close

Enter

Log in using OpenID

Dr Sarah Martins da Silva, Consultant Gynaecologist and NRS

embedDownload
TRANSLATIONAL RESEARCH
IN MALE INFERTILITY
Dr Sarah Martins da Silva
Consultant Gynaecologist and NRS Fellow
Assisted Conception Unit
Ninewells Hospital
Dundee
INTRODUCTION
• Infertility defined as inability to conceive after 12
months regular intercourse without contraception
– 50% conceive within 3 months
– 72% within 6 months
– 85% within 12 months
• Primary or secondary
GENERAL CONCEPTS: FEMALE
 Ovulating?
 Regular cycle
 D21 progesterone
 If not - ovulation induction – clomiphene, tamoxifen,
letrozole, gonadotrophin (rFSH)
 Tubal patency?
 Hysterosalpingogram / HyCoSy
 Diagnostic laparoscopy + HTB
 If not - can it be fixed surgically?
GENERAL CONCEPTS: MALE
• Diagnostic semen analysis
Parameter
Lower Reference
Limit
Semen Volume (ml)
1.5
Total sperm number (106 per ejaculate)
39
Sperm concentration (106 per ml)
15
Total motility (PR + NP,%)
40
Progressive motility (PR, %)
32
Vitality (live spermatozoa, %)
58
Sperm morphology (normal forms,%)
4
Other consensus threshold values
pH
>7.2
MAR test (motile spermatozoa with bound particles,
%)
<50
GENERAL CONCEPTS: MALE
• Diagnostic semen analysis
Parameter
Lower Reference
Limit
Semen Volume (ml)
1.5
Total sperm number (106 per ejaculate)
39
Sperm concentration (106 per ml)
15
Total motility (PR + NP,%)
40
Progressive motility (PR, %)
32
Vitality (live spermatozoa, %)
58
Sperm morphology (normal forms,%)
4
Other consensus threshold values
pH
>7.2
MAR test (motile spermatozoa with bound particles,
%)
<50
GENERAL CONCEPTS: MALE
• Diagnostic semen analysis
Parameter
Lower Reference
Limit
Semen Volume (ml)
1.5
Total sperm number (106 per ejaculate)
39
Sperm concentration (106 per ml)
15
Total motility (PR + NP,%)
40
Progressive motility (PR, %)
32
Vitality (live spermatozoa, %)
58
Sperm morphology (normal forms,%)
4
Other consensus threshold values
pH
>7.2
MAR test (motile spermatozoa with bound particles,
%)
<50
GENERAL CONCEPTS: MALE
• Diagnostic semen analysis
Parameter
Lower Reference
Limit
Semen Volume (ml)
1.5
Total sperm number (106 per ejaculate)
39
Sperm concentration (106 per ml)
15
Total motility (PR + NP,%)
40
Progressive motility (PR, %)
32
Vitality (live spermatozoa, %)
58
Sperm morphology (normal forms,%)
4
Other consensus threshold values
pH
>7.2
MAR test (motile spermatozoa with bound particles,
%)
<50
NOMENCLATURE
Azoospermia
No sperm in ejaculate
Oligozoospermia
Total number / concentration of sperm
below reference limit
Asthenozoospermia
% progressive motile sperm below
reference limit
Teratozoospermia
% morphologically normal sperm below
reference limit
Oligoasthenozoospermia
Oligoteratozoospermia
Asthenoteratozoospermia
Oligoasthenoteratozoospermia
NOMENCLATURE
Azoospermia
No sperm in ejaculate
Oligozoospermia
Total number / concentration of sperm
below reference limit
Asthenozoospermia
% progressive motile sperm below
reference limit
Teratozoospermia
% morphologically normal sperm below
reference limit
Oligoasthenozoospermia
Oligoteratozoospermia
Asthenoteratozoospermia
Oligoasthenoteratozoospermia
CLINICAL PROBLEM
• 1:7 couples affected, 3.5 million people in UK
• Male factor underlying >50% cases subfertility
• Currently no treatment to boost sperm motility in vitro / no
drug available for subfertile male
• Limited therapeutic options for couples
• Treatment rests with Artificial Reproduction Technology
(ART)
– Expensive
– Invasive
– No guarantee of success
• Unmet clinical need
DRUG DISCOVERY
• High throughput screening: time and resource efficient
method of identifying potential compounds as new drugs
• Unique resource: University of Dundee Drug Discovery
Unit
– Biotech and pharma capabilities
– Remit for drug discovery in neglected diseases and
novel therapeutic targets
– Libraries of thousands of small molecular compounds
specifically designed for ‘lead-like’ characteristics
SPERM MOTILITY
• Sperm motility correlated both to spontaneous conception
and ART success – attractive drug target
• Limited understanding
• Difficulties and challenges in sperm research
– Very small cells
– Motile
– Virtually no cytoplasm – post-translational modification
main mechanism of intra-cellular signalling
– Gametes – unable to culture
– Knock-out mice infertile
CALCIUM
• Alteration in [Ca2+]i central to sperm motility and function
• [Ca2+]i relates to IVF fertilisation rates
(Alasmari et al. 2013)
• pH ([H+]i) also crucial to sperm motility and fertility
• [Ca2+]i and [H+]i primarily determined by ion channels and
transporters of the sperm plasma membrane
• Calcium stores also contribute to [Ca2+]i: hyperactivated
motility
CatSper CHANNELS
• CatSper ion channels recently demonstrated in human
sperm
(Lishko et al. 2011; Strunker et al. 2011)
•
•
•
•
•
Cation channel of Sperm
Sperm specific
Principle piece of flagellum
Permeable to calcium
CatSper activation
– Progesterone
– Prostaglandins
– Increase in intracellular pH
Publicover & Barratt 2011
FLIPR CALCIUM 3 ASSAY KIT
• Cells incubated with
calcium sensitive dye
• Masking dye remains
outside the cell (and
blocks background
fluorescence)
• Release of calcium into
the cytoplasm in
response to stimulation:
dye binds to the
intracellular calcium and
becomes fluorescent
FLEXSTATION 3
•
384 well microplate reader
•
Integrated 16 channel pipettor
• Directly transfers user-defined reagents to read plate
• Adjustable dispensor height / velocity to prevent cell
dislodging
• Tituration option
Base reader optics system
• Excitation and emission wavelengths set to optimise
readout
•
3.6mmol progesterone
SPERM ASSAY
• Healthy volunteer donors
• Density gradient centrifugation
• Capacitated 80% cells x 2.5h
• Incubate 100 million cells
in 5ml x2 FLIPR Calcium 3 dye x 60mins
• Wash off (700g x 5mins)
• Resuspend in BSA-free Flexstation buffer
• Add 250,000 cells (50ml) per well of 384 microplate
• Centrifuge microplate (700g x 3 mins) RT
• FlexStation assay: 100s, readings every 2s
• After 20s, progesterone / screening compound added
DDU ION CHANNEL LIBRARY
•
•
•
•
3242 compounds
11 plate assay
SP screen (10mmol)
Raw data analysed
using SoftMax Pro
(AUC function)
• Data further assessed
using Activity Base to
calculate percentage
effect (PE)
• Plotted as frequency
distribution
Median
SCREEN OUTCOME FOR
ION CHANNEL LIBRARY
OUTPUT
RESULT
% OF TOTAL
No. of compounds screened
3242
-
Putative hits >25% activation
252
7.7
Putative hits >50% activation
74
2.3
Putative hits >75% activation
20
0.06
HIT COMPOUNDS
• 74 compounds rescreened
• Standard 10 point half-logarithm concentration response
curve to test for potency
• 67 returned concentration response curves, although low
potency
• 48/67 compounds passed purity and integrity testing
(LC-MS)
– 9 failed due to purity <90%
– 10 failed due to incorrect ion mass
• Chemical analysis and SAR grouping
– 5 distinct groups with similar chemical structure
COMPUTER-ASSISTED
SPERM ANALYSIS (CASA)
• Computerised assessment of sperm motility parameters
–
–
–
–
–
–
–
VAP: Smoothed Path Velocity (microns/sec)
VCL: Track Velocity (microns/sec)
VSL: Straight Line Velocity (microns/sec)
ALH: Amplitude of Lateral Head Displacement (microns)
BCF: Beat Cross Frequency (hertz)
LIN: Linearity (ratio of VSL/VCL)
STR: Straightness (ratio of VSL/VAP)
IVF: SPERM SELECTION
1 Cumulus penetration
2 Zona pellucida
3 Sperm-oocyte fusion
KREMER TEST
1
2
3
ELECTROPHYSIOLOGY
HIT ANALYSIS
• Analysis for direct sperm motility effect (CASA, Kremer):
1/5 compound increased motility
**
*
**
* **
DONOR SAMPLES
**
*
*
PATIENT SAMPLES (n=8)
PATCH CLAMP
ELECTROPHYSIOLOGY
• Potassium channel blocker: indirect effect on
CatSper by alteration of sperm cell membrane
potential
DDU CHEMOGENOMIC LIBRARY
•
•
•
•
•
223 compounds
Single plate assay
SP screen (40mmol)
Raw data analysed using SoftMax Pro (AUC function)
Data further assessed using Activity Base to calculate
percentage effect (PE)
OUTPUT
RESULT
% OF TOTAL
No. of compounds screened
223
-
Putative hits >25% activation
25
11.2
Putative hits >50% activation
14
6.3
Putative hits >75% activation
7
3.1
MOST POTENT ‘HIT’
• Trequinsin
• Class 3 PDEi
40mM Trequinsin
60
50
RFU
)
U
F
R
(
e
c
n
e
s
e
r
o
u
lF
40
30
10mM progesterone
20
40mM BRL
10
0
-10
-20
0
10
20
30
40
50
Time (s)
60
70
80
90
100
MOTILITY ASSAY
CASA
TOTAL MOTILITY (%)
70
*
*
60
*
*
*
50
40
30
20
KREMER
10
PROGRESSIVE MOTILITY (%)
0
TIME 0
20 min
40 min
60 min
90 min
120 min
60
**
50
*
**
**
40
**
*
**
30
**
20
10
0
TIME 0
20 min
40 min
CONTROL
60 min
90 min
TREQUINSIN
120 min
ELECTROPHYSIOLOGY
• Trequinsin and progesterone have direct effect on
CatSper
• Trequinsin also blocks potassium channels, thus
having a dual effect on CatSper activation
CONCLUSIONS
• Robust HTS assay developed
• Holds promise for drug development for male
subfertility – in vitro
• Aim to expand screening – new compound library(s)
• Overall aim to develop in vivo prescribable for
infertile men
THANK YOU
1/--pages
Report inappropriate content