Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for nonobstructive azoospermia

Purpose: Our objective was to apply ooplasmic round spermatid nuclear injections for the treatment of nonobstructive azoospermia. Materials: Participants were nine azoospermic men who had previously undergone diagnostic testicular biopsy. Spermatogenetic arrest was diagnosed at the round spermatid stage (n=6) or primary spermatocyte stage (n=3). A second (therapeutic) testicular biopsy was performed and round spermatid nuclei were recovered from all the participants. Results: Forty-nine mature oocytes were successfully injected with nuclei and then cultured for 72 hr. Twenty-four embryos were transferred to nine women. No pregnancy was achieved. Conclusions: Round spermatids can be recovered from therapeutic testicular biopsy material of men negative for round spermatids in previous routine diagnostic testicular biopsy specimens. Round spermatid nuclear injections may play a role in the treatment of nonobstructive azoospermia.     

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.     

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.     

Cryopreserved Immature Mouse Oocytes: A Chromosomal and Spindle Study

Purpose: Cryopreservation of human oocytes might provide an alternative approach to freezing supernumerary embryos obtained during IVF. This process, performed on immature denuded prophase I mouse oocytes, was investigated. Methods: We first investigated the capacity of frozen, immature, murine oocytes to continue in vitro maturation after thawing. We then evaluated the risk to offspring from chromosomal damage by cytogenetical and cytological (spindle) analysis. Finally, we attempted to determine the reasons for and the stage of maturation failure. Results: A total of 700 immature oocytes was frozen, 629 (90%) were recovered intact after thawing, and 53% extruded the first polar body, versus 74% for the control group. Freezing was not accompanied by an increase in aneuploidy in maturing oocytes (18 and 15% for thawed and control oocytes, respectively). Consequently, the first meiotic division occurred normally, without an increase in nondisjunction. Spindle analysis demonstrated only a few abnormalities (15 and 2% for thawed and control oocytes, respectively) incompatible with further development. Oocytes arrested during in vitro maturation were mainly at the metaphase I stage (64 and 76% for thawed and control oocytes, respectively). Whereas 17% of thawed oocytes were blocked before the formation of the first meiotic spindle, this never occurred in the control group. Conclusions: Immature murine oocytes can withstand cryopreservation, which is encouraging for future human application of this technique.     

Developmental potential and ultrastructural injuries of metaphase II (MII) mouse oocytes after slow freezing or vitrification

Purpose: The purpose of this study was to determine the developmental ability and ultrastructure of MII mouse oocytes after cryopreservation by slow freezing or vitrification.Methods: Ovulated MII mouse oocytes were allocated to slow frozen, vitrified and control groups. Oocytes in the slow frozen and vitrified groups were cryopreserved using 1,2 propandiol (PROH) and ethylene glycol (EG) respectively as cryoprotectants. After thawing, the surviving MII oocytes in both cryopreserved groups and the control group were inseminated and their developmental ability was compared. The ultrastructure of MII oocytes in both cryopreserved groups was assessed immediately after thawing and 10 h post insemination at the pronuclear stage, and compared with that of the control group.Results: The survival rates were nearly identical in both cryopreserved groups. The fertilization rates were also identical and comparable to that of the control group. The further development of vitrified oocytes was similar to that of the control oocytes, whereas it was severely limited in the slow-frozen oocytes. In the slow-frozen MII oocytes, the intermediate filaments were destroyed and the oolemma and microvilli were also modified. At the pronuclear stage deterioration of mitochondria and the presence of numerous vacuoles were also observed within the ooplasm. In the vitrified MII oocytes, the intermediate filaments were the only structures affected and these cytoskeletal elements were reorganized at the pronuclear stage.Conclusions: Vitrification results in less ultrastructural damage and better post fertilization development of MII mouse oocytes than slow freezing.     

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.     

Live birth after SrCl2 oocyte activation in previous repeated failed or low fertilization rates after ICSI of frozen-thawed testicular spermatozoa: case report

Purpose

To report a live birth resulting after strontium chloride (SrCl₂) oocyte activation in a couple with complete fertilization failure or low fertilization rates following intracytoplasmic sperm injection (ICSI) of frozen-thawed testicular spermatozoa.

Methods

The couple underwent ICSI of frozen-thawed testicular spermatozoa. After ICSI, the oocytes were artificially activated by SrCl₂ because the results of fertilization were not satisfactory in the previous cycles. The main outcome measures were fertilization, pregnancy, and birth.

Results

In the first and second cycles performed previously at another clinic, fertilization rates were 9.1 % and 0.0 %, respectively. In the third cycle, 31 metaphase II oocytes were retrieved. After sperm injection, all of the oocytes were stimulated using SrCl₂ for activation. Sixteen oocytes were fertilized (51.6 %), and a single embryo was transferred into the uterus on Day 3. A healthy girl weighing 2750 g was born at 40 weeks of gestation by caesarean section.

Conclusions

This result suggests that SrCl₂ could be useful for oocyte fertilization in case of repeated complete fertilization failure or low fertilization rates following ICSI of frozen-thawed testicular spermatozoa.     

Optimal use of fresh and frozen-thawed testicular sperm for intracytoplasmic sperm injection in azoospermic patients

PURPOSE: To optimize the use of fresh and frozen-thawed testicular biopsy specimens from patients with azoospermia. METHODS: Fifty-one patients suffering from obstructive and non-obstructive azoospermia underwent testicular sperm extraction (TESE). The specimens were divided and used for either in vitro maturation or freezing for a future intracytoplasmic sperm injection (ICSI) cycle. RESULTS: At initial testicular sperm extraction, very few motile spermatozoa were seen. After 24 h of in vitro maturation, sperm motility increased remarkably, with a maximum motility rate seen between 48 and 72 h of culture. Motile spermatozoa were observed up to 120 h in culture. In the 22 fresh ICSI cycles, a total of 294 oocytes were injected using motile sperm and 212 oocytes demonstrated normal 2PN formation (fertilization rate, 72.1%). In 36 frozen-thawed ICSI cycles, a total of 454 oocytes were injected and 302 oocytes became 2PN (66.5%). On day 3, high quality embryos were observed in 54.2% of fresh cycles and 54.1% of frozen cycles (P > 0.05). The clinical pregnancy rate did not show a significant difference between using fresh (59%) and frozen (55.5%) testicular biopsy sperm for ICSI (P > 0.05), but the embryo implantation rates did differ significantly between fresh (29.5%) and frozen-thawed (22.2%) cycles (P < 0.05). A total of 33 healthy babies have been born from 22 women, giving birth after 58 embryo transfer attempts (38%). CONCLUSION: The freezing and in vitro culturing of testicular biopsy tissue is a very reliable approach for the management of testicular biopsy specimens from azoospermic patients, and offers the possibility of several treatments of IVF/ICSI from a single sample.     

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.     

Vitrification negatively affects the Ca2+-releasing and activation potential of mouse oocytes,but vitrified oocytes are potentially useful for diagnostic purposes

Research questionTo what extent does vitrification affect the Ca²⁺-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa?DesignThe effect of mouse oocyte vitrification on Ca²⁺ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca²⁺ store content of the endoplasmic reticulum was determined at different time points during the vitrification–warming procedure. Finally, the Ca²⁺ pattern induced by cryoprotectant exposure was determined.ResultsAfter human sperm injection into mouse oocytes, Ca²⁺ dynamics but not fertilization rates were significantly altered by vitrification warming (P < 0.05). Ca²⁺ dynamics in response to SrCl₂ or ionomycin were also altered by oocyte vitrification. In contrast, activation and blastocyst rates after SrCl₂ exposure were not affected (P > 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified–warmed oocytes (P < 0.05); blastocyst rates were not affected (P > 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca²⁺ store content. Oocytes rapidly recovered during warming and recovery in Ca²⁺-containing media; a threshold area under the curve of Ca²⁺ dynamics to obtain activation rates above 90% was determined.ConclusionsVitrified–warmed mouse oocytes display reduced Ca²⁺-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca²⁺-signalling machinery in vitrified–warmed human oocytes is required.     

Resumption of meiosis-I tissue to enucleated preovulatory oocytes: a preliminary report

Purpose : Ovarian tissue banking may be the best strategy to preserve female fertility. But optimal method to obtain viable mature oocytes remains challenging. In order to bypass the long in vitro oocyte growth period, we developed this study to test whether reconstruction of thawed primordial oocytes with enucleated preovulatory germinal vesicle (GV) oocytes could induce dictyate nuclei to undergo chromosomal condensation and meiotic maturation. Methods : Isolated primordial oocytes from thawed mouse ovarian tissue were reconstructed with enucleated GV oocytes. After electrofusion and in vitro maturation, the reconstituted oocytes were assessed for first polar body extrusion, cytoskeleton configuration, and chromosome abnormalities. Results : Primordial oocytes from thawed ovarian tissue showed a high survival rate. Following transfer and electrofusion, they could be fused with enucleated GV oocytes (35.6%, 36/101) and extruded a first polar body (52.8%, 19/36). These mature oocytes showed a normal spindle configuration and chromosome number. Conclusions : We successfully established a mouse cell model to prove that omitting the whole growth and maturation period by transfer of primordial oocytes to developmentally older enucleated oocytes would bypass the long growth period required to the preovulatory stage. Polar body extrusion could also ensue after in vitro growth. This study provided an alternative approach for future investigations in oocyte maturation.     

The effects of coculture with autologous cryopreserved endometrial cells on human in vitro fertilization and early embryo morphology: A randomized study

Purpose: The aim of this study was to examine the influence of endometrial cells on the fertilization rate and early embryonic morphology following routine in vitro fertilization (IVF). Cryopreservation with subsequent thawing allowed the use of autologous somatic cells, thus minimizing the risk of transmission of infective agents. Interpatient variability was eliminated by randomizing oocytes from each cycle into the control or coculture group. Results: Two hundred ninety-four oocytes from 24 IVF cycles (21 patients) were included in the study (145 coculture and 149 control). The normal fertilization rate of control oocytes (56.4%) was not significantly different from that of oocytes cocultured with endometrial cells (61.4%). The mean number of blastomeres in cocultured embryos (3.65) was not significantly different from the number in control embryos (3.46) 2 days after insemination, but the proportion of embryos with minimal or no fragmentation was significantly higher in the coculture group [34/84 (40.5%) vs.17/80 (21.3%);P<0.01]. Conclusions: The inclusion of cryopreserved autologous endometrial cells in routine clinical IVF procedures does not influence fertilization or the early cleavage rate but may reduce the extent of embryo fragmentation during the early cleavage divisions.     

Enhanced cryosurvival of bovine blastocysts produced in vitro in serum-free medium

Purpose : Culture systems affect the development of IVP embryos and consequently their cryosurvival potential. The viability of postthawed bovine IVP embryos developed from IVM/IVC medium in the presence or absence of serum was compared. Methods : Cumulus-oocyte complexes were matured in IVM medium supplemented with or without serum. Some oocytes were evaluated for nuclear maturation status and others were inseminated with semen. Presumptive zygotes were cultured in IVC medium supplemented with or without serum for 9 days. Blastocysts were cryopreserved with 1.5 M ethylene glycol in PBS. Results : No difference was observed in the nuclear maturation status and cleavage rates in both groups, but significantly (P < 0.05) higher in blastocyst rates in the serum-supplemented group. After freezing, survival of blastocysts was higher in the serum-free group. At 36 h culture after thawing, blastocysts developed without serum had significantly (P < 0.05) higher cell number than those cultured with serum. Conclusions : We conclude that serum-free culture system enhances the viability of frozen-thawed bovine embryos.     

Clinical Assisted Reproduction: Prolonged Culture of Human Cryopreserved Embryos with Recombinant Human Leukemia Inhibitory Factor

Purpose: To evaluate the efficiency of recombinant humanleukemia inhibitory factor (LIF) in the prolonged culture ofhuman cryopreserved-thawing embryos. Methods: After thawing, all embryos were divided into fourgroups: (1) Human tubal fluid (HTF), (2) HTF + LIF, (3)M3^TH medium, and (4) M3^TH medium plus LIF. Followingprolonged culture, embryo development in each groupwas compared. Results: In embryo development from about the 2– to 4–cellto 9– to 16–cell stage, there were nonsignificant differencesbetween each group. There was lower morula formation ratein group 1 (6.9%) than those in other groups (23.2%, 19.7%,23.1%). The lower blastocyst formation in group 1 and 3(0%, 0%) than those in group 2 and 4 (11.0%, 12.8%)were noted. Conclusions: LIF is beneficial for preimplantation embryos.LIF does not influence the early embryo development.LIF-supplemented HTF provided a similar culture environmentfor thawing embryos as LIF-supplemented M3^TH medium.Supported by China Medical College Hospital, Taiwan     

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes

Purpose

The aim of the present study was to investigate the fertilizing capacity of fresh, frozen-thawed and freeze-dried canine spermatozoa.

Methods

After canine spermatozoa were injected into mouse oocytes, the rates of oocyte activation, male pronuclear formation and chromosomal aberrations were investigated.

Results

The rates of oocyte activation were comparable (90.6–100%), no matter the sperm type injected. The percentage of male pronuclear formation was higher (P?<?0.001) in the freeze-dried spermatozoa (92.3%) than the fresh (61.5%) and frozen-thawed (69.2%) spermatozoa. However, the chromosomal damage in the oocytes injected with freeze-dried spermatozoa was higher (72.9%: P < 0.001) than with fresh (26.9%) and frozen-thawed (21.4%) spermatozoa.

Conclusions

These data indicate using mouse oocytes that freeze-dried canine spermatozoa may potentially fertilize canine oocytes although chromosomal damage is frequently generated.     

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.     

Live birth after frozen-thawed oocytes matured in vitro in a PCOS patient: a model for improving implantation rates in IVM cycles and objectively assessing the real potential of development of frozen oocytes matured in vitro

Over the past 20 years, in vitro maturation (IVM) of oocytes has emerged in the strategy of infertility treatment, with the main indication being in patients suffering from polycystic ovarian syndrome (PCOS). More recently, IVM has been proposed as an option for fertility preservation in women having to undergo gonadotoxic treatments. However, despite the increasing application of IVM, the potential of development of in vitro matured oocytes after thawing remains ill-established and few pregnancies have been reported so far. We report herein a case of live birth after frozen-thawed oocytes matured in vitro and embryo transfer during an artificial cycle in a 29-year-old patient with primary infertility due to PCOS.

The present case demonstrates that the transfer of frozen-thawed IVM oocytes during an artificial cycle in PCOS patients is feasible and leads to pregnancy and live birth. This strategy may also be an interesting option to objectively assess the developmental potential of these oocytes after freezing and thawing, which is a major concern for physicians who include the IVM approach in their fertility preservation program.     

Efficacy of cryopreservation of embryos generated by intracytoplasmic sperm injection with spermatozoa from frozen testicular tissue

Purpose

To evaluate the viability of frozen embryos generated by intracytoplasmic sperm injection (ICSI) with frozen testicular spermatozoa.

Methods

A total of 68 fresh embryo transfer (ET) cycles and 85 subsequent frozen-thawed ET (FET) cycles were grouped according to the source of spermatozoa: fresh testicular spermatozoa (TESE) or frozen-thawed testicular spermatozoa (t-TESE).

Results

There were no significant differences in the age of female patients, number of oocytes, or fertilization rates in fresh ET cycles with TESE (TESE-fresh ET) versus t-TESE (t-TESE-fresh ET). The rate of embryo survival after thawing (95.7 % vs. 94.0 %) was similar in frozen ET cycles (FET) with TESE (TESE-FET) and with t-TESE (t-TESE-FET). While there were significant differences in the proportion of good quality embryos, no statistical differences were found in the pregnancy or clinical abortion rates between the two groups. Moreover, delivery rates were not significantly different.

Conclusions

Although the proportion of good quality embryos was affected by cryopreservation of testicular tissue, embryo survival rate was not. As well, subsequent pregnancy could be achieved successfully via t-TESE-FET cycles. Therefore, FET is not affected by the cryopreservation of testicular tissue, and avoids further oocyte retrieval and TESE procedures.     

Comparison of in-vitro outcomes from cryopreserved oocytes and sibling fresh oocytes

In Italy, the restrictive IVF law generalizes the indication for oocyte freezing for surplus oocytes in 78.5% of in-vitro assisted reproductive cycles. With a view to understanding better what the prospects for intracytoplasmic sperm injection (ICSI) on frozen-thawed oocytes might be, the consequences of freeze-thaw procedures on fertilization, cleavage rates and embryo quality obtained from frozen-thawed oocytes were studied and compared with the results obtained from sibling fresh oocytes. Eleven IVF and 29 ICSI on 76 and 169 fresh oocytes were performed and the corresponding 40 ICSI on 221 sibling frozen-thawed oocytes. There was no difference in terms of fertilization rate between fresh and sibling frozen-thawed oocytes. The cleavage rate (98.0 and 94.4% with fresh oocytes in IVF and ICSI; 77.3% with frozen-thawed oocytes in ICSI; P < 0.001) and embryo quality (grade I embryos over total embryos: 36.7 and 22.2% with fresh oocytes in IVF and ICSI; 12.1% with frozen-thawed oocytes in ICSI; respectively P < 0.001 and P < 0.05) were statistically lower after oocyte cryopreservation. The significant decrease in meiotic spindle retrieval rate before freezing (62.4%) and after thawing procedures (43.4%; P < 0.001) suggests that cryoconservation induces irreversible damage to microtubule repolymerization. The consequences of oocyte cryopreservation procedures on embryo development are reviewed.     

Pregnancies and births resulting from in vitro matured oocytes fertilized with testicular spermatozoa

Purpose: In vitro maturation (IVM) of immature human oocytes is an attractive option for the treatment of infertility. Similarly, intracytoplasmic sperm injection (ICSI) followed by testicular fine needle aspiration (TEFNA) is an important treatment for primarily male-factor infertility. This report highlights the combination of these two advanced assisted reproduction techniques, namely IVM and fertilization with TEFNA-retrieved spermatozoa by ICSI to overcome both of male and female infertility problems.Methods: Before immature oocyte retrieval (IOR), gonadotropin stimulation was given for 3 or 5 days. Following IVM, and mature oocytes were inseminated by ICSI followed by TEFNA.Results: Four couples with five completed treatment cycles were performed, and total of 36 immature oocytes were retrieved. Following 36 to 48 h of culture, 32 (88.89%, 32/36) oocytes became mature. The mature oocytes were inseminated with TEFNA-retrieved sperm, and 18 (56.25%, 18/32) oocytes were fertilized normally following ICSI. Eleven embryos were transferred in five cycles and two pregnancies and two singleton births were achieved in two patients.Conclusions: This result demonstrates that the successful pregnancies and live births can be established from embryos produced from {in vitro} matured oocytes that fertilized with testicular sperm.