Introduction
Gonadotropins include two pivotal reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH). During in vitro fertilization and embryo transfer (IVF-ET) therapy, FSH is used for ovarian stimulation, but the role of LH, as well as the timepoint and criteria to add LH in the controlled ovarian stimulation (COS) process, remains controversial [
1‐
3]. Although LH is known to promote follicular maturation and induce ovulation [
4], its other functions in the reproductive process remain unclear.
After combining with the luteinizing hormone/chorion gonadotropin receptor (LHCGR) in the cell membrane, LH can activate the downstream signaling pathway [
5]. The LHCGR is primarily expressed in follicular granulosa cells and can promote hormone-dependent steroidogenesis [
6]. However, since the LHCGR is also found in nongonadal but reproductively relevant tissue, such as uterine tissues, LH might be involved in roles other than follicular growth [
7]. Therefore, additional clinical and biological studies are required to explore the functions of LH to better understand the entire reproductive process.
During IVF-ET therapy, FSH is used to stimulate follicular growth. With follicular development, the serum LH level can increase and may cause a premature LH surge and ovulation, which can be suppressed by gonadotropin-releasing hormone (GnRH) analogs (commonly agonists and antagonists) [
8]. The mechanisms of IVF protocols using GnRH analogs as agonists or antagonists are different. When a GnRH analog is administered as an agonist, there is a flare-up of FSH and LH, but the GnRH analog receptors are later downregulated to a low level in the pituitary gland [
9]. Endogenous gonadotropins remain at a lower level and for a longer duration with agonist use compared to antagonist use. When GnRH is used as an antagonist, it can rapidly inhibit gonadotropin secretion by reverse binding to the GnRH receptor [
10]. Thus, its advantage is rapid and temporary suppression of pituitary secretion. Therefore, the LH level may vary and play different roles depending on the IVF regimen used.
To explore the effect of different regimens on the LH level and clinical outcome, we herein compared the LH distribution of agonist and flexible antagonist regimens and assessed the effects of the LH level on the ovulation trigger day (LHOTD) on the overall clinical outcome of both regimens.
Materials and methods
Study design and participants
This retrospective cohort study was conducted after approval by the ethics committee of the First People’s Hospital of Yunnan Province (No. KHLL2020-KY013). Patients undergoing their first cycle with controlled ovarian stimulation using a GnRH analog and fresh embryo transfer (ET) at the university-affiliated hospital from January 2017 to May 2020 were included. The exclusion criteria were as follows: 1) the presence of polycystic ovary syndrome (PCOS), luteinized unruptured follicle syndrome, and other endocrinology disorders; 2) the use of oral contraceptives in the last 3 months; 3) a self-reported history of a family genetic disorder or an abnormal chromosomal karyotype; and 4) incomplete medical records. The clinical information was deidentified. Simple randomization was used to select the agonist or antagonist regimen. However, for patients with normal ovarian reserve and the desire to have more oocytes retrieved, the agonist tended to be used. For patients with a potential high ovarian response or low anti-Müllerian hormone (AMH) level (less than 2 ng/mL), antagonists tended to be used. Finally, pituitary suppression was initiated after the patient learned about the difference between agonists and antagonists and signed the informed consent form.
Agonist regimen procedure
The agonist pituitary suppression regimen was initiated at the mid-luteal phase. A total of 1.25 mg of the long-acting triptorelin acetate (Diphereline, Ipsen Pharma Biotech, Signes, France; Decapeptyl, Ferring GmbH, Kiel, Germany) was injected intramuscularly (IM). After approximately 14 days, when the estradiol (E2) level was < 50 pg/mL, the endometrial thickness was < 5 mm, and no ovarian cysts were noted, COS was initiated by an FSH (rFSH, Gonal-F, Merck-Serono, Aubonne, Switzerland) subcutaneous (SC) injection once a day. Ultrasound and serum hormone examinations were conducted from the 6th day of COS. When there were two dominant follicles with a diameter of > 18 mm, the serum LH, E2, and progesterone levels and the endometrial thickness (EMT) were recorded. Then, 5,000 IU of exogenous human chorionic gonadotropin (hCG) was injected at 10 PM to trigger the ovulation process.
Antagonist regimen procedure
In the flexible antagonist regimen, exogenous FSH (rFSH, Gonal-F, Merck-Serono, Aubonne, Switzerland) was SC injected daily from the 2nd day of menses to start COS. From the 4th day, when the E2 level was > 300 pg/mL, the diameter of the dominant follicle was > 14 mm or the LH level was > 10 mIU/mL, a 0.25 mg SC injection of cetrorelix acetate (Cetrotide, Merck Europe B.V., Idron, France) or ganirelix acetate (Orgalutran, Merck Sharp & Dohme B. V., Ravensburg, Germany) was injected daily until the diameters of the two dominant follicles were > 18 mm. Subsequently, the ovulation trigger was administered by injecting exogenous hCG.
IVF/ICSI-ET
Thirty-six hours after the ovulation trigger was administered, transvaginal ultrasound-guided puncture and oocyte retrieval were performed. According to male sperm motile measurements, conventional IVF or ICSI was conducted. The statuses of the zygote and embryo were monitored and recorded. The presence of two pronuclei (2PN) on the first day was considered normal fertilization. The number of 2PN divided by the number of oocytes retrieved was defined as the 2PN rate. On the 3rd day, the original 2PN embryos, with 6–8 blastomeres and cell debris < 20%, were considered the optimal choice for ET. β-hCG was tested on the 14th day after ET. Luteal support was continued to the 8th week if the β-hCG test was positive.
Data collection
Baseline characteristics, including age, body mass index (BMI), AMH level, and antral follicle count (AFC), were recorded. Primary or secondary infertility, causes of female infertility, history of parturition or miscarriage, and methods of fertilization (conventional IVF versus ICSI) were recorded. The LHOTD was collected on the day of the ovulation trigger, and all serum hormones were measured by a UniCel DxI 800 Access Immunoassay System (Beckman Coulter, CA, USA). Medical records were also reviewed to document the levels of estradiol and progesterone, as well as the endometrial thickness, on the ovulation trigger day.
After ET, follow-up visits were cancelled if the β-hCG test was negative. If the β-hCG test was positive, a clinical pregnancy test was conducted at the 5th–6th week. The detection of a viable sac(s) was defined as a clinical pregnancy. An extrauterine sac was defined as an ectopic pregnancy. Pregnancy loss during the first trimester was defined as early pregnancy loss. If pregnancy continued past the 12th week, it was defined as an ongoing pregnancy. The primary outcomes of the study were the clinical pregnancy and live birth rates.
Statistical analysis
Statistical analyses were conducted with SPSS (version 26.0, Armonk, NY, USA). A
P value of < 0.05 via a two-tailed test was considered statistically significant. The cycles were assigned into tertile groups based on the LHOTD in each agonist and antagonist regimen (T1, T2, and T3). To compare the three LHOTD groups, normally distributed continuous data, such as age, BMI and the number of oocytes retrieved, are expressed as the mean ± standard deviation (SD) and were compared by one-way ANOVA. Nonnormally distributed continuous data, such as the AMH level, AFC, indicators on ovulation trigger day and 2PN rate, are expressed as the median (25
th–75
th percentiles) and were compared by the Kruskal–Wallis test. Categorical data, such as gravidity and parity, medical history, fertilization method, the number of embryos transferred and clinical outcomes, are demonstrated as counts (percentage) and were analyzed by the chi-square (χ
2) test. The multiple pairwise comparison
P value was adjusted by the Bonferroni method. A stepwise progressive multivariate regression model [
11] was introduced to assess the effect of the LHOTD on the clinical pregnancy and live birth rates. Finally, a total of 3 models were developed to account for the important information of each IVF-ET treatment stage as comprehensively as possible to eliminate confounding factors. Model 1 included age, BMI, the AMH level, and the AFC. In Model 2, primary infertility, the cause of female infertility, and history of parturition or miscarriage were included. In Model 3, we added the ovulation trigger day indicators, including progesterone, E2, EMT, the number of oocytes retrieved, fertilization method, and the number of embryos transferred, on the basis of Model 2. Mantel‒Haenszel stratification analysis [
12] was used to demonstrate that the LHOTD of the antagonist regimen was not correlated with the clinical outcome.
Discussion
In the present study, we compared the clinical outcomes among three different LHOTD groups with agonist and antagonist regimens. The results showed a disparate correlation between the LHOTD and clinical outcomes in both regimens. With effective pituitary suppression, the LHOTD maintains a low level and dense distribution and is an independent factor affecting the clinical pregnancy and live birth rates in women receiving an agonist regimen. In contrast, the LHOTD in the flexible antagonist regimen group showed a relatively high level and scattered distribution compared with the agonist regimen group and exhibited no correlation with the clinical outcome overall.
The limitations of our study included its single-center research and retrospective design. However, based on the large sample size and multiple statistical methods used, bias and confounders were eliminated as much as possible. Furthermore, the use of a stepwise multivariate logistic regression method demonstrated that there were significant differences in both clinical pregnancy and live birth rates in the agonist regimen after eliminating confounders at different phases. Moreover, agonist and antagonist regimens were both included in the same period to analyze the correlation between the LHOTD and clinical outcomes. These results revealed the effects of different pituitary-suppression methods on patients undergoing IVF. However, the cause of these results remains unclear, and we intend to continue exploring the LH mechanism affecting outcomes in the future.
At present, whether LH is connected to IVF outcomes remains controversial, irrespective of the regimen used. Agonists were the first GnRH analogs and were introduced to suppress a premature LH surge in the pituitary gland. Due to the persistence of agonist desensitization, the serum LH level can be suppressed throughout the entire COS process. Westergaard et al. reported that the LH level on the 8
th day of ovarian stimulation was positively correlated with the clinical outcomes, with a high LH level associated with a low early miscarriage rate and a high live birth rate [
13]. Similar results were also reported in a retrospective study by Humaidan et al., which showed that the serum LH level on the 8
th day of COS had a significant impact on the ovarian response and clinical outcomes, suggesting that the LH level should not be too low during COS [
14]. Our previous study demonstrated that profoundly suppressed LH levels on the day of COS initiation were correlated with a higher early miscarriage rate and adverse IVF outcomes [
15]. However, Balasch et al. showed that the LH level on the 7
th day of stimulation was not correlated with the clinical outcomes, including clinical pregnancy, early pregnancy loss, and ongoing pregnancy [
16]. Furthermore, in a study of 246 cycles, the LH level during COS was not connected to the ovarian response and clinical outcomes [
17]. Moreover, in a study by Esposito et al., the mean LH level in periovulation was not correlated with clinical pregnancy or spontaneous abortion [
18]. Research on the effect of the LHOTD on clinical outcomes using an agonist regimen has rarely been previously reported. To the best of our knowledge, the present study is the first to report a positive correlation of the LHOTD with clinical outcomes using an agonist regimen.
Since the discovery of GnRH-antagonist function in pituitary suppression [
19], the antagonist regimen has been gradually introduced into COS. It has the advantages of fewer injections for patients, shorter stimulation days, avoidance of the adverse effects of agonists [
20], and adequate prevention regarding premature LH surges [
21]. Furthermore, the clinical outcomes of COS are similar between agonist and antagonist regimens [
22,
23]. However, the effect of the LHOTD on clinical outcomes using antagonist regimens remain controversial. The influences of LH have been reported at different stages of COS. On the COS initiation day, higher LH levels may be beneficial to endometrial maturation [
24]. In contrast, other research reported a reduced chance of successful pregnancy when a higher LH level occurred during the early follicular phase [
25]. In patients with PCOS, the Day 2 or 3 basal LH level was unrelated to the clinical outcome [
26]. At the mid-follicular phase, a study reported that the profound suppression of LH leads to a higher ongoing pregnancy rate after the antagonist is administered [
27]. However, another study reported different results [
28]: at the periovulatory phase, a fixed GnRH antagonist regimen showed that a low LHOTD was associated with low ongoing pregnancy and high miscarriage rates [
29]. This was in contrast to the research conducted by Ramachandran et al. [
30], who reported that the LHOTD was not related to pregnancy outcomes. With regard to LH levels during the process of ovarian stimulation, there was a study in which no difference in clinical outcomes was reported between the maximal LH level during COS > 4 mIU/mL and < 4 mIU/mL groups [
31], whereas another study showed that the group with a minimal LH level during COS < 0.5 mIU/mL achieved a worse prognosis [
32]. Our results showed that the LHOTD was not associated with clinical outcomes in the flexible antagonist regimen group except for the group of women aged less than 35 years and the excessively suppressed LHOTD group. These findings implied that more LH may be needed in younger women or those with extremely low LH levels undergoing a flexible antagonist regimen.
Interestingly, the LHOTD showed diverse effects on outcomes in different analog regimens. The aim of both regimens was to inhibit a premature LH surge; however, the mechanisms in the regimens were different, causing the circulating LH level to be discrepant (Fig.
1A). In the agonist regimen group, the LHOTD showed a denser distribution (Fig.
1B) and a lower level compared with normal physiology. It is posited that LH is essential for follicular development [
33], and its level should not be too low in the COS process [
34]. Thus, the profound suppression of LH might lead to insufficient and impaired LH functions. In contrast, the LH level during COS was suppressed slightly in the flexible antagonist regimen group and showed a scattered curve (Fig.
1C). The results of LH distribution were more similar to normal physiological LH levels [
35]. Adequate LH levels thus support the entire COS and subsequent reproductive process, which might explain why the LHOTD with the antagonist regimen showed no correlation with the clinical pregnancy and live birth outcomes.
The agonist regimen inhibited ~ 90% of patient LH levels to a limit of < 1.34 mIU/mL, while 90% had an LH level > 1 mIU/mL in the antagonist regimen (Fig.
2). These results might be caused by the diverse mechanisms of pituitary suppression using agonists or antagonists. A GnRH agonist has an extremely high affinity for the GnRH receptor relative to wild-type GnRH in the pituitary gland, and after the transient surge of gonadotropin, the pituitary gland becomes desensitized to GnRH and stops secreting endogenous LH for a long period [
36]. However, this process is not easy to control, as the pituitary response to agonists is specific to the individual. Thus, a patient’s pituitary gland may be profoundly suppressed by an agonist causing low LH levels, and these patients may be more suitable for exogenous LH supplementation. In contrast, GnRH antagonists can reversibly bind to the GnRH receptor [
37] and inhibit the signaling pathway regulating gonadotropin secretion. The suppression process is mild, controlled, and transient, and the occurrence of LH deficiency is therefore less likely. Further experiments will need to clarify the reasons for these findings.
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