Superovulation with exogenous gonadotropins does not inhibit the luteinizing hormone surge

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FERTLTY AND STERLTY Copyright e 1988 The American Fertility Society Printed in U.S.A. Superovulation with exogenous gonadotropins does not inhibit the luteinizing hormone surge Anna Glasier, M.D.* Samuel S.

1 FERTLTY AND STERLTY Copyright e 1988 The American Fertility Society Printed in U.S.A. Superovulation with exogenous gonadotropins does not inhibit the luteinizing hormone surge Anna Glasier, M.D.* Samuel S. Thatcher, Ph.D. E. Jean Wickings, Dr.rer.Medic Stephen G. Hillier, Ph.D. David T. Baird, D.Sc. Department of Obstetrics and Gynaecology, University of Edinburgh, Centre for Reproductive Biology, Edinburgh, Scotland The administration of human chorionic gonadotropin to women undergoing superovulation with exogenous gonadotropins was delayed in order to document the occurrence of a surge of luteinizing hormone (LH). An LH surge was seen to occur in 10 of 10 women receiving clomiphene citrate (CC) and pulsatile human menopausal gonadotropin (hmg); in 10 of 12 women treated with pulsatile hmg alone; and in 12 of 14 women treated with single daily injections of hmg without ce. The height of the surge was attenuated in all cycles and the timing of its onset was significantly earlier among women receiving single daily injections of hmg. Possible mechanisms for these findings are discussed. Fertil Steril 49:81, 1988 Superovulation with exogenous gonadotropins is reported to result in the failure of a luteinizing hormone (LH) surge in both monkeys1,2 and women.3,4 n most superovulation regimes, human chorionic gonadotropin (hcg) is given to stimulate oocyte maturation. The timing of hcg administration depends on predefined threshold levels of estradiol (E2) and the diameters of developing follicles' and precedes the estimated day of the spontaneous LH surge in most women. Withholding hcg, Messinis et al. 5 demonstrated the presence of an LH surge in 12 of 12 women treated with a combination of clomiphene citrate (CC) and human menopausal gonadotropin (hmg) administered in a pulsatile manner. A permissive role for CC and the pulsatile nature of hmg administration were both proposed as possible explanations for the presence of a surge in these women. We have treated three groups of women with pulsatile hmg, with and without CC, and with Received June 8,1987; revised and accepted October 13, * Reprint requests: Dr. Anna Glasier, Department of Obstetrics and Gynaecology, University of Edinburgh, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, Scotland. hmg alone given as a single daily injection to determine whether the development of an LH surge is facilitated by either the mode of administration of hmg or by the additional use of CC. Patients MATERALS AND METHODS Twenty-seven women (mean age, 30 years; range, 24 to 37 years) with regular spontaneous menstrual cycles undergoing in vitro fertilization (VF) and embryo transfer (ET), because of tubal infertility (n = 13) or a partner with oligozoospermia (n = 14), were treated in the following manner. Group A (Oligozoospermia) hmg 225 V/day as a single daily intramuscular injection was administered from day 3 of the cycle for 5 days. When urinary estrone excretion-measured in a single early morning urine specimen (EMV)-was >1 ~g/gm creatinine/24 hours, with the diameter of the leading follicle measuring more than 16 mm in ultrasound scan, 5000 V hcg was given intramuscularly. Laparoscopic oocyte recovery was performed 34 to 36 hours after hcg. Glasier et a. LH surge during superovulation 81

2 Group B (Tubal nfertility) Thirteen women were randomly allocated to one of two treatment schedules. Nine women completed both treatments. Schedule hmg 225 Vlday was given subcutaneously into the anterior abdominal wall by pulsatile infusion pumps starting on day 2 or 3 of the cycle. Pulse frequency was either one pulse every 2 hours (pulse amplitude, V hmg) or every 3 hours (pulse amplitude, 28.1 V hmg), depending on availability of pumps. f an LH surge had not been identified by day 17 or 18, 5000 V hcg was administered intramuscularly and hmg administration stopped. Laparoscopic oocyte recovery was carried out 32 hours after the start of a spontaneous LH surge or 34 to 36 hours after hcg. Schedule hmg was administered, hcg given, and laparoscopy performed in a manner identical to that in schedule, but all women received 150 mg CC orally for 5 days ffom the hmg start day. Monitoring The response to treatment was monitored by daily measurements of urinary estrone and pregnanediol excretion in early morning specimens of urine and results were corrected to reflect 24-hour values by determining the ratio to creatinine.7 Daily ultrasound scans were performed using a real-time sector scanning machine (3.5 MHz Diasonics DS-RF, BMS [Scotland] Ltd., Strathclyde) to determine follicle number and diameter. Daily venous blood samples were collected at 8:30 A.M. for LH estimation and sampling frequency was increased for the women in group B to 6 hourly when urinary estrone excretion exceeded 60 J,g/gm creatinine/24 hours or when the diameter of the largest follicle exceeded 16 mm. Serum was stored at -20 a C for later estimation of progesterone (P) and gonadotropins. The start of an LH surge was identified when the LH concentration in one blood sample showed an % increase relative to the mean value of the four previous samples. 8 The presence of a surge was confirmed by a combination of three criteria: (1) a continued rise in LH concentration; (2) a rise in urinary pregnanediol levels following the surge to a level of> 1 mglgm creatinine that was sustained for 82 Glasier et al. LH surge during superovulation more than 24 hours; and (3) a rise in serum P concentration to about 1.4 nglml (also sustained for more than 24 hours). Hormone Assays Serum LH concentrations were measured in a rapid radioimmunoassay (RA).9 The results were expressed in V of the standard preparation RP 68/40. The sensitivity of the assay was 2 V; the intra- and interassay variations were 2.9 and 12.7%, respectively. P concentrations in serum were measured by specific RA following extraction with petroleum ether (40 a C to 60 a C). The detection limit of the assay was nglml and the intra-assay variation was 10%; all samples were analyzed in the same assay.lo Vrinary estrone-3-g1ucuronide levels were measured by specific RA. The sensitivity of the assay was 6 ng; the intra- and interassay variations were 7.9 and 15.8%, respectively. Pregnanediol was measured in urine by gas chromatography following hydrolysis, extraction, and acetylation of the sampley Vrinary steroids were expressed as a ratio of the creatinine concentration, measured colourimetrically, in the EMV sample. Statistical Analysis Results were analyzed using Student's t-test for paired log-transformed data. RESULTS Group A (Oligospermia-Single Daily njections of Human Menopausal Gonadotropin) A spontaneous LH surge was detected in 12 of the 14 women in group A before the criteria for hcg administration was reached (Fig. 1A). The surge started at a mean of 7.9 ± 0.8 days (range, 5.0 to 10.0). The 2 remaining women received hcg on day 11 of the cycle. A mean peak value for the height of the surge cannot be calculated because of the infrequency of blood sampling at this time. Group B (Tubal Disease-Pulsatile Human Menopausal Gonadotropin with Clomiphene Citrate) A spontaneous LH surge was identified in all ten women; the onset was at a mean of 10.5 ± 1.1 days (range, 10 to 13) with a mean peak LH value of 25.0 Fertility and Sterility

3 rn'"n A ~~22!iUlllay CLollllpt\enll rna/day 350 B o-o/,-a 8,, 5 hmg2uulo'y 50 :::: 40 " ~ 30 '" \ a iii ii i i e DA'iS Of CYCLE Figure 1 (A) A spontaneous LH surge started on day 8 of the cycle in one woman treated with daily injections of hmg. Pregnanediol excretion rose accordingly on day 9, with the increase maintained. (B) An LH surge occurred on day 12 of the cycle in one woman treated with pulsatile hmg and CC. A rise in serum progesterone is seen on day 11. ± 5.0 lull (range, 15.7 to 62.6). Results from a single woman are shown in Figure 1B. Group B (Tubal Disease-Pulsatile Human Menopausal Gonadotropin Alone) A spontaneous LH surge was detected in 10 of the 12 women receiving pulsatile hmg without CC (Fig. 2B). The onset of the surge occurred at a mean of 9.2 ± 1.1 days (range, 8 to 14), with a mean peak LH value of 29.3 ± 6.0 lull (range, 13.4 to 47.3). Results from an individual woman in whom no surge occurred are shown in Figure 2A. There was no significant difference in either serum LH or follicle-stimulating hormone (FSH) concentrations over 5 days preceding the onset of the surge among the three treatment groups, although LH levels tended to be higher among women taking CC during the days of actual CC administration. During the 5 days before the LH surge, urinary estrone levels were, however, significantly higher among women receiving pulsatile hmg and CC than among those treated with pulsatile hmg alone (P < 0.05) or daily hmg injections (P < 0.02) (Fig. 3). Mean estrone concentrations before and after the surge and pregnandiol concentrations after the surge for all three groups are shown in Figure 3. Figure 2 (A) No increase in urinary pregnanediol/creatinine ratio ( ) is seen during 14 days of treatment with hmg alone until the patient is given 5000 U hcg (t) on days 16 to 17 of the cycle. (B) A spontaneous LH surge started on day 10 in one woman treated with pulsatile hmg alone. A rise in serum progesterone ( ) confirms the identification of the surge. The day of the surge was significantly earlier (P < 0.001) in the group of women treated with single daily injections of hmg when compared with women receiving pulsatile hmg with or without CC. Table 1 shows the day of the surge and mean peak value of LH for both groups of women, comparing the results with those obtained during previous studies in our program 5 of women treated A l 1/\ Y \ }/ \ ~3 0 \ 2 ~.oo ~J./\ '~ ;:: 200 / 0 ~ i:i q... q 1 > fi q"' ili :];"" U~i ~200 A-6 L~ S ~ i:::1~""~~ 200 ~ to o,r9-,,,, i DAYS RELATVE TO LH SURGE Figure 3 Mean ± SEM urinary estrone ~g/gm creatinine and urinary pregnanediol mg/gm creatinine around the LH surge in all three treatment groups. (A) Pulsatile hmg + CC. (B) Pulsatile hmg alone. (C) Daily hmg. Glasier et al. LH surge during superovulation 83

Table 1 Day of LH Surge and Mean Peak Value for Each Treatment Cycle Compared with Spontaneous Cycles Treatment LH surge/total women Day of onset Peak value Spontaneous cycles" CC onlyb CC +

4 Table 1 Day of LH Surge and Mean Peak Value for Each Treatment Cycle Compared with Spontaneous Cycles Treatment LH surge/total women Day of onset Peak value Spontaneous cycles" CC onlyb CC + pulsatile FSHh from day 6 CC + pulsatile hmg from day 6 CC + pulsatile hmg from day 2 Pulsatile hmg only from day 2 hmg only from day 2 (daily injection) 9/9 10/10 10/ ± ± 0.3 (NS) 13.2 ± 0.4 (NS) 12.8 ± 0.3' 10.5 ± 1.1 C 9.2 ± Ld 7.9 ± 0.8 e 58 ± 5 53 ± 6 NS 34 ± 4 d 28 ± 3 e 29.3 ± 5 e 25.0 ± 5 e paired t-test) 'P < 0.05; dp < 0.01; ep < NS, not significant. " Data from reference 12. b Data from reference 5. Significance of the difference with spontaneous cycles (unwith CC alone, CC and gonadotropins from day 6, and in spontaneous cycles.12 DSCUSSON There recently have been a number of reports at variance with the original observation that superovulation regimes using exogenous gonadotropins inhibit the onset of the LH surge5,13 even when administered without CC. 14,15 Even the Norfolk group that originally reported inhibition of the surge3.4 recently described the identification of a surge in up to 20% of women occurring "prematurely" (that is, before the administration of exogenous hcg).16 The fact that the height of the surge is always attenuated and may occur either very early in the cycle or else may not have occurred by the time of exogenous hcg administration suggests that, in earlier studies, the LH surge may have been simply missed rather than absent. The LH surge may have occurred earlier in the group treated with single daily injections of hmg as compared with pulsatile administration because injections were stopped routinely after 5 days of treatment, while pulsatile hmg was continued according to the patient's response. Messinis et al,5 suggested that either the administration of hmg in a pulsatile manner or the additional use of CC may facilitate the development of a surge. n this study, we have shown that a surge will occur in 22 of the 26 cycles without CC treatment, regardless of the mode of administration of gonadotropins. The administration of CC did, however, result in much higher levels of estrone excretion being reached before the onset of the LH surge. Despite this difference, there were no significant differences in either the number of follicles aspirated or the number of oocytes obtained among the three treatment groups. This observation supports that of Templeton et al,17 84 Glasier et al. LH surge during superovulation n a recent article, it was claimed that an LH surge failed to occur in five of ten women treated with "pure" FSH from day 2 of the cycle.18 However, an increase of LH concentration of over 100% on day 10 was observed in the five women who were designated as having failed to produce an LH surge, although there was no evidence of luteinization, as indicated by a subsequent rise in the concentration of progesterone. Thus, in all women treated with FSH, a discharge of LH occurred about day 10 at a time coincidental to the preovulatory LH surge. t was suggested that the smaller antral follicles (10 to 15 mm in diameter) secreted a substance(s) that drastically modified the magnitude of the midcycle LH surge. nhibin is secreted in large amounts by the developing follicles during cycles stimulated with gonadotropin.19,2o n large amounts, follicular fluid inhibits the secretion of LH and it may be that inhibin will delay the generation of an LH surge.21 t has been suggested that the preovulatory follicle(s) secretes another factor that prevents the positive feedback effect of estrogen.22,23 However, the nature of this factor and its possible role in the control of the LH surge in the normal cycle is obscure. Acknowledgments. We are grateful to the Chelsea Hospital for Women, London, UK, for supplying the reagents for the LH assays; to the National nstitute of Biological Standards and Control, London, UK, for the standard preparation for the LH assays; to Dr. Padmananda Samarajeewa, Ph.D., Department of Biochemistry, University College, London, UK, for supplying the reagents for the urinary estrone-3-glucuronide assays; to Sisters A. Michie and H. Hillier for management of patients; to Tom McFetters and Ten Pinner for illustrations; and to Margaret Harper for typing the manuscript. This work was supported, in part, by MRC Programme grant no. G REFERENCES 1. Shenken RS, Hodgen GD: Follicle stimulating hormone induced ovarian hyperstimulation in monkeys: blockade of Fertility and Sterility

5 the luteinizing hormone surge. J Clin Endocrinol Metab 57:50, Collins R, Williams RF, Hodgen GD: Endocrine consequences of prolonged ovarian hyperstimulation: hyperprolactinaemia, follicular atresia and premature luteinization. Fertil Steril 40:436, Ferraretti AP, Garcia JE, Acosta AA, Jones GS: Serum luteinizing hormone during ovulation induction with human menopausal gonadotropin for in vitro fertilization in normally menstruating women. Fertil Steril40:742, Garcia JE, Jones GS, Acosta AA, Wright G Jr: Human menopausal gonadotropin/human chorionic gonadotropin follicular maturation for oocyte aspiration: phase, Fertil Steril 39:167, Messinis E, Templeton AA, 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, 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, Metcalf MG, Livesey JH: Use of small samples of urine to monitor gonadotrophins in menopausal women. Clin Chim Acta 94:287, Testart J, Frydman R, Feinstein MC, Thebault A, Roger M, Scholler R: nterpretation of plasma luteinizing hormone assay for the collection of mature oocytes from women: definition of a luteinizing hormone surge-initiating rise. Fertil Steril 36:50, Djahanbakhch 0, McNeilly AS, Hobson BM, Templeton AA: A rapid method luteinizing hormone radioimmunoassay for the prediction of ovulation. Br J Obstet Gynaecol 88:1016, Backstrom CT, McNeilly AS, Leask RM, Baird DT. Pulsatile secretion of LH, FSH, prolactin, oestradiol and progesterone during the menstrual cycle. Clin Endocrinol 17:29, Chamberlain J, Contractor SF. A gas-liquid chromatographic method for the rapid estimation of pregnanediol and allopregnanediol in non-pregnancy urine. J Obstet Gynecol 101:649, Djahanbakhch 0, McNeilly, AS, Warner PM, Swanston la, Baird, DT. Changes in plasma levels of prolactin, in relation to those of FSH, oestradiol, androstenedione and proges- terone around the preovulatory surge of LH in women. J Clin Endocrinol 20:463, Navot D, Margalioth EJ, Laufer N, Mor-Josef S, Schenker JG. Asynchronous ovulation in human menopausal gonadotropin induction of ovulation for in vitro fertilization. Fertil Steril 42:6, 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, Messinis E, Templeton AA, Baird DT: Endogenous luteinizing hormone surge in women during induction of multiple follicular development with pulsatile follicle stimulating hormone. Clin Endocrino24:193, Van Uem JFHM, Garcia JE, Liu HC, Rosenwaks Z: Clinical aspects with regard to the occurrence of an endogenous LH surge in gonadotropin-induced normal menstrual cycles. J n Vitro Fert Embryo Transfer 3:345, Templeton AA, Messinis E, Baird DT: Characteristics of ovarian follicles in spontaneous and stimulated cycles in which there was an endogenous luteinizing hormone surge. Fertil Steril 46:1113, Messinis E, Templeton AA: Endocrine and follicle characteristics of cycles with and without endogenous luteinizing hormone surges during superovulation induction with pulsatile follicle-stimulating hormone. Hum Reprod 2:11, McLachlan R, Robertson DM, Healy DL, dekretser DM, Burger HG: Plasma inhibin levels during gonadotropin-induced ovarian hyperstimulation for VF: a new index of follicular function? Lancet 1:1233, Tsonis CG, Messinis E, Templeton AA, McNeilly AS, Baird DT: Gonadotropic stimulation of inhibin secretion into peripheral blood by the human ovary during the follicular and early luteal phase of the cycle. J Endocrinol Metab. n press 21. Stillman RJ, Williams RF, Lynch A, Hodgen GD: Selective inhibition of follicle stimulating hormone by porcine follicular fluid extracts in the monkey: effects on midcycle surges and pulsatile secretion. Fertil Steril 40:823, Retton V, Siler-Khodr TM, Paverstein CJ, Smith CG, Asch RH: Effects of porcine follicular fluid on gonadotropin concentrations in rhesus monkeys. J Clin Endocrinol Metab 54:500, Littman BA, Hodgen GD: Human menopausal gonadotropin stimulation in monkeys: blockade of the luteinizing hormone surge by a highly transient ovarian factor. Fertil Steril 41:440, 1984 Glasier et al. LH surge during superovulation 85

I.E.Messinis 1,4, S.Milingos 1, K.Zikopoulos 2, G.Hasiotis 3, K.Seferiadis 3 and D.Lolis 2

I.E.Messinis 1,4, S.Milingos 1, K.Zikopoulos 2, G.Hasiotis 3, K.Seferiadis 3 and D.Lolis 2 Human Reproduction vol.13 no.9 pp.2415 2420, 1998 Luteinizing hormone response to gonadotrophinreleasing hormone in normal women undergoing ovulation induction with urinary or recombinant follicle stimulating