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Objective To investigate the effects of cryoprotectants on the expression of AQP7 in oocytes. Design Experimental animal study. Setting University-based research laboratory. Animal(s) Adult female C57BL/6J mice. Intervention(s) In metaphase II (MII) oocytes obtained from adult female C57BL/6J mice and from donations by fertile women, the mouse oocytes were treated with human tubal fluid medium containing 8% ethylene glycol (EG), 9.5% dimethylsulfoxide (DMSO), and 0.5 M sucrose, respectively; 293T cells transfected with GFP-hAQP7 expression vector were treated with the same solutions. Main Outcome Measure(s) AQP7 expression in oocytes examined by reverse-transcriptase-nested polymerase chain reaction and immunofluorescence, changes in the volume of mouse oocytes treated with different solutions calculated to determine their permeability to water, and survival rates of vitrified oocytes. Result(s) AQP7 is expressed in human and mouse oocytes. Cryoprotectants, including EG, DMSO, and sucrose, up-regulated AQP7 expression in mouse oocytes and 293T cells transfected with GFP-hAQP7 expression vector. Compared with other cryoprotectants, DMSO stimulated higher expression of AQP7, and this was associated with faster cell volume recovery and lower survival rates of vitrified oocytes. Conclusion(s) DMSO up-regulates AQP7 expression in mouse oocytes more than EG. This may facilitate water diffusion and reduce the time for oocytes to reach osmotic balance with the cryoprotectant solution during cryopreservation.

Objective To investigate the possible roles of cyclin B1/cyclin-dependent kinase (cdc2) and Polo-like kinase 1 (Plk1) in the pathogenesis of endometriosis. Design A case-control study. Setting University hospital. Patient(s) Patients with or without endometriosis were diagnosed by pathological examination or laparoscopy. The patients with the following criteria within the past 6 months were excluded: endocrine or inflammatory diseases, pregnancy or lactation, hormonal therapy, and neoplasm in the uterine cavity. Intervention(s) Eutopic and ectopic endometria were obtained at the time of surgery. Blood was collected on the same day as surgery. Main Outcome Measure(s) The mRNA/protein expression and localization of cyclin B1, cdc2, and Plk1 in endometrium, and serum levels of E 2 and P. Result(s) The expression levels of cyclin B1 and Plk1, but not cdc2, in ectopic endometria were significantly higher than in eutopic endometria. The immunohistochemical staining of cyclin B1 and Plk1 was detected in the nuclei of ectopic and eutopic endometrial cells. Furthermore, ectopic endometrial expression levels of cyclin B1 or Plk1 were positively correlated with serum E 2 levels. Conclusion(s) Cyclin B1 and Plk1 may play important roles in the pathogenesis of endometriosis by mediating ectopic endometrial cell proliferation under regulation of ovarian hormones.

Objective To investigate c-fos and matrix metalloproteinase-9 (MMP-9) expression in the endometrium from women with or without endometriosis throughout the menstrual cycle, and to explore the correlation of c-fos expression with MMP-9 expression and 17β-E 2 levels in serum. Design Molecular studies in human tissue. Setting A women's hospital in China. Patient(s) Fifty-five premenopausal women (25 with endometriosis and 30 without endometriosis) undergoing laparoscopic surgery or hysterectomy. Intervention(s) Eutopic and ectopic endometrium tissue were obtained at the time of surgery. Peripheral sera were also collected on the same day. Main Outcome Measure(s) Immunohistochemical localization of c-fos in the endometrium, c-fos and MMP-9 protein levels in the endometrium, and 17β-E 2 levels in the serum. Result(s) c-fos protein was predominantly located in the nuclei of glandular epithelial cells and stromal cells. c-fos and MMP-9 protein levels in paired eutopic and ectopic endometria from women with endometriosis were significantly higher than those in the endometrium from women without endometriosis. No significant difference in c-fos or MMP-9 protein levels was observed between paired eutopic and ectopic endometria. c-fos protein levels in endometrium positively correlated with endometrial MMP-9 levels and serum 17β-E 2 levels. Conclusion(s) Expression of c-fos in the human endometrium may be regulated by 17β-E 2 , and c-fos may be involved in development of endometriosis by promoting MMP-9 gene expression and subsequently the invasive potential of endometrial explants.

Objective To assess the effects of autologous mature follicular fluid (FF) on the in vitro maturation (IVM) outcome of human cumulus-removed oocytes from routine stimulated ovaries, particularly on the IVM co-culture outcome with autologous cumulus cells. Design Prospective comparison study. Setting A provincial reproductive medicine and research center. Patient(s) A total of 196 germinal vesicle (GV) and 192 metaphase I (MI) stage oocytes were recruited from 151 infertile women by ovarian stimulation for routine intracytoplasmic sperm injection (ICSI) program. Intervention(s) Two novel IVM systems assisted with either autologous mature FF alone or both autologous mature FF and autologous cumulus cells were developed to mature in vitro MI and GV stage oocytes, respectively. A standard IVM system was used as the control. Main Outcome Measure(s) Cumulative IVM rate of oocytes and the subsequent fertilization, cleavage, and preimplantation embryonic development. Result(s) By adding autologous mature FF to the medium of the IVM system, the nuclear maturation of human cumulus-removed oocytes was significantly promoted in both speed and rate, but not the corresponding fertilization, cleavage, and preimplantation embryonic development. Conclusion(s) Autologous mature FF favors the IVM of human cumulus-removed oocytes, including the IVM co-culture with autologous cumulus cells, mainly in nuclear, but not cytoplasmic, maturation.

Objective To explore the importance of follicle-stimulating hormone receptor (FSHR) in granulosa cells in the ovarian response to gonadotropin stimulation. Design Prospective study. Setting A women's hospital in China. Patient(s) One hundred infertile women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (rFSH). Intervention(s) These women were divided into three groups: poor, moderate, and high responders, according to the number of follicles with diameter ≥14 mm. The FSHR expression at both mRNA and protein levels was determined by either reverse transcription–polymerase chain reaction or Western blot in granulosa cells. E 2 concentrations in serum and FSH levels in serum/follicular fluid (FF) were measured by electrochemiluminescence immunoassay. Main Outcome Measure(s) Relative expression of mRNA and protein of FSHR in granulosa cells, serum E 2 , FSH level in serum and FF, and the number of mature follicles. Result(s) The expression of FSHR, at both the mRNA and protein levels, was significantly different among the three groups, with the lowest expression in the poor responders. The level of FSHR protein was positively correlated with the peak level of serum E 2 and the number of mature oocytes. FSH levels in FF and the dosage of rFSH used were significantly different among the three groups, with the highest values in the poor responders. Conclusion(s) Different levels of FSHR expression in granulosa cells result in different ovarian response, and lower expression of FSHR may account for poor ovarian response to gonadotropin stimulation, which suggests the critical role of FSHR in the ovarian response to gonadotropin stimulation.

Objective To establish a mouse model for endometriosis and to evaluate roles of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in the formation of disease. Design Experimental laboratory study. Setting A women's hospital in China. Patient(s) and Animal(s) Ten women with endometriosis and 10 control women, as well as ICR mice. Intervention(s) Endometrial fragments were transplanted in the peritoneal cavities of mice at minilaparotomy. Transplants were observed and then removed for the assessment of morphology and immunohistochemical staining of VEGF and MMP-2. Main Outcome Measure(s) Observation of transplants, expression of VEGF and MMP-2. Result(s) On days 1 and 2, glandular and stromal cells were viable at the margins of transplants. On day 3, the transplants were surrounded by mesothelial cells, and the endometrial glands and stromal cells were clearly viable at the interface. The scores of VEGF and MMP-2 of viable glandular cells of transplants were increased compared with the ones before transplantation. The scores of VEGF and MMP-2 of transplants from women with endometriosis were higher than those of control women. Conclusion(s) Endometrial transplants from the patients with endometriosis express more VEGF and MMP-2 than endometrium in control women, suggesting that VEGF and MMP-2 may expedite the formation of endometriosis in its early stage.

Objective To determine matrix metalloproteinase 2 (MMP-2) levels in sera and peritoneal fluids of women with or without endometriosis-associated infertility and to determine whether MMP-2 had correlation with 17β-E 2 and P in the sera and peritoneal fluids of the patients with endometriosis. Design A case–control study. Setting A women's hospital in China. Patient(s) Forty patients with endometriosis-associated infertility undergoing laparoscopy and 18 women undergoing tubal ligation. Intervention(s) Sera and peritoneal fluids were collected from women with or without endometriosis. Main outcome measure(s) Matrix metalloproteinase 2, 17β-E 2 , and P concentration. Result(s) Compared with the control women, the patients with endometriosis had higher MMP-2 levels in their sera and peritoneal fluids. Serum and peritoneal fluid MMP-2 levels were higher in the proliferative phase than in the secretory phase. The level of MMP-2 in the patients was correlated positively with 17β-E 2 in serum and peritoneal fluid and negatively with P. Conclusion(s) The results demonstrated a correlation between MMP-2 concentrations and steroid hormones in sera and peritoneal fluids of the patients with endometriosis-associated infertility. Estradiol might up-regulate MMP-2, resulting in the formation of endometriosis. Progesterone might down-regulate MMP-2 to inhibit the formation and development of endometriosis.

Objective To evaluate cardiovascular and metabolic features of offspring conceived by in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI). Design Literature review and meta-analysis. Setting Not applicable. Patient(s) Offspring from IVF-ICSI versus natural conception. Intervention(s) None. Main Outcome Measure(s) Systolic and diastolic blood pressure (SBP and DBP), cardiovascular function, body mass index (BMI), and lipid and glucose profiles. Result(s) We included 19 studies that had recruited 2,112 IVF-ICSI and 4,096 naturally conceived offspring, ranging from childhood to early adulthood. The blood pressure levels of IVF-ICSI offspring were statistically significantly higher than those of naturally conceived offspring (weighted mean differences and confidence intervals: 1.88 mm Hg [95% CI, 0.27, 3.49] for SBP and 1.51 mm Hg [95% CI, 0.33, 2.70] for DBP). In addition, cardiac diastolic function was suboptimal and vessel thickness was higher among IVF-ICSI offspring. Compared with the metabolism of naturally conceived offspring, IVF-ICSI offspring displayed comparable BMI, lower low-density lipoprotein cholesterol levels, and higher fasting insulin levels. Conclusion(s) Children conceived by IVF-ICSI manifested a minor yet statistically significant increase in blood pressure without the clustering of increased BMI or impaired lipid metabolism by early adulthood. Our findings indicate a risk of cardiovascular disease among IVF-ICSI offspring, which calls for longer-term follow-ups and further investigation.

Objective To examine epithelial Na + channel (ENaC) expression in endometrium of overweight/obese women with polycystic ovary syndrome (PCOS) during the window of implantation, and to explore the mechanism linking leptin-mediated reduction of γ-ENaC to low endometrial receptivity. Design Controlled, prospective, clinical, experimental study. Setting University-based infertility center. Patient(s) Blood and endometrium samples were collected from 12 control women and 12 overweight/obese PCOS patients. Pregnancy outcomes were obtained from 245 women with male-factor infertility (533 cycles) and 57 infertile women with PCOS (120 cycles) who underwent intrauterine insemination. Intervention(s) Human endometrial biopsies. Main Outcome Measure(s) Expression of ENaC mRNA and protein in endometrium. Result(s) The expression of γ-ENaC decreased in the secretory phase endometrium of PCOS patients who showed increased serum leptin levels. In cultured endometrial cells (Ishikawa cells), leptin dose-dependently down-regulated the expression of γ-ENaC and reduced the JAr spheroid attachment rate, which could be blocked by knockdown of STAT3, a signal in the pathway of leptin receptor activation. The overweight/obese PCOS patients with increased serum leptin levels showed a significantly increased biochemical pregnancy rate, suggesting that high leptin might attenuate endometrial receptivity and increase very early pregnancy loss. Conclusion(s) High serum leptin may reduce endometrial receptivity by activating the STAT3 signal pathway and down-regulating γ-ENaC expression in the endometrium. These results provide valuable new insights into the molecular mechanisms linking abnormal ENaC gene expression to early pregnancy loss in overweight/obese PCOS patients.

The growth differential factor-9 (GDF-9) gene, an oocyte-specific factor, was screened in 106 Chinese women with diminished ovarian reserve (DOR), and three single-nucleotide polymorphisms, c.G169A, c.C447T and c.G546A, were detected. We found GDF-9 c.G546A, but not c.G169A or c.C447T, to be correlated with the poor ovarian stimulation and in vitro fertilization outcomes in women with DOR.

Objective To evaluate the cholesterol metabolism linked to assisted reproductive technology (ART) by analyzing the expression levels and DNA methylation patterns of the insulin-induced gene (INSIG), sterol regulatory element-binding protein (SREBP), and SREBP cleavage-activating protein in the fetus and placenta. Design Experimental research study. Setting An IVF center, university-affiliated teaching hospital. Patient(s) Four patients groups were recruited: pregnancies after IVF/intracytoplasmic sperm injection (ICSI) (n = 55), natural pregnancies (n = 40), multifetal reduction after IVF/ICSI (n = 56), and multifetal reduction after controlled ovarian hyperstimulation (COH) (n = 42). Intervention(s) Expression and DNA methylation of INSIG-SREBP- SREBP cleavage-activating protein in the fetus and placenta samples were determined. Main Outcome Measure(s) The expression and DNA methylation patterns were tested by real-time quantitative polymerase chain reaction (PCR) and pyrosequencing. Result(s) In the ICSI treatment group, significantly higher levels of triglycerides and apolipoprotein-B were observed in cord blood compared with controls. Meanwhile, in ICSI-conceived fetuses, the expression of INSIG1 was significantly higher, and methylation rates were lower, than in the IVF and control groups. Furthermore, in the placenta, the INSIG1 and SREBF1 transcripts were also significantly higher with lower methylation rates in the ICSI group than in the IVF and control groups. Conclusion(s) Our results indicated that the dysregulation of INSIG1 and SREBF1 caused by ART were observed not only in the fetus but also in the placenta, primarily in the ICSI group. However, the long-term sequelae of this dysregulation should be closely followed.

Objective To evaluate whether the genomic imprinting status of assistant reproductive technology (ART)–conceived offspring is stable. Design Prospective clinical observational study. Setting In vitro fertilization (IVF) center, university-affiliated teaching hospital. Patient(s) Sixty ART-conceived babies (30 IVF and 30 intracytoplasmic sperm injection [ICSI]) and 60 naturally conceived babies. Intervention(s) Collection of umbilical cord blood and peripheral blood samples. Main Outcome Measure(s) Expression profile was examined by microarray and real-time reverse-transcription polymerase chain reaction (PCR), allele-specific expression was studied by direct sequencing after PCR, and DNA methylation status was investigated by sodium bisulfite sequencing. Result(s) Hierarchic clustering demonstrated no obvious clustering between the ART- and naturally conceived offspring, suggesting similar genomic imprinting expression between the two groups. Three differentially expressed genes were identified in ART-conceived offspring, with PEG10 and L3MBTL up-regulated and PHLDA2 down-regulated. Allele-specific expression of the differentially expressed imprinted genes was maintained in the majority of the ART- and naturally conceived offspring. However, in one ICSI case, monoallelic expression of L3MBTL was disrupted and all CpGs were completely unmethylated. These were not inherited from the parents. Conclusion(s) The global profile of imprinting is stable in children conceived through ART. However, imprinting of a few specific imprinted genes may be vulnerable in a fraction of ART-conceived children.

Objective To examine the effect of assisted reproductive technology on the stability of DNA methylation at differentially methylated regions (DMRs) in twins conceived by IVF. Design Prospective clinical observational study. Setting IVF center, university-affiliated teaching hospital. Patient(s) Fifty-nine pairs of twins were recruited, including 29 pairs conceived through IVF and 30 pairs of naturally conceived twins. Intervention(s) Collection of umbilical cord blood samples. Main Outcome Measure(s) DNA was extracted from umbilical cord blood. Two maternally methylated regions (KvDMR1 and PEG1 ) and one paternally methylated region ( H19/IGF2 DMR) were analyzed using bisulfite-based technologies. Result(s) Although H19/IGF2 DMR and KvDMR1 showed slightly more variable levels of methylation in IVF cases than in spontaneous cases, methylation indices did not reveal significant differences at three DMRs between IVF-conceived and naturally conceived twins. Conclusion(s) Our results suggest no significant increase in imprint variability at these DMRs, but the greater variance in the IVF twins has a biologically meaningful consequence and may be a topic for future investigation. Large cohorts are needed to systematically assess the potential epigenetic risk in twins conceived with IVF.

Objective To investigate whether the embryotoxic effect of peritoneal fluid (PF) from infertile women with mild endometriosis on mouse oocytes and embryos is associated with changes in embryonic epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and their receptors. Design Experimental animal study. Setting University-based research laboratory. Animal(s) Adult ICR mice. Intervention(s) Peritoneal fluid was obtained from fertile women with no endometriosis (PF-NE) and infertile women with mild endometriosis (PF-E). In vitro fertilization was performed, and mouse two-cell stage embryos were cultured in human tubal fluid medium with or without PF. Main Outcome Measure(s) Rates of fertilization, cleavage, and blastulation. The embryonic EGF and IGF-I levels in culture medium were analyzed by enzyme-linked immunosorbent assay, and EGF receptor, IGF-I receptor, and phosphorylated extracellular signal-regulated protein kinases (p-ERK) expression was determined by immunofluorescence and confocal microscopy. Result(s) When oocytes and embryos were cultured in media with PF-E, the fertilization capability of oocytes and the development potential of embryos were decreased. The levels of embryonic EGF, IGF-I, and their receptors were increased. However, p-ERK of the postreceptor signal transduction pathway was down-regulated. Conclusion(s) Endometriotic PF may attenuate oocyte and embryo development by impairing embryonic growth factor/receptor/signal transduction, resulting in endometriotic infertility.

Objective To report an unusual case of congenital cervical atresia and tubal factor infertility, achieving triplet pregnancy through transmyometrial embryo transfer (ET) resulting in the delivery of healthy twins after multifetal pregnancy reduction (MFPR). Design Case report and literature review. Setting University hospital. Intervention(s) Controlled ovarian hyperstimulation, oocyte retrieval, in vitro fertilization, transmyometrial ET, MFPR procedure for triplet pregnancy. Patient(s) A 33-year-old patient with endometriosis-associated and tubal factor infertility who underwent uterovaginal canalization surgery for congenital cervical atresia 9 years ago. Main Outcome Measure(s) Successful delivery after transmyometrial ET followed by MFPR. Result(s) A total of three IVF cycles and four procedures of transmyometrial ET were performed. In the third cycle a triplet pregnancy was achieved. MFPR was performed 35 days after embryos transfer, two healthy babies delivered at 31 weeks gestation. Conclusion(s) With aggressive therapy, successful pregnancy is possible in similar patients.

Objective To investigate the fertilization ability, chromatin structure, and DNA integrity of spermatozoa after being cryopreserved within an empty zona pellucida (ZP). Design Prospective study. Setting Reproductive research center in a university-affiliated hospital. Patient(s) Normozoospermic patients. Intervention(s) Spermatozoa were cryopreserved within the ZP or with traditional methods. Main Outcome Measure(s) The sperm recovery rate, sperm motility, fertilization ability, chromatin structure, and DNA integrity were assessed before and after cryopreservation. Result(s) Significantly higher sperm recovery rate was identified for the spermatozoa cryopreserved within the ZP than those cryopreserved with traditional methods, but the motility recovery was similar. Frozen-thawed samples showed increased damage to the sperm chromatin and DNA, which were assessed by acridine orange test (AO) and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay; however, no difference of chromatin and DNA integrity was observed for spermatozoa cryopreserved within the ZP or with traditional methods. In addition, the fertilization ability, as assessed by injecting spermatozoa into hamster oocytes, was similar for spermatozoa cryopreserved with different cryopreservation methods. Conclusion(s) Cryopreservation of spermatozoa within an empty ZP results in higher sperm recovery rate, and the post-thaw sperm functions of spermatozoa cryopreserved within the ZP are comparable with spermatozoa cryopreserved with traditional methods.

Objective To investigate the risks of chromosome mutation after ART for couples with comparable genetic backgrounds. Design Prospective clinical observational study. Setting In vitro fertilization center at a tertiary-care, university-affiliated teaching hospital. Patient(s) Ninety-seven male children whose fathers have normal spermatogenesis were recruited, including 19 babies conceived through IVF, 18 babies conceived through intracytoplasmic sperm injection (ICSI), and 60 naturally conceived babies, as well as the babies' fathers. Intervention(s) Collection of peripheral and umbilical cord blood samples. Main Outcome Measure(s) The Yq genetic status of the babies and fathers according to 13 Y-specific markers covering four azoospermia factor (AZF) subregions, the karyotype, and the neonatal examination. Result(s) We found that all children had a normal 46, XY karyotype, but de novo Y-chromosome microdeletions were identified in 1 (5.3%) of 19 IVF offspring and in 3 (16.7%) of 18 ICSI offspring. The incidence of de novo Y-chromosome microdeletion in male children conceived through ICSI or IVF was statistically significantly higher than that in those conceived naturally (10.8% vs. 0). In four babies with microdeletion, one was complicated, with hypospadias. Conclusion(s) Our results, for the first time, indicate that risks of gene mutation may increase in the ART offspring, even though their fathers have normal spermatogenesis and genetic background. Hence, intense attention should be placed on genetic safety in the ART children, and the benefits and risks of adopting ART should be balanced gingerly.

WITHDRAWN TABLE 1. compared the results between the two groups Group A Group B P value cycles 59 30 Age of female 30.73 4.16 29.60 3.36 0.201 Cycles of no embryo to transfer 7 3 1.000 D0 oocytes 17.44 8.03 18.53 10.26 0.583 2PN/D0 oocytes 57.05%(587/1029) 57.19%(318/556) 0.955 D3 embryos 491 249 D3 embryos /D0 oocytes 47.72%(491/1029) 47.78%(249/556) 0.264 good blastocysts 147 135 good blastocysts /D3 embryos 29.94% (147/491) 54.22% (135/249) 0.000 genetic balance embryos 142 82 genetic balance embryos/D3 embryos 28.92% (142/491) 32.93% (82/249) 0.262 available embryos 88 82 available embryos /D3 embryos 17.92% (88/491) 32.93% (82/249) 0.000 ongoing pregnancy patients 20 18 ongoing pregnancy ratespregnancy rates 38.46% (20/52) 69.23% (18/26) 0.000 P-92 Tuesday, October 21, 2014 CELL FREE FETALDNATESTINGAND INFERTILITY PATIENTS: WILL DISCORDANT GENDER RESULTS BE MORE COMMON THAN PREVIOUSLY EXPECTED? A PLEA FOR CAUTION AND RESEARCH. S. Tsai, N. M. Grindler, E. Beaver, R. Longman, A. Cooper. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Washington University, St. Louis, MO; Division ofMaternal Fetal Medicine and Ultrasound, Department of Obstetrics and Gynecology, Washington University, St. Louis, MO. OBJECTIVE: Noninvasive prenatal testing by analysis of cell free fetal DNA (cffDNA) in maternal circulation is a rapidly evolving field with clinical applications including detection of fetal aneuploidy, Rh genotyping and fetal gender. CffDNA originates from sloughing trophectoderm cells and clears maternal plasmawithin hours of delivery. Prior studies have confirmed similar levels of circulating cffDNA in maternal circulation for IVF pregnancies compared to naturally occurring pregnancies. Our institution has been tracking gender discordant cffDNA results. Our objective was to analyze these patients to determine the risk factors for discordant gender cffDNA results. DESIGN: Descriptive case series. FERTILITY & STERILITY MATERIALS AND METHODS: Patient #1: 37yo G1, singleton pregnancy conceived after IVF utilizing endometrial cell coculture, derived from previously frozen maternal endometrial biopsy cells; cffDNA testing at 12.5 weeks. Patient #2: 36yo G4P0, heterotopic pregnancy conceived after the transfer of 3 embryos during a fresh IVF cycle. Underwent laparoscopic right salpingectomy at 7.6 weeks; cffDNA testing at 12.3 weeks. Patient #3: 33yo G1, spontaneously conceived dichorionic, triamniotic triplet pregnancy with demise of 2 of the fetuses diagnosed on ultrasound at 8.3 weeks. cffDNA testing at 20.3 weeks. Patient #4: 35yo G3P1, spontaneously conceived singleton pregnancy; cffDNA testing at 10.2 weeks. RESULTS: Patient #1: cffDNA 46,XX, ultrasound (US) male fetus; amniocentesis mosaic 45X/46XY; normal healthy male at birth. Patient #2: cffDNA 46,XY, US female fetus, healthy female at birth. Patient #3: cffDNA 46,XY, US female fetus, IUGR at term, healthy female at birth. Patient #4: cffDNA 46,XX, US male fetus; healthy male at birth. CONCLUSION: ART pregnancies represent 1-2% of live births in the US. Yet, ART and multiple pregnancies are overrepresented in this case series. We postulate that this discordance may be a result of multiple implantation sites and continued sloughing trophectoderm despite removal or demise, underlying mosaicism not clinically detected, or alterations in implantation from laboratory techniques and/or gamete/embryo micromanipulation. We believe this case series should heighten awareness, caution and further research in this unique patient population with regard to gender discordance in cffDNA testing. Supported by: 1K12HD063086-01 (ARC). P-93 Tuesday, October 21, 2014 WHICH IS BETTER FOR ROBERTSONIAN TRANSLOCATION’S PGD CYCLES,BLASTOMERE FISH OR TROPHECTODERM ARRAY? J. Huang, Y. Lian, N. Zhao, P. Liu, J. Qiao. Peking University Third Hospital, Beijing, China. OBJECTIVE: To explore a better protocol of the PGD cycles for Robertsonian translocations. DESIGN: Retrospective study. MATERIALS ANDMETHODS: From January 2012 to June 2013, a total of 89 PGD cycles for Robertsonian translocations were done in our reproductive center, including 59 blastomere FISH cycles (defined as GroupA) and 30 trophectoderm array cycles (defined as Group B). In group A, one blastomere was biopsied at D3, then FISHed by two chromosomal probes. After diagnosed and blastocyst culture, one or two blastocysts were transferred in each fresh cycle or one or two surplus blastocysts were transferred in several thawing cycles. In group B, several trophectoderm cells were biopsied at D5 or D6, then diagnosed by CGH or SNP arrays with 24 chromosomes. All the transfer cycles in groupBwere in thawing cycles with single blastocyst transferred. Compared the oocytes number, 2PN, D3 embryos, genetic balance embryos, good blastocysts, available embryos and ongoing pregnancy rates between the two groups.

in the follicular fluid (FF) collected during Intra cytoplasmic sperm injection (ICSI) to the ovarian response, endometrial thickness, embryological and clinical outcomes. DESIGN: Cross sectional observational study. MATERIALS ANDMETHODS: Couples who presented to a university affiliated fertility center for ICSI secondary to isolated male factor infertility during a one-year period were approached for participation. Women under the age of 38 year who consented and from whom we obtained FF samples for analysis (94) were included in the study. The FF samples were centrifuged, and stored in liquid nitrogen. Two organochlorine pesticides (OCPs) Lindane and DDT, Three Organophosphate (OPs) Chlorpyrifos, Diazinon, Malathion, one Chloroacetanilide (Pretilachlor) and two Pyrethroid (Bioallethrin and bCyfluthrin) were examined in the FF using Gas Chromatography/Mass Spectrometry. Correlations between the level of the above pesticides and ovarian response, endometrial thickness, embryological and clinical outcomes were done. Multiple regression analysis was used to correlate the values of the pesticides to the outcomes. RESULTS: The mean age of study participants was 31.5 6.2 years. Baseline FSH of study participants was 7.6 2.6, and Estradiol 59.3 10.5. There were significant negative correlations between FF concentrations of the 8 examined pesticides on the endometrial thickness (Adjusted r1⁄40.2, P1⁄40.0001). However, Pretilachlor, chloropyrifos, B-cyfluthrin, and Diazinon were the only toxic agents that negatively correlated with the number of the oocytes retrieved (adjusted r1⁄40.07, P1⁄40.0001). Fertilization and early embryos cleavage rates were negatively correlated with Pretilachlor and B-cyfluthrin P < 0.05. Moreover, high concentrations of Lindane, DDT, Diazinon , and chloropyrifos were significantly associated with fewer number of implanted embryos (adjusted r1⁄40.2, P1⁄4 0.006, 0.02 , 0.0001, and 0.006, respectively). CONCLUSION: High FF concentrations of toxic pesticides including OCPs, OPs, Chloroacetanilide, Pyrethroid may adversely affect the different stages of ICSI process and consequently its embryological outcome. Still more data is needed to evaluate the effect of these pesticides on the clinical outcome.

Objective To assess the expression patterns of SAM68 in the testes of azoospermic patients with normal and abnormal spermatogenesis. Design Retrospective study and in vitro study. Setting University hospital. Patient(s) Testicular biopsies of azoospermic men with normal spermatogenesis (OAZ; n=20), with maturation arrest at the spermatocyte stage (MA; n=20), and with Sertoli cell–only syndrome (SCOS; n=10). Intervention(s) No interventions with patients. Knockdown of Sam68 was performed in the GC-2spd(ts) cell line. Main Outcome Measure(s) SAM68 expression was analyzed using quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry analysis in tissues. Moreover, Sam68 was knocked down in GC-2spd(ts) cells. Cell viability was measured using the MTT assay, and the apoptosis rate was detected using flow cytometry with the Annexin V–FITC kit. Result(s) Using qRT-PCR, the expression level of testicular SAM68 mRNA in MA and SCOS patients was statistically reduced compared with in OAZ patients. In addition, using qRT-PCR, Western blot, and immunohistochemistry analyses, mRNA and protein expressions of SAM68 were absent or barely detectable in testicular tissues in 45% (9 of 20) of patients with MA and in all patients with SCOS. Furthermore, decreased expression of Sam68 suppressed germ cell proliferation and induced apoptosis in transfected GC-2spd(ts) cells. Conclusion(s) Deficient SAM68 expression was observed in the human testis with MA at the spermatocyte stage and SCOS. These results may offer new perspectives on the molecular basis of abnormal spermatogenesis.

Objective To report the first clinical experience of preimplantation genetic diagnosis (PGD) for X-linked agammaglobulinemia (XLA), which resulted from a point mutation in Bruton's tyrosine kinase (BTK) gene. Design Case report. Setting Clinical IVF laboratory. Patient(s) A couple carrying BTK gene mutation (c.832C>T) that is associated with XLA. Intervention(s) Day 3 embryos, obtained by IVF with intracytoplasmic sperm injection, underwent blastomere biopsy and DNA testing via nest polymerase chain reaction and restriction enzyme analysis to detect the presence of a maternal BTK mutation. Main Outcome Measure(s) Successful PGD for a BTK gene mutation and transfer of healthy embryos. Result(s) The patient received two PGD cycles. In total, 19 oocytes were fertilized, 15 embryos were suitable for embryo biopsy, 6 were diagnosed as unaffected, 6 were carriers, 2 were mutant, and the remaining 1 did not provide any diagnosis. In two treatment cycles, two ETs of three embryos each were performed. Unfortunately no pregnancy was achieved. Conclusion(s) We are the first to report a case of PGD for XLA. The result indicates that PGD is feasible for this genetic disease and may represent a viable alternative to prenatal diagnosis with the subsequent option of terminating an affected pregnancy.