In Vitro Fertilization Control Research Articles

P-260 In Vitro Fertilization (IVF) using magnetotactic sperm cells and their prospects for assisted in vivo reproduction

Abstract Study question Are bovine Magnetotactic Sperm Cells (MSC) able to fertilize an oocyte in-vitro and Which would be the main requirements for translating this technology to in-vivo? Summary answer IVF using highly purified MSC result in embryo development until blastocyst stage. We demonstrate different alternatives to control multiple MSC, individually and as a swarm. What is known already Medical microrobots have appeared as the new generation of drug delivery vehicles. They are tiny devices in the order of a few micrometers, able to move in biological environments by different means, physical, chemical, or biohybrid. They are functionalized to deliver targeted therapies for drugs, cells, or other payloads. In assisted reproduction, sperm-hybrid microcarriers have been pioneered by our group, and various in vitro demonstrations have been done to assist sperm cells with motion deficiencies or low count and to deliver anticancer drugs for treating, for example, cervical and ovarian cancer, but so far in-vitro fertilization has never been shown. Study design, size, duration Experimental quantitative study design. A total of 520 bovine cumulus-oocyte complexes were used for in vitro fertilization. The analyzed sample groups were using bovine frozen sperm cells, MSC, or by evaluating parthenogenetic activation. A power of 0.98 with an a-error of 0.05 was used for the IVF experiments. Sperm samples in the control as well as the micromotor groups have been analyzed for different sperm parameters. Participants/materials, setting, methods Bovine MSC are created by thawing frozen sperm cells and adding polystyrene microparticles. Different sperm parameters were evaluated to check the effects of the particles in the cells. After developing a purification method for the MSC, bovine ovaries were collected from a slaughterhouse nearby and, after in vitro maturation, were used for the IVF experiments. Ultrasound, and photoacoustic imaging setups with a set of electromagnets were employed for real-time manipulation and visualization of MSC. Main results and the role of chance Sperm viability was significantly higher in the MSC group compared to the control group 6h after formation (72.3 ± 5.0% vs 35.0 ± 4.1%), and after 24 hours, both viabilities decreased drastically to 16.6 ± 3.9% and 7.7 ± 1.7% respectively, probably a possible local antioxidant effect by the microparticles through the formation of a protein corona. In the case of motility, the MSC group presented significantly higher motility up to 6h (64.0 ± 1.5%), and in all the measured times, the motility of the MSC group was higher than the control group. Purification of the micromotor sample was performed, but an average of 63 free sperm was still on the MSC sample. IVF experiments were performed, and blastocyst development was observed in IVF control (2x106 sperm/ml), medium concentration (same concentration as MSC group), and the MSC groups (275.000 sperm/ml). No blastocyst formation was observed in the parthenogenetic, parthenogenetic + particles, and low-concentration groups (number of free sperm). The MSC group was significantly different in cleavage and blastocyst rates compared to the low concentration groups, with p-values of 0.04552 and 0.005865, respectively, which suggest that fertilization may have occurred by the micromotors instead by the free sperm in the sample. Limitations, reasons for caution More studies of these MSC in the different gynecological tissues and on embryo development are needed to ensure the safety of future human translation. In addition, developing a detection system for micromotors in deep tissue is needed to perform in vivo experiments. Wider implications of the findings MSC show an alternative to efficiently guide efficiently sperm cells in different biological environments and can be a promising alternative method for assisted fertilization, and the treatment of a variety of gynecological diseases. They can also be used as exploratory tools to learn more about sperm migration in-vivo, among others Trial registration number European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program No. 853609

Optimizing oocyte electroporation for genetic modification of porcine embryos: Evaluation of the parthenogenetic activation

In reproductive biology, understanding the effects of novel techniques on early embryo development is of paramount importance. To date, the effects of electrical activation on oocytes prior to in vitro fertilization (IVF) are not well understood. The aim of this study was to investigate the effects of oocyte electroporation prior to IVF on embryo development and to differentiate between true embryos and parthenotes by using a TPCN2 knock-out (KO) male to evaluate the presence of the KO allele in the resulting blastocysts. The study consisted of three experiments. The first one examined oocyte electroporation with and without subsequent IVF and found that electroporated oocytes had higher activation rates, increased occurrence of a single pronucleus, and no effect on sperm penetration. Cleavage rates improved in electroporated oocytes, but blastocyst rates remained constant. Genotype analysis revealed a significant increase in the proportion of parthenotes in the electroporated groups compared to the IVF control (30.2 % vs. 6.8 %). The second experiment compared two electroporation media, Opti-MEM and Nuclease-Free Duplex Buffer (DB). DB induced higher oocyte degeneration rates, and lower cleavage and blastocyst rates than Opti-MEM, while parthenogenetic formation remained consistent (60.0 and 48.5 %). In the third experiment, the timing of electroporation relative to IVF was evaluated (1 h before IVF, immediately before IVF and 7 h after IVF). Electroporation immediately before IVF resulted in higher activation rates and different pronuclear proportions compared to the other timing groups. The penetration rate was higher in the immediate electroporation group, and cleavage rate improved in all electroporated groups compared to the control. Blastocyst rates remained constant. Genotyping revealed no significant differences in parthenote proportions among the timing groups, but these were higher than the control (56.25 %, 63.89 %, 51.61 %, 2.44 %, respectively), and showed higher mutation rates when electroporation was performed 7 h after IVF. Overall, this comprehensive study sheds light on the potential of electroporation for creating genetically modified embryos and the importance of media selection and timing in the process, the best media being the Opti-MEM and the more efficient timing regarding mutation rate, 7 h post-IVF, even when the parthenote formation did not differ among electroporated groups. Further studies are needed to reduce the parthenogenetic activation while maintaining high mutation rates to optimize the use of this procedure for the generation of gene-edited pig embryos by oocyte/zygote electroporation.

ERAP, KIR, and HLA-C Profile in Recurrent Implantation Failure.

The mother’s uterine immune system is dominated by uterine natural killer (NK) cells during the first trimester of pregnancy. These cells express killer cell immunoglobulin-like receptors (KIRs) of inhibitory or activating function. Invading extravillous trophoblast cells express HLA-C molecules, and both maternal and paternal HLA-C allotypes are presented to KIRs. Endoplasmic reticulum aminopeptidase 1 (ERAP1) and 2 (ERAP2) shape the HLA class I immunopeptidome. The ERAPs remove N-terminal residues from antigenic precursor peptides and generate optimal-length peptides to fit into the HLA class I groove. The inability to form the correct HLA class I complexes with the appropriate peptides may result in a lack of immune response by NK cells. The aim of this study was to investigate the role of ERAP1 and ERAP2 polymorphisms in the context of KIR and HLA-C genes in recurrent implantation failure (RIF). In addition, for the first time, we showed the results of ERAP1 and ERAP2 secretion into the peripheral blood of patients and fertile women. We tested a total of 881 women. Four hundred ninety-six females were patients who, together with their partners, participated in in vitro fertilization (IVF). A group of 385 fertile women constituted the control group. Women positive for KIR genes in the Tel AA region and HLA-C2C2 were more prevalent in the RIF group than in fertile women (p/pcorr. = 0.004/0.012, OR = 2.321). Of the ERAP polymorphisms studied, two of them (rs26653 and rs26618) appear to affect RIF susceptibility in HLA-C2-positive patients. Moreover, fertile women who gave birth in the past secreted significantly more ERAP1 than IVF women and control pregnant women (p < 0.0001 and p = 0.0005, respectively). In the case of ERAP2, the opposite result was observed; i.e., fertile women secreted far less ERAP2 than IVF patients (p = 0.0098). Patients who became pregnant after in vitro fertilization embryo transfer (IVF-ET) released far less ERAP2 than patients who miscarried (p = 0.0032). Receiver operating characteristic (ROC) analyses indicate a value of about 2.9 ng/ml of ERAP2 as a point of differentiation between patients who miscarried and those who gave birth to a healthy child. Our study indicates that both ERAP1 and ERAP2 may be involved in processes related to reproduction.

Perinatal Outcomes of In Vitro Fertilization Versus Natural Pregnancy in Very Low Birth Weight Infants

Objective: In vitro fertilization (IVF) pregnancy is increasing. The recent reviews have reported the perinatal outcomes of IVF were preterm birth, low birth weight, small for gestational age, congenital malformations, neurologic disorders and epigenetic defects. We aimed to analyze the perinatal outcomes of IVF compared with natural pregnancy on very low birth weight infants. Methods: Our study population was derived from Neonatal Intensive Care Unit of the Gangnam CHA Medical Center from 2010 to 2014, consisting of singleton live births in very low birth weight infants. We grouped IVF group (n=24) and control group (natural pregnancy, n=112). We analyzed two groups about maternal characteristics, neonatal characteristics, and outcomes (retinopathy of prematurity [ROP], bronchopulmonary dysplasia [BPD], periventricular leukomalacia [PVL], necrotizing enterocolitis [NEC], death). Results: Maternal age was significantly older in IVF group (34.9±0.9 vs. 33.0±0.4, P=0.03). The Apgar score 1 minute of the IVF group was significantly lower than control group (4.0±0.3 vs. 4.8±0.2, P=0.03). But there were no other significant differences of neonatal morbidities. In univariate logistic regression analysis with IVF, the odds ratio of maternal age was 1.13 (95% confidence interval: 1.01-1.27, P=0.04). The relative risk of RDS, BPD, PVL, and ROP was increased, but it was not significant in univariate and multivariate logistic regression. Conclusion: As the maternal age increased, IVF pregnancy was significantly higher. The relative risk of RDS, BPD, PVL, and ROP was high in IVF group, but it was not significant in univariate and multivariate logistic regression.

The effect of varicocele on the outcome of in vitro fertilization

Objective To investigate the effect of varicocele on the outcome of in vitro fertilization (IVF). Methods The 139 patients in vitro fertilization cycle were diagnosed by B-ultrasound, and 323 normal semen treated by IVF as control group. The rates of fertilization, cleavage rate, high quality embryo rate and pregnancy outcome in the IVF group were compared with those in the control group. Results There was statistically significant difference in fertilization rate, cleavage rate, high-quality embryo rate, embryo rate, clinical pregnancy rate between the IVF group and control group(P 0.05). Conclusions Varicocele can lead to the decline of IVF embryo quality, which has no obvious effect on pregnancy rate of abortion. Key words: Varicocele; Fertilization in Vitro

Association between sperm mitochondrial ND2 gene variants and total fertilization failure

ABSTRACTThe objective of this study was to explore the association of sperm mitochondrial ND2 (MT-ND2) gene variants with total fertilization failure (TFF). A retrospective comparative study of 246 cases of fresh in vitro fertilization (IVF) cycles or half-intracytoplasmic sperm injection cycles in the Han Chinese population was performed from July 2011 to May 2017. A total of 59 cases undergoing TFF, and 187 control cases with normal fertilization (fertilization rates >50%) were included. The sperm mitochondrial genovariation was determined using nested sequencing. A total of 32 homoplasmic variants and 47 heteroplasmic variants of MT-ND2 gene were observed in this study. There were no significant differences in the frequencies of the 32 homoplasmic variants of MT-ND2 gene between the TFF and control groups. A total of 53 pair-wise comparisons were performed, and the general characteristics of the IVF failure and control subjects were adjusted in logistic models. Data suggested that there were no significant differences in the frequencies of point 4914, 5320, and 5426 heteroplasmic variants of MT-ND2 gene between the TFF and control groups. In addition, no significant difference was observed in the frequency of mtDNA haplogroup D or haplogroup G between the IVF failure group and the normal fertilization group. This study suggests that the MT-ND2 gene variants might not be associated with TFF.Abbreviations: ATP: adenosine triphosphate; dNTP: deoxy-ribonucleoside triphosphate; FADH2: flavin adenine dinucleotide; FDR: false discovery rate; FSH: follicle-stimulating hormone; IVF: in vitro fertilization; LH: luteinizing hormone; MTATP6: mitochondrially encoded ATP synthase 6; MTCYB: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MT-ND2: mitochondrial ND2; NADH: nicotinamide adenine dinucleotide; ND2: NADH dehydrogenase subunit 2; OXPHOS: oxidative phosphorylation; PCR: single nucleotide polymorphisms; SNPs: single nucleotide polymorphisms; TFF: total fertilization failure

Hyper response to ovarian stimulation affects the follicular fluid metabolomic profile of women undergoing IVF similarly to polycystic ovary syndrome.

During in vitro fertilization (IVF), the hyper response to controlled ovarian stimulation (COS) is a common characteristic among patients diagnosed with polycystic ovary syndrome (PCOS), although non-diagnosed patients may also demonstrate this response. In an effort to investigate follicular metabolic characteristics associated with hyper response to COS, the present study analyzed follicular fluid (FF) samples from patients undergoing IVF. FF samples were obtained from patients with PCOS and hyper response during IVF (PCOS group, N = 15), patients without PCOS but with hyper response during IVF (HR group, N = 44), and normo-responder patients receiving IVF (control group, N = 22). FF samples underwent Bligh and Dyer extraction, followed by metabolomic analysis by ultra-performance liquid chromatography mass spectrometry, considering two technical replicates. Clinical data was analyzed by ANOVA and chi-square tests. The metabolomic dataset was analyzed by multivariate statistics, and the significance of biomarkers was confirmed by ANOVA. Clinical data showed differences regarding follicles production, oocyte and embryo quality. From the 15 proposed biomarkers, 14 were of increased abundance in the control group and attributed as fatty acids, diacylglycerol, triacylglycerol, ceramide, ceramide-phosphate, phosphatidylcholine, and sphingomyelin. The PCOS patients showed increased abundance of a metabolite of m/z 144.0023 that was not attributed to a class. The clinical and metabolic similarities observed in the FF of hyper responders with and without PCOS diagnosis indicate common biomarkers that could assist on the development of accessory tools for assessment of IVF parameters.

The role of Kisspeptin in bovine in vitro embryo production

The aim of this study was to investigate the effect of Kisspeptin (Kp) on the medium used in different stages of in vitro production of bovine embryo (IVEP), evaluating cleavage (CR) and blastocyst (BR) rates. The study was divided into three experiments that analyzed, respectively, the action of Kp on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of bovine embryos. In experiment 1, the oocytes were matured in IVM medium and distributed into the following treatments: maturation (IVM Control, n = 102), maturation with addition of 10-7 M Kp (Kp 10-7 IVM, n = 90), and hormone-free maturation luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with the addition of 10-7M Kp (No hormones + Kp 10-7, n = 84), following maturation to normal stages of IVEP. In experiment 2, the oocytes were fertilized in IVF medium, in the following treatments: TALP-FERT without Kp (Control IVF, n = 103) and TALP-FERT with the addition of 10-7M Kp (Kp 10-7 IVF, n = 119), usually following the other steps. Finally, in the third experiment, the oocytes passed through all phases and were divided into IVC in two treatments: SOF medium without Kp (Control IVC, n = 109) and SOF medium with the addition of 10-7M Kp (Kp 10-7, N = 106). The data were analyzed by PROC GLIMMIX of the SAS program. In experiment 1, the means of CR and BR were similar (P &gt; 0.05) between treatments (IVM Control76.47% and 37.25%, Kp 10-7 MIV80% and 33.33%, and No hormones + Kp 10-770.24% and 30.95%, respectively). In experiment 2, the means of CR were similar for the IVF Control and Kp 10-7 IVF groups (P &gt; 0.05), 76.70% and 86.55% respectively. But, the mean of the BR of the group Kp 10-7 IVF was 38.66%, which was higher (P &lt; 0.05) than that of the FIV Control group, which was 31.07%. In the third experiment, the means of CR and BR (P &gt; 0.05) were similar between the IVC Control and Kp 10-7 IVC groups (CR 83.50% and 78.30%, and BR 26.60% and 23.60%, respectively). Although at this concentration of 10-7M during IVC no change in embryo production is seen, Kp presents the same performance as both gonadotrophins in oocyte maturation and modulates the fertilization process, providing more blastocysts. With these findings, it can be seen that Kp presents a regulatory action on bovine reproduction, and can be an excellent tool to maximize IVEP indexes.

80 Cumulus Cells Reflect the Success of IVF of Porcine Oocytes Treated with Dimethylthiourea

The aim of this study was to evaluate the effect of the antioxidant dimethylthiourea (DMTU) as a supplement during the collection and washing of cumulus–oocyte complexes (COC) on the apoptosis in cumulus cells (CC) and the competition for oocyte development in vitro. The COC were obtained by follicular aspiration. The aspiration and washing medium was TCM-199 with 10% porcine follicular fluid, 0.3 mM sodium pyruvate, and penicillin/streptomycin. Treated groups were supplemented with 2 or 20 mM DMTU. After 44 h of in vitro maturation (IVM), the percentage of nuclear maturation was determined by staining of the denuded oocytes with Hoechst 33442 (n = 357). After 18 h of in vitro fertilization (IVF) with 15°C refrigerated spermatozoa from boars of proven fertility, the suspected zygotes were stained with Hoechst 33342 to evaluate the percentages of sperm penetration (SP), monospermic penetration (MP), male pronucleus formation (MPN), and IVF efficiency (monospermic oocytes/total oocytes) (n = 260). In post-IVM CC, flow cytometry was performed after staining with annexin V-propidium iodide to evaluate apoptosis and viability (n = 90,000). Data were analysed with Flowing V 2.5.1 (http://flowingsoftware.btk.fi/). A comparison of proportion among categories was used (Fisher’s exact test), and significant differences were determined at P &lt; 0.05. No effects were observed in the percentages of nuclear maturation. Treatments with DMTU significantly increased the SP (Control: 35%; 2 mM: 72.9%; 20 mM: 67.8%) without modifying the MP. Although in both treatments, the amount of MPN was always greater than in the control, the percentages of MPN formation did not show significant differences. The 20 mM treatment significantly increased the efficiency of IVF (control: 22.2%; 2 mM: 31.9%; 20 mM: 45.7%). In the CC of the treated groups, a significant increase in viability (control: 7.4%; 2 mM: 12.2%; 20 mM: 14.7%) and a decrease in early apoptosis (control: 60.4%; 2 mM: 55.5%; 20 mM: 57.1%) were observed. Late apoptosis decreased in the 20 mM treatment (27.3%) compared with the other experimental groups (control: 31.3%; 2 mM: 31.2%). The addition of DMTU during COC collection and washing reduce the ROS-mediated apoptosis in CC and optimize the efficiency of IVF. Besides, CC quality reflects the effect of DMTU on oocytes, so viability and apoptosis analysis on CC constitutes an interesting noninvasive method to assess oocyte competence.

Effectiveness of in vitro fertilization in women with previous tubal sterilization

ObjectiveThe objective was to determine the effectiveness of in vitro fertilization (IVF) on live-delivery rates in women who had previously undergone tubal sterilization. Study designWe examined first IVF live deliveries for women aged 20–44 years at their first embryo transfer (ET) with history of hospital admission for tubal sterilization in Western Australia (WA). The ET cycles (n=178) were ascertained over the period of 1996 to 2010 using WA hospital records. A control group of subfertile women matched by age was randomly selected (n=178). We used Kaplan–Meier curves and life-table analysis to evaluate the cumulative live-delivery rates. ResultsAn overall cumulative live-delivery rate in women who had undergone previous tubal sterilization (31%) was comparable to that of subfertile controls (34%) within the first 24 months. Younger women (aged 20–34 years) with previous sterilization (34%) were slightly more likely to deliver an IVF live baby than older women (aged 35–39 and 40–44 years) (33% and 22%, respectively), although this difference was not statistically significant (p=.449). ConclusionWomen who desire fertility after a tubal sterilization procedure and undergo IVF have rates of pregnancy similar to age-matched subfertile IVF control patients. ImplicationsIn vitro fertilization success in women who had undergone previous tubal sterilization is similar to that of the subfertile controls and thus does not depend on past fertility. Age is the most important predictive factor in achieving pregnancy.

Increase of CD69, CD161 and CD94 on NK cells in women with recurrent spontaneous abortion and in vitro fertilization failure.

Recurrent spontaneous abortion (RSA) and in vitro fertilization (IVF) failure with unknown causes are the controversial issues that are probably related to the immune system. To compare circulating NK cells expressing activation and inhibition surface markers between patients with RSA and IVF failure with those of healthy multiparous and successful IVF control women, respectively. In this case-control study peripheral blood samples were collected from 43 patients who included 23 women with RSA and 20 with IVF failure, plus 43 healthy control women comprising of 36 normal multiparous women and seven women with successful IVF. The expression of CD69, CD94 and CD161 surface markers on CD56+NK cells were assessed using specific monoclonal antibodies by flowcytometry. The percentage of NK cells increased significantly in patients with RSA and in women with IVF failure in comparison to healthy multiparous and successful IVF control groups (p<0.001). The overall expression of CD69, CD94, CD161 were also increased significantly on NK cells in both patient groups compared to control groups (p<0.001). Elevated expression of CD69 and CD161 on NK cells can be considered as immunological risk markers in RSA and IVF failure. However, it is not clear if high expression of CD94 on peripheral blood NK cells is related to abnormal activity of endometrial NK cells.

Fertility preservation outcomes may differ by cancer diagnosis.

CONTEXT:Cancer survival has improved significantly and maintaining fertility is both a major concern and an important factor for the quality of life in cancer patients.AIMS:To explore differences in oocyte stimulation for fertility preservation (FP) patients based on cancer diagnosis.SETTINGS AND DEIGN:Between 2005 and 2011, 109 patients elected to pursue FP at a single institution.MATERIALS AND METHOD:In vitro fertilization (IVF) outcome variables between four cancer diagnostic groups (breast, gynecologic, lymphoma/leukemia and other) and age-matched male factor or tubal factor infertility IVF control group were compared.STATISTICAL ANALYSIS:ANOVA and Chi-square analyses were employed to compare variables between the groups that were normally distributed. Kruskal–Wallis with subsequent Mann–Whitney U-test were used for data that were not normally distributed.RESULTS:Women with gynecologic malignancies were significantly older than the women in the other three groups, but tended to have a better ovarian response. Women with hematologic malignancies were most likely to have been exposed to chemotherapy and had the longest stimulations with a similar number of oocytes retrieved. The age-matched IVF controls had higher peak estradiol levels, number of oocytes obtained, and fertilization rates when compared to cancer patients with or without a history of prior chemotherapy.CONCLUSIONS:Factors including age, type of cancer and chemotherapy exposure, can influence response to ovarian stimulation. Discussing these findings with patients presenting for FP may aid in setting realistic treatment expectations.

The search for biomarkers of human embryo developmental potential in IVF: a comprehensive proteomic approach

The objective of these studies was to identify differentially expressed peptides/proteins in the culture media of embryos grown during in vitro fertilization (IVF) treatment to establish their value as biomarkers predictive of implantation potential and live birth. Micro-droplets of embryo culture media from IVF patients (conditioned) and control media maintained under identical culture conditions were collected and frozen at -80°C on Days 2-3 of in vitro development prior to analysis. The embryos were transferred on Day 3. The peptides were affinity purified based on their physico-chemical properties and profiled by mass spectrometry for differential expression. The identified proteins were further characterized by western blot and ELISA, and absolute quantification was achieved by multiple reaction monitoring (MRM). We identified up to 14 differentially regulated peptides after capture using paramagnetic beads with different affinities. These differentially expressed peptides were used to generate genetic algorithms (GAs) with a recognition capability of 71-84% for embryo transfer cycles resulting in pregnancy and 75-89% for those with failed implantation. Several peptides were further identified as fragments of Apolipoprotein A-1, which showed consistent and significantly reduced expression in the embryo media samples from embryo transfer cycles resulting in viable pregnancies. Western blot and ELISA, as well as quantitative MRM results, were confirmatory. These results demonstrated that peptide/protein profiles from the culture medium during early human in vitro development can discriminate embryos with highest and lowest implantation competence following uterine transfer. Further prospective studies are needed to establish validated thresholds for clinical application.

Enhancement of peripheral blood CD56dim cell and NK cell cytotoxicity in women with recurrent spontaneous abortion or in vitro fertilization failure

Recent studies support the concept that NK cells play an important role in the success or failure of embryo implantation. Recurrent spontaneous abortion (RSA) is the most common complication of pregnancy. Some couples suffer from infertility of unknown cause. In vitro fertilization (IVF) is one of the useful treatment methods used for treatment of this type for infertility with variable outcomes. The aim of this study was to compare the percentage of peripheral blood CD56+ (CD56dim and CD56bright) cells and the level of NK cell cytotoxicity in patients with RSA and patients with IVF failure with those of healthy multiparous and successful IVF control women. In this case–control study peripheral blood samples from 43 patients, which included 23 women with RSA and 20 with IVF failure, plus 43 healthy control women comprising 36 normal multiparous women and 7 women with successful IVF, were collected. The percentage of peripheral blood NK cells (CD56+) was identified by flow cytometry, then peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation (Ficol-Hypaque) and incubated with NK-sensitive K562 cells. The NK cell cytotoxicity level was determined by lactate dehydrogenase (LDH) release assay. The percentage of CD56dim cells and the level of peripheral blood NK cell cytotoxicity in RSA patients and women with IVF failure were significantly higher than in both the healthy multiparous and successful IVF control groups (P<0.001). The findings of the present study suggest that increases in the percentage of CD56dim cells and NK cytotoxicity in peripheral blood may be important contributing factors for both RSA and IVF failure.

Chromosomal abnormalities in spontaneous abortion after assisted reproductive treatment

BackgroundWe evaluated cytogenetic results occurring with first trimester pregnancy loss, and assessed the type and frequency of chromosomal abnormalities after assisted reproductive treatment (ART) and compared them with a control group. We also compared the rate of chromosomal abnormalities according to infertility causes in ICSI group.MethodsA retrospective cohort analysis was made of all patients who were referred to the Genetics Laboratory of Fertility Center of CHA Gangnam Medical Center from 2005 to 2009 because of clinical abortion with a subsequent dilation and evacuation (D&E) performed, and patients were grouped by type of conception as follows: conventional IVF (in vitro fertilization) (n = 114), ICSI (intracytoplasmic sperm injection) (n = 140), and control (natural conception or intrauterine insemination [IUI]) (n = 128). Statistical analysis was performed using SPSS software.ResultsA total 406 specimens were referred to laboratory, ten abortuses were excluded, and in 14 cases, we did not get any spontaneous metaphase, chromosomal constitutions of 382 specimens were successfully obtained with conventional cytogenetic methods. Overall, 52.62% of the miscarriages were found to be cytogenetically abnormal among all patients, the frequency was 48.4% in the control group, 54.3% of miscarriages after ICSI and 55.3% after conventional IVF (p = 0.503). The most prevalent abnormalities were autosomal trisomy, however, nine (11.69%) sex chromosome aneuploidy were noted in the ICSI group vs. four (6.45%) and two (3.23%) cases in the conventional IVF group and control group. We compared chromosomal abnormalities of miscarriages after ICSI according to infertility factor. 55.71% underwent ICSI due to male factors, 44.29% due to non-male factors. ICSI group having male factors showed significantly higher risk of chromosomal abnormalities than ICSI group having non-male factors (65.8% vs. 34.2%, p = 0.009, odds ratio = 1.529, 95% CI = 1.092-2.141).ConclusionsThere is no increased risk of chromosomal abnormalities due to ART was found with the exception of a greater number of sex chromosomal abnormalities in the ICSI group with male factor infertility. Therefore, these alterations could be correlated with the underlying parental risk of abnormalities and not with the ICSI procedure itself.

Chromosomal abnormalities in spontaneous abortions after assisted reproductive treatment: 5 year experiences from 2005 to 2009

OBJECTIVE: To evaluate cytogenetic results occurring first trimester spontaneous abortion after different assisted reproductive treatment (ART), and to understand whether ART affect early pregnancy loss resulting from chromosomal defects or not. DESIGN: Retrospective study. MATERIALS AND METHODS: A chart review was made of all patients were referred for cytogenetic evaluations of clinical abortions with a subsequent dilatation and evacuation from 2005 to 2009. Patients were grouped by type of conception; conventional in vitro fertilization (IVF) (n=114), intracytoplasmic sperm injection (ICSI) (n=140) and control including natural conception and intrauterine insemination (n=128), and causes of infertility. The types and frequencies of chromosomal anomalies were evaluated in each group. Statistical analysis was performed using SPSS (Chicago, IL) software. RESULTS: Out of total 406 specimens referred to laboratory, 382 specimens were successfully analyzed by conventional cytogenetic methods. Overall, 52.6% of the miscarriages were found to be cytogenetically abnormal. The incidences of chromosome anomalies in miscarriages after ICSI and after IVF were 54.3% and 55.3% and no difference from in controls (48.4%, p=0.503). The most prevalent anomalies were autosomal trisomies. Meanwhile, sex chromosome aneuploidies were noted in the ICSI group (9 cases, 11.7%) comparing to the IVF group (4 cases, 6.5%) and control group (2 cases, 3.2%). We also compared chromosomal anomalies of miscarriages after ICSI according to infertility factors, among these cases, ICSI group with male factors, showed significantly high risk of chromosomal anomalies than ICSI group with non-male factors (65.8% vs. 34.2%, p=0.009, odds ratio=1.529). CONCLUSION: There is no increase risk for chromosomal anomalies in ART groups except for higher incidence of sex-chromosome anomalies in ICSI group. However, it seems to be correlated with the underlying parental risk of abnormality and not with the ICSI procedure itself.

Miscarriage rates after dehydroepiandrosterone (DHEA) supplementation in women with diminished ovarian reserve: a case control study

BackgroundDehydroepinadrosterone (DHEA) supplementation improves pregnancy chances in women with diminished ovarian reserve (DOR), by possibly reducing aneuploidy. Since a large majority of spontaneous miscarriages are associated with aneuploidy, one can speculate that DHEA supplementation may also reduce miscarriage rates.MethodsWe retroactively compared, utilizing two independent statistical models, miscarriage rates in 73 DHEA supplemented pregnancies at two independent North American infertility centers, age-stratified, to miscarriages reported in a national U.S. in vitro fertilization (IVF) data base.ResultsAfter DHEA supplementation the miscarriage rate at both centers was 15.1% (15.0% and 15.2%, respectively). For DHEA supplementation Mantel-Hänszel common odds ratio (and 95% confidence interval), stratified by age, was significantly lower, relative to odds of miscarriage in the general IVF control population [0.49 (0.25-0.94; p = 0.04)]. Miscarriage rates after DHEA were significantly lower at all ages but most pronounced above age 35 years.DiscussionSince DOR patients in the literature are reported to experience significantly higher miscarriage rates than average IVF patients, the here observed reduction in miscarriages after DHEA supplementation exceeds, however, all expectations. Miscarriage rates after DHEA not only were lower than in an average national IVF population but were comparable to rates reported in normally fertile populations. Low miscarriage rates, comparable to those of normal fertile women, are statistically impossible to achieve in DOR patients without assumption of a DHEA effect on embryo ploidy. Beyond further investigations in infertile populations, these data, therefore, also suggest the investigations of pre-conception DHEA supplementation in normal fertile populations above age 35 years.

Efficiency of SCNT Buffalo (Bubalus bubalis) Embryos in Different Culture Medium and Analysis of mRNA Expression of Insulin-like Growth Factors During Embryogenesis

Growth factors in culture media are known to affect the embryo production rates in in vitro production cultures. To improve the efficiency of somatic cell nuclear transfer (SCNT) derived embryos in Indian buffaloes (Bubalus bubalis), embryos were cultured in three different culture mediums viz. Group-A; TCM-199 + FBS, Group-B; TCM-199 + Poly vinyl alcohol (PVA) and Group-C; CR1aa + BSA. Embryo production rate and expression level of insulin-like growth factor genes (IGF-1, IGF-1R, IGF-2 and IGF-2R) were analysed in embryo culture. Cleavage and blastocyst production rates were 62.5% and 22.3% in Group-A, 53.8% and 13.0% in Group-B and 62.0% and 19.2% in Group-C respectively, whereas in in vitro fertilization (IVF) control cultured in TCM-199 plus 10% FBS, rates were 79.1% and 29.4%. Relative gene expression of SCNT embryos was compared with that in IVF control. IGF-1 and IGF-2 mRNA expression at blastocyst stage was up-regulated (p ≤ 0.05) in all culture groups, while IGF-1R and IGF-2R expression was down regulated (p ≤ 0.05) in Group-B and Group-C. In conclusion, the higher mRNA levels at certain stages in different culture conditions affected in vitro development of SCNT embryos. These results show that the transcript level of the insulin-like growth factor genes was significantly altered by in vitro culture condition. Culture medium TCM-199 with 10% FBS produced higher number of embryos and was able to co-op with gene expression of IVF control. Differences continue to be observed between SCNT cultured and IVF embryos, and until these differences are minimized, aberrations in SCNT embryonic development will continue to arise.

281 ATTEMPTS FOR ESTABLISHMENT OF PORCINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS

The porcine embryonic stem (ES) cells could be a useful tool for the production of transgenic animals and the study of developmental gene regulation. Even though the efficiency of establishment of ES cells from in vivo blastocysts is relatively high, especially in mice, it is difficult and expensive to obtain in vivo embryos in domestic animals. Recent development of techniques in the production of embryos in vitro could be a useful source for the establishment of ES cells. However, the morphology and cell quality of in vitro-produced embryos are inferior to those of their in vivo counterparts. Although many attempts have been made to establish ES cells from in vitro-produced embryos, the overall efficiency is extremely low because of the poor embryo quality. However, aggregation of in vitro-produced embryos was developed to increase the number of cells in the inner cell mass (ICM) of blastocysts and could be useful in the application to ES cell establishment. Therefore, in this study, we attempted to derive porcine ES cells by using aggregation of in vitro-produced embryos by in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Embryos at the 4-cell stage were produced by culturing embryos for 2 days after IVF and SCNT. After removal of the zona pellucida with acid Tyrode’s solution, three 4-cell-stage embryos (IVF3X) from IVF and two 4-cell-stage embryos (NT2X) from SCNT were aggregated by co-culturing them in an aggregation plate followed by culturing to the blastocyst stage. Embryos from IVF (IVF control) and SCNT (NT control) were also cultured to the blastocyst stage. All blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Two primary colonies were formed in the IVF control group (3.9%), whereas four primary colonies were formed in the IVF3X group (12.5%). One primary colony was formed in the NT2X group (20%), although no colony was formed in the NT control group. One of the IVF3X lines gradually disappeared after sub-passing, and the NT2X line also disappeared. Two ES-like cell lines derived from the IVF control were maintained up to 14 passages, and three ES-like lines from IVF3X were also maintained for more than 14 passages. These cells morphologically resembled human ES cells (flat and single layered) and expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, Oct-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. These results indicated that a porcine ES cell line could be established from in vitro-produced aggregated blastocysts. Further research is required to establish ES cell lines from SCNT embryos and characterize the differentiation and developmental abilities of these porcine ES-like cells. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

Adolescents conceived by IVF: parenting and psychosocial adjustment

A follow-up study was conducted in mid-adolescence on parenting and the child's psychosocial development after in vitro fertilization (IVF). The first phase of the study had compared 31 IVF families and 31 families with a naturally conceived child when the children were 2 years old (Colpin et al., 1995). Of these, 24 IVF families and 21 control families participated again when the children were 15-16 years old. Fathers, mothers and adolescents completed questionnaires assessing parenting style and stress, and adolescent psychosocial adjustment. No significant differences were found in self- or adolescent-reported parenting style, or in parenting stress between IVF mothers and mothers in the control group, nor between IVF fathers and fathers in the control group. Neither did we find significant differences in self- or parent-reported behavioural problems between adolescents conceived by IVF and those conceived naturally. Comparison of behavioural problems between IVF adolescents informed or not informed about the IVF conception did not reveal significant differences. Parenting and 15-16-year-old adolescents' psychosocial adjustment did not differ significantly between IVF families and control families. This study is, to the best of our knowledge, the first psychosocial follow-up in mid-adolescence, and adds to the evidence that IVF children and their parents are well-adjusted. Large-scale studies in adolescence are needed to support these findings.