Endometrial preparation For FET

 1 
Dr Astha Gupta, Fellow in reproductive medicine, IHR
 WHY

WHA
HOW
T
2
 WHY??
 Cryopreservation and subsequent replacement
of Frozen embryo is integral part of ART
programme.

 According to ICMART world report 2006- FET

comprised 27.4 % of all initiated IVF cycles.

 Elective single embryo transfer leads to

cumulative live birth rate per oocyte retrieved
same as double embryo transfer and reduces rate
of multiple pregnancy.(McLernon et al, BMJ,
2010)
3
 WHY??
 Significant improved pregnancy rate in cryopreserved cycle
due to better endometrial receptivity and synchronisation.(8, 5
in on1)
 Prevention of OHSS
 PGS and PGD
 Premature LH surge
 Lab preference

4
Success of FET
Transfer of
embryo in
window of
receptivity.

ua t e on Go
d eq isati c od
A ron oni l
ch bry tria em qua
n
sy f em ome nt. bry lity
o end me o
p
and evelo
d

5
 What
 Endometrial thickness > 7mm

 Triple line endometrium

 Adequate uterine blood flow

6
 HOW??
A) Natural FER cycle
 TRUE
 MODIFIED

B) Artificial
 E+P
 E + P+ GnRH agonist

C) Ovulation induction
 Clomiphene citrate
 Letrozole
 HMG

7
 Natural cycle
 Uses patient own sex steroid production from growing follicle
 Success depends on accurate determination of ovulation time
and endometrial receptivity.

8
 Monitoring
 Serial USG- from d10-12 of cycle

 Endometrial thickness
 Follicular development
 Time the testing for LH surge
 Detection of ovulation

 LH( blood and urine) for detection of LH surge-

 Ovulation 36hrs after LH surge

 Urinary LH increases 21hrs after blood LH surge.
 LH levels >180% of baseline indicates ovulation will happen next day(ref 14,
15 1)

9
 FET
 Day of ovulation corresponds to day of pickup
 3-5 days after ovulation day depending on stage of embryo.

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 Advantages of NC-FET
 No medication required
 More physiological

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 Disadvantages
 Only in women with regular ovulatory cycle
 Variability in timing of LH surge, both in cycles and patient
 Once or twice daily measurement required
 Urinary LH KIT-
 Difficult to interpret by patient
 Variable threshold
 30% false negative( ref 16 1)

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 Modified Natural cycle
 Ovulation triggered by HCG administration.

 MONITORING-

USG-Hcg administered when dominant follicle reaches 17-18

mm
 Ovulation 36 hrs after Hcg

13
 Advantages of Modified
NC-FET
 LH monitoring not required.
 Less frequent USG monitoring
 No difference in pregnancy rate, live birth rate compared to
NC-FET

Weissman et al,2011, reprod Biomed Online

Greoenwoud et al, 2013, Human Reprod Update

 Fatemi HM et al concluded in 2010 that Hcg negatively

affects the rate of ongoing pregnancy (18 1)

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 Disadvantage of Modified
NC-FETs
 Unexpected ovulation- difficult time adjustment for thawing
for embryo
 Cycle cancellation – 7-12%( 18 1)

15
 Artificial cycle( E+P)
 Exogenous hormones used to mimic endocrine characteristics
of natural cycle( oestrogen + progesterone)

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 Oestrogen
 Endometrial development and suppression of dominant follicle
development.

 Timing-
 Administration at start of cycle
 Earliest possible
 Before fourth day
 Duration of oestrogen exposure may vary from 6-38 days( atleast
12 -14 days) before progesterone supplementation: mimic
physiological variable follicular phase.
 Shorter oestrogen supplementation(<6 days) leads to higher
abortion rate and breakthrough bleeding occurs after >40 days of
supplementation. 17
 Routes
Oral tablet

Transdermal plasters

creams

Transvaginal ring

18
 Oral
 Estradiol valerate ( progynova, endofert)
 Micronised estrogen (17 beta estradiol)

Regimes –
 Constant dose(CD)
 Increasing dose(ID)

19
 Regimes( Increasing dose)
 4mg/day Day 2-5
 6 mg/day Day 6 till pregnancy test positive
 Usg on day13
 Progesterone added when endometrium >8mm
 Dose increased to 8mg when ET< 8mm.

Nekoo et al, 2014, Journal of family and reproductive health.

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Madero et al, 2016,human reprod

6 mg/day
2 mg/day 4 mg/day
Day 13
Day 1-7 Day 8-12
onwards

21
 Norfolk protocol

2 mg of
micronised 4 mg day Day 6- 1 mg/day Day 14-
estradiol valerate 13 28
Day 1-5

22
 Liao et al ,2014, Int J Clin Exp Pathol

 2 mg/day Day1-4
 4 mg/day Day5-8
 6 mg/day Day9-12
 USG on day12
 Progesterone added when endometrium >8mm
 Dose increased to 8mg when ET< 8mm.

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 1 mg Day1-5
 2 mg Day6-9
 6 mg Day10-13
 2 mg Day14-17
 4mg Day 18-26
 1 mg on day27-28

Kalem et al ,2015, J Turk Ger Gyanecol Assoc

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 Constant dose
 4-8 mg daily from Day2-3 onwards

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 CD vs ID
NO
STATISTICALL
Y
SIGNIFICANT
DIFFERENCE
IN ANY OF
THE
REPRODUCTIV
E OUTCOME
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 Disadvantages of oral route
 Undergoes first pass metabolism in liver to estriol
 Circulatory estrogen decreases by 30%
 Estradiol valerate : estrone ratio- .2

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 Transdermal( patch, cream)
INCREASING DOSE
 50 microgram/every three days Day 1-6
 100microgram(1 plaster) Day 7-9
 200microgram(1 plaster) Day 10-11
 100microgram daily (400)Day 12-14
 100microgram(1 plaster) Day 15-17
 200microgram(1 plaster) Day 18-28

Kalem et al ,2015, J Turk Ger Gyanecol Assoc

28
 Prato et al, 2002,fert and sterlt

100 200 300

microgram day microgram day microgram day
1-7 8-10 11 onwards

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 Madero et al, 2016,human reprod
 50 microgram on Day1-3
 100 microgram on Day 4-6
 150 microgram Day7 onwards

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 Constant dose
 150 microgram every 3 days from day1 till ET.

 Oestrogel gel (17 beta estradiol)-

 80 gm(.06%)
 One dose in spatula delievers 2.5 gm gel containing 1.5mg of
estradiol
 Results in 25% of those serum estradiol concenteration after
same oral dose intake

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 Transdermal route
ADVANTAGES-

 Escape first pass metabolism

 More physiological estradiol: estrone ratio-1
 Avoid systemic side effects

DISADVANTAGES-
 Fluctuation in estrogen concenteration

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ORAL vs TRANSDERMAL
 No stastically significant difference in LBR after oral route for
CD or ID
 No stastically significant difference in LBR after transdermal
route for CD or ID.
 Higher biochemical pregnancy rate in CD (both for oral and
transdermal route)- stastically siginficant in oral route.

 Madero et al, 2016,human reprod

33
 Transvaginal
A)Vaginal tablet (17 beta estradiol)

Dose-
 1-2 mg/day
Liao et al in 2014: added vaginal estrogen on day12 or

continued oral estrogen in patients with ET <8 mm.

B) Polysiloxane impregnated estradiol rings –

Provide a serum E2 concenteration of 60 pg/ml.
Replaced every 3 months.

C) Vaginal cream 34
 Transvaginal
Advantages-
 Comparable implantation and clinical pregnancy rate per
transfer cycle.
 Skips first pass metabolism and hence higher bioavailibility:
used in patients with inadequate endometrium
Disadvantages-
 Damages effect of vaginal progesterone used for luteal
Support.
 Local irritation

35
 Progesterone
 ROUTE-
 IM
 Vaginal suppositories or rings
 Vaginal gel
 Oral tablet

 TIME –
 Depends on endometrial thickness(> 8mm)
 Independent of duration of estrogen exposure( 38 39 1)

36
 Progesterone
 IM- natural progesterone in doses of
50 mg on day 14
100 mg day 15-26
 Vaginal- micronised

100 or 200 mg on day14

300 or 600 mg day 15-26
Oral route-
 Inefficient absorption due to hepatic hydroxylation and
conjugation .
 Very low serum progesterone( <10 microgram/l) with marked
fluctuations.
 Not used for endometrial preparation
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 Progesterone
Vaginal ring
 Rubber vaginal ring with 1 gm of natural progesterone.
 Releases 10 mg/ 24 hrs
 Once placed-releases stable hormone amount for 3 months.
 Less plasma levels but sufficient intrauterine progesterone
levels( 60-100 nmol/l) which allow embryo implantation and
pregnancy.

38
 Vaginal vs IM route
 No difference in pregnancy rate between two(37 1).
 IM route results in 5fold higher progesterone levels compared
to vaginal route
 Vaginal route preferred because of absence of hepatic first
pass metabolism.
 Vaginal route induces endometrial secretary pattern that
resembles natural cycle.
 In IM route, only 43% of endometrium is in phase . Rest have
asynchronous maturation.( 43 1)
 Vaginal route as better compliance without local irritation

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 Advantages of E+ P
 Time of thawing embryo and transfer can be planned
 Useful in patient with irregular cycles
 No difference in clinical, ongoing pregnancy and live birth
rate between NC-FET and AC-FET

Groenewoud et al, 2013, Hum Reprod Update

Kalem et al ,2016, J Turk Ger Gynaecol Association

40
 Disadvantages
 E + P doesn’t ensure complete pituitary suppression.
 Premature luteinisation(5%) leads to early endometrial
progesterone exposure and incorrect calculation of timing of
thawing and transfer.
 Less physiological due to exogenous drug use.

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 E +P+ GnRHa
 Useful in patient with remaining ovarian function.
 Suppresses ovarian function completely before inducing E+P.
 GnRHa single im depot administered on day21 of previous cycle.
 E2 started 14 days after agonist.

DISADVANTAGES
 Expensive
 Side effects of agonists
 Delays the resumption of menses if FET fails.

ADVANTAGES
 Less cancellation rate(4.5%) compared to NC-FET(17.4%).

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 Simmilar implantation, pregnancy rate and abortion rate as
compared to without agonist.

Glujovsky et al, 2010, Cochrane Database

Nekoo et al, 2014, Journal of Family and Reproductive health

NC-FET and GnRH agonist supported AC-FET- no difference

in clinical pregnancy and live birth rate
Hill et al, 2010, Fertil Steril
Groenewoud et al, 2013, Human Reprod Update:
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OVULATION INDUCTION

Letrozole

Clomiphene
citrate

HMG

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 Letrozole

Prevents the
Releases HPO Increase FSH
a selective conversion of
from negative release from
aromatase androgen to
feedback anterior
inhibitor estrogen in
effect pitutary
granulosa cell

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 Letrozole
 Increases follicular sensitivity to FSH due to raised androgen
levels.
 Short t1/2 life (2 days)- no antiestrogenic effect on
endometrium and cervix
 Intact negative feedback mechanism- monofollicular
development
 Reduces effect of supraphysiological E2 levels on
endometrium.

46
 Regime
 Dose- 2.5 mg D3-D7
 Usg on day 10
 Hcg 10,000 IU when follicle >=17 mm and endometrium >=7
mm
 Progesterone started on day of hcg trigger.

Sibai etal, 2015, middle east fertility society journal

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 Letrozole Vs AC-NET
 Lower endometrial thickness- attributed to lower oestrogen
level due to aromatase inhibition.
 Higher pregnancy rate (non significant).
 Lower abortion rate (non significant).
 Higher ongoing pregnancy rate (significant).
 Better endometrial receptivity
 Increased endometrial VEGF and vascularisation.
 Increased integrin expression in surface and glandular
epithelium.

Sibai etal, 2015, middle east fertility society journal

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 Letrozole
 Moderate ovarian stimulation with E2 levels same as normal
cycle.
 Higher midluteal progesterone level- lower abortion rate(non
significant)
 Li et al in march 2014 reported significant higher clinical
pregnancy rate and lower abortion rate.

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 Gonadotropins
 Regime-
 Start on Day 2: 37.5 -75 IU
 Day 6-7: USG and serum LH, FSH, progesterone and 17beta estradiol
 if no dominant F is recruited , the dose is increased at increments 37.5
U every week to a maximum 225 U/d .When single dominant F of 10-
12 mm the dose is maintained
 Hcg: when follicle >= 17mm and ET >= 7mm and estradiol >150
pg/ml.
 Ovulation day: 2 days after hcg(in absence of spontaneous LH surge).
otherwise 1 day after.
 Scheduled ET date postponed for 1 day if spontaneous LH surge seen.

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 NC-FET VS HMG
 No stastistical difference in
 IR per embryo
 LBR per ET
 Endometrial thickness

Peeraer et all, 2015, hum Reproduc( RCT)

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 Clomiphene citrate
 Dose- 100 mg D 2-6.
 E2 - 2mg/d until D of HCG
 HCG – at follicular size (20-24 mm) and E thickness ≥8mm:
HCG 10000 .

Cochrane review 2008 –

 No difference in outcomes in AC-FET and CC regimes.
 No difference in outcomes in AC-FET + GnRHa and CC
regimes.

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 CC + hmg
 CC 100 mg D 2-6.
 HMG 150 IU daily from Day6

Van der Auwera et al in 1994 compared CC + HMG vs HMG .

IR was found to be significantly higher in HMG group compared
to CC + HMG.

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Role of l arginine and sildenafil
High blood flow impedance of uterine radial arteries (>.81)

impairs the growth of the glandular epithelium

decrease in vascular endothelial growth factor (VEGF) expression,

Low VEGF levels cause poor vascular development

decreases blood flow in the endometrium.

The vicious circle leads to a thin endometrium.

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Mechanism of action
L arginine(a
substrate of NO)

NO

activating cyclic
guanosine
monophosphate
(cGMP)

relaxation of
vascular smooth
muscle

55
 Sildenafil
citrate(type 5
specific
phosphodiesterase
inhibitor)

prevents the
breakdown of
cGMP

potentiates the
effect of NO on
vascular smooth
muscle

56
 Role of l arginine and sildenafil
 Tasaki et al, 2010, fert stert : l arginine and sildenafil citrate
significantly improved uterine A RI and endometrial
thickness in the patients with a thin endometrium.
 Firouzabadi et al, 2013, Iran J Reprod Med : sildenafil vs
control
 Significantly higher endometrial thickness
 Significantly higher triple line pattern
 Higher chemical pregnancy and implantation rate (non
significant)

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 Role of aspirin
 Reduces uterine vascular resistance and improved uterine flow
rate.
 Improves pregnancy rate
 Schisterman et al,2009, Curr Opin Obstet Gynecol conducted
a review of the most recent meta-analyses concerning aspirin
and IVF :the current evidence is insufficient to base
recommendations regarding the use of LDA treatment in
conjunction with IVF. 

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 A systematic review and meta analysis by Khairy et al,2007,
fert and stert-
 No significant improvement in pregnancy rate, live birth rate,
miscarriage rate.
 Weckstein et al in 1997, fert and stert- no difference in
endometrial thickness but significant increase in pregnancy
rate, implantation rate.

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 Role of G-CSF
 MECHANISM OF ACTION-
 Alters expression of endometrial genes important for
implantation.
 Endometrial vascular remodelling
 Local immune modulation
 Alteration of cellular adhesion pathway
 Increase in regulatory T cells and dendritic cells.

 DOSE- 30 MU( 300 microgram/ml) 6-12 hrs before hcg

injection.

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 Review by Cavalcante et al in 2015, Iran J
Reprod Med

 Gleicher et al in 2013 and Kunicki et al in 2014 concluded a

significant improvement in endometrial thickness in group
who received G- CSF as compared to controls.
 LI et al in 2013 found no significant difference in endometrial
thickness before and after infusion
 Barad et al in 2014 concluded that use of G-CSF did not
improve IR and pregnancy rate.

61
 GnRH Agonist in
Adenomyosis
 Adenomyosis affects normal implantation
 Disrupts myometrial architecture
 Alters uterine peristaltic activity- interfere with sperm
transport.
 Overexpression of aromatasep450, decrease in integrin beta3,
LIF and osteopontin
 Alteration in HOX 10 gene
 Impairs endometrial receptivity and blastocyst-endometrial
implantation.

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 Role of GnRHa
 Direct antiproliferative action
 Reduces inflammation and angiogenesis
 Hypoestrogenic effects

 DOSE- 3.75 mg/month for 3months

 Estradiol valerate started 4 weeks after last dose GnRHa

PRETREATMENT WITH GNRHa RESULTS IN HIGHER CLINICAL

PREGNANCY RATE IN PATIENTS WITH ADENOMYOSIS.

Park et al, 2016, Clin Exp Reproduc Med

63
 CONCLUSION
 Programmed AC-FET is the most convenient due to flexibility of scheduling
and limited monitoring.
 AC-FET have not been shown to be superior to NC- FET or Modified NC-FET
with regards to pregnancy rate
 Mode of administration of oestrogen have little significance with regards to
pregnancy rate in AC-FET.
 Mode of administration of progesterone does not seem to influence pregnancy
rate.
 No difference in clinical pregnancy rate when AC-FET = GnRHa is compared
to AC-FET or NC-FET.

Based on current published literature, it is impossible to recommend any one

method of endometrial preparation in FET over other.
Glujovsky et al, 2010, Cochrane database

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THANKS
 65 