Edited by: Panagiotis Drakopoulos, University Hospital Brussels, Belgium
Reviewed by: Gurkan Bozdag, Hacettepe University, Turkey; Mehtap Polat, Anatolia IVF Center, Turkey
*Correspondence: Yuan Li,
This article was submitted to Reproduction, a section of the journal Frontiers in Endocrinology
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To explore the association between serum LH levels and the cumulative live birth rate (CLBR) within one complete cycle, and the impact of serum LH levels on the live birth rate (LBR) after the initial embryo transfer (ET) considering different ET strategies (fresh or freeze-all).
A retrospective cohort study.
University-affiliated reproductive center.
1480 normogonadotrophic women who underwent COS with GnRH antagonist protocol for the first IVF/ICSI attempt.
The sample was stratified into low and higher LH groups according to serum LH peak levels of <4 (Group A) and ≥4 IU/L (Group B) during COS. Patients were also sub-grouped into conventional fresh/frozen ET cycles and freeze-all cycles.
The LBR after the initial embryo transfer and the CLBR within one complete cycle.
The numbers of day-3 high-quality embryos, the numbers of embryos available, and the other pregnancy outcomes after the initial ET.
In the whole cohort, the CLBRs decreased significantly in the low (63.1% vs. 68.3%,
In comparison with higher LH levels, low LH levels decrease the CLBRs per oocyte retrieval cycle for normogonadotrophic women who underwent COS using GnRH antagonists. This discrepancy may arise due to the significant detrimental effect of low LH levels on the LBRs after fresh embryo transfers.
Currently, the gonadotropin-releasing hormone (GnRH) antagonist protocol has already been one of the mainstream controlled ovarian stimulation (COS) protocols because of its convenience, safety, and comparable efficacy compared with the classical GnRH agonist long protocol (
Luteinizing hormone (LH) not only plays a central role in follicle development, ovulation, and steroidogenesis (
Concerning the LH threshold during COS using GnRH antagonists, by setting an absolute LH value or LH quartiles on a fixed predefined day, some scholars conclude that LH concentrations do not influence cycle outcomes (
To date, the number of studies investigating the effect of serum LH concentrations on pregnancy outcomes in GnRH antagonist-treated cycles remains limited. There is not yet a report about the association between LH levels and cumulative live birth rates (CLBRs), and little is known about the influence of LH levels on live birth rates (LBRs) when performing different embryo transfer (ET) strategies. This study’s objective was to address these considerations.
In this retrospective cohort study, we obtained data from patients who underwent COS using GnRH antagonist protocol for the first
All the patients enrolled were with good ovarian reserve, stimulated with GnRH antagonist protocol followed by either fresh ET or cryopreservation of all embryos, and were followed up until the treatment cycle was completed. Namely, all the embryos were used up, or a live birth was achieved. The inclusion criteria were as follows: (1) patients with good ovarian reserve, meaning age ≤38 years, basal serum FSH <10 IU/L, and antral follicle count (AFC) ≥6, (2) regular menstrual cycle, 21-35d, (3) body mass index (BMI) <30 kg/m2, (4) at least one embryo was available. The exclusion criteria were as follows: patients with a diagnosis of polycystic ovary syndrome, diabetes mellitus, hypogonadotropic amenorrhea, genital system tumors, abnormal uterine cavity morphology (i.e., Müllerian malformations, submucosal myoma, severe intrauterine adhesion, adenomyosis), or those who underwent preimplantation genetic testing. We also excluded the patients who happened a premature LH surge.
Ovarian stimulation was initiated with an individualized dose of 150-225 IU recombinant FSH (rFSH: Gonal-F®, Merck Serono) on day 3 of the menstrual cycle. Gonadotrophin dosage adjustment was allowed according to the follicular development monitored by serial transvaginal ultrasound scans and hormone measurements after 4-5 days of fixed-dose rFSH. Pituitary downregulation was performed with a flexible GnRH antagonist protocol. In brief, 0.125-0.25 mg cetrorelix acetate (Cetrotide®, Merck Serono, Geneva, Switzerland) was given when the leading follicular diameter ≥14 mm since stimulation day 5. The dosage and duration adjustment of the antagonist was allowed in terms of the clinician’s experience and discretion, based on the patient’s characteristics, follicular development, and subsequent LH levels. Recombinant LH (rLH: Leuveris®, Merck Serono) was supplemented when the follicles’ growth is slow or not synchronized with the hormone measurements from the day of GnRH-antagonist administered. Triggering of final oocyte maturation was performed with 0.2 mg of triptorelin (Decapeptyl®, Ferring) plus 2000 IU recombinant hCG (Ovitrelle®, Merck Serono) as soon as at least three follicles of 17 mm were visible, followed by ovum pick-up 34-36 hours later. The retrieved oocytes were fertilized by IVF or ICSI according to the status of the sperm.
As a routine clinical procedure in our center, all blood samples were drawn early in the morning, between 8 am and 10 am. Serum hormone profiles were measured as follows: (i) on the initial day of stimulation cycle, (ii) 4-5 days after the gonadotrophins administration, (iii) then every 1 to 2 days according to the individual follicular development and endocrine profile until the day of triggering. We recorded the LH peak levels and times of LH lower than 1.2 IU/L during the entire stimulation.
The hormone levels were analyzed at the central laboratory of Chao-Yang Hospital with an electrochemiluminescence immunoassay kit (Roche Diagnostics GmbH, Mannheim, Germany). The detection limits were 0.1 IU/L for FSH and LH, 5.00 pg/mL for E2, and 0.05 ng/mL for P. The inter-assay and intra-assay coefficients of variation were 2.46~4.55% and 5.10%~8.11% for E2, 3.78%~5.66% and 3.78%~5.92% for P, 3.79%~5.48% and 2.26%~5.16% for FSH, and 3.16%~5.66% and 3.12%~4.67% for LH, respectively.
Two good-quality cleavage embryos were routinely transferred or vitrified on the third day after ovum pick-up, and the remaining embryos were cultured for 2-3 more days for blastocyst vitrification. A good-quality embryo was defined as follows: the number of cells on day 3 was 7-9 cells, <20% fragmentation, and regular-sized cells. Freeze-all procedures were performed only in patients with a high risk of ovarian stimulation syndrome, serum P level exceeding 1.5 ng/mL during COS, or those with inadequate endometrial morphology or thickness. If available, up to two cleavage embryos were transferred in the first frozen-thawed embryo transfer (FET) cycle. Luteal phase support was administered in both fresh transfer and freeze-all cycles until 9-10 weeks after conception as described previously (
This study’s primary outcomes were the LBR after the initial ET (i.e., fresh ET or the first FET in the freeze-all cycle) and the CLBR within one complete treatment cycle. We defined the LBR as the delivery of a live infant born after 24 completed weeks of gestation. The secondary outcomes included the numbers of day-3 high-quality embryos, the numbers of embryos available, and the other pregnancy outcomes after the initial ET. We defined biochemical pregnancy as serum β-hCG level >15 IU/L at 12-14 days after embryo transfer. The implantation rate was calculated as the number of visible gestational sacs divided by the number of embryos transferred. We defined the clinical pregnancy as a pregnancy diagnosed by ultrasonographic visualization of one or more gestational sacs or definitive clinical signs of pregnancy at 7-8 gestational weeks and the early pregnancy loss as spontaneous pregnancy loss before 12 gestational weeks. These definitions are in accordance with the latest revision of “The International Glossary on Infertility and Fertility Care, 2017” (
Patients were stratified into two groups according to serum LH peak levels below or above 4 IU/L during the entire COS period. Comparisons between groups were carried out using the Student’s
The methods of searching and group divisions are shown in
Flow chart and group division of this study.
The patients’ baseline characteristics are summarized and described in
Baseline patient characteristics.
Characteristics | Group A | Group B |
|
---|---|---|---|
n=666 | n=814 | ||
Age (y), mean ± SD | 31.48 ± 3.61 | 32.18 ± 3.77 | <0.001 |
BMI (kg/m2), mean ± SD | 22.81 ± 3.41 | 22.84 ± 3.62 | 0.857 |
AFC, mean ± SD | 15.80 ± 7.61 | 15.97 ± 7.63 | 0.675 |
Hormone profile at baseline, mean ± SD | |||
E2 (pg/mL) | 47.95 ± 19.00 | 49.20 ± 18.34 | 0.201 |
FSH (IU/L) | 6.50 ± 2.12 | 6.77 ± 1.90 | 0.163 |
LH (IU/L) | 3.90 ± 2.67 | 4.75 ± 2.29 | <0.001 |
Infertility diagnosis, n (%) | 0.303 | ||
Primary | 403 (60.5%) | 471 (57.9%) | |
Secondary | 263 (39.5%) | 343 (42.1%) | |
Duration of infertility (y), median (IQR) | 2.5 (2-4) | 3 (2-4) | 0.942 |
Cause of infertility, n (%) | 0.962 | ||
Male factor | 56 (8.4%) | 69 (8.5%) | |
Female factor | 314 (47.1%) | 375 (46.1%) | |
Combined factors | 241 (36.2%) | 305 (37.5%) | |
Unexplained factor | 55 (8.3%) | 65 (8.0%) |
BMI, body mass index; AFC, antral follicle count; E2, estradiol; FSH, follicle-stimulation hormone; LH, luteinizing hormone.
COS and IVF/ICSI-ET parameters per LH group in the whole population are provided in
Parameters of ovarian stimulation and embryo transfer.
Characteristics | Group A | Group B |
|
---|---|---|---|
n=666 | n=814 | ||
Duration of stimulation (days) | 9.86 ± 1.46 | 9.77 ± 1.40 | 0.223 |
Total gonadotrophin |
2297.54 ± 788.52 | 2327.99 ± 804.57 | 0.465 |
Total antagonist dose (mg) | 0.375 (0.125-0.5) | 0.5 (0.25-0.625) | <0.001 |
rLH supplementation |
375 (150-900) | 300 (75-750) | <0.001 |
Percentage of rLH |
561 (84.2%) | 614 (75.4%) | <0.001 |
Hormone profile at trigger day | |||
E2 (pg/mL) | 3614.13 ± 2118.49 | 3583.40 ± 2257.76 | 0.789 |
P (ng/mL) | 0.91 ± 0.58 | 0.96 ± 0.69 | 0.229 |
LH (IU/L) | 2.11 ± 1.01 | 3.13 ± 1.92 | <0.001 |
Frequencies of LH below 1.2 IU/L | |||
0 | 268 (40.2%) | 495 (60.8%) | <0.001 |
1 | 224 (33.6%) | 246 (30.2%) | 0.161 |
≥2 | 174 (26.1%) | 73 (9.0%) | <0.001 |
FORT |
0.54 ± 0.25 | 0.52 ± 0.24 | 0.091 |
IVF | 454 (68.2%) | 537 (66.0%) | 0.371 |
ICSI or IVF - ICSI split | 212 (31.8%) | 277 (34.0%) | |
Fertilization rate (IVF) | 0.62 ± 0.21 | 0.60 ± 0.24 | 0.069 |
Fertilization rate (ICSI) | 0.74 ± 0.20 | 0.76 ± 0.21 | 0.256 |
No. of oocytes retrieved | 14.46 ± 6.96 | 14.21 ± 7.17 | 0.492 |
No. of MII oocytes | 9.84 ± 5.62 | 9.56 ± 5.56 | 0.589 |
No. of good-quality embryos on day 3 | 4.29 ± 3.49 | 4.23 ± 3.50 | 0.769 |
No. of total embryos available | 4.23 ± 2.41 | 4.18 ± 2.38 | 0.691 |
|
|||
Fresh ET | 208 (31.2%) | 233 (28.6%) | 0.275 |
Frozen-thawed ET | 458 (68.8%) | 581 (71.4%) | |
Endometrial thickness | 9.56 ± 1.96 | 9.42 ± 1.87 | 0.162 |
No. of embryos transferred | 1.95 ± 0.31 | 1.97 ± 0.32 | 0.316 |
Cleavage embryos transferred | 599 (89.9%) | 731 (89.8%) | 0.931 |
Blastocyst | 67 (10.1%) | 83 (10.2%) |
Data are mean ± SD, median (IQR), or n (%). E2, estradiol; P, progesterone; rLH, recombinant LH; MII, metaphase II; ET, embryo transfer.
FORT= follicle output rate, calculated as the number of 16-22 mm preovulatory follicles/the number of 3-8 mm antral follicles on the third day of the menstrual cycle.
We also divide patients according to the ovarian response.
In the whole cohort, the overall CLBR per oocyte retrieval cycle decreased significantly in the low LH group than in the higher LH group (420/666, 63.1% vs. 556/814, 68.3%,
Pregnancy outcomes of the low and higher LH groups stratified by embryo transfer strategy.
The pregnancy outcomes of patients with a different ovarian response in the low and higher LH groups after different ET strategies were summarized and described respectively in
Pregnancy outcomes of the low and higher LH groups based on different ovarian responses and embryo transfer strategies.
Oocytes Count | 4-9 | 10-15 | >15 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Fresh cycle, N=210 | Freeze-all cycle, N=209 | Fresh cycle, N=159 | Freeze-all cycle, N=317 | Fresh cycle, N=59 | Freeze-all cycle, N=513 | |||||||
Group A, n=98 | Group B, n=112 | Group A, n=86 | Group B, n=123 | Group A, n=77 | Group B, n=82 | Group A, n=129 | Group B, n=188 | Group A, n=27 | Group B, n=32 | Group A, n=243 | Group B, n=270 | |
Biochemical pregnancy rate, % (n) | 51.0% (50) | 59.8% (67) | 50.0% (43) | 54.5% (67) | 51.9% (40) | 59.8% (49) | 52.7% (68) | 64.9% (122)c | 55.5% (15) | 62.5% (20) | 68.7% (167) | 66.3% (179) |
Implantation rate, % (n/N) | 35.3% (65/184) | 37.5% (84/224) | 26.8% (44/164) | 31.7% (73/230) | 32.1% (50/156) | 37.2% (61/164) | 34.3% (87/254) | 38.0% (132/347) | 30.2% (16/53) | 38.1% (24/63) | 43.1% (206/478) | 41.6% (222/534) |
Clinical pregnancy rate, % (n) | 40.8% (40) | 53.5% (60)b | 38.4% (33) | 48.0% (59) | 39.0% (30) | 52.4% (43)b | 47.3% (61) | 53.2% (100) | 44.4% (12) | 56.3% (18) | 58.8% (143) | 55.6% (150) |
Early pregnancy loss rate, % (n) | 28.0% (14) | 14.9% (10)b | 27.9% (12) | 14.9% (10)b | 25.0% (10) | 16.3% (8) | 11.8% (8) | 21.3% (26) | 20.0% (3) | 10% (2) | 18.0% (30) | 16.2% (29) |
Live birth rate, % (n) | 36.7% (36) | 50.9% (57)a | 36.0% (31) | 46.3% (57) | 38.9% (30) | 50.0% (41) | 46.5% (60) | 51.1% (96) | 44.4% (12) | 56.3% (18) | 56.4% (137) | 54.8% (148) |
Cumulative live birth rate, % (n) | 46.9% (46) | 59.8% (67)b | 45.3% (39) | 54.5% (67) | 61.0% (47) | 68.2% (56) | 60.5% (78) | 63.3% (119) | 74.1% (20) | 84.4% (27) | 77.8% (189) | 80.0% (216) |
aP=0.039; bP<0.1; cP=0.030.
The results of multivariate logistic regression analyses are presented in
Multivariate logistic regression analysis by treatment outcome.
Outcome | Variable | COR (95% CI) | P value | AOR (95% CI) | P value |
---|---|---|---|---|---|
|
Low LH* | 0.792 |
0.034 | 0.756 |
0.014 |
No of oocytes retrieved | 1.090 |
<0.001 | 1.091 |
<0.001 | |
|
Low LH* | 0.577 |
0.005 | 0.532 |
0.002 |
P level at trigger day | 0.688 |
0.103 | 0.601 |
0.042 | |
Endometrial thickness | 1.170 |
0.003 | 1.191 |
0.002 | |
No of embryo transferred | 1.932 |
0.008 | 2.014 |
0.009 | |
|
Low LH* | 0.622 |
0.016 | 0.596 |
0.017 |
No of oocytes retrieved | 1.107 |
<0.001 | 1.108 |
<0.001 | |
|
Low LH* | 0.922 |
0.517 | 0.918 |
0.508 |
|
Low LH* | 0.896 |
0.414 | 0.864 |
0.307 |
No of oocytes retrieved | 1.091 |
<0.001 | 1.081 |
<0.001 |
*To evaluate the LH effect on pregnancy outcomes, the higher LH group was taken as reference. aAdjusted for ET strategy (fresh vs. freeze-all), female age, infertility duration, baseline FSH and LH levels, total Gn dose, dose of GnRH antagonists and rLH supplementation, E2 levels at trigger day, and oocytes count. bAdjusted for dose of GnRH antagonists, progesterone and LH levels at trigger day, endometrial thickness at trigger day, and number of embryos transferred. cAdjusted for dose of GnRH antagonists, type and duration of infertility, baseline LH levels, E2 and LH levels at trigger day, and oocytes count. dAdjusted for the total Gn dose, dose of rLH supplementation, E2 levels at trigger day, oocytes count, number and stage of embryos transferred. eAdjusted for female age, duration of infertility, baseline FSH and LH levels, dose of Gn and rLH supplementation, E2 levels at trigger day, and oocytes count.
CLBR, cumulative live birth rage; LBR, live birth rate; FET, frozen-thawed embryo transfer.
The present study assessed the association between serum LH levels and the CLBR per oocyte retrieval cycle and LBR after the initial ET in reproductive-aged normogonadotrophic women in GnRH antagonist IVF/ICSI cycles, in consideration of different ET strategies, for the first time. Our results suggest that low LH levels significantly decrease the likelihood of CLBs by utilizing all fresh and frozen embryos from one stimulated cycle. Furthermore, the harmful effect of low LH mainly exists in fresh embryo transfers.
In this study, we divided the patients into low and higher LH groups arbitrarily at the cut-off value of 4 IU/L according to our former experience (
We have already known that profoundly LH suppression is detrimental for patients undergoing either GnRH agonist or GnRH antagonist-treated cycles (
While the role of LH in ovarian stimulation is universally accepted, a question remains whether serum LH levels influence pregnancy outcomes. Some authors failed to find any significant difference between different LH groups regarding the implantation rates, clinical pregnancy rates, and ongoing pregnancy rates (
The underlying mechanism by which low LH levels seem to reduce pregnancy rates has not been fully elucidated. We are not able to perform single nucleotide polymorphism analysis in routine clinical practice yet. But we can look for clues from the existing clinical data. Expect for the quality of embryos transferred, a slow luteinization process, a delayed corpus luteum function, or a poor endometrium receptivity will also decrease the LBR after fresh ETs. Accordingly, our data suggest that low serum LH levels during COS might affect the patients’ corpus luteum function or endometrium receptivity, then cause the asynchrony between the embryo and the endometrium, potentially resulting in implantation failure and poor reproductive outcomes.
The biological activity of LH is conferred primarily through binding to the specific LH receptor (LHCG-R), which is mainly expressed on ovarian theca, mural granulosa, and luteal cells (
To the best of our knowledge, this paper is the first to investigate the association between LH levels during the entire COS process and reproductive outcomes, considering both the conventional fresh/frozen ET strategy and the freeze-all strategy. The long-term follow-up of the cohort allowed us to provide information on the most clinically meaningful outcomes, i.e., CLBR, which could reflect the utilization of all the embryos obtained from the stimulation cycle. Most importantly, our analysis revealed that low LH’s detrimental effect was pronounced only in fresh ET cycles. The utilization of elective FET has increased significantly in recent years due to the introduction of the GnRH agonist trigger protocol and improvements in cryopreservation techniques. However, whether or not the freeze-all policy should be offered to the overall IVF population remains controversial (
The main weakness of this study is its retrospective nature. To minimize confounding potential, we strictly selected patients according to inclusion and exclusion criteria and adjusted our analysis for multiple variables. The confounding factors, either previously known to affect LBRs or varied significantly between the study groups, were all mentioned. Only the variables showing a tendency of association with CLBs or LBs in the univariate analyses (
In summary, our study demonstrated that LH levels during COS with the GnRH antagonist protocol were individualized, and the low serum LH levels were associated with a decreased CLBR per oocyte retrieval cycle and LBR after fresh ET. Currently, individually tailored ovarian stimulation is advocated. Although needing further confirmation, our results indicate that more attention should be paid to LH levels and activities, especially when the follicular development was asynchronous with serum hormone profiles when performing COS, to adjust the medication regimen accordingly and to aim towards LH levels in an optimal range in an attempt to maximize the reproductive outcomes. In the future, basic research focusing on different LH levels in assisted reproduction is needed. Meanwhile, whether the freeze-all strategy was superior to the fresh transfer strategy for patients with improper LH levels remains to be examined.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by the Ethics Committee of Chao-Yang Hospital. The patients/participants provided their written informed consent to participate in this study.
YYL contributed to data collection, analysis and interpretation, manuscript drafting, and critical discussion. SL contributed to the critical discussion and revision of the manuscript. LH contributed to the analysis and interpretation of the data. HS and YHR contributed to the critical discussion of the manuscript. HML, TYW, and HHL contributed to the data collection and revision of the manuscript. YL contributed to the study design, supervision, interpretation of data, and critical discussion of the manuscript. All authors contributed to the article and approved the submitted version.
This study was supported by the 2018 Fertility Research Program of Young and Middle-aged Physicians-China Health Promotion Foundation.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank the staff from the Medical Center for Human Reproduction of Chao-Yang Hospital for their assistance with the data collection and all the participants in this study.
The Supplementary Material for this article can be found online at:
Distribution of the categories of the number of oocytes retrieved (<4, 4-9, 10-15, >15) of the low and higher LH groups.