High-quality Cleavage Embryo versus Low-quality Blastocyst in Frozen-thawed Cycles: Comparison of Clinical Outcomes.

This study compared the clinical outcomes of the frozen-thawed cycles of high-quality cleavage embryos with low-quality blastocysts to provide a reference for the choice of frozen-thawed embryo transfer schemes and to improve clinical pregnancy rates. A retrospective analysis was performed on the clinical data of patients undergoing frozen-thawed embryo transfer at the Reproductive Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from 2016 to 2017. In total, 845 cases were divided into a high-quality cleavage embryo group (group A) and a low-quality blastocyst group (group B). Each group was further divided into subgroups based on the number of transplants. Group A was categorized into two subgroups comprising of 94 cases in subgroup A1 (1 high-quality 8-cell group) and 201 cases in subgroup A2 (2 high-quality 8-cell group). Group B was divided into four subgroups consisting of 73 cases in subgroup B1 (D53BC group), 65 cases in subgroup B2 (D54BC group), 110 cases in subgroup B3 (D63BC group), and 282 cases in subgroup B4 (D64BC group). The pregnancy outcomes and neonatal outcomes between the groups were compared. The clinical pregnancy rates (56.72% and 60.00%) and live birth rates (47.76% and 46.15%) in subgroups A2 and B2 showed no significant differences, but these rates were significantly higher in subgroups A2 and B2 than in the rest subgroups (P<0.05). The multiple birth rate (26.32%) in the subgroup A2 was significantly higher than that in the rest subgroups (P<0.05). There were no statistically significant differences in the abortion rates among all groups (P>0.05). In terms of neonatal outcomes, there were no statistically significant differences in the proportion of premature births, sex ratios, and birth defects among the low-weight and gigantic infants (P>0.05). Transplanting two high-quality cleavage embryos during the frozen-thawed embryo transfer cycles could significantly increase clinical pregnancy rates and live birth rates, but at the same time, it also increased the risks of multiple births and complications to mothers and infants. The D54BC subgroup had the most significant advantages among all groups (P<0.05). The rest low-quality blastocysts had clinical outcomes similar to the single high-quality cleavage embryo group.

Developmental Potential and Clinical Value of Embryos with Abnormal Cleavage Rate.

To investigate the developmental potential and clinical value of embryos with abnormal cleavage rate, a retrospective analysis was performed on 66 635 2-prokaryotic (2PN) and 1-pronuclear (1PN) embryos. The embryos were given conventionally in vitro fertilization (IVF) treatment and continuously cultured on the day 3 (D3) at the Reproductive Medicine Center, Tongji Medical College, Huazhong University of Science and Technology from January 2016 to December 2017. The embryos were separated into the day-2 (D2) undivided group with 106 cases, the arrested development group with 3482 cases, the blastomere reduction group with 541 cases, and the control group with 62 506 cases, respectively. The blastocyst utilization rates of these three abnormal groups were 2.83%, 10.86% and 6.84%, respectively, which were significantly different from that in control group (39.46%). Furthermore, 2 cases of anabiosis and 1 case of live birth were found in D2 undivided group. In arrested development group, there were 55 cases of anabiosis, 11 cases of clinical pregnancy in single-embryo transplantation (including 6 cases of live birth), and 25 cases of clinical pregnancy in combination with one normal embryo transplantation (including 23 cases of live births and 15 cases of dizygotic twins under B-ultrasound). There were 13 case of anabiosis in blastomere reduction group: there was 1 case of single embryo transplantation and clinical pregnancy was obtained; there were also 6 cases of clinical pregnancy in combination with one single normal embryo transplantation (including 5 cases of live births and 2 cases of dizygotic twins under B-ultrasound). In conclusion, embryos with abnormal cleavage rate still have the potential to continue to develop, and have certain blastocyst utilization rate and live birth.

[Microdrop-vitrification for epididymal spermatozoa without cryoprotectants].

OBJECTIVE: To explore the feasibility and safety of microdrop-vitrification for epididymal spermatozoa obtained by percutaneous epididymal sperm aspiration (PESA) without cryoprotectants. METHODS: We treated the epididymal sperm samples from 22 patients by conventional freezing (Group 1) and microdrop-vitrification without cryoprotectants (Group 2), and evaluated the effectiveness of the two methods by comparing their revival rate, retrieval rate and incidence of sperm nuclear DNA fractures. RESULTS: Motile sperm were found in all but 1 case in Group 1. The revival rates of the frozen sperm were low in both Groups 1 and 2 ([18.16 +/- 9.38]% vs [21.99 +/- 10.95]%, P > 0.05), but statistically significant differences were shown between the two groups in the retrieval rate ([58.39 +/- 12.67]% vs [70.82 +/- 14.94]%, P < 0.01). Before freezing, nuclear DNA fractures existed in the epididymal sperm samples of all the 22 patients, comet sperm were seen after unicellular gel electrophoresis, and the incidence of sperm nuclear DNA fracture was (26.68 +/- 9.45)%. After freezing, no increase was observed in the incidence of sperm nuclear DNA fracture in either Group 1 or 2 ([28.68 +/- 12.54]% vs [27.64 +/- 10.70]%, P > 0.05). CONCLUSION: Microdrop can be used as a suitable freezing carrier for a low number of sperm, and cryoprotectant-free vitrification with microdrop may be a simple, safe and effective method for the cryopreservation of a low number of epididymal sperm.

[Cryopreservation by cryoloop damages human immature oocytes].

OBJECTIVE: To examine the influence of cryoloop on the spindle and chromosome configurations of human oocytes cryopreserved in the germinal vesicle (GV) and metaphase II (M II) stages, as well as on the survival rate and potential for in vitro maturation (IVM). METHODS: GV oocytes were randomly assigned into a control group (matured in vitro into the M II stage), a GV cryopreserved group (cryopreserved in the GV stage and then matured in vitro), and an M II cryopreserved group (matured in vitro and cryopreserved in the M II stage). After cryopreservation and IVM, immunostaining of the tubulin and chromatin was performed followed by visualization using laser scanning confocal microscopy (LSCM). RESULTS: A significantly higher survival rate was observed in the GV cryopreserved group than in the M II , but the maturation rate showed no significant difference between the GV cryopreserved group and the control (P > 0.05). Compared with the control group, there was a statistically significant decrease in the rates of normal meiotic spindles and chromosomes in the GV cryopreserved group (P < 0.05). A significantly lower rate of normal spindles was noted in the M II cryopreserved group than in the control, but no statistical difference was shown in the rate of normal meiotic chromosomes between the two groups (P > 0.05). CONCLUSION: Cryopreservation by cryoloop has a damaging effect on the spindle and chromosome of human oocytes in the GV and M II stages.

[Nuclear status of day 2 preembryos affects embryo quality and implantation potential].

OBJECTIVE: To assess the effects of the nuclear status of day 2 preembryos on day 3 embryo quality and implantation potential and to weigh its clinical value in the human in-vitro fertilization-embryo transfer (IVF-ET) program. METHODS: Embryos obtained from 409 fresh conventional IVF-ET/ICSI cycles from July to October 2006 were assessed retrospectively. Day 2 preembryos were classified according to the number of nuclei in each blastomere in 3 groups: grade A with only mononucleated blastomeres, grade B with one or more blastomeres containing no visible nucleus, and grade C with one or more multinucleated blastomeres. Comparisons were made of the rates of arrested embryos and excellent embryos on day 3 as well as of the pregnancy outcome and implantation potential of those in whom cohorts of similar nuclear scoring embryos were transferred. RESULTS: There were fewer arrested embryos and more excellent embryos on day 3 in grade A than in grade B and C (P < 0.01), and so were there in grade B than in grade C (P < 0.01). Among the 234 cycles in which all the transfer embryos were derived from a similar day 2 nuclear scoring, 51 cycles originated from grade A embryos (group A) and 183 from grade B (group B), with similar clinical pregnancy rates between the two groups, while the implantation rate was higher in group A than in B (P < 0.05). CONCLUSION: Day 2 nuclear scoring can be used to predict the devel- opment and implantation potential of embryos. A combined evaluation of day 2 nuclear scoring and day 3 embryo morphology helps identify the most viable embryos and reduce the number of embryos for transfer.

[Influence of maternal age on meiotic spindle and chromosome configuration of oocytes].

OBJECTIVE: To study the effect of maternal age on meiotic spindle and chromosome configuration of oocytes. METHODS: Spindle and chromosome configuration was examined in day 1 unfertilized human oocytes after in vitro fertilization (IVF) and intracytoplasmic sperm injection(ICSI) by immunocytochemistry and visualized by laser confocal microscopy. RESULTS: Statistically significant differences were observed on normal spindle and chromosome configurations of oocytes between 25-29 maternal age group (33% and 31%, respectively), and 30-34 age group (P< 0.05) as well as 35-40 age group(0%, P<0.01). The incidence of abnormal spindle and chromosome configurations of oocytes from 30-34 and 35-40 maternal age groups was much higher than that of oocytes from 25-29 age group (P<0.01, P<0.05). CONCLUSION: Incidence of abnormal spindle and chromosome configuration of oocytes is related to maternal age. It could be an important reason of age related oocyte aneuploidy.

[Comparison between the results of ICSI with fresh and with frozen-thawed sperm obtained by PESA to treat azoospermia].

OBJECTIVE: Retrospective study of the results of ICSI (intracytoplasmic sperm insemination) with frozen sperm obtained by PESA (percutaneous epididymal sperm aspiration) was performed in 27 patients. METHODS: With conventional freezing method, sperm from diagnosing PESA and the remaining motile sperm after treating cycle were frozen. After frozen-thawed and ICSI process, fertilization rate, implantation rate, clinical pregnancy rate were compared and other outcomes including pregnant combinations and parameters of newborns of experimental group (which used frozen-thawed sperm) and control group (which used fresh PESA sperm) were analyzed respectively. RESULTS: One hundred and sixty three and 1 157 oocytes of stage M II were injected respectively in the experimental group (15 cycles) and control group (100 cycles), and fertilization rate of experimental group was prominently higher than that of control group (84.05% vs 73.29%, P < 0.05), while implantation rate and clinical pregnancy rate were of no difference from the control, respectively (23.07% vs 15.73%; 53.33% vs 37.00%, P > 0.05). The differences in newborn's weights between two groups were of no statistical significance (P > 0.05). In the experimental group, eight clinical pregnancies were achieved including 5 live deliveries and 3 ongoing pregnancies, 37 clinical pregnancies including 30 deliveries with only 1 fetal death, 3 ongoing pregnancies and 4 abortions in the control group. Neither vital pregnant combinations nor neonate malformations were found in both groups. CONCLUSION: ICSI using frozen-thawed sperm obtained by PESA is an economic effective and safe method to treat azoospermia. Recovering rates of frozen sperm form PESA should be further increased.

[Vitrification of human cleaved embryos in vitro fertilization-embryo transfer].

OBJECTIVE: To assess the outcome of thawing human cleaved embryos from cryopreservation by vitrification. METHODS: A total of 957 day 2 or day 3 embryos from 219 patients were thawed after vitrification with ethylene glycol 5.5 solution and 0.25 ml straw between Jan 2003 and Jun 2005, 514 embryos were recovered and transferred in 178 patients. RESULTS: The survival rate of thawing embryos and the clinical pregnancy rate after transfer was 72.2% and 19.7% respectively. Twenty-two healthy babies were born from 16 deliveries, including 12 girls and 10 boys, 6 pregnancies ended in miscarriage and another 13 are ongoing. CONCLUSION: Vitrification method is an alternative for cryopreservation of human cleaved embryos because of high effectiveness, convenience and good cost efficiency.

[Mechanism of insulin-like growth factor-I affecting adhesion of trophoblast cells in vitro].

OBJECTIVE: To investigate the mechanism of insulin-like growth factor-I (IGF-I) affecting adhesion of trophoblast cells in vitro. METHODS: Trophoblast cells were obtained from early gestation at artificial abortion to set up the in vitro trophoblast cell adhesion model. The trophoblast cells were incubated with or without 10 nmol/L IGF-I and were divided into three groups (10 nmol/L IGF-I, 10 nmol/L IGF-I + alpha v beta3Ab, and control). The amount of adhered cells was assessed by examining absorbency using enzyme-linked immunoassay. Morphological changes were studied using scanning electron microscopy. The expression of phosphorylated focal adhesion kinase was determined by immunocytochemistry. RESULTS: After serum-starved trophoblast cells were incubated only with IGF-I, the mean absorbency was 0.491 +/- 0.049, obviously higher than control 0.198 +/- 0.022 and the difference was dramatic (P < 0.01). When cells were pre-treated with antibody against alpha v beta3 integrin and then incubated with IGF-I, the mean absorbency was only 0.184 +/- 0.031, distinctly lower than that incubated with IGF-I, and the difference was significant (P < 0.01), however, compared with control, there was no significant difference (P > 0.05). Scanning electron microscopy highlighted a dramatic increase in lamellipodial formation and extension in the IGF-I treated cells compared with control. Immunocytochemistry staining showed phosphorylated focal adhesion kinase was expressed in the trophoblast cells treated with IGF-I. CONCLUSIONS: 10 nmol/L IGF-I can significantly stimulate trophoblast cells adhesion to fibronectin, but antibody against alpha v beta3 integrin obviously blocks its adhesion. IGF-I can stimulate lamellipodial formation and extension at the adhesion sites, and promote adhesion of trophoblast cells to fibronectin by activating phosphorylated focal adhesion kinase.

[Microorganism contamination in in vitro fertilization-embryo transfer system and their sources].

OBJECTIVE: To investigate the incidence of microorganism contamination in in vitro fertilization-embryo transfer (IVF-ET) and to determine the sources of microorganism. METHODS: Two thousand one hundred and seventy-four cycles of in vitro fertilization from January 1999 to June 2003 were evaluated retrospectively and bacterial cultures were performed in 61 semen samples from asymptomatic men with normal semen parameters and in 34 follicle fluid samples from infertility women through oocyte picking up procedures. RESULTS: Microorganisms were found in 11 cases. The incidence of their contamination in IVF culture system was 0.51% and the most common microorganisms were Escherichia coli and fungi. Microorganisms were detected in 97% of unprocessed semen, 10% in processed semen, 6% in semen mixed with media and 9% in follicle fluid. CONCLUSIONS: The incidence of microorganism contamination was 0.51% and the most common microorganisms were Escherichia coli and fungi. Semen may have the potential to contaminate IVF culture system.