Embryo scoring stage wise gametes .......

Embryo scoring

1. Selection of Embryo - Scoring Right

2. Learning Objectives Oocyte and embryo morphology Molecular and cellular anatomy of the oocyte Embryo morphology -cell number, fragmentation, cell size Embryo scoring Oocyte, zygote and cleavage-stage embryo scoring Cumulus-oocyte complex scoring Zona pellucida, polar body, pervitelline space, ooplasmic and vaculorisation scoring Pronuclei scoring Advanced methods for studying embryo viability Preimplantation genetic diagnosis and screening Dynamic assessment of embryonic development by time-lapse imaging Which embryo selection method should be offered to the patients? Evaluating options 02

3. Oocyte and Embryo Morphology 03

4. Cellular anatomy of the oocyte 04 Oocyte - surrounded by a thick extracellular matrix (the zona pellucida), ZP in turn is covered in layers of granulosa cells (the corona radiata) Ooplasm – cytoplasm of the ova Nucleus - germinal vesicle, and the nucleolus - germinal spot Oocyte - structure Gray, H. (2105). Gray's anatomy: The anatomical basis of clinical practice. Edinburgh: Churchill Livingstone/Elsevier. 41 st Edition

5. Molecular anatomy of the oocyte 05 Mia Nicolacoudis , CC BY 4.0 <https://creativecommons.org/licenses/by/4.0>, via Wikimedia Commons

6. Development of Embryo (Days 1 – 5) 06 https://basicmedicalkey.com/cleavage-and-implantation/ . Accessed on 16 Dec 2020

7. Embryo morphology -cell number, fragmentation, cell size 07 Three scoring parameters for quality: cell number, cell symmetry and fragmentation D3 human embryos with good developmental potential should develop to the 7–8 cell stage The cellular debris during cleavage – fragmentation Uneven blastomere - higher degree of aneuploidy/multinuclear rate – adverse outcomes Hardarson et al. Hum Reprod . 2001 Feb;16(2):313-8. Kong et al. PLoS One. 2016; 11(4): e0153697

8. 08 Embryo scoring

9. Oocyte scoring – Total oocyte scoring (TOS) 09 Lazzaroni-Tealdi et al. PLoS One. 2015; 10(12): e0143632. 6 parameters: ( i ) Oocyte shape; (ii) oocyte size; (iii) ooplasm characteristics; (iv) structure of the perivitelline space (PVS); (v) zona pellucida (ZP); and (vi) polar body (PB) morphology. Each parameter graded as worst (-1), average (0), or best (1), creating a TOS by adding up individual parameter assessments. The maximal TOS of an oocyte, therefore, could be a +6, the lowest a -6

10. Oocyte scoring – Dale 10 Wilding et al. J Assist Reprod Genet. 2007 Aug; 24(8): 350–358

11. Zygote scoring – Berzinova (1/2) 11 Berzinova et al. Reprod Biol Endocrinol. 2009; 7: 9 Criterion 1: Morphology of nucleolar precursor bodies (NPBs) Pattern " O“: Same number of small NPBs distributed in the nucleus or large NPBs with polar distribution between the two pronuclei. Pattern " Other", Zygotes with non-symmetrical alignments of NPB.

12. Zygote scoring – Berzinova (2/2) 12 Berzinova et al. Reprod Biol Endocrinol. 2009; 7: 9; Shoukir et al. Hum Reprod. 1997; 12 (7): 1531-1536 Criterion 2: Assessment of 1 st mitotic division Early cleavage (EC) – Two blastomeres after 23-27h of insemination [ Shoukir et al. cut-off: 25h ] No early cleavage (NEC) – Does not reach this stage with intact nuclear membranes Better outcomes with O pattern and EC zygotes

13. Zygote scoring – Scott 13 Scott et al. Reprod Biomed Online. 2003; 6(2): 201-214. Z1 - Equal pronuclei. Equal number and size of nucleoli, aligned in both pronuclei at the pronuclear junction. The absolute number of nucleoli ranges between three and seven. (A) Z2 - Equal pronuclei. Equal number and size of nucleoli, scattered in both pronuclei. The absolute number of nucleoli ranges between three and seven. (B) Z3 - Equal pronuclei. Equal number and even or (and) uneven size of nucleoli,aligned in one pronucleus at the pronuclear junction. The other pronucleus with randomly scattered nucleoli. The absolute number of nucleoli ranges between three and seven. (C) Z4 - Unequal or separated pronuclei. (D)

14. Zygote scoring – Depa-Martynow 14 Depa Martynow et al. Folia Histochem Cytobiol . 2007; 45 Suppl 1: S85-89. Based on three criteria Presence of a cytoplasmic halo Nuclear size and alignment NPB number and distribution A – Zygote with cytoplasmic halo B – Zygote without cytoplasmic halo

15. Zygote scoring – Senn 15 Senn et al. Hum Reprod. 2006; 21(1): 234-239. Zygotes initially graded based on Proximity Orientation and centering of the pronuclei Cytoplasmic halo Number and polarization of NPBs Cumulated pronuclear score (CPNS): sum of scores assigned to the six parameters is calculated for each zygote Lower CPNS values of frozen-thawed zygotes may indicate the freezing damage to zygotes CPNS may be used as a single predictor tool for implantation of both fresh and frozen-thawed zygotes

16. Cleavage stage (D3) embryo scoring – Honk Kong assisted reproduction center grading 16 https://www.hkarc.com.hk/Blastocyst_Culture_en.html Accessed on 16 Dec 2020

17. Cleavage stage (D3) embryo scoring – Turner 17 Lim et al. Open Journal of Obstetrics and Gynecology, 7, 1271-1281 Grade A—regular or only slightly irregular blastomeres, with or without minor fragments Grade B—irregular blastomeres, with or without minor fragments Grade C—irregular or regular blastomeres, up to 30% fragments Grade D—irregular or regular blastomeres, more than 30% fragments and fragmented (usually unsuitable for transfer)

18. Cleavage stage (D3) embryo scoring – Depa-Martynow 18 Depa-Martynow et al. Folia Histochem Cytobiol . 2007; 45 Suppl 1: S85-89 Grade A: embryos with 8 blastomeres and a maximum 20% of cytoplasmic fragmentation Grade B: embryos with 8 blastomeres and over 20% cytoplasmic fragmentation Grade C: 4-6 cell embryos with a maximum 20% fragmentation Grade D: 4-6 cell embryos and over 20% fragmentation

19. Cleavage stage (D3) embryo scoring - Stenson 19 Score Description 3 ≥ 10 – 20% fragmentation, even or uneven blastomeres 2 > 20 – 50% fragmentation, even or uneven blastomeres 1 Fragmentation precluded counting blastomeres Cleavage arrest or morphologically abnormal embryo Stenson et al. Reprod Biomed Online. 2010; 21(1): 118-125

20. Blastocyst (D5) embryo scoring – Gardner (1/3) 20 Gardner et al. Fertil Steril . 2000; 73(6): 1155-1158. The non-invasive evaluation of embryos at the early stages of their development (until day three following fertilization) Three components Expansion and hatching manner of blastocysts Inner cell mass (ICM) Trophoectoderm (TE)

21. Blastocyst (D5) embryo scoring – Gardner (2/3) 21 Gardner et al. Fertil Steril . 2000; 73(6): 1155-1158. Expansion Grade Blastocyst development and stage status 1 Blastocoel cavity less than half the volume of the embryo 2 Blastocoel cavity more than half the volume of embryo 3 Full blastocyst, cavity completely filling the embryo 4 Expanded blastocyst, cavity larger than the embryo, with thinning of the shell 5 Hatching of the shell 6 Hatched out of the shell

22. Blastocyst (D5) embryo scoring – Gardner (3/3) 22 Gardner et al. Fertil Steril . 2000; 73(6): 1155-1158. ICM grade Inner cell mass quality A Many cells, tightly packed B Several cells, loosely grouped C Very few cells TE Grade TE quality A Many cells, forming a cohesive layer B Few cells, forming a loose epithelium C Very few large cells

23. Cumulus Oocyte Complex (COC) scoring 23 » oocytes surrounded by ≥5 layers of compact cumulus cells A » oocytes surrounded by 2-4 layers of compact cumulus cells B C Bakri et al. Saudi J Biol Sci. 2016 Jan; 23(1): S50–S55 » oocytes surrounded by < 2 layers of cumulus cells OR half naked oocyte

24. Zona pellucida, polar body, perivitelline space, ooplasmic and vacuolization scoring ZP Scoring: No specific benefit in measuring zona thickness to predict effect/outcome PB scoring: oocytes with an abnormally large polar body should not be inseminated, due to the risk of oocyte aneuploidy PVS scoring: Presence of inclusions in the perivitelline space is anomalous – cannot predict prognosis Cytoplasmic scoring: clustering of organelles - associated with lower implantation potential. smooth-surfaced endoplasmic reticulum – risk of abnormal outcome Vacuolization: large vacuoles (<14 mm in diameter) are associated with fertilization failure 24 ESHRE. Hum Reprod . 2011 Jun;26(6):1270-83

25. Pronuclei scoring system (ESHRE consensus) 25 ESHRE. Hum Reprod . 2011 Jun;26(6):1270-83

26. Advanced methods for studying embryo viability 26

27. Preimplantation genetic diagnosis (PGD) and screening (PGS) 27 » PGD can be applied to embryos at different stages: zygotes (polar body biopsy), cleavage stage embryos (blastomere biopsy) or blastocysts (trophectoderm biopsy) 02 » PGD uses polymerase chain reaction (PCR)-based techniques to diagnose specific genetic mutations or fluorescence in situ hybridization (FISH) to diagnose chromosomal abnormalities or to sex the embryos for patients carrying X-chromosome-linked diseases 01 Ajduk et al. F1000 Biology Reports 2012, 4:11 (doi:10.3410/B4-11) » PGS involving FISH assessment of a limited number of chromosomes in one blastomere of a cleavage stage embryo (ineffective)- replaced by technologies based on comparative genomic hybridization (CGH) or single nucleotide polymorphism (SNP) arrays 03

28. Time Lapse Microscopy (TLM) 28 Chen et al. Fertil Steril . 2013; 99(4): 1035-1043. Records regular time interval photographs of a cell or an embryo over a period of several hours This system is composed of four parts; a florescent/phase contrast microscope, a digital camera which records real-time images, computer software to control the camera and an incubator as an environment for preservation of the natural condition for cells or embryos. Assessment of the oocyte/embryo developmental potential during fertilization, cleavage, development of the blastocyst, hatching and subsequent changes at intervals of 5-6 days, by the selection of credible morphological criteria and flexible evaluation using TLM instead of time point analysis may improve IVF success and reduce the risk of multiple pregnancies

29. 29 Which embryo selection method should be offered to the patients?

30. Evaluating Options 30 Factors in deciding evaluating options Expertise of the clinician in that technique Affordability of the patient Size of the clinic & scope of its services provided TLM is claimed to have at least two advantages. It provides undisturbed culture conditions and significantly more embryo observation points. It is a sophisticate option PGD is an invasive technique, often requires elective cryopreservation, off-site testing, and delayed transfer due to the pending biopsy results. The technology is not error free and may result in the loss of embryos Kovac et al. J Assist Reprod Genet . 2019 Apr;36(4):603-605 Scoring based on Morphology is still the most common method that is being employed. It is non-invasive but less accrate

31. 31 SUMMARY

32. 32 SUMMARY 1 Scoring techniques help prognosticate the outcome and helps in selection of good oocyte/ zygote/embryo for implantation in IVF 3 Other less common morphology-based scoring include Zona pellucida, polar body, perivitelline space, ooplasmic and vacuolization scoring 5 TLM – non-invasive/ more sophisticate. Records regular time interval photographs of a cell or an embryo over a period of several hours 2 Methods of morphology-based scoring: Oocyte scoring Zygote scoring Cleavage stage embryo scoring (D3) Blastomere stage (D5) embryo scoring 4 PGD / PGS – invasive technique. Helps in diagnosis of genetic mutations and chromosomal anomalies. Risk of embryo loss is unavoidable 6 Factors in deciding evaluating options 1. Expertise of the clinician in that technique. 2. Affordability of the patient 3. Size of the clinic & scope of its services provided