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Gonadotropin suppression with oral contraceptives before in vitro fertilization
FERTILITY AND STERILITY
Vol. 53, No.2, February 1990
Copyright" 1990 The American Fertility Society
Printed on acid-free paper in U.S.A.
Gonadotropin suppression with oral contraceptives before in vitro fertilization*
Yael Gonen, M.D.t William Jacobson, Ph.D. Robert F. Casper, M.D.:j: Division of Reproductive Science, Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada
One hundred eighty-one stimulation cycles in which gonadotropin suppression with oral contraceptives (OCs) preceded induction of follicle stimulation (study group) and 113 stimulation cycles without pituitary suppression (control group) were compared. The mean length of ovarian suppression was 35.3 ± 0.9 days. No spontaneous luteinizing hormone (LH) surges occurred when the use of OC preceded ovarian hyperstimulation, whereas in the control group the incidence ofLH surges was 19.5%. The mean amount of human menopausal gonadotropin required was significantly lower in the study group than in the control group (8.9 ± 0.4 and 10.9 ± 0.4 ampules, respectively). Significantly more follicles ~ 1.5 cm in diameter were seen on the day before oocyte retrieval and significantly more oocytes were retrieved per attempt in the group with OC pretreatment. Our data clearly demonstrate that OCs are useful in in vitro fertilization stimulation protocols to facilitate scheduling of cycles and to prevent spontaneous LH surges. Fertil Steril53:282, 1990
Modification of the standard ovulation induction protocols, in the form of suppression of endogenous gonadotropins, has recently been widely employed in in vitro fertilization and embryo transfer (IVF -ET) to prevent spontaneous luteinizing hormone (LH) surges, to increase scheduling efficiency, and to improve outcome. Most attention has been received by gonadotropin-releasing hormone-agonists (GnRH-a) for gonadotropin supReceived June 6, 1989; revised and accepted September 27, 1989. * Supported by a grant from the Medical Research Council of Canada to R.F.C. and by a Fellowship Training Grant to Y.G. from Wyeth Pharmaceutical Company, Toronto, Ontario, Canada. t Visiting clinical research fellow. Present address: Department of Obstetrics and Gynecology, Carmel Hospital, Haifa, Israel. :j: Reprint requests: Robert F. Casper, M.D., Toronto General Hospital, 6-240 EN, 200 Elizabeth Street, Toronto, Ontario, M5G2C4.
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pression, although progestins alone or oral contraceptives (OCs) may also be used. Induction of a transient hypogonadotropic state with synthetic steroids has been suggestedl - 3 to synchronize follicular development and to improve estradiol (E 2 ) response to controlled ovarian hyperstimulation. Frydman and colleagues2 have successfully used a programed cycle and a "fixed" day for human chorionic gonadotropin (hCG) administration for oocyte retrieval without compromising pregnancy rates compared with those of a fully monitored group. However, Mashiach et al. 4 found that the day of hCG administration differs according to both the duration of gonadotropin suppression and the protocol used for follicular maturation. The objective of this study was to examine the use of OCs for gonadotropin suppression before the initiation of follicular hyperstimulation in comparison with ovarian hyperstimulation without previous pituitary suppression. We determined whether the length of gonadotropin suppression was corre-
Fertility and Sterility
lated with E2 response, amount of drugs required for follicle stimulation, the day ofhCG administration, or the pregnancy rate and outcome. MATERIALS AND METHODS
The routine use of OC for gonadotropin suppression was initiated in our in vitro fertilization (IVF) program at the Toronto General Hospital in July 1988 to facilitate scheduling of up to 15 cycles per week. From July to December 1988 inclusive, 173 infertile couples had undergone gonadotropin suppression with OCs before induction of ovulation in 181 stimulation cycles. Ninety-eight patients who underwent 113 stimulation cycles without previous pituitary suppression between October 1987 and June 1988 inclusive, served as a control group. The same stimulation protocol was used for induction of ovulation and age, gravidity, parity, and the cause of infertility were similar in both groups. In the study group, 50 /lg ethinyl estradiol (EE 2) and 1 mg of ethynodiol diacetate (Demulen 50; Searle, Oakville, Ontario, Canada) were administered daily for periods ranging from 18 to 61 days. The duration of OC use was determined by the availability of an opening in the IVF program each week. The third day after the last day of OC, regardless of when withdrawal bleeding actually occurred, was considered day 1 of the stimulation cycle. In all the patients (study and control groups), ovarian follicular stimulation was induced with clomiphene citrate (CC) (Serophene; Serono, Randolph, MA) 100 mg/day, cycle days 5 to 9 and human menopausal gonadotropins (hMG, Pergonal; Serono) 75 to 150 IV intramuscular (1M) daily starting on cycle day 5 or 6, until at least two follicles 1.8 to 2.0 cm in the greatest diameter were observed simultaneously with E2 levels of 600 pmol/ L per follicle :2: 1.5 cm. Human chorionic gonadotropin (hCG, Profasi; Serono) 5,000 IV 1M was routinely used to trigger the final stage of follicular maturation. Follicular development was monitored by daily determination of serum E2 and LH and serial ultrasound measurement with a transvaginal 7 MHz transducer (Bruel and Kjaer, Type 8538, Naerum, Denmark). Oocyte collection was performed 34 hours after hCG administration by transvaginal ultrasonically guided follicle aspiration, which has been used in our program since October 1987. One hundred fifty-eight of the 181 study cycles which proceeded to oocyte retrieval were evaluated
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with respect to the length of follicular stimulation, the amount of hMG required and serum E2 response, the number of oocytes retrieved per attempt, embryos transferred, and fertilization and cleavage rates. The pregnant and nonpregnant groups were compared and the correlation between the duration of treatment with OCs and each of these cycle parameters was analyzed. In addition, the study and control groups were compared with respect to incidence of LH surges, defined as an increase in LH concentration of > 100% compared with the mean of the previous 2 days. We also compared the length of induction of ovulation, the amount of hMG required, E2 response, the number of follicles :2: 1.5 cm measured on the day before oocyte retrieval, the number of oocytes obtained per attempt, and the pregnancy and abortion rates. Due to personnel and technique changes in our IVF laboratory in July 1988, we believed that comparison of fertilization and cleavage rates between the two groups were not valid and are not discussed in our results. Estradiol Assay
Estradiol (E 2) was measured by a solid phase radioimmunoassay (RIA) using 1251-labeled E2 (CoatA-Count Estradiol) purchased from Diagnostic Products Corporation (Los Angeles, CA). Estradiol values were expressed in pmol/L (pg/mL X 0.003671). Intra-assay and interassay coefficients of variations were also determined directly in serum pools placed at 2 or 3 levels of the working range, respectively. The intra-assay coefficients of variation at 290 pmol/L and 1,000 pmol/L E2 was 5.6% and 4.0%, respectively. The interassay coefficients of variation at 280 pmol/L, at 500 pmol/L, and at 2,500 pmol/L was 12%, 9.5%, and 7.5%, respectively. . Statistical Evaluation
The data obtained were compared by Student's t-test for unpaired results. Fertilization and cleavage rates were analyzed using the nonparametric Mann-Whitney V-test on rank data. Pregnancy and cancellation rates were compared by x 2 test. When cycle parameters were analyzed according to the length of OC treatment, differences between the groups in parametric data were examined by one-way analysis of variance, whereas fertilization and cleavage rates were compared by KruskalWallis test for nonparametric data. Correlation
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JEdS
Table 1
Cycle Parameters in Controlled Cycles (no OC) and Cycles with Gonadotropin Suppression with OC
No. of cycles No. of patients No. of cancellations No. ofLH surges No. of retrievals No. ofhMG required/cycle No. offollicles ;:,: 1.5 cm on day after hCG DayofhCG No. of oocytes/attempt E2 day 9 (pmol/L) E2 day hCG (pmol/L) E2 after hCG (pmol/L) Pregnancy rate/ET Abortion rate a
b
OC
NoOC
181 173 23 (12.7) a 0 158 8.9± 0.4' 6.6± 0.2 12.1 ± 0.1 4.7± 0.2 1,312 ± 70 4,256 ± 137 5,725 ± 214 22/108 (20.4) 2/22 (9.1)
113 98 18 (15.9) 22 (19.5) 95 10.9 ± 0.4 6.0± 0.2 12.3 ± 0.1 4.1 ± 0.2 1,420 ± 80 4,424 ± 157 5,353 ±200 12/76 (15.8) 4/12 (33.3)
Values in parentheses are percents. NS, not significant.
was checked by linear regression analysis. P values < 0.05 were regarded as statistically significant. Data are presented throughout this paper as means ± standard error (±SE) of the mean.
RESULTS A decision to use routine OC gonadotropin suppression to facilitate scheduling in our IVF program since July 1988 allowed the study of consecutive unselected patients undergoing controlled ovarian hyperstimulation. Table 1 presents cycle parameters in the study and in the control groups. One hundred fifty-eight (87.3%) of the 181 study cycles, in which gonadotropin suppression with OC preceded induction of ovulation, proceeded to transvaginal oocyte retrieval. Twenty-three cycles (12.7%) were cancelled, 87% of them due to a failure of E2 to increase by at least twice each day after the first significant rise from baseline or because of a drop in E2 of ~30% after hCG. The other 13% were cancelled because of poor follicular development or for other factors not related to ovarian stimulation. The mean length of ovarian suppression did not differ between cycles that were cancelled and those that reached oocyte retrieval (36 ± 2.4 and 35.3 ± 0.9 days, respectively). In the control group, 95 (84.1 %) of the 113 stimulation cycles proceeded to oocyte retrieval, whereas 18 cycles (15.9%) were cancelled, 83.3% of them due to suboptimal E2 response as described above or poor follicular development, and 16.7% due to an LH surge of unknown time of onset. The mean amount of hMG required to achieve optimal E2 response and
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Pvalue
NS b P
, Values are means ± SE.
follicular development was significantly lower when OC administration preceded induction of ovulation compared with the group without previous pituitary suppression (8.9 ± 0.4 and 10.9 ± 0.4 ampules, respectively; P < 0.0005). Although the average cycle day for hCG administration did not differ between the two groups (12.1 ± 0.1 and 12.3 ± 0.1), almost all OC-suppressed patients went to oocyte retrieval during the midweek since the OC was stopped on Saturday or Sunday, resulting in a day 1 on Tuesday or Wednesday. The mean number of follicles ~ 1.5 cm on the day before ovum retrieval was significantly greater in the study than in the control group (6.6 ± 0.2 and 6.0 ± 0.2, respectively; P < 0.05) and significantly more oocytes were retrieved per attempt in the study than in the control group (4.7 ± 0.2 and 4.1 ± 0.2, respectively; P < 0.05). The E2 levels on cycle day 9, the day of hCG administration and the day after hCG, did not differ between the two groups. No spontaneous LH surges occurred in the study patients whereas in the control group, the incidence of LH surge was 19.5% (P < 0.001). One hundred and eight embryo transfers (ETs) in the study group resulted in 22 clinical pregnancies for a success rate of 20.4% per ET, whereas 76 ETs in the control group resulted in 12 clinical pregnancies (15.8% per ET). All pregnancies were documented by transvaginal ultrasound with confirmation of fetal heart activity. Early abortion occurred in 2 pregnancies (9.1 %) in the study group and in 4 pregnancies (33.3%) in the control group. Table 2 compares the cycle parameters of study patients who conceived with study patients who
Fertility and Sterility
Table 2
Comparison of Pregnant and Nonpregnant Patients in Gonadotropin Suppression Group
Age (years) No. of days on OC No. ofhMG required No. of follicles ;00 1.5 cm on day after hCG DayofhCG No. of oocytes/attempt No. of embryos/transfer Fertilization rate/oocyte Cleavage rate/oocyte E2 day 9 (pmoljL) E2 day hCG (pmoljL) E2 after hCG (pmoljL) a
Nonpregnant (n = 136)
Pregnant (n = 22)
Pvalue
33.3 ± 0.3 a 35.1 ± 0.9 8.9± 0.4 6.7± 0.2 12.1 ± 0.1 4.8± 0.2 2.7 ± 0.2 39.9± 3.1 36.9 ± 3.1 1,323 ± 76 4,228 ± 144 5,672 ± 231
34.1 ± 0.6 36.3 ± 2.8 9.0± 0.9 6.5± 0.5 12.1 ± 0.2 4.4 ± 0.5 3.1 ± 0.3 75.2 ± 4.9 72.8± 4.9 1,243 ± 168 4,429 ± 424 6,054 ±589
NS b NS NS NS NS NS NS P < 0.0005 P< 0.0005 NS NS NS
Values are means ± SE.
did not. For the most part, the cycle parameters appear to be similar for both groups. The mean duration of treatment with OC did not differ between the pregnant and nonpregnant group (36.3 ± 2.8 and 35.1 ± 0.9 days, respectively). No difference was found in the number of ampules of hMG required for follicle stimulation between the pregnant and nonpregnant group (9.0 ± 0.9 versus 8.9 ± 0.4 ampules, respectively), nor in the E2 response on the day ofhCG administration (4,429 ± 424 versus 4,228 ± 144 pmoljL). The mean number offollicles ~ 1.5 cm on the day before the oocyte retrieval, the cycle day of hCG administration, as well as the mean number of oocytes retrieved and embryos transferred, did not differ. Both, the fertilization and the cleavage rates per oocyte recovered were significantly higher (P < 0.0005) in the pregnant than in the nonpregnant group (Table 2). No correlation by regression analysis was found between the length of OC use and fertilization rate (r2 = 0.05 and r2 = 0.001 in the pregnant and the nonpregnant group, respectively) or in other cycle parameters in the pregnant or in the nonpregnant group. Table 3 summarizes cycle parameters according to the duration of treatment with OCs before the induction of ovulation. No statistically significant difference in pregnancy or cancellation rates was found between cycles with previous gonadotropin suppression for .:::;;3 weeks compared with 4, 5, or 6 or more weeks of suppression. Of all cycle parameters analyzed, the only difference was found in the mean E2 levels on cycle day 9 between the group of patients treated with OC for .:::;;21 days and those treated for ;?:43 days (1,586 ± 270 and 980 ± 90 pmoljL, respectively; P < 0.05). E2 levels on the
Vol. 53, No.2, February 1990
b
NS, not significant.
day of hCG administration or the following day, however, did not differ among the five groups. There was no difference in the other cycle parameters. DISCUSSION
The use of gonadotropin suppression before ovulation induction has been associated with an increased number of oocytes collected, improved fertilization and pregnancy rates,5,6 and a decreased incidence of endogenous LH surges. As a result, cycles in which gonadotropin suppression has been achieved result in a decreased cancellation rate in those programs which routinely cancel LH surge cycles. 6-8 Most attention has been received by GnRH -a, although OCs or progestins alone may also be used for gonadotropin suppression. The administration of GnRH -a has several disadvantages and side effects. Many patients experience hot flushes, headaches, and fatigue,9 especially with long-term protocols. Multiple follicular stimulation requires higher doses of gonadotropins and longer regimens, resulting in higher cost and greater inconvenience to the patients. 1O There is an increased incidence of ovarian hyperstimulation syndrome9,1l,12 and ovarian cyst formation,9 as well as the need for luteal phase support. The recently reported13 lower survival of cryopreserved embryos after thawing and the relatively high abortion rate 6,9 in pregnant patients receiving GnRH-a during IVF cycles suggests that poor quality embryos may be produced in these cycles. In an effort to assess the potential benefits from the use of OC for pituitary suppression, we evaluated 181 stimulation cycles in which gonadotropin suppression with OCs preceded induction of ovula-
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r Table 3
Effect of the Length of OC Gonadotropin Suppression on Cycle Parameters, Fertilization, and Pregnancy Rates
No.ofretrievals Mean length of OC use (days) Mean age of patients (years) No. ofhMG required DayofhCG E2 day 9 (pmol/L) E2 day of hCG (pmol/L) E2 day after hCG (pmol/L) Mean no. of follicles ~ 1.5 cm No.ofoocytes/attempt No. of embryos/transfer Fertilization rate/oocyte Cleavage rate/oocyte No. of pregnancies Pregnancy rate/ET
29 to 35 days
36 to 42 days
16
36
27
36
43
20.1 ± 0.2" 33.6 ± 0.1 9.9 ± 1.6 11.8 ± 0.2 1,586 ± 270 4,316 ± 464 5,721 ± 743 7.0 ± 0.7 5.4 ± 0.9 3.6 ± 0.4 32.9 ± 9.1 32.2 ± 9.2 2
24.5 ± 0.4 34.3 ± 0.5 8.8± 0.9 12.1 ± 0.2 1,495 ± 173 4,251 ± 289 5,654 ±420 6.5± 0.5 4.6± 0.4 2.6± 0.3 49.2 ± 5.7 47.3 ± 5.7 6 6/26 (23.1)
31.6 ± 0.4 32.8± 0.8 8.7± 1.0 12.0 ± 0.2 1,254 ± 128 4,273 ± 244 5,885 ±356 6.9± 0.5 4.6± 0.6 2.4± 0.4 37.1 ± 7.4 36.6± 7.5 2 2.17 (11.8)
38.5 ± 0.3 33.5 ± 0.6 8.8± 0.6 12.0 ± 0.2 1,447 ± 158 4,395 ± 332 6,072 ±517 6.6± 0.4 4.7 ± 0.4 2.9± 0.3 47.4 ± 6.3 44.7 ± 6.3 4 4/24 (16.7)
49.5 ± 0.8 32.8± 0.5 9.0± 0.7 12.4 ± 0.2 980 ± 90 4,112 ±266 5,404 ±444 6.5 ± 0.4 4.7± 0.4 2.8± 0.2 48.5± 5.6 42.5 ± 5.5 8 8/31 (25.8)
2/10 (20)b
tion. Our data show that the use of OCs allows flexibility in patient scheduling and facilitates efficient use of IVF -ET personnel as a result of reasonably predictable timing of oocyte retrieval during the middle of the week in almost all cases in this study. The use of OCs for gonadotropin suppression prevents the occurrence of spontaneous LH surges to a similar extent as GnRH-a use. In addition, we observed a reduction in the amount of hMG required for follicular stimulation and maturation, in contrast to the markedly increased requirement for hMG in GnRH-a suppressed cycles. The very low spontaneous abortion rate (9.1 %) in the presence of an acceptable pregnancy rate of 20.4% per ET suggests that no adverse effect on embryo quality occurred with OC gonadotropin suppression. Considering the low cost of OCs in comparison with GnRH-a, the simplicity of use, the greater patient acceptance (oral administration versus daily subcutaneous injections), the reduction in hMG needed for hyperstimulation, and the absence of side effects in this study, our results clearly demonstrate that consideration should be given to the use of OCs as an alternative to GnRHa in IVF stimulation protocols. Furthermore, we did not use luteal phase support with progesterone or hCG in any of the patients reported in this study with no obvious effect on cycle length or pregnancy rates. Our data demonstrate that gonadotropin suppression with OCs synchronizes follicular development and is associated with a uniform response to ovarian hyperstimulation. In contrast to Mashiach et al.,4 the results of present series show that the
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days
22 to 28 days
" Values are means ± SE.
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~43
0;21 days
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b
Values in parentheses are percents.
length of treatment with OCs does not appear to have any adverse effect on follicular recruitment, cycle cancellation, or pregnancy rate. In our study of 181 cycles, we did not find a more profound suppression leading to poor ovarian stimulation period or increased cancellation rate when OC was administered for longer periods of time. Mashiach et al. 4 reported that the latent phase, which represents a period of ovarian insensitivity, was prolonged and directly correlated to the duration of OC suppression. Studying only 16 patients in each group, these authors 4 concluded that suppression with an OC for 30 days compared with 15 days, resulted in a greater cancellation of oocyte retrievals, a lower fertilization rate, and a lower pregnancy rate. The reason for the difference between Mashiach's study4 and ours is not clear, but may be related to the smaller number of patients in the previous report or the use of a different OC with more potent progestin. However, we believe that a more likely explanation is the fact that day 1 of the cycle was designated to be the day after the last OC pill, compared with a day 1, in our study, that was 3 days after the last pill. Benadiva et al. 14 reported that women with suppressed pituitary function may need to return to a certain threshold level of FSH for early follicular recruitment, if a desirable ovarian response is to be achieved during ovarian hyperstimulation. Klein and Mishell 15 observed that the follicular phase is prolonged after discontinuing OC and suggested that a brief interval is needed to reestablish normal ovarian-pituitary relationships in a spontaneous cycle. It is possible that, in the present study, a de-
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lay in the initiation of ovarian hyperstimulation until day 5 of the cycle resulted in a more responsive pituitary-ovarian axis. Thus, it is possible that the length of ovarian recovery and different designation of day 1 after the gonadotropin suppression, rather than the length of OC treatment prior to induction of ovulation may play the main role in successful and uniform response to ovarian hyperstimulation. In summary, the suppression ofthe hypothalamic-pituitary-ovarian axis by OCs is an acceptable tool to manipulate the cycle for IVF. Our data have shown that the method can result in an acceptable pregnancy rate with low cancellation and abortion rates, as well as reduced costs. No correlation was found between the length of ovarian suppression and the other cycle parameters which were analyzed. It is concluded that the length of treatment with OCs does not appear to have any adverse effect on response to induction of ovulation or pregnancy rate and outcome. However a short delay in the initiation of ovarian hyperstimulation to allow return of pituitary-ovarian responsiveness is likely important. It is our belief, that the use of OCs for gonadotropin suppression prior to induction of follicular hyperstimulation for IVF should be considered as an alternative to the use of GnRH -a.
Acknowledgment. The authors thank Ms. Mary Coates for her help with the preparation of the manuscript.
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REFERENCES 13. 1. Jones GS, DeMoraes Ruehsen M, Johanson AJ, Raiti S, Blizard RM: Elucidation of normal ovarian physiology by exogenous gonadotropin stimulation following steroid pituitary suppression. Fertil Steril20:14, 1969 2. Frydman R, Forman R, Rainhorn J-D, Belaisch-Allart J, Hazout A, Testart J: A new approach to follicular stimulation for in vitro fertilization: programed oocyte retrieval. Fertil SteriI46:657, 1986 3. Patton PE, Burry KA, Wolf DP, Kiessling AA, Craemer MJ: The use of oral contraceptives to regulate oocyte retrieval. Fertil Steril49:716, 1988 4. Mashiach S, Dor J, Goldenberg M, Shalev J, Blankstein J,
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Rudak E, Shoam Z, Finelt Z, Nebel L, Goldman B, BenRafael Z: Protocols for induction of ovulation. The concept of programmed cycles. Ann NY Acad Sci 541:37,1988 Neveu S, Hedon B, Bringer J, Chinchole J-M, Arnal F, Humeau C, Cristol P, Viala J -L: Ovarian stimulation by a combination of a gonadotropin-releasing hormone agonist and gonadotropins for in vitro fertilization. Fertil SteriI47:639, 1987 Meldrum DR, Wisot A, Hamilton F, Gutlay AL, Kempton W, Huynh D: Routine pituitary suppression with leuproide before ovarian stimulation for oocyte retrieval. Fertil Steril 51:455, 1989 Porter RN, Smith W, Craft IL, Abdulwahid NA, Jacobs HS: Induction of ovulation for in vitro fertilization using buseralin and gonadotropins. Lancet 2:1284,1984 Fleming R, Haxton MJ, Yates RWS, Conagham C, Coutts JRT: A simple procedure for the induction of multiple follicular growth with blockade of the LH surge in normal women. (Abstr.) Presented at the 67th Annual Meeting of the Endocrine Society, Baltimore, Maryland, June 19 to 21, 1985. Caspi E, Ron-EI R, Golan A, Nachum H, Herman A, Soffer Y, Weinraub Z: Results of in vitro fertilization and embryo transfer by combined long-acting gonadotropin-releasing hormone analog D-Trp-6-luteinizing hormone-releasing hormone and gonadotropins. Fertil SteriI51:95, 1989 MacLachlan V, Besanko M, O'Shea F, Wade H, Wood C, Trounson A, Healy D: A controlled study ofluteinizing hormone-releasing hormone agonist (buseraline) for the induction of folliculogenesis before in vitro fertilization. N Engl J Med 320:1233, 1989 Golan A, Ron-EI R, Herman A, Weinraub Z, Soffer Y, Caspi E: Ovarian hyperstimulation syndrome following DTrp-6 luteinizing hormone-releasing hormone microcapsules and menotropin for in vitro fertilization. Fertil Steril 50:912, 1988 Ezratty V, Cohen J: Ovarian hyperstimulation syndrome with GnRH agonist. (Abstr.) Presented at the VI World Congress In Vitro Fertilization and Alternate Assisted Reproduction, Jerusalem, April 2 to 7,1989 Von Steirteghem AC, Van Den Abbeel E, Smitz J, Camus M, Khan I, Staessen C, Devroey P: Cryopreservation of supernumerary embryos after GIFT and IVF in Clomid-hMG and Buserelin-hMG stimulated cycles. (Abstr.) Presented at the VI World Congress In Vitro Fertilization and Alternate Assisted Reproduction, Jerusalem, April 2 to 7,1989 Benadiva CA, Ben-Rafael Z, Blasco L, Tureck R, Mastroianni LJ, Flickinger GL: Ovarian response to human menopausal gonadotropin following suppression with oral contraceptives. Fertil Steril50:516, 1988 Klein T A, Mishell DR: Gonadotropin, prolactin and steroid hormone levels after discontinuation of oral contraceptives. Am J Obstet GynecoI127:585, 1977
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Vol. 53, No.2, February 1990
Copyright" 1990 The American Fertility Society
Printed on acid-free paper in U.S.A.
Gonadotropin suppression with oral contraceptives before in vitro fertilization*
Yael Gonen, M.D.t William Jacobson, Ph.D. Robert F. Casper, M.D.:j: Division of Reproductive Science, Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada
One hundred eighty-one stimulation cycles in which gonadotropin suppression with oral contraceptives (OCs) preceded induction of follicle stimulation (study group) and 113 stimulation cycles without pituitary suppression (control group) were compared. The mean length of ovarian suppression was 35.3 ± 0.9 days. No spontaneous luteinizing hormone (LH) surges occurred when the use of OC preceded ovarian hyperstimulation, whereas in the control group the incidence ofLH surges was 19.5%. The mean amount of human menopausal gonadotropin required was significantly lower in the study group than in the control group (8.9 ± 0.4 and 10.9 ± 0.4 ampules, respectively). Significantly more follicles ~ 1.5 cm in diameter were seen on the day before oocyte retrieval and significantly more oocytes were retrieved per attempt in the group with OC pretreatment. Our data clearly demonstrate that OCs are useful in in vitro fertilization stimulation protocols to facilitate scheduling of cycles and to prevent spontaneous LH surges. Fertil Steril53:282, 1990
Modification of the standard ovulation induction protocols, in the form of suppression of endogenous gonadotropins, has recently been widely employed in in vitro fertilization and embryo transfer (IVF -ET) to prevent spontaneous luteinizing hormone (LH) surges, to increase scheduling efficiency, and to improve outcome. Most attention has been received by gonadotropin-releasing hormone-agonists (GnRH-a) for gonadotropin supReceived June 6, 1989; revised and accepted September 27, 1989. * Supported by a grant from the Medical Research Council of Canada to R.F.C. and by a Fellowship Training Grant to Y.G. from Wyeth Pharmaceutical Company, Toronto, Ontario, Canada. t Visiting clinical research fellow. Present address: Department of Obstetrics and Gynecology, Carmel Hospital, Haifa, Israel. :j: Reprint requests: Robert F. Casper, M.D., Toronto General Hospital, 6-240 EN, 200 Elizabeth Street, Toronto, Ontario, M5G2C4.
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pression, although progestins alone or oral contraceptives (OCs) may also be used. Induction of a transient hypogonadotropic state with synthetic steroids has been suggestedl - 3 to synchronize follicular development and to improve estradiol (E 2 ) response to controlled ovarian hyperstimulation. Frydman and colleagues2 have successfully used a programed cycle and a "fixed" day for human chorionic gonadotropin (hCG) administration for oocyte retrieval without compromising pregnancy rates compared with those of a fully monitored group. However, Mashiach et al. 4 found that the day of hCG administration differs according to both the duration of gonadotropin suppression and the protocol used for follicular maturation. The objective of this study was to examine the use of OCs for gonadotropin suppression before the initiation of follicular hyperstimulation in comparison with ovarian hyperstimulation without previous pituitary suppression. We determined whether the length of gonadotropin suppression was corre-
Fertility and Sterility
lated with E2 response, amount of drugs required for follicle stimulation, the day ofhCG administration, or the pregnancy rate and outcome. MATERIALS AND METHODS
The routine use of OC for gonadotropin suppression was initiated in our in vitro fertilization (IVF) program at the Toronto General Hospital in July 1988 to facilitate scheduling of up to 15 cycles per week. From July to December 1988 inclusive, 173 infertile couples had undergone gonadotropin suppression with OCs before induction of ovulation in 181 stimulation cycles. Ninety-eight patients who underwent 113 stimulation cycles without previous pituitary suppression between October 1987 and June 1988 inclusive, served as a control group. The same stimulation protocol was used for induction of ovulation and age, gravidity, parity, and the cause of infertility were similar in both groups. In the study group, 50 /lg ethinyl estradiol (EE 2) and 1 mg of ethynodiol diacetate (Demulen 50; Searle, Oakville, Ontario, Canada) were administered daily for periods ranging from 18 to 61 days. The duration of OC use was determined by the availability of an opening in the IVF program each week. The third day after the last day of OC, regardless of when withdrawal bleeding actually occurred, was considered day 1 of the stimulation cycle. In all the patients (study and control groups), ovarian follicular stimulation was induced with clomiphene citrate (CC) (Serophene; Serono, Randolph, MA) 100 mg/day, cycle days 5 to 9 and human menopausal gonadotropins (hMG, Pergonal; Serono) 75 to 150 IV intramuscular (1M) daily starting on cycle day 5 or 6, until at least two follicles 1.8 to 2.0 cm in the greatest diameter were observed simultaneously with E2 levels of 600 pmol/ L per follicle :2: 1.5 cm. Human chorionic gonadotropin (hCG, Profasi; Serono) 5,000 IV 1M was routinely used to trigger the final stage of follicular maturation. Follicular development was monitored by daily determination of serum E2 and LH and serial ultrasound measurement with a transvaginal 7 MHz transducer (Bruel and Kjaer, Type 8538, Naerum, Denmark). Oocyte collection was performed 34 hours after hCG administration by transvaginal ultrasonically guided follicle aspiration, which has been used in our program since October 1987. One hundred fifty-eight of the 181 study cycles which proceeded to oocyte retrieval were evaluated
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with respect to the length of follicular stimulation, the amount of hMG required and serum E2 response, the number of oocytes retrieved per attempt, embryos transferred, and fertilization and cleavage rates. The pregnant and nonpregnant groups were compared and the correlation between the duration of treatment with OCs and each of these cycle parameters was analyzed. In addition, the study and control groups were compared with respect to incidence of LH surges, defined as an increase in LH concentration of > 100% compared with the mean of the previous 2 days. We also compared the length of induction of ovulation, the amount of hMG required, E2 response, the number of follicles :2: 1.5 cm measured on the day before oocyte retrieval, the number of oocytes obtained per attempt, and the pregnancy and abortion rates. Due to personnel and technique changes in our IVF laboratory in July 1988, we believed that comparison of fertilization and cleavage rates between the two groups were not valid and are not discussed in our results. Estradiol Assay
Estradiol (E 2) was measured by a solid phase radioimmunoassay (RIA) using 1251-labeled E2 (CoatA-Count Estradiol) purchased from Diagnostic Products Corporation (Los Angeles, CA). Estradiol values were expressed in pmol/L (pg/mL X 0.003671). Intra-assay and interassay coefficients of variations were also determined directly in serum pools placed at 2 or 3 levels of the working range, respectively. The intra-assay coefficients of variation at 290 pmol/L and 1,000 pmol/L E2 was 5.6% and 4.0%, respectively. The interassay coefficients of variation at 280 pmol/L, at 500 pmol/L, and at 2,500 pmol/L was 12%, 9.5%, and 7.5%, respectively. . Statistical Evaluation
The data obtained were compared by Student's t-test for unpaired results. Fertilization and cleavage rates were analyzed using the nonparametric Mann-Whitney V-test on rank data. Pregnancy and cancellation rates were compared by x 2 test. When cycle parameters were analyzed according to the length of OC treatment, differences between the groups in parametric data were examined by one-way analysis of variance, whereas fertilization and cleavage rates were compared by KruskalWallis test for nonparametric data. Correlation
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JEdS
Table 1
Cycle Parameters in Controlled Cycles (no OC) and Cycles with Gonadotropin Suppression with OC
No. of cycles No. of patients No. of cancellations No. ofLH surges No. of retrievals No. ofhMG required/cycle No. offollicles ;:,: 1.5 cm on day after hCG DayofhCG No. of oocytes/attempt E2 day 9 (pmol/L) E2 day hCG (pmol/L) E2 after hCG (pmol/L) Pregnancy rate/ET Abortion rate a
b
OC
NoOC
181 173 23 (12.7) a 0 158 8.9± 0.4' 6.6± 0.2 12.1 ± 0.1 4.7± 0.2 1,312 ± 70 4,256 ± 137 5,725 ± 214 22/108 (20.4) 2/22 (9.1)
113 98 18 (15.9) 22 (19.5) 95 10.9 ± 0.4 6.0± 0.2 12.3 ± 0.1 4.1 ± 0.2 1,420 ± 80 4,424 ± 157 5,353 ±200 12/76 (15.8) 4/12 (33.3)
Values in parentheses are percents. NS, not significant.
was checked by linear regression analysis. P values < 0.05 were regarded as statistically significant. Data are presented throughout this paper as means ± standard error (±SE) of the mean.
RESULTS A decision to use routine OC gonadotropin suppression to facilitate scheduling in our IVF program since July 1988 allowed the study of consecutive unselected patients undergoing controlled ovarian hyperstimulation. Table 1 presents cycle parameters in the study and in the control groups. One hundred fifty-eight (87.3%) of the 181 study cycles, in which gonadotropin suppression with OC preceded induction of ovulation, proceeded to transvaginal oocyte retrieval. Twenty-three cycles (12.7%) were cancelled, 87% of them due to a failure of E2 to increase by at least twice each day after the first significant rise from baseline or because of a drop in E2 of ~30% after hCG. The other 13% were cancelled because of poor follicular development or for other factors not related to ovarian stimulation. The mean length of ovarian suppression did not differ between cycles that were cancelled and those that reached oocyte retrieval (36 ± 2.4 and 35.3 ± 0.9 days, respectively). In the control group, 95 (84.1 %) of the 113 stimulation cycles proceeded to oocyte retrieval, whereas 18 cycles (15.9%) were cancelled, 83.3% of them due to suboptimal E2 response as described above or poor follicular development, and 16.7% due to an LH surge of unknown time of onset. The mean amount of hMG required to achieve optimal E2 response and
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Pvalue
NS b P
, Values are means ± SE.
follicular development was significantly lower when OC administration preceded induction of ovulation compared with the group without previous pituitary suppression (8.9 ± 0.4 and 10.9 ± 0.4 ampules, respectively; P < 0.0005). Although the average cycle day for hCG administration did not differ between the two groups (12.1 ± 0.1 and 12.3 ± 0.1), almost all OC-suppressed patients went to oocyte retrieval during the midweek since the OC was stopped on Saturday or Sunday, resulting in a day 1 on Tuesday or Wednesday. The mean number of follicles ~ 1.5 cm on the day before ovum retrieval was significantly greater in the study than in the control group (6.6 ± 0.2 and 6.0 ± 0.2, respectively; P < 0.05) and significantly more oocytes were retrieved per attempt in the study than in the control group (4.7 ± 0.2 and 4.1 ± 0.2, respectively; P < 0.05). The E2 levels on cycle day 9, the day of hCG administration and the day after hCG, did not differ between the two groups. No spontaneous LH surges occurred in the study patients whereas in the control group, the incidence of LH surge was 19.5% (P < 0.001). One hundred and eight embryo transfers (ETs) in the study group resulted in 22 clinical pregnancies for a success rate of 20.4% per ET, whereas 76 ETs in the control group resulted in 12 clinical pregnancies (15.8% per ET). All pregnancies were documented by transvaginal ultrasound with confirmation of fetal heart activity. Early abortion occurred in 2 pregnancies (9.1 %) in the study group and in 4 pregnancies (33.3%) in the control group. Table 2 compares the cycle parameters of study patients who conceived with study patients who
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Table 2
Comparison of Pregnant and Nonpregnant Patients in Gonadotropin Suppression Group
Age (years) No. of days on OC No. ofhMG required No. of follicles ;00 1.5 cm on day after hCG DayofhCG No. of oocytes/attempt No. of embryos/transfer Fertilization rate/oocyte Cleavage rate/oocyte E2 day 9 (pmoljL) E2 day hCG (pmoljL) E2 after hCG (pmoljL) a
Nonpregnant (n = 136)
Pregnant (n = 22)
Pvalue
33.3 ± 0.3 a 35.1 ± 0.9 8.9± 0.4 6.7± 0.2 12.1 ± 0.1 4.8± 0.2 2.7 ± 0.2 39.9± 3.1 36.9 ± 3.1 1,323 ± 76 4,228 ± 144 5,672 ± 231
34.1 ± 0.6 36.3 ± 2.8 9.0± 0.9 6.5± 0.5 12.1 ± 0.2 4.4 ± 0.5 3.1 ± 0.3 75.2 ± 4.9 72.8± 4.9 1,243 ± 168 4,429 ± 424 6,054 ±589
NS b NS NS NS NS NS NS P < 0.0005 P< 0.0005 NS NS NS
Values are means ± SE.
did not. For the most part, the cycle parameters appear to be similar for both groups. The mean duration of treatment with OC did not differ between the pregnant and nonpregnant group (36.3 ± 2.8 and 35.1 ± 0.9 days, respectively). No difference was found in the number of ampules of hMG required for follicle stimulation between the pregnant and nonpregnant group (9.0 ± 0.9 versus 8.9 ± 0.4 ampules, respectively), nor in the E2 response on the day ofhCG administration (4,429 ± 424 versus 4,228 ± 144 pmoljL). The mean number offollicles ~ 1.5 cm on the day before the oocyte retrieval, the cycle day of hCG administration, as well as the mean number of oocytes retrieved and embryos transferred, did not differ. Both, the fertilization and the cleavage rates per oocyte recovered were significantly higher (P < 0.0005) in the pregnant than in the nonpregnant group (Table 2). No correlation by regression analysis was found between the length of OC use and fertilization rate (r2 = 0.05 and r2 = 0.001 in the pregnant and the nonpregnant group, respectively) or in other cycle parameters in the pregnant or in the nonpregnant group. Table 3 summarizes cycle parameters according to the duration of treatment with OCs before the induction of ovulation. No statistically significant difference in pregnancy or cancellation rates was found between cycles with previous gonadotropin suppression for .:::;;3 weeks compared with 4, 5, or 6 or more weeks of suppression. Of all cycle parameters analyzed, the only difference was found in the mean E2 levels on cycle day 9 between the group of patients treated with OC for .:::;;21 days and those treated for ;?:43 days (1,586 ± 270 and 980 ± 90 pmoljL, respectively; P < 0.05). E2 levels on the
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b
NS, not significant.
day of hCG administration or the following day, however, did not differ among the five groups. There was no difference in the other cycle parameters. DISCUSSION
The use of gonadotropin suppression before ovulation induction has been associated with an increased number of oocytes collected, improved fertilization and pregnancy rates,5,6 and a decreased incidence of endogenous LH surges. As a result, cycles in which gonadotropin suppression has been achieved result in a decreased cancellation rate in those programs which routinely cancel LH surge cycles. 6-8 Most attention has been received by GnRH -a, although OCs or progestins alone may also be used for gonadotropin suppression. The administration of GnRH -a has several disadvantages and side effects. Many patients experience hot flushes, headaches, and fatigue,9 especially with long-term protocols. Multiple follicular stimulation requires higher doses of gonadotropins and longer regimens, resulting in higher cost and greater inconvenience to the patients. 1O There is an increased incidence of ovarian hyperstimulation syndrome9,1l,12 and ovarian cyst formation,9 as well as the need for luteal phase support. The recently reported13 lower survival of cryopreserved embryos after thawing and the relatively high abortion rate 6,9 in pregnant patients receiving GnRH-a during IVF cycles suggests that poor quality embryos may be produced in these cycles. In an effort to assess the potential benefits from the use of OC for pituitary suppression, we evaluated 181 stimulation cycles in which gonadotropin suppression with OCs preceded induction of ovula-
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r Table 3
Effect of the Length of OC Gonadotropin Suppression on Cycle Parameters, Fertilization, and Pregnancy Rates
No.ofretrievals Mean length of OC use (days) Mean age of patients (years) No. ofhMG required DayofhCG E2 day 9 (pmol/L) E2 day of hCG (pmol/L) E2 day after hCG (pmol/L) Mean no. of follicles ~ 1.5 cm No.ofoocytes/attempt No. of embryos/transfer Fertilization rate/oocyte Cleavage rate/oocyte No. of pregnancies Pregnancy rate/ET
29 to 35 days
36 to 42 days
16
36
27
36
43
20.1 ± 0.2" 33.6 ± 0.1 9.9 ± 1.6 11.8 ± 0.2 1,586 ± 270 4,316 ± 464 5,721 ± 743 7.0 ± 0.7 5.4 ± 0.9 3.6 ± 0.4 32.9 ± 9.1 32.2 ± 9.2 2
24.5 ± 0.4 34.3 ± 0.5 8.8± 0.9 12.1 ± 0.2 1,495 ± 173 4,251 ± 289 5,654 ±420 6.5± 0.5 4.6± 0.4 2.6± 0.3 49.2 ± 5.7 47.3 ± 5.7 6 6/26 (23.1)
31.6 ± 0.4 32.8± 0.8 8.7± 1.0 12.0 ± 0.2 1,254 ± 128 4,273 ± 244 5,885 ±356 6.9± 0.5 4.6± 0.6 2.4± 0.4 37.1 ± 7.4 36.6± 7.5 2 2.17 (11.8)
38.5 ± 0.3 33.5 ± 0.6 8.8± 0.6 12.0 ± 0.2 1,447 ± 158 4,395 ± 332 6,072 ±517 6.6± 0.4 4.7 ± 0.4 2.9± 0.3 47.4 ± 6.3 44.7 ± 6.3 4 4/24 (16.7)
49.5 ± 0.8 32.8± 0.5 9.0± 0.7 12.4 ± 0.2 980 ± 90 4,112 ±266 5,404 ±444 6.5 ± 0.4 4.7± 0.4 2.8± 0.2 48.5± 5.6 42.5 ± 5.5 8 8/31 (25.8)
2/10 (20)b
tion. Our data show that the use of OCs allows flexibility in patient scheduling and facilitates efficient use of IVF -ET personnel as a result of reasonably predictable timing of oocyte retrieval during the middle of the week in almost all cases in this study. The use of OCs for gonadotropin suppression prevents the occurrence of spontaneous LH surges to a similar extent as GnRH-a use. In addition, we observed a reduction in the amount of hMG required for follicular stimulation and maturation, in contrast to the markedly increased requirement for hMG in GnRH-a suppressed cycles. The very low spontaneous abortion rate (9.1 %) in the presence of an acceptable pregnancy rate of 20.4% per ET suggests that no adverse effect on embryo quality occurred with OC gonadotropin suppression. Considering the low cost of OCs in comparison with GnRH-a, the simplicity of use, the greater patient acceptance (oral administration versus daily subcutaneous injections), the reduction in hMG needed for hyperstimulation, and the absence of side effects in this study, our results clearly demonstrate that consideration should be given to the use of OCs as an alternative to GnRHa in IVF stimulation protocols. Furthermore, we did not use luteal phase support with progesterone or hCG in any of the patients reported in this study with no obvious effect on cycle length or pregnancy rates. Our data demonstrate that gonadotropin suppression with OCs synchronizes follicular development and is associated with a uniform response to ovarian hyperstimulation. In contrast to Mashiach et al.,4 the results of present series show that the
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days
22 to 28 days
" Values are means ± SE.
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~43
0;21 days
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b
Values in parentheses are percents.
length of treatment with OCs does not appear to have any adverse effect on follicular recruitment, cycle cancellation, or pregnancy rate. In our study of 181 cycles, we did not find a more profound suppression leading to poor ovarian stimulation period or increased cancellation rate when OC was administered for longer periods of time. Mashiach et al. 4 reported that the latent phase, which represents a period of ovarian insensitivity, was prolonged and directly correlated to the duration of OC suppression. Studying only 16 patients in each group, these authors 4 concluded that suppression with an OC for 30 days compared with 15 days, resulted in a greater cancellation of oocyte retrievals, a lower fertilization rate, and a lower pregnancy rate. The reason for the difference between Mashiach's study4 and ours is not clear, but may be related to the smaller number of patients in the previous report or the use of a different OC with more potent progestin. However, we believe that a more likely explanation is the fact that day 1 of the cycle was designated to be the day after the last OC pill, compared with a day 1, in our study, that was 3 days after the last pill. Benadiva et al. 14 reported that women with suppressed pituitary function may need to return to a certain threshold level of FSH for early follicular recruitment, if a desirable ovarian response is to be achieved during ovarian hyperstimulation. Klein and Mishell 15 observed that the follicular phase is prolonged after discontinuing OC and suggested that a brief interval is needed to reestablish normal ovarian-pituitary relationships in a spontaneous cycle. It is possible that, in the present study, a de-
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lay in the initiation of ovarian hyperstimulation until day 5 of the cycle resulted in a more responsive pituitary-ovarian axis. Thus, it is possible that the length of ovarian recovery and different designation of day 1 after the gonadotropin suppression, rather than the length of OC treatment prior to induction of ovulation may play the main role in successful and uniform response to ovarian hyperstimulation. In summary, the suppression ofthe hypothalamic-pituitary-ovarian axis by OCs is an acceptable tool to manipulate the cycle for IVF. Our data have shown that the method can result in an acceptable pregnancy rate with low cancellation and abortion rates, as well as reduced costs. No correlation was found between the length of ovarian suppression and the other cycle parameters which were analyzed. It is concluded that the length of treatment with OCs does not appear to have any adverse effect on response to induction of ovulation or pregnancy rate and outcome. However a short delay in the initiation of ovarian hyperstimulation to allow return of pituitary-ovarian responsiveness is likely important. It is our belief, that the use of OCs for gonadotropin suppression prior to induction of follicular hyperstimulation for IVF should be considered as an alternative to the use of GnRH -a.
Acknowledgment. The authors thank Ms. Mary Coates for her help with the preparation of the manuscript.
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8.
9.
10.
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Rudak E, Shoam Z, Finelt Z, Nebel L, Goldman B, BenRafael Z: Protocols for induction of ovulation. The concept of programmed cycles. Ann NY Acad Sci 541:37,1988 Neveu S, Hedon B, Bringer J, Chinchole J-M, Arnal F, Humeau C, Cristol P, Viala J -L: Ovarian stimulation by a combination of a gonadotropin-releasing hormone agonist and gonadotropins for in vitro fertilization. Fertil SteriI47:639, 1987 Meldrum DR, Wisot A, Hamilton F, Gutlay AL, Kempton W, Huynh D: Routine pituitary suppression with leuproide before ovarian stimulation for oocyte retrieval. Fertil Steril 51:455, 1989 Porter RN, Smith W, Craft IL, Abdulwahid NA, Jacobs HS: Induction of ovulation for in vitro fertilization using buseralin and gonadotropins. Lancet 2:1284,1984 Fleming R, Haxton MJ, Yates RWS, Conagham C, Coutts JRT: A simple procedure for the induction of multiple follicular growth with blockade of the LH surge in normal women. (Abstr.) Presented at the 67th Annual Meeting of the Endocrine Society, Baltimore, Maryland, June 19 to 21, 1985. Caspi E, Ron-EI R, Golan A, Nachum H, Herman A, Soffer Y, Weinraub Z: Results of in vitro fertilization and embryo transfer by combined long-acting gonadotropin-releasing hormone analog D-Trp-6-luteinizing hormone-releasing hormone and gonadotropins. Fertil SteriI51:95, 1989 MacLachlan V, Besanko M, O'Shea F, Wade H, Wood C, Trounson A, Healy D: A controlled study ofluteinizing hormone-releasing hormone agonist (buseraline) for the induction of folliculogenesis before in vitro fertilization. N Engl J Med 320:1233, 1989 Golan A, Ron-EI R, Herman A, Weinraub Z, Soffer Y, Caspi E: Ovarian hyperstimulation syndrome following DTrp-6 luteinizing hormone-releasing hormone microcapsules and menotropin for in vitro fertilization. Fertil Steril 50:912, 1988 Ezratty V, Cohen J: Ovarian hyperstimulation syndrome with GnRH agonist. (Abstr.) Presented at the VI World Congress In Vitro Fertilization and Alternate Assisted Reproduction, Jerusalem, April 2 to 7,1989 Von Steirteghem AC, Van Den Abbeel E, Smitz J, Camus M, Khan I, Staessen C, Devroey P: Cryopreservation of supernumerary embryos after GIFT and IVF in Clomid-hMG and Buserelin-hMG stimulated cycles. (Abstr.) Presented at the VI World Congress In Vitro Fertilization and Alternate Assisted Reproduction, Jerusalem, April 2 to 7,1989 Benadiva CA, Ben-Rafael Z, Blasco L, Tureck R, Mastroianni LJ, Flickinger GL: Ovarian response to human menopausal gonadotropin following suppression with oral contraceptives. Fertil Steril50:516, 1988 Klein T A, Mishell DR: Gonadotropin, prolactin and steroid hormone levels after discontinuation of oral contraceptives. Am J Obstet GynecoI127:585, 1977
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