An Incidental Detection of a Rare UPD in SNP-Array Based PGT-SR: A Case Report.

Uniparental disomies (UPD) refers to the inheritance of both homologs of a chromosome from only one parent with no representative copy from the other parent. UPD was with an estimated prevalence of 0.15‰ in population. Current understanding of UPD was limited to subjects for which UPD was associated with clinical manifestation due to imprinting disorders or recessive diseases. Segmental UPD was rare, especially for a segmental UPD with a combination of hetero- and isodisomy. This paper presents a couple with reciprocal translocation 46,XY, t(14;22)(q32.3;q12.2) for PGT-SR. Among 8 biopsied blastocysts, one euploid blastocyst (No.4) with segmental loss of heterozygosity (LOH)(22) [arr[hg19] q12.1q22.3 (28,160,407 - 35,407,682)] was detected by B allele frequency. We found the chromosome contained both UPiD(22) [arr[hg19] q12.1q22.3 (28,160,407 - 35,407,682) ×2 hmz mat] and UPhD(22) [arr[hg19] q22.3qter(35,407,682 - 51,169,045) ×2 htz mat] by haplotype analysis. UPDtool software confirmed the result. What's more, the segmental UPD and reciprocal translocation shared the same breakpoint, chr22q12.1 (28,160,407), while the breakpoint between iso- and heterodisomy was chr22q22.3 (35,407,682). We reported the first segmental UPD with a combination of hetero- and isodisomy, which may result from aneuploidy rescue. This case emphasizes the importance of the combination of comprehensive chromosome screening and haplotype analysis to reduce the risk of misdiagnosis.

GDF9His209GlnfsTer6/S428T and GDF9Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

BACKGROUND: Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. METHODS: Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. CONCLUSIONS: GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.

Impact of dehydroepiandrosterone sulfate and free androgen index on pregnancy and neonatal outcomes in PCOS patients.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder associated with infertility and pregnancy complications. The pathogenesis of PCOS and its impact on reproductive function may be influenced by the source of androgens, including testosterone, free androgen, dehydroepiandrosterone sulfate (DHEAS). However, the differential effects of these androgen on pregnancy and neonatal outcomes and the cut-off value of East Asian population with PCOS remain unclear. METHODS: A retrospective cohort study was conducted at the Reproductive Medicine Center of the First Affiliated Hospital of Sun Yat-sen University from January 2015 to November 2022, involving 636 cycles of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). Subgroup analyses were performed using cut-off values of 6.4 for free androgen index (FAI), 9.5 µmol/L for DHEAS. Pregnancy and neonatal outcomes were compared between groups. Restricted cubic spline (RCS) was used to identify significant cut-off values affecting pregnancy. RESULTS: Higher FAI levels (> 6.4) were associated with decrease in clinical pregnancy rate (PR) (50.61% vs. 41.66%, p = 0.024), live birth rate (LBR) (42.42% vs. 32.35%, p = 0.011). When DHEAS levels exceeded 9.5 µmol/L, there was a significant decrease in clinical PR (51.27% vs. 42.73%, P = 0.039), LBR (42.73% vs. 32.73%, P = 0.012). Negative correlations were also observed between DHEAS levels and cumulative pregnancy rate (70.57% vs 56.62% p = 0.002) and cumulative live birth rate (CLBR) (59.35% vs 43.37%, p = 0.0007). Both FAI and DHEAS elevated is associated with the lowest clinical pregnancy rate (37.84%). Conversely, when solely FAI is elevated, the pregnancy rate increases to 52.38%, while an elevation in DHEAS alone is associated with a pregnancy rate of, both of which are lower than when neither FAI nor DHEAS are elevated (60.68%). The live birth rates exhibit a similar trend (30.00% vs 40.00% vs 41.83% vs 44.48%). RCS revealed a significant decrease in CPR and CLBR when DHEA levels exceeded 7.69 umol/L, while the cut-off value of FAI was 6.36 for CPR and CLBR. CONCLUSION: In conclusion, PCOS patients with biochemical hyperandrogenism show unsatisfactory clinical PR and CLBR when undergoing assisted reproductive technology (ART). This may be attributed to the influence of both adrenal-derived DHEAS and ovarian-derived FAI on the unfavorable pregnancy outcomes.

Higher Cumulative Live Birth Rate but Also Higher Late Miscarriage Risk in Non-Obese Women with Polycystic Ovary Syndrome Undergoing the First IVF/ICSI Cycle.

Purpose: To determine the impact of polycystic ovary syndrome on in vitro fertilization/intracytoplasmic sperm injection and embryo transfer outcomes while analyzing the influencing factors. Patients and Methods: A retrospective cohort study comprised 4839 patients who underwent their first cycle of IVF/ICSI treatment from January 2016 to December 2021. Cumulative pregnancy rates, cumulative live birth rates, and late miscarriage rates compared between the PCOS group and control group. Subgroup analysis and binary regression were used to analyze the influence of BMI on clinical outcomes among individuals diagnosed with PCOS. Results: Non-obese PCOS patients exhibited higher cumulative pregnancy rates, cumulative live birth rates, and late miscarriage rates compared to the control group with the normal BMI population (84.7% vs71.2%, P < 0.001; 74.1% vs 61.6%, P < 0.001; 4.1% vs 2.0%, P = 0.002), but there was no significant difference in early miscarriage rates between the two groups. Conclusion: Non-obese PCOS patients demonstrated a notably higher cumulative live birth rate but also a higher risk of late miscarriage compared to non-PCOS females with a normal BMI.

Knowledge-embedded spatio-temporal analysis for euploidy embryos identification in couples with chromosomal rearrangements.

BACKGROUND: The goal of the assisted reproductive treatment is to transfer one euploid blastocyst and to help infertile women giving birth one healthy neonate. Some algorithms have been used to assess the ploidy status of embryos derived from couples with normal chromosome, who subjected to preimplantation genetic testing for aneuploidy (PGT-A) treatment. However, it is currently unknown whether artificial intelligence model can be used to assess the euploidy status of blastocyst derived from populations with chromosomal rearrangement. METHODS: From February 2020 to May 2021, we collected the whole raw time-lapse videos at multiple focal planes from in vitro cultured embryos, the clinical information of couples, and the comprehensive chromosome screening results of those blastocysts that had received PGT treatment. Initially, we developed a novel deep learning model called the Attentive Multi-Focus Selection Network (AMSNet) to analyze time-lapse videos in real time and predict blastocyst formation. Building upon AMSNet, we integrated additional clinically predictive variables and created a second deep learning model, the Attentive Multi-Focus Video and Clinical Information Fusion Network (AMCFNet), to assess the euploidy status of embryos. The efficacy of the AMCFNet was further tested in embryos with parental chromosomal rearrangements. The receiver operating characteristic curve (ROC) was used to evaluate the superiority of the model. RESULTS: A total of 4112 embryos with complete time-lapse videos were enrolled for the blastocyst formation prediction task, and 1422 qualified blastocysts received PGT-A ( n = 589) or PGT for chromosomal structural rearrangement (PGT-SR, n = 833) were enrolled for the euploidy assessment task in this study. The AMSNet model using seven focal raw time-lapse videos has the best real-time accuracy. The real-time accuracy for AMSNet to predict blastocyst formation reached above 70% on the day 2 of embryo culture, and then increased to 80% on the day 4 of embryo culture. Combing with 4 clinical features of couples, the AUC of AMCFNet with 7 focal points increased to 0.729 in blastocysts derived from couples with chromosomal rearrangement. CONCLUSION: Integrating seven focal raw time-lapse images of embryos and parental clinical information, AMCFNet model have the capability of assessing euploidy status in blastocysts derived from couples with chromosomal rearrangement.

Letrozole During Frozen Embryo Transfer in Women With Polycystic Ovarian Syndrome: A Randomized Controlled Trial.

OBJECTIVE: To compare live-birth rates between letrozole application and artificial cycle for endometrium preparation during frozen embryo transfer (FET) cycle among women with polycystic ovarian syndrome (PCOS). METHODS: A randomized controlled trial was conducted. Women with PCOS were randomized to letrozole application for ovulation induction compared with artificial cycle for endometrial preparation during FET. The primary outcome was live-birth rate per embryo transfer. Secondary outcomes included pregnancy-related outcomes, perinatal outcomes, and maternal complication rates. Assuming α=0.05 and 80% power, 186 patients per group were required to demonstrate a difference of 15% in live-birth rate: 205 patients (at least) per group were randomized to allow for a 10% dropout rate. RESULTS: Four hundred twenty patients were enrolled from 2018 to 2021. Two hundred ten patients were assigned to the letrozole application group, and 210 were assigned to the artificial cycle group. There was no difference in the live-birth rate (42.4% vs 42.9%, P =>.99). There was no difference in secondary outcomes, including clinical pregnancy rate (51.4% vs 56.2%, P =.378), implantation rate (51.8% vs 55.8%, P =.401), and miscarriage rate (8.6% vs 11.0%, P =.511). For perinatal outcomes, singleton birth weight was significantly higher in the artificial cycle group (3,108±56 g vs 3,301±58, P =.018), and the incidence of gestational diabetes mellitus (GDM) was significantly higher in letrozole application group (14.6% vs 5.6%, P =.050). The other outcome was no difference in maternal complications. CONCLUSION: There was no difference in pregnancy outcomes between letrozole application compared with artificial cycle for endometrial preparation in women with PCOS who underwent FET. The risk of GDM was higher in the letrozole application group, and the singleton birth weight was lower in the artificial cycle group. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1800014746.

The dynamic expression of SOX17 in germ cells from human female foetus and adult ovaries after specification.

Background: SOX17 has been identified as a critical factor in specification of human primordial germ cells, but whether SOX17 regulates development of germ cells after sex differentiation is poorly understood. Methods: We collected specimens of gonadal ridge from an embryo (n=1), and ovaries of foetuses (n=23) and adults (n=3). Germ cells were labelled with SOX17, VASA (classic germ cells marker), phosphohistone H3 (PHH3, mitosis marker) and synaptonemal complex protein 3 (SCP3, meiosis marker). Results: SOX17 was detected in both cytoplasm and nucleus of oogonia and oocytes of primordial and primary follicles from 15 to 28 gestational weeks (GW). However, it was exclusively expressed in cytoplasm of oogonia at 7 GW, and in nucleus of oocytes in secondary follicles. Co-expression rates of SOX17 in VASA+ germ cells ranged from 81.29% to 97.81% in foetuses. Co-staining rates of SOX17 and PHH3 or SCP3 were 0%-34% and 0%-57%, respectively. Interestingly, we distinguished a subpopulation of SOX17+VASA- germ cells in fetal ovaries. These cells clustered in the cortex and could be co-stained with the mitosis marker PHH3 but not the meiosis marker SCP3. Conclusions: The dynamic expression of SOX17 was detected in human female germ cells. We discovered a population of SOX17+ VASA- germ cells clustering at the cortex of ovaries. We could not find a relationship between mitosis or meiosis and SOX17 or VASA staining in germ cells. Our findings provide insight into the potential role of SOX17 involving germ cells maturation after specification, although the mechanism is unclear and needs further investigation.

Storage time does not influence pregnancy and neonatal outcomes for first single vitrified high-quality blastocyst transfer cycle.

RESEARCH QUESTION: Does blastocyst storage time have an impact on pregnancy and neonatal outcomes following the first single vitrified/warmed high-quality blastocyst transfer cycle for young women? DESIGN: Retrospective cohort study in a university-affiliated reproductive medical centre. RESULTS: A total of 2938 patients undergoing their first frozen embryo transfer (FET) cycle with a single high-quality blastocyst (Day 5: 3BB and above; Day 6: 4BB and above) transferred were divided into five groups: Group A with storage time ≤3 months (n = 1621), Group B with storage time of 4-6 months (n = 657), Group C with storage time of 7-12 months (n = 225), Group D with storage time of 13-24 months (n = 104), and Group E with storage time of 25-98 months (n = 331). After adjusting for confounding factors by multivariate logistic regression, there were no significant differences in live birth rate [Group A as reference; Group B: adjusted odds ratio (aOR) 0.954 (95% CI 0.791- 1.151); Group C: aOR 0.905 (95% CI 0.674-1.214); Group D: aOR 0.727 (95% CI 0.474-1.114); Group E: aOR 1.185 (955 CI 0.873-1.608)], ß-human-chorionic-gonadotropin-positive rate, clinical pregnancy rate and miscarriage rate between Group A and the other groups. Among all singletons born after FET, there were no significant differences with regards to gestational age, preterm birth, birthweight, low birthweight, high birthweight and macrosomia. CONCLUSION: Long-term cryostorage of human vitrified high-quality blastocysts does not affect pregnancy or neonatal outcomes.

Pregnancy and neonatal outcomes of monozygotic twins resulting from assisted reproductive technology: a 10-year retrospective study.

BACKGROUND: Monozygotic twins (MZTs) are associated with high risks of maternal and fetal complications. Even with the widely used elective single embryo transfer (SET), the risk of MZTs following assisted reproductive technology (ART) treatments remains. However, most studies of MZTs focused on the relevant etiology, with few studies describing pregnancy and neonatal outcomes. METHODS: This retrospective cohort study included 19,081 SET cycles resulting from in-vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), preimplantation genetic testing (PGT) and testicular sperm aspiration (TESA) performed between January 2010 and July 2020 in a single university-based center. A total of 187 MZTs were included in this investigation. The main outcome measures were the incidence, pregnancy and neonatal outcomes of MZTs. Multivariate logistic regression analysis was performed to figure out the risk factors for pregnancy loss. RESULTS: The overall rate of MZTs from ART treatment in SET cycles was 0.98%. No significant difference was found in the incidence of MZTs among the four groups (p = 0.259). The live birth rate of MZTs in the ICSI group (88.5%) was significantly more favorable than in the IVF, PGT and TESA groups (60.5%, 77.2% and 80%, respectively). IVF resulted in a significantly increased risk of pregnancy loss (39.4%) and early miscarriage (29.5%) in MZT pregnancies compared to ICSI (11.4%, 8.5%), PGT (22.7%, 16.6%) and TESA (20%, 13.3%). The total rate of twin-to-twin transfusion syndrome (TTTS) in MZTs was 2.7% (5/187); however, the TESA group had the highest rate at 20% and was significantly higher than the PGT group (p = 0.005). The four ART groups had no significant effect on the occurrence of congenital abnormalities or other neonatal outcomes in newborns from MZT pregnancies. Multivariate logistic regression analysis revealed that infertility duration, cause of infertility, the total dose of Gn used, history of miscarriages, and the number of miscarriages were not related to the risk of pregnancy loss (p > 0.05). CONCLUSIONS: The rate of MZTs was similar among the four ART groups. The pregnancy loss and the early miscarriage rate of MZTs was increased in IVF patients. Neither the cause of infertility nor the history of miscarriage was correlated with the risk of pregnancy loss. MZTs in the TESA group had a higher risk of TTTS, placental effects influenced by sperm and paternally expressed genes may play a role. However, due to the small total number, studies with larger sample sizes are still needed to validate these result. Pregnancy and neonatal outcomes of MZTs after PGT treatment seem to be reassuring but the duration of the study was short, and long-term follow-up of the children is needed.

Identification of common genetic polymorphisms associated with down-regulated gonadotropin levels in an exome-wide association study.

OBJECTIVE: To investigate whether common genetic polymorphisms are associated with gonadotropin levels after down-regulation with daily gonadotropin-releasing hormone agonist and whether the polymorphisms of candidate variants influence the ovarian response to exogenous gonadotropins. DESIGN: Genetic association study. SETTING: University-affiliated in vitro fertilization center. PATIENTS: Subjects enrolled in an exploratory exome-wide association study (n = 862), a replication exome-wide association study (n = 86), and a classifier validation study (n = 148) were recruited from September 2016 to October 2018, September 2019 to September 2020, and January 2021 to December 2021, respectively. The included patients were aged ≤40 years and had a basal follicle-stimulating hormone (FSH) ≤12 IU/L. INTERVENTIONS: All participants received a luteal phase down-regulation long protocol. Genome DNA was extracted from the peripheral blood leukocytes. For the exploratory and replication cohorts, exome sequencing was conducted on a HiSeq 2500 sequencing platform. The multiplex polymerase chain reaction amplification technique and next-generation sequencing also were performed in the exploratory and replication cohorts. For the samples of the validation cohort, Sanger sequencing was performed. MAIN OUTCOME MEASURES: The primary endpoint was the gonadotropin levels after down-regulation, and the secondary endpoints were hormone levels and follicle diameters during stimulation, the total dose of FSH, duration of FSH stimulation, number of oocytes retrieved, and clinical pregnancy rate. RESULTS: In the exploratory cohort, we identified that FSHB rs6169 (P=2.71 × 10-24) and its single-nucleotide polymorphisms in high linkage disequilibrium were associated with the down-regulated FSH level. The same locus was confirmed in the replication cohort. Women carrying the C allele of FSHB rs6169 exhibited higher average estradiol level during stimulation (P=6.82 × 10-5), shorter duration of stimulation, and less amount of exogenous FSH (Pduration=0.0002; Pdose=0.0024). In the independent validation set, adding rs6169 genotypes into the prediction model for FSH level after down-regulation enhanced the area under the curve from 0.560 to 0.712 in a logistic regression model, and increased prediction accuracy by 41.05% when a support vector machine classifier was applied. CONCLUSION: The C allele of FSHB rs6169 is a susceptibility site for the relatively high level of FSH after down-regulation, which may be associated with increased ovarian FSH sensitivity.

Pregnancy outcomes in patients with polycystic ovary syndrome who conceived after single thawed blastocyst transfer: a propensity score-matched study.

BACKGROUND: It remains unclear whether polycystic ovary syndrome (PCOS) is an independent risk factor for pregnancy complications in women undergoing assisted reproductive technology (ART) treatment. For the integrative treatment of PCOS patients, it is still important to investigate the pregnancy outcomes of PCOS patients after adjusting for potential biases, such as body mass index, embryo quality and endometrial preparation method. METHODS: This retrospective cohort study ultimately included a total of 336 PCOS patients who conceived after single thawed blastocyst transfer in the PCOS group and 2,325 patients in the control group from January 2018 to December 2020. A propensity score matching (PSM) model was used, and 336 PCOS patients were matched with 336 patients in the control group. RESULTS: Before PSM, no differences in the miscarriage rate, pregnancy complication rate, preterm birth rate, or live birth rate were found between the PCOS group and the control group. After PSM, the late miscarriage rate of the PCOS group was significantly higher than that of the control group (3.3% vs. 0.6%, P = 0.040), although the early miscarriage rates were similar (14.0% vs. 13.7%). The rates of pregnancy complications, preterm birth and live birth in the PCOS group were comparable to those in the matched control group (P = 0.080, P = 0.105, P = 0.109, respectively). The neonatal weights of male infants and female infants were similar between the two groups (P = 0.219, P = 0.169). Subgroup analysis showed that PCOS patients with homeostasis model assessment of insulin resistance (HOMA-IR) levels ≥ 2.49 had a significantly increased risk of preterm birth compared with those with HOMA-IR levels < 1.26 and 1.26 ≤ HOMA-IR levels < 2.49 (26.0% vs. 6.0% vs. 9.8%, P = 0.005). PCOS patients with total testosterone levels ≥ 0.7 ng/ml had a higher early miscarriage rate but a lower late miscarriage rate than those with total testosterone levels < 0.7 ng/ml (29.4% vs. 12.3%, 0% vs. 3.6%, respectively, P = 0.032). CONCLUSIONS: PCOS is an independent risk factor for late miscarriage in patients conceived after a single thawed blastocyst transfer, even after adjusting for biases. Among PCOS patients, insulin resistance and hyperandrogenism are associated with a higher risk of preterm birth and early miscarriage, respectively.

Which type of chromosomal mosaicism is compatible for embryo transfer: a systematical review and meta-analysis.

PURPOSE: Chromosomal mosaicism becomes a common phenomenon in Preimplantaion genetic testing (PGT). This meta-analysis was conducted to study which feature of chromosomal mosaicism was compatible for embryo transfer. METHODS: After searching the database PubMed, Embase, CCTR and related reviews up until May 2021. Two reviewers extracted relevant information and assessed study quality by the Newcastle-Ottawa scale independently. Summary Odd Radios (OR) were calculated using fixed- or random-effects models for clinical outcomes. A network meta-analysis compared the clinical outcomes of different chromosomes. RESULTS: A total of six studies with 1,106 cycles of single mosaic embryo transferred were included. Significant results of implantation rate (IR), miscarriage rate (MR), and ongoing pregnancy/live birth rate (OP/LBR) were observed when comparing embryos with mosaicism level < 50% and ≥ 50% [OR 1.42, 95% CI (1.06, 1.89); OR 0.45, 95% CI (0.27, 0.75); OR 1.74, 95% CI (1.28, 2.37)], and embryos with mosaicism with only affecting segmental chromosome(s) and only involving whole chromosome(s) [OR 1.31, 95% CI (1.01, 1.71); OR 0.57, 95% CI (0.36, 0.93); OR 1.51, 95% CI (1.15, 2.00)]. Embryos with only mosaic gains or losses had significant higher IR and OP/LBR than complex mosaicism [Gains vs complex: OR 1.75, 95% CI (1.20, 2.54); OR 1.73, 95% CI (1.16, 2.58). Losses vs complex: OR 1.90, 95% CI (1.34, 2.71); OR 2.10, 95% CI (1.44, 3.07)]. Mosaic embryos with only one chromosome involved had significant favorable outcomes of IR and OP/LBR than with three or more chromosomes involved [OR 1.76, 95% CI (1.23, 2.52); OR 1.86, 95% CI (1.25,2.78)]. Chr. 7, Chr. 2, Chr. 1, Chr. 18, Chr. 11, Chr. X, Chr. 13, Chr. 14, Chr. 12, and Chr. 9 were considered as prioritized chromosomes of mosaic embryos for transfer. CONCLUSIONS: This analysis support the embryos with mosaicism level ≥ 50%, whole chromosome(s) involved, multiple mosaic abnormalities were associated with worse pregnancy outcomes. Mosaicism level of 50% could be used as a threshold to assess the mosaic embryos.

Impact of metabolic disorders on endometrial receptivity in patients with polycystic ovary syndrome.

The present study investigated the expression of endometrial receptivity-related molecules in patients with polycystic ovary syndrome (PCOS) and different androgen status, insulin resistance (IR) levels, and body mass indexes (BMI) to identify the mechanism underlying their effects on pregnancy outcomes. The present study recruited 43 participants from November 2020 to January 2021, which were classified into five groups: i) Hyperandrogenemia (HA) combined with impaired glucose tolerance group (n=8); ii) HA combined with diabetes mellitus group (n=8); iii) HA combined with non-IR (NIR) group (n=10); iv) non-HA (NHA) androgen combined with IR group (n=8); and v) NHA combined with NIR group (n=9). In addition, according to their BMIs, patients were sub-grouped into lean/normal (n=27), overweight (n=8) or obese (n=8) groups. The mRNA expression levels of endometrial receptivity-related molecules were detected using reverse transcription-quantitative PCR. In addition, flow cytometry was used to determine the phenotype and percentage of uterine natural killer cells (uNK). According to the results, patients with PCOS and IR status, HA and obesity (BMI ≥24 kg/m2) demonstrated significantly decreased mRNA expression levels of adiponectin, adiponectin receptor (AdipoR)1, AdipoR2, adapter protein containing PH domain, PTB domain and leucine zipper motif 1, estrogen receptor (ER) α, ERß, progesterone receptor (PR), IL-15, integrin ß3 avß3, and insulin-like growth factor binding protein-1, but increased mRNA expression levels of IL-6 and IL-8 compared with NHA + NIR group or lean/normal group, respectively. In addition, obese patients with PCOS demonstrated increased mRNA expression levels of PR compared with overweight patients. This suggested that insulin resistant status, HA, and obesity could alter the endometrial receptivity of patients with PCOS, which may explain poorer embryo implantation and pregnancy outcomes in clinics.

A moderately extended time interval between hCG administration and oocyte retrieval is good for most patients with oocyte retrieval scheduled on the same day: a retrospective cohort study.

Evidence is limited regarding the time intervals between human chorionic gonadotropin (hCG) administration and oocyte retrieval in controlled ovarian hyperstimulation cycles, and it is difficult to determine proper schedules to optimise outcomes for patients undergoing oocyte retrieval on the same day. We aimed to identify correlations between factors pertaining to treatment outcomes and time intervals to facilitate working schedules of ART centres. Our study included 2509 patients who underwent ICSI cycles. Based on different time intervals between hCG administration and oocyte retrieval, all cycles were divided into four groups: group 1 (34.00-35.99 hours), group 2 (36.00-36.99 hours), group 3 (37.00-37.99 hours) and group 4 (38.00-39.32 hours). Female age, basal FSH level, Gn starting stimulation dosage and total Gn dosage of group 1 were significantly higher than those of other groups. E2 level on hCG day and number of follicles aspirated were significantly higher in group 4 than in the other groups. Number of oocytes retrieved, oocyte retrieval rate, cleavage rate and number of usable embryos were positively correlated with the time interval, even after adjusting for female age, basal FSH level, E2 on hCG day and number of follicles aspirated. A fixed hCG administration time matching arranged oocyte retrieval is good enough for most patients to achieve maximal treatment outcomes. For patients with lower treatment expectations (expected no. of oocyte retrieval ≤3), moderately delayed oocyte retrieval would be more appropriate.Impact StatementWhat is already known on this subject? The time interval between hCG administration and OPU during COH is essential for ART treatment outcomes, but different intervals were reported in previous studies.What the results of this study add? Fixed hCG administration time matching arranged OPU is good enough for most patients to achieve maximal outcomes. For patients with lower treatment expectations (expected no. of oocyte retrieval ≤3), moderately delayed oocyte retrieval would be required.What the implications are of these findings for clinical practice and/or further research? We studied whether the oocytes and pregnancy outcomes changed along with extended time intervals, and there is no need to adhere to an exact interval for every patient. Therefore, it would help clinicians develop more reasonable time schedules for fertility centres and patients undergoing oocyte recovery on the same day.

The Therapeutic Potential of Small Activating RNAs for Colorectal Carcinoma.

Small double-strand RNAs have been recognized as master regulators of gene expression. In contrast to the evolutionary conserved RNA interference machinery, which degrades or inhibits the translation of target mRNAs, small activating RNA (saRNA) activates the specific gene in a target dependent manner through a similar mechanism as RNAi. Recently, saRNA mediated expression regulation of specific genes has been extensively studied in cancer researches. Of particular interest is the application of the RNA mediated gene activation within colorectal cancer (CRC) development, due to the high incidence of the CRC. In this review, we summarize the current knowledge of saRNA mediated genetic activation and its underlying mechanisms. Furthermore, we highlight the advantages of the utilization of saRNAs induced gene expression as an investigating tool in colorectal cancer research. Finally, the possibility and the challenge of the saRNA application as a potential therapy for colorectal cancer are addressed.

What was the fate of human embryos following long-term cryopreservation (≥12 years) and frozen embryo transfer?

STUDY QUESTION: Do human embryos survive long-term cryopreservation (CP) (≥12 years) and implant after frozen embryo transfer (ET)? SUMMARY ANSWER: Human embryos remain usable after long-term CP. WHAT IS KNOWN ALREADY: Several cohort studies have reported the live birth rate or neonatal outcomes of human embryos after CP for up to 5 years. Only a few case reports have described successful live births from human embryos after long-term CP up to 12 years. STUDY DESIGN, SIZE, DURATION: This retrospective observational study in China included 20 patients (128 embryos) from March 2016 to April 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty patients who had at least one live birth during their previous IVF/ICSI treatments and had surplus embryos cryopreserved were observed. Data concerning frozen embryo recovery, pregnancy and obstetric outcomes following frozen ET were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 128 embryos of 20 patients were observed. The embryo storage duration was 12.0-17.1 years, with a mean of 13.9 ± 1.73 years. In all, 115 embryos were thawed to transfer, with a survival rate of 74%. Sixty embryos were further cultured, which resulted in 20 blastocysts with a blastocyst formation rate of 33%. There were 21 cleavage-stage embryos and 13 blastocysts transferred in a total of 12 and 11 cycles, respectively, which resulted in one biochemical pregnancy, one first trimester miscarriage, two ectopic pregnancies, three singletons and one case of twins, with a clinical pregnancy rate of 25% (D3 ET) and 36% (blastocyst transfer) and a live birth rate of 17% (D3 ET) and 27% (blastocyst transfer). Two of the four patients who had live birth developed gestational diabetes mellitus. One of the five live births was a preterm delivery. LIMITATIONS, REASONS FOR CAUTION: The sample size was small due to the unique study population, and all the embryos underwent slow freezing. The fate of long-term cryopreserved embryos after vitrification is still unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results provide evidence to support the use of embryos after extended CP to preserve patients' fertility. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Key Research and Development Programme of China (2016YC1000205) and the Guangzhou Scientific Programme (201508020006). None of the authors has any conflicts of interest to declare.

Imprinting Status in Two Human Parthenogenetic Embryonic Stem Cell Lines: Analysis of 63 Imprinted Gene Expression Levels in Undifferentiated and Early Differentiated Stages.

Human parthenogenetic embryonic stem cells (hPESCs) represent a source of histocompatible tissues for transplantation and carry two copies of the maternal genome, but lack the paternal genome. In this study, we selected 63 known human imprinted genes to investigate the imprinting status of hPESC. The expression level of these genes, including 27 maternally and 36 paternally imprinted were illustrated in hPESC and human embryonic stem cells (hESCs) derived from fertilized embryo lines. The expression activity changes of these genes were analyzed in undifferentiated and early differentiated hPESC lines. In addition, the methylation status of four differentially methylated regions (DMRs) of the imprinted genes was analyzed in undifferentiated and early differentiated hPESC and hESC lines. As a result, we found that all the maternally imprinted genes were expressed at similar levels in the undifferentiated hPESC lines and the hESC lines, except ZNF264 and ATP10A. Twenty-one analyzed paternal imprinted genes were expressed at the same level in two separated hPESC lines as well as compared with the hESC lines, whereas 15 other paternal imprinted genes were significantly downregulated or inactivated in hPESC lines as compared with the hESC line. During prolonged passage, the expression levels of the majority of imprinted genes remained stable in two hPESC lines. The four DMRs, including PEG3/ZIM2 (DMRs), SNURF/SNRPN DMRs, and KVDMR1 DMRs are highly methylated in the genes of two undifferentiated hPESCs and its embryonic bodies (EBs), whereas the genes of the undifferentiated hESCs and its EBs are half methylated. During the early differentiation stage, the imprinted genes showed the same expression trend and the expression levels of H19, IGF2, SLC22A2, SLC22A3/SLC22A18, and CPA4 were significantly upregulated in both hPESC lines. As conclusion, hPESCs show a substantial degree of epigenetic stability with respect to some imprinted genes.

Activation of KLF4 expression by small activating RNA promotes migration and invasion in colorectal epithelial cells.

RNA activation mediated by small double-stranded RNAs targeting promoter sequence named small activating RNAs (saRNAs) is one of the mechanisms for gene activation. Artificial regulation of gene expression through RNA activation does not affect the alteration of the genomic DNA sequences or exogenous plasmid DNA, therefore it is a relative manageable approach for gene perturbation. KLF4 is a member of zinc-finger transcription factors and its functions in colorectal cells are still controversial. In order to elucidate the functions of KLF4, we synthesized saRNAs that target the promoter regions of KLF4 and transfected into varied colorectal epithelial cell lines. We found the KLF4 gene expression is specifically increased in the human normal epithelial cell NCM460 and colorectal epithelial cancer cell Caco-2 and HCT116, but not in other human colorectal epithelial cell lines. In addition, we observed that saRNAs induced overexpression of KLF4 could promote cell migration/invasion in NCM460 and HCT116 cell lines. This effect is mediated partly by inducing EMT and facilitating nuclear translocation of ß-catenin.

Effect of letrozole on moderate and severe early-onset ovarian hyperstimulation syndrome in high-risk women: a prospective randomized trial.

BACKGROUND: Ovarian hyperstimulation syndrome is an iatrogenic complication of controlled ovarian stimulation. Early ovarian hyperstimulation syndrome occurs during luteal phase of controlled ovarian stimulation within 9 days after human chorionic gonadotropin trigger and reflects an acute consequence of this hormone on the ovaries. Late ovarian hyperstimulation syndrome occurs 10 or more days after human chorionic gonadotropin trigger and reflects increased endogenous human chorionic gonadotropin levels following pregnancy. Human chorionic gonadotropin stimulates granulosa-lutein cells to produce vascular endothelial growth factor messenger RNAs, which in turn raises serum vascular endothelial growth factor concentration and increases vascular permeability in women with ovarian hyperstimulation syndrome. Efforts to reduce the incidence and severity of ovarian hyperstimulation syndrome after oocyte retrieval, and in particular primary prevention efforts, are vital to prevent thrombogenesis and other serious complications. OBJECTIVE: The objective of the study was to compare the efficacy of letrozole, an aromatase inhibitor, with aspirin in primary prevention of early ovarian hyperstimulation syndrome and to compare vascular endothelial growth factor levels between groups. STUDY DESIGN: Participants in this prospective randomized trial included 238 participants undergoing cryopreservation of the whole embryos after oocyte retrieval with at least 1 of the following high-risk factors for ovarian hyperstimulation syndrome: oocyte retrieval ≥25; estradiol level ≥5000 pg/mL on the day of human chorionic gonadotropin administration; and clinical or ultrasonographic evidence of ovarian hyperstimulation syndrome on the day of oocyte retrieval, such as ultrasonographic evidence of ascites. After human chorionic gonadotropin triggering, experimental (119 cases) and control (119 cases) groups received letrozole and aspirin, respectively, for 5 days. The 5 categories of ovarian hyperstimulation syndrome include no, yes-mild, yes-moderate, yes-severe, and yes-critical. The primary outcome was the incidence and severity of early ovarian hyperstimulation syndrome. The secondary outcome included vascular endothelial growth factor level both on the second and seventh day after the human chorionic gonadotropin trigger, and clinical and laboratory features of ovarian hyperstimulation syndrome symptoms. RESULTS: The incidence of ovarian hyperstimulation syndrome was significantly higher in women receiving aspirin, compared with letrozole (90.2% vs 80.4%, P = .044). Moderate and severe ovarian hyperstimulation syndrome was also higher in the aspirin group, 45.1%, compared with the letrozole group, 25.0% (P = .002). Moreover, the duration of luteal phase was shortened in letrozole group compared with aspirin group (8.1 ± 1.1 days vs 10.5 ± 1.9 days, P < .001). The vascular endothelial growth factor level was significantly higher in the letrozole-treated group than aspirin-treated group (0.49 ± 0.26 vs 0.42 ± 0.22, P = .029). CONCLUSION: Letrozole was more effective than aspirin in decreasing the incidence of moderate and severe early-onset ovarian hyperstimulation syndrome. Our results indicate that ovarian hyperstimulation syndrome might be caused through a luteolytic effect rather than through modulation of vascular endothelial growth factor, racing by a decline in estradiol and termination of early-onset ovarian hyperstimulation syndrome in advance in high-risk women with cryopreservation of the whole embryos.

Comparison of a xeno-free and serum-free culture system for human embryonic stem cells with conventional culture systems.

BACKGROUND: Elimination of all animal components during derivation and long-term culture of human embryonic stem cells (hESCs) is necessary for future applications of hESCs in clinical cell therapy. METHODS: In this study, we established the culture system of xeno-free human foreskin fibroblast feeders (XF-HFF) in combination with chemically defined medium (CDM). XF-HFF/CDM was compared with several conventional culture systems. The hESCs cultured in different media were further characterized through karyotype analysis, pluripotency gene expression, and cell differentiation ability. RESULTS: The hESCs in the XF-HFF/CDM maintained their characteristics including typical morphology and stable karyotype. In addition, hESCs were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. RT-PCR analysis shown that the stem cell markers OCT3/4, hTERT, SOX2, and Nanog were present in the cell line hESC-1 grown on XF-HFF/CDM. Furthermore, the results of cell growth and expression of bFGF, Oct-4, and hTERT indicated that XF-HFF/CDM had better performance than human serum-matrix/CDM and XF-HFF/human serum. CONCLUSION: The comparison of different xeno-free culture conditions will facilitate clarifying the key features of self-renewal, pluripotency, and derivation and will shed light on clinic applications of hESCs.