Embryology III PERIMPLANTATION DEVELOPMENT autumn 2024 Zuzana Holubcová Department of Histology and Embryology zholub@med.muni.cz Embryo preparation for for implantation Pre- and periimplantation embryo development Embryo hatching - blastocyst´s escape from a porous glycoprotein coat – Zona pellucida - a key requirement for successful embryo implantation and establishing pregnancy - D6-D7 in humans, in the uterus in vivo, but can happen spontaneously in vitro (uterine effectors not crically required) - preceded by blastocyst expansion and followed by embryo attachment to the endometrium - failure to hatch suggests compromised embryo fitness - hatching delay might cause the embryo to miss the uterine receptive window Embryo hatching site Multiple site hatching Hatching site abembryonic poleembryonic pole U-shaped 8-shaped Embryo hatching mechanism - relatively unknown - 2 forces - 1) physical = mechanical pressure exerted on ZP by blastocyst expansion - 2) chemical = enzymatic digestion of ZP ←lytic enzymes secreted by embryo´s mural TE e.g. trypsin-like Ser-proteases, Cys-proteases, matrix metalloproteinases (MMPs), tissue plasminogen activators (tPAs), cathepsins... ←proteases from uterine milieu - strypsine, lysine - Stages: 1) ZP softening, thinning, and local rupture 2) gradual widening of ZP perforation 3) penetration of leading TE cells through ZP 4) active movement of cellular mass outside of the ZP Role of cytokines? Role of TE cytoskeleton? Role of desmosomes? Mechanism of embryo hatching Mechanism of embryo hatching ❖ „Zona-breaker cells” - TE cells located at points of hatching lining and protruding through ZP - not epithelial-like morphology but a “plump” appearance - specialized TE projections that first invade ZP (“hatching pioneers”) TE epithelial-like cells ZP-breaker cells Sathanathan 2003 Mechanism of embryo hatching ❖ „Zona-breaker“ cells - have surface microvilli interacting with the ZP and large bundles of contractile tonofilaments - contain lysosomes and secrete vesicles that interact with the ZP Sathanathan 2003 - intense vacuolisation related to secretolytic activity Mechanism of embryo hatching ❖ „Zona-breaker“ cells Mechanism of embryo hatching - proteomics (LC-MS) - ZP breakers (first hatching cells) compared to TE cells inside ZP - upregulation of specific proteins in the zona breaching cells Almagor et al 2020 Hatching TETE inside ZP SERPINs (Ser-protease inhibitors) Paraoxonase Myosin N-acetylmuramoyl -L-alanine amidase Galectin Actin Histidine-rich glycoproteinLipocallin Gamma butyrobetaine hydroxylase Lupus La protein ❖ „Zona-breaker“ cells Assisted hatching - ZP hardening occurring during in vitro culture and freezing/thawing reduces the chance of spontaneous hatching - lower hatching success in conventional IVF cycles (50%) than after ICSI which leaves small openning in ZP - assisted hatching (AH) technique used to soften, open, or remove ZP to rescue blastocyst with hatching problems - AH D3 prevents blastocyst expansion! - D3/D5-6 - AH: a) Mechanical b) Chemical c) Laser-assisted - controversial (↑implantation rate but not LBR) Monozygotic twinning „compacted ICM“ - different focal planes ICM splitting Sutherland et al 2019 ICM Monozygotic twinning Expanded blastocyst natural hatching Herniation of non-expanded blastocyst in assisted hatching and FET cycles risk of ICM splitting Hatching through multiple opennings Monozygotic twinning - genetically identical same sex twins - epigenetic marks account for phenotypic discordance Craig et al 2020 (adapted) ICM splitting after implantation ICM splitting before implantation Whole blastocyst splitting at hatching - associated with ART! ~75%~20% ~3% Monozygotic twinning - genetically identical same sex twins - epigenetic marks account for phenotypic discordance Craig et al 2020 (adapted) ICM splitting after implantation ICM splitting before implantation Whole blastocyst splitting at hatching - associated with ART! ~75%~20% ~3% herniation ❖Acardiac twin - atrophic acardiac twin may compromise development of a healthy twin Twinning pathologies Monozygotic twinning ❖Conjoined („Siamese“) twins - nature/degree of union variants ← partial splitting of primitive node/streak - monochorionic, monoamniotic (2-5% MZ twins) - aberrant axial (right-left/ craniocaudal) asymmetric patterning - impaired expression of signaling molecules leads to laterally defects Monozygotic twinning ❖Parasitic twin = asymmetrical twin/unequal conjoined twin - one twin underdeveloped and attached to its healthy twin ❖Vanishing twin - 1 in 8 multigestation - loss of a twin before 12 wg - fetal competion for space and nutrition - non-developing embryo dies in utero and is partially or completely reabsorbed Blastocyst in vitro fusion Twinning pathologies - genetic chimerism - genetically distinct cell with one organism - early embryo fusion? Twinning pathologies ❖Seskvizygotic twins Case report: Gabett et al, NEJM 2019. - monochorionic diamniotic twins with discordant sex - 46XX/46XY chimerism in both children - share 78% of paternal DNA - caused by dispermic fertilization? Maternal zonolytic factors - proteinase strypsin (ISP-1) and lysis (ISP-2) - proteases and protease inhibitors secreted by the oviduct and uterine lining - facilitate embryo hatching in vivo - protease activity is suppressed in the oviduct to prevent ectopic pregnancy - human homologous not yet identified ! - uterine secretion regulated by P4 (↑) and E2 (↓,) during menstrual cycle - co-expressed in uterine glands during the perimplantation period - form homo-/hetero-tetramer complexes with ZP-lysis activity Mechanism of embryo hatching ❖ Maternally-derived zonolytic factors - cKO mice lacking epithelial/mesenchymal estrogene receptor (ER) Winuthayanon et al. 2015 - absence of ER in uterine lining leads to elevated protease activity, premature ZP dissolution and embryo lysis by day 2 Premature ZP dissolution - untimely dissolution of ZP rarely observed in vitro - cause unknown - risk of blastomere separation and compaction failure - linked to poor embryo development and Emma Whitney Embryo-endometrial signaling Embryo-endometrial signaling Biomarkers predicting success of implantation in spent IVF culture medium? (GM-SCF?, hyaluronan?) sHLA-G ❖ Paracrine signaling Evans 2020 (modified) - 6 CBG genes encoding biologically active -subunit in humans evolved as a result of duplication of LHB gene - structural similarity with LH (and FSH) −  - unit - identical −  - unit - ~80% homology with LH - different glycosylation - provides specificity - binds to common LH/hCG receptor ❖hCG - hCG can mimic the bioactivity of LH Embryo-endometrial signalling P4 suppress uterine inflammation and promote establishment of pregnancy Embryo-secreted hCG sustains corpus luteum and P4 production ❖hCG Embryo-endometrial signalling 5 isoform, differing activities ➢ Classical (regular) hCG ← preimplatation embryo and syncytiotrophoblast ➢ Hyperglycosylated hCG (hCG-H) - lower activity but longer half-life than regular hCG ← preimplatation embryo ← extravillous cytotrophoblast during early pregnancy ← malignant hCG-producing tumors ➢ Sulphated hCG ← pituitary gland - assumed to supplement pituitary LH functions ➢ Free  unit of classical hCG ➢ Free  unit of hyperglycosylated hCG ← preimplatation embryo ← malignant hCG-producing tumors ❖hCG TGF receptor Embryo-endometrial signalling *urine of pregnant women contains also proteolytically processed („nicked“) forms of hCG (hCGn), hCG- (hCG-n), and core fragment of hCG- (hCG-cf), ❖hCG 50% regular hCG 40% HCG-H 10% hCG- A) rec-hCG = pure regular hCG B) purified urinary hCG - molecular heterogenity (2.2-12% hCG-H)≠ trigger shotsecretion 8 cells 50% of Glc consumed by a blastocyst is not oxidized but converted to lactate (Warburg effect) Miazzoto et al 2020 >50% Instead of being only a metabolic by-product, lactate serve as a key embryo-derived signal augmenting implantation David Gardner Embryo-endometrial signalling ❖Lactate ↓pH Gardner 2015 Facilitates embryo hatching, endometrial tissue disaggregation and trophoblast invasion Embryo-endometrial signalling ❖Lactate Gardner 2015 Induces angiogenesis and increases vascular permeability ↓pH Embryo-endometrial signalling ❖Lactate Gardner 2015 Modulates the immune response of the the mother ↓pH „host-defence“ phenotype „healing“ phenotype Embryo-endometrial signalling ❖sHLA-G Embryo-endometrial signalling - soluble form of HLA-G - HLA-G is expressed on TE and extravillous trophoblast (EVT) cells (and tumor cells) - HLA-G plays a key role in establishing maternal-fetal immune tolerance - sHLA-G has systemic immunomodulation effects - present in conditioned (spent) embryo culture medium - documented as a positive implantation predictor of IVF embryos - in tumors, concentration reflects invasiveness and metastasis potential Ferreira et al 2017 ❖Extracellular vesicles Embryo-endometrial signalling - cell-derived membranous nano-sized vesicles - secreted by both uterus and embryo (TE/ICM) - different size, origin, and cargo - known to contain proteins, lipids, and ncRNAs - packaged content alters during the uterine cycle and embryo development - embryotrophic effects - TE uptake of endometrium-secreted exosomes promotes TE adhesion and invasion enhance embryo implantation ❖miRNA Embryo-endometrial signalling Liang et al 2017 - transcriptional regulator of gene expression - involved in intercellular communication, including embryo-maternal crosstalk - both embryo- and endometrium-derived - different types and multiple packing forms ❖GM-CSF Embryo-endometrial signalling Choi et al 2024 - cytokine synthesized in the female reproductive tract, including endometrium - not expressed in the embryo - uncertainty over its positive effect on embryo development and implantation - commercially available embryo culture medium supplemented with GM-CSF ❖Hyaluronan (=hyaluronic acid, HA) Embryo-endometrial signalling - high-molecular-mass polysacharide found in ECM - HA secretion by invasive trophoblast and synthesis in endometrial tissue can promote trophoblast penetration during embryo implantation BUT it does not act as a „glue“ promoting embryo adhesion and attachment to the endometrium - HA supplementation of embryo culture/transfer media - uncertainity over its positive effect on embryo development and implantation Seeding into fertile ground..... .....to be continued