Adverse impact of elevated progesterone levels on human chorionic gonadotropin trigger day on blastocyst transfer outcomes in gonadotropin-releasing hormone agonist cycles

AbstractObjective

Dose an elevated serum progesterone (P) level on the human chorionic gonadotropin (hCG) trigger day have a negative effect on clinical pregnancy outcomes for embryos transferred at different stages of development in long-acting gonadotropin-releasing hormone agonist (GnRHa) in vitro fertilization-embryo transfer (IVF-ET) cycles?

Study Design

This was a noninterventional, retrospective, observational, single-centre cohort study. A total of 1951 patients received long-acting GnRHa for pituitary downregulation in IVF-ET cycles at Nanjing Drum Tower Hospital from January 2018 to December 2020. The serum P levels on the day of hCG administration were measured, together with other cycle parameters, to explore the relationship between P levels and the clinical pregnancy rate (CPR) of different embryos transferred.

Results

When the serum P level on the hCG day was higher than 1.5 ng/mL, the CPR did not decrease significantly. There was no correlation between the CPR of cleavage-stage embryo transfer and the serum P level on the hCG day. In addition, the interaction analysis suggested that the CPR of patients undergoing blastocyst transfer decreased as serum P levels on the hCG day increased. Progesterone levels on the day of hCG administration were closely related to the CPR of blastocyst transfer rather than cleavage-stage embryo transfer.

Conclusion

An increased serum P level on the day of hCG administration did not affect the CPR of cleavage-stage embryo transfer, but it reduced the CPR of blastocyst transfer cycles.

KeywordsIntroductionProblems with endometrial receptivity account for approximately 2/3 of embryo implantation failures[Ruiz-Alonso M. Galindo N. Pellicer A. Simon C. What a difference two days make: “personalized” embryo transfer (pET) paradigm: a case report and pilot study.]. Studies have suggested that supraphysiological levels of oestrogen and progesterone (P) in controlled ovarian hyperstimulation (COH) cycles are able to disrupt the endometrial environment, leading to abnormal endometrial receptivity[Macklon N.S. van der Gaast M.H. Hamilton A. Fauser B.C.J.M. Giudice L.C. The impact of ovarian stimulation with recombinant FSH in combination with GnRH antagonist on the endometrial transcriptome in the window of implantation., Bourgain C. Ubaldi F. Tavaniotou A. Smitz J. Van Steirteghem A.C. Devroey P. Endometrial hormone receptors and proliferation index in the periovulatory phase of stimulated embryo transfer cycles in comparison with natural cycles and relation to clinical pregnancy outcome., Kolibianakis E.M. Devroey P. The luteal phase after ovarian stimulation.]. As early as 1991, Schoolcraft et. al.[Schoolcraft W. Sinton E. Schlenker T. Huynh D. Hamilton F. Meldrum D.R. Lower pregnancy rate with premature luteinization during pituitary suppression with leuprolide acetate.] and Silverberg et. al.[Silverberg K.M. Burns W.N. Olive D.L. Riehl R.M. Schenken R.S. Serum progesterone levels predict success of in vitro fertilization/embryo transfer in patients stimulated with leuprolide acetate and human menopausal gonadotropins.] reported that higher preovulatory serum P levels were strongly associated with a lower clinical pregnancy rate (CPR) and an increased pregnancy loss rate for in vitro fertilization-embryo transfer (IVF-ET). However, the effect of increased serum progesterone levels on the day of human chorionic gonadotropin (hCG) administration on clinical outcomes of COH cycles remains controversial.Persistently elevated P levels play a crucial role in both initiating the endometrial transition towards receptivity for embryo implantation and maintaining further embryo invasion[Two pathways of progesterone action in the human endometrium: implications for implantation and contraception., Blakemore J.K. Kofinas J.D. McCulloh D.H. Grifo J. Serum progesterone trend after day of transfer predicts live birth in fresh IVF cycles.]. Several studies have reported that increased P levels, usually defined as greater than 1.5 ng/mL[Bosch E. Labarta E. Crespo J. Simón C. Remohí J. Jenkins J. et al.Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles., Griesinger G. Mannaerts B. Andersen C.Y. Witjes H. Kolibianakis E.M. Gordon K. Progesterone elevation does not compromise pregnancy rates in high responders: a pooled analysis of in vitro fertilization patients treated with recombinant follicle-stimulating hormone/gonadotropin-releasing hormone antagonist in six trials., Venetis C.A. Kolibianakis E.M. Bosdou J.K. Tarlatzis B.C. Progesterone elevation and probability of pregnancy after IVF: a systematic review and meta-analysis of over 60 000 cycles., Xu B. Li Z. Zhang H. Jin L. Li Y. Ai J. et al.Serum progesterone level effects on the outcome of in vitro fertilization in patients with different ovarian response: an analysis of more than 10,000 cycles.], occur on the hCG day in approximately 6-30% of COH cycles. Increased serum P level (> 1.5 ng/mL) on the day of hCG administration are associated with significantly lower ongoing pregnancy rates in IVF-ET cycles[Bosch E. Labarta E. Crespo J. Simón C. Remohí J. Jenkins J. et al.Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles., Venetis C.A. Kolibianakis E.M. Bosdou J.K. Tarlatzis B.C. Progesterone elevation and probability of pregnancy after IVF: a systematic review and meta-analysis of over 60 000 cycles., Fanchin R. de Ziegler D. Taieb J. Hazout A. Frydman R. Premature elevation of plasma progesterone alters pregnancy rates of in vitro fertilization and embryo transfer., Harada T. Yoshida S. Katagiri C. Takao N. Ikenari T. Toda T. et al.Reduced implantation rate associated with a subtle rise in serum progesterone concentration during the follicular phase of cycles stimulated with a combination of a gonadotrophin-releasing hormone agonist and gonadotrophin., Shulman A. Ghetler Y. Beyth Y. Ben-Nun I. The significance of an early (premature) rise of plasma progesterone in in vitro fertilization cycles induced by a “long protocol” of gonadotropin releasing hormone analogue and human menopausal gonadotropins., Bosch E. Valencia I. Escudero E. Crespo J. Simón C. Remohí J. et al.Premature luteinization during gonadotropin-releasing hormone antagonist cycles and its relationship with in vitro fertilization outcome.,

M.T. Connell G. Patounakis M.W. Healy A.H. DeCherney K. Devine E. Widra et al. Is the effect of premature elevated progesterone augmented by human chorionic gonadotropin versus gonadotropin-releasing hormone agonist trigger? Fertility and sterility. 106 3 2016 584 589.e1 e1

]. Studies by Papanikolaou et al.[Papanikolaou E.G. Kolibianakis E.M. Pozzobon C. Tank P. Tournaye H. Bourgain C. et al.Progesterone rise on the day of human chorionic gonadotropin administration impairs pregnancy outcome in day 3 single-embryo transfer, while has no effect on day 5 single blastocyst transfer.] and Huang et al.[Huang Y. Wang E.-Y. Du Q.-Y. Xiong Y.-J. Guo X.-y. Yu Y.-P. et al.Progesterone elevation on the day of human chorionic gonadotropin administration adversely affects the outcome of IVF with transferred embryos at different developmental stages.] have focused on the effect of elevated serum P levels on the possibility of clinical pregnancy in cleavage-stage embryo and blastocyst transfers. The results suggested that when the P level reached the range of 1.0-1.5 ng/mL on the day of hCG administration, the CPR of cleavage-stage embryo transfer decreased, while that of blastocyst transfer was the opposite. Adverse effects on blastocyst transfer outcomes were observed only when the P concentration reached 1.75 ng/mL or above.

To define the effect of serum P levels on the clinical outcomes of embryo transfer in fresh cycles on the hCG day of COH cycles, we conducted a retrospective study of long-acting gonadotropin-releasing hormone agonist (GnRHa) IVF-ET cycles from 2018 to 2020 in our reproductive medicine centre. At the same time, we aimed to further explore whether increased serum P level on the day of hCG administration have different effects on the clinical outcomes of different types of embryos transferred (cleavage-stage embryos or blastocysts).

Material and methodsPopulation characteristics and study design

This was a retrospective cohort study of patients undergoing long-acting GnRHa IVF-ET cycles. Women treated with the first long-acting GnRHa IVF-ET from January 2018 to December 2020 at the reproductive medicine centre of Nanjing Drum Tower Hospital were enrolled in our study. Before starting IVF cycle, each couple was informed that their basic information and treatment data may be used for subsequent research. Each couple was informed and provided written informed consent. We obtained approval from the ethics committee of Nanjing Drum Tower Hospital. Because this is a retrospective study, the ethics committee approved that we do not need additional re informed consent. Patients who were younger than 45 years and underwent their first IVF-ET treatment in our hospital were selected. The exclusion criteria were as follows: (1) intracytoplasmic sperm injection (ICSI) cycles; (2) uterine-related diseases, such as adenomyosis and endometriosis; or (3) endometrium-related diseases, such as endometrial polyps and endometrial dysplasia. A total of 1951 IVF-ET cycles were included in our retrospective analysis.

Controlled ovarian stimulation protocol

The early follicular-phase long-acting GnRHa long (EFLL) protocol was applied for these patients. A long-acting GnRHa (triptorelin acetate, decapeptyl, Ferring GmbH, 1.875 mg or 3.75 mg) was administered in the early follicular phase. The levels of oestradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH) , and P, and the diameter and number of follicles were measured 28 or 35 days after GnRHa downregulation. Some patients who did not meet the criteria for pituitary downregulation were postponed to 32 or 40 days to be measured again. After the pituitary downregulation criteria were met, 75-300 IU recombinant FSH (rFSH, Gonal-F, Merck Sereno) with or without 75-150 IU recombinant LH (rLH, Luveris, Merck Sereno) or 75-300 IU human menopausal gonadotropin (HMG, Menotropins for Injection, Livzon Pharm) was injected daily. The starting dose of gonadotropin (Gn) was based on the patient’s age, body mass index (BMI), basal FSH level, antral follicle count (AFC) and anti-Mullerian hormone (AMH) level. Follicular growth and serum hormone (FSH, LH, E2, P) changes were monitored regularly during the cycle, and the Gn dosage was adjusted accordingly. Ovulation was induced with 10000 IU hCG when there were 1-2 dominant follicles that reached 18 mm. When the number of follicles on the trigger day was more than 15 and the E2 level was more than 5000 pg/mL, 250 μg recombinant hCG (rhCG, Merck Sereno) or 5000 IU hCG was used to avoid ovarian hyperstimulation syndrome (OHSS). Serum P levels were routinely measured and recorded on the hCG trigger day and one day later. The oocytes were collected 36-38 hours after triggering. Mature oocytes (metaphase II, MⅡ) were cultured to produce cleavage-stage embryos or blastocysts after fertilization. If there were a large number of high-quality embryos on the 3rd day (> 3 for patients less than 38 years old; > 4 for patients over 38 years old), we usually cultured the embryos to blastocysts and conducted the blastocyst transfer on the 5th day. If the number of high-quality embryos was small on the 3rd day, we usually directly transferred the cleavage-stage embryos. When the P level on the hCG day was > 3.0 ng/mL, whole embryo freezing was conducted.

Embryo transfer and pregnancy detection

Except for some reasons, fresh embryos were transferred under the guidance of abdominal ultrasound on the 3rd (cleavage-stage embryo) or 5th day (blastocyst) after oocyte retrieval. After pregnancy, luteal support was continued until two months after embryo transfer. Biochemical pregnancy was defined as a positive serum β-human chorionic gonadotropin (β-hCG) test (concentration above 200 mIU/mL) measured 12-14 days after embryo transfer. Ultrasound examination was performed 30 days after embryo transfer. Clinical pregnancy was defined as the presence of a gestational sac. The patients were continuously followed up to assess any abnormalities in pregnancy and until live birth.

Statistical analysis

A smooth curve fit analysis was conducted to detect the relationship between the P levels on the hCG day and the CPR or live birth rates. All data were divided into 2 groups according to different P levels on the hCG day. To solve the problem of the low number of patients in Group 2 and control for confounding variables, the propensity score matching was conducted. In addition, an interaction analysis was employed in the two groups of different embryos transferred. All our data analyses were performed with R (http://www.R-project.org) and EmpowerStats software (www.empowerstats.com, X&Y solutions, Inc. Boston MA). P < 0.05 was considered statistically significant.

ResultsAssociation of P levels on the hCG day and the CPRAs shown in Figure 1, the results of the smooth curve fitting showed that the CPR slightly decreased with increasing serum P levels on the hCG day, without statistical significance. We divided all data into 2 groups: Group 1, comprising P levels less than or equal to 1.5 ng/mL, and Group 2, comprising P levels greater than 1.5 ng/mL (Table 1). The two groups were similar in terms of the patient’s age, BMI, basal FSH levels, and AFC. The total dose and days of Gn stimulation were similar between the two groups. On the trigger day, the serum P level was significantly higher in Group 2 than in Group 1 (1.81 ± 0.28 ng/mL vs 0.59 ± 0.36 ng/mL). The number of oocytes retrieved, and the total number of available embryos (day-3 embryos above grade II) were significantly higher in Group 2 than in Group 1. The CPR and live birth rate of Group 1 were similar to those of Group 2. To solve the problem of the low number of patients in Group 2 and control for confounding variables, the propensity score matching was conducted for these 2 groups. There was also no significant difference in the CPR between the two groups after propensity score matching (Table. S1).Figure thumbnail gr1

Figure 1The illustrated curved line shows the relation between the P level on the hCG day and the CPR. The area between two dotted lines is expressed as the 95% CI.

Table 1Characteristics of patients with different serum P levels (1.5 ng/mL) on hCG day.

Smooth fitting curve analysis of the P levels on the hCG day and CPR for different types of transferred embryosTo explore the relationship between the P levels on the hCG day and clinical outcomes of different transferred embryo types in IVF-ET cycles, we divided all enrolled cycles into 2 groups according to different types of embryos transferred: Group A, cleavage-stage embryos, and Group B, blastocysts. In 1659 cycles, patients received cleavage-stage embryo transfer, while in 292 cycles, patients received blastocyst transfer. The basal and clinical characteristics of the patients in these two groups are listed in Table S2. We further constructed a smooth fitting curve. As shown in Figure 2, the CPR of the patients transferred with blastocysts decreased obviously as the P level on the hCG day gradually increased, while the CPR of patients receiving cleavage-stage embryo transfer did not decrease significantly.Figure thumbnail gr2

Figure 2The illustrated curved line shows the relation between the P level on the hCG day and the CPR of different types of embryos transferred. The area between two dotted lines is expressed as the 95% CI.

Statistical interaction between different embryos transferred and the P levels on the hCG day on pregnancy outcomesFigure 2 presents the association between the P levels on the hCG day and the CPR, stratified by the types of embryos transferred. The red line, which represents blastocyst transfer, showed a decline in the CPR with increasing P levels after adjusting for covariables. In contrast, there was no such association in cleavage-stage embryo transfer cycles. In addition, a test for the interaction between the P levels on the hCG day and types of embryos transferred on the CPR was statistically significant (Table 2, P=0.02).

Table 2Effect modification of P levels on the hCG day on CPR according to different types of embryos transferred.

Adjust for: Age; BMI; Basal FSH; Basal progesterone; AFC; Total Gn dose; Gn duration; P level after trigger day; Number of oocytes retrieved; Number of total embryos; Number of transferred embryos.

DiscussionMost studies suggest that a higher serum P level on the hCG day of IVF-ET cycles affects clinical pregnancy outcomes. A systematic review including 63 studies and more than 60000 IVF/ICSI cycles reported that higher P levels in the late follicular phase are detrimental to the CPR with fresh embryo transfer[Venetis C.A. Kolibianakis E.M. Bosdou J.K. Tarlatzis B.C. Progesterone elevation and probability of pregnancy after IVF: a systematic review and meta-analysis of over 60 000 cycles.]. Furthermore, the study by Bosch et. al. revealed an association between increased P levels (above 1.5 ng/mL) on the day of hCG administration and a lower CPR with fresh embryo transfer. Data from 4032 IVF/ICSI cycles indicated that ongoing pregnancy rates are higher among patients with serum P levels ≤ 1.5 ng/mL [Bosch E. Labarta E. Crespo J. Simón C. Remohí J. Jenkins J. et al.Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles.].According to this result, we first grouped all the data by the P level on the hCG day (≤ 1.5 ng/mL and > 1.5 ng/mL). In contrast to those of previous studies, the results of our study comprising 1951 IVF-ET cycles indicated that there was no significant reduction in the CPR when the P level was greater than 1.5 ng/mL. The correlation between the P level on the hCG day and the live birth rate was also analysed through smooth curve fitting and interaction effect analysis (Fig. S1 and Table. S3). The live birth rate was similar to the CPR. Patients who received the GnRHa and GnRH antagonist regimens were included in a previous study by Bosch et al.[Bosch E. Labarta E. Crespo J. Simón C. Remohí J. Jenkins J. et al.Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles.]. For patients who underwent COH with the GnRH antagonist protocol in our reproductive medicine centre, the whole embryo freezing strategy was routinely employed. Therefore, only patients who received long-acting GnRHa pituitary downregulation treatment were included in this study. The former study reported ongoing pregnancy rates of 24.2% when the P level on the hCG day was greater than 1.5 ng/mL and 38.4% when the P level was less than 1.5 ng/mL; both rates were significantly lower than those in our present study. With increasing advances in IVF-ET technology, the cut-off values for P levels on the hCG day need to be continuously updated. At the same time, the conclusion of our study was similar to those of several other studies [Shapiro B.S. Daneshmand S.T. Garner F.C. Aguirre M. Hudson C. Thomas S. Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders., Yding Andersen C. Bungum L. Nyboe Andersen A. Humaidan P. Preovulatory progesterone concentration associates significantly to follicle number and LH concentration but not to pregnancy rate.] which reported that a higher P level on the hCG day was not associated with the CPR.To solve the problem of increased P levels on the hCG day, two alternative strategies are currently considered: whole embryo freezing and blastocyst transfer[Corti L. Papaleo E. Pagliardini L. Rabellotti E. Molgora M. La Marca A. et al.Fresh blastocyst transfer as a clinical approach to overcome the detrimental effect of progesterone elevation at hCG triggering: a strategy in the context of the Italian law.]. However, frozen-thawed embryo transfer might bring more economic pressure to patients, delay the time of achieving pregnancy in a single cycle, and bring additional complications risks during pregnancy and in newborns [Pinborg A. Wennerholm U.B. Romundstad L.B. Loft A. Aittomaki K. Soderstrom-Anttila V. et al.Why do singletons conceived after assisted reproduction technology have adverse perinatal outcome? Systematic review and meta-analysis., Pinborg A. Henningsen A.A. Loft A. Malchau S.S. Forman J. Andersen A.N. Large baby syndrome in singletons born after frozen embryo transfer (FET): is it due to maternal factors or the cryotechnique?., Wennerholm U.-B. Henningsen A.-K.- A. Romundstad L.B. Bergh C. Pinborg A. Skjaerven R. et al.Perinatal outcomes of children born after frozen-thawed embryo transfer: a Nordic cohort study from the CoNARTaS group., Ishihara O. Araki R. Kuwahara A. Itakura A. Saito H. Adamson G.D. Impact of frozen-thawed single-blastocyst transfer on maternal and neonatal outcome: an analysis of 277,042 single-embryo transfer cycles from 2008 to 2010 in Japan., Kaser D.J. Melamed A. Bormann C.L. Myers D.E. Missmer S.A. Walsh B.W. et al.Cryopreserved embryo transfer is an independent risk factor for placenta accreta., Sazonova A. Kallen K. Thurin-Kjellberg A. Wennerholm U.-B. Bergh C. Obstetric outcome in singletons after in vitro fertilization with cryopreserved/thawed embryos., The longer-term health outcomes for children born as a result of IVF treatment. Part II–Mental health and development outcomes., Meister T.A. Rexhaj E. Rimoldi S.F. Scherrer U. Sartori C. Effects of perinatal, late foetal, and early embryonic insults on the cardiovascular phenotype in experimental animal models and humans.]. Therefore, blastocyst transfer is thought to be an alternative way to improve the impaired clinical outcomes for elevated P levels on the hCG day [Papanikolaou E.G. Kolibianakis E.M. Pozzobon C. Tank P. Tournaye H. Bourgain C. et al.Progesterone rise on the day of human chorionic gonadotropin administration impairs pregnancy outcome in day 3 single-embryo transfer, while has no effect on day 5 single blastocyst transfer., Huang Y. Wang E.-Y. Du Q.-Y. Xiong Y.-J. Guo X.-y. Yu Y.-P. et al.Progesterone elevation on the day of human chorionic gonadotropin administration adversely affects the outcome of IVF with transferred embryos at different developmental stages.]. In addition, we conducted the analysis in two groups according to the type of embryo transfer, cleavage-stage embryo transfer and blastocyst transfer, and we found a decline in the CPR with increasing P levels on the hCG day in blastocyst transfer cycles, which suggested that blastocyst transfer did not overcome the possible adverse effects of higher P levels on the hCG day. At the same time, there were some previous studies that supported our opinion [Corti L. Papaleo E. Pagliardini L. Rabellotti E. Molgora M. La Marca A. et al.Fresh blastocyst transfer as a clinical approach to overcome the detrimental effect of progesterone elevation at hCG triggering: a strategy in the context of the Italian law., Ochsenkühn R. Arzberger A. von Schönfeldt V. Gallwas J. Rogenhofer N. Crispin A. et al.Subtle progesterone rise on the day of human chorionic gonadotropin administration is associated with lower live birth rates in women undergoing assisted reproductive technology: a retrospective study with 2,555 fresh embryo transfers., Elgindy E.A. Abou-Setta A.M. Mostafa M.I. Blastocyst-stage versus cleavage-stage embryo transfer in women with high oestradiol concentrations: randomized controlled trial.]. In a recent meta-regression analysis, Venetis et al.[Venetis C.A. Kolibianakis E.M. Bosdou J.K. Tarlatzis B.C. Progesterone elevation and probability of pregnancy after IVF: a systematic review and meta-analysis of over 60 000 cycles.] found no evidence for the influence of the developmental stage of the embryos at the time of transfer (cleavage-stage vs. blastocyst) on the association between elevated P levels and successful pregnancy. To provide the basis for the clinical selection of embryo transfer, we preliminarily explored the possible cut-off value of the hCG day P level. In Figure 2, when the P level was higher than 1.15 ng/mL, the red line (blastocyst transfer) was below the blue line (cleavage-stage embryo transfer). A further test of the interaction between the P levels on the hCG day and types of embryos transferred on the CPR of the two subgroups (divided by the P level: 1.15 ng/mL) was statistically significant in the subgroup with the higher P level (Table. S4, P3.0 ng/mL, which occurred in approximately 2.5% of total cycles. If the P level was greater than 3.0 ng/mL, the conclusions from this study may not apply. Furthermore, this hypothesis needs to be further confirmed by clinical prospective randomized controlled trials.There are still some weaknesses in the present study. Studies have suggested that increased P levels on the day of hCG (greater than 1.5 ng/mL) do not affect the clinical pregnancy outcomes of patients with high ovarian responsiveness[Griesinger G. Mannaerts B. Andersen C.Y. Witjes H. Kolibianakis E.M. Gordon K. Progesterone elevation does not compromise pregnancy rates in high responders: a pooled analysis of in vitro fertilization patients treated with recombinant follicle-stimulating hormone/gonadotropin-releasing hormone antagonist in six trials., Requena A. Cruz M. Bosch E. Meseguer M. García-Velasco J. High progesterone levels in women with high ovarian response do not affect clinical outcomes: a retrospective cohort study.]. Different cut-off values of P levels on the hCG day have been determined for different levels of ovarian responsiveness: 1.5 ng/mL in poor ovarian responders, 1.75 ng/mL in intermediate ovarian responders, and 2.75 ng/mL in high ovarian responders[Xu B. Li Z. Zhang H. Jin L. Li Y. Ai J. et al.Serum progesterone level effects on the outcome of in vitro fertilization in patients with different ovarian response: an analysis of more than 10,00

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