THE EFFECT OF FLUID FLOW ON HUMAN SPERM FERTILITY PARAMETERS
by
Kari Rappa
A Thesis Submitted to the Faculty of
College of Engineering
In Partial Fulfillment of the Requirements for the Degree of
Master of Science
Florida Atlantic University
Boca Raton, FL
August 2017
Copyright by Kari Rappa
ii
ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Asghar, for all the guidance he has given me while I pursue my degree, and also my supervisory committee, Dr. Oscar Curet and Dr.
Mirjana Pavlovic for their support. I am also grateful for all the amazing faculty and staff at Florida Atlantic University who continue to make this a great educational institution.
iv ABSTRACT
Author: Kari Rappa
Title: THE EFFECT OF FLUID FLOW ON HUMAN SPERM FERTILITY
Institution: Florida Atlantic University
Thesis Advisor: Dr. Waseem Asghar
Degree: Master of Science
Year: 2017
Current sperm processing methods used in assisted reproductive technologies can cause damage to the sperm cell. New ways that mimic the natural guidance mechanisms present in the female genital tract may offer ways to sort sperm with better fertility parameters. Sperm that respond to these cues may have better over sperm health. Human sperm exhibit positive rheotaxis by orienting and swimming against the fluid released by the female genital tract. At certain flow rates sperm can actively orient and swim against the flow. Sperm retrieved that exhibit positive rheotaxis have higher motility and better morphology than the original semen sample.
v THE EFFECT OF FLUID FLOW ON HUMAN SPERM FERTILITY PARAMETERS
List of Tables ...... viii
List of Figures ...... ix
1. INTRODUCTION ...... 1
1.1 Natural sperm selection and guidance ...... 1
1.2 Infertility and sperm characteristics ...... 3
1.2.1 Sperm concentration and motility ...... 4
1.2.2 Sperm morphology, ROS and DNA integrity ...... 5
1.3 Current sperm processing techniques for ART ...... 6
1.4 Purpose ...... 7
2. MATERIALS AND METHODS ...... 8
2.1 Device Fabrication ...... 8
2.2 Sperm Preparation ...... 9
2.3 Sperm Sorting ...... 9
2.4 Sperm concentration ...... 10
2.5 Sperm velocity analysis ...... 11
2.6 Morphology assessment ...... 12
2.7 Hyaluronic acid assay binding analysis ...... 12
2.8 Statistical Analysis ...... 12
3. RESULTS ...... 13
3.1 Sperm orientation in differential fluid flow ...... 13
vi 3.2 Sperm velocity in differential fluid flow ...... 15
3.3 Sperm motility and retrieval analysis ...... 18
3.4 Sperm velocity ...... 21
3.5 Hyaluronic acid binding analysis ...... 22
3.6 Morphology analysis ...... 23
4. DISCUSSION ...... 24
5. REFERENCES ...... 30
vii LIST OF TABLES
Table 1: VCL, VAP, VSL before flow in the differential fluid flow chip ...... 16
Table 2: VCL, VAP, VSL after flow in the differential fluid flow chip ...... 17
Table 3: Sperm velocity data for stock, control and flow after retrieval from constant
fluid flow chip ...... 21
viii LIST OF FIGURES
Figure 1: Anatomical depiction of a sperm cell. Originally published in: Borg, Claire
L.; Wolski, Katja M., Sep 15, 2009, Human Reproduction Update, 16(2).
Reproduced with permission from Oxford University Press...... 5!
Figure 2: Differential fluid flow chip. 4 mm diameters sperm inlet. 76 µm channel
height. 28.5 mm long channel ...... 8!
Figure 3: Constant fluid flow and sperm collection chip.4 mm diameter sperm inlet.
6 mm by 2.5 mm elliptical collection chamber. Channel 76 µm high and 8 mm
long...... 9!
Figure 4: Depiction curvilinear velocity (VCL): solid line. Average path velocity
(VAP): dotted line. Straight line velocity (VSL): arrow...... 11!
Figure 5: Sperm position before flow in the differential fluid flow chip ...... 13!
Figure 6: Sperm position during fluid flow in the differential fluid flow chip. The
flow is going from left to right ...... 14!
Figure 7: Percentage of sperm at different flow rate that oriented and progressively
swam against flow ...... 14!
Figure 8: Sperm paths before flow ...... 15!
Figure 9: Sperm paths during flow. Flow is from left to right ...... 16!
Figure 10: VCL, VAP, VSL before and after flow in the differential fluid flow
chip...... 17!
ix Figure 11: Concentation of million sperm/mL and type of motility observed in each
experimental group. (PR) progressive motility. (NP) non-progressive motility.
(IM) immotility. (PR+NP) total motility ...... 18!
Figure 12: Percent of sperm recovered from the collection chip after one hour ...... 19!
Figure 13: Motility type of sperm recovered from constant fluid flow collection chip.
(PR) progressive motility. (NP) non-progressive motility. (IM) immotility ...... 20!
Figure 14: VCL, VAP, VSL for stock, control, and flow...... 22!
Figure 15: Percent of sperm in stock, control, and flow that bound to hyaluronic
acid ...... 22!
Figure 16: Percent of normal sperm present in stock, control, and flow ...... 23!
x 1. INTRODUCTION
1.1 Natural sperm selection and guidance
A successful pregnancy occurs when sperm travel through the female genital tract
(FGT) and fertilize the oocyte in the oviduct. Only about one sperm per every million sperm ejaculated makes it to the oviduct (Eisenbach & Giojalas, 2006). Before fertilization, sperm undergo a maturation process called capacitation. This process happens asynchronously, with only 10% of sperm being capacitated at any given time
(Eisenbach & Giojalas, 2006). Capacitation allows the sperm to respond to various stimulants, penetrate the cumulus cells around the egg, bind the sperm receptors, and undergo the acrosome reaction (Eisenbach & Giojalas, 2006; Gaffney, Gadêlha, Smith,
Blake, & Kirkman-Brown, 2011). The acrosome reaction is the release of proteolytic enzymes from the head of the sperm that allows the sperm to penetrate the egg coat
(Eisenbach & Giojalas, 2006). The small amount of sperm that are capable of fertilizing the oocyte indicates the FGT has a stringent sperm selection process that incorporates both long-range and short-range mechanisms.
The long-range mechanisms include the natural contractions and movement of the
FGT and outward fluid flow from the oviduct and cervix, which causes sperm to exhibit positive rheotaxis (Cerezales, Boryshpolets, Eisenbach, & others, 2015; Henkel & Schill,
2003; Miki & Clapham, 2013). Human sperm exhibit different types of swimming behavior depending on location in the female reproductive tract (Gaffney et al., 2011;
Gillies, Cannon, Green, & Pacey, 2009). Shortly after ejaculation, sperm movement is
1 linear and progressive, but once it reaches the fallopian tubes it becomes quicker and more erratic (Cerezales et al., 2015; Eisenbach & Giojalas, 2006; Gillies et al., 2009).
Sperm tend to swim near surfaces and walls with or without the presence of flow due to steric repulsion, hydrodynamic forces, and flagellar beat (Elgeti, Kaupp, & Gompper,
2010; Kantsler, Dunkel, Blayney, & Goldstein, 2014; Tung et al., 2015). The flagella beat in a chiral pattern, resulting in a helical swim pattern that allows the sperm to explore the full surface of the channel (Ishimoto & Gaffney, 2015; Kantsler et al., 2014). The chiral flagella beat traces out a path that when it aligns with a surface, turns the sperm head into the surface, trapping it (Ishimoto & Gaffney, 2015; Kantsler et al., 2014). In shear flow conditions, the tail of a trapped sperm at a surface explores and experiences areas of greater flow velocity than the head, which helps orient their head against the flow
(Ishimoto & Gaffney, 2015; Kantsler et al., 2014; Sarkar, 1984). Additionally, sperm with their head downstream of flow in a channel will make a U-turn for the wall when exposed to shear flow (Sarkar, 1984). Once the sperm get to the lower isthmus of the oviduct, they are affected by the oviductal flow. The flow is increased up to four hours after coitus, and the sperm at the storage site orient to this flow and swim against it (Miki
& Clapham, 2013). The sperm that have been capacitated show an increased progression and swim rate through the viscous oviductal fluid (Gaffney et al., 2011). Highly viscous fluid and surface-confined space causes sperm to slither swim (swimming in one plane), which results in the sperm swimming 50% faster (Gaffney et al., 2011; Nosrati, Driouchi,
Yip, & Sinton, 2015).
The short-range mechanisms are positive thermotaxis and chemotaxis (Cerezales et al., 2015; Eisenbach & Giojalas, 2006). The temperature in the oviduct is warmer
2 closer to the egg, creating a temperature gradient that the sperm respond orient and swim towards. (Cerezales et al., 2015; Eisenbach & Giojalas, 2006). Sperm are also known to orient and swim towards chemoattractant gradients (Cerezales et al., 2015; Eisenbach &
Giojalas, 2006). Progesterone is a known chemoattractant released by the egg and it is believed that there may be more (Cerezales et al., 2015; Eisenbach & Giojalas, 2006;
Gatica et al., 2013). When sperm sense a thermal or chemical gradient, sperm exhibit hyperactivation and the flagella begins beating asymmetrically with greater intensity to head in the direction of the attractant (Cerezales et al., 2015). Sperm hyperactivation is characterized by an increased amplitude and asymmetrical flagellar bending and is believed to be mediated through calcium influx via CatSper calcium channels (Cerezales et al., 2015; Eisenbach & Giojalas, 2006; Suarez, 2008; Zhang et al., 2016).
Sperm who exhibit rheotaxis have shown no evidence of hyperactivation or calcium influx, and thus it is believed to be a purely passive, physical process (Zhang et al., 2016). Long-range mechanisms must be vigorous because of the long distance they have to send a signal. Fluid flow is easier to establish and stabilize across a long range than a chemical or thermal gradient which must be controlled at every point (Zhang et al.,
2016). Rheotaxis also appears to be a more efficient system. Approximately 50% of sperm exhibit rheotaxis versus less than 10% for chemotaxis and thermotaxis (Bahat et al., 2003; Cohen-Dayag, Tur-Kaspa, Dor, Mashiach, & Eisenbach, 1995; Zhang et al.,
2016).
1.2 Infertility and sperm characteristics
Infertility affects roughly 48.5 million couples worldwide and 30-50% of these cases are caused by male factor infertility (Boivin, Bunting, Collins, & Nygren, 2007;
3 Mascarenhas, Flaxman, Boerma, Vanderpoel, & Stevens, 2012; Ombelet, Cooke, Dyer,
Serour, & Devroey, 2008). Assisted reproductive technologies (ART), such as intracytoplasmic sperm injection (ICSI), intrauterine insemination (IUI) and in vitro fertilization (IVF) offer these couples the opportunity to start families. However, only about a third of ART cycles result in a birth (Center For Disease Control, n.d.). Sperm processing is an important part of the ART cycle and there are several factors to consider when determining the quality of sperm, such as concentration, motility, morphology,
DNA integrity and levels of reactive oxygen species (ROS). Infertile men tend to have abnormal sperm parameters, such as low concentration, abnormal morphology and elevated levels of DNA damage and ROS (Larson-Cook et al., 2003; Pasqualotto,
Sharma, Nelson, Thomas, & Agarwal, 2000).
1.2.1 Sperm concentration and motility
Sperm concentration and motility are two easily identifiable indicators of quality.
Men who have had a partner become pregnant within 12 months post-contraception stoppage have a fifth centile concentration of 15 million cells/mL (12-16 million cells/mL
95% confidence interval) (Cooper et al., 2010). There is an increase in pregnancy frequency as the concentration rises up to roughly 40 million cells/mL, but there is no association with increased pregnancy with concentrations about 40 million cells/mL
(Bonde et al., 1998). In addition to concentration, the percent motility of the sample is also taken into consideration. There are three types of motility: progressive motility, non- progressive motility and immotility. Progressive (PR) motility refers to sperm that travel in mostly straight lines or large circles. Non-progressive (NP) motility refers to sperm that do not have forward progression or move in tight circles. Immotility refers to sperm
4 that do not move. Total motility is the sum of progressive and non-progressive motile sperm. Men who have a pregnant partner within 12 months have a fifth centile total motility percent of 40% (Cooper et al., 2010). Sperm with a high/normal motility have pregnancy rates above 50% (Cooper et al., 2010; Donnelly, Lewis, McNally, &
Thompson, 1998). Sperm motility can also be assessed by computer-aided sperm analysis
(CASA). CASA analysis has been shown to be a predictor of fertility and pregnancy rates
(Fréour, Jean, Mirallié, Dubourdieu, & Barrière, 2010; Garrett, Liu, Clarke, Rushford, &
Baker, 2003; Hirano et al., 2001; Larsen et al., 2000).
1.2.2 Sperm morphology, ROS and DNA integrity
Figure 1: Anatomical depiction of a sperm cell. Originally published in: Borg, Claire L.; Wolski, Katja M., Sep 15, 2009, Human Reproduction Update, 16(2). Reproduced with permission from Oxford University Press. Sperm are considered morphologically normal if the head and tail (Figure 1) are normal according to the requirements of the strict Tygerberg method or the WHO criteria
(Cooper et al., 2010; Menkveld et al., 2001). Morphology affects the ability to swim up- stream through the genital tract. Sperm with larger heads create more drag and have a more difficult time swimming against flow, while those with more elongated heads tend to swim better in Newtonian fluids (Gillies et al., 2009). Morphologically normal sperm are associated with higher rates of pregnancy and implantation than abnormal sperm (De
5 Vos et al., 2003; Grow et al., 1994). Additionally, high levels of ROS cause oxidative stress which can cause morphological defects in addition to decreases in DNA integrity, sperm motility and sperm viability (Pasqualotto et al., 2000; Wright, Milne, & Leeson,
2014). DNA fragmentation is also found to coexist with low concentration samples and morphologically abnormal samples (Wright et al., 2014).
1.3 Current sperm processing techniques for ART
Sperm must be removed from the seminal plasma within one hour after ejaculation to limit damage from non-sperm cells (World Health Organization, 2010).
The most frequently used methods of sperm processing are sperm washing, direct swim- up (DSW) and discontinuous density gradient centrifugation (DGC). These processes are used to remove debris, non-germ cells, and dead sperm in order to create a concentrated sperm sample that has a high percentage of morphologically normal and motile cells. The type of processing method used depends on the characteristics of the ejaculate. Sperm washing yields the largest amount of sperm if the semen sample is good quality, but DGC and DSW are more often used if there is an abnormality (World Health Organization,
2010). DSW aims to select motile sperm by making them swim up out of a medium.
DSW provides a lower amount of sperm compared to washing, but it selects for motility and is good for low motility samples (World Health Organization, 2010). DGC is similar to swim up in that it also selects for motile cells, but it is more consistent because it is easier to standardize than DSW (World Health Organization, 2010). These methods are good for sorting motile and morphologically normal sperm, but they all involve a centrifugation step which can be damaging to sperm cells. Centrifugation creates a sperm pellet that can also include inflammatory cells and immature sperm, which produce ROS
6 and can cause DNA fragmentation in the healthy sperm cells (R. J. Aitken, Bronson,
Smith, & De Iuliis, 2013; González-Marín, Gosálvez, & Roy, 2012). These conventional methods also do not work well on sperm samples that have low sperm counts, low motility, or cryopreserved samples that have reduced motility (Knowlton, Sadasivam, &
Tasoglu, 2015). Roughly 55% of sperm used for ICSI contains normal DNA (Ramos, de
Boer, Meuleman, Braat, & Wetzels, 2004). DNA damage is correlated with lower IVF rates, irregular pre-implantation development, early loss of pregnancy and increased birth defects in children conceived through ART (R. John Aitken & De Iuliis, 2007;
Tremellen, Miari, Froiland, & Thompson, 2007; Wright et al., 2014). It is important to find alternative methods to sperm sorting. The best methods for sperm sorting should isolate large numbers of motile and morphologically normal sperm and eliminate harmful substances such as other dead cells, leukocytes, ROS that can cause damage (Henkel &
Schill, 2003).
1.4 Purpose
Microfluidic technologies are increasingly being used to study sperm sorting and are advantageous in that they can mimic that environment of the female genital tract.
Different microfluidic devices have successfully selected healthy sperm by flow, thermal gradients, biochemical gradients, microgrooves and passive processes which rely on the sperm motility. This project will determine which flow rate causes the most sperm to orient and swim against the flow. It also aims to collect motile and morphologically normal sperm using fluid flow in a low-cost microfluidic device
7 2. MATERIALS AND METHODS
2.1 Device Fabrication
The design for the chip was created in AutoCAD 2015 and uploaded to the UCP
Software for cutting. The PMMA (McMaster-Carr, Atlanta, GA and ePlastics, San Diego,
CA 1.5 mm and 3 mm thick) and the DSA (3M, St. Paul, MN, 76 µm) were cut using a
VLS 2.30 laser cutter (VersaLaser, Scottsdale, AZ). The differential fluid flow chip consisted of 1.5 mm PMMA cut into a 28.5mm x 8 mm piece. A 4 mm diameter sperm inlet was cut into the piece 28.5 mm away from a 0.764 mm diameter fluid flow inlet.
This was then attached to a piece of DSA which had a 4 mm diameter sperm inlet and a
22.4 mm x 4 mm channel cut into it. This was then attached to a 75 mm x 25 mm glass slide (Figure 2).
Figure 2: Differential fluid flow chip. 4 mm diameters sperm inlet. 76 µm channel height. 28.5 mm long channel
8 The constant fluid flow and sperm collection chip was made with 3 mm thick PMMA cut into 75 mm x 25 mm. A sperm inlet of 4 mm diameter was cut 8 mm away from an elliptical sperm collection chamber (long axis 6 mm, short axis 2.4 mm). The fluid flow inlet with diameter 1.98 mm was cut 13 mm away from the sperm inlet. This piece of
PMMA was then attached to a piece of 76 µm DSA with same dimensions of PMMA and a 13 mm x 2 mm long channel from flow inlet to sperm inlet (8 mm from elliptical collection chamber to sperm inlet). Three pieces of 3 mm thick PMMA were then cut into dimensions of 10.6 mm x 75 mm with a 4 mm diameter sperm inlet. These three pieces were attached by 76 µm DSA and stacked on top of the sperm inlet of the bottom PMMA piece (Figure 3).
Figure 3: Constant fluid flow and sperm collection chip.4 mm diameter sperm inlet. 6 mm by 2.5 mm elliptical collection chamber. Channel 76 µm high and 8 mm long. 2.2 Sperm Preparation
Human sperm in 1 mL vials and 0.5 mL canes were purchased from California
Cryobank, Fairfax, VA and Cryos International, Orlando, FL and stored in liquid nitrogen. Sperm was thawed at 37 C for 15 minutes before use.
2.3 Sperm Sorting
HTF-HEPES (InVitroCare, Frederick, MD) solution supplemented with 1% BSA
(FisherSci, Fair Lawn, NJ) was filled into a 10 mL syringe (Becton, Dickson and
9 Company, Franklin Lake, NJ). A 17 gauge blunt needle (SAI, Lake Villa, IL) attached to
0.90” OD tubing (Cole-Parmer, Vernon Hills, IL) was attached to the syringe. The syringe was then placed on the syringe pump (New Era Pump Systems, East
Farmingdale, NY) and pumped in fluid until the channel was full. The pump was then stopped and allowed to reach an equilibrium state where no flow occurred. A 4 µL sample of semen was then loaded into the sperm inlet of the differential flow chip. The sperm were allowed to swim with no flow for a period of 10 minutes to allow an ample amount of sperm into the channel. At that point, the syringe pump was turned on at a flow rate of either 2 µL/min, 4 µL/ min, 5 µL/min, 6 µL/min, 8 µL/min and 10 µL/min.
The amount of sperm that oriented and swam against the flow was manually counted and recorded. A constant rate of 3 µL/min was used to sort sperm in the collection chip. The collection chamber was filled with 1% HTF-BSA and then covered with DSA. The syringe pump was turned on and to fill the collection chip was with 1% HTF-BSA. A 10
µL stock sample of semen was loaded into the 4 mm diameter by 15 mm high sperm inlet of the collection chip. The control group used the same chip under the same conditions minus the flow. The chips were then left to incubate for an hour before collection sperm from the collection chamber.
2.4 Sperm concentration
Sperm from the stock, control, and flow experimental groups were counted using
Makler chamber (Sefi Medical, Israel) as per the instructions and labeled as motile, non- progressively motile, or immotile. Each count was taken at least twice and the average of that count used as data point.
10 2.5 Sperm velocity analysis
The differential fluid flow chip was placed on a light microscope stage and recorded using IC Capture (The Imaging Source, Charlotte, NC) 5 mm from the sperm inlet before and after flow for one minute at 30 fps. The sperm collected from the collection chip were prepared per WHO guidelines. A 11 µL sample was placed on a glass slide and covered with a 24 mm x 24 mm to give a depth of approximately 20 µL.
The slide was then recorded using a Nikon DS-Fi3 camera with NIS-Elements software
(Nikon) attached to a light microscope for one minute at 25 fps. The videos were then uploaded to ImageJ (National Institute of Health) and analyzed using the CASA plugin to obtain the curvilinear velocity (VCL), average path velocity (VAP) and the straight-line velocity (VSL) (Figure 4).
VCL VA P
VSL
Figure 4: Depiction curvilinear velocity (VCL): solid line. Average path velocity (VAP): dotted line. Straight line velocity (VSL): arrow.
11 2.6 Morphology assessment
Sperm were recovered from the collection chip after one hour. A 2 µL sample from the stock, control, and flow groups was then placed on a pre-stained GoldCyto SB sperm blue slide (Fertility Stuff, Murphy, NC) and covered with a 24 mm x 24 mm coverslip. The slide was then heated at 60 C for at least five minutes. The sperm were then examined at 1000x and checked for normal or abnormal morphology per the WHO guidelines: (head must be smooth, regularly contoured, oval, 40-70% acrosomal region, no large vacuoles, no more than two vacuoles; midpiece must be slender, regular, same length as head, major axis aligned with the head, not larger than one-third size of the head; principal piece must have a uniform diameter, thinner than midpiece, approximately 45 µm long, no sharp angles) (World Health Organization, 2010).
2.7 Hyaluronic acid assay binding analysis
Sperm were recovered from the collection chip after one hour. A 10 µL sample was placed on the HBA slide (CELL-VU, New York, NY). Readings were to take place no earlier than 10 minutes after incubation. If bound sperm was more populous, that amount was counted first until the number 100 was reached or the whole grid counted.
The same number of squares would then be used to count the number of unbound sperm.
The amount of bound sperm was divided by the total amount of sperm counted to get the percent that were bound.
2.8 Statistical Analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA) for all three groups, and a two-tailed t-test assuming unequal variance was used for between two groups. A p-value of less than 0.05 was considered statistically significant.
12 3. RESULTS
3.1 Sperm orientation in differential fluid flow
In order to create a simple microfluidics device that can quickly sort motile and morphologically normal sperm, the flow rate with the best sperm response must be chosen. Videos taken of sperm in the channel before and after flow were analyzed to determine which fluid flow rate would be best for sorting sperm, the number of sperm that oriented and swam against the direction of the flow were counted. Before flow, sperm are oriented in random directions (Figure 5). After flow, some of the sperm orient against the source direction of flow (Figure 6).
Figure 5: Sperm position before flow in the differential fluid flow chip
13
Figure 6: Sperm position during fluid flow in the differential fluid flow chip. The flow is going from left to right
90.00% 80.00% 70.00% 60.00% 50.00% 40.00% Percent 30.00% 20.00% 10.00% 0.00% 2 4 5 6 8 10 Fluid Flow Rate in uL/min
Figure 7: Percentage of sperm at different flow rate that oriented and progressively swam against flow The percentage of sperm that orient their head towards the flow and actively swim against the flow increases from 2 µL/min (61.85% ± 7.08%) to 4 µL/min (67.14% ±
9.63%) before steadily decreasing for the 5 µL/min (41.58% ± 15.28%), 6 µL/min
14 (26.39% ± 7.59%), 8 µL/min (21.04% ± 6.89%), and 10 µL/min flow rates (10.95% ±
9.32%). A one-way ANOVA showed these results to be significantly different across all groups (p < 0.05). A two-tailed t-test assuming unequal variances was analyzed between the 2 µL/min and 4 µL/min, 2 µL/min and 5 µL/min, and the 4 µL/min and 5 µL/min flow rates. None of these three groups are significantly different from each other (p <
0.05)
3.2 Sperm velocity in differential fluid flow
Sperm motility parameters curvilinear velocity (VCL), average path velocity
(VAP) and straight-line velocity (VSL) were measured before and after flow. The paths of the sperm before and after flow can generated by the CASA plugin can be seen in
Figure 8 and Figure 9 respectively.
Figure 8: Sperm paths before flow These paths show the random linear progression of sperm that is normally exhibited in the absence of flow.
15
Figure 9: Sperm paths during flow. Flow is from left to right The paths that are curved are a result of a sperm with its head facing downstream turning to head upstream. The erratic/squiggly paths are from sperm that were trying to swim but were being pushed back by the flow. Table 1 and Table 2 show the before and after velocities of sperm in the differential fluid flow chip.
Table 1: VCL, VAP, VSL before flow in the differential fluid flow chip
Flow velocity µm/s µL/min VCL VAP VSL 2 52.31 ± 1.97 27.46 ± 1.14 23.50 ± 2.86 4 48.84 ± 1.97 26.64 ± 1.68 25.00 ± 1.27 5 59.59 ± 10.14 26.15 ± 0.63 17.62 ± 2.64 6 57.35 ± 2.69 26.67 ± 0.86 23.33 ± 1.32 8 50.22 ± 4.54 26.43 ± 2.15 24.67 ± 2.12 10 52.70 ± 4.94 26.98 ± 0.62 22.15 ± 0.62
16 Table 2: VCL, VAP, VSL after flow in the differential fluid flow chip
Flow velocity µm/s µL/min VCL VAP VSL 2 54.64 ± 0.64 26.50 ± 1.66 20.65 ± 1.72 4 52.61 ± 1.89 29.43 ± 4.58 24.45 ± 8.06 5 55.85 ± 0.84 31.62 ± 8.03 28.04 ± 10.26 6 55.26 ± 6.12 31.95 ± 1.94 28.93 ± 1.81 8 54.87 ± 5.11 31.41 ± 2.27 28.64 ± 2.27 10 49.09 ± 1.01 30.51 ± 3.74 26.35 ± 6.58
Before flow, there is no significant difference of VCL and VAP between the six flow rates (p < 0.05). 5 µL/min has a significantly lower VSL than the other five flow rates in the before flow condition (p < 0.05). After flow, there is no significant difference in
VCL, VAP and VSL between the six flow rates. A graph of the velocities can be seen in
Figure 10.
80.00
70.00
60.00 VCL Before 50.00 VCL After
m/s m/s 40.00
µ VAP Before 30.00 VAP After 20.00 VSL Before
10.00 VSL After
0.00 2 4 5 6 8 10 µL/min
Figure 10: VCL, VAP, VSL before and after flow in the differential fluid flow chip.
17 3.3 Sperm motility and retrieval analysis
50 45 40 35 30 25 Stock 20 Control 15 Million Sperm/mL Million Flow 10 5 0 PR NP IM PM + NP Motility Type
Figure 11: Concentration of million sperm/mL and type of motility observed in each experimental group. (PR) progressive motility. (NP) non-progressive motility. (IM) immotility. (PR+NP) total motility
The total stock concentration (59 ± 17x106/mL) is significantly larger than both the total control (7.0 ± 9.0x106/mL) and flow (11 ± 6x106/mL) concentrations (p < 0.05).
There is no significant difference in total concentration between the control and the flow.
The stock concentration also has a significantly larger concentration of PR (17 ±
12x106/mL), NP (6.5 ± 3x106/mL), IM (36 ± 11x106/mL) and total motile (23 ±
14x106/mL) than the control and flow groups (p < 0.05). The flow group has a significantly higher concentration of PR sperm (8.6 ± 4.5x106/mL) and total motile sperm
(9.6 ± 5.2x106/mL) than the control (3.3 ± 3x106/mL and 5.2 ± 5.7x106/mL respectively)
(p < 0.05). There is no significant difference between the control and flow in the concentration of NP (1.9 ± 3.6x106/mL and 1.0 ± 1.2x106/mL) and IM (1.8 ± 5.0x106/mL and 1.2 ± 1.5x106/mL) (p > 0.05).
18 30%
25%
20%
15% Control
10% Flow Percent Recoverd Percent
5%
0% Total M PR Type of Sperm Recoverd
Figure 12: Percent of sperm recovered from the collection chip after one hour
The percent of sperm recovered from the control and flow groups is 11.88% ±
14.94% and 18.26% ± 10.31%. The high level of variance within the two groups makes it so that there is no significant difference (p > 0.05). The flow group also has a significantly larger recovery of motile (16.24% ± 8.78%) (p < 0.05) and progressively motile (14.54% ± 7.66%) (p < 0.001) as compared to the control, whose values are 8.75%
± 9.75% and 5.54% ± 5.09% respectively.
19 100% 90% 80% 70% 60% 50% Stock
Percent 40% Control 30% Flow 20% 10% 0% PR NP IM Motility Type
Figure 13: Motility type of sperm recovered from constant fluid flow collection chip. (PR) progressive motility. (NP) non-progressive motility. (IM) immotility Sperm motility was assessed based upon motile, non-progressively motile and immotile. The average percent motile for stock, control, and experimental group is
27.66% ± 11.43%, 46.85% ± 36.60% and 82.98% ± 15.06% respectively. The percent of motile sperm found in the flow group is significantly higher than that found in both the control and stock groups (p < 0.05). The average percent of non-progressively motile sperm in the stock, control and flow groups is 12.50% ± 4.86%, 22.15% ± 26.96% and
8.03% ± 9.99% respectively. There is no significant difference in non-progressively motile sperm between the stock and the control group or in the stock and the flow group
(p < 0.05). The control group did have significantly more non-progressively motile sperm than the flow group (p < 0.05). The total motility (M+NP) was significantly different between all three groups, 40.17% ± 13.68%, 69.00% ± 40.39% and 91.02% ± 9.49% for the stock, control and flow group respectively (p < 0.05). The average percent of immotile sperm for stock, control and flow groups were 59.83% ± 13.68%, 9.95% ±
20 17.86%, and 8.98% ± 9.49%. The stock has a significantly higher amount of immotile sperm compared to both the control and flow groups (p < 0.05).
3.4 Sperm velocity
To determine swimming speed and linearity, the sperm kinematics need to be known. Table 3 below shows the average kinematics of the stock, control, and flow groups.
Table 3: Sperm velocity data for stock, control and flow after retrieval from constant fluid flow chip
VCL VAP VSL LIN WOB STOCK 48.26 ± 0.23 36.73 ± 6.02 32.40 ± 6.21 0.88 ± 0.06 0.76 ± 0.12 CONTROL 55.67 ± 4.77 44.51 ±11.38 36.64 ± 9.67 0.82 ± 0.05 0.78 ± 0.23
FLOW 64.23 ±2.62 52.09 ±4.95 46.13 ±3.69 0.89 ± 0.09 0.81 ± 0.09
Flow group is significantly larger in VCL, VAP, VSL than the control and stock.
The control VCL, VAP. VSL is also significantly larger than the stock. There is no significant difference between the linearity (describes path curvature) or the wobble (how many times the head crosses over the VAP). Figure 13 shown below indicates a clear increase in the velocities from stock to control to flow.
21 80
70
60
50
40 Stock um/sec 30 Control Flow 20
10
0 VCL VAP VSL Type of Velocity
Figure 14: VCL, VAP, VSL for stock, control, and flow. 3.5 Hyaluronic acid binding analysis
120%
100%
80%
60%
40%
Percent of Sperm Bound of Sperm Percent 20%
0% Stock Control Flow Sperm Experimental Group
Figure 15: Percent of sperm in stock, control, and flow that bound to hyaluronic acid
The percent of sperm that bound to the hyaluronic acid is 83.9% ± 6%, 89.3% ±
15% and 90.7% ± 6% for the stock, control and flow groups respectively. There is an increase in the amount of sperm that bind in the control and flow groups, but the difference is not significant.
22 3.6 Morphology analysis
10% 9% 8% 7% 6% 5% 4%
Percent Normal Percent 3% 2% 1% 0% Stock Control Flow Experimental Group
Figure 16: Percent of normal sperm present in stock, control, and flow The average values for the percent of the sperm sample that are morphologically normal is 5.11% ± 1.26%, 4.25% ± 3.81% an 5.48% ± 3.40% for the stock, control and flow groups respectively. The flow group did have a larger percent of normal, but the result is not significant when compared to the other groups.
23 4. DISCUSSION
It is important to continue to search for way to improve sperm processing procedures in order to create the best pregnancy outcomes. A good sperm processing technique will be able to get a good sample of motile and morphologically normal sperm.
It is known that sperm exhibit positive rheotaxis to various flows through a passive, physical process and is one of the natural guidance mechanisms to the egg (Eisenbach &
Giojalas, 2006; Kantsler et al., 2014; Miki & Clapham, 2013; Zhang et al., 2016).
Therefore, it can be used to process motile and morphologically normal sperm. The best flow rate determined by using the differential fluid flow chip was chosen to be 3 µL/min.
Although the 4 µL/min had a greatest percent that responded, the forward progression was not as high as that for 2 µL/min. The flow rate of human oviductal fluid is not known, but mouse studies have shown that the average oviductal fluid flow rate is 18.0 ±
1.6 µm/s (Tung, Ardon, G. Fiore, S. Suarez, & Wu, 2014). This value is approximately equal to the fluid flow rate of 3 µL/min. The paths of the sperm before flow follow a relatively straight curvilinear path (Figure 8). After flow, the sperm that were already swimming upstream continue to do so. Sperm whose heads were facing downstream from flow made in arc like trajectory to turn themselves to face into the flow (Figure 9). This shows the sperm are responding to the flow near a surface with a chiral flagellar pattern.
The sperm tale is in a spot of higher shear flow than the head is at a surface, thus, the hydrodynamic forces and steric repulsion help turn the sperm to face upstream (Ishimoto
& Gaffney, 2015; Kantsler et al., 2014). As the fluid flow rate raised above 5 µL/min,
24 majority of the sperm were being swept away. There was no significant difference between the sperm kinematics before and after flow. For the lower flow rates, this is most likely due to the sperm being able to actively swim against the flow, but the flow impeding some forward progress. The higher flow rates having higher VCL, VAP and
VSL after flow can be explained by the amount of sperm that were being swept away.
These sperm could not be edited out of the CASA software program, and thus were counted as having a sperm velocity without swimming movement. These sperm should have actually not been included in the analysis. Additionally, the sperm channel height was 76 µm, which means that the sperm could possibly swim in and out of frame, so not all sperm tracks may have been accounted for.
The average total concentration of sperm in the donated samples is well above the lower reference limit; however, the average total motility is only about 40%, the lower reference value, and the average progressive motility is below the 2.5 percentile (Cooper et al., 2010; World Health Organization, 2010). These low numbers are most likely due to the cryopreservation process and/or the thawing process. It is well known that cryopreservation has an effect on sperm motility, and it is estimated that only about 50% of cryopreserved sperm survive the process (O’Connell, McClure, & Lewis, 2002; World
Health Organization, 2010). The experiments were also all done at room temperature, which also decreases motility parameters as opposed to performing experiments at 37°C.
The average concentrations of the control and flow groups are less than the concentrations of normal sperm samples after DGC and DSW (Ghaleno et al., 2014; Ng,
Liu, & Baker, 1992; World Health Organization, 2010). These cryopreserved samples have a low motility, and if the control and flow concentration averages are compared
25 with low motility sperm sample concentrations after DGC and DSW, then the results are comparable (Ng et al., 1992). The recovery of progressively motile sperm from the flow group is slightly less than that of DGC (18-19%), but is higher than that that of swim-up
(5%) (Ng et al., 1992). The control group has a recovery of progressively motile sperm similar to that of swim-up (Ng et al., 1992). Thus, even though the concentrations of the flow and control were relatively similar, the flow was able to sort and recover sperm with higher motility. Out of the sperm that were recovered, the flow group had a significantly higher motility with more progressively motile sperm than both the stock and control.
The average motility and progressive motility of the flow is also larger than found in samples after DGC and similar to what is found in DSW (Ghaleno et al., 2014; Ng et al.,
1992). The control group also had percent motility and percent progressive motility at levels similar to DGC. The higher percent of non-progressive and immotile sperm in the control group versus the flow group can partially be explained by the flow itself. In the control, sperm just have to be able to swim down the channel, but in flow, they have to swim the channel length and against the flow. In addition, it is possible that a small volume/capacity in the device leads to contamination of dead sperm cells, and that increasing the capacity of the device can prevent contamination (Sarkar, 1984).
Sperm kinematics need to be analyzed before and after collection in order to understand how flow affects sperm velocity, swimming behavior and motility. The sperm that were analyzed after collection were limited to a 20 µm deep chamber that effectively kept them in the xy-plane. The flow group had velocities significantly higher than the control and stock group. An increase in velocity is expected because only strong, fast sperm will be able to travel the length of the channel against stream. The increase of
26 velocity in the control group over the stock group is also expected because sperm that travel down microfluidic channels have larger velocities than the semen sample (Asghar et al., 2014; Tasoglu et al., 2013). From this it is clear that sperm still maintain their increased velocity after being removed from flow. DGC separates sperm with VSL of 56
µm/s and VAP of 59 µm/s (Ng et al., 1992). DSW is able to separate sperm with a VSL of 69 µm/s and VAP of 73 µm/s (Ng et al., 1992). While DSW may be better at sorting sperm with higher VSL and VAP, the difference between DGC and the constant fluid flow chip is minimal.
Hyaluronic acid binding is a test of sperm maturity and is a good indicator of how many sperm have gone through capacitation. The cells surrounding the egg, the cumulus oophorous, are surrounded by a hyaluronic gel matrix. Only capacitated sperm cells will be able to penetrate this matrix to get to the egg, this HA binding is a good indicator of fertilizing capacity of the sperm (Gaffney et al., 2011; Huszar et al., 2006). According to
HBA! sperm hyaluronan binding assay, the upper half of sperm samples had an average of 93% ± 2.6% binding, and the lower half had an average of 70% ± 18% binding.
Compared to the top half, both the control and the flow groups are similar to the standard.
The stock sample is significantly less than that of the standard. There was no significant difference between the experimental groups in the percent of sperm that bound, suggesting that rheotaxis does not depend on sperm being capacitated. This also goes along with the finding that rheotaxis is most likely a passive and physical process
(Ishimoto & Gaffney, 2015; Kantsler et al., 2014; Zhang et al., 2016).
The average percent of normal sperm in the stock sample is just above the lower reference value. Cryopreservation can also cause morphological abnormalities, which
27 could potentially be the cause of the low percent normal sperm in these samples
(O’Connell et al., 2002). The three groups do not show any statistically significant difference between morphologically normal forms. Sperm tails without a head are still able to swim up-stream, as well as sperm with broken necks, and other morphological abnormalities. Fluid flow does not appear to be able to select morphologically normal sperm, in contrast to DGC and DSW, which are relatively efficient (Ghaleno et al., 2014;
Ng et al., 1992).
The constant fluid flow chip was able to sort sperm with greater motility characteristics than the stock sample. Compared to the most commonly used sperm processing techniques, the percent retrieval and percent motile were similar. However, the small amount that are recovered, and the even smaller amount that are morphologically normal suggests that this device would be best used for IVF or ICSI rather than IUI. The deficiency of sorting morphological sperm is something that should be worked on in the future, as currently, the determination of normal depends on the technician examining the sample and can vary from clinic to clinic. Creating a device that has a much larger volume may help prevent cells from contaminating the collection well.
Additionally, sperm enter a slither motion, increasing their forward linear progression, when traversing through the viscous fluid of the narrow oviduct. Future research can focus on using different viscosities of in the low process. Chemotaxis is known to be a short-range mechanism, but adding it into a fluid flow chip can help determine how sperm behavior changes from the long-range to short-range guidance mechanisms. With
40-50% of infertility cases being caused by male impact infertility, it is important to
28 continue research methods to sort and select healthy sperm without the risk of genetic defects.
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