FERTILITY AND STERILITY Vol. 58, No.

3, September 1992
Copyright IV 1992 The American Fertility Society Printed on acid~free paper in U.S.A.

The impact of embryonic development and endometrial maturity

on the timing of implantation*

Paul A. Bergh, M.D.

Daniel Navot, M.D.t

Department of Obstetrics and Gynecology, and Reproductive Science, Mount Sinai Medical Center, New York, New York

Objective: To gain insight into the peri-implantation period in the human and to answer the
question whether timing of nidation is dependent on the stage of embryonic development, endometrial
maturation, or a possible dialogue between the two.
Design: Seventy-five women underwent embryo transfer (ET) throughout 93 cycles. Thirty-three
ETs resulted in viable pregnancies and deliveries. These pregnancy cycles were used for embryonic
signal detection. Embryos of identical age were transferred onto hormonally and histologically defined
endometria of different maturational stages (days 15 to 19). Human chorionic gonadotropin (heG)
was measured by a hypersensitive chemiluminescence assay in maternal serum every 1 to 5 days to
detect the first embryonic signal.
Results: Individual linear regressions of heG versus embryonic age and endometrial maturation
were performed on 33 viable pregnancy cycles (r2 = 90.5% to 99.9%, P < 0.02 to 0.002). First signal
detection was restricted to an embryonic age of 7.1 ± 0.28 (mean ± SD) days (range 6.6 to 7.4)
irrespective of endometrial maturation. The pattern of heG detection was triphasic, described by
a sigmoidal curve with the maximal slope corresponding to an heG doubling time of 15.9 hours.
Embryo transfers on cycle day 19 had a steeper slope of heG detection than days 15 and 16
(P < 0.05).
Conclusions: First embryonic signal detection (presumed window of implantation) extends be-
tween cycle days 20 and 24. Implantation is dependent on embryonic age and is independent of
endometrial maturation within this window. The timing and sigmoidal pattern of heG detection
coincides with structural changes of the implantation bed. The steeper slope of late ETs may repre-
sent a compensatory mechanism for late maternal recognition of pregnancy for corpus luteum
rescue. Fertil Steril 1992;58:537-42

Key Words: Human implantation, embryonic development, endometrial maturity, embryonic

signal, human chorionic gonadotropin

Failure of sustained implantation is the crucial
event that differentiates between fertile and non-
fertile cycles in the reproductive process. However,
nidation remains one ofthe least explored early em-
bryonic phenomena, despite the new insights gained
by over a decade of experience with human in vitro
fertilization (IVF). Specifically, IVF has allowed
* Presented at the 46th Annual Meeting of the American Fer-
tility Society, Washington, DC, October 15 to 18, 1990.
t Reprint requests: Daniel Navot, M.D., Division of Repro-
ductive Endocrinology, Mount Sinai Medical Center, One Gustave
Levy Place, Box 1175, New York, New York 10029.
close scrutiny of the early phases of folliculogenesis,
oocyte maturation, sperm-egg interaction, and em-
bryonic development, whereas the peri-implantation
events have remained relatively inaccessible. To
date, the data pertaining to uterine receptivity and
embryonic-uterine interactions have been limited to
the window of embryo transfer (ET) derived from
the ovum donation model (1, 2). In these studies,
endometrial receptivity has been found to be tem-
porally restricted to days 17 to 19 of a normalized
28-day cycle. Recently, the period of endometrial
receptivity for 2 to 3-day-old embryos has been ex-
tended to days 15 through 20 of the cycle (3). Mor-

Vol. 58, No.3, September 1992 Bergh and Navot Timing of human implantation 537
phological evidence on the actual time of implan-
tation has been restricted to the classical obser-
vations of Hertig and associates (4,5) who have de-
lineated the time of implantation as occurring
between 5.5 and 6 days after ovulation. Although
their study included anatomical specimens of im-
planting embryos, the author's capacity to accurately
pinpoint the time of ovulation or embryonic age was
limited. To evaluate the relative roles of endometrial
maturation and embryonic development on implan-
tation, one ofthese variables must be kept constant.
Our study was designed to answer a fundamental
question in the physiology of implantation regarding
the timing of nidation and its dependence on the
stage of embryonic development, endometrial mat-
uration, or a possible dialogue between the two. In
this study, identical age embryos were transferred
onto hormonally and histologically defined endo-
metria of different maturational stages. We have as-
sumed that the first embryonic signal (human cho-
rionic gonadotropin [hCG 1) detection is indicative
of and is temporally related to embryo implantation.
The absence of exogenous hCG contamination in
this model allowed for detection of the first embry-
onic signal using a very sensitive hCG assay.
MATERIALS AND METHODS
The study population consisted of 33 women who
conceived and delivered subsequent to transfer with
self or donated ova. From an initial population of
75 patients undergoing 93 ET cycles, only the 33
viable pregnancy cycles (35.5%) were analyzed. The
study was approved by the Institutional Review
Board, and all patients signed an appropriate in-
formed consent before being included in this study.
Twenty-two conceptions were achieved with fresh
embryos, whereas 11 followed cryopreservation in
natural cycles. All patients underwent a preparatory
cycle that served to assess the adequacy of their spe-
cific cycle. During the preparatory cycle, in addition
to hormonal monitoring, timed endometrial biopsies
were performed. In-phase endometrial biopsies were
a prerequisite for initiation of the actual transfer
cycle (6, 7).
All ETs were normalized to day 15, corresponding
to the day of progesterone (P) initiation in simulated
cycles or to the day after the luteinizing hormone
(LH) surge in natural cycles (8). The actual day of
transfer was arbitrary. In ovum donation cycles, ini-
tiation of P in the recipient on the day of hCG ad-
ministration to the donor resulted in a day 19 trans-
fer. Initiation on the day that eggs became available
538 Bergh and Navot Timing of human implantation
or when fertilization was confirmed would result in
transfers on days 17 and 16, respectively. Transfers
were performed with 42 to 46-hour-old embryos
throughout normalized cycle days 15 to 19, a window
that was previously shown to allow subsequent suc-
cessful implantation (3). Eight additional patients
who had blastocysts transferred served as separate
controls for the possible detection of hCG produced
by embryos in the uterine cavity before implanta-
tion. Human chorionic gonadotropin was measured
in maternal serum every 1 to 5 days starting before
ET. A hypersensitive chemiluminescence assay was
used for hCG dimer determinations (Amerlite,
Amersham Int, Arlington Heights, IL) (9). The in-
tra-assay and interassay coefficients of variation
were 5.8% and 8.7%, respectively. The lowest limit
of detection was determined as 3 SDs (SD = 0.3
lUlL) above the mean value (0.1 lUlL) in 45 serum
samples before ET (First International Reference
Preparation 75/537). The cross-reactivity ofhCG in
this assay with human LH, human follicule-stimu-
lating hormone, and human thyroid-stimulating
hormone was <1.0%. Values are expressed as means
±SD.
In each individual cycle, serial serum hCG mea-
surements were evaluated for first signal detection.
A value :2 1.0 lUlL was considered first signal. In
all cycles, linear regression analysis after log trans-
formation was performed up to an embryonic age of
24 days. Either embryonic age or cycle day was used
as the independent variable and the natural log of
the hCG as the dependent variable. Analysis ofvari-
ance and Student's t-test were applied as appropri-
ate. Level of significance was set at P < 0.05.
RESULTS
To elucidate the relative contribution of endo-
metrial maturational stage on the timing of first
embryonic signal detection, cycles were placed into
five groups according to the endometrial age (nor-
malized cycle day 15 to 19) at the time of ET. The
number of cycles per group was distributed as fol-
lows: day 15, 3 cycles; day 16, 11 cycles; day 17, 11
cycles; day 18, 5 cycles; day 19, 3 cycles. Figure 1A
illustrates the results of this regression analysis us-
ing the normalized embryonic age as the indepen-
dent variable. The graph clearly depicts the con-
vergence between the five groups at the time of
earliest signal detection (hCG = 1.0 lUlL), corre-
sponding to an average embryonic age of 7.1 ± 0.28
days (range 6.6 to 7.4 days). The range of average
r2 between groups was 90.5% to 99.9%, P < 0.02 to
Fertility and Sterility
I
 10000
cepts at hCG = 1.0 varied between day 20.3 for day
15 transfers, day 21.4 for day 16 transfers, day 21.6
1000 hi 81...1
for day 17 transfers, and day 23.0 and day 24.0 for
~...
Q 100
transfers performed on days 18 and 19, respectively .
e Although the slopes of the regressions between the

.
~
f.)
10 A five groups remained unchanged, there was a sig-
nificant difference in the cycle day on which the
first embryonic signal was detected (P < 0.05).
Analysis of the pattern of hCG detection disclosed
0 a triphasic rise in hCG. This is illustrated in Figure
0 10 15 20 25
2 with the use of a quartic least square polynomial
Embryonic Ale (days)
regression described by the equation:
10000 0

+ 0.263x2 - (1.32

1'---1
In (y) = 3.95 - 1.86x X 1O-2)x3
1000
Im,lalltati.a
....,.;a Wld.w
...Q 100
...
..,
Although the level of hCG was significantly ele-
..
~
U
10
vated compared with baseline by embryonic age 6
to 8 days, there was essentially no rise in hCG, with
levels hovering around 1 to 2 IU jL. An appreciable
rise of hCG, with a doubling time of 15.9 hours, was
IB 20 22 24 26 21 30 32 then noted between an embryonic age of 8 to 11
days. After day 11, the rate of hCG detection re-
Normalized Cycle Day
mained steady with an average doubling time of 1.3
days.
Day IS Day 16 Da, 17 DIY II Day I'
AT, ,'.0 . .9.9.9 Av, r'.0 . .962 At'. ,'.0.J52 Av, ,1.0 . .916 AT, ,.-0.90S Seventeen blastocysts served as separate controls
o v for the possible detection of hCG produced by em-
bryos in the uterine cavity before implantation.
Figure 1 (A), The upper panel depicts linear regressions of These blastocysts were transferred on days 20 to 22
heG detection against embryonic age, Individual regressions are
grouped according to the day of ET corresponding to endometrial
and had a mean hCG level of 0.38 ± 0.27 IU jL
maturational stage. The horizontal line at heG = 1.0 lUlL rep- throughout the following week. These values are
resents 3 SD above the lower limit of heG detection, which was similar to background hCG levels and remain well
arbitrarily defined as an unequivocal embryonic signal. All the
regressions converge at an heG of 1.0 lUlL, coinciding with an
below the signal detection threshold of 1.0 IU jL.
embryonic age of 7.1 ± 0.28 days. (B), The lower panel illustrates
the same regressions using endometrial maturation as the ordi-
nate. The regressions intersect with the first embryonic signal 1000
mark over a range of endometrial maturation stages that define
0
8
the presumed window of implantation. @ 0

100 0
0
~
... 0
0 0
0
;l 0
0.002. An ANOV A of embryonic age at time of first e 10
embryonic signal detection revealed no significant
differences between the five groups (P = 0.83) (Fig.
lA). This figure also illustrates a gradual increase
.
CJ
U
<> 0
r2=O.914

in the rate of hCG detection (slope of regressions) 0 0 0

0
according to the day of ET, i.e., endometrial mat- oL-~--~~--~~--~~--~~--~~--~~

urational stage. The apparent direct correlation be- 4 5 7 8 9 10 11 lZ 13 14 15 16 17

tween day of transfer and rate of hCG detection did
not attain statistical significance (ANOVA, P = 0.06).
Regressions were then performed using endometrial
maturation (normalized cycle day) as the indepen-
dent variable (Fig. IB). The distribution of inter-
Embryonic Age (days)
Figure 2 The triphasic pattern of heG detection is illustrated
by the quartic, least square polynomial regression described by
the function: (In (y) = 3.95 - 1.86x + 0.263x2 - (1.32 X 1O-2)x3
+ (2.28 X 1O-4)x4; r2 = 0.914).

Vol. 58, No.3, September 1992 Bergh and N avot Timing of human implantation 539
 The use of cryopreserved embryos together with
the fresh embryos in the analysis was justified by
the similar intercepts (P = 0.43) with a difference
of only 0.08 days at the first embryonic signal (heG
= 1.0) and virtually identical slopes (P = 0.69) for
cryopreserved and fresh embryos.
DISCUSSION
We have previously demonstrated that transfers
with 2-day-old embryos on cycle days 15 through 19
are conducive of sustained implantation (1, 3). This
established window of ET alludes to the actual win-
dow of implantation, namely, if a 6-day-old embryo
is capable of nidation, then the window of implan-
tation would extend from day 19 to day 23. In the
present study, we have demonstrated that the de-
tection of the first embryonic signal occurs between
cycle days 20 and 24. The actual signal detection
was dependent on embryonic age and independent
of the endometrial maturational stage within the
presumed window of implantation. The mean em-
bryonic age at first signal detection was 7.1 ± 0.28
days. Thus, on the one end of the spectrum, cycle
day 15 transfers with 2-day-old embryos resulted in
the perception of heG on day 20, whereas on the
other end of the spectrum, transfers on day 19 re-
sulted in the perception of heG on day 24 (Fig. 1B).
Within the window of endometrial receptivity,
beyond having a permissive role, the endometrium
has no apparent impact on the timing of implan-
tation. An appreciable shift in the time of first signal
detection relative to endometrial maturation would
imply an active dialogue between embryo and en-
dometrium. From the embryonic point of view, a
less than optimal endometrial milieu would call for
an embryonic diapause, namely, a physiological
maturational arrest, which is well described in other
mammals (10, 11). Thus the embryo appears to be
the principal determinant of the timing of nidation.
Although the study design does not allow definite
conclusions to be made, our observations do not
support the concept of a possible embryonic diapause
in the human.
The classical, in vivo, morphological data of Her-
tig and associates (4, 5) have delineated the time of
implantation between 5.5 to 6 days after ovulation.
Buster et al. (12) have recovered blastocysts from
the uterine cavity in natural cycles an average of 4.5
days after ovulation. Their observations are consis-
tent with Hertig's data, placing implantation at an
embryonic age of 5 to 6 days.
540 Bergh and Navot Timing of human implantation
In vitro data on embryonic development and heG
production has described blastocyst formation on
day 5 and the earliest detection of heG secretion at
an embryonic age of 7 to 8 days (13-15). In vitro
embryonic development and initial secretion of heG
appears to trail the in vivo data. These in vitro cul-
tured embryos secrete heG at one order of magni-
tude below the level of the in vivo implanted embryos
at approximately the same age (13-15). This may
be a reflection of suboptimal in vitro conditions and/
or the relatively poor quality embryos used in these
studies because only surplus embryos or those con-
sidered unsuitable for transfer were examined.
Previous in vivo studies of natural cycles have
placed the first detection of heG between 6 to 11
days after ovulation (16-18). In vivo studies of IVF
cycles report similar findings with first detection of
heG between 6.5 to 10 days (19, 20) after ovum re-
trieval. Whereas these latter studies offer the ad-
vantage of a more accurate assessment of embryonic
age, they are hampered by the use of heG for ovu-
lation induction, which produces measurable levels
of serum heG for 9 to 14 days after intramuscular
injection (21, 22).
In the above studies, heG detection varied over
an exceptionally wide range of presumed embryonic
ages, despite the fact that embryonic/endometrial
maturational stages were constant. In contrast, our
results, in which endometrial maturational stage was
intentionally varied over a 5-day period while em-
bryonic age was held constant, show a remarkably
consistent pattern of detection. Systemic detection
of heG has been traditionally equated with proof of
implantation (23, 24). We have assumed that the
detection of the first embryonic signal (heG) is in-
dicative of and temporally related to embryo im-
plantation. Indeed, our data suggest that the first
signal detection occurs on day 7,1 day after the pre-
sumed day of implantation as deduced from previous
morphological studies (4, 5). The eight patients in
our study who had expanded blastocysts transferred
served as an in vivo control for heG signal detection
from preimplantation embryos in the uterine cavity.
The lack of signal detection is consistent with our
assumption that preimplantation heG production
cannot be detected in the systemic circulation.
The triphasic pattern of systemic heG detection
is consistent with Hertig and collaborators' mor-
phological observations (4, 5, 25). That is, by 6 to 8
days after ovulation when the embryo is superficially
implanted and the endometrium is just beginning
to show evidence of progestational hyperplasia, heG
levels may reach the threshold of systemic detection
Fertility and Sterility
through dilated but noncongested capillary sinu-
soids. In addition, definitive trophoblast formation
is limited to the area of endometrial contact (4, 25).
Although heG is being produced, the quantity de-
tectable in maternal serum is surely restricted by
the lack of an effective uteroplacental circulation.
This phase of nidation is characterized by the de-
tectable but flat heG levels at an embryonic age of
6 to 8 days (Fig. 2). The second phase is character-
ized by the abrupt rise in heG detection that prob-
ably marks the rapid maturation of the uteropla-
cental circulation and heG uptake from the
edematous interstitium rather than accelerated
production of trophoblastic heG (18). This period
corresponds to our observation of the rapid doubling
time of 15.9 hours at embryonic ages 8 to 10 days.
By 11 to 12 days after ovulation, this circulation is
well established as evidenced by the confluence of
maternal sinusoids with lacunar spaces (4, 25). Once
this relationship is fully established, the slower but
continued steady rise in heG detection reflects the
equilibrium between trophoblast production and
transfer to the maternal circulation. Figure lA il-
lustrates a gradual increase in the rate of heG de-
tection (slope of regressions) according to the day
of ET. This may correlate with the increased vas-
cularization and permeability of the more advanced
endometrium (4). From a teleological viewpoint, an
accelerated recognition of heG with advancing
endometrial maturity would compensate for late
implantation and effect timely corpus luteum
rescue.
We have shown that within a previously defined
window of endometrial receptivity, the timing of
embryo implantation is dependent on embryonic
developmental stage and is independent of endo-
metrial maturation. The pattern and timing of heG
detection reflects the corresponding structural
changes of the implantation bed. The relatively
higher rate of detection of systemic heG after late
transfers may represent a maternal compensatory
mechanism. The unique in vivo model used in this
study allowed insight into the peri-implantation pe-
riod in the human, delineating embryonic-endo-
metrial interactions that are conducive of sustained
implantation.
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