The significance of the ratio in follicle-stimulating hormone and luteinizing hormone in induction of multiple follicular growth

FERTILITY AND STERILITY Copyright ~ 1985 The American Fertility Society

Vol. 43, No.3, March 1985 Printed in U.s A.

The significance of the ratio in follicle-stimulating hormone and luteinizing hormone in induction of multiple follicular growth

Rob E. Bernardus, M.D. Georgeanna Seegar Jones, M.D. Anibal A. Acosta, M.D. Jairo E. Garcia, M.D. Hung-Ching Liu, Ph.D. Debra L. Jones Zev Rosenwaks, M.D. * The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia

Among the patients enrolled in the Norfolk In Vitro Fertilization Program there were 32 who had been stimulated according to the basic stimulation protocol using two ampules of human menopausal gonadotropin (hMG) daily. Because of their inadequate response, 23 of these 32 patients were stimulated subsequently with a combination of two ampules of "pure" follicle-stimulating hormone (FSH) and two ampules of hMG on cycle days 3 and 4. The remaining nine patients received four ampules of "pure" FSH only on cycle days 3 and 4. Stimulation was continued with hMG in both FSH regimens. Ten thousand units of human chorionic gonadotropin was used for final maturation. Parallel with the increase in the ratio of exogenous FSH to luteinizing hormone, an increase in oocyte recovery was observed, as well as an improvement in transfer and pregnancy rates. It was concluded that FSH enrichment had a beneficial effect in these patients. Fertil Steril 43:373, 1985

Hormonal control of multiple follicular growth in an in vitro fertilization program is directed toward maximizing the yield of fertilizable 00cytes and enhancing successful implantation and early embryonic development in utero. Hyperstimulation of the ovaries results in higher recovery of fertilizable oocytes and subsequent higher transfer and pregnancy rates than occurs in the

Received May 17, 1984; revised and accepted November 13, 1984. *Reprint requests: Zev Rosenwaks, M.D., Director, The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, 304 Medical Tower, Norfolk, Virginia 23507. Vol. 43, No.3, March 1985

natural cycle. However, a standard protocol for stimulation which ensures success still does not exist. Accountable for that is the patient's individual response to a certain drug regimen, governed by yet many unknown factors. In an effort to improve the outcome in patients who did not respond adequately to our basic stimulation protocol with human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG), the ratio in exogenous follicle-stimulating hormone (FSH) to luteinizing hormone (LH) was changed in the early follicular phase. Therefore, "pure" FSH was used on cycle days 3 and 4. This article reports the effect on oocyte recovery and subsequent outcome of the addition of "pure" FSH administered concurrently with hMG/hCG. Bernardus et al. Ratio in human gonadotropins in IVF

373

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Table 1. Drug Regimens Used in 32 Patients

MATERIALS AND METHODS

Among the patients who applied to the program for Vital Initiation of Pregnancy at the Eastern Virginia Medical School, there were 32 regularly ovulating women who were initially stimulated according to a stimulation protocol utilizing two ampules of hMG (Pergonal, Serono Laboratories, Inc., Randolph, MA) daily from cycle day 3. This type of induction will be referred to as the "2hMG" protocol (Table 1). Twenty-seven of these 32 women had an intractable tubal defect, 3 presented with a male factor, 1 patient had extensive treatment for endometriosis, and 1 patient had idiopathic infertility. Most patients were between 31 and 35 years of age. Because of their inadequate response to plasma estradiol (E 2) and/or failure in establishing a pregnancy, 23 of these women were stimulated with a combination of FSH (Urofollitropin, Serono) and hMG on cycle days 3 and 4. This type of stimulation will be referred to as the "combination FSHlhMG" protocol (Table 1). Two ampules of FSH were injected at 9:00 A.M. and two ampules of hMG were administered at 4:00 P.M. on cycle days 3 and 4. The mean interval between both treatment cycles was 8.9 months (range, 2 to 17 months). Eight of those 23 patients were considered low responders on the basis of their E2 responses when stimulated with the 2-hMG protocol. Another nine patients were given FSH only on cycle.days 3 and 4. This type of stimulation will be further referred to as the "4-FSH" protocol. Two ampules of "pure" FSH were administered at 9:00 A.M. and at 4:00 P.M., respectively, on cycle days 3 and 4 (Table 1). The mean interval between treatment cycles was 9.1 months (range, 4 to 15 months). Six patients (66.6%) were considered low responders because of their E2 response under 2-hMG stimulation. It was because of this poor E2 response that these patients were considered for the 4-FSH protocol. "Pure" FSH contains 75 IU of human urinary FSH and < 1 IU of LH in each vial. In both FSH regimens~ stimulation was continued with hMG thereafter. In aU stimulation protocols, final follicular maturation was induced with 10,000 IU of hCG administered 50 to 52 hours after the last hMG dose. Laparoscopy for follicle aspiration was scheduled 36 to 38 hours after hCG administration. Patient clinical and 374

Bernardus et al. Ratio in human gonadotropins in NF

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3

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5

6

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2FSH 2FSH

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Daily ratio of FSHtoLH on eyele days 3: and 4

1501150

300/150 300/< 1

biochemical monitoring in both groups was as previously described in detail. 1 • 2 In brief, starting from cycle day 3, blood was collected daily for E2 determinations. Starting on cycle day 6, ultrasonographic and pelvic examinations were performed daily. A clinical shift was considered to have occurred when the karyopyknotic index showed 30% or more cornified cells in the vaginal wash and a cervical score of 4 was reached. 2 Each of the following parameters scored 1 point if (1) the volume of cervical mucus was ~ 2 ml, (2) spinnbarkeit was ~ 10 cm, (3) there was a clear acellular aspect to the mucus, and (4) dilatation of the external cervical os was obtained. According to the E2 levels on the day of discontinuation of hMG, the patients were categorized as high (E 2 > 600 pg/ml), normal (E 2 between 300 and 600 pg/ml) , and low responders (E2 < 300 pg/ml). In the low responders' group, hMG was discontinued after 3 days of clinical shift; in the normal responders' group, no further stimulation was given on the day of the clinical shift. In the high responders, regardless of the occurrence of clinical shift, further hMG was suspended when E2 values reached 600 pg/ml. Laparoscopy was performed under general anesthesia. Pneumoperitoneum was achieved with 100% carbon dioxide. Follicular aspiration was performed by the method -of Jones et al. 3 Oocytes were classified according to the morphologic criteria of Veeck et al. 4 Plasma E2 and plasma progesterone were measured by radioimmunoassay kit (Pantex, Santa Monica, CA), as previouslydescribed. 2 Comparisons of oocyte recovery, fertilization, and transfer rates were made between the cycles stimulated with 2-hMG and the subsequent cycles in which the ratio of exogenous FSHlLH was Fertility and Sterility

changed in the early follicular phase by the use of "pure" FSH. Statistical analysis was performed by Student's t-test. RESULTS

The total oocyte recovery per cycle increased significantly (P < 0.05) when "pure" FSH was used, as compared with that in cycles induced with hMG only (Table 2). In the combination FSH/hMG and in the 4-FSH groups the mean number of potentially fertilizable oocytes (i.e., mature and immature oocytes combined) was 3.1 and 3.9 per cycle, respectively, while in the 2hMG group the result was 1.1 per cycle (Table 2). The number of oocytes that actually were fertilized was 2 oocytes per cycle in the combination FSHIhMG group and 2.7 in the 4-FSH group. Corresponding results in the previous 2-hMG cycles were 1.0 and 0.7 per cycle, respectively (Table 2). The number of oocytes transferred per cycle in the combination FSH/hMG group was 2.4-fold when compared with the 2-hMG cycles. In the 4-FSH group, 3.1 times more oocytes were transferred per cycle than in the corresponding cycles induced with hMG alone (Table 2). No pregnancies occurred in these 32 patients when only hMG was used. In the 23 cycles stimulated with combined FSHIhMG the pregnancy rate per transfer was 27.7%. When only FSH was used in the first 2 days of stimulation, the pregnancy rate per cycle was 28.5% (Table 2). No case of severe clinical hyperstimulation (grade 3) was observed. 5 When the type of oocyte retrieved was considered, the number of preovulatory oocytes per cycle increased from 0.9 ± 0.6 (mean ± standard deviation [SD]) in the 2-hMG group to 1.6 ± 1.1 (mean ± SD) in the combination FSHIhMG group (P < 0.05). The number of immature oocytes in-

creased from 0.3 ± 0.7 (mean ± SD). to 1.5 ± 1.4 (mean ± SD) when FSH was added (P < 0.05). Although there was a minimal increase in the absolute number of atretic oocytes per cycle when the combination of FSH/hMG was employed, the atretic oocytes decreased from 45.4% of all retrieved oocytes with 2-hMG to 29.5% of all oocytes with the combination of F.8H/hMG regimen (Fig. 1). In the 4-FSH-initiated cycles versus the 2hMG-alone cycles (Fig. 2), only a slight increase was seen in the mean number of preovulatory oocytes retrieved per cycle: from 0.8 ± 0.7 (mean ± SD) in the 2-hMG cycles to 1.0 ± 1.0 (mean ± SD). However, the number of immature oocytes increased significantly (P < 0.05) from 0.4 ± 0.7 (mean ± SD) to 3.2 ± SD when FSH was used. There was no significant increase in the absolute number of atretic oocytes in the 4-FSH group. However, the proportion of atresia as compared with that in the 2-hMG regimen decreased from 52% to 33.3% of all retrieved oocytes. Comparisons between the peripheral steroid concentrations of the different stimulation protocols did not reveal significant differences on any given cycle day when concentrations were synchronized around the day of laparoscopy, designated as day + 1 (Figs. 3 to 6). DISCUSSION

The use of FSH for follicular growth has been studied in the human 6 , 7 as well as in the monkey.8 It has been established that either pituitary 6 or urinary FSH7 , 8 can induce multiple follicular growth and ovulation will occur when hCG is administered. In a study 9 of the importance of varying FSHI LH ratios in hMG it was documented that the

Table 2. Comparisons of Oocyte Recovery, Results ofFertilization in Vitro and Embryo Transfer in Cycles Induced According to the Three Stimulation Protocols (see Table lr

No. of cycles Oocyte recovery/cycle Fertilized oocytes/cycle Fertilized preovulatory oocytes/ cycle Fertilized immature oocytes/ cycle Oocytes transferred/cycle Pregnancies/transfer

2-hMG

Combination FSHIhMG

2-hMG

4-FSH

23 2.2 ± 0.3 1.0 ± 0.1 0.7 ± 0.1

23 4.4 6 ± 0.6 2.0 6 ± 0.3 1.2 b ± 0.2

9 2.5 ± 0.4 0.7 ± 0.2 0.4 ± 0.1

9 6.7 6 ± 1.4 2.7 b ± 0.8 1.2 b ± 0.5

0.3 ± 0.08

0.8 b ± 0.3

0.3 ± 0.2

1.5 6 ± 0.6

0.7 ± 0.1 0112

1.8 b ± 0.3 5/18 (27.7%)

0.7 ± 0.2 0/5

2.26 ± 0.6 217 (28.5%)

aResults are means ± standard error of the mean. Fertilization resulting in polyspermia was excluded. 6p < 0.05. Vol. 43, No.3, March 1985

Bernardus et al. Ratio in human gonadotropins in IVF

375

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hMG preparation with the lowest LH content or with the highest FSH/LH ratio was significantly less effective in inducing ovulation. The ratios of FSH to LH used were 751530, 75/84, and 82/28, respectively, as measured by bioassay. Furthermore, these preparations were given to oligoovulatory and anovulatory women and were administered as a single injection on day 1 of an arbitrary cycle, followed by 10,000 IV of hCG on day 9. Other studies showed that the FSH/LH ratio was of little importance. lO , 11 Our results do not substantiate these findingsP Moreover, the results presented of an increasing FSH/LH ratio Preovulatory

Figure I Yield of different types of 00cytes in 23 patients stimulated with both the 2-hMG regimen and the combination FSH/hMG regimen on cycle days 3 and 4 (see Table 1) .

from 150/150 in the 2-hMG group to 300/150 in the combination FSHIhMG group, to 3001< 1 in the 4-FSH regimen are suggestive for a dose-related enhancing role of FSH in follicular recruitment and development. However, it should be emphasized that the low LH contamination in the "pure" FSH was measured by radioimmunoassay and not by bioassay. Furthermore, the higher fertilization and transfer rates in the cycles enriched with FSH, concurrent with better pregnancy rates, indicate that enhanced multiple follicular development did not adversely affect the quality of the oocytes or corpus luteum function. In addi-

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Bernardus et a1. Ratio in human gonadotropins in IVF

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Figure 2 Yield of different types of 00cytes in nine patients stimulated with both the 2-hMG regimen and the 4-FSH regimen on cycle days 3 and 4 (see Table 1).

Fertility and Sterility

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tion, when the proportion of atresia was considered in the cycles with FSH, a decrease was noted, as compared with the cycles with hMG only. While the management of the FSH-enriched regimens was essentially the same as was established once for the basic hMG protocol, the results in terms of pregnancies achieved in the cycles with FSH indicate the validity of the method employed. However, the significant increased yield of immature oocytes in the FSH-enriched cycles may point out the possibility of a further improvement. It is tempting to speculate that 1 day of extra stimulation or discontinuation of hMG at a higher plasma E2 level may have resulted in a better yield of preovulatory oocytes. Retrospec-

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tive analysis of the cycles, especially with the 4-FSH regimen, supports this assumption. The surprising finding of similar E2 profiles in spite of significant differences in multiple follicular recruitment, as judged by ultrasound and subsequent follicular aspiration, may be indicative of the fact that peripheral estrogen concentrations are mainly a reflection ofthe most dominant follicle and not of all growing follicles. These data are not in accordance with those published by Venturoli et al. 7 A possible explanation might be the fact that they stimulated patients with polycystic

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tL Figure 4 Serum progesterone in 23 patients (mean ± sm stimulated in two subsequent cycles with 2-hMG and combination FSHI hMG on cycle days 3 and 4, respectively (see Table 1). Vol. 43, No.3, March 1985

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377

ovaries, who are known to be very responsive to ovulation induction with exogenous gonadotropins. It still remains to be determined whether the differences observed in the different stimulation protocols are caused by either the increase in FSH content in the early follicular phase and/or the delayed administration of LH, as is apparent in the 4-FSH group. In addition, the increased pregnancy rates when FSH was added might well be secondary to the improved yield and transfer rates. In conclusion, addition of "pure" FSH in the early follicular phase in patients who did not respond adequately on hMGIhCG alone on previous occasions resulted, in our hands, in a beneficial effect in terms of oocyte recovery, transfer rate, and pregnancy rate. . Acknowledgment. The "pure" FSH was a gift from Serono Laboratories, Inc., Randolph, MA. REFERENCES 1. Jones HW Jr, Jones GS, Andrews MC, Acosta A, Bundren C, Garcia J, Sandow B, Veeck L, Wilkes C, Witmyer J, Wortham JE, Wright G: The program for in vitro fertilization at Norfolk. Fertil Steril 38:14, 1982 2. Garcia JE, Jones GS, Acosta AA, Wright G Jr: Human menopausal gonadotropin/human chorionic gonadotropin follicular maturation for oocyte aspiration: Phase II, 1981. Fertil SteriI39:174, 1983

378

Bernardus et at. Ratio in human gonadotropins in IVF

3. Jones HW.Jr, Acosta AA, Garcia J: A technique for the aspiration of oocytes from human ovarian follicles. Fertil Steril 37:26, 1982 4. Veeck LL, Wortham JWE Jr, Witmyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW Jr: Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil Steril 39:594, 1983 5. Schenker JG, Polishuk WZ: Ovarian hyperstimulation syndrome. Obstet Gynecol 46:23, 1975 6. SchoemakerJ, Wentz AC, Jones GS, Dubin NH, Sapp KC: Stimulation of follicular growth with "pure" FSH in patients with anovulation and elevated LH levels. Obstet Gynecol 51:270, 1978 7. Venturoli S, Paradisi R, Fabbri R, Magrini 0, Porch E, Flamigni C: Comparison between human urinary folliclestimulating hormone and human menopausal gonadotropin treatment in polycystic ovary. Obstet Gynecol 63:6, 1984 8. Schenken RS, Hodgen GD: Follicle-stimulating hormone induced ovarian hyperstimulation in monkeys: blockade of the luteinizing hormone surge. J Clin Endocrinol Metab 57:50, 1983 9. Jacobson A, Marshall JR: Ovulatory response rate with human menopausal gonadotropins of varying FSH/LH ratios. Fertil Steril 20:171, 1969 10. Crooke AC, Butt WR, Bertrand PV, Morris C: Treatment of infertility and secondary amenorrhea with folliclestimulating hormone and chorionic gonadotropin. Lancet 2:636,1967 11. Taymor ML, Sturgis SH: Induction of ovulation with human postmenopausal gonadotropin. II. Probable causes of overstimulation. Fertil Steril 17:736, 1966 12. Jones GS: Update on in vitro fertilization. Endocr Rev 5:62,1984

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