Association between occupational testicular radiation exposure and lower male sex ratio of offspring among orthopedic surgeons.

BACKGROUND: Exposure to occupational radiation can lower the male sex ratio. However, specific radiation exposure to the testes has not been evaluated.
OBJECTIVE: This study aimed to examine the association between testicular radiation exposure and lower male sex ratio in children.
METHODS: A comprehensive questionnaire survey was administered to 62 full-time male doctors with children aged < 10 years at 5 hospitals. Based on the possibility of testicular radiation exposure 1 year before the child's birth, participants were assigned to 3 groups as follows: RT (orthopedic surgery), RNT (cardiology/neurosurgery), and N (others). Intergroup differences in the proportion of female children were ascertained, and the female sex ratio (number of female/total number) of each group was compared against the standard value of 0.486. Multivariate logistic regression analysis with a generalized estimating equation was used to model the effects on the probability of female birth while controlling for the correlation among the same fathers.
RESULTS: The study population included 62 fathers and 109 children, 49 were female: 19/27, 11/30, and 19/52 in the RT, RNT, and N group, respectively; the RT group had the highest proportion of females (p = 0.009). The p values for comparisons with the standard sex ratio (0.486) were 0.02, 0.19, and 0.08 for the RT, RNT, and N groups, respectively. Based on the N group, the adjusted odds ratios for the child to be female were 4.40 (95% confidence interval 1.60-2.48) and 1.03 (0.40-2.61) for the RT and RNT groups, respectively.
CONCLUSIONS: Our results imply an association between testicular radiation exposure and low male sex ratio of offspring. Confirmatory evidence is needed from larger studies which measure the pre-conceptional doses accumulated in various temporal periods, separating out spermatogonial and spermatid effects.

Introduction

The number of radiation-generating medical procedures has increased over time, especially for spinal surgery. It includes minimally invasive procedures, such as lateral lumbar interbody fusion and percutaneous pedicle screw insertion. Thus, there have been concerns on the compounded radiation exposure of surgeons [1-4]. Although several clinical studies have reported that doses of exposure are within the recommended limits, the current radiation exposure limits are extrapolated from the values reported in studies of individuals who survived the atomic bombings in Hiroshima and Nagasaki [5-7]. Thus, the limited evidence on the long-term health-related effects of low-dose radiation raises concerns on the development of cataract, thyroid diseases, and cancer [8-12].

Despite an increased interest in protection against radiation, there is insufficient motivation among orthopedic surgeons to assess the use of protection against radiation because the direct effects of radiation exposure are not tangible and may not be discernible for several years [13, 14]. Thus, there is an explicit need to induce evidence-based and awareness-based behavioral changes to urge orthopedic surgeons to take action to avoid radiation exposure.

This study focused on the association between radiation exposure and the sex ratio of the offspring as an indicator of the potential effect of radiation exposure. We hypothesized that testicular radiation exposure skews the sex ratio of the offspring of male doctors toward the female sex by decreasing the ratio of sex-determining sperms. Occupational radiation exposure has been previously suggested to lower the male sex ratio of the offspring of male radiologists, male orthopedic surgeons, and young male cardiologists [15-17]. However, these studies did not evaluate testicular radiation exposure. Moreover, previous studies administered questionnaire surveys where non-responder-induced selection bias is a major limitation. Therefore, we conducted this study to examine the association between testicular radiation exposure and higher female sex ratio using a comprehensive survey of at-risk groups.

Materials and methods

Study design and setting

We conducted a case-control study using data obtained from a comprehensive survey of full-time doctors at 5 private hospitals in the Kyushu region, Japan—Shinkomonji Hospital, Fukuokawajiro Hospital, Shinyukuhashi Hospital, Shinmizumaki Hospital, and Shintakeo Hospital. Each study center was a designated emergency care and training hospital. Therefore, these hospitals have the unique advantage of being provided with many opportunities for treating trauma cases and employing many young, full-time doctors. The survey was conducted in February 2017, with the permission of the director of each hospital. The publication of the results was approved by the certified ethics committee of the main research institution, Shinkomonji Hospital. Information was collected from the study participants via individual interviews, conducted by medical clerks. The information was anonymized and sent to the researchers. Provision of responses to the survey was regarded as provision of tacit consent for study participation; the opt-out consent process was used.

Study population and data collection

The study population comprised all full-time male doctors with at least one child aged <10 years at the time of the survey. In consideration of the psychological burden of the respondents, details of any deceased children were not included. The exclusion criterion was refusal for study participation. The survey was conducted in February 2017 by the secretary of the medical office in each hospital. Physicians with more than one child were counted more than once; therefore, the number of participants included in the analysis is the number of children, not the number of physicians. There were two rationales for limiting the age of the children to 10 years: one was to reduce recall bias, and the other was to limit the study inclusion eligibility to at-risk groups. Because of the increasing medical radiation exposure over time [1], the younger generation is more likely to be exposed to radiation. Moreover, younger and less experienced physicians tend to have higher radiation exposure [5].

Outcome of interest and exposure

The outcome of interest was defined as having a female child. The participants were assigned to three groups based on the departments they belonged to a year before the birth of the child, which were as follows: departments that used medical radiation and had a high possibility of testicular radiation exposure (RT group), departments that used medical radiation but had a low possibility of testicular radiation exposure (RNT group), and departments that did not use medical radiation, including medical students (N group). Particularly, the RT group included doctors from the department of orthopedic surgery, wherein radiation is used primarily during surgery, whereas the RNT group included doctors from cardiology/neurosurgery, wherein radiation is used during catheterization.

In all study centers, apron-type protectors were generally used in the operating room, and coat-type protectors were used in the catheterization room. A coat-type protector provides full circumferential coverage of the torso, whereas the apron-type protector covers only the front of the torso. Thus, using apron-type protectors leave the testes unprotected against lateral and posterior radiation exposure. In addition, the physicians primarily face the source of the radiation in the catheterization room, whereas the surgeons deliver radiation at various angles and positions in the operating room. Therefore, we speculated that orthopedic surgeons are at a greater risk of experiencing lateral and posterior radiation exposure that could potentially affect the testes (Fig 1). The radiation dose to the testes considered herein is similar to that in the study by Funao et al. [18]. They reported that during a single interbody fusion, the average radiation exposure of an unshielded surgeon to scattered radiation in the genital area was 0.15 mSv, whereas the average radiation exposure of a radiographer away from the surgical field was 0.05 mSv—a difference of approximately 0.1 mSv/procedure.

Fig 1

Percutaneous pedicle screw insertion in a two-way C-arm position.

The surgeon wore an apron-type protector, leaving the lateral and posterior torso unprotected. The radiation source of the anteroposterior fluoroscopy was on the floor to ensure that the lower half of the torso is exposed to scattered rays.

The rationale for the selection of a cut-off date of exactly 1 year before the birth of the child was to localize the period at risk of changes that led to a lower male sex ratio. It takes approximately 74 days for the spermatogonia to differentiate and mature into sperm [19]. We hypothesized, albeit without supporting evidence, that testicular radiation exposure reduces the male sex ratio of the child by decreasing the ratio of sex-determining sperms. Accordingly, we estimated that the period at risk was 2–3 months before conception. This duration is related to the period after which the primary spermatocyte has differentiated into the secondary ‘male’ and ‘female’ spermatocytes and the period when the mature sperm has migrated to the epididymis where it is stored. In other words, considering that the gestation period is approximately 280 days, the period at risk of a decrease in the male sex ratio of the child due to testicular radiation exposure began approximately one year before the child’s birthday.

Statistical analyses

We described the paternal age at the child’s conception and the proportion of female children by the paternal exposure status. We used Fisher’s exact test to compare the proportion of female children between groups. A single-sample test of proportion was used to compare the observed female sex ratio (number of female children/total number of children) of each group with the standard ratio of 0.486 in Japan [20]. Furthermore, multivariate logistic regression analysis was conducted to model the effects of the exposure variables (RT and RNT) on the probability of having a female child. We used generalized estimating equations as a control for the correlation of observations among the same fathers. We included the paternal age at the child’s conception in the analytical model. Statistical analyses were performed using Stata version 15.1 (Stata Corporation, College Station, TX, USA).

Results

The study population comprised 62 fathers and 109 children, and the responses were obtained from all eligible doctors (response rate, 100%). There were 27, 30, and 52 participants in the RT, RNT, and N groups, respectively. There was no observable difference in paternal age at the child’s conception (Table 1). Overall, 49 children (45%) were female: 19 (70%) in the RT group, 11 (37%) in the RNT group, and 19 (37%) in the N group. A significantly higher proportion of females was observed in the RT group than in the other groups (p = 0.009).

Table 1

Paternal age at the child’s conception and the proportion of female children.

	Total, N = 109	Paternal exposure status			P value
		Na, n = 52 (48)	RTb, n = 27 (25)	RNTc, n = 30 (28)
Paternal age at the child’s conception, years	35.2 (6.1)	36.2 (7.3)	33.3 (3.9)	35.1 (5.3)
Proportion of female children	49 (45)	19 (37)	19 (70)	11 (37)	0.009

Data are presented as means (standard deviations) and numbers (%).

aN, physicians working in departments that did not use medical radiation, including medical students.

bRT, physicians working in departments that used medical radiation and had a high possibility of testicular radiation exposure.

cRNT, physicians working in departments that used medical radiation but had a low possibility of testicular radiation exposure.

The female sex ratios (number of female children/total number of children) were 0.70, 0.37, and 0.37 for the RT, RNT, and N groups, respectively (Table 2). The two-sided p values for the comparison with the national standard value of 0.486 were 0.02, 0.19, and 0.08 for the RT, RNT, and N groups, respectively.

Table 2

Female sex ratio of each paternal status and comparison with the national standard value.

Paternal exposure status	Female sex ratio of the childa	P valueb
Total	0.45 (0.36–0.54)	0.45
Nc	0.37 (0.23–0.50)	0.08
RTd	0.70 (0.53–0.88)	0.02
RNTe	0.37 (0.19–0.54)	0.19

Data are presented as ratios (95% confidence intervals).

aSex ratio was calculated as the number of female children dividided by the total number of children.

bP values were calculated using a single-sample test of proportion based on the Japanese standard value of 0.486.

cN, physicians working in departments that did not use medical radiation, including medical students.

dRT, physicians working in departments that used medical radiation and had a high possibility of testicular radiation exposure.

eRNT, physicians working in departments that used medical radiation but had a low possibility of testicular radiation exposure.

The crude odds ratios for the child to be female on the basis of the ratio in the N group were 4.00 (95% confidence interval [CI], 1.50–10.7) and 0.99 (95% CI, 0.39–2.48) for the RT and RNT groups, respectively, with adjusted odds ratios of 4.40 (95% CI, 1.60–12.1) and 1.03 (95% CI, 0.40–2.61), respectively (Table 3).

Table 3

Crude and adjusted odds ratios of having a female child of the RT and RNT groups.

Paternal exposure status	N	Unadjusted OR (95% CI)	P value	Adjusted OR (95% CI)a	P value
Nb	52	Reference	—	Reference	—
RTc	27	4.00 (1.50, 10.7)	0.006	4.40 (1.60, 12.1)	0.004
RNTd	30	0.99 (0.39, 2.48)	0.98	1.03 (0.40, 2.61)	0.96

OR, odds ratio; CI, confidence interval.

aAdjusted OR values were estimated from a logistic regression model adjusted for paternal age at conception.

bN, physicians working in departments that did not use medical radiation, including medical students.

cRT, physicians working in departments that used medical radiation and had a high possibility of testicular radiation exposure.

dRNT, physicians working in departments that used medical radiation but had a low possibility of testicular radiation exposure.

Discussion

Key results and clinical implications

The results of a survey of 5 private hospitals suggested that working in the department of orthopedic surgery—which entailed a high chance of testicular radiation exposure—at the time of conception was associated with a lower sex ratio, i.e., increase in female offspring (crude odds ratio 4.0, adjusted odds ratio 4.4). These results support the hypothesis that testicular radiation exposure skews the sex ratio of offspring towards females and motivate an investigation of the association between occupational testicular radiation exposure and lower sex ratio. The younger generation, who might be exposed to radiation more frequently, is also the generation that is likely to have children; thus, sex ratio may be of great concern to them. The results of this study contribute to raising awareness on radiation exposure among the younger generation and promoting protection against radiation.

Comparison with other studies

Our results are consistent with the inconclusive evidence on occupational exposure of healthcare workers to medical X-rays. Hama et al. [15] administered a questionnaire survey among 700 male radiologists working in university hospitals, with a response rate of 89%, and reported a low percentage of male children (48.5%). Interestingly, upon limiting the subjects to those who responded “yes” to the question “Have you ever received a dose of radiation higher than the maximum recommended by the International Commission on Radiological Protection?”, the percentage of male children was 34.5% (30/87), which is similar to what we identified in the RT group (30%). Zadeh et al. [16] conducted a questionnaire survey among 504 orthopedic surgeons (response rate, 66%) and reported that 47.0% of their children were male. Pillarisetti et al. [17] conducted a web-based survey of 8,000 cardiologists (response rate, 6%) and noted a similar trend, with 49.9% of the children being male. However, these results are limited by selection biases because they used questionnaires with voluntary responses.

The effect of radiation exposure other than medical X-ray exposure on the sex ratio of the offspring of healthcare workers remains controversial. Jablon & Kato [21] studied 1172 Japanese men exposed in utero to the atomic bomb and reported that exposure had no effect on the sex ratio of their children. Shea & Little [22] also found that in a study of 7678 children, in which a questionnaire was used to determine whether the father had been X-rayed in the 12 months prior to conception, the father’s exposure had no effect on the sex ratio of the child. In a study of nearly 150,000 children in India, Koya et al. [23] observed no difference in the sex ratio of children born in natural high-level radiation areas and those in natural normal-level radiation areas. Moreover, Dickinson et al. [24] reported a sex ratio (number of male children/number of female children) of 1.101 in a study of approximately 10,000 children whose fathers had worked in a nuclear facility before conception. Scherb et al. [25] found a sharp increase in the male birth rate after 1986 in Russia and Cuba; Cuba imported 60% of their food from the Soviet Union and attributed this finding to radioactive contamination caused by the Chernobyl disaster. In particular, the results of the two studies cited above are inconsistent with our findings. The discrepancy in results may be due to differences in various factors (e.g., source dose, frequency of exposure, continuous versus intermittent exposure, external versus internal exposure, and amount of testicular exposure) that may have affected the sex ratio of offspring.

Possible explanations of our findings

It is unclear whether the Y chromosome is more radiosensitive than the X chromosome, which could result in a higher concentration of X-chromosome-bearing sperm (X-CBS) in the testes and thereby a higher probability of female births that could decrease the sex ratio. However, a previous study [26] revealed that male mice exposed to competitive risks had lower proportion of Y-CBS. A study [27] in dogs indicated that the ingestion of environmental toxicants might cause bias in the sex ratio of the sperm toward X-CBS. Various factors have been suggested to reduce the sex ratio of sperms in non-human mammals.

As mentioned earlier, physicians perform operations while facing the table with a coat-type protector in the catheterization room, whereas surgeons perform surgery in a variety of positions with an apron-type protector in the operating room. The position of the surgeon is strongly associated with radiation exposure, as chest exposure at a 90° angle to the source position was reported to have twice as high radiation exposure as that in the forward-facing position [28]. Because lead protectors have a near-100% shielding effect [28, 29], the amount of radiation exposure varies greatly between the areas covered by the protector and those that are exposed. Therefore, it was clear that the RT group possessed a higher risk for testicular radiation exposure than the RNT group. In this study, the sex ratio in the RT group was notably lower, whereas that in the RNT group was similar to that in the N group. These results imply that testicular radiation exposure could possibly reduce the sex ratio of the sperm.

Strengths and limitations

A key strength of our study was the use of complete enumeration data of at-risk populations. A comprehensive survey allowed us to present associations without non-respondent bias.

However, this study had several limitations. First, possible radiation exposure might have been misclassified, and to date, there is no available measurement for actual testicular radiation exposure. Because radiation exposure is influenced by various factors, such as the radiation dose and surgeon’s protective actions, this poses a limitation to defining testicular radiation exposure based solely on information on the department to which the father belonged. Furthermore, department affiliation was only available at one time point, i.e., one year before the birth of the child. The assumption of the period at risk could have led to misclassification if the personnel’s department had changed within that period. In addition, because we could not determine the timing of exposure, we cannot differentiate between spermatid effects, which we hypothesized, and spermatogonial effects. Second, there was the possible presence of unmeasured confounders, as we have only adjusted for paternal age at the child’s conception. Ideally, a randomized controlled trial would provide a more valid comparison. However, it is ethically impossible to enforce and verify this hypothesis. Furthermore, no strong risk factors affecting the sex ratio of the offspring have been reported, and it can be inferred that the impact of unmeasured confounders is small [30]. Third, because the outcomes in this study are not rare, the odds ratios cannot be approximated to risk ratios (i.e., they cannot indicate the strength of the risk of the RT group). Thus, the odds ratios reported in this study should be interpreted with caution, as they are only a measure of the presence or absence of an association. Finally, although the results were highly statistically significant, they are based on a quite small sample, and the play of chance, or some unidentified confounding factor, cannot be ruled out. The fact that the sex ratio was higher in the RNT and N groups might imply a chance effect. In addition, evidence for a causal relationship between testicular radiation and decreased sex ratio of the offspring was not strong. Therefore, while interpreting the results of this study, it is important to note that the results do not support the practice of intentional testicular radiation exposure for gender selection.

Conclusions

Herein, we reported a possible association between testicular radiation exposure and low male sex ratio of the offspring. Confirmatory evidence is needed from larger studies which measure the pre-conceptional doses accumulated in various temporal periods, separating out spermatogonial and spermatid effects.

Minimal data set used in the analysis.

(XLSX)

Click here for additional data file.

4 May 2021

PONE-D-21-09546

Association between occupational testicular radiation exposure and lower male sex ratio of offspring among orthopedic surgeons

PLOS ONE

Dear Dr. Hijikata,

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Reviewer #2: No

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Reviewer #1: GENERAL COMMENTS TO THE AUTHORS:

This paper describes the results of a survey of full-time male doctors employed in 3 different professions with access to radiation with regards to the sex of their 109 children. The doctors responded to a survey asking questions about work 1 year ahead of child’s birth. The authors speculate that inadequate protection from x-ray radiation from fluoroscopy during orthopedic surgery could have an effect on the sex ratio of children born after radiation exposure. This conclusion is not supported by the data and a giant leap is made from a small survey with absolutely no information on the nature and magnitude of radiation exposures to the conclusion that “results imply an association between testicular radiation exposure and low male sex ratio of offspring. This result might help young orthopedic surgeons to recognize the risks of radiation exposure and to take protective action against it.”

There are multiple factors that could affect the ratio of boys to girls and the biological mechanism for this association has not been proposed in this paper. Before any conclusions are made, it would be necessary to understand the magnitude of radiation exposures in this group of physicians and the amount of scatter radiation to the testes from fluoroscopic procedures during orthopedic surgery.

SPECIFIC COMMENTS TO THE AUTHORS:

INTRODUCTION

Line 60:

“[8-13].”: Ref #12 is not appropriate.

Line 67:

“sex ratio of the offspring”: The authors need to cite literature (if exist) that this concern is justified.

MATERIALS AND METHODS

Line 81:

“a retrospective longitudinal analysis”: Should use standard epidemiological study designs, e.g. cohort study.

Line 87:

“a questionnaire survey”: I am not sure I agree. Even questionnaires require consent. Do you have an IRB approval for this study? After reaching the paper through, I think it is required.

Line 100:

“recall bias”: Recall bias only happens in case-control studies; therefore, this cannot be a longitudinal study.

Line 119:

“lateral and posterior radiation exposure.”: How much of the radiation dose could be received this way? Could you please give us an idea? What is the mean cumulative, badge-based dose for study participants? What percentage of it do you think could be due to scatter radiation.

DISCUSSION

Line 181:

“comprehensive”: Why does this study qualify to be called “comprehensive”?

Line 187:

“who are exposed”: If this is correct, there are bigger concerns about the effects of radiation in those physicians than changing sex ratio.

Line 193:

“89%”: What was the response rate in this study?

Line 246:

“imply a chance effect.”: What were the badge doses?

CONCLUSIONS

Line 252:

This is a case-control study: you started with cases (male offspring) and controls (female offspring) and then interviewed them about place of work 1 year before child’s birth. However, because outcomes are not rare (~50%), odds do not approximate risks. This is most likely due to biased sample selection and recall bias.

REFERENCES

Line 312:

“12.”: It is hard to understand why this reference was chosen. See Ashmore et. al. 2010, which addresses problems with Canadian NDR.

Reviewer #2: General comments

This is an interesting and generally well written examination of sex ratio in offspring of orthopedic surgeons. The findings, although apparently quite strong, are somewhat outweighed by the absence of quantitative dose information. The authors use of the 12 months preceding birth is an odd one. If the sex ratio changes are a spermatid effect then the relevant period is from 12 to 9 months before the birth of a child, while if the changes are a spermatogonial effect than then anything prior to 12 months before birth should be considered. The Discussion is a little skimpy, and the “Comparison with other studies” section could usefully consider the findings in other studies, which are mostly null or in the opposite direction to the present study. I think the authors should tone down the conclusions in the Abstract and at the end of the Discussion in the light of the problems with this study and the lack of support of their findings from other studies. Although the MS is generally clear and well written, the language is occasionally non-idiomatic and would benefit from services of a native English speaker.

Detailed comments (page, line)

p.3 l.48-49 This sentence (“This result …”) should probably be toned down in the light of the problems with this study and the lack of support of these findings from other studies.

p.7 l.130-137 The use of one year before the birth of the child as the relevant period is odd. If the sex ratio changes are a spermatid effect, as the authors seem to hypothesize then the relevant period is from 12 to 9 months before birth (i.e. 3 months before conception), since the process of sperm maturation is about 2-3 months. If spermatogonial effects are more relevant then it would be anything before 12 months before birth (i.e., more than 3 months before conception). The analysis needs to be changed to reflect these biological data.

p.11 l.190-p.12 l.205 The statement “The results of this study are consistent with the available evidence” is not altogether correct. The study of Jablon & Kato (Am J Epidemiol 1971 93 253-8) of the offspring of Japanese atomic bomb survivors found no paternal-radiation-associated change in sex ratio. Dickinson et al (J Epidemiol Commun Health 1996 50 645-52) found some evidence of elevation of male:female sex ratio in offspring of British nuclear workers receiving >10 mSv 90-day preconceptional dose (so in the opposite direction to that implied by this study) but there was no association with total preconceptional dose. Shea et al (Am J Epidemiol 1997 145 546-51) studied a general UK population and found that the sex ratio in exposed fathers was the same as in unexposed fathers; however, there was no dosimetry in this study. Koya et al (Radiat Environ Biophys 2015 54 453-63) observed no change in sex ratio with father’s preconceptional dose in the Kerala high background radiation area of India. Scherb et al (Environ Health 2013 12 63) document a jump in the sex ratio, with increased proportion of male births, post 1986 in Russia and Cuba, which they attribute to radioactive contamination from the Chernobyl nuclear accident; however there was no dosimetry in this study, but nevertheless the results point in the opposite direction to those in the present study. The study of Hama et al that the authors discuss is interesting, but it is based on a self-selected survey sample and the possibility of selection bias cannot be discounted. The very small reduction in sex ratio in the study of Pillarisetti et al is probably not significant. The Discussion section could usefully discuss all these findings.

p.13 l.233-234 This sentence (“First, exposure variables … exposure.”) is somewhat misleading. Quantitative radiation exposure variables are not available in the present study, or at least not given. The only measure of exposure is the type of job performed, which is probably a poor surrogate. It might be best if this is rephrased as “First, the fact of possible radiation exposure may be misclassified, and there is no measurement of actual testicular radiation exposure.”

p.13 l.238-239 The sentence “Therefore it is not possible…” should probably be removed, as it can be inferred from what is said previously, assuming the sentence above is modified in the say suggested.

p.14 l.254-255 This sentence (“This result …”) should probably be toned down in the light of the problems with this study and the lack of support of these findings from other studies.

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18 Jun 2021

Response to Reviewers:

Reviewer #1

Thank you for your important remarks. Our responses to your comments are found below.

GENERAL COMMENTS TO THE AUTHORS:

This paper describes the results of a survey of full-time male doctors employed in 3 different professions with access to radiation with regards to the sex of their 109 children. The doctors responded to a survey asking questions about work 1 year ahead of child’s birth. The authors speculate that inadequate protection from x-ray radiation from fluoroscopy during orthopedic surgery could have an effect on the sex ratio of children born after radiation exposure. This conclusion is not supported by the data and a giant leap is made from a small survey with absolutely no information on the nature and magnitude of radiation exposures to the conclusion that “results imply an association between testicular radiation exposure and low male sex ratio of offspring. This result might help young orthopedic surgeons to recognize the risks of radiation exposure and to take protective action against it.”

There are multiple factors that could affect the ratio of boys to girls and the biological mechanism for this association has not been proposed in this paper. Before any conclusions are made, it would be necessary to understand the magnitude of radiation exposures in this group of physicians and the amount of scatter radiation to the testes from fluoroscopic procedures during orthopedic surgery.

Response

Thank you for your valuable suggestions. As you pointed out, the sample size of this study is small, and the methods are not rigorous; therefore, we understand that we can only report only exploratory results. We have improved the content of the manuscript (particularly the interpretation of the results) and included a review of the existing evidence. We would love to hear from you concerning this.

SPECIFIC COMMENTS TO THE AUTHORS:

INTRODUCTION

Line 60:

“[8-13].”: Ref #12 is not appropriate.

Response

We read the study by Ashore (2010) and realized that there was a problem with the Canadian NDR. Ref #12, Sent (2001), has been deleted as it was cited for background information only and does not relate significantly to the content of the manuscript. Thank you very much for providing the reference. It was very helpful.

Line 67:

“sex ratio of the offspring”: The authors need to cite literature (if exist) that this concern is justified.

Response

Thank you for this plausible suggestion. The paper that prompted us to conduct this study is Zadeh HG (1997), modified Ref #16 (Ref. #17 before revision). This reference has been cited in the manuscript (lines 72-74, page 4 of the 'Manuscript'), and its contents have been presented and compared with the results of the present study in the Discussion (lines 219-221, page 12 of the 'Manuscript').

MATERIALS AND METHODS

Line 81:

“a retrospective longitudinal analysis”: Should use standard epidemiological study designs, e.g. cohort study.

Response

Thank you for your suggestion. Your comment on recall bias below helped us to understand that this was a case-control design. We have changed longitudinal analysis to case-control study (line 83, page 5 of the 'Manuscript').

Line 87:

“a questionnaire survey”: I am not sure I agree. Even questionnaires require consent. Do you have an IRB approval for this study? After reaching the paper through, I think it is required.

Response

Thank you for your question. On June 8, 2021, an additional ethical review was carried out by the certified ethics committee of the main research institution, Shinkomonji hospital, regarding the ethical appropriateness of the analysis and publication of the results using the dataset currently in our possession. The study was approved. We have added this information in the study design and setting (lines 90-91, page 5 of the 'Manuscript').

Line 100:

“recall bias”: Recall bias only happens in case-control studies; therefore, this cannot be a longitudinal study.

Response

Thank you for this remark. We recognized that a case-control design was most appropriate, as the study measured the presence or absence of the outcome (female sex of the child) at the time of the survey and measured the presence or absence of the exposure (opportunity for testicular radiation exposure) retrospectively. As mentioned above, we have revised the study design section (line 83, page 5 of the 'Manuscript').

Line 119:

“lateral and posterior radiation exposure.”: How much of the radiation dose could be received this way? Could you please give us an idea? What is the mean cumulative, badge-based dose for study participants? What percentage of it do you think could be due to scatter radiation.

Response

Thank you for these very important questions. As we mentioned in the Discussion, we are aware that one serious limitation of this study was the inability to measure the actual radiation dose. Since we were unable to provide additional data, we cited the study by Funao et al. (modified ref. 18), wherein the radiation dose was similar to ours (lines 131-136, page 7 of the 'Manuscript').

DISCUSSION

Line 181:

“comprehensive”: Why does this study qualify to be called “comprehensive”?

Response

Thank you for your question. We referred to the study as ‘comprehensive,’ because we had data for the entire cohort, as all subjects in the study settings were included. However, following your remarks, we understood that the study is a case-control study. We have deleted the term ‘comprehensive’ since it is not appropriate in a case-control design i.e., the source cohort is not captured (lines 198, page 11 of the 'Manuscript').

Line 187:

“who are exposed”: If this is correct, there are bigger concerns about the effects of radiation in those physicians than changing sex ratio.

Response

Thank you for pointing this out. The phrase ‘who are exposed’ was clearly an exaggeration. We have replaced it with a more conservative term, ‘who might be exposed.’ There is no doubt that inexperienced doctors (as we were) tend to rely more on radiography in clinical practice, and the results of ref.5 suggest this. To confirm that the younger generation is more exposed to radiation, radiation exposure should be measured. The Society for MIST (https://s-f-mist.com/en/), of which we are members, has taken the lead in conducting a survey to determine the extent to which radiation damage is actually occurring; publication of the results is pending.

Line 193:

“89%”: What was the response rate in this study?

Response

Thank you for your question. Although the sample size was small, the response rate was 100%. It has been stated in the Results that ‘the responses were obtained from all eligible doctors.’ We have also added the percentage of responses (lines 170, page 9 of the 'Manuscript').

Line 246:

“imply a chance effect.”: What were the badge doses?

Response

Thank you for your question. We could not obtain information on the actual radiation dose around the testes for each group, as the rate of badge wearing was very low in the target population, and the badges were never worn around the testes. The term ‘chance effect’ was used because we could not explain the mechanism by which the sex ratio skewed towards males in the RNT and RT groups. Therefore, it was most likely a ‘chance effect.’

CONCLUSIONS

Line 252:

This is a case-control study: you started with cases (male offspring) and controls (female offspring) and then interviewed them about place of work 1 year before child’s birth. However, because outcomes are not rare (~50%), odds do not approximate risks. This is most likely due to biased sample selection and recall bias.

Response

Thank you for these important remarks. As you pointed out, this is a case-control study, and the outcomes are not rare. Therefore, the odds ratio cannot be interpreted as a risk ratio. This means that therefore, we cannot conclude from the results that ‘the RT group was four times more likely to have daughters.’ However, we believe that the odds ratios were useful in determining whether there was an association. We also believe that there was no significant selection bias, at least in the target population, as all respondents were available at the time of the survey. Furthermore, although participants had to recall information from up to 11 years prior to the study, it was easy for them to recall their department at that time; therefore, the recall bias was small.

The manuscript has been revised substantially to bring it closer to a reasonable interpretation. First, the Key results and clinical implications were revised for a better interpretation of the results (lines 198-208, page 11-12 of the 'Manuscript'). Second, to avoid misunderstanding about the odds ratios, we have added a limitation to the Strengths and limitations section (lines 289-293, page 15 of the 'Manuscript'). Finally, the claims made in the Conclusions have been modified (lines 51, page 3 and, lines 303-304, page 16 of the 'Manuscript').

REFERENCES

Line 312:

“12.”: It is hard to understand why this reference was chosen. See Ashmore et. al. 2010, which addresses problems with Canadian NDR.

Response

Ref #12 has been deleted. Thank you for pointing this out.

Reviewer #2

Thank you for your valuable suggestions that have helped us improve our manuscript. Our responses to each of your comments are presented below.

General comments

This is an interesting and generally well written examination of sex ratio in offspring of orthopedic surgeons. The findings, although apparently quite strong, are somewhat outweighed by the absence of quantitative dose information. The authors use of the 12 months preceding birth is an odd one. If the sex ratio changes are a spermatid effect then the relevant period is from 12 to 9 months before the birth of a child, while if the changes are a spermatogonial effect than then anything prior to 12 months before birth should be considered. The Discussion is a little skimpy, and the “Comparison with other studies” section could usefully consider the findings in other studies, which are mostly null or in the opposite direction to the present study. I think the authors should tone down the conclusions in the Abstract and at the end of the Discussion in the light of the problems with this study and the lack of support of their findings from other studies. Although the MS is generally clear and well written, the language is occasionally non-idiomatic and would benefit from services of a native English speaker.

Response

Thank you very much for summarizing the results and highlighting the issues. We will respond to your comments in more detail below. This first submitted version of the manuscript was edited by Editage (https://www.editage.jp/). We sent it to Editage for proofreading again.

Detailed comments (page, line)

p.3 l.48-49 This sentence (“This result …”) should probably be toned down in the light of the problems with this study and the lack of support of these findings from other studies.

Response

Thank you for pointing this out. The interpretation of our results was exaggerated. We have substantially revised the Key results and clinical implications (lines 198-208, page 11-12 of the 'Manuscript') and Conclusions (lines 51, page 3 and lines 303-304, page 16 of the 'Manuscript') to understate the interpretation of the results.

p.7 l.130-137 The use of one year before the birth of the child as the relevant period is odd. If the sex ratio changes are a spermatid effect, as the authors seem to hypothesize then the relevant period is from 12 to 9 months before birth (i.e. 3 months before conception), since the process of sperm maturation is about 2-3 months. If spermatogonial effects are more relevant then it would be anything before 12 months before birth (i.e., more than 3 months before conception). The analysis needs to be changed to reflect these biological data.

Response

Thank you very much for your valuable comments. As you pointed out, we stated the rationale for using the one year cut-off poorly, and the inaccuracy of the rationale was not fully explained. We hypothesized that the period at risk was the period after the secondary spermatocyte formation, when the sperm carries either the X or Y chromosome. AS you pointed out, the period at risk is not a time point, but a period with a range of 2–3 months (the period during which the secondary spermatocyte becomes sperm and is stored in the epididymis). Therefore, limiting the measurement of an exposure to a single time point may lead to misclassification. However, we believe that the magnitude of misclassification was small because it is rare for a personnel’s department affiliation to be changed within 2–3 months. Furthermore, limiting the exposure measurement to ‘one year ago’ makes it easier to be explained. Most parent do not forgets their child's birthday; therefore, we thought that it would be very easy to answer the question: ‘What department were you in exactly one year before your child's birthday?.’ We are unsure whether this was responsible for the 100% response rate.

We have added more accurate information about spermatogenesis in the Outcome of interest and exposure section (lines 143-153,　page 7-8 of the 'Manuscript'). In addition, the possibility of a misclassification of the exposure has been added to the limitations (lines 280-284, page 15 of the 'Manuscript').

p.11 l.190-p.12 l.205 The statement “The results of this study are consistent with the available evidence” is not altogether correct. The study of Jablon & Kato (Am J Epidemiol 1971 93 253-8) of the offspring of Japanese atomic bomb survivors found no paternal-radiation-associated change in sex ratio. Dickinson et al (J Epidemiol Commun Health 1996 50 645-52) found some evidence of elevation of male:female sex ratio in offspring of British nuclear workers receiving >10 mSv 90-day preconceptional dose (so in the opposite direction to that implied by this study) but there was no association with total preconceptional dose. Shea et al (Am J Epidemiol 1997 145 546-51) studied a general UK population and found that the sex ratio in exposed fathers was the same as in unexposed fathers; however, there was no dosimetry in this study. Koya et al (Radiat Environ Biophys 2015 54 453-63) observed no change in sex ratio with father’s preconceptional dose in the Kerala high background radiation area of India. Scherb et al (Environ Health 2013 12 63) document a jump in the sex ratio, with increased proportion of male births, post 1986 in Russia and Cuba, which they attribute to radioactive contamination from the Chernobyl nuclear accident; however there was no dosimetry in this study, but nevertheless the results point in the opposite direction to those in the present study. The study of Hama et al that the authors discuss is interesting, but it is based on a self-selected survey sample and the possibility of selection bias cannot be discounted. The very small reduction in sex ratio in the study of Pillarisetti et al is probably not significant. The Discussion section could usefully discuss all these findings.

Response

Thank you very much for all the reference-by-reference information you have provided. In the manuscript, we only cited previous studies on occupational exposure to medical X-rays among healthcare workers. We have reviewed the papers you suggested and have made significant additions and revisions to the Comparison with other studies section for a more in-depth discussion (lines 228-247, page 13 of the 'Manuscript').

p.13 l.233-234 This sentence (“First, exposure variables … exposure.”) is somewhat misleading. Quantitative radiation exposure variables are not available in the present study, or at least not given. The only measure of exposure is the type of job performed, which is probably a poor surrogate. It might be best if this is rephrased as “First, the fact of possible radiation exposure may be misclassified, and there is no measurement of actual testicular radiation exposure.”

Response

Thank you for your detailed remarks. We have revised the sentence (lines 275-277, page 15 of the 'Manuscript').

p.13 l.238-239 The sentence “Therefore it is not possible…” should probably be removed, as it can be inferred from what is said previously, assuming the sentence above is modified in the say suggested.

Response

We have deleted the sentence as suggested.

p.14 l.254-255 This sentence (“This result …”) should probably be toned down in the light of the problems with this study and the lack of support of these findings from other studies.

Response

Following your remarks, we have toned down our conclusions. Thank you very much for reading the manuscript in detail and for your constructive comments. We have revised in the Conclusions (lines 51, page 3 and, lines 303-304, page 16 of the 'Manuscript').

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.

9 Aug 2021

PONE-D-21-09546R1

Association between occupational testicular radiation exposure and lower male sex ratio of offspring among orthopedic surgeons

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: General comments

This is an interesting and generally well written examination of sex ratio in offspring of orthopedic surgeons. The paper is much improved on the first version. However, there are still quite a few weaknesses, both in the presentation of the Tables and particularly in the Discussion.

Detailed comments (page, line)

p.3 l.51 I would replace the final sentence by “Confirmatory evidence is needed from larger studies which measure the pre-conceptional doses accumulated in various temporal periods, separating out spermatogonial and spermatid effects.”.

p.8 l.146-153 What evidence is there in support of this hypothesis, that sex ratio results entirely from spermatid effects? If this is (as I suspect) a largely post hoc hypothesis then this should be made clear.

p.8 l.148 I assume that by “2-3 months” is meant “2-3 months before conception”.

p.8 l.151-153 Is this sentence (“This means that…”) correct? Surely it is the period 1-3 months before conception rather than the date of birth that is meant.

Tables 1, 2, 3 It is needlessly confusing that in Table 1 the proportion of female children is given, whereas in Table 2 it is effectively the proportion of male children that is given. Although the labelling of Table 3 does not make this clear, it must be the case that it is the odds ratio of female:male births that is given. The labelling of Table 3 must be improved, and all three Tables use more or less the same measure (males/total or females/total, it does not matter which).

p.12 l.211 – p.13 l.227 The sentence on p.12 l.211-212 (“The results of this study are consistent …”) should be removed or at least toned down. The evidence cited in support of this statement in the rest of the para is really rather weak. As previously noted by the referee the study of Hama et al that the authors discuss is interesting, but it is based on a self-selected survey sample and the possibility of selection bias cannot be discounted. The very small reduction in sex ratio in the study of Pillarisetti et al is probably not significant. These weaknesses and limitations of this evidence must be discussed here.

p.13 l.224-227 This sentence (“The results of this study, obtained …”) repeats what is said at the beginning of the Discussion, and should be removed.

p.13 l.246-247 This sentence (“Therefore, it should be noted …”) is rather odd. Are the authors really implying that the effects observed are not due to the radiation alone, but to some other occupational factor specific to healthcare workers? Obviously if the effects are related to radiation exposure (rather than some unknown factor specific to this occupational group) there is no reason why they should not apply to all radiation exposed groups. If the findings relate to some factor specific to this occupational group then this considerably lessens their scientific interest, and begs the question as to whether radiation exposure is really the cause of what has been observed in this study.

p.15 l.276-277 There should be discussion at this point of the limitations of the data in relation to timing of exposure. In particular the data make it quite difficult to differentiate between spermatid effects (which the authors assume, as above, but possibly based on no strong prior body of data) and spermatogonial effects. This hypothesis could also be discussed.

p.15 l.293 – p.16 l.294 The sentence is not terribly clear, and the authors should also highlight the role that confounding by some unknown factor may play. So perhaps rephrase as: “Finally, although the results were highly statistically significant, they are based on a quite small sample, and the play of chance, or some unidentified confounding factor, cannot be ruled out.”.

p.16 l.303-304 I would replace the final sentence by “Confirmatory evidence is needed from larger studies which measure the pre-conceptional doses accumulated in various temporal periods, separating out spermatogonial and spermatid effects.”.

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Submitted filename: Hijikata et al PLoS ONE re-review 2021.docx

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21 Aug 2021

Response to Reviewers:

Reviewer #2

General comments

This is an interesting and generally well written examination of sex ratio in offspring of orthopedic surgeons. The paper is much improved on the first version. However, there are still quite a few weaknesses, both in the presentation of the Tables and particularly in the Discussion.

Response: Thank you for your insightful comments. We have revised the manuscript in line with your suggestions. We hope that our edits and the responses we have provided below satisfactorily address all the issues and concerns you have noted and that the revised manuscript is now suitable for publication.

Detailed comments (page, line)

p.3 l.51

I would replace the final sentence by “Confirmatory evidence is needed from larger studies which measure the pre-conceptional doses accumulated in various temporal periods, separating out spermatogonial and spermatid effects.”.

Response: Thank you for this comment. We have accordingly replaced the final sentence (p.3 l.47-49).

Revise: Confirmatory evidence is needed from larger studies which measure the pre-conceptional doses accumulated in various temporal periods, separating out spermatogonial and spermatid effects.

p.8 l.146-153

What evidence is there in support of this hypothesis, that sex ratio results entirely from spermatid effects? If this is (as I suspect) a largely post hoc hypothesis then this should be made clear.

Response: Thank you for your comment. To clarify, this is not a post-hoc hypothesis. There is a widespread rumor among orthopeadic surgeons in Japan that the children of orthopedic surgeons tend to be female. If this is indeed true, we would like to understand why this is so. Thus, we formulated this hypothesis and designed this study to test it. However, as you have pointed out, this hypothesis has no supportive evidence, and we have accordingly made this clear (p.7 l.141).

Revise: We hypothesized, albeit without supporting evidence, that…

p.8 l.148

I assume that by “2-3 months” is meant “2-3 months before conception”.

Response: Yes, and per your comment, we have revised this in the manuscript for clarity (p.8 l.143-144).

Revise: 2–3 months before conception.

p.8 l.151-153

Is this sentence (“This means that…”) correct? Surely it is the period 1-3 months before conception rather than the date of birth that is meant.

Response: We apologize for the unclear text. We have now corrected this to "The risk period began one year before the child’s birthday” (p.8 l.146-149).

Revise: In other words, considering that the gestation period is approximately 280 days, the period at risk of a decrease in the male sex ratio of the child due to testicular radiation exposure began approximately one year before the child's birthday.

Tables 1, 2, 3

It is needlessly confusing that in Table 1 the proportion of female children is given, whereas in Table 2 it is effectively the proportion of male children that is given. Although the labelling of Table 3 does not make this clear, it must be the case that it is the odds ratio of female:male births that is given. The labelling of Table 3 must be improved, and all three Tables use more or less the same measure (males/total or females/total, it does not matter which).

Response: Thank you very much for your meaningful comments. We apologize for the confusing presentation of our tables. Per your comment, we have presented all calculated sex ratios in this study as female sex ratio and have accordingly revised the relevant sections (i.e., Abstract [p.2 l.33-34 and l.41], Mateials and Methods [p.8 l.155-156], Results [p.10 l.183, l.185], and Tables 1 and 2). Moreover, in other instances, we have clearly indicated whether we are referring to female or male sex ratio (p.4 l.70, p.5 l.75, p.7 l.139, l.142, p.8 l.148 and l.155). We have also revised the title of Table 3 to clearly indicate that the odds ratios of having female children are presented (p.11 l.207).

p.12 l.211 – p.13 l.227

The sentence on p.12 l.211-212 (“The results of this study are consistent …”) should be removed or at least toned down. The evidence cited in support of this statement in the rest of the para is really rather weak. As previously noted by the referee the study of Hama et al that the authors discuss is interesting, but it is based on a self-selected survey sample and the possibility of selection bias cannot be discounted. The very small reduction in sex ratio in the study of Pillarisetti et al is probably not significant. These weaknesses and limitations of this evidence must be discussed here.

Response: Thank you for your significant remarks. We have revised the tone of this particular sentence (p.12 l.234) and corrected the last sentence of the paragraph (p.13 l.245-246).

Revise: Our results are consistent with the inconclusive evidence…

However, these results are limited by selection biases because they used questionnaires with voluntary responses.

p.13 l.224-227

This sentence (“The results of this study, obtained …”) repeats what is said at the beginning of the Discussion, and should be removed.

Response: We have deleted this sentence per your comment.

p.13 l.246-247

This sentence (“Therefore, it should be noted …”) is rather odd. Are the authors really implying that the effects observed are not due to the radiation alone, but to some other occupational factor specific to healthcare workers? Obviously if the effects are related to radiation exposure (rather than some unknown factor specific to this occupational group) there is no reason why they should not apply to all radiation exposed groups. If the findings relate to some factor specific to this occupational group then this considerably lessens their scientific interest, and begs the question as to whether radiation exposure is really the cause of what has been observed in this study.

Response: Thank you for pointing this out. We believe that radiation affects not only medical professionals but also the general population. We understand that our phrasing was misleading and have deleted the sentence accordingly.

p.15 l.276-277

There should be discussion at this point of the limitations of the data in relation to timing of exposure. In particular the data make it quite difficult to differentiate between spermatid effects (which the authors assume, as above, but possibly based on no strong prior body of data) and spermatogonial effects. This hypothesis could also be discussed.

Response: We appreciate your comments. As you have pointed out, we have discussed the limitations of our data in relation to timing exposure (p.15 l.301-303).

Revise: In addition, because we could not determine the timing of exposure, we cannot differentiate between spermatid effects, which we hypothesized, and spermatogonial effects.

p.15 l.293 – p.16 l.294

The sentence is not terribly clear, and the authors should also highlight the role that confounding by some unknown factor may play. So perhaps rephrase as: “Finally, although the results were highly statistically significant, they are based on a quite small sample, and the play of chance, or some unidentified confounding factor, cannot be ruled out.”.

Response: Per your comment, we have rephrased this for clarity (p.16 l.312-314).

Revise: Finally, although the results were highly statistically significant, they are based on a quite small sample, and the play of chance, or some unidentified confounding factor, cannot be ruled out.

p.16 l.303-304

I would replace the final sentence by “Confirmatory evidence is needed from larger studies which measure the pre-conceptional doses accumulated in various temporal periods, separating out spermatogonial and spermatid effects.”.

Response: Thank you for this suggestion. We have accordingly revised this (p.16 l.322-324).

Revise: Confirmatory evidence is needed from larger studies which measure the pre-conceptional doses accumulated in various temporal periods, separating out spermatogonial and spermatid effects.

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.

19 Dec 2021

Association between occupational testicular radiation exposure and lower male sex ratio of offspring among orthopedic surgeons

PONE-D-21-09546R2

Dear Dr. Hijikata,

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Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. 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 #2: Yes

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

Reviewer #2: Yes

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4. 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 #2: No

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

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Reviewer #2: Yes

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6. 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 #2: General comments

This is an interesting and generally well written examination of sex ratio in offspring of orthopedic surgeons. The paper is much improved on the second version, and has met all my concerns.

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Reviewer #2: No

21 Dec 2021

PONE-D-21-09546R2

Association between occupational testicular radiation exposure and lower male sex ratio of offspring among orthopedic surgeons

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