Pregnancy: Prospective follow-up study of 55 children born after subzonal insemination and intracytoplasmic sperm injection

In 163 couples referred for assisted fertilization, pregnancywas established by subzonal insemination (SUZI), intracytoplasmicsperm injection (ICSI) or a combination of both techniques.These couples agreed to participate in a prospective study thatincluded a prenatal diagnosis and clinical follow-up of thechildren. No cytogenetic aberrations were observed in 43 childrentested. In 23 pregnancies occurring after SUZI, 15 women gavebirth to 21 children. After replacement of combined SUZI andICSI embryos, 10 pregnancies resulted in 8 deliveries with 10children. Transfer of ICSI embryos led to 130 pregnancies endingin 20 deliveries with 24 children, with many others still ongoingsuccessfully. In total, 55 children have been examined: 29 boysand 26 girls. One child from a singleton pregnancy presentedmultiple congenital malformations; one twin child presenteda quadriparesis. In this observational study on a limited numberof children, the incidence of major malformations was not differentfrom the incidence in the general population.     

Subzonal insemination, partial zona dissection or intracytoplasmic sperm injection? An easy decision?

This review aims to analyse and compare the results to dateof subzonal insemination (SUZI), partial zona dissection (PZD)and intracytoplasmic sperm injection (ICSI) to evaluate criticallywhether it is now possible to replace SUZI and PZD by ICSI.It appears that ICSI is a much more efficient assisted reproductiontechnique than SUZI and PZD for resolving cases of severe maleinfertility and/or repeated failure of conventional in-vitrofertilization (IVF). For ICSI compared with SUZI and PZD, fertilization(49.4, 17.7 and 16.8% respectively), percentage of patientsreaching embryo transfer (91.0, 55.1 and 23.3% respectively),percentage of transfers performed with two or three embryos(83.3% ICSI and 39.3% SUZI), pregnancy rate per embryo replacement(28.2, 18.7 and 16.5% respectively) and pregnancy rate per oocyteretrieval (24.8, 10.3 and 3.8% respectively) are all improved.In addition, cases of severely impaired semen characteristics,which were condemned to infertility for life with conventionalIVF, SUZI or PZD, can now be treated and resolved efficientlywith ICSI.     

High fertilization and implantation rates after intracytoplasmic sperm injection

Previously reported better fertilization rate after intra-cytoplasmicsingle sperm injection (ICSI) than after subzonal inseminationof several spermatozoa was confirmed in a controlled comparisonof the two procedures in 11 patients. Intracytoplasmic sperminjection was carried out in 150 consecutive treatment cyclesof 150 infertile couples, who had failed to have fertilizedoocytes after standard in-vitro fertilization (IVF) proceduresor who were not accepted for IVF because not enough motile spermatozoawere present in the ejaculate. A single spermatozoon was injectedinto the ooplasm of 1409 metaphase II oocytes. Only 117 oocytes(8.3%) were damaged by the procedure and 830 oocytes (64.2%of the successfully injected oocytes) had two distinct pronucleithe morning after the injection procedure. The fertilizationrate was not influenced by semen characteristics. After 24 hof further in-vitro culture, 71.2% of these oocytes developedinto embryos, which were transferred or cryopreserved. Only15 patients did not have embryos replaced. Three-quarters ofthe transfers were triple-embryo transfers. High pregnancy rateswere noticed since 67 pregnancies were achieved, of which 53were clinical, i.e. a total and clinical pregnancy rate of 44.7%and 35.3% per started cycle and 49.6% and 39.2% per embryo transfer.A total of 237 supernumerary embryos were cryopreserved in 71treatment cycles.     

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: 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.     

Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm injection cycles

From October 1992 to December 1994, 2732 cycles of treatmentby intracytoplasmic sperm injection (ICSI) were carried outin couples mainly with severe male-factor infertility. The overallfertilization rate in these 2732 cycles was 71% of intact oocytes.However, in 76 (72 couples) of these cycles, none of the injectedoocytes became fertilized,so the total fertilization failurerate was 3% (76/2732 cycles). Details of these 76 cycles wereanalysed. The results show that total fertilization failureafter ICSI may be explained by different factors related to(i) semen characteristics (only immotile or round-headed spermatozoafor ICSI) or (ii) the oocytes (number, abnormal morphology,damage after ICSI). Of 26 couples, 22 achieved fertilizationin their subsequent ICSI cycles. In conclusion, total fertilizationfailure after ICSI for the treatment of severe malefactor infertilitywas mainly caused by the poor viability of the spermatozoa usedfor injection; it was also associated with a low number andpoor quality of oocytes. Repeated ICSI treatment may be usefulor necessary in couples with total fertilization failure.     

Andrology: The effect of sperm preparation methods on the fertilization rate of oocytes micro-inseminated by subzonal sperm injection

Three methods were used to prepare spermatozoa for subzonalinjection into mature oocytes. In method A, the washed spermsuspension was incubated for 18 h in modified T6 culture medium.Method B consisted of incubating the sperm suspension for 6h in regular T6 culture medium. In method C, the sperm suspensionwas incubated for 6 h in regular T6 culture medium containing20% (v/v) follicular fluid. The percentages of acrosome-freespermatozoa and fertilization rates were compared for 42 treatmentcycles assigned randomly to the three sperm preparation methods.The sperm suspensions prepared by methods A and C each had significantlyhigher proportions of acrosome-free spermatozoa compared tosuspensions prepared by method B. The fertilization rates ofoocytes micro-injected with spermatozoa prepared by methodsA and C were significantly higher than for method B. Eight clinicalpregnancies resulted from 28 cycles in which embryo replacementoccurred. We conclude that the fertilization rate followingsubzonal sperm injection is related directly to the percentageof acrosome-free spermatozoa in the sperm suspension used formicro-injection     

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.     

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.     

In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection.

About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected approximately 4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.     

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.     

Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection1

The aim of this study was to determine why oocytes remain unfertilizedor develop three pronuclei after intracytoplasmic sperm injection(ICSI). Unfertilized and abnormally fertilized oocytes werefixed in glutaraldehyde,stained with Hoechst 33342 and examinedby fluorescence microscopy to identify oocyte, sperm and polarbody DNA.One-pronuclear oocytes were considered to be unfertilized.Atotal of 285 unfertilized oocytes were examined (104 ICSI cycles).Overall, 83% of these oocytes were not activated (still at metaphaseII) while 17% had activated and formed a single (female) pronucleus.About 66% of the unfertilized, metaphase II oocytes containeda swollen sperm head, indicating that the oocyte was correctlyinjected but had failed to activate and complete the secondmeiotic division. Premature chromosome condensation of the spermDNA was evident in 6% of these metaphase II oocytes (4% of theunfertilized oocytes). The swollen sperm head was located amongthe oocyte chromosomes in 5%of the metaphase II oocytes. Othercauses of failed fertilization in the metaphase II oocytes werethe failure of sperm head decondensation (11%) and ejectionof the spermatozoon from the oocyte (23%). A similar patternwas observed in one-pronuclear oocytes (52%, swollen sperm head;28%, intact, undecondensed sperm head; 20%, ejection of thespermatozoon), which indicates that asynchronous pronucleardevelopment does not explain the presence of one-pronuclearoocytes. A total of 41 threepronuclear oocytes were examinedand all had a single polar body, which indicates that the retentionof the second polar body leads to the formation of the thirdpronucleus.In conclusion, this study demonstrates that: (i)the major cause of fertilization failure after ICSI is failureof oocyte activation; (ii) ejection of the spermatozoon intothe perivitelline space is not a major cause of fertilizationfailure;and (iii) sperm head decondensation and oocyte activationafter ICSI can occur independently.     

Clustering of male infertility in the families of couples treated with intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is an effective treatment modality for male factor infertility, but it could promote the transgenerational transmission of genetic defects causing gametogenic failure. Cytogenetic and molecular techniques permit the diagnosis of some infertility-causing genetic aberrations, but many more probably evade detection with currently available technology. The analysis of the recurrence pattern of infertility in infertile couples' families could define the importance of heritable factors in the pathogenesis of human infertility. We have subjected 621 consecutive infertile couples treated with ICSI in a single institution to a comprehensive genetic workup including documentation of the family history, karyotyping and various DNA tests. In all, 1302 fertile couples served as controls. Of the infertile couples 6.4% were shown to have a fertility problem with a definite genetic basis. Male, but not female fertility problems displayed a distinct pattern of familial aggregation. In addition, the infertile couples had fewer siblings than the fertile controls, a finding compatible with suboptimal fertility already among the infertile couples' parents. In summary, our data indicate that male factor infertility should be considered a potentially heritable condition. The recurrence risk for infertility in the offspring of couples treated with ICSI might be substantial.     

Fertiliza1tion and early embryology: Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection

Human oocytes that failed to display signs of fertilizationby 44 h after intracytoplasmic sperm injection (ICSI) were processedfor electron microscopic analysis. All oocytes were arrestedat metaphase II. The first polar body contained intact corticalgranules and chromosome clumps, which were not surrounded bya nuclear envelope but still associated with microtubules. Whena second globular body was present, it always showed the sameultrastructure, indicating that it had originated from fragmentationof the first polar body and not from the resumption of the secondmeiotic division. The most prominent organelles of the oocytecytoplasm were the smooth endoplasmic reticulum and mitochondria.In the oocyte cortex, cortical granules were intact, with nosigns of incipient or incomplete cortical reaction. Oocyte chromosomeswere found in the oocyte periphery near the locality of thefirst polar body extrusion. They consisted of dense aggregatesof chromatin associated with microtubules. The chromatin ofthe injected spermatozoon was demembranated and partially decondensed.In some cases, vesicular and tubular structures, apparentlyof oocyte origin, were associated with the periphery of thesperm chromatin mass but they never formed a continuous layer.These data suggest that fertilization failure after ICSI isbasically a failure of oocyte activation.     

Treatment of severe male immunological infertility by intracytoplasmic sperm injection

A total of 29 infertile couples (group A) with male antispermantibodies detected by the mixed antiglobulin reaction (MAR)and partly by flow cytometry (n = 21) were treated using anintracytoplasmic sperm injection (ICSI) technique to assistfertilization. In all, 22 of them had shown a poor fertilizationrate (6%) in previous in-vitro fertilization (IVF) treatments.The fertilization and cleavage rates in ICSI, 79 and 89% respectively,were similar to those in a MAR-negative group (group B; n =20) injected because of male infertility (68 and 93% respectively).A third group (group C; n = 37) with male immune infertilitywas treated by conventional IVF. All these couples had at leastone oocyte fertilized, but the overall fertilization rate (44%)in group C was significantly poorer (P < 0.001) than thatin the two ICSI groups. However, the embryo quality was lowerin group A compared with that in the other groups. A total of13 pregnancies resulted in group A (46%), of which five endedin miscarriage. None of the six pregnancies (30%) in group Baborted during the first trimester. These results reveal, forthe first time, that ICSI offers a good chance of fertilizationfor couples with male immunological infertility. However, post-fertilizationevents may compromise these results because of factors not yetclearly understood.     

Examination of the safety of intracytoplasmic injection procedures by using bovine zygotes

We have evaluated the safety of intracytoplasmic sperm injection(ICSI) procedures by using bovine zygotes. Bovine zygotes wereinjected with a small amount (2–3 pl) of either mediumalone or medium containing polyvinylpyrrolidone (PVP) (sham-ICSI,without spermatozoon) using the same procedure as ICSI, andthe subsequent in-vitro embryonic development and embryo quality(number of cells/blasto-cyst) were examined. Control zygoteswhich had not been injected were similarly evaluated after in-vitrodevelopment The sham-ICSI of either medium alone or medium containingPVP into bovine zygotes had no harmful effects on the rate ofnormal fertilization and on the rate of development to hatchedblastocyst stage compared with those of controls (P > 0.05).In addition, no harmful effects were observed in the numberof cells per blastocyst (embryo quality). The results suggest,for the first time, that the ICSI procedures currently usedfor animal and human ICSI are neither detrimental to embryonicdevelopment nor detrimental to embryo quality.     

The usefulness of a piezo-micromanipulator in intracytoplasmic sperm injection in humans

Intracytoplasmic sperm injection (ICSI) has wide clinical application. In order to achieve good results with this method, it is important to restrict the possibility of oocyte injury as much as possible, and securely inject spermatozoa into the ooplasm. For this purpose, we clinically applied piezo-ICSI, which employs a micromanipulator with piezoelectric elements, to humans, and compared the results with those obtained by conventional ICSI. Conventional ICSI and piezo-ICSI were used in 279 cycles and 335 cycles respectively. Piezo-ICSI showed significantly more favourable results, with a survival rate of 88.1% (conventional ICSI: 81.4, P < 0.001), a fertilization rate of 79.4% (conventional ICSI: 66.4%, P < 0.001), and a pregnancy rate of 23.1% (conventional ICSI: 14.9%, P < 0.05). In piezo-ICSI, the needle used is not sharpened and has a flat tip. However, deformation of the oocyte during insertion of the needle is restrained by vibration of the piezo, and the oolemma is punctured readily and securely by the piezo pulse, at the site where the spermatozoon is injected. Piezo-ICSI is a promising new technique for human ICSI that should improve the survival, fertilization and pregnancy rates after ICSI.     

Successful twin pregnancy in a dual-transplant couple resulting from in-vitro fertilization and intracytoplasmic sperm injection: case report

There are numerous reports of successful pregnancy following liver transplantation. Little information is available regarding the incidence and management of infertility in transplant recipients, particularly the use of artificial reproductive technologies. We present a case of a successful twin pregnancy resulting from in-vitro fertilization with intracytoplasmic sperm injection (IVF/ICSI) in a liver transplant recipient, whose partner was a renal transplant recipient with severe oligozoospermia. With careful evaluation and monitoring, and the involvement of appropriate consultants, artificial reproductive technologies can be safely used in transplant recipient couples experiencing infertility.     

Successful pregnancy in an infertile patient with conservatively treated endometrial adenocarcinoma after transfer of embryos obtained by intracytoplasmic sperm injection.

A rare case of successful pregnancy in a woman with early-stage endometrial adenocarcinoma conservatively treated is presented. The patient, having polycystic ovaries, was initially diagnosed with hyperplasia of the endometrium and treated with several cycles of ovulation induction following intrauterine insemination. Then dilatation and curettage were carried out when hysteroscopy was performed. The histology report identified a well-differentiated adenocarcinoma of the endometrium. After repeated endometrial curettage, in-vitro fertilization and embryo transfer were introduced for immediate treatment of the patient's infertility in order to avoid the risk of recurrence of neoplastic endometrial lesions by oestrogens. A single pregnancy was achieved after transfer of the embryos obtained after intracytoplasmic sperm injection. This was performed due to the poor semen characteristics (asthenozoospermia). The patient delivered a healthy normal male infant at term. A transvaginal ultrasound examination 2 months after delivery showed a smooth, linear endometrium. Moreover, the histology report after endometrial biopsy was free of any malignancies. The patient now desires another pregnancy. We conclude that conservative treatment of early-stage endometrial adenocarcinoma in young women wishing to preserve fertility should be considered in carefully selected cases. Assisted reproductive technologies may be helpful for immediate achievement of pregnancy in such patients.     

Successful combination of transrectal electroejaculation and intracytoplasmic sperm injection in the treatment of anejaculation

We describe the case of a couple whose infertility was causedby the absence of seminal emission following retroperitonealsurgery for testicular cancer. Ejaculate could be retrievedfrom the husband by rectal probe electroejaculation (RPE) butsperm quality was so poor that conventional in-vitro fertilizationwas impossible. With intracytoplasmic sperm injection of spermatozoaretrieved by RPE — a combination not reported previously— we were able to induce a pregnancy with successful outcome.