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JOURNAL FÜRFERTILITÄT UND REPRODUKTION
JOURNAL FÜRFERTILITÄT UND REPRODUKTION
Krause & Pachernegg GmbH · VERLAG für MEDIZIN und WIRTSCHAFT · A-3003 Gablitz
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KASTELIC DEffect of multiple pipette use on the outcome of
intracytoplasmatic sperm injection (ICSI)
Journal für Fertilität und Reproduktion 2002; 12 (2) (Ausgabefür Schweiz), 19-29
Journal für Fertilität und Reproduktion 2002; 12 (2) (Ausgabefür Österreich), 20-33
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J. FERTIL. REPROD. 2/200220
D. Kastelic
EFFECT OF MULTIPLE PIPETTE USEON THE OUTCOME OF INTRACYTO-PLASMATIC SPERM INJECTION (ICSI)
SUMMARY
Performing intracytoplasmic sperminjection (ICSI), it is customary to useone injection pipette for sperm selec-tion, immobilisation and injection.The objective of this prospectiverandomised study was to documentthe relationship between fertilizationrates, embryo quality and pregnancyrates and blastocyst development ofthe remaining embryos in cycleswhen one pipette was used for thewhole procedure vs. cycles wherethe pipettes were changed at inter-vals. In this study 58 patients (in 60cycles) who had 6 or more oocytesretrieved and who required ICSI dueto severe male infertility were ran-domly allocated into two groups. InGroup 1 ICSI was performed usingone injection pipette for sperm selec-tion, immobilization, sperm aspirationand for oocyte injection. In Group 2one injection pipette was used for
sperm selection and immobilization,then the pipette was replaced forinjection. This pipette was changedafter every four oocytes injected.
Fertilization rates were significantlylower in Group 1 (63.7 %) where asingle pipette was used than in Group2 (73.4 %) where the injection pipettewas changed. There was no statisticaldifference in embryo cleavage, mor-phological score, pregnancy rates orimplantation rates between groups.
It can be concluded that using oneinjection pipette for the ICSI proce-dure resulted in poorer fertilizationrates, but no effect was observedwhen comparing cleavage rates,pregnancy rates, implantation ratesor blastocyst development from theremaining embryos. These observa-tions are based on a small numberof cycles and a more extensive studywould be required to evaluate thegain from changing the injectionpipette during ICSI.
INTRODUCTION
Intracytoplasmic sperm injection(ICSI) was first performed almost onedecade ago [1] and has now evolvedinto a highly successful procedureroutinely applied in many IVF labo-ratories all over the world. This tech-nique gives couples with severe maleinfertility a chance to father theirown biological child. The efficacy ofthe ICSI procedure depends upon anumber of biological and technicalfactors. Spermatozoa from fresh orfrozen/thawed ejaculates and fromtesticular biopsy or epididymalaspirates give high fertilization andpregnancy rates after ICSI [2, 3].
Mechanical immobilisation of thespermatozoa before injection playsan important role in achieving con-sistently high fertilization rates [4].Immobilisation is commonly per-formed by pressing the sperm tail
Zusammenfassung
Bei der Durchführung der intrazyto-plasmatischen Spermieninjektion(ICSI) wird üblicherweise eine Injek-tionspipette für die Selektion derSamenzelle sowie deren Immobili-sierung und Injektion in die Eizellebenutzt. In dieser prospektiven ran-domisierten Studie wurde das Ver-hältnis zwischen Fertilisierungsrate,Embryonenqualität und Schwanger-schaftsrate sowie Blastozystenent-wicklung der verbliebenen Embryo-nen für Behandlungszyklen, beidenen eine einzige Pipette für dengesamten Prozeß verwendet wurde,im Vergleich zu Behandlungszyklenuntersucht, bei denen die Pipettenausgewechselt wurden. Im Rahmendieser Studie wurden 58 Patienten(in 60 Behandlungszyklen), denen6 oder mehr Eizellen entnommenwurden, und eine ICSI aufgrundschwerer männlicher Infertilität an-gezeigt war, nach dem Zufallsprinzip
in zwei Gruppen eingeteilt. In Grup-pe 1 wurde die ICSI mittels einerInjektionspipette durchgeführt, diesowohl für die Selektion der Samen-zelle, als auch deren Immobilisie-rung, Aspiration und Einbringung indie Eizelle verwendet wurde. InGruppe 2 wurde eine Injektions-pipette für Selektion und Immobili-sierung der Samenzelle benutzt, fürdie Spermieninjektion wurde eineweitere Pipette verwendet. DiesePipette wurde nach erfolgter Injekti-on von jeweils vier Eizellen ausge-tauscht.
In Gruppe 1, in der eine einzigePipette benutzt wurde, waren dieFertilisierungsraten signifikant niedri-ger (63,7 %) als im Vergleich zuGruppe 2 (73,4 %) (P< 0,05), in derdie Injektionspipette ausgetauschtwurde. Bezüglich der Furchungssta-dien und der Morphologie des
Embryos, der Schwangerschafts- undder Implantationsrate war keinestatistische Differenz zwischen denbeiden Gruppen festzustellen.
Daraus kann gefolgert werden, daßdie Verwendung einer einzigenInjektionspipette bei der Durchfüh-rung des ICSI-Verfahrens niedrigereFertilisierungsraten zur Folge hatte,jedoch keine Auswirkungen beimVergleich der Teilungsraten, Schwan-gerschaftsraten, Implantationsratenoder Blastozystenentwicklung derverbliebenen Embryonen festzustel-len waren. Diese Beobachtungenbasieren auf einer geringen Anzahlvon Behandlungszyklen und eswürde einer umfangreicheren Studiebedürfen, um den Vorteil, der sichdurch einen Austausch der Injektions-pipette während einer ICSI ergebenkönnte, zu bewerten.
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For personal use only. Not to be reproduced without permission of Krause & Pachernegg GmbH.
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against the bottom of the injectiondish using the tip of the injectionpipette. This disruption to the spermmembrane is thought to simulate thedisruption which occurs at sperm-oocyte-fusion in natural fertilizationand is believed to be important foroocyte activation and for the releaseof sperm cytosolic factors involved inoocyte activation and decondensationof the spermatozoa [4, 5]. It has alsobeen suggested that immobilising thespermatozoon before injection mayprevent interference by the spermwith the cytoskeleton and metaphasespindle of the oocyte [6]. Injection ofimmobilised spermatozoa withouttail breaking results in significantlylower fertilization [7]. By performingaggressive sperm immobilisationprior to ICSI with testicular or epidi-dymal spermatozoa Palermo et al. [8]concluded that a positive effect maysuggest a different membrane consti-tution in these spermatozoa.
FACTORS AFFECTING ICSIOUTCOME
Oocyte morphology
There is some debate about the effectof the morphology of oocyte on ferti-lization and embryo quality in ICSI[9]. Some authors did not find anycorrelation between oocyte morpho-logy, fertilization rate and embryoquality [10, 11], while other authorshave reported that oocyte morpho-logy has a significant influence onfertilization rates and embryo quality[9, 12, 13]. In addition, Alikani et al.[14] observed a higher rate of mis-carriage in women with dysmorphicoocytes.
Aspiration of cytoplasm during theprocedure
It is considered that aspiration of theooplasm, following puncture of theoocyte and prior to sperm injection,is an integral part of the ICSI proce-dure and an essential step for oocyte
activation [15, 16]. Cytoplasmicaspiration may provide a mechanicalstimulus to induce an importantrelease of intracytoplasmic calciumstores [17]. Nagy et al. [18] observedthat breakage of the oolemma maybe due to differences in the oolemmaor to the sharpness of the injectionpipette. This may have an influenceon the developmental capacity of amicroinjected oocyte. Vanderzwalmenet al. [6] concluded in a comparisonof ICSI pipettes with or without aspike that the presence of a spike onthe injection pipette facilitates pas-sage through the zona pellucida butnot through the oolemma since theydid not observe any difference infertilization rates or damage rateswhen using pipettes with or withouta spike.
Site of sperm deposition
The site of sperm deposition in theoocyte has been examined by manyauthors. Nagy et al., [18] concludedthat the orientation of the oocyte atICSI and the deposition of the spermnear the meiotic spindle affects theembryo development rate. In evalu-ating the orientation of the openingof the injection pipette some outhorsreported better embryonic develop-ment [19] and pregnancy rates [20]when the opening was directed to-wards the animal pole, thus injectingthe sperm towards the direction ofthe meiotic stage MII (MII) spindle.Hardarson et al., [21] found in theirstudy that in 93 % of oocytes the MIIspindle was located in the samehemisphere as the polar body (theanimal pole). They therefore con-cluded, that it might be advantageousfor the sperm to be injected at themidline between the two hemispheresof the oocyte in the direction of thevegetal pole in order to avoid damageto the MII spindle.
Microtools
Design and quality of the microtoolsare an important factor for a success-ful ICSI [22]. ICSI requires that eachgamete, sperm and oocyte should be
manipulated individually. A singlesperm is selected and injected intothe oocyte cytoplasm using two typesof microtool. A holding pipette isused to hold the oocyte in a desiredposition, and an injection pipette isused for sperm immobilisation, aspi-ration and injection into the oocyte.
Vanderzwalmen et al. [6] studied theinfluence of the shape and size of thepipettes’ aperture on embryo qualityand on the rate of degeneratedoocytes. They observed that in caseswhere the diameter of the injectionpipette was between 9 and 10 mi-crons there was a higher percentageof degenerated oocytes and poorquality embryos. Svalander et al. [15]also concluded that the quality ofmicrotools may influence the outcomeof the ICSI procedure in severalways; a blunt injection pipette candamage the oocyte by compression,whereas a pipette with a too largediameter could cause an overload ofinjected fluid.
Although, it may be customary dur-ing the ICSI procedure to use a singleinjection pipette for the entire pro-cedure, i.e. sperm immobilisation andinjection of the sperm into the oocyte,the efficiency of the injection pipetteby the last injected oocyte, is ques-tionable. No studies regarding theusing of a single pipette versus morethan one pipette for a single proce-dure have been reported. This maybe an aspect of the ICSI techniquewhich could affect outcome, eitheras a result of loss of sharpness due torepeated injection or a deteriorationin the quality of the pipette becauseof accumulation of debris at the tip.
In order to determine whether thereis any influence regarding the usageof a single vs. multiple injectionpipettes our current study was de-signed to compare the outcome afterICSI with two different techniques:
� Using the same injection pipettefor sperm selection and immobili-sation and for the injection of allthe oocytes for an individual pa-tient (Group 1);
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� Changing the injection pipetteafter sperm selection and immobi-lisation and after four oocytes hadbeen injected (Group 2).
The outcome of ICSI in both groupswas compared, including oocytesurvival rates, fertilization rates,embryo cleavage, pregnancy andimplantation rates.
MATERIALS AND METHODS
Selection of patients
A total of 58 couples requiring ICSItreatment due to severe male infertil-ity were included in the study, eachof whom had six or more metaphaseII stage oocytes (MII) retrieved. Twocouples had a repeat ICSI treatment,yielding a total number of 60 treat-ment cycles performed between Feb-ruary and May 2000 in the Infertilitycentre, Women’s Hospital Postojna,Slovenia.
Ovarian stimulation
All female patients were treated witha combination of a gonadotrophin-releasing hormone analogue (GnRH,Decapeptyl® 0.1 mg s.c/daily, Ferring,Kiel, Germany) from day 3–9 of themenstrual cycle and with humanmenopausal gonadotropin (hMG;Pergonal® 75; Serono, Aubonne,Switzerland) from day 5. The dose ofPergonal was adjusted for individualpatients. Ultrasound examinationswere performed from day 10 of thecycle onwards. When at least threefollicles equal to or larger than 18mm in diameter were recorded byultrasound, ovulation was inducedwith 10 000 IU human ChorionicGonadotropin (hCG; Profasi®, Serono,Aubonne, Switzerland). Intravaginallyadministered micronized progester-one 100 mg x 3/day (Utrogestan;Asta Medica, Wien, Austria) wasused for luteal support for 60 daysafter oocyte retrieval. Oocytes wereretrieved 36 hours after hCG adminis-tration under transvaginal ultrasoundguidance.
Study groups
The patients who had 6 or more MIIoocytes were randomly allocatedinto one of the two groups for ICSI.Patients older than 42 years or thosewith abnormal endocrinology wereexcluded from the study. Group 1included twenty-eight patients (30cycles), where only one injectionpipette was used for sperm selection,immobilization, sperm aspirationand oocyte injection. Group 2 in-cluded thirthy patients (30 cycles), inwhich one injection pipette was usedfor sperm selection, immobilizationin the microsedimentation dish andfor sperm transfer into PVP in theinjection dish. Then the pipette wasreplaced by a new one for the injec-tion of no more than four oocytes.The injection pipette was changedafter every four injected oocytes untilICSI was accomplished for all of theoocytes for that patient.
Oocyte preparation
Immediately after follicular aspira-tion the oocyte cumulus complex(OCC) was washed with pre-equili-brated flushing medium containingheparin (Cat.No. 1084 5060, Medi-Cult, Jyllinge, Denmark) using a cen-tre-well dish (#3025,Falcon®, NewJersey, USA) without oil. After wash-ing, the OCC were transferred intothe centre of the dish and incubatedin pre-equilibrated Universal IVFmedium (Cat.No. 10311010, Medi-Cult) for 2–3 hours in a CO2 incuba-tor (BB6220, Heraeus, Hanau, Ger-many) at 37°C in an atmosphere of5.3 % CO2 in air until the ICSI proce-dure. No more than six oocytes wereincubated together.
The oocytes were denuded of cumu-lus just prior to ICSI. Two differentconcentrations of hyaluronidase(Cat.No. 10110010; Medi-Cult) wereused for removal of cumulus cells;80 IU/ml and 1:1 dilution in SPM(SPM, Cat.No. 10700060, Medi-Cult)to give 40 IU/ml hyaluronidase. Onedroplet of each strength of hyaluroni-dase and five droplets of sperm prep-aration medium were placed under
the pre-equilibrated liquid paraffin(Cat.No.1010 0060; Medi-Cult) in adish (Falcon®, #3001). No more thansix oocytes were denuded at once.
Denudation took place firstly in 80 IU/ml hyaluronidase using a glass pipettewith an inner diameter of 900 µm(Oovum pick-up set, Cat.No.1681;Repromed®, International Medical,Zutphen, Neederland). The oocyteswere then transferred to 40 IU/mlhyaluronidase and aspiration of almostall of the cumulus cells was completedby a second pipette with an openingof 160 µm diameter (Inspection setCat.No.1670; Repromed®). After nomore than 30 seconds the oocyteswere washed 5 times in SPM andincubated for a further 15 min beforethe ICSI procedure was performed.
After denudation the stage of maturityof the oocytes was determined underthe inverted microscope e.g. welldefined germinal vesicle (GV), meta-phase I stage (MI), metaphase II stage(MII) when the first polar body (PB)had been extruded and oocytes withempty zona pellucida. Oocytes werethen incubated in a CO2 incubatorfor at least 20 min. Morphologicallynormal appearing mature oocytes(MII stage) were transferred into theinjection dish just prior to the ICSIprocedure.
Sperm assessment
Before the treatment cycle began,semen samples were assessedaccording to WHO [23] recommen-dations and male patients with severemale infertility were categorised as:� severe oligo- and/or astheno- and/
or teratozoospermia or a combina-tion of them (< 1 x 106/ml, < 50 %motility and < 5 % normal mor-phology according to Kruger’s strictcriteria respectively);
� obstructive or non-obstructiveazoospermia (testicular spermextraction-TESE was performed).
No karyotype abnormalities werefound in the ten men who underwenttesticular biopsy because of theirazoospermia.
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Semen preparation
The male partners were asked toabstain from sexual activity for 3–6days before the day of ICSI and askedto provide a split ejaculate on themorning of oocyte retrieval. For allfresh ejaculated samples sperm con-centration and motility from the firstpart of the ejaculate were assessedby microscopy using a Makler count-ing chamber.
Sperm samples were diluted 1:1 withSPM and centrifuged at 200 x g for5 minutes. The pellet was recoveredand transferred into the microsedi-mentation dish as described byWalmsley et al. [24]. One largedroplet of SPM covering up to ¼ ofthe dish (Falcon®, #1006) was placedon the bottom with two separatedstrips of 5 µl of polyvinyl pyrrolidone(PVP, Cat.No. 1090 5000; Medi-Cult)and covered with pre-equilibratedliquid paraffin. One droplet (20–50µl) of concentrated sperm pellet wasadded into SPM and another dropletof 1 µl of the sperm pellet was addedto the PVP. The sperm was then incu-bated for 0.5–1 hour in a CO2 incu-bator, the incubation period depend-ed upon the quality of sperm sample.In cases where sperm had been re-covered from testicular biopsy and incases of severe oligospermia up tothree dishes were prepared.
In order to limit exposure of theoocytes to suboptimal conditionsoutside the incubator as much aspossible, sperm selection and immo-bilisation was performed in advanceand all selected sperm were trans-ferred to the PVP droplet in the in-jection dish prior to ICSI.
If the sperm for ICSI was recoveredfrom testicular biopsies the procedurewas performed on the same day asICSI by open biopsy under localanaesthesia. A piece of excised tes-ticular tissue (0.5 x 0.5 x 0.5 mm)was placed in a petri dish with 0.2ml of SPM and dissected into smallpieces using fine scissors. The pres-ence of spermatozoa was assessedunder an inverted microscope and
when spermatozoa were found allthe surrounding medium withoutpieces of the biopsied tissue wastransferred into a tube (Falcon®,#2003) with 0.3 ml of SPM. Thesperm suspension was mixed using avortex and incubated for up to 2.5hours at 37°C, depending upon theviability of the sperm. After incuba-tion the tube with testicular spermwas gently shaken and the suspen-sion layered under pre-equilibratedparaffin oil in the microsedimentationdish and incubated for a further 0.5to 1 hour before ICSI. Any remainingsuspension was frozen to avoid theneed for repeat surgery in future ICSIattempts [25].
Electroejaculation (EEJ) was performedin four patients with spinal cordinjury. The procedure was performedon the morning of oocyte collectionusing a rectal probe electrode devel-oped by Seager [26]. The antegradeportion of the ejaculate was obtainedin splits. The first part of the ejaculatewas assessed and prepared as freshejaculates and used for ICSI proce-dure. The 2nd and 3rd parts of theejaculate were cryopreserved if therewas at least 100 000 motile sperma-tozoa in the ejaculate. Sperm con-centration and motility were assessedprior to sperm preparation. Spermmorphology was assessed later in theandrology laboratory.
Sperm injection
ICSI procedure was carried out onthe heated stage (37° C) of a Diaphot300 inverted microscope (Nikon,Tokyo, Japan) at 200 x magnificationusing Hoffman modulation contrastoptics. The microscope wasequipped with a video camera(Panasonic, Osaka, Japan) so that allmanipulations could be monitoredand recorded (Panasonic, Japan).
A Narashige micromanipulator sys-tem was used (Narashige, Tokyo,Japan) with a MO-188 Joystick andMM-188 three dimensional motordrive for the holding side and IM6Microinjector (Cell Tram-Microinjec-
tor Eppendorf, Hamburg, Germany)for the injection side.
Holding pipettes with an angle of 30°and opening of 15 µm (Cat. No.13903015, Laboratoire CCD, Paris,France) and spiked injection pipetteswith an opening of 8 µm and angleof 30° (Cat. No. 13813008, Labora-toire CCD, Paris, France) were used.Holding and injection pipettes werealigned under low magnification withthe use of an extra dish for filling thetips of both pipettes. The holdingpipette was filled with liquid paraffinand then with SPM medium by capil-lary action. The injection pipette wasfilled with a small amount of PVPsolution.
Sperm selection and immobilization:motile sperm which appeared tohave normal morphology, as couldbe observed in our optics means,were selected immediately beforethe ICSI procedure, from the cleanedges of the microsedimentationdroplets or from a PVP droplet afterincubation. The sperm was trans-ferred into a clean PVP drop andimmobilized. The tip of the injectionpipette was placed over the tail ofsperm and pressed against the tail byquick, controlled downward move-ment of the injection pipette usingthe motor-driven coarse control ofthe micromanipulator. When thepipette touched the sperm tail half-way between head and the tip of thetail, the tail was gently squashedbetween the injection pipette andthe bottom of the dish. The numberof sperm selected and immobilisedwas greater than the number ofoocytes for injection. Up to 6 sper-matozoa were aspirated into theinjection pipette and transferred intothe PVP drop in the injection dish.For patients in Group 2, the injectionpipette was changed at this point,whereas, in Group 1 the same pipettewas used for the remainder of theprocedure.
All ICSI procedures were performedin injection dishes (Falcon®, #1006).One strip of PVP was put into the
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centre of the dish and an extra stripof PVP was placed close to the edgeof the dish. Four small droplets of10 µl SPM were positioned near thestrip of PVP, covered with pre-equili-brated liquid paraffin, and warmedin a CO2 incubator. Oocytes weretransferred to the injection dish. Upto four oocytes were placed intoeach injection dish, one per droplet.The exposure time outside the incu-bator was not more than 5 minutesin each group. The number of injec-tion dishes prepared per patientdepended upon the number oocytesto be injected. The injection dish,containing up to four oocytes and 5–6immobilised spermatozoa in PVPwas placed on the heated stage ofthe inverted microscope.
The injection pipette was loweredinto the PVP droplet and a singleimmobilized sperm was aspirated tailfirst into the pipette. The injectionpipette containing the sperm wasthen moved from the PVP dropletinto the first droplet containing anoocyte. The oocyte was held by slightnegative pressure exerted on theholding pipette, and was rotatedusing both pipettes so that the polarbody was located at the 11 o’clockposition. With the polar body in thisposition the injection pipette wasalways situated with the bevel facing12 o’clock. The tip of the injectionpipette was moved close to theoocyte at 3 o’clock and a checkmade to ensure that it was in thesame plane as the oocyte equatorialplane. The sperm was then gentlypositioned close to the bevelled tip.The injection pipette was introducedinto the oocyte until the tip was onethird to one half way across theoocyte. Negative pressure was appliedto the injection pipette and gentlesuction carefully applied in order topuncture the oolemma. Puncture ofthe oolemma was evidenced by therapid and free flow of ooplasm intothe injection pipette. The pressure onthe pipette was immediately reversedand with positive pressure ooplasmand sperm were slowly ejected into
the oocyte, transferring as little PVPas possible. The injection pipette waswithdrawn gently and the injectedoocyte then released from the hold-ing pipette. This procedure was re-peated for each oocyte.
Injected oocytes were replaced inthe incubator (37°C, 5.3 % CO2) intheir injection dish. After ICSI proce-dure was completed, injected oocyteswere washed in four droplets of pre-equilibrated IVF medium underparaffin oil and transferred into 0.5 mlIVF medium under 0.5 ml liquidparaffin in a 4-well dish (Falcon®,#3654). All oocytes from both groupswere incubated in a CO2 incubatorovernight. All ICSI procedures wereperformed by one operator.
Assessment of ICSI outcome (survivalof oocytes post injection, fertilization,embryo development and pregnancydetection)
Injected oocytes were first observedduring the ICSI procedure. Allmorphologically normal oocytes inwhich introduction of the sperm intothe ooplasm proceeded smoothlywere recorded and pre-selected.The injected oocytes were observed16–18 hours after microinjection, forevidence of survival and fertilization.The criteria for normal fertilizationwere the presence of two PB togetherwith two clearly visible pronuclei(2PN). On a few occasions one orthree PN were detected, and this wasrecorded.
Twenty-four to twenty-six hours afterICSI the fertilized oocytes were as-sessed for early cleavage and embryoswhich had cleaved to 2-cells wereidentified. Forty-four to forty-eighthours after ICSI, the embryos werescored according to the classificationof Giorgetti et al. [27]: cleavage stage(1 point), less than 10 % of fragmen-tation (1 point), no irregular blasto-meres (1 point) and four blastomereson day 2 (1 point). A score of 4 wasregarded as representing perfect mor-phology.
A maximum of three selected embryoswas transferred into a dish with M3medium (Cat.No.10330010, Medi-Cult) and loaded with approx. 20 µlmedium into a Frydman doublelumen catheter (Cat.No.130DL45,Laboratoire, CCD, Paris, France). Allremaining embryos were cultured inM3 medium and examined on day 5for blastocyst formation as describedby Muggleton-Harris et.al. [28].
Pregnancy was detected by measur-ing serum concentration of B-subunithCG (human Chorionic Gonado-tropin) 16 days after ET. Where thepregnancy test was positive an ultra-sound scan was performed 6 weekslater to confirm the presence of aclinical pregnancy. Clinical pregnan-cies were defined by the observationof a yolk sac, foetal pole and foetalheart activity on scan. Ultrasoundscan also identified early miscarriage,with the loss of at least one amnioticsac, and the presence of an extra-uterine pregnancy necessitating sur-gical intervention.
The implantation rate was expressedas the ratio between the number offoetuses with heart beat and thenumber of embryos transferred.Clinical miscarriage and extrauterinepregnancies were included for calcu-lation of the implantation rate.Embryo quality score (EQS) was cal-culated as the proportion of gestationsacs to number of embryos trans-ferred to patients who conceived[29].
Statistical analysis
Quantitative variables were summedup by their means ± SD. Paired Stu-dent’s test was used for comparingmeans between two groups, Chi-squared test and Fisher’s exact testwere applied for possible relation-ships between qualitative variables.A P value < 0.05 was considered assignificant. Analysis was completedusing Statistics Package for socialScience (SPSS) 6.1 for Windows 95.
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RESULTS
The study included 58 patients whounderwent 60 cycles of ICSI treat-ment. In Group 1 fresh ejaculatedsperm was used for 21 patients with2 couples undergoing a second cycle.In two couples where the men hadspinal cord injury sperm were recov-ered on the day of ICSI by EEJ, in fivepatients fresh testicular sperm wasused for the procedure. In Group 2twenty three patients underwent ICSIcycles with fresh ejaculated sperm,in two couples the sperm were re-covered after EEJ and for five couplesfresh testicular spermatozoa was used.The mean age of the female patientsin group 1 was 31.7 ± 3.9 years(range 25–41 ) and in group 2 31.2 ±4.3 years (range 26–41). Meanmaternal age, the duration of stimu-lation and the mean dose of humanmenopausal gonadotropin (hMG)ampoules did not differ statisticallybetween the two groups (Table 1).
A comparative number of oocyteswas retrieved in both groups, with nostatistical difference: a total of 306oocytes in Group 1 and 305 oocytesin Group 2. In Group 1, 240 oocyteswere in MII stage (78.4 %), 34 in GVstage (11.1 %), and 17 in MI stage(5.5 %), with 15 oocytes having anempty zona (5 %). In Group 2, 244oocytes were in MII stage (80 %),38 in GV stage (12.4 %) and 12 in MI(4 %). Eleven oocytes had an emptyzona (3.6 %). There were no statisti-cally significant differences in the twogroups between the number of oocytesat MII stage and those at other stagesof maturation.
The percentage of normally fertilizedoocytes with two PN was significantlylower in Group 1 (63.7 %) where oneinjection pipette was used for theentire micromanipulation procedurecompared to Group 2 (73.4 %) whereat least three injection pipettes wereused (Chi-square; P = 0.023). Therewas no apparent effect of the numberof injection pipettes used the abnor-
mally fertilized oocytes with one PNin Group 1 0.4 % vs. 0.5 % in Group2, or three PN 0.8 % vs. 1.2 % aswell as on the proportion of oocytesthat remained intact after ICSI: 97 %in Group 1 vs. 95 % in Group 2.
Early cleavage embryos were assessedafter 24–26 hours. Nine out of 153normally fertilized oocytes with twoPN in Group 1 (5.8 %) and sixteenout of 179 in Group 2 (8.9 %) hadcleaved to the 2-cell stage andassessed as early cleavaging embryos.No significant difference was ob-served between two groups. Therewas also no significant difference inthe cleavage rates per injected
oocyte (61.3 % vs. 67.6 %) or in thenumber of blastomeres on day 2(Table 2).
The morphological score on day 2was similar for both groups. InGroup 2 patients had slightly moreembryos with the highest score of 4,but the difference was not statisticallysignificant (Table 3).
Embryo transfer was performed in all60 cycles. In Group 1 84 embryosand in Group 2 85 embryos weretransferred. The mean number ofembryos transferred was 2.8 for bothgroups with a maximum of 3 embryosbeing replaced. In Group 1 a single
Table 1. Clinical data of the female patientsGroup 1 Group 2 P value
No. of cycles 30 30Female age (years)* 31.7 ± 3.9 31.2 ± 4.3 0.618Range 25–41 26–41Duration of stimulation (days)* 15.2 ± 1.7 16.1 ± 2.1 0.067Total doses of HMG** (ampoules) 23.5 ± 2.6 24.6 ± 7.6 0.559*mean ± SD; **HMG-human menopausal gonadotrophin
Table 2. Fertilization and embryo cleavage rates after ICSI in cycles in which oneinjection pipette was used (Group 1) compared with cycles in which the injectionpipette was changed after every fourth oocyte (Group 2)
Group 1 Group 2 P value
No. of oocytes retrieved (range) 306 (6–16) 305 (7–15)Mean ± SD/cycle 10.2 ± 2.8 10.2 ± 2.09 0.959No. of oocytes injected (Metaphase II)(%) 240 (78.4) 244 (80) 0.26No. of damaged oocytes (%) 7 (3) 12 (5) 0.262 PN* normally fertilized (%) 153 (63.7) 179 (73.4) 0.023**1 PN abnormally fertilized (%) 1 (0.4) 1 (0.5)3 PN abnormally fertilized (%) 2 (0.8) 3 (1.2)No. of cleaved embryos/oocytes 147 (61.3) 165 (67.6)Percentage of cleaved embryos/fertilized 96.0 92.2No. of 2–3 cell stage embryos (%) 44 (29.9) 55 (33.3)No. of ≥ 4 cell stage embryos at ET (%) 103 (70) 110 (66.6)*PN = pronuclei; **Chi-square (P < 0.05)
Table 3. Morphological score* of embryos on day 2 after ICSI procedure in Group1 and 2.Embryo score* Group 1 n (%) Group 2 n (%)
Score 4 47 (32) 58 (35)Score 3 68 (46) 81 (49)Score 2 28 (19) 18 (11)Score 1 04 (3) 08 (5)* Giorgetti (1995); with score 4 a perfect morphology was represented (Chi-square 4.77;
P = 0.189; Distribution of the embryo score is not different in both groups)
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embryo was transferred in one cycle,two embryos in 4 cycles, two ofwhich were elective 2 embryo trans-fers, and in 25 cycles three embryoswere transferred. In Group 2 twoembryos were transferred in five cy-cles and three embryos in 25 cycles.
No difference was observed betweenthe two groups. Eleven patients inGroup 1 and 13 patients in Group 2had positive pregnancy tests withraised βhCG; all were confirmed asclinical pregnancies on ultrasoundscan. Thus, the pregnancy rate be-tween the two groups did not differ:36.7 % vs. 43.3 %. In Group 2 onemiscarriage, one extrauterine preg-nancy and one multiple pregnancywere observed. The implantation rate
which was calculated for all cycles,was similar in both groups: 13.1 %for Group 1 vs. 16.5 % for Group 2.Similarly, there was no difference inembryo quality score (EQS) calculatedfrom the number of gestation sacsper number of embryos transferred inpregnant women between the twogroups: 37.9 % in Group 1 vs. 40 %in Group 2. The ongoing pregnancyrate was 36.7 % for both Groups(Table 4).
The relationship between fertilizationrates (P < 0.05) and implantationrates (not significant) when 6 to 9oocytes were injected and more than9 oocytes were injected are describedin Table 5. No difference was observedbetween the two groups.
All not transferred embryos werecultured to day 5 and assessed fordevelopment to expanded blastocyst.In Group 1 six embryos out of 63remaining embryos (9.5 %) developedto blastocyst compared with ten em-bryos out of 80 remaining embryosfrom Group 2 (12.5 %). This was notsignificantly different (Table 6).
DISCUSSION
The aim of this study was to evaluateone particular technical aspect of theICSI procedure in relation to outcome.In our centre it is customary for oneinjection pipette to be used for spermselection, immobilization, spermaspiration and oocyte injection, in-dependent of the number of oocytesavailable for injection. It was antici-pated that a difference might bedetected where a single injectionpipette was used for the entire pro-cess compared with cycles where apipette was used to inject not morethan four oocytes.
Density gradient centrifugation wasthe standard technique used in ourlaboratory to recover motile, morpho-logically normal spermatozoa [30]. Ithas been reported that the percentageof spermatozoa with normal morphol-ogy significantly increases in ejacu-lated sperm samples treated by den-sity gradients [31, 32]. In addition,spermatozoa of better nuclear con-sistency were observed from densitygradient preparations [33]. In casesof low sperm count and low motility,e. g. spermatozoa harvested fromtesticular tissue, frozen-thawed sam-ples or severe oligospermic samples,density gradient preparations may becontra-indicated because valuablespermatozoa may be lost [24]. Den-sity gradient centrifugation was notused for sperm preparation in thisstudy. Spermatozoa of patients withsevere male infertility may possessanomalies in the DNA which cannotbe observed using conventionalsemen analysis, e.g. number of sper-matozoa, motility and morphology
Table 5. Relationship between fertilization rates and implantation rates according to thenumber of oocytes injected (6–9 and more than 9 oocytes) in Group 1 and 2.No. of Group 1 Group 2oocytes injected FR* (%) IR** (%) FR* (%) IR** (%)
6–9 104/164 (63.5) 10/64 (15.6) 134/181 (74.1) 11/68 (16.2)> 9 49/76 (64.5) 1/20 (5.0) 45/63 (71.4) 3/17 (17.6)Total 153/240 (63.7) 11/84 (13.1) 179/244 (73.4) 14/85 (16.5)* FR: Fertilization Rate; **IR: Implantation Rate
Table 6. Blastocyst development of embryos remaining after embryo transfer on day 5in Group 1 and 2
Group 1 Group 2(No. of cycles =26) (No. of cycles = 24)
No. of remaining embryos 63 80Mean/cycle 2.4 3.3No. of blastocyst/n (%) 6/63 (9.5)* 10/80 (12.5)** Chi-square: P = 0.60; the percentage blastocyst formation of the remaining embryos after ET
not significant.
Table 4. Pregnancy and embryo implantation rates after ICSI in Group 1 and 2Group 1 Group 2
No. of cycles (= transfers) 30 30No. of transferred embryos in all cycles
(Mean ± SD) 84 (2.8 ± 0.4) 85 (2.8 ± 0.4)No. of transferred embryos in pregnant cycles
(Mean ± SD) 29 (2,6 ± 0,7) 35 (2,7 ± 0,5)No. of clinical pregnancies (%) 11 (36.7) 13 (43.3)No. of implantation/transferred embryos (%) 11/84 (13.1) 14/85 (16.5)No. of ongoing pregnancies (%) 11 (36.7) 11 (36.7)Embryo quality score* 11/29 (37.9) 14/35 (40)* Embryo quality score = No. of gestation sacs/No. embryos transferred to patients who
conceived [29]
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[34]. Where ICSI is performed withspermatozoa which have DNA dam-age, fertilization and the first cleavagestages occur but the genome of thespermatozoa may not be capable ofcompleting embryogenesis and blasto-cyst development [35].
Thus, all semen samples were pre-pared by microsedimentation. Theorigin of the sperm was either freshejaculated, fresh testicular spermato-zoa or EEJ. Other studies have shownno difference in ICSI outcome fromejaculated or testicular sperm [36].Similarly no difference in ICSI out-come was found for sperm of differentorigin and for this reason it was notconsidered necessary to analyse theresults separately. It has been ob-served that in microsedimentationdroplets motile spermatozoa moveaway from debris to the edge of
droplets so that the ICSI operator canavoid aspiration of debris, erythro-cytes or other cells. In addition, thetechnique allows visualisation ofmotile spermatozoa [24].
Some authors use injection pipettesof different diameters for sperm se-lection and for ICSI in semen sampleswith severe oligoasthenozoospermicand testicular sperm. Vanderzwalmenet al. [6] used pipettes of 15 and 30microns diameters for sperm aspira-tion from droplets containing largenumber of immotile spermatozoa,debris and other cells. However,their results cannot be comparedwith the results obtained in this study,since Vanderzwalmen and colleaguesaltered a number of the technicalaspects of ICSI for the purpose oftheir study e.g. diameter of injectionpipette and cytoplasm aspiration.
Tucker and colleagues [37] reportedthe use of extra flat-ended 10 microninjection pipettes for the aspiration ofsperm harvested from testicular tissuefrom droplets of medium covered withparaffin oil. Hlinka et al. [38] usedmodified microtools for ICSI andclaimed to have obtained increasedfertilization rates. However, theirstudy was based on the use of a mod-ified pipette to avoid using PVP forsperm immobilisation and is there-fore not comparable with our study.
Fujii and colleagues [39] looked atthe effect of a concentration of 3%PVP versus 8% PVP, in order to avoidthe use of extra injection pipettes foraspiration of motile spermatozoa incases of severe oligoasthenozoosper-mia. As previously explained, thelatter contain relatively large amountof sperm debris and/or other cells.
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In the present study, motile sperma-tozoa were selected for injectionfrom large numbers of immotile sper-matozoa, debris and/or other cells.Where the sperm is surrounded by alot of debris this may block the injec-tion pipette or become attached tothe outside of the pipette. Techniqueshave been suggested as a means ofovercoming the technical difficultiesof pipette blocking or becoming cov-ered with debris; e.g. rubbing theinjection pipette against the holdingpipette, against the oocyte, or againstthe oil at the edge of medium dropletto remove debris [40]. In Group 1 ofthis study the pipettes were rubbedagainst the bottom of the dish to cleanthem. The same injection pipette wasthen used for injecting all the patient’soocytes, and it was considered thata possible loss of sharpness withrepeated use might have an effect onICSI outcome. This requires furtherinvestigation, since we did not ob-serve that debris interfered with theinjections in our study. In the presentstudy blocking of injection pipettewas observed to be a problem withGroup 1. Thus, all cases where thepipette became blocked during theprocedure were excluded from thestudy. In Group 2 where the injectionpipette was changed after spermimmobilisation, care was taken toavoid exposure to the air outside thedroplet in order to avoid the risk ofcontamination or drying.
The percentage of damaged oocytesafter ICSI was very low in the currentstudy, 3 % vs. 5 %. Thus the damagerate was within the acceptable rangefor ICSI, which is less than the 10 %quoted by Van Steirteghem et al. [41]and the ESHRE Task Force [42]. Weexpected a higher damage rate whena single injection pipette was usedfor the ICSI procedure, due to thepossibility of losing its sharpnessduring the procedure. However, nocorrelation was found between thenumber of injecting pipettes perOCC and number of damaged OCC.Thus, we assume that damage doesnot necessarily arise because of loss
of sharpness of the pipette. Analysisof our data showed that oocytedamage occurred irrespective of thenumber of injecting pipettes used fora single procedure. However, as sug-gested by Nagy et al. [18] damageappears to reflect the properties ofthe oolemma.
The rate of abnormal fertilization wasrelatively low (< 1 %) in both groups.A small number of multipronucleateoocytes were observed in bothgroups, supporting the theory thatICSI outcome is not related to theorigin of spermatozoa or the numberof injection pipettes used per cycle.If it is assumed that multipronucleateembryos developed because an extraspermatozoon attached to the outsidewall of the pipette and is injected intothe oocyte we might have expectedto observe a higher incidence of 3PNin Group 1. This was not the case.Some triploid embryos arise after ICSI,probably because of the failure of theoocyte to extrude second PB [43].
In both groups we observed a verylow rate (< 1%) of activated oocytesi. e. with a single pronucleus. Inthese cases the formation of the malepronucleus might not occur due todefects in the sperm cell itself, suchas impaired microtubule nucleationand elongation, and/or compromisedsperm aster function [44]. Anotherexplanation for the observation ofone PN was given by Van der Waster-laken et al. [20]. They suggested thatthe development of a single pro-nucleus might be due to the effect ofPVP or the deposition of the spermfar from the meiotic spindle duringICSI. Nagy et al. [45] reported theearly appearance of a single PN fivehours after injection and the PN haddisappeared before they made theirnext observation, 16 hours after ICSI.Geriss et al. [46] reported a very lowincidence of one PN (3.9 %) afterintroduction of sperm immobilisationwhereas Chen et al. [47] reported alow incidence of one PN when dif-ferent techniques of sperm immobili-sation were compared. They observedthat when immobilisation was per-
formed by compressing the mid-pieceof the spermatozoon, the one PNrate was 1.5 % compared with 3 %when immobilisation was performedat the tip of the tail. Barak et al. [48]reported on a successful birth of amale baby, developed from a singlepronucleated embryo, formed byinjection of spermatid.
The higher rate of normally fertilizedoocytes (2PN) in Group 2 of thisstudy, where the injection pipetteswere changed, may be the result of areduction in injury to the oolemmaor cytoskeleton by frequent changesof pipette. Analysis of the data fromthese cycles, where between 6 and16 oocytes were injected, shows nodifference in the fertilization rates ordamage rates between the firstoocytes injected and the last. If thereduction in fertilization rates oroocyte damage was due to a loss ofsharpness of the injection pipettewith repeated use it would beexpected that in Group 1 there wouldbe more damage towards the end ofthe procedure than at the start. Thiswas not the case. When comparingthe time taken to complete each pro-cedure it was found that the intervalbetween the first and the last inject-ed oocytes in Group 1 was between8 and 20 minutes. In Group 2, whenthe injection pipette was replacedafter every four injected oocytes, thetime interval between the first andthe last injected oocytes, was up to45 minutes. The time that any groupof oocytes was kept out of the incu-bator was the same for Group 1 andGroup 2. The extra time for Group 2related to the change of pipettes, andthe oocytes remained in the incuba-tor during these periods of time. Thedifference in fertilization rates be-tween Groups 1 and 2 was statisti-cally significant but there was nosignificant difference in implantationrates between the two groups when 6to 9 oocytes or more than 9 oocyteswere injected (Table 5). In Group 1,we observed very low implantationrates for embryos when more than 9oocytes were injected. However, due
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to the small numbers of cases ana-lysed this difference in implantationrates was not shown to be statisticallysignificant (P = 0.22).
Based upon our scoring system forembryos, these data suggest thatchanging the pipette after spermselection and immobilisation gives atrend towards better quality embryosand more spare embryos with thepotential for freezing. This may be areflection of better developmentalcapacity from ICSI cycles where theinjection pipette is changed aftersperm immobilisation. The total rela-tively low rate of blastocyst develop-ment may be due to the fact thatembryos with highest score of 4 werealways selected for transfer and onlyembryos with scores of 3, 2 and 1were available for extended culture.It may be even due to the sequentialculture medium, that might not beoptimal for this purpose.
It can be concluded that using oneinjection pipette for an entire ICSIprocedure resulted in poorer fertili-zation rates, but there was no appar-ent effect on cleavage rates andpregnancy rates. It is possible thatthe greatest benefit is obtained fromchanging the injection pipette once –after all sperm have been immobi-lised and transferred to PVP. Whenmore embryos are produced due to ahigher fertilization rate, it increasesthe chance of the patient for preg-nancy per cycle including the chancefollowing freezing-thawing. Theobservations reported from this studyare based on a small number ofcycles and a more extensive studywould be required to evaluate thepotential gain in fertilization, embryoquality and implantation from chang-ing the injection pipette during ICSIprocedure. The study would need tobe extended in order to establishwhether using the same injectionpipette for sperm selection, immobi-lisation and injection for all theoocytes for an individual patient hasa negative influence on the develop-mental capacity of the injectedoocytes.
ACKNOWLEDGEMENTS
I would like to thank to Dr. PrimozReš and all members of the InfertilityCenter (Women’s Hospital Postojna,Slovenia) for their assistance duringthis study. I am grateful to Dr. YonaBarak (Herzliya Medical Center;Herzliya, Israel) for her critical com-ments during the study. I wish tothank to Dr. Linda Gregory (CARU,University Hospital of Wales, Cardiff,UK) for the suggestions received inpreparing the manuscript. I am verygrateful to Dr. Kay Elder (Bourn HallClinic, Bourn, Cambridge, UK) forencouragement during the study andfor correcting this manuscript. I wishto thank Marc van den Bergh (ErasmeULB, Brussels, Belgium) for statisticalevaluation of these data. Furthermore,I wish to thank to Laboratoire C.C.D.,Paris, France for support in part of thepipettes in this work.
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BSc Darja Kastelic, MAS
Studium der Biologie an der Universität Ljubljanavon 1975 bis 1982. Von 1987 bis 1994 Weiterbil-dung zur klinischen Embryologin an der Universitäts-klinik für Gynäkologie und Geburtshilfe Ljubljana.1994 bis 2001 Aufbau und Leitung des IVF-Laborsam Infertility Center Postojna. 1998 bis 2000 Studium
zum Master of Advanced Studies in Clinical-Embryology (MAS); DonauUniversität Krems, ALPHA, und Bourn Hall Clinic. Die vorliegende Arbeit istTeil der für die Graduierung notwendigen Master Thesis. Seit Februar 2001Aufbau und Leitung des IVF-Labors am Kinderwunsch Institut Dobl.
Korrespondenzadresse:Darja Kastelic, MASDas Kinderwunsch-InstitutA-8143 Dobl, Am Sendergrund 12e-mail: [email protected]
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