In recent decades, in vitro fertilization-embryo transfer (IVF-ET) has grown rapidly throughout the world, becoming an important method of treating infertility. The treatment process revolves around controlled ovarian hyperstimulation (COH). Gonadotropin-releasing hormone antagonist (GnRH-ant) protocols are widely used due to their advantages of short stimulation time, low costs, and a lower incidence of ovarian hyperstimulation syndrome (OHSS) (1–3). GnRH-ant binds to specific receptors on the pituitary gland and inhibits endogenous Luteinizing hormone (LH). It can prevent the appearance of early follicular LH surge, thereby inhibiting premature follicle production and reducing the cycle cancellation rate, which brings a new choice for clinical ovulation induction programs (4).

LH is a glycoprotein hormone secreted by the pituitary gland, which plays an important role in estrogen synthesis, follicle development, and ovulation induction (5).On the one hand, high LH levels are harmful to pregnancy outcomes in both the natural and ovarian stimulation cycles. Too little LH, on the other hand, is linked to pregnancy loss (6, 7). Scholars generally agree that an adequate level of LH is required for follicular development. According to some studies, the LH window has a range of 1.2–5 IU/L (8, 9). The team led by Professor Li Yuan proposed that LH≥4IU/L be considered the critical value, and antagonists should be considered when the threshold was exceeded (10). However, there is no consensus on the appropriate value of LH during COH with antagonist protocol.

At present, antagonist protocols are mainly divided into fixed and flexible protocols (11). The starting day of GnRH antagonist administration (i.e., both the fixed and flexible protocols) is mainly based on the day of ovarian stimulation, the diameter of the follicles, the estradiol levels, or a combination of these parameters (3). However, LH levels on the antagonist administration day have received less attention.

A retrospective analysis was used in this study. We divided the LH level on the antagonist administration day into four groups according to the quartile method and compared the laboratory indicators and pregnancy outcomes among the four groups.

Statistical methods were used to analyze the effects of LH level on the antagonist administration day on laboratory indicators and estimate the cut-off values of LH on antagonist administration day for predicting various pregnancy outcomes,so as to illustrate that the LH Level on the antagonist administration day could be used as a predictor of the reproductive outcomes in women with normal ovarian function.

A total of 1032 patients were included in the initial analysis, and 854 patients were included in the final study after applying the exclusion criteria. No cycle cancellation due to unexpected premature ovulation was reported among patients of groups.

Age, BMI, infertility type, infertility years, causes of infertility,basal FSH level, basal E2 level, basal T level, started Gn dose, time of antagonist administration, and fertilization method were not significantly different among the four groups. Basal LH levels were significantly different among the four groups. ( Table 1 ).

Laboratory indicators and pregnancy outcomes were compared among the four groups. There were no significant differences among the four groups in terms of duration of Gn, endometrium on trigger day, number of embryos (D3),number of high-quality embryos,fresh CPR,OHSS rate and Cumulative CPR.There were significant differences among the four groups in terms of total Gn dosage, E2 on trigger day, P on trigger day,LH on trigger day, number of retrieved oocytes, number of 2PN embryos, number of blastocysts, Number of ET and fresh ETR. ( Table 2 ).

Pearson chi-square test was used to verify the correlation between LH on antagonist administration day and basal LH level,LH on trigger day,endometrium on trigger day,number of oocytes retrieved,number of 2PN embryos,number of embryos (D3),number of blastocysts,number of ET and number of high-quality embryos.The results showed that LH on antagonist administration day had a significant correlation with basal LH level,LH on trigger day,number of oocytes retrieved,number of 2PN embryos,number of blastocysts,number of ET. ( Figures 2 , 3 )

Linear regression analysis revealed that there is a significant positive correlation between LH on antagonist administration day and basal LH level (p<0.05, Figure 4 ), LH on trigger day (p<0.05, Figure 5 ), number of oocytes retrieved (p<0.05, Figure 6 ), number of 2PN embryos (p<0.05, Figure 7 ), number of blastocysts (p<0.05, Figure 8 ), number of ET (p<0.05, Figure 9 ).

The optimal cut-off value of LH on antagonist administration day of various pregnancy rates was analyzed by ROC curve.The results showed that the optimal cut-off value of LH on antagonist administration day was 4.18IU/L using Fresh ETR as the standard (AUC=0.559; P=0.003; Figure 10 ). Using Fresh CPR as the criterion, the optimal cut-off value for evaluating LH on antagonist administration day was 3.99IU/L (AUC=0.515; P=0.534; Figure 11 ). Using OHSS as the criterion, the optimal cut-off value for evaluating LH on antagonist administration day was 4.63IU/L (AUC=0.605; P=0.36; Figure 12 ). Using Cumulative CPR as the criterion, the optimal cut-off value for evaluating LH on antagonist administration day was 4.66IU/L(AUC=0.557; P=0.005; Figure 13 ).

Total dosage of Gn in Q4 group was the least, but the number of oocytes retrieved was the most.Although there was no difference in the number of high-quality embryos among the four groups, both Fresh CPR and Cumulative CPR in group Q4 were higher than those in the other three groups.The results were not statistically significant, but they did give us some insight.In predicting the optimal cut-off value of LH on antagonist administration day in multiple pregnancy rates,Almost all the optimal cut-off value are greater than 4, which is similar to the view of Professor Li Yuan’s team (10). Their study concluded that LH≥4IU/L be considered the critical value, and antagonists should be considered when the threshold was exceeded.Our study suggests that fresh ETR would be increased if adding antagonists after LH Level on the antagonist administration day > 4.18IU/L, Cumulative CPR would be increased if adding antagonists after LH Level on the antagonist administration day > 4.66IU/L.

Current research on the relationship between LH levels, ovarian reactivity, and pregnancy outcome during ovulation stimulation has yielded inconclusive results.According to Benmachiche et al. (16) a low LH level on the trigger day was associated with a lower rate of continued pregnancy and live birth and an increased rate of early abortion.Lahoud et al. (6) discovered that mid-follicular LH levels were related to ovarian reactivity but not to live birth rate.Another study (17) found that patients with low basal LH levels (≤3U/L) had no special ovarian responsiveness during ovulation induction but had a poorer pregnancy outcome than those with LH≥3U/L.

LH level is of great significance for maintaining a more appropriate follicle development environment and better receptivity of implanted endometrium in the COH regimen (18, 19). The premature addition of GnRH-ant, excessive use of GnRH-ant, or GnRH-ant usage beyond recommended days may lead to excessive ovarian suppression, thereby resulting in low serum LH levels and a relative lack of estrogen that might affect the growth and development of oocytes. Excessive inhibition of LH level was not found in this study. However, if GnRH-ant is added too late or the dose is insufficient, it may lead to high LH levels and an early LH surge, resulting in decreased follicle quality, reduced pregnancy rate, premature ovulation, and cycle cancellation (20). The increase in serum P level induced by an LH surge can also affect the expression of genes related to endometrial receptivity, thus, affecting embryo implantation (21). Another study reported that an increase in serum P level during the late follicular phase affected not only the embryo quality (22, 23) but also reduced implantation and clinical pregnancy rates (21, 24, 25). However, there are also views that the increase of serum P level at the late follicular stage does not affect embryo quality and cumulative live birth rate (26). This study found that as LH on the antagonist administration day increased, so did LH on the trigger day and P on the trigger day. However, there were no significant differences between the four groups in terms of the number of high-quality embryos and fresh CPR.

Current antagonist protocols are divided into fixed and flexible regimens and are mainly based on Gn stimulation time, follicle development size, and estrogen levels. However, little attention has been paid to LH levels on the antagonist administration day. A study suggested that LH levels can be used as an indicator for the addition of antagonists during COS. Patients with persistently low LH levels (LH<4.0 IU/L) may not require an antagonist (10). It is suggested that the implantation and pregnancy rates decreased with an increased antagonist dosage (27). A study stated that the number of natural killer cells and the expression level of perforin in endometrium were increased in patients treated with GnRH-Ant, hence suggesting that GnRH-ant may reduce endometrial receptivity (28). However, a randomized controlled trial showed that administration of GnRH-ant during the proliferative phase did not affect endometrial receptivity and embryo implantation; the pregnancy rates were not significantly different when compared with controls (29). Studies on the negative effects caused by elevated LH levels have mostly focused on embryo quality and endometrial receptivity. Therefore, the main observation indicators of this study were fresh ETR, fresh CPR,Cumulative CPR,the number of high-quality embryos, and OHSS rate. Although there was no significant difference in the number of high-quality embryos,fresh CPR and Cumulative CPR among the four groups, However, we give the optimal cut-off value of LH on antagonist administration day affecting different pregnancy rates,This has important guiding significance for clinical work.

This is a manuscript with LH on antagonist administration day as the main object of study, and LH on antagonist administration day ‘s significance in pregnancy outcomes of GnRH-ant protocols had not received enough attention before.As this was a retrospective study, many confounding factors limit the generalization of the findings to a certain extent. The sample size is not very large, which weakens the credibility of the study. Additionally, we only included patients undergoing IVF-ET due to female tubal factors, but did not include patients with other common clinical diseases such as PCOS and EMS.In the future, we can consider increasing the sample size for corresponding research.In addition, the lack of studies on abortion rate and live birth rate of pregnancy indicators is also a pity, which can be considered to supplement data based on later follow-up.

The LH Level on the antagonist administration day could be used as a predictor of the reproductive outcomes in women with normal ovarian function.There was a significant positive correlation between LH on the antagonist administration day and number of oocytes retrieved,number of 2PN embryos, number of blastocysts.LH on the antagonist administration day could predict Fresh CPR, OHSS and Cumulative CPR to some extent.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Reproductive Medicine Ethics Committee of the Affiliated Hospital of Shandong University of Chinese Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

CP contributed to study design, data collection, statistical analysis and drafting of the manuscript. KW assisted with data collection and interpretation and reviewed the analyzed results. HW provided ART-related clinical theory and technical support. CP and KW reviewed the analyzed results and revised the manuscript. All authors contributed to the article and approved the submitted version.