In Vitro Maturation of Immature Oocyte in Patients with PCOS

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Mark Perloe, MD:  “I’m here with Dr. Seang Lin Tan at the Advances in Infertility Treatment in Fort Lauderdale, Florida, and we’ve heard a lot of papers presented on how IVF rates are improving.  We’ve noticed some of the differences in dosing between European programs and programs in the United States but one of the things we’ve realized is we haven’t been able to bring the cost of IVF down, we haven’t really improved the safety, and certain patients are still at significant risk for OHSS.  I found your presentation of the alternative very interesting, and I wonder if you could comment on your work?” 

Seang Lin Tan, MD:  “Clearly IVF success rates have improved significantly over the last ten to fifteen years and today we can safely say that the result of IVF in good infertility programs are such that a woman has a better chance of pregnancy going through IVF than she has going through a spontaneous menstrual cycle and  natural conception even if she’s totally fertile.  The problem, however, is we pay a price to have such good success rates and the price that we pay is that there’s a higher incidence of multiple pregnancy and ovarian hyperstimulation syndrome.  I think as we move into the new millennium while we are looking at further improvements in IVF success rates we need to also look at ways to reduce these complications.  A number of years ago we identified young women and women with polycystic ovaries seen on ultrasound scan.  I mean women who just have an ultrasound appearance of PCO and who do not necessarily need to have a full clinical and biochemical picture of polycystic ovarian syndrome with irregular menstrual cycles at normal hormonal levels and so on as being at a particularly high risk of developing OHSS.  So one of the strategies that we have attempted to try to achieve in the last few years is how to treat these people more successfully while reducing complication rates.” 

Mark Perloe, MD:  “When we looked at in vitro maturation of follicles our experience initially started after giving HCG on the day of retrieval.  We had some eggs that were mature but we also had some that were germinal vesicle eggs and these GV embryos that matured never gave us pregnancies.  How did you think the experience might be different in looking at earlier, smaller follicles?” 

Seang Lin Tan, MD:  “I think GV oocytes in the context of a standard IVF cycle where ovarian stimulation has been employed is about as different as in a non-stimulative cycle.  In our case, we perform a baseline ultrasound scan to count the number of antral follicles in the ovaries as well as identify whether the patient has polycystic ovaries or not.  Provided they have polycystic ovaries and/or the antral follicle count is more than twenty, we offer them the alternative of having IVM or IVF.  Now clearly we can also offer IVM in patients who have a lower antral follicle count but the pregnancy rate is correspondingly lower.  What we then do is at the beginning of the menstrual cycle, either a natural cycle or a hormonally induced menstrual cycle, we perform a baseline ultrasound scan on day two or three of the cycle.  We then perform a repeat ultrasound scan between day six to eight of the cycle.  At that time we make sure what the endometrial lining is like, and we look at whether there is a dominant follicle or not because we wish to avoid one.  Then if everything else is normal, we give them a single injection of HCG in a dose of 10,000 IU and thirty six hours later we perform oocyte retrieval in the natural menstrual cycle.” 

Mark Perloe, MD:  “The HCG receptors are present in the theca at that time but we’ve heard other speakers say that HCG receptors or LH receptors are not present within granulosa cells on day eight so how are we seeing the oocyte respond in resuming myosis, and how are we seeing the separation of the oocyte from the wall of the follicle if the receptor isn’t present?” 

Seang Lin Tan, MD:  “I think what our data clearly suggest is that they are receptor expressions and what is happening is we find if we do not give HCG when the appearance of the GV oocyte at the time of oocyte retrieval looks a little different as compared to those where HCG has been given, for example, 45% of the oocytes that are collected at the time of retrieval if they’ve had HCG pre-priming they would have undergone GVBD.  They would mature very quickly within 24 hours and by 48 hours over 80% of them would have reached metaphase II stage in comparison to those who have not had HCG pretreatment, and the incidence of maturation to M2 stage is about 69%.  So the pretreatment of HCG both accelerates the development of M2 oocytes as well as increases the percentage of GV oocytes that reaches M2.” 

Mark Perloe, MD:  “Now you had noted that at 24 hours many of these are M2 but some take the 48 hours.  You’re looking at them at 24-hour intervals so your embryologists are doing ICSI twice and you’ve resorted to ICSI on these.  Why ICSI as opposed to regular in vitro fertilization?” 

Seang Lin Tan, MD:  “With conventional IVF practice the shell, so to speak, may be a little harder if you have undergone in vitro culture.  Now our traditional practice still is to perform ICSI for all these patients.  We’ve had some recent data suggesting that provided there has been no abnormal semen analysis at all previously then the fertilization rates, whether you do ICSI or not, is probably the same but because our program is still relatively new we therefore hesitate to change too much at the same time so we’ve still been doing ICSI.” 

Mark Perloe, MD:  “Is there information that those that mature on the first day behave differently than those on the second?” 

Seang Lin Tan, MD:  “We don’t have any information on this yet.  We would perform a single embryo transfer two or three days after fertilization.  We start the patient on estrogen supplementation on the day of oocyte retrieval and we start progestogen supplementation on the day of ICSI.” 

Mark Perloe, MD:  “Now we’ve heard about estrogen perhaps preventing the expression of vitronectin receptors and Dr. Lessey’s work was quite interesting in that area.  Are you continuing the estrogen throughout the luteal phase or after transfer or do you maintain estrogen just up until transfer?” 

Seang Lin Tan, MD:  “We maintain estrogen right through until the pregnancy test is done.  I guess that’s a little different in the IVM cycle versus in the natural cycle because here we have given no hormonal stimulation and we have aspirated autosites so we have emptied all the granulosa cells so I think that that is a distinctive difference there.” 

Mark Perloe, MD:  “You adjust the dose of estrogen based on the thickness of the endometrium on day eight?” 

Seang Lin Tan, MD:  “On the day of egg retrieval so if on the day of egg retrieval the endometrial thickness is less than 6 mm we would give a dose of estrogen, 10-12 mg of estradiol valerate.  However, if the endometrial thickness is more than 8 mm then the dose of estradiol valerate is 6 mg.” 

Mark Perloe, MD:  “How are these excess embryos behaving after cryopreservation?” 

Seang Lin Tan, MD:  “The same as with conventional IVF; we find that the implantation rate with IVM embryos is lower.  Overall, we are getting about a 9.5% implantation rate as compared to a 17% implantation rate with IVF but the overall pregnancy rate with IVM, provided the patient has more than ten immature oocytes collected, is about a 30% clinical pregnancy rate per cycle.” 

Mark Perloe, MD:  “You have looked at other factors besides the initial antral follicle count to help you decide and predict who might be a good candidate.  What were those factors and are you still using them?” 

Seang Lin Tan, MD:  “We routinely measure the intraovarian stromal maximum velocity to blood flow but we have found that is not as predictive in IVM as compared to conventional IVF and probably the reason is because there is no hormonal medication being used.  In conventional IVF, we believe that Vmax is predictive of ovarian stimulation success rates because if there is better blood flow there is more delivery of hormones to the target cells whereas in IVM because you’re not actually giving any medications I think it probably does make a difference.” 

Mark Perloe, MD:  “In terms of the blood flow around individual follicles, at what stage is that first demonstrated and have you been able to see a ring of follicular blood flow on these follicles?” 

Seang Lin Tan, MD:  “Not in IVM, in IVF, yes.  In IVM the follicles are too tiny for blood flow to be meshed separately around each follicle.  The endometrial thickness we would like to achieve would be ideally above 10 mm in diameter at the time of embryo transfer.” 

Mark Perloe, MD:  “With further research in this area, where are you looking to take us?” 

Seang Lin Tan, MD:  “At the moment we are now able to collect immature oocytes from about 50% of antral follicles.  Clearly the single most important clinical step forward would be if we were able to increase the percentage of egg recovery to the 75%-80% that we would get in conventional IVF.  I believe this is possible given the fact that ultrasound technology is constantly improving so that we are now able to see even smaller follicles more clearly on ultrasound scan.  We are now in the process of manufacturing a finer needle and by combining it with a better ultrasound machine we hope in the next year or two to increase the percentage of follicles on whom we can get immature oocytes.  At the other extreme, hopefully, we’ll be able to increase the maturation rate of GV oocytes to M2 oocytes from the 75%-80% that we are currently observing to closer to 90%.  Those would be the two main advances, ideally and obviously we would like to combine this with a preimplantation genetic diagnosis program so that we can reduce the number of embryos that we transfer and by doing so reduce the multiple pregnancy rate.” 

Mark Perloe, MD:  “Thank you so much.” 

Seang Lin Tan, MD:  “You’re most welcome."