Female Reproductive Tract, Semen Analysis, and Embryo Culture Study Guide

Female Reproductive Tract and COC Harvest

Learning Objectives and Induction Areas

• Anatomical Overview: Understand the anatomy of the female reproductive tract in monovular species.

• Ovarian Cell Types: Provide detailed insight into the structure of key ovarian cell types including ovarian follicles, corpus luteum, corpus albicans, and Cumulus Oocyte Complexes (COCs).

• Uterine Structure: Detailed insight into the structure of the uterus.

• Harvesting Procedures: Harvest COCs from ovarian follicles using needle aspiration.

• Cell Identification: Recover COCs from pooled follicular aspirates and identify the different cell types present.

• Laboratory Skills: Gain experience with culture plates and handle COCs using various pipette types.

Introduction to Monovular Species Models

• Species Selection: The practical utilizes sheep (ovine) and cow (bovine) tissue.

• Model Suitability: These species are effective models for human female reproductive function because the size and architecture of adult ovine/bovine ovaries reflect those of pre-pubertal/adolescent girls and women.

• Oocyte Dimensions: Oocytes from these species are similar in size to human oocytes.

• Applications: In vitro matured (IVM) oocytes from these species are used for In Vitro Fertilisation (IVF) and In Vitro Embryo Production (IVEP) in research and commercial embryo transfer.

• Developmental Timelines: The time scales required to derive blastocysts following IVM and IVF in ovine/bovine models closely match human developmental timelines.

PPE and Safety Requirements

• Protective Clothing: Lab coats, disposable aprons, and safety glasses are mandatory.

• Hand Protection: Double-gloving (wearing two pairs of gloves) is required.

• Sharps Safety: Extreme care must be taken with needles and syringes to avoid needle-stick injuries.

• Hygiene: Hands must be washed thoroughly after the practical session.

Reproductive Cycle Identification

• Stages of the Reproductive Cycle:

  • Follicular phase.

  • Ovulation.

  • Early luteal phase.

  • Mid luteal phase.

  • Late luteal phase.

  • Pre-pubertal ovary.

• Anatomical Variants: Observed differences in follicle size and ovarian architecture between sheep and cows identify the specific cycle stage.

Protocols for COC Recovery

• Follicle Aspiration:

  • Use a sterile needle and syringe.

  • Priming the syringe: Use $0.5$ to $1.0\,ml$ of oocyte holding medium ($H199$).

  • Handling: Hold the ovary in the non-dominant hand and the syringe in the dominant hand.

  • Angle: Push the needle into the follicle with the point facing upwards while sucking with the syringe to prevent fluid loss.

  • Strategy: Aspirate the largest follicles first to prevent accidental bursting.

• Processing Aspirates:

  • Pool aspirates into a Universal tube.

  • Draw a grid on the underside of a clean Petri dish to facilitate counting.

  • Transfer cell-rich media from the bottom of the tube to the Petri dish using a Pasteur pipette.

  • Allow the dish to settle for $5\,minutes$ before searching for COCs under a stereomicroscope.

• Transfer and Culture:

  • Use a micropipette set to $20\,\mu l$ to recover individual COCs.

  • Transfer COCs into a 4-well culture dish containing $500\,\mu l$ of fresh media per well.

  • Wait to transfer COCs into well 4, which serves as a medium reference well.

  • Calculate recovery rate: $\frac{\text{Number of COCs Recovered}}{\text{Number of Follicles Aspirated}} \times 100$.

Semen Analysis Practical Series

Objectives and Introduction

• Bovine as a Human Substitute: Bovine semen is used as a surrogate because its spermatozoa are more homogeneous than human semen, which contains a mix of sperm, immature germ cells, and somatic cells (epithelial and white blood cells).

• Key Parameters: Evaluation of sperm count, concentration, and motility.

• Isolation Techniques: Isolation of viable spermatozoa using "swim-up" and Density Gradient Centrifugation (DGC).

Macroscopic and Microscopic Examination

• Straw Processing: Cryopreserved semen straws contain approximately $200\,\mu l$. Cut the sealed end first into an Eppendorf, then the plugged end.

• Macroscopic Indicators:

  • Volume: Measured with a $200\,\mu l$ pipette.

  • pH: Applied to pH strips; color should stabilize in under $30\,seconds$.

  • Viscosity: Observed as semen drops from a plastic pipette. High viscosity is indicated by a "thread" longer than $2\,cm$.

• Morphology Smear Preparation:

  • Place a $5\,\mu l$ droplet near the edge of a frosted-edge microscope slide.

  • Use a second slide to spread the droplet via capillary action and draw it across to form a smear.

  • Label slides with pencil only to prevent the label from washing off during staining.

The Makler Counting Chamber

• Components:

  1. Lower Part: Metal base ($A$), handles ($H$), and an optical glass disc ($D$) surrounded by four pins ($P$) that are exactly $10\,\mu m$ high.

  2. Upper Part: A circular cover glass ($C$) in a metal ring with a grid of $100$ squares (each $0.1\,mm \times 0.1\,mm$).

• Principles: The space within a row of 10 squares is exactly $\frac{1}{1,000,000}\,ml$.

• Motility Categories (WHO 2021 Guidance):

  • Category a (Rapid Progressive): $> 25\,\mu m/s; active linear or large circle movement.\n * **Category b (Slow Progressive)**: 5$to$< 25\,\mu m/s.\n * **Category c (Non-progressive)**: < 5\,\mu m/s; active tail movement but no progression (e.g., small circles).\n * **Category d (Immotile)**: No movement.\n* **Calculations**:\n * Standard concentration: C = \frac{N}{3} \times 10^6\,ml^{-1}$(where$N is the count from three strips of 10 squares).\n * Low concentration: If count is too low for strips, count the entire grid and use C = N \times 10^5.\n\n## Staining and Vitality Assessment\n* **Vitality (Eosin-Nigrosin Live/Dead Staining)**:\n 1. Add 50\,\mu l semen to an Eppendorf.\n 2. Add 100\,\mu l$Eosin (Reagent 1) and mix for$30\,seconds.\n 3. Add 150\,\mu l Nigrosin (Reagent 2) and mix.\n 4. Examine at x40$magnification. Count a minimum of$100 sperm. Unstained cells are viable; stained cells are dead.\n* **Morphology (Kwik-Diff)**:\n * Performed in a fume cupboard with double gloves.\n * Dip slide 10 times (1 second each) into Fixative (Stain 1).\n * Dip 10 times into Stain 2, then 10 times into Stain 3.\n * Rinse with distilled water and air dry for 15\,minutes$. Analyze$50$to$100$spermatozoa at$x40 magnification.\n\n## Sperm Isolation Procedures\n* **Swim-up Method**:\n * Pipette 500\,\mu l of warm sperm wash into a tube.\n * Hold at a 45^{\circ} angle and underlay semen beneath the wash using a glass pipette.\n * Incubate in a hotbox at 39^{\circ}C$for$45\,minutes. Recover the haze (motile sperm) from the top.\n* **Density Gradient Centrifugation (DGC)**:\n * Overlay 1\,ml$of$45\%$gradient solution onto$1\,ml$of$90\% gradient solution.\n * Very carefully overlay the semen on top.\n * **Centrifugation 1**: 700g$for$20\,minutes.\n * Discard supernatant and retrieve the pellet.\n * **Centrifugation 2**: Resuspend pellet in 1\,ml$sperm wash, centrifuge at$500g$for$5\,minutes.\n * Discard supernatant to leave a minimal volume (50$to$100\,\mu l).\n\n# Embryo Culture and Collection\n\n## Sterile Technique and Lab Safety\n* **Sanitization**: Disinfect areas with ethanol before/after work.\n* **Identification**: Use three points of reference for labeling: patient name, date of birth, and unique ID number.\n* **Handling Hygiene**: Change gloves often; never "double-dip" in stock solutions (always use aliquots).\n\n## Culture Dish Preparation\n* **Fertilisation Dish**: 4-well dish with medium and 250\,\mu l mineral oil overlay.\n* **Culture Dish**: 60\,mm$dish with$20\,\mu l drops of media overlaid with mineral oil.\n* **Equilibration**: Dishes must be incubated to reach appropriate temperature and gas concentration.\n* **Overlay Rules**: Fill oil from the side to avoid moving culture drops. Ensure no air gap between oil and lid is sealed; gas exchange is required.\n\n## Mouse Embryo Development and Manipulation\n* **Group Culture**: In mice, a higher blastocyst yield is obtained via group culture rather than individual culture.\n* **Bead Practice**: Use an EZ-Grip pipette to move polystyrene beads in a clockwise direction between drops as a simulation for embryo handling.\n* **Washing Technique**: Move zygotes through two "wash drops" before placing them in the final culture drop.\n\n## Embryo Morphology and Grading\n* **Cleavage-Stage (Day 3) Grading**:\n * **Grade 4**: Blastomeres equal in size; < 10\% fragmentation.\n * **Grade 3**: Slightly uneven blastomere sizes; 10$to$20\% fragmentation.\n * **Grade 2**: Majority of blastomeres are different sizes; 20$to$50\% fragmentation.\n * **Grade 1**: Non-stage specific sizes; > 50\% fragmentation.\n* **Blastocyst-Stage (Day 5) Grading**:\n * **Expansion Status (Score 1-6)**: From Early Blastocyst (1$: blastocoel$< 50\%$volume) to Hatched Blastocyst ($6: evacuated from the Zona Pellucida).\n * **Inner Cell Mass (ICM) (Grade A-D)**: \n * **A**: Prominent, many compacted cells.\n * **B**: Cells larger and more loosely adhered.\n * **C**: Very few cells visible.\n * **D**: No visible cells or necrotic.\n * **Trophectoderm (TE) (Grade A-D)**:\n * **A**: Many small identical cells in a continuous layer.\n * **B**: Fewer cells with gaps.\n * **C**: Few small cells mixed with large cells.\n * **D**: Very few or degenerate cells.\n\n# Assisted Reproduction Technology (ART)\n\n## In Vitro Maturation (IVM)\n* **Medium Composition**: Maturation medium requires energy substrates, salts, and surge levels of gonadotrophic hormones (LH and FSH).\n* **Incubation Conditions**: Humidified incubator at 38.5^{\circ}C$to$39^{\circ}C$($6\%$$CO_2$in air) for$24\,hours.\n* **Selection**: Select COCs with a complete layer of cumulus oophorous cells and high morphological quality.\n\n## Oocyte Denudation\n* **Definition**: The process of stripping cumulus cells from the oocyte to assess nuclear maturation or prepare for ICSI (Intra-Cytoplasmic Sperm Injection).\n* **Enzymatic Loosening**: COCs are briefly exposed to Hyaluronidase (Hyase).\n* **Mechanical Removal**: Gentle, repeated pipetting using pipettes with progressively decreasing bore sizes.\n* **Purpose for ICSI**: Denudation allows visualization of the polar body. The polar body is typically positioned at the "North" or "South" pole during sperm injection to minimize damage.\n* **Optimal Temperature**: Hyase works optimally at 37^{\circ}C$$.