Talk delivered at the Nordic IVF Laboratory Meeting, Oslo, Norway, September 8th, 2018

1. Sandro C. Esteves
ANDROFERT & University of Campinas (UNICAMP),
Campinas, BRAZIL
Aarhus University, DENMARK
Oocyte number and
success in ART –
The “ART Calculator”

2. Esteves, 2
Sandro Esteves, MD., PhD.
• Medical and Scientific Director, ANDROFERT, Campinas, BRAZIL
• Collaborating Professor, Department of Surgery, University of Campinas
(UNICAMP), BRAZIL
• Research Collaborator, American Center for Reproductive Medicine, Cleveland
Clinic, USA
• Research Collaborator, Genetic Unit, Universidad Autónoma de Madrid, SPAIN
• Honorary Professor of Reproductive Endocrinology, Faculty of Health, Aarhus
University, DENMARK
Declare receipt of honoraria for lectures from Merck, Besins, Gedeon
Richter, and Lilly
Affiliation & Disclosure

3. 1. Importance of oocyte number and female age in
ART success
2. Estimation of individualized oocyte number for
modelling patient-oriented strategies as a
means to increase ART efficiency
Lecture Outline
Esteves, 3

4. Modified from Smith et al. JAMA 2015
156,947 UK women
(257,398 IVF cycles;
cumulative LBR using
fresh and frozen ET)
With increased women’s age in ART programs,
we should maximize reproductive outcomes and shorten TTLB
Esteves, 4

5. Quality Dimensions
1. Effectiveness (Technical aspects to deliver the best outcome)
2. Safety (risks, adverse effects, lab quality, etc.)
3. Patient centeredness (patient values and preferences)
Success in ART
The bottom line
• Evaluate properly
• Give a fair estimate of outcome
• Develop a time-limited treatment plan
Dancet et al. Hum Reprod 2011; Mainz. Int J Qual Health Care 2003
Esteves, 5

6. AMH and AFC predict ovarian response but not LBR
AMH~AFC>FSH>Age
Predictors of Poor
Response
Predictors of Excessive
Response
Accuracy for Non-Pregnancy
Prediction ~ 50-55%
Modified from Broer et al. Fertil Steril 2009 Modified from Broer et al. Fertil Steril 2009Modified from Broer et al. Hum Reprod Update 2011
Accuracy ~0.82 Accuracy ~0.78
Esteves, 6

7. Female age and number of oocytes can predict LBR
Modified from McLernon et al. BMJ 2016
Modified from Sunkara et al. Hum Reprod 2011
Modified from De Geyter et al. Swiss Med Wkly 2015
Esteves, 7

8. Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number
POSEIDON GROUP
Poseidon Group. Fertil Steril 2016
Esteves, 8

9. Low prognosis groups
Young Older
Adequate
ovarian
reserve
Poor
ovarian
reserve
Courtesy of Chloe Xilinas, EXCEMED
Poseidon Group. Fertil Steril 2016; Humaidan et al. F1000Research 2016
2
2
Esteves, 9

10. Prevalence of low prognosis patients according
to POSEIDON criteria
5% 30%
10% 55%
47%
Poseidon
1-4
53%
Non Poseidon
N=432 – YEAR 2017
Source: ANDROFERT
Esteves, 10

11. Modified from Drakopoulos et al. Hum Reprod 2016
Low Prognosis Owing to Decreased Number of Oocytes
and thus lower CLBR
Poseidon Patients
Age-adjusted CLBR strongly influenced by oocyte number
* Age adjusted (OR: 0.9; 95% Cl: 0.9-1.01)
21.7%
39.7%
50.5%
61.5%p=0.002
p=0.02
p=0.01
Esteves, 11

12. Group 1
Young & Suboptimal/Low
Oocyte Number
Group 2
Old & Suboptimal/Low
Oocyte Number
Group 3
Young & Expected Low
Oocyte Number
Group 4
Old & Expected Low
Oocyte Number
High
embryo
aneuploidy
risk
Low
embryo
aneuploidy
risk
Few embryos generated
Reduced Cumulative Live Birth Rate
Poseidon
Patients
Esteves, 12

13. Esteves et al., ESHRE 2018; P-394
The older the women the lower the probability of euploid
embryos, thus increasing the risk of NO transferable embryos
Euploidy probability vs Female age
Cycles with no transferable embryos
Courtesy of Chloe Xilinas, EXCEMED
Courtesy of Ubaldi & Rienzi. Genera
Jan 2012 – Dec 2013
Esteves, 13

14. Estimation of Age-dependent Decrease in Blastocyst
Euploidy by Next Generation Sequencing - Development of
a Novel Prediction Model
Quadratic model on age significant (Prob>ChiSquare=0.028);
derived model was log[p(t)/(1−p(t))]=7.575-0.21⋅age-0.011⋅(age-38.41)2,
where p(t) is the probability of a blastocyst being euploid for age (t) in years.
Term Estimate Std Error Wald
ChiSquare
Prob >
ChiSquare
Lower
95%
Upper
95%
Intercept
7.5746663 1.028021 54.290324 <.0001* 5.5597808 9.5895518
age
-0.212288 0.026273 65.284544 <.0001* -0.263783 -0.160793
(age-
38.4082)*(age-
38.4082)
-0.01059 0.004834 4.7979866 0.0285* -0.020066 -0.001114
Esteves, Carvalho, Martinhago, Melo, Bento, Humaidan & Alviggi. Panminerva Medica 2018 ahead of print
Esteves, 14

15. Euploidy probability vs Female age
Measure
Training Validation
Number of rows
1,036 260
Sum of
Frequencies 1,036 260
-LogLikelihood
588.50165 139.70103
Number of
Parameters 3 3
BIC
1197.6537 295.89356
AICc
1183.028 285.50206
Generalized
RSquare 0.1162863 0.1893375
Validation by randomly splitting 80% of the data
for training and 20% for testing
Esteves, Carvalho, Martinhago, Melo, Bento, Humaidan & Alviggi. Panminerva Medica 2018 ahead of print
Esteves, 15

16. Esteves et al., ESHRE 2018; O-007
Progressive decline* in blastocyst euploidy
probability as a function of female age
Analysis of 1,220 trophectoderm biopsies from 436 patients undergoing
ICSI and PGT-A with NGS
1.2 1.9 2.6 3.4 4.4
5.5
6.7
8.2
9.8
11.6
13.6
15.7
17.9
20.2
22.4
24.5
26.6
Geometric mean of the yearly variation: 13.6%
*The % decrease in blastocyst euploidy
probability from year (t) to year (t+1) was
defined as the ratio p(t+1)/p(t) x 100
Esteves, 16

17. Number of oocytes needed to maximize live births should be
individualized
Female age is a critical factor to
be taken into account
Patient-oriented strategies can
be used to achieve the
individualized oocyte number
Esteves, 17

18. Introduced an Intermediate Marker of Success in ART:
the Ability to Retrieve the Number of Oocytes Needed to Obtain
at Least One Euploid Blastocyst for Transfer in Each Patient
Poseidon Group. Fertil Steril 2016; Humaidan et al. F1000Research 2016
What is new in POSEIDON?
Introduced the Concept of ‘Low Prognosis’ in ART
Combined Oocyte Quality and Quantity for Identification
and Stratification of the ’Low Prognosis’ Patient
Included ’Hypo-responders’ as a Distinct Category
of ’Low Prognosis’ Patients
Introduced an Intermediate Marker of Success in ART:
the Ability to Retrieve the Number of Oocytes Needed to Obtain
at Least One Euploid Blastocyst for Transfer in Each Patient
Esteves, 18

19. Courtesy of Ubaldi & Rienzi (GENERA; Jan 2012-Dez 2013)
Source: ANDROFERT; Jan 2015-Sept 2016 (PGT-A by NGS)
Live birth (%)
Availability of euploid embryos maximizes IVF efficiency by
offsetting the negative effect of age on implantation and
pregnancy
Esteves, 19

20. http://www.members.groupposeidon.com/Calculator
Estimation can be done by means of a mathematical function
taking into account relevant predictive factors
Input variables
• Maturation rate
• 2PN Fertilization rate
• Cleavage or Blastulation rate
• Euploidy rate per age group
Adjusted according to maternal
and paternal age, sperm source,
sperm and oocyte status,
and type of embryo transfer
(fresh; FET)
Output variable
Y (N oocytes)
Function
Y = f(X)
Esteves, 20

21. There are two ways to do that…
the HARD WAY the EASY WAYand
Esteves, 21

22. The easy way
http://www.members.groupposeidon.com/Calculator
Esteves, 22

23. age p N (P=0.8) N (P=0.9)
28 0.6748 2 3
29 0.6665 2 3
30 0.6538 2 3
31 0.6365 2 3
32 0.6144 2 3
33 0.5873 2 3
34 0.5549 2 3
35 0.5174 3 4
36 0.4750 3 4
37 0.4283 3 5
38 0.3784 4 5
39 0.3268 5 6
40 0.2753 5 8
41 0.2259 7 9
42 0.1803 9 12
43 0.1399 11 16
44 0.1056 15 21
45 0.0775 20 29
Minimum number (N) of blastocysts required to achieve at least one euploid blastocyst for
transfer, with probabilities (P) of 0.8 and 0.9, where “p” is the model-estimated probability
of a blastocyst being euploid for female age in years
Esteves, Carvalho, Martinhago, Melo, Bento, Humaidan & Alviggi. Panminerva Medica 2018 ahead of print
Esteves, 23

24. Model
Estimates (No.
embryos by
female age)
Esteves, Carvalho, Martinhago, Melo, Bento, Humaidan & Alviggi. Panminerva Medica 2018 ahead of print
Esteves, 24

25. http://www.members.groupposeidon.com/Calculator
Esteves, 25

26. The most optimal treatment strategy is planned with the
mindset to achieve the POSEIDON marker of success
GnRH analogue regimen
Gonadotropin dose and drug type
Trigger strategy
Adjuvant therapy
Combined strategies (AccuVit; Duostim, etc.)
Personalized use of laboratory technology
Personalized luteal phase support
Esteves, 26

27. Agonist or Antagonist +
rFSH alone or rFSH + LH
Agonist or Antagonist + rFSH + LH
GnRH Antagonist + Low dose rFSH (100-175 IU) GnRH Antagonist + rFSH + LH
Poseidon
G1 Poseidon
G2 Poseidon
G3 Poseidon
G4
Non
Poseidon
patients
Andersen et al. (eds.) Research Topic Frontiers in Endocrinology. In preparation
It is important to use the right FSH starting dose and adapt it during
the cycles to optimize the number of oocytes retrieved and at the
same time minimize risks such as OHSS and cycle cancellation
Esteves, 27

28. Esteves, Yarali, Ubaildi et al. Validation of ART Calculator Consortium;
Unpublished data
No detrimental effects on embryo ploidy in patients
who retrieve more oocytes
Response Number Euploid Blastocysts
Distribution Binomial
Estimation Method Logistic Regression
Validation Method Validation Column
Probability Model Link Logit
Generalized Regression for number
Euploid Blastocysts Model Launch
Lasso [x] Adaptive
Term Estimate Std Error
Wald
ChiSquare
Prob >
ChiSquare
Lower
95%
Upper
95%
Intercept 6.2769104 0.6256681 100.64771 <0.0001* 5.0506235 7.5031972
Age Female -0.182507 0.152275 143.64894 <0.0001* -0.212352 -0.152662
Number
Blastocysts
0.0302516 0.0189943 2.536584 0.1112 -0.006977 0.0674797
Parameter Estimates for Original Predictors
3,108 Trophectoderm Biopsies
1,109 patients
Euploidy probability vs. number
of blastocysts by age group
Esteves, 28

29. Higher ovarian response than that achieved with mild/natural
ovarian stimulation does not increase risk of embryo aneuploidy
Term Estimate Std Error
Wald
ChiSquare
Prob >
ChiSquare
Lower
95%
Upper
95%
Intercept 3.8117137 1.5331832 6.1809102 0.0129* 0.8067298 6.8166876
Age Female -0.22129 0.019748 125.56789 <0.001* -0.259996 -0.182585
typeOSGrou
ped[Convent
ional-Other]
0.065727 0.33909 0.0375714 0.8463 -0.598877 0.7303311
Response Number Euploid Blastocysts
Distribution Binomial
Estimation Method Adaptive Lasso
Validation Method Validation Column
Probability Model Link Logit
Adaptive Lasso with Validation Column Model Summary
Parameter Estimates for Original Predictors
1,632 Trophectoderm Biopsies by NGS
631 patients
Euploidy probability vs Female Age, by OS Type
Esteves, Yarali, Ubaildi, et al. Validation of ART Calculator Consortium;
Unpublished data
Esteves, 29

30. Administration of an inappropriately low gonadotrophin dose
may lead to the growth of a low number of follicles resulting
in an ‘iatrogenic’ poor or suboptimal response
• Type of Gonadotropin
• FSH Dose
• Add LH
Courtesy of Chloe Xilinas, EXCEMED
Esteves, 30

31. Unpublished data; Source: ANDROFERT
Current Practice – Flexibility of iCOS
Rec-hFSH
Starting Dose Distribution (%)
% cycles with
Dose Adaptation after Sd5
53.4%
Esteves, 31

32. Current Practice – Flexibility of iCOS
% cycles
by Trigger Method
% cycles
with LH Activity Supplementation
12%
Dual trigger
43%
GnRH analog
45%
hCG
57%
Rec-LHc
43%
No-LH
Unpublished data; Source: ANDROFERT
Esteves, 32

33. Target number of oocytes difficult to achieve by a single OS cycle
http://www.members.groupposeidon.com/Calculator
Courtesy of Chloe Xilimas, EXCEMED
Esteves, 33

34. Haahr, Esteves & Humaidan. Reprod Biol Endocrinol 2018
Management of Poseidon poor prognosis patients
<AFC <5 and/or AMH <1.2 ng/ml
Group 3, young (AGE <35) Group 4, old (AGE ³35)
Poor reserve - good quality Poor reserve - poor quality
Reason for poor response:
• Poor ovarian reserve
• Asynchronous development
(Genetic polymorphism of FSH-R; LH-R; V-LH-β)
iCOS Treatment:
• Long GnRHa protocol
• GnRH antagonist (E2, OCP)
• DuoStim ?
• Stimulation with 300 IU/d rFSH
• Androgens ?
Embryo Transfer strategy:
• Fresh transfer
• Oocyte/embryo accumulation and FET
Measure of success:
In average, a total of 4-5 oocytes are needed
to obtain one euploid embryo
Reason for poor response:
• Poor ovarian reserve
• Asynchronous development
• High Aneuploidy rate
(Genetic polymorphism of FSH-R; LH-R; V-LH-β)
iCOS Treatment:
• Long GnRHa protocol
• GnRH antagonist (E2, OCP)
• DuoStim
• Stimulation with 300 IU/d rFSH
• Androgens ?
Embryo Transfer strategy:
• Fresh transfer
• Oocyte/embryo accumulation, PGS?
and FET (Oocyte donation)
Measure of success:
In average, a total of 12 oocytes are needed
to obtain one euploid embryo
Esteves, 34

35. Modified from Ubaldi et al. Fertil Steril 2016
Dual stimulation
Courtesy of Chloe Xilinas, EXCEMED
rFSH or rFSH+LH rFSH or rFSH+LH
Esteves, 35

36. Male age!
Does it matter?

37. Ramajit Raghav,
94 yo.
Indian farmer claims to be the oldest dad in the world, and says he wants more…

38. Female age is key

39. Review Criteria
An extensive search of studies examining the influence of paternal age on
ART outcomes was performed using PubMed and MEDLINE. The start date
for the search was January 2008, and the end date was March 2018.
The overall strategy for study identification and data extraction was based on
the following keywords: “paternal age”, “male age factor”, “masculine age
factor”, “male ageing”, “paternal ageing”, “reproductive techniques”,
“assisted”, “ICSI”, “in vitro fertilization” “sperm injections”, “intracytoplasmic”,
“IVF”, with the filters “humans” and “English language.” Data that were solely
published in conference or meeting proceedings, websites or books were not
included. Citations dated outside the search dates were only included if
provided conceptual content.
ESTEVES, 39CLÍNICA DE ANDROLOGIA E LABORATÓRIO DE REPRODUÇÃO HUMANA
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40. Study Country Number Main findings Age cutoff
Frattarelli
2008
USA 1023
Decline in semen volume and total motile sperm
number
45
Bellver 2008 Spain 2698
Negative association between age and semen volume,
sperm concentration, and total progressive motility
NR
Girsh, 2008 Israel 484 Male age higher in non-pregnant group; Conventional
semen parameters lower in men aged >50
50
Ferreira, 2010 Brazil 1024 No effect of age on sperm concentration and motility NR
Duran 2010 USA 408
Semen volume, sperm motility and fertilization rates
inversely correlated with age
NR
Nijs, 2011 Belgium 278
No correlation between male age and sperm
concentration, progressive motility, and DFI
NR
Varshini, 2011 India 504 DFI increased in men older than 40 years 40
Stone, 2013 USA 5081
Total sperm numbers fall by ~2% per year, morphology
by ~0.8% per year, and total sperm motility by ~2%
35
Alshahrani,
2014
USA 472
No significant differences in conventional semen
parameters. Higher DFI in men >40y
40
Beguería, 2014 Spain 4887
Semen volume and %motile sperm decreased with a
mean reduction of 0.22 ml and 1.2% every 5 years
NR
Kaarouch,
2018
Morocco 204
No effect on conventional semen parameters; Higher
DFI and sperm aneuploidy in older men
40
Paternal age X semen parameters
Bertoncelli Tanaka et al. Panminerva Med. 2018 Jul 18. [Epub ahead of print]

41. Author Country
Number
(cycles)
Method
Egg donation
model
Fertilization Embryo
development
Pregnancy
Miscarriage/
stillbirth
Live birth Age cutoff
Gu, 2012 China 103 IVF Yes No effect NR No effect No effect No effect NA
Robertshaw, 2014 USA 237 IVF Yes NR NR NR
Increase in pregnancy loss
with aging
26% lower odds of LB with each 5-
year increase in age
NR
Wu, 2015 China 9991 IVF No No effect No effect
Lower in men >36y than
<32 y (women 30-34y)
No effect No effect 36
Girsh, 2008 Israel 484 ICSI Yes No effect Decreased Decreased NR NR 40-50
Ferreira, 2010 Brazil 1024 ICSI No NR No effect
Decreased by 5% for
each year of age in
oligozoospermia
No effect NR NR
Tsai, 2013 Taiwan 184 ICSI No No effect No effect No effect No effect No effect NA
Ramasamy, 2014 USA 1046 ICSI No NR NR Decreased NR NR 50
Beguería, 2014 Spain 4887 ICSI Yes No effect No effect No effect No effect No effect NA
Frattarelli, 2008 USA 1023 IVF and ICSI No No effect
Reduced blastocyst
formation
No effect Increased miscarriage Decreased 55
Bellver, 2008 Spain 2698 IVF and ICSI Yes No effect No effect No effect No effect NR NA
Campos, 2008 Spain 915 IVF and ICSI Yes No effect No effect Decreased No effect NR 39
Luna, 2009 USA 672 IVF and ICSI Yes Decreased
Reduced blastocyst
formation
No effect No effect NR 40* and 60‡
Duran, 2010 USA 519 IVF and ICSI Yes Decreased No effect No effect No effect No effect 50
Ghuman, 2016 UK 7104 IVF and ICSI No NR NR No effect No effect NR NA
Meijerink, 2016 Netherlands 7051 IVF and ICSI No No effect No effect No effect NR NR NA
McPherson, 2017 Australia 2215 IVF and ICSI No No effect NR See LBR NR
10% lower pregnancy and LBR in
women aged 35 years with
partners >40 years than women
aged 35 years with partners <30
years
40
Tiegs, 2017 USA 573 IVF and ICSI Yes Decreased No effect No effect No effect No effect 45
Chapuis, 2017 France 2491 IVF and ICSI No No effect
Reduced blastocyst
formation
Decreased in ICSI but
not with IVF
No effect — 50
Park, 2017 Korea 452 ICSI No No effect No effect Decreased NR Decreased 46
Kaarouch, 2018 Morocco 83 IVF No Decreased Decreased Decreased Increased NR 40
Bertoncelli Tanaka et al. Panminerva Med. 2018 Jul 18. [Epub ahead of print]
Paternal age X IVF outcomes
ESTEVES, 41

42. ESTEVES, 42
ESTEVES, 42
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43. ESTEVES, 43
Esteves, Yarali, Ubaldi et al; ART Calculator Validation Consortium, Unpublished
data
Paternal age and
blastocyst genetic status
1070
patients
TB + NGS
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44. Age
CLÍNICA DE ANDROLOGIA E LABORATÓRIO DE REPRODUÇÃO HUMANA
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Cho, Esteves, Agarwal. Curr Opin Obstet Gynecol 2016
Finding the link

45. Aitken, Reproduction 2014; 147:S1-S11
Mechanism of oxidative-induced sperm DNA damage
OGG1: 8-oxi guanine glicosylase
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Activity of OGG1 decreases with advanced paternal
age, thus resulting in 8-oxi guanine accumulation

46. Sperm chromatin repair mechanisms
CLÍNICA DE ANDROLOGIA E LABORATÓRIO DE REPRODUÇÃO HUMANA
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Champroux et al. Basic and Clin Androl 2016; 26:17
OGG1 is deficient
in oocytes;
8OHdG carried to
embryos by
fertilizing sperm
8OHdG highly
mutagenic (G-T
transversion)

47. proband schizophrenic Kong et al. Nature 2012; 488:471
proband a parent of an autistic case
♦ proband autistic
An exponential model estimates
paternal mutations doubling every
16.5 years
Paternal age and germline mutations
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48. Guanine
particularly
vulnerable
Oxidative stress causes harm to DNA
No.oxidizedregions
Chromosome size
Imunoprecipitação 8OHdG
chrX
chrY
chr15
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49. Impact of paternal age on
neurodevelopmental status of naturally
conceived offspring
Autism
Bipolar disease/schizophrenia
Marfan syndrome
Paternal age (yrs)
Oddsratio
Bipolar disease
Frans et al. Arch Gen
Psychiatry 2008;
65:1034
Paternal age (yrs)
Oddsratio
Autism
Reichenberg et al. Arch
Gen Psychiatry 2006;
63:1026
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50. POSEIDON–based
Stratification
FSH Starting Dose
Gonadotropin Dose
Adaptation
Flexible OS
(eg. LH, Duostim,
trigger)
Maximize oocyte yield
to achieve estimated
N oocytes for at least
1 euploid embryo
(ART Calculator)
Reduced
Time to Live Birth
Conclusions
Esteves, 50

51. 51
iART
Esteves, 51

52. www.groupposeidon.com
http://www.members.groupposeidon.com/Calculator
Esteves, 52