the statistical results of lesions in Figure 2, as follows:

Figure 2. Placental histopathological lesions in GDM

The inflammatory lesions group revealed no lesion in controls and only once per each of
the two subgroups villitis and amnionitis, respectively.

The degenerative lesions group has the strongest histological significance. Among this
lesion types subgroups, the thickening of trophoblastic basement membrane is the peak
of the group, being 4 times higher than in controls, in which it is absent. Villous fibrosis
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 and stromal edema are only one-fold higher than in controls, in which they are absent.
Fibrinoid necrosis is 3 times higher than in controls. Placental calcifications,
ubiquitously placed, are twice as much, while hyaline degeneration is 0.3 times higher
than in controls.

The proliferative lesions group shows a 4 times higher peak than the control group, in
which they lack, through the intra- and perivillous fibrosis subgroup. Syncytial changes
by agglutination are 3 times higher towards the controls, while Hofbauer cells
hyperplasia is 2 times higher than in controls, in which is absent. We have found the
chorangiosis equal to the controls.

The circulatory lesions group has its peak in the nucleated platelets subgroup, that are 4
times higher than in controls, in which they lack, unlike the edema of the intima and
chorion is which lesions were absent in controls, and only once were higher. Interstitial
(intervillous) haemorrhages were found 3-fold higher than in controls.

Clinical data of babies born to mothers with GDM

The parameters considered in this study were comparatively analysed between SG and CG.
No significant differences were recorded between SG and CG regarding the physical parameters
of foetal growth and development, i.e. weight, height and cranial perimeter.
It is not about IUGR in these cases, it is about growth and development which are greater than
GA.
Foetal suffering degree (basically hypoxic and metabolic), impacting on foetal growth and
development, and prematurity, significantly more frequent in this category, have also influenced
other postnatal evolution parameters, such as: neonatal adaptation (APGAR score), need of
resuscitation at birth and need of mechanical ventilation within first 24 hours of life in the SG as
compared with the CG.

PID SCORE

As noticed in the analysis of histological changes of placenta for both PIH and GDM, there are
more common than specific changes between the two conditions, as well as between pathological
and control placentas (most of the latter being normal). These changes allowed for the
development of a score to assess the Placental Insufficiency Degree (PID).

As seen in Table 6, this score uses a scale comprising 4 degrees of placental insufficiency, each
of them associated with certain histological changes assessed by the anatomic pathologist that
examines each placenta after birth; according to the presence or absence of changes, 0 or 1
values are introduced in the grid, thus resulting by addition an ascending score (IV-th degree
providing the highest lesion and postnatal hypoxia risks). At the onset of hypoxic symptoms in
the newborn within early postnatal period an overall retrospective assessment of the degree of
foetal distress during pregnancy is performed, which may guide the neonatologist towards a
rapid therapeutic conduct.
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 PID
(PLACENTAL INSUFFICIENCY
HISTOLOGICAL CHANGES OF THE
DEGREE SCORE)
PLACENTA st
I II III IVth
Degree Degree Degree Degree
THROMBOSES AND/OR HAEMORRHAGES 0 0 0 1
OF THE VILLI OR DECIDUA
SYNCYTIAL HYPERPLASIA (KNOT, 0 0 1 1
BUNDLE, BRIDGE)
MFI VILLOUS AND/OR INTERVILLOUS 0 1 1 1
FIBRINOID
MFI AND/OR VILLI STRUCTURAL CHANGES 1 1 1 1

PLACENTAL INSUFFICIENCY SCORE 1 2 2 4

Table 6 PID (Placental Insufficiency Degree) Score

This score we have called PID Score has been built so that to be quickly applied in both the
pathological conditions within pregnancies with PIH and GDM, and any pathological situation
of the mother that would lead to placental insufficiency with major consequences for foetal
growth, development and oxygenation.

The PID Score also provides the opportunity to validate our hypothesis that placenta is an
important witness of pathological events during prenatal period critical for postnatal evolution,
particularly for hypoxic events occurring after birth. We have therefore used the PID Score in all
the studies cases vs. controls to ascertain the sensitivity and specificity of this grading scale.

The comparative analysis of PID Score showed highly significant differences (p<0.001) between
the studied pathological placentas (PIH + GDM) and control placentas. This finding allowed us
to consider scores sensitivity and specificity as the most important validating indicator for the
present thesis results. With 100% sensitivity and 42.86% specificity for a 95% confidence
interval, the PID Score we have developed may be a valuable guide for assessing placental
insufficiency, with great practical value and important assessment ratio for the prognosis of
severity of child postnatal hypoxic suffering.

With positive predictive value of 63.64% and negative predictive value of 100%, the PID Score
has its utmost utility in predicting the absence of placental insufficiency (100%), but it also has
high predictive value for the presence of placental insufficiency (63.64%).

In conclusion, we can state that the PID Score developed based on this thesis is of value for the
anatomic pathologist practitioner in the overall retrospective assessment of placenta, providing

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him with an useful and rapid mean to assess placental insufficiency due to MFI suffering, as
well as for the neonatologist, because of the important ratio of early prognosis of newborn
hypoxia severity after birth, according to the degree of foetal distress during pregnancy.

CONCLUSIONS

1. PIH and GDM are a real top seed of maternal diseases with major impact on foetus and
newborn by the morphological changes in the placenta.

2. The work hypothesis was that placenta is an important witness of the events occurring in the
prenatal period, its morphological changes being a marker of pathological events in the peri-
and postnatal period.

3. Morphological research of placenta has been focused on the MFI, i.e. the exchange structure
the injury of which generates the perinatal pathology and the postnatal suffering of the newborn.
It is thus argued, by personal researches, that the direct consequence of morphological changes
in the MFI is hypoxemia and foetal/neonatal hypoxia, with immediate and future consequences
in the development, especially the neurologic one.

4. Research on histological changes in PIH was made on 34 placentas from mothers with this
pathology that gave birth to intrauterine growth restriction and/or preterm children. An equal
number of placentas from mothers without PIH were studied, following the case-control study
design.

5. Results of histological study of placenta in PIH showed a significant incidence of

degenerative and proliferative lesions (p<0.05) and a moderate incidence of circulatory lesions
(p<0.001), either associated or not, as compared with lesions of placentas in the control group.

6. Immunohistochemical staining for PIH revealed retarded villous development mainly due
to hypoxia. Villous agglutination with compromised MFI is proved by reactions of Ac anti-CK7,
very intense in the villous cytotrophoblasts membranes, intense in the fibrinoid necrosis areas
and moderate in all the types of syncytial clusters (buds, knots, bridges) in the terminal villi that
became avascular, which shows the impairment of oxygen, nutrient and residue exchanges in the
MFI of a high number of villi.

7. Compared with the control group, the newborns from mothers with PIH had significantly
higher incidence (p<0.05) of adaptation problems and neonatal pathology.

8. By collating the results of research on morphological changes in the placenta with that on
clinical manifestations of newborns from mothers with PIH, we have noticed that the major
impact of morphological changes in the placenta on the MFI involves:

reduction in the number of terminal villi accompanied by reduction or atrophy of their

capillaries;
reduced foetal oxygenation, with increased incidence of neonatal hypoxic pathology;
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 reduced foetal nutrition with direct consequences on foetal growth and development by
increased IUGR incidence, either associated or not with prematurity.

9. The results of histological research on placenta in GDM have shown a significant incidence
of degenerative lesions in the MFI structures, as each case had at least one histological
degenerative change towards the controls. Although significant compared with the control group,
the incidence of proliferative and circulatory lesions was moderate (against that of degenerative
lesions).

10. Immunohistochemical staining for GDM allowed the detection of epitopic fragments
expressed after lesion degeneration through chronic suffering of MFI.

11. Compared with the control group, the newborns from mothers with GDM had significantly
higher incidence (p<0.05) of adaptation problems and neonatal pathology.

12. By collating the results of research on morphological changes in the placenta with that on
clinical manifestations of newborns from mothers with GDM, we have noticed that the major
impact of morphological changes in the placenta on the MFI involves:

alteration of placental metabolic and hormonal functions by the increase in number of

terminal villi and their capillaries;
unbalanced exacerbation of foetal nutrition with direct consequences on foetal growth
and development (foetal macrosomia);
paradoxically, reduced foetal oxygenation due to degenerative lesions in MFI, with
significant increase of neonatal hypoxic pathology, leads to the large for gestational age
newborn, with perinatal hypoxia and difficult adaptation due to metabolic vulnerabilities
(the giant with feet of clay).

13. The microscopy results recorded similar changes (and/or common) in PIH and GDM
placentas for the three main lesion groups, particularly in the MFI. This has allowed for the
development of a morphological score that enables assessment of PID for the examined
placentas and validation of research results. The GIP Score, with 100% specificity and 42.86%
sensitivity and a high predictivity ratio for the presence of placental insufficiency (63.64%),
provides good opportunity to estimate postnatal evolution of high-risk newborns from mothers
with PIH and GDM, as placental insufficiency causes the impossibility of placenta to ensure the
oxygen and nutrient needs of a growing foetus.

14. The application of PID score to studied cases has certainly shown the significant positive
correlation between PID and peri and postnatal hypoxic disorders, especially for high scores;
there is a great degree of agreement between high scores of SARNAT Grading Scale and high
scores of PID (3). In the case of lower scores, the PID score becomes an important
morphologic argument for clinical suspicion and prevention of neurologic disorders starting later,
particularly in the newborns with apparently favourable postnatal development.

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