Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer

The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new opportunities for noninvasive prenatal diagnosis (NIPD). However, the extremely low levels of cfDNA within a high background of the maternal DNA in maternal circulation necessitate highly sensitive molecular techniques for its reliable use in NIPD. In this proof of principle study, we evaluated the earliest possible detection of cfDNA in the maternal plasma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, amplification, magnetics). Blood samples were collected from in vitro fertilization (IVF) patients at 2 to 6 weeks following embryo transfer (i.e., 4 to 8 week pregnancies) and plasma DNA was extracted. The genomic regions of both X and Y chromosome-specific sequences (AMELX and AMELY) were concurrently amplified in two sequential PCRs; first by conventional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene sequences were counted by a flow cytometer. Our results showed that the pregnancies yielding boys had significantly higher plasma AMELY gene fractions (0.512 ± 0.221) than the ones yielding girls (0.028 ± 0.003) or non-pregnant women (0.020 ± 0.005, P= 0.0059). Here, we clearly demonstrated that the BEAMing technique is capable of reliably detecting cfDNA in the blood circulation of 4-week-pregnant women, which is only two weeks after the embryo transfer. BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders.     

Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality

To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150–200 nm) by Nanosight^® and electron microscopy. Zygotes were cultured in the presence of 3x10⁵EVs/mL, 1.5x10⁵EVs/mL or 7.5x10⁴EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7–9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.     

Toxic Metal and Trace Element Concentrations in Blood and Outcome of In Vitro Fertilization in Women

Biological Trace Element Research - The aim of this study was to investigate the association of trace element and toxic metal concentrations in blood and the outcome of in vitro fertilization...     

Deregulated HOXB7 Expression Predicts Poor Prognosis of Patients with Esophageal Squamous Cell Carcinoma and Regulates Cancer Cell Proliferation In Vitro and In Vivo

Background

We observed abnormal HOXB7 expression in esophageal squamous cell carcinoma (ESCC) previously. This study was to evaluate the prognostic significance of HOXB7 and reveal the potential mechanism.

Methods

Immunohistochemistry was used to confirm the abnormal expression of HOXB7 in ESCC. The prognostic significance of HOXB7 expression was analyzed in two independent cohorts. RNAi was used to establish two stable HOXB7-knockdown cell strains. CCK8 assay, cell growth curve assay, colony formation assay, flow cycle analysis and tumorigenicity assay in nude mice were employed to investigate the effect of HOXB7 on proliferation in vitro and in vivo.

Results

Immunohistochemistry confirmed the abnormal expression of HOXB7 in ESCC compared with paracancerous mucosa (18/23 vs. 9/23, p=0.039). HOXB7 expression was positively correlated with the T stage, lymph node metastasis and TNM stage. The median survival of patients with high HOXB7 expression was significantly shorter than that with low expression (45 months vs. 137 months, p = 0.007 for cohort 1; 19 months vs. 34 months, p = 0.001 for cohort 2). Multivariate survival analysis showed that HOXB7 expression was another independent prognostic factor (HR [95% CI] = 0.573 [0.341–0.963], p = 0.036 for cohort 1; HR [95%CI] = 0.543 [0.350–0.844], p = 0.024 for cohort 2). Experiments in vitro and in vivo showed that after knockdown of HOXB7, the proliferation rate dropped, growth rate descended, colony-formation ability reduced, G1-phase arrest occurred and the tumorigenicity reduced remarkably.

Conclusions

HOXB7 could promote cancer cell proliferation and might be an independent prognostic factor for patients with ESCC.     

牛卵母细胞体外受精及体外发育的研究

本研究利用从屠宰场收集到的牛卵巢,获取卵母细胞,经体外成熟后进行体外受精,其受精率为73±2％。受精卵分别用发育培养基TCM—199+10％FCS(MA)、颗粒细胞单层(MB)和卵管上皮单层(MC)进行培养,其8—16细胞的发育率分别为52％、54％和61％;而囊胚的发育率分别为20％、28％和42％。相比较而言,MB和MC培养基内突破8—16细胞阻断发育到囊胚的发育率优于MA。     

我国被子植物体外受精研究——十年回顾

简要回顾了十余年来我国被子植物体外受精领域的研究历程和主要成就.我国科学家在性细胞的分离, 尤其是生活胚囊和雌配子的分离方面走在国际前列. 在一定程度上我国科学家的开拓性工作引起了国际同行的广泛兴趣和重视, 推动了该领域的迅速发展, 为实现被子植物真正意义的体外受精做出了独特的贡献.十年来, 我国已建立了自己独特的被子植物离体受精实验技术系统并利用该系统与国际同行合作进行了一系列研究, 尤其在探讨配子相互作用、卵细胞激活、避免多精入卵的机制等方面做出了创新性工作, 为正确认识体外受精系统的优越性与局限性, 也为深入探讨受精机制提供了有价值的新资料.目前正以体外受精操作系统结合细胞生物学和分子生物学等多种手段对受精过程中的一些重要事态与机理做进一步探讨.     

我国被子植物体外受精研究—十年回顾

简要回顾了十余年来我国被子植物体外受精领域的研究历程和主要成就。我国科学家在性细胞的分离 ,尤其是生活胚囊和雌配子的分离方面走在国际前列。在一定程度上我国科学家的开拓性工作引起了国际同行的广泛兴趣和重视 ,推动了该领域的迅速发展 ,为实现被子植物真正意义的体外受精做出了独特的贡献。十年来 ,我国已建立了自己独特的被子植物离体受精实验技术系统并利用该系统与国际同行合作进行了一系列研究 ,尤其在探讨配子相互作用、卵细胞激活、避免多精入卵的机制等方面做出了创新性工作 ,为正确认识体外受精系统的优越性与局限性 ,也为深入探讨受精机制提供了有价值的新资料。目前正以体外受精操作系统结合细胞生物学和分子生物学等多种手段对受精过程中的一些重要事态与机理做进一步探讨     

In Vitro Chick Embryo Cell Response to Strain MC29 Avian Leukosis Virus

Strain MC29 avian leukosis (myelocytomatosis) virus induced infection, elaboration of virus, and morphological alteration in chick embryo cells in vitro. Virus liberation began within 18 hr, morphological change was detectable at about 40 hr, and the cultures could be completely altered within 80 hr after infection. Altered cells were about half the volume and grew at approximately twice the rate of uninfected elements. The output of virus estimated by electron microscopy was about 140 particles per cell per hr. Deoxyribonucleic acid remained constant, but ribonucleic acid increased in both infected and control cells in adjustment to culture environment. The rates of uptake and incorporation of ³H-uridine and the incorporation of ³H-thymidine increased in the infected cells with onset of morphological change but were unaffected by processes of infection and virus elaboration per se. Incorporation of a ¹⁴C-amino acid mixture was slightly greater in the infected than in control cells. The speed of continuity of infection and massive morphological alteration constitute a unique response to avian tumor viruses, and the system gives promise of singular value for detailed studies of the processes of infection and morphological change.     

Awake EEG Disregulation in Good Compared to Poor Sleepers

This study was designed to test a disregulation model of sleep deprivation by assessing the ability of good sleepers compared to poor sleepers to shift daytime EEG patterning to changing environmental demands. Ten good and ten poor sleepers were identified from a sample of 110 college students who completed the Pittsburgh Sleep Quality Inventory (PSQI). EEG and SCR were recorded during a five task assessment session, including: (1) pre-baseline, (2) eyes open at rest, (3) eyes closed at rest, (4) sensory attentiveness (listening to an audio book clip), and (5) cognitive effort (a higher level cognitive flexibility task). A significant Group × Task interaction, F (3, 16) = 4.81, p = . 01 was attained on the theta data. Specifically, for good sleepers, theta decreased from the “eyes open at rest” to the “sensory attentiveness” tasks, while poor sleepers showed the opposite pattern. This pattern of theta suppression was found in 70% of the good sleepers and only 20% of the poor sleepers. No between group differences were noted in the SCR data, supporting a brain disregulation model, rather than a general psychophysiological stress model. Partial funding for this research was provided by the office of Research, Grants, and Contracts at the University of Tennessee at Martin.     

Live Birth Sex Ratio after In Vitro Fertilization and Embryo Transfer in China - An Analysis of 121,247 Babies from 18 Centers

In order to study the impact of procedures of IVF/ICSI technology on sex ratio in China, we conducted this multi-center retrospective study including 121,247 babies born to 93,895 women in China. There were 62,700 male babies and 58,477 female babies, making the sex ratio being 51.8% (Male: Female  = 107∶100). In univariate logistic regression analysis, sex ratio was imbalance toward females of 50.3% when ICSI was preformed compared to 47.7% when IVF was used (P<0.01). The sex ratio in IVF/ICSI babies was significantly higher toward males in transfers of blastocyst (54.9%) and thawed embryo (52.4%) when compared with transfers of cleavage stage embryo (51.4%) and fresh embryo (51.5%), respectively. Multiple delivery was not associated with sex ratio. However, in multivariable logistic regression analysis after controlling for related factors, only ICSI (adjusted OR = 0.90, 95%CI: 0.88–0.93; P<0.01) and blastocyst transfer (adjusted OR = 1.14, 95% CI: 1.09–1.20; P<0.01) were associated with sex ratio in IVF/ICSI babies. In conclusion, the live birth sex ratio in IVF/ICSI babies was influenced by the use of ICSI, which may decrease the percentage of male offspring, or the use of blastocyst transfer, which may increase the percentage of male offspring.     

Improved in Vitro Fertilization Outcomes after Treatment of Subclinical Hypothyroidism in Infertile Women

ObjectiveTo assess the effect of treatment of maternal subclinical hypothyroidism on infertility outcome.MethodsBetween April 1, 2006, and April 22, 2007, we conducted a prospective, randomized trial in infertile women with subclinical hypothyroidism who elected to undergo in vitro at the Shatby University Hospital for Women in Alexandria, Egypt. Patients were randomly assigned to 2 groups: treatment group (group A) and placebo group (group B). Male-factor infertility was ruled out. One month before the assisted reproduction technology procedure, group A underwent levothyroxine treatment with a dosage of 50 to 100 mcg daily, while group B started placebo. All patients underwent controlled ovarian stimulation. Patients who achieved pregnancy were followed up throughout their pregnancy until delivery. Levothyroxine treatment and placebo were maintained throughout pregnancy in group A and group B, respectively.ResultsMean thyrotropin value was significantly lower in group A than in group B (1.1 ± 0.3 mIU/L vs 4.9 ± 0.7 mIU/mL, respectively). Mean number of retrieved oocytes was similar in both groups (6.19 ± 0.74 [group A] vs 6.08 ± 0.79 [group B]). Miscarriage rate was significantly lower in group A than in group B (9% vs 13%, respectively), and the clinical pregnancy rate and delivery rate were significantly higher in group A than in group B (35% and 10% vs 26% and 3%, respectively), indicating that the quality, not the quantity, of retrieved oocytes was more important.ConclusionThese preliminary data suggest that levothyroxine supplementation should be recommended to achieve clinical pregnancies in women with subclinical hypothyroidism who are undergoing in vitro fertilization–intracytoplasmic sperm injection. (Endocr Pract. 2010:16: 792-797)     

Hospital Mental Health Admissions in Women after Unsuccessful Infertility Treatment and In Vitro Fertilization: An Australian Population-Based Cohort Study

Objective

To examine the association between in vitro fertilization (IVF) and later admission to hospital with a mental health diagnosis in women who remained childless after infertility treatment.

Methods

This was a population-based cohort study using linked administrative hospital and registry data. The study population included all women commencing hospital treatment for infertility in Western Australia between the years 1982 and 2002 aged 20–44 years at treatment commencement who did not have a recorded birth by the end of follow-up (15 August 2010) and did not have a hospital mental health admission prior to the first infertility admission (n=6,567). Of these, 2,623 women had IVF and 3,944 did not. We used multivariate Cox regression modeling of mental health admissions and compared women undergoing IVF treatment with women having infertility treatment but not IVF.

Results

Over an average of 17 years of follow-up, 411 women in the cohort were admitted to hospital with a mental health diagnosis; 93 who had IVF and 318 who did not. The unadjusted hazard ratio (HR) for a hospital mental health admission comparing women who had IVF with those receiving other infertility treatment was 0.50 (95% confidence interval [CI] 0.40–0.63). After adjustment for age, calendar year and socio-economic status the HR was 0.56 (95% CI 0.44–0.71).

Conclusions

IVF treatment is associated with a reduced risk of hospital mental health admissions in women after unsuccessful infertility treatment. This may be explained by the healthy cohort effect.     

In Vitro Embryogenesis in Unfertilized Embryo Sacs of Oryza sativa L.

Haploid rice plantlets were induced from cultured ovaries in our previously reported experiment. The present paper is an embryological study on this subject. Young flowers of two japonica cultivars were excised and cultured just in the same manner as before. Liquid medium used for float culture was N6+3% sucrose+0.125 ppm MCPA. The inoculated materials were checked to be at late uninuclcate pollen stage which corresponded mainly to the uuinuelcate embryo sac stage, but as well as some 2- or 4-nucleate embryo sacs. Samples were fixed at 23 days intervals in acetomethanol (1:3), stained in toto with diluted Ehrlich's hematoxylin and sectioned by paraffin method for microscopical observation. 4 days after inoculation most of the embryo sacs developed up to 8-nucleate stage with polarized differentiation of the egg apparatus, central cell and antipodals. From 7th day on, proembryos of various sizes and shapes appeared in the micropylar region of some embryo sacs; some consisted of meristcmatic ceils, others were highly vacuo]ated. One-celled as well as linear multicellular suspensors atypical of in vivo zygote proembryos were observed, ttowever, it was uncertain whether the proembryos originated from the egg cell, the synergids, or the differentiating egg apparatus as a whole. Another peculiar event occured during culture was the formation of endosperm-like free nuclei from the unfertilized polar nuclei in some embryo sacs. Sometimes the free nuclei were numerous and showed a tendency of cell formation in localized areas. 12–15 days after inoculation, the proembryos developed into mieroseopieal ealli with globular or pearlike shape, wbieh continued enlarging to visible size with naked eyes at about 18–24 th day. Further growth eventually led the ealli protruding out the ovary wall beyond 32–35 the day. These observations indicate that the embryo sue, similarly as the pollen, can be induced to embryogenesis in vitro. This may open a new way to study the mechanism controlling gametophytie and sporophytie developmental pathways of embryo sac and provide means for large-scale production of "embryo sac plants" in future.     

体外受精-胚胎移植技术妊娠后流产的危险因素分析

目的:探讨体外受精-胚胎移植技术妊娠后流产的危险因素。方法:采用t检验、受试者工作特征曲线(Receiver operating characteristic curve,ROC曲线)以及二元Logistic回归分析分别对2015年1月至2017年12月我中心行体外受精-胚胎移植技术(In vitro fertilization and embryo transfer,IVF-ET)及卵胞浆内单精子注射技术(Intracytoplasmic sperm injection,ICSI)后妊娠的242例临床妊娠患者临床参数包括年龄、身体质量指数(Body mass index,BMI)、基础卵泡生成素(Follicle stimulating hormone,FSH)、黄体生成素(Luteinizing hormone,LH)、雌二醇(Estradiol,E2)水平、促性腺激素(Gonadotropins,Gn)总用量、Gn天数、人绒毛膜促性腺激素(Human choionic gonadotophin,HCG)日LH、E2、黄体酮(progesterone,P)水平、移植日子宫内膜厚度以及移植后14-16天血HCG浓度进行回顾性分析。结果:流产组与继续妊娠组年龄、移植后14-16天血HCG浓度分别31.44±4.40岁和29.59±3.94岁、396.96±377.66 IU/L和702.85±496.91 IU/L,差异具有统计学意义(P<0.05)。通过ROC曲线对各临床参数分析后求得各参数的cut-off值并以此为标准分组,结果显示不同年龄(29.41%和9.42%)以及移植后14-16天血HCG浓度(4.20%和22.76%)分组流产率比较差异具有显著的统计学意义(P<0.01)。二元Logistic回归分析上述参数与流产率的相关性,结果提示仅年龄、移植后14-16天血HCG水平与妊娠后流产率有显著相关性(P=0.01,P=0.001)。结论:年龄>33岁、移植后14-16天血HCG浓度≤582.6IU/L是IVF/ICSI妊娠后流产的独立危险因素,对此类患者进行治疗时应考虑到其流产的风险并提前采取预防措施避免不良妊娠结局。     

Intrauterine Instillation of Human Chorionic Gonadotropin with Intrauterine Insemination Catheter Around the Golden Time of Embryo Transfer Does Not Improve In Vitro Fertilization /Intracytoplasmic Sperm Injection Outcomes in Infertile Women: A Randomized Controlled Trial

Background:We set out to explore the effect of intrauterine human chorionic gonadotropin (hCG) instillation by intrauterine insemination (IUI) catheter before embryo transfer (ET) on assisted reproductive technologies (ART) outcomes of infertile women.Methods:One hundred women with infertility who were scheduled for in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles were included in the study. They were randomly devoted to two groups: experimental (n= 50) and control (n= 50). In the experimental group, 500 IU hCG passed into the internal cervical orifice via IUI catheter within 15 minutes before the transfer of fresh or vitrified cleavage-stage embryos. The control group underwent the same ET procedure without prior injection of hCG. Results:None of the outcomes showed a statistically significant difference between the two groups. In the intervention and control groups, respectively, biochemical pregnancies rates were 26% and 18%, implantation rates were 13.5% and 8.6%, clinical pregnancies rates were 22% and 14%, ongoing pregnancies rates were 18% and 14%, and live birth rates were 14% and 12%.Conclusion:Intrauterine injection of hCG via IUI catheter is not recommended in a clinical routine setting at this stage. Future efforts are warranted to further refine the applicability of this modality.Key Words: Assisted reproductive technologies, Embryo transfer, Human chorionic gonadotropin, Intrauterine insemination catheter, Randomized clinical trial