Human Reproduction vol.14 no.8 pp.

19441948, 1999
Luteal support with micronized progesterone following
in-vitro fertilization using a down-regulation protocol
with gonadotrophin-releasing hormone agonist: a
comparative study between vaginal and oral
administration
S.Friedler
1
, A.Raziel, M.Schachter, D.Strassburger,
I.Bukovsky and R.Ron-El
IVF and Infertility Unit, Department of Obstetrics and Gynecology,
Assaf Harofeh Medical Center, Zerin 70300, Israel
1
To whom correspondence should be addressed
This study aimed to compare the efcacy of micronized
progesterone administered as luteal support following
ovulation induction for in-vitro fertilization (IVF)
embryo transfer in cycles using gonadotrophin-releasing
hormone agonist, either orally (200 mg4/day) or
vaginally (100 mg2/day) and to characterize the luteal
phase hormonal prole during such treatments. Atotal of 64
high responder patients requiring intracytoplasmic sperm
injection due to male factor infertility were prospectively
randomized into two treatment groups. Patients treated
orally or vaginally were comparable in age (31.9 6.1
versus 30.6 5.2; mean SD), number of oocytes
retrieved (17 8.2 versus 18 7.0), and number of
embryos transferred (3.1 1.2 versus 2.7 0.9) per
cycle. Following low dose vaginal treatment, a signi-
cantly higher implantation rate (30.7 versus 10.7%,
P < 0.01), but similar clinical pregnancy rate (47.0 versus
33.3%) and ongoing pregnancy rate (41.1 versus 20.0%)
was observed, compared with oral treatment. In concep-
tion cycles, luteal serum progesterone and oestrogen con-
centrations did not differ between the treatment groups.
In non-conception cycles, late luteal progesterone con-
centrations were signicantly lower following vaginal
treatment. As low dose micronized progesterone adminis-
tered vaginally is simple, easy and well tolerated, it could
be recommended as the method of choice for luteal support,
especially for high responder patients at risk for ovarian
hyperstimulation syndrome.
Key words: luteal endocrine prole/luteal support/micronized
progesterone
Introduction
The need for luteal support following ovulation induction for
in-vitro fertilization (IVF)embryo transfer in cycles using
gonadotrophin-releasing hormone agonist (GnRHa) has
become a consensus.
Methods of luteal phase support include corpus luteum
stimulation to secrete endogenous oestrogen and progesterone
by serial injections of human chorionic gonadotrophin (HCG)
(Smith et al., 1989; Ballaisch-Allart et al., 1990) or with
exogenous replacement of progesterone (Van Steirteghemet al.,
1944 European Society of Human Reproduction and Embryology
1988; Smitz et al., 1988). Although a meta-analysis of random-
ized trials of luteal support (Soliman et al., 1994) has shown
a preference for HCG, it cannot be recommended in patients
with high response because it may aggravate the risk of ovarian
hyperstimulation syndrome (OHSS).
Micronized progesterone induces endometrial maturation
and leads to pregnancies following IVFembryo transfer or
during cycles of oocyte donation. Micronized progesterone is
absorbed after oral or vaginal administration but various
protocols for its administration have been proposed in the
literature (Salat-Baroux et al., 1988; Devroey et al., 1991;
Smitz et al., 1992a; Pouly et al., 1996) and the optimal
effective route or dosage for luteal phase support following
ovulation induction using GnRHa down-regulation for IVF
has not yet been established.
The aim of our study was to compare the efcacy of
micronized progesterone administered as luteal support, either
orally or vaginally at different dosages, and to characterize
the luteal phase hormonal prole during such treatments.
Materials and methods
Study population
The study population included 64 couples with male factor infertility
requiring intracytoplasmic sperm injection (ICSI), using fresh
ejaculated spermatozoa, treated at Assaf Harofeh Medical Centers
IVF unit. All patients included in this study were high responders,
with serum oestradiol concentration of 2500 pg/ml on the day of
HCG administration and all had 1 embryo available for intrauterine
transfer following IVF/ICSI.
Ovulation induction and oocyte retrieval
Ovulation induction and oocyte retrieval were performed according
to a routine protocol using GnRHa (DTRP
6
, Decapeptyl 3.75 mg
i.m.; Ferring, Malmo, Sweden) suppression, with human menopausal
gonadotrophin (HMG) (Pergonal

; Teva, Petah Tikva, Israel) for

ovarian stimulation. Oocytes were retrieved 36 h after administration
of 5000 IU of HCG (Chorigon

; Teva), by vaginal ultrasound guided

follicular puncture and those at metaphase II were selected for ICSI.
ICSI procedure, embryo transfer and pregnancy evaluation
ICSI was performed according to previously described methodology
(Van Steirteghem et al., 1993) using, preferably, motile spermatozoa.
Fertilization was assessed on the following day, 1618 h post sperm
injection. If two distinct pronuclei were observed, then fertilization
was judged to have occurred. Embryonic cleavage and morphological
quality was assessed ~24 h later and embryo transfer was performed.
Our policy is to limit the number of embryos transferred to three,
except in older women (age 38 years) or in cases with recurrent
failures of implantation.
Luteal support with micronized progesterone
Luteal support
The patients included in this study were prospectively randomized by
order of embryo transfer into two groups of 32 patients each, for luteal
support with micronized progesterone [Utrogestan

; Basins Iscovesco
(CTS), Paris, France] administered either orally (200 mg4/day) or
vaginally (100 mg2/day), starting the day following embryo transfer
(1), until serum HCG measurement 14 days following embryo
transfer. The minimal dosage administered vaginally represented the
dosage used successfully (Salat-Baroux et al., 1988) for luteal support
in cycles following transfer of cryopreservedthawed embryos. Only
clinical pregnancies including sonographic demonstration of a gesta-
tional sac were counted.
Hormonal prole of the luteal phase
The day of the ovulatory stimulus was considered to be day 0 of the
luteal phase. Oestradiol and progesterone concentrations in the serum
were measured by microparticle enzyme immunoassay (MEIA),
performed using the AxSYM system (Dainabot Co., Tokyo, Japan),
an automated immunoassay analyser specically designed to
accommodate MEIA (Smith et al., 1993). This method utilizes a
microparticle coupled with each ligand (oestradiol, progesterone) as
a competitor against the ligand in the sample. The accuracy of
serum progesterone measurement was validated as reported elsewhere
(Momoeda et al., 1998). Blood samples were obtained on the morning
of days 0, 5, 8, 11 and 15. Luteal phase endocrine proles
were analysed in conception and non-conception cycles better to
demonstrate the effect of exogenous administration of progesterone
in the presence and absence of endogenous early concentrations
of HCG.
Statistical analysis
Statistical evaluation was performed using Students t-test,
2
test
and Fishers exact test, where appropriate. Difference was considered
signicant at P 0.05. Results are expressed as mean SD.
Results
The two groups were comparable in age (31.9 6.1 versus
30.6 5.2), number of oocytes retrieved per cycle (17 8.2
versus 18 7.0), and number of embryos transferred per
cycle (3.1 1.2 versus 2.7 0.9), orally versus vaginally
respectively.
Luteal phase endocrine proles including mean concentra-
tions of oestradiol, progesterone and oestradiol/progesterone
ratio in the serum, according to conception and non-conception
cycles in the two treatment groups, are depicted in Figure
1ac. Regarding luteal serum oestradiol concentrations no
signicant differences were noted between the groups. Serum
oestradiol concentrations decreased constantly until day 11
in all groups, with no signicant difference, although following
oral treatment a tendency for lower oestradiol concentrations
was noted both in conception and non-conception cycles. After
day 11, oestradiol concentrations increased in conception
cycles, in both treatment groups, probably reecting the rise
in HCGconcentrations affecting corpus luteumfunction. Luteal
serum progesterone concentrations declined gradually with no
signicant difference between the groups until day 8 post HCG
administration. Later on, in conception cycles, progesterone
concentrations increased, probably reecting the rise in endo-
genous HCG concentrations stimulating the corpora lutea,
with no signicant difference between the treatment groups.
1945
Comparing non-conception cycles at day 11 and day 15,
signicantly higher (P 0.032) serum progesterone concentra-
tions were found using 800 mg micronized progesterone and
oral compared to vaginal treatment of 200 mg micronized
progesterone. Oestradiol/progesterone ratios reected the
serum oestradiol concentrations, being higher from day 8
onwards after vaginal treatment but with no signicant differ-
ence between conception and non-conception cycles.
The clinical outcome in the two treatment groups is described
in Table I. Asignicantly higher (P 0.01) rate of implantation
was observed following luteal support administered vaginally
compared with oral treatment, although the differences in the
pregnancy, miscarriage and ongoing pregnancy rates were not
signicant statistically.
No side-effects resulting from vaginal administration of
micronized progesterone, such as local intolerance or pruritus,
were reported in this study. Following oral treatment two
patients complained of somnolence.
Discussion
Ovarian stimulation cycles preceded by GnRHa down-
regulation necessitate luteal phase supplementation (Smitz
et al., 1988; Smith et al., 1989; Bellaisch-Allart et al., 1990;
Buvat et al., 1990; Herman et al., 1990). This prevents
luteal phase insufciency caused by prolonged suppression of
gonadotrophin excretion leading to insufcient stimulation of
the corpora lutea and impairment in production of late luteal
oestradiol and progesterone (Calogero et al., 1987; Smitz et al.,
1987, 1988). Recently, it was suggested that luteal phase
deciency may result not from insufcient gonadotrophin
stimulation but from failure of a malfunctioning corpus luteum
to react to stimulation and produce progesterone (Momoaeda
et al., 1998). However, this study was conducted in natural
cycles, whereas after ovulation induction, when pituitary down-
regulation precedes ovarian stimulation, insufcient gonadotro-
phin stimulation seems a more plausible aetiology for the
luteal phase insufciency. Signicant improvement of all
endometrial features showing impairment of progesterone
bioavailability (i.e. endometrial histological maturation
assessed by histology, ultrastructure by electron microscopy
and oestradiol and progesterone receptor distribution by
immunocytochemistry) has been reported after luteal phase
supplementation of progesterone (Bourgain et al., 1994). Luteal
phase supplementation by progesterone increased the preg-
nancy rate signicantly following IVF according to a meta-
analysis (Soliman et al., 1994). Vaginal administration leads
to lower serum concentrations of progesterone but has fewer
side-effects compared to its administration either orally or
progesterone in oil by i.m. injection (Miles et al., 1994).
Principal metabolites found in blood following oral
administration of micronized progesterone are 17-alpha-hydro-
xyprogesterone, 11-desoxycorticosterone and 20-dihydro-
progesterone. Vaginal administration causes synchronous
secretory maturation in the endometrium and elicits only minor
changes in plasma concentrations of psychotropic metabolites,
such as 5-alpha and 5-beta-pregnanolone, which are known to
act directly on GABA receptors of the central nervous system
S.Friedler et al.
Figure 1. Luteal phase endocrine prole in conception and non-conception cycles using luteal support with micronized progesterone
administered orally or vaginally. (a) Luteal phase serum oestradiol. (b) Luteal phase serum progesterone. (c) Luteal phase serum
oestradiol(E2)/progesterone(P) ratio.
Table I. Clinical outcome of intracytoplasmic sperm injection, according to luteal support administered
vaginally or orally
Orally Vaginally P
a
value
Implantation rate (%) 10/93 (10.7) 28/91 (30.7) 0.01
Pregnancy rate/embryo transfer 10/30 (33.0) 16/34 (47.0) NS
Miscarriages (%) 4/10 (40.0) 2/16 (12.5) NS
Ongoing pregnancy rate/embryo transfer 6/30 (20.0) 14/34 (41.1) NS
a
By Fishers exact test.
NS not signicant.
(CNS) (sedative/hypnotic, anxiolytic or anti-epileptic effects).
The notion of the rst uterine pass effect, referring to a
mechanism of direct vagina to uterus transport of progesterone,
was suggested by De Ziegler (De Ziegler et al., 1994, 1995)
and has gained further support in the literature since then
(Miles et al., 1994; Balasch et al., 1996; Buletti et al.,
1946
1997; Fanchin et al., 1997; Cicinelli et al., 1998). The exact
mechanism of transport is not yet claried; however in an
ex-vivo uterine perfusion model a time dependent, wave-like
progression of
14
C-progesterone from cervix to fundus was
observed reminiscent of passive diffusion (Buletti et al., 1997).
A countercurrent distribution mechanism could be involved
Luteal support with micronized progesterone
with diffusion from vaginal/uterine vein to artery, as
progesterone concentrations in the uterine artery were found
to be higher than in the radial artery following vaginal
administration (Cicinelli et al., 1998).
The optimal effective dosage for luteal support has not
been established yet. In women lacking ovarian function and
receiving oestradiol, even a dose of progesterone as low as
45 mg/48 h, as given by a sustained release vaginal gel,
caused complete secretory changes in endometrial glands
(day 20) and stroma (day 24) as indicated by endometrial
biopsies. This was despite low (15 ng/ml) plasma progester-
one concentrations (Fanchin et al., 1997). In the context of
clinical luteal phase support following IVF, use of between
200 and 600 mg progesterone/day has been reported.
Endometrial preparation for cryopreservedthawed embryo
transfer was successfully performed using a protocol com-
bining oestrogen (transdermal and oral preparations) with
micronized progesterone given vaginally at a daily dose of
100 mg (Salat-Baroux et al., 1988). The efcacy of 400 mg/day
micronized progesterone administered orally or vaginally has
been compared with HCG injected every third day following
embryo transfer at a dose of 1500 IU (Buvat et al., 1990).
When serum peak oestradiol concentration was 2700 pg/ml,
implantation rate was 56 times higher with vaginal administra-
tion, compared to oral treatment (26 versus 5%). Pregnancy rate
and implantation rate with vaginal treatment were both similar
to those following HCG injections.
Further studies comparing natural progesterone adminis-
tered i.m. or vaginally reported a signicantly lower early
spontaneous abortion rate, resulting in higher ongoing preg-
nancy rate/embryo transfer following vaginal administration.
However, the daily dosage of natural progesterone used in
these studies was 600 mg/day micronized progesterone given
vaginally compared with 50 mg/day progesterone in oil given
i.m. Vaginal administration resulted in a better ongoing preg-
nancy rate (30.5 versus 19.1%) as well as a lower rate of early
spontaneous abortions (2.9 versus 9.1%) (Devroey et al., 1991;
Smitz et al., 1992b).
Another study compared luteal support using 90 mg
progesterone gel administered vaginally with 300 mg/day
micronized progesterone taken orally and found no difference
regarding pregnancy rate/embryo transfer, rate of spontaneous
abortions or number of babies born per embryos replaced
(Pouly et al., 1996).
Therefore we examined the efciency of this low daily
dosage of micronized progesterone in the context of luteal
support following ovulation induction using GnRHa/HMG.
Our results indicate that supplementation of luteal phase
with a low dose micronized progesterone absorbed vaginally,
100 mg twice daily, leads to signicantly higher embryo
implantation rates compared to oral ingestion. In these cycles
the endometrium was receptive in spite of lower plasma
progesterone concentrations, representing probably higher
local tissue progesterone concentrations achieved by vaginal
administration, and reecting the strong uterine rst-pass effect
(Cornet et al., 1991; De Ziegler et al., 1994, 1995; Miles
et al., 1994; Balasch et al., 1996; Buletti et al., 1997; Fanchin
et al., 1997; Cicinelli et al., 1998).
1947
The luteal endocrine prole in our patients is similar to
those reported elsewhere (Smitz et al., 1987; Herman et al.,
1996), showing a decline in oestradiol and progesterone
production until day 8 post-HCG. Later on, probably due
to endogenous secretion of HCG in conception cycles, the
corpora lutea are stimulated to produce further oestrogen and
progesterone indicated by the rise in their serumconcentrations.
Our data show clearly that, in spite of lower serum progesterone
concentrations following vaginal administration, the concep-
tion rate is not impaired. Indeed the superior clinical outcome
obtained using a lower dose of micronized progesterone
administered vaginally indicates better bioavailability of
progesterone absorbed vaginally.
Specic techniques should be used to determine plasma
concentrations of progesterone after oral administration, since
progesterone metabolites which are produced in signicant
amounts may interfere with the radioimmunoassay (Nahoul
et al., 1987, 1993), thus causing difculty in comparing the
oral and the vaginal administration of micronized progesterone.
The method of plasma progesterone measurement used in this
study is based on MEIA, a technique that minimizes cross-
reaction with the metabolites of orally ingested progesterone,
allowing reliable comparison of plasma progesterone con-
centrations between the two treatment groups.
The tendency to lower late luteal phase oestradiol concentra-
tions following oral administration could reect a different
pattern/rate of steroid metabolism in this group. As conceptions
have occurred over a wide range of luteal phase oestradiol/
progesterone serum ratio, this parameter may not have a clear
predictive value regarding establishment of conception. This
nding concurs with previous reports showing that endometrial
morphology is not altered by extreme shifts in luteal phase
oestradiol concentrations and oestradiol/progesterone ratios
(De Ziegler et al., 1992, 1993).
As low dose micronized progesterone absorbed vaginally is
an easy, well tolerated and simple method of luteal support, it
could be recommended as the method of choice for luteal
support, especially for high responder patients at risk for OHSS.
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Received on November 19, 1998; accepted on May 4, 1999