Embryology III PERIMPLANTATION DEVELOPMENT autumn 2024 Zuzana Holubcová Department of Histology and Embryology zholub@med.muni.cz Placentation and establishment of pregnancy Embryo nutrition 3) Hemotrophic nutrition - 12 wg to term - nutritients obtained from maternal blood - oxidative phosphorylation 1) Cytotrophic nutrition - from conception (D0) to D10 pc - use of maternal cellular sources - quite metabolism 2) Histiotrophic nutrition - D11 pc -12 wg - nutritients obtained from maternal uterine gland secretions - glycolysis, low 02 environment Perimplantation stage TrophectodermInner cell mass Cytotrophoblast EpiblastHypoblast Amnionic sac Day 7-8 Syncitiotrophoblast (primitive ectoderm)(primitive endoderm) Blaminar embryonic disc Primitive yolk sac Placentation Key steps of placenta development: 1) Trophoblast differentiation and invasion 2) Developing of chorionic villi (branching and angiogenesis) 3) Angiogenesis and remodeling of maternal vasculature Papuchova and Latos 2022 - establishing of a stable maternal-fetal interface in specialized ephemeric organ = placenta - contribution of both maternal and fetal cells Giant cells - multinuclated terminal differentiation stadium of EVT - in contact with myometrium, looses invasive capacity - invade stroma, uterine glands, and spiral arteries - interact with uNK cells and macrophages - mediate induction of immune tolerance - initiate process of vascular remodelling Villous Cytotrophoblast (vCTB) - key role in vascular remodeling Trophoblast differentiation - develop anchoring villi system - erode maternal tissue - direct contact with maternal blood - responsible for deep placentation Development of chorionic villus • Villous cytotrophoblast - proliferative CTB cells make protrusions penetrating primitive syncitium primary villi Carlson 2009 + branching and infiltration by hypoblast-derived extraembryonic mesoderm (ExM) secondary villi tertiary villi + vascularization • Cytotrophoblastic shell - continuous external CTB layer - encapsulates fetus at maternal-fetal interface - anchors embryo to decidua - defects (thin, premature, disorganized structure) can lead to spontaneous miscarriage Carlson 2009 • Cytotrophoblastic column - rows of proliferative cells connecting tertiary villi to cytotrophoblastic shell - cease mitosis and undergo endoreplicative cycles (polyploidization) at distal ends → extravillous trophoblast (EVT) Development of chorionic villus • Extravillous trophoblasts (EVTs) ← differentiation of villous CTB - single cells detaching from tips of anchoring villi (cell column trophoblast) - migrate through stroma and invade uterine glands, and spiral arteries Sternberg et al 2021 Knofler et al 2013 Development of chorionic villus - transformation of narrow high-resistence maternal vessels to highly dilated low resitence conduits Knofler et al 2019 Vascular remodeling - iEVT - induce apoptosis and cellular dedifferentiation of the smooth layer - eEVT - adopt vascular adhesion phenotype - interdigitate into endotelial layer and replace maternal endotelial cells - form trophoblast plugs Trophoblast plugs - Temporary occlusions of spiral arteries made by endovascular EVT → low 02 environment ➢ Promotion of angiogenesis and vasculogenesis ➢ Enhancement of placenta development ➢ Prevention of ROS-induced damage Vascular remodeling O´Brian and Wang 2023 Disintegration of trophoblast plugs at the end of first trimestr → perfusion with highly oxigenated maternal blood Vascular remodelling Vascular remodeling Dimitriadis et al 2023 - during the first trimester of pregnancy, narrow, high resistance and low capacity uterine spiral arteries are transformed to wide, high-flow an low resistance vessels capable to provide hemotrophic nutrition during later stages of pregnancy - vessel dilatation: 0.25 → 2-3 mm (3x ↑ 02 tension) = chorionic vesicle - layer encapsulating embryo - consist from trophoblast cells and extraembryonic mesoderm Chorion 5 wg ➢ primary and secondary villi project uniformly from entire outer surface ➢ tertiary villi formed asymmetrically, preferentially close to decidua basalis ➢ villi at embryonic pole grow and branch (chorion frondosum) ➢ villi at abembryonic pole atrophy (chorion leave) 4th month ➢ chorion laeve fuses with decidua parietalis ➢ chorion frondosum fuses with decidua basalis forming discoid placenta Chorion Uteroplacental circulation Calson 2012 - direct contact of endometrial/fetal tissue with maternal blood - chorionic villi bathed in maternal blood filling intervillous space - capable to support fetal development for ~ 42 gw (placental senescence) ❖Hemochorial placenta overall surface 12-14 m2 Function of placenta ❖ Oxigenation ❖ Nutrition ❖ Excretion ❖ Immunity ❖ Endocrine function - hCG, hPL ❖ Anabolism (glycogen, cholesterol, fatty acids) Placental barrier - prevents direct contact of maternal and fetal blood cells 1st trimestr 3rd trimestr - basal lamina (ECM) - cytotrophoblast (CTB) forms a continous layer beneath the syncithiotrophoblast (SCT) - basal lamina (ECM) - discontinuous CTB layer beneath thin STB cytoplasmic layer Placental barrier - transcellular gradient transfer across STB syncitium via facilitated diffusion O´Brian and Wang 2023 STB cytosol Placental barrier defects of placental vasculature Erytroblastosis fetalis = Hemolytic Disease of the Newborn (HDN) fetal blood cell´s hemolysis small numbers of fetal cells can escape to maternal circulation mother develops antibodies against allogenic epitopes hemorrhage during previous delivery (e.g. Rh factor, Kell) paracellular transfer Placental barrier - defects in placental vasculature small numbers of fetal cells can escape to maternal circulation Microchimerism = presence of cells from one individual in another genetically distinct individual Boddy et al 2015 - bi-directional cell trafficing between fetus and mother during pregnancy - positive and negative effects Peri-implantation embryo Lyonization = X-Chromosome inactivation - one of the X chromosomes in female embryos is packed into transcriptionaly inactive heterochromatin - demethylation of X-inactivation center on X-chromosome (XIST) → translation of long non-coding RNA (lncRNA) that coats the entire X chromosome and associates with chromatin remodelling proteins inducing epigenetic silencing of genes encoded on one of the two X chromosomes in female cells lncRNA Lyonization = X-Chromosome inactivation Talon et al 2019 Transient reactivation in ICM of preimplantation mouse blastocyst - occurs randomly in peri-implantation female embryos - in PGCs X chromosome reactivate Lyonization = X-Chromosome inactivation - dosage compensation of X-coded genes between female and male cells - influences severity of X-linked diseases in females → sexual dimorphismus in placental function Lyonization = X-Chromosome inactivation - some genes are known to escape X inactivation → increased gene expression in females relative to males e.g. sex-dependent expression of SMS gene and synthesis of antioxidant spermine - males more susceptible to nutritional insults - greater fetus to placenta weight in females Gong et al 2018 Maxwell et al 2023 Gastrulation 3rd week pc - Establishment of 3 germ layers Early derivatives inner cell mass trophoectoderm epiblast hypoblast cytotrophoblast syncytiotrophoblast extraembryonic endoderm extraembryonic mesoderm yolk sac endoderm embryonic ektoderm embryonic mesoderm embryonic endoderm allantoic endoderm neural crest primordial germ cells villous cytotrophoblast extravillous cytotrophoblast Germ layer derivatives Calson 2012 Clinical pregnancy Lim et al 2021 „heart“ beat detection by ultrasound Ongoing clinical pregnancy - placenta and organ systems fully formed Live birth - gestational age 38-42 weeks - ~ 50 cm, 3-3.5 kg - eutrophic - signs of fetal maturity Congenital anomalies - major anomalies present in ~3% of liveborn infants - induced by genetic and/or environmental factors Prevention of congenital anomalies Estimated RISK with respect to maternal age ❑ Non-invasive diagnotic methods ➢ Anamnesis ➢ Ultrasound ➢ Biochemical testing -hCG, AFP, PAPP-A, eE3 ➢ cfDNA ❑ Invasive diagnotic methods ➢ PGT ➢ Chorionic villous sampling ➢ Amniocentesis ➢ Fetal blood sampling ➢ Induced abortion 15-18 wg11-13 wg Pregnancy complications linked to placenta pathology ➢ Pre-eclampsia ➢ Gestational trophoblastic diseases ➢ Pregnancy loss after 12 wg - clinical miscarriage - silent/missed miscarriage ➢Fetal growth restriction ➢Fetal macrosomia ➢ Preterm labour ➢ Still birth ➢Ceasarian section ➢Placenta accreta „fetoreduction“ = elective reduction of number of fetuses in high risk multifetal monochorial pregnancies Pre-eclampsia Dimitriadis et al 2023 - life-threatening disease of pregnancy caused by placenta dysfunction - exclusive to humans, 1-5% pregnancies (20+wg) - leading cause of maternal and neonatal morbidity - survivors have long-term complications and high risk of later development of metabolic and cardiovascular diseases - complex multisystem disease - manifested by - sudden-onset hypertension - proteinuria - edema - complications - HELLP syndrome = Hemolysis , Elevated Liver enzymes and Low Platelet count - Eclampsia - seisures and coma - Haemorhagic stroke - Placental abruption - Renal failure ➢ preterm pre-eclampsia with early onset < 34 wg ➢ preterm pre-eclampsia with late onset ≥ 34 wg ➢ term pre-eclampsia ≥ 37 wg ➢ post-partum pre-eclampsia - diagnosed after delivery Pre-eclampsia Dimitriadis et al 2023 - caused by placenta dysfunction caused by defective or insufficient (“shallow“) placentation placental malperfusion (ischemia and reperfusion injury), disregulated release of placental factors, angiogenic imbalance, maternal endothelia dysfunction, reduced vasodilatation and systemic inflammation ? poor invasion and/or premature senescence of the trophoblast ? STB stress ? inadequate spiral artery remodelling ? incomplete plugging and premature onset of blood perfusion ? imune imbalance at maternal-fetal interface ? excessive blood clotting ❖ Proposed mechanisms: Pre-eclampsia Exact etiology? Lian et a l 2022 Role of miRNAs Pre-eclampsia ❖ Antiphosholipid syndrom (APS) - autoimmune disorder associated with pregnancy complication including pre-eclampsia - caused by antiphospholipid antibodies (APA) raising risk of blood clotting ❖ Systemic Lupus Erythematosus (SLE) - autoimmune disease in which immune system mistakenly attacks nuclear and cytoplasmic antigens in the body - multisystem inflammation and increased risk of infertility and pregnancy complication including preeclampsia Poor peripherial circulation Pre-eclampsia - autoantibody effect on fetal maternal interface Meroni et al 2018 - heterogenous group of pregnancy-related disorders arising from abnormal proliferation of placental trophoblast Gestation Trophoblastic diseases Lok et al 2021 Molar pregnancy = Hydatidoform mole (mola hydatidosa) - overproliferation and dysmorphysim of chorionic villi - vascular obliteration and nodular swellings - embryo is absent or non-viable cause or consequence of trophoblastic transformation? Molar pregnancy Froeling and Seckl 2014Froeling and Seckl 2014 Partial Mole Complete Mole Molar pregnancy KHDC3L-mutated or - imbalance of genomic imprinting, dominance of growth-promoting paternal genes Molar pregnancy ➢ Clinical manifestation: - Vaginal bleeding - Uterus enlargement - Extremely elevated hCG - Hyperemesis Gravidarum - Theca lutein cyst - Hyperthyreoidism - Tachycardia - High blood pressure RISK of developing post molar tumor ! Choriocarcinoma - Malignant trophoblastic cancer - consists of CTB and SCT cells at the absence of chorionic villi - Elevated production of hCG - Metastasis spreading typically to lungs Preceded by ← molar pregnancy ← abortion ← normal gestation ← ectopic pregnancy ← de novo = germ cell tumour = gestational choriocarcinoma Research of peri-implantation development Zhou et al 2021 Fixed hysteroscopy samples Drug-treated hESCs or iPSC (induced trophoblast stem cells iTSC) Research of peri-implantation development „14 day rule“ 1979 - Ethics Advisory Board of the US Department of Health, Education and Welfare - embryos shall not be kept alive in vitro longer than 14 days after fertilisation or the stage of development that is equivalent to when embryos finish implantation Research of peri-implantation development „14 day rule“ Magdalena Zernizka-Goetz - introduction of culture system capable to support embryonic development past implantation stage Bedzhov et 2014 Shahbazi et 2016D13 pc D8.5 pc - recapitulation of key features of periimplantation embryo development CGB, HLA-G- ECM coated dishes, no endometrium! Research of peri-implantation development „14 day rule“ 2021 - drop of 14 day limit Research of peri-implantation development Papuchova and Latos 2022 ❖ Gastruloids Research of peri-implantation development - stem cell-based models recapitulating gastrulation process - 2D/3D structures generated by aggregation of pluripotent mouse/human stem cells - trackable and scaleable culture Drug treatment - mimic features of early mammalian development and generate embryonic cell types Sullivan and Santos 2023 Research of peri-implantation development ❖ Gastruloids Ex utero development Uterus transplantation - uterus agenesis - anatomical malformations - previous hysterectomy