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Characteristics of ovarian follicles in spontaneous and stimulated cycles in which there was an endogenous luteinizing hormone surge*
FERTIIJTY AND STERILITY Copyright c 1986 The American Fertility Society
Vol. 46, No.6, December 1986 Printed in U.SA.
Characteristics of ovarian follicles in spontaneous and stimulated cycles in which there was an endogenous luteinizing hormone surge *
Allan Templeton, M.D. t:J: Ioannis E. Messinis, M.D.t David T. Baird, D.Sc.§ University of Aberdeen, Aberdeen, and University of Edinburgh, Edinburgh, Scotland
The growth of ovarian follicles was assessed with the use of ultrasound in spontaneous cycles and in cycles stimulated with clomiphene citrate (CC) alone, CC plus pulsatile human menopausal gonadotropin, and CC plus pulsatile folliclestimulating hormone (FSH). At the time of the onset of the luteinizing hormone surge (LH), the size of the leading follicle did not differ significantly between the spontaneous and the stimulated cycles, although it was larger in the CCIFSH cycles. During the two days before the LH surge onset, the growth rate was faster in the stimulated than the spontaneous cycles. It is suggested that despite the provocation of extremely high plasma-estradiol levels and multiple follicular development, the leading follicle in stimulated cycles ovulated at a size equal to or greater than that in spontaneous cycles. The reason for the higher follicle size in the CCIFSH cycles is, as yet, unclear. Fertil Steril46:1113, 1986
Ultrasound scanning of the ovaries and measurement of estrogen levels in blood or urine are the two main parameters used to assess follicular growth in in vitro fertilization (IVF) programs. It is surprising that there is a lack of information concerning the optimum follicle size at which human chorionic gonadotropin (hCG) should be given in hyperstimulated cycles. In anovulatory women, clomiphene citrate (CC) and human menopausal gonadotropin (hMG) Received April 24, 1986; revi8ed and accepted August 13, 1986. *Supported by grant G426375 from the Medical Research Council. tDepartment of Obstetrics and Gynaecology, University of Aberdeen. :j:Reprint requests: Professor Allan Templeton, Department of Obstetrics and Gynaecology, University of Aberdeen, Foresterhill, Aberdeen AB9 2ZD, Scotland. §Department of Obstetrics and Gynaecology, University of Edinburgh. Vol. 46, No.6, December 1986
stimulate the development of follicles, which in general are larger than those in spontaneous cycles,I-4 although in some cases, due to premature administration of hCG, they have been found to be smaller. 5 We have shown recently, in contrast, that when multiple follicular development was induced with CC in normally cycling women, the size of the leading follicle at the time of the luteinizing hormone (LH) surge onset was similar to that seen in spontaneous cycles. 6 To date, the literature lacks data that compare the pattern offollicular growth before the onset of the endogenous LH surge in gonadotropin-induced cycles in normal women and in spontaneous cycles. This information would be of value in timing the administration of hCG as close as possible to the LH surge onset, because premature administration of hCG may have detrimental effects on follicle growth and oocyte maturation. 7 This study compares follicular dimensions in cycles induced in normally cycling women with Templeton et al. Ovarian follicles in stimulated cycles
1113
CC, CC plus pulsatile hMG, and CC plus pulsatile follicle-stimulating hormone (FSH) with those in spontaneous cycles. In all cycles an endogenous LH surge occurred.
during one spontaneous cycle. All gave informed consent. Ultrasonic scans of the ovaries started on cycle day 7. When the leading follicle reached 15 to 16 mm, blood samples for LH measurement were taken every 6 hours. The ultrasound scans of the ovaries were performed by two operators. A real-time sector scanning machine (3.5 MHz) was used (Diasonics DSlRF, SonotroniDiasonics, Les Ulis, France). The follicles were measured with the use of calipers on screen with a resolution of 1 mm. For each follicle, a diameter was calculated as the mean of three dimensions. Measurement ofE 2 in blood was done with RIA, described elsewhere. 13 Sensitivity for E2 was 35 pg/ml. Interassay and intraassay coefficients of variation were 11.2% and 6.5%, respectively. Statistical analysis of the results was performed with the use of Student's t-test.
MATERIALS AND METHODS
Twenty-seven women were studied in two groups. The first group included 16 normally cycling women with tubal infertility who requested IVF and embryo transfer (ET). The treatment protocol has been reported. 8 Superovulation was induced with CC (9 cycles), CC plus hMG (11 cycles) and CC plus "pure" FSH (11 cycles). Each treatment regimen was given to the same woman only once. Four women had all three regimens in three different cycles, seven women were treated in two cycles, and the remaining five were treated in one cycle. The dose of CC in all cycles was 150 mg/day from cycle days 2 to 6. The hMG (Pergonal, Serono Laboratories, UK), containing 75 IU FSH and 75 IU LH/ampule and "pure" FSH (Metrodin, Serono Laboratories), containing 75 IU FSH/ampule, were injected subcutaneously in a pulsatile manner (28 IU every 3 hours) via a pump,9 starting on day 6. Daily ultrasonic scans of the ovaries and measurements of total estrogen in 24 hours urine collections lO were made from day 6 to assess follicular growth. Also, daily blood samples were taken for LH measurement with rapid radioimmunoassay (RIA).l1 When urinary total estrogen values reached the level of 60 J.Lg/24 hours or the leading follicle reached a diameter of 17 to 18 mm, blood samples for LH measurement were taken every 6 hours. Plasma was stored at - 20°C for subsequent measurement of LH12 and estradiol (E 2). Laparoscopy for follicle aspiration and egg recovery was done 34 to 36 hours after the LH surge onset (data not shown). The second group included 11 volunteer women with normal ovulatory cycles who were studied
RESULTS
An endogenous LH surge occurred in all spontaneous and treated cycles, as previously reported. 8 , 14 The day of the cycle on which the endogenous LH surge started (mean ± standard error [SED did not differ significantly among the four groups (spontaneous-13.6 ± 0.6, range, 11 to 17 days; CC-13.7 ± 0.3, range 12 to 15 days; CCIhMG-12.8 ± 0.2, range 12 to 15 days; and CC/FSH-13.2 ± 0.4, range, 11 to 16 days). Table 1 shows that the size of the largest follicle (leading) (mean ± SE) in the CC and the CC/ hMG cycles on the day of onset of the LH surge (21.0 ± 0.5, range, 19 to 24 mm; and 20.8 ± 0.3, range, 19 to 23 mm, respectively) was similar to that of the dominant follicle in the spontaneous cycles (19.7 ± 0.6; range, 18 to 24 mm). In contrast, the size of the leading follicle in the CC/ FSH cycles (23.3 ± 0.9; range, 18 to 28 mm) was significantly greater than in the other three
Table 1. Follicle Diameter (mm) Estimated by Ultrasound (Mean Measurement of Three Dimensions) on the Day of the Onset of Endogenous LH Surge in Cycles Induced with the Administration to Normal Women ofCC, CC plus Pulsatile hMG, or CC plus Pulsatile FSH: Comparison with Spontaneous Cycles a Cycles Spontaneous (n = 11) CC (n = 9) CC/hMG (n = 11) CC/FSH (n = 11)
Mean
19.7 21.0 20.8 23.3
± ± ± ±
±
First follicle SE Range
0.6 b 0.5 b 0.3 b 0.9
Second follicle
Third follicle
Fourth follicle
Fifth follicle
18.8 ± 0.7 b 19.2 ± O.4 b 21.6 ± 0.7
16.1 ± 1.3 18.3 ± 0.3 19.4 ± 0.9
14.8 ± 1.0 16.5 ± 0.5 17.7 ± 1.0
15.4 ± 0.7 16.1 ± 0.9
18--24 19--24 19--23 18--28
aAIl values are mean ± SE. bp < 0.05 (differences from CC/FSH cycles).
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Fertility and Sterility
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·5
·4
·3 ·2 -1 Days from LH Surge Onset
o
Figure 1 Rate of growth of the leading follicle estimated on ultrasound examination (mean ± SE) during the 5 days before the onset of endogenous LH surge in 11 normal spontaneous cycles (0) and in cycles induced in normal women with ee (n = 9) (e), ee plus pulsatile hMG (n = 11) (~), and ee plus pulsatile FSH (n = 11) (A). *p < 0.05 (differences from the other three groups).
groups (P < 0.05). On the same day, the secondlargest follicle was significantly larger in the CCI FSH (21.6 ± 0.7 mm) than in the CCIhMG (19.2 ± 004 mm) and the CC-only cycles (18.8 ± 0.7 mm) (P < 0.05). The third and fourth follicles, in order, were also larger in the CC/FSH cycles than in the other two groups of induced cycles, but the difference was not statistically significant. Follicular fluid volume (mean ± SE) of the leading follicle obtained at aspiration was significantly greater in the CCIFSH (12.8 ± 1.3 ml, n = '10) than in the CCIhMG (904 ± 0.8 ml, n = 11) and the CC-only cycles (9.1 ± 1.1 ml, n = 7) (P < 0.05). There was no significant difference between the CCIhMG and the CC-only cycles. There was good correlation between the ultrasonic diameter of the leading follicle on the day before laparoscopy and the diameter of the same follicle estimated from the aspirated follicular fluid volume using the following formula: volume = 4/3 Tr"s (r = 0.91, n = 28). Figure 1 shows the size and the rate of growth of the leading follicle during the 5 days before the onset of LH surge (day 0) in the spontaneous group and the three groups of hyperstimulated cycles. Follicular dimensions did not differ significantly among the four groups from day - 5 to day - 2 before the onset of the surge. Also, the VoL 46, No.6, December 1986
growth rate was similar in the four groups during the same period. However, the growth rate during the last two days before the LH surge onset was faster and the size of the leading follicle greater in the CC/FSH cycles than in the other three groups (P < 0.05) . The increase in follicular diameter (a diameter) (mean ± SE) from day - 2 to day 0 was significantly higher in the CCIFSH cycles (5.7 ± 004 mm) than in the spontaneous (2.7 ± 0.2 mm) (P < 0.001), the CCIhMG (4.0 ± 0.5 mm), and the CConly cycles (3.8 ± 0.5 mm) (P < 0.05) (Fig. 2). Also, the difference was significant between the CCIhMG and the spontaneous cycles (P < 0.05) and between the CC-only and the spontaneous cycles (P < 0.05) (Fig. 2). In Table 2 the size of the leading follicle in all groups at the onset of the LH surge is shown in relation to the plasma E2 levels on the same day (mean ± SE). The E2 levels in the spontaneous cycles (236 ± 21 pg/ml) were significantly lower than in the three treatment groups (P < 0.001). Also, the E2 levels in the CC-only cycles (950 ± 133 pg/ml) were significantly lower than in the CCIhMG (2164 ± 249 pg/ml) and the CC/FSH cycles (2092 ± 190 pg/ml) (P < 0.001). There was *
6 5
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...
4
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..
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o SP
CC
CC/hMG
CC/FSH
Figure 2 Increase in diameter of the leading follicle estimated on ultrasound examination (~ diameter) from day - 2 before the onset of endogenous LH surge to the day of LH surge onset in 11 normal spontaneous cycles (SP) and in cycles induced in normal women with ee (n = 9), ee plus pulsatile hMG (n = 11), and ee plus pulsatile FSH (n = 11). *Differences from SP (P < 0.001), ee, and eelhMG (P < 0.05). **Differences from SP (P < 0.05).
Templeton et aI. Ovarian follicles in stimulated cycles
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Table 2. Ultrasonic Diameter (mm) (Mean Measurement of Three Dimensions) of the Leading Follicle and Plasma E2 Levels (pglml) on the Day of the Onset ofEndogenous LH Surge in Cycles Induced with the Administration to Normal Women ofCC, CC plus Pulsatile hMG, or CC plus Pulsatile FSH: Comparison with Spontaneous Cyclesa Follicle diameter
Cycles
19.7 21.0 20.8 23.3
Spontaneous (n = 11) CC (n = 9) CClhMG (n = 11) CC/FSH (n = 11) a All
0.6 b ± 0.5 b ± 0.3 b ± 0.9 ±
236 950 2164 2092
± 21 c 133d ± 249 ± 190
±
values are mean ± SE.
bp < 0.05 (differences from CC/FSH cycles). cp < 0.001 (differences from the three treatment groups). dp < 0.001 (differences from CC/hMG and CCIFSH cycles).
.no significant difference between the CClhMG and the CC/FSH cycles. DISCUSSION
In this study, ultrasound measurements were performed by only two operators, to minimize variation. 15, 16 The good correlation between ultrasound measurements and the actual follicle fluid volume verify the accuracy of the estimates, which is better than that previously reported from our department. 6 The rate of growth of the leading follicles from day - 5 to day - 2 was similar in all four groups. In a previous study, we showed that during treatment with CClhMG and CCIFSH the plasma concentration of FSH during the 5 days up to the LH surge was double that in normal and CC alone cyclesP These findings, together with the present results, suggest that above a certain minimal level the rate of growth of a large antral follicle is independent of the concentration ofFSH. The reason for the accelerated rate of growth during the final 48 hours in the group treated with FSH is uncertain. It is not known whether the increase in diameter is due solely to accumulation of fluid or whether it also reflects an increased number and/or size of granulosa cells. This is the first study that compares follicular growth among cycles induced with the administration to normal women of CC, CC plus pulsatile hMG, and CC plus pulsatile FSH and also between them and spontaneous cycles. The results show that there was no difference in the time at which the endogenous LH surge started and in the minimum size of the leading follicle between the hyperstimulated and the spontaneous cycles, although in the CC/FSH cycles the mean size was greater. In agreement with our previous data,6 1116
Templeton et al.
Ovarian follicles in stimulated cycles
these results suggest that during multiple follicular stimulation the relationship between the leading follicle and the pituitary, concerning the time of onset of midcycle LH surge, remains intact. Although the endogenous LH surge is the result of the positive feedback effect of E2 on the hypothalamic-pituitary unit,18 in the present study, great differences in E2 levels were found between the spontaneous cycles and the three treatment groups at the onset of the LH surge. This suggests that in cycles with multiple follicular development, regardless of the circulating E2 levels, the positive feedback effect of E2 on the midcycle LH discharge is under the control of the growing follicles. Although the mechanism is not clear, one can postulate that unspecified factors produced by the follicles synchronize the onset of the LH surge with the time at which follicle development has reached an appropriate stage, corresponding to the size of the preovulatory follicle in spontaneous cycles. However, the possibility that the treatment regimen used in this study for ovarian hyperstimulation contributed to the timing of the endogenous LH surge cannot be excluded. Previous data have shown that during ovarian hyperstimulation with daily injections of hMG only, an endogenous LH surge is detected in some women at a time when the leading follicle is not mature, as assessed by its ultrasonic dimensions. 19 Therefore, it still remains to be determined whether administration of pulsatile hMG or FSH without CC would result in an appropriately timed endogenous LH surge in relation to follicular size. In conclusion, this study shows that when normally cycling women are superovulated with CC or combinations of CC with pulsatile hMG or FSH, follicle sizes similar to those in spontaneous cycles are achieved before the endogenous LH surge starts. In other words, it seems likely that the onset of the LH surge in these and the spontaneous cycles is regulated by the same mechanisms. Acknowledgments. We thank Mrs. Lin Christie for hormone assays and Sheila Lawson, M.B., Ch.B., for her help with the ultrasound examinations. We also thank Sister Alison Michie for help in managing the patients.
REFERENCES 1. Robertson RD, Picker RH, Wilson PC, Saunders DM: Assessment of ovulation by ultrasound and plasma estradiol determinations. Obstet Gynecol 54:686, 1979 Fertility and Sterility
2. Ylostalo P, Ronnberg L, Jouppila P: Measurement of the ovarian follicle by ultrasound in ovulation induction. Fertil Steril 31:651, 1979 3. Queenan JT, O'Brien GD, Bains LM, Simpson J, Collins WP, Campbell S: Ultrasound scanning of ovaries to detect ovulation in women. Fertil Steril 34:99, 1980 4. Funduk-kurjak B, Kurjak A: Ultrasound monitoring of follicular maturation and ovulation in normal menstrual cycle and in ovulation induction. Acta Obstet Gynecol Scand 61:329, 1982 5. O'Herlihy C, De Crespigny LC, Lopata A, Johnston I, Hoult I, Robinson H: Preovulatory follicular size: a comparison of ultrasound and laparoscopic measurements. Fertil Steril 34:24, 1980 6. Messinis IE, Templeton A: Urinary oestrogen levels and follicle ultrasound measurements in clomiphene induced cycles with an endogenous luteinizing hormone surge. Br J Obstet Gynaecol 93:43, 1986 7. Williams RF, Hodgen GD: Disparate effects of human chorionic gonadotropin during the late follicular phase in monkeys: normal ovulation, follicular atresia, ovarian acyclicity and hypersecretion of follicle-stimulating hormone. Fertil Steril 33:64, 1980 8. Messinis IE, Templeton A, Baird DT: Endogenous luteinizing hormone surge during superovulation induction with sequential use of clomiphene citrate and pulsatile human menopausal gonadotropin. J Clin Endocrinol Metab 61:1076, 1985 9. Sutherland lA, White S, Chambers GR, Rothwell D, Mason WP, Tucker M, Jacobs HS: A miniature infuser for the pulsatile administration of LHRH. J Biomed Eng 6:129,1984 10. Brown JB, MacLeod SC, MacNaughton C, Smith MA, Smyth B: A rapid method for estimating estrogens in urine using a semi-automatic extractor. J Endocrinol 42:5,1968
11. Djahanbakhch 0, McNeilly AS, Hobson BM, Templeton AA: A rapid luteinizing hormone radioimmunoassay for the prediction of ovulation. Br J Obstet GynaecoI88:1009, 1981 12. McNeilly AS, Hagen C: Prolactin, TSH, LH and FSH response to a combined LHRH/TRH test at different stages of the menstrual cycle. Clin Endocrinol (OxO 3:427,1974 13. Backstrom CT, McNeilly AS, Leask RM, Baird DT: Pulsatile secretion of LH, FSH, prolactin, oestradiol and progesterone during the menstrual cycle. Clin Endocrinol (OxO 17:29, 1982 14. Messinis IE, Templeton A, Baird DT: Endogenous luteinizing hormone surge in women during induction of multipIe follicular development with pulsatile follicle stimulating hormone. Clin Endocrinol (OxO 24:193, 1986 15. Prins GS, Vogelzang RL: Inherent sources of ultrasound variability in relation to follicular measurements. J In Vitro Fert Embryo Transfer 1:221, 1984 16. Eissa MK, Hudson K, Docker MF, Sawers RS, Newton JR: Ultrasound follicle diameter measurement: an assessment of interobserver and intraobserver variation. Fertil Steril 44:751, 1985 17. Messinis IE, Templeton AA, Baird DT: Comparison between clomiphene plus pulsatile human menopausal gonadotrophin and clomiphene plus pulsatile follicle stimulating hormone in induction of multiple follicular development in women. Hum Reprod 1:223, 1986 18. Yen SSC: The human menstrual cycle. In Reproductive Endocrinology, Edited by SSC Yen, RB Jaffe. Philadelphia, WB Saunders Co, 1978, p 126 19. Vargyas JM, Morente C, Shangold G, Marrs RP: The effect of different methods of ovarian stimulation for human in vitro fertilization and embryo replacement. Fertil Steril 42:745, 1984
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Vol. 46, No.6, December 1986
Templeton et al. Ovarian follicles in stimulated cycles
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Vol. 46, No.6, December 1986 Printed in U.SA.
Characteristics of ovarian follicles in spontaneous and stimulated cycles in which there was an endogenous luteinizing hormone surge *
Allan Templeton, M.D. t:J: Ioannis E. Messinis, M.D.t David T. Baird, D.Sc.§ University of Aberdeen, Aberdeen, and University of Edinburgh, Edinburgh, Scotland
The growth of ovarian follicles was assessed with the use of ultrasound in spontaneous cycles and in cycles stimulated with clomiphene citrate (CC) alone, CC plus pulsatile human menopausal gonadotropin, and CC plus pulsatile folliclestimulating hormone (FSH). At the time of the onset of the luteinizing hormone surge (LH), the size of the leading follicle did not differ significantly between the spontaneous and the stimulated cycles, although it was larger in the CCIFSH cycles. During the two days before the LH surge onset, the growth rate was faster in the stimulated than the spontaneous cycles. It is suggested that despite the provocation of extremely high plasma-estradiol levels and multiple follicular development, the leading follicle in stimulated cycles ovulated at a size equal to or greater than that in spontaneous cycles. The reason for the higher follicle size in the CCIFSH cycles is, as yet, unclear. Fertil Steril46:1113, 1986
Ultrasound scanning of the ovaries and measurement of estrogen levels in blood or urine are the two main parameters used to assess follicular growth in in vitro fertilization (IVF) programs. It is surprising that there is a lack of information concerning the optimum follicle size at which human chorionic gonadotropin (hCG) should be given in hyperstimulated cycles. In anovulatory women, clomiphene citrate (CC) and human menopausal gonadotropin (hMG) Received April 24, 1986; revi8ed and accepted August 13, 1986. *Supported by grant G426375 from the Medical Research Council. tDepartment of Obstetrics and Gynaecology, University of Aberdeen. :j:Reprint requests: Professor Allan Templeton, Department of Obstetrics and Gynaecology, University of Aberdeen, Foresterhill, Aberdeen AB9 2ZD, Scotland. §Department of Obstetrics and Gynaecology, University of Edinburgh. Vol. 46, No.6, December 1986
stimulate the development of follicles, which in general are larger than those in spontaneous cycles,I-4 although in some cases, due to premature administration of hCG, they have been found to be smaller. 5 We have shown recently, in contrast, that when multiple follicular development was induced with CC in normally cycling women, the size of the leading follicle at the time of the luteinizing hormone (LH) surge onset was similar to that seen in spontaneous cycles. 6 To date, the literature lacks data that compare the pattern offollicular growth before the onset of the endogenous LH surge in gonadotropin-induced cycles in normal women and in spontaneous cycles. This information would be of value in timing the administration of hCG as close as possible to the LH surge onset, because premature administration of hCG may have detrimental effects on follicle growth and oocyte maturation. 7 This study compares follicular dimensions in cycles induced in normally cycling women with Templeton et al. Ovarian follicles in stimulated cycles
1113
CC, CC plus pulsatile hMG, and CC plus pulsatile follicle-stimulating hormone (FSH) with those in spontaneous cycles. In all cycles an endogenous LH surge occurred.
during one spontaneous cycle. All gave informed consent. Ultrasonic scans of the ovaries started on cycle day 7. When the leading follicle reached 15 to 16 mm, blood samples for LH measurement were taken every 6 hours. The ultrasound scans of the ovaries were performed by two operators. A real-time sector scanning machine (3.5 MHz) was used (Diasonics DSlRF, SonotroniDiasonics, Les Ulis, France). The follicles were measured with the use of calipers on screen with a resolution of 1 mm. For each follicle, a diameter was calculated as the mean of three dimensions. Measurement ofE 2 in blood was done with RIA, described elsewhere. 13 Sensitivity for E2 was 35 pg/ml. Interassay and intraassay coefficients of variation were 11.2% and 6.5%, respectively. Statistical analysis of the results was performed with the use of Student's t-test.
MATERIALS AND METHODS
Twenty-seven women were studied in two groups. The first group included 16 normally cycling women with tubal infertility who requested IVF and embryo transfer (ET). The treatment protocol has been reported. 8 Superovulation was induced with CC (9 cycles), CC plus hMG (11 cycles) and CC plus "pure" FSH (11 cycles). Each treatment regimen was given to the same woman only once. Four women had all three regimens in three different cycles, seven women were treated in two cycles, and the remaining five were treated in one cycle. The dose of CC in all cycles was 150 mg/day from cycle days 2 to 6. The hMG (Pergonal, Serono Laboratories, UK), containing 75 IU FSH and 75 IU LH/ampule and "pure" FSH (Metrodin, Serono Laboratories), containing 75 IU FSH/ampule, were injected subcutaneously in a pulsatile manner (28 IU every 3 hours) via a pump,9 starting on day 6. Daily ultrasonic scans of the ovaries and measurements of total estrogen in 24 hours urine collections lO were made from day 6 to assess follicular growth. Also, daily blood samples were taken for LH measurement with rapid radioimmunoassay (RIA).l1 When urinary total estrogen values reached the level of 60 J.Lg/24 hours or the leading follicle reached a diameter of 17 to 18 mm, blood samples for LH measurement were taken every 6 hours. Plasma was stored at - 20°C for subsequent measurement of LH12 and estradiol (E 2). Laparoscopy for follicle aspiration and egg recovery was done 34 to 36 hours after the LH surge onset (data not shown). The second group included 11 volunteer women with normal ovulatory cycles who were studied
RESULTS
An endogenous LH surge occurred in all spontaneous and treated cycles, as previously reported. 8 , 14 The day of the cycle on which the endogenous LH surge started (mean ± standard error [SED did not differ significantly among the four groups (spontaneous-13.6 ± 0.6, range, 11 to 17 days; CC-13.7 ± 0.3, range 12 to 15 days; CCIhMG-12.8 ± 0.2, range 12 to 15 days; and CC/FSH-13.2 ± 0.4, range, 11 to 16 days). Table 1 shows that the size of the largest follicle (leading) (mean ± SE) in the CC and the CC/ hMG cycles on the day of onset of the LH surge (21.0 ± 0.5, range, 19 to 24 mm; and 20.8 ± 0.3, range, 19 to 23 mm, respectively) was similar to that of the dominant follicle in the spontaneous cycles (19.7 ± 0.6; range, 18 to 24 mm). In contrast, the size of the leading follicle in the CC/ FSH cycles (23.3 ± 0.9; range, 18 to 28 mm) was significantly greater than in the other three
Table 1. Follicle Diameter (mm) Estimated by Ultrasound (Mean Measurement of Three Dimensions) on the Day of the Onset of Endogenous LH Surge in Cycles Induced with the Administration to Normal Women ofCC, CC plus Pulsatile hMG, or CC plus Pulsatile FSH: Comparison with Spontaneous Cycles a Cycles Spontaneous (n = 11) CC (n = 9) CC/hMG (n = 11) CC/FSH (n = 11)
Mean
19.7 21.0 20.8 23.3
± ± ± ±
±
First follicle SE Range
0.6 b 0.5 b 0.3 b 0.9
Second follicle
Third follicle
Fourth follicle
Fifth follicle
18.8 ± 0.7 b 19.2 ± O.4 b 21.6 ± 0.7
16.1 ± 1.3 18.3 ± 0.3 19.4 ± 0.9
14.8 ± 1.0 16.5 ± 0.5 17.7 ± 1.0
15.4 ± 0.7 16.1 ± 0.9
18--24 19--24 19--23 18--28
aAIl values are mean ± SE. bp < 0.05 (differences from CC/FSH cycles).
1114
Templeton et al. Ovarian follicles in stimulated cycles
Fertility and Sterility
25
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..5 ...
20
] "0
15
.. is'" ..
:! E
u..
10 O~
·5
·4
·3 ·2 -1 Days from LH Surge Onset
o
Figure 1 Rate of growth of the leading follicle estimated on ultrasound examination (mean ± SE) during the 5 days before the onset of endogenous LH surge in 11 normal spontaneous cycles (0) and in cycles induced in normal women with ee (n = 9) (e), ee plus pulsatile hMG (n = 11) (~), and ee plus pulsatile FSH (n = 11) (A). *p < 0.05 (differences from the other three groups).
groups (P < 0.05). On the same day, the secondlargest follicle was significantly larger in the CCI FSH (21.6 ± 0.7 mm) than in the CCIhMG (19.2 ± 004 mm) and the CC-only cycles (18.8 ± 0.7 mm) (P < 0.05). The third and fourth follicles, in order, were also larger in the CC/FSH cycles than in the other two groups of induced cycles, but the difference was not statistically significant. Follicular fluid volume (mean ± SE) of the leading follicle obtained at aspiration was significantly greater in the CCIFSH (12.8 ± 1.3 ml, n = '10) than in the CCIhMG (904 ± 0.8 ml, n = 11) and the CC-only cycles (9.1 ± 1.1 ml, n = 7) (P < 0.05). There was no significant difference between the CCIhMG and the CC-only cycles. There was good correlation between the ultrasonic diameter of the leading follicle on the day before laparoscopy and the diameter of the same follicle estimated from the aspirated follicular fluid volume using the following formula: volume = 4/3 Tr"s (r = 0.91, n = 28). Figure 1 shows the size and the rate of growth of the leading follicle during the 5 days before the onset of LH surge (day 0) in the spontaneous group and the three groups of hyperstimulated cycles. Follicular dimensions did not differ significantly among the four groups from day - 5 to day - 2 before the onset of the surge. Also, the VoL 46, No.6, December 1986
growth rate was similar in the four groups during the same period. However, the growth rate during the last two days before the LH surge onset was faster and the size of the leading follicle greater in the CC/FSH cycles than in the other three groups (P < 0.05) . The increase in follicular diameter (a diameter) (mean ± SE) from day - 2 to day 0 was significantly higher in the CCIFSH cycles (5.7 ± 004 mm) than in the spontaneous (2.7 ± 0.2 mm) (P < 0.001), the CCIhMG (4.0 ± 0.5 mm), and the CConly cycles (3.8 ± 0.5 mm) (P < 0.05) (Fig. 2). Also, the difference was significant between the CCIhMG and the spontaneous cycles (P < 0.05) and between the CC-only and the spontaneous cycles (P < 0.05) (Fig. 2). In Table 2 the size of the leading follicle in all groups at the onset of the LH surge is shown in relation to the plasma E2 levels on the same day (mean ± SE). The E2 levels in the spontaneous cycles (236 ± 21 pg/ml) were significantly lower than in the three treatment groups (P < 0.001). Also, the E2 levels in the CC-only cycles (950 ± 133 pg/ml) were significantly lower than in the CCIhMG (2164 ± 249 pg/ml) and the CC/FSH cycles (2092 ± 190 pg/ml) (P < 0.001). There was *
6 5
** **
E
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...
4
III
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..
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u..
o SP
CC
CC/hMG
CC/FSH
Figure 2 Increase in diameter of the leading follicle estimated on ultrasound examination (~ diameter) from day - 2 before the onset of endogenous LH surge to the day of LH surge onset in 11 normal spontaneous cycles (SP) and in cycles induced in normal women with ee (n = 9), ee plus pulsatile hMG (n = 11), and ee plus pulsatile FSH (n = 11). *Differences from SP (P < 0.001), ee, and eelhMG (P < 0.05). **Differences from SP (P < 0.05).
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Table 2. Ultrasonic Diameter (mm) (Mean Measurement of Three Dimensions) of the Leading Follicle and Plasma E2 Levels (pglml) on the Day of the Onset ofEndogenous LH Surge in Cycles Induced with the Administration to Normal Women ofCC, CC plus Pulsatile hMG, or CC plus Pulsatile FSH: Comparison with Spontaneous Cyclesa Follicle diameter
Cycles
19.7 21.0 20.8 23.3
Spontaneous (n = 11) CC (n = 9) CClhMG (n = 11) CC/FSH (n = 11) a All
0.6 b ± 0.5 b ± 0.3 b ± 0.9 ±
236 950 2164 2092
± 21 c 133d ± 249 ± 190
±
values are mean ± SE.
bp < 0.05 (differences from CC/FSH cycles). cp < 0.001 (differences from the three treatment groups). dp < 0.001 (differences from CC/hMG and CCIFSH cycles).
.no significant difference between the CClhMG and the CC/FSH cycles. DISCUSSION
In this study, ultrasound measurements were performed by only two operators, to minimize variation. 15, 16 The good correlation between ultrasound measurements and the actual follicle fluid volume verify the accuracy of the estimates, which is better than that previously reported from our department. 6 The rate of growth of the leading follicles from day - 5 to day - 2 was similar in all four groups. In a previous study, we showed that during treatment with CClhMG and CCIFSH the plasma concentration of FSH during the 5 days up to the LH surge was double that in normal and CC alone cyclesP These findings, together with the present results, suggest that above a certain minimal level the rate of growth of a large antral follicle is independent of the concentration ofFSH. The reason for the accelerated rate of growth during the final 48 hours in the group treated with FSH is uncertain. It is not known whether the increase in diameter is due solely to accumulation of fluid or whether it also reflects an increased number and/or size of granulosa cells. This is the first study that compares follicular growth among cycles induced with the administration to normal women of CC, CC plus pulsatile hMG, and CC plus pulsatile FSH and also between them and spontaneous cycles. The results show that there was no difference in the time at which the endogenous LH surge started and in the minimum size of the leading follicle between the hyperstimulated and the spontaneous cycles, although in the CC/FSH cycles the mean size was greater. In agreement with our previous data,6 1116
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Ovarian follicles in stimulated cycles
these results suggest that during multiple follicular stimulation the relationship between the leading follicle and the pituitary, concerning the time of onset of midcycle LH surge, remains intact. Although the endogenous LH surge is the result of the positive feedback effect of E2 on the hypothalamic-pituitary unit,18 in the present study, great differences in E2 levels were found between the spontaneous cycles and the three treatment groups at the onset of the LH surge. This suggests that in cycles with multiple follicular development, regardless of the circulating E2 levels, the positive feedback effect of E2 on the midcycle LH discharge is under the control of the growing follicles. Although the mechanism is not clear, one can postulate that unspecified factors produced by the follicles synchronize the onset of the LH surge with the time at which follicle development has reached an appropriate stage, corresponding to the size of the preovulatory follicle in spontaneous cycles. However, the possibility that the treatment regimen used in this study for ovarian hyperstimulation contributed to the timing of the endogenous LH surge cannot be excluded. Previous data have shown that during ovarian hyperstimulation with daily injections of hMG only, an endogenous LH surge is detected in some women at a time when the leading follicle is not mature, as assessed by its ultrasonic dimensions. 19 Therefore, it still remains to be determined whether administration of pulsatile hMG or FSH without CC would result in an appropriately timed endogenous LH surge in relation to follicular size. In conclusion, this study shows that when normally cycling women are superovulated with CC or combinations of CC with pulsatile hMG or FSH, follicle sizes similar to those in spontaneous cycles are achieved before the endogenous LH surge starts. In other words, it seems likely that the onset of the LH surge in these and the spontaneous cycles is regulated by the same mechanisms. Acknowledgments. We thank Mrs. Lin Christie for hormone assays and Sheila Lawson, M.B., Ch.B., for her help with the ultrasound examinations. We also thank Sister Alison Michie for help in managing the patients.
REFERENCES 1. Robertson RD, Picker RH, Wilson PC, Saunders DM: Assessment of ovulation by ultrasound and plasma estradiol determinations. Obstet Gynecol 54:686, 1979 Fertility and Sterility
2. Ylostalo P, Ronnberg L, Jouppila P: Measurement of the ovarian follicle by ultrasound in ovulation induction. Fertil Steril 31:651, 1979 3. Queenan JT, O'Brien GD, Bains LM, Simpson J, Collins WP, Campbell S: Ultrasound scanning of ovaries to detect ovulation in women. Fertil Steril 34:99, 1980 4. Funduk-kurjak B, Kurjak A: Ultrasound monitoring of follicular maturation and ovulation in normal menstrual cycle and in ovulation induction. Acta Obstet Gynecol Scand 61:329, 1982 5. O'Herlihy C, De Crespigny LC, Lopata A, Johnston I, Hoult I, Robinson H: Preovulatory follicular size: a comparison of ultrasound and laparoscopic measurements. Fertil Steril 34:24, 1980 6. Messinis IE, Templeton A: Urinary oestrogen levels and follicle ultrasound measurements in clomiphene induced cycles with an endogenous luteinizing hormone surge. Br J Obstet Gynaecol 93:43, 1986 7. Williams RF, Hodgen GD: Disparate effects of human chorionic gonadotropin during the late follicular phase in monkeys: normal ovulation, follicular atresia, ovarian acyclicity and hypersecretion of follicle-stimulating hormone. Fertil Steril 33:64, 1980 8. Messinis IE, Templeton A, Baird DT: Endogenous luteinizing hormone surge during superovulation induction with sequential use of clomiphene citrate and pulsatile human menopausal gonadotropin. J Clin Endocrinol Metab 61:1076, 1985 9. Sutherland lA, White S, Chambers GR, Rothwell D, Mason WP, Tucker M, Jacobs HS: A miniature infuser for the pulsatile administration of LHRH. J Biomed Eng 6:129,1984 10. Brown JB, MacLeod SC, MacNaughton C, Smith MA, Smyth B: A rapid method for estimating estrogens in urine using a semi-automatic extractor. J Endocrinol 42:5,1968
11. Djahanbakhch 0, McNeilly AS, Hobson BM, Templeton AA: A rapid luteinizing hormone radioimmunoassay for the prediction of ovulation. Br J Obstet GynaecoI88:1009, 1981 12. McNeilly AS, Hagen C: Prolactin, TSH, LH and FSH response to a combined LHRH/TRH test at different stages of the menstrual cycle. Clin Endocrinol (OxO 3:427,1974 13. Backstrom CT, McNeilly AS, Leask RM, Baird DT: Pulsatile secretion of LH, FSH, prolactin, oestradiol and progesterone during the menstrual cycle. Clin Endocrinol (OxO 17:29, 1982 14. Messinis IE, Templeton A, Baird DT: Endogenous luteinizing hormone surge in women during induction of multipIe follicular development with pulsatile follicle stimulating hormone. Clin Endocrinol (OxO 24:193, 1986 15. Prins GS, Vogelzang RL: Inherent sources of ultrasound variability in relation to follicular measurements. J In Vitro Fert Embryo Transfer 1:221, 1984 16. Eissa MK, Hudson K, Docker MF, Sawers RS, Newton JR: Ultrasound follicle diameter measurement: an assessment of interobserver and intraobserver variation. Fertil Steril 44:751, 1985 17. Messinis IE, Templeton AA, Baird DT: Comparison between clomiphene plus pulsatile human menopausal gonadotrophin and clomiphene plus pulsatile follicle stimulating hormone in induction of multiple follicular development in women. Hum Reprod 1:223, 1986 18. Yen SSC: The human menstrual cycle. In Reproductive Endocrinology, Edited by SSC Yen, RB Jaffe. Philadelphia, WB Saunders Co, 1978, p 126 19. Vargyas JM, Morente C, Shangold G, Marrs RP: The effect of different methods of ovarian stimulation for human in vitro fertilization and embryo replacement. Fertil Steril 42:745, 1984
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