Dr. José A. Horcajadas
Associate Professor, U. Pablo de Olavide, Seville, Spain Research Associate Professor, EVMS, Norfolk, Virginia, US CSO, Recombine Europe, Barcelona, Spain
Assessing Endometrial Receptivity:
Endometrial Markers of Implantation
Genomics of Human Embryo Receptivity
Heraklion (Crete), 5th MSRM Annual Meeting, April, 27-29 2006
Dr. José A. Horcajadas
Fundación IVI (FIVI)-Instituto Universitario IVI (IUIVI) and University of Valencia
Spain
Median number of follicles on the day of hCG All Subjects Evaluable
0 3 6 9 12 15 18
>=11 mm >=15 mm >=17 mm
ganirelix 0.25 mg ganirelix 2 mg buserelin
Median serum values for LH, FSH, Estradiol and Progesterone
Simón et al. (2005) Hum Reprod 12:3318-27
Median endometrial da-ng according to Noyes criteria
Simón et al. (2005) Hum Reprod 12:3318-27
Expression of E-‐receptor (a) and P-‐receptor (A and B)
for luminal epithelium, glandular, and stromal -ssue
SEM evalua*on
• Blinded evalua*on by single assessor
• Selec*on of 10 random areas of endometrial surface
• Coun*ng per area:
– Total number of cells
– Number of cells with developing pinopods
– Number of cells with fully developed pinopods – Number of cells with regressing pinopods
– Number of cells with cilia
– Number of cells with microvilli
– Number of other cells
SEM evalua*on pinopods
Percentage of cells exhibi*ng any type of pinopods
0 2 4 6 8 10 12
hCG +2 hCG +7
% ganirelix 0.25 mg
ganirelix 2 mg buserelin
natural
EMBRYONIC IMPLANTATION
MOLECULAR DIALOGUE
-
Health embryo at blastocyst stage
Endometrial Receptivity To improve the receptivity
in the endometria under stimulated cycles To know the bases of Infertility
To select the best embryo/s
EMBRYONIC IMPLANTATION
Pregnancy
IS THE ENDOMETRIUM A MINOR PLAYER?
Endometrium Ovary
Fertilization Gametes…
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
20 22 24 26 28 30 32 34 36 38 40 42 44
Aneuploidy is the major cause of loss of implanta-on poten-al with AMA
Reprogene-cs data 2012-‐2015, n= 13,400 PGS cycles by array CGH and blastocyst biopsy Aneuploid blastocysts
years
Source: CDC
Infertility increases with age due to Chromosome abnormalities
Source: Reprogenetics (N = 18,000 PGS cycles of aCGH), CDC
Women are reproducing later:
Birth to first-time mothers by age
(per 1000 women)
Delayed reproduc-on =
infer-lity by aneuploidy
Biopsy stage:
before arrays
• 30% postmeio-c
abnormali-es undetected
• Triple amount of cells to analyze vs. blastocysts
• 30%a to 60%b loss of implanta-on poten-al
• PGD can compensate the damage but does not reach its poten-al
• Non detrimentala
• Not all embryos reach
blastocyst, and not all at the same day
a Scott et al (2013), b Mastenbroek et al. (2007)
Normal Trisomy Monosomy
Normal DNA
Embryo
DNA
Array Compara-ve
Genome Hybridiza-on
46,XY
46,XX+7-‐10
Evolution of PGS:
Reprogenetics data
Reprogenetics PGS procedures a year in US. *2015 annualized 0
2000 4000 6000 8000 10000
day 5, NGS day 5 aCGH
8% day 3
PGS: Relevance of mitocondrial DNA
PGS: Relevance of mitocondrial DNA
Implanta-on process
14 15 16 17 18 19 20 21
Bergh P, Navot D.
Fertil Steril 1992; 58: 537-42
CLINIC IMPLANTION WINDOW
LH+2 LH+7
LH+0
Receptive endometrium features
» Morphological markers
» Biochemical markers
» Gene expression pattern
WHAT IS THE ENDOMETRIAL RECEPTIVITY?
14 15 16 17 18 19 20 21
Bergh P, Navot D.
Fertil Steril 1992; 58: 537-42
CLINIC IMPLANTION WINDOW
LH+2 LH+7
LH+0
GENE EXPRESSION PROFILING OF HUMAN ENDOMETRIAL RECEPTIVITY AT THE TIME OF IMPLANTATION
EXPERIMENTAL DESIGN
MICROARRAY
AFFIMETRIX HG-U95A 12,686 genes
RNA ISOLATION TWO ENDOMETRIAL
BYOPSIES
LH+2
LH+7
Riesewijk et al. Molec Human Reprod 9, 253-64, 2003
FIVE WOMEN
Caucasian
Fertile women with normal cycles 23–39 years
body mass index:19-25 kg/m2
GENES REGULATED DURING HUMAN ENDOMETRIAL RECEPTIVITY
Up at LH+7 Down at LH+7
Strong (>10) 22 5
Medium (5-10) 47 12
Weak (3-5) 84 41
153 58
Results (>3.0 fc in 4 out of 5)
LH+2 LH+7
PCA 1
-1500 -1000 -500 0 500 1000 1500 2000
-3000 -2000 -1000 0 1000 2000 3000 4000
11756
9064
8669
9068 9286
9068
11756
8669
9064
9286
Principal Component Analysis
LH+2 vs Lh+7 samples
Based on 2000 random genes
GENE EXPRESSION PROFILING OF HUMAN ENDOMETRIAL
RECEPTIVITY AT THE TIME OF IMPLANTATION
Window of Implantation in
Natural Cycles
14 15 16 17 18 19 20 21
Bergh P, Navot D.
Fertil Steril 1992; 58: 537-42
LH+2 LH+7
LH+0
Window of Implantation in COS Cycles
14 15 16 17 18 19 20 21
hCG+2 hCG+7
hCG+0
STUDIES OF THE GENE EXPRESSION
PROFILE OF THE ENDOMETRIUM UNDER COS
- Gene expression profile of the endometrium during
the WOI in women under treatment with agonists and
different doses of antagonist and in comparison to
natural cycle
- Gene expression profile of the endometrium during the WOI in women under treatment with agonists in comparison to natural cycle
STUDIES OF THE GENE EXPRESSION
PROFILE OF THE ENDOMETRIUM UNDER COS
- Gene expression profile of the endometrium during the WOI in women under treatment with agonists and different doses of antagonist and in comparison to natural cycle
STUDIES OF THE GENE EXPRESSION
PROFILE OF THE ENDOMETRIUM UNDER COS
GENE EXPRESSION PROFILING OF WINDOW OF
IMPLANTATION IN NATURAL AND STIMULATED CYCLES
EXPERIMENTAL DESIGN
FIVE ENDOMETRIAL
BYOPSIES AT EACH TIME POINT
MICROARRAY
AFFIMETRIX HG-133A
>22,000 genes 50 WOMEN
Caucasian
Fertile women with normal cycles 23–39 years
body mass index:19-25 kg/m2
LH Day 1 2 3 4 5 6 7 8 9
hCG Day 1 2 3 4 5 6 7 8 9
GeneSpring 7.0 GEPAS
HISTOLOGICAL CONTROL:
BLIND DATING BY NOYES CRITERIA
PCA OF THE ENDOMETRIAL BIOPSIES
FROM LH+1 TO LH+9 AND hCG+1 TO hCG+9
- Principal Component Analysis (PCA) integrates the gene expression data of thousand of genes randomly selected to establish relationships between samples.
- This analysis allows to distribute the endometrial samples in a three dimensional space according to their gene expression profile.
- Those samples with similar gene expression patterns cluster together in this type of analysis.
Day LH/hCG+1
PRINCIPAL COMPONENT ANALYSIS (PCA)
Day LH+1/hCG+1
Day LH/hCG+3 Day LH+3/hCG+3
PRINCIPAL COMPONENT ANALYSIS (PCA)
Day LH/hCG+5 Day LH+5/hCG+5
PRINCIPAL COMPONENT ANALYSIS (PCA)
Day LH+7/hCG+7
PRINCIPAL COMPONENT ANALYSIS (PCA)
Day LH/hCG+9 Day LH+9/hCG+9
PRINCIPAL COMPONENT ANALYSIS (PCA)
Natural/LH vs IVF across the WOI
PRE-RECEPTIVE RECEPTIVE
PRINCIPAL COMPONENT ANALYSIS (PCA)
Journal of Clinical Endocrinology of Metabolism 2008, 93:4500-4510
Fertility and Sterility, 90:2152-2164
OTHER STUDIES IN STIMULATED CYCLES
USING MICROARRAYS
Haouzi et al., 2009 Biology of Reproduction
PUBLICATIONS USING GENE EXPRESSION MICROARRAYS UNTIL 2007
Human Reprod Update 2007
Today >250 publications in PubMed
asking for endometrium and gene
expression microarray
Development of a customized microarray for endometrial receptivity evaluation:
The ERA: Endometrial Receptivity Array HOW TO APPLY THIS INFORMATION TO
THE CLINIC
Pre-existing Tests for Endometrial Receptivity Evaluation
- E-tegrity Test: Antibodies for Integrin β3 (Lessey et al., 1995)
-EFT Quick Guide: Cyclines through the menstrual
cycle (Kliman et al., 2006)
New England Journal of Medicine 2006;354:2463-72.
INTEGRATING THE AVAILABLE INFORMATION
INTO A MOLECULAR TOOL FOR ENDOMETRIAL EVALUATION
GENE EXPRESSION DATA
NATIONAL INSTITUTE OF BIOINFORMATIC
BIOINFORMATICS FOR FUNTIONAL GENOMICS
Endometrial Evaluation
Predictor
EEP
Low Density
ERA
Endometrial Receptivity
Array
15k
Probe group: 206 + 28 + 76: 310 genes – 17 genes =
238 genes / 738 probes
Número total de spots:15.744 spots Control Agilent: 536 spots
Spots útiles: 15.208 spots
8 replicates = 738 x 8 = 5.904 spots Empty sponts = 9.304
8x15K
Endometrial Receptivity Array (ERA)
Design
Endometrial Receptivity Array (ERA) Hierarchical clustering
R
NR
Endometrial Receptivity Array (ERA)
Transcriptomic Signature
Endometrial Receptivity Array (ERA)
Comparison with Histology
- It includes 238 genes selected for a single experiment performed over 25 different endometrial biopsies in 2006
- It doesn’t contain some genes of unknown function but useful as biomarkers
- It doesn’t contain genes discovered in other gene expression platforms
- It uses Agilent customized gene expression microarray
Can the ERA be improved?
ENDOMETRIAL RECEPTIVITY ARRAY
- Including genes/biomarkers discovered by other researchers and method (Review of the literature since 2005, 196 genes has been found, only 54 shared with ERA)
- Including genes related wiith immune response, very relevant in some disorders such as implantation failure
- Defining the transcriptomic signature of other ethnicities
- New design of oligos, more robust, less false positive or negative - New platform of analysis (digital PCR)
- This new method allows the analysis of 100 samples in 4 hours
- This new method can reduce the price 5 times (to less than 100 euros)
A NEW METHOD OF “ER” ANALYSIS
VALIDATION OF OLIGONUCLEOTIDES
PREDICTION
NR
PREDICTION
PR
PREDICTION
R
Non-receptive or
LH +2
Pre-receptive or
LH +5
Receptive or LH +7
ER Map ER Grade
Fluidgim analysis: heat map
Gene: COL16A1
Fluidgim analysis: Melting curves
Fluidgim analysis: Ct data
0 5 10 15 20 25 30 35 40 45 50 1
7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151 157
Total explained variance (%)
Principal component
ER Map ER Grade
PRINCIPAL COMPONENTS ANALYSIS
16 Principal components
explain 93.47% of the total variance
ER Map ER Grade
Total variance explained
Component
Initial Eigenvalues Extraction Sums of Squared Loadings
Total % of variance Cumulative % Total % of variance Cumulative %
1 67,412 42 42 64,421 40,263 40,263
2 20,316 13 55 19,367 12,104 52,368
3 18,914 12 67 19,345 12,091 64,459
4 11,381 7 74 13,450 8,406 72,865
5 6,278 4 78 5,212 3,257 76,122
6 4,673 3 81 4,736 2,960 79,082
7 4,313 3 83 3,736 2,335 81,417
8 3,054 2 85 2,965 1,853 83,270
9 2,586 2 87 2,814 1,759 85,029
10 2,136 1 88 2,742 1,714 86,743
11 1,939 1 89 2,051 1,282 88,024
12 1,683 1 90 1,993 1,245 89,270
13 1,399 1 91 1,909 1,193 90,463
14 1,343 1 92 1,693 1,058 91,521
15 1,127 1 93 1,664 1,040 92,561
16 1,001 1 93 1,458 ,911 93,472
PRINCIPAL COMPONENTS ANALYSIS
ER Map ER Grade
0 5 10 15 20 25 30 35 40 45 50
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Total explained variance (%)
Principal component
PRINCIPAL COMPONENTS ANALYSIS
16 Principal components
explain 93.47% of the total variance
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Cumulative % of total explained variance
Principal component
ER Map ER Grade
PRINCIPAL COMPONENTS ANALYSIS
93.47%
ER Map ER Grade
DISCRIMINANT ANALYSIS
16 components
Classificacion Resultsa
Time
Predicted Group Membership
Total
NR PreR R PostR
Original Count NR 15 0 0 0 15
PreR 1 47 3 0 51
R 0 8 35 1 44
PostR 0 0 1 13 14
Unclassified
cases 2 2 3 4 11
% NR 100,0 0,0 0,0 0,0 100,0
PR 2,0 92,2 5,9 0,0 100,0
R 0,0 18,2 79,5 2,3 100,0
PR 0,0 0,0 7,1 92,9 100,0
Unclassified
cases 18,2 18,2 27,3 36,4 100,0
a. 88,7% de casos agrupados originales clasificados correctamente.
ER Map ER Grade
DISCRIMINANT ANALYSIS
16 components +
24 genes with biological significance
Classificacion Resultsaa
Time
Predicted Group Membership
Total
NR PreR R PostR
Original Count NR 14 0 0 0 14
PreR 0 49 0 0 49
R 0 2 36 1 39
PostR 0 0 0 14 14
Unclassified
cases 2 3 1 3 9
% NR 100 0 0 0 100
PreR 0 100 0 0 100
R 0 5,1 92,3 2,6 100
PostR 0 0 0 100 100
Unclassified
cases 22,2 33,3 11,1 33,3 100
a. 97,4% of original cases correctly classified
ER Map ER Grade
- There is a high number of genes, with a define pattern, involved in endometrial receptivity (WOI genes)
- There is a high number of WOI genes that are aberrantly expressed in stimulated cycles at the time of implantation (LH+7 in natural cycles and hCG+7 in COS cycles)
CONCLUSIONS
- Microarray technology and other “omics” techniques are good tool for analyzing gene expression profile of the endometrium at the time of
implantation to compare optimal versus non optimal conditions (infertility or subfertility)
