Pregnancy outcomes of day 5 embryo transfer in patients at high risk of developing ovarian hyperstimulation syndrome and analysis of factors affecting blastocyst formation.

OBJECTIVES: To investigate the effects of day 5 embryo transfer (D5ET) compared with day 3 embryo transfer (D3ET) in patients at high risk of developing ovarian hyperstimulation syndrome (OHSS); to analyse factors affecting blastocyst formation. METHODS: Patients at high risk of developing OHSS underwent either D3ET or D5ET. RESULTS: A total of 253 patients received D3ET; 263 received D5ET. The number of embryos transferred was lower in the D5ET group than in the D3ET group. There were no between-group differences in pregnancy or live birth rates. Implantation rate was higher, and multifetation rate lower, in the D5ET group compared with the D3ET group. In addition, the incidence of moderate or severe OHSS was lower in the D5ET group than in the D3ET group. The woman's age, gonadotrophin dosage and insemination method were associated with the quality of blastocyst formation. CONCLUSIONS: In patients with a high risk of developing OHSS, compared with D3ET, D5ET decreased the multifetation rate and the incidence of moderate or severe OHSS, but did not affect the pregnancy or live birth rate. Women of a younger age, who have had an appropriate gonadotrophin dose and insemination by in vitro fertilization, are suitable candidates for blastocyst transfer.

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.

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.

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.

Effect of coincubation time of sperm-oocytes on fertilization, embryonic development, and subsequent pregnancy outcome.

Several studies have reported improved IVF by shortening the time of sperm-oocyte coincubation from 16-18 hours to 1-4 hours. The objective of this study was to examine the advantages and disadvantages of a shortened sperm-oocyte coincubation time in order to assess the effects of this insemination method for clinical IVF practice. Two insemination methods, the shortened method (4 hours) and the standard method (16-18 hours) of coincubation of sperm-oocytes for two groups of patients based on the quality of sperm were compared. Group I, was composed of couples without male factor; Group II, involved couples with mild male factor. Fertilization, good quality embryos, clinical pregnancy, and implantation rates were compared by two different insemination methods. In Group I, fertilization, clinical pregnancy, and implantation rates were not different between the two insemination methods. However, the polyspermy rate was significantly higher (P < 0.05) in the shortened (7.3%) than in the standard (4.1%) insemination method. In Group II, the fertilization rate was significantly lower (P < 0.05) using the shortened insemination method (62.6%) compared to the standard insemination method (68.7%). When fertilization failed with the shortened insemination method, the clinical pregnancy and implantation rates were 34.7% and 24.1%, respectively, from the rescue intracytoplasmic sperm injection (ICSI). The live birth rate from the rescue ICSI was 32.0% with normal infants. The duration of sperm-oocyte coincubation does not affect fertilization, embryo quality, clinical pregnancy, and implantation rates. However, fertilization rates will decrease with the shortened insemination method when the sperm parameters are poor. From the results of the present study we suggest that the combination of the shortened sperm-oocyte coincubation and rescue ICSI method may be an efficient method for IVF treatment in order to prevent fertilization failure when sperm parameters were poor as mild male factor.

[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.

Effects of cumulus cells on vitreous cryopreservation of human mature oocytes and clinical pregnancy outcomes.

The purpose of this study was to explore the effects of cumulus cells on vitreous cryopreservation of human mature oocytes and clinical pregnancy outcomes. The study was divided into group A (cumulus cells were removed from the oocytes before freezing) containing 24 participants and 193 oocytes and group B (cumulus cells were retained with the oocytes before freezing) containing 26 participants and 240 oocytes. Based on no significant differences in age, duration of infertility, infertile causes, and number of retrieved oocytes between both groups when oocytes were retrieved from infertile women, we found that the survival rate of post thaw oocytes (88% vs 58%), cleavage rate (80% vs 56%), and high-quality embryo rate (75% vs 59%) were significantly higher in group B than in group A. Under the conditions that there were no significant differences between the 2 groups in the general status of the participants undergoing embryo transfer, the embryo implantation rate (37% vs 15%) and the clinical pregnancy rate (50% vs 17%) were significantly higher in group B than in group A, all with Ps < .05. We conclude that the retention of cumulus cells can improve the developmental competence of vitrified-thawed human mature oocytes and clinical pregnancy outcomes.

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.

[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.

Clinical outcome of emergency egg vitrification for women when sperm extraction from the testicular tissues of the male partner is not successful.

The development of an effective oocyte cryopreservation system will have a significant impact on the clinical practice of reproductive medicine. However, the important option of emergency oocyte cryopreservation has yet to be well documented. In this report, we review the cases of 15 women with male partners who were diagnosed with nonobstructive azoospermia and for whom testicular sperm extraction on the day of oocyte retrieval failed. Emergency oocyte vitrification was performed and after two months, the vitrified oocytes were warmed and the surviving oocytes inseminated with frozen-thawed donor sperm by intracytoplasmic sperm injection (ICSI). A total of 117 mature oocytes from the 15 women were vitrified and warmed. The post-warming survival rate was 84.6% (99/117), and the fertilization rate following ICSI was 83.8% (83/99). We selected 30 embryos for transfer to 15 patients, 8 of whom became pregnant. The clinical pregnancy rate was 53.3% (8/15) and the implantation rate was 30.0% (9/30). Nine healthy live births resulted from 8 pregnancies. These results indicate that emergency oocyte vitrification is an effective rescue technique that can be applied clinically with acceptable pregnancy and live birth rates when testicular sperm extraction from the male partner failed on the day of oocyte retrieval. These results also highlight another important option for oocyte cryopreservation through the use of vitrification technology.

[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.

Effects of the copper intrauterine device on the expression of cyclooxygenase-1 and -2 in the endometrium.

OBJECTIVE: To examine the expression levels of cyclooxygenase (COX)-1 and COX-2 in the endometrium before and after insertion of the copper intrauterine device (Cu-IUD). METHODS: Ten patients were investigated. Two endometrial biopsies were taken from the uterus of each patient. The first biopsy was taken prior to insertion of the Cu-IUD, and the second was taken 1 month after insertion on the same day of the menstrual cycle and from the same location. The levels of COX-1 and COX-2 mRNA and protein in the endometrium were determined using reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: Before insertion, expression of COX-2 mRNA and proteins was 0.399+/-0.014 and 14.75+/-1.31, respectively. Post insertion, expression of COX-2 mRNA and proteins was 0.563+/-0.041 and 18.61+/-1.93, respectively. A significant increase (P<0.05) of COX between pre and post insertion of the Cu-IUD was only seen with COX-2. There was no significant change in the level of COX-1 mRNA or proteins before and after insertion of the Cu-IUD. CONCLUSION: COX-2 is the primary isoenzyme stimulating overproduction of prostaglandins in the endometrium after the insertion of Cu-IUDs.

[Effects of intrauterine contraceptive device on expression of vascular endothelial growth factor, kinase insert domain-containing receptor and microvessel density in endometrium].

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) and its receptor, kinase insert domain-containing receptor(KDR) and microvessel density (MVD) in endometrium from women wearing fixed copper-intrauterine contraceptive device (IUD, FCu-IUD) or fixed indomethacin-releasing copper-IUD (FICu-IUD). METHODS: Twenty healthy women were divided into two study groups: 10 cases wearing the FCu-IUD (FCu-IUD group), 10 cases wearing the FICu-IUD (FICu-IUD group). Immunohistochemical technique was used to determine the expression of VEGF and KDR in endometrium, and the microvessel density (MVD) was counted. The expression of VEGF mRNA was determined by in situ-hybridization. RESULTS: Before insertion of FCu-IUD, the expression of VEGF and KDR proteins was 0.357 +/- 0.032 and 0.215 +/- 0.029 respectively. After insertion of FCu-IUD, the expression of VEGF and KDR proteins was 0.568 +/- 0.027 and 0.244 +/- 0.022 respectively, significantly higher than before insertion (P < 0.05). The expression of VEGF mRNA was 0.359 +/- 0.022 before insertion of FCu-IUD, after insertion of FCu-IUD, the expression of VEGF mRNA was 0.425 +/- 0.019 (P < 0.05). There were no significant changes in the level of VEGF protein and mRNA, as well as KDR in endometrium before and after insertion of FICu-IUD. Compared with before insertion of FCu-IUD 15.4 +/- 2.8, a significant increase in MVD was observed after insertion of FCu-IUD 19.8 +/- 4.8, and the expression of VEGF protein was positively correlated with MVD (r = 0.847, P < 0.01). MVD counts were not different significantly before and after insertion of FICu-IUD. CONCLUSIONS: FCu-IUD can enhance the expression of VEGF and KDR in the endometrium. FICu-IUD can inhibit the activity of VEGF and KDR by releasing indomethacin. VEGF and KDR may be related to the structural and functional changes of microvessels in endometrium after insertion of FCu-IUD or FICu-IUD.