miR-188-3p-targeted regulation of ATG7 affects cell autophagy in patients with nonobstructive azoospermia.

BACKGROUND: Nonobstructive azoospermia (NOA) is one of the most difficult forms of male infertility to treat, and its pathogenesis is still unclear. miRNAs can regulate autophagy by affecting their target gene expression. Our previous study found that miR-188-3p expression in NOA patients was low. There are potential binding sites between the autophagy gene ATG7 and miR-188-3p. This study aimed to verify the binding site between miR-188-3p and ATG7 and whether miR-188-3p affects autophagy and participates in NOA by regulating ATG7 to influence the autophagy marker genes LC3 and Beclin-1. METHODS: Testicular tissue from 16 NOA patients and 16 patients with normal spermatogenesis and 5 cases in each group of pathological sections were collected. High-throughput sequencing was performed to detect mRNA expression differences. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemical staining and immunofluorescence were used to detect protein localization and expression. Autophagosome changes were detected by electron microscopy. The targeting relationship between miR-188-3p and ATG7 was confirmed by a luciferase assay. RESULTS: ATG7 protein was localized in the cytoplasm of spermatogenic cells at all levels, and the ATG7 gene (p = 0.019) and protein (p = 0.000) were more highly expressed in the NOA group. ATG7 expression after overexpression/inhibition of miR-188-3p was significantly lower (p = 0.029)/higher (p = 0.021) than in the control group. After overexpression of miR-188-3p, the ATG7 3'UTR-WT luciferase activity was impeded (p = 0.004), while the ATG7 3'UTR-MUT luciferase activity showed no significant difference (p = 0.46). LC3 (p = 0.023) and Beclin-1 (p = 0.041) expression in the NOA group was significantly higher. LC3 and Beclin-1 gene expression after miR-188-3p overexpression/inhibition was significantly lower (p = 0.010 and 0.024, respectively) and higher (p = 0.024 and 0.049, respectively). LC3 punctate aggregation in the cytoplasm decreased after overexpression of miR-188-3p, while the LC3 punctate aggregation in the miR-188-3p inhibitor group was higher. The number of autophagosomes in the miR-188-3p mimic group was lower than the number of autophagosomes in the mimic NC group. CONCLUSIONS: LC3 and Beclin-1 were more highly expressed in NOA testes and negatively correlated with the expression of miR-188-3p, suggesting that miR-188-3p may be involved in the process of autophagy in NOA. miR-188-3p may regulate its target gene ATG7 to participate in autophagy anDual luciferase experiment d affect the development of NOA.

Analysis of Related Causes for No Embryos Transferred and Corresponding Coping Measures in Assisted Reproductive Technology.

OBJECTIVE: To analyze the related causes for no embryos transferred in assisted reproductive technology (ART) in order to provide corresponding coping measures for infertile couples. STUDY DESIGN: The data of 607 couples who underwent ART and had no embryos transferred in our reproductive center between January 2010 and January 2014 were retrospectively analyzed. RESULTS: The cycles of no embryos transferred accounted for 3.99% (607/15,224) of total cycles. Of those, complete fertilization failure, oocyte retrieval failure, and complete abnormal fertilization accounted for 28.3% (172/607), 25.7% (156/607) and 22.24% (135/607), respectively. The incidence of complete abnormal fertilization was higher in IVF than in ICSI (p<0.05). In both IVF and ICSI cycles, the incidences of no embryos transferred were higher in the patients retrieving ≤3 oocytes than in the patients retrieving >3 oocytes (p<0.05). In IVF cycles the incidences of no embryos transferred were higher in the patients with primary infertility than in those with secondary infertility (p<0.05). CONCLUSION: The main causes of no embryos transferred are complete fertilization failure, oocyte retrieval failure, and complete abnormal fertilization. Retrieving adequate number of mature oocytes is the key to success of ART. Patients who experienced complete abnormal fertilization in IVF or the patients with primary infertility who experienced complete fertilization failure or normal fertilization without cleavage should receive ICSI in the next treatment.

Effects of Vitrification on Outcomes of In Vivo-Mature, In Vitro-Mature and Immature Human Oocytes.

BACKGROUND/AIMS: To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. METHODS: Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15), in vitro-mature group (group B, n = 88) and immature group (group C, n = 85), and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16), group B (n = 25) and group C (n = 25) for detecting DNA injury. RESULTS: After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P < 0.05), but there were no statistical differences in fertility rate, high-quality embryo rate, blastulation rate and aneuploidy rate amongst the three groups (all P > 0.05). Zona pellucida density (ZPD) was significantly lower in group A than in groups B and C both before and after vitrification (all P < 0.05). ZPD was significantly higher after thawing than before vitrification (all P < 0.05), but zona pellucida thickness (ZPT) was not significantly changed in all the three groups (all P > 0.05). Rate of comet cells was significantly lower in group A than in groups B and C (all P < 0.01). Comet tail was significantly longer in group C than in groups B and A (all P < 0.05). CONCLUSION: In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT.

Role of PAFAH1B1 in human spermatogenesis, fertilization and early embryonic development.

Spermatogenesis, fertilization and subsequent embryonic development are complex processes that require tight regulation. The PAFAH1B1 gene plays important roles in these reproductive events in mice, but its expression and roles in human reproduction have not been investigated. Expression analysis of testicular tissue by reverse transcription quantitative PCR and immunohistochemistry revealed varied expression levels among samples of different spermatogenic abilities (as assessed by the Johnsen score), with protein expression restricted to spermatogonia, spermatocytes and spermatids. Immunofluorescence on spermatozoa showed expression over the acrosome and midpiece regions of ejaculated samples, whereas a high proportion of percutaneous epididymal sperm aspiration-derived spermatozoa showed expression restricted to the midpiece. Analysis for PAFAH1B1 mRNA also revealed different expression levels among unfertilized oocytes, zygotes, cleavage stage embryos and blastocysts, with protein localized at the membrane level in oocytes and zygotes, and gradually distributing within the cytoplasm of cleavage stage embryos and blastocysts. Interestingly, microinjection of PAFAH1B1 siRNA into zygotes significantly (P = 0.024) increased fragmentation formation rates in subsequent embryonic development stages. Altogether, these are the first results to support a role for PAFAH1B1 in human spermatogenesis and early embryonic development.

YTHDF2 regulates MSS51 expression contributing to mitochondria dysfunction of granulosa cells in polycystic ovarian syndrome patients.

RESEARCH QUESTION: Granulosa cells (GCs) dysfunction plays a crucial role in the pathogenesis of polycystic ovary syndrome (PCOS). It is reported that YTH domain-containing family protein 2 (YTHDF2) is upregulated in mural GCs of PCOS patients. What effect does the differential expression of YTHDF2 have in PCOS patients? DESIGN: Mural GCs and cumulus GCs from 15 patients with PCOS and 15 ovulatory controls and 4 cases of pathological sections in each group were collected. Real-time PCR, Western Blot, immunohistochemistry, and immunofluorescence experiments were conducted to detect gene and protein expression. RNA immunoprecipitation assay was performed to evaluate the binding relationship between YTHDF2 and MSS51. Mitochondrial morphology, cellular ATP and ROS levels and glycolysis-related gene expression were detected after YTHDF2 overexpression or MSS51 inhibition. RESULTS: In the present study, we found that YTHDF2 was upregulated in GCs of PCOS patients while MSS51 was downregulated. YTHDF2 protein can bind to MSS51 mRNA and affect MSS51 expression. The reduction of MSS51 expression or the increase in YTHDF2 expression can lead to mitochondrial damage, reduced ATP levels, increased ROS levels and reduced expression of LDHA, PFKP and PKM. CONCLUSIONS: YTHDF2 may regulate the expression of MSS51, affecting the structure and function of mitochondria in GCs and interfering with cellular glycolysis, which may disturb the normal biological processes of GCs and follicle development in PCOS patients.

Effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection in mouse mature oocytes.

Better pregnancy outcomes can be obtained by human mature oocyte vitrification, but many problems remain to be resolved in human mature oocyte vitrification. Since mature oocyte development possesses its own maturity cycle, there should be the optimal timing for mature oocyte vitrification. The purpose of this study was to observe the effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection (ICSI) in vitrified mouse mature oocytes and explore its possible mechanism. Mouse oocytes were randomly divided into three groups according to different frozen timing including Groups A, B, and C in which oocytes were vitrified within 2 h after ovum pick-up, and 3-4 and 5-6 h after ovum pick-up, respectively. Spindle-related parameters were measured, ICSI was performed. The spindle occurrence rate of vitrified-thawed oocytes was 98.4% in Group A, 82.3% in Group B, and 75.8% in Group C, without statistical differences between pre-vitrification and post-thawing and among the three groups (P > 0.05). The angles between the polar body and spindle were larger after thawing than before vitrification (P < 0.01). The spindle retardance values were lower after thawing than before vitrification in Groups B and C (P < 0.05), but higher in Group A (P < 0.05). The spindle retardance values before vitrification were higher in Group B than in Groups A and C (P < 0.05), but the spindle retardance value, oocyte survival and two-cell rate after thawing were higher in Group A than in Groups B and C (P < 0.05). There were no statistical differences in ICSI fertility rate between the three groups (P > 0.05). The damage on the spindle is the slightest and embryo quality is the highest in the mouse oocytes vitrified within 2 h after ovum pick-up. The spindle retardance value is more valuable than the spindle occurrence rate in the evaluation of vitrified-thawed oocyte quality, and is positively correlated with embryo quality.

Effects of human cumulus cells on in vitro fertilization outcomes and its significance in short-term insemination.

OBJECTIVE: To observe the effects of cumulus cells on in vitro fertilization. STUDY DESIGN: Oocytes were retrieved from 47 patients (> 10/patient) who underwent short-term insemination from August 2009 to June 2010. The oocytes from each patient were divided into a cumulus cell-free group (cumulus cells were removed from the incubation medium 4 hours after coincubation of male and female gametes) with 389 oocytes and a cumulus cell group (cumulus cells were retained with the gametes until fertilization was evaluated 16-18 hours after co-incubation) with 402 oocytes. RESULTS: Polyspermic fertilization was 0.96 +/- 1.14 in the cumulus cell-free group and 0.47 +/- 0.72 in the cumulus cell group with p < 0.05. There were no significant differences in normal fertilization (5.96 g 1.73 vs. 6.55 +/- 3.72), 1PN fertilization (0.06 +/- 0.25 vs. 0.09 +/- 0.28), fertilization failure (1.34 +/- 1.17 vs. 1.45 +/- 1.84), cleavage (6.06 +/- 2.04 vs. 6.51 +/- 3.94), high-quality embryo (3.94 +/- 1.79 vs. 4.74 +/- 3.45) and usable embryo (5.06 +/- 1.86 vs. 5.68 +/- 3.98) between cumulus cell-free group and cumulus cell group, all with p > 0.05. CONCLUSION: In our study short-term insemination (4 hours) causes a statistical increase in polyspermic fertilization. In order to ensure correct oocyte fertilization and reduction of polyspermic fertilization, it is better to retain the cumulus cells for 16-18 hours.

Effect of modified single cell fixation method on cell-nuclear areas and fluorescence in-situ hybridization signals.

BACKGROUND: Fluorescence in situ hybridization (FISH) is an irreplaceable method in pre-implantation genetic diagnosis. We explored the effects of a modified single cell fixation method on the cell-nuclear area and FISH signal. METHODS: From January 2006 to March 2008, the blastomeres with marked nuclei from D3 embryos were selected. Cells were fixed with three different methods. The effects of the three methods on the cell-nuclear areas and FISH signals were then analyzed. RESULTS: The cell fixation rate was higher in conventional (Group B, 94.85%) and modified (Group C, 95.79%) Tween-20/HCl + methanol/glacial acetic acid methods than in the methanol/glacial acetic acid method (Group A, 86.73%) with p < 0.05. The complete signal rates in group A, B, and C were 95.3%, 93.5%, and 93.4%, respectively, with p > 0.05. The mean cell-nuclear areas in groups A, B, and C were 55.3, 46.2, and 49.5 microm3, respectively, with p < 0.05 in group A compared with group B or C, but with p > 0.05 between Group B and C. There was no significant difference in signal overlap and splitting rates between the three groups. CONCLUSIONS: Modified Tween-20/HCl + methanol/glacial acetic acid method fails to increase FISH signal overlap and splitting rates. It is simple and its fixation time is short. It can be widely used in clinical practice.

Day 3 embryo transfer may have better pregnancy outcomes in younger than 35-year-old patients with poor ovarian response.

OBJECTIVE: To explore the pregnancy outcomes of embryo transfer with D2 or D3 embryos in patients with poor ovarian response. METHODS: The pregnancy outcomes of 620 patients who had poor ovarian response and underwent the first in vitro fertilization-embryo transfer (IVF-ET) were retrospectively analyzed. Of the 620 cycles, all available fresh D2 embryos were used in 365 cycles (day 2 embryo transfer) and all available fresh D3 embryos were used in 255 cycles (day 3 embryo transfer) without superfluous embryos for freezing. RESULTS: There was a significant difference in clinical pregnancy rate between day 2 (32.73 %) and day 3 (50.83 %) embryo transfer in younger than 35-year-old patients, but no significant differences in implantation rate, live birth rate and spontaneous abortion rate (P > 0.05). There were similar pregnancy outcomes between day 2 and 3 embryo transfer in 35-year and older patients. CONCLUSION: D3 embryo transfer may have better pregnancy outcomes in younger than 35-year-old patients with poor ovarian response.

[Pregnancy outcomes of day-2 versus day-3 embryo transfer in poor responders].

OBJECTIVE: To investigate whether the day of embryo transfer (day 2 or day 3) affects clinical pregnancy outcomes in poor responder patients. METHODS: We retrospectively analyzed the pregnancy rates of 265 initial fresh cycles of in vitro fertilization-embryo transfer (IVF-ET), all transferred on day 2 (n = 169) or day 3 (n = 96) irrespective of quality because of an extremely low number of available embryos. RESULTS: Among the poor responders aged < 35 years, a higher rate of clinical pregnancy was achieved in the day-3 than in the day-2 group (50% vs 32.43% ; RR = 0.65, 95% CI: 0.43 - 0.99), and among those aged years, the two groups showed similar pregnancy outcomes. CONCLUSION: Shortening the time of embryo culture has no obvious benefit for the pregnancy outcome. For the poor responders under 35 years of age, day-3 embryo transfer may afford an even higher rate of clinical pregnancy.

Potential biomarkers for clinical outcomes of IVF cycles in women with/without PCOS: Searching with metabolomics.

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrinological and metabolic disorder which is the common cause of female infertility. The dysmetabolism displayed in it has not been completely ascertained. Metabonomics may shed light on understanding many small molecule endogenous metabolites and their associated metabolic pathways. Objective: To analyze the different metabolites and related metabolic pathways in follicular fluid and embryo culture fluid of PCOS and non-PCOS groups. Finding markers predictable for clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) treatment. Population and sample: 60 women who underwent IVF-ET were selected, including 30 with PCOS and 30 with the fallopian tubal issues only. We collected the first tube follicular fluid (FF) of all patients at the time of oocyte pick up and the waste embryo culture medium (ECM) after D3 high-quality embryo transplant. Methods: All samples were performed nontargeted Ultra High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-QE-MS) analysis. Related metabolic pathways were screened by KEGG annotation. To search potential indicators, the logistic regression was made combined with clinical data. Mean outcome measures: Predictive performance of markers of clinical outcomes (pregnancy rate, delivery rate, live birth rate, miscarriage rate) of assisted reproductive technology (ART). Results: Comparing the PCOS group against the non-PCOS group, we found 11 significantly different metabolites in the FF and 56 in the ECM. There are a total of 11 kinds of biomarkers associated with clinical outcomes. Androsterone sulfate, Glycerophosphocholine, and Elaidic carnitine seem robust to predict the abortion rate of the PCOS group, with an AUC of 0.941, 0.933, 0.933, respectively. The glycerol phospholipid metabolic pathway is enriched in both the follicular fluid and embryo culture fluid. Conclusions: The differential metabolites were mainly a variety of lipids. Some of them can predict clinical outcomes to a certain extent.

Effect of ambient air pollutants on in vitro fertilization-embryo transfer pregnancy outcome in Zhengzhou, China.

With the acceleration of China's urbanization and industrialization, air pollution has become a major environmental problem. Retrospective data analysis of 6564 patients who underwent IVF-ET in the center for reproductive medicine of the First Affiliated Hospital of Zhengzhou University from 2015 to 2020. Different stages were selected from 90 days before oocyte retrieval to 35 days after transfer and divided into five exposure periods. Multivariate logistic regression was used to analyze the relationship between six ambient air pollutants (PM2.5, PM10, NO2, SO2, CO and O3) and the IVF-ET pregnancy outcome. The results showed that air pollutants can significantly affect the IVF pregnancy outcome. The harmful effects of ambient air pollutants are more obvious in the patients aged < 35 years, single embryo transfer and cleavage stage embryo transfer.

[Importance of aneuploidy screening in preimplantation genetic diagnosis for the couples of chromosome translocation carriers].

OBJECTIVE: To determine the importance of aneuploidy screening in preimplantation genetic diagnosis for the couples of chromosome translocation carriers. METHODS: To perform 11 prenatal genetic diagnosis (PGD) cycles for 7 couples of chromosome translocation carriers from January 2006 to March 2009 in the Reproductive Medical Center, First Affiliated Hospital of Zhengzhou University. To re-analyze the well-fixed, non-multinuclear and non-debris nuclei using the probes of LSI 13, 18, 21, CEPX, CEPY to detect the aneuploidy rate which come from the PGD cycles of the couples of chromosome translocation carriers. The euploid embryo was defined as two fluorescence in situ hybridization (FISH) signals of LSI 13, 18, 21 respectively and two signals of CEPX, or one signal of CEPX and one signal of CEPY. The other abnormal signals were defined as aneuploid embryo. RESULTS: (1) A total of 130 nuclei from 11 PGD cycles got the integrated re-FISH signals. Nine hundred and thirty-seven FISH signals were analyzed, including 304 signals from 38 euploid nuclei and the others from 92 aneuploid nuclei. (2) The number of the aneuploid nuclei from grade I, II and III embryo was 20 (22%), 36 (39%), and 36 (39%). The number of the euploid nuclei from grade I, II and III embryo was 13(34%), 17 (45%), and 8 (21%). There was no significant difference of aneuploidy rate between the embryos form different grades (P > 0.05). However, the rate of aneuploid nucleus from good quality embryos (grade I + grade II) was 60% (59/92). (3) The euploidy rate was 71.4% (30/42) from balanced embryos, while 9.1% (8/88) from unbalanced embryos. There was significant difference between them (χ² = 53.4, P < 0.05). The rate of aneuploidy from balanced embryos was lower than those from unbalanced embryos (P < 0.05). CONCLUSIONS: Since higher rate of aneuploidy was detected in embryos of the couples of chromosome translocation carriers. It is advisable to recommend the FISH re-analysis for aneuploidy screening to preimplantation genetic diagnosis for the couples of chromosome translocation carriers.

Can Time-Lapse Incubation and Monitoring Be Beneficial to Assisted Reproduction Technology Outcomes? A Randomized Controlled Trial Using Day 3 Double Embryo Transfer.

Objective: To determine if the application of time-lapse incubation and monitoring can be beneficial to clinical outcomes in assisted reproductive technology. Methods: A total of 600 patients were equally randomized to three groups, namely, conventional embryo culture and standard morphological selection (CM group), time-lapse culture and standard morphological selection (TLM group), and time-lapse culture and morphokinetic selection (TLA group). Notably, 424 undergoing fresh autologous in vitro fertilization cycles were analyzed, 132 patients in the CM group, 158 in the TLM group, and 134 in the TLA group. Main outcomes included clinical outcomes, embryo development rates, and perinatal outcomes. Results: Clinical pregnancy rates in the time-lapse groups were significantly higher than in the CM group (CM 65.2% vs. TLM 77.2% vs. TLA 81.3%). Implantation rates and live birth rates were significantly higher for the TLA group (59.7 and 70.9%) compared with the CM group (47.7 and 56.1%) but not compared with the TLM group (55.4 and 67.1%). There was no statistical difference in miscarriage and ectopic pregnancy rates among the three groups. Overall, birth weight was significantly higher in the time-lapse groups (CM 2,731.7 ± 644.8 g vs. TLM 3,066.5 ± 595.4 g vs. TLA 2,967.4 ± 590.0 g). The birth height of newborns in the TLM group was significantly longer than that of the CM group and TLA group (CM 48.3± 4.4 cm vs. TLM 49.8± 2.3 cm vs. TLA 48.5± 2.7 cm). Conclusion: Time-lapse incubation and monitoring have a significant benefit on clinical pregnancy rates and on overall birth weights while morphokinetic analysis is not necessary. Clinical Trial Registration: [www.ClinicalTrials.gov], identifier [NCT02974517].

Different expression and localization of aquaporin 7 and aquaporin 9 in granulosa cells, oocytes, and embryos of patients with polycystic ovary syndrome and the negatively correlated relationship with insulin regulation.

OBJECTIVE: To investigate the expression of aquaporin 7 (AQP7) and aquaporin 9 (AQP9) in the granulosa cells of patients with polycystic ovary syndrome (PCOS) and healthy women and detect their localization in oocytes at the germinal vesicle (GV), metaphase I (MI), MII, embryo, and blastocyst stages and the in vitro response to insulin stimulation. DESIGN: Randomized, assessor-blinded study. SETTING: Reproductive medical center. PATIENT(S): A total of 40 women (aged 20-38 years) comprising 29 cases of primary infertility and 11 cases of secondary infertility, of whom 17 had an initial diagnosis of PCOS and three received a PCOS diagnosis after an infertility examination. INTERVENTION(S): Controlling different concentrations of insulin and different treatment times in cultures of normal human granulosa cells in vitro. MAIN OUTCOME MEASURE(S): Expression of AQP7 and AQP9 genes and proteins in granulosa cells detected by real-time quantitative polymerase chain reaction, and localization in oocytes at the GV, MI, MII, embryo, and blastocyst stages by Western blot, immunohistochemical, and immunofluorescence assays, and concentrations of insulin in follicular fluid by enzyme-linked immunosorbent assay. RESULT(S): The expression levels of the AQP7 mRNA and protein in the granulosa cells of patients with PCOS were higher than found in healthy controls. We found AQP7 protein expressed in human oocytes at GV, MI, MII, embryo, and blastocyst stages; it was mainly located in the nucleoplasm. In the PCOS group, the expression level of AQP9 mRNA and protein in granulosa cells was lower, and AQP9 protein was expressed in oocytes at the GV, MI, MII, embryo, and blastocyst stages; it was localized on the nuclear membrane. Compared with healthy women, the insulin expression in patients with PCOS was higher. In cultures of normal human granulosa cells in vitro, the expression of AQP7 and AQP9 mRNA and protein decreased with the increase in insulin concentration; expression statistically significantly decreased when the insulin concentration was 100 nmol/L, and after 6 to 24 hours of exposure the lowest expression levels were found at 12 hours. CONCLUSION(S): The different localization and expression of AQP7 and AQP9 between the two groups suggests that they might be involved in oocyte maturation and embryonic development through different regulatory pathways. The expression levels of AQP7 and AQP9 were negatively correlated with insulin regulation, suggesting that insulin might affect the maturation of PCOS follicles by changing AQP7 and AQP9 expression.

[Effects of cryopreservation time and thawing method of human oocyte vitrification on the outcome of assisted reproduction].

OBJECTIVE: To evaluate the effects on pregnancy outcome of freezing time from oocyte retrieval and thawing method for metaphaseII human oocytes vitrification. METHODS: From Mar 2007 to Mar 2009, the clinical outcome of 30 infertile women undergoing vitrified-thawing oocytes of in vitro fertilization-embryo transfer (IVF-ET) in the Reproductive Medical Center of the First Affiliated Hospital of Zhengzhou University was studied retrospectively, including 21 women with double fallopian tube obstruction and 9 women's husband azoospermia. All infertile women were divided into three groups, including 5 cases in group A (freezing between 4 and 5 hours from oocyte retrieval and conventional thawing method), 9 cases in group B (freezing within 2 hours from retrieval and conventional thawing method) and 16 cases in group C (freezing within 2 hours from retrieval and improved thawing method). The vitrified oocytes were preserved for 2 months to 1 year and thawed for Intracytoplasmic sperm injection (ICSI) and embryo transfer. The outcome of IVF and pregnancy were recorded. RESULTS: (1) The rates of oocyte survival was (65 ± 33)% in group B and (72 ± 23)% in group C and the rate of transfer cycle was 9/9 in group B and 16/16 in group C, which were all significantly higher than (16 ± 17)% of oocyte survival and 1/5 of transfer cycle in group A (P = 0.001, 0.021). However, the rate of oocyte survival and transfer cycle between group B and group C did not reach statistical difference (P > 0.05). The rate of implantation and clinical pregnancy of (33 ± 38)% and 9/16 in group C were significantly higher (4 ± 11)% and 1/9 in group B (P = 0.033, 0.040). (2) The mean age of women in group C were (28.6 ± 2.1) in oneself oocyte, (28.0 ± 4.6) in donor oocyte and (28.1 ± 3.4) in donor sperm. The rate of oocyte survival was (73 ± 25)%, (88 ± 10)% and (66 ± 25)%. The rate of fertilization rate was (84.6 ± 0.9)%, (79.3 ± 2.0)% and (82.8 ± 15.0)%. The rate of implantation was (20.0 ± 44.7)%, (33.0 ± 0.1)%, (41.6 ± 41.7)%. The rate of clinical pregnancy was 1/5 in oneself cycles, 3/3 in donor oocyte cycles, 5/8 banked donor sperm cycles in group C. All above clinical parameters were not statistically different (P > 0.05). (3) In group A, one women underwent IVF-ET and no clinical pregnancy was observed. One women pregnancy was terminated at two months in group B. The clinical pregnancies rate of group C was 9/16, late abortion occurred in 1 woman, the other 8 women underwent term pregnancy, including 5 male infants and 4 female infants. All of infants showed normal Karyotype. Live-birth rates per warmed oocyte was 5.9%(8/135). The mean gestational weeks and birth weight of the infants were (39.4 ± 0.9) weeks and (3574 ± 569) g, respectively. CONCLUSIONS: Embryo quality and clinical outcome of thawing cycles could be significantly improved when oocyte vitrification was performed within 2 hours from oocyte retrieval and improved thawing method.

[Clinical application of oocyte vitrification in failed testicular sperm extraction cycles: report of 8 cases].

OBJECTIVE: To evaluate the clinical application value of oocyte vitrification in failed testicular sperm extraction cycles in non-obstructive azoospermia (NOA) patients. METHODS: We retrospectively analyzed the clinical data of 8 women undergoing oocyte frozen-thawing cycles by vitrification because of failed testicular sperm extraction from their NOA husbands and no banked donor sperm on the day of oocyte retrieval. The oocytes were cryopreserved by vitrification with cryotop and thawed 2 months later. The surviving metaphase II (MII) oocytes were injected with the banked donor sperm of the same blood type as the husbands by intracytoplasmic sperm injection (ICSI) for fertilization. The rates of oocyte survival, fertilization, cleavage, good embryos and pregnancy were evaluated. RESULTS: Sixty oocytes were vitrified and 47 (78.3%) survived after thawing, of which 41 MII oocytes underwent ICSI and 33 (80.5%) of them were fertilized. The rates of cleavage and good embryos were 81.8% (27/33) and 59.3% (16/27) respectively. Fifteen of the embryos were transferred to the 8 patients, with 1.9 +/- 0.8 per cycle, of which 5 (33.3%) were confirmed by ultrasound to have been implanted and 5 resulted in clinical pregnancy (62.5%), all singleton without miscarriage. Three normal boys and 1 normal girl were already born, with the pregnancy time of (39 + 4 +/- 0.4) wk and newborn body weight of (3787.5 +/- 513.7) g, respectively. CONCLUSION: Vitrification of oocytes in failed testicular sperm extraction cycles is a promising technique for preserving female fertility, which, with ICSI of banked donor sperm, may result in satisfactory clinical outcomes.

[Application of sperm fluorescence in situ hybridization in preimplantation genetic diagnosis].

OBJECTIVE: To investigate the role of sperm fluorescence in situ hybridization (FISH) in preimplantation genetic diagnosis (PGD) for male chromosomal disorders. METHODS: From Jul. 2006 to Aug. 2008, FISH was performed in sperm and embryo of 9 infertile couples due to male chromosomal abnormality including 7 couples with Robertsonian translocation, one couple with reciprocal translocation and one couple with Klinefelter's syndrome. Correlation analysis was performed between sperm and embryo FISH results. RESULTS: (1) FISH analysis of 8568 sperms showed 24 sperms had no fluorescence signals. The rate of normal/balanced sperm of carriers were 85.71% (6045/7053)in seven Robertsonian translocation, 30.42% (306/1006) in one reciprocal translocation and 68.76% (350/509) in Klinefelter's syndrome. (2) A total of 158 embryos were biopsied, of which 135 embryos were successfully fixed for FISH. A hundred and one embryos exhibit informative signal including 36 normal/balanced embryos and 75 abnormal embryos. Twenty-one embryos were transferred and one couple obtained successful term pregnancy. The rate of normal/balanced embryo were 29.0% (31/107) in 7 carriers of Robertsonian translocation, 6.3% (1/16) in one reciprocal translocation and 33.3% (4/12) in Klinefelter's syndrome. (3) A positive correlated relationship was found between the percentage of normal embryo and the percentage of normal sperm (r = 0.75, P = 0.02). CONCLUSION: It is advisable to recommend the sperm FISH analysis for being routinely incorporated into the genetic screening offered prior to preimplantation genetic diagnosis.

Comparative study on the pregnancy outcomes of in vitro fertilization-embryo transfer between patients with different ovarian responses (a STROBE-compliant article).

There have been few studies on large-sample data of cleavage-stage embryo and blastocyst transfers. We compared the pregnancy outcomes of patients with different ovarian responses after the transfer of different numbers of embryos in different developmental stages.Patients were divided into 3 groups including low response group, medium response group, and high response group according to different ovarian responses. Patients in each group were further divided into 4 subgroups including group A: transfer of 1 D3 embryo, group B: transfer of 2 D3 embryos; group C: transfer of 1 D5 blastocyst; and group D: transfer of 2 D5 blastocysts.In low ovarian responders, the implantation rate, clinical pregnancy rate and live birth rate were significantly lower in the group A than in the groups B and C. In medium ovarian responders, the implantation rate was significantly higher, but the multiple pregnancy rate was significantly lower in the group C than in the group B. The multiple pregnancy rate was significantly higher in the group D than in the group C. In high ovarian responders, the implantation rate was significantly lower, but the multiple pregnancy rate was significantly higher in the group B than in group C.Based on the above results, the single blastocyst transfer is preferable for the patients with different ovarian responses.

Identification and characterization of intestine microRNAs and targets in red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in the innate immunity system of invertebrates, especially in the aspect of antivirus. In the present study, high-throughput small RNA Illumina sequencing systems were used to identify differentially expressed miRNAs (DEMs) from the intestines of Procambarus clarkii that were infected with white spot syndrome virus (WSSV). As a result, 39 known and 12 novel miRNAs were identified in both NG and WG small RNA libraries. Seven DEMs were determined to be involved in the antiviral innate immunity in the intestines of P. clarkii. The results of the target gene predictions of the DEMs showed that the putative target genes of these 7 DEMs are related to tight junctions, vascular smooth muscle contraction regulation of the actin cytoskeleton, focal adhesion, RNA transport, mRNA surveillance, viral carcinogenesis, and Salmonella infection. These results provide theoretical insights for future studies on the antiviral immunity of crustaceans.