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Impact of mild ovarian stimulation on implantation
Juan A GarciaGarcia--VelascoVelasco IVIIVI--MadridMadrid Rey Juan Carlos University, Madrid, Spain
Window of Implantation
hCGLH+0 injection STIMULATEDNATURAL CYCLES CYCLES
14 15 16 17 18 19 20 21
hCG+2LH+2 hCG+7LH+7 Bergh P, Navot D. Fertil Steril 1992; 58: 537‐42
Implantation
Hydrosalpinx/ Endom. cavity COH
Trombophilias
Embryo Endometrium (morphologic, genetic) (morphologic, genetic)
Embryo transfer
1 3/4/2009
Factors involved
Controlled Ovarian Hyperstimulation
High levels of estradiol
ENDOMETRIUM ? EMBRYO ?
LOW IMPLANTATION RATES IN HIGH RESPONDER PATIENTS • Clinical studies showing low IR in high responder patients (Pellicer et al. Hum Reprod 1989; Simón C et al. Hum Reprod 1995; Pellicer at al. Fertil Steril 1996; Valbuena et al, Hum Reprod 1999)
• Endometrial receppytivity but not embryo qqyuality is affected (Simón C et al. Hum Reprod 1995; Valbuena et al, Hum Reprod 1999)
• Evidence of altered endocrine milieu in the periimplantation period (Pellicer A et al. Fertil Steril 1996)
• Increased IR when E2 levels were lower in subsequent cycles (Simón C et al. Fertil Steril 1998; 70:234-9)
• Extremely high E2 levels are embryotoxic for the embryo (Valbuena et al. Fertil Steril 2001)
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• Receptive endometrium features
» Morphological markers » Biochemical markers » Gene expression pattern
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GarciaGarcia--VelascoVelasco et al, 1999
The evolution of technology
The evolution of technology
1983 2008 3995 US$ 300 US$
4 3/4/2009
The evolution of technology
MICROARRAY TECHNOLOGY
Reproduction 160 • Microarray analysis, first described in 1995 (Schena, Science) 4.567 140
• Based120 on nucleic acid complementarity. 100 • It allows80 to analyze thousand of genes in one single experiment. Serie1
60 1.390 • Increasing40 number of papers are published every year using this technology. 289 20 • Gene0 Ontology recognizes not only genes but also pathways that may be involved.
1
COH does have an impact on endometrium
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EFFECT OF CONTROLLED OVARIAN HYPERSTIMULATION IN IVF ON ENDOMETRIAL GENE EXPRESSION PROFILES
Caucasian EXPERIMENTAL DESIGN Fertile women with normal cycles 18–35 years body mass index:19‐25 kg/m2
FIVE WOMEN FIVE WOMEN
BiBioopsi iees Biopsies
LH+7 1 LH+2 1
hCG+7 2 LH+7 2
Comparison: COH CYCLE Comparison: NATURAL CYCLE LH+7 vs hCG+7 LH+2 vs LH+7 Horcajadas et al. Mol Hum Reprod 2005
EFFECT OF CONTROLLED OVARIAN HYPERSTIMULATION IN IVF ON ENDOMETRIAL GENE EXPRESSION PROFILES
PCA ANALYSIS
LH+2 LH+7 IVF+7 Comparative results
LH7 / LH2 DNA fragments FC>2.0
Up 894 down 505
hCG+7/LH+7 Up 281 9 115
Down hCG+7/LH+7 227 0 277
LIST OF THE TEN MOST UP‐ AND DOWN‐REGULATED GENES AT hCG+7 versus LH+7
Fold Funtional Name Change Category hypothetical protein FLJ22390 38,87 Unknown Troponin C, show 30,89 Structural protein matrix metalloproteinase 26 16,96 Enzyme
major histocompatibility complex, class II, DO beta 12,23 Immune response differentially expressed in hematopoietic lineages 11,89 Inhibitor Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5 11,88 Inhibitor UP
Calpain 6 11,80 Glycoprotein Galanin 11,79 Neuropeptide Sorbitol dehydrogenase 11,55 Enzyme Branched chain keto acid dehydrogenase E1, beta polypeptide (maple syrup urine disease) 10,32 Enzyme
Fold Name Change Funcional Category Cartilage oligomeric matrix protein (pseudoachondroplasia, epiphyseal dysplasia 1, multiple) -58,55 Structural protein dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing protein 2) -31,25 Immune response
leukemia inhibitory factor (cholinergic differentiation factor) -23,02 Cytokine
Mucin 16 -13,61 Membrana protein insulin-like growth factor binding protein 1 -11,99 Regulatory protein glutathione peroxidase 3 (plasma) -11,81 Enzyme DOWN solute carrier family 15 (oligopeptide transporter), member 1 -10,62 Transporter progestagen-associated endometrial protein (placental protein 14, pregnancy-associated endometrial alpha-2-globulin, alpha uterine protein) -9,83 Secreted protein ATP-binding cassette, sub-family C (CFTR/MRP), member 3 -9,58 Transporter Calponin 1, basic, smooth muscle -9,26 Muscle protein
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VALIDATION STUDIES BY QUANTITATIVE‐PCR
Q-PCR endometrial RNA A 14
12
10
8 C: LH+2 A: LH+7 6 B: hCG+7
4 normalized RNA values normalized RNA 2
0 glycodeline Gluthatione Perox.-3 Transcobalamin Dipeptidylpeptidase 4
Q-PCR endometrial RNA A 60
50
40
C: LH+2 30 A: LH+7 B: hCG+7
20 normalized RNA values normalized RNA 10
0 Squalene_epoxidase secretoglobin1Dmember2 CXCL13
2
Not all protocols are created equal
Study of the endometrial development in oocyte donors treated with either high‐ or standard‐dose GnRH antagonist compared to treatment with a GnRH agonist or in natural cycles
EXPERIMENTAL DESIGN
Caucasian Fertile women with normal cycles Antagonist 18–35 years body mass index:19‐25 kg/m2 Agonist Biopsies
standddard‐dose hCG+2 12 ganirelix hCG+7
high‐dose 9 hCG+2 ganirelix hCG+7
hCG+2 10 Buserelin hCG+7
LH+2 12 Natural cycle LH+7
Simón et al. Hum Reprod 2005
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Aims of the study
‐ Number- of follicles on the day of hCG
‐ Serum hormone values at different times of the stimulated cycle
‐ Endometrial dating of biopsies by Noyes method
‐ Expression of Estradiol and Progesterone receptors (immunohst)
‐ Scanning electron microscopy
‐ Genomic studies by microarray
‐ Validation of genomic assays by Q‐PCR
Simón et al. (2005) Hum Reprod 12:3318-27
Clinical Design Natural cycle hCG +2 +7 LH +2 LH +7 biopsy biopsy biopsy biopsy rFSH
ganirelix (0.25 mg) Progesterone
biopsy biopsy biopsy biopsy rFSH
buserelin Progesterone
biopsy biopsy biopsy biopsy rFSH ganirelix (2.0 mg) Progesterone
Median # of follicles on the day of hCG All Subjects
18 ganirelix 0.25 mg ganirelix 2 mg 15 buserelin
12
9
6
3
0 >=11 mm >=15 mm >=17 mm
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LH and P rises LH ≥ 10 IU/L ; P ≥ 1 ng/mL prior during LH rise LH+P rise LH rise LH+P rise ganirelix 0.25 mg0000 ganirelix 2 mg1100 buserelin NA NA 4 4
Subject Day LH value P value ganirelix 2 mg 0027 6 31.0 1.2 buserelin 0008 hCG 10.0 1.14 0014 hCG 10.8 1.25 0030 hCG 11.0 2.47 0035 9 13.8 1.01
Median serum hormones Note: days 1‐6‐8: subjects with 8 days of recFSH
5IU/L LH 2800 pg/mL Estradiol 2400 4 2000 3 1600
2 1200 800 1 400 0 0 day 1 day 6 day 8 day hCG hCG day 1 day 6 day 8 day hCG hCG hCG +2 +7 hCG +2 +7
10 IU/L FSH 1000 ng/mL Progesterone 8 100 6 10 4
2 1
0 0,1 day 1 day 6 day 8 day hCG hCG day 1day 6day 8day hCG hCG hCG +2 +7 hCG +2 +7 0.25 mg ganirelix 2 mg ganirelix buserelin
Median endometrial thickness All Subjects 15
ganirelix 0.25 mg 12 ganirelix 2 mg
9 buserelin
6
3
0 hCG +2 hCG +7
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Endometrial dating Mean (SEM) ‐ according to Noyes criteria
10 POD ganirelix 0.25 mg
8 ganirelix 2 mg buserelin natural 6
4
2
0 hCG +2 hCG +7
POD = post ovulatory day
E‐receptor (α) Mean H‐scores (SEM) 4 H-score ganirelix 0.25 mg ganirelix 2 mg buserelin 3 natural
2
1
0 Luminal Luminal Glandular Glandular Stromal Stromal hCG+2 hCG+7 hCG+2 hCG+7 hCG+2 hCG+7
P‐receptor (A and B) Mean H‐scores (SEM) ganirelix 0.25 mg 4 H-score ganirelix 2 mg buserelin 3 natural
2
1
0 Luminal Luminal Glandular Glandular Stromal Stromal hCG+2 hCG+7 hCG+2 hCG+7 hCG+2 hCG+7
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Scanning electron microscopy: sample hCG+7ganirelix (2 mg group)
Antag (HD and SD) and NC 6% vs Buserelin 2.9% High‐dose ganirelix treatment can produce the required epithelial response.
SEM evaluation pinopods
Percentage of cells exhibiting any type of pinopods
12 % ganirelix 0.25 mg 10 ganirelix 2 mg buserelin 8 natural
6
4
2
0 hCG +2 hCG +7
Principal Component Analyses of 33 endometrial samples
Using 500 randomly selected genes.
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Comparison of the microarray (Panel A) and the Q‐PCR (Panel B) data for four selected genes
A
Negative values indicate absence of expression of this gene as determined by microarray
B
Comparison of the microarray studies on the gene expression pattern of the stimulated cycles compared to the natural cycles
WINDOW OF IMPLANATION GENES Normally up- Normally down- Number of Treatment and regulation sense regulated regulated genes (n = 894) (n = 504) Leuprolide (agonist) Up 281 9 115 First
STUDY Down 277 227 0 Ganirelix 0.25 mg/day (antagonist) Up 22 0 4 55% Down 69 46 0 Ganirelix 2 mg/day (antagonist) Up 88 0 7 21% Down 24 15 1 Buserelin long protocol (agonist) Second STUDY Up 22 3 4 70% Down 100 76 2
Simón et al. Hum Reprod 2005
3
Endometrial changes through WOI
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GENE EXPRESSION PROFILING DURING THE WINDOW OF IMPLANTATION IN NATURAL versus STIMULATED CYCLES
EXPERIMENTAL DESIGN
FIVE ENDOMETRIAL 50 WOMEN AFFIMETRIX BYOPSIES AT EACH TIME POINT HG‐133A >22,000 genes
LH Day 1 2 3 4 5 6 7 8 9
hCG Day 1 2 3 4 5 6 7 8 9
PRE‐RECEPTIVE RECEPTIVE
Caucasian Fertile women with normal cycles 23–39 years HISTOLOGICAL CONTROL: body mass index:19‐25 kg/m2 BLIND DATING BY NOYES CRITERIA
PCA OF THE ENDOMETRIAL SAMPLES FROM LH+1 TO LH+9
LH+1 LH+3 LH+5
LH+7 LH+9
Day LH/hCG+1
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Day LH/hCG+3
Day LH/hCG+5
Day LH/hCG+7
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Day LH/hCG+9
BIOLOGICAL PROCESSES OVER‐EXPRESSED DAILY in NATURAL CYCLES
MOST DIFFERENTIATED FUNCTIONALITIES IN RECEPTIVE ENDOMETRIUM IN NATURAL versus STIMULATED CYCLES
NATURAL CYCLE STIMULATED CYCLE GO biological process GO biological process GO terms over‐expressed: GO termsterms over‐‐expressed:
1. antigen processing, endogenous antigen via MHC class I 1.1. mitotic checkpointcheckpoint 2. antigen presentation, endogenous antigen 2.2. antigenantigen processing, endogenousendogenous antigenantigen viavia MHC classclass II 3. complement activation, classical pathway 3.3. spindlespindle organization organization and and biogenesis biogenesis 4. response to drug 44.. antigenantigen presentation,, endogenousendogenous antigenantigen 5. regulation of DNA metabolism 5.5. mitotic sistersister chromatid chromatid segregation segregation 6. mitosis 6. regulationregulation of DNA metabolism 7. DNA replication 7.7. microtubule‐based‐based movement movement 8. small GTPase mediated signal transduction 8. cellcell division 9. cell division 9. phosphoinositide‐‐mediatedmediated signaling signaling 10. negative regulation of progression through cell cycle 10.10. DNA‐‐dependentdependent DNADNA replicationreplication 11. skeletal development 11.11. regulationregulation of of development development 12. DNA repair 12.12. nucleotide metabolism metabolism 13. amino acid metabolism 13.13. DNA repairrepair 14. cytoskeleton 14.14. cellcell proliferationproliferation 15.15. regulationregulation of of signal signal transduction transduction 16.16. carboxyliccarboxylic acid acid metabolism metabolism 17.17. positive regulation regulation of of cellular cellular process process 18.18. negative regulationregulation of of cellular cellular physiological physiological process process
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Clinical applications Diagnosis Gene targeting Protein analysis Diagnostic method must be nonnon--invasiveinvasive andhifbd at the time of embryo trans fer
Endometrial fluid
CONCLUSIONS
- Specific gene pattern can be identified daily in the luteal phase by gene expression analysis. - Biological pathways regulating the shift from pre to receptive endometrium have been identified. - There are WOI ggpyenes, pathways and biolog ical processes disregulated in the receptive endometrium in stimulated versus natural cyclescycles.. - The antagonist regimens resembled more closely
natural cycles when compared to agonist regimen
Future strategies
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C. Simón Fundación IVI (FIVI) A. Pellicer Instituto Universitario IVI (IUIVI) Universidad de Valencia
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