How to select best embryo?

1. Revealing Human Embryos:
Morphology And Contents
April 21, 2017
Ragaa Mansour, M.D., Ph.D.
Director, The Egyptian IVF-ET Center

2. Successful reproduction has always been
considered as a ̋ Miracle ̏
Cow uterus
The ancient Egyptians
had a ̋ Neteru ̏ of
childbirth and creator
of the ̋ Ka.̏
(Meskhenet)
with a symble of cow
uterus
Westcard Papyrus
Fifth Dynasty (2494 BC)

3. de Graaf described a bisected ovary, with the follicles
thought to be the actual ova.
In recent times:
1675

4. In 1827 von Baer in St. Petersburg discovered the true ovule
within the Grafian follicle.
150 years later

5. In 1939, Gregory Pincus was the first to show how rabbit
oocytes aspirated from their follicles would begin their
maturation in- vitro.
He also was the first to extract oocytes from excised human
ovaries and mature them in-vitro.
100 years later

6. 1952 – 1957
In Edinburgh
1957 - 1960
In California
- PMS and hCG
- Endocrine control of
ovulation, and artificial
insemination
15 years later
Robert Edwards
Repeated pincus work but on mouse oocytes

7. ̋ Nature 1962 ̏
̋ Lancet 1965̏
Bob Edwards was confident enough to work with
Human Oocytes
Molly Rose
(UK)
Howard &
Georgina Johns
(USA)
Dr. Victor Lewis
(UK)
He asked clinical doctors for slithers or wedges of ovaries from
patients with PCO, or excised ovaries for other conditions.

8. ̋ That evening in 1969 we watched in delight virtually all stages
of human fertilization in vitro ̏ .
Bob Edwards surprised the world unaccustomed to the
idea of human fertilization in-vitro
Robert Edwards
Nature,1969

9. Edwards telephoned steptoe, and ever since their long and
revolutionary collaboration started.
Steptoe PC, Laparoscopy and ovulation. The Lancet 1968

10. Oocytes came in a steady stream and were successfully
fertilized and grown beautifully to the stage of blastocyst.
̋ Naturȅ 1971- Human blastocysts grown in culture
Steptoe PC, Edwards RG, Purdy JM

11. ̋ The first clinical pregnancy arose after the transfer of a single blastocyst ̏
Two years later the first IVF baby was born
Lancet 1976, Steptoe PC, Edwards RG. Reimplantation
of a human embryo with subsequent tubal pregnancy

12. ̋ We are indeed enmeshed in some of the most fundamental
evolutionary stages of our existence as we pass from oocyte to
blastocyst and to implantation ̏.
And gradually IVF Labs worldwide increased steadily
̋ Robert Edwards
̏
2 PN
blastocyst
morula

13. How to select the best? I- Morphology
- Good quality MII oocyte has
regular size, clear moderately
granulate cytoplasm, a small
perivitelline space, colorless
zona, and intact PB (1)
- Heavily fragmented PB, dense
central granulation, and vacuoles
> 14µm are negative predictors of
embryo quality, IR, and PR (2)
- 6.6% of 1000 ICSI cycles had
abnormal oocyte morphology (3)
(1) Ebner et al., 2003 (2) Ebner et al., 2005 (3) Mansour et al.,1998

14. Scoring for fertilization
• At 16-20 hours post insemination two centrally aligned PN, with 2
PB located near the PN axis is a good sign. Any deviation could lead
to reduced morphology. (1,2)
• However, PN morphology is an unstable factor within the
dynamic process of fertilization. And recently it was suggested
to stop scoring PN all together. (3)
(1) Edwards and Beard , 1997 (2) Gianaroli et al., 2003 (3) Allan Handyside, 2016

15. 3 PN zygots should be spotted and cultured separately
● Human triploids originate mostly from dispermic fertilization and
less frequently from digynic oocytes.
● It often develops to partial hydatiform mole. Occasionally they can
develop and reach term with early neonatal death.

16. Evaluation of the cleaving embryos
- number of balstomeres, equal
in size and symmetrical with a
visible nucleus and no
fragmentation.
- Multinucleation, unequal
blastomeres, as well as large
amount of fragmentation are
potential source of aneuploidy.
(Johanson et al., 2003, Ziebe et al., 1997, van Royen et al., 1999, Hardarson et al., 2001

17. Embryos should follow a specific time line for development
Thomas Ebner
D5
D4
D3
D2
D1
D0
0 h
23-29 h
66-70 h
114-120 h
18-24 h
42-44 h
90-100 h

18. Extended culture
Advances in our knowledge of in-vitro culture conditions
and the development of better culture media facilitated
extended culture.
(Gardner et al., 1998)

19. Cohesive trophectoderm
with numerous sickle-
shaped cells
Extended culture facilitated Embryo Selection
Tightly packed slightly
oval inner cell mass >
4500 µm.
(Richter et al., 2001, Kovacic et al., 2004, Gardner et al., 2000
Viable Blastocyst

20. Cochrane database, March 2016 (27 RCT)
Clinical pregnancy rate
39-46%
OR 1.3
Moderate quality evidence
36%
Live birth rate
32 – 42%
OR 1.48
Low quality evidence
29%
Cumulative pregnancy rate
NO Difference
OR 0.89
Very low quality evidence
Multiple pregnancy rate
Miscarriage rate
NO Difference
OR 1.05, 1.15
low quality evidence
Freezing rate
37 -46% 60%
OR 0.48
Low quality evidence
Cancelled ET
2 -4%
OR 2.5
Moderate quality evidence
1%

21. Time lapse imaging
Multicenter study on > 1500 embryos of known outcome: →
50.6% of embryos that resulted in pregnancy were
categorized as non suitable, using the time laps.
(kirkegaard et al 2014)
Precise evaluation of morphology and finding a
dynamic marker of embryo quality

22. Time lapse imaging
Cochrane database, 2015
There is no evidence of a difference in the live
birth, miscarriage, still birth, or clinical pregnancy
rate per couple randomly assigned to either time
lapse system or conventional incubator.

23. We need an intelligent computer software to analyze precise
pattern and timing of embryo division and solve the algorithm
of embryo selection.
The algorithm of embryo selection
EEVA

24. II. Contents
are these beautiful embryos euploid?
Rabinowitz et al., 2012
More than half of human embryos generated during IVF
contain aneuploid cells.
274 embryos, only 27.7% were euploid.
We do not know

25. Yes !
(Hasold et al 1996, Vialard et al, 2001) Rabinowitz et al., 2012
(Gabriel et al 2011) Santiago Monne 2016
PGDIS, Bolognia
Are we actually causing aneuploidy as we do IVF/ICSI?
Allan Handyside 2016
- Analysis of large data from oocyte donor cycles that underwent PGS
in different centers showed different aneuploidy rates and even
between doctors from the same center.
- Aneuploidy is the most common type of chromosome abnormality,
and it is the leading cause of implantation failure, miscarriage, and
congenital anomalies in humans.

26. segregation of a whole chromosome to the same pole as its homologue
(non-disjunction), or chromatid precocious separation errors.
Aneuploidy
The presence of extra or missing chromosomes

27. Preimplantation Genetic Testing (PGT)
Obtaining a biopsy from the embryo for genetic testing before its
transfer into the uterus. It is indicated for :
1- patients at high risk of transmitting a genetic abnormality to
their children:
a) all monogenic defects.
b) carriers of balanced translocations, or
numerical chromosomal anomalies.
2- aneuploidy screening

28. Clinical
PR/per ET
# ET procedures
Cycles
(OR)
Procedures
27%
14,482 (72%)
20,207
Aneuploidy Screening
29%
4,758 (78%)
6,096
Single gene defects
27%
3,219 (64%)
5,027
Chromosomal anomalies
25%
955 (76%)
1,263
Sexing for X linked diseases
29%
494 (73%)
676
Social sexing
Indications of genetic testing
(ESHRE PGD Consortium) Moultou et al 2014

29. Meta analysis of aneuploidy screening
Lee et al., 2015 (Hum Reprod)
 19 articles: 3 RCTs, and 16 observational.
 Overall, there are potential benefits of aneuploidy screening
resulting in a higher IR.
 The three RCTs done for good prognosis patients
demonstrated clinical benefit in terms of improved clinical
PR and the use of single ET.

30.  A new and powerful technique to help us select the best
embryos is karyomapping.
 It works well for translocation diagnosis in addition to
aneuploidy screening.
Karyomapping
Allan Handyside, 2016
(PGDIS meeting)

31. Karyomapping is like a finger print.
It detects single
gene defect, and
chromosomal
aneuploidy

32. Rechitsky & Kuliev, 2015

33. Mitochondrial Content
Another important indicator for implantation potential of an
embryo is the mitochondrial content.
In 2014 a microarray platform was developed to allow
simultaneous assessment of aneuploidy and quantification of
mitochondrial DNA content in human embryos.
Konstantinidis et al., 2014

34. Diez-Juan et al., 2015
E Fragouli et al., 2015
High mt DNA copy number in embryos is indicative of a lower
embryo quality and is associated with lower implantation rates.
Less is Better

35. The Future: ̋ Single Euploid Embryo Transfer ̏
̋ Holy Grail in IVF ̏
The technology is going there →
PGD and PGS plus mitochondrial DNA at the same time →
69-72% implantation rates
Allan Trounson 2016
Allan Handyside, 2016
Rechitsky & Kulieve, 2015

36. What about ?
- If a patient has no blastocyst (2-4%)
or
- If a patient has no euploid embryos (10%)
Chchrane data base, 2016
Dagan Wells, 2016
Santiago Mumme,2016
PGDIS meeting

37.  Give all the embryos a chance.
 Transfer every single embryo.
Allan Handyside, 2016
(PGDIS meeting)
Wise Advice

38. Give every single embryo a chance
 A mosaic embryo has a potential of making a
healthy baby (about 50% of the potential of a
normal embryo). It resulted in a 16% live birth rate.
Dagan Wells, 2016
Santiago Munne,2016
PGDIS meeting

39. Analysis of the embryo surroundings
I- spent embryo culture medium
1- The protein translated from specific gene expression
products (proteomics)
2- Metabolic biomarkers
3- RNA-based negative regulators of gene expression
(micro RNA).
II- Cumulus cells.

40. Micro RNA
Three micro RNA (mi R-3729, mi R-191, mi R-645) were
differentially expressed on day 5 of embryos that had successful
pregnancy.
However the analysis is challenging and more research is ongoing .
-Bagga et al., 2005
-Rodgaard et al, 2015
-Calliano et al, 2015

41. Biochemical Markers for embryo viability
I- Glycolytic activity
- Both pyruvate and glucose uptake
significantly increase in cleaving embryos
that developed to blastocyst stage.
- Glucose (but not pyruvate) uptake
significantly increase in blastocysts
of highest grade.
- Gardner et al., 2001

42. Biochemical Markers for embryo viability
II- Amino acid turnover
- Leucin
The only amino acid depleted in the cleaving embryos
that progressed to blastocyst.
- Alanine
Has a striking net appearance due to involvement in
disposal of embryo toxic ammonium ions.
-Houghton et al., 2002

43. Biochemical criteria of metabolic activity
Unfortunately the required techniques for detection of
biochemical markers such as ultramicroflurescence, and high-
performance liquid chromatography are not usually available in
standard IVF labs, thus limiting their application in routine work.
Thomas Ebner, 2010

44. Conclusions
1- Morphology and rate of cleavage are still the foremost
methods of embryo selection.
2- The developmental fate of an embryo is largely dictated by
the quality of the oocyte.
3- PN morphology is an unstable factor within the dynamic
process of fertilization.

45. Conclusions
4- The time of the first mitotic cleavage is a reliable indicator of
embryo viability.
5- Multinucleation and asymmetrical cleavage are documented
to reduce viability.
6- Extended culture facilitated selection from embryos that
reached blastocyst stage.

46. Conclusions
7- The implantation and pregnancy rates with blastocyst transfer
are significantly higher than cleavage stage transfer.
8- An intelligent software algorithm to analyze images from time
laps systems is needed.
9- Detection of biochemical markers, proteomics, and micro
RNA are not currently applicable for routine work in IVF
Labs.

47. Conclusions
10 - The future is for genetic testing.
aneuploidy screening + single gene defect
+ mt DNA content at the same time.
(69-72% implantation rates)

48. Conclusions
11 -1n poor prognosis patients we have to give every single
embryo a chance.
12- Combining morphological evaluation, cytogenetical
analysis, and embryo metabolism will increase the ability
to select the most viable embryo out of a pool.

49. Everyone is a miracle in miniature with a personal
history of men and women who lived, loved, and gave
birth to others ever since the creation of mankind.
Harvey Graham 1950
No body is genetically perfect