Embryology Notes PDF

Isabella Ronchi Embryology Introduction The periods of human embryology can be divided according to: Medical point of view: 1st, 2nd and 3rd trimester Embryological point of view: o Period of t...

Isabella Ronchi Embryology Introduction The periods of human embryology can be divided according to: Medical point of view: 1st, 2nd and 3rd trimester Embryological point of view: o Period of the egg: from fertilisation to implantation (conceptus of pre-implantation embryo). o Period of the embryo: from implantation to the 8th week o Period of the foetus: from the end of the 8th week Pregnancy lasts 38 weeks if counted from fertilisation or 40 weeks counted from the last menstrual period. The embryo is a curved object that changes its shape over time. Some axes can be distinguished: ADULT EMBRYO Anterior Ventral Posterior Dorsal Superior Cranial/rostral Inferior Caudal Sections can be transverse, median (sagittal) or coronal (frontal).  Isabella Ronchi Gametogenesis The phases of gametogenesis are: 1. Primordial germ cells and their migration.: in the 2nd week primordial germ cells are inside the embryo, at the level of the epiblast. During the 3rd week they migrate in the wall of the yolk sac. From week 5 to 6 they go back in the embryo, in the area where the gonads will form. Migration is a complex mechanism that requires dynamic rearrangement of the cytoskeleton. While they migrate, cells interact with each other and with substrates in the extracellular matrix. Anomalies and misdirection of primordial germ cells to extragonadal sites can cause teratomas, because they are pluripotent germ cells that can give rise to different tissues. Teratomas are neoplasms of mixed differentiation, with tissues representing all embryonic layers. They may contain several different types of tissue, such as hair, muscle and bone. Teratomas can be mature or immature, or sometimes a mix of mature and immature cells. Teratomas usually occur in the ovaries in women, the testicles in men and the tailbone in children (sacrococcygeal teratomas). They may also occur in the CNS, chest or abdomen. They can be benign or malignant. Large sacrococcygeal teratomas can cause a variety of complications before and after birth. They can grow rapidly in the foetus and they require high blood flow, resulting in foetal heart failure, a condition known as hydrops. This manifests as dilation of the heart, collection of fluid in tissues of the body, including the skin and body cavities such as around the lungs (pleural effusion), around the heart (pericardial effusion) and/or in the abdominal cavity (ascites). If neglected, hydrops can also be dangerous for the mother, resulting in similar symptoms of swelling, hypertension and fluid in the lungs with shortness of breath. In addition to hydrops, which can occur in approximately 15% of very large sacrococcygeal teratomas, these tumours can cause polyhydramnios (too much amniotic fluid), foetal urinary obstruction, bleeding into the tumour or rupture of the tumour with bleeding into the amniotic space. Dystocia could also be a consequence (a condition where the foetus cannot be delivered due to the size of the tumour). In adults, sacrococcygeal teratomas may not cause symptoms. In some cases, they cause progressive lower back pain, weakness and abnormalities due to obstruction of the genitourinary and gastrointestinal tracts. Such symptoms include constipation and increased frequency of stools or urinary tract infections. In rare cases, sacrococcygeal tumours cause paralysis of the legs and tingling or numbness. 2. Increase in number of PGCs by mitosis: the following phases of gametogenesis have a different pacing in males and females. In the ovary, by the 5th month of gestation all 7 million oogonia have entered the prophase of the first meiotic division (primary oocytes surrounded by primordial follicles). At birth they decrease to 2 million, at puberty 40.000 and only 400 will be ovulated. In males PGCs that reach the gonads are called spermatogonia. They proliferate during the early embryonic period. They are dormant from the 6th week until puberty. Intensive proliferation goes on after puberty throughout life.  Isabella Ronchi 3. Reduction in chromosomal number by meiosis: meiosis leads to a reduction in the number of chromosomes and a reassortment of paternal and maternal chromosomes. For each spermatogonia there will be 4 viable gametes, whilst in females each oogonium gives 1 viable gamete. Oogonia begin meiosis by the 5th month of foetal gestation. Meiosis in females is synchronous and slow. The first meiotic division is completed at ovulation, so completion might take up to 50 years. The second meiotic division is only completed if the egg is fertilised. Males have a surge of testosterone at puberty, which causes the maturation of somatic cells (Sertoli cells) into seminiferous tubules. Meiosis in males is asynchronous: not all spermatogonia enter meiosis at the same time. The first meiotic division lasts 8 days and the second meiotic division lasts 16 days. Spermiogenesis takes 24 hours days. In the broadest sense spermatogenesis begins with proliferation of spermatogonia, which takes 16 days. The total is 64 days. 4. Structural and functional maturation of egg and sperm. Spermatogenesis At puberty Sertoli cells differentiate into a system of seminiferous tubules. The dormant PGCs resume development, divide several times by mitosis and then differentiate into spermatogonia. Spermatogenesis takes place in the seminiferous tubules. The wall of the seminiferous tubules can be divided into a basal compartment and in an adluminal compartment. Spermatogonia undergo proliferation in the basal compartment and morphological changes in the adluminal compartment. The basal compartment and the adluminal compartment are separated by junctional processes of Sertoli cells. Spermatogonia become primary spermatocytes once they enter the first meiotic division. While primary spermatocytes enter meiosis I, they become antigenically different from the body of the male who is containing them. Therefore, they have to be kept separated from the blood vessels in the stroma by the blood-testis barrier to avoid an immune response. When the first meiotic division is completed, they become secondary spermatocytes. The second meiotic division gives rise to spermatids. Spermatids undergo a series of morphological changes, in a process called spermiogenesis, that gives them their final shape. There’s a reduction in size of the nucleus and a change in its shape, thanks to the condensation of chromosomal material (histones are replaced by protamines). The cytoplasm is eliminated as a residual body and the Golgi apparatus is condensed at the apical end of the nucleus, taking the name of acrosome. Extruded cytoplasm is phagocytosed by Sertoli cells. Finally, a flagellum forms on the other side of the centriole, and is responsible for the motility of the spermatozoon. The flagellum contains the axoneme (9x2 + 2 microtubules). Deficiency  Isabella Ronchi in the axonemal structure can cause defects in sperm motility and often leads to male infertility. Leydig cells are unicellular endocrine cells in the stroma of the seminiferous tubules, they secrete androgen and testosterone. Sertoli cells have many important functions: o They maintain the blood-testis barrier and o They secrete other proteins, such as isolate the antigenically different haploid inhibin for feedback loop to the germ cells from the male adult immune hypothalamus, Mullerian-inhibiting factor system and retinal-binding proteins o They secrete androgen-binding proteins o They maintain and coordinate o They phagocytose residual bodies of sperm spermatogenesis cells o They secrete a tubular fluid Adjoining segments of the seminiferous tubules present different stages of maturation of spermatozoa, because meiosis is asynchronous. Because spermatocytes are connected to each other and to Sertoli cells, in a certain segment of the tubule the same association of cells is visible (stage). The appearance of the same association in the same segment is a spermatogenic cycle. The distance along the tubule between the same stage is called a spermatogenic wave. At the end of spermiogenesis and spermiation (the process by which mature spermatids are released from the supporting Sertoli cells into the lumen of the seminiferous tubules), mature spermatids are not yet motile. Spermatozoa are propelled in the epididymis by fluid pressure generated in the seminiferous tubules and by muscle contraction and ciliary movements. Spermatozoa further mature and acquire motility in the epididymis and are stored in its lower part. Freshly ejaculated sperms are unable or poorly able to fertilise, they must first undergo a series of changes known as “capacitation reaction”, which occurs in the female reproductive tract. Capacitation is associated with removal of sperm plasma membrane proteins, reorganisation of plasma membrane lipids and proteins. The main causes of male infertility related to spermatozoa are: o Number: less than 10 million/ml o Motility  Isabella Ronchi o Abnormal sperms (5-20% abnormal o Endocrine disorders spermatozoa in the ejaculation of a healthy o Environmental pollutants man) o Cigarette smoking o Altered genome o Obstruction of genital duct system o Medication and drugs Oogenesis Oogenesis consists in the maturation of the oocyte and of the follicle. Together they form a morpho- functional integrated unit. In the cortex of the ovary there are ovarian follicles in different stages of maturation. When the oogonia reach the ovary, they get in contact with support cells and together they form primordial follicles, which enter into meiosis 1. By the end of the 5th month of gestation they have all entered meiosis 1 and they are called primary oocytes. The follicular cell and the oocyte interact with one another, allowing the oocyte to be maintained in meiosis 1. Interactions are based on the concentration of cAMP and cGMP. They are both produced in the follicular cells and transferred in the cytoplasm of the oocyte. There’s a higher concentration of cAMP in the cytoplasm of the oocyte, because it inhibits the Maturation Promoting Factor (MPF). Moreover, cGMP inhibits phosphodiesterase, whose role is to transform cAMP into 5’AMP, which is important for the progression of meiosis. Each month 5-12 primordial follicles begin folliculogenesis. The zona pellucida is formed: it’s a specialised extracellular matrix, made of glycoproteins (Zonula proteins 1-4) and glycosaminoglycans, located  Isabella Ronchi between the oocyte and the follicular cells. Follicular cells become isoprismatic (cuboidal). The oocyte and the follicular cells communicate across the zona pellucida through gap junctions and microvilli. Some primary follicles proceed their development and become growing follicles (or late primary follicles). They become multilayered, forming the granulosa, which expresses FSH receptors. The granulosa is supported by a basal lamina called membrana granulosa, which acts as a barrier to capillaries from the stroma. The stroma is organised in theca folliculi, divided into theca interna (highly vascularised and glandular, androgen producing and LH factor expressing) and theca externa, made of a connective tissue-like capsule for protection. Since only one follicle can be ovulated, some growing follicles degenerate and some enlarge mainly by taking up fluids. Antral follicles (or secondary follicles or pre-Graafian follicles) have fluid filled cavities which merge to form an antrum. This cavity is filled with liquor folliculi, rich in hyaluronic acid. The formation of the antrum separates cells surrounding the oocyte (cumulus oophorus) from those adjacent to the theca (granulosa cells). FSH receptors on the granulosa cells trigger the production of estrogens. The theca interna produces testosterone. The size of the follicles in this stage is about 200 µm and the follicular cells are arranged in 6-10 layers. Only one of the growing follicles will continue its maturation. At the beginning of the cycle as many as 50 follicles start maturation, but only 3 obtain the size of 8 mm. The initial growth is hormone- independent, but continuation requires minimum tonic levels of FSH. A dominant large follicle becomes independent from FSH and secretes inhibin, which lowers the levels of FSH. Non-dominant follicles degenerate. This one follicle is called Graafian follicle and it gets very close to the surface of the ovary. 10 to 12 hours before ovulation meiosis resumes following the LH surge. The cumulus oophorus cells respond to LH, shutting down gap junctions. cGMP levels increase, promoting the transformation of cAMP into 5’ AMP. The MPF is activated, therefore meiosis I is completed and meiosis II begins and arrests in metaphase. The secondary oocyte is formed. Completion of the first meiosis leads to the formation of the first polar body. The secondary oocyte is contained in a big tertiary follicle (2 cm), which protrudes on the surface of the ovary like a blister (the region is called stigma). In the meantime hyaluronic acid and water are accumulated in the antrum, increasing the pressure. Follicular cells produce large amounts of estradiol, which prepares the female genital tract for gamete transport.  Isabella Ronchi Ovulation takes place 38 hours after the LH and SFH surge. An inflammatory reaction in the outer follicle causes the rupture of the outer follicular wall. The ovulated complex consists of: oocyte, zona pellucida, corona radiata, sticky matrix containing cells of the cumulus oophorus. Ovulation can lead to mittleschmerz , a variable amount of abdominal and pelvic pain. The cause is a slight bleeding in the abdominal peritoneal cavity or the enlargment of the follicle before ovulation. The ovulated complex is captured by the fimbriae of the Fallopian tubes. It is then transported (3-4 days) by the contraction of the muscular wall of the uterine tube and movement of cilia. Transport can be divided in two phases: o Slow transport in the ampulla (72 hours), because fertilisation happens here o Rapid phase (8 hours) from the isthmus to the uterus The rest of the cells in the remaining granulosa and the thecal cells continue producing hormones and transform into the corpus luteum. As soon as ovulation takes place the theca and granulosa collapse (corpus haemorrhagicum). These cells undergo leutinisation: they increase in size, accumulate lipid droplets, produce progesteron and estrogen. This is important for the preparation of the endometrium (inner lining of the uterus) for possible implantation of an embryo. The corpus luteum is functional for approximately 2 weeks. If no fertilisation happens, it turns into menstrual corpus luteum. It regresses and ceases hormone production after 10 days. It finally transforms into corpus albicans, made of scar tissue. If fertilisation happens, it becomes gravidic corpus luteum, which remains functional for about 5-6 months and it produces hormones (stimulated by Human Chorionic Gonadotropin, produced by cyncytiotrophoblast of the blastocyst). After 6 months it starts to regress, because the placenta is developed enough to produce hormones.  Isabella Ronchi Embryology – 1st and 2nd week Fertilization Ejaculation is a rapid transit through the ductus deferens and urethra. Seminal fluid (2-6 ml, pH 7.2-7.8) is enriched by prostatic secretion and seminal vesicle secretion, which contains fructose to provide energy to the spermatozoa. Spermatozoa retain their function in the female reproductive tract for around 80 hours. Once they’re deposited in the vagina, they need to undergo several processes before they reach the egg: 1. The first barrier they encounter the difference in pH. The vagina is acidic (pH = 4.3), but the buffering capacity of the seminal fluid can increase the pH to 6.0-6.5, the optimal value for motility. Buffering only lasts a few minutes, enough time for the sperm to approach the cervix of the uterus. 2. The following barrier is the cervical mucus in the cervical canal, which is usually very thick and viscous. In the ovulation phase, the mucus becomes more fluid to facilitate the passage of spermatozoa. In the fast phase of passage, sperm can reach the tube within 5 to 20 minutes after ejaculation. These spermatozoa are less likely to fertilize the egg because they don’t spend enough time in the genital tract to mature. This phase is mostly due to the contraction of the female reproductive tract. The majority of the spermatozoa will reach the uterine tube thanks to their swimming and can take up to 2-4 days. 3. Only several hundreds reach the tube and they bind to the isthmus for 24 hours. Here, the capacitation reaction takes place. Cholesterol and glycoproteins are removed from the spermatozoa, exposing molecules that are able to bind to the zona pellucida. 4. In the period of hyperactivity, the spermatozoa break free of the bond with the tubal epithelium. The combination of muscle contraction and swimming brings them to the ampullary portion of the tube, where fertilization usually takes place. The movement of the cilia and the contractions of the female genital tract push the oocyte towards the uterus, so the spermatozoa have to swim against the current to go towards the ovary. 5. When they reach the oocyte, the spermatozoa need to cross the corona radiata, made of follicular cells of the cumulus oophorus. The release of hyaluronidase from the acrosome catalyses the degradation of hyaluronic acid, the most important component of the follicular fluid. The energetic swimming of the spermatozoa also provides mechanical help in this step. 6. Some spermatozoa manage to cross the corona radiata and they find themselves in contact with the zona pellucida. Binding to the zona pellucida is species specific and is regulated by zonal proteins, in humans ZP3. Binding of these proteins cases a reaction in the acrosome (the Golgi apparatus containing enzymes and covering the nucleus of the spermatozoon). Calcium flows into the cytoplasm of the head of the spermatozoon, intracellular  Isabella Ronchi pH increases and the sperm plasma membrane fuses with the anterior region of the acrosome membrane. Acrosin is released, allowing the spermatozoon to enter the perivitelline space. 7. Finally, the plasma membrane of the spermatozoon fuses with the plasma membrane of the egg; the head, midpiece and tail sink into the egg. A protein called Izumo on the sperm membrane identifies and binds to a corresponding protein, Juno, on the egg membrane. This marks the first stage of fertilization. Within minutes the remaining Juno proteins on the egg membrane are ejected within bubble-like extracellular vesicles. This prevents other sperm cells from entering the egg and interfering with fertilization. The prevention of polyspermy consists in two phases: a fast block and a permanent block. The fast block consists in membrane depolarization (from -70 to +10), which lasts a few minutes and prevents adhesion. The permanent block consists in an increase of calcium concentration, that causes the fusion of the cortical granules with the plasma membrane, releasing their content in the perivitelline space. The perivitelline space swells and becomes harder, so that other spermatozoa that had managed to get into it get stuck. Finally, the zona reaction takes place: sperm receptors proteins (ZP) are hydrolysed and spermatozoa cannot attach anymore. There are changes in the plasma membrane of the egg as well. 8. The entrance of the spermatozoon in the oocyte triggers the completion of meiosis II and the formation of the second polar body. Paternal mitochondria are eliminated either through active elimination (enzymes) or through dilution. The egg is metabolically activated: maternal mRNA is recruited and there is an intensification of egg respiration and metabolism. The sperm nucleus decondenses due to a higher permeability of the nuclear membrane and to loss of protamine. The two pronuclei are formed. When they fuse together and mix their genetic material, the zygote is formed, restoring the normal diploid number. Cleavage Cleavage is a process that begins about 30 hours after fertilization, through which the number of cells increases, without increasing the size of the zygote. With each successive subdivision, the nuclear/cytoplasmic ratio increases. Mammalian cleavage takes a few days; it starts in the fallopian tube and continues down the uterine cavity. The cells formed by cleavage are called blastomeres. When there are about 8-9 cells, compaction begins: it’s a process that maximises cell-to-cell contact, mediated by cell- surface adhesion glycoproteins, including the E-cadherin-catenin complex. After 3 days of cleavage, there are about 16 blastomeres and the zygote is called morula. Two populations of cells can be distinguished: o The trophoblast made of the outer flattened cells connected by tight junctions o The inner cell mass or embryoblast made of the inner blastomeres connected by gap junctions Sodium driven water transport causes the formation of a liquid filled cavity, called blastocoel. When this cavity is formed, the morula is called blastocyst. The zona pellucida remains intact up to this stage, and is important in the communication with the mother. It has many functions: o It promotes maturation of the oocyte o It acts as barrier, allowing only one and the follicle sperm to enter and preventing polyspermy after fertilization  Isabella Ronchi o It initiates acrosomal reaction o It prevents premature implantation o It acts as a filter during cleavage o It acts as an immunological barrier o It keeps together blastomeres between the mother and the o It facilitates differentiation of antigenically different embryo trophoblastic cells The embryo takes charge at the beginning of cleavage: most maternal RNA is degraded by the 2-cell stage, because the DNA of the embryo starts being transcribed and translated. The first example of the embryo taking charge is the formation of the two separate cell lines and the formation of a specialization of the plasma membrane and of the blastomeres. The blastomeres start expressing an apical and a basolateral domain. As the number of cells increases, some of them are going to lose the apical domain, and only those in contact with the zona pellucida maintain it. There are four lineage-specific transcription factors, which play an important role in the formation of the first lineages in the blastocyst. Until the 16-cell stage, cells can still transform: they can become both an inner mass cell or a trophoblast cell. Developmental potency is the types of cell to which a precursor can give rise. Developmental fate is the type of cells to which a precursor normally gives rise. A mitotic error at the cleavage state may result in a mosaic cleavage-state embryo. When forming a blastocyst, the mosaic cell line does not become isolated and persists throughout the trophectoderm and inner cell mass. Implantation The embryo stays in the ampullary portion of the Fallopian tube for 3 days and takes 8 hours to cross the isthmus (progesterone relaxes utero-tubal junction). As long as the zona pellucida is there, implantation cannot take place. Around 6-7 days after fertilization hatching of the blastocyst begins. Microvilli from the trophoblast extend to the zona pellucida and release proteases enzymes. The zona pellucida is digested and the embryo slides out of it. The blastocyst is now able to attach to the mucosa of the uterus. In the meantime, at the end of the menstrual cycle the uterine mucosa is being prepared for implantation. There must be a suitable cellular and nutritional environment and the formation of an immunoprivileged site. There are three states of implantation:  Isabella Ronchi o Apposition: it can only occur in the period of time known as “implantation window”. This reception- ready phase of the endometrium lasts 4 days and comes 6 days after the LH peak. In this stage the blastocyst can still be eliminated by being flushed out. o Adhesion: in many sites of the endometrium, the glycocalyx of the endometrial epithelium becomes thinner, microvilli disappear to prepare a flattened surface and pinopodes appear. Trophoblastic cells and endometrial cells express adhesion molecules. The blastocyst implants on the side of the inner cell mass. During apposition and adhesion, there’s an important process of regulation of immunotolerance. The trophoblast mediates exchanges with the mother through the production of an early pregnancy factor (immunosuppressant protein). The maternal environment, as a matter of fact, could reject the embryo, because it is a semi allograft, meaning that the portion of genetic information coming from the father isn’t recognised as self by the mother. Leukocytes infiltrated the endometrium produce interleukin 2, which prevents recognition of the embryo as a foreign body. o Invasion: the blastocyst invades the endometrium, but it’s important that it doesn’t reach the myometrium. The trophoblastic cells differentiate into two different layers. The inner portion is called cytotrophoblast. The portion of the trophoblast that comes in contact with the epithelium of the endometrium is called syncytiotrophoblast. It’s highly invasive and it produces enzymes that digest the epithelium and the lamina propria of the endometrium. Active finger-like processes extending from the syncytiotrophoblast then penetrate between the separating endometrial cells and pull the embryo into the endometrium. After 9 days, the blastocyst is completely embedded in the uterine wall and the syncytiotrophoblast surrounds the embryo. Small holes called lacunae start forming in the syncytiotrophoblast as it continues to expand. On day 12, capillaries in the endometrium surrounding the developing embryo dilate, giving rise to maternal sinusoids. Enzymes within the syncytiotrophoblast begin to erode the lining of sinusoids and uterine glands. This allows anastomosis between the maternal sinusoids and the lacunar network. While the blastocyst erodes the uterine wall, there might be some vaginal bleeding, which is usually misinterpreted as an abnormal menstrual cycle, if the woman isn’t aware that she is pregnant. A plug of acellular material, called the “coagulation plug”, seals the small hole where the blastocyst implanted.  Isabella Ronchi Adjacent cells of the endometrial stroma respond to the presence of the blastocyst and to the progesterone secreted by the corpus luteum by differentiating into metabolically active secretory cells, called decidual cells. This response is called decidual reaction. The endometrial glands in the vicinity also enlarge, and the local uterine wall becomes more highly vascularized and oedematous. The uterine wall is maintained in a favourable state by the progesterone secreted by the corpus luteum. When an embryo implants, cells of the trophoblast produce the hormone human chorionic gonadotropin (hCG), which supports the corpus luteum and thus maintains the supply of progesterone. hCG can be detected in maternal blood by day 8 and in urine by day 10. From this moment on, the endometrium is called decidua, and it’s divided into: - Decidua basalis, on the side where the embryo implanted - Decidua capsularis, covering the amniotic sac - Decidua parietalis, on the other side from where the embryo implanted The normal implantation site for the blastocyst is the posterior wall of the uterine cavity. Occasionally, a blastocyst can implant in the peritoneal cavity, on the surface of the ovary, within the oviduct, or at an abnormal site in the uterus. These ectopic pregnancies might present with abnormal uterine bleeding, pelvic pain, abdominal pain and massive first trimester bleeding. 95-98% of ectopic pregnancies take place in the Fallopian tubes and they lead to tube rupture and haemorrhage. In ectopic pregnancies hCG is produced at a slower rate, which is why exams might result in a false negative. Ectopic pregnancies are often confused with appendicitis (if the implantation site is the right tube) and with miscarriages. Transvaginal ultrasonography is very helpful for detecting early tubal pregnancies. The main risk factors are: pelvic inflammatory disease (scar tissue in the uterine tubes), endometriosis and smoking (the cilia are affected). Becoming bilaminar Even before implantation occurs, cells of the embryoblast begin to differentiate into two epithelial layers. By day 8, the embryoblast consists of a distinct external layer of columnar cells, called the epiblast, and an internal layer of cuboidal cells, called the hypoblast. The resulting two-layer embryoblast is called the bilaminar embryonic disc. Soon after the embryonic disc has formed, the amniotic cavity appears as fluid begins to collect between the cells of the epiblast and overlying trophoblast. A layer of hypoblast cells expands toward the embryonic pole, and differentiates into a thin membrane, called Heuser’s membrane or exocoelomic membrane. This membrane and the cells of the hypoblast together form the walls of the primitive yolk sac. Simultaneously, the extraembryonic mesoderm forms, filling the remainder of the blastocyst cavity with  Isabella Ronchi loosely arranged cells. Large cavities begin to appear in the extraembryonic mesoderm and they eventually fuse together to form one single cavity, the extraembryonic coelom or chorionic cavity. By day 12, the primary yolk sac is displaced (and eventually degenerates) by the second wave of migrating hypoblast cells, which forms the secondary yolk sac. By day 13, the embryonic disc with its dorsal amnion and ventral yolk sac is suspended in the chorionic cavity solely by a thick stalk of extraembryonic mesoderm, the connecting stalk. During the first week, the embryo eliminates wastes and receives nutrients by diffusion. In the second week, the embryo cannot rely on simple diffusion only anymore, so the uroplacental circulation is developed. The foetus delivers urea, uric acid, water and carbon dioxide. The mother provides oxygen and nutrients (carbohydrates, amino acids, lipids, hormones, vitamins, iron…). Unfortunately, harmful substances can enter the uroplacental circulation too, such as drugs, poisons and viruses. Miscarriage Spontaneous abortions or miscarriages are pregnancy losses that occur before the 20th week of gestation. It is most common during the 3rd week after fertilization. Approximately 25-30% of recognized pregnancies end in miscarriages, usually during the first 12 weeks. A spontaneous abortion occurring several days after the first missed period is often mistaken for delayed menstruation. This is also why women may not even be aware that they are pregnant. More than 50% of known SAs result from chromosomal abnormalities. Other causes may be: failure of the blastocyst to implant (poorly developed endometrium and immunotolerance). After the 10th gestational week, 25-40% of SAs are related to foetal causes, 25-35% to placental causes and 5-10% to maternal causes.  Isabella Ronchi Embryology – 3rd week Gastrulation Gastrulation is the process whereby the bilaminar embryonic disc undergoes reorganisation to form a trilaminar disc. Approximately 15 days after fertilisation, a thickened structure starts to form along the midline in the epiblast, near the caudal end of the bilaminar embryonic disc. This is called the primitive streak. At this stage, the formation of the primitive streak defines the major body axes of the embryo, including the cranial/rostral end (towards the head) and the caudal end (towards the tail), as well as the left and right side of the embryo. Over the course of the next day, the primitive streak elongates to occupy half the length of the embryonic disc, and the primitive groove becomes deeper. The cranial end of the primitive streak is expanded into a structure called the primitive node. It contains a depression, called the primitive pit, which is continuous with the primitive groove. On day 16, epiblast cells migrate inwards towards the streak, detach from the epiblast and slip into the interior of the embryonic disc. This process is known as invagination. The hypoblast cells are eventually completely replaced by the new cell layer, which is referred to as the definitive endoderm. Migrating cells undergo a process called epithelial-to-mesenchymal transformation (EMT): an epithelium consists in a sheet of regularly shaped cells tightly interconnected to one another at their lateral cell surfaces, whilst a mesenchyme consists of much more irregularly shaped and loosely connected cells. During EMT, epiblast cells detach from their neighbours as they extend footlike processes called pseudopodia, which allow them to migrate through the primitive streak. Snail is one of the many factors involved in EMT: it is able to repress epithelial features, for example the expression of cytokeratin, desmoplakin and E-cadherin. By day 16, some epiblast cells migrating through the primitive streak diverge into the space between the epiblast and the nascent definitive endoderm to form a third germ layer, the intraembryonic mesoderm. The mesodermal cells spread everywhere in the space between the endoderm and the epiblast, with the exception of the oropharyngeal membrane and cloacal membrane, at the two extremities of the embryo.  Isabella Ronchi The mouth and the anal and urogenital openings are going to be in these locations. The different subdivisions of the mesoderm are: o Prechordal plate: a meso-endodermal structure located caudally to the oropharyngeal membrane in the midline. It interacts with the anterior visceral membrane. This region will be important to trigger the development of the head and the rostral part of the CNS. o Notochord: a tubular structure elongating rostral to the primitive streak. While the notochord elongates the primitive streak starts regressing. At the beginning the notochord is a hollow tube, which converts to a flattened plate and then to a solid rod. First, the ventral floor of the tube fuses with the underlying endoderm and the two layers break down, leaving behind the flattened notochordal plate. The notochordal plate then completely detaches from the endoderm, and its free end fuses as it rolls up into the mesoderm-containing space between ectoderm and endoderm, changing it to a solid rod called the notochord. It plays an important inductive and patterning role in the early embryo (axial skeleton, neural plate). The only physiological remnant of the notochord in the adult body is the nucleus polposus of the intervertebral discs. Some pieces of the notochord may remain close to the base of the scalp or somewhere in the vertebral column. In these two cases, malignant cancers, called chordomas, develop. They grow very slowly and they can cause compression of brain and spinal structures or symptoms such as headache and double vision. o Paraxial mesoderm: it develops on the sides of the notochord, the regions of the embryo that are going to form the head and the trunk. The trunk portion is going to go through a process of segmentation that will form somites. The head portion will remain unsegmented and will form the head mesenchyme, with the contribution of the neural crest and the prechordal plate. o Intermediate mesoderm: it develops on the sides of the paraxial mesoderm. It will form the kidneys, gonads, urinary system and part of the genital system. o Lateral plate mesoderm: it develops on the sides of the intermediate mesoderm. It’s going to be divided into the splanchnic mesoderm and the somatic mesoderm. It will form the lining of body cavities. o Primitive heart field or cardiogenic mesoderm: located rostrally to the oropharyngeal membrane.  Isabella Ronchi Once the formation of the definitive endoderm and mesoderm is complete, epiblast cells no longer migrate towards the primitive streak. At this point, the remaining cells of the epiblast are referred to as ectoderm. It will give rise to the neural tube and to the outer epithelium of the body. Formation of the neural plate is induced by the primitive node and the notochord. As a result of neural induction, ectodermal cells differentiate into a thick plate of pseudostratified columnar neuroepithelial cells. This process is called neurulation. The neural plate forms first at the cranial end of the embryo and then differentiates in a cranial- to-caudal direction. The neural plate folds during the fourth week to form a neural tube, the precursor of the CNS. The lateral lips of the neural plate also give rise to an extremely important population of cells, the neural crest cells, which are sometimes considered as a 4th germinal layer, as they give rise to the peripheral nervous system, but contributes to the formation of the heart, the eye and the skin. The neural plate is broad cranially and is tapered caudally. The expanded cranial portion gives rise to the brain, while the narrow caudal portion gives rise to the spinal cord. Induction, commitment and regionalisation of the neural plate are guided by the notochord and the prechordal plate. The notochord starts producing some signalling molecules (Noggin, Chordin, Shh) towards the ectoderm. BMP-4 keeps ectodermal cells in their state, but the secretion of those molecules inhibits the effect of BMP-4, so the ectodermal cells can change into the neural plate. The process of gastrulation ends with the formation of the tail bud. It originates from the remnants of the primitive streak, which undergoes to a process called secondary neurulation. The last part of the neural tube proliferates and then undergoes a process of cavitation. This sort of tube connects with the rest of the neural tube. In humans secondary neurulation is not a prominent process. Because gastrulation takes place in a rostro-to-caudal manner, the caudal region is still gastrulating while the layers of the cephalic region begin their specific differentiation. Environmental factors, mutations and maternal diabetes with elevated insulin levels can cause sirenomelia (caudal dysplasia). The lowest part of the body looks like a mermaid, because the lower limbs, urogenital system and lumbar vertebrae fail to develop completely. Defects are also present in the last portion of the digestive and urinary tracts. The formation of body axes depends on the confinement of some molecules and factors in the rostral or caudal region. At the beginning, all factors (Nodal, BMPs, Wnts, FGFs, retinoic acid) are expressed in the same way in the entire epiblast. When the prechordal plate forms, it communicates with the epiblast and produces some factors that restrict the action of other factors to specific regions. The reason why the primitive streak develops in the caudal portion is because there’s a higher concentration of those factors. o In the head region Wnt, Nodal and BMP are decreased o In the trunk region Wnt and Nodal increase, while BMP decreases o In the tail region Wnt, Nodal and BMP increase  Isabella Ronchi Sonic Hedgehog signalling molecule Sonic Hedgehog (Shh) is one of the signalling pathways that are used during development for intercellular communication. Hh is important for the organogenesis of almost all organs in mammals, as well as in regeneration and homeostasis. Furthermore, Hh signalling is disrupted in diverse types of cancer. There are three types of Hh proteins: Shh, Indian-Hedgehog (Ihh) and Desert-Hedgehog (Dhh). Shh has particularly marked roles in nervous system cell type specification and limbs patterning, whereas Ihh has important roles in skeletal development, mainly endochondral ossification. Dhh is restricted to the gonads, including granulosa cells of ovaries and Sertoli cells of the testis. Several evidences demonstrate that embryogenesis and tumorigenesis have common characteristics, where both processes depend on coordinated mechanisms of proliferation, differentiation and migration. Inactive signalling occurs in the absence of Shh ligand, wherein PTCH1 inhibits SMO resulting in GLI1 sequestration in the cytoplasm by SUFU. In the presence of Shh, PTCH1 suppression of SMO is abrogated resulting in the nuclear accumulation of GLI1 and activation of target genes that promote several oncogenic properties of tumour cells. Inhibition of the Shh pathway is primarily directed at inhibition of SMO and GLI1 with many of these compounds in clinical trials for solid cancer. Most activity of Shh signalling pathways takes place at the level of the primary cilium. Primary cilia are fundamental for normal cell signalling during development and homeostasis. Alcohol is a powerful teratogen, that mainly alters the genes expressed in the midline of the body. Drinking while pregnant might lead to failure of activation of Hox genes in the baby and deficiency of activation of Shh pathway. The main consequences in the body are cyclopia and holoprosencephaly (the brain fails to separate in two hemispheres). Symmetry of the embryo In the adult body, the position of some organs is not symmetrical. In mammalian embryos, the current earliest known manifestation of asymmetry involves the beating of the cilia in the primitive node. Node cells contain a monocilium, which is motile in central nodal cells and non-motile in peripheral nodal cells. The motile cilia beat only in one direction. Symmetry breaking molecules are swept on the left side of the embryo, triggering an asymmetric cascade of gene expression. For example, the ciliary currents sweep Shh, retinoic acid and FGF8 to the left, triggering the expression of Nodal and Lefty, which in turn activate homeoboxes. Pitx2 is a homeobox, which contains the transcription factor for establishing left- sideness. Its expression is repeated on the left side of the heart, stomach and gut primordia as these organs are assuming their normal asymmetrical position in the body. 5HT (serotonin) is more concentrated on the left side, whilst MAO (Monoamine Oxidase) is more concentrated on the right side. The mechanosensory model of nodal flow says that Lrd (left-right dynein) is a molecular motor important to move cargos towards the minus end of microtubules, but also in bending cilia and flagella, creating a sliding force between microtubules. The nodal vesicular parcels model says that the morphogens are packaged inside membrane vesicles, and moved across the node by nodal flow to establish a left-right gradient of morphogens. Abnormalities in the ciliary movement at the node may cause:  Isabella Ronchi o Situs inversus totalis: inversion of all the organs with respect to the right-left axis (i.e., the stomach on the right, liver on the left, appendix on the left…). It doesn’t lead to any complications. It’s caused by a mutation in the Lrd gene. o Kartagener syndrome: situs inversus totalis + immotile respiratory cilia and sperm flagella. It’s caused by mutations in dynein genes and deficiency in ciliary dynein arms.  Isabella Ronchi Embryology – 4th week By the end of the 3rd week, the embryo has a rostro-caudal axis, a dorso-ventral axis, symmetric bilaterality and three tissue layers. The organogenetic period goes from the 4 th to the 8th week. All major internal and external structures are established. They all have minimal function, except for the cardiovascular system. The heart starts beating around day 21-22 and can be heard by Doppler ultrasonography during the 5th week. Tissues and organs grow and differentiate very rapidly during this period and are therefore more susceptible to teratogens, which can lead to the development of major abnormalities. Thalidomide was a powerful anti- nausea drug, that created birth defects, for example very short upper (if drug started in the 4th week) and lower (If drug started in the 5th week). From 1962 it became mandatory to test new drugs on pregnant animals as well to test their effect on foetuses. Segmentation After gradients of gene products have been established, further development along the rostro-caudal axis is mediated by segmentation genes, which are zygotic-effect genes and they function to subdivide the embryo into smaller and smaller regions along the axis. Segmentation involves the paraxial mesoderm of the trunk region and the formation of somites. 42-44 pairs of Somitomeres are formed (one on each side). Somitomeres 1-7 don’t form somites and the more caudal are going to disappear. So, the final count of somites is 35-37. Segmentation is guided by opposite gradients of proliferating factors (FGF8, more concentrated in the caudal portion) and differentiating factors (retinoic acid, more concentrated in the rostral region) and gene expression. Genes oscillate in their expression from rostral to caudal (permissive-non permissive state): for a somite to form the wavefront must reach them when they are in a permissive state. Segmentation along the cranio-caudal axis becomes less and less evident with development, with the exception of the vertebral column and the spinal cord. Each somite forms a: o Sclerotome: involved in the formation of intervertebral discs, distal ribs, neural arch, meninges. Each vertebra is formed by two adjoining sclerotomes. Arthrotomes contribute to the formation of intervertebral discs, vertebral joint surfaces and proximal ribs. o Dermatome: involved in the formation of the dermis and the blade of the scapula. o Myotome: involved in the formation of intrinsic back muscles and limb muscles. It’s divided into the epimere (dorsal), that will give rise to epaxial muscles and hypomere (ventral), that will give rise to the hypaxial muscles. The syndetome gives rise to the tendons of epaxial musculature.  Isabella Ronchi Some myoblasts from the hypomere are going to migrate in the forming limbs. The muscles of the limbs derive from progenitor myogenic cells that migrate into the limb bud from the ventral portion of the myotome. The muscle progenitors initially form two major muscle masses as the limb bud forms. The ventral muscle mass gives rise mainly to the flexors, pronators and adductors, whereas the dorsal muscle mass gives rise mainly to extensors, supinators and abductors. Axial bones derive from the somites, limb bones from the lateral plate mesoderm and craniofacial bones come from the mesoderm of the head and neural crest cells. Segmentation of the paraxial mesoderm also affects the neural tube. The neural tube adjacent to somites will be divided into functional segments: the spinal cord segments, called neuromeres. Each segment innervates the cutaneous territory (dermatome), the muscular territory (myotome) and the bony-tendinous territory (sclerotome) originating from the adjacent somite. This connection will be maintained all throughout adult life, no matter the distance between the spinal cord and the innerved organs. Folding of the embryo During the 4th week, the embryo folds. The embryo is suspended in the chorionic cavity by the connecting stalk and it has dorsally the amniotic sac and ventrally the yolk sac. The embryo becomes a tubular structure, forming a tube within a tube. The outer tube is due to the formation of the body wall that delimits the chorionic cavity; the vitelline sac is also partially integrated in the embryo and it will form the primitive intestine. The embryonic disc and the amnion grow vigorously, but the yolk sac hardly grows at all. The developing notochord, neural tube and somites stiffen the dorsal axis of the embryo; therefore, most of the folding is concentrated in the thin, flexible outer rim of the disc. Forward growth of the neural plate causes the thin cranial rim of the disc to fold under, forming the ventral surface of the future face, neck and chest. Starting on about day 23, a similar process of folding commences in the caudal region of the embryo as the rapidly lengthening neural tube and somites overgrow the caudal rim of the yolk sac. The thin caudal rim of the embryonic disc, which contains the cloacal membrane, folds under and becomes part of the ventral surface of the embryo. The connecting stalk (which connects the caudal end of the embryonic disc to the developing placenta) is carried cranially until it merges with the neck of the yolk sac, which has begun to lengthen and constrict. The root of the connecting stalk contains a slender endodermal hind-gut diverticulum called the allantois. The right and left sides of the embryonic disc flex sharply ventrally, constricting and narrowing the neck of the yolk sac. At the head and tail ends of the embryo these lateral edges of the embryonic disc make contact  Isabella Ronchi with each other and then zip up towards the site of the future umbilicus. When the edges meet, the ectodermal, mesodermal and endodermal layers on each side fuse with the corresponding layer on the other side. As a result, the ectoderm of the original embryonic disc covers the entire surface of the three-dimensional embryo except for the future umbilical region, where the yolk sac and the connecting stalk emerge. When the cranial, caudal and lateral edges of the embryo meet and fuse, the cranial and caudal portions of the endoderm are converted into blind-ending tubes, the future foregut and hindgut. At first, the central midgut region remains broadly open to the yolk sac. However, as the gut tube forms, the neck of the yolk sac is gradually constricted, reducing its communication with the midgut. By the end of the 6 th week, the gut tube is fully formed, and the neck of the yolk sac has been reduced to a slim stalk called vitelline duct. The cranial end of the foregut is capped by the oropharyngeal membrane, which ruptures at the end of the 4th week to connect the future oral cavity to the foregut. The caudal end of the hindgut is capped by the cloacal membrane, which ruptures during the 7th week of development to form the urogenital and anal openings. The lateral plate mesoderm splits into two layers: the somatopleaura (parietal layer), which associates with the ectoderm and the splanchnopleaura (visceral layer), which associates with the endoderm. The splanchnic mesoderm surrounds the gut tube and the somatic mesoderm forms the body wall. The space between these layers is originally open to the chorionic cavity. However, when the folds of the embryo fuse along the ventral midline, this space is enclosed within the embryo and becomes the intraembryonic coelom, mesothelium derived from the mesoderm (parietal and visceral serosae). Organs are suspended in the coelomatic cavity by mesenteries, formed by the splanchnic mesoderm. The ventral mesentery will disappear quickly, while the dorsal mesentery remains in many organs and it contains vessels and nerves directed to the viscera. Most triploblastic animals have a fluid-filled space somewhere between the body wall and the gut. Such a cavity can provide numerous functional advantages. The intraembryonic coelom acquires a U shape appearance and different portions of it are going to form the body cavities lined by a serosa (pericardial, peritoneal and pleural). The somatopleaura is closed laterally in the most rostral region and it’s open caudally, where it allows communication between the intraembryonic and extraembryonic cavities. The advantages of having an intraembryonic coelomatic cavity include: o Organs formed inside a coelom can freely move, grow and develop, independently of the body wall while fluid cushions and protects them from shocks o Peristalsis of the gut does not affect the body wall o Movements of the body wall during locomotion do not distort the internal organs o Organs can be attached to each other by ligaments so that they can be suspended in a particular order while still being able to move freely within the cavity o Space for growth of new membranes of the species inside the maternal body o Formation of an efficient circulatory system Formation of the cardiovascular system  Isabella Ronchi Cranial to the oropharyngeal membrane, a second important structure has begun to appear: the horseshoe- shaped cardiogenic area, which will give rise to the heart. Cranial to the cardiogenic area, a third important structure forms: the septum transversum. This structure appears on day 22 as a thickened bar of mesoderm. The septum transversum forms the initial partition separating the coelom into thoracic and abdominal cavities and gives rise to part of the diaphragm and the ventral mesentery of the stomach and duodenum. If on one side complete closure doesn’t take place, the pleural cavity and the peritoneal cavity keep communicating, causing Associated diaphragmatic hernia. The organs in the abdominal cavity exert a lot of pressure, so if there is an opening, some organs will protrude in the thoracic cavity, compressing the lungs. The baby will be born with hypoplastic lungs and dyspnoea. Because of the median folding, the heart region becomes ventral, in front of the foregut. The septum transversum, primordial heart and pericardial coelom and oropharyngeal membrane move on the ventral surface of the embryo. Their sequence is inverted 180°. Folding in the rostral region leads to the fusion of the two endocardial tubes, which form a single tubular heart, surrounded by the pericardial cavity. The formation of primitive blood vessels begins at the beginning of the 3 rd week. The formation of primitive blood vessels begins in the extraembryonic mesoderm of the yolk sac, in the connecting stalk and chorion. Two days later blood vessels start to form also in the embryo. Blood vessel formation takes place by vasculogenesis and angiogenesis. Vasculogenesis is the formation of vessels ex novo from precursors. Mesenchymal cells differentiate into smooth muscle cells, endothelial cells and the other components of blood vessels. Angiogenesis consists in budding from existing vessels (this process is also present in adults during the menstrual cycle, wound healing and tumours). Tumours require blood supply to grow, so low oxygen environments trigger angiogenesis (production of Vascular Endothelium Growth Factor A binding to VEGF-R2 on adjacent endothelial cells). The new vessels are leaky and facilitate intravasation and metastasis of tumour cells. Also, other local cells (e.g., neutrophils, lymphocytes, macrophages) produce angiogenic factors. Vasculogenic mimicry refers to the ability of cancer cells to organise themselves into vascular-like structures to obtain nutrients and oxygen independently of normal blood vessels or angiogenesis. In the tubular heart we can recognize an inflow portion and an outflow portion. The blood pumped out from the heart is medium oxygenated.  Isabella Ronchi The inflow portion includes: o Umbilical veins (richly oxygenated); o Vitelline veins (poorly oxygenated); o Cardinal veins (poorly oxygenated). The outflow portion includes: o Pharyngeal arch arteries (dorsal aortae), give rise to the vitelline arteries (blood to the sac and to the primitive intestine); o Umbilical arteries (from the body of the embryo to the placenta); o Segmental arteries (perfuse the body of the embryo).  Isabella Ronchi Embryology Maternal adnexa The embryo must establish a “parasitic” relationship with the mother, in order to acquire oxygen and nutrients and to eliminate wastes. It also must avoid being rejected as a foreign body. These requirements are fulfilled by the placenta and extraembryonic membranes that surround the embryo and serve as an interface with the mother. These structures are: the placenta and the chorion (deriving from the trophoblast) and amnion, yolk sac, allantois and extraembryonic mesoderm (deriving from the inner cell mass). Amniotic sac and amniotic fluid After folding, the embryo is completely surrounded by the amniotic sac. Its main functions are: o It protects from mechanical injury o It protects the foetus from adhesion o It accommodates growth of the embryo o It acts as a barrier to infections. It has o It allows foetal movement and muscular bacteriostatic properties. development o It maintains a relative constant o It allows foetal respiratory movements. temperature The ribcage expands and relaxes, even o It assists in maintaining homeostasis of though the foetus cannot exchange oxygen fluids and electrolytes with the outside. The amniotic membranes have anti-inflammatory and anti-angiogenic properties, so they can be used to cover wounds and burns, as they reduce pain and inflammation and enhance the wound healing process, by providing a matrix for migration and proliferation of cells. Amniotic fluid comes first from maternal plasma through the decidua parietalis, and then from the chorionic plate. From the 11th week the kidneys of the foetus start to work and they produce a sort of primitive urine, which is released into the amniotic sac. In the late pregnancy the foetus produces 500 ml of urine per day. The amount of amniotic fluid in a healthy woman is 30 ml by the 10 th week, 350 ml by the 20th week and 700-1000 ml by week 33-34. Every three hours all the amniotic fluid is absorbed by the amnio-chorionic membrane into maternal tissues and some of it is also swallowed by the foetus. Swallowing amniotic fluid promotes the development of the digestive tract, foetal circulation and placenta. The amniotic fluid contains: foetal epithelial cells, proteins, carbohydrates, hormones, pigments, electrolytes, lipids and foetal lung fluids. Because it contains foetal cells, amniocentesis is useful to detect chromosomal abnormalities, CNS abnormalities, omphalocele, duodenal or oesophageal atresia and Rh disease. A reduced amount of amniotic fluid is called oligohydramnios. The causes might be: diminished placental blood flow, preterm rupture of the amnio-chorionic membrane, renal agenesis or urinary tract obstruction. Foetal complications include: pulmonary hypoplasia, growth restriction, facial distortion (Potter syndrome). An increased amount of amniotic fluid is called polyhydramnios. Maternal factors include diabetes, cardiac problems and infection. Foetal factors are: oesophageal or duodenal atresia, anencephaly (poor development of the brain) and infection. Sometimes the amniotic membrane might even rupture, producing amniotic bands what could wrap around part of the foetus’ body. This can lead to constriction or amputation of portion of the body.  Isabella Ronchi Yolk sac (vitelline sac) In other animals that don’t have a placenta, the yolk sac is the major source of nutrition. In humans it is relevant because part of it is incorporated in the body of the embryo for the development of the digestive tract. The formation of the vitelline stalk or canal (omphalomesenteric duct) allows communication between the embryo and the yolk sac. It will be incorporated in the umbilical cord. Allantois It’s an evagination of the yolk sac into the connecting stalk. In other animals it’s important for the elimination of waste and for oxygen exchanges, so the allantois is very big. In humans blood vessels connecting to the placenta form around the allantois. The umbilical circulatory arc is formed by arteries and veins that connect the embryo to the placenta. It will also form the urachus, continuous with the urinary bladder. Chorion and placenta The extraembryonic tissues of the embryo and the maternal endometrium cooperate for the development of the placenta. The extraembryonic mesoderm that is part of the chorion is also called chorionic plate. The chorion is made of the cytotrophoblast and the extraembryonic mesoderm. The side of the endometrium where the embryo has implanted is called decidua basalis, while the opposite side is the decidua capsularis. Chorionic villi will grow more on the side of the embryo (chorion frondosum) than the opposite side (chorion leave). In the secondary villi there’s a core of mesoderm, where a capillary network will form to exchange nutrients with the blood contained in the maternal lacunae in the syncytiotrophoblast. The tertiary villi form only on the side of the decidua basalis and they will give rise to branches. The branches float in the maternal lacunae. The syncytiotrophoblast erodes the wall of the spiral arteries of the endometrium. A lot of maternal blood flows into the lacunae at low pressure. Foetal blood has a lower concentration of oxygen and a lower partial pressure of oxygen than maternal blood. Foetal hemoglobin has higher affinity for oxygen. The tertiary villi present capillaries, which will connect to the system of the umbilical veins and arteries. The cytotrophoblast at the apex of some tertiary villi start to proliferate and it surrounds the syncytiotrophoblast (cytotrophoblastic shell). Blood enters in the intervillous spaces from spiral arteries in a pulsatile way, it spurs against the chorionic plate and slows down. The floating villi exchange gases and nutrients. The blood is then picked up by the open ends of the uterine veins that also penetrate the cytotrophoblastic shell. The intervillous spaces contain 150 mL of blood and they are replenished 3-4 times/minute. By the 5th month of gestation, the cytotrophoblast cells disappear, so substances only need to cross the endothelium and the syncytiotrophoblast. The main exchanges are:  Isabella Ronchi From mother to foetus From foetus to mother o Oxygen o Carbon dioxide o Water, electrolytes o Water, electrolytes o Nutrients o Urea, uric acid o Hormones o Creatinine o Antibodies (passive immunity) o Bilirubin o Vitamins o Hormones o Iron o Red blood cell antigens o Harmful substances Infectious diseases can be transmitted through the placenta, but also when the foetus is passing through the birth canal at delivery. An example is Zika virus, which can lead to microcephaly, ocular abnormalities, intrauterine growth restriction and foetus demise. There are several pathways by which Zika virus and other TORCH (Toxoplasma gondii, other, rubella virus, cytomegalovirus, herpes simplex virus) pathogens might be vertically transmitted. The placenta can be considered as an endocrine organ. It produces human chorionic somatotropin or placental lactogen. It’s similar to the growth hormone and it’s produced by the syncytiotrophoblast to regulate maternal metabolism and blood glucose. The foetal side of the placenta lacks the Major Histocompatibility complex in the syncytiotrophoblast and in the villous cytotrophoblast. However, it is present in foetal tissues and placental stroma. There is also a sort of paralysis in the mother’s immune system, at least a selective repression to a response to foetal antigens. There is also a decidual immune barrier and molecules produced on the foetal placental surface that inactivate maternal immune cells locally. If the placenta doesn’t work properly, this can lead to Intra Uterine Growth Retardation (IUGR). The amnion and the smooth chorion (chorion leave) fuse to form the amnio-chorionic membrane, which will fuse with the decidua parietalis after the disappearance of the decidua capsularis. The foetal side consists in the chorion frondosum (chorionic plate and villi) and it’s shiny due to the amnion. The maternal side consists in the decidua basalis, covered by foetally-derived cytotrophoblastic shell. It’s dull and divided into around 35 lobes, each lobe with cotyledons (main stem villus and its branches). The placenta is connected to the embryo by the umbilical cord, formed by the vitelline duct and the connecting stalk.  Isabella Ronchi Pathological placentas If the syncytiotrophoblast is too invasive and touches the myometrium: o Placenta accreta: abnormal adherence of chorionic villi to the myometrium (partial or complete absence of the decidua basalis). Pregnancy and birth are normal, but after birth the placenta fails to detach and attempts at removal may cause haemorrhages that are difficult to control. o Placenta increta: penetration of the myometrium. o Placenta percreta: complete penetration of the myometrium to reach the perimetrium; can result in attachment to rectum or bladder. Placenta previa is the attachment of the placenta at the level of the internal uterine os. Blood vessels may rupture during late pregnancy. The mother may bleed to death and the foetus may suffer because of reduced blood supply. Placenta abruption is partial or complete premature separation of the placenta from the uterine wall. The main causes are: trauma, smoking, hypertension, use of drugs. The symptoms are: abrupt painful bleeding in 3rd trimester, foetal distress and possible diffuse intravascular coagulation. The mother may bleed to death and the foetus may suffer because of reduced blood supply. Vasa previa happens when blood vessels run over or in close proximity to the cervical os. This can lead to painless vaginal bleeding, foetal bradycardia (because the vessels are compressed and the foetus doesn’t receive enough blood) and vessel rupture. Choriocarcinoma is a carcinoma of the trophoblast during pregnancy, abortion or hydatiform mole. The hydatiform mole is a condition due to a problem in the development of chorionic villi, which have a grapelike aspect. This is not a functional placenta; foetal tissue doesn’t develop or only partially develops. Choriocarcinomas can spread to the liver and to the brain. The mole is usually of paternal origin, and the maternal chromosomes are usually inactivated. Hydatiform mole may present with vaginal bleeding, uterine enlargement, pelvic pressure/pain, excessive production of hCG and hyperemesis. Twinning o Dizygotic twins: two oocytes are fertilised. There are two amnions, two chorions and two placentas. The placentas and chorions may fuse if blastocysts implant close. If the two placentas are too close, one embryo might steal blood from the other. The twins can have different sex and they are genetically alike as brothers and sisters. o Monozygotic twins: one oocyte is fertilised. The most common type of twinning begins at the blastocyst stage, by the end of the first week. One blastocyst develops two inner cell masses, two amniotic sacs, one placenta, one chorionic sac. Sometimes separation takes place earlier, from 2- blastomeres to the morula stage. This leads to the formation of two blastocysts, so two chorionic sacs, 2 amniotic sacs and two placentas. Another possibility is separation of the embryonic disc, which  Isabella Ronchi leads to the formation of one placenta, one amniotic sac and one chorion. Two primitive streaks are formed, but their different angulation and non-separation due to aberrant hypoblast configuration. This is the situation that is most likely to give rise to conjoined twins.