Comparative study of pregnancy outcomes between day 3 embryo transfer and day 5 blastocyst transfer in patients with progesterone elevation

Abstract

Objective

To explore the effects of progesterone (P) elevation on pregnancy outcomes of day 3 embryo and day 5 blastocyst transfer.

Methods

Clinical outcomes (pregnancy and ectopic pregnancy rates) following day 3 embryo and day 5 blastocyst transfer cycles were retrospectively analysed. Day 3 embryo and day 5 blastocyst transfer cycles were divided into normal P level (P ≤ 1.5 ng/ml) and P elevation group (P > 1.5 ng/ml), based on the serum P level on the day of human chorionic gonadotropin (hCG) administration.

Results

A total of 2868 cycles were analysed. In day 3 embryo transfer cycles (n = 2345), the clinical pregnancy rate was significantly higher in the normal P level group compared with the P elevation group (55.4% versus 46.7%, respectively) and the ectopic pregnancy rate was significantly lower in the normal P level group compared with the P elevation group (2.8% versus 7.9%, respectively). In day 5 blastocyst transfer cycles (n = 523), there were no significant differences in the clinical pregnancy and ectopic pregnancy rates between the two groups, based on the P level.

Conclusion

These preliminary findings suggest that day 5 blastocyst transfer should be adopted for patients with P elevation on the day of hCG administration.

Keywords

Progesterone elevation pregnancy outcome day 3 embryo transfer day 5 blastocyst transfer

Introduction

Under physiological status, the progesterone (P) level rapidly rises after ovulation, which leads to the development of secretory-phase endometrium. Under superovulation, the P level in the late follicular phase often rises ahead of time, due to the use of exogenous gonadotropin and the development of multiple ovarian follicles.¹ The elevated P level in the late follicular phase can decrease the implantation rate of cleavage-stage embryo transfer used for in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI).^2–5 During superovulation, high levels of steroid hormones may increase the risk of ectopic pregnancy.⁶ The effects of P elevation in the late follicular phase on the pregnancy outcomes of different-stage embryo transfer are uncertain, particularly in terms of ectopic pregnancy rates. This current preliminary study retrospectively analysed the IVF cycles, including day 3 embryo transfer and day 5 blastocyst transfer cycles, that were performed over a 2-year period in a reproductive centre, in order to explore the effects of the P level in the late follicular phase on the clinical pregnancy rate and ectopic pregnancy rate of different-stage embryo transfers.

Patients and methods

Patient population

This retrospective study analysed data from consecutive women undergoing IVF cycles, including day 3 embryo transfer cycles and day 5 blastocyst transfer cycles, which were performed at the Reproductive Medical Centre, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China between January 2010 and December 2011. Inclusion criteria were: (i) age ≤38.0 years; (ii) first or second IVF cycle; (iii) basal follicle-stimulating hormone (FSH) < 10 mIU/ml. Exclusion criteria were: (i) uterine lesions such as uterine malformation, polyps and intrauterine adhesion; (ii) three failed individual IVF cycles; (iii) either of the couple had chromosomal abnormalities. Primary infertility was considered as infertility in a couple who had never got pregnant; secondary infertility was defined as a failure to conceive following a previous pregnancy.

According to the serum P level on the day of human chorionic gonadotropin (hCG) administration, the day 3 embryo transfer and day 5 blastocyst transfer cycles were divided into a normal P level group (P level ≤ 1.5 ng/ml) and a P elevation group (P level > 1.5 ng/ml). The clinical pregnancy rate and ectopic pregnancy rate were compared between the normal P level and P elevation groups.

All study methods were approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University. All patients enrolled into the study provided written informed consent to participate.

Serum hormone measurements

Blood samples were obtained from the cubital vein on the day of hCG administration. Samples that were not used immediately were stored at 2–8℃, then used within 24 h. Blood samples were centrifuged at 2000 g for 10 min at 25℃. Serum levels of FSH, luteinizing hormone (LH), 17β-oestradiol (E₂) and P were measured on the day of hCG administration using an automated Elecsys Immunoanalyzer (Roche Diagnostics, Mannheim, Germany).

IVF treatment

Downregulation of reproductive hormone levels was performed with gonadotropin releasing hormone agonist, administered in the luteal phase (Decapeptyl®; Ferring Pharmaceuticals, Kiel, Germany; 0.05 mg intramuscular [i.m.] injection once daily until the day of hCG administration). When downregulation reached the standard level (FSH < 5.0 IU/l; E₂ < 50 pg/ml; LH < 5.0 IU/l; P < 1.0 ng/ml; maximum diameter of antral follicles <10 mm), recombinant FSH (Gonal-f®; Serono, Madrid, Spain; 150 – 225 IU, i.m. injection once daily for 10–14 days) or human menotrophin (MENOPUR®; Ferring Pharmaceuticals; 150 – 225 IU, i.m. injection once daily for 10–14 days) was given to induce superovulation. Transvaginal ultrasonography was undertaken using a Philips HDI 4000 Ultrasound Machine (Philips Healthcare, Best, The Netherlands) and when it showed that follicular development had reached the expected stage, a single i.m. injection of 250 µg hCG (Ovidrel®; Merck Serono, Geneva, Switzerland) was administered. Oocytes were collected by transvaginal ultrasound-guided puncture 36–37 h after hCG administration. IVF was performed according to the semen status. On day 3, embryos were evaluated,⁷ then two or thee high-quality embryos were transferred or were used for blastocyst culture. On day 5, blastulation status was observed and one or two blastocysts were transferred.

Clinical pregnancy

Clinical pregnancy was diagnosed when B-mode ultrasound showed a gestation sac and fetal heart beat at 35 days after embryo transfer. An ectopic pregnancy was diagnosed when B-mode ultrasound showed a gestation sac with or without an embryo bud and heart beat outside the uterine cavity, or an ectopic pregnancy lesion was found during laparoscopy at 35 days after embryo transfer.

Statistical analyses

All statistical analyses were performed using the SPSS® statistical software package, version 12.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Independent-sample t-test was used to analyse the differences between parameters with mean values; χ²-test was used to analyse the differences between parameters presented as rates. A P-value < 0.05 was considered statistically significant.

Results

This study analysed the data from 2868 IVF cycles, including 2345 day 3 embryo transfer cycles and 523 day 5 blastocyst transfer cycles. The overall ectopic pregnancy rates were 3.4% (43/1272) in day 3 embryo transfer cycles and 2.1% (five of 236) in day 5 blastocyst transfer cycles.

In day 3 embryo transfer cycles, there were no significant differences in age, duration of infertility, proportion of patients with primary infertility, body mass index (BMI), proportion of patients without fallopian tube lesions, endometrial thickness and number of embryos transferred between the normal P level group (P ≤ 1.5 ng/ml) and the P elevation group (P > 1.5 ng/ml) (Table 1). E₂ and P levels at the time of hCG administration were significantly higher in the P elevation group compared with the normal P level group (P < 0.001 for both comparisons). Clinical pregnancy rates were 55.4% (1132/2045) in the normal P level group and 46.7% (140/300) in the P elevation group (P < 0.001) (Table 2). Ectopic pregnancy rates were 2.8% (32/1132) in the normal P level group and 7.9% (11/140) in the P elevation group (P = 0.002).

Table 1.

Demographic and clinical data for patients undergoing day 3 embryo transfer cycles and day 5 blastocyst transfer cycles, stratified according to their serum progesterone (P) levels on the day of treatment with human chorionic gonadotropin (hCG).

Characteristic	Day 3 embryo transfer cycles n = 2345			Day 5 blastocyst transfer cycles n = 523
Characteristic	P ≤ 1.5 ng/ml n = 2045	P > 1.5 ng/ml n = 300	Statistical significance	P ≤ 1.5 ng/ml n = 435	P > 1.5 ng/ml n = 88	Statistical significance
Age, years^a	30.17 ± 2.90	30.79 ± 3.79	NS	30.16 ± 4.21	30.24 ± 3.19	NS
Duration of infertility, years^a	4.32 ± 2.88	4.51 ± 3.11	NS	4.09 ± 2.88	3.03 ± 2.12	NS
Primary infertility, n (%)^b	1079/2045 (52.8)	143/300 (46.7)	NS	211/435 (48.5)	39/88 (44.3)	NS
Body mass index, kg/m^2a	22.26 ± 3.03	21.84 ± 2.64	NS	22.05 ± 2.78	22.65 ± 3.78	NS
No fallopian tube lesions^b	521/2045 (25.5%)	65/300 (21.7%)	NS	190/435 (43.7%)	35/88 (39.8%)	NS
Endometrial thickness, mm^a	12.42 ± 2.28	12.41 ± 2.41	NS	12.15 ± 2.23	12.14 ± 2.62	NS
Embryos transferred, n^a	2.11 ± 0.31	2.11 ± 0.37	NS	1.12 ± 0.32	1.24 ± 0.43	NS
E₂ on hCG administration, pg/ml^a	5276.73 ± 3231.19	7173.09 ± 2710.04	P < 0.001^c	7291.12 ± 2586.89	8127.69 ± 2835.14	NS
P on hCG administration, ng/ml^a	0.73 ± 0.36	1.91 ± 0.38	P < 0.001^c	0.74 ± 0.32	1.93 ± 0.39	P < 0.001^c

Data presented as mean ± SD or n (%) of patients.

Independent-samples t-test.

χ²-test.

Compared with normal P level group (P ≤ 1.5 ng/ml).

E₂, 17β-oestradiol; NS, no statistically significant difference (P ≥ 0.05).

Table 2.

Pregnancy outcomes for patients undergoing day 3 embryo transfer cycles and day 5 blastocyst transfer cycles, stratified according to their serum progesterone (P) level on the day of treatment with human chorionic gonadotropin (hCG).

Outcome	Day 3 embryo transfer cycles n = 2345			Day 5 blastocyst transfer cycles n = 523
Outcome	P ≤ 1.5 ng/ml n = 2045	P > 1.5 ng/ml n = 300	Statistical significance	P ≤ 1.5 ng/ml n = 435	P > 1.5 ng/ml n = 88	Statistical significance
Clinical pregnancy	1132/2045 (55.4)	140/300 (46.7)	P < 0.001^a	199/435 (45.7)	37/88 (42.0)	NS
Ectopic pregnancy	32/1132 (2.8)	11/140 (7.9)	P = 0.002^a	3/199 (1.5)	2/37 (5.4)	NS

Data presented as n (%) of patients.

Compared with normal P level group (P ≤ 1.5 ng/ml); χ²-test.

NS, no statistically significant difference (P ≥ 0.05).

In day 5 blastocyst transfer cycles, there were no significant differences in age, duration of infertility, proportion of patients with primary infertility, BMI, proportion of patients without fallopian tube lesions, endometrial thickness, number of embryos transferred and E₂ levels at the time of hCG administration between the normal P level group (P ≤ 1.5 ng/ml) and the P elevation group (P > 1.5 ng/ml) (Table 1). The P level at the time of hCG administration was significantly higher in the P elevation group compared with the normal P level group (P < 0.001). There were no significant differences in clinical pregnancy and ectopic pregnancy rates between the normal P level group and the P elevation group (Table 2).

Discussion

The pregnancy outcomes of IVF are related to many factors such as age, embryo quality and endometrial receptivity. The P level is closely associated with clinical pregnancy rates following IVF.^2–5 Melo et al.⁸ reported that there were no significant differences in fertilization rate, cleavage rate, implantation rate and clinical pregnancy rate among recipients based on whether the oocytes came from donors with or without an elevated P level on the day of hCG administration, suggesting that P elevation did not affect oocyte quality. An analysis of 4000 IVF/ICSI cycles indicated that serum P elevation (P > 1.5 ng/ml) on the day of hCG administration significantly decreased the clinical pregnancy rates.⁵ In this current study, the clinical pregnancy rate was also significantly decreased in the P elevation group of day 3 embryo transfer cycles compared with the normal P level group. This may be associated with the fact that an earlier P elevation stimulates a premature transition to secretory-phase endometrium, which in turn might negatively affect endometrial receptivity.

There have been different reports regarding the effects of P elevation on blastocyst transfer. Papanikolaou et al.⁹ performed a prospective study of 483 patients and found that P elevation affected the pregnancy outcomes of day 3 embryo transfer, but failed to affect the pregnancy outcomes of day 5 blastocyst transfer. An investigation of the endometria of natural and stimulated cycles found that there was no earlier endometrial development in the mid-luteal phase of stimulated cycles.¹⁰ In this current study, there was no significant difference in the clinical pregnancy rates between the normal P level group and the P elevation group following day 5 blastocyst transfer. This might be due to the P elevation on the day of hCG administration not affecting day 5 endometrium.

The incidence of ectopic pregnancy is ∼1.9 % in natural pregnancies, while in assisted reproductive technology (ART), it is about 2.1–11%.¹¹ This higher rate of ectopic pregnancy in patients receiving ART might be due to the presence of fallopian tube disease. The exact cause of ectopic pregnancy is unclear in IVF embryo transfer, but diseases of the fallopian tubes (such as salpingitis) or a history of fallopian tube surgery are associated with a higher risk for ectopic pregnancy.^12,13 Ectopic pregnancy following ART is associated with the depth of the transplantation tube into the uterine cavity, the fluid volume in the transplantation tube, the velocity of embryo transfer, the number of embryos transferred, the patient’s body position and embryo transmigration in the uterine cavity.¹⁴

The ectopic pregnancy rate was demonstrated to be significantly higher in superovulation cycles (1.8%) than in oocyte-donation cycles (0.5%).⁸ There has been a lack of conclusive evidence concerning the effects of P elevation in the late follicular phase on ectopic pregnancy. Paltieli et al.¹⁵ stimulated isolated fallopian tubes with E₂, human menopausal gonadotropin, P, LH and FSH, then measured ciliary beat frequency; Paltieli’s group found that a high concentration of P could lead to ciliary functional disorder, speculating that high concentrations of P may be one of the main causes of ectopic pregnancy in superovulation. An analysis of endometrial functional genomics in 12 patients undergoing oocyte donation found marked changes in endometrial gene expression profiling (including 13 endometrial receptivity-related genes) in patients with P elevation on the day of hCG administration.¹⁶ A comparison of endometrial gene expression between natural and stimulated cycles in the same patient found marked changes in endometrial gene expression profiling in stimulated cycles; these changes affected endometrial receptivity.¹⁷ Under physiological status, the development of cleavage-stage embryos takes place in the fallopian tubes. Therefore, the transferred cleavage-stage embryos do not immediately implant, but return to the fallopian tubes for development and then do not re-enter the uterine cavity for implantation until the blastula stage.

In this current study, embryo transfer was performed by the same doctors, the depth of the transplantation tube into the uterine cavity was always 1.5 cm and the fluid volume in the transplantation tube was always 2 µl. These current results demonstrated that in the day 3 embryo transfer cycles, levels of P and E₂ on the day of hCG administration were significantly higher in the P elevation group than in the normal P level group; and the ectopic pregnancy rate was significantly increased in the P elevation group compared with normal P level group. In contrast, in the day 5 blastocyst transfer cycles, there were no significant differences in the level of E₂ on the day of hCG administration or in the ectopic pregnancy rate between the P elevation group and normal P level group. The current findings in the day 3 embryo transfer cycles suggest the following: (i) higher levels of P and E₂ can induce uterine smooth muscle contraction that allows transferred day 3 embryos to enter the fallopian tubes readily; (ii) high P level-induced changes in endometrial gene expression negatively affect embryo implantation in the uterine cavity; (iii) high P level-induced ciliary movement disorder does not facilitate the normal return of embryos to the uterine cavity. The current findings in the day 5 blastocyst transfer cycles suggest that blastocysts are more readily implanted (because they do not need to re-enter the fallopian tubes for further development) and that P elevation has no effect on the physiological status of day 5 endometrium.

Does blastocyst transfer decrease the risk of ectopic pregnancy? Because blastocysts do not need to re-enter the fallopian tubes for further development, the ectopic pregnancy rate should be decreased. In this current study, the ectopic pregnancy rate was 3.4% in day 3 embryo transfer cycles and 2.1% in day 5 blastocyst transfer cycles. Although the ectopic pregnancy rate was slightly lower in day 5 blastocyst transfer cycles than in day 3 embryo transfer cycles, it was not possible to undertake a statistical comparison of the difference because of the disparity between the numbers of cycles undertaken in the two groups. Future research will investigate the causes of ectopic pregnancy in a prospective study that directly compares day 3 embryo transfer cycles with the same number of day 5 blastocyst transfer cycles.

In conclusion, these current results suggest that P elevation in the late follicular phase can decrease the clinical pregnancy rate and increase the ectopic pregnancy rate in day 3 embryo transfer cycles, but day 5 blastocyst transfer appears to avoid these negative effects of P elevation. Therefore, day 5 blastocyst transfer should be adopted in the patients who have high P levels on the day of hCG administration and are unwilling to give up fresh embryo transfer. However, since the number of blastocyst transfer cycles was smaller than the number of day 3 embryo transfer cycles in this current study, studies with larger sample sizes will be needed to confirm these preliminary results.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

References

Qiao

Wang

. Serum progesterone concentration on day of HCG administration and IVF outcome. Reprod Biomed Online 2008; 16: 627–631.

Azem

Tal

Lessing

. 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: 368–372.

Venetis

Kolibianakis

Papanikolaou

. Is progesterone elevation on the day of human chorionic gonadotrophin administration associated with the probability of pregnancy in in vitro fertilization? A systematic review and meta-analysis. Hum Reprod Update 2007; 13: 343–355.

Bosch

Valencia

Escudero

. Pemature luteinization during gonadotropin-releasing hormone antagonist cycles and its relationship with in vitro fertilization outcome. Fertil Steril 2003; 80: 1444–1449.

Bosch

Labarta

Crespo

. 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: 2092–2100.

Zhang

Sun

. Ectopic pregnancy in frozen-thawed embryo transfer: a retrospective analysis of 4,034 cycles and related factors. Syst Biol Reprod Med 2013; 59: 34–37.

Veek

. An atlas of human gametes and conceptuses, London: Parthenon, 1999, pp. 215–215.

Melo

Meseguer

Garrido

. The significance of premature luteinization in an oocyte-donation programme. Hum Reprod 2006; 21: 1503–1507.

Papanikolaou

Kolibianakis

Pozzobon

. 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. Fertil Steril 2009; 91: 949–952.

10.

Bourgain

Devroey

. The endometrium in stimulated cycles for IVF. Hum Reprod Update 2003; 9: 515–522.

11.

David

. Textbook of assisted reproductive techniques, London: Matin Dunitz, 2001, pp. 658–659.

12.

Bouyer

Coste

Shojaei

. Risk factors for ectopic pregnancy: a comprehensive analysis based on a large case-control, population-based study in France. Am J Epidemiol 2003; 157: 185–194.

13.

Pisarska

Carson

. Incidence and risk factors for ectopic pregnancy. Clin Obstet Gynecol 1999; 42: 2–8.

14.

Strandell

Thorburn

Hamberger

. Risk factors for ectopic pregnancy in assisted reproduction. Fertil Steril 1999; 71: 282–286.

15.

Paltieli

Eibschitz

Ziskind

. High progesterone levels and ciliary dysfunction – a possible cause of ectopic pregnancy. J Assist Reprod Genet 2000; 17: 103–106.

16.

Labarta

Martínez-Conejero

Alamá

. 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: 1813–1825.

17.

Haouzi

Assou

Mahmoud

. Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients. Hum Reprod 2009; 24: 1436–1445.