Infertility and subfertility affect a significant proportion of human beings who have a desire to become pregnant. Approximately 12% of the reproductive-aged population (6.7 million couples) in the USA faced impaired fecundity according to statistics published in 2013 by the Centers for Disease Control and Prevention (CDC), of which one-third showed the cause as the man, in another one-third the cause as the woman, leaving the last one-third as unidentified [1]. The leading cause of male infertility is abnormal or lack of sperm, which is usually associated with low sperm motility and flawed sperm function resulting in an inability to fertilize an oocyte [1, 2]. In today’s practice, one of the best techniques to treat male infertility is a method in assisted reproductive techniques (ART) called intracytoplasmic sperm injection (ICSI). This method in which a single viable sperm is injected directly into the oocyte provides numerous advantages with fewer drawbacks [3]. It decreases the number of sperm needed for fertilization and bypasses in vivo barriers including the female genital tract. However, this technique requires more careful selection of the sperm because bypassing the in vivo environment also eliminates the natural selection of sperm. ART has recently replaced the natural selection of sperm which has been existent since before the evolution of humans. This is a possible reason why clinical pregnancy and live birth rates are lower than expected, compared to implanted embryo numbers achieved through ART. Thus, a clinically applicable, efficient and inert (not to damage sperm) instrument was needed to find the most fertilizable sperm for ICSI.

Another issue with ICSI was the necessity of separation of motile sperm from the rest of the semen. Traditional methods used in common practice for this separation are centrifugation, swim-up, and density gradient centrifugation (DGC). The most commonly used criteria to evaluate the usefulness of these methods are the recovery rates, motility, morphology, and DNA integrity of the sperm.

A newly developed technique named microfluidic sperm sorting or sperm chip technique or microfluidic chip has been added to these techniques recently, and it offers more than just sperm selection. It presents a design that mimics the filtration function of the female genital system, which is responsible for the natural selection of sperm. Unlike the unprocessed samples, sperm preparation methods show higher rates of motility and better morphology, as well as less DNA fragmentation in the collected sperm [4]. This new-generation spermatozoon selection method also gives the chance to select spermatozoa with lower DNA fragmentation indexes. Moreover, when a combination of DGC and swim-up techniques is compared to the microfluidic sperm sorting, the latter was proved to provide lower rates of DNA fragmentation [4, 5]. Although conventional sperm preparation techniques can improve sperm function and morphology, they are thought to be limited in preparation of genetically enhanced samples. The microfluidic chip, however, may make sperm selection better in functionality, morphology and genetics [6].

In this study, we aimed to identify the effect of the microfluidic sperm sorting technique on clinical pregnancy rates in ICSI treatment in patients with unexplained infertility.

A total of 213 patients who met the eligibility criteria were included in the study. Table 1 shows the comparison between two groups in terms of patients’ demographic and clinical characteristics. The mean maternal age was comparable between the sperm chip and control groups (34.9 $\pm$ 5.0 and 35.2 $\pm$ 6.2, respectively (p = 0.689)). The mean age of men was also similar between the groups (37.1 $\pm$ 5.4 and 37.4 $\pm$ 6.3, respectively, p = 0.715). Body mass index (kg/m${}^2$) was evaluated between study and control groups (23.4 $\pm$ 3.5 vs 23.8 $\pm$ 3.5, respectively, p = 0.488).

When sperm concentrations were compared, both the sperm chip and control groups had similar results (33.2 $\pm$ 26.8 and 32.6 $\pm$ 28.6 million/mL, respectively, p = 0.473).

Differences were detected between two groups regarding COH parameters and results, as shown in Table 2. Total FSH doses administered in the sperm chip and control groups were 1908.4 $\pm$ 796.1 IU/L and 1863.3 $\pm$ 779.8 IU/L, respectively (p = 0.677). The average number of stimulation days was statistically shorter for the sperm chip group than the control group (8.7 $\pm$ 1.8 days and 9.4 $\pm$ 2.2 days, respectively, p = 0.011). The mean number of retrieved oocytes and MII oocyte number were also similar between the groups (p = 0.429 and p = 0.633, respectively). The mean number of Day-1 fertilization of oocytes did not differ significantly between the groups (5.5 $\pm$ 3.8 and 5.1 $\pm$ 4.4, p = 0.566). The mean total number of embryo obtained after ICSI between study vs control group (1.5 $\pm$ 0.5 vs 1.6 $\pm$ 0.5, p = 0.070) and embryo transfer day did not show significant difference between Day-3 and Day-5. When subgroup analysis was carried out between the groups in regards to embryo development, although the grade of embryos transferred on Day-3 was comparable between the groups, grade 1 embryos were transferred significantly more in the sperm chip group on Day-5 transfers (75.8% vs 55.9, p = 0.050) (Table 2).

Fertilization rate of embryos transferred on Day-3 and Day-5 were compared between the groups and no significant difference was observed (Day-3: 3.7 $\pm$ 2.5 vs 4.9 $\pm$ 4.2 (p = 0.114) and Day-5: 6.4 $\pm$ 4.1 vs 5.3 $\pm$ 4.5 (p = 0.183)).

Clinical pregnancy rate did not show significant difference between the groups (Day-3: 38.8% vs 42.3% (p = 0.255) and Day-5: 48.5% vs 44.1% (p = 0.621). Miscarriage rate did not show significant difference between the groups (Day-3: 11.1% vs 9.6 % (p = 0.343) and Day-5: 10.6 % vs 8.4 % (p = 0.434).

Live birth rate did not show significant difference between the groups (Day-3: 27.8% vs 32.6% (p = 0.164) and Day-5: 37.8% vs 35.6% (p = 0.712)) (Table 3).

The number of oocytes retrieved was a significant predictor for clinical pregnancy (p = 0.004). However, maternal age, paternal age and sperm concentration did not predict clinical pregnancy (Table 4). Additionally, this association with the number of oocytes retrieved for clinical pregnancy was found to be still significant after adjustment of confounding factors in multivariate linear regression analysis (p = 0.012).

In our study, a relatively recent technique, the sperm chip technique, was compared to one of the traditional approaches. The ICSI procedures were performed with males having normal semen analysis. Although use of the sperm chip technique contributed to the development of good quality embryos reaching to the blastocyst stage, clinical pregnancy rates were not found to be significantly higher in the sperm chip group and use of this technique did not increase clinical pregnancy rates in the ICSI procedure.

There are several reports in the literature evaluating the factors that affect ART success. Clinical variables such as maternal age, total sperm count, total motile sperm count, sperm motility, embryo quality and sperm selection method are among these predictors of IVF/ICSI treatment success [9, 10].

The sperm chip is a technique developed as an alternative to other sperm preparation techniques and best imitates natural sperm selection in the female genital tract. Sperm can be prepared with this method to use during intrauterine insemination (IUI), IVF and ICSI procedures. In other words, the sperm chip technique was developed to imitate the female genital system and provide great convenience for clinicians, especially in male infertility cases and recurrent unsuccessful previous ART attempts [11, 12, 13]. A recent study by Ozcan et al. [14] showed that use of microfluidic sperm-sorting chip for sperm selection in infertile cases with male factor might improve IVF success rates.

For patients with oligozoospermia, ICSI is a vital technique since only a few sperm are adequate for the procedure. In ICSI, the process of oocyte selection is disregarded and fully left to the ICSI performer [15]. As clinical success following ICSI depends on obtaining the healthiest and the most viable sperm cells, sperm chip technique enables us to select sperm having the best DNA quality and being the least exposed to free oxygen radicals.

Another factor for predicting success is the sperm DNA Fragmentation Index (DFI). It was shown that high DFI is associated with failed pregnancies following IVF/ICSI cycles [16, 17, 18]. Although another study published later showed that high DFI had no effect on fertilization rate, quality of embryo or clinical pregnancy rate, it was indicated that the spontaneous abortion rate was statistically higher in those with abortion of over 27% [19]. It was also shown that DFI had little to no effect on fertilization and early embryo development. However, it had an obvious negative effect on blastocyst development phase and embryo implantation [20, 21]. When ejaculate is added into the sperm chip channels, sperm with the least DNA fragmentation will sort at the highest percentage and therefore will have the highest chance for fertilization [22]. Thus, the most clinically usable, highly motile sperm with nearly undetectable levels of DNA fragmentation could be selected by sperm chip technique [4, 23]. It is possible to speculate that sperm chip techniques may increase clinical pregnancy rate by eliminating the negative effect of DFI by selecting healthy sperm. In other words, the number of good quality embryos that can reach the blastocyst stage was increased significantly by the use of techniques. Success in obtaining good quality embryos with the use of sperm chip techniques may be explained by the lower exposure of the sperm to oxidant factors. In traditional methods, during processing of the sperm sample, the protocol may extend to 2 hours and this causes a prolonged exposure to the free oxygen radicals already produced as a result of the procedure. On the other hand, a sperm chip technique that lacks centrifugation and is completed in one step protocol only takes 30–45 minutes [24]. Reduced procedure time results in an important drop in the sample’s exposure time to free oxygen radicals, enabling prevention of possible DNA damage [25].

One of the most important indications for the sperm chip technique use is recurrent IVF failures. Yildiz et al. [13] found a significant improvement in the fertilization rates in infertility patients who had previously failed more than twice in fertility treatment. In contrast, we did not obtain increased success regarding fertilization rate comparing to controls although our study population included the patients with previous failure histories rather than patients coming to our clinic for the first time.

Despite many existent studies showing the potential of sperm chip technique microscopically when it comes to sperm morphology, motility and DNA integrity, there are not many studies in which clinical pregnancy rates were reported [4, 6, 26, 27]. In a recent randomized controlled study with 122 patients, grade 1 embryo count was found to be significantly higher in sperm chip group, similar to our results. This study failed to show a significant difference in clinical pregnancy or live birth rates [28]. Similarly, we found that use of sperm chip contributes positively to the quality of embryo to be transferred on the 5th day but did not increase the clinical pregnancy rate.

Relatively small sample size and the undetermined cumulative pregnancy rate per patient due to failed reaching of all the frozen embryos data (even if the total and transferred embryo counts were similar) are the main limitations of our study.

In addition, we did not investigate the effect of sperm motility on fertilization and clinical pregnancy rates because sperm motility might cause a potential bias favoring the sperm chip group. Thus, we included patients with similar sperm motility and we only found that the number of oocytes retrieved was a significant predictor of clinical pregnancy rates, even after adjusting multivariable regression models for potential confounders.

The sperm chip techniques were found as clinically useful for increasing the development of good quality embryos that reach the blastocyst stage. However, we found no increase in clinical pregnancy rates. Further research in a larger study population is necessary to determine whether sperm chip technique would be an option to increase success in ICSI treatment.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

SO—protocol/project development; data collection or management, manuscript writing/editing; HGC—protocol/project development, data collection or management, data analysis, manuscript writing/editing; YK— protocol/project development, data collection or management; EK—protocol/project development, data collection or management; MG—manuscript writing/editing; JY—manuscript writing/editing; EB—protocol/project development, data collection or management, data analysis, manuscript writing/editing. All authors read and approved the final manuscript.

The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was taken from all couples in the study. The study was approved by Acibadem Mehmet Ali Aydinlar University Medical Research Ethics Committee (ATADEK 2017-15/16). Approval was obtained from ClinicalTrials.gov with NCT03355937 approval number.

The authors would like to thank the participants of this study and to staff of Acibadem Fulya Hospital Embryology Laboratory for their help to access the data.

This research received no external funding.

The authors declare no conflict of interest.