IN VITRO FERTILIZATION

Optimization of estradiol supplementation during the

luteal phase improves the pregnancy rate in women
undergoing in vitro fertilization– embryo transfer cycles
Krzysztof Lukaszuk, M.D., Ph.D.,a,b Joanna Liss, Ph.D.,a Mariusz Lukaszuk, M.D.,a and
Bozena Maj, M.Sca
a
INVICTA Fertility and Reproductive Center and b Department of Endocrinology, Institute of Obstetrics and Gynaecology,
Medical University, Gdansk, Poland

Objective: To evaluate the influence of different E2 supplementation doses during the luteal phase on implantation
and pregnancy rates in women undergoing intracytoplasmic sperm injection (ICSI) cycles.
Design: Prospective, randomized study.
Setting: A private IVF unit.
Patient(s): One hundred sixty-six women younger than 40 years who were undergoing IVF with long protocol
controlled ovarian hyperstimulation (COH). A total of 231 cycles were investigated. Group 1 (P only) included
80 cycles, group 2 (P and 2 mg of E2) included 73 cycles, and group 3 (P and 6 mg of E2) included 78 cycles.
Intervention(s): Supplementation in the luteal phase with different doses of E2 (0, 2, or 6 mg/d).
Main Outcome Measure(s): Serum E2 and P levels in the late luteal phase, and implantation rate and pregnancy
rate (PR) were documented. The data were analyzed with regard to the entire study population and further
stratified according to the E2 dose used.
Result(s): Significantly higher implantation rate and PR were recorded in those who received low dose E2
supplementation compared with no substitution (PR 23.1% vs. 32.8%). The best implantation and pregnancy
results were found significantly in the group with high dose E2 supplementation (PR 51.3%).
Conclusion(s): For women treated with a long GnRH analogue protocol for COH, addition of a high dose of E2
to daily P supplementation significantly improved the IVF– embryo transfer results. (Fertil Steril威 2005;83:
1372– 6. ©2005 by American Society for Reproductive Medicine.)
Key Words: E2 supplementation, luteal support, prospective randomized study, pregnancy rate, IVF– embryo
transfer

The results of IVF programs are still something to be improved.
The technology behind IVF is very effective up to embryo
transfer. The last stage—the implantation phase—is still a prob-
lem, and pregnancy rates (PR) are largely unpredictable.
The role of P supplementation in the luteal phase of down-
regulated cycles is well established. Standard doses and routes
of administration (p. vag. or i.m.) are routinely used worldwide
(1). The role of E2 in the luteal phase is unclear and still under
evaluation. Results published by Ghosh et al. (2) did not con-
firm an obligatory role of E2 with regard to implantation. Also
in non-human species the influence of the luteal phase E2 level
on implantation is controversial (3– 6).
The levels of serum E2 in human cycles decrease in the
late luteal phase (7). It has been confirmed that this decline
Received July 5, 2004; revised and accepted November 20, 2004.
Reprint requests: Joanna Liss, Ph.D., INVICTA Fertility and Reproductive
Center, ul. Podwale Grodzkie 2B, 80-895 Gdansk, Poland (FAX: 48-
58-7631476; E-mail: Joanna.Liss@invicta.pl).
adversely affects the implantation rate (8). Baird et al. (9)
found a higher E2 level (on day 12 after ovulation) in natural
conception cycles than in nonconception cycles. Stewart et
al. (10) confirmed the same results in insemination concep-
tion cycles, as soon as 6 days after ovulation. Fahri et al. (11)
improved their PR with the administration of 2 mg of oral E2
daily in the luteal phase.
In the present prospective, randomized study we evaluated
the effect of adding different doses of E2 to the luteal support
protocol on PRs in women treated by means of long stimu-
lation protocol IVF cycles.
MATERIALS AND METHODS
Selection of Subjects
A total of 166 women treated by means of ICSI in our IVF unit
between March 2002 and March 2003 were included in the
study. The indications for ICSI included male factor infertility
(66; 39.8%), tubal factor (36; 21.7%), anovulation (20; 12.1%),
endometriosis (6; 3.6%), immunological (2; 1.2%), and un-

1372 Fertility and Sterility姞 Vol. 83, No. 5, May 2005 0015-0282/05/$30.00
Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2004.11.055
explained factors (19; 11.4%). In 17 cases (10.2%) there
were mixed factors.
The women were randomized to three groups: no E2,
2 mg, or 6 mg of E2 daily, starting from the day of oocyte
pickup and lasting for the entire of the luteal phase. All
women took monophasic oral contraceptives (OC) for 3
weeks from the first day of the pre-ICSI cycle.
Stimulation Protocol
All women used our long protocol of pituitary suppression
with the GnRH agonist nafarelin (Pharmacia Upjohn,
Kalamazoo, MI), starting on day 14 of the cycle. Fourteen
days later (7 days after the end of OC administration) the
administration of urinary gonadotropins (Menogon, Ferring,
Kiel, Germany) for ovarian stimulation was started accord-
ing to the step-down protocol (225 IU for 3 days and 150 IU
for the following days), regardless of the woman’s age, basal
serum FSH concentration, and presence or absence of poly-
cystic ovaries (PCO) in ultrasonography.
Monitoring of follicular growth was carried out by means
of a day 8 ultrasonographic scan and assay of serum E2.
Oocyte pick-up was performed 35 hours after the adminis-
tration of 10,000 or 7,500 IU of hCG (Choragon, Ferring,
Kiel, Germany or Pregnyl, Organon, Oss, The Netherlands).
A maximum of two embryos was transferred on day 3 in
women younger than 36 years and three embryos were
transferred in older women. Embryo transfer was performed
atraumatically using a Soft Frydman Set catheter (Labora-
toire CCD, Paris, France) under ultrasonographic guidance,
with a full bladder.
All subjects received luteal phase support with natural
micronized P (Utrogestan, Laboratoires Besins-Iscovesco,
Paris, France), 600 mg/d vaginally in three divided doses,
starting on the day of oocyte pick-up. The women were
randomly allocated to daily doses of 0, 2, or 6 mg of E2
during the entire luteal phase. We did not use placebo.
Verification of pregnancy status and quality was carried
out on day 16 after egg collection by means of assay of
serum hCG, P, and E2. Clinical pregnancy was defined as the
presence of a gestational sac on ultrasonography at 5 weeks
and 2 days of gestation. The fetal heart was evaluated at 6
weeks and 3 days. Each time, serum P concentrations were
checked and P supplementation was regulated accordingly.
Specimen Collection and Preparation: Hormone Analysis
Fasting venous blood samples (7 mL) were collected asep-
tically without any additives between 8:00 AM and noon on
day 12 after embryo transfer. The blood was allowed to clot
at room temperature and the serum was separated by cen-
trifugation. The samples were stored at ⫺20°C until ana-
lyzed.
Estradiol, P, and hCG levels were determined by chemi-
luminescence immunoassays (Immulite, DPC, Los Angeles,
Fertility and Sterility姞
CA) carried out according to the manufacturer’s suggestions.
The sensitivities were: E2, 15 pg/mL; P, 0.2 ng/mL; hCG, 1.1
mIU/mL. The intra-assay and interassay coefficients of vari-
ation were less than 10% in all assays.
Power Calculation and Statistical Analysis
The statistical package StatSoft, Inc. (2001) (Tulsa, OK)
STATISTICA (data analysis software system), version 6
(www.statsoff.com) was used for data analysis. Clinical
characteristics were analyzed by using Student’s unpaired t
test. Values are reported as mean ⫾ SD.
According to power calculations, a minimum of 80 cycles
in each arm of the study would be needed to show a signif-
icant difference in PR, which was the primary outcome
measure of this randomized study. It was calculated for the
difference in PR of 25%, which we received in pilot studies
(data not published).
The significance level (␣) was set at 0.05 with a power of
0.95. An interim analysis of the number of pregnancies
achieved showed that sufficient numbers were already avail-
able to reach the required power level. Hence, recruitment
was stopped and analysis of the results commenced.
The ␹2 test was used to assess differences between groups
with regard to different rates of development. A value of
P⬍.05 was considered statistically significant.
RESULTS
During the study period, 231 cycles were included and
prospectively randomized. Group 1 (P only) included 80
cycles, group 2 (P and 2 mg of E2) included 73 cycles, and
group 3 (P and 6 mg of E2) included 78 cycles.
Table 1 shows the characteristics of the groups. No dif-
ferences were found with regard to mean age, cause of
infertility, duration of infertility, or presence of primary or
secondary infertility.
Table 2 presents the characteristics of the ICSI cycles. No
differences were found in the mean number of oocytes
retrieved, the mean number of oocytes fertilized, or the mean
number of embryos transferred.
In the whole group of 166 women we achieved 80 clinical
pregnancies (48.2%). The highest PR (51.3%) was achieved
in the group with the highest degree of E2 supplementation
(Table 2). The implantation rate also increased with E2
supplementation, the highest rate (29.9%) being in group 3.
We measured the levels of hCG, E2, and P on day 16 after
oocyte pick-up. The E2 and P data were divided according to
pregnancy status. The results are presented in Table 3.
The levels of E2 and P in pregnant vs. nonpregnant women
significantly differed in each group. The E2 levels in non-
pregnant women did not significantly differ in groups 1 and
2, but the mean level in group 3 was significantly higher
1373
 TABLE 1
Characteristics of the treatment groups.

Group 1 Group 2 Group 3

Variable (no E2) (2 mg E2) (6 mg E2) P value

No. of subjects 50 47 69
No. of cycles 80 73 78
Mean (⫾ SD) age 32.1 ⫾ 4.5 31.7 ⫾ 3.9 31.1 ⫾ 3.7 NS
No. of subjects with indicated cause
of infertility
Tubal factor 14 13 9
Male factor 16 21 29
Endometriosis 4 1 1 NS
Anovulation 8 4 8
Immunological factor 1 0 1
Unexplained 4 5 10
Mixed factor 3 3 11
Mean (⫾ SD) duration of infertility (y) 4.8 ⫾ 2.9 4.9 ⫾ 2.9 4.3 ⫾ 2.7 NS
NS ⫽ not significant.
Lukadzuk. E2 supplementation in IVF luteal phase. Fertil Steril 2005.

(P⬍.001). The mean level of E2 in pregnant women was
significantly higher in group 3 than in groups 1 and 2
(P⬍.001). There was no significant difference in the mean
E2 level in groups 1 and 2 in pregnant women (P⫽.15).
Progesterone levels did not differ between groups.
DISCUSSION
The role of E2 in the human luteal phase is still under
evaluation. It is especially interesting, and probably different
from normal, in IVF cycles, where oocyte aspiration disrupts
the luteal function of the ovaries (12). Many early reports
indicate no relationship between midluteal E2 levels and IVF
program outcomes (13, 14) or lack of benefit of luteal phase
E2 supplementation (15).
Muasher et al. (13) found that a luteal phase E2 level
decrease on day 10 after oocyte pickup lowers the chance of
pregnancy. Sharara and McClamrock (16) tried to predict
implantation rate and PR according to the size of the E2 peak

TABLE 2
In vitro fertilization cycle characteristics of the investigated groups and comparison of results of
ICSI in the three groups.

Group 1 Group 2 Group 3

Variable (no E2) (2 mg E2) (6 mg E2) P value

No. of cycles 80 73 78
No. of transfers 78 70 76
Mean (⫾ SD) hMG dose (IU) 25.8 ⫾ 10.4 26.1 ⫾ 6.1 26.2 ⫾ 6.8 NS
Mean (⫾ SD) no. oocytes retrieved 8.6 ⫾ 5.3 10.3 ⫾ 4.5 9.2 ⫾ 4.5 NS
Mean (⫾ SD) fertilization rate (%) 5.4 ⫾ 3.7 6.7 ⫾ 3.8 6.1 ⫾ 3.6 ⬍.05
(72.2) (72.7) (72.9)
Mean (⫾ SD) no. of embryos transferred 2.2 ⫾ 0.7 2.3 ⫾ 0.6 2.1 ⫾ 0.6 NS
No. of pregnancies 18 23 39 ⬍.001
Pregnancy rate (%) 23.1 32.8 51.3 ⬍.001
Implantation rate (%) 9.8 17.8 29.9 ⬍.001
Multiple pregnancy rate of pregnancies (%) 0 30.4 25.6 ⬍.001
Ectopic pregnancy (%) 1 (5.5) 1 (4.3) 0 NS
Spontaneous abortion rate (%) 4 (22.2) 4 (17.4) 5 (12.8) NS
Note: NS ⫽ not significant.
Lukadzuk. E2 supplementation in IVF luteal phase. Fertil Steril 2005.

1374 Lukadzuk et al. E2 supplementation in IVF luteal phase Vol. 83, No. 5, May 2005
 TABLE 3
The results of E2 and P levels in day 16 after pick-up in investigated groups.

Group 1 Group 2 Group 3

(without E2) (2 mg E2) (6 mg E2)

Estradiol (pg/mL) (mean ⫾ SD)

Pregnancy 280.8 ⫾ 212.3 404.9 ⫾ 197.1 974.0 ⫾ 132.1
NS P⬍.001
P⬍.001
No pregnancy 134.8 ⫾ 66.8 153.2 ⫾ 54.9 268.9 ⫾ 26.8
NS P⬍.001
P⬍.001
Progesterone (ng/mL) (mean ⫾ SD)
Pregnancy 71.4 ⫾ 15.9 72.3 ⫾ 27.2 79.1 ⫾ 37.4
NS NS
NS
No pregnancy 14.5 ⫾ 4.6 15.1 ⫾ 2.9 16.6 ⫾ 4.1
NS NS
NS
Note: NS ⫽ not significant. P value determined by Student’s unpaired t test.
Lukadzuk. E2 supplementation in IVF luteal phase. Fertil Steril 2005.

to midluteal phase ratio. They affirmed that the worst prog-
nosis was for those women in whom this ratio was greater
than 5.
Aktan et al. (17) confirmed higher peak and midluteal E2
levels in pregnant than in nonpregnant women and they also
reported that the rate of decrease from the E2 peak before
oocyte pickup to the midluteal level was similar in pregnant
and nonpregnant women. They suggested that the higher
midluteal E2 level in pregnant women was only the result of
a higher peak E2 level. Unfortunately, the results were not
based on the E2 level on an appropriate midluteal day, which
could have been found if some preliminary day-to-day luteal
phase E2 level had been measured.
We found that a real and significant E2 level decrease
starts on day 8 (unpublished data). This is probably why
Aktan et al. (17) found no correlation between the decrease
in E2 concentrations and PR.
Fujimoto et al. (18) attempted to make clinical decisions
on the basis of midluteal E2 levels in unsuccessful IVF first
cycles. They achieved a significant improvement in PR (al-
most 2.5 times higher) in the randomized group who were
given supplemental P and hCG vs. the group given supple-
mental P alone during the luteal phase. In the P ⫹ hCG
group the midluteal phase E2 concentration was 15 times
higher than in the P-only group.
We also estimated the effectiveness of E2 supplementation
with regard to implantation rate and PR. Supplementation
with hCG may cause ovarian hyperstimulation syndrome
(OHSS) and make it impossible to confirm pregnancy ac-
cording to the hCG level. Human chorionic gonadotropin
also plays other roles, not only in the elevation of E2 levels.
Hence we decided to supplement E2 levels by directly
using the effector E2 valerate. We gave the different E2 doses
starting on the day of oocyte pickup.
The investigated groups did not differ with regard to age,
duration of infertility treatment, diagnosed causes of infer-
tility, day 3 hormonal status (FSH, LH, E2), gonadotropin
dose and duration of stimulation, peak E2 level, number of
retrieved oocytes, fertilization rates, and number of embryos
transferred. On the other hand there was a significant differ-
ence between the three groups with regard to PR (P⬍.001).
There was also a difference in the implantation rate, which
significantly increased with E2 treatment—almost twice as
much when we added 2 mg/d and more than three times
when the dose was 6 mg (P⬍.001).
It is still not confirmed that E2 in the luteal phase is
necessary for implantation. In some investigations concern-
ing donor oocyte programs pregnancies have been achieved
without E2 supplementation (19, 20).
Circulating E2 concentrations are higher in normal fertile
women with spontaneous pregnancy cycles than in unsuc-
cessful cycles (10). It has been suggested that secretion of
hCG by preimplantation embryos could stimulate E2 produc-
tion. It is possible that the better implantation rates in hCG-
supported cycles than in P-only supported cycles are a re-
flection of this mechanism.
Our results suggest that E2 supplementation could help
embryos that insufficiently stimulate E2 production, or where

Fertility and Sterility姞 1375

there is a weak maternal reaction. In our study we achieved
the best results in the group given 6 mg of E2 daily. In our
opinion it is an appropriate dose, with no threat of OHSS or
liver decompensation. The safety of luteal phase E2 supple-
mentation has not been thoroughly investigated (21).
Acknowledgments: The authors thank Nicholas Bolton, M.D., for revising
the language of the manuscript.
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