Sperm Selection by Microfluidic Separation Improves Embryo Quality
Phase: NA
Conditions: Sperm DNA Fragmentation, Embryo Quality, Fertility Disorders, Infertility, Infertility, Male, Infertility Unexplained
Sponsor: University of California, San Francisco

 
 
 
Sperm Selection by Microfluidic Separation 
Improves Embryo Quality 
 
 
NCT03085433 
 
 
Document Date: 3/17/2017 

Title: Microfluidic Separation Reduces Sperm DNA Damage and Improves Embryo Quality
Background/Significance
More than 70 million couples worldwide are infertile and up to 40 million are actively seeking 
infertility care. In the year 2013, a total of 160,521 assisted reproductive technology (ART) 
procedures were performed in the United States (Sunderam et al, 2015). Isolation of motile and 
morphologically normal sperm is an integral part of assisted reproduction.  Traditional sperm 
processing for assisted reproduction involves centrifugation and “swim up” techniques that 
employ a density gradient to isolate motile sperm. However, studies have suggested that 
centrifugation induces reactive oxygen species and DNA damage (Malvezzi et al, 2014; Wang 
Assist Reprod Genet, 2014). Increased sperm DNA damage has been associated with poor 
outcomes in assisted reproduction, including lower fertilization rates, impaired embryo 
progression, and decreased pregnancy rates (Virro et al, 2004; Benchaib et al 2007; Simon et al 
2014). 
In contrast, microfluidic-based sperm sorting has the capability of selectively isolating highly 
motile, morphologically normal sperm with high DNA integrity from an unprocessed semen 
sample (Shirota et al 2016; Asghar et al, 2014; Tasoglu et al 2013). In semen samples from 
healthy male volunteers split into standard processing via centrifugation and swim-up procedure 
compared with microfluidic sperm sorting, a significantly higher percent motility and lower rate of 
sperm DNA fragmentation was detected with microfluidic sperm sampling (Shirota et al 2016). 
While the microfluidic sperm sorting technique has thus proven to be an efficient and reliable 
means of sperm preparation compared with the centrifugation and swim-up procedure, its use in 
clinical practice has not been rigorously studied.  We aim to compare traditional preparation and 
microfluidic sperm sorting on assisted reproductive technology outcomes including oocyte 
fertilization and embryo quality in subjects electing to undergo in vitro fertilization for infertility.
Preliminary Studies:
The microfluidic sperm sorting device has been shown to isolate highly motile, morphologically 
normal, and high DNA integrity sperm from unprocessed human semen. We have utilized the 
microfluidic sperm chip to select sperm for intracytoplasmic sperm injection (ICSI) in 26 cases of 
patients with poor prior ART outcome after standard sperm processing and selection. In these 
poor prognosis cases, we had 73% fertilization rate and 59% good quality day 3 embryos. 
Furthermore, among those patients who have received an embryo transfer, 58% (7 out of 12) 
have had a positive pregnancy test. While we cannot compare these data statistically to the prior 
failed cycle (due to concerns regarding regression to the mean), these data compare favorably to 
age-matched controls. However, given that this is a highly selected population, we believe a 
randomized controlled trial is necessary to rigorously explore the utility of sperm selection with 
the microfluidic sperm chip.
Hypothesis:
Subjects randomized to sperm preparation by microfluidic sperm sorting will obtain higher quality 
day 3 embryos than those randomized to o traditional sperm processing.
Study Aims:
1) Primary Aim: To compare day 3 embryo quality resulting from fertilization of oocytes by sperm 
selected by microfluidic sperm sorting compared to traditional sperm preparation methods.
2) Secondary Aim: To compare fertilization rates, blastulation rates, and pregnancy rates for 
subjects randomzied to sperm selection by microfluidic sperm sorting compared to traditional 
sperm preparation methods.
Design:
This is a randomized controlled trial of couples undergoing in vitro fertilization for unexplained 
infertility. Couples will be randomized to sperm selection by the clinical standard of centrifugation 

and density-gradient processing compared to the microfluidic sperm sorting chip. Subjects and 
investigators performing outcome assessment will be blinded to treatment assignment.
Inclusion Criteria:
The target population includes couples planning in vitro fertilization (IVF) with or without 
intracytoplasmic sperm injection for unexplained infertility at the UCSF Center for Reproductive 
Health with a history of poor embryo quality as defined by < =40% high quality D3 embryos in a 
prior IVF cycle. All eligible couples will be asked to join the study. 
Exclusion Criteria:
Male partner with severe oligoasthenospermia (concentration < 5 x 10^6 
spermatozoa/mL; motility< 10%)
Female partner with anovulation (PCOS, FHA)
Female partner age >41
Female partner AFC< 7
Female partner with obstructed fallopian tubes (assessed in all patients prior to IVF)
Use of oocyte donor
Either Partner:
Cancer diagnosis in either partner
Any significant disease or psychiatric disorder that would interfere with consenting process
Treatment History:
History of >1 prior cycle cancellation due to poor response
Treatment Plan:
Embryo co-culture
Use of adjunctive non-gonadotropin medications to improve embryo quality: growth hormone, 
sildenafil
Study Procedures:
Couples meeting eligibility criteria will be offered enrollment. Randomization will be performed by 
the study coordinator upon initiation of controlled ovarian stimulation. Neither study clinician nor 
subject will be aware of treatment allocation. Ovarian stimulation will be performed with standard 
treatment chosen by primary physician.  The day of ovulation trigger will be determined by the 
primary physician. The andrology lab will be notified of allocation to conventional sperm 
preparation versus microfluidic sperm sorting on the day of ovulation trigger. Oocyte retrieval is 
performed  36 hours after ovulation trigger. On the day of oocyte retrieval, the male partner will 
produce a semen sample per usual clinic protocol. A semen analysis will be performed on all 
samples for assessment of volume, motility, concentration, and morphology prior to semen 
processing according to WHO criteria using a light microscope, consistent with usual clinic 
protocol. Following the semen analysis, the semen of those assigned to microfluidic sperm 
sorting will be pipetted into the inlet chamber of the sorting chip. The microfluidic sperm sorting 
device is a flow and chemical-free microfluidic single use “chip” with an inlet sample chamber 
connected to an outlet collection chamber by a microfluidic channel. The sorted sperm are 
collected from the outlet.
The semen of those assigned to standard processing will undergo conventional centrifugation 
and density gradient separation. ICSI or IVF will be performed by an embryologist blinded to 
treatment allocation with the processed sperm according to the primary physician’s orders. After 
16-18 hours, fertilization will be evaluated by the existence of two pro-nuclei. Fertilized embryos 
will be cultured to day 3 and evaluated by an embryologist blinded to treatment allocation for 
embryo quality. Cell number will be assessed in addition to blastomere symmetry and 
fragmentation (grade 1-6, highest quality to lowest). Those whose treatment plan involves a day 3 
embryo transfer (determination made by primary physician) will undergo embryo transfer per 
clinic standard on day 3. Otherwise, embryos will be cultured to the blastocyst stage prior to 

transfer. Blastocysts will be graded by Gardner Criteria, per clinic protocol. All women who 
undergo an embryo transfer will have a serum pregnancy test (beta HCG) 14 days later. Those 
with a positive serum test will repeat testing 48 hours later. Pregnancy ultrasounds will be 
scheduled at 5 weeks 5 days per clinic standard of care.
Device:
FERTILE: microfluidic sperm sorter
The device is labware. It is not an implant and is not purported to support or sustain human life. It 
is a flow and chemical free microfluidic single-use chip with an inlet chamber for receiving 
untreated semen samples and an outlet for collection of normal, healthy sperm for use in assisted 
reproductive technology procedures in andrology and embryology laboratories. It does not 
present any potential for serious risk to the health, safety, or welfare of participants.
Statistical Methods:
As this is a randomized controlled trial, known and unknown confounders are assumed to be 
evenly distributed between treatment groups. However, at the completion of enrollment, baseline 
characteristics will be assessed between groups and chi squared or t-tests will be performed, as 
appropriate to ensure groups are evenly distributed with respect to the most important clinical 
parameters such as age, duration of infertility, gravity/parity, prior infertility treatment, and ovarian 
reserve (AFC). The primary outcome, day 3 high quality embryo proportion will be defined as 
proportion of all viable embryos on day 3 with at least 6 cells and fragmentation/symmetry scores 
of 1-2. This proportion will be compared between groups with a t-test. An ANOVA will be 
performed to obtain and age and AFC-adjusted comparison of day 3 high quality embryo 
proportion between groups. Secondary outcomes will include fertilization and pregnancy rates. 
Fertilization rate will be defined as number of 2PN embryos per oocyte retrieved. Pregnancy rate 
will be defined as clinical pregnancy (ultrasound demonstrating gestational sac with yolk sac) per 
transfer. These secondary outcomes will be evaluated with a t-test.
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