Objective: To investigate the effect of low progesterone (P) level on the day of human chorionic gonadotropin (hCG) injection on the outcome of in vitro fertilization (IVF) with GnRH- agonist (GnRH-a) long protocol.
Methods: A retrospective study was conducted in a reproductive medicine center of University-affiliated teaching hospital. 1115 women included were divided into two groups according to the P level: Group 1 (233 women with P level ≤ 0.5 ng/ml, and Group 2 (882 women with P level >0.5, ≤ 1.5 ng/ml). No. of oocyte retrieved, fertilization rate, implantation rate, and clinical pregnancy rate were compared between the two groups.
Results: Compared with Group 2, Group 1 had significantly decreased number of oocyte retrieved (9.84 ± 4.01 vs. 11.83 ± 4.06, P=0.000) and significantly increased implantation rate (39.4% vs. 34.1%, P=0.037). Group 1 had an increased trend of clinical pregnancy rate (55.8% vs. 49.9%, P=0.109) compared with that of Group 2. However, the fertilization rates were similar between Group 1 and Group 2 (76.6% vs. 77.0%, P=0.725).
Conclusions: Low P level on the day of hCG injection may have a trend towards higher implantation and clinical pregnancy rates after IVF cycles with GnRH-a long protocol. Therefore, we should not worry about the low P on hCG injection day.
Progesterone, In vitro fertilization, Pregnancy outcome
Progesterone (P) can be synthesized and secreted by the ovary and the adrenal glands. In the women ovary, granulosa cells and theca cells produce P. In the later stage of follicular, P is produced by mature granulosa cells before the luteinizing hormone (LH) peak , and plays a key role in the regulation ovulation .
During the process of controlled ovarian stimulation (COS), the premature LH surge could be avoidable with GnRH- agonist (GnRH-a) / GnRH-ant . So the elevation of P level would be avoided in most COS cycles because of the reduced LH level. However, P elevation during late follicular phase still occurs in about 38% of all in vitro fertilization cycles [4-5].
Until now, many studies have attempted to explore the impact of late follicular high-level P on the pregnancy outcome after IVF, and suggested that a higher P level have detrimental effect on the endometrial receptivity and negatively affect the pregnancy rate following IVF [6-14]. So during the clinical practice, embryo transfer would be cancelled if the P level is higher than a threshold.
The above researches seem to finally answer the question on whether a high P level have negative effect on the pregnancy outcome after COS or not. However, few studies have assessed the influence of low P level on IVF outcome.
In addition, animal experiments have indicated that the P level during the late follicular phase is important for the maturation of oocyte , fertilization of oocyte , and luteinization of theca / granulosa [17,18]. Additionally, P is important to support the endometrium in the luteal phase. Therefore, we suppose that low level P in the phase of late follicular may compromise pregnancy outcome after IVF cycles.
Until now, two clinical trails [19,20] have assessed the impact of low P level on the pregnancy outcome after IVF. One study included 254 women undergoing COS with GnRH-a protocol, and showed significant lower clinical pregnancy rate in infertile women with low level P (<0.7 ng/ml) . The other study compared pregnancy outcome of IVF among women with low P level, normal P level and high P level, and similarly demonstrated that low P level on the day of hCG injection impair live birth in patients with GnRH-ant protocol followed fresh embryo transfer . Considering the studies, which assess the association between low P level and pregnancy outcome after COS, were limited. So, the present study was aimed to study whether low P level on the day of hCG injection has detrimental effect on the pregnancy outcome after IVF with GnRH-a protocol.
Materials and Methods
Study population and design
The present study was reviewed and approved by the Institutional Review Board and the Ethics Committee of Xiangya Hospital, Changsha, China. The study was based on the Declaration of Helsinki, as revised in 1983. All participants in the study have obtained informed consent.
This was a retrospective cohort study, which was conducted at the Reproductive Medicine Center of Xiangya hospital, Central South University (Changsha, China). Women undergoing fresh IVF-ET with GnRH-a protocol between January of 2014 and May of 2015 were included in the present study. The inclusion criteria including: (1) 20-35 years old; (2) the first COS for IVF; (3) GnRH-a long protocol; (4) tubal factor infertility; (5) P level was ≤ 1.5ng/ml on the day of HCG injection. Exclusion criteria including: (1) endometrial polyp; (2) uterine anomaly; (3) donor oocytes or cryopreserved embryos. The included women were divided into two groups according to the concentration of P: Group 1: ≤ 0.5 ng/ml; Group 2: >0.5 ng/ml to ≤ 1.5 ng/ml.
Ovulation induction and IVF-ET procedures
All women used the long protocol with GnRH-a down regulation. GnRH-a (Triptorelin, sc 0.1 mg/day) was used beginning on the 21st day of the cycle prior to stimulation. Gn (FSH/HMG) and reduced dose of GnRH-a (Triptorelin, sc 0.05 mg/day) were given on the 2nd day of menstruation or later, and at this moment the serum estradiol concentration (E2) level should be ≤ 50 pg/ml, LH <5 mIU/ml, all follicle <9 mm. The dosage of Gn (FSH/HMG) ranged from 150 to 225 IU, according to the women age, AMH, and the number of total antral follicle. Oocytes were triggered with 10000 IU of hCG when the largest two follicles ≥ 18 mm. Oocytes were retrieved 36 hours after hCG injection, and followed by conventional IVF. No more than two highquality embryos were transferred 3 days after oocyte retrieval. The luteal phase was supported using 80 mg progesterone on oil intramuscular injection daily lasting 60 days. The presence of a gestational sac with heart activity via ultrasound 28-35 days after embryo transfer was considered to be clinical pregnancy.
P assessment immunoassay
Electrochemiluminescence immunoassay was used to measure the P levels on the day of hCG injection, with the measured sensitivity and total imprecision of 0.03 ng /ml and <5%, respectively.
Continuous data were recorded as Mean ± SD, and the Student’s t-test/ One-Way ANOVA were selected to conduct the statistics. Categorical data was reported as numbers and the Chi-Square test was used to compare the percentage. The women recruited in the present study were divided into 2 groups according to the level of P on the hCG injection day. SPSS16.0 (SPSS Inc, USA) was implied to carry out the statistical analysis, and the difference was considered to be significance when the P<0.05.
A total of 1115 IVF cycles (1115 women) were included in the present study. The average clinical pregnancy rate (PR) per transfer cycle was 51.1%. The average P level on the day of hCG injection was 0.78 ng/ml with a range from 0.03 ng/ml to 1.50 ng/ml. Other demographic date, such as age, infertile duration, BMI, COS days, E2 level, LH level on the day of hCG injection, the number of oocyte retrieved were summarized in Table 1.
|Characteristics||Mean ± SD|
|Age (years)||29.81 ± 3.13|
|Infertility (years)||4.28 ± 2.94|
|BMI (kg/m2)||21.73 ± 2.88|
|Length of stimulation (days)||11.05 ± 2.01|
|Total dose of Gn(IU)||1975.30 ± 684.34|
|Endometrial thickness on HCG day(mm)||10.91 ± 2.16|
|E2 on HCG day(pg/ml)||3557.45 ± 1935.64|
|LH on HCG day (IU/L)||1.67 ± 1.01|
|P on HCG day (ng/mL)||0.78 ± 0.31|
|No. of oocyte retrieved||11.42 ± 4.13|
|No. of MII oocyte||8.46 ± 3.01|
|No. of embryos||8.78 ± 3.81|
|No. of high quality embryos||5.57 ± 3.30|
|No. of embryos transferred||1.99 ± 0.09|
|No. of gestational±sac||0.70 ± 0.77|
|Kind of infertility|
|Primary infertility||38.6% (430/1115)|
|Secondary infertility||61.4% (685/1115)|
Table 1:Characteristics of study group (n=1115).
As hypothesized before, BMI, the number of oocyte/ MII oocyte obtained, the number of embryo/ high quality embryo, E2 level, LH levels on the day of hCG injection were different between Group 1 and Group 2 (Table 2). Nonetheless, our analysis still showed that there was no significantly difference in the oocyte maturation rates (74.4% vs. 74.1%, P=0.708), fertilization rates (76.6% vs. 77.0%, P=0.725,) and clinical pregnancy rates (55.8% vs. 49.9%, P=0.109) between the Group 1 and the Group 2. However, Group 1 has a significantly higher embryo implantation rate compared with that of the Groups 2 (39.4% vs. 34.1%, P=0.037) (Table 3).
|Characteristics||Group 1 (n=233)||Group2 (n=882)||P|
|Age (years)||29.75 ± 3.40||29.84 ± 3.08||0.714|
|Infertility (years)||4.63 ± 3.35||4.19 ± 2.82||0.067|
|BMI (kg/m2)||22.12 ± 3.20||21.63 ± 2.79||0.032|
|Length of stimulation (days)||11.10 ± 2.56||11.03 ± 1.84||0.700|
|Total dose of Gn (IU)||1940.02 ± 760.31||1984.62 ± 662.97||0.377|
|Endometrial thickness on HCG day (mm)||10.97 ± 2.06||10.89 ± 2.19||0.644|
|E2 on HCG day (pg/ml)||2447.85 ± 1401.10||3850.58 ± 1951.67||0.000|
|LH on HCG day (IU/L)||1.43 ± 0.76||1.73 ± 1.05||0.000|
|No. of oocyte retrieved||9.84 ± 4.01||11.83 ± 4.06||0.000|
|No. of MII oocyte||7.32 ± 3.00||8.76 ± 2.95||0.000|
|No. of embryos||7.54 ± 3.58||9.11 ± 3.80||0.000|
|No. of high quality embryos||4.96 ± 3.09||5.73 ± 3.33||0.001|
|No. of embryos transferred||1.98 ± 0.13||1.99 ± 0.08||0.197|
|No. of gestational sac||0.78 ± 0.79||0.68 ± 0.76||0.072|
Table 2: Characteristics of group 1 and group 2.
|Group 1 (n=233)||74.4%||76.6%||39.4%||55.8%|
|Group 2 (n=882)||74.1%||77.0%||34.1%||49.9%|
Table 3: Comparison of IVF outcome between group 1 and group 2.
In order to evaluate the effect of P level on the day of hCG injection on the pregnancy outcome of IVF-ET, the effect was further valuated at a threshold increment of 0.1 ng/ml. There was no significantly difference in the oocyte maturation rate, oocyte fertilization rate, embryo implantation rate, and clinical pregnancy rate amongst groups (Table 4).
|Oocyte maturation rate [%(n)]||Fertilization
|Clinical pregnancy rate [%(n)]|
|≤ 0.2||27||77.7 (199/256)||84.4 (216/256)||45.3 (24/53)||59.3 (16/27)|
|>0.2 to ≤ 0.3||46||75.2 (319/424)||73.8 (313/424)||41.8 (38/91)||58.7 (27/46)|
|>0.3 to ≤ 0.4||72||74.2 (520/701)||75.3 (528/701)||41.0 (59/144)||59.7 (43/72)|
|>0.4 to ≤ 0.5||88||73.3 (668/911)||76.7 (699/911)||35.1 (61/174)||50.0 (44/88)|
|>0.5 to ≤ 0.6||113||74.5 (905/1215)||77.0 (936/1215)||36.0 (81/225)||51.3 (58/113)|
|>0.6 to ≤ 0.7||131||74.1 (1072/1447)||76.0 (1100/1447)||32.7 (85/260)||47.3 (62/131)|
|>0.7 to ≤ 0.8||136||74.9 (1155/1542)||77.4 (1193/1542)||33.6 (91/271)||51.5 (70/136)|
|>0.8 to ≤ 0.9||130||74.4 (1143/1537)||77.5 (1191/1537)||37.7 (98/260)||53.8 (70/130)|
|>0.9 to ≤1.0||93||73.6 (886/1203)||76.6 (921/1203)||35.5 (66/186)||53.8 (50/93)|
|>1.0 to ≤ 1.1||100||73.6 (913/1240)||76.4 (947/1240)||38.0 (76/200)||55.0 (55/100)|
|>1.1 to ≤ 1.2||65||72.2 (559/774)||75.6 (585/774)||33.1 (43/130)||44.6 (29/65)|
|>1.2 to ≤ 1.3||56||73.1 (538/736)||77.4 (570/736)||25.9 (29/112)||41.1 (23/56)|
|>1.3 to ≤ 1.5||58||75.1 (558/743)||79.3 (589/743)||26.1 (30/115)||39.7 (23/58)|
Table 4: Clinical outcome and progesterone on the day of hCG injection.
Binary logistic regression analysis was implied to evaluate the effect of women age, duration of infertility, BMI, endometrial thickness, E2, LH, P levels on the day of hCG injection, number of oocyte/MII oocyte retrieved, and the number of embryos/ high quality embryo on clinical pregnancy. The analysis indicated that duration of infertility, P level were negatively correlated with clinical pregnancy, and that increased length of stimulation, endometrial thickness and the number of high quality embryo were associated with improved clinical pregnancy rates (Table 5).
|Duration of infertility||-0.035||0.097|
|Length of stimulation||-0.080||0.027|
|Total dose of Gn||0.000||0.019|
|P on hCG day||-0.439||0.029|
|No. of good quality embryos||0.063||0.001|
aClinical pregnancy served as dependent variable; Method = Backward stepwise (Likelihood Ratio)
cPvalues. R values were considered statistically non-significant unless P<0.05.
Table 5: Hierarchical regression analysis for variables predicting cigarette and alcohol use from the reasons for quitting, number of quit methods, and barriers to cessation.
So far, two studies have referred to the effect of low P level on the day of hCG injection on the pregnancy outcome after IVF cycles [19,20]. One included 254 women undergoing 296 IVF cycles with GnRH-ant protocol, and the other was with GnRH-ant protocol. To the best of our knowledge, this study is the largest with respect to sample size with 1115 IVF cycles, aiming to assess the effect of low P level on the hCG injection day on pregnancy outcome during COS in a long protocol with GnRH-a suppression.
As we all known, high P level on the day of hCG injection have detrimental effect on the embryo implantation via impairing endometrial receptivity. In our reproductive medicine center, we mostly cancelled embryo transfer if the P was higher than 1.5 ng/ml on the day of hCG injection.
By comparing the pregnancy outcome of these two groups, our results indicated that low P level may have no effect on the oocyte maturation, fertilization and clinical pregnancy. However, Group 1 had higher embryo implantation rate (P<0.05) and showed a trend towards higher clinical pregnancy rate (P>0.05) than Group 2, even the women in Group 1 have higher BMI, less number oocytes retrieved and embryos than that of women in Group 2. Our study showed obviously contrary results with two previous researches, one done by Santos-Ribeiro et al., which indicated that low P level on the day of hCG injection has detrimental impact on the pregnancy outcome of IVF using GnRH-ant for pituitary down- regulation ; another study, which including 254 women undergoing COS with GnRH-a pituitary suppression, also came to the same conclusion that low P range (<0.7 ng/ml) results in a lower pregnancy rate .
Additionally, the present study further evaluated the effect of P level on the day of hCG injection at a threshold increment of 0.1 ng/ml on IVF outcome, and the results did not find any significantly difference in the oocyte maturation rate, oocyte fertilization rate, embryo implantation rate and clinical pregnancy rate amongst groups. In a binary logistic regression model, P level on the day of hCG injection was an independent predictive factor for clinical pregnancy. The present study demonstrated an increased trend in pregnancy rate when P level decreased.
We analyzed the possible explanations for the above results as below: (i) Total dose of exogenous Gn, length of stimulation, stimulation protocol, oocyte maturation rate, and fertilization rate were same between Group 1 and Group 2. So, the difference in embryo implantation rate and clinical pregnancy rate would not be associated with oocyte quality and oocyte fertilization competence. (ii) E2 level of Group 2 was significantly higher compared with that of Group 1. Previous studies believed supraphysiologic E2 level have detrimental effect on the endometrial receptivity and resulted in impairment of embryo implantation [20-22]. In the present study, the higher E2 level may impair the endometrial receptivity even if the endometrial thickness is similar between the two groups. (iii) Previous researches including clinical trials and basic animal experiments all indicated that P is critical and necessary for the oocyte maturation and fertilization. Our present study found low P level on the day of hCG injection was associated with increased embryo implantation rate and increased clinical pregnancy rate. So, we suppose that the low P level in the IVF cycle is still above the lowest threshold of oocyte development, maturation and fertilization. (iv) The women in the present study were all undergone ovary stimulation with GnRH-a long protocol, whereas two previous trails used GnRH-ant protocol, which may contribute to the difference of the results.
This study has some limitations, one is that the sample size was not large enough, and another limitation is that the evaluation index is the fertilization rate, implantation rate and clinical pregnancy rate rather than the live birth rate, which may be a better observation. In spite of the limitations, the present study is of interest in that few studies have reported with contrary results.
In general, the present study demonstrated that low P level on the day of hCG injection have a trend towards a higher embryo implantation and clinical pregnancy after IVF cycles with GnRH-a long protocol. Therefore, we should not worry about the low P level on the day of hCG injection.
The authors declare that they have no conflict of interest.
This project was supported by the National Natural Science Foundation of China (Grant No. 81401269), and the class General Financial Grant from the China Postdoctoral Science Foundation (Grant No. 2017M620360).
Zhao Jing designed the study, performed the analyses and wrote the initial draft of the manuscript; Hao Jie, Wang Yonggang and Xu Bin collected the data of patients; Li Yanping performed the quality assessment of included studies; All authors contributed to the design, interpretation of the results and critical revision of the manuscript.
2. Chabbert-Buffeta N, Skinner DC, Caraty A, Bouchard P. Neuroendocrine effects of progesterone. Steroids. 2000 Oct 1;65(10-11):613-20.
3. Felberbaum R, Diedrich K. Ovarian stimulation for in-vitro fertilization/intracytoplasmic sperm injection with gonadotrophins and gonadotrophin-releasing hormone analogues: agonists and antagonists. Human Reproduction. 1999 Sep 1;14(suppl_1):207-21.
4. Ubaldi F, Camus M, Smitz J, Bennink HC, Van Steirteghem A, Devroey P. Premature luteinization in in vitro fertilization cycles using gonadotropin-releasing hormone agonist (GnRH-a) and recombinant folliclestimulating hormone (FSH) and GnRH-a and urinary FSH. Fertility and Sterility. 1996 Aug 1;66(2):275-80.
5. Bosch E, Valencia I, Escudero E, Crespo J, Simon C, Remohi J, Pellicer A. Premature luteinization during gonadotropin-releasing hormone antagonist cycles and its relationship with in vitro fertilization outcome. Fertility and Sterility. 2003 Dec 1;80(6):1444-9.
6. Kiliçdag EB, Haydardedeoglu B, Cok T, Hacivelioglu SO, Bagis T. Premature progesterone elevation impairs implantation and live birth rates in GnRH-agonist IVF/ ICSI cycles. Archives of gynecology and obstetrics. 2010 Apr 1;281(4):747-52.
7. M Kolibianakis E, A Venetis C, Bontis J, C Tarlatzis B. Significantly lower pregnancy rates in the presence of progesterone elevation in patients treated with GnRH antagonists and gonadotrophins: a systematic review and meta-analysis. Current Pharmaceutical Biotechnology. 2012 Mar 1;13(3):464-70.
8. 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. Fertility and Sterility. 2012 Jun 1;97(6):1321-7.
9. 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. Human Reproduction Update. 2013 Sep 1;19(5):433-57.
10. Santos-Ribeiro S, Polyzos NP, Haentjens P, Smitz J, Camus M, Tournaye H, et al. Live birth rates after IVF are reduced by both low and high progesterone levels on the day of human chorionic gonadotrophin administration. Human Reproduction. 2014 Aug 1;29(8):1698-705.
11. 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. Reproductive Biomedicine Online. 2012 May 1;24(5):511-20.
12. Li R, Qiao J, Wang L, Zhen X, Lu Y. Serum progesterone concentration on day of HCG administration and IVF outcome. Reproductive Biomedicine Online. 2008 Jan 1;16(5):627-31.
13. Özçakir HT, Levi R, Tavmergen E, Göker EN. Premature luteinization defined as progesterone estradiol ratio> 1 on hCG administration day seems to adversely affect clinical outcome in long gonadotropinreleasing hormone agonist cycles. Journal of Obstetrics and Gynaecology Research. 2004 Apr;30(2):100-4.
14. Bosch E, Labarta E, Crespo J, Simon C, Remohi J, Jenkins J, Pellicer A. Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles. Human reproduction. 2010 Aug 1;25(8):2092- 100.
15. Borman SM, Chaffin CL, Schwinof KM, Stouffer RL, Zelinski-Wooten MB. Progesterone promotes oocyte maturation, but not ovulation, in nonhuman primate follicles without a gonadotropin surge. Biology of Reproduction. 2004 Jul 1;71(1):366-73.
16. Zelinski-Wooten MB, Hess DL, Wolf DP, Stouffer RL. Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys. Fertility and Sterility. 1994 Jun 1;61(6):1147- 55.
17. Hibbert ML, Stouffer RL, Wolf DP, Zelinski-Wooten MB. Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates. Proceedings of the National Academy of Sciences. 1996 Mar 5;93(5):1897- 901.
18. Natraj US, Richards JS. Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. Endocrinology. 1993 Aug 1;133(2):761-9.
19. Levy MJ, Smotrich DB, Widra EA, Sagoskin AW, Murray DL, Hall JL. The predictive value of serum progesterone and 17-OH progesterone levels onin vitro fertilization outcome. Journal of Assisted Reproduction and Genetics. 1995 Mar 1;12(3):161-6.
20. Simon C, Cano F, Valbuena D, Remohi J, Pellicer A. Clinical evidence for a detrimental effect on uterine receptivity of high serum oestradiol concentrations in high and normal responder patients. Human Reproduction. 1995 Sep 1;10(9):2432-7.
21. Valbuena D, Jasper M, Remohí J, Pellicer A, Simón C. Ovarian stimulation and endometrial receptivity. Human Reproduction. 1999 Dec 1;14(suppl_2):107-11.
22. Horcajadas JA, Riesewijk A, Polman J, van Os R, Pellicer A, Mosselman S, Simón C. Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Molecular Human Reproduction. 2004 Mar 1;11(3):195-205.