Abstract
Purpose
This study aimed to investigate which patient and cycle characteristics may affect the number of mature oocytes and cryopreservable blastocysts in the GnRH analog trigger cases.
Methods
This was a retrospective cohort study of 2749 GnRHa trigger cycles in patients at risk of OHSS, including a group of PGT patients, between 2011 and 2020 at Istanbul Memorial Hospital, ART and Reproductive Genetics Center. Patient and cycle characteristics were evaluated using the Generalized Linear Mixed Model (GLMM). The number of mature oocytes and the number of cryopreservable blastocysts were evaluated.
Results
A one-unit increase in female age, daily gonadotropin dose, E2 level on day 2, and LH level on trigger day significantly decreased the number of mature oocytes retrieved (p < 0.001) and the number of cryopreservable blastocysts as p < 0.001, p < 0.001, p < 0.001, and p = 0.003, respectively. The duration of GnRH antagonist use also decreased the number of mature oocytes retrieved (p < 0.001) but not the number of cryopreservable blastocysts.
Conclusion
The GLMM used in our study showed that a one-unit increase in female age, daily gonadotropin dose, E2 level on day 2, and LH level on trigger day significantly decreased the number of mature oocytes retrieved and the number of cryopreservable blastocysts.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10815-022-02702-x.
Keywords: GnRHa trigger, Mature oocytes, Blastocyst
Introduction
Ovarian hyperstimulation syndrome (OHSS) is a life-threatening complication of treatment with assisted reproductive technology (ART). The administration of gonadotropin-releasing hormone analogs (GnRHa) to induce final oocyte maturation is an effective strategy to prevent OHSS [1–3]. This strategy is not limited to cases with polycystic ovary syndrome (PCOS). It can also be used in patients undergoing the freeze-all procedure for various reasons, for example, social egg freezing, preimplantation genetic testing (PGT), fertility preservation in cancer patients, and patients whose hormonal and ultrasonographic findings in the late follicular phase indicate hyperresponses and, thus, the risk of OHSS.
Although the GnRHa trigger is currently recognized as a safer strategy, there is concern that some patients may respond sub-optimally to the GnRHa trigger, resulting in a lower number or absence of mature oocytes and cryopreservable blastocysts [4–7].
There is insufficient information in the literature about the patient and cycle-specific characteristics that could negatively affect the number of mature oocytes and the number of cryopreservable blastocysts.
In our study, we included all these patient and cycle parameters in a generalized linear mixed model (GLMM) to assess which patient and cycle characteristics affect the number of mature oocytes and the number of cryopreservable blastocysts in GnRH-analog trigger cases.
Materials and methods
Patient population
This retrospective cohort study was conducted between August 2011 and December 2020 at the Istanbul Memorial Hospital ART and Reproductive Genetics Center and approved by the Institutional Review Board of Istanbul Memorial Hospital (number: 25.06.2021/004). The data were taken from our center-specific aura database. Two thousand four hundred seventy-nine cycles in which GnRHa was used to avoid the risk of OHSS were evaluated. In 1146 cases, it was their first ART cycle, while in 1603 cases, previous ART cycles had been performed. The policy of our center is to eliminate any risk of OHSS. Therefore, GnRHa triggers and the freeze-all strategy were used in cases with high ovarian reserve, whether or not they had a history of hyperresponse or OHSS. Patients at risk of OHSS were informed that GnRHa triggers would be administered, no embryo transfer would be performed, and a freeze-all strategy would be used. In addition, the study also included cases whose hormonal and ultrasonographic findings in the late follicular phase indicated a hyper-response and OHSS risk and for whom the trigger strategy was therefore changed from recombinant human chorionic gonadotropin (rhCG) to GnRHa, as well as patients undergoing PGT for aneuploidy testing who had evidence of a possible hyperresponse. After giving verbal and written information, each patient signed a consent form agreeing to a freeze-all procedure.
Patients with a history of hyper-response in their previous cycles were identified. However, if it was their first cycle, anti-Mullerian hormone (AMH) and/or antral follicle count (AFC) were considered. The total number of follicles on day 2 and the number of follicles above 11 mm on trigger day were routinely recorded. Factors that indicated the use of the GnRHa trigger were a high AMH level (> 3 ng/ml) and/or a high AFC (> 12 or more in each ovary) on day 2. However, the concordance of AMH level and AFC on day 2 was not observed in every patient, so the main indication for a GnRHa trigger was the number and size of follicles on the trigger day (> 15 follicles over 11 mm).
Severe male infertility cases with cryptozoospermia or azoospermia, which could affect fertilization rate and embryo development, were excluded. A detailed history of any pretreatments, diagnosis of hypothalamic amenorrhea, and/or hypothalamic dysfunction was recorded.
Patient-specific factors included female age, body mass index (BMI), AMH level, day 2 luteinizing hormone level (LH), progesterone level (P), estradiol level (E2), and duration of GnRH antagonist use. On the GnRHa trigger day, E2 and LH levels were determined. In some cases, trigger day was postponed by 1 day despite the sufficient size of the leading follicle(s). This was to allow further growth of the middle-sized follicles. P levels were then recorded on trigger day (n = 458). In addition, on the day after the trigger, LH and P levels were recorded in cases where the LH level was below 0.5 IU/L on the trigger day, as recommended in some studies [4, 6, 8]. The total number of days of controlled ovarian stimulation (COS), the duration of GnRH antagonist use, infertility factors, and the additional LH preparation used were included in the analyses.
The number of obtained oocytes (COC), mature oocytes (MII), normally fertilized oocytes with 2PN, and the number of good and top-quality cryopreservable blastocysts were recorded. The mean number of mature oocytes in the study population was 15.9 (min–max 1–60), the mean number of blastocysts in the study population was 6.9 (min–max 1–37), and the mean blastocyst formation rate from PN2 stage to blastocyst stage was 53%.
Ovarian stimulation
A short GnRH antagonist protocol was used in all cases, whether they had received pretreatment. Recombinant follicle-stimulating hormone (rFSH) (Gonal-F, Merck, Switzerland) was used. However, as there is insufficient data in the literature on the use of recombinant luteinizing hormone (rLH) in cases with FSH-only preparations and that have low LH levels in the late follicular phase, a combination of rFSH and rLH (Luveris, Merck, Switzerland) was used (n = 590).
The initial dose of gonadotropins ranged from 75 to 225 IU, depending on the BMI of the patients, the response of the ovaries to any previous stimulation(s), and the number of AFC, and the initial dose was adjusted according to the response of the individual patient. Oocyte pick-up (OPU) was performed 36 h after injection of 0.4 mg GnRHa (Lucrin, Abbott Laboratories, USA) or 2 ampoules (0.2 mg) GnRHa (Gonapeptyl, Ferring, Switzerland) under transvaginal ultrasound guidance. Although studies in the literature have not shown significant differences in the number of retrieved oocytes and top and good-quality embryos between patients receiving different GnRHa doses (0.4 mg vs. 0.2 vs. 0.1 mg), we opted for the higher dose to avoid the possible risk of the patient not self-administering a fully effective dose [9–11].
The freeze-all procedure was used and so these cases did not receive any medication after OPU. However, in cases with enlarged ovaries, a GnRH antagonist (Cetrotide, Merck, Switzerland) was administered for 5 days to reduce the volume of the ovaries and prevent ovarian torsion.
COC collection
On the day of the OPU, the follicles were aspirated one by one. The gynecologist who performed the collection informed the embryologist about the size of the follicle for each puncture. This is the routine protocol at our center to follow the embryonic development of each egg retrieved from small, medium, and large follicles.
A second embryologist assisted in the procedure to document the process of isolation, identification, and positioning of the cumulus oocyte complexes (COCs) in the culture dish. The oocytes were incubated at 37 °C and 6% CO2 until denudation. COCs were denuded both enzymatically and mechanically 3 h after OPU by pipetting the oocytes in hyaluronidase solution (Irvine Scientific, USA). ICSI was performed approximately 40 h after GnRHa administration under 400 × magnification using Olympus IX70 and Olympus IX71 inverted microscopes. A single-step culture medium (Life Global®, Brussels, Belgium) supplemented with 10% Plasmanate (Life Global®, Brussels, Belgium) was used for embryo culture. Fertilization was checked 16–18 h after ICSI, and blastocysts were scored according to Gardner classification (114–120 h after ICSI) and vitrified based on final morphology and score.
The number of collected oocytes, mature oocytes, and cryopreserved blastocysts was recorded.
Vitrification
A routine blastocyst stage freeze-all procedure was performed in all analog trigger cases. On day 5 or day 6, good and top-quality blastocysts (at least 3BB) were vitrified using Kitazato vitrification media and Cryotops® as carriers.
Statistical analyses
The NCSS (Number Cruncher Statistical System) Statistical Software (Utah, USA) program was used for the statistical analyses. In the evaluation of the data obtained from the study, the Shapiro–Wilk test and boxplot diagrams were used with regard to the agreement of the descriptive statistical methods (mean, standard deviation, median, IQR) and the variables with a normal distribution. The one-way ANOVA test for group comparison of continuous variables and Pearson’s chi-square test for group comparison of categorical variables were applied. The generalized linear mixed model (GLMM) was used for univariate and multivariate evaluations of the risk factors influencing the number of mature oocytes and the number of cryopreservable blastocysts. The multicollinearity of the linear models was assessed based on variance inflation factor (VIF) values. Significance was assessed at a level of p < 0.05.
Results
Table 1 shows the number of cases and the criteria evaluated with the generalized linear mixed model.
Table 1.
The number of cases and the criteria evaluated by generalized linear mixed model
| Characteristics | Number | Median (IQR) |
|---|---|---|
| Female age (year) | 2749 | 31 (28, 35) |
| BMI (kg/m2) | 2749 | 24.1 (21.5, 27.6) |
| AMH level (ng/mL) | 2722 | 3.99 (2.63, 6.02) |
| Additional LH preparation used (n) | 2749 | 590 (21.5) |
| Day2 progesterone level (µg/L) | 2698 | 0.2 (0.1, 0.3) |
| Day2 E2 level (ng/L) | 2711 | 37 (28, 48) |
| Day2 LH level (IU/L) | 2707 | 6.6 (5.1, 8.6) |
| Cycle length (day) | 2746 | 9 (8, 10) |
| Total gonadotropin dosage used (IU) | 2749 | 1925 (1425, 2325) |
| Daily gonadotropin dosage used (IU) | 2749 | 213.89 (157.5, 243.18) |
| Trigger day E2 level (ng/L) | 2746 | 3544.5 (2531, 4976) |
| Trigger day LH level (IU/L) | 2749 | 1.8 (1.1, 2.9) |
| Number of follicles ˃ 11 mm on trigger day (n) | 2749 | 15 (12, 18) |
| Maturation rate (%) | 2749 | 88 (78.6, 95.2) |
| Fertilization rate (%) | 2749 | 84.6 (73.9, 92.3) |
| Number of frozen blastocysts (n) | 2749 | 6 (4, 9) |
| Post-trigger day progesterone level (µg/L) | 477 | 11.3 (8.3, 14.8) |
| Post-trigger day E2 level (ng/L) | 97 | 6321 (4287, 8008) |
| Post-trigger day LH level (IU/L) | 485 | 46 (34, 62) |
| Duration of GnRH antagonist use (day) | 2749 | 5 (4, 6) |
Table 2. shows the evaluation of univariable and multivariable factors in relation to the number of mature oocytes.
Table 2.
Evaluation of univariable and multivariable factors in relation to the number of mature oocytes
| Characteristics | Number of mature oocytes | |||
|---|---|---|---|---|
| Univariable | Multivariable | |||
| Beta (%95 CI) | p | Beta (%95 CI) | p | |
| Female age (year) | − 0.336 (− 0.393, − 0.279) | < 0.001* | − 0.202 (− 0.255, − 0.150) | < 0.001* |
| BMI (kg/m2) | 0.113 (0.052, 0.173) | < 0.001* | 0.277 (0.217, 0.338) | < 0.001* |
| Infertility factors | < 0.001* | < 0.001* | ||
| Male factor | 0.836 (− 0.026, 1.699) | 0.057 | 0.114 (− 0.577, 0.804) | 0.746 |
| Ovulatory dysfunction | 2.17 (1.291, 3.049) | < 0.001* | 1.026 (0.298, 1.755) | 0.006* |
| Genetic factor | 2.441 (1.26, 3.622) | < 0.001* | 2.046 (0.933, 3.160) | < 0.001* |
| Tubal factor | 1.457 (− 0.15, 3.064) | 0.075 | 0.388 (− 0.894, 1.671) | 0.553 |
| Endometriosis | 1.743 (− 1.739, 5.225) | 0.326 | 2.035 (− 0.701, 4.771) | 0.145 |
| Combined factor | 2.586 (1.599, 3.572) | < 0.001* | 1.296 (0.493, 2.100) | 0.002* |
| Unexplained | Reference | – | ||
| Duration of infertility (year) | − 0.099 (− 0.178, − 0.021) | 0.013* | 0.004 (− 0.061, 0.070) | 0.901 |
| Day2 FSH level (mIU/mL) | − 0.872 (− 1.122, − 0.622) | < 0.001* | ||
| Day2 progesterone level (µg/L) | 1.335 (0.625, 2.044) | < 0.001* | 1.755 (1.008, 2.501) | < 0.001* |
| Day2 E2 level (ng/L) | − 0.018 (− 0.031, − 0.004) | 0.011* | − 0.040 (− 0.052, − 0.027) | < 0.001* |
| Day2 LH level (IU/L) | 0.451 (0.369, 0.532) | < 0.001* | 0.209 (0.131, 0.287) | < 0.001* |
| Trigger day E2 level (ng/L) | 0.002 (0.002, 0.002) | < 0.001* | 0.002 (0.002, 0.002) | < 0.001* |
| Trigger day LH level (IU/L) | − 0.275 (− 0.416, − 0.134) | < 0.001* | − 0.698 (− 0.823, − 0.572) | < 0.001* |
| Post-trigger day progesterone level (µg/L) | 0.405 (0.31, 0.5) | < 0.001* | ||
| Post-trigger day E2 level (ng/L) | 0.001 (0.001, 0.002) | < 0.001* | ||
| Post-trigger day LH level (IU/L) | 0.019 (− 0.007, 0.045) | 0.150 | ||
| Duration of GnRH antagonist use (day) | − 0.294 (− 0.519, − 0.068) | 0.011* | − 0.497 (− 0.756, − 0.238) | < 0.001* |
| Total gonadotropin dosage used (IU) | − 0.002 (− 0.002, − 0.001) | < 0.001* | ||
| Daily gonadotropin dosage used (IU) | − 0.027 (− 0.031, − 0.023) | < 0.001* | − 0.025 (− 0.030, − 0.021) | < 0.001* |
| Cycle length (day) | 0.178 (0.003, 0.352) | 0.046* | − 0.038 (− 0.232, 0.158) | 0.704 |
| Additional LH preparation used (n) | − 1.347 (− 2.042, − 0.652) | < 0.001* | − 0.348 (− 0.974, 0.278) | 0.275 |
| AMH level (ng/mL) | 0.194 (0.109, 0.279) | < 0.001* | 0.051 (− 0.021, 0.123) | 0.163 |
| Number of follicles ˃ 11 mm on trigger day (n) | 0.084 (0.034, 0.135) | 0.001* | 0.030 (− 0.012, 0.073) | 0.162 |
Variables with a value of p < 0.200 in the univariable evaluations were included in the multivariable analyses
GLMM generalized linear mixed model
*p < 0.05
In this statistical model, according to multivariable analyses, a one-unit increase in BMI, LH level on day 2, progesterone level on day 2, and E2 level on trigger day significantly increased the number of mature oocytes, with a value of 0.277 (p < 0.001), 0.209 (p < 0.001), 1.755 (p < 0.001), and 0.002 (p < 0.001), respectively. The presence of ovulatory dysfunction, genetic factors, and combined infertility factors (two or more) significantly increased the number of mature oocytes with a value of 1.026 (p = 0.006), 2.046 (p < 0.001), and 1.296 (p < 0.002), respectively. Conversely, a one-unit increase in the female age, the E2 level on day 2, the LH level on trigger day, the duration of GnRH antagonist use, and the daily gonadotropin dose significantly decreased the number of mature oocytes with a value of 0.202 (p < 0.001), 0.04 (p < 0.001), 0.698 (p < 0.001), 0.497 (p < 0.001) and 0.025 (p < 0.001), respectively.
Higher female age, higher E2 level on day 2, higher LH level on trigger day, longer duration of GnRH antagonist use, and consequently higher daily gonadotropin dosage may have led to a decrease in the number of mature oocytes.
Table 3 shows the evaluation of univariable and multivariable factors in relation to the number of cryopreservable blastocysts.
Table 3.
Evaluation of univariable and multivariable factors in relation to the number of cryopreservable blastocysts
| Characteristics | Number of cryopreservable blastocyst | |||
|---|---|---|---|---|
| Univariable | Multivariable | |||
| Beta (%95 CI) | p | Beta (%95 CI) | p | |
| Female age (year) | − 0.194 (− 0.226, 0.163) | < 0.001* | − 0.151 (− 0.184, − 0.118) | < 0.001* |
| BMI (kg/m2) | − 0.012 (− 0.045, 0.022) | 0.486 | ||
| Infertility factors | 0.014* | 0.007* | ||
| Male factor | − 0.062 (− 0.543, 0.418) | 0.799 | − 0.246 (− 0.683, 0.192) | 0.270 |
| Ovulatory dysfunction | 0.592 (0.102, 1.081) | 0.018* | 0.561 (0.107, 1.014) | 0.015* |
| Genetic factor | 0.903 (0.247, 1.56) | 0.007* | 0.338 (− 0.370, 1.047) | 0.349 |
| Tubal factor | 0.399 (− 0.495, 1.293) | 0.382 | 0.357 (− 0.458, 1.171) | 0.391 |
| Endometriosis | 0.507 (− 1.43, 2.444) | 0.608 | 1.062 (− 0.681, 2.804) | 0.232 |
| Combined factor | 0.428 (− 0.122, 0.977) | 0.127 | 0.410 (− 0.093, 0.914) | 0.110 |
| Unexplained | Reference | |||
| Duration of infertility (year) | − 0.128 (− 0.171, 0.085) | < 0.001* | − 0.054 (− 0.095, − 0.013) | 0.010* |
| Day2 FSH level (mIU/mL) | − 0.385 (− 0.531, − 0.24) | < 0.001* | ||
| Day2 progesterone level (µg/L) | 0.431 (0.038, 0.823) | 0.031* | 0.396 (− 0.069, 0.861) | 0.095 |
| Day2 E2 level (ng/L) | − 0.009 (− 0.017, 0.002) | 0.018* | − 0.016 (− 0.023, − 0.008) | < 0.001* |
| Day2 LH level (IU/L) | 0.216 (0.171, 0.262) | < 0.001* | 0.133 (0.084, 0.182) | < 0.001* |
| Trigger day E2 level (ng/L) | 0.001 (0.001, 0.001) | < 0.001* | 0.001 (0.001, 0.001) | < 0.001* |
| Trigger day LH level (IU/L) | − 0.119 (− 0.198, − 0.04) | 0.003* | − 0.255 (− 0.333, − 0.178) | < 0.001* |
| Post-trigger day progesterone level (µg/L) | 0.187 (0.127, 0.247) | < 0.001* | ||
| Post-trigger day E2 level (ng/L) | 0 (0, 0.001) | 0.092 | ||
| Post-trigger day LH level (IU/L) | 0.012 (− 0.004, 0.028) | 0.142 | ||
| Duration of GnRH antagonist use (day) | 0.006 (− 0.119, 0.132) | 0.924 | ||
| Total gonadotropin dosage used (IU) | − 0.001 (− 0.001, 0.001) | < 0.001* | ||
| Daily gonadotropin dosage used (IU) | − 0.01 (− 0.013, − 0.008) | < 0.001* | − 0.004 (− 0.006, − 0.001) | 0.003* |
| Cycle length (day) | 0.082 (− 0.015, 0.179) | 0.096 | ||
| Additional LH preparation used (n) | − 0.276 (− 0.664, 0.111) | 0.162 | ||
| AMH level (ng/mL) | 0.013 (− 0.034, 0.060) | 0.590 | ||
| Number of follicles ˃ 11 mm on trigger day (n) | 0.044 (0.016, 0.072) | 0.002* | 0.022 (− 0.004, 0.049) | 0.096 |
Variables with a value of p < 0.200 in the univariable evaluations were included in the multivariable analyses
GLMM generalized linear mixed model
*p < 0.05
In this statistical model, ovulatory dysfunction, a one-unit increase in LH level on day 2, and a one-unit increase in E2 level on trigger day significantly increased the number of cryopreservable blastocysts with a value of 0.561 (p = 0.015), 0.133 (p < 0.001) and 0.001 (p < 0.001) respectively. A one-unit increase in female age, E2 level on day 2, LH level on trigger day, infertility duration, and daily gonadotropin dosage significantly decreased the number of cryopreservable blastocysts with a value of 0.151 (p < 0.001), 0.016 (p < 0.001), 0.255 (p < 0.001), 0.054 (p = 0.010), and 0.004 (p = 0.003) respectively.
Evaluation of the LH values on day 2 and on the trigger day revealed 26 cases with extremely low LH values on day 2 (< 1 IU/L) and on the trigger day (< 0.1 IU/L). Examination of the cases with extremely low LH on day 2 revealed that of these 7 cases, 6 had different pre-treatments. The oocyte maturation rate ranged from 0 to 90%. In one case that had been treated with depot agonists for adenomyosis for 2 months, the LH level on day 2 was 0.1 IU/L, and no mature oocytes were retrieved. Another case with hypothalamic dysfunction and secondary amenorrhea due to diabetes mellitus and rapid weight loss had a LH level of 0.1 IU/L on day 2, and no mature oocytes were retrieved. No fertilization was observed in 2 cases. If we had been able to predict these outcomes at the time in these two high OHSS–risk cases, we would have canceled their cycles, thus avoiding unnecessary patient distress. In this hyper-responder group, the mean AMH level was 3.9 ng/ml. The mean oocyte maturation rate, fertilization rate, and the rate of blastocyst/normally fertilized oocytes were 58.4%, 58.2%, and 30.8%, respectively. In contrast, in our routine ART program, in cases without OHSS risk where an hCG trigger was therefore used, the mean oocyte maturation rate, fertilization rate, and blastocyst/normally fertilized oocyte rate were significantly higher at 86.5%, 84.7%, and 71.1%, respectively.
Table 4 shows patient and cycle characteristics of cases with low LH levels on day 2 (LH < 1 IU/L).
Table 4.
Patient and cycle characteristics of cases with extremely low LH levels (LH < 1 IU/L) on day 2
| Case no | Female Age | Pretreatment | BMI | AMH | Day2 LH level | Trigger Day LH level | Mean duration of GnRH antagonist | No of Total oocytes (COC) | No of Mature oocytes (MII) | No of Fertilized oocytes (PN2) | Oocyte maturation rate | Oocyte fertilization rate | Mean cryopreservable blastocysts | Cryo blast/fertilized oocyte rate |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Case1 | 30 | OC pills (1 month) | 25.9 | 1.2 | 0.7 | 1.4 | 6 | 7 | 5 | 4 | 71.4% | 80% | 3 | 75% |
| Case2 | 30 | Lupron Stop | 22.9 | 2.1 | 0.5 | 0.2 | 4 | 22 | 20 | 14 | 90.9% | 70% | 6 | 42.8% |
| Case3 | 37 | Lupron Stop | 28.5 | 2.3 | 0.4 | 1.4 | 4 | 14 | 9 | 7 | 64.3% | 77.8% | 2 | 28.8% |
| Case4 | 42 | OC pills (3 months) | 20.1 | 7.1 | 0.1 | 0.8 | 6 | 11 | 10 | 10 | 90.9% | 100% | 4 | 40% |
| Case5 | 33 | Lupron Stop | 18.9 | 1.3 | 0.1 | 0.5 | 7 | 12 | 10 | 9 | 83.3% | 90% | 4 | 44.4% |
| Case6 | 32 | Depot GnRH agonist (2 months) | 30.1 | 5.5 | 0.1 | 0.1 | 5 | 2 | – | – | – | – | – | – |
| Case7 | 30 | None | 16.9 | 8.2 | 0.1 | 1.5 | 4 | 10 | – | – | – | – | – | – |
| TOTAL | 34.4 | 5/6 | 23.3 | 3.9 | 0.28 | 0.84 | 5.1 | 11.1 | 7.7 | 6.3 | 58.4% | 58.2% | 2.7 | 30.8% |
OC oral contraceptives
Table 5 shows patient and cycle characteristics of cases with extremely low LH levels (LH < 0.1 IU/L) on trigger day. There were 19 cases that had extremely low LH levels (LH < 0.1 IU/L) on trigger day. Of these 19 cases, 4 cases had received pretreatments. One case had taken OCPs for 1 month and another for 3 months. One case had used medroxyprogesterone acetate, whereas another case had been administered 2 months depot GnRH analogs for adenomyosis. The mean AMH level of these 19 patients was 5.1 ng/ml, the number of COC was 31.2, and the mean number of cryopreservable blastocysts was 8.7. Of these 19 cases, only one had no mature oocytes. This case had an extremely low LH level of 0.1 IU/L on day 2, and a history of 2-month depot agonist. In cases with lower LH levels on trigger day, female age was younger (30.3 years), AMH level was significantly higher (5.8 ng/ml), BMI was lower (23.7 kg/m2), and the total number of oocytes was significantly higher (n = 25).
Table 5.
Patient and cycle characteristics of cases with extremely low LH levels (LH < 0.1 IU/L) on trigger day
| Case no | Female age | Pretreatment | BMI | AMH Level | Day2 LH Level | Trigger day LH level | Mean duration of GnRH antagonist | No of Total oocyte (COC) | Mature oocyte (MII) | No of Fertilized oocyte (2PN) | Oocyte maturation rate | Oocyte fertilization rate | Mean cryopreservable blastocyst | Cryo blast/fertilized oocyte rate |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Case1 | 28 | OC pills (1 month) | 24 | 5.1 | 2.9 | 0.1 | 5 | 23 | 17 | 15 | 73.9% | 88.2% | 5 | 33.3% |
| Case2 | 20 | None | 18.2 | 5.6 | 5.5 | 0.1 | 6 | 42 | 33 | 27 | 78.6% | 81.8% | 8 | 29.6% |
| Case3 | 31 | None | 24.9 | 6.2 | 5.6 | 0.1 | 5 | 24 | 16 | 12 | 66.7% | 75% | 1 | 8.3% |
| Case4 | 30 | None | 24.5 | 6.5 | 5 | 0.1 | 6 | 55 | 48 | 38 | 87.3% | 79.2% | 13 | 34.2% |
| Case5 | 39 | None | 24.9 | 2 | 3.6 | 0.1 | 7 | 24 | 23 | 18 | 95.8% | 78.3% | 4 | 22.2% |
| Case6 | 30 | None | 19.8 | 11 | 5.6 | 0.1 | 7 | 67 | 38 | 33 | 56.7% | 86.8% | 15 | 45.5% |
| Case7 | 31 | None | 20.7 | 6.4 | 8.8 | 0.1 | 6 | 38 | 28 | 24 | 73.7% | 85.7% | 12 | 50% |
| Case8 | 33 | None | 19.5 | 2.7 | 12.2 | 0.1 | 5 | 6 | 6 | 5 | 100% | 83.3% | 2 | 40% |
| Case9 | 34 | None | 25 | 4 | 5.2 | 0.1 | 5 | 46 | 34 | 32 | 73.9% | 94.1% | 23 | 71.9% |
| Case10 | 28 | OC pills (1 month) | 29.3 | 1.8 | 6.8 | 0.1 | 4 | 21 | 18 | 14 | 85.7% | 77.8% | 11 | 78.6% |
| Case11 | 28 | None | 23.6 | 5 | 4.5 | 0.1 | 6 | 21 | 20 | 20 | 95.2% | 100% | 7 | 35% |
| Case12 | 32 | None | 24.8 | 3.2 | 6.3 | 0.1 | 4 | 9 | 9 | 5 | 100% | 55.6% | 2 | 40% |
| Case13 | 30 | None | 24.1 | 3.1 | 6.8 | 0.1 | 6 | 27 | 22 | 21 | 81.5% | 95.5% | 13 | 61.9% |
| Case14 | 30 | MPA | 25.6 | 10.2 | 7.6 | 0.1 | 5 | 36 | 26 | 22 | 72.2% | 84.6% | 9 | 40.9% |
| Case15 | 25 | None | 25 | 4.4 | 9.4 | 0.1 | 6 | 31 | 13 | 9 | 41.9% | 69.2% | 4 | 44.4% |
| Case16 | 34 | None | 20.5 | 4 | 6.7 | 0.1 | 6 | 34 | 30 | 27 | 88.2% | 90% | 9 | 33.3% |
| Case17 | 27 | None | 20.9 | 6.8 | 10.3 | 0.1 | 7 | 34 | 27 | 21 | 79.4% | 77.8% | 11 | 52.4% |
| Case18 | 26 | None | 22.1 | 4.9 | 6.8 | 0.1 | 6 | 23 | 18 | 12 | 78.3% | 66.7% | 8 | 66.7% |
| Case19 | 32 | Depot GnRH agonist (2 months) | 30.1 | 5.5 | 0.1 | 0.1 | 5 | 2 | – | – | – | – | – | – |
| Total | 29.8 | 4/19 | 23.1 | 5.1 | 6.6 | 0.1 | 5.5 | 31.2 | 23.1 | 19.7 | 74% | 85.3% | 8.7 | 44.2% |
OC oral contraceptives
Discussion
Several studies have emphasized the pivotal role of the GnRHa trigger in ovulation induction, not only for the prevention of OHSS but also for a variety of patient groups such as oocyte donors, cancer patients, patients with low ovarian reserve, and patients with immature oocytes or empty follicle syndrome [12–17]. However, there is a lack of detailed investigation of patient and cycle-specific parameters in GnRHa trigger cases that may influence the number of mature oocytes and cryopreservable blastocysts.
In our study, we investigated a number of specific factors that could affect the number of mature oocytes retrieved and the number of cryopreservable blastocysts.
Firstly, in this GLMM, the number of mature oocytes obtained was evaluated. According to multivariable analyses, a one-unit increase in BMI, LH level on day 2, progesterone level on day 2, and E2 level on trigger day significantly increased the number of mature oocytes. These characteristics in GnRHa-triggered patients with a high number of mature oocytes indicate that these patients are very likely to be patients with PCOS. On the other hand, a one-unit increase in female age, E2 level on day 2, LH level on trigger day, duration of GnRHa use, and daily gonadotropin dosage used significantly decreased the number of mature oocytes.
Secondly, the number of cryopreservable blastocysts was evaluated. Our generalized linear mix model analyses of data of GnRHa trigger patients showed that the presence of ovulatory dysfunction, a one-unit increase in LH level on day 2, and a one-unit increase in E2 level on trigger day significantly increased the number of cryopreservable blastocysts. These results confirm that in hyperresponder patients with ovulatory dysfunction and higher day 2-LH and trigger day-E2 levels, there are higher numbers of retrieved oocytes and cryopreservable blastocysts. However, a one-unit increase in female age, E2 level on day 2, LH level on trigger day, duration of infertility, and daily gonadotropin dosage significantly decreased the number of cryopreservable blastocysts. Although the mean age of the women in our study was 31.5 years, the age range was from 18 to 46 years, with 369 cases ≥ 38 years, of whom 51 were over 42 years old. These older patients with a relatively lower ovarian reserve required a relatively higher daily dose of gonadotropin, which significantly reduced the number of cryopreservable blastocysts. This decrease in the number of cryopreservable blastocysts is not unexpected in older patients, as higher aneuploidy rates are associated with advancing female age.
Although our results are consistent with those of other studies showing that AMH and the number of follicles > 11 mm are indicators of high ovarian reserve, they were not found to indicate a higher number of mature oocytes in our study [18–20]. In the multivariable analysis used in our study, neither the AMH value nor the number of follicles > 11 mm was a significant parameter influencing the number of mature oocytes retrieved or the number of cryopreservable blastocysts. The number of follicles larger than 11 mm on the trigger day does not necessarily correspond to the number of mature oocytes that can be retrieved. Follicle size and the homogeneity of follicular growth are the most important parameters influencing the number of mature oocytes retrieved. This can be explained by the results of previous studies by Shapiro et al. (2022) and Kahraman et al. (2017) [21, 22]. Shapiro found that punctures of follicles with a diameter of 12.5 mm rarely resulted in good-quality blastocysts [21]. Furthermore, Kahraman et al. found that the highest rate of top- and good-quality blastocysts was achieved in embryos obtained from large follicles in homogeneous cycles [22].
In the extension of the study, a small number of cases (n = 26) with extremely low day 2 (< 1 IU/L) (n = 7) and/or trigger day LH values (< 0.1 IU/L) (n = 19) were investigated. Further examination of the cases with extremely low LH values on day 2 revealed that the mean oocyte maturation rate, fertilization rate, and blastocyst/normally fertilized oocyte rate were 58.4%, 58.2%, and 30.8%, respectively. Whereas, in our routine ART program, in cases without OHSS risk, where the hCG trigger was therefore used, the mean oocyte maturation rate, fertilization rate, and blastocyst/normally fertilized oocyte rate were significantly higher at 86.5%, 84.7%, and 71.1%, respectively. Therefore, low LH values on day 2 must be considered a risk factor for low oocyte maturation, fertilization, and blastocyst development rates. In the two cases where fertilization was not observed, if we could have predicted these outcomes in these two cases with high OHSS risk, we would have cancelled their cycles, thus avoiding unnecessary patient distress.
Meyer et al. (2015) have shown that the vast majority of suboptimal responses to the GnRH agonist trigger are the result of hypothalamic suppression, including long-term OCP use, low BMI, and other causes, and noted that women with classically defined hypothalamic amenorrhea are at risk for suboptimal response to the GnRH agonist trigger [7]. Similarly, in our two patients with extremely low LH levels on day 2, there was a history of hypothalamic dysfunction and secondary amenorrhea due to diabetes mellitus and rapid weight loss in one patient, as well as a history of 2 months of pretreatment with depot agonists.
When our 19 cases with a value of LH < 0.1 IU/L were examined more closely, it was found that they had the highest ovarian reserve and had therefore received a lower initial dose of gonadotropin (1928 IU total dose) and a longer duration of GnRH antagonist injection (5.2 days). These factors were among the underlying causes of severely suppressed LH levels on trigger day. In these cases with extremely low LH levels on trigger day, the women were younger, their AMH levels were significantly higher, their BMI was lower, and their total number of oocytes was significantly higher. Meyer et al. (2015) argued that the level of LH on the day of the ovulation trigger is the single most valuable marker to help clinicians assess the risk of a patient’s suboptimal response to the GnRHa trigger [7]. However, our results showed that these hyperresponder patients had a statistically significantly higher number of cryopreservable blastocysts (8.1) compared to other LH groups due to the high number of oocytes retrieved, despite the low oocyte maturation rates.
Lu et al. (2016) reported that patients with suboptimal response to GnRHa trigger as defined by a serum LH < 15 IU/L on the morning after GnRHa trigger had significantly lower LH levels on the day of the trigger and a significantly lower number of mature oocytes retrieved compared to those with adequate response [23]. In contrast, Benmachiche et al. (2019) found no significant effect of post-trigger level LH on the number of mature oocytes, which is consistent with the findings of Yding Andersen et al. [11, 24]. On the other hand, Kummer et al. (2013) showed that peak E2, post-trigger LH, and progesterone level, as well as the magnitude of the LH increase, were independent predictors of the total number of oocytes retrieved and mature oocytes [6]. However, Dunne et al. and Meyer et al. both argue that post-trigger LH levels are not predictive of oocyte maturation, and, therefore, measurement is not necessary [7, 25]. Unfortunately, there is still no consensus on the optimal serum LH threshold levels after the trigger [5, 23]. In our study, the post-trigger LH and P levels were recorded in 458 cases where the LH level was < 0.5 IU/L on the trigger day. A low post-trigger LH level was defined as LH 15 IU/L, and a low post-trigger P level as 3.5 ng/ml [6]. No significant correlation was found between these levels and the oocyte maturation rate or the number of cryopreservable blastocysts. Only 5 cases had a post-trigger LH level of less than 15 IU/L; 8 patients had a low post-trigger P level of less than or equal to 3.5 ng/ml; 3 patients had both low LH and P levels. In these patients who have a particularly high risk of OHSS, the GnRHa trigger was administered.
There is insufficient data in the literature on the use of rLH in cases with FSH-only preparations that have low LH levels in the late follicular phase. Precise LH levels under which oocyte quality and subsequent embryonic development competence may be negatively affected have not yet been identified. In our study, 587 cases with low LH levels (LH 1 < IU/L) during the late follicular phase were observed. The rLH was administered during the late follicular phase to reduce the possibility of inadequate estradiol synthesis and a reduced fertilization rate due to the profound suppression of peripheral LH activity. This variable was taken into account in our analyses. Our results showed that the additional administration of LH had no effect on the number of mature oocytes retrieved or the number of cryopreservable blastocysts, suggesting that the additional administration of rLH is of no benefit in the treatment of these patients.
The limitation of our study is its retrospective design. Its strengths are a large number of cases and the wide range of patient and cycle specific parameters evaluated with the GLMM model.
Although AMH and the number of follicles > 11 mm are indicators of high ovarian reserve, they were not found to be significant parameters influencing the number of mature oocytes retrieved or the number of cryopreserved blastocysts. The GLMM used in our study showed that a one-unit increase in female age, the daily gonadotropin dose, and the LH level on trigger day significantly reduced the number of mature oocytes retrieved and the number of cryopreservable blastocysts.
Supplementary Information
Below is the link to the electronic supplementary material.
Author contribution
Semra Kahraman and Yucel Sahin contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Yucel Sahin, Ipek Nur Balin Duzguner, and Soner Duzguner. The first draft of the manuscript was written by Semra Kahraman, Yucel Sahin, and Ipek Nur Balin Duzguner commented on previous versions of the manuscript. Semra Kahraman, Yucel Sahin, and Ipek Nur Balin Duzguner read and approved the final manuscript.
Data Availability
The datasets generated and/or analysed during the current study are not publicly available due to patient privacy and hospital policy but are available from the corresponding author on reasonable request.
Declarations
Ethics approval
Ethical approval was waived by the local Ethics Committee of Istanbul Memorial Hospital in view of the retrospective nature of the study.
Consent
After verbal and written information, consent form was obtained from all individual participants included in the study accepting controlled ovarian stimulation, oocyte pick-up procedure, and freeze-all procedure.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated and/or analysed during the current study are not publicly available due to patient privacy and hospital policy but are available from the corresponding author on reasonable request.