Transcript Slayt 1

CURRENT APPROACHES IN SEVERE
MALE INFERTILITY
Prof. Semra Kahraman M.D.
Bio.Çağrı Beyazyürek, Zafer Candan, Sevil Ünal,
Semra Mılık, Assoc. Prof. Semih Özkan M.D.
Istanbul Memorial Hospital,
ART and Reproductive Genetics Center
Istanbul, Turkey
SEVERE MALE INFERTILITY
Genetic factors
Preimplantation genetic diagnosis
Surgical sperm recovery techniques
Sperm DNA fragmentation
Derivation of gamete cells from
embryonic stem cells
Development of artificial gametes
Male Infertility and Genetics
Structural Chromosomal Abnormalities
Translocations (Robertsonian, Reciprocal, Cryptic)
Duplication, inversion, insertion
Numerical Chromosomal Abnormalities
Gain or loss of entire chromosomes
Micro or macrodeletions on Y chromosome
Gene defects
GENETIC FACTORS
İMH ART and Reproductive Genetics Center
Karyotype analysis of 1935 infertile men with severe
oligozoospermia or azoospermia
1214 cases: Non-obstructive azoospermia (NOA)
721 cases: Severe oligoasthenoteratozoospermia
(OAT) (total sperm concentration in the whole
ejaculate< below 5 million).
(1364 cases: Y-microdeletion analysis)
RESULT
In cases with severe male
factor infertility the incidence
of having an abnormality in
at least one test (karyotype
analysis or Y-microdeletions)
is 16,6% in our study.
Distribution of normal and abnormal
karyotypes in infertile men
İMH ART and Reproductive Genetics Center
NOA
KARYOTYPE
OAT
%
n=1214 (%62)
TOTAL
%
n=721(37.2)
%
n=1935
Normal
973
80.15
645
89.46
1618
83.6
Klinefelter’s
133
10.95
5
0.69
138
7.13
Mosaic Klinefelter
10
0.82
6
0.83
16
0.83
Other sex chromosome mosaicism
(45,X/46,XY)
13
1.07
2
0.28
15
0.78
0.90
0
0.00
11
0.57
10
0.82
1
0.14
11
0.57
Reciprocal Translocation
12
0.98
13
1.8
25
1.29
Robertsonian Translocation
1
0.08
12
1.66
13
0.67
Inversions
4
0.33
1
0.14
5
0.26
Markers
1
0.08
1
0.14
2
0.10
Other Abnormalities
4
0.33
1
0.14
5
0.26
Total Abnormalities
199
16.4
42
5.83
241
12.45
42
3.46
34
4.72
76
3.93
45,X male 45,X,tas(Y;2)(p11.3;qter)
46,XX males
45,X male (1)
46,XX males (10)
Other Sex Chromosomal Abnormalities
(isoXq, idic Y and 47,XYY)
Normal variable features/
Heterochromatin Polymorphisms

Klinefelter Syndrome was the most frequently detected abnormality (57.3% of detected abnormalities)
Chromosomal Variants
Heterochromatin polymorphism is
considered as a variant of a normal
karyotype, but is more frequent in
infertile men.
More attention must be directed to
infertile men with heterochromatin
polymorphism
Y chromosome microdeletion results in
severe male infertility
NOA (%)
n=1041
OAT (%)
n=323
TOTAL (%)
n=1364
NORMAL
942
317
1259
DELETED
99 (9.5)
6 (1.85)
105 (7.7)
AZFa
4
0
4 (3.8)
AZFb
2
2
4 (3.8)
AZFc
48
4
52 (49.5)
AZFbc
28
0
28 (26.6)
AZFabc
17
0
17 (16.3)
AZFc deletion 82%
Y-microdeletion rate in several studies
Country
Patient Number
%
Our study
1364
7.7
India
83
9.6
Spain
50
16.0
Japon
63
15.8
USA, Australia
50
20.0
USA
108
7.0
France
53
9.4
Finland
201
9.0
China
101
11.0
Slovenia
226
4.4
Taiwan
94
11.7
New Zealand
65
7.7
Patients having both karyotype
abnormality and Y-micro-deletion
Karyotype Abnormalities
Regions deleted on Y-chromosome
N%
47,XXY
Partial AZFa, AZFb and AZFc
1
Y complete
7
46,XX male SRY+ (4)
46,XX male SRY- (2)
45,X male SRY+ (1)
del complete b, c (2)
46,X,del(Y)(q11.2)
del complete b, del partial c (1)
3
del complete b, c, sy 160 (1)
idic Y(p)
del Y complete (1)
3
del partial a, del complete b, c (1)
46,XY(92%)/45,X(8%)
del complete b, c
45,X(22%),46,XY(78%)
del complete b, c
45,X(43%),46,XY(57%)
del complete b, c
mos45,X[9]/46,XY[40]/47,XYY[1]
del complete b, c, sy 160
mos45,X(50%)/46,Xi(Y)(p11.1)(50%)
del Y complete, sry+
mos45,X(24%)/46,X,idic(Yp)(72%)
del complete b, c
mos45,X(52%)46,X,idicY(p)(48%)
del complete b, c, sy 160
45,X(30%))/46,XidicYp(70%)
del complete b, c, sy 160
TOTAL
8
22 (9.1.%)
Why Y-chromosome Micro-deletion
analysis before TESE procedure?
The deletion types of AZF a,b and c loci on Yq11, are the potential
prognostic factors in patients planned to undergo TESE/mic-TESE
procedures.
cAZF c = Approximately 50% of the cases, mature spermatozoa
cAZF b = Nearly impossible to find mature spermatozoa
cAZF b+c and c AZF a+b+c = Total absence of testicular spermatozoa
Y-microdeletion and Mic-TESE
Sperm
recovery
Result
Mic-TESE
30 patients / 35
cycles
partial a (2)
partial c (25)
complete bc (2)
partial abc (1)
14 patients/
16 mic-TESE
SRR:(48.4%)
9 pregnancies
PR:(64.2/patient)
1 unembryonic/
1 clinical abort
8 babies
Ejaculate
4
partial c (3)
partial b (1)
+
2 pregnancy,
1 ET cancellation
ELSI
1
complete c
+
No pregnancy
TESA
1
Complete c
+
No pregnancy
Retreival type
n=41
Deletion
Sperm retreival rates of TESE patients
with same micro-deletions
partial c (n=15)
(152,157,158,254,255)
Same deleted regions
(variable phenotypic
expression)
Sperm found (n=6)
SRR: 40%
No sperm (n=9)
SRR:60%
Conclusions
The high frequencies of cytogenetic
abnormalities and Y micro deletions
definitely suggest the need for genetic
screening and counselling in severe male
factor cases.
Karyotyping should be regarded as a
mandatory part of the pre-treatment
screening process for all men referred for
ICSI.
Y-deletion analysis test is neccessary before
deciding TESE procedure.
Preimplantation Genetic
Diagnosis for Male Infertility
(Aneuploidy)
Golden Standarts
Biopsy: Single Blastomere
Fixation: Hypotonic+Fixative
method
Hybridization: FISH at least 9
chromosomes:
13,15,16,17,18,21,22,X,Y
Analysis at least 2
rounds+recheck
Transfer on day 4
Indications and combined factors in
1000 PGD cycles
MF
13.2%
RIF
(43.5%)
3.8%
10.2%
RPL
12%
1.1% %9.2 5.2%
5.8%
9.3%
15.9%
AMA
14%
Male Factor and PGD (n=433)
29.6%
RIF
n=129
8.5%
27.2%
2.5% 20.6% 11.6%
RPL
AMA
Sperm Source
Source
n
Ejaculate
308
(71.1%)
mic-TESE/TESA
125
(28.9%)
Total
433
Number of cycles
160
146
140
120
34%
100
76
80
66
*
60
55
20.4%
40
33
20
12
0
NOA
VA
SOAT
OAT
AT
IT
PGD for Only Male Factor
Infertility
PGD
Without PGD
129
263
Female age (years)
30.5 ±3.5
28.3±3.9
ns
Mean MII oocytes
13.2 ±5.1
12.9±5.5
ns
Fertilization rate (%)
73.5
70.3
ns
Mean embryos
transfered
2.4
3.1
<0.05
Pregnancy rate (%)
58%
48.7%
Abortion rate
7%
19.7%
28.6%
13.8%
No. of cycles
(%)
Implantation rate (%)
P-value
ns
<0.05
<0.05
Istanbul Memorial Hospital ART and Genetics Center
Effect of additional factors on the outcome of PGD cycles for male infertility
80
70
72
60
64
60
58
MF
%
50
38
40
MF+AMA
26
30
20
10
0
abnormal embryos
clinical pregnancy rate
MF+AMA+RIF
Distributing of Chromosomal Abnormality
n
Cycles
Mean maternal age, (min-max)
Embryos diagnosed
Normal
Abnormal
Aneuploid
Monosomy
Trisomy
Complex Aneuploidy
Others
Haploidy/polyploidy
%
433
33.5 (20-47)
40
60
77.8
34.6
30.4
30.8
4.2
22.2
PGD RESULTS
Ejaculated vs Testicular Sperm
(Maternal ages below 38)
Ejaculate
(SOAT)
Testicular
Sperm (NOA)
Cycles initiated
50
45
Embryos diagnosed as
abnormal, (%)
56
62
Clinical pregnancy, %
64*
44.8
Conclusion
The results of our study shows that the rate
of aneuploidy is as high as 60% in patients
with severe male factor infertility
Aneuploidy rate increases with the presence
of other combined contributing factors such
as AMA, RIF and RSA
PR dramatically decreases as more
indications are combined with male infertility.
Preimplantation Genetic
Diagnosis (PGD)
Translocations
Probes
PGD-translocations n=104
Locus Spesific (LSI) (200-500kb)
Centromeric (CEP) (alpha satellite p11-q11)
Telomeric (Tel) (60-170kb)
Whole Chromosome Painting Probes (WCP)
PGD for Male translocation Carriers
n=104
Robertsonian
Reciprocal
Patients
29
48
Cycles
37
67
Biopsied embryos
215
418
With conclusive
results
186
366
104 (55.9%)
291 (79.5%)
Normal
82 (44.1%)
75 (20.5%)
Mean maternal
age
32.7 (23-45)
32.5 (20-47)
ET cycles
31 (83.8%)
48 (%71.6)
10/31 (32.2%)
11/48 (22.9%)
Abnormal
PR/ET cycles
Sperm FISH Aneuploidy screening for translocation
cases. Is There Any Interchromosomal Effect?
(n=5)
Disomy rates
Karyotype
13
0
18
21
(27.4) 0.2
XY
1
1
46,XY,rcpt(9;18)(p13.3;q21.3)
2
46,XY,rcpt(3;17)(p13;q23)
0.6
0
0
1
3
46,XY,rcpt(11;15)(p12;p13)
1
0
0
1
4
45,XY robt(13;14)(q10;q10)
(9.2)
0.1
4
0.2
5
45,XY robt(13;14)(q10;q10)
(9.5)
0.2
0.4
0.3
Conclusion
. PGD should be a viable alternative for
translocation carriers to reduce miscarriages
Spermatozoa FISH testing may be used as an
indicator of aneuploidy and segregation rate
in gametes in translocation carriers and can
give good approximation of success in a
PGD cycle
Aneuploidy screening should be a part of
genetic evaluation if female partner is >38
years
Micro-Dissection
TESE Procedures in
Azoospermic Patients
SPERM RECOVERY in NOA
PATIENTS (n=1023)
İMH Andrology Unit
Micro-TESE: (NOA)
729
TESA
294
Sperm recovery rate in NOA Cases
375 / 729 = % 51.4
Results of Mic-TESE
Patients with first Mic-TESE trials :
– No of patients : 591
– Sperm recovery: 354
– No sperm
: 237
Sperm recovery rate: 354 / 591 = (%60)
Patients with previously conventional TESE trial
with no sperm recovery :
– No of patients: 60
– Sperm recovery: 32
– No sperm: 28
Sperm recovery rate: 32 / 60 = %53.3
Secondary MicroTESE success
rates in patients
Secondary MicroTESE n=242
Sperm recovered: 178
No Sperm
Success rate
:
64
:178/242=73.5%
TESE/mic-TESE
TESE 36% vs Mic-TESE (Schlegel) 68%
Klinefelter’s Syndrome
No of cases: 65 Micro-TESE cases
Sperm found: 26
No sperm
: 39
Success rate 26/65 = % 40
Surgical sperm recovery rate
according to hystopathology
Sertoli cell only (Germ cell aplasia)
No of cases: 56
Sperm found: 20
No sperm
: 36
Success rate: 20/56 = % 35
Surgical sperm recovery rate
according to hystopathology
Maturation arrest
Total number of cases : 37
Sperm found
: 19
No sperm found
: 18
Success rate
: 19 / 37 = 51%
Conclusion
Mic-TESE is one of the most recent and
important advance in surgical sperm
retrieval techniques. It’s success in
sperm retrieval and less complication
rates made this technique is the most
preferable procedure in sperm retrieval.
Conclusion
Mic-TESE procedure has been you
used in our clinic since June 2002. micTESE operation improved our sperm
retrieval rates and fulfilled some of our
patients hopes of having biologically
their own offsprings.
Sperm DNA
Fragmantation
• Meta-analysis:SCSA, performed in
semen, cannot predict the outcome
of ICSI
(Evenson D. 2006)
Sperm DNA fragmantation/TUNEL TEST
Terminal deoxynucleotidyl transferase mediated dUTP
nick-end labeling assay.
To detect the DNA damage, accounting for apoptotic
sperms
At least 500 sperm are count for evaluation
The clinical value of TUNEL in predicting of IVF/ICSI
outcomes in terms of fertilization rate and clinical
outcome is not clear yet;
– < 20 % low degree of sperm DNA damage group
– ≥ 20 % high degree of sperm DNA damage group
Representative Images of TUNEL Asssay
Green stained ones are
apoptotic sperms, high
DNA fragmentation
Tunel TEST in RIF Cases with
Low Sperm Motility
DNA Fragmantation
>20%
<20%
18
19
Mean Male Age
35.7
37.6
Mean Sperm Concentration (mil/ml)
20.6
33.9
Mean Total Sperm Motility (%)
23.0
30.4
Mean Progressive Motile Sperm (%)
3.3
5.2
No of patients
Tunel TEST in RIF Cases with
Low Sperm Motility
≥ 20%
< 20%
18
19
Mean ♀ Age
32.5 ± 6.4
32.2 ± 4.7
ns
Mean ♂ Age
35.7 ± 6.6
37.3 ± 5.1
ns
Fertilization Rate
75.2 %
83.1 %
ns
Slow Growing Embryo on Day3
30.1%
32.6%
ns
Mean of ET Number
2.5
2.4
ns
Implanatation Rates
20 %
19.5 %
ns
Clinıcal Pregnancy Rates
25 %
30 %
ns
DNA Fragmentation
n
p
210 mouse oocyctes (ICSI):
65 fertilization
Nayernia et al.,2006
7 transgenic births
This study shows the higher plasticity potential of adult stem
cells, like hES cells
In April 2007, Nayernia declared that his team obtained the
bone marrow stem cells, called mesenchymal stem cells,
from four adult men who were about to undergo bone
marrow transplants
BMS cells are able to differentiate to early germ cells,
primordial germ cells (PGCs) and even spermatogonial stem
cells (SSC) and spermatogonia in vitro and in vivo.
In-vitro generated artificial
gametes: ultimate solution
Patients with absent
gametes or gonads
Somatic cell haploidization.
Converting somatic cells from
mitotic division to meiotic division
directly.
De-differentiating somatic cells
into embryonic stem cell and redifferantiating ES cells into
gametes.
Extracting adult stem cell and redifferentiating them into gametes.
ASSISTED REPRODUCTIV
TECHNIQUES
DIRECTOR:
Prof. SEMRA KAHRAMAN MD.
IVF CLINIC
SEMRA KAHRAMAN MD.
GÜVENÇ KARLIKAYA MD.
HALE KARAGÖZOĞLU MD.
AYNUR ERŞAHİN MD.
MÜSTECEP KAVRUT MD.
MUSTAFA ACET MD.
NUR DOKUZEYLÜL MD.
ŞEREF SARICA MD.
CRYO / EMBRYO / ANDROLOGY
CO CULTURE LABORATORY
SEVIL UNAL Bio.
HAKAN YELKE Bio.
GÜNSELİ CENGİZ Bio.
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ŞEBNEM ÜNVER Bio.
ÖZLEM YUVACAN Bio.
FERHAT CENGİZ Bio.
SERKAN SELİMOĞLU Bio.
ANDROLOGY
Assoc. Prof. SEMİH ÖZKAN MD.
REPRODUCTIVE GENETICS
FRANCESCO FIORENTINO
PhD. GÜLAY ÖZGÖN MD.
MOLECULAR GENETICS
BAHAR İSMAİLOĞLU Bio.
SELMA DEMİR
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