The influence of oocyte maturity and embryo quality on pregnancy rate in a program for in vitro fertilization-embryo transfer*

Transcription

1 FERTILITY AND STERILITY Copyright The American Fertility Society Printed on acid-free paper in U.S.A. The influence of oocyte maturity and embryo quality on pregnancy rate in a program for in vitro fertilization-embryo transfer* George A. Hill, M.D.t Melanie Freeman, M.S. Maria Cristina Bastias, M.D. B. Jane Rogers, Ph.D. Carl M. Herbert III, M.D. Kevin G. Osteen, Ph.D. Anne Colston Wentz, M.D. Center for Fertility and Reproductive Research, Vanderbilt University Medical Center, Nashville, Tennessee An important factor influencing the pregnancy rate after in vitro fertilization-embryo transfer (IVF-ET) appears to be the number of embryos transferred to the uterus. In this study, the influence of oocyte maturity and embryo quality on pregnancy rate was assessed in patients undergoing IVF-ET. Ovarian hyperstimulation was performed by human menopausal gonadotropin (hmg [n = 29)), clomiphene citrate (CC)/hMG (n = 81), and hmg/follicle-stimulating hormone (FSH [n = 13]) protocols. Oocyte maturity was graded on a scale from 1 to 5 based on the morphology of the ooplasm, cumulus mass, corona radiata, and membrana granulosa cells. Embryos were graded according to the symmetry of the blastomeres and the presence or absence of fragmentation. Mature preovulatory oocytes yielded the highest fertilization rates. No differences were found among the protocols in terms of fertilization rate, embryo quality, or pregnancy rate. When all protocols were combined, patients who conceived had a significantly higher number of embryos transferred than those who did not conceive (3.6 ± 0.1 [mean± SEM] versus 2. 7 ± 0.1). When embryo quality was compared, there was no difference in the number of"b" embryos transferred between patients who conceived and those who did not (1.2 ± 0.2 versus 1.2 ± 0.1), but the patients who conceived had significantly more "A" embryos transferred (1.6 ± 0.3 versus 0.8 ± 0.1). These data suggest that the treatment protocol did not determine embryo quality. Furthermore, the increase in pregnancy rates seen with an increase in embryos transferred is the result of the transfer of more "A" embryos. Fertil Steril52:801, 1989 Multiple factors have been found to be important in influencing the pregnancy rate of patients undergoing in vitro fertilization-embryo transfer (IVF-ET). These have included the etiology of infertility/ age of the patient,2 the type of ovarian stimulation, 2 follicular phase estradiol (E2) levels,2 3 the number of oocytes collected,2 and the number of oocytes fertilized. 2 These factors affect Received April17, 1989; revised and accepted July 13, * Presented at the VI World Congress, In Vitro Fertilization and Alternate Assisted Reproduction, Jerusalem, Israel, April 2to 7,1989. t Reprint requests: George A. Hill, M.D., Rm. D-3223 Medical Center North, Vanderbilt University, Nashville, Tennessee the number of embryos that develop and, subsequently, the number of embryos transferred. The number of embryos transferred appears to be the most important factor in influencing the pregnancy rate. 4 Multiple investigators have examined various stimulation protocols to determine which protocol might be superior. 5 6 Differences in the number of follicles developing, the number of oocytes collected, the rate of fertilization, of cleavage, and of ET have been reported. Improved rates of pregnancy have also been attributed to changes in stimulation protocols. 7 8 This study sought to determine the effect of oocyte maturity on fertilization rates and embryo Hill et al. Oocyte maturity and embryo quality in IVF 801

2 Table 1 Serum E 2 Levels on HCG Days -1, 0, and+ 1, Separated by Whether the Patient Conceived or Did not Conceive Day-1 DayO Day+1 Protocol Pregnant Not pregnant Pregnant Not pregnant Pregnant Not pregnant hmg 596±59 623±65 1,004 ± ±95 1,227 ± 270 1,188± 109 CC/hMG 907±69 802±45 1,371 ± 98 1,154±59 1,755 ± 138b 1,414± 72 hmg/fsh 515 ± ± ± ± ± ± 118 Combined 819 ± ± 35 1,264 ± 89b 1,055 ±48 1,612 ± 125b 1,308 ± 57 Values are means ± SEM. b P < 0.05 compared with nonpregnant. quality, as well as the effect of embryo quality on subsequent pregnancy rates in patients undergoing IVF-ET. In addition, three different protocols for ovarian hyperstimulation were examined to determine if any one protocol resulted in significantly improved embryo quality. MATERIALS AND METHODS One hundred twenty-three consecutive patients admitted to the IVF-ET program at Vanderbilt University Medical Center in Nashville, Tennessee, between October 1987 and August 1988, who subsequently underwent ET, were included in this study. The age of these patients ranged from 28 to 43 years. Ovarian hyperstimulation was performed by one of three protocols as previously described 5 9 : human menopausal gonadotropin (hmg; Pergonal; Serono Laboratories, Inc., Randolph, MA; n = 29), clomiphene citrate (CC; Serophene; Serono Laboratories, Inc.)/hMG (n = 81), and hmg/follicle-stimulating hormone (FSH; Metrodin; Serono Laboratories, Inc.; n = 13). Once adequate follicular maturation had been obtained, human chorionic gonadotropin (hcg; LyphoMed, Melrose Park, IL) was administered, and oocyte retrieval was performed 35 hours later by transvaginal aspiration with ultrasound guidance. Follicular fluids and follicular washes were examined microscopically with a dissecting stereo microscope situated inside an isolette having a controlled, humidified atmosphere of 37"C and 5% C0 2 Oocytes were graded for maturity (1 to 5) on the basis of the morphological characteristics of the cumulus mass, the corona radiata, the ooplasm, and the detached membrana granulosa cells. Grade 1 ("immature") oocytes exhibited a dense, compact cumulus, if present, and a very adherent, compact layer of corona cells. The ooplasm, if visible, revealed the presence of the germinal vesicle. The membrana granulosa cells were compact and nonaggregated. These oocytes were inseminated 24 to 30 hours after retrieval. Grade 2 ("nearly mature") oocytes exhibited an expanded cumulus mass and a slightly compact corona radiata. The membrana granulosa cells were expahded and well aggregated. These oocytes were inseminated 6 or 24 hours after retrieval, based on the compactness of the corona cells. Grade 3 ("mature or preovulatory") oocytes exhibited a very expanded cumulus and a very radiant corona radiata, revealing a distinct zona pellucida. The ooplasm was clear, and the membrana granulosa cells were expanded and well aggregated. These oocytes were inseminated 6 hours after retrieval. Grade 4 ("postmature") oocytes exhibited a very expanded cumulus often having clumps. The corona radiata was radiant, yet often clumped and irregular or incomplete. The zona pellucida was very visible and the ooplasm either slightly granular or dark. The membrana granulosa cells were small and relatively nonaggregated. These oocytes were inseminated 6 hours after retrieval. Grade 5 ("atretic") oocytes rarely had an associated cumulus mass. The corona radiata, if present, was clumped and very irregular. The ooplasm was dark and frequently misshapen and the zona pellucida very visible. The membrana granulosa cells were very small clumps of cells. These oocytes were inseminated 6 hours after retrieval. Oocytes were placed in insemination media (Ham's F-10 medium [Gibco, Grand Island, NY] + 15% maternal serum) and inseminated with 375,000 total motile spermatozoa (125,000 motile sperm/ml). Twenty-four hours later the embryos were examined for evidence of fertilization and transferred to new media. A maximum of four embryos was transferred to the uterus approximately 48 hours after aspiration. Before the transfer, the embryos were graded according to the symmetry of the blastomeres and the presence or absence of fragmentation (A: symmetrical, no fragmentation; B: <10% fragmentation and/or asymmetrical; C: 802 Hill et al. Oocyte maturity and embryo quality in IVF Fertility and Sterility

3 Table 2 Fertilization Rate Among the Three Protocols Based on Oocyte Maturity at the Time of Follicular Aspiration Protocol Oocyte maturity hmg CC/hMG hmg/fsh Total /4 (O)b 0/12 (0) I 0/0 (0) 0/16 (0) I 17/27 (63)< 67/120 (56)6 10/12 (83) 94/159 (59)' 43/55 (78)d 142/166 (86)h 21/23 (91)k 206/244 (84)h 32/58 (55)e 105/156 (67)' 8/14 (57) 145/228 (64)' 0/4 <W 1/9 (ll)i 0/0 (O) 1/13 (8) 1 Total 92/148 (62) 315/463 (68) 39/49 (80) 446/660 (68) Numbers in parentheses are percent values. b P < 0.05 compared with oocytes graded as 2, 3, or 4. : P < 0.05 compared with oocytes graded as 1 or 5. P < 0.01 compared with oocytes graded as 1, 4, or 5. e P < 0.05 compared with oocytes graded as 1, 3, or 5. 1 P < compared with oocytes graded as 2, 3, or 4., ~ P < 0.01 compared with oocytes graded as 1, 3, or 5.. P < compared with oocytes graded as 1, 2, 4, or 5. ~ P < compared with oocytes graded as 1, 3, or 5. ~ P < 0.01 compared with oocytes graded as 1, 3, or 4. P < 0.05 compared with oocytes graded as 4. 1 P < compared with oocytes graded as 1, 3, or 4. 10% to 25% fragmentation; D: >25% fragmentation). Statistical Analysis The data are expressed as mean ± standard error of the mean (SEM). Analysis was performed using Student's t-test, analysis of variance, and x 2 with Yate's correction where appropriate. Significance was defined as P < RESULTS A total of 123 consecutive cycles of IVF-ET using three different stimulation protocols were evaluated. The E 2 levels on hcg days -1 and 0 in patients who conceived were not significantly different from those in patients who did not conceive when examined by protocol. On the day after hcg, patients who were treated with CC/hMG and conceived had significantly higher E 2 levels than those who were treated with CC/hMG who did not conceive. There were no differences in the E 2 levels in conception versus nonconception cycles in patients treated with either hmg or hmg/fsh on day +1. However, when all protocols were combined, the patients who conceived had higher E 2 levels on hcg days 0 and+ 1 than those who did not conceive (Table 1). A total of 660 oocytes were retrieved in this group of patients (5.4 per patient). A mean of 5.1 ± 0.4 (SEM), 5.8 ± 0.3,* and 3.8 ± 0.4* oocytes per patient were retrieved in the hmg, CC/hMG, and hmg/fsh groups, respectively. The overall fertil- * P< ization rate (68%), as well as the fertilization rate based on oocyte maturity, are shown in Table 2. The highest fertilization rate was seen in those oocytes graded as 3, followed by the ones graded as 4 and 2. There was no fertilization seen in any oocytes graded as 1 and the fertilization rate for those graded as 5 was very low (Table 2). When the total number of embryos transferred was examined by protocol, only the patients stimulated with CC/hMG who conceived had significantly more embryos transferred than those who did not conceive. There was no significant difference in the patients treated with either hmg or hmg/fsh due to the smaller number of patients in these groups. However, when all stimulation protocols were combined, the patients who conceived had significantly more embryos transferred than those who did not conceive (Table 3). When the number of embryos transferred was examined according to the grade of the embryo, there were no significant differences in the number of "B" embryos received in patients who conceived and those who did not. However, patients treated with hmg or CC/hMG who conceived received significantly more "A" embryos than those not conceiving. Patients treated with hmg/fsh who conceived received more "A" embryos than those who did not conceive, but this was not statistically significant because of the small number of patients in this group. When all protocols were combined, patients who conceived received significantly more "A" embryos than those who did not conceive (Table 3). No ovarian stimulation protocol was found to be superior when the number of "A" embryos, "B" embryos, or total embryos transferred was examined (0.7 ± 0.2, 1.1 ± 0.1, 0.6 ± 0.2 ["A" embryos]; Hill et al. Oocyte maturity and embryo quality in IVF 803

4 Table 3 Total Number of Embryos Transferred and Embryos Transferred That Were Classified as "A" or "B" Separated by Whether the Patient Conceived or Did Not Conceive" Total "A" "B" Protocol Pregnant Not pregnant Pregnant Not pregnant Pregnant Not pregnant hmg 3.0 ± ± ± 0.7b 0.5 ± ± ± 0.2 CC/hMG 3.7 ± 0.1b 2.8± ± 0.3b 0.9 ± ± ± 0.1 hmg/fsh 3.5 ± ± ±0 0.6± ± ± 0.2 Combined 3.6 ± 0.1b 2.7 ± ± 0.3b 0.8 ± ± ± 0.1 "Values are means± SEM. 1.1 ± 0.2, 1.2 ± 0.1, 1.1 ± 0.2 ["B" embryos]; 2.6 ± 0.2, 3.0 ± 0.1, 2.8 ± 0.3 [total embryos] for hmg, CC/hMG, and hmg/fsh, respectively). When oocyte maturity at aspiration was examined to determine if a particular maturity was more likely to become an "A" embryo, oocytes graded as either 2 or 3 were significantly more likely to become "A" embryos than oocytes graded as 4 (24 of 62 [39%] and 72 of 199 [36%] versus 26 of 127 [20%] for oocytes graded as 2, 3, or 4, respectively [P < 0.001]). When this was evaluated according to stimulation protocol, the differences were not significant. The pregnancy rate was not significantly different among the three protocols (Table 4). Among patients who conceived, 19 of 26 (73%) had received at least one "A" embryo compared with 46 of 97 (47%) of those who did not conceive (P < 0.05). Of the patients who did not receive an "A" embryo, only 8 of 58 (14%) conceived; in contrast, 6 of 31 (19%) patients who received one "A" embryo and 13 of 34 (38%) who received more than one "A" embryo conceived (Table 4). DISCUSSION Multiple factors have been reported to affect the outcome of pregnancy in patients undergoing IVF- Table 4 Total Number of Patients Who Conceived and Patients Who Conceived Based on the Number of"a" Embryos Replaced" Protocol hmg CC/hMG hmg/fsh Total No"A" 4/29 (14) 1/17 (6) b 20/81 (25) 7 /35 (20) 2/13 (15) 0/6 (0) l"a" 0/5 (0) 4/20 (20) 2/6 (33) >l"a" 3/7 (43) 10/26 (38) 0/1 (0) Total 26/123 (21) 8/58 (14)' 6/31 (19) 13/34 (38) "Numbers in parentheses are percent values. b P < 0.05 compared with patients who received more than one"a". 'P < 0.01 compared with patients who received more than one"a". b P < 0.01 compared with nonpregnant. ET. Protocols differ in terms of numbers of oocytes retrieved, oocytes fertilized, and embryos transferred. Numerous protocols have been examined to find a protocol that would prove superior in terms of pregnancy. 5-9 In this report, the ovarian response to hyperstimulation, the fertilization rate, and the number and quality of embryos transferred were examined. Several differences were noted when patients who conceived were compared with those who did not conceive. The serum E 2 on the day of hcg and the day after hcg was significantly higher in those patients who conceived compared with those who did not. This finding is compatible with the observation that the height and pattern of E 2 response is important, with high responders having a higher pregnancy rate than those with intermediate or low responses. 2 The higher E 2 seen in patients who conceive may also result from an increased number of follicles that develop, which subsequently leads to increased numbers of oocytes aspirated and embryos transferred. These factors vary depending on the stimulation protocol, as evidenced by a significantly higher number of oocytes retrieved per patient in the CC/hMG group compared with the hmg/fsh group. The hmg group also had more oocytes retrieved per patient than the hmg/fsh group, but this was not statistically significant. The data presented in this report clearly demonstrate that the maturity of the oocyte affects the subsequent outcome in terms of both oocyte fertilization and embryo quality. V eeck et al. 10 described the appearance of immature and preovulatory oocytes and reported a higher fertilization rate for preovulatory oocytes when compared with immature oocytes that had been matured in vitro. The classification system described in this report further divides the oocyte into five stages of maturity. Those graded as mature preovulatory (grade 3) have a higher fertilization rate than those graded as mature (grade 2) or postmature (grade 4), with those graded as immature (grade 1) or atretic 804 Hill et al. Oocyte maturity and embryo quality in IVF Fertility and Sterility

5 (grade 5) being the lowest. Oocyte maturity was also important in determining the quality of the embryo. Oocytes graded either 2 or 3 were more likely to develop into an "A" embryo than oocytes graded as 4, suggesting that it is better to have an oocyte that is slightly immature or mature rather than one that is slightly overmature. The E 2 level has also been found to be important in the development of the embryo. Dor et al. 11 found better embryo quality and improved pregnancy rates when the E 2 continued to rise after the administration of hcg; however, they found no differences in the mean daily E 2 levels between conception and nonconception cycles. This fact may be beneficial in managing clinical ovarian hyperstimulation protocols, because this would suggest that better results would be obtained by erring on the side of stopping a little early rather than late.. The number of embryos transferred is known to be important in subsequent pregnancy rates. 2 This report examines not only the number of embryos transferred, but also examines the quality of those embryos transferred. Patients who conceived had significantly more embryos transferred. However, this increase in embryos transferred in the patients who conceived appeared to be secondary to an increase in the number of "A" embryos transferred, because the number of "B" embryos transferred did not differ between the patients who conceived and those who did not conceive. When the protocols were examined, no protocol proved superior in terms of producing "A'; embryos. The "A" embryos transferred are clearly related to the pregnancy rate, because patients receiving more than one "A" embryo had a pregnancy rate of 38%, compared with 19% when one "A" embryo was transferred and 14% when no "A" embryos were transferred. Puissant et al. 12 have developed a scoring system for grading embryos similar to the system detailed in the current report. Using points to score embryos based on the appearance of the blastomeres and the presence or absence of fragmentation, Puissant et al. 12 found higher pregnancy rates associated with higher embryo scores, which reflected better quality embryos. Claman et al. 13 also examined the effect of embryo quality on pregnancy rate by looking at cleavage rates and fragmentation of the embryo. These authors found higher pregnancy rates when ET included at least one embryo of the four-cell stage by 40 hours postinsemination. The presence of faster cleaving embryos appeared to be the important factor. Pregnancy rates were low regardless of the number of embryos transferred when all embryos transferred were cleaving slowly. Although fragmentation of the embryos resulted in a lower pregnancy rate in the report by Claman et al., 13 this was not statistically significant. The studies of both Puissant et al. 12 and Claman et al. 13 support our data and suggest that pregnancy rates are higher when at least one good quality embryo is transferred. The data in the current report differ somewhat from the data of Claman et al. 13 in regard to fragmentation. Claman et al. 13 found no effect of fragmentation on subsequent pregnancy rates; however, it is difficult to ascertain from their data what proportion of the fragmented embryos were fast or slow cleaving. The data in the current study do support the data of Dor et al., u which report higher pregnancy rates in patients receiving unfragmented embryos. The important point in all these reports appears to be that embryo quality is an important variable in determining subsequent pregnancy rates in patients undergoing IVF-ET. Multiple factors may be of benefit in evaluating the embryo in the future. One of these is plateletactivating factor, which has been shown to be produced in higher amounts by embryos that subsequently resulted in a pregnancy. 14 The production of embryo-derived platelet-activating factor was related to the stimulation protocol, the E 2 level, and the morphology and cell number of the embryo. This represents a biochemical means of accessing embryo quality and may prove useful along with the microscopic evaluation of the embryo in accessing the probability of a given embryo producing a pregnancy. Other factors have also been postulated to be of benefit as a noninvasive way to evaluate the embryo. 15 Whether or not these become clinically beneficial awaits further studies. In summary, although multiple factors play a role in the outcome of IVF-ET, the number of quality embryos transferred is very important. The maturity of the oocyte at aspiration is important in subsequent embryo quality, and this study indicates that embryo quality can be related to pregnancy rate, in that the transfer of higher numbers of better quality embryos is associated with a higher pregnancy rate. Future studies need to be performed to access which parameter best reflects the quality of the embryo. REFERENCES 1. Edwards RG, Fishel SB, Cohen J, Fehilly CB, Purdy JM, Slater JM, Steptoe PC, Webster JM: Factors influencing Hill et al. Oocyte maturity and embryo quality in IVF 805

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