Prolactin at moderately increased levels confers a neuroprotective effect in non-secreting pituitary macroadenomas.

CONTEXT: Prolactin, a hormone synthesized by the anterior pituitary gland demonstrates promise as a neuroprotective agent, however, its role in humans and in vivo during injury is not fully understood.
OBJECTIVE: To investigate whether elevated levels of prolactin attenuate injury to the retinal nerve fiber layer (RNFL) following compression of the optic chiasm in patients with a prolactin secreting pituitary macroadenoma (i.e., prolactinoma). DESIGN SETTING AND PARTICIPANTS: A retrospective cross-sectional study of all pituitary macroadenoma patients treated at a single institution between 2009 and 2019. MAIN OUTCOME MEASURE(S): Primary outcome measures included RNFL thickness, mean deviation, and prolactin levels for both prolactin-secreting and non-secreting pituitary macroadenoma patients.
RESULTS: Sixty-six patients met inclusion criteria for this study (14 prolactin-secreting and 52 non-secreting macroadenoma patients). Of 52 non-secreting macroadenoma patients, 12 had moderate elevation of prolactin secondary to stalk effect. Patients with moderate elevation in prolactin demonstrated increased RNFL thickness compared to patients with normal prolactin levels (p < 0.01). Additionally, a significant positive relation between increasing levels of prolactin and RNFL thickness was identified in patients with moderate prolactin elevation (R = 0.51, p-value = 0.035). No significant difference was identified between prolactinoma patients and those with normal prolactin levels.
CONCLUSIONS: Moderately increased serum prolactin is associated with increased RNFL thickness when compared to controls. These associations are lost when serum prolactin is < 30 ng/ml or elevated in prolactinomas. This suggests a neuroprotective effect of prolactin at moderately increased levels in preserving retinal function during optic chiasm compression.

Introduction

Pituitary adenomas account for approximately 15% of all intracranial tumors [1]. These tumors can cause hormonal derangements by either the over- or under-production of pituitary hormones. If the tumor is sufficiently large, patients can present with a stereotyped vision loss secondary to tumor impingement on the optic nerves, chiasm or tract [1, 2]; and prolonged compression of these structures leads to a characteristic thinning of the retinal nerve fiber layer (RNFL) [3]. For many patients, decompression of the anterior visual pathway by tumor removal (with surgery) or tumor shrinkage (with medication) can result in rapid visual recovery–although the factors that predict which patients experience recovery and to what extent they will recover remains an active area of investigation. RNFL thickness–a surrogate for retinal ganglion cell axonal health–has been used as a biomarker for indexing various mechanisms of delayed axonal degeneration [4], including white matter injury following anterior visual pathway compression [5-7]. Here, we investigate the relation between serum hormone levels of prolactin, RNFL thickness and visual function in a retrospective cohort of pituitary macroadenoma patients.

Prolactin (PRL), a hormone synthesized in the anterior pituitary gland and associated with lactation, also demonstrates diverse physiologic functions, including processes that mediate neuroprotection. Prolactin has been implicated in oligodendrocyte progenitor cell proliferation [8], neurotrophic factor release [9], and increased white matter volume [10]. Together, these studies highlight the various neuroprotective roles of PRL; yet PRL’s role in humans and in vivo during injury is not fully understood. By studying the effects of compression on visual pathway structures, we can explore the relation between increasing PRL levels and retinal ganglion cell axonal health.

Prolactin can be elevated one of two ways: 1) excess production from lactotroph cells secondary to growth of a prolactinoma, or 2) a decrease in the inhibition of prolactin secretion resulting from a physiologic block of dopamine delivery from the hypothalamus through the pituitary stalk–a phenomenon known as “stalk effect”. This phenomenon is seen with mass effect from non-secreting macroadenomas. [11, 12]

The present study identified two groups for investigation: patients with macro-prolactinomas (PRO), and control patients with non-secreting (NS) macroadenomas. The control group was further subdivided into those non-secreting macroadenomas with hyperprolactinemia (NS+) from stalk effect, and those non-secreting macroadenoma cases without hyperprolactinemia (NS). This natural variation in serum prolactin allows for the study of varying levels of prolactin on measures of retinal ganglion cell axonal integrity and visual function. We hypothesized that increased serum levels of PRL are associated with a decrease in secondary injury from compression as measured by RNFL thickness.

Materials and methods

All patients with pituitary tumors treated at the University of Rochester Multidisciplinary Pituitary Clinic between 2009 and 2019 were evaluated for inclusion in this study. Inclusion criteria for both the prolactinoma and control groups were age greater than 18, pituitary tumor >1 cm in any dimension (i.e., macroadenoma), serum prolactin level recorded at time of diagnosis, and ophthalmologic testing performed at or after diagnosis. Both Male and Female patients were included in the study, as defined by their biological sex on chart review. Macroadenomas were defined based on volume, independent of the cell of origin or serum prolactin level as is standard in the literature [13]. Of 2,728 patients treated at our institution (including both micro and macroadenoma patients), 239 were identified to have both Humphrey 24–2 perimetry and spectral domain optical coherence tomography (SD-OCT), which measures the peripapillary retinal nerve fiber layer. Of those patients, 66 were macroadenoma patients. Control patients were chosen from the population of non-secreting pituitary tumor patients. The study protocol was approved by the institutional review board of the University of Rochester and the need for consent was waived. All data were anonymized before being accessed.

Power analysis

A power analysis was conducted based on the initial hypothesis that elevated levels of prolactin in prolactin-secreting macroadenoma patients will demonstrate attenuation of injury to the retinal nerve fiber layer compared with non-secreting macroadenoma patients. Previous research indicates that pre-operative mean RNFL thickness in pituitary macroadenoma patients (standard deviation) is 81.9 μm (8.8) [14]. We anticipated a similar mean RNFL thickness for non-secreting pituitary macroadenoma patients in this study. For patients with elevated prolactin, we anticipated a mean RNFL thickness to be closer to the control group mean of 91.9 μm (9.7), as reported in Moon et al. [14]. A total sample size of 30 participants (15 per group) was found to provide 80% power to detect a 10 μm difference in mean RNFL thickness between the two groups, using a Welch’s T-test and a 5% significance level.

Tumor characteristics

All patients had pituitary tumors greater than 1 cm in any direction. Tumor size was identified by a single trained investigator and the largest dimension in any direction was recorded. Tumors not originating in the pituitary gland were excluded.

Measurement of serum prolactin levels

Serum prolactin was measured as part of the routine clinical workup for each patient via FDA approved Roche Elecsys Prolactin II Assay (Electrochemiluminescence Immunoassay [ECLIA]; Roche Diagnostics; Indianapolis, IN) with a reference range of 4.8–23.3ng/ml [15]. This assay demonstrates increased sensitivity to detect the concentration of monomeric prolactin by avoiding false elevation secondary to reactivity with macroprolactin [16]. Our clinical laboratory is a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory and accredited by the College of American Pathologists and New York State Department of Health. Briefly–antigen-specific monoclonal antibodies were coated onto beads and mixed with the PRL sample to allow an immune reaction to occur. Unbound sample was then washed away and a second monoclonal antibody with an electrochemiluminescent probe was added to the mixture to bind the PRL-antibody complex. An electrode was then introduced to the sample, which generates quantifiable electrochemiluminescence via an oxidation-reduction reaction that directly correlates to the amount of PRL present. All data for this study were recorded prior to any treatments including dopamine agonists and/or surgery.

Ophthalmologic data

All included subjects had ophthalmologic examination data which included RNFL evaluation with SD-OCT–optic disc cube 200 × 200 protocol as described previously [7]. Measures of RNFL thickness were reported as a function of clock hour position of the fovea and subsequently grouped into anatomical quadrants based on a standard division of the visual field (e.g., for right eyes; superior quadrant: 11 and 1 o’clock; nasal quadrant: 2, 3, and 4 o’ clock; inferior quadrant: 5 and 7 o’ clock; and the temporal quadrant: 8, 9, and 10 o’clock). The 12 o’clock and 6 o’clock positions were excluded from analysis secondary to nontemporal overlap of retinal ganglion cell projections [17]. The temporal RNFL quadrant demonstrates increased sensitivity to injury with pituitary tumors secondary to its association with crossing retinofugal fibers at the level of the optic chiasm [14]. Average peripapillary RNFL values were obtained for each eye and within each quadrant (prolactinoma group n eyes = 27; control group n eyes = 104). One subject from the prolactinoma group had RNFL data only from one eye. Automated perimetry testing was performed using Humphrey Field Analyzer (24–2 SITA-Standard algorithm); Carl Zeiss Meditec, Dublin, CA, USA. Visual function was assessed using mean deviation–recorded from the Humphrey automated perimetry test for 27 eyes in the prolactinoma group and 104 eyes in the control group.

Statistical analysis

Statistical analysis was performed using R (version 3.6.1). Normality was assessed using the Shapiro-Wilks test. Parametric data were analyzed using one-tailed Welch’s t-test where the alternative hypothesis stated that the prolactinoma group’s retinal layer measurements would be greater than the controls. Eyes were treated independently under the assumption that compressive injury may not be equal in both eyes, thus PRL’s assumed effects may also vary in each eye. Nonparametric data were analyzed using the Mann–Whitney U test. A one-way ANOVA was used to assess differences in average RNFL thickness between prolactinoma patients, non-secreting controls with elevated PRL due to stalk effect (NS+), and controls with normal PRL (NS). Pearson linear regression was used to analyze mean deviation and prolactin levels for each group. Mahalanobis distance was calculated to identify outliers in the mean deviation data. Multiple linear regression analysis was performed to assess the effects of PRL, age, and tumor volume on RNFL thickness in the PRO group. A p-value <0.05 was considered statistically significant.

Results

Out of the 239 patients who were identified to have formal ophthalmologic testing, a total of 66 met inclusion criteria for the study. This includes 14 prolactinoma patients and 52 non-secreting pituitary tumor patients. Of the non-secreting pituitary tumors, 12 were identified to have stalk effect, and 40 patients had normal levels of serum prolactin. See Fig 1.

Fig 1

The patient selection flow chart.

Abbreviations: OCT, optical coherence tomography.

There was no significant difference in biological sex between patients in the PRO and Control groups (Table 1). Age at diagnosis for the PRO (43.2 ± 18.8 years) was significantly younger than the Control group (59.0 ± 15.0 years) (p-value = 0.0076). Median serum prolactin levels for the prolactinoma group (744.1 ng/ml) were significantly greater than the controls (14.2 ng/ml) (p-value < 0.00001) (Table 1). There was no significant difference in tumor size between the PRO (22.1 mm ± 14.7) and control groups (25.1 ± 8.9; p-value = 0.20). See Table 1. Of the patients in the PRO group, 22 eyes underwent visual field testing with mean deviation measurements. Of the Control group 51 eyes had mean deviation data available from visual field testing. Ophthalmologic data revealed that there was no difference between mean deviation of the PRO (-4.4 ± 4.2 dB) and control groups (-6.9 ± 8.0 dB) (p-value = 0.79) (Table 2). Average RNFL thickness was similar in both groups (Table 2), however, the temporal quadrant of RNFL was significantly thinner in the control group than the prolactinoma group (p-value = 0.04).

Table 1

Characteristics of the study population including mean deviation recorded by Humphrey 24–2 automated perimetry.

For mean deviation PRO n = 22 eyes; Controls n = 51 eyes. Welch’s T-test was used for normally distributed data, Mann-Whitney U test was performed for non-normally distributed data, and Chi-squared was used for nominal data.

	PRO Group	Controls	P-value
	Mean (SD)	Mean (SD)
Participants, n	14	52
Eyes, n	27	104
Age at diagnosis	43.2 (18.8)	59.0 (15.0)	0.0076
Male/Female	9/5	29/23	0.33
Prolactin at diagnosis (ng/ml) †	744.1	14.2	<.00001
Mean deviation (dB)††	-4.4 (4.2)	-6.9 (8.0)	0.79
Tumor Size (mm)	22.1 (14.7)	25.1 (8.9)	0.20

OCT = optical coherence tomography;

† Median value reported;

†† different n

Table 2

RNFL thickness measurements recorded from SD-OCT optic disc and macular cube scan protocols.

One-sided Welch T-test was used to determine significance. The data demonstrate a statistically significant difference in RNFL thickness between PRO and control groups in the temporal quadrant.

		PRO Group	Controls
		Mean (SD)	Mean (SD)
RNFL Eyes, n		27	104
RNFL Average (μm)		82.7 (15.6)	81.5 (19.5)
RNLF Quadrants (μm)	Superior	104.4 (23.1)	102.3 (25.1)
	Nasal	63.7 (10.9)	65.2 (13.3)
	Temporal	57.1 (10.8) *	52.1 (14.0)
	Inferior	105.4 (26.7)	105.7 (27.3)

RNFL = retinal nerve fiber layer;

*p-value <0.05

Further analysis of RNFL, assessing the relation between PRO patients and both NS (n = 80 eyes, mean PRL = 13.1 ng/ml) and NS+ (n = 24 eyes, mean PRL = 50.5 ng/ml) patients, revealed a significant difference between the groups (p-value = 0.0027)–See Table 3. Post-hoc analysis also demonstrated a significant difference between RNFL thickness of NS+ and NS (p-value = 0.0018). See Fig 2. This analysis was repeated for tumor size, which identified no significant differences between the groups. In plotting the mean deviation data, a single data point varied from the rest. Prior to running statistical analysis on the data set, Mahalanobis distance was used–which identified an extreme outlier in the mean deviation data for the NS+ group (See S1 Fig). This was validated post-hoc using the extreme studentized deviate test (z-score 3.66, p <0.05). Mahalanobis distance and extreme studentized deviate test did not identify any other outliers in either the PRO or NS groups. The extreme outlier was removed from the mean deviation analysis.

Table 3

Subject characteristics by group including visual ability as mean deviation recorded by Humphrey 24–2 automated perimetry (PRO n eyes = 22, controls NS+ n eyes = 18, controls NS n eyes = 33) and SD-OCT average RNFL thickness (PRO n eyes = 27, controls NS+ n eyes = 24, and NS n eyes = 80).

One way ANOVA was used to determine significance and Chi-squared was used for nominal data.

	PRO	Controls		p- value
		NS+	NS
Subjects, n	14	12	40
Males/Females	9/5	5/ 7	24/16	0.45
Age at diagnosis	43.2 (18.8)	55.3 (18.6)	60.2 (13.8)	0.0044
Prolactin (ng/ml)	2453.3 (3907.6)	50.5 (21)	13.1 (7.2)	0.00013
Tumor size (mm)	22.1 (14.7)	26.4 (7.6)	24.6 (10.0)	0.55
Mean deviation (dB)	-4.4 (4.2)	-4.4 (6.1)	-7.1 (8.7)	0.28
Average RNFL Thickness (um)	82.7 (15.6)	91.7 (19.1)	78.4 (15.6)	0.0027

OCT = optical coherence tomography; value reported as mean (st. deviation)

Fig 2

Average RNFL distribution of PRO and controls which were further divided into those with hyperprolactinemia as determined clinically as > 30 ng/ml (NS+) and those with normal prolactin (NS).

For RNFL analysis PRO eyes n = 27, NS+ eyes n = 24, and NS eyes n = 80. RNFL did differ significantly between the groups (p-value 0.0027) using one-way ANOVA. Post Hoc (Tukey) analysis demonstrated significant difference between the NS+ and NS groups (p-value 0.0018). **p-value < 0.01.

Pearson correlation analysis of prolactin as a function of mean deviation revealed little to no association in the PRO group (R = -0.23, R2 = 0.051, p-value = 0.31). However, among control participants subdivided by stalk effect, a significant inverse correlation with mean deviation was identified for the NS group (R = -0.47, R2 = 0.23, p-value = 0.0053) and a significant direct correlation for the NS+ group (R = 0.51, R2 = 0.26, p-value = 0.035). See Fig 3.

Fig 3

Pearson linear regression analysis was performed for all subjects with the controls divided by subgroups (NS+ and NS).

Analysis of PRL as a function of mean deviation in the PRO group revealed little negative association (R = -0.23, p-value = 0.31). When analyzing the controls by subgroups, NS+ demonstrated a significant correlation between mean deviation and serum PRL level (R = 0.51, p-value = 0.035) while the NS group demonstrated an inverse correlation that approached significance (R = -0.47, p-value = 0.0053).

Discussion

In this study, we demonstrate a positive relation between prolactin and RNFL thickness during anterior visual pathway injury from pituitary masses. This trend was significant when assessing PRL levels and RNFL for non-secreting pituitary tumors with moderately elevated PRL levels (NS+) due to stalk effect. We also demonstrate a significant relation between serum PRL and mean deviation. Together, these data suggest that at moderately elevated levels, PRL may confer neuroprotection against injury with preservation of RNFL thickness.

It is also important to note that in this study we focused on the relation between prolactin and RNFL thickness at the time of diagnosis–an approach that is agnostic to treatment type (e.g. open vs trans-sphenoidal tumor resection or medical therapy). This cross-sectional approach removes confounding factors related to the potential effects of treatment on both RNFL thickness and visual function, and the role that decompression plays in mediating recovery. Thus, an important distinction is that our data demonstrate the potential for preservation of RNFL thickness as opposed to facilitating recovery. Further research is necessary to determine the impact of serum prolactin on neural recovery after injury–which might inform therapeutic approaches for central nervous system pathologies other than pituitary tumors.

Our study is novel in that we assess the effects of elevated PRL in two hyperprolactinemia states; non-secreting tumors that cause stalk effect and moderately elevated PRL levels, and prolactinoma patients with extremely elevated PRL levels. One previous study investigating changes in vision as a function of hormone status in patients with pituitary macroadenomas focused primarily on medically treated functional pituitary tumors only and demonstrated no significant relationship in 6 patients [18]. Hyperprolactinemia from stalk effect was not considered.

One limitation of the current study is the small sample size. Clinical use of OCT at our institution is limited to patients who experience “vision loss” as their chief complaint. Additionally, as one of the largest regional referral centers for pituitary tumor patients in the United States [19], many patients complete their initial ophthalmologic workup outside of our academic medical center. Our findings, specifically with respect to the relation between moderate elevation in PRL and RNFL thickness warrants further study both in a larger cohort of patients and in a prospective fashion.

PRL has been studied for its neuroprotective role in the retina and in white matter. In the retina, PRL receptors have been identified in the retinal pigment epithelium and found to be protective against cell death from oxidative stress [20]. Additional evidence has identified PRL as a trophic factor that regulates glial-neuronal interactions and protects against retinal degeneration [21]. Whether these mechanisms are triggered by PRL during compressive injury has not been elucidated, but provides a possible route for the protective characteristics observed in this study. PRL’s role in white matter injury has been studied most recently in multiple sclerosis patients and in mouse models of spinal cord injury. These studies attribute increased oligodendrocyte proliferation and remyelination after injury to increased PRL levels [8] and increased white matter volume [10]. We have previously shown that remyelination is possible after compressive injuries to the anterior visual pathway [22] and that measures of diffusion known to correlate with myelination are sensitive to varying levels of serum prolactin in a patient with empty-sella syndrome [7]. Due to PRL’s role in remyelination, this provides another possible mechanism for its protective quality following compressive injuries.

OCT derived measurements of RNFL thickness have been used extensively to diagnose and follow various pathologies of the visual system including compressive injuries [5, 6, 14, 23]. Pre-operative RNFL measurements have also been shown to predict post-operative visual outcomes for pituitary tumor patients [6]. These measurements may serve as biomarkers for injury along the visual pathway where thinning indicates ongoing injury and disease progression [7, 24]. This allows for RNFL measurements to be used to quantify the efficacy of neuroprotective agents, like PRL. Our results demonstrate that moderately increased serum PRL levels are associated with increased RNFL thickness and mean deviation.

We demonstrated that moderately elevated PRL levels like those found in patients with stalk effect were more strongly correlated with RNFL thickness whereas the extremely elevated levels of PRL in the PRO group conferred no preservation of RNFL thickness. Notably, the control group in our dataset was statistically older than the PRL group. Given that RNFL thickness has been shown to decrease slowly with age it may be expected for the controls whose average age was greater than the prolactinoma group to have thinner retinas [25, 26]. Additionally, a 24-hour PRL collection study found that older patients had a lower pulse mass and lower peak values of PRL secreted [27], although average PRL values overall did not significantly decline with age. Taken together, our data demonstrates that even with the added variable of age there is a preservation of retinal thickness in the group with mildly elevated prolactin. In other words, regardless of the natural retinal thinning and PRL changes that may come with age, older patients with moderately elevated PRL had greater RNFL thickness than patients with significant hyperprolactinemia. In the NS group, PRL was inversely associated, suggesting that PRL at these lower levels may also result in worsening visual function during injury. Additional studies of visual function are needed to investigate this possibility.

When analyzing mean deviation, the PRO group demonstrated little to no association with PRL. While there was no observed increase in visual function (as measured by mean deviation) at increasingly higher levels of prolactin in the PRO group, this does not exclude the possibility that elevated PRO prevents further decline in visual function. This may also indicate that PRL is protective within a certain range. Physiologically PRL demonstrates both inhibitory and excitatory actions. In the hypothalamus, elevated PRL levels inhibit the secretion of gonadotrophin-releasing hormone. Further investigation is required to elucidate whether a similar mechanism may be involved in prolactin’s neuroprotective function. Alternatively, chronic, extremely elevated levels of PRL in the PRO group can cause receptor desensitization and downregulation thus preventing the neuroprotective potential. The kinetics of the human prolactin receptor have shown it to behave with its agonist in a bell-shaped fashion implying supersaturation at high levels and decreased pathway activation at low levels [28]. Thus, the moderately elevated levels of PRL may work at peak PRL receptor activity without desensitization. Additionally, proteolytic cleavage of PRL generates active peptides (vasoinhibins/16K PRL)–which have been shown to have effects on vasculature by promoting vasopressin release, and on neurons by inhibiting neurite outgrowth [29]. High levels of PRL would in turn result in increased levels of vasoinhibins, whose function may prevent or oppose the neuroprotective action of PRL.

In summary, this study demonstrates attenuation of injury to the retina during anterior visual pathway injury with moderately elevated prolactin levels in non-secreting pituitary tumor patients. We show that hormone status, specifically prolactin, a known neuroprotective agent, may influence the structure-function relationship of the visual system during injury. Additional studies with larger sample sizes that include visual function tests could further elucidate the role of prolactin in preserving function after injury.

Outlier identification was determined using Mahalanobis distance.

A) Classic Mahalanobis distance is plotted for each data point of the NS+ group mean deviation with a threshold of 3. B) A QQ plot of the Mahalanobis distance and Chi-square quantile further identifies outliers with an extreme outlier at the 7th quantile corresponding to the 18th data variable in A.

(DOCX)

Click here for additional data file.

23 Feb 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tudor C. Badea, M.D., M.A., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study by Paul et al. aims to investigate whether there is a causal relationship between serum prolactin (PRL) levels and the thickness of the retinal nerve fiber layer in patients that suffered optic chiasma compression associated with macroadenomas. This is interesting clinical study. The PRL field of research started decades ago, but in vivo studies are certainly missing to better understand its complex and intricated roles. In general, the study makes sense, data are a bit scarse, but it is inherent to the nature of retrospective studies. I consider that it is worth publishing in Plos One, but I have some concerns that need to be addressed before recommending acceptance with no revision. In particular, some aspects about the statistics, data analysis and presentation, and discussion need to be addressed.

Major comments:

- Even if several effects of PRL are now known to do not depend on the sex, main ones do depend on the sex. It is therefore necessary to clearly study this issue when one deals with PRL. Biological sex is not mentioned in the Materials and Methods section. P.7 lines 171-172: the authors state “There was no difference in male/female make-up between the PRO and Control group”. Which figure does support this data? Line 171 p.7, the “male/female” terms refer to animals. Please correct.

- page 9, lines 206-208: What is the rationale to eliminate what the authors consider as “extreme outliers”? Working with confidence intervals may be a good way to solve this type of issues. In this line, I did not find any estimate of sample size to reach statistically significant conclusions. Even if the study is retrospective, such estimate is necessary. At least to get an idea of the power of the statistical data.

- p.7, lines 175-176: what does this “Of the patients in the PRO group, 22 eyes had mean deviation measurements” mean? Similar comment for the following sentence “Of the Control group 51 eyes had mean deviation data available.”?

p.2 line 55: The authors state that “Of 52 non-secreting macroadenoma patients, 12 had moderate elevation of prolactin secondary to stalk effect”. Do the authors have direct evidence of that, I mean, for their own data? Please, clarify.

p.3 line 94-95: The authors state that “we investigate the relation between serum hormone levels of prolactin, RNFL thickness and visual function in a retrospective cohort of pituitary macroadenoma patients.” How visual function was studied?

- p.13, lines 289-291: The authors state that “While there was no observed increase in visual function at increasingly higher levels of prolactin in the PRO group, […]”, but I cannot find any data supporting that. RNFK thickness is not a measure of visual function.

p.7, line 181: What is the physiological relevance of the temporal quadrant of the retinal nerve fiber layer?

Please define the superior, nasal, temporal, and inferior quadrants in the Methods section.

- About the tumor characteristics, the authors state that “Tumor size was identified by a single trained investigator and the largest dimension in any direction was recorded” (p.5, lines 135-136). The tumor size should be measured. Where are they recorded?

- Please detail the PRL assay (p.5 line 139).

- p.5-6 (lines 145 and 149): the number of tested eyes in the control group does not match. Please correct.

- p.6, line 158: what are normal levels of serum PRL?

- Please discuss why a moderate increase in serum PRL levels is beneficial, while a greater increase is not, from a mechanistic point of view. In this line, p.13, lines 293-295, the authors mention that opposite actions of PRL may be due to the fact that PRL exerts both inhibitory and excitatory actions and that PRL regulates gonadotropin release. This seems to imply that the opposite actions of PRL on RFNL are due to the activation of opposite signaling pathways, which may happen, but what directs the resulting effect of PRL. Why at high doses, the neurotoxic effects would prevail and at moderate doses, the neuroprotective effects would be more important? It seems to me that this explanation does not take into account that the PRL receptors respond to their agonist with a bell-shape curve. I am referring to the fact that at high concentrations of PRL, PRL receptors desensitize. Conversely, small amount of agonist activates poorly the receptor. This may explain why the neuroprotective effect is lost if patients have too much or too little of PRL. In addition, PRL can be transformed into active peptides (vasoinhibins/16K prolactin), which also have neuronal effects. Please, complete the discussion to fully discuss this crucial issue for the study. At last, what are the actions of gonatropin on RNFL/neuronal survival?

- p. 12, lines 283-283: The authors mention that their control group is older than their PRL group. Then they discuss the implication of aging in RNFL thickness, but could they also include that PRL levels also decrease with age? It would reinforce their point that physiological PRL levels are neuroprotective and that extreme serum PRL levels, either too high or too low, are detrimental for neurons.

- p.4 lines 104-108: Please add references that support the two ways through which serum PRL can increase in humans.

- p.4 lines 11-112: I do not understand the logic behind the use of NS+ and NS in the control group… Does N stand for normal and S for… serum?

- Table 1: The p value for the age at diagnosis needs to be checked and aligned.

Minor comments:

- p.4, line 102: “[…] visual pathway structures, we can explore […]”. A coma seems to be missing.

- p.5, lines 126-130: The institution's reference sounds repetitive.

- It would be easier to detect the figures and tables in the Result section if they were written in bold.

- Please regroup all figure legends, including table legends at the end of the manuscript.

- p.9 lines 203: “Post-hoc” should be written in italics.

- p.10, line 238: Please correct typo “[…] our data demonstrate that […]”.

- p.11 line 260, please add a coma before but.

- p.13, lines 293-294: please add a coma after hypothalamus.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Stéphanie C. Thébault

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

2 May 2022

Responses to Highlighted Reviewer’s Comments:

1. Even if several effects of PRL are now known to not depend on the sex, main ones do depend on the sex. It is therefore necessary to clearly study this issue when one deals with PRL. Biological sex is not mentioned in the Materials and Methods section. P.7 lines 171-172: the authors state “There was no difference in male/female make-up between the PRO and Control group”. Which figure does support this data? Line 171 p.7, the “male/female” terms refer to animals. Please correct

Response: We have included a statement acknowledging biological sex in the methods section. We also refer readers to Table 1, which includes demographic data on biological sex. Male and Female are used to describe biological sex as documented in the medical chart.

p.5 lines 130-131

Both Male and Female patients were included in the study, as defined by their biological sex on chart review.

p.8 lines 213-214

There was no significant difference in biological sex between patients in the PRO and Control groups (Table 1).

2. What is the rationale to eliminate what the authors consider as “extreme outliers”? Working with confidence intervals may be a good way to solve this type of issues. In this line, I did not find any estimate of sample size to reach statistically significant conclusions. Even if the study is retrospective, such estimate is necessary. At least to get an idea of the power of the statistical data.

Response: Re: Outlier analysis. We have provided further context for the use of this method within the body of the paper, emphasizing the how this is the most objective way to identify a potentially erroneous value. Additional post-hoc validation was also performed.

p.10 lines 251-257

In plotting the mean deviation data, a single data point varied from the rest. Prior to running statistical analysis on the data set, Mahalanobis distance was used – which identified an extreme outlier in the mean deviation data for the NS+ group (See Supplemental Data). This was validated post-hoc using the extreme studentized deviate test (z-score 3.66, p <0.05). Mahalanobis distance and extreme studentized deviate test did not identify any other outliers in either the PRO or NS groups. The extreme outlier was removed from the mean deviation analysis.

Re: Sample size estimate and power analysis. A power analysis was performed based on data publicly available in the literature and added to the methods section of the manuscript.

p.5 lines 140 -150

Power analysis: A power analysis was conducted based on the initial hypothesis that elevated levels of prolactin in prolactin-secreting macroadenoma patients will demonstrate attenuation of injury to the retinal nerve fiber layer compared with non-secreting macroadenoma patients. Previous research indicates that pre-operative mean RNFL thickness in pituitary macroadenoma patients (standard deviation) is 81.9 µm (8.8) (Moon et al.). We anticipated a similar mean RNFL thickness for non-secreting pituitary macroadenoma patients in this study. For patients with elevated prolactin, we anticipated a mean RNFL thickness to be closer to the control group mean of 91.9 µm (9.7), as reported in Moon et al. A total sample size of 30 participants (15 per group) was found to provide 80% power to detect a 10 µm difference in mean RNFL thickness between the two groups, using a Welch’s T-test and a 5% significance level.

References:

Moon CH, Hwang SC, Kim BT, Ohn YH, Park TK. Visual prognostic value of optical coherence tomography and photopic negative response in chiasmal compression. Invest Ophthalmol Vis Sci. 2011;52(11):8527-33.

3. p.7, line 181: What is the physiological relevance of the temporal quadrant of the retinal nerve fiber layer? Please define the superior, nasal, temporal, and inferior quadrants in the Methods section.

Response: We have included additional text that better defines the quadrants, their structural/functional relationship to the visual field, and increased sensitivity of the temporal RNFL quadrant to chiasmal compression.

p.6 lines 170-179

Measures of RNFL thickness were reported as a function of clock hour position of the fovea and subsequently grouped into anatomical quadrants based on a standard division of the visual field (e.g., for right eyes; superior quadrant: 11 and 1 o’clock; nasal quadrant: 2, 3, and 4 o’ clock; inferior quadrant: 5 and 7 o’ clock; and the temporal quadrant: 8, 9, and 10 o’clock). The 12 o’clock and 6 o’clock positions were excluded from analysis secondary to nontemporal overlap of retinal ganglion cell projections (Schneider et al.). The temporal RNFL quadrant demonstrates increased sensitivity to injury with pituitary tumors secondary to its association with crossing retinofugal fibers at the level of the optic chiasm (Moon et al.).

References:

Schneider CL, Prentiss EK, Busza A, Matmati K, Matmati N, Williams ZR, et al. Survival of retinal ganglion cells after damage to the occipital lobe in humans is activity dependent. Proc Biol Sci. 2019;286(1897):20182733.

Moon CH, Hwang SC, Kim BT, Ohn YH, Park TK. Visual prognostic value of optical coherence tomography and photopic negative response in chiasmal compression. Invest Ophthalmol Vis Sci. 2011;52(11):8527-33.

4. About the tumor characteristics, the authors state that “Tumor size was identified by a single trained investigator and the largest dimension in any direction was recorded” (p.5, lines 135-136). The tumor size should be measured. Where are they recorded?

Response: Tumor size is reported in Table 1. The text has been updated to better reflect this.

p.8 line 217-218

There was no significant difference in tumor size between the PRO (22.1 mm ± 14.7) and control groups (25.1 ± 8.9; p-value = 0.20). See Table 1.

5. Please detail the PRL assay (p.5 line 139).

Response: Additional text has been added to describe the PRL assay.

p.6 lines 160-166

Briefly – antigen-specific monoclonal antibodies are coated onto beads and mixed with the PRL sample to allow an immune reaction to occur. Unbound sample is then washed away and a second monoclonal antibody with an electrochemiluminescent probe is added to the mixture to bind the PRL-antibody complex. An electrode is then introduced to the sample, which generates quantifiable electrochemiluminescence via an oxidation-reduction reaction that directly correlates to the amount of PRL present.

6. p.5-6 (lines 145 and 149): the number of tested eyes in the control group does not match. Please correct

Response: We are very grateful to the reviewer for seeing this--we identified a typo in the submitted manuscript. The total number of eyes in the prolactinoma group is 27, and for the control group 104. The manuscript text has been updated to reflect this.

7. p.6, line 158: what are normal levels of serum PRL?

Response: The normal reference range for prolactin obtained in our laboratory is 4.8-23.3ng/ml.

p.6 line 159-160.

Serum prolactin was measured via Electrochemiluminescence Immunoassay (ELISA) with a reference range of 4.8-23.3ng/ml…

8. Please discuss why a moderate increase in serum PRL levels is beneficial, while a greater increase is not, from a mechanistic point of view. In this line, p.13, lines 293-295, the authors mention that opposite actions of PRL may be due to the fact that PRL exerts both inhibitory and excitatory actions and that PRL regulates gonadotropin release. This seems to imply that the opposite actions of PRL on RFNL are due to the activation of opposite signaling pathways, which may happen, but what directs the resulting effect of PRL. Why at high doses, the neurotoxic effects would prevail and at moderate doses, the neuroprotective effects would be more important? It seems to me that this explanation does not take into account that the PRL receptors respond to their agonist with a bell-shape curve. I am referring to the fact that at high concentrations of PRL, PRL receptors desensitize. Conversely, small amount of agonist activates poorly the receptor. This may explain why the neuroprotective effect is lost if patients have too much or too little of PRL. In addition, PRL can be transformed into active peptides (vasoinhibins/16K prolactin), which also have neuronal effects. Please, complete the discussion to fully discuss this crucial issue for the study. At last, what are the actions of gonadotropin on RNFL/neuronal survival?

Response: We appreciate the reviewer’s suggestion and have bolstered the discussion section to address these key issues – particularly with respect to proposed mechanisms that may be responsible for prolactin’s effect at moderate levels. With regards to the role of gonadotropin on RNFL/neuronal survival, we felt commenting on this would be beyond the scope of the current article.

p.15 lines 370-380

Alternatively, chronic, extremely elevated levels of PRL in the PRO group can cause receptor desensitization and downregulation thus preventing the neuroprotective potential. The kinetics of the human prolactin receptor have shown it to behave with its agonist in a bell-shaped fashion implying supersaturation at high levels and decreased pathway activation at low level (Kinet et al.) Thus, the moderately elevated levels of PRL may work at peak PRL receptor activity without desensitization. Additionally, proteolytic cleavage of PRL generates a 16K PRL called vasoinhibin – which has been shown to have effects on vasculature by promoting vasopressin release, and on neurons by inhibiting neurite outgrowth (Castillo et al.). High levels of PRL would in turn result in increased levels of vasoinhibin, whose function may prevent or oppose the neuroprotective action of PRL.

References:

Kinet S, Bernichtein S, Kelly PA, Martial JA, Goffin V. Biological properties of human prolactin analogs depend not only on global hormone affinity, but also on the relative affinities of both receptor binding sites. J Biol Chem. 1999;274(37):26033-43.

Castillo X, Melo Z, Varela-Echavarria A, Tamariz E, Arona RM, Arnold E, et al. Vasoinhibin Suppresses the Neurotrophic Effects of VEGF and NGF in Newborn Rat Primary Sensory Neurons. Neuroendocrinology. 2018;106(3):221-33.

9. p. 12, lines 283-283: The authors mention that their control group is older than their PRL group. Then they discuss the implication of aging in RNFL thickness, but could they also include that PRL levels also decrease with age? It would reinforce their point that physiological PRL levels are neuroprotective and that extreme serum PRL levels, either too high or too low, are detrimental for neurons.

Response: We appreciate the reviewer’s suggestion to include this info and have added it to the discussion, along with relevant citations.

p.14 lines 351-358

Additionally, a 24-hour PRL collection study found that older patients had a lower pulse mass and lower peak values of PRL secreted (Roelfsema et al.), although average PRL values overall did not significantly decline with age. Taken together, our data demonstrates that even with the added variable of age there is a preservation of retinal thickness in the group with mildly elevated prolactin. In other words, regardless of the natural retinal thinning and PRL changes that may come with age, older patients with moderately elevated PRL had greater RNFL thickness than older patients with significant hyperprolactinemia.

References:

Roelfsema F, Pijl H, Keenan DM, Veldhuis JD. Prolactin secretion in healthy adults is determined by gender, age and body mass index. PLoS One. 2012;7(2):e31305.

10. p.4 lines 104-108: Please add references that support the two ways through which serum PRL can increase in humans.

Response: Two references have been added to support the mechanisms by which PRL can increase in humans.

Serri O, Chik CL, Ur E, Ezzat S. Diagnosis and management of hyperprolactinemia. CMAJ. 2003;169(6):575-81.

Mancini T, Casanueva FF, Giustina A. Hyperprolactinemia and prolactinomas. Endocrinol Metab Clin North Am. 2008;37(1):67-99, viii.

11. p.4 lines 11-112: I do not understand the logic behind the use of NS+ and NS in the control group… Does N stand for normal and S for… serum?

Response: NS stands for “non-secreting.” The text has been updated to better reflect this and to eliminate any confusion. NS+ represents “non-secreting” macroadenoma patients with an elevation of prolactin secondary to stalk effect. See p.4 lines 110-112. Thank you for this request to clarify.

12. Table 1: The p value for the age at diagnosis needs to be checked and aligned.

Response: This value was missing a leading zero. This has been corrected. Thank you.

Responses to Minor Reviewer’s Comments:

p.4, line 102: “[…] visual pathway structures, we can explore […]”. A coma seems to be missing.

Corrected, thanks.

p.5, lines 126-130: The institution's reference sounds repetitive.

The institution reference has been removed.

It would be easier to detect the figures and tables in the Result section if they were written in bold.

All references to figures are now in bold type.

Please regroup all figure legends, including table legends at the end of the manuscript.

Table Legends remain with the Tables in accordance with PLOS One Formatting guidelines.

p.9 lines 203: “Post-hoc” should be written in italics.

Corrected, thanks.

p.10, line 238: Please correct typo “[…] our data demonstrate that […]”.

Corrected, Thank you.

p.11 line 260, please add a coma before but.

Done.

p.13, lines 293-294: please add a coma after hypothalamus.

Done.

Responses to Editor’s Comments:

Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

We have addressed all editorial comments in the body of the manuscript.

p.5 lines 136-138

The study protocol was approved by the institutional review board of the University of Rochester and the need for consent was waived. All data were anonymized before being accessed.

Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

The Caption for the Supplemental Figure has been added at the end of the manuscript.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

16 May 2022

20 Jun 2022

Responses to Reviewer’s Comments:

1. Please specify the antibody (manufacturer, ref.) used for the PRL assay.

Additional Editor Comments (if provided): Please provide the additional information requested by the reviewer. In particular, please describe not just the source of the antibody used, but the evidence demonstrating it specificity (western blot analysis, immunostaining of transfected cells, analysis in KO mice, etc.). If that evidence is derived from other published papers, please provide the references to them.

Response: The manuscript has been updated to include the PRL assay manufacturer (Roche Diagnostics; Indianapolis, IN) and evidence of its sensitivity to detect prolactin (see new reference below, [16]). Of note, we have also included text to clarify that PRL testing was conducted in a CLIA certified clinical laboratory as part of the routine clinical workup for pituitary tumor patients at our institution.

16. Fahie-Wilson M, Bieglmayer C, Kratzsch J, Nusbaumer C, Roth HJ, Zaninotto M, et al. Roche Elecsys Prolactin II assay: reactivity with macroprolactin compared with eight commercial assays for prolactin and determination of monomeric prolactin by precipitation with polyethylene glycol. Clin Lab. 2007;53(5-6):301-7.

Page 6, lines 149-156

Measurement of Serum Prolactin Levels: Serum prolactin was measured as part of the routine clinical workup for each patient via FDA approved Roche Elecsys Prolactin II Assay (Electrochemiluminescence Immunoassay [ECLIA]; Roche Diagnostics; Indianapolis, IN) with a reference range of 4.8-23.3ng/ml [15]. This assay demonstrates increased sensitivity to detect the concentration of monomeric prolactin by avoiding false elevation secondary to reactivity with macroprolactin [16]. Our clinical laboratory is a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory and accredited by the Colleague of American Pathology and New York State Department of Health.

2. In the discussion about the potential mechanistic explanation for the opposite roles of PRL according to its levels, the "vasoinhibin" term should be used in plural. This was the whole point renaming them vasoinhibins instead of 16K prolatin, which infers that only 16 kDa N-terminal fragments are produced.

Response: The text has been updated per the reviewers’ comments.

Page 15, lines 346-350

Additionally, proteolytic cleavage of PRL generates active peptides (vasoinhibins/16K PRL) – which have been shown to have effects on vasculature by promoting vasopressin release, and on neurons by inhibiting neurite outgrowth [32]. High levels of PRL would in turn result in increased levels of vasoinhibins, whose function may prevent or oppose the neuroprotective action of PRL.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Jul 2022

Prolactin at moderately increased levels confers a neuroprotective effect in non-secreting pituitary macroadenomas

PONE-D-21-39870R2

Dear Dr. Paul,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Tudor C. Badea, M.D., M.A., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

25 Jul 2022

PONE-D-21-39870R2

Prolactin at moderately increased levels confers a neuroprotective effect in non-secreting pituitary macroadenomas

Dear Dr. Paul:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tudor C. Badea

Academic Editor

PLOS ONE