12-17 Strategies to improve embryo quality: Assisted hatching and remo

Lesson 17 of 19

Strategies to improve embryo quality: Assisted hatching and removal of fragments

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Noelia Grau, PhD Clinical embryologist. IVF Laboratory

Index

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FragmentationAssisted hatchingAssisted hatching + Aspiration of fragmentsConclusions

Fragmentation

FragmentationWhat is it?How is it classified?How does it originate?What are its effects?Assisted hatchingHow is it done?Tricks and tipsWhen is it done?Good for something?Assisted hatching + Aspiration of fragmentsWhat for?StudiesHow is it done?Indications for assisted hatching and fragment aspirationConclusions

Fragmentation

What is it?

• Cell portions without a nucleus, described in 1970 in human embryos
• Also present "in vivo" and in other species
• 75% of 79,000 embryos generated in IVF lab present fragmentation
• Appears in mitotically active embryos
• Fragments can lyse, move, and be episodic
• It is related to lower embryo viability

Fragmentation

What is it?

Fragmentation

How is it classified?

BEFORE: MINOR or MAJOR NOW: degree of fragmentation

0%

5%

10%

15%

25%

>40%

Fragmentation

How is it classified?

• TYPE I: few small fragments, associated with 1 cell.
• TYPE II: small fragments associated with 1 or + cells.
• TYPE III: small fragments distributed and associated with several cells.
• TYPE IV: large fragments distributed and associated with several asymmetric cells.
• TYPE V: granular fragments and associated with cytoplasmic contraction.

Fragmentation

How is it classified?

D2: Cells ≥45 µm D3: Cells ≥40 µm

Fragmentation

How does it originate?

• High levels of free radicals due to high sperm concentrations
• pH or temperatura shock
• Ovarian stimulation protocols
• Cytoplasm excess
• Low follicular oxygen
• Apoptosis in granulosa cells
• Patient-dependent

Fragmentation

What effects does it have?

• A = no frag.
• B = < 25% frag.
• C = 25 a 50% frag-
• D = > 50% frag.

Fragmentation

What effects does it have?

Mean embryos with lysed cells: Yes 0-5% frag: 1.7% If> 35% frag .: 11.4%

Fragmentation mean: In D3 embryos: 15.4% In transferred embryos: 8.6%

Fragmentation

What effects does it have?

Type II: elimination of selected blastomeres (maintains embryo viability if one cell is abnormal)

Assisted hatching

• Fragmentation

What is it? How is it classified? How does it originate? What are its effects?

• Assisted hatching

How is it done? Tricks and tips When is it done? Good for something?

• Assisted hatching + Aspiration of fragments

What for? Studies How is it done? Indications for assisted hatching and fragment aspiration

• Conclusions

Assisted hatching

How is it done?

Tyrodes Acid

• Tyrodes acid medium (2.4 pH)
• Pipette 10-12 µm
• Mouth suction
• Acid is spewed
• We aspirate medium
• Displace embryo
• Wash embryo

Assisted hatching

How is it done?

Laser

• Shoot laser light through lens
• Irradiation time setting (1.5-1.8 ms)
• ZP thermolysis
• Precise, cylindrical holes
• Displace embryo
• Wash embryo

Assisted hatching

Tricks and tips

• Hole size: 15-20 µm
• If Tyrodes acid:
• Place embryo and lower AH pipette by blowing
• If no hole is formed, stop
• Time to break ZP: 2-5 seconds
• A lot of expertise
• If laser:
• Better many short shots than one long shot
• Equipment maintenance

Assisted hatching

When is it done?

• Thickened ZP (≥ 18 µm)
• Advanced maternal age (≥ 39 years)
• High fragmentation (≥ 15%)
• Warmed embryos
• With previous failed cycles

Assisted hatching

Assisted hatching

Good for something?

• Cohen et al., 1992: Embryos with ZP thicker than 15 µm with AH improve implantation capacity
• Grace et al., 2007: Better prognosis if AH is performed in patients with implantation failure
• Ge et al., 2008: Better pregnancy and implantation rate when AH is done to thawed embryos
• Figueira et al., 2012: In vitrified oocytes the AH technique increases the implantation rate
• Wan CY et al., 2014: Improves results after frozen transfer
• Figueira Rde C et al., 2012: Improves the results in vitrified oocytes
• Xinmei Lu et al., 2019: Improves results in frozen transfers

YES

• Hurst et al., 1998: AH does not increase the outcomes in IVF cycles with good prognosis
• Frydman et al., 2006: AH does not benefit patients of advanced maternal age
• Valojerdi et al., 2010: Worse pregnancy and implantation rate if we do AH to warmed embryos
• Figueira Rde C et al., 2010: AH does not improve outcomes in women of advanced age

NO

Assisted hatching

Good for something?

• Pregnancy rate • Implantation rate • Multiple pregnancy rate • LB rate • Miscarriage rate

12 RCT2574 cycles

Assisted hatching

Good for something? In vitrified embryos…

METHODOLOGY:

• 1h after warming
• More time complicates aspiration
• If aspirating lysed cell, use Tyrodes

Assisted hatching

Good for something? In blastocyst…

a) AH near ICM b) AH opposite ICM A % ICM extrusion (P<.001) A % complete hatching (P = .002) It is advantageous to hatch in the nearby ICM area to perform invasion into the endometrium.

Assisted hatching

Drawbacks

• Damage to any blastomere during AH
• Increase in the rate of formation of monozygotic twins
• If the ZP has small holes (less than 5µm) trapped embryos

Assisted hatching

Good for something? We do not agree ….

• Methods to perform AH: acid, laser, pronase
• Hole length and thickness: partial, total dissection ...
• Hole location
• Quality and embryo stage
• Moment in which the technique is performed
• Experimental design: prospective, retrospective
• Work protocols and ZP hardening
• Type of laser, number of shots and exposure time

Assisted hatching and fragment aspiration

• Fragmentation

What is it? How is it classified? How does it originate? What are its effects?

• Assisted hatching

How is it done? Tricks and tips When is it done? Good for something?

• Assisted hatching + Aspiration of fragments

What for? Studies How is it done? Indications for assisted hatching and fragment aspiration

• Conclusions

Assisted hatching and fragment aspiration

What for?

OBJECTIVES:

1. To reestablish cell contact, promoting compaction, cavitation and blastocyst formation
2. To avoid transmission of cellular degradation

Assisted hatching and fragment aspiration

Studies

• Alikani et al., 1999: Fragment aspiration alters the course of development of certain embryos and improves their implantation potential.
• Keltz et al., 2006: Fragmented embryos with fragment aspiration obtain the same clinical outcomes as non-fragmented embryos.
• Valojerdi et al., 2006: Fragment aspiration has a positive effect on fragmented embryos and produces good quality blastocysts
• Seok-Gi Kim et al. , 2018: Early aspiration of fragments on D2 improves pregnancy and implantation rates.

YES

• Halvaei et al., 2016: Fragment aspiration does not improve ART outcomes in non-selected patients.
• Safari et al., 2017: Fragment aspiration does not improve outcomes in cycles with warmed embryos.

NO

Assisted hatching and fragment aspiration

How is it done?

Assisted hatching and fragment aspiration

Indications for assisted hatching and fragment aspiration.

GROUP A: without AH + FA because ≤10% frag., <39y and <2 attempts. GROUP B: no AH + FA despite> 10% frag., ≥39y and / or ≥2 attempts. GROUP C: with AH + FA because> 10% frag., ≥39y and / or ≥2 attempts.

Assisted hatching and fragment aspiration

Indications for assisted hatching and fragment aspiration.

<10% frag

Conclusions

• Fragmentation is a very common abnormality in embryos
• Both the percentage and pattern of embryo fragmentation directly affect pregnancy and implantation rates.
• The elimination of fragments facilitates the communication between cells and therefore a normal compaction and the formation of blastocysts.
• The use of assisted hatching combined with fragment aspiration can increase the implantation capacity of the embryos to be transferred.

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