Patterns of serum-luteinizing hormone surges in stimulated cycles in relation to injections of human chorionic gonadotropin*

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1 FERTILITY AND STERILITY Copyright ov 1990 The American Fertility Society Printed on acid-free paper in U.S.A. Patterns of serum-luteinizing hormone surges in stimulated cycles in relation to injections of human chorionic gonadotropin* Vera Baukloh, M.S. t Robert Fischer, M.D. OlafNaether, M.D. Heinz-G. Bohnet, M.D. Institute for Hormone and Fertility Research, Hamburg, Federal Republic of Germany Endogenous-luteinizing hormone (LH) surges may complicate the management of in vitro fertilization cycles. To investigate the effects of LH surges after hormonal stimulation 53 IVF cycles were analyzed by assessing LH levels three times daily until egg collection. In 43% the LH rise started before the planned exogenous trigger for ovulation was given, in 11% the rise occurred simultaneously with and in 45% after the injection of human chorionic gonadotropin. Three main patterns of serum LH surges were identified: (A) low-lh tonus with straight increase to maximum; (B) low tonus with elevation before straight increase; (C) high tonus with large variations but no prominant peak. These patterns were not related to the follicular estradiol increase, luteal steroid concentrations or resulting pregnancy rates. Fertil Steril53:69, 1990 The luteinizing hormone (LH) surge occurring in the natural cycle is the trigger for the final maturation of the metaphase II oocyte and for the initiation of follicular rupture. It occurs. after a sustained rise in estradiol (E 2) levels, with a slight decrease thereafter just before or concomitently with the onset of the LH surge. 1 This pattern has also been observed in gonadotropin-induced cycles, 2 3 although the LH surge is markedly attenuated. Several authors 4-6 have observed lower pregnancy rates after in vitro fertilization (IVF) and embryo transfer (ET) procedures in cases where an endogenous LH discharge was identified as compared with human chorionic gonadotropin (hcg) triggered cycles. This led to the policy of cancelling treat- Received March 13, 1989; revised and accepted August 24, *Supported by Serono Diagnostics, Freiburg, FRG. t Reprint requests: V. Baukloh, Institute for Hormone and Fertility Disorders, Lornsenstrasse 4, D-2000 Hamburg, Federal Republic of Germany. ment cycles with endogenous LH surges before hcg injection in many IVF groups. Luteinizing hormone has been shown to accelerate the final oocyte maturation in vitro by approximately one-half hour. 7 However, oocytes which had not been exposed to LH or to hcg have been reported by Templeton et al. 8 to show normal fertilization rates. Spontaneous LH surges occur in approximately 20% of cycles stimulated by clomiphene citrate (CC) plus human menopausal gonadotropin (hmg) The availability of monoclonal antibodies specific for LH enables distinction between endogenous LH and hcg administered for timed oocyte retrieval. We have therefore used a monoclonal antibody based radio-immuno assay (RIA) to determine serum LH profiles in IVF patients during CC plus hmg-treated cycles and have assessed the relationship between endogenous LH surges and the success rates of the therapy. MATERIALS AND METHODS From October 1987 to June 1988 a total of 151 patients were subjected to CC plus hmg stimula- Baukloh et al. Patterns of serum LH surges 69

2 tion for IVF therapy. In these 64 endogenous LH surges were observed, 30 occurred before the injection of hcg, and 34 at the same time as or after hcg administration. In 53 of these patients who completed their IVF cycle up to ET, serum LH concentrations were closely monitored around the time of expected LH surge (day 10 to 14 of the cycle). Blood samples were taken in the morning (7 A.M. to 8 A.M.), early afternoon (1 P.M. to 2 P.M.), and evening (10 P.M. to 12 P.M.) until the day of the follicular puncture. Luteinizing hormone concentrations were determined by RIA (LH Maiaclone, Serono Diagnostics, Freiburg, FRG). The interassay and intra-assay coefficients of variation were 7.7% and 5.0%, respectively, the lower limit of detection was 0.3 miu /ml. The assay exhibits very low cross-reactivity with hcg ( % at 5 IU/mL hcg). This high specificity allows the measurement of LH even after the injection of hcg for ovulation induction. The stimulation protocol was as follows: CC (Dyneric, Merrell Dow, Riisselsheim, FRG) 100 mg/d from day 2 of the cycle until day 6, hmg 150 IU per day (Pergonal, Serono) from day 5 until a sufficient ovarian response was achieved (at least three developing follicles~ 16 mm in diameter, and E2 at least 300 pg/ml per follicle ~ 16 mm) with daily monitoring starting on cycle day 8. In cases where hcg was injected before an LH surge oocyte collection was performed 36 hours later. If an endogenous LH surge was detected before the planned hcg injection follicular puncture was performed 30 hours after the first significant rise in serum LH levels. The onset of the LH surge was defined as the first point at which the serum LH exceeded the baseline level by at least two standard deviations. When an endogenous LH rise was observed an additional dose of 5000 IU hcg was given as soon as the surge onset was verified (2 to 10 hours after the first positive serum sample). Embryo transfer was performed usually 2 days after the oocyte pick-up when four-cell-stages were noted. At this time as well as 4 and 8 days later serum samples were obtained for measurements of E 2 and progesterone (P) to assess the quality of the luteal phases. For the purpose of this study the data were evaluated according to three different criteria: I. Time relationship between the onset of the LH surge and hcg injection: LH surge starts 1. before, 2. simultaneously with, or 3. after hcg injection. II. Form of the LH surge: A1 rapid increase with maximum/tonus> 4, A2 rapid increase with maximum/tonus < 4, B1 small LH elevation over tonus for some hours before further rapid increase, B2 blunted LH peak with return to baseline before start of rapid increase, C high variation of LH tonus with only one blunted peak. III. Type of follicular E2 curve: Plateau increase 20% to 30%, leap increase 50% to 60%, and steady increase 40% to 50% on the day of LH surge. The definition of E2 response patterns was based on the criteria proposed by Nader et al. 12 who described two different E2 patterns associated with an endogenous LH surge (plateau and leap type). Statistics The data were evaluated by comparison of group means by analysis of variance (ANOVA) and by x2 tests fo:r: frequency analysis where appropriate. Steroid concentrations E2 and P were log-transformed13 before ANOV A. RESULTS Of the 151 patients treated during the study period October 1987 to June 1988, 13% showed no LH increase during CC plus hmg stimulation (Fig. 1), the remainder had LH surges before (20%), concomitant with (23%), or after (44%), the hcg administration. The pregnancy rates in these four groups were not significantly different (25%, 23%, 18%, and 25% of egg collections). The lengths of the follicular phases of the first two groups (surge before hcg injection, and surge simultaneously with hcg injection, days) were significantly shorter than in the others (no increase, and surge after hcg injection, days; P < ). In the 53 cycles studied in detail in this investigation the same tendencies for follicular phase lengths were observed but the differences were not statistically significant. In the majority of cases (73%) the initiation of the LH peak was detected in the morning samples. In these cycles the onset of the LH surge had occurred during late night/early morning (between 10 P.M. and 7:30 A.M.). In only 10% of the cases the surge started later in the morning between 7:30 A.M. and 1:30 P.M., and in 17% in the afternoon to early evening hours. During the study period (October 1987 to June 1988) no clear-cut seasonal changes in the daytime 70 Baukloh et al. Patterns of serum LH surges Fertility and Sterility

3 ~ Ill II Ill IG (,)... :l c 20 0 ;: :I.. ~ Ill 10 i5 :l 11.. (Averige cycle diy of folliculir puncture) Cycle day of follicular puncture LH surge. no increase (n 20) before HCG Injection (n 30) simultaneously to HCG Injection ( n 39) after HCG injection (n 67l Figure 1 Distribution of endogeneous LH surges and average follicular phase length in 151 clomiphene plus hmg-stimulated cycles between October 1987 and June , number of pregnancies; D, average cycle day of follicular puncture. of LH surge onset occurred. However, during the spring months (March to May 1988), 95% of the LH elevations were noted in the early morning hours while during the other months this proportion was significantly lower (61 %; x 2 = , P < 0.025). The subclassification according to the time relationship between hcg injection and onset of the LH surge (criterium I) revealed pregnancy rates not statistically different between subgroups (17%, 17% and 42%, x 2 = , P > 0.1). The same was true for subgroups of different LH patterns (criterium Il-pregnancy rates: 32%, 18%, 0%, 22%, and 100%), and for those of different follicular E2 curves (criterium III-pregnancy rates: 30%, 31%, and21%). LH Parameters The time between the onset of the LH surge and hcg injection was significantly different for subgroups in all three criteria but the time between the LH surge onset and occurrence of the maximum value only differed significantly in I (time relationship between LH surge onset and hcg injection) and II (type of LH surge, Table 1) as did the periods until follicular puncture and insemination of obtained oocytes. The LH tonus and variation (standard deviation [SD]) as well as the maximum value were similar between subgroups of criterium I and III. As expected by the definition of subgroups in II (form of LH surge) all LH parameters were significantly different between groups (Fig. 2A to E). The LH tonus was higher in subgroups A2, B2, and C than in A1 and B1 while the highest variation in LH concentrations was also seen in C. The time between the onset of the LH surge and the occurrence of the maximum value was similar in subgroups B1, B2, and C (22.2, 20.2, and 18.8 hours) and significantly higher than in subgroups A1 and A2 (9.9 and 10.4 hours). Length of Follicular Phases There were no significant differences between the length of follicular phases for any of the criteria analysed. The day on which follicular puncture was performed varied from cycle day 11 to 15 in all subgroups. The evaluation of the follicular E 2 response revealed significant differences up to the day of the LH surge onset between subgroups according to criterium I (Table 2). The E 2 levels for all follicular cycle days were highest in subgroup 3 (LH surge after hcg injection) and lowest in subgroup 2 (LH surge simultaneous with hcg) although the E 2 increase paralleled those seen in subgroup 1 (LH surge onset before hcg). In the subgroups analysed by the type of E 2 response (criterium III) no significant differences in follicular E 2 concentrations were revealed for any day of the cycle. Between subgroups according to criterium II (form oflh surge) significant differences were evident for E 2 levels at all cycle days (Fig. 2A to E). Lowest E 2 levels were seen in subgroups B1 and B2 with a steep increase early in the cycle flattening after the LH surge (Fig. 2C and D). The highest E 2 levels throughout the follicular phase occurred in subgroup C with a steep increase until the LH surge and a slight decrease thereafter. Subgroups A1 and A2 showed intermediate E 2 levels with a flattening curve from the day before the LH surge Baukloh et al. Patterns of serum LH surges 71

4 Table 1 Time Relationships Between Onset of LH Surge and HCG Injection, LH Maximum, Follicular Puncture, and Insemination of Obtained Oocytes in Subgroup II (Type oflh Surge)a Time until A1 A2 (hours) (n = 25) (n = 11) B1 B2 c (n = 5) (n = 9) (n = 3) F HCG injection 3.26 ± ± 4.09 LHmaximum 9.92 ± ± 3.11 Follicular puncture ± ± 2.10 Insemination of oocytes ± ± ± ± ± P< ± ± ± P< ± ± ± P< ± ± ± P<0.005 a Values are means± standard error. onset to the day of LH surge and a further increase thereafter. Number of Obtained Oocytes, Fertilization, and Embryo Transfer Rates No significant differences were observed between any of the subgroups when the number of oocytes obtained, and the rates of fertilization and ETs were compared (data not shown). Five to eight oocytes were inseminated and 43% to 71% of these were transferred as embryos. Likewise there were no differences in the number of transfers with one or two versus transfers with three or more embryos between subgroups of any of the analyzed criteria. Similarly the cell stages observed at the time of ET did not differ between subgroups. Luteal Phases Luteal phase E 2 as well as P concentrations on the day of ET and 4 and 8 days later were not significantly different in subgroupings I (timing oflh surge onset) and III (follicular E 2 increase type). Between subgroups of criterium II (type of LH surge) also no significant differences were seen in luteal phase steroid concentrations. However, the highest concentrations for both E 2 and P were observed in C (E pg/ml, P 139 ng/ml on day 8) and A2 (E pg/ml, P 97 ng/ml on day 8) »... ajatlft 25l LH-max*, """! ~ b ~ ~ ~ Cycle d~y r!(ahye to onset of LH s~rge L.J ~. ' -~ =11-. l ;l ' t)lll~~osi t<0.05 pc0.01 -l l Figure 2 Follicular E 2 and LH levels in five subgroups according to the type of LH surge. A1 rapid increase, maximum/ tonus > 4; A2, rapid increase, maximum/tonus < 4; B1, small LH elevation over tonus before further rapid increase; B2, blunted LH peak, return to baseline before start of rapid in '!rease; C, high variation of LH tonus, only one blunted peak. Comparison of Conception Cycles with those not Resulting in Pregnancy The cycles resulting in pregnancies showed no difference to the "unsuccessful" cycles with regard to follicular or luteal steroid levels as well as follicular serum LH concentrations. A striking difference between the two groups was seen in the time period between the onset of the LH surge and the time of oocyte insemination. This interval was significantly shorter (Fig. 3, F = , P < 0.025) in 12 cycles with intact pregnancies (40.74 hours) and conceptions resulting in early abortions (36.03 hours) than in the 38 cycles not resulting in pregnancy (47.88 hours). The number of embryos transferred was significantly higher in conception cycles (4.2 ± 0.35) than in "no pregnancy" cycles (2.9 ± 0.26; t = , P < 0.01) although the number of obtained oocytes was similar (5.93 ± 0.52 and 5.87 ± 0.37, respectively). Likewise the number of transfers with three or more embryos was higher in the former group (13 of 15 transfers) as compared with the un- 72 Baukloh et al. Patterns of serum LH surges Fertility and Sterility

5 Table 2 Follicular E 2 Response in Subgroup I (Time Relationship Between Onset of LH Surge and HCG Injection) Relative to the Day of LH Surge Onset a Cycle day< LH surgeb (8) 624 (16) 882 (22) 1324 (23) 1859 (23) 2212 (19) (3) 743 (6) 1204 (6) 1386 (6) 2060 (6) (19) 890 (24) 1294 (24) 1719 (24) 2313 (24) F p <0.001 <0.001 <0.001 a Values are geometric means; values in parentheses are number of measurements. b LH surge: 1, before; 2, simultaneously with; 3, after hcg injection <0.050 <0.050 c Cycle day 0 = day of LH surge onset. d NS, not significant NSd successful cycles (21 of 38 cycles) but this difference did not reach statistical significance. DISCUSSION High tonic urinary LH levels during the follicular phase of stimulated cycles have been associated with a reduced chance of pregnancy after IVF.14 This was not verified for serum LH levels in the present study because there were no differences in the LH tonus of pregnancy and no pregnancy cycles after IVF. However, the same group ofinvestigators10 reported an LH tonus of 9.9 ± 1.0 miu /ml in serum of their seven patients treated by the standard stimulation regimen. The highest LH tonus observed in our study was 8.2 miu /ml (mean ± SD: 3.6 ± 1.4 miu /ml) that is still below the level observed in their study. The occurrence of an endogenous LH surge as observed in 64 IVF cycles after CC plus hmg stimulation in this study represented a follicular phase l1meunt11 lh mu1mum tontcular puncture (onsetollhsurge) lniiciiiflgllllltflft t2) o--olboltlollll"'traut l ln ll -I'IOIIfligiiiiiCY(II ll) Figure 3 Intervals between onset of LH surge and time of hcg injection, time until LH maximum, time of follicular puncture and insemination of oocytes in conception and "unsuccessful" IVF cycles. 1 day shorter than expected in comparison with 87 cycles in which ovulation was triggered by hcg injection (Fig. 1). This shorter period of follicular maturation obviously had no detrimental effect on oocyte quality since the pregnancy rates were not different between. exogenously (hcg) and endogenously (LH) triggered cycles. In vitro fertilization cycles resulting in pregnancy were not managed differently from other cycles until oocyte recovery (Fig. 3). Oocytes finally establishing a conception were inseminated about 8 hours earlier in relation to the LH surge onset than oocytes not leading to a pregnancy. Therefore the time oocytes are allowed to mature in vitro in relation to the onset of the LH surge may be more decisive for their fertilizability than the period they are exposed to LH in vivo. The low pregnancy rates in cycles with an endogenous LH discharge reported by other authors4-6 may have been because of problems in the timing ofthe onset of the LH surge. This might be caused by different definitions of a significant LH rise. If LH levels are not determined frequently enough small elevations may be missed resulting in luteinized follicles and overmature oocytes at the time of recovery. Basically three different types of serum LH surges were identified in the 53 cases studied in detail around the time of the expected surge onset until follicular puncture. These subgroups differed in the time periods between onset of the LH surge and the hcg injection, time until the maximum LH value was observed, time until follicular puncture and insemination of oocytes (Table 1). The characteristics of these subgroups were: (A) (A1 and A2), low LH tonus with straight increase after surge onset; (B) (B1 and B2), low tonus with small but significant LH elevation before the start of the real Baukloh et al. Patterns of serum LH surges 73

6 peak; (C), relatively high tonus with large variations but no prominant peak. Nevertheless, these parameters had no influence on the pregnancy rates that were not statistically different between the subgroups. These findings are in good agreement with the results of Macnamee et al.10 who also found three principal types of LH secretion in urine: the biphasic type (corresponding to B2), the plateau type (corresponding to B1), and the sharp rise type (corresponding to A1 and A2). Zech and colleagues15 identified six different patterns of urinary LH in their 55 patients stimulated with CC plus follicle-stimulating hormone (FSH) and reported pregnancies only in cases where there was an LH surge registered either before or after the hcg injection. Interestingly they had no pregnancy in cases with very low, very high, or strongly fluctuating urinary LH levels in which there was also no LH surge observed. This last category seems to correspond with our subgroup C with only blunted serum LH peak and no further increase but in all the three cases of this subgroup pregnancy was established. Although urinary LH levels were not recorded in our study it appears from these findings that serum and urinary LH concentrations are not well correlated in CC plus hmg-stimulated cycles. The follicular E2 concentrations were significantly different in the subgroups of different serum LH patterns (Fig. 2) for any cycle day but not between subgroups divided according to the E2 increase on the day of LH surge onset (criterium III). Therefore it is not the E2 concentration16 but rather the combination of follicle number and growth rate17 in relation to the dynamics of E 2 increase2 12 which seems to be associated with the type of LH surge in CC plus hmg-stimulated cycles. In the present study the plateau type of follicular E2 response was associated in 61% with the A type, in 26% with the B type of LH surge, and all C type LH patterns occurred in this category. Both the leap and steady increase E2 types had appro ximately 75% type A and 25% type B LH surges. The trigger for ovulation by hcg injection may have accelerated the secretion of LH because the maximum value after onset of the LH surge was already observed 7.6 hours later whereas the differences between LH surge onset and maximum were 19.6 hours and 13.9 hours in the other subgroups (data not shown). No significant differences were identified in the luteal phases of LH subgroups stressing the fact that LH secretion does not seem to have a decisive effect after ovulation.18 The important function of LH is to initiate the resumption of oocyte meiosis at the end of the follicular phase. This may be prematurely triggered by high tonic levels or small elevations before the definite onset of the surge14 19 as suggested by subgroup B (B1 and B2) in the present study. Therefore LH concentrations must be closely monitored throughout the follicular phase in every stimulation regimen that does not involve suppression of LH secretion by gonadotropin-releasing hormone analogues.10 An LH surge occurs in the majority of CC plus hmg-stimulated cycles with about 45% starting only after the injection of hcg. Because the type of the LH surge has no effect on the probability of conception the precise identification of the surge onset, if occurring before hcg triggering, is sufficient to allow proper timing of follicular puncture and insemination of oocytes. REFERENCES 1. 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. 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