Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection

FERTILITY AND STERILITY VOL. 72, NO. 2, AUGUST 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection Dimitris Loutradis, M.D., Peter Drakakis, M.D., Konstantinos Kallianidis, M.D., Spyridon Milingos, M.D., Spyridon Dendrinos, M.D., and Stylianos Michalas, M.D. IVF Unit, Alexandra Maternity Hospital, First Department of Obstetrics and Gynecology, Athens University Medical School, Athens, Greece Objective: To evaluate the relation of oocyte morphology with embryo quality and pregnancy rates (PRs) after intracytoplasmic sperm injection (ICSI). Design: Retrospective study of patients undergoing ICSI. Setting: University Hospital IVF Center. Patient(s): Sixty-eight patients who underwent ICSI and had transfer of good-quality embryos (grade 3), 60 patients with transfer of both good- and poor-quality embryos (grade 3 and grade 2), and 18 patients with transfer of poor-quality embryos (grade 2). Intervention(s): Comparison of the outcome of ICSI in the three groups of patients and the relation of oocyte morphology to embryo quality. Main Outcome Measure(s): Oocyte morphology and embryo quality (grade). Fertilization, cleavage, and pregnancy rates. Serum E 2 on the day of hcg administration. Result(s): Oocytes with poor morphology (dark cytoplasm; many vacuoles or fragments in cytoplasm) led to poor-quality embryos and consequently to lower PRs (5.5% versus 29.4%). Serum E 2 on the day of hcg administration was significantly higher in the group with good-quality embryos compared with that with poor-quality embryos (2,047 135.7 versus 1,651 164.8 pg/ml, respectively). Conclusion(s): Serum E 2 on the day of hcg administration is a marker of embryo quality. Oocyte morphology correlates well with embryo quality and PRs after ICSI. (Fertil Steril 1999;72:240 4. 1999 by American Society for Reproductive Medicine.) Key Words: ICSI, oocyte morphology, embryo quality, pregnancy rate Received March 20, 1998; revised and accepted March 22, 1999. Reprint requests: Dimitris Loutradis, M.D., Sirinon 62, P. Faliron 17561, Greece. 0015-0282/99/$20.00 PII S0015-0282(99)00233-2 Intracytoplasmic sperm injection (ICSI) has improved the outcome for couples with male factor infertility (1). Mammalian models, used in conjunction with micromanipulation systems, are ideal for conducting fertilization studies. The requirements for maturational events necessary for gamete fusion, such as oocyte activation, capacitation, the acrosome reaction, and interaction between sperm and egg membranes, have been brought into a new perspective since fertilization has been examined through micromanipulation (2, 3). In conventional IVF, oocyte maturity is assessed indirectly. Thus, the nuclear maturity and the morphologic appearance of the oocytes themselves cannot be examined (3). In ICSI, on the other hand, the morphologic structure of the denuded oocytes can be assessed in a more detailed and precise manner. In mammals, the preovulatory surge of gonadotropins initiates a series of events that lead to the maturation and subsequent ovulation of one or more oocytes (4). The occurrence of a complex sequence of maturational changes during the process of follicle growth confers on the oocytes the capacity to undergo normal fertilization and subsequent embryonic development (5). Gross morphologic alterations in the nucleus are an inadequate index of complete maturation of the oocyte; physiologic full maturation appears to require cytoplasmic changes as well. However, the intrafollicular mechanism involved in the regulation of nu- 240

TABLE 1 Hormonal profiles of the patients in groups A, B, and C at day 2 to day 4 of the cycle. Variable (n 68) (n 69) (n 18) Age (y) 31.7 0.5 31.0 0.4 30.0 0.4 FSH level (miu/ml) 6.4 0.3 6.6 0.3 6.3 0.5 LH level (miu/ml) 6.2 0.4 6.3 0.5 5.2 0.7 PRL level (ng/ml) 13.3 0.7 11.5 0.7 13.2 3.2 Free T level (pg/ml) 1.8 0.1 1.8 0.2 1.5 0.3 Total T level (ng/ml) 0.6 0.1 0.6 0.1 0.5 0.1 Serum E 2 on the day of hcg administration (pg/ml)* 2,407 135.7 2,082 124.2 1,651 164.8 Note: grade-3 embryos; group B grade 3 2 embryos; group C grade-2 embryos. All values are means SEM. P values for all group comparisons (except for serum E 2 levels) were not statistically significant by ANOVA. * P.05 for all group comparisons by the 2 test. clear and cytoplasmic processes for maturation is not well known (6 8). ICSI bypasses all physiologic sperm screening mechanisms and leads to fertilization with microsurgical epididymal sperm aspiration (MESA), testicular sperm excision (TESE), or round spermatid nuclear injection (ROSNI) with results similar to those of ejaculated sperm (9, 10). Furthermore, the biologic characteristics of the resulting embryos have not yet been fully assessed. In this work, we tried to investigate the relation between oocyte development and ultimate fetal potential. We assessed the oocytes for nuclear and cytoplasmic maturity precisely and evaluated the resulting fertilization rate, the quality of the embryos derived from ICSI, the cleavage rate and the cell stage at the time of transfer, and the pregnancy rate (PR). MATERIALS AND METHODS A total of 155 couples with a diagnosis of severe male factor infertility with similar indices according to Kruger s criteria were treated by ICSI. The stimulation protocol was as follows. On day 21 of the previous cycle, a baseline ultrasound scan was performed and buserelin acetate intranasal spray was begun at a dose of 100 g five times daily. GnRH agonist administration was continued until hcg administration. For all patients, the extent of ovarian suppression was evaluated by ultrasound scan and serum E 2 ( 40 pg/ml) before starting exogenous gonadotropin administration (about 15 days after administering the spray). After gonadotropin administration and follow-up, hcg was given when at least two follicles were 17 mm and serum E 2 was 400 pg/ml. ICSI was performed only on oocytes in metaphase II. ICSI was performed following conventional techniques (11). Nuclear maturity was assessed by extrusion of the first polar body in the perivitelline space. Maturity of the cytoplasm was defined as clear cytoplasm with uniform structure and homogeneous fine granularity; immaturity of the cytoplasm was defined as cytoplasmic abnormalities, such as vacuoles or fragments (oocytes with cytoplasmic incorporations or spots). Embryos were scored and chosen for transfer based on rapid cleavage, absence of fragmentation, and even size of blastomeres (good quality 3; poor quality 2) (12). consisted of 68 women who had embryo quality of grade 3. consisted of 69 women who had embryo quality 3 and 2, and group C had 18 women with grade-2 embryos. The patients hormonal profile, including FSH, LH, PRL, free T, and total T, was evaluated in all groups. Data were analyzed using analysis of variance (ANOVA) and 2 test, where appropriate. Institutional review board approval was obtained. RESULTS The hormonal profiles of the patients in the three groups are presented in Table 1. The women in the three groups were similar in age and their hormonal profiles were comparable. Serum E 2 concentrations on the day of hcg administration were statistically significantly different (Table 1). The quality of the oocytes is described in Table 2. Upon stripping of the cumulus and assessment of nuclear and cytoplasmic maturity, there was a striking difference in the quality of oocytes among the three groups. A polar body (nuclear maturity) in the perivitelline space could not be observed in 11.3% of group-a oocytes, 16.3% of group-b oocytes, and 22.7% of group-c oocytes. The difference was statistically significant between all groups. On assessment of cytoplasmic maturity of oocytes, 1.9% were found to have abnormal cytoplasm in group A, 4.1% in group B, and 7.8% in group C; the difference was statistically significant. The remainder of the oocytes were in the germinal vesicle stage or with broken or empty zonae (Table 2). FERTILITY & STERILITY 241

TABLE 2 Quality of oocytes retrieved in groups A, B, and C. Variable (grade-3 (grade 3 2 (grade-2 No. of oocytes retrieved 889 826 228 No. of oocytes suitable for ICSI 650 (73.1) 552 (66.8) 131 (57.2) No. of oocytes unsuitable for ICSI 239 (26.9) 274 (33.2) 97 (42.8) Lack of polar body 101 (11.4) 135 (16.4)* 52 (22.9) Lack of germinal vesicle 96 (10.8) 87 (10.6)* 22 (9.7) Broken zona 17 (1.9) 12 (1.4) Empty zona 8 (0.8) 6 (0.7) Dark cytoplasm 2 (0.2) 22 (2.7) 17 (7.5) Many vacuoles 15 (1.7) 11 (1.3) 2 (0.9) Fragments in cytoplasm 1 (0.1) 4 (1.8) Dark cytoplasm, many vacuoles, and fragments in cytoplasm 17 (1.9) 34 (4.0)* 23 (10.2) Note: Values are n or n (%). All P values were determined by the 2 test with continuity correction. P.001 versus group A. P.1 versus group B. P.05 versus group B. The fertilization rate was similar in the three groups (Table 3). A difference in the cleavage rate among the three groups was observed. At the time of ET, some differences were observed in the cleavage stage of embryos derived from each patient group. More embryos in group A developed to 4 cells (65.9%) than in groups B (61.3%) and C (53.4%) (P.05 [group A versus group B]; P.05 [group A versus group C and group B versus group C]). In addition, the proportion of embryos at the 2-cell stage was higher in group C than in groups A and B (30.6% versus 12.7% and 23.6%, respectively; P.05 for group A versus groups B and C) (Table 4). No more than three to four embryos were replaced per patient. The clinical PR was 29.4% in group A, 15.9% in group B, and 5.5% in group C. Thus, a higher rate was achieved in patients who received good-quality embryos at ET (Table 3). DISCUSSION Our results in this study show that the quality of the embryos at the time of ET has a critical effect on the outcome of ICSI, as was the case with conventional IVF. At the 4- to 8-cell stage of the development of human embryos, the embryonic genome is expressed by maternal encoded genomic products stored in the oocyte during the later stages of development in the ovary (13). The aim of every stimulation protocol is to yield as many mature and as few immature oocytes as possible; that is, to attain the best possible synchronization of follicular maturation. For nuclear maturation, an attractive hypothesis has been formulated; that gap junction mediated transmission of follicular-cell cyclic adenosine 3-5 monophosphate (camp) to the oocyte inhibits oocyte maturation (14), whereas gonadotropin stimulation ultimately terminates cumulus oocyte communication and initiates resumption of meiosis by interruption of direct transfer of camp to the oocyte (14, 15). Oocytes matured in vitro appear to undergo nuclear maturation, but are not cytoplasmically mature. For assessment of full maturation of ova matured in vitro, it is necessary to examine not only their fertilizability, but also their potential for early embryonic development (in our study, fertilization rates were similar in all groups) (Table 3). After sperm penetration, the sperm undergo a sequence of nuclear differentiation steps (nuclear membrane breakdown, chromatic decondensation, nuclear swelling, DNA synthesis, and, finally, pronuclear formation). These changes occur in TABLE 3 Fertilization rate, cleavage rate, and PRs by group. Variable (grade-3 (grade 3 2 (grade-2 Fertilization rate 76.1 74.2 72.5 Cleavage rate 99.5 99.0 92.6 Pregnancy rate 29.4 15.9* 5.5 Note: Values are percentages. P values for all group comparisons for the fertilization rate and cleavage rate were not statistically significant. P.05 versus groups A and B. P.05 versus group B. 242 Loutradis et al. Egg morphology and embryo quality Vol. 72, No. 2, August 1999

TABLE 4 Cleavage stage of embryos at ET by group. Stage (grade-3 (grade 3 2 (grade-2 Cleavage to 2 cells 12.7 23.6* 30.6* Cleavage to 3 cells 16.3 7.6* 4.6* Cleavage to 4 cells 65.9 61.3 53.4* Cleavage to 5 6 cells 5.0 7.5 11.3 Note: All values represent the percentage of embryos cleaved. Within each cleavage category, all group comparisons were P.05 by the 2 test with continuity correction unless otherwise indicated. P.05 versus group B. fully mature oocytes, but not in immature oocytes, as we have presented in this study, where even immature cytoplasmic oocytes have the potential to be fertilized. Thus, the maturational stage of the cytoplasm is partially controlled by the sperm nucleus, but also by other cytoplasmic constituents. Moor et al. (16) and Ainsworth et al. (17) demonstrated that oocytes require a specific intrafollicular steroid environment for the inductive signals of meiotic resumption and the completion of the full maturational process. Steroids exert a significant influence on the synthesis of cytoplasmic factors that induce normal decondensation of the sperm head and formation of the male pronucleus (18). Yoshimura et al. (19) investigated the influence of ovarian steroidogenesis on oocyte maturation and fertilization. Cyanoketone was used to block ovarian steroid synthesis. Similar results showing that ovarian steroid production is not essential for the resumption of meiosis but participates in cytoplasmic maturation of the oocyte have been obtained in the perfused rabbit ovary. The addition of E 2 to the perfusate reverses the adverse effects of cyanoketone on fertilizability (19). The data provide evidence that E 2 administered in conjunction with gonadotropin and cyanoketone confers on intrafollicular oocytes the ability to undergo normal fertilization. This observation is in accordance with the proposal that estrogen may be the major steroidal signal during the critical early inductive phase of oocyte maturation (20). In our study, we considered the influence of the serum E 2 level during ovulation induction on cytoplasmic maturity. A significantly lower E 2 value was found in group C compared with the other groups. Furthermore, a higher E 2 level is correlated with successful fertilization and an enhanced cleavage rate of oocytes associated with pregnancy after IVF (21). These data suggest that estrogen-rich follicles are the source of ova that undergo successful fertilization and eventuate in pregnancy. Or data with ICSI, in which we had the chance to evaluate the quality of oocytes more accurately, support the hypothesis that E 2 plays a fundamental role in the process of oocyte maturation leading to successful embryogenesis. In this study, we investigated a new marker for assessment of the quality of the stimulation protocol. A limitation of our study was that we used a hormone preparation for ovulation induction, and therefore we cannot determine precisely which component was responsible for the affected oocytes. In addition, the criteria for hcg administration to trigger nuclear maturity are under consideration; a doubleblind study is necessary to identify the exact timing of hcg administration. It is clear that in ICSI, different sperm indices do not affect the fertilization rate, PR, or the outcome of pregnancy as long as a morphologically well-shaped motile sperm is used for injection (22). Although poor-quality embryos failed to demonstrate the same PR as those derived from mature oocytes, the cause was not the quality of the sperm, but the quality of the oocyte and particularly the maturity of the cytoplasm. In summary, by using ICSI we found that accurate assessment of oocyte maturity, both nuclear and cytoplasmic, demonstrated a significantly higher PR from good quality oocytes compared with those with poor morphology. Our observation is not in accordance with the data of De Sutter et al. (23), who did not find any deviation from the normal distribution of embryo quality from oocytes with abnormalities. Therefore, additional studies with different stimulation protocols are required to estimate the follicular environment (hormonal milieu and growth factors) that would be helpful to identify the ideal protocol to produce the greatest number of mature oocytes. References 1. VanSteirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;2:1061 6. 2. Sofikitis NV, Kanakas N, Mantzavinos T, Antypas S, Loutradis D, Agapitos F, et al. 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