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Northern California Fertility Medical Center (L.P.L.), Roseville, California 95661; Center for Women’s Health and Reproduction (A.T.F.), Department of Obstetrics and Gynecology, University of Illinois, Chicago, Illinois 60612; Department of Obstetrics and Gynecology (M.A.F.), Division of Human Reproduction and Infertility, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; Center for Women’s Health (D.G.M.), Oklahoma City, Oklahoma 73120; and Center for Women’s Medicine (B.A.L.), Greenville Hospital System, Greenville, South Carolina 29617
Address all correspondence and requests for reprints to: Bruce A. Lessey, Center for Women’s Medicine, 890 West Faris Road, Suite 470, Greenville, South Carolina 29617. E-mail: blessey{at}ghs.org.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Apoptosis is regulated by various gene products including cytokines, interleukins, and steroid hormones. Many promoters and inhibitors of apoptosis have been described. TNF-
and the apoptosis-related protein bax have been shown to promote apoptosis, whereas the presence of bcl-2 and bcl-x appears to inhibit this process (4). In a rat endometrial cell line, a decrease in progesterone (P4) or the administration of antiprogestins results in apoptosis of the epithelial cells without gross change in the stromal component. Conversely, addition of P4 prevents apoptosis in this model (5).
Apoptosis was first described in the human endometrium by Hopwood and Levison in 1975 (6). Later studies by Tabibzadeh et al. (7) confirmed that apoptosis is rare in the proliferative endometrium, whereas the number of apoptotic cells progressively increases in secretory endometrium and peaks during the menstrual phase. As the human endometrium undergoes cyclical changes during the menstrual cycle to prepare for blastocyst implantation, apoptosis may play an important part in this process of tissue remodeling and allow rapid placental development (8). The purpose of the present study was to examine those factors that support the luteal phase and reduce the apoptosis known to occur before menstruation. Using a prospective, controlled, and randomized study design, we examine for the first time the effects of exogenous human chorionic gonadotropin (hCG) and P4 on endometrial apoptosis during the late luteal phase.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Twelve healthy, reproductive-age women (ages 20–34 yr) with regular menstrual cycles (range, 26–32 d) were enrolled for this study (Table 1
), and each was compensated for her participation. The clinical investigations reported in this study were conducted in accordance with the guidelines in The Declaration of Helsinki, and all protocols used in this study were approved by the Institutional Review Board at the University of North Carolina-Chapel Hill. The number of subjects (n = 12) was chosen as the initial cohort to determine whether differences could be demonstrated between two commonly used treatment regimens for luteal-phase support, using each volunteer as her own control. All patients had at least one previous pregnancy, but none had been pregnant in the 6 months preceding entry into the study, and none were currently breastfeeding. Patients had received no hormonal therapy in the 30 d before the study, and none had received Depo-Provera in the past 9 months. None of the women were taking prescription medication, and all were considered healthy. Each woman was asked to practice nonhormonal contraception in each biopsy cycle. Additional samples, timed to the mid-secretory or menstrual phase, were also obtained from fertile volunteers.
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All endometrial biopsies were timed to the urinary LH surge, and each was performed 12 d after the urinary LH kit turned positive (LH + 12, d 26). All 12 patients underwent an endometrial biopsy in a natural (control) cycle. At the time of the first visit, each participant was randomized to one of two groups. Randomization was performed by drawing an envelope with one of 12 cards designating either hCG or P4 treatment. The first group received P4 suppositories, 200 mg intravaginally daily starting on d LH + 4–13 (d 18–27). The other group received a single im injection of 10,000 IU hCG on the fifth day after the LH surge (d 19). All participants then returned on d 12 after the urinary LH surge in the treatment cycle for repeat endometrial biopsy at which time serum was obtained to measure P4 levels.
Gel electrophoresis for DNA fragmentation
Electrophoretic analysis of DNA fragmentation was performed on each endometrial tissue. Tissues analyzed were snap-frozen immediately after collection and stored at –80 C to prevent nonspecific activation of DNases. Tissues were gently homogenized in 0.5 ml homogenization buffer [10 mM EDTA, 50 mM Tris (pH 8.0), 0.5% sodium lauryl sarcosine, 0.5 mg/ml proteinase K] and then incubated at 50 C for several hours. RNase A (0.5 mg/ml) was added, and homogenates were incubated for 1 h. The homogenates were extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (25/24/1, vol/vol). DNA was precipitated by the addition of 0.1 vol 3 M sodium acetate and 3 vol ice-cold ethanol and incubated at –80 C for at least 2 h. DNA was pelleted by centrifugation at 14,000 rpm for 15 min at 4 C, washed with 0.2 ml ice-cold 80% ethanol, air dried, resuspended in 100 µl Tris-EDTA (pH 8.0), and quantitated by absorbance at 260 nm. DNA fragmentation was analyzed by using the ApoAlert LM-PCR ladder assay kit (Clontech Laboratories, Palo Alto, CA) because of its high sensitivity and low background. The slides were examined for the characteristic ladder pattern produced by 120- to 180-bp nuclear fragments.
Immunohistochemistry
Immunostaining for three apoptosis-related proteins was performed on formalin-fixed sections. The antibodies used and the relative dilution for each antibody is shown in Table 2. Paraffin-embedded sections of each sample (5 µm) were pretreated by placing the slides in a microwave in 0.01 M citrate buffer on high for 15 min. Sections were deparaffinized in xylene and rehydrated in ethanol with increasing concentrations of water. Endogenous peroxidase activity was quenched with 5% hydrogen peroxide for 30 min. Nonspecific binding sites were blocked with 2% normal goat serum (NGS) for 30 min at room temperature.
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Assessment of staining intensity and distribution was made using the semiquantitative HSCORE scoring system by a single blinded observer. HSCORE was calculated using the following equation: HSCORE =
Pi (i + 1), where i is the 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 0–100%. Low intraobserver (r = 0.983; P < 0.0001) and interobserver (r = 0.994; P < 0.0001) differences for HSCORE in uterine tissues have been reported previously using this technique (9).
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method
The TUNEL assay was performed on formalin-fixed, paraffin-embedded tissues using the ApopTag in situ detection kit (Chemicon International, Temecula, CA). Tissues were cut 5 mm thick and placed on Superfrost Plus slides. The sections were deparaffinized in xylene and rehydrated in graded ethanol washes. The slides were washed in PBS. The tissue was pretreated with proteinase K for 15 min at room temperature, and then the slides were washed in two changes of distilled H2O. Endogenous peroxidases were quenched in 3% hydrogen peroxide in PBS for 5 min, and the slides were washed in PBS. Equilibration buffer was applied to the sections for a few minutes at room temperature. The buffer was gently removed, and working strength terminal deoxynucleotidyl transferase enzyme was placed on the sections and incubated for 1 h at 37 C. Slides were incubated for 10 min in stop/wash buffer and then washed in PBS. The sections were incubated for 30 min at room temperature with antidigoxigenin conjugate and then washed in numerous changes of PBS. The color was developed by applying peroxidase substrate to the sections. The color development was monitored by looking at the slides under the microscope. The slides were developed for 3–5 min in small batches to control the time. The color development was stopped by washing the slides in distilled H2O for at least 5 min. The sections were counterstained with methyl green, dehydrated, and mounted. The semiquantitative HSCORE method was used to assign the degree of apoptosis in each sample.
RIA determinations
Serum P4 levels were determined using an Immulite 100 instrument (Diagnostic Products Corp., Los Angeles, CA) (analytical sensitivity, 0.1 ng/ml; calibration range, 0.2–40 ng/ml; inter- and intraassay precision range, 6–16%) (see package insert at http://www.dpcweb.com/package_inserts/immulite_1000/imm1000_dom.html). Serum P4 levels were presented as nanomoles per milliliter using the conversion factor of 3.026 from SI units.
Statistical analysis
Comparisons for serum P4 concentrations and the numbers of apoptotic bodies identified by TUNEL were compared between control and treatment (hCG or P4) using ANOVA with post hoc Scheffé’s correction. Comparisons of immunohistochemical staining patterns for bcl-2, bcl-x, and bax were also made using ANOVA comparing the semiquantitative HSCORE for staining intensity, a numerical score ranging from 0–4. Significance was based on a 95% confidence interval and a P value of <0.05.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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). Gel electrophoresis of extracted DNA demonstrated characteristic laddering indicative of apoptosis in all samples in the control group. No characteristic laddering was demonstrated in samples from either treatment group. Typical appearance of the laddering results is shown in Fig. 1 and are compared with menstrual endometrium and mid-secretory endometrium (LH + 8). From these results it appeared that DNA fragmentation based on DNA laddering appears in the late luteal phase (control cycles) and in menstrual endometrium but was not seen in the mid-secretory phase or in the late-secretory phase samples from treatment cycles in women receiving hCG or P4 support.
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. Analysis of the TUNEL method estimated the degree of apoptosis using HSCORE determinations. In the control cycles, the mean HSCORE was 2.95 ± 0.46 (SE) compared with 1.3 ± 0.34 in the hCG-treated group and 0.1.9 ± 0.25 in the P4 group. Based on ANOVA analysis with Scheffé’s correction, TUNEL scores were significantly higher (epithelial cells) in control cycles compared with hCG-treated cycles (P = 0.04) but not different from P4-treated cycles (P < 0.21). There was no significant difference between hCG and P4 treatment groups (P = 0.64) with regard to TUNEL results. These results are shown in Fig. 3A and Table 3.
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, the mean serum P4 was highest in the hCG treatment arm and lowest in the control group, although this difference was only of borderline statistical significance (P = 0.08).
We next examined the endometrium of each patient for one of three apoptosis-related proteins. These included bcl-2, bcl-x, and bax proteins. Using immunohistochemistry, each sample was stained for each antigen and the results presented using the semiquantitative HSCORE that is a measure of intensity and distribution of each antigen. Because most of the differences noted in the TUNEL staining occurred in the epithelial compartment and because stromal staining was noted to be highly variable from sample to sample, we report here only the HSCORE for each apoptosis marker in the epithelial compartment. The overall results are summarized in Table 3. Treatment with either hCG or P4 significantly increased epithelial bcl-2 compared with untreated controls. There was no statistical difference in bcl-x immunostaining between groups. bax was significantly lower in the P4-treated group compared with the untreated controls, although this decrease was not observed in the hCG treatment group. The immunohistochemical appearance of these three antigens in typical samples from control and treatment cycles is shown in Fig. 4, showing the modest increase in bcl-2 (A vs. B), the decrease in bax immunostaining (C vs. D), and the comparable appearance of bcl-x (E vs. F).
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Discussion |
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The induction of apoptosis at the time of menses has physiological significance and provides a mechanism for effective removal of spent endometrium during menses in lieu of successful embryonic implantation (11). Apoptosis typically peaks during menstruation and may be associated with elevated cytokine production, notably TNF- (7). Withdrawal of P4 has been shown to increase the number of apoptotic endometrial cells in vivo (12) and is associated with marked increase in other menstruation-related proteins and enzymes including the matrix metalloproteinases (13). This study has demonstrated that treatment with exogenous P4 or mid-luteal hCG administration decreases the number of apoptotic cells as evidenced by DNA fragmentation and by development of apoptotic bodies (TUNEL staining). Based on the TUNEL results, it appeared that P4 treatment was less effective in reducing this particular marker of apoptosis, although both treatments appeared to dramatically reduce the presence of laddering in endometrial samples after treatment. Although serum P4 levels tended to be highest in the hCG group, tissue levels (within the endometrium) of P4 may also be high in the group receiving vaginal P4 treatment. These differences noted between these two techniques may also reflect a difference in sensitivity of the two methods, with laddering being less sensitive than TUNEL staining for showing evidence of DNA fragmentation.
The delay in menstruation that occurs with pregnancy is a result in part of the timely rescue of the CL by embryo-derived hCG. The postponing of the apoptotic-mediated demise of the endometrium is likely a prerequisite for survival of the conceptus. This sentinel event may be critical and delay of CL rescue may be the basis for the increase in pregnancy loss when implantation is delayed beyond the normal window of implantation (14). Women with infertility or recurrent pregnancy loss likely experience delayed implantation that is correctable, using the types of treatments described in this study.
Based on animal data, it appears that P4 transforms the proliferative endometrium into a secretory structure that is capable of maintaining the nascent pregnancy. Progesterone induces the expression of numerous proteins to support implantation should it occur (15) but also sets the stage of menstruation when P4 levels fall. In the present study, we studied the endometrium at a time before menstruation when apoptosis is already beginning, on d 26, corresponding to LH + 12. Although the signal for apoptosis is not entirely understood, these results suggest that the apoptotic process can be forestalled in the presence of luteal-phase support in the form of exogenous P4 or hCG.
The induction of apoptotic pathways is part of well-orchestrated events in many tissues (16). The bcl family of gene products have been proposed as major regulators of apoptosis, with bcl-2 serving a protective function and bax and bcl-x favoring apoptosis (4). Tabibzadeh et al. (7, 11) have previously shown that bcl-2 is diminished in the endometrium at the time of menstruation. This factor protects cells from apoptosis from a variety of stimuli. In this study, the progression toward apoptosis depended solely on the sustained availability of P4 directly, or indirectly through hCG administration. Under the influence of both treatments, epithelial bcl-2 expression was increased relative to the untreated controls. Others have shown that the addition of P4 can change the ratio of bcl gene products in favor of apoptosis-inhibiting products (12), and it may be the ratio of the bcl gene products that is an important signaler for apoptotic events.
In our study subjects, the persistent expression of bcl-2 is in contrast with the findings of Koh et al. (17), who proposed that persistence of bcl-2 staining in the luteal phase was a result only of the presence of lymphocytes in the stroma. Others point out that stromal bcl-2 expression may account for the low levels of apoptosis in this compartment (11). Dahmoun et al. (18) note that there is some discrepancy between the glandular and stromal apoptosis, with epithelial activity increasing 2 ds before menstruation whereas stromal apoptosis increased after menses had been initiated. There was a reciprocal relationship between epithelial and stromal bcl-2 during this transition as well. In examination of other apoptosis-related peptides, we found less consistent results. bcl-x is a member of the bcl-2 family that may interact to regulate apoptosis (16). Overall, there was no significant change in bcl-x between control and treatment conditions.
Exogenous treatment with hCG has been shown to alter endometrial morphology in other primate species and that the response to hCG depended on concomitant P4 action from the CL (10, 19). It is possible that the action of P4 or hCG on different cell types may regulate apoptosis differentially depending on the levels of P4 and its receptor, PR. It is known from animal studies that in pregnancy, cell death from apoptosis occurs only in the decidua, despite high levels of P4 (20). In the human, this process has been demonstrated in the cytotrophoblast of normal pregnancies (21) and is likely the basis for the tissue remodeling required during implantation and placentation.
The other protein examined in this study was bax. bax also maintains an extensive homology to bcl-2 but favors apoptosis, accelerating apoptotic cell death and counteracts the anti-apoptotic effects of bcl-2 (22). Comparing treatment with untreated controls we found that BAX expression was significantly reduced in the P4-treated women but only slightly diminished in the hCG treatment group. These results are in relative agreement with the results of TUNEL immunostaining, which showed a marked decrease in the P4-treated group compared with a more modest decrease in the hCG-treated group. Additional studies will be necessary to sort out the relative importance of each protein, but based on these data, the reciprocal expression of bcl-2 and bax appear to be the most consistent finding, with the overall reduction in DNA fragmentation noted in these studies.
The significance of these findings lies in the demonstration of a human model for the study of endometrial apoptosis. Such studies may lead to a better understanding of the molecular events that regulate this important process but may also facilitate the design of better methods for assisted reproductive technologies. Although the timing of implantation is vital to the success of naturally occurring pregnancies (14), embryos replaced during assisted reproductive technologies cycles may not be as well synchronized with the endometrium or the CL. These data suggest that both exogenous P4 and hCG can influence the endometrium of the late luteal phase and prevent or delay the start of endometrial apoptosis. Support of the endometrium or the CL is likely to be critical for successful implantation in both natural cycles and in the setting of in vitro fertilization with embryo transfer. Finally, understanding the timing and regulation of apoptosis in normal cycles may suggest additional strategies to target the endometrium for purposes of contraception. Based on these results, a single injection of mid-luteal hCG may be as effective as daily vaginal P4 at prevention of endometrial apoptosis.
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Acknowledgments |
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Footnotes |
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First Published Online January 25, 2005
Abbreviations: CL, Corpora lutea; P4, progesterone; hCG, human chorionic gonadotropin; NGS, normal goat serum; P4, progesterone; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
Received October 29, 2004.
Accepted January 12, 2005.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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. Endocr J 2:87–95
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