Effect of parental and ART treatment characteristics on perinatal outcomes.

STUDY QUESTION: Do parental characteristics and treatment with ART affect perinatal outcomes in singleton pregnancies? SUMMARY ANSWER: Both parental and ART treatment characteristics affect perinatal outcomes in singleton pregnancies. WHAT IS KNOWN ALREADY: Previous studies have shown that singleton pregnancies resulting from ART are at risk of preterm birth. ART children are lighter at birth after correction for duration of gestation and at increased risk of congenital abnormalities compared to naturally conceived children. This association is confounded by parental characteristics that are also known to affect perinatal outcomes. It is unclear to which extent parental and ART treatment characteristics independently affect perinatal outcomes. STUDY DESIGN, SIZE, DURATION: All IVF clinics in the Netherlands (n = 13) were requested to provide data on all ART treatment cycles (IVF, ICSI and frozen-thawed embryo transfers (FET)), performed between 1 January 2000, and 1 January 2011, which resulted in a pregnancy. Using probabilistic data-linkage, these data (n = 36 683) were linked to the Dutch Perinatal Registry (Perined), which includes all children born in the Netherlands in the same time period (n = 2 548 977). PARTICIPANTS/MATERIALS, SETTING, METHODS: Analyses were limited to singleton pregnancies that resulted from IVF, ICSI or FET cycles. Multivariable models for linear and logistic regression were fitted including parental characteristics as well as ART treatment characteristics. Analyses were performed separately for fresh cycles and for fresh and FET cycles combined. We assessed the impact on the following perinatal outcomes: birth weight, preterm birth below 37 or 32 weeks of gestation, congenital malformations and perinatal mortality. MAIN RESULTS AND THE ROLE OF CHANCE: The perinatal outcomes of 31 184 out of the 36 683 ART treatment cycles leading to a pregnancy were retrieved through linkage with the Perined (85% linkage). Of those, 23 671 concerned singleton pregnancies resulting from IVF, ICSI or FET. Birth weight was independently associated with both parental and ART treatment characteristics. Characteristics associated with lower birth weight included maternal hypertensive disease, non-Dutch maternal ethnicity, nulliparity, increasing duration of subfertility, hCG for luteal phase support (compared to progesterone), shorter embryo culture duration, increasing number of oocytes retrieved and fresh embryo transfer. The parental characteristic with the greatest effect size on birth weight was maternal diabetes (adjusted difference 283 g, 95% CI 228-338). FET was the ART treatment characteristic with the greatest effect size on birth weight (adjusted difference 100 g, 95% CI 84-117) compared to fresh embryo transfer. Preterm birth was more common among mothers of South-Asian ethnicity. Preterm birth was less common among multiparous women and women with 'male factor' as treatment indication (compared to 'tubal factor'). LIMITATIONS, REASONS FOR CAUTION: Due to the retrospective nature of our study, we cannot prove causality. Further limitations of our study were the inability to adjust for mothers giving birth more than once in our dataset, missing values for several variables and limited information on parental lifestyle and general health. WIDER IMPLICATIONS OF THE FINDINGS: Multiple parental and ART treatment characteristics affect perinatal outcomes, with birth weight being influenced by the widest range of factors. This highlights the importance of assessing both parental and ART treatment characteristics in studies that focus on the health of ART-offspring, with the purpose of modifying these factors where possible. Our results further support the hypothesis that the embryo is sensitive to its early environment. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Foreest Medical School, Alkmaar, the Netherlands (grants: FIO 1307 and FIO 1505). B.W.M. reports grants from NHMRC and consultancy for ObsEva, Merck KGaA, iGenomics and Guerbet. F.B. reports research support grants from Merck Serono and personal fees from Merck Serono. A.C. reports travel support from Ferring BV. and Theramex BV. and personal fees from UpToDate (Hyperthecosis), all outside the remit of the current work. The remaining authors report no conflict of interests. TRIAL REGISTRATION NUMBER: N/A.

The preconception diet is associated with the chance of ongoing pregnancy in women undergoing IVF/ICSI treatment.

BACKGROUND: Subfertility and poor nutrition are increasing problems in Western countries. Moreover, nutrition affects fertility in both women and men. In this study, we investigate the association between adherence to general dietary recommendations in couples undergoing IVF/ICSI treatment and the chance of ongoing pregnancy. METHODS: Between October 2007 and October 2010, couples planning pregnancy visiting the outpatient clinic of the Department of Obstetrics and Gynaecology of the Erasmus Medical Centre in Rotterdam, the Netherlands were offered preconception counselling. Self-administered questionnaires on general characteristics and diet were completed and checked during the visit. Six questions, based on dietary recommendations of the Netherlands Nutrition Centre, covered the intake of six main food groups (fruits, vegetables, meat, fish, whole wheat products and fats). Using the questionnaire results, we calculated the Preconception Dietary Risk score (PDR), providing an estimate of nutritional habits. Dietary quality increases with an increasing PDR score. We define ongoing pregnancy as an intrauterine pregnancy with positive heart action confirmed by ultrasound. For this analysis we selected all couples (n=199) who underwent a first IVF/ICSI treatment within 6 months after preconception counselling. We applied adjusted logistic regression analysis on the outcomes of interest using SPSS. RESULTS: After adjustment for age of the woman, smoking of the woman, PDR of the partner, BMI of the couple and treatment indication we show an association between the PDR of the woman and the chance of ongoing pregnancy after IVF/ICSI treatment (odds ratio 1.65, confidence interval: 1.08-2.52; P=0.02]. Thus, a one-point increase in the PDR score associates with a 65% increased chance of ongoing pregnancy. CONCLUSIONS: Our results show that increasing adherence to Dutch dietary recommendations in women undergoing IVF/ICSI treatment increases the chance of ongoing pregnancy. These data warrant further confirmation in couples achieving a spontaneous pregnancy and in randomized controlled trials.

Neuronal differentiation of embryonic stem cells.

Neuronal differentiation from totipotent precursors in vitro, is thought to require two signals: first a biophysical state (cellular aggregation) followed by a biochemical signal (retinoic acid treatment). In investigating the properties of retinoic acid-differentiated embryonic stem cell lines. However, we noted that retinoic acid treatment without prior aggregation, is sufficient to induce expression of the neuronal markers GAP-43 and NF-165. In agreement, immunohistochemistry revealed the presence of GAP-43 positive cells in these embryonic stem cell monolayers after three days of retinoic acid (RA) treatment. Furthermore an NF-165 positive subpopulation of cells was clearly observed after 4-5 days of RA treatment. The expression of these neuronal markers coincided with the appearance of electrically excitable cells, as assayed with whole cell patch clamp recording. We conclude that for neuronal differentiation of totipotent embryonic stem cells in vitro, one biochemical signal, i.e. retinoic acid treatment, is sufficient.

The role of receptor protein tyrosine phosphatase alpha in neuronal differentiation of embryonic stem cells.

In the present study, we have investigated the function of the receptor protein tyrosine phosphatase alpha (RPTP alpha) in the neuronal differentiation of E14-embryonic stem (E14-ES) cells. RNAase protection and western blot analysis revealed that E14-ES cells up regulate RPTP alpha expression upon neuronal differentiation with retinoic acid. Overexpression of RPTP alpha, by stable DNA transfection, and subsequent differentiation with retinoic acid, resulted in a temporally enhanced expression of the neuronal markers GAP-43 and NF-164. Electrophysiological experiments demonstrated that RPTP alpha overexpression also enhanced the development of neurotransmitter responses during differentiation. These results indicate that RPTP alpha plays an important role in the cascade of molecular events that lead to the formation of neurons.

Regulation of growth and differentiation in early development: of mice and models.

In this article we describe some of the fundamental processes occurring during early murine development, introduce cellular models used to investigate these processes and review some well-known factors that may be involved in their control. These include transforming growth factor beta, retinoic acid and leukaemia inhibitory factor. Refinements to the culture conditions of embryonic stem and embryonal carcinoma cells have enabled us to test the effects of these factors on growth and differentiation and in particular to establish that their interaction may determine the ultimate developmental state of the cell population. Preliminary studies using neutralizing antibodies in embryos are described that suggest that deregulation of normal expression can lead to a failure to implant. Insights into the events underlying normal embryonic development and implantation, yielded by the type of study described here, may contribute to an understanding of the mechanisms causing early embryonic loss and the role of toxicants in this process.

Culture of bovine embryos to the blastocyst stage using Buffalo rat liver (BRL) cells.

A comparison was made between the development of in vitro matured and fertilized bovine oocytes in co-culture with bovine oviduct epithelial (BOE) cells or with Buffalo rat liver (BRL) cells. Both cell types supported development from the 1-cell to the blastocyst stage with equal efficiencies (4.4% for BRL cells, 4.0% for BOE cells). Medium conditioned by either cell type supported development to the blastocyst stage as efficiently as co-cultures (6.4 and 7.3% blastocysts for BOE and BRL conditioned medium, respectively). A higher percentage of blastocyst development was found when embryos were cultured closely apposed in small drops of BRL-conditioned medium compared with larger volumes (20.5 versus 7.0%). The ability of BRL-conditioned medium to support embryonic development was dependent on the duration of the conditioning period (optimum 24 to 48 h), and was not lost when the medium was stored at -20 degrees C for extended periods. The effects were independent of the conditions used to promote maturation in vitro and the procedure for fertilization. With 2 different methods to produce embryos in culture, both the BRL cell co-culture and BRL-conditioned medium in microdrops supported embryo development to the blastocyst stage. The use of the BRL cell line reduces the variability associated with primary BOE cell cultures.

Use rate and assisted reproduction technologies outcome of cryopreserved semen from 629 cancer patients.

OBJECTIVE: To assess the use rate and assisted reproductive technologies (ART) outcome of the cryopreserved semen of cancer patients with an average follow-up of 7 years (range, 2-23 years). DESIGN: Retrospective data analysis. SETTING: University-affiliated andrology and reproduction center. PATIENT(S): Six hundred twenty-nine male cancer patients who were referred for semen cryopreservation between 1983 and 2004. INTERVENTION(S): Review of patient characteristics and ART outcome. MAIN OUTCOME MEASURE(S): Use rate and live births using cryopreserved semen. RESULT(S): A total of 749 semen samples from 557 men were preserved. Ninety-one patients died during follow-up, and another 29 requested disposal. Forty-two patients requested the use of their banked semen. ART data were available for 37 patients. A total of 101 ART cycles (32 IVF, 53 intracytoplasmic sperm injection [ICSIs], nine cryo-ET, and seven intrauterine inseminations [IUIs]) were performed, resulting in, respectively, 8, 16, 2, and 1 pregnancies. Pregnancies rates for IVF and ICSI were significantly higher than those for IUI. CONCLUSION(S): So far, 7.5% of the cancer survivors have used their banked semen, which led to live births in 49% of the couples. Semen cryopreservation is a reliable method to preserve fertility potential and gives couples a reasonable chance of achieving parenthood.

One normal child and a chromosomally balanced/normal twin after intracytoplasmic sperm injection in a male with a de-novo t(Y;16) translocation.

A balanced translocation t(Y;16)(q11.21;q24) is described in a male with severe oligoasthenoteratozoospermia (OAT). Before having a chromosome investigation, the patient and his partner had undergone intracytoplasmic sperm injection (ICSI) treatment resulting in the birth of a healthy 46,XX child. After detection of the t(Y;16) translocation, the couple opted for further ICSI treatment, although they were extensively counselled on the risk of having chromosomally unbalanced offspring. This treatment resulted in a twin pregnancy, one with a 46,XX karyotype and the other a 46,X,t(Y;16) (q11.21;q24) karyotype, the same as the father. After an uncomplicated pregnancy two healthy children were born. We conclude that patients with a Y/autosome translocation as a cause of OAT can have chromosomally normal children after ICSI treatment.

Transforming growth factor-beta in the early mouse embryo: implications for the regulation of muscle formation and implantation.

In a search for functions of transforming growth factor-beta during early embryonic development we used two different experimental approaches. In the first we made use of embryonic stem (ES) cells. ES cells in culture differentiate to derivatives of all three germ layers and mimic some aspects of organogenesis when grown as aggregates in suspension to form embryoid bodies. Differentiation proceeds further when the embryoid bodies attach to suitable substrates. Muscle and neuronal cells are among the most readily identified cell types then formed. We examined the effect of all-trans retinoic acid (RA) and members of the transforming growth factor-beta family (TGF-beta 1, TGF-beta 2) under these conditions in an assay where single aggregates formed in hanging microdrops in medium supplemented with serum depleted of lipophilic substances which would include retinoids. Endoderm-like cells formed under all conditions tested. RA at concentrations of 10(-8) M and 10(-7) M induced the formation of neurons but in the absence of RA or at concentrations up to 10(-9) M, neurons were not observed. Instead, beating muscle formed in about one-third of the plated aggregates; this was greatly reduced when RA concentrations increased above 10(-9) M. Immunofluorescent staining for muscle specific myosin showed that two muscle cell types could be distinguished: elongated, non-contractile myoblasts and mononucleate flat cells. The mononucleate flat cells appeared to correspond with rhythmically contracting muscle. The number of non-contractile myoblasts increased 3-fold over controls in the presence of 10(-9) M RA. TGF-beta s increased the number of contractile and non-contractile muscle cells by a factor 3 to 7 over controls, depending on the TGF-beta isoform added and the muscle cell type formed. TGF-beta 2 also invariably increased the rate at which contracting muscle cells were first observed in replated aggregates. The stimulatory effect of TGF-beta s on the formation of mononucleate flat cells was completely abrogated by RA at 10(-9) M while the number of myoblasts under similar conditions was unchanged. These data suggest that a complex interplay between retinoids and TGF-beta isoforms may be involved in regulation of differentiation in early myogenesis. In the second approach, neutralizing polyclonal rabbit antibodies specific for TGF-beta 2 were injected into the cavity of mouse blastocysts 3.5 days post coîtum (pc). After 1 day in culture, embryos were transferred to pseudopregnant females. The number of decidua, embryos and resorptions were counted at day 8.5-9.5 pc.(ABSTRACT TRUNCATED AT 400 WORDS)

ESHRE guidelines for good practice in IVF laboratories. Committee of the Special Interest Group on Embryology of the European Society of Human Reproduction and Embryology.

Education has always been a priority for the European Society of Human Reproduction and Embryology (ESHRE). Many efforts have been dedicated to promoting knowledge of techniques, procedures and strategies in order to ensure use of the highest quality practices in reproductive medicine. The need to develop a set of guidelines was a logical consequence that found its first expression in 1990, when Focus on Reproduction (vol. 1, pp. 10-38) published the first guidelines which were distributed among the membership. Five years later a new, more complete edition with several novel techniques and developments appeared in Human Reproduction (vol. 10, pp. 1246-1271). Both have proved to be invaluable references. Five more years have now passed. The necessity to produce current guidelines for good IVF laboratory practice has provided the strongest motivation. This originated from the increasing awareness that embryologists have a duty to prevent unintentional incidents that might result from poor practice in the laboratory. Therefore, the Embryology Special Interest Group (SIG) undertook to draw up guidelines aimed at giving support and guidance to the laboratory staff. All the aspects required to provide a safe working system were taken into consideration by members of the SIG and their effort produced this document. We hope that it will assist staff in achieving the best clinical outcome for their patients.

Nuclear transfer and electrofusion in bovine in vitro-matured/in vitro-fertilized embryos: effect of media and electrical fusion parameters.

In this study, micromanipulation and electrofusion conditions for the cloning of in vitro-produced bovine embryos (here after termed IVM/IVF embryos) derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes were established. The effect of DC field strength on the fusion rate was tested in a model system using pronuclear stage embryos in which a cytoplasmic vesicle was removed and reinserted. Efficient fusion (80%) was obtained by applying a pulse of 1.75 kV/cm for 40 microseconds. In vitro development of manipulated pronuclear stage embryos was as efficient as that of unmanipulated control embryos. Different fusion media were compared in the cloning procedure, using IVM oocytes as recipients and blastomeres from day 6 IVM/IVF donor embryos. Zimmermann cell fusion medium reduced the lysis of nuclear transfer embryos compared to F300 (5% vs. 25%). The effects of drugs disrupting the microfilaments and microtubuli were determined. Neither the addition of cytochalasin B (CCB) for 1 hr in the postfusion medium nor incubation of donor blastomeres with nocodazole had a significant effect on the fusion or cleavage rate of the nuclear transfer embryos. Additional experiments demonstrated that there was no difference in developmental potential between nuclear transfer embryos allowed to develop in vitro or in vivo and that the embryos gave a 15% pregnancy rate in recipient cattle.

Sperm analysis in a subfertile male with a Y;16 translocation, using four-color FISH.

Sperm analysis was performed in a male with oligoasthenoteratozoospermia (OAT) and a reciprocal t(Y;16) (q11. 21;q24), using four-color FISH. Intracytoplasmic sperm injection (ICSI) treatment in this patient had resulted in the birth of one chromosomally balanced and two chromosomally normal children. To assess the risk of having a chromosomally unbalanced conception after ICSI, morphologically normal spermatozoa were studied with a set of probes allowing detection of all segregation variants. There were 51% normal or balanced sperm cells. The fraction of sperm products resulting from alternate and adjacent I segregation was 87%, 12% were products of 3:1 disjunction, and the other 1% had other types of aneuploidy. If morphologically abnormal cells were also included in the FISH analysis, nearly 90% of all the spermatozoa were unbalanced. We conclude that although the majority of males with a Y/autosome translocation are infertile due to azoospermia, our patient produces sufficient morphologically and chromosomally normal spermatozoa to have chromosomally normal or balanced offspring after ICSI. Assuming that ICSI with an unbalanced spermatozoon from this patient would result in a nonviable embryo in many cases, the combination of in vitro and subsequent in vivo selection probably results in a risk of unbalanced offspring of much less than 50%. Hence, FISH studies on the sperm of translocation carriers are useful for estimating the risk of having unbalanced offspring after ICSI and in understanding the mechanisms underlying infertility in such carriers.

Stage-specific appearance of the mouse antigen TEC-3 in normal and nuclear transfer bovine embryos: re-expression after nuclear transfer.

Bovine embryos, recovered from the uterus in vivo or derived from in vitro matured and in vitro fertilized oocytes, were investigated for the presence of the developmentally regulated mouse antigen TEC-3 by indirect immunofluorescence. During preimplantation embryo development TEC-3 is expressed on bovine morulae and blastocysts. It is absent from unfertilized and fertilized oocytes, and from all stages before the 32-cell stage. The finding that TEC-3 is not expressed before the onset of embryonic transcription, which occurs at the eight-cell stage in the bovine, but only when the embryonic genome is active, makes it a potential marker for studying nuclear reprogramming after nuclear transfer. Nuclear transfer embryos were made by electrical fusion of blastomeres from morulae derived in vivo with enucleated metaphase II oocytes. Indirect immunofluorescence with the TEC-03 antibody showed that the TEC-3 antigen, present on blastomeres of the morula stage embryo, disappeared after fusion and was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage. These data suggest that the bovine embryonic nucleus may be able to revert to the equivalent of an earlier developmental stage when transferred to ooplasm, and is then capable of following the normal developmental program.