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Progesterone receptor membrane component 1 (PGRMC1) expression in fetal membranes among women with preterm premature rupture of the membranes (PPROM)
Placenta 35 (2014) 331e333
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Short communication
Progesterone receptor membrane component 1 (PGRMC1) expression in fetal membranes among women with preterm premature rupture of the membranes (PPROM) L. Feng a, B.C. Antczak a, L. Lan b, C.A. Grotegut a, J.L. Thompson a, T.K. Allen c, A.P. Murtha a, * a b c
Department of Obstetrics and Gynecology, Duke University, Durham, NC, USA Department of Biostatistics and Bioinformatics, Duke University, Durham, NC, USA Department of Anesthesia, Duke University, Durham, NC, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 9 March 2014
PGRMC1 function is implicated in maintaining fetal membrane (FM) integrity. PGRMC1 was detectable primarily in the cytoplasm of FM cells and was actively regulated in FMs and relevant for PGRMC1mediated progesterone action. By cell type, PGRMC1 expression was higher in amnion and chorion compared with decidua. By clinical phenotype, PGRMC1 expression was higher among preterm-no-labor and term-no-labor subjects compared to PPROM. PGRMC1 expression appears to be diminished in PPROM subjects. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: PPROM PGRMC1 Expression Fetal membrane cells
1. Introduction PPROM is the result of FM remodeling and weakening. Chorion thinning in PPROM may be a result of apoptosis and extracellular matrix (ECM) degradation [1,2]. Progesterone inhibits basal and TNFa-induced apoptosis in FMs [3]. Progesterone also protects cytotrophoblast from cell death and attenuate cytokine-induced matrix metalloproteinase 9 (MMP9) activities [4,5]. However, nuclear progesterone receptors are negative in the chorion and amnion indicating potential non-classic mechanisms for progesterone [4,6]. PGRMC1 is highly expressed in these cells and partially mediates the inhibition of TNF induced MMP9 activity by progestins [4]. PGRMC1 promotes cancer and granulosa cell survival [7e10]. The anti-apoptotic and antiinflammatory functions of PGRMC1 suggest its potential roles in maintaining FM integrity. We hypothesize that PGRMC1 regulation may modulate apoptosis in FM cells and alters ECM remodeling contributing to PPROM. Therefore, we aim to investigate if PGRMC1 expression in FM varies by cell layer or clinical status.
* Corresponding author. Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Box 3967, Duke University Medical Center, Durham, NC 27710, USA. Tel.: þ1 919 681 5220; fax: þ1 919 681 7861. E-mail addresses: [email protected], [email protected] (A.P. Murtha). http://dx.doi.org/10.1016/j.placenta.2014.03.008 0143-4004/Ó 2014 Elsevier Ltd. All rights reserved.
2. Methods Subjects were prospectively enrolled into this Duke University IRB-approved protocol and all subjects provided written consent to participate. The study groups were classified as: uncomplicated-term-C-section-without-labor (TNL; n ¼ 9); preterm-without-labor (PTNL; n ¼ 10) and PPROM (n ¼ 10). Samples were collected for immunohistochemistry and Western blot (WB) respectively as previously described [11]. For each subject, one sample was collected from the site of rupture labeled [R] and the other was collected from an area distant to rupture site [D]. 2.1. Immunohistochemistry UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen kit was used following the manufacturer’s instruction. PGRMC1 antibody (Sigma) and normal rabbit IgG as negative control were used at 1:200 dilutions [4]. Ten images per slide were taken. The relative staining intensity was evaluated using a 5-point scoring system (Supplement Fig. 1) [12]. 2.2. WB PGRMC1 (1:2000); b-actin (1:6000) and secondary goat anti-rabbit IgG (1:2000) from Cell Signaling Technology were used. Image J was used for densitometry and data are presented as ratio after normalized to b-actin. Three replicates were performed for each sample. 2.3. Statistical analysis Multivariate ordinal logistic regression analysis was used to determine relative PGRMC1 expression adjusting for reader, site and measurements (the 10 scores per slides). The decidual layer and PPROM were selected as the reference groups for cell type and clinical phenotype, respectively. Adjusted ORs and 95% CI were estimated to assess the relative odds of PGRMC1 staining. For WB data, KruskaleWallis test was used for three-group comparison and the ManneWhitney test was used for two-group comparisons with significance defined as P < 0.05.
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3. Results The subject characteristics are summarized in Supplement Table 1. PGRMC1 was predominantly found in the cytoplasm of cells with occasional nuclear staining in all layers of FMs (Fig. 1). The agreement between readers ranged from 0.51 to 0.66, which is considered moderate to good agreement using weighted-Kappa statistic. The distribution of PGRMC1 varied considerably by clinical group and cell type. When clinical phenotypes were combined, PGRMC1 expression was the highest in amnion and the lowest in decidua (P < 0.05). When the cell types were combined, PGRMC1 expression was the lowest in PPROM subjects and the highest in PTNL subjects (P < 0.05) (Fig. 1 and Table 1A). When PGRMC1 expression was compared within each layer among PPROM, TNL and PTNL subjects, PGRMC1 expression was the highest in PTNL subjects and lowest in PPROM subjects in chorion or decidua layer (same as the combined data). In the amnion layer, PGRMC1 expression was the lowest in TNL instead of PPROM subjects (Table 1B). Table 2 summarized the comparison of PGRMC1 expression between [R] and [D] within each clinical cohort. PGRMC1 expression was lower at [R] in PPROM, higher at [R] in TNL and not different in PTNL. WB analysis also showed that PGRMC1 expression in full thickness FMs was significantly lower in PPROM subjects compared to PTNL and TNL. There was no significant difference in PGRMC1 expression in PTNL and TNL subjects (Fig. 2).
Table 1A Relative PGRMC1 staining in fetal membranes by cell type or clinical status. Predictor Cell type Decidual cells Chorion cells Amnion cells Clinical phenotype PPROM (N ¼ 10) TNL (N ¼ 9) PTNL (N ¼ 10)
Adjusted OR (95% CI)
P for trend
1.0 1.64 (1.4e1.9) 4.71 (3.9e5.7)
<0.0001
1.0 1.35 (1.15e1.59) 4.66 (3.84e5.65)
<0.0001
The covariates of the multivariate ordinal logistic model include reader, measurements, cell layer and site of collection (rupture vs. distant). The decidual cells are the reference group for the cell type and PPROM is the reference group for clinical phenotype.
Table 1B Relative PGRMC1 staining in fetal membranes by clinical phenotype in each cell layer. Clinical phenotype
PPROM (N ¼ 10) TNL (N ¼ 9) PTNL (N ¼ 10)
Amnion
Chorion
Decidua
Adjusted OR (95% CI)
Adjusted OR (95% CI)
Adjusted OR (95% CI)
1.0 0.69 (0.51e0.95) 1.93 (1.34e2.78)
1.0 2.74 (2.08e3.60) 8.74 (6.33e12.1)
1.0 1.19 (0.90e1.57) 4.92 (3.56e6.80)
The covariates of the multivariate ordinal logistic model include reader, measurements, cell layer and site of collection (rupture vs. distant). PPROM is the reference group in each cell layer.
4. Discussion We report here for the first time the localization and expression of PGRMC1 in FMs among pathologic conditions. PGRMC1 alters several survival signaling proteins, including the Akt protein kinase and the cell death-associated protein IkB [13]. PGRMC1 mediates cytokine-induced MMP expression and activity in cytotrophoblast cells [4]. MMPs are zinc-dependent endopeptidase enzymes involved in ECM remodeling. Significantly elevated levels of MMP9 expression and activity have been identified in FMs of PTL and PPROM patients [14,15]. Our findings indicate that PGRMC1 may be an important mediator of progesterone function in FMs.
Table 2 Relative PGRMC1 staining in fetal membranes by collection site. Site of collection
Distant Rupture
PPROM (N ¼ 10)
TNL (N ¼ 9)
PTNL (N ¼ 10)
Adjusted OR (95% CI)
Adjusted OR (95% CI)
Adjusted OR (95% CI)
1 0.653 (0.506e0.844) P ¼ 0.001
1 1.272 (1.032e1.567) P ¼ 0.024
1 0.936 (0.707e1.238) P ¼ 0.641
The covariates of the multivariate ordinal logistic model includes reader, measurements, cell layer.
Fig. 1. Representative images to show the differences of PGRMC1 expression in fetal membranes from each cohort. The PGRMC1 staining in PTNL and TNL cohort is much more intense compare to PPROM using specific antibody against PGRMC1. A is amnion layer; C is chorion layer; D is decidua layer. High power image demonstrate the nuclear staining and cytoplasmic staining. Abbreviations: PTNL, preterm not in labor; TNL, term not in labor; PPROM, preterm premature rupture of membranes.
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therapeutic approach for preterm birth although further study is needed. 5. Authors’ roles Dr. Liping Feng carried out all of the molecular studies for the manuscript and wrote the manuscript. Brian Antczak and Dr. Jennifer Thomson helped with collecting the human tissues and scoring the IHC staining. Lan Lan performed the statistical analysis. Dr. Terrence Allen and Chad Grotegut participated in discussions and consulted on the manuscript. Dr. Amy Murtha participated in studying design, data interpretation and writing the manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.placenta.2014.03.008. References Fig. 2. Relative expressions of PGRMC1 in fetal membranes collected from different clinical cohorts determined by western blotting. A, Representative image of western blotting. Positive control is the recombinant PGRMC1 protein with His-Tag modification. The molecular weight is slightly higher than 28 kDa. B, Densitometry is used to quantify the intensity of western blotting bands. The ratios of band intensities between PGRMC1 protein and B-actin are calculated to allow for normalization of the data. Each bar represents the average SEM of all samples for each cohort. There were statistically significant differences (P ¼ 0.009) between three groups. When two groups are compared, PGRMC1 expression is statistically significant less in PPROM compared to TNL and PTNL (P ¼ 0.004 and 0.017 respectively) groups. There were no statistical significant differences between TNL and PTNL (P ¼ 0.675); Abbreviations: PTNL, preterm not in labor; TNL, term not in labor; PPROM, preterm premature rupture of membranes.
Furthermore, diminished PGRMC1 at the rupture site among PPROM subjects may be associated with the FM rupture. In TNL subjects PGRMC1 expression was higher at the rupture site and no difference in PTNL subjects. We speculate three potential biases that limit our ability to draw firm conclusions. First, PTNL patients usually are complicated by pathological conditions such as preeclampsia and the implications of these conditions on PGRMC1 expression are unknown. However, it is not possible to obtain PTNL FM from uncomplicated pregnancy; Second, none of TNL and PTNL patients experienced labor which may impact these changes. And it is not established whether PGRMC1 expression is altered in the setting of labor; Finally, the rupture site tissues we collected from cesarean delivery might not yet exhibit biochemical and mechanical characteristics of rupture at the time of surgery. Using immunohistochemistry allows us to quantify and localize PGRMC1 protein expression in each cell layer. Simultaneously, we considered the weaknesses of immunohistochemistry for protein quantification. Therefore, we minimized staining batches with randomly mixed clinical groups. Multiple images for each slide were evaluated by two independent blinded evaluators to limit the impact of staining and processing variability. Finally, results of WB further support the findings that PGRMC1 expression is the lowest in FMs from PPROM subjects. Targeting PGRMC1 can be a specific
[1] Canzoneri BJ, Feng L, Grotegut CA, Bentley RC, Heine RP, Murtha AP. The chorion layer of fetal membranes is prematurely destroyed in women with preterm premature rupture of the membranes. Reprod Sci 2013;20:1246e54. [2] Menon R, Fortunato SJ. The role of matrix degrading enzymes and apoptosis in rupture of membranes. J Soc Gynecol Investig 2004;11:427e37. [3] Luo G, Abrahams VM, Tadesse S, Funai EF, Hodgson EJ, Gao J, et al. Progesterone inhibits basal and TNF-alpha-induced apoptosis in fetal membranes: a novel mechanism to explain progesterone-mediated prevention of preterm birth. Reprod Sci 2010;17:532e9. [4] Allen TK, Feng L, Grotegut CA, Murtha AP. Progesterone receptor membrane component 1 as the mediator of the inhibitory effect of progestins on cytokine-induced matrix metalloproteinase 9 activity in vitro. Reprod Sci 2014;21:260e8. [5] Murtha AP, Feng L, Yonish B, Leppert PC, Schomberg DW. Progesterone protects fetal chorion and maternal decidua cells from calcium-induced death. Am J Obstet Gynecol 2007;196. 257 e251e255. [6] Merlino A, Welsh T, Erdonmez T, Madsen G, Zakar T, Smith R, et al. Nuclear progesterone receptor expression in the human fetal membranes and decidua at term before and after labor. Reprod Sci 2009;16:357e63. [7] Peluso JJ, Liu X, Saunders MM, Claffey KP, Phoenix K. Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1. J Clin Endocrinol Metab 2008;93:1592e9. [8] Peluso JJ. Non-genomic actions of progesterone in the normal and neoplastic mammalian ovary. Semin Reprod Med 2007;25:198e207. [9] Crudden G, Loesel R, Craven RJ. Overexpression of the cytochrome p450 activator hpr6 (heme-1 domain protein/human progesterone receptor) in tumors. Tumour Biol 2005;26:142e6. [10] Peluso JJ, Romak J, Liu X. Progesterone receptor membrane component-1 (PGRMC1) is the mediator of progesterone’s antiapoptotic action in spontaneously immortalized granulosa cells as revealed by PGRMC1 small interfering ribonucleic acid treatment and functional analysis of PGRMC1 mutations. Endocrinology 2008;149:534e43. [11] fortner K, Ransom C, canzoneri B, Bentley R, Feng L, Lan L, et al. Bacteria localization and chorion thinning among preterm premature rupture of membranes. PloS One 2013;9(1). http://dx.doi.org/10.1371/journal.pone.0083338. e83338. [12] Kraus DM, Feng L, Heine RP, Brown HL, Caron KM, Murtha AP, et al. Cigarette smoke-induced placental adrenomedullin expression and trophoblast cell invasion. Reprod Sci 2014;21:63e71. [13] Debose-Boyd RA. A helping hand for cytochrome p450 enzymes. Cell Metab 2007;5:81e3. [14] Fortunato SJ, Menon R. Distinct molecular events suggest different pathways for preterm labor and premature rupture of membranes. Am J Obstet Gynecol 2001;184:1399e406. [15] Xu P, Alfaidy N, Challis JRG. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in human placenta and fetal membranes in Relation to preterm and term labor. J Clin Endocrinol Metab 2002;87:1353e61.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Short communication
Progesterone receptor membrane component 1 (PGRMC1) expression in fetal membranes among women with preterm premature rupture of the membranes (PPROM) L. Feng a, B.C. Antczak a, L. Lan b, C.A. Grotegut a, J.L. Thompson a, T.K. Allen c, A.P. Murtha a, * a b c
Department of Obstetrics and Gynecology, Duke University, Durham, NC, USA Department of Biostatistics and Bioinformatics, Duke University, Durham, NC, USA Department of Anesthesia, Duke University, Durham, NC, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 9 March 2014
PGRMC1 function is implicated in maintaining fetal membrane (FM) integrity. PGRMC1 was detectable primarily in the cytoplasm of FM cells and was actively regulated in FMs and relevant for PGRMC1mediated progesterone action. By cell type, PGRMC1 expression was higher in amnion and chorion compared with decidua. By clinical phenotype, PGRMC1 expression was higher among preterm-no-labor and term-no-labor subjects compared to PPROM. PGRMC1 expression appears to be diminished in PPROM subjects. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: PPROM PGRMC1 Expression Fetal membrane cells
1. Introduction PPROM is the result of FM remodeling and weakening. Chorion thinning in PPROM may be a result of apoptosis and extracellular matrix (ECM) degradation [1,2]. Progesterone inhibits basal and TNFa-induced apoptosis in FMs [3]. Progesterone also protects cytotrophoblast from cell death and attenuate cytokine-induced matrix metalloproteinase 9 (MMP9) activities [4,5]. However, nuclear progesterone receptors are negative in the chorion and amnion indicating potential non-classic mechanisms for progesterone [4,6]. PGRMC1 is highly expressed in these cells and partially mediates the inhibition of TNF induced MMP9 activity by progestins [4]. PGRMC1 promotes cancer and granulosa cell survival [7e10]. The anti-apoptotic and antiinflammatory functions of PGRMC1 suggest its potential roles in maintaining FM integrity. We hypothesize that PGRMC1 regulation may modulate apoptosis in FM cells and alters ECM remodeling contributing to PPROM. Therefore, we aim to investigate if PGRMC1 expression in FM varies by cell layer or clinical status.
* Corresponding author. Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Box 3967, Duke University Medical Center, Durham, NC 27710, USA. Tel.: þ1 919 681 5220; fax: þ1 919 681 7861. E-mail addresses: [email protected], [email protected] (A.P. Murtha). http://dx.doi.org/10.1016/j.placenta.2014.03.008 0143-4004/Ó 2014 Elsevier Ltd. All rights reserved.
2. Methods Subjects were prospectively enrolled into this Duke University IRB-approved protocol and all subjects provided written consent to participate. The study groups were classified as: uncomplicated-term-C-section-without-labor (TNL; n ¼ 9); preterm-without-labor (PTNL; n ¼ 10) and PPROM (n ¼ 10). Samples were collected for immunohistochemistry and Western blot (WB) respectively as previously described [11]. For each subject, one sample was collected from the site of rupture labeled [R] and the other was collected from an area distant to rupture site [D]. 2.1. Immunohistochemistry UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen kit was used following the manufacturer’s instruction. PGRMC1 antibody (Sigma) and normal rabbit IgG as negative control were used at 1:200 dilutions [4]. Ten images per slide were taken. The relative staining intensity was evaluated using a 5-point scoring system (Supplement Fig. 1) [12]. 2.2. WB PGRMC1 (1:2000); b-actin (1:6000) and secondary goat anti-rabbit IgG (1:2000) from Cell Signaling Technology were used. Image J was used for densitometry and data are presented as ratio after normalized to b-actin. Three replicates were performed for each sample. 2.3. Statistical analysis Multivariate ordinal logistic regression analysis was used to determine relative PGRMC1 expression adjusting for reader, site and measurements (the 10 scores per slides). The decidual layer and PPROM were selected as the reference groups for cell type and clinical phenotype, respectively. Adjusted ORs and 95% CI were estimated to assess the relative odds of PGRMC1 staining. For WB data, KruskaleWallis test was used for three-group comparison and the ManneWhitney test was used for two-group comparisons with significance defined as P < 0.05.
332
L. Feng et al. / Placenta 35 (2014) 331e333
3. Results The subject characteristics are summarized in Supplement Table 1. PGRMC1 was predominantly found in the cytoplasm of cells with occasional nuclear staining in all layers of FMs (Fig. 1). The agreement between readers ranged from 0.51 to 0.66, which is considered moderate to good agreement using weighted-Kappa statistic. The distribution of PGRMC1 varied considerably by clinical group and cell type. When clinical phenotypes were combined, PGRMC1 expression was the highest in amnion and the lowest in decidua (P < 0.05). When the cell types were combined, PGRMC1 expression was the lowest in PPROM subjects and the highest in PTNL subjects (P < 0.05) (Fig. 1 and Table 1A). When PGRMC1 expression was compared within each layer among PPROM, TNL and PTNL subjects, PGRMC1 expression was the highest in PTNL subjects and lowest in PPROM subjects in chorion or decidua layer (same as the combined data). In the amnion layer, PGRMC1 expression was the lowest in TNL instead of PPROM subjects (Table 1B). Table 2 summarized the comparison of PGRMC1 expression between [R] and [D] within each clinical cohort. PGRMC1 expression was lower at [R] in PPROM, higher at [R] in TNL and not different in PTNL. WB analysis also showed that PGRMC1 expression in full thickness FMs was significantly lower in PPROM subjects compared to PTNL and TNL. There was no significant difference in PGRMC1 expression in PTNL and TNL subjects (Fig. 2).
Table 1A Relative PGRMC1 staining in fetal membranes by cell type or clinical status. Predictor Cell type Decidual cells Chorion cells Amnion cells Clinical phenotype PPROM (N ¼ 10) TNL (N ¼ 9) PTNL (N ¼ 10)
Adjusted OR (95% CI)
P for trend
1.0 1.64 (1.4e1.9) 4.71 (3.9e5.7)
<0.0001
1.0 1.35 (1.15e1.59) 4.66 (3.84e5.65)
<0.0001
The covariates of the multivariate ordinal logistic model include reader, measurements, cell layer and site of collection (rupture vs. distant). The decidual cells are the reference group for the cell type and PPROM is the reference group for clinical phenotype.
Table 1B Relative PGRMC1 staining in fetal membranes by clinical phenotype in each cell layer. Clinical phenotype
PPROM (N ¼ 10) TNL (N ¼ 9) PTNL (N ¼ 10)
Amnion
Chorion
Decidua
Adjusted OR (95% CI)
Adjusted OR (95% CI)
Adjusted OR (95% CI)
1.0 0.69 (0.51e0.95) 1.93 (1.34e2.78)
1.0 2.74 (2.08e3.60) 8.74 (6.33e12.1)
1.0 1.19 (0.90e1.57) 4.92 (3.56e6.80)
The covariates of the multivariate ordinal logistic model include reader, measurements, cell layer and site of collection (rupture vs. distant). PPROM is the reference group in each cell layer.
4. Discussion We report here for the first time the localization and expression of PGRMC1 in FMs among pathologic conditions. PGRMC1 alters several survival signaling proteins, including the Akt protein kinase and the cell death-associated protein IkB [13]. PGRMC1 mediates cytokine-induced MMP expression and activity in cytotrophoblast cells [4]. MMPs are zinc-dependent endopeptidase enzymes involved in ECM remodeling. Significantly elevated levels of MMP9 expression and activity have been identified in FMs of PTL and PPROM patients [14,15]. Our findings indicate that PGRMC1 may be an important mediator of progesterone function in FMs.
Table 2 Relative PGRMC1 staining in fetal membranes by collection site. Site of collection
Distant Rupture
PPROM (N ¼ 10)
TNL (N ¼ 9)
PTNL (N ¼ 10)
Adjusted OR (95% CI)
Adjusted OR (95% CI)
Adjusted OR (95% CI)
1 0.653 (0.506e0.844) P ¼ 0.001
1 1.272 (1.032e1.567) P ¼ 0.024
1 0.936 (0.707e1.238) P ¼ 0.641
The covariates of the multivariate ordinal logistic model includes reader, measurements, cell layer.
Fig. 1. Representative images to show the differences of PGRMC1 expression in fetal membranes from each cohort. The PGRMC1 staining in PTNL and TNL cohort is much more intense compare to PPROM using specific antibody against PGRMC1. A is amnion layer; C is chorion layer; D is decidua layer. High power image demonstrate the nuclear staining and cytoplasmic staining. Abbreviations: PTNL, preterm not in labor; TNL, term not in labor; PPROM, preterm premature rupture of membranes.
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therapeutic approach for preterm birth although further study is needed. 5. Authors’ roles Dr. Liping Feng carried out all of the molecular studies for the manuscript and wrote the manuscript. Brian Antczak and Dr. Jennifer Thomson helped with collecting the human tissues and scoring the IHC staining. Lan Lan performed the statistical analysis. Dr. Terrence Allen and Chad Grotegut participated in discussions and consulted on the manuscript. Dr. Amy Murtha participated in studying design, data interpretation and writing the manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.placenta.2014.03.008. References Fig. 2. Relative expressions of PGRMC1 in fetal membranes collected from different clinical cohorts determined by western blotting. A, Representative image of western blotting. Positive control is the recombinant PGRMC1 protein with His-Tag modification. The molecular weight is slightly higher than 28 kDa. B, Densitometry is used to quantify the intensity of western blotting bands. The ratios of band intensities between PGRMC1 protein and B-actin are calculated to allow for normalization of the data. Each bar represents the average SEM of all samples for each cohort. There were statistically significant differences (P ¼ 0.009) between three groups. When two groups are compared, PGRMC1 expression is statistically significant less in PPROM compared to TNL and PTNL (P ¼ 0.004 and 0.017 respectively) groups. There were no statistical significant differences between TNL and PTNL (P ¼ 0.675); Abbreviations: PTNL, preterm not in labor; TNL, term not in labor; PPROM, preterm premature rupture of membranes.
Furthermore, diminished PGRMC1 at the rupture site among PPROM subjects may be associated with the FM rupture. In TNL subjects PGRMC1 expression was higher at the rupture site and no difference in PTNL subjects. We speculate three potential biases that limit our ability to draw firm conclusions. First, PTNL patients usually are complicated by pathological conditions such as preeclampsia and the implications of these conditions on PGRMC1 expression are unknown. However, it is not possible to obtain PTNL FM from uncomplicated pregnancy; Second, none of TNL and PTNL patients experienced labor which may impact these changes. And it is not established whether PGRMC1 expression is altered in the setting of labor; Finally, the rupture site tissues we collected from cesarean delivery might not yet exhibit biochemical and mechanical characteristics of rupture at the time of surgery. Using immunohistochemistry allows us to quantify and localize PGRMC1 protein expression in each cell layer. Simultaneously, we considered the weaknesses of immunohistochemistry for protein quantification. Therefore, we minimized staining batches with randomly mixed clinical groups. Multiple images for each slide were evaluated by two independent blinded evaluators to limit the impact of staining and processing variability. Finally, results of WB further support the findings that PGRMC1 expression is the lowest in FMs from PPROM subjects. Targeting PGRMC1 can be a specific
[1] Canzoneri BJ, Feng L, Grotegut CA, Bentley RC, Heine RP, Murtha AP. The chorion layer of fetal membranes is prematurely destroyed in women with preterm premature rupture of the membranes. Reprod Sci 2013;20:1246e54. [2] Menon R, Fortunato SJ. The role of matrix degrading enzymes and apoptosis in rupture of membranes. J Soc Gynecol Investig 2004;11:427e37. [3] Luo G, Abrahams VM, Tadesse S, Funai EF, Hodgson EJ, Gao J, et al. Progesterone inhibits basal and TNF-alpha-induced apoptosis in fetal membranes: a novel mechanism to explain progesterone-mediated prevention of preterm birth. Reprod Sci 2010;17:532e9. [4] Allen TK, Feng L, Grotegut CA, Murtha AP. Progesterone receptor membrane component 1 as the mediator of the inhibitory effect of progestins on cytokine-induced matrix metalloproteinase 9 activity in vitro. Reprod Sci 2014;21:260e8. [5] Murtha AP, Feng L, Yonish B, Leppert PC, Schomberg DW. Progesterone protects fetal chorion and maternal decidua cells from calcium-induced death. Am J Obstet Gynecol 2007;196. 257 e251e255. [6] Merlino A, Welsh T, Erdonmez T, Madsen G, Zakar T, Smith R, et al. Nuclear progesterone receptor expression in the human fetal membranes and decidua at term before and after labor. Reprod Sci 2009;16:357e63. [7] Peluso JJ, Liu X, Saunders MM, Claffey KP, Phoenix K. Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1. J Clin Endocrinol Metab 2008;93:1592e9. [8] Peluso JJ. Non-genomic actions of progesterone in the normal and neoplastic mammalian ovary. Semin Reprod Med 2007;25:198e207. [9] Crudden G, Loesel R, Craven RJ. Overexpression of the cytochrome p450 activator hpr6 (heme-1 domain protein/human progesterone receptor) in tumors. Tumour Biol 2005;26:142e6. [10] Peluso JJ, Romak J, Liu X. Progesterone receptor membrane component-1 (PGRMC1) is the mediator of progesterone’s antiapoptotic action in spontaneously immortalized granulosa cells as revealed by PGRMC1 small interfering ribonucleic acid treatment and functional analysis of PGRMC1 mutations. Endocrinology 2008;149:534e43. [11] fortner K, Ransom C, canzoneri B, Bentley R, Feng L, Lan L, et al. Bacteria localization and chorion thinning among preterm premature rupture of membranes. PloS One 2013;9(1). http://dx.doi.org/10.1371/journal.pone.0083338. e83338. [12] Kraus DM, Feng L, Heine RP, Brown HL, Caron KM, Murtha AP, et al. Cigarette smoke-induced placental adrenomedullin expression and trophoblast cell invasion. Reprod Sci 2014;21:63e71. [13] Debose-Boyd RA. A helping hand for cytochrome p450 enzymes. Cell Metab 2007;5:81e3. [14] Fortunato SJ, Menon R. Distinct molecular events suggest different pathways for preterm labor and premature rupture of membranes. Am J Obstet Gynecol 2001;184:1399e406. [15] Xu P, Alfaidy N, Challis JRG. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in human placenta and fetal membranes in Relation to preterm and term labor. J Clin Endocrinol Metab 2002;87:1353e61.