Role of Ca2+ ionophore in ICSI failed fertilization

Transcription

1 Role of Ca2+ ionophore in ICSI failed fertilization Liow Swee Lian DVM, PhD O & G Partners Fertility Centre Gleneagles Hospital SINGAPORE

2 Process of Fertilization Generated by Foxit PDF Creator Foxit Software

3 The fertilization calcium wave. The calcium wave initiates at the point of sperm entry and crosses the oocyte as a tsunami-like wave. McDougall et al (2000)

4 Calcium influx at fertilization Small elevation of calcium just beneath the plasma membrane Rapid influx of calcium Subcortical calcium increase No local change in cytoplasmic calcium concentration Swann et al (1994)

5 Oocyte activation failure ICSI most efficient procedure in treating severe male infertility, failed IVF and unexplained infertility 1 5% ICSI cycles = Partial/complete failed fertilization Fertilization failures Minor causes incorrect immobilization/ injection of sperm expulsion of injected sperm from the oocyte partial decondensation of sperm head Principal cause Deficiency in mechanism of oocyte activation

6 IMMOBILIZATION OF SPERM Is MANDATORY even with totally non-motile but viable sperm Sperm cytosolic factor Release of intracellular Ca 2+ Ca 2+ oscillations Resumption of meiosis in oocyte Sperm head nuclear decondensation Pronuclear formation

7 Localization of PLC zeta in normal fertile and infertile sperm Kashir et al (2010) Hum Reprod Update

8 Inositol 1,4,5-triphosphate (InsP3) increases at fertilization after microinjection of PLC-δ

9 Calcium inducing ability between fertile and infertile sperm Kashir et al (2010) Hum Reprod Update

10 PLC zeta A Sperm-Oocyte Activating factor Histine > Proline point mutation (Heytens et al, 2009) Microinjection of wild type and mutant PLC zeta into mouse oocytes Resulting Ca2+ releasing patterns

11 Ca 2+ Oscillations/Spikes Number of calcium spikes - determine OA efficiency Has much influence on embryo development Sum of elevated Ca2+ most important rather than normal pattern of Ca2+ oscillation

12 Fertilization Failure Origins: or Mouse Oocyte Activation Test (MOAT) Sperm activation potential/ability Human sperm injected into mouse MII oocytes Endpoint pronuclear formation and 2-cell mouse embryos

13 Fertilization Failure Origins: or Human Oocyte Activation Test (HOAT) Useful in Total Fertilization Failure sperm or oocyte problem Proven donor sperm injected into patient s fail-to-fertilize oocytes Husband s sperm injected into donor IVM/ unfertilized oocytes End-point: PN formation

14 Artificial Oocyte Activation 1. Electro-activation by Electrical pulses 2. Chemical activation ionomycin/ca 2+ ionophore 3. Recombinant activating factor/crna PLC zeta Liow SL 2010

15 Electro-activation of Oocytes + Platinum electrode + e 250 um - - Platinum electrode Activation medium: 0.3 M Mannitol, 0.05 mm CaCl 2, 0.1 mm MgSO 4 Activation parameter: 100 usec duration, 1500 mv/cm Liow SL 2010

16 Artificial Oocyte Activation A Calcium ionophore 2. An antibiotic produced by Streptomyces chartreusis NRRL Capable of extracting Ca2+ from the bulk of an aqueous phase (culture medium) into an organic phase (cytoplasm) 4. Facilitates Ca2+ transport across cell membrane 5. Less efficient in Ca2+ transport 6. 2 mol of A23187 : 1 mol of Ca+ Ionomycin 1. Calcium ionophore 2. An antibiotic produced by Streptomyces conglobatus ATCC Capable of extracting Ca2+ from the bulk of an aqueous phase (culture medium) into an organic phase (cytoplasm) 4. Facilitates Ca2+ transport across cell membrane 5. More efficient Ca2+ transport 6. 1 mol of ionomycin : 1 mol of Ca2+

17 Artificial Oocyte Activation Ca 2+ ionophore / ionomycin 1. Prepare ionomycin solution [1:10 (v/v)] 2. First Activation: 30 min after ICSI, place ICSI oocytes into 10 um ionomycin droplet ; Incubate for 10 minutes 3. Wash the oocytes in IVF droplets 4. Second Activation: 30 min after first activation, place oocytes into 10 um ionomycin droplet, incubate for 10 minutes 5. Wash oocytes in IVF droplets; Transfer to culture droplets

18 Artificial Oocyte Activation Ca 2+ ionophore / ionomycin Add 90 ul IVF medium FINAL WORKING SOLUTION = 10 um ionomycin 10ul of 100 um ionomycin + 90 ul IVF medium 10 ul stock solution (1M) ionomycin 100 ul 100uM ionomycin

19 Proven fertile donor Globospermic man Globospermic + oocyte activation with ionomycin

20 Summary Oocyte activation is essential in promoting release of meiotic arrest in the oocyte A sperm-oocyte activating factor (SOAF) eg PLC zeta Sperm deficient in SOAF poor fertilization /total fertilization failure Artificial oocyte activation can be used to treat fertilization failure.