Embryology I OOGENESIS autumn 2024 Ovulation and luteinization Zuzana Holubcová Department of Histology and Embryology zholub@med.muni.cz Ovarian cycle Preovulatory follicle Gradient of oocyte-secreted factorsGradient of gonadotropins COC microenvironment maintained by morphogenic gradient of oocyte-secreted factors (GDF9, BM15, BMP6, BMP8,...) - prevention of apoptosis - expansion of COCs - prevention of premature luteinisation → supplement to IVM culture? Ovulation induction ❖LH surge - begins ~36h before ovulation - initiates molecular signaling leading to (1) COC expansion (2) meiosis resumption and oocyte maturation (3) rupture of Graafian follicle a release of egg (4) luteinization of emptied follicle Ovulation induction ❖LH surge peptides produced by mural GCSLH stimulate thecal and mural GCs csignal transduced by EGF-like peptides produced by mural GCS signal transduced by EGF-like peptides produced by mural GCS csignal transduce d by EGF- like peptides produced by mural GCS oocyte maturation Epiregulin Amphiregulin Betacellulin Russell and Robker 2007 Cumulus expansion - LH/hCG - induced morphostructural changes of cumulus cells surrounding the oocyte - cumulus oocyte complex (COC) - enlargement, loosening and mucification of cumulus Cumulus expansion - LH surge triggers expression of HA syntase and Glc uptake - synthesis and deposit of mucoelastic matrix in extracellular space leads to cumulus cells distancing with Gap junction maintained - further distancing leads to retraction of TZP → oocyte meiotic maturation - cumulus ECM composed of strands of hyaluronic acid (HA) and crosslinking proteins, glycoproteins and proteoglycans (TSG-6, versican, II HC, Ptx3) required for cumulus retention and stability - ↑HA → ECM water attraction → ↑ follicular fluid pressure Cumulus expansion - Cumulus cells play role in (1) deposition of molecular factors (2) ovulation (3) fertilization (4) embryo passage through oviduct Cumulus expansion https://clinicalgate.com/transport-of-gametes-and-fertilization/ COC detachment from follicular wall Ovulation Coagulation Acquisition of viscoelastic properties facilitates COCs release through follicle rupture Ovulation - rupture of Graffian follicle and expulsion of COC containing secondary oocyte (MII oocyte, egg) and granulosa cells of corona radiata Ovulation - vasodilatation - perforation of capillars - increased permeability ❖Chemotaxis LH Prior LH surge After LH surge release of proinflamatory and antiinflamatory factors ❖Vascular changes local edema Ovulation ❖Follicle rupture - HA activates CD44 receptor on cumulus cells → migratory and invasive phenotype - COC separation from follicular wall and adhesion to apex epithelium - proteolytic degradation of tunica albuginea - role of lysosomal matrix metalloproteinases and ADAMTS-1 - just before ovulation apical region becomes avascular (ischemia) Ovulation ▪ Progesterone → stimulates production of collagenase by theca cells → dissolution of connective tissue → displace cortisol from its binding protein → ↑ cortisol mitigates inflamatory reaction PGE2 → stimulates plasminogen activator → plasminogen converted to plasmin → activation of collagenase → dissolution of connective tissue PGF2a → induces rupture of lysosomes → follicle and ovarian contractions ↑ ❖Follicle rupture ▪ Prostaglandinds Ovulation Ovulation - stigma (macula pellucida) = avascular spot left on the ovarian surface Transgenic mouse Myr-Tomato H2B-GFP Ovulation Melina Schuh Thomas et al 2024 - ex vivo imaging of ovulation in isolated mouse follicles Ovulation Melina Schuh Transgenic mouse Myr-Tomato H2B-GFP Thomas et al 2024 Ovulation Melina Schuh Transgenic mouse Myr-Tomato H2B-GFP Thomas et al 2024 Luteinisation Alvino 2011 terminal differentiation of follicular GCs and theca cells to LUTEAL CELLS corpus hemorhagicum corpus luteum Luteinisation Talbott and Davis 2017 = proces of lipid accumulation - synthesis of progesterone (P4) in response to LH - dependent of bioavailability of cholesterol Corpus luteum Corpus albicansCorpus hemorhagicum Corpus luteum - vascularization of GCs layer - fibroblast proliferation - basement mebrane break down - GCS grow and become vacuolozed - GCS acquire LH receptor and start steroidogenesis - cell cycle arrest (p27, p21, cycD) - proliferation of sER and structural modification of mitochondria (CL) Corpus luteum - cyst-like „yellowish“ structure 2-5cm - temporary endocrine organ - arises from ovulatory follicle - secretion of steroid hormones - PROGESTERON (and estradiol)* - feed-back loop inhibition of GnRH, FSH and LH * + relaxin, inhibin Hyperplasia of endometrium ensuring suitable environment for implantation Corpus luteum NO FERTILIZATION NO IMPLANTATION LUTEOLYSIS (-hCG) (+hCG) ↓progesterone ischemic necrosis of endometrium constriction of spiral arteries ~10 weeks ~14 days Corpus luteum graviditatis Corpus albicans - provides progestagenic support to early pregnacy ~9 days Accumulation of prostaglandins? menstruation bleeding Corpus luteum dysfunction ❖Luteal support - administration of progesteron and/or hCG to complement CL secretion - encouraging the uterine lining to support implantation - normal P4 production by CL peaks 4 days after ovulation and drops after next 9 days - Supraphysiological level of steroids (FSH) and prevention of LH release during ovarian stimulation for IVF cause GnRH suppression and may cause corpus luteum dysfunction Monitoring ovulation - Transvagnal Sonographic Folliculometry → sexual intercourse, IUI timing - from preovulatory follicles - in central anesthesia - ultrasound guidance - transvaginally - each follicle invaded by a hollow needle and COCs are aspirated - performed by accredited IVF specialist (MD) Oocyte retrieval in ART https://www.youtube.com/watch?v=0h3LaaL97e0 https://www.youtube.com/watch?v=sbu4la4bzjc https://www.youtube.com/watch?v=RTnJlTrOMXo Oocyte retrieval in ART Muter et al 2023 Oocyte retrieval in ART ❖ Putative markers of oocyte quality - cell growth/survival/apoptosis, steroidogenesis, intercellular signalling, lipid metabolism, ECM formation, vesicle trafficing, inflammatory factors,.. ▪ follicular fluid content ▪ cumulus cell phenotype - number of layers, compacteness, color, blood clot presence - reaction to enzymatic treatment ▪ cumulus cells gene expresion - biochemic/proteomic profile, presence of specific cytokines, ox-red potential,.. Oocyte retrieval in ART - no oocytes obtained from preovulatory follicles during IVF procedure despite normal hormonal response and ultrasound monitoring - usually technical problem during COCs aspiration and/or hCG trigger administration - genuine absence of oocyte very rare ((„genuine EFS“ - 0.0016%) - Possible cause? A) COC stick to follicle wall due to insufficient LH/hCG trigger which causes COC expansion and detachment B) Oocyte degeneration within follicle Chen et al., 2017. Empty follicle syndrome (EFS) - genetic predisposition for proapoptotic genes expression in GCs - defect ZP leading to impaired GCs-oocyte communication Anovulation ❖ Luteinized unruptured follicle (LUF) - failure of Graaffian follicle to rupture after LH peak - egg trapped inside the peristent follicle with thick wall - altered endocrine profile - low FSH and LH, high progesteron - altered folliculogenesis dynamics - earlier follicular selection, faster and longer growth - progressive loss of cystic appearance and luteinization without ovulation - thick echogenic endometrium - mature/premature LUFs - high recurrency, sub-/in-fertility - associated with endometriosis (73%!), pelvis inflamatory disease and excessive used of NSAIDs (!) Anovulation ❖ Luteinized unruptured follicle - role of COX2 - production of prostagladins required for follicular maturation and rupture Geng et al 2022 Anovulation cyst Hormonally active ❖ Ovarian cysts Anovulation ❖Polycystic ovary syndrom • 2 out of 3 Rotterdam criteria: - follicle arrest in antral stage, multiple growth ceasing follicles (and ovarian cysts) - reduced ovulation rates, oligo-/a-menorhea - hyperandrogenism Anovulation ❖Polycystic ovary syndrom • Role of androstenedione in folicular cyst formation Okutsu et al 2008 - oocyte degeneration - increased GC apoptosis - premature luiteinisation of GCS • Role of neuroendocrine impairment ? - hyperactivity of GnRH neurones? - role of kisspeptin? kisspeptin Ruddenklau and Campbell 2019 ❖Clomiphene (p.o.) - non-steroid selective modulator of estrogen receptors in hypotalamus - induce release of GnRH (↑FSH, LH) - long clearance, negative impact on endometrial lining - used in an-/oligo-ovulation disorders (e.g. PCOS) - RISK OF MULTIPLE PREGNANCY! ❖ Letrozole (p.o.) - aromatase inhibitor (blocks estrogen synthesis) - negative loop increases GnRH production and thus FSH a LH levels - anticancer drug (breast cancer), gynecomastia treatment - „off-label“ use for induction of ovulation - lower impact on endometrium, more expensive Pharmacological induction of ovulation Letrozole Androgens aromatase ↓ ❖Bromocriptine (p.o.) - ergoline derivate and dopamin agonist - reduces production of prolactin by pituitary gland - treatment of anovulation caused by hyperprolactinemia Pharmacological induction of ovulation ↑Prolactin - decreased GnRH production - granulosa cell dysfunction - inhibition of corpus luteum function - endometrium dysfunction ❖Metformin (p.o.) - antidiabetic drug (DM 2) - stimulation of ovulation in insulin-resistant PCOS - empirical treatment - gastorintestinal side effects Pharmacological induction of ovulation ❖Combined contraceptive pills (p.o.) - estrogen + progesterone - feedback inhibition of FSH and LH secretion from pituitary gland - inhibition of ovulation, suppression of endometrial growth, thickening of cervical mucous - resumption of fertility after discontinuation - relief from dysmenorhea, prevention of endometriosis recurrence - ↓risk of endometrial cancer - ↑ risk of breast cancer Pharmacological inhibition of ovulation Can suppression of ovulation have a protective effect against ovarian aging by preventing tissue damage? Pharmacological inhibition of ovulation Mouse model of ovulation supression - daily administration of OC 2-12m - OC-treated mice - no significant difference if follicle number compared to control - higher number of oocytes after stimulation - more living fetuses after spontaneous mating - reduced amount of brownish foamy fibrous tissues control OC-administration - tissue damage caused by ovulation cycle might contribute to age-related fertility decline