Reproductive BioMedicine Online (2015) 30, 157–165

w w w. s c i e n c e d i r e c t . c o m
w w w. r b m o n l i n e . c o m

ARTICLE

Comparison of progesterone measurement on

day of, and day after, HCG administration in
IVF–embryo transfer cycles
Liu Liu a,b, Lijuan Zhao a, Tin Chiu Li a,b, Haiyan Zhu a, Xiaona Lin a,
Xiaoying Jin a, Xiaomei Tong a, Songying Zhang a,*

a
Center of Reproductive Medicine, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hang Zhou, China;
b
Department of Obstetric and Gynecology, Prince of Wales Hospital, The Chinese University of Hong Kong
* Corresponding author. E-mail address: zhangsongying@126.com (S Zhang).

Dr Liu Liu is an MD student in reproductive medicine in Zhejiang University, China. She has been conducting clini-
cal research under the supervision of Professor TC Li in Sheffield and Hong Kong. Her particular interest is en-
dometrial receptivity in women undergoing IVF treatment.

Abstract The aim of this study was to identify whether progesterone measurement on the day after HCG administration has any
additional clinical value, over that obtained from progesterone measurement on the day of HCG administration. This was a single-
centre, non-interventional retrospective, observational, cohort of a consecutive series of 1457 ovarian stimulation cycles leading to
fresh embryo transfer cycles between January 2011 and May 2013. Progesterone was found to rise and increase rapidly by about five-
fold over a 24-h period. The result of logistic regression analysis suggests that progesterone measurement around the time of HCG
administration is one of the three significant (P < 0.001) variables affecting clinical pregnancy rate. The predictive value of proges-
terone measurement on the day of HCG administration could be improved by having a further measurement 24-h later. If the pro-
gesterone levels on both days were normal, the implantation rate was 36%, but if the progesterone level on both days were high,
the implantation rate dropped to 22%. Progesterone measurement should be considered on the day of HCG administration and also
the day after HCG administration to provide clinically useful information on whether or not embryos should be frozen and transfer
deferred to a subsequent cycle.
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

KEYWORDS: implantation rate, IVF/ET, progesterone

http://dx.doi.org/10.1016/j.rbmo.2014.10.017
1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
158
Introduction
Several studies (Al-Azemi et al., 2012; Bosch et al., 2010;
Huang et al., 2012; Lahoud et al., 2012; Papanikolaou et al.,
2012; Wu et al., 2012; Xu et al., 2012) and a recent meta-
analysis (Venetis et al., 2013) have suggested that elevated
progesterone on the day of human chorionic gonadotrophin
(HCG) administration is associated with adverse pregnancy out-
comes in women undergoing IVF and embroyo transfer treat-
ment. One possible cause is that elevated progesterone on
the day of HCG administration disturbs endometrial devel-
opment (Van Vaerenbergh et al., 2011; Xu et al., 2012) and
gene expression (Labarta et al., 2011; Li et al., 2011; Van
Vaerenbergh et al., 2011), leading to impaired endometrial
receptivity and reduced implantation rate (Al-Azemi et al.,
2012; Xu et al., 2012).
Some investigators have examined progesterone levels on
the day after HCG administration. Develioglu et al. (1999) re-
ported that high progesterone levels on the day after HCG
administration (>6 ng/ml) was associated with an earlier down-
regulation of P receptor expression, as well as accelerated
glandular development and pinopode expression, resulting in
earlier closure of the implantation window and decreased preg-
nancy rates. Another report of 114 cycles also showed that
high serum progesterone level on the day after HCG admin-
istration was associated with reduced pregnancy rates (Burns
et al., 1994). Furthermore, a recent report in unstimulated
cycles suggested that the progesterone elevation for 2 days
or more before the luteinizing hormone (LH) surge impaired
the clinical pregnancy rate of frozen-thawed embryo trans-
fers in natural cycles, whereas progesterone elevation only
on the day of LH surge did not have such an adverse effect,
suggesting that serial progesterone measurements around the
time of LH surge or HCG administration is more informative
than a single measurement (Lee et al., 2014). It is not known,
however, if progesterone measurement on the day after HCG
administration is as informative, more informative or less in-
formative than progesterone measurement on the day of HCG
administration.
In this study, the value of progesterone measurement on
the day of HCG administration was compared with the mea-
surement on the day after HCG administration in an IVF–
embryo transfer programme; specifically, the objective was
to determine if progesterone measurement on the day after
HCG administration had any additional clinical value, com-
pared with that obtained from progesterone measurement on
the day of HCG administration.
Materials and methods
Patients’ characteristics
This was a non-interventional retrospective, observational,
cohort study of patients undergoing gonadotrophin-releasing
hormone agonist IVF–embryo transfer treatment. Patients were
treated at a single centre in the Sir Run Run Shaw hospital,
Hangzhou, China, between January 2011 and May 2013. All
patients provided written informed consent. During the study
period, it was routine practice to obtain two blood samples,
24 h apart, around the time of HCG administration for
L Liu et al.
oestrogen and progesterone measurement. Both samples were
obtained in the morning, the first one 12–14 h before HCG ad-
ministration (day of HCG administration) and the second
sample was obtained 24 h later, 12–14 h after HCG adminis-
tration (day after HCG administration). It was also routine
policy to consider fresh embryo transfer if the number of re-
trieved oocytes was less than or equal to 15, and the pro-
gesterone concentration was less than or equal to 2.0 ng/ml
on the day of HCG administration. Otherwise, patients would
be advised to have all their embryos frozen for subsequent
frozen–thawed embryo transfer.
Protocol for ovarian stimulation
The protocol for ovarian stimulation has been described pre-
viously (Liu et al., 2013). In summary, two ovarian stimula-
tion protocols, long gonadotrophin-releasing hormone (GnRH)
agonist protocol and short GnRH agonist protocol were used.
Daily injections of recombinant FSH (rFSH, Gonal-F; Serono
Laboratories, Switzerland; or Puregon; N.V. Organon, the Neth-
erlands) (150–300 IU, daily) were started from the third or fifth
day of the menstrual cycle, and the dose was adjusted ac-
cording to follicle development. When a cohort of three or
more follicles reached a mean diameter of 16–18 mm or more,
HCG (6500–10,000 IU; Serono; Denmark) was given to trigger
ovulation. No more than three embryos were transferred on
day 3 after oocyte retrieval during the study period. Excess
available embryos were cryopreserved for subsequent FET
cycles. Four grades of embryos were defined (Veeck, 1999).
Embryos with a normal cleavage rate (six to eight cells on the
third day) and less than 20% fragmentation were defined as
‘high-quality’. Luteal phase support consisted of intramus-
cular progesterone in oil (Xianju Pharmaceutical factory,
China) at 40 mg/day for 1 day, beginning from the night
of oocyte retrieval and then at 80 mg/day for at least
2 weeks.
Definition of outcome
Pregnancy was considered to have occurred if serum beta-
HCG concentration 12 days after embryo transfer was 10 IU/L
or more. Clinical pregnancy was defined by the observation
of a gestational sac with or without a fetal heartbeat on ul-
trasound evaluation 35 days after embryo transfer. The number
of sacs was taken as the number of successful implanta-
tions. Biochemical pregnancy referred to pregnancy, which
did not progress to clinical pregnancy, but with either (a) two
serum samples (at least 2 days apart) both with beta-HCG level
of 10 IU/L or more, and with the second measurement higher
than the initial one, or (b) one sample with level 25 IU/L or
more. Clinical spontaneous abortion was defined when a preg-
nancy failed to progress after an intrauterine gestational sac
had been detected by pelvic ultrasonography.
Definition of elevated progesterone
In this study, high progesterone level on the day of HCG ad-
ministration was defined as one above the 90th centile for that
Progesterone level around the time of HCG injection 159

A B Overall (n = 5828)

500

Overall (n = 5828)
400
1,200

1,000

Frequency
300

800
Frequency

200
600

400
100

200

0 0
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 0.00 10.00 20.00 30.00 40.00 50.00
Serum progesterone on day of HCG (ng/ml) Serum progesterone on day after HCG (ng/ml)

C D Fresh embryo transfer cycles (n = 1457)

Fresh embryo transfer cycles (n = 1457) 125

80

100
Frequency

60
Frequency

75

40
50

20
25

0 0
0.00 0.50 1.00 1.50 2.00 2.50 0.00 5.00 10.00 15.00 20.00 25.00
Serum progesterone on day of HCG (ng/ml) Serum progesterone on day after HCG (ng/ml)

Figure 1 (A) The frequency distribution of progesterone on the day of HCG administration in the entire population (n = 5828). The
median was 1.4 ng/ml and 90th centile was 2.5 ng/ml; (B) the frequency distribution of progesterone on the day after HCG admin-
istration in the entire population (n = 5828). The median was 6.9 ng/ml and the 90th centile was 15.4 ng/ml; (C) the frequency dis-
tribution of progesterone on the day of HCG administration in cycles with fresh embryo transfer (n = 1457). The median was 1.1 ng/
ml and the 90th centile was 1.7 ng/ml; (D) the frequency distribution of progesterone on the day after HCG administration in cycles
with fresh embryo transfer (n = 1457). The median was 5.5 ng/ml and the 90th centile was 9.5 ng/ml.

particular day, which was 1.7 ng/ml. Similarly, high proges-
terone level on the day after HCG administration was defined
as one above the 90th centile for that particular day, which
was 9.5 ng/ml. The cut-off value of 1.7 ng/ml on the day of
HCG administration was consistent with several previous
reports, in which the progesterone level on the day of HCG
administration was considered as elevated when the result
was greater than or equal to 1.5 ng/ml (Bosch et al., 2010;
Huang et al., 2012; Li et al., 2011; Liu et al., 2013; Ochsenkühn
et al., 2012). As no data have been published on what con-
stitutes elevated progesterone level on the day after HCG ad-
ministration, a trend analysis was conducted to ascertain if
the cut-off value of progesterone on the day after HCG
administration chosen (9.5 ng/ml) could separate groups with
significantly different outcome (clinical pregnancy rate).
Patterns of progesterone profile
According to the serial progesterone concentrations on the
day of HCG administration and on the day after HCG admin-
istration, patients were categorized into four groups: group
NN, progesterone level normal on the day of HCG and day after
HCG administration; group NH, progesterone level normal on
the day of HCG but high on the day after HCG administra-
tion; group HN, progesterone level high on the day of HCG
160
but normal on the day after HCG administration; group HH,
progesterone level high on the day of HCG and day after HCG
administration.
Hormonal measurements
The first oestrogen and progesterone measurement was ob-
tained 12–14 h before HCG administration, and the second
measurement for oestrogen and progesterone was obtained
12–14 h after HCG administration. Hormone assay was carried
out within 2 h of collection, according to the manufactur-
er’s instructions on a Beckmancoulter immunoassay ana-
lyzer (DXI800, California, USA). The inter-assay coefficients
of variation for oestradiol and progesterone were 12.0% and
10.0%, respectively, and the intra-assay coefficients varia-
tion for oestradiol and progesterone were 8.0% and 6.3%, re-
spectively. This assay was used for the duration of the study.
All the assays were carried out in the same laboratory, within
the IVF unit.
Statistical analysis
All statistical analyses were conducted using Statistical Package
for the Social Sciences Version 16.0 (IBM Corp., New York, NY,
USA). Groups were compared using one-way analysis of vari-
ance for continuous variables, and chi-squared test for cat-
egorical variables, as appropriate. Logistic regression analysis
was used to examine the variables associated with clinical
pregnancy rate. P < 0.05 was considered to indicate signifi-
cance. The primary end-point for the comparison of results
between groups was the implantation rate as it is not af-
fected by the number of embryos transferred.
Ethics
The study was approved by the Hospital Ethics Com-
mittee on 6 May 2014, as a service evaluation project as it
Overall (n = 1457)
60 48.5 51.0
(46.2−53.8) (48.4−52.6)
Clinical pregnancy rate (%)
40
20
0 lower implantation rate and tended towards a lower clinical
≤ 4.00 4.01−6.00
Serum progesterone on day after HCG (ng/ml)
Figure 2 A trend analysis using Mantel–Haenszel test of the re-
lationship between clinical pregnancy rate and increasing serum
progesterone levels on the day after HCG administration in the
overall study population. *P < 0.05 for comparison with all pre-
vious progesterone level intervals; the vertical bars represent 95%
confidence interval for each group.
L Liu et al.
was a retrospective study with no extra intervention
required.
Results
In total, 5828 consecutive ovarian stimulation cycles (carried
out in 5549 patients) for IVF between January 2011 and May
2013 were selected for inclusion in the study. Among these
5828 cycles, 1457 cycles had fresh embryo transfers (carried
out in 1453 patients), and the remaining 4371 cycles (75.0%)
did not proceed to fresh embryo transfer because there were
either the progesterone level on the day of HCG administra-
tion was less than or equal to 2 ng/ml but more than 15 oocytes
retrieved (3187 cycles), or the progesterone level on the day
of HCG administration was over 2 ng/ml (1184 cycles). The
1457 cycles that used fresh embryos for transfer were in-
cluded in the final analysis of this study. The frequency dis-
tribution of serum progesterone concentration on the day of,
and day after, HCG administration for the entire population
(n = 5828) and selected subpopulation (n = 1457) is shown in
Figure 1. The clinical features and treatment outcomes of
the selected subpopulation (n = 1457) are shown in Table 1.
The embryo implantation rate in women with normal pro-
gesterone on the day of HCG administration (35.2%) was sig-
nificantly (P < 0.05) higher in women with elevated
progesterone on the day of HCG administration (26.6%). Simi-
larly, the clinical pregnancy rate and implantation rate in
women with normal progesterone on the day after HCG ad-
ministration (49.1% and 35.3%) were significantly (both
P < 0.05) higher than women with elevated progesterone on
the day after HCG administration (39.3% and 25.7%),
respectively.
The clinical pregnancy rate in the four groups of partici-
pants according to their progesterone level on the day after
HCG administration (group 1, less than the 30th centile; group
2, 30th to 60th centile; group 3, 60th to 90th centile; and group
4, over the 90th centile) was analysed with the use of Mantel–
Haenszel method and the results are shown in Figure 2. Clini-
cal pregnancy rate in group 4 (39.0%) was found to be
significantly (P < 0.05) lower than the other three groups, in-
50.0 dependently or combined, whereas no difference was found
(48.2−52.0) 39.0* in the results between groups 1, 2 and 3.
(31.4−47.1) The clinical features and outcome of treatment of the four
subgroups of participants according to the pattern of pro-
gesterone levels on the day of, and day after, HCG adminis-
tration are presented in Table 2. Women in group HH, with
high progesterone measurements on both days (day of HCG
and day after HCG administration), seemed to be younger,
had a higher number of oocytes retrieved, produced a higher
number of embryos compared with the group NN (control
group, normal progesterone on both days), but achieved a
6.01−9.50 >9.50 pregnancy rate. In comparing groups NN and NH (both had
normal progesterone on the day of HCG administration), it
was found that women in group NH (high progesterone on the
day after HCG administration) had significantly (P < 0.05, re-
spectively) lower implantation rate (26.9%, respectively) than
women in group NN (36.0%, respectively). In comparing groups
HN and HH (both had high progesterone on the day of HCG
administration). It was found that women in group HH (high
progesterone on the day after HCG administration) also seemed
Table 1 Clinical features and treatment outcomes in women with normal or elevated progesterone if progesteronea measurement on the day of, and on day after, HCG admin-

Progesterone level around the time of HCG injection

istration are separately considered alone.
Overall P-value on day of HCG P-value on day after HCG
Variables
Normal Elevated P value Normal Elevated P value

Number of fresh 1457 1309 148 1312 145

embryos
Age (years)b 32.0 ± 4.8 31.8 ± 4.6 30.7 ± 3.8 <0.05 31.9 ± 4.5 30.0 ± 3.6 <0.001
Cause of infertility Tubal (%) 49.3 50.9 50.0 NS 51.1 48.3 NS
Male (%) 23.0 22.4 23.0 NS 22.3 24.1 NS
Endometriosis (%) 5.2 4.6 4.7 NS 4.5 5.5 NS
Anovulation (%) 6.8 6.8 7.4 NS 6.9 6.9 NS
Unexplained (%) 15.7 15.4 14.9 NS 15.3 15.2 NS
Basal endocrinological profile FSH (IU/L)b 8.1 ± 1.8 8.1 ± 1.9 8.2 ± 1.8 NS 8.1 ± 1.9 7.5 ± 1.5 <0.001
Oestradiol (pg/ml)b 43.7 ± 1.9 43.4 ± 18.9 46.2 ± 19.7 NS 43.7 ± 18.8 42.8 ± 19.9 NS
Stimulation Protocol Long (%) 77.5 70.7 79.1 NS 71.5 72.4 NS
Short (%) 22.5 29.3 20.9 NS 28.5 27.6 NS
Fertilization methods IVF (%) 65.0 65.6 65.5 NS 65.8 64.1 NS
ICSI (%) 35.0 34.4 34.5 NS 34.2 35.9 NS
Dosage of recombinent FSH vials (IU)b 2245.3 ± 632.4 2237.3 ± 641.0 2305.5 ± 562.7 NS 2230.0 ± 644.5 2385.1 ± 515.2 <0.05
Recombinent FSH duration (days)b 9.1 ± 1.5 9.1 ± 1.4 9.3 ± 1.5 NS 9.0 ± 1.4 9.8 ± 1.3 <0.001
Number of oocytes retrievedb 8.4 ± 4.1 8.2 ± 4.1 10.0 ± 3.8 <0.05 8.0 ± 4.0 12.4 ± 3.2 <0.001
Endometrial thickness on the day 10.1 ± 2.0 10.1 ± 2.0 10.3 ± 2.0 NS 10.1 ± 2.0 10.1 ± 1.8 NS
of HCG administration (mm)b
Number of available embryosb 6.3 ± 3.6 6.2 ± 3.5 7.5 ± 3.5 <0.001 6.0 ± 3.4 9.3 ± 3.2 <0.001
Number of embryos transferred per 1.8 ± 0.4 1.8 ± 0.4 1.8 ± 0.4 NS 1.8 ± 0.4 1.8 ± 0.4 NS
fresh embryo transferb
Number of high-quality embryos 1.6 ± 0.5 1.6 ± 0.5 1.7 ± 0.5 NS 1.6 ± 0.5 1.7 ± 0.5 NS
transferred per fresh embryo transferb
Positive pregnancy rate per 54.2 54.9 48.0 NS 55.0 47.6 NS
fresh embryo transfer (%)
Clinical pregnancy rate per 48.1 48.9 41.2 NS 49.1 39.3 <0.05
fresh embryo transfer (%)
Embryo implantation rate 34.4 35.2 26.6 <0.05 35.3 25.7 <0.05
per fresh embryo transfer (%)
Biochemical pregnancy rate per 10.3 10.9 14.8 NS 10.7 17.5 NS
positive pregnancy cycle (%)
Clinical spontaneous abortion rate 9.1 9.8 4.9 NS 9.3 8.5 NS
per clinical pregnancy cycle (%)
Ectopic pregnancy rate per 18.7 19.3 11.5 NS 18.8 17.5 NS
clinical pregnancy cycle (%)

ICSI = intracytoplasmic sperm injection.

NS = not significant.

161
a
For definition of normal and high progesterone, refer to text.
b
Results are presented as mean ± standard deviation.
 162
Table 2 Clinical features and treatment outcomes in women with four different patterns of progesteronea profile.
Group NN Group NH Group HN Group HH P value
Variables

Number of fresh embryo transfers 1204 105 108 40

Age (years)b 31.9 ± 4.6 30.1 ± 3.7d 31.0 ± 3.9 29.7 ± 3.5d <0.001
Cause of infertility Tubal (%)c 51.1 48.2 50.8 48.0 NS
Male (%)c 22.3 23.8 21.9 24.3 NS
Endometriosis (%)c 4.5 5.9 4.8 5.8 NS
Anovulation (%)c 6.8 6.2 7.0 6.9 NS
Unexplained (%)c 15.3 15.9 15.5 15.0 NS
Basal endocrinological profile FSH(IU/L)b 8.1 ± 1.9 7.4 ± 1.6d 8.3 ± 1.9 7.9 ± 1.2 <0.05
Oestradiol (pg/ml)b 43.5 ± 18.7 41.6 ± 20.3 46.3 ± 20.2 46.0 ± 18.7 NS
Stimulation protocol Long (%)c 70.8 70.3 79.4 78.0 NS
Short (%)c 29.2 29.7 20.6 22.0 NS
Fertilization methods IVF (%)c 65.8 63.9 66.2 64.7 NS
ICSI (%)c 34.2 36.1 33.8 35.3 NS
Dosage of recombinent FSH vials (IU)b 2225.7 ± 650.2 2388.0 ± 525.6 2273.2 ± 77.9 2379.0 ± 504.0 NS
Recombinent FSH duration (days)b 9.0 ± 1.4 9.8 ± 1.4 9.2 ± 1.6 9.6 ± 1.2 NS
Number of oocytes retrievedb 7.9 ± 4.0 12.3 ± 3.2e 9.0 ± 3.5d 12.7 ± 3.3e,f <0.001
Endometrial thickness on the day of HCG 10.1 ± 2.0 10.0 ± 1.9 10.3 ± 2.0 10.3 ± 1.7 NS
administration (mm)b
Number of available embryosb 5.3 ± 3.0 7.3 ± 2.7e 5.5 ± 2.9 6.7 ± 2.6e,f <0.001
Number of embryos transferred per fresh 1.8 ± 0.4 1.8 ± 0.4 1.8 ± 0.4 1.8 ± 0.4 NS
embryo transferb
Number of high-quality embryos transferred 1.6 ± 0.5 1.7 ± 0.5 1.6 ± 0.5 1.7 ± 0.5 NS
per fresh embryo transferb
Positive pregnancy rate per fresh embryo transfer (%)c 55.4 49.5 50.0 42.5 NS
Clinical pregnancy rate per fresh embryo transfer (%)c 49.8 40.0 42.6 37.5 NS
Embryo implantation rate per fresh embryo transfer (%)c 36.0 26.9d 28.2d 22.2d <0.05
Biochemical pregnancy rate per positive pregnancy cycle (%)c 10.3 19.2 14.8 11.8 NS
Clinical miscarriage rate per clinical pregnancy cycle (%)c 9.7 11.9 4.3 6.7 NS
Ectopic pregnancy rate per clinical pregnancy cycle (%)c 19.4 19.0 10.9 13.3 NS

Group NN = progesterone level normal on the day of HCG and day after HCG administration; group NH = progesterone level normal on the day of HCG but high on the day after HCG admin-
istration; group HN = progesterone level high on the day of HCG but normal on the day after HCG administration; group HH = progesterone level high on the day of HCG and day after HCG
administration.
NS = not significant.
a
For definition of normal and high progesterone, refer to text.
b
Results are presented as mean ± standard deviation, one-way analysis of variance test.
c
Chi-squared test.
P < 0.05, compared with Group NN, post-hoc group-by-group pairwise comparison.

L Liu et al.
d
e
P < 0.001, compared with Group NN, post-hoc group-by-group pairwise comparison.
f
P < 0.001, post-hoc group-by-group pairwise comparison between HN and HH.
Progesterone level around the time of HCG injection 163

Table 3 Logistic regression analysis of factors affecting clini- Discussion

cal pregnancy rate. The regression score of independent
univariables prior to the selection of the model. In this study, we have confirmed previous observations that
Score P value
elevated progesterone on the day of HCG administration is
Variables associated with a reduction in implantation and clinical preg-
nancy rates (Al-Azemi et al., 2012; Huang et al., 2012; Lahoud
Age (years) 17.4 <0.001 et al., 2012; Liu et al., 2013; Ochsenkühn et al., 2012;
Number of oocytes retrieved 2.8 NS Papanikolaou et al., 2012; Venetis et al., 2013; Wu et al., 2012;
Number of available embryos 4.8 NS Xu et al., 2012), although a number of other investigators did
Endometrial thickness on the day of HCG 0.07 NS not reach the same conclusion (Fanchin et al., 1997; Martínez
administration (mm) et al., 2004). This may be related to the cut-off level chosen
Oestradiol level on the day of HCG 0.7 NS to define high or normal progesterone or the ovarian re-
administration (pg/ml) sponse to ovarian stimulation. In addition, we also found that
Progesterone level on the day of HCG 1.9 NS progesterone rises rapidly just before and after HCG admin-
administration (ng/ml) istration, and increased by about five-fold over a 24-h period.
Oestradiol level on the day after HCG 0.9 NS The mechanism of P elevation on the day of HCG adminis-
administration (pg/ml) tration and the relationship to the LH activity has been ad-
Progesterone level on the day after HCG 2.1 NS dressed in a recent review, which suggested that premature
administration (ng/ml) luteinization is a misnomer and that the elevated
Sum of progesterone levels on the day of 2.3 NS progesterone is direct consequence of FSH stimulation and
and the day after HCG administration multiple follicle development (Kasum et al., 2014).
(ng/ml) The result of logistic regression analysis in our study sug-
Dosage of recombinent FSH vials (IU) 1.1 NS gests that progesterone measurement is one of the three sig-
Recombinent FSH duration (days) 1.0 NS nificant variables affecting clinical pregnancy rate. Among the
Number of embryos transferred per fresh 11.8 <0.001 three different progesterone measurements, the most infor-
embryo transfer mative was the sum of the two measurements on the day of
Number of high-quality embryos transferred 22.7 <0.001 and on the day after HCG administration, followed by pro-
per fresh embryo transfer gesterone measurement on the day after HCG administra-
tion, then followed by progesterone measurement on the day
NS = not significant.
of HCG administration. Our results suggest that the predic-
tive value of a single progesterone measurement on the day
of HCG administration could be further improved by having
an additional progesterone measurement on the following day.
to have lower implantation rate and clinical pregnancy rate If the progesterone levels were high on both days (day of HCG
(22.2% and 37.5%, respectively) than women in group HN > 1.7 ng/ml; day after HCG > 9.5 ng/ml), the embryo implan-
(28.2% and 42.6%, respectively), but the difference did not tation rate was the lowest (22.2%) compared with the rest of
reach statistical significance. the three groups. Specifically, when compared with women
To further determine the relative contribution of the pro- who had normal progesterone on both days (implantation rate
gesterone measurement on the day of HCG administration 36.0%), the reduction was 38.3%, which was both statisti-
and the day after HCG administration on the clinical preg- cally and clinically significant. More importantly, our results
nancy rate, logistic regression analysis was carried out by highlight the clinical value of having an additional proges-
using clinical pregnancy rate as the dependent variable, and terone measurement the day after HCG administration. In the
following factors as independent variables: age, the number subgroup of women whose progesterone levels on the day of
of oocytes retrieved, the number of embryos obtained, en- HCG administration were normal but progesterone levels on
dometrial thickness on the day of HCG administration, EHCG, the day after HCG administration were high, the implanta-
PHCG, EHCG+1, PHCG+1, sum of PHCG and PHCG+1, total rFSH dose tion rate (26.9%) was lower than those who had normal pro-
used, duration of rFSH stimulation, the number of embryos gesterone on both days (36.0%), a reduction of 25%, which is
transferred per embryo transfer, and the number of high- statistically and clinically significant. In this situation, we think
quality embryos transferred per embryo transfer. The re- the option of freezing all embryos for later transfer should
gression model selected the following variables in decreasing also be considered and discussed.
order of importance: the number of high-quality embryos Women with high progesterone levels on the day after HCG
transferred per embryo transfer, the age of participants, and administration seem to be significantly younger, with a sig-
sum of PHCG and PHCG+1. Had the sum of PHCG and PHCG+1 been nificantly higher number of available embryos for transfer
removed as an independent variable, PHCG+1 would be se- (Table 2). One would expect that, in this group of woman,
lected instead as the third variable in the model. In addi- embryo quality should be better and hence the implanta-
tion, if PHCG+1 was also removed from the list of independent tion rate should be higher; in contrast, the implantation
variables, PHCG would be selected as the third variable rate in this group of women was lower despite their advan-
instead. The result of logistic regression analysis is pre- tage of being younger and having more embryos available. The
sented in Tables 3 and 4. Adjustment for clustering owing finding is consistent with the notion that reduced implanta-
to multiple cycles in the same patient (four patients) was tion rate observed in this group of women is caused by the
carried out, and the results remained the same (data not adverse effect of high levels of steroid hormones on the
shown). endometrium.
164 L Liu et al.

Table 4 The multivariable selected in each step of the stepwise forward logistic re-
gression model.
Wald P value Standardized
Variables
coefficients
selected

Step one Number of high-quality embryos 24.0 <0.001 0.423

transferred per fresh embryo transfer
Constant 18.7 <0.001 3.377
Step two Age (years) 13.9 <0.001 1.078
Number of high-quality embryos 21.3 <0.001 0.440
transferred per fresh embryo transfer
Constant 3.0 NS 0.300
Step three Sum of progesterone levels on the day 13.8 <0.001 1.119
of and the day after HCG
administration (ng/ml)
Age (years) 21.8 <0.001 1.106
Number of high-quality embryos 25.8 <0.001 0.392
transferred per fresh embryo transfer
Constant 10.8 <0.001 0.069

NS = not significant.

Although a number of studies have suggested that proges- Acknowledgements

terone on the day of HCG administration should be consid-
ered high when it is more than approximately 1.5 ng/ml
(approximately 5 nmol/l) (Bosch et al., 2010; Huang et al.,
2012; Li et al., 2011; Liu et al., 2013; Ochsenkühn et al., 2012),
no studies have indicated what constitutes high progester-
one level the day after HCG administration. The findings in
our study suggest that a progesterone level of more than
9.5 ng/ml (approximately 30 nmol/l) is considered as abnor-
mally high, resulting in a significant reduction in implanta-
tion rate. In an unstimulated natural cycle, however, the peak
progesterone level occurs in the mid-luteal phase, and
30 nmol/L is a widely accepted cut-off level used to indi-
cate ovulation.
One possible weakness of our study is the retrospective
nature and that this study involved a selected population of
women with progesterone levels of less than or equal to
2 ng/ml on the day of HCG administration and less than or
equal to 15 oocytes retrieved. Nevertheless, it did demon-
strate that, in this selected population, an additional pro-
gesterone measurement on the day after HCG administration
is clinically useful, over and above the predictive value ob-
tained from the progesterone measurement on the day of HCG
administration. On the other hand, the strength of this study
includes the large number of patients recruited in a single
centre, with all blood samples obtained consistently in the
morning, with the same hormone assay used throughout the
study. In addition, the study was completed in a relatively
short period of time ( 2 years) during which, there was no
change in laboratory protocol or personnel, and the implan-
tation rate and clinical pregnancy rate were stable. All these
factors together enabled meaningful and reliable analysis of
implantation rate and pregnancy rate.
To conclude, we have found that progesterone measure-
ment should be considered not only on the day of HCG
administration but also the day after HCG administration to
provide clinically useful information on whether or not embryos
should be frozen and transfer deferred to a subsequent cycle.
This study was funded by the National Natural Science Foun-
dation of China (Number: 81270657), the Major Science and
Technology Projects of Zhejiang (Number: 2011C13037) and
the Program for Zhejiang Leading Team of S & T Innovation,
P. R. China (Number: 2011R50013-26).
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Declaration: The authors report no financial or commercial con-
flicts of interest.
Received 4 May 2014; refereed 26 October 2014; accepted 28 October
2014.