What medium should I use for diluting PureSperm 100?

PureSperm Buffer is the best media to use when diluting PureSperm 100. It has been optimised and ionically balanced to best match PureSperm 100.

Is there any human serum albumin in PureSperm 100?

Our lab research has shown that the yield of motile sperm recovered from the gradient was approximately the same, regardless of whether the gradient layers contained human serum albumin or not. Therefore, we have not deemed it necessary to include any protein in PureSperm 100. The colloid in these products eliminates aggregation of sperm and reduces their sticking to the centrifuge tube. However, human serum albumin must be added to the “wash” solution, as provided in PureSperm Wash.

Are antibiotics included in PureSperm 100?

No, PureSperm 100 does not contain antibiotics. They are not included for the following reasons:

1. The gradient will remove most, if not all, of the bacterial contamination present in the ejaculate, provided that the retrieval of the sperm pellet is carried out according to the instructions given in the package insert.
2. Antibiotics commonly used in cell culture media are toxic to sperm.

What should be used for washing the sperm pellet after the PureSperm 100 gradient centrifugation?

We recommend PureSperm Wash for washing the sperm pellet after gradient centrifugation. The ionic balance of PureSperm Wash lies between that of the gradient and commonly used fertilisation media and will not therefore cause premature hyperactivation of the sperm. PureSperm Wash is also ready-to-use and contains an appropriate level of human serum albumin.

Why do I need to buy a separate product, such as PureSperm Buffer, to dilute PureSperm 100? Could I use PureSperm Wash instead?

You can use PureSperm Wash to dilute PureSperm. However, a comparison in our laboratories has shown that you will obtain a better motile sperm yield by using PureSperm Buffer which has been optimised for this purpose.

Does one need to incubate PS100 and PS40/80 in 5-6% CO², 37°C before use?

The most important thing is to avoid temperature fluctuation in the sperm sample. Ejaculate has a temperature of approximately room temperature or slightly higher. We therefore recommend equilibrating PureSperm to room temperature, no more. The preparation is also processed at room temperature, so this means that an equivalent temperature is maintained.

How do I use PureSperm Buffer?

PureSperm Buffer is used to dilute PureSperm 100 to obtain the layers of different densities required for a discontinuous density gradient. PureSperm Buffer has been carefully formulated to give optimal results when used according to the manufacturer´s instructions. It is ready-to-use, no additives being required.

Why do I need to use PureSperm Buffer to dilute PureSperm 100?

A comparative analysis has shown that you will obtain a better yield of motile sperm by using PureSperm Buffer for diluting PureSperm than by using non-optimised products. Commercially available culture media, washing media, or salt solutions such as Earle’s ´Balanced Salt Solution have not been optimised for this purpose and also require other ingredients need to be added before use, thus increasing the cost.

Why do I need to buy a separate product, such as PureSperm Buffer to dilute PureSperm 100? Could I use PureSperm Wash instead?

Yes, you can use PureSperm Wash to dilute PureSperm 100. However, a comparison in our laboratories has shown that you will obtain a better yield of motile sperm from using PureSperm Buffer which has been optimised for this purpose.

Should I equilibrate PureSperm Buffer before use?

If a bottle of PureSperm Buffer has been previously opened and then stored in the refrigerator, it should be brought up to room temperature before use to avoid cold-shocking the sperm. It is not necessary to equilibrate PureSperm Buffer in the CO² incubator before use.

Are there any antibiotics in PureSperm Buffer ?

No, PureSperm Buffer does not contain antibiotics. Many commonly used antibiotics are toxic to sperm. Furthermore, antibiotics such as penicillin, have a very short half-life in solution. When used according to the manufacturer´s instructions, the PureSperm density gradient will enable an uncontaminated sperm preparation to be obtained from the semen sample.

PureSpermBuffer can be used to dilute viscous samples. The outcome of a sperm preparation can be affected by an increased osmolality in the ejaculate. A simple way to minimize the increase of osmolality is by early dilution of the ejaculate. PureSpermBuffer is the perfect buffer to be used for this purpose. Why is PSB good to use?

The prostate secretion contains large amounts of proteolytic enzymes (PSA) while the seminal vesicle secretion contains large amounts of proteins (semenogelin), peptides and prostaglandins which causes the gel formation at ejaculation when the secretions are mixed. After that the proteolytic enzymes start breaking down the large proteins and the gel will liquify. When you add a buffered salt solution you basically extend the solution and increase the space between the large proteins (semenogelin)and the complexes that they make-up break apart and the gel becomes liquified faster

Reference: Holmes, Björndahl, L., and Kvist, U. (2019). Possible factors influencing post‐ejaculatory changes of the osmolality of human semen in vitro. Andrologia, 51(11), e13443–n/a. https://doi.org/10.1111/and.13443

You simply add PureSpermBuffer to the ejaculate (dilution 1+3), 1 part PureSpermBuffer and 3 parts sample (e.g. 0.5 ml PureSperm Buffer + 1.5 ml semen sample).

Incubate for 15-30 minutes at 37°C, mix using a pipette, and the sample is ready for preparation (preferably using a density gradient).

What tools do I need to perform the sperm preparation using Speedikit?

You only need a centrifuge and some sterile pipettes. PureSperm Speedikit includes the prefilled tubes for both separation and washing of the sample.

Do I need an incubator for performing a sperm preparation with Speedikit?

No, the whole procedure is performed at room temperature.

Can one use the same protocol for SpermCatch as we have for PVP products?

Yes, you can use SpermCatch in the same way as products containing PVP.

What’s the difference between SpermCatch and PVP?

SpermCatch does not contain any plastic, it’s made of hyaluronic acid which is a natural component.

How do I best prepare my ICSI dishes using SpermCatch?

Dishes must be prepared quickly to avoid osmolarity changes in the media. Only make two at a time. It is convenient to have two dishes per patient.

What survival rates should we expect after freezing/thawing?

The number of sperm cells surviving a freezing procedure can vary significantly between samples. Analyses on freezing/thawing donor samples however has shown average survival rates of between 65-70%. (“Survival” has been defined as the number of motile sperm cells after thawing compared to the number of motile sperm cells before freezing.)

Can I use Sperm CryoProtec for TESE?

Yes, you can. There is no need to add anything; it’s as simple and easy as using Sperm CryoProtec with ejaculated sperm.

Do I need to add egg-yolk to Sperm CryoProtec before use?

No, you don’t. Sperm CryoProtec is a ready-to-use freezing medium which doesn’t require any additives. Just follow the instructions and your sperm will be able to be safely frozen and thawed.

Does Sperm CryoProtec contain antibiotics?

No, none of our products contains any antibiotics. Adding antibiotics would significantly reduce the shelf life of our products and could “mask” post-sterilisation contamination of the product

How do I get the optimal freezing temperature?

-Optimal freezing temperature can be obtained by using CryoFloater. The cryofloater provides a stable raft with the correct and constant height above the nitrogen surface, thereby guaranteeing optimal freezing temperature and the best possible result.

Free of charge when you order our cryoprotectant, Sperm CryoProtec

What is the optimal thawing temperature?

We recommend thawing the straw or vial of frozen sperm in water heated to 37°C, as this method yields better results than thawing at room temperature. Instead of using a traditional water bath, you can preheat a 50 ml tube of water in the incubator to 37°C. This approach is simpler and more convenient, as it eliminates the need to constantly monitor and maintain the temperature of a water bath

How do I best perform the incubation before freezing?

 -Place the straws on a cold steel tray when equilibrating in the refrigerator prior to cryopreservation. This way the straws will remain cold until they are placed in the nitrogen vapour on the cryofloater. Use forceps when moving the straws.

The time range for incubation is 10-60 minutes. Any difference between 10 and 60 minutes? The survival results, when incubating for 60 minutes compared to 10, shows a slight increase when incubated for 60 minutes. The major difference however is, between not incubating and incubating for 10 minutes.

What is the optimal time on the CryoFloater in LN₂?

-The recommended time is 10-30 minutes. Measuring the temperature on the floater, it goes down quite fast, and 10 minutes is more than enough but, in order to make it more flexible, we recommend the range of 10-30. No difference in result with regards to survival rate has been found in our tests between 10 and 30 minutes but it will give you the chance to take the coffee you so desperately need!

Can Blastocysts cultured in any culture media be vitrified with your kits?

VitriBlast /ThermoBlast kit can be used to freeze blastocysts that have been cultured in any culture media.

What device should I use with the VitriBlast kit?

Any device can be used but our general recommendation is to use a closed-system.

Is DMSO more harmful than other cryoprotectants?

No. In general, DMSO is actually one of the least harmful organic solvents. It has a very low toxicity and is not genotoxic, nor mutagenic or carcinogenic.

DMSO is an FDA approved drug used to treat interstitial cystitis, but has been (and is still) widely used to treat various diseases including arthritis, local pains, inflammation, infections (herpes), autoimmune diseases, and gastric ulcer.

In embryology, DMSO was reported to protect membranes better than other cryoprotectants. It is less toxic than propylene glycol and is the standard component of the world’s most successful vitrification mixtures (both human and zoological).

How do I store DMSO?

-DMSO is solid below +18°C and needs to be above this temperature to reach liquid form. If there is a shortage of time, it can be warmed in the hand or warmed in the incubator.

Do I need to mix the solutions each time before use?

 -The EG and DMSO can be pre-mixed in the bottles (VB 2 and VB 3) and stored in the refrigerator for up to two weeks. However, it is important to know that the volumes in the VB 2 and 3 are not EXACTLY 10 mL, there is always a little surplus. Therefore, the volumes in the VB 2 and 3, must be measured and the surplus discarded prior to adding the EG and DMSO in order to achieve the correct concentrations.

Why should I collapse the blastocyst before vitrification?

-Collapsing the blastocyst will improve the results. If laser is used, shoot as far from inner cell mass

(ICM) as possible and ensure both the zona and the trophectoderm are breached.

If an ICSI-pipette or other sharp instrument is used, puncture right through the trophoblast cell layer into the blastocoele and be sure to puncture as far as possible from the ICM.

The pipette should be inserted at the one o’clock position and exit through the blastocyst at the 11 o’clock position. Collapsing is optional when vitrifying early blastocysts with smaller blastocoele cavities.

What is Endometrial Microbiome?

The endometrial microbiome is a community of microorganisms, primarily bacteria, that reside within the uterus. Similar to the vaginal microbiome, Lactobacillus species typically dominate the endometrial microbiome. Lactobacillus produces lactic acid, creating an acidic environment that inhibits the growth of pathogenic bacteria. This acidic environment helps to maintain a healthy balance of microorganisms within the uterus. Dysbiosis, or an imbalance of the endometrial microbiome, can lead to increased maternal immune activation, making it more difficult to suppress immune responses against the developing embryo and potentially compromising pregnancy outcomes.

Who should test for EMT?

Women experiencing recurrent pregnancy loss, implantation failure, unexplained infertility, or those undergoing assisted reproductive technologies (ART) may benefit from an EMT. Additionally, women with a history of chronic endometritis could consider testing to assess the health of their endometrial microbiome.

Given that the microbial environment significantly influences the implantation process, EMT testing can also be beneficial for women undergoing intrauterine insemination (IUI) to potentially increase success rates.

Why is it better to take the test from the endometrium (endometrial luminal fluid) instead of the vagina?

Recent studies have shown that vaginal and endometrial microbiomes share many similarities, but approximately 20% of patients exhibit distinct bacterial profiles. Sampling directly from the endometrial cavity provides a more accurate representation of the microbial community within the uterus. This specificity is crucial for making appropriate treatment choices.

How can a contamination from the vaginal microbiome be avoided?

To minimize vaginal contamination during endometrial sampling, healthcare providers typically use specialized pipettes designed to collect fluid directly from the endometrial cavity.

Before collection, the vagina and cervical canal should be washed with sterile cotton balls and dried.

There is a possibility of bacteria adhering to the surface of the collection pipette from the cervix. When transferring the sample into the collection tube using the pipette, ensure that the pipette’s surface does not come into contact with the tube, allowing only the collected specimen to be transferred.

Why is an EMT test needed? Can we not, treat directly with antibiotics and Lactobacillus instead without testing?

Antibiotics and probiotics are commonly used to treat endometrial imbalances.

The EMT can identify the specific microorganisms present and their relative abundance and, depending on the test results, suggest examples of appropriate treatment options in combination with antibiotics and supplements.

It goes without saying that it is nonsense to administer antibiotics when the microbiota is normal, and even in dysbiosis it is important to select and treat with the correct antibiotic depending on the species of bacteria detected.

In addition, the indiscriminate use of antibiotics without testing can upset the delicate balance of the microbiome and lead to antibiotic resistance. Additionally, many cases do not improve with treatments based on supplements alone.

Selecting the appropriate antimicrobials according to test results and combining the treatment strategy with supplements is critical in terms of improving implantation rates.

Why is the Varinos test method preferable compared to others?

The Varinos test method offers several advantages over traditional methods, making it a preferred choice for microbial analysis. Here are the key reasons:

1. High Sensitivity

• Lower Detection Limit: Varinos tests exhibit exceptionally high sensitivity, meaning they can detect even minute quantities of bacteria that may go undetected by other methods. This increased sensitivity is crucial for identifying low-abundance microorganisms that could be significant in various biological processes.

2. Species-Level Identification

• Beyond Genus: Unlike traditional 16S rRNA sequencing, which primarily identifies bacteria at the genus level, Varinos technology allows for species-level identification. This granular level of detail provides more accurate and informative insights into microbial communities.
• Comprehensive and Cost-Effective: Varinos’ proprietary technology enables comprehensive species-level identification at a lower cost compared to other methods, making it a more accessible and cost-effective option for researchers and clinicians.

3. Validated by Extensive Research

• Peer-Reviewed Publications: The effectiveness and reliability of the Varinos test method have been extensively validated through numerous peer-reviewed publications. This body of research provides strong evidence supporting the accuracy and precision of the technology.