Author Response
Reviewer #1 (Public Review):
This study optimized a protocol for analyzing microplastics (MPs) in bovine and human follicular fluid. The authors identified the most common plastic polymers in the follicular fluid and assessed the impact of polystyrene beads on bovine oocyte maturation based on the concentration of MPs in follicular fluid. The authors found a decrease in maturation rate in the presence of polystyrene beads and conducted proteomic analysis of oocytes treated with and without MPs, revealing protein alterations.
Strengths:
• The optimization of the protocol for analyzing MPs in follicular fluid, which is important for future research in this area.
• Investigating the effects of MPs on oocyte maturation and proteomic profiles is significant.
Thank you for the summary and for highlighting our manuscript’s strengths. Weaknesses:
• The effects of polystyrene beads on oocyte maturation and proteomic profiles are not directly demonstrated, and insufficient analysis is performed to support the claims made in the manuscript.
We disagree with this statement, as we have shown that the oocyte maturation is affected by the PS beads, which clearly have some effects on the zona pellucida as well, all supported by well thought experimental analysis. Regarding the proteomics data, as suggested to be emphasized by reviewer 3, in the oocyte maturation experiment the PS exposure was performed using cumulus-oocyte-complexes and we believe that the cumulus cells might have a protective role (to a certain extent) to the oocyte. At first, we have performed different methods to try and check incorporation of PS beads into oocyte and cumulus cells but, unfortunately, we could not validate a protocol for that. Therefore, although we have seen some changes on proteomics, indeed we were not able to directly demonstrate which pathways could have been responsible for the decreased oocyte maturation and increased zona pellucida fragility.
• The use of polystyrene beads does not fully mimic the concentration and interaction of MPs in follicular fluid, which warrants careful interpretation and discussion.
We are aware that the concentration of polystyrene (PS) used in our experiments (0.01ug/mL and 0.1ug/mL) did not fully represent the PS concentrations found in human and bovine follicular fluid (FF) (0.0013 and 0.0043 ug/mL). We note though that PS is not the only MPs detected in the FF and, in this study we selected PS concentrations that were in the range of the total MPs found in FF (0.102 and 0.025 ug/mL, for human and bovine, respectively). We will carefully re-read and revise the manuscript in order to ensure that we are not at risk of misguiding readers on the environmental relevance of the chosen experimental concentrations. Nevertheless, we firmly believe that our study was performed using a substantially more realistic concentration than the overwhelming majority of existing studies, which tend to use hundreds of thousands of times more plastic than what is naturally occurring (as described by Mills et al. - https://doi.org/10.1186/s43591-023-00059-1).
• A major weakness is the lack of mechanism. Determining the cause of meiotic arrest (decreased maturationrate) would be needed to strengthen the paper. Are spindle morphology, chromosome morphology/alignment and/or spindle assembly checkpoint mechanism perturbed in MPs-treated oocytes?
• Functional assays to validate one or more of the pathways suggested by the proteomic analysis would be necessary to strengthen the paper.
We appreciate that understanding the mechanisms underlying the observed changes is important, however, prior to this work, little was known about the effects of MPs on reproductive health. As such, the experimental plan for this work was focused on providing an assessement of the extent to which MPs occur in reproductive systems, and the effect of these MPs on general metrics of oocyte health and function. It is only with this baseline knowledge that experiments aimed at studying the mechanisms underlying these changes can/should be designed, which we will certainly consider for future research.
• The analysis of broken zona pellucida is not sufficiently convincing. Definitely the breakage of zona pellucida is most likely a result of oocyte denudation. However, this may indicate increased fragility of polystyrene beads-treated oocytes. Investigating cytoskeletal components in oocytes treated with or without polystyrene beads would strengthen this paper.
Indeed, the reviewer is correct that the breakage of the zona pellucida happened during denudation. Yet, because all groups were processed in the exact same way, the differences we observed between our experimental and control groups clearly indicate that the PS beads are causing some form of damage to the zona pellucida, or indirect effects through cumulus-oocyte interactions, irrespective of the initial breakage. This is a question we want to answer in future experiments.
• The percentage of degenerated oocytes in the control group is abnormally high which raises concern that the oocytes are not healthy.
The reviewer is correct in noting that the baseline number of degenerated oocytes is high. This is unlikely to be due to oocyte health, and is more likely attributed to the fact that the students that were working on this experiment had a period of adaptation to learn to work with these cellular types. In this regard, it is important to mention that we designed the experiment such that this effect was evenly distributed throughout all of the groups. In other words, the technique refinement did not introduce any systematic bias into the data. Thus, while the baseline number of degenerated oocytes is high, we are confident that the effects of MPs are robust.
• The small font size of the figures (such as Fig. 1C) affects the quality of the manuscript.
Thank you for pointing this out. We will improve readability of all our figures for a resubmission.
• Finally, the authors should cite previous publications on the effects of MPs on female reproduction, as this is not a novel area of research, despite the use of different concentrations. For example, "Polystyrene microplastics lead to pyroptosis and apoptosis of ovarian granulosa cells via NLRP3/Caspase-1 signaling pathway in rats (DOI: 10.1016/j.ecoenv.2021.112012)".
Yes, absolutely. We we will include this interesting and relevant work in our revised mansucript.
Reviewer #2 (Public Review):
This study presents valuable findings including the use of an improved method of Raman spectroscopy to measure accumulation of microplastics in ovarian follicular fluid obtained from cows and women and demonstration that experimental direct exposure of bovine eggs to biologically relevant levels of polystyrene, a microplastic found in both cows and women's follicular fluid, negatively influenced ova maturation status and the abundance of proteins involved in oxidative stress, DNA damage, apoptosis, and oocyte maturation.
Thank you for the summary and for highlighting our manuscript’s strengths.
The evidence supporting the claims of the authors is solid but inclusion of human population from which the follicular fluid was obtained (e.g., demographics, reason for assisted reproduction),
Agreed. We will include all information regarding the reason for IVF, age, BMI, and IVF outcomes in the revised manuscript.
and details about quality control for proteome profiling experiments (i.e., peptide count cut-off for significant proteins) would have strengthened the study. The work will be of interest to exposure scientists, reproductive toxicologists, regulatory scientists, and reproductive health clinicians.
For protein identification, the default settings of MaxQuant were used. In brief, proteins are only considered as identified with at least one unique or razor peptide. Razor peptides are non-unique and assigned to a single protein to ensure that they are only used once for identification. Additionally, a false discovery rate of 1% was applied using a decoy sequence database approach. Quantification was performed on proteins with at least two different peptides. We will include this information in the revised manuscript.
Reviewer #3 (Public Review):
The study from Grechi et al showed that emerging environmental microplastics (MPs) are present in both human and bovine follicular fluid. Moreover, based on the characterization and quantification data, authors treated bovine oocytes with environmentally relevant levels of polystyrene (PS) MPs and found that PS MPs interfered with oocyte maturation in vitro. This study is novel, particularly the first part of MP characterization and quantification, and for the first time confirms the presence of MPs in follicular fluid of humans and large farm animals. These results provide a possible mechanism by which the female infertility rate has been increasing in both humans and large farm animals.
Thank you for the summary and for highlighting our manuscript’s novelty.
The session of exposing MPs to bovine and related oocyte health evaluation can be further improved. For example, authors examined the morphology of the oocyte zona pellucida (ZP) and degeneration and stained oocyte DNA to determine the meiotic maturation status. However, a much more comprehensive oocyte health evaluation can be performed including but not limited to the examination of oocyte spindle morphology, meiotic division, fertilization, early embryo development, mitochondria, and accumulation of ROS. These additional endpoints can provide more robust evidence to determine the impact of MPs on oocyte health.
We agree with the reviewer that a more comprehensive oocyte health evaluation can be performed. Doing so, however, is beyond the scope of any single study as there are many different pathways and mechanisms by which MPs may be affecting oocytes and attempting to include all of these experiments in a single study is simply not feasible. Indeed, we plan on continuing along this line of work in future experiments.
While the oocyte proteomic analysis identified altered proteins, more functional studies and causation experiments can be performed.
As noted in our reply to reviewer 1, we appreciate that understanding the mechanisms underlying the observed changes is important, however, prior to this work, little was known about the effects of MPs on reproductive health. As such, the experimental plan for this work was focused on providing an assessement of the extent to which MPs occur in reproductive systems, and the effect of these MPs on general metrics of oocyte health and function. It is only with this baseline knowledge that experiments aimed at studying the mechanisms underlying these changes can/should be designed, which we will certainly consider for future research.
In addition, authors exposed cumulus-oocyte-complexes (COCs) but not denuded oocytes with MPs, it is crucial to determine whether MPs accumulate in cumulus cells or oocytes or both as well as the compromised oocyte quality is caused by the direct effect of MPs or the indirect impact on somatic cumulus cells to cause a secondary effect on the oocytes.
As stated previously, at first, we have performed different methods to try and check incorporation of PS beads into oocyte and cumulus cells but, unfortunately, we could not validate a protocol for that. Therefore, although we have seen some changes on proteomics, indeed we were not able to directly demonstrate which pathways could have been responsible for the decreased oocyte maturation and increased zona pellucida fragility, and what is the possible role of the cumulus cells on it.
