In this webinar,
Laura Van Os, MSc, Embryologist at IVF-Spain, Alicante, Spain, has talked about sperm and how it is prepared for ART treatment. Laura provided standard and more advanced techniques used for selecting sperm, explained how the sperm vitrification process looks like and provided some of the main causes that can lead to male infertility or sub-fertility.
Laura started with a statement that the contribution of the male factor to fertility is huge, and 48.5 million couples are said to be infertile worldwide. The male factor accounts for around 30-40% of infertility cases nowadays, and this is if we only consider cases where there is only a male factor involved. Very often, there are both female and male factors involved. If we considered these cases as well, the percentage would go up to 50%.
Causes of male infertility
The first subject that Laura has thoroughly explained was the main causes of male fertility. Laura mentions that we can categorize them into 3 groups. They depend on the source of the problem:
These occur when there are some hormonal problems. This means that the actual sperm factory is working properly or will be able to work properly. However, the signals that should arrive from the brain to start producing sperm cells are the ones, which are missing. Within this group, we would have:
-
- hypogonadotropic, hypogonadism – this means that the brain areas responsible for producing hormones through the hypothalamus, the hypothesis, pituitary gland are not producing the hormones such as GnRH, FSH and LH. Since they fail to produce these hormones, the testicles are not receiving the proper signal, and this is why the whole system is shut down
- hyperprolactinaemia – would be a particular event where the high levels of a hormone called prolactin have an inhibitory effect on these glands in the brain, and so again, the whole system eventually shuts down because of this hormonal innovation
- pharmacological, some medicines can produce the same effect as prolactin and some substances like example anabolic steroids, which can also shut down the whole sperm production machinery
In the second group there are:
- testicular problems – these are the ones that affect the sperm factory. Within this group we would find:
- varicocele – a common, prevalent condition in the male population. Varicocele means that the veins in the testicles are alerts, and for this reason, the blood pools in the testicles. The temperature rises, which is always bad for the testicles and the sperm counts go down, and testosterone levels go down
- cryptorchidism – is quite prevalent in the population, and this refers to undescended testicles after birth. This tends to resolve on its own or with a little surgery. However, if both testicles do not descend aside, this might provoke later fertility problems during adulthood
- testicular cancer – the treatment for this cancer is chemotherapy or radiotherapy. The disease and also the treatment can impair sperm production, so it is very important to freeze sperm before such a treatment even though physicians sometimes forget about the fertility issues
- physical trauma in your testicles or surgery on your testicles can make the tissue change, and this can also impair sperm production
- genetical conditions can prevent normal sperm production, for example, Klinefelter syndrome would be such a case where a male will have an extra X chromosome, so they have an XXY karyotype, and these men physically are completely normal, they are healthy, but they are not producing sperm in a normal way
- Y chromosome microdeletions, which can lead to no sperm production or little sperm production
- Primary Ciliary Dyskinesia, Sertoly cell syndrome occur less frequently infections – could lead to no sperm production or lower sperm production
- environmental factors can also impair sperm production, for example, exposure to heat. The testicles have a lower temperature than the rest of the body. If you were exposed to them continuously to a source of heat, this would also lead to impaired sperm production
- autoimmune diseases can lead to sperm-producing antibodies which will attack your own sperm cells, so this would also be a fertility problem
The final, third group is:
- post testicular – where the testicles are producing sperm in a normal way, the signalling is working, but the body fails to expel the sperm cells and to the exterior and thus to reach a viable pregnancy naturally.
In this group, we can highlight:
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- pharmacological – there might be some medicines that impair normal sexual function
- retrograde ejaculation – a part of the sperm ends up in the bladder, so it is lost
- genetical conditions, such as Young syndrome or Congenital bilateral absence of the vas deferens, which is associated with some mutations of cystic fibrosis, but it does not mean that you need to have the disease of an absence of the ducts. People might not know that they do not have those ducts
- obstructions of the ejaculatory ducts
- vasectomy – people who underwent a vasectomy will not be able to have sperm cells in their ejaculate
- nerve injuries or spinal cord injuries, which could also prevent normal sexual function
- systemic diseases
Alterations in basic sperm analysis
Other issues mentioned by Laura are kinds of alterations that can occur and will be visible on a sperm analysis result. When a couple arrives at an IVF clinic, and they’ve been trying to have a baby unsuccessfully for more than a year, the first thing that will be done in any fertility clinic is to perform a basic sperm analysis. We’ll look at the sperm count, motility, morphology and pH, white blood cells, volume and colour.
pH and volume can be important if there is an obstruction problem.
If the volume is low and the pH is acidic, we might suspect there might be an absence of vas deferens. This is usually associated with the function of the seminal vesicle, which produces alkaline, which is the basic component of the sperm. This is why the sperm would have an acidic pH.
If we see a high quantity of white blood cells, more than one million per millilitre,
we might have a suspicion that there is an infection going on. If there is some blood in the sample, if it has a reddish colour, this could give us a hint that something is going on.
To assess the fertility itself, we will mainly look at the sperm count, motility and morphology. According to the World Health Organization (WHO), the normal standard would be to have
more than 15 million per millilitre in a sperm sample or a
total of more than 39 million sperm cells. If it is
less than this, it
would be considered Oligozoospermia.
The
percentage of sperm cells which can move from one point to another should be above 32%. If the
values are lower than this, it will be considered as
Asthenozospermia. When it comes to morphology,
4 % of normal shaped cells in the sample is okay, l
ess than this would be considered as Teratozoospermia.
The threshold values that the WHO set in their last manual, there’s a new one about to come out, but this is the one we’re currently using. If you have values that are not that close to these, perhaps you just have bad lifestyle habits, you’re smoking, drinking too much, perhaps you could lose some pounds or gain some pounds, you’re exercising too much or taking some substances which are affecting your sperm quality. Therefore, it might not mean there is a big condition going on, this might just be an unnatural deviation from normality. Sperm quality also decreases with age. A general checkup can never do any harm.
In cases where there is a c
oncentration of less than 5 million per millilitre or something like this,
we might advise performing some hormonal analysis. We would check the
testosterone levels, FSH and prolactin, and so on, and perhaps recommend the patient to have a physical exam done by a neurologist.
Basic sperm selection techniques
The techniques that we use as a routine are:
- Density gradients
- Swim-up
- Selection of sperm cells under 400x the microscope
In
density gradients, we place the
raw sample on top of 2 or 3 layers of media with different densities, and we place a tube and the centrifuge, and
during the centrifugation, the sperm cells will separate from the other components. The sperm cells will be washed to another centrifugation, and then afterwards, we put some clean media on top, and
the sperm cells which can move the fastest and strongest will swim up through the tube. We need to wait, and we will have to take an aliquot from the upper part of the tube. These are the fastest and the strongest sperm cells.
If we’re going to perform ICSI, we will place it under the microscope, and with amplification of 400 times, we will select the best looking sperm cells for fertilization. If we’re going to fertilize it through IVF, we will just adjust the concentration of the aliquot.
Alterations in advanced sperm analysis
With advanced paternal age, the rate of miscarriages increases, the rate of fertilization decreases, the quality of the embryos decreases. Therefore, some more advanced sperm analysis is required. We perform molecular tests in the routine analysis as part of advanced sperm analysis. What we test for is sperm
DNA fragmentation and apoptosis.
Sperm DNA fragmentation refers to the breaks that the genetic code of the sperm has. It is normal to a certain extent that sperm will have a little bit of fragmentation, the egg can repair it to a certain extent. However, if this fragmentation is high, it will affect embryo development. You do not want to fertilize the eggs with fragmented sperm.
It is tested through a Sperm Chromatin Dispersion Assay through flow cytometry, and
if the DNA fragmentation index is less than 15%, we would consider the DNA fragmentation to be normal. If it’s between 15 and 30%, we would consider it to be pre-pathological, and above 30%, it would be pathological.
Apoptosis is also checked through flow cytometry. Apoptosis means programmed cell death. This means that
there are sperm cells that are moving freely, they look completely alive, but in fact, they have started the process of dying. Again, you do not want to fertilize the egg with a dying sperm cell. To a certain extent, it is normal to have some apoptotic cells, and probably during basic preparation, we will remove some of them, but for some patients, this is increased, and when we have a result of more than 30%, these are markers that are also sometimes increased with paternal age.
Additional sperm selection techniques
Some additional techniques are used for sperm selection, such as:
MACS stands for
magnetic-activated cell sorting, and this
technique is used to select sperm cells with no apoptosis. The technique is based on the fact that apoptotic sperm cells, so sperm cells which are dying.
They have a lipid in their membranes that is called phosphatidylserine. this lipid happens to bind to a molecule which is called annexing 5. In the MACS technique, we have a column that is coated with micro fluids with annexing 5. When you pass the sperm cell through the column the apoptotic sperm cells will bind to it, and you will obtain a subset of sperm cells that are not apoptotic. It’s a way of selecting sperm cells that show no apoptosis for men who have apoptotic levels increased.
PICSI/ Sperm slow are based on the same principle. When we do the SCSA test, not only do we learn about the DNA fragmentation index, but we can also see if there is a higher proportion of a mature cell. This also tells us something about the integrity of the sperm cell, and it is very easy and fast to use the technique. PICSI dishes are dishes coated with hyaluronic acid, and this is based on the principle that mature sperm cells will express receptors for hyaluronic acid. When you put a droplet of sperm in the dish, the mature sperm cells will be binding to the hyaluronic acid, so they will have a very characteristic movement where the head is stuck but trying to move, whereas some sperm cells will be moving around completely freely. The sperm cells moving freely are the immature ones and those we don’t want to take.
It is the same with
Sperm slow, which is media-rich in hyaluronic acid. You will look for these sperm cells that seem to be stuck to the hyaluronic acid.
IMSI allows us to select sperm cells according to their morphology. We start preparing the sample for a regular ICSI, but we place it under a microscope with higher amplification. In an
ICSI procedure, you have 400 times amplification, while
during IMSI, you have 6 000 times amplification. You can see the sperm cells better, and you can see if they have back walls or not. We can select them a bit better according to how the sperm cells look like.
The setback of this technique is that it takes more time to perform ICSI. Also, if an embryologist is not used to collecting the sperm with such a high amplification, the embryologist might do some dexterity.
Laura, later on, talked about the
DNA compaction, the genetic code of the sperm is compacted inside the cells thanks to protamines, protamine-1 and protamine-2. In fertile men, these proteins have a ratio of 1-1, which means they have the same quantity of protamine-1 and protamine-2, but in sub-fertile men, it has been seen that this rate is altered. It might be possible that protamine transition is not working correctly, and this could explain some cases of previous fertilization problems previous failed IVF cycles.
SpermXpression tool allows us to find out if a patient is expressing a different ratio of these protamines. It’s a very easy to use kit, it is sent home to you.
- Microfluidics and DNA fragmentation
In recent years, we have seen the rise of the use of microfluidics, and these are systems that allow us to select sperm cells that have no DNA fragmentation from the raw sample. They have pairs of microwells connected with microchannels, and the
sperm cells with no DNA fragmentation have a characteristic movement, they will be able to migrate through these microchannels. You place the raw sample on one end, and you collect it after some time of culture. The sample that arrives at the other end of the channel is the one that you will use for fertilization.
We use two kinds of chips: we will use a high volume, so we’ll use 850 microliters and will retrieve around 500 microliters, this allows us to retrieve quite a high volume when we process a sperm sample, and so we will be able to use it for intrauterine insemination as well as for any ICSI or IVF procedure.
With the other one, we’re using a very little volume, so we will be able to use it with ICSI. The good thing about this is that this allows us to select for DNA fragmentation and also that it avoids centrifugation. The setbacks would be that perhaps it’s not the best system for samples with very low concentration or severe sperm samples and also not for frozen sperm samples.
Sperm sources
The main source for sperm is
ejaculation, which will be used in the majority of cases. For some men, it might be necessary to use alternative sources, such as
testicles and epididymis. We can extract the sperm cells from the testicles or the veins through aspiration with a small needle, or we can do it with a small surgery. The sperm cells are produced in the testicles, and they later mature in the epididymis in a process that lasts for 2-2.5 months.
We would need to extract the sperm that way in cases where patients underwent a vasectomy, in patients who have
difficulties collecting samples and in patients who have an
absence of vas deferens.
Some patients seem to have a better quality of sperm in the testicles, so with less DNA fragmentation. This is giving them a better prognosis than they have in the ejaculate. For some patients, during storage or maturation, the sperm gets damaged.
Fertilization
Artificial oocyte activation is indicated for patients who have had recurrent fertilization failure. This means that less than 1/3 of their mature eggs are fertilized in previous IVF cycles when sperm is introduced in an egg or when a sperm naturally fertilizes an egg. It triggers calcium ionophore oscillations, and this triggers a molecular pathway that eventually will lead to fertilization.
Some fertilization failures, although not all, maybe due to the lack of this activation of calcium. This failure to activate may be due to egg factors or sperm factors.
Calcium ionophore is the most commonly used artificial oocyte activation. It’s very easy to use protocol. You have to culture the embryos in calcium ionophore rich media for around 10-15 minutes right after ICSI. We do this with 100% of eggs in case of previous fertilization failure, so if patients come to us, and previously none of their eggs fertilized, we will use calcium for culture for all of their eggs. However, it’ll be used in 50% of eggs in cases where only around less than 1/3 of the eggs were fertilized and see if it’s helping.
Sperm vitrification
The last important aspect of Laura’s presentation was
sperm freezing, which is becoming a very common technique in IVF clinics, and as Laura said nowadays, we are working a lot with frozen sperm samples. In the past, we have put a lot of effort into improving sperm freezing protocols to try to get the best results. In a way, it happened with eggs and embryos, there was a transition where initially we were using a slow freezing technique with eggs and embryos, and slowly this was completely modified in Europe by vitrification which is ultra-rapid freezing. Most clinics are still using the slow freezing method, but nowadays the protocol of ultra freezing the sperm has been improved to avoid glass formation.
Currently, we have been successfully using this for around 2 years with really good results.
When we see a good quality sperm after thawing the samples,
afterwards we have the same results as with fresh sperm. In terms of blastocyst rate and pregnancy rate, sperm cells that were thawed after vitrification looked less damaged and less affected than the ones which were thawed after slow freezing. It might be the future in Europe’s cryopreservation of sperm samples.
