Andrology: Intracytoplasmic sperm injection (ICSI) for severe semen abnormalities: dissecting the tail of spermatozoa at the tip

Recently, several investigators have emphasized that damagingthe membrane of spermatozoa by compressing the mid-piece orcutting the mid-portion of the tail prior to injection yieldsbetter results than using motile spermatozoa in intracytoplasmicsperm injection (ICSI). Here we report our experience usinga modified immobilization technique of dissecting the tail ofthe spermatozoon at the tip in 78 cycles on 60 patients. In55 treatment cycles purely using this modified technique, 468mature oocytes were injected. A total of 35 oocytes (7.5%) wereinjured. Of the intact oocytes, 282 (65.1%) were normally fertilizedand 266 (94.3%) subsequently cleaved. A single pronucleus wasobserved in 16 (3.7%) oocytes, and three pronuclei were notedin 11 (2.5%) oocytes. Embryo transfers were performed in 54cycles, and 18 women (32.7%) achieved clinical pregnancies.In 23 cycles, we compared the effects of these three immobilizationtechniques on the sibling oocytes obtained from the same patientregarding normal fertilization, abnormal fertilization, andembryo cleavage and quality. The results were comparable amongthem. Seven pregnancies (30.4%) were achieved in this series.Dissecting a sperm tail at the tip is easily and quickly performedand achieves permanent immobilization. Compression of the mid-pieceis also easy, but usually takes several actions to achieve immobilization.Cutting the tail at the mid-portion requires more skill. Therefore,dissecting the tail of the spermatozoon at the tip may providean alternative method to immobilize the spermatozoon permanentlyprior to ICSI.     

Andrology: CASE REPORT Pregnancy after intracytoplasmic sperm injection of spermatozoa extracted from frozen-thawed testicular biopsy

The case report illustrates the successful application of anew method of sperm extraction from a frozen-thawed testicularbiopsy specimen within an established programme of intracytoplasmicsperm injection.     

Andrology: The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters

High success rates have been reported for the use of intracytoplasmicsperm injection (ICSI) in alleviating essentially andrologicalinfertility. However, neither the relationship between any ofthe sperm parameters and the result of ICSI nor the minimalsperm requirements for ICSI have been investigated so far. Inthis paper, our objective was therefore to study the relationshipbetween three basic sperm parameters (total sperm count, spermmotility and morphology) and the outcome of ICSI by retrospectiveanalyses of fertilization, embryo development and pregnancyrates in 966 micro-injection cycles, performed with ejaculatedsemen. The results showed that there was no important influencefrom either the type or the extent of sperm impairment on theoutcome of ICSI. Even in the most extreme cases of male-factorinfertility, where cryptozoospermia or total astheno- or totalteratozoospermia was diagnosed in the initial semen sample,high fertilization and pregnancy rates were obtained by ICSI.Only one condition had a strongly negative influence on theresult of ICSI: where an immotile (presumably dead) spermatozoonwas injected into the oocyte. Thus the only ultimate criterionfor successful ICSI is the presence of at least one living spermatozoonper oocyte in the pellet of the treated semen sample used formicro-injection.     

Intracytoplasmic sperm injection: a state of the art technique

Of the micromanipulation techniques developed in the twentiethcentury, intracytoplasmic sperm injection (ICSI) has been themajor breakthrough in the field of assisted fertilization. Thisarticle reviews the indications for the use of ICSI, its clinicalapplication, the establishment of an ICSI programme includingprotocol and the results obtained since the introduction ofICSI and the potential risks. In addition, intracytoplasmicspermatid injection is briefly discussed.     

Andrology: Pregnancy after intracytoplasmic sperm injection of metaphase II oocytes with spermatozoa from a man with complete retrograde ejaculation

Retrograde ejaculation is an uncommon cause of infertility,which has been treated successfully with different kinds ofartificial reproduction technique, e.g. cervical cap artificialinsemination by husband, intra-uterine and intraperitoneal insemination,standard in-vitro fertilization, pronuclear stage transfer andgamete intra-Fallopian transfer. All these techniques requirea minimal number and motility of spermatozoa obtained afterpost-masturbation voiding. In some cases, only very few spermatozoawith very poor or no motility are found in the urine voidedimmediately after masturbation. In such a case, where no morethan 14 spermatozoa were recovered over a 3 h search, intracytoplasmicsperm injection of metaphase II oocytes led to the developmentand replacement of three fair embryos, resulting in an ongoingtwin pregnancy. This technique opens up perspectives for thetreatment of men with complete retrograde ejaculation and quasi-azoospermicpost-voiding specimens.     

Andrology: Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration

Intracytoplasmic sperm injection (ICSI) has been successfulin cases of extreme oligoasthenozoospermia in achieving pregnanciesvia in-vitro fertilization (IVF) with the lowest imaginablesperm counts. In azoospermia caused by congenital bilateralabsence of the vas deferens (CBAVD), it has been shown thatepididymal spermatozoa can be retrieved in large numbers, butfertilization rates using conventional IVF are low. Furthermore,no fertilization has ever been possible using testicular spermatozoawith conventional IVF. In the most extreme case of absence ofthe epididymis, spermatozoa can only be retrieved from maceratedtesticular biopsy specimens. In such cases, all that can beseen are free-floating Sertoli cells with many spermatids attached,and only occasional spermatozoa per high power field which haveonly the barest, occasional, slightly twitching motion. Theobjective of the present study was to determine whether ICSIcould achieve better results than conventional IVF with microsurgicalaspiration of spermatozoa (MESA). ICSI (using epididymal ortesticular spermatozoa) from men with CBAVD or irreparable obstructiveazoospermia, achieved good fertilization and normal embryosin 82% of cases, compared to 19% with conventional IVF. Therewas an overall fertilization rate of 45%, with 85% progressingto normally cleaving embryos using ICSI, compared to 6.9% usingconventional IVF. The pregnancy rate with ICSI/MESA was 47%per stimulated cycle (normal delivery rate was 30%), comparedto 4.5% with conventional IVF. These results were achieved inpatients who had consistently failed to fertilize in previouscycles with MESA and conventional IVF. We conclude that althoughcomplex mechanisms (facilitated by epididymal passage) may berequired by spermatozoa for conventional fertilization of humanoocytes (whether in vivo or in vitro), no such mechanisms arerequired for fertilization after direct microinjection. Becauseof the consistently good results using epididymal spermatozoawith ICSI in comparison to conventional IVF, and also the goodresults in extreme cases requiring testicular tissue spermatozoa,ICSI may be man dated for all future MESA patients with CBAVD,or with irreparable obstructive azoospermia.     

Andrology: Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection

In this report, we present the results of our first 100 consecutivecycles of intracytoplasmic sperm injection (ICSI). Overall,fertilization occurred in 98% of cycles and embryos were transferredin 94% (2.6 embryos per cycle). About 50% of patients had embryosfrozen. The overall fertilization rate was 71%, of which 4%were abnormally fertilized (three pronuclei). A total of 30clinical pregnancies were established (32% per transfer), resultingin 18 singleton, six twin and one triplet ongoing pregnancies.The implantation rate per embryo was 15%. There were no significantdifferences in the fertilization or pregnancy rates betweenpatients Who had only occasional motile spermatozoa in the ejaculate,semen that was too poor for routine in-vitro fertilization (IVF),or who had failed routine IVF and/or subzonal sperm injection(SUZI). A group of 18 patients were treated with both ICSI androutine IVF on their first cycle because of the high likelihoodof failed fertilization due to poor sperm morphology (<20%normal). In this group, ICSI oocytes had a fertilization rateof 76% compared to only 15% for the routine IVF (control) oocytes,and six patients conceived after transfer of ICSI embryos (33%),indicating that ICSI can be used successfully on 50% of theoocytes if fertilization failure is expected. Similarly, patientswho had failed to become pregnant with SUZI achieved excellentresults after ICSI. There were no significant differences betweenICSI and routine IVF in the proportions of grade 1, 2 or 3 embryoson day 3 post-oocyte recovery. In conclusion, we have achievedresults comparable to those reported from Belgium and we havefound that ICSI is universally applicable to all forms of severemale factor infertility. ICSI produces fertilization, pregnancyand freezing rates comparable to routine IVF with normozoospermicsamples and has none of the drawbacks of other assisted fertilizationtechniques.     

Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection

The fertilization rates and further development of 528 humanmetaphase IT oocytes directly injected by a single spermatozoonwere analysed with respect to their morphological features atthe light microscopy level at the time of retrieval. The deviationsof oocyte morphology which were most frequently observed, afterremoval of cumulus cells, were dark incorporations, dark zonapellucida, large peri-vitelline space, spots, vacuoles, refractilebodies and irregular shape. These deviations correlated neitherwith the fertilization rate nor with the embryo quality score,as compared to ‘ideal’ oocytes. Since the majorityof oocytes displayed deviations from the ‘ideal’morphotype but were still fertilized and developed in cultureat a normal rate, they were probably as normal as ‘ideal’oocytes. Since some of these morphotypes, such as refractilebodies, have been shown to be associated with failure of fertilization,it seems that intracytoplasmic sperm injection may be an appropriatemethod of treatment for couples in whom repeated failure ofin-vitro fertilization is associated with the retrieval of dysmorphicoocytes in the presence of normal semen characteristics.     

Intracytoplasmic sperm injection in the mouse

Intracytoplasmic sperm injection (ICSI) into mouse oocytes involvesa very low survival rate. This study was designed to determinewhy ICSI frequently fails in mice. Metaphase II oocytes wereobtained from superovulated 4–6 week old F₁ hybrid mice.Spermatozoa were retrieved from the epididymis of 12–14week old F₁ hybrid mice. The spiked microinjection pipette usedto inject a spermatozoon into the ooplasm had outer and innerdiameters of 10 and 8 µm respectively. The oocytes usedin the first part of the study were not activated (group 1).Some oocytes were incubated with calcium ionophore for 5 min(group 2). The injected oocytes were evaluated 6, 20, 48 and72 h after injection. A total of 143 eggs in each group underwentICSI. In group 1, sperm heads escaped into the perivitellinespace. In all, 63 (47%) of the remaining oocytes were damagedduring the injection or had degenerated by the first evaluation.The survival rate was 53%, but fertilization did not occur.In group 2, 31 oocytes (22%) were damaged during microinjectionor soon degenerated. Two oocytes underwent accidental subzonalinsemination. Six oocytes were fertilized (4.2%) among the 78%of survivors. After injection, the sperm tail was found in thecytoplasm (27 and 31% in groups 1 and 2 respectively), the perivitellinespace (45% in both groups) or protruding through the zona pellucida(28 and 23% respectively). More oocytes degenerated when thetail remained in the cytoplasm, i.e. 78% in group 1 and 36%in group 2.     

Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa

In 25 patients (14 suffering from obstructive azoospermia, sixfrom non-obstructive azoospermia, three from astheno-azoospermiaand two from absence of ejaculation) spermatozoa were extractedfrom testicular biopsies. Intracytoplasmic sperm injection (ICSI)with fresh testicular spermatozoa was performed in 18 cases;spermatozoa in excess were cryopreserved in pills. No pregnancieswere achieved. In the remaining seven patients, testicular spermatozoawere retrieved and cryopreserved during a diagnostic testicularbiopsy. After thawing, sperm motility was assessed in 17 cases(68%), and 18 ICSI with cryopreserved testicular spermatozoawere performed. The mean two-pronuclear (2PN) fertilizationrate was 59%, the mean cleavage rate was 92%, and six clinicalpregnancies were achieved, all of them still ongoing (pregnancyrate 33%). A comparison of the results of ICSI carried out withfresh or cryopreserved testicular spermatozoa showed that themean 2PN fertilization rates per cycle (53 compared with 55%),mean cleavage rates per cycle (99 compared with 96%) and embryoquality were not significantly different In conclusion, cryopreservationof testicular spermatozoa is feasible, even in patients withnon-obstructive azoospermia, and the results of ICSI with frozen-thawedtesticular spermatozoa are similar to those obtained using freshtesticular spermatozoa. Cryopreservation of testicular spermatozoamay avoid repetition of testicular biopsies to retrieve spermatozoafor successive ICSI cycles in patients in whom the only sourceof motile spermatozoa is the testicle.     

Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection

In this study the fertility and outcome of intracytoplasmic sperm injection (ICSI) using megalohead spermatozoa from the ejaculates and testicles was evaluated. Seventeen males with megalohead and pinhead sperm forms in their ejaculate were studied in 22 cycles. A high number of sperm heads without tails and abundant round spermatid forms were commonly observed. Round-headed spermatozoa were seldom accompanied by these severely abnormal spermatozoa. The majority of megalohead spermatozoa were observed to have multiple tails, were predominant in the sample, and were used for ICSI. Ejaculated megalohead spermatozoa were used for ICSI in 15 cycles, while testicular spermatozoa were used in seven cycles where there were no vital spermatozoa or spermatozoa of low vitality in the ejaculate. The same abnormal morphology was observed in the testicles as in the ejaculated spermatozoa in the same males. Mean (+/- SD) low motility 4.7 +/- 5.6% and sperm count (3.8 +/- 4.19 x 10(6)) were common findings in these severely teratozoospermic patients. A low fertilization rate (43.2%) was achieved by using megalohead sperm forms (group I, n = 17) in comparison with the control group (60.2%) which had zero normal sperm morphology according to strict criteria (group II, n = 30) (P <0.01). Furthermore, a low pregnancy rate (9.1%) was obtained in the megalohead sperm group in comparison with the control group (40%) (P <0.05). Low fertilization and pregnancy rates may be due to a high incidence of chromosomal abnormalities from severely defective spermatozoa in the ejaculate. Couples should be counselled and warned about possible low fertilization and pregnancy rates with ICSI when only pinhead and megalohead forms with a high number of sperm heads without tails are present in the ejaculate.     

Andrology: Preferences for intracytoplasmic sperm injection versus donor insemination in severe male factor infertility: a preliminary report

With the advent of intracytoplasmic sperm injection (ICSI),our programme noted a drop in the number of couples using donorinsemination (DI) for severe male factor infertility. Over thefirst 8 months in which our infertility programme offered bothtreatments, 27 consecutive couples scheduled for ICSI and 15consecutive couples scheduled for DI were evaluated Since allpatients in our infertility programme beginning in-vitro fertilization(TVF) with planned ICSI or starting DI undergo a semi-structuredpsychological interview, the psychologist's clinical notes aswell as the medical chart were reviewed and coded retrospectivelyto determine factors related to a couple's treatment choice.Couples who chose IVF-ICSI over DI had a higher occupationalstatus and included husbands with higher educational levels.Their most common motivation was to have the husband's biologicalchild (93% of couples in the ICSI group). The most common motivationfor choosing DI (60% of DI couples) was that IVF was not financiallyaffordable. Choice of treatment was not related to psychologicaladjustment, the husband having prior biological children, orhis risk of passing on a genetic defect to offspring. Thesepreliminary data raise the concern that, with the success ofICSI, DI may change in the USA from being an option dictatedby semen quality to a second choice treatment utilized for economicreasons.     

Andrology: Percutaneous epididymal sperm aspiration for obstructive azoospermia

We report the use of percutaneous epididymal sperm aspirationas a simpler and more acceptable alternative to microscopicepididymal sperm aspiration for patients with obstructive azoospermiain whom bypass surgery is not feasible or has not been successful.Some contamination of the aspirate with blood is inevitable,but with careful sperm preparation techniques this can be reducedsubstantially in the final aliquot used for assisted conception.Spermatozoa with active forward progression may be used forgamete intra-Fallopian transfer treatment, but when this capacityis absent intracytoplasmic sperm injection is recommended. Threepregnancies were obtained in seven couples and a set of twinshas been delivered.     

Andrology: Successful fertilization and establishment of pregnancies after intracytoplasmic sperm injection in patients with globozoospermia

Globozoospermia or round-headed spermatozoa is a rare type ofteratozoospermia where the acrosome is absent resulting in maleinfertility with no known therapy. A few studies have shownthat round-headed spermatozoa cannot bind to or penetrate thezona pellucida, and no normal fertilization has been observedin in-vitro fertilization (IVF) after insemination of humanoocytes with round-headed spermatozoa. In this study, the fertilizationcapacity of round-headed spermatozoa after intracytoplasmicsperm injection (ICSI) into human oocytes has been examined.In pre-clinical experiments, 45 oocytes were injected; 41 oocyteswere intact after injection, 15 oocytes were fertilized normally,and 13 of these 15 oocytes developed further in vitro. ICSIwas carried out in 11 treatment cycles of seven infertile coupleswith globozoospermia. Normal fertilization and embryo transferoccurred in four cycles (three patients). Positive serum humanchorionic gonadotrophin was observed in three cycles (two patients);one patient had a pre-clinical abortion and the other patientbecame pregnant twice: the first pregnancy was ectopic and thesecond pregnancy is a twin pregnancy which is currently at 16weeks of gestation.     

Andrology: Reconsideration of testicular biopsy and follicle-stimulating hormone measurement in the era of intracytoplasmic sperm injection for non-obstructive azoospermia?

To determine the possibility of finding motile spermatozoa andspermatids in patients with high serum follicle stimulatinghormone (FSH) and spermatogenetic disorders proven by pathology,100 cases of male infertility were reviewed. Of these, 71 patientswere found to have non-obstructive azoospermia or severe primaryspermatogenetic disorders, and 20 had obstructive azoospermia.A prospective study of the most recent 51 cases was conducted.Multiple testicular tissue biopsies were examined by a pathologistand a well-trained gynaecological technician. The findings ofspermatozoa, spermatids and serum FSH concentrations were comparedamong six different histological groups. It was concluded that51.2% of the non-obstructive azoo-spermic and failed spermatogeneticpatients had spermatids and even motile ‘shaking’spermatozoa and should be re-evaluated. In the non-obstructiveazoospermic patients here, almost all the motile spermatozoaand spermatids were found in patients with a serum FSH concentrationof <30 mIU/ml. It is suggested that a testicular biopsy shouldbe conducted in every case of non-obstructive azoospermia andspermatogenetic disorder, even in those patients with elevatedserum FSH concentrations.     

Outcome of intracytoplasmic sperm injection with testicular spermatozoa in obstructive and non-obstructive azoospermia

From 1 August 1993 until 30 September 1994, 69 couples sufferingfrom azoospermia underwent testicular sperm extraction and intracytoplasmicsperm injection. In 50 couples with obstructive azoospermiaa total of 631 meta-phase-II oocytes were injected after testicularsperm extraction yielding a 2-PN fertilization rate of 57%.In female patients <40 years of age an ongoing pregnancyrate per transfer of 42% (14/33) was obtained. So far, eighthealthy babies have been born, including two singletons andthree twin gestations. In 19 couples with non-obstructive azoospermiaa total of 264 metaphase-II oocytes were injected after testicularsperm extraction, yielding a 2-PN fertilization rate of 58%.An ongoing pregnancy rate per transfer of 31% (5/16) was established.So far, six healthy babies have been born including one singleton,one twin and one triplet gestation.     

High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy

In cases requiring microsurgical epididymal sperm aspiration(MESA) for congenital absence of the vas deferens (CAVD) orirreparable obstructive azoospermia, often no spermatozoa canbe retrieved from the epididymis, or there may even be no epididymispresent. We wished to see whether testicular biopsy with testicularsperm extraction (TESE) in such cases could yield spermatozoathat would result in successful fertilization and pregnancy(despite the absence of epididymal spermatozoa) using intracytoplasmicsperm injection (ICSI). In the same setting during the same2-week period, 28 patients with CAVD or irreparable obstructionwere treated; 16 consecutive fresh MESA—ICSI cycles and12 cycles which required testicular biopsy with testicular spermextraction (TESE—ICSI) were performed. Normal two-pronuclearfertilization rates were similar in both groups: 45% for epididymalspermatozoa and 46% for testicular biopsy-extracted spermatozoa.Cleavage rates were also similar (68% for epididymal and 65%for testicular spermatozoa). The ongoing pregnancy rates inthis series were 50 and 43% respectively. We conclude that epididymalspermatozoa and testicular spermatozoa yield similar fertilization,cleavage and ongoing pregnancy rates using ICSI. When epididymalspermatozoa cannot be retrieved, a testicular biopsy can beperformed and the few barely motile spermatozoa thus obtainedcan be used for ICSI. It appears that all cases of obstructiveazoospermia can now be successfully treated.     

Andrology: Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes

The chromosome constitution of human spermatozoa was determinedafter injecting individual spermatozoa into mouse oocytes. Ofa total 279 eggs arrested at first cleavage metaphase, 200 (71.7%)were suitable for the analysis of sperm chromosomes. Incidencesof spermatozoa with numerical and structural chromosome aberrationswere 1.3 and 6.9% respectively in spermatozoa with normal headmorphology, showing values comparable with those found in previousstudies using the hamster oocyte-human sperm fusion system.The ratio of X- to Y-bearing spermatozoa did not differ significantlyfrom the expected 1:1 ratio. The incidence of structural chromosomeaberrations was about four times higher in spermatozoa withamorphous, round and elongated heads (26.1%) than in those withmorphologically normal heads, whereas the incidence of aneuploidywas not significantly different between the two groups. No increasein chromosome aberrations was found in spermatozoa with largeheads. The same was true for spermatozoa with small heads. Althoughthe sample size used in this study is rather small, the resultsnevertheless indicate that some morphological abnormalitiesin the sperm heads are associated with their chromosome defects.     

Andrology: Factors affecting pentoxifylline stimulation of sperm kinematics in suspensions

Sperm suspensions were prepared either by the ‘swim-up’technique or by a Percoll gradient method before pentoxifyllinewas added to improve sperm motion characteristics. They weresubsequently washed and then incubated with oocytes in in-vitrofertilization (IVF) programmes. The curvilinear velocity andlateral head displacement were 20% higher in Percoll gradient-separatedsamples compared with samples prepared by the swim-up technique(P > 0.001), sperm motion characteristics being assessedby computer-assisted semen analysis. The dose-response studyin which samples were separated by Percoll gradient/swim-upmethod showed that pentoxifylline gave maximum enhancement ofsperm motion characteristics at a concentration of 2.8 mM/l,when the curvilinear velocity and lateral head displacementwere significantly increased (P > 0.001). However, when pentoxifyllinewas removed by washing, the enhanced motion characteristicswere reduced or lost in the process. Surprisingly, washing hada significant effect on the control samples, the motion characteristicsbeing Increased significantly (P > 0.001). This study castsdoubt on the usefulness of pentoxifylline in IVF programmes.     

Fertilization and early embryology: Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen--thawed epididymal spermatozoa

In seven patients who did not become pregnant following microsurgicalepididymal sperm aspiration (MESA) and intracytoplasmic sperminjection (ICSI), a subsequent ICSI was performed using previouslycryopreserved supernumerary epididymal spermatozoa without re-operatingon the husband. During the original MESA procedure a mean spermconcentration of 12.3x10⁶/ml was achieved. The supernumeraryspermatozoa were cryopreserved for later use. After thawingfrozen epididymal spermatozoa a mean concentration of 1.9x10⁶spermatozoa/ml was obtained in straws containing a total volumeof sperm suspension of 250 µl. From 68 intact oocytesinjected with frozen—thawed epididymal spermatozoa, atwo pronuclear fertilization rate of 45% and a cleavage rateof 82% were obtained. A total of 17 embryos were replaced inthe seven patients, resulting in two ongoing singleton pregnanciesand one twin delivery. Six embryos were cryopreserved. In conclusion,it would appear mandatory to cryopreserve supernumerary spermatozoaduring a MESA in order to avoid subsequent further scrotal surgery.