Johnson et al’s (1) efforts to rescue and utilize immature oocytes generated during ovarian stimulation cycles which would otherwise be wasted are commendable. This author’s experience in a similar endeavor (2) suggests that ovarian stimulation regimes may generate approximately 20% immature oocytes (1, 2), which is a significant loss if efforts are not made to rescue and utilize them.
The poor maturation rate of germinal vesicle-stage (GV) oocytes reported by Johnson et al. with cumulus cell (CC) but not granulosa cell (GC) co-culture was probably because 24 hours is insufficient to attain maturity. GV oocytes may take up to 48 hours to mature even in specialized IVM medium (Medi-Cult, Denmark) containing FSH, hCG and follicular fluid with cumulus cell co-culture (3). Johnson et al’s findings of poor outcome after IVM led them to conclude their extended culture techniques with and without cumulus cells (CC) in standard medium is inefficient and a waste of resources.
This author’s preliminary observations suggests a high level of maturity of metaphase I oocytes (12/12) in ultra micro-drop (UMD) culture of about 1.5 to 2µL (4) within 24 hours in standard medium (Cleavage Medium, Cook IVF, Ireland). GV oocytes that matured under similar conditions were somewhat lower at 24 hours (39.4%, 13/33) but improved marginally by 48 hours of IVM (48.5%, 17/30). Under similar conditions of IVM, but with autologous GC and or CC co-culture in a 2.5 to 3.0 µl of medium UMD culture system, a higher proportion of GV oocytes attained maturity (54.5%, 6/11, p=0.5981) at 24 hours and (81.8%, 9/11, p=0.1138) 48 hours. Paracrine factors released into the UMD co-culture milieu by the egg, GC and CC concentrated within the ultra volume of the culture system may be involved in the maturation of GV oocytes. This author’s findings in UMD+GC+CC co-culture were somewhat higher at 24h (54.5% vs 23.8%) but similar at 48 hours of IVM (81.8% vs 85.7%) to that obtained with specialized IVM medium+CC+follicular fluid+FSH+hCG supplementation (3) but cytoplasmic maturity in the former remains to be elucidated. Although these data are preliminary, it appears reasonable to suggest that efforts to rescue immature oocytes from stimulated cycles be continued.
Jaffar Ali, PhD
IVF Laboratory, RMU, WSH
King Fahad Medical City
Kingdom of Saudi Arabia
1. Johnson JE, Lee Higdon HL, Boone WR. Effect of human granulosa cell co-culture using standard culture media on the maturation and fertilization potential of immature oocytes. Fertil Steril 2008; 90: 1674-9.
2. Ali J, Joshi HN, Al-Badr M, Shahata MAM, Abdulkader A, Al-Natsha S, Flamerzi M et al. Subzonal insemination of immature oocytes generated during intracytoplasmic injection cycles. Med. Sci. Res. (UK) 1998; 26:389-91.
3. Zhu XM, Zhu YM, Xu CM, Qian YL, Jin F, Huang HF. Autologous mature follicular fluid:its role in in vitro maturation of human cumulus-removed oocytes. Fertil Steril 2008; 90:1094-102
4. Ali J. Continuous ultra micro-drop (cUMD) culture yields higher pregnancy and implantation rates than either larger-drop culture or fresh-medium replacement. Clin. Embryologist. 2004; 7:1-23.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.04.027
The Authors Respond:
We would like to thank Dr. Ali for his interest in our research and appreciate his supportive comments. Our goal in attempting to mature MI and GV oocytes was to decrease oocyte wastage and, hopefully, provide more viable embryos for our IVF patients’ use for ET or cryopreservation. While our process of using standard culture media proved inefficient in these respects (few immature oocytes matured, fertilized and then cleaved to embryos), it is heartening to know that, as we theorized, the use of specialized culture medium (such as medium supplemented with autologous granulosa cells and mature follicular fluid) and techniques (such as continuous ultra micro-drop culture) can improve the nuclear maturation rates of immature oocytes. It is disappointing that, as yet, we have been less successful in attaining cytoplasmic maturation of immature oocytes and unfortunately, the production of additional usable embryos remains low. Perhaps future research will overcome this obstacle as well.
Jane E. Johnson, MS
William R. Boone, PhD
Department of Obstetrics and Gynecology
Greenville Hospital System University Medical Group
Greenville, South Carolina
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.04.028