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. 2020 May 1;15(5):e0232156. doi: 10.1371/journal.pone.0232156

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility

Aula Asali 1,*, Netanella Miller 1, Yael Pasternak 1, Vita Freger 2, Michael Belenky 2, Arie Berkovitz 1,3,4
Editor: Marco Aurélio Gouveia Alves5
PMCID: PMC7194411  PMID: 32357155

Abstract

Purpose

To examine the efficacy of motile sperm organelle morphology examination (MSOME) and intracytoplasmic morphologically-selected sperm injection (IMSI) for unexplained infertility.

Methods

This historical study, included 271 couples with primary, unexplained infertility/male subfertility, treated at an outpatient, IVF clinic, 2015–2018. These couples underwent MSOME after ≥3 failed intrauterine insemination (IUI) cycles and ≥1 failed IVF-ICSI cycle. They proceeded to intracytoplasmic morphologically-selected sperm injection (IMSI) within 6 months of MSOME. IMSI is conducted on the day of oocyte pick-up with a fresh semen sample. Pregnancy and delivery rates were analyzed.

Results

The cohort was divided based on percentage of normal cells at MSOME: Group A included 55 with no normal cells, Group B, 184 with 0.5%≤ normal cells ≤1.5% and Group C, 32 with ≥2% normal cells. Normal spermatozoa were found in 49 (89%) of Group A after extensive search. Group A had higher pregnancy rate (62.7%) compared to B (47.2%, P = 0.05) and C (28.1%, P = 0.002). Group B had higher pregnancy rate than C (p = 0.045). Delivery rate was higher in Group A (52.1%) compared to B (34.1%, p = 0.023) and C (21.9%, p = 0.007). Pregnancy and delivery rates were higher in A compared to B+C (p = 0.018, p = 0.01, respectively).

Conclusions

MSOME may be useful for evaluating unexplained infertility. IMSI can be recommended for men with <2% normal spermatozoa at MSOME.

Introduction

Unexplained infertility refers to the absence of a definable cause for a couple's failure to conceive after 12 months despite thorough evaluation, or after 6 months among women 35 and older [1]. It affects 10–30% of infertile/sub-fertile couples [2,3].

In contrast to clearly diagnosed conditions, such as ovulatory disorders, unexplained infertility may encompass situations that are overlooked by conventional infertility assessment, or are undetectable using current diagnostic techniques. A systematic review concerning treatment options for couples with unexplained infertility concluded that current data were not compelling enough to indicate a specific treatment modality that would significantly increase the chance of conception and suggested that these couples should be assessed and treated individually [2,4].

Motile sperm organelle morphology examination (MSOME) is performed in real-time using an inverted light microscope equipped with high-power Nomarski optics, enhanced with digital imaging to achieve magnification up to 6300× [5]. Thus, MSOME enables detecting subtle sperm organellar malformations in motile spermatozoa that an embryologist might consider normal for fertilization at 200× to 400× magnification [5]. Using this technique together with a micromanipulation system has allowed the introduction of a modified intracytoplasmic sperm injection (ICSI) procedure, known as intracytoplasmic morphologically-selected sperm injection (IMSI) [6]. When the spermatozoa with normal morphology and motility selected for ICSI are detected under a magnification of ×400, in IMSI the motile spermatozoa are selected under magnification up to 6300×[5].

To the best of our knowledge, there are no data regarding the role of MSOME and possibly IMSI in evaluating couples with unexplained infertility. This study examined the efficacy of IMSI in unexplained infertility when conception did not occur after at least one IVF-ICSI cycle. We examined also the possible benefit of IMSI in relation to MSOME results.

Materials and methods

This historical study included 271 couples. The inclusion criteria were couples with primary unexplained infertility/male subfertility who had experienced at least three failed IUI cycles and at least one failed IVF-ICSI cycle, from 2015 through 2018 in Assuta Medical Center, Rishon LeZion, Israel. All couples underwent IMSI within 6 months of the initial MSOME. IMSI is conducted on the day of oocyte pick-up with a fresh semen sample.

Since 2015, when MSOME was incorporated in our lab, we offer MSOME evaluation to couples with unexplained infertility who had at least one failed IVF-ICSI cycle. Male subfertility was defined as a total motile sperm count ≥1 × 106 [7].

Data were collected regarding the first IVF-IMSI cycle after examination. Information regarding transferred frozen embryos of IVF-IMSI cycles (cumulative pregnancy rate) were included, as well.

Exclusion criteria were Mullerian abnormality or submucosal or subserosal fibroids >7 cm, despite regular menstrual cycles, because of the negative effect of these complications on implantation and pregnancy outcomes [8]; ovulation abnormalities; tubal pathology; and male infertility (when the total motile sperm count was <1 × 106, because IUI treatment does not benefit this group).

Data concerning routine semen analysis, percentage of normal cells at MSOME, basic demographic characteristics of the female partner, number of retrieved oocytes, number of transferred embryos, day of transfer and pregnancy outcomes (abortion rate, delivery rate) were collected from electronic medical records.

Clinical pregnancy, defined as missed abortion, blighted ovum or fetal pole with heartbeat, was considered a positive result. Chemical pregnancy (when the level of hCG is initially elevated enough to produce a positive result on a pregnancy test but then declines before a gestational sac is detected with ultrasound) was considered a negative result.

The cohort was divided into 3 groups according to the percentage of normal cells at MSOME. Group A included 55 cases with no normal cells at MSOME. Group B included 184 cases with 0.5% to 1.5% normal cells at MSOME and Group C consisted of 32 cases of at least 2% normal cells at MSOME. This reflects the normal MSOME, as a fertile male has an average of 2% normal cells (laboratory data, unpublished).

The primary outcomes of cumulative pregnancy rate and delivery rate were compared between the three subgroups of the cohort (A, B and C), in order to define the population who may benefit most from IMSI.

Spermatozoa collection and processing for MSOME

Sperm samples were obtained by masturbation or by using a spermicide-free polyurethane condom. After being allowed to liquefy at room temperature for 20 minutes, the samples were loaded on PureCeption Sperm Separation Media (Sage In-Vitro Fertilization, Inc. Trumbull, CT, USA) gradient of 1 mL 40% v/v (upper phase) and 1 ml 80% v/v (lower phase). This consisted of a sterile colloidal suspension of silane-coated silica particles in HEPES-buffered human tubal fluid containing 10 mg/l gentamicin. It was centrifuged for 20 minutes at 300 relative centrifugal field at room temperature. The upper liquid was then removed and the pellet was re-suspended in Quinn's Sperm Washing Medium (Sage In-Vitro Fertilization, Inc.) and centrifuged again for 5 minutes at 600 rpm at room temperature. After this procedure was performed twice, the pellet was re-suspended in sperm washing medium at a spermatozoa concentration of approximately 107/ml. A 4 μl droplet of polyvinyl pyrrolidone (PVP) 10% solution (Sage In-Vitro Fertilization, Inc.) was placed in a glass-bottom tissue culture dish (World Precision Instruments, Sarasota, FL, USA) and covered with paraffin oil (Sage In-Vitro Fertilization, Inc.). Next, 1 μl of the sample suspension was loaded onto the PVP droplet. The droplet containing the spermatozoa was examined under 6000x magnification using an inverted phase contrast microscope Nikon Eclipse Ti (Nikon Instruments, Inc., Melville, NY, USA) equipped with differentiated interference contrast, and an Invenio 3SII camera. The magnification was achieved by using an oil-covered 100x lens, while the additional 60x magnification was provided by DeltaPix software (Smorum, Denmark).

Then, 200 motile spermatozoa along the borders of the droplet were examined for size, shape and presence of nuclear vacuoles and disorders, Fig 1.

Fig 1. Sperm head morphology at 6000x magnification.

Fig 1

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A. Normal morphology. B. Large acrosomal vacuole. C. Equatorial and post-acrosomal vacuoles. D. Wide head. E. Narrow head. F. Highly vacuolated head.

The percentage of normal sperm head shapes was calculated based on the following criteria: length 4.75 ± 0.28 μm and width 3.28 ± 0.20 μm; symmetric oval shape; smooth texture; no more than 2 acrosomal vacuoles comprising <4% of the sperm head area; no post-acrosomal or deep vacuoles; centrally located midpiece and no regional disorders such as cytoplasmatic extrusions or invaginations [9–11].

Ethics statement

The study was approved by the Assuta Medical Center Ethics Committee. Due to the retrospective nature of the study, informed consent was not required.

Data will be made available upon reasonable request to the corresponding author.

Statistical analysis

Data are described as mean, standard deviation, minimum and maximum for continuous variables and as percentage of total for nominal parameters. In univariate analyses, Chi-Square test or Fisher's Exact test were used for non-metric parameters, each when appropriate. One-way analysis of variance or Kruskal-Wallis non-parametric test was used to compare three groups at different levels of MSOME, each by matching the rules. Bonferroni post hoc comparisons were used (to adjust P-values when several dependent or independent statistical tests were performed simultaneously on a single data set), The alpha used for the Bonferroni Correction was 0.05/3 = 0.017. Logistic regression was used to evaluate the effect of parameters that were statistically different by pregnancy rate in the univariate analysis. The multinomial significance levels were set at 0.05. All statistical analyses were performed using SPSS-25.

Results

The cohort included 312 couples who underwent MSOME after at least one failed IVF-ICSI attempt. Among them, 68 (21.8%) couples had isolated teratozoospermia (no normal spermatozoa in MSOME), 205 (65.7%) had male subfertility and 39 (12.5%) had normozoospermia. Among the 312 couples, 41 did not complete follow-up or underwent IMSI more than 6 months after the MSOME examination. Thus, 271 proceeded to IMSI within 6 months of the initial MSOME.

The basic characteristics of the cohort are shown in Table 1. The mean age of the male partners was 37.6 ± 5.8 years. The female partners were a mean age of 35.5 ± 5.5 years.

Table 1. Basic characteristics of the cohort.

Characteristic Mean ± SD Minimum Maximum
Age of male partner, years 37.6 ± 5.8 24 60
Age of female partner, years 35.5 ± 5.5 23 45
BMI of female partner 23.35 ± 4.12 16 37
Volume of the sample, ml 2.78 ± 1.5 0.2 9
Concentration, 106/ml 57.28 ± 47.7 0 250
Total motile count 75.92 ± 85.26 0.15 501.9
Motility, % 45.98 ± 18.54 3 93
Percentage of normal cells at MSOME 1.32 ± 1.5 0 8
Number of oocytes picked up 7.92 ± 4.5 1 24
Number of transferred embryos 2.13 ± 0.717 1 4
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Results of the logistic regression on pregnancy rate are shown in Table 2. Older female age and higher percentage of normal cells at MSOME had negative effects on pregnancy (p = 0.001 and p = 0.006, respectively). The day of embryo transfer had a positive effect on pregnancy (p = 0.009). No correlations were found between pregnancy and age of male partner (p = 0.101), BMI of female partner (p = 0.817), number of oocytes retrieved (p = 0.173) or number of transferred embryos (p = 0.1).

Table 2. Logistic regression on pregnancy rate.

Parameter Significance Odds ratio 95% CI for OR
Lower Upper
Male partner age, years 0.101 1.080 0.985 1.185
Female partner age, years 0.001* 0.840 0.758 0.931
BMI of female 0.817 1.008 0.940 1.082
Percentage of normal cells at MSOME 0.006* 0.374 0.186 0.754
Number of oocytes picked up 0.173 1.066 0.972 1.168
Number of transferred embryos 0.1 1.438 0.933 2.215
Day of transfer 0.009* 1.929 1.178 3.158
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*Significant difference.

CI, confidence interval, OR, odds ratio.

Cleaved embryos were transferred in 236 cases (91.8%) and blastocysts were transferred in 21 (8.2%). The cumulative pregnancy rate of the entire cohort was 46.9% (126 couples conceived after the first IVF-IMSI cycle). Four couples (3.2% of pregnancies) had multiple gestations.

Comparison between the three subgroups of the cohort

The cohort was grouped according to the percentage of normal cells at MSOME. Table 3 shows the demographic and clinical characteristics of the 3 groups. Group A included 55 cases in which no normal cells were found at MSOME, but normal cells were found during IMSI at ovum pick-up for 49 (89.1%). Group B included 184 couples and Group C 32 couples.

Table 3. Demographic and clinical characteristics of the three sub-groups.

Variable Group A (n = 55) Group B (n = 184) Group C (n = 32) P-value
Female partner age, years 36.1 ± 5.4 35.5 ± 5.6 34.1 ± 5.2 0.264
Male partner age, years 38.3 ± 4.9 37.6 ± 5.9 36.3 ± 6.2 0.283
BMI of female partner 24 ± 4.5 23.3 ± 3.98 22.8 ± 4.24 0.447
Sample volume, ml 2.72 ± 1.89 2.9 ± 1.5 2.3 ± 1 0.131
% motility 44.4 ± 18.8 46.3 ± 17.9 46.7 ± 21.9 0.785
Total motile count 41.8 ± 51.0 82.6 ± 85.2 96.3 ± 114.98 0.003*
Number of oocytes picked up 9.3 ± 4.7 7.65 ± 4.4 7.19 ± 3.99 0.06
Number of transferred embryos 2.26 ± 0.7 2.12 ± 0.7 1.97 ± 0.7 0.176
Day of transfer 2.98 ± 0.5 3.1 ± 0.6 3.25 ± 0.7 0.16
Pregnancy rate 62.7% 47.2% 28.1% 0.008**
Abortion rate 8.3% 11% 6.3% 0.657
Delivery rate (includes ongoing pregnancy) 52.1% 34.1% 21.9% 0.015*
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*Significant difference between Group A and B, and Group A and C.

**Significant difference between the three groups.

A significant difference was found between the 3 groups regarding cumulative pregnancy rate (p = 0.008) and delivery rate (p = 0.015). We found higher pregnancy rate in Group A (62.7%) compared to Group B (47.2%, p = 0.05) and Group C (28.1%, p = 0.002) and in Group B compared to C (47.2% vs. 28.1%, p = 0.045). Higher delivery rate was found in Group A (52.1%) compared to B (34.1%, p = 0.023) and C (21.9%, p = 0.007). No differences in delivery rates were found between Groups B and C (34.1% vs. 21.9%, respectively, p = 0.174).

Total motile count differed between groups (p = 0.003). It was lower in group A than in Group B (p = 0.005) and Group C (p = 0.011).

No difference was found regarding the age of the male partner (p = 0.283), the female partner (p = 0.264) or BMI (p = 0.447). No difference was found between groups in sample volume (p = 0.131), motility of the spermatozoa (p = 0.785), number of retrieved oocytes (p = 0.06), number of transferred embryos (p = 0.176), transfer day (p = 0.16) or abortion rates (8.3% vs. 11% vs. 6.3%, p = 0.657).

Comparison between subgroup A and the other study groups

When comparing pregnancy rates between Group A and the rest of the cohort (Groups B and C together), Group A experienced significantly higher pregnancy and delivery rates compared to B+C (62.7% vs. 44.3%, p = 0.018 and 52.1% vs. 32.2%, p = 0.01), respectively. In addition, in Group A more oocytes were picked-up compared to the rest of the cohort (9.3 ± 4.7 vs. 7.58 ± 4.4, p = 0.01). Total motile count was higher in group B+C (84.6 ± 90.03) than in Group A (41.84 ± 51.1, p = 0.001). No difference was found regarding abortion rate (8.33% vs. 10.24%, p = 0.69).

Discussion

The motile sperm organelle morphology examination (MSOME) was introduced by Bartoov, et al. in 2001 [12]. This examination is based on morphological analysis of isolated motile spermatozoa in real-time, at high magnification (up to 6300×) [13]. Thus, MSOME can detect subtle organellar malformations in motile spermatozoa that an embryologist might consider normal for fertilization at 200× to 400× magnification [5], especially when a normal sperm nucleus is a significant factor for successful implantation and pregnancy in ICSI procedures [13,14].

We hypothesized that an unrecognized male factor is involved in the etiology of some cases of unexplained infertility. MSOME, as described above, is more sensitive for detecting subtle sperm head abnormalities. Our cohort included couples who had at least one unsuccessful, conventional IVF-ICSI cycle, assuming that an unknown male factor was not solved in the previous cycle. In support of this assumption, a previous study [15] found an increase in implantation and pregnancy rates using IMSI over conventional ICSI for patients with previously unsuccessful ICSI, with no difference in the results of the first cycle with ICSI vs. IMSI.

To the best of our knowledge, this is the first study to evaluate the utility of MSOME and possible IMSI for unexplained infertility. We found that the percentage of normal cells at MSOME affects pregnancy rates. With IMSI, the lower the percentage of normal cells, the higher the pregnancy rate. An explanation for this apparent contradiction is that a lower percentage of normal cells in MSOME necessitates an extensive search under ultra-magnification to find the highest quality spermatozoa.

This process is not performed for routine procedures, but is performed for IMSI.

Couples with higher percentage of normal cells at MSOME do not benefit from the extensive search; they have enough normal sperm for fertilization.

In addition, men with higher percentage of normal cells in the ejaculate can be managed with routine IVF-ICSI. But, when there are only a few normal spermatozoa in the ejaculate, the chance of finding them in routine IVF-ICSI is low.

We found that even in Group A (no normal spermatozoa with MSOME), normal spermatozoa were found in 89% of the couples after an extensive search on the day of oocyte pick-up.

When dividing the cohort according to the percentage of normal cells at MSOME, we found a significantly higher pregnancy rate in Group A as compared to Group B or C and as compared to the rest of the cohort (Groups B+C). These results are in accordance with the above explanation: the lower the percentage of normal cells at MSOME the more extensive is the search for high quality spermatozoa, which occurs when IMSI is performed on the day of oocyte pick-up.

A significantly higher pregnancy rate was found in Group B vs. C, as well. Group B can be defined as male subfertility, for which an extensive search for high quality spermatozoa is also conducted; thus, these couples benefit from IMSI also.

The delivery rate in Group A was higher than that in B and C and there was no significant difference between Groups B and C. This further indicates that men with no normal cells at MSOME benefit the most from IMSI.

Based on the assumption that unexplained infertility might be partially explained by covert male factors, O’Neill et al. [4] designed a treatment algorithm based on sperm chromatin integrity to guide the management of couples with apparently unexplained infertility. They found that when these couples failed IUI but had normal sperm DNA fragmentation, IVF resulted in a clinical pregnancy rate of 12.7%. Those with abnormal sperm DNA fragmentation underwent ICSI with ejaculated spermatozoa, resulting in a higher clinical pregnancy rate of 18.7%. Couples with abnormal sperm DNA fragmentation who failed ICSI with ejaculated spermatozoa, achieved a clinical pregnancy rate of 31.0% with surgically retrieved spermatozoa. Despite the high pregnancy rate using ICSI with surgically retrieved spermatozoa, it is an invasive procedure, while MSOME is not.

We found significantly lower total motile count in group A as compared to B and C. Spermatozoa are separated based on density gradient, where morphologically normal spermatozoa have higher densities [16]. Couples with low percentage of normal cells at MSOME, have high percentage of spermatozoa with severe morphological abnormalities; thus, fewer motile spermatozoa are extracted.

According to our results, the younger the female partner, the higher the pregnancy rate. This has been described previously [17]. In addition, transfer of blastocysts resulted in higher pregnancy rates as compared to transferring cleaved embryos. This finding was previously reported in a meta-analysis that showed live birth rate and other outcomes, including pregnancy rate, after fresh IVF-ICSI were significantly improved with blastocyst transfer as compared to cleavage-stage embryo transfer. These results were attributed to better selection of high-quality embryos for implantation [18].

This study is the first to evaluate the role of MSOME and IMSI in treating couples with unexplained infertility. The most significant limitation of the study is the retrospective design.

Conclusion

Hidden male factor may play a role in the etiology of unexplained infertility. MSOME may be a useful tool for diagnosing hidden male factor and should be considered when evaluating couples with unexplained infertility who did not conceive after the first cycle. IMSI may provide an advantage to couples with unexplained infertility and none or fewer than 2% normal spermatozoa at MSOME, because the extensive search to find high-quality cells might overcome the hidden male factor which was the obstacle to achieving pregnancy in the previous cycles. Importantly, this method is noninvasive, unlike when spermatozoa are surgically retrieved. Further studies that compare IMSI vs. ICSI among males with severe, isolated teratozoospermia diagnosed by MSOME are needed to strengthen these primary results.

Supporting information

S1 Data

(XLS)

Click here for additional data file. (40.5KB, xls)
S2 Data

(XLSX)

Click here for additional data file. (44.6KB, xlsx)

Acknowledgments

The authors express their appreciation to Faye Schreiber for important English language contributions and editing, and to Navah Jelin for contributing to the statistical analysis.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors received no specific funding for this work

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Decision Letter 0

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22 Oct 2019

PONE-D-19-23244

Unexplained infertility treatment, should we change our approach? - An eight-year follow-up study

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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: This case-control study aims to examine the usefulness of a diagnostic test (MSOME) and therapeutic approach (IMSI), by comparing the outcomes of pregnancy and delivery rates, in a population of couples with unexplained infertility and at least one failed IVF cycle. The cases underwent diagnostic testing and therapeutic approach, the controls did not receive testing and received standard of care. The cases were divided based on MSOME into 3 groups: A (0% normal cells), B (1% normal cells), C (2+% normal cells). When IMSI was used to select the sperm for injection, pregnancy and delivery rates were highest for those groups with the least normal cells by MSOME A>B>C. Controls had pregnancy and delivery rates similar to group C.

Overall, there is a need for additional study in the utility of IMSI, though I would argue, at least at its present level of detail, it’s not clear that this study investigates a population distinct from the male infertility and prior ICSI failure populations that have previously been presented (MSOME and IMSI Reviewed in 2013, PMID 23948449). The study acknowledges the limitations of it being retrospective, with some detail unobtainable. The study does admirably study cumulative pregnancy and delivery rate, including fresh and frozen transfers.

1. Major: Criticism of many unexplained infertility studies stems from differences in inclusion/exclusion criteria for ‘unexplained’ couples. Additional detail in the materials and methods on page 4 (or supplement) regarding the ovulatory, male factor, uterine/tubal/endometriosis, and ovarian reserve workup of the cases and controls would strengthen the ability of the reader to interpret generalizability to their unexplained infertility/mild male factor population. In the discussion it is recognized as a limitation that this level of detail appears unavailable in the control group. But is the case group all unexplained infertility or would some of them qualify as male factor fertility? The novelty of this study is using the technique in an unexplained infertility population rather than male factor infertility, so an ability to differentiate between them is critical to the claim of novelty.

2. Major: studying the pregnancy rate per couple has some advantages, but additional detail regarding the representation of fresh/frozen embryos/numbers of transfers would be appreciated. Additionally, the length of follow-up per couple is inherently different over an overall 8 year timespan. Consider reporting average length of treatment in couples? Do I assume correctly that only the first pregnancy represented by the embryos generated in an IMSI cycle would be represented in the data? Additionally, how was pregnancy defined in your electronic database? Home test, serum?

3. Major: I would presume the comparison of outcomes between controls collected 2010-2018 and cases 2015-2018 would generally find that controls had lower pregnancy/delivery rates, at least with US trends; I don’t know if the same can be extrapolated to other countries. What would a sensitivity analysis comparing the controls from 2015-2018 to cases from 2015-2018 show? On a related question, how was the decision made to include the number of cases you did? Was a power analysis done, or was it a convenience sample based on introduction of an EMR in 2010 and including all cycles that met criteria?

4. Major: The title does not inform the reader of the study design, population, or intervention of this study, and the eight-year follow-up was only for the controls, not the cases. Title revision strongly recommended (consider consulting the STROBE guideline).

5. Minor: I’m not sure I understand/agree with the logic behind the ‘explanation for this apparent contradiction…that a lower percentage of normal cells in MSOME necessitates an extensive search under ultra-magnification to find the highest quality spermatozoa.’ The criteria by which ‘highest quality’ are judged would not seem to be different in group A and B, but the extensiveness of the search has been a major criticism of the IMSI technique and a concern with the time the oocytes wait to be injected.

6. Minor: Bonferroni corrections used for multiple comparisons is great, but it’s not clear what alpha was used to judge significance in the different cases; I like to be able to see how close the p-value is to the alpha.

7. Minor: is the IMSI technique success related to the embryologist/andrologist examining the sperm? I can’t imagine a single embryologist/andrologist performed all the studies, was there a bias in the most experienced performing those for Group A where difficulty in finding normal sperm was anticipated?

**********

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: No

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 May 1;15(5):e0232156. doi: 10.1371/journal.pone.0232156.r002

Author response to Decision Letter 0

18 Dec 2019

1. Major: Criticism of many unexplained infertility studies stems from differences in inclusion/exclusion criteria for ‘unexplained’ couples. Additional detail in the materials and methods on page 4 (or supplement) regarding the ovulatory, male factor, uterine/tubal/endometriosis, and ovarian reserve workup of the cases and controls would strengthen the ability of the reader to interpret generalizability to their unexplained infertility/mild male factor population. In the discussion it is recognized as a limitation that this level of detail appears unavailable in the control group. But is the case group all unexplained infertility or would some of them qualify as male factor fertility? The novelty of this study is using the technique in an unexplained infertility population rather than male factor infertility, so an ability to differentiate between them is critical to the claim of novelty.

Thank you for your comment.

All the included couples in the case group and control group had unexplained infertility, and had experienced at least three failed IUI cycles and at least one failed IVF-ICSI cycle. Unexplained infertility was an inclusion criterion. The missing data in the control group was not related to the diagnosis of unexplained infertility or the treatment they underwent.

According to a recent manuscript published in Human Reproduction, subfertility was defined as “couples trying to conceive for at least 12 months. Selected subfertile couples had regular menstrual cycles, at least one patent fallopian tube and a total motile sperm count >1 × 106” (PMID 30395266), which enabled them to try IUI. Therefore, they can be added to the unexplained infertility group. I added the definition of male subfertility to the methods section, page 4. “Male subfertility was defined as a total motile sperm count >1 × 106” [8].”

We emphasized this in the methods section and added exclusion criteria, pages 4,5.

“Exclusion criteria were Mullerian abnormality or submucosal or subserosal fibroids >7 cm, despite regular menstrual cycles, because of the negative effect of these complications on implantation and pregnancy outcomes [9]; ovulation abnormalities; tubal pathology; and male infertility (when the total motile sperm count was <1 × 106, because IUI treatment does not benefit this group).”

2. Major: studying the pregnancy rate per couple has some advantages, but additional detail regarding the representation of fresh/frozen embryos/numbers of transfers would be appreciated. Additionally, the length of follow-up per couple is inherently different over an overall 8 year timespan. Consider reporting average length of treatment in couples? Do I assume correctly that only the first pregnancy represented by the embryos generated in an IMSI cycle would be represented in the data? Additionally, how was pregnancy defined in your electronic database? Home test, serum?

The pregnancy rate represented in the data is the cumulative pregnancy rate (pregnancy from fresh and frozen embryos). We included the results of the first IMSI only, because we assumed that if there was a hidden male factor it would be resolved in the first IMSI cycle.

I added the definition to the methods section, page 5: “Clinical pregnancy, defined as missed abortion, blighted ovum or fetal pole with heartbeat, was considered a positive result. Chemical pregnancy was considered a negative result”.

3. Major: I would presume the comparison of outcomes between controls collected 2010-2018 and cases 2015-2018 would generally find that controls had lower pregnancy/delivery rates, at least with US trends; I don’t know if the same can be extrapolated to other countries. What would a sensitivity analysis comparing the controls from 2015-2018 to cases from 2015-2018 show? On a related question, how was the decision made to include the number of cases you did? Was a power analysis done, or was it a convenience sample based on introduction of an EMR in 2010 and including all cycles that met criteria?

I added the following explanation to the statistical analysis section. Page 8.

“Based on the assumption of a 12% difference in pregnancy rates between groups, we calculated that a sample size of 500 couples would be sufficient to provide a power of 80% to show an effect, at two-tailed alpha of 5%.”

We assumed that the pregnancy rate among couples with unexplained infertility who failed a previous IVF-ICSI and had a hidden male factor would be similar to that of couples with their first treatment, which is described in the literature as 40-45%. The pregnancy rate among the control group is 28%, therefore, the difference we used in calculating the sample size was 12%.

The control group included 285 couples, in which only 61 were in 2015-2018, because of the small sample size we included cases from 2010-2015. The cumulative pregnancy rate among those 61 cases was 24.6%, delivery rate 8.2%, abortion rate 16.39%.

4. Major: The title does not inform the reader of the study design, population, or intervention of this study, and the eight-year follow-up was only for the controls, not the cases. Title revision strongly recommended (consider consulting the STROBE guideline).

Thank you for your comment. We changed the title to:

“The possibility of integrating motile sperm organelle morphology examination

(MSOME) and intracytoplasmic morphologically-selected sperm injection (IMSI) in

treating couples with unexplained infertility: a historical, case-control study”

5. Minor: I’m not sure I understand/agree with the logic behind the ‘explanation for this apparent contradiction…that a lower percentage of normal cells in MSOME necessitates an extensive search under ultra-magnification to find the highest quality spermatozoa.’ The criteria by which ‘highest quality’ are judged would not seem to be different in group A and B, but the extensiveness of the search has been a major criticism of the IMSI technique and a concern with the time the oocytes wait to be injected.

The maximum time from providing the semen sample to oocyte injection is four hours. Couples with higher percentage of normal cells at MSOME do not gain benefit from the extensive search, they have enough normal sperm for fertilization.

In addition, men with higher percentage of normal cells in the ejaculate can be managed with routine IVF ICSI . But, when there are only a few normal spermatozoa in the ejaculate, the chance of finding them in routine IVF ICSI is low. I added this explanation to Page 15.

6. Minor: Bonferroni corrections used for multiple comparisons is great, but it’s not clear what alpha was used to judge significance in the different cases; I like to be able to see how close the p-value is to the alpha.

The alpha used for the Bonferroni Correction was 0.05/3=0.017. In pregnancy rates between groups A and C, p=0.002. In delivery rate between groups A and C, p=0.007, and for groups A and B, p=0.023. For total motile counts between groups A and B p=0.005, between groups A and C p=0.011. I added this to the statistical analysis section, Page 8.

7. Minor: is the IMSI technique success related to the embryologist/andrologist examining the sperm? I can’t imagine a single embryologist/andrologist performed all the studies, was there a bias in the most experienced performing those for Group A where difficulty in finding normal sperm was anticipated?

We have 5 embryologists in our lab. Each one examines the sperm randomly. That means that all 5 embryologists examined each group in the study, without any particular preference.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file. (20.9KB, docx)

Decision Letter 1

Marco Aurélio Gouveia Alves

13 Feb 2020

PONE-D-19-23244R1

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility: a historical, case-control study

PLOS ONE

Dear Dr. asali,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Mar 29 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

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  • An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The revised version of the manuscript has somewhat improved but there are still several issues to be addressed, particularly concerning the experimental approach.

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

Reviewers' comments:

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 #1: All comments have been addressed

Reviewer #2: (No Response)

**********

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 #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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 #1: Yes

Reviewer #2: Yes

**********

5. 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

Reviewer #2: Yes

**********

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 #1: Comments from prior review have been addressed sufficiently. Limitations of the study design are appropriately acknowledged, details sufficient for the reader to evaluate outcomes.

Reviewer #2: In this manuscript entitled: “The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility: a historical, case-control study”. Authors asked themselves if the IMSI technique can be used successful on cases of couples with unexplained infertility.

In general, authors don´t explain very well their results and the manuscript is not easy to read. First at all, a general advice for following submission is to include line number. This will not be only beneficial for the reviewers; authors can follow recommendation easier.

One of my concerns is why authors make a difference between MSOME and IMSI. MSOME is not a previous procedure that is part of the process of IMSI? When you read the title and the manuscript it seems that there are 2 different procedures.

Is control group a real control? I am concern about this topic. The only information that we have about them is the reproductive outcome, but we don´t know nothing about the % or normal spermatozoa or the rest of seminogram values. Authors should consider delete this group and built the manuscript discussion making the comparison between groups A, B and C. As they stated: group C is the normal/average group and further comparisons should be done with this group. What we know about the proposed control group?

Authors show beneficial effect of IMSI versus ICSI. This expected result agrees with previous reports that highlight the beneficial outcome of IMSI. Nevertheless, it is hard to understand why the group with higher percentage of anormal spermatozoa had the higher reproductive outcome. The only reason that could explain this contradiction is that the physician made bigger effort to find a normal cell and performed later the ICSI. Once you finish to read the manuscript the conclusion that the reader made is that IMSI is beneficial for those patients with severe morphological sperm problems. But since physician are selecting normal spermatozoa why the rest of group are not improving? Bigger effort should be made to explain this contradictory result.

Minor comments:

Introduction Section:

Please delete this sentence “One study found that ICSI resulted in a similar, cumulative live birth rate as compared with IVF for couples with non-male factor infertility [7]” It doesn´t apport information.

Material and Methods:

Please rephrase it: All couples underwent MSOME when IMSI was done within 6 months of the MSOME. What this means you performed a MSOME analysis and later before 6 months you performed an IMSI. Or maybe did you make a MSOME and you freeze the spermatozoa and later or you performed an IMSI. I am sure this is not the message that you want to send but it is rather confusing. Please clarify.

When you wrote “IVF/ICSI cycles. Male subfertility was defined as a total motile sperm count >1 × 106” What you really mean is less than a million?

Please defined what do you consider “Chemical pregnancy”

When you described the sperm selection “The upper liquid was then removed and the pellet was re-suspended in Quinn's Sperm Washing Medium (Sage In-Vitro Fertilization, Inc.) and centrifuged again for 5 minutes at 600 rpm at room temperature. “What do you mean for the pellet? The spermatozoa that can be found on the bottom of the tube? Do you take the spermatozoa and semen contaminants that can be found on the 40% fraction? Please specify this point.

It is possible to insert in table-3 information about the concentration of the ejaculates. It is the only reason that can explain why different groups that have the same % of motile spermatozoa and same ejaculate volume have different values of total motile count.

Statistical analysis:

Please explain the assumption of a 12% difference in pregnancy rates between groups. How did you calculate this? There is a big gap between the number of couples included in each group. Did the author have this in mind when they performed the statistic? Did you balance statistically this difference?

**********

7. 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: Jamie Peregrine, MD, MS

Reviewer #2: No

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 May 1;15(5):e0232156. doi: 10.1371/journal.pone.0232156.r004

Author response to Decision Letter 1

24 Feb 2020

Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

Re: PONE-D-19-23244R1: The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility

Dear Professor Alves,

Please find below, our response to the additional comments of reviewer 2. Thank you.

Reviewer #2

1. One of my concerns is why authors make a difference between MSOME and IMSI. MSOME is not a previous procedure that is part of the process of IMSI? When you read the title and the manuscript it seems that there are 2 different procedures.

Thank you for your comment. MSOME and IMSI are two different procedures. MSOME is an advanced semen analysis that examines the morphology of motile spermatozoa under ultra-magnification of 6300x. It is the process of looking for normal spermatozoa using an inverted light microscope equipped with high-power Nomarski optics, enhanced with digital imaging to achieve magnification up to 6300×. IMSI is the micromanipulation of the spermatozoa found after extended search and injected into the oocyte.

2. Is control group a real control? I am concern about this topic. The only information that we have about them is the reproductive outcome, but we don´t know nothing about the % or normal spermatozoa or the rest of seminogram values. Authors should consider delete this group and built the manuscript discussion making the comparison between groups A, B and C. As they stated: group C is the normal/average group and further comparisons should be done with this group. What we know about the proposed control group?

Thank you for your comment. According to your recommendation we deleted the control group.

3. Authors show beneficial effect of IMSI versus ICSI. This expected result agrees with previous reports that highlight the beneficial outcome of IMSI. Nevertheless, it is hard to understand why the group with higher percentage of anormal spermatozoa had the higher reproductive outcome. The only reason that could explain this contradiction is that the physician made bigger effort to find a normal cell and performed later the ICSI. Once you finish to read the manuscript the conclusion that the reader made is that IMSI is beneficial for those patients with severe morphological sperm problems. But since physician are selecting normal spermatozoa why the rest of group are not improving? Bigger effort should be made to explain this contradictory result.

You are correct, this concept is difficult to understand. Our cohort included couples who had at least one unsuccessful, conventional IVF-ICSI cycle, assuming that moderate and mild male factor abnormalities can be solved in the first IVF cycle (enough normal spermatozoa in the ejaculate). Failure to achieve pregnancy in the first or second cycle is not due to unresolved male factor and therefore an extended search will not enhance the implantation rate. In contrast, with severe teratozoospermia requires an extended search to be resolved, failure in the previous cycles can be explained by an unresolved, unknown male factor. In support of this assumption, a previous study found an increase in implantation and pregnancy rates using IMSI over conventional ICSI for patients with previously unsuccessful ICSI, with no difference in the results of the first cycle with ICSI vs. IMSI (15. Klement AH, et al. Intracytoplasmic morphologically selected sperm injection versus intracytoplasmic sperm injection: a step toward a clinical algorithm. Fertil Steril 2013;99:1290-3. doi: 10.1016/j.fertnstert.2012.12.020).

This is explained in page 12, second paragraph.

4. Minor comments:

Introduction Section:

Please delete this sentence “One study found that ICSI resulted in a similar, cumulative live birth rate as compared with IVF for couples with non-male factor infertility [7]” It doesn´t apport information.

We deleted the sentence.

5. Material and Methods:

Please rephrase it: All couples underwent MSOME when IMSI was done within 6 months of the MSOME. What this means you performed a MSOME analysis and later before 6 months you performed an IMSI. Or maybe did you make a MSOME and you freeze the spermatozoa and later or you performed an IMSI. I am sure this is not the message that you want to send but it is rather confusing. Please clarify.

MSOME is the examination of the semen. According to this examination, we divided the cohort into 3 groups according to the percentage of normal spermatozoa in the semen. In our cohort, we included only couples who had MSOME and proceeded to IMSI within 6 months from the MSOME.

Couples who want to proceed to IMSI provide a new semen sample on the day of oocyte pick-up. This was clarified in the Abstract and Methods.

6. When you wrote “IVF/ICSI cycles. Male subfertility was defined as a total motile sperm count >1 × 106” What you really mean is less than a million?

Male subfertility was defined as a total motile sperm count ≥1 × 106.

Male infertility was defined as a total motile sperm count was <1 × 106

7. Please defined what do you consider “Chemical pregnancy”.

When the level of hCG is initially elevated enough to produce a positive result on a pregnancy test but then declines before a gestational sac is detected with ultrasound.

I added this to the Methods section, page 5

8. When you described the sperm selection “The upper liquid was then removed and the pellet was re-suspended in Quinn's Sperm Washing Medium (Sage In-Vitro Fertilization, Inc.) and centrifuged again for 5 minutes at 600 rpm at room temperature. “What do you mean for the pellet? The spermatozoa that can be found on the bottom of the tube? Do you take the spermatozoa and semen contaminants that can be found on the 40% fraction? Please specify this point.

The pellet is the cellular fraction at the bottom of the tube. Generally, the semen itself will remain above the upper layer and debris and immotile cells will get "stuck" between the upper and lower layers. Most of the motile cells will pass through both layers and reach the bottom of the tube, forming the pellet.

9. It is possible to insert in table-3 information about the concentration of the ejaculates. It is the only reason that can explain why different groups that have the same % of motile spermatozoa and same ejaculate volume have different values of total motile count.

The difference in total motile count is that this parameter was calculated after sample preparation in which spermatozoa are separated based on density gradient, where morphologically normal spermatozoa have higher densities. Couples with low percentage of normal cells at MSOME, have high percentage of spermatozoa with severe morphological abnormalities; thus, fewer motile spermatozoa are extracted.

This was explained on page 14.

10. Statistical analysis:

Please explain the assumption of a 12% difference in pregnancy rates between groups. How did you calculate this? There is a big gap between the number of couples included in each group. Did the author have this in mind when they performed the statistic? Did you balance statistically this difference?

We did this calculation in order to estimate the sample size of the study group and the control group. Based on your previous comment we deleted the control group.

Attachment

Submitted filename: Response to reviewer 23-02-2020.docx

Click here for additional data file. (20.9KB, docx)

Decision Letter 2

Marco Aurélio Gouveia Alves

16 Mar 2020

PONE-D-19-23244R2

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility

PLOS ONE

Dear Dr. asali,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Apr 30 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

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

Reviewers' comments:

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 #1: (No Response)

Reviewer #2: All comments have been addressed

**********

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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

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Reviewer #1: I agree with reviewer 2’s recommendation and the authors’ decision to remove the ‘control’ group.

A few other minor comments:

Page 2: ‘2010-2018’. Now that the control group is removed, isn’t the study only 2015-2018?

Page 3: ‘Using this technique together with a micromanipulation system has allowed the introduction of a modified intracytoplasmic sperm injection (ICSI) procedure, known as intracytoplasmic morphologically-selected sperm injection (IMSI)’. Can you say something about ICSI in the second paragraph to introduce this more familiar acronym sooner? Introducing it in the current state is confusing.

Page 4: ‘All couples underwent MSOME when IMSI was within 6 months of the initial MSOME’. This is unclear.

Page 4: ‘at least one failed IVF-ICSI cycle’ vs ‘several, previous failed IVF/ICSI cycles’. To whom was MSOME offered, and who was included in the study? Also if IVF/ICSI and IVF-ICSI the same, recommend consistency.

Page 10: ‘Group A 10 included 55 cases in which no normal cells were found at MSOME, but normal cells were found during IMSI at ovum pick-up for 49 (89.1%). Group B included 184 couples and Group C 32 couples.’ Did all Group B and Group C patients have normal cells found for IMSI? If not, an argument could be made that maybe MSOME is just not predictive of IMSI, even if <6 months, so I would include the information if you have it.

Page 12: consider modifying as bolded: ‘Thus, MSOME can detect subtle organellar malformations in motile spermatozoa that an embryologist might consider normal for fertilization at 200× to 400× magnification [5], especially important when a normal sperm nucleus is a significant factor for successful implantation and pregnancy in ICSI procedures [13,14].

Page 13: the argument is strengthened, but make sure formatting (IVF-ICSI or IVF/ICSI or IVF ICSI) is consistent.

Page 13: It is confusing to have 2 definitions for ‘male subfertility’: both >1 million cells and Group B.

Reviewer #2: Authors adressed correctly all my questions and concerns. Moreover, modifications were performed on the manuscript.

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Reviewer #1: Yes: Jamie Peregrine

Reviewer #2: No

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PLoS One. 2020 May 1;15(5):e0232156. doi: 10.1371/journal.pone.0232156.r006

Author response to Decision Letter 2

23 Mar 2020

Response to reviewer-

Page 2: ‘2010-2018’. Now that the control group is removed, isn’t the study only 2015-2018?

Thank you for your comment. You are right. I changed this in the abstract.

Page 3: ‘Using this technique together with a micromanipulation system has allowed the introduction of a modified intracytoplasmic sperm injection (ICSI) procedure, known as intracytoplasmic morphologically-selected sperm injection (IMSI)’. Can you say something about ICSI in the second paragraph to introduce this more familiar acronym sooner? Introducing it in the current state is confusing.

I added a paragraph in page 3 explaining the difference between ICSI and IMSI: "When the spermatozoa with normal morphology and motility selected for ICSI are detected under a magnification of ×400, in IMSI the motile spermatozoa are selected under magnification up to 6300×[5]".

The advantage of IMSI is that the sperm is detected under magnification up to 6300× which can detect subtle organellar malformations in motile spermatozoa that an embryologist might consider normal for fertilization at 200× to 400× magnification (ICSI). This explanation is written in page 3 and page 12.

Page 4: ‘All couples underwent MSOME when IMSI was within 6 months of the initial MSOME’. This is unclear.

I changed this sentence to: All couples underwent IMSI within 6 months of the initial MSOME.

Page 4: ‘at least one failed IVF-ICSI cycle’ vs ‘several, previous failed IVF/ICSI cycles’. To whom was MSOME offered, and who was included in the study? Also if IVF/ICSI and IVF-ICSI the same, recommend consistency.

MSOME was offered to couples who had at least one failed IVF-ICSI cycle. I changed this. And changed IVF/ICSI to IVF-ICSI.

Page 10: ‘Group A 10 included 55 cases in which no normal cells were found at MSOME, but normal cells were found during IMSI at ovum pick-up for 49 (89.1%). Group B included 184 couples and Group C 32 couples.’ Did all Group B and Group C patients have normal cells found for IMSI? If not, an argument could be made that maybe MSOME is just not predictive of IMSI, even if <6 months, so I would include the information if you have it.

All the couples in group B and C had normal cells for IMSI.

Page 12: consider modifying as bolded: ‘Thus, MSOME can detect subtle organellar malformations in motile spermatozoa that an embryologist might consider normal for fertilization at 200× to 400× magnification [5], especially important when a normal sperm nucleus is a significant factor for successful implantation and pregnancy in ICSI procedures [13,14].

I did this.

Page 13: the argument is strengthened, but make sure formatting (IVF-ICSI or IVF/ICSI or IVF ICSI) is consistent.

Thank you. I changed to IVF-ICSI.

Page 13: It is confusing to have 2 definitions for ‘male subfertility’: both >1 million cells and Group B.

All the couples in the cohort had unexplained infertility or male subfertility, when male subfertility is defined as a total motile sperm count ≥1 × 106 (written in page 4), that means that each group may have cases of male subfertility.

Attachment

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Click here for additional data file. (14.2KB, docx)

Decision Letter 3

Marco Aurélio Gouveia Alves

9 Apr 2020

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility

PONE-D-19-23244R3

Dear Dr. asali,

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

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With kind regards,

Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

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Reviewers' comments:

Acceptance letter

Marco Aurélio Gouveia Alves

22 Apr 2020

PONE-D-19-23244R3

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility

Dear Dr. Asali:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

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Thank you for submitting your work to PLOS ONE.

With kind regards,

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on behalf of

Dr. Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

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