Product Manual - Nidacon Your Swedish IVF
Contents
1. 0 Nidacon Nidation Conception 1 The building of a nidus a nest as with birds 1 The union of male and female gametes the sperm and egg 2 Implantation of the fertilized ovum zygote and the building of a nest in the endometrium 2 An impression or idea the placenta n T ES z E T En ee b _ a T di T A E i e ok r e PT een aft Li TT e de a P E AU AA kA Lal D r is f LES Je DK Ak Gesi uw Pe A d s n T nml qe a wv CAR wk P pr En d jy oe e DUNS T an pr ye A as e 1 P e E g en 3 r e HE WD ld e d me td tee Deeg OG fae NN Aw n e TM Xt m a si re ai J TW 7 S E e IW I L us p er as a F i 1 E J4 va k a bie e a i we i D E za Ee E Se Ae ue to fn Lors ke i A A el e de P Es e LO P m ets g Pos as L s LE er La lt Aa Ci E gp s ch a aa E a a kee gt j i Aa sd D E BR F e s a P la d IW A ST ER TE d P A Ce i e a d Ka j kat e e a aJ Ca T Ce z i k A v BS hee Des KT Y ee pue al i ke E zi P i s E ee iy tT re ET Pula Ta Bei a e TZ Fee e cr ue ana V 4 E 2 wi e e i LA endi a JJ S ch CES LE he 2 p jea r1 P H DEP e os Se A ITA ag A S de Mm OA A ech dl passt cT A d kafi F n a Gw TT Wa ed al Pe A D e o Th A e F P E DAT UM di 11 ba t del j pi EC m i es J a e wa Fl2. Note Using drops reduces the risk of loosing the blastocyst The blastocyst tends to float on the viscous solution 3 It is also important to incubate solution 3 under the same conditions as the other two solutions 15 Attach the tube to the Cryocane for storage in hence the use of 1 mL liquid nitrogen 26 Y Nidacon Vinficaton and warming of blastocysts Warming procedure 1 Label the NUNC dish with the patient ID and each respective well with each solution number i e 4 9 6 Pipette the warming media 4 5 and 6 as described below Well 1 ThermoBlast 4 1000 uL Wel 2 ThermoBlast 5 1000 uL Well 3 ThermoBlast 6 1000 uL o Incubate at 37 C in 5 6 CO for 30 minutes 4 Carefully detach the cryoloop from the cryo tube making sure not to touch the inside of the tube with the cryoloop the blastocyst may be lost Unscrewing the top and moving the cryoloop from the tube are the most risk filled moments during this procedure 5 Immerse only the loop in the surface of solution 4 Let the blastocyst fall off Identify its presence in the well and incubate for 2 minutes on the heating stage 2 minutes includes time for locating the blastocyst Transfer the blastocyst to solution 5 Let the blasto cyst simply sink to the bottom do not wash Incubate for 3 minutes in solution 5 Transfer the blastocyst to solution 6 Incubate for 5 minutes Transfer the blastoc
3. thus diminis hing damage due to ice crystal formation seminal plasma as well as ROS and their sources thereby ensuring optimal recovery of motile sperm on thawing Disposable sterile cryopreservation vials or plastic straws Scissors CryoFloater Nidacon Seria spen ssmmole SINUS uL uL uL oon 2100 700 400 2200 138 433 2300 ot 467 2400 800 500 2500 833 ES 2600 867 567 2700 900 600 2800 a 635 2900 967 667 3000 1000 Example 300 uL Sperm Sample 3 100 uL SCPII 1 The incubation time before freezing can be reduced to 15 minutes but we recommend 60 minutes Y Nidacon 19 Freezing of spermatozoa Processed ejaculate 1 Add 1 part of Sperm CryoProtec II to 3 parts of sample see dilution table ensuring thorough mixing after adding each drop 2 Fill straws with sperm suspension or aliquot into vials 5 Equilibrate straws or vials in refrigerator for 30 60 minutes d Unprocessed ejaculate 1 Measure the volume of the ejaculate 2 Mix ejaculate with SCPII dilution 1 3 see table ensuring thorough mixing after adding each drop in order to avoid osmotic shock 3 Transfer 0 8 1 8 ml of the mixture to 2 ml cryovials 1 2 5 H lt 4 Place the straws horizontally in nitrogen vapour above the liquid nitrogen surface on a piece of styro foam CryoFloater Leave for 30 minutes Transfer the straws quickly into the liquid nitrogen and thereafter store in liquid
4. FDA approved Limulus Amoe bocyte Lysate LAL test using a quantitative spec trophotometric method in order to obtain real values with the units EU mL according to the U S Pharmacopoeia The test is done by the accredited laboratory of the Microb iology Institute at the Sahlgrenska University Hospital of Gothenburg Sweden Biological analyses Human sperm test The biological efficacy assay involves assessment of yield motility viability measured both subjectively and using computer assisted sperm analysis Hamilton Thorne IVOS Each batch is tested biologically using human semen samples The samples are separated into two parts the one part being used for control and the second part being used for preparing sperm with the new batch The test batch results are compared with the results from the control The analyses provide a count of the sperm per mL the sperm activity is graded and the activity is also QUALITY ASSURANCE CERTIFICATE d Colloidal Stock Sperm PureSperm 100 k y y l i De f Si Botech 250PS100VD22 Production date 22 April 2015 Expiry date quality of this parti gt cular bate procedures atch was tested and evaluated objectively by stri rin O uality Control Procedures r medical devices The gent quality control Accepted values Test results Physical analyses EH at roni temperat ure in cjr 7 Osmolality mOsm Seid eina 300 315 Comply Sterility ond
5. S Zavaczki Z Hansch E Vigue L Fertil Steril 2003 Jun 79 Suppl 3 1616 24 Why the WHO Recommendations for Eosin Nigrosin Staining Techniques for Human Sperm Vitality Assessment Must Change Lars Bj rndahl et al Journal of Andrology Vol 25 No 5 September October 2004 Washed paraffin oil becomes toxic to mouse embryos upon exposure to sunlight Provo M B amp Herr C 1998 Theriogenology 49 214 Phenol red in tissue culture media is a weak estrogen Implica tions concerning the study of estrogen responsive cells in culture Y Berthois et al Proc Natl Acad Sci 1986 Vol 83 pp 2496 2500 Effects of 17 3 estradiol on in vitro maturation of pig oocytes in protein free medium Qing Li et al Journ of Repr and Development 2004 Vol 50 No3 Impact of estrogenic compounds on DNA integrity in human spermatozoa Evidence for cross linking and redox cycling ac tivities L E Bennets et al Mutation Research 641 2008 1 11 Oogenesis in cultures derived from adult human ovaries A Bukovsky et al Repr Biol and Endocr 2005 3 17 doi 10 1186 1477 7827 3 17 Estrogenic compounds and oxidative stress in human sperm and lymphocytes in the Comet assay Anderson D et al Mutat Res 2003 544 2 3 173 8 The use of two density gradient centrifugation techniques and the swim up method to separate spermatozoa with chromatin and nuclear anomalies D Sakkas Manicardi GC Tomlinson Human Repr 2000 May 15 5 11
6. apart from each other horizontally You then have two good slides 4 Air dry the smears lips 1 Usea pencil to mark your sample slides since the stain will remove permanent markers Morphology Staining The sperm will be stained in a darker colour blue and the background will be lighter Consequently the shape size and integrity of the sperm can easily be determined using 100x objective oil immersion microscopy Light microscope 40 100 x objectives Pipette 5 Dip the dry smears into the staining solution for 8 seconds O Rinse in double distilled water changing the water 3 times Let slides air dry lying flat 7 Mount the slides with coverslips and DPX or equivalent mounting fluid and let them dry com pletely before examination 8 Examine using a bright field 100 x objective under oil immersion 9 Classify at least 200 sperm classification according to the 2002 NAFA and ESHRE SIGA manual on Basic Semen Analysis Y Nidacon 29 References References Penicillin degradation products inhibit in vitro granulopoiesis Neftel KA et al Br J Haematol 1983 54 2 255 60 Adverse reactions following intravenous pc g treatment to de gradation of the drug in vitro Neftel et al Kliniche Wochen Schrift 1984 62 25 29 Effects of f Lactam antibiotics on profilerating eukaryotic cells Neftel et al Antimicrobial Agents and Chemotherapy 1987 p 1657 1661 The antibiotic streptomycin assessed in
7. assurance control helps maintain our standard for low endotoxin levels and also ensures our products are free from microbiological contamination There have been several reports of paraffin oils becoming embryo toxic after exposure to light on the laboratory bench As a precaution against any possible light induced changes NidOil is packaged in amber screw top bottles A prospective randomized study to compare four different mineral oils used to culture human embryos in IVF ICSOI therapy Presented at ESHRE 2008 by Dr C Sifer Paris No of cycles 127 126 120 DIS GQE day 3 Comparison between 1 Mineral Oil CryoBioSystem 3 Liquid Paraffin MediCult 3 NidOil Nidacon 4 Ovoil Vitrolife Recommendations before use NidOil should be equilibrated in the same way as the culture medium before use to avoid differences in New Quality Assurance test Many questions has been raised lately to whether the oil that is used for coverage in embryo culture can actually damage the embryo All oil batches today from different manufactures are tested for sterility endotoxins and a mouse embryo assay showing blastocyst development This is apparently not enough since damage to cultures has been observed with an approved batch of oil One answer could be peroxidation of the oil which has been investigated in several publications and found to be harmful to fertilisation and embryo development when over a certain level It ha
8. from the ejaculate Combine sperm pellets if double procedure has been used 8 Centrifuge at 500 x g for 10 minutes Do not use the brake 9 Aspirate PureSperm Wash super natant leaving as little liquid as possible above the pellet If no pellet is seen leave the bottom 0 25 mL fluid 10 Resuspend the sperm pellet in a suitable volume of media The sample is now ready for use Immotile dead sperm debris epithelial cells leucocytes bacteria Immature amp senescent sperm Motile sperm Y Nidacon Denstty Gradient Preparation callate trie cerne lo ace wie Correct e IOTes USE mie estela Rpm VE g 1 118 x 2 x 10 g the centrifugal force r rotational radius the distance mm from the centre 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 800 1 118 x 165 x 10 1275 Conversion table concert between times gravity x g and centrifuge rotor speed RPM http cabinet weblog com pt arquivo TROO40dh4 Centrifuge speed pdf G Force RPM calculator http drycake com calculator gforce php C lips 1 Gradients should be layered immediately prior to i When retrieving the pellet after the gradient centri use but the different density solutions of PureSperm can be prepared in advance provided t
9. morphology Vitrification procedure using the cryoloop Additional equipment Cryocane for storage of cryo tubes Crystalwand Vial Clamp for holding the cryo tube Cryoloop Hampton Research Note If the additives are stored in the refrigerator remove them in good time prior to use DMSO turns solid below 18 C The additives can be stored outside the refrigerator in the supplied packaging even after opening If urgent the DMSO bottle can be warmed by holding the bottle in your hand 1 Label the NUNC dish with the patient ID and each well with corresponding solution number ie 1 2 and 3 rial makes it possible to freeze cells while at the same time avoiding the formation of intracellular ice crystals The use of the vitrification technique results in a very homogenous structure an amorphous crystalline structure Work on a heated stage at all times when manipulating the blastocyst Do not let the blastocyst remain exposed to the microscope light during incubation Liquid nitrogen reservoir Liquid nitrogen Culture dishes NUNC 4 well Heated stage Inverted microscope Hatching blastocyst after vitrification and warming Pipette the vitrification media as described below When adding the DMSO and Ethylene glycol EG which are included in the kit to solutions 2 and 3 pipette the two up and down a few times to obtain optimal mixing of the media Well e VitriBlast Well e VitriBlast 2 DM
10. must be adjusted very specifically to avoid ionic shock and subsequent hyperactivation Product composition The component salts of Nidacon s products are balanced with specific regard to the ion composition of both the ejaculate and the female reproductive tract This balance ensures a smooth transition from ejaculate to fertilisation medium via the gradient and wash Buffer The zwitterion buffer HEPES is included to maintain the pH of the PureSperm gradient and PureSperm Wash while working with the sperm on the bench Fluids desig ned to maintain pH in a CO environment i e in the incubator are unsuitable for use outside the incubator as they do not possess sufficient buffering capacity to maintain the pH Fluctuations in pH and temperature are detrimental to sperm survival on the bench In addition HEPES has an anti oxidant effect reducing reactive oxygen species ROS which can be damaging in the sperm preparation Glucose Glucose is a component of PureSperm products Glucose is the primary energy substrate available to sperm in the human female repro ductive tract Product composition Antibiotics Antibiotics are not included in our products for several rea sons Penicillin G a commonly used antibiotic in cell culture medium only lasts for approximately 10 days in aqueous solution being inactivated after this time and the degradation products are cell toxic Furthermore this antibiotic is ineffec
11. nitrogen Do not touch the straw with your hand Place the vials in the fridge 4 5 C for 30 min Freeze the vials horizontally in the freezer or in nitrogen vapour above the liquid nitrogen surface on a piece of styrofoam CryoFloater Leave for 30 min Store in liquid nitrogen 20 Y Nidacon Thawing procedure processed ejaculate 1 Remove the straws from the liquid nitrogen tank 2 Place straws in water at 37 C for 30 secs 3 Dry surface of straw 4 Cut one end of straw 5 Hold the straw over a tube with 5 mL PureSperm Wash and cut the other end of the straw Any sperm suspension remaining in the straw can be expelled using a pipette f U gt Thawing procedure unprocessed ejaculate 1 Remove the vials from the liquid nitrogen tank 2 Place vials in water at 37 C until all ice crystals are gone approximately 2 3 min 3 Dilute the thawed material with 0 5 ml Pure Sperm Wash 4 Prepare the thawed material on a 40 and 80 PureSperm density gradient Use 1 ml of each for the gradient and layer not more than 1 ml of the thawed ejaculate onto the gradient AY fl 2 3 9 9 7 3 fee oa ane y Vr Freezing of spermatozoa Centrifuge at 500 x g for 10 minutes Do not use thebrake Aspirate PureSperm Wash supernatant leaving as much liquid as required for desired concentration If no pellet is seen leave the bo
12. required sperm concentration The sample is now ready for analysis or use lips 1 If you have a viscous sample be extra careful when you re move the upper layer after incu bation It is easy sample and disrupt the v layers semen to get hold of the 18 Q Nidacon Sperm CryoProtec II Background The cryoprotectant in SpermCryoProtec II is glycerol the proportion being reduced as far as possible to minimize toxicity to sperm while still providing cryoprotection Recommendations Although it is possible to freeze unprocessed semen we recommend that you prepare the ejaculate using a PureSperm density gradient This method removes Reagents and Equipment Sperm CryoProtec II and PureSperm Wash Sterile pipettes Disposable sterile centrifuge tubes e g Falcon 2075 Dilution table Sperm Sample Seri Sperm Sample uL uL uL 100 DE 1100 200 67 100 300 100 1300 400 do 1400 500 167 1500 600 200 1600 700 SE 1700 800 en 1800 900 300 1900 1000 599 2000 For other volumes than those listed calculate Volume Sperm Sample 3 Volume SCPII Tips 1 To avoid osmotic shock for the sperm it is import ant to slowly mix Sperm CryoProtec II with your sperm sample but don t mix for longer than 5 minutes since glycerol is toxic to cells at RT Freezing of spermatozoa Moreover a high concentration of glucose is present as an osmotic agent to reduce intracellular water
13. the reagent is oxidised to Fe3 by peroxidise present in the Functional analysis Efficacy test used to prove the efficacy and function of the products Visual control constant visual control during production filling labeling and final control of chosen ready packages shelf life Packaging Shelf life 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 Therefore most of the products have a shelf life of one to two years at room temperature Packaging The packaging for Nidacon s products has received the same care and attention to detail as the design of the products Bottles For most of our products we have chosen borosilicate glass instead of sodium silicate glass to avoid the leaching of sodium from the bottles into the contents during the long shelf life Research in our laboratory has shown that sufficient sodium ions can leach from a sodium silicate bottle to have a negative effect on the development of two cell mouse embryos Therefore we avoid exposing all cells to raised sodium ion levels in the products by packaging in borosilicate glass Sperm Morfo Stain 150ml are wire Gert wg UE All ingredients are chosen for their temperature tolerance and their stability in aqueous solution Rigorous shelf life testing has been carried out in Nidacon s laboratory to ensure that
14. the theoretical stability of the salt formulations is matched by their actual stability when combined in the product Stoppers Based on embryo toxicity testing of three types of commercially available rubber stoppers approved for pharmaceutical use today Nidacon chose silicone rubber as the material for the stoppers We found that both natural latex rubber and butyl rubber are toxic to embryos preventing development and possibly causing embryonic death Silicone rubber did not have any detrimental effect allowing embryonic development and hatching to proceed as usual Therefore stoppers made from pharmaceutical silicone rubber were chosen for our products This convenience includes ease of transportation and long shelf life 6 Y Nidacon Background Under normal physiological circumstances sperm under go a series of maturation changes after ejaculation which enables them to negotiate the different sections of the female reproductive tract and eventually locate and fertilise the egg If sperm are to be used for ART it is essential that any product which is used for sperm preparation must match the sperm s physiological requirements as closely as possible If sperm are stimulated excessively particularly ionically they become hyper active a process which results in the sperm using up its energy resources and dying before fertilisation is achieved Therefore the pH and osmolality of the sperm solutions
15. 12 6 Recovery and survival of sperm is higher with PureSperm den sity gradient than swim up in neat and cryo preserved thawed semen specimen P Raganathan A Agarwal Fertility amp Sterility 2001 Elimination of bacteria from human serum during sperm pre paration using density gradient centrifugation with a novel tube insert Fourie J et al 2012 Andrologia 44 513 517 Physiologic ICSI Hyaluronic acid HA favours selection of spermatozoa without DNA fragmentation and with normal nucleus resulting in improvement of embryo quality Parmegiani L Cognigni GE Bernardi S et al Fertility and Ste rility 2009 Advance online publication Vitrification of mouse and human blastocysts using a novel cryoloop container less technique Lane M et al 1999 Fertility and Sterility Vol 72 No 6 pp1073 1078 Perinatal outcome of blastocyst transfer with vitrification using cryoloop A 4 year follow up study Takahashi K et al 2005 Fertility and Sterility Vol 84 No 1 pp88 92 Artificial Shrinkage of blastocoele using either microneedle or laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts Mukaida T et al 2006 Human Reproduction Vol 21 No 12 pp3246 3252 Obstetric outcomes of transfer of vitrified blastocysts Wikland M et al 2010 Human Reproduction Vol 25 No 7 pp 1699 1707 30 Y Nidacon MN Contacts N e ma Produ
16. PS e ges dl P 5 7 IX s HS I 2 s Pi A ef i A he P e eg m LI lt a p ra A A A D we a gt ae La i Pa ds E x e Ge am E i k mII Luis mm 23 n r1 a n _ d dis m D BR a i T d e F d 5 e 4 r e 1 _ he CH A P ef P a EY FRA Ee l ei p e D TE LI 4 z pe d s a F t on E LAT TR a L La Pr ec D AJ b e Contents nasa A nee ae nen ee Ee 4 o 5 SS 6 A o o E 6 a aE Oe Te ers eeu LE ove Ee 7 Products Ordering Informante 8 o A II ee ere eee ee eae 9 12 se lea natal eo e E E E A A en AA 13 Density gradient preparato cometa tem ode ta ainia 14 15 Ee 16 lazo nets O PE En o Deacon igen ecto nee 17 EP ETE E E 0 ene ane eee eae 18 eeben 19 21 Eege ER Ehre 23 Vitrification and warming of blastocgsts s 24 27 PP e dup e M DUM D EU 28 Morphology ta ENT 29 A ee eee 30 We Sl Contents Introduction e Quality Introduction Nidacon International AB manufactures and sells Medical Devices mainly for Assisted Reproduction Technologies ART with IVE ICSI and insemination IUI solutions The company was founded in 1996 by Assoc Prof Paul V Holmes MSc PhD DrMedSc an embryologist and endocrinologist from the Dept of Obstetrics and Gynaecology at Sahlgrenska University Hospital in Gothenburg Sweden Quality Nidacon is certified according to SS EN ISO 9001 implemen ted 2000 12 15 and SS EN I
17. PureSperm Wash does not contain any antibiotics and since swim up cannot guarantee removal of bacterial contamination it is recommended to add antibiotics Reagents and Equipment PureSperm Wash Round bottomed centrifuge tubes Disposable sterile conical centrifuge tubes Procedure 1 Transfer 1 mL of liquefied semen to a sterile round bottomed centrifuge tube If the sample is too viscous try diluting it with PureSperm Buffer before 2 Use a new pipette to carefully layer 1 5 mL PureSperm Wash over the semen 5 Without disturbing the layers place the centrifuge tube at a 45 angle into a CO incubator at 37 C for 60 minutes 4 Carefully remove the uppermost 0 5 1 0 mL of medium containing motile sperm using a sterile pipette V PureSperm Wash is a salt solution balanced and adjusted for the nutrition and long survival of human sperm It functions exceedingly well in this role when using swim up to prepare sperm for ART We recommend that you add Penicillin at a concentration of 100 U mL Sterile pipettes CO incubator Bench top centrifuge with swing out rotor 5 Place this fluid in a sterile conical centrifuge tube containing 5 mL PureSperm Wash O Centrifuge at 500 x g for 10 minutes Do not use the brake 7 Aspirate the supernatant leaving no more than 2 mm depth of liquid above pellet 8 Resuspend the sperm pellet in a suitable volume of medium to obtain the
18. SO EG Well amp VitriBlast 3 DMSO EG 24 Y Nidacon 1000 pL 850 uL JoL To IL 700 uL 150 uL 150 uL Vinficaton and warming of blastocysts Vitrification procedure using the cryoloop 5 Incubate at 37 C in 5 6 CO for 30 minutes maximum for 1 hour Longer time makes it difficult to create a film on the loop 4 During the 30 minute incubation of the dish collapse the blastocyst This can be done either by laser Fertilase red 5 see pictures below or by using an ICSI pipette Laser If laser is used shoot as far from inner cell mass ICM as possible The laser beam shoots vertically aim as illustrated below Be sure that you create a hole through the zona and the trophectoderm ICSI pipette 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 the11 o clock position 5 Remove the NUNC dish from the incubator and place it on a heating stage make sure the heat controller is set high enough to obtain 37 C in the media 6 Place the punctured and collapsed blastocyst in solution 1 Start the stop watch 7 After 1 5 2 minutes transfer the blastocyst by aspira ting solution 2 into the pipette tip collect the blastocyst from solution 1 and transfe
19. SO 13485 implemented 2003 08 15 The management system secures continued development of the organisation We register our products according to the valid directives and requirements 0434 for all different countries This also ensures our high quality on the market and it shall continue to be our beacon Nidacon intends to always maintain the high quality of its products and in order to achieve this all batches are Nidacon considers many different factors when desig ning its products We hope that the attention to detail has helped to create products which will lead to better results We aim to work in close relation with our customers they are the cornerstones of our research department We take pride in the develop ment of our products and make sure we respond to the needs of our customers and research colleagues All our products are developed in close cooperation with professionals in the different fields One of the first products to result from the company s research and development PureSperm was introduced onto the market in November 1996 It has gained rapid acceptance and is now the global market leader for isolation and preparation of sperm used in human assisted reproduction It was the first product of its kind to achieve both 510 k clearance from the US FDA and CE marking with the Euro pean authorities tested at Nidacon before they are cleared for the market Sterility controls are performed on each b
20. a battery of in vitro tests for reproductive toxicology K Lemiere et al Toxicology in Vitro 2007 21 1348 1353 An aminoglycoside antibiotic gentamicin induces oxidative stress reduces antioxidant reserve and impairs spermatogene sis in rats K Narayana J Tox Sci 2007 33 1 85 96 Bacterial contamination and sperm recovery after semen preparation by density gradient centrifugation using silane coated silica particles at different g forces C M Nicholson L Abramsson S E Holm and E Bjurulf Human Reproduction Vol 15 No 3 662 666 March 2000 Contamination by seminal plasma factors during sperm selec tion Bj rndahl L Mohammadieh M Pourian M S derlund I Kvist U J Androl 2005 Mar Apr 26 2 170 3 Platelet activating factor significantly enhances intrauterine in semination pregnancy rates in non male factor infertility W Roudebush A Toledo H Kort D Mitchell Leef C Elsner J Massey Fertility and Sterility Volume 82 Issue 1 Pages 52 56 An alternative to PVP for slowing sperm prior to ICSI Balaban B Lundin K et al Hum Reprod 2003 Sep 18 9 1887 9 Detrimental effects of polyvinylpyrrolidone on the ultrastruc ture of spermatozoa Strehler E Baccetti B Sterzik K Capitani S Collodel G De Santo M Gambera L Piomboni P Hum Re prod 1998 Jan 13 1 120 3 Hyaluronic acid binding by human sperm indicates cellular ma turity viability and unreacted acrosomal status Huszar G Ozenci CC Cayli
21. atch manu factured the endotoxin level is measured and biological efficacy tests are carried out A batch is only released for sale if it meets specific criteria Each batch is accompanied by a quality assurance certi ficate which records the results of the tests Using this rigorous quality control system we ensure that each batch meets the correct standards Consequently the customers are secure in the knowledge that our products are reliable and will provide good results when used correctly 4 Y Nidacon Quality Assurance tests Physical analyses pH tested on every batch during production and after bottled product at room temperature in air Osmolality tested on every Quality Assurance expressed as a percentage of the total sperm All data are recorded from before and after the separation and purification and are compared to the control i e using an earlier already approved batch Human Sperm Survival test for oil batch during production and after Sterility and toxin analyses Microbiological growth control performed after production of a batch and involve bacterial and fungal growth assays The assays are performed under a period of 2 3 weeks in order to be able to detect any growth They are done by the Bacteriological Laboratory of Sahlgrenska University Hospital Gothenburg Swe den an accredited indepen dent state laboratory Endotoxin detection This assay is done with an
22. ction d Vice President E ES Mr Hakan Nilsson j Marketing Ms Magda Alic Holmes Mr Oscar Rymo Regulatory Affairs Manisha Olausson gr Production Py Technical Assistant Y Miho Nilsson Mr Sean Graham ai Product development ei Marketing wae Ms Anna Nil ng Laessker Mr Mauricio Lucena Executive Medical Director MD Dr Ewa Ustyanowska Holmes MD Finance Ms Kristina Wright zm Product Specialist ASES ONE O E Logistics Mr Dennis Johansson Sr Chief Executive Officer Pa gr Marketing Y Dr Paul V Holmes Mr Anders Edvardsson Animal Product Specialist Y Ms Emma Holmes OP Quality Assurance Ms Marina Danilova If you need further information or Ms Ann Sofie Forsberg have any comments regarding Product Specialist the information in the manual pmi a please contact Tel 46 31 703 06 30 Fax 46 31 40 54 15 S Nidacon Fl jelbergsgatan 16 B S 431 37 M lndal Sweden Phone 46 31 703 06 30 Fax 46 31 40 54 15 contact nidacon com www nidacon com Anfang reklam 1000 ex 2015
23. es The different cell types contained in semen are normal motile sperm juvenile sperm and senescent sperm no fertilisation function and sperm with DNA breaks Epithelial cells from the male reproductive tract male immune cells and cell debris detritus are also present in the semen as are bacteria and possibly viruses Moreover the seminal fluid contains biologicals such Scientific basics as sperm decapacitating factors and reactive oxygen species ROS both of which negatively affect fertilisation After ejaculation in vivo normal sperm quickly migrate from the liquefied semen into the uterine cervix of the female thereby separating themselves from adverse affects of the factors previously mentioned In the andrology laboratory of an IVF clinic separation of the normal motile sperm from seminal fluid and its contents can be achieved by using either a discontinuous density gradient or a swim up Positive features of a discontinuous density gradient according to Nidacon Feature Separates motile sperm from other cell types Separates out immature aged and dying sperm Separates out morphologically abnormal sperm Separates out sperm with damaged chromatin Removes bacteria and viruses Density Gradients Swim up 4 4 4 4 Ex 4 4 If the density gradient has been prepared correctly the sperm pellet should contain only functional fertile sperm The use of two density gradient centrifugati on
24. hat they are stored at 4 C and brought up to room temperature before use 1 Viscous samples can be treated with PureSperm Buffer You simply add PureSperm Buffer to the ejaculat dilution 1 3 1 part PureSperm Buffer and 3 parts sample incubate for 15 30 minutes at 37 C and the sample is ready for use fugation care must be taken to avoid contaminating the pellet with components of the ejaculate or upper gradient layer Therefore we recommend that you use a new pipette after removing most of the gradient to avoid contamination for example by bacteria Y Nidacon 15 5peeaikKit PureSperm Speedikit Background We especially recommend PureSperm SpeediKit for the smaller clinics or for IUI clinics PureSperm SpeediKit is a rapid and efficient alternative to sperm preps using density gradient centrifugation Everything is included in a convenient kit form for quick sperm preparation all based on the effective centrifugation through a single layer Reagents and Equipment Ready to use tubes of PureSperm Unilayer and PureSperm Wash included in the kit Procedure 1 Use a sterile pipette to carefully layer liquified semen up to 1 5 mL on top of the PureSperm Unilayer If you have a sample volume greater than 1 5 mL use two tubes 2 Centrifuge at 300 x g for 30 minutes Do not use the brake 5 Use a new sterile pipette to aspirate the supernatant leaving about 5 mm of liquid above the pellet If no pellet is
25. ic salt solution Optimized for washing the sperm recovered from density gradient preparations for use in swim up procedures for extension of sperm prior to IUI or as a medium for maintaining sperm Shelf life 1 year QA Sterility Osmolality Endotoxin pH Human Sperm Survival PureSperm Speedikit A kit that provides you with the compo nents required to prepare 10 sperm samples for TUI It contains ready to use tubes for a single layer colloid centrifugation and ready to use tubes with Pure Sperm Wash for the washing of the pellet after centrifuga tion A perfect product for the small clinic 10 patients kit Shelf life 1 year 10 Y Nidacon QA Sterility Osmolality Endotoxin E pH HumanSperm Survival N maa m n E Se E a i CH gd d E p ei i al Ve Components Centrifuge tube containing the ProInsert PRP Pellet Retrieval Pipette Components NaCl MgSO EDTA Purified water HEPES Lactate Pyruvate O KH gt PO4 Glucose NaHCO3 ASA human serum albumin Components Silane Coated Silica KCl NaCl Glucose Pyruvate EDTA MgSO Citrate Lactate HEPES Purified water NaHCOs hSA human serum albumin Sperm CryoProtec Sterile salt solution containing glycerol optimised for freezing both gradient prepared sperm and for unprocessed ejacu lates Nidacon recommends the nitrogen vapour freezing technique since this technique seem
26. media PureSperm Wash PRP Pellet Retrieval Pipette Procedure for using Prolnsert 1 Open a Prolnsert Kit and remove the centrifuge tube containing the ProInsert 2 Use a sterile pipette to add 2 mL of PureSperm 80 to the outer channel The gradient material will run down into the ProInsert chamber out through a hole at the bottom of the chamber and down the wall of the centrifuge tube to form a layer at the bottom of the centrifuge tube 3 Repeat number 2 above using a new sterile pipette tip and PureSperm 40 again via the outer channel 4 Useanew sterile pipette tip to carefully layer liquefied semen up to 1 5 mL again via the outer channel Take care not to touch the edges of the central channel with the semen 5 Capthe tube and centrifuge at 300 x g for 20 minutes Do not use the centrifuge brake Calculate the correct RPM for your centrifuge 6 Add 5mL PureSperm Wash to a centrifuge tube not illustrated Prolnsert risking to re contaminate Since the gradient will include many risky contaminants it is crucial that the sperm pellet is not re contaminated The ProInsert will reduce this re contamination risk The insert is included in the centri fuge tube from the start and after the preparation the pellet can be retrieved through a channel which leads all the way down to the pellet without coming in contact with the now contaminated gradient Bench top ce
27. mete zygote and pre embryo culture in the incubator or during mani pulations outside the incubator No additi ves UV protective packaging Shelf life 2 years CE QA Density Sterility Endotoxin Human Sperm Survival Mouse Embryo assay Peroxides analyses VitriBlast Kit for vitrification of blastocysts based on well tested formulations Numerous publications demonstrate the effectiveness regarding both survival and pregnancy rates Shelf life 12 months QA Sterility Osmolality Endotoxin pH Mouse Embryo assay ThermobBlast Kit optimised for warming blastocysts vitrified with VitriBlast Kit Ready to use solutions Shelf life 12 months QA Sterility Osmolality Endotoxin pH Mouse Embryo assay Y Nidacon Components NaCl MgSO IG KH DCH Glucose NaHCOs Pyruvate Lactate EDTA Purified water ERES Hyaluronic acid ASA human serum albumin Components Light paraffin oil Components NaCl MgSO Glucose Pyruvate Ficoll HEPES Sucrose DMSO Vase KH DCH NaHCO EDTA Purified water Lactate Ethyleneglycol hSA human serum albumin Components NaCl MgSO Glucose Pyruvate Purified water HEPES ICI KH5 PO NaHCOs EDTA Sucrose Lactate ASA human serum albumin Background A normal semen sample ejaculate is made up of seminal fluid which contains a number of different cells cell debris microbiological and biological substanc
28. ntrifuge with swing out rotor Sterile pipettes 7 Attach the sperm retrieval pipette from the ProInsert Kit to a 1 2 mL syringe not illustrated 8 Pass the pipette slowly into the Prolnsert via the central channel down to the sperm pellet see graphic be careful not to disrupt the pellet Aspirate the sperm pellet Retract the pipette until the tip of the pipette is safely above the liquid surface aspirate a little air and retract the pipette from the central channel This procedure is to ensure that no contents in the pipette will be lost during transfer to the PureSperm Wash Transfer the pellet to the tube containing PureSperm Wash Centrifuge at 500 x g for 10 minutes Do not use the brake 9 Aspirate the PureSperm Wash supernatant leaving as little liquid as possible above the pellet If no pellet is seen leave the bottom 0 25mL fluid 10 Re suspend the sperm pellet in a suitable volume of culture transfer medium e g PureSperm Wash to obtain the required sperm concentration for IVF ICSI or IUI The sperm sample is now ready for analysis or use y y Y Nidacon i Swim up PureSperm Wash Background For most situations Nidacon recommends using a discon tinuous density gradient for preparing human sperm from semen However many customers at some time need to use the swim up technique and the most ideal product for this purpose is PureSperm Wash Recommendations Since
29. purification of human sperm Shelf life 2 years CE QA Sterility Osmolality Endotoxin pH Human Sperm Survival PureSperm 40 PureSperm 80 Ready to use density gradient solutions 40 and 80 They make work in the lab easier and minimizes the risk for mistakes Shelf life 2 years CE QA Sterility Osmolality Endotoxin pH Human Sperm Survival PureSperm Buffer Balanced salt solution designed specifically for diluting PureSperm 100 to make up the layers of different densities for a dis continuous gradient The optimised formu lation of PureSperm Buffer is designed to maximise sperm survival during gradient centrifugation Shelf life 2 years CE QA Sterility Osmolality Endotoxin pH Human Sperm Survival Q Nidacon Products Components Silane Coated Silica Purified water NaCl CaCl Glucose KCI EDTA HEPES Components Silane Coated Silica KCI NaCl Citrate Glucose Lactate Pyruvate HEES EDTA Purified water Components NaCl EDTA KCI Citrate HEPES Glucose Lactate Purified water Pyruvate Products ProInsert The ProInsert reduces the risk of reconta mination of the sperm pellet during sperm retrieval after a density gradient separation The ProInsert is a safe and easy to use device designed for use with Nidacon products Shelf life 2 years QA Efficacy test MEA Sterility Human Sperm Survival PureSperm Wash Sterile isoton
30. r it to solution 2 8 Incubate on the heating stage for EXACTLY 2 minutes Start the stopwatch and observe when 2 minutes is approaching It is easier to start the stopwatch and let it run towards 2 minute This removes the stress of the beeping noise when using a countdown timer While incubating proceed to step 9 Do not let the blastocyst remain exposed to the microscope light during the incubation Y Nidacon 25 Vitnfication and warming of blastocysts Vitrification procedure using the cryoloop 9 During the 2 minute incubation prepare 2 x 10 uL 13 Immerse in liquid nitrogen drops of solution 3 in the middle of the dish see diagram below The droplets evaporate quickly It is better to prepare them as late as possible 10 Attach the loop to the Crystalwand 11 At the end of 2 minutes transfer the blastocyst by aspirating solution 3 from the well into the pipette tip collect the blastocyst from solution 2 and transfer it to solution 3 in the 10 uL droplet The blastocyst must remain in solution 3 for 30 45 seconds in cluding the time in the loop 14 Attach the cryo tube to the Vial Clamp and immerse the tube in the liquid nitrogen allowing it to fill Carefully insert the cryoloop into the tube and keep the cryoloop in liquid nitrogen during the entire procedure Use the Crystalwand to close the tube 12 Coat the loop with solution 3 from the unused 10 uL droplet and place the blastocyst on the loop
31. rotec II Sperm CryoFloater Optimised for freezing sperm Sperm vapour freezing device SpermCatch Slowing down sperm prior to ICSI Culture Products Butze MISI SUA HEI Warming of vitrified blastocysts Overlay during embryo culture Ordering information Cat No Description DIAC Cen IN OX Description DIC PSK 020 PureSperm 40 80 2x20mL SCP 020 Sperm CryoProtec II 2 x 20 mL PS40 100 PureSperm 40 O0 rine NO 100 Nidoil to seal PS80 100 PureSperm 80 100 mL NO 300 Nidoil 300 mL PS100 100 Pures 9 100 1 n ndis dif MSAN SVS 010 SpermVitalStain 2x10mL PS100 250 PureSperm 100 2 510 ale SMS 250 Sperm MorfoStain 250mL P5100 1000K PureSperm 100 4x250mL VBK 010 VitriBlast Kit Spe MUO Teal PSB 100 PureSperm Buffer 100 mL PSW 100 PureSperm Wash 100 mL TBK 010 ThermoBlast Kit 4 x 10 mL PSW 020 PureSperm Wash 2 20 mL PI15 5 ProInsert 5 kits PSSK 070 PureSperm SpeediKit 10 patient preps CPV 001 Cryofloater vial i 5C 100 SpermCatch 6 x 100 uL CEN OO Cryofloater straw il 10 items or more on one purchase order gives 5 discount We have distributors in most countries for a complete list take a look at our web page www nidacon com 8 Y Nidacon s H A Purelparm 4U PureSperm 100 Sterile silane coated silica colloid in an isotonic salt solution It is optimised for the preparation of discontinuous density gradi ents for the separation and
32. s a solution without PVP contains instead hyaluronic acid which is a natural component Several studies have shown that SpermCatch gives the same or even better results than PVP Since SpermCatch is a solution containing hyaluronic acid see the following reference for the advantages ref 21 Sterile pipettes ICSI equipment Petri dish 7 Fill your injection pipette with SpermCatch to avoid the sperm sticking to the inside of your pipette It will also help you to make a controlled injection 8 Immobilise the individual sperm by using the injection pipette to knick the sperm tail 9 Aspirate the immobilised sperm 10 Move to one of the oocyte droplets Focus on the oocyte and position the oocyte with the holding pipette Bring down your injection pipette and inject the sperm Make sure that the oolemma is broken before you expel the sperm SpermCatch 1 Itis convenient to have two dishes per patient 25 Y Nidacon Nido Background Mineral oil to overlay the embryo culture is used extensi vely in IVF laboratories NidOil is a paraffin oil which has been specifically chosen and then treated in our production facilities to ensure that its purity and hand ling characteristics are suitable for using as an overlay when culturing gametes and embryos NidOil does not require washing before use and it is neither too sticky nor too viscous to facilitate pipetting NidOil Our stringent quality
33. s also been shown that peroxidase Q Nidacon Impl rate 215 30 0 24 6 se Jo po temperature and gaseous content between the compo nents of the culture system level in oil can increase over time due to exposure to light or high temperature storage The peroxidase test in now included in our Quality Assurance Certificate which comes with every batch and we also test the raw material before we make the order In this way we hope to be able to provide you with an oil for your cultures that is safe to use and still practical with the long shelf life and room temperature storage before opening If you have any questions regarding our tests please let us know 23 Vitrification and warming of blastocysts VitriBlast Kit ThermoBlast kKit Background The formation of intracellular ice crystals is a major problem during the cooling and warming of cells These ice crystals have detrimental effects on cell survival rates Vitrification which is the rapid freezing of cellular mate Recommendations VitriBlast can be used with different types of vitrifica tion devices like the cryotop the cryoloop or the high security straw HSV The method described below is with the cryoloop but the same protocol can be used for all devices Reagents and Equipment VitriBlast and ThermoBlast Kit Sterile pipettes Device for vitrification CO incubator Stopwatch Vitrified and warmed blastocyst with excellent
34. s to provide the best results after thawing Shelf life 1 year CE QA Sterility Endotoxin pH Recovery rate after freezing and thawing CryoFloater A floating device used when cryo preser ving semen or prepared sperm in liquid ni trogen It provides a constant distance between the sample and the nitrogen sur face to standardize the freezing rate QA Visual inspection Sperm VitalStain One step staining technique for assessment of sperm vitality one of the basic tools in semen analysis Shelf life 2 years CE QA pH Functional analysis Sperm MorfoStain Classic Romanovsky stain It is a one step stain optimised for assessment of sperm morphology Shelf life 2 years CE QA Morphological test Q Nidacon Components NaCl KCl HEPES Glucose MgSO KH gt PO4 Components Polyethylene foam Components NaCl Eosin Purified water Components Methanol Eosin Y Methylene B Products EDTA NaHCOs Lactate Glycerol Pyruvate Purified water Nigrosine Formalin May Grunwald Giemsa Azur B Ll Products 12 SpermCatch For slowing down sperm prior to ICSI without using polyvinylpyrrolidone PVP To avoid ICSI injection of PVP it contains only natural products for increasing the viscosity Shelf life 1 year QA Sterility Osmolality Endotoxin pH Human Sperm Survival NidOil Sterile light paraffin oil for use as an overlay during ga
35. seen after centrifugation remove all fluid except the lowest 0 5 mL 4 Use a new pipette to aspirate the pellet or the lowest 0 5 mL O1 of PureSperm colloid followed by rinsing the sperm with PureSperm Wash The kit contains both the PureSperm colloid and the PureSperm Wash for 10 patients already dispensed in centrifuge tubes You do not need an incubator Bench top centrifuge with swing out rotor Sterile Pasteur pipettes O Transfer sperm pellet to the tube containing PureSperm Wash Resuspend the sperm O Centrifuge at 500 x g for 10 minutes Do not use the brake Use a new pipette to aspirate the supernatant If no pellet is seen leave the bottom 0 25 mL fluid 8 Resuspend the pellet in the remaining Pure Sperm Wash The sperm preparation is now ready for use 16 Y Nidacon ProInsert Background A density gradient will effectively remove lymphocytes epithelial cells abnormal or immature sperm cell debris seminal fluid bacteria and to some extent viruses When performing a density gradient the only means of retrieving the sperm pellet at the bottom of the tube is to either go directly through the layers disrupt them and risking to re contaminate the pellet or to remove as much as possible of the layers go through the remaining gradient and still Reagents and Equipment Centrifuge tube containing the ProInsert Centrifuge tube for washing
36. techniques and the swim up method to seperate spermatozoa with chromatin and nuclear DNA anomalies D Sakkas et al 2000 Human Reprod Initial sample Sediment Final prep Paired t test Swim up ZU IA LOS 0 6 PureSperm gradient Jog A SS PRA mo P 0 001 The mean percentage of spermatozoa positive to Chromomycin A3 decreased presence of protamine General care and use i Allsolutions should be brought up to room tempera ture before use to avoid the temperature fluctuations which are detrimental to sperm survival Q Nidacon 1 Openandreseal bottles in a laminar air flow bench using sterile techniques to avoid contamination 1 Store all opened bottles at 2 8 C after re sealing Lo Denstty Gradient Preparation PureSperm 100 PureSperm 40 PureSperm 80 PureSperm Buffer PureSperm Wash Recommendations If you have a sample with a high volume gt 3mL you can prepare two PureSperm gradients for each semen sample This reduces the risk of overloading a single gradient Reagents and Equipment PureSperm 100 plus PureSperm Buffer or PureSperm 40 and 80 Sterile Pasteur pipettes Procedure 1 14 If you use PureSperm 100 dilute with PureSperm Buffer to make your gradient solutions for example add 2 mL PureSperm Buffer to 8 mL PureSperm 100 to obtain 10 mL 80 PureSperm Add 6 mL PureSperm Buffer to 4 mL PureSperm 100 to obtain 10 mL 40 PureSperm Instead you can use the ready to
37. the unstained white live cells Pipette Test tube 7 Cover this slide with a second microscope slide and when the drop is evenly spread between the two slides pull them apart from each other horizontally You then have two good slides 8 Air dry the two slides and examine If you want to store for later use mount the slides with DPX or equivalent mountant and a cover slide 9 Examine using a bright field 40 x objective or a 100 x objective under oil immersion 10 Count 200 sperm the white unstained are classified as alive and the red or pink are classified as dead Sperm coloured only at the neck region are classified as alive 28 Q Nidacon Sperm MorfoStain Background The technique is based on the principle that sperm with different characteristics will stain so that one can differen tiate between them The numerical estimation of ab normal sperm in an ejaculate can aid in the judgement of whether and which kind of infertility treatment will be necessary Reagents and Equipment Coplin jar or similar Microscope slides Procedure 1 Make a semen smear on a microscope slide using your conventional method or the method recommended by Nidacon 2 Transfer a 20 uL drop onto a labelled microscope slide with a pipette making a string line of fluid in the middle of the slide 5 Cover this slide with a second microscope slide and when the drop is evenly spread between the two slides pull them
38. tive against some of the bacteria most commonly found in semen Streptomycin and gentamycin are cytotoxic Gentamicin in particular has been shown to be toxic to embryos Therefore it seems prudent to avoid including potenti ally spermicidal components in sperm preparation fluids Moreover bacterial contamination in the ejaculate is removed by density gradient preparation Therefore the absence of antibiotics in the gradient will not be detrimen tal to the sperm preparation and avoids exposing the sperm to potentially toxic compounds Adaitives and Phenol Red No preservatives or unstable ingredients are added to Nidacon products In addition we have decided not to use phenol red in our media since it has been proven to have estrogenic effects Gametes have receptors for estrogen and they can be affected by its presence For instance it has been shown that estrogen inhibits sperm motility and the acrosome reaction Products e Ordering information Sperm VitalStainTM Sperm MorfoStainTM Diagnostic Products RM aaa e Sperm Preparation Gradient Preparation Swim up Unilayer PureSperm 100 PureSpemm 40 80 PureSperm Wash PureSperm Speedikit Gradient stock solution Ready to use gradient Optimised washing medium UI Sperm preparation kit ProlnsertTM Gradients simplified PureSperm Buffer Optimised PureSperm dilutant PureSperm Wash Optimised washing medium Sperm CryoP
39. toxin analyses Niort a irerobiologica growth Control N Endfatexig fenster during eaten ue Wan quantitative at assay EU mi lt 3 00 Comply Biological analyses Human sperm Survival after 13 hrs d Com ply Date of release 20 May 2035 hi rl d V CLIII eh eit CHE Girl oe Teratec ced daps y fi T Pobre ven Wa P UT Cm e Gn e A o Paul M Holma CEQ ames MSc Php Orense Marina Danfioyg Quality Assurance Coordinator Doc POO 10 4 Vari 2 Dare 2015 02 42 sample Q Nidacon Prepared sperm are covered by oil and incubated overnight in 37 C 5 696 CO Percentage of motile sperm on day 2 Mouse Embryo Assay MEA for bottles etc is used to assess the in vitro growth and deve lopment of pre implan tation embryos exposed to the test item The assay predicts embryo toxicities in medical devi ces or related products to be used for assisted repro ductive technology ART Mouse Embryo IVF Assay for media a sensitive assay mimicking the human IVF procedure The preferred assay to screen assisted reproductive tech nology supplies for toxici ties impairing male and female gamete fecundability and subsequent embryo development capacity Peroxide analyses the perox ide level is measured using a QuantiChrom Peroxide Assay The improved method utilises the chromogenic Fe3 xylenol orange reaction in which a purple complex is formed when Fe2 provided in
40. ttom 0 10 mL fluid The sample is now ready for use Centrifuge at 300 x g for 20 min Aspirate everything except the pellet and 4 6 mm of the PureSperm 80 layer Use a new pipette to aspirate the pellet Transfer to a new tube containing 4 ml PureSperm Wash Centrifuge at 500 x g for 10 min Aspirate PureSperm Wash supernatant and the sample is now ready for use S Y Nidacon 23 al SpermCatch Background SpermCatch is an alternative to PVP polyvinyl pyrrolidone which today is the most common substance used for slowing down sperm prior to ICSI How ever PVP has been reported to cause problems such as da maging the sperm plasma membrane It may also interfere with sperm nucleus decondensation Reagents and Equipment SpermCatch NidOil Injection media Procedure 1 Place a 10 uL drop of SpermCatch in the middle of a petri dish 2 Place 4 drops of 10 uL injection media around the SpermCatch drop in the petri dish 5 Immediately cover the drops with NidOil 4 Incubate for 30 minutes in CO environment at 37 C 5 Add 1 uL of prepared sperm suspension to the middle of the SpermCatch drop 6 Incubate for 10 minutes in CO environment at 37 C lips i ICSI dishes must be prepared quickly to avoid osmolarity changes in the media Only make two at a time SpermCatch i
41. use PureSperm 40 and PureSperm 80 solutions Use a sterile pipette to add 2 mL of 80 PureSperm to a conical tube Use a new pipette to carefully layer 2 mL 40 PureSperm on top of the 80 layer It is important not to disrupt the two layers and to maintain a sharp interface Layer the liquefied semen onto the gradient We recommend that you dont take more than 1 5 mL gradient or you risk overloading the gradient and not getting a good result Before centrifugation After centrifugation Seminal plasma _ Upper raft Gradient upper layer A ower ratt Gradient lower layer Sperm pellet selected motile population provides security when handling tubes or recovering sperm pellets and provides two tubes to balance the centrifuge rotor PureSperm Wash Sterile 2 mL and 10 mL pipettes Bench top centrifuge with swing out rotor 5 Centrifuge at 300 x g for 20 minutes Make sure that your centrifuge uses the correct g force use equation p 13 Do not use the brake 6 Aspirate in a circular movement from the surface everything except the pellet and 4 6 mm of the 80 PureSperm layer If no pellet is seen after centrifuga tion remove all fluid except the lowest 0 5 mL 7 Use a new pipette to aspirate the pellet or the lowest 0 5 mL Transfer sperm pellet to a new tube and resus pend pellet in 5 mL PureSperm Wash Always use a new tube with PureSperm Wash to avoid contamina tion
42. yst to culture medium and allow the blastocyst time to reexpand Wait 1 to 4 hours before final assessment If the blastocyst has not re expanded after 4 hours the chance of reexpansion is low Y Nidacon 3 Vitality Staining Sperm VitalStain Background Sperm vitality should be determined in semen samples with 5096 or more immotile spermatozoa according to the WHO laboratory manual for the examination of human sperm SpermVitalStain uses the eosin nigrosine technique in a one step method to establish the percentage of live Reagents and Equipment Light microscope 40 100 x magnification Microscope slides Procedure 1 Shake the bottle of Sperm VitalStain before use 2 Take an equal amount of Sperm VitalStain and the sperm sample eg 50 uL SVS 50 uL sample Use for example an eppendorf tube 5 Mix well 4 Leave for 30 seconds at room temperature 5 Prepare a slide using your conventional method or use the method recommended by Nidacon O Transfer a 20 uL drop onto a labelled microscope slide with a pipette making a string line of fluid in the middle of the slide 6 a lips 1 The 100x objective with immersion oil will give you a very clear picture of stained versus unstained sperm spermatozoa It is based on the principle that dead cells i e those with damaged plasma membranes will take up the eosin and stain red Nigrosine provides the background to facilitate visualisation of
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