FERTILITY AND STERILITY®
`VOL. 71, NO. 4, APRIL 1999
`Copyright ©1999 American Society for Reproductive Medicine
`Published by Elsevier Science Inc.
`Printed on acid-tree paper in USA.
`
`Oral versus intramuscular progesterone for
`in vitro fertilization: a prospective
`randomized study
`
`Frederick L. Licciardi, M.D., Andrea Kwiatkowski, B. S.N., R.N.C.,
`Nicole L. Noyes, M.D., Alan 8. Berkeley, M.D., Lewis L. Krey, Ph.D., and
`Jamie A. Grifo, M.D., Ph.D.
`
`Program for In I/itro Fertilization, Reproductive Surgery and Infertility, Department of Obstetrics and
`Gynecology, New York University School of Medicine, New York, New York
`
`Objective: To evaluate the efficacy of oral micronized progesterone compared with 1M progesterone in oil
`for luteal support in patients undergoing IVF who are treated with a GnRH agonist.
`Design: Randomized prospective clinical trial.
`Setting: University-based IVF center.
`Patient(s): Women <40 years of age who were undergoing IVF with luteal GnRH pituitary down-regulation.
`Inter'vention(s): Patients were randomized to receive either oral micronized progesterone (200 mg three
`times daily) or IM progesterone (50 mg daily).
`Main Outcome Measure(s): Progesterone levels at standardized days 21 and 28, and pregnancy and embryo
`implantation rates.
`Result(s): Day 21 progesterone levels were 77.6 : 13.2 ng/mL in the IM group and 81.5 : 16.2 ng/mL in
`the oral group. Day 28 progesterone levels were 76.3 : 15.0 ng/mL in the IM group and 53.6 : 10.1 ng/mL
`in the oral group. The clinical pregnancy rates were 57.9% and 45.8% for the IM and oral groups, respectively.
`The implantation rate per embryo was significantly higher in the IM group (40.9%) than in the oral group
`(1 8. 1%).
`C0nclusi0n(s): When used according to our protocols, oral progesterone and IM progesterone result in
`comparable levels of circulating progesterone. However, oral progesterone results in a reduced implantation
`rate per embryo. (Fertil Steril® 1999',71:614—8. ©1999 by American Society for Reproductive Medicine.)
`Key Words: Oral micronized progesterone, in vitro fertilization, implantation
`
`Progesterone supplementation of the luteal
`phase is prescribed routinely for women under-
`going IVF. The most common routes of admin-
`istration are IM injection and vaginal supposi-
`tory. Progesterone delivered by IM injection
`can lead to marked inflammation at the injec-
`tion site, resulting in redness, pain, and even
`sterile abscess formation. Although vaginal
`suppositories are easier to tolerate, the suppos-
`itory material may escape from the vagina,
`leading to inconvenience and uncertainty as to
`the dosage of progesterone absorbed. Proges-
`terone taken orally would avoid these potential
`complications. This prospective randomized
`study examined the use of oral micronized pro-
`gesterone for luteal support after IVF and com-
`pared its efficacy to that of IM progesterone.
`
`MATERIALS AND METHODS
`
`Patients were recniited through signs placed
`in the waiting areas at our clinic that explained
`the study. The inclusion criteria were the use of
`GnRH down-regulation and age <40 years.
`Patients were assigned to receive either IM or
`oral progesterone supplementation according
`to a randomization table. The protocol was
`approved by the institutional board of research,
`and all patients gave informed consent before
`entering the study.
`
`Patients were prescribed either progesterone
`in oil (50 mg IM daily) or micronized proges-
`terone (200 mg orally three times daily) begin-
`ning on day 15 of an IVF cycle. The day of
`oocyte retrieval was normalized to day 14, and
`
`InnoPharma Exhibit 1095.0001
`
`Received July 20, 1998;
`revised and accepted
`November 2, 1998.
`Presented at the 54th
`Annual Meeting of the
`American Society for
`Reproductive Medicine,
`San Francisco, California,
`October 4-9, 1998.
`Reprint requests: Frederick
`L. Licciardi, M.D., Program
`for In Vitro Fertilization,
`Reproductive Surgery and
`Infertility, Department of
`Obstetrics and
`Gynecology, New York
`University School of
`Medicine, 660 First
`Avenue, 5th Floor, New
`York, NY 10016 (FAX: 212-
`265-7853).
`
`0015-0282/99/$20.00
`PII S0015-0282(98)00515-9
`
`614
`
`

`

`semm samples for progesterone were drawn in the morning
`on days 21 and 28, before the administration of any medi-
`cations on those days. All patients underwent ovarian stim-
`ulation using luteal phase GnRH down-regulation followed
`by stimulation with IM FSH, hMG, or a combination of FSH
`and hMG. Embryo transfers were performed on day 3 after
`oocyte retrieval. Embryos were graded on a scale of 1-4,
`with 1 being the highest quality. Deductions in grade scores
`were based on blastomere asymmetry and increasing degrees
`of fragmentation.
`
`All the oral progesterone was supplied by a single source.
`Each 200-mg oral capsule contained 200 mg of micromzed
`progesterone Umted States Pharmacopeia and 140 mg of
`methocel E4M. After the two powders were titrated together
`using geometric dilution, they were placed in a size-zero
`gelatin capsule (Eli Lilly, Indianapolis, IN).
`
`Progesterone assays were performed using the Immulyte
`system. The intra-assay and interassay coefficients of varia-
`tion were 8. l%—l3% and 6.9%—l3%, respectively. The man-
`ufacturer of this progesterone assay system has not per-
`formed an evaluation of cross-reactivity with the most
`abundant metabolites of oral progesterone, 501- and 5(3-
`pregnanolone. We therefore tested for cross-reactivity by
`spiking 5-ng/mL progesterone standards with 30, 120, and
`240 ng/mL of each of these two progestins.
`
`Statistical comparisons were made using a standard soft-
`ware program. The Mann-Whitney U test was used for
`comparison of numbers and levels. Rates were compared
`using the X2 test, with the Yates’ correction when necessary.
`For smaller groups, Fisher’s exact test was used. A P value
`of <.05 was considered statistically sigmficant.
`
`RESULTS
`
`Nineteen patients received IM progesterone and 24 pa-
`tients received oral progesterone. There were no statistically
`sigmficant differences between the two groups in mean age,
`response to stimulation, or retrieval outcome. Moreover, the
`number and quality of the embryos and the climcal preg-
`nancy rate were not sigmficantly different between the two
`groups (Tables 1 and 2). There were two miscarriages in the
`oral group, one of which was chromosomally abnonnal, and
`no miscarriages in the M group.
`
`A statistically sigmficant difference was observed in the
`implantation rate per embryo. The patients who received oral
`progesterone demonstrated a greater than twofold lower im-
`plantation rate per embryo compared with the patients who
`received IM progesterone (Table 2). This lower implantation
`rate was reflected in a lower multiple pregnancy rate in the
`oral group. We imtially intended to enroll more patients;
`however, the study was terminated for ethical reasons be-
`cause the differences in implantation rates were highly sta-
`tistically sigmficant.
`
`FERTILITY & STERILITY®
`
`I
`
`
`
`1
`
`Intramuscular versus oral progesterone for IVF: comparison
`of stimulation data.
`
`Route of administration
`
`Variable
`
`IM (11 = 19)
`
`P0 (11 = 24)
`
`34.5 : 0.57
`11.9 : .275
`
`1,964 : 230.3
`15.8 : 1.43
`10.8 : 1.20
`3.47 : .193
`
`Age (y)
`Day of hCG administration
`E2 level on day of hCG
`administration
`No. of oocytes
`No. of embryos
`No. of embryos transferred
`No. of highest-quality
`1.45 : .083
`1.64 : .095
`embryos
`1.91 : .072
`1.86 : .099
`Mean embryo quality
`Note: Values are means : SEM. All differences were not statistically
`significant. P0 = oral.
`
`34.9 : .075
`11.3 : .502
`
`1,770 : 173.2
`13.7 : 1.10
`10.1 : .867
`3.46 : .170
`
`Overall, there was no difference in the circulating pro-
`gesterone levels between the IM and oral groups. This re-
`mained true when we examined the subgroup of patients
`who were not pregnant and therefore did not have the en-
`hanced progesterone level that accompames a pregnancy. It
`was of interest, however, that on day 28, four patients in the
`oral group, none of whom were pregnant, had progesterone
`levels of <20 ng/mL (Table 3). Patients with E2 levels of
`>l,000 pg/mL were given 5,000 U of hCG, and those with
`lower levels were given 10,000 U. There were no differences
`in progesterone levels, implantation rates, or pregnancy rates
`between the two dosage groups.
`
`There was little cross-reactivity of 501- and 5B-preg-
`nanolone in our progesterone assay. The 3 compound was
`slightly more reactive than the or compound, but the maxi-
`mum cross-reactivity was 1.3% (Table 4).
`
`
`
`Intramuscular versus oral progesterone for IVF: differences
`in pregnancy results.
`
`Route of administration
`
`Variable
`
`IM (11 = 19)
`
`P0 (11 = 24)
`
`11/19 (57.9)
`
`No. of clinical pregnancies/no. of
`oocyte retrievals (%)
`No. of patients with multiple
`implantation/total no. of
`pregnant patients (%)
`No. of higher-order multiple
`implantations
`Implantation rate per embryo (%)
`Note: P0 = oral.
`* Three sets of quadruplets and one set of triplets.
`T P = .004 (versus IM).
`
`9/11 (81.8)
`
`4*
`40.9
`
`11/24 (45.8)
`
`4/11 (36.3)
`
`0
`18.1T
`
`615
`
`lnnoPharma Exhibit 1095.0002
`
`

`

`
`
`Intramuscular versus oral progesterone for IVF: differences
`in circulating progesterone levels (mg/mL).
`
`Route of administration
`
`IM
`
`P0
`
`19
`77.6 : 13.2
`76.3 : 15.0
`
`8
`54.3 : 6.87
`28.5 : 2.29
`
`24
`81.5 : 16.2
`53.6 : 10.1
`
`13
`66.2 : 19.63
`28.9 : 5.06
`
`1 (19.0)
`
`2 (10.8, 16.7)
`
`Variable
`
`All patients
`No. of patients
`Mean -- SEM P4 level on day 21
`Mean -- SEM P4 level on day 28
`Nonpregnant patients only
`No. of patients
`Mean -- SEM P4 level on day 21
`Mean -- SEM P4 level on day 28
`No. of patients with P4 level of
`<20 ng/mL on day 21 (P4
`levels of individual patients)
`No. of patients with P4 level of
`<20 ng/mL on day 28 (P4
`levels of individual patients)
`
`4 (5.2, 9.0, 9.1,
`18.0)
`Note: Differences between groups were not statistically significant.
`
`1 (18.0)
`
`DISCUSSION
`
`This prospective randomized study demonstrated that oral
`progesterone is associated with a significantly lower implan-
`tation rate per embryo compared with 1M progesterone when
`it is used for luteal support for IVF. This difference was
`observed without differences in circulating progesterone
`levels.
`
`Over the past two decades, exogenous gonadotropins
`have been used to induce ovulation in infertile patients, and
`supplemental luteal progesterone has been used in an attempt
`to improve uterine receptivity in such cycles. The scientific
`foundation for progesterone use is that elevated luteal levels
`of estrogen decrease the incidence of embryo implantation,
`which is one proposed mechanism of postcoital hormonal
`contraception. Using the mouse model, it has been demon-
`strated clearly that increases in the progesterone-to-estrogen
`ratio negate the effects of estrogen alone on the endometrium
`and allow for implantation (1). Therefore, the high levels of
`E2 that are produced during ovarian hyperstimulation may
`interfere with implantation unless supplemental progester-
`one is given.
`
`Despite this theoretic benefit of luteal progesterone, and
`its wide use for such purposes, randomized studies have not
`shown that progesterone supplementation of gonadotropin-
`induced cycles (IVF or other) improves pregnancy rates in
`humans (2, 3). The situation is different, however, in gonad-
`otropin treatment cycles that use GnRH pituitary down-
`regulation, because luteal phases become short (4) and IVF
`pregnancy rates are at least half as high as when progester-
`one supplements are not used (4, 5). Progesterone is neces-
`
`sary in this situation because GnRH agonists cause prema-
`ture luteolysis (6, 7) by suppressing pituitary release of
`gonadotropins for up to 12 days after their discontinuation.
`In addition, GnRH agonists result in a decrease in the num-
`ber of LH receptors found on granulosa cells, and they have
`the direct effect of suppressing granulosa cell E2 and pro-
`gesterone production (8).
`
`The methods most often used to increase progesterone
`levels in IVF cycles include hCG administration and proges-
`terone supplementation. Human chorionic gonadotropin in-
`creases both E2 levels and progesterone levels, whereas
`progesterone has no effect on E2 levels (9). However, both
`have been shown to be equally effective in supporting the
`luteal phase of patients undergoing IVF who are treated with
`a GnRH agonist, as measured by pregnancy rates (4).
`
`More recently, oral micronized progesterone has been
`evaluated for use as luteal support (10). Preliminary infor-
`mation about luteal oral progesterone has been derived from
`studies that examined the use of oral progesterone in post-
`menopausal hormone replacement therapy (11). Substituting
`natural progesterone for the synthetic progestins commonly
`used in hormone therapy would have the advantage of avoid-
`ing the androgenic and psychotropic side effects that are
`associated with those medications. Oral progesterone ini-
`tially was regarded as clinically ineffective because of poor
`intestinal absorption and rapid metabolism caused by the
`intestinal mucosa, intestinal flora, and a first-pass effect from
`the liver.
`
`in-
`Micronizing progesterone (creating microspheres)
`volves processing the compound into a fine powder and
`suspending it in an oil carrier, increasing its bioavailability.
`Despite micronization, the intestinal absorption of oral pro-
`gesterone is limited. Thus, the actions of the intestines and
`liver, coupled with limited absorption, result in a level of
`bioavailability that has been reported to be < 10% (12). This
`is increased somewhat when the drug is taken with food (12).
`
`Low circulating progesterone levels seem to be sufficient
`to counter the negative effects of estrogen replacement on
`the endometrium (13, 14). Histologic examination reveals
`
`
`
`Cross—reactivity of the lmmulite progesterone assay with
`5a— and 53—pregnano|one.
`
`Progesterone assay cross-reactivity at 5 ng/mL
`
`Concentration
`added (ng/mL)
`
`50¢-pregnanolone
`(%)
`
`5 B-pregnanolone
`(%)
`
`30
`120
`240
`
`0.67
`0.60
`0.60
`
`1.3
`1. 1
`0.7
`
`* A 0.67% rate of cross-reactivity means that 0.67% of 30 ng/mL of
`50¢-pregnanolone was measured as progesterone.
`
`616
`
`Licciardi et al.
`
`Oral Versus IM progesterone for IVF
`
`Vol. 71, No. 4, April 1999
`
`|nnoPharma Exhibit 1095.0003
`
`

`

`that the epithelium may not be converted to a secretory
`pattern; however, glandular cells do show mitotic arrest, and
`therefore hyperplasia is halted. Certainly, for infertility ther-
`apy, secretory conversion is desired. Therefore, increasing
`the dosage and decreasing the dosing interval should allow
`for adequate endometrial progesterone exposure.
`
`When progesterone is given orally, progesterone levels
`have been reported to peak at approximately 2 hours, with a
`following half-life of approximately 2 hours (15). In addi-
`tion, the absorption of oral progesterone shows considerable
`intersubject variability, so that some patients have high se-
`mm levels and others have low levels after taking the same
`dose (16). We attempted to overcome these potential prob-
`lems by using a dosing interval of 8 hours, rather than the
`standard 12 hours, and by using a novel sustained-release
`methylcellulose vehicle. Methylcellulose forms a matrix
`around compounds that protects against stomach degrada-
`tion, creating a sustained-release effect in the small intestine
`and aiding the absorption of the medication.
`
`The use of oral progesterone for luteal support in patients
`undergoing IVF who are taking GnRH has been limited. A
`poor outcome was reported by Buvat et al. (17), who dem-
`onstrated that oral micromzed progesterone in oil (100 mg at
`8 A.M., 100 mg at noon, and 200 mg at 8 P.M.) produced a
`climcal pregnancy rate of 23% and an implantation rate per
`embryo of 7.5%, compared with rates of 45% and 19%,
`respectively, for IM progesterone (the difference was statis-
`tically sigmficant). However, Pouly et al. (10) reported that
`oral progesterone (100 mg in the morning and 200 mg in the
`evemng) resulted in a climcal pregnancy rate of 25% and an
`implantation rate of 29.9%, compared with rates of 28.8%
`and 35.3%, respectively, for vaginal progesterone gel (the
`difference was not statistically sigmficant). The rates in our
`study are consistent with those of Pouly et al. (10).
`
`Although there were overall differences in progesterone
`levels with the two routes of admimstration, and no differ-
`ences in pregnancy rates, the number of patients in our study
`was too small to detect statistically sigmficant differences in
`these areas. Overall, morning levels of progesterone were
`not different between the two groups, although the lowest
`levels were found in those patients who received the oral
`drug.
`
`The rapid degradation of orally admimstered progester-
`one results in a high concentration of circulating metabolites,
`including deoxycorticosterone, estrone, and E2. The most
`common metabolites, 5a and 5B reduced pregnanolones,
`circulate in concentrations that are higher than that of pro-
`gesterone itself (15, 18). Earlier assays of progesterone have
`measured these two compounds, resulting in erroneous ele-
`vations of the perceived circulating progesterone levels. Our
`testing showed that these compounds were not detected by
`the Immulite assay system, so that we believe that we ob-
`tained an accurate impression of the levels of circulating
`progesterone.
`
`We can only guess at the cause of oral progesterone’s
`negative effects on embryo implantation. Overall, progester-
`one levels were not lower in the oral group; however, this
`group did contain the patients with the lowest progesterone
`levels. The progesterone metabolites, circulating at high
`levels, may bind to progesterone receptors and interfere with
`normal progesterone action by interfering with transcription
`cofactor or DNA binding. Alternatively,
`the 5d and 5B
`reduced pregnanolones are known to have high affimty for
`'y-aminobutyric acid receptors (19). Such receptors are
`present in the reproductive tract (20), and their activation
`may adversely affect pregnancy outcome.
`
`Although we imtially intended to enroll a larger number
`of patients in each group of the study, a disparity in the
`implantation rates led us to end the study prematurely for
`ethical reasons. The small number of patients lowers the
`power of this study and restricts our ability to comment on
`the usefulness of oral progesterone. Because there was a
`large difference between the two groups, however, we be-
`lieve that we are justified in concluding that the difference in
`the implantation rate per embryo is significant when our
`protocols and method of admimstering oral micromzed pro-
`gesterone are used.
`
`Acknowledgments The authors thank Mortimer Levitz, M.D., and the staff
`of his laboratory for their assistance and expertise in performing the hor-
`monal immunoassays.
`
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`618
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`Licciardi et al.
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`Oral Versus IM progesterone for IVF
`
`Vol. 71, No. 4, April 1999
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`|nnoPharma Exhibit 1095.0005
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