Search

Purpose: To assess whether embryo transfer (ET) technique can influence the clinical pregnancy rate (CPR) and its correlation with the embryo transfer difficulty., Design: This single center retrospective cohort analysis of fresh and frozen single blastocyst transfers performed between January 2016 and December 2021 included fresh and frozen single blastocyst transfers performed during the study timeframe. Direct technique was the only one used from January 2016 to September 2017. From September 2017 to March 2019, the choice between the two techniques was given by randomization, due to a clinical trial recruitment. From April 2019, only the afterload technique was used. Preimplantation genetic testing cycles and gamete donation procedures and cycles performed with external gametes or embryos were excluded. CPR was the primary outcome, while difficult transfer rate the secondary one. Univariate and multivariate logistic regressions were performed., Results: During the period, 8,189 transfers were performed. CPR of the afterload group resulted significantly higher compared to the direct group (44.69% versus 41.65%, OR 1.13, 95% CI 1.02-1.25, p = 0.017) and the rate of difficult transfers two-thirds lower (9.06% versus 26.85%, OR 0.27, 95% CI 0.24-0.31, p < 0.001)., Conclusion: Our study demonstrated that CPR is significantly affected by the ET technique. In particular, with the afterload protocol, both CPR and easy transfer rates increased., Trial Registration: http://clinicaltrials.gov registration number: NCT05364528, retrospectively registered on 3rd of May 2022., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Study Question: Are trophectoderm biopsy or other pre-vitrification features or laboratory practices associated with differences in blastocyst post-warming behavior (degeneration, re-expansion and live birth after single embryo transfer (SET))?, Summary Answer: Blastocyst morphology, day of full development and artificial shrinkage (either laser-assisted or biopsy-induced) are the pre-vitrification parameters/practices most strongly associated with post-warming behavior and implantation potential while there was no association with trophectoderm biopsy., What Is Known Already: Since the introduction of vitrification, the adoption of cycle segmentation, freeze-all and SET policies, as well of trophectoderm biopsy-based aneuploidy testing (i.e. pre-implantation genetic testing for aneuploidies (PGT-A)), the number of blastocysts vitrified worldwide has increased greatly. Previous studies already defined generally high blastocyst cryo-survival rates after vitrification-warming (>95%), along with a positive correlation between blastocyst re-expansion and morphology with implantation. Additionally, artificial shrinkage has been outlined as a potentially beneficial procedure, while the association between embryo cryo-survival and trophectoderm biopsy is still unclear., Study Design, Size, Duration: Cohort study conducted at two IVF centers (1 and 2). A total of 2129 consecutive SETs using vitrified-warmed blastocysts in either non-PGT or PGT-A cycles between June 2016 and August 2017 were included. A freeze-all strategy was in place and three main pre-vitrification practices were used: (i) no biopsy and no artificial shrinkage (Clinic 1); (ii) trophectoderm biopsy and vitrification of collapsed blastocyst within 30 min (Clinics 1 and 2); and (iii) no biopsy but laser-assisted artificial shrinkage (Clinic 2). The primary outcome was the blastocyst degeneration rate. Overall, 2108 surviving blastocysts were graded at 1.5 h after warming for degeneration (absent or partial) and re-expansion (full, partial or absent) grades and post-warming morphological quality. Logistic regression analyses were conducted to assess the association of any pre-vitrification feature with blastocyst post-warming behavior. The logistic regressions conducted upon live birth after either untested or euploid SET also included the post-warming characteristics., Participants/materials, Setting, Methods: Center 1 is a private IVF facility, while center 2 is the IVF facility of a University hospital. In non-PGT cycles, ICSI with blastocyst culture up to full-expansion and vitrification were performed. At center 1 the untested blastocysts were vitrified when still expanded, while at center 2 they underwent laser-assisted artificial shrinkage. In PGT-A cycles, in both clinics, trophectoderm biopsy (which involves laser-assisted shrinkage) was done without previous zona-opening on Day 3, and vitrification was performed within 30 min whilst the blastocyst remained collapsed. A qPCR-based chromosome analysis was conducted. Only SETs were performed (euploid-SET in case of PGT-A). Any cycle-, laboratory- and embryo-based feature which could impact blastocyst post-warming behavior was included in the analyses as putative confounder., Main Results and the Role of Chance: The overall degeneration rate was 1% (N = 21/2129). The results were consistent among different vitrification/warming operators or kits used, as well as any other IVF laboratory-related parameter. Blastocyst artificial shrinkage (either laser-assisted or biopsy-induced) involved a lower risk of degeneration after warming (odds ratio (OR) [95% CI] = 0.26 [0.09-0.79]). Conversely, both poor morphological quality pre-vitrification and taking 7 days to reach full blastulation resulted in a significantly higher risk (OR [95% CI] = 11.67 [3.42-39.83] and 4.43 [1.10-20.55], respectively). Importantly, trophectoderm biopsy did not show any association with blastocyst cryo-survival/degeneration. Overall 2.5% (N = 53/2108) blastocysts failed to re-expand post-warming. The only parameters significantly associated with no blastocyst re-expansion post-warming were average (OR [95% CI] = 4.96 [2.20-11.21]) or poor (OR [95% CI] = 19.54 [8.39-45.50]) morphological quality and taking 7 days to reach full blastulation (OR [95% CI] = 3.19 [1.23-8.29]), as well as prevention of spontaneous hatching pre-vitrification (OR [95% CI] = 0.10 [0.01-0.85]). The post-warming features of the survived blastocyst (i.e. degeneration and re-expansion scores and morphological quality) showed no significant association with vitrified blastocyst competence (i.e. live birth) when corrected for pre-vitrification ones (i.e. morphological quality, day of full development and, for untested SET, maternal age at oocyte retrieval). Of note, poor-quality blastocysts pre-vitrification showed a high overall cryo-survival rate post-warming 92.8% (N = 116/125), but the live birth rates were only 2.1% (N = 1/48) and 7.3% (N = 5/68) after untested and euploid SET, respectively., Limitations, Reasons for Caution: This study is not randomized and the populations of patients undergoing either non-PGT or PGT-A cycles were different. Centers 1 and 2 adopted different pre-vitrification practices for non-biopsied blastocysts, according to their own laboratory policy. To this regard, multivariate logistic regression analyses were conducted for all outcomes under investigation., Wider Implications of the Findings: Pre-vitrification features may be used to assist selection of competent embryos, moreover, these results allay concern that trophectoderm biopsy might be associated with impaired blastocyst quality or competence after vitrification/warming., Study Funding/competing Interest(s): None., Trial Registration Number: None.

Purpose: This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos., Methods: Mice oocytes (n = 40) and embryos (8 cells, n = 35 and blastocysts, n = 165), bovine embryos (2PN, n = 35), and MII oocytes (n = 84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates., Results: Ninety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos' survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts' rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts' rates., Conclusion: This novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.

Purpose of Review: The purpose is to determine the efficiency and efficacy of oocyte cryopreservation by slow freezing versus vitrification, recent data collected from the Italian National Assisted Reproductive Technology Register during the period 2009-2014 will be presented and reviewed. The data on oocyte cryopreservation were also compared with the results obtained with embryo cryopreservation and relative IVF with fresh oocytes., Recent Findings: During the period 2009-2014 preservation of oocytes by vitrification had a significantly higher survival rate, implantation, and pregnancy rate than slow freezing; however, there are still large variations in success rates among centers in relation to the number of procedures performed., Summary: Vitrification has now become the method of choice for oocyte cryopreservation because of better results than slow freezing, but still requires a more standardized utilization. The transfer of fresh or cryopreserved embryo still shows a statistically significant better performance than transfers with embryos obtained with cryopreserved oocytes. Only in a few centers with much experience in cryopreservation are the results between transfers of frozen embryos or embryos obtained from oocyte cryopreservation comparable.

Purpose: Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration., Methods: Samples were studied by light and transmission electron microscopy., Results: We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed., Conclusions: This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism., Competing Interests: Compliance with ethical standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Conflict of interest The authors declare that they have no conflict of interest.

It is well documented that oocyte vitrification using open systems provides better results than closed systems. However, its use is limited owing to risks of contamination posed by direct exposure to liquid nitrogen and cross-contamination when stored in liquid nitrogen tanks. A device that produces clean liquid air (CLAir) having similar a temperature as liquid nitrogen and a sterile storage canister device (Esther) that keeps samples sealed in their own compartment while in regular liquid nitrogen tanks were developed. The following experiments were performed: temperature measurements, bioburden tests, vitrification and storage experiments with mice embryos and human oocytes. Results showed similar cooling rates for liquid nitrogen and liquid air. Bioburden tests of CLAir and Esther showed no contamination, while massive contamination was found in "commercial" liquid nitrogen and storage canisters. Mice blastocysts had a survival rate of over 90%, with 80% hatching rate after vitirification in CLAir and 1 week storage in Esther, similar to the fresh (control) results. Human oocytes vitrified in CLAir and in liquid nitrogen for three consecutive vitrification/warming cycles showed 100% survival, seen as re-expansion in both groups. These new systems represent a breakthrough for safe vitrification using open systems and a safe storage process generally., (Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: This study is a retrospective collection of aggregated data from all the Italian ART centers reporting to the Italian National Register from cycles started between January 2005 and December 2013., Methods: Data from both slow freezing (SF) and vitrification (V) were assessed for the period 2007-2013, while during the years 2005-2006 cryopreservation was exclusively performed by SF., Results: In the study period, a total of 2,526,024 oocytes were retrieved (from 378,543 retrievals), of which 1,346,061 (53.3 %) were inseminated in fresh cycles and 214,481 (8.5 %) were cryopreserved. Cryopreserved oocytes were used in 24,173 cycles yielding 19,453 transfer cycles (80.5 % of the thawing/warming cycles) and 3043 clinical pregnancies (15.6 % per transfer). A significant difference in implantation (8.7 vs 12.9 % OR 1.30 CI 1.20-1.40) and pregnancy rates per transfer (12.2 vs 14.9 % OR 1.34 CI 1.23-1.46) was found between SF and V. Complete outcome data was available for 2708 pregnancies (89.8 %), leading to 1882 deliveries and 2152 live births. Neonatal major congenital anomalies were 0.9 % (20/2152)., Conclusions: A wide variation in pregnancy rates were found among different centers and lower rates were reported in donor cycles and in centers with more experience.

Objective: To compare mature human oocytes cryopreservation with slow freezing (SF) and vitrification (VT) in infertile couples., Design: Retrospective study of national Italian data submitted during the period 2007-2011., Setting: National ART registry., Patient(s): Infertile patients with supernumerary oocytes., Intervention(s): Thawing or warming of cryopreserved oocytes and ICSI., Main Outcome Measure(s): oocyte survival, fertilization, implantation and clinical pregnancy rate between SF and VT., Result(s): A total of 14,328 cycles with 11,599 transfers, 1,850 pregnancies, 1,168 deliveries and 1,342 babies born were analyzed from 146 reporting centers (range of cycles 1-1,255 per center). The SF oocytes' survival rate was lower than in VT (51.1% vs. 63.1%). Fertilization rate was significantly higher in SF than in VT (SF 71.6% vs. VT 70.1%). VT showed a significantly higher pregnancy rate, both per started cycle (14.4% vs. 12.0%) and per transfer (18.0% vs. 14.8%), and implantation rate (9.5% vs. 8.1%) than SF. However, the range and median pregnancy rate per started cycle were, respectively, 0%-50% and 7.7% in SF and 0%-100% and 6.7% in VT., Conclusion(s): VT showed a statistically significant higher performance than SF. As with other ART procedures, the results are not homogeneous among clinics and protocols, but the confirm the clinical value of oocyte cryopreservation in infertile patients., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To analyze the fetal and neonatal outcomes of pregnancies achieved with fresh and/or frozen oocytes in the same group of patients., Design: Observational study and comparative analysis., Setting: Research unit of an academic medical center., Patient(s): A group of 855 women with cryopreserved oocytes and their resulting 954 assisted reproductive technology clinical pregnancies were enrolled and followed up during the same time period and in the same clinical setting; the outcomes of 197 pregnancies from frozen/thawed oocytes were compared with 757 obtained from fresh sibling oocyte cycles., Intervention(s): None., Main Outcome Measure(s): Pregnancies were followed until delivery, and neonatal data (up to 28 days after delivery) were collected., Result(s): No significant differences were found between the use of fresh and frozen oocytes in the rates of therapeutic abortions for fetal anomaly (1.5% vs. 0.8%) and ectopic pregnancies (3.6% vs. 2.9%), but a significantly higher rate of spontaneous abortions at ≤ 12 weeks (17.6% vs. 26.9%) was observed in the frozen/thawed oocytes group. No statistical differences were found in major anomalies at birth (2.8% vs. 4.6%). Despite no difference in gestational age at delivery, the mean birth weights were significantly lower with fresh oocyte pregnancies, both in singleton (2,725 ± 727 g) and twins (2,128 ± 555 g), than with frozen-thawed oocytes (3,231 ± 615 g and 2,418 ± 492 g, respectively). However, the analysis of the 63 patients who obtained pregnancies both in fresh and thawed cycles (138 pregnancies) showed no differences in the abortion rate and in the mean birth weight., Conclusion(s): These results provide strong support to the notion that fetal and perinatal complications and congenital anomalies do not differ between pregnancies from frozen-thawed and fresh oocytes. The significantly lower mean birth weight observed with pregnancies from fresh oocytes supports similar observations reported for pregnancies from embryo cryopreservation and requires further prospective studies., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the safety of a new ultravitrification closed device., Design: Ultravitrification research., Setting: Private assisted reproduction center., Animal(s): Microorganisms (bacteria)., Intervention(s): A styrofoam container holding 1,000 mL of liquid nitrogen (LN2) was contaminated with Pseudomonas aeruginosa and Escherichia coli. Forty closed devices (Ultravit) and 20 open devices (Cryotop) loading approximately 0.5 μL of antibiotic-free medium were plunged into this contaminated LN2 for 5-10 seconds and then inoculated into selective culture dishes. Colony-forming units were analyzed and counted after an overnight incubation at 37°C., Main Outcome Measure(s): Detection of micro-organisms in different devices after incubation., Result(s): There was no contamination in any of the closed devices, whereas in 45% of open devices these bacteria were present., Conclusion(s): With this study we demonstrated, in an experimental model using contaminated LN2, that this new closed device is a safe system that does not allow cell contact with LN2, avoiding cell contamination., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the efficacy of a new ultravitrification technique with a low concentration of cryoprotectants., Design: Ultravitrification research., Setting: Private assisted reproduction center., Patient(s): Oocytes donated voluntarily with the aim of research., Intervention(s): Ultravitrification with different protocols of 100 mature oocytes and 100 immature oocytes divided in four groups to determine which is the adequate cryoprotectant concentration and the appropriate cooling solution., Main Outcome Measure(s): Human oocytes survival rate with low concentration of cryoprotectants by ultravitrification technique., Result(s): We obtained higher survival rates with slush nitrogen than with liquid nitrogen (92% vs. 56%) and better results with 2 M of cryoprotectants than with 1.5 M (92% vs. 60%). The best protocol was 2 M PrOH + 0.5 M sucrose + slush nitrogen with a mature oocytes survival rate of 92% (23 of 25) and immature of 88% (22 of 25)., Conclusion(s): This ultravitrification technique is a new option to preserve human oocytes that avoids the use of a high cryoprotectant concentration while obtaining a high survival rate with a concentration of cryoprotectants typical of slow freezing., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the efficacy of oocyte cryopreservation by a single slow-cooling protocol involving sucrose (0.2 mol/L) in the freezing solution., Design: Observational comparison of the clinical outcome in fresh and frozen thawed cycles., Setting: Public and private IVF centers., Patient(s): Infertile couples undergoing IVF treatment., Intervention(s): Use of a maximum three oocytes in fresh cycles, as established by local law, and cryopreservation and later use of surplus oocytes. Likewise fresh cycles, maximum three thawed oocytes were used per cycle. All thawed oocytes were microinjected., Main Outcome Measure(s): Embryologic and clinical parameters of fresh and thawed cycles., Result(s): Two thousand forty-six patients underwent 2,209 oocyte retrievals involving oocyte cryopreservation. Overall, the survival rate of thawed oocytes was 55.8%. In 940 thaw cycles, the mean numbers of inseminated oocytes and fertilization rates were significantly decreased vs. fresh cycles outcomes (2.6 ± 0.7 vs. 2.9 ± 0.2 and 72.5% vs. 78.3%, respectively), as were the rates of implantation (10.1% vs. 15.4%), pregnancy rates per transfer (17.0% vs. 27.9%), and pregnancy rates per cycle (13.7% vs. 26.2%). Differences in clinical outcome were found among centers. A pregnancy rate per thawing cycle above 14% was achieved by most clinics. Fifty-seven retrievals involving oocyte cryopreservation achieved a pregnancy after fresh embryo replacement. Implantation and pregnancy rates per embryo transfer and per thawing cycles were 17.5%, 28.6%, and 24.6%, respectively., Conclusion(s): Under the conditions tested, the clinical outcome of oocyte slow-cooling cryopreservation is reduced compared with fresh cycles. Nevertheless, in cases of inapplicability of embryo cryopreservation, oocyte cryopreservation should be offered to patients with surplus oocytes., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Over the last few years, there has been renewed interest and scientific debate concerning human oocyte cryopreservation. The aim of this study was to analyse the clinical data coming from our long experience of slow-freezing oocytes. Between 2001 and 2007, 1280 thawing cycles were carried out using oocytes previously frozen by means of a slow 1,2 propaniedol+sucrose protocol. A total of 7585 oocytes were thawed, of which 4409 survived and 3622 were microinjected; 144 clinical pregnancies were obtained. The number of thawing cycles increased from 19 in 2001 to 268 in 2007, and the number of thawed oocytes from 197 to 1652. Although the survival rate was significantly lower in the period 2002-2005 than in the period 2006-2007, pregnancy and implantation rates steadily improved from respectively 6.7% and 2.4% in 2001 to 15% and 8.2% in 2007. Our data demonstrate a clinically important improvement in oocyte crypreservation over the years in a Centres with proved experience, and can be offered as a standard of care not only before cancer treatment but also for couples refusing embryo crypreservation or in countries with very restrictive limitations on embryo or zygote freezing.

Background: The aim of the present study is to investigate cryopreservation of oocytes in patients refusing embryo cryopreservation for ethical reasons, patients from whom no sperm could be retrieved and patients with enough oocytes to yield a number of fresh and cryopreserved embryos to transfer., Methods: A total of 2900 oocytes out of 6216 retrieved were cryopreserved in 286 patients undergoing 303 cycles. The reasons for cryopreservation were because no sperm was found in 16 cycles, for ethical or personal reasons in 80, and in 207 only supernumerary oocytes were frozen. In 159 cycles, the oocytes were thawed and the surviving metaphase II oocytes microinjected., Results: A total of 1087 oocytes were thawed, 760 (69.9%) survived and 687 were microinjected. We obtained 368 (53.5%) normally cleaved embryos, 331 were transferred and 37 were cryopreserved. One hundred and forty-five transfers (range 1-3 embryos/patient) were performed and 18 (12.4%) pregnancies were obtained. Twelve patients delivered 13 healthy children, and six first trimester abortions were observed (33.3%)., Conclusion: Although a low implantation rate was observed and a higher abortion rate than in fresh cycles, our results show that in sibling oocytes, the process of cryopreservation apparently does not affect the fertilization and cleavage rate. In this group of patients, producing a large number of mature gametes, oocyte cryopreservation gives the couple extra chances to achieve a pregnancy within a single retrieval and is a good effort towards reducing the number of embryos cryopreserved and enhancing our experience in this new technology.

Objective: To report the first birth after transfer of cryopreserved embryos generated by intracytoplasmic sperm injection of cryopreserved testicular spermatozoa into cryopreserved human oocytes., Design: Case report., Setting: Tertiary center for reproductive technology., Patient(s): A 36-year-old woman with primary infertility of 3 years' duration and a 37-year-old man with congenital bilateral absence of the vas deferens., Intervention(s): Cryopreservation of human embryos after oocytes and sperm thawing., Main Outcome Measure(s): Live birth., Result(s): A healthy, normal female infant with a birth weight of 2,950 g was born by cesarean section at 38 weeks' gestation, with normal 6-month follow-up., Conclusion(s): Embryo cryopreservation can lead to successful results, even with the use of cryopreserved gametes.

Objectives: Conventional sperm freezing procedures need the addition of a relatively large volume of cryoprotectant. The dilution of extremely poor sperm suspensions from ejaculate or testicular tissue may make the recovery of viable spermatozoa difficult at the moment of the intracytoplasmic sperm injection (ICSI) procedure. The cryopreservation of a few spermatozoa in empty zonae pellucidae is an interesting solution for crypto-azoospermic infertile men. We have modified this procedure by filling empty human zonae with TEST Yolk Buffer, an optimal cryoprotective medium, in order to analyse the number of zonae lost after thawing, the number of recovered spermatozoa per zona after thawing, and the sperm motility rate before freezing and after thawing., Materials and Methods: Fifty empty human zonae pellucidae previously filled with TEST Yolk Buffer were injected with 750 motile spermatozoa from ten infertile men (15 spermatozoa per zona). Sterile straws containing two zonae each were frozen following a two-phase protocol., Results: All of the zonae and 445/750 spermatozoa (59%) were recovered. The mean number (+ SD) of spermatozoa per zona was 8.9 +/- 1.9 (range: 5-12). The recovery rate of motile spermatozoa was 73% (327/445), with a mean number of motile spermatozoa per zona of 6.5 +/- 1.7 (range: 3-10)., Conclusions: The cryopreservation of a small number of motile spermatozoa within empty zonae pellucidae using TEST Yolk Buffer as a freezing medium is possible without any major loss of spermatozoa and with an appreciable maintenance of sperm motility. This procedure seems to avoid: i) uncertain sperm retrieval after a laborious and time-consuming search on the day of oocyte aspiration; ii) the need for a repeated testicular biopsy; and iii) the need for heterologous insemination or oocyte cryopreservation (11).