J Assist Reprod Genet (2013) 30:161–195 DOI 10.1007/s10815-013-9940-1

Abstracts

Published online: 5 February 2013 # Springer Science+Business Media New York 2013

Natural Cycle IVF and Mild Stimulation

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A new concept: the oocyte to baby rate, to evaluate the biological efficiency of in vitro fertilization

Timing of oocyte retrieval in natural cycles LB Cai, XQ Qian, T Feng, ZJ Yan, L Ma, JY Liu Yan Gao Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China; E-mail: [email protected]

State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; E-mail: [email protected]

Objectives: To investigate factors related to pre-ovaluation in natural cycle, and decide a proper timing for oocyte retrieval. Methods: The patient basal background including basal blood oestradiol (E2), FSH and LH concentrations, together with follicles diameters were tested on menstrual cycleday 3; Diameters of follicles were confirmed by ultrasonography on day 8 to day 11; And blood LH, E2 and progesterone (P) were taken every morning when follicule diameter ≥14 mm; Maturation was triggered using 0.2 mg of GnRHa intramuscular injection at 20:30 the day of follicule diameter ≥15 mm as well as E2 concentration at 1100 pmol/L, and the oocytes were retrieved after 34–36 h. Results: Pre-ovulation has a relationship with basal E2 concentration, P and LH concentration on HCG day, rather than basal LH or FSH concentration or follicle diameter. Conclusions: Timing of oocyte retrieval should be taken according to patient’s basal E2 concentration, P and LH level and her regular ovulation pattern.

Objectives: As a new relevant standard of success in assisted reproduction, the oocyte to live babies born (LBB) in relation to the number of oocytes retrieved and used can assess the real biological efficiency of assisted reproduction technology (ART). Methods: Retrospective analysis of the live birth rate in relation to total number of oocytes collected. In patients with remained frozen embryo, The final LBB was estimated. According age, the patients divided into three groups (group1, <35 years, group 2, 35∼39 years and group3, ≥40 years). Two types of analyses were carried out; first, we assessed the overall oocyte to baby rate for each age group, and then the rate according to the oocyte yield within each age group. Results: A total of 1,865 oocyte retrievals cycles yielded 22,861 oocytes. The total number of transferred and frozen embryos was 7,652 (a utilization rate of 33.4 %). The overall oocyte to LBB rate was 5.2 % (1183 babies born). When analyzed by groups, there was no increase in oocyte to LBB rate with increased of the number of oocytes retrieved. Conclusions: During ART cycles, only approximately 5 % of fresh oocytes produce a baby. The time has come to

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investigate new methods of oocyte viability assessment and consider changing current ART practice to recruit fewer oocytes. Mild ovarian stimulation maybe consider as a valid alternatives protocol to the current ones. A-3 Impact of basal FSH/LH ratio on the outcome of IVF/ICSI Y Meng, Y Mao, W Wang, Y Cui, JY Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, China; Email: [email protected] Objectives: To investigate the predictive significance of basal FSH/LH ratio on the outcomes of IVF/ICSI in infertile couples. Methods: 2167 stimulating IVF/ICSI cycles from Jan. 2008 to Dec. 2008 were studied retrospectively, due to male factor, fallopian tube factors, endometriosis or unexplained infertility, whose average age was under 40. Based on bFSH level, all the patients and cycles were divided into following two groups: normal ovarian function (bFSH≤8.5U/L) and abnormal ovarian function (bFSH > 8.5 u/L), then each group, in turn, was divided into quartiles according to age (≤29, 30∼34 and 35∼40 years-old). Results: Women younger than 29 years old with FSH/LH≤1 showed significantly lower dosage of gonadotrophins than those in FSH/LH ratio > 1; Regardless of age, the number of oocytes retrieved was significantly more in the women with normal ovarian function in FSH/LH≤1 than those in FSH/LH >1, moreover at ages younger than 35 years old, patients with higher FSH/LH ratio showed significantly fewer number of retrieved oocytes than women with lower FSH/LH ratio, while for female with abnormal ovarian function, it can be only found in groups of age 30–34; and the same trend on oocyte at retrieval was also found in the other groups. Conclusions: The basal FSH/LH ratio may be a good predictor of ovarian response while selecting proper stimulation protocol and dosage of start. But we cannot predict the outcome of IVF/ICSI only by basal FSH/LH ratio. A-4 Is modified natural cycle IVF an option for patients with poor ovarian reserve? Nalini Mahajan, Shalu Gupta, Shivani Singh Mother and Child Hospital, New Delhi, India; E-mail: [email protected]

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Objectives: Do Modified Natural Cycle (MNC)-IVF justify the procedure being offered to women with POR who were not ready to accept donor eggs. Methods: 21 women in the age group of 25–44 years getting regular periods, who responded poorly in previous IVF cycles and were not willing for donor oocytes, were offered MNC-IVF. Follicular monitoring was started from cycle day 6–8. GnRH antagonist and 75 IU of urinary gonadotrophin was started when the follicle size reached 12–14 mm and ovulation trigger was given when follicle reached 18 mm. OPU was scheduled 34 h post trigger. ICSI was performed on all MII oocytes and Day 2 ET was done. Luteal phase support was given with oral Dydrogesterone 10 mg twice a day and injection of HCG 2000iu, 2 and 5 days post ET. UPT and Serum β HCG was done 14 days after ET. Results: 21 women, with average age of 35.6±4.4 years, went through 35 cycles of MNC-IVF. Average AMH, AFC and FSH were 0.8±0.6 ng/ml, 3.3±1.3 and 11.1±6.7 IU/L respectively. Six (17.1 %) out of 35 cycles were cancelled due to premature ovulation. 29 (82.8 %) cycles reached the OPU stage. Only in one cycle, no oocyte could be retrieved. Average gonadotrophin ampoules used per cycle were 4.06± 0.93 and women needed average 5.06±0.93 days of antagonist. With the fertilization rate of 78.1 %, 21 (60 %) cycles reached embryo transfer stage. Number of oocytes retrieved per pick up was 1.17±0.46 and embryo transfer per pick up was 75 %. Six women had Bhcg>50iu/l on 14th day post ET, making the biochemical pregnancy rate 17.1 % per cycle and 28.6 % per women enrolled. Ongoing pregnancy rate was 11.4 % per cycle and 19 % per women. Conclusions: MNC-IVF can definitely be offered to women with previously failed conventional IVF cycles due to poor response, who are averse to going in for donor oocytes. A-5 Biological efficiency of natural cycle IVF is higher than controlled ovarian stimulation IVF Markus Nitzschke IMI Instituto Mexicano de Infertilidad, Mexico; E-mail: [email protected] Objectives: In order to measure the real biological efficiency of ART cycles, the oocyte to clinical pregnancy rate (OCPR) can be calculated, where clinical pregnancies are reported in relation to the number of oocytes retrieved and used. Many authors argue that compared to controlled ovarian stimulation IVF (COSIVF), the efficiency of Natural Cycle IVF (NC-IVF) is rather low. Methods: We would like to prove the opposite. In 2010, 78 patients with 115 cycles were scheduled for NCIVF, defined

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in this study as IVF/ICSI only using NSAIDs to control premature ovulation and induce ovulation with HCG or GnRH-agonists. We calculated the biological efficiency of NC-IVF using the OCPR, comparing our data with recently published data about the OCPR in COS-IVF (Patrizio and Sakkas, 2010). Results: Patients were 22–39 years of age (average 33.7± 3.7). Overall cancellation rate due to premature ovulation was 17.4 % (20/115). In 75.8 % (72/95) of aspirations, oocytes were retrieved. 83.3 % (60/72) of oocytes were mature and 80.0 % (48/60) of oocytes were successfully fertilized by ICSI. In 83.3 % (40/48) of the cases an embryo was transferred 2–4 days after aspiration. Transfer rate per aspiration was 42.1 % (40/95). Clinical pregnancy rate/transfer was 30.0 % (12/40). The overall OCPR was 16.6 %. Patrizio and Sakkas reported an OCPR of 3.8 % in their best group consisting of oocyte donors (all < 32 years). Conclusions: Even though a direct comparison is not possible as different studies cannot directly be compared, the great difference in the OCPR suggests a higher biological efficiency in NC-IVF compared with COS-IVF.

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Results: Because the data distribution was characterized by skewness of the distribution, the LH level was transformed as logarithm (log (LH)), and after that, the data was normal distribution. The serum LH levels on each time point were listed in table 1, and the value is represented as median (upper quartile, low quartile). The LH level on the day of hCG was significantly higher in letrozole group and low dose Gn group than in other three groups. Conclusions: Among these different mild stimulation protocols, the serum LH dynamics were totally different. Although the Day 5 LH level in CC cycles was higher than in LE cycles, the LH levels on the day before and of hCG administration was significantly lower than LE cycles, and premature LH surge was very rare in CC cycles. Using GnRH antagonist can suppress LH level significantly. Using LH level on stimulation Day 5 can predict the occurrence of premature LH surge. A-7 Luteal-phase ovarian stimulation in IVF: a new sight of eliminating premature LH surge and OHSS

A-6 The variation of circulating LH level in mild ovarian stimulation Rui Huang, Xiaoyan Liang, Cong Fang, Yu Deng, Chaomin Yue The Sixth Affiliated Hospital of Zhongshan University, Guangzhou, China; E-mail: [email protected] Objectives: Monitoring serum LH level is a main trend in IVF treatment, especially in mild stimulation because no GnRH agonist was utilized and premature LH surge might occur abruptly and unexpectedly. This study is to evaluate the serum LH level variation in different mild ovarian stimulation protocols and to predict LH surge in advance. Methods: Eight Hundred nine mild stimulation IVF cycles in patients with diminished ovarian reserve or repeated IVF failure from Jun. 2010 to Aug. 2012 were included, among which clomiphene citrtate (plus gonadotropin in some cases) was utilized in 279 cycles; letrozole in 29 cycles, low-dose gonadotropin (no more than 150 IU/d, started from cycle day 5) in 216 cycles, GnRH antagonist coadministrated in the other 285 cycles. The Basal LH level, serum LH levels on the Day 5 of stimulation, the day before hCG administration, and the day of hCG administration were compared among these groups and in GnRH antagonist co-administrated cycles, LH levels on the day of antagonist administration were also analyzed.

Yanping Kuang, Qingqing Hong, Qiuju Chen, QifengLyu, Ai Ai, Yonglun Fu Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, P.R. of China; E-mail: [email protected] Objectives: Ovarian stimulation, usually carried out with exogenous gonadotropins for in vitro fertilization-embryo transfer (IVF-ET), sometimes elicits a premature LH rise during the mid-follicular phase of stimulation cycles and results into compromised pregnancy outcomes. The precaution of premature LH rise makes the stimulation complex and costly. The existing healthy antral follicles in the luteal phase provide the possibility of performing ovarian stimulation during the luteal phase but it is not identified in the routine settings. We assessed the feasibility of initiating ovarian stimulation in the luteal phase with human menopausal gonadotrophin (HMG) and letrozole in women undergoing IVF/ICSI treatment. Methods: A cohort trial was conducted in infertile women undergoing IVF/ICSI treatment. Ovarian stimulation was initiated with HMG 225 IU and letrozole 2.5 mg every day after spontaneous ovulation. Ovulation was induced with GnRH agonist 100 μg. The top-quality embryos were cryopreserved for a later transfer. The primary outcome was the number of oocyte retrieved. Secondary outcomes were the pregnancy rate and implantation rate in frozen-thawed embryo transfers (FET).

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Results: Of 242 women enrolled, all participants succeeded to obtain oocytes and 227 women had top-quality embryos to cryopreserve. The total number of oocytes retrieved was 13.1, producing top-quality embryos 4.8. The clinical pregnancy rate and implantation rate were 57.26 % (71/124) and 42.55 % (100/235). Moreover, no case occurred the premature LH surge. No case complained of signs of moderate/severe ovarian hyperstimulation syndrome (OHSS) during the stimulation cycles. Conclusions: Luteal-phase ovarian stimulation is feasible to produce competent oocytes/embryos, with optimal pregnancy outcome in FET cycles. More important, it uncovers a new sight of eliminating premature LH surge and OHSS and may challenge current IVF practices. A-8 The pregnancy outcomes in intrauterine insemination treatment with Gonadotrophin low-dose step-up protocols JY Liu, W Wang, JY Liu Center of Clinical Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing, China; Email: [email protected] Objectives: To identify baseline predictors of ovarian responses in Gonadotrophin low-dose step-up protocols. Methods: The clinical outcomes were analysed in intrauterine insemination cycles with Gonadotrophin low-dose step-up protocols to identify baseline predictors of ovarian responses. Results: 1) The pregnancy rates, twins rates and abortion rates were 8.1 %, 9.09 % and 18.18 % respectively. Only one OHSS case happened and no multiple in all cases. 2) There was statistically significant in weight, BMI, antral follicle count and Gonadotrophin amount between IUI and poor response groups. Conclusions: The pregnancy outcomes in intrauterine insemination treatment with Gonadotrophin low-dose step-up protocols is better for the anovulatory women are resistant to other therapy and maybe affected by prediction parameters, including weight, BMI, antral follicle count etc.

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Objectives: We aimed to compare between the ovulation induction effect of some medications in terms of stimulation time, and number of maturing follicles for Intrauterine Insemination (IUI) treatment of a cohort of Saudi infertile patients. Methods: During a 24-month period, 301 consecutive Saudi women with infertility underwent IUI in an infertility clinic at King Saud University, in Saudi Arabia. The major clinical outcome was pregnancy rate (PR), which was correlated with the type of ovulation stimulation medication used and the number and size of maturing follicles at the insemination time. Results: The human-derived menopausal gonadotrophins (HMG), Merional, and the recombinant ovulation induction hormone (rFSH), Puregon needed comparable time before the follicles are ready for IUI. Pregnancy rate of Puregon was about double that of Merional, 13.28 % and 7.14 % respectively, though the difference did reach significance (P value: 0.158). The best pregnancy results occurred when the follicles took about 12–13 days, but the difference was not significant yet (P value: 0.129). As far as follicles size and number on the hCG day, three follicles of at least 15-mm diameter had better results that one or two, though the difference is not significant (P value: 0.673). However, three follicles of at least 18-mm diameter had a significantly better pregnancy rate, 42.86 %, in comparison to 1 or 2 follicles of at least the same size, 9.87 % and 7.5 % respectively (Pvalue of 0.02). Conclusions: Merional and Puregon have comparable stimulation and PR results in IUI treatment, though Puregon is slightly better. The best PR occurred when the follicles matured within 12–13 days, about the same as in natural cycles. PR in case of three maturing follicles on the hCG day is better than 1 or 2, especially when the diameter is at least 18 mm.

In Vitro Maturation (IVM) A-10 Natural Cycle IVF combined with IVM

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Jin-Ho Lim

Stimulation Medication and patient response as prognostic factors in IUI-treated infertile Saudi patients

Maria Fertility Hospital, Seoul, Korea; E-mail: lim@ mariababy.com

Ahmed M. Isa

Objectives: IVM has been developed to prevent the side effects of ovarian hyperstimulation syndrome. The efficiency and safety of IVM treatment as one of infertility treatments has been already proven. The clinical pregnancy rate of IVM reached 35–40 %/ET, and more than 3000 healthy

King Saud University, College of Medicine, OB GYNE Department, Assisted Conception Unit, Saudi Arabia; E-mail: [email protected]

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IVM babies have been born in the world. However, the main indication of IVM was PCOS patients with irregular anovulatory cycles. To expand the indication of IVM for patients with regular ovulatory cycles, we designed the new procedure what we like to call ‘Natural cycle IVF combined with IVM (Natural IVF/M)’. Methods: We check baseline ultrasound on MCD #2–3. If the number of AFC is more than 7–8, we recommend Natural IVF/M for patients with regular cycles. When the leading follicle reaches 12–14 mm in diameter, we give 10,000 IU of HCG and collect the oocytes 36–38 h later. The matured oocytes collected at the time of OPU are inseminated by ICSI on the same day, and the immature oocytes are cultured in vitro for 24–48 h. In vitro matured oocytes are then inseminated by ICSI subsequently. On day 3 or 4 after OPU, the best 1–3 embryos are selected for transfer. Results and Conclusions: We could confirm that the Natural IVF/M together with IVM are an efficient treatment especially for younger patients with acceptable clinical pregnancy rate and we could do embryo transfer in almost every case. A-11 Mitochondria enhancement and transfer in order to improve maturation competence of oocytes Yoshiharu Morimoto IVF Osaka, Japan; E-mail: [email protected] Objectives: IVM technique has been developed and spread to the world. However, the maturation rate of oocytes remains low. Enhancement of mitochondrial function may contribute to solve the issue. Our strategy is to improve the mitochondrial function by reduction of reactive oxygen species (ROS) in the whole body and enhance the improvement of mitochondrial function in oocytes by mitochondrial transfer. Methods: Mitochondrial dynamics was investigated using transmission electron microscope, confocal laser scanning microscope, Cell Voyager CV 1000 (Time-lapse florescence confocal laser scanning microscope) and scanning electrochemical microscopy. Results: By our ultra-structural observation of oocytes in maturation process during IVM procedure, mitochondria have shown their unique appearances. In the germinal vesicle stage oocyte, mitochondria were dense and scattered in cytoplasm. In the second metaphase stage, mitochondria increased in number and aggregated in the center of cytoplasm. The appearance of mitochondrial distribution changed dynamically at each stage of maturation.

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Conclusions: Mitochondrial dynamics such as amount, distribution, CCO activity and relation with cytoskeletons as a basic information for mitochondrial transfer has been clarified in human oocytes. For the next step, we need to decide timing in maturation process, position and donor organs for mitochondrial transfer. Enhancement and improvement of mitochondrial function in the whole body and oocytes would open the new window not only in clinical IVM procedure, but for cases with poor quality oocytes and aging. A-12 Natural cycle IVM/IVF: A Mexican experience Pablo Díaz Spíndola IECH Fertility Center, Monterrey, Mexico; E-mail: pablo. [email protected] Objectives: In Mexico, all infertility treatments are self-paid by the patients. There is not private or public insurance (social security) that can be used to treat infertility issues. This same thing occurs in most Latin American countries. In 2010, the INEGI (Mexican National statistics office) reported that one out of six couples has an infertility problem, and 20 % of these couples will require a complex treatment such as IVF, to solve their infertility issue. In a country with a population of more than 118 millions a total of 500,000 couples require IVF as their ideal treatment. However, accordingly to RED LARA in 2009 only 4588 IVF cycles were performed in 25registered fertility centers in Mexico. Furthermore, only 42 cycles per one million couples were performed in Mexico. The main reason for this lack of treatment is basically economic, since most of the couples are unable to pay for these treatments. Methods: Since 2011 our fertility center offers Natural cycles combined with IVM/IVF. During our initial cases, Dr. Ri Chen-Chian and Dr. Pasquale Patrizio supervised our embryo lab and clinical practice, respectively. Results: Even though, our fertility center has had only 29 cases since then, a reported 75 % reduction in the cost of conventional IVF was achieved. Furthermore, in 2012 patients with PCOS (or with other anovulatory disorder) were also included in the protocol using rFSH priming with mixed results (more definite results are still pending). Conclusions: Even though this protocol has an enormous potential in our country, it requires greater disclosure of their benefits. Besides the low costs (due to friendlier stimulation protocols), less complications (such as an ovarian hyperstimulation syndrome) and ethical dilemmas (embryo freezing) are seen.

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A-13 Comparison of the application for two kinds of in-vitro matured culture media Ting Feng Human Clinical Reproductive Medicine Center, the First Affiliated Hospital of Nanjing Medical University, China; E-mail: [email protected] Objectives: The application was compared between the self-made medium and commercial medium in order to provide the basis for the optimization of the in-vitro oocyte matured system. Methods: Using the cycles of ICSI in corresponding period as the controls (C1 and C2), 110 and 108 clinical cycles of IVM using the self-made medium (A) and the commercial medium (B) were collected, respectively, and the differences between A and B were compared in in-vitro matured, embryo development, embryo implantation, clinical pregnancy and outcomes. Results: In the situation of non-difference between the two control group, the in-vitro mature and embryo development in A were better than those in B, and the rate of transplanted embryo as well as the high-quality embryo showed the significant difference (P<0.05 or P<0.01). Although the rate of the embryo implantation, pregnancy and live birth in A were lower than those in B, actually, the difference were not significant due to the fact that the index in C2 (2006 or later) was also better than C1, which were associated with the improvement of the clinical therapy scheme and the use of hysteroscope as well. The abortion in A was lowest among all the groups (10.53 %), there was significant difference between the A and B (P<0.05) while the rate in C2 (the control for B) was obviously lower than that in C1 (the control for A), which suggested that the embryo cultured from the self-made medium might reduce the abortion. Conclusions: The application of the self-made IVM medium showed better effect which might be associated with the additional adding the mHFF and E2. The system needs to be further optimized in order to ensure it should be effective, safe and cheap.

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Objectives: Poor ovarian response to gonadotropin stimulation is not rare and is a key factor to reduce pregnancy rate (PR). Advanced age, previous ovarian surgery, low AMH and elevation of day3 FSH are related to poor ovarian response. Numerous strategies have been suggested to improve outcome of poor responders, and growth hormone administration is reported to achieve more oocytes and increase fertilization rate. Our study is to analyze pregnancy outcome of co-treatment with growth hormone in poor responders of IVF. Methods: In order to verify the effects of co-treatment of GH during COS in poor responders undergoing IVF/ET, we conducted a retrospective study with GH co-treatment in IVF (group A, n=94) matched with non-GH control (group B, n=90). Results: The E2 level on hCG day (pg/ml), number of oocyte retrieved, number of embryo obtained was significantly higher in group A than group B, respectively. Moreover, the pregnancy rate was also significantly higher in group A than group B (31.9 % vs. 16.7 %, p<0.05). Further analysis of poor responders with age <40, we observed the beneficial effect of GH co-treatment group was significant increased in pregnancy rate (37.7 % vs. 20.3 %, p<0.05) and implantation rate (15.1 % vs. 11.9 %, p<0.05) compared to GH (−) group, respectively. However, this advantage was not shown in poor responders with age >40. Conclusions: This study demonstrated in IVF/ET of poor responders, E2 level, the number of oocyte retrieved, number of good quality embryo obtained as well as PR were satisfactory achieved in GH co-treatment cycles. GH may improve the PR at least in poor responders with age <40.GH may enhance the oocyte quality by accelerating and coordinating cytoplasmic and nuclear maturation. The enhancement of endometrial receptivity by GH awaits further investigation. A-15 Moderate IVF treatment for advanced reproductive age women Peter S. Uzelac

Poor Responders A-14 The use of growth hormone (GH) in poor responders Chii-Ruey Tzeng Center for Reproductive Medicine & Sciences, Taipei Medical University Hospital, Taipei, Taiwan; E-mail: [email protected]

Marin Fertility Center, Greenbrae, CA, USA; E-mail: [email protected] Objectives: A growing body of literature suggests that, among some infertility patient populations, mild approaches in assisted reproductive technology (ART) may result in similar outcomes when compared to those utilizing controlled ovarian hyperstimulation (COH). Given the well-recognized decline in success of conventional in vitro fertilization (IVF) with age, it is unclear whether mild

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approaches in ART should be considered for women older than 35 years. It is the aim of this paper to characterize the role of natural cycle IVF, minimal stimulation IVF and in vitro maturation (IVM) for women of advanced reproductive age. Methods: A review and summary of the literature on mild approaches in ART for women over the age of 35 years is presented. Results: Available evidence regarding natural cycle IVF and its variants suggest that it should not be routinely offered to women of advanced reproductive age, especially over the age of 39. Data from centers well-experienced with minimal stimulation IVF report per cycle live birth rates for several subgroups of women older than 35 years that are comparable to those in the 2010 US Assisted Reproductive Technology National Summary Report. Limited data on IVM suggests that it can be considered up until the age of 40. Conclusions: Recent additions to the literature provide new insight into the efficacy of mild approaches in ART to women of advanced reproductive age. Suboptimal study designs, heterogeneity of protocols and variation in center experience make definitive conclusions difficult however there are several treatment options which would appear to be reasonable for select patient populations. Future studies addressing patient satisfaction, dropout rates and cost will further define the role of mild approaches in ART for this group of women.

the basal characteristics, clinical outcomes and cycle cancellation rate between the two groups; second we analyzed the reasons for cycle cancellation. Results: There were no significant differences in terms of age, duration and cause of infertility, basal FSH, AFC and BMI in the two groups. Although the clinical pregnancy rate per embryo transfer, implantation rate and miscarriage rate were comparable (p>0.05) in the two groups, the accumulative clinical pregnancy rate was significantly higher in UltrashortGnRH agonist protocol (25.1 % vs. 14.5 %; p< 0.05). Besides, we found the cycle cancellation rate was significantly higher in mild ovarian stimulation protocol (44.5%vs17.5 %; p<0.05). Furthermore, we analyzed the reasons for cycle cancellation, and we noted that the rate of the endometrial factors related to cycle cancellation in the mild ovarian stimulation protocol were higher than in ultrashortGnRH agonist protocol (22.4%vs7.8 %; P < 0.05), while other factors were similar in the two groups. Conclusions: The ultrashortGnRH agonist protocol would improve accumulative clinical pregnancy rate, decrease the cycle cancellation rate and relieve the treatment time as well as psychological stress in the poor ovarian responders. Therefore, the ultrashortGnRH agonist protocol may be a valid alternative treatment strategy for the poor ovarian responders. A-17 The value of progesterone level on HCG day in prediction no available embryo with minimal stimulation

A-16 Comparison of utrashortGnRH agonist and mild stimulation protocol on IVF outcomes in poor ovarian responders according to Bologna criteria Yun Sun

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1

Department of Reproductive Medicine, Renji Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China; 2 Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China; E-mail: [email protected] Objectives: To assess the efficacy of UltrashortGnRH agonist protocol in poor ovarian responders undergoing in vitro fertilization and embryo transfer. Methods: A retrospectively analysis was performed in 342 women (401 IVF cycles) with poor ovarian response diagnosed by the latest Bologna criteria between January 2009 and August 2011. Among this patients, 254 patients (291 IVF cycles) adopted UltrashortGnRH agonist protocol (group A); while 88 patients (110 IVF cycles) adopted mild ovarian stimulation protocol (group B). First we compared

Yi Zheng, Bufang Xu, Yun Feng Ruijin hospital affiliated to the Shanghai Jiao Tong University School of Medicine, China; E-mail: zhengshiningrain@ 126.com Objectives: To estimate the value of progesterone level on the day of human chorionic gonadotrophin (HCG) administration in prediction no available embryo on the 3rd day after oocyte retrieval in minimal stimulation IVF cycles, and find out the relevant factors which may co-influence the outcome of IVF. Methods: retrospective compare the progesterone level on the day of HCG of 58 cycles having no optimum embryo to transfer with 224 cycles having available embryo, both by minimal stimulation. Then analyze the relevant factors of progesterone level on the day of HCG. Results: HCG day’s progesterone level in cycles with no available embryo was significantly lower than those with available embryo (1.35±0.12 ng/ml vs. 1.91±0.11 ng/ml, p<0.05). And the progesterone level on the day of HCG was correlated with the same day’s estradiol level, number of

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obtained oocyte and the final fertilization rate (p<0.05). The progesterone level before ovulation can facilitate LH/FSH surge’s formation and the effect of estradiol, which have an important role of follicle development and maturation. Conclusions: progesterone level on HCG day of minimal stimulation cycles with no available embryo was much lower than those with available embryos, reflecting ovary’s different response extent and the quality of oocytes, which may be meaningful in predict IVF’s outcome of minimal stimulation.

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Conclusions: Taken together, these results reveal a specific, SETinitiated, PP2A-mediated, pathway that leads to increased lyase activity of P450c17 and testosterone biosynthesis. Since our previous studies have demonstrated the overexpression of SET in ovaries of polycystic ovary syndrome (PCOS) patients, this regulatory pathway may contribute to the hyperandrogenemia associated with PCOS. A-19

Polycystic Ovary Syndrome (PCOS)

Metabolic and Reproductive Characteristics of the Prenatally Androgenized Female Rats during Puberty

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Xiao-nan Yan

SET regulates androgen production by PP2A in the cultured preantral follicles

State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, China; E-mail: yanxiaonan@ 126.com

Ling-ling Gao, Xiao-qiang Liu, Bo-qunXu, Shi-wen Jiang, Yu-gui Cui, Jia-yin Liu* The State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, China; E-mail: [email protected] Objectives: SET has multiple cell functions including nucleosome assembly, histone binding, transcription control, and cell apoptosis. In ovaries SET is predominantly expressed in theca cells and oocytes. In this study mouse preantral follicle culture model is applied to investigate the role of SET in ovarian androgen biosynthesis. Methods: The recombinant adenoviral vectors AdCMVSET and AdH1-SiRNA/SET are constructed to overexpress and knockdown SET expression respectively. The culture media of infected follicles are collected to evaluate the testosterone production. Results: Our results show that SET overexpression in theca cells stimulates testosterone production whereas SET knockdown decreases testosterone production. Moreover, we find that PP2A activity is inhibited by SET overexpression, but stimulated by SET knockdown. Treatment with PP2A inhibitor okadaic acid (OA) leads to increased testosterone synthesis, while treatment with PP2A activators 1, 9dideoxyforskolin and FTY720 results in decreased testosterone synthesis. Furthermore, knockdown of PP2A in theca cells confirms the key role of PP2A in the testosterone synthesis, and OA is able to block the AdH1-SiRNA/SET-mediated inhibition on testosterone production. The central role of PP2A in SET-mediated regulation of testosterone production is confirmed by the finding that SET promotes the lyase activity of P450c17 and PP2A inhibits its lyase activity.

Objectives: Due to the heterogeneity in clinical symptoms, the early pathophysiological mechanisms responsible for the manifestations of polycystic ovarian syndrome (PCOS) remain unclear. Clinical, experimental, and genetic evidence supports a potential role of epigenetics and fetal programming in the pathogenesis of PCOS. Methods: To investigate the role of prenatal androgen exposure in the development of metabolic and reproductive derangements in rats during puberty, pregnant rats were injected with 5α-dihydrotestosterone (DHT) and their female offspring were observed from postnatal 4 to 8 weeks. Results: The prenatally androgenized (PNA) rats exhibited an increased number of atretic follicles and cystic follicles compared with control. The elevated fasting glucose, fasting insulin, and leptin levels were detected in the PNA rats at the age of 5 to 8 weeks. Following intraperitoneal insulin tolerance test (IPITT), the PNA group exhibited significantly higher 30- and 60-min glucose levels than the control group from postnatal 5 to 8 weeks. Prenatal DHT treatment significantly decreased insulin-stimulated phosphorylation of the IRS1 and AKT in the skeletal muscle of PNA rats. The abundance of IRS1 and IRS2 was decreased in the skeletal muscle and liver after stimulation with insulin in the PNA group, whereas no decrease of insulin signaling was detected in the adipose tissue. Conclusions: These findings validate the contribution of prenatal androgen exposure to both metabolic and reproductive derangements of PCOS in pubertal female rats. The impaired insulin signaling through IRS and AKT may have been involved in the development of insulin resistance during puberty.

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A-20 PCOS related to fetal origins of disease Nannan Zhao State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China; Email: [email protected] Objectives: Polycystic ovary syndrome (PCOS) is one of the most common etiology of subfertility in women. The prevalence of PCOS in women of reproductive age is 5– 10 %. Although assisted reproductive technology (ART) has been widely used in the past 30 years to help these patients to have babies, the characteristics of PCOS, hyperandrogenism and insulin resistance remain to have continued effects to these fetuses during pregnancy. Researchers are more and more concerning the safety of fetus or offspring driven from PCOS patients who have received ART treatment. Our study’s objective is to know whether the abnormal endocrine environment has relationship with adverse perinatal outcomes (gestational diabetes mellitus, GDM), hypertensive disease during) and some Fetal borne diseases (cardiovascular disease, diabetes) and the mechanism involved in. Methods: Placental specimens were collected from PCOS pregnant women treated with ART (experimental group) and pregnant women who got ART treatment for the other etiological factors (control group), then matched based on parity, maternal age, gestational weeks at delivery, and infant sex. The maternal part were collected and dissected immediately following delivery. A gene Chip Affymetrix HG-U133 plus 2.0 Array was utilized to view and analyze the global gene expression alteration between the two groups. Results and Conclusions: By mean of qRT-PCR, we certified microarray data from 10 of the dys-regulated genes, 5 of which we located through immunohistochemistry, more precisely in the placenta.

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effective monitor of the oxidative burden within cellular compartment, reducing hydrogen peroxide and alkyl hydrogen peroxide related to oxidative stress and apoptosis. In our previous study, PrxIV was found expression in oocyte cumulus oophorus complex. However, the role of Prx4 in follicle development has not been documented. There are hydrogen peroxide damage in estrogen production and pathologic dysfunction such as cell apoptosis in polycystic ovary syndrome (PCOS) pathophysiology. In this study, we explored the effect of Prx4 on follicle development and possible role in the pathophysiology of PCOS. Methods: (1) Different expression of PrxIV between PCOS ovaries and nomal control was detected by immunohistochemistry, immunofluorescence and western blot. (2) The levels of PrxIV expression in mice oocyte cumulus oophorus complex at different growing stages were detected by immunohistochemistry and western blot. Results: (1) PrxIV expression was located in granulosa cells and oocytes in human and mice ovaries. (2) It is showed that PrxIV protein expression dramatically increased in oocyte cumulus oophorus complex with the growth of mice follicles; (3) Compared with the normal, PrxIV protein expression decreased in ovaries in PCOS patients. Conclusions: Oocytes regulate follicular development by interacting with oocyte cumulus oophorus complex, in which apoptosis play an important role. Derangement of apoptotic activity and follicle maturation failure were observed in PCOS ovary tissues. Prx4, as an anti-apoptotic factor, located in oocyte cumulus oophorus complex may promote follicles development. Additional studies are required to explore the mechanism of Prx4 in the pathophysiology development of polycystic ovary syndrome. A-22 CAG and GGN repeats polymorphism of androgen receptor gene and epigenetic Influence in women with Polycystic ovary syndrome (PCOS) C Yuan, J Shen, C Gao, L Gao, Y Cui, J Liu

A-21 Peroxiredoxin IV expression in PCOS ovaries Y Qian State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China; Email: [email protected] Objectives: Peroxiredoxin IV, a typical 2-Cys Prx, is one of members of Peroxiredoxin (Prx) family. PrxIV provides an

First Affiliated Hospital of Nanjing Medical University, Nanjing, China; E-mail: [email protected] Objectives: The human androgen receptor (AR) gene contains 2 polymorphic trinucleotide (CAG and GGN) repeats sequence in exon 1, which can transcript into Glutamine (Gln) and Glycine (Gly) respectively. The number of CAG and GGN repeats may confer differential receptor activity, and specific ranges of variants have been correlated with androgen-sensitive disease processes. Polycystic ovary syndrome (PCOS) is a female condition characterized by infertility, oligomenorrhea and androgen excess, many features

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of which are effected through the AR. In Epigenetics, X chromosomes in women are partially inactivity. The aim of our investigation is obtaining the differences of CAG and GGN repeat numbers between PCOS and control groups. Methods: We compared frequency distributions of CAG and GGN repeat alleles and their pattern of expression via Xinactivation analysis among 120 age matched women with regular menstruation and 120 infertile women with PCOS, all of Han Chinese. HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction (HELP) was used to analysis the polymorphic trinucleotide (CAG and GGN) repeats sequence for finding the differences between PCOS and control group excluding the factor of X chromosome inactivity. Results: There were no significant difference of CAG repeat numbers between PCOS patients and controls. The numbers of GGN repeat and homozygote in control group were significantly more than PCOS group (p<0.05). Conclusions: The shorter GGN allele may influence the disease process leading to PCOS, in the contrary, there is no association in CAG repeat number between PCOS patients and controls depends our data. A-23 Testosterone and TNFα impair insulin signaling and insulin stimulation of glucose uptake in mouse granulose cells Y Liu, F Diao, Y Cui, J Liu The State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, China; E-mail: [email protected] Objectives: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive-aged women, leading to symptoms of menstrual disorders, oligo-ovulation or anovulation, hyperandrogenism, and is associated with insulin resistance. Hyperandrogenism and inflammation play important roles in the etiology of PCOS. Both of testosterone (T) and tumor necrosis factor-α (TNFα) can induce insulin resistance in muscle cells and adipocytes. However, their functions in mouse granulose cell glucose uptake, ATP and lactate production have not been determined. The present study aims to determine whether T and TNFα play roles in insulin resistance of granulose cells. Methods: Primary mouse granulose cells were subjected to insulin for 30 min after 24-h pre-exposure to TNFα (0, 1, 10, 100 ng/ml), 24-h pre-exposure to low (10-10, 10-9 mol/L)

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doses of testosterone or 72-h high (10-8, 10-7 mol/L) doses, then glucose uptake and production of ATP and lactate were measured. Protein phosphorylation of insulin receptor substrate-1 (IRS-1, Tyr608), serine-threonine kinase (Akt, Ser473) were measured by Western blot with signal intensity measured by immunofluorescence. Results: 10-8 mol/L testosterone and 10 ng/ml TNFα decreased glucose uptake, ATP production and insulinstimulated phosphorylation of Akt (Ser473) and IRS-1 (Tyr608). Physiological does of testosterone has the adverse effect. Conclusions: Hyperandrogenism and TNFα can induce insulin resistance in mouse granulose cell by effect the insulin signaling pathway, while testosterone in physiological does can increase glucose uptake, ATP production, phosphorylation of AKT (Ser473) and IRS-1 (Tyr608). A-24 Effects of upregulation of HSP27 expression on oocyte development and maturation derived from polycystic ovary syndrome Lingbo Cai, Xiang Ma, Shan Liu, Jinjuan Liu, Wei Wang, Yugui Cui, Wei Ding, Yundong Mao, Huiping Chen, Jie Huang, Jiayin Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China; E-mail: [email protected] Objectives: Heat shock protein 27 (HSP27) is a heat shock protein family member which can inhibit apoptosis. Our previous studies reported down-regulated HSP27 in ovarian tissue derived from women with polycystic ovary syndrome (PCOS); however, the exact effect of HSP27 on oocyte maturation and developmental competence in PCOS is unclear. Here, we investigate the effect of testosterone on HSP27 expression and mouse oocyte maturation. The effect of HSP27 over-expression was also studied in vitro using oocytes derived from PCOS patients. Methods: Oocyte maturation was evaluated by morphological observation. Mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and embryonic developmental competence was evaluated. Critical apoptotic factors and cytokines were measured at both the mRNA and protein level. Our in vitro studies using mice oocytes revealed that no effect of DHT treatment on HSP27 expression. Results: Overexpression of HSP27 lowered the maturation rate of oocytes derived from PCOS patients. Meanwhile,

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fertilization rate and high quality embryo rate were similar between the HSP27 overexpressing group and controls; however, the blastocyst formation rate in this group was significantly higher than control. Conclusions: Expression analysis revealed that the oocytesecreted factors, Bmp15 and Gdf9, and the apoptotic-related regulators, Caspase 3, 8 and 9, were all significantly decreased in HSP27 overexpressing oocytes. In conclusion, upregulation of HSP27 inhibits oocyte maturation from PCOS patients, but improves embryonic developmental potential.

Conclusions: NR4A1 plays a role in the regulation of apoptosis of pre-adolescent ovarian GCs, its mechanism could be by mitochondrial energy metabolism. It provides us significant information for understanding the pathological mechanism of follicular dysplasia in PCOS.

A-25

Jing Wang, Yundong Mao, Wei Wang, Wei Ding, Jie Huang, Feiyang Diao, Xiang Ma, Yugui Cui, Jiayin Liu

Orphan nuclear receptor, NR4A1, regulated the apoptosis of mouse ovarian granulosa cells Fan Jiang, Mei Li, Yu-Gui Cui, Jia-Yin Liu State key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; E-mail: [email protected] Objectives: The orphan nuclear receptor NR4A1 is a member of the steroid hormone nuclear receptor superfamily with no identified endogenous ligand. NR4A1 is an immediately responding gene which induced by multiple extracellular stimulations. NR4A1 plays roles in regulation of cell survival, differentiation and apoptosis, in a cell-type specific manner. In our previous study, we found that NR4A1 was down-regulated in patients with polycystic ovary syndrome (PCOS). Methods: Mouse NR4A1 small interfering RNA (siRNA) and recombinant adenoviruses over-expressing NR4A1 were respectively transfected into the cultured preadolescent mouse ovarian GCs. Cell viability of GCs was measured by MTT assay, and the cell-cycle progression and apoptosis rate of GCs were assessed by flow cytometry after an infection period of 48 h. Expression of the apoptosisrelated factors, Bcl-2 family (Bcl-2, Bax) and Caspase family (Caspase-3, Caspase-9), were detected by real-time quantitative PCR and western blot analysis. Results: The apoptosis rate of pre-adolescent mouse GCs in the NR4A1 interference group was significantly lowered (P < 0.05), while apoptosis rate was increased in the NR4A1 over-expression group (P<0.05). Expression of apoptosis-related factors Bcl-2, Bax, Caspase 3 and Caspase 9 significantly increased at both mRNA level and protein level (P<0.05). ATP synthase activity and ATP content, showing mitochondrial energy metabolism, were significantly reduced after over-expression of NR4A1 in the GCs (P<0.01).

A-26 Polycystic ovary syndrome might increase ectopic pregnancy risk: an analysis of ectopic pregnancy risk with assisted reproductive technology procedures

State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China; E-mail: [email protected] Objectives: The incidence of ectopic pregnancy (EP) after assisted reproductive technology (ART) was higher than that in the general population. The possible risk factors of EP following ART might include female infertility status especially tubal distortion, ART procedure, embryo implantation potential and abnormal hormonal condition. Methods: EP was defined as either ectopic only or heterotopic pregnancy. The main outcome measures were age, previous EP, fresh or frozen-thawed cycles, natural or hyperstimulation cycles and infertility factors including male factor, tubal pathologies, endometriosis, PCOS, other ovulatory dysfunction excluding PCOS, uterine factor, unexplained infertility and other infertility factors Results: In comparison with patients of non-PCOS and non-tubal factor infertility, the risk of EP was significantly increased in patients with PCOS and tubal factor infertility by 2.25 and 1.85 respectively (adjusted odds ratio [adjusted OR] 2.25; adjusted OR 1.85, P<0.05). EP after frozen-thawed embryo transfer cycles was significantly decreased. Taken patients of non-PCOS and non-tubal factor as reference, the risk of EP of patients with only tubal factors, only PCOS, and combined PCOS and tubal factors was significantly increased by 1.92, 2.82 and 3.60 respectively. Conclusions: In this study, PCOS and tubal factor infertility was found to significantly increase the risk of EP in ART procedure, while the frozen-thawed embryo transfer cycles act as a protective factor. It was observed that the risk of EP of women with both PCOS and tubal factor was higher than women with only tubal factor, which further validated the possible relationship between PCOS and EP.

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A-26 SET regulates transcription of key steroidogenic enzyme CYP17A1 and production of DHEA in ovarian theca cells from mouse Xiao-qiang Liu, Chao Gao, Jun Ye, Chun Yuan, Ling-ling Gao, Yu-gui Cui, Jia-yin Liu Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China; Email: [email protected] Objectives: Transcriptional regulating factor SET, a multitasking protein, is widely expressed in various tissues and mediates diverse biological processes. We previously found that expression of SET gene was up-regulated in polycystic ovaries by using microarray. In this study, the effects of SET overexpression and/or silencing on steroidogenic enzyme CYP17a1 were assessed in ovarian theca cells. Methods: Recombinant adenovirus AdCMV-SET and AdSiRNA-SET are constructed to enhance and/or knockdown the expression of SET in theca cells, respectively. The expression patterns of CYP17a1 were subsequently analyzed by qRT-PCR. Moreover, concentrations of DHEA in the spent medium were measured by Enzyme-Linked immunosorbent Assay (ELISA. Results: Our results show that overexpression of SET in theca cells stimulates the expression of CYP17a1, leading to increased DHEA production. Conversely, knockdown of SET exhibits a significant decrease in CYP17a1 expression and DHEA production. Conclusions: Collectively, our results outline a previously unrecognized role for SET in the transcriptional regulation of CYP17a1 in ovarian theca cells. Modulation of this steroidogenic enzyme by SET could affect the production of DHEA, and then influence the capacity of the ovarian theca cells to produce androgen. This study may help us to further explore the mechanism of SET on the hyperandrogenism of PCOS. A-27 SET Up-regulated Key Steroidogenic Enzymes and Androgen Production in PCOS BoqunXu, Yugui Cui, LinglingGao, Kai Xue, Mei Li, Yuan Zhang, FeiyanDiao, Xiang Ma, Jiayin Liu State Key laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, China; Email: [email protected]

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Objectives: In previous study by microarray, we found that the expression of SET gene was up-regulated in polycystic ovary syndrome (PCOS) ovaries. Some evidence suggested that SET gene was essential for regulating both the promoter activity of CYP17 and the biological activity of P450c17. Methods: In present study, the PCOS and normal ovaries were collected to assay the expression and cellular localization of SET by Western Blot and immunohistochemistry. We employed adenovirus-mediated manipulation procedure to over-express and knock-down SET gene in mouse follicles cultured in vitro, and then observed androgen secretion and steroidogenic enzyme expression in theca cells. Results: Level of SET protein in PCOS ovaries was triple higher than those in normal ovaries (P<0.01). SET was expressed predominantly in theca cells and oocytes of the human ovarian follicles. Compared with the follicles infected with AdCMV (control group), testosterone level in culture medium of the AdCMV-SET infection group was significantly increased. SET expression was dramatically down-regulated in theca cells infected by AdSiRNA-SET, while testosterone level in the media significantly decreased when compared with control group (1.49 ng/ml and 5.64 ng/ml, P<0.05). The expression levels of CYP17 and HSD3B2 were significantly increased in the theca cells of SET over-expressed (P<0.05), while they were significantly decreased in the AdSiRNA-SET treated group (P<0.05). Conclusions: SET plays a positive role in regulating ovarian androgen biosynthesis by enhancing transcription of steroidogenic enzyme genes CYP17 and HSD3B1. A-28 SNPs in insulin receptor gene, +168715 C>T, +176506 C>T and +176525 G>A, in Chinese patients with polycystic ovary syndrome Cunsi Yin, Weijun Pan, Yugui Cui, Chun Yuan, Chao Gao, Li Gao, Jiayin Liu Center of Clinical Reproductive Medicine, Ma’anshan Municipal Health Hospital for Women and Children, Ma’anshan 243000, China; E-mail: [email protected] Objectives: To investigate the single nucleotide polymorphisms (SNPs) of tyrosine kinase domain of insulin receptor (INSR) gene in Chinese patients with polycystic ovary syndrome (PCOS). Methods: 96 patients with PCOS and 48 normal women as controls. Frequency of genotypes of a single nucleotide polymorphism of the insulin receptor gene in patients and controls. Results: (1) The occurring frequency of SNPs, +168715 C>T, in Exon 17 of INSR gene in PCOS group (63.5 %)

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was significantly higher than that in the control group (45.8 %) (p=0.043). The difference was much more obvious between fat patient group and non-fat patient group (p= 0.039). (2) There existed polymorphism, +176506 C>T and +176525 G>A, in the front 85 bp and 56 bp of exon 22 of INSR, in two groups. The occurring frequency of polymorphism in these two points was same. The occurring frequency of polymorphism in PCOS group (64.6 %) was also higher than that in control group (47.9 %), which was not significant (p=0.055). Compared among subgroups, the occurring frequency of polymorphism in two points in the non-fat group of patients with PCOS was significantly higher than that in the control group (p=0.042). Conclusions: The SNPs in INSR, (+168715 C>T, +176506 C>T and +176525 G>A), were possibly correlated with the risk of PCOS, especially in non-fat patients with PCOS.

Pregnancy Loss A-29 Assessment and treatment of repeated implantation failure Jichun Tan Shengjing Hospital Affiliated to China Medical University, Shenyang, China 110014; E-mail: [email protected] At present, IVF-ET technology has become important for infertility treatment. The individual reports indicated the clinical pregnancy rate of IVF-ET can be up to 60 % even higher, but its overall pregnancy rate is still relatively low. Successful embryo implantation and pregnancy are depended on many factors, such as a well-functioning endometrium, a normal healthy embryo, skilled transfer technique, appropriate luteal support. Repeated implantation failure (RIF) is diagnosed when good-quality embryos repeatedly fail to implant after transfer in several IVF treatment cycles. The causes for RIF involve endometrium receptivity, embryo and multifactorial causes. Abnormal uterine anatomy, non-receptive endometrium, and the medical condition of the mother can reduced the endometrium receptivity. Embryo quality can originate from either paternal sperm factors or from the oocyte and culture environment. Other factors included on the woman’s age, the indication for IVF, the treatment protocol employed, number of embryos transferred, unskilled transfer technology, and companied with endometriosis, hydrosalpinx. Accordingly, the investigation and treatment of patients with RIF should focus on risk factors of endometrium receptivity and embryo

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quality and may improve our ability to treat RIF and improve IVF results in general. A-30 The mechanism of embryonic implantation Loy Lan Too Kempas Medical Centre, Johore, Malaysia; E-mail: [email protected] The implantation is a complex process accomplished by synchronization and interactions between embryo and Endometrium by local exchange of signals including a number of cytokines, growth factors, cell–cell and Cell–matrix changing. The uterus undergoes dynamic changes during the early embryonic implantation. The first change is regulated by estrogen and progesterone, this hormonal change characterized primarily by changes in both the luminal and glandular epithelial cell in preparation for blactocyst apposition and attachment. The second change is the steroid induced change by embryonic signals, the chorionic gonadotropin induced the glandular epithelium responds by increasing transcriptional and post-translation modifications of secretory proteins and the stromal fibroblast. The third change is the trophoblast invasion and remodeling of the endometrial stromal compartment, this dramatically alter the morphological and functional characteristics of the uterine endometrium, such as interlukin-1(IL-1) system, interleukin-18(IL-18) system, matrix Metalloproteinases (MMP) and vascular endothelial growth factor (VEGF) system. So, an implantation is a process that requires a communication between the uterus and embryo in the early stage of embryo implantation, the endometrium consists of three consecutive stage which is the apposition stage, the adhesion stage and the invasive stage. Finally, the implantation, not only the morphological changed but also the biochemical changed, its changing including the plasma membrane transformation, as well as the adhesion molecules, cytokines, growth factors and proteinases. A-31 IVF twin pregnancy: A case report of a complete hydatidiform mole coexistent with a viable fetus LK Ng, SL Lee, SL Yu, HK Tan Singapore General Hospital, Dept of Obstetrics and Gynaecology, Outram Road, Singapore 169608; E-mail: [email protected]

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Introduction: Twin pregnancy is common with IVF programs. Twin pregnancy comprising of a complete hydatidiform mole and coexisting fetus is a rare incident, occurring in 1 per 22,000 to 100,000 of normal pregnancies. The incidence of this event to occur in IVF pregnancies is thought to be similar to that normal pregnancy. This is the first case we encounter in our department. Case report: Patient was a 33 year old Chinese, G1P0, undergoing her third cycle of IVF program. Her antenatal blood results were normal. At 8.4 weeks post ET (embryo transfer), she developed hyperemesis, per vaginal bleeding and cough. Ultrasound scan performed then showed a viable fetus of 38 mm CRL concurring with 10.4 weeks menstrual age. Posterior to gestational sac was a 5.6 cm inhomogeneous hyperechoic/cystic area, containing several small cystic spaces of 3–5 mm and a 2.8 cm tubular cystic area. No colour flow detected. Impression was a blood clot. At 11.6 weeks menstrual age, the fetus measured 52 mm. Posterior to gestational sac was another sac completely filled with 8.3 cm echogenic mass. It contained 3 cysts measuring 3.7 cm, 2.2 cm and 1.4 cm as well as multiple smaller cysts of 4–12 mm. Impression was molar pregnancy. At 12.7 weeks menstrual age, the fetus measured 66 mm. The placenta of the other gestational sac with multiple cysts developed into snowstorm appearance, a typical characteristics of mole. At 16 weeks gestation, the mole had increased manifolds measuring 16× 9 cm. The pregnancy was terminated.

permeability (CPP)), TJ components expression (such as ZO1, CLDN4, CLDN8 and OCLN) and TJ protein location. Results: We observed TJ structure under transmission electron microscope, and TJ proteins location in BeWo cells by Immunofluorescence. By culturing a monolayer hypoxia model of BeWo cells in vitro using 250 μmol/L CoCl2, we founded that TJ function was sharply altered compared with control group including a decreased TER and a increased CPP, as well as a decrease in ZO1 and CLDN4 expression, a increase in CLDN8 expression for exposure a series time. Meanwhile, the arrangement of these three proteins were altered after 24 h exposure of CoCl2. CONCLUSION: A vitro chemical hypoxia model induced by 250 μmol/L CoCl2 was established in culturing trophoblast cell monolayer. Conclusions: Our data for the first time demonstrated that hypoxia leaded to a TJ dysfunction in trophoblast cell might mediated by HIF-1α accumulation and VEGF production. This may be a potential mechanism involved in PE generation and evolution.

A-32

Zi-Jiang Chen, CSRM

Tight junction dysfunction induced by CoCl2-mimic hypoxia in human trophoblast cell through HIF-1αVEGF axis

Center for Reproductive Medicine, Shandong Provincial Hospital, Shandong University, Jinan, China; National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, China; The Key Laboratory for Reproductive Endocrinology, Ministry of Education of the People’s Republic of China, Jinan, China; Shandong Provincial Key Laboratory of Reproductive Medicine, Jinan, China; Center for Reproductive Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; E-mail: chenzijiang@ vip.163.com

Haijun Zhao, Yuan Zhang, Yugui Cui, Jiayin Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing, China; E-mail: [email protected] Objectives: Placenta is a critical organ that protects the fetus and sustain pregnancy. Placenta hypoxia is related with many pregnancy complication. Tight junction (TJ) barrier is exit in placenta and plays a mainly part in implementation of placental transportation. Methods: After a detected of TJ configuration and TJ location, we examed how TJ changes undergoing cobalt chloride (CoCl2)-mimic hypoxia in vitro culturing BeWo monolayer in terms of TJ function (for instance, transepithelial electrical resistance (TER) and cell paracellular

Others A-33 Multiple pregnancies after assisted reproduction in China

Objectives: To analyze the current situation of multiple pregnancies after ART in China (mainland). Methods: Data after IVF (2006–2009) from 25 reproductive centers in China (mainland) were collected, the perinatal parameters, neonatal parameters were analyzed by statistic software. Results: 2006–2009, there are 127,614 transfer cycles in total after IVF, 49,932 transfer cycles got clinical pregnancies (39.13 %). There are 13,767 twin pregnancy (27.57 %)

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and 1,506 Triplets or more multiple pregnancy (3.02 %) in these clinical pregnancies. Conclusions: Multiple pregnancy rate was high, selective single blastocyst transfer need to be encouraged. A-34 INVO fast track in ART Elkin Lucena, MAbby Moran, Angela M. Saa, Carlos Pulido, Oscar Lombana, Eliana Bonilla Fertility and Sterility Colombian Center – CECOLFES, Bogota, Colombia; E-mail: [email protected]

Objectives: We assessed the outcome of this procedure, employing the specially designed INVOcellTM device, in combination with a mild ovarian stimulation protocol; with the aim of evaluate its usefulness as an alternative treatment option for infertile couples without severe male factor. Methods: One hundred twenty-five cycles were performed (June/2009–May/2011). In average 6.5 oocytes per cycle were retrieved, and a mean of 4.2 was placed per INVOcellTM device. The device was preloaded with 1.1 mL of pre-gazed and pre-warmed culture medium, and a count of 35.000 to 50.000 motile spermatozoa. After 72 h of one step culture, the device was removed from the vaginal cavity.

Results: Groupsa ≤29 30–34 35–39 ≥40 Total

Cycles (n) 17 54 48 6 125

Transfersb (n) 16 (94,1) 51 (94,4) 43 (89,6) 4 (66,7) 114 (91,2)

Retrievalc 7,53 6,52 6,23 5,5 6,5

INVOcelld 4,59 3,8 4,27 5 4,2

Cleavagee 2,7 (58,7) 2,8 (73,7) 2,5 (58,5) 1,5 (30) 2,6 (63)

ETf 2.3 2.3 2.0 1.5 2,1

Pregnancyg 10 (58,8) 22 (40,7) 17 (35,4) 1 (16,66) 50 (40)

Note: INVO = Intravaginal culture of oocytes; ET = Embryo Transfer. Values in parentheses are percentages. a Ranges of ages. bCycles that ended up in transfer. cMean number of retrieved oocytes per punction. dMean number of oocytes placed for fertilization per INVO cell device (cycle). eMean number of retrieved viable proper developed embryos per INVOcell device (cycle). fMean number of transferred embryos per cycle. gNumber of clinical pregnancies per cycle.

Conclusions: INVO is a simple procedure that does not require complex laboratory equipment. INVO does not require complicated maintenance, INVO produces highquality embryos that result in a good rate of clinical pregnancy per cycle (40 %) and a lower rate of multiple gestations (<12 %). INVO has good acceptance because of patient involvement during the initial stage of embryo development, with every positive and significant psychological effects. INVO makes it possible to offer treatment to infertile couples in small medical facilities practically anywhere. All these factors lower the cost of treatment, placing it within reach of a larger population of infertile couples. A-35 Perioperative medical managements for prevention of OHSS Yunxia Cao Reproductive Medicine Centre of The 1st affiliated hospital of Anhui Medical University, Hefei, China; E-mail: [email protected]

Objectives: To evaluate the effect of perioperative medical managements for prevention of OHSS. Methods: To compare some different protocols of control ovarian stimulation including mild stimulation protocol and to know the occurrence rate of OHSS. Some measurements around peri oocytes retrieval have been taken to prevent the occurrence of OHSS and evaluate the effects of these measures, which include decreasing the dosages of Gn, using GnRH antagonist for triggering, coasting, whole embryos crypreservation, using album, hydroxyethyl starch, cabergoline, etc., and also to compare the results of these measurements and the effectiveness. Results: From all these measures, we conclude that which methods are better and which is the best. We will choose them to be used in clinical prevention and treatment of OHSS. Also, we can choose which protocols of control ovarian stimulation are more applicative for the patients. Conclusions: From the data and comparing results, we will evaluate the protocol of controlled ovarian stimulation and choose more applicative one in clinical work and how to make the protocol individualization. Some better measures will be chosen to prevent the capable occurrence of OHSS and decrease the rate of OHSS.

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A-36 Altered glucose metabolism in endometriotic stromal cells Xiaochen Qi, Hui Ji, Li Shu, Zhen Hou, Yanxin Zhang, Wei Wang, Yugui Cui, Jiayin Liu, Yundong Mao Jiangsu Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210005, China; Center of Clinical Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; Email: [email protected] Objectives: To evaluate the metabolic phenotype and glycolytic genes’ expression of endometrial stromal cells from women with and without endometriosis. Methods: : Our prospective study include a total of 36 women undergoing laparoscopic examination during the proliferative phase of menstrual cycle,23 of them were histologically diagnosed as endometriosis (eutopicendometrium: n=10; endometrioma tissues: n=13) and 13 without endometriosis (normal endometrium: n=13). Stromal cells were separated from the samples. The glucose consumption and lactate production were tested by colleting culture medium from cultured primary endometrial stromal cells in vitro. Expression of glycolytic genes were quantified by quantitative real-time polymerase chain reaction (qPCR) in endometrial stromal cells (normal control: n=13, eutopic: n=10, endometrioma: n=13). Results: Endometrioticstomal cells had higher glucose consumption and lactate production compared with eutopic endometrium with endometriosis and without endometriosis, between whom there was no difference. The GLUT1, GLUT3, HKI, PKM, PDHB and SDHB mRNA expression were decreased in ectopic stromal cells compared with eutopic stromal cells. The HIF1α, PDK1 and LDHA mRNA expression were increased in ectopic stromal cells. Conclusions: Abnormal glycolytic genes’ expression in endometriotic stromal cells is in favor of an enhanced glycolytic metabolism. It performed with increased glucose uptake and accumulated lactate production. The altered glucose metabolism may be crucial for the survival of ectopic stromal cells. A-37 Age and body mass index as prognostic factors in infertile Saudi women treated with intra-uterine-insemination (IUI) Saleh AlAsiri King Saud University, Faculty of Medicine, Saudi Arabia; E-mail: [email protected]

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Objectives: To determine the most responsible factors underlying the success of IUI treatment of infertile Saudi women, and to examine whether clinical outcomes were related to patient demographic factors such as age and BMI, along with the number of IUI cycles completed. Methods: During a 24-month period, 301 consecutive Saudi women with infertility underwent IUI in an infertility clinic at King Saud University, in Saudi Arabia. The major clinical outcome was pregnancy rate (PR), which was correlated with patient’s age and BMI, along with the number of IUI’s to determine whether they could be used as prognosticators of success. Results: A p-value of 0.225 (highest 14.89 % and lowest 4.16 % PR) indicated that PR between all age groups were not statistically significant. Only one pregnancy at 44 years old occurred among 24 women over 41 years. A p-value of 0.788 (highest 13.04 % and lowest 7.84 % PR) indicated that there was no significant difference between different BMI groups. Although there was a trend suggesting that first treatment cycle was the most successful, difference among cycle 1, 2 and 3 or more did not reach statistical significance. Conclusions: The PR remained approximately 10 % from 19 to 40 years of age and declined to 4 % after 40. There was positive but not statistically significant correlation of PR with patient’s BMI from 8 % to 13 %. There was a negative but not statistically significant correlation of PR with number of IUI from 13 % to 8 %. A-38 Study on cytokines profiling in endometrial secretomics and clinical outcomes in in vitro fertilization—embryo transfer cycles P Zhang, Z Niu, Q Chen, A Zhang, D Wu, Y Feng Center of Reproductive Medicine, Ruijin Hospital, Shanghai Jiaotong University, China; E-mail: zhangpinggui86@126. com Objectives: To investigate the relationship between cytokines profiling in endometrial secretomics and clinical outcomes in in vitro fertilization-embryo transfer cycles. Methods: Patients (63 cycles) undergoing endometrial secretomics immediately before embryo transfer in vitro fertilization were enrolled. Clinical outcomes of the study participants who underwent endometrial secretion aspiration were compared with 171 controls matched for important prognostic variables. Endometrial secretions samples were analysed by a multiplex immunoassay. Patients of study were divided into two groups according to the follow-up results, 17 kinds mediators in each groups were analysed respectively.

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Results: Patient’s general characteristics, ovarian stimulation dates and clinical outcomes were analysed between study and control groups, there were no difference (P> 0.05). Study group was divided into two parts based on clinical pregnancy. To compare 17 kinds soluble meditors in two groups, IL-13 (9.76±5.04 vs 18.37±17.82), IFN-r (7.50±2.62 vs 49.97±85.95), MCP-1 (17.5±9.63 vs 49.97± 85.95) in pregnancy group were significantly lower than that in non-pregnancy (P<0.05). Conclusions: Endometrial secretion aspiration prior to embryo transfer during in vitro fertilization did not have a detectable effect on clinical pregnancy. The rising expression of IL-13, IFN-r, and MCP-1 in endometrial secretions is against to pregnancy in embryo implantation window, furthermore, higher level of IL-13 and MCP-1 in endometrial secretions has a negative association with embryo implantation.

significantly higher than 42.5 % (79/186) in stage III/IV. Pregnancy rate of 46.6 % (125/268) with history of surgery was significantly higher than 27.6 % (16/58) with no history of surgery (P<0.05). Pregnancy rate of 48.2 % (79/164) in long-term scheme was higher than 38.3 % (62/162) in short-term scheme, but there was no significant (p = 0.075). (4) Multivariable logistic regression: Clinical pregnancy rate of infertility year no more than 5 years, stage I/II, history of surgery proved stage I/II and stage III/IV was significantly higher compared with infertility year more than 5 year, stage III/IV and no history of surgery respectively (adjusted OR and 95 %CI: 2.003, 1.263–3.175; 1.899, 1.11–3.248; 3.769, 1.802–7.887 and 1.985, 1.033–3.815, P<0.05). Conclusions: Factors that affect clinical pregnancy rate of IVF in endometriosis related infertility were infertility year, stage and surgery.

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Research on the factors that affect clinical pregnancy rate of in-vitro fertilization in endometriosis related infertility

Regulation of STAT3 by miR-106a is linked to cognitive impairment in ovariectomized mice M. Zhang, Y Ye, J Cong, D Pu, J Liu, G Hu, J Wu

Z. Hou Department of Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, China; E-mail: [email protected] Objectives: To evaluate the factors that affect clinical pregnancy rate of in-vitro fertilization (IVF) in endometriosis related infertility. Methods 326 cycles of IVF in endometriosis related infertility from 2007 to 2011 were retrospectively investigated in Department of Reproductive Medicine of the First Affiliated Hospital of Nanjing Medical University. Factors that affected clinical pregnancy rate of IVF were analyzed by bivariate analysis and multivariable logistic regression. Factors included age, body mass index (BMI), basic follicle-stimulating hormone (FSH), antral follicle count (AFC), cancinoma antigen (CA)-125 and CA-199, endometriotic stage and history of surgery, stimulation scheme. Results: (1) Pregnancy rate: There are 141 pregnant cycle and 185 non-pregnant cycle, pregnancy rate was 43.3 % (141/326). (2) Basic parameters: Statistical analysis revealed that there is no significant difference in age, BMI, basic FSH, AFC, CA-125 and CA-199 between clinical pregnancy group and non-pregnancy group (P>0.05). (3) Bivariate analysis: Clinical pregnancy rate of 50 % (87/174) in the group of infertility year less than 5 years was significantly higher than 35.5 % (54/152) in the group of more than 5 years. Pregnancy rate of 56.1 % (46/82) in stage I/II was

State Key Laboratory of Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing Medical University, 210029, Nanjing, China; E-mail: jie.wuyale@ gmail.com Objectives: MicroRNAs are abundantly expressed in the brain and play an important role in disorders of the brain, including cognitive impairment and Alzheimer’s disease (AD). A growing body of evidence suggests the JAK/STAT signaling pathway plays a key role in the pathogenesis of AD. Therefore, we characterized the expression and role of miR-106a and JAK/STAT signaling in an ovariectomized (OVX) mouse model of cognitive impairment. Methods: Escape latency and time spent in the platform quadrant were tested by the Morris water maze test. We investigated the expression of microRNAs, mRNAs, and protein in the hippocampus using Quantitative real-time PCR and Western blotting. MiR-106a was noted to be highly over-expressed in the hippocampus at 12 weeks post-OVX and the cell lineSH-SY5Y cells was selected for further study. Results: We demonstrated that cognitive impairment were significantly reduced at 12 weeks post-OVX, compared with age-matched controls. MiR-106a was upregulated, and was inversely correlated with STAT3 and phospho-STAT3 in the hippocampus at 12 weeks post-OVX. Transfection

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of SH-SY5Y cells with a miR-106a mimic reduced the expression of STAT3 mRNA, compared to control cells transfected with a scrambled mimic. STAT3 and phospho-STAT3 protein expression were upregulated and down regulated by a miR-106a inhibitor or miR106a mimic, respectively, indicating that miR-106a negatively regulates STAT3. Luciferase reporter gene assays confirmed that miR-106a directly targets the 3′ untranslated region of STAT3. Conclusions: This study suggests that miR-106a negatively regulates STAT3 activation, and also that miR-106a may provide a marker of onset or potential therapeutic target for cognitive disturbances.

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areas. TNFα (1 mg/mL) remarkably promoted hESCs survival (p < 0.05), which was suppressed by sTNFR:Fc (200 ng/mL, p<0.05), not by TNFα-mAb. sTNFR:Fc or TNFα-mAb suppressed the ICAM-1, MMP-1 and VEGF expressions induced by TNFα. TNFα (1 mg/mL) also promoted the invasiveness of hESCs which was inhibited by sTNFR:Fc (200 ng/mL) Conclusions: TNFα-inhibitors can suppress the proliferation, cell cycle and invasiveness of hESCs. A-42 Serial ultrasound findings in complete hydatidiform mole: case report

A-41 LK Ng, SL Lee, LK Tan, HK Tan Biological effects of the TNFα inhibitors on endometriosis: Experimental study in vivo and in vitro Ying Liu, Liang Sun, Yugui Cui, Zhen Hou, Li Shu, Xiaocheng Qi, Chao Gao, Yuan Zhang, Yundong Mao, Jiayin Liu The State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, China; E-mail: [email protected] Objectives: Tumor necrosis factor-α (TNF-α), a secretory factor, plays an important role in the pathogenesis of endometriosis. Etanercept, as a TNFα receptorII: IgG Fc fusion protein (sTNFR:Fc), and anti-tumor necrosis factor-α monoclonal antibody (TNFα-mAb) both are TNFα inhibitors. To investigate the biological effects of TNFα inhibitors on endometriosis, we used sTNFR: Fc in a mouse model of endometriosis in vivo and compared the effects of sTNFR:Fc and TNFα-mAb in human eutopic endometrial stromal cells (hESCs) in vitro. Methods: Using a surgically induced mouse model of endometriosis, we assessed the effects of sTNFR:Fc and the recombinant murine TNFα (rmTNFα) on the ectopic endometritic lesion by detect fluorescence intensity and areas. The effects of sTNFR:Fc and TNFα-mAb on TNFα-induced proliferation, cell cycle and invasiveness of hESCs were further evaluated by Edu, flow cytometry and invasion assay respectively, and the ICAM-1, MMP-1, VEGF productions were examined by real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA). Results: sTNFR:Fc (3 mg/kg and 10 mg/kg) caused a regression on the fluorescence intensity of endometriotic lesions in the mouse model (p<0.05), but not on the lesion

Singapore General Hospital, Dept of Obstetrics and Gynaecology, Outram Road, Singapore 169608; E-mail: ng.lay. [email protected] Introduction: Hydatidiform moles are a common complication of pregnancy. Its incidence varies in different countries, occurring 1 in 1000 pregnancies in the US and 1 in 100 pregnancies in Indonesia. Several studies have concluded that the sensitivity of ultrasound for accurately predicting hydatidiform mole is in the region of 44 % to 50 %. It is because in the early stages of development hydatidiform mole resembles a missed miscarriage or an embryonic pregnancy, with no ultrasound features suggestive of mole. We present a case of complete hydatidiform mole with serial transvaginal images taken in the early first trimester. Case report: Patient was a 48 years old gravid 3, para 2 Chinese woman. The following ultrasound scans were performed: (i) 4.9 weeks menstrual age : A 13×11×19 mm irregular thick-walled cystic area seen in upper endometrial cavity, suggestive of an early gestation sac. (ii) 5.3 weeks menstrual age: IUGS measuring 27×5×9 mm concurring with 5+ weeks. No fetal pole or heart pulsations of note. Findings were suggestive of anembryonic pregnancy. (iii) 5.9 weeks menstrual age: An irregular cystic area measuring 19×14×10 mm containing echogenic material and small amount of fluid was seen on ultrasound scan. Findings were suggestive of a non-viable pregnancy. (iv) 6.9 weeks menstrual age: Gestational sac had increased many fold measuring 91×43×90 mm. It contained multiple cystic areas. Irregular solid areas were also noted on its walls. Ultrasound diagnosis: Unable to exclude molar pregnancy. She underwent evacuation of uterus. The uterus was 16 weeks size, product of conception and vesicular components were seen. The patient was referred for chemotherapy.

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A-43 Spontaneous uterine scar dehiscence mimicking funneling in cervical canal: Case report Lee Yin Kuan Singapore General Hospital; E-mail: lee.yin.kuan@sgh. com.sg Introduction: Cesarean section which is the most common operation in obstetrics is on a rising trend in most countries. As a result of this operation, scar dehiscence or uterine rupture may occur in subsequent pregnancy. We present the ultrasound findings in a case of spontaneous scar dehiscence in the late third trimester. Case report: Patient was a 32 year old gravida 2, para 1 Chinese woman. Her first delivery was by lower segment cesarean section (LSCS). She had major depressive disorder and schizophrenia. She had booked in another institution for her second pregnancy. The findings of ultrasound scans were unremarkable. She was admitted at 35+6 weeks as a result of per vaginal bleeding. There was no contraction pain and fetal movements were felt. On examination, the abdomen was soft and there was no scar tenderness. Speculum revealed stale blood, no fresh bleeding was noted. CTG was reactive and no contractions were detected. Patient opted for discharge. Three days later, she presented in our hospital with persistent mild per vaginal bleeding. Again, there was no contraction pain, abdomen was soft and there was no scar tenderness. Vaginal examination revealed the os to be 1 cm, and cardiotocography was reactive. An ultrasound scan which was performed revealed a low lying placenta. The amniotic sac appeared to balloon inferiorly. Impression is there is funneling in cervical canal and it measured 8.4×7.8 cm. The os was not clearly demonstrated, but impression was it was not dilated. Patient was advised and consented for emergency LSCS. Operative findings: there was no funneling in cervical canal. Uterine scar dehiscence measuring 8 x 6 cm was present, and the low-lying anterior placenta was bulging over dehiscence. A-44 Genetic polymorphisms in the TGF-β1 gene and HPVpositive high grade cervical intraepithelial neoplasia risk Juan Miao, Yishan Dong, Zhixue You, Delan Zhou, Bo Ding, Shilong Fu, Suping Han Department of Gynecology and Obstetrics, The First Affiliated Hospital, Nanjing Medical University, China; E-mail: [email protected]

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Objectives: Transforming growth factor-β1 (TGF-β1) plays a pivotal role in inflammation and immune responses, which controls the human papillomavirus (HPV) clearance and escape of immune surveillance. This study was aimed to clarify the influence of TGF-β1 polymorphism on HPVpositive high grade cervical intraepithelial neoplasia (CIN2-3) in Chinese population. Methods: Case–control study of women with HPVpositive high grade cervical intraepithelial neoplasia (CIN2-3) (n=181) and women without high-grade CIN (n=192). Two nonsynonymous single nucleotide polymorphisms (SNP) in TGF-β gene (rs1800469:C509T and rs1982073:T869C) were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Results: Multivariate analysis found TC/CC genotypes of TGF-β1 rs1982073:T869C to be associated with a statistically significantly higher risk of HPV-positive CIN2-3 (OR=1.593, 95 % CI=1.018–2.493), compared with the TT genotype, after adjustment for age, parity, abortive times, smoking status and marital status. However, no significant association was observed for TGF-β1 C509T polymorphism. The stratified analysis showed that the increased risk with TC/CC genotypes was more pronounced in women with parity being ≤1 (OR=1.911, 95 % CI=1.127–3.240), and never smokers (OR=1.715, 95 % CI=1.066–2.760). Conclusions: These findings suggest that functional SNP rs1982073 in TGF-β1 could contribute to the susceptibility of HPV positive high-grade CIN in Chinese population. This is an initial study to indicate that TGF-β1 T869C (rs1982073) might contribute to HPV-positive high-grade CIN. A-45 Expression level of AMH in cumulus cells was related to mouse oocyte quality Yi-Hui Zhang, Yu-Gui, Cui, Ri-Cheng Chian, Jia-Yin Liu Clinical Center of Reproduction Medicine, the First Affiliated Hospital, Nanjing Medical University, China; E-mail: [email protected] Objectives: In females, Anti-Mullerian hormone (AMH) is expressed only in the granulosa cells of follicles. It has been confirmed that serum AMH levels can predict the number of antral follicles. Yet, it is still controversial whether AMH has a relationship to the quality of the oocytes. Methods: In this study, mouse cumulusoocyte complex were cultured in vitro, then downregulated or upregulated

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the expression of AMH gene using adenovirus infection. Expression of a series of genes related to oocyte quality were examined by qPCR. Results: We found that the downregulated or upregulated expression of AMH in cumulus cells significantly changed EP3, YEAF, H3A and CCNB2 expression in oocytes. Conclusions: Our findings revealed that AMH in cumulus cells can affect oocyte quality. The mechanism of relationship between AMH in cumulus cells and oocyte quality will be investigated further. A-46 Effects of different ovarian stimulation protocols for endometriosis on the outcomes of IVF-ET Xiaoyan Xu, Lin Kong, Jichun Tan*, Hua Guan, Shasha Wang The Center for Reproductive Medicine, Shengjing Hospital Affiliated to China Medical University, Shenyang, China, 110014, China; E-mail: [email protected] Objectives: To investigate the effects of different ovarian stimulation for endometriosis on outcomes of IVF-ET. Methods: A retrospective analysis was performed on 177 infertile women (altogether 198 cycles) with endometriosis following IVF-ET treatment. These patients were divided into 4 groups based on different COH protocols, minimal ovarian stimulation group (group A, n=49), short COH group (group B, n=73 cycles), long COH group (group C, n=42 cycles) and prolonged COH group (group D, n=34 cycles). Set up statistical database. Then the hormone level, the requirement of gonadotrophin (Gn), the number of oocytes obtained, the implantation rate and the clinical pregnancy rate were analyzed. Results: Among the 4 groups, there were no significant differences in patient’s age, years of infertility, number of retrieved oocytes and transplanted embryos (P>0.05). The dosage of Gn in group A (5.9±5.1) was lower than that in groups B (33.2±9.2), C (34.2±11.4) and D (38.9±12.4), respectively (P<0.05); and the dosage of Gn in group D was significantly higher compared with that in group A, B and C, respectively (P<0.05). The clinical pregnancy rate of group C (43.2 %) was significantly higher compared with the group A (30.6 %), group B (24.2 %) and group D (17.6 %), respectively, P<0.05. Conclusions: Although the long COH protocol can achieve higher pregnancy rate, but the dosage of Gn increased significantly, and the dosage of Gn in minimal ovarian stimulation protocol little, and can obtain satisfactory pregnancy rate.

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Preimplantation Genetic Diagnosis (PGD) A-47 Aneuploidy in human pre-implantation embryos Kangpu Xu Weill Cornell Medical College of Cornell University, New York, USA; E-mail: [email protected] Interests in chromosomal copy numbers at various stages of human pre-implantation embryonic development have grown stronger recently since one of the main difficulties in improving outcome of assisted reproductive technologies is to identify healthy-looking as well as euploidy embryos. Aneuploidy arises during gametogenesis in both females and males, apparently more often in oocytes than in spermatozoa and is clearly related to female age. Aneuploidy occurs during preimplantation embryonic development. ART procedures are also believed to contribute the mistakes, including ovarian stimulation regimen and embryo culture techniques. To complicate the matter more, evidence exist that self-correction of first meiotic error occurs in the second meiotic division. After fertilization, human embryos undergo a very tricky developmental period when some or a majority of blastomeres may not have the same number of chromosomes. Mosaicism among individual cells within an embryo seems to be common, at least in the first 2–4 days of development. The frequency of mosaicism at any particular stages, however, is debatable since solid data are lacking. Aneuploidy embryos may be less frequent and mosaicism appears to be less common at blastocyst stages. This implies a selection process or, again, selfcorrection mechanisms exist during compaction and blastulation. Understand the mechanisms that control female and male meiosis and embryogenesis and determine the time and frequencies of aneuploidy at every stage of development may help us to target the best stages and utilize the best technologies to identify euploidy embryos for embryo transfer. A-48 The genetics of hydatidiform moles: recent advances and role of ART in preventing moles Rima Slim Departments of Human Genetics and Obstetrics and Gynecology, McGill University Health Centre, Montreal H3G 1A4, Canada; E-mail: [email protected] Hydatidiform mole is a human pregnancy with no embryo. The paradoxical nature of this aberrant pregnancy has

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fascinated and puzzled scientists in all civilizations. Common HMs occur once in every 600 pregnancies in western countries but have 2 to 10 times higher frequencies in Asian countries. Common moles are sporadic, not recurrent, and have complex multifactorial etiologies. By studying rare familial forms of recurrent hydatidiform moles (RHMs), two maternal-effect genes responsible for this condition have been identified. NLRP7 codes for a nucleotide oligomerization domain-like receptor protein and is mutated in 60 to 80 % of RHMs cases. KHDC3L codes for a KH domain containing 3-like, subcortical maternal complex member, and is mutated in 5 to 14 % of NLRP7negative patients. Patients with two mutations in any of these two genes usually fail to have normal pregnancies. The exact molecular functions of these two genes and how they lead to moles is not fully understood. Data from various groups indicate that mutations in these two genes do not affect ovulation, fertilization, but abnormalities in the embryos of the patients manifest after the formation of the zygote and/or during early development. These observations are in line with the emerging views about the mechanisms leading to all types of moles and are corroborated with a recent report on a successful pregnancy after oocyte donation in a patient with two NLRP7 mutations. In addition, because these two genes are not present in mice, gathering ART data and observations on the early development of embryos from these patients, and on patients with moles in general, will play a pivotal role in our understanding of their roles in early postzygotic development.

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Methods: We Recruited 6 couples with mean maternal age of 34.5 years (Rang 28–42 years) who suffered from postimplantation RM/RIF for more than two times . Thawing their preserved frozen embryos, cleavage stage biopsy was done; then embryos were subjected to vitrification cryopreservation once more. One blastomere from each embryo was taken and subjected to whole genome amplification and sequential array comparative genomichybridization (aCGH) analysis. Single euploid embryo was transferred into the frozen transfer cycles. Results: Ten out of 28 biopsy embryos were diagnosed as euploid. Six frozen cycles of single embryo transfer resulted into four successful clinical pregnancies (including two live births and two ongoing pregnancies), one biochemical pregnancy and one implantation failure. Conclusions: Though cleavage stage pre-implantation genetic screening (PGS) is still controversial. Our pilot clinical study suggests encouraging results. Twice vitrification procedure seemed little negative influence on the viability of the embryos. And array CGH procedure was easy scheduled by the genetic diagnostic laboratory because frozen cycles were performed. The patients suffering from RM/RIF are always afraid of embryo transfer with troubling heart. This strategy is a possibly potential alternative clinical treatment for the couples suffering from RM/RIF in the progress of in vitro fertilization. A-50 Pregnancies after transfer of vitrified biopsied blastocysts

A-49 Yan Hao, Ping Zhou, Zhiguo Zhang, Yunxia Cao Frozen embryo aneuploidy screening is a potential clinical pathway for the couples suffering from postimplantation recurrent miscarriage and/or implantation failure in the progress of in vitro fertilization: a pilot study Jiandong Shen, Wei Wu, Lingbo Cai, Xueping Sun, Yugui Cui, Jiayin Liu The State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, PR China; E-mail: [email protected] Objectives: To explore and evaluate a clinical treatment pathway for the couples that had experienced postimplantation recurrent miscarriage and/or implantation failure (RM/RIF) in the progress of in vitro fertilization. However, they still have many frozen embryos for transfer.

Centre for Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, P. R. of China; E-mail: [email protected] Objectives: To show two cases describing the successful pregnancies after transplantation of vitrified biopsied embryos after preimplantation genetic diagnosis. Methods: Descriptive cases of report of two couples, one of which is a carrier of translocations of paternal origin with 46, XY, t(4,22) and the other is a carrier of sex chromosomal abnormality of paternal origin with 46, XXY. Results: These two patients became pregnant, and are presently ongoing. Conclusions: This report shows that vitrification may be a better method for cryopreservation of biopsied human embryos. Frozen–thawed blastocysts obtained from vitrified biopsied embryos can be successfully pregnancy.

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Kato Ladies Clinic, Tokyo, 2Shinbashi Yume Clinic, Tokyo, St. Luke Clinic, Oita, Japan; E-mail: f-aono@towako-kato. com

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Successful PGD for late infantile neuronal ceroidlipofuscinosis achieved by combined chromosome and TPP1 gene analysis Jiandong Shen, David Cram, Wei Wu, Lingbo Cai, Xiaoyu Yang, Xueping Sun, Yugui Cui, Jiayin Liu The State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, PR China; E-mail: [email protected] Objectives: Late infantile Neuronal CeroidLipofuscinosis (NCL-2) is a severe debilitating autosomal recessive disease caused by mutations in the TPP1 gene. There is no cure and treatments are largely ineffective, resulting in early childhood death. A couple with two affected children presented for reproductive genetic counseling and chose to undertake IVF and PGD to avoid the possibility of another affected child. Biochemical analysis of TPP1 enzyme activity found low levels in probe and indicting defective TPP1 genes in both partners. However, DNA testing revealed only one of the two mutations in the mother that was inherited by the proband. Methods: Linkage analysis identified 5 informative linked STR markers to aid the genetic diagnosis. Following IVF, 5 cleavage-stage embryos were biopsied and blastomeres were first subjected to whole genome amplification, then a series of downstream PCR analyses to diagnose the TPP1 gene genotype and finally array CGH to assess the chromosomal status of each embryo. Results: Two unaffected euploid embryos were identified for transfer. A single expanded blastocyst was transferred on day 5 resulting in an ongoing pregnancy. Confirmatory prenatal diagnosis by amniocentesis showed concordance of the embryo and fetal diagnosis. Ultrasound scans revealed normal fetal parameters and a healthy pregnancy ensured. Conclusions: This is the first successful report of PGD for NCL-2 using a strategy of simultaneous single gene PGD and array CGH to identify an unaffected and chromosomally normal embryo for transfer.

Objectives: Aggressive chemotherapy and radiotherapy have greatly enhanced the life expectancy of young cancer patients, but these treatments cause massive destruction of the ovarian reserve resulting in infertility or sterility. However, oocyte cryopreservation can preserve their fertility of these patients after cancer treatment. We applied a minimal ovarian stimulation protocol using clomiphene citrate (CC) to retrieve mature oocytes, and the cryotop vitrification method to cryopreserve them. Methods: Eighty-two unmarried hematopoietic defect patients with informed consent who underwent the CC cycle from January, 2007 to September, 2012. Fifty mg CC was administered from cycle day 3 and 75 IU HMG was administered every other day from day 8 until the leading follicle developed to 18 mm in diameter. Administration of CC was then stopped, and 300 μg GnRH-agonist was given as a maturation trigger. Oocytes were retrieved from 30 to 36 h following the administration of the GnRH-agonist using a narrow gauge needle. The retrieved oocytes were denuded before vitrification. The cryotop method was used to vitrify the oocytes. Results: The type of cancers included acute and chronic leukemia, malignant lymphoma, aplastic anemia, and myelodysplastic syndrome. The mean age of the patients was 27.2(± 5.1, S.D.). The mean number of retrieved oocytes per cycles was 3.8, and the mean number of morphologically normal cryopreserved oocytes per cycle was 3.2. The mean numbers of oocyte retrieval cycles per patient was 1.8. The mean number of morphologically normal cryopreserved oocytes per patient was 5.8. No patients had any adverse side effects. Conclusions: Our data showed that the minimal ovarian stimulation protocol using CC for oocyte retrieval with a narrow gauge needle was a safe, simple, effective method of preserving fertility for unmarried cancer patients. A-53 The efficacy and safety of application of trehalose to human oocytes vitrification

Fertility Cryopreservation Z. Zhang, Y Cao A-52 Oocyte cryopreservation for unmarried female blood cancer patients

Reproduction Medicine Centre, First Affiliated Hospital of Anhui Medical University, Hefei, China; E-mail: zzg100ster@ gmail.com

Fumihito Aono1, Shokichi Teramoto2, Osamu Kato1, Keiichi Kato1, Takafumi Utsunomiya3

Objectives: To explore the efficacy and safety of intracellular trehalose used in human oocytes vitrification.

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Methods: Fresh human oocytes that were obtained from IVF/ICSI patients, were randomly distributed into four groups: 1) Intra- and extracellular trehalose [0.15 M intraand 0.5 M extracellular trehalose+15 % ethylene glycol (EG)+ 15 % dimethylsulphoxide (DMSO)]; 2) Extracellular trehalose [0.5 M extracellular trehalose + 15 % EG+ 15 % DMSO]; 3) Sucrose control [0.5 M sucrose + 15%EG+ 15 % DMSO]; 4) Fresh control (without vitrification). The first three groups were chosen to undergo freezing and thawing. Trehalose was introduced into oocytes by microinjection. After oocytes were thawed, all survival oocytes were fertilized by ICSI. When the embryo developed to blastocyst, we tested the chromosomal status of human embryos by fluorescence in situ hybridization (FISH) (13,15,16,18,21,22,X,Y). Survival of human oocytes cryopreserved in each group, and fertilization rate, cleavage rate, blastocyst rate, the high qulility embryo rates of day 3 and day 5–6, and the blastocyst aneuploidy rate. Results: The fertilization rates and cleavage rates was significantly higher (P<0.05) in the intra- and extracellular trehalose group (94.7 % and 96.3 %) than in the extracellular trehalose group (65.9 % and 66.7 %) and sucrose group (83.9 % and 28 75.7 %). The percentage of high-quality blastocyst of day 5–6 were also significantly different (P< 0.05) among the three groups (55.6 % vs0% vs11.1 %, respectively). The percentage of abnormal embryo of the intra- and extracellular trehalose group was higher than the other three groups, but there were no statistically significance between the four groups. Conclusions: (1) Intracellular trehalose can improve human oocytes vitrification effect, and improve freeze-thawed oocytes developmental potential; (2) The aneuploidy rate of intra- and extracellular trehalose group is not higher than the control group, but still need to increase the number of embryos and chromosome to further demonstrate. A-54 Successful cryopreservation using CryoTop for the vitrification of blastocysts with blastomere biopsy Li Zhu, Chen-Hui Ding, Li Tao, Yu-Liang Liu, Yan-Hong Zeng, Yan-Wen Xu, Can-Quan Zhou Reproductive Medical Centre, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; E-mail: [email protected] Objectives: To study the effect of vitrification on the development potential of blastocysts after blastomere biopsy. Methods: Pre-implantation genetic diagnosis (PGD) was performed for both single gene diseases and chromosomal

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abnormalities. An open Cryotop system was used to vitrify blastocysts. The outcome parameters were survival rates after warming, clinical pregnancy rates and implantation rates. Clinical outcome was compared to control group with thawed blastocyst transfer from conventional IVF or ICSI. Results: There were no statistical differences between the PGD and non-PGD groups with regards to the implantation rates and clinical pregnancy rates (32.1 % vs 36.5 %, 43.75 % vs 51.76 %). Significant higher proportions of embryos at early blastocyst stage (42.59 % vs 21.68 %, P<0.001) or hatched/hatching stage (24.07 % vs 4.07 %, P<0.001) were found in PGD group. To investigate the implantation potential of vitrified blastocyst at early stages, PGD groups were subdivided according to the number of early blastocyst in transferred embryos. The implantation rates were 28.13 %, 31.43 % and 35.0 % in the groups with all embryos at early stage, part of at early stage, or non at early stage respectively, and no significant differences were found. Conclusions: CryoTop vitrification is an effective way to preserve surplus blastocysts derived from biopsied embryo at cleavage stage. Early blastocysts subjected to blatomere biopsy have implantation potential, and are worthy for cryopreservation. A-55 Effect of the water extraction of CuscutaChinensis and Epimedium Herb applied to the freezability of rat epididymal sperm Xiao-Wei Nie Department of Reproductive Medicine, First Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, China; E-mail: [email protected] Objectives: To observe the effect of the water extraction of CuscutaChinensis and Epimedium Herb applied to the rat sperm cryopreservation and optimize rat sperm cryopreservation system. Methods: Extracts of CuscutaChinensis and Epimedium Herb were isolated and purified. The water extracts were applied in the rat epididymal sperm cryopreservation. Sperm motility index (SMI) and sperm abnormality rate were assessed by Eosin Y staining. Results: Cryoprotectants added with water extract of CuscutaChinensis and Epimedium Herb can improve sperm motility index after thawing. Conclusions: Water extract of ofCuscutaChinensis and Epimedium Herb has a protective effect on rat sperm cryopreservation.

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A-56 Cryo-storage time had no influence on post thaw survival and pregnancy outcome in vitrified human oocytes Lingbo Cai Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, China; E-mail: cai_ [email protected] Objectives: To evaluate the impact of cryopreservation storage duration on oocyte survival, implantation competence, and pregnancy outcome. Methods: Retrospection of oocyte cryopreservation and thawed cycles in our center, base on storage time, from 2006.3 to 2010.10, 162 Oocyte thawed cycles were divided into three groups, group1, storage time less than 365 days, group 2, between 366 and 730 days and group 3, longer than 731 days. Oocyte survive rate, fertilization rate and clinical outcome were compared among three groups. We also examined the correlations between storage time and oocyte survive rate. Results: During 2006.3∼2010.10, there were 162 oocyte thawed cycles and 1158 vitrified oocytes, the clinical pregnancy rate was 44.74 % per transfer and implantation rate was 28.75 %, miscarriage rate was 13.25 %. Group 1 had 76 thawed cycles and 683 oocytes, in Group2 there was 62 cycles and 355 oocytes, in group3, there was 24 thawed cycles and 120 oocytes, We compared outcomes of three groups and found that there were similar results in oocyte survive rate (81.41 %, 80.28 % and 81.67 %), clinical pregnancy rate (42.86 %, 43.33 % and 54.54 %), miscarriage rate (10.0 %, 15.38 % and 16.67 %) among three groups. Correlation examination found that there was no correlation between storage time and oocyte survive rate. Conclusions: Cryo-storage duration did not adversely affect post thaw survival and pregnancy outcome in oocyte vitrification patients. A-57 Vitrification and warming temperature affects morphology, zonapellucida and developmental competence of mature human oocytes Ruihuan Gu, Yun Feng, Yijuan Sun, Aijun Zhang, Xiaowei Lu Center of Reproductive Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; E-mail: [email protected]

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Objectives: Oocyte cryopreservation has wide clinical implications for assisted reproductive technology, such as the preservation of women’s fertility, establishment of oocyte banks and so on. The objective of the present study was to determine the changes in morphology, zonapellucida and developmental competence of vitrified mature human oocytes after loading of cryoprotectants at two different temperatures. Methods: Oocytes, obtained from forty women undergoing infertility treatment in Ruijin Hospital, were vitrified after loading of cryoprotectants at either 37 °C or room temperature (RT, 24–26 °C). Oocyte morphology was evaluated before and after vitrification using OCTAX EyeWare software. The time required for enzyme-digestion of the zonapellucida was noted. Furthermore, embryo development and clinical pregnancy were evaluated after intracytoplasmic sperm injection. Results: In all, 480 oocytes were vitrified. The equilibration time with cryoprotectants was significantly shorter at 37 °C than at RT. Oocyte survival, fertilization and cleavage rates were similar between the two temperatures; however, embryo quality, implantation rate and pregnancy rate were significantly higher at 37 °C. Oocyte morphology were similar irrespective of the equilibration temperature. The time required for zonapellucida dissolution was significantly longer when the mature oocytes were equilibrated with cryoprotectants at RT. Conclusions: The results of the present study indicate that vitrification/warming at 37 °C reduces equilibration time, improves embryo quality, implantation rate and pregnancy rate, reduces vitrification related changes in the dissolution time of the zonapellucida and has no effect on oocyte morphology. A-58 Clinical outcome of vitrified-warmed blastocyst comes from low grade D3 embryo and blastocyst from vitrifiedwarmed cleavage-stage embryo transfers in ART patients Y Qiu, CL Xu, CY Li, ZH Deng, H Yang, Y Zou, W Liu, MM Qiu, LZ Xun, J Wang Reproductive Medical Centre, Third Affiliated Hospital, Guangxi Medical University, Nanning Second people’s Hospital, Nanning, Guangxi, China; E-mail: nnqying@ sina.com Objectives: This study sought to evaluate the outcome of vitrified-warmed blastocyst and blastocyst from vitrifiedwarmed cleavage-stage embryo transfers in patients undergoing ART treatment.

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Methods: We compared the clinical results of embryo transfer on the vitrified-warmed blastocyst and blastocyst from vitrified-warmed cleavage-stage after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Results: Our data showed that blastocyst from vitrifiedwarmed cleavage-stage on day 5 did significantly increase clinical pregnancy rate (65.31 % vs 48.15 %, p<0.05) and implantation rate (48.72 % vs 30.2 %, p<0.05) in patients in comparison with vitrified-warmed blastocyst stage embryo transfer under all years old. Under 35 years of age, vitrified-warmed blastocyst got slower clinical pregnancy rate compared with blastocyst from vitrified-warmed cleavage-stage (74.00%vs 37.50 %, p<0.05).In patients older than 35 years of age, the clinical pregnancy rate after vitrified-warmed blastocyst transfer was the same compared with blastocyst from vitrified-warmed cleavagestage (40.00 % vs 42.86 %, p>0.05). In blastocyst from vitrified-warmed cleavage-stage group, it was found that resulted significantly increasing clinical pregnancy rate (74.000 % vs 42.86 %) under 35 years of age compared with older than 35 years of age. However, in blastocyst from vitrified-warmed cleavage-stage group, it was found that resulted the same clinical pregnancy rate (37.50%vs 40.00 %) under 35 years of age compared with older than 35 years of age. Conclusions: The results of study indicate that blastocyst from vitrified-warmed cleavage-stage can have good clinical pregnancy outcomes. Embryo of low grade on D3 got clinical pregnancy rate acceptable.

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1 expressions are correlate with the follicle development in endometriosis patients. Methods: 36 infertile patients with stage III and IV endometriosis (group A) and 35 tubal and/or male factor infertile patients (group B) were included in this study. The patients with number of mature oocyte ≤4 were considered as poor responders. According to this, the two groups were further divided into four subgroups. Luteinized granulosa cells was collected from follicle fluid by Percoll method, and Stepone real-time PCR was used to analysis the expression level of inhibin α, MMP-2 and TIMP-1. Results: The expression of inhibin α in endometriosis group and poor responders in control group were significantly lower than those of the normal responders in control group, in which the alternation of inhibin α expression was not correlated to the variables of follicle development. Moreover, MMP-2 levels were lower and TIMP-1 levels were higher in both endometriosis and tubal/male infertility poor responders compared to normal ovarian responders. Interestingly, the correlation between MMP-2 or TIMP-1 expression and variables of follicle development in tubal or male infertility patients was observed, but it lost in endometriosis patients. Conclusions: Therefore, the preliminary results suggest that aberrance of MMP-2 and TIMP-1 expression in luteinized granulosa cells might be associated with impairment of follicle development in endometriosis-associated infertility. A-60 Adiponectin gene polymorphisms may not be associated with idiopathic premature ovarian failure

Reproductive Endocrinology A-59 Expression of inhibin α, matrix metallpproteinase-2 and tissue inhibitors of metallpproteinase-1 in luteinized granulosa cells: comparison between women with and without endometriosis

Yuqin Ye, Danhua Pu, Jiayin Liu, Fanghong Li, Yugui Cui, Jie Wu State Key Laboratory of Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing Medical University, 210029, Nanjing, China; E-mail: jie.wuyale@ gmail.com

R Tan, L Liu, D Pu, Y Cui, S Jiang, J Liu, J Wu State Key Laboratory of Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing Medical University, 210029, Nanjing, China; E-mail: jie.wuyale@ gmail.com Objectives: To assess the differences in expression of inhibin α, MMP-2 and TIMP-1 in luteinized granulosa cells from women with and without endometriosis undergoing IVF, and determine whether inhibin α, MMP-2 and TIMP-

Objectives: To investigate the relationship between adiponectin gene polymorphisms and idiopathic premature ovarian failure (POF) in Chinese women. Methods: One hundred twenty idiopathic POF and 104 healthy controls were recruited in this study. All subjects were analyzed at the SmaI and BsmI loci of the adiponectin gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Results: The frequency of the T allele of the SmaI polymorphisms was 71.7 % in POF women, which was similar to the controls (71.6 %) (p = 0.994). And for the BsmI

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polymorphisms, the G allele was 31.25 % in POF patients and 26.9 % in control women (p=0.315). In addition, there was no significant difference among the three genotypes (wild type genotype, mutant heterozygote and homozygote) of the SmaI and BsmI polymorphisms, in the prediction value of POF. By haplotype analysis, haplotype TT as reference, there were no significant difference in the frequency of haplotype GT and TG between POF patients and controls (GT: OR=1.11, 95% CI=0.70–1.73, and TG: OR= 1.28, 95 % CI=0.82–2.01, respectively). Conclusions: Our results suggest that the SmaI and BsmI polymorphisms of adiponectin gene might not be responsible for idiopathic POF, at least in Chinese population. More researches are needed to determine whether the findings are generalizable to other populations. A-61 Effect of Bisphenol-A on human endometrial cells proliferation and its possible mechanism D Qin, Y Cui, L Jian, L Wang, L Gao, J Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; Email: [email protected] Objectives: Bisphenol A (4,4′-dihydroxy-2,2-diphenylpropane; BPA) is an endocrine disruptor that affects the reproductive health of wildlife and possibly of humans. Evidence suggests that BPA associated with anomalous reproductive tract development, cancer, and pregnancy loss and so on. Methods: In this study, we investigated the proliferation and viability of human endometrial stroma cells (hESCs) and RL95-2 cells in vitro after exposure to concentrations of BPA. Results: BPA induced the cell cycle arrest at concentrations of 10−4 mol/L after a 24 h incubation period. It also induced a concentration-dependent (10-8, 10-6, 104 mol/L) decrease in steroid receptors mRNA and or protein expression in hESCs and RL95-2 cells. Further research show, the ERα protein expression in RL95-2 cells was no significant difference after pretreatment with ICI182, 780. To discern the mechanism of BPA action, we detected the MAPK and PI3K/AKT signals in RL95-2 cells. Activation of ERK1/2 phosphorylation was observed. Conclusions: In light of these results, it is possible that BPA could have effects hESCs through GPR30 receptor, representing a strong support to the growing recognition of the adverse effects of BPA on human health.

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A-62 In silico study of bisphenol-A binding to human sex hormone binding globulin (hSHBG) as an agonist J Hu, Y Cui, C Gao, J Luo, J Liu China Pharmaceutical University, Nanjing 210000, China & State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, 210029, China; E-mail: [email protected] Objectives: Bisphenol A is an organic compound consisting of two phenolic rings connected by a single carbon carrying two methyl groups. SHBG was initially uncovered by its ability to bind estradiol and testosterone. Sex hormonebinding globulin (SHBG) or sex steroid-binding globulin (SSBG) is a glycoprotein that binds to sex hormones such as testosterone and estradiol. The SHBG inhibits the function of these hormones. Thus, bioavailability of sex hormones is influenced by the level of SHBG. Methods: Computational modeling, simulation and analysis were performed using the program MOE. protein he coordinates for the docking studies were taken from a crystal structure of hSHBG bound with an estradiol (PDB ID: 1LHU). Testosterone bound structure were modified from the structure of hSHBG binding with dihydrotestosterone (PDB ID: 1D2S). Results: The position and orientation of estradiol docked in silico into the hSHBG were the same as that in the crystal structure with a free energy of binding −30.56 kcalmol-1. There are four hydrogen bonds between the ligand and the receptor residues Ser42, Asp65, Asn82 and Lys134 make hydrogen bonds with hSHBG. The residues Asn82 and Lys134 make hydrogen bonds with two oxygen atoms of estradiol. The free binding energy of BPA docked into the binding pocket of the hSHBG is −25.13 kcalmol-1. Conclusions: The mode of interactions and binding energy of BPA were similar to that of estradiol and testosterone. This reveals that BPA can bind to hSHBG as an agonist of estradiol and testosterone. A-63 Different expression of Angiogenic Cytokines in placenta subjected to assisted reproductive technology Chanjuan Li, Yuan Zhang, Yugui Cui, Haijun Zhao, Jiayin Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; Email: [email protected]

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Objectives: Recently, several studies focused on the safety of fetus or offspring derived from assisted reproductive technology (ART) which might be suspected to result in more maternal and infant complications than nature conceiving. Placenta is critical organ for both fetus and mother with many physical functions, among which vascular angiogenesis is one of the most important functions directly affected fetal development. Accordingly, we hypothesized that the expression of angiogenic cytokine in placenta from ART treatment may have altered, which be associated with the complications of offspring. Methods: Human Angiogenesis Antibody Array of 43 antibodies was used to examine differences in angiogenic cytokine of placenta derived from in vitro fertilization and natural pregnancy. Four differentially expressed cytokines were then certified by Western Blotting and immunohistochemistry analysis. Results: Forty-three angiogenic cytokine were identified in the placenta from ART: 12 up-regulated and 31 had no change. The up-regulated angiogenic cytokines such as VEGFR3, bFGF, IFNG and MMP1, were confirmed by Western Blot. Immunohistochemistry analysis showed those cytokins were expressed and localized in the placental vascular endothelial cells, or in the cytoplasm and membrane of syncytiotrophoblast cells. Conclusions: This is the first study of comparing expression of angiogenic cytokines in placenta derived from in vitro fertilization and natural pregnancy. Differentially expressed angiogenic cytokines, such as VEGFR3, VEGFD, bFGF, Tie2 and MMP1, may alter placental functions in angiogenesis and partially elucidate pathogensis of the placenta from ART, which may bring about negative perinatal outcomes.

flow cytometry, and proliferation using MTT. HSP10 expression was detected by real-time PCR. HSP10 gene was then down-regulated in the cultured mouse ovarian GCs by AdCMV-H1-SiRNA/HSP10, or over-expressed by AdCMV-HSP10. PD98059 (p-ERK inhibitor) was used to treat cells to induce the high apoptosis index. The critical apoptotic and proliferative factors, including p-ERK, Bcl-2, Bax, caspase9, caspase3 and Ki67, were monitored by realtime RT-PCR, and Western blot. Results: Compared with the control groups, the apoptosis index of testosterone-treated mouse GCs was significantly increased (P<0.05), while HSP10 expression was decreased (P < 0.05). In the AdCMV-H1-SiRNA/HSP10-treated groups, cell viability was decreased (P<0.05), and cell cycle was arrested by G2. The expression of p-ERK, Bcl-2, Ki67 and ratio of Bcl-2/Bax was lowered, while expression of those apoptotic factors, including Bax, caspase9, caspase3 was increased (P<0.05). Compared with control group, Bcl2 expression in the mouse GCs of over-expressing HSP10 group was increased (P<0.05), while reduction of p-ERK, Bcl-2 and elevation of caspase9, caspase3 induced by PD98059 were significantly suppressed (P<0.05). Conclusions: Hyperandrogenic condition induced apoptosis of mouse GCs cultured in vitro, and its mechanism could be that testosterone reduced HSP10 expression by reducing Bcl-2 expression and increasing Bax expression.

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Jian Hu, Xiao-qiang Liu, Chun Yuan, Jia-yin Liu

Effect of HSP10 on apoptosis induced by testosterone in the cultured mouse ovarian granulosa cells

Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China; Email: [email protected]

Kao-Kao Zhao, Yu-Gui Cui, Xiang Ma, Fei-Yang Diao, Jia-Yin Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China; Email: [email protected] Objectives: To investigate the effect of HSP10 on apoptosis induced by testosterone in the mouse ovarian granulose cells (GCs), so as to define the possible role of HSP10 in ovarian pathological development of polycystic ovary syndrome (PCOS) and hyperandrogenic conditions. Methods: The cultured mouse ovarian GCs treated with testosterone (10-5 mol/L) were assessed for apoptosis using

A-65 In silico study of bisphenol-A and related compounds binding to human sex hormone binding globulin (hSHBG) as agonists

Objectives: Calculated mode and free energies of bisphenolA and its related compounds bind to hSHBG by in silico study, the results were compared with two natural ligands of hSHBG, indicating that BPA and related compounds could bind to hSHBG as antagonists. Methods: Molecular simulation software MOE2009.10 Results: These compounds have been successfully docked in silico into the ligand binding site of hSHBG using the software MOE2009.10. The structures have been compared with estradiol and DHT bound structure of hSHBG. It is found that the binding energies of selected bisphenols, binding to hSHBG, were similar with estradiol and DHT. This reveals that some bisphenols could bind to hSHBG as agonists of estradiol and DHT, and

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the binding modes and energies are related to their delicate structural differences. Conclusions: We conclude that our present study indicates that some bisphenols are capable of binding to hSHBG and producing adverse effects by affecting the efficient level of hSHBG and steroids binding to hSHBG. Since bisphenols are released to the environment, taken into account the huge production of these compounds, especially BPA, these ubiquitous chemicals would have adverse impact on human beings and many other species for a long time.

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Conclusions: Activin A promoted the secretion of estradiol of ESCs by increasing the expression of P450arom via ALK4-Smads pathway, which might be an important reason for promoting ectopic foci survival and development.

Reproductive Biology A-67 How advances in ovarian physiology are improving embryo quality in human ARTs

A-66 David F. Albertini Activin A stimulates endometrial stromal cells of endometriosis to express aromatase via ALK4-Smads pathway Juan Zheng, Zhen Hou, Yugui Cui, Yundong Mao, Jiayin Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China; Email: [email protected] Objectives: Activin A was one of the altered expression cytokine, which up-regulated in the women with endometriosis. Investigation of Activin A in human endometrial stromal cells (ESCs) will explain their involvement in human endometriosis pathophysiology. Methods: Real time-PCR and Western blot were used to determine the different expression of P450arom in endometrial and endometriotic cells. ESCs were cultured in vitro and stimulated with various concentrations of Activin A for different time. To determine whether Activin A-induced P450arom expression is via ALK4-smads signaling pathway independent of PGE2, ESCs were treated with Smad4siRNA, to down-regulate smad4 expression, SB431542, an inhibitor of ALK4, and H89, an inhibitor of PKA. To observe the phosphorylation of Smad3 and its subsequent nuclear translocation, the protein of the cytoplasmic and nuclear were extracted separately. Results: P450arom was more abundant in eutopic and ectopic stromal cells from patients with endometriosis compared to women without endometriosis (p<0.01). Activin A can induce the expression of P450arom mRNA and protein in ESCs from women with endometriosis (p<0.01). Moreover, neither estradiol production nor the expression of P450arom mRNA and protein were affected by the Activin A in the ESCs from women without endometriosis. Either activin A or testosterone can induce a pronounced level of estradiol, and they have synergistic effect (p<0.05).

Molecular and Integrative Physiology, University of Kansas Medical Center, USA; E-mail: [email protected] Objectives: To review the major advances in ovarian physiology over the last decade that have direct bearing on attempts to increase embryo quality in human ARTs. Methods: Compare the state of knowledge on the regulation of follicle and oocyte maturation prior to and following 2002 based on a literature survey on animal and human studies. Results: After nearly 20 years of controlled ovarian hyperstimulation, using natural or recombinant gonadotropins as a driving force for maturation and oocyte retrieval, the clinical practice of human ARTs has failed to acknowledge insights into ovarian physiology that promise to improve both embryo quality and live birth outcomes. The philosophy that “more is better” is being re-examined in the context of three fundamental principles in ovarian physiology that collectively emphasize the importance of maintaining ovarian function under the least perturbing conditions while maintaining access to adequate numbers of oocytes retrieved. The first advance derives from recognition of an intraovarian signaling system that deploys a complex array of paracrine and autocrine factors responsible for follicle activation and synchronization of oogenesis with folliculogenesis both prior to and after the expression of gonadotropin sensitivity. Secondly, while FSH is a driving force for follicle growth and maturation both in vivo and in vitro, excessive FSH stimulation results in a loss of oocyte quality manifest in truncation of, and disturbances in, the growth and maturative phases of oogenesis. Thirdly, discoveries on the EGF and ANF signaling pathways mediated by g-protein modulation in both granulosa and germ cell compartments of the cumulus-oocyte complex cast a secondary role on LH during ovulation that must be factored into attempts to obtain quality embryos from in vitro matured oocytes. Conclusions: Excessive use of gonadotropins likely compromises embryo quality as a result of desynchronizing the

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communication pathways that integrate the processes of oogenesis and folliculogenesis. Both in vivo and in vitro ARTs will benefit from careful titration of the extrinsic and intrinsic factors that mediate the link between oocyte and embryo quality. A-68 Cumulus cells gene expression analysis to identify competent oocytes and embryos P. Patrizio Yale University Fertility Center- New Haven, CT-USA; [email protected] The standard protocols of controlled ovarian stimulation (COS) results in the production of multiple oocytes and embryos; however, as confirmed by many recent reports, in fresh non-donor cycles only about 20 % of the embryos transferred and 5 % of the oocytes retrieved and fully utilized produce a live birth. This is because the majority of the oocytes and the embryos obtained with standard COS protocols are chromosomally or genetic abnormal. To improve the biological efficiency of the process it is imperative a) to have a better understanding of the genetic pathways important for oocyte competence; b) to implement methods for the selection of competent gametes and embryos for transfer; and c) to perfect protocols of COS. A number of new technologies have the potential to improve ART results, but many are currently only used in research. These methodologies include, a) the use of array comparative genomic hybridization (aCGH) for blastomeres or for trophectoderm biopsies (from blastocyst-stage embryos) or for polar bodies of oocytes to detect aneuploidies; b) the use of geneexpression profiling by microarray and real-time PCR of cumulus cells to detect aneuploid oocytes; c) and proteomics and metabolomics of individual embryos. In earlier studies we demonstrated that oocyte gene-expression of in vitro matured oocytes is incomplete or missing for genes involved in mRNA processing, protein synthesis and homeostatic proteins (ferritins, calcium ions and growth-factor homeostasis). In another study, after CGH analysis of the 1st polar body, aneuploid oocytes were compared to those chromosomally normal and 327 genes were found to display statistically (P<0.05) significant differences in gene expression levels. Real-time PCR analysis confirmed abnormal expression of genes involved in spindle assembly and chromosome alignment, as well as others regulating cellular metabolism. Recent studies on cumulus cells transcriptome allowed the detection of a wide range of molecules associated to a stagespecific signature as well as genes involved in many cellular

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processes such as metabolic control and resumption of meiosis, thus identifying possible new molecular markers of oocyte competence. Several cumulus cells encoded genes have been associated with chance of conception (such as PTGS2, VCAN, PTX3, SPSB2 and TP53I3), providing further information on pathways involved in oocytecumulus cell interaction. Cumulus cells surrounding aneuploidy oocytes have been characterized as displaying a unique genetic profile, with statistically significant down regulation (P<0.05) of two genes, SPSB2 and TP53I3, as confirmed by real-time PCR experiments. The expression of SPSB2 was also found to display a strong trend towards upregulation in the CCs of oocytes that produced a healthy live birth (P=0.05). However, the regulatory mechanisms of chromatin state are still poorly understood. Among the regulatory molecules, long non-coding RNAs (lncRNA’s) represent an interesting group of RNAs that do not template for protein synthesis. They recruit chromatin modifiers to mediate transcriptional changes in processes ranging from X-inactivation (XIST) to imprinting (H19) to modulation of developmental processes in germ cells. Current research is aiming at assessing the expression levels of lncRNAs in the cumulus oophorus cells to identify lncRNAs that are critical for both oocyte nuclear and cytoplasmatic maturation and the process of oocyte aging. Finally, emerging proteomic technologies may soon allow the selection of developmentally competent embryos based on their unique protein and secretome profile. In summary, the oocytes are the most important variable for the reproductive success. Understanding the bi-directional communication between the oocyte and its surrounding cumulus cells and developing platforms to detect noninvasively competent gametes would represent the next breakthrough in ART. A-69 Culture media for oocyte maturation and embryo culture Teraporn Vutyavanich Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; E-mail: [email protected] In vitro culture of embryos involves more than just choosing the appropriate culture media that that meets the minimal nutritional, pH and osmolality requirement of the embryos. Human embryos possess considerable degree of plasticity and they can adapt to different culture media and techniques currently in use. However, adaptation comes at a cost and it may result in epigenetic and metabolic changes and/or

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abnormal gene expression. A recent study showed that the birth weights of the infants were significantly different depending on which culture media the embryos were exposed to, despite the fact that they were cultured for 2–3 days before transfer. Currently there are two opposing views of culture media formulations: the stage-specific sequential media, which are based on “back to the nature” approach and the complex monoculture media which allow embryos to choose what they want during development. Available data do not support the advantage of one media over the other. Recently a commercial medium supplemented with granulocytesmacrophage colony-stimulating factor (GM-CSF) is available. The medium was claimed to significantly increase live birth rate (by 28 %) in patients with previous miscarriages. More researches are needed to confirm this finding, and to assess its long-term effect on the offspring. The use of culture media for human embryonic stem cell to enhance blastocyst development is another area for research. In vivo, the developing embryo is moving along the female genital tract and is surrounded by a thin film of tubal or uterine fluid. To mimic this condition, the embryos are usually cultured in microdrops (20–50 μl) under an oil overlay. There are currently 3 static and 2 dynamic formats for embryo culture, i.e. monoculture or sequential media where embryos are transferred to a new dish on day three, and continuous uninterrupted monoculture where embryos are left in place without media renewal. The dynamic format involves microfluidic culture device, where there is a continuous medium renewal without embryo movement or embryos are cultured in a dish with moving platform. There are a variety of media for in vitro maturation (IVM) of oocytes. Basically, they contain salt solutions, energy substrates, amino acids and supplemented steroid hormones. Conventional IVM media are available commercially and are widely used. A study in our center showed that supplemented blastocyst medium is as good as conventional IVM medium to support maturation and developmental competence of human immature oocytes. A-70 Oxygen requirements for pre-implantation development in vitro John X. Zhang Department of Obstetrics and Gynecology, Feinburg School of Medicine, Northwestern University, Chicago, Illinois, USA; E-mail: [email protected] Studies using animal models have suggested that lower oxygen concentrations than that in the atmosphere may

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benefit preimplantation development in vitro. However, it is only recently that evidence emerged from human studies in support of this notion. Incubation under reduced oxygen concentrations requires specialized equipment. To utilize exiting incubators without the capability of regulating oxygen concentration, a desiccator was connected to a pressurized gas tank containing 5 % O2, 5 % CO2and 90 %N2, and placed in a regular incubator with 5 % CO2 in air. The treatment outcome of IVF-ET procedures in patients under 35 years of age 12 months before (172 cycles) and after (165 cycles) the use of this desiccator incubation system was compared. All patient demographics and ovarian stimulation characteristics were indistinguishable. However, clinical pregnancy rates increased from 48.3 % before the use of the desiccator to 57.0 % (p< 0.05, Chi squared test), and implantation rates increased from 35 % to 44 % (p<0.05). When 32 day-5 transfers before the desiccator were compared with 44 day-5 transfers after the desiccator, clinical pregnancy rates and implantation rates did not differ (59 % vs 61 %, and 52 % vs 50 %). These suggest that reduced oxygen concentration benefits pre-implantation development in vitro, particularly during the first 2 days of development after IVF. This was later confirmed using frozen and thawed one-cell mouse embryos. A-71 Effect of Hsp27 on the early embryonic development Shan Liu, Xiaonan Dai, Lingbo Cai, Xiang Ma, Jinjuan Liu, Shiwen Jiang, Jiayin Liu, Yugui Cui State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, China; E-mail: liushan6016515@ 163.com Objectives: Our previous studies showed that the heat shock protein 27(Hsp27) expressed in oocyte of growth follicles was down-regulated in PCOS ovaries. We found that down-regulation of Hsp27 improved the maturation of mouse oocytes and increased early apoptosis of oocytes. In this study, we observed the effect of Hsp27 on the mouse early embryo development. Methods: By microinjection with AdCMV-Hsp27 or AdSiRNA-Hsp27 adenovirus into cytoplasm of mouse zygotes, the blastocyst morphology was observed, and cell apoptosis of blastocysts was detected by TUNEL. After cultured in vitro for 96 h, blastocysts were analyzed Hsp27 expression by real time PCR and immunofluorescent. The blastocyst formation rate and embryo quality were evaluated.

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Results: The expression of Hsp27 significantly increased in embryos of the Hsp27 over-expressed group, while it was significantly suppressed 75 % in embryos of the SiRNA group (p<0.05). The cell apoptotic rate of blastocysts insignificantly changed (p>0.05). The blastocyst formation rate and embryo quality were insignificantly changed after Hsp27 over-expression or gene silencing (p>0.05). Conclusions: Over-expression or down-regulation of Hsp27 in zygotes, as a single factor, did not significantly affect the subsequent embryonic development. A-72 Altered global gene expressions of human embryoid bodies treated with 10-6 mol/L bisphenol A Jian Luo, Yugui Cui, Chunyan Jiang, Boxian Huang, Song Ning, Aiqin Chen, Chao Gao, Li Gao, Jiayin Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China; Email: [email protected] Objectives: To detect the global gene expressions of human embryoid bodies after disposing by bisphenol A. Methods: Human ES cells were grown in suspension without feeder cells and inhibiting factor bFGF to induce their differentiation into embryoid bodies (EBs). After the EBs were treated with 10-6 mol BPA in 7 days, a gene chip was utilized to analyze its genes. We used the same way to deal with the EBs in order to get more sample. UsingRealtime PCR, to verify some of the differentially expressed genes. Results: Compare with the control group which dealt with DMSO in 7 days, there were 158 genes upregulated and 458 down regulated with a threshold fold change over 2.0. Analysis these differentially expressed genes with MAS, a software about Bio-molecular functional annotation system. The results suggested that they were involved in cell differentiation, molecular functions, germinal layer development and signaling pathways. EBs was a powerful tool for learning human early embryonic development. Treat the EBs with BPA should help us to understand the impact of BPA on early embryonic development and its possible mechanism of action. Conclusion: Differentiation of EBs from human ES cells could be a useful model for dectecing global gene expression changes in response to BPA, which could help us to study the effect or the mechanism of BPA on early human embryonic development.

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A-73 Biological effects of the TNFα inhibitors on endometriosis: Experimental study in vivo and in vitro Ying Liu, Liang Sun, Yugui Cui, Zhen Hou, Li Shu, Xiaocheng Qi, Chao Gao, Yuan Zhang, Yundong Mao, Jiayin Liu The State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, China; E-mail: [email protected] Objectives: To investigated the effects of anti-tumor necrosis factor α (TNFα) on the regression of experimental endometriosis in vitro, and to evaluate the effects on the cell proliferation, cell cycle, and invasiveness of eutopic endometrial stromal cells (hESCs) from endometriosis patients in vivo. Methods: Using the homologous and variant endometrium explant mouse model of endometriosis, we studied TNFα receptorII: IgG Fc fusion protein (sTNFR:Fc) and the recombinant murine TNFα (rmTNFα) effects on the endometritic lesion by detect fluorescence intensity, areas. The cell proliferation and cell cycle of hESCs treated with sTNFR:Fc and rmTNFα were evaluated by the Edu proliferation assay and MTT. The effects of TNFα and TNFα inhibitors on the invasion of hESCs were examined by using an invasion assay, and the genes and proteins production of ICAM-1, MMP-1, VEGF were determined by real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Results: Etanercept (3 mg/kg and 10 mg/kg) caused a regression on the fluorescence intensity of endometriotic lesions in the mouse model (p<0.05), but not on the lesion areas. TNFα (1 mg/mL) remarkably promoted hESCs survival (p<0.05), which was suppressed by sTNFR:Fc (200 ng/mL), but not by TNFα-mAb. sTNFR:Fc or TNFα-mAb markedly suppressed the expressions of ICAM-1, MMP-1 and VEGF induced by TNFα in vitro. TNFα (1 mg/mL) also promoted the invasiveness of hESCs which was inhibited by sTNFR:Fc (200 ng/mL) and TNFα-mAb (1000 ng/mL). Conclusion: TNFα-inhibitors can suppress the proliferation and cell cycle of hESCs, and inhibit invasiveness of hESCs. A-74 Bisphenol A may increase the risks of fetal origins Parkinson’s disease Boxian Huang Department of Life Science and Technology, China Pharmaceutical University, China; E-mail: [email protected]

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Objectives: To date, the mechanism of Parkinson’s disease remains unclear. Current studies tend to combine the factors involved in hereditary, genetic susceptibility and the environmental toxins, which are all potential causes of fetal origins diseases. Our study’s objective is to know whether Environmental Endocrine Disruptors (EED) can cause many neurodegenerative diseases, including ALS, Huntington’s disease, and Parkinson’s disease. We have developed an assay to identify bisphenol A (BPA) that promote neuronal degeneration that may increase the risks of fetal origins Parkinson’s disease. Methods: The model from EB to DA was treated with vary of does-windows and time-windows of BPA, and then we test the expression of all the three germ layers’ markers. such as embryoid bodies, Neuronal Progenitor cells, dopaminergic neurons. The methods involved in the test include real-time PCR, differentiation of human embryonic stem cells into DA Neurons, microarray, immunostaining and flow cytometry analysis, neurophysiological monitoring, high performance liquid chromatography and so on. Results: BPA is a kind of EED and DA is a hallmark of Parkinson’s disease, we confirm activity of BPA treated with human neurons can decrease the number of DA in offspring, and at the same time establish a kind of toxicological testing model; The expression of all the three germ layers marker are also down regulated. The electrophysiological properties of BPA treated with DA neurons are decreased. Conclusions: We first developed a toxicological testing model of DA-derived from human embryonic stem cell and confirmed BPA may increase the risks of fetal origins Parkinson’s disease. A-75 Embryo culture and quality control in assisted reproduction Howaida Hashim Kingdom Hospital/Consulting Clinics; E-mail: hhashimh@ aol.com Objectives: In all types of areas, the need of improving the quality of any creation, procedure or service in order to advance client satisfaction, is assuming larger proportions. Methods: One of the main questions in an IVF Unit, Why were my embryos of such poor quality? While a good IVF clinic is good at making good embryos, sometimes we will encounter patients whose embryos are all of poor quality. Improvement of embryo quality during in-vitro culture can be achieved by understanding and controlling the requirements of gametes and embryos. The success of an IVF lab is very dependent on the conditions in the lab.

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Results: The implementation of an integral quality system leads to increase standardization and efficiency of all procedures as well as improves transparency and trace ability of all actions performed in the laboratory and subsequently the quality of service provided will improve significantly. Conclusions: While abnormal embryos from patients with multiple failures probably may not benefit from standardization and require specialized therapy, Quality management system within the ART Laboratory is a necessity.

Male Factor and Spermotogenesis A-76 Conventional treatment of male infertility in the era of ART Aucky Hinting Faculty of Medicine Airlangga University, Surabaya, Indonesia; E-mail: [email protected] Objectives: The management of infertile males remains a problem for clinicians due to the limitation of effective treatment. Although ART, especially ICSI, provides a huge contribution in overcoming male infertility problems, not all clinics possess the ability to perform ICSI and not all patients are able and willing to participate in ICSI program particularly those in developing countries. Therefore, conventional treatment still plays a role in the management of male infertility. Methods: Mini review. Results: Conventionally, infertile males may receive counseling, medical and surgical treatment, toxic and environmental factors elimination, or empirical treatment. Medical treatment can be divided into pathophysiological-based specific treatment and empirical treatment, which is merely based on hypothetical approach. Specific treatment in several conditions, such as sexual dysfunction, endocrine abnormalities, and acute infection, may provide effective treatment results. However, the proportion of those abnormalities in male infertility is low. Most of male infertility are idiopathic, those with untreatable conditions, or those who have failed treatment which can only be treated empirically. Basically, hypothetical approach for such treatments are spermatogenesis stimulation, epididymal function improvement, sperm transport improvement and sperm metabolic stimulation. Conclusions: A follow-up study (36 months) in the author’s clinic indicates that natural pregnancy rate from specific

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treatment was 53.4 % and from empirical treatment was 31.5 %. Current literatures also show that ART pregnancy rate still depends on sperm factors, hence the need for conventional treatment in the ART era. Unfortunately most of the studies in male infertility are poorly controlled. In the future, more randomized controlled trial will be needed. A-77 Expression of Calretinin in the testes of rats at different development stages Xiaonan Dai State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, China; E-mail: daixiaonan_ [email protected] Objectives: Testicular steroidogenesis was mainly regulated by LH secreted by anterior pituitary, while androgen production also subtly regulated by neuroendocrine system and paracrine. Our previous studies showed that the protein profile of human testes changed when endogenous testosterone production was suppressed by exogenous androgen, including many regulators such as calretinin (CALB2). We supposed that CALB2 participated in regulating androgen production in testes. In this study, we observed the different expression of CALB2 in testes of rats at different development stages. Methods: Male SD rats at three development stages (3 to 4 weeks as early maturity group; 16 weeks as middle-aged group; over 12 months as aged group) were used in this study. Sera testosterone concentration was detected by radioimmunoassay (RIA). Cellular location of CALB2 in testes was observed by immunohistochemistry. Expression of CALB2 in mRNA level and protein level was quantificationally analyzed by qPCR and western blot. Results: Compared to the early maturity group, sera testosterone level in the middle-aged group was significantly higher (P<0.01), and expressions of CALB2 were significantly increased (P<0.05). Interestingly, rats of the aged group had lower sera testosterone level and lower testicular expression levels of CALB2 than the middle-aged group (P<0.05). Conclusions: Calretinin is positively correlated with serum sera testosterone level, and differently expressed in testes of rats at different development stages, which may can illustrate suggested that calretinin regulated testosterone production as an important paracrine and autocrine factor.

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A-78 KiSS-1 and GPR54 in hypothalamus and testes of rats at different development stages Yugui Cui, Xuesong Wang, Zhaohui Zhang, Guipeng Ding, Xueling Hu, Jiayin Liu State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; Email: [email protected] Objectives: Loss of functional mutations of the gene encoding GPR54, the putative receptor for the KiSS-1-derived peptide kisspeptin, has been found to be associated with abnormal puberty onset and hypogonadotropichypogonadism both in rodents and human. It was suggested that GPR54 and KiSS-1 system are key elements in the regulatory network of the gonadotropic axis. Methods: Interaction between KiSS-1 and GnRH agonist NMDA on the gene expression was studied. The expression of kisspeptin and GPR54 in rat testis from different developmental stages was determined using immunohistochemistry. Results: Persistent expression of KiSS-1 mRNA was detected in the hypothalamus of the infantile, prepubertal, pubertal, and adult rats. The maximum expression was found in the pubertal stage. Injection of kisspeptin into the lateral cerebral ventricle markedly increased sera testosterone production. The expression of LH and FSH mRNA in the pituitary also increased after the central administration of kisspeptin. NMDA down regulated KiSS-1 mRNA in the hypothalamus, while MK801, an antagonist of NMDA receptor, showed upregulatory expression. Both kisspeptin and GPR54 were detected in germ cells in testes. Rat testis at pubertal stage showed the highest expression of KiSS-1 gene. Conclusions: In conclusion, KiSS-1 and GPR54 were likely important genes related to pubertal onset and reproductive activity, they were expressed not only in the central nervous system, but also in testis, with a unique pattern during pubertal development. KiSS-1 may mediate regulation of neurotransmitters, such as NMDA, in stimulation of GnRH releasing. KiSS-1 and GPR54 may also directly involve in spermatogenesis. A-79 Effects of the Yangjing capsule extract on steroidogenesis and apoptosis in mouse Leydig cells Dalin Sun, Yugui Cui, Baofang Jin, Xindong Zhang, Xiaoyu Yang, Chao Gao Institute of Andrology, Nanjing University of Chinese Medicine, Nanjing 210046, China; E-mail: [email protected]

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Objectives: In this study, we investigated the effects of the Yangjing capsule extract on steroidogenesis and apoptosis in the cultured mouse Leydig tumor cells (MLTC-1). Yangjing capsule is an innovative formulas based on traditional Chinese Medicine, which has been tried to treat male infertile and hypogonidism. MLTC-1 cells were cultured and treated with the Yangjing capsule extract for 24 h. Testosterone level in medium was measured by radioimmunoassay. Methods: Expression of key steroidogenic enzymes (StAR, CYP11A1, HSD3B) in cells was examined using real-time RT-PCR and immunoblotting. Secondly, MLTC-1 cells were treated for 48 h in a serum-free medium. Cell viability was measured by MTT assays. Cell cycle and apoptosis were analyzed using flow cytometry. Expression of activated caspase-3 was analyzed using RT-PCR and a colorimetric protease assay. The Yangjing capsule extract promoted testosterone production, while increased the expression of StAR, CYP11A1, and HSD3B mRNAs and proteins, when compared with control. H89, a PKA inhibitor, significantly inhibited those stimulated effects. The Yangjing capsule extract improved the cell viability of MLTC-1, decreased the number of cells in G0/G1 phase, and increased the number of cells in S-phase. Cell apoptosis was inhibited, expression of caspase-3 was inhibited. Conclusions: These results showed that Yangjing capsule extract can promote steroidogenesis, and have protective effects in Leydig cells, which provide preliminary evidence showing the therapeutic effects of Yangjing capsule in male infertile and hypogonidism.

Stem Cells A-80 Normal human embryonic stem cell lines were derived from microsurgical enucleated tripronuclear zygotes Chunyan Jiang, Lingbo Cai, Boxian Huang, Juan Dong, Aiqin Chen, Song Ning, Lianju Qin, Yugui Cui, Jiayin Liu National Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, 210029, China; Email: [email protected] Objectives: Tripronuclear zygotes (3PN), which have three copies of each chromosome, occur approximately 5–7 % in human IVF cycles. Diploid and triploid human embryonic stem (hESC) cell lines have been derived from triploid zygotes. Some strategies have been tried to rescue diploidy by removing the extra male pronucleus of the tripronuclear zygote. However, whether human embryonic stem cells can be derived from rescued 3PN zypotes is not well-known.

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Methods: We restored 433 diploid zygotes from triploid zygotes donated by 224 couples, of which 22 blastocysts were developed. Results: After whole zonapellucida free embryo being plated onto feeder cells to grow and passage, 2 hESC lines (CCRM-hESC-22 and CCRM-hESC-23) were generated and both of them carried the normal 46, XY karyotype. The hESC lines were then characterized by morphology, expansion in vitro, and expression of specific markers including alkaline phosphatase, OCT4, SSEA4, TRA-1-60 and TRA-1-81. The pluripotency of the cell lines was confirmed by embyoid body formation and differentiation in vitro and teratoma production in vivo. Conclusions: Our study suggests that corrected 2PN embryos by removal of extra pronuclei have a normal development potential by giving rise to the normal hESC lines, although blastocysts formation rate were not improved. A-81 Regenerative effects of transplanted mesenchymal stem cells in rat model of thin endometrium Jing Zhao, Yanping Li, Qiong Zhang, Yonggang Wang Reproductive Medicine Center, Xiangya Hospital, Central South University, Changsha, Hunan, China; E-mail: [email protected] Objectives: thin endometrium has detrimental effect on embryo implantation. This study was to evaluate the effect of administration of mesenchymal stem cells derived from rat bone marrow (BMSCs) on the thin endometrium. Methods: A thin endometrium model was created in 40 female adult rats. The rats were randomized into four groups: culture medium and BMSCs (1*107/ml) administrated to the thin emdometrium models by the way of veancaudai or uterine in situ transplanted. After 16 days, the uteri were harvested and stained with the hematoxylineosin, and the expression of endometrial receptivity markers (vimentin, cytokeratin, LIF, CD34, and ανβ3) and inflammatory cytokines (interleukin (IL)-1, IL-6, IL10, interferon (IFN)-γ, and vascular endothelial growth factor (VEGF)) were analyzed. Results: The results indicate a statistically significant increased endometrial thickness in stem cell-treated rats compared with control group. The expression of vimentin, cytokeratin, LIF, CD34, and ανβ3 in stem cell-treated rats were also improved compared with control groups; We also found that BMSCs upregulated the expression of VEGF, IL6, IL-10, and downregulated that of IL-2, IFN-γ. Transplantation of MSC with i.v. administration achieved a thicker endometrium compared with in-situ transplanted group.

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Conclusions: Transplantation of BMSCs has beneficial effects on reconstruction of thin endometrium in a rat model. Engrafted MSCs may regulate cytokines and inhibit inflammation to prevent the thin endometrium progression. A-82 Differentiation of rat bone marrow mesenchymal stem cells into endometrial cells Yonggang Wang Xiangya Hospital, China; E-mail: [email protected] Objectives: We explored to induce bone marrow mesenchymal stem cells into endometrial cells. We try to provide experimental foundation for treatment of thin endometrium by transplantation of stem cells. Methods: Rat bone marrow mesenchymal stem cells and human endometrium stromal cells were isolated and identified. BMSCs were induced by co-culture with human endometrium stromal cells in the Transwell plate, adding E2 (1× 10-7 mol/L) and growth factors (10 ng/ml TGF-β, 10 ng/ml

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EGF, 10 ng/ml PDGF-BB). After the induction, on the 7th day, immunofluorescence was used to test the special protein keratin. In the common culture plate we divided the cells into three groups. Group co-culture: BMSCs and hECs were cultured together; Group BMSCs: only BMSCs; GrouphECs: only hECs. And each group was cultured in three ways. After the induction, on the 14th and 20th day, Immune-fluorescence was used to test the special proteinkeratin. The protein of decidualization PRL and IGFBP1 were also tested by ELISA on the 3rd and 19th days. Results: Rat bone marrow mesenchymal stem cells and human endometrium stromal cells were isolated. After induction the keratin of BMSCs or human endometrium stromal cells was negative. The expression of PRL and IGFBP1 had no different with co-culture and single culture after 3 or 19 days culture (P>0.05). Conclusions: 1) Bone marrow mesenchymal stem cells were differentiate into endometrial stromal cells in the condition; 2) E2, growth factors and cAMP analog can induce Bone marrow mesenchymal stem cells differentiate into endometrial stromal cells, and the ability of them is similar; 3) In our experiment. Bone marrow mesenchymal stem cells hadn’t differentiate into endometrial epithelial cells.