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IVF News Autumn 2016

Contents
    • Optimize your sperm preparation
    • The importance of storing the sperm
    • The 2016 Ig Nobel Prize Winner
    • Frequently asked questions
    • Processing of semen by density gradient centrifugation selects spermatozoa with longer telomeres for assisted reproduction techniques
    • Tips & tricks
    • New distributors
    • Upcoming events
    • Who to contact
Web408090
Optimize your sperm preparation

PureSperm 90 has joined the PureSperm family and is now available. This product makes the line of ready-to-use products more complete and it is now possible to perform different density gradient preparations very easily.

All of the ready-to-use PureSperm products have also been updated and their formulations optimized.

The raw material, the silane coated silica is still our own unique material. It is produced by Nidacon and only used for the PureSperm products.

But is it necessary to have different density gradient preparations? No, it’s not necessary, if 40/80 is used or 40/90 for all your patients, you will have very good preparations, regardless.

It is, however, possible to use 40/80 for inseminations, donors, samples where you need a high yield and use 40/90 for samples where a higher percentage of motile sperm is desired. As shown in the two graphs the difference is not large but sometimes it’s the small things that make a difference.

Product Specialist – Ms. Ann-Sofie Forsberg


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The importance of storing the sperm sample in Room Temperature and avoiding temperature fluctuations

It has proven to be of great importance for the outcome that the sperm sample is maintained in a temperature as constant as possible and that the temperature should be around room temperature. We here intend to further clarify with references to publications on the subject.

  • Activation of enzymes

The ejaculate contains enzymes which start working directly after ejaculation. This results in an increase in osmolality.

If the sperm sample is stored in an incubator (37°C), the enzymes work more efficiently and the increase of the osmolality will occur much faster. The difference in osmolality between the ejaculate and the gradient or swim-up medium will be higher than if the sample had been stored at room temperature. As is well known, sperm do not like shocks (cold-shock, dilution shock, osmolality shock and so forth)

Ref: Osmotic shock induces structural damage on equine spermatozoa plasmalemma and mitochondria. L. Gonzalez-Fernandez et al, Theriogenology 78 (2012) 415-422

Similar studies have been performed on a number of different species. A PhD student at Nidacon will soon publish results with human sperm where this is confirmed. The results so far also show negative effects on motility and DNA-integrity.

  • Temperature fluctuation

In the reference below, temperature fluctuation and motility is discussed. The conclusion is that the slightest jump up or down in temperature has a negative effect on motility, and causes hyperactivity.

Ref: Behavioral mechanism of human sperm in thermotaxis: a role for hyperactivation, Sergii Boryshpolets et al, Hum Rep, 2015

The findings of this study show that the fraction of hyperactivated spermatozoa is tightly dependent on the temperature and, especially, on temperature changes. This is because hyperactivation events could be generated as a result of temperature fluctuations. With the extreme temperature sensitivity of human spermatozoa and their response to even subtle temperature changes (Bahat et al., 2012), it is of outmost importance to maintain a well-controlled constant temperature in studies of sperm motility

  • The WHO-manual

The WHO-manual recommends storage of sperm samples in constant temperature.

However, it does define the temperature to be between 22-37°C, but still constant in that interval. ”Avoid changes in temperature”.

With above in mind, the optimal storage temperature is room temperature, from ejaculation until the washed sperm are introduced to the oocyte.

Animal Product Specialist – Ms. Anna Niläng Laessker 


underwear
 The 2016 Ig Nobel Prize Winner

The Ig Nobel Prizes honour achievements that make people LAUGH, and then THINK. The prizes are intended to celebrate the unusual, honour the imaginative — and spur people’s interest in science, medicine, and technology

REPRODUCTION PRIZE [EGYPT] — The late Ahmed Shafik, for studying the effects of wearing polyester, cotton, or wool trousers on the sex life of rats, and for conducting similar tests with human males.

Effects of different types of textiles on male sexual activity

Ahmed Shafik European Urology,vol 24 no 3,1993

Abstract: The effect of different types of textile underpants on sexual activity was studied in 50 men. All the subjects were potent and sexually active. They were divided into 5 equal groups: 4 tests and 1 control. Each of the 4 test groups were dressed in one type of textile underpants made of 100% polyester, 50/50% polyester/cotton mix, 100% cotton, or 100% wool. Sexual behaviour was assessed before and after 6 and 12 months of wearing the pants, and 6 months after their removal. Behavioural response was rated as potent if the subject’s penis became erect, entered the vagina, and ejaculated. The rate of potent intromission (I) to mounts (M) (I/M ratio) was determined. The electrostatic potentials (EP) generated on the penis and scrotum was measured by an electrostatic kilovolt-meter. The I/M ratio at 6 and 12 months of wearing the polyester and polyester/cotton mix pants was significantly reduced compared to the pre-test levels and the controls (p <. 001). The reduction was more manifest in the pure polyester than in the polyester/cotton mix group, and at the 12-month than at the 6-month examination.

The polyester-containing pants generated EP, which may induce electrostatic fields in the intrapenile structures and could explain the diminished sexual activity. The cotton and wool textiles did not generate EP. Thus, polyester underpants could have an injurious effect on human sexual activity.

Conclusion:

If you are thinking about buying underpants for your husband or anyone else for Christmas, make sure that it’s of the right material.

http://www.improbable.com/ig/


telomeros
Processing of semen by density gradient centrifugation selects spermatozoa with longer telomeres for assisted reproduction techniques Qingling Yang et al,

 

Abstract

The ends of eukaryotic chromosomes contain specialized chromatin structures called telomeres, the length of which plays a key role in early human embryonic development. Although the effect of sperm preparation techniques on major sperm characteristics, such as concentration, motility and morphology have been previously documented, the possible status of telomere length and its relation with sperm preparation techniques is not well-known for humans. The aim of this study was to investigate the role of density gradient centrifugation in the selection of spermatozoa with longer telomeres for use in assisted reproduction techniques in 105 samples before and after sperm processing. After density gradient centrifugation, the average telomere length of the sperm was significantly longer (6.51 ± 2.54 versus 5.16 ± 2.29, P < 0.01), the average motile sperm rate was significantly higher (77.9 ± 11.8 versus 44.6 ± 11.2, P < 0.01), but average DNA fragmentation rate was significantly lower (11.1 ± 5.9 versus 25.9 ± 12.9, P < 0.01) compared with raw semen. Additionally, telomere length was positively correlated with semen sperm count (rs = 0.58; P < 0.01).

Conclusion:

Density gradient centrifugation is a useful technique for selection of sperm with longer telomeres.


 Frequently asked questions

How do I calibrate my centrifuge to make sure that I use the correct g-force?

To achieve the optimal result using a density gradient, it is critical, to make sure that your centrifuge uses the correct g-force. You can either use the equation and do the calculation or use the RCF  nomograph

Rpm = v[ (g/(1.118 x r)] x 10³

r = rotational radius, the distance (mm) from the center of the rotor to the bottom of a centrifuge tube in the bucket when raised to horizontal position

For example; to achieve 300 x g when radius = 165 mm the centrifuge speed must be:

Rpm = v[ (300/(1.118 x 165)] x 10³ = 1275

By using the RCF Nomograph  Bild

 

gforce

 

Using the RCF Nomograph

To calibrate the centrifuge to achieve the correct g force, place a straightedge on the nomograph connecting the known Radius of rotation (cm) and the desired G-force (g). The point at which the straightedge intersects the Speed of rotation (rpm) axis is the rpm.
For example, if the rotating radius is 14 cm and the G-force (g) is, 300g (Nidacon
protocol), the relative centrifugal force between 1300 – 1500 rpm.


tipsandtricks
Tips & Tricks

We hope you have had some use of the tips&tricks that we have started to send to you.

Our hope is to provide you with the small details that can make a big difference but are difficult to find in literature and not often talked about at conferences.

If you have any questions regarding small details, how to make the most of the procedure or product, please let us know, most likely, many others have asked themselves the same thing.

If you are not receiving our monthly tips&tricks, send us an e-mail.

New distributors

polycompany

Polycompany International group S.A is a new Nidacon distributor in Chile. We would like to welcome Alejandro Mucientes and colleagues at Polycompany International group S.A as our new distributor in Chile.


in-vitro-life

In Vitro Life is a new Nidacon distributor in Perú. We would like to welcome Marco Arturo Escobar Aguilar and colleagues at In Vitro Life as our new distributor in Perú.

IVF News Spring 2016

Contents
    • New family member.
    • Why male marathoners might be the best men around.
    • Zika virus and its effect on assisted reproduction techniques.
    • Freezing Gradient-Prepared sperm compared with Freezing Whole ejaculate followed by Gradient-Preparation.
    • Frequently asked questions.
    • Every small step matters.
    • New distributors
    • Upcoming events.
New family member

We have the great pleasure to announce that we are extending our line of density gradient products with a new gradient concentration.

Introducing PureSperm® 90! The launch of this new family member will take place at the ESHRE meeting in Helsinki, 2016.

 

Greater flexibility

You will now have a wider selection of density gradients to choose from depending on what you want to use the final sperm preparation for.
The difference in the results, for example, between a 40/80 and a 40/90 gradient is that the higher concentration will give a higher percentage motile sperm but a slightly lower yield. The overall difference is not very large but will give a more specific separation depending on the treatment chosen for the patient.

Improved composition

We have also made some minor changes in the composition of salts to all three concentrations of our gradients in order to optimize the sperm preparations even further. The overall result of this change is a higher progressive motility in the final sperm preparation.
The clinical tests have shown good results with no significant differences in fertilization or pregnancy rate but the overall feedback is that the new composition is better and it will be the gradient of choice for the test clinics from now on.

Same quality
The new PureSperm®90 is guaranteed to give the same high quality sperm preparation as the PureSperm®40 and PureSperm®80. The only difference will be, as can be expected, that a gradient with a PureSperm® 90 as the bottom layer will give a higher percentage motile sperm but a slightly lower yield.
So in conclusion, you will get a wider selection of even higher quality products for your lab.

Available in July
The new products will be available at ESHRE, more exactly on the 1st of July. PureSperm®90 will so far only be available in 100 ml bottles and the price will be the same as for PureSperm® 80. Please let us know if you have any questions regarding these new products.

Product Specialist – Ms. Ann-Sofie Forsberg


Why male marathoners might be the best men around

Long distance runners are hot-scientifically speaking. It’s not just that running makes men fit and athletic: long –distance running might be the mark, natural-selection-wise, of a high quality male, according to a new research, one that women would do well to select as a mate.

 

The study, conducted by the University of Cambridge’s Division of Biological Anthropology, analysed a group of 542 long distance runners (439 men and 103 women). They found that the best performing runners were likely exposed to higher levels of prenatal testosterone in their mother’s blood.

The scientists assessed this using the “2D:4D ratio” – the length of the ring finger versus the index finger, since people who have been exposed to higher levels of testosterone have a longer fourth finger in comparison to the second.

Prenatal testosterone exposure, which “androgenizes “the foetus, is also associated with traits such a stronger sex drive, higher sperm count and better cardiovascular efficiency in men. Bluntly speaking, better long-distance running skills might signal a higher quality male – at least in terms of reproductive fitness.

You know what the say – better runner, longer ring finger


Zika virus and its effect on assisted reproduction techniques

The authorities in United States are warning of the risk for the ZIKA virus and the possible pathological effects of the virus during embryonic and fetal development. This decision has been made as a result of children from endemic viral areas being born with microcephaly.

 

Implications for IVF

For infertility patients it is a very difficult decision to postpone treatment for an undetermined period, since their chance of getting pregnant decreases with time.

Nidacon suggests the following to minimize risks:

  1. Blastocyst Vitrification

By Vitrification of Blastocyst and subsequent frozen storage, you can postpone transfer of the healthy embryo to when the epidemic has decreased or is under control. Many clinics have excellent results with VitriBlast vitrification and high survival rates after ThermoBlast warming.

  1. Cleaning of the Semen sample

A PureSperm® density gradient will effectively remove lymphocytes, epithelial cells, abnormal, immature and senescent sperm, cell debris, seminal fluid, bacteria and, to some considerable extent, also viruses.

It is important to avoid any re-contamination of the separated sperm and we therefore recommend using the ProInsert (1, 2)

Test for Zika virus is recommended before any treatment.

  1. Patient Communication

Inform the patient that semen handling procedures are merely risk reduction methods, while the risk itself cannot be totally eliminated.

An informed consent must be signed by the couple wherein the potential consequences of the technique inadequacy are acknowledged.

Ref:

Elimination of bacteria from human semen during spermpreparation using density gradient centrifugation with a novel tube insert

  1. Fourie, N. Loskutoff & C. Huyser, Johannesburg, South Africa,

Andrologia, 2011, pp 513-517.

Use of a novel washing method combining multiple density gradients and trypsin for removing human immunodeficiency virus-1 and hepatitis C virusfrom semen.

Naida M. Loskutoff, Ph.D., Carin Huyser, Ph.D.,b Raksha Singh, B.Tech, David L. Walker, M.Sc., Alan R. Thornhill, Ph.D., Lynn Morris, Ph.D.,d and Lynne Webber, M. Med. Path.Virol., Johannesburg, South Africa,

Fertility and Sterility, Vol. 84, No. 4, October 2005, pp 1001-1010.

The symposium, “Prevention of Communicable Diseases in Assisted Reproduction”

The Association of Colombian Centers in Human Reproduction will arrange the symposium in Bogota, Colombia on the 6th of May 2016. Carin Huyser from The University of Pretoria, South Africa will give a lecture on Prevention of infections in the ART program, from protocol to practice.

Together with Nidacon, Cecolfes will arrange a workshop on the preparation of sperm and the freezing of sperm.

KAM, Latin America – Mr. Mauricio Lucena


Freezing Gradient-Prepared sperm compared with Freezing Whole ejaculate followed by Gradient-Preparation.

Introduction

To obtain the best survival results when freezing and thawing sperm, you need a suitable cryo-protective medium. Certainly, the medium is not all that is needed. A suitable protocol for freezing and thawing affects the results significantly.

The objective of this study was to determine whether there are differences regarding the numbers of sperm and their motility when (1) freezing gradient-prepared sperm or (2) freezing whole ejaculates followed by gradient-preparation after thawing.

Method

Protocol 1:

Motility and number of sperm were determined in the unprepared ejaculates (n=12). Thereafter, the ejaculates were split equally in two halves.

The first half was prepared on a PureSperm® gradient (2ml 40% and 2ml 80%), centrifuged at 300 x g for 20 minutes and, thereafter, transferred to 5ml PureSperm® Wash (PSW), and centrifuged for 10 min at 500 x g. The upper 4ml of PSW was discarded. After preparation, the sperm suspensions were frozen and thawed using SpermCryoProtec™ (SCP) and the protocol recommended for this product (for the protocol, see Nidacon.com). After thawing, the straw contents were extended in 5ml PSW and centrifuged for 10 min at 500 x g.

Protocol 2:

The second half of the ejaculates was directly frozen using SCP (Nidacon.com for protocol). When thawing, all samples were extended in 1ml PSW. (Extending in this way is important, since the sperm need to restore density prior to the gradient preparation). Thereafter, the thawed, extended ejaculates were prepared as in protocol 1.

Sperm motility and number of motile sperm were assessed for the two different treatments.

Results

  • Motility was 73% when gradient preparing sperm prior to freezing and 61% when using gradient after thawing. (Figure 1).

  • Number of motile sperm was more than double when using protocol 1, i.e. gradient preparing sperm samples prior to freezing (Figure 2).

Conclusion

  1. Preparing sperm prior to freezing is preferable to freezing sperm before preparation. The recovery was more than doubled when following Protocol 1 in this experiment. Also, the percentage motile sperm was better when using Protocol 1.
  2. If still using Protocol 2, it is important to extend the thawed sperm sample in PureSperm® Wash prior to gradient preparation, in order to restore the sperm density, when using density gradients.

 


 

Frequently asked questions

How long can I use the product after opening?

The same shelf-life applies even after opening. If you, for example, have a bottle of PureSperm®100 with a shelf–life that says 17Jan 2017, you open the bottle and, thereafter, keep it in the fridge and use it until the 17th of January 2017. Of course, you must use aseptic procedures when handling the bottle.

How can the products be so stable?

All ingredients are chosen for their temperature tolerance and their stability in aqueous solutions.
Rigorous shelf life testing before and after opening has been carried out to ensure that the theoretical stability of the salt formulation is matched by the actual stability when combined in the product.


 

Every small step matters

In ecology, sustainability is the capacity to endure; it is how biological systems remain diverse and productive indefinitely. In more general terms, sustainability is the endurance of systems and processes. The principle for sustainability is sustainable development, which includes the four interconnected domains: ecology, economics, politics and culture.

Nidacon believes in the importance of our legacy and works actively to make a positive imprint and to take responsibility for our impact on our world. A more conscious use of resources is essential in order to maintain healthy ecosystems and environments. Having a well-functioning system for waste recycling was therefore a natural step for us atNidacon.

We investigated our express transports and came to the conclusion that we should compensate for carbon emissions. Therefore from now on, we will pay an extra fee for every package that is shipped from us. UPS has a program called “UPS Carbon Neutral”which we will join and, for all other transports, we will compensate by planting trees through WWF.

Since we are all helping couples to fulfill their dreams of having a family, we feel that we have an extra responsibility for the children in the world. We will start by supporting an organization called “We care”. Today they have three ongoing projects for “street children” in Nepal. We put a lot of effort into choosing which organizations/projects we support but are open to your suggestions. Please, contact us with your thoughts!

We are proud to be a member of an organization called CSR West Sweden which focuses on global sustainability within all the four interconnected domains mentioned above. We attended a very interesting seminar based on KPMG’s study on Global sustainability “Megaforces” . To ensure our continual development we will keep attending seminars, courses and conferences with this focus.

These are just small steps but every step matters!

“The Nidacon team”


New distributors

We would like to welcome Dr. Holger Kayser and colleagues at Esco Micro Pte. Ltd as our new distributor in Germany, Austria and Switzerland.

 

Esco represents innovation and forward-thinking designs, which are all coupled with the highest standard quality since 1978. The Esco Group of Companies remains dedicated in delivering innovative solutions for the clinical, life sciences, research, industrial, laboratory, pharmaceutical and IVF community.

 

 

We would also like to welcome Catherine Broberg and colleagues at E.in Art as our new distributor for Ghana, Nigeria, Uganda, Senegal, Burkina Faso, Ivory Coast, Cameroun, Benin, Mali, Niger, Togo, Congo, Gabon and Rwanda.

 

 

E. in Art stands for excellence in assisted reproduction and offers a full service and organizational package. The mission of E in ART is to provide a customized approach to improve the success rate of infertility clinics and ART centers.

 


Upcoming events

 

Meet us at ESHRE 2016!
The 32nd Annual Meeting of the European Society of Human Reproduction & Embryology will this year be held at Messukeskus Expo and Convention Centre in Helsinki Finland.
Of course Nidacon will be present at the exhibition as every year. Do not forget to stop by our booth at location C152 for news and product updates!


 

IVF News Spring 2015

Contents
    • PureSperm SpeediKit, now a 10-patient kit.
    • Ovulation affects your shopping and choice of men
    • ProInsert– new version
    • Efficacy of density gradient separation on the preservation of sperm DNA integrity.
    • New collaboration
    • Nidacon products used for breeding world champions!

 

PureSperm SpeediKit, now a 10-patient kit.

PureSperm Speedikit is a quick and efficient alternative for sperm preparation using a single layer of PureSperm colloid followed by rinsing the sperm with PureSperm Wash.

The price per preparation will be lower which is beneficial for our customers.

The kit is designed for the smaller clinic. All you need is a centrifuge and pipettes. It is very easy to use even if you are not an embryologist.
Until now the kit has consisted of PureSperm colloid and PureSperm Wash for 5 patients, already dispensed in centrifuge tubes, plus semen collection tubes. We have now removed the semen collection tubes and added more single-layer/ wash tubes which results in a kit for 10 patients instead of 5.
The price per preparation will be lower which is beneficial for our customers. This new kit has the same long shelf life, 12 months. It can be stored at room temperature all the time.


Ovulation affects your shopping and choice of men

New research from The University of Texas at San Antonio (UTSA) College of Business suggests women seek more options in dating partners near ovulation when they are most fertile which may lead them to also seek a greater variety of products and services.

“Just like a fisherman casting a wide net, ovulating women seek to cast a wide net into the dating pool and expand the number of potential suitors they have to choose from”, says Kristina M. Durante, UTSA marketing assistant professor and lead investigator of the study. “And, this desire for variety in men at ovulation triggers a variety seeking mind-set that carries over into desire for variety in products.” Forthcoming in the April 2015 issue of the Journal of Consumer Research, “Playing the Field: The Effect of Fertility on Women’s Desire for Variety” provides some of the first evidence that choice behaviour in our personal relationships may influence choice behaviour in the market-place. Durante and then UTSA visiting assistant professor Ashley Rae Arsena focused their predictions on previous research that finds that ovulation canshift women’s mating psychology. Durante and Arsena conducted four studies that included 553 female participants in the U.S. between 18 and 40 years of age who were not pregnant or taking hormonal contraceptives. The studies found that women’s desire for new options in men triggered a variety seeking mind-set that led women to also desire variety in products. Loyalty to a romantic partner reduced the desire for product variety, suggesting that loyalty in romantic relationships can translate to brand loyalty.

Journal Reference 1. Kristina M. Durante, Ashley Rae Arsena. Playing the Field: The Effect of Fertility on Women’s Desire for Variety. Journal of Consumer Research, 2014; 000 DOI: 10.1086/679652


ProInsert– new version

From the beginning of May, the content in each pouch of ProInsert will change. The washing tube and the extra pipette will be removed.

Most of you already have tubes that can be used for washing and the extra pipette has also been considered expendable by many users. This change has made it possible for us to lower the price and hopefully make the product affordable for more users. The price is now 29 Euros for a package of 5 instead of 35. The new price was actually in place on the 1st of January.
This product not only minimizes the risk for contamination of the pellet after centrifugation, it also makes it easier to prepare the gradien and will save you a lot of time.


Efficacy of density gradient separation on the preservation of sperm DNA integrity.

Efficacy of density gradient separation on the preservation of sperm DNA integrity.
1Gabriella Donà, 2Alessandra Andrisani, 3Decio Armanini, 1Luciana Bordin*.. 1Department of Molecular Medicine-Biological Chemistry, University of Padova, Italy; 2 Department of Medicine-Endocrinology, University of Padova, Italy; 3Department of Woman Health and Children Health, University of Padova, Italy.

Sperm preparation represents one of the most delicate step in the assisted reproduction techniques (ART) as reported in a recent study which compared the effect of different commercial buffers according to their capacity to induce acrosome reaction (AR), paying particular attention to cell survival at the end of the capacitating incubation. Interestingly, PSW-Nidacon buffer was particularly suitable for sperm preparation and incubation, since cells reaching the AR was almost three-five fold compared with results obtained with other commercial buffers. PSW-Nidacon also preserved cells from apoptosis (only 3.5±1.4% of total cells were not viable) compared with the great number observed with the other ones study (Andrisani et al, 2014).

Besides inducing AR, sperm preparation must maintain the integrity of DNA within the cell, avoiding DNA degeneration/fragmentation. Critical step for preserving DNA integrity seems to be sperm isolation by gradient centrifugation, controversially accused to be one of the inductors of DNA fragmentation (Aitken et al., 2014). For this reason we evaluated sperm DNA integrity after PS-Nidacon gradient (40/80) centrifugation in samples from 8 healthy volunteers. Preliminary results showed that PS gradient and PSW incubation preserved sperm nucleus as indicated by the low response to propidium iodide (PI) both at T0 and after 1h in capacitating buffer. On the other hand, when an aliquot of the same sperm preparation was incubated in PSW in the presence of H2O2, PI labelling was clearly visible, thus confirming that ROS-induced oxidative assault dramatically increases nuclear envelop degeneration (Fig. 1)

To evaluate cell viability sperm samples were centrifuged and exposed to DNA-specific fluorochrome propidium iodide (PI) 12 μM for 10 min at room temperature. Sperm was washed with PBS, fixed with 2% (w⁄v) paraformaldehyde in PBS, incubated overnight at 4°C on poly-l-lysine-treated slides and mounted. At least 200 cells were evaluated for each sample and fluorescence was detected with the UltraView LCI confocal system.

 

In addition, to assess DNA status the single gel electrophoresis COMET was also performed in these samples. In figure 2 COMET patterns of samples incubated in the absence or presence of H2O2 were shown. Oxidative assaults, besides denaturing nuclear membrane, induced high DNA fragmentation (percentages of sperm presenting DNA fragmentation were <10% at both T0 and 1h incubation vs 60% of H2O2-treated cells), as shown by the luminescent code of the comet left by DNA during electrophoresis.

Sperm were suspended in 0.7 % (w/v) low-melting-point agarose at a concentration of 4×105 cells/ml, applied to the surface of a microscope slide pre-coated with 1% agarose, to form a microgel and allowed to stand at 4°C for 20 min.

The slides were treated with lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, pH 10, containing 1% Triton X-100 and 10% dithiothreitol) in the dark for 1 h at 4°C.

The slides were then placed in a horizontal electrophoresis unit and allowed to equilibrate for 20 min with buffer (300 mM NaOH, 1 mM Na2EDTA, pH 13) before electrophoresis (25 V, 300 mA) for 15 min.

When electrophoresis was complete, the slides were treated with neutralizing solution (0.4 M Tris-HCl, pH 7.5) for 5 min, fixed with methanol at 4°C for 3 min, then stained with propidium iodide 15 µM and mounted with a coverslip. Fluorescence was detected with the UltraView LCI confocal system.

Controversial opinions have been recently raised among those who deny (De Lamirande et al., 2012) and those who assent that the use of gradient centrifugation may induce DNA fragmentation due to potential contaminating compounds present in commercial solutions (Aitken et al., 2014). However, in our present and previous studies (Andrisani et al., 2014), both PS gradient and PSW prevented sperm from oxidative assault induced by ROS generation and, concomitantly, did not induce the increase of DNA fragmentation which remained at the same, or even lower, levels commonly observed with other separation/incubation methods (Xue et al., 2014). In addition, PS gradient, correctly utilised as recommended by the manufacture, completely prevents sperm contamination by other cells (leucocytes, immature sperm, debris et al,) as evidenced by our and other (Aitken et al., 2014) studies, thus guaranteeing the best preparation for ART.

Many comparisons have been made between the two most utilised techniques for sperm separation: density gradient centrifugation and swim up centrifugation. This last has been widely demonstrated to be by far less efficacious in eliminating non-sperm components (e.g. debris, bacteria) and contaminating substances (e.g. the prostatic zinc) from the semen (Bjorndahl et al., 2005). I addition, swim up methods induce higher level of ROS generation in the samples (Aitken et al., 2014) thus compromising both DNA fragmentation, as indicated in the present preliminary study, and the final ability of cells to undergo AR (Donà et al., 2011).

Bibliography

      • Aitken RJ, Finnie JM, Muscio L, Whiting S, Connaughton HS, Kuczera L, Rothkirch TB, De Iuliis GN. Potential importance of transition metals in the induction of DNA damage by sperm preparation media. Hum Reprod. 2014 Oct 10;29(10):2136-47.
      • Andrisani A, Donà G, Ambrosini G, Bonanni G, Bragadin M, Cosmi E, Clari G, Armanini D, Bordin L. Effect of various commercial buffers on sperm viability and capacitation. Syst Biol Reprod Med. 2014 Mar 27.
      • Björndahl L, Mohammadieh M, Pourian M, Söderlund I, Kvist U. Contamination by seminal plasma factors during sperm selection. J Androl. 2005 Mar-Apr;26(2):170-3.
      • De Lamirande E, San Gabriel MC, Zini A. Human sperm chromatin undergoes physiological remodeling during in vitro capacitation and acrosome reaction. J Androl. 2012 Sep-Oct;33(5):1025-35.
      • Donà G, Fiore C, Andrisani A, Ambrosini G, Brunati A, Ragazzi E, Armanini D, Bordin L, Clari G. Evaluation of correct endogenous reactive oxygen species content for human sperm capacitation and involvement of the NADPH oxidase system. Hum Reprod. 2011;26(12):3264-73.
      • Donà G, Fiore C, Tibaldi E, Frezzato F, Andrisani A, Ambrosini G, Fiorentin D, Armanini D, Bordin L, Clari G. Endogenous reactive oxygen species content and modulation of tyrosine phosphorylation during sperm capacitation. Int J Androl. 2011;34(5):411-9.
      • Malvezzi H, Sharma R, Agarwal A, Abuzenadah AM, Abu-Elmagd M. Sperm quality after density gradient centrifugation with three commercially available media: a controlled trial. Reprod Biol Endocrinol. 2014 Dec 2;12:121.
      • Xue X, Wang WS, Shi JZ, Zhang SL, Zhao WQ, Shi WH, Guo BZ, Qin Z. Efficacy of swim-up versus density gradient centrifugation in improving sperm deformity rate and DNA fragmentation index in semen samples from teratozoospermic patients. J Assist Reprod Genet. 2014 Sep;31(9):1161-6.

 

New collaboration

Nidacon has previously worked with EggCentris for all our QC testing but since they have closed down that part of their business, we are now collaborating with Embryotools in Barcelona, Spain. We are certain that Embryotools will provide us with the same high quality testing as Egg-Centris did and they will also be a useful partner for us in our research.

A presentation of the company;
Embryotools is a privately owned company that has been founded by two embryologists, Gloria Calderón, PhD and Nuno Costa-Borges, PhD. Both have in common a huge passion about assisted reproduction and hold an international reputation in the IVF field with several remarks along their careers. While Gloria was a member of the team that obtained the first IVF babies in Spain in the 80’s, Nuno carried out projects in animal research that led to the first successfully cloned animals in Spain and the first horses in Europe from biopsied embryos selected for gender selection. Combining more than 30 years’ experience in both clinical embryology and animal reproduction, they have decided to move away from the daily IVF routine when still working at IVI Barcelona Located at Barcelona Scientific Park (PCB), one of the areas of highest scientific and technical activities in Spain, Embryotools facilities include offices and state of the art laboratories equipped with the most advanced technology and equipment.

QC TESTING SERVICES
Embryotools offers a wide range of specialized and advanced services for testing all type of raw materials, equipment, culture media, devices and other disposable items that are used in the IVF Laboratory. All tests are designed by PhD level scientists with several years of experience with human and mouse embryos, giving suppliers and users’ full con-fidence in the materials from batch to batch.
Among other services, Embryotools offers mouse embryo assays (MEAs) with different degrees of sensitivity and information accor-ding to products’ and customer’s require-ments. In particular, time-lapse based MEAs, allowing detection of any potential toxic effect sooner than with any other assay.

IVF TRAINING
As a reference center in IVF training, Embryotools provides embryologists around the world the opportunity to attend advan-ced hands-on training courses. All sessions are run in a real IVF environment, with the most modern technologies and equipment available for hands-on, including time-lapse incubators, inverted microscopes equipped with adaptive electronic condensers, latest laser systems, micromanipulators of diffe-rent brands, pH meters, temperature, CO2 and humidity probes for the most advance level of QC & QA, different brands of standard and benchtop incubators.

IVF CONSULTING
Finally, Embryotools also provides indepen-dent scientific and clinical consulting services to reproductive centers to improve results with best practices and protocols. Embryo-tools also assists manufacturers on develo-ping and optimizing new products for IVF based on a long term experience and deep understanding of IVF in-dustry needs. Another important area of Em-bryotools work is related to validation of new products or devices ready to be released to the IVF market through scientific basic studies performed using the mammalian model.
For further information, please contact: Embryotools SL : info@embryotools.com


Nidacon products used for breeding world champions!

The sperm from the famous jump horse Casall, ridden by Olympic silver medalist Rolf-Göran Bengtsson, are worth gold.

To breed offspring from this horse is a very costly and delicate business. There-fore the sperm are handled with great care. The AI-veterinarians have chosen to use Nidacon products for the handling of semen from this horse. We are very proud!


Fun facts about Nidacon!

Do you know that of all men working at Nidacon, 57 % are left handed! In the world it’s about 11%.


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