Insight to IVF A Laboratory Perspective

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Wesley Campbell
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1 Insight to IVF A Laboratory Perspective

2 Laboratory Environment Need homeostatic, non-toxic, nurturing environment Warm laboratory to minimise temperature fluctuations Assure nothing in lab is toxic Keep lab free of VOCs No cardboard in lab Air new equipment outside of lab Use VOC free paints Check VOCs using VOC measuring device Take care with cleaning reagents within the laboratory Embryologists contributing to environment No perfume, lotions, make up

3 IVF Laboratory: Optimal culture conditions for optimal pregnancy results Commitment to Single Embryo Transfer Embryo culture Cryopreservation of sperm, eggs, embryos ovarian tissue Manage patient expectations through flexibility and choice

4 Function of the Embryology Laboratory Culture media preparation Egg collections (oocyte retrieval; egg harvesting; EPU; VPU) Sperm Preparation (and sometimes extraction) Insemination Embryo Culture Embryo biopsy (as required) Embryo transfer Gamete/embryo freezing and thawing/vitrification and warming With respect to our patients With the highest attention to detail With extensive witnessing procedures

5 Culture conditions and quality control Most common: Complex Sequential Culture media used: Complex means it provides all nutrients including amino acids and protein for optimal embryo growth Sequential as it mimics the environment in the fallopian tubes then uterus thus the culture media embryos are exposed to is changed at appropriate times of development adapting to embryo s requirements Importance of temperature and ph Controlled environment essential for optimal embryo growth therefore we use IVF Chambers Mincs Strict QC/QA Program

6 What influence does specific culture media and environment have on our embryos? What is our end point in IVF?

7 What is in culture media? Water Salts Energy substrates: glucose, pyruvate, lactate Buffer NaHCO3/MOPs Non-essential Amino Acids Essential Amino Acids Glutamine Chelators mainly EDTA Macromolecules: Albumin/Hyaluronan Antibiotics Phenol Red?

8 What is the purpose of culture media? Assist in preparing gametes for fertilisation and grow embryos to then be placed into uterus

Differences between oviduct and uterus in mammalian embryos

2 mm Oxygen concentration 8 % 1.5 % Carbon dioxide conc.

9 Differences between oviduct and uterus in mammalian embryos Component Oviduct Uterus Glucose concentration 0.5 mm 3.15 mm Pyruvate concentration 0.32 mm 0.10 mm Lactate concentration 10.5 mm 5.2 mm Oxygen concentration 8 % 1.5 % Carbon dioxide conc. 12% 10% ph Glycine concentration Alanine concentration Serine concentration

10 Differences in the physiology of the mammalian embryo for development from the zygote to the blastocyst stage. Precompaction stage Postcompaction stage Low biosynthetic activity High biosynthetic activity Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient) Pyruvate-based metabolism Maternal genome Single cell Low ability to maintain cellular homeostasis Totipotent Glucose-based metabolism Embryonic genome Transporting epithelium Complex systems for maintenance of cellular homeostasis Differentiation into inner cell mass and trophectoderm

11 Oviduct pyruvate lactate glucose non-ess AA Uterus pyruvate lactate glucose non-ess AA essential AA

12 Does culture media matter if your patients are getting pregnant? Evidence that there are differences in DNA methylation patterns in placental and umbilical blood samples from IVF children compared to naturally conceived. There is an association with ART and specific imprinting disorders Subfertility/Infertility, hormone stimulation, in vitro culture, uterine environment appear to influence the proper establishment and maintenance of genomic imprints in the developing epigenome.

13 Epigenetics & Imprinting Epigenetics: Chromatin modifications that regulate gene activity that are not due to DNA sequence changes (Saitou et al. 2012) Imprinted genes are genes whose expression is determined by the parent that contributed them Imprinted genes violate the usual rule of inheritance that both alleles in a heterozygote are equally expressed. Genomic imprinting is a specialized epigenetic mechanism that employs repressive modifications to silence one parental allele, while activating modifications on the other parental allele enable expression Hirasawa & Feil 2010 DNA methylation and histone modifications are two epigenetic mechanisms that alter the functional state of chromatin, activating or repressing gene expression

14 In Vitro Embryo culture Culture Media Does culture media effect the health of the embryo? Suboptimal media may compromising imprint maintenance. Mouse embryos cultured in deficient media displayed biallelich19 expression in postimplantation extraembryonic tissues (Sasaki et al. 1995) Whitten's but not potassium simplex optimized medium plus amino acids (KSOMaa) cultured embryos exhibited biallelic H19 expression (Doherty et al. 2000). Extremely important for culture media to provide all the nutritional requirements when required Metabolites and cofactors are important regulators of the epigenome Many imprinted genes are involved in growth and metabolism

15 Culture Environment There is growing evidence that the environment in which an embryo develops can effect its metabolism, epigenetic alterations and developmental potential; all aspects of environment important! Culture conditions have been shown in mouse models to impact epigenetic patterns of the embryo and especially the placenta. Specifically, culture at atmospheric (20%) oxygen tension as compared to physiologic (5%) oxygen tension resulted in marked differences in global gene expression, in particular genes involved in cell growth and maintenance, relative to embryos developed in vivo. Don t stress gametes or embryos! Maintain homeostatic environment!

and/or viability post implantation Exposing preimplantation embryo to stressors that impair function of

16 Healthy adults from IVF should be our end point: Embryos do have the ability to adapt to less than optimal culture conditions however this comes at a cost, culminating in incrementally lower rates of blastocyst and/or viability post implantation Exposing preimplantation embryo to stressors that impair function of mitochondria not only alters survival to the blastocyst and resultant viability but subsequent fetal growth and health

Number of follicles does not equal number of eggs retrieved Once the follicular fluid has been aspirated the

17 Egg Retrieval Follicle Scan: eggs are within the follicles. Eggs are retrieved trans-vaginally with the use of a Ovum needle attached to a vaginal ultrasound probe. Number of follicles does not equal number of eggs retrieved Once the follicular fluid has been aspirated the eggs are removed from the fluid in an IVF chamber and placed into culture media. Average egg number is around 8-10 Quality not quantity, everyone is different!

18 Egg Maturation Only mature eggs will fertilise Germinal Vesicle Metaphase I Metaphase II Mature Egg

19 Maturation

20 Semen Parameters SPERM CONCENTRATION >15 MILLION / PER ML Natural Variations SPERM MOTILITY >50% Swimming forward Different grades of motility SPERM MORPHOLOGY <96% Abnormal forms considered Normal Genetic Lifestyle ANTI-SPERM ANTIBODIES

21 Sperm Retrieval Ejaculation Most common! - Motile sperm have to be separated from the non motile and abnormal sperm using a two part gradient system and centrifugation. - Sperm are then washed with culture media and are ready for insemination. If indicated: Retrograde Ejaculation Sperm extraction

Sperm Retrieval Testicular biopsy: Males that may require intervention for sperm retrieval: Obstructive azoospermia eg: Vasectomy Congenital Absence of Vas deference (CAV) Non-obstructive azoospermia

22 Sperm Retrieval Testicular biopsy: Males that may require intervention for sperm retrieval: Obstructive azoospermia eg: Vasectomy Congenital Absence of Vas deference (CAV) Non-obstructive azoospermia eg: Kleinfelters Y-chromosome deletion Sertoli cell only Virtual Azoospermia Depending on the aetiology sperm can be retrieved by: Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle), Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle) Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved) Can be performed under local anaesthetic unless incision required then need general anaesthetic

23 Seminiferous Tubules from Testicular Biopsy

25 Insemination Depends on sperm quality or patient history (previous IVF cycles) Standard Insemination (IVF) Microinjection (ICSI)

Microinjection (ICSI) In cases where semen quality is sub-optimal

Intra-Cytoplasmic Sperm Injection (ICSI).

26 Microinjection (ICSI) In cases where semen quality is sub-optimal the sperm are injected into the egg using a procedure called Intra-Cytoplasmic Sperm Injection (ICSI). A single sperm is immobilized and drawn into a fine pipette for injection into the egg. The egg is held steady using a holding pipette. The sperm is expelled into the cytoplasm of the egg and the pipette is withdrawn from the egg.

27 ICSI

28 Fertilisation

29 Fertilisation (Day 1) Fertilised egg Unfertilised Abnormal (2 Pronuclei Visible) (>2 PN) eg. - Standard Insemination more than 1 sperm has entered - ICSI Second polar body hasn t been extruded by egg as above

30 Pronuclei appear

31 Sometimes things don t go quite to plan One pronuclei

oolemma Lock and key defective: eg egg may not have ZP3 receptor so sperm cannot bind; ~64% of

32 Failed Fertilisation Can be due to the sperm, egg or both Standard Insemination: Sperm egg interaction fails due to adhesion, binding, penetration of zona pellucida or fusion with oolemma Lock and key defective: eg egg may not have ZP3 receptor so sperm cannot bind; ~64% of males with abnormal sperm parameters have defective sperm-zona interactions Most can be overcome with ICSI

Defective sperm decondensation Eggs may have abnormal chromosome complements that don t allow

33 Failed Fertilisation with ICSI: ICSI: Sperm able to activate egg or egg receptive to activation? Cascade of reactions required for fertilisation and pronuclear formation. Defective sperm decondensation Eggs may have abnormal chromosome complements that don t allow further development Spindle which allows the chromosomes to split appropriately may be abnormal and be a cause of failed fertilisation.

34 Abnormal Fertilisation 3+ pronuclei Standard IVF insemination: 2+ sperm fertilising egg Failure of egg polyspermy mechanism: egg has several mechanisms to prevent more than one sperm entering however in some instance they may fail. ICSI: Failure of second polar body extrusion? Polyploidy, mainly triploidy, usually due to polyspermy, occurs in about 2-3% of all human pregnancies and ~15% of miscarriages.

35 Embryo Development Day 2 Embryo starts dividing 24 hours post sperm insemination Division during pre-implantation development is called cleavage Day 2 embryo are expected to be at the 2-4 cell stage 2-cell embryo 4-cell embryo

36 Day 3 Embryo expected to be at the 6-8 cell stage Switch from maternal to embryonic control Quality of embryo dependent on: Cell Stage Development Fragmentation 8-cell embryo

37 Embryo Fragmentation - During cell division fragments of cytoplasm break off.? Programmed cell death? Cytoskeletal and spindle defects - Extensive fragmentation have decreased blastulation rate, >15% fragmentation blastocyst rate declines - Correlation with fragmentation and embryos that have inherent defects

38 Day 4 Embryo at the Morula stage (mulberry like in appearance) Embryo undergoes rapid cleavage

39 Day 5 Blastocyst Not all Embryo can develop to the blastocyst stage! Therefore is used as a selection tool of most viable embryo Embryo differentiates into 2 different cell types Inner Cell Mass (ICM) Trophectoderm (TE) >60 cells Quality determined by Approx no. cells within the 2 cell types Degeneration present Expansion TE ICM

40 Blastocysts come in different shapes and sizes!

41 Embryo Development Day 1 Day 2 Day 3 Day 4 Day 5

44 Embryo Transfer Ultrasound guided Embryo is aspirated into tip of catheter Embryo injected into uterus

Cryopreservation There are two methods of embryo cryopreservation Slow Freezing Vitrification Nature 305, 707-709 (1983).

45 Cryopreservation There are two methods of embryo cryopreservation Slow Freezing Vitrification Nature 305, (1983). Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Alan Trounson & Linda Mohr Cryobiology Aug;21(4): Vitrification as an approach to cryopreservation.fahy GM, MacFarlane DR, Angell CA, Meryman HT.

46 Slow Freezing Was main method of cryopreservation in IVF for many years Still used in some clinics with good results Controlled step wise decrease in temperature. Lethal intracellular freezing can be avoided if cooling is slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid.

Vitrification A process of converting a material into a glass-like amorphous solid

addition of heat, or by mixing with an additive Embryos suitable for freezing are

implantation rates of >35% for blastocysts that have been frozen, stored at -196 o

47 Vitrification A process of converting a material into a glass-like amorphous solid which is free of any crystalline structure, either by the quick removal or addition of heat, or by mixing with an additive Embryos suitable for freezing are vitrified to be used during a Frozen Embryo Transfer cycle Vitrification gives implantation rates of >35% for blastocysts that have been frozen, stored at -196 o C and warmed. Survival Rates in excess of 90% for oocytes, cleavage embryos and blastocysts

48 Cryoprotectants The major players in penetrating cryoprotectants: Ethylene Glycol Dimethyl sulfoxide 1,2-Propanediol Time vs temperature vs concentration Single vs cocktail

Cryoprotectants Food Chem Toxicol. 2010 Jul;48(7):1905-12. doi: 10.1016/j.fct.2010.04.032. Epub 2010 Apr 28.

49 Cryoprotectants Food Chem Toxicol Jul;48(7): doi: /j.fct Epub 2010 Apr 28. Assessment of the genotoxicity of three cryoprotectants used for human oocyte vitrification: dimethyl sulfoxide, ethylene glycol and propylene glycol. Aye M 1, Di Giorgio C, De Mo M, Botta A, Perrin J, Courbiere B. Chinese Hamster Ovary cell line Results showed: DMSO was not genotoxic EG not directly genotoxic PrOH produced DNA damage leading to chromosome mutations

50 Safety of Vitrification Fully open system theoretical risk of contamination is >500,000 transfers Mol Hum Reprod Mar 31. pii: gav013. [Epub ahead of print] Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification. De Munck N, Petrussa L, Verheyen G, Staessen C, Vandeskelde Y, Sterckx J, Bocken G, Jacobs K, Stoop D, De Rycke M, Van de Velde H. Hum Reprod Aug;28(8): doi: /humrep/det107. Epub 2013 Apr 16. Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions. Vanderzwalmen P 1, Connan D, Grobet L, Wirleitner B, Remy B, Vanderzwalmen S, Zech N, Ectors FJ.

51 Which embryos do you freeze? Every lab has their own criteria!

52 Increase in Freeze All Cycles why? PGS has something to do with this Research to show that transferring embryos in an non-stimulated cycle improves outcomes for mother and baby: singleton pregnancies following frozen versus fresh embryo transfers were significantly less likely to be complicated by perinatal mortality [relative risk (RR) 0.68 (95% confidence interval (95%CI) )] small for gestational age [RR 0.45 (95%CI )], preterm birth [RR 0.84 (95%CI )], low birthweight [RR 0.69 (95%CI )] antepartum haemorrhage [RR 0.67(95%CI )].

53 Oocyte Cryopreservation

54 Ovarian Tissue Feasible fertility preservation alternative to oocyte freezing?

Test for genetic mutation and transfer unaffected embryos PGD/PGS 3 different types of genetic testing: Aneuploidy screening 24 chromosome screening SNP, array CGH, NGS

55 Test for genetic mutation and transfer unaffected embryos PGD/PGS 3 different types of genetic testing: Aneuploidy screening 24 chromosome screening SNP, array CGH, NGS Chromosome rearrangement testing SNP, array CGH, NGS or FISH Single gene testing Single gene disorders e.g Cystic Fibrosis, SMA, Huntington's Karyomapping, PCR or SNP+aneuploidy

56 Embryo Biopsy Day 3 Blastocyst

57 Day 3 Embryo biopsy

58 Blastocyst biopsy

59 Day 3 or Blastocyst Biopsy? Day 3 biopsy - Day 3 embryos show high levels of chromosome abnormality and mosaicism - Biopsy of one cell may not be representative of the remainder of the embryo - Only one cell to analyse Blastocyst biopsy has been associated with: - Higher implantation rate than Day 3 - Lower aneuploidy rate than Day 3 - High implantation rates regardless of maternal age - Reduced mosaicism compared with Day 3 - More cells available for analysis

~50% euploid Aneuploidy rate continues to decline through pregnancy

60 Aneuploidy difference according to stage Blastocysts display significantly lower aneuploidy rates Day 3: ~20-25% euploid Day 5: ~50% euploid Aneuploidy rate continues to decline through pregnancy Spontaneous abortion ~50% aneuploid Stillbirth ~4% aneuploid Livebirth ~0.3% aneuploid

61 100 Blastocyst Euploidy Rate by Age R² = < Euploidy rate (%) Linear (Euploidy rate (%))

62 Day 3 versus Day 5 biopsy 24 chromosome screening data Age Implantation (%) Day 3 Day 5 Aneuploidy (%) Implantation (%) Aneuploidy (%)

63 24 chromosome screening Why do it? Which embryo should be transferred? How many transfers would be needed to select the right one?

64 With the knowledge we have today we do all we can do to assure the health of our patients, the babies resulting from our programs and the adults they will become. Thank you!

Understanding eggs, sperm and embryos. Marta Jansa Perez Wolfson Fertility Centre

Understanding eggs, sperm and embryos Marta Jansa Perez Wolfson Fertility Centre What does embryology involve? Aims of the embryology laboratory Creation of a large number of embryos and supporting their