Mistake in our news "Effect of various commercial buffers on sperm viability and capacitation".

News   •   Apr 15, 2019 15:49 CEST

There was a mistake in the photo, the product is PureSperm Wash and not PureSperm Buffer as it appears in the photo. We apologize for the mistake.

Andrisani A, Donà G, Ambrosini G, Bonanni G, Bragadin M, Cosmi E, Clari G, Armanini D, Bordin L

Infertility affects 10-15% of couples trying to conceive and assisted reproduction techniques (ART) such as intrauterine insemination (IUI) and in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) can increase the possibility of conception. Sperm preparation protocols currently available for assisted conception include density gradient separation and washing methods, that are aimed at isolating and capacitating as much motile sperm as possible for subsequent oocyte fertilization

Sperm preparation in washing buffers is a very important step in maintaining the correct physiological activities of cells. The procedure widely adopted to assess cell viability is normally restricted to evaluation of flagellum motility/hyperactivation, a parameter which is insufficient for sperm characterization.

Recent studies emphasized the importance of the evaluation of novel parameters, such as biochemical analysis of the endogenous content of reactive oxygen species (ROS) and the level of Tyr-phosphorylation of the cells, as mostly efficacy features depicting the correct status of sperm. (Donà et al., 2011). In fact, only when the above mechanisms are correctly satisfied, sperm may be considered capacitated and can undergo acrosome reaction. Capacitation is a set of alterations leading to the acrosome reaction (AR), an exocytotic process mediated by hydrolytic enzymes (e.g acrosin) that are released to allow sperm to fertilize the oocyte. What is to be underlined is that one of the most delicate step in the ART procedure is represented by the correct sperm preparation, including initial washing and final incubation leading to the precious achievement of the capacitated status.

A recent study (Andrisani et al, 2014.) compared the effect of different commercial buffers according to their capacity to induce ROS production, the Tyr-P of the head and consequently, AR, paying particular attention to the cell survival at the end of 2h incubation. Interestingly, PSW-Nidacon was by far the best medium for sperm preparation: in fact, cells reaching the AR (67.2±7.9%) 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 (Fig. 1).


(Andrisani et al., 2014)

Sperm, incubated for 180 min in capacitating conditions in different buffers, were analysed for Tyr-P pattern, AR and viability (NVC) by immunofluorescence cytochemistry as described in Methods. Number of cells expressed as % of the total amount of cells showing Tyr-P in any part of the cell-body, or in the head, were detected and reported as Tyr-P cells and Tyr-P head, respectively. Percentage of cells undergoing acrosome reaction or not viable cells (AR and NVC, respectively) were also reported.

Spontaneous AR percentages were also evaluated in each sample in the absence of A23187 and values reported (SAR).

**p<0.0001 and *p<0.02, comparing each sample against T0 (ANOVA followed by Dunnett’s post hoc test). Values are expressed as the mean ±SD.

The study also addressed the well-known problem on how buffers must be stored, since human serum albumin, glucose, lactate etc. promote bacterial/fungal contamination, thus resulting in increased buffer ROS production with a consequent fast sperm denaturation and inability to reach the capacitated state.

Capacitation implies marked reorganization of membrane architecture, due to the activity of extracellular proteins which have the task of extracting cholesterol and reorganizing membrane in specialized microdomains, also called rafts, capable of remodelling and reorganizing themselves to undergo acrosome reaction.

Once capacitation has occurred, these rafts migrate from the flagellum, where they are found extensively, to the peri-acrosomal region where they presumably allow interaction with the oocyte . The buffers used in the ART for sperm preparation must induce optimal conditions to achieve AR, an, once more, PSW clearly showed the best membrane reorganization to achieve the potential AR (Fig. 2).

PSW-Nidacon is an optimal sperm medium to prepare sperm to undergo the potential successive AR. And to prevent time-dependent denaturation.

In order to avoid any complications, it is recommendable to use fresh buffer avoiding prolonged storage.

(Andrisani et al., 2014)

Sperms, incubated for up to 180 min in capacitating conditions in different buffers, were analysed for CTB labeling by immunofluorescence cytochemistry A) T0, control non-capacitated cells; sperm incubated in B) PSW-Nidacon ; C) PSW-Nidacon* D) Number of cells expressed as % of the total amount of cells showing CTB labeling in flagellum, head, or both as transient conditions, were detected and reported.

†† p<0.0001 and †p<0.02, comparing each sample against T0 (ANOVA). Values are expressed as the mean ±SD.

  • 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. [Epub ahead of print]
  • 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. 

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Effect of various commercial buffers on sperm viability and capacitation.

News   •   Apr 15, 2019 14:00 CEST

Andrisani A, Donà G, Ambrosini G, Bonanni G, Bragadin M, Cosmi E, Clari G, Armanini D, Bordin L

Infertility affects 10-15% of couples trying to conceive and assisted reproduction techniques (ART) such as intrauterine insemination (IUI) and in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) can increase the possibility of conception. Sperm preparation protocols currently available for assisted conception include density gradient separation and washing methods, that are aimed at isolating and capacitating as much motile sperm as possible for subsequent oocyte fertilization

Sperm preparation in washing buffers is a very important step in maintaining the correct physiological activities of cells. The procedure widely adopted to assess cell viability is normally restricted to evaluation of flagellum motility/hyperactivation, a parameter which is insufficient for sperm characterization.

Recent studies emphasized the importance of the evaluation of novel parameters, such as biochemical analysis of the endogenous content of reactive oxygen species (ROS) and the level of Tyr-phosphorylation of the cells, as mostly efficacy features depicting the correct status of sperm. (Donà et al., 2011). In fact, only when the above mechanisms are correctly satisfied, sperm may be considered capacitated and can undergo acrosome reaction. Capacitation is a set of alterations leading to the acrosome reaction (AR), an exocytotic process mediated by hydrolytic enzymes (e.g acrosin) that are released to allow sperm to fertilize the oocyte. What is to be underlined is that one of the most delicate step in the ART procedure is represented by the correct sperm preparation, including initial washing and final incubation leading to the precious achievement of the capacitated status.

A recent study (Andrisani et al, 2014.) compared the effect of different commercial buffers according to their capacity to induce ROS production, the Tyr-P of the head and consequently, AR, paying particular attention to the cell survival at the end of 2h incubation. Interestingly, PSW-Nidacon was by far the best medium for sperm preparation: in fact, cells reaching the AR (67.2±7.9%) 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 (Fig. 1).

(Andrisani et al., 2014)

Sperm, incubated for 180 min in capacitating conditions in different buffers, were analysed for Tyr-P pattern, AR and viability (NVC) by immunofluorescence cytochemistry as described in Methods. Number of cells expressed as % of the total amount of cells showing Tyr-P in any part of the cell-body, or in the head, were detected and reported as Tyr-P cells and Tyr-P head, respectively. Percentage of cells undergoing acrosome reaction or not viable cells (AR and NVC, respectively) were also reported.

Spontaneous AR percentages were also evaluated in each sample in the absence of A23187 and values reported (SAR).

**p<0.0001 and *p<0.02, comparing each sample against T0 (ANOVA followed by Dunnett’s post hoc test). Values are expressed as the mean ±SD.

The study also addressed the well-known problem on how buffers must be stored, since human serum albumin, glucose, lactate etc. promote bacterial/fungal contamination, thus resulting in increased buffer ROS production with a consequent fast sperm denaturation and inability to reach the capacitated state.

Capacitation implies marked reorganization of membrane architecture, due to the activity of extracellular proteins which have the task of extracting cholesterol and reorganizing membrane in specialized microdomains, also called rafts, capable of remodelling and reorganizing themselves to undergo acrosome reaction.

Once capacitation has occurred, these rafts migrate from the flagellum, where they are found extensively, to the peri-acrosomal region where they presumably allow interaction with the oocyte . The buffers used in the ART for sperm preparation must induce optimal conditions to achieve AR, an, once more, PSW clearly showed the best membrane reorganization to achieve the potential AR (Fig. 2).

PSW-Nidacon is an optimal sperm medium to prepare sperm to undergo the potential successive AR. And to prevent time-dependent denaturation.

In order to avoid any complications, it is recommendable to use fresh buffer avoiding prolonged storage.

(Andrisani et al., 2014)

Sperms, incubated for up to 180 min in capacitating conditions in different buffers, were analysed for CTB labeling by immunofluorescence cytochemistry A) T0, control non-capacitated cells; sperm incubated in B) PSW-Nidacon ; C) PSW-Nidacon* D) Number of cells expressed as % of the total amount of cells showing CTB labeling in flagellum, head, or both as transient conditions, were detected and reported.

†† p<0.0001 and †p<0.02, comparing each sample against T0 (ANOVA). Values are expressed as the mean ±SD.

  • 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. [Epub ahead of print]
  • 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. 

The aim of this study was to examine the effects of four commercial sperm washing buffers on sperm viability and capacitation. Distinguishing each particular pathological state of the sperm sample would be helpful to select the preferred buffer treatment since both ROS production and membrane reorganization can be significantly altered by commercial buffers.

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Comparison of outcomes after vitrification using DMSO or PrOH.

News   •   Apr 01, 2019 14:00 CEST

Trichomona vaginalis a latent intruder in unexplained infertility?

News   •   Mar 18, 2019 14:00 CET

The incidence of T. vaginalis infection has been increasing around the world over the past decade. This has lead to insight of the importance of this microorganism as a serious problem in reproductive health outcomes including pelvic inflammatory disease, pregnancy complications and an increased risk of HIV acquisition.

P-038 Evaluation of the effects of different in vitro incubation conditions on sperm DNA integrity

News   •   Mar 04, 2019 14:00 CET

M. Bungum; N. Forsell; A. Giwercman  

Author Affiliations - Skane University Hospital, Reproductive Medicine Centre, Malmö, Sweden 


Abstract

Introduction:

Prolonged in vitro incubation of spermatozoa has been shown to have adverse effects on sperm motility, vitality as well as on DNA integrity. Knowledge regarding how shorter incubation periods prior to the IVF/ICSI procedure affect semen quality is however limited. The aim of the present study was to examine if sperm DNA integrity was affected during incubation in three different conditions for 2 hours after sperm preparation prior to the IVF/ICSI procedure.

Materials and Methods:

Density gradient centrifuged samples from two hundred men undergoing infertility work-up were included in the study. Following gradient centrifugation one reference sample was frozen immediately. Thereafter samples were divided into three aliquots and incubated for two hours in either1) room temperature (23-24 °C); 2) in a 37°C humidified incubator with 6%CO2 and 5%O2 or 3) in a 37°C humidified incubator with atmospheric air. The Sperm Chromatin Structure Assay (SCSA) was used to assess the extent of sperm DNA damage. Sperm DNA fragmentation was expressed as DNA fragmentation index (DFI).

Results:

A statistically significant increase in DFI was seen in density gradient prepared samples incubated for 2 hours at 37°C, 6%CO2 and 5%O2 compared to the reference sample taken immediately after preparation. This was the case also for samples incubated at 37°C in atmospheric air. Moreover, statistically significant lower DFI levels were seen in the group incubated at room temperature compared to those incubated at 37°C, 6%CO2 and 5%O2 or at 37°C in atmospheric air.

Conclusions:

In order to prevent against further sperm DNA damage after density gradient preparation prior to the IVF/ICSI procedure, spermatozoa should be stored at room temperature.


In order to prevent against further sperm DNA damage after density gradient preparation prior to the IVF/ICSI procedure, spermatozoa should be stored at room temperature.

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The device that will make your sperm preparation easier

News   •   Feb 18, 2019 14:00 CET

Sperm preparation from a viscous semen sample

News   •   Feb 04, 2019 14:00 CET

It can be extremely difficult to obtain good yields of motile sperm from highly viscous semen samples.

What happens in the semen between ejaculation and fertilization?

News   •   Jan 14, 2019 14:00 CET

It is a common misconception that the ejaculate is presented as a homogenous fluid at ejaculation and offers a stable and good environment for the sperm. The reality is that the sperm are ejected together with the zinc-rich prostatic secretion in the first ejaculate fraction.

Nidacon will be closed for Christmas Holidays from December 24th until January 1st 2019

News   •   Dec 18, 2018 11:06 CET

Tips and Tricks December 2018 - About Shelf Life

News   •   Dec 06, 2018 09:23 CET

Consideration about shelf life of Nidacon products

Nidacon is conscious of customer requirements and always tries to provide products which are convenient. This convenience includes ease of transportation and long shelf life

Tips and Tricks

Nidacon products have a shelf life of one to two years From Production at room temperature. Notice that after distribution the remaining shelf life will be shorter. We try to ensure you have the longest possible shell life.

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About Nidacon International AB

Nidacon a Swedish company, manufactures and markets medical devices, mainly for Assisted Reproduction Technologies (ART).

We continually strive to improve the outcome of Assisted Reproduction Technologies (ART), with more pregnancies, by developing superior media systems for clinics, patients, and the animal breeding industry.

Address

  • Nidacon International AB
  • Flöjelbergsgatan 16 B,
  • 431 37 Mölndal
  • Sweden

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