Effect of exposed-to-air frequency of cryopreserved embryo on clinical outcomes of vitrified-warmed embryo transfer cycles: a retrospective analysis of 9,200 vitrified-warmed transfer cycles.

BACKGROUND: Cryopreservation of embryos plays a major role in the in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. However, the storage condition of the cryopreserved embryo can change temporarily due to repeated retrieval of the embryo from the liquid nitrogen (LN2) tank during the practical application during cryopreservation. Whether the implantation potential of a cryopreserved embryo will be damaged when the cane containing it is temporarily exposed to air due to the transfer between the LN2 tank and LN2 container is yet to be elucidated. Also, whether the exposed-to-air frequency (EAF) of cryopreserved embryos influences the clinical outcomes is unclear. OBJECTIVE: To investigate whether the EAF of cryopreserved embryo affects the clinical outcomes of vitrified-warmed embryo transfer. METHODS: A total of 9200 vitrified-warmed embryo transfer cycles were included in this study. All cycles were divided into five groups according to different EAFs (2, 4, 6, 8, or ≥ 10). Post-warming survival rates and clinical outcomes, including implantation, clinical pregnancy and live birth rates were investigated. Kruskal-Wallis test and Pearson's chi-squared tests were used to compare the patient characteristics and clinical outcomes among the five groups. Furthermore, multivariate logistic regression analyses were conducted to investigate the association between EAF and clinical outcomes. RESULTS: No significant differences were observed in the positive HCG rate, implantation rate and live birth rate (P > 0.05) among five EAF groups with respect to D3 embryo, D5 blastocyst and D6 blastocyst. Post-warmed survival rate of D3 embryos (P = 0.015) differed significantly among the five EAF groups, but it was not EAF-dependent. Although clinical pregnancy was different among the five groups with respect to D5 blastocyst (P = 0.042), multivariate logistic regression analysis adjusted for confounding variables suggested that EAF did not adversely affect clinical pregnancy or live birth. CONCLUSION: These findings indicated that human vitrified embryos in the open system could be repeatedly retrieved from the LN2 tank without affecting the implantation potential of the embryo.

A prospective randomized comparison of early embryo cleavage kinetics between two media culture systems.

OBJECTIVE: To investigate whether early embryo cleavage kinetics were affected by type of culture media. METHODS: In this prospective sibling-split study, 620 oocytes from 37 patients were randomly allocated into two groups: Cook group and Vitrolife group. Oocytes/embryos in Cook group, would be cultured with Cook sequential culture medium, while oocytes/embryos in Vitrolife group, would be cultured with Vitrolife sequential culture medium. Time-lapse imaging technology was used to calculate exact timing of early embryo cleavage events which included time to 2PN breakdown, cleavage to 2-, 3-, 4-, 5- cell and the time duration in the 2-,3-cell stage. Then these timing of early embryo cleavage events were compared between Cook group and Vitrolife group. Moreover, fertilization rate, cleavage rate, high quality embryo rate, usable blastocyst rate, pregnancy rate and implantation rate of these two groups were also analyzed. RESULTS: The results showed there were no differences in all timing of early embryo cleavage events between the two groups. In addition, the two groups were similar in fertilization rate (Cook 71.0% vs. Vitrolife 71.3%, P>0.05), cleavage rate (Cook 98.1% vs. Vitrolife 98.2%, P>0.05), high quality embryo rate (Cook 52.1% vs. Vitrolife 52.7%, P>0.05), usable blastocyst rate (Cook 29.7% vs. Vitrolife 28.0%, P>0.05), pregnancy rate (Cook 46.7% VS. Vitrolife 50.0%, P>0.05) and implantation rate (Cook 30.3% VS. Vitrolife 29.0%, P>0.05). CONCLUSIONS: Morphokinetics used for embryo selection are not affected by the two different culture media.

[Incidence and management of monozygotic twin conceived by assisted reproductive techniques].

OBJECTIVE: To analysis the incidence and management of monozygotic twin (MZT) conceived by assisted reproductive techniques (ART). METHODS: A retrospective analysis of clinical pregnancies and MZT that resulted from ART was performed in Reproductive Medical Center, the First Affiliated Hospital, Wenzhou Medical University between January 2011 and January 2014. RESULTS: A total of 5 908 pregnancies were diagnosed: 2 012 twins, 157 high-order multiple pregnancy (HOMP), including 4 quadruplets. Overall, 51 MZT pregnancies were identified of them including 32 cases HOMP and 19 cases MZT. The incidence of MZT resulting from cleavage-stage embryo transfer was similar to blastocyst transfer (P = 0.960). The percent of MZT resulting from in vitro fertilization [0.93% (28/3 022)], frozen-thawed embryo transfer [0.87% (13/1 502)] and intracytoplamic sperm injection [0.72% (10/1 384)] did not show statistical significance (P = 0.794). The expectantly managed MZT was associated with a significantly greater likelihood of miscarriage [6/19 vs 5.11% (101/1 976)], and low birth weight infant [73.91% (17/23) vs 42.89% (1 453/3 388), P < 0.01], when compared with dizygotic twin (DZT) did not undergo selective embryo reduction (SER). In monozygotic (MZ)-triplets with SER to 2 fetuses or to 1 fetus, there was no cases of preterm birth or low birth weight infant observed in MZ-triplets with SER to 1 fetus; when compared with MZ-triplets with SER to 2 fetuses, the low birth weight infant [56.00% (14/25), P = 0.021] has statistical significance. The likelihood of the survival of two babies was lower in MZ-triplets with SER to 2 fetuses when compared with non-MZ triplets with SER to 2 fetuses [42.86% (9/21) vs 75.21% (91/121), P = 0.003]. CONCLUSIONS: The incidence of MZT pregnancies following ART is high. It plays a significant role in the occurrence of HOMP. MZT pregnancies are at an increased risk of adverse outcomes, it should transform to a single embryo thansfer (SET) program to reduce them incideuce. Reduction of MZT contained in multiple pregnancies appears to be a safe option.

[Impact of male reproductive tract infection on semen quality].

OBJECTIVE: To investigate the association of male reproductive tract infection (RTI) with semen parameters and sperm DNA damage. METHODS: We classified 1 084 males attending the infertility clinic into an RTI group (n = 300) and a non-RTI control group (n = 784). According to the WHO standards, we obtained routine semen parameters, detected sperm morphology, and determined the sperm DNA fragmentation index (DFI) by sperm chromatin structure assay. RESULTS: There were statistically significant differences between the RTI and control groups in the semen volume ( [2.58 ± 1.20] vs [3.00 ± 2.10] ml), grade a + b sperm ([50.6 ± 17.2] vs [53.2 ± 15.8]%), grade d sperm ( [39. 8 ± 17.8] vs [36.5 ± 16.2]%), and total sperm count ([218.5 ± 185.0 ] vs [278.5 ± 375.5 ] x 10(6)/ejaculate) (all P < 0.05), but not in the males' age, sperm concentration or pH value (P > 0.05). The percentage of morphologically normal sperm was significantly lower ([3.46 ± 2.90] vs [4.61 ± 3.60%, P < 0.05) but the DFI was markedly higher in the RTI group than in the control ([19.4 ± 11.4] vs [15.2 ± 8.8]% , P < 0.01). The percentage of the cases with DFI > 30% was remarkably higher (13.0 vs 5.74% ) while that of the cases with DFI < 10% dramatically lower in the former than in the latter (16.0 vs 28.0%). The level of seminal plasma elastase was correlated negatively to sperm concentration, sperm count, and the percentage of morphologically normal sperm (P < 0.05) but positively to DFI and grade d sperm (P < 0.05 or P < 0.01). CONCLUSION: Male reproductive tract infection not only affects semen parameters and sperm morphology but also causes serious sperm DNA damage.

[Comparision of in vitro maturation applied in PCOS and non-PCOS patients undergo stimulated and unstimulated protocols].

OBJECTIVE: To compare the laboratory and clinical results between unstimulated in vitro maturation (IVM) and IVM converted from in vitro fertilization (IVF) in polycystic ovarian syndrome (PCOS) and non-PCOS patients. METHODS: We divided 591 IVM cycles in the First Affiliated Hospital of Wenzhou Medical Univesity from Jan. 2008 to Dec. 2013 into 4 groups: group A1B1, PCOS patients underwent unstimulated IVM protocol, 240 cycles; group A1B2, PCOS patients underwent IVM converted from conventional stimulated IVF protocol, 153 cycles; group A2B1, non-PCOS patients underwent unstimutlated IVM protocol, 103 cycles; group A2B2, non-PCOS patient underwent IVM converted from conventional stimulated IVF protocol, 95 cycles. Multiple linear regression method and binary logistic regression method were used to assess the influence of PCOS and protocols for IVM on laboratory and clinical outcomes. RESULTS: The mean number of oocytes retrieved was positively related with PCOS [partial regression coefficient (B) = 3.37, P < 0.01]. The maturation rate of oocytes was positively related with hCG-prime prior to oocyte aspiration (B = 0.05, P = 0.010). High-quality embryo rate was positively related with PCOS and IVM converted from IVF (B = 0.08, P = 0.010; B = 0.09, P = 0.001), as well as implantation rate related with them (B = 0.07, P = 0.010; B = 0.10, P < 0.01). PCOS and IVM converted from IVF improved hCG positive (hCG>10 U/L) rate (OR = 1.636, 95%CI: 1.113-2.204, P < 0.05; OR = 1.861, 95%CI: 1.307-2.649, P < 0.05) and the clinical pregnancy rate (OR = 1.507, 95%CI: 1.041-2.240, P < 0.05; OR = 1.881, 95%CI: 1.312-2.696, P < 0.05). IVM converted from IVF protocol decreased the spontaneous abortion rate (OR = 0.490, 95%CI: 0.245-0.978, P < 0.05). Multiple gestation rate and ectopic pregnancy rate were not affected by PCOS condition and protocol used (P > 0.05). CONCLUSIONS: PCOS and IVM converted from IVF protocol improved the high-quality embryo rate, implantation rate, hCG positive rate and clinical pregnancy rate. IVM converted from IVF protocol reduced the spontaneous abortion rate. PCOS patients may be more suitable for the IVM treatment. No matter PCOS or non-PCOS patients, IVM converted from IVF protocol had better pregnancy outcome than that of unstimulated cycle.

Reproductive outcomes in patients with high levels of sperm DNA fragmentation using testicular sperm for intracytoplasmic injection: a retrospective analysis.

This study aims to compare the embryological and clinical parameters of intracytoplasmic sperm injection (ICSI) cycles using testicular versus ejaculated sperm in male patients with elevated sperm DNA fragmentation (SDF). A total of 73 ICSI cycles were examined in couples where the male partner exhibited high levels of SDF. ICSI was performed using either ejaculated or testicular sperm. The primary outcomes were rates of blastocyst formation, high-quality embryo development, and clinical pregnancy. The DNA fragmentation index (DFI) for testicular sperm (16.81 ± 17.51) was significantly lower than that of ejaculated sperm (56.96 ± 17.56). While the blastocyst formation rate was significantly higher in the testicular sperm group compared to the ejaculated sperm group, no statistically significant differences were noted in fertilization rate (72.15% vs. 77.23%), rate of high-quality embryo formation (47.17% vs. 46.53%), clinical pregnancy (50% vs. 56.52%), Cumulative pregnancy (70.2% vs. 55.6%), or live birth rate (43.75% vs.43.48%). Testicular spermatozoa have no additional advantage over ejaculated spermatozoa except for blastocyst quality in patients with high SDF, the use of testicular spermatozoa for the first ICSI cycle in male infertility patients with high SDF should be undertaken after much consideration at present.

[Clinical study on in vitro maturation of immature oocytes transferred from conventional in vitro fertilization.].

OBJECTIVE: To investigate clinical effect of in vitro maturation (IVM) of immature oocytes transferred from conventional in vitro fertilization embryo transfer (IVF-ET) cycles. METHODS: From January 2008 to June 2009, medical documents of 155 infertile patients underwent IVF-ET in the Reproductive Medical Center of First Affiliated Hospital of Wenzhou Medical College were analyzed retrospectively. If more than 20 oocytes were monitored after 5 - 7 days of ovulation induction or follicular developmental retardation were confirmed after 8 - 13 days of ovulation induction, according to patients' wish, IVM were transferred in 60 cycles (group A). In the mean time, IVF was continued in 95 cycles (group B). The mean dosage of gonadotropin, the cancellation rate of cycles, the mean numbers of oocytes retrieved and maturation, the rate of fertilization and excellent quality embryos, pregnancy outcome and the incidence rate of ovarian hyperstimulation syndrome (OHSS) were compared and analyzed. RESULTS: The rates of embryo transfer were 92% (55/60) in group A and 63% (60/95) in group B, which showed significant differences (P < 0.05). In group A, the mean dosage of the gonadotropin, the mean number of oocytes retrieved, the cleavage rate and OHSS rate were (1030 +/- 468) U, 10 +/- 6, 82.2% (231/281) and 0, and were (1544 +/- 338) U, 14 +/- 4, 94.0% (502/534) and 35% (21/60) in group B, respectively, all data above exhibited statistical difference between two groups (P < 0.05). However, the rates of fertilization and excellent quality embryos had no significant differences between two groups (P > 0.05). In group A, the rate of clinical pregnancy per transfer was 53% (29/55) and multiple pregnancy was 14% (4/29), and were 47% (28/60) and 32% (9/28) in group B, they all had no significant differences (P > 0.05). CONCLUSION: IVM of immature oocytes used in conventional IVF cycles not only obtained a high clinical pregnancy rate, but also reduced gonadotropin using dosage and avoided OHSS completely.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

Simulated Microgravity Using a Rotary Culture System Compromises the In Vitro Development of Mouse Preantral Follicles.

BACKGROUND: Growing cells in simulated weightlessness condition might be a highly promising new technique to maintain or generate tissue constructs in a scaffold-free manner. There is limited evidence that microgravity condition may affect development of ovarian follicles. The objective of the present study was to investigate the effects of simulated microgravity on the in vitro development of mouse preantral follicles. METHODS AND RESULTS: Ovarian tissue from 14-day-old mice, or preantral follicles mechanically isolated from 14-day-old mouse ovaries were cultured at a simulated microgravity condition generated using a rotating wall vessel apparatus. Follicle survival was assessed quantitatively using H&E staining. Follicle diameter and oocyte diameter were measured under an inverted microscope. Ultrastructure of oocytes was evaluated using transmission electron microscopy. We observed that simulated microgravity compromised follicle survival in vitro, downregulated PCNA and GDF-9 expressions, and caused ultrastructural abnormalities in oocytes. CONCLUSION: This study showed for the first time that three-dimensional culture condition generated by simulated microgravity is detrimental to the initial stage development of mouse preantral follicles in vitro. The experimental setup provides a model to further investigate the mechanisms involved in the in vitro developmental processes of oocytes/granulosa cells under the microgravity condition.

Prediction of clinical pregnancy in vitrified-warmed single blastocyst transfer cycles by pre-freeze morphology.

BACKGROUND: The selection of blastocyst warmed for transfer is based on pre-freeze morphology in vitrified-warmed single blastocyst transfer cycles. But, it is controversial which parameter of blastocyst morphology most closely related to the clinical outcomes. OBJECTIVE: To estimate the effect of blastocoele expansion, trophectoderm (TE) morphology grade, and inner cell mass (ICM) morphology grade on clinical pregnancy in vitrified-warmed single blastocyst transfers. MATERIALS AND METHODS: There were 172 vitrified-warmed single blastocyst transfer cycles during the year 2012 included in this analysis. Comparison of clinical results between pregnancy and no pregnancy group based on patient and blastocyst morphology characteristics was done. Then stepwise logistic regression analysis was used to select the best morphological predictor for clinical pregnancy. Last, comparison of patient characteristics and clinical outcomes separated by the best independent morphological predictor was done. RESULTS: Comparison of clinical results between pregnancy and no pregnancy group and logistic regression showed the clinical pregnancy rate was affected by ICM. Comparison of patient characteristics separated by ICM grade, ICM grade A cycles got higher clinical pregnancy rate than ICM grade B cycles (54.3% vs. 35.0% respectively, p=0.037). CONCLUSION: Blastocyst with good ICM morphology could increase clinical pregnancy rate in vitrified-warmed single blastocyst transfer cycles.

Slow-controlled freezing versus speed-cooling for cryopreservation of whole guinea pig ovaries.

The objective of this study was to evaluate the feasibility of whole-ovary perfusion, and to compare the effects of speed-cooling and slow-controlled freezing of whole guinea pig ovaries. Slow-freezing and speed-cooling procedures were performed after perfusion of guinea pig ovaries with cryoprotectants. Ink perfused via the vascular pedicles was present in the microvessels around various follicles at various stages of development in the cortical and medullar regions, thereby confirming that perfusion was effective. Vascular damage was essentially confined to the cannulated artery. Based on histological examination, there were (mean ± SEM) 93.1 ± 4.2, 79.0 ± 2.0, and 54.7 ± 8.5% healthy follicles in the fresh, slow-freezing and speed-cooling groups, respectively (each group differed from the other two, P < 0.05). Trypan blue staining of isolated follicles confirmed that cellular damage was greater following speed-cooling than slow-freezing (58.6 vs 29.2%, P < 0.05). Based on a TUNEL assay, speed-cooling caused more apoptotic granulosa and theca cells in antral follicles than slow-freezing. In conclusion, the present study provided evidence that guinea pig whole ovaries could be perfused with cryoprotectant and cryopreserved in vitro. Furthermore, the slow-freezing protocol resulted in less cellular damage in thawed tissues than speed-cooling.