Human chorionic gonadotropin levels after ovulation triggering as predictors of clinical pregnancy after intracytoplasmic sperm injection: a prospective cohort study

ABSTRACT This study aimed to assess the correlation between human chorionic gonadotropin (HCG) levels after ovulation triggering and clinical pregnancy following intracytoplasmic sperm injection (ICSI), affected by HCG formulation and dose. The study was a prospective cohort study of 100 women who underwent ICSI and controlled ovarian stimulation (COS) using a fixed gonadotropin-releasing hormone (GnRH) antagonist protocol. Women were divided according to the type of ovulation triggering into three groups: (1) urinary HCG (uHCG) group (n = 33); (2) recombinant HCG (rHCG) group (n = 33); and (3) dual trigger group (n = 34). The three groups were compared to each other taking into consideration the HCG levels and their correlations to clinical pregnancy following ICSI. Clinical pregnancy could be predicted by serum HCG levels 34 h after ovulation triggering by uHCG, rHCG and dual trigger at cutoff levels of 73.72, 57.35 and 22.37 mIU/ml, respectively, with sensitivities of 95%, 94.7% and 87%, and specificities of 76.9%, 92.9% and 90.9%, respectively. Follicular fluid (FF) HCG levels could predict clinical pregnancy after ovulation triggering by uHCG, rHCG and dual trigger at cutoff levels of 21.58 mIU/ml, 20.82 mIU/ml and 8.66 mIU/ml, respectively, with sensitivities of 95%, 94.7% and 91.3%, and specificities of 92.3%, 92.9% and 90.9%, respectively. In conclusion, serum and FF HCG levels after ovulation triggering could predict clinical pregnancy following ICSI at variable cutoff levels according to HCG formulation and dose.


Introduction
In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are important subsets of assisted reproductive technology (ART) that contribute to the treatment of infertility.Success of IVF/ICSI cycles depends, in part, on retrieval of sufficient number of good-quality mature oocytes [1,2].Final oocyte maturation occurs when the oocyte resumes meiotic division and develops to metaphase II (MII) stage when it becomes capable of fertilization, division and blastocyst formation [3].In the endogenous menstrual cycle, oocyte maturation is triggered by luteinizing hormone (LH) surge [4].
The LH and human chorionic gonadotropin (HCG) are dimeric glycoprotein hormones that have a significant homology.They are 100% identical in the α subunit and 85% identical in the β subunit and stimulate the same receptors [5].Therefore, in ART cycles, HCG is commonly utilized to trigger final oocyte maturation via a LH-like surge [6].In addition, at a minimal dose, HCG can act as a dual trigger when combined with gonadotropin-releasing hormone agonist (GnRHa) in antagonist protocols to minimize the risk of ovarian hyperstimulation syndrome (OHSS) and achieve luteal phase support [7].Moreover, HCG has a key role in adjusting the endometrium for implantation [8].
The HCG has being extracted from urine of pregnant women for a long time and known as urinary HCG (uHCG).Because of its biological origin, uHCG has some limitations concerning the presence of impurities and variations between batches.Therefore, recombinant HCG (rHCG) has been produced in the ovarian cells of Chinese hamsters using recombinant DNA technology to overcome these limitations with superior purity, batch-to-batch consistency, and better control of the final amount in varied doses.Urinary-derived products remain popular due to affordability, accessibility and aging population, particularly in low-income countries and Europe, and increasing demand for older women [9][10][11].
A synthetic peptide called GnRHa, which is modeled after the hypothalamus GnRH, interacts with the GnRH receptor to cause it to respond biologically by releasing LH and FSH.However, continued stimulation with GnRHa after the initial 'flare' of gonadotropins results in GnRH receptor down-regulation and pituitary desensitization.Only GnRH antagonist IVF/ICSI cycles or cycles in which no GnRH analog was used during stimulation will allow for the administration of GnRHa for ovulation triggering [12].
Although ICSI became the gold standard of IVF treatment, it is complicated and has highcost, and different success rates have been reported for various infertility problems.Therefore, detecting predictors for ICSI outcome is necessary for clinicians to determine the suitable treatment plan for each case [13][14][15][16].Insufficient research has been done on the clinical importance of HCG levels following ovulation triggering, and there have been inconsistent findings [17].Assessment of serum HCG levels is simple, cheap and can be done early before embryo transfer.Consequently, it may serve as a reliable indicator of clinical pregnancy after ICSI.Therefore, we aimed in this study to assess the correlation between both serum and follicular fluid (FF) HCG levels after ovulation triggering and clinical pregnancy following intracytoplasmic sperm injection (ICSI), affected by HCG formulation and dose.

Study design
This was a prospective cohort study of women underwent ICSI between March 2021 and July 2022 in Royal Center, Mansoura, Egypt.The Mansoura Faculty of Medicine Institutional Research Board (MFM-IRB) evaluated and approved the study protocol (Code No. MD.20.09.370), and the trial was registered with ClinicalTrials.gov,identifier NCT05969834.The STROBE cohort reporting guidelines was used [18].The main inclusion criterion was women who have undergone controlled ovarian stimulation (COS) through fixed GnRH antagonist protocol with ovulation triggering, according to the risk of OHSS, by (1) uHCG; (2) rHCG; or (3) a combination of GnRHa and HCG (dual trigger).Three groups of women were formed according to the type of trigger; uHCG group (n = 33), rHCG group (n = 33) and dual trigger group (n = 34); and the three groups were compared to each other.

COS protocol
The COS utilized the fixed GnRH antagonist protocol.On the second day of the menstrual cycle, ovarian stimulation with a gonadotropin preparation was initiated after confirming absence of ovarian cysts by ultrasound scanning.Gonadotropin doses were determined according to the patient's age, BMI, basal FSH and estradiol (E2).Beginning on the seventh day of the menstrual cycle and continuing until the day of oocyte maturation triggering, a 0.25 mg/day subcutaneous dose of the GnRH antagonist preparation (Cetrotide®, Cetrorelix 0.25 mg, Merck Serono, Darmstadt, Germany) was administered.

Assessment of serum and follicular fluid (FF) HCG levels
On the day of the ova pickup, serum and FF were collected.Blood samples were collected 34 h after HCG injections (ovulation triggering).The FF was collected from several follicles during ova pickup (at 36-38 h following triggering), and all bloody samples were discarded.For 10 min at 3000 rpm, samples were centrifuged to separate the serum from the cells.Serum and FF HCG were assessed with Sandwich Immunoassay for the quantitative determination of total β-HCG levels using an automated system (Access 2, Beckman Coulter, USA).

ICSI protocol
At 36-38 h following triggering, oocytes were collected by transvaginal aspiration of follicles under transvaginal sonography (TVS) guidance.After that, the endometrium was prepared for embryo transfer (ET) by the same regimen for all women in the three groups through administration of natural progesterone vaginal supplement (Prontogest®, Progesterone 400 mg, vaginal pessaries, MARCYRL, Egypt) in a dose of 400 mg every 12 h + 4 mg/day estradiol valerate oral supplement (Cyclo-Progynova® white tablets, Bayer Pharma, Leverkusen, Germany).
The ICSI procedures were carried out exactly as stated previously [20].Oocyte quality was assessed according to the modification of Xia's criteria [21,22] as follows: Fertilization was confirmed 18-20 h following the ICSI procedure.Embryo division and grading were assessed on day 2 or 3 according to Stensen et al. [23].On day 5, blastocysts were evaluated using Gardner scoring criteria described by Gardner et al. [24,25].On day 5 after oocyte retrieval, at least one healthy embryo was transferred transcervically using ultrasound guidance.Extra good-quality embryos were cryopreserved.Luteal phase support was maintained for the first 2 weeks after ET with the same regimen that was started on the day of ova pickup.

Documentation of pregnancy
On day 14 after ET, a quantitative serum β-HCG assay was performed, and a result of ≥50 mIU/ml was regarded as conclusive evidence of biochemical pregnancy.Positive pregnancy test cases were scanned with TVS between 4 and 6 weeks after ET in order to provide evidence of clinical intrauterine pregnancy, wherein one or more gestational sacs are located within the uterine cavity and contains a fetal pole and heart activity.

Outcome measures
The primary outcome measure of this study was the clinical pregnancy rate while the secondary outcome measures were oocyte maturation rate (OMR), oocyte quality, fertilization rate, and percentage of top-quality embryos.The clinical pregnancy rate was calculated by dividing the number of clinical pregnancies by the number of ET procedures.The OMR was determined for each case by dividing the number of mature oocytes (MII oocytes) by the total number of retrieved oocytes.The oocyte quality was assessed according to the modification of Xia's criteria.The fertilization rate was determined for each case by dividing the number of fertilized oocytes by the number of injected oocytes.The percentage of top-quality embryos was determined for each couple by dividing the number of top-quality embryos by the number of fertilized oocytes.

Sample size calculation
The G*Power 3.1.9.2 was utilized to figure out the ideal sample size (linear multiple regression: Fixed model, single regression coefficient; twotailed significance; alpha error probability = 0.02; power = 98%, number of predictors = 2; allocation ratio for groups = 1).According to population size in a previous study by Levy et al. [26], a minimum of 99 participants (33 in each group) were required to identify a difference of at least 20% between our study groups.

Statistical analysis
The IBM® SPSS® Statistics, version 21.0 for Windows, was used to compile all of the final tabulations.Numbers and percentages were used to show the categorical data, whereas the mean and standard deviation were used to characterize continuous data.The Chi-square test was utilized to evaluate the comparability of categorical variables.When comparing two groups with normally distributed continuous data, the Student t test was utilized.When comparing continuous data from more than two groups, we employed the Analysis of Variance (ANOVA) test.Tukey's test was used to perform post-hoc analysis for continuous data between more than two groups.Receiver operator characteristics (ROC) test was used to assess the accuracy of tests of interest including sensitivity, specificity and area under curve, and level of significance (P value) was considered of statistical significance if less than 0.05.

Results
The study included a total of 100 participants.Figure 1 shows a flow chart to the study from enrollment to analysis.Final triggering of oocyte maturation was performed via uHCG in 33 women (uHCG group), rHCG in 33 women (rHCG group), and dual trigger in 34 women (dual trigger group).Table 1 compares the three groups based on their demographic and hormonal characteristics.There were no statistically significant differences between the three groups in the age, BMI, parity and number of previous miscarriages.The infertility duration, type and cause were similar in the three groups.Also, the basal serum AMH, FSH, LH and E2 levels were comparable among the three groups.
Table 2 shows the stimulation characteristics, ICSI characteristics and ICSI outcomes of the three groups.Among the three groups, the total administered gonadotropin dose was comparable and no statistically significant difference was observed in the stimulation days.Serum HCG level after triggering was significantly different among the three groups, and the Post-Hoc analysis revealed that the uHCG group had significantly higher level than both the rHCG group (P < 0.001, not presented in tables) and the dual trigger group (P < 0.001,  not presented in tables), and the level was also significantly higher in the rHCG group than in the dual trigger group (P = 0.002, not presented in tables).The FF HCG level was considerably different among the three groups, and the Post-Hoc analysis revealed that the level was significantly higher in the uHCG group than in both the rHCG group (P < 0.001, not presented in tables) and the dual trigger group (P < 0.001, not presented in tables), and moreover, the level was significantly higher in the rHCG group than in the dual trigger group (P = 0.042, not presented in tables).
Although the number of collected oocytes and the OMR were not substantially different among the three groups, the rHCG group had significantly higher quality of mature oocytes (P < 0.001), and the Post-Hoc analysis revealed that the significant difference is between the rHCG group and both the uHCG group (P < 0.001, not presented in tables) and the dual trigger group (P = 0.006, not presented in tables).There were no statistically significant differences between the three groups in the fertilization, cleavage, top division, blastulation and top blastulation rates.The number of transferred embryos was two embryos in all cases except two cases, where only one embryo was transferred (one case was in the rHCG group and the other was in the dual trigger group).In all the three groups, the clinical pregnancy rate was comparable (Table 2).
The accuracy of serum and FF HCG levels in predicting clinical pregnancy are shown in Table 3 and Figures 2 and 3. Clinical pregnancy could be predicted by serum HCG levels 34 h after ovulation triggering by uHCG, rHCG and dual trigger at cutoff levels of 73.72, 57.35 and 22.37 mIU/ml, respectively, with sensitivities of 95%, 94.7% and 87%, and specificities of 76.9%, 92.9% and 90.9%, respectively.The FF HCG levels could predict clinical pregnancy after ovulation triggering by uHCG, rHCG and dual trigger at cutoff levels of 21.58, 20.82 and 8.66 mIU/ml, respectively, with sensitivities of 95%, 94.7% and 91.3%, and specificities of 92.3%, 92.9% and 90.9%, respectively.

Discussion
The ICSI is an important subset of ART that contributes to treatment of infertility [27].The HCG can act as a dual trigger in a low dose in combination with GnRHa in antagonist protocols to minimize the risk of OHSS and achieve a luteal phase support [28].Insufficient research has been made on the clinical importance of HCG levels following ovulation triggering, and there have been inconsistent findings [17].Therefore, we aimed to evaluate the correlation between HCG levels and clinical pregnancy following ICSI, taking into considerations the HCG formulation and dose.To achieve this aim, we conducted this prospective study on 100 women from couples who were subjected to ICSI procedure and were divided according to type of ovulation trigger.The current study found no statistically significant differences between the three groups in terms of the number of collected oocytes or OMR, however, the quality of mature oocytes had significant higher values among rHCG group than the other two groups.Similar to the current study, some researches have not been able to demonstrate that the dual trigger could increase the quantity of retrieved oocytes [29][30][31].Contrary to the current study, Nakagawa et al. showed that the number of oocytes retrieved was significantly higher among uHCG group.However, he came in agreement with us regarding the OMR [32].Also, Li et al. against our study, reported higher number of oocytes retrieved among the dual trigger group with no difference between HCG group and dual trigger group regarding the quality of mature oocytes [33].A recent metaanalysis, in particular, verified a significant rise in the quantity of oocytes retrieved following the dual trigger [34].
The studied groups had comparable fertilization and cleavage rates.This came in agreement with the previous studies by Nakagawa et al. and Li et al. [32,33].All embryos were transferred at day 5 and neither the number of transferred embryos nor the clinical pregnancy rate differed significantly across the three groups.Likely, Nakagawa et al. and Youssef et al. reported no differences between uHCG, rHCG and dual triggers regarding ICSI outcomes including number of transferred embryos and clinical pregnancy rate [32,35].Li et al. and Qiu et al. did not find additional benefit of dual trigger over HCG alone on pregnancy outcome [33,36].
Contrary to the current study, Hu et al. in their meta-analysis which included 14 RCTs found that clinical outcome of ICSI improved significantly among dual trigger group and clinical pregnancy rates were higher [34].Also, Mizrachi et al. in another recent systematic review concluded that higher number of transferred embryos and clinical pregnancy were associated with dual trigger [37].They explained their results by the FSH surge that is induced by dual trigger and the GnRHa that improves endometrial receptivity.
The comparison between uHCG and rHCG regarding the outcome showed that both groups were comparable.Madani et al. and Yang et al. have not reported significant differences between the rHCG and uHCG groups regarding implantation rate, chemical pregnancy rate, or clinical pregnancy rate, and these findings are consistent with the current study [38,39].However, Youssef et al. in their meta-analysis favored rHCG and reported higher clinical pregnancy rates with rHCG than uHCG [35].On the contrary, Mosaad et al. and Liu et al. proposed that biochemical and clinical pregnancy rates were significantly higher among uHCG group [31,40].
We tried to evaluate the accuracy of both serum and FF HCG levels in prediction of clinical pregnancy after ICSI and we outlined cutoff values with high sensitivities and specificities in the total cohort and in each group separately.HCG level is useful in prediction of clinical pregnancy after ICSI, but they failed to outline precise cutoff value [41,42].Other studies reported significant positive correlation between serum HCG level and clinical pregnancy outcome after ICSI with no specific cutoff value [43][44][45].Zhang et al. and Mizrachi et al., on the other hand, failed to demonstrate the value of serum HCG level in prediction of chemical or clinical pregnancy after ovulation trigger by HCG alone [17,46].

Zhang et al. and Hassan et al. found that serum
The study had the advantage of being prospective.Also, as far as we are aware, this study is one of the few studies that assessed the accuracy of FF HCG level in predicting clinical pregnancy.Another strength point is the strict inclusion criteria that eliminate the effect of any other factor that can affect the outcomes of the study.Moreover, the study has taken into consideration the effect of the HCG dose and formula when evaluating HCG levels for prediction of clinical pregnancy.
This study had a number of limitations, including that it did not use randomization and the fact that it was conducted on a relatively small population.Another limitation point is the lack of evaluation of other outcomes with clinical significance such as implantation rate, multiple pregnancy rate and ongoing pregnancy rate, but we aimed first to assess if there is association between HCG levels and clinical pregnancy, then we will evaluate other parameters in future larger studies.
In conclusion, serum and FF HCG levels after ovulation triggering could predict clinical pregnancy following ICSI at variable cutoff levels according to HCG formulation and dose.

Figure 2 .
Figure 2. ROC curves to test accuracy of serum HCG to predict clinical pregnancy.(a) ROC curve to test accuracy of serum HCG to predict clinical pregnancy in total cohort, (b) ROC curve to test accuracy of serum HCG to predict clinical pregnancy in uHCG group, (c) ROC curve to test accuracy of serum HCG to predict clinical pregnancy in rHCG group, (d) ROC curve to test accuracy of serum HCG to predict clinical pregnancy in dual trigger group.

Figure 3 .
Figure 3. ROC curves to test accuracy of follicular fluid HCG to predict clinical pregnancy.(a) ROC curve to test accuracy of follicular fluid HCG to predict clinical pregnancy in total cohort, (b) ROC curve to test accuracy of follicular fluid HCG to predict clinical pregnancy in uHCG group, (c) ROC curve to test accuracy of follicular fluid HCG to predict clinical pregnancy in rHCG group, (d) ROC curve to test accuracy of follicular fluid HCG to predict clinical pregnancy in dual trigger group.

Table 1 .
Demographics and basal hormonal characteristics of the study groups.
a Expressed as mean ± SD, and P value was calculated by the analysis of variance (ANOVA) test.b Expressed as frequency and percentage, and P value was calculated by the Chi-square test.AMH, antimullerian hormone; BMI, body mass index; E2, estradiol; FSH, follicle stimulating hormone; LH, luteinizing hormone; PCOS, polycystic ovary syndrome; rHCG, recombinant human chorionic gonadotropin; uHCG, urinary human chorionic gonadotropin.

Table 2 .
Stimulation and ICSI characteristics and outcomes in the study groups.
a Expressed as mean ± SD, and P value was calculated by the analysis of variance (ANOVA) test.b Expressed as frequency and percentage, and P value was calculated by the Chi-square test.ET, embryo transfer; rHCG, recombinant human chorionic gonadotropin; uHCG, urinary human chorionic gonadotropin.

Table 3 .
Accuracy of serum and follicular fluid HCG levels in predicting clinical pregnancy.