Window of Implantation:

Natural vs stimulated cycles

Orhan Bukulmez, M.D.

Associate Professor and Division Director
University of Texas Southwestern Medical Center
Division of Reproductive Endocrinology and Infertility
Department of Obstetrics and Gynecology
Dallas, Texas.
 WOI
• Synchronization of implanting blastocyst and
receptive endometrium
• Thought to be limited to days 7 to 9 or 6 to 10
days post ovulation
• Endometrium may stay receptive as short as few
hours to as long as 2 days
• Blastocyst stage transfer after 5 days of
replacement doses of P4 or LH+7days result in
acceptable implantation rates
 WOI biomarkers from the recent past
• Pinopods
• αvβ3 integrin
• LIF
• IGFBP-1
• HOXA10
• MUC1
• EMX2
• CSF-1
• PR downregulation
• Others
Achache H, Revel A. Hum Reprod Update 2006;12:731
 WOI Markers from today
(LH+2 vs LH+7 or per hCG trigger??)
• cDNA microarrays of hundreds of genes with little overlap
between studies- hence the used term “signature”
• Most recent full transcriptome analysis without using known
platforms or probes
• Intense bioinformatics-ongoing validation studies
• Proteomics from endometrium or from endometrial secretions
(no clear correlation with microarrays)*,~
• Micro RNA arrays**
• Nucleolar channel system formation (premature in COS)***
• The assumption is endometrial receptivity issues is responsible
from majority of IVF failures.
*Garrido-Gomez T, et al. Hum Reprod 2014;29(9): 1957
~ Li M-Q, Jin, L-P In J Clin Exp Pathol 2013;6:1964
**Galliano D & Pellicer A. Fertil Steril 2014;101(6):1531
*** Zapantis et al Hum Reprod 2013;28:3292
 Controlled ovarian stimulation
• Premature P4 increase without premature LH surge (>1.5 ng/ml)
• Early maturation of endometrium-Dating??
• Abnormal hormonal milieu: No correlation between serum E2
levels and endometrial receptivity also poor correlation with
USG findings- 17OHP?, DHEAS?, very high E2?
• Increased serum cytokine levels in COS
• Perceived differences between agonist vs antagonist protocols
• Potential differences according to which trigger agent(s) used
• Alters cDNA microarray results
• Observation that Frozen ET may be better than Fresh ET in terms
of outcomes
Fresh autologous< Fresh donor oocyte IVF cycles: SART
data 2008-2010 adjusted for age, #eggs,#ETs

Yeh et al Fertil Steril 2014;102:399

Fertil Steril High responders
 What about the low end of the graph for P4?
2723 cycles, 19-36 y/o, 2006-2012, first or second cycle fresh ET, adjusted for #Ets,
D3vs D5, peak E2, FSH dose and #COCs

Is certain amount of
P4 elevation
required like in
natural cycles?

Ribeiro-Santos et al Human Reprod 2014; 29(8):1698

High P4 levels in the beginning of stimulation

• Disturbed luteolysis described in ARA/DOR/poor responder

• (P4 may be above 0.3 ng/ml)
– Beckers NG et al Fertil Steril 2002;78:291
• Increased P4 during initiation of stimulation (>1.6 ng/ml)
associated with low OPRs
– Kolibianakis EM at al Hum Reprod 2004;19:1525
• If P4 is >1.6, then pretreatment GnRH antagonist for 3 days
leads to decreased P4 and comparable PRs to groups with
normal P4 levels
– Blockeel C et al Curr Pharm Biotechnol 2011;12:423
 Trigger agent?

• Dual trigger with agonist+trigger dose hCG

results in better IR, CPR and LBR in antagonist
cycles
– Lin M-H et al Fertil Steril 2013;100(5):1296
• hCG trigger vs agonist trigger with 1500 IU
hCG at 35 h:Endometrial cDNA microarray
results comparable in a small egg donor study
– Humaidan P et al Hum Reprod 2012;27(11):3259
 DATING IS OUT!

Out of phase biopsy results timed with LH kit (d21-22 & 26-27)
Poorly discriminates fertile of infertile females: EMB should not
be routine in assessment of infertile couples
 JCEM 2008;93(11):4500-4510

Microarray platform for transcriptomics

Natural cycle LH+1 to LH+9 EMB (25)
COS hCG+1 to hCG+9 (24)- Long Lupron, no LPS (No P4 levels available)
2 days of delay in activation and repression of two clusters of genes (in the hundreds each)
in hCG+7 vs LH+7

JCEM 2004;89(11):5742-5752

15 egg donors: 5 natural cycle, 7 antagonist (with our without LPS), 3 agonist with LPS
EMB: Day 21 (LH+8, LH+1 considered to be day of ovulation) hCG+9
Microarray platform, histology, pinopodes, ER and PR content
COH: H/E dating advanced for 1 or 2 days vs natural cycles
 Gene expression profile: Natural vs COS
• Same patient (n=21) in natural and then in COS cycle
• EMB LH+2 and LH+7 (?) and the OPU date (hCG+2)
and on day of ET (hCG+5) (?)

Haouzi et al Hum Reprod 2009;24:1436

Natural
Gene expression (cDNA microarray) profiles
stimulated vs nonstimulated
• Next generation, high-throughput RNA sequencing screens entire
transcriptome
• No probes (cDNAs), hence no hybridization
• Offers advantages over microarrays: absolute transcript levels,
transcript variants, currently unannotated transcribed regions,
unlimited sensitivity hence enables the detection of rare
transcripts- enormous amount of data even making
bioinformatics more complex (>15,000 gene transcripts)
• LH+2 (n=6) vs LH+7 (n=6) biopsies, validation with RT-PCR and WB
• Extensive data, more predicted genes in WOI, decreased HOXA10
expression– more EMBs, more analysis is needed
 Human Genome Complexity
• 20,000 genes – About 2% of the DNA is
exons
• 30-60% of the genes undergo alternative
splicing of its mRNA-hence multiple
isoforms of a protein (PR with at least 2,
fibronectin with at least 20 isoforms).
• 50% of predicted genes with unknown
function
• 50 million variants (CNVs) in humans
among more than 19,000 genes- too
many or too few of the dosage-sensitive
genes, which may be responsible for a
substantial amount of human phenotypic
variability, complex behavioral traits and
disease susceptibility
• Non-coding RNAs like miRNAs-fine tuning
the translation process
 Is non-invasive endometrial stripe
assessment totally out?
• Non-invasive: Tv-USG
• Various thickness cut-off values reported to
predict “receptive endometrium”
• 8-12 mm trilaminar endometrial stripe on the
day of hCG administration or the day of P4
start?
• Seemed to work for FET cycles
 What about the embryo?
• Holy Grail: Non-invasive embryo assessment to select an
embryo with 100% chance of healthy live birth
• Invasive methodology via PGS suggests high aneuploidy rates
in embryos and transferring embryos tested normal with
various whole genome assessment platforms can increase
pregnancy rates- not well tested in poor prognosis population
• Positives and negatives of each assay: Array-CGH, SNP arrays,
now next generation sequence-
– Does it influence cumulative PRs from one fresh cycle if freeze all and
FET strategy is used?
– Stay tuned!
44.9% of the transferrable blastocyst showed aneuploidy in good
prognosis patients
Yang et al Mol Cytogenet 2012;5:24
 Non-invasive embryo selection?
• Use of sperm selection techniques like IMSI?
• Aspiration of blastocele fluid for DNA analysis?
• Omics of blastocyst culture media?
• Embryo time-lapse monitoring?
What about the Embryo transfer Technique?

• It is estimated that poor embryo transfer

technique may account for as much as 30% of
all failures in assisted reproduction
– Cohen, 1998
 Embryo and Endometrium: Do they talk to
each other
• Embryo enters endometrial cavity up to 72 h prior
to implantation
• In vitro data performed by using blastocyst
condition media support the potential presence of
soluble factors modulating endometrial gene
expression in a differential matter according to the
“quality of the blastocyst” ( implanted vs not-
implanted)
Cuman C et al. Preimplantation human blastocysts release factors that differentially alter
human endometrial epithelial cell adhesion and gene expression relative to IVF success.
Hum Reprod 2013;28(5):1161-1171
 Frozen embryo transfer (FET)
• Great improvements within the past decade
• Even better pregnancy rates than fresh
• Better pregnancy outcomes- Lower SGA, PTB
• Lower birth defects rates than fresh

Barnhart Fertil Steril 2014;102

Shapiro BS et al Fertil Steril 2014;102
Pinborg A Hum Reprod Update 2013:19:87
 Figure 3 Trends in estimated numbers of live births with fresh transfer and FET. These estimates were
calculated by multiplying the reported numbers of cycles and the respective birth rates on SART's national
report, and summing across age groups.

Bruce S. Shapiro , Said T. Daneshmand , Forest C. Garner , Martha Aguirre , Cynthia Hudson

Clinical rationale for cryopreservation of entire embryo cohorts in lieu of fresh transfer
Fertility and Sterility, Volume 102, Issue 1, 2014, 3 - 9

http://dx.doi.org/10.1016/j.fertnstert.2014.04.018
Figure 2 Trends in RR for live birth per transfer in FET vs. fresh transfer by SART age group. An RR exceeding 1.0 indicates
greater birth rate with FET. By 2012 the birth rate per transfer with FET exceeded that for fresh transfer in the four oldest
age group. RR above 1.0 inicates greater birth rate with FET

Bruce S. Shapiro , Said T. Daneshmand , Forest C. Garner , Martha Aguirre , Cynthia Hudson

Clinical rationale for cryopreservation of entire embryo cohorts in lieu of fresh transfer

Fertility and Sterility, Volume 102, Issue 1, 2014, 3 - 9

http://dx.doi.org/10.1016/j.fertnstert.2014.04.018
 Fresh vs FET

EP rates lower in blast FETs as compared to fresh day 3 ET Huang F&S 2014;102:1345
 Pooled estimate on the risk of PTB in singletons born after IVF/ICSI in frozen/thawed cycles
versus singletons born after IVF/ICSI in fresh cycles. τ2 = 0.0138.

Pinborg A et al. Hum. Reprod. Update 2013;19:87-104

© The Author 2012. Published by Oxford University Press on behalf of the European Society of
Human Reproduction and Embryology. All rights reserved. For Permissions, please email:
journals.permissions@oup.com
 Pooled estimate on the risk of PTB in singletons born after IVF/ICSI in frozen/thawed cycles
versus SC singletons in the general population. τ2 = 0.0240.

Pinborg A et al. Hum. Reprod. Update 2013;19:87-104

© The Author 2012. Published by Oxford University Press on behalf of the European Society of
Human Reproduction and Embryology. All rights reserved. For Permissions, please email:
journals.permissions@oup.com
 NEJM 2012;366:1803

• IVF&ICSI FETs combined compared with births

from fertile women no difference in birth defects
 What do we need?
• New technologies for non-invasive assessment
of endometrial receptivity
• Further consideration of freeze all strategies
• Better and non-invasive embryo assessment