Transcript and 2

New biomarkers in ovulation induction.
INTERNATIONAL SYMPOSIUM ON REPRODUCTIVE MEDICINE1
PCOS,OVULATION INDUCTION AND FERTILITY PRESERVATION :NEW
TRENDS 2010 JUNE 4-6,2010 MARMARA HOTEL INSTABUL,TURKEY.
Prof. DIMITRIS LOUTRADIS
First Department of Obstetrics and Gynaecology
University of Athens, Medical School
Biomarkes in ovarian stimulation
Background 1
1. Controlled ovarian stimulation (COS) using gonadotropins is a
widely used treatment in most assisted reproduction techniques
2. Several markers as E2 or U/S have been studied and proposed to
predict COS outcome
3. Day 3 serum FSH seems to be the best predictive marker for
ovarian
function,
even
though
a
significant
intra-individual
variation from cycle to cycle and potential differences in the
bioactivity of FSH measured should be taken into consideration
Biomarkers in ovarian stimulation
Background 2
4.
Various parameters which could be used to estimate patient’s
ovarian reserve are easily measurable, minimally invasive and
inexpensive, such as inhibin and AMH
5.
Genetic differences might also be an important factor determining
the ovarian response to gonadotropin stimulation. However, other
factors proposed to affect ovarian response to FSH are the
distribution of FSH isoforms and the single nucleotide
polymorphisms
The purpose is to summarize the currently available evidence,
which indeed supports the concept that
different genotypes in terms of polymorphisms can decisively influence
the ovarian response to FSH stimulation
Biomarkers in Ovarian Stimulation
Single Nucleotide Polymorphisms (SNPs) of
FSH
Single Nucleotide Polymorphisms (SNPs) of
ESR1 and ESR 2
A model of a polygenic analysis of three
different loci
(ESR1,ESR2,and FSHR).
FSH receptor gene polymorphisms have a role for different
ovarian response to stimulation in patients entering
IVF/ICSI-ET programs
We examined
79 subfertile women
35 “poor responders” (PR)
44 “ovarian dysfunction” (OD)
46 normo-ovulatory women (GR)
To investigate the associations between
polymorphisms and hormonal profiles
the
receptor
 We determined the prevalence of Ser680Asn polymorphisms
of the FSHR gene
 We correlated the variants with the clinical characteristics of
assisted reproductive technologies (ART) in “poor responders”
(PR), “good responders” (GR) and “ovarian dysfunction” (OD)
Greek patients
Loutradis D et al, Assist Reprod Genet, 2006
520 bp 
413 bp 
RFLP analysis of the Asn680Ser FSHR variant. Following BsrI digestion
of the PCR products, 2.5% agarose gel electrophoresis revealed the
patterns presented. Lanes 1-4 present two bands, 520 and 413 bp,
corresponding to the Asn/Ser variant, Lanes 4-7 present a 413 bp
band corresponding to the Ser/Ser variant, and Lanes 8-11 present a
520 bp band corresponding to the Asn/Asn variant.
M represents a 100-bp molecular size marker
Frequency distribution of different allelic variants in
125 women, according to ovarian function
Data are number of subjects
Asn/Ser
Ser/Ser
Asn/Asn
Total
Ovarian
Dysfunction
10 (22.7%)
20 (45.4%)
14 (31.8%)
44
Poor
Responders
15 (42.8%)
10 (28.5%)
10 (28.5%)
35
Good
responders
24 (52.1%)
12 (26.0%)
10 (21.7%)
46
Frequency distribution of FSHR genotypes at position 680
in different study groups
Study
Asn/Asn
(%)
Asn/Ser
(%)
Ser/Ser
(%)
Subjects
(type)
Subjects
(No)
Origin
Gender
Conway, 1999
25
52
23
PCOS
93
UK
Females
Perez Mayorga,
2000
29
45
26
IVF patients
161
Germany
Females
Sudo, 2002
41
47
12
Ovulatory and anovulatory
522
Japan
Females
De Castro, 2003
31
50
19
IVF patients
102
Spain
Females
Laven, 2003
16
44
40
Normogonadotropic
anovulatry infertile
148
Germany
Females
Sunblad, 2004
32
45
23
Ovulatory
44
Argentine
Females
Falconer, 2005
35
24
41
Infertile women
68
Sweden
Females
Greb, 2005
34
49
18
Ovulatory
125
Germany
Females
Jun, 2006
42
46
12
IVF patients
263
Korean
Females
Klinkert, 2006
38
45
17
IVF patients
105
Dutch
Females
Loutradis, 2006
27
39
34
IVF patients
125
Greece
Females
Biological and clinical characteristics of 97 patients that
underwent IVF/ICSI (grouped according to FSRH polymorphism)
Asn/Ser
Ser/Ser
Asn/Asn
29 ± 3
31 ± 3
33 ± 3
FSH (mIU/mL)
7.4 ± 3.4 b
9.0 ± 3.5 b
8.5 ± 2.1 b
LH (mIU/mL)
4.9 ± 2.2 a
7.9 ± 1.5 a
6.5 ± 4.0 a
PRL (ng/mL)
10.1 ± 1.9 a
Age (y)
14.8 ± 3.9
a
12.6 ± 2.8 a
Estrogen levels on HCG
admin (pg/mL)
2733 ± 1792 c
1583 ± 1181 c
1273 ± 602 c
Total rFSH dose (IU)
3070 ± 1494
3512 ± 1499
3105 ± 992
11 ± 0
11 ± 1
11 ± 0
Stimulation days
Number of follicles
8.7 ± 2.8 a
6.6 ± 2.0
a
5.7 ± 2.9
Number of oocytes
7.7 ± 2.8 a
6.0 ± 2.5
a
5.3 ± 3.3 a
Mature oocytes (%)
69.5 ± 2.4
67.5 ± 12.6
90.9 ± 10.8
Fertilization Rate (%)
98.0 ± 5.4
98.0 ± 2.5
94.8 ± 6.1
3/44
3/30
2/23
Pregnancies (n)
Kruskal Wallis test for the analysis of variance. Superscripts indicate: a: p<0.001, b: p<0.05, c: p<0.01
a
Biological and clinical characteristics of 97 patients that
underwent IVF/ICSI, grouped according to ovarian response
Ovarian Dysfunction (OD)
Poor responders (PR)
Good responders (GR)
Asn/Ser
Ser/Ser
Asn/Asn
Asn/Ser
Ser/Ser
Asn/Asn
Asn/Ser
Ser/Ser
Asn/Asn
Age (y)
27 ± 3
33 ± 2
27 ± 4
31 ± 3
32 ± 2
34 ± 2
28.3 ± 2.6
28.8 ± 1.8
32.9 ±
2.4
FSH (mIU/ mL)
15.5 ±
5.2
14.2 ±
1.9
13.6 ± 1.2
5.5 ± 0.7
8.0 ±
0.5a
8.1 ± 0.5
6.9 ± 0.8
6.4 ± 1.4
7.5 ± 0.9
10.2 ±
0.9
8.5 ± 2.0
10.1 ± 1.3
3.8 ± 0.6
7.8 ± 1.3
2.1 ± 0.5
4.6 ± 1.3 a
7.4 ± 1.1 a
9.8 ± 1.6
a
a
a
E2 (pg/mL)
2806
± 304a
746
± 84 a
1344
± 16 a
595
± 164
557
± 149
728
± 294
4055
± 1080 a
2995
± 130 a
1796
± 406 a
Gonadotropin
Dose (IU)
3522
± 548
4483
± 244b
3439
± 479
4873
± 764
4720
± 1009
3787
± 968c
1849
± 218 c
1858
± 246 c
2322
± 427 c
9.0 ± 0.7
5.0 ± 0.7
4.0 ± 0.0 b
5 ± 0.8 a
5 ± 0.8 a
3 ± 0.6 a
11 ± 0.8 a
9 ± 0.7 a
9 ± 0.6 a
b
b
8.0 ± 0.7
4.0 ± 0.7
4.0 ± 0.0 b
4 ± 0.6 a
4 ± 0.9 a
2 ± 0.5 a
10 ± 0.6 a
9 ± 0.7 a
9 ± 0.7 a
b
b
0/5
1/8
0/3
0/15
0/10
1/10
3/24
2/12
1/10
LH (mIU/ mL)
Follicles (#)
Oocytes (#)
Pregnancies
a
a
a
Kruskal Wallis test for the analysis of variance. Superscripts indicate: a: p<0.001, b: p<0.05, c: p<0.01
CONCLUSIONS
 This preliminary study shows that there is a tendency for
OD patients to present the Ser/Ser allelic variant
(44.5%), whereas GR patients present more often the
Asn/Ser allelic variant (p<0.05). This latter genotype
gives more follicles and oocytes, while the serum E2 was
higher either in the GR or the OD patients
 The FSH level on day 3 of menstrual cycle was slightly
elevated in the Ser/Ser and Asn/Asn genotypes
Estrogen Receptor 1 and 2 gene polymorphisms have a
role for different ovarian response to stimulation in
patients entering IVF/ICSI-ET programs (1)
• Purpose :
To examine the frequency distribution of the Pvu II and
Rsa I polymorphisms of the estrogen receptor 1
(ESR1) and estrogen receptor 2 (ESR2) genes
respectively, in Greek patients undergoing IVF.
• Methods :
The hormonal profiles and treatment of all patients
were analyzed and both ESR1 and ESR2
polymorphisms were examined using real-time PCR.
Estrogen Receptor 1 and 2 gene polymorphisms have a
role for different ovarian response to stimulation in
patients entering IVF/ICSI-ET programs
 The ESR1 intron 1 contains two single-nucleotide
polymorphisms (SNP’s) at the PvuII (T/C) and XbaI
(A/G) restriction sites, and the first locus is
reportedly associated with susceptibility to
endometriosis and COH/Pregnancy outcome of IVF.
 The ESR2 RsaI (G/A) locus is linked to ovulatory
dysfunction of unknown etiology.
REAL-TIME PCR FOR PvuII POLYMORPHISM
OF ESR1 GENE
The probe is designed to recognize the polymorphism (C), on the highest temperature, while the
absence of the polymorphism (T) appears on a lower temperature.
The green curves represent the homozygotes for the polymorphism (CC).
The blue curves represent the heterozygotes for the polymorphism (CT).
The red curves represent the homozygotes without the polumorfism (TT).
Oligonucleotide Primers and Probes Used for
RT-PCR Assays for ESR2 gene
REAL-TIME PCR FOR RsaI POLYMORPHISM
OF ESR2 GENE
The probe is designed to recognize the polymorphism (A), on the highest temperature,
while the absence of the polymorphism (G) appears on a lower temperature.
The red curves represent the heterozygotes for the polymorphism (GA).
The blue curves represent the homozygotes without the polumorfism (GG).
CONCLUSIONS
• This study shows that CC allelic variant (ESR1) is associated with
similar pregnancy rates compared to CT,TT allelic variant (25% vs
22% ,21%). Moreover, the former genotype demonstrates lower E2
serum levels on the day of hCG administration.
• The GA allelic variant (ESR2)is associated with higher pregnancy
rate compared to GG(24.6 vs 14.3%).
As it emerges from the data of ESR1 and ESR 2 genes in the COS
outcome, the weight of these factors are probably low.
Rationale
A model of a polygenic analysis of three different loci
(ESR1,ESR2,and FSHR)
Genetic studies contribute towards our understanding of disease
pathogenesis and hold the promise of improving our ability to individualize
treatment for patients.
•
Could be evidence that a polygenic model, including the well studied
ESR1,ESR2 and FSHR genes involve in the COS and pregnancy
outcome.
•
The aim of our study is to analyze three different loci( polymorphisms in
ESR1,ESR2,and FSHR gene), in order to explore their involvement in ART
outcome in Greek patients.
In order to investigate the susceptibility of the genetic
factors in ovarian response we development a model of
a polygenic analysis of three different loci
(ESR1,ESR2,and FSHR).
ESR2 locus was excluded from the study because the
distribution of genotype was unequal (GG 101,GA 7
only one woman with AA genotype).
Genetic profile ESR1/FSHR genes in association with
number of oocytes and pregnancy rates, in 109 women
undergoing IVF/ICSI
Mean
oocyte
numbe
r
±SD
Number
of women
carrying ≤
3 oocytes
(Ν=21)
Number
of women
carrying >
4 oocytes
(Ν=88)
%genotype
%genotype
with
≤3 oocytes
with
>4 oocytes
Number
of
women
with
(+)
pregnanc
y (Ν=26)
CC/SS
8±4
0
4
0,0%
100,0%
2
CC/SA
7±2
1
12
7,7%
92,3%
CC/AA
5±2
3
4
42,8%
TC/SS
7±3
2
8
TC/SA
6±3
7
TC/AA
7±3
TT/SS
Number
of
women
with
(-)
pregnanc
y (Ν=83)
(Ν=10
9)
%genotyp
e
with
(+)
pregnanc
y
%genotype
with
(-)
pregnancy
2
4
50%
50%
4
9
13
30,8%
69,0%
57,1%
0
7
7
0%
100%
20,0%
80,0%
2
8
10
20%
80%
26
21,2%
78,8%
10
23
33
30%
70%
1
14
6,7%
93,3%
2
13
15
13%
87%
7±3
1
7
12,5%
87,5%
2
6
8
25%
75%
TT/SA
5±4
4
8
33,3%
66,7%
2
10
12
17%
83%
TT/AA
7±4
2
5
28,6%
71,4%
2
5
7
28,5%
71,0%
TOTAL
6±3
21
88
19,3%
80,7%
26
83
109
23,9%
76,1%
total
ESR/FSH
Genotype distribution in poor responder of ESR1 and FSHR
loci in combination, in the population that presented positive
clinical pregnancy and the population that did not achieve
clinical pregnancy. Note: SS=Ser/Ser, SA=Ser/Asn,
AA=Asn/Asn.
(+)
PREGNANCY
(-)
PREGNANCY
TOTAL
POOR
ESR/FSH
CC/SS
%genotype with
(+) pregnancy
%genotype with
(-) pregnancy
not present in poor responders
CC/SA
IIII
IIII
0%
100%
CC/AA
IIIII
IIIII
0%
100%
CT/SS
IIII
IIII
0%
100%
IIIIIIIIII
IIIIIIIIIIIII
23,07%
76,92%
III
III
0%
100%
II
III
33%
66,60%
TT/SA
IIIIII
IIIIII
0%
100%
TT/AA
III
III
0%
100%
37
41
9,75%
90,24%
CT/SA
III
CT/AA
TT/SS
TOTAL
I
4
Genotype distribution of ESR1 and FSHR loci in combination,
in the population in good responder that presented positive
clinical pregnancy and the population that did not achieve
clinical pregnancy. Note: SS=Ser/Ser, SA=Ser/Asn,
AA=Asn/Asn.
(+)
PREGNANCY
(-)
PREGNANCY
TOTAL
GOOD
ESR/FSH
%genotype with
(+) pregnancy
%genotype with
(-) pregnancy
CC/SS
I
Ι
ΙΙ
50%
50%
CC/SA
I
ΙΙ
ΙΙΙ
33,30%
66,60%
CC/AA
not present in good responders
Ι
100%
ΙΙΙIII
ΙΙΙΙΙΙΙΙΙΙ
40%
60%
I
ΙΙΙΙΙΙI
ΙΙΙΙΙΙΙΙ
13%
87%
TT/SS
I
ΙΙ
ΙΙΙ
33,30%
66,60%
TT/SA
IΙ
ΙΙI
ΙΙΙΙΙ
40%
60%
TT/AA
I
Ι
100%
TOTAL
12
33
36,36%
CT/SS
I
CT/SA
IIII
CT/AA
21
63,63%
Genotype distribution of ESR1 and FSHR loci in combination,
in the population that presented positive clinical pregnancy
and the population that did not achieve clinical pregnancy.
Note: SS=Ser/Ser, SA=Ser/Asn, AA=Asn/Asn.
(+)
PREGNANCY
(-)
PREGNANCY
TOTAL
TOTAL
ESR/FSH
%genotype with
(+) pregnancy
%genotype with
(-) pregnancy
CC/SS
2
2
4
50%
50%
CC/SA
4
9
13
31%
69%
CC/AA
0
7
7
0%
100%
CT/SS
2
8
10
20%
80%
CT/SA
10
23
33
30,00%
70,00%
CT/AA
2
13
15
13%
87%
TT/SS
2
6
8
25%
75,00%
TT/SA
2
10
12
17%
83%
TT/AA
2
5
7
29%
71%
TOTAL
26
83
109
24%
76%
 FSHR: The Asn/Ser allelic variant genotype gives more
follicles and oocytes, while the serum E2 was higher either
in the GR or the OD(FSH elevate) patients.
 ESR1:CC allelic variant (ESR1) is associated with similar
pregnancy rates compared to CT,TT allelic variant .
 Polygenic model, ESR1,and FSHR:
- Good responder CT/SA higher pregnancy rate.(10/26)
- Poor responder CT/SA higher pregnancy rate. (3/4)
- Poor prognosis CC/AA :Women with CC/AA combination have 1,5-2,4
times more risk to be poor responders in comparison with women that don’t carry this
combination.
Conclusions
 Polygenic model, ESR1, and FSHR:
- Good responder CT/SA higher pregnancy rate.(10/26)
- Poor responder CT/SA higher pregnancy rate. (3/4)
-Poor prognosis in general population CC/AA.
On the basis of our
data we conclude , that
there is evidence that a
polygenic trait of ESR1, and FSHR demonstrate convincingly that SNPs
genes can be used as markers to predict ovarian response
patients.
in ART
Rationale for hyperadrogeanic
• The aim of our study is to analyze two different loci
( polymorphisms in ESR1and FSHR gene), in order to explore
their involvement in ART outcome in Greek hyperadrogeanic
patients.
PCOS
Like
(n=20)
Good
Responders
(n=28)
Age
33±5
33±5
Number of attempts
1±1
1±0
FSH mIU/ml
6,3±1,9
6,5±1,5
LH mIU/ml
5,2±4,2
4,7±3,2
LH/FSH
0,8±0,3
0,6±0,6
PRL ng/ml
10,9±5,3
15,3±7,3
Δ4 Andr.ng/ml
6,6±3,1
1,8±0,8
DHEAS μg/ml
6,2±1,7
2,6±0,9
0,9±1,1
0,5±0,2
Stimulation days
10±2
10±1
Total FSH dose
2750±1487
2750±1125
Peak E2
2400,0±1126,0
1411,0±1074,9
Number of follicles
8±3
10±3
Number of oocytes
7±3
8±3
Number of pregnancies
4
11
Pregnancy rate
20 %
39,3%
T.Testosterone ng/ml
Distribution of genotype
PCOS-Like
(n=20)
Good Responders
(n=28)
ESR1-CC(%)
18.8
14.3
ESR1-CT(%)
50
57.1
ESR1-TT(%)
31.3
28.6
ESR2-GG(%)
93.8
92.9
ESR2-GA(%)
6.3
7.1
ESR2-AA(%)
0
0
FSHR-Ser/Ser(%)
37.5
17.9
FSHR-Ser/Asn(%)
37.5
60.7
FSHR-Asn/Asn(%)
25
21.4
PCOS-Like hyperandrogenemia
Good Responders = ≥5 oocytes and/or FSH≤10 and/or peak E2≥1500
Polygenic study of ESR1 FSHR in
Good responder .
The genetic profile of the women that had clinical pregnancy in
each group ,not considering each locus separately ,but in
combination too good responders.
ESR1/FSHR
Pregnancies (Ν=11)
TOTAL
(Ν=28)
Pregnancies
CC/SS
I
Ι
100%
CC/SA
I
ΙΙΙ
33,30%
CC/AA
No Pregnancies
66,60%
Not present in good responders
CT/SS
I
Ι
100%
CT/SA
IIII
ΙΙΙΙΙΙΙΙΙΙ
30%
70%
CT/AA
I
ΙΙΙΙΙ
20%
80%
TT/SS
I
ΙΙΙ
33,30%
66,60%
TT/SA
I
ΙΙΙΙ
25%
75%
TT/AA
I
Ι
100%
Polygenic study of ESR1 FSHR in PCOS like
The genetic profile of the women that had clinical pregnancy in each group
,not considering each locus separately ,but in combination too PCOS like
ESR/FSH
Pregnancies
(Ν=4)
No Pregnancies
(Ν=16)
Total (Ν=20)
Pregnancies
No Pregnancies
CC/SS
I
I
0%
100%
CC/SA
I
I
0%
100%
CC/AA
I
I
0%
100%
III
IIIIII
50%
50%
CT/SA
II
II
0,00%
100,00%
CT/AA
III
III
0%
100%
TT/SS
II
II
0%
100
III
IIII
33.3%
95%
CT/SS
TT/SA
TT/AA
III
I
Absent of this genotype
PCOS like
The present study analyze a polygenic model , including the studied of ESR1,and FSHR.
Conclusions
 Polygenic model, ESR1, and FSHR:
- Good responder CT/SA higher pregnancy rate.(10/26)
- Poor responder CT/SA higher pregnancy rate. (3/4)
-Poor prognosis no pregnancy CC/AA.
- Hyperadrogeanic patients CT/SS (3/4).
On the basis of our
data we conclude , that
there is evidence that a
polygenic trait of ESR1,and FSHR demonstrate convincingly that SNPs
genes can be used as markers to predict ovarian response
hyperadrogeanic patients.
in