Case report: Pregnancy resulting from intracytoplasmic injection of spermatozoa from a frozen-thawed testicular biopsy specimen

A testicular biopsy specimen was taken in connection with scrotalexploration of a healthy 35 year old man who had azoospermia.Bilateral severe scarring of unknown aetiology was found inthe exploration, and no epididymal spermatozoa could be obtained.Spermatozoa from the fresh biopsy specimen were used for intracytoplasmicsperm injection (ICSI) on the same day. Two-embryo transferresulted in biochemical pregnancy. The rest of the biopsy specimenwas frozen as small pieces of tissue using glycerol as a cryoprotectant.ICSI was then performed with spermatozoa prepared from the frozen-thawedtissue. One embryo was obtained and transferred. The transferresulted in pregnancy, and a living fetus was seen in ultrasoundscans at the seventh and 16th weeks of pregnancy. It is possibleto avoid repeated testicular biopsies by using cryopreservationof testicular tissue.     

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.     

Pregnancy resulting from intracytoplasmic injection of cryopreserved spermatozoa recovered from testicular biopsy

We report a clinical pregnancy occurring in a 31 year old patientfollowing intracytoplasmic sperm injection (ICSI) of cryopreservedspermatozoa obtained from a testicular biopsy. This was thecouple‘s second attempt at an ovarian stimulated cycleresulting in the collection of 17 metaphase II ova which wereall injected with progressively motile spermatozoa. A fertilizationrate of 58% and a cleavage rate of 90% were achieved. This reportis our first case of ICSI using cryopreserved testicular spermatozoawhich resulted in normal fertilization, embryo development andan on-going singleton pregnancy.     

Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa

A comprehensive study is presented of a series of 124 infertilemen undergoing testicular sperm retrieval for intracytoplasmicsperm injection (ICSI). In this study we correlated the histologicalchanges observed in the testicular tissue with the results ofthe wet preparation and the outcome after ICSI using testicularspermatozoa. In all patients with normal spermatogenesis andhypospermatogenesis spermatozoa were recovered from the wetpreparation. The sperm recovery rate was 84% in patients withincomplete germ-cell aplasia and maturation arrest, while inpatients with complete germ-cell aplasia or maturation arrestthis figure was 76%. In these patients more specimens were sampledand fewer spermatozoa were recovered. Since no spermatozoa wererecovered in only 10 patients, ICSI with testicular sperm wasperformed in the remaining 114 couples (91.9%). The normal fertilizationrate was 57.8%. The fertilization rate was significantly lowerin couples among whom the husband showed germ-cell aplasia andmaturation arrest. Overall, 55.2% of normally fertilized oocytesdeveloped into embryos showing 50% of anucleate fragments. Therewere no major differences between the different histologicalcategories in terms of embryonic development in vitro. The overallpregnancy rates per testicular sperm extraction (TESE) procedure,per ICSI procedure and per transfer were respectively 36.3,39.5 and 43.7%. The overall implantation rate per embryo (sacs/embryosreplaced) was 20.3%. A lower implantation rate was observedin couples among whom the husband had maturation arrest (notstatistically significant). The above data show that testicularbiopsies may have an important therapeutic role in the managementof infertility in azoospermic patients.     

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.     

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.     

Testicular biopty gun needle biopsy in collecting spermatozoa for intracytoplasmic injection, cryopreservation and histology.

Using testicular spermatozoa from either open biopsy (29 cycles) or biopty gun needle biopsy (49 cycles), a total of 81 intracytoplasmic sperm injection (ICSI) cycles among 57 couples were carried out from January, 1994 to September, 1997. In six cycles, no spermatozoa were obtained, and in three cycles spermatozoa from both needle and open biopsies were used. The fertilization (37% after open and 41% after needle biopsy) and pregnancy rates (29% per embryo transfer compared with 16% per embryo transfer) were similar after both open and needle biopsies. Five pregnancies were achieved among the 14 couples with non-obstructive azoospermia of the male partner, four of these after needle biopsy. It was possible to use cryopreserved testicular spermatozoa after both needle and open biopsies, and one pregnancy started after using cryopreserved testicular spermatozoa in both groups. Histological needle biopsy was carried out in 62 cases, and they were all diagnostic, giving 15-20 cross-sections of seminiferous tubuli per biopsy. Testicular needle biopsy using a 14 gauge biopsy needle gave a sufficient amount of tissue and spermatozoa for ICSI, cryopreservation and histology, even in non-obstructive azoospermia. This technique is simpler and cheaper than open biopsy and, hence, it can be regarded as the optimal method for the retrieval of testicular spermatozoa.     

Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men

In non-obstructive azoospermia spermatozoa can usually onlybe isolated from the testicles, and thus the most promisingtreatment model is testicular sperm extraction (TESE). Hormoneconcentrations, testicular volume determinations and testicularbiopsy results are not uniform enough to select potential candidatesfor successful TESE and intracytoplasmic sperm injection (ICSI)approaches in advance. The aim of this study was to assess theefficacy of using ICSI with testicular spermatozoa in casesof non-obstructive azoospermia and to compare the inclusioncriteria and sperm existence in the testicles in sperm obtainableand non-obtainable groups. All men showed either complete orincomplete (n = 14) maturation arrest in spermatogenesis, severehypospermatogenesis (n = 10) or Sertoli cell-only syndrome (n= 5) in their testicular biopsies. Only 14 out of a total of29 men provided enough spermatozoa for the ICSI procedure, whileno spermatozoa were found in the testicular samples of the remaining15 men. Out of 123 oocytes obtained from 14 females, 101 wereinjected with the husbands' testicular sperm cells. Total fertilizationfailure was observed in three cases. Of 39 oocytes fertilized,38 cleaved. The fertilization and cleavage rates were 38.6 and97.4% respectively. The pregnancy rate was 20.7% per initiatedcycle. In the group from whom spermatozoa were obtainable, thepregnancy rate was 42.9% per initiated cycle and 54.5% per embryotransfer. A total of six pregnancies were achieved, of whichtwo Were twins and four were singletons. One singleton pregnancyresulted in abortion in the first trimester. There was no statisticaldifference concerning the serum follicle stimulating hormoneconcentration, testicular volume and biopsy results in groupsin which spermatozoa were obtainable or not. In conclusion,although the association of TESE with ICSI obtained pregnanciesfor some patients with non-obstructive azoospermia, furtherstudies are needed to determine the inclusion criteria for successfulTESE.     

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.     

Enzymatic digestion of testicular tissue may rescue the intracytoplasmic sperm injection cycle in some patients with non-obstructive azoospermia

Recovery of testicular spermatozoa from azoospermic patientswith testicular failure, followed by intracytoplasmic sperminjection (ICSI) is a recent advance in the treatment of maleinfertility. In most cases, free spermatozoa are recovered fromtesticular tissue after mechanical mincing of multiple biopsies.Testicular sperm retrieval, however, remains unsuccessful in30–50% of male patients suffering from Sertoli cell-onlysyndrome and maturation arrest. In this study, a strategy wasdeveloped in order to maximize the chance of sperm retrievalin difficult cases of testicular failure. The ultimate stepwas the use of enzymatic procedures (collagenase type IV) todissociate the testicular tissue completely. Testicular tissueof 41 patients for whom no spermatozoa were found after mechanicalmincing of the testicular tissue was investigated. In 14 outof the 41 cases (34%), enough spermatozoa for ICSI were foundafter fine mincing of multiple biopsies and several hours' searchin the cell suspension treated with the erythrocyte-lysing buffer(ELB). In 27 out of the 41 patients, no spermatozoa were foundeven after the use of ELB. In seven out of these 27 failures(26%), spermatozoa for ICSI were retrieved after enzymatic dissociationof the residual minced tissue pieces, thus making ICSI possibledespite failure to find spermatozoa with conventional mincing.From this study, we may conclude that enzymatic digestion oftesticular tissue is easy to perform, is not time-consumingand constitutes a successful method in reducing the sperm recoveryfailures in patients with non-obstructive azoospermia.     

Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection

In this study we investigated whether morphology and chromatinanomalies in human spermatozoa can influence fertilization afterintracytoplasmic sperm injection (ICSI). We examined unfertilizedoocytes, using the fluorochrome Hoechst 33342, to determinewhether a relationship exists between failure of fertilizationand sperm chromatin quality. Sperm chromatin packaging qualitywas assessed using the chromomydn A3 (CMA3) fluorochrome, andthe presence of DNA damage in spermatozoa, using in-situ nicktranslation. Normal males present sperm parameters with a normalmorphology of >20%, CMA3 fluorescence of <30% and exhibitendogenous nicks in <10% of their spermatozoa. When patientswere separated according to these values no difference was observedin their fertilization rates after ICSL When the unfertilizedICSI oocytes were examined, we found that patients with CMA3fluorescence of <30% and nicks in <10% of their spermatozoahad only 17.5 and 21.6% respectively of their unfertilized oocytescontaining spermatozoa that remained condensed. In contrast,patients with higher CMA3 and nick values had a significantlyhigher number, 412 and 48.9%, of their unfertilized oocytescontaining condensed spermatozoa. Sperm morphology did not showany such pattern. The percentage of spermatozoa which had initiateddecondensation in unfertilized oocytes was not influenced bymorphology, CMA3 fluorescence or nicks. In light of these resultswe postulate that poor chromatin packaging and/or damaged DNAmay contribute to failure of sperm decondensation after ICSIand result in failure of fertilization.     

Obstetric outcome of 424 pregnancies after intracytoplasmic sperm injection

An evaluation of the outcome of pregnancies resulting from intracytoplasmicsperm injection for severe male factor infertility was conductedby analysing the data obtained from the patients and/or theirobstetrician/gynaecologist on standardized questionnaires. Thedata from 424 pregnancies between April 1991 and September 1994were analysed. Early pregnancy loss before 16 weeks occurredin 99 cases (23.3%), including 48 clinical abortions (11.3%),47subclinical pregnancies (11.1%) and four ectopic pregnancies(0.9%). Vanishing twins and triplets, which could be regardedas early embryonic wastage, were found in 36 cases (8.5%). Onepregnancy was interrupted at week 15 of gestation because ofanhydramnios, and four pregnancies (0.9%) ended in spontaneouslate abortions before 26 weeks. A total of 320 pregnancies (75.5%)resulted in the birth of at least one child; 222 of these (69.3%)were singletons, 93 were twins (29.1%) and five were triplets(1.6%). The problems of prematurity and low birthweight wereespecially related to the multiplicity of pregnancies. Furthermore,from among the total of 423 babies born, we have observed threecases of stillbirth and five cases of neonatal mortality. Theperinatal mortality rate was therefore 18.9 per 1000 births.The results of this study show that the obstetric outcome ofthese pregnancies was similar to that obtained after conventionalin-vitro fertilization and other assisted reproduction techniques.     

Pregnancies achieved after frozen-thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissues from non-obstructive azoospermic men.

The use of frozen-thawed testicular tissue as a source of spermatozoa for intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia yields favourable fertilization and pregnancy rates while avoiding both repetitive biopsies and unexpected cycle cancellations. Spermatozoa were obtained from frozen-thawed testicular biopsy specimens from 67 non-obstructive azoospermic men. Following fertilization, supernumerary two pronuclear (2PN) oocytes were frozen. After thawing, 17 cycles of embryo transfer were carried out with a mean number of 2.7 embryos and a mean cumulative embryo score (CES) of 18.3 per transfer. The clinical pregnancy and implantation rates per transfer in these cycles (23.5 and 8.3% respectively) were comparable to those of fresh embryo transfers (35.7 and 12.7% respectively) with a mean number of 2.7 embryos and a mean CES of 28.7 per transfer. Abortion rates, although higher with cryopreserved 2PN oocytes were not significantly different. With this approach, cryopreservation of supernumerary 2PN oocytes can be used to improve the cumulative pregnancy rates in a severely defective spermatogenetic population. To our knowledge, these are the first pregnancies reported which have been obtained by the transfer of cryopreserved pronuclear oocytes obtained from ICSI using cryopreserved testicular spermatozoa.     

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.     

Andrology: Pregnancy achieved by intracytoplasmic sperm injection using cryopreserved semen from a man with testicular cancer

A successful pregnancy was achieved by intracytoplasmic sperminjection (ICSI) using cryopreserved semen from a man with testicularcancer. He was a victim of right testicular seminoma, and wasazoospermic after right orchidectomy and radiotherapy. The wifehad had three successive failures of intrauterine insemination(IUI) using semen that was cryopreserved before radiotherapy.The couple then underwent in-vitro fertilization (IVF) treatmentICSI was performed because the sperm motility was extremelypoor after thawing. Eight of 12 injected oocytes had normalfertilization and embryo cleavage. After replacement of fourembryos, a singleton pregnancy developed. She delivered a healthymale baby at 39 weeks gestation. In addition to IUI and IVF,ICSI further provides male patients with cancer an unprovedchance of fathering a child. Any men diagnosed with cancer whohave not yet finished their families should have their spermatozoafrozen before treatment, regardless of its quality.     

Simultaneous bilateral tubal pregnancy after intracytoplasmic sperm injection

Since the advent of assisted reproductive technology, the concernabout ectopic implantation of embryos has increased dramatically.Simultaneous bilateral tubal pregnancy is the least common typeof ectopic implantation of two embryos. In this report we presentthe first case of simultaneous bilateral tubal pregnancy afterintracytoplasmic sperm injection (ICSI) and embryo transfertreatment. The present case had no risk factor for ectopic pregnancy.Therefore, for early diagnosis and management of such cases,close clinical follow-up and routine ultrasonography followingICSI are necessary.     

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.     

High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia

Thirty-two infertile couples with obstructive and non-obstructiveazoospermia were included in this study. Testicular sperm extraction(TESE) was performed in 16 obstructive azoospermic cases wheremicrosurgical sperm aspiration (MESA) or percutaneous spermaspiration (PESA) were impossible because of totally destroyedepididymis and 16 non-obstructive azoospermia cases with severespermatogenetic defect where the testicles were the only sourceof sperm cells. A total of 288 oocytes was obtained from 32females and 84% were injected. The fertilization rates (FR)with 2 pronuclei (PN) and cleavage rate were 50.8 and 68.2%respectively. A total of 15 pregnancies was achieved (53% perembryo transfer), nine from the obstructive and six from thenon-obstructive group. Four pregnancies resulted in clinicalabortion (26.6%). The ongoing pregnancy rate was 39.2% per embryotransfer (ET) and 343% per started cycle. A high implantationrate was also achieved (26.6% in non-obstructive and 30% inobstructive azoospermia group). Using testicular spermatozoain combination with ICSI in both obstructive and non-obstructiveazoospermic groups, high implantation and pregnancy rates canbe achieved.     

Birth of a healthy neonate following the intracytoplasmic injection of testicular spermatozoa from a patient with Klinefelter's syndrome

Klinefelter's syndrome is one of the known causes of azoospermia or cryptoazoospermia, and it may present in non-mosaic (47,XXY) or mosaic (47,XXY/46,XY) form. The likelihood of finding spermatozoa in the ejaculate or testicular tissue of patients with mosaic Klinefelter's syndrome is low, and with the non-mosaic form, even lower. We describe a patient with non-mosaic Klinefelter in whom initially non-motile spermatozoa were derived from searching the ejaculate. Ten mature oocytes were injected, but none was fertilized. Subsequently, testicular biopsy was undertaken in order to collect spermatozoa for oocyte injection. Fifteen motile sperm cells were found and injected. Nine oocytes were fertilized and cleaved; three embryos were transferred into the uterine cavity. The woman conceived and following a normal pregnancy delivered a healthy child. Genetic analysis of the neonate disclosed a normal 46,XY karyotype. Non-motile spermatozoa in the ejaculate did not prove their fertilization potential, but their presence did not exclude finding motile, fertile spermatozoa in the testicular tissue in a non-mosaic Klinefelter patient. This report is further evidence that normal spermatozoa with fertilization potential are produced in the testes of patients with Klinefelter's syndrome.     

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.