To the Editor:
Jee et al. (1) report that in their hands a prematuration culture using a phosphodiesterase type 3 (PDE3) inhibitor does not improve developmental competence of immature oocytes. We and others previously reported a beneficial effect of this system (2-6). Fundamental differences in experimental design between their work and ours may explain this discrepancy.
1. We employed a mouse model designed specifically to approximate the cytoplasmic immaturity that hinders IVM in the clinical setting. Oocytes derived from small antral follicles in women are cytoplasmically compromised compared to oocytes derived from larger preovulatory follicles. Follicular development in mice is more synchronized than in humans, so to model cytoplasmic immaturity we retrieved immature oocytes from mice primed with eCG for 24h, rather than 48h (2, 6). Jee et al. retrieved oocytes 48h after priming, when they already have achieved cytoplasmic maturation in vivo. These oocytes would not be expected to benefit from additional cytoplasmic maturation in vitro. Indeed, exposing cytoplasmic mature oocytes at GV-stage to PDE3 inhibitor for an extended period of time in culture would render them post-mature. Jee’s paper, therefore, reported the effects of postmaturation not prematuration culture on developmental competence.
2. The authors implied that differences between their IVF results and ours may be explained by differences in mice IVF systems. However, as mentioned above, their results cannot be compared with ours, since we used oocytes at an earlier stage of development. Results from in vivo matured oocytes best reflect IVF system quality, which is lacking in the study by Jee et al. Our IVF experiments, unlike those of Jee et al, were performed in several replicates and with rigorous statistical analysis and our in vivo controls resulted in >80% 2-cells, ≥70% blastocysts (2, 6), acceptable for mouse IVF.
3. The authors made incorrect statements regarding our published works. They state that prematuration culture of human oocytes with PDE3 inhibitors has been applied only during a 24-h period. We have cultured oocytes in prematuration media for 24, 48 and 72 hours (4, 5), and more recently showed that embryos could be obtained from human oocytes prematured for 48 hours (4).
4. The authors incorrectly claim that our human study (4) did not show differences in oocyte maturation and embryonic development between prematured oocytes and controls: we observed a 20% higher maturation rate in human oocytes prematured in PDE3 inhibitor compared to controls. Moreover, we obtained significantly more embryos of good quality on day 3 post-ICSI from prematured oocytes compared to controls (83% vs 53%), rates similar to in vivo matured human oocytes (88%).
In conclusion, the report by Jee et al that PDE3 inhibitors do not improve IVM is unfounded. We suggest that they consider employing a more appropriate experimental paradigm and more rigorous statistical analysis to avoid flawed conclusions.
Daniela Nogueira, Ph.D. Laboratoire de Biologie de la Reproduction, IFREARES Toulouse, France
Leen Vanhoutte, B.Sc. Department of Reproductive Medicine Ghent University Hospital Ghent, Belgium
1. Jee BC, Chen HY, Chian RC. Effect of a phosphodiesterase type 3 inhibitor in oocyte maturation medium on subsequent mouse embryo development. Fertil Steril. Published online June 11, 2008. doi:10.1016/j.fertnstert.2008.03.041.
2. Vanhoutte L, Nogueira D, Gerris J, Dhont M and De Sutter P. Effect of temporary nuclear arrest by Phosphodiesterase 3-Inhibitor on morphological and functional aspects of in vitro matured mouse oocytes. Mol Reprod Dev 75:1021-1030.
3. Zeng HT, Yao SZ, Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes. Hum ReprodRon-El RFriedler SSchachter MRaziel ACortvrindt RMeiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development. Biol Reprod. 2006;74:177-84.
4. Ron-El R, Raziel A, Cortvrindt R, Smitz J. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development. Biol Reprod. 2006;74:177-84.
5. Nogueira D, Albano C, Adriaenssens T, Cortvrindt R, Bourgain C, Devroey P, Smitz J. Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor in vitro. Biol Reprod. 2003;69: 1045-52.
6. Nogueira D, Cortvrindt R, De Matos DG, Vanhoutte L, Smitz J. Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro. Biol Reprod. 2003; 69:2045-52.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2008.12.110
The Authors Reply:
We have recently reported that a prematuration culture (PMC) using a phosphodiesterase type 3 (PDE3) inhibitor (cilostamide) does not improve in vitro developmental competence of immature mouse oocytes (1). Nogueira and Vanhoutte claimed that our negative findings about the effect of cilostamide have clear discrepancy because other researchers have reported a beneficial effect of this system using murine model (2, 3).
Nogueira et al. (2) demonstrated for the first time that the maturation rate after treatment with the PDE3 inhibitor (Org 9935) was similar to that in non-arrested controls, but the fertilizability was significantly improved, and the capacity for embryonic development was enhanced in mouse IVM system. Subsequently, Vanhoutte et al. (3) reported same beneficial effect using different PDE3 inhibitor (cilostamide). Nogueira and Vanhoutte pointed that there were fundamental differences in experimental design between their work and ours, which may explain those discrepancy.
They firstly argued that the time of retrieval was different, i.e., we obtained cumulus-oocyte complexes (COC) 48h after gonadotropin injection but 24h in previous two reports (2,3). They postulated that no effectiveness of PMC with cilostamide in our study could be attributed to longer duration of gonadotropin exposure; immature oocytes retrieved 48h later after gonadotropin injection could be cytoplasmically matured than those retrieved 24h later, thus additional culture by PMC could result in over-maturation(?) of them. However, this assumption is still premature because none of the researchers have directly compared the effectiveness of PMC according to duration of gonadotropin exposure. In general, without PMC, immature oocytes retrieved 48h later after gonadotropin injection have better developmental competence than those retrieved 24h later; if PMC with cilostamide has a positive effect in immature oocytes retrieved 24h later after gonadotropin injection, IVM outcomes should also be compared between immature oocytes retrieved 24h later after gonadotropin injection combined with additional 24h’s PMC and those retrieved 48h later without additional PMC. If the outcomes are similar between the two groups, the impact of PMC will be meaningless.
We have several published data with regards IVM and cryopreservation from mouse model (4-8). Although we did not include the results from in vivo matured oocytes in our study (1), we do not believe that our mice IVM/IVF systems are not acceptable.
We did not describe specifically that PMC of ‘human’ oocytes with PDE3 inhibitors was applied only during a 24h period in ‘work by Nogueira et al.’ (9) in our manuscript. In the Introduction section, we mentioned that ‘PMC with PDE3 inhibitors (including mouse, cattle, and human) has been applied only during 24h periods’ and we subsequently introduced the good work by Nogueira et al. (9) comparing 24h and 48h’s exposure; the authors concluded that a 24h PMC period is the most effective in preserving embryonic integrity.
Nogueira and Vanhoutte also claimed that we incorrectly mentioned their work on human study (9). In their study, the maturation rates (up to 30h) were similar between 24h (67%) and 48h PMC (67%) and those maturation rates were similar to CEO control without PMC (63%) but higher than denuded control without PMC (46%). Indeed, the same trend was also found at observation after maturation up to 48h (74%, 73%, 71%, 54%). In their study, two control groups were used because CEOs undergone PMC as undenuded state but subsequent IVM was performed as denuded state. Clearly, maturation rate after 24h PMC was higher by 20% when compared with denuded control but only by 3% when compared with intact CEO control. Since most of current IVM procedure was accomplished as undenuded, intact CEO state, their results may be insignificant in clinical setting. In general, denuded immature oocytes have lower maturation and development potential than intact CEO. Their work merely indicated that IVM outcome from intact CEO without PMC is similar to IVM of denuded CEO with PMC. Therefore, from Nogueira’s stidy (9), it can be concluded that if CEOs are not denuded, PMC would be unnecessary.
In a work by Shu et al. (10), it was evident that PMC treatment using cilostamide (20 μM for 24 h) had no effect on fertilization and embryonic development of human COCs (collected without any stimulation). However, they demonstrated that combined treatment of cilostamide with forskolin would enhance the blastocyst formation rates, although significant differences were not reached.
Taken together with previous two reports (9,11), there still has been no concrete evidence to demonstrate the beneficial effect of PMC with PDE3 inhibitors on the developmental competence in human GV-stage oocytes.
Byung Chul Jee, MDDepartment of Obstetrics and Gynecology
Seoul National University Bundang Hospital
Seongnam, Korea Ri-Cheng Chian, Ph.D.
Department of Obstetrics and Gynecology
Montreal, Quebec, Canada
1. Jee BC, Chen HY, Chian RC. Effect of a phosphodiesterase type 3 inhibitor in oocyte maturation medium on subsequent mouse embryo development. Fertil Steril;in press.
2. Nogueira D, Cortvrindt R, De Matos DG, Vanhoutte L, Smitz J. Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro. Biol Reprod. 2003; 69:2045-52.
3. Vanhoutte L, Nogueira D, Gerris J, Dhont M and De Sutter P. Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes. Mol Reprod Dev 2008;75:1021-1030.
4. Xu L, Wang Y, Zhou P, Cao YX, Huang TH, Chian RC. Cytogenetic analysis of in vivo and in vitro matured oocytes derived from naturally cycling and stimulated mice. Syst Biol Reprod Med 2008;54(3):155-62.
5. Huang JY, Chen HY, Tan SL, Chian RC. Effect of choline-supplemented sodium-depleted slow freezing versus vitrification on mouse oocyte meiotic spindles and chromosome abnormalities. Fertil Steril 2007;88(4 Suppl):1093-100.
6. Huang JY, Chen HY, Park JY, Tan SL, Chian RC. Comparison of spindle and chromosome configuration in in vitro- and in vivo-matured mouse oocytes after vitrification. Fertil Steril 2007;in press.
7. Ruvolo G, Huang JY, Chen HY, Ding H, Jee BC, Chian RC. Chromosome aneuploidy and DNA fragmentation in mouse oocytes matured in vitro. Fertil Steril 2007;submitted.
8. Wang Y, Ock SA, Chian RC. Effect of gonadotrophin stimulation on mouse oocyte quality and subsequent embryonic development in vitro. Reprod Biomed Online 2006;12(3):304-14.
9. Nogueira D, Ron-El R, Friedler S, Schachter M, Raziel A, Cortvrindt R, et al. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development. Biol Reprod 2006;74:177–84.
10. Shu YM, Zeng HT, Ren Z, Zhuang GL, Liang XY, Shen HW, Yao SZ, Ke PQ, Wang NN. Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes. Hum Reprod 2008; 23:504-13.
11. Vanhoutte L, De Sutter P, Nogueira D, Gerris J, Dhont M, Van der Elst J. Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase 3-inhibitor. Hum Reprod 2007;22:1239–46.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2008.12.111