Long-term storage of vitrified oocytes does not affect pregnancy and live birth rates: analysis of 5362 oocyte donation cycles.

RESEARCH QUESTION: Does long-term storage of vitrified oocytes affect laboratory and reproductive outcomes after intracytoplasmic sperm injection? DESIGN: Retrospective cohort study including 41,783 vitrified-warmed oocytes from 5362 oocyte donation cycles between 2013 and 2021. Five categories of storage time were established to analyse its effect on clinical and reproductive outcomes (≤1 year [reference group], 1-2 years, 2-3 years, 3-4 years and >4 years). RESULTS: The mean number of warmed oocytes was 8.0 ± 2.5 oocytes. Oocyte storage time ranged from 3 days to 8.2 years (mean: 0.7 ± 0.9). Mean oocyte survival (90.2% ± 14.7% overall) did not significantly decrease with longer storage time after adjusting for confounders (88.9% for time >4 years, P = 0.963). A linear regression model did not show a significant effect of oocyte storage time on fertilization rate (about 70% in all time categories) (P > 0.05). Reproductive outcomes after the first embryo transfer were statistically comparable across storage times (P > 0.05 for all categories). Longer term oocyte storage (>4 years) did not affect the chances of clinical pregnancy (OR 0.700, 95% CI 0.423 to 1.158, P = 0.2214) or live birth (OR 0.716, 95% CI 0.425 to 1.208, P = 0.2670). CONCLUSIONS: Oocyte survival, fertilization rate, pregnancy and live birth rates are not affected by the time spent by vitrified oocytes in vapour-phase nitrogen tanks.

Exonic genetic variants associated with unexpected fertilization failure and zygotic arrest after ICSI: a systematic review.

Fertilization failure (FF) and zygotic arrest after ICSI have a huge effect on both patients and clinicians, but both problems are usually unexpected and cannot be properly diagnosed. Fortunately, in recent years, gene sequencing has allowed the identification of multiple genetic variants underlying failed ICSI outcomes, but the use of this approach is still far from routine in the fertility clinic. In this systematic review, the genetic variants associated with FF, abnormal fertilization and/or zygotic arrest after ICSI are compiled and analyzed. Forty-seven studies were included. Data from 141 patients carrying 121 genetic variants affecting 16 genes were recorded and analyzed. In total, 27 variants in PLCZ1 (in 50 men) and 26 variants in WEE2 (in 24 women) are two of the factors related to oocyte activation failure that could explain a high percentage of male-related and female-related FF. Additional variants identified were reported in WBP2NL, ACTL9, ACTLA7, and DNAH17 (in men), and TUBB8, PATL2, TLE6, PADI6, TRIP13, BGT4, NLRP5, NLRP7, CDC20 and ZAR1 (in women). Most of these variants are pathogenic or potentially pathogenic (89/121, 72.9%), as demonstrated by experimental and/or in silico approaches. Most individuals carried bi-allelic variants (89/141, 63.1%), but pathogenic variants in heterozygosity have been identified for PLCZ1 and TUBB8. Clinical treatment options for affected individuals, such as chemical-assisted oocyte activation (AOA) or PLCZ1 cRNA injection in the oocyte, are still experimental. In conclusion, a genetic study of known pathogenic variants may help in diagnosing recurrent FF and zygotic arrest and guide patient counselling and future research perspectives.

No difference in morphokinetics between male and female preimplantation embryos from ART.

RESEARCH QUESTION: Do morphokinetic parameters vary between male and female preimplantation embryos? DESIGN: This was a retrospective cohort study of 175 cycles between March 2018 and June 2021 at two reproductive centres. It included time-lapse data from 92 female and 83 male preimplantation embryos exclusively issued from fresh oocyte donation and undergoing intracytoplasmic sperm injection (ICSI). Only fresh elective single-embryo transfers on day 5 were assessed, and the sex of the embryo was confirmed at birth. The morphokinetic parameters analysed were measured in hours post-insemination (hpi). A two-tailed Student's t-test was used to compare the morphokinetics between embryo sexes and a value of P < 0.05 was considered statistically significant. RESULTS: Following strict inclusion criteria to avoid poor-quality preimplantation embryos, no significant differences were found in morphokinetic parameters when comparing cycles that resulted in female versus male live births for the following: time to pronuclear fading (22.1 ± 2.4 versus 22.4 ± 2.9 hpi; P = 0.52); time to the 2-cell stage (24.6 ± 2.5 versus 25.0 ± 2.5 hpi; P = 0.34); time to the 3-cell stage (35.3 ± 3.3 versus 35.8 ± 3.1 hpi; P = 0.28); time to the 4-cell stage (36.3 ± 3.4 versus 36.9 ± 3.7 hpi; P = 0.20); time to the 5-cell stage (47.9 ± 4.6 versus 48.0 ± 4.8 hpi; P = 0.88); time to the 8-cell stage (54.0 ± 6.5 versus 54.1 ± 6.5 hpi; P = 0.91); time to the start of blastulation (86.3 ± 14.6 versus 85.7 ± 15.5 hpi; P = 0.78); and time to the full blastocyst stage (93.0 ± 16.9 versus 93.2 ± 17.2 hpi; P = 0.94). CONCLUSIONS: There are no significant differences in morphokinetics between male and female preimplantation embryos.

How long can the sperm wait? Effect of incubation time on ICSI outcomes.

In sperm processing for IVF/ICSI incubation times differ considerably both between and within assisted reproduction facilities. There is no established consensus on the optimal sperm incubation timings to maximize pregnancy rates, and the few studies addressing this association rely on manual and operator-dependent methods for time recording. The present retrospective cohort study includes 1169 ICSI cycles using fresh semen processed by swim-up. An operator-independent, radiofrequency-based system was used to record sperm incubation times: from sample collection to swim-up (T1, 0.35 ± 0.26); from swim-up to ICSI (T2, 3.30 ± 2.2); and total time from sample collection to ICSI (T, 3.66 ± 2.26). In oocyte donation cycles, we observed a significant negative effect of T1 on fertilization rate (FR; generalized linear modelling regression, coeff. -0.20, p = 0.001); however, after analysing all times by deciles and by adjusted logistic regression, none of the time intervals had a significant effect on pregnancy (biochemical, clinical, and ongoing) and live birth (LB) rates (p > 0.05 for all outcomes). In cycles using the patient's oocytes, we observed a negative effect of T2 (ordinal regression, coeff. -0.25, p = 0.011) and T (-0.33, p = 0.005) on the mean morphological score of the embryo cohort. In these cycles, a trend associating longer values of T with higher LB rates was identified (OR = 1.47, p = 0.050), although this difference is likely not clinically significant. In conclusion, while longer sperm incubation in vitro may impact slightly both FRs and embryo morphology after ICSI, no adverse effects were detected on the reproductive outcomes.

Altered mitochondrial function in spermatozoa from patients with repetitive fertilization failure after ICSI revealed by proteomics.

BACKGROUND: Unexplained fertilization failure (FF), occurring in 1-3% of intracytoplasmic sperm injection (ICSI) cycles, results in both psychological and financial burden for the patients. However, the molecular causes behind FF remain largely unknown. Mass spectrometry is a powerful technique to identify and quantify proteins across samples; however, no study so far has used it to dissect the proteomic signature of spermatozoa with FF after ICSI. OBJECTIVE: To investigate whether sperm samples from patients suffering repetitive FF after ICSI display alterations in their protein content. MATERIAL AND METHODS: Seventeen infertile men were included: 5 patients presented FF in ≥3 consecutive ICSI cycles, while 12 patients had a fertilization rate >75% (controls). Individual sperm samples were subjected to 2D-LC-MS/MS. Both conventional and novel statistical approaches were used to identify differentially abundant proteins. Additionally, analysis of mitochondrial and proteasomal abundance and activity were performed, using Western blot, FACS analysis of JC-1 staining and AMC-peptide fluorometric assay. RESULTS: Four proteins presented lower abundance (FMR1NB, FAM209B, RAB2B, and PSMA1) in the FF group compared to controls, while five mitochondrial proteins presented higher abundance in FF (DLAT, ATP5H, SLC25A3, SLC25A6, and FH) (p < 0.05). The altered abundance of mitochondrial DLAT and proteasomal PSMA1 was corroborated by Western blot. Of relevance, novel stable-protein pair analysis identified 73 correlations comprising 28 proteins within controls, while different mitochondrial proteins (ie, PDHA2, PHB2, and ATP5F1D) lost >50% of these correlations in specific FF samples pointing out specific mitochondrial deregulations. DISCUSSION: This is the first proteomic analysis of spermatozoa from patients who resulted in fertilization failure after ICSI. The altered proteins, most of them related to mitochondrial function, could help to identify diagnostic/prognostic markers of fertilization failure and could further dissect the molecular paternal contribution to reach successful fertilization. CONCLUSION: Sperm samples from patients with FF after ICSI present altered abundance of different proteins, including mainly mitochondrial proteins.

Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI.

STUDY QUESTION: Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? SUMMARY ANSWER: New mutations in the PLCZ1 sequence are associated with FFs after ICSI. WHAT IS KNOWN ALREADY: FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. STUDY DESIGN, SIZE, DURATION: We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into two groups based on oocyte evaluation 19 h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19 h after injection. MAIN RESULTS AND THE ROLE OF CHANCE: In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500 L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. LIMITATIONS, REASONS FOR CAUTION: Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. WIDER IMPLICATIONS OF THE FINDINGS: PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.

Sperm telomere length in donor samples is not related to ICSI outcome.

PURPOSE: Variations in sperm telomere length (STL) have been associated with altered sperm parameters, poor embryo quality, and lower pregnancy rates, but for normozoospermic men, STL relevance in IVF/ICSI is still uncertain. Moreover, in all studies reported so far, each man's STL was linked to the corresponding female partner characteristics. Here, we study STL in sperm donor samples, each used for up to 12 women, in order to isolate and determine the relationship between STL and reproductive outcomes. METHODS: Relative STL was determined by qPCR in 60 samples used in a total of 676 ICSI cycles. Univariable and multivariable statistical analyses were used to study the STL effect on fertilization rate; embryo morphology; biochemical, clinical, and ongoing pregnancy rates; and live birth (LB) rates. RESULTS: The average STL value was 4.5 (relative units; SD 1.9; range 2.4-14.2). Locally weighted scatterplot smoothing regression and the rho-Spearman test did not reveal significant correlations between STL and the outcomes analyzed. STL was not different between cycles resulting or not in pregnancy and LB (Mann-Whitney U test, p > 0.05). No significant effect of STL on reproductive outcomes was found, with the OR for each unit increase in STL (95% CI) of 0.94 (0.86-1-04), 0.99 (0.9-1.09), 0.98 (0.89-1.09), and 0.93 (0.8-1.06) for biochemical, clinical, and ongoing pregnancy and LB, respectively. The multilevel analysis confirmed that the effect of STL on fertilization; biochemical, clinical, and ongoing pregnancy; and LB was not significant (p > 0.05). CONCLUSION: After addressing STL independently from female variables, results show that STL measurement is not useful to predict reproductive outcomes in ICSI cycles using donor semen.

Is there an association between PAWP/WBP2NL sequence, expression, and distribution in sperm cells and fertilization failures in ICSI cycles?

Successful fertilization in mammals depends on the sperm's ability to initiate intracellular Ca2+ oscillations in the egg, a phenomenon that is elicited by Sperm-oocyte activating factors (SOAFs), whose quantitative and/or qualitative defect might result in fertilization failure. One such proposed factor is Post-acrosomal WW domain-binding protein (PAWP/WBP2NL), although its ability to activate human oocytes has been questioned and its implication in human fertilization failure remains unknown. Here, we sought to determine if PAWP/WBP2NL expression and distribution in sperm cells associate with low/complete fertilization failure in males participating in intracytoplasmic sperm injection (ICSI) cycles. This prospective study was conducted on eight couples referred for elective ICSI with either the woman's own (n = 4) or a donor eggs (n = 4). Eight sperm donor samples used in ICSI, which resulted in normal fertilization rates, were used as the control group. For each male patient and donor sperm, PAWP/WBP2NL sequence, protein expression, and cellular distribution were analyzed by PCR amplification-sequencing, Western blot, and immunofluorescence, respectively. PAWP/WBP2NL was present in all samples, and no significant differences were detected between patients with fertilization failure and donors in sequence variants or mean protein expression, or in the proportion of PAWP/WBP2NL-positive sperm. In conclusion, no clear association between PAWP/WBP2NL protein expression in sperm and fertilization outcome in ICSI were observed from this cohort.