Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa

Aim: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). Methods: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. Results: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P < 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. Conclusion: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

Relationship between ATP Content and the Developmental Potential of Human Oocytes

To investigate the relationship between the ATP content in human oocytes and the deve-lopmental potential of human oocytes, unfertilized oocytes from clinical IVF and immature oocytes unsuitable for ICSI were collected. The ATP content of these unfertilized and in vitro matured eggs were determined quantitatively by measuring the luminescence using an ATP-dependent bioluminescence assay. The result showed that the ATP content of unfertilized oocytes was higher than in vitro matured ones (2.20±0.67 pmol vs 1.72±0.49 pmol, P<0.05 ). In unfertilized oocytes, the ATP content of those whose fertilization rates (FR) was higher than 50 % was 2.43±0.60 pmol, which was significantly different from those whose FR was less than 50 % (1.72±0.56 pmol), while in in vitro matured oocytes, the ATP content of those whose FR more than 50 % was 1.8±0.44 pmol, slightly higher than those less than 50 % (1.55±0.40 pmol), without statistical significance. There was a tendency that the ATP content of oocytes of pregnant patients was higher than those of controls, but the sample number was too small to show any significance in statistics. Briefly, there was positive correlation between the ATP content in oocytes and developmental potential of oocytes.

In vitro maturation of human oocytes maintaining good development potential for rescue intracytoplasmic sperm injection with fresh sperm

BACKGROUND The outcomes of the use of commercial in vitro maturation(IVM)medium to culture immature oocytes obtained from conventional ovulation induction,followed by rescue intracytoplasmic sperm injection(RICSI),are not ideal.It is thus difficult to widely adopt this approach in clinical practice.Therefore,it is necessary to explore methods for improving the clinical outcome of IVM.AIM To study the effect of sperm on the developmental potential of in vitro-matured oocytes in conventional culture.METHODS This was a retrospective study of patients whose immature oocytes were harvested from conventional oocyte stimulation cycles and underwent ICSI at our hospital between June 2018 and August 2020.RICSI was performed using sperm collected on the day of oocyte harvest(old)and sperm collected on the day of RICSI(fresh)and oocytes matured in vitro after 24 h of culture in conventional medium.The rates of in vitro oocyte maturation,normal fertilization,normal cleavage,day-3 top-quality embryos,and useful blastocyst formation were compared between the two groups.RESULTS In total,102 germinal vesicle(GV)-stage immature oocytes were cultured in the old sperm group.In the fresh sperm group,122 GV-stage immature oocytes were collected and cultured in vitro for 24 h.There were no significant differences in the general conditions of males and females between the two groups(P>0.05).The oocyte maturation,normal fertilization,and normal cleavage rates of the old and fresh groups were 51.0%vs 55.7%,61.5%vs 64.7%,and 93.8%vs 93.2%,respectively.None of the rates differed significantly(P>0.05)between the two groups.However,the day-3 top-quality embryo and useful blastocyst rates of the old and fresh sperm groups were 16.6%vs 63.4%;6.67%vs 34.6%,respectively.The day-3 top-quality embryos and useful blastocyst rates of the old sperm group were significantly lower than those of the fresh group(P<0.05).CONCLUSION In vitro maturation with conventional culture medium combined with the use of fresh sperm collected on the day of RICSI is an easy-to-implement strategy for patients whose oocytes are completely or mostly immature.

Vitrification versus Slow Freezing of Human Oocytes: Effects on Ultrastructure and Developmental Potential

Objective:This study compared spindles,cytoskeleton,and developmental potential between vitrified and slow-frozen oocytes using PolScope and electron microscopy.Methods:Oocytes were randomly divided into control,slow freeze-thaw,and vitrification freeze-thaw groups(0,1,and 3 h).PolScope was used to observe spindles,angle of spindles to the first polar bodies,surface areas of oocytes,and lining and outer ret of zona pellucida.Surfaces and ultrastructure of oocytes were observed by scanning electron microscopy and transmission electron microscopy.These measures were used to characterize the impacts of two freezing methods on the developmental capacity of human oocytes.Results:The visible frequency of spindle formation was 92.4%,56.4%,11.2%,24.8%,and 61.1%in control group,slow freeze-thaw group,and the three vitrification freeze-thaw groups(0,1,and 3 h),respectively.Compared to oocytes in the slow freeze group,the angle of the spindle to the first polar body in oocytes in the 3-h vitrification freeze-thaw group was less(37.3°vs.68°,P=0.023).No significant differences were observed between the surface area of oocytes,or lining and outer ret of oocyte zona pellucida between freeze-thaw in these same two groups.Microvilli appeared normal.However,protrusions on oocyte surfaces were increased,and microvilli were laid down on the membrane surface in the 3-h vitrification freeze-thaw group in comparison to the slow-freeze group.Similar comparisons showed better recovery of perivitelline space and mitochondria between the 3-h vitrified and slow-frozen groups.Bipronuclear(2PN)fertilization rate observed in the slow-freeze group(65.7%)was lower than the rate seen in controls(79.2%,P=0.041).No significant differences were observed in 2PN fertilization,cleavage,and blastocyst formation rates between the 3-h vitrification freeze-thaw and control groups.Conclusions:Results suggest that vitrification freeze-thaw for oocyte cryopreservation was a better choice than slow freeze-thaw.

Embryonic, genetic and clinical outcomes of fresh versus vitrified oocyte: A retrospective cohort study

Objective:To compare embryonic development,ploidy status and clinical outcomes between fresh and frozen-thawed oocytes.Methods:This retrospective cohort study evaluated 83 fertilization cycles including both fresh and frozen oocytes from 79 patients at the HP Fertility Center of Hai Phong International Hospital of Obstetrics and Pediatrics in Vietnam.The patient underwent several ovarian stimulation cycles to accumulate a certain number of oocytes that would be vitrified.In the last oocyte retrieval,all patient’s oocytes including both frozen and fresh would be fertilized.The outcomes included the rates of oocyte survival,cleavage embryo,blastocyst,ploidy status,pregnancy,biochemical pregnancy and clinical pregnancy.Results:The oocyte survival rate after thawing was 96.5%.No statistically significant difference was found when comparing fresh and frozen oocytes regarding fertilization rate(78.1%vs.75.5%,P=0.461),usable cleavage embryo rate(86.9%vs.87.2%,P=0.916)but usable blastocyst rate was found higher statistically in the frozen oocyte group(44.4%vs.54.0%,P=0.049).The percentages of euploid,aneuploid and mosaic embryos between the fresh group and the vitrified group had no significant differences(33.8%vs.31.6%,P=0.682;51.0%vs.54.2%,P=0.569;15.2%vs.12.4%,P=0.787;respectively).The rates of pregnancy,biochemical pregnancy and clinical pregnancy had no statistical difference(68.8%vs.64.8%,P=0.764;12.5%vs.3.6%,P=0.258;37.5%vs.46.4%,P=0.565).17 Mature oocytes are the minimum to have at least one euploid embryo.Conclusions:Oocyte vitrification does not affect embryonic,genetic and clinical results.The number of mature oocytes should be considered for fertilization in some cases.

Effects of vitrification and cryostorage duration on single-cell RNA-Seq profiling of vitrified-thawed human metaphase Ⅱ oocytes

Oocyte cryopreservation is widely used for clinical and social reasons.Previous studies have demonstrated that conventional slow-freezing cryopreservation procedures,but not storage time,can alter the gene expression profiles of frozen oocytes.Whether vitrification procedures and the related frozen storage durations have any effects on the transcriptomes of human metaphase Ⅱ oocytes remain unknown.Four women(30–32 years old)who had undergone IVF treatment were recruited for this study.RNA-Seq profiles of 3 fresh oocytes and 13 surviving vitrified-thawed oocytes(3,3,4,and 3 oocytes were cryostored for 1,2,3,and 12 months)were analyzed at a single-cell resolution.A total of 1987 genes were differentially expressed in the 13 vitrifiedthawed oocytes.However,no differentially expressed genes were found between any two groups among the 1-,2-,3-,and 12-month storage groups.Further analysis revealed that the aberrant genes in the vitrified oocytes were closely related to oogenesis and development.Our findings indicated that the effects of vitrification on the transcriptomes of mature human oocytes are induced by the procedure itself,suggesting that long-term cryostorage of human oocytes is safe.

Effect of Incubation Temperature and Warming Solutions on the Viability and Maturation of Vitrified-thawed Human Immature Oocytes

Objective To investigate the viability and maturation of frozen-thawed human immature oocytes exposed to different temperature of vitrification and warming solutions. Methods The immature oocytes in germinal vesicle （GV） and matephase I （MI ） stages were collected from our ICSI patients and exposed to different temperature of vitrification and warming solutions before frozen in the freezing/thawing procedures. The different temperature groups were as follows： Group A, equilibration solution at 37℃, vitrification solution at room temperature, warming solution at 37℃; Group B, both vitrification and warming solution at room temperature; Group C, both vitrification and warming solutions at 37℃; Group D, the frozen-thawed oocytes and the fresh oocytes were cultured for in vitro maturation. The survival rate and maturation rate were compared among groups. The oocytes were examined using immunofluorescent stainingand confocal microscopy to check the spindle configuration and chromosome arrangement. Results The survival rates and MII rates of GV stage oocytes in groups A, B, C were 100%（15/15）, 81.3%（13/16）, 68.8%（11/16） and 33.3%（2/6）, 83.3%（10/12）,72.7%（8/11）, respectively. The survival rate of group C was significantly lower than that of the control （P〈0.05）. The normal spindle and chromosome configuration were only observed in group B, with the rates of 20% （2/20） and 10% （1/10）, respectively. The survival rates of MI stage in groups A, B, C were 71.4%（10/14）, 100% （12/12） and 83.3%（10/12）, no significantly difference from that of the contro1（100%, 14/14）. The MII rates of MI stage in groups A, B and C were 0%（0/14）, 66.7%（8/12） and 80% （8/10）, respectively. The MⅡ rate in group A was significantly lower than that in other groups （P〈0.01）. Only one oocyte in group C was found with normal spindle and chromosome configurations. Conclusion The appropriate operation temperature of vitrification and warming solutions can improve the outcomes of the vitrified-thawed human immature oocytes.

Preliminary Research on the Effect of Linolenic Acid on Human Oocyte Maturation

Objective:To investigate the effects of exogenousα-linolenic acid(ALA)on in vitro maturation(IVM)and developmental competence of human oocytes.Methods:Experiment 1 examined the effects of ALA at different concentrations(0[control],10,50,100,and 200μmol/L)in the IVM medium on oocyte maturation.Treatment with 50μmol/L ALA significantly accelerated oocyte maturation(P<0.05)and resulted in significantly higher mitochondrial DNA(mtDNA)copy number compared to the control.Hence,50μmol/L ALA was selected for combination with follicular fluid(FF)to investigate oocyte developmental potential.mtDNA of the matured oocyte was analyzed by real-time polymerase chain reaction.Experiment 2 investigated the effects of FF with optimal ALA concentration(Group A:ALA+FF+IVM medium)or without ALA(Group B:FF+IVM medium)on oocyte maturation,fertilization,2 pronuclear cleavage,and embryo and blastocyst development.Malondialdehyde(MDA)content and superoxide dismutase(SOD)activity were measured for maturation medium from Group A,Group B,and Group C(control group,IVM medium only).Results:Treatment with 50μmol/L ALA obviously accelerated oocyte maturation(P<0.05)and resulted in significantly higher mtDNA copy number(P<0.05)in the matured oocytes compared to the control(0μmol/L ALA).Supplementation of 50μmol/L ALA and FF(Group A)obviously increased the total maturation rate than FF-treated group(Group B)which has higher(P<0.05)total maturation rate than that of Group C.However,no significant differences were observed in fertilization,embryo availability,and blastocyst production among Group A,B,and C.Treatment with 50μmol/L ALA decreased the level of MDA(P<0.05),but had no effect on the activity of SOD in the IVM medium.Conclusions:Our results suggested that the treatment with 50μmol/L ALA during IVM improves maturation in human oocytes.It is also likely to improve embryo availability and blastocyst formation.This might be associated with the alteration of mtDNA replication(increased mtDNA copy number)and the reduction of oxidative stress(reduced MDA level).