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Micromanuplation Techniques (ICSI, AH, PGD..)

Micromanuplation Techniques (ICSI, AH, PGD..). BASAK BALABAN, BSc American Hospital of Istanbul Assisted Reproduction Unit. AMERICAN HOSPITAL. Evolution of ICSI. IVF - Low fertilization & CPR for male factor subfertility Late 1980s.....

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Micromanuplation Techniques (ICSI, AH, PGD..)

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  1. Micromanuplation Techniques(ICSI, AH, PGD..) BASAK BALABAN, BSc American Hospital of Istanbul Assisted Reproduction Unit AMERICAN HOSPITAL

  2. Evolution of ICSI • IVF- Low fertilization & CPR for male factor subfertility Late 1980s..... • PZD (Partial zona dissection), ZD (zona drillling): male factor subfertility • SUZI (Subzonal microinjection of spermatozoa into the PVS): male factor subfertility 1992....... • ICSI (Intracytoplasmic sperm injection): severe male factor infertility

  3. Evolution of ICSI • ICSI Intracytoplasmic injection of one motile spermatozoon for each Metaphase II oocyte **Improved modifications into the procedure • Reducing the concentration of hyaluronidase used for cumulus-corona radiata removal • Selecting a motile spermatozoon that was immobilized prior to the injection • Aspiration of cytoplasm to ensure rapture of the oocyte membrane Joris et al., HR 1998

  4. Equipment needed for ICSI MICROMANUPLATOR Joysticks, motor controls Joystick, heater Joris et al., HR 1998

  5. Equipment needed for ICSI Joris et al., HR 1998

  6. Indications of ICSI ESHRE Capri Workshop Group, HR 2007

  7. Distribution of ICSI applications ESHRE Capri Workshop Group, HR 2007

  8. Outcome of ICSI • Perinatal Risks: IVF& ICSI outcomes are similar (Neri 2006) Twins & singletons born after IVF & ICSI have a 2-fold increased risk for perinatal mortality, preterm delivery and low birth weight and a 3-fold risk for very low birthweight (Helmerhorst, Jackson 2004). Vanishing twins after MET, or infertility itself can be the cause? (Pinborg 2005, Schieve 2002). ESHRE Capri Workshop Group, HR 2007

  9. Congenital Abnormalities IVF&ICSI similar malformation rates(Hansen, Lie 2005), but higher when compared with general population (Katalinic 2004, Belva 2007) ESHRE Capri Workshop Group, HR 2007

  10. Outcome of ICSI • Long-term follow up: Singleton IVF&ICSI 5 year-olds are comparable with their naturally concieved peers. In 150 8-year old ICSI children, pubertal staging,neurological status, need for more remedial therapy, surgery or hospitalization were comparable with 147 matched spontaneously concieved controls(Belva 2007). However considering higher risk of congenital (male-urogenital) anomalies in relation to sperm quality and source, particular interest should be given to the use of sperm from testicular biopsy with non-obstructive pathology(Wennerholm 2006) ESHRE Capri Workshop Group, HR 2007

  11. Outcome of ICSI • Imprinting diseases: Reports of Angelman and Beckwith-Wiedeman syndromes in children born after IVF&ICSI suggest a possible risk of in-vitro culture procedures(Maher 2003), and hormone stimulation(mild COH suggested). A systematic survey aimed at those syndromes and defined phenotypes linked to imprinting genes may clarify whether epigenetic anomalies play a role in ART(or subfertility) more often than in general population(Ludwig 2005) ESHRE Capri Workshop Group, HR 2007

  12. Genetic and Epigenetic Charecteristics of ICSI children Obstetric outcome: 229 ICSI, 194 naturally conceived singleton PR were age matched(37.8) No diff. in: Uncomplicated vaginal, caesarean delivery,Gestational age, frequency of low birth rate, malformations Long term (5 years) physical and psychological outcome: No diff. in the full scale IQ assess ment, general health e.g. Common diseases, chronic illnesses, surgical intervention, physical development Natural cycle results taken from National Vital Stat. Report 2002,2004 Palermo et al.,RBM Online 2008

  13. Genetic and Epigenetic Charecteristics of ICSI children Palermo et al.,RBM Online 2008

  14. ASSISTED HATCHINGThe older patient, certain embryos , thawed embryos, or everyone ?

  15. WHY PERFORM ASSISTED HATCHING ? • The ratio of lysin production to ZP thickness could determine whether the embryo will lyse the zona and perform the ***HATCHING****procedure • Suboptimal culture conditions and or advanced maternal age may cause deficiencies in lysin secretion which could impaireffective hatching • The trophectoderm of some embryos may not be able to secrete the hatching factor and lysin production could be influenced by the patient’s age(Cohen J. 1992) • Uterin lysins action could also be impaired in some patients(Mandelbaum J.1996 )

  16. INDICATIONS FOR ASSISTED HATCHING • AH has been proposed as a method for improving the capacity of the embryos to implant by facilitating the hatching process(Cohen 1992 ) • Women/embryos with poor prognosis * Advanced age * Thick ZP (more then 15 microns-Cohen J. 1992 ) * Repeated implantation patients * Embryos with extensive fragmentation * Frozen-thawed embryos with cell death

  17. WHEN TO PERFORM ASSISTED HATCHING ? • When breaches are made in the ZP of early cleavage IVF embryos,embryonic cell loss may occur through the zona as a result of uterine contractions after replacement of the embryos • It is advisable to manipulate the embryos for AH after the adherence between blastomeres has increased just before compaction(6-8 cell stage) (Dale,1991) • It is very critical to achieve zona slitting or drilling with rotating the embryo until the area where the largest PVS is visible in order not to give any harm to blastomeres near the zona border

  18. METHODS OF ASSISTED HATCHING • Mechanical (partial zona dissection ) • Chemical (Acid Thyrode, Pronase) • Laser assisted hatching

  19. PARTIAL ZONA DISSECTION • The zona pellucida is pierced with a very thin glass microneedle through both sides,the needle tip position being controlled in perivitelline space by eye • Then the suction of the holding pipette is stopped and the holding pipette is rubbed against the trapped area of the zona until this area has been completely abraded.

  20. THREE DIMENTIONAL PARTIAL ZONA DISSECTION • Differs from conventional partial dissection by an additional second cut for a cross shaped opening rather then a single slit opening • The second cut is made by passing through the ZP under the first slit opening and ending in the same positions as the first cut.A cross-shaped opening can be seen when rotating the embryo and focusing on the surface of ZP • Beneficial particularly for PGD and clinically proved to be safe and efficient Cieslak J. F&S 1999.

  21. Clinical outcome of three-dimensional partial zona dissection and conventional partial zona dissection P = .0834 Cieslak Fertil Steril 1999

  22. ACID TYRODE’S ASSISTED HATCHING • The micropipette with acidic Tyrode’s solution is brought close to the ZP and the solution is expelled over a small area until ZP is dissolved through to the inside • The embryos have to be rinsed in fresh medium for several times after the procedure to avoid acidic solution

  23. Zona thinning of cleavage stage embryos or total zona removal of blastocysts by pronase • Enzymatic digestion of the ZP for blastocyst stage embryos first described by Fong (1997) • Blastocysts transferred to 10IU/ml pronase solution and incubated for 1 minute at 37oC and 5 % CO2 for initial stretching and softening of the ZP • After the observation of zona expansion and the increase in PVS they were transferred to culture media and washed several times just before the complete dissappearance of the zona Fong et al. HR1997

  24. Zona manipulated blastocyst transfers on day 5 Fong et al. HR 1998

  25. ZI Blastocyst Transfer ZF Blastocyst Transfer p Value Mean female Age (y) 31.5 31.8 NS Duration of infertility (y) 8.0 7.3 NS # of blastocysts transferred 3.3 3.5 NS Clinical PR/ET 38.1% 48.7% <0.05 Implantation/embryo 21.6% 30.8% <0.05 Multiple PR 48.5% 53.9% NS ZONA INTACT VS ZONA FREE BLAST TRANSFER Urman & Balaban, F&S 2002

  26. ** It is important that the laser is accurately controlled and produces precise ZP openings without thermal or mutagenic effects Contact lasers Non-contact lasers **Safety and efficacy of both systems have been demonstrated in clinical practice **Laser AH enables a rapid no-touch microdrilling which might be efficient, precise, safer and chemical free for AH LASER ASSISTED HATCHING

  27. A comparison between quarter, partial and total laser assisted hatching in selected infertility patients Mantoudis et al Hum Reprod 2001 P<0.001

  28. A comparison of four different techniques of assisted hatching Balaban et al. HR 2002 Retrospective, selected patients

  29. AH results in better CPR and IR Feng et al.,F&S 2008 in press Retrospective, selected group, Laser or AT is superior to PZD Hseih 2002, Makrakis 2006, Lanzendorf 2007 shows superiority of LAH

  30. AH for cryo-thawed embryos Gabrielsen HR 2004: AH sig. increased IR (AT) Ng HR 2005: No difference in IR(Laser) Petersen RBM 2006: No difference in CPR&IR (Laser) Sifer HR 2006: No dif.in IR(pronase) Valojerdi F&S 2007: LAH sig.increased CPR&IR Ge RBM 2008: LAH sig. İncreases CPR Ng 2008: LAH sig.increases IR&OPR

  31. LAH improves CPR&IR in FET No dif. in patient,or thawed embryo charecteristics Balaban et al.,HR 2006

  32. 3D-PZD PZD Laser Acid Tyrode’s Total ZP removal Pronase thinning

  33. Assisted Hatching on assisted conception (IVF&ICSI)(Review) Seif MMW et al., Cochrane Database of Systematic Reviews 2006- CD001894 • 23 randomised control trials consisting of 2668 women reported on 849 pregnancy outcomes, primary outcome LBR, secondary outcome CPR, IR,MPR,miscarriage, ectopic p, MZ twinning, congenital & chromosomal abn., embryo damage

  34. CPR 1st.attempt: No dif. Repeat attempt: Sig. in favourof AH Age: No diff.for <35/>35 Prognosis: Sig. improved CPR for good and poor prognosis patients Cochrane review, 2006

  35. Live birth rate 1st./repeated attempt: Nosig. difference ICSI/IVF: No difference Method of AH: No difference Prognosis: No difference Age: Insufficient data Cochrane review, 2006

  36. Miscarriage rate Cochrane review, 2006 No dif. in any subgroups

  37. MPR No sig. diff. for: 1st./repeat attept, prognosis, Age:insuffcient data Cochrane review, 2006

  38. Other secondary outcomes • MZT:4 trials; *Hurst:2/3 for AH- 0/3 in control *Lanzendorf, Ng, Isik: No MZT • Ectopic Pregnancy: 3 trials; *Lanzerdorf:1 in control, 0 in AH *Hellebaut, Hurst: 0 • Congenital and/or chromosomal abnormalities: 2 trials by Lanzendorf and Hurst reported absence • Embryo damage: 3 trials: Hurst, Lanzendorf, Stein reported absence • Blastocyst development: No trials

  39. Prospective randomised trials published after the cochrane data • Ma S F&S 2006: Similar CPR, sig. higher IR in woman >35 • Frydman HR 2006: No dif. in CPR and LBR for women >37 and <3PIF • Sagoskin F&S 2007: No dif. in CPR and LBR for good prognosis patients • Dayal F&S 2007: Improved CPR and IR for woman with 1 PIF • Valojerdi F&S 2007: No dif. in CPR and IR for women >37 and >2 PIF • Ge HS RBM Online 2007: No dif. in CPR and IR for unselected groups

  40. Preimplantation Genetical Diagnosis/Screening • High risk PGD(PGD): Patients of transmitting a genetic or chromosomal abnormality to their children, which includes single gene defects(autosomal recessive/ dominant, X linked disorders), and chromosomal abnormalities(translocations, structural aberrations, etc..) • Low risk PGD(PGS): Infertile patients undergoing IVF with the aim of increasing the IVF PRs.(Advanced maternal age, RIFs, normal karyotypes with repeated miscarriages) ESHRE PGD Guidelines Thornhill et al.,HR 2005

  41. Stages of embryo biopsy • Polar body (1st.and/2nd.): 1st.PB removal 36-42hr post-HCG injection(Verlinsky 1990), and/ 2nd.PB removal 18-22hr. Post-insemination. ** Limitations: Only allows maternally derived chromosomal aneuploidies& translocations. Paternal distribution cannnot be diagnosed, technically risky if PBs are fragmented) • Cleavage stage blastomere biopsy: Applied on the morning of day 3 post insemination(6-8cell stage). It’s acceptable to exclude the biopsy of poor quality embryos ** (A single biopsied blastomere don’t represent the actual karyotype.While approx. 30% abnormal embs. are mosaics, mosaicism alone causes only 5% of diag.errors as in most mosaics almost all cells are abnormal. Colls, 2007) • Blastocyst (trophoectoderm) biopsy: On the morning of day 5/6 post-insemination. ** (Clinical application very recent, limited data: De Boer,2004. Appplicability on a large scale needs validation) ESHRE PGD Guidelines Thornhill et al.,HR 2005 PGDIS Guidelines RBM Online 2007

  42. Recommendations • Insemination: Only ICSI for PCR cases(to eliminate the risk of paternal contamination), ICSI & IVF for FISH cases • Culture medium: Standard IVF culture medium with single embryo culture • Biopsy medium: Calcium, magnesium free medium. Media suppl. with sucrose to shrink the cells and allow more space in ZP • No.of cells to remove: No concensus.. The decision to remove 1/2 cells is based on cell no. and accuracy & reliability of the diagnostic test used. If 2 cells are going to be removed, it’s recommended only for embryos ≥6 cell embryos. (Goessen et al., HR 2008 decreased blast. formation, similar LBR. With two cells removed, efficiency of PCR analysis reduced, although FISH analysis not effected. De Vos et al., HR 2009 decreased blast. formation, and LBR).

  43. 1st. & 2nd. PB Biopsy Montag et al., RBM Online 2008

  44. Blastomere biopsy by pushing Blastomere biopsy by aspiration Wang et al.,F&S 2008

  45. Trophectoderm biopsy De boer et al.,F&S 2004

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