Recombinant LH supplementation improves cumulative live birth rates in GnRH antagonist protocol: a multi-center retrospective study using a propensity score-matching analysis.

Abstract

Background: Luteinization hormone (LH) is critical in follicle growth and oocyte maturation. However, the value of recombinant-LH (r-LH) supplementation to recombinant-follicle stimulation hormone (r-FSH) during controlled ovarian stimulation in Gonadotrophin releasing hormone (GnRH) antagonist regimen is controversial. 

Methods: The multicenter retrospective cohort study recruited 899 GnRH antagonist cycles stimulated with r-LH and r-FSH in 3 reproductive centers, and matched to 2652 r-FSH stimulating cycles using propensity score matching (PSM) for potential confounders in a 1:3 ratio. The primary outcome was the cumulative live birth rate (CLBR) per complete cycle.

Results: The baseline characteristics were comparable in the r-FSH/r-LH and r-FSH groups after PSM. The r-FSH/r-LH group achieved higher CLBR than the r-FSH group (66.95% VS 61.16%, p=0.006). R-LH supplementation also resulted in higher 2-pronuclear embryo rate, usable embryo rate, live birth rate in both fresh embryo transfer cycles and frozen-thawed embryo transfer (FET) cycles. No significant differences were found in the moderate and severe ovarian hyperstimulation syndrome (OHSS) rate, and cycle cancel rate in prevention of OHSS.

Conclusions: R-LH supplementation to r-FSH in GnRH antagonist protocol was significantly associated with higher CLBR, live birth rate in fresh and FET cycles, and improved embryo quality without increasing OHSS rate and cycle cancel rate.

Background

Controlled ovarian stimulation (COS) is the first and critical procedure of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Gonadotrophin releasing hormone (GnRH) antagonists are applied to conveniently down-regulate the pituitary to reduce the endogenous gonadotropins without the flare-up effect. Thus, the follicle development is under the control of the exogenous gonadotropins.

According to the “two cell-two gonadotropins” theory, follicle stimulating hormone (FSH) synergizes with luteinization hormone (LH) to promote folliculogenesis and oocyte maturation. FSH stimulates granulosa cells to produce estradiol from androgen transformed from cholesterol by the theca cells in response to LH stimulation. It has been proved that the recombinant FSH (r-FSH) alone after pituitary down-regulation can induce follicle development, which might be due to the residual endogenous LH secretion. However, lacking LH supplementation may undermine follicle development [1], and affect the decidualization of endometrium and implantation of embryos [2]. Consequently, the addition of exogenous recombinant LH (r-LH) in combination with FSH is likely to promote the normal development of follicles. While conflicting opinions regarding this question have been published.

A real-world study involving 9787 cycles suggests that LH supplementation to moderate and severe poor ovarian responders can improve the cumulative live birth rate (CLBR) [3]. When comparing the 2nd ICSI cycles stimulated by r-FSH and r-LH in GnRH antagonist protocol with the 1st cycles stimulated with r-FSH of 228 cycles, the 2nd cycles achieved higher implantation rates [4]. A study involving 320 cycles receiving GnRH antagonist treatment [5] and another study recruiting 1565 cycles [6] conclude that r-LH supplementation is beneficial in pregnancy rate, live birth rate, fertilization rate, and implantation rate. Another meta-analysis concludes that r-LH combined with r-FSH can achieve higher ongoing pregnancy rates [7].

On the other side, some meta-analyses suggest that r-LH supplementation is not beneficial in the pregnancy rates in women in GnRH antagonist cycles [8–10]. No beneficial effect of LH addition during COS was found in young women with diminished ovarian reserve [11].

The above studies have not achieved consensus on the value of LH supplementation. Furthermore, the effects on embryo development and CLBR are less systematically evaluated. Thus, the present study aims to investigate whether patients with GnRH antagonist protocol benefit from the r-LH supplementation in embryo development, live birth rates in fresh cycles and frozen-thawed embryo transfer (FET) cycles, as well as the cumulative live birth rate (CLBR) in the complete cycles in multiple centers.

Methods

Study population and design

This multicenter retrospective cohort study recruited the IVF/ICSI cycles using GnRH antagonist protocol conducted at the Reproductive Medicine Center of the Sixth Affiliated Hospital of Sun Yat-sen University, Northwest Women’s and Children’s Hospital, and Jiangsu Provincial Hospital from January 2014 to December 2018. Only the first oocyte retrieval cycles of a patient and the subsequent FET cycles were included. Other inclusion criteria were autologous gametes and r-FSH used. Cycles were excluded if (1) the female partner underwent recurrent spontaneous abortions, hydrosalpinx, intrauterine adhesion, uterine malformation, submucosal myoma, adenomyosis, and thyroid dysfunction; (2) chromosomal abnormalities in either of the spouses; (3) human menopausal gonadotropin, letrozole or clomiphene was used for ovarian stimulation; (4) in vitro matured oocytes, frozen-thawed oocytes, embryo biopsies. We analyzed the oocyte retrieval cycles, fresh embryo transfer cycles, FET cycles and complete cycles (when at least a live birth was achieved or all embryos were transferred) from the included cycles. All cycles were followed up until December 2020.

This project was approved by Ethics Committees at the Sixth Affiliated Hospital of Sun Yat-sen University (2020ZSLYEC-295), Northwest Women’s and Children’s Hospital (2019013), and Jiangsu Provincial Hospital (2020-SR-046). The written informed consents were waived due to the retrospective nature of this study.

Controlled ovarian stimulation procedures and embryo evaluation

Fixed GnRH antagonist protocol (GnRH antagonist started on day 5 of r-FSH stimulation) and flexible GnRH antagonist protocol (GnRH antagonist started when the mean diameter of dominant follicles reached 12mm) [12] were adopted for controlled ovarian stimulation following the routine of the three reproductive centers. R-FSH of 100 to 300 IU/day was administered according to the individual characteristics, such as Anti-Mullerian hormone, basal FSH, LH, estradiol, progestin, and antral follicle counts (AFC) on days 2-3 of the menstrual cycle. Whether r-LH was supplemented was determined by the specialties.

Follicular growth was evaluated by serum concentration of estradiol, progestin, FSH, and LH, and transvaginal ultrasonography. Once the diameters of three dominant follicles ≥ 17mm or the diameters of two dominant follicles ≥ 18mm, human chorionic gonadotrophin was injected, and oocytes were retrieved after 36-38h. Most fresh embryos were transferred on day 3. Frozen-thawed embryo transfer was adopted when there was the risk of ovarian hyperstimulation syndrome (OHSS) [13], thin endometrium, elevated progesterone, and requirement of patients. The cleavage embryos were evaluated by Scott’s criteria [14]: grades I and II with ≥ 4 cells were usable; with ≥ 6 cells were of good quality. Blastocysts (days 5 and 6) were evaluated by Gardner’s system [15]: blastocysts graded as 3-6, and inner cell mass and trophectoderm assessed as AA, AB, AC, BA, BB, BC, CA, or CB were usable embryos, grade 3-6 blastocysts graded with AA, AB, BA, and BB were good-quality embryos.

Luteal phase support was performed with oral or vaginal progesterone until 8 weeks of gestation.

Outcome measures

The primary outcome was the CLBR which referred to the probability of achieving at least 1 live birth in a complete cycle.

Embryo outcomes were represented by the number of oocyte retrieval, number of 2 pronuclear (2PN) embryo and 2PN embryo rate after IVF, number of 2PN embryo and 2PN embryo rate after ICSI, usable embryo number and rate, good-quality embryo number and rate, mild/moderate OHSS [13] rate and cycle cancel rate in prevention of OHSS.

Pregnancy outcomes followed fresh embryo transfer and FET were clinical pregnancy rates, and live birth rate. Clinical pregnancy was defined as the observation of gestational sac(s) through a transvaginal probe. Live birth was defined as a live-born infant(s) after 28 gestational weeks.

Statistical analysis

PSM and data analysis were conducted with SAS version 9.4 (SAS Institute, Cary, NC, USA). Patients who underwent ovarian stimulation with r-FSH/r-LH and r-FSH were randomly matched with the 1:3 nearest neighbor matching method. The covariates included maternal age, paternal age, maternal body mass index (BMI), infertility factors, infertility type, basal FSH, AFC, and fertilization type.

Continuous variables that followed normal distribution were summarized as mean values (± standard deviations, SDs), and compared by student’s t-test. Data with skewed distribution were described as medians (quartiles) and Mann–Whitney U test was adopted for comparisons. Categorical variables were described as counts (percentages) and compared by Pearson Chi-square test. P < 0.05 was regarded as statistically significant.

Results

The process of inclusion and exclusion was shown in Figure. 1. A total of 6,621 oocyte retrieval cycles met the inclusion criteria, 899 cycles of which received r-FSH/r-LH administration. After PSM, 884 oocyte retrieval cycles received r-FSH/r-LH treatment were matched with 2,652 r-FSH stimulated cycles, 293 fresh embryo transfer cycles with r-FSH/r-LH treatment were matched with 879 r-FSH stimulated cycles, 753 FET cycles with r-FSH/r-LH treatment were matched with 2,259 r-FSH stimulated cycles, 702 complete cycles with r-FSH/r-LH treatment were matched with 2,106 r-FSH stimulated cycles.

Baseline characteristics

The baseline characteristics of the oocyte retrieval cycles are presented in Table 1. The parental ages, female BMI, infertility factors, infertility type, AFC, and fertilization type were not statistically significant after PSM. The basal FSH of the r-FSH/r-LH group was close to that of the r-FSH group (6.48 [5.48, 7.60] VS 6.26 [5.32, 7.52]), although the difference was significant (p = 0.012). Table 2 shows the baseline items of fresh embryo transfer cycles, Table 3 shows that of the FET cycles, and Table 4 displays the baseline characteristics of the complete cycles. The distributions of these characteristics were comparable in two groups after PSM.

Embryo development

The comparisons of embryo development between the r-FSH/r-LH and the r-FSH group are presented in Table 5. The r-FSH/r-LH group retrieved less oocytes than the r-FSH group (10.00 [7.00, 14.00] VS 12.00 [7.00, 17.00], p < 0.001). While the number of 2-pronuclear embryos was comparable whether fertilized by IVF or ICSI. Thus, the r-FSH/r-LH group presented higher 2-pronuclear embryo rate than the r-FSH group (IVF 2-pronuclear rate: 90.00% [77.78%, 100.00%] VS 86.96% [75.00%, 100.00%], p < 0.001; ICSI 2-pronuclear rate: 81.82% [62.50%, 93.75%] VS 75.00% [60.00%, 88.89%]). The numbers of usable embryos and good-quality embryos were comparable in the two groups. While the usable embryo rate in 2-pronuclear embryos was higher in the r-FSH/r-LH group [92.31% (75.00%, 100.00%) VS 87.50% (66.67%, 100.00%), p < 0.001]. No significant difference existed in the good-quality embryo rates of the two groups. Furthermore, the mild or moderate OHSS rate was similar in these groups (p = 0.864), 3.05% in the r-FSH/r-LH group versus 2.94% in the r-FSH group. The cycle cancel rate in prevention of OHSS was not statistically different in the two groups (r-FSH/r-LH group: 30.88%, r-FSH group: 27.53%, p = 0.055).

Pregnancy outcomes

After fresh embryo transfer (Table 6), 50.85% of r-FSH/r-LH cycles, and 49.49% of r-FSH cycles achieved clinical pregnancy (p = 0.686). While the live birth rate in fresh cycles was significantly higher in the r-FSH/r-LH group than in the r-FSH group (39.93% VS 28.10%, p < 0.001).

As for FET cycles (Table 7), the clinical pregnancy rate in the r-FSH/r-LH group was 63.75% versus 61.97% in the r-FSH group, no significant difference was observed. While the r-FSH/r-LH group obtained a significantly higher live birth rate than the r-FSH group (51.53% VS 43.16%, p < 0.001).

The CLBR in complete cycles (Table 8) of the r-FSH/r-LH group was also statistically higher than that of the r-FSH group (66.95% VS 61.16%, p = 0.006).

Discussion

Numerous clinical practices have proved that r-FSH alone is capable of inducing satisfactory follicle development during controlled ovarian stimulation. Although LH is critical for follicle growth and oocyte maturation, the benefit of r-LH supplementation in the GnRH antagonist regimen remains disputable. In this multicenter retrospective cohort study, we investigated the effects of r-LH supplementation on the whole process of IVF/ICSI in the same cohort for the first time. The r-LH supplementation was found to be associated with improved embryo developments, live birth rates in both fresh and frozen-thawed embryo transfer cycles, and the CLBR in complete cycles, perhaps by improving the quality of retrieved oocytes. The occurrence rates of OHSS and cycle cancel in prevention of OHSS were not increased.

The effects of r-LH supplementation on embryo development, OHSS rate, and cycle cancel rate have not been clearly investigated. Although a randomized study reported less OHSS incidence and lower cycle cancel rate in the r-LH supplementation group down-regulated by GnRH agonist [16], no significant differences were observed in the present study. The different conclusion might be attributed to the GnRH antagonist protocol in our study that reduced the OHSS occurrence compared with the GnRH agonist protocol [17].

A prospective randomized study, which focused on the GnRH antagonist administered cycles, reported that the number of oocytes retrieved was similar whether r-LH was supplemented or not [18]. While another prospective randomized study showed the number of oocytes recovered was relatively lower in the r-FSH/r-LH group (5.33 ± 4.8 VS 7.00 ± 3, p > 0.05), whose trend was consistent with the results of our study [19]. The estradiol level on the HCG day was also lower in the r-FSH/r-LH group (1,228.00 ± 830.59 VS 2,640.22 ± 1,221 pg/ml, p < 0.01) [19]. The above phenomenon suggested that r-LH supplementation did not help to improve ovarian response.

On the other side, elevated fertilization rate [6] and good quality embryo rate were observed when r-LH was supplemented [20]. LH was proved to promote folliculogenesis through (i) facilitating the synthesis of androgens for production of estradiol and induction of FSH receptor expression in the granulosa cells [21]; (ii) recruiting local growth factors, such as EGF, GDF9, and TGF-β to promote oocyte maturation [19, 22]; (iii) decreasing cumulus apoptosis rate [19]; (iv) resumption of meiosis and ovulation [23, 24].

Our results were in accordance with the function of LH. The data showed the r-LH supplementation was associated with an increased normal fertilization rate (2-pronuclear embryo rate of both IVF and ICSI), usable embryo rate, and live birth rate in FET cycles. This may represent that the appropriate concentration of r-LH facilitates nuclear and cytoplasmic maturation, thus contributing to better fertilization and embryogenesis.

Furthermore, exposure to low endogenous LH by down-regulation leads to stagnation of endometrium growth [25], decreasing endometrium receptivity [2], and decreasing implantation rate [25]. While the disturbance can be rescued by LH receptor stimulation through mid-cycle HCG supplementation [25]. The present study seems to coincide with this conclusion that the live birth rate was improved in the r-FSH/r-LH group. The findings were also in accordance with previous studies that a higher live birth rate was achieved when r-LH was supplemented in GnRH antagonist protocol [4–7]. However, the clinical pregnancy rates in fresh and FET cycles were comparable in the two groups, indicating a higher miscarriage rate in the r-FSH group, which might suggest the unsatisfactory developmental potential of embryos.

The impact of r-LH supplementation on CLBR is less investigated perhaps because of the complicated calculation. There is only one real-world study focusing on the poor ovarian responders and reporting the CLBR of moderate and severe poor ovarian responders is improved when r-LH was provided [3]. Our study suggests the CLBR is elevated in r-FSH/r-LH stimulated patients with GnRH antagonist pituitary down-regulation, perhaps through the elevated oocyte quality, promoted embryo developmental potential, optimized decidualization and receptivity.

Based on the consensus on LH supplementation among the Asia Pacific Fertility Advisory Group in 2011 [26], LH supplementation has been recommended to patients with central ovarian failure, poor ovarian response history with < 4 oocytes with FSH ≥ 300 IU/day, and unsatisfactory response to current COS cycle. Furthermore, patients aged > 35 years should consider r-LH supplementation due to the potential poor or suboptimal response, and the decreased bioactivity of endogenous LH [26]. Our study, on the other side, provides new evidence on the value of r-LH supplementation to common patients receiving COS through GnRH antagonist protocol.

The intrinsic nature of retrospective research is the shortage of the present study. However, the PSM adjusts the unbalance of demographic characteristics and reduces the bias as much as possible.

The strengths of this study include (i) the primary outcome, CLBR, is most concerned by patients and physicians, and evaluates the whole process of COS. This is the first study investigating the effects of r-LH supplementation on CLBR in patients undergoing the GnRH antagonist protocol. (ii) the multicentral study makes the conclusion more reliable and suitable for generalization.

Conclusions

In conclusion, r-LH supplementation to r-FSH in GnRH antagonist protocol was significantly associated with higher CLBR, live birth rate in fresh and FET cycles, and better embryos without increasing OHSS rate and cycle cancel rate. The effects might be achieved through the elevated oocyte quality, promoted embryo developmental potential, optimized decidualization and receptivity.

Abbreviations

r-LH: Recombinant Luteinization hormone; GnRH: Gonadotrophin releasing hormone; r-FSH: Recombinant-follicle stimulation hormone; PSM: Propensity score matching; CLBR: Cumulative live birth rate; FET: Frozen-thawed embryo transfer; OHSS: Ovarian hyperstimulation syndrome; COS: Controlled ovarian stimulation; IVF: in vitro fertilization; ICSI: Intracytoplasmic sperm injection; AFC: Antral follicle counts; 2PN: 2 pronuclear; BMI: Body mass index; SD: Standard deviation.

Declarations

Ethics approval and consent to participate

The present study obtained approvals from the Ethics Committees at the Sixth Affiliated Hospital of Sun Yat-sen University (2020ZSLYEC-295), Northwest Women’s and Children’s Hospital (2019013), and Jiangsu Provincial Hospital (2020-SR-046). The written informed consents were waived by the ethics committees because it is a retrospective study.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request

Competing interests

The authors declare that they have no competing interests.

Funding

The study is supported by the Fertility Research Program of Young and Middle-aged Physicians in 2019.

Authors' contributions

MW analyzed and interpreted the patient data, and was a major contributor to the manuscript. RH, XL, YM, WS collected the patients’ clinical data and contributed to the essay writing. QL designed the study and took part in the result interpretation. All authors read and approved the final manuscript.

Acknowledgements

Not applicable

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