Hanwang Zhang

Serum progesterone level effects on the outcome of in vitro fertilization in patients with different ovarian response: an analysis of more than 10,000 cycles

OBJECTIVEnTo investigate the relationship between serum P levels on the day of hCG administration and pregnancy outcomes in different responders undergoing IVF.nnnDESIGNnRetrospective study.nnnSETTINGnTeaching hospital.nnnPATIENT(S)nA total of 11,055 women who underwent their first IVF/intracytoplasmic sperm injection cycles and a subgroup of 4,021 women undergoing frozen-embryo transfer (FET) cycles.nnnINTERVENTION(S)nPatients underwent IVF-ET with the long GnRH agonist protocol. The ovarian response was classified as high (≥ 20 oocytes; n = 2,023), poor (≤ 4 oocytes; n = 827), or intermediate (remaining cases; n = 8,205) according to the number of oocytes retrieved. Clinical outcomes of IVF-ET and FET cycles were analyzed according to plasma P levels.nnnMAIN OUTCOME MEASURE(S)nOngoing pregnancy rates (PRs).nnnRESULT(S)nOngoing PRs in fresh cycle were inversely associated with serum P levels on the day of hCG administration for all patients. Different P threshold concentrations were determined according to different ovarian response: We proposed a serum P level of 1.5 ng/mL as the threshold for poor responders, 1.75 ng/mL for intermediate responders, and 2.25 ng/mL for high responders. Our study does not show negative results for elevated P levels on oocyte performance in terms of fertilization, cleavage rate, or PR of FET cycles within different ovarian responses, offering no evidence for a detrimental effect of high P on oocyte quality.nnnCONCLUSION(S)nElevated P levels on the day of hCG administration negatively influence PR regardless of different ovarian responses, although increased P threshold concentration is associated with better ovarian responses. The detrimental effect of P elevation on PR seems to be unrelated to oocyte quality in all responders.

Hsa-miR-222 Is Involved in Differentiation of Endometrial Stromal Cells in Vitro

Decidualization is a critical step during embryo implantation and characterized by the differentiation of endometrial stromal cells (ESCs) into decidual cells. Because miRNAs are important determinants of cellular fate specification, in this study, the miRNA expression in ESCs during in vitro decidualization was profiled by using a microarray. Significance analysis of microarrays revealed that 49 miRNA genes were differently (>2-fold) expressed between the noninduced ESCs and induced ESCs with a false discovery rate of 0. The expression variance of hsa-miR-222, 221, 143, 101, 30d, 30c, 181b, 27b, 29b, 507, and 23a was validated by using quantitative PCR (P < 0.05). Based on microRNA (miRNA) and mRNA expression variance and predicted target genes of miRNAs, a bioinformatic model of miRNAs controlling ESCs differentiation was formulated. Finally, we proved that down-regulation of has-miR-222 could decrease the number of cells in S phase during ESCs differentiation (P < 0.05). Antisense oligonucleotides of has-miR-222 could increase reporter gene expression by targeting the 3 untranslated regions of CDKN1C/p57kip2 mRNAs as well as increase CDKN1C/p57kip2 protein levels (P < 0.05). In conclusion, our results suggest that a subset of miRNAs play a key role in gene reprogramming during ESCs decidualization and that hsa-miR-222 participates in ESC differentiation by regulating ESCs terminally withdrawing from the cell cycle.

Is frozen embryo transfer cycle associated with a significantly lower incidence of ectopic pregnancy? An analysis of more than 30,000 cycles

OBJECTIVEnTo analyze the incidence of ectopic pregnancy (EP) in fresh compared with frozen-thawed cycles.nnnDESIGNnRetrospective cohort study.nnnSETTINGnTeaching hospital.nnnPATIENT(S)nThirty-one thousand nine hundred twenty-five women undergoing inxa0vitro fertilization-embryo transfer (IVF-ET) from January 2006 to Decemberxa02013.nnnINTERVENTION(S)nFresh IVF-ET compared with frozen-thawed ET (FET).nnnMAIN OUTCOME MEASURE(S)nIncidence of EP with fresh IVF-ET compared with frozen-thawed ET cycles, clinical pregnancy rate, and rate of EP per clinical pregnancy.nnnRESULT(S)nFor the fresh IVF cycles, 19,173 patients underwent oocyte retrieval; 15,042 had an ET, 6,431 of these patients (42.7%) had a clinical pregnancy, and among these 297 (1.97%) appeared to have an EP. The group of patients undergoing frozen-thawed ET (12,752 patients) included 12,255; there were 5,564 pregnancies (45.4%) and 124 ectopic implants (1.01%). The incidence of an EP per clinical pregnancy was 4.62% for the fresh transfer group compared with 2.22% for the frozen-thawed cycle group; this difference was statistically significant. In addition, the fresh ET cycles had the highest risk of EP, followed by day-3 embryo FET cycles; blastocyst FET cycles had the lowest risk of EP, and the differences were all statistically significant.nnnCONCLUSION(S)nFrozen-thawed ET cycles were associated with a statistically significantly lower risk of EP when compared with fresh cycles. These findings are consistent with ovarian stimulation being associated with an increased risk of EP.

Presence of hepatitis B virus in oocytes and embryos: a risk of hepatitis B virus transmission during in vitro fertilization

OBJECTIVEnTo define the risk of hepatitis B virus (HBV) transmission through oocytes and embryos from chronic HBV carriers.nnnDESIGNnLaboratory-based study.nnnSETTINGnResearch laboratory in a university hospital.nnnPATIENT(S)nThirty-one couples with hepatitis B surface antigen (HBsAg)-negative women and HBsAg-positive men, 41 couples with HBsAg-positive women and HBsAg-negative men, and 39 seronegative couples.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nHepatitis B virus DNA and RNA analyses in oocytes and embryos, and the location of virus particles containing HBsAgs.nnnRESULT(S)nHepatitis B virus DNA was detected in 3 of 18 male HBsAg-positive/female HBsAg-negative couples (and in 13 of 84 embryos) and 3 of 14 male HBsAg-negative/female HBsAg-positive couples (and in 15 of 71 oocytes and embryos). Hepatitis B virus RNA was detected in 9 of 13 male HBsAg-positive/female HBsAg-negative couples (and in 39 of 52 embryos) and 8 of 17 male HBsAg-negative/female HBsAg-positive couples (and in 30 of 63 oocytes and embryos). The HBsAg, which is present in the nuclei and cytoplasm of oocytes and embryos, was detected in 6 of 10 male HBsAg-negative/female HBsAg-positive couples (and in 13 of 20 oocytes and embryos). Hepatitis B virus DNA, HBV RNA, and HBsAg were not found in 135 oocytes and embryos from 39 seronegative couples.nnnCONCLUSION(S)nThe presence of HBV in oocytes and embryos suggests the possibility of vertical transmission of HBV via the germ line.

Regulation of endometrial receptivity by the highly expressed HOXA9, HOXA11 and HOXD10 HOX-class homeobox genes

STUDY QUESTIONnAre other HOX genes, in addition to HOXA10, involved in endometrial receptivity?nnnSUMMARY ANSWERnThe highly expressed HOXA9, HOXA11 and HOXD10 genes also appear to be involved in endometrial receptivity.nnnWHAT IS KNOWN ALREADYnWithin the HOX family of homeobox transcription factor genes are the leading candidates for the regulation of embryonic implantation. A crucial role of HOXA10 in endometrial receptivity has been well established.nnnSTUDY DESIGN, SIZE, DURATIONnTo identify HOX candidate genes, we performed data mining on all 39 human HOX genes in the Human body index gene expression database of normal human tissue. The temporal and spatial expression pattern of four highly expressed HOX genes in the human endometrium was determined. To further investigate the function of these Hox genes, we used a robust in vivo mouse model in which we blocked maternal Hox gene expression.nnnPARTICIPANTS/MATERIALS, SETTING AND METHODSnAnalysis of a gene expression profile set in the public domain consisting of 504 samples representing 95 different normal human tissues, showed that in addition to HOXA10, also HOXA9, HOXA11, HOXB6 and HOXD10 mRNA showed increased expression in the human endometrium (16 samples). The temporal and spatial expression pattern of these four HOX genes throughout the menstrual cycle was determined in the endometrium from 27 female patients eligible for IVF-embryo transfer with a normal cycle by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. The role of maternal Hoxa9, Hoxa11 and Hoxd10 was assessed in a mouse implantation model by expression knockdown using RNA interference. Forty mice were transfected with Hoxa9-, Hoxa11- or Hoxd10-specific small hairpin RNA (shRNA) constructs or a vector control by injection into the uterine horn at Day 2 after vaginal plug detection (Day 1) (160 mice in total). The effects were examined by qRT-PCR and western blot at Day 4 and litter sizes counted at Day 9 of pregnancy.nnnMAIN RESULTS AND THE ROLE OF CHANCEnHOXA10, HOXA9, HOXA11 and HOXD10 all showed increased expression during the mid-secretory phase of the menstrual cycle (P < 0.01). Knockdown of Hoxa9, Hoxa11 and Hoxd10 in the murine uterus resulted in significantly reduced average implantation rates (P < 0.01) and, with regard to four Hox target genes, also correlated with a significantly increased empty spiracles homolog 2 (Emx2) and insulin-like growth factor binding protein-1 (Igfbp1), and decreased integrin β3 (Itgb3) and leukemia inhibitory factor (Lif), expression (P < 0.01).nnnLIMITATIONS, REASONS FOR CAUTIONnMenstrual cycle stage was not confirmed by serum hormone analysis. We verified the absence of significant differences in stage-specific expression of the reference genes used in our study (ACTB/Actb and GAPDH/Gapdh) and therefore possible limitations of this approach were minimized. In addition, the translatability of our data from a mouse model to patients needs to be investigated further.nnnWIDER IMPLICATIONS OF THE FINDINGSnWe provide evidence that three other HOX genes in addition to HOXA10 are involved in endometrial receptivity, and that part of their function is asserted through several known HOX target genes, suggesting the presence of a central HOX signal transduction pathway.

Effect of Dehydroepiandrosterone Administration in Patients with Poor Ovarian Response According to the Bologna Criteria

Background Dehydroepiandrosterone (DHEA) is now widely used as an adjuvant to IVF treatment protocols in poor responders. However, clinical evidence for DHEA on improvement of ovarian response and IVF outcome is still limited, the validity of the results of the earlier studies, especially the varied inclusion criteria, is a subject of debate. Recently, the ESHRE Working Group developed a new definition, the Bologna criteria. The aim of the current study was to investigate the potential effect of DHEA treatment on in vitro fertilization (IVF) outcome of poor ovarian responders that fulfill the Bologna criteria. Methods This study investigated 386 poor ovarian responders that fulfill the Bologna criteria. Patients underwent IVF-ET treatment with the GnRH antagonist protocol. The study group contained 189 patients, who received 75 mg of DHEA daily (25 mg three times daily) before the IVF cycle. The control group was composed of 197 patients who received infertility treatment, but did not receive DHEA. The IVF outcome parameters in each group were compared. Results The study and control groups did not show statistically significant differences in terms of patient demographics characteristics, mean numbers of oocytes retrieved, mature oocytes, fertilization rate, cleavage rate, or embryo availability. While the DHEA group demonstrated significantly higher implantation rates (18.7% vs. 10.1%; P<0.01) and ongoing PRs (26.7% vs. 15.8%; P<0.05) as compared with the control. Conclusions DHEA pre-treatment does not significantly increase oocyte yield. However, the ongoing PRs in this subgroup of women are significantly higher after DHEA administration, suggesting that DHEA may increase IVF results by improving oocyte and embryo quality.

Administration of calcitonin promotes blastocyst implantation in mice by up-regulating integrin β3 expression in endometrial epithelial cells

STUDY QUESTIONnDoes exogenous calcitonin improve the efficiency of implantation in mice by increasing uterine receptivity?nnnSUMMARY ANSWERnThe administration of calcitonin could improve the efficiency of implantation by increasing the expression of several receptivity-related genes in endometrial epithelial cells (EECs).nnnWHAT IS KNOWN ALREADYnCalcitonin is one of the biomarkers of uterine receptivity, which is transiently produced in the uterine epithelia during the period of implantation both in humans and mouse.nnnSTUDY DESIGN, SIZE, DURATIONnHormone-replaced mice were used for in vivo experiments. To evaluate the effect of calcitonin on uterine receptivity, the expression of endometrial genes was analyzed 36 h after i.p. injection of 0.5 IU calcitonin in a treatment group versus saline in the control. To evaluate the effect of calcitonin on implantation efficiency in vivo, two groups received 0.5 IU or 2 IU calcitonin (i.p.) 24 h before embryo transfer, and a control group received saline (i.p.) (n = 18 mice per group). Implantation sites were counted 7 days after embryo transfer. The RL95-2 human endometrial carcinoma cell line was used to study the mechanisms underlying the effect of calcitonin on gene expression in the endometria. Using an in vitro model of endometrium-trophoblast interaction, established with RL95-2 cells and JAR (human choriocarcinoma cell line) trophoblast, endometrial receptivity was evaluated by comparing attachment and outgrowth of JAR spheroids in control and treatment groups.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnUterine receptivity in ovariectomized mice was induced by injection of estradiol and progesterone. Expression of eight genes in murine endometrium and RL95-2 cells was analyzed by real-time RT-PCR, western blot, immunohistochemical analysis, flow cytometry and enzyme-linked immunosorbent assay. We tested the effects of a protein kinase C inhibitor, matrigel and an antibody against integrin αvβ3 using RL95-2 cells and performed attachment and outgrowth assays using the in vitro model of endometrium-trophoblast interaction. Implantation efficiency was evaluated by counting the implantation sites after embryo transfer.nnnMAIN RESULTS AND THE ROLE OF CHANCEnCalcitonin up-regulated αvβ3 in RL95 cells, which in turn resulted in increased levels of the leukemia inhibitory factor (LIF) and heparin binding-epidermal growth factor (HB-EGF) mRNA (both P < 0.01 versus control) and protein (both P < 0.05 versus control). The attachment and expansion of JAR spheroids was promoted by pretreatment of EECs with calcitonin (P < 0.05 versus control) together with significantly increased expression of αvβ3, LIF and HB-EGF. Moreover, the injection of calcitonin in the preimplantation phase increased the total number of implantation sites in treatment groups (55 in control versus 78 and 85 in 0.5 and 2 IU groups, respectively). Compared with the control group (3.11 ± 2.14), the average number of implantation sites in the 2 IU calcitonin treatment group increased (4.72 ± 1.87, P = 0.022).nnnLIMITATIONS, REASONS FOR CAUTIONnExperiments were performed in mice and human cell lines but not in primary cultures of human endometrial cells.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe findings presented here have important implications, in that calcitonin administration (currently used for treatment of hypercalcemia or osteoporosis) may have clinical benefits in assisted reproduction programs, by facilitating endometrial receptivity and embryo implantation. However, further studies are required to confirm these findings.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis work was supported by National Science Foundation of China (No. 81170619). There are no financial or commercial conflicts in this study.

Blastocyst culture and cryopreservation to optimize clinical outcomes of warming cycles

Surplus embryos available for cryopreservation in fresh cycles are considered as having good potential for future use. However, the optimal stage of embryo cryopreservation remains unclear. In this study, 1190 patients with surplus embryos on day 3 were divided into two groups: cleavage-stage embryo cryopreservation (control group) and blastocyst cryopreservation (blastocyst group). The clinical outcomes of the subsequent warming cycles were evaluated. The proportion of cycles with blastocyst formation was 73.8% in the blastocyst group. Although in the blastocyst group, the cancellation rate of blastocyst transfer was increased due to lack of blastocysts available for cryopreservation, the blastocyst group achieved significantly higher rates of clinical pregnancy/cycle (43.2% versus 34.9%; P=0.003), pregnancy/transfer (59.5% versus 35.4%; P<0.001) and implantation (46.5% versus 22.2%; P<0.001) from the first warming cycle compared with the control group. In an embryo-number classified analysis, the clinical pregnancy rate was also higher in the blastocyst group. However, the cumulative pregnancy was similar between the two groups. Blastocyst culture as an embryo selection tool will not improve embryo viability but it will help patients to achieve pregnancy more quickly. Extended culture of surplus embryos to the blastocyst stage for cryopreservation optimizes the clinical outcomes.

The relationship between polycystic ovary syndrome, glucose tolerance status and serum preptin level

BackgroundPolycystic ovary syndrome (PCOS) is linked to obesity, impaired glucose tolerance and diabetes. Recently, studies have found that preptin enhances insulin secretion in rats and might play a role in the pathogenesis of diabetes and PCOS in humans. The aim of this study was to evaluate the relationship between PCOS, glucose tolerance status, and serum preptin level.MethodsThis study was conducted in a university-affiliated hospital from October 2010 to August 2011. Anthropometric parameters, sex hormone concentrations, blood pressure, lipid profiles, fasting glucose and insulin, 2-h blood glucose after glucose overloaded (2hOGTT), glycosylated haemoglobin (HbA1c), homeostasis model assessment-insulin resistance index (HOMA-IR), and serum preptin of the samples were analyzed.ResultsSixty-three PCOS patients, including 33 women with normal glucose tolerance (NGT) and 30 women with impaired glucose tolerance (IGT), and 63 patients without PCOS, including 35 women with NGT and 28 women with IGT were recruited in this study. For patients with and without PCOS, women with IGT had higher serum preptin levels compared with women with NGT. Preptin levels in PCOS patients were higher compared with patients without PCOS, but the difference was not significant. Fasting serum preptin levels correlated positively with TG, SBP, DBP, FBG, 2hOGTT, and HOMA-IR in simple regression analysis of the pooled data. While in multiple stepwise regression analysis, preptin levels were independently related with glucose tolerance, but not with PCOS.ConclusionsIrrespective of PCOS status, women with IGT had higher serum preptin levels compared with women with NGT. Preptin levels are related with glucose tolerance status, but not with PCOS status.

Myeloid ecotropic viral integration site 1 (MEIS) 1 involvement in embryonic implantation

BACKGROUND The HOXA10 homeobox gene controls embryonic uterine development and adult endometrial receptivity. The three-amino-acid loop extension (TALE) family homeobox genes like myeloid ecotropic viral integration site 1 (MEIS) provide enhanced target gene activation and specificity in HOX-regulated cellular processes by acting as HOX cofactors. METHODS AND RESULTS Analysis of an Affymetrix data set in the public domain showed high expression of MEIS1 in human endometrium. MEIS1 expression was confirmed during the human menstrual cycle by RT–PCR and in situ hybridization and was increased during the secretory compared with proliferative phase of the cycle (P = 0.0001), the time of implantation. To assess the importance of maternal Meis1 expression in a mouse model, the uteri of Day 2 pregnant mice were injected with Meis1 over-expression or small interfering RNA (siRNA) constructs. Blocking Meis1 expression by siRNA before implantation significantly reduced average implantation rates (P = 0.00001). Increased or decreased Meis1 expression significantly increased or decreased the expression of integrin β3, a transcriptional target of HOXA10 and an important factor in early embryo-endometrium interactions (P = 0.006). Manipulating Meis1 expression before implantation also dramatically affected the number of pinopodes, uterine endometrial epithelial projections that develop at the time of endometrial receptivity. CONCLUSIONS The results suggest that in mouse, meis1 contributes to regulating endometrial development during the menstrual cycle and establishing the conditions necessary for implantation.