Comparison of Reproductive Outcomes in ICSI Cycles Using Sperm Chip Technique and Density Gradient Technique in Men with Normal Semen Analysis

Abstract

Background: Microfluidic sperm sorting procedure or sperm chip technique is a chemical-free method of selecting sperm using a disposable chip. It is a new gentle alternative for sperm processing which has been produced to obtain sperm with higher rates of motility and better morphology, as well as to reduce DNA fragmentation in sperm with high DNA fragmentation to nearly undetectable levels. We aimed to evaluate sperm chip techniques on clinical pregnancy rates in patients who underwent intracytoplasmic sperm injection (ICSI). Methods: The patients in whom fresh embryo transfer (ET) on Day-3 or 5 after ICSI had been performed were analyzed in this prospective randomized cohort study. Results: Of those, 102 patients underwent ICSI with sperm isolated using sperm chip technique (study group) while 111 patients underwent ICSI with sperm isolated using swim-up technique (control group). No significant difference was observed between the groups in terms of fertilization rate in patients who underwent ET on the 3rd or 5th day. In the patients having ET on Day-3, Grade 1 embryos were obtained similarly between the sperm chip group and the control group. Grade 1 embryos transferred on Day-5 were observed significantly more frequently in the study group (p = 0.050). However, clinical pregnancy rates did not show significant differences between the groups in patients who were transferred on both the 3rd and 5th days. Conclusions: Although sperm selection using by sperm chip technique provides advantage in terms of blastocyst quality, use of this technique does not enhance success in terms of clinical pregnancy. Clinical Trial Registration: Approval was obtained from ClinicalTrials.gov with NCT03355937 approval number.

Full text

Clin. Exp. Obstet. Gynecol. 2023; 50(3): 46
https://doi.org/10.31083/j.ceog5003046
Copyright: © 2023 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.
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Original Research
Comparison of Reproductive Outcomes in ICSI Cycles Using Sperm
Chip Technique and Density Gradient Technique in Men with Normal
Semen Analysis
Selin Ozaltin1, Hale Goksever Celik2,*, Yucel Kocyigit3, Emre Kar4, Mete Gungor1,
John Yeh5, Ercan Bastu6,7
1Department of Obstetrics and Gynecology, Acibadem Mehmet Ali Aydinlar University Acibadem Maslak Hospital, 34752 Istanbul, Turkey
2Department of Obstetrics and Gynecology, Acibadem Mehmet Ali Aydinlar University Acibadem Fulya Hospital, 34752 Istanbul, Turkey
3Department of Obstetrics and Gynecology, Acibadem Fulya Hospital, 34349 Istanbul, Turkey
4Department of Obstetrics and Gynecology, Istanbul City Hospital, 34480 Istanbul, Turkey
5Department of Obstetrics and Gynecology, University of Harvard, Cambridge, MA 02138, USA
6Department of Obstetrics and Gynecology, Biruni University, 34010 Istanbul, Turkey
7Department of Obstetrics and Gynecology, UMass Chan Medical School, Worcester, MA 01655, USA
*Correspondence: hgoksever@yahoo.com (Hale Goksever Celik)
Academic Editor: Johannes Ott
Submitted: 6 January 2023 Revised: 12 February 2023 Accepted: 13 February 2023 Published: 1 March 2023
Abstract
Background: Microfluidic sperm sorting procedure or sperm chip technique is a chemical-free method of selecting sperm using a dis-
posable chip. It is a new gentle alternative for sperm processing which has been produced to obtain sperm with higher rates of motility
and better morphology, as well as to reduce DNA fragmentation in sperm with high DNA fragmentation to nearly undetectable levels.
We aimed to evaluate sperm chip techniques on clinical pregnancy rates in patients who underwent intracytoplasmic sperm injection
(ICSI). Methods: The patients in whom fresh embryo transfer (ET) on Day-3 or 5 after ICSI had been performed were analyzed in this
prospective randomized cohort study. Results: Of those, 102 patients underwent ICSI with sperm isolated using sperm chip technique
(study group) while 111 patients underwent ICSI with sperm isolated using swim-up technique (control group). No significant difference
was observed between the groups in terms of fertilization rate in patients who underwent ET on the 3rd or 5th day. In the patients having
ET on Day-3, Grade 1 embryos were obtained similarly between the sperm chip group and the control group. Grade 1 embryos trans-
ferred on Day-5 were observed significantly more frequently in the study group (p= 0.050). However, clinical pregnancy rates did not
show significant differences between the groups in patients who were transferred on both the 3rd and 5th days. Conclusions: Although
sperm selection using by sperm chip technique provides advantage in terms of blastocyst quality, use of this technique does not enhance
success in terms of clinical pregnancy. Clinical Trial Registration: Approval was obtained from ClinicalTrials.gov with NCT03355937
approval number.
Keywords: infertility; sperm chip; microfluidic sperm sorting; assisted reproductive techniques (ART); in vitro fertilization (IVF);
intracytoplasmic sperm injection (ICSI); clinical pregnancy rates
1. Introduction
Infertility and subfertility affect a significant propor-
tion of human beings who have a desire to become preg-
nant. Approximately 12% of the reproductive-aged popula-
tion (6.7 million couples) in the USA faced impaired fecun-
dity 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 uniden-
tified [1]. The leading cause of male infertility is abnor-
mal or lack of sperm, which is usually associated with low
sperm motility and flawed sperm function resulting in an in-
ability 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 intracy-
toplasmic sperm injection (ICSI). This method in which a
single viable sperm is injected directly into the oocyte pro-
vides 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 ex-
istent since before the evolution of humans. This is a pos-
sible reason why clinical pregnancy and live birth rates are
lower than expected, compared to implanted embryo num-
bers 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 sepa-
ration of motile sperm from the rest of the semen. Tradi-
tional methods used in common practice for this separation
are centrifugation, swim-up, and density gradient centrifu-

gation (DGC). The most commonly used criteria to evalu-
ate 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. Un-
like 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 fragmen-
tation 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 prepara-
tion techniques can improve sperm function and morphol-
ogy, they are thought to be limited in preparation of genet-
ically enhanced samples. The microfluidic chip, however,
may make sperm selection better in functionality, morphol-
ogy 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 infer-
tility.
2. Materials and Methods
2.1 Participants
In this randomized prospective cohort study, data of
213 patients who had undergone ICSI procedure with mi-
crofluidic chip or swim-up technique at Acibadem Fulya
Hospital for 2 years were analyzed. According to our power
analysis results, sample population was selected. Of those,
the microfluidic chip technique was used for 102 patients
and the swim-up technique was used for 111 patients.
Patients were randomized by giving odd and even
numbers to use microfluidic chip technique and swim-up
technique. 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 Commit-
tee (ATADEK 2017-15/16). Approval was obtained from
ClinicalTrials.gov with NCT03355937 approval number.
Inclusion criteria were as follows: (1) women aged be-
tween 18 and 43 years, (2) regular menstrual cycles (men-
strual cycles of 25–34 days), (3) normal body mass index
(BMI) of 19.3–28.9 kg/m2, (4) no metabolic or endocrine
disorders, (5) normal hormone panel, (6) normal World
Health Organization (WHO) sperm parameters (volume,
concentration, total number, total and progressive motility,
morphology), (7) couples undergoing the ICSI cycle, (8)
normal uterine cavity documented by hysterosalpingogra-
phy or hysteroscopy, (9) embryo transfers on Day-3 or 5,
(10) fresh embryo transfers.
The exclusion criteria were as follows: (1) history
of cytotoxic chemotherapy and/or radiotherapy, (2) history
of ovarian surgery, (3) patients with sperm count ≤1 mil-
lion/mL, (4) patients undergoing in vitro fertilization (IVF)
cycle, (5) motile sperm rate ≤10%, (6) uterine or endome-
trial or tubal pathology, (7) frozen/thawed embryo transfers
(8) paternal age >50 years and (9) women aged >43 years.
The demographic and clinical data including age,
BMI, infertility duration, prior IVF cycle numbers, and
sperm parameters were recorded. The ovarian stimulation
parameters such as total gonadotropin dose, stimulation du-
ration, number of oocytes retrieved, fertilization rate, Day-3
and Day-5 quality of embryos obtained after ICSI proce-
dure, grade of embryos at transfer day were analyzed and
compared between the study and control groups. Results
of ovarian stimulation were also compared between the pa-
tients in different groups regarding embryo transfer day.
The fertilization rate and quality of embryos were evalu-
ated as the primary outcomes, while clinical pregnancy rate
and live birth rates were secondary outcomes.
2.2 Controlled Ovarian Stimulation Protocol (COH)
Antagonist protocol was used for all patients under-
going the fresh ICSI cycle. The COH started with recom-
binant follicle stimulating hormone (FSH) on the second or
third day of the menstrual cycle by evaluation by transvagi-
nal ultrasonography. FSH dose was determined according
to the patient’s age, BMI, antral follicle count (AFC), hor-
mone levels and previous attempt history [7]. The infertility
specialist (author: EB) monitored ovarian follicular devel-
opment using transvaginal ultrasound at 1–3 day intervals.
Patients were treated with a daily 0.25 mg gonadotropin re-
leasing hormone (GnRH) antagonist (Cetrotide, 0.25 mg;
Merck KGaA, Darmstadt, Germany) given from stimula-
tion day 6 onwards (fixed regimen). A 250 mg dose of
human chorionic gonadotropin (hCG) (Ovitrelle, Merck
KGaA, Darmstadt, Germany) was injected to achieve fol-
licular maturation when the leading follicles reached 17–18
mm in diameter. Oocyte retrieval took place 34–36 h af-
ter hCG injection and metaphase II (MII) oocytes were fer-
tilized by ICSI and cultured until the day of transfer in a
commercially available culture medium. Embryo forma-
tion was controlled after 16–18 hours and monitored for
replication state until embryo transfer day. Embryos were
graded according to the Istanbul Embryo Grading Consen-
sus, including grade 1, 2 and 3 [8].
For Day-3 embryos: a grade-1 embryo was classi-
fied as having <10% fragmentation with equally sized blas-
tomeres; a grade-2 embryo was classified as having 10–
25% fragmentation with equally sized blastomeres; while
a grade-3 embryo was classified as having >25% fragmen-
tation with blastomeres of various sizes.
2

Table 1. The comparison of the patients regarding their demographic and clinical characteristics.
Characteristics All population (n = 213) Sperm chip group (n = 102) Control group (n = 111) pvaluea
mean ±SD or number (%)
Maternal age (years) 35.0 ±5.6 34.9 ±5.0 35.2 ±6.2 0.689
Maternal age (years)
0.707≤35 112 (52.6) 55 (53.9) 57 (51.4)
>35 101 (47.4) 47 (46.1) 54 (48.6)
Paternal age (years) 37.2 ±5.9 37.1 ±5.4 37.4 ±6.3 0.715
Paternal age (years)
0.285≤37 113 (53.1) 58 (56.9) 55 (49.5)
>37 100 (46.9) 44 (43.1) 56 (50.5)
Body mass index (kg/m2) 23.6 ±3.5 23.4 ±3.5 23.8 ±3.5 0.488
Infertility duration (months) 47.6 ±45.5 51.4 ±47.6 44.2 ±43.4 0.295
Previous IVF cycle number 2.5 ±1.9 3.3 ±2.2 1.8 ±1.1 <0.001
Previous IVF cycle number
<0.0011 77 (36.2) 20 (19.6) 57 (51.4)
≥1 136 (63.8) 82 (80.4) 54 (48.6)
Sperm concentration (million/mL) 32.8 ±27.7 33.2 ±26.8 32.6 ±28.6 0.473
Sperm concentration (million/mL)
0.075≤10 58 (27.2) 22 (21.6) 36 (32.4)
>10 155 (72.8) 80 (78.4) 75 (67.6)
Abbreviations: SD, standard deviation.
a,p<0.05 accepted as statistically significant; Bold, Statistically significant. Independent sample t-test for continuous variables
or chi-square test for categorical variables were used.
For Day-5 embryos: a grade-1 embryo was classified
as having many prominent and easily discernible cells that
form a cohesive epithelium; a grade-2 embryo was clas-
sified as having many easily discernible cells that form a
loose epithelium; a grade-3 embryo was classified as hav-
ing few, difficultly discernible cells.
According to their morphologies, embryos were trans-
ferred on the third or fifth days. If there were 4 or more good
quality embryos on Day-3, we decided to transfer them on
Day-5. Vaginal micronized progesterone (Progestan 200
mg; Koçak, Tekirdağ, Turkey) or vaginal progesterone gel
(Crinone 8%, Merck Serono, Italy) was administered daily
after the oocyte retrieval.
2.3 Semen Preparation
The couples were informed about the requirement of
2–5 days of abstinence before sample collection. Semen
samples were collected by masturbation into a sterile con-
tainer that was labeled. Samples were incubated at 37 °C
for 60 minutes.
2.4 Centrifuge and Swim-Up Technique
The liquefied semen sample was diluted in a one-to-
one ratio in a culture medium and centrifuged at 1500 rpm
for 10 minutes. Subsequently, the supernatant was dis-
carded and fresh culture medium of 1 mL was laid and incu-
bated for one hour at 37 °C at an angle of 45°. Finally, the
supernatant was discarded again, and the remaining sperm
were used for ICSI.
2.5 Microfluidic Chip Technique
In this technique, a microfluidic sperm chip (Fer-
tile Chip®, Koek Biotechnology, Izmir, Turkey) was used
for sperm sorting. This disposable system contains poly-
methylmethacrylate and two-sided sticky film that has
micro-flow free channels. A sperm sample was added into
the 1 mL channel opening and the microfluidic chip was
incubated for 30 minutes at 37 °C. Spermatozoa which
reached to the exit were collected for ICSI.
2.6 Density Gradient Technique
A two-layer density gradient was applied by layering
90% medium under 50% medium in a conical centrifuge
tube. Following fluidification, 1 mL of the semen sample
was layered over the top layer and centrifuged at 1400 rpm
for 10 minutes.
After centrifugation, most of the leukocytes, dead
sperm and other components of the plasma are removed
gently. Then the pellet was put into a new sterile tube and
resuspended in 3 mL of medium to remove the gradient
medium. Centrifugation was repeated. Finally, the pellet
was resuspended in a sterile medium.
2.7 Embryo Transfer and Outcome Measurements
During the study period, one embryo was transferred
to patients <35 years of age in their first two IVF attempts;
two embryos were transferred only after first 2 or more
failed IVF attempts. In patients who are aged above 35
years, either one or two embryos could be transferred re-
3

Table 2. Comparison of the patients regarding their ovarian stimulation parameters and results.
Characteristics All population (n = 213) Sperm chip group (n = 102) Control group (n = 111) pvaluea
mean ±SD or number (%)
Total gonadotropin dose (IU/L) 1884.9 ±786.2 1908.4 ±796.1 1863.3 ±779.8 0.677
Stimulation duration (days) 9.1 ±2.0 8.7 ±1.8 9.4 ±2.2 0.011
Stimulation duration (days)
0.005≤9 134 (62.9) 74 (72.5) 60 (54.1)
>9 79 (37.1) 28 (27.5) 51 (45.9)
Number of oocytes retrieved 10.1 ±7.0 9.7 ±6.2 10.4 ±7.6 0.429
Number of oocytes retrieved
0.513≤10 133 (62.4) 66 (64.7) 67 (60.4)
>10 80 (37.6) 36 (35.3) 44 (39.6)
Metaphase II oocyte number 6.8 ±5.2 7.0 ±5.1 6.6 ±5.3 0.633
Metaphase I oocyte number 1.0 ±1.4 0.9 ±1.2 1.1 ±1.6 0.358
Day-1 pronucleus 5.3 ±4.1 5.5 ±3.8 5.1 ±4.4 0.566
Day-2 pronucleus 4.9 ±3.9 4.7 ±3.2 5.0 ±4.4 0.460
Number of obtained embryos after ICSI procedure 1.5 ±0.5 1.5 ±0.5 1.6 ±0.5 0.070
Embryo transfer day
0.087Day-3 88 (41.3) 36 (35.3) 52 (46.8)
Day-5 125 (58.7) 66 (64.7) 59 (53.2)
Grade of embryos transferred on Day-3
0.145
Grade 1 46 (52.3) 16 (44.4) 30 (57.7)
Grade 2 24 (27.3) 9 (25) 15 (28.8)
Grade 3 18 (20.5) 11 (30.6) 7 (13.5)
Grade of embryos transferred on Day-5
0.050
Grade 1 83 (66.4) 50 (75.8) 33 (55.9)
Grade 2 18 (14.4) 8 (12.1) 10 (16.9)
Grade 3 24 (19.2) 8 (12.1) 16 (27.1)
Clinical pregnancy rate (%) 42.3 (90/213) 41.2 (42/102) 43.2 (48/111) 0.760
Abbreviations: SD, standard deviation; ICSI, intracytoplasmic sperm injection.
a,p<0.05 accepted as statistically significant; Bold, Statistically significant. Independent sample t-test for continuous variables
or chi-square test for categorical variables were used.
gardless of previous IVF attempts, in accordance with the
Turkish legislation of embryo transfer policy.
On the 10th day following embryo transfer, serum
beta-hCG level was measured. If the beta-hCG level was
>10 mIU/mL, it was considered as positive and patients
with such levels were regarded as chemically pregnant.
Clinical pregnancy was confirmed by the presence of a ges-
tational sac on the 10th day after the positive result. Ongo-
ing pregnancy was defined as the presence of fetal cardiac
activity beyond 12 weeks of amenorrhea.
2.8 Statistical and Power Analysis
IBM SPSS Statistics (IBM Corp. Released in 2017.
IBM SPSS Statistics for Windows, Version 25.0, Armonk,
NY, USA) was used for statistical analysis. The sample size
was calculated to prevent type-II errors. Inclusion of a mini-
mum of 204 couples was calculated in the study with at least
102 couples per group, for a power analysis with a 0.5% er-
ror margin and 90% reliability. Categorical variables were
expressed as numbers and percentages, whereas continu-
ous variables were expressed as mean and standard devia-
tion. Comparison of continuous variables between groups
was performed, and the Student’s ttest was used when the
assumption for the precondition of a parametric distribu-
tion was met. The chi-square test was used for comparison
of categorical variables. To determine risk factors affect-
ing clinical pregnancy, logistic regression analysis was used
to evaluate variables found to be statistically significant by
univariate analysis. For the multivariate analysis, the pos-
sible factors identified by univariate analyses were further
entered into the logistic regression analysis to determine in-
dependent predictors of patient outcome. A p-value of 0.05
was considered as statistically significant.
3. Results
A total of 213 patients who met the eligibility criteria
were included in the study. Table 1shows the comparison
between two groups in terms of patients’ demographic and
clinical characteristics. The mean maternal age was com-
parable between the sperm chip and control groups (34.9 ±
5.0 and 35.2 ±6.2, respectively (p= 0.689)). The mean age
of men was also similar between the groups (37.1 ±5.4 and
4

Table 3. Comparison of the patients according to different embryo transfer days and whether spermchip is used or not.
Characterists
Embryo transfer on Day-3 Embryo transfer on Day-5
Sperm chip group (n = 36) Control group (n = 52) pvalueaSperm chip group (n = 66) Control group (n = 59) pvaluea
mean ±SD or number (%) mean ±SD or number (%)
Day-1 pronucleus 3.7 ±2.5 4.9 ±4.2 0.114 6.4 ±4.1 5.3 ±4.5 0.183
Day-2 pronucleus 3.1 ±2.2 4.6 ±4.3 0.044 5.5 ±3.4 5.5 ±4.5 0.969
Number of obtained embryos after ICSI procedure 1.5 ±0.5 1.6 ±0.5 0.599 1.5 ±0.5 1.6 ±0.5 0.054
Grade
0.145 <0.050
Grade 1 16 (44.4) 30 (57.7) 50 (75.8) 33 (55.9)
Grade 2 9 (25) 15 (28.8) 8 (12.1) 10 (16.9)
Grade 3 11 (30.6) 7 (13.5) 8 (12.1) 16 (27.1)
Clinical pregnancy rate (%) 38.8 (14/36) 42.3 (22/52) 0.255 48.5 (32/66) 44.1 (26/59) 0.621
Miscarriage rate (%) 11.1 (4/36) 9.6 (5/52) 0.343 10.6 (7/66) 8.4 (5/59) 0.434
Live birth rate (%) 27.8 (10/36) 32.6 (17/52) 0.164 37.8 (25/66) 35.6 (21/59) 0.712
Abbreviations: SD, standard deviation; ICSI, intracytoplasmic sperm injection.
a,p<0.05 accepted as statistically significant; Bold, Statistically significant. Independent sample t-test for continuous variables or chi-square test for categorical variables were used.
Table 4. Predictive factors to determine clinical pregnancy rate in ICSI procedure.
Factor Univariate analysis Multivariate analysis
Odds ratio 95% Confidence interval pvalueaOdds ratio 95% Confidence interval pvaluea
Maternal age (years) 0.60 0.34–1.03 0.064 0.72 0.33–1.57 0.409
Paternal age (years) 0.72 0.42–1.24 0.238 1.05 0.49–2.24 0.900
Sperm concentration (million/mL) 1.16 0.63–2.14 0.639 1.41 0.74–2.69 0.302
Sperm chip use 0.92 0.53–1.58 0.760 0.91 0.52–1.60 0.736
Number of oocytes retrieved 2.31 1.31–4.08 0.004 2.19 1.19–4.04 0.012
Abbreviations: ICSI, intracytoplasmic sperm injection.
a,p<0.05 accepted as statistically significant; Bold, Statistically significant. Univariate and multivariate linear logistic regression analysis were
used.
5

37.4 ±6.3, respectively, p= 0.715). Body mass index
(kg/m2) was evaluated between study and control groups
(23.4 ±3.5 vs 23.8 ±3.5, respectively, p= 0.488).
When sperm concentrations were compared, both the
sperm chip and control groups had similar results (33.2 ±
26.8 and 32.6 ±28.6 million/mL, respectively, p= 0.473).
Differences were detected between two groups regard-
ing COH parameters and results, as shown in Table 2. To-
tal FSH doses administered in the sperm chip and control
groups were 1908.4 ±796.1 IU/L and 1863.3 ±779.8 IU/L,
respectively (p= 0.677). The average number of stimula-
tion days was statistically shorter for the sperm chip group
than the control group (8.7 ±1.8 days and 9.4 ±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 ±3.8 and 5.1 ±4.4, p
= 0.566). The mean total number of embryo obtained after
ICSI between study vs control group (1.5 ±0.5 vs 1.6 ±0.5,
p= 0.070) and embryo transfer day did not show signifi-
cant 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 trans-
ferred 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 signifi-
cant difference was observed (Day-3: 3.7 ±2.5 vs 4.9 ±
4.2 (p= 0.114) and Day-5: 6.4 ±4.1 vs 5.3 ±4.5 (p=
0.183)).
Clinical pregnancy rate did not show significant dif-
ference 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 be-
tween 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 pre-
dictor for clinical pregnancy (p= 0.004). However, mater-
nal age, paternal age and sperm concentration did not pre-
dict clinical pregnancy (Table 4). Additionally, this associ-
ation with the number of oocytes retrieved for clinical preg-
nancy was found to be still significant after adjustment of
confounding factors in multivariate linear regression anal-
ysis (p= 0.012).
4. Discussion
In our study, a relatively recent technique, the sperm
chip technique, was compared to one of the traditional ap-
proaches. 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 alterna-
tive 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 intrauter-
ine 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 recur-
rent unsuccessful previous ART attempts [11–13]. A recent
study by Ozcan et al. [14] showed that use of microflu-
idic 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 tech-
nique since only a few sperm are adequate for the proce-
dure. 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 [16–18]. Although another study pub-
lished later showed that high DFI had no effect on fertil-
ization rate, quality of embryo or clinical pregnancy rate, it
was indicated that the spontaneous abortion rate was statis-
tically higher in those with abortion of over 27% [19]. It
was also shown that DFI had little to no effect on fertiliza-
tion and early embryo development. However, it had an ob-
vious 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 frag-
mentation 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 un-
detectable 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
6

good quality embryos with the use of sperm chip techniques
may be explained by the lower exposure of the sperm to ox-
idant 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, en-
abling 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 ob-
tain increased success regarding fertilization rate compar-
ing 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 re-
ported [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 re-
sults. 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 cu-
mulative pregnancy rate per patient due to failed reaching
of all the frozen embryos data (even if the total and trans-
ferred 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 be-
cause sperm motility might cause a potential bias favor-
ing 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 regres-
sion models for potential confounders.
5. Conclusions
The sperm chip techniques were found as clinically
useful for increasing the development of good quality em-
bryos 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 suc-
cess in ICSI treatment.
Availability of Data and Materials
The datasets used and/or analyzed during the current
study are available from the corresponding author on rea-
sonable request.
Author Contributions
SO—protocol/project development; data collection
or management, manuscript writing/editing; HGC—
protocol/project development, data collection or manage-
ment, data analysis, manuscript writing/editing; YK—
protocol/project development, data collection or manage-
ment; EK—protocol/project development, data collection
or management; MG—manuscript writing/editing; JY—
manuscript writing/editing; EB—protocol/project devel-
opment, data collection or management, data analysis,
manuscript writing/editing. All authors read and approved
the final manuscript.
Ethics Approval and Consent to Participate
The study was performed in accordance with the eth-
ical 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 Commit-
tee (ATADEK 2017-15/16). Approval was obtained from
ClinicalTrials.gov with NCT03355937 approval number.
Acknowledgment
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.
Funding
This research received no external funding.
Conflict of Interest
The authors declare no conflict of interest.
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