Live birth rate per transfer is not impacted by the proportion of smooth endoplasmatic reticulum aggregates oocytes.

Background: Despite the data published to date, prognostic factors and the clinical impact of ICSI cycles with smooth endoplasmatic reticulum aggregates (SERa) positive oocytes remain unclear. Objective: Are the clinical outcomes of an ICSI cycle impacted by the proportion of oocytes with SERa? Materials and Methods: Retrospective study (2016-2019), including data from 2468 ovum pick-ups, performed in a tertiary university hospital. Cases are categorised based on the rate of SERa positive oocytes compared to the total number of MII oocytes: 0% (n=2097), <30% (n=262) and ≥30% (n=109). Main outcome measures: Patient characteristics, cycle characteristics and clinical outcomes are compared between the groups. Results: Compared to SERa negative cycles, women with ≥30% SERa positive oocytes are older (36.2y vs. 34.5y, p<0.001), have lower anti-mullerian hormone levels (AMH) (1.6ng/ml vs. 2.3ng/ml, p<0.001), have received more gonadotropins (3227U vs. 2858IU, p=0.003), have a lower number of good quality day 5 blastocysts (1.2 vs. 2.3, p<0.001) and face more blastocyst transfer cancellation (47.7 vs. 23.7%, p<0.001). Women with <30% SERa positive oocytes are younger (33.8y, p=0.04), have higher AMH levels (2.6ng/ml, p<0.001), have more oocytes retrieved (15.1, p<0.001), have a higher number of good quality day 5 blastocysts (3.2, p<0.001) and have less transfer cancellations (14.9%, p<0.001) compared to SERa negative cycles A multivariate analysis shows no significant difference in cycle outcomes between the categories. What is new?: Treatment cycles with ≥30% SERa positive oocytes are less likely to result in an embryo transfer when only non-SER oocytes are used. However, live birth rate per transfer is not affected by the proportion of SERa positive oocytes.

Assisted oocyte activation does not overcome recurrent embryo developmental problems.

STUDY QUESTION: Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER: AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY: The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION: This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE: We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION: Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS: Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER: NCT03354013.

No difference in cumulative live birth rates between cleavage versus blastocyst transfer in patients with four or fewer zygotes: results from a retrospective study.

STUDY QUESTION: Is the cumulative live birth rate (CLBR) per oocyte collection cycle (OCC) comparable after cleavage-stage or blastocyst-stage transfer in combination with supernumerary blastocyst vitrification on Day 5 (D5) in patients with four or fewer zygotes on Day 1? SUMMARY ANSWER: The CLBR in a fresh blastocyst-transfer or cleavage-stage transfer policy followed by vitrification on D5 is comparable in patients with four or fewer zygotes. WHAT IS KNOWN ALREADY: Blastocyst transfer enhances the self-selection of the embryo and shortens the time to pregnancy in patients with normal or high ovarian response. Whether these advantages are also present in patients with a low ovarian response and/or a limited number of available zygotes is a continuous debate. STUDY DESIGN SIZE DURATION: This was a retrospective, observational cohort study of 2359 consecutive OCCs between January 2014 and December 2018. According to a shift in transfer policy in our center, 571 OCCs had been scheduled for a fresh transfer on Day 3 (D3) and 1788 on D5. The D5 group was matched to the D3 group by propensity score (PS) matching according to multiple maternal baseline covariates. After PS matching, there were 571 OCCs in each group. PARTICIPANTS/MATERIALS SETTING METHODS: OCCs scheduled for a D3 transfer (n = 571) or for a D5 transfer (n = 1788) were matched by PS matching in a 1:1 ratio accounting for potential confounding factors associated with CLBR. The model included patient characteristics, such as maternal age and cycle rank, as well as treatment characteristics such as GnRH analog regimen and ovarian response. Embryological variables included the number of zygotes and the number of 6- to 7- and 8-cell embryos on D3. The delivery outcomes of the fresh treatment cycle and the consecutive vitrified-warmed embryo transfers were analyzed up to the first live birth. The primary endpoint of this study was CLBR per OCC. Secondary outcomes were live birth rate per fresh transfer and embryo implantation rate per transferred embryo. MAIN RESULTS AND THE ROLE OF CHANCE: The CLBR per OCC was comparable between the D5 and D3 groups (16.8% versus 17.7%, respectively, P = 0.600). Live birth rates per OCC did not differ between a cleavage-stage transfer and blastocyst-stage transfer policy (15.2% versus 12.4%, respectively, P = 0.160). In the D5 group, 201 cycles did not result in a blastocyst to perform an embryo transfer or cryopreservation; in the D3 group, only 59 cycles did not have an embryo transfer because of poor embryo quality (35.2% versus 10.3%, respectively; P < 0.001). A significantly higher number of fresh double embryo transfers were performed in the D3 group compared to D5 (23.8% versus 7.0%, respectively, P < 0.001). LIMITATIONS REASONS FOR CAUTION: Although adjusted for important confounders in the PS matching, BMI and embryo quality of the transferred embryo(s) were not taken into account. This study is limited by its retrospective design and is a single-center study, which may limit the generalizability of our findings. WIDER IMPLICATIONS OF THE FINDINGS: The CLBR in a fresh blastocyst-transfer or cleavage-stage transfer policy followed by vitrification on D5 is comparable. A fresh embryo transfer on D3 can still be considered in patients with a poor ovarian response and/or limited number of zygotes when combined with blastocyst vitrification without impacting the overall CLBR of the cycle. STUDY FUNDING/COMPETING INTERESTS: No external funding was obtained for this study. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: This retrospective study was approved by the local ethical committee at Ghent University Hospital (B 670201731234).

A stepwise approach to move from a cleavage-stage to a blastocyst-stage transfer policy for all patients in the IVF clinic.

STUDY QUESTION: Is a stepwise change management approach an efficacious method to move from a Day 3 transfer policy to a Day 5 transfer policy for all patients in an IVF program? SUMMARY ANSWER: A stepwise change from a Day 3 to a Day 5 transfer policy maintained the live birth rates per oocyte collection cycle (OCC) of the IVF program, with increased single embryo transfer (SET) and reduction of twin pregnancies. WHAT IS KNOWN ALREADY: Evidence has shown that the probability of a live birth following IVF with a fresh embryo transfer (ET) is significantly higher after blastocyst-stage transfer than after cleavage-stage transfer. Blastocyst culture and transfer are usually performed in cases of good prognosis patients but many centers keep transferring cleavage-stage embryos for most of their patients because of the higher transfer cancelation rate in a blastocyst transfer policy. STUDY DESIGN SIZE DURATION: In January 2012, a Day 5 embryo culture and blastocyst transfer policy including vitrification of supernumerary Day 5 blastocysts were implemented in a stepwise approach. The retrospective descriptive single-center analysis involving a preintervention phase consisted of Day 3 ETs and Day 3 slow freezing from 2010 until 2012. The postintervention phase involved a 6-year period from 2012 until 2017 in which three consecutive changes in the transfer policy were made, each over a 2-year period, based on the number of zygotes on Day 1. The primary outcome was live birth delivery rate per OCC during the stepwise change. PARTICIPANTS/MATERIALS SETTING METHODS: All patients with at least one zygote available on Day 1 were scheduled for a fresh transfer, either on Day 3 or 5. Cycles with preimplantation genetic testing, freeze-all and oocyte donation cycles and cycles with a Day 2 transfer in the preintervention period were excluded. In the preintervention group, all cycles were scheduled for Day 3 transfer (n = 671 OCC) and slow freezing of the remaining Day 3 embryos. In the postintervention period, three periods were analyzed: period 1 (n = 1510 OCC; 1-9 zygotes: Day 3 transfer and >9 zygotes: Day 5 transfer); period 2 (n = 1456 OCC; 1-4 zygotes: Day 3 transfer and >4 zygotes: Day 5 transfer) and period 3 (n = 1764 OCC; Day 5 transfer). All remaining embryos underwent extend culture and were vitrified on Day 5, if developed to at least an early blastocyst. Data were analyzed using a mixed regression model with patient as a random factor. MAIN RESULTS AND THE ROLE OF CHANCE: In the preintervention group, all OCC were scheduled for a Day 3 transfer. In period 1, period 2 and period 3, 20.9%, 61.5% and 100% of the OCCs were scheduled for a Day 5 transfer, respectively. More transfers per OCC were canceled in the postintervention period 2 and period 3 compared to the preintervention period (5.3% and 18.7% versus 3.4%, respectively; P < 0.0001). The mean number of embryos used per transfer decreased gradually after the introduction of the Day 5 transfer policy, from 1.62 ± 0.65 in the preintervention group to 1.12 ± 0.61 in period 3 (P < 0.0001). The percentage of SET cycles increased from 48.4% in the preintervention group to 54.6%, 73.8% and 87.8% in period 1, period 2 and period 3, respectively (P < 0.0001). The mean number of cryopreserved surplus embryos was significantly lower in period 3 compared to the preintervention group (1.29 ± 1.97 versus 1.78 ± 2.80; P < 0.0001).Pregnancy and live birth delivery rate per fresh transfer, respectively, were significantly lower in the preintervention group (26.7% and 19.1%) as compared to period 3 (39.3% and 24.2%) (P < 0.0001). Twin pregnancy rate decreased gradually from 11.0% to 8.2%, 5.7% and 2.5% in the preintervention group, period 1, period 2 and period 3, respectively (P < 0.0001). Live birth rate and cumulative live birth delivery rates per OCC were significantly higher in group 2 compared to the preintervention period (25.6% and 35.8% versus 18.5% and 25.9%, respectively). Similar live birth and cumulative live birth delivery rates per OCC were achieved between the preintervention period and period 3 (18.5% and 25.6% versus 19.7% and 24.9%; respectively). LIMITATIONS REASONS FOR CAUTION: The primary limitation is the retrospective design of the study. The allocation of the cycles was done by the number of zygotes available without taking into account both embryological and clinical prognostic factors. Furthermore, the analysis was restricted to cycles where the standard transfer policy was followed. Embryos which were in the morula or compaction stage were not vitrified or cultured to Day 6, which could have contributed to the slight, not statistically significant, drop in live birth rate per OCC in group 3. WIDER IMPLICATIONS OF THE FINDINGS: Live birth and cumulative live birth delivery rate per OCC in an unselected patient population is maintained in a Day 5 transfer policy compared to a Day 3 transfer policy. Additionally, a significantly reduction in twin pregnancy rate and a significant increase in SET were observed in a Day 5 transfer policy. For centers wanting to make the step from Day 3 to Day 5, this study provides a practical stepwise change management approach. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: None.

Blastocyst transfer for all? Higher cumulative live birth chance in a blastocyst-stage transfer policy compared to a cleavage-stage transfer policy.

BACKGROUND: In an unselected patient population, what is the cumulative live birth rate per oocyte collection cycle in a blastocyst-stage transfer policy compared to a cleavage-stage transfer policy? METHODS: A retrospective cohort analysis of 1656 IVF and ICSI cycles was performed in two timeframes between January 2010 and December 2016. Transfer was scheduled, either on day 3 (n=729) or on day 5 (n=927). In this study, the main outcome measure was cumulative live birth rate per oocyte collection cycle including fresh and frozen embryo transfers in both groups. RESULTS: The cumulative live birth rates per oocyte collection cycle were comparable between patients with cleavage-stage transfers (day 3 group) and those with blastocyst-stage transfers (day 5 group) (23.7% versus 25.5%, respectively; p = 0.42). After controlling for confounders, there was a 34% increased chance of live birth with blastocyst-stage transfer policy compared with cleavage-stage transfer policy (odds ratio (OR) =1.34; 95% confidence interval (CI), 1.051 to 1.704; p = 0.018). CONCLUSION: In an unselected patient cohort, the cumulative live birth chance per oocyte collection cycle is higher in a blastocyst-stage transfer policy compared to a cleavage-stage transfer policy.

Seasons in the sun: the impact on IVF results one month later.

BACKGROUND: Several retrospective studies have evaluated seasonal variations in the outcome of IVF treatment. Some also included weather conditions, mostly temperature and hours of daylight. The results were conflicting. METHODS: In a retrospective study we analysed all fresh cycles (N = 9865) that were started between January 1, 2007 and December 31, 2012. Because some patients were included more than once, correction for duplicate patients was performed. We focused on individual variables provided as monthly results by our national meteorological institute. We evaluated if weather conditions determined by temperature, rain and sunshine at the start of ovarian stimulation had an effect on the outcome of IVF in terms of number of mature and fertilized oocytes, pregnancy and live birth rates. We shifted the results in IVF outcome to the weather results of one month earlier, as we supposed that the selection of good quality oocytes might start in the weeks before ovarian stimulation is initiated. RESULTS: There was a clear trend towards better results when the "early" weather conditions (one month before the treatment cycle) were good. There was a statistically significant correlation between the number of rainy days (Pearson Correlation -0.326; p < 0.01) and the rain flow (Pearson Correlation -0.262; p < 0.05) on the one hand and the live birth rate per cycle on the other. The live birth rate per cycle was statistically different between cohorts of patients that were stratified into quartiles of sunshine hours (p < 0.01) and of number of rainy days (p < 0.05) during the month before the start of ovarian stimulation. CONCLUSIONS: Weather conditions during the month before IVF treatment have an impact on live birth rate.

Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization.

STUDY QUESTION: What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation? SUMMARY ANSWER: Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation. WHAT IS KNOWN ALREADY: Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce. STUDY DESIGN, SIZE, DURATION: This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37°C under 6% CO(2). The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the Mann-Whitney U-test. MAIN RESULTS AND THE ROLE OF CHANCE: Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P < 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P < 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P < 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes. LIMITATIONS, REASONS FOR CAUTION: The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation. WIDER IMPLICATIONS OF THE FINDINGS: Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.

Fertilization, pregnancy and embryo implantation rates after ICSI in cases of obstructive and non-obstructive azoospermia.

The aetiology of azoospermia can be grossly divided into obstructive and non-obstructive causes. Although in both cases testicular spermatozoa can be used to treat male fertility, it is not well established whether success rates following intracytoplasmic sperm injection (ICSI) are comparable. Therefore, a retrospective analysis of fertilization, pregnancy and embryo implantation rates was performed following ICSI with testicular spermatozoa in obstructive or non-obstructive azoospermia. In total, 193 ICSI cycles were carried out with freshly retrieved testicular spermatozoa; in 139 cases of obstructive and 54 cases of non-obstructive azoospermia. The fertilization rate after ICSI with testicular spermatozoa in non-obstructive azoospermia was significantly lower than in obstructive azoospermia (67.8% versus 74.5%; P = 0.0167). Within the non-obstructive group, the fertilization rate in the group of maturation arrest (47.0%) was significantly lower than in case of Sertoli cell-only (SCO) syndrome (71.2%) or germ cell hypoplasia (79. 5%). Embryo quality on day 2 after ICSI was similar for all groups. Pregnancy rates per transfer between obstructive (36.8%) and non-obstructive groups (36.7%) were similar. In cases of maturation arrest the pregnancy rate per transfer was lowest (20.0%) although not significantly different from SCO syndrome or hypoplasia groups. Embryo implantation rates were not different between the obstructive (19.6%) and non-obstructive groups (25.8%), and were lowest in cases of germ cell hypoplasia (15.8%). This retrospective analysis shows that although fertilization rate after ICSI with testicular spermatozoa in non-obstructive azoospermia is significantly lower than in obstructive azoospermia, pregnancy and embryo implantation rates are similar.

Fertilization, pregnancy and embryo implantation rates after ICSI with fresh or frozen-thawed testicular spermatozoa.

This study aimed to determine whether fertilization and implantation rates after intracytoplasmic sperm injection (ICSI) with fresh or frozen-thawed testicular spermatozoa were comparable. Between 1 January 1996 and 31 December 1996, 65 ICSI cycles with testicular spermatozoa and 35 cycles with frozen-thawed testicular spermatozoa were carried out. In 50 out of 65 ICSI cycles, testicular spermatozoa could be retrieved and in 34 out of 35 cycles carried out with frozen-thawed testicular spermatozoa, motile spermatozoa could be recovered. The fertilization rate after ICSI with frozen-thawed testicular spermatozoa was significantly lower (71.1%; P < or = 0.008) than with fresh testicular spermatozoa (79.3%). The pregnancy rate was similar for both groups (38.2 and 26.5 %). The implantation rate per transferred embryo, however, was significantly lower in the frozen-thawed rather than in the fresh testicular sperm group (9.1 versus 24.6%; P = 0.001). The live birth rate per transferred embryo was also higher in the group in which fresh testicular spermatozoa were used (18.8 versus 7.9% P = 0.043). This retrospective study shows that is possible to achieve a high fertilization rate after ICSI with both fresh and frozen-thawed testicular spermatozoa but implantation and live birth rates per transferred embryo, however, are significantly lower after ICSI with frozen-thawed than with fresh testicular spermatozoa.

Quality of frozen-thawed testicular sperm and its preclinical use for intracytoplasmic sperm injection into in vitro-matured germinal-vesicle stage oocytes.

OBJECTIVE: To study the effects of cryopreservation on the quality of human testicular spermatozoa and the efficiency of intracytoplasmic sperm injection (ICSI) with frozen-thawed testicular sperm into metaphase II oocytes in vitro-matured from the germinal-vesicle stage oocyte. DESIGN: Preclinical freezing study on supernumarary testicular spermatozoa after ICSI. SETTING: Tertiary IVF center coupled with an institutional research environment. PATIENT(S): Twenty-nine patients undergoing excisional testicular biopsy for ICSI. INTERVENTION(S): Isolated testicular spermatozoa were cryopreserved and thawed; frozen-thawed motile testicular spermatozoa were microinjected. MAIN OUTCOME MEASURE(S): Prefreezing and post-thawing motility and viability, survival rate, fertilization rate, cleavage rate, and embryo quality after ICSI. RESULT(S): Mean percentage motility decreased from 21% before freezing to 6% after thawing. Vitality was impaired to a similar extent, decreasing from 68% to 22% (32% recovery rate). Injection of frozen-thawed testicular spermatozoa into in vitro-matured oocytes resulted in a fertilization rate of 50.9%. Cleavage rate was severely impaired. Half of the fertilized oocytes became arrested in the one-cell stage. CONCLUSION(S): Despite the low quality of the fresh testicular spermatozoa, a high percentage of prepared testicular sperm fractions showed survival and motility after the freezing and thawing process. Injection of frozen-thawed testicular sperm into matured oocytes resulted in fertilization rates comparable with these with fresh testicular sperm, but cleavage rates were severely impaired, which might be due to source of oocytes used for ICSI.

Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue.

To maximize the total number of spermatozoa which can be retrieved from a testicular biopsy, four mechanical methods for preparation were compared. In all, 17 biopsies were divided into four equal fractions, which were weighed individually and prepared by rough shredding (control), fine mincing, vortexing and crushing (electrical Potter). After resuspension in an erythrocyte-lysing buffer, removal of the remaining tissue pieces, washing and centrifugation, the following parameters were evaluated: number of spermatozoa per 100 mg tissue, percentage motility, percentage vitality and percentage normal morphology (strict Kruger criteria). After further purification by discontinuous Percoll centrifugation, the same parameters were assessed again. This procedure was efficient in obtaining only spermatozoa in the final fraction. Before Percoll centrifugation, only minor differences between methods were observed. Percoll centrifugation generally resulted in a pronounced loss of sperm cells, partly because of the abnormal dimensions of the eliminated cells. After Percoll centrifugation, treatment of the testicular tissue by fine mincing was the most effective method in terms of the total number of motile spermatozoa and percentage normal morphology.