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Advanced sperm functional testing

Advanced Sperm Functional Testing

Figure 1: Sperm DNA Fragmentation testing (SDF). TUNEL assay, Promega.

Figure 2: Sperm Chromatin condensation analysis using the Aniline Blue stain.

Figure 3: Seminal Oxidative Reductive Potential testing system. Mioxsys system.

Sperm DNA Fragmentation and Oxidative Reductive Potential Testing

Male infertility is a relatively common medical condition affecting up to 12% of men globally. The Centres for Disease Control and Prevention (CDC) report a 9.4% infertility rate among men in the USA. Male partners are found to be solely responsible for 20–30% of infertility cases and contribute to approximately 50% of cases overall. Many clinicians rely on conventional semen analyses as a surrogate measure of a man’s ability to father a child. However, this approach seems to be an oversimplification of the assessment of male fertility potential due to large intra- and inter-individual variations in conventional semen parameters and is unable to precisely predict the likelihood of pregnancy. Advanced tests of sperm function have been proposed as alternative methods that can enhance the diagnostic accuracy of male infertility particularly in cases of unexplained infertility, one or more abnormal semen parameters, recurrent pregnancy loss or failure of intrauterine insemination.

At Cape Fertility we offer advanced sperm functional testing which includes the assessment of Sperm DNA Fragmentation, Sperm Chromatin Condensation and seminal Oxidative- Reductive Stress testing.

The integrity of genetic material in the sperm is crucial for successful fertilisation and normal embryo development. Sperm DNA fragmentation (SDF) is a term used to describe the genetic material within the sperm, which could impact the fertility potential of the male, IVF/ICSI outcomes and has been shown to be associated with miscarriage. A conventional semen analysis cannot assess the sperm at the molecular level and, as result aid the detection of DNA fragmentation. The ORP test is novel in the area of infertility. The MiOXSYS system provides an estimation of the static oxidation-reduction potential (sORP). It represents the integrated measure of the existing balance between total oxidants and reductants in a biological system.

Studies in the literature have shown that:

Abnormal Sperm DNA fragmentation and oxidative stress levels affects blastocyst development
The higher the DNA fragmentation levels, The higher chances of failed assisted conception treatment and miscarriage
Sperm DNA fragmentation is higher in sub-fertile men with abnormal sperm parameters
Men with normal sperm parameters are also found to have high sperm DNA fragmentation and abnormal levels of oxidative stress

Advantages of the Sperm functional testing

Provides a comprehensive analysis of sperm DNA integrity, Sperm chromatin packaging and seminal oxidative reductive potential that may help to identify men who are at risk of subfertility
Provides information that helps in the clinical diagnosis, management and treatment of male fertility
Provides prognostic value in assessing the outcome of assisted conception treatment

High rates of sperm DNA fragmentation and pregnancy potential

Normal, healthy pregnancies do occur in couples where the male partner has high percentage of sperm with fragmented DNA, although the chances are significantly reduced, as the percentage of sperm bearing low levels of DNA fragmentation is much lower
Embryos derived from sperm with highly fragmented DNA have poor prognosis
DNA fragmentation could result in initiation of apoptosis (natural cellular death) and mutations resulting in blastocyst arrest, miscarriage and abnormalities in the offspring
Spermatozoa with high DNA fragmentation fertilising younger oocytes than older oocytes carry a better prognosis of successful pregnancy, as the are much more efficient at DNA repair of defective sperm

Causes of Sperm DNA Fragmentation

In men, the major contributing factor for sperm DNA fragmentation is oxidative stress, which can be associated with one or more of the following:

Infection
Pyrexia
Elevated testicular temperature
Recreational drugs
Smoking
Alcohol
Stress
Diet
Environmental and occupational pollutants
Advanced chronological age
Varicocoele

Indications for men who may benefit from sperm functional testing

Unexplained infertility
Arrested embryo development
Poor blastocyst development
Multiple failed IVF/ICSI treatments
Recurrent miscarriage
Advanced chronological age
Varicocoele
Poor semen parameters
Exposure to harmful substances

Treatment of abnormal sperm functional testing

It depends essentially on the cause. If the damage is caused by free radicals, a change in lifestyle and diet designed to protect against oxidative stress may help reduce the levels of DNA fragmentation. Other treatment options include:

Antibiotics in the co-existence of an infection
Life style changes – drugs, smoking and occupation
Diet – fresh foods, particularly those containing antioxidants and vitamin C&E
Varicocoele surgery
Testicular aspiration of sperm (DNA damage occurs at the post-testicular level, hence testicular sperm may have a better DNA integrity than ejaculated sperm)
ICSI rather than IVF

Initiatives to reduce the levels of fragmentation or oxidative stress can be assessed by undertaking a second test three months later.

In Vitro Fertilisation (IVF) – What Happens in the Laboratory Day 0 to Day 6

By Embryologists – Gloria Raidani and Hughlene Baker

The goal of every IVF journey is to succeed in having one healthy baby. Everyone’s’ IVF journey is a unique and different experience.

What happens in the IVF laboratory is very important and requires the embryologist to collect as much information as possible about your embryos in order to select the best embryos for your Embryo transfer and freezing.

Herewith a detailed explanation as to what happens in the laboratory from Day 0 up till Day 6.

DAY 0

The egg retrieval day is what the embryologists call Day 0. Egg retrieval is performed using transvaginal ultrasound guide in a theatre under sedation by your doctor.

Screening and collection of the oocytes is performed by the embryologist. At this time the retrieved eggs are counted and assessed for maturity/quality.

The sperm that will be used to inseminate the eggs is received from your partner in the morning and washed (processed) to make it ready for insemination.

Should the sperm that you wish to use be frozen, the straws or vials containing the sperm will also be thawed and processed in the same way.

Our laboratory predominantly performs Intracytoplasmic Sperm Injection (ICSI). This is a procedure where the sperm is injected individually into a mature egg.

This procedure requires a high powered microscope and micromanipulation skills using specialised needles by the embryologist. The ICSI procedure is performed at least 3 to 6 hours after the eggs have been aspirated.

The eggs are individually assessed to make sure that they are at the right maturity stage for the ICSI procedure to take place. Only mature eggs can be injected with sperm since immature eggs will not fertilise.

After ICSI the oocytes are placed in a specially designed culture medium covered with oil, that contains proteins, amino acids and enzymes that mimic the fluid in the fallopian tubes needed for embryo development. Should you have requested to have your eggs grown in our time-lapse Incubator, they will be placed in the Embryoscope(c).

DAY 1 (LABORATORY CHECK)

The injected eggs are evaluated for fertilisation. This process takes place 16 – 20 hours after ICSI.The presence of 2 pronuclei (two discs) inside the egg – one from the egg and one from the sperm- indicates normal fertilisation and the fertilised eggs are called zygotes.

At times, the embryologist may not see the presence of pronuclei in the eggs and that does not always mean that the egg has not fertilised. After fertilisation check, the dish containing the eggs is placed back into the incubator.

DAY 1 (PATIENT REPORT FROM LAB)

You will receive an update:

* Confirming the number of oocytes retrieved the previous day

* The number of mature eggs that were injected with sperm

* The number of eggs showing normal fertilisation

* Notification of when you will receive the next update for embryo development

DAY 2 (NO LABORATORY EVALUATION)

The fertilised eggs start to divide in to cells and become embryos.

Most of the embryos will have between 2 to 4 cells.

Embryologists are very protective over the growing embryos and will not expose them to temperature variations as this may have an effect on embryo development.

DAY 2 (NO REPORT)

You will not receive an update on Day 2 as the embryologists do not perform embryo evaluations on this day.

DAY 3 (CLEAVAGE STAGE)

Embryos at this stage have developed further and vary from 6 cells, 8 cells, Multicell embryos and compacting embryos.

On this day the Embryologist will transfer the divided embryos into a new petri dish with media supporting further development of the embryo.

The embryos are graded and images of them captured for further evaluation.

Day 3 (PATIENT REPORT FROM LAB) You will receive:

An update on the developing embryos which states how far the embryos are growing

– A confirmation time of when your embryo transfer will take place on Day5. Our laboratory performs more Day 5 than Day 3 embryotransfers.

A day 3 embryo transfer would depend on the number and quality of the embryos that you have.

DAY 4 (NO LABORATORY EVALUATION)

No evaluation of embryos occurs at the Morula stage because the embryos resemble a cell mass that has no distinct features that are easy to grade.

This stage is the transition between the cleavage stage embryo and a blastocyst on day 5.

DAY 4 (NO REPORT)

You will not receive an update on Day 4 as the embryologists do not perform embryo evaluations on this day.

DAY 5 (BLASTOCYST STAGE)

Most embryos will have reached this stage, the Blastocyst stage. The embryo will have increased in size and be more developed. The Blastocyst has certain structures which the embryologist expect to see while performing an assessment:

The Inner cell Mass (ICM) which is the foetal component and the trophectoderm cells (TE ) which is the placental component.

The blastocyst will be graded according to the following factors:

* the size of the embryo in terms of expansion
* appearance of the ICM
* appearance of the TE cells

The embryos are loaded by the embryologist into a catheter and transferred into the uterine cavity by the doctor. The remaining good quality blastocysts are frozen.

Should your cycle be a freeze all cycle where no Embryo transfer takes place, grading of the embryos will be performed by the Embryologists and the best quality embryos will be frozen.

Any remaining viable embryos that are not fully developed are cultured further for possible freezing on Day 6.

DAY 5 (DAY OF EMBRYO TRANSFER)

Images of the embryos are taken on Day 5 as well and sent to your doctor for discussion before the embryo transfer.

Once you and your doctor have discussed the embryo results and agreed to how many embryos are for transfer, the Embryologist will select the best embryo/s .

The embryo transfer procedure does not require any anaesthesia.

DAY 6 (FINAL LAB CHECK)

The remaining embryos are assessed and graded and the suitable embryos will be frozen.

The laboratory will only freeze good quality blastocysts as the lower grade embryos will give a poor success rate at thawing.

DAY 6 (FINAL PATIENT LAB REPORT)

A final e-mail will be sent to you indicating:- Number and grade of the embryo/s transferred

– Number and grade of the remaining embryos frozen, if any of the remaining embryos were of good quality to freeze

Warm wishes for your pregnancy test 🙂

GENETIC SCREENING

Should your IVF process be for genetic testing, the embryos will be cultured until day 5 and day 6 where they will be assessed to see if they have formed a blastocyst.

To perform genetic testing, several cells from the trophectoderm layer are extracted. This procedure is called embryo biopsy. The extracted cells are sent to a genetics lab for analysis.

Should you be having a fresh embryo transfer, the biopsy will be performed on day 5 and the cells sent away for testing immediately so as to get the results on time for a day 6 embryo transfer.

Most cases susbsequently have a freeze all cycle where all the biopsied embryos are frozen. After the results are received a few days later, you will have the normal embryo/s thawed and transferred.

Every clinic has a different system that they use to grade embryos.

We have an adapted grading system that you may request should you wish to study what the grades of your blastocyst mean.

Environment and Infertility

Definition

Xenoestrogens are substances in the environment that mimic the action of estrogen. Xeno comes form the Greek word Xeno what means foreign and estrogen is one of the two female hormones. The other female hormone is progesterone. So Xenoestrogens are substances that have the same effect on the human body as one of the female hormones. Some examples are BPA, Dioxine and Pyrethoids.

There has been a great concern that Xenoestrogens have a significant effect on infertility specifically in endometriosis and male infertility.

BPA

One of the most common Xenoestrogens is BPA (Bisphenol A). BPA is used in plastic to make it stronger. It is used mainly in plastic bottles, trays, on the inside of cans, and is also found on receipts and other rolled paper therefore everyone is exposed.

We can avoid exposure to BPA by not using plastic bottles and cans. Using glass, porcelain, and stainless steel are better options and are BPA free. Heating plastics releases more BPA or BPA substitutes so if you are storing food or drinks in plastic containers, put it on a glass or porcelain plate before microwaving or adding boiling water. Don’t leave reusable water bottles in the car in the summer where it gets baked.

Dioxin

Dioxin (another famous xenoestrogen) is a toxic byproduct of industrial and consumer processes. The main sources of dioxins are waste incineration. Dioxins are subsequently released into the environment, contaminating fields and crops. Livestock eat the crops and the dioxin enters their tissue. Humans then eat the contaminated animal products and become exposed to the Dioxin. There is strong suggestion that Dioxin is a major contributor to the increase in endometriosis.

Pyrethoid pesticides

A south African study looked at the effect of Pyretoids (a pesticide used in many insecticides) The results are suggestive of decreased ovarian reserve associated with exposure to pyrethoid pesticides. This means that these Pyretoids have a significant effect on egg quality and quantity in women.

Air Pollution

Air pollution is another important factor. A study has looked at women who lived close to high ways compared to women who lived in more rural areas. These women were 21 percent more likely to report secondary infertility than women who lived farther away, and that increase was statistically significant, researchers report in the journal Human Reproduction (this is a very important
fertility Journal)

Sperm Quality

The testis is one of the most sensitive organs of the human body and male reproductive system malfunction seems to be a good sensitive marker of environmental hazards. Over the past 50 years, human sperm concentration decreased drastically from 113 to 61 million/mL, which represents almost 50% decrease. Evidence showed that human semen quality has been also declining during the last decades, in particular in the United States and Europe. For example it has been estimated that the sperm count in American males is decreasing by 1.5% each year. A study in China found that city-dwelling men had higher levels of abnormally shaped sperm than their rural counterparts. Their sperm also swam more slowly. These dramatic effects on sperm count are very important and should be treated with great caution. There are different causes for this problem and we have definitely not found all the culprits. However the following have been shown to be contributors to the decline in sperm counts:

Air pollution
Heavy metals
Xenoestrogens
Increase in lifestyle diseases like diabetes, obesity and hypertension.

Smoking

Infertility rates in both male and female smokers are about double the rate of infertility found in nonsmokers. The effect is dose dependend: The risk for fertility problems increases with the number of cigarettes smoked daily. Smoking during pregnancy also can lead to growth restriction of the baby before birth. Children born with lower-than- expected birth weights are at higher risk for medical problems later in life (such as diabetes, obesity, and cardiovascular disease). Children whose parents smoke are at increased risk for sudden infant death syndrome (SIDS) and developing asthma. So somking in pregnancy has long term health efects on the offspring.

Obesity

One of the best-established connections between obesity and reproductive problems is the link between obesity and infertility. Obesity decreases the rates of successful pregnancy in natural conception cycles. In women who are undergoing IVF obesity reduces the rates of pregnancy as well.

What to do?

Stop smoking
Moderation in alcohol
Avoid obesity, hypertension and diabetes
Do not consume sugary drinks
Don’t use plastic bottles or cans
Use fresh food (fruits, vegetables, meat, fish) in stead of processed food.

There is currently no certainty on the hereditary effects on the fertility of the offspring although there is concern on certain diseases.