alpesh optimising icsi (including imsi)
TRANSCRIPT
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Optimising ICSI (Including IMSI)
Alpesh DoshiHead of Embryology
Research Instruments Workshop Jordan March 2012 1
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Indications for ICSI• Male factor
– Oligozoospermia (<10 million/ml)– Asthenozoospermia (<40% progressive motility)– Teratozoospermia (<3% normal forms)– Antisperm Antibodies (>35% IgA or IgG)– Globozoospermia– Ejaculatory disorders (retrograde, electroejaculation)– Congenital absence of the Vas deferens– Obstruction of ejaculatory ducts– Failed Vasectomy reversal– Non obstructive/Obstructive Azoospermia
• Poor/ No Fertilisation after IVF (Zona receptor binding)
• PGD-Single gene defects –Paternal Contamination
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Equipment
• Micromanipulators• AntiVibration tables/ Platforms• Microtools
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Equipment Location
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Equipment Micro Tools
Spike/ Non spike, Angle, Internal/ Outer diameter, MEA test, CE marked, packaging, Cost.
Avoid making your own !
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Equipment
Select carefullyConsider:
•Ease of use•Reliability•Service available locally
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Sperm preparation techniques
• Sperm Preparation Techniques need to be:
-Swim up, Density gradients (300g force)• PESA- Wash, Mini density gradient• Testicular (TESE) sperm preparation-
Milking/washing, Mini DG (Rare)- ELB, Collagenase txt, Pentoxifylline (if necc)
• Frozen sperm- Long Incubation post thaw showed High DNA damage (Dalzell et al 2004) incubation time post thaw showed High DNA damage (Dalzell et al 2004)
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• Sperm Selection– At 400X – PVP used to slow down movement– Normal Sperm selected– Morphology of head, motion pattern and light
refraction are considered– Immotile and poorly progressive sperm have
centrosomal damage (Sathananthan and Trounson 2000)
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• Sperm Selection– For PESA/TESA and severe oligospermia
• At 400X • Use long drops of the sperm suspension (washed well)• Select sperm and deposit into the PVP holding droplet. • How long should this take?
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Fert Rate Pregnancy Rate
30mins-1hr 54.2% 41.2%
1hr-2hr 46.3% 36.6%
2hr-3hr 28.0% 26.8%
>3hrs 25.4% 21.2%
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The Procedure - Some considerations Searching for testicular sperm >1000 NOA TESE/ ICSI Cycles
D Monahan et al, Oral presentation ESHRE 2011
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Pentoxifylline/ Theophylline ....• Phosphodiesterase inhibitor/ Caffeine
derivative.• Mangoli et al 2011 (Fertil steril) : Higher FR
and CPR in Pentox group compares to HOS• DeMendosa et al 2000 (Fertil Steril)• Kovacic et al 2006 (J Androl)• Griveau et al 2006 (RBM online) • Gioretti et al 2005(RBM online)
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CRGH Protocol• Pentoxifylline/theophylline (Gynemed- Germany)
working solution made fresh (1mg/ml)• Warm to 37°C.• Add 1 :1 ratio of sperm suspension/ Pentox • Isolate motile and morphologically normal
spermatozoa into PVP drop.• Wash in PVP, immobilise and Inject. • Do not exceed 10 mins in pentox solution.
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Video (Pentox)
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The Procedure - Some considerations HEPES or culture media?
-Time consideration, pH, toxicity- balance!• Morgia et al 2006 (Fertil Steril)- sign higher
triploidy, damage rate with oocytes exposed to hepes. Sign lower good quality embryos, lower implantation and pregnancy rate in hepes group.
- Exposure to PVP • Hlinka et al 1998 (Hum Reprod) sign higher
fert rate with no PVP used -Immobilisation method
No advantage of aggressive swipe techniques (A Velaers et al ESHRE 2011)
One gentle swipe
“accurate set up of micro tools is essential”
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Polar Body positioning
• Anifandis et al (2010)- Reprod sci.- Sign higher fertilisation rate, good quality
embryos and pregnancy rate with oocytes injected with PB at 11 o’clock compared to 6, 7 or 12 o’clock
• Woodward et al (2008) RBM Online- Highest frequency of normal fertilised oocytes and
good quality embryos with injection in/ near the plane of spindle ie at 3, 4, 8 and 9 o’clock
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At CRGH: Post hCG...• Egg collection- 37 hrs • Sperm collection- 37 hrs• Sperm preparation -37.5 hrs• Sperm incubation - up to 40-41 hrs(min 1 hr inc)• Denudation -40 hrs (39 if large egg no/TESE sperm)• ICSI- 41 hrs• Large egg no’s or TESE sperm – start ICSI at 40 hrs• ICSI complete by 41.5- 42 hrs• Fert check 16-18 hrs post ICSI
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The Procedure - Some considerations Timing of ICSI ( Dozortsey et al Fertil Steril 2004)
Fert rates increase with time elapsed post hCG with optimal at >41 hrs.
Highest implantation rates achieved at 37-41 hours post hCG
Lower implantation rates achieved at <37hrs or >41hrs are due to metabolic incompetence either metabolic immaturity or post maturity.
“Don’t leave your ICSI cases till the end of the day”
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The Oocyte....
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Oocyte preparation• VEC 36-37 hrs post hCG• Preincubation of 2-4 hrs resulted in improved
maturation of oocytes, fertilisation and embryo quality.(Isiklar et al 2004)
• Denudation using Hyaluronidase (10-80IU/ml)– Higher conc and exposure time induces
parthenogenesis (Van de velde et al 1997)
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Microscopic Evaluation of morphology and maturity
• Evaluated under x400 mag• 10-12% of oocytes immature (GV, M1).
Score and separate at denudation• In vitro matured oocytes. Very poor fert &
preg rates (De Vos 1999, Nagy 1996). High chromosomal abnormailities (Picton H. personal communication.)
• Metaphase II oocytes graded according to cytoplasmic & polar body integrity.(Xia 1997, Serhal 1998)
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Temperature and the spindle
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A DROP IN TEMPERATURE CAN EQUATE TO A DEPOLYMERIZED SPINDLEA DROP IN TEMPERATURE CAN EQUATE TO A DEPOLYMERIZED SPINDLE
–Temperature fluctuations can induce de polymerisation and hence
non disjunction of chromosomes leading to aneuploidies (Wang et al 2001)
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Temperature and spindle • Pickering et al (1990) Fert Steril.
– Microtubule disorganisation, reduction in spindle size, complete lack of spindle seen in all oocytes when exposure time was 30 mins to rtp.
• Almeida & Bolton (1995) Zygote– 77% of oocytes had spindle disruption when
exposed for 2 mins at rtp. Chromosomal dispersal in 50%. Effect irreversable when time exceeded 10 mins.
• Wang et al (2001) Human Reprod.– Spindle depolymerisation by 5 mins when
exposed to to rtp.
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Individual testing in different labs based on size of drops, type and diameter of dishes and room temp.
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Injection..• Aspiration volume of cytoplasm into pipette:
-Dumoulin et al 2001- sign reduced blast rate with >6pl of cytoplasm aspirated
-Hiraoka et al (2011) ESHRE oral pres: Sign higher Fertilisation rate in oocytes with less cytoplasm aspirated.
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It’s not getting the sperm in, it’s getting the right sperm in that matters
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• Finding the best sperm
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“Physiologic ICSI”: Hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo qualityLodovico Parmegiani et al Fertil Steril 2010
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IMSI
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• Finding the best sperm - IMSI
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IMSI Workstation Digital Camera Objective
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• Finding the best sperm - IMSI
30Gris Reproduccion, Barcelona
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IMSI grading
3131
GRADE I
No vacuoles
GRADE II < 2 vacuoles
GRADE III
> 2 vacuoles or at least one large vacuole
GRADE IV
Large vacuole and abnormal head shapes or other
abnormalities
Sperm was selected using Vanderzwalmen et al., (2008) grading system
III
III
IV
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Meta analysis- IMSI Vs ICSI
– 37 studies in literature– Only 3 were comparative or randomised. – Outcomes: Fertilisation, implantation, Pregnancy
and Miscarriage rates.
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Souza Setti et al 2010 RBM Online
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34Souza Setti et al 2010 RBM Online
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Meta-analysis showed:Outcome ICSI IMSI Conclusion
Fertilisation Rate
76.7% 75.7% NS
Top Quality Embryos (2 studies)
27.7% 41.2% StatisticallySignificant
Implantation Rate
10.5% 21.9% Statistically Significant
Pregnancy Rate
26.6% 47.6% Statistically Significant
Miscarriage Rate
29% 14.7% Statistically Significant
Souza Setti et al 2010 RBM Online
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Clinical Outcome of IMSI: A prospective Randomised Study
Balaban et al (2011)- RBM Online
Unselected Population -No Significant Difference seen in outcome measures.
Severe Male factor Group - Significantly higherImplantation
rates.
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Study Significant Findings (MSOME & IMSI vs. ICSI)
Souza Setti et al, 2010 Significantly higher pregnancy and implantation and significantly lower miscarriage rate in IMSI group
Figueira et al, 2010 Significantly lower aneuploidy rate in IMSI group
Wilding et al, 2010 64.8% of sperm selected in ICSI had significant DNA fragmentation. Embryo quality, pregnancy and implantation rate higher in IMSI group
Monquat et al, 2010 Swim up sperm have less nuclear vacuolation than DGS
Vanderzwalmen et al, 2008 Presence of nuclear vacuoles reduces PR, higher pregnancy rate in IMSI group. Proposed sperm grading scheme
Antinori et al 2008 +ve correlation for pregnancy and miscarriage in OAT group
Bartoov et al, 2002 Significantly higher pregnancy rate in IMSI group
Bartoov et al, 2002 Describe MSOME. Sperm with vacuole occupying >4% of nuclear area abnormal and not used for injection
Bartoov, 2001 First reported IMSI. Magnification x6000.
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Future.....
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• Nasum
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SEMFixed
TEMFixed
PolarizedNative
NomarskiNative
InformationExternal InternalCourtesy: Dr Sergei Yakovenko, Altravita
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Future Aim
to understand how the methods of external spermatozoa observation (such as Nomarsky, Hoffman, SEM) reflect the internal structure of spermatozoa (NASUM and TEM).
Courtesy: Dr Sergei Yakovenko, Altrvita
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Nasum (Native assesment of sperm ultra morphology)
• Simultaneous use of Nomarsky and Hoffmans contrast
• Resolution increased by circular polarised light• Additional lenses give a total magnification of
x 20000 (including video zoom)
Courtesy: Dr Sergei Yakovenko, Altrvita
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99 ,102
Courtesy: Dr Sergei Yakovenko, Altrvita