Alpesh optimising icsi (including imsi)

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

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

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

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”

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”

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)

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.

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

• 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

IMSI

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• Finding the best sperm - IMSI

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IMSI Workstation Digital Camera Objective

• Finding the best sperm - IMSI

30Gris Reproduccion, Barcelona

IMSI grading

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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

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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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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

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.....

• Nasum

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SEMFixed

TEMFixed

PolarizedNative

NomarskiNative

InformationExternal InternalCourtesy: Dr Sergei Yakovenko, Altravita

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

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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Courtesy: Dr Sergei Yakovenko, Altrvita

Thank you

Alpesh.doshi@uclh.nhs.uk