This webinar reviews the impact of sperm quality on assisted conception and discusses how sperm populations and individual sperm can be selected to optimize outcomes.

Webinar Author

Webinar Transcript: Improving Outcomes Through Better Handling, Preparation and Selection of Sperm

In this webinar, Dave Morroll, PhD, Director of Clinical Support, discusses how we might seek to improve the outcome of ART treatments by focusing on the male partner and, more specifically, on the handling and preparation of sperm, together with sperm selection.

Recorded: 2nd June 2020
Duration: 30 min

Overview:

  1. Introduction
  2. Sperm DNA Damage – Causes and Treatments
  3. Laboratory Effects on Sperm DNA Fragmentation – Positive and Negative
  4. Sperm Preparation
  5. Sperm Selection
  6. Summary

Introduction

We should all know by now that the male contribution to sub-fertility is significant and possibly increasing, yet we have perhaps treated the sperm side of a treatment cycle poorly. Certainly, I believe that there’s generally insufficient attention on the way we handle and prepare the sperm samples.

Let’s remind ourselves of just what has specialized in fascinating cell the mature sperm is. In the process of spermiogenesis, when a round spermatid metamorphosizes into a motile DNA delivery system largely devoid of cytoplasm, the cell’s nucleus is remodelled and that reflects the function of the sperm.

Looking more closely, the chromatin is repackaged and complexed with protamines rather than histones. The doughnut loop model allows the genetic material to become densely packed into a small nucleus forming the large part of the sperm head and this packaging protects the DNA.

Mature sperm are released and stored in the epididymis before ejaculation.

The sperm produced can be counted, their shape assessed, and their motility evaluated. The WHO 2010 Edition defines how many sperm should be present and what proportion should be swimming and have a normal shape. These reference values indicate a lower level of the range of values seen in men who became fathers, and this is taken as a guide to assessing the chances of any individual man having problems conceiving. But the semen analysis is purely descriptive; it describes the ejaculate and the prognostic value of this has been repeatedly questioned. There are several factors of play here, but we’ll look more closely at sperm DNA fragmentation.

Sperm DNA Damage – Causes and Treatments

How is sperm DNA fragmentation caused and what can we do about it?

As already mentioned, the standard semen analysis is ubiquitous but not particularly accurate and so, for the most part, is considered only a rough guide for differentiating ‘probably fertile’ from ‘probably sub-fertile’ groups of patients.

Additionally, tests may help to offer up more useful information and improve our ability to determine the true fertility potential – not only fertilization potential and but ongoing competence of the embryo generated so it can develop into a healthy pregnancy.

As we’ve already mentioned, the health or integrity of the sperm’s DNA would seem a logical marker for fertility potential and data supports this. However, DNA fragmentation tests may measure differing aspects of DNA damage under different conditions. The tests are not standardized, with varying thresholds used, and these add up to make the clinical effectiveness of testing somewhat debatable.

Let’s look briefly at the various tests. A very interesting review by Ribas Maynou J. & Benet J. last year gave a far more detailed and eloquent description of the different tests and types of DNA damage they measure than I can manage in the time available. But the key message is that TUNEL, SCSA, Halo and Comet tests clearly do not detect the same types of fragmentation.

More importantly, perhaps, these various types of fragmentation seemingly have some different effects on the reproductive processes. DNA damage may occur in the testes or in the genital tract and be caused by a range of external and internal factors.

It’s also important to stress that DNA damage may occur after ejaculation and the procedures we have in the laboratory can either protect or expose the sperm cells to further DNA damage.

Another way of performing an enhanced assessment of sperm qualities is to determine the percentage that can bind to hyaluronan. Remember that hyaluronan is a major component of the extracellular matrix of the cumulus mass and so a sperm’s ability to bind with it is clearly of biological importance.

Work by Huszar et al showed that this ability is associated with plasma membrane remodelling, cytoplasmic extrusion and attainment of nuclear maturity during spermiogenesis. Subsequent studies have also demonstrated that hyaluronan binding tends to deselect sperm with DNA damage which, logically, would be a useful function for the cumulus to perform. The hyaluronan binding assay, which evaluates the proportion of sperm in an ejaculate that expresses the receptors to hyaluronan can be easily combined with a standard semen analysis and has been reported to correlate with treatment outcomes including fertilization, cleavage and pregnancy rate. The same principle can be used not only to assess the quality of a sperm sample but also select individual sperm for injection of ICSI.

So, we now have a picture of sperm quality, in part, being related to the spermiogenic processes, repackaging of chromatin and to damage or fragmentation of the DNA. But what impact does this have on the reproductive process and outcomes of treatment?

I was co-author on a practice guideline for the British Fertility Society in 2013 when we reviewed the evidence for measures of sperm quality, including sperm DNA fragmentation in both natural and assisted conception. We found a number of methods, thresholds and reported impacts which subsequent reviews have also found.

Whilst there is evidence of an effect on fertilization, embryo development (especially after the point of paternal DNA activation) and chance of pregnancy etc., the overall conclusion was that it was the association between DNA damage and miscarriage or pregnancy loss that was the most compelling. We’ll see later how deselection of DNA damaged sperm may reduce the risk of miscarriage.

Accepting that DNA damage in sperm is a problem then we should examine what we can do to address it. Let’s first look at the impact of DNA damage in sperm and what the man himself can do.

Sperm can be sensitive to what the man consumes; general diet, alcohol, caffeine, recreational drugs, and medicines can all affect sperm quality and DNA integrity. Similarly, exposure to environmental pollutants may damage sperm, which may be especially relevant to specific jobs. More positively, there is some evidence that oral antioxidants can help alleviate DNA damage. Exposing the testes to high temperatures certainly does not help, so controlling scrotal temperature is important and this is mirrored by controlling temperature during processes in our labs. And, amongst a whole range of reasons why smoking is a bad habit, the damage to the DNA integrity of the man’s sperm is another. Of course, this has implications for conception, miscarriage and maybe even for the offspring.

Finally, a drastic measure (and one I am absolutely not advocating, by the way) is to find a young partner. This is a rather flippant way of highlighting the fact that we know sperm DNA damage can be corrected by the oocyte around the time of fertilization, but the efficacy of this is very much a function of the quality of the oocyte. Since oocyte quality is also age-dependent, men with poor sperm DNA may have better outcomes with younger partners. (In the interest of fairness, I should also point out that this works both ways – women with poorer quality oocytes might have better outcomes with men with better sperm quality and older men have higher DNA damage on the whole).

Laboratory Effects on Sperm DNA Fragmentation – Positive and Negative

That’s looked at what the causes and remedies are in the man himself; now I’ll turn my attention to the lab effect and there are a number of factors we need to consider here.

1.) Time; duration of abstinence, interval between sample production and preparation, and time between preparation and use.

2.) Temperature: we’ve spoken about scrotal temperature but temperature during handling and storage of samples also has an impact.

3.) Sperm preparation: which should select a population of sperm that is genetically healthier.

4.) And finally, there are techniques to select those sperm used in treatment at the individual level.

Now let’s look at these lab effects in a little more detail.

We’re perhaps used to suggesting that a man abstains from any sexual activity for around two to five days before providing a sample. We now know, however, that longer abstinence is associated with higher levels of DNA damage.

Conversely, reducing abstinence can reduce the proportion of damaged sperm as shown in this paper by Gosalvez and colleagues at the University of Madrid. They reported that ejaculation every 24 hours resulted in reduced DNA fragmentation, with the final three-hour abstinence giving even better results.

Their study used volunteers with normal semen parameters so I would be concerned about such short abstinence for clinical work – especially in those men with low sperm numbers – but the general principle that shorter abstinence may help reduce the impact of DNA fragmentation is worthy of consideration.

Once we have the sample, we should prepare it as soon as is practicable. Matsuura and colleagues reported that DNA fragmentation increased spontaneously over time in the ejaculate and this was accelerated at 37 degrees.

Sperm Preparation

Let’s move on to look at sperm preparation techniques. As I’ve already mentioned, sperm preparation should isolate a motile, morphologically normal population of sperm but this should also have lower rates of DNA fragmentation. But do the various methods employed give different effects?

There’s a range of sperm preparation techniques available and I do not have time in this presentation to address all of them so I’ve focused on the three simplest and most widely-used; the washing concentration, the swim-up and the density gradient.

Diluting semen with culture media and centrifugation is, of course, by far the simplest method but because it compacts the healthy sperm into a pellet with non-sperm cells and poor-quality, and dead sperm, there’s a real risk of reactive oxygen species damaging healthy sperm. This, together with the fact that there is no selective process to refine the population of sperm, means that the washing concentration should be a last resort and is not generally recommended.

The swim-up, and other sperm migration techniques rely on motility so are best for samples of generally high quality. As with the washing concentration, it’s generally not recommended that the sperm samples be washed, then a swim-up performed, but rather medium layered over whole semen and the collected sperm washed.

Density gradients may optimize yields and are preferred when handling samples from patients with potentially infectious diseases. Though many publications show that swim-up and density gradient centrifugation are equally efficient in removing sperm cells with compromised DNA, there seems to be a balance towards density gradients being the most efficient method. Enciso et al in 2011 suggested they’re more efficient in removing sperm with single strand breaks.

According to a Björndahl and colleagues in 2005, swim up and density gradient centrifugation also produced different levels of contamination with seminal components in the final sperm preparation. They used the prostatic secretion zinc as a marker of soluble seminal components and demonstrated a time-dependent diffusion of zinc from semen into the overlaying swim-up medium, and the final zinc concentration in swim up preparations was greater than that after density gradient centrifugation.

The WHO summarizes the choice simply as the direct swim-up being ideal for normal zoospermic examples, and density gradients being suited especially to sub-optimal samples.

Ultimately, the choice of method appeared not to impact outcomes according to Boomsma and colleagues. That is, perhaps, unsurprising as – apart from yields – both of these commonly used techniques perform similarly, especially considering their ability to deselect DNA-damaged sperm. This is observed across a whole range of other methods such as this report from Thomas Ebner, which is just one example amongst many.

Interestingly, this deselection is not so effective in frozen thawed sperm, at least when using a swim-up technique according to this 2011 study by Meseguer M, et al.

Although I’ve suggested that all sperm preparation methods are effective, we should perhaps also look at how we handle the sperm.

The first important point to highlight is that sperm are not oocytes or embryos. This may seem blindingly obvious, and yet we’ve traditionally held sperm in conditions more suited to the oocyte. For example, some sperm-washing media are buffered to pH 7.3 – 7.4 or so, and yet we know that a higher pH promotes sperm motility as Achikanu C. and colleagues beautifully demonstrated in 2018. Nadia de Rosa and Matt Tomlinson reported motility in sperm-washing media of varying composition and highlighted that motility was dependent on pH and concentration of bicarbonate, but the impact of these two factors was independent of one another.

This is a really important consideration when designing specialized andrology media as we must support the selection of a healthy population of competent sperm that can capacitate and undergo the acrosome reaction in order to successfully fertilize the oocyte. The addition of suitable antioxidants will also protect the sperm against damage.

As highlighted by Achikanu, de Rosa and others, we can build andrology media foremost based on the needs of the sperm. Elements might include:

– Albumin, which is found in high concentrations in the oviduct. As well as antioxidant effects which protect the sperm from DNA damage, it’s well known that albumin support sperm motility and is needed to capacitation.

– Bicarbonate is also found in high concentration in seminal plasma in the oviduct. Bicarbonate and albumin in combination prepare the sperm membrane for the acrosome reaction.

– Alkaline pH also mimics the sperms natural environment. In combination with higher levels of bicarbonate, this drives out protons which elevates the intracellular pH, increases intracellular calcium levels and that, in turn, induces hyper activation.

Using ORIGIO’s Gradient Series as an example, how does this translate him to the media? There is:

– Higher pH, controlled using the zwitterion HEPES

– Additional albumin in the sperm washing medium at 10 milligrams (rather than the usual 5 milligrams).

– A range of antioxidants (in addition to the HSA which, as already mentioned, itself has antioxidant properties).

For poor samples we can, of course, artificially stimulate motility (this can be particularly useful in testicular sperm, for example). A range of compounds can be used including theophylline, caffeine, myoinositol and 2-deoxyadenosine, though pentoxifylline is perhaps the most widely used.

The ability to select good, motile sperm in testicular samples is of special interest in the DNA damage story since it’s been reported that using testicular sperm alleviates the negative effect of DNA damage in men with persistent high levels of DNA fragmentation in their ejaculate. For example, Greco and co-workers reported no improvement in embryological markers but pregnancy and implantation rates were significantly improved. This was echoed by Esteves and colleagues in 2017 although a very recent review by Jarvi questioned the strength of the evidence of benefit.

Sperm Selection

We’ve looked at the selection of a population of sperm via various preparation techniques and the possible impact of handling of storage. Now let’s look at the selection of individual sperm.

This is important because for ICSI we’ve usually chosen the sperm for injection based on shape, size, and motility; in other words, it’s a visual check. But we know that morphologically normal sperm in samples from men with oligoasthenoteratozoospermia (in other words those men that most need ICSI) have elevated rates of aneuploidy and DNA damage compared with normal sperm in normozoospermic cases. Put simply, the visual check is not very robust.

If we choose the wrong sperm (even if it looks normal) we may compromise the chance of a successful cycle and the effect may not just be on the chance of successful fertilization; compromised DNA integrity can impact embryo development, implantation or miscarriage risk. So, what can we do to augment our sperm selection?

One approach is IMSI; using enhanced optics to examine the ultrastructure of sperm and looking for the presence of vacuoles. Setti and colleagues suggested in 2010 that IMSI doesn’t improve fertilization but, as discussed in the last slide, may improve post-fertilization events, leading to higher pregnancy rates and lower chance of miscarriage. A later Cochrane review by Teixeira in 2013 reported that any evidence of benefit of IMSI was of low quality and that the RCTs didn’t really support the clinical use of IMSI. In the same year, however, Setti’s group published a second review again supporting the possible role of IMSI in overcoming later paternal effects.

The other option for individual sperm selection is hyaluronan binding. I discussed this at the beginning of the presentation and highlighted its association with sperm maturity and DNA integrity. I described the hyaluronan and binding assay and the same principle can be used to choose a single sperm before injection into the oocyte. This can be done in two formats; viscous hydrogel containing hyaluronan – Sperm Slow – or a dish with a dried plaque of hyaluronan which is rehydrated and used – the PICSI dish.

Once the plaque of the PICSI dish is rehydrated, sperm can be added and allowed to swim across the plaque. Those expressing receptors to hyaluronan may bind to the surface in the circular plaque and then be chosen, aspirated into an ICSI pipette and used for the injection.

In Sperm Slow, sperm are introduced to a drop of the hyaluronan-rich medium and, if expressing receptors, get trapped in the hydrogel and then lose progressive motility and shudder on the spot (and these, again, can be selected and used in the injection process).

In the US, Katie Worrilow and associates performed a large, multi-centre, randomized controlled study of standard ICSI, compared with ICSI using a PICSI dish, to select the sperm and, interestingly, in cycles where the man exhibited low hyaluronan-binding (as demonstrated by a low HBA score of under 65%), the miscarriage rate was significantly decreased when individual sperm for injection were selected, based on their ability to bind hyaluronan.

A much larger study in the UK, directed by David Miller of the University of Leeds showed a significant reduction in chance of miscarriage when using a PICSI dish for sperm selection and this effect was particularly seen in older women. The live birth rate was slightly higher (but not statistically significant) but for individual couples, lowering the risk of miscarriage is extremely relevant. It should be noted that the study was performed on a broad population and sperm selection may not be relevant for all couples.

If we take all of the studies together it’s clear that hyaluronan-binding tends to de-select DNA-damaged sperm, and we know sperm that is DNA-damaged is linked to the risk of miscarriage, as we discussed earlier.

So, it seems an entirely cohesive argument that hyaluronan-binding might reduce miscarriage risk as demonstrated by the Miller and Worrilow multi-centre studies.

Summary

– Sperm nucleus contains densely packed DNA but this can still be prone to damage.

– When it is damaged, that damage can negatively affect reproductive processes leading to poorer outcomes.

– There’s a range of causes of this DNA damage but sperm preparation effectively reduces the proportion of damaged sperm.

– But all the good work can easily be undone by poor handling and storage in the laboratory.

– Part of this is being cognizant and how optimal conditions for sperm are not the same as for eggs, with high pH and bicarbonate levels being particularly positive.

– IMSI may help late paternal effects.

– Hyaluronan-binding techniques deselect DNA-damaged sperm and can help reduce the risk of a miscarriage.

About the Presenter: David Morroll PhD

David has worked as a Clinical Embryologist since 1986, training in Manchester, where he also completed his doctorate studies. He has since managed laboratories in a number of UK IVF units including those in Nottingham, London and Leeds, and consulted to a number of clinics overseas. He joined ORIGIO as Director of Embryology in November 2011.

He has previously served as Chair of the UK Association of Clinical Embryologists (ACE) and Association of Biomedical Andrologists (ABA), and was involved with several working groups, notably the HFEA Expert Group on Multiple Births after IVF.

David has a keen interest in quality management, including ISO certification and External Quality Assurance. He has also consulted to IVF units in Malta, India, the Philippines, and Zimbabwe. In January 2019, David was appointed to the role of Director of Clinical Support, focusing on post-marketing clinical studies, QA/complaints, and laboratory audits.

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