Prevalence of an endogenous LH rise following final GnRH antagonist dose in the IVF antagonist protocol.

K. Løssl: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. N. Friis Wang: Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. J.W. Bogstad: Writing – review & editing, Supervision, Project administration, Investigation. T.V. Dam: Writing – review & editing, Project administration, Methodology, Investigation. N. Bülow: Writing – review & editing, Project administration, Methodology, Investigation. J.A. Bøtkjær: Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation. M.R. Petersen: Writing – review & editing, Validation, Supervision, Methodology, Investigation. L.B. Dessing: Writing – review & editing, Resources, Project administration, Methodology, Investigation. B. Oxlund-Mariegaard: Writing – review & editing, Project administration, Methodology, Investigation. M.M. Celicanin: Writing – review & editing, Resources, Project administration, Methodology, Investigation. A. Pinborg: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization. L.S. Mamsen: Writing – review & editing, Resources, Project administration, Methodology, Conceptualization. L.C. Poulsen: Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation. C. Yding Andersen: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

This work was supported by an unrestricted grant of 150,000 DKK from 10.13039/501100003358 Gedeon Richter , which was used to cover study costs. The funder was not involved in the design, execution, or interpretation of the study.

A total of 203 women were assessed for eligibility, of whom 115 agreed to participate and 100 completed the study between January 2023 and June 2024. A total of 15 women were excluded for the following reasons: use of urinary-derived gonadotropins (n = 6), GnRHa triggering (n = 3), logistical issues (n = 3), lack of follicle development (n = 1), spontaneous pregnancy (n = 1), and medication administered incorrectly (n = 1). Overall, 17% (17/100) of the included women had an LH rise > 10 IU/L from the day of hCG trigger until OPU. Baseline characteristics were similar in the two groups ( Table 1 ). When the GnRH-ant was initiated on stimulation day 5, none of the women (0/22) had an LH rise > 10 IU/L. When the GnRH-ant was initiated on stimulation day 6 and the last dose was administered at 8 am on trigger day, 13% (7/56) of patients had an LH rise. In contrast, when the GnRH-ant was initiated on stimulation day 6 at 10 pm, with last dose given in the evening before the trigger day, 45% (10/22) of patients had an LH rise. All 17 women with an LH rise had the rise detected in the blood sample drawn on the day after hCG trigger (range 10.2 – 32.6 IU/L). Out of the 17 cases two also had an LH level > 10 IU/L on trigger day and three on the day of OPU. None of the women had LH levels > 10 IU/L in the FF collected at OPU, but the LH concentration was significantly higher in the group of women with an LH rise (median (IQR) 3.36 (2.30–3.85) vs. 1.0 (1.0–1.33) IU/L, p = <0.001). Four patients received Pergoveris; in these patients, the LH concentration on trigger day was 2.7 IU/L (IQR 2.0–4.6), compared with 1.5 IU/L (IQR 0.8–2.4) in patients treated with rFSH (n = 96). This difference was not statistically significant (p = 0.602). Table 1 Baseline characteristics. Endogenous LH rise (n = 17) No LH rise (n = 83) P-value Age at inclusion, median (IQR) 36.0 (34.0–37.0) 34.0 (31.0–37.0) 0.166 Weight (kg), median (IQR) 68.0 (63.0–74.5) 64.07.8 (59.0–74.0) 0.466 BMI (kg/m 2 ), median (IQR) 22.7 (20.8–26.2) 23.5 (21.1–27.5) 0.509 Duration of infertility, months, median (IQR) 18.0 (12.0–24.0) 24.0 (18.0–30.0) 0.07 AMH (pmol/L), median (IQR) 11.0 (6.0–22.0) 14.0 (9.3–21.0) 0.465 Cycle length (days), median (IQR) 27.0 (26.0–28.0) 28.0 (27.0–30.0) 0.058 Previous IVF/ICSI treatments, median (IQR) 0.0 (0.0–0.0) 0.0 (0.0–1.0) 0.557 Primary cause of infertility, n (%) 0.129 Male factor Female factor No male partner Unexplained 6/17 (35%) 4/17 (24%) 6/17 (35%) 1/17 (6%) 36/83 (43%) 14/83 (17%) 13/83 (16%) 20/83 (24%) LH rise is defined as a plasma LH level > 10 IU/L measured from day of ovulation trigger until oocyte retrieval. BMI: Body-mass index, AMH: Anti-Müllerian hormone Baseline characteristics. LH rise is defined as a plasma LH level > 10 IU/L measured from day of ovulation trigger until oocyte retrieval. BMI: Body-mass index, AMH: Anti-Müllerian hormone The group of women with endogenous LH rise was characterized by a significantly shorter duration of stimulation (median (IQR) 8.0 (7.0–9.0) vs. 9.0 (8.0–10.0) days, p = 0.008), and a shorter duration of GnRH-ant administration (4.0 (3.0–4.0) vs. 5.0 (4.0–6.0) days, p = <0.001) ( Table 2 ). Further, women with LH rise had significantly fewer oocytes retrieved, but in contrast, showed a significantly higher fertilization rate, and a higher rate of day 2 embryos per retrieved oocyte, while the total number of blastocysts per oocyte retrieved did not differ significantly ( Table 2 ). A total of 63 patients had a fresh embryo transfer with either a day 2 embryo (n = 8) or a day 5 blastocyst (n = 55), while 14 patients had elective freeze-all due to no blastocyst development before day 6 (n = 5), OHSS risk (n = 7), endometriosis (n = 1), and suspicion of endometrial asynchrony (n = 1). 27 patients had no blastocyst suitable for transfer and/or freezing. Pregnancy rates did not differ significantly between groups ( Table 2 ). Table 2 Cycle characteristics and cycle outcomes. Endogenous LH rise (n = 17) No LH rise (n = 83) P-value Antral follicle count, median (IQR) 10 (7.0–18.0) 14.5 (10.0–20.0) 0.180 Gonadotropin (rFSH) starting dose (IU), median (IQR) 225.0 (150.-300.0) 225.0 (162.5–300.0) 0.542 Total gonadotropin dose (IU), median (IQR) 1500.0 (1200.0–2400.0) 2000.0 (1445.0–2550.0) 0.099 Duration of stimulation, days, median (IQR) 8.0 (7.0–9.0) 9.0 (8.0–10.0); 0.008 Number of days with GnRH antagonist, median (IQR) 4 (3.0–4.0) 5.0 (4.0–6.0) < 0.001 Oocytes, median (IQR) 5.0 (3.0–7.0) 8.0 (5.0–10.0) 0.005 2PN oocytes, median (IQR) 4.0- (2.0–6.0) 3.0 (2.0–6.0) 0.637 Embryos on D2, median (IQR) 3.0 (2.0–5.0) 4.0 (2.0–6.0) 0.757 Blastocysts on D5, median (IQR) 2.0 (1.0–3.0) 2.0 (1.0–4.0) 0.529 Total number blastocysts, median (IQR) 2.0 (1.0–3.0) 2.0 (0.5–4.0) 0.937 No. of fertilised oocytes per retrieved oocyte (2PN rate), median (IQR) 0.7 (0.6–0.8) 0.5 (0.3–0.7) 0.020 No. of day 2 embryos per retrieved oocyte, median (IQR) 0.7 (0.6–0.9) 0.5 (0.3–0.7) 0.013 No. of blastocysts per retrieved oocyte, median (IQR) 0.3 (0.1–0.5) 0.3 (0.2–0.5) 0.127 Patients with no oocytes retrieved, n (%) 0 (0%) 0 (0%) NA Patients with no 2PN oocytes, n (%) 0 (0%) 8/83 (10%) 0.182 Patients with fresh transfer, n (%) Cleavage stage embryo, n (%) Blastocyst, n (%) 13/17 (76%) 4/17 (24%) 9/17 (53%) 50/83 (60%) 4/83 (5%) 46/83 (55%) 0.207 Patients with a first frozen embryo transfer*, n (%) 1/17 (6%) 13/83 (16%) 0.290 Positive hCG**, n (%) Positive hCG per transfer, n (%) 8/17 (47%) 8/14 (57%) 42/83 (51%) 42/63 (67%) 0.790 0.499 Clinical pregnancy, n (%) Clinical pregnancy per transfer, n (%) 6/17 (35%) 6/14 (43%) 32/83 (39%) 32/63 (51%) 0.801 0.591 LH rise is defined as a plasma LH level > 10 IU/L measured from day of hCG trigger until oocyte pick-up. 2PN: Two-pronuclear, D2: Day 2 after oocyte pick-up, D5: Day 5 after oocyte pick-up, Fresh embryo transfer: embryo transfer in the IVF/ICSI cycle with ovarian stimulation. A good quality day 2 embryo is defined as: 2–6 cells, < 20% fragmentation, cell size according to cell number, no known multinucleation. A good quality blastocyst: ≥ 3BB day 5 or ≥ 4BB day 6. A clinical pregnancy was defined as a pregnancy documented by ultrasound showing a gestational sac in the uterus. *A total of 14 patients had elective freeze-all due to OHSS risk (n = 7), day 6 blastocyst(s) only (n = 5), endometriosis (n = 1), and suspicion of premature luteinization (n = 1). **Positive hCG was defined as hCG > 3 IU/L. 1fullfilling the clinics criteria for transfer or vitrification. Cycle characteristics and cycle outcomes. LH rise is defined as a plasma LH level > 10 IU/L measured from day of hCG trigger until oocyte pick-up. 2PN: Two-pronuclear, D2: Day 2 after oocyte pick-up, D5: Day 5 after oocyte pick-up, Fresh embryo transfer: embryo transfer in the IVF/ICSI cycle with ovarian stimulation. A good quality day 2 embryo is defined as: 2–6 cells, < 20% fragmentation, cell size according to cell number, no known multinucleation. A good quality blastocyst: ≥ 3BB day 5 or ≥ 4BB day 6. A clinical pregnancy was defined as a pregnancy documented by ultrasound showing a gestational sac in the uterus. *A total of 14 patients had elective freeze-all due to OHSS risk (n = 7), day 6 blastocyst(s) only (n = 5), endometriosis (n = 1), and suspicion of premature luteinization (n = 1). **Positive hCG was defined as hCG > 3 IU/L. 1fullfilling the clinics criteria for transfer or vitrification. Plasma progesterone concentrations on the day of hCG trigger, the day after trigger, and the day of OPU were significantly lower in the group of women with endogenous LH rise ( Table 3 ). A significant inverse correlation between plasma progesterone and LH was present on the day after hCG trigger (ρ = −0.604, p < 0.001) ( Fig. 1 ). Spearman correlation analyses were also performed to evaluate associations between progesterone on trigger+ 1 and stimulation parameters. Progesterone on trigger+ 1 was positively correlated with stimulation duration (ρ = 0.40, p < 0.001), total antagonist days (ρ = 0.39, p < 0.001), and follicle counts ≥ 10 mm (ρ = 0.35, p < 0.001), ≥ 14 mm (ρ = 0.34, p < 0.001), and ≥ 16 mm (ρ = 0.21, p = 0.043). In the subgroup of patients scanned on trigger day, similar positive associations were observed for follicle counts ≥ 10 mm (ρ = 0.41, p = 0.004), ≥ 14 mm (ρ = 0.43, p = 0.002), and ≥ 16 mm (ρ = 0.34, p = 0.017). Furthermore, progesterone on trigger+ 1 was correlated with progesterone trigger day (r = 0.60, p < 0.001). Table 3 Plasma hormone concentrations from trigger day until day of OPU, and follicular fluid LH level on the day of OPU. Endogenous LH rise (n = 17) No LH rise (n = 83) P-value Plasma, trigger day Estradiol (nmol/L) Progesterone (nmol/L) FSH (IU/L) LH (IU/L) 4.8 (3.0–6.1) 1.0 (0.8–1.2) 14.6 (10.1–19.7) 3.2 (2.1–6.6) 5.4 (3.6–7.3) 2.3 (1.4–3.4) 15.1 (11.3–19.9) 1.3 (1.0–2.2) 0.185 < 0.001 0.799 < 0.001 Plasma, trigger day + 1 day Estradiol (nmol/L) Progesterone (nmol/L) FSH (IU/L) LH (IU/L) 4.7 (3.3–8.1) 8.3 (5.0–9.4) 12.3 (9.8–16.5) 13.1 (11.5–20.3) 7.4 (5.4–9.2) 14.2 (10.2–20.0) 11.7 (9.0–15.6) 3.1 (1.8–4.8) 0.024 < 0.001 0.506 < 0.001 Plasma, day of OPU Estradiol (nmol/L) Progesterone (nmol/L) FSH (IU/L) LH (IU/L) 2.5 (1.7–3.3) 10.9 (9.4–16.8) 9.6 (6.1–11.0) 5.9 (4.0–8.3) 2.8 (1.9–3.8) 22.1 (16.1–29.4) 7.6 (6.2–10.8) 1.5 (1.0–2.6) 0.404 < 0.001 0.580 < 0.001 Follicular fluid 1 , day of OPU LH (IU/L) 3.4 (2.3–3.9) 1.0 (1.0–1.33)  10 IU/L measured from day of ovulation trigger until oocyte pick-up. OPU: oocyte pick-up. LH: luteinizing hormone, FSH: follicle stimulating hormone. Fig. 1 Correlation between plasma progesterone and LH on the day after hCG trigger. Legend: Scatter plot showing the relationship between plasma progesterone (nmol/L) and LH (IU/L) on the day after hCG trigger. Each point represents an individual patient. A significant inverse correlation was observed (Spearman’s rho = −0.604, p < 0.001), indicating that higher progesterone levels were associated with lower LH concentrations. Plasma hormone concentrations from trigger day until day of OPU, and follicular fluid LH level on the day of OPU. Results are presented as median (IQR). LH rise is defined as a plasma LH level > 10 IU/L measured from day of ovulation trigger until oocyte pick-up. OPU: oocyte pick-up. LH: luteinizing hormone, FSH: follicle stimulating hormone. Correlation between plasma progesterone and LH on the day after hCG trigger. Legend: Scatter plot showing the relationship between plasma progesterone (nmol/L) and LH (IU/L) on the day after hCG trigger. Each point represents an individual patient. A significant inverse correlation was observed (Spearman’s rho = −0.604, p < 0.001), indicating that higher progesterone levels were associated with lower LH concentrations. The Backward LR multivariable logistic regression analysis ( Table 4 ) identified that the duration of GnRH antagonist administration significantly reduced the likelihood of an endogenous LH rise with an OR= 0.532 (95% CI: 0.318–0.892, p = 0.017) showing that one additional day of antagonist treatment almost halved the odds of an LH rise. Higher plasma progesterone levels on trigger day and a higher number of oocytes retrieved also seemed to reduce the likelihood of an endogenous LH rise, but these results were only borderline significant ( Table 4 ). Table 4 Potential predictors of endogenous LH rise in the GnRH antagonist protocol. OR (EXP(B)) 95% CI P-value Number of GnRH antagonist days 0.532 0.318 – 0.892 0.017 Plasma progesterone on trigger day 0.460 0.211 – 1.002 0.051 Number of oocytes retrieved 0.792 0.624 – 1.006 0.056 Logistic regression analyses using backward LR (Likelihood Ratio). Dependent variable: LH rise > 10 IU/L (Yes/No). Step 1 included the four potential predictor variables: plasma progesterone on trigger day, number of GnRH antagonist days, duration of stimulation, and number of oocytes retrieved (the best proxy of pre-ovulatory follicles on trigger day). Three variables were kept in the final model (step 2) as shown above. OR: Odds ratio, CI: confidence interval, GnRH: Gonadotropin releasing hormone. Potential predictors of endogenous LH rise in the GnRH antagonist protocol. Logistic regression analyses using backward LR (Likelihood Ratio). Dependent variable: LH rise > 10 IU/L (Yes/No). Step 1 included the four potential predictor variables: plasma progesterone on trigger day, number of GnRH antagonist days, duration of stimulation, and number of oocytes retrieved (the best proxy of pre-ovulatory follicles on trigger day). Three variables were kept in the final model (step 2) as shown above. OR: Odds ratio, CI: confidence interval, GnRH: Gonadotropin releasing hormone. A sensitivity analysis using an LH cutoff of > 8 IU/L was conducted. We found that 20 patients had an LH > 8 IU/L. Consistent with the main analysis, these patients had fewer oocytes retrieved (median (IQR 25–75) 5.0 (3.75–6.25) in the group with LH > 8 IU/L versus 8.0 (5.75–10) (p = 0.001) in the group with LH < 8 IU/L).

This was a prospective, observational study conducted between January 2023 and June 2024 at two fertility clinics in Denmark. The Danish National Committee on Health Research Ethics and the Capital Region of Denmark, Research and Innovation, Legal Department approved the study (Journal number: H-3–2013–201). Informed consent was obtained from all participants. The study included 100 women undergoing IVF/ICSI in the GnRH-ant protocol with a hCG trigger. Inclusion criteria were age 18–41 years, indication for IVF or ICSI, and ovarian stimulation with recombinant (r)FSH. Exclusion criteria were GnRHa- or dual trigger, hypogonadotropic hypogonadism, severe comorbidities, and testicular sperm aspiration or -extraction. Women could be included only once undergoing an IVF/ICSI cycle. All patients received standard IVF/ICSI treatment with the fixed GnRH-ant protocol with hCG (Ovitrelle® 250 µg/6500 IU) administered for final maturation of follicles when 2–3 follicles reached ≥ 17–18 mm. Stimulation was performed with individually dosed rFSH (Gonal-F (n = 53), Bemfola (n = 37), Rekovelle (n = 6), Pergoveris (n = 4)). The GnRH-ant was administered i) from Stimulation Day 5 including day of trigger at 8 am (n = 22, Clinic 1, standard clinical practice), ii) from Stimulation Day 6 including day of trigger at 8 am (n = 56, Clinic 2), or iii) from Stimulation Day 6 at 10 pm, last dose the evening before trigger day (n = 22, Clinic 2). The dosing strategy in Clinic 2 was based on patient/physician preference. Blood samples were collected at three time points: 1) on the day of hCG trigger between 8am and 2pm according to patient preference i.e., close to last GnRH-ant dose if administered in the morning and up to approximately 16 h after last dose if administered at 10 pm the evening before trigger day, 2) on the day after hCG trigger between 8am and 2pm according to patient preference i.e., approximately 24–40 h after last GnRH-ant dose, and 3) on the day of OPU at 8 am, i.e., approximately 48–60 h after last GnRH-ant dose. Plasma was analysed for concentrations of LH, FSH, progesterone, and estradiol. The hormonal measurements were not used to guide the IVF/ICSI treatment but analysed retrospectively after study completion. Follicular fluid (FF) was obtained from the first aspirated follicle (17–22 mm) without visible blood contamination during OPU, avoiding dilution with flushing medium. FF was centrifuged and the supernatant stored at −80°C and later analysed in one batch for the concentration of LH. Hormone measurements were performed using Elecsys assays (Roche Diagnostics). The Elecsys LH assay shows negligible cross reactivity with FSH and hCG (<0.1%). The coefficient of variation (CV) for LH was < 5%, FSH < 6%, progesterone < 10%, estradiol < 7%, and Anti-Müllerian hormone (AMH)  10 IU/L in any of the three plasma samples (day of hCG trigger, day after hCG trigger, and day of OPU). Since the LH level was measured only once daily from trigger day to OPU, we chose in this ‘opening study’ to use a relatively low LH cut-off level, as the measured LH level most likely does not represent the peak level of the day. Secondary outcomes included proportion of women with LH > 10 IU/L in FF, the plasma levels of other reproductive hormones, number of follicles on trigger day, number of oocytes retrieved, the number of fertilized (2PN) oocytes, the number of cleavage-stage embryos (at least two cells) on day 2 after OPU, the number of blastocysts (Gardner score 1–6) on day 5 and in total, and the number of patients with at least one embryo transfer, a positive hCG test, and a clinical pregnancy in the groups with and without an endogenous LH rise. Categorical variables were presented as counts (%), and continuous variables as mean ± standard deviation (SD) if normally distributed, or median with interquartile range, (IQR, 25th-75th percentile) if not. Normality was assessed using the Shapiro–Wilk test. Group comparisons were performed using independent t -tests for normally distributed continuous variables, Wilcoxon rank-sum tests for non-normally distributed continuous variables, and Fisher’s exact or chi-square tests for categorical variables, as appropriate. A simple linear correlation between plasma progesterone and LH levels were performed. A multivariable logistic regression (backward likelihood ratio) was used to identify potential predictors of LH rise (yes/no), including number of GnRH-ant days, stimulation duration, progesterone on trigger day, and oocytes retrieved. Oocytes retrieved served as a proxy for preovulatory follicles, as only half of the women had follicle measurements on trigger day. A sensitivity analysis using an LH threshold of > 8 IU/L was conducted. Statistical significance was set at p < 0.05. All analyses were conducted using SPSS version 29.0.1.0 and RStudio version 2025.05.0.

Our study reveals that approximately one in six women (17 out of 100) undergoing treatment with a fixed GnRH-ant protocol experience an LH rise between hCG-induced final follicle maturation and OPU. This indicates that a substantial proportion of women in the GnRH-ant protocol undergo some kind of ‘dual trigger’ without the clinician’s awareness. Clinically, this implies that the final follicular maturation in these women differ significantly from what is anticipated. Although our study, which included 100 women, is too small to fully assess the consequences of this endogenous LH rise, it highlights a previously unrecognized aspect of the standard GnRH-antagonist protocol. We defined an LH rise by a significant pituitary LH release, i.e., a plasma LH level exceeding 10 IU/L. Since LH was measured only once daily from trigger day to OPU, we chose in this study to use a relatively low LH cut-off level as the measured LH level most likely would not represent the peak level of the day. It is therefore likely that the measured LH values reflect either the ascending or descending phase of the rise rather than its peak [9] , [10] . On the day of hCG trigger representing the ‘baseline’ value in our dataset, as no previous blood sampling was performed, the mean (SD) LH level was 2.5 (3.6) IU/L for the whole cohort of 100 patients. When the two patients with an LH rise > 10 on trigger day were excluded, the mean (SD) of LH on trigger day was 2.0 (1.7) IU/L. Thus the 10 IU/L cut-off level corresponded to the mean + 2 SD (whole cohort) or mean + 3 SD (cohort excluding the two patients with an LH rise on trigger day). However, we acknowledge that no standard definition of the onset of the LH surge exists [11] . The FF concentrations of LH were significantly higher in women with an endogenous serum LH rise; however, in no case did they exceed 10 IU/L. FF was collected at OPU, approximately 36 h after the hCG trigger. By this time, LH concentrations in FF have already declined to less than one-third of the levels observed 12–17 h into the ovulatory process [12] . Therefore, FF LH at OPU does not reflect earlier peri-ovulatory levels [12] . In general, LH appears to enter the follicle more rapidly than exogenous hCG [12] , suggesting that elevated LH concentrations may exert physiological effects; although, likely less pronounced than those of a full mid-cycle LH surge. The induction of oocyte maturation occurs during the first 12–17 h of final follicular maturation, representing a key phase of ovulation induction that may be influenced by the presence of LH [13] . We observed that all women with endogenous LH rise initiated the GnRH-ant treatment on stimulation day 6, whereas no LH rises were observed when the GnRH-ant was initiated on stimulation day 5. In addition, the duration of GnRH-ant exposure may influence the occurrence of an LH rise as women who experienced a rise had significantly shorter exposure to the GnRH-ant. However, these findings should be interpreted with caution and warrant further investigation in a standardised study design. Women with an LH rise showed similar baseline characteristics including AMH and AFC compared to those without a rise, but had fewer oocytes retrieved. Interestingly, however, the fertilization rate (2PN) and number of day 2 cleavage-stage embryos per oocyte retrieved were significantly higher in the LH rise group. These observations should be interpreted with caution, as the study was not powered to assess cycle outcomes. However, the finding warrants further investigation and may support the hypothesis that dual trigger is associated with improved functional competence of oocytes. Additionally, women with an LH rise exhibited significantly reduced progesterone levels from trigger day to OPU compared to those without an LH rise. This observation challenges the current understanding that subtle increases in progesterone are the primary trigger of the gonadotropin surge from the pituitary [14] , [15] , [16] . On the day following the trigger, both progesterone and estradiol levels were significantly higher in women without an LH rise. A strong inverse correlation was also observed between plasma progesterone and LH levels on the day after trigger ( Fig. 1 ). Notably, LH rises were almost entirely absent when progesterone exceeded 10 nmol/L (3.1 ng/mL), suggesting that elevated progesterone suppresses the LH secretion. This aligns with findings from progestin-primed ovarian stimulation protocols, where progesterone is used to prevent premature LH release [17] , [18] , [19] . Together, these results point to a need for further investigation into the role of progesterone during the periovulatory period. The results suggest that initiating GnRH-ant treatment no later than stimulation day 5 may prevent an endogenous LH rise. However, as discussed above, the suppression of an endogenous LH rise may well be explained by higher plasma progesterone levels supressing the endogenous LH secretion as the effect of last GnRH-ant administration ceases. Nonetheless, this study focuses for the first time on a topic that could potentially influence the results of the widely used GnRH-ant protocol, but larger studies are needed to confirm whether the timing of GnRH-ant initiation influences cycle characteristics and outcomes and thus directly or indirectly the prevalence of an LH rise, and to determine whether an endogenous LH rise is ultimately beneficial for the oocyte quality. This exploratory study has several limitations. While different GnRH-ant initiation strategies were used—on stimulation day 5 or 6—the data suggesting that this variation may have influenced the prevalence of LH rises is at most hypothesis generating, but it is nonetheless an observation that deserves attention in future studies. Additionally, hormone measurements were performed once daily, which limits the ability to capture the full hormonal dynamics, particularly the peak and duration of the LH rise [20] , [21] . Although a single LH measurement > 10 IU/L may not definitively indicate a true surge, it reflects a significant increase over baseline (trigger-day) LH levels and is consistent with late follicular phase LH values reported in previous GnRH-ant and GnRHa cycles [22] . Also, it is unlikely that our single measurements coincided with peak LH values, meaning true peak levels may have been underestimated. Another limitation is that this study was not powered to assess clinical outcomes such as the number of high-quality embryos or cumulative pregnancy and live birth rates. In conclusion, to the best of our knowledge, this study is the first to demonstrate that approximately 17% of women undergoing IVF with a fixed GnRH-ant protocol experience an endogenous LH rise > 10 IU/L between the final GnRH-ant dose and OPU. The potential impact of such endogenous LH activity occurring in parallel with hCG-induced final oocyte maturation as well as the duration and peak levels requires further studies. In this cohort, an LH rise was associated with shorter GnRH-ant exposure, lower progesterone levels from trigger day to OPU, and fewer retrieved oocytes. The significantly higher fertilization rate and number of cleavage-stage embryos per oocyte retrieved in women with LH rise warrants future studies.

One of the most widely used protocols for ovarian stimulation for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment is the gonadotropin releasing hormone antagonist (GnRH-ant) protocol which was introduced in the late 1990’es [1] . The ‘fixed’ GnRH-ant protocol includes daily GnRH-ant injections (i.e., ganirelix 0.25 mg) from a ‘fixed’ stimulation day, i.e., from stimulation day 5 or 6 to suppress the endogenous gonadotropin surge and prevent premature ovulation. However, the initial study on luteinizing hormone (LH) pharmacokinetics showed that serum levels return to pretreatment values approximately 24 h after the last GnRH-ant administration in the natural cycle [2] . As a result, some women undergoing ovarian stimulation with supraphysiological levels of oestradiol may experience endogenous LH and FSH release as the inhibitory action of the last administered GnRH-ant disappears. This release could either coincide with the exogenous hCG trigger administered after the last GnRH-ant dose or emerge during the subsequent 36 h leading up to oocyte pick-up (OPU). This would not result in premature ovulation but result in a trigger with both exogenous and endogenous LH activity (i.e., a dual trigger), which could affect oocyte competence, follicular dynamics, steroid production, and overall cycle outcomes. Interestingly, this topic remains largely unexplored, and to our knowledge, no studies have been published on the subject. In the natural menstrual cycle, the midcycle surge of both FSH and LH initiates ovulation. While exogenous hCG effectively triggers ovulation through activation of LH/hCG receptors [3] , a study has demonstrated that a GnRH agonist (GnRHa) trigger, which induces a combined LH and FSH surge, yields a higher proportion of mature oocytes compared to hCG trigger alone [4] . Moreover, several studies have reported improved outcomes using a dual trigger (GnRHa + hCG) compared to hCG alone [5] , [6] , [7] , [8] , suggesting an enhanced oocyte maturation potential when endogenous gonadotropins are elevated between trigger and OPU. The frequency and impact of any concurrent endogenous LH and FSH rise after the last GnRH-ant administration alongside the exogenous hCG activity is therefore interesting to investigate. The primary aim of this study was to determine the prevalence of an endogenous LH rise, representing an endogenous gonadotropin release, in the fixed GnRH-ant protocol as the effect of the final GnRH-ant disappears. Secondly, we aimed to identify potential differences in characteristics of women and cycles with or without an LH rise with regards to steroidogenesis, oocyte competence, and cycle outcomes.

The authors have no conflicts of interest in relation to the present work. However, KL has received consulting fees from Ferring Pharmaceuticals, given lectures for Ferring, Merck and Gedeon Richter, and received support for attending meetings and travel expenses from Ferring, Merck and Gedeon Richter. NFW has given lectures for Ferring and received support for attending meetings and travel expenses from Gedeon Richter. NB has received consulting fees from Ferring Pharmaceuticals, given lectures for Gedeon Richter, and received support for attending meetings and travel expenses from Theramex. BO has received support for attending meetings and travel expenses from Gedeon Richter and Ferring. AP has received grants or contracts and consulting fees from Gedeon Richter, Ferring, Merck and Cryos, given lectures for Gedeon Richter, Ferring, Merck and Organon, and received support for attending meetings and travel expenses from Gedeon Richter. CYA has received consulting fees from Ferring and IBSA, given lectures for Ferring and IBSA, and received support for attending meetings and travel expenses from Ferring and IBSA. JWB, TVD, JAB, MRP, LBD, MMC, LSM, and LCP have no conflicts of interest to declare.