Exam Notes on Oxidative Stress and Antioxidants PDF

Oxidative Stress and Antioxidants Male Infertility: ○ Male factor involved in 50% of infertility cases; 40-80% are linked to Reactive Oxygen Species (ROS). ○ ROS are essential for sperm functions (e.g., capacitation, acrosome reaction), but excess ROS cau...

Oxidative Stress and Antioxidants Male Infertility: ○ Male factor involved in 50% of infertility cases; 40-80% are linked to Reactive Oxygen Species (ROS). ○ ROS are essential for sperm functions (e.g., capacitation, acrosome reaction), but excess ROS causes damage like poor motility, abnormal morphology, DNA damage, and cell death. insights. Cervical Mucus Migration Tests: ○ Mimics the natural female tract. ○ High sperm migration correlates with better in vivo fertilization outcomes. Sperm DNA Fragmentation Importance: DNA damage reduces fertility, cannot be repaired by sperm but only by the oocyte. Key Factors: ○ Increases with high temperatures (37°C). ○ Fewer cysteines in Protamine 1 lead to higher DNA fragmentation. ○ Protamine 1 tightly compacts DNA for stability; cysteine bonds ensure protection. Cryopreservation Process: Cooling, freezing, thawing with cryoprotectants like glycerol. Challenges: 50-60% sperm survive freezing, with reduced functionality. Vitrification Rapid Cooling: Avoids ice crystal formation, preserving motility/viability (>50%) even without cryoprotectants. Key Points to Remember Oxidative stress plays a major role in male infertility. Lifestyle factors influence DNA fragmentation. Cryopreservation and vitrification impact sperm viability and lifespan. Sperm Transport Initial Deposition: Sperm deposited in the vagina during intercourse; >99% lost due to leakage. Cervical Entry & Crypts: Sperm enter cervix within minutes; some survive hours in cervical crypts, nourished by mucus.  Uterine & Oviduct Transport: Sperm move via uterine contractions, reaching oviduct in 2-7 hours by self-propulsion. Uterotubal Junction: Regulates sperm entry into the oviduct. Sperm Selection & Survival Cervical Mucus: Filters sperm, allowing passage only during fertile window. Uterine Selection: Morphologically abnormal sperm are eliminated. Oviducal Binding: Sperm form a reservoir on epithelial cells for gradual release. Capacitation: Occurs in the uterus/oviduct, preparing sperm for fertilization. Oocyte Transport Ovulation: Oocyte released into the peritoneal cavity. Fimbrial Pickup: Fallopian tube fimbriae capture the oocyte. Oviducal Transport: Ciliary movement and contractions move the oocyte to the ampulla. Cumulus Cells: Aid oocyte adhesion and transport. Fertilization Site Ampullary-Isthmic Junction: Sperm and oocyte meet here. Sperm Activation: At ovulation, sperm detach from oviducal cells and swim to the oocyte. Factors Influencing Transport Hormones: Estrogen and progesterone regulate cervical mucus and motility. Uterine Contractions: Assist sperm transport but are not essential. Chemoattractants: Guide sperm to the oocyte, possibly involving progesterone. 1. Sperm Capacitation Sperm undergoes a series of physiological changes after ejaculation, known as capacitation, which enhances its ability to fertilize the oocyte. This process involves the removal of glycoprotein coats and changes in the sperm membrane, allowing for increased motility and the ability to undergo the acrosome reaction. 2. Acrosome Reaction  Upon reaching the zona pellucida (the outer layer of the oocyte), the sperm undergoes the acrosome reaction, where enzymes are released from the acrosome (a cap-like structure on the sperm). These enzymes help digest the zona pellucida, facilitating sperm penetration. 3. Sperm-Oocyte Fusion Once the sperm penetrates the zona pellucida, it binds to the oocyte's plasma membrane. The fusion of the sperm and oocyte membranes allows the sperm nucleus to enter the oocyte, leading to the formation of a zygote. 4. Cortical Reaction The entry of the sperm triggers the cortical reaction in the oocyte, where cortical granules release their contents into the perivitelline space. This reaction modifies the zona pellucida, preventing additional sperm from binding and entering, thus ensuring monospermy (fertilization by a single sperm). 5. Completion of Meiosis The oocyte, which is arrested in metaphase II of meiosis, completes its second meiotic division upon fertilization. This results in the formation of a second polar body and the mature ovum, which contributes half of the genetic material to the zygote. 6. Zygote Formation The fusion of the sperm nucleus and the oocyte nucleus forms the diploid zygote, which contains genetic material from both parents. The zygote then begins the process of cleavage, leading to the formation of a multicellular embryo. Molecular Mechanisms Fertilization involves various molecular signals and interactions, including: Sperm-Zona Pellucida Interaction: Specific receptors on the sperm surface bind to glycoproteins in the zona pellucida, initiating the acrosome reaction. Calcium Oscillations: Following fertilization, calcium ions are released within the oocyte, which are crucial for activating developmental processes and the completion of meiosis.Acrosome Reaction and Role of ZPs (Short Answer for Exam) Role of ZPs (Short Answer for Exam) The zona pellucida (ZP) is made of glycoproteins (ZP1, ZP2, ZP3, ZP4 in humans). ZP3 and ZP4 are essential for sperm binding and triggering the acrosome reaction. They interact with specific receptors on the sperm, inducing the release of enzymes that help the sperm penetrate the zona pellucida and reach the oocyte for fertilization Stages of Implantation: Hatching: ○ Blastocyst escapes the zona pellucida to expose the trophoblast for uterine interaction. Apposition: ○ Initial loose contact between blastocyst and endometrial surface. ○ Occurs during the "receptive window" (6-10 days post-ovulation). Adhesion: ○ Firm attachment through cell surface receptors (integrins, cadherins, selectins). ○ Pinopodes: § Increase endometrial surface area and absorb fluid, bringing the blastocyst closer. § Regulated by progesterone and estrogen. Invasion: ○ Trophoblast cells invade the endometrium, establishing maternal-fetal connections. Trophoblast Differentiation: Cytotrophoblast: Proliferative precursor cells. Syncytiotrophoblast: Invasive, multinucleated cells that digest maternal extracellular matrix (via MMPs) for deep embedding. Functions of Syncytiotrophoblasts: Anchoring: Secure the embryo in the endometrium. Vascular Connection: Invade maternal blood vessels for placenta formation and nutrient/oxygen exchange. Immune Modulation: Prevent maternal immune rejection of the embryo When and how our epigenetic profile is set? How stable is our epigenomic profile? Our epigenetic profile is set and modified throughout our lifetime, with critical periods during early development. The process is influences by various factors: Epigenetic Reprogramming The epigenome undergoes significant changes during early development: · Fetal germ cells: Low methylation levels · Embryo-Blastocyst: Low methylation · Developing fetus: Increasing methylation · Mature gametes: High methylation · Zygote: High methylation This cycle of methylation demethylation is crucial for proper development and cellular differentiation. Our epigenetic profile is relatively stable but can be influences by various factors throughout life such as: 1. Environmental factors: diet, lifestyle and exposure to toxin can affect epigenetics 2. Medications: some drugs can alter epigenetic patterns 3. Periconception environment: the condition during early embryonic development can have lasting effects on the epigenome Hormonal Regulation by the Anterior Pituitary Gland (Top Panel): FSH (Follicle-Stimulating Hormone): Peaks during the early follicular phase (days 1–7), stimulating follicular recruitment and growth. LH (Luteinizing Hormone): A surge in LH (around day 14) triggers ovulation, the release of the mature egg from the dominant follicle. 2. Ovarian Hormones (Middle Panel): Estradiol (Estrogen): Ø Produced by developing follicles. Ø Rises during the follicular phase and peaks just before ovulation. Ø Responsible for the proliferation (thickening) of the endometrium. · Progesterone:  Ø Secreted by the corpus luteum after ovulation. Ø Peaks during the luteal phase (days 14–28), stabilizing the endometrium to support potential implantation. 3. Ovarian Events (Third Panel): Follicular Phase (Days 1–14): Ø Recruitment of several follicles, followed by selection of a dominant follicle, which grows and releases estradiol. Ø Ovulation (Day 14): Triggered by the LH surge, the mature follicle releases an oocyte (egg). · Luteal Phase (Days 15–28): Ø After ovulation, the ruptured follicle transforms into the corpus luteum, producing progesterone. Ø If no pregnancy occurs, the corpus luteum degenerates into the corpus albicans. 4. Uterine Endometrial Changes (Bottom Panel): Menstrual Phase (Days 1–5): Shedding of the uterine lining due to the drop in progesterone and estrogen levels. Proliferative Phase (Days 6–14): Under the influence of estrogen, the endometrium regenerates and thickens. Secretory Phase (Days 15–28): Progesterone from the corpus luteum prepares the endometrium for potential implantation, with glands secreting nutrients for a fertilized egg. Circulating Factors 1. Endocrine - Hormones ○ Ovarian Estrogen & Progesterone: § Govern conception transitions and impact growth factors, transcription factors, cytokines, and cell cycle regulators. § Receptors (ERα/β, PR-A/B) expressed in the endometrium. ○ Estrogen Role: § Prepares the uterus by promoting endometrial growth and PR expression. ○ Progesterone Role: § Post-ovulation, it supports gland secretion and enables the implantation window. ○ Species Differences:  § Humans rely on progesterone; rodents require an additional estrogen surge. 2. Human Chorionic Gonadotropin (hCG): ○ Secreted by the embryo; affects trophoblasts (autocrine) and maternal tissues (paracrine). Interleukin (IL)-1α and β are key components of the IL-1 system, which also includes the receptor antagonist (IL-1ra) and two receptors (IL-1RtI and IL-1RtII). They are expressed throughout the menstrual cycle, mainly in the surface epithelium. IL-1 plays a crucial role in embryo implantation by responding to the receptive endometrium, triggering a second wave of cytokines. For example, in mice, blocking the IL-1 receptor before implantation reduces the number of embryos that implant, highlighting IL-1's role in this process. IGF I and IGF II are ligands that bind to their receptors IGF1R and IGF2R, respectively, and are regulated by six binding proteins. IGFBP-1 modulates the effects of IGF1 and IGF2, affecting both mitogenic and metabolic processes. It is localized to predecidual stromal cells in the late secretory- phase endometrium and decidual cells during pregnancy. IGFBP-1 interacts with IGF2, which is synthesized by trophoblasts. Both IGF2 and IL-1β can inhibit the activity of IGFBP-1+ 1. Mucin 1 (MUC1): ○ Barrier protein; reduces locally at the implantation site. 2. Osteopontin: ○ Upregulated by progesterone; supports adhesion through receptor binding. Implantation Process 1. Uterine Preparation: ○ Progesterone transitions the uterus to a receptive state. 2. Blastocyst Activation: ○ Mediated by factors like HB-EGF, initiating attachment and invasion. 3. Adhesion and Invasion: ○ Integrins, laminin, and enzymes like metalloproteinases regulate blastocyst invasion. 4. Decidualization: ○ Stromal cell transformation supports implantation; prostaglandins and VEGF aid vascularization. Extracellular Vesicles (EVs) in Implantation 1. Definition & Types:  ○ Nano-sized vesicles (exosomes, microvesicles, apoptotic bodies) carrying DNA, RNA, and proteins. 2. Maternal-Fetal Communication: ○ Blastocyst to Endometrium: § EVs modulate immune tolerance and promote receptivity. ○ Endometrium to Blastocyst: § Endometrial EVs enhance blastocyst development and attachment. 3. Functions: ○ Modulate endometrial receptivity, facilitate trophoblast invasion, regulate immunity, and promote angiogenesis. 4. Bioactive Cargo: ○ miRNAs: E.g., miR-30d enhances adhesion. ○ Proteins: LIF, VEGF aid receptivity and angiogenesis. ○ Lipids: Signal pathway mediators. Cleavage Stage Description: Rapid mitotic divisions of the zygote form a solid ball of cells (morula). Timing: 3-4 days post-fertilization, 16-32 cell stage. Compaction As the morula develops, the cells begin to compact, forming tight junctions between them. This process is crucial for the subsequent differentiation of cells. Compaction leads to the polarization of cells, with outer cells becoming more flattened and inner cells becoming rounded. Mechanism: Key Molecules: E-cadherin: Drives compaction, relocates to cell junctions. α/β-catenins: Link E-cadherin to actin cytoskeleton. JAM-1: Detected from 8-cell stage. Requirement: Calcium-dependent process. Formation of the Blastocoele Following compaction, the cells begin to secrete fluid into the intercellular spaces, leading to the formation of a fluid-filled cavity known as the blastocoele. The accumulation of fluid causes the morula to expand and transforms it into a blastocyst. Mechanism: Na/K-ATPase generates ion gradients. Water movement via aquaporins (AQPs). Tight junctions prevent fluid loss. Differentiation of Cell Types Trophoblast: Outer cells forming the placenta, facilitating implantation. Inner Cell Mass (ICM): Internal cluster forming the embryo proper. Hatching Process: Blastocyst breaks free from the zona pellucida, allowing uterine implantation. Embryo Metabolism Overview Embryo metabolism involves biochemical processes critical for growth, development, and implantation. It transitions from anaerobic reliance to more diverse energy use as the embryo develops. Key Stages of Metabolism 1. Pre-Compaction (Early Stage) Low Oxidation: Pyruvate, lactate, and amino acids are minimally oxidized. Energy Use: High ATP:ADP ratio inhibits glycolysis via phosphofructokinase (PFK). 2. Blastocyst Stage Energy Shift: Increased oxygen consumption and glucose utilization.  Glycolysis: Higher flux through glycolysis as AMP levels rise and ATP:ADP ratio drops. Key Metabolic Processes Energy Sources Early reliance on anaerobic metabolism using maternal nutrients. Post-implantation, metabolic activity increases with oxidative phosphorylation and expanded substrate use (glucose, fatty acids, amino acids). Glucose Metabolism Purpose: Provides energy and pentose sugars for nucleic acids, phospholipids, and amino acids. Amino Acid Metabolism Functions: Protein synthesis, cellular functions, and energy (e.g., glutamine, aspartate). Special Role: Glycine buffers intracellular pH. Fatty Acid Oxidation Provides additional energy, particularly in later stages. Metabolic Regulation Insulin: Enhances glucose uptake. Growth Factors: IGF stimulates growth and metabolism. Oxygen: Regulates anaerobic vs. aerobic metabolism based on availability. Developmental Stages and Metabolism Pre-Implantation Relies on maternal nutrients. Energy demand is low, focused on cleavage and early development. Post-Implantation Metabolic demand rises with maternal connection. Wider use of substrates (glucose, fatty acids, amino acids). Key Shifts in Metabolism Pre-implantation: Anaerobic pathways dominate; low activity.  Post-implantation: Oxidative phosphorylation and glycolysis support higher energy needs. Energy Pathways in Preimplantation Development (summary) The embryo relies on two main glycolytic pathways to generate energy: 1. Aerobic Glycolysis: Also known as the TCA or Krebs cycle, it requires oxygen and takes place in the mitochondria. It is efficient and provides more ATP per molecule of glucose. 2. Anaerobic Glycolysis: Known as the Embden-Meyerhof pathway, it occurs in the cytoplasm and does not require oxygen. It produces less ATP but is faster. Nutrient Use During Development Stages 1. Early Preimplantation Stages (Zygote to Morula): ○ Carboxylic Acid-Based Metabolism Dominates: Pyruvate and lactate are the primary energy sources, with minimal glucose uptake. ○ This stage emphasizes oxidative metabolism (aerobic), ensuring the embryo has sufficient energy for cell divisions and early development. 2. Later Stages (Blastocyst Formation): ○ Glucose Uptake Increases: As the embryo progresses to compaction and forms a blastocyst, it starts relying more on glucose metabolism. ○ Glycolysis becomes the predominant pathway, supporting the energy demands for differentiation and preparation for implantation. Role of Amino Acids Protein Synthesis: Building blocks for cell structures and functions. Metabolism: Provide intermediates for energy and biosynthetic pathways. pH Regulation: Some amino acids act as buffers, maintaining the embryo's optimal internal environment. Energy Source: Certain amino acids, like glutamine and aspartate, can be used as energy substrates. Embryo Viability and Biomarkers  The nutrient levels and their metabolites reflect how healthy and viable an embryo is. Secreted Factors: Molecules released by the embryo can also indicate its developmental potential. These metrics are useful as biomarkers in assessing embryo quality during in vitro fertilization (IVF) or related procedures. 1. Sperm Capacitation Sperm undergoes a series of physiological changes after ejaculation, known as capacitation, which enhances its ability to fertilize the oocyte. This process involves the removal of glycoprotein coats and changes in the sperm membrane, allowing for increased motility and the ability to undergo the acrosome reaction. 2. Acrosome Reaction Upon reaching the zona pellucida (the outer layer of the oocyte), the sperm undergoes the acrosome reaction, where enzymes are released from the acrosome (a cap-like structure on the sperm). These enzymes help digest the zona pellucida, facilitating sperm penetration. 3. Sperm-Oocyte Fusion Once the sperm penetrates the zona pellucida, it binds to the oocyte's plasma membrane. The fusion of the sperm and oocyte membranes allows the sperm nucleus to enter the oocyte, leading to the formation of a zygote. 4. Cortical Reaction The entry of the sperm triggers the cortical reaction in the oocyte, where cortical granules release their contents into the perivitelline space. This reaction modifies the zona pellucida, preventing additional sperm from binding and entering, thus ensuring monospermy (fertilization by a single sperm). 5. Completion of Meiosis The oocyte, which is arrested in metaphase II of meiosis, completes its second meiotic division upon fertilization. This results in the formation of a second polar body and the mature ovum, which contributes half of the genetic material to the zygote.  6. Zygote Formation The fusion of the sperm nucleus and the oocyte nucleus forms the diploid zygote, which contains genetic material from both parents. The zygote then begins the process of cleavage, leading to the formation of a multicellular embryo. 1. Cell Cleavage Definition**: Cell cleavage refers to the series of rapid mitotic divisions that occur in the early embryo after fertilization. Process**: · Begins shortly after fertilization, typically within 24-30 hours. · The zygote undergoes successive divisions without significant growth, resulting in smaller cells called blastomeres. - Cleavage patterns can be classified as: · Holoblastic Cleavage: Complete division of the egg (typical in humans). · Meroblastic Cleavage: Partial division of the egg (seen in species with yolk-rich eggs). Outcome**: Cleavage leads to the formation of a multicellular structure known as the morula, which consists of 16-32 cells. 2. Blastulation - Definition**: Blastulation is the process that follows cleavage, leading to the formation of the blastocoel and the transition from morula to blastula or blastocyst. - Process**: · The morula develops a fluid-filled cavity called the blastocoel. · Cells begin to reorganize, resulting in the formation of the blastula (or blastocyst in mammals). - Significance**: This stage is crucial for the subsequent implantation into the uterine wall. 3. Blastocyst Structure The blastocyst is a key structure in early embryonic development, characterized by its distinct components: a. Blastocoel Cavity Description**: A fluid-filled cavity that forms within the blastocyst. Function**: Provides a space for cell migration and differentiation, and is essential for the implantation process. b. Inner Cell Mass (ICM) Description**: A cluster of cells located at one pole of the blastocyst. Function**: - The ICM is the source of embryonic stem cells, which can differentiate into all cell types of the body. - It eventually develops into the embryo proper and some extraembryonic tissues. c. Trophoblast - Description**: The outer layer of cells surrounding the blastocyst. - Function**: · The trophoblast plays a critical role in implantation into the uterine wall. · It develops into part of the placenta, facilitating nutrient and gas exchange between the mother and the developing embryo. d. Zona Pellucida - Description**: A glycoprotein layer that surrounds the oocyte and early embryo. - Function**: · The zona pellucida protects the embryo and prevents polyspermy (fertilization by multiple sperm).It degenerates during the blastocyst stage and is replaced by trophoblastic cells, allowing for implantation into the uterine lining. Decidualization · Transformation of endometrial stromal fibroblasts into secretory decidual cells · Occurs in mid-luteal phase, independent of pregnancy · Progesterone-driven process Key Hormones in Implantation and Early Pregnancy Estrogen: stimulates proliferation and differentiation of uterine epithelial cells Progesterone: stimulates stromal cells, critical for pregnancy maintenance until placenta takes over (around 12 weeks) hCG: synthesized by trophoblast cells, prolongs progesterone secretion Implantation Failure Major limiting factor in ART success Causes: 1. Endometrial factors: thin endometrium, altered expression of adhesive molecules, immunological factors 2. Embryonic factors: genetic abnormalities, sperm defects, embryonic aneuploidy, zona hardening The Sperm - Common sperm issues: Oligospermia: Low sperm count. Asthenospermia: Poor sperm motility. Teratospermia: Abnormal sperm morphology. Azoospermia: Absence of sperm Two types: Obstructive Azoospermia [OA] occurs when there is a physical blockage or obstruction in the reproductive tract that prevents the sperm from being present in the semen Non-Obstructive Azoospermia [NOA] occurs when the testes can't produce enough sperm due to factors like hormonal imbalances, genetic conditions or damage to the testicular tissue - Sperm retrieval techniques: PESA (Percutaneous Epididymal Sperm Aspiration). TESE (Testicular Sperm Extraction). The Oocyte (Ovarian reserve refers to the reproductive potential left within a woman's two ovaries based on number and quality of eggs) Controlled Ovarian Hyperstimulation (COH): is important in ART, the goal is to stimulate the ovaries to produce multiple follicles and increase the chances of retrieving viable oocytes. Protocols and doses depend on: Age (older women may have decreased ovarian reserve that can affect stimulation) , body weight, ovarian reserve is assessed through: - FSH (levels indicate ovarian function), - LH (important for follicle development), - AMH (reflects the number of remaining follicles), and antral follicle count (the number of visible follicles in the ovaries during ultrasound scans, providing an estimate of ovarian reserve). Monitoring includes: Ultrasound scans, serum estrogen levels rise as follicles develop, indicating ovarian response and progesterone levels are monitored to assess luteal phase support and overall hormonal balance. Triggering ovulation: Administer hCG when at least 3 follicles reach >17mm diameter to ensure that follicles are mature enough to release oocytes. Egg retrieval occurs ~36 hours post-hCG injection this is to ensure that oocytes are collected just before ovulation occurs. 1. Definition of Stem Cells: Self-Renewal: Stem cells can divide and produce exact copies of themselves over long periods. This means they can replenish themselves, maintaining a consistent supply of stem cells throughout the body or in a laboratory setting. Potency: Potency refers to the stem cell's ability to develop into various types of cells in the body. Different stem cells have different levels of potency: Ø Totipotent: Can produce all cell types, including extra-embryonic tissues (e.g., the placenta). These are found in very early embryos. Ø Pluripotent: Can develop into almost any cell type in the body but not extra-embryonic tissues. Example: embryonic stem cells. Ø Multipotent: Can develop into a limited range of cell types related to a specific function or tissue. Example: hematopoietic stem cells, which form blood cells. · Differentiation: This is the process by which stem cells mature and transform into specialized cells with specific functions, like muscle cells, nerve cells, or skin cells. Differentiation allows stem cells to contribute to growth, healing, and tissue regeneration by replacing damaged or dead cells with healthy ones. · Functions: · Build embryos and tissues (development). · Repair tissues (regeneration). 2. Sources of Stem Cells:  Pre-implantation embryos: Early-stage embryos before implantation in the uterus. The inner cell mass (ICM) of the blastocyst contains pluripotent stem cells which can differentiate into any cell type. These cells are crucial for research and therapeutic applications due to the ability to develop into all three germ cells. Post-implantation embryos: Later stages of embryonic development. They are composed of more differentiated cells compared to the pre implantation embryos. The development includes formation of the embryonic disc and establishment of the basic body plan. These cells are less pluripotent but still have potential for specific differentiation pathways. 3. Types of Human Pluripotent Stem Cells: Human Embryonic Stem (hES) Cells: Derived from early embryos, specifically from the inner cell mass of blastocysts. Are pluripotent can differentate into many cell types and have unlimited renewal in vitro. Induced Pluripotent Stem Cells (iPSCs): Reprogrammed somatic (adult) cell, are pluripotent, generated by specific transcription factors. Induced Pluripotent Stem Cells (iPSCs) 4. What is Epigenetic Memory? When adult cells are reprogrammed into iPSCs, they retain some "memory" of their original identity. This memory comes from their previous methylation patterns—chemical modifications to DNA that influence which genes are active or silent. For example: If a fibroblast (a type of connective tissue cell) is reprogrammed into an iPSC, the iPSC might "remember" its fibroblast origin due to retained methylation patterns. As a result, it might more readily differentiate back into a fibroblast compared to other cell types. 1. Methylation Effects: Stable Methylation Patterns in Differentiation: In fully differentiated cells (like muscle or nerve cells), specific methylation patterns ensure that only the genes required for that cell’s function are active, while others are turned off. This stability helps maintain the specialized identity and function of the cell. Impact on Reprogramming Efficiency: During the process of creating iPSCs, removing these methylation patterns is crucial to make the cell pluripotent again. However, if methylation patterns are stubborn or inconsistently removed, they can reduce the efficiency of reprogramming or result in iPSCs with limited pluripotency. Influence on Differentiation Potential: After reprogramming, the remaining or altered methylation patterns can affect how iPSCs differentiate into other cell types. If methylation patterns aren’t reset properly, the iPSCs might struggle to form certain types of cells or may favor differentiation into specific lineages (like the original cell type). Pluripotency Testing 1. Methods of Differentiation: Directed differentiation into three germ layers (endoderm, mesoderm, ectoderm). Functional assays like teratoma formation or lineage-specific differentiation tests. 2. Importance of Functional Tests: Molecular markers provide insights but cannot replace functional assays for confirming pluripotency. 1. Genetic Instability: Culture-adapted hES cells may develop chromosomal abnormalities or mutations affecting self- renewal and differentiation balance. 2. Epigenetic Alterations: Selective pressures in culture can lead to changes in the genome or epigenome, requiring continuous quality monitoring. 3. Cancer Risk: Disruption in the balance between self-renewal, differentiation, and death can lead to uncontrolled proliferation resembling cancer. Summary Human pluripotent stem cells are derived from early embryos or reprogrammed somatic cells. Characterization involves both molecular markers and functional assays. Applications span regenerative medicine, drug discovery, and disease modeling but face challenges like genetic instability and regulatory hurdles in clinical translation. Hypothalamic-Pituitary-Ovarian (HPO) Axis Overview The Hypothalamic-Pituitary-Ovarian (HPO) axis is a critical endocrine system that regulates female reproductive function, including the menstrual cycle, ovulation, and fertility. 1. Components of the HPO Axis a. Hypothalamus Function**: The hypothalamus is the control center for the HPO axis. Secretion**: Produces and secretes Gonadotrophin Releasing Hormone (GnRH). GnRH is released in a pulsatile manner, which is essential for stimulating the pituitary gland. b. Pituitary Gland Location**: Situated at the base of the brain, connected to the hypothalamus. Function**: The pituitary gland responds to GnRH by releasing gonadotrophins. Gonadotrophins Released**: Follicle Stimulating Hormone (FSH): Stimulates the growth and maturation of ovarian follicles. Plays a role in the regulation of the menstrual cycle. Luteinising Hormone (LH): Triggers ovulation and the formation of the corpus luteum. Stimulates the production of estrogen and progesterone from the ovaries. 2. Regulation of the HPO Axis The HPO axis operates through a feedback mechanism involving hormones: Negative Feedback: Estrogen and progesterone produced by the ovaries exert negative feedback on the hypothalamus and pituitary gland to regulate the secretion of GnRH, FSH, and LH. Positive Feedback: In certain phases of the menstrual cycle, high levels of estrogen can lead to a surge in LH, triggering ovulation. 3. Disruptions to the HPO Axis Disruptions in the HPO axis can lead to various reproductive issues, including: Effects on Secondary Sexual Characteristics: Delayed or absent development of secondary sexual characteristics (e.g., breast development, menstrual cycle initiation). Fertility Issues:  Irregular menstrual cycles, anovulation (failure to ovulate), and infertility can result from hormonal imbalances or disruptions in the axis. Common causes of disruption may include: Stress, excessive exercise, nutritional deficiencies, hormonal disorders, and certain medications. Ovarian Cycle Follicular Development: Primordial follicles: the earliest stage of ovarian follicles, consisting of primary oocytes surrounded by a single layer of granulosa cells. Granulosa cells secrete estrogen which is crucial for regulation of the menstrual cycle, they provide support and nourish the oocyte and have FSH receptors that allow them to respond to FSH during later stages of the follicular development. Stages of Maturation: Progress from primordial → primary → antral stages. Early stages are FSH-independent; antral follicles require FSH and form a fluid-filled cavity (antrum). Ovulation: Trigger: as follicles mature there is a rise in estrogen levels lead to an LH surge. LH Surge Effects: Completes meiosis I (forms a secondary oocyte). As there is rapid follicle growth, granulosa cells lose the FSH/estrogen receptors. Granulosa cells develop progesterone receptors. Process: The mature follicle ruptures, releasing the secondary oocyte into the fallopian tube. Corpus Luteum: Develops from the remaining granulosa cells after ovulation Produces progesterone to maintain the uterine lining Lifespan of 14 days unless pregnancy occurs then degenerates into the corpus albicans Suppresses FSH and LH, to prevent further follicular development during the luteal phase Endometrial Cycle Menstrual Phase (Days 1–5)  Hormonal Changes: Progesterone drops at the end of the luteal phase. Effects: ○ Spiral arterioles spasm, causing reduced blood flow. ○ The functional layer of the endometrium sheds, leading to menstrual bleeding. Proliferative Phase (Days 5–14) Hormonal Changes: Estrogen levels rise as ovarian follicles develop. Effects: ○ Endometrium grows and thickens, preparing for implantation. ○ Progesterone receptors form in preparation for the next phase. Secretory Phase (Days 14–28) Hormonal Changes: Progesterone is secreted by the corpus luteum after ovulation. Effects: ○ Endometrium becomes secretory, producing nutrients for potential embryo. ○ Spiral arterioles develop to improve blood supply. ○ Myometrial contractions are suppressed to support implantation. Clinical Applications Contraception Combined Oral Contraceptive Pill (COCP) maintains constant estrogen and progesterone levels Inhibits ovulation through negative feedback on FSH and LH Subfertility Assessment Check baseline FSH and LH on days 2-5 of cycle Measure peak progesterone levels 7 days before next period Polycystic Ovarian Syndrome (PCOS) Characterized by polycystic ovaries, hyperandrogenism, and oligo/amenorrhea Treatment includes ovulation induction, weight loss, and endometrial protection Pregnancy and Menopause 1. Early Pregnancy hCG Production: The implanted blastocyst produces hCG to sustain early pregnancy.  hCG Role: Maintains the corpus luteum, ensuring progesterone production until the placenta takes over (~12 weeks). 2. Menopause Ovarian Changes: Ovaries stop ovulating, ending menstrual cycles. Hormonal Changes: Estrogen levels drop, causing symptoms like hot flashes, night sweats, mood changes, and sleep issues. 3. Hormone Replacement Therapy (HRT) Purpose: Relieves menopausal symptoms and prevents osteoporosis. Risks: ○ Estrogen increases VTE risk. ○ Combined estrogen-progestogen therapy may raise breast cancer risk, requiring regular monitoring. What is the window of implantation? The window of implantation is the brief period (~6–10 days after fertilization) when the uterine lining (endometrium) is ready to support a fertilized egg (blastocyst), crucial for successful pregnancy. Timing: Occurs during the luteal phase after ovulation, when the endometrium becomes receptive for blastocyst attachment. Endometrial Changes: Increased blood vessel permeability, tissue swelling, and secretory glands produce nutrients for the embryo. Receptivity: The endometrium transitions from prereceptive (non-adhesive) to receptive, allowing effective embryo attachment. Signaling: The blastocyst signals to maintain progesterone from the corpus luteum, supporting implantation. If Missed: Implantation failure leads to a non-receptive phase, and the endometrium sheds during menstruation. What is the receptive endometrium? The receptive endometrium is the uterine lining's prepared state for implantation of a fertilized egg (blastocyst), essential for pregnancy. Timing: Occurs in the luteal phase during the implantation window (days 20–24 of a 28-day cycle). Hormonal Influence: Progesterone (from the corpus luteum) and estrogen prepare the endometrium.  Morphological Changes: ○ Thickened stromal lining. ○ Developed glands secrete nutrients and growth factors. ○ Increased blood vessel growth for better nutrient delivery. Cellular Changes: ○ Shorter microvilli on epithelial cells aid attachment. ○ Reduced surface negative charge eases blastocyst adhesion. ○ Stromal cells transform into decidual cells to support the embryo. Signaling: Specific molecules attract the blastocyst and promote implantation while suppressing inhibitory signals. Duration: Lasts ~3 days; without implantation, the endometrium reverts and sheds during menstruation.