Comparison of microfluidic and swim-up sperm separation methods for IVF

During fertilization in vivo, the ejaculated spermatozoa is being selected very intensely. From the initial number of hundreds of millions, only a few tens or hundreds sperm cells reach the ovulated oocyte. During fertilization in vitro, sperm cells suitable for utilization must be selected carefully. Choice of a suitable sperm separation method for ICSI is an important step and there are several methods available currently [1]. Some of these methods are commonly used (swim-up, density gradient centrifugation - DGC), some are already obsolete (IMSI) and some are applied in specific cases (MACS). In present time, the methods of microfluidic chips are more and more popular. Nevertheless, before implementation of a new method to operation of a laboratory, it is appropriate to verify its efficiency. Standard parameters, such as concentration, motility or morphology of spermatozoa, are not sufficient for an objective evaluation of separation efficiency. In about 30% of male infertility cases, the cause cannot be detected by these parameters [2,3]. DNA fragmentation represents such morphologically undetectable damage of spermatozoa most often. Correct selection of a sperm cell with non-damaged DNA is one of the prerequisites for achieving successful fertilization and embryo development in assisted reproductive technologies. Data from the literature suggest that the frequency of spermatozoa with massive DNA fragmentation is a marker of sperm quality and also possible predictor of fertility [4]. The tested microfluidic separation method was presented as a very effective and gentle sperm separation system [5,6,7]. Many reports indicate that microfluidic separation is a beneficial technique to remove spermatozoa with fragmented DNA and provides higher IVF outcomes compared to standard sperm selection techniques [5,6,8]. The possible beneficial effects of this technique in clinical application are still debatable. Recent analyses reported that microfluidic chip had a positive effect on total number of grade 1 embryos after ICSI, but in other IVF parameters, this method did not improve the reproductive outcomes [5].

The aim of our study was to compare the conventional swim-up sperm separation method with the new microfluidic chip method with emphasis on reducing the concentration of sperm cells with fragmented DNA.

Based on a standard examination of spermiogram, normozoospermia was found in 8 patients, while results of 2 patients were classified as asthenozoospermia (Tab. 1). Patients with very low sperm concentration (oligozoospermia) were not included in this study intentionally. In oligozoospermic patients, it is often very difficult to obtain sufficient amount of spermatozoa to perform the separation techniques and final results of the Halosperm test can be affected negatively by the very low number of sperm cells analysed. The risk of error caused by a very low number of sperm cells analysed is significantly increased.

In nine patients, the number of spermatozoa with fragmented DNA was very low already before processing (DFI below 30%), only one patient showed a higher value (patient No. 9, DFI 43%) (Tab. 2). After separation by the swim-up method, this parameter decreased in all the patients, to 48% of the initial DFI value on average (15.4% - 69%). If the same samples were processed using the FERTILE system, the DFI values were always lower in comparison with the swim-up method in all the patients. Using the FERTILE method, the values of DFI were reduced to 38% of the initial values in the ejaculate on average (6.3% - 66%). The separation with microchip was more efficient than the swim-up separation method by 10.5 % on average (2% - 27.7%) (Tab. 3). Nevertheless, no statistically significant difference was found in the number of fragmented spermatozoa between the swim-up separation method and the FERTILE method (Fig. 1). When expressed in absolute numbers, the mean DFI in the separated samples decreased from 18.2% in the unprocessed samples to 8.7% in the samples separated by the swim-up method and to 6.6% in the samples separated by the FERTILE method (Tab. 2). Efficiency of the FERTILE method was then better than efficiency of the swim-up method by 2.1%.

Figure 1

In the patient No. 9 (asthenozoospermia; 56 mil/ ml, progressive motility 29%, non-progressive motility 10%, normal morphology 5%), the initially high DFI value (43%) was decreased to 14% by the swim-up method and to 11% by the FERTILE method (Tab. 2).

The present study evaluated the extent to which using of the microfluidic chip for spermatozoa separation discarded the spermatozoa with fragmented DNA in contrast to the classical swim-up method. The method of sperm separation using the micro-fluidic chips is increasingly utilized and is often presented as a very gentle method which significantly decreases the proportion of spermatozoa with fragmented DNA [1,11]. In this study, we presented that the DFI values of unprocessed samples were significantly higher than after the swim-up or FERTILE separations. However, no significant differences were detected in the DFI values between the separations by swim-up and FERTILE method (Figure 1). A study comparing the density gradient centrifugation (DGC) and the microfluidic method (FERTILE) and their effect on separation of spermatozoa with non-fragmented DNA has been published recently [6]. Unlike our experiment, this work revealed a significant difference in efficiency of the separation methods, with a significant decrease of the spermatozoa with fragmented DNA after utilization of the microfluidic chip when compared to the DGC method. This can be due to utilization of the DGC method, which is a method less gentle than the swim-up method used in the present study, and the observed difference can be caused by the negative effect of the DCG method on sperm DNA integrity. We work primarily with the swim-up method at it has been reported to be more gentle and significantly decreased proportion of fragmented spermatozoa when compared to the DGC [12].

A study on DNA integrity and comparison of the swim-up method and a sperm separation method using the microfluidic chips has already been published. It has reported a significantly lower proportion of spermatozoa with fragmented DNA after the separation using the microfluidic chips than after the swim-up separation method [11]. However, the study was realized with the Sperm Sorter Qualis system which uses laminar flow for separation of spermatozoa. Therefore it is a different principle of separation and in this respect maybe more efficient than the FERTILE system tested in the present study.

It is remarkable that separation by the FERTILE method did not decrease the DFI below 10%, not even in the patient with a high DFI value (see Results table 2, patient No. 9). This finding suggests that despite being able to select spermatozoa better due to its strict selection criteria, this separation system is not specific for spermatozoa with fragmented DNA.

It has been reported previously, that using of the microfluidic chip method significantly reduced total sperm number suitable for utilization in comparison to other methods like swim-up, DGC or MACS [1]. It is not considered a problem for ICSI cycles, but it should be taken into account for the classical IVF or IUI in the context of the limited input volume (max 10 μl). When the conventional swim-up method and the FERTILE were compared with focus on embryological parameters, only a significantly higher proportion of good blastocysts was observed after using the microfluidic chip FERTILE. No differences were found in neither of the other parameters [5]. Correct selection of good spermatozoa with intact DNA is very important. It is well known that sperm DNA fragmentation affects negatively total efficiency of the IVF methods [13].

The FERTILE separation method is a simple, undemanding and efficient. It requires no additional instrumentation and it can be realized only with basic laboratory equipment without utilization of other chemicals. In our study, spermatozoa after separation were examined for DNA fragmentation, which was lower after utilization of the microfluidic chip method than after the swim-up method in all the patients. Nevertheless, absolute difference of the DFI value was better for the FERTILE method than for the swim-up by just 2.1%. The difference between the FERTILE and the swim-up methods was not statistically significant in the observed parameter.