Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones, acting on the same receptor (LHCGR) (1). These gonadotropins were considered equivalent at the molecular level for long time, until the demonstration of specific intracellular-mediated signaling (2). In vitro models of human granulosa cells demonstrated that hCG is more potent than LH in inducing cyclic adenosine monophosphate production (cAMP) production (2), while the latter leads to preferential ERK1/2 and AKT pathways activation (2). Thus, although LH and hCG activate different kinetics (2, 3), whether and how they differently influence in vivo response remains unclear (4).

In humans, follicle-stimulating hormone (FSH) and LH act in concert to stimulate folliculogenesis and ovulation. Therefore, these gonadotropins are used in the controlled ovarian stimulation (COS) in order to produce relatively high oocyte number to be used fresh or after cryopreservation (5) to obtain pregnancies. The physician identifies the presumably most appropriate regimen, in terms of gonadotropin-releasing hormone (GnRH) analog protocol, FSH formulation, starting FSH dose, and combination of different gonadotropins, following the evaluation of demographic, anthropometric, and ovarian reserve profiles (6–8). Generally, FSH is selected as standard treatment, and hCG or LH may be added. The knowledge of human physiology provides a rationale for LH activity supplementation during COS. Although in vitro and animal models provided the evidences of hormone-specific actions, the choice of the optimal gonadotropin combination to be used in COS is not well standardized and remains entrusted to clinician’s decision. Especially, the pregnancy hormone hCG is generally used to obtain LH-like activity and support of multi-follicle growth since decades (9). With this in mind, human menopausal gonadotropin (hMG) is commonly used as preparation with LH-like activity, due to the presence of LH and/or hCG molecules. hMG alone and hCG/LH + FSH were repeatedly proposed (10, 11) but some unfavorable results, in particular in terms of number of oocytes retrieved (12, 13), provided concerns about the usefulness of addition of “LH activity.”

Currently, the gonadotropin market offers a wide choice, including urinary and recombinant preparations of FSH, LH, hCG, and hMG alone or in various combinations, recently further enriched by biosimilars. This palette of competitor drugs, registered for the same indication but biochemically and physiologically different, introduced the concept of “personalized” assisted reproductive technology (ART) schemes, which is very attractive for patients and doctors but not supported by solid evidence and largely industry-promoted. These gonadotropins show different kinetics in in vitro models, but no clear in vivo differences in COS are available so far. Most studies have been tried to answer the question of what is the best gonadotropin combinations, although inconclusive results were achieved, not sufficient to guide a really evidence-based, personalized choice in ART. Indeed, no powerful, properly designed, controlled prospective clinical trials are available to support the rationale of any COS scheme so far. As a matter of fact, the design of randomized clinical trials is challenging in this setting, due to the peculiar emotional situation and heterogeneity of the infertile population together with the time and costs required. Thus, 64 meta-analyses have been published to compare different ART approaches and outcomes (Table 1). However, each review is focused on a specific single comparison (e.g., hMG vs. FSH, GnRH agonist vs. antagonists, etc.) in a peculiar clinical setting. In particular, 25 systematic reviews compared the efficacy of different GnRH analogs, 17 compared urinary and recombinant FSH preparations, and only 6 evaluated the efficacy of LH supplementation to FSH (Table 1). None of these comparisons provided a comprehensive analysis of entire process, from oocyte recruitment to live birth rate, and their conclusions are rarely translated in clinical practice. In fact, no accepted guideline exists in this field of medicine in which registered indications and reimbursability of gonadotropins by the national health care systems are guided by costs rather than scientific evidence/clinical outcome.

Having in mind physiology and the different in vitro effects of LH and hCG, in this work, we addressed the question whether LH, LH-like activity, and hCG could have different results on COS outcomes. To this purpose, we evaluated the efficacy of LH or hCG plus FSH or hMG alone, compared to what is considered the standard care for COS, i.e., the use of FSH alone, using a meta-analytic approach. This is the first meta-analysis in which all gonadotropin combinations are considered. Moreover, a full-spectrum evaluation of all ART endpoints is provided, to recognize when and how LH, LH-activity, and hCG influence ART outcomes.

We performed a meta-analysis according to the Cochrane Collaboration and PRISMA statement. The meta-analysis was accepted in the International Prospective Register of Systematic Reviews (PROSPERO; registration n. CRD42016048404) prior to commencing the study, ensuring transparency and originality of the review process.

Of the 2,117 publications initially identified, 1,602 remained after duplicates removal. According to the strategy research, we identified 196 potentially relevant studies, based on the information given in the abstract. All trials were thoroughly appraised for eligibility in the meta-analysis and methodological quality. Seventy studies were included in the final analysis (Table 2; Figure 1).

This is the first meta-analysis comparing comprehensively the efficacy of the mostly used gonadotropin combinations in ART. We find that the administration of FSH alone during COS retrieves higher oocyte number than either LH supplementation or hMG use. However, the combined use of FSH + LH reduces the FSH dose required for oocyte retrieved, while hMG leads to higher FSH dose needed. Interestingly, FSH + LH increases the pregnancy rate of about 1.20 fold, in spite of lower number of oocyte retrieved compared to FSH alone, whereas hMG does not. On the contrary, FSH + hCG treatment does neither change final oocytes number, nor FSH dose required for each oocyte, nor pregnancy rate. Although live birth rate is usually considered a better endpoint than pregnancy rate to evaluate ART outcome, it is not reported in many studies included and our meta-analysis does not show significant difference in live birth rate. All these differences are modest but, although apparently not clinically relevant, they are useful to better understand in vivo the overall effects of the different gonadotropin regimens.

These results suggest that gonadotropin preparations differently influence COS outcome, providing some evidence for ART personalization and improvement and leading to different results compared to those of previous meta-analyses. This difference could be due to the wide range of studies evaluated, which are focused on different endpoints and patient characteristics. FSH + LH treatment is linked to a relatively lower number of oocytes retrieved but higher pregnancy rate. The addition of LH or LH-activity might increase the selective pressure exerted on follicular selection exerted by the two gonadotropins together, compensated by improved oocyte quality. Indeed, the differences between FSH alone and FSH + LH or LH activity are lost, at least in terms of MII oocyte number. Moreover, the use of hMG leads to a higher embryos number and implantation rate compared to FSH alone. These results confirmed that the higher pregnancy rate found when FSH + LH or hMG are used together with GnRH agonist protocol, instead of FSH alone, is due to a positive effect of better oocyte quality on fertilization and embryo implantation. On the contrary, FSH + hCG treatment does not change ART outcomes compared to FSH alone, suggesting that LH and hCG result in different actions in vivo in the presence of FSH, reflecting in vitro observations (3). The overall model (Figure 5) shows a progressively better outcome when FSH is used together with LH or LH-activity (such as hMG). Thus, LH and hCG action in vivo is different in women undergoing COS, with LH improving oocyte maturation and quality, and therefore pregnancy rate, more than hCG, reflecting previous in vitro data.

Luteinizing hormone and hCG are characterized by specific molecular and biochemical features; they interact with distinct binding sites of the same receptor (25–27), resulting in lower dissociation rate by hCG than LH binding (28). Gonadotropin-specific ligand-receptor features imply different gene expression and intracellular signaling in vitro, whereby LH triggers higher levels of ERK1/2- and AKT-pathway activation than hCG, which, in turn, mediates more potent cAMP increase in human primary granulosa cells (2). Downstream effects of gonadotropins’ signaling consist in LH-related proliferative and anti-apoptotic signals, vs. high steroidogenic potential and pro-apoptotic activity of hCG in vitro, in both human and goat primary granulosa cells (3, 29). In particular, cell death was described as a result of the intracellular cross-talk among cAMP/protein kinase A (PKA)-mediated steroidogenic and pro-apoptotic pathways (30) preferentially activated by FSH and hCG, in steroidogenic cells in vitro (31).

Interestingly, our analysis of the literature reveals that LH addition to FSH treatment for ART provides lower oocyte numbers than other treatments, probably as a result of higher follicular selection (which is apoptosis-mediated). In this regard, few speculative considerations should be done. First, COS cycles are far from being a physiologic hormonal regimen; they are optimized for multi-follicular maturation in order to obtain the highest number of healthy oocytes (32), subjecting ovaries to treatments with pre-designed, high doses of exogenous hormones, which change the natural endocrine milieu of the woman. As a result, a mono-ovulatory species becomes multi-ovulatory, deviating from the natural, cyclic balance between gonadotropins and steroid hormones (33) and, thereby, life/death signals, a situation clearly different from ovarian physiology. On the other hand, FSH and LH are naturally produced to regulate mono-follicular selection, growth and maturation. The message provided by in vitro studies is that highly steroidogenic gonadotropins, i.e., FSH and hCG, mediate apoptotic stimuli in granulosa cells via cAMP/PKA-pathway (2, 3, 29–31). In the ovarian setting of a multi-follicular maturation as in COS, stimulation is a potent signal for early tertiary follicle recruitment (34) and triggering steroidogenesis, results in estrogen over-production which, in turn, induces more pronounced multi-follicular survival and maturation (35) than that inducible by LH treatment.

The ART outcome obtained with hMG reflects the heterogeneity typical of this compound. hMG derives from post-menopausal or pregnant women and contains both FSH and LH activities (36). LH activity is provided by residual LH molecules and by hCG supplementation, leading to high variability of the product (37). Moreover, given the high steroidogenic potential of hCG demonstrated in vitro (2, 31), which is more similar to that of FSH rather than LH (31), it is not surprising that ART outcome does not change whether hMG is used instead of FSH, except in GnRH agonist protocols, where high oocyte numbers might possibly occur as a positive effect of the flare-up phase on follicle recruitment. The discrepancy provided by GnRH-agonist and -antagonist protocols was not demonstrated by previous meta-analyses, likely due to strict inclusion criteria focused specifically on the evaluation of the analog instead of gonadotropins combination. The most recent meta-analysis on this field suggests only a significant adverse events occurrence reduction when GnRH antagonists are used (38).

This study suggests that GnRH antagonist protocol may be disadvantageous for oocytes quality, although the addition of LH seems to compensate, at least in part, this negative effect. FSH alone allows higher number of oocytes retrieved than FSH + LH, in GnRH agonist, but not antagonist protocols. GnRH antagonist is linked to lower FSH doses required for each oocyte retrieved, in the presence of LH. Moreover, pregnancy rate is higher by hMG than FSH treatment in GnRH agonist, but not antagonist protocols. This reflects the different mechanism of action and possibly different effects among GnRH analogs, which was hypothesized, although largely debated (39). GnRH analogs are differently used in clinical practice. In particular, GnRH agonists are generally proposed in women with BMI <25 kg/m² (40), in poor responders (38, 41), and/or as a final trigger to minimize the ovarian hyperstimulation syndrome (OHSS) occurrence (42). Overall, GnRH antagonist is linked to reduced COS duration and overall medical costs of the stimulation phase and is recommended when a mild stimulation is required, such as for hyper-responder women (38, 43) or PCOS patients (44). These results support the hypothetical difference between agonists and antagonists, which was never demonstrated by previous meta-analyses (Table 1).

With this in mind, the cost-effectiveness evaluation currently remains the main variable useful to guide the clinician choice in the setting of the personalized therapy (45). However, the assessment of ART costs is particularly challenging, and the consideration of both COS-related and pregnancy/infant-associated medical costs is mandatory. Several studies evaluated the ART medical costs alone, considering the cumulative gonadotropin dosages used, the cycle cancelation rate and the risk of adverse events. The FASTT study suggested that IUI was the cheapest/efficient first-line treatment (46), while the FORT-T trial suggested better cost-effectiveness results when sequential traditional embryo transfer is selected (47). Crawford et al. (48) recently evaluated the overall ART costs in 14,398 cycles, suggesting that sequential embryo transfer is more expensive, concerning the procedure costs, but markedly cheaper overall, reducing multiple live births and total, final expenses. Although each study seems to be conclusive, these results remain challenging, and international or national consensus on the best COS approach is not reached so far. Moreover, the gonadotropin combination is not generally considered in this cost-effectiveness evaluation, limiting the strength of these suggestions. Our results suggest a reduced FSH dose needed for each oocyte retrieved when the combination of FSH + LH was used for COS. Thus, the gonadotropin combination should be considered in the cost-saving evaluation of a specific ART procedure. The overall charge, even when LH, hCG, or hMG are used in addition/substitution to FSH, must be considered according to the local reimbursement system. Finally, no study so far evaluated the “weight” of gonadotropin-producing companies on the clinician’s decision.

The main limit of this meta-analysis is the heterogeneity of studies included as suggested by the elevated I² score. Couple infertility represents a challenging clinical condition, difficult to define according to strict clinical criteria. Indeed, different inclusion and exclusion criteria are used in each trial, making the comprehensive comparison of these results difficult. As a confirmation, a recent phase III single-blind, randomized, parallel-group clinical trial performed on 939 poor responder women did not find any safety and efficacy differences between FSH alone and FSH + LH (49). This reinforces the knowledge of a high heterogeneity of studies in ART setting, in which also the women classification as poor responders could mask the different gonadotropin effects in vivo. The relative high risk-of-bias of the studies included, as shown in Figure 4, represents an important limit that should be carefully considered to design further appropriate studies. However, although the pharmacological approach to ART is evaluated, no publication biases are evident at funnel plots analyses (data not shown). As highlighted by previous meta-analyses, we found high selection and allocation biases, confirming the finding that more than 80% of clinical trials did not apply any blinding technique (50). This high percentage is probably due to the difficulty in applying these procedures to ART, in which over 30 therapeutic complex approaches are currently available.

In conclusion, we found that different performance in ART is depending on gonadotropin combination used for COS, reflecting the physiological role of these molecules as previously indicated by in vitro data. This leads to important implication for clinical practice, where pregnancy rate or oocyte numbers might be the preferentially selected outcome. Especially, LH addition to FSH decreases FSH need and progressively improves ART outcomes and pregnancy rate. In GnRH agonist protocols, a better pregnancy rate is obtained by FSH + LH and hMG treatment. FSH + hCG or hMG alone are equally effective compared to FSH alone on pregnancy rate.

DS and LC searched and evaluated separately the studies. All authors participated to the analysis, discussion of the results, and manuscript preparation.

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.