Loss of MIG-6 results in endometrial progesterone resistance via ERBB2

Female subfertility is highly associated with endometriosis. Endometrial progesterone resistance is suggested as a crucial element in the development of endometrial diseases. We report that MIG-6 is downregulated in the endometrium of infertile women with endometriosis and in a non-human primate model of endometriosis. We find ERBB2 overexpression in the endometrium of uterine-specific Mig-6 knockout mice (Pgrcre/+Mig-6f/f; Mig-6d/d). To investigate the effect of ERBB2 targeting on endometrial progesterone resistance, fertility, and endometriosis, we introduce Erbb2 ablation in Mig-6d/d mice (Mig-6d/dErbb2d/d mice). The additional knockout of Erbb2 rescues all phenotypes seen in Mig-6d/d mice. Transcriptomic analysis shows that genes differentially expressed in Mig-6d/d mice revert to their normal expression in Mig-6d/dErbb2d/d mice. Together, our results demonstrate that ERBB2 overexpression in endometrium with MIG-6 deficiency causes endometrial progesterone resistance and a nonreceptive endometrium in endometriosis-related infertility, and ERBB2 targeting reverses these effects.

More incisive data are then shared from a mouse model: in uterine-specific Mig-6 knock-out mice data are presented to demonstrate that MIG-6 loss results in endometrial progesterone resistance via ERBB2. Specifically, Mig-6 loss exacerbates development of endometriosis and implantation failure in the mouse model and there is an associated increased expression in ERBB2. When there is additional ablation of ERBB2 the phenotype is rescued. The supplementary data are useful inclusions.
The microarray studies reveal genes observed to be differentially expressed in Mig-6d/d mice reverted to normal expression amounts with additional Erbb2 ablation in Mig-6d/dErbb2d/d mice.
The authors conclude that their data support MIG-6-induced ERBB2 overexpression contributing to to endometrial progesterone resistance and thus a role in endometriosis-related infertility. In their mouse model, targeting ERBB2 reverses the phenotype.
Significance to the field: From a clinical perspective the focus is on endometriosis and mechanisms underpinning associated female subfertility where there are many lines of evidence suggesting progesterone resistance. Usually the impact of progesterone resistance is reported against a context of impact upon decidualization: herein there are very interesting data presented concerning the impact on epithelial proliferation, and consequently endometrial function of relevance to fertility/ subfertility.

Context with published literature:
The introduction is succinct presenting the case of endometrial progesterone-resistance as a phenomenon in women with endometriosis. This is supported with published data from animal models. The dearth of data concerning mechanisms involved is highlighted and thus where the present data make a highly important contribution. The data build further upon earlier work that indicate Mig-6 is a progesterone-regulated gene that mediates progesterone repression of estrogen action in the mouse uterus (Jeong JW, et al. Endocrinology, 2005;Jeong JW, et al. Proc Natl Acad Sci ,2009).

Methodology:
Research ethical approvals are in place for human and animal studies. The "phenotyping" of participants is a crucial component of this study pertaining to progesteroneresistance in women with endometriosis. The dating of the endometrium from women with and without endometriosis is based upon histology. If available, further support for important menstrual cycle staging would be provided by serum sex hormone (estradiol, progesterone) at the time of endometrial biopsy. Please clarify if any other commonly present uterine myometrial confounders, also reported to be associated with potential progesterone-resistance, i.e. small uterine leiomyoma and adenomyosis were present in any of the participants from whom endometrium was sampled. RT-q-PCR and immunohistochemistry (IHC) are standard. For the IHC please clarify negative controls employed. For the microarray studies: how was quality of RNA established?
Results/ figures: Circulating serum progesterone concentrations are highest in the early and mid-secretory (ES, MS) phases with a physiological decline in notably progesterone in the late-secretory (LS) phase. In Figure 1A it is noted MIG-6 expression is highest in the ES: please comment further here as if a progesterone-induced gene -would not levels also be expected to be higher in MS stage? In Figure 1A even though in the figure legend it is stated n> 3 per stage please give n= per stage. In Figure 1B please clarify in legend the stage of the menstrual cycle H-scores were measured. Please enhance legends of figures 2B; 2C; 4E to explain the fluorescence photomicrographs. Figure 3: provides very clear support in the mouse for the impact upon epithelial proliferation and rescue of implantation. Figure 6: depicts the proposed molecular mechanisms of MIG-6 function in the uterus. This summarises the important contributions in the manuscript and notably the impact upon epithelial proliferation. The further pathways/ mechanisms precisely responsible for subfertility in women with endometriosis still require to be established: hence this should be made clear. Supplementary data illustrate an impressive reversal of endometrial epithelial hyperplasia.

Reviewer #2 (Remarks to the Author):
In endometriosis patients, overall P4-effects on the endometrium are diminished (P4 resistance). MIG-6 is one of the P4-induced genes in the endometrium, mediating anti-proliferation effects of P4 on the epithelial cells. In this study, the authors investigate the functions of MIG-6 in the pathogenesis of endometriosis and endometriosis-associated fertility problems. The authors discovered that MIG-6 expression was reduced in the endometrium of infertile women diagnosed with endometriosis. In the induced-endometriosis models of non-human primates and mice, MIG-6 expression in the eutopic endometrium progressively decreased as endometriosis lesions grew. In the mouse endometrium, the deletion of Mig-6 by Pgr-Cre blunted the epithelial response to P4, but the co-deletion of Erbb2 corrected the epithelial defects. Therefore, the authors concluded that MIG-6 downregulation and subsequent ERBB2 upregulation contribute to the P4 resistance of the endometriotic endometrium.
The authors have already shown that MIG-6 is a crucial mediator of P4 action in the endometrium by utilizing the same mouse model. Thus, this study's primary finding is that P4 represses endometrial epithelial proliferation in part by repressing ERBB2 expression via upregulation of MIG-6. Given P4 resistance is one of the hallmarks, all P4-regulated genes, including MIG-6, are expected to be downregulated in the eutopic and ectopic endometria of endometriotic women. The current study confirms this idea. MIG-6 is a negative regulator of EGF pathways known to reduce the expression levels EGFRs, including ERBB2, by promoting internalization and degradation via binding to the intracellular domain. Thus, MIG-6 downregulation and subsequent ERBB2 overexpression confirm the well-accepted molecular functions of MIG-6. Overall, this study supports the well-accepted concept that the loss of P4 pathway activity causes endometriosis. The impact would have been higher if this study provided insights into the mechanism of MIG-6 downregulation in the endometrium by endometriosis progression. The study's impact would also increase by testing If ERBB2 null mutation can rescue PGR-null mice's endometrial phenotypes.
The fundamental role of P4 in the endometrium is to counteract against E2 actions. The authors should have analyzed the effects of E2 alone in the endometrium of normal and mutant mice. Without this baseline, the P4 effect cannot be accurately evaluated. The expression patterns of PGR and ESR1 in the uterus under different hormonal conditions should also be compared among mice in different genotypes.
The inclusion of Erbb2 d/d mice would help understand the role of MIG-6 and ERBB2 in normal uterine functions.
Since Pgr-Cre is expressed in the ovary, the reproductive phenotypes of Mig-6 d/d and Mig6 d/d Erbb2 d/d mice cannot be attributed solely to the uterine defects. The ovarian phenotypes of knockout mice should be included in the study.
The recombination efficiency of Pgr-Cre is not 100%. Incomplete deletion would increase when 4 floxed alleles are simultaneously targeted. To establish the dispensability of MIG6 and ERBB2 in implantation, the loss of ERRB2 and MIG-6 in the implantation sites of double knockout mice should be tested by IHC. Figure S2 indicates mice who received the endometriosis-inducing surgery showed two distinct responses; the decidual response was retained and lost. The authors should analyze the correlations between the number of implantation sites and the number/size of endometriosis lesions. Figure 2B. As assessed by the brightness in the areas with no tissues (e.g., epithelial lumen and large vessels), the background signal levels are significantly different among pictures. How did the authors adjust the background levels in different samples? The methods do not describe the adjustment of the background. The difference between EGFR and ERBB2 likely reflects the difference in the tissues expressing these receptors. The authors may consider including EGFR IHC. Figure 3 analyzes the expression levels of epithelial genes. As Figure 3B shows, the epithelium amount is significantly higher in Mig-6 d/d mice than the other two genotypes. The analyses would be more accurate if the transcript levels are normalized against epithelial-genes, such as Ecadherin and Keratin-18.
The rationale of the experiment presented in Figure 4 D and E is unclear. P4 resistance, including the downregulation of MIG-6, is a consequence of endometriosis development, as shown in Figure  1 D and E. Do the authors propose an alternative model in which P4 resistance causes endometriosis?

Reviewer #3 (Remarks to the Author):
This study implicates MIG-6 as a critical mediator of endometriosis that functions through ERBB2. The study describes and refines several novel animal models and represents a major new contribution to the field of endometriosis research. The rescue of MIG-6 phenotypes by simultaneous ERBB2 inactivation is a stunning and elegant genetic result that strongly substantiates the scientific claims made in this study. The authors deserve credit for this result and also for the inclusion of data from human specimens and also from a non-human primate model of endometriosis that together make for a very strong study that clearly merits publication in Nature Communications. Also, these results are a significant advance over prior work on MIG-6 that has largely been limited to mouse genetic models. The manuscript is well-written and the results clearly presented, and the conclusions well-substantiated by the data. The supplementary data is of very high quality and adds to the study. However, there are some minor technical and scientific issues that merit some attention prior to publication: Introduction-clear and succinct, but the authors should present more background information on MIG-6 for a general readership; e.g. type of protein, regulation, biological context, etc.  Finally, is ERBB2 expresion aberrant in the human and primate models?
We sincerely thank the editor and the reviewers for carefully reviewing this manuscript and for the insightful comments, which have helped improve the quality of the manuscript. We are pleased with the positive comments of the reviewers.

POLICIES AND FORMS REQUIRED FOR RESUBMISSION
We have uploaded "Editorial policy checklist" and "Reporting summary".

DATA AND CODE AVAILABILITY
Our microarray datasets have been deposited to Gene Expression Omnibus (accession code: GSE138185). It is described in "Data Availability" section.
To maximize the reproducibility of research data, we have provided an excel file containing the raw data.

We also provided uncropped versions of blots for Supplementary Figures 4 and 5 in Supplementary Figures 13.
We have replaced our bar graphs with plots that feature information about the distribution of the underlying data.

ORCID
We provided ORCID for all authors.

Overall Comment
This is a clearly written manuscript addressing the issue of endometrial progesterone resistance. The main objective is to provide data on role of MIG-6 in endometrial progesterone-resistance and thereby contribute to understanding further the molecular mechanisms responsible for endometrial progesterone resistance, notably in the context of endometriosis.
It is very interesting to see these data identifying the impact of disturbed progesterone signalling upon epithelial proliferation, rather than solely decidualization: the latter so often the case.
Data are presented from human endometrial biopsies and two animal models (non-human primate (NHP, baboon) and mouse) permitting interrogation of endometrial responses to modulation of a progesterone-induced gene, MIG-6. In endometrium from infertile women with endometriosis, NHP model of endometriosis, MIG-6, is downregulated. In contrast, in mice modeled with endometriosis and with Mig-6 deficient endometrium an increase of endometriosis development and implantation failure is described.
Observational data in Figure 1 present the case for the association of loss of MIG-6 and the presence of endometriosis in women and the NHP (baboon).
More incisive data are then shared from a mouse model: in uterine-specific Mig-6 knock-out mice data are presented to demonstrate that MIG-6 loss results in endometrial progesterone resistance via ERBB2. Specifically, Mig-6 loss exacerbates development of endometriosis and implantation failure in the mouse model and there is an associated increased expression in ERBB2. When there is additional ablation of ERBB2 the phenotype is rescued. The supplementary data are useful inclusions.
The microarray studies reveal genes observed to be differentially expressed in Mig-6d/d mice reverted to normal expression amounts with additional Erbb2 ablation in Mig-6d/dErbb2d/d mice.
The authors conclude that their data support MIG-6-induced ERBB2 overexpression contributing to to endometrial progesterone resistance and thus a role in endometriosisrelated infertility. In their mouse model, targeting ERBB2 reverses the phenotype.
Significance to the field: From a clinical perspective the focus is on endometriosis and mechanisms underpinning associated female subfertility where there are many lines of evidence suggesting progesterone resistance. Usually the impact of progesterone resistance is reported against a context of impact upon decidualization: herein there are very interesting data presented concerning the impact on epithelial proliferation, and consequently endometrial function of relevance to fertility/ subfertility.

Context with published literature:
The introduction is succinct presenting the case of endometrial progesterone-resistance as a phenomenon in women with endometriosis. This is supported with published data from animal models. The dearth of data concerning mechanisms involved is highlighted and thus where the present data make a highly important contribution. The data build further upon earlier work that indicate Mig-6 is a progesterone-regulated gene that mediates progesterone repression of estrogen action in the mouse uterus (Jeong JW, et al. Endocrinology, 2005;Jeong JW, et al. Proc Natl Acad Sci ,2009).
Response: We are pleased with the positive comment.

Methodology:
C1. Research ethical approvals are in place for human and animal studies.
The "phenotyping" of participants is a crucial component of this study pertaining to progesterone-resistance in women with endometriosis.

R1. Thank you
C2. The dating of the endometrium from women with and without endometriosis is based upon histology. If available, further support for important menstrual cycle staging would be provided by serum sex hormone (estradiol, progesterone) at the time of endometrial biopsy. C3. Please clarify if any other commonly present uterine myometrial confounders, also reported to be associated with potential progesterone-resistance, i.e. small uterine leiomyoma and adenomyosis were present in any of the participants from whom endometrium was sampled.

Results/ figures:
C5. Circulating serum progesterone concentrations are highest in the early and mid-secretory (ES, MS) phases with a physiological decline in notably progesterone in the late-secretory (LS) phase. In Figure 1A it is noted MIG-6 expression is highest in the ES: please comment further here as if a progesterone-induced gene -would not levels also be expected to be higher in MS stage? (PMID: 15845616). Mig-6 was identified as a rapidly induced gene by acute P4 treatment (4 hours), but its induction was not continued with chronic P4 treatment (40 hours). As we observed in the mouse uterus, the expression of MIG-6 was significantly increased in human endometrium at early secretory phase compared to proliferative phase, but this induction was not observed at Mid-secretory phase. These results suggest that MIG-6 is an acute P4 response gene in both the human and mouse endometrium.

R5. This is a very important comment. We used transcriptomic analysis to identify alterations in gene expression after acute and chronic P4 treatment in the mouse uterus
C6. In Figure 1A even though in the figure legend it is stated n> 3 per stage please give n= per stage. Fig. 1A. In addition, the bar graph has been replaced with dot plots to clearly show the sample number as well as the data distribution.

R6. We have provided exact sample number for
C7. In Figure 1B please clarify in legend the stage of the menstrual cycle H-scores were measured.

R7. The samples were from early secretory phase women.
C8. Please enhance legends of figures 2B; 2C; 4E to explain the fluorescence photomicrographs. Figure  C9. Figure 3: provides very clear support in the mouse for the impact upon epithelial proliferation and rescue of implantation.

R9. Thank you for this encouraging comment.
C10. Figure 6: depicts the proposed molecular mechanisms of MIG-6 function in the uterus. This summarises the important contributions in the manuscript and notably the impact upon epithelial proliferation. The further pathways/ mechanisms precisely responsible for subfertility in women with endometriosis still require to be established: hence this should be made clear.

R10.
We agree with the reviewer. We have added this comment for the further study in the Discussion. " Figure 8 depicts our proposed molecular mechanisms of MIG-6 function in endometrial P4 resistance and associated infertility and how P4 responsiveness and fertility can be restored. However, the further pathways and mechanisms precisely responsible for subfertility in women with endometriosis still require to be established." (See Line 336 -339).

Overall Comment
In endometriosis patients, overall P4-effects on the endometrium are diminished (P4 resistance). MIG-6 is one of the P4-induced genes in the endometrium, mediating antiproliferation effects of P4 on the epithelial cells. In this study, the authors investigate the functions of MIG-6 in the pathogenesis of endometriosis and endometriosis-associated fertility problems. The authors discovered that MIG-6 expression was reduced in the endometrium of infertile women diagnosed with endometriosis. In the induced-endometriosis models of non-human primates and mice, MIG-6 expression in the eutopic endometrium progressively decreased as endometriosis lesions grew. In the mouse endometrium, the deletion of Mig-6 by Pgr-Cre blunted the epithelial response to P4, but the co-deletion of Erbb2 corrected the epithelial defects. Therefore, the authors concluded that MIG-6 downregulation and subsequent ERBB2 upregulation contribute to the P4 resistance of the endometriotic endometrium.
The authors have already shown that MIG-6 is a crucial mediator of P4 action in the endometrium by utilizing the same mouse model. Thus, this study's primary finding is that P4 represses endometrial epithelial proliferation in part by repressing ERBB2 expression via upregulation of MIG-6.
Given P4 resistance is one of the hallmarks, all P4-regulated genes, including MIG-6, are expected to be downregulated in the eutopic and ectopic endometria of endometriotic women. The current study confirms this idea. MIG-6 is a negative regulator of EGF pathways known to reduce the expression levels EGFRs, including ERBB2, by promoting internalization and degradation via binding to the intracellular domain. Thus, MIG-6 downregulation and subsequent ERBB2 overexpression confirm the well-accepted molecular functions of MIG-6.
Overall, this study supports the well-accepted concept that the loss of P4 pathway activity causes endometriosis.
Response: We are pleased with the positive comment.
C1. The impact would have been higher if this study provided insights into the mechanism of MIG-6 downregulation in the endometrium by endometriosis progression.

R1. We found that the attenuation of MIG-6 in endometriosis and MIG-6 loss causes infertility due to an implantation failure in the mouse uterus. Our animal models suggest that endometriosis results in MIG-6 loss in the eutopic endometrium of women with endometriosis. Endometriosis changes immune function, cytokines and inflammatory signals that may lead to progesterone resistant conditions including MIG-
6 loss in the eutopic endometrium. However, we could not demonstrate the mechanism of MIG-6 loss in the endometrium over endometriosis progression. Identification of the mechanism how endometriosis leads to MIG-6 downregulation in the endometrium is important to understand the pathophysiology of progesterone resistance in endometriosis-related infertility.
C2. The study's impact would also increase by testing If ERBB2 null mutation can rescue PGRnull mice's endometrial phenotypes.
R2. Taking the Reviewers' advice, we have generated Pgr cre/cre Erbb2 f/f and Pgr cre/f Erbb2 f/f mice to determine the effect of ERBB2 ablation in PRKO mice. However, ERBB2 null mutation did not rescue the phenotype of implantation failure in PRKO mice (see Suppl. Fig. 08).
C3. The fundamental role of P4 in the endometrium is to counteract against E2 actions. The authors should have analyzed the effects of E2 alone in the endometrium of normal and mutant mice. -Without this baseline, the P4 effect cannot be accurately evaluated. Fig. 1E; Fig. 3C R3. Taking Fig. 09). Therefore, we have not included the E2 alone condition in molecular analysis.
C4. The expression patterns of PGR and ESR1 in the uterus under different hormonal conditions should also be compared among mice in different genotypes.

R4. In previous study, we showed that the expressions of PGR and ESR1
were not changed in the uteri of Mig-6 d/d mice compared to control mice at GD 3.5. As we expected, the expression levels of PGR and ESR1 were not changed in the uteri of Erbb2 d/d and Mig-6 d/d Erbb2 d/d mice compared to control mice at GD 3.5 (Suppl. Fig. 06).
C5. The inclusion of Erbb2 d/d mice would help understand the role of MIG-6 and ERBB2 in normal uterine functions. Figure 4, 5, 6, S6, S7, S9 and S11. Erbb2 d/d mice show normal uterine phenotypes like control mice.

R5. Taking the Reviewer's advice, we have included Erbb2 d/d mice in
C6. Since Pgr-Cre is expressed in the ovary, the reproductive phenotypes of Mig-6 d/d and Mig6 d/d Erbb2 d/d mice cannot be attributed solely to the uterine defects. The ovarian phenotypes of knockout mice should be included in the study. Figure 4A). These data indicate that

Mig-6 d/d Erbb2 d/d and Erbb2 d/d mice have a normal ovarian function.
C7. The recombination efficiency of Pgr-Cre is not 100%. Incomplete deletion would increase when 4 floxed alleles are simultaneously targeted. To establish the dispensability of MIG6 and ERBB2 in implantation, the loss of ERRB2 and MIG-6 in the implantation sites of double knockout mice should be tested by IHC. Figure 4, S4, and S5. These data support appropriate recombination efficiency of Pgr-Cre.

R7. We have confirmed the loss of MIG-6 and ERBB2 expression in the uteri of Mig-6 d/d and Mig-6 d/d Erbb2 d/d mice at GD3.5 by IHC, RT-qPCR and Western blot analyses as shown in
C8. Figure S2 indicates mice who received the endometriosis-inducing surgery showed two distinct responses; the decidual response was retained and lost. The authors should analyze the correlations between the number of implantation sites and the number/size of endometriosis lesions.
R8. This is a very insightful comment. We have examined the possible correlation between the number of implantation sites and the number of endometriosis lesions. However, no significant correlation exists between the number of implantation sites and the number of endometriosis lesions (Suppl. Fig. 03).
C9. Figure 2B. As assessed by the brightness in the areas with no tissues (e.g., epithelial lumen and large vessels), the background signal levels are significantly different among pictures. How did the authors adjust the background levels in different samples? The methods do not describe the adjustment of the background.
R9. The images were taken with exactly the same parameters, including exposure time, and the background of images was not adjusted. We think the background is darker in some images because MIG-6 proteins are expressed in cytoplasm.
C10. Fig. S3. The finding that EGFR is not overexpressed in the uterus of MIG-6 d/d mice is intriguing. The difference between EGFR and ERBB2 likely reflects the difference in the tissues expressing these receptors. The authors may consider including EGFR IHC. Fig. S4. As in the Western blot result, the results of EGFR IHC show that level of EGFR was not changed in in the uteri of Mig-6 d/d mice.

R10. Taking the Reviewer's advice, we have included EGFR IHC in
C11. Figure 3 analyzes the expression levels of epithelial genes. As Figure 3B shows, the epithelium amount is significantly higher in Mig-6 d/d mice than the other two genotypes. The analyses would be more accurate if the transcript levels are normalized against epithelial-genes, such as E-cadherin and Keratin-18.

R11. Taking the Reviewer's advice, we have confirmed our RT-qPCR results by normalizing against E-cadherin gene expression (see below results
). In addition, there should not be many epithelial cells in normal post-implantation stage (gestation day 5.5) control mice (Fig. 4B, a and b) and Mig-6 d/d Erbb2 d/d mice (Fig. 4B, e and f). However, the phenotype of implantation failure in Mig-6 d/d mice revealed more epithelial cells due to no luminal closure at GD 5.5.
C12. The rationale of the experiment presented in Figure 4 D and E is unclear. P4 resistance, including the downregulation of MIG-6, is a consequence of endometriosis development, as shown in Figure 1 D and E. Do the authors propose an alternative model in which P4 resistance causes endometriosis?
R12. Yes, we determined the effect of MIG-6 loss on endometriosis development. According to Sampson's theory of retrograde menstruation, menstrual blood containing endometrial cells flows backward through the fallopian tubes and into the pelvic cavity rather than out of the body. These endometrial cells that should have been shed during menstruation can then lead to implantation and further spreading of endometriosis lesions. We found that Mig-6 d/d Rosa26 mTmG/+ mice had a significantly increased incidence of endometriotic lesions compared to control mice. Therefore, MIG-6 loss in eutopic endometrium by endometriosis accelerates progress of this disease.

Overall Comment
This study implicates MIG-6 as a critical mediator of endometriosis that functions through ERBB2. The study describes and refines several novel animal models and represents a major new contribution to the field of endometriosis research. The rescue of MIG-6 phenotypes by simultaneous ERBB2 inactivation is a stunning and elegant genetic result that strongly substantiates the scientific claims made in this study. The authors deserve credit for this result and also for the inclusion of data from human specimens and also from a non-human primate model of endometriosis that together make for a very strong study that clearly merits publication in Nature Communications. Also, these results are a significant advance over prior work on MIG-6 that has largely been limited to mouse genetic models. The manuscript is well-written and the results clearly presented, and the conclusions well-substantiated by the data. The supplementary data is of very high quality and adds to the study. However, there are some minor technical and scientific issues that merit some attention prior to publication: Response: We are pleased with the positive comment. We have tried our best to address minor technical and scientific issues in the manuscript.
C1. Introduction-clear and succinct, but the authors should present more background information on MIG-6 for a general readership; e.g. type of protein, regulation, biological context, etc.

R1.
We have added more detailed information on MIG-6 to the Introduction.
C2. Fig. 1. Does the gradual loss of MIG-6 in the non-human primate model correlate with important biological parameters (proliferation rate by Ki67, secretory changes, etc.)?
R2. We observed increased ERBB2 in the eutopic endometrium from the same baboons over time and with endometriosis progression. There are reverse correlations between MIG-6 and ERBB2 proteins in Figure 6C.
C3. Fig. 2. Since panel A represents quantitation of the images in panel b, it might make more sense to switch panel order. Fig. 3.

R3. Taking the Reviewer's advice, we have switched the panel order in
C4. Fig. 4 and elsewhere. It would be worthwhile for the authors to present data on ER and PR expression in their various models as this could be related to the underlying biology and observations.
R4. Taking the Reviewer's advice, we have examined the levels of PGR and ESR1 in the uteri from our mutant mice at GD 3.5 (Suppl. Fig. 6) as well as ovariectomized mice treated with E2 plus P4 for 3 days (Suppl. Fig. 7). The results have been added to the manuscript.
C5. Finally, is ERBB2 expression aberrant in the human and primate models?
R5. This is a very important question. We have examined the expression of ERBB2 in the induced endometriosis of non-human primates. We observed increased ERBB2 in the eutopic endometrium from the same baboons over time and with endometriosis progression. There are reverse correlations between MIG-6 and ERBB2 proteins in Figure 6.

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): As a previous reviewer I consider the points I raised in review have been addressed. I have a further minor comment: Concerning: Page 30 from 42 (in combined pdf) lines 731-4 Figure 1. MIG-6 expression in the endometrium of women with endometriosis and nonhuman primate, baboon model. (A), RT-qPCR analysis of MIG-6 gene expression in endometrium from women with and without endometriosis during the menstrual cycle (n ≥ 3 for each group). Further clarity in legend is required as two sets of RT-qPCR analysis of MIG-6 gene expression are presented.
Overall: the major finding in current manuscript is an important addition to the field, i.e. that progesterone represses endometrial epithelial proliferation, involving ERBB2 expression, via upregulation of MIG-6. These current data presented build further upon earlier work that indicate Mig-6 is a progesterone-regulated gene that mediates progesterone repression of estrogen action in the mouse uterus. As noted in my previous review, usually the impact of progesterone resistance is reported in the context of impact upon decidualization. These are very interesting data concerning the impact on epithelial proliferation, and consequently endometrial function of relevance to fertility/ subfertility.

Reviewer #2 (Remarks to the Author):
The authors satisfactorily addressed the critiques of the reviewers. Nevertheless, this reviewer recommends a minor revision as new data in the revised manuscript suggest a novel mechanism that has not been discussed before; the PGR-MIG6-ERBB2 signaling pathway makes a positive feedback loop. In previous studies, the authors showed P4 signal transduction from PGR to ERBB2 via MIG6. Surprisingly, the loss of MIG6 repressed the expression of PGR in both uterine epithelial and stromal cells when mice were treated with E2+P4 for 3 days ( Figure S7). This observation indicates that the repression of ERBB2 by MIG6 potentiates P4 action on the uterine cells by maintaining the PGR expression. Therefore, the authors should discuss the potential significance of the feedback loop in pregnancy and endometriosis. In addition, the loss of ERBB2 restored PGR expression in both epithelial and stromal cells of the MIG6 null uterus, although ERBB2 expression is restricted to the epithelium. This observation confirms the notion that epithelial-stromal tissue communications regulate PGR in the mouse uterus (PMID: 10727249, PMID: 26858409). Thus, the discussion will be deepened if the authors address the role of cross-talk between epithelial and stromal cells in the PGR-MIG6-ERBB2 signaling pathway. This reviewer also recommends the