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6/24/2019 1 SPERM, OOCYTES AND EMBRYOS SPERM, OOCYTES AND EMBRYOS SPERM, OOCYTES AND EMBRYOS SPERM, OOCYTES AND EMBRYOS IN THE IVF LAB IN THE IVF LAB IN THE IVF LAB IN THE IVF LAB

JILL MELLON, BSc.(Hons), MSc. Lab Director PCRM Burnaby JASON AU, BSc., MSc. Lab Supervisor PCRM Burnaby

www.pacificfertility.ca 1

We will discuss: We will discuss: We will discuss: We will discuss:

Biopsy of embryos for Genetic Testing 4

• Purpose
• Procedure

www.pacificfertility.ca 2

Sperm 1

• Basics of Semen Analysis - what it tells us
• Most suitable treatment - guidelines

Oocytes 2

• Ovarian stimulation to produce more eggs
• Egg numbers and quality

IVF and ICSI 3

• IVF
• ICSI
• Embryo Development

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INTRODUCTION INTRODUCTION INTRODUCTION INTRODUCTION

All staff have at least a BSc. in a Science subject and some of us have a Masters in Clinical Embryology Combined experience of more than 60 years (not all me!) Training is on site and extensive: normally ONE year for Andrologist and TWO years for Embryologist Known within the Clinic for our attention to detail which borders on OCD Compassionate and caring people with an unhealthy interest in our results!

www.pacificfertility.ca 3

Basic Semen Analysis

• 1. Sperm
• 1. Sperm
• 1. Sperm
• 1. Sperm

www.pacificfertility.ca 4

• Abstinence (2-5 days)
• Time to when examination is performed (30 mins)
• Volume = semen volume (>1.5mL)
• Concentration = how many millions per ml (>15)
• Sperm Motility = good, fair, poor (≥40%)
• Morphology = normal or abnormal (≥4%)

Semen analysis is a good non-invasive test to assist the Physician in directing optimal treatment

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WHO Guideline WHO Guideline WHO Guideline WHO Guideline

“Data characterizing the semen quality of fertile men, whose partners had a time to pregnancy of 12 months

• r less, provided the reference ranges

for this manual. Raw data from between about 400 and 1900 semen samples, from recent fathers in eight countries on three continents, were used to generate the reference ranges.”

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Sperm Morphology Sperm Morphology Sperm Morphology Sperm Morphology

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WHO Laboratory Manual for the Examination and Processing of Human Semen. 2010.

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NOTE: Sperm production takes 65-80 days illness may affect the sample – therefore repeat if necessary Always consider male and female factors (such as duration of infertility, patient age and partner age, previous pregnancy) first choice of treatment must include consideration of all factors REI recommendation is for what will give highest chance of success in our IVF Lab, and does not always mean other treatments cannot work Recommended treatments: intrauterine insemination, Standard IVF insemination, and intracytoplasmic sperm injection.

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Sperm Sperm Sperm Sperm Sperm Washing for Insemination Sperm Washing for Insemination Sperm Washing for Insemination Sperm Washing for Insemination

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YouTube “PCRM: The IVF Laboratory” https://www.youtube.com/watch?v=XFE-kd0nJRM

6/24/2019 5 Pre-wash Post-wash

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Sperm Washing for Insemination Sperm Washing for Insemination Sperm Washing for Insemination Sperm Washing for Insemination Pregnancy Rates by Treatment Type Pregnancy Rates by Treatment Type Pregnancy Rates by Treatment Type Pregnancy Rates by Treatment Type

www.pacificfertility.ca 10

12% 14% 8% 5% 3% 25% 21% 13% 8% 0% 57% 52% 43% 38% 15% 0% 10% 20% 30% 40% 50% 60% 70% <35 35-37 38-40 41-42 >43 Clinical Pregnancy Rate Age Groups IUI-H IUI-D IVF/ICSI

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As back up for a treatment cycle, or when the patient will be unavailable during treatment or has problems producing a sample For cancer patients, sperm can be frozen pre-treatment to preserve

• fertility. This includes adolescent

patients Transgender patients can freeze sperm before their gender re-assignment Sperm can be frozen indefinitely and stored in liquid nitrogen, with no effect on its quality. Semen has a cryoprotectant solution added before being frozen in “straws” using a controlled rate freezer. Sperm freezing is done for many reasons:

www.pacificfertility.ca 11

Sperm Freezing Sperm Freezing Sperm Freezing Sperm Freezing

• 2. Oocytes (Eggs)
• 2. Oocytes (Eggs)
• 2. Oocytes (Eggs)
• 2. Oocytes (Eggs)

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Hill, M.A. (2019, May 19) Embryology Female gametogenesis.jpg. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Female_gametogenesis.jpg

GV MI MII

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www.pacificfertility.ca 13

Egg Retrieval Egg Retrieval Egg Retrieval Egg Retrieval Egg Masses Egg Masses Egg Masses Egg Masses – – – – Cumulus Oocyte Complex Cumulus Oocyte Complex Cumulus Oocyte Complex Cumulus Oocyte Complex

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Average number of eggs retrieved in <35yo = 16 Average number of eggs retrieved in >40yo = 9

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Oocytes Maturity Oocytes Maturity Oocytes Maturity Oocytes Maturity

www.pacificfertility.ca 15

Zhang P, Zucchelli M, Bruce S, et al. Transcriptome profiling of human pre-implantation development. PLoS ONE. 2009;4(11):e7844.

Good quality oocyte Poor quality oocyte

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Oocytes Quality Oocytes Quality Oocytes Quality Oocytes Quality

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We will discuss: We will discuss: We will discuss: We will discuss:

Biopsy of embryos for Genetic Testing 4

• Purpose
• Procedure

www.pacificfertility.ca 17

Sperm 1

• Basics of Semen Analysis - what it tells us
• Most suitable treatment - guidelines

Oocytes 2

• Ovarian stimulation to produce more eggs
• Egg numbers and quality

IVF and ICSI 3

• IVF
• ICSI
• Embryo Development

A calculated number of highly motile sperm is added to a dish containing the eggs. This is incubated overnight and checked the following morning for signs of fertilization Used where semen analysis is in the normal range Considered to be more “natural” and less invasive than ICSI

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IVF

6/24/2019 10 A single sperm is injected into a single mature egg. Reasons where the preferred treatment may be ICSI

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ICSI (Intracytoplasmic Sperm Injection) ICSI (Intracytoplasmic Sperm Injection) ICSI (Intracytoplasmic Sperm Injection) ICSI (Intracytoplasmic Sperm Injection)

Low concentration of sperm No normal sperm Extracted sperm Poor sperm motility To optimize (not guarantee) fertilization – for example for biopsy and genetic testing

IVF/ICSI IVF/ICSI IVF/ICSI IVF/ICSI – – – – EMBRYO DEVELOPMENT EMBRYO DEVELOPMENT EMBRYO DEVELOPMENT EMBRYO DEVELOPMENT

www.pacificfertility.ca 20

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IVF/ICSI IVF/ICSI IVF/ICSI IVF/ICSI -

• Embryo Development: Blastocysts

Embryo Development: Blastocysts Embryo Development: Blastocysts Embryo Development: Blastocysts

www.pacificfertility.ca 21

YouTube “Reproductive System, Part 4 - Pregnancy & Development: Crash Course A&P #43” https://www.youtube.com/watch?v=BtsSbZ85yiQ

IVF/ICSI IVF/ICSI IVF/ICSI IVF/ICSI – – – – TRANSFER AND FREEZE TRANSFER AND FREEZE TRANSFER AND FREEZE TRANSFER AND FREEZE

Only a proportion of these will become blastocysts We very rarely transfer more than 2 embryos, and recommend transfer of only 1 in patients up to age 37 The embryo with best “morphology” is transferred and others which make good quality blastocysts are vitrified Damage or loss are rare when they are warmed, and pregnancy rates are the same

www.pacificfertility.ca 22

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Freezing (Vitrification) The process takes 30-40 minutes for up to 4 embryos Embryos are stored in straws, within small tanks filled with liquid nitrogen

www.pacificfertility.ca 23

IVF/ICSI FREEZE (VITRIFICATION) IVF/ICSI FREEZE (VITRIFICATION) IVF/ICSI FREEZE (VITRIFICATION) IVF/ICSI FREEZE (VITRIFICATION)

Thawing (Warming) The process takes around 30 minutes for each embryo and then the embryo is given a few hours to recover before being transferred. Immediately post-thaw 3 hours post-thaw

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IVF/ICSI FREEZE AND THAW IVF/ICSI FREEZE AND THAW IVF/ICSI FREEZE AND THAW IVF/ICSI FREEZE AND THAW

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www.pacificfertility.ca 25

IVF/ICSI IVF/ICSI IVF/ICSI IVF/ICSI – – – – PREGNANCY RATES FRESH VS. FROZEN PREGNANCY RATES FRESH VS. FROZEN PREGNANCY RATES FRESH VS. FROZEN PREGNANCY RATES FRESH VS. FROZEN

10 20 30 40 50 60 <35 years 35-37 years 38-40 years

Fresh V. Frozen Transfer - CPR 2018

fresh ET frozen ET

Why PGT?

• Previous pregnancy losses
• Advanced maternal age
• Familial genetic condition
• Multiple failed transfers from IVF cycles

Reduce TTP Save storage of abnormal embryos Family planning Surrogate community Frozen donor eggs

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• 4. Embryo Biopsy for preimplantation genetics testing
• 4. Embryo Biopsy for preimplantation genetics testing
• 4. Embryo Biopsy for preimplantation genetics testing
• 4. Embryo Biopsy for preimplantation genetics testing

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Biopsy Biopsy Biopsy Biopsy

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Thank you to our amazing team! Thank you to our amazing team! Thank you to our amazing team! Thank you to our amazing team!

www.pacificfertility.ca 28

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WE WELCOME YOUR WE WELCOME YOUR WE WELCOME YOUR WE WELCOME YOUR QUESTIONS! QUESTIONS! QUESTIONS! QUESTIONS!