Early pregnancy loss is defined as the termination of pregnancy before 20 weeks of gestation or a foetal weight of less than 500g. It is a very common incident with the majority of spontaneous miscarriages happening within the first 12 weeks of pregnancy. It accounts for over 50,000 hospital admissions in the UK annually where 85% of spontaneous miscarriages occur within the first trimester, (Newson, 2015). The maximum chance of getting pregnant with optimum conditions is 30-40% during a normal menstrual cycle but subsequently it usually results in an early loss of pregnancy, (Macklon et al, 2002). There are many factors which contribute to this loss, chromosomal, endocrine and uterine abnormalities as well as many lifestyle factors.
Before any early embryo development fertilisation must occur to form a conceptus. Fertilisation is the combination of two different gametes to produce a conceptus with a diploid set of chromosomes. Fertilisation occurs in the ampulla region of the uterine tube. The facilitation of the movement of the newly formed conceptus is provided by the cilia present in the uterine tube which help transport the conceptus down the tube in uterine fluid ready for implantation into the endometrium. Within 24 hours of fertilisation quick mitotic cell divisions happen, known as cleavage, (Schoenwolf & Larsen, 2009). These allow the subdivision of cells but prevent the conceptus growing due to the zona pellucida preventing any further growth. The subdivisions of cells are blastomeres. The cells continue to divide but by 16-32 cells a process known as compaction occurs where the conceptus is known as a morula. This is due to the fact that there is a polar phenotype developing, an apical and basal domain. These domains allow the differentiation of two new cell types, trophoblasts and inner cell mass. Trophoblasts are the outer layer of cells surrounding the conceptus which provides nutrients before the establishment of the placenta and then later on develops into the placenta. The inner cell mass are a small cluster of cells which will eventually become the definitive structures of the foetus, lying in the blastocoelic cavity, (figure 1). Together these make the blastocyst which is what will leave the uterine tube by the end of the 6th day after fertilisation, and will subsequently implant into the endometrium, (Watson & Barcroft, 2001).
Figure 1 shows the steps undertaken to form a blastocyst once fertilisation has happened, (https://ib-biology2010-12.wikispaces.com/Human%20Reproduction)
Before implantation, the blastocyst must hatch out of zona pellucida to facilitate implantation. The zona pellucida is vital for the maintenance of the conceptus but also to prevent 2 genetically different conceptuses from interacting with one another. However, the stretched zona pellucida develops a crack allowing the blastocyst to use enzymes to squeeze out by a process known as Zona hatching. The blastocyst is now able to directly interact with the endometrium (Schoenwolf & Larsen, 2009).
Changes in Endometrium
Implantation is the attachment of a conceptus to the endometrium lining of the uterus for successful growth and survival.
For successful implantation, both the blastocyst and the endometrium need to be ready for the changes which will subsequently occur. The endometrium is constantly changing due to the menstrual cycle under the influence of hormones, primarily as progesterone and oestrogen. Subsequently, there is only a small period of receptivity of the endometrium to allow implantation of the blastocyst, the implantation window lasting for only 4 days, (Elnashar & Aboul-Eneih, 2004). During the early stages of implantation and pregnancy, progesterone is needed to maintain the endometrium and allowing it to be in a receptive state ready for the conceptus.
Endometrial microvilli briefly fuse to become large ectoplasmic protrusions, known as pinopodes; they are initiated by the influence of progesterone to help endocytose uterine fluid, decreasing the overall volume of the uterus, bringing in the walls closer for the conceptus to adhere and remain fixed on the wall. They help to show how receptive the endometrium is towards a conceptus in a human, (Nikas & Psychoyos, 1997).
There is also an increase in secretions from epithelial glands which is also due to increasing progesterone levels. The secretions help to secrete nutrients into the uterine cavity, used by conceptus whilst there has been no decidualisation or placenta formation yet.
Progesterone is constantly being secreted by the corpus luteum to maintain pregnancy by helping to change the receptiveness of the endometrium. Decidualisation is a process in which the endometrium thickens and becomes vascularised to support the blastocyst when it attaches to the wall. The main difference is the stromal cell as the overall cell morphology changes going from a small spindle shape to a much larger, plumper secretory decidual cell. These decidual cells form a pericellular rim of extracellular matrix which is required for the trophoblasts to move towards arteries and myometrium. It is under the influence of a protein, Insulin like growth factor binding protein-1 (IGFBP-1), (King, 2000). The decidua remains during the first trimester but will be subsequently replaced by the placenta later on.
The Luteo-placental shift is a very important event which happens in regards to the production of progesterone and oestrogen. Originally the corpus luteum secretes mainly progesterone during the early stages, but the corpus luteum has a very short life span of only a couple of weeks. This would mean that after a few weeks, there would be a significant drop in progesterone secretion leading to loss of conceptus as the endometrium cannot maintain the pregnancy, (Johnson & Everitt, 2007). Human Chorionic Gonadotrophin hormone (hCG) is produced by the synctiotrophoblast and helps to rescue the corpus luteum, binding to the corpus luteum receptors producing progesterone, until the seventh week of pregnancy. By the seventh week the placenta is developed and will take over progesterone secretion throughout the rest of the pregnancy, (Finlayson, 2007).
Implantation begins when the blastocyst comes into contact with the uterine wall and tends to happen in 3 steps- : Apposition, Adhesion and Invasion.
Apposition is a temporary loose connection between the blastocyst and the endometrium. The blastocyst will adhere to a region on the endometrium where there is a high surface area. In this way it will allow for more wall exposure when implanting e.g. a dip on the wall. There must have been sufficient lysis of the zona pellucida to allow direct contact between the trophoblasts of the blastocyst and the decidua, (Carson, 2000). Mucins are glycoproteins found at the apical surface of the uterine epithelia. MUC1 is under the influence of progesterone and is an anti-adhesive molecule inhibiting cell to cell interaction, (Hoffman et al, 2013). It acts as a barrier between the mother and foetus and usually the levels decreases in most animals during implantation. However in humans, MUC1 expression levels are at its highest during implantation and as a direct result human MUC1 has another function not seen in other animals. It allows a platform for blastocyst interaction with the endometrium, providing a suitable attachment site; hence the increasing in MUC1 levels during implantation, (Dharmaraj et al, 2009).
It is a stronger form of attachment by the trophobasts being able to adhere to the endometrium, by penetrating into the wall using protrusions. There is constant communication between the blastocyst and the endometrium to signal change in the stromal cells (decidualisation). Multiple signalling molecules need to be secreted from either the endometrium or the blastocyst to allow advantageous interactions. Selectins are one of the most important molecules secreted by the trophoblasts as they allow cell to cell communication. L-selectin allows direct physiological interactions with oligosaccharide receptors present on the endometrium. Leukaemia Inhibitory factor (LIF) promotes active attachment of the conceptus to the endometrial wall as there is a form of communication between the uterus and the blastocyst. The uterine lining has glandular epithelial cells which secrete LIF whilst the blastocyst contains LIF receptors, (Charnock-Jones et al, 1994). These all act under the influence of progesterone allowing the control and guidance of the blastocyst to the endometrium, (Fritz, 2014). E-cadherins are necessary during both blastocyst formation but also during the control and guidance of the trophoblast invasion into the endometrial lining. It is expressed in maternal epithelium to form a permeability barrier between the maternal immune cells and the embryonic molecules in the decidual zone, (Van Mourik et al, 2009).
Invasion of the trophoblast allows the cells to proliferate and penetrate further and completely into the endometrium wall. As the blastocyst further penetrates the cells differentiate further into synctiotrophoblasts and cytotrophoblasts. Synctiotrophoblast are the fusion of trophoblast whilst cytotrophoblasts maintains their cellular structure, further producing trophoblasts. The synctiotrophoblasts further invades until it reaches the uterine stroma below the decidua allowing the conceptus to embed within it. As soon as the synctiotrophoblast come into contact with maternal blood, chorionic villi are formed, initiating the start of placentation.
Placentation is the formation of the placenta from the synctiotrophoblasts and the cytotrophoblasts from the conceptus, (figure 2a and 2b). The chorionic villi degrade the uterine decidua, absorbing all its nutrients to supply to the conceptus. The synctiotrophoblast cells will become the surface cover of the placenta acting as a barrier between the foetus and the mother, whilst fusion of cytotrophoblast forms the main body of the placenta. The placenta is completely formed by the end of the first trimester allowing full nutrient, gas and waste exchange between the foetus and the mother, (Pacific Research Center For Early Human Development, 2007).
Figure 2a and 2b: The formation of a placenta from the synctiotrophoblasts and the cytotrophoblasts, (http://humanphysiology2011.wikispaces.com/15.+Reproductive+Physiology)
Causes of early pregnancy loss
Chromosomal abnormalities are the main cause of early pregnancy loss affecting 50-70% of people. It is most commonly due to abnormalities in the chromosomal number i.e. aneuploidy with trisomies being the most prevalent but also monosomy and polyploidy cause significant damage to the conceptus, (Cheng et al, 2014). It has been shown that the majority of mutations arise randomly within the conceptus itself, leading to the formation of a new de novo mutation as both of the parental karyotypes are normal, (Romero et al, 2015). This means that both of the parents have the normal number of chromosomes needed for a successful conceptus but there must have been a mutation within the conceptus which had arisen to form the anomalous chromosomal number, leading to a spontaneous abortion. There is direct link between the likelihood of chromosomal imbalance and an increase in maternal age due to an ageing oocyte. This is due to the fact that there is no new oocyte formation during a woman’s lifetime. The increased frequency of non-disjunction developing due to the ageing oocyte further proves the theory of how maternal age is vital in chromosomal aberrations. In a study carried out by the Atlanta Down syndrome project, results show that trisomy 21 is more likely to transpire over the age of 35 due to either meiosis I nondisjunction (4x greater) or due to meiosis II nondisjunction (5x greater), (Allen et al, 2008). The importance of maternal age is backed up by these results, the chances of having a healthy foetus is down to the age of the oocyte.
Approximately up to 15% of early pregnancy loss is due to endocrine disorders, (Women’s Health and education center, 2014). Progesterone is needed to maintain and develop the endometrium to allow for successful pregnancy. It inhibits the smooth muscles of the uterus to prevent premature expulsion of the conceptus as it tries to implant, relaxing the smooth muscles in the body, (Finlayson, 2007). Progesterone signals to the endometrium to begin changing allowing for implantation by altering the morphology of the underlying stromal cells. During the luteo-placental shift, there is a change in production of progesterone from the corpus luteum to the placenta. If an abnormality occurs during the luteo-placental shift, the levels of progesterone decrease at the beginning of implantation and results in the endometrium not being able to fully support the conceptus, leading to its termination, (Schindler, 2004).
Uterine Anatomical Abnormalities
Figure 3: Types of uterine abnormalities (Kaproth-Joslin et al, 2013)
The most frequent uterine abnormality which causes early pregnancy loss is the presence of a septum within the uterine cavity, causing up to 55% embryo loss, (Morgan & Yang, 2015). A septum is a fibrous wall which may extend partially into the uterus or all the way down into the cervix. As it is, there is limited space within the uterus for the foetus to develop so the presence of the septum further makes it difficult for the uterus to hold the foetus. This causes the uterus to release the foetus, resulting in subsequent miscarriage. Treatment to correct the uterus is by surgical removal of the septum, (Selvaraj & Selvaraj, 2010).
A Bicornuate uterus is a unification defect of the Müllerian ducts causing the indentation of the uterine surface to form two separate endometrial cavities known as horns of the uterus, representing 25% of uterine abnormalities, (Luijkx and Gaillard, 2015). There is a significant decrease in area for a foetus to develop, making it an incredibly high risk pregnancy which requires continuous monitoring but more often leads to miscarriage instead, ( Adeyemi et al, 2013).
A Didelphys uterus is failure of the fusion of the Müllerian ducts to form 2 hemiuteri, (Lewis & Levine, 2010). Due to the formation of the 2 hemiuteri, there is a lack of uterine volume and blood flow to each compartment, making it a hostile environment for the conceptus resulting in miscarriage. These are the main important uterine anatomical abnormalities; the rest can be seen in figure 3.
Maternal Immune Dysfunction
Antiphospholipid syndrome is an autoimmune disease characterised by the presence of three abnormal antibodies (aPL) in the blood, anti- β2 glycoprotein I, lupus anticoagulant and anticardiolipin. The presence of aPL affects the body in two ways. Firstly it affects the maturation, mobility and invasiveness of the trophoblast. They cause defective placentation to arise as a result of loss of trophoblast invasion into the decidual cells but also anti- β2 glycoprotein I triggers an inflammatory response in the trophoblast, compromising its survival, (Kwak-Kim et al, 2013). If this case happened, the mother would lose her foetus and the only treatment would be regulation of these antibodies if a subsequent pregnancy happened. Secondly these antibodies attack normal proteins on the cell plasma membrane initiating the activation of the coagulation system. This activation causes multiple thrombi to form which blocks the major blood vessels within the placenta to the conceptus, preventing any blood flow causing death, (Rose & Ho, 2014). It also increases the chance of pregnancy complications for the mother, mainly pre-eclampsia or early labour. Treatment would be aspirin as it is used commonly for thrombophilic disorders as it inhibits the formation of thromboxane A2, required for platelet aggregation in blood clot formation.
Smoking affects the implantation process as it damages the function and the viability of the oocyte. Either active or passive smoking directly affects the developing foetus as the major metabolites of nicotine, cadmium and cotinine have been detected in follicular fluid, surrounding the oocyte and in the follicle. Cadmium exposure increases the chance of aneuploidy and decreases the likelihood of the cell reaching metaphase II, damaging the DNA. Second hand tobacco smoke has a detrimental effect on the foetus as it concentrates within the foetal circulation, (Meeker, 2013). Carbon monoxide obtained from the tobacco smoke displaces oxygen, irreversibly binding to haemoglobin in the blood. Foetal haemoglobin has a higher affinity for oxygen at lower partial pressures, resulting in more carbon monoxide getting into the foetal bloodstream preventing sufficient gas exchange. This would cause asphyxiation and death of the foetus. Also a greater risk of aneuploidy due to sperm damage is paternal smoking, which may lead to a spontaneous abortion if there is chromosomal imbalance.
Foetal Alcohol Spectrum Disorder (FASD) is a prevalent group of disorders caused by alcohol abuse during pregnancy. It is widely seen in parts of the world but in some countries, rates are record high e.g. in western cape, South Africa, 0.75 out of every 1000 live births has some form of FASD, (WHO,2011). Not all embryos are fortunate and survive; many spontaneously abort due to foetal hypoxia, as alcohol is an oxygen dependent metabolism, requiring oxygen to break it down. This oxygen is usually intended for the foetus to survive, but is instead sacrificed. There is also a hypothesis that because acetaldehyde, one of the main metabolites of alcohol is a mutagen, it inhibits RNA synthesis, leading to conceptus death due to lack of protein and DNA synthesis, (Windham et al, 1997).
Diet is vital in maintaining a successful pregnancy along with folic acid supplement. Folic acid is important both preconception and post conception as it prevents a large number of congenital abnormalities especially neural tube defects. Low folic acid levels during pregnancy increases the risk of preterm delivery, pre-eclampsia and spontaneous abortions. Hyperglycaemia may be damaging to the developing foetus as over nutrition of glucose may result in macrosomia of baby, making it predisposed to be insulin resistant (diabetes) or obese. Therefore constant monitoring of blood glucose level during pregnancy is vital to keep the baby healthy and to reduce the risk of congenital diabetes, (Yessoufou & Moutairou, 2011).
The causes of early pregnancy loss are mainly due to genetic, endocrine, environmental and dietary changes. They are all linked to one another and rely heavily upon the maternal reproductive system and just one slight imbalance can easily lead to the loss of pregnancy. However, there is also a link between early pregnancy loss and the role of implantation during pregnancy which can either be caused by failure of the maternal support system or due to the foetus itself. Some of these factors may be predicted and can be corrected with the right help and intervention to prevent recurrent loss of embryo. Each part of fertilisation, implantation and maintenance of pregnancy is susceptible as a target contributing to the loss of embryo. Spontaneous abortions within the first trimester are indeed a common occurrence and regularly happen without the mother knowing, often acting as a first warning that there may be possible issue with the maintenance of a future full term pregnancy.
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