Effect of the Yuzpe regimen of emergency contraception on markers of endometrial receptivity

Elizabeth G. Raymond1,5, Laurie P. Lovely2, Mario Chen-Mok1, Markku Seppälä3, Robert J. Kurman4 and Bruce A. Lessey2

1 Family Health International, Research Triangle Park, NC, 2 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, NC, USA, 3 Department of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland and 4 Departments of Gynecology and Obstetrics and of Pathology, Johns Hopkins Hospital, Baltimore, MD, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This exploratory study was designed to determine whether treatment with the Yuzpe regimen of emergency contraception altered endometrial integrin expression or other markers of uterine receptivity. Nineteen parous women were followed for two menstrual cycles. In the second cycle, each participant took 100 mg ethinyl oestradiol and 1 mg norgestrel on the day of the urinary luteinizing hormone (LH) surge and repeated the dose 12 h later. In both cycles, endometrial biopsy, phlebotomy and vaginal sonogram were performed 8–10 days after the urinary LH surge. No significant difference was found between untreated and treated cycles in most measures of endometrial histology or in endometrial expression of ß3 integrin subunit, leukaemia inhibitory factor, glycodelin, or progesterone receptors assessed by immunohistochemical techniques. Five statistically significant changes were noted in treated cycles: a reduction in endometrial MUC-1 expression, an increase in endometrial oestrogen receptor, lower luteal phase serum oestrogen concentration, reduced endometrial thickness, and greater proportion of glandular supranuclear vacuoles. The relationship of these findings to the contraceptive action of the Yuzpe regimen is unclear.

Key words: emergency contraception/endometrial receptivity/integrins/leukaemia inhibitory factor/oestrogen receptors


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although the Yuzpe regimen of emergency contraception was first described a quarter of a century ago (Yuzpe et al., 1974Go), the mechanism by which it achieves its contraceptive effect remains unclear. Delay or absence of ovulation has been documented in some women after treatment (Ling et al., 1983Go; Rowlands et al., 1986Go; Swahn et al., 1996Go) but this effect appears insufficient to explain fully the observed efficacy of the regimen (Trussell and Raymond, 1999Go). Another theory is that the regimen disrupts maturation of the endometrium and consequently inhibits implantation of the fertilized ovum. Several studies have shown that treatment causes out-of-phase or asynchronous development of endometrial glands and stroma in some patients (Yuzpe et al., 1974Go; Ling et al., 1979Go, 1983Go), and that use, shortly after ovulation, results in a decrease in the endometrial concentrations of oestrogen and progesterone receptors (Kubba et al., 1986Go). However, other research has failed to confirm these findings (Swahn et al., 1996Go). Furthermore, it is not clear whether these effects would be sufficient to prevent implantation.

To investigate this issue further, we designed this exploratory study to evaluate the effect of the Yuzpe regimen on a panel of endometrial proteins that have recently been identified as possible mid-luteal phase markers of receptivity to implantation. The best characterized of these proteins is the ß3 subunit of {alpha}vß3 integrin. This cell adhesion molecule is one of several integrins that appear in the endometrium during the mid-secretory phase (Lessey et al., 1992Go, 1994Go). Its expression has been reported to be reduced or delayed in some women with various types of infertility, including those with endometrial histological delay (Lessey et al., 1992Go), in some infertile women with grossly normal endometrial histology (Lessey et al., 1995Go), and in women taking oral contraceptives (Somkuti et al., 1996Go). We also measured leukaemia inhibitory factor (LIF), glycodelin (PP14, PEP), the cell surface mucin MUC-1, oestrogen receptor (ER), and progesterone receptor (PR). LIF and glycodelin normally reach maximal concentrations in the endometrium in the mid-luteal phase, and some studies have suggested that production of these proteins is also reduced in some infertile women and in women with out-of phase endometria (Klentzeris et al., 1994Go; Hambartsoumian, 1998Go). Low serum concentrations of glycodelin have also been found in the luteal phase of women with retarded endometrial development (Joshi et al., 1986Go; Klentzeris et al., 1994Go) and women with recurrent pregnancy loss (Tulppala et al., 1995Go). In contrast, ER and PR normally decrease at the time of expected embryo implantation (Lessey et al., 1988Go., 1996Go). Abnormal persistence of PR appears to be associated with infertility (Lessey et al., 1996Go). MUC-1 is present during the mid-secretory phase of the cycle, but its role in the implantation process in humans remains uncertain. In animals, this protein has been thought to inhibit implantation, but in women, it is most abundant during the mid-secretory phase of the cycle (Aplin, 1997Go).

Along with these biochemical markers, we assessed other characteristics of the endometrium that may be associated with uterine receptivity. These included endometrial echogenicity as measured by ultrasound, which has been associated with out-of-phase endometria as defined by biopsy (Grunfeld et al., 1991Go), and endometrial thickness. We also measured the density of ultrastructural elements called pinopodes in the endometrium, using scanning electron microscopy. Pinopodes appear on the surface of endometrial epithelial cells during the mid-luteal phase of most natural menstrual cycles but appear early in stimulated cycles (Nikas and Psychoyos, 1997Go).

Our premise was that a demonstrable effect of the emergency contraception regimen on these markers would constitute indirect evidence that the regimen may render the endometrium unable to accept implantation of an embryo.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
This study was approved by the Institutional Review Boards at Family Health International and the School of Medicine at the University of North Carolina at Chapel Hill. All participants gave signed informed consent.

Parous women aged 18–35 years who had normal menstrual cycles, who had no contra-indications to the use of oral contraceptives, and who were using a non-hormonal method of contraception (but not an intrauterine device) were invited to participate in the study. Data were not collected on number of previous deliveries, miscarriages or history of infertility. Each woman was followed for two consecutive menstrual cycles. In both cycles, she was instructed to monitor her urine daily starting on the 11th day of the cycle using a commercially available luteinizing hormone (LH) detection kit (Ovuquik®; Quidel, San Diego, CA, USA). In the first study cycle, no treatment was given. In the second cycle, she was told to take two pills each containing 50 mg ethinyl oestradiol and 0.5 mg norgestrel (Ovral®; Wyeth Ayerst, Philadelphia, PA, USA) on the day she detected LH in her urine and to repeat this dose 12 h later. This day corresponds approximately to the day before or the day of ovulation (Miller and Soules, 1996Go) and was chosen because the regimen should be expected to have a contraceptive effect when administered on this day. In both cycles, participants returned 8, 9, or 10 days after the LH surge for endometrial biopsy, vaginal sonogram, and phlebotomy.

Immunohistochemistry
The ß3 integrin subunit, LIF, MUC-1, glycodelin, glandular ER{alpha} and glandular PR were assayed by immunohistochemical techniques as previously reported (Lessey et al., 1995Go, 1996Go). The antibodies used for these studies included: Mab SSA6 (ß3) (Lessey et al., 1992Go), Mab N-18 (LIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal rabbit antibody CT-1 (MUC-1; Daniel Carson, Newark, DE, USA), Mab HC-20 (ER{alpha}; Santa Cruz Biotechnology), Mab B39 (PR; Abbott Laboratories, Chicago, IL, USA), and affinity-purified polyclonal antiglycodelin IgG, 6 µg/ml (rabbit) (Kamarainen et al., 1996Go). The slides were stained in a single batch to eliminate interassay variability. For control stainings, the same isotype, non-immune rabbit IgG (6 µg/ml) was used. Normal secretory endometrium constituted a positive control tissue, exhibiting strong staining with active antiglycodelin IgG and no staining with the non-immune rabbit IgG tested at the same concentration.

Endometrial biopsies were snap frozen on liquid nitrogen and stored at –80°C until use. Immunoperoxidase staining was performed on 5 µm cryosections. Tissue sections were placed onto poly-L-lysine coated slides, fixed in –20°C acetone for 10 min, and stained using Vectastain Elite® ABC kits (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine (DAB; Sigma Chemical Co., St Louis, MO, USA) was used as the chromagen. Primary antibody was placed on cryosections following blocking with 1% bovine serum albumin in phosphate buffered saline (PBS) and allowed to bind at room temperature for 1 h. A PBS pH 7.2–7.4 rinse was followed by secondary antibody consisting of biotinylated goat anti-mouse antibody for 30 min. Following a PBS rinse, the endogenous peroxidases were quenched with a 30 min incubation with 0.3% H2O2 in absolute ethanol, followed by a 30 min rehydration in PBS. Avidin:biotinylated horseradish peroxidase macromolecular complex (ABC) was then incubated on the sections for 30 min before adding diaminobenzidine for 3 min to complete the reaction. Samples were subsequently washed in PBS and mounted. The resulting staining was evaluated on a Nikon microscope, and photomicrographs were made using Kodak 100 ASA film.

Immunohistochemistry was performed on each of the biopsies and the results were determined by a single blinded observer (B.A.L.). For this analysis, staining intensity was assigned using a semi-quantitative HSCORE as previously described (Lessey et al., 1995Go). The HSCORE was calculated using the following equation:


where i = intensity of staining with a value of 1, 2, or 3 (weak, moderate or strong respectively) and Pi is the percentage of stained epithelial cells for each intensity, varying from 0 to 100%.

Electron microscopy
Fixation
Biopsy samples were rinsed gently in PBS to remove blood and tissue debris before fixation. Samples were placed in a fixative containing 2% paraformaldehyde, 2.5% glutaraldehyde in 0.15 mol/l sodium phosphate buffer, pH 7.4, and stored at 4°C overnight or until processed. After fixation, each sample was rinsed three times for 10 min each rinse with 0.15 mol/l Karlsson and Schultz sodium phosphate buffer, pH 7.4. Post-fixation of biopsies was performed in 1% osmium tetroxide in the same buffer for 1 h.

Dehydration and critical point drying
After osmium post-fixation, the samples were rinsed three times with deionized water and dehydrated through a graded series of ethanols (30%, 50%, 75%, 95%, 100%). Following dehydration, the samples were dried in the Balzers CPD 020 Critical Point Drier (Balzers Union Ltd, Principality of Liechtenstein) using liquid carbon dioxide as the transition fluid. The dried endometrial biopsies were mounted on aluminium scanning electron microscope (SEM) stubs with colloidal silver paste, allowing the silver paste to dry, then sputter coated with gold-palladium alloy (60:40) to a thickness of 20 nm using the Hummer X Sputter Coater® (Anatech Ltd, Alexandria, VA, USA).

Scanning electron microscopy
The samples were observed with a SEM using an accelerating voltage of 20 kV, a working distance of 25 mm, and a specimen tilt of 40° (Cambridge Stereoscan S200; LEO Electron Microscopy Inc., Thornwood, NY, USA). The density of pinopodes was recorded as none, few, some, many, or ubiquitous.

Immunoassays
Serum obtained at the time of biopsy was analysed for oestrogen, progesterone, and glycodelin. The oestradiol and progesterone assays were done using commercial kits obtained from Diagnostic Products Corporation (Los Angeles, CA, USA). Both assays were based on solid phase methodology (antibody-coated tubes) and I125-labelled steroids. They did not require extraction or purification of the samples. Assays were done according to the manufacturer's instructions with known control samples of low, medium, and high hormone content included in each assay for quality control. Statistical analysis was done by computer using the STATLIA software (Brendan Scientific Co., Grosse Pointe Farms, MI, USA) and the four-parameter logistic method of curve fitting. The detection limit was 8 pg/ml for oestradiol and 20 pg/ml for progesterone. Interassay variations for both assays were ~9% and intra-assay variations were 6%. Glycodelin concentration was determined using an immunofluorometric assay (IFMA) (Koistinen et al., 1996Go). The IFMA used monoclonal antibodies to glycodelin to coat microtitre wells and secondary labelling was performed using europium chelate. Experimental samples of 25 µl of cell culture supernatants were added to previously prepared microtitre plates and incubated overnight at room temperature. Samples were washed and europium-labelled antibody was used as secondary antibody. After subsequent washing, enhancement solution was added and fluorescence was measured with a fluorometer. Results are presented as averages of triplicates in ng/ml of media.

Sonographic determinations of endometrial thickness
Sonograms were performed transvaginally (General Electric model RT3200, Brookfield, WI, USA). The appearance of the endometrium was classified according to a described system (Smith et al., 1984Go) and the thickness determined at its greatest dimension in the mid-fundus.

Endometrial histology
Endometrial biopsies were evaluated and dated using Noyes' criteria (Ferenczy, 1994). Specific cellular features relevant to endometrial dating were recorded individually, including mitoses (present or absent), subnuclear and supranuclear vacuoles (absent or present in more or less than 50% of glands), stromal oedema (present or absent), and predecidual reaction (none, around spiral arterioles only, around spiral arterioles and beneath surface epithelium, or confluent). The date assigned was based on the most advanced element of both epithelial and stromal components. Histology that differed by 3 or more days from the date calculated from the date of the LH surge was considered to be out of phase (Peters and Wentz, 1992Go).

Sample size determination
The target sample size of 20 women was based on cost and logistical considerations. This sample size provided 80% power to detect a reduction of 0.47 points ({alpha} = 0.05, one-sided) in the ß3 integrin subunit HSCORE between untreated and treated cycles, calculated using the Wilcoxon sign-rank test (Al-sunduqchi, 1990Go) and assumptions about the mean HSCORE and SD in both cycles taken from a previous study (Lessey et al., 1995Go).

Analysis
Differences were analysed between untreated and treated cycles using Wilcoxon sign-rank tests, McNemar's tests, or sign tests, as appropriate. One-sided tests were used for comparisons of the ß3 integrin subunit and LIF, because treatment was expected to decrease concentrations of both proteins, and two-sided tests were used for all other comparisons. For all tests, StatXact® (Cytel Software Corporation, Cambridge, MA, USA) was used to calculate exact P values and a significance level of 0.05 was used. No adjustments were made for multiple comparisons because of the exploratory nature of this analysis.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We recruited 33 women, of whom eight did not return after the initial screening interview, four did not ovulate by day 17 of the first cycle, one developed a medical condition during the first cycle that prevented her from continuing, and insufficient tissue was obtained from another participant. Thus 19 women completed both study cycles and were included in the analyses.

Participants had a median age of 30 years (range 20–34). Most (15/19) were Caucasian. All were parous and it was at least 6 months since the end of their last pregnancy. Only two smoked. They ranged in weight from 47.3–97.7 kg, with a median of 61.4 kg. No participant took any medications during either study cycle that were likely to have affected the study results.

In the treated cycles, all participants took the emergency contraceptive pills on the day they detected LH in their urine except two, who took the pills the following day. All participants took the two doses of pills 11 or 12 h apart except one, who took the doses 24 h apart. No participant vomited within 2 h after taking the pills, suggesting that the hormones were absorbed in all cases.

Endometrial dating was not possible in two participants in the treated cycle because of inadequate biopsy samples. In the other 17 participants, the biopsies were out of phase by at least 3 days in four women in the untreated cycle and in six women in the treated cycle [not significant (NS)]. Three women had out-of-phase endometria in both cycles. Asynchrony between glands and stroma was noted in only one woman, in her untreated cycle (NS). When cellular features used in dating the endometria were examined separately, no significant differences were noted in mitoses or stromal oedema, subnuclear vacuoles, or predecidual reaction. We did note a significant difference in supranuclear vacuoles (P = 0.0005), however, which were more prevalent in specimens from treated cycles.

Changes in pinopode density between treated and untreated cycles were not significant (NS). Mean endometrial thickness as measured by ultrasound was significantly lower in the treated cycle (7.58 mm; SD 2.04) than in the untreated cycle (9.79 mm; SD 3.68; P = 0.01). However, no relationship was observed between endometrial echo patterns and treatment: in 11/19 cases, the patterns were the same in both cycles; in 4/19 the pattern was categorized as `A' in the untreated cycle and `B' in the treated cycle; and in the remaining 4/19, it was `B' in the untreated cycle and `A' in the treated cycle.

Endometrial ß3 integrin subunit and LIF expression in the epithelial component of the endometrium as measured by immunohistochemistry staining was not significantly reduced in biopsy specimens from the treated cycles compared to untreated cycles (Table IGo). Immunohistochemical PR and glycodelin were not significantly different in the two cycles. However, endometrial ER was significantly higher and MUC-1 was significantly lower in treated cycles. Interestingly, serum oestradiol concentrations were significantly lower in treated cycles, but serum progesterone and glycodelin were not affected by treatment.


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Table I. Endometrial and serum test results
 
Based on both endometrial histology and luteal phase serum progesterone concentrations in our study, it appeared that treatment did not prevent ovulation in any participants.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study found no substantial evidence that treatment with the Yuzpe regimen of emergency contraception on the day of or the day after the urinary LH surge prevents ovulation, alters endometrial structure, or consistently affects endometrial proteins thought to be associated with uterine receptivity. Endometrial expression of ß3 integrin subunit and LIF, which have both been found to be decreased in infertile patients (Lessey et al., 1995Go; Cullinan et al., 1996Go; Hambartsoumian, 1998Go), were not significantly reduced by emergency contraceptive pill treatment; in fact, concentrations of both were higher (but not significantly so) in treated cycles. Serum and tissue concentrations of glycodelin, another marker of endometrial receptivity (Westergaard et al., 1998Go), also were the same in untreated and treated cycles.

We did note five statistically significant differences in treated cycles compared with untreated cycles: a reduction in endometrial MUC-1 expression, an increase in endometrial ER, lower luteal phase serum oestrogen concentration, reduced endometrial thickness, and greater proportion of glandular supranuclear vacuoles. However, the relationship between these changes and the contraceptive action of the emergency contraceptive treatment is unclear. Although in humans, endometrial MUC-1 normally increases in the mid-luteal phase, this protein seems to inhibit implantation in animals (Aplin, 1997Go) and the potential significance of a reduction of expression is not understood. ER normally decreases in the mid-luteal phase (Lessey et al., 1988Go) but an association between persistence of ER and failure of implantation or infertility has not been demonstrated. Endometrial thickness has been found not to correspond to endometrial development in the luteal phase (Grunfeld et al., 1991Go). Since we did not adjust for the multiple statistical comparisons in our analysis, it is possible that some of these changes may have resulted by chance.

Our results differ from those of several earlier studies of the endometrial effects of the Yuzpe regimen. In the first published study of the regimen (Yuzpe et al., 1974Go), it was reported that biopsies taken on the first day of bleeding after treatment showed marked asynchrony between glands and stroma. It was postulated that treatment early in the cycle worked by causing failure of implantation (Yuzpe et al., 1974Go). Two other early studies also showed asynchrony and/or out-of-phase features in biopsies from most of 12 women treated at or shortly after ovulation (Ling et al., 1979Go, 1983Go). A subsequent study found reduced concentrations of endometrial oestrogen and progesterone receptors 3 days following treatment among eight women treated 2 days after the LH surge (Kubba et al., 1986Go).

In contrast, a more recent study found no endometrial phase abnormalities in eight women treated 2 days after the LH surge (Swahn et al., 1996Go). That study did find significantly more vacuolated cells (similar to our results) and wider diameters of gland lumina, but the authors interpreted these effects as clinically insignificant. Two previous studies have evaluated integrins (Taskin et al., 1994Go) and glycodelin (Young et al., 1994Go) in women treated with the emergency contraceptive pill regimen. No disturbances of integrin expression were found, but glycodelin concentrations were reduced by treatment. However, both studies had design flaws: the treatment was given long after the end of the fertile period, when the regimen would not be expected to be effective, and the proteins may not have been evaluated on the appropriate day of the luteal phase (Lessey, 1994Go).

The reason for the differences between our primarily negative findings and the striking structural endometrial abnormalities seen by early researchers is not clear. In our study, the time in the cycle of both treatment and evaluation was different from that in most patients in previous studies; perhaps the effects of treatment are extremely time-specific. A more likely explanation is that, unlike earlier studies, we interpreted our biopsies in a masked fashion; thus our findings were unlikely to have been influenced by preconceived hypotheses and knowledge of whether or not the woman had received treatment.

Both the endometrial histology and the luteal phase serum progesterone concentrations in our study indicated that treatment did not prevent ovulation in any participants. This finding was not surprising, since treatment was given after LH was detected in urine. This timing corresponds to the day before or the day of ovulation, which is probably too late to prevent ovulation. In some cases, participants may have already ovulated when they took the regimen. Since we made no attempt to document ovulation with serial sonograms, it remains possible that treatment induced a luteinized unruptured follicle syndrome, though this seems to be an unlikely mechanism for this form of contraception.

Our results leave a puzzling gap in our understanding of the mechanism of action of this therapy. Considering that maximal fertility occurs on the days in the cycle when women were treated in our study, in order for the Yuzpe regimen to be able to prevent 75% of expected pregnancies, it must have some contraceptive effect when taken on or after those days (Trussell and Raymond, 1999Go). However, our study confirmed that it does not prevent ovulation when taken at that time (Ling et al., 1979Go), and we also found no striking effects on the endometrium. Perhaps the regimen affects endometrial function in ways undetectable by the tests we chose to perform, or it could cause important changes earlier or later in the cycle than when we performed our tests. For example, the levonorgestrel-releasing intrauterine device has been shown to cause `inappropriate' premature glycodelin expression, which has been postulated to contribute to its contraceptive effect by blocking spermatozoa–egg interactions (Mandelin et al., 1997Go). If the emergency contraceptive pills caused a similar effect, we might just have missed it because of the timing of our specimen collection. Alternatively, the emergency contraception regimen may affect sperm transport, tubal motility or the fertilization process itself. Data are not currently available to assess these possibilities.


    Acknowledgments
 
We gratefully acknowledge the contributions of Hannu Koistinen for assistance with the glycodelin assays and Robert Bagnall for his expertise and technical assistance with scanning electron microscopy. Support for this study was provided by Family Health International (FHI) with funds from the Mellon Foundation and the United States Agency for International Development (USAID), Cooperative Agreement Number AID/CCP-A-00–95–00022–02. Laboratory facilities and personnel also received support from NIH grants HD 35041 and HD 34824 (BL), which are part of the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation (U01 HD 34824); cooperative agreement (U54HD35041), which is part of the Specialized Cooperative Centers Program in Reproduction Research; and the Academy of Finland and the Finnish Federation of Life and Pension Insurance Companies. The views expressed in this article do not necessarily reflect those of FHI or any of the funding agencies.


    Notes
 
5 To whom correspondence should be addressed at: Family Health International, Research Triangle Park, NC, USA. Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 2, 2000; accepted on July 10, 2000.