|Year : 2015 | Volume
| Issue : 2 | Page : 80-85
Do increased levels of progesterone and progesterone/estradiol ratio on the day of human chorionic gonadotropin affects pregnancy outcome in long agonist protocol in fresh in vitro fertilization/intracytoplasmic sperm injection cycles?
Neeta Singh, Simran Deep Kaur, Nisha Malik, Neena Malhotra, P Vanamail
Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi, India
|Date of Submission||17-Jan-2015|
|Date of Decision||27-Feb-2015|
|Date of Acceptance||23-Mar-2015|
|Date of Web Publication||12-Jun-2015|
Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences, Room No. 3090 A, Teaching Block, 3rd Floor, New Delhi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The effect of elevated levels of serum progesterone (P 4 ) and estradiol (E 2 ) on the day of human chorionic gonadotropin and their cut-off value on in vitro fertilization (IVF) outcomes is still not clear. Aims: The aim was to evaluate the association between serum P 4 , E 2 and progesterone/estradiol ratio (P 4 /E 2 ) on pregnancy outcome in IVF/intracytoplasmic sperm injection (ICSI) cycles with long agonist protocol. Setting and Design: Retrospective, single center, cohort study. Materials and Methods: A review of complete data of 544 women undergoing fresh IVF/ICSI cycles (539 cycles) with long agonist protocol from January 2012 to February 2014 was done. Data were stratified into Three groups according to the number of oocytes retrieved: low (≤4 oocytes obtained), intermediate (5-19 oocytes obtained), and high ovarian response (≥20 oocytes obtained). Statistical Analysis: Fishers exact test/Chi-square was carried for comparing categorical data. Receiver operating characteristics analysis was performed to determine the cut-off value for P 4 and P 4 /E 2 detrimental for pregnancy. Results: A negative association was observed between pregnancy rate (PR) and serum P 4 and P 4 /E 2 levels with no effect on fertilization and cleavage rate. The overall cut-off value of serum P 4 and P 4 /E 2 ratio detrimental for pregnancy was found to be 1.075 and ≥0.35, respectively. Different P 4 threshold according to the ovarian responders were calculated, 1.075 for intermediate and 1.275 for high responders. Serum E 2 levels were not found to be significantly associated with PR. Conclusion: Serum P 4 levels and P 4 /E 2 ratio are a significant predictor for pregnancy outcome without affecting cleavage and fertilization rate while serum estradiol levels do not seem to affect PR.
Keywords: Estradiol, in vitro fertilization/intracytoplasmic sperm injection, progesterone, progesterone/estradiol ratio
|How to cite this article:|
Singh N, Kaur SD, Malik N, Malhotra N, Vanamail P. Do increased levels of progesterone and progesterone/estradiol ratio on the day of human chorionic gonadotropin affects pregnancy outcome in long agonist protocol in fresh in vitro fertilization/intracytoplasmic sperm injection cycles?. J Hum Reprod Sci 2015;8:80-5
|How to cite this URL:|
Singh N, Kaur SD, Malik N, Malhotra N, Vanamail P. Do increased levels of progesterone and progesterone/estradiol ratio on the day of human chorionic gonadotropin affects pregnancy outcome in long agonist protocol in fresh in vitro fertilization/intracytoplasmic sperm injection cycles?. J Hum Reprod Sci [serial online] 2015 [cited 2020 Jun 4];8:80-5. Available from: http://www.jhrsonline.org/text.asp?2015/8/2/80/158606
| Introduction|| |
Pregnancy outcomes during in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles may be influenced by supra-physiological concentrations of estradiol (E 2 ) and progesterone (P 4 ).  The current use of both Gonadotropin-releasing hormone (GnRH) agonist and antagonist, highly effective to prevent luteinizing hormone (LH) surge, has limited the need for determination of serum P levels. Still, studies have shown a rise in P 4 levels in 35% (5-35%) of GnRH agonists cycles and 38% (20-38%) with GnRH antagonists ,, but the effect on IVF outcome is still unclear. The mechanisms that account for this P 4 rise are not clearly understood. Recently, this elevated P 4 has been linked with the number of mature follicles and secretion of P 4 in late follicular phase. ,, Several questions have been raised regarding this P 4 elevation at the time of human chorionic gonadotropin (hCG) administration. The underlying mechanism of its effect on IVF success rate is not yet clear. Several studies-deny any association between P 4 and pregnancy rate (PR) ,, while others show a negative association. ,, In several studies, the increasing P 4 levels have shown an adverse effect on oocyte maturation, fertilization, or early cleavage  while other studies have denied the concept of poor embryo quality ,, and found it to be associated with impaired endometrial receptivity as a consequence of disturbed endometrial development and maturity , and gene expression.  Different detrimental cut-off values of P 4 and P 4 /E 2 has also been determined, but a consensus could not be reached.
This study aims to investigate the relationship between P 4 and E 2 levels on the day of hCG administration with PR, oocyte number, fertilization and cleavage rate in GnRH long agonist protocol in nondonor fresh IVF/ICSI cycles.
| Materials and methods|| |
This is a retrospective, single-center cohort study of patients undergoing IVF/ICSI treatment. Complete data of 544 women who underwent nondonor fresh IVF/ICSI cycles between January 2012 and February 2014 with long agonist protocol was reviewed. The primary or combined indications for fertility treatment were tubal pathology (45.1%), male subfertility (22.7%), endometriosis (10.7%), polycystic ovarian syndrome (5.9%), unexplained infertility (14.4%). To eliminate the confounding factors that might affect the outcome, following inclusion criteria was kept: Age < 40 years, follicle stimulating hormone (FSH) <10, antimullerian hormone (AMH) >1, fresh cycles (frozen cycles excluded). The study involves no violation of animal or human rights.
Patients underwent controlled ovarian hyperstimulation (COH) with use of a GnRH agonist long protocol. The pituitary down-regulation was achieved by subcutaneous injection of 1 mg of leuprolide acetate daily from the midluteal phase of the preceding cycle. Ovarian stimulation was done with 150-300 IU recombinant FSH follitropin alpha (Gonal-F, Merck Serono) SC (subcutaneously) and dose adjusted according to the response. Recombinant Chorionic Gonadotropin alpha (250 mg; Ovitrelle) was given to trigger ovulation when at least two-three leading follicles reached a mean diameter of 18 mm. Serum P 4 and E 2 levels were measured on the day of hCG administration by the chemiluminescent immunoassay using Access 2 Immunoassay system (Beckman Coulter) in the same laboratory. Oocyte pickup was done 36 h after hCG administration. Oocytes were cultured in G-IVF plus media (VITROLIFE) containing 10% of human serum albumin with gentamicin as an antibacterial agent and inseminated with motile sperm prepared by the two-layer percoll gradient method. Fertilization was defined as oocytes with two pronuclei 16-20 h after insemination. Embryos were transferred to G-IVF plus media and were classified by blastomere equalization and cytoplasmic fragment. Day 3 or 5 embryo transfer was done depending upon the number of embryos, and excess good-quality embryos were cryopreserved for subsequent frozen embryo transfer cycles with different grades of embryos (1-3). An ongoing pregnancy was defined as the pregnancy test done after 14 days of embryo transfer with a positive heartbeat by ultrasound at 6 weeks of gestation.
Grouping of high, intermediate and poor ovarian responders
Patients were categorized into 3 groups according to the number of oocytes retrieved: Low (≤4 oocytes), intermediate (5-19 oocytes) or high ovarian response (≥20 oocytes).
Six groups according to P 4 levels
Group A <1.0, group B: 1.0-1.25, group C 1.26-1.50, group D 1.51-1.75, group E 1.76-2.0 and group F > 2.0.
Five groups according to E 2 levels
Group A (<1000 pg/ml), group B (1000-2000 pg/ml), group C (2000-3000 pg/ml), group D (3000-4000 pg/ml), and group E (>4000 pg/ml).
All the statistical analyses were carried out using Statistical package for the social sciences (SPSS) IBM version 19.0 (Chicago, Illinois). Data were expressed as mean, standard deviation (SD) or frequencies and percentages. For comparing categorical data, Chi-square/Fishers exact test was carried out as appropriate. Receiver operating characteristics (ROC) analysis was performed to determine the cut-off value for P 4 and P 4 /E 2 at an approximately equivalent sensitivity and specificity, which may discriminate between pregnancy and nonpregnancy. A P < 0.05 was considered to be statistically significant.
| Results|| |
A total of 544 ovarian stimulation cycles were included in the study. Of these, 5 cycles need to be cancelled as no oocyte could be retrieved. Of the total 539 cycles with long agonist protocol, 319 (59.1%) were conventional IVF cycles and 220 (40.8%) were ICSI cycles. Of all, 146 (27.08%) clinical pregnancies were noted. Baseline characteristics such as age, body mass index, serum AMH, FSH, LH (follicular phase), total gonadotropin dose, endometrial thickness on day of hCG, E 2 and P 4 on the day of hCG, number of oocytes retrieved, fertilization rate, cleavage rate and PR for all the subjects among the different ovarian responders are shown in [Table 1].
|Table 1: Baseline characteristics of subjects in three groups according to ovarian response|
Click here to view
Overall, the mean age of the patients in our study was 31.6 ± 3.6 years (21-40). Average (SD) values of P 4 among different ovarian responders (≤4 oocytes, 5-19 oocytes and ≥20 oocytes) were 1.06 (0.7), 1.3 (0.8) and 1.4 (0.8), respectively, with an increasing value among high responders. Serum E 2 levels and numbers of mature oocytes retrieved also showed an increasing trend in high responders with a statistically significant difference between the three groups (P < 0.01).
We analyzed the correlation between the serum P 4 levels on fertilization rate, cleavage rate and PR among the different groups [Table 2]. It was found that PR was affected significantly with the change in serum P 4 levels. The total number of oocytes retrieved also varied significantly between the groups showing increasing trend with P 4 values.
|Table 2: Comparison of fertilization rate, cleavage rate, pregnancy rate and number of oocytes retrieved in different groups according to serum P4 levels|
Click here to view
The trend of PR according to P 4 levels is as shown in [Figure 1]. PR showed a significantly decreasing trend (Chi-square trend in proportion = 8.25; P = 0.004) with increasing level of P 4 with maximum PR in the range of 1.01-1.25, but after a serum P level of 2 ng/ml, relatively stable effect was seen.
|Figure 1: Trend of pregnancy rate with 95% confidence limits according to P4 levels|
Click here to view
To assess an optimum P 4 level for an equivalent sensitivity and specificity for pregnancy status, ROC analysis was carried out. The area under curve (AUC) 0.58 (95% confidence interval [CI]: 0.53-0.63) was significant (P = 0.006). Overall, P 4 level was found to be 1.075 for an equivalent sensitivity and specificity value of 55% [Figure 2] with positive and negative predictive value of 31.3% and 77%, respectively. For intermediate and high responders, the cut-off value was found to be 1.075 for an equivalent sensitivity and specificity value of 56% and 1.275 for the sensitivity and specificity value of 62%, respectively, while it could not be predicted for low responders as AUC was not statistically significant.
|Figure 2: Receiver operating characteristic curve for defining optimal detrimental cut-off value for P4 on human chorionic gonadotropin day|
Click here to view
[Table 3] shows the correlation of E 2 on the day of hCG with fertilization, cleavage and PR. Bivariate logistic regression analysis revealed that the variable E 2 on day of hCG alone is not a significant predictor of pregnancy status, fertilization rate and cleavage rate although the number of oocytes retrieved increased significantly with an increase in E 2 levels.
|Table 3: Comparison of fertilization rate, cleavage rate, pregnancy rate and number of oocytes retrieved in different groups according to serum E2 levels|
Click here to view
Impact of P 4 /E 2 ratio on the day of human chorionic gonadotropin
To assess threshold level of P 4 to E 2 ratio (P 4 [ng/ml] ×1000/E 2[pg / ml] ) for PR, ROC analysis was carried out. AUC 0.58 (95% CI: 0.53-0.64) was found to be statistically significant (P = 0.003). For an equivalent sensitivity and specificity value (56%) the corresponding value of P 4 /E 2 ratio was found to be 0.35 [Figure 3]. PR (31.4%) among the patients having ≤ 0.35 P 4 /E 2 ratio was significantly (P = 0.047) higher compared with 23.4% observed among the patients having value >0.35. However, fertilization and cleavage rates were not significantly (P > 0.05) different between the two categories. Similar ROC analysis was carried out according to the ovarian responders. As AUC was not significant, therefore P 4 /E 2 cut-off value could not be predicted.
|Figure 3: Receiver operating characteristic curve for defining optimal detrimental cut-off value for P4/E2 ratio on human chorionic gonadotropin day|
Click here to view
| Discussion|| |
This study has analyzed the correlation between serum P 4 , E 2 and P 4 /E 2 ratio on hCG day with PR. As a secondary outcome, the effect on cleavage and fertilization rate was also studied. The results have shown that serum P 4 levels and P 4 /E 2 ratio is a significant predictor for PR while the E 2 levels had no significant association. PR showed a significantly decreasing trend with an increasing level of P 4 and P 4 /E 2 levels. Different cut-off levels for P 4 and P 4 /E 2 were determined among different ovarian responders. Fertilization and cleavage rate were not affected by either P 4 or E 2 levels.
The potential effect of type of responders on the association between P 4 elevation and probability of pregnancy was also explored. Earlier there have been three studies in which data were analyzed according to the type of ovarian response. , Recent study by Xu et al. has concluded that there is a significant decrease in ongoing PR and implantation rate with P elevation in all ovarian responses to COH.  Our study has also shown results in agreement with that study and different cut-off values for P 4 among intermediate and high responders were predicted.
A positive correlation between number of mature follicles and secretion of P 4 in late follicular phase have been reported in earlier studies.  In the present study also, an increasing pattern of serum P level was observed with a significant (P < 0.05) difference between the three types of responders thus supporting the concept that increasing P 4 levels is a reflection of the number of follicles and not due to premature luteinization.
Till now, there have been numerous studies which have evaluated the association of P 4 elevation with PR with conflicting results. Our results were in agreement with a recent meta-analysis published in 2013 which have reported a detrimental effect of P 4 elevation on PR in range of 0.8-1.1 ng/ml (odds ratio: 0.79).  The present study has used ROC analysis which is a preferred method to identify optimal thresholds to define these detrimental cut-offs and the value was found to be 1.075 for an equivalent (53%) level of sensitivity and specificity.
Serum E 2 concentration on the day of hCG alone was not found to be a significant predictor of pregnancy status which is in consensus with previous studies by Kyrou et al. (2012) and Yu Ng et al. ,
Not only the P 4 levels alone but also P 4 /E 2 has nowadays been considered to be a reasonable option for prediction of pregnancy outcome. A prospective cohort study by Elgindy published in 2012 have found a cut-off levels of P 4 >1.5 ng/ml and P 4 /E 2 >0.55 as detrimental to pregnancy.  In our study, P 4 /E 2 ratio ≤0.35 was found to be associated with a significantly higher PR compared to those having value >0.35 although no significant association with fertilization and cleavage rates was found. [Table 4] are summarizes the most recent studies with different threshold cut-offs of serum P 4 and P 4 /E 2 levels. ,,,,,,,,
|Table 4: Recent studies showing the cut - off levels of P4 or P4/E2 levels in women undergoing fresh IVF cycles|
Click here to view
Either cryopreservation of pronuclear or cleavage stage embryos or blastocyst transfer has been suggested as a strategy to overcome this problem. A recent study by Corti et al. in 2013 has concluded that fresh blastocyst transfer does not completely overcome the detrimental effects of progesterone rise at hCG on pregnancy outcome. 
| Conclusion|| |
The present study has shown a negative association of increasing P 4 levels and P 4 /E 2 with PR. It still remains uncertain whether freezing the embryos and transferring them later in frozen-thawed cycles will be a solution to improve the pregnancy outcome when P 4 levels are high. More randomized studies are required to evaluate the effectiveness of frozen cycles in comparison to fresh cycles to overcome the detrimental effect of elevated P 4 or P 4 /E 2 ratio.
| Acknowledgments|| |
We acknowledge the patients who participated and the colleagues who supported us while conducting this study.
| References|| |
Wu Z, Li R, Ma Y, Deng B, Zhang X, Meng Y, et al.
Effect of HCG-day serum progesterone and oestradiol concentrations on pregnancy outcomes in GnRH agonist cycles. Reprod Biomed Online 2012;24:511-20.
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. Hum Reprod 2010;25:2092-100.
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. Fertil Steril 2003;80:1444-9.
Harada T, Katagiri C, Takao N, Toda T, Mio Y, Terakawa N. Altering the timing of human chorionic gonadotropin injection according to serum progesterone (P) concentrations improves embryo quality in cycles with subtle P rise. Fertil Steril 1996;65:594-7.
Fleming R, Jenkins J. The source and implications of progesterone rise during the follicular phase of assisted reproduction cycles. Reprod Biomed Online 2010;21:446-9.
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. Fertil Steril 2012;97:1321-7.e1-4.
Martínez F, Coroleu B, Clua E, Tur R, Buxaderas R, Parera N, et al.
Serum progesterone concentrations on the day of HCG administration cannot predict pregnancy in assisted reproduction cycles. Reprod Biomed Online 2004;8:183-90.
Urman B, Alatas C, Aksoy S, Mercan R, Isiklar A, Balaban B. Elevated serum progesterone level on the day of human chorionic gonadotropin administration does not adversely affect implantation rates after intracytoplasmic sperm injection and embryo transfer. Fertil Steril 1999;72:975-9.
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. Reprod Biomed Online 2011;23:187-95.
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. J Assist Reprod Genet 1996;13:207-11.
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. Fertil Steril 1993;59:1090-4.
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. Fertil Steril 2012;98:347-54.
Schoolcraft W, Sinton E, Schlenker T, Huynh D, Hamilton F, Meldrum DR. Lower pregnancy rate with premature luteinization during pituitary suppression with leuprolide acetate. Fertil Steril 1991;55:563-6.
Bustillo M, Stern JJ, Coulam CB. Serum progesterone at the time of human chorionic gonadotrophin does not predict pregnancy in in-vitro
fertilization and embryo transfer. Hum Reprod 1995;10:2862-7.
Silverberg KM, Martin M, Olive DL, Burns WN, Schenken RS. Elevated serum progesterone levels on the day of human chorionic gonadotropin administration in in vitro
fertilization cycles do not adversely affect embryo quality. Fertil Steril 1994;61:508-13.
Fanchin R, Righini C, Olivennes F, de Ziegler D, Selva J, Frydman R. Premature progesterone elevation does not alter oocyte quality in in vitro
fertilization. Fertil Steril 1996;65:1178-83.
Van Vaerenbergh I, Fatemi HM, Blockeel C, Van Lommel L, In′t Veld P, Schuit F, et al.
Progesterone rise on HCG day in GnRH antagonist/rFSH stimulated cycles affects endometrial gene expression. Reprod Biomed Online 2011;22:263-71.
Labarta E, Martínez-Conejero JA, Alamá P, Horcajadas JA, Pellicer A, Simón C, et al.
Endometrial receptivity is affected in women with high circulating progesterone levels at the end of the follicular phase: A functional genomics analysis. Hum Reprod 2011;26:1813-25.
Fanchin R, Righini C, Olivennes F, Ferreira AL, de Ziegler D, Frydman R. Consequences of premature progesterone elevation on the outcome of in vitro
fertilization: Insights into a controversy. Fertil Steril 1997;68:799-805.
Gordon K, Kolibianakis E, Griesinger G, Yding Andersen C, Witjes H, Mannaerts B. Impact of elevated progesterone at the end of the follicular phase during treatment with recombinant FSH and ganirelix: A combined analysis. Hum Reprod 2012;27:302-37.
Venetis CA, Kolibianakis EM, Bosdou JK, Tarlatzis BC. Progesterone elevation and probability of pregnancy after IVF: A systematic review and meta-analysis of over 60 000 cycles. Hum Reprod Update 2013;0:1-25.
Kyrou D, Al-Azemi M, Papanikolaou EG, Donoso P, Tziomalos K, Devroey P, et al.
The relationship of premature progesterone rise with serum estradiol levels and number of follicles in GnRH antagonist/recombinant FSH-stimulated cycles. Eur J Obstet Gynecol Reprod Biol 2012;162:165-8.
Yu Ng EH, Yeung WS, Yee Lan Lau E, So WW, Ho PC. High serum oestradiol concentrations in fresh IVF cycles do not impair implantation and pregnancy rates in subsequent frozen-thawed embryo transfer cycles. Hum Reprod 2000;15:250-5.
Elgindy EA. Progesterone level and progesterone/estradiol ratio on the day of hCG administration: Detrimental cutoff levels and new treatment strategy. Fertil Steril 2011;95:1639-44.
Azem F, Tal G, Lessing JB, Malcov M, Ben-Yosef D, Almog B, et al
. Does high serum progesterone level on the day of human chorionic gonadotropin administration affect pregnancy rate after intracytoplasmic sperm injection and embryo transfer? Gynecol Endocrinol 2008;24:368-72.
Seow KM, Lin YH, Hsieh BC, Huang LW, Huang SC, Chen CY, et al.
Characteristics of progesterone changes in women with subtle progesterone rise in recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonist cycle. Gynecol Obstet Invest 2010;70:64-8.
Yu N, Yang J, Yin TL, Zhao QH. Effect of serum estradiol and progesterone level and progesterone/estradiol ratio on the day of human chorionic gonadotropin administration in pregnancy outcome of in vitro
fertilization-embryo transplantation. J Clin Rehabil Tissue Eng Res 2010;14:5763-6.
Rezaee Z, Ghaseminejad A, Forootan M, Hosseinipoor T, Forghani F. Assessment of serum progesterone level on the day of HCG injection in infertile polycystic ovarian syndrome patients referred to women′s hospital, Tehran, 2009. Int J Fertil Steril 2012;5:231-4.
Cetinkaya ES, Berker B, Aytac R, Atabekoglu C, Sonmezer M, Ozmen B. The value of the progesterone-to-estradiol ratio on the day of hCG administration in predicting ongoing pregnancy and live birth rates in normoresponders undergoing GnRH antagonist cycles. Eur J Obstet Gynecol Reprod Biol 2013;170:452-7.
Bu Z, Zhao F, Wang K, Guo Y, Su Y, Zhai J, et al.
Serum progesterone elevation adversely affects cumulative live birth rate in different ovarian responders during in vitro
fertilization and embryo transfer: A large retrospective study. PLoS One 2014;9:e100011.
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. Eur J Obstet Gynecol Reprod Biol 2013;171:73-7.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]