Improving Success in ART: How to define it and key strategies

to get the best outcomes

Kiev, Ukraine, 21 September 2018

How to define subgroups for COS

Filippo Maria Ubaldi M.D., Ph.D.

GENERA, Centers for Reproductive Medicine, Italy

Objectives

❑ Show the importance of the number of the oocytes to maximize the CLBR

❑ Show the impact of the female age on embryo chromosomal constitution

❑ Discuss ovarian reserve biomarkers and their role in IVF

❑ Show individualized ovarian stimulation strategies to maximize the CLBR

What is the aim of IVF?

Live birth of an

healthy baby

Cumulative live birth rate per started cycle

What is the measure of

success in IVF ?

Have the baby as fast aspossible in the most effective

and efficient way

Effective & efficient IVF approach

❑ Maximize the number of oocytes and consider fresh+frozen ET

(cumulative live birth rates)

Choose the best treatment strategy (iCOS) with the most effective drug

❑ Optimize the lab technologies

- good cryopreservation programme

- blastocyst colture

- select the blastocyst with the highest implantation potential

❑ Van der Gaast et al-2006 - 13 oocytes; below and above PRs are compromised (n=7,422)

❑ Verberg et al-2009 - 5 for mild stimulation and 10 oocytes for conventional stimulation (meta-analysis ;

mild-313 cycles; conventional-279 cycles)

❑ Bosch et al-2011 - LBR increase up to 15 oocytes maximize the chances of pregnancy (n=7954)

❑ Sunkara et al-2011 - LBR increase up to 15 oocytes; plateaus between 15-20 and decline steadily beyond

20 (n=400,135)

❑ Ji et al-2013 - Optimum - 15 oocytes for LBR below and above PRs are compromised; however,

cumulative LBR increase with increasing oocyte number (n=2,455)

❑ Fatemi et al-2013 - A high ovarian response 18 oocytes does not jeopardize LBR in fresh ET’s and even is

associated with increased cumulative PR (Engage; n=1,506)

Live Birth Rate per fresh embryo transfers

The number of the oocyte is a key factorto maximize cumulative pregnancy rates

The more oocytes retrieved, the higher is the CLBR

The more oocytes retrieved, the higher is the CLBR

Increasing the number of the oocytes it increases live

birth rate with frozen cycles

Increasing the number of the oocytes it increases the

risk of OHSS

Retrospective population-based

registry study including 39387 women

undergoing 77956 fresh IVF cycles

(2007–2013) and 36270 FRET

The increased number of oocyte retrieveddosn’t get worse oocyte competence

Me

ann

um

be

ro

fM

II o

ocy

tes

Mean number of euploid blastocyst

Pearson’s correlationR=0,426P<0,01

Colamaria, Ubaldi oral presentation, ESHRE 2015

27,6%

35,2%

34,8%

Me

ann

um

be

ro

fM

II o

ocy

tes

Mean number of blastocyst

Pearson’s correlationR=0,636P<0,01

915 PGT-A cycles (2610 blastocysts, 24 chr analysis, one Centre), mean female age 39,2 years

The increased number of oocyte retrieveddosn’t get worse oocyte competence

Euploidy rate is independent from the numberof obtained blastocyst but not from the age

Number of blastocysts

% normal embryos

egg donors <35 y 35-37 y 38-40 y 41-42 y >42 years

1-3 58% 61% 51% 39% 22% 13%

4-6 62% 60% 52% 38% 23% 17%

7-10 65% 62% 51% 36% 21% 14%

>10 68% 63% 55% 37% 25% n/a

N. = 4,747 cycles and 29,803 embryos. (Modified from Munne)Ata, Munne et al. (2012) Reprod Biomed Online and unpublished data

Oocyte number and miscarriage rate

Sunkara SK et al. Hum Reprod, 2014

No increase in miscarriage rate with the increase of the oocyte number

Stratified by age and oocyte number1-3 - 4-9 - 10-14 - > 15

Overall association

STUDY QUESTION: Is there an association between the

number of oocytes retrieved for IVF, and perinatal and

obstetric outcomes?

SUMMARY ANSWER: No significant associa-tion was

found between the number of oocytes and perinatal

outcomes, while an association was found for placenta

praevia

Increased number of oocytes retrieved doesn’tget worse perinatal and obstetric outcomes

… so we must maximize the oocyte yield …

… but not all patients are the same

What we really need to

know is, how to define the

right individual treatment

for the right patient…

…and the right ovarian

stimulation strategy

Define the strategy according to the age and the ovarian reserve

Age=

Oocyte quality

Ovarian reserve=

Oocyte quantity

Heffner N Engl J Med, 2004 CDC, 2016 10 y.o. 20 y.o. 30 y.o.

Embryo euploidy and maternal age

Conclusion(s): Our results confirm the known

inverse relationship between advanced maternal

age (>35 years) and embryo euploidy

Result(s): We analyzed 37711 embryos: 22599 day-3

and 15112 day-5 embryos

Number of oocyte to obtain an euploid blastocyst and female age

05

101520253035

Do

no

rs

<35

36

-37

38

-39

40

41

42

43

44

Mean # of oocytes

Female age

Oo

cyte

s

With the courtesy of Dr Bosch, IVI Valencia, PGT-A cycles 2016 (n=1145)

<35 39-40 42-43

Euploidy rate 60% Euploidy rate 30% Euploidy rate 20%

2 blastocysts≈

4 fertilizedoocytes

≈

5 MII oocytes≈

6 COCs≈

3 blastocysts≈

6 fertilizedoocytes

≈

8 MII oocytes≈

10 COCs≈

5 blastocysts≈

10 fertilizedoocytes

≈

14 MII oocytes≈

18 COCs≈

GENERA, unpublished data

We should choose the right approachfor the right patient to yield the

optimal number of oocyte toobtain (at least) one euploid embryo

We have to predict the ovarian reserve

An ideal biomarker of ovarian reserve should have:

❑ ability to measure ovarian follicle number and oocyte quality

❑ reflect the functional capacity of the ovary

❑ require a minimum number of measurements with few

restrictions on the timing of the sample

❑ must be reliable(Rosen et al., 2012)

Biomarkers to predict the ovarian reserve

To date, no biomarker has been shown to have all these requirements

Biomarkers to predict the ovarian reserve

❑ 57 Trials; 4,786 women

❑ AUC

– Female age = 0.61

– AFC = 0.79

– AMH = 0.81

– AFC & AMH = 0.85

Broer et al., FS 2013

Ovarian Reserve TestsPrediction of Poor Ovarian Response

❑ 55 Trials; 5705 women

❑ AUC

– Female age = 0.61

– AFC = 0.76

– AMH = 0.78

– AFC & AMH = 0.80

Broer et al-HRU 2013

Prediction of Poor Ovarian ResponsePrediction of Excessive Response

AMH and AFC as predictors of the ovarian reserve

AMH and AFC:

preferred methods for predicting the ovarian reserve

with a varied degree of precision

(Hendriks, 2005; Iliodromiti, 2014; La Marca, 2010; Lukaszuk, 2013; Polyzos, 2013)

AMH and AFC can predict low and high ovarian response

Polyzos et al, 2013

Scatter plots and correlations for log 10 primary follicle counts vs ovarian reserve test results (Hansen et al 2011)

…Although AMH level is an objective test, and therefore should not have the inter-operator variability issues

associated with subjective determination of AFC (Arce et al., 2013), values from manual assays have been shown

to vary between assays as well as laboratories, owing to differences in site-specific processes (Iliodromiti, 2014).

Improper storage and handling of samples, delayed centrifugation, storage at room temperature, can drastically

affect AMH levels (Nelson, 2013) In addition, interference by the serum complement that binds to the assay

antibodies in fresh samples can lead to variability in AMH assays (Craciunas, 2014; Rustamov, 2012, 2014).

AMH Values May Be Falsely Lowered By Storage

Conditions, Assay

Processing and Length of Time Stored

Julie S. Rhee, MD1, Emily A. Seidler, MD1, Elizabeth Eklund, BS2,Amber R. Cooper, MD MSCI1

Limits and pitfalls of ovarian reserve biomarkers:AMH assessment in the real world

AMH assessment: reliability in the real world

Nap

les

Vic

en

za

The same serum sample, assayed in 3 different labs of 3 different cities

FS 26 10 27 5.9 9.8 6.5

SM 36 5 6 0.5 0.9 3.9

EA 38 20 5 0.9 3.4 1.6

AS 31 6 11 1.6 2.5 3.4

Ro

me

Italy Name AgeDay of

cycle AFCAMH ng/ml

Lab 1 Lab 2 Lab 3

LP 29 3 15 3.4 1.7 2.5

MP 31 9 22 2.4 11.1 4.4

AFC: pros and cons

Number of follicles

a little normal a lot

< about 10 about 10 – about 15 > about 15

… but let’s take also into consideration age and if possible AMH

r-FSH 300 IUI + r-LH 150 IU r-FSH 225 IUI r-FSH 150 IUI

Clinical common sense of AFC: let’s use AFC with a pragmatic approach to chose the correct starting dose of gonadotropins

The ovarian response could be influencedby specific genetic characteristics of gonadotrophins and their receptors

EXON 10

LHCGR 291Asn/Ser (rs12470652)could affect number of

oocytes/FSH consumption

LHCGR 312 Ser/Asn (rs2293275)N variant may render the LHCGR

more sensitive to the ligand

Two single nucleotide polymorphisms of LHCGRaffect sensitivity to exogenous gonadotrophins

Alviggi C, et al.. Hm Repod. 2016 (suppl)

Lindgren I, et al.. Hum Reprod 2016

FSHR Asn680Ser (rs6166) Less oocyte and increased

FSH consumption

FSHR −29G/A (rs1394205)Poor ovarian response

FSHR Thr307Ala (rs6165) Less oocyte and increased

FSH consumption

NCBI-SNP database, more than 730 SNPs in the FSHR

gene. The most studied SNPs:

Greb, et al. JCEM, 2005

Gromoll & Simoni TEM 2005Alviggi et al., 2016

Swapna S et al., JCEM 2011

FSHR genotype (position 680)

Number of FSH amps needed

for COS and OPU in women

according to the FSH-R genotype

RCT: total FSH dose required (left) and serum E2 concentration (right) in normo-ovulatory women undergoing COS, grouped according to N680S

genotype for the FSH receptor gene (Behre et al.,2005)

The Ser/Ser genotype of the

FSHR gene at position 680

cause a differential E2

response to FSH

Forest plots of differences among FSHR (rs6166) genotype carriers in relation to the number of oocytes retrieved

(A) (rs6166) A homozygotes versus G homozygotes (C) (rs6166) heterozygotes versus G homozygotes

Clinical effects of SNPs significantly related to COS outcome

Alviggi et al., Hum Reprod Update 2018

In case of prediction of hyper-response

normal

Main Objective: safety of the patient

▪ GnRH-antagonist protocol

▪ Low starting dose r-FSH

▪ GnRH-agonist trigger

La Marca and Sunkara. Human Reprod Update, 2014

Modified from La Marca and Sunkara, Hum Reprod Update 2014

In case of prediction of normo-response

normal

Main Objective: maximize success

▪ GnRH agonist or antagonist ?

▪ Daily r-FSH starting dose ? (200-225 IU)

In case of prediction of hypo-response

normal

Modified from La Marca and Sunkara, Hum Reprod Update 2014

▪ GnRH antagonist + max r-FSH

▪ DuoStim

▪ Egg donation

Main Objective: try to maximizethe # of oocyte/menstrual cycle

Can we overcome POR by increasing the FSH dose?YES in group 1-2 POSEIDON: hypo-responders

WHY? ~30%

Reasons for Poor Response:

• FSH dose does not reach threshold

• Genetic polymorphism of FSH-R; LH-R

Increase the dose of FSH

(Perez-Mayorga, 2000; De Castro,

2003; Jun, 2005; Behre 2005)

Add r-LH to FSH

(Ferraretti, 2004; De Placido,

2005; Alviggi, 2013)

▪ Women, with normal ovarian

reserve who show sub-optimal or

unexpected poor response to

exogenous FSH

▪ These women require an

increased cumulative FSH dose

(>2500-3000 IU) & more days of

stimulation (FSH hypo-sensitivity)

GROUP 1 -2

Patients <35 (Group 1) or >35 years (Goup

2) with adequate ovarian reserve

parameters (AFC ≥5; AMH ≥1.2 ng/ml)

and with an unexpected poor or

suboptimal ovarian response

Can we overcome POR by increasing the FSH dose?NO in group 3-4 POSEIDON: poor ovarian reserve

GROUP 3 - 4

Patients <35 y (Group 3) or >35

years (Group 4) with poor ovarian

reserve pre-stimulation para-meters

(AFC <5; AMH <1.2 ng/ml)

▪ Normo or hyper-gonado-tropicwomen, with reduced ovarianreserve and <3-4 oocytesretrieved

▪ In these women higher doses ofgonadotropins will notcompensate the absen-ce offollicles…

… and then how to increase the number of

oocytes to maximize live birth rates ?

Baerwald et al Hum Reprod Update 2013 Adams GP et al., J Reprod Fertil, 1992

n. of follicles >5 mm diameter of the largest follicle Baerwald et al, Fertil Steril 2003

2 148 20 26Dayof cycle

Follicle waves in the ovarian cycle

51 patients with poor ovarian

reserve (AMH <1,5 ng/ml, AFC < 6

follicles and/or < 5 oocyte retrieved

in previous COH) undergoing ICSI

treatment and PGT-A

51 patients started the first stimulationcycle

6 excluded:-no response to the

stimulation

45 patients to egg retrieval

43 patients to egg retrieval

18 FP stimulationcycles with euploid

blastocystobtained

42 patientsperforming FP ICSI

42 patientsperforming LP ICSI

2 excluded:-2 no sperm available

23 LP stimulationcycles with euploid

blastocystobtained

Primary outcome measure:

euploid blastocyst rate.

Secondary outcome measures:

#of retrieved COCS and MII oocytes

300 IU + 150 IU r-FSH + rLH

GnRH-atrigger

GnRH antagonist

OPU

(GnRH antagonist)

GnRH-atrigger OPU

STOP

300 IU + 150 IU r-FSH + rLH

DuoStim increases the oocyte yields per ovarian cycle

MII oocytesthat did notreach blasto-cyst stage

MII oocytesthat madeaneuploidblastocyst

MII oocytesthat madeeuploidblastocyst

Huge intraovarian follicle waves variabilitySimilar laboratory results between FPS and LPS

Similar oocyte competence after FPS and LPS

Study question: Are the mean numbers of blastocysts obtained from sibling cohorts of oocytes

recruited after follicular-phase (FPS) and luteal-phase-stimulations (LPS) in the same ovarian

cycle (DuoStim approach) similar?

Study Design, Size and Duration: Case-control study conducted in paired follicular phase- and

luteal phase-derived cohorts of oocytes collected after DuoStim at 2 private IVF clinics

between October 2015 and December 2017

Summary answer: The follicles recruited during the anovulatory phase of the ovarian cycle

may be rescued through LPS and originate larger cohorts of oocytes with comparable

competence than paired-FPS-derived ones.

Similar oocyte competence after FPS and LPS

The most remarkable achievement with the DuoStim approach…

96,5% 91,3%

73,9%

42,3%

99,4% 97,4%90,3%

65,5%

0,0%

20,0%

40,0%

60,0%

80,0%

100,0%

120,0%

cycles with ≥1 MII oocyte

cycles with ≥1 fertilized oocyte

cycles with ≥1 blastocyst

cycles with ≥1 euploid blastocyst

Comparison between FPS alone vs DuoStim FPS DuoStimP<0.01

P<0.01

P<0.01

GENERA unpublished data, 2018

Take home messages

❑ AMH and AFC are methods for predicting the ovarian reserve

with a varied degree of precision.

❑ Ovarian reserve markers are useful to predict ovarian response

❑ FSHR and LHR polymorphisms can affect COS outcome

❑ Individualized COS according to the ovarian reserve of the patient

can maximize Cumulative Live Birth Rate

❑ The female age is correlated with the oocyte and blastocyst

euploidy rate and with the ovarian reserve

Thank you for your attention

Grazie per la vostra attenzione

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