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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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Email: contact@nidacon.com

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