Sperm Selection Methods in the 21st century

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Denis A Vaughan1, 2 and Denny Sakkas 1.

1. Boston IVF, 130 Second Avenue, Waltham MA

2. Beth Israel Deaconess Medical Center/Harvard Medical School

Correspondence: dvaughan@bostonivf.com

Running Title: Sperm Selection Methods

Key Words: sperm selection; Intracytoplasmic sperm injection (ICSI); in vitro fertilization (IVF);
paternal; semen analysis; paternal age

Summary Sentence: This manuscript reviews the efficacy of both available and emerging sperm
selection techniques to improve the pool of sperm available for assisted reproductive
technologies.

Conference Presentation: This manuscript summarizes a presentation from the 4th Biomarker
Meeting: Personalized Reproductive Medicine; Biomarkers for the Assessment of Ovarian
Reserve, Gametes, Embryos, Endometrium and Pregnancy held on April 12th and 13th, 2018 in
Valencia, Spain.

ABSTRACT

Natural sperm selection in humans is a rigorous process resulting in the highest quality sperm

reaching, and having an opportunity to fertilize, the oocyte. Relative to other mammalian

species, the human ejaculate consists of a heterogeneous pool of sperm, varying in

characteristics such as shape, size and motility. Semen preparation in assisted reproductive

technologies (ART) has long been performed using either a simple swim up method or density

gradients. Both methodologies provide highly motile sperm populations, however neither

replicate the complex selection processes seen in nature. A number of methods have now been
developed to mimic some of the natural selection processes that exist in the female

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reproductive tract. These methods attempt to select a better individual, or population of,

spermatozoa when compared to classical methods of preparation. Of the approaches already

tested, platforms based upon sperm membrane markers, such as hyaluronan or annexin V,

have been used to either select or deselect sperm with varied success. One technology that

utilizes the size, motility and other characteristics of sperm to improve both semen analysis and

sperm selection is microfluidics. Here, we sought to review the efficacy of both available and

emerging techniques, that aim to improve the quality of the sperm pool available for use in

ART.

INTRODUCTION

The contribution of male factor to global infertility has not been well defined, although it is

thought to play a role in up to 50% of couples presenting for infertility treatment[1]. Semen

analysis has long been utilized as the primary investigative tool when assessing male factor in

an infertility setting, in spite of its poor positive predictive value[2].

It has long been known that the quality of the paternal genome can influence reproductive

outcome, particularly when examining animal research. A series of landmark studies by Bernard

Robaire’s group, showed that, in rats, paternal exposure to chemotherapeutics had adverse

effects on offspring [3, 4]. In the rat, chronic low-dose paternal exposure to the

chemotherapeutic drug, cyclophosphamide, increased pre- and post-implantation losses and

malformations[4]. Further studies in mice have also shown that ancestral environmental

exposures such as toxicants, abnormal nutrition or even stress can promote the epigenetic
transgenerational inheritance of disease and phenotypic variation [5]. In particular, epigenetic

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transgenerational inheritance of disease and phenotypic variation has been well described

using the common pesticide, vinclozolin. This pesticide has been shown to specifically affect

sperm and can induce changes in DNA methylation, non-coding RNA (ncRNA) and histone

retention. It appears to leave different epigenetic marks, which have been called epimutations,

and it is these that lead, at least in part, to the transgenerational phenotypes[6].

Semen quality and trends over time have attracted much interest, with conflicting data

published in the literature. A systematic review by Levine et al[7] reported a significant decline

in sperm counts over a 40-year period, while other studies refute this[8]. Of greater concern are

the consistent reports that sperm from older males negatively affects both the perinatal

outcomes[9] and future health of the offspring[10, 11].

Human epidemiological studies have suggested a relationship between advanced paternal age

at conception and adverse neurodevelopmental outcomes in offspring, including an increased

risk for conditions such as autism and schizophrenia[10, 11]. In a recent study, over 40 million

documented live births were examined from the Centers for Disease Control and Prevention

and the National Center for Health Statistics database, between 2007 and 2016, in the USA. The

study investigated the impact of advanced paternal age on maternal and perinatal outcomes

and found that increasing paternal age was associated with higher rates of premature birth, low

birth weight, and low Apgar scores[9]. Although many of the above referenced studies have

linked paternal inheritance patterns to advancing male age, the quality of the paternal genome

used in Assisted Reproduction Technologies (ART) could be of even greater concern[12]. Firstly,

intracytoplasmic sperm injection (ICSI) allows the fertilization of eggs with sperm that may not
be successful in vivo. Secondly, the ICSI technique itself has been thought to play a role as

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studies in non-human primates have shown that DNA decondensation was delayed at the apical

portion of the sperm head and that it was possible that this asynchronous male DNA

decondensation could be related to the persistence of the sperm acrosome and perinuclear

theca after ICSI [13]. Recent studies also posit that offspring, resulting from ART where ICSI was

used, possess significantly lower median sperm concentrations, total sperm counts and total

motile sperm counts when compared to spontaneously conceived peers[14].

Natural Sperm Selection

In vivo, fewer than 500 [15] ejaculated sperm successfully navigate the path through the

vagina, cervical canal and uterus to ultimately reach the ampulla of the fallopian tube, where

fertilization occurs. In some species sperm inseminated in the female reproductive tract months

before ovulation may still successfully fertilize an oocyte [16], however, in humans the

fertilization window is narrower and oocytes are usually fertilized within hours of ovulation[17].

Human sperm have been shown to survive in the human female reproductive tract for up to six

days before fertilization[18],

In assisted reproductive technologies (ART), if one could replicate the rigorous in vivo selection

process in vitro, one would hypothesize that the quality of the sperm used for ART procedures

may be improved, impacting the overall success of IVF. Despite this theory, we have been

largely unsuccessful in this pursuit, with sperm selection methods for the past 40 years largely

relying on a trusted examination of motility by swim-up, or forced passage through a

differential gradient[19].
It has been shown that sperm quality may be affected by evolutionary pressures. Nascimento et

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al[20] demonstrated a correlation between mating type and sperm motility: sperm from

polygamous (multi-partner) primate species swim faster and with greater force than sperm

from polygynous (single partner) primate species.

Despite the lower motility of sperm in humans, the natural monthly fecundity rate in young,

healthy, heterosexual couples is 25-30%. We know that, in an infertile population, higher sperm

concentration results in a better fertilization rate[21] however, in the setting of IVF and, in

particular, ICSI this relationship may not be as important.

Current approaches to Sperm Selection

A number of sperm selection techniques have been developed that can be broadly categorized

into methods based on sperm density, membrane surface charge, morphology, motility,

membrane integrity or nuclear integrity (Table 1). Although all may improve sample quality,

none have convincingly and consistently demonstrated an improvement in clinical outcomes

that would argue for the routine clinical application of such techniques. The two tests that have

been most utilized are both based on selection of sperm according to their membrane integrity.

The first uses magnetic activated cell sorting (MACS) with colloidal superparamagnetic

microbeads conjugated with annexin V. The sample is passed through a column containing

annexin V microbeads and sperm expressing externalized phosphatidylserine (PS) are

deselected. Using this technique, sperm remaining after de-selection have been shown to be of

higher quality based on nuclear DNA integrity and a number of other markers[22]. Clinically,

however, the technique has failed to show a consistent improvement in pregnancy rates. A

meta-analysis by Gil et al[23] found a statistically significant improvement in pregnancy rates

when compared to density centrifugation and swim up techniques, however a RCT by Romany

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et al[24] did not find any improvement in clinical outcome when utilizing MACS selected sperm.

The most widely evaluated sperm selection technique has been one that selects sperm that

bind to hyaluronic acid (HA), which is the main component of the extracellular matrix of the

cumulus oophorus. Only mature sperm, which express receptors for hyaluronic acid, can bind

to the oocyte and have the opportunity to fertilize. Therefore, intuitively it makes sense that

those sperm expressing the hyaluronic acid receptor would be of the highest quality, making it

a natural selection mechanism. Indeed, sperm which bind to hyaluronic acid are more likely to

be of normal morphology, have lower levels of DNA fragmentation and lower rates of

chromosomal aneuploidy[25, 26]. There are a variety of available commercial products which

select sperm on the basis that they express HA receptors, including PICSI® dishes, SpermCatch™

and SpermSlow™.

Theoretically, these positive selection characteristics should translate into superior fertilization

rates and, ultimately, better clinical outcomes when HA positive sperm are selected for ICSI and

fertilization. However, the data regarding HA is conflicting. Parmegiani et al. [27] reported

higher rates of fertilization (92 Vs 86%) and more high-quality embryos (36 Vs 24%) in HA

selected sperm, compared to controls. However Van Den Bergh et al. [28] did not find any

difference in fertilization rates. Regarding clinical pregnancy rates, a large multicenter

randomized control trial, performed by Worrilow et al[29], did not show a statistically

significant difference in implantation rate nor clinical pregnancy rate but did note a significant

reduction in miscarriage rate (3.3% HA-selected versus 15.1% in controls). A recent large

randomized clinical trial [30] found that PICSI did not significantly increase the term livebirth
rate compared with standard ICSI, but a significant decrease was observed in miscarriage rates

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among couples in the PICSI group. Ultimately, no difference in live birth has been reported

conclusively in the literature [31-33] and a Cochrane review in 2014[34] concluded that data

was insufficient to conclude that HA selected sperm improve outcomes in ART.

Future approaches to Sperm Selection

Microfluidics

The most rapidly developing field for sperm selection related to the field of ART is the

adaptation of microfluidics-based technologies to sperm selection and preparation. One of the

first attempts in this area was performed by Smith and Takayama who published a series of

papers showing that microfluidics was a viable approach to improving, not only sperm quality,

but laboratory efficiency [35, 36]. Microfluidics involves the study and control of fluids, ranging

from picoliters to microliters, inside micrometer-sized channels [37]. It is ideally suited to

sperm as it can be used to simulate the geometry of micro-confined regions within the female

reproductive tract, thereby allowing for biomimicry-based selection approaches that are more

representative of the in vivo selection environment as long as other characteristics, such as

temperature and fluid composition, also reflect the in vivo environment.

Several different approaches have been employed within the microfluidic sphere which have

yielded promising results. Some of these include selections by flow[38], electrophoresis[39] and

chemical gradients[40]. In addition, several microfluidic devices manipulate either hydrostatic

and/or capillary forces to aid in selection or identification of the most mobile and robust

spermatozoa[41]. For example, Takayama and Smith used two parallel laminar flow channels
where non-motile spermatozoa and debris would flow along their initial streamlines and exit

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one outlet, whereas motile spermatozoa had an opportunity to swim into a parallel stream and

exit a separate outlet. They found that both motility (98%) and morphology (22%) improved

significantly compared to density gradient preparations.[35]

Some of the potential advantages of utilizing a microfluidics platform include scalability,

increased automation and reduced turnaround times for male infertility diagnosis and

treatment. There are several commercially available sperm selection methods founded on the

principals of microfluidics such as that by DxNow, based on work done by Demirci and his

group[41].

A number of microfluidic platforms have already undergone some initial testing and preliminary

studies suggest that selection yields a progressive motile portion of male gametes with better

morphology and improved nuclear DNA integrity[42] . Parella et al also reported a small cohort

of couples undergoing ICSI after sperm separation with the microfluidics platform which

achieved a respectable ongoing pregnancy rate (5/7: 71%). The same study postulated that this

may be due to improvement in the ability to generate euploid embryos after microfluidic sperm

selection[42].

Relying solely on the motility characteristics and size to sort sperm by microfluidics will most

likely be surpassed by further innovations to these systems. Already, we have seen the addition

of chemo-attractants to further help separate sperm in a microfluidics platform[43, 44].

Addition of cumulus cells in a microfluidic chamber, to act as the chemoattractant, increased

the percentage of motile sperm from 58.5% to 82.6%[45]. To closer mimic the in vivo

environment, interactive culture systems could be developed whereby sperm are cultured with
oviductal or fallopian tube cells. In a recent study by Ferraz et al[46], bovine embryos were

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cultured in a microfluidic chamber in addition to oviductal feeder cells. The authors concluded

that cultured embryos demonstrated in vivo characteristics, as culture using “oviduct-on-a-

chip” yielded 50% of zygotes with no discernible difference in gene expression pattern to in vivo

zygotes.

Microfluidics integrated with optics

A further improvement to sperm selection by microfluidics will likely be the integration of

optics into the selection process[47].

One of the most interesting applications for integrating microfluidics and optics may be the use

of Raman Spectroscopy, which has already been shown to distinguish sperm with improved

nuclear integrity [48, 49]. Microfluidics, paired with dynamic, high-speed imaging has allowed

for more accurate observation and quantification of the dynamics of sperm movement in a

setting that mimics their natural environment, by simultaneously visualizing the processes at

small length scales and fast timescales. With on chip digital imaging approaches, within

microfluidic systems, label free 3D imaging of sperm is possible[50]. These methods employ a

digital optoelectronic sensor, such as a complementary metal-oxide semiconductor (CMOS)

chip, to digitally reconstruct the volume of the cell in 3D space by recording the interference

pattern difference between a reference light-wave and a light-wave scattered by the

specimen[50].

Segerink’s group have been at the forefront of microfluidic technology to assess sperm,

developing a microfluidic system to trap and perform electrical analysis at the single sperm
level. They characterized the sperm flagellar beat frequency by electrical impedance monitoring

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and studied the effect of various stimuli on beat frequency. A clear difference in beat frequency

and flagellar beat angle (FBA) was observed between non-hyperactivated and hyperactivated

sperm cells[51].

Indeed, just as microfluidics holds promise in human assisted reproductive technologies, the

applications in livestock reproduction are vast. In vitro creation of bovine embryos has

increased exponentially in recent years and now comprises the majority of embryos

transferred[52]. Microfluidics has not yet been adopted into commercial livestock reproduction

largely owing to the difficulties experienced when attempting large scale production of such

small scale (micron) products[53].

Future application of microfluidics

It is the opinion of these authors that research involving various microfluidics platforms appears

the most promising of the emerging sperm selection techniques. It can be applied to a number

of facets in the ART laboratory, from sperm selection automation to its inclusion for non-

invasive preimplantation genetic testing of spent media. It also appears to have great potential

and applicability in the world of livestock reproduction[54]. Possible future applications are

summarized in Figure 1. Analysis, sorting and selection may all be integrated into microfluidic

platforms. Analysis of count and motility could be performed using simple chips that could be

examined by smartphone applications[55]. Rapidly developing single cell molecular analytical

platforms may also be used in the future to gain better insights into methylation levels of

sperm, RNA and protein expression[56-58]. The greatest benefit will be observed in sorting and
selection of sperm. Microfluidic chips designed to challenge sperm size, motility and binding

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capabilities will all be able to identify sperm with individual characteristics associated with

better reproductive outcome[35, 36, 38, 59, 60]. Further improvement of the sperm population

per individual patient will also be obtained by using chemoattractants, feeder cells and/or

optics[45, 61, 62]all integrated within a single apparatus, aimed at delivering highly fertile and

reproductively competent male germ cells.

Clinical Validation of Sperm Selection

The promise of these emerging technologies (Table 1 and Figure 1), including microfluidics and

sperm selection still await numerous validation studies and large-scale clinical trials. In fact, the

first reports using microfluidics in human ART in the early 2000s by Takayama and Smith[35, 36]

have taken a long time to gain impetus. One of the overriding difficulties, in trying to prove that

sperm selection strategies in ART will be successful, is that success will largely be based on

improved live birth rates. The relative maternal contribution to a successful live birth far

outweighs that of the paternal contribution and complicates the ability to prove that sperm

selection can have a dramatic improvement[63]. It is not however impossible to show and we

are already seeing paternal influences gaining greater notoriety in their impact on live birth

rates both after natural conception and IVF[9].

Conclusion

Despite the advances in ART over the past 40 years, IVF still remains a relatively inefficient

process. We have previously demonstrated that the chance of a live birth per oocyte retrieved
is only about 5%[64]. Similarly, the sperm qualities which make in vivo fertilization successful

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remain elusive despite extensive research efforts. Ideally, a sperm selection method that would

provide each egg with a population of sperm or a single sperm that is better selected for

reproductive potential will ultimately improve live birth outcomes. This simple goal in IVF is

needed as many of the naturally occurring sperm selection methodologies that evolution has

put in place are bypassed[63]. In particular for ICSI, which now comprises the majority of IVF

cycles worldwide[65], few in vivo barriers remain for sperm selection. It is human nature to

embrace hope in the absence of reliable alternatives, however it is imperative that rigorous

clinical trials be performed from the outset, before these technologies are routinely

implemented in the laboratory.

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 Downloaded from https://academic.oup.com/biolreprod/advance-article-abstract/doi/10.1093/biolre/ioz032/5364542 by Macquarie University user on 26 February 2019
Figure 1. Analysis, sorting and selection of sperm using microfluidics and other associated

technologies for the future. Abnormal (red), normal (orange) and highly selected (green) sperm

are shown. Sperm analysis will be performed “point of care” or using platforms providing

relevant molecular characteristics of sperm to individualize patient care. Sperm sorting and

selection will be improved by gross selection in microfluidic chambers based on size (depicted

by circles and cylinders and specific motility characteristics. Further selection will be performed

with the addition of chemoattractants or antibodies (**) aimed at specific markers shown to

improve reproductive outcomes. Finally, integrated optics may non-invasively examine nuclear

and other cellular characteristics to further improve selection in cases of ICSI. References to

relevant technologies are in parentheses.

Table 1. Summary of sperm analysis and selection modalities currently being adopted in clinical

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research and trial settings. References of studies relating to clinical utility are provided.

Selection Method Sperm Sperm References

Mechanism analysis Selection
Density Gradient
Separation
  [19]

Surface charge Electrophoresis

Zeta Method
  [66]

Morphological Motile sperm

characteristics organelle [67]
morphology  
examination
(MSOME) [68]

Intracytoplasmic  
sperm injection [69]
(ICSI)

Intracytoplasmic
morphologically  
selected sperm
(IMSI)
Motility
characteristics
Microfluidics   [35, 36, 41, 42]

Membrane
Integrity
Hypo-osmotic
swelling test
  [70]

(HOST)
Membrane
proteins
Hyaluronic acid
(HA)
  [25-34]

[23, 24]
Magnetic activated
cell sorting  
(MACS)
DNA chromatin
integrity
DNA
fragmentation
  [71]