ВРТ / ИКСИ в позднем репродуктивном возрасте

Advanced Maternal Age
Definition
Chronological : Age above 35yrs
Biological Ovarian reserve markers
FSH
AMH
AFC
Response to stimulation
All parameters should be included in the definition

Introduction
Worldwide tendency to postpone pregnancy until later reproductive life :
Building up a career
Late marriages & increased divorce
Lack of social incentives to support parenthood
Misleading idea that ART can overcome fertility decline with aging
Patients of AMA comprise 10-20 % of IVF population
ICSI cycles after 40 yrs : Europe 25%
Egypt 10%

Oocyte pool decreases from 1-2 millions at birth to
300,000 - 500,000 at puberty to few hundred eggs at menopause
Faddy et al. 1992
300 - 400 oocytes will be ovulated during reproductive years
Decline of fertility starts 10 - 13 yrs before menopause & is an expression of accelerated ovarian ageing
Helen S. et al. 2003 HR

How Does Biology Work ?
Aging is associated with fertility decay due to a decline in both ovarian reserve and oocyte / embryo competence mainly as a result of:
Spindle & meiotic errors
Reduced mitochondrial activity
Increased aneuploidy Pellestor et al. Hum Genet 2003, 2005
Oocyte aneuploidy increases with age
< 35 yrs 10% 40 yrs 30%
43 yrs 40% > 45 yrs 90%
Capalbo et al 2017, Franasiak JM et al. FS 2014
This explains the sharp increase in chromosomal defects after 35yrs
from 2.5 - 3 / 1000 births to 37 BJOG International 2010

Infertility Work-up in AMA
Recommended after 6 months of seeking pregnancy SOGC 2011
Exclude age related factors : fibroids, endometriosis , polyps
Multi –marker approach to evaluate ovarian reserve
Broekmans et al. 2006
TSH, PRL, Tubal patency , HS
Hypertension, DM
History of previous radiation / chemotherapy
Details about previous IVF trials : poor response or
cancellation

Ovarian Reserve Markers
Correlate with egg quantity / not quality
Predict response to stimulation
Poor predictors for pregnancy
Help in determining FSH dose & stimulation protocol
Fauser B et al.2007
FSH : D3 FSH ( > 14 IU /L ) : First sign of ovarian aging , usually occurs between 35 & 40 yrs
AFC : FSH sensitive / correlates with primordial follicles number
AMH : Produced by GCs of antral & pre-antral follicles

Ovarian Reserve Tests
& Age
24 - 33y 34 - 38y 39 y
AMH ng/ml 2.1 1.6 1.1
AFC 11 10 7
FSH IU/L 6.9 7.4 7.9
Imog et al. 2011

Poor Responders
ESHRE Criteria Bologna: at least 2 criteria Ferraretti et al. HR 2011
1-Age > 40yrs presenting with other risk factors for PR
2-Previous POR ≤ 3 oocytes with standard dose stimulation
3- Abnormal ovarian reserve (AFC <5-7 fol. or AMH < 0.5-1.1 ng/ml )
Or : Any age + 1 cycle ≤3 eggs retrieved + abnormal reserve tests
Any age + 2 previous cycles ≤3 eggs using max. stimulation

POSEIDON Groups of Poor Response
Patient- Oriented Strategies Encompassing IndividualizeD Oocyte Number
New measure : Number of oocytes needed to retrieve to obtain at least one euploid embryo
Changed definition of PR to have homogenous population
4 Groups based on:
Age and expected embryo euploidy rate
< 35 yrs 60% , 40 - 42 yrs 30% > 42 yrs 15 %
Ovarian reserve biomarkers ( AFC, AMH )
Previous response & oocyte quality
Thor Haahr et al . 2015

POSEIDON Groups
I < 35 yrs , AFC ≥ 5 , AMH ≥ 1.2 ng /ml , < 9 retrieved eggs
young pt . with unexpected poor/ suboptimal response
II > 35 yrs, AFC ≥ 5 , AMH ≥ 1.2 ng /ml , < 9 retrieved eggs
older pts. with unexpected suboptimal response
III < 35 yrs , AFC < 5, AMH < 1.2 ng /ml , < 5 retrieved eggs
young with poor reserve
IV > 35 yrs ,AFC <5, AMH < 1.2 ng /ml , < 5 retrieved eggs
older pts. with poor reserve
This changes the prognosis of women with the same number as well as different oocyte yields

Treatment Strategies in Practice
Current strategies
Maximizing ovarian response
Natural or mild stimulation IVF protocol
Personalized COS
Freeze - all embryos
Enhancement of embryo selection via BC – stage PGT-A &
frozen single ET
Endometrial scratching
Oocyte cryopreservation / medical & social freezing
Egg donation
Future solutions : Mitochondrial transfer
Chromosome therapy
In vitro generation of gametes
In vitro activation
Ubaldi F J. Frontiers in Gin Endocrinology 2019

Maximizing Ovarian Response
Keep in mind :
Gns can not generate follicles ex-novo
Max. threshold dose of Gns 300 IU FSH /d ± 75-150 IU LH/d
Still OHSS can occur
Antagonist / Agonist protocols are equally effective in PRs
RCOG Guidelines 2019
hCG trigger at 16mm fol. Size: less premature luteinization , doubles CPR above 43y from 7.7% t0 15.5% Wu et al. 2015
D2 transfers give better IVF outcome

Mini-dose Long Agonist Protocol
D21 D1 D6 Day of
hCG
Limited oocytes available for fertilization
Longer time –to-pregnancy
Higher cancellation rate
Progestin
Down regulation
Wessman et al . Fertil Steril 2003
100μg triptorelin
225IU FSH
Individualized dose FSH/LH
25-50 μg agonist

Minimal Stimulation
D1 D3 D6 D7 D11 D13
↑ OPU 36-37 hrs
D8 D10 Agonist
Trigger at fol. size 16mm
Ibuprufen 600mg on day of agonist
If LH rise : early OCP
Vitrification : oocytes /embryos
BC freezing
FET : natural / HRT cycle
Modified Dr J. Zhang
Letrozole 2.5 mg bd X 5
R- FSH 150 IU
D6,8,10
CC25 mg/d X 4

Combined r- FSH & r-LH 2:1
Role of LH in folliculogenesis & implantation
Stimulates the pathway P4 → Androgens → Estrogens
Enhances ovarian sensitivity to FSH leading to final maturation & successful luteinization by hCG
Increases IR & CPR Patermor 2007 / Reprod Biomed Online
LH polymorphism ( V-βLH ) shorter half life
Causes ovulatory dysfunction , POF, ↑PRL , Infertility & Endometriosis
Mafra et al. 2010 / Obstetr Gynecol Reprod Biol.
Greater needs for r-FSH > 2500IU Alviggi et al 2009
RCOG Guidelines 2019 : Gns dose > 300 IU is not recommended for predicted PRs

Duostim Protocol de Ziegler 2015
2 GnRH- antagonist protocols ( 300 IU FSH /d)
COS 1 : start D6 post OCPs COS2 : start right after OPU1
Triggering : GnRH-a in COS 1&2
Results : Similar number of eggs & BC in COS1 & 2
Twice as many oocytes & BC in a 4-week time frame
No statistically significant difference in number of retrieved MII oocytes 5.7 ±3.3 vs 5.1 ± 3.4 or
Biopsied euploid blastocysts / stimulated cycle 44.8% vs 46.9% in LP stimulation vs FP. F. M Ubaldi 2016
Only cost effective when it increases CLBR by 20 %
( now hardly reaches 18 % )

Double Ovulation Trigger
GnRH-a with a reduced or standard dose of hCG 40 hrs and 34hrs prior to OPU respectively Kasum et al 2016
Indications :1- EFS
K. Deepika et al. 2015 / Journal of Human Reprod Sciences
R. Beck-Fruchter et al. 2012 HR
2- Poor responders
Significant increase in number of retrieved eggs , M II eggs, FR, IR , PR & LBR Oliveira et al 2016 J BRA Assist Reprod

Natural & Modified Natural Cycle
Natural : Less cost & side effects with more natural hormonal environment but, fewer oocytes/ embryos and less success rate
LBR In PRs ≤ 35y 2.5% 36-39 y 2.4% ≥ 40y 1 % Polyzos et al. 2012
Modified NC
250 μg Cetrorelix is started concomitantly with 150IU r-FSH when the leading follicle 13-14mm till hCG injection
Cancellation Rate: 12/45= 26.7% vs 3/45= 6.7% in antagonist arm
CPR / cycle / ET similar
LBR / ET similar
Total FSH dose less Kadoch et al .
Segawa et al. Fertil Steril 2009
RCOG Guidelines 2019: Modified NC is probably not trecommended over conventional stimulation for expected PRs

Freeze - all Policy
Rationale : Improves endometrial receptivity
↓ OHSS & MPR
↑ Cumulative PR and cost effectiveness in high responders
Indications : OHSS prevention
Increased P4 on day of hCG ( >2.5 ng /ml ) Xu et al. 2012
Dual stimulation in poor responders
Outcome : FAP v Fresh D3 ET n= 530
LBR 46.34% v 35.9 % OPR 39.7% v 31.1 %
Roque et al 2016 In AMA No RCTs . 4 small retrospective studies showing no benefit

Freeze - all Policy For Poor Responders
n= 83,000 1st cycle Acharya et al 2018
Cumulative PR
Fresh ET Frozen ET
32 % 15 %
LBR 25 % 10 %
Advantages of FAP: Decrease preterm birth & LBW
Pelkonen et al. HR 2010
Disadvantages : Evidence of benefit is limited to high responders
Cumulative outcome is not different in poor responders
Increases time to pregnancy
Questionable cost effectiveness in PRs
Higher risk of LGA ( RR1.54) & PET ( RR1.29 )
Maheshwari et al HR Update 2018

Oocyte cryopreservation : eggs, ovarian tissue , IVM oocytes
Postpone parenthood: Pre-cancer Rx
Social issues ( after 37 yrs : Doyle et al 2019)
It is a reproductive insurance , reducing the need for egg donation
Egg donation :
Especially after many failed euploid ETs
> 35 % PR in females in their 50s US data
Associated immunological problems impairing placentation

Preimplnatation Genetic Testing of Aneuploidies PGT-A
Aim : Avoid ET of aneuploid embryos & Increase PR /ET especially after SET to prevent multiple gestation , miscarriage & vital chromosomal syndromes
Ubaldi FM et al. HR 2015 , Dahdouh & Chen MA 2015
Types of PGT-A :
Comprehensive chromosome testing CCT
Comparative genome hybridization array CGH -a
Single nucleotide polymorphisms - array SNP-a
Next generation sequencing NGS
Types of PGT-A :
Cleavage stage biopsy D3
PBs biopsy D 0-1
Tropho-ectoderm biopsy D 5-7 ( Mosaicism 28-90% )

Embryo Genetic Screening
MA & SR Mastenbroek et al 2013 HR Update
9 RCTs
FISH & D3 biopsy
PGS significantly lowered LBR for women of AMA 13-23 % vs 26 % without PGS
PGS in good prognosis women & in RIF gave similar outcomes
Technical drawbacks & chromosomal mosaicism underlie this inefficacy of PGS
New PGS approaches should be evaluated carefully
Rubio et al 2017 D2 PGT-A in women 38-41y : Sign. higher CLBR using PGT-A 52.9% vs 24.2 % / first ET
36 % vs 21.9% / patient
Dramatically less miscarriage rate 2.7 % vs 39 % in controls
Polar body testing CCT did not increase LBR in 205 women 36-40y 24% with or without testing Verpoest et al . HR 2018

Endometrial Scratching
Mechanisms :
Induce endometrial decidualization Timeya T. et al J. Reprod & Infertility 2014
Provokes secretion of cytokines & GFs Liand Hao 2009
Recruits stem cells to the endometrium Taylor 2004 , Du & Taylor 2007
How? Biopsy ( Pipelle sampler ) , scratch , hysteroscopy ± versa point
Raziel et al 2007, Narvekar et al 2010
Biopsy / Scratch are superior to hysteroscopy
2 SR : Potdar et al 2012, El-Toukhy et al. 2013

Endometrial Scratching
Timing : one week before the treatment cycle
Barash et al 2003 , Raziel et al. 2007, Zhou et al 2008
Immediately before starting ovarian stimulation
In follicular phase of index cycle ( no benefit ! )
Karimzad et al . 2010. Zhou et al 2008
Before triggering ovulation
Not on OPU day Cochrane SR 2015, Nastri et al 2012
Can improve IVF outcome Botdar et al 2012 SR & MA
Doubles LBR 48.9 % vs 22.5% Barash A. 2003 FS , Zhou L 2008
Doubles PR & LBR 66.7% vs 30.3 %
Timeya T. et al J. Reprod & Infertility 2014
Pipelle study : Same LBR 26.1 % Sarah Lensen et al 2016
MA & SR 2019 / HR Open n= 2537 : It remains unclear whether we should do scratching prior to IVF N E Hoogenhuijze et al. 2019
Awaiting results from SCRaTCH and Endometrial Scratch Study

Mitochondrial DNA Transfer
Oocyte mitochondria: Functionally immature
6000 in germ cells to 300,000-400,000 in MII
Decrease after fertilization & with embryo development
Chappel S. 2013
Primarily inherited from the mother
mtDNA susceptible to mutations ( has no histones , introns , repair enzymes )
Leese 2012, Bentov et al 2011
Leading to energetic stress in the oocyte or embryo or
Myopathy, encephalopathy, lactic acidosis , stroke later in
life Moont S. et al. 2013

In Vitro Activation
Tanaka & Kawamura 2017

AUGMENT 3 centers
Autologous germline mitochondrial energy transfer from ovarian cortex
( less prone to progressive mutations as mtDNA from somatic cells)
Dori C. Woods et al. 20
Steps : Isolation of egg precursor cells by magnet – assisted cell sorting
Extraction of mt.
Injection of mt. into autologous oocytes
CPR/ cycle 22% CPR/ ET 32 % n = 60
Fakih MH et al. 2015
AUGMENT: Might be beneficial in ageing oocytes & RIF via in vitro differentiation ( IVD) of oogonial stem cells .
Silvestris et al HR 2018

AUGMENT
E. Labarta et al 2019 FS : Triple blind RCT in PRs .
mean age 36+ ys n= 250 control
n= 253 AUGMENT arm
Conclusion : Mitochondrial injection does not benefit
Developmental capacity of treated oocytes or
Euploidy status of embryos nor
PR
So AUGMENT should not be considered a novel way of ovarian rejuvenation in poor prognosis pts with bad embryos

Adjuvant Therapy
GH Stimulates FSH receptor expression on granulosa cells Increases intra-ovarian IGF-1
4-18 IU sc /d from day of stimulation ( 7. 5 IU /d from D6 )
7/9 studies : No change or significant improvement
J.Dor et al HR . Eftekhar et al Archives of OBGYN 2013
↑ collected eggs 5.9 vs 3.7 ↑ PR 25.7vs 11.4 % ( P=NS) Chung-Hoon et al
Recently :Duffy et al. ↑ CPR MA 2013
Kolibianakis et al. ↑ PR MA 2009
Kyrou et al. ↑ LBR MA2009
Sustained release GH 20 mg D2+ midluteal + late luteal doses in PRs >39y = Sign. more mature oocytes Choe SA et al. 2018
No clear evidence of benefit in PRs Yue- Ming Xu et al 2019

Adjuvant Therapy Cont...
Androgens : DHEA / Testosterone
Augment FSH receptor expression
Promote GCs development
Increase pre-antral & antral follicles
Cochrane MA 2015 : 1496 PRs
No evidence of benefit on CPR
Safety ?
Nagels HE et al. 2015 , Sunkara et al. 2011

Anti -estrogen, Aromatase inhibitors :
Better embryo quality, less eggs Baart et al 2007
↑Endometrial receptivity Devroey 2004
Letrozole ---- better endometrium & folliculo- genesis
Can be used in mild protocol but probably not recommended by RCOG guidelines 2019
100 mg CC or 5mg Letrozole +150-225IU r-FSH Vs conventional protocol
Comparable results , ↑PR > 37yrs , Less cost
Yoo et al 2011 Clin Exp. Reprod Med
Aromatase inhibitors cab be used with high dose FSH
Schoolcraft et al .2008, G Velasco et a2005
CC alone or with Gns or Gns alone are equally recommended in PRs RCOG Guidelines 2019

Potential Future Approaches to Treat AMA Inferility
Minimally / non-invasive embryo biopsy : investigate leftover IVF products ( proteomics, metaboloites, nucleic acids )
Spindle chromosomal complex transfer
Chromosome therapy
Isolation of oogonial stem cells OSCs
Induced ( somatic ) pluripotent cells
In vitro generation of new gametes : Ovarian cortex
Bone marrow stem cells
Silvisters et al. HR 2018, Kuwamora et al 2019

Age and IVF Outcome
Age 20-30yrs 31-35 36-40 41…..
FR 67% 68% 70% 67%
G1,2 Es 54% 52% 53% 55%
PR 58% 55% 46% 26%
CPR 51% 46% 38% 18%
Miscarriage R 8% 12% 19% 35%
S. Sunkara ,T. El Toukhy 2017

Cycle Cancellation & LBR ≥ 40 yrs
Initiated cycles Cancellation Rate LBR %
Klipstein et al .2005 2750 19.9% 9.7
Tsafrir et al. 2007 1217 16.6 % 4.7
Serour et al. 2010 2386 16% 6.7
ESHRE 2010 8.6

Outcome of IVF in AMA
Number of eggs needed to find one euploid embryo
35-37 yrs 5 eggs
38-40 yrs 7
41-42 yrs 10
> 42 yrs 20 Vaiarelli et al . 2018
Implantation potential on euploid BC is independent of maternal age 45-50 %
Cimadomo D. et al. 2018
Implantation Rate as a function of maternal age
25-29 y 18.2 % 30-34 y 16.1%
35-39 y 15.3% 40-44 y 6.1 %
ASRM Practice Committee 2006

IVF Outcome in AMA
LBR after IVF Canadian ART data < 35 yrs 37.4% 35-39 yrs 26.5 % > 40yrs 11.4 %
J. Gunby FS 2011
LBR /cycle 6.7 % ( range 10% - 0.5% )
40-42 y 7.4%
> 43 y 1.1% Serour et al. 2014
HEFA 2014 LBR 38-39y 19.2 /cycle 40-42y 12.7%
43-44y 5.1% ≥ 45 yrs 1.5 %
When to stop IVF in AMA ? at ≥ 45 yrs .
Mehmet at al . 2013
Still reasonable LBR up to 44 yrs
Most pregnancies occur within the first 3 cycles
After 45 stop ART procedures using patients’ own oocytes

Pregnancy in AMA
Increased risk of spontaneous miscarriage
17 %- 28% 25-39 yrs
34% -52 % ≥40 yrs Canadian Data
53% ≥ 45yrs ( 10% at 25-29yrs )
Norwegian data n=421,201 BMJ 2019
Increased risk of maternal & obstetrical complications :
Maternal death Ectopic Hypertension & PET
Gestational diabetes Prematurity IUGR
Fetal and neonatal death Operative delivery
Smajdor et al. 2008

Pregnancy in AMA
Increased risk of aneuploidy & other chromosomal abnormalities Down’s risk 1 : 204 at 35 y
1 : 65 at 40 y
1 : 2 at 45y
Hook et al. Obst Gynecol
Some authors link pregnancy after 35 yrs , usage of fertility drugs & androgens with an increased risk of developing ovarian cancer.
Cochrane review 2019: The risk is slightly higher in nulliparous and for borderline tumors
Age , infertility itself , parity , genetics & BMI should be taken into consideration.

So ,What is the Magic Formula ?
None
Fertility preservation ( oocyte / ovarian tissue freezing)
Careful assessment of oocyte reserve
Optimization of ovarian stimulation through individualizing treatment plan: iCOS , oocyte /embryo banking via duoStim protocol and enhanced embryo selection for SET by PGT-A
Realistic expectations based on proper counseling and the best available practice
Egg donation

Conclusions
ATR outcome is adversely affected by AMA
Launching social campaigns & educating young generations are important to promote awareness of age impact upon fertility
Properly counsel females regarding risks of pregnancy especially above 40 yrs
Ovarian stimulation must be tailored individually
Androgen adjuvant therapy and day 2 ET increase CPR in PRs
In virto activation and mitochondrial transfer are developing areas in ART
Egg donation is the last efficient alternative

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