- Email: [email protected]
Prematurely condensed chromosomes and meiotic abnormalities in unfertilized human oocytes after ovarian stimulation with and without gonadotropin-releasing hormone agonist
FERTILITYAND STERILITY@
Vol. 61, No. 5, May 1997
Copyright ’ 1997AmericanSocietyforReproductive Medicine
Printed on acid-free paper in U. S. A.
Published by ElsevierScienceInc.
Prematurely condensed chromosomes and meiotic abnormalities in unfertilized human oocytes after ovarian stimulation with and without gonadotropin-releasing hormone agonist
Catherine Racowsky, Ph.D.* Angela L. Prather, B.S. Malinda K. Johnson, B.S. Division of Reproductive University of Arizona,
Sandra P. Olvera, B.S. Timothy J. Gelety, M.D.
Endocrinology
and Infertility,
Department
of Obstetrics and Gynecology,
College of Medicine,
Tucson, Arizona
Objective: To investigate the incidence of meiotic abnormalities, aneuploidy, and prematurely condensed sperm chromosomes in failed fertilized oocytes after controlled ovarian hyperstimulation (COH). Design: Retrospective analysis of air-dried preparations of unfertilized oocytes. Setting: University hospital-based infertility clinic. Patient(s): Thirty-three patients undergoing IVF having only tubal factor as the cause of infertility. Twelve patients (13 cycles) underwent treatment with hMG alone (-GnRH agonist [GnRH-a]), and 21 patients (24 cycles) underwent treatment with leuprolide acetate (LA) and hMG (+GnRH-a group). Intervention(s): Standard IVF-ET treatment cycle for ovarian stimulation using hMG with or without LA. Main Outcome Measure(s): The meiotic stage, ploidy, and the presence of prematurely condensed sperm chromosomes were determined in 161 air-dried preparations of unfertilized oocytes. Result(s): Significantly more unfertilized oocytes were at metaphase II in the -GnRH-a group as compared with the +GnRH-a group, with significantly fewer exhibiting meiotic aberrations. Aneuploidy rates did not differ between groups. However, significantly more oocytes in the +GnRH-a group revealed prematurely condensed sperm chromosomes than in the -GnRH-a groupConclusion(s): The use of GnRH-a for COH does not have an impact on aneuploidy rates in failed fertilized oocytes. However, the higher incidence of meiotic aberrations and prematurely condensed sperm chromosomes in the unfertilized population indicates that some retrieved oocytes exhibit incomplete nuclear and cytoplasmic maturation after the use of this agonist. (Fertil Sterile 1997;67:932-8. 0 1997 by American Society for Reproductive Medicine.) Key Words: GnRH-a, prematurely condensed sperm chromosomes, oocyte meiotic abnormalities, IVF failures
The formation of a cyte with a haploid quires a coordinated events that ensures
developmentally competent oochromosomal complement reand finely tuned sequence of both nuclear and cytoplasmic
Received October 2, 1996; revised and accepted January 22, 1997. * Reprintrequests:CatherineRacowsky,Ph.D.,Divisionof ReproductiveEndocrinologyand Infertility,Departmentof Obstetrics and Gynecology,Collegeof Medicine,Universityof Arizona, Tucson,Arizona 85724 (FAX: 520-626-2768). 932
maturation of the gamete. When controlled ovarian hyperstimulation (COH) is used for the purpose of IVF’, the resulting multiple follicular development typically arises from the addition of follicles at different stages of development (see review by Taymour [ll). Consequently, oocytes retrieved for M? often are at various stages of maturity, and even when the timing of insemination is adjusted according to the maturity of the cumulus-corona cells, fertilization rates are generally suboptimal. The addition of GnRH agonists (GnRH-a) to COH 0015-0282/97/$17.00 PI1 SOOX-0282(97)00125-8
regimens for IVF has several recognized advantages, including reduced cancellation rates, increased ease of scheduling, and fewer premature luteinizations (2, 3). In addition, the increased number of oocytes obtained per retrieval generally results in an increase in the number of embryos available for transfer (4). Moreover, a meta-analysis of randomized studies has demonstrated a twofold increase in the pregnancy rate when GnRH-a treatment has been used in conjunction with COH (51, and use of this agonist has been shown to result in improved embryo quality (2,6) and reduced spontaneous abortion rates (7). Use of GnRH-a results in suppression of androgen production by the ovaries (81, which in turn promotes maturation of secondary follicles that otherwise would become atretic. Accordingly, the quality of these extra oocytes may be compromised, as has been suggested previously based on the implantation rate per embryo transferred after the use of this agonist (9). The possibility exists, therefore, that, despite a typical increase in oocyte quantity after use of GnRH-a in COH protocols, oocyte quality may not be optimum in all oocytes of the retrieved cohort. In an attempt to assess further the quality of oocytes harvested after the use of GnRH-a in IVF patients, we have compared results for patients stimulated with gonadotropins alone or with gonadotropins plus GnRH-a. We have assessed oocyte maturity and fertilization rates at retrieval. In addition, because cytogenetic analysis of failed fertilized oocytes provides some insight into the overall quality of a retrieved cohort (lo), we have investigated the meiotic abnormality rate in these failures for both groups. Furthermore, because the phenomenon of premature sperm chromosome condensation is associated with oocyte cytoplasmic immaturity (ll), we have determined the relative incidence of prematurely condensed sperm chromosomes in the unfertilized oocytes of both patient groups.
levels by rapid RIA. Human chorionic gonadotropin (Profasi, 10,000 USP U IM; Serono Laboratories) was given when at least three follicles were observed with a lead diameter of 217 mm and a serum E2 2 500 pg/mL (1,840 pmol/L) was achieved. Transvaginal aspiration was performed 34 to 35 hours later. The COH regimen used for the patients in the +GnRH-a group (n = 21, representing 24 cycles) has been described in detail (12). Briefly, GnRH-a (leuprolide acetate; TAP Pharmaceuticals, North Chicago, IL) was begun on cycle day 21(1 mg SC twice daily) after spontaneous or induced withdrawal bleeding and/or 8 days after detection of an urinary LH surge. On day 31, transvaginal sonography was performed and serum Ez was assayed to ensure ovarian suppression (no ovarian cyst > 10 mm in diameter and Ez < 30 pg/mL 1110 pmol/LI). The GnRH-a was reduced to 0.5 mg/d SC, and hMG (300 IU/d IM) was administered for 5 days. Follicular development was evaluated after 5 days by transvaginal sonography and measurement of peripheral serum Ez by rapid RIA. The hMG dosage was then individualized based on follicular response, which was monitored at l- to 3-day intervals. Human chorionic gonadotropin (10,000 USP U) was administered IM when three or more follicles had a mean diameter of 218 mm and when a serum E2 2 500 pg/mL (1,840 pmol/L) was achieved. Ultrasound-guided transvaginal sonography was performed 34 to 35 hours later. After aspiration, all oocytes were scored for maturity using standard procedures for assessment of corona-cumulus expansion. Oocytes were scored for the presence of pronuclei (PN) 16 to 18 hours postinsemination. Those without cleavage and those with 1 or more than 2 PNs at the time of ET were set aside for preparation of air-dried spreads. Oocytes were air dried as soon as possible after ET, with the majority (88%) being air dried 48 to 60 hours postinsemination and the remainder being air dried between 61 and 80 hours.
MATERIALS AND METHODS Patients and IVF’Procedures
Preparation and Analysis of Air-Dried Oocyte Chromosome Spreads
The present study was approved by the institutional Human Subjects Committee at the University of Arizona Health Sciences Center and included 33 patients (age 26.7 to 36.9 years) having only a diagnosis of tubal factor as the cause of infertility. Twelve of these patients, representing 13 cycles, underwent COH with only hMG (Pergonal, 225 to 300 IU IM daily; Serono Laboratories, Randolph, MA) from cycle day 3 to 11 (the -GnRH-a group). Follicular development was evaluated by transvaginal sonography and measurement of peripheral serum Ez
Preparations of air-dried oocytes were made using either the protease procedure (13) or Dyban’s procedure (14) and then aged at room temperature for 2 to 3 days before being stained with Wright’s stain (13). Select preparations were photographed using a Cytovision 2.21 Imaging System (Applied Imaging, Santa Clara, CA) with a Zeiss Axioskop (Carl Zeiss Inc., Tempe, AZ). Spreads were analyzed under phase contrast illumination using a Nikon Labophot-2 compound microscope and classified as exhibiting 111 nondegenerating metaphase II (MID
Vol. 67, No. 5, May 1997
Racowsky
et al.
Oocyte abnormalities
in IVF failures
933
Figure 1 Air-dried preparations of failed fertilized human oocytes showing oocyte chromosome configurations. (A), Metaphase II chromosomes of a haploid oocyte (n = 23) showing 6 sperm heads that probably were bound to the zona pellucida before air drying; (B), degenerating MII; (C), a diploid MI1 chromosome configuration revealing failure of segregation of homologous chromosomes at anaphase I; (D), mitotic chromosomes of an oocyte that underwent developmental arrest postsyngamy. Magnification, x350.
chromosomes (Fig. 1A); [21 degenerating MI1 chromosomes (Fig. 1B); 131a meiotically aberrant configuration; 141 1 or >2 PNs; or 151mitotic chromosomes arrested postsyngamy (Fig. lD>. The meiotically aberrant configurations included MI chromosomes, or chromosomes at either diploid MI1 (Fig. 10 or failed anaphase I. When MI1 chromosomes could be counted accurately, ploidy was determined. To avoid errors in assessment of hypohaploidy because of chromosome scattering, the count was included only if the chromosomes were within a clearly defined region overlying the cytoplasm of the oocyte. For all oocytes, the presence of decondensing sperm heads and prematurely condensed sperm chromosomes was noted. Statistical
Analyses
Data were expressed as the means 2 SEM. For mean comparisons, analysis of variance was applied using the Statistical Analysis System @AS), with homogeneity of variance being tested using Bartlet’s 934
Racowsky
et al.
Oocyte abnormalities in IVF failures
test. Differences in proportions were tested using Fisher’s exact test. Linear regression analysis was applied where indicated. For all analyses, a probability of P < 0.05 was considered statistically significant, and all P values were two-tailed. REXJLTS
Patient age did not differ between the two groups (32.5 2 0.7 versus 33.12 0.6 years for -GnRH-a and +GnRH-a groups, respectively). Significantly more ampules of hMG were used by the +GnRH-a patients, as compared with those not using GnRH-a (33.1 2 1.1 versus 27.8 2 1.9 ampules; P < 0.014). The use of GnRH-a resulted in significantly more oocytes being retrieved per patient (13.6 + 1.7 versus 8.9 k 1.2; P < 0.051, with preovulatory oocytes being retrieved with the highest frequency in both groups (61.8% + 8.0% versus 64.5% + 3.2% for -GnRHa and +GnRH-a, respectively). The distribution of grades of oocyte maturity was not affected significantly by stimulation protocol, except that more Fertility and Sterility@
postmature oocytes were retrieved in the +GnRH-a group (3.8% 2 1.4% versus 0.8% +- 0.8%; P < 0.04). Use of GnRH-a significantly enhanced the rate of fertilization of those oocytes that were graded as preovulatory at retrieval (75.5% 2 4.7% versus 42.5% + 10.7%; P < 0.002), as well as the overall rate of fertilization (59.1% 2 6.8% versus 35.8% t 6.8%; P < 0.002). Table 1 shows the results of the chromosomal analyses performed. There was no significant difference between the two patient groups regarding the proportion of air-dried oocytes that were analyzable (83.8% and 88.9% for -GnRH-a and +GnRH-a, respectively). The remaining oocytes could not be analyzed because of excessive chromosome overlapping or scattering. Significantly more oocytes were at MI1 in the -GnRH-a group than in the +GnRH-a group (P < 0.0411, with significantly fewer oocytes exhibiting meiotic aberrations (P < 0.026). However, there was no significant difference between the two patient groups regarding the proportion of oocytes that exhibited 1 or >2 PNs, that had undergone postsyngamy arrest, revealing mitotic chromosomes, or that were achromosomal. Likewise, the proportions of MI1 oocytes that exhibited degenerate chromosomes or that were aneuploid did not differ between the two patient groups. The overall aneuploidy rate among failed fertilized oocytes obtained from all patients in this study was 48.9%, and the hyperhaploidy rate was 42.9%. Of all oocytes examined, 16 contained decondensing sperm heads (Fig. 2A), and there was no difference between patient groups in the proportion of oocytes exhibiting this phenomenon. Prematurely condensed sperm chromosomes were observed in oocytes from both patient groups and in oocytes at all stages of maturity at retrieval. The distribution of oocytes with prematurely condensed sperm chromosomes by oocyte maturity was as follows: 35% in
Table 1 Chromosome Analysis of Unfertilized Human
Oocytes Obtained After Retrieval From Patients Treated With or Without GnRH-a
Nos. air-dried oocyte preparations Nos. analyzable oocyte preparations* Nos. MII$ Nos. meiotically aberrant$ Nos. 1 PN$ Nos. >2 PNP Nos. postsyigamy arrest* Nos. achromosomal$
-GnRH-a
+GnRH-a
62 52 (83.9)
99 88 (88.9)
44 (84.6) 3 (5.8) 1 (1.9) 0 (0) 2 (3.8) 1 (1.9)
61 (69.3) 17 (19.3) 2 (2.3) 3 (3.4) 4 (4.5) l(1.1)
P
NSI 0.041 0.026
NW
NSt
NSP
NW
* Percentage of air-dried oocytes is given in parentheses. ? NS, not significant. $ Percentage of analyzable oocytes is given in parentheses. Vol. 67, No. 5, May 1997
preovulatory, 30% in intermediate, 20% in immature, and 15% in postmature oocytes. Although the majority of oocytes with prematurely condensed sperm chromosomes were at MI1 (71.4%), several oocytes containing prematurely condensed sperm chromosomes had failed to emit their first polar body. All but one of the prematurely condensed sperm chromosome configurations were at various stages of formation of the presynthetic gap phase (G,) (Fig. 2B-E), the remaining one being at G2 with clearly defined duplicated chromatids (Fig. 2F). Significantly more analyzable oocytes in the +GnRH-a group revealed prematurely condensed sperm chromosomes than in the -GnRH-a (20.2% +_3.2% versus 2.6% ? 1.2%; P < 0.0371, and this difference between treatment groups was also upheld when only the MI1 oocytes were considered (P < 0.050). Furthermore, a significant positive correlation was observed between the number of +GnRH-a oocytes exhibiting prematurely condensed sperm chromosomes and the total number of oocytes retrieved (P < 0.008). DISCUSSION
The results of this study confirm and extend those of previous investigations that have assessed the use of GnRH-a for COH on oocyte quality. Treatment with GnRH-a required use of more ampules of hMG for COH and resulted in a greater number of oocytes retrieved. Cytogenetic analysis of the fertilization failures revealed that although the meiotic aneuploidy rate was not affected significantly by treatment with the agonist, the incidence of both meiotic aberrations and prematurely condensed sperm chromosomes in these failed fertilized oocytes was enhanced in the +GnRH-a group. Cytogenetic analysis of IVF failures is considered invaluable because information obtained provides insight into the incidence of meiotic errors (15, 16) and the occurrence of both anomalies in the physiology (17) and abnormalities in the fertilization process (11, 18). From a clinical standpoint, such information could be of prognostic value for subsequent cycles of treatment, particularly if patient-specific oocyte pathologies are identified (19). Accordingly, the present study was conducted to contribute to the body of literature that addresses the incidence of chromosomal anomalies in failed fertilized oocytes after GnRH-a treatment (15, 20). It was impossible to classify into a meiotic stage 14.5% of the 161 oocytes processed, which is consistent with the 10% to 20% loss rate observed by some workers (15, 18), although others have reported an attrition rate as high as 35% (20). Oocytes were lost from the study because of the well-known difficulties Racowsky
et
al. Oocyte abnormalities in IVF failures
935
Air-dried preparations of human oocytes showing (A) a decondensing sperm head; and (B-F) various degrees of condensation of sperm chromatin. (B), Prematurely condensed sperm chromosomes observed in early prophase of the presynthetic gap phase (Gil; (0, prematurely condensed sperm chromosomes in midprophase of G1; (D), MI1 chromosomes of an oocyte exhibiting a prematurely condensed chromosome in late prophase of Gi; (E), an oocyte exhibiting prematurely condensed sperm chromosomes in late prophase of Gi, the chromatids of which k~rrozu)are interspersed with the MI1 oocyte chromosomes; (F), an arrested MI1 oocyte exhibiting prematurely condensed sperm chromosomes at Gz; the duplicated sperm chromatids (arrow) are intermingled with the oocyte chromosomes. Magnification, x350. Figure 2
encountered in air-drying human oocytes. These include technical problems associated with 111inadequate dispersion or overscattering of chromosomes (15,211 and 121structural anomalies associated with chromosome fragmentation and chromatid separation (22). Meiotically aberrant oocytes, arising from failure to emit the first polar body (i.e., diploid MI1 oocytes), are observed relatively frequently when unfertilized oocytes are analyzed. In the present study, the overall incidence was 14.2%, which is higher than the 7% to 9% incidence reported previously (21, 23). The major contributors to this elevated incidence of diploids were patients in the +GnRH-a group, from whom more oocytes were retrieved. Taken together, these observations are consistent with the previous conclusion (21,231 that a positive correlation exists between the number of oocytes retrieved and the incidence of diploid oocytes. Several previous studies have reported the pres936
Bacowsky
et al.
Oocyte abnormalities
in IVF failures
ence of prematurely condensed sperm chromosomes in air-dried human oocytes (see review by Tejada et al. [181). This phenomenon is associated with an asynchrony between oocyte nuclear and cytoplasmic maturation such that cytoplasmic chromosome condensing factors remain, leading to induction of condensation of the sperm nucleus (24). Accordingly, the presence of prematurely condensed sperm chromosomes indicates an immaturity of the oocyte. The results of the present study reveal that 15.9% of all oocytes examined had prematurely condensed sperm chromosomes present in their cytoplasm, which is consistent with the range of frequencies previously reported for ostensibly unfertilized human oocytes (reviewed by Tejada et al. [ND. However, when the data were stratified by stimulation protocol, GnRHa treatment was associated with a significant elevation in the incidence of this phenomenon. Indeed, such an abnormality in fertilization was observed in Fertility and Sterility@
as many as 20% of oocytes examined in this patient group but was present in only 3% of oocytes from patients treated without agonist. The occurrence of prematurely condensed sperm chromosomes did not correlate with 111insufficient sperm parameters, because all couples with male factor infertility were excluded from this study; and 121the grade of the cumulus-oocyte maturity at retrieval. Likewise, our observations do not support an association between a higher frequency of prematurely condensed sperm chromosomes and idiopathic infertility (25) or FSH stimulation (18), because all patients included in this study exhibited bilateral tubal factor and FSH was used to stimulate only 17% of the patients whose oocytes exhibited this fertilization abnormality. The fact that prematurely condensed sperm chromosomes were observed in both MI1 oocytes and oocytes failing to emit the first polar body demonstrates that the presence of this anomaly is not limited to those oocytes that fail to complete nuclear meiotic maturation. A higher incidence of diploid oocytes and prematurely condensed sperm chromosomes was observed in the +GnRH-a group, indicating impairment of the nuclear and cytoplasmic maturation processes in some oocytes. These effects were unlikely to be because of patient age, because there was no significant difference between the -GnRH-a and +GnRHa groups in patient age. The possible mechanisms that mediate these GnRH-a-associated perturbations in development remain to be elucidated. At least two possibilities exist. First, the agonist may act directly at the ovarian level to disrupt the follicular environment, rendering it incompatible with normal developmental events of the oocyte. In support of this possibility, not only have GnRH and GnRHa been shown to have direct modulatory effects on granulosa cell function in a variety of mammalian species (23), but also GnRH-a has been shown to inhibit P production and to enhance E2 production by human granulosa cells cultured in vitro after in vivo exposure to the agonist (23). However, evidence against a possible direct deleterious action of the agonist on the follicle is provided by [ll the previous observation that follicular atresia was not increased in women treated with GnRH-a (23); and [2] the present observation that there was no significant increase in the incidence either of degenerate oocytes retrieved or of MI1 oocytes exhibiting degenerate chromosomes in air-dry spreads. An alternative, and more likely, explanation for the increased frequency of diploid oocytes and oocytes exhibiting prematurely condensed sperm chromosomes in the +GnRH-a group relates to the increased number of oocytes retrieved from patients who used this stimulation regimen. Under the in-
fluence of GnRH-a, “additional” follicles are recruited to increase the size of the retrieval cohort. Such additional follicles may arise from either those that otherwise would undergo atresia (9) or those that are in the young growing pool (21). Regardless, these follicles represent a suboptimal population that contains fewer mature oocytes. Taken together, the present findings indicate that within the larger cohort of oocytes retrieved after GnRH-a treatment, the quality of at least some oocytes is compromised, as manifested by an increased incidence of meiotic aberrations and prematurely condensed sperm chromosomes. Based on available data, it is more likely that these phenomena result from the addition into the recruited cohort of secondary follicles containing less mature oocytes rather than from an adverse effect of GnRH-a either directly on the oocyte itself or indirectly through perturbation of the follicular environment. Accordingly, in cycles involving the use of GnRH-a, it may be prudent to delay administration of hCG until the lead follicles are larger than the accepted size in cycles of COH not using GnRH-a.
Vol. 67, No. 5, May 1997
Racowsky
Acknowledgment. We thank Mark Stevens, B.A., for assisting in preparation of the photographic plates.
REFERENCES 1. Taymour ML. The regulation of follicle growth: some clinical implications in reproductive endocrinology. Fertil Steril 1996;65:235-47. 2. Ron-El R, Herman A, Golan A, Nachum H, Soffer Y, Caspi E. Gonadotropins and combined gonadotropin-releasing hormone agonist-gonadotropins protocols in a randomized prospective study. Fertil Steril 1991;55:574-8. 3. Kubik CJ, Guzick DS, Berga SL, Zeleznik AJ. Randomized, prospective trial of leuprolide acetate and conventional superovulation in first cycles of in vitro fertilization and gamete intrafallopian transfer. Fertil Steril 1990;54:836-41. 4. Tan SL, Kingsland C, Campbell S, Mills C, Bradfield J, Alexander N, et al. The long protocol of administration of gonadotropin-releasing hormone agonist is superior to the short protocol for ovarian stimulation for in vitro fertilization. Fertil Steril 1992;57:810-4. 5. Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA. The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 1992;58:888-96. 6. Chetowski RJ, Rode RA, Burruel V, Nass TE. The effect of pituitary suppression and the woman’s age on embryo viability and uterine receptivity. Fertil Steril 1991;56:1095-103. Grochowski D, Wolczynski S, Kuczynski, Kulikowski M, Szamatowicz M. The results of an in vitro fertilization program: two regimens of superovulation. Gynecol Endocrinol 1995;9: 59-62. Cedars MI, Surrey E, Hamilton F, Lapolt P, Meldrum DR. Leuprolide acetate lowers circulating bioactive luteinizing hormone and testosterone concentrations during ovarian stimulation for oocyte retrieval. Fertil Steril 1990;53:62731. et al.
Oocyte abnormalities
in IVF failures
937
9. Liu HC, Lai YM, Davis 0, Berkeley AS, Graf M, Grifo J, et al. Improved pregnancy outcome with gonadotropin releasing hormone agonist (GnRH-a) stimulation is due to the improvement in oocyte quantity rather than quality. J Assist Reprod Genet 1992;9:338-44. 10. De Sutter P, Dhont M, Vanekerckhove D. Hormonal stimulation for in vitro fertilization: a comparison of fertilization rates and cytogenetic findings in unfertilized oocytes. J Assist Reprod Genet 1992;9:254-8. 11. Schmiady H, Kentenich H. Premature chromosome condensation after in vitro fertilization. Hum Reprod 1989;4:68995. 12. San Roman GA, Surrey ES, Judd HL, Kerin JF. A prospective randomized comparison of luteal phase versus concurrent follicular phase initiation of gonadotropin-releasing hormone agonist for in vitro fertilization. Fertil Steril 1992;58:744-9. 13. Racowsky C, Kaufman ML, Dermer RA, Homa ST, Gunnala S. Chromosomal analysis of meiotic stages of human oocytes matured in vitro: benefits of protease treatment before fixation. Fertil Steril 1992;57:1026-33. 14. Dyban AP. An improved method for chromosome preparations from preimplantation mammalian embryos, oocytes or isolated blastomeres. Stain Technol 1983;58:69-72. 15. Tejada MI, Mendoza MR, Corcostegui B, Benitc JA. Chromosome studies in human unfertilized oocytes and uncleaved zygotes after treatment with gonadotropin-releasing hormone analogs. Fertil Steril 1991;56:874-80. 16. Pieters MHEC, Geraedts JPM, Dumoulin JCM, Evers JLH, Bras M, Kornips FHAC, et al. Cytogenetic analysis of in vitro fertilization (IVF) failures. Hum Genet 1989;81:367-70. 17. Papadopoulos G, Randall J, Templeton AA. The frequency of chromosome anomalies in human unfertilized oocytes and
938
Racowsky
et al.
Oocyte abnormalities in IVF failures
18.
19.
20.
21.
22.
23.
24.
25.
uncleaved zygotes after insemination in vitro. Hum Reprod 1989;4:568-73. Tejada MI, Mendoza MR, Corcostegui B, Benito JA. Factors associated with premature chromosome condensation (PC0 following in vitro fertilization. J Assist Reprod Genet 1992; 9:61-7. Eichenlaub-Ritter U, Schmiady H, Kentenich H, Soewarto D. Recurrent failure in polar body formation and premature chromosome condensation in oocytes from a human patient: indicators of asynchrony in nuclear and cytoplasmic maturation. Hum Reprod 1995; 10:2343-g. Delhanty JDA, Penketh RJA. Cytogenetic analysis of unfertilized oocytes retrieved after treatment with the LHRH analogue, buserelin. Hum Reprod 1990;5:699-702. Tarin JJ, Pellicer A. Consequences of high ovarian response to gonadotropins: a cytogenetic analysis of unfertilized human oocytes. Fertil Steril 1990;54:665-70. Plachot M, Veiga A, Montagut J, de Grouchy J, Calderon G, Lepretre S, et al. Are clinical and biological IVF parameters correlated with chromosomal disorders in early life? Hum Reprod 1988;3:627-35. Pellicer A, Tarin JJ, Miro F, Sampaio M, De 10s Santos MJ, Remohi J. The use of gonadotropin releasing hormone analogues (GnRHa) in in vitro fertilization: some clinical and experimental investigations of a direct effect on the human ovary. Hum Reprod 1992;7 Suppl 1:39-47. Badenas J, Santalo J, Calafell JM, Estop AM, Egozcue J. Effect of the degree of maturation of mouse oocytes at fertilization: source of chromosome imbalance. Gamete Res 1989;24:205- 18. Selva J, Martin-Pont B, Hugues JN, Rince P, Fillion C, Herve F, et al. Cytogenetic study of human oocytes uncleaved after in-vitro fertilization. Hum Reprod 1991;6:709-13.
Fertility and Sterility@
Vol. 61, No. 5, May 1997
Copyright ’ 1997AmericanSocietyforReproductive Medicine
Printed on acid-free paper in U. S. A.
Published by ElsevierScienceInc.
Prematurely condensed chromosomes and meiotic abnormalities in unfertilized human oocytes after ovarian stimulation with and without gonadotropin-releasing hormone agonist
Catherine Racowsky, Ph.D.* Angela L. Prather, B.S. Malinda K. Johnson, B.S. Division of Reproductive University of Arizona,
Sandra P. Olvera, B.S. Timothy J. Gelety, M.D.
Endocrinology
and Infertility,
Department
of Obstetrics and Gynecology,
College of Medicine,
Tucson, Arizona
Objective: To investigate the incidence of meiotic abnormalities, aneuploidy, and prematurely condensed sperm chromosomes in failed fertilized oocytes after controlled ovarian hyperstimulation (COH). Design: Retrospective analysis of air-dried preparations of unfertilized oocytes. Setting: University hospital-based infertility clinic. Patient(s): Thirty-three patients undergoing IVF having only tubal factor as the cause of infertility. Twelve patients (13 cycles) underwent treatment with hMG alone (-GnRH agonist [GnRH-a]), and 21 patients (24 cycles) underwent treatment with leuprolide acetate (LA) and hMG (+GnRH-a group). Intervention(s): Standard IVF-ET treatment cycle for ovarian stimulation using hMG with or without LA. Main Outcome Measure(s): The meiotic stage, ploidy, and the presence of prematurely condensed sperm chromosomes were determined in 161 air-dried preparations of unfertilized oocytes. Result(s): Significantly more unfertilized oocytes were at metaphase II in the -GnRH-a group as compared with the +GnRH-a group, with significantly fewer exhibiting meiotic aberrations. Aneuploidy rates did not differ between groups. However, significantly more oocytes in the +GnRH-a group revealed prematurely condensed sperm chromosomes than in the -GnRH-a groupConclusion(s): The use of GnRH-a for COH does not have an impact on aneuploidy rates in failed fertilized oocytes. However, the higher incidence of meiotic aberrations and prematurely condensed sperm chromosomes in the unfertilized population indicates that some retrieved oocytes exhibit incomplete nuclear and cytoplasmic maturation after the use of this agonist. (Fertil Sterile 1997;67:932-8. 0 1997 by American Society for Reproductive Medicine.) Key Words: GnRH-a, prematurely condensed sperm chromosomes, oocyte meiotic abnormalities, IVF failures
The formation of a cyte with a haploid quires a coordinated events that ensures
developmentally competent oochromosomal complement reand finely tuned sequence of both nuclear and cytoplasmic
Received October 2, 1996; revised and accepted January 22, 1997. * Reprintrequests:CatherineRacowsky,Ph.D.,Divisionof ReproductiveEndocrinologyand Infertility,Departmentof Obstetrics and Gynecology,Collegeof Medicine,Universityof Arizona, Tucson,Arizona 85724 (FAX: 520-626-2768). 932
maturation of the gamete. When controlled ovarian hyperstimulation (COH) is used for the purpose of IVF’, the resulting multiple follicular development typically arises from the addition of follicles at different stages of development (see review by Taymour [ll). Consequently, oocytes retrieved for M? often are at various stages of maturity, and even when the timing of insemination is adjusted according to the maturity of the cumulus-corona cells, fertilization rates are generally suboptimal. The addition of GnRH agonists (GnRH-a) to COH 0015-0282/97/$17.00 PI1 SOOX-0282(97)00125-8
regimens for IVF has several recognized advantages, including reduced cancellation rates, increased ease of scheduling, and fewer premature luteinizations (2, 3). In addition, the increased number of oocytes obtained per retrieval generally results in an increase in the number of embryos available for transfer (4). Moreover, a meta-analysis of randomized studies has demonstrated a twofold increase in the pregnancy rate when GnRH-a treatment has been used in conjunction with COH (51, and use of this agonist has been shown to result in improved embryo quality (2,6) and reduced spontaneous abortion rates (7). Use of GnRH-a results in suppression of androgen production by the ovaries (81, which in turn promotes maturation of secondary follicles that otherwise would become atretic. Accordingly, the quality of these extra oocytes may be compromised, as has been suggested previously based on the implantation rate per embryo transferred after the use of this agonist (9). The possibility exists, therefore, that, despite a typical increase in oocyte quantity after use of GnRH-a in COH protocols, oocyte quality may not be optimum in all oocytes of the retrieved cohort. In an attempt to assess further the quality of oocytes harvested after the use of GnRH-a in IVF patients, we have compared results for patients stimulated with gonadotropins alone or with gonadotropins plus GnRH-a. We have assessed oocyte maturity and fertilization rates at retrieval. In addition, because cytogenetic analysis of failed fertilized oocytes provides some insight into the overall quality of a retrieved cohort (lo), we have investigated the meiotic abnormality rate in these failures for both groups. Furthermore, because the phenomenon of premature sperm chromosome condensation is associated with oocyte cytoplasmic immaturity (ll), we have determined the relative incidence of prematurely condensed sperm chromosomes in the unfertilized oocytes of both patient groups.
levels by rapid RIA. Human chorionic gonadotropin (Profasi, 10,000 USP U IM; Serono Laboratories) was given when at least three follicles were observed with a lead diameter of 217 mm and a serum E2 2 500 pg/mL (1,840 pmol/L) was achieved. Transvaginal aspiration was performed 34 to 35 hours later. The COH regimen used for the patients in the +GnRH-a group (n = 21, representing 24 cycles) has been described in detail (12). Briefly, GnRH-a (leuprolide acetate; TAP Pharmaceuticals, North Chicago, IL) was begun on cycle day 21(1 mg SC twice daily) after spontaneous or induced withdrawal bleeding and/or 8 days after detection of an urinary LH surge. On day 31, transvaginal sonography was performed and serum Ez was assayed to ensure ovarian suppression (no ovarian cyst > 10 mm in diameter and Ez < 30 pg/mL 1110 pmol/LI). The GnRH-a was reduced to 0.5 mg/d SC, and hMG (300 IU/d IM) was administered for 5 days. Follicular development was evaluated after 5 days by transvaginal sonography and measurement of peripheral serum Ez by rapid RIA. The hMG dosage was then individualized based on follicular response, which was monitored at l- to 3-day intervals. Human chorionic gonadotropin (10,000 USP U) was administered IM when three or more follicles had a mean diameter of 218 mm and when a serum E2 2 500 pg/mL (1,840 pmol/L) was achieved. Ultrasound-guided transvaginal sonography was performed 34 to 35 hours later. After aspiration, all oocytes were scored for maturity using standard procedures for assessment of corona-cumulus expansion. Oocytes were scored for the presence of pronuclei (PN) 16 to 18 hours postinsemination. Those without cleavage and those with 1 or more than 2 PNs at the time of ET were set aside for preparation of air-dried spreads. Oocytes were air dried as soon as possible after ET, with the majority (88%) being air dried 48 to 60 hours postinsemination and the remainder being air dried between 61 and 80 hours.
MATERIALS AND METHODS Patients and IVF’Procedures
Preparation and Analysis of Air-Dried Oocyte Chromosome Spreads
The present study was approved by the institutional Human Subjects Committee at the University of Arizona Health Sciences Center and included 33 patients (age 26.7 to 36.9 years) having only a diagnosis of tubal factor as the cause of infertility. Twelve of these patients, representing 13 cycles, underwent COH with only hMG (Pergonal, 225 to 300 IU IM daily; Serono Laboratories, Randolph, MA) from cycle day 3 to 11 (the -GnRH-a group). Follicular development was evaluated by transvaginal sonography and measurement of peripheral serum Ez
Preparations of air-dried oocytes were made using either the protease procedure (13) or Dyban’s procedure (14) and then aged at room temperature for 2 to 3 days before being stained with Wright’s stain (13). Select preparations were photographed using a Cytovision 2.21 Imaging System (Applied Imaging, Santa Clara, CA) with a Zeiss Axioskop (Carl Zeiss Inc., Tempe, AZ). Spreads were analyzed under phase contrast illumination using a Nikon Labophot-2 compound microscope and classified as exhibiting 111 nondegenerating metaphase II (MID
Vol. 67, No. 5, May 1997
Racowsky
et al.
Oocyte abnormalities
in IVF failures
933
Figure 1 Air-dried preparations of failed fertilized human oocytes showing oocyte chromosome configurations. (A), Metaphase II chromosomes of a haploid oocyte (n = 23) showing 6 sperm heads that probably were bound to the zona pellucida before air drying; (B), degenerating MII; (C), a diploid MI1 chromosome configuration revealing failure of segregation of homologous chromosomes at anaphase I; (D), mitotic chromosomes of an oocyte that underwent developmental arrest postsyngamy. Magnification, x350.
chromosomes (Fig. 1A); [21 degenerating MI1 chromosomes (Fig. 1B); 131a meiotically aberrant configuration; 141 1 or >2 PNs; or 151mitotic chromosomes arrested postsyngamy (Fig. lD>. The meiotically aberrant configurations included MI chromosomes, or chromosomes at either diploid MI1 (Fig. 10 or failed anaphase I. When MI1 chromosomes could be counted accurately, ploidy was determined. To avoid errors in assessment of hypohaploidy because of chromosome scattering, the count was included only if the chromosomes were within a clearly defined region overlying the cytoplasm of the oocyte. For all oocytes, the presence of decondensing sperm heads and prematurely condensed sperm chromosomes was noted. Statistical
Analyses
Data were expressed as the means 2 SEM. For mean comparisons, analysis of variance was applied using the Statistical Analysis System @AS), with homogeneity of variance being tested using Bartlet’s 934
Racowsky
et al.
Oocyte abnormalities in IVF failures
test. Differences in proportions were tested using Fisher’s exact test. Linear regression analysis was applied where indicated. For all analyses, a probability of P < 0.05 was considered statistically significant, and all P values were two-tailed. REXJLTS
Patient age did not differ between the two groups (32.5 2 0.7 versus 33.12 0.6 years for -GnRH-a and +GnRH-a groups, respectively). Significantly more ampules of hMG were used by the +GnRH-a patients, as compared with those not using GnRH-a (33.1 2 1.1 versus 27.8 2 1.9 ampules; P < 0.014). The use of GnRH-a resulted in significantly more oocytes being retrieved per patient (13.6 + 1.7 versus 8.9 k 1.2; P < 0.051, with preovulatory oocytes being retrieved with the highest frequency in both groups (61.8% + 8.0% versus 64.5% + 3.2% for -GnRHa and +GnRH-a, respectively). The distribution of grades of oocyte maturity was not affected significantly by stimulation protocol, except that more Fertility and Sterility@
postmature oocytes were retrieved in the +GnRH-a group (3.8% 2 1.4% versus 0.8% +- 0.8%; P < 0.04). Use of GnRH-a significantly enhanced the rate of fertilization of those oocytes that were graded as preovulatory at retrieval (75.5% 2 4.7% versus 42.5% + 10.7%; P < 0.002), as well as the overall rate of fertilization (59.1% 2 6.8% versus 35.8% t 6.8%; P < 0.002). Table 1 shows the results of the chromosomal analyses performed. There was no significant difference between the two patient groups regarding the proportion of air-dried oocytes that were analyzable (83.8% and 88.9% for -GnRH-a and +GnRH-a, respectively). The remaining oocytes could not be analyzed because of excessive chromosome overlapping or scattering. Significantly more oocytes were at MI1 in the -GnRH-a group than in the +GnRH-a group (P < 0.0411, with significantly fewer oocytes exhibiting meiotic aberrations (P < 0.026). However, there was no significant difference between the two patient groups regarding the proportion of oocytes that exhibited 1 or >2 PNs, that had undergone postsyngamy arrest, revealing mitotic chromosomes, or that were achromosomal. Likewise, the proportions of MI1 oocytes that exhibited degenerate chromosomes or that were aneuploid did not differ between the two patient groups. The overall aneuploidy rate among failed fertilized oocytes obtained from all patients in this study was 48.9%, and the hyperhaploidy rate was 42.9%. Of all oocytes examined, 16 contained decondensing sperm heads (Fig. 2A), and there was no difference between patient groups in the proportion of oocytes exhibiting this phenomenon. Prematurely condensed sperm chromosomes were observed in oocytes from both patient groups and in oocytes at all stages of maturity at retrieval. The distribution of oocytes with prematurely condensed sperm chromosomes by oocyte maturity was as follows: 35% in
Table 1 Chromosome Analysis of Unfertilized Human
Oocytes Obtained After Retrieval From Patients Treated With or Without GnRH-a
Nos. air-dried oocyte preparations Nos. analyzable oocyte preparations* Nos. MII$ Nos. meiotically aberrant$ Nos. 1 PN$ Nos. >2 PNP Nos. postsyigamy arrest* Nos. achromosomal$
-GnRH-a
+GnRH-a
62 52 (83.9)
99 88 (88.9)
44 (84.6) 3 (5.8) 1 (1.9) 0 (0) 2 (3.8) 1 (1.9)
61 (69.3) 17 (19.3) 2 (2.3) 3 (3.4) 4 (4.5) l(1.1)
P
NSI 0.041 0.026
NW
NSt
NSP
NW
* Percentage of air-dried oocytes is given in parentheses. ? NS, not significant. $ Percentage of analyzable oocytes is given in parentheses. Vol. 67, No. 5, May 1997
preovulatory, 30% in intermediate, 20% in immature, and 15% in postmature oocytes. Although the majority of oocytes with prematurely condensed sperm chromosomes were at MI1 (71.4%), several oocytes containing prematurely condensed sperm chromosomes had failed to emit their first polar body. All but one of the prematurely condensed sperm chromosome configurations were at various stages of formation of the presynthetic gap phase (G,) (Fig. 2B-E), the remaining one being at G2 with clearly defined duplicated chromatids (Fig. 2F). Significantly more analyzable oocytes in the +GnRH-a group revealed prematurely condensed sperm chromosomes than in the -GnRH-a (20.2% +_3.2% versus 2.6% ? 1.2%; P < 0.0371, and this difference between treatment groups was also upheld when only the MI1 oocytes were considered (P < 0.050). Furthermore, a significant positive correlation was observed between the number of +GnRH-a oocytes exhibiting prematurely condensed sperm chromosomes and the total number of oocytes retrieved (P < 0.008). DISCUSSION
The results of this study confirm and extend those of previous investigations that have assessed the use of GnRH-a for COH on oocyte quality. Treatment with GnRH-a required use of more ampules of hMG for COH and resulted in a greater number of oocytes retrieved. Cytogenetic analysis of the fertilization failures revealed that although the meiotic aneuploidy rate was not affected significantly by treatment with the agonist, the incidence of both meiotic aberrations and prematurely condensed sperm chromosomes in these failed fertilized oocytes was enhanced in the +GnRH-a group. Cytogenetic analysis of IVF failures is considered invaluable because information obtained provides insight into the incidence of meiotic errors (15, 16) and the occurrence of both anomalies in the physiology (17) and abnormalities in the fertilization process (11, 18). From a clinical standpoint, such information could be of prognostic value for subsequent cycles of treatment, particularly if patient-specific oocyte pathologies are identified (19). Accordingly, the present study was conducted to contribute to the body of literature that addresses the incidence of chromosomal anomalies in failed fertilized oocytes after GnRH-a treatment (15, 20). It was impossible to classify into a meiotic stage 14.5% of the 161 oocytes processed, which is consistent with the 10% to 20% loss rate observed by some workers (15, 18), although others have reported an attrition rate as high as 35% (20). Oocytes were lost from the study because of the well-known difficulties Racowsky
et
al. Oocyte abnormalities in IVF failures
935
Air-dried preparations of human oocytes showing (A) a decondensing sperm head; and (B-F) various degrees of condensation of sperm chromatin. (B), Prematurely condensed sperm chromosomes observed in early prophase of the presynthetic gap phase (Gil; (0, prematurely condensed sperm chromosomes in midprophase of G1; (D), MI1 chromosomes of an oocyte exhibiting a prematurely condensed chromosome in late prophase of Gi; (E), an oocyte exhibiting prematurely condensed sperm chromosomes in late prophase of Gi, the chromatids of which k~rrozu)are interspersed with the MI1 oocyte chromosomes; (F), an arrested MI1 oocyte exhibiting prematurely condensed sperm chromosomes at Gz; the duplicated sperm chromatids (arrow) are intermingled with the oocyte chromosomes. Magnification, x350. Figure 2
encountered in air-drying human oocytes. These include technical problems associated with 111inadequate dispersion or overscattering of chromosomes (15,211 and 121structural anomalies associated with chromosome fragmentation and chromatid separation (22). Meiotically aberrant oocytes, arising from failure to emit the first polar body (i.e., diploid MI1 oocytes), are observed relatively frequently when unfertilized oocytes are analyzed. In the present study, the overall incidence was 14.2%, which is higher than the 7% to 9% incidence reported previously (21, 23). The major contributors to this elevated incidence of diploids were patients in the +GnRH-a group, from whom more oocytes were retrieved. Taken together, these observations are consistent with the previous conclusion (21,231 that a positive correlation exists between the number of oocytes retrieved and the incidence of diploid oocytes. Several previous studies have reported the pres936
Bacowsky
et al.
Oocyte abnormalities
in IVF failures
ence of prematurely condensed sperm chromosomes in air-dried human oocytes (see review by Tejada et al. [181). This phenomenon is associated with an asynchrony between oocyte nuclear and cytoplasmic maturation such that cytoplasmic chromosome condensing factors remain, leading to induction of condensation of the sperm nucleus (24). Accordingly, the presence of prematurely condensed sperm chromosomes indicates an immaturity of the oocyte. The results of the present study reveal that 15.9% of all oocytes examined had prematurely condensed sperm chromosomes present in their cytoplasm, which is consistent with the range of frequencies previously reported for ostensibly unfertilized human oocytes (reviewed by Tejada et al. [ND. However, when the data were stratified by stimulation protocol, GnRHa treatment was associated with a significant elevation in the incidence of this phenomenon. Indeed, such an abnormality in fertilization was observed in Fertility and Sterility@
as many as 20% of oocytes examined in this patient group but was present in only 3% of oocytes from patients treated without agonist. The occurrence of prematurely condensed sperm chromosomes did not correlate with 111insufficient sperm parameters, because all couples with male factor infertility were excluded from this study; and 121the grade of the cumulus-oocyte maturity at retrieval. Likewise, our observations do not support an association between a higher frequency of prematurely condensed sperm chromosomes and idiopathic infertility (25) or FSH stimulation (18), because all patients included in this study exhibited bilateral tubal factor and FSH was used to stimulate only 17% of the patients whose oocytes exhibited this fertilization abnormality. The fact that prematurely condensed sperm chromosomes were observed in both MI1 oocytes and oocytes failing to emit the first polar body demonstrates that the presence of this anomaly is not limited to those oocytes that fail to complete nuclear meiotic maturation. A higher incidence of diploid oocytes and prematurely condensed sperm chromosomes was observed in the +GnRH-a group, indicating impairment of the nuclear and cytoplasmic maturation processes in some oocytes. These effects were unlikely to be because of patient age, because there was no significant difference between the -GnRH-a and +GnRHa groups in patient age. The possible mechanisms that mediate these GnRH-a-associated perturbations in development remain to be elucidated. At least two possibilities exist. First, the agonist may act directly at the ovarian level to disrupt the follicular environment, rendering it incompatible with normal developmental events of the oocyte. In support of this possibility, not only have GnRH and GnRHa been shown to have direct modulatory effects on granulosa cell function in a variety of mammalian species (23), but also GnRH-a has been shown to inhibit P production and to enhance E2 production by human granulosa cells cultured in vitro after in vivo exposure to the agonist (23). However, evidence against a possible direct deleterious action of the agonist on the follicle is provided by [ll the previous observation that follicular atresia was not increased in women treated with GnRH-a (23); and [2] the present observation that there was no significant increase in the incidence either of degenerate oocytes retrieved or of MI1 oocytes exhibiting degenerate chromosomes in air-dry spreads. An alternative, and more likely, explanation for the increased frequency of diploid oocytes and oocytes exhibiting prematurely condensed sperm chromosomes in the +GnRH-a group relates to the increased number of oocytes retrieved from patients who used this stimulation regimen. Under the in-
fluence of GnRH-a, “additional” follicles are recruited to increase the size of the retrieval cohort. Such additional follicles may arise from either those that otherwise would undergo atresia (9) or those that are in the young growing pool (21). Regardless, these follicles represent a suboptimal population that contains fewer mature oocytes. Taken together, the present findings indicate that within the larger cohort of oocytes retrieved after GnRH-a treatment, the quality of at least some oocytes is compromised, as manifested by an increased incidence of meiotic aberrations and prematurely condensed sperm chromosomes. Based on available data, it is more likely that these phenomena result from the addition into the recruited cohort of secondary follicles containing less mature oocytes rather than from an adverse effect of GnRH-a either directly on the oocyte itself or indirectly through perturbation of the follicular environment. Accordingly, in cycles involving the use of GnRH-a, it may be prudent to delay administration of hCG until the lead follicles are larger than the accepted size in cycles of COH not using GnRH-a.
Vol. 67, No. 5, May 1997
Racowsky
Acknowledgment. We thank Mark Stevens, B.A., for assisting in preparation of the photographic plates.
REFERENCES 1. Taymour ML. The regulation of follicle growth: some clinical implications in reproductive endocrinology. Fertil Steril 1996;65:235-47. 2. Ron-El R, Herman A, Golan A, Nachum H, Soffer Y, Caspi E. Gonadotropins and combined gonadotropin-releasing hormone agonist-gonadotropins protocols in a randomized prospective study. Fertil Steril 1991;55:574-8. 3. Kubik CJ, Guzick DS, Berga SL, Zeleznik AJ. Randomized, prospective trial of leuprolide acetate and conventional superovulation in first cycles of in vitro fertilization and gamete intrafallopian transfer. Fertil Steril 1990;54:836-41. 4. Tan SL, Kingsland C, Campbell S, Mills C, Bradfield J, Alexander N, et al. The long protocol of administration of gonadotropin-releasing hormone agonist is superior to the short protocol for ovarian stimulation for in vitro fertilization. Fertil Steril 1992;57:810-4. 5. Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA. The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 1992;58:888-96. 6. Chetowski RJ, Rode RA, Burruel V, Nass TE. The effect of pituitary suppression and the woman’s age on embryo viability and uterine receptivity. Fertil Steril 1991;56:1095-103. Grochowski D, Wolczynski S, Kuczynski, Kulikowski M, Szamatowicz M. The results of an in vitro fertilization program: two regimens of superovulation. Gynecol Endocrinol 1995;9: 59-62. Cedars MI, Surrey E, Hamilton F, Lapolt P, Meldrum DR. Leuprolide acetate lowers circulating bioactive luteinizing hormone and testosterone concentrations during ovarian stimulation for oocyte retrieval. Fertil Steril 1990;53:62731. et al.
Oocyte abnormalities
in IVF failures
937
9. Liu HC, Lai YM, Davis 0, Berkeley AS, Graf M, Grifo J, et al. Improved pregnancy outcome with gonadotropin releasing hormone agonist (GnRH-a) stimulation is due to the improvement in oocyte quantity rather than quality. J Assist Reprod Genet 1992;9:338-44. 10. De Sutter P, Dhont M, Vanekerckhove D. Hormonal stimulation for in vitro fertilization: a comparison of fertilization rates and cytogenetic findings in unfertilized oocytes. J Assist Reprod Genet 1992;9:254-8. 11. Schmiady H, Kentenich H. Premature chromosome condensation after in vitro fertilization. Hum Reprod 1989;4:68995. 12. San Roman GA, Surrey ES, Judd HL, Kerin JF. A prospective randomized comparison of luteal phase versus concurrent follicular phase initiation of gonadotropin-releasing hormone agonist for in vitro fertilization. Fertil Steril 1992;58:744-9. 13. Racowsky C, Kaufman ML, Dermer RA, Homa ST, Gunnala S. Chromosomal analysis of meiotic stages of human oocytes matured in vitro: benefits of protease treatment before fixation. Fertil Steril 1992;57:1026-33. 14. Dyban AP. An improved method for chromosome preparations from preimplantation mammalian embryos, oocytes or isolated blastomeres. Stain Technol 1983;58:69-72. 15. Tejada MI, Mendoza MR, Corcostegui B, Benitc JA. Chromosome studies in human unfertilized oocytes and uncleaved zygotes after treatment with gonadotropin-releasing hormone analogs. Fertil Steril 1991;56:874-80. 16. Pieters MHEC, Geraedts JPM, Dumoulin JCM, Evers JLH, Bras M, Kornips FHAC, et al. Cytogenetic analysis of in vitro fertilization (IVF) failures. Hum Genet 1989;81:367-70. 17. Papadopoulos G, Randall J, Templeton AA. The frequency of chromosome anomalies in human unfertilized oocytes and
938
Racowsky
et al.
Oocyte abnormalities in IVF failures
18.
19.
20.
21.
22.
23.
24.
25.
uncleaved zygotes after insemination in vitro. Hum Reprod 1989;4:568-73. Tejada MI, Mendoza MR, Corcostegui B, Benito JA. Factors associated with premature chromosome condensation (PC0 following in vitro fertilization. J Assist Reprod Genet 1992; 9:61-7. Eichenlaub-Ritter U, Schmiady H, Kentenich H, Soewarto D. Recurrent failure in polar body formation and premature chromosome condensation in oocytes from a human patient: indicators of asynchrony in nuclear and cytoplasmic maturation. Hum Reprod 1995; 10:2343-g. Delhanty JDA, Penketh RJA. Cytogenetic analysis of unfertilized oocytes retrieved after treatment with the LHRH analogue, buserelin. Hum Reprod 1990;5:699-702. Tarin JJ, Pellicer A. Consequences of high ovarian response to gonadotropins: a cytogenetic analysis of unfertilized human oocytes. Fertil Steril 1990;54:665-70. Plachot M, Veiga A, Montagut J, de Grouchy J, Calderon G, Lepretre S, et al. Are clinical and biological IVF parameters correlated with chromosomal disorders in early life? Hum Reprod 1988;3:627-35. Pellicer A, Tarin JJ, Miro F, Sampaio M, De 10s Santos MJ, Remohi J. The use of gonadotropin releasing hormone analogues (GnRHa) in in vitro fertilization: some clinical and experimental investigations of a direct effect on the human ovary. Hum Reprod 1992;7 Suppl 1:39-47. Badenas J, Santalo J, Calafell JM, Estop AM, Egozcue J. Effect of the degree of maturation of mouse oocytes at fertilization: source of chromosome imbalance. Gamete Res 1989;24:205- 18. Selva J, Martin-Pont B, Hugues JN, Rince P, Fillion C, Herve F, et al. Cytogenetic study of human oocytes uncleaved after in-vitro fertilization. Hum Reprod 1991;6:709-13.
Fertility and Sterility@