Session 16: ICSI - Q&A
Questions for Dr. Dean Morbeck
- Min Xu What about the 2PN/mature egg for regular IVF groups? If mature eggs can be calculated by subtracting any immature and degenerated egg from total eggs on day 1.
- Eva Schenkman: This number may not be truly accurate as some eggs could have matured late overnight that may not have been mature on Day 0. So still may not be truly equivalent.
- Ivette Vanrell: I am interested in your opinion on aspiration ICSI vs pressure ICSI. Thanks!!
- Dean Morbeck: Aspiration, stir and stretch ICSI all can work well. The key is good control when breaking the membrane and assuring the sperm “dives” into the cytoplasm.
- (For Dean) Min Xu University of Michigan: How is the total fertilization failure rate between two groups (conventional insemination vs ICSI)
- Dean Morbeck: Total fertilization failure is typically 2-3% higher for IVF than ICSI. Numbers vary but I’ve always seen 3-5% failed fertilization rate for IVF and 2-3% for ICSI.
- Jayaprakash Divakaran Do you have any data to show the fertilization rate between a group of patients who definitely needed ICSI due to male factor in comparison with those who had normal seminogram?
- Dean Morbeck: My 6th slides shows ICSI fertilization rate (per egg collected - not injected) for cases where the indication was a male factor or a female factor. I also included unexplained cases. In general, the fertilization rate per egg collected is 55% for male factor cases vs 60% for female factor cases where there the semen analysis was normal.
Questions for Dr. Caroline McCaffrey
- Jacques Cohen: Thanks Caroline and Congratulations. The most intriguing point for me from your work is the extremely low rate of complete fertilization failure (specially in the conventional IVF group). What is your “secret” when doing regular IVF?
- Sinan Özkavukcu: How many motile sperm/egg do you put into each droplet for IVF?
- Al Maravilla Are the semen samples processed differently between groups
- Michael Reed Do you adjust final sperm insemination concentration per oocyte based on morphological score, e.g. higher concentration for very severe morphology?
- Liesl Nel-Themaat: Can you talk about ploidy rates and how it relates ICSI vs conventional insemination?
Questions for Dr. Joe Conaghan
- Min Xu: (Michigan)What is the usable blastocyst rate from this tech? (is it different from the blast rate from the “traditional ICSI)?
- Nadia Elakabawy -Although I see the no aspiration technique gives a higher fertilization rate. I see it as harsh to eggs. Did you follow up the blastocyst formation and implantation rate? Thank you for the wonderful presentation.
- Kimball Pomeroy: Joe, did you look at embryo quality with the analysis of ICSI improvement?
- Dee Kini: Did you compare pregnancy rates instead of only fertilisation rate? Dr. Dee Kini.
- Remya A K: the technique seem to give a lot of stress to oocyte
- Teresa Ernest: Were the ages of the patients eggs more or less the same injected per embryologist? Could this have affected fertilization rates if for example, some got more donor eggs and younger patients compared to older patients?
- Tony Anderson: It appears in the video you were aspirating a bit. Yes/No?
- Answered live
- Erin Fischer: Yes, in the second video you do see the sperm move back a bit before going into the egg to assist with forward projection, but it is after the membrane has broken.
- Sumiyya Younas ( Pakistan): Do we need to pop the membrane from the opposite side of the oocyte in our teacher icsi?
- Sonal Vaidya: Will this technique cause over disruption of the ooplasm and risk degeneration of oocytes?
- Mariana Agatão: Dr. Conaghan, do you prefer to “move the needle around” inside the oocyte other than aspirating a little bit of its ooplasma until the membrane pops?
- MAURO CAIAZZO: A question for Joe conaghan, how much is the degeneration rate from the teacher? Using the this method
- Erin Fischer: Less than 2%
- Kelly Ketterson: Less than 2.5%
Questions for Dr. Karl Swann
- Lisa Leonard-Johnson: Karl what are your thoughts on using calcium ionophore activation on blastocyst development, do you think it can be negatively affected?
- Kimball Pomeroy: What percent of injected human oocytes become activated with PLC zeta RNA?
- Junaid Kashir: If PLCzeta is injected in the correct range (which varies between species) then a very high percentage of injected oocytes manage to activate and successfully fertilise. In mice, if PLCzeta is injected in the correct range with high quality PLCzeta protein/RNA, then the success rate is 80-95%
The success rate is comparable to ICSI with sperm.
- Jayaprakash Divakaran (UAE): How do you measure the calcium Oscillation?
- Junaid Kashir: Usually this is using a calcium sensitive dye which is either injected or introduced into the oocyte. This dye increases in intensity in the presence of calcium, and decreases as calcium decreases, allowing us to image calcium changes in real time. Other methods have also correlated cytoplasmic
- Sung Tae Kim (Ohio): Any relation between variable PLC localization and sperm abnormality?
- Junaid Kashir: Generally, levels and localization patterns seem to exhibit very strong correlations with sperm parameters. Lower levels of PLCzeta and abnormal localisation correspond to abnormal sperm (low motility, abnormal morphology, and low count).
A big example is globozoospermia, which has mostly been studied in relation to PLCzeta. Levels of PLCzeta are absent, and if sperm do exhibit some PLCzeta, the localisation is abnormal.
There are also many other conditions and factors now being associated with PLCzeta abnormalities. For More detail you can refer to some very recent papers by our lab:
- Yu Sun: Do you inject PLC-zeta to all oocytes of patients? How do you decide on which oocytes to inject with PLC?
- Junaid Kashir: Currently, PLCzeta has not been used to treat patients. All data in humans is purely experimental, and oocytes/embryos only examined as far as the blastocyst stage.
- ali zain: how can we read about efficacy and dose rates of CA ionophore injection
- Junaid Kashir: There are many reviews by myself and Karl, as well as Drs Ebner and Montag discussing this issue.
- Carolina Petrella: Is there a way to look at sperm morphology and correlate it with Oocyte activation success
- Junaid Kashir: Generally, abnormal sperm morphology correlates very strongly to abnormalities in PLCzeta levels and localization patterns
- Kimball Pomeroy Have you tried to use ethanol to activate human oocytes?
- Junaid Kashir: Many studies have previously succesfully done this, and while fertilisation is succesful, development is very poor. Very few clinics would clear using an alcohol for patients.
- Ciler CELIK-OZENCI: Regarding FF; what do you think about the possible role of centriole carried by sperm to the oocyte? oocyte has centriolar material but no centriole. does sperm centriole have a place in oocyte activation?
- Junaid Kashir: The centriole of course would play a large role in syngamy. This would play a significant role in oocyte activation, but is more driven by oocyte activation rather than driving oocyte activation.
Events such as sperm head decondensation and nuclear condensation/envelope breakdown are also events that are calcium-dependent (as as such influenced by PLCzeta in theory).
Cell division rates, and profiles of gene expression are directly influenced by levels of PLCzeta and calcium release. If calcium levels or PLCzeta levels are too high,, cell division occurs faster, and if low then cell division is slow as well.
Calcium release and levels of PLCzeta seem to be required in a specific window for ideal embryogenesis to occur
- Philip Chan Can we use pin prick to activate the oocyte?
- Junaid Kashir: Mechanical activation is more successful in animal models. In humans this seems to be more damaging than useful
- David McCulloh: Pin prick is unlikely to work since the ICSI pipette is doing that even with sperm. I suspect that 30% failure is not due to failing to get into the egg (full prick) so the sperm seems much more effective than the prick (this is true for many species)
- Karl Swann: So called ‘mechanical activation’ will only cause a single Ca2+ increase. The sperm usually triggers Ca2+ oscillations that last for hours. We need to generate Ca2+ oscillations to improve activation and later development.
- Shaden Baddar(UAE): Will using PLC zeta improve fertilization rates for cases with Globo-zoospermia?
- Junaid Kashir: This has been successfully done by many groups where globozoospermia sperm fertilisation can be rescued by using calcium ionophore, which has resulted in live births. With PLCzeta, calcium release and fertilisation can rescue fertility, but development past the blastocyst stage has not yet been studied
- Robert Heudes: would you be able to isolate micro vesicles (MV) or EV as a transporter of PLC Zeta. this was not the case..at no point was EVs or MVs mentioned as transporter during this presentation?
- Junaid Kashir: This is something being investigated by ours and many other labs, but the issue is still reliably making PLCzeta in a recombinant form. There is much still to be done to make this in a reliable manner. Following this, examining the right composition of said EVs and MVs would be also important. So a good idea in theory, but practically very demanding and would take a long time.
- Sung Tae Kim Is artificial oocyte activation reagent (Ionophore) available in the US?
- Junaid Kashir: Yes. you can purchase from Sigma Aldrich
- Richard Rawlins: Overuse of ICSI is tied to desire to avoid having to tell patients there has been fertilization failure during IVF, but in my experience directing 7 different labs, it has also been linked to the fact that in the USA there is a separate CPT code for ICSI and the clinic can bill an additional charge for using it. Clearly it is not necessary for all ART cases. RGR
- David McCulloh: Yes this is definitely a difficult situation to defend…
- Charles Muller: Karl, Is there a likely relationship between PLCzeta activity and acrosome reaction or cytosol alkalinization? ie, any concerns about ICSI vs natural insemination?
- Andrew Gordon: For Karl - Any value going back to the Hamster Oocyte Penetration Test?
- Karl Swann: This test will not tell you whether the particular sperm you use for ICSI has enough PLCzeta.
MC: And… how can we get Sr2+?
Karl Swann: Sr2+ does not cause Ca2+ oscillations or activate human oocytes. If it did, no one would ever have bothered with Ca2+ ionophores.
- Yu Sun: Why don't' all labs use piezodrill for ICSI?
- Karl Swann: Piezodrill can be tricky to learn compared with conventional ICSI. It involves more expensive equipment and requires the use of mercury or fluorinert. In principle it’s still probably better to do ICSI.
- Liesl Nel-Themaat: Can anybody talk about mechanical activation (excessive mixing of the cytoplasms) during ICSI in patients with prior fertilization failure?
- Karl Swann: Mechanical activation will not generate Ca2+ oscillations. Excessive mixing of cytoplasms is probably causing one single large Ca2+ increase.
- Jimmy Portella (Peru): What is your opinion about Piezo ICSI?
- Liesl Nel-Themaat Piezo is mainly used for mouse ICSI since it is very hard to penetrate the mouse oolemma. Not sure it has any value in humans.
- Dean Morbeck: Piezo has been used extensively in Japan and shows value for flattening the learning curve. There are no reports illustrating this nor have any clinics outside of Japan provided evidence that piezo ICSI is superior to mechanical ICSI. However- since Piezo is now readily available for clinical ART laboratories outside of Japan, it’s use may increase and we may learn if it is of value.
- Min Xu: has anyone tried zn chelator on human egg activation?
- Karl Swann: The zinc chelator TPEN has been tried but only with limited numbers. In mouse eggs TPEN is not as good at activating development as stimuli that cause Ca2+ oscillations.
- Barbara Podsiadly My question is in relation to 3PN rate. Ours is approaching 8% in IVF and 3% in ICSI. Do you think this is solely due to sperm number inseminated for IVF or are there other factors we should be looking at in the lab to try and reduce this. Obviously for ICSI this is an egg factor .. but just wanted to see if there was something else we can do for IVF.
- Dean Morbeck: A high 3PN or polyspermy rate is most commonly due to inseminating with too many sperm. Concentration of sperm is typically no higher than 200,000 motile sperm per mL. If you still see high 3PN rates, try reducing the sperm concentration further.
- Sung Tae Kim: Is that powder, right? How about HFEA (GM 508)?
- Karl Swann: The GM508 is a ready to use solution with ionophore made up in medium.