Gynecol. Endom'nol. 6 (1992) 135-139
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Comparison of ovarian response in the same women with the same or different lots of human menopausal gonadotropin M . P. Diamond*,M . L. Polant, M . Blanchette", C. M . Mazure', A . H . DeCherney" and B. Lunenfeld*tt Yale University School of Medicine, Departments of Obstetrics and Gynecology and $Psychiatry, Division of Reproductive Endocrinology, New Haven, Connecticut, U S A %tanford University, Stanford, California, USA; %heba Medical Center, Ramat Gan, Israel Key words: HUMAN MENOPAUSAL GONADOTROPIN, OVULATION INDUCTION,FOLLICLES, OOCYTES, BIOEQUIVALENCE
ABSTRACT p > 0.05) in all three cycles were also similar. There was no sign$cant dgerence in the day ofhCGadministration ( A 1 , 9 . 1 f 0 . 2 ; A 2 , 9 . 0 f O . l ; B ,9 . 2 f 0 . 2 ) o r t h e total number ofampules o f h M G utilized on Day 9 (AZ, l . l f 0 . 4 ; A 2 , 0 . 6 f 0 . 2 ; B , 0 . 8 f 0 . 2 ) . Thisstudy extends our previous observations in ovulatory women that ovarian response to h M G induction ofsuperovulation is similar in successive cycles and demonstrates in vivo consistency ofhuman ovarian response to d@erent lots o f PergonaP.
A variation in the bioactivity of dgerent production lots o f human menopausal gonadotropin (hMG) has been suggested. Therefore, we evaluated ovarian response to h M G in 1 4 women during three separate ZVF cycles. Thefirst two cycles were peformed with the same lot (#03310027; Cycles A1 and A2); the third cycle utilized dgerent lots O f h M G (Cycle B). Zn all cycles, h M G was administered 3 ampules/day beginning cycle Day 3 and continued f p r at least 6 days. Estradiol and ultrasound evaluations were pe$.ormed on D a y 3, and then daily, beginning on cycle Day 8. Fourteen women completed all three cycles. There were no sign$cant dgerences in baseline estradiol and ultrasound. Estradiol levels on D a y 8 ( A l , 7 5 4 f 130; A2, 700 f 107; B, 520 f 80 pg/ml, analysis of variance p > 0.5) and on D a y 9 (AZ, 1 0 5 1 f 144; A2, 1 1 4 0 f 155; B, 840 f 124pg/ml, p > 0.05) were similar as well. The numberofsmall (1.0-1.4 cm)follicles, large ( 2 1.5 cm) follicles, and totalfollicles (Day 8: total A l , 5.5 f 0.9; A2, 4 . 0 f 0 . 7 ; B , 4.5f0.9,p>O.O5;Day9:total A l , 6 . 7 k 0 . 9 ; A2, 6.9f0.8; B, 6.9+ 0.9,
INTRODUCTION Successful pregnancy outcomes in assisted reproductive surgical procedures occur in only a small percentage of cycles. Consequently, most couples employing these techniques undergo repetitive cycles of ovarian stimulation. The choice of stimulation protocols for multiple follicular recruitment in these subsequent cycles has been a source of previous evaluation by our group, using the criteria of
Correspondence:Dr M. P. Diamond, Vanderbilt University Medical Center, Department of Obstetrics and Gynecology, C-1100, MCN, Nashville, Tennessee 37232, USA
135
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Clinical intraindividual hMG bioactivity
serum estradiol levels, estradiol patterns, and timing of human chorionic gonadotropin (hCG) administration’”. These studies have suggested similarity in response to the same stimulation protocol. The current report extends these previous findings, by evaluating, in a new group of patients, the following parameters: follicular development as assessed by ultrasound and estradiol determinations, oocyte quality and ferthzation, and pre-embryo development. Additionally, since there have been suggestions that discrepancies in ovarian response may be due to variations in bioactivity in different lots of human menopausal gonadotropin (hMG)4,comparison is made between repetitive stimulation with the same or H e r e n t hMG lots (Pergonal, Serono Laboratories, Braintree, MA).
identified follicles on ultrasound examination so that there were less than two follicles of 1 1.5 cm. Oocyte recovery and maturity grading have been previously described5. Insemination of preovulatory oocytes was performed 6-8h after aspiration; immature oocytes were preincubated for 24 h before insemination. Oocytes were examined 14-18 h later for fertilization, as evidenced by identi6cation of two or more pronuclei, and 42 h later for cleavage. Normally fertilized oocytes were transferred approximately48 h afier recovery. Statistical analysis was performed using the paired t-test, X’ analysis, and analysis of variance. Sigdicance was defined as p < 0.05. All data are expressed as mean -+ SEM.
RESULTS MATERIALS AND METHODS A prospective study was initiated to record the results of multiple follicular development in: Women who received human menopausal gonadotropin (hMG, Pergonal, Serono, Braintree, MA) from t h e same lot (#03310027) in two successive in virto fertilization (IVF) cycles (Cycles A1 and A2),and (2) The same women after a third cycle of stimulation with hMG from a different lot (Cycle B). Arrangements were made for purchase by patients of hMG from the same lot at a local pharmacy (for Cycles A1 and A2). No financial support was provided for the performance of this study. In all cycles, the hMG stimulation protocol consisted of the administration of 3 ampules/day from Days 3 to 7. O n Day 8, daily measurement of serum estradiol and follicular dmensions by ultrasound (ADR,3000 S/L, Tempe, Arizona) was initiated. Stimulation was continued (with 2, 3, or 4 ampules/day) until at least two follicles had diameters 2 1.5 cm, and estradiol levels 2 400 pg/m. Human chorionic gonadotropin (hCG, ProGsF, Serono, Braintree, MA), 10 000 IU was administered approximately 24 h after the last hMG dose. Oocyte recovery was scheduled for 36 h after hCG administration. In all cycles, oocyte recovery was cancelled if either of the following developed afier hCG administration: precipitous (greater than 30%) drop in estradiol level, or disappearance of the previously
136
The results of 23 women were available for comparison of the outcome of the two hMG cycles utilizing stimulation with Pergonal lot #03310027 (Table 1). There was no significant difference in ovarian response to stimulation in these two cycles (A1 and A2),as assessed on cycle Day 8, cycle Day 9, or the day of hCG administration when assessed by either estradiol level or follicular development. There was also no difference in patient management on Day 9 as assessed by ampules of hMG administered on Day 9. In the initial cycle, one woman did not receive hCG (peak estradiol level 82 pg/ml) and two women were dropped after hCG administration because of a fall in estradiol. Thus, 20 women were available for assessment of oocyte recovery and fertilization, and pre-embryo cleavage. There was no si@cant difference in the number ofoocytes recovered (Table 1)or in oocyte maturation between Cycles A1 and A2 (x’ = 1.36, NS). There was also no difference in the mean number of oocytes which fertilized, or subsequent cleavage. Polyploidy was observed in three preembryos in Cycles Al, and six in Cycle A2 (NS). Finally, there was no difference in the cleavage stage prior to transfer or cryopreservation (x’ = 3.88, NS). Two women conceived in Cycle
A2. During the course of this study, 14 of the women described above also underwent hMG stimulation, utilizing the same stimulation protocol, but a different lot of Pergonal. Comparing these three cycles by the same parameters, ANOVA showed no significant Werences in
Gynecological Endocrinology
Clinical intraindividual hMG bioactivity
Diamond et al.
Table 1 Response to hh4G stimulationin two successive cycles using the same Pergonal lot (#03310027) in 23 women Cycle A1
Estradiol @g/ml) Day 8 Day 9 Day of hCG Follicles on Day 8 1.0-1.4 cm 2 1.5 cm
p
790f91 6 9 3 f 5 7 NS l l 0 0 f 114 1108f 108 NS 1142f 129 1115& 107 NS 4.1 f 0.7 1.0f0.3 5.1 f 0 . 7
3.5 k 0.6 1.0k0.3 4.5 f 0 . 6
NS NS NS
3.5 kO.6 3.3rt0.5 6.7f0.7
3 . 6 f 0.6 3.6f0.4 7.1 f 0 . 6
NS NS NS
Follicles on day of hCG administration 1.0-1.4 cm 3.7f0.6 3.5 f 0 . 5 2 1.5 cm 3.3f0.4 4.0f0.4 total 7.0f0.7 7.4f0.8
NS NS NS
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Cycle A 2
Follicles on Day 9 1.0-1.4cm . 1 1 . 5 cm total
Ampules of hMG on Day 9
0.8 f 0.3
0.5 f 0.2
NS
Day of hCG number of oocytes number of oocytes fertilized number of cleaved pre-embryos NS = not significant
9.1 f 0 . 2 5.9 f 0.6 3.0 f 0.3
9.0f0.1 4.6 f 0.7 2.5 f 0.4
NS NS NS
3.0 f 0.4
2.2 f 0.4
NS
ovarian response, as assessed by estradiol level or follicular development, hMG adrmnistration on Day 9, or day of hCG administration (Table 2). Oocytes were recovered in 11 women in each of these three cycles, with no significant differences observed in the number of oocytes recovered, the number fertilized, or pre-embryo cleavage. There was also no dfference in oocyte maturation = 3.67, NS), polyploid ferthzation ( F = 0.89, NS) or stage of pre-embryo cleavage at the time of pre-embryo transfer or cryopreservation = 2.41, NS).
(x’
(x2
DISCUSSION Clinical response to gonadotropin stimulation for multiple follicular development is influenced by the pre-existing gonadotropin and sex steroid hormonal milieu, the presence of ovarian cysts, the
Gynecological Endocrinology
dose of gonadotropin adrmnistered, and the timing (cycle day) ofadrninistration6.An additionalpotential source of variation is the demonstrated differences in measurement ofluteinizinghormone (LH) levels when assessed in animal studies by radioimmunoassay and bi~assay’.~. Differences in bioactivity and immunoactivity have also been described in serum samples of normal subjects9Jo, women with polycystic ovarian women with endometriosis12,and in hMG lots uthzed for ovulation induction4.Consequently, it has been suggested that failure to respond to gonadotropin stimulation may be due to a ‘bad’lot of hMG. Our study addresses this issue. Aniong the 23 women prospectively recruited into this project, we were able to accumulate a complete set of data on 14. These indviduals underwent three in vitro f e d z a t i o n (IVF) cycles, two with hMG lot #03310027, and a thud cycle with hMG fi-om a different lot obtained from their local pharmacy. The clinical ovarian response to the stimulationwas assessed by serum estradiollevel and fohcular development throughout the fohcular phase; no significant differences were noted. Additionally, there were also no significant differences in subsequent stages of the IVF process among these couples, including number of oocytes harvested, oocytes ferthzed or embryos replaced. Thus, in contrast to reported differences whch may exist in hMG lots in LH and follicle stimulating hormone (FSH) by immuno- and bioactivity, we were unable to demonstrate differencesin clinical response to different lots of Pergonal in this limited number of patients. While we d d not show a significant difference across hMG lots for a variety ofvariables that affect in vitro fertiLization/embryotransfer (IVF/ET) outcome, we recognize that a sample size of 14 may not be sufficient to demonstrate the potential differences in the dependent variables measured. Consequently, we have used power analysis as a method to estimate how many women would be needed to show a difference across hMG lots given the effect size seen in our sample of 14. The probabhty that one would find a sigmficant difference across groups when there is a dfference to be found is represented by power. Ths probability (power) is a function of 01 level (the risk of mistakenly concluding that there is a Merence when there is not), effect size (the difference one finds between groups in terms of the population
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Table 2 Response to the same hMG stimulation protocol of 14 women undergoing three IVF cycles, two with the same lot of Pergonal(#03310027; Cycles A1 and A2),and the third with a Merent lot (Cycle B) Cycle A1
Cycle A2
Cycle B
P
Estradiol (pg/ml) Day 8 Day 9 Day of hCG
754 f 130 1051 f 144 l l l O f 166
700 f 107 1140 f 155 1145 f 142
520 f 80 840 f 124 861 f 112
NS NS NS
Follicles on Day 8 1.0-1.4 cm 2 1.5 cm total
4.5 f 0.9 0.9 f 0.3 5.5 f 0.9
3.1 f 0.7 0.9 f 0.3 4.0 f 0.7
3.6 f 0.8 2.6 f 0.4 4.5 f 0.9
NS NS NS
Follicles on Day 9 1.0-1.4 cm 2 1.5 cm total
3.9 f 0.7 2.9 f 0.7 6.7 f 0.9
3.2 f 0.6 3.7 f 0.4 6.9 f 0.8
3.7 f 0.5 3.2 f 0.6 6.9 f 0.9
NS NS NS
Follicles on day of hCG 1.0-1.4 cm 2 1.5 cm total
4.1 f 0.8 3.1 f 0.8 7.2 f 1.0
3.1 f 0.6 3.9 f 0.4 7.1 f 0.7
3.1 f0.5 3.6 f0.4 6.7 f 0.9
NS NS NS
Ampules of hMG on Day 9
1.1 f 0.4
0.6 f 0.2
0.8
* 0.2
NS
Day of hCG number of oocytes number of oocytes fertilized number of cleaved pre-embryos NS = not significant
9.1 f 0.2 5.5 f 0.8 3.2 f 0.5 2.5 0.3
9.0 f 0.1 4.5 f 1.1 2.3 f 0.5 1.9 f 0.3
9.2 f 0.2 5.8 f 0.8 3.4 f 0.8 3.0 f 0.7
NS NS NS NS
*
standard deviation between groups divided by the standard deviation within groups), and sample size (more subtle effects requiring more subjects). Power analysis is the relationship of all four of these parameters. We have reasoned that with the effect size found in our sample, a traditional 01 level of 0.05, and an acceptablepower value ofat least 80%, we could provide an estimate of the number of subjects required to show a difference across hMG lots, if in fact such a difference existed. Using the data in our sample of estradiol values on Day 8 across the three conditions of Cycles A l , A2 and B as an example, a relatively modest effect size of 0.249 is found. Thus, for three independent groups, a total n of over 150 women would be needed to show a possible difference in estradiol level on Day 8. Our study has used a repeated
138
measure design which utilizes the same women across a l l three treatments, thus reducing the variance within groups and potentially reducing the sample size needed. Nonetheless, considerably more women would be required to show a difference between groups assuming this effect size is maintained. In summary, the similar responses described in this report extend the observations of previous reports that response to stimulation in successive cycles tends to be consistent. Knowledge of this tendency is important when considering future plans of ovulation induction for women who have M e d to conceive in previous cycles'-3. Thus, if clinical response in prior cycles was adequate, stimulation with the same protocol can be recommended in subsequent cycles.
Gynecological Endocrinology
Clinical intraindividual h M G bioactivity
Diamond et al.
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REFERENCES 1. Diamond, M. P., Webster, B. W., Gamer, C. H., Vaughn, W. F., Mason, W. S., Herbert, C. M., Osteen, K. G., Rogers, B. J. and Wentz, A. C. (1985). Selection of superior stimulation protocols for follicular development in a program for in vitro fertilization. Fertil. Steril., 43, 251 2. Diamond, M. P., DeCherney, A. H., Hill, G. A., Nero, F. and Wentz, A. C. (1987). Response to repetitive cycles of ovulation induction in the same women. J . In Vitro Fertil. Embryo Transfer, 4, 251 3. Diamond, M. P., DeChemey, A. Ji,,i:aretto, F and Lunenfeld, B. (1989). Multiple consecutive cycles of ovulation induction with human menopausal gonadotropins. Gynecol. Endocrinol., 3, 237 4. Stone, B., Tun, T., Quinn, K. and ivlarn, R . (1988). Analysis of responses of in vitro fertilizatinr, (IVF) patients to different batches of human menopausal gonadotropins (hMG) during controlled ovarian hyperstimulation (COH). Presented at The American Fertility Society, Atlanta, GA, October, p. 5120 5. Laufer, N., DeChemey, A. H., Haseltine, F. P., Polan, M . L . , Mezer, H . C . , Dlugi, A.M., Sweeney,D., Nero, F. andNafiolin, F. (1983).The use ofhigh-dose human menopausal gonadotropins in an in vitro fertilization program. Fertil. Steril., 40, 734 6. Diamond, M. P. and Wentz, A. C. (1986). Ovulation induction with human menopausal gonadotropins. Obstet. Gynecol. S u w . , 41, 480 7. Dufau, M. L., Hodgen, G. D., Goodman, A. L. and Catt, K. J. (1977). Bioassay of circulating
Gynecological Endocrinology
luteinizing hormone in the rhesus monkey: comparison with radioimmunoassayduring physiological changes. Endocrinology, 100, 1557 8. Neill, J. D., Dailey, R. A , , Tsou, R. C. and Reichert, L. E. Jr. (1977). Immunoreactive LHlike substances in serum of hypophysectomized and prepubertal monkeys: inactive in an in vitro LH bioassay. Endom’nology, 100, 856 9. Dufau, M. L., Beitins, I. Z., McArthur, J. W. and Catt, K. J. (1976). Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and irnmunoreactive scnim LH levels in normal subjects. J . Clin. Endocrinol. Metab., 43, 658 10. Romani, P., Robertson, D. M. and Diczfalusy, E. (1977). Biologically active luteinizing hormone (LH) in plasma. 11. Comparison with immunologically active LH levels throughout the human menstrual cycle. Acta Endocrinol., 84, 697 1 1 . Lobo, R . A., Kletzky, 0. A., Campeau, S. D. and di Zerega, G. S. (1983).Elevated bioactive luteinizing hormone in women with the polycystic ovary syndrome. Fertil. Steril., 39, 674 12. Meldrum, D. R., Tsao, Z., Monroe, S. E., Braunstein, G. D., Sladek, J., Lu, J. K. H., Vale, W., Kivier, J., Judd, H. L. and Chang, R. J. (1984). Stimulation of LH fiagments with reduced bioactivity following GnRH agonist administration in women./. Clin. Endocrinol. Metab., 58,755-7 13. Lobo, R . A., Shoupe, D., Chang, S. P. and Campau, J. (1984). The control ofbioactive luteinizing hormone secretion in women with polycystic ovary syndrome. A m . J . Obstet. Gynecol., 148, 423
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Comparison of ovarian response in the same women with the same or different lots of human menopausal gonadotropin M . P. Diamond*,M . L. Polant, M . Blanchette", C. M . Mazure', A . H . DeCherney" and B. Lunenfeld*tt Yale University School of Medicine, Departments of Obstetrics and Gynecology and $Psychiatry, Division of Reproductive Endocrinology, New Haven, Connecticut, U S A %tanford University, Stanford, California, USA; %heba Medical Center, Ramat Gan, Israel Key words: HUMAN MENOPAUSAL GONADOTROPIN, OVULATION INDUCTION,FOLLICLES, OOCYTES, BIOEQUIVALENCE
ABSTRACT p > 0.05) in all three cycles were also similar. There was no sign$cant dgerence in the day ofhCGadministration ( A 1 , 9 . 1 f 0 . 2 ; A 2 , 9 . 0 f O . l ; B ,9 . 2 f 0 . 2 ) o r t h e total number ofampules o f h M G utilized on Day 9 (AZ, l . l f 0 . 4 ; A 2 , 0 . 6 f 0 . 2 ; B , 0 . 8 f 0 . 2 ) . Thisstudy extends our previous observations in ovulatory women that ovarian response to h M G induction ofsuperovulation is similar in successive cycles and demonstrates in vivo consistency ofhuman ovarian response to d@erent lots o f PergonaP.
A variation in the bioactivity of dgerent production lots o f human menopausal gonadotropin (hMG) has been suggested. Therefore, we evaluated ovarian response to h M G in 1 4 women during three separate ZVF cycles. Thefirst two cycles were peformed with the same lot (#03310027; Cycles A1 and A2); the third cycle utilized dgerent lots O f h M G (Cycle B). Zn all cycles, h M G was administered 3 ampules/day beginning cycle Day 3 and continued f p r at least 6 days. Estradiol and ultrasound evaluations were pe$.ormed on D a y 3, and then daily, beginning on cycle Day 8. Fourteen women completed all three cycles. There were no sign$cant dgerences in baseline estradiol and ultrasound. Estradiol levels on D a y 8 ( A l , 7 5 4 f 130; A2, 700 f 107; B, 520 f 80 pg/ml, analysis of variance p > 0.5) and on D a y 9 (AZ, 1 0 5 1 f 144; A2, 1 1 4 0 f 155; B, 840 f 124pg/ml, p > 0.05) were similar as well. The numberofsmall (1.0-1.4 cm)follicles, large ( 2 1.5 cm) follicles, and totalfollicles (Day 8: total A l , 5.5 f 0.9; A2, 4 . 0 f 0 . 7 ; B , 4.5f0.9,p>O.O5;Day9:total A l , 6 . 7 k 0 . 9 ; A2, 6.9f0.8; B, 6.9+ 0.9,
INTRODUCTION Successful pregnancy outcomes in assisted reproductive surgical procedures occur in only a small percentage of cycles. Consequently, most couples employing these techniques undergo repetitive cycles of ovarian stimulation. The choice of stimulation protocols for multiple follicular recruitment in these subsequent cycles has been a source of previous evaluation by our group, using the criteria of
Correspondence:Dr M. P. Diamond, Vanderbilt University Medical Center, Department of Obstetrics and Gynecology, C-1100, MCN, Nashville, Tennessee 37232, USA
135
Diamond et al.
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Clinical intraindividual hMG bioactivity
serum estradiol levels, estradiol patterns, and timing of human chorionic gonadotropin (hCG) administration’”. These studies have suggested similarity in response to the same stimulation protocol. The current report extends these previous findings, by evaluating, in a new group of patients, the following parameters: follicular development as assessed by ultrasound and estradiol determinations, oocyte quality and ferthzation, and pre-embryo development. Additionally, since there have been suggestions that discrepancies in ovarian response may be due to variations in bioactivity in different lots of human menopausal gonadotropin (hMG)4,comparison is made between repetitive stimulation with the same or H e r e n t hMG lots (Pergonal, Serono Laboratories, Braintree, MA).
identified follicles on ultrasound examination so that there were less than two follicles of 1 1.5 cm. Oocyte recovery and maturity grading have been previously described5. Insemination of preovulatory oocytes was performed 6-8h after aspiration; immature oocytes were preincubated for 24 h before insemination. Oocytes were examined 14-18 h later for fertilization, as evidenced by identi6cation of two or more pronuclei, and 42 h later for cleavage. Normally fertilized oocytes were transferred approximately48 h afier recovery. Statistical analysis was performed using the paired t-test, X’ analysis, and analysis of variance. Sigdicance was defined as p < 0.05. All data are expressed as mean -+ SEM.
RESULTS MATERIALS AND METHODS A prospective study was initiated to record the results of multiple follicular development in: Women who received human menopausal gonadotropin (hMG, Pergonal, Serono, Braintree, MA) from t h e same lot (#03310027) in two successive in virto fertilization (IVF) cycles (Cycles A1 and A2),and (2) The same women after a third cycle of stimulation with hMG from a different lot (Cycle B). Arrangements were made for purchase by patients of hMG from the same lot at a local pharmacy (for Cycles A1 and A2). No financial support was provided for the performance of this study. In all cycles, the hMG stimulation protocol consisted of the administration of 3 ampules/day from Days 3 to 7. O n Day 8, daily measurement of serum estradiol and follicular dmensions by ultrasound (ADR,3000 S/L, Tempe, Arizona) was initiated. Stimulation was continued (with 2, 3, or 4 ampules/day) until at least two follicles had diameters 2 1.5 cm, and estradiol levels 2 400 pg/m. Human chorionic gonadotropin (hCG, ProGsF, Serono, Braintree, MA), 10 000 IU was administered approximately 24 h after the last hMG dose. Oocyte recovery was scheduled for 36 h after hCG administration. In all cycles, oocyte recovery was cancelled if either of the following developed afier hCG administration: precipitous (greater than 30%) drop in estradiol level, or disappearance of the previously
136
The results of 23 women were available for comparison of the outcome of the two hMG cycles utilizing stimulation with Pergonal lot #03310027 (Table 1). There was no significant difference in ovarian response to stimulation in these two cycles (A1 and A2),as assessed on cycle Day 8, cycle Day 9, or the day of hCG administration when assessed by either estradiol level or follicular development. There was also no difference in patient management on Day 9 as assessed by ampules of hMG administered on Day 9. In the initial cycle, one woman did not receive hCG (peak estradiol level 82 pg/ml) and two women were dropped after hCG administration because of a fall in estradiol. Thus, 20 women were available for assessment of oocyte recovery and fertilization, and pre-embryo cleavage. There was no si@cant difference in the number ofoocytes recovered (Table 1)or in oocyte maturation between Cycles A1 and A2 (x’ = 1.36, NS). There was also no difference in the mean number of oocytes which fertilized, or subsequent cleavage. Polyploidy was observed in three preembryos in Cycles Al, and six in Cycle A2 (NS). Finally, there was no difference in the cleavage stage prior to transfer or cryopreservation (x’ = 3.88, NS). Two women conceived in Cycle
A2. During the course of this study, 14 of the women described above also underwent hMG stimulation, utilizing the same stimulation protocol, but a different lot of Pergonal. Comparing these three cycles by the same parameters, ANOVA showed no significant Werences in
Gynecological Endocrinology
Clinical intraindividual hMG bioactivity
Diamond et al.
Table 1 Response to hh4G stimulationin two successive cycles using the same Pergonal lot (#03310027) in 23 women Cycle A1
Estradiol @g/ml) Day 8 Day 9 Day of hCG Follicles on Day 8 1.0-1.4 cm 2 1.5 cm
p
790f91 6 9 3 f 5 7 NS l l 0 0 f 114 1108f 108 NS 1142f 129 1115& 107 NS 4.1 f 0.7 1.0f0.3 5.1 f 0 . 7
3.5 k 0.6 1.0k0.3 4.5 f 0 . 6
NS NS NS
3.5 kO.6 3.3rt0.5 6.7f0.7
3 . 6 f 0.6 3.6f0.4 7.1 f 0 . 6
NS NS NS
Follicles on day of hCG administration 1.0-1.4 cm 3.7f0.6 3.5 f 0 . 5 2 1.5 cm 3.3f0.4 4.0f0.4 total 7.0f0.7 7.4f0.8
NS NS NS
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Cycle A 2
Follicles on Day 9 1.0-1.4cm . 1 1 . 5 cm total
Ampules of hMG on Day 9
0.8 f 0.3
0.5 f 0.2
NS
Day of hCG number of oocytes number of oocytes fertilized number of cleaved pre-embryos NS = not significant
9.1 f 0 . 2 5.9 f 0.6 3.0 f 0.3
9.0f0.1 4.6 f 0.7 2.5 f 0.4
NS NS NS
3.0 f 0.4
2.2 f 0.4
NS
ovarian response, as assessed by estradiol level or follicular development, hMG adrmnistration on Day 9, or day of hCG administration (Table 2). Oocytes were recovered in 11 women in each of these three cycles, with no significant differences observed in the number of oocytes recovered, the number fertilized, or pre-embryo cleavage. There was also no dfference in oocyte maturation = 3.67, NS), polyploid ferthzation ( F = 0.89, NS) or stage of pre-embryo cleavage at the time of pre-embryo transfer or cryopreservation = 2.41, NS).
(x’
(x2
DISCUSSION Clinical response to gonadotropin stimulation for multiple follicular development is influenced by the pre-existing gonadotropin and sex steroid hormonal milieu, the presence of ovarian cysts, the
Gynecological Endocrinology
dose of gonadotropin adrmnistered, and the timing (cycle day) ofadrninistration6.An additionalpotential source of variation is the demonstrated differences in measurement ofluteinizinghormone (LH) levels when assessed in animal studies by radioimmunoassay and bi~assay’.~. Differences in bioactivity and immunoactivity have also been described in serum samples of normal subjects9Jo, women with polycystic ovarian women with endometriosis12,and in hMG lots uthzed for ovulation induction4.Consequently, it has been suggested that failure to respond to gonadotropin stimulation may be due to a ‘bad’lot of hMG. Our study addresses this issue. Aniong the 23 women prospectively recruited into this project, we were able to accumulate a complete set of data on 14. These indviduals underwent three in vitro f e d z a t i o n (IVF) cycles, two with hMG lot #03310027, and a thud cycle with hMG fi-om a different lot obtained from their local pharmacy. The clinical ovarian response to the stimulationwas assessed by serum estradiollevel and fohcular development throughout the fohcular phase; no significant differences were noted. Additionally, there were also no significant differences in subsequent stages of the IVF process among these couples, including number of oocytes harvested, oocytes ferthzed or embryos replaced. Thus, in contrast to reported differences whch may exist in hMG lots in LH and follicle stimulating hormone (FSH) by immuno- and bioactivity, we were unable to demonstrate differencesin clinical response to different lots of Pergonal in this limited number of patients. While we d d not show a significant difference across hMG lots for a variety ofvariables that affect in vitro fertiLization/embryotransfer (IVF/ET) outcome, we recognize that a sample size of 14 may not be sufficient to demonstrate the potential differences in the dependent variables measured. Consequently, we have used power analysis as a method to estimate how many women would be needed to show a difference across hMG lots given the effect size seen in our sample of 14. The probabhty that one would find a sigmficant difference across groups when there is a dfference to be found is represented by power. Ths probability (power) is a function of 01 level (the risk of mistakenly concluding that there is a Merence when there is not), effect size (the difference one finds between groups in terms of the population
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Clinical intraindividual hMG bioactivity
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Table 2 Response to the same hMG stimulation protocol of 14 women undergoing three IVF cycles, two with the same lot of Pergonal(#03310027; Cycles A1 and A2),and the third with a Merent lot (Cycle B) Cycle A1
Cycle A2
Cycle B
P
Estradiol (pg/ml) Day 8 Day 9 Day of hCG
754 f 130 1051 f 144 l l l O f 166
700 f 107 1140 f 155 1145 f 142
520 f 80 840 f 124 861 f 112
NS NS NS
Follicles on Day 8 1.0-1.4 cm 2 1.5 cm total
4.5 f 0.9 0.9 f 0.3 5.5 f 0.9
3.1 f 0.7 0.9 f 0.3 4.0 f 0.7
3.6 f 0.8 2.6 f 0.4 4.5 f 0.9
NS NS NS
Follicles on Day 9 1.0-1.4 cm 2 1.5 cm total
3.9 f 0.7 2.9 f 0.7 6.7 f 0.9
3.2 f 0.6 3.7 f 0.4 6.9 f 0.8
3.7 f 0.5 3.2 f 0.6 6.9 f 0.9
NS NS NS
Follicles on day of hCG 1.0-1.4 cm 2 1.5 cm total
4.1 f 0.8 3.1 f 0.8 7.2 f 1.0
3.1 f 0.6 3.9 f 0.4 7.1 f 0.7
3.1 f0.5 3.6 f0.4 6.7 f 0.9
NS NS NS
Ampules of hMG on Day 9
1.1 f 0.4
0.6 f 0.2
0.8
* 0.2
NS
Day of hCG number of oocytes number of oocytes fertilized number of cleaved pre-embryos NS = not significant
9.1 f 0.2 5.5 f 0.8 3.2 f 0.5 2.5 0.3
9.0 f 0.1 4.5 f 1.1 2.3 f 0.5 1.9 f 0.3
9.2 f 0.2 5.8 f 0.8 3.4 f 0.8 3.0 f 0.7
NS NS NS NS
*
standard deviation between groups divided by the standard deviation within groups), and sample size (more subtle effects requiring more subjects). Power analysis is the relationship of all four of these parameters. We have reasoned that with the effect size found in our sample, a traditional 01 level of 0.05, and an acceptablepower value ofat least 80%, we could provide an estimate of the number of subjects required to show a difference across hMG lots, if in fact such a difference existed. Using the data in our sample of estradiol values on Day 8 across the three conditions of Cycles A l , A2 and B as an example, a relatively modest effect size of 0.249 is found. Thus, for three independent groups, a total n of over 150 women would be needed to show a possible difference in estradiol level on Day 8. Our study has used a repeated
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measure design which utilizes the same women across a l l three treatments, thus reducing the variance within groups and potentially reducing the sample size needed. Nonetheless, considerably more women would be required to show a difference between groups assuming this effect size is maintained. In summary, the similar responses described in this report extend the observations of previous reports that response to stimulation in successive cycles tends to be consistent. Knowledge of this tendency is important when considering future plans of ovulation induction for women who have M e d to conceive in previous cycles'-3. Thus, if clinical response in prior cycles was adequate, stimulation with the same protocol can be recommended in subsequent cycles.
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Clinical intraindividual h M G bioactivity
Diamond et al.
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REFERENCES 1. Diamond, M. P., Webster, B. W., Gamer, C. H., Vaughn, W. F., Mason, W. S., Herbert, C. M., Osteen, K. G., Rogers, B. J. and Wentz, A. C. (1985). Selection of superior stimulation protocols for follicular development in a program for in vitro fertilization. Fertil. Steril., 43, 251 2. Diamond, M. P., DeCherney, A. H., Hill, G. A., Nero, F. and Wentz, A. C. (1987). Response to repetitive cycles of ovulation induction in the same women. J . In Vitro Fertil. Embryo Transfer, 4, 251 3. Diamond, M. P., DeChemey, A. Ji,,i:aretto, F and Lunenfeld, B. (1989). Multiple consecutive cycles of ovulation induction with human menopausal gonadotropins. Gynecol. Endocrinol., 3, 237 4. Stone, B., Tun, T., Quinn, K. and ivlarn, R . (1988). Analysis of responses of in vitro fertilizatinr, (IVF) patients to different batches of human menopausal gonadotropins (hMG) during controlled ovarian hyperstimulation (COH). Presented at The American Fertility Society, Atlanta, GA, October, p. 5120 5. Laufer, N., DeChemey, A. H., Haseltine, F. P., Polan, M . L . , Mezer, H . C . , Dlugi, A.M., Sweeney,D., Nero, F. andNafiolin, F. (1983).The use ofhigh-dose human menopausal gonadotropins in an in vitro fertilization program. Fertil. Steril., 40, 734 6. Diamond, M. P. and Wentz, A. C. (1986). Ovulation induction with human menopausal gonadotropins. Obstet. Gynecol. S u w . , 41, 480 7. Dufau, M. L., Hodgen, G. D., Goodman, A. L. and Catt, K. J. (1977). Bioassay of circulating
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luteinizing hormone in the rhesus monkey: comparison with radioimmunoassayduring physiological changes. Endocrinology, 100, 1557 8. Neill, J. D., Dailey, R. A , , Tsou, R. C. and Reichert, L. E. Jr. (1977). Immunoreactive LHlike substances in serum of hypophysectomized and prepubertal monkeys: inactive in an in vitro LH bioassay. Endom’nology, 100, 856 9. Dufau, M. L., Beitins, I. Z., McArthur, J. W. and Catt, K. J. (1976). Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and irnmunoreactive scnim LH levels in normal subjects. J . Clin. Endocrinol. Metab., 43, 658 10. Romani, P., Robertson, D. M. and Diczfalusy, E. (1977). Biologically active luteinizing hormone (LH) in plasma. 11. Comparison with immunologically active LH levels throughout the human menstrual cycle. Acta Endocrinol., 84, 697 1 1 . Lobo, R . A., Kletzky, 0. A., Campeau, S. D. and di Zerega, G. S. (1983).Elevated bioactive luteinizing hormone in women with the polycystic ovary syndrome. Fertil. Steril., 39, 674 12. Meldrum, D. R., Tsao, Z., Monroe, S. E., Braunstein, G. D., Sladek, J., Lu, J. K. H., Vale, W., Kivier, J., Judd, H. L. and Chang, R. J. (1984). Stimulation of LH fiagments with reduced bioactivity following GnRH agonist administration in women./. Clin. Endocrinol. Metab., 58,755-7 13. Lobo, R . A., Shoupe, D., Chang, S. P. and Campau, J. (1984). The control ofbioactive luteinizing hormone secretion in women with polycystic ovary syndrome. A m . J . Obstet. Gynecol., 148, 423
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