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+
:	Andrology & Gynecology: Current Research Editors
Dr. Srivastava N.

June 11th, 2013

Thank you for your e-mail dated June 5th, 2012 regarding our submitted paper, �Microfluidic device increases sperm concentration by optimization of chamber structure.� We appreciate the valuable comments provided by the reviewers. 
We have addressed the reviewer�s comments and incorporated the revisions in our revised manuscript with green highlights. The line, page, and figure numbers indicate those of the revised manuscript unless mentioned otherwise. 
We hope that you find this manuscript suitable for publication in Andrology & Gynecology: Current Research. We greatly appreciate your review of the revised manuscript.

	Koji Matsuura, Ph. D.
Lecturer,
Cardiovascular Physiology, 
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 
Okayama University, Okayama, 700-8530, Japan
Phone: +81-86-251-8966 0Fax: +81-86-251-8966
	Email: kojimatu@md.okayama-u.ac.jp
Sincerely yours,
Koji Matsuura

 
Reviewer comments: AGCR-13-32

Reviewer 1: 
General: 
COMMENT
This technique improves the sperm concentration of patients requiring ICSI. 

RESPONSE: Thank you for your comment. We believe the time required for ICSI could be reduced by increasing the sperm count before the treatment.

COMMENT
The number of samples used to optimize the technique is not mentioned in materials and methods.

RESPONSE: Thank you for your comment. The number of samples has been described on the bottom of page 4 line 3. We considered that the total of two semen sample was sufficient for this experiment, because the enrichment ratio was not dependent on the difference of the semen sample.

COMMENT
There are formatting mistakes throughout the article which needs to be corrected.

RESPONSE: Thank you for your comment. We have now checked the formatting and have corrected the mistakes.

COMMENT
The title says �sub fertility� but the technique is applied on normal male.

RESPONSE: Thank you for your comment. We have revised the title to �Microfluidic device increases sperm concentration by optimization of chamber structure.�

COMMENT
Specific: 
The work presented here is very interesting and if optimized adequately can definitely help the patients worldwide. In the developing countries, the infertile couples are not able to get themselves treated, main reason being lack of resources. If this technique can be optimized plus the cost is effective it can be beneficial to the patients. More work needs to be done here in future and this should be applied to large samples and also in a clinical setup.

RESPONSE: Thank you for your comment. We believe that a prototype of this device could be produced, and we should aim to conduct clinical studies to validate this device and identify whether it can fulfill the requirements for routine clinical use in ART laboratories.


Reviewer 2: 

COMMENT
General: The manuscript suffers from poor grammar and syntax. The manuscript is not fulfilling the general requirements of a scientific paper. The author is not following the general rules writing of a scientific paper. Many items in the general structure of the manuscript are missing.
The abstract is not written in the normal format; Objectives, Methodology, Results and conclusion.
The �Discussion� section is missing.

RESPONSE: Thank you for your comment. Although the reviewer has suggested a structured abstract in comment 1, this was not reflected in the revised manuscript, because a structured abstract is not required by this journal. We have separated �Results and discussion� into �Results� and �Discussion.� We have used an English editing service to check the entire manuscript in detail to correct the points raised by the reviewer.


Reviewer 3: 
General: In my opinion this device could be of interest in the field of assisted reproduction. However, the following aspects should be considered in order to make this device usable in the IVF lab routine.
COMMENT
You stated �the use of this device can increase sperm concentration without strong mechanical damages such as pellet preparation using centrifugation� however, this is not enough to improve ICSI outcome. It would be more useful if it could select sperm motility too, such as the �pellet and swim up� method does. In fact, the important aspect in sperm preparation in order to gain a good ICSI outcome is not only the recovery of a good number of sperm but also the recovery of good motile sperm. So, the use of this device could be useful only in increasing sperm concentration and a second method of selection should be used to select sperm motility, (this point should be considered) 
RESPONSE: Thank you for your comment. We have added the following discussion related to this point on the bottom of page 6 lines 1�8.

In this study, the concentration of human sperms was increased 10-fold, demonstrating that the microfluidic device could be useful for subfertile men considering IVF. However, to improve the outcome of ICSI, good motile sperms with highly progressive velocities should be collected and injected, in addition to increasing the number of motile sperms recovered. At present, motile sperms can be collected under a microscope using a glass capillary to search for them in the chamber (b) shown in Fig. 1B. Further development of the chip device and the protocol is required that would enable collection of good motile sperms that could swim up in the chip device after concentrating the sperms on the bottom of the chamber. 

COMMENT
You cannot state that �the success rate of ICSI would be increased� or �This microfluidic device reduces ICSI treatment time� or �This microfluidic device may also increase the success rate of IVF� in absence of any scientific evidence! These are just hypothesis. Before stating this you should demonstrate this with scientific data. I am not sure that it would reduce ICSI treatment time since a second method of selection should be used to select sperm motility (as reported above)

RESPONSE: Thank you for your comment. We have deleted the statements �the success rate of ICSI would be increased� and �This microfluidic device may also increase the success rate of IVF�. We have revised �This microfluidic device reduces ICSI treatment time� to �the average time taken for porcine sperm injection into porcine oocyte was significantly reduced� on page 3 lines 10�11.

COMMENT
I am not sure that this device may decrease the cost of an IVF treatment. You should consider that at least one device should be provided per each couple, since the re-use of the device would be never admitted. So, the cost of the device should be very competitive in order to convince the customer to the used the device instead of centrifugation. Only in this way it could be preferred to another technique.

RESPONSE: Thank you for your comment. In general, the total cost of this device would be determined by several factors such as material and fabrication costs, patent fees, and labor costs, which are always changing. Therefore, because of this, we decided that the discussion of the cost should not be mentioned throughout this manuscript. We have therefore deleted the last sentence of the abstract.

COMMENT
English language should be revised. 

RESPONSE: Thank you for this comment. We have gone over the manuscript in detail to correct these by English editing service.

Specific:
COMMENT
You reported that �Human semen was supplied by a healthy fertile male in his 30s�.  More details concerning patient�s selection should be listed (patient number, mean age, sperm count and motility) since the device could be not useful under a certain threshold of sperm concentration. Eg. Male partner semen characteristics are: sperm count e"15x106/ml with sperm mobility of A+Be"50% according to the World Health Organization criteria and normal morphology e"4% according to Kruger s strict criteria& 

RESPONSE: Thank you for your comment. We have now included the semen characteristics used in this study (sperm count and motility) on page 4 lines 5�6.

COMMENT
You stated: �Samples were incubated at room temperature or 37�C for 0.5�1h to liquefy the semen (7) and then diluted with distilled water�. Is dilution always required before the injection into the flow chamber? Which is the maximum volume that can be injected in the flow chamber? The procedure is not clear and it is important in order to evaluate the possible use of this device in IVF lab routine. Centrifugation allows concentrating sperm present in large volumes, allowing maximum sperm retrieval. 

RESPONSE: Thank you for this comment. We have adjusted the sperm count in the semen from > 107 cells/ml to 1.0 ( 104 or 1.0 ( 105 cells/ml for the experiments, and we used distilled water to reduce sperm motility for counting under a microscope. However, dilution is not always required for application of this device. The reason for the dilution is described on page 4 lines 8�10. We have also added the maximum volume on the bottom of page 4 lines 6�8.

COMMENT
Furthermore, you should consider that seminal plasma is detrimental for sperm motility, for this reason sperm washing with the addition of a culture media is performed in IVF. Seminal plasma has to be removed and spermatozoa have to be resuspended with culture media since seminal plasma could impair sperm motility. Thus, the passage in centrifuge(05ABDMQSUotuyz{��������<	=	[	\	k	�	�	�	�	

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RESPONSE: Thank you for your comment. We have deleted the sentence �The Use of this device~� in the revised version of the manuscript, as suggested by the reviewer.










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