Human Reproduction vol.11 no.3 pp.651-654, 1996
CASE REPORT
A sporadic case of delayed implantation after in-vitro
fertilization in the human?
J0rgen Grinsted1 and Birthe Avery2
'The Fertility Clinique, 0sterbrogade 62, DK-2100 Copenhagen
and ''Department of Reproduction, Royal Veterinary and
Agricultural University, BUlowsvcj 13, DK-1870
Frederiksberg C, Denmark
2
To whom correspondence should be addressed
A possible case of delayed implantation after in-vitro
fertilization (TVT) is described. The patient was sterilized
in 1981, and made fertile again by tubal anastomosis in
1988. In 1990 and 1992 the patient had two right-sided
tuba! pregnancies, the first was treated with prostaglandin
instillation, the second with salpingectomy. In connection
with the salpingectomy in 1992, the left tube was observed
to be constricted in the middle part and with phimosis of
the ostium. In 1994 three IVF embryos were transferred,
but 15 days after the transfer, serum human chorionic
gonadotrophln (HCG) was negative (<10 IU/ml). Seven
weeks after the embryo transfer, menstruation was still
missing, and the serum HCG was now positive (329 IU/
ml). Subsequent ultrasound scans were compatible with an
intrauterine pregnancy, progressing normally, but 5 weeks
delayed compared with the oocyte aspiration. The pregnancy was successfully carried to term. Such a long delay
hi detection of HCG, in association with a normal pregnancy, has not been described in the literature before.
Key words: diapause/pregnancy diagnosis/prolonged gestation
Introduction
Delayed implantation, or diapause, is a well described
phenomenon in several species of mammals and marsupials,
but does not occur in reptiles and birds, because their eggs do
not implant (Sandell, 1990; Mead, 1993). Delayed implantation
can be induced by lactation, stress, hibernation and the season.
In delayed implantation, the embryo becomes temporarily
arrested at the blastocyst stage of development In lactating
mice and rats, delayed implantation is obligatory, and in
addition, it is easy to induce experimentally by ovariectomy
on day 4 of pregnancy. Delayed implantation in mice and rats
has thus been thoroughly studied (Nieder and Weitlauf, 1984;
Weitlauf, 1985; Oswald and McClure, 1987; Paria etal, 1992,
1993a, 1993b; Tsujii and Nakatsuji, 1992; Weitlauf and Knisley,
1992; Das et al, 1994; and many others). Other examples of
species with delayed implantation are marsupials (Bryant and
Rose, 1986; Curlewis et al, 1986; Shaw and Renfree, 1986;
© European Society for Human Reproduction and Embryology
Fletcher et al, 1988), rabbits, badgers, weasels, minks
(Martinet and Allain, 1985; Stoufflet et al., 1989), roe deer
(Flint et al, 1994), the Japanese brown bear (Tsubota and
Kanagawa, 1993), some bats (Kimura et al., 1987; Van der
Merwe and van Aarde, 1989; Funakoshi, 1993; Bernard and
Bojarski, 1994), the western spotted skunk (Enders et al.,
1986; Berria et al, 1989; Rozell and Mead, 1993), the giant
panda (Monfort et al, 1989; Zeng et al, 1992), seals (Temte,
1985; Skinner and Westlin van Aarde, 1989; Reijnders, 1990)
and the horse (Vandeplassche, 1986). In the study of excessively
prolonged pregnancies in the mare, Vandeplassche (1986)
concludes that a diapause of 3—5 weeks is rare, but should be
considered to be a reasonable differential diagnosis. In the
human, a few cases of delayed implantation have been
described after ovarian stimulation and ovulation induction, or
in-vitro fertilization (IVF) treatment; however, these pregnancies were only delayed by < 2 weeks, as measured by detection
of serum human chorionic gonadotrophin (HCG), with most
of them ending as spontaneous abortions (Naaktgeboren et al.,
1986, 1987; Hamori et al, 1989). In another report, a tubal
pregnancy presented itself by a delayed appearance of HCG
(Beghin et al, 1992).
When prolonged gestation occurs in the human, one should
first try to rule out causes other than delayed implantation,
and critically re-evaluate more likely factors, such as a deviant
menstrual pattern and reliability of the date for the last
menstruation, occurrence of a bleeding not recognized as
menstrual, cessation of progesterone pill treatment as a cause
of delayed ovulation, and first date for a positive pregnancy
test. We present a case here which is highly indicative of a 5
week delay of implantation after IVF treatment To our
knowledge, this is the first report in the literature.
Case report
A 36 year old woman, born in 1958, gravida 7, parity 4, with
regular menstruations (3/28) and with a 6 year history of
secondary infertility, was admitted to the IVF clinic in 1994
for IVF treatment
Her previous history was as follows: she had delivered four
children, in 1974 (girl, 3000 g/50 cm), 1976 (girl, 3800 g/51
cm), 1979 (girl, 3740 g/52 cm) and 1981 (boy, 3080 g/51 cm)
after uneventful pregnancies. The third delivery was apparently
delayed by 5 weeks, the fourth by 2 weeks, based on the date
of the last menstruation. In 1981 she was sterilized by
laparoscopy with bipolar diathermia. In 1988 reversal of
the sterilization was performed by laparotomy with tubal
651
J.Grinsted and B-Avery
re-anastomosis. In 1990 the patient was admitted with a
positive pregnancy test and 8 weeks of menostasis, caused
by a right-sided tubal pregnancy, which was treated with
laparoscopic prostaglandin instillation. In 1991 she underwent
laparoscopy for loosening of adherences between the omentum
and the left salpinx. At that time, both tubes were patent. From
1991 to 1994 she was treated - 2 0 times with intrauterine
inseminations. In 1992 a right-sided salpingectomy was performed, due to another tubal pregnancy. The left tube was
observed to be slender, constricted in the middle, with partial
phimosis of the ostium, but with fimbriae. In 1994 she
experienced a 7 week period of menostasis; however, no signs
of pregnancy could be detected, either by ultrasonography or
by serum HCG measurement (<10 IU/1).
The patient had thus continuously tried to obtain a viable
pregnancy since the tubal re-anastomosis in 1988. Because of
that, she was admitted for IYF treatment in October, 1994. On
November 15,11 oocytes were aspirated and fertilized (day 0).
Nine of the oocytes cleaved, of which three were transferred
to the uterus. The patient was diagnosed non-pregnant, 15
days after oocyte aspiration (November 30), based on a serum
HCG <10 IU/1. The coital history in that period was as
follows. In November she had intercourse once in the period
between the embryo transfer and the negative pregnancy test
After that she did not have intercourse until after she was
proven pregnant in January. This was due to the fact that she
felt very depressed about not being pregnant, and in addition
she became troubled by nausea. During that period she contacted the IVF clinic several times, because of missing menses
after the embryo transfer. Ultrasound scans showed that the
endometrium was in the secretory phase, indicating that no
ovulation had occurred. Another ultrasound scan was performed 7 weeks after oocyte aspiration (January 2), and
the HCG was measured. An ultrasound scan now showed
endometrium typical of early pregnancy and compatible with
early implantation, and the serum HCG was positive at 329
IU/1. The following day (January 3), the serum HCG was 632
IU/1. At 8 weeks after oocyte aspiration (January 11), a
10 mm gestational sac and a yolk sac were demonstrated. Two
subsequent ultrasound scans on January 25 and February 24,
corresponding to 10 and 14 weeks after oocyte aspiration,
showed one live fetus that had grown from a crown-rump
length of 8.1 mm to 56 mm during the 4 week period. The
calculated age of the fetus 10 and 14 weeks after oocyte
aspiration would then be 12 and 16 pregnancy weeks; however,
the age of the fetus calculated from the crown—rump length
was 6 ± 1 weeks and 12 ± 1 weeks respectively (Figures 1
and 2). The pregnancy continued normally, but was delayed
by 4-6 weeks in relation to the IVF treatment, with the
expected date for the delivery being between September 4 and
16, instead of August 8, 1995. On September 7, 1995 a normal
boy was delivered (4450 g/58 cm). The birth occurred 42
weeks and 3 days after the embryo transfer, corresponding to
44 weeks of pregnancy, or 38-40 weeks of pregnancy corrected
for crown—rump length.
Discussion
In the human it is virtually impossible to prove that a
discrepancy between the expected and actual length of gestation
652
Figure 1. Ultrasonographic image recorded 10 weeks after embryo
transfer. An intrauterine fetus was observed with a crown-rump
length of 8.1 mm, corresponding to a pregnancy of 6 ± 1 weeks.
According to the date for oocyte aspiration, this pregnancy should
have been 12 weeks old.
Figure 2. Ultrasonographic image recorded 14 weeks after embryo
transfer observation of the same pregnancy as in Figure 1, but
4 weeks later. The crown—rump length is now 56 mm,
corresponding to a pregnancy of 12 ± 1 weeks. According to the
date of oocyte aspiration, the pregnancy should have been 16
weeks old.
is caused by delayed implantation. In animal breeding, it is
the rule rather than the exception, to know the exact dates of
heat and breeding, and hence the expected day of parturition.
In the human such information is rarely available or reliable.
In the case of our patient, she had a long history of infertility,
with tubal sterilization and re-anastomosis, two extrauterine
pregnancies, resulting in a right-sided salpingectomy, a malformed left oviduct, possibly with adherences, although
described as being patent in 1991, and 20 intrauterine inseminations which did not result in a pregnancy supported by the
remaining left oviduct. This was why she was admitted for
IVF treatment. After embryo transfer and before the negative
pregnancy test she had intercourse once, but was not sexually
active until after she was diagnosed pregnant. In addition,
menstruation was not restored, she developed nausea, and
Diapause in the human
the endometrium remained in the secretory phase until an
intrauterine pregnancy was demonstrated 5 weeks after embryo
transfer. All of this points toward a case of delayed implantation, and not a spontaneous conception, since she did not have
intercourse in the critical period.
During in-vitro cultivation, human blastocysts secrete HCG
(Fishel et al., 1984), but it is only after implantation that HCG
is detected in the maternal blood. A positive pregnancy test is
defined by the arbitrarily chosen serum {5-HCG concentration
>10 IU/1. This value is significantly higher than the low basic
concentration of circulating HCG (<4 IU/1) present in the
serum and urine of men and non-pregnant women. HCG values
>10 IU/1 are usually reached 3-7 days after implantation,
which in turn occurs 6-8 days after conception (Bergh and
Navot, 1992; review by Chard, 1992), meaning that our patient,
with an HCG value <10 IU/1 on November 30, 15 days postaspiration, should have been diagnosed as non-pregnant With
HCG values of 329 and 632 IU/1 measured on January 2 and
3 (7 weeks post-aspiration), the HCG concentration could
theoretically have been >10 IU/1 around December 27, if a
doubling time of 1.2-1.5 days was used for the calculation
(Hamori et al, 1989; Pellicer et al, 1991; Bergh and Navot,
1992; Zegers-Hochschild et al, 1994). Demonstration of an
intrauterine 10 mm gestation sac and a yolk sac on January
11 (8 weeks post-aspiration) corresponds with a 3.5-4 but not
an 8 week old conceptus, as demonstrated by Pellicer et al.
(1991), who determined the time-frames for the first detection
of the embryonic sac, yolk sac and heart beats by ultrasonography in pregnancies from patients treated with FVF or
artificial insemination. The two subsequent ultrasound scans
on January 11 and February 24, showing crown-rump lengths
of 8.1 mm (6 ± 1 weeks) and 56 mm (12 ± 1 weeks)
respectively, correspond well with the previous findings.
The mechanisms behind delayed implantation are complex
and differ between species, and are in general not well
understood (review by Mead, 1993). Hormones, like prolactin,
progesterone and oestrogen seem to be involved in the control.
Sometimes the same hormone has opposite effects in two
different species, which makes it impossible to extrapolate
results from one species to the other. The uterus, however,
plays an important role in diapause, and LIF (leukaemia
inhibitory factor), which is synthesized both by the uterus and
by the blastocyst, appears to be important for implantation, at
least in the human and mice (Charnock-Jones et al., 1994;
Stewart, 1994). A possible explanation for the delayed
implantation in our patient could be the following: since no
menstruation occurred after the negative HCG test 15 days
post-aspiration, the corpus luteum must have been rescued. A
negative HCG means that the embryo is not implanted; HCG,
however, is being produced by the blastocyst, and although
not systemically detectable, each of the three transferred
embryos in this case should have been producing HCG, which
could be sufficient for prevention of luteolysis by a locally
mediated effect on the corpus luteum. The progesterone
concentration could be low, and might induce the embryo(s)
to enter diapause. A rise in progesterone could result in
reactivation of the dormant embryo, followed by implantation,
which could explain the time lapse of ~5 weeks. The rise
could be triggered by a new ovulation; however, this was not
demonstrated since ultrasound scans from the embryo transfer
and onwards showed endometrium in the secretory phase. This
case supports the findings of Edwards (1988), who reported a
case in which the human implantation window was still open
when three IVF embryos were replaced 1 week after the
normal time for replacement, and with the uterus being in the
secretory phase.
Although delayed implantation in the human has not been
described before, there is no reason to believe that it does not
exist An estimate of the incidence is therefore not available.
In the horse, which has an average gestation period of 336
days, with a physiological variation of 320-350 days, the
incidence of extremely prolonged pregnancies (+30-63 days)
has been estimated to be ~ 1 % (Vandeplassche, 1986).
The demonstration of a pregnancy, obtained under controlled
clinical circumstances and excessively prolonged by 5 weeks,
as judged by HCG measurement and ultrasound scan, allows
that the differential diagnosis: delayed implantation, although
rare, should be considered when prolonged gestation is
observed in the human.
Acknowledgements
This study was partly supported by the Animal Biotechnology
Research Center. We thank Mrs Inger Heinze for producing copies
of the photographs.
References
Beghin, C , Dhont, M., Merchiers, E. and Vandekerckhove, D. (1992) Delayed
implantation. A case report. J. Reprod. MetL, 37, 482-484.
Bergh, P.A. and Navot, D. (1992) The impact of embryonic development and
endometrial maturity on the timing of implantation. FertiL SteriL, 58,
537-542.
Bernard, R.T.F. and Bojarski, C. (1994) Effects of prolactin and hCG
treatment on luteal activity and the conceptus during delayed implantation
in Schreibers' long-fingered bat (Miniopterus schreibersii). J. Reprod.
FertiL, 100, 359-365.
Berria, M., DeSantis, M. and Mead, R.A. (1989) Lesions to the anterior
hypothalamus prevent the melatonin-ihduced lengthening of delayed
implantation. Endocrinology, 125, 2897-2904.
Bryant, S.L. and Rose, R.W. (1986) Growth and role of the corpus luteum
throughout delayed gestation in the potoroo, Potorous tridactylus. J. Reprod.
FertiL, 76,409-414.
Chard, T. (1992) Pregnancy tests: a review. Hum. Reprod., 7, 701-710.
Charnock-Jones, D.S., Sharkey, A.M., Fenwick, P. and Smith, S.K. (1994)
Leukaemia inhibitory factor mRNA concentration peaks in human
endometrium at.the time of implantation and the blastocyst contains mRNA
for the receptor at this time. / . Reprod FertiL, 101, 421-426.
Curlewis, J.D., White, A.S., Loudon, A.S. and McNeilly, A.S. (1986) Effects
of lactation and season on plasma prolactin concentrations and response to
bromocriptine during lactation in the Bennett's wallaby (Macropus
rufogriseus rufogriseus). J. Endocrinol., 110, 59—66.
Das, S.K., Wang, X.N., Paria, C.B., Damm, D., Abraham, 1A. et aL (1994)
Heparin-binding EGF-like growth factor gene is induced in the mouse
uterus temporally by the blastocyst solely at the site of its apposition: a
possible ligand for interaction with blastocyst EGF-receptor in implantation.
Development, 120, 1071-1083.
Edwards, R.G. (1988) Human uterine endocrinology and the implantation
window. Ann. N.Y. Acad. Set, 541, 445-454.
Enders, A.C., Schlafke, S., Hubbard, N.E. and Mead, R.A. (1986)
Morphological changes in the blastocyst of the western spotted skunk
during activation from delayed implantation. BioL Reprod., 34, 432—437.
Fishel, S.B., Edwards, R.G. and Evans, CJ. (1984) Human chorionic
gonadotropin secreted by preimplantation embryos cultured in vitro. Science,
223, 816-818.
653
J.Grinsted and B-Avery
Fletcher, T.P., Jetton, A.E. and Renfree, M.B. (1988) Influence of progesterone
and oestradiol-17 beta on blastocysts of the tammar wallaby (Macropus
eugenii) during seasonal diapause. J. Reprvd. Fertii, 83, 193-200.
Flint, A.P., Krzywinski, A., Sempere, AJ., Mauget, R. and Lacroix, A. (1994)
Luteal oxytocin and monestry in the roe deer Capreolus capreolus. J. Reprvd.
Fertii., 101,651-656.
Funakoshi, K. (1993) Ecology and reproductive pattern in bats. J. Reprod
Dev., 39, J109-J115.
Hamori, M., Stuckensen, J.A., Rumpf, D., Kniewald, T., Kniewald, A. and
Marquez, M. (1989) Early pregnancy wastage following late implantation
after in-vitro fertilization and embryo transfer. Hum. Reprod, 4, 714-717.
Kimura, K., Takeda, A. and Uchida, T.A. (1987) Changes in progesterone
concentrations in the Japanese long-fingered bat, Miniopterus screibersii
fuliginosus. J. Reprod Fertii., 80, 59-63.
Martinet, L. and Allain, D. (1985) Role of the pineal gland in the photoperiodic
control of reproductive and non-reproductive functions in mink (Mustela
visort). Ciba Found. Symp., 117, 170-187.
Mead, R.A. (1993) Embryonic diapause in vertebrates. J. Exp. Zool., 266,
629-641.
Monfort, S.L., Dahl, K.D., Czekala, N.M., Stevens, L., Bush, M. and Wildt,
D.E. (1989) Monitoring ovarian function and pregnancy in the giant panda
(Ailuropoda melanoleuca) by evaluating urinary bioactive FSH and steroid
metabolites. /. Reprod. Fertii, 85, 203-212.
Naaktgeboren, N., Devroey, P., Wisanto, A., Tracy, E. and Van Steirteghem,
A.C. (1986) Endocrine profiles in early pregnancies with delayed
implantation. Hum. Reprod., 1, 9-14.
Naaktgeboren, N., Devroey, P., Wisanto, A. and Van Steirteghem, A.C. (1987)
Pregnancies without corpus luteum rescue. Ann. BioL Clin., 45, 456-459.
Nieder, G l . and Weitlauf, H.M. (1984) Regulation of glycolysis in the mouse
blastocyst during delayed implantation. J. Exp. Zool., 231, 121-129.
Oswald, C. and McClure, RA. (1987) Energy allocation during concurrent
pregnancy and lactation in Norway rats with delayed and undelayed
implantation. /. Exp. Zool., 241, 343-357.
Paria, B.C., Jones, K.L., Flanders, K.C. and Dey, S.K. (1992) Localization
and binding of transforming growth factor-beta isoforms in mouse
preimplantation embryos and in delayed and activated blastocysts. Dev.
BioL, 151, 91-104.
Paria, B.C., Huet-Hudson, YM. and Dey, S.K. (1993a) Blastocyst's state of
activity determines the 'window' of implantation in the receptive mouse
uterus. Proc. Natl. Acad. Sci. USA, 90, 10159-10162.
Paria, B.C., Das, S.K., Andrews, G.K. and Dey, S.K. (1993b) Expression of
the epidermal growth factor receptor gene is regulated in mouse blastocysts
during delayed implantation. Proc. NatL Acad. Sci. USA, 90, 55-59.
Pellicer, A., Calatayud, C , Miro, F., Castellvi, R.N., Ruiz, A. et al (1991)
Comparison of implantation and early development of human embryos
fertilized in vitro versus in vivo using transvaginal ultrasound. J. Ultrasound
Med., 10,31-35.
Reijnders, PJ. (1990) Progesterone and oestradiol-17 beta concentration
profiles throughout the reproductive cycle in harbour seals (Phoca vitulina).
J. Reprod. Fertii., 90, 403-409.
Rozell, M.D. and Mead, R.A. (1993) Effect of melatonin on pituitary secretion
of prolactin in vitro during delayed implantation and the periimplantation
period in the spotted skunk. J. Exp. Zool., 267, 524-532.
Sandell, M. (1990) The evolution of seasonal delayed implantation. Q. Rev.
BioL, 65, 23-^2.
Shaw, G. and Renfree, M 3 . (1986) Uterine and embryonic metabolism after
diapause in the tammar wallaby, Macropus eugenii. J. Reprod. FertiL, 76,
339-347.
Skinner, J.D. and Westlin van Aarde, L.M. (1989) Aspects of reproduction in
female Ross seals (Ommatophoca rossii). J. Reprod. FertiL, 87, 67-72.
Stewart, C.L. (1994) Leukaemia inhibitory factor and the regulation of preimplantation development of the mammalian embryo. MoL Reprod Dev.,
39, 233-238.
Stoufflet, I., Mondain-Monval, M., Simon, P. and Martinet, L. (1989) Patterns
of plasma progesterone, androgen and oestrogen concentrations and in-vitro
ovarian steroidogenesis during embryonic diapause and implantation in the
mink {Mustela vison). J. Reprod. FertiL, 87, 209-221.
Temte, J.L. (1985) Photoperiod and delayed implantation in the northern fur
seal (Callorhinus ursinus). J. Reprod FertiL, 73, 127-131.
Tsubota, T. and Kanagawa, H. (1993) Morphological characteristics of the
ovary, uterus and embryo during the delayed implantation period in the
Hokkaido brown bear (Ursus arctosyesoensis). J. Reprod Dev., 39,325-331.
Tsujii, H. and Nakatsuji, N. (1992) Effect of delayed implantation of blastocyst
on fetal weight in embryo-transferred rats. Jap. J. FertiL SteriL, 37,380-383.
654
Vandeplassche, MM. (1986) Delayed embryonic development and prolonged
pregnancy in mares. In Morrow, D.A. (ed.). Current Therapy in
Theriogenology 2. Diagnosis, Treatment, and Prevention of Reproductive
Diseases in Small and Large Animals. W.B.Saunders, USA. pp. 685-689.
Van der Merwe, M. and van Aarde, RJ. (1989) Plasma progesteron
concentrations in the female Natal clinging bat (Minopterus screibersii
natalensis). J. Reprod. FertiL, 87, 665-669.
Weitlauf, H.M. (1985) Changes in the rate of translation with reactivation of
delayed implanting mouse embryos. J. Exp. Zool., 236, 309-312.
Weitlauf, H.M. and Knisley, K.A. (1992) Changes in surface antigens on
preimplantation mouse embryo. BioL Reprod, 46, 811-816.
Zegers-Hochschild, F, Altieri, E , Fabres, C , Fernandez, E., Mackenna, A.
and Orihueli, P. (1994) Predictive value of human chorionic gonadotrophin
in the outcome of early pregnancy after in-vitro fertilization and spontaneous
conception. Hum. Reprod., 9, 1550-1555.
Zeng, G.Q., Zhang, X., Jiang, G.T., Liu, W.X., Xie, Z. and Liu, N.L. (1992)
Studies on the reproductive biology of giant panda: II. Changes of
progesterone and chorionic gonadotropin-like substance in urine of giant
pandas during pregnancy. Acta ZooL Sin., 38, 429—434.
Received on August 3, 1995; accepted on November 29, 1995