Post-ovulatory aging affects mitochondria, spindle and protein metabolism in mouse oocytes.

In brief: Post-ovulatory aging (POA) results in a decline in oocyte quality and embryonic developmental capacity although the underlying mechanisms remain elusive. This study provides comprehensive mRNA expression profiles of fresh and aging oocytes in mice for the first time. Abstract: POA impairs the quality of mammalian oocytes with harmful effects on the developmental potential of the embryo. This is a major problem for humans since it is associated with low rate of natural fertility, with high rate of spontaneous abortion and low efficiency of in vitro fertilization. However, the molecular mechanisms underlying this process remain unclear and new methods are demanded to control POA. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis on fresh and aging MII mouse oocytes and compared their global RNA transcription patterns. Nine hundred and twenty-one differentially expressed genes (DEGs) were identified. Five hundred and sixty-nine genes were downregulated, while 356 were upregulated in the group of aging oocytes. Gene ontology (GO) enrichment analysis demonstrated that a series of DEGs were significantly enriched involving mitochondrial functions, spindle functions and protein metabolism. The results of qPCR and a series of functional tests further confirmed that the disorder of mitochondrial functions, spindle functions and impairment of protein metabolism were actually involved in the progression of POA. In this study, panoramic mRNA expression profiles of fresh and aging oocytes were depicted and fully validated. Our data will provide a useful resource for further research on the regulation of gene expression of POA and suggest potential strategies to delay and reverse POA.

Comparison of clinical outcomes of frozen-thawed D5 and D6 blastocysts undergoing preimplantation genetic testing.

BACKGROUND: This study aimed to analyze the clinical outcomes of blastocyst which undergo the preimplantation genetic testing (PGT) transplantation from frozen-thawed D5 and D6. In addition, the effect of blastocyst grade on clinical and neonatal outcomes was also investigated in this study. METHODS: The pregnancy and miscarriage rates of 1130 cycles of frozen embryo transfer, including 784 D5 frozen embryos and 346 D6 frozen embryos in the Reproductive Hospital of Shandong University from January to December 2020 were analyzed. Gardner blastocyst scoring was used for blastocyst evaluation. RESULTS: The pregnancy rate of D5 blastocyst was significantly higher, whereas the miscarriage rate of D5 blastocyst was lower, than that of D6 blastocyst tissue biopsy. No significant difference was observed in birth weight and low birth weight of D5 blastocyst and D6 blastocyst, preterm birth, gestational age, and neonatal sex. Frozen-thawed D5 blastocysts have higher pregnancy success rates and lower miscarriage rates compared to D6 blastocysts. CONCLUSION: Therefore, both blastocyst grade and embryo biopsy date must be considered when transferring frozen embryos.

Efficiency and safety of laser-assisted hatching on vitrified-warmed blastocyst transfer cycles: a prospective control trial.

To clarify the efficiency and safety of laser-assisted hatching (LAH) application on vitrified-warmed blastocyst transfer (VBT) cycles, we designed the non-randomized concurrent control trial included 4039 VBT cycles in the Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, during the even days from November 2014 to December 2015. The VBT cycles were divided into LAH group (n = 1932) and non-LAH group (n = 2107) according to the date of blastocyst thawing. Laser-partial zona pellucida dissection was performed on all blastocysts thawed on that day every 4 days, and those blastocysts were assigned to the LAH group. There were a higher biochemical pregnancy rate (66.87% vs 63.69%; P = 0.034; rate ratio for LAH vs non-LAH group [RR], 1.050; 95% confidence interval [CI], 1.004-1.098) and an increased live birth rate (48.81% vs 45.51%; P = 0.036; RR, 1.072; 95% CI, 1.005-1.145) with comparable ectopic pregnancy, twin or multiple pregnancies, spontaneous abortion and birth defect rates of the LAH group than those of the non-LAH group. Subgroup analysis showed that live birth rate, birth defect rate, and other pregnancy outcomes were comparable for patients younger than 35 years when blastocyst transfer, patients with endometrium thickness less than 0.9 cm during ovulation or the initiation of progesterone treatment, ICSI blastocysts, AC or BC blastocysts according to Gardner morphological criteria and day 5 blastocysts of the LAH group than it of non-LAH group. LAH could be performed selectively on vitrified-warmed blastocysts before transfer for better pregnancy outcomes. Trial registration number: ChiCTR2000032975. Date of registration: May 17, 2020. Retrospectively registered.

Mutation analysis of tubulin beta 8 class VIII in infertile females with oocyte or embryonic defects.

Variants of tubulin beta 8 class VIII (TUBB8) have been shown to be associated with female infertility characterized by oocyte or embryonic defects. To further investigate the mutational spectrum of TUBB8 and the prevalence of variants, we performed Sanger sequencing of TUBB8 on a total of 115 infertile females who had undergone repeated in vitro fertilization cycles with oocyte or embryonic defects and 200 healthy controls. A total of 31 variants which were absent from the controls were identified in 36 unrelated individuals, accounting for a large proportion of this cohort (31.3%). All of the variants including heterozygous/homozygous missense variants and a heterozygous frameshift insertion variant were at conserved sites and predicted to be deleterious. Besides, these variants had diverse phenotypic effects, including not only oocyte maturation arrest, fertilization failure, and early embryonic arrest, but also multi-pronuclei (MPN) formation, which is a new phenotype associated with TUBB8 variants. Overall, this study reveals a large number of variants of the TUBB8 gene in infertile females with oocyte or embryonic defects. Our results not only broaden the mutational and phenotypic spectra of TUBB8 variants, but also further confirm the critical role of TUBB8 in oocyte maturation, fertilization, and early embryonic development.

Complex preimplantation genetic tests for Robertsonian translocation, HLA, and X-linked hyper IgM syndrome caused by a novel mutation of CD40LG gene.

PURPOSE: To perform complex preimplantation genetic tests (PGT) for aneuploidy screening, Robertsonian translocation, HLA-matching, and X-linked hyper IgM syndrome (XHIGM) caused by a novel mutation c.156 G>T of CD40LG gene. METHODS: Reverse transcription PCR (RT-PCR) and Sanger sequencing were carried out to confirm the causative variant of CD40LG gene in the proband and parents. Day 5 and D6 blastocysts, obtained by in vitro fertilization (IVF) with intracytoplasmic sperm injection, underwent trophectoderm (TE) biopsy and whole genomic amplification (WGA) and next generation sequencing (NGS)-based PGT to detect the presence of a maternal CD40LG mutation, aneuploidy, Robertsonian translocation carrier, and human leukocyte antigen (HLA) haplotype. RESULTS: Sanger sequencing data of the genomic DNA showed that the proband has a hemizygous variant of c. 156 G>T in the CD40LG gene, while his mother has a heterozygous variant at the same position. Complementary DNA (cDNA) of CD40LG amplification and sequencing displayed that no cDNA of CD40LG was found in proband, while only wild-type cDNA of CD40LG was amplified in the mother. PGT results showed that only one of the six tested embryos is free of the variant c.156 G>T and aneuploidy and having the consistent HLA type as the proband. Meanwhile, the embryo is a Robertsonian translocation carrier. The embryo was transplanted into the mother's uterus. Amniotic fluid testing results are consistent with that of PGT. A healthy baby girl was delivered, and the peripheral blood testing data was also consistent with the testing results of transplanted embryo. CONCLUSIONS: The novel mutation of c. 156 G>T in CD40LG gene probably leads to XHIGM by nonsense-meditated mRNA decay (NMD), and complex PGT of preimplantation genetic testing for monogenic disease (PGT-M), aneuploidy (PGT-A), structural rearrangement (PGT-SR), and HLA-matching (PGT-HLA) can be performed in pedigree with both X-linked hyper IgM syndrome and Robertsonian translocation.

Over Ten-Cell Good Embryo Transfers on Day Three have Equivalent Clinical Outcomes with Those of Eight-Cell Embryos in Female Patients Aged ≤35 Years: A Retrospective Cohort Study.

BACKGROUND/AIM: To compare the clinical outcomes between > 10- and 8-cell good quality embryos on day 3. METHODS: A retrospective study of a cohort of 459 patients was included in the fresh embryo transfer (ET) cycle group from January 2009 to April 2016. In this group, 2 good quality embryos on day 3, were transferred on corresponding dates, in 75 patients (> 10-cell embryos), and in 384 patients (8-cell embryos). Seven hundred and forty four patients, with 1 blastocyst transferred derived from > 10-cell (n = 183) or 8-cell (n = 561) good quality embryos on day 3, were assigned in the frozen ET (FET) group. RESULTS: In the ET group, the clinical pregnancy and live birth rates of the > 10-cell transfer patients were comparable with those of the 8-cell transfer group (62.67 vs. 69.27%, 60.00 vs. 59.90%, respectively); however, the miscarriage rate of > 10-cell transfers was significantly lower than that of 8-cell transfers (4.26 vs. 13.53%, p < 0.05). In the FET group, there were no statistically significant differences found in the clinical pregnancy, live birth, and miscarriage rates between one > 10-cell-derived and one 8-cell-derived blastocyst transfers (71.04 vs. 65.78%, 59.02 vs. 54.19%, and 16.92 vs. 17.62%, respectively). CONCLUSIONS: The results suggested that > 10- and 8-cell, good quality embryos on day 3, had comparable viability and clinical outcomes.

Comparison of two protocols of blastocyst biopsy submitted to preimplantation genetic testing for aneuploidies: a randomized controlled trial.

PURPOSE: To compare the effectiveness of two protocols of blastocyst biopsy submitted to preimplantation genetic testing for aneuploidies (PGT-A). METHODS: This is a randomized controlled trial of a cohort of 221 patients undergoing PGT-A. 106 female patients aged ≤ 40 years with no less than 8 mature oocytes retrieved and ≥ 3 good-quality embryos on day 3 were randomly assigned to the day-3 hatching-based TE biopsy. The remaining 115 females aged ≤ 40 years with ≥ 8 MII oocytes obtained and no less than 3 high-quality embryos on day 3 were assigned to the TE biopsy without hatching group (also called the new biopsy group). The primary outcome was measured by a live birth after the first embryo transfer. RESULTS: The live birth rate did not differ significantly between the two groups (50.00% vs. 59.26%, P > 0.05, OR 1.46; 95% CI 0.78-2.70). There was no significant between-group difference in the rates of implantation, clinical pregnancy, and miscarriage. However, the frozen blastocyst rate was significantly lower in the day-3 hatching-based TE biopsy compared with the new biopsy group (47.54% vs. 53.96%, P < 0.05, OR 1.29; 95% CI 1.08-1.56). CONCLUSIONS: Our study provides strong evidence that the new blastocyst biopsy method exhibits advantages over day-3 hatching-based TE biopsy method. Using this method, we were able to obtain more blastocysts to perform trophectoderm biopsy in patients subjected to PGT-A.

Using time-lapse technology to explore vacuolization in embryos on Day 3 and Day 4.

PURPOSE: To investigate the occurrence and development state of embryo vacuoles between the 8-cell and morula stages, and to explore how vacuoles affected the development of embryos. METHODS: A retrospective study of a cohort of 422 patients undergoing conventional in vitro fertilization or intracytoplasmic sperm injection. With the help of time-lapse imaging, the development processes and outcomes of good quality embryos with or without vacuoles were analyzed. RESULTS: Vacuole positive embryos had significantly lower blastulation rate and good quality blastulation rate than vacuole negative embryos, p < 0.05. Compared to vacuole negative embryos, the number of best and good quality blastocysts was significantly reduced, while the number of fair and discarded ones was significantly increased, p < 0.05. The average starting time of vacuolization was 73.7 ± 9.3 h after insemination. The proportion of blastomeres affected by vacuoles was associated with embryonic developmental potential. CONCLUSIONS: Vacuolization on Day 3 and Day 4 was frequently observed and was detrimental to embryo development. The proportion of blastomeres affected by vacuoles may be an indicator of embryo developmental potential.

Conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI): which is preferred for advanced age patients with five or fewer oocytes retrieved?

PURPOSE: To investigate the clinical outcomes of conventional IVF and ICSI in female patients aged 40 years and over with no more than five oocytes retrieved and non-male factor infertility. METHODS: A retrospective study of a cohort of 644 patients undergoing IVF/ICSI treatment. The 534 female patients aged ≧ 40 years with no more than five oocytes retrieved and non-male factor infertility undergoing their first conventional IVF cycles were assigned in IVF group. The rest of 110 patients aged 40 years and over with no more than five oocytes retrieved and non-male factor infertility undergoing first ICSI cycles were recruited in ICSI group. RESULTS: Our results showed the clinical pregnancy, live birth and miscarriage rates were similar between the IVF and ICSI groups (21.59% vs. 13.25%, P > 0.05; 12.16% vs. 6.02%, P > 0.05; 43.68% vs. 54.55%, P > 0.05; respectively), however, the implantation and cumulative live-birth rates were significantly higher in the IVF compared to the ICSI group (15.11% vs. 7.75%, 14.59% vs. 5.56%, P < 0.05), though the IVF group had a lower normal fertilization rate (61.56% vs. 76.00%, P < 0.001). CONCLUSIONS: Our study provides strong evidences that the conventional IVF exhibits advantages over the ICSI method in non-male factor infertility for advanced age patients with five or fewer oocytes retrieved.

Morphokinetic parameters from a time-lapse monitoring system cannot accurately predict the ploidy of embryos.

PURPOSE: This study aimed to test whether there is an association between embryo morphokinetic parameters and ploidy status. METHODS: Patients with high risk of aneuploidy were analyzed by time-lapse microscopy combined with preimplantation genetic screening (PGS). Accordingly, 256 blastocysts from 75 patients were subjected to trophectoderm biopsy and microarray comparative genomic hybridization (array-CGH). Blastocyst development process was analyzed using time-lapse images. RESULTS: Morphokinetic parameters: tPNf, t2, t3, t4, t5, t8, t9, tcom, tM, tSB, tB, tEB, CC1, CC2, CC3, S2, S3, t5-t2, and tB-tSB showed no significant difference in euploid embryos compared to aneuploid counterparts. In addition, two risk models based on previously published morphokinetic parameters failed to segregate euploid from aneuploid embryos. CONCLUSIONS: Morphokinetic parameters subjected to investigation in the present study failed to improve the chance of selecting euploid embryos.

[Application of calcium ionophore A23187 in ICSI for globozoospermia: A report of 2 cases and review of the literature].

OBJECTIVE: To investigate the pathogenesis of globozoospermia, fertilization ability of round-headed sperm, and the application value of assisted oocyte activation in intracytoplasmic sperm injection (ICSI) for the wives of glohozoospermia men. METHODS: We collected oocytes from the wives of 2 globozoospermia patients and randomly divided them into two groups after ICSI to receive calcium ionophore A23187-activation and conventional treatment, respectively. We reviewed the relevant literature published at home and abroad, and discussed the etiology of globozoospermia, fertilization ability of round-headed sperm, and treatment options for this disease. RESULTS: Quality embryos were obtained in the A23187-activation group while no fertilized oocytes, oocyte cleavage, quality embryos, or blastular formation were found in the conventional treatment group. Both women achieved pregnancy and gave birth to healthy neonates after transfer of the quality embryos from the A23187-activation group. CONCLUSION: Calcium ionophore A23187 can be applied to ICSI for the wives of globozoospermia men and bring about desirable clinical outcomes. Meanwhile, attention should be paid to its safety.

Day 3 ET, single blastocyst transfer (SBT) or frozen-thawed embryo transfer (FET): which is preferable for high responder patients in IVF/ICSI cycles?

PURPOSE: To compare the clinical outcomes after day 3 embryo transfer, day 5 single blastocyst transfer (SBT) and frozen-thawed embryo transfer (FET) in high responder patients (>15 retrieved oocytes) undergoing IVF/ICSI treatment. METHODS: A retrospective analysis of three embryo transfer strategies for the high responder patients in IVF/ICSI cycles. The 1041 high responder patients diagnosed as primary infertility with more than 15 oocytes retrieved were recruited in Day 3 ET group, 308 patients with more than 15 oocytes retrieved first transferred with one blastocyst in SBT group and 425 patients with more than 15 oocytes retrieved in fresh cycle, first transferred with one frozen-thawed blastocyst were assigned in FET group. RESULTS: In the high responder patients, the clinical pregnancy rate after day 5 SBT was significantly lower than that of day 3 ET (43.18% VS 57.16%, p < 0.05). In addition, the clinical pregnant rate and implantation rate of FET cycles were significantly higher than SBT cycles (59.06% vs. 43.18% and 64.70% vs. 47.40%, p < 0.05). The multiple pregnancy rate in FET cycles was markedly lower than that of day 3 ET (2.35% VS 34.97%, p < 0.05). CONCLUSIONS: FET was the preferable strategy for the high responder patients in IVF/ICSI cycles to obtain both desirable clinical outcome and lower multiple pregnancy rates.

Pregnancy with oocytes characterized by narrow perivitelline space and heterogeneous zona pellucida: is intracytoplasmic sperm injection necessary?

PURPOSE: This retrospective study analyzed fertilization protocols and pregnancy outcomes for oocytes with with narrow perivitelline space and heterogeneous zona pellucid (NPVS/HZP). METHODS: In 63 in-vitro fertilization cycles filled with NPVS/HZP oocytes (abnormal oocytes group) and 521 cycles with normal oocytes (normal oocytes group), major clinical and laboratory parameters were recorded and compared in different fertilization cycles (conventional IVF cycles, rescue ICSI cycles, and traditional ICSI cycles). RESULTS: NPVS/HZP oocytes meant lower MIIoocytes rates in both IVF and ICSI cycles compared with normal oocytes (p < 0.05). The 2PN rates for abnormal oocytes were significantly lower than those for normal oocytes in both conventional IVF cycles (58.8% VS 71.3%, P < 0.05) and rescue ICSI cycles (58.0% VS 78.0%, P = 0.0000). The high-quality embryo rates in normal oocytes groups were significantly higher than those in abnormal oocytes groups in different fertilization cycles (52.2% VS 35.0%, P < 0.01; 42.9% VS 23.9%, P < 0.001; 50.6% VS 31.0%, P = 0.0000, respectively). No clinical pregnancy was obtained from abnormal oocytes in 11 conventional IVF cycles. The clinical pregnancy rates in rescue ICSI and traditional ICSI cycles were comparatively lower in abnormal oocytes groups, but there was no significant difference as compared with normal oocytes groups (35.0% VS 48.1% and 26.7% VS 50.7%, P > 0.05, respectively). CONCLUSIONS: Retrieval of oocytes characterized by NPVS/PZP from cycle to cycle was one of the reasons for obscure infertility. ICSI may be the right way to avoid fertilization failure and get pregnancy in women with NPVS/HZP oocytes.

Confocal microscopic analysis of the microfilament configurations from human vitrification-thawed oocytes matured in vitro.

BACKGROUND: The alteration in microfilaments of human oocytes by vitrification has not been understood well. OBJECTIVE: To evaluate the effect of vitrification on the microfilaments of human in vitro matured oocytes and the time needed for the repair. MATERIALS AND METHODS: Human in vitro matured oocytes were divided into the control group (Group 1) and the vitrified group. The vitrified oocytes were further divided into four sub-groups that were cultured after thawing for 1 h, 2 h, 3 h and 4 h, respectively (Groups 2, 3, 4 and 5). RESULTS: The survival rate of oocytes was 87.4% after vitrification and thawing. The percentage of oocytes with a normal configuration of microfilaments in Group 2 and Group 3 was significantly lower than that of the fresh control group (Group 1), whereas the percentage of oocytes with normal microfilament configuration in Group 4 and Group 5 was comparable to the control group. CONCLUSION: Vitrification alters microfilament structure of oocytes, which takes at least 3h after thawing for the repair and recovery.

Intracytoplasmic Sperm Injection May Not Improve Clinical Outcomes Despite Its Positive Effect on Embryo Results: A Retrospective Analysis of 1130 Half-ICSI Treatments.

Objective: To explore the clinical application value of half-ICSI treatment for infertility in assisted reproductive technology. Method: A retrospective analysis of 1130 half-ICSI treatments was conducted at the Affiliated Reproductive Hospital of Shandong University from January 2011 to December 2015. Patients with low fertilization rates in previous cycles, primary infertility for >5 years with unexplained reason, or secondary infertility for >5 years without fallopian tube factor were involved in this study. The 2PN rate, high-quality embryo rate, oocyte utilization rate, and clinical outcomes were compared between IVF insemination group (IVF group) and ICSI insemination group (ICSI group). The clinical outcome of half-ICSI insemination treatment, grouped according primary and secondary infertility, was also analyzed. Results: Compared with IVF, ICSI resulted in a significantly higher 2PN rate (74.8% vs. 62.9%), high-quality embryo rate (54.6% vs. 51.7%), and oocyte utilization rate (35.9% vs. 32.8%; P<0.05). Among the 884 fresh-embryo transfer cycles, there were no notable differences in clinical pregnancy rate, live birth rate, or neonatal abnormality rate between the IVF and ICSI groups. Among the 792 primary infertility cycles, ICSI resulted in a significantly higher 2PN rate, high-quality embryo rate, and oocyte utilization rate compared with IVF (75.3% vs. 62.4%, 54.3% vs. 50.8%, 36.4% vs. 32.6%, P<0.05). For the 338 secondary infertility cycles, ICSI resulted in a significantly higher 2PN rate (73.6% vs. 63.9%, P<0.05) compared with IVF, but there were no notable differences in other laboratory results. Moreover, the biochemical pregnancy rate of the ICSI group was significantly lower than for IVF in secondary infertility cycles (49.3% vs. 65.6%; P<0.05). A total of 89 cycles (7.9%) with complete IVF fertilization failure showed a low second polar body (2PB) rate (33.6%) after a 5-h short-time fertilization period, including 34 cycles (3.0%) with no 2PB oocytes observed in the IVF group. Conclusion: ICSI insemination improved laboratory results compared with IVF insemination, however, fresh-embryo transfer of ICSI originated embryos did not improve clinical pregnancy and live birth rates. Rescue ICSI has been successfully applied in clinical IVF insemination to avoid fertilization failure. Therefore, as an extra intervention, it is suggested that ICSI be used judiciously.

Non-Assisted Hatching Trophectoderm Biopsy Does Not Increase The Risks of Most Adverse Maternal and Neonatal Outcome and May Be More Practical for Busy Clinics: Evidence From China.

OBJECTIVE: This study was conducted in order to investigate whether non-assisted hatching trophectoderm (TE) biopsy increases the risks of adverse perinatal outcomes in livebirths following elective single cryopreserved-thawed blastocyst transfer. PATIENTS AND METHODS: A total of 5,412 cycles from 4,908 women who achieved singleton livebirths between 2013 and 2019 were included in this retrospective cohort study. All embryos in this study were fertilized by intracytoplasmic sperm injection (ICSI) and cryopreserved through vitrification. The main intervention is to open the zona pellucida (ZP) of day 5/6 blastocyst immediately for biopsy without pre-assisted hatching. The main outcome measures are the common maternal and neonatal outcomes, including hypertensive disorders of pregnancy (HDPs), gestational diabetes mellitus (GDM), abnormal placentation, abnormalities in umbilical cord and amniotic fluid, preterm birth, cesarean section, low birth weight, postpartum hemorrhage, and prolonged hospital stay (both mothers and infants). The generalized estimation equation (GEE) was used to control the effects of repeated measurements. The non-conditional logistic regression model was used to examine the associations between embryo biopsy status and each adverse perinatal event. Given that the selection bias and changes in learning curve might affect the results, we selected 1,086 similar (matching tolerance = 0.01) cycles from the ICSI group via propensity score matching (PSM) for second comparisons and adjustment (conditional logistic regression). RESULTS: After adjusting for confounders, we confirmed that the non-assisted hatching protocol did not increase the risks of most adverse maternal and neonatal outcomes. Despite this, there were increased risks of GDM (aOR: 1.522, 95% CI: 1.141-2.031) and umbilical cord abnormalities (aOR: 11.539, 95% CI: 1.199-111.067) in the biopsy group. In the second comparisons after PSM, GDM incidence in the biopsy group was still higher (7.26% vs. 5.16%, P = 0.042), yet all measurement outcomes were equally likely to occur in both groups after the second adjustment. CONCLUSIONS: The non-assisted hatching TE biopsy does not increase the risks of most adverse perinatal outcomes. However, there is a higher GDM incidence in the biopsy group, and this association warrants further study. Considering its safety and simplicity, the non-assisted hatching protocol has the potential to become the preferred option for TE biopsy, especially in busy clinics and IVF laboratories.

Evaluation of the developmental potential of metaphase I oocytes from stimulated intracytoplasmic sperm injection cycles.

The objective of the present study was to evaluate the developmental potential and clinical application value of metaphase I (MI) oocytes obtained from stimulated intracytoplasmic sperm injection (ICSI) cycles. ICSI was performed on MI oocytes immediately after denudation (Group A), or on in vitro-matured (IVM) oocytes following culture; oocytes in culture were further divided into two groups, being cultured for either 3-5 h (Group B) or 24-28 h (Group C). Metaphase II oocytes from the same cycle(s) isolated for ICSI served as the control group (Group D). The rates of normal fertilisation, cleavage and high-quality embryos were compared among the four groups. High-quality embryos were transferred whenever possible, and pregnancy rates were evaluated. Results showed that normal fertilisation rates for Groups B, C and D were significantly higher than that of Group A (68.6%, 57.8%, 74.5% and 30.1%, respectively; P<0.01). The rate of high-quality embryos in Group B was comparable with Group D; the rate for Group C was significantly lower than that of the other groups (P<0.05). Two clinical pregnancies were achieved after transfer of embryos from IVM oocytes. In vitro maturation of MI oocytes for a short period of time may increase the number of available embryos; however, overnight in vitro culture of MI oocytes did not improve results.

Application of improved single blastomere fixation technique in preimplantation genetic diagnosis.

To modify a fixation method improving the intensity and clarity of the single blastomeric signal detection by fluorescence in situ hybridization (FISH) in preimplantation genetic diagnosis. 333 cycles of assisted reproduction with preimplantation genetic diagnosis FISH (PGD-FISH) performed in our hospital were analyzed and a total of 3452 single blastomeres were obtained. For the conventional fixation method, the blastomeres were kept in 0.1% sodium citrate with 0.2 mg/ml bovine serum albumin (BSA) for 2-5 min. FISH was performed and the internal relationship between embryo quality and fixed rate, signal detection rate, and signal determination rate was explored. With the modified method, 91.54% of blastomeres were fixed, while 88.30% were fixed with the conventional method. The signal detection rate was significantly increased for the modified group than for the conventional group (compared 98.53% with 94.78%, P < 0.001). Especially, the signal determination rate also showed a significant difference between the two methods (compared 90.51% with 74.17%, P < 0.001). After the development of the fixation method, the fixation efficiency and the signal determination rate were greatly improved, providing more definite diagnosis for the patient. It will hopefully allow more assisted reproduction programs to offer their patients preimplantation genetic diagnosis with FISH.

Resveratrol improves in vitro maturation of oocytes in aged mice and humans.

OBJECTIVE: To evaluate the effects of resveratrol on oocyte maturation in aged mice and humans. DESIGN: Experimental laboratory study. SETTING: University-based reproductive medicine center. PATIENT(S): A total of 64 women 38-45 years of age undergoing intracytoplasmic sperm injection (ICSI) and 48-52-week-old female C57BL/6J mice. INTERVENTION(S): In vitro culture in the presence of three different concentrations of resveratrol (0.1, 1.0, and 10 µm) or dimethylsulfoxide. MAIN OUTCOME MEASURE(S): Parameters of oocyte nuclear maturation, fertilization, immunofluorescence intensity of mitochondria, and normal morphology of spindle and chromosome of oocytes undergoing in vitro maturation (IVM) in aged mice and humans; blastocyst formation and levels of SRIT1, CAT, SOD1, and GPX4 gene expressions in aged mice. RESULT(S): Resveratrol at 1.0 µm significantly increased first polar body emission rate in oocytes derived from aged mice and humans, and an increased percentage of fertilization and blastocyst formation was observed in aged mice. In addition, immunofluorescence intensity of mitochondria and normal morphology of spindle and chromosome of oocytes undergoing IVM were notably improved compared with control samples in aged mice and human. Furthermore, the use of resveratrol exhibited enhanced expression patterns of SRIT1, CAT, SOD1, and GPX4 in aged mice. CONCLUSION(S): Resveratrol induced oocyte maturation and blastocyst formation in aged mice, and improved oocyte maturation and quality was examined in aged humans. In conclusion, 1.0 µm resveratrol was the appropriate concentration in IVM medium.