CLINICAL ASSISTED REPRODUCTION: Development of Blastocyst-Stage Embryos After Round Spermatid Injection in Patients with Complete Spermiogenesis Failure

Purpose: Our purpose was to evaluate the progression of embryos derived from round spermatid injection to the blastocyst stage and compare the results with those obtained by the use of testicular or epididymal spermatozoa.Methods: Thirty-eight patients with azoospermia enrolled in this study. In 29 patients with obstructive or nonobstructive azoospermia, spermatozoa were recovered from epididymis or testis. In the remaining nine cases with nonobstructive azoospermia, only round spermatids were found in seven, whereas in two of the patients, there were no elongated or round spermatids. Six of these cases underwent round spermatid injection.Results: Twenty-one of 29 patients with injection of spermatozoa underwent embryo transfer on day 3, and 10 pregnancies (47.6%) were obtained. In eight cycles, embryos were further cultured for delayed transfer. In six cases undergoing round spermatid injection, no transfer was performed on day 3 and extended culture with delayed embryo transfer was applied. The mean number of fertilized oocytes and mean number of embryos on day 3 and also the fertilization rate and mean number of good-quality embryos on day 3, mainly grade 1 or 2, were statistically significantly higher in the spermatozoa group than the round spermatid injection group. Compared to the spermatozoa group, the number of arrested embryos was significantly higher and the number of blastocyst-stage embryos and number of good-quality blastocysts were significantly lower in the spermatid injection group. No blastocysts developed in two spermatid cycles and embryo transfer was not possible, and in the remaining four cycles, after at least one blastocyst transfer, no pregnancies were achieved. However, in eight cycles with extended culture in the spermatozoa group, embryo transfers were achieved in all and three pregnancies, for a pregnancy rate of 37.5%, were obtained after blastocyst transfer.Conclusions: Our preliminary results showed that round spermatid injection was associated with a significantly lower fertilization and embryo development rate and a significantly higher developmental arrest rate compared with the injection of spermatozoa. Extended culture and delayed embryo transfer did not improve the clinical outcome after round spermatid injection, and these results suggested a developmental failure in embryos preventing successful implantation after round spermatid injection.     

Poor outcome with round spermatid injection in azoospermic patients with maturation arrest

OBJECTIVE: To compare the outcome of intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI), both obtained by testicular sperm extraction (TESE), and to compare the results of fresh versus frozen ROSI. DESIGN: Retrospective study.Setting: An IVF unit at a university hospitalPatient(s): Eighteen infertile couples with nonobstructive azoospermia.Intervention(s): TESE with ROSI or ICSI of mature spermatozoa into metaphase II oocytes was performed. The resulting embryos were transferred to female partners. The spare round spermatids were frozen. MAIN OUTCOME MEASURE(S): Fertilization and cleavage rates, embryo quality, and clinical pregnancy rates. RESULT(S): Seventeen ROSI cycles and six ICSI cycles were compared. Fertilization rate following ROSI (44.9%) was significantly lower than with ICSI (69%). A significantly higher rate of cleavage arrest occurred following ROSI (40.8%) as compared to ICSI (8.2%). The morphology of embryos resulting from ROSI was significantly poorer. No pregnancies were achieved following ROSI as compared to a 50% clinical pregnancy rate in the ICSI group. The fertilization and cleavage rates following ROSI with fresh versus frozen-thawed spermatids were comparable. CONCLUSION(S): In azoospermic patients with maturation arrest at the stage of round spermatids the efficiency of ROSI appears to be extremely poor. The role of ROSI in the treatment of nonobstructive azoospermia should be reevaluated.     

Efficiency of Using Frozen-Thawed Testicular Sperm for Multiple Intracytoplasmic Sperm Injections

Purpose: To compare fertilization and pregnancy rates of fresh and frozen-thawed testicular sperm injections (TESE-ICSI). Methods: Sperm collected from the testes of 28 azoospermic patients by an open testicular biopsy technique was used for initial ICSI or cryopreserved. Results: Fresh-sperm ICSI treatment (28 cycles) resulted in a 58.1% fertilization rate and a 32.1% clinical pregnancy rate per embryo transfer, while frozen-thawed sperm (24 subsequent cycles) had rates of 54.5 and 29.2%, respectively. The PR was lower using frozen-thawed sperm from nonobstructive azoospermia patients (9.1%) than from obstructive azoospermia patients (46.2%). PR declined to 0% upon the fourth ICSI attempt. Conclusions: Fertilization, embryo cleavage, and pregnancy rates were unaffected by fresh or frozen-thawed sperm use. A 57.1% cumulative clinical PR was achieved using the latter. The PR was significantly lower using frozen-thawed sperm from nonobstructive azoospermia patients than from obstructive azoospermia patients.     

Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.

OBJECTIVE: To report a case of nonobstructive azoospermia in which round spermatids recovered from thawed testicular tissue were used for injection. DESIGN: Case report. SETTING: Reproductive Medicine Unit, S.I.S.ME.R. PATIENT(S): A 33-year-old azoospermic man. INTERVENTION(S): Intracytoplasmic sperm injection with frozen-thawed spermatids. MAIN OUTCOME MEASURE(S): Fertilization, embryo cleavage, pregnancy, and delivery. RESULT(S): Birth of a healthy, chromosomally normal girl. CONCLUSION(S): Frozen-thawed testicular round spermatids from a patient with a history of incomplete spermatogenesis can maintain their viability and their capacity to fertilize and to lead to full-term pregnancy.     

Correlation between adjusted late spermatid score and sperm count in normozoospermic and oligozoospermic men

ObjectiveTo correlate late spermatid score and sperm count in men with normal semen analysis and oligozoospermia undergoing intracytoplasmic sperm injection (ICSI) to set the average threshold of spermatids/tubule of testicular tissue needed for normal sperm count/ml of semen.Material and methodsThis study was conducted on 24 normozoospermic subjects and 18 oligozoospermic patients who underwent wide bore needle biopsy because of failed sperm collection at the day of ICSI. Clinical data were reviewed and testicular biopsy slides were examined for Johnson score and adjusted late spermatid score.ResultsThe optimum cut off value for adjusted late spermatid score in predicting a sperm count >20 million/ml was 20.75 late spermatid per tubule with 100% specificity. Model equations were produced to predict the sperm count using adjusted late spermatid score and vice versa.ConclusionThis work supports the hypothesis that late spermatid score is a simple and reliable method for quantitation of spermatogenesis and it correlates well with the sperm count. The value of late spermatids needed for normal sperm count in this report is different from some other reports. The better standardization of work in this study may help resetting a new late spermatid threshold for normal sperm count.     

A novel culture system for mouse spermatid maturation which produces elongating spermatids capable of inducing calcium oscillation during fertilization and embryonic development

Purpose

To establish an in vitro culture system for mouse round spermatids that models spermiogenesis and enables the assessment of oocyte activation ability.

Methods

Round spermatids and Sertoli cells were isolated from testicular tissues of B6D2F1 male mice and co-cultured in the presence of testosterone and recombinant FSH. Cultured spermatids were examined for morphology and condensation of nuclei, fertilization and development rate, and Ca²⁺ oscillation pattern after ICSI.

Results

The cultured spermatids elongated and resembled normal elongating spermatids in terms of both morphology and nuclear condensation. No significant differences in fertilization and development rates were observed between fresh and cultured elongating spermatids. Moreover, cultured spermatids showed similar Ca²⁺ oscillation patterns to fresh elongating spermatids during an initial stage in oocyte activation.

Conclusions

These data suggest that a co-culture system of spermatids and Sertoli cells, supplemented with testosterone and recombinant FSH, supports normal differentiation of round spermatids into elongating spermatids, as assessed by their morphology, nuclear condensation, and oocyte activation ability.     

Update on surgical sperm recovery – The European view

Surgical sperm recovery has become a well-established procedure to obtain spermatozoa for intra-cytoplasmic sperm injection (ICSI). Although a tendency exists to treat all azoospermic patients by ICSI using surgically retrieved sperm, vasovasostomy remains the gold standard for post-vasectomy azoospermia. In men with obstructive azoospermia in whom vasovasostomy is not indicated, sperm can be easily obtained by either aspiration from epididymis or testis, or a testicular biopsy. In about half of men with non-obstructive azoospermia, sperm may be obtained by testicular biopsy but unfortunately no accurate tests are currently available to predict successful recovery. In these patients, not only recovery rates are limited but also the chance to establish an ongoing pregnancy is decreased compared to men with normal spermatogenesis. When no spermatozoa are recovered after testicular sperm extraction (TESE), the use of donor sperm or adoption is indicated. Given the extremely low pregnancy rates, ICSI using round spermatids is not an option and remains unlawful in some countries.     

Update on surgical sperm recovery--the European view

Surgical sperm recovery has become a well-established procedure to obtain spermatozoa for intra-cytoplasmic sperm injection (ICSI). Although a tendency exists to treat all azoospermic patients by ICSI using surgically retrieved sperm, vasovasostomy remains the gold standard for post-vasectomy azoospermia. In men with obstructive azoospermia in whom vasovasostomy is not indicated, sperm can be easily obtained by either aspiration from epididymis or testis, or a testicular biopsy. In about half of men with non-obstructive azoospermia, sperm may be obtained by testicular biopsy but unfortunately no accurate tests are currently available to predict successful recovery. In these patients, not only recovery rates are limited but also the chance to establish an ongoing pregnancy is decreased compared to men with normal spermatogenesis. When no spermatozoa are recovered after testicular sperm extraction (TESE), the use of donor sperm or adoption is indicated. Given the extremely low pregnancy rates, ICSI using round spermatids is not an option and remains unlawful in some countries.     

Intracytoplasmic injection of fresh and cryopreserved testicular spermatozoa in patients with nonobstructive azoospermia—a comparative study

Objective: To compare the outcome of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed testicular spermatozoa in patients with nonobstructive azoospermia.Design: Retrospective analysis of consecutive ICSI cycles.Setting: In Vitro Fertilization Unit, Assaf Harofeh Medical Center.Patient(s): Eighteen with nonobstructive azoospermia in whom testicular sperm was found after testicular sperm extraction.Intervention(s): Testicular sperm retrieval, cryopreservation, and ICSI with fresh or frozenthawed testicular spermatozoa.Main Outcome Measure(s): Two-pronuclear fertilization; embryo cleavage rates, mean number of embryos transferred per cycle, and their relative quality, embryo implantation, clinical pregnancy, and ongoing pregnancy rates (PRs) per ET.Result(s): No statistically significant differences were noted in all parameters examined between ICSI cycles with fresh or cryopreserved testicular spermatozoa from the same nine patients and comparing all ICSI cycles performed; with fresh (25 cycles) and thawed (14 cycles) testicular spermatozoa, respectively: two-pronuclear fertilization, 47% versus 44%; embryo cleavage rates, 94% versus 89%; implantation rates, 9% versus 11%; and clinical PR, 26% versus 27%. The delivery or ongoing PR using fresh sperm was better (21% versus 9%), but the difference did not reach statistical significance. The cumulative clinical PRs and ongoing PRs per testicular sperm extraction procedure were 36% and 24%, respectively.Conclusion(s): Testicular sperm cryopreservation using a simple freezing protocol is promising in patients with nonobstructive azoospermia augmenting the overall success achieved after surgical sperm retrieval. (Fertility Sterility 1997;68:892-7. C 1997 by American Society for Reproductive Medicine.)     

Reproductive capacity of the nucleus of the male gamete after completion of meiosis

Purpose Our purpose was to investigate the possibility of achieving fertilization and subsequent normal embryonic development by injecting round spermatid nuclei into rabbit oocytes.Results Two- to four-cell-stage embryos developed after round spermatid nuclear injections into rabbit ooplasma could further develop in vitro up to the expanding blastocyst stage or in vivo up to complete gestation.Conclusion The current findings show that the haploid set of chromosomes of round spermatid can pair with the chromosomes of the ootid to participate in complete fertilization and subsequent embryonic and fetal development. In addition, we suggest that postmeiotic modifications of the round spermatid are not required for the pairing of male gamete chromosomes with those of the ootid.     

Comparison of the Media for Isolation and Storage of Round Spermatid Nuclei Before Intracytoplasmic Injection

Purpose: This study investigated whether K ⁺ -rich medium is better than pure NaCl solution or Na ⁺ -rich cell culture medium for handling round spermatid nuclei prior to injection into oocytes (ROSNI). Methods: Round spermatids of the mouse were isolated and stored in isotonic NaCl, a cell culture medium (CZB), or a nucleus isolation medium (NIM) before injection into oocytes. The rates of normal fertilization, embryonic development in vitro, and birth of normal offspring after transfer of embryos to foster mothers were determined. Results: In vitro development of ROSNI-produced zygotes to blastocysts was the same when naked spermatid nuclei were exposed briefly to three media. However, a long (60-min) exposure of the nuclei to NA ⁺ -rich medium was detrimental. In K ⁺ -rich NIM naked spermatid nuclei best retained their ability to participate in normal embryonic development. Conclusion: NIM was better than Na ⁺ -rich medium for retaining isolated spermatids competent to participate in normal embryonic development.     

Intracytoplasmic Sperm Injection with Testicular Spermatozoa in Men with Azoospermia

Purpose : The aim of the study was to gain an insight into the optimal management of the infertile couple with the husband suffering from azoospermia. Methods : One hundred and forty-two intracytoplasmic sperm injection (ICSI) cycles performed with testicular extracted spermatozoa were retrospectively analysed. The following factors were investigated for their possible influence on fertilization, cleavage, damage, pregnancy, and ongoing pregnancy rates; the use of fresh, cryopreserved, and preincubated (24 h) spermatozoa and the etiology of the husbands' azoospermia (obstructive and nonobstructive). All microinjections were performed with apparently normal spermatozoa—a head with a tail of normal length. In 116 cycles at least two embryos were available for transfer. Results : The overall fertilization, clinical pregnancy, and ongoing pregnancy rates obtained for the 116 cycles were 65.0, 30.2, and 22.4% respectively. Similar outcomes were obtained for cycles using fresh testicular and cryopreserved testicular spermatozoa. Similarly, no significant differences were obtained between the cycles using spermatozoa from obstructive or nonobstructive azoospermic patients. An increase in motility after a 24-h preincubation was observed, and although this group was relatively small (n = 17), a significant improvement in fertilization (73.7%) and pregnancy (53.9%) rate was obtained when the testicular sample was preincubated for 24 h. This improvement prevailed in the obstructive azoospermic group, but was less pronounced in nonobstructive patients. Conclusions : This study shows that the outcome of fresh and frozen–thawed testicular spermatozoa in ICSI is comparable, obstructive and nonobstructive etiologies perform the same, and that preincubation of testicular spermatozoa results in increased fertilization and pregnancy rates. All testicular biopsies are therefore performed the day before oocyte retrieval, superfluous spermatozoa cryopreserved, and the remaining testicular homogenate preincubated for the 24 h prior to oocyte retrieval. With this regime, most azoospermic patients are treated successfully, irrespective of the use of fresh or frozen–thawed spermatozoa from obstructive or nonobstructive cases.     

Somatic mutational analysis of DAX1 in testes from men with idiopathic azoospermia

Mutations in the orphan nuclear receptor DAX1 (NR0B1) cause X-linked adrenal hypoplasia congenital (AHC), a disorder characterized by primary adrenal failure, hypogonadotropic hypogonadism. and azoospermia. We tested the hypothesis that DAX1 somatic mutations in human testis may cause azoospermia. DAX1 sequencing analysis in 15 testicular biopsy samples from men with idiopathic nonobstructive azoospermia did not reveal mutations in the coding region of the gene. We conclude that somatic abnormalities in DAX1 are absent or uncommon in these patients.     

Intracytoplasmic sperm injection with testicular spermatozoa is less successful in men with nonobstructive azoospermia than in men with obstructive azoospermia

OBJECTIVE: To assess the efficiency of intracytoplasmic sperm injection (ICSI) using testicular spermatozoa in cases of nonobstructive azoospermia. DESIGN: Retrospective case series. SETTING: Tertiary university-based infertility center. PATIENT(S): Overall, 595 couples were included. In 360 couples, the man had normal spermatogenesis. In 118, 85, and 32 couples the man had germ-cell aplasia, maturation arrest, and tubular sclerosis/atrophy, all with focal spermatogenesis present. INTERVENTION(S): We performed 911 ICSI cycles using fresh sperm obtained after testicular biopsies: 306 ICSI cycles used testicular sperm from men with nonobstructive azoospermia, and 605 ICSI cycles used testicular sperm from men with obstructive azoospermia. MAIN OUTCOME MEASURE(S): Fertilization, cleavage, implantation, and pregnancy rates. RESULT(S): Overall, the 2PN fertilization rate was lower in the nonobstructive group: 48.5% vs. 59.7%. There were no differences in in vitro development or in the morphological quality of the embryos. In the nonobstructive group, a total of 718 embryos were transferred (262 transfers) vs. 1,525 embryos in the obstructive group (544 transfers). Both the clinical implantation rate and clinical pregnancy rate per cycle were significantly lower in the nonobstructive group compared with the obstructive group: 8.6% vs. 12.5% and 15.4% vs. 24.0%, respectively. CONCLUSION(S): A statistically significant lower rate of fertilization and pregnancy results from ICSI with testicular sperm from men with nonobstructive azoospermia, compared with men with obstructive azoospermia.     

The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei

Objective: To compare the oocyte-activating ability of whole human round spermatids and their isolated nuclei.

Design: Prospective study using sibling oocytes from patients undergoing spermatid conception and intracytoplasmic sperm injection treatment cycles.

Setting: Private assisted reproduction laboratories and a university department.

Patient(s): Couples with male infertility.

Intervention(s): Sibling oocytes were injected either with whole round spermatids or with their isolated nuclei, followed by artificial triggering of oocyte activation with calcium ionophore. Other sibling oocytes were injected either with isolated spermatid cytoplasm or with whole mature spermatozoa.

Main Outcome Measure(s): Numbers of activated oocytes and cleaving embryos.

Result(s): After oocyte activation was boosted with calcium ionophore, whole spermatids and isolated spermatid nuclei were equally effective in supporting oocyte activation and the formation of pronuclei, whereas no control oocyte was activated under the same conditions after injection of isolated spermatid cytoplasmic compartments. Cleavage rates were lower after the injection of isolated spermatid nuclei than after the injection of whole spermatids.

Conclusion(s): The factor responsible for human oocyte activation after round spermatid injection is associated with spermatid nuclei. The requirement for the artificial trigger (calcium ionophore) suggests that this factor is identical to the male gamete activity previously characterized as calcium oscillator.     

The significance of karyotyping and azoospermia factor analysis in patients with nonobstructive azoospermia or oligozoospermia

ObjectiveWe present our study about the significance of karyotyping and azoospermia factor(AZF) analysis in patients with azoospermia or oligozoospermia.Materials and methodsWe retrospectively reviewed 141 Taiwanese patients with nonobstructive azoospermia and 45 Taiwanese patients with oligozoospermia at MacKay Memorial Hospital, Taiwan, from 2010 to 2021 to determine the significance of karyotyping and azoospermia factor analysis. The karyotyping was analyzed using the Giemsa banding method. The AZF microdeletions were determined using multiplex polymerase chain reaction using primers specifically flanking the AZF subregions.ResultsWe found that 7.80% of patients with nonobstructive azoospermia had AZF microdeletions and 19.86% of patients with nonobstructive azoospermia had chromosomal anomalies or polymorphic variations. Furthermore, 4.44% of patients with oligozoospermia had AZF microdeletions, and 4.44% of patients with oligozoospermia had chromosomal anomalies or polymorphic variations.ConclusionIn this study, 25.53% of patients with nonobstructive azoospermia and 8.88% of patients with oligozoospermia had abnormal findings. The significance of karyotyping and azoospermia factor analysis is more critical in patients with nonobstructive azoospermia than patients with oligozoospermia. Both karyotyping and AZF analysis could prevent delayed treatment for male infertility through accurate diagnosis and appropriate treatment. The number of our patients with AZFc microdeletion was also higher than that of patients with AZFa or AZFb. The spermatogenic potential may gradually decline in patients with AZFc microdeletion. The earlier is the diagnosis, the earlier will be the retrieval of testicular spermatozoa.     

Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring

Purpose: This study was performed to determine whether frozen-thawed mouse round spermatids can fertilize oocytes and contribute to normal embryo development. Methods: Freshly collected mouse testicular cells were frozen in PBS containing 7.5% glycerol and 7.5% fetal bovine serum. After thawing and removal of the cryoprotectants, round spermatids were selected and injected individually into mature oocytes which had been previously activate with Sr ²⁺ -containing Ca ²⁺ -free medium. Results: After thawing, 75–85% of testicular cells were alive. About 90% of the oocytes were fertilized by intracytoplasmic injection of frozen-thawed round spermatids; 11% (17/150) of embryos transferred to foster mothers developed into normal offspring. Conclusions: Mouse round spermatids can be cryopreserved for production of normal offspring.     

Using a vortex mixer for testicular sperm collection

OBJECTIVE: To evaluate 2 methods of processing testicular tissue for the retrieval of viable sperm from men with nonobstructive azoospermia. STUDY DESIGN: Fresh testicular tissue was obtained from nonobstructive azoospermia patients using a biopsy needle. The specimens were divided into 2 fractions. All specimens were minced and immersed in human tubal fluid (HTF). The first fraction was filtered through a nylon filter and incubated for 3 hours. The supernatant was centrifuged, resuspended in HTF and analyzed. The second fraction was immediately vortexed for 5 minutes and filtered through a nylon filter. The supernatant was centrifuged, resuspended in HTF and analyzed. RESULTS: Spermatozoa were obtained in 13 of 24 cases (54.2%) using the vortex method and in 5 of 24 cases (20.8%) with the nylon filter method. CONCLUSION: The vortex mixing method may be a better option than the conventional method for processing testicular tissue for sperm collection.     

Intracytoplasmic Injection Using Spermatids and Subsequent Pregnancies: Round Versus Elongated Spermatids

The purpose of this clinical trial study was to report the outcome of intracytoplasmic injection of round (RS) and elongated (ES) spermatids retrieved from the testis of nonobstructive men. Seven and three cycles using RS and ES injections were performed, respectively. Only one cycle utilizing the late stage of ES (Sd2) resulted in an on-going pregnancy. The remarkable low success rate following RS microinjection was established in this study.